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Identification and Expression of Invertase Genes in Populus

Permanent Link: http://ufdc.ufl.edu/UFE0021733/00001

Material Information

Title: Identification and Expression of Invertase Genes in Populus
Physical Description: 1 online resource (149 p.)
Language: english
Creator: Bocock, Philip N
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: colinearity, invertase, poplar, sugar
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Plant Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Invertase (EC 3.2.1.26) plays a key role in carbon utilization as it catalyzes the irreversible hydrolysis of sucrose into glucose and fructose. These sugars can act as both metabolic fuel and as signaling compounds directly affecting resource allocation in the plant and indirectly influencing the expression of genes responsive to shifts in hexose and sucrose availability. The invertase family in plants is composed of two sub-families thought to have distinct evolutionary origins and can be distinguished by their pH optima for activity: acid invertases and neutral/alkaline invertases. The acid invertases apparently originated in eubacteria and are targeted to the cell wall and vacuole, while neutral/alkaline invertases apparently originated in cyanobacteria and function in the cytosol. The recently sequenced genome of Populus trichocharpa (Torr. & Gray) allowed us to identify the genes encoding invertase in this woody perennial. Here we describe the identification of eight acid invertase genes; three of which belong to the vacuolar targeted group (PtVIN1-3), and five of which belong to the cell wall targeted group (PtCIN1-5). Similarly, we report the identification of 16 neutral/alkaline invertase genes (PtNIN1-16). Expression analyses using whole genome microarrays and RT-PCR reveal evidence for expression of all invertase family members. An examination of the microsyntenic regions surrounding the poplar invertase genes reveals extensive colinearity with Arabidopsis invertases. We also find evidence for expression of a novel intronless vacuolar invertase (PtVIN1), which apparently arose from a processed PtVIN2 transcript that re-inserted into the genome. To our knowledge, this is the first intronless invertase found in plants. The response of two poplar vacuolar invertases (PtVIN2 and -3) to exogenous sugar treatments was examined and compared to those of the Arabidopsis, maize and rice vacuolar invertase orthologs. We found that PtVIN2 and -3 exhibit a reciprocal response to both light and exogenous sugar treatments whereby PtVIN2 expression is repressed under both light and high sugar conditions while PtVIN3 is induced under the same conditions. This reciprocal response has been previously documented in other plant systems including Arabidopsis, maize and rice. An examination of the microsyntenic chromosomal regions containing vacuolar invertase reveals extensive colinearity between Arabidopsis and poplar, but does not include rice and maize. The conserved colinear structure of the chromosomal segments containing the vacuolar invertases in Arabidopsis and poplar, coupled with the conserved reciprocal responses of these vacuolar invertases to sugar in Arabidopsis, poplar, maize and rice, has led to the hypothesis that the reciprocal nature of this sugar response arose in an ancient genome duplication event that occurred prior to the monocot and eudicot divergence on the evolutionary tree. To better understand the role of invertase in sucrose export and sink development, yeast invertase (SUC2) was ectopically expressed in a Populus tremula x P. alba hybrid. Despite the efficacy of this transgenic approach in other plant systems resulting in whole-plant shifts in carbon allocation and dramatic changes in sugar accumulation; transgenic poplars showed no whole-plant carbon allocation shifts. Metabolic analyses revealed that while most of the transgenic poplar lines did not exhibit the expected alterations in sugar accumulation, intermediates in the glyoxylate and the tricarboxylic acid cycles did fluctuate relative to the non-transgenic controls.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Philip N Bocock.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Davis, John M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021733:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021733/00001

Material Information

Title: Identification and Expression of Invertase Genes in Populus
Physical Description: 1 online resource (149 p.)
Language: english
Creator: Bocock, Philip N
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: colinearity, invertase, poplar, sugar
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Plant Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Invertase (EC 3.2.1.26) plays a key role in carbon utilization as it catalyzes the irreversible hydrolysis of sucrose into glucose and fructose. These sugars can act as both metabolic fuel and as signaling compounds directly affecting resource allocation in the plant and indirectly influencing the expression of genes responsive to shifts in hexose and sucrose availability. The invertase family in plants is composed of two sub-families thought to have distinct evolutionary origins and can be distinguished by their pH optima for activity: acid invertases and neutral/alkaline invertases. The acid invertases apparently originated in eubacteria and are targeted to the cell wall and vacuole, while neutral/alkaline invertases apparently originated in cyanobacteria and function in the cytosol. The recently sequenced genome of Populus trichocharpa (Torr. & Gray) allowed us to identify the genes encoding invertase in this woody perennial. Here we describe the identification of eight acid invertase genes; three of which belong to the vacuolar targeted group (PtVIN1-3), and five of which belong to the cell wall targeted group (PtCIN1-5). Similarly, we report the identification of 16 neutral/alkaline invertase genes (PtNIN1-16). Expression analyses using whole genome microarrays and RT-PCR reveal evidence for expression of all invertase family members. An examination of the microsyntenic regions surrounding the poplar invertase genes reveals extensive colinearity with Arabidopsis invertases. We also find evidence for expression of a novel intronless vacuolar invertase (PtVIN1), which apparently arose from a processed PtVIN2 transcript that re-inserted into the genome. To our knowledge, this is the first intronless invertase found in plants. The response of two poplar vacuolar invertases (PtVIN2 and -3) to exogenous sugar treatments was examined and compared to those of the Arabidopsis, maize and rice vacuolar invertase orthologs. We found that PtVIN2 and -3 exhibit a reciprocal response to both light and exogenous sugar treatments whereby PtVIN2 expression is repressed under both light and high sugar conditions while PtVIN3 is induced under the same conditions. This reciprocal response has been previously documented in other plant systems including Arabidopsis, maize and rice. An examination of the microsyntenic chromosomal regions containing vacuolar invertase reveals extensive colinearity between Arabidopsis and poplar, but does not include rice and maize. The conserved colinear structure of the chromosomal segments containing the vacuolar invertases in Arabidopsis and poplar, coupled with the conserved reciprocal responses of these vacuolar invertases to sugar in Arabidopsis, poplar, maize and rice, has led to the hypothesis that the reciprocal nature of this sugar response arose in an ancient genome duplication event that occurred prior to the monocot and eudicot divergence on the evolutionary tree. To better understand the role of invertase in sucrose export and sink development, yeast invertase (SUC2) was ectopically expressed in a Populus tremula x P. alba hybrid. Despite the efficacy of this transgenic approach in other plant systems resulting in whole-plant shifts in carbon allocation and dramatic changes in sugar accumulation; transgenic poplars showed no whole-plant carbon allocation shifts. Metabolic analyses revealed that while most of the transgenic poplar lines did not exhibit the expected alterations in sugar accumulation, intermediates in the glyoxylate and the tricarboxylic acid cycles did fluctuate relative to the non-transgenic controls.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Philip N Bocock.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Davis, John M.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021733:00001


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IDENTIFICATION AND EXPRESSION OF INVERTASE GENES IN POPULOUS


By

PHILIP N. BOCOCK


















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

































2007 Philip N Bocock

































To my wife, Traci









ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. John Davis, who has helped to deepen my knowledge

of molecular biology and has taught me how to ask questions. Without his endless help, I would

not be here. I would also like to acknowledge my committee members: Drs. Curt Hannah, Karen

Koch, Tim Martin and Gary Peter for their valuable advice throughout this undertaking. Also, I

would especially like to thank Drs. Li-Fen Huang and Karen Koch whose collaboration have

made this project possible. I am also grateful to other faculty, staff, and graduate students for

their help and encouragement, especially Alison Morse, Kathy Smith, Chris Dervinis, Mike

Reed, Gogce Kayihan, Gustavo Ramirez, Rocio Diaz, John Mayfield and Diego Fajardo.

Without the support of my family this endeavor would have never been accomplished. I

must especially thank my wife, Traci, and our two children, Isaiah and Edward, for their love,

encouragement and support that have gotten me through this education.









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ..............................................................................................................4

L IS T O F T A B L E S ................................................................................. 8

LIST O F FIG U RE S ................................................................. 9

ABSTRACT ................... ............... ......... ... ...... ... .............

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ................................... .................13

P o p lar as a M o d el .......................................................................... 13
C arbon Transport and U utilization ............................................................................ ...... 13
S u c ro se ........................................................................14
Sucrose C leaving E nzym es............................................................................ ................... 14
A cid In v e rta se ................................................................................................................... 1 5
Proteinaceous Inhibitors of Acid Invertase ..................... ......................................16
Structure of A cid Invertases ........................................................................... 18
Neutral/Alkaline Invertase (Cytosolic Invertase) ................................. ............... 19
Regulation of Invertase: ...... ..... ............... ............... ........ .... ..... ........19
S u g a rs .............. .... ...............................................................1 9
H o rm o n e s ................................................................2 0
W wounding and Pathogens ...................... .... ......... .. .......................... ............... 20
Environm mental Stim uli ..................................................... ....... ... 21
Invertase Investigations in Transgenic Plants..................................... ........................ 22
Research Objectives.......... .......... .................................. 23

2 EVOLUTION AND DIVERSITY OF INVERTASE GENES IN Populus trichocarpa.......27

In tro d u ctio n .......... .... .. ......... ............................................................................................ 2 7
M materials and M methods ...................................... .. .......... ....... ...... 29
Plant M material ............................................................... .. ........ 29
BLAST Searches and DNA Annotation ...........................................................30
Construction of Sequence Sim ilarity Trees ................................................. ............... 30
Identification of G ene D uplication........................................................... ............... 30
Isolation of RN A and RT-PCR A ssay.................................... .......................... ......... 31
Q uantitative R T-PCR .................. ................... ........ ................................ .... ... 31
G enom ic D N A Isolation ......................................................................... ...................32
M icroarray D esign and A analysis ......................................................... ............... 32
R e su lts ............... ............ ................................................ ................ 3 3
Identification of Poplar Invertase Genes .............. ......................................... 33
Structure of the Poplar Invertase Genes ..............................................34
Expression of the Poplar Invertase Gene Family ....................... ...................35









Evolutionary Development of the Poplar Invertase Family ................. ............... 35
Putative Evidence for Origins of PtVIN ............................... ... ........................ 39
D iscu ssio n ................... ...................3...................9..........

3 RECIPROCAL SUGAR REGULATION IS CONSERVED AMONG VACUOLAR
INVERTASAES OF POPLAR, ARABIDOPSIS, MAIZE AND RICE ...............................56

In tro du ctio n ................... ...................5...................6..........
M materials and M methods ................................... ... .. .......... ....... ...... 58
Plant Material ................................. .......................58
Sequence A lignm ents and Sim ilarity Trees ........................................ .....................59
R N A E extraction ................... .......................................... ............. .... ........59
Quantitative RT-PCR in Poplar and Pine.............................................. .................. 59
Quantitative RT-PCR in Arabidopsis ............... .. ......... .... ..................... 60
Sugar Treatments for Poplar, Arabidopsis, Pine and Rice...........................................61
Light-Dark Treatments for Poplar, Pine and Arabidopsis.............................................61
R esults..................................... ... .. ...... ........ ........ ........................ 62
Predicting Gene Orthology Using Protein Sequence Similarity ..................................62
A Chromosome Duplication Event is Responsible for the Two Member VIN Family
in P oplar and A rabidop sis ........................................................ ........................... ... 64
Sugar Response Demonstrates Conservation of Gene Function in VINs........................65
R eciprocal R response in L ight............................................................... .....................66
D iscu ssio n ................... ...................6...................7..........

4 OVEREXPRESSION OF YEAST INVERTASE IN POPLAR ........................................76

Intro du action ............. ...... ..76......................................
M materials and M methods .................. ..................... ................ ...................... 78
Plant Material, Transgenesis, and Growth Conditions.................................................78
V sector C onstruction.................................................... ........ .......... .. .............78
Construction of Similarity Trees .............................. ......... ... ................... 79
Isolation of RNA, Generation of cDNA and Real-Time PCR Assay.............................79
Genomic DNA Isolation and PCR Amplification............................ ............... 80
Metabolic Profiling: Extraction, Separation and Identification ....................................81
Construction and Experimental Design of Grafts ................................. ............... 81
Measurement of Photosynthesis and Respiration.....................................................82
Protein Extraction ...................................................................... ........ 83
Total Invertase A activity A ssay .............. ..... ..... .......... .. ............................... 84
Detection of Invertase Activity in Native Polyacrylamide Gels ..............................84
Sugar and Starch Determination...................................................... 84
Results and D discussion ................. ..... .... ..... .. .. ...... .............. 85
Construction of Overexpressing Yeast Invertase Vectors....................................85
Expression of SUC2 in Poplar................ ............................. ................. ............... 86
Y east Invertase A ctive in Cytosol .............................................. ............... ....87
Metabolic Profiling Reveals Alterations in CwSUC2 and VacSUC2 Transgenic
L in es..... . ..........................................................88



6









Metabolic Profiling Reveals Alterations in Sugar Accumulation in CytSUC2
Transgenic L ines ................................... .............................. ........... 89
Whole-Plant Phenotypes are not Apparent ............. ............................................ 90
C including R em arks ................. .................................. ................ ............... 90

5 CONCLUSIONS ................... ......... .. ...... ... ..................102

APPENDIX

A REPRESSION OF INVERTASE IN POPULUS........................... ......... ............. 106

Introduction ......... ... ............................ ......... ............. ........ 106
M materials and M ethods ............. .......................... ............................................... 107
Plant Material, Transgenesis, and Growth Conditions.................................................107
V sector C onstruction........................ ..... .................... ............ ........ .............. 107
Genomic DNA Isolation and PCR Amplification.................................... ................ 108
R e su lts ........................................................................................................1 0 9
Construction of RN A i V ector................................................ ............................ 109
Insertion of Target Gene Sequence .................................. 109
Further W ork ............................................................... .... .... ......... 110

B REGULATION OF INVERTASE: A"SUITE" OF TRANSCRIPTIONAL AND POST-
TRANSCRIPTIONAL M ECHANISM S ................................................... .................. 115

A b stra ct ............................. ......... ... .. ................................................... 1 1 5
Introduction.............. ...... ..... ............................. ......................116
Compartmentalization of Vacuolar Invertases in Precursor Protease Vesicles (PPV)......... 117
W all-A associated K inase (W A K ) .................................................................................... 119
O their K inases A affecting Invertases......................................................................... ...... 120
Differential Sugar Regulation of Invertases .............. ................................................ 121
R N A T urn O ver and D ST ............................................... ............................. ................... 124
Invertase Inhibitors ....................................................... ............ .. ............ 125
S u m m ary ................... ...................1...................2.........7
A know ledgem ents ...................................................................... .. .. ....... ............. .. 128

L IST O F R E F E R E N C E S ..................................................................................... ..................13 1

B IO G R A PH IC A L SK E T C H ......................................................................... .. ...................... 149









LIST OF TABLES


Table page

2-1 Nomenclature and chromosomal location of 24 poplar invertase genes.............................44

2-2 Primers used in RT-qPCR and RT-PCR......................................... ........ ... ............... 49

3-1 Percent similarity of predicted VINs from poplar, Arabidopsis, rice and maize ..................71

4-1 Metabolites altered in CwSUC2 and VacSUC2 overexpressing plants.............................99

4-2 Metabolites altered in CytSUC2 overexpressing plants ................................. ...............100

4-3 Summary of phenotypic experiments performed. .................... .... ..................101

A-i Primer sequences used for -200 bp target sequence cloning. ....................................113

A-2 Summary of RNAi constructs made ..................................................... ................... 114









LIST OF FIGURES


Figure page

1-1 Sucrose synthesis, transport and metabolism in source and sink cells...............................24

1-2 Protein inhibitors of cell wall invertases share strong sequence similarity with
inhibitors of pectin methylesterase .............. ... ........ ........................ .................. 25

1-3 Representation of cell wall and vacuolar isoforms of carrot invertase...............................26

2-1 Exon-intron structures of predicted invertase genes.............................................................45

2-2 Invertase am ino acid sim ilarity trees ....................................................................... 46

2-3 Expression of poplar invertase genes ......................................................... .................... 47

2-4 Evolutionary development of the poplar acid invertase family.................... ............ 48

2-5 Amino acid alignment of poplar invertases...................... .................... ...............55

3-1 Protein sim ilarity tree of V IN s ............................... ............................................................... 72

3-2 Phylogenetic representation of relevant plant species ........................................................73

3-3 Conservation of specific VINisoform transcript repression under sugar treatments
acro ss tax a ...............................................................................................74

3-4 Conservation of reciprocal regulation of VINtranscript under dark and light treatments
acro ss tax a ...............................................................................................7 5

4-1 Schematic representation of yeast invertase overexpressing constructs targeted to the
cytosol, cell w all and v acu ole ........................................ .............................................93

4-2 S. cerevisiae SUC2 in relationship to other plant invertases.....................................95

4-3 Relative expression of double 35S driven SUC2 in root, stem and leaf.................... ........ 96

4-4 Relative expression of SUC2 transcript in overexpressing transgenic events in leaf............97

4-5 CytSUC2 transgenic plants display increased invertase activity due to presence of
tran sg en e .................................................................................9 8

A-1 Schematic representation of pCAPT vector used for RNAi silencing ...........................111

A-2 Schematic representation of RNAi directed constructs...................................................... 112

B-l Recent additions to mechanisms controlling invertases ............................................. 129









B-2 Protein inhibitors of cell wall invertases share strong sequence similarity with
inhibitors of pectin m ethylesterase ............. ............ .................. ........ .................... 130









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

IDENTIFICATION AND EXPRESSION OF INVERTASE GENES IN POPULOUS

By

Philip N. Bocock

December 2007

Chair: John M. Davis
Major: Plant Molecular and Cellular Biology

Invertase (EC 3.2.1.26) plays a key role in carbon utilization as it catalyzes the irreversible

hydrolysis of sucrose into glucose and fructose. These sugars can act as both metabolic fuel and

as signaling compounds directly affecting resource allocation in the plant and indirectly

influencing the expression of genes responsive to shifts in hexose and sucrose availability. The

invertase family in plants is composed of two sub-families thought to have distinct evolutionary

origins and can be distinguished by their pH optima for activity: acid invertases and

neutral/alkaline invertases. The acid invertases apparently originated in eubacteria and are

targeted to the cell wall and vacuole, while neutral/alkaline invertases apparently originated in

cyanobacteria and function in the cytosol. The recently sequenced genome of Populus

trichocharpa (Torr. & Gray) allowed us to identify the genes encoding invertase in this woody

perennial. Here we describe the identification of eight acid invertase genes; three of which

belong to the vacuolar targeted group (PtVIN1-3), and five of which belong to the cell wall

targeted group (PtCIN1-5). Similarly, we report the identification of 16 neutral/alkaline invertase

genes (PtNIN1-16). Expression analyses using whole genome microarrays and RT-PCR reveal

evidence for expression of all invertase family members. An examination of the microsyntenic

regions surrounding the poplar invertase genes reveals extensive colinearity with Arabidopsis









invertases. We also find evidence for expression of a novel intronless vacuolar invertase

(PtVINI), which apparently arose from a processed PtVIN2 transcript that re-inserted into the

genome. To our knowledge, this is the first intronless invertase found in plants. The response of

two poplar vacuolar invertases (PtVIN2 and -3) to exogenous sugar treatments was examined and

compared to those of the Arabidopsis, maize and rice vacuolar invertase orthologs. We found

that PtVIN2 and -3 exhibit a reciprocal response to both light and exogenous sugar treatments

whereby PtVIN2 expression is repressed under both light and high sugar conditions while

PtVIN3 is induced under the same conditions. This reciprocal response has been previously

documented in other plant systems including Arabidopsis, maize and rice. An examination of the

microsyntenic chromosomal regions containing vacuolar invertase reveals extensive colinearity

between Arabidopsis and poplar, but does not include rice and maize. The conserved colinear

structure of the chromosomal segments containing the vacuolar invertases in Arabidopsis and

poplar, coupled with the conserved reciprocal responses of these vacuolar invertases to sugar in

Arabidopsis, poplar, maize and rice, has led to the hypothesis that the reciprocal nature of this

sugar response arose in an ancient genome duplication event that occurred prior to the monocot

and eudicot divergence on the evolutionary tree. To better understand the role of invertase in

sucrose export and sink development, yeast invertase (SUC2) was ectopically expressed in a

Populus tremula x P. alba hybrid. Despite the efficacy of this transgenic approach in other plant

systems resulting in whole-plant shifts in carbon allocation and dramatic changes in sugar

accumulation; transgenic poplars showed no whole-plant carbon allocation shifts. Metabolic

analyses revealed that while most of the transgenic poplar lines did not exhibit the expected

alterations in sugar accumulation, intermediates in the glyoxylate and the tricarboxylic acid

cycles did fluctuate relative to the non-transgenic controls.









CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW

Current population growth combined with increasing development and urbanization

worldwide is putting strain on the terrestrial ecosystem's main carbon sink, forests. One way to

combat this increasing demand of decreasing forest resources is to manipulate carbon

sequestration patterns in trees in order to direct carbon allocation into the most desirable organs

such as stems, to meet industrial demands, or roots to help increase long term carbon storage

capacity in the soil. Unfortunately, these mechanisms are not well understood. Carbon

sequestration is not part of a single pathway, but a culmination of many pathways dealing with

carbon absorption through photosynthesis, carbon loss through respiration, carbon transport

through the vascular system, carbon partitioning within a cell, and carbon allocation into long-

term storage compounds such as lignin.

Poplar as a Model

The perennial tree, poplar, has emerged as a model species for physiology and genetics

research. Poplar is amenable to transformation, can be clonally propagated and has a haploid

genome size of 480 million base pairs which is roughly four times larger than Arabidopsis, has

been sequenced and encodes approximately 45,000 genes (Bradshaw and Stettler, 1995;

Bradshaw et al., 2000; Tuskan et al., 2006). Poplar also is an economically important tree on the

global market with interest from industry on its improvement.

Carbon Transport and Utilization

Plants utilize carbon by precisely partitioning the reduced carbon obtained through

photosynthesis into different locations within the cell and subsequently allocating it throughout

the plant (Figure 1-1). Photosynthesis reduces carbon dioxide into sugars that can be ushered into

glycolysis and the tricarboxylic acid cycle for the production of ATP and NADH, or the sugars









can be used as a source of carbon for the synthesis of primary and secondary metabolites

essential for the growth and development of the plant. Alternatively, these sugars can be

converted into starches, triacyl glycerides or polypeptides for long-term storage (Sturm, 1999).

These same sugars are also used to synthesize sucrose, the primary sugar used for carbon

transport in higher plants. Sucrose can be transiently stored in the vacuole and then exported to

the non-photosynthesizing sink tissues via the phloem. In source cells, sucrose is synthesized in

the cytosol, stored for later use in the vacuole, travels to neighboring cells through

plasmodesmata, and eventually enters the phloem via plasmodesmata or from the surrounding

apoplast (Turgeon and Hepler, 1989; Grusak et al., 1996).

Sucrose

Sucrose and its glucose and fructose cleavage products are primary molecules in

carbohydrate translocation, sugar signaling, and osmotic maintenance. Sucrose is a non-reducing

disaccharide composed of an al-p2 linked glucose and fructose. The cleavage of a single sucrose

molecule in solution results in two molecules of hexoses thereby doubling the osmotic potential

of the solution. By compartmentalizing sucrose and/or the hexoses in various cellular

compartments, osmotic gradients are realized, providing the basis for short-distance transport

from cell to cell, as well as long-distance transport from organ to organ via the phloem as first

proposed by Munch (1930). Sucrose and its component hexoses also provide sugar signals that

trigger numerous biological pathways playing roles in cell division, expansion, differentiation

and maturation (Koch, 2004).

Sucrose Cleaving Enzymes

Plants possess two enzymes that have the capability of cleaving sucrose: sucrose synthase

(EC 2.4.1.13) and invertase (EC 3.2.1.26). Sucrose synthase is a glycosyl transferase catalyzing a

reversible reaction converting sucrose into UDP-glucose and fructose in the presence of UDP.









Invertase, in contrast, hydrolyzes sucrose into glucose and fructose in an irreversible reaction.

The free energy of these two reactions are -4.18 KJ and -27.6 KJ, respectively (Ap Rees, 1984).

The invertase family can be grouped according to two properties: cellular location and pH

optima for activity (Winter and Huber, 2000). The first group, known as the acid invertases, has

a pH optimum of between 4.5 and 5.0 and consists of an insoluble, extracellular, cell wall-bound

form and a soluble form located in the lumen of the vacuole (Haouazine-Takvorian et al., 1997;

Sherson et al., 2003). The second group, known as the neutral/alkaline invertases, has a pH

optimum of about 7.0-7.8. The neutral/alkaline invertases are entirely soluble and appear to be

located in the cytosol (Chen and Black, 1992; Van den Ende and Van Laere, 1995).

Acid Invertase

Acid invertases are P-fructofuranosidases that hydrolyze not only sucrose, but also other 3-

fructose containing oligosaccharides such as raffinose and stachyose. The acid invertase Km for

sucrose is in the low mM range and activity can be inhibited by Tris buffer, heavy metal ions

such as Hg2+ and Ag2+, as well as other divalent cations such as Mg2+, Ca2+, and Zn2+ (Krausgrill

etal., 1996; Tymowska-Lalanne and Kreis, 1998b).

Acid invertases have also been shown in a variety of species to be glycosylated. Some

examples are in radish (Faye and Ghorbel, 1983), maize (Doehlert and Felker, 1987), carrot

(Lauriere et al., 1988; Stommel and Simon, 1990; Unger et al., 1992), and tobacco (Weil and

Rausch, 1990). In yeast, there are two isoforms of acid invertase that are encoded by a single

gene, but arise from differential splicing of exons. One isoform is glycosylated and targeted to

the extracellular space, while the other is non-glycosylated and is located in the cytosol. This

glycosylation seems to have no effect on the enzymatic properties of acid invertase (Faye et al.,

1981), but is instead required for its transport to the secretary pathway where it is directed to the









apoplastic and vacuolar spaces (Carlson and Botstein, 1982; Perlman et al., 1982; Bergh et al.,

1987). In contrast to yeast, the invertase isoforms in plants are encoded by different genes.

Invertase can range in molecular mass from a 48 kD monomeric form found in radish

(Faye et al., 1981) to a 450 kD oligomeric form found in lily pollen (Singh and Knox, 1984),

however, most molecular masses are around 60 kD. Analyses of some invertases under

denaturing conditions on SDS gels show the presence of proteolytic fragments. A 70 kD

vacuolar invertase monomer from mung bean hypocotyls was processed into a 30 kD N-terminal

fragment and a 38 kD C-terminal fragment (Arai et al., 1991). In carrot, a 68 kD monomer of

vacuolar invertase was processed into an N-terminal fragment of 43 kD and a C-terminal

fragment of 25 kD (Unger et al., 1992; Unger et al., 1994). The role of this fragmentation is

unknown, but under native conditions these fragments are tightly associated.

Proteinaceous Inhibitors of Acid Invertase

Cell wall and vacuolar invertase activity can be regulated by a family of proteinaceous

inhibitors known as cell wall inhibitor of fructosidase (CIF) and vacuolar inhibitor of

fructosidase (VIF), or collectively as C/VIF (Rausch and Greiner, 2004). Although CIF are cell

wall invertase inhibitors, they are broadly active against both cell wall and vacuolar invertases.

In contrast, VIF inhibition is specific to vacuolar invertases. Neither of the inhibitors affect

fungal invertases indicating a minimal role for these interactions in pathogen defense (Greiner et

al., 1998; Greiner et al., 1999; Link et al., 2004). The C/VIF related protein family is not highly

conserved, and in Arabidopsis, sequence identities range from roughly 20 % to 40 % for

approximately 14 family members (Rausch and Greiner, 2004). The C/VIF related protein family

also contains pectin methylesterase inhibitors (PMEI). PMEI are indistinguishable from the

C/VIF by sequence alone and retain nearly identical structures to the C/VIF (Giovane et al.,

2004; Hothorn et al., 2004a; Hothorn et al., 2004b; Di Matteo et al., 2005).









Important implications arise from the capacity of these related proteinaceous inhibitors to

distinguish between their targets. Arabidopsis has only eight putative acid invertases (six cell

wall and two vacuolar), but more than 60 pectin methylesterase (PME)-related genes based on

sequence similarity (Micheli, 2001; Sherson et al., 2003; Rausch and Greiner, 2004; De Coninck

et al., 2005). X-ray crystollagraphy has revealed that CIF is conformationally stable over a broad

pH and temperature range, however, the invertase/inhibitor complex is only stable at an acidic

pH. This indicates that binding is determined not by conformational changes, but rather by pH-

induced changes at the interface of the invertase/inhibitor complex (Hothorn et al., 2004a;

Hothorn and Scheffzek, 2006). In contrast, the highly similar structure of the PMEI was found to

undergo large structural rearrangements (Figure 1-2) under these same conditions suggesting

PMEI uses a different mode of binding than does the CIF (Hothorn et al., 2004b). PMEI contains

a flexible a-hairpin that is important both in dimer formation and in binding of PME (Hothorn et

al., 2004b). Chimeral domain swap experiments of the a-hairpin domain between PMEI and CIF

indicate that this domain of PMEI is necessary and sufficient for activity against PME; however,

the corresponding a-hairpin in NtCIF is not sufficient for invertase inhibition (Hothorn et al.,

2004b). The approximately 28 amino acid residues encoding this a-hairpin may be key in

distinguishing these two classes of inhibitors (Hothorn et al., 2004b).

Analysis of PMEI and CIF crystal structures not only clarifies interactions between these

inhibitors and their targets, but also aids our understanding of how distinct functions can arise for

proteins sharing very similar structures and ancestry. It is worth noting, however, that the

crystallographic analyses were performed on the cell wall invertase inhibitor and thus do not

explain the apparently narrower substrate specificity of the vacuolar invertase inhibitor. These

data indicate that the VIF may use a different mode of action than the CIF. It is also of interest









that two proteins in Arabidopsis previously annotated based on sequence similarity as cell wall

invertases (AtcwINV3&6), may actually be fructan exohydrolases (FEH). FEH protein

sequences are nearly identical to those of demonstrated acid invertase family members, yet the

recombinant enzymes are completely inactive against sucrose (De Coninck et al., 2005). It has

yet to be determined if the C/VIF family can distinguish between FEH and the known invertase

and PME substrates. Given the level of sequence diversity within the inhibitor family it seems

likely that biochemical and structural analyses will be required to resolve these questions.

Structure of Acid Invertases

Acid invertases are initially synthesized as pre-proteins containing a leader sequence that is

cleaved upon entry into the secretary pathway, the mature peptide sequence, and in the case of

the vacuolar invertases, a short C-terminal extension (Figure 1-3). The leader sequence consists

of two parts: a signal peptide and an N-terminal extension of unknown function, but is thought to

play a role in protein folding, targeting, or activity (Sturm, 1999). The signal peptide is required

for entry into the endoplasmic reticulum and from there to the apoplast (Blobel, 1980) or to the

vacuole if the required vacuolar targeting domain is present (Neuhaus and Rogers, 1998). The

vacuolar targeting domain is thought to be located either in the C-terminal extension or in the N-

terminal propeptide which is longer in vacuolar invertases than in cell wall invertases.

Plant acid invertases have two common features in their sequences. Towards the N-teminal

end of the protein is the P-fructosidase domain, NDPN, which is usually encoded by a mini-exon

of only 9 nucleotides. Towards the C-terminal end of the protein is another conserved sequence

consisting of the amino acids WECXDF. In potato, Bournay (1996) observed that under cold

stress, the mini-exon of the potato pCD1 11 cell wall invertase encoding the NDPN domain is

skipped in an alternative splicing event.









Neutral/Alkaline Invertase (Cytosolic Invertase)

Neutral/alkaline invertases are located in the cytosol and have a pH optimum in the neutral

to slightly alkaline range. In contrast to the acid invertases, neutral/alkaline invertases are not

glycosylated and hydrolyze only sucrose indicating that they are not fructofuranosidases.

Neutral/alkaline invertase activity is strongly inhibited by its reaction products; however, activity

is not inhibited by heavy metal ions indicating a different catalytic site than that of the acid

invertases. To date, neutral/alkaline invertases have not been found in association with a protein

inhibitor.

Regulation of Invertase

Sugars

Sugars play important roles in plants not only as fuel for metabolism, but they also

generate osmotic pressure and act as signaling molecules for various metabolic pathways. Sugars

have been shown to act as repressors of genes related to sugar acquisition and mobilization as

well as activators of genes related to sugar storage and utilization (Koch, 1996; Rolland et al.,

2002; Halford and Paul, 2003). As one of only two enzymes able to cleave sucrose, invertase is

an important player in sugar signaling. The majority of acid invertases have been shown to be

sugar induced rather than repressed (Roitsch, 1999; Roitsch and Ehness, 2000). This is consistent

with their proposed roles in carbon use versus carbon acquisition (Koch, 1996; Rolland et al.,

2002; Koch, 2004). However, some isoforms of vacuolar invertase have been shown to be

inhibited by sugars. This has been the case in tomato where transcripts for vacuolar invertase

TIV1 were shown to be reduced after treatment with 20 mM glucose (Godt and Roitsch, 1997b).

In maize, transcript as well as activity for vacuolar invertase Ivrl was also shown to be repressed

in the presence of 4 % glucose (Xu et al., 1996).









Hormones

Phytohormones play a central role in controlling growth, differentiation and development

in plants. As such, these hormones are particularly involved in regulating sink strength (Kuiper,

1993) and carbohydrate partitioning (Brenner and Cheikh, 1995). Invertases have been shown to

be regulated by the full set of phytohormones (Roitsch et al., 2003). In most cases this is related

to an increased demand for carbohydrates as a result of the hormone stimulated growth. In a

promoter study of the Lin5 invertase promoter linked to a reporter gene in tomato, it was shown

that gibberellic acid, auxin, and ABA all increased GUS activity indicating induction of this

isoform by these hormones (Proels et al., 2003). Work has also been done in maize

demonstrating that ABA increases the accumulation of the vacuolar invertase Ivr2 activity

(Trouverie et al., 2003). Brassinosteroids (Goetz et al., 2000), zeatin (Godt and Roitsch, 1997b),

ethylene (Linden et al., 1996) and cytokinin (Ehness and Roitsch, 1997) have all also been

shown to alter transcript accumulation of various invertase isoforms in plants.

Wounding and Pathogens

The wounding of plants is a common occurrence that can result in water loss as well as

pathogen infection. Response to wounding therefore often involves mechanisms intended to

counter osmotic stress as well as pathogen attack (Reymond and Farmer, 1998; Reymond et al.,

2000). As invading organisms alter sugar levels and effectively increase the sink of the tissue

being invaded, increases in invertase activity and expression logically follows. It is often difficult

to distinguish the invertase activity of the invading pathogen from that of the host plant (Ruffner

et al., 1992). In 1990, it was shown that carrot extracellular invertase transcript accumulates

dramatically after bacterial infection in the roots and leaves as well as after mechanical

wounding (Sturm and Chrispeels, 1990a). Results obtained from studying infection by biotrophic

fungi (Fotopoulos et al., 2003), necrotrophic fungi (Benhamou et al., 1991) and viruses (Herbers









et al., 2000) all show increases in invertase activity. Wounding has been shown in numerous

systems to correlate with increases in invertase activity and transcript level. An increase in either

one or both of these has been demonstrated in carrot (Sturm and Chrispeels, 1990a; Ramloch-

Lorenz et al., 1993), red goosefoot (Ehness and Roitsch, 1997), tomato (Ohyama et al., 1998),

sugar beet (Rosenkranz et al., 2001) and pea (Zhang et al., 1996).

Environmental Stimuli

The partitioning and allocation of photoassimilates can be affected by several

environmental factors such as temperature, gravity, light, wounding, drought and nutrient

availability (Wardlaw, 1990). As would be expected, these stimuli have all been demonstrated to

alter invertase transcript abundance and/or activity levels. Cold temperatures have stimulated

invertase transcripts that were undetectable at higher temperatures in potato tubers (Pressey and

Shaw, 1966) and Jerusalem artichoke tubers (Goupil et al., 1988). In sweet potato (Huang et al.,

1999) and tulip (Balk and de Boer, 1999), cold temperatures were shown to dramatically increase

the activity of invertase. Alternative splicing has also been shown to occur in potato resulting in

the elimination of the 0-fructosidase motif (Bournay et al., 1996). Invertase transcripts are also

induced and peak at one hour after gravistimulation (Kaufman et al., 1985; Wu et al., 1993a; Wu

et al., 1993b). Far-red light also increases cell wall invertase activity in radish (Zouaghi and

Rollin, 1976) and wheat (Krishnan et al., 1985). Salinity generally causes a reduction in sink

enzyme activities. These in turn could contribute to observed increases in sucrose of source

leaves and decreases in photosynthesis from both feedback inhibition and sugar-repression of

photosynthetic genes. Experiments in tomato revealed that in a salt sensitive strain, growth and

photosynthesis were not positively correlated as they were in the salt tolerant strain. In this salt

sensitive strain, growth and photosynthesis were both negatively correlated with glucose,

fructose and sucrose accumulation in young and old leaves, suggesting a blockage in their use for









growth. In the salt tolerant variety, transient increases in these same sugars were accompanied by

increases in acid invertase activity (Balibrea et al., 2000).

Invertase Investigations in Transgenic Plants

Invertase's irreversible reaction combined with its location in a variety of cellular

compartments makes it a key component in the carbon utilization strategy of the plant and thus

an ideal target for manipulation to understand carbon allocation and partitioning.

Several groups working with tomato (Dickinson et al., 1991), tobacco (Sonnewald et al.,

1991) and potato (Heineke et al., 1992) all saw very similar results after overexpressing the yeast

invertase, SUC2, in the apoplast, cytosol, and vacuole. These invertase overexpressors all had the

common theme of turning source tissues into sink tissues and reducing the sucrose available for

export. All plants demonstrated stunted growth and reduced root formation, including a reduction

in the number of tubers produced in the case of potato. These tubers also had an increase in the

protein to starch ratio. The mature (source) leaves in all three plants accumulated starches in

addition to simple sugars, while in the case of the cytosolic overexpressors, these accumulations

were also seen in the young (sink) leaves. Leaves were curved indicating rapid cell expansion or

division. The plants also demonstrated bleaching and necrotic regions that appeared to be linked

to the source state of the leaf. These lesions began at the leaf margin and moved inward while

also being accompanied by a reduction in photosynthesis.

The cell wall and vacuolar invertases have been repressed in several plant species,

including tomato (Ohyama et al., 1995), potato (Zrenner et al., 1996), and carrot (Tang and

Sturm, 1999). However, the results from these experiments were more varied than those of the

invertase overexpressors, although the theme of increased sucrose content accompanied by a

decrease in hexoses pervaded. In tomato these alterations in sugar accumulations appeared in

both the fruit and leaves. In addition to altered sugar levels, the fruit had elevated rates of









ethylene evolution. The wounding induced activity of invertase was also suppressed in the

transgenic plants.

The results from repressing the cell wall and vacuolar invertases in carrot are probably the

most interesting. Growth was shown to be altered at very early stages of development. The

transgenic, cotyledon-stage embryos were still masses of cells while the control plants had

already developed two to three leaves and one primary root. However, when these plantlets were

grown on media supplemented with hexoses, their growth returned to normal. After maturation

and transfer to soil, the plants expressing the cell wall antisense cDNA had a much bushier

appearance and accumulated more sucrose and starch than the controls. The taproot size was also

reduced and contained lower levels of carbohydrates than that of the controls. The dry weight

ratio of leaf to root shifted from 1:3 in the control to 17:1 in the cell wall antisense transgenics.

The vacuolar antisense plants also had increased numbers of leaves, although the tap root

developed normally, yet slightly smaller. The leaf to shoot ratio in these plants was 1.5:1.

Research Objectives

The purpose of this research was to investigate invertase contributions to carbon allocation

and partitioning in a woody perennial. To achieve this goal, the invertase family was identified

and annotated using the sequenced genome ofPopulus trichocarpa. Expression characteristics of

the invertase family were then examined across multiple tissues as well as through various

exogenous treatments including auxin, nitrogen-supplemented fertilizer and wounding. The

response of two poplar vacuolar invertases (PtVIN2 and -3) to exogenous sugar treatments was

examined and compared to the sugar responses ofArabidopsis, maize and rice. A reverse genetic

approach was also employed by ectopic expression of yeast invertase in transgenic poplar. In

conjunction, RNAi was used to target endogenous poplar invertases for down-regulation.
































Figure 1-1. Sucrose synthesis, transport and metabolism in source and sink cells as well as
invertase localization. This cartoon demonstrates apoplastic phloem loading in the
source leaf and apoplastic or symplastic phloem unloading in the sink tissue. In this
scheme, sucrose (S) is synthesized in the cytosol from triose-phosphates (TP).
Sucrose then enters the apoplast where it is actively transported into the phloem by a
sucrose proton symporter. This phloem loading step can be affected by activity levels
of the apoplastically located cell wall invertase (CIN) which hydrolyzes sucrose into
glucose (G) and fructose (F). Sucrose can be unloaded from the phloem either
symplastically or apoplastically. From the apoplast, sucrose can enter the sink cell
directly via a sucrose proton symporter, or be hydrolyzed by CIN into glucose and
fructose. In the sink cells, sucrose can be cleaved by sucrose synthase (SUSY) into
UDP-glucose (UDPG) and fructose, or hydrolyzed by a neutral/alkaline invertase
(NIN). Sucrose can also enter the vacuole where it can then be hydrolyzed by a
vacuolar invertase (VIN). After phosphorylation by hexokinase (HK), the hexoses can
enter respiration. Figure adapted from Rausch et al. (2004).


































Figure 1-2. Protein inhibitors of cell wall invertases (CIF, cell wall inhibitor of fructosidase)
share strong sequence similarity with inhibitors of pectin methylesterase (PMEI,
pectin methylesterase inhibitor), but crystal structures indicate important differences
in flexibility. The top panel depicts CIF. The oval denotes an a-hairpin thought
responsible for binding specificity of CIF and PMEI. The a-hairpin in CIF is rigid at
all pHs and temperatures tested. The bottom panel shows PMEI in three different
conformations that demonstrate the flexibility of the PMEI a-hairpin. (Hothorn et al.,
2004a; Hothorn et al., 2004b; Hothorn and Scheffzek, 2006). Images were
constructed by Protein Explorer (Martz, 2002). Figure adapted from Huang et al.
(2007).









cell wall invertase:


O 7, NDPNG WECXDF

vacuolar invertase:

NDPNG WECXDF

t I


signal N-terminal
peptide propeptide


mature
polypeptide


C-terminal
extension


Figure 1-3. Representation of cell wall and vacuolar isoforms of carrot invertase (Sturm, 1999).









CHAPTER 2
EVOLUTION AND DIVERSITY OF INVERTASE GENES IN POPULUS TRICHOCARPA

Introduction

Invertase (EC 3.2.1.26), also known as a P-fructofuranosidase, catalyzes the irreversible

hydrolysis of sucrose into glucose and fructose, indicating this enzyme has a key role in carbon

utilization. These sugars are synthesized in photosynthesizing source leaves and transported to

non-photosynthesizing sink tissues. Sucrose is the primary form of sugar transport in most

plants, establishing this disaccharide and its glucose and fructose components as central to plant

growth and development.

The invertase family is composed of two smaller sub-families distinguished by their pH

optima for activity and are thought to have distinct evolutionary origins in plants (Winter and

Huber, 2000). The acid invertase sub-family is targeted to either the cell wall or vacuole

(Haouazine-Takvorian et al., 1997; Sherson et al., 2003) and is believed to have originated from

respiratory eukaryotes and aerobic bacteria (Sturm and Chrispeels, 1990b). The neutral/alkaline

invertases appear to be localized to the cytosol (Chen and Black, 1992; Van den Ende and Van

Laere, 1995) and are found in cyanobacteria, where the family is thought to have originated

(Vargas et al., 2003), green algae and plants. The presence of these two sub-families reflect the

hypothesized origin of green algae and higher plants; the endosymbiotic event in which a

cyanobacteria invaded a non-photosynthetic, respiratory eukaryote (Mereschkowsky, 1905;

Margulis, 1981; Margulis and Sagan, 2003).

Members of the acid invertase sub-family share enzymatic and biochemical properties as

well as sequence similarity. Both vacuolar and cell wall targeted isoforms are P-

fructofuranosidases that can hydrolyze fructose-containing compounds other than sucrose, such

as raffinose and stachyose. Acid invertases are closely related to a class of enzymes known as









fructan exohydrolases (FEH). FEHs are difficult to distinguish from acid invertases by sequence

alone and are thought to have evolved from acid invertases even though FEHs exclusively break

down fructans and cannot hydrolyze sucrose (Van den Ende et al., 2002). It was recently

discovered that two previously annotated cell wall invertases from Arabidopsis (AtcwlNV3 and

AtcwlNV6) may, in fact, be FEHs (De Coninck et al., 2005).

Much less is known about the neutral/alkaline invertase sub-family due to purification

difficulties and low, unstable enzymatic activity (Sturm and Tang, 1999; Roitsch and Gonzalez,

2004). This sub-family likely has a different mode of action than the acid invertase sub-family

since sucrose is the sole substrate and they are not inhibited by heavy metals, elements that

strongly inhibit the acid invertases (Roitsch and Gonzalez, 2004).

An important driver of species origination and diversification is gene duplication.

Duplication makes it possible for a gene to acquire new functions without losing the function of

the progenitor gene (Kramer et al., 1998; Lynch and Conery, 2000; Sankoff, 2001; Becker and

Theissen, 2003; Irish, 2003; Litt and Irish, 2003; Zahn et al., 2005). Gene duplication can occur

in tandem, through the duplication of a chromosomal segment, an entire chromosome, or through

genome duplication (Otto and Whitton, 2000; Wendel, 2000; Adams and Wendel, 2005).

Chromosomal duplications result in conserved, colinear locus order that can be used to infer

ancestry of loci of interest both within and between species (Lynch and Conery, 2000).

Poplar is known to have undergone at least two genome duplication events in its

evolutionary history (Sterck et al., 2005; Tuskan et al., 2006). The first genomic duplication

event occurred prior to the divergence of Arabidopsis and poplar and is known as the "eurosid"

duplication event (Bowers et al., 2003; Blanc and Wolfe, 2004; De Bodt et al., 2005; Zahn et al.,

2005; Tuskan et al., 2006). The second genome duplication event occurred after the divergence









of Arabidopsis and poplar but prior to the divergence of the Populus and Salix genera and is

referred to as the "salicoid" event (Tuskan et al., 2006). Arabidopsis is known to have undergone

at least one genomic duplication event after the divergence with poplar (Bowers et al., 2003;

Blanc and Wolfe, 2004; De Bodt et al., 2005; Zahn et al., 2005; Tuskan et al., 2006). By

knowing the timing and location of genomic duplication events, the evolutionary development of

a variety of gene families can be predicted in these species.

The recently released sequence of the poplar genome (Tuskan et al., 2006) opens the door

for systematic analysis of metabolically important gene families in a model tree. Here, I report

the identification of the poplar invertase gene family and show expression data from both

microarray and reverse transcription-polymerase chain reaction (RT-PCR) experiments. I show

that acid and neutral/alkaline invertase genes are regulated in vegetative or floral organs. An

examination of the microsyntenic regions surrounding the poplar invertase genes reveals

extensive colinearity with Arabidopsis invertases and allows orthologous and paralogous

relationships among genes to be inferred.

Materials and Methods

Plant Material

Rooted softwood cuttings of Populus trichocarpa (Torr. & Gray) (genotype "Nisqually-1")

were planted in 8 L pots and placed in a fan- and pad-cooled greenhouse with natural light

augmented with full spectrum fluorescent lighting during the winter to give a day length of 15 h.

Plants were grown on an ebb-and-flow flood bench system with a daily supply of Peters

Professional 20-10-20 water-soluble fertilizer diluted to a final concentration of 4 mM

nitrogen. Plants were grown until 80 cm tall at which point microarray and quantitative RT-PCR

(RT-qPCR) experiments were performed. Floral organs used in RT-PCR experiments were









harvested from a Populus deltoides growing locally on the University of Florida-Gainesville

campus.

BLAST Searches and DNA Annotation

The Populus acid and neutral/alkaline invertase gene families were identified using the

tBLASTn function of the Joint Genome Institute's database (http://www.jgi.doe.gov/) and the

previously described genes AtvaclNVI and -2 (Atlg62660, Atlg12240), AtcwlNVl-6

(At3g13790, At3g52600,Atlg55120, At2g36190, At3g13784, At5gl1920), from Arabidopsis

and InvDC1-5 (Accession #s X69321, X78424, X78423, Y18707, Y18706) from carrot (Lee and

Sturm, 1996; Sturm, 1996; Sherson et al., 2003). The resulting nucleotide sequences were

annotated using a combination of protein alignments with known Arabidopsis and carrot

invertases as well as the predictions of GENSCAN (http://genes.mit.edu/GENSCAN.html)

(Burge and Karlin, 1997).

Construction of Sequence Similarity Trees

Predicted amino acid sequences were aligned using CLUSTALW

(http://clustalw.genome.jp) to construct similarity trees using the TREEVIEW program (Page,

1996). PAUP (Swofford, 1993) was used for bootstrap analysis with 100,000 iterations.

Identification of Gene Duplication

Gene duplications were identified as "recent" or "ancient" by Tuskan et al. (2006) where

"recent" and "ancient" refer to the "salicoid" and "eurosid" duplication events, respectively.

Briefly, the poplar and Arabidopsis genomes were reconstructed into conserved syntenic

segments that were subsequently compared with a variant of the algorithm described by Hokamp

et al. (2003). Tandem duplications were defined as neighboring gene models with high sequence

identity on a chromosomal segment.









Isolation of RNA and RT-PCR Assay

Total RNA was extracted using standard methods (Chang, 1993), DNase-treated and

purified on RNAeasy QIAGEN columns (Valencia, CA). Purified RNA (5 [tg) was used to

synthesize cDNA using a mixture of 500 ng oligo-dT, 100 ng random primers, and M-MLV-RT

as per manufacturer's instructions (Invitrogen, Carlsbad, CA), with the exception that the DTT

was excluded. PCR reactions were run with a single step at 94 C for 3 min, and then 35 cycles

of 94 C (30 s), 57 C (30 s), and 72 C (4 min), and a single step at 72 C for 10 min. Actin was

used to verify integrity of cDNA template. All primers are listed in Table 2-2.

Quantitative RT-PCR

Gene expression was analyzed using the SYBR Green kit (Stratagene, La Jolla, CA) and

Mx3000P thermo-cycler (Stratagene) as per manufacturer's instructions. Briefly, 1 [tl of the

synthesized cDNA and 0.15 tl of a 0.25 [tM solution of each primer were used for each 50 [tl

RT-qPCR reaction. Primers were designed using NetPrimer (Premier Biosoft International, Palo

Alto, CA) software and synthesized by Invitrogen. Each real-time PCR reaction was performed

in triplicate (technical replicates) on four individual plants (biological replicates) and carried out

for 40 cycles with annealing, extension, and melting temperatures of 55 'C, 72 'C, and 95 'C,

respectively. Melting curves were generated to check the specificity of the amplified fragments.

In the case of PtVIN2, an extension temperature of 79 C with the fluorescence reading taken at

the end of the run was used to correct for a spurious primer-dimer amplicon. Changes (n-fold) in

gene expression relative to the geometric mean (Vandesompele et al., 2002) of three control

genes encoding actin, ubiquitin and ubiquitinL (Brunner et al., 2004) were determined using the

program DART-PCRv1.0 (Peirson et al., 2003).









Genomic DNA Isolation

Genomic DNA was isolated from poplar shoot tip tissue. Approximately 250 mg of tissue

was ground in liquid nitrogen and added to a buffer containing 0.3 M sucrose, 10 mM Tris (pH

7.9), 1 mM EDTA, and 4 mg/mL diethyldithiocarbamic acid. Samples were spun (20,800 rcf)

and pellets resuspended in a buffer containing 100 mM Tris (pH 7.9), 500 mM NaC1, 20 mM

EDTA, 1 % SDS, 0.1 % 2-mercaptoethanol, and 100 [tg/ml proteinase K. Samples were

incubated at 65 C for 1 h, spun (20,800 rcf) and supernatant extracted with chloroform.

Isopropanol was used to precipitate the DNA at room temperature, spun (20,800 rcf), and

resuspended in Tris-EDTA buffer. One tl RNase A was added to DNA sample and incubated at

37 C for 30 min. The chloroform extraction was repeated, followed by ethanol precipitation in

presence of sodium acetate at -80 C for 1 h. Samples were spun for 10 min (20,800 rcf), air

dried and resuspended in Tris-EDTA buffer. Ten ng of genomic DNA was used in PCR reactions

as previously described.

Microarray Design and Analysis

Poplar whole-genome 60-mer oligonucleotide microarrays (three different 60-mer probes

per gene model) were designed by NimbleGen (Madison, WI) in collaboration with Oak Ridge

National Laboratory. Labeling, hybridization and scanning were carried out by NimbleGen using

standard procedures (Quesada et al. unpublished data). Briefly, signal intensity detected for each

probe was log2-transformed, normalized, and contrasted to a set of 20 negative control probes. A

mixed-model analysis of variance (ANOVA) was applied to each individual probeset with gene

as a fixed effect, and probe as a random effect. P-values were adjusted for false discovery rate

(Benjamini and Hochberg, 1995), with the modifications reported by Storey and Tibshirani

(Storey and Tibshirani, 2003). All the analyses described were carried out using SAS and JMP

software (SAS Institute, Cary, NC). For contrasting the transcript abundance between treatments,









individual probes that gave no signal on any array based on comparison to the negative controls

were excluded, and a complete mixed ANOVA model was used that included gene and tissue

type as fixed effects, while probe ID, plant, and the interaction of tissue by plant were treated as

random effects. Microarray data have been deposited in the Gene Expression Omnibus (GEO)

database (www.ncbi.nlm.nih.gov/geo/); accession number GSE6422.

Results

Identification of Poplar Invertase Genes

I used Arabidopsis and carrot invertase genes as queries to identify 24 putative invertase

genes (Table 2-1), eight in the acid invertase sub-family and 16 in the neutral/alkaline invertase

sub-family, and followed the rice invertase nomenclature (Hirose et al., 2002; Ji et al., 2005).

The distinct origin of the acid and neutral/alkaline sub-families (Sturm, 1999; Vargas et al.,

2003) is reflected in their intron/exon structures (Figure 2-1) and amino acid alignments (Figure

2-5). The acid invertases can be further subdivided into two well-supported clades, "a" and "P",

which are inferred to be cell wall and vacuolar targeted, respectively (Figure 2-2A). This

inference is based on sequence similarity to the Arabidopsis invertases. The neutral/alkaline

invertases also subdivide into two clades (a and 0) that are supported both by bootstrap analysis

(Figure 2-2B) and intron/exon structure (Figure 2-1B), however the functional implications of

this sub-division are not clear.

Four (PtNIN13-16) of the 16 neutral/alkaline invertases are encoded by seemingly

incomplete ORFs (data not shown). PtNIN13 is missing ORFs for the first and last exons. In the

case of PtNIN14 and -15, a portion of the ORF encoding the third exon is missing as well as

ORFs corresponding to the first and last exons. Finally, PtNIN16 contains a short ORF encoding

a portion of the third exon. In all cases, genomic sequence was examined for several Kb in either

direction until neighboring genes were identified in order to verify that the sequences were truly









missing. Evidence of gene expression was obtained for all four of these "partial" genes using one

or more of the methods listed in Table 2-1. Further work will need to be performed to understand

their evolution.

Structure of the Poplar Invertase Genes

The acid invertase sub-family is encoded by seven exons whose locations are generally

conserved in plants (Tymowska-Lalanne and Kreis, 1998b). This family also contains PtVIN1

which contains no introns. All eight genes encode the motifs NDPNG and WECXDF, which are

essential for catalytic activity and are conserved in this gene family (Sturm and Chrispeels,

1990b) (Figure 2-5A). Except for PtVIN1, the NDPNG motif is partially encoded by a mini-exon

encoding the tripeptide DPN, one of the smallest known exons in plants (Bournay et al., 1996).

There are several key features that distinguish the 0 clade of the poplar acid invertases

from the a clade with a highly significant bootstrap value of 100 %. The first two features are N-

terminal and C-terminal extensions, both proposed to play a role in targeting to the vacuole

(Sturm, 1999). Third is the conserved WECXDF domain that contains one of the three

carboxylate groups required for activity (Pons et al., 1998; Alberto et al., 2004); the X in this

domain is a proline in the a clade and a valine in the 0 clade (Figure 2-5A).

The neutral/alkaline invertase sub-family also clusters into a and 1 clades based on amino

acid alignments (Figure 2-2B, Figure 2-5B) that are distinct and well supported (Figure 2-2B).

The members of the a clade (PtNIN1-6) are encoded by six exons with conserved locations

(Figure 2-1B), whereas the six members of the 1 clade (PtNIN7-12) are encoded by four exons

(Figure 2-1B). The different intron/exon structures and the different number of exons between

the clades, suggests that the a and 1 clades arose from different ancestral genes.









Expression of the Poplar Invertase Gene Family

Expression of the poplar invertase family in mature leaves, young leaves, nodes,

internodes, and roots was examined using whole genome microarrays where evidence for

expression was detected for 16 of the 24 genes (Figure 2-3A). RT-qPCR with gene specific

primers designed against PtCIN3, PtVIN3, PtNIN3 and PtNIN9 was then used to validate the

microarray data using the same samples that were analyzed in the microarrays (Figure 2-3B).I

then obtained evidence for transcript-level regulation ofPtNIN7, -10, -11 and -16 in additional

microarray analyses of responses to nitrogen availability (PtNIN7 and -11) (data not shown) and

exogenous auxin (PtNINIO and -16) (data not shown).

Analyses of microarrays provided evidence for expression of all but four invertase genes

(PtCIN1 and -2, PtVIN1, PtNIN13). One possible explanation for this result is that these four

invertase genes are not transcribed in the organ(s) examined. Alternatively, the gene may have

been transcribed in the organ(s) examined but transcript abundance was not distinguishable from

background on the arrays. To test the first hypothesis, RT-PCR was carried out on a larger

number of poplar organs. I detected transcripts for PtCINJ and -2 in floral organs and not organs

used in the array analysis (Figure 2-3C). This is consistent with previous results in tomato (Godt

and Roitsch, 1997a), carrot (Lorenz et al., 1995), and Arabidopsis (Tymowska-Lalanne and

Kreis, 1998a; Sherson et al., 2003) where certain cell wall invertases are expressed solely in

floral organs. I detected transcripts for PtVINJ and PtNIN13 using RT-PCR in nearly all organs

tested (Figure 2-3C). This indicates that the transcripts of these two genes were below the

detection level of the arrays.

Evolutionary Development of the Poplar Invertase Family

Determining orthology and paralogy between genes can be a useful tool in elucidating

gene function. The shared evolutionary history between Arabidopsis and poplar, as well as the









extensive research previously conducted in the Arabidopsis invertase gene family, makes

Arabidopsis invertases ideal candidates to compare with poplar invertases in order to determine

orthology. Amino acid alignments between poplar and Arabidopsis invertases (Figure 2-2,

Figure 2-5) reveal little about orthology since the Arabidopsis invertases are more similar to each

other than they are to their presumed poplar orthologs. Consequently, I examined the

chromosomal segments encoding invertases to infer orthology since the linear order of ORFs are

often conserved after duplication events.

I examined paralogous regions of the poplar genome, as defined by Tuskan et al. (2006),

containing the neutral/alkaline invertase sub-family members and found that five of the 16 poplar

neutral/alkaline invertases appear to have originated in the recent salicoid genome duplication

event (Table 2-1). No evidence was found for tandem duplications (defined as identical, adjacent

genes on the chromosome), or for expansion of the neutral/alkaline family as a result of the more

ancient eurosid genome duplication event that occurred in the common ancestor of poplar and

Arabidopsis. Therefore, the salicoid duplication event can explain the growth of the poplar

neutral/alkaline invertase family relative to the Arabidopsis gene family (16 versus 9 members,

respectively).

To establish orthology between the poplar acid invertase sub-family and the more widely

studied Arabidopsis acid invertase sub-family, I compared the genomic organization of the two

species' acid invertase sub-families and found substantial conservation of microsyntenic regions

(Figure 2-4A). These conserved, microsyntenic regions suggest that the common ancestor to

poplar and Arabidopsis contained two cell wall invertases and one vacuolar invertase (Figure 2-

4B). I hypothesize that these three progenitor invertases (VINa, CINa, and CINb) underwent a









series of duplication events prior to and subsequent to the speciation event between poplar and

Arabidopsis, as outlined in Figure 2-4B.

Vacuolar invertases PtVIN2 and -3 lie within conserved, colinear chromosomal segments

(Figure 2-4B), indicating that a chromosomal duplication event gave rise to these two invertases.

Similarly, the two Arabidopsis vacuolar invertases (AtvaclNV] and -2) reside in a conserved,

colinear arrangement (Figure 2-4A), also indicative of a chromosomal duplication event.

Conservation can also be observed across the poplar/Arabidopsis species divide. For example, a

nuclear apical meristem (NAM) family ORF lies downstream of the vacuolar invertases on

relevant chromosomal segments in both species (Figure 2-4A). In addition, loci encoding a

thylakoid luminal related protein, a 3-oxoacyl-ACP synthase III protein and a flavin containing

monoxygenase are located on colinear chromosomal segments with both the poplar and

Arabidopsis vacuolar invertases (Figure 2-4A). The colinearity of multiple loci across

Arabidopsis and poplar suggests that a genomic duplication event of a progenitor vacuolar

invertase and surrounding loci occurred in a common ancestor of poplar and Arabidopsis giving

rise to the two vacuolar invertases (Figure 2-4A, B).

Two additional chromosomal duplication events can be identified through colinearity. The

conservation of loci encoding a kinesin motor related protein, ubiquitin-2, and a 40S ribosomal

protein S9 can be seen surrounding AtcwlNV2 and -4 as well as PtCIN3 and the tandem pair

PtCIN1 and -2. The conservation of this chromosomal segment between Arabidopsis and poplar

is again indicative of a duplication event giving rise to these genes prior to the Arabidopsis and

poplar speciation event (Figure 2-4B). Because the presence of the tandem pair PtCINJ and -2

occurs only in poplar, duplication likely occurred after the poplar and Arabidopsis speciation

event.









The microsyntenic segment containing the poplar cell wall invertases PtCIN4 and -5 is

more difficult to interpret (Figure 2-4B). This chromosomal segment contains a locus encoding a

C2H2 zinc finger family protein that is shared on two chromosomal segments in Arabidopsis that

also contain AtcwlNV3 and the tandem pair AtcwlNVI and -5. This poplar segment also contains

a DEAD box RNA helicase downstream of the tandem invertase pair that is conserved on the

AtcwlNV3 chromosomal segment but absent from the tandem AtcwlNVI and -5 chromosomal

segment. The simplest explanation of the development of these invertases is a tandem

duplication event prior to the poplar/Arabidopsis speciation event (duplication of CINa, Figure

2-4B). Following the speciation event, a chromosomal duplication event occurred only in

Arabidopsis to give rise to a tandem pair; AtcwlNV3 and the other member which was lost.

Poplar-specific innovations include a tandem duplication giving rise to the PtCIN1 and -2

pair (Table 2-1, Figure 2-4) and the appearance of PtVINI, the third poplar vacuolar invertase,

described in more detail in the next section. I hypothesize PtVIN1 arose from a processed PtVIN2

mRNA that was inserted in trans- into the poplar genome and therefore is not colinear with any

other invertase.

Arabidopsis innovations include AtcwlNV3, which apparently arose from a duplication of

the chromosomal segment containing AtcwlNV1 and -5. This duplication was subsequently

followed by the loss of one of the proposed two new invertases resulting in the single AtcwlNV3.

The sixth and final cell wall invertase in Arabidopsis, AtcwlNV6, poses a bit of a conundrum.

The lack of colinearity with other regions of the Arabidopsis genome and its isolation on the

protein similarity tree (Figure 2-2A) makes it difficult to derive a putative progenitor. I

hypothesize that AtcwlNV6 arose from a gene duplication event followed by functional

divergence, as this is the most common mode of gene family diversification. However, because









genes are frequently rearranged and/or lost after chromosomal duplication events, the absence of

colinearity cannot rule out a chromosomal duplication event. Some other technique will need to

be employed to define the origin ofAtcwlNV6.

Putative Evidence for Origins of PtVIN1

In its recent evolutionary history, poplar has acquired an innovation in the acid invertase

family. PtVIN1 is an intronless invertase that clusters with the 0 group, indicating vacuolar

targeting (Figure 2-1, 2-2). An intronless invertase, to my knowledge, has not previously been

reported. This gene retains all of the characteristics relevant to the acid invertase family with the

exception of introns (Figure 2-5A).

PtVIN1 consists of a single 1.6 Kb ORF with several possible upstream TATAA boxes and

a 3' polyadenylation signal (data not shown). I verified the sequence obtained from the Joint

Genome Institute's database by designing gene specific primers (Table 2-2) flanking the entire

ORF of PtVIN1 and cloning and sequencing PCR products amplified from both purified P.

trichocarpa genomic DNA as well as cDNA generated from DNase-treated RNA. PtVIN1

transcript was detected in all tissues examined (Figure 2-3C).

I speculate that PtVIN1 arose relatively recently in poplar evolutionary history as a

processed transcript of PtVIN2 (86 % identity at the nucleotide level and 77 % identity at the

amino acid level) that was reverse transcribed and reinserted into the genome. This phenomenon

is unusual but not unprecedented with examples being found in the alcohol dehydrogenase gene

family in the genus Leavenworthia (Charlesworth et al., 1998), as well as numerous examples in

human, rat, and dog (Coulombe-Huntington and Majewski, 2007).

Discussion

In my study of the poplar genome, I identified 24 putative invertase genes: eight acid and

16 neutral/alkaline invertases. The poplar genome encodes ca. 45,000 genes which, when









compared to ca. 27,000 genes in Arabidopsis represents an approximately 1.6-fold increase in

gene number (Tuskan et al., 2006). This total gene number expansion is not the result of unique

poplar genes, but rather the expansion of specific gene families (Tuskan et al., 2006).

Interestingly, the expansion of poplar's invertase gene family relative to Arabidopsis and rice is

not consistent between the two invertase sub-families. Arabidopsis contains 17 members: eight

acid and nine neutral/alkaline invertases (Vargas et al., 2003; Ji et al., 2005), whereas the rice

genome encodes 19 invertase genes: eleven acid and eight neutral/alkaline invertases (Ji et al.,

2005). Thus, the overall increase in poplar invertase gene numbers is driven primarily by an

increase in the neutral/alkaline invertases.

One explanation for the expansion of the neutral/alkaline invertase sub-family in poplar

may lie in the apparent lack of an active phloem loading step in this group of woody perennial

plants. Turgeon and Medville (1998) suggest that in willow, and likely poplar (both are members

of the Salicaceae), photoassimilate accumulation in the source phloem is accomplished via an

uninterrupted, symplastically connected sucrose concentration gradient between the mesophyll

cells and the sieve element-companion cell complex (SE-CCC). This is in contrast to both

apoplastic phloem loaders and symplastic phloem loaders where the solute concentration in the

SE-CCC is dramatically higher than the solute concentration in the surrounding apoplastic space

and mesophyll cytoplasm. Turgeon and Medville (1998) propose that regulation of the sucrose

concentration gradient from the mesophyll cells to the SE-CCC in poplar could be maintained in

the cytoplasm and/or vacuole. This could implicate both neutral/alkaline invertases (thought to

be cytoplasmically located) and vacuolar invertases as key components of this pathway. The

growth of the poplar neutral/alkaline invertase subfamily occurred in the recent genomic

duplication event (salicoid) that took place prior to the willow/poplar speciation event (Tuskan et









al., 2006). This makes it likely that willow also contains the expanded neutral/alkaline invertase

sub-family.

Of the 16 neutral/alkaline invertases identified in poplar, four (PtNIN13-16) are missing

significant portions of their coding regions. The sequences found in these four neutral/alkaline

invertases retain conserved intron/exon splice sites, conserved sequence motifs and the ability to

be transcribed (data not shown). This transcriptional evidence appears to rule out these genes

from being defined as "pseudogenes" under traditional definitions of the term in which

pseudogenes are transcriptionally and translationally silent and therefore not subject to selection

(Li et al., 1981). However, there are examples of transcribed pseudogenes in eukaryotic

organisms including human, mouse, silk moth, Arabidopsis and liverwort (Balakirev et al.,

2003). McCarrey and Riggs (1986) propose that pseudogenes could act as negative regulators of

transcription of their progenitor genes by providing antisense RNA to hybridize with the sense

RNA of the progenitor. Troyanovsky and Leube (1994) identified cis elements in the promoter

region of a pseodogene derived from human cytokeratin 17 that can interact with distal elements

in the promoter of the functional gene to regulate transcriptional activity. Thus, PtNIN13-16 may

have biologically relevant functions in gene regulation, as opposed to invertase enzyme activity,

and may still be subjected to selection.

ClustalW and bootstrap analysis of the acid invertase families of poplar and Arabidopsis

clearly divide the acid invertases into the a clade (cell wall invertases) and the 0 clade vacuolarr

invertases). It has recently been reported that two of six cell wall invertases in Arabidopsis may

not be invertases, but rather fructan exohydrolases (FEHs) (De Coninck et al., 2005). FEH

protein sequences are nearly identical to those of demonstrated acid invertase proteins but FEH

does not use sucrose as a substrate (De Coninck et al., 2005). As I did not test recombinant cell









wall invertase activity in this work, I cannot rule out that one or more of the five PtCIN genes

described here may encode an FEH.

To my knowledge, this work is the first report of a plant with more than two vacuolar

invertases. Analysis of the microsyntenic regions surrounding these vacuolar invertases in poplar

and Arabidopsis identified colinearity that I use to postulate that these genes arose from an

ancient gene duplication event in a common ancestor of these two species. In contrast, I found no

colinearity in the microsyntenic region containing PtVIN1 with regions in Arabidopsis or

elsewhere in the poplar genome. This, along with the lack of introns in PtVIN1, leads me to

hypothesize that PtVIN1 arose from a processed PtVIN2 mRNA that was inserted in trans- into

the poplar genome.

The absence of an intronless vacuolar invertase in any other organism studied to date

suggests that PtVIN1 arose recently in evolutionary history. It will be interesting to see if this

invertase anomaly extends to Populus species other than the sequenced Populus trichocarpa

genome. Work performed by Coulombe-Huntington and Majews (2007) on intron loss in

mammalian systems found that genes susceptible to intron loss tend to be involved in

housekeeping functions and expressed at high levels. This is consistent with the high expression

patterns of PtVIN2, the putative progenitor to the intronless PtVIN]. Because PtVIN1 has

retained all the features necessary for transcription, such as an intact TATAA binding site and no

indels or stop codons, it appears that there has been some level of selection in favor of the

expression of PtVIN1. The hypothesis that PtVIN1 originated as a reverse transcribed, processed

transcript is supported by the concomitant loss of all six introns from the presumed progenitor

locus PtVIN2.









One distinguishing feature of the expression of the invertase family is the floral specificity

of certain cell wall isoforms. Tomato Lin7 is floral specific with highest expression in the stamen

(Godt and Roitsch, 1997b). In carrot, InvDC2 was expressed solely in floral buds (Lorenz et al.,

1995) and in Arabidopsis, AtcwlNV2 was found to be floral specific (Tymowska-Lalanne and

Kreis, 1998a). Poplar is a dioecious tree and, as I only collected floral material from a female

tree, I did not examine invertase expression in the male floral organs. Even so, I identified two

floral specific cell wall isoforms in PtCIN1 and -2.

The recently released assembly of the poplar genome (Tuskan et al., 2006) opens the door

for analyses of metabolically important gene families in a model tree. In this study of the

invertase gene family, I identified 24 putative gene family members and obtained evidence for

expression of all members. A better understanding of the roles that individual invertase isoforms

play in carbon utilization will be important in dissecting the functional implications of invertase

gene family evolution and diversity.



















Table 2-1. Nomenclature and chromosomal location of 24 poplar invertase genes.


Gene name
Acid invertases
PtCINI
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVINI
PtVIN2
PtVIN3
Neutral/alkaline invertases
PtNINI


PtNIN2
PtNIN3
PtNIN4
PtNIN5
PtNIN6
PtNIN7
PtNIN8
PtNIN9
PNINI10
PNINI11
PfNIN12
PtNIN13a
PtNIN14b
PtNIN15b
PtNIN16c


Predicted compartment JGIv1.1 gene model name


Cell wall
Cell wall
Cell wall
Cell wall
Cell w ll
Vacuole
Vacuole
Vacuole


gwl XVI 2453 1
gwl XVI 2454 1
estExtfgenesh4 pg CLGVI1370
estExt_genesh4_pg C_LG_V11536
eugene3 00061607
fgenesh4pm C_LG_111000407
estExt_fgenesh4_pg C_LG_1110902
estEt Genewsel v1 C LG XV2841

estExt Genewisel v1 C LG V1112120
eugene3 00130058
gwl VIII 2341 1
gwl X 3512 1
gwl 1312491
gwl 6649 1
estExt_fgenesh4_pg C_LG_V1531
eugene3 00190739
fgenesh4_pg C_LG_IV001415
fgenesh4_pm C_LG_11000804
gwl IX 1371 1
eugene3 00410102
gwl XIV 1765 1
gwl XIV 4326 1
gwl 376 8 1


Linkage group location Expression Support Type of gene duplication Duplicate gene


16 5729453
16 5739912
6 13897519
6 15233568
6 15229483
3 11913972
3 108433683
15 9251445

8 6388303
13 512039
8 1096197
10 20965604
scldl31 616042
scfld66 160259
516687404
19 9274739
4 15428994
2 13356400
9 2392685
scld41 1148890
14 3660215
14 11708730
scfld376 31617
17 1427133


PtCIN2
PtCIN1

PtCIN5
PtCIN4
PtVIN2
PtVIN3
PtVIN2


PtNIN5
PtNIN4
PtNIN3
PtNIN2


PtNIN12
PtNIN11
PtNIN13
PtNIN9
PtNIN8
PtNINIO


aMissing first and last exon, Missing first, last, and portions of third exons, 'Contains only portion ofthird exon

Type of predicted duplication event (if any) is noted. E: EST support; M: Microarray support; R:

RT-PCR support; n/a: not assigned.


IV I/


""`''"""" '"













PtVINI
PtVIN2 1 m
PtVIN3
PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5 -I I



B

PtNIN1
PtNIN2S U U -_
PtNIN3
PtNIN4 U
PtNIN5 -
PtNIN6
PtNIN7
PtNIN8I
PtNINI9
PtNIN100 : nl
PtNIN11
PtNIN12



Figure 2-1. Exon-intron structures of predicted invertase genes with complete exons (20 of 24).
PtNIN13-16 are not represented here as the complete sequence could not be
determined. A) Acid invertases. B) Neutral/alkaline invertases. Exons whose
junctions have been verified (using EST and/or sequencing data) are represented by
solid black boxes, exons whose junctions have not been verified are represented by
empty boxes. Introns are represented by black lines.














AtcwlNV5


AtcwlNV1
(At3G13790)
AtcwlNV4
(At2G36190)
AtcwlNV2
(At3G52600)
PtCIN3 10/
PtCIN2 100


(AtlG12240)


At3G05820 At1G56560


Figure 2-2. Invertase amino acid similarity trees based on full length sequences. A) Protein
similarity tree of eight poplar and eight Arabidopsis acid invertases. a denotes cell
wall invertases (PtCIN1-5) and 0 denotes vacuolar invertases (PtVIN1-3). B) Protein
similarity tree of 12 poplar and 9 Arabidopsis neutral/alkaline invertases. The a clade
(PtNIN1-6) and 0 clade (PtNIN7-12) are marked. PtNIN13-16 are not included.
Bootstrap values are reported as a percentage of 100,000 repetitions. Branch lengths
denote protein similarity.


AtcwlNV6(FEH)
(At5G11920)


AtvaclNV1
(AtlG62660)


















SPtCIN3

PtCIN4 a

PtCIN5 I

PtVIN2



PtNIN2


PtNIN3

PtNIN4

PtNIN5

PtNIN6

PtNIN8

PtNIN9

PtNIN12 P

PtNIN14

PtNIN15


PtCIN3 PtVIN3
12 12
08 08
06I 06
02 02
0 0
ML YL R IN N ML YL R IN N


PtNIN3


PtNIN9


12 12
08 08



ML YL R IN N ML YL R IN N


C

P. trichocarpa P. deltoides

0- 0
LL


E E .
4 0 N
o |, Ei a o 0
W X M -J cn U M. M.
PtCINI
PtCIN2

PtVINI


PtNIN13


Actin


Figure 2-3. Expression of poplar invertase genes. A) Heat map showing relative expression of
poplar invertase genes in five organs. If transcript for a gene was significantly
detected at an FDR of 10 % (contrasted with the array negative control probes) in at
least one organ, then data from all organs was included on the heat map. Dark blue
denotes high expression and light blue denotes low expression. B) RT-qPCR
validation of microarray data. Relative transcript levels detected by RT-qPCR (black
bars) and microarray analysis (grey bars) in mature leaves (ML), young leaves (YL),
roots (R), internodes (IN), and nodes (N). In order to compare the microarray and RT-
qPCR platforms, RNA was re-extracted from the same tissues used in the microarray
experiment. First strand cDNA was then synthesized and used as template in the RT-
qPCR reactions. The highest and lowest relative transcript estimates were assigned
values of 1 and 0, respectively. Intermediate values were then adjusted relative to
their difference between the highest and lowest transcript estimate. C) Expression of
PtCIN1,2, PtVIN1 and PtNIN13 in various organs using cDNA generated from P.
trichocarpa (vegetative organs) and P. deltoides (floral organs) as template. Contrast
and resolution of images were adjusted in order to maximize visibility of bands. Actin
cDNA was amplified to verify integrity of the RT-PCR template in each reaction.


--r ~1



















Trehalose-6-phosphate
phosphatase Musficopper oxidase NAM

Flawn containing 3-oxoacyl-ACP PtVIN3 Thylakoid luminal
monoxygease synthase prom related

PtVlN2
CC-NBS-LRR class
disease resistance

AtvadNVI


AtvaoNV2


Kmesm motor related 22 Kb

PtCIN2


PtCIN1


11.6 Kb PX domain
UBQ2 m contamng protem

40S nbosomal
protein S9
m


PtCIN3

AtcwlVR2


AtcwlVR4


F-box family C2H2 zinc finger famlyprotein


Endomembrane protein 70


AtcwlVRI AtcwlVR5
DEAD box RNA he.hase

AtcwlVR3
24Kb

PtCIN4 PtCIN5


4 Kb


B


WNa CINa CINb


1 a


WNa CINb


SSpeciation even

Poplar Arabidopsis

_rW~Rt>_ 9M"" >& -mini>_ -11W -slo s-NNo

WNa CNb WNa
wN.- Ci"Ngene paI r
Tnsrense oon of Tandem duphcaton
pro is t... n ptI


PtWN1 ptC/N4 ptCIN5 PtCIN1 PtC.N2
PtWN2 ptCtN3


AtvacbNVI A bAtcwlNV3

Atvac/NV2 AtcwINVI Atcw.NV5


a
CINb

CINb





Atcw-NV2 Atcw-NV6
AtcwlNV4


Figure 2-4. Evolutionary development of the poplar acid invertase family. A) Colinear

chromosomal segments containing poplar invertase ORFs (checkered) and

Arabidopsis invertase ORFs (solid). Conserved ORFs between chromosomal

segments are depicted with identical shading; PX, phox; NAM, nuclear apical

meristem; ACP, acyl carrier protein. B) Cartoon depicting hypothesized development

of the acid invertase sub-family in poplar and Arabidopsis.











Table 2-2. Primers used in RT-qPCR and RT-PCR.
Gene name Primer sequence Tm(C) Amplicon size (base pairs)
PtVIN1_RTqPCR_3' TCCTGGCCTCAAAATTTGC 58 216
PtVIN1_RTqPCR_5' GCCCTGGTTCCACCTATAGTT 58
PtVIN2_RTqPCR_3' CGCATCTTCGTCTTCTTGTG 57 189
PtVIN2_RTqPCR_5' CCCAGCTATAGTCCTGCCC 57
PtVIN3_RTqPCR_3' GCTTGGAAAATGACTCTGTAGGTC 59 197
PtVIN3_RTqPCR_5' ATACACTCCCTTGCTAGACAACC 57
PtCIN3_RTqPCR_3' TTGGTAGAATAGATGGTATAGCCCC 61 224
PtCIN3_RTqPCR_5' TGTTAAAGTTTCTCCCAGTCTTGG 60
PtNIN3_RTqPCR_3' TGGGGCGAGCATCTCCT 59 148
PtNIN3_RTqPCR_5' TGCTGATGGTTTTGACATGTTC 58
PtNIN8_RTqPCR_3' GTTTCAACAATGAGCAAGCG 57 211
PtNIN8_RTqPCR_5' GATTTATCTCTAGCAGAAACTCCAG 56
PtNIN9_RTqPCR_3' GGCATGAAGGCGTTTAGTG 56 393
PtNIN9_RTqPCR_5' CACGATCCTGTCAGGAACAG 56
PtNIN10 RT PCR 3' GCTTCCCAACATATCTGCCG 60 824
PtNIN10 RT PCR 5' TCGCCCGGAGGTACAGAAT 60
PtNIN13 RT PCR 3' GCTCCCCTACATACCTGCCA 60 629
PtNIN13 RT PCR 5' TCGCCCAGAAGTGCAGAGA 60
PtNIN14 RT PCR 3' TGTTATTTAATTCAGTGAAATTCAGCA 60 334
PtNIN14 RT PCR 5' GTTCTATATGACTTGCATCGTCAAAAT 61
PtNIN15 RT PCR 3' TGTTATTTAATTGAGTGAAATTCAGCC 61 241
PtNIN15 RT PCR 5' CTCTATGACTCGCATCGTCAAAAG 61
PtNIN16 RT PCR 3' CTAGCCATGAAGGAATTTGATCTTC 61 201
PtNIN16 RT PCR 5' ATGCTCTTTGTCAATGATGGAAC 59














PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


< A>


< B >


QTLS NkJV H
QTLS N*LN H
QSLSw KK[TH
PHT MQEKSSY
---NETD*PY
GYPQN SLSWQ
DYPWNNSILSWQ
-YNWTN AFSWQ


F



:Ii m-GS
HIi -GS
PIIYI GS


<*D>
AN

PSVN S .3. RV
D NI LS
G TRP A
DPDL ** Q


P jD.. KT
NITI TDE 3


DKN
DKN
DEN


ES
ES
ES


S SQ Y P
S.Q VPY
*IQ SVY
CS SAM
HO S *V
* S *YP
* S *YP
* S *Y*


G IA


GIEG
G I


MADTSPLLPFSHSLGP-----------GSTYSSIRWRSKIVLLLVFSGLFLVPLIVSIAS
MADPSPLLPVSNSLEPSYSPAPEGAVSAGCPATHLRRSKKVLIAVFSGLLVVSLILATIN
-MDTNPSHTSSDPPYTPLLD-------NPSPARIRRPFNGFAAILASLIFLLSLVALIIN


-------------------LF FVLNGEA IYSEY
-LF IYPQF
--------KFPLLFA FVL NGVE IYLRY
-------------------VF CCV-SS 1EVE LENNGCQNFQ
F-------------------------- I CVL H SH S ------
NDNGFKQHVQYLQEDDQNVSFSPPKETTK I RP--SRGS SEK LGAQEK
NNN-GGRHVQYHSQEDEDASLATPKE ET LPYSR SEK LGAQVK
QSQ------ESLPEQNQNRSPSTPRPTESFSKPEPR---- GQ PKPFFSDKVS


95
94
94
95
85
167
179
161


155
154
154
154
144
226
238
220


RTGYHFQPPRMWINDPNAPMYYKGLYHLFYQYNPKGAVWG
RTGYHFQPPRNWINDPNAPMYYKGLYHLFYQYNPKGAVWG
RTGYHFQPPKNWINDPNGPLYYKGLYHLFYQYNPKGAVWG
RTIFHFQPPRNWLNDPNGPMWYKGVYHLFY IGALFG
FQPPKNWMNDPNGPMYYKGVYHLFYQY IGAVWG
RTIFHFQP KNWMNDPNGPLYYKG Y IFYQMNPHAAVWG
RTAFHFQP NWMNDPNGPLYYKG YH FYQYNPHAAVWG
RTAYHFQPFKNWMNDPDGPLFMKGIYHLFYQYNPRAVWG












PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


PtCIN1
PtCIN2
PtCIN3
PtCIN4
PtCIN5
PtVIN1
PtVIN2
PtVIN3


A PQ K




QQDS D KW-LQ

GEDEDDKWf


NH
NH
YH A




AS
P


<* E >






SI


F
.S F
H T

S IS


PGQLWI-EEL
PGQLW LKP
PGQLWjAEEL


FSS KEPFIPKWAK LD SGKD
FSS KPF PKWA LD G KA
FPSK IPF PKWAK LD G KA

LN DIL I --PSWTIPQLISKs

DKKALEST SNVDFSCSTSGG--- A
DRKAIERT SNVEFSCSTNGG----SH
EISETKHEKY ------CSGG----


PGLP;L
PGG L



1S GPGL


R------
RRFLASI


213
212
212
213
203
285
297
280


273
272
272
273
263
344
357
340


D T
T F
DV F





FA A











PtCIN1 504
PtCIN2 510
PtCIN3 503
PtCIN4 500
PtCIN5 490
PtVIN1 573 I
PtVIN2 586 I
PtVIN3 563 F




PtCIN1 564
PtCIN2 570
PtCIN3 563
PtCIN4 559
PtCIN5 550
PtVIN1 632 K
PtVIN2 645 K
PtVIN3 622 K

B

PtNIN9 1
PtNIN11 1
PtNIN8 1
PtNIN12 1
PtNIN7 1
PtNIN10 1
PtNIN2 1
PtNIN5 1
PtNIN1 1
PtNIN3 1
PtNIN4 1
PtNIN6 1


PtNIN9 55
PtNIN11 55
PtNIN8 54
PtNIN12 54
PtNIN7 56
PtNIN10 1
PtNIN2 1
PtNIN5 56
PtNIN1 1
PtNIN3 55
PtNIN4 56
PtNIN6 56


LT
LT
LT
IPQ
PR
E-I
E-
T-


HKe

II
MKLTLRSIDHSVESFAGGR ITSV YP^IIAV


HKLSV LVDHSIIBaaaES AC IGR TSVMAAIY K

IEFQfOIM EID LJSF OGG IG:


P PS ---------
LP PENRGGENPRNE
SIKVP S
HKENFI--------
K STTfRRKPHL----
QI F RRYSNEQ--------
NI F RPYSNEQQ-------
I E F HPFIFDQN




-MSSLDGDVSQNGS KSVDAHP EIEDLDF DKPPRP EQCD ----
-MSSINVDVSLKGS RNAETLCD EIEEMDF DRPPRP DQCD ----

ID-EIIDKETVG NGSSVWS SEMDDIDF DKPK-- IESFD SLSE
---- DGKEMGG RNVSSVCS SEMDDFDL LDKPK-- IEQSFDESLSEL
MSPIAAMDVCQNAS KNFEAAGS FEIDSEFLR--LSDKPRP EKSFDESFS---
------------------------------------------------------------
------------------------------------------------------------
----- IRPCRFFLSKKNRVFFNIHHSLTSNLIGNQFNFEKNKIFFTYPFRILGSRTIFK
------------------------------------------------------------
---. ATIDAVLQILSGAGPRSFSSDLCFNNLDrAFR-SKHIKYV KRASRHMKMLEC
-----.TIEAILQILSGAGPCVFSSDPCFRSIDITFSSKLH KRV KWASRCMKMFECI
-ATIKTVLQILSGGLPCPHRFDLSFGGLNSVLSICSD KR1NIGLVYIKLNNGM



LNELFG-VPLLSPRPSSRAESNFRL DG YSP SGFN------------------
LSELSTGLPIPSPRPSSRVENNFRL NCPS SGFN------------------
IGLARG------------------- NFETTNSPG SGFN------------------
IGLARG------------------ FETTYSP GSGFN------------------
-------------------ENSFRI ENISPA SGFN------------------
------------------------------------------------------------
------------------------------------------------------------
EAQKSFCAPYISFGQSRLITGDFRGASIBASBASQVRIFSTSVETRVNDNNFERIYVQNG
------------------------------------------------------------
SVQQNCIGKHWFKRSGDGDLSVNAT KIQL RCKCQiAERVSGVTTEGGNGTWFVDSAK
NVLQNGIGNHWFKGLGDRDRSVNAT INQLIRCKIPQAERVSGVTE-GGNGTWFVDGAN
RLLGKCRSRGVG---AVTSRGKVKCIES RCKCQRAESFGGATANEWSPVSLPVNGV













TPRSQYG--F
TPLSQFG--V
TPASSARNSF
-TPASSTRNSF
TPR---SCGF


IDKDENVLGD
-----------
-IDKDENVLGD


IGIKPLVVER


PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6



PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6



PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6



PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6


WS K



I- D G
RI GVLVD CESVNRENL

---- E- l----- c- -


SPKREES EKEAWKLi vYcs'


SSS

NSTS


' -AVFRFISIAL


D RNS
D THN
D RKT
:D RKT
HjDRNI
DSDLEK



GS
GD
V GD
SE


E EVKNILK YDLSW ID
EIV NF LK L QS W i D 1
SIDIIQ A iQ
VRFLT LQ *~ l D 0

-VAANDPGD PND* 5
ILQSH


QSWE IHCY
-- KFL LQSW* VDC
IRFL LQSW* TMDH
IRFL LQSW* TMDH
E OIRNI *TL L SWEKTMD ,


FW 'LAA T
FWILRA K

7 WW LLRAYT
TA
WWIILL~T
5WILA I


L BYTI N I T I


I BYS S N I T I
L IL *Y 1N m m P



C IL *115m m P
C IL S I I I F
CG SL EZP Q GMLLLLSGDF


104 TH
105 TH
87 PH
87 PH
86 SH
1---
1 ---
136 IES
1 ---
173 SVD
175 SVD
131 ITQ


TLNLNGAVN--TPGVLELGDTQQLMREKEVLTSNGSANKEEESLATNGAVGTGRDASRKV
TLNQNGAVTGEHTDCFGAWDAQQLTREKEGFASKAALNQEKESLATNGAVGTGRDASPKV
HG --------------------------------------ATNIFEKGSFALKGNE


FGESIGRVPVD*





* IGRA. 3S
* IGRA. 3S
* IGRA. 3S
* IGRA. 3S
* A.


B
F
F
F
F
F

FL
L
L
L
L
L
L











C D


I..RMG
I..MG

SI M ID R M G
SIDRIMG
I..MG
SMIDRI**
CI.. MI
CI.. MI
CI.. MI
CI.. MI
CI.. MI
CI.. MI


PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6


PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6


PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6


PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6


456
439
439
437
350
338
479
297
530
533
488


F ITK' HEL
NMF 4TK
C IVK
NMF RU VK
F D TT H
F EKR L
S* S
S* S
L LS

I *NR LS
DI A.NR
I A


IFQ
IFQ
I.
* nEY IDLIN
*H YYID
*H I.WL
*H Y .
IL


FAA. T
F *C T







FLA


IA.Q A. LA.TLKD P
IA.QA.EAER A. E

E u.ILATLKDPY
IA. *A.DAE 3D A.


E F














IS I
IS L
IS L
I S LE
* FWIBY5
* FWIBY5
* F BY
TI 'PDK


L LP

LLFVN I


ClQ W MMAGYLVKMMLD LS
YQTS IMAGYLVAKMLEP L



FQTWS iAGlVAKTS LENP


i sK SS
isWIGLIMLE
iQTsIGLALLDP


QIKPPMRRSH F-
QIKPPIRRSN --
QINPVLKRSS C-
QKPVLRRSSW C
QTHLVKRS C-
KSARSRLTRSNS SF
EFCVCG NT
EICVCG NT
EICVC
NAFSC SNP
SAFSC THP
IN ---AL A


PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6


PtNIN9
PtNIN11
PtNIN8
PtNIN12
PtNIN7
PtNIN10
PtNIN2
PtNIN5
PtNIN1
PtNIN3
PtNIN4
PtNIN6


Figure 2-5. Amino acid alignment of poplar invertases. A) Amino acid alignment of acid
invertases. Black shading corresponds to identical residues, grey shading corresponds
to similar residues. A-F denote highly conserved motifs as defined by Pons et al.
(1998) which are thought to play roles in activity. Asterisks mark completely
conserved residues thought to be involved in substrate binding or catalysis (Alberto et
al. 2004). B) Amino acid alignment of neutral/alkaline invertases. A-O correspond to
amino acids that are consistently different between a and 0 group invertases.
Asterisks show amino acids most likely to correspond to active residues for
neutral/alkaline invertases, assuming equivalence with the catalytic residues of
unsaturated glucuronyl hydrolase (UGL) from Bacillus sp. (Itoh et al. 2004). Black
shading corresponds to identical residues, grey shading corresponds to similar
residues. Alignment produced with CLUSTALW and figure produced with
BOXSHADE.


S..GQA
S..GQA
S..GQA
S..GQA
L eVKQA

OS.GQA

*KIK OS.
S..GQ


RCSRVA SQI
RCSRGAASQI
KCSRF ASQI
KRGQKNS KPF
NRGQKNS KTFW
KRARKIF QPF









CHAPTER 3
RECIPROCAL SUGAR REGULATION IS CONSERVED AMONG VACUOLAR
INVERTASES OF POPLAR, ARABIDOPSIS, MAIZE AND RICE

Introduction

Sugars play an essential role in plant cell growth and development as they act as fuel for

metabolism, generate osmotic pressure for short and long distance transport and act as signaling

molecules for various metabolic pathways (Koch, 1996; Rolland et al., 2002; Halford and Paul,

2003). Sucrose is the primary form of sugar transport in most plants, establishing this

disaccharide and its glucose and fructose cleavage products as central to plant growth and

development.

Invertase (EC 3.2.1.26), also known as P-fructofuranosidase, irreversibly hydrolyzes

sucrose into glucose and fructose, and is positioned to play a central role in both carbon

metabolism and sugar signaling. Several invertase isozymes have been identified from plant

species as either soluble (readily extractable from cytosol or vacuole), or insoluble (bound to cell

wall components). Vacuolar and cell wall invertases show optimal activity at an acidic pH and

are also called acid invertases. The mature acid invertases are glycosylated and located in the

cellular compartment their name implies (e.g. cell wall or vacuolar).

Vacuolar invertases (VIN), like their cell wall localized family members, are thought to

have evolved from respiratory eukaryotes and aerobic bacteria (Sturm and Chrispeels, 1990a).

VINs have been identified and characterized to various levels in many different plant species

including, but not limited to, Arabidopsis, carrot, maize, poplar, rice, tobacco and tomato

(Tymowska-Lalanne and Kreis, 1998b; Ji et al., 2005; Bocock et al., 2007). To my knowledge,

with the exception of poplar (containing three), all plants studied to date encode only two VINs.

Of poplar's three VINs, one contains no introns and is thought to be a recent evolutionary









innovation stemming from a processed transcript inserted in trans back into the genome (Bocock

et al., 2007).

VIN transcripts have been found to be reciprocally regulated by sugar. In maize, Ivrl was

found to be transcriptionally repressed after treatment with exogenous glucose under the same

conditions that induced transcription of Ivr2 (Xu et al., 1996). In tomato, the VIN, TIV1, was also

found to be repressed after treatment with exogenous sugar, however, induction of the second

tomato VIN under the same conditions was not shown (Godt and Roitsch, 1997a).

An important driver of species origination and diversification is gene duplication.

Duplication makes it possible for a gene to acquire new functions without losing that of the

progenitor gene (Kramer et al., 1998; Lynch and Conery, 2000; Sankoff, 2001; Becker and

Theissen, 2003; Irish, 2003; Litt and Irish, 2003; Zahn et al., 2005). Gene duplication can occur

in tandem, through the duplication of a chromosomal segment, an entire chromosome, or through

genome duplication (Otto and Whitton, 2000; Wendel, 2000; Adams and Wendel, 2005).

Genome duplications give rise to copies of linked genes in a particular order that can be used to

infer ancestry of loci of interest both within and between species (Lynch and Conery, 2000).

Poplar, Arabidopsis, maize and rice are all known to have undergone multiple genome

duplication events in their evolutionary history. Poplar and Arabidopsis have each undergone a

genome duplication event that post-dates their divergence on the evolutionary tree (Bowers et

al., 2003; Sterck et al., 2005; Tuskan et al., 2006). Poplar and Arabidopsis also share at least two

genome duplication events that pre-date their evolutionary divergence and are referred to as the 0

and y genome duplication events (Bowers et al., 2003; De Bodt et al., 2005; Tuskan et al., 2006).

On the monocot side of the evolutionary tree, maize is known to have undergone two genome

duplication events, one post- and the other pre-dating its divergence with rice (Blanc and Wolfe,









2004). The y event referred to previously is thought to have occurred prior to the monocot-

eudicot divergence. Some evidence suggests that the y event may even pre-date the gymnosperm

divergence, although this conclusion requires more data (Bowers et al., 2003). The timing and

location of genomic duplication events can be utilized to understand the evolutionary

development of a variety of gene families.

In this work, the sequenced genomes of poplar, Arabidopsis, and rice are compared in

order to test possible scenarios for the evolutionary development of the VIN family. I also take

advantage of the unique reciprocal sugar regulation that occurs in this family to analyze the

functional conservation of VINin monocots and eudicots. This evidence indicates that the two

VINs so common throughout the plant world arose at, or prior to the y genome duplication event.

I also examined the reciprocal sugar response in Pinus taeda, a gymnosperm, but was unable to

validate the presence of this reciprocal response.

Materials and Methods

Plant Material

Populus trichocarpa (genotype "Nisqually-1") and Pinus taeda were grown in 8 L pots in

a fan- and pad-cooled greenhouse with natural light augmented with full spectrum fluorescent

lighting during the winter to give a day length of 15 h. Greenhouse temperatures ranged from 20-

35 C. At noon, light intensity in the greenhouse averaged 500-700 iE/m2/min PAR, which is

one-half the light intensity outside the greenhouse. Plants were grown on an ebb-and-flow flood

bench system with a daily supply of Peters Professional 20-10-20 water-soluble fertilizer

diluted to a final concentration of 4 mM nitrogen. Plants were grown to a height of 60-100 cm

prior to experimentation.

Arabidopsis plant material was prepared as described by Huang (2006). Briefly,

Arabidopsis thaliana (Col-0) seeds were sterilized and soaked in water for 4-5 d at 4C in the









dark. Seeds were sown on half-strength Murashige and Skoog (MS) medium, pH 5.8

(GIBCOBRL, U.S.A.) solidified with 0.16% (w/v) phytagel. The typical sugar supplement for

enhanced plant growth was omitted. Plantlets were cultured under cycles of 12 h light/dark at 25

C. All plant material was immediately frozen in liquid nitrogen after harvesting, and stored at -

80 C prior to RNA extraction.

Sequence Alignments and Similarity Trees

Predicted amino acid sequences were aligned using CLUSTALW

(http://clustalw.genome.jp) to construct similarity trees using the TREEVIEW program (Page,

1996). PAUP (Swofford, 1993) was used for bootstrap analysis with 100,000 iterations.

RNA Extraction

Total RNA was extracted from poplar and pine leaves using standard methods (Chang,

1993), DNase-treated and purified on RNAeasy QIAGEN columns (Valencia, CA). Total RNA

was extracted from Arabidopsis plants and rice embryos using the RNeasy Plant Mini Kit

(QIAGEN) and DNase treated (DNA-free Kit, Ambion, Austin, TX) according to manufacturers'

instructions.

Quantitative RT-PCR in Poplar and Pine

Poplar and pine cDNA was synthesized from purified RNA (5 [tg) using a mixture of 500

ng oligo-dT, 100 ng random primers, and M-MLV-RT as per manufacturer's instructions

(Invitrogen, Carlsbad, CA), with the exception that the DTT was excluded. Gene expression was

analyzed using the SYBR Green kit (Stratagene, La Jolla, CA) and Mx3000P thermo-cycler

(Stratagene) as per manufacturer's instructions. Briefly, 1 [tl of synthesized cDNA and 0.15 [tl of

a 0.25 [tM solution of each primer were used for each 50 [tl RT-qPCR reaction. Primers were

designed using NetPrimer (Premier Biosoft International, Palo Alto, CA) software and

synthesized by Invitrogen. Primer sequences are as follows: PtVIN1 forward, 5'-









GCCCTGGTTCCACCTATAGTT-3'; reverse, 5'-TCCTGGCCTCAAAATTTGC-3' (yields 216

bp product); PtVIN2 forward, 5'-CCCAGCTATAGTCCTGCCC-3'; reverse, 5'-

CGCATCTTCGTCTTCTTGTG-3' (yields 189 bp product); PtVIN3 forward, 5'-

ATACACTCCCTTGCTAGACAACC-3'; reverse, 5'-GCTTGGAAAATGACTCTGTAGGTC-

3'(yields 197 bp product); PtaedaVIN1 forward, 5'-TGATTCCCGACCGCTGG-3'; reverse, 5'-

ATTAGCCTCCGACTTCACCC-3' (yields 185 bp product); PtaedaVIN2 forward, 5'-

GGGCTGCGGTATGATTATGG-3'; reverse, 5'-CTGGAGCAGCACATTCTCG-3' (yields 261

bp product). Each real-time PCR reaction was performed in triplicate (technical replicates) on

four individual plants (biological replicates) in poplar and six individual plants in pine and

carried out for 40 cycles with annealing, extension, and melting temperatures of 55 C, 72 C,

and 95 C, respectively. Melting curves were generated to check the specificity of the amplified

fragments. In the case of PtVIN2, an extension temperature of 79 C with the fluorescence

reading taken at the end of the run was used to correct for a spurious primer-dimer amplicon.

Changes (n-fold) in gene expression relative to the geometric mean (Vandesompele et al., 2002)

of three control genes encoding actin, ubiquitin and ubiquitin_L (Brunner et al., 2004) were

determined using the program DART-PCRv1.0 (Peirson et al., 2003).

Quantitative RT-PCR in Arabidopsis

Real time quantitative RT-PCR in Arabidopsis was conducted as described by Huang

(2006). Briefly, 200 ng of RNA were used in 25 al reactions with Taq-Man one-step RT-PCR

Master Mix reagents (Applied Biosystems, Foster City, CA) according to manufacturer's

instructions.









Sugar Treatments for Poplar, Arabidopsis, Pine and Rice

Mature leaves (LPI 12) from four greenhouse grown poplars (biological replicates, one leaf

per plant, n=4) were covered in aluminum foil for three to five days. Leaves were then excised

and taken to a dark room where aluminum foil was removed. Leaves were lightly rubbed with a

carborundum suspension to remove the cuticle and subsequently cut in half (lengthwise). The

two leaf halves were placed in 1 % (w/v) mannitol (negative control) and 1 % (w/v) glucose,

respectively, and incubated in the dark at room temperature on a shaker. After a 16 h incubation

period, leaves were frozen in liquid nitrogen and RNA extracted.

Sugar treatments in Arabidopsis were conducted on two week old plants that were placed

in the dark for three days, after which they were transferred to 1/2 strength liquid MS minimal

media supplemented with 1 % (w/v) mannitol or glucose and incubated in the dark at room

temperature on a shaker. After a 16 h incubation period, plants were harvested.

Greenhouse grown Pinus taeda branches containing fully mature needles were wrapped in

aluminum foil for five days. Branches were then excised and taken to a dark room where

aluminum foil was removed. Needles were removed and then lightly rubbed with a carborundum

suspension to remove the cuticle. Needles from a single branch were then divided in half

between solutions of 1 % (w/v) mannitol and 1 % (w/v) glucose, respectively, and incubated in

the dark at room temperature on a shaker. After a 16 h incubation period, needles were frozen in

liquid nitrogen and RNA extracted. Needles were used from six different plants (biological

replicates, n=6).

Light-Dark Treatments for Poplar, Pine and Arabidopsis

Mature leaves (LPI 12) from four greenhouse grown poplars (biological replicates, n=4)

were covered in aluminum foil for five days. After the fifth day, half of the leaves were

uncovered and exposed to sunlight for approximately 5 h. At noon, wrapped and exposed leaves









were harvested and RNA extracted. Greenhouse grown Pinus taeda branches containing fully

mature needles from six plants (biological replicates, n=6) were wrapped in aluminum foil for

five days. After the fifth day, half of the branches were uncovered and exposed to sunlight for

approximately 5 h. At noon, needles from the wrapped and exposed branches were harvested and

the RNA extracted. Treatments in Arabidopsis were conducted on two week old plants that were

placed in the dark for three days, after which half were exposed to light for approximately 5 h

before plants were harvested and RNA extracted.

Results

Predicting Gene Orthology Using Protein Sequence Similarity

The hypothesis that the reciprocal regulation of VIN, first identified in the maize VINs Ivrl

(ZmlVR1) and Ivr2 (ZmlVR2),(Xu et al., 1996) is conserved not only in monocots, but also in

eudicots was tested. To my knowledge, all plants studied to date contain two VINs, with the

exception of poplar, which contains three. Poplar's PtVIN1 is thought to be a very recent

innovation specific to poplar since it has no introns and likely arose through a processed

transcript of PtVIN2 inserting into the genome in trans (Bocock et al., 2007). I predict that the

poplar VINs PtVIN2 and -3 (not PtVIN1) are the likely othologs of ZmlVR1 and -2. Arabiodopsis

and rice both contain only two VINs and are referred to here as AtvaclVR1, AtvaclVR2, OsVIN1

and OsVIN2, respectively (Haouazine-Takvorian et al., 1997; Ji et al., 2005).

Nucleotide and protein sequences were obtained from the various databases (Joint Genome

Institute, poplar; NCBI, Arabidopsis and maize; TIGR, rice and maize). I then translated the

nucleotide sequences and constructed amino acid alignments between the four species (data not

shown). I discovered that ZmIVR2 (accession # CAD91358) as it exists in NCBI, is missing

information on the N-terminal end. In order to obtain more complete sequence, I searched the

TIGR Maize database. I found that ZmIVR2 corresponds to TIGR contig AZM5_84630.









Subsequent analysis of the contig in GENESCAN (http://genes.mit.edu/GENSCAN.html) (Burge

and Karlin, 1997) allowed me to identify an additional 42 amino acids on the N-terminal end.

Based on amino acid alignments with the other VINs, I estimate that there are still roughly 140

amino acids missing from the N-terminal end (data not shown).

To obtain VIN sequences for pine, I searched for Pinus taeda ESTs on NCBI using

AtvacIVR1 and -2 as queries. The ESTs were then aligned and assembled using the Sequencher

program (Gene Codes, Ann Arbor, MI). This technique resulted in two, distinct contigs referred

to hereafter as PtaedaVIN1 and PtaedaVIN2. The lack of a third contig resulting from the

assembled ESTs indicates that pine, like the other plant species mentioned above, encodes only

two VINs. The nucleotide sequences were translated into amino acid sequences and protein

alignments revealed that PtaedaVIN1 and -2 are missing roughly 130 and 270 amino acids from

their N-terminal ends, respectively (data not shown).

The amino acid sequences were then analyzed in an attempt to identify the vacuolar

orthologs between species. As can be seen in Table 3-1, OsVIN1 and ZmIVR1 appear to be

orthologs as they are more similar to each other (82 % similarity) than they are to their family

members within each species. This pattern is repeated between OsVIN2 and ZmIVR2 (88 %

similarity) indicating that these two genes are also orthologous, based on sequence similarity. In

the case of poplar, Arabidopsis and pine, orthology cannot be determined. In Arabidopsis and

pine, the VINs are most similar to their family member within the species rather than their

putative orthologs in other species. In contrast, the poplar VINs in comparison to each other are

less similar than compared to any other VIN (Table 3-1).

These alignments were then used to generate a bootstrapped protein similarity tree (Figure

3-1). As expected from percent similarity, OsVIN1 and ZmIVR1 cluster together as do OsVIN2









and MzIVR2. However, the poplar, Arabidopsis and pine VINs tend to cluster within each

species rather than with their orthologs (Figure 3-1). Using this data, it was predicted that

ZmlVRI and OsVIN1 are orthologs whose transcripts are sugar repressed while ZmlVR2 and

OsVIN2 encode sugar induced transcripts. It is not straightforward to predict orthology between

poplar, Arabidopsis and pine using this approach due to the lack of obvious pairwise clustering

of protein sequences among species.

A Chromosome Duplication Event is Responsible for the Two-Member VIN Family in
Poplar and Arabidopsis

Large scale chromosomal or genome duplications are responsible for the growth of many

gene families (Sankoff, 2001). Chromosomal segments arising from genome duplication events

often retain a conserved linear order of loci. Conservation of loci order on a chromosomal

segment is not only evidence of gene family expansion through large scale chromosomal

duplication, but it also can serve as another method to be used in predicting orthology, and

therefore function. Figure 3-2A depicts the locations of several putative large scale or genomic

chromosomal duplications overlayed on an evolutionary tree outlining the development of

poplar, Arabidopsis, maize, pine and rice.

In a previous work, I examined the chromosomal segments containing the VINs in poplar

and Arabidopsis and identified neighboring open reading frames (ORF) that were used to

establish colinearity between the species (Bocock et al., 2007). In summary, I showed that poplar

and Arabidopsis VINs lie within conserved, colinear chromosomal segments (Figure 3-2B)

(Bocock et al., 2007). These data indicate that a chromosomal duplication event is responsible

for the development of the two-member VIN family in poplar and Arabidopsis. Conservation of

colinearity between the two species indicates that the duplication event must have occurred prior

to poplar and Arabidopsis divergence, likely in duplication event 0 or y (Figure 3-2A).









I hypothesized that the duplication event giving rise to the two member VIN family

occurred prior to the eudicot and monocot divergence, which would explain the conserved two

member VINfamily size in maize and rice. To address this question, I examined the

chromosomal segments containing the VINs in maize and rice to identify neighboring ORFs that

could be used to establish colinearity between maize, rice and the eudictos. Unfortunately, I was

unsuccessful in identifying neighboring ORFs in maize as the contigs containing ZmlVR1 and -2

were not large enough to contain any ORFs other than the VINs. In rice, I was able to identify

numerous ORFs upstream and downstream of OsVIN1 and -2 and compare these to the eudicots.

No evidence of colinearity was found between rice and the eudicots. This finding does not

support the hypothesis, however it also does not necessarily contradict the hypothesis as it is

quite common for small insertions, deletions and rearrangements to occur subsequent to genome

duplication events (Sankoff, 2001). If the VINs truly arose from the y event, then there have been

approximately 221-300+ million years (Blanc and Wolfe, 2004) for rearrangements to occur and

obscure the duplication event.

Sugar Response Demonstrates Conservation of Gene Function in VINs

Based on amino acid sequence similarity, I hypothesized that OsVIN1 and ZmlVR1 are

orthologs and likely retain the same transcript repression to exogenous sugar. Similarly, I

predicted OsVIN2 and ZmlVR1 to be orthologous and retain the same transcript induction to

exogenous sugar. In poplar and Arabidopsis, the link is a bit more confusing. I was able to use

conservation of colinearity to establish chromosomal duplication as the likely originator of the

two VINs, however I was unable to use it to establish orthology.

I next examined the response of the VINs to exogenous sugar treatment to address the

above questions. Leaves from greenhouse-grown poplars were wrapped in foil for approximately

three to five days in order to clear the leaves of any carbohydrate reserves. The leaves were then









excised, treated with a suspension of carborundum to remove the cuticle, and incubated

overnight in solutions of either 1 % mannitol (to serve as an osmotic and wounding control) or

glucose in the dark. Transcripts for PtVIN1,2 and -3 were then assayed. I found that, like

ZmlVR1, transcripts for PtVIN2 were repressed in the presence of sugar (Figure 3-4A).

Transcripts for PtVIN3 were induced in the presence of sugar (data not shown). PtVIN1, the

intronless poplar invertase discussed earlier showed no sugar response at all. Transcripts for

PtVIN1 were also found to be two orders of magnitude less abundant than its presumed

progenitor, PtVIN2 (data not shown). I speculate that PtVIN1 lost at least some of the regulatory

features of PtVIN2 when it re-inserted into the genome.

Huang (2006) examined Arabidopsis plants for this sugar response. Whole plants grown on

sugar free media were transferred to the dark for approximately three days to clear the

carbohydrate reserves. The plants were then transferred to 1 % mannitol or glucose in the dark.

After incubation in the solutes, transcripts for AtvaclVR1 and -2 were assayed. Similar to

ZmlVR1 and PtVIN2, transcripts for AtvaclVR2 were found to be repressed (Figure 3-4A).

AtvaclVR1 was found to be induced under these conditions (data not shown).

In a similar fashion, Huang (unpublished data) treated rice embryos with 3 % sucrose,

which in poplar, Arabidopsis and maize incurs the same VIN response as glucose (data not

shown). OsVIN1 transcripts were found to be down-regulated as expected from OsVINI's

similarity to ZmlVR1 (Figure 3-4A).

Reciprocal Response in Light

Exogenous sugar treatments serve as an in vitro assay to mimic photosynthesis conditions

in vivo by recreating the high sugar environments associated with active photosynthesis. I

hypothesized that the VINs repressed by sugar would also be repressed by light, while the sugar

induced VINs would be induced by light. Leaves of greenhouse grown poplar were covered for









three to five days to clear the leaves of any starch reserves. Half of the foil covered leaves were

then uncovered and exposed to light. At noon on the same day, the covered and light exposed

leaves were harvested and transcripts associated with PtVIN2 and -3 were assayed. Similarly,

Arabidopsis plants grown on /2 strength MS media lacking sugars were placed in the dark for

three to five days. Half of the plants were then moved to the light and allowed to photosynthesize

for approximately 5 h. The plants were then harvested and transcripts for AtvaclVR1 and -2 were

assayed. As expected, light was found to repress transcripts for PtVIN2 and AtvaclVR2 while

inducing transcripts for PtVIN3 and AtvaclVR1 (Figure 3-3).

Discussion

In this study, I utilized newly released sequence information to establish orthology

between VINs in poplar, Arabidopsis, maize, pine and rice. I was able to establish clear orthology

between ZmlVR1 and OsVIN1 as well as ZmlVR2 and OsVIN2 using available nucleotide data,

however establishing orthology between the other species proved more difficult.

Through an examination of gene colinearity on chromosomal segments encoding the VIN

loci, I was able to establish that a chromosomal duplication gave rise to the two-member VIN

family in poplar and Arabidopsis. I was unable to establish conservation of colinearity between

the eudicots and rice, however a lack of sequence information from maize made it impossible to

compare the two monocots. The fact that the maize and rice genomes both appear to encode only

two VINs makes it likely that the chromosome duplication event giving rise to the two VINs in

the eudicots occurred prior to the eudicot-monocot divergence in the y event. Convergent

evolution in the monocots could serve as an alternate explanation for the appearance of the two-

member VIN family in the monocots. This can only be ruled out by identifying a conservation of

colinearity on the VIN chromosomal regions between rice and another monocot, such as maize or

sorghum, as more genomic sequence becomes available. The grasses retain considerable









conservation of colinearity (Bennetzen and Ma, 2003), so it is not unreasonable to expect that the

predicted colinear regions will be identified.

It will be more difficult to test colinearity between the eudicot lines and those of the

monocot lines due to the extent of time and evolutionary change since their divergence. The

conservation of function described in this paper, however does go a long way in establishing

orthology between the monocot and eudicot VINs. While the conservation of reciprocal sugar

response cannot rule out the explanation of convergent evolution, the likelihood that only two

VIN family members would be encoded by such a diverse array of plant genomes and also retain

the same functions for such a long evolutionary time frame makes the convergent evolution

hypothesis extremely unlikely.

This work also describes the identification of two EST contigs from the gymnosperm,

Pinus taeda, that are highly similar to previously described VINs in other species. I name these

two contigs PtaedaVIN1 and PtaedaVIN2. These two putative VINs had good EST coverage for

nearly the entire coding region. As I was unable to find any ESTs that assembled into a third VIN

contig, I believe that PtaedaVIN1 and -2 are either the only two VINs encoded by the Pinus taeda

genome, or, at the very least, the only two significantly expressed VINs in this species. In order

to identify conservation of VIN function between the gymnosperms and angiosperms, I

conducted the all the same sugar and light/dark experiments as done for poplar and Arabidopsis.

I was unable to identify the same reciprocal response that was seen in poplar and Arabdidopsis.

It is unclear whether this is because the P. taeda VINs do not have the reciprocal regulation

feature as do the angiosperms, or that conditions tested did not effectively reveal these responses.

The possibility remains that additional experimentation could define a differential sugar

responsiveness in pine under somewhat different conditions.









In this study, I show that the reciprocal sugar regulation of VINs first identified in maize

(Xu et al., 1996) is conserved in at least three eudicot species. Sugar treatments were conducted

on poplar and Arabidopsis and demonstrate the reciprocal response in these two species. I also

demonstrate that sugar repressed OsVIN1 transcripts, as predicted based on its sequence

similarity to the sugar-repressed ZmlVR1. A search of the literature also revealed that TIV1, one

of tomato's two vacuolar invertases is also repressed after treatment with glucose (Godt and

Roitsch, 1997a). Collectively, these data help to establish this sugar response as highly conserved

throughout plants.

Poplar and Arabidopsis plants were subjected to a light/dark experiment intended to mimic

the exogenous sugar treatment experiments. The purpose of this is two-fold. First, demonstrating

a commonality between exogenous sugar applications and light treatments helps to establish the

sugar-induced VINs as "feast" genes. Feast genes are those that are induced in response to sugars

and are thought to play roles in storage processes and carbon utilization. These genes would also

be up-regulated in actively photosynthesizing cells (Koch, 1996). This helps us to place these

sugar up-regulated genes in a more general context regarding their role in plant growth and

development. The second purpose of establishing the commonality of transcript response

between the light/dark treatment and the exogenous sugar treatment would be the ease of the

assay. For plants that are less amenable to experimental manipulation than poplar and

Arabidopsis, the light/dark treatment provides another tool for testing the reciprocal VIN

response.

Advances in genome sequencing are rapidly increasing available genomic sequence. This

abundance of data is allowing new types of questions to be asked as well as application of

functional data to the larger story of plant evolution. In this study of the VIN gene family, I have









added to our knowledge of the response of VINs to sugar and light treatments. I have tied this

into a larger, evolutionary story and discussed how this gene family has developed with the

evolution of plants. Hopefully this work has achieved its goal of aiding our understanding of how

plants process and utilize carbohydrates obtained through photosynthesis.













Table 3-1. Percent similarity of predicted VINs from poplar, Arabidopsis, rice and maize.
Protein names are in bold with the corresponding gene model names below.
PtVIN2 PtVIN3 AtvaclVR1 AtvaclVR2 OsVIN1 OsVIN2 IMzlVR1 MzlVR2 IPtaedaVIN1 PtaedaVIN2
PtVIN2 69 77 76 77 69 72 76 74 74
estExt fgenesh4 pg C LG 1110902
PtVIN3 69 71 71 67 71 70 71 70
estExt Genewisel v1 C LG XV2841
AtvaclVR1 85 70 67 69 71 73 71
Atlg62660
AtvaclVR2 70 70 69 72 73 71
At1g12240
OsVIN1 77 82 76 74 73
Os04g45290 1
OsVIN2 74 88 70 69
Os02g01590
MzlVR1 76 73 75
P49175
MzlVR2 69 70
AZM5 84630
PtaedaVIN1 89

PtaedaVIN2













OsVIN2 1

100

100

ZmlVR1 100 8 PtVIN3
91


100
OsVIN1
PtVIN2



01 AtvaclVR2 AtvacIVR1


Figure 3-1. Protein similarity tree of VINs. Bootstrap values are reported as a percentage of
100,000 repetitions. Branch lengths denote protein similarity with the exception of
ZmIVR2, PtaedaVIN1 and -2 where the first -140-230 amino acids have not yet been
sequenced.


PtaedaVIN2


PtaedaVIN1


ZmlVR2

































flavin-containing 3-oxoacyl-facyl-halose-6-phosphate
monooxygenase carrier-protein] rhostase Th-lakoid no apical meristem (NAM)
family protein synthaselll 45Kb phosphatase Multicopperoxidase 32 Kb Thylakoi n family protein

Trehalose-6-phosphate PtVIN2 Multicopper oxidase no apical meristem
ph htase uo o (NAM) family protein

flavin-containing 3-oxoacyl-[acyl- PtVIN3
monooxygenase disease resistance proteiarrier-protein] no apical meristem (NAM)
family protein (CC-NBS-LRR class) snthase Il family protein

flavncontainingvaclNV
monooxygenase disease resistance protein Thylakoid no apical meristem (NAM)
family protein (CC-NBS-LRR class) lumenal protein- family protein 4Kb

AtvaclNV2


Figure 3-2. A) Phylogenetic representation of relevant plant species. Stars represent large scale
duplication events as proposed by Blanc and Wolfe (2004), Van de Peer (2004), and
Sterck et al. (2005). Greek letters denote duplication events as described by Bowers
et al. (2003). There is some uncertainty as to the location of the y event relative to the
divergence of the gymnosperms and angiosperms, this is denoted by "?". B)
Microcolinearity between poplar and Arabidopsis VINs. Boxes depict genes. Poplar
VINs are checkered, and Arabidopsis VINs are solid, all other members of a particular
gene family are depicted with matching patterns. Labels appear over the first
appearance of a certain gene family member. Panel B adapted from Bocock et al.
(2007).















PtVIN2 AtvaclVR2
120 120
100 100 Rice embryos
Suc
80 80
z z
E 60 E 60 0% 3
40 40 OsVINI
20 20
0 20 Actin
0- 0
Mannitol Glucose Mannitol Glucose

Figure 3-3. Conservation of specific VIN isoform transcript repression under sugar treatments
across taxa. A) PtVIN2, AtvaclVR2 and OsVIN1 transcripts are repressed by sugar.
Bars represent the mean level of transcript; error bars denote SEM; n=4 and n=3 for
PtVIN2 and AtvaclVR2, respectively. Rice panel contributed by L-F Huang. Suc,
sucrose.











PtVIN2


Dark Light


AtvaclVR2


PtVIN3

-I-


Light


AtvaclVR1


Dark Light
Dark Light


Dark Light


Figure 3-4. Conservation of reciprocal regulation of VINtranscript under dark and light
treatments across taxa. A) Poplar's PtVIN2 is repressed by light while PtVIN3 is
induced by light. Bars represent the mean level of transcript; error bars denote
standard error (SEM); n=3. B) Arabidopsis' AtvaclVR2 is repressed by light while
AtvaclVRI is induced by light. Bars represent the mean level of transcript; error bars
denote SEM; n=3. Panel B contributed by L-F Huang (2006).









CHAPTER 4
OVEREXPRESSION OF YEAST INVERTASE IN POPLAR

Introduction

Plants fix carbon and obtain energy through photosynthesis whereby sunlight is harnessed

and used to reduce and fix carbon obtained from CO2. As photosynthesis does not occur in all

plant cells, carbohydrates synthesized in the photosynthesizing "source" cells must be

transported to the non-photosynthesizing "sink" cells. The primary products of photosynthesis

are starch and sucrose. While starch serves primarily as a means of storage, sucrose plays a

central role in the transport of photoassimilates throughout the plant.

Sucrose, as the primary form of sugar transport in most plants, acts as a major player in

plant growth and development. Sucrose is a non-reducing disaccharide made of a molecule of

glucose covalently bonded to a molecule of fructose, and is found in multiple compartments

within the cell. In source cells, sucrose is synthesized in the cytosol, can be stored in the vacuole

for later use, can travel to neighboring cells through plasmodesmata, and eventually enters the

phloem via plasmodesmata or from the surrounding apoplast depending on whether the plant is a

symplastic or apoplastic loader of phloem (Turgeon, 1989; Grusak et al., 1996).

The composition of sucrose from two hexoses means sucrose plays roles not only as a

transport molecule, but also in osmotic maintenance and sugar signaling. The cleavage of a

single sucrose molecule in solution results in two molecules of hexoses thereby doubling the

osmotic potential of the solution. By cleaving sucrose and compartmentalizing the resulting

hexoses in various cellular compartments, osmotic gradients are created providing the basis not

only for short-distance transport from cell to cell, but also for long-distance transport from organ

to organ via the phloem as first proposed by Minch (1930). Sucrose and its component hexoses









provide sugar signals triggering numerous biological pathways playing roles in cell division,

expansion, differentiation and maturation (Koch, 2004).

The enzymes that cleave sucrose are influential players of plant metabolism and

development. Invertase (EC 3.2.1.26) is one of only two enzymes able to cleave sucrose.

Invertase, also known as P-fructofuranosidase, hydrolyzes sucrose into two hexoses, glucose and

fructose, in an irreversible reaction. Invertase is found in multiple cellular compartments and can

be divided into three sub-families base on cellular localization. Invertases localized to the

vacuole and apoplast are also known as acid invertases based upon their pH optimum for activity

(Haouazine-Takvorian et al., 1997; Sherson et al., 2003). The third sub-family of invertase is

localized to the cytosol and are also known as neutral/alkaline invertases due to their pH

optimum (Chen and Black, 1992; Van den Ende and Van Laere, 1995).

Several groups in the past have utilized transgenic forms of plants to study the role of

invertase in sucrose translocation. Invertase derived from yeast has been overexpressed in the

apoplast in Arabidopsis (Von Schaewen et al., 1990), potato (Heineke et al., 1992), tobacco

(Sonnewald et al., 1991) and tomato (Dickinson et al., 1991). Overexpression in this

compartment resulted in numerous growth defects summarized by stunted growth, inhibition of

photosynthesis, accumulation of leaf starch, and necrotic lesions on leaves followed by overall

yellowing of the leaf (Von Schaewen et al., 1990; Dickinson et al., 1991; Sonnewald et al.,

1991; Heineke et al., 1992). Sonnewald et al. (1991) also overexpressed yeast derived invertase

in the vacuolar and cytosolic compartments of tobacco resulting in similar phenotypes to the

apoplastic overexpressing tobacco plants.

Here I describe the overexpression of yeast invertase in Populus, a deciduous, perennial

tree species. While the previously mentioned work has done much to establish the role of









invertase in carbon allocation and partitioning, it is still unknown what roles invertase may play

in a deciduous, perennial plant. The deciduous, perennial nature of poplar requires numerous

cycles of sugar movement and carbon sequestration in storage organs such as the root and stem

in the winter, followed by remobilization of these stored carbon compounds the following spring.

It is also thought that poplar may employ a unique mode of sucrose movement into the phloem

of source leaves analogous to that of poplar's closely related cousin, willow (both members of

Salicaceae) (Turgeon and Medville, 1998). A reverse genetic approach was utilized here to

directly manipulate invertase levels in three subcellular compartments, so that the effects of

ectopic invertase expression could be assessed in poplar.

Materials and Methods

Plant Material, Transgenesis, and Growth Conditions

Hybrid poplar clone, INRA 717-1-B4 (P. tremula x P. alba) was placed into sterile culture

prior to Agrobacterium-mediated transformation (Leple et al., 1992). Individual clones from

independent lines were clonally propagated as softwood cuttings under mist, transferred to 8 L

pots and grown to a height of 60-100 cm prior to experimentation in a fan- and pad-cooled

greenhouse with natural light augmented with full spectrum fluorescent lighting during the

winter to give a day length of 15 h (Lawrence et al., 1997). Greenhouse temperatures ranged

from 20-35 C. At noon, the light intensity in the greenhouse averaged 500-700 iE/m2/min PAR,

which is one-half the light intensity outside the greenhouse. Plants were grown on an ebb-and-

flow flood bench system with a daily supply of Peters Professional 20-10-20 water-soluble

fertilizer diluted to a final concentration of 4 mM nitrogen.

Vector Construction

The cell wall targeted SUC2 construct (CwSUC2) was obtained by a generous donation

from Uwe Sonnewald (Institut fur Biologie der Universitat Erlangen-Nurnberg, Erlangen,









Germany). The cytosolic targeted SUC2 construct (CytSUC2) was PCR amplified without the N-

terminal signal peptide from the CwSUC2 construct using the primer sequences: CytSUC2

forward, 5'-CACCATGACAAACGAAACTAGCGATAG-3'; CytSUC2 reverse, 5'-

CAGGTAACTGGGGTCGGGAGAA-3'. The vacuolar targeted SUC2 construct (VacSUC2) was

designed by adding a vacuolar targeting domain from tobacco chitinase to the 3' end of the

CwSUC2 construct in two PCR steps. The first step used the primer sequences: CwSUC2

forward, 5'-CACCATGGATGTTCACAAGGAAGTTA-3'; VacSUC2 reverse_step_l, 5'-

GTCCAACAAACCATTACCTTTTACTTCCCTTACTTGGAACTTGTCAAT-3'. The second

step used the CwSUC2 forward primer again, and the primer VacSUC2 reverse_step_2, 5'-

TCACATCGTATCTACCAAGTCCAACAAACCATTACCTTTTACTTCC-3'. Cycling

parameters for all PCR reactions were 95 C for 5 min to activate DNA polymerase, then 35

cycles of 95 C for 30 s, 60 C for 30 s and 72 C for 2 min, followed by a final step of 72 C for

5 min. The above PCR amplicons were cloned into the pENTR/D-TOPO vector (Invitrogen,

Carlsbad, CA) and then sub-cloned into Gateway destination vectors by LR recombination

reaction for expression in E. coli and Agrobacterium. The Gateway destination vectors were

kindly donated by G. Tuskan (Oak Ridge National Laboratory, Oak Ridge, TN).

Construction of Similarity Trees

Amino acid sequences were aligned using CLUSTALW (http://clustalw.genome.jp) to

construct similarity trees using the TREEVIEW program (Page, 1996). The PAUP (Swofford,

1993) program was used for bootstrap analysis with 1,000 iterations.

Isolation of RNA, Generation of cDNA and Real-Time PCR Assay

Total RNA was extracted using standard methods (Chang, 1993), DNase-treated and

purified in RNAeasy QIAGEN columns (Valencia, CA). Purified RNA (5 ltg) was used to









synthesize cDNA using a mixture of 500 ng oligo-dT, 100 ng random hexamer primers, and M-

MLV-RT as per manufacturer's instructions (Invitrogen), with the exception that the DTT was

excluded.

Gene expression was analyzed using the SYBR Green kit (Stratagene, La Jolla, CA) and

Mx3000P thermo-cycler (Stratagene) as per manufacturer's instructions. Briefly, each reaction

was run in triplicate and contained 1 [tl of synthesized cDNA along with 0.15 [tl of each 0.25 |tM

primer in a final reaction volume of 50 tl. Primers were designed using NetPrimer (Premier

Biosoft International) software and synthesized by Invitrogen. Primer sequences were as follows:

SUC2 forward, 5'-TTTGAGTTGGTTTACGCTG-3'; SUC2 reverse, 5'-

TATTTTACTTCCCTTACTTGG-3' (428 bp product). Cycling parameters were 95 C for 10

min to activate DNA polymerase, then 40 cycles of 95 C for 30 s, 46 C for 30 s and 72 C for 1

min. Melting curves were generated to check the specificity of the amplified fragments. Changes

(n-fold) in gene expression relative to the geometric mean (Vandesompele et al., 2002) of three

control genes encoding actin, ubiquitin and ubiquitin_L (Brunner et al., 2004) were determined

using the program DART-PCRv1.0 (Peirson et al., 2003).

Genomic DNA Isolation and PCR Amplification

To confirm transgene presence, genomic DNA was isolated from poplar leaves using the

Plant DNAeasy Kit (Qiagen) as per manufacturer's instructions. Approximately 25 ng of DNA

was used as template for PCR. Transgene presence was confirmed by PCR using nptll-specific

primers with the following sequences: forward, 5'-ATCCATCATGGCTGATGCAATGCG-3';

reverse, 5'-CCATGATATTCGGCAAGCAGGCAT-3' (253 bp of T-DNA insertion). Cycling

parameters were 30 cycles of 94 C for 1 min, 58 C for 1 min and 72 C for 1 min. Amplicons

were then separated on 1 % (w/v) agarose gels and stained with ethidium bromide.









Metabolic Profiling: Extraction, Separation and Identification

For metabolic profiling by GC-MS, three biological replicates for each of the selected

transgenic lines (CwSUC2-2, -14, CytSUC2-18, -50 and VacSUC2-20, -30) and a non-transgenic

(NT) control line were randomized in the greenhouse and grown to a final height of 1 m prior to

the harvesting and pooling of LPI 10-12 leaf tissues. Metabolites were analyzed in pooled leaf

tissues using GC-MS where chromatogram peaks were evaluated via their retention times and

mass spectra as described in Morse et al. (2007).

Construction and Experimental Design of Grafts

Plants were grown under normal conditions until approximately 1 m in height and were

then used as the source for rootstock and scion buds. A bud from scion material was attached to

the rootstock with Parafilm approximately 5 cm above the soil surface. The lateral shoots that

emerged from the rootstock after grafting were removed to facilitate growth of the scion. After

the grafts took (approximately 2 weeks), Parafilm was removed as well as all plant material

above the graft and leaves below the graft inducing the scion bud to break. The scion was

maintained as the only growing stem on the plant and measurements were taken regularly during

growth. Grafting combinations were conducted such that each combination had three replicates

(in some cases, one replicate was lost; these are noted). Grafting combinations are as follows

(scion/rootstock): CwSUC2-2/NT (n=2), CwSUC2-2/CwSUC2-2 (n=2), CwSUC2-14/NT,

CwSUC2-14/CwSUC2-14 (n=2), VacSUC2-20/NT, VacSUC2-20/VacSUC2-20 (n=2), VacSUC2-

30/NT (n=2), VacSUC2-30/VacSUC2-30, NT/CwSUC2-2, NT/CwSUC2-14, NTIVacSUC2-20,

NT/VacSUC2-30, NT/NT. Plants were randomized in the greenhouse and grown to a final height

of approximately 1 m.









Measurement of Photosynthesis and Respiration

Photosynthesis parameters were measured on a mature leaf (LPI 12) from each plant of the

grafted plants described previously. Light saturated net photosynthesis was measured with a

portable photosynthesis system (Li-6400, Li-Cor, Lincoln, NE) equipped with a red/blue LED

light source under the following chamber conditions: chamber [CO2] 380 [tmol mol-1,

photosynthetic photon flux density 2000 [[mol m-2 s-1, leaf temperature 18 to 22 C, vapor

pressure deficit < 1.5 kPa, air flow rate 500 t[mol s-. Measurements were recorded when the sum

of the coefficients of variation for [CO2], [H20], and flow rate for a 30 s running period dropped

below 0.3 %. Immediately following the light-saturated net photosynthesis measurement, a net

photosynthesis versus leaf internal CO2 concentration (A-Ci) curve was generated by taking

measurements at nine additional levels of reference [CO2] ranging from 50 [tmol mol-1 to 1800

[tmol mol-1, with all other chamber conditions remaining constant. The parameters for maximum

rate of carboxylation of Rubisco (Vcmax) and maximum rate of electron transport (Jmax) were

derived from the A-Ci curves with a program (Photosyn Assistant, Dundee Scientific, Dundee,

UK) that fits a model proposed by Farquhar et al. (1980) and modified by von Caemmerer and

Farquhar (1981), Sharkey (1985), Harley and Sharkey (1991) and Harley et al. (1992).

Parameters were adjusted to a standard temperature of 200C using the methods described in

Walcroft et al. (1997). Statistical differences were assessed by ANOVA using the GLM

procedure of the SAS Version 8.0 statistical software package (SAS Institute, Cary, NC).

To measure foliage respiration, four biological replicates for each of the transgenic lines

CytSUC2-18 and -50 and a NT control line were randomized in the greenhouse and grown to a

final height of approximately 1 m on an ebb-and-flow flood bench system with a daily supply of

Peters Professional 20-10-20 water soluble fertilizer diluted to a final concentration of 10 mM









nitrogen. At noon, plants were moved to a dark growth chamber maintained at 35 C while

respiration measurements were conducted. Measurements were recorded on both a sink (LPI 5)

and source leaf (LPI 12) from each plant. Respiration was measured with the same

photosynthesis system used for photosynthesis measurements. The photosynthesis leaf chamber

conditions were as follows: chamber [CO2] 380 [[mol mol-1, leaf temperature 34 to 36 C, air

flow rate 200 [tmol s'. Measurements were recorded when the sum of the coefficients of

variation for [CO2], [H20], and flow rate for a 30 s running period dropped below 0.3 %.

Statistical differences were subjected to ANOVA using the Mixed procedure of the SAS Version

8.0 statistical software package (SAS Institute).

Protein Extraction

Poplar leaves were frozen and ground in liquid nitrogen. Total protein was extracted by

sonicating 300 mg ground tissue for 30 s in 1 ml of cold extraction buffer containing 50 mM

Hepes-KOH, pH 7.4, 2 mM EDTA, 2 mM EGTA, 5 mM DTT, 100 |iM PMSF and 0.3 %

DIECA. Supernatant was collected after centrifugation at 3,220 g for 20 min at 4 OC and is

referred to as the soluble fraction. The pellet was then washed four times with 5 ml extraction

buffer and resuspended in 2.5 ml extraction buffer containing 1 M NaCl and incubated overnight

at 4 C. Supernatant was collected after centrifugation at 3,220 g for 20 min at 4 OC and is

referred to as the insoluble fraction. Soluble and insoluble fractions were desalted with extraction

buffer using a 30 kD cutoff Centricon (Millipore, Billerica, MA) in order to concentrate protein,

remove the NaCl from the insoluble fraction and remove the invertase inhibitor from both

fractions. Proteins were quantified using a BCA Protein Assay Kit (Pierce, Bonn, Germany) as

per manufacturer's instructions for the microplate procedure.









Total Invertase Activity Assay

Total protein extracts were assayed for total invertase activity using a modification of the

method of Arnold (1965). Briefly, protein extracts (25 il of soluble or insoluble fractions) were

added to 155 pl of 60 mM phosphate-citrate buffer, pH 4.5. Assays were brought up to 30 OC and

at time zero, 20 pl of 1 M sucrose was added. Reactions were stopped at 30 min by adding 600

lil of a modified Sumner's reagent containing 1 % (w/v) 3,5 dinitrosalicylic acid, 0.05 % (w/v)

Na2SO3, 1 % (w/v) NaOH and 0.2 % (v/v) phenol. Samples were then incubated at 95 OC for 10

min and absorbance read at 540 nm.

Detection of Invertase Activity in Native Polyacrylamide Gels

SUC2 activity was detected using a native invertase activity gel assay (Gabriel and Wang,

1969). Briefly, protein extracts in 100 mM Tris-phosphate, pH 6.7, 0.1 % (w/v) bromophenol

blue, 10 % (v/v) glycerol, and 0.1 % (v/v) Triton X-100 were loaded on a 10 % (w/v)

polyacrylamide gel. The gel and running buffers were 100 mM Tris-phosphate, pH 6.7. After

running overnight at 40 V and 4 C, gels were incubated in a 100 mM sucrose, 100 mM NaOAc,

pH 5.0 solution for 30 min at 30 OC. After a quick rinse in distilled water, gels were developed

by incubating in a 95 C solution of 500 mM NaOH containing 0.1 % (w/v) 2,3,5-

triphenyltetrazolium chloride, giving rise to red bands at positions of invertase activity.

Sugar and Starch Determination

For soluble sugar (glucose, fructose and sucrose) determinations, ground, frozen tissue

samples were extracted using double distilled water and clarified via the Carrez method to

remove proteins. Glucose, fructose and sucrose were calculated from the spectrophotometric

measurement of NADPH production using a D-glucose/D-fructose sugar assay kit as per

manufacturer's instructions (Boehringer Mannheim, Germany). Starch was solubilized from









ground, frozen samples using dimethylsulfoxide and hydrochloric acid and assayed using a

Boehringer Mannheim starch assay kit as per manufacturer's instructions.

Results and Discussion

Construction of Overexpressing Yeast Invertase Vectors

To further define the role of invertase in the different cellular compartments (apoplast,

cytosol and vacuole) in poplar, I created three constructs designed to target and express the yeast

invertase gene, SUC2, in those compartments via Agrobacterium-mediated transformation

(Figure 4-1). A plasmid containing a cell wall targeted SUC2 was received through a generous

donation from Uwe Sonnewald (Institut fur Biologie der Universitat Erlangen-Numrberg,

Erlangen, Germany). This construct contains a fusion of SUC2 (nucleotides 64-1765) (Taussig

and Carlson, 1983) with the signal peptide of potato proteinase inhibitor II (nucleotides 1-230)

(Keil et al., 1986). The gene fusion was PCR amplified and recombined into an overexpression

vector using the Gateway (Invitrogen) cloning system and is hereafter referred to as CwSUC2. In

order to create a vacuolar targeted SUC2 gene fusion, the vacuolar targeting domain from

tobacco chitinase (Neuhaus et al., 1991) was fused to the C-terminal end of the CwSUC2

construct and is hereafter referred to as VacSUC2. To direct accumulation of SUC2 in the

cytosol, the coding region of SUC2 was PCR amplified without any signal peptide from the

original CwSUC2 construct and subsequently recombined into the overexpression vector and is

hereafter referred to as CytSUC2. All constructs were driven by two, identical constitutive

CaMV 35S promoters linked in tandem and terminated with the OCS terminator (Figure 4-1).

Yeast invertase was used in these constructs for three reasons: first, SUC2 is known to be

active over a broad pH range (Goldstein and Lampen, 1975), which is important considering the

pH differences between the acidic vacuole and apoplast as compared to the more neutral cytosol.

Secondly, this invertase is not inhibited by the endogenous invertase inhibitors present in plants









(Greiner et al., 1998; Greiner et al., 1999; Link et al., 2004; Huang et al., 2007). Lastly, SUC2

has been expressed in a variety of plants to date including Arabidopsis (Von Schaewen et al.,

1990), potato (Heineke et al., 1992), tobacco (Von Schaewen et al., 1990; Sonnewald et al.,

1991; Ding et al., 1993; Sonnewald et al., 1993; Tomlinson et al., 2004) and tomato (Dickinson

et al., 1991), with significant results.

The two major sub-families of plant invertases are the acid and neutral/alkaline invertases.

The acid invertases are thought to have evolved from respiratory eukaryotes and aerobic bacteria

(Sturm and Chrispeels, 1990a) while the neutral/alkaline invertases are thought to have

originated from cyanobacteria (Vargas et al., 2003). SUC2, coming from yeast, a eukaryote,

indicates that SUC2 is more closely related to the plant acid invertase sub-family than the

neutral/alkaline sub-family. The acidic pH optimum for activity of SUC2 also supports this

conclusion. To further address this, amino acid alignments of SUC2 with endogenous poplar

invertases were examined (Figure 4-2A). Additionally, a protein similarity tree was constructed

using full length sequences of invertases included from Arabidopsis, carrot, corn, poplar, potato,

tomato, various micro-organisms, as well as levanase and inulinase (related enzymes provided as

outliers) (Figure 4-2B). These data indicate that although SUC2 may be evolutionarily more

closely related to the acid invertases than the neutral/alkaline invertases, amino acid sequences

have diverged such that SUC2 sequence is as different from the acid invertases as it is from the

neutral/alkaline invertases (Figure 2B). This lack of sequence similarity from the plant invertases

is likely the reason behind the lack of SUC2 inhibition from the endogenous plant invertase

inhibitors.

Expression of SUC2 in Poplar

In order to verify the constitutive nature of the double 35S (2x35S) promoter in poplar, I

analyzed expression of the 2x35S::SUC2 construct in three different organs with RT-qPCR.









Plants representing three separate transgenic events were tested in order to average out any line

specific effects. I found that the transgene driven under the double 35S promoter is expressed at

highest levels in leaf (Figure 4-3). Expression levels of the transgene in root and stem are

approximately 50 % of the level of expression of that in leaf (Figure 4-3). RNA from leaf

material was extracted and transgene expression levels analyzed in order to identify the highest

expressing lines of each cellular compartment directed construct (Figure 4-4). The two highest

expressing lines from each compartment were then selected for further experimental analysis.

Yeast Invertase Active in Cytosol

To test that the accumulation of SUC2 transcripts translate to increases in invertase

activity, total protein was extracted and fractionated according to solubility from leaf. The

insoluble fraction yielded no detectable increase in total invertase activity in any of the

transgenic lines examined (data not shown). In the soluble fraction, as much as two fold

increases of total invertase activity were detected in the CytSUC2-18 line (Figure 4-5A), but,

unexpectedly, not in any of the VacSUC2 or CwSUC2 constructs (data not shown). This indicates

that the SUC2 transcript accumulation seen in the cell wall and vacuolar targeted constructs

either did not translate into increased activity or that the increase occurred in the wrong cellular

compartment.

One explanation for the lack of total invertase activity increases in the CwSUC2 and

VacSUC2 transgenic lines could be that the endogenous plant invertases were down-regulated in

order to compensate for the exogenous increases in invertase activity. To address this, an

invertase assay was employed that does not detect plant invertase activity, but can detect SUC2

activity (Sonnewald et al., 1991). Total protein extracts from the insoluble and soluble fractions

were separated by electrophoresis on a non-denaturing polyacrylamide gel and then assayed for

activity (see Materials and Methods). Activity was detected only in the soluble fraction of lines









CytSUC2-18 and -50 (Figure 4-5B). This indicates that the total soluble invertase activity

increases seen in Figure 4-5A are a result of the expression of ectopic SUC2. This also indicates

that the lack of activity seen in the cell wall and vacuolar constructs is due to the lack of an

active form of SUC2 in the targeted compartment and not necessarily due to down-regulation of

plant invertase activity.

Metabolic Profiling Reveals Alterations in CwSUC2 and VacSUC2 Transgenic Lines

To determine whether SUC2 expression altered endogenous levels of sugars and any of the

metabolic pathways they feed, leaf tissue from three biological replicates from each of the

selected transgenic lines and NT controls were subjected to metabolite analysis using gas

chromatography-mass spectrometry (GC-MS) with electron impact ionization. As was expected

due to lack of detectable invertase activity alterations, the CwSUC2 and VacSUC2 transgenic

lines showed no significant changes in the levels of sucrose, glucose or fructose. Surprisingly, 12

metabolites were found to be significantly altered in at least one of those transgenic lines (Table

4-1). For three of those metabolites, the exact structures could not be determined and are referred

to by their retention index followed by their key mass/charge (m/z) ratios. Many of the other nine

metabolites can be found in, or are associated with, both the glyoxylate and TCA cycles. This

would indicate that even though I could not detect an increase in invertase activity, metabolic

shifts were still occurring in these transgenic poplars. If the glyoxylate cycle is indeed stimulated

in the CwSUC2 and VacSUC2 transgenic lines, this could account for my inability to detect shifts

in glucose and fructose as one end product of the glyoxylate cycle is glucose. As the glyoxylate

cycle occurs in specialized peroxisomes called glyoxysomes, it is possible that the metabolic

consequences of SUC2 expression are restricted to cellular compartments that represent a small

proportion of the total metabolite pool measured in these studies. One possible way to test this









hypothesis would be to isolate peroxisomes and evaluate metabolic shifts in these specific

compartments.

Metabolic Profiling Reveals Alterations in Sugar Accumulation in CytSUC2 Transgenic
Lines

As would be expected from the increased invertase activity that accumulated in the

CytSUC2 transgenic lines (Figure 4-5), metabolite analysis revealed alterations in the levels of

sucrose as well as its hydrolysis products: glucose and fructose (Table 4-2). The transgenic line

containing the greatest increases of invertase activity (CytSUC2-18) also expressed the greatest

reduction in sucrose levels by nearly 30 %. CytSUC2-50 also revealed reduced levels of sucrose

(20 % reduction), however this was not found to be a statistically significant shift and could be

explained by the lower levels of invertase activity evident in line -50 compared to line -18

(Figure 4-5).

One would expect that increases in invertase activity would result not only in lower levels

of sucrose, but also in increased levels of glucose and fructose since these compounds would be

produced by sucrose hydrolysis. Surprisingly, this was not the case for the CytSUC2 lines where

analyses of both -18 and -50 revealed reductions in glucose and fructose levels (Table 4-2).

Interestingly, the greatest decreases in hexose abundance occurred in line -50, which had the

lowest invertase activity of the two lines. This may indicate that sucrose levels are directly

related to alterations in invertase activity, whereas observed hexose reductions are indirectly

linked to invertase activity. Also of note are the 50 % reductions in malic and glyceric acid levels

in the two CytSUC2 lines (Table 4-2).

One possible explanation for the reductions in hexoses as well as glyceric and malic acids

may lie in increased respiration rates. To test this hypothesis, CytSUC2-18 and -50 plants were

propagated and grown along with NT control plants with a high nutrient fertilizer in a









greenhouse. Plants were then moved to a warm (35 C) growth chamber and kept in the dark

while leaf respiration rates were measured using a portable photosynthesis system from both

source (LPI 12) and sink (LPI 5) leaves. Respiration rates remained unchanged between the

CytSUC2 lines and the NT control plants (data not shown).

Whole-Plant Phenotypes Are Not Apparent

Phenotypes of clonally propagated SUC2 and NT plants were compared at whole-plant, -

organ and molecular-levels (Table 4-3). I detected no differences in propagation efficiency,

growth rate, plant architecture or plant size when grown under controlled greenhouse conditions

(data not shown). One strategy to increase whole-plant differences was to graft transgenic

material onto non-transgenic. This allows me to specifically target SUC2 expression to the root

and/or crown of the tree thereby increasing the sink of the roots while not affecting the ability of

the source organs from photosynthesizing and exporting sucrose. CwSUC2 and VacSUC2 lines

(scion) were grafted onto NT rootstock as well as NT plants (scion) onto CwSUC2 and VacSUC2

rootstock. Control grafts were also performed for all lines (transgenic onto itself, and NT onto

itself) and whole plant phenotypes including growth rates, plant architecture and plant size were

measured as well as photosynthetic rates. In all cases, the transgene was found to have no

detectable effect relative to the NT control (data not shown). Unfortunately the CytSUC2 plants

were excluded from this experiment due to problems in propagation that were unrelated to the

transgenic constructper se.

Concluding Remarks

My results indicate that it was possible to successfully transform poplar with the yeast

invertase gene, SUC2. In contrast to the many other plant species transformed with this gene (see

Introduction) I saw no whole-plant altering phenotypes. This is likely due to the relatively low

level of induction as compared to what was seen in other plant species. In the transformant









containing the highest SUC2 activity (CytSUC2-18), I saw approximately a two-fold induction in

total activity (Figure 4-5A). In contrast, tobacco and potato transgenics expressing the same

SUC2 gene saw as much as 50- and 100-fold increases in activity, respectively (Sonnewald et al.,

1991; Heineke et al., 1992). Even with the modest increases in invertase activity seen in poplar, a

subtle metabolic phenotype could be detected in transgenic lines CytSUC2-18 and -50. While the

reduction in sucrose is to be expected, the reduction in glucose and fructose is counter-intuitive.

Increased respiration could be one explanation of the decrease in hexoses I observed. This

hypothesis would also be supported by the more dramatic reductions observed in malic acid.

Respiration was measured, but no significant changes were detected. This may simply be

because respiration was not altered significantly enough to be measurable. Alternatively,

respiration may not have been altered at all, in which case the metabolic shifts observed may

have resulted from some other metabolic pathway.

The SUC2 constructs targeted to the cell wall and vacuole pose a conundrum. While

transcripts were detected for the CwSUC2 and VacSUC2 transgenes, invertase activity was not

detected either in the total activity assay or in the native gel assay. This would seemingly

indicate that the transcript simply did not result in detectable levels of mature, active protein.

However, a metabolic phenotype was evident in these transgenics. Since no increase in total

activity was found, one potential explanation for this metabolic phenotype may reside in altered

workloads of endogenous invertase to exogenous yeast invertase. The increase in activity

resulting from the transgene may have led to a compensatory down-regulation of the plants'

endogenous invertase activity. Minimal changes to sugar levels would thus have been likely. The

lack of detectable activity in the native gel makes this a tenuous hypothesis, however. This

hypothesis could only be correct if the yeast invertase underwent some sort of post-translational









modification in the cell's secretary system rendering the yeast invertase inactive in the native gel

assay, much like the undetectable plant invertases. If this were true, the in vivo increases in

activity must be quite small. Collectively, my data indicate that the transgenic poplar lines did

give rise to metabolic phenotypes, but that these were not straightforward to interpret. Also,

these transgenics did not show detectable whole-plant phenotypes potentially due to the

relatively subtle increases of SUC2 dependent invertase activity.










Cytosolic targeted invertase
ATA
I ISUC2 TAG



Cellwall targeted invertase
ATG
ISP SUC2 TAGe



Vacuolar targeted invertase
ATG
SP SUC2 TAG

I Chitinase
VTD

Figure 4-1. Schematic representation of yeast invertase overexpressing constructs
targeted to the cytosol, cell wall and vacuole. Block arrows represent 35S
promoter, solid black box represents SUC2 coding region, brick filled box
represents the signal peptide (SP) taken from potato proteinase inhibitor II,
cross-hatched box represents the vacuolar targeting domain (VTD) taken from
tobacco chitinase.












WQRTAFHFQIEEN P YKG WI
VHRTGYHFQNPRH P YKG--L~
----------GD PLNY--DQ'
------------ TSADALNY---NQ\
--------- INKG LWDEKDAKE


G ID SKI

A----- AFLL-hU GE1K
----- LACLMKEFP P E
DT EGTP IFMIT TNI


A
PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


177 NIELLSGIIHGVP GI E DEYPVKTGQNGLT NGPQIKHIIKTS-%DDRHI
177 K KAKHP H QGGI EFPDIYP LSGENGLBP \GQNjKH LKVS- RY
125 -- VDIQMII NL DGF FPILLVT CIL IDRRMG QALF S
123 ------PEQN LI KL SDG F FPLLCA CSIIDRRMGIYGY- QALF
142 W*EPSQKW A AQD KIEIS SiDLKtWKLE SFANEGFIGYQYECG EVPT QD


EGA ADKIGKWYIDNPE GIGIRYDYGIFYA KT YIQ GR ILWGIIG*SDSEVA
SDKKKDKYFIDEGL GWAGLRLDYGNFYAKT F P TNR LWGMAN SDDPQK
SREI DGSKNLNRAINNRLStLSFHIREYY I NE Y YKTE YITEAT
CAKQ KPELDGKEF RIEKRITaLSYHIQIYY LIFQ NN YYKTE YIHTAV
PS VISFISINIGAPAGGSFNQYFISFNGTHFEADNQ l DFG DIYALQIFFNT


NiKK WSLQG RTBVLDTKTGSIL QWPIEEYESLRLKSKNF IEYKAGSAVXLEID
DKWgIGIQL RK LD- SGKIL QWPA E EKLRGHNVQLS QMDQGNHVEVKLII
FN--IYPEQ SW DWIEEGGY IGN QP DFRFFTLG LWS SSLGTIKQNE
NFN--VIPES DW FDF MLRGGY IGNSP DFRWFLVG CVLSSLVTrAQAT
PTYISILGIAWASNWEYSAFVPTIPWRSS SL KFSLNTEYQ PETELINLKAEPIL


ATDIVAE D ----RKAIERTAESNIEFSCSTNGASHGA PFGLLVLADDDT
TAFDVDVT FSSLDKAEPFDPKWAKLD DVC QK SKDPGGPFGLL L EN E
IESKWD G-----------NMP CYPLESEDWI T SDPKN PWIYHNG
D EERWE G-----------EMP K TY PLEHEWLTFDPKN RW YHNG
SAGPWS TNT -------TLTKANSYNjDLSNST TLEFELAVNTTQTIKSIF


EYTiB AKBNNGS KTFFCTDQ RSSVANDBRIGSVPVLE EK----IL
EFT FK IKVLLCSD ASS KELYKPSFAGFVDVDL DKS ---- L
SW LFT DCM IDRMELAQ I K QJHIPEIYDT F ---- KQ R
W L GRPQIAKS Q S PE YDG --- KQAR
LS KGLEDPEEY RMGFEVSA SFFLIRGNSrVK KENPYF N SVNNQPFE


117
118
85
83
97


LKVKYMNP PPGIGAKDFRDPTTAUKTSEK GSKINTIIALV DTEDFI
REVKPDDNPI NDANVNGSAFRDPTT A HG-H GSRRKH ALR FK
DPDFGEEIG --LT--------- L GDLQ
LVDFGAMIG ------------LYI RTRD ALK
WT*NTPESEEQYIS -------------- S YTEYQPVL NSTQ PKVF


236
236
178
176
202


296
296
238
236
262


356
355
296
294
322


412
415
345
343
375












PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


PtVIN2
PtCIN1
PtNIN2
PtNIN10
S cerSUC2


B









Levanase



S mutans GSSIVR

Eco




01


468
471
401
399
435


HIBIESELQG
HLwES GE
YQETVAGFITS1
KYQ*SIAGY V
NBMLYKVLDi


cUTSRVYP R GSAULFNFN -EAG SKIWNINSAFIR
SSRVYP KALYFN -ETI NNAWS NTPVMN
NPE MFEDYDLBEFCVC-GL GRKRCSRAARSQI
NPSNLL EDKSARSRL-R ------------
LYFNDG DSTNTYFMTTGLGS GIDNLFYIDKFQV


527 PYSNEQQ
530 VPVKS--
460 LV-----

495 REVK---


PtVIN2
PtVIN1
AtvaclNV2
AtvaclNV1
carrotlnv Dc5


li K121VR


Figure 4-2. S. cerevisiae SUC2 in relationship to other plant invertases. A) Amino acid
alignment of SUC2 and select poplar invertases. Black shading corresponds to
identical residues, grey shading corresponds to similar residues. Alignment
produced with CLUSTALW and figure produced with BOXSHADE. B)
Amino acid similarity tree using full length sequences. Depicts phylogenetic
relationship of S. cerevisiae SUC2 (in bold) with selected invertases from
Arabidopsis, carrot, corn, poplar, potato and tomato. Invertases from various
micro-organisms are also included along with the closely related enzymes,
levanase and inulinase, provided as outliers. Clades I-IV represent vacuolar,
cell wall, a-neutral/alkaline and B-neutral/alkaline invertases, respectively. a-
and p- designations of the neutral/alkaline invertases represent two distinct
clades as described by Bocock et al. (2007). Bootstrap values of the major
clades are depicted and reported as a percentage of 1,000 repetitions. Branch
lengths denote protein similarity.












Double 35S driven SUC2 expression


140

120

S100
I-i
8 80

60

40

20

0-
root stem leaf

Organ



Figure 4-3. Relative expression of double 35S driven SUC2 in root, stem and leaf. Bars
represent the mean level of SUC2 transcript across three separate transgenic
events; error bars denote standard error, n=3.



















Relative expression of VacSUC2


120


-m-mn-m--m-


18 50 16 2 21 25 17 28 31 12 6 9 36 NT 19
Transgenic line


160
140
120
100
80
60
40
20
0
30 20 29 15 21 38 27 36 24 25 34 NT 7
T20
Transgenic line


Relative expression of CwSUC2


14u

120

100

80

60

40

?0


2 14 25 26 28 11 3 10
Transgenic line


Figure 4-4. Relative expression of SUC2 transcript in overexpressing transgenic events

in leaf. A) Cytosolic targeted construct. B) Cell wall targeted construct. C)

Vacuolar targeted construct. In all panels, bars represent mean level of SUC2

transcript; error bars denote standard error when available, n=3.


raT .


Relative expression of CytSUC2











Total invertase activity (soluble
fraction)
140 .


NT Cyt-18 Cyt50
Transgenic line


IS

0 LO LO


S

t- t-LO LO
5 -% 51 -zz
0000zz


stmurn'


Figure 4-5. CytSUC2 transgenic plants display increased invertase activity due to
presence of transgene. A) Soluble proteins were extracted from a source leaf
(LPI 12) and assayed for invertase activity. Bars represent mean invertase
activity, error bars denote standard error, n=2. B) Protein extracts (20 [g total
protein each lane) were separated using non-denaturing gel electrophoretic
conditions and assayed for activity.Top panel: No invertase activity is
detected in NT plants or in the insoluble fraction (IS). Activity can be seen
(red bands) in the soluble fraction (S) of CytSUC2 lines -18 and -50 in both
biological reps. Bottom panel: Coomassie stain of gel to show loading.












Table 4-1. Metabolites altered in CwSUC2 and VacSUC2 overexpressing plants
expressed as a ratio of gg/g FW (glucose equivalents) of transgenic/NT
controls.
VacSUC2-20/NT VacSUC2-30/NT CwSUC2-2/NT CwSUC2-14/NT
5 Carbon-sugar alcohol 1.08 1.18* 1.17* 1.18t
Iditol 1.11 1.29* 1.21* 1.16
Glucoside (m/z 434) 1.08 1.55t 1.21 1.37
16.25-273 (Unknown compound) 0.74 0.56 0.52t 0.45t
alpha-keto-Glutaric acid 1.11 1.27 1.63* 2.43*
Maleic acid 1.13 1.25 1.42* 1.33
Citric acid 1.21 1.25 1.39* 0.79
10.79-184 (Unknown compound) 1.08 0.58* 0.35* 0.28*
Fumaric acid 1.43 2.48 1.58* 1.32
Serine 0.88 0.87 0.89 0.69*
Carbamoyl-phosphate 0.97 0.62t 0.35* 0.35*
15.7-221 327 (Unknown compound) 0.5* 0.62* 0.51* 0.42*
*=significant at 0.05
significant at 0.10










Metabolites altered in CytSUC2 overexpressing plants expressed as a ratio of
pg/g FW (sorbitol equivalents) of tran s


CytSUC2-18/NT CytSUC2-50/NT
Fructose 0.83* 0.80*
Sucrose 0.71* 0.82
Myoinositol 0.68* 0.96
Glucose 0.75 0.70t
Galactose 0.65t 0.61t
Glyceric acid 0.52t 0.57t
Malic acid 0.52t 0.52t
*=significant at 0.05
t=significant at 0.10


Table 4-2.


.











Table 4-3. Summary of phenotypic experiments performed.
Trait Tested Plants Tested Significant Changes Found
PCR verification of transgene Cyt, Cw, Vac Cyt, Cw, Vac
Transcriptional changes Cyt, Cw, Vac Cyt, Cw, Vac
Invertase activity Cyt, Cw, Vac Cyt
Sugars (fructose, glucose, sucrose) (root, stem, leaf) Cyt, Cw, Vac Cyt (leaf)
Metabolites Cyt, Cw, Vac Cyt, Cw, Vac
Rootability Cyt, Cw, Vac no
Growth rates Cyt, Cw, Vac no
Starch (leaf, root and stem) Cyt, Cw, Vac no
Respiration Cyt no
Growth rates after grafting Cw, Vac no
Photosynthetic rates after grafting Cw, Vac no









CHAPTER 5
CONCLUSIONS

As a first step in examining the role invertase plays in carbon allocation and partitioning,

the invertase family was identified in Populus trichocarpa, a woody, perennial tree species

whose genome was recently sequenced (Tuskan et al., 2006) (Chapter 2). The identification of

eight acid invertase genes; three of which belong to the vacuolar targeted group (PtVIN1-3), and

five of which belong to the cell wall targeted group (PtCIN1-5) is described. Similarly, I report

the identification of 16 neutral/alkaline invertase genes (PtNINI-16). Expression analyses using

whole genome microarrays and RT-PCR revealed evidence for expression of all invertase family

members. Evidence is also reported for expression of a novel intronless vacuolar invertase

(PtVIN1), which apparently arose from a processed PtVIN2 transcript that re-inserted into the

genome. An examination of the microsyntenic regions surrounding the poplar invertase genes

revealed extensive colinearity with Arabidopsis invertases, indicating strong evolutionary

conservation in the development of the invertase family between the two species. To further

determine if the evolutionary conservation of the invertase family, demonstrated by conserved

colinearity, extended to the functional level, vacuolar invertases were selected as a case study

(Chapter 3). Vacuolar invertases have a well documented reciprocal response to sugar treatment

in which one invertase is up-regulated while the other is down-regulated after sugar treatment in

maize (Xu et al., 1996). The conserved, colinear structure of the chromosomal regions

containing these vacuolar invertase genes in Arabidopsis and poplar did not extend to maize and

rice. However, the reciprocal response was found to occur in all four plant species. One

explanation for this is that the function of these genes has been conserved throughout evolution.

An alternative hypothesis is that this reciprocal response is an example of parallel evolution that

arose independently in the different plant lineages. Finally, invertase activity in poplar was









manipulated with transgenesis by ectopically expressing yeast invertase in three cellular

compartments (apoplast, cytosol and vacuole) (Chapter 4). This doubled invertase activity in the

cytosol, but did not alter invertase activity in other cellular compartments. Subtle shifts in carbon

partitioning within the cell were observed, but no detectable alterations in carbon allocation

between tissues. Specific invertase genes identified in Chapter 2 were also targeted for repression

via RNAi (Appendix A).

The overall objective of determining contributions by invertase to carbon allocation and

partitioning in a woody perennial was met in the following ways:

* Poplar contains three vacuolar invertases, one of which is a novel, intronless gene found
only in poplar.

* Poplar contains five cell wall invertases.

* Poplar contains 16 neutral/alkaline invertases that clearly divide into two, structurally
distinct groups and represents a significant expansion in gene number over that of
Arabidopsis and rice.

* Poplar and Arabidopsis acid invertases share substantial similarities in their chromosomal
arrangement indicating that chromosomal duplication can explain much of the
development of the invertase family.

* Poplar and Arabidopsis vacuolar orthologs identified through shared colinearity both retain
a conserved, reciprocal response to exogenous sugar treatment that is also found in the
monocots, maize and rice.

* Invertase activity in poplar can be increased through the introduction of exogenous yeast
invertase.

* An increase in invertase activity shifts carbon partitioning within the cell.

This research has opened up several new questions that would be valuable to be addressed

in future research. The first question is to address how invertase fits into the hypothesized lack of

active phloem loading model proposed by Turgeon and Medville (1998) for poplar and similar

species. One possibility could be that the expansion of the neutral/alkaline invertase sub-family









in poplar may contribute to the movement of photoassimilates through the mesophyll cells and

into the phloem.

A second question that arises from this research deals with the significance and

development of the intronless PtVIN1. It is unknown when in poplar's evolutionary history

PtVIN1 arose. When this question is addressed, it may also help us to understand the function of

this intronless gene.

The third question deals with the activity of the cell wall invertase sub-family members. It

has recently been reported that two of the six cell wall invertases in Arabidopsis may not be

invertases, but rather fructan exohydrolases (FEHs) (De Coninck et al., 2005). FEH protein

sequences are nearly identical to those of demonstrated acid invertase proteins, but FEH does not

use sucrose as a substrate (De Coninck et al., 2005). It is likely that at least one poplar cell wall

invertase is in fact an FEH, but analysis of recombinant cell wall invertase activity was outside

the scope of research presented here.

The fourth question is why the poplars ectopically expressing yeast invertase lack whole-

plant phenotypes. Several groups overexpressed yeast derived invertase in the apoplast of

Arabidopsis (Von Schaewen et al., 1990), potato (Heineke et al., 1992), tobacco (Sonnewald et

al., 1991) and tomato (Dickinson et al., 1991). Sonnewald et al. (1991) also overexpressed yeast-

derived invertase in the vacuolar and cytosolic compartments of tobacco. In all cases,

overexpression resulted in numerous growth defects that included stunted growth, inhibition of

photosynthesis, accumulation of leaf starch and formation of necrotic lesions on leaves followed

by overall yellowing of the leaf (Von Schaewen et al., 1990; Dickinson et al., 1991; Sonnewald

et al., 1991; Heineke et al., 1992). The lack of phenotypes might be due to the relatively modest

induction of activity (two-fold) as compared to these other plants. This may have resulted from









inadvertent selection and propagation of the most vigorous transgenic plants, and hence those

least markedly altered.

Fifth, and finally, valuable work could be performed on poplar plants with a reduction in

invertase activity. As described in Appendix A, transgenic poplars were produced encoding

constructs designed to repress specific endogenous invertase genes through the use of RNAi.

These plants have been confirmed to express the transgene, however they have not been screened

for reduced expression of the targeted invertase gene or even a reduction in overall invertase

activity. The phenotype of these lines is normal and thus the cost of intensive phenotypic

screening must be weighed against the likely benefit of the approach.

The purpose of this research was to investigate invertase contributions to carbon allocation

and partitioning in a woody perennial. To achieve this goal, the invertase family was identified

and annotated using the sequenced genome ofPopulus trichocarpa. Several novel features were

identified in the poplar invertase family. These include a three-member vacuolar invertase sub-

family, an expressed, intronless vacuolar invertase as well as an expansion of the neutral/alkaline

invertase family relative to that of Arabidopsis and rice. The response of two poplar vacuolar

invertases (PtVIN2 and -3) to exogenous sugar treatments was examined. A reciprocal regulation

of transcript first documented in maize was also found to occur, similar to that of Arabidopsis

and rice. A reverse genetic approach was also employed by ectopic expression of yeast invertase

in transgenic poplar resulting in a subtle shift in carbon partitioning as shown by metabolic

analyses. In conjunction, RNAi was used to target endogenous poplar invertases for down-

regulation.









APPENDIX A
REPRESSION OF INVERTASE IN POPULOUS

Introduction

The cell wall and vacuolar invertases have been repressed in several plant species,

including tomato (Ohyama et al., 1995), potato (Zrenner et al., 1996), and carrot (Tang et al.,

1999). The results from these experiments were more varied than those of the invertase

overexpressors, although the theme of increased sucrose content accompanied by a decrease in

hexoses pervaded. In tomato these alterations in sugar accumulations appeared in both the fruit

and leaves. In addition to altered sugar levels, the fruit also had elevated rates of ethylene

evolution. In carrot, growth was shown to be altered at very early stages of development. The

transgenic, cotyledon-stage embryos were still masses of cells while the control plants had

already developed two to three leaves and one primary root. However, when these plantlets were

grown on media supplemented with hexoses, their growth returned to normal. After maturation

and transfer to soil, the plants expressing the cell wall anti-sense cDNA had a significantly

increased shoot to root ratio and accumulated more sucrose and starch than the controls. The

vacuolar anti-sense plants had increased numbers of leaves, while tap root size was slightly

decreased.

Here I describe the design of vectors aimed at endogenous invertase repression via RNAi

in Populus, a deciduous, perennial tree species. While the previously mentioned work has done

much to establish the role of invertase in carbon allocation and partitioning, it is still unknown

what roles invertase may play in a deciduous, perennial plant system. The deciduous, perennial

nature of poplar requires numerous cycles of sugar movement and carbon sequestration in

storage organs such as the root and stem in the winter, followed by remobilization of these stored

carbon compounds the following spring. It is also thought that poplar may employ a unique









mode of sucrose movement into the phloem of source leaves analogous to that of poplar's

closely related cousin, willow (both members of Salicaceae) (Turgeon and Medville, 1998).

These added levels of complexity that can be seen by utilizing the model tree, poplar, may offer

us new insights into the function of invertase.

Materials and Methods

Plant Material, Transgenesis and Growth Conditions

Hybrid poplar clone, INRA 717-1-B4 (P. tremula x P. alba) was placed into sterile culture

prior to Agrobacterium-mediated transformation (Leple et al., 1992) on media supplemented

with glucose and fructose. Individual clones from independent lines were clonally propagated as

softwood cuttings under mist, transferred to 8 L pots and grown to a height of 60-100 cm prior to

experimentation in a fan- and pad-cooled greenhouse with natural light augmented with full

spectrum fluorescent lighting during the winter to give a day length of 15 h (Lawrence et al.,

1997). Greenhouse temperatures ranged from 20-35 C. At noon, the light intensity in the

greenhouse averaged 500-700 iE/m2/min PAR, which is one-half the light intensity outside the

greenhouse. Plants were grown on an ebb-and-flow flood bench system with a daily supply of

Peters Professional 20-10-20 water-soluble fertilizer diluted to a final concentration of 4 mM

nitrogen.

Vector Construction

The pCAPT binary vectors for RNAi was constructed as described by Filichkin et al.

(2007). Briefly, the vector was constructed using the pART27 backbone (Gleave, 1992). The

GATEWAY Conversion System (Invitrogen) was used to incorporate the proper recombination

sites and genes for positive and negative selection. This consists of an attR recombination site

flanking a ccdB gene and a chloramphenicol resistance gene. The vector contains spectinomycin

and kanamycin (NPTII) resistance genes for selection in bacteria and plants, respectively.









Approximately 200 bp target fragments of acid invertase genes from poplar (primer sequences

listed in Table A-i) were PCR amplified using primers with tails encoding attB recombination

sites. GATEWAY entry clones were created using BP Clonase (Invitrogen) mediated

recombination. The entry clones were finally cloned into the pCAPT binary vector using LR

Clonase (Invitrogen) according to manufacturer's instructions. All constructs were sequenced.

Genomic DNA Isolation and PCR Amplification

To clone target gene sequences and transgene presence, genomic DNA was isolated from

poplar shoot tip tissue. Approximately 250 mg of tissue was ground in liquid nitrogen and added

to a buffer containing 0.3 M sucrose, 10 mM Tris (pH 7.9), 1 mM EDTA, and 4 mg/mL

diethyldithiocarbamic acid. Samples were spun (20,800 rcf) and pellets resuspended in a buffer

containing 100 mM Tris (pH 7.9), 500 mM NaC1, 20 mM EDTA, 1 % SDS, 0.1 % 2-

mercaptoethanol, and 100 [tg/ml proteinase K. Samples were incubated at 65 C for 1 h, spun

(20,800 rcf) and supernatant extracted with chloroform. Isopropanol was used to precipitate the

DNA at room temperature, spun (20,800 rcf), and resuspended in Tris-EDTA buffer. One tl

RNase A was added to DNA sample and incubated at 37 C for 30 min. The chloroform

extraction was repeated, followed by ethanol precipitation in presence of sodium acetate at -80

C for 1 h. Samples were spun for 10 min (20,800 rcf), air dried and resuspended in Tris-EDTA

buffer. Approximately 25 ng of genomic DNA was used as template for PCR. Cycling

parameters were 95 C for 5 min to activate DNA polymerase, then 35 cycles of 95 C for 30 s,

annealing temperature for 30 s and 72 C for 1 min followed by a final step at 72 C for 5 min.

The annealing temperature was set to five degrees below the melting temperature for the primer

pair (Tm listed in Table A-i). Amplicons were then separated on 1 % (w/v) agarose gels and

stained with ethidium bromide. Transgene presence was confirmed by PCR using nptll-specific









primers with the following sequences: forward, 5'-ATCCATCATGGCTGATGCAATGCG-3';

reverse, 5'-CCATGATATTCGGCAAGCAGGCAT-3' (253 bp of T-DNA insertion). Cycling

parameters were 30 cycles of 94 C for 1 min, 58 C for 1 min and 72 C for 1 min. Amplicons

were then separated on 1 % (w/v) agarose gels and stained with ethidium bromide.

Results

Construction of RNAi Vector

To repress endogenous poplar acid invertase genes, I used a binary vector, pCAPT,

graciously donated by Stephen DiFazio (West Virginia University, Morgantown, WV) (Figure

A-i). The pCAPT vector is designed for the cloning and insertion of a target sequence. A single

GATEWAY cassette (Invitrogen) is located upstream of an inverted repeat of the octapine

synthase (OCS) terminator. Transcription is controlled by two CaMV 35S promoters linked in

tandem. This transcript results in a hairpin formed by the inverted repeat of the OCS terminator.

Transitive silencing of the inserted target gene fragment then occurs via RNAi (Filichkin et al.,

2007).

Insertion of Target Gene Sequence

The acid invertase gene family was selected to be used for RNAi directed gene silencing.

Using annotated sequences described in Chapter 2, nucleotide alignments were constructed using

the eight acid invertases in poplar. Two gene specific primer pairs per gene were designed such

that a -200 bp fragment from both the N-terminal and C-terminal ends of the gene could be PCR

amplified from genomicDNA (Figure A-2; Table A-i). These fragments were then cloned into

the pCAPT binary vector which was then sequenced to verify insertion in the proper orientation.

The pCAPT binary containing the target gene insert was then grown in Agrobactierium and used

to transform poplar trees as outlined in the Materials and Methods. Shoot tips from regenerated

transgenic poplars were used as a source of genomic DNA to screen for the presence of the









transgene using primers directed against the NPTII reporter gene as outlined in the Materials and

Methods. Table A-2 contains a complete list of all pCAPT vectors and regenerated transgenic

poplars that were successfully made.

Further Work

The next step in this project is to screen the approximately 30 separate transgenic events

per construct in order to verify the effectiveness of the RNAi vector and rank the transgenic

plants according to target gene transcript levels. Transcripts from the other acid invertase family

members then need to be quantified to check for the specificity of the RNAi mediated gene

silencing as well as to screen for compensatory increases in related gene transcripts in case of

functional redundancy. Total invertase activity should then be assayed to verify that a

successfully silenced invertase gene results in decreased invertase activity. Phenotyping could

then be done where one would expect increases in available sucrose, decreases in available

hexoses, as well as whole-plant phenotypes such as alterations in growth rates.









RB 35S gfp R1 R2 OCS-R PIV2 OCS-F nos5' NPTII nos3' LB
Gene Fragment.


Figure A-i. Schematic representation of pCAPT vector (16041 bp) used for RNAi silencing. R1
and R2, attR recombination sites flanking a ccdB gene and a chloramphenicol
resistance gene from the GATEWAY vector conversion cassette (Invitrogen). RB and
LB, right and left T-DNA borders, respectively; 35S, double CaMV 35S promoter
linked in tandem; gfp, fragment of GFP coding sequence; PIV2, potato intron; OCS-R
and OCS-F, octopine synthase terminator fragments in reverse (R) and forward (F)
orientations, respectively; NPTII, neomycin phosphotransferase II; gene fragment,
location of targeted invertase sequences. Diagram not to scale, adapted from
(Filichkin et al., 2007).











PtCIN2 ~3.6kb


Figure A-2. Schematic representation of RNAi directed constructs. Endogenous PtCIN2
demonstrates strategy of RNAi construct design. PtCIN2 exons are represented by
black boxes and introns by black lines. The 5' and 3' -200 bp-oligos to be targeted by
RNAi constructs are marked by black lines below their respective locations. The
RNAi constructs containing the -200 bp-oligos are depicted below the PtCIN2
schematic. Grey block arrows represent 35S promoter, solid black box represents
endogenous -200bp-oligo to be targeted by RNAi machinery, dotted block arrows
represent OCS terminator oriented in opposing directions and separated by a short
potato intron (grey box) in order to form hairpin in vivo. Figure not drawn to scale.


5' construct
5' construct


3' constructace r
3' construct


~
I
d


I I


m I










Table A-1. Primer sequences used for -200 bp target sequence cloning.
Primer Name Primer Sequence Tm (oC)
PtCIN1 NT forward CACCATGGGCTATGCCACACAC 61
PtCIN1 NT reverse CGTTGATCCAGTGCCTTGGAGG 66
PtCIN1 CT forward CACCGAGAGTTTTGGAGC 53
PtCIN1 CT reverse AACATTCATGACAGGCGT 51
PtCIN2 NT forward CACCATGGCTATATGCCAAACAC 61
PtCIN2 NT reverse GTTGATCCAGTTCCTAGGAGGCT 61
PtCIN2 CT forward CACCGAGAGTTTTGGAGC 53
PtCIN2 CT reverse TTCATTGCGTGGATTCTCTC 56
PtCIN3 NT forward CACCATGGCTTTGTTAAAGTTTCTC 62
PtCIN3 NT reverse GTTGATCCAGTTCTTAGGAGGCTG 61
PtCIN3 CT forward CACCGAAAGTTTTGGAG 48
PtCIN3 CT reverse GACATTCATCACAGGCAC 48
PtCIN4 NT forward CACCTTCTTGGTTGGTTTATGC 59
PtCIN4 NT reverse GGTTCTATATGACTGCTTTTCTTGC 59
PtCIN4 CT forward CACCGAGAGTTTTGGTGGG 58
PtCIN4 CT reverse TTGATTTGGGCTTTGTTCATG 59
PtCIN5 NT forward CACCGGAGATATCTGTTATTTGG 58
PtCIN5 NT reverse ATTCATCCAGTTTTTAGGAGGTTG 59
PtCIN5 CT forward CACCTAGTTGAGAGTTTTGGTGGT 60
PtCIN5 CT reverse CTAAAGGTGTGGCTTCCTTCG 59
PtVIN2 NT forward CACCCTTCCAGTTTCCAATTCCTTA 65
PtVIN2 NT reverse AACAAAGTCTCGGGTTTCGC 61
PtVIN2 CT forward CACCCGTTGCTGAGTTTGAGTTA 62
PtVIN2 CT reverse TGAGGCTGCCATTATTTCCTTTG 63
PtVIN3 NT forward CACCCCCACCATACACTCCCTTG 67
PtVIN3 NT reverse TGGTGATACCCCTTGAGCCACTCC 68
PtVIN3 CT forward CACCAGTTTTGCTCAAGGAG 56
PtVIN3 CT reverse TTGGTCAAATAGGAAAGGATGG 59










Table A-2. Summary of RNAi constructs made. NT and CT, N-terminal and C-terminal
constructs, respectively; X denotes possession of the RNAi construct (pCAPT) and
transgenic plant expressing the construct.
Gene pCAPT Transgenic Plant
PtCINI, NT X X
CT X
PtCIN2, NT X
CT X X
PtCIN3, NT X
CT X X
PtCIN4, NT X
CT X
PtCIN5, NT X X
CT X X
PtVIN2, NT X
PtVIN3, NT X X
CT X X









APPENDIX B
REGULATION OF INVERTASE: A "SUITE" OF TRANSCRIPTIONAL AND POST-
TRANSCRIPTIONAL MECHANISMS

This appendix contains a review that resulted from a collaboration between Dr. Li-Fen

Huang, Dr. Karen Koch and myself. It was published in Functional Plant Biology. While I took

part in the construction and editing of the entire manuscript, some parts I did not write. I did not

write the PPV compartmentalization, differential sugar regulation, or RNA turnover sections. I

also originate the idea for the first figure. All other parts are my original work.

Abstract

Recent evidence indicates that several mechanisms can alter invertase activity and, thus,

affect sucrose metabolism and resource allocation in plants. One of these mechanisms is the

compartmentalization of at least some vacuolar invertases in precursor protease vesicles (PPV),

where their retention could control timing of delivery to vacuoles and hence activity. PPV are

small, ER-derived bodies that sequester a subset of vacuolar-bound proteins (such as invertases

and protease precursors) releasing them to acid vacuoles in response to developmental or

environmental signals. Another newly-identified effector of invertases is wall-associated kinase

2 (WAK2), which can regulate a specific vacuolar invertase in Arabidopsis (AtvaclNV1) and

alter root growth when osmolyte supplies are limiting. WAKs are ideally positioned to sense

changes in the interface between the cell wall and plasma membrane (such as turgor), because

the N-terminus of each WAK extends into the cell wall matrix (where a pectin association is

hypothesised) and the C-terminus has a cytoplasmic serine/threonine kinase domain (signaling).

Still other avenues of invertase control are provided by a diverse group of kinases and

phosphatases, consistent with input from multiple sensing systems for sugars, pathogens, ABA

and other hormones. Mechanisms of regulation may also vary for the contrasting sugar responses

of different acid invertase transcripts. Some degree of hexokinase involvement and distinctive









kinetics have been observed for the sugar-repressed invertases, but not for the more common,

sugar-induced forms examined thus far. An additional means of regulation for invertase gene

expression lies in the multiple DST (Down STream) elements of the 3'-untranslated region for

the most rapidly repressed invertases. Similar sequences were initially identified in small auxin-

up RNAs (SAUR) where they mediate rapid mRNA turnover. Finally, the invertase inhibitors,

cell wall- and vacuolar inhibitors of fructosidase (CIF and VIF, respectively) are

indistinguishable by sequence alone from pectin methylesterase inhibitors (PMEI); however,

recent evidence suggests binding specificity may be determined by flexibility of a short, N-

terminal region. These recently characterized processes increase the suite of regulatory

mechanisms by which invertase and, thus, sucrose metabolism and resource partitioning can

be altered in plants.

Introduction

The capacity to use sucrose is essential to most plant cells, because this sugar is typically

the long-distance transport form for carbohydrates. Invertase (EC 3.2.1.26) irreversibly

hydrolyses sucrose into glucose and fructose, and is thus positioned to play a central role in both

carbon metabolism and sugar signaling. Glucose and fructose are hexoses that serve as an energy

source for respiration, provide metabolites for synthetic processes, generate osmotic pressure in

growing tissues and function as metabolic signals affecting the expression of many downstream

genes (Koch, 2004). Given the importance of sucrose cleavage and the diverse roles for products

of invertase activity, it is not surprising that multiple avenues have developed for the regulation

of the invertases.

Several invertase isozymes have been identified from plant species as either soluble

(readily extractable from cytosol or vacuole), or insoluble (bound to cell wall components).

Vacuolar and cell wall invertases show optimal activity at an acidic pH and thus are also called









acid invertases. The mature acid invertases are glycosylated and located in the cellular

compartment their name implies (e.g. cell wall or vacuolar).

Invertase is regulated at transcriptional and post-translational levels by many different

factors including hormones, sugars, pathogens, oxygen availability and proteinaceous inhibitors

(Zeng et al., 1999; Long et al., 2002; Link et al., 2004; Trouverie et al., 2004; Voegele et al.,

2006). However, important new methods of control have also been identified by recent work on

the invertase path of sucrose use. These are reviewed here and include protein trafficking,

signaling cascades, transcript turnover and inhibitor binding.

Compartmentalization of Vacuolar Invertases in Precursor Protease Vesicles (PPV)

A new level of regulation has been revealed for vacuolar invertase activity by recent

evidence for their localization in a class of minute vesicles called precursor protease vesicles

(PPV) (Rojo et al., 2003). These organelles surround the large, central vacuole, as well as the

smaller, protein-storage vacuoles (Chrispeels and Herman, 2000; Hayashi et al., 2001; Rojo et

al., 2003). The PPV are best known for their roles as storage sites for precursor proteases that are

later released into vacuoles, where the low pH activates maturation of the protease precursors

(Chrispeels and Herman, 2000; Hayashi et al., 2001; Schmid et al., 2001). These vesicles are

plant-specific ER bodies, formed from dilated cisternae of the ER and surrounded with

ribosomes (Chrispeels and Herman, 2000; Hayashi et al., 2001). PPVcontain a distinct

population of proteins that include protein disulfide isomerase (PDI), binding protein [(BiP)

HSP-70] (Schmid et al., 2001), PYK10 3-glucosidase (Matsushima et al., 2003) and precursor

proteins for diverse cysteine proteases such as responsive to dehydration-21 (RD21) (Hayashi et

al., 2001), cysteine endopeptidase [CysEP (from castor bean)] (Schmid et al., 2001) and vacuolar

processing enzyme gamma (VPEy) (Chrispeels and Herman, 2000; Rojo et al., 2003).









Presence of vacuolar invertases in PPV is supported by localization of at least one vacuolar

invertase to these compartments in Arabidopsis (Rojo et al., 2003). Collectively, the PPV

contents, including the vacuolar invertase(s), are released into the vacuolar lumen by a fusion of

these organelles with the tonoplast. The process is distinct from the autophagic engulfment of

other vesicles by the vacuole (Chrispeels and Herman, 2000; Hayashi et al., 2001). When the

PPV contents enter acidic vacuoles, the acid invertase moves into a low-pH, high-sucrose

environment optimal for its activity.

Previous evidence indicates that before vacuolar fusion, PPV are not acidic enough to

support appreciable acid invertase activity. A low pH would also activate maturation of the

VPEy cysteine protease (Hayashi et al., 2001; Schmid et al., 2001) and, thus, rapidly degrade the

invertase. The AtvacINV2 vacuolar invertase is indeed a target of VPEy after this protease

matures in the acidic central vacuole (Rojo et al., 2003). However, the rate of proteolytic

degradation of invertase by VPEy is modest in the vacuole and probably a result of dilution in

the larger compartment. It is feasible that co-retention of both the VPEy protease and vacuolar

invertase in PPV could be a mechanism of retaining their activities in reserve for later purposes.

Once the PPV fuses with the central vacuole, action of the maturing protease on the invertase

would limit the duration of any increases in sucrose-cleaving activity that arose from the release

of PPV contents into the acidic vacuole. Such pulse-type mechanisms of regulation facilitate fine

control, and are evident for some of the sucrose synthases (Hardin et al., 2003), and for

increments of starvation-induced autophagy (Rose et al., 2006).

The impact of vacuolar invertase retention by PPV could vary considerably. The portion of

total invertase in these organelles at any given time could be significant under some

circumstances and limited in others (Rojo et al., 2003). It is also unclear whether all vacuolar









invertases follow a PPV path to acid vacuoles and whether this might vary with conditions or

plant development. In Arabidopsis, one or both vacuolar invertases may be involved, because the

proteins are virtually identical (Rojo et al., 2003; Carter et al., 2004). Still another factor is likely

to be the timing and extent of PPV fusions with acid vacuoles. This can occur during senescence

(Schmid et al., 2001; Rojo et al., 2003), salt stress (Hayashi et al., 2001) and possibly during

diverse, pre-senescent phases of development (Rojo et al., 2003). Recent evidence has indicated

PPV may contribute to pathogen responses (Rojo et al., 2004) and aspects of seed development

such as seed coat formation (Nakaune et al., 2005), seed protein properties (Gruis et al., 2004)

and remobilization of seed structures including the megagametophyte (He and Kermode, 2003),

endosperm (Schmid et al., 2001) and nucellar tissue (Greenwood et al., 2005).

Wall-Associated Kinase (WAK)

Recent work has shown that a wall-associated kinase (WAK) can play a role in the

regulation of vacuolar invertase thus establishing a cross-compartmental link between WAK and

vacuolar invertase(s) (Kohorn et al., 2006b).AT-DNA null allele of WAK2 reduced vacuolar

invertase activity in Arabidopsis roots by 62% and decreased levels of a specific vacuolar

invertase transcript (AtvaclNV1) by over 50%. The wak2-1 null allele also reduced plant growth

under low nutrient conditions. Unlike effects on vacuolar invertase, the cell wall isoforms were

unaltered by the wak2-1 null allele, indicating either that other WAKs may regulate cell wall

invertases, or that WAK-based signals mediate only vacuolar forms (Kohorn et al., 2006b). This

work further supports the role of vacuolar invertases in resource partitioning as well as turgor

maintenance and cell wall expansion (Koch, 2004).

Wall-associated kinases proteins are hypothesized to serve as sensors of cellular turgor, the

extent of cell wall loosening, and/or the degree of cell expansion (Zhang et al., 2005; Kohom et

al., 2006b). WAKs are ideally positioned to do so, because they span the plasma membrane









(Figure B-1). Each of the five WAK genes encodes a transmembrane protein with an active

cytoplasmic serine/threonine kinase domain on the C-terminus and a distinctive extracellular

domain on the N-terminus. The extracellular domain is similar to the vertebrate epidermal

growth factor-like domain. Evidence indicates this extracellular domain may be bound to pectin

in the cell wall, which would provide a link to the extracellular matrix (He et al., 1999; Wagner

and Kohorn, 2001; Decreux and Messiaen, 2005; Kohorn et al., 2006a). This physical tie

between the extracellular matrix and the cytoplasm could provide an anchor, or reference point

enabling the WAK to play a role in sensing or transmitting information on the status of the cell

wall relative to the plasma membrane. Such information could be invaluable to adjustment of

cell expansion or turgor. Different members of the WAK family are expressed at organ junctions,

in shoot and root apical meristems, in expanding leaves, and in response to environmental stimuli

such as wounding and pathogen attack (Wagner and Kohorn, 2001). Antisense reduction of

WAK1-5 protein levels also established the necessity of these genes for expansion of leaf cells

(cellular division was unaffected) (Lally et al., 2001; Wagner and Kohorn, 2001).

The WAK family appears to be part of a larger, 22-member WAK-like (WAKL) family in

Arabidopsis and is widespread in the plant kingdom (Verica and He, 2002). Protein gel blots

usingWAK1 antibodies indicate related proteins in pea, tobacco and maize (He et al., 1996; Gens

et al., 2000). WAKL expressed sequence tags have also been identified in tomato, soybean and

rice (Verica and He, 2002), but a functional relationship between WAK and WAKL has yet to be

shown.

Other Kinases Affecting Invertases

Invertase can be activated by mycorrhization, pathogen infection and wounding, as well as

various hormones (Sturm and Chrispeels, 1990a; Benhamou et al., 1991; Ehness et al., 1997;

Hall and Williams, 2000; Blee and Anderson, 2002; Pan et al., 2005). Diverse kinases appear to









regulate activity of both cell wall and vacuolar invertases in many of these instances (Ehness et

al., 1997; Pan et al., 2005). The serine/threonine kinase inhibitor, staurosporine, prevents the

accumulation of invertase transcripts that typically occurs during defence responses (Ehness et

al., 1997). In contrast, the same inhibitor, and also two other serine/threonine kinase inhibitors

(K252a and H7), increase the extent to which ABA induces both vacuolar and cell wall

invertases (Pan et al., 2005). However, the tyrosine protein kinase inhibitor, quercetin, strongly

suppresses the ABA induction of acid invertases (Pan et al., 2005) indicating that multiple types

of kinases with differing modes of regulation are involved in modulating invertase activity.

Phosphatases have also been implicated in the regulation of invertases (Ehness et al., 1997;

Pan et al., 2005). Pan et al. (2005) demonstrated that expression and activity of both vacuolar

and cell wall invertases increased in response to ABA and acid phosphatase. These data, in

conjunction with the kinase inhibitor data, indicate that reversible protein phosphorylation is

playing some role in the ABA signaling network (Pan et al., 2005). The phosphatase inhibitor,

endothall, also induces cell wall invertase transcripts when added to cell suspensions of

Chenopodium rubrum (Ehness et al., 1997). This work on invertase is consistent with other

ongoing analyses of both ABA and defense-signaling cascades (Roitsch et al., 2003; Roitsch and

Gonzalez, 2004).

Differential Sugar Regulation of Invertases

Despite the widespread use of invertase expression as a sugar response marker in yeast

(Ahuatzi et al., 2004; Kig et al., 2005), the regulation of these genes has been difficult to dissect

in plants. Not only are there many more individual invertase genes (Tymowska-Lalanne and

Kreis, 1998a; Sturm, 1999; Sherson et al., 2003), but they are also differentially regulated (Xu et

al., 1996; Tymowska-Lalanne and Kreis, 1998a; Huang, 2006), and sugar response mechanisms

vary (Roitsch et al., 1995; Xiao et al., 2000; Huang, 2006). All plant species studied to date have









two vacuolar invertases (Haouazine-Takvorian et al., 1997), with several cell wall invertases.

Fully-sequenced genomes indicate six putative cell wall invertases in Arabidopsis (Tymowska-

Lalanne and Kreis, 1998a; Sherson et al., 2003) and nine in rice (Ji et al., 2005). Additional

complexity has been introduced by recent evidence that two of the putative cell wall invertase

sequences may encode fructan exohydrolases (a closely-related enzyme) (De Coninck et al.,

2005). Similar invertase family structures are also evident in poplar, maize, potato and tomato

(Fridman and Zamir, 2003; Huang, 2006); P. N. Bocock, unpubl. data).

The majority of these invertases are sugar-induced rather than repressed (Roitsch, 1999;

Roitsch and Ehness, 2000), which is consistent with their roles in carbon use v. carbon

acquisition by plants (Koch, 1996; Rolland et al., 2002; Koch, 2004). Both vacuolar and cell wall

invertases enhance use of carbohydrate resources by cleaving imported sucrose at sites of growth

or storage (Winter and Huber, 2000; Koch, 2004). These invertases can also amplify sugar

signals to other genes that respond to changes in hexose availability (Koch, 2004).

The identities of sugars that induce invertases remain unclear, however. Evidence thus far

indicates limited involvement of the classic hexokinase-mediated sensing system in up-

regulation of invertases by sugars. Neither overexpression of the key hexokinase gene, AtHXKl,

nor its antisense reduction reportedly affects expression of a sugar-induced AtcwlNV1 (P-fructl)

gene for cell wall invertase in Arabidopsis (Xiao et al., 2000). Non-hexokinase mechanisms of

sugar sensing may play more widespread roles in sugar-induced gene expression than previously

recognized (Purcell et al., 1998; Rolland et al., 2002; Sinha et al., 2002; Lalonde et al., 2004;

Halford, 2006). Although signaling systems differ between plants and yeast, the hexokinase-

based sensing in yeast is largely linked to sugar repression rather than induction (Johnston, 1999;

Palomino et al., 2005; Kim et al., 2006), and a balance between the two is hypothesized to









mediate responses (Ronen and Botstein, 2006). In plants, mechanisms of sugar sensing have thus

far been studied mainly by following responses of genes down-regulated by glucose (e.g.

photosynthesis, seed germination) (Smeekens, 2000; Rolland et al., 2002; Rolland and Sheen,

2005). Another consideration is that genes known to be sucrose- rather than hexose-responsive

are often expressed in or near the vascular system (Rook et al., 1998), as are many invertases

(Andersen et al., 2002; Wachter et al., 2003).

Some acid invertase genes are sugar-repressed. This is less common, but occurs

consistently in all species examined thus far. The singular repression of one vacuolar invertase

by sugars is conserved across gene families in maize (Xu et al., 1996), tomato (Godt and

Roitsch, 1997b), rice (Huang, 2006) and poplar (P. N. Bocock, unpubl. data). At least some

degree of involvement has been indicated for the hexokinase-mediated sensing system in this

repression. Contributions from this mechanism are supported by data from metabolizeable and

non-metabolizeable glucose analogues tested in Arabidopsis for their capacity to repress the

AtvaclNV2 vacuolar invertase (Huang, 2006). Another feature of invertase repression by sugars

is its rapid down-regulation, with responses evident in minutes rather than hours (Huang, 2006).

Still another factor may be the demonstrated compartmentalization of the sugar-repressed

AtvacINV2 invertase protein in the precursor protease vesicles [see section on PPV (Rojo et al.,

2003)], implying a possible relationship between the PPV and the observed sugar responses.

Finally, additional insights into the sugar repression of some invertases may lie in their

respective roles. Demands for osmotic constituents (two hexoses from one sucrose) may well

dominate sucrose partitioning in response to specific developmental and/or stress signals.

Expression of at least one vacuolar invertase under these conditions could be highly

advantageous, and need not be viewed as separate from its role in sucrose import. Sucrose









entering cells is often cleaved first in the vacuole, thus giving vacuolar invertase a prominent role

in sucrose partitioning (Koch, 2004). Theoretically, expression of a starvation-tolerant invertase

could confer an import priority for certain cells or tissues under stress, as well as favor

immediate allocation of incoming resources to osmotic constituents. A similar scenario could

hold for key aspects of development that rely heavily on osmotic constituents for cellular

expansion. Data thus far are consistent with contributions by both the sugar-induced and -

repressed vacuolar invertases to import-based osmotic support of expansion sinks. Examples

include root elongation in Arabidopsis (Stessman, 2004; Huang, 2006; Kohorn et al., 2006b;

Sergeeva et al., 2006), petiole growth in sugar beet (Gonzalez et al., 2005), expansion of ovaries

and silks in maize (Andersen et al., 2002), enlargement of Agrobacterium-induced galls

(Wachter et al., 2003) and growth of diverse fruits and vegetables such as tomatoes, carrot roots

and newly-forming potato tubers (Koch and Zeng, 2002).

RNA Turn Over and DST

At least one mechanism of post-transcriptional regulation of invertase is also indicated for

the sugar-repressed vacuolar invertases. In both rice and Arabidopsis, the 3' untranslated regions

of the sugar-repressed genes OsVIN1 and AtvaclNV2 carry apparent downstream (DST) elements

implicated in rapid turnover of plant mRNAs (Huang, 2006). The DST are highly conserved and

can mediate sequence-specific decay of short-lived mRNA in vivo (Newman et al., 1993;

Sullivan and Green, 1996). The DST are especially notable for their role in rapid destabilization

of small auxin-up RNAs (SAUR) (Newman et al., 1993; Gil and Green, 1996; Johnson et al.,

2000; Feldbrugge et al., 2002).

Additional evidence is also consistent with altered mRNA longevity for the sugar-

repressed form of vacuolar invertase. Although glucose rapidly decreases mRNA levels of the

AtvaclNV2 vacuolar invertase in vivo (within 30 min), the promoter alone shows a contrasting









induction by sugars (Huang, 2006). When transcription is blocked by cordycepin, however, a

glucose-enhanced rate of decay is evident for AtvaclNV2 mRNA (Huang, 2006). Glucose-based

destabilization of mRNAs thus seems a likely contributor to the rapid repression observed for the

AtvaclNV2 vacuolar invertase (Huang, 2006).

Invertase Inhibitors

Cell wall and vacuolar invertase activity can be regulated by a family of proteinaceous

inhibitors known as cell wall inhibitor of fructosidase (CIF) and vacuolar inhibitor of

fructosidase (VIF), or collectively as C/VIF reviewed by Rausch and Greiner (2004). Although

CIF are cell wall invertase inhibitors, they are broadly active against both cell wall and vacuolar

invertases. In contrast, VIF inhibition is specific to vacuolar invertases. Neither of the inhibitors

affect fungal invertases indicating a minimal role for these interactions in pathogen defense

(Greiner et al., 1998; Greiner et al., 1999; Link et al., 2004). The C/VIF related protein family is

not highly conserved, and in Arabidopsis, sequence identities range from roughly 20 to 40% for

-14 family members (Rausch and Greiner, 2004). The C/VIF related protein family also contains

pectin methylesterase inhibitors (PMEI). PMEI are indistinguishable from the C/VIF by

sequence alone and retain nearly identical structures to the C/VIF (Giovane et al., 2004; Hothorn

et al., 2004a; Hothorn et al., 2004b; Di Matteo et al., 2005).

Important implications arise from the capacity of these related proteinaceous inhibitors to

distinguish between their targets. Arabidopsis has only eight putative acid invertases (six cell

wall and two vacuolar), but more than 60 pectin methylesterase (PME)-related genes based on

sequence similarity (Micheli, 2001; Sherson et al., 2003; Rausch and Greiner, 2004; De Coninck

et al., 2005). X-ray crystollagraphy has revealed that CIF is conformationally stable over a broad

pH and temperature range, however, the invertase/inhibitor complex is only stable at an acidic

pH. This indicates that binding is determined not by conformational changes, but rather by pH-









induced changes at the interface of the invertase/inhibitor complex (Hothorn et al., 2004a;

Hothorn and Scheffzek, 2006). In contrast, the highly similar structure of the PMEI was found to

undergo large structural rearrangements (Figure B-2) under these same conditions suggesting

PMEI uses a different mode of binding than does the CIF (Hothorn et al., 2004b). PMEI contains

a flexible a-hairpin that is important both in dimer formation and in binding of PME (Hothorn et

al., 2004b). Chimeral domain swap experiments of the a-hairpin domain between PMEI and CIF

indicate that this domain of PMEI is necessary and sufficient for activity against PME; however,

the corresponding a-hairpin in NtCIF is not sufficient for invertase inhibition (Hothorn et al.,

2004b). The -28 amino acid residues encoding this a-hairpin may be key in distinguishing these

two classes of inhibitors (Hothorn et al., 2004b).

Analysis of PMEI and CIF crystal structures not only clarifies interactions between these

inhibitors and their targets, but also aids our understanding of how distinct functions can arise for

proteins sharing very similar structures and ancestry. It is worth noting, however, that the

crystallographic analyses were performed on the cell wall invertase inhibitor and thus do not

explain the apparently narrower substrate specificity of the vacuolar invertase inhibitor. These

data indicate that the VIF may use a different mode of action than the CIF. It is also of interest

that two proteins in Arabidopsis previously annotated based on sequence similarity as cell wall

invertases (AtcwINV3&6), may actually be fructan exohydrolases (FEH).FEH protein sequences

are nearly identical to those of demonstrated acid invertase family members, yet the recombinant

enzymes are completely inactive against sucrose (De Coninck et al., 2005). It has yet to be

determined if the C/VIF family can distinguish between the FEH and the known invertase and

PME substrates, and given the level of sequence diversity within the inhibitor family it seems

likely that biochemical and structural analyses will be required to resolve these questions.









Summary

Compartmentalization of vacuolar invertase in PPV introduces a new dimension to

regulation of invertases in vivo (Rojo et al., 2003; Koch, 2004). The PPV sequester at least some

of the vacuolar invertase(s) (Rojo et al., 2003) and other proteins (especially protease precursors)

(Chrispeels and Herman, 2000; Hayashi et al., 2001) for later release into acid vacuoles. It is

currently unclear whether these invertases are active inside PPV before vesicle fusion with

acidic, sucrose-containing vacuoles.

Wall-associated kinases can regulate vacuolar invertases (Kohom et al., 2006b). At least

one of them (WAK2) regulates a specific vacuolar invertase (AtvacINV1) that predominates in

Arabidopsis roots. IfWAK2 is dysfunctional, vacuolar invertase activity drops to less than 50%

in roots, and growth is impaired under low-osmolyte conditions. The WAK kinases are ideally

positioned to serve as status sensors for the interface between the plasma membrane and cell

wall, because each WAK has an N-terminus in the extracellular matrix (possibly associated with

pectin), and a C-terminal serine/threonine kinase domain in the cytoplasm (signaling capacity).

Diverse kinases and phosphatases have been implicated in the regulation of invertases,

which is consistent with the range of signaling networks that affect them. These extend from

sugar and ABA sensing, to interactions with pathogens and symbionts.

Contrasting responses to sugars within the invertase gene family may also be mediated by

different mechanisms. Some degree of hexokinase involvement and distinctive kinetics have

been observed for the sugar-repressed invertases (Huang, 2006), but not for the more common,

sugar-induced forms examined thus far (Xiao et al., 2000).

DST elements implicated in mRNA turnover also lie in the 3' untranslated region of the

most rapidly repressed invertases. Their sequences resemble those in SAUR where they mediate

mRNA destabilization.









The invertase inhibitors, CIF and VIF share high sequence similarity with PMEI, but

evidence from crystal structures and chimeral proteins suggests that binding specificity may be

determined by flexibility of a short, N-terminal region.

Acknowledgements

We thank the National Science Foundation, Metabolic Biochemistry Program, GrantAward

No. MCB-0080282; the Department of Energy,Office of Science, Office of Biological and

Environmental Research, Grant Award No. DE-AC05-00OR22725; and the University of

Florida Experiment Station for funding.









Cell Plasma \ .
\wall-membrane





.....E .xt.race.lular....
.................. STK domain



Transmembrane
domain

WAK
___________________________________.


nucleus









PPVs


Figure B-1. Recent additions to mechanisms controlling invertases include the wall associated
kinases (WAK), precursor protease vessicles (PPV), and vacuolar processing
enzymes (VPE). At least one WAK (WAK2) can regulate activity of a specific
vacuolar invertase (AtvaclNV1) in Arabidopsis. The WAK are ideally positioned to
detect alterations in the interface between the cell wall and plasma membrane (e.g.
turgor or expansion), because their extracellular terminus resides in the cell wall
matrix (possibly anchored in pectin) and their cytoplasmic tail includes a kinase
domain. This kinase can apparently trigger cascades regulating transcription of
vacuolar invertase and other effectors of its activity. Also, at least some vacuolar
invertase(s) reach the vacuole through PPV, where they can be sequestered for
extended periods. Timing of activity may be determined by when the enzyme is
released to an acidic, sucrose-storing environment. The PPV also releases protease
precursors such as the vacuolar processing enzyme [VPEy(A)], into the acidic
vacuole which then act upon vacuolar invertases (e) to limit the duration of their
activity.


/i'






































Figure B-2. Protein inhibitors of cell wall invertases (CIF, cell wall inhibitor of fructosidase)
share strong sequence similarity with inhibitors of pectin methylesterase (PMEI,
pectin methylesterase inhibitor), but crystal structures indicate important differences
in flexibility. The top panel depicts CIF. The oval denotes an a-hairpin thought
responsible for binding specificity of CIF and PMEI. The a-hairpin in CIF is rigid at
all pHs and temperatures tested. The bottom panel shows PMEI in three different
conformations that demonstrate the flexibility of the PMEI a-hairpin. (Hothorn et al.,
2004a; Hothorn et al., 2004b; Hothorn and Scheffzek, 2006). Images were
constructed by Protein Explorer (Martz, 2002).









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BIOGRAPHICAL SKETCH

Philip Bocock was born May 20, 1979 in Wilmington, Delaware. He was raised in Austin,

Texas where he attended Westwood High School. He graduated high school in 1997 and entered

Texas A&M University in September of the same year. He earned his Bachelor of Science in

biochemistry and genetics accompanied with a minor in forest science in May of 2001.

In the fall of 2001, Philip began work on a PhD in the Plant Molecular and Cellular

Biology Program at the University of Florida. At the University of Florida, Philip entered the

laboratory of Dr. John Davis and began conducting research on carbon allocation and

partitioning using the model tree, Populus. Under the tutelage of Dr. John Davis, Philip

identified the members of the poplar invertase gene family and subsequently employed a variety

of techniques to seek to understand the role this enzymatic family plays in carbon allocation and

partitioning in poplar. The work detailed in this dissertation was entirely conducted in the

laboratory of Dr. John Davis.





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1 IDENTIFICATION AND EXPRESSION OF INVERTASE GENES IN POPULUS By PHILIP N. BOCOCK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Philip N Bocock

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3 To my wife, Traci

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4 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. John Da vis, who has helped to deepen my knowledge of molecular biology and has taught me how to as k questions. Without his endless help, I would not be here. I would also like to acknowledge my committee members: Drs. Curt Hannah, Karen Koch, Tim Martin and Gary Peter for their va luable advice throughout this undertaking. Also, I would especially like to thank Drs. Li-Fen Huang and Karen Koch whose collaboration have made this project possible. I am also grateful to other faculty, staff, and graduate students for their help and encouragement, especially Al ison Morse, Kathy Smith, Chris Dervinis, Mike Reed, Gogce Kayihan, Gustavo Ramirez, Roci o Diaz, John Mayfield and Diego Fajardo. Without the support of my family this end eavor would have never been accomplished. I must especially thank my wife, Traci, and our two children, Isaiah and Edward, for their love, encouragement and support that have gotten me through this education.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW..............................................................13 Poplar as a Model.............................................................................................................. .....13 Carbon Transport and Utilization...........................................................................................13 Sucrose........................................................................................................................ ............14 Sucrose Cleaving Enzymes.....................................................................................................14 Acid Invertase................................................................................................................. ........15 Proteinaceous Inhibitors of Acid Invertase.....................................................................16 Structure of Acid Invertases............................................................................................18 Neutral/Alkaline Invertase (Cytosolic Invertase)...................................................................19 Regulation of Invertase:....................................................................................................... ...19 Sugars......................................................................................................................... .....19 Hormones....................................................................................................................... .20 Wounding and Pathogens................................................................................................20 Environmenta l Stimuli.....................................................................................................21 Invertase Investigations in Transgenic Plants.........................................................................22 Research Objectives............................................................................................................ ....23 2 EVOLUTION AND DIVERSITY OF INVERTASE GENES IN Populus trichocarpa.......27 Introduction................................................................................................................... ..........27 Materials and Methods.......................................................................................................... .29 Plant Material................................................................................................................. .29 BLAST Searches and DNA Annotation..........................................................................30 Construction of Sequence Similarity Trees.....................................................................30 Identification of Gene Duplication..................................................................................30 Isolation of RNA and RT-PCR Assay.............................................................................31 Quantitative RT-PCR......................................................................................................31 Genomic DNA Isolation..................................................................................................32 Microarray Design and Analysis.....................................................................................32 Results........................................................................................................................ .............33 Identification of Popl ar Invertase Genes.........................................................................33 Structure of the Poplar Invertase Genes..........................................................................34 Expression of the Poplar Invertase Gene Family............................................................35

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6 Evolutionary Development of the Poplar Invertase Family............................................35 Putative Evidence for Origins of PtVIN1 ........................................................................39 Discussion..................................................................................................................... ..........39 3 RECIPROCAL SUGAR REGULATION IS CONSERVED AMONG VACUOLAR INVERTASAES OF POPLAR, ARABIDOPSIS MAIZE AND RICE.................................56 Introduction................................................................................................................... ..........56 Materials and Methods.......................................................................................................... .58 Plant Material................................................................................................................. .58 Sequence Alignments and Similarity Trees....................................................................59 RNA Extraction...............................................................................................................59 Quantitative RT-PCR in Poplar and Pine........................................................................59 Quantitative RT-PCR in Arabidopsis ..............................................................................60 Sugar Treatments for Poplar, Arabidopsis Pine and Rice..............................................61 Light-Dark Treatments for Poplar, Pine and Arabidopsis ...............................................61 Results........................................................................................................................ .............62 Predicting Gene Orthology Usi ng Protein Sequence Similarity.....................................62 A Chromosome Duplication Event is Responsible for the Two Member VIN Family in Poplar and Arabidopsis ............................................................................................64 Sugar Response Demonstrates Conservation of Gene Function in VIN s........................65 Reciprocal Response in Light..........................................................................................66 Discussion..................................................................................................................... ..........67 4 OVEREXPRESSION OF YEAST INVERTASE IN POPLAR............................................76 Introduction................................................................................................................... ..........76 Materials and Methods.......................................................................................................... .78 Plant Material, Transgenes is, and Growth Conditions....................................................78 Vector Construction.........................................................................................................78 Construction of Similarity Trees.....................................................................................79 Isolation of RNA, Generation of cDNA and Real-Time PCR Assay..............................79 Genomic DNA Isolation a nd PCR Amplification...........................................................80 Metabolic Profiling: Extraction, Separation and Identification......................................81 Construction and Experimental Design of Grafts...........................................................81 Measurement of Photosynthesis and Respiration............................................................82 Protein Extraction............................................................................................................83 Total Invertase Activity Assay........................................................................................84 Detection of Invertase Activity in Native Polyacrylamide Gels.....................................84 Sugar and Starch Determination......................................................................................84 Results and Discussion......................................................................................................... ..85 Construction of Overexpressi ng Yeast Invertase Vectors...............................................85 Expression of SUC2 in Poplar.........................................................................................86 Yeast Invertase Active in Cytosol...................................................................................87 Metabolic Profiling Reveals Alterations in CwSUC2 and VacSUC2 Transgenic Lines.......................................................................................................................... ...88

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7 Metabolic Profiling Reveals Alterations in Sugar Accumulation in CytSUC2 Transgenic Lines..........................................................................................................89 Whole-Plant Phenotype s are not Apparent......................................................................90 Concluding Remarks.......................................................................................................90 5 CONCLUSIONS..................................................................................................................102 APPENDIX A REPRESSION OF INVERTASE IN POPULUS.................................................................106 Introduction................................................................................................................... ........106 Materials and Methods.........................................................................................................107 Plant Material, Transgenes is, and Growth Conditions..................................................107 Vector Construction.......................................................................................................107 Genomic DNA Isolation a nd PCR Amplification.........................................................108 Results........................................................................................................................ ...........109 Construction of RNAi Vector........................................................................................109 Insertion of Target Gene Sequence...............................................................................109 Further Work.................................................................................................................110 B REGULATION OF INVERTASE: A"SUI TE" OF TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL MECHANISMS..............................................................................115 Abstract....................................................................................................................... ..........115 Introduction................................................................................................................... ........116 Compartmentalization of Vacuolar Invertases in Precursor Proteas e Vesicles (PPV).........117 Wall-Associated Kinase (WAK)..........................................................................................119 Other Kinases Affecting Invertases......................................................................................120 Differential Sugar Regulation of Invertases.........................................................................121 RNA Turn Over and DST.....................................................................................................124 Invertase Inhibitors........................................................................................................... ....125 Summary........................................................................................................................ .......127 Acknowledgements...............................................................................................................128 LIST OF REFERENCES.............................................................................................................131 BIOGRAPHICAL SKETCH.......................................................................................................149

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8 LIST OF TABLES Table page 2-1 Nomenclature and chromosomal location of 24 poplar invertase genes...............................44 2-2 Primers used in RT-qPCR and RT-PCR................................................................................49 3-1 Percent similarity of predicted VINs from poplar, Arabidopsis rice and maize..................71 4-1 Metabolites altered in CwSUC2 and VacSUC2 overexpressing plants.................................99 4-2 Metabolites altered in CytSUC2 overexpressing plants......................................................100 4-3 Summary of phenotypic experiments performed................................................................101 A-1 Primer sequences used for ~200 bp target sequence cloning.............................................113 A-2 Summary of RNAi constructs made...................................................................................114

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9 LIST OF FIGURES Figure page 1-1 Sucrose synthesis, transport an d metabolism in source and sink cells..................................24 1-2 Protein inhibitors of cell wall invert ases share strong sequence similarity with inhibitors of pectin methylesterase....................................................................................25 1-3 Representation of cell wall and v acuolar isoforms of carrot invertase..................................26 2-1 Exon-intron structures of pr edicted invertase genes..............................................................45 2-2 Invertase amino acid similarity trees.....................................................................................46 2-3 Expression of popl ar invertase genes....................................................................................47 2-4 Evolutionary development of the poplar acid invertase family.............................................48 2-5 Amino acid alignment of poplar invertases...........................................................................55 3-1 Protein similarity tree of VINs..............................................................................................72 3-2 Phylogenetic representati on of relevant plant species...........................................................73 3-3 Conservation of specific VIN isoform transcript repr ession under sugar treatments across taxa.................................................................................................................... ......74 3-4 Conservation of reciprocal regulation of VIN transcript under dark and light treatments across taxa.................................................................................................................... ......75 4-1 Schematic representation of yeast invertase overexpressing constructs targeted to the cytosol, cell wall and vacuole............................................................................................93 4-2 S. cerevisiae SUC2 in relationship to other plant invertases.................................................95 4-3 Relative expression of double 35S driven SUC2 in root, stem and leaf................................96 4-4 Relative expression of SUC2 transcript in overexpressing transgenic events in leaf............97 4-5 CytSUC2 transgenic plants display incr eased invertase activity due to presence of transgene...................................................................................................................... ......98 A-1 Schematic representation of pCAP T vector used for RNAi silencing...............................111 A-2 Schematic representation of RNAi directed constructs......................................................112 B-1 Recent additions to mech anisms controlling invertases.....................................................129

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10 B-2 Protein inhibitors of cell wall invertases share st rong sequence similarity with inhibitors of pectin methylesterase..................................................................................130

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IDENTIFICATION AND EXPRESSION OF INVERTASE GENES IN POPULUS By Philip N. Bocock December 2007 Chair: John M. Davis Major: Plant Molecula r and Cellular Biology Invertase (EC 3.2.1.26) plays a key role in carbon utilization as it catalyz es the irreversible hydrolysis of sucrose into glucose and fructose. Th ese sugars can act as bo th metabolic fuel and as signaling compounds directly affecting resource allocation in the plant and indirectly influencing the expression of genes responsive to shifts in hexose and sucrose availability. The invertase family in plants is composed of two s ub-families thought to have distinct evolutionary origins and can be distinguished by their pH optima for activity: acid invertases and neutral/alkaline invertases. The acid invertases appare ntly originated in eubacteria and are targeted to the cell wall and vacuole, while neutra l/alkaline invertases appa rently originated in cyanobacteria and function in the cyto sol. The recently sequenced genome of Populus trichocharpa (Torr. & Gray) allowed us to identify the genes encoding inve rtase in this woody perennial. Here we describe the identification of eight acid invertase genes; three of which belong to the vacuolar targeted group ( PtVIN1-3 ), and five of which belong to the cell wall targeted group ( PtCIN1-5 ). Similarly, we report the identifi cation of 16 neutral/a lkaline invertase genes ( PtNIN1-16 ). Expression analyses using whole genome microarrays and RT-PCR reveal evidence for expression of all invertase family members. An examination of the microsyntenic regions surrounding the poplar invertase genes reveals extensive colinearity with Arabidopsis

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12 invertases. We also find evidence for expres sion of a novel intronle ss vacuolar invertase ( PtVIN1 ), which apparently arose from a processed PtVIN2 transcript that re-inserted into the genome. To our knowledge, this is the first intr onless invertase found in plants. The response of two poplar vacuolar invertases ( PtVIN2 and -3 ) to exogenous sugar treatments was examined and compared to those of the Arabidopsis maize and rice vacuolar i nvertase orthologs. We found that PtVIN2 and -3 exhibit a reciprocal response to bot h light and exogenous sugar treatments whereby PtVIN2 expression is repressed under both light and high sugar conditions while PtVIN3 is induced under the same conditions. This reciprocal response has been previously documented in other plant systems including Arabidopsis maize and rice. An examination of the microsyntenic chromosomal regions containing vac uolar invertase reveals extensive colinearity between Arabidopsis and poplar, but does not include rice and maize. The conserved colinear structure of the chromosomal segments containing the vacuolar invertases in Arabidopsis and poplar, coupled with the conserved reciprocal responses of these v acuolar invertases to sugar in Arabidopsis poplar, maize and rice, has le d to the hypothesis that the re ciprocal nature of this sugar response arose in an anci ent genome duplication event that occurred prior to the monocot and eudicot divergence on the evol utionary tree. To be tter understand the role of invertase in sucrose export and sink development, yeast invertase ( SUC2 ) was ectopically expressed in a Populus tremula x P. alba hybrid. Despite the efficacy of this transgenic approach in other plant systems resulting in whole-plan t shifts in carbon allocation an d dramatic changes in sugar accumulation; transgenic poplars showed no whol e-plant carbon allocation shifts. Metabolic analyses revealed that while most of the tran sgenic poplar lines did not exhibit the expected alterations in sugar accumulation, intermediate s in the glyoxylate and the tricarboxylic acid cycles did fluctuate relative to the non-transgen ic controls.

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13 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Current population growth combined with increasing development and urbanization worldwide is putting strain on th e terrestrial ecosystems main carbon sink, forests. One way to combat this increasing demand of decreasing forest resources is to manipulate carbon sequestration patterns in trees in order to direct carbon allocation into th e most desirable organs such as stems, to meet industrial demands, or roots to help increase long term carbon storage capacity in the soil. Unfortunately, these mechanisms are not well understood. Carbon sequestration is not part of a single pathway, but a culmination of many pathways dealing with carbon absorption through photosyn thesis, carbon loss through respiration, carbon transport through the vascular system, carbon partitioning w ithin a cell, and carbon allocation into longterm storage compounds such as lignin. Poplar as a Model The perennial tree, poplar has emerged as a model species for physiology and genetics research. Poplar is amenable to transformati on, can be clonally propagated and has a haploid genome size of 480 million base pairs whic h is roughly four times larger than Arabidopsis, has been sequenced and encodes approximatel y 45,000 genes (Bradshaw and Stettler, 1995; Bradshaw et al. 2000; Tuskan et al. 2006). Poplar also is an econom ically important tree on the global market with interest fr om industry on its improvement. Carbon Transport and Utilization Plants utilize carbon by precisely parti tioning the reduced carbon obtained through photosynthesis into different locations within the cell and subsequently allocating it throughout the plant (Figure 1-1). Photosynthe sis reduces carbon dioxide into s ugars that can be ushered into glycolysis and the tricarboxylic acid cycle for the production of ATP and NADH, or the sugars

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14 can be used as a source of carbon for the sy nthesis of primary and secondary metabolites essential for the growth and development of th e plant. Alternatively, these sugars can be converted into starches, triacyl glycerides or polypeptides fo r long-term storage (Sturm, 1999). These same sugars are also used to synthe size sucrose, the primar y sugar used for carbon transport in higher plants. Sucros e can be transiently stored in the vacuole and then exported to the non-photosynthesizing sink tissues via the phloe m. In source cells, sucrose is synthesized in the cytosol, stored for later use in the v acuole, travels to neighboring cells through plasmodesmata, and eventually enters the phl oem via plasmodesmata or from the surrounding apoplast (Turgeon and Hepler, 1989; Grusak et al. 1996). Sucrose Sucrose and its glucose and fructose cleav age products are pr imary molecules in carbohydrate translocation, sugar signaling, and osmo tic maintenance. Sucr ose is a non-reducing disaccharide composed of an 12 linked glucose and fructose. The cleavage of a single sucrose molecule in solution results in two molecules of hexoses thereby doubling the osmotic potential of the solution. By compartmentalizing sucros e and/or the hexoses in various cellular compartments, osmotic gradients are realized, pr oviding the basis for short-distance transport from cell to cell, as well as long-distance trans port from organ to organ via the phloem as first proposed by Mnch (1930). Sucrose and its compone nt hexoses also provide sugar signals that trigger numerous biological pathways playing roles in cell division, expansion, differentiation and maturation (Koch, 2004). Sucrose Cleaving Enzymes Plants possess two enzymes that have the capab ility of cleaving sucros e: sucrose synthase (EC 2.4.1.13) and invertase (EC 3.2.1.26). Sucrose syntha se is a glycosyl tr ansferase catalyzing a reversible reaction converting su crose into UDP-glucose and fruc tose in the presence of UDP.

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15 Invertase, in contrast, hydrolyzes sucrose into glucose and fructo se in an irreversible reaction. The free energy of these two reactions are .18 KJ and .6 KJ, respectively (Ap Rees, 1984). The invertase family can be grouped according to two properties: cel lular location and pH optima for activity (Winter and Huber, 2000). The first group, known as the acid invertases, has a pH optimum of between 4.5 and 5.0 and consists of an insoluble, extr acellular, cell wall-bound form and a soluble form located in the lumen of the vacuole (Haouazine-Takvorian et al. 1997; Sherson et al. 2003). The second group, known as the ne utral/alkaline inve rtases, has a pH optimum of about 7.0-7.8. The neutra l/alkaline invertases are entirely soluble and appear to be located in the cytosol (Chen and Black, 1992; Van den Ende and Van Laere, 1995). Acid Invertase Acid invertases are -fructofuranosidases th at hydrolyze not only su crose, but also other fructose containing oligosaccharides such as raffinose and stachyose. The acid invertase Km for sucrose is in the low mM range and activity can be inhibited by Tris buffer, heavy metal ions such as Hg2+ and Ag2+, as well as other divale nt cations such as Mg2+, Ca2+, and Zn2+ (Krausgrill et al. 1996; Tymowska-Lalanne and Kreis, 1998b). Acid invertases have also been shown in a variety of species to be glycosylated. Some examples are in radish (Faye and Ghorbel, 1983), maize (Doehlert a nd Felker, 1987), carrot (Lauriere et al. 1988; Stommel and Simon, 1990; Unger et al. 1992), and tobacco (Weil and Rausch, 1990). In yeast, there ar e two isoforms of acid invertas e that are encoded by a single gene, but arise from differential splicing of exons. One isoform is glycosylated and targeted to the extracellular space, while the other is non-gly cosylated and is located in the cytosol. This glycosylation seems to have no effect on the enzymatic properties of acid invertase (Faye et al. 1981), but is instead required for its transport to th e secretory pathway where it is directed to the

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16 apoplastic and vacuolar spaces (C arlson and Botstein, 1982; Perlman et al. 1982; Bergh et al. 1987). In contrast to yeast, the invertase isoforms in plants are encoded by different genes. Invertase can range in molecular mass from a 48 kD monomeric form found in radish (Faye et al. 1981) to a 450 kD oligomeric form f ound in lily pollen (Singh and Knox, 1984), however, most molecular masses are around 60 kD. Analyses of some invertases under denaturing conditions on SDS gels show the presence of proteolytic fragments. A 70 kD vacuolar invertase monomer from mung bean hypoc otyls was processed into a 30 kD N-terminal fragment and a 38 kD C-terminal fragment (Arai et al. 1991). In carrot, a 68 kD monomer of vacuolar invertase was processed into an N-te rminal fragment of 43 kD and a C-terminal fragment of 25 kD (Unger et al. 1992; Unger et al. 1994). The role of this fragmentation is unknown, but under native conditions these fragments are tightly associated. Proteinaceous Inhibitors of Acid Invertase Cell wall and vacuolar invertase activity can be regulated by a family of proteinaceous inhibitors known as cell wall in hibitor of fructosidase (CIF ) and vacuolar inhibitor of fructosidase (VIF), or collect ively as C/VIF (Rausch and Gr einer, 2004). Although CIF are cell wall invertase inhibitors, they are broadly active agai nst both cell wall and v acuolar invertases. In contrast, VIF inhibition is specific to vacuol ar invertases. Neither of the inhibitors affect fungal invertases indicating a minimal role for th ese interactions in pathogen defense (Greiner et al. 1998; Greiner et al. 1999; Link et al. 2004). The C/VIF related protein family is not highly conserved, and in Arabidopsis sequence identities range fr om roughly 20 % to 40 % for approximately 14 family members (Rausch and Grei ner, 2004). The C/VIF related protein family also contains pectin methylesterase inhibitors (PMEI). PMEI are indistinguishable from the C/VIF by sequence alone and retain nearly id entical structures to the C/VIF (Giovane et al. 2004; Hothorn et al. 2004a; Hothorn et al. 2004b; Di Matteo et al. 2005).

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17 Important implications arise from the capacity of these related proteinaceous inhibitors to distinguish between their targets. Arabidopsis has only eight putative acid invertases (six cell wall and two vacuolar), but more than 60 pectin methylesterase (PME)related genes based on sequence similarity (Micheli, 2001; Sherson et al. 2003; Rausch and Greiner, 2004; De Coninck et al. 2005). X-ray crystollagraphy has revealed that CIF is conforma tionally stable over a broad pH and temperature range, however, the invertase /inhibitor complex is only stable at an acidic pH. This indicates that binding is determined not by conformational cha nges, but rather by pHinduced changes at the interface of th e invertase/inhibitor complex (Hothorn et al. 2004a; Hothorn and Scheffzek, 2006). In contrast, the highl y similar structure of the PMEI was found to undergo large structural rearrangements (Figur e 1-2) under these same conditions suggesting PMEI uses a different mode of binding than does the CIF (Hothorn et al. 2004b). PMEI contains a flexible -hairpin that is important both in dimer formation and in binding of PME (Hothorn et al. 2004b). Chimeral domain swap experiments of the -hairpin domain between PMEI and CIF indicate that this domain of PM EI is necessary and sufficient for activity against PME; however, the corresponding -hairpin in NtCIF is not sufficien t for invertase inhibition (Hothorn et al. 2004b). The approximately 28 amino acid residues encoding this -hairpin may be key in distinguishing these two cla sses of inhibitors (Hothorn et al. 2004b). Analysis of PMEI and CIF crystal structures not only clarifies interactions between these inhibitors and their targ ets, but also aids our understanding of how distinct functions can arise for proteins sharing very similar st ructures and ancestry. It is worth noting, however, that the crystallographic analyses were performed on th e cell wall invertase inhi bitor and thus do not explain the apparently narrower s ubstrate specificity of the vacuolar invertase inhibitor. These data indicate that the VIF may use a different mode of action than the CIF. It is also of interest

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18 that two proteins in Arabidopsis previously annotated based on sequence similarity as cell wall invertases (AtcwINV3&6), ma y actually be fructan exohydrolases (FEH). FEH protein sequences are nearly identical to those of demonstrated acid i nvertase family members, yet the recombinant enzymes are completely inactive against sucrose (De Coninck et al. 2005). It has yet to be determined if the C/VIF family ca n distinguish between FEH and the known invertase and PME substrates. Given the leve l of sequence diversity within the inhibitor family it seems likely that biochemical and structural analyses will be required to re solve these questions. Structure of Acid Invertases Acid invertases are initially synthesized as pr e-proteins containing a le ader sequence that is cleaved upon entry into the secretory pathway, th e mature peptide sequence, and in the case of the vacuolar invertases, a short C-terminal exte nsion (Figure 1-3). The le ader sequence consists of two parts: a signal peptide and an N-terminal extension of unknown function, but is thought to play a role in protein folding, targeting, or activ ity (Sturm, 1999). The signal peptide is required for entry into the endoplasmic reticulum and from there to the apoplast (Bl obel, 1980) or to the vacuole if the required vacuolar targeting domain is present (Neuhaus and Rogers, 1998). The vacuolar targeting domain is thought to be located either in the C-terminal extension or in the Nterminal propeptide which is longer in vacuol ar invertases than in cell wall invertases. Plant acid invertases have two common featur es in their sequences. Towards the N-teminal end of the protein is the -fructosidase domain, NDPN, which is usually encoded by a mini-exon of only 9 nucleotides. Towards the C-terminal e nd of the protein is another conserved sequence consisting of the amino acids WECXDF. In pot ato, Bournay (1996) obser ved that under cold stress, the mini-exon of the potato pCD111 cell wall invertase encoding the NDPN domain is skipped in an alternative splicing event.

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19 Neutral/Alkaline Invertase (Cytosolic Invertase) Neutral/alkaline invertases are located in the cytosol and have a pH optimum in the neutral to slightly alkaline range. In contrast to the ac id invertases, neutral/alkaline invertases are not glycosylated and hydrolyze only sucrose indicat ing that they are not fructofuranosidases. Neutral/alkaline invertase activity is strongly inhibited by its re action products; however, activity is not inhibited by heavy metal ions indicating a different catalytic site than that of the acid invertases. To date, neutral/alkaline invertases ha ve not been found in association with a protein inhibitor. Regulation of Invertase Sugars Sugars play important roles in plants not only as fuel for metabolism, but they also generate osmotic pressure and ac t as signaling molecules for vari ous metabolic pathways. Sugars have been shown to act as repressors of genes related to sugar acquisition and mobilization as well as activators of genes related to suga r storage and utiliza tion (Koch, 1996; Rolland et al. 2002; Halford and Paul, 2003). As one of only two en zymes able to cleave sucrose, invertase is an important player in sugar si gnaling. The majority of acid inve rtases have been shown to be sugar induced rather than repressed (Roitsch, 199 9; Roitsch and Ehness, 2000). This is consistent with their proposed roles in carbon use ve rsus carbon acquisition (Koch, 1996; Rolland et al. 2002; Koch, 2004). However, some isoforms of v acuolar invertase have been shown to be inhibited by sugars. This has been the case in to mato where transcripts for vacuolar invertase TIV1 were shown to be reduced after treatmen t with 20 mM glucose (Godt and Roitsch, 1997b). In maize, transcript as well as activity for vacuolar invertase Ivr1 was also shown to be repressed in the presence of 4 % glucose (Xu et al. 1996).

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20 Hormones Phytohormones play a central role in contro lling growth, differentiation and development in plants. As such, these hormones are particular ly involved in regulati ng sink strength (Kuiper, 1993) and carbohydrate partitioning (Brenner and Ch eikh, 1995). Invertases have been shown to be regulated by the full set of phytohormones (Roitsch et al. 2003). In most case s this is related to an increased demand for carbohyd rates as a result of the horm one stimulated growth. In a promoter study of the Lin5 invertase promoter linked to a repo rter gene in tomato, it was shown that gibberellic acid, auxin, and ABA all incr eased GUS activity indica ting induction of this isoform by these hormones (Proels et al. 2003). Work has also been done in maize demonstrating that ABA increases the accumulation of the vacuolar invertase Ivr2 activity (Trouverie et al. 2003). Brassinosteroids (Goetz et al. 2000), zeatin (Godt and Roitsch, 1997b), ethylene (Linden et al. 1996) and cytokinin (Ehness and Ro itsch, 1997) have all also been shown to alter transcript accumulation of various invertase isoforms in plants. Wounding and Pathogens The wounding of plants is a common occurren ce that can result in water loss as well as pathogen infection. Response to wounding therefore often involves mechanisms intended to counter osmotic stress as well as pathoge n attack (Reymond and Farmer, 1998; Reymond et al. 2000). As invading organisms alter sugar levels a nd effectively increase the sink of the tissue being invaded, increases in invertase activity and e xpression logically follows. It is often difficult to distinguish the invertase activity of the invadi ng pathogen from that of the host plant (Ruffner et al. 1992). In 1990, it was shown that carrot extr acellular invertase transcript accumulates dramatically after bacterial infection in the roots and leaves as we ll as after mechanical wounding (Sturm and Chrispeels, 1990a). Results obt ained from studying infection by biotrophic fungi (Fotopoulos et al. 2003), necrotrophic fungi (Benhamou et al. 1991) and viruses (Herbers

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21 et al. 2000) all show increases in invertase activity. Wounding ha s been shown in numerous systems to correlate with increases in invertase activity and transcript level. An increase in either one or both of these has been demonstrated in carrot (Sturm and Chrispeels, 1990a; RamlochLorenz et al. 1993), red goosefoot (Ehness and Roitsch, 1997), tomato (Ohyama et al. 1998), sugar beet (Rosenkranz et al. 2001) and pea (Zhang et al. 1996). Environmental Stimuli The partitioning and allocation of photoassimilates can be affected by several environmental factors such as temperature, gravity, light, wounding, drought and nutrient availability (Wardlaw, 1990). As w ould be expected, these stimuli have all been demonstrated to alter invertase transcript abundan ce and/or activity levels. Cold temperatures have stimulated invertase transcripts that were undetectable at higher temperat ures in potato tubers (Pressey and Shaw, 1966) and Jerusalem artichoke tubers (Goupil et al. 1988). In sweet potato (Huang et al. 1999) and tulip (Balk and de Boer, 1999), cold temperatures were s hown to dramatically increase the activity of invertase. Alternative splicing has also been shown to occur in potato resulting in the elimination of the -fructosidase motif (Bournay et al. 1996). Invertase transcripts are also induced and peak at one hour after gravistimulation (Kaufman et al. 1985; Wu et al. 1993a; Wu et al. 1993b). Far-red light also increases cell wall invertase activity in radish (Zouaghi and Rollin, 1976) and wheat (Krishnan et al. 1985). Salinity generally causes a reduction in sink enzyme activities. These in turn could contribut e to observed increases in sucrose of source leaves and decreases in photos ynthesis from both feedback inhi bition and sugar-repression of photosynthetic genes. Experiments in tomato revealed that in a salt sensit ive strain, growth and photosynthesis were not positively correlated as they were in the salt tolerant strain. In this salt sensitive strain, growth and photosynthesis we re both negatively correlated with glucose, fructose and sucrose accumulation in young and old leaves, suggesting a bloc kage in their use for

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22 growth. In the salt tolerant variety, transient in creases in these same sugars were accompanied by increases in acid invert ase activity (Balibrea et al. 2000). Invertase Investigations in Transgenic Plants Invertases irreversible reaction combined with its location in a variety of cellular compartments makes it a key component in the ca rbon utilization st rategy of the plant and thus an ideal target for manipulation to un derstand carbon allocation and partitioning. Several groups working with tomato (Dickinson et al. 1991), tobacco (Sonnewald et al. 1991) and potato (Heineke et al. 1992) all saw very similar resu lts after overexpr essing the yeast invertase, SUC2 in the apoplast, cytosol, and vacuole. These invertase overexpressors all had the common theme of turning source tissues into sink tissues and reducing the sucrose available for export. All plants demonstrated stunted growth and reduced root forma tion, including a reduction in the number of tubers produced in the case of potato. These tubers also had an increase in the protein to starch ratio. The mature (source) leav es in all three plants accumulated starches in addition to simple sugars, while in the case of the cytosolic overexpressors, these accumulations were also seen in the young (sink) leaves. Leaves were curved indicating rapid cell expansion or division. The plants also demonstrated bleaching a nd necrotic regions that appeared to be linked to the source state of the leaf These lesions began at the leaf margin and moved inward while also being accompanied by a reduction in photosynthesis. The cell wall and vacuolar invertases have been repressed in several plant species, including tomato (Ohyama et al. 1995), potato (Zrenner et al. 1996), and carrot (Tang and Sturm, 1999). However, the results from these ex periments were more varied than those of the invertase overexpressors, alt hough the theme of increased sucr ose content accompanied by a decrease in hexoses pervaded. In tomato these alterations in sugar accumulations appeared in both the fruit and leaves. In addition to altered sugar levels, the fruit had elevated rates of

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23 ethylene evolution. The wounding induced activity of invertase was also suppressed in the transgenic plants. The results from repressing the cell wall and vac uolar invertases in carrot are probably the most interesting. Growth was shown to be altere d at very early stages of development. The transgenic, cotyledon-stage embr yos were still masses of cells while the control plants had already developed two to three leaves and one primary root. Ho wever, when these plantlets were grown on media supplemented with hexoses, their growth returned to normal. After maturation and transfer to soil, the plan ts expressing the cell wall anti sense cDNA had a much bushier appearance and accumulated more sucrose and starch than the controls. The taproot size was also reduced and contained lower leve ls of carbohydrates than that of the controls. The dry weight ratio of leaf to root shifted fr om 1:3 in the control to 17:1 in the cell wall antisense transgenics. The vacuolar antisense plants also had increa sed numbers of leaves, although the tap root developed normally, yet slightly smaller. The le af to shoot ratio in these plants was 1.5:1. Research Objectives The purpose of this research was to investig ate invertase contributi ons to carbon allocation and partitioning in a woody perennial. To achieve this goal, the invertase family was identified and annotated using the sequenced genome of Populus trichocarpa Expression characteristics of the invertase family were then examined acr oss multiple tissues as well as through various exogenous treatments including auxin, nitrogen -supplemented fertilizer and wounding. The response of two poplar vacuolar invertases ( PtVIN2 and -3 ) to exogenous sugar treatments was examined and compared to the sugar responses of Arabidopsis maize and rice. A reverse genetic approach was also employed by ectopic expression of yeast invertase in transgenic poplar. In conjunction, RNAi was used to target e ndogenous poplar invertases for down-regulation.

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24 NIN VIN CIN CIN LIGHT NIN VIN CIN CIN NIN VIN CIN CIN LIGHT Figure 1-1. Sucrose synthesis, transport and metabolism in source and sink cells as well as invertase localization. This cartoon demonstrates apoplasti c phloem loading in the source leaf and apoplastic or symplastic phloem unloading in the sink tissue. In this scheme, sucrose (S) is synthesized in the cytosol from triose-phosphates (TP). Sucrose then enters the apoplast where it is actively transported into the phloem by a sucrose proton symporter. This phloem loadi ng step can be affect ed by activity levels of the apoplastically located cell wall inve rtase (CIN) which hydrolyzes sucrose into glucose (G) and fructose (F). Sucrose can be unloaded from the phloem either symplastically or apoplastic ally. From the apoplast, su crose can enter the sink cell directly via a sucrose proton symporter, or be hydrolyzed by CIN into glucose and fructose. In the sink cells, sucrose can be cleaved by sucrose synthase (SUSY) into UDP-glucose (UDPG) and fructose, or hydr olyzed by a neutral/alkaline invertase (NIN). Sucrose can also enter the vacuol e where it can then be hydrolyzed by a vacuolar invertase (VIN). After phosphoryl ation by hexokinase ( HK), the hexoses can enter respiration. Figure adapted from Rausch et al. (2004).

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25 Figure 1-2. Protein inhibitors of cell wall invertases (CIF, cell wall inhibitor of fructosidase) share strong sequence similarity with inhibi tors of pectin methylesterase (PMEI, pectin methylesterase inhibito r), but crystal structures i ndicate important differences in flexibility. The top panel de picts CIF. The oval denotes an -hairpin thought responsible for binding specifi city of CIF and PMEI. The -hairpin in CIF is rigid at all pHs and temperatures tested. The botto m panel shows PMEI in three different conformations that demonstrate the flexibility of the PMEI -hairpin. (Hothorn et al. 2004a; Hothorn et al. 2004b; Hothorn and Scheffzek, 2006). Images were constructed by Protein E xplorer (Martz, 2002). Figur e adapted from Huang et al. (2007).

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26 Figure 1-3. Representation of cell wall and vacu olar isoforms of carro t invertase (Sturm, 1999).

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27 CHAPTER 2 EVOLUTION AND DIVERSITY OF INVERTASE GENES IN POPULUS TRICHOCARPA Introduction Invertase (EC 3.2.1.26), also known as a -fructofuranosidase, catalyzes the irreversible hydrolysis of sucrose into glucose and fructose, indicating this enzyme has a key role in carbon utilization. These sugars are synthesized in photo synthesizing source leaves and transported to non-photosynthesizing sink tissues. Sucrose is the primary form of sugar transport in most plants, establishing this disaccharide and its gluc ose and fructose components as central to plant growth and development. The invertase family is composed of two smaller sub-families distinguished by their pH optima for activity and are thought to have distinct evolutionary origins in plants (Winter and Huber, 2000). The acid invertase sub-family is targeted to either th e cell wall or vacuole (Haouazine-Takvorian et al. 1997; Sherson et al. 2003) and is believed to have originated from respiratory eukaryotes and aerobi c bacteria (Sturm and Chrispeel s, 1990b). The neutral/alkaline invertases appear to be localized to the cy tosol (Chen and Black, 1992; Van den Ende and Van Laere, 1995) and are found in cyanobacteria, wher e the family is thought to have originated (Vargas et al. 2003), green algae and plants. The presen ce of these two sub-families reflect the hypothesized origin of green algae and higher plants; the endosymbiotic event in which a cyanobacteria invaded a non-photosynthetic, respiratory eukaryote (Mereschkowsky, 1905; Margulis, 1981; Margulis and Sagan, 2003). Members of the acid invertas e sub-family share enzymatic and biochemical properties as well as sequence similarity. Both vacuolar and cell wall targeted isoforms are fructofuranosidases that can hydr olyze fructose-containing compounds other than sucrose, such as raffinose and stachyose. Acid invertases are closely related to a cl ass of enzymes known as

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28 fructan exohydrolases (FEH). FEHs are difficult to distinguish fr om acid invertases by sequence alone and are thought to have e volved from acid invertases even though FEHs exclusively break down fructans and cannot hydrolyze sucrose (Van den Ende et al. 2002). It was recently discovered that two previously anno tated cell wall invertases from Arabidopsis ( AtcwINV3 and AtcwINV6 ) may, in fact, be FEHs (De Coninck et al. 2005). Much less is known about the ne utral/alkaline invert ase sub-family due to purification difficulties and low, unstable enzymatic activity (Sturm and Tang, 1999; Roitsch and Gonzalez, 2004). This sub-family likely has a different mode of action than the acid invertase sub-family since sucrose is the sole substrate and they ar e not inhibited by heavy metals, elements that strongly inhibit the acid invertases (Roitsch and Gonzalez, 2004). An important driver of species originati on and diversification is gene duplication. Duplication makes it possible for a gene to acqui re new functions withou t losing the function of the progenitor gene (Kramer et al. 1998; Lynch and Conery, 2000; Sankoff, 2001; Becker and Theissen, 2003; Irish, 2003; Litt and Irish, 2003; Zahn et al. 2005). Gene duplication can occur in tandem, through the duplication of a chromoso mal segment, an entire chromosome, or through genome duplication (Otto and Whitton, 2000; Wendel, 2000; Adams and Wendel, 2005). Chromosomal duplications result in conserved, colinear locus order that can be used to infer ancestry of loci of interest both within and between species (Lynch and Conery, 2000). Poplar is known to have undergone at le ast two genome duplication events in its evolutionary history (Sterck et al. 2005; Tuskan et al. 2006). The first genomic duplication event occurred prior to the divergence of Arabidopsis and poplar and is k nown as the eurosid duplication event (Bowers et al. 2003; Blanc and Wolfe, 2004; De Bodt et al. 2005; Zahn et al. 2005; Tuskan et al. 2006). The second genome duplication ev ent occurred after the divergence

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29 of Arabidopsis and poplar but prior to the divergence of the Populus and Salix genera and is referred to as the salicoid event (Tuskan et al. 2006). Arabidopsis is known to have undergone at least one genomic duplication event after the divergence with poplar (Bowers et al. 2003; Blanc and Wolfe, 2004; De Bodt et al. 2005; Zahn et al. 2005; Tuskan et al. 2006). By knowing the timing and location of genomic duplicat ion events, the evoluti onary development of a variety of gene families can be predicted in these species. The recently released sequence of the poplar genome (Tuskan et al. 2006) opens the door for systematic analysis of metabolically important gene families in a model tree. Here, I report the identification of th e poplar invertase gene family a nd show expression data from both microarray and reverse transcrip tion-polymerase chain reaction (RT-PCR) experiments. I show that acid and neutral/alkaline invertase genes ar e regulated in vegetative or floral organs. An examination of the microsyntenic regions su rrounding the poplar i nvertase genes reveals extensive colinearity with Arabidopsis invertases and allows orthologous and paralogous relationships among genes to be inferred. Materials and Methods Plant Material Rooted softwood cuttings of Populus trichocarpa (Torr. & Gray) (genotype Nisqually-1) were planted in 8 L pots and placed in a fanand pad-cooled greenhouse with natural light augmented with full spectrum fluorescent lighting dur ing the winter to give a day length of 15 h. Plants were grown on an ebband-flow flood bench system with a daily supply of Peters Professional 20-10-20 water-soluble fertilizer dilute d to a final concentration of 4 mM nitrogen. Plants were grown until 80 cm tall at which point microarray and quantitative RT-PCR (RT-qPCR) experiments were pe rformed. Floral organs used in RT-PCR experiments were

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30 harvested from a Populus deltoides growing locally on the Univer sity of Florida-Gainesville campus. BLAST Searches and DNA Annotation The Populus acid and neutral/alkaline invertase gene families were identified using the tBLASTn function of the Joint Genome Institu tes database (http://www.jgi.doe.gov/) and the previously described genes AtvacINV1 and -2 (At1g62660, At1g12240), AtcwINV1-6 (At3g13790, At3g52600,At1g55120, At2g36190, At3g13784, At5g11920), from Arabidopsis and InvDC1-5 (Accession #s X69321, X78424, X78423, Y18707, Y18706) from carrot (Lee and Sturm, 1996; Sturm, 1996; Sherson et al. 2003). The resulting nucleotide sequences were annotated using a combination of protein alignments with known Arabidopsis and carrot invertases as well as the predictions of GENSCAN (http://genes.mit.edu/GENSCAN.html) (Burge and Karlin, 1997). Construction of Sequence Similarity Trees Predicted amino acid sequences were aligned using CLUSTALW (http://clustalw.genome.jp) to construct simila rity trees using the TR EEVIEW program (Page, 1996). PAUP (Swofford, 1993) was used for boot strap analysis with 100,000 iterations. Identification of Gene Duplication Gene duplications were identified as recent or ancient by Tuskan et al. (2006) where recent and ancient refer to the salicoid and eurosid duplication events, respectively. Briefly, the poplar and Arabidopsis genomes were reconstructe d into conserved syntenic segments that were subsequently compared with a variant of the algori thm described by Hokamp et al. (2003). Tandem duplications we re defined as neighboring gene models with high sequence identity on a chromosomal segment.

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31 Isolation of RNA and RT-PCR Assay Total RNA was extracted using standard methods (Chang, 1993), DNase-treated and purified on RNAeasy QIAGEN columns (V alencia, CA). Purified RNA (5 g) was used to synthesize cDNA using a mixture of 500 ng olig o-dT, 100 ng random primers, and M-MLV-RT as per manufacturers instructi ons (Invitrogen, Carlsbad, CA), with the exception that the DTT was excluded. PCR reactions were run with a single step at 94 C for 3 min, and then 35 cycles of 94 C (30 s), 57 C (30 s), and 72 C (4 min), and a single step at 72 C for 10 min. Actin was used to verify integrity of cDNA temp late. All primers are listed in Table 2-2. Quantitative RT-PCR Gene expression was analyzed using the SYBR Green kit (Stratagene, La Jolla, CA) and Mx3000P thermo-cycler (Stratagene) as pe r manufacturers instructions. Briefly, 1 l of the synthesized cDNA and 0.15 l of a 0.25 M solution of each primer were used for each 50 l RT-qPCR reaction. Primers were designed using NetP rimer (Premier Biosoft International, Palo Alto, CA) software and synthe sized by Invitrogen. Each realtime PCR reaction was performed in triplicate (technical re plicates) on four individual plants (b iological replicates) and carried out for 40 cycles with annealing, extension, and melting temperatures of 55 C, 72 C, and 95 C, respectively. Melting curves were generated to check the specificity of the amplified fragments. In the case of PtVIN2 an extension temperature of 79 C with the fluorescence reading taken at the end of the run was used to correct for a spur ious primer-dimer amplicon. Changes (n-fold) in gene expression relative to the geometric mean (Vandesompele et al. 2002) of three control genes encoding actin, ubiquiti n and ubiquitin_L (Brunner et al. 2004) were determined using the program DART-PCRv1.0 (Peirson et al. 2003).

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32 Genomic DNA Isolation Genomic DNA was isolated from poplar shoot tip tissue. Ap proximately 250 mg of tissue was ground in liquid nitrogen and added to a buffe r containing 0.3 M sucrose, 10 mM Tris (pH 7.9), 1 mM EDTA, and 4 mg/mL diethyldithiocarbamic acid. Samples were spun (20,800 rcf) and pellets resuspended in a buffer containi ng 100 mM Tris (pH 7.9) 500 mM NaCl, 20 mM EDTA, 1 % SDS, 0.1 % 2-mercaptoethanol, and 100 g/ml proteinase K. Samples were incubated at 65 C for 1 h, spun (20,800 rcf) and supern atant extracted with chloroform. Isopropanol was used to preci pitate the DNA at room temp erature, spun (20,800 rcf), and resuspended in Tris-EDTA buffer. One l RNase A was added to DNA sample and incubated at 37 C for 30 min. The chloroform extraction was re peated, followed by ethanol precipitation in presence of sodium acetate at -80 C for 1 h. Samples were spun for 10 min (20,800 rcf), air dried and resuspended in Tris-EDTA buffer. Ten ng of genomic DNA was used in PCR reactions as previously described. Microarray Design and Analysis Poplar whole-genome 60-mer oligonucleotide mi croarrays (three di fferent 60-mer probes per gene model) were designed by NimbleGen (M adison, WI) in collabor ation with Oak Ridge National Laboratory. Labeling, hybridization and s canning were carried out by NimbleGen using standard procedures (Quesada et al. unpublished data). Briefly, signal intensity detected for each probe was log2-transformed, normalized, and contrast ed to a set of 20 negative control probes. A mixed-model analysis of variance (ANOVA) was a pplied to each individual probeset with gene as a fixed effect, and probe as a random effect. P-values were adjusted for false discovery rate (Benjamini and Hochberg, 1995), with the modifi cations reported by St orey and Tibshirani (Storey and Tibshirani, 2003). All the analyses described were carried out using SAS and JMP software (SAS Institute, Cary, NC). For contras ting the transcript abundance between treatments,

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33 individual probes that gave no signal on any array based on compar ison to the negative controls were excluded, and a complete mixed ANOVA m odel was used that included gene and tissue type as fixed effects, while probe ID, plant, and the interaction of tissue by plant were treated as random effects. Microarray data have been de posited in the Gene E xpression Omnibus (GEO) database (www.ncbi.nlm.nih.gov/ge o/); accession number GSE6422. Results Identification of Poplar Invertase Genes I used Arabidopsis and carrot invertase gene s as queries to identify 24 putative invertase genes (Table 2-1), eight in the acid invertase su b-family and 16 in the neutral/alkaline invertase sub-family, and followed the rice invertase nomenclature (Hirose et al. 2002; Ji et al. 2005). The distinct origin of the acid and neut ral/alkaline sub-families (Sturm, 1999; Vargas et al. 2003) is reflected in their intron/exon structures (Figure 2-1) and amino acid alignments (Figure 2-5). The acid invertases can be further subdivided into two we ll-supported clades, and which are inferred to be cell wall and vacuolar targeted, respectively (Figure 2-2A). This inference is based on sequence similarity to the Arabidopsis invertases. The neutral/alkaline invertases also subdivide into two clades ( and ) that are supported both by bootstrap analysis (Figure 2-2B) and intron/exon structure (Figure 21B), however the functional implications of this sub-division are not clear. Four ( PtNIN13-16 ) of the 16 neutral/alkaline inve rtases are encoded by seemingly incomplete ORFs (data not shown). PtNIN13 is missing ORFs for the first and last exons. In the case of PtNIN14 and -15 a portion of the ORF encoding the third exon is missing as well as ORFs corresponding to the first and last exons. Finally, PtNIN16 contains a short ORF encoding a portion of the third exon. In all cases, genomic sequence was examined for several Kb in either direction until neighboring genes we re identified in order to verify that the sequences were truly

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34 missing. Evidence of gene expression was obtained fo r all four of these partial genes using one or more of the methods listed in Table 2-1. Furthe r work will need to be performed to understand their evolution. Structure of the Poplar Invertase Genes The acid invertase sub-family is encoded by seven exons whose locations are generally conserved in plants (Tymowsk a-Lalanne and Kreis, 1998b). This family also contains PtVIN1 which contains no introns. All eight genes encode the motifs NDPNG and WECXDF, which are essential for catalytic activity and are conserved in this gene family (Sturm and Chrispeels, 1990b) (Figure 2-5A). Except for PtVIN1 the NDPNG motif is part ially encoded by a mini-exon encoding the tripeptide DPN, one of the smallest known exons in plants (Bournay et al. 1996). There are several key featur es that distinguish the clade of the poplar acid invertases from the clade with a highly signifi cant bootstrap value of 100 %. The first two features are Nterminal and C-terminal extensi ons, both proposed to play a role in targeting to the vacuole (Sturm, 1999). Third is the conserved WECXDF domain that contains one of the three carboxylate groups requir ed for activity (Pons et al. 1998; Alberto et al. 2004); the X in this domain is a proline in the clade and a valine in the clade (Figure 2-5A). The neutral/alkaline invertase s ub-family also clusters into and clades based on amino acid alignments (Figure 2-2B, Figure 2-5B) that are distinct and well s upported (Figure 2-2B). The members of the clade ( PtNIN1-6 ) are encoded by six exons with conserved locations (Figure 2-1B), whereas the six members of the clade ( PtNIN7-12 ) are encoded by four exons (Figure 2-1B). The different in tron/exon structures and the diffe rent number of exons between the clades, suggests that the and clades arose from different ancestral genes.

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35 Expression of the Poplar Invertase Gene Family Expression of the poplar invertase family in mature leaves, young leaves, nodes, internodes, and roots was examined using whole genome microarrays where evidence for expression was detected for 16 of the 24 genes (Figure 2-3A). RT-qPCR with gene specific primers designed against PtCIN3, PtVIN3 PtNIN3 and PtNIN9 was then used to validate the microarray data using the same sa mples that were analyzed in th e microarrays (Figure 2-3B).I then obtained evidence for tran script-level regulation of PtNIN7 -10 -11 and -16 in additional microarray analyses of responses to nitrogen availability ( PtNIN7 and -11 ) (data not shown) and exogenous auxin ( PtNIN10 and -16 ) (data not shown). Analyses of microarrays provided evidence for expression of all but four invertase genes ( PtCIN1 and 2 PtVIN1 PtNIN13 ). One possible explanation for th is result is that these four invertase genes are not transcribe d in the organ(s) examined. A lternatively, the gene may have been transcribed in the organ(s) examined but tr anscript abundance was not distinguishable from background on the arrays. To test the first hypot hesis, RT-PCR was carried out on a larger number of poplar organs. I detected transcripts for PtCIN1 and -2 in floral organs and not organs used in the array analysis (Figure 2-3C). This is consistent wi th previous results in tomato (Godt and Roitsch, 1997a), carrot (Lorenz et al. 1995), and Arabidopsis (Tymowska-Lalanne and Kreis, 1998a; Sherson et al. 2003) where certain cell wall invert ases are expressed solely in floral organs. I dete cted transcripts for PtVIN1 and PtNIN13 using RT-PCR in nearly all organs tested (Figure 2-3C). This indicates that the transcripts of these two genes were below the detection level of the arrays. Evolutionary Development of the Poplar Invertase Family Determining orthology and paral ogy between genes can be a us eful tool in elucidating gene function. The shared e volutionary history between Arabidopsis and poplar, as well as the

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36 extensive research previ ously conducted in the Arabidopsis invertase gene family, makes Arabidopsis invertases ideal candidates to compare with poplar invertases in order to determine orthology. Amino acid alignments between poplar and Arabidopsis invertases (Figure 2-2, Figure 2-5) reveal little about orthology since the Arabidopsis invertases are more similar to each other than they are to their presumed popl ar orthologs. Conseque ntly, I examined the chromosomal segments encoding invertases to infe r orthology since the lin ear order of ORFs are often conserved after duplication events. I examined paralogous regions of th e poplar genome, as defined by Tuskan et al. (2006), containing the neutral/alkaline i nvertase sub-family members and found that five of the 16 poplar neutral/alkaline invertases appear to have orig inated in the recent salicoid genome duplication event (Table 2-1). No evidence was found for tande m duplications (defined as identical, adjacent genes on the chromosome), or for expansion of the neutral/alkaline family as a result of the more ancient eurosid genome duplication event that oc curred in the common ancestor of poplar and Arabidopsis Therefore, the salicoid duplication ev ent can explain the growth of the poplar neutral/alkaline invertase family relative to the Arabidopsis gene family (16 versus 9 members, respectively). To establish orthology between th e poplar acid invertase sub-family and the more widely studied Arabidopsis acid invertase sub-family, I compared the genomic organization of the two species acid invertase sub-families and found subs tantial conservation of microsyntenic regions (Figure 2-4A). These conserved, microsyntenic regions suggest that the common ancestor to poplar and Arabidopsis contained two cell wall i nvertases and one vacuol ar invertase (Figure 24B). I hypothesize that these th ree progenitor invertases ( VINa CINa and CINb ) underwent a

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37 series of duplication events prio r to and subsequent to the spec iation event between poplar and Arabidopsis as outlined in Figure 2-4B. Vacuolar invertases PtVIN2 and -3 lie within conserved, colinear chromosomal segments (Figure 2-4B), indicating that a chromosomal duplication event gave rise to these two invertases. Similarly, the two Arabidopsis vacuolar invertases ( AtvacINV1 and -2 ) reside in a conserved, colinear arrangement (Figure 2-4A), also indi cative of a chromoso mal duplication event. Conservation can also be observed across the poplar/ Arabidopsis species divide. For example, a nuclear apical meristem (NAM) family ORF lie s downstream of the v acuolar invertases on relevant chromosomal segments in both specie s (Figure 2-4A). In a ddition, loci encoding a thylakoid luminal related protein, a 3-oxoacyl-AC P synthase III protein and a flavin containing monoxygenase are located on colinear chromoso mal segments with both the poplar and Arabidopsis vacuolar invertases (Figure 2-4A). The colinearity of multiple loci across Arabidopsis and poplar suggests that a genomic duplic ation event of a progenitor vacuolar invertase and surrounding loci occurred in a common ancestor of poplar and Arabidopsis giving rise to the two vacuolar i nvertases (Figure 2-4A, B). Two additional chromosomal duplication events can be identified through colinearity. The conservation of loci encoding a kinesin motor related protein, ubiquitin-2, and a 40S ribosomal protein S9 can be seen surrounding AtcwINV2 and -4 as well as PtCIN3 and the tandem pair PtCIN1 and -2 The conservation of this chromosomal segment between Arabidopsis and poplar is again indicative of a duplication event giving rise to these genes prior to the Arabidopsis and poplar speciation event (Figure 2-4B). B ecause the presence of the tandem pair PtCIN1 and -2 occurs only in poplar, duplication lik ely occurred after the poplar and Arabidopsis speciation event.

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38 The microsyntenic segment containi ng the poplar cell wall invertases PtCIN4 and -5 is more difficult to interpret (Figure 2-4B). This chromosomal segment contains a locus encoding a C2H2 zinc finger family protein that is shared on two chromosomal segments in Arabidopsis that also contain AtcwINV3 and the tandem pair AtcwINV1 and -5 This poplar segment also contains a DEAD box RNA helicase downstream of the tande m invertase pair that is conserved on the AtcwINV3 chromosomal segment but absent from the tandem AtcwINV1 and -5 chromosomal segment. The simplest explanation of the de velopment of these invertases is a tandem duplication event prior to the poplar/ Arabidopsis speciation event (duplication of CINa Figure 2-4B). Following the speciation event, a chro mosomal duplication even t occurred only in Arabidopsis to give rise to a tandem pair; AtcwINV3 and the other member which was lost. Poplar-specific innovations include a ta ndem duplication giving rise to the PtCIN1 and -2 pair (Table 2-1, Figure 24) and the appearance of PtVIN1 the third poplar vacuolar invertase, described in more detail in the next section. I hypothesize PtVIN1 arose from a processed PtVIN2 mRNA that was inserted in transinto the poplar genome and theref ore is not colinear with any other invertase. Arabidopsis innovations include AtcwINV3 which apparently arose from a duplication of the chromosomal segment containing AtcwINV1 and -5. This duplication was subsequently followed by the loss of one of the proposed tw o new invertases resulting in the single AtcwINV3. The sixth and final cell wall invertase in Arabidopsis AtcwINV6, poses a bit of a conundrum. The lack of colinearity w ith other regions of the Arabidopsis genome and its isolation on the protein similarity tree (Figure 2-2A) makes it difficult to deri ve a putative progenitor. I hypothesize that AtcwINV6 arose from a gene duplication event followed by functional divergence, as this is the most common mode of gene family diversification. However, because

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39 genes are frequently rearranged an d/or lost after chromosomal dupl ication events, the absence of colinearity cannot rule out a chromosomal duplica tion event. Some other technique will need to be employed to define the origin of AtcwINV6 Putative Evidence for Origins of PtVIN1 In its recent evolutionary hi story, poplar has acquired an i nnovation in the acid invertase family. PtVIN1 is an intronless invertase that clusters with the group, indicating vacuolar targeting (Figure 2-1, 2-2). An intronless invertas e, to my knowledge, has not previously been reported. This gene retains all of the characteristics relevant to the acid invertase family with the exception of introns (Figure 2-5A). PtVIN1 consists of a single 1.6 Kb ORF with several possible upstream TATAA boxes and a 3 polyadenylation signal (data not shown). I verified the sequence obtained from the Joint Genome Institutes database by designing gene specific primers (Table 2-2) flanking the entire ORF of PtVIN1 and cloning and sequencing PCR produc ts amplified from both purified P. trichocarpa genomic DNA as well as cDNA gene rated from DNase-treated RNA. PtVIN1 transcript was detected in all ti ssues examined (Figure 2-3C). I speculate that PtVIN1 arose relatively recently in popl ar evolutionary history as a processed transcript of PtVIN2 (86 % identity at the nucleotide level and 77 % identity at the amino acid level) that was reverse transcribed and reinserted into th e genome. This phenomenon is unusual but not unprecedented with examples being found in the alcohol dehydrogenase gene family in the genus Leavenworthia (Charlesworth et al. 1998), as well as numerous examples in human, rat, and dog (Coulombe-H untington and Majewski, 2007). Discussion In my study of the poplar genome, I identified 24 putative invertase genes: eight acid and 16 neutral/alkaline invertases The poplar genome encodes ca. 45,000 genes which, when

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40 compared to ca. 27,000 genes in Arabidopsis represents an approximate ly 1.6-fold increase in gene number (Tuskan et al. 2006). This total gene number expans ion is not the result of unique poplar genes, but rather the expansio n of specific gene families (Tuskan et al. 2006). Interestingly, the expansion of poplars invertase gene fa mily relative to Arabidopsis and rice is not consistent between the two invertase sub-families. Arabidopsis contains 17 members: eight acid and nine neutral/alka line invertases (Vargas et al. 2003; Ji et al. 2005), whereas the rice genome encodes 19 invertase genes: eleven acid and eight neutral/alk aline invertases (Ji et al. 2005). Thus, the overall increase in poplar invert ase gene numbers is driven primarily by an increase in the neutral/alkaline invertases. One explanation for the expansion of the neut ral/alkaline invertase sub-family in poplar may lie in the apparent lack of an active phloe m loading step in this group of woody perennial plants. Turgeon and Medville (1998 ) suggest that in willow, and likely poplar (both are members of the Salicaceae ), photoassimilate accumulation in the source phloem is accomplished via an uninterrupted, symplastically c onnected sucrose concentration gradient between the mesophyll cells and the sieve element-companion cell comple x (SE-CCC). This is in contrast to both apoplastic phloem loaders and symplastic phloem loaders where the solute concentration in the SE-CCC is dramatically higher th an the solute concentration in the surrounding apoplastic space and mesophyll cytoplasm. Turgeon and Medville (1998) propose that regulation of the sucrose concentration gradient from the mesophyll cells to the SE-CCC in poplar could be maintained in the cytoplasm and/or vacuole. This could imp licate both neutral/alkalin e invertases (thought to be cytoplasmically located) and vacuolar inve rtases as key components of this pathway. The growth of the poplar neutral/a lkaline invertase subfamily o ccurred in the recent genomic duplication event (salicoid) that took place prio r to the willow/poplar speciation event (Tuskan et

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41 al. 2006). This makes it likely that willow also co ntains the expanded neutral/alkaline invertase sub-family. Of the 16 neutral/alkaline invertas es identified in poplar, four ( PtNIN13-16 ) are missing significant portions of their coding regions. The sequences found in these four neutral/alkaline invertases retain conserved intr on/exon splice sites, conserved se quence motifs and the ability to be transcribed (data not shown). This transcriptional evidence appears to rule out these genes from being defined as pseudogenes under trad itional definitions of the term in which pseudogenes are transcriptionally and translationally silent and therefore no t subject to selection (Li et al. 1981). However, there are examples of transcribed pseudogenes in eukaryotic organisms including human, mouse, silk moth, Arabidopsis and liverwort (Balakirev et al. 2003). McCarrey and Riggs (1986) pr opose that pseudogenes could ac t as negative regulators of transcription of their progenito r genes by providing antisense RNA to hybridize with the sense RNA of the progenitor. Troyanovs ky and Leube (1994) identified cis elements in the promoter region of a pseodogene derived from human cytokeratin 17 that can interact with distal elements in the promoter of the functional gene to regulate transcrip tional activity. Thus, PtNIN13-16 may have biologically relevant func tions in gene regulation, as oppos ed to invertase enzyme activity, and may still be subjected to selection. ClustalW and bootstrap analysis of th e acid invertase fam ilies of poplar and Arabidopsis clearly divide the acid invertases into the clade (cell wall invertases) and the clade (vacuolar invertases). It has recently been reported that two of six ce ll wall invertases in Arabidopsis may not be invertases, but rather fruc tan exohydrolases (FEHs) (De Coninck et al. 2005). FEH protein sequences are nearly iden tical to those of demonstrated acid invertase proteins but FEH does not use sucrose as a substrate (De Coninck et al. 2005). As I did not test recombinant cell

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42 wall invertase activity in this work, I cannot rule out that one or more of the five PtCIN genes described here may encode an FEH. To my knowledge, this work is the first report of a plant wi th more than two vacuolar invertases. Analysis of the micr osyntenic regions surrounding these vacuolar invertases in poplar and Arabidopsis identified colinearity that I use to postu late that these genes arose from an ancient gene duplication event in a common ancestor of these two species. In contrast, I found no colinearity in the microsyntenic region containing PtVIN1 with regions in Arabidopsis or elsewhere in the poplar genome. This, along with the lack of introns in PtVIN1, leads me to hypothesize that PtVIN1 arose from a processed PtVIN2 mRNA that was inserted in transinto the poplar genome. The absence of an intronless vacuolar invert ase in any other organism studied to date suggests that PtVIN1 arose recently in evolutionary history. It will be interesting to see if this invertase anomaly extends to Populus species other than the sequenced Populus trichocarpa genome. Work performed by Coulombe-Huntin gton and Majews (2007) on intron loss in mammalian systems found that genes susceptibl e to intron loss tend to be involved in housekeeping functions and expresse d at high levels. This is cons istent with the high expression patterns of PtVIN2 the putative progenitor to the intronless PtVIN1. Because PtVIN1 has retained all the features necessary for transcription, such as an intact TATAA binding site and no indels or stop codons, it appears that there has been some level of selection in favor of the expression of PtVIN1 The hypothesis that PtVIN1 originated as a revers e transcribed, processed transcript is supported by the c oncomitant loss of all six introns from the presumed progenitor locus PtVIN2

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43 One distinguishing feature of the expression of th e invertase family is the floral specificity of certain cell wall isoforms. Tomato Lin7 is floral specific with hi ghest expression in the stamen (Godt and Roitsch, 1997b). In carrot, InvDC2 was expressed solely in floral buds (Lorenz et al. 1995) and in Arabidopsis AtcwINV2 was found to be floral specific (Tymowska-Lalanne and Kreis, 1998a). Poplar is a dioecious tree and, as I only collected floral ma terial from a female tree, I did not examine invertase expression in the male floral organs. Even so, I identified two floral specific cell wall isoforms in PtCIN1 and -2 The recently released assembly of the poplar genome (Tuskan et al. 2006) opens the door for analyses of metabolically important gene families in a model tree. In this study of the invertase gene family, I identified 24 putativ e gene family members and obtained evidence for expression of all members. A better understanding of the roles that indivi dual invertase isoforms play in carbon utilization will be important in dissecting the functional implications of invertase gene family evolution and diversity.

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44 Table 2-1. Nomenclature and chromosomal location of 24 poplar invertase genes. Gene namePredicted compartmentJGIv1.1 gene model nameLinkage group locationExpression SupportType of gene duplicationDuplicate gene Acid invertases PtCIN1 Cell wallgw1.XVI.2453.116: 5729453Rtandem PtCIN2 PtCIN2 Cell wallgw1.XVI.2454.116: 5739912E, Rtandem PtCIN1 PtCIN3 Cell wall 6: 13897519E, M, Rn/a PtCIN4 Cell wallestExt_fgenesh4_pg.C_LG_VI15366: 15233568E, M, Rtandem PtCIN5 PtCIN5 Cell walleugene3.000616076: 15229483Mtandem PtCIN4 PtVIN1 Vacuolefgenesh4_pm.C_LG_III0004073: 11913972Rinsertion PtVIN2 PtVIN2 VacuoleestExt_fgenesh4_pg.C_LG_III09023: 108433683E, M, Rancient PtVIN3 PtVIN3 VacuoleestExt_Genewise1_v1.C_LG_XV284115: 9251445E, M, Rancient PtVIN2 Neutral/alkaline invertases PtNIN1 CytosolestExt_Genewise1_v1.C_LG_VIII21208: 6388303E, M, Rn/a PtNIN2 Cytosoleugene3.0013005813: 512039E, M, Rrecent PtNIN5 PtNIN3 Cytosolgw1.VIII.2341.18: 1096197E, M, Rrecent PtNIN4 PtNIN4 Cytosolgw1.X.3512.110: 20965604E, M, Rrecent PtNIN3 PtNIN5 Cytosolgw1.131.249.1scfld131: 616042E, M, Rrecent PtNIN2 PtNIN6 Cytosolgw1.66.49.1scfld66: 160259E, M, Rn/a PtNIN7 CytosolestExt_fgenesh4_pg.C_LG_V15315:16687404E, M, Rn/a PtNIN8 Cytosoleugene3.0019073919: 9274739E, M, Rrecent PtNIN12 PtNIN9 Cytosolfgenesh4_pg.C_LG_IV0014154: 15428994E, M, Rrecent PtNIN11 PtNIN10 Cytosolfgenesh4_pm.C_LG_II0008042: 13356400M, Rrecent PtNIN13 PtNIN11 Cytosolgw1.IX.1371.19: 2392685E, M, Rrecent PtNIN9 PtNIN12 Cytosoleugene3.00410102scfld41: 1148890E, M, Rrecent PtNIN8 PtNIN13aCytosolgw1.XIV.1765.114: 3660215Rrecent PtNIN10 PtNIN14bCytosolgw1.XIV.4326.114: 11708730Mn/a PtNIN15bCytosolgw1.376.8.1scfld376: 31617E, M, Rn/a PtNIN16cCytosoleugene3.0017014017: 1427133Mn/aaMissing first and last exon; bMissing first, last, and portions of third exons; cContains only portion of third exon. Type of predicted duplication ev ent (if any) is noted. E: EST support; M: Microa rray support; R: RT-PCR support; n/a: not assigned.

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45 A PtVIN1 PtVIN2 PtCIN5 1Kb 1Kb 1Kb 1KbPtCIN1 PtVIN3 PtCIN4 PtCIN2 PtCIN3 B PtNIN1 PtNIN2 PtNIN8 PtNIN9 PtNIN7 PtNIN10 PtNIN11 PtNIN12 PtNIN3 PtNIN5 PtNIN4 PtNIN6 1Kb 1Kb PtNIN1 PtNIN2 PtNIN8 PtNIN9 PtNIN7 PtNIN10 PtNIN11 PtNIN12 PtNIN3 PtNIN5 PtNIN4 PtNIN6 1Kb 1Kb Figure 2-1. Exon-intron structures of predicted invert ase genes with complete exons (20 of 24). PtNIN13-16 are not represented here as th e complete sequence could not be determined. A) Acid invertases. B) Ne utral/alkaline invertases. Exons whose junctions have been verified (using EST and/or sequencing data) are represented by solid black boxes, exons whose junctions ha ve not been verified are represented by empty boxes. Introns are re presented by black lines.

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46 A 59 100 95 54 85 10 0 100 100 100 100 100 100 100 PtCIN4 PtCIN1 PtCIN2 PtCIN3 PtCIN5 AtvacINV2 (At1G12240) PtVIN3 PtVIN1 PtVIN2 AtvacINV1 (At1G62660) AtcwINV6(FEH) (At5G11920) AtcwINV3(FEH) (At1G55120) AtcwINV5 (At3G13784) AtcwINV1 (At3G13790) AtcwINV4 (At2G36190) AtcwINV2 (At3G52600) B 100 100 51 86 65 65 100 100 69 93 93 100 83 100 100 100 51 PtNIN10 PtNIN7 At1G35580 At4G09510 PtNIN8 PtNIN12 At1G72000 At1G22650 At5G22510 PtNIN6 PtNIN4 PtNIN3 At3G065600 At3G05820 At1G56560 PtNIN1 PtNIN5 PtNIN2 PtNIN9 PtNIN11 At4G34860 PtNIN1 PtNIN5 58 100 100 51 86 65 65 100 100 69 93 93 100 83 100 100 100 51 PtNIN10 PtNIN7 At1G35580 At4G09510 PtNIN8 PtNIN12 At1G72000 At1G22650 At5G22510 PtNIN6 PtNIN4 PtNIN3 At3G065600 At3G05820 At1G56560 PtNIN1 PtNIN5 PtNIN2 PtNIN9 PtNIN11 At4G34860 PtNIN1 PtNIN5 100 100 51 86 65 65 100 100 69 93 93 100 83 100 100 100 51 PtNIN10 PtNIN7 At1G35580 At4G09510 PtNIN8 PtNIN12 At1G72000 At1G22650 At5G22510 PtNIN6 PtNIN4 PtNIN3 At3G065600 At3G05820 At1G56560 PtNIN1 PtNIN5 PtNIN2 PtNIN9 PtNIN11 At4G34860 PtNIN1 PtNIN5 58 Figure 2-2. Invertase amino acid similarity trees based on full length sequences. A) Protein similarity tree of ei ght poplar and eight Arabidopsis acid invertases. denotes cell wall invertases (PtCIN1-5) and denotes vacuolar invertas es (PtVIN1-3). B) Protein similarity tree of 12 poplar and 9 Arabidopsis neutral/alkaline invertases. The clade (PtNIN1-6) and clade (PtNIN7-12) are marke d. PtNIN13-16 are not included. Bootstrap values are reported as a percen tage of 100,000 repetitions. Branch lengths denote protein similarity.

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47 A PtCIN3 PtNIN6 PtNIN8 PtNIN9 PtNIN12 PtNIN14 PtNIN15 PtNIN1 PtNIN2 PtNIN3 PtNIN4 PtNIN5 PtCIN5 PtVIN2 PtVIN3 PtCIN4M a tu r e l e a f Yo u n g l e a f I n te r n o d e N o d e R o o t B PtCIN30 0.2 0.4 0.6 0.8 1 1.2 MLYLRINN PtVIN3 0 0.2 0.4 0.6 0.8 1 1.2 MLYLRINN PtNIN30 0.2 0.4 0.6 0.8 1 1.2 MLYLRINN PtNIN90 0.2 0.4 0.6 0.8 1 1.2 MLYLRINN C Immature StemPtNIN13Root Xylem Phloem Leaf Shoot Tip Catkin Pre Zygotic Floral Post Zygotic FloralPtCIN1 PtCIN2 Actin PtVIN1 P. trichocarpaP. deltoides Figure 2-3. Expression of poplar invertase genes. A) Heat ma p showing relative expression of poplar invertase genes in five organs. If transcript fo r a gene was significantly detected at an FDR of 10 % (contrasted w ith the array negative control probes) in at least one organ, then data from all organs was included on the heat map. Dark blue denotes high expression a nd light blue denotes low expression. B) RT-qPCR validation of microarray data. Relative tran script levels detected by RT-qPCR (black bars) and microarray analysis (grey bars) in mature leaves (ML), young leaves (YL), roots (R), internodes (IN), a nd nodes (N). In order to compare the microarray and RTqPCR platforms, RNA was re-extracted from the same tissues used in the microarray experiment. First strand cDNA was then synthe sized and used as template in the RTqPCR reactions. The highest and lowest rela tive transcript estimates were assigned values of 1 and 0, respectively. Intermediate values were then adjusted relative to their difference between the hi ghest and lowest transcript estimate. C) Expression of PtCIN1, 2, PtVIN1 and PtNIN13 in various organs us ing cDNA generated from P. trichocarpa (vegetative organs) and P. deltoides (floral organs) as template. Contrast and resolution of images were adjusted in order to maximize visibility of bands. Actin cDNA was amplified to verify integrity of the RT-PCR template in each reaction.

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48 A AtcwIVR5 AtcwIVR1 AtcwIVR5 AtcwIVR1 AtcwIVR3 AtcwIVR3 PtCIN5 PtCIN4 24 Kb PtVIN3 PtVIN3 AtvacINV1 AtvacINV1 AtvacINV2 AtvacINV2 32 Kb PtVIN2 45 Kb AtcwIVR2 AtcwIVR4 PtCIN3 PtCIN3 4 Kb 4 Kb PtCIN2 PtCIN1 22 Kb 11.6 Kb AtcwIVR5 AtcwIVR1 AtcwIVR5 AtcwIVR1 AtcwIVR3 AtcwIVR3 PtCIN5 PtCIN4 24 Kb PtVIN3 PtVIN3 AtvacINV1 AtvacINV1 AtvacINV2 AtvacINV2 32 Kb PtVIN2 45 Kb AtcwIVR2 AtcwIVR2 AtcwIVR4 AtcwIVR4 PtCIN3 PtCIN3 4 Kb 4 Kb PtCIN2 PtCIN1 22 Kb 11.6 Kb NAM Thylakoidluminal protein related Multicopperoxidase Flavincontaining monoxygenase CC-NBS-LRR class disease resistance 3-oxoacyl-ACP synthaseIII Trehalose-6-phosphate phosphatase Kinesinmotor related 40S ribosomal protein S9 UBQ2 F-box familyEndomembraneprotein 70 C2H2 zinc finger family protein PX domain containing protein DEAD box RNA helicase B VINa VINa VINa CINa CINb CINa CINa CINb CINb Chromosomal duplicationTandem duplicationChromosomal duplicationSpeciation eventPoplarArabidopsis Tandem duplication VINa VINa CINa CINb CINb VINa VINa CINa CINb CINb PtVIN1 PtVIN2 PtCIN4 PtCIN3 PtCIN1 PtCIN2 PtVIN3 AtvacINV1 AtvacINV2 AtcwINV1 AtcwINV6 (unknown ancestry) AtcwINV2 AtcwINV4 Trans-reinsertion of processed transcript Chromosomal duplication & subsequent loss of gene pair AtcwINV3 AtcwINV5 PtCIN5 CINa CINa CINa VINa VINa VINa CINa CINb CINa CINa CINb CINb Chromosomal duplicationTandem duplicationChromosomal duplicationSpeciation eventPoplarArabidopsis Tandem duplication VINa VINa CINa CINb CINb VINa VINa CINa CINb CINb PtVIN1 PtVIN2 PtCIN4 PtCIN3 PtCIN1 PtCIN2 PtVIN3 AtvacINV1 AtvacINV2 AtcwINV1 AtcwINV6 (unknown ancestry) AtcwINV2 AtcwINV4 Trans-reinsertion of processed transcript Chromosomal duplication & subsequent loss of gene pair AtcwINV3 AtcwINV5 PtCIN5 CINa CINa CINa Figure 2-4. Evolutionary development of the poplar acid invertase family. A) Colinear chromosomal segments containing popl ar invertase ORFs (checkered) and Arabidopsis invertase ORFs (solid). Conserved ORFs between chromosomal segments are depicted with identical shading; PX, phox; NAM, nuclear apical meristem; ACP, acyl carrier protein. B) Cartoon depicting hypothesized development of the acid invertase sub-family in poplar and Arabidopsis.

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49 Table 2-2. Primers used in RT-qPCR and RT-PCR. Gene namePrimer sequence Tm (C) Amplicon size (base pairs) PtVIN1_RTqPCR_3'TCCTGGCCTCAAAA TTTGC58216 PtVIN1_RTqPCR_5'GCCCTGGTTCCACCTATAGTT58 PtVIN2_RTqPCR_3'CGCATCTTCGTCTTCTTGTG57189 PtVIN2_RTqPCR_5'CCCAGCTATAGTCCTGCCC57 PtVIN3_RTqPCR_3'GCTTGGAAAATGACTCTGTAGGTC59197 PtVIN3_RTqPCR_5'ATACACTCCCTTGCTAGACAACC57 PtCIN3_RTqPCR_3'TTGGTAGAATAGATGGTATAGCCCC61224 PtCIN3_RTqPCR_5'TGTTAAAGTTTCT CCCAGTCTTGG60 PtNIN3_RTqPCR_3' TGGGGCGAGCATCTCCT59148 PtNIN3_RTqPCR_5' TGCTGATGGTTTTGACATGTTC58 PtNIN8_RTqPCR_3' GTTTCAACAATGAGCAAGCG57211 PtNIN8_RTqPCR_5' GATTTATCTCTAGCAGAAACTCCAG56 PtNIN9_RTqPCR_3' GGCATGAAGGCGTTTAGTG56393 PtNIN9_RTqPCR_5' CACGATCCTGTCAGGAACAG56 PtNIN10_RT_PCR_3'GCTTCCCAACATATCTGCCG60824 PtNIN10_RT_PCR_5'TCGCCCGGAGGTACAGAAT60 PtNIN13_RT_PCR_3'GCTCCCCTACATACCTGCCA60629 PtNIN13_RT_PCR_5'TCGCCCAGAAGTGCAGAGA60 PtNIN14_RT_PCR_3'TGTTATTTAATTCAGTGAAATTCAGCA60334 PtNIN14_RT_PCR_5'GTTCTATATGACTTGCATCGTCAAAAT61 PtNIN15_RT_PCR_3'TGTTATTTAATTGAGTGAAATTCAGCC61241 PtNIN15_RT_PCR_5'CTCTATGACTCGCATCGTCAAAAG61 PtNIN16_RT_PCR_3'CTAGCCATGAAGGAATTTGATCTTC61201 PtNIN16_RT_PCR_5'ATGCTCTTTGTCAATGATGGAAC59

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50 A PtCIN1 1 -----------------------------------------------------------PtCIN2 1 -----------------------------------------------------------PtCIN3 1 -----------------------------------------------------------PtCIN4 1 -----------------------------------------------------------PtCIN5 1 -----------------------------------------------------------PtVIN1 1 MADTSPLLPFSHSLGP-----------GSTYSSIRWRSKIVLLLVFSGLFLVPLIVSIAS PtVIN2 1 MADPSPLLPVSNSLEPSYSPAPEGAVSAGCPATHLRRSKKVLIAVFSGLLVVSLILATIN PtVIN3 1 -MDTNPSHTSSDPPYTPLLD-------NPSPARIRRPFNGFAAILASLIFLLSLVALIIN PtCIN1 1 -------------------------M MAMP HT L S V L A LFA LL FVLT N NG A EA SHK IYSEY PtCIN2 1 -------------------------MAMP NT L S V L A LFA L FFVLS N NG A EA SHK IYPQF PtCIN3 1 -------------------------MAL LKF L P V L A LFA LL FVLS N NGVEA SHK IYLRY PtCIN4 1 -------------------------M E I LAVFL VG LCCV L QSS GI EVE A LENNGCQNFQ PtCIN5 1 -------------------------M E I SVIWV VG FCVL LV DH GVQ ASH Q S SR -----PtVIN1 50 NDNGFKQHVQYLQEDDQNVSFSPPKETTK P QI L RP G --SRG V SA GV SEK AN VNL K GAQEK PtVIN2 61 NNN-GGRHVQYHSQEDEDASLATPKE MA K P ET L LP AG YSRG V SA GV SEK AN VNL K GAQVK PtVIN3 53 QSQ------ESLPEQNQNRSPSTPRPTESFSKPEPR----G V AQ GV SPK SN P S FFSDKVS < A> < B > < C > PtCIN1 95 VSKDLINW ES LE P AIYPSKWFD NY GCWSGSATVLPNG E PVI F YTGI VDKN N S QIQN Y AVP PtCIN2 94 VSKDLINW ES LE P ALYPSKWFD NY GCWSGSATILPNG E PVI F YTGI ADKN N S QIQN Y AVP PtCIN3 94 VSKDLINW ES LE P AIYPSKWFD NY GCWSGSATILPNG E PVI F YTGI VDEN N R QIQN Y AVP PtCIN4 95 VS Y DLINWIHL NH AL C PT EP YDIN S CWSGSATILP GKG PVILYTGI DA N -HC QVQNMAMP PtCIN5 85 VS Y DLVNWVHID H AIYPT QPS DINGCWSGS T TILP GEK P A ILYTGI D T KN H QVQNLAVP PtVIN1 167 VSRDLINW F HL PL AI VSDE WFDING V WTGSATIL L NG KI VMLYT -GS TN ESV QVQNLA Y P PtVIN2 179 VSKDLI H WLHL PL AM VAD KWYD K NG V WTGSATILPDG KI VMLYT -GS TN ESV QVQNLA Y P PtVIN3 161 VS T DLI H WL Y L PF AM V P D HWYDING V WTGSATLLPDG QI MMLYT -GS TN ESV QVQNLA Y P < D> PtCIN1 155 ANLSDP Y LREWVK PDD NPIV N P DAN V N G SA FRDPTTAW W A DGHWRILIGSRR K H R G VA PtCIN2 154 ANLSDP Y LREWVK PDD NPIV N P DVS V N G SA FRDPTTAW W A DGHWRILIGSRRNH V G VA PtCIN3 154 AN S SDP Y LREWVK PDD NPIV Y P DPS V N A SA FRDPTTAW RVDGHWRILIGSKK RDR G IA PtCIN4 154 K NLSDP F L E EWIKF AQ NPIM T PP D GVEG NN FRDPTTAW LSH DGKW S VIIGS WN N NQ G MA PtCIN5 144 K NLSDPLLKEW K K SPY NPLM T P ID GID PDL YRDPTTAW Q G P D KI WRVIVGS QI N GH G -R A PtVIN1 226 AD HN DPLL LK WVKY SG NPVL VS P P GID PND FRDPTTAW YTS EGKWRI T IGSK A N NT G IA PtVIN2 238 AD HD DPLL LK WVKY SG NPVL V PP P GI G A KD FRDPTTAW KTS EGKWRIIIGSK I NK T G IA PtVIN3 220 ANLSDPLL I DWVKY PN NPVI T PP N G T E TDE FRDPTTAW M G P DG T WRI T IGSRHNK SIG LS

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51 <* E > PtCIN1 213 Y LYRS K DF KK W V K AKH PLHSV Q GTGMWECPDFYPVSLSG E NGLD P SV M G Q NVKHVLK V SL PtCIN2 212 Y LYRS R D LKK W A K TKH PLHSV QR TGMWECPDFFPVS SF G E NGLD P SVNG Q NVKH A LK V SL PtCIN3 212 Y LYRS L DF KK W F K AKH PLHSV Q GTGMWECPDFFPVSLS SE DGLDTSV G G S NVRHVLK V SL PtCIN4 213 ILYRS E DF F NW T K YQD PL Y S TER TGMWECPDFYPVSV NST DGVDTSV LNAG VKHVMK A S F PtCIN5 203 ILYRS K DF V NW T R IDS PLHS SGK T E MWECPDFFPVS T S ST NGVDTS S Q DKST KHVLK A S F PtVIN1 285 LVY D T E DF I NF KLSG-V LH G V P GTGMWEC V DFYPVS K TG Q NGLDTS A NG PH VKHVVK T SL PtVIN2 297 LVY D T E DF I NY ELLSGI LH G V PK TGMWEC V DFYPVS K TG Q NGLDTSVNG P QVKHVIK T SL PtVIN3 280 LVY Q T S NF TT Y ELLEGV LHAV P GTGMWEC V DFYPVAI N G ST GLDTS AY G AG IKHVLK A SL < F > PtCIN1 273 D M TR Y EYY T MGTYD K K K DKYFPD EGL VD GWA GLR L DYG N FYASKTFFD P STNRRILWGW A PtCIN2 272 D L TR Y EYY T LGTYD N K K EKYFPD EGL VD GWA GLR L DYG N FYASKTFFD P STNRRILWGWV PtCIN3 272 D L TR Y EYY T IGTYD E K K DRYYPD EAL VD GWA GLRYD C G N FYASKTFFD P STNRRILWGW A PtCIN4 273 N S-HDYY M IGTY VPEI EKY I PDN DFTGT GM D LRYD H G K FYASKTFFD SVK NRRILWGWV PtCIN5 263 N H-HDYY I LGSY MPEN DKF SV E TNF MD S GV D LRYDYG K FYASKTFFD G A M NRRILWGWI PtVIN1 344 D DV RKD S Y A LGTYD D K TG KWYPDN PE ID V GIGI ML DYG M FYASKTFYD QDKG RRVLWGWV PtVIN2 357 D DD RHDYY A LGTY AD K VG KWYPDN PE ID V GIGIRYDYG I FYASKTFYD Q S KG RRVLWGWI PtVIN3 340 D D TKRD H Y A IG V YD PVT DKW T PDN PKE D V GIGL QV DYG R YYASKTFYD QN TQRRILWGWI PtCIN1 333 NESD DPQK D K DKGWAGIQ L IPRKVWLD -P SGKQLLQWPV A ELEKLR G HNVQ LS N QM L DQ G PtCIN2 332 NESDA VQQ D T DKGWAGI LL IPRKVWLD -P SGKQLLQWPV A ELEKLR G HNVQ LS N QM L DQ G PtCIN3 332 NESDS VQQ D K NKGWAGIQ L IPRRVWLD -P SGKQLLQWPV A ELEKLR S HNVQ LR N QK L YQ G PtCIN4 331 NESDS IE DDMDKGWSGLQ S IPRHIWLD RSGKQLVQWPIEEI N KLH G K K V S F L D KK I DSE PtCIN5 321 NESDSE S DDI K KGWSGLQ S IPR T V L L SK N GKQIVQWPV K EIEKLR S KNV S F H D KK L KS G PtVIN1 404 A ESDTE V DDV K KGWA S LQ G IPR T I L LD T KT SS NLLQWPVEEVERLR L K GKE F N N IE V KT G PtVIN2 417 G ESDSE VA DV K KGWA S LQ G IPR T V V LD T KTG S NLLQWPVEEVE S LR L K SK NF N N IE V KA G PtVIN3 400 NETDTE T DDLDKGWA S VQ T IPRKV LY D N KTG T NILQWPVEEIE G LR L R STD F TEIV V GP G PtCIN1 392 NH VEV K VITAAQADVDVTF SFSS LD K AE PF D PKWAK LDA LD VC A QK G S KDP GGLGPFGLL PtCIN2 391 NH VEV K VITAAQADVDVTF SFSS LD K AE PF D PKWAK LDA LD VC A QK G S KAP GGLGPFGLL PtCIN3 391 YH VEV KG ITAAQADVDVTF SFPS LD K AE PF D PKWAK LDA LD VC A QK G S KAQ GGLGPFGLL PtCIN4 390 SI F EV QG ITAAQADVEV V FEL PE L QET E FL N --LTA VD PQL LC SDAN A SI KG R LGPFGLL PtCIN5 380 SVLEV PG ITASQADVDVSFEL LN LE D AE IL D --PSWT D PQL LC S QK K A SV RG K LGPFGLL PtVIN1 464 SVM P L E L DG A T Q L DI AAE FEL DKKALESTA E SNVDFSCSTSGG---A AQ RGALGPFGLL PtVIN2 477 S A V P L E L DG A T Q L DI VAE FEL DRKAIERTA E SNVEFSCSTNGG---A SH RGALGPFGLL PtVIN3 460 SVV P L D I GQ A T Q L DI FAE FEI EI I SETKHEKY-----G CSGG---A VD R S ALGPFGLL PtCIN1 452 TLASE N L E EFTPVFFRVFKA A D KHKVLLCSD AR-------S SSLG K EL Y K P SFAGFVDV PtCIN2 451 TLASE N L E EFTPVFFRVFKA V D KHKVLLCSD ARRFLASLNS SSLG E EL Y K P SFAGFVDV PtCIN3 451 TLASE K L E EFTPVFFRVFKA A D KHKVLLCSD AR-------S SSLG VG L Y K PP FAGFVDV PtCIN4 448 TLAT KD L T E Q T A IFFRIFKG LKG-YV VLMCSDQS ------RSAL RD EV D K T TYGAFIDI PtCIN5 438 AF AT KD L K E Q T A IYFRIFR SNHK-YI VLMCSDQS ------RSSV RE EL D K T TYGAFVDM PtVIN1 520 V LA D D S L A E H T S VYF Y V A KG NNGT HK TFF CTDQS ------RSSVA N DV K K EI YG S YV P V PtVIN2 533 V LA D D D L T EYTPVYF F V A KG NNGSL K TFF CTDQS ------RSSVA N DV R K EI YG S YV P V PtVIN3 510 V VA DQT L S E L TPIFFR PVNTT E GIVETYF CADET ------R FVSQI DL LNSV YG ST V P V

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52 PtCIN1 504 D LT D K KLSLRSLIDHSVVESFGAGGR IA ISSRVYPTIAVFE N AHLYVFNNGSETITV EN L PtCIN2 510 D LT D K KLSLRSLIDHSVVESFGAGGRT A ITSRVYPTIAVFE K AHLYVFNNGSETITV EN L PtCIN3 503 D LT D K KLTLRSLIDHSVVESFGAGGRT V ITSRVYP I IAVFD K AHLFVFNNGSETVTV E TL PtCIN4 500 D PQR E N ISLR -S LDHSIIESFGG E GR AC IT N RVYP K LAI Q E E ARLFIFNNGT L SVTISSL PtCIN5 490 D PRH E I ITLRSLIDHSIVESFGG E GR AC ITTR A Y AK LAI HKQ A Y LF A FNNGT S SV K IS R L PtVIN1 573 LE-G EKLSVR I LVDHSIVESFA Q GGRT V ITSRVYPT R AIY GA ARLFLFNNA I E ATVT SSL PtVIN2 586 LE-G EKLSVR I LVDHSIIESFA Q GGRT C ITSRVYPT R AIY GS ARLFLFNNATE AGVT SSL PtVIN3 563 FTDEK FQ MR V LV REID L L SFA Q GGR RV ITSRIYPT K AIY GD ARLFLFNNAT GVNVK ATL PtCIN1 564 NAWSMNT PV MN VP VK S--------PtCIN2 570 NAWSMN LPV MN VP IK NRGGENPRNE PtCIN3 563 NAWSM KVPV MN VP VK S--------PtCIN4 559 NAWSMN K A Q IN HKENFI-------PtCIN5 550 NAWSM KN A Q I VSTT K RRKPHL---PtVIN1 632 KI W Q MNSA F I RRYSNEQ-------PtVIN2 645 KI W N MNSA F I RPYSNEQQ------PtVIN3 622 KI W E LNSA F I HPF L FDQN------B PtNIN9 1 -MSSLDGDVSQNGS L KSVDAHPA L AEIEDLDF S R I LDKPPRP LN ME R Q R SCDE R ----S PtNIN11 1 -MSSINVDVSLKGS L RNAETLCD M AEIEEMDF S R I FDRPPRP LN MD R Q R SCDE R ----S PtNIN8 1 ----M DA T KETVG L MNGSSVWS I SEMDDIDF S R L SDKPK-LN IE R K R SFDE R SLSEL S PtNIN12 1 ----M DG T KEMGG L RNVSSVCS I SEMDDFDL S R L LDKPK-LN IE R Q R SFDE R SLSEL S PtNIN7 1 MSPIAAMDVCQNAS V KNFEAAGS I FEIDSEFLR--LSDKPRP VN VE R K R SFDE R SFS--PtNIN10 1 -----------------------------------------------------------PtNIN2 1 -----------------------------------------------------------PtNIN5 1 ----M RP S CRFFLSKKNRVFFN L HHSLTSNL S GNQFNFEKNK Q FFTYPFRILGSRTIFK PtNIN1 1 -----------------------------------------------------------PtNIN3 1 ----M AT S DAVLQ V LSGAGPRSFSSDLCFNNLD L AFR-SKH I KYV K K R ASRHMKMLEC S PtNIN4 1 ----M AT T EAILQ V LSGAGPCVFSSDPCFRS S D L TFSSKLH I KRV K K R ASRCMKMFEC S PtNIN6 1 ----M AT S KTVLQ V LSGGLPCPHRFDLSFGGLNSVLSICSD V KRR K NIGLVY K KLNNGM PtNIN9 55 LNELFG-VPLLSPRPSSRAESNFRL IDHL DG L YSP G R R SGFN-----------------PtNIN11 55 LSELSTGLPIPSPRPSSRVENNFRL IDHL NC L PSP G R R SGFN-----------------PtNIN8 54 IGLARG------------------ID NFETTNSP G G R SGFN-----------------PtNIN12 54 IGLARG------------------ID TFETTYSP G G R SGFN-----------------PtNIN7 56 -------------------ENSFRI IDHL EN L SPA G R R SGFN-----------------PtNIN10 1 -----------------------------------------------------------PtNIN2 1 -----------------------------------------------------------PtNIN5 56 EAQKSFCAPYISFGQSRLITGDFRGASI V AS V ASQVR K FSTSVETRVNDNNFERIYVQNG PtNIN1 1 -----------------------------------------------------------PtNIN3 55 SVQQNCIGKHWFKRSGDGDLSVNAT I K RL QL L RCKCQ K AERVSGVTTEGGNGTWFVDSAK PtNIN4 56 NVLQNGIGNHWFKGLGDRDRSVNAT I N RL QL L RCK G PQAERVSGVTE-GGNGTWFVDGAN PtNIN6 56 RLLGKCRSRGVG---AVTSRGKVKC IDR WES M RCKCQ K AESFGGATANEWSPVSLPVNGV

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53 PtNIN9 96 --------------------------------------------------TPRSQYG--F PtNIN11 97 --------------------------------------------------TPLSQFG--V PtNIN8 77 --------------------------------------------------TPASSARNSF PtNIN12 77 --------------------------------------------------TPASSTRNSF PtNIN7 79 --------------------------------------------------TPR---SCGF PtNIN10 1 -----------------------------------------------------------PtNIN2 1 -----------------------------------------------------------PtNIN5 116 IGIKPLVVER----------------------------------------IDKDENVLGD PtNIN1 1 -----------------------------------------------------------PtNIN3 115 TLNLNGAVN--TPGVLELGDTQQLMREKEVLTSNGSANKEEESLATNGAVGTGRDASRKV PtNIN4 115 TLNQNGAVTGEHTDCFGAWDAQQLTREKEGFASKAALNQEKESLATNGAVGTGRDASPKV PtNIN6 113 HG------------------------------------------ATNIFEKGSFALKGNE PtNIN9 104 E TH P A V A EAWD A LRRSLV V FRG Q PVGTIAA L D N T -G E Q LNYDQVFVRDFVPSALAFLM N G PtNIN11 105 E TH P T V A EAWE A LRRSLVYFRG E PVGTIAA L D N S -E E Q VNYDQVFVRDFVPSALAFLM N G PtNIN8 87 E PH PMV A DAWE A LRRSLVFFRG Q PVGTIAA Y D HASE EVLNYDQVFVRDFVPSALAFLM N G PtNIN12 87 E PH PMV A DAWE A LRRSLVYFRG Q PVGTIAA Y D HASE EVLNYDQVFVRDFVPSALAFLM N G PtNIN7 86 E SH PMV V DAWE S LRRTLVYFR SQ PVGTIAA L D H S -V E E LNYDQVFVRDFVPSALAFLM N G PtNIN10 1 ---MV D EAWE R L N KS Y VYFKG K PVGTLAA M D T S -A D A LNY N QVFVRDFVPTGLA C LM KE PtNIN2 1 ------------------M Y C G S PVGTVAA N D PGDKMP LNYDQVFVRDFVPSALAFLL R G PtNIN5 136 E ESR I GVLVD D CESVNRENLD G GQEVE I VSPKREES EI EKEAWKL L N D A V VMYC G SP V GT PtNIN1 1 -----------------------------------------------------------PtNIN3 173 SVD P TEE EAWE L LR D SVV H Y C G S PIGTIAA N D P T SSS VLNYDQVFIRDFIPSGIAFLL K G PtNIN4 175 SVD PI EE EAWE L LR N SMVYY C G S PIGTIAA N D P T SSS VLNYDQVFIRDFIPSGIAFLL K G PtNIN6 131 E TQS I EE EAWD L LR A SVV C Y C G N PIGTIAA N D PNSTS ILNYDQVFIRDFIPSGIAFLL K G A PtNIN9 163 -E P EIVKNFILKTL R LQSWEK K ID R F HL G E GVMPASFKVL HD PV RNS E ------T L M AD PtNIN11 164 -E P EIVKNFILKTL R LQSWEK K ID R F QL G E GVMPASFKVL HD PV THN E ------T L M AD PtNIN8 147 -E P EIVKQFLLKTL H LQ G WEK R ID R F KL G E G A MPASFKVL HD PI RKT D ------S L V AD PtNIN12 147 -E P DIVK H FLLKTL Y LQ G WEK R ID R F KL G E G A MPASFKVL HD PI RKT D ------S L V AD PtNIN7 145 -E H EVVRNFLLKTL H LQS R EK M VD Q F KL G A GVMPASFKVL HH P DRNI E ------T L M AD PtNIN10 56 PP E P EIVRNFLLKTL H LQ GL EK R VD N FT L G E GVLPASFKVL YDSDLEK E ------T L LV D PtNIN2 43 -E G EIVKNFLL HA L Q LQSWEKTVDCYS P GQGLMPASFKV R T V PLD -D -NNL EEVLD P D PtNIN5 196 --VAANDPGDK M PLNYD QSWEKTVDCYS P GQGLMPASFKV R T V PLD -D -SKF EEVLD P D PtNIN1 1 ---M EIVKNFLL H TL Q LQSWEKTVDCYS P GQGLMPASFKV K T V PLD GS D -GGF EEVLD P D PtNIN3 233 -E Y DIVRNFLL H TL Q LQSWEKTMDC H S P GQGLMPASFKV R T F PLD GD D -SAT EEVLD P D PtNIN4 235 -E Y DIVRNFLL H TL Q LQSWEKTMDC H S P GQGLMPASFKV R T VR LD GD D DFAT EEVLD P D PtNIN6 191 -E Y DIVRNFIL Y TL Q LQSWEKTMDCYS P GQGLMPASFKV R T V PLD SE D -SAT EEVLDAD B PtNIN9 215 FGESAIGRVAPVDSG F WWI F LLRAY T K S TGD T SL A E M PE C Q K GMRLIL S LCLSEGFD T FP PtNIN11 216 FGESAIGRVAPVDSG F WWI F LLRAY T K S TGD T SL A EKPE C Q K GMRLIL S LCLSEGFD T FP PtNIN8 199 FGESAIGRVAPVDSG F WWIILLRAY T K S TGD L SL A E T PE C Q K GMRLIL T LCLSEGFD T FP PtNIN12 199 FGESAIGRVAPVDSG F WWIILLRAY T K S TGD L SL A ERPE C Q K GMKLIL T LCLSEGFD T FP PtNIN7 197 FGESAIGRVAPVDSG F WWIILLRAY T K S TGD S SL A E M PE C Q R GMRLIL N LCLSEGFD T FP PtNIN10 110 FG A SAIGRVAPVDSG F WWIILLR S Y I K R T R D YA L L DRPEVQ N GMKLIL K LCLSDGFD T FP PtNIN2 98 FGESAIGRVAPVDSG L WWIILLRAYGK L TGD YA LQER V DVQTGIKLIL N LCL A DGFD M FP PtNIN5 251 FGESAIGRVAPVDSG L WWIILLRAYGK L TGD YA LQER V DVQTGIKLIL N LCLTDGFD M FP PtNIN1 57 FGESAIGRVAPVDSG L WWIILLRAYGK I TGD YA LQER V DVQTGIRL G L N LCLSDGFD M FP PtNIN3 290 FGE A AIGRVAPVDSG L WWIILLRAYGK C SGD L SVQER I DVQTGIKMIL R LCL A DGFD M FP PtNIN4 293 FGE A AIGRVAPVDSG L WWIILLRAYGK C SGD L SLQER I DVQTGIKMIL R LCL A DGFD M FP PtNIN6 248 FGE A AIGRVAPVDSG L WWIILLRAYGK C SGD L SVQER V DVQTGMKMIL R LCL A DGFD M FP

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54 C D E F PtNIN9 275 TLL CA DG C CMIDRRMGV Y G Y PIEIQALFF M ALRCA LL LLK Q DEEGKE F VE R I TK RL H ALS PtNIN11 276 TLL CA DG C CMVDRRMGV Y G Y PIEIQALFF M ALRCA LL LLK Q DEEG N E F VE R I TK RL H ALS PtNIN8 259 TLL CA DG CS MIDRRMGI Y G Y PIEIQALFF M ALR S A CS LLK H DEEGKE C IE R I VK RL H ALS PtNIN12 259 TLL CA DG CS MIDRRMGI Y G Y PIEIQALFF M ALR S A SS MLK H D Q EG N E F IE R I VK RL H ALS PtNIN7 257 TLL CA DG C CMIDRRMGV Y G Y PIEIQALFF M ALRCA LI LLK Q DDEGKE F VD R V AT RL H ALS PtNIN10 170 TLL CA DG CS MIDRRMGI Y G Y PIEIQALFY F ALRCAK Q MLK P E L DGKE F IE R I EK RI T ALS PtNIN2 158 SLL VT DG S CMIDRRMGI H G H PLEIQALFY S ALR SS R E ML VVN D GS K N LV RA INNRL S ALS PtNIN5 311 SLL VT DG S CMIDRRMGI H G H PLEIQ -----------MI VVN D GS K N LV RA INNRL S ALS PtNIN1 117 TLL VT DG S CMIDRRMGI H G H PLEIQALFY S ALRCAR E ML IVN DE T K N LV AA INNRL S ALS PtNIN3 350 TLL VT DG S CMIDRRMGI H G H PLEIQALFY S AL L CAK E ML AP ED GSA DLL RA LNNRL V ALS PtNIN4 353 TLL VT DG S CMIDRRMGI H G H PLEIQALFY S AL L CAR E ML AP ED GSA DLI RA LNNRL V ALS PtNIN6 308 TLL VT DG S CMIDRRMGI H G H PLEI E ALFY S AL L CAR E ML AP ED GSA DLI RA LNNRL V ALS G H IJ PtNIN9 335 FHMR S YYWIDLK Q LNDIYRYKTEEYSHTAVNKFNV I PD S LPEWIFDFMP V HGGY F IGNV S PtNIN11 336 FHMR S YYWIDLK Q LNDIYRYKTEEYSHTAVNKFNV I PD S LPEWIFDFMP V RGGY F IGNV S PtNIN8 319 YHMR S YFWLD FQQ LNDIYRYKTEEYSHTAVNKFNV I PD S IPDWVFDFMP T RGGY F IGNV S PtNIN12 319 YHMR S YFWLD FQQ LNDIYRYKTEEYSHTAVNKFNV I PD S IPDWVFDFMP I RGGY F IGNV S PtNIN7 317 YHMR N YFWLDMK Q LNDIYRYKTEEYSHTAVNKFNV M PD S LPDWVFDFMP T RGGY F IGNV S PtNIN10 230 YHI QT YYWLD FTQ LN N IYRYKTEEYSHTAVNKFNV I PE S IPDWVFDFMP L RGGYLIGNV S PtNIN2 218 FHIREYYWVDMR K INEIYRYKTEEYS TE A T NKFNIYPE Q IP S WL M DWIP EE GGYLIGNL Q PtNIN5 359 FHIREYYWVDM NK IN V IYRYKTEEYS TE A T NKFNIYPE Q IP S WL M DWIP EE GGYLIGNL Q PtNIN1 177 FHIREYYWVDMR K INEIYRY N TEEYS TD AVNKFNIYPD Q IP S WL V DWIP EE GGYLIGNL Q PtNIN3 410 FHIREYYWIDLR K LNEIYRYKTEEYS YD AVNKFNIYPD Q V SP WL V EWMP NQ GGYLIGNL Q PtNIN4 413 FHIREYYWIDLR K LNEIYRYKTEEYS YD AVNKFNIYPD Q I SP WL V EWMP NQ GGYLIGNL Q PtNIN6 368 FHIREYYWIDLK K LNEIYRY T TEEYS YD AVNKFNIYPD Q IP P WL V EFMP N KGGYLIGNL Q K PtNIN9 395 PA K MDFRWF C LGNC I AILSSLATPEQ ST AIMDLIE S RWEELVGEMPLKV I YPAIESHEWR PtNIN11 396 PA R MDFRWF C LGNC I AILSSLATPEQ ST AIMDLIE S RWEELVGEMPLKV I YPAIESHEWR PtNIN8 379 PA R MDFRWF A LGNC I AILSSLAT H EQ AM AIMDLIEARWEELVGEMPLKI A YPAIESHEWR PtNIN12 379 PA R MDFRWF A LGNC I AILSSLAT H EQ AM AIMDLIEARWEELVGEMPLKI A YPAIESHEWR PtNIN7 377 PA R MDFRWF C LGNC V AILSSLATPEQ AS AIMDLIE S RWEELVGEMPLKICYPALESHEWR PtNIN10 290 PA R MDFRWF L VGNC V AILSSL V TP A Q AT AIMDLVE E RWEDLIGEMPLKI T YPALE G HEWR PtNIN2 278 PAHMDFRFFTLGN L W S VVSSLGTP K Q NE AVL N LIE S KWDDLVG N MPLKICYPALES E DWR PtNIN5 419 PAHMDFRFFTLGN L W S VISSLGTP KHNE AIL N LIEAKWDDLVG N MPLKICYPALE HE DWR PtNIN1 237 PAHMDFRFFTLGN L WAIVSSLGT SK Q NE GIL N LIEARWDDLMG H MPLKICYPALE YE EWR PtNIN3 470 PAHMDFRFFSLGN I W S VVS G LAT R DQ SN AILDLIEAKW S DLVADMPLKICYPALE GQ EW Q PtNIN4 473 PAHMDFRFFSLGN I W S IVS G LAT R DQ SN AILD F IEAKW S DLIADMPLKICYPALE GQ EW Q PtNIN6 428 PAHMDFRFFTLGN L W S IVSSLAT L DQ SH AILDLIEAKW A ELVAEMPIKICYPALE GQ EWR L M PtNIN9 455 IVTG C DPKNTRWSYHNGGSWP V LLW L LTAACIK T GRP Q IARRAIELAETRL V KD N WPEYY PtNIN11 456 IVTG C DPKNTRWSYHNGGSWP V LLW L LTAACIK T GRP Q IARRAIELAETRL I KD N WPEYY PtNIN8 439 IVTG C DPKNTRWSYHNGGSWP V LLW L LTAACIK T GRP Q IARKAIDLAETRL L KD S WPEYY PtNIN12 439 IVTG C DPKNTRWSYHNGGSWP V LLW L LTAACIK T GRP Q IARKAIDLAETRL L KD G WPEYY PtNIN7 437 T VTG C DPKNTRWSYHNGGSWP V LLW L LTAACIK T GRP Q IARRAIELAESRLSKDHWPEYY PtNIN10 350 LVTG F DPKNTRWSYHNGGSWP M LLW L LSAACIKVGRP Q IAKRAIELAE Q RLSKD G WPEYY PtNIN2 338 IITGSDPKNT P WSYHNGGSWPTLLW QF T L ACMKM D R ME LA Q KAI A LAE K RL QV DHWPEYY PtNIN5 479 IITGSDPKNT P WSYHNGGSWPTLLW QF T L ACIKM N R VE LA Q KAI A LAE K RL QV DHWPEYY PtNIN1 297 IITGSDPKNT P WSYHNGGSWPTLLW QF T L ACIKMGKP E LA Q KAI A LAETRLS M D Q WPEYY PtNIN3 530 IITGSDPKNT P WSYHNAGSWPTLLW Q LT V ACIKM N RP E IA A RAVDIAE K RISRDKWPEYY PtNIN4 533 IITGSDPKNT P WSYHNAGSWPTLLW Q LTAACIKM N RP E LA A RAVEIAE K RISRDKWPEYY PtNIN6 488 IVTGSDPKNT A WSYHNGGSWPTLLW Q LT V ACIKM N RP E IA E RAV Q L V E R RISRDKWPEYY

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55 N O PtNIN9 515 D G K L GRFVGKQAR K FQTWSIAGYLVAKMLLEDPSH LG MV A LEEDK Q M KPPMRRSH S W T FPtNIN11 516 D G K L GRFIGKQAR KS QTWSIAGYLVAKMMLEDPSH LGT V A LEEDK Q M KPPIRRSN S W T -PtNIN8 499 D G K L GRYIGKQAR K YQTWSIAGYLVAKMMLEDPSH LG MI S LEEDK Q M NPVLKRSS S W T CPtNIN12 499 D G K L GRYVGKQAR K YQTWSIAGYLVAKMMLEDPSH LG MI S LEEDR Q M KPVLRRSS S W T CPtNIN7 497 D G K L G L YVGKQAR K FQTWSIAGYLVAKMMLEDPSH LG MI S LEEDK Q I THLVKRSA S W T CPtNIN10 410 D G K T GRYVGKQAR K YQTWSIAGYLVAKMMVE N PS NLL MI S LEEDK KSARSRLTRSNS T SF PtNIN2 398 D T R S GKFIGKQ S R L YQTWTVAGFL TS KVLLE N P E K AS LL FW DED Y DLL EFCVCG L NT S G R PtNIN5 539 D T R T GKFIGKQ S R L YQTWTVAGFL TS KILLE N P Q R AS LL FW DED Y ELL EICVCG L NT S G R PtNIN1 357 D T R S GRFIGKQ S R L FQTWTI S GFL TS KMLLE N P D K AS LL F LEED Y ELL EICVCA LS K T G R PtNIN3 590 D T K K ARFIGKQAR L FQTWSIAGYLVAKLLL A DPS AAR ML VT DED P ELV NAFSCM IS SNP R PtNIN4 593 D T K K ARFIGKQA HL FQTWSIAGYLVAKLLL A DPS AAR ML V MDED P ELV SAFSCM IS THP R PtNIN6 548 D T K R ARFIGKQA HL FQTWSI S GYLVAKL F L AN PS AAK I FVN EED P ELV N---AL IS ANP R PtNIN9 -------------PtNIN11 -------------PtNIN8 -------------PtNIN12 -------------PtNIN7 -------------PtNIN10 470 -------------PtNIN2 458 K RCSRVAA R SQI LV PtNIN5 599 K RCSRGAA K SQI LV PtNIN1 417 K KCSRFAA R SQI LV PtNIN3 650 R KRGQKNS K KPF IV PtNIN4 653 R NRGQKNS K KTF MV PtNIN6 605 R KRARKIF K QPF IV Figure 2-5. Amino acid alignment of poplar i nvertases. A) Amino acid alignment of acid invertases. Black shading corresponds to id entical residues, grey shading corresponds to similar residues. A-F denote highly conserved motifs as defined by Pons et al. (1998) which are thought to play roles in activity. Asterisks mark completely conserved residues thought to be involved in substrate bi nding or catalysis (Alberto et al. 2004). B ) Amino acid alignment of neutral/al kaline invertases. A-O correspond to amino acids that are consis tently different between and group invertases. Asterisks show amino acids most likel y to correspond to act ive residues for neutral/alkaline invertases, assuming equi valence with the catalytic residues of unsaturated glucuronyl hydrolase (UGL) from Bacillus sp. (Itoh et al. 2004). Black shading corresponds to identical residues, grey shading corresponds to similar residues. Alignment produced with CLUSTALW and figure produced with BOXSHADE.

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56 CHAPTER 3 RECIPROCAL SUGAR REGULATION IS CONSERVED AMONG VACUOLAR INVERTASES OF POPLAR, ARABIDOPSIS MAIZE AND RICE Introduction Sugars play an essential role in plant cell growth and development as they act as fuel for metabolism, generate osmotic pressure for shor t and long distance transp ort and act as signaling molecules for various metabolic pathways (Koch, 1996; Rolland et al. 2002; Halford and Paul, 2003). Sucrose is the primary form of sugar tr ansport in most plants, establishing this disaccharide and its glucose and fructose cleav age products as central to plant growth and development. Invertase (EC 3.2.1.26), also known as -fructofuranosidase, irreversibly hydrolyzes sucrose into glucose and fructose, and is posi tioned to play a central role in both carbon metabolism and sugar signaling. Several invertase isozymes have been identified from plant species as either soluble (readily extractable from cytoso l or vacuole), or insoluble (bound to cell wall components). Vacuolar and cell wall invertases show optimal activity at an acidic pH and are also called acid invertases. The mature acid invertases are gl ycosylated and located in the cellular compartment their name im plies (e.g. cell wall or vacuolar). Vacuolar invertases ( VIN ), like their cell wall localized family memb ers, are thought to have evolved from respiratory eukaryotes and aerobic bacteria (Sturm and Chrispeels, 1990a). VIN s have been identified and characterized to va rious levels in many different plant species including, but not limited to, Arabidopsis carrot, maize, poplar, rice, tobacco and tomato (Tymowska-Lalanne and Kreis, 1998b; Ji et al. 2005; Bocock et al. 2007). To my knowledge, with the exception of poplar (containing three) all plants studied to date encode only two VIN s. Of poplars three VIN s, one contains no introns and is t hought to be a recent evolutionary

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57 innovation stemming from a proces sed transcript inserted in trans back into the genome (Bocock et al. 2007). VIN transcripts have been found to be reci procally regulated by sugar. In maize, Ivr1 was found to be transcriptionally re pressed after treatment with exogenous glucose under the same conditions that induced transcription of Ivr2 (Xu et al. 1996). In tomato, the VIN, TIV1, was also found to be repressed after treatment with exoge nous sugar, however, induction of the second tomato VIN under the same conditions was not shown (Godt and Roitsch, 1997a). An important driver of species originati on and diversification is gene duplication. Duplication makes it possible for a gene to acqui re new functions withou t losing that of the progenitor gene (Kramer et al. 1998; Lynch and Conery, 2000; Sankoff, 2001; Becker and Theissen, 2003; Irish, 2003; Litt and Irish, 2003; Zahn et al. 2005). Gene duplication can occur in tandem, through the duplication of a chromoso mal segment, an entire chromosome, or through genome duplication (Otto and Whitton, 2000; Wendel, 2000; Adams and Wendel, 2005). Genome duplications give rise to copies of linked genes in a particular order that can be used to infer ancestry of loci of inte rest both within and between species (Lynch and Conery, 2000). Poplar, Arabidopsis maize and rice are all known to have undergone multiple genome duplication events in their evol utionary history. Poplar and Arabidopsis have each undergone a genome duplication event that pos t-dates their divergence on th e evolutionary tree (Bowers et al. 2003; Sterck et al. 2005; Tuskan et al. 2006). Poplar and Arabidopsis also share at least two genome duplication events that predate their evolutionary divergence and are referred to as the and genome duplication events (Bowers et al. 2003; De Bodt et al. 2005; Tuskan et al. 2006). On the monocot side of the evolutionary tree, maize is known to have undergone two genome duplication events, one postand the other pre-dating its divergen ce with rice (Blanc and Wolfe,

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58 2004). The event referred to previous ly is thought to have occu rred prior to the monocoteudicot divergence. Some evidence suggests that the event may even pre-date the gymnosperm divergence, although this conclusi on requires more data (Bowers et al. 2003). The timing and location of genomic duplication events can be utilized to understand the evolutionary development of a variety of gene families. In this work, the sequenced genomes of poplar, Arabidopsis and rice are compared in order to test possible scenarios for the evolutionary development of the VIN family. I also take advantage of the unique reciprocal sugar regulation that occurs in this family to analyze the functional conservation of VIN in monocots and eudicots. This evidence indicates that the two VIN s so common throughout the plant world arose at, or prior to the genome duplication event. I also examined the reciprocal sugar response in Pinus taeda a gymnosperm, but was unable to validate the presence of th is reciprocal response. Materials and Methods Plant Material Populus trichocarpa (genotype Nisqually-1) and Pinus taeda were grown in 8 L pots in a fanand pad-cooled greenhouse with natural light augmented with full spectrum fluorescent lighting during the winter to give a day length of 15 h. Greenhouse temperatures ranged from 2035 C. At noon, light intensity in the greenhouse averaged 500-700 E/m2/min PAR, which is one-half the light intensity outsi de the greenhouse. Plants were grown on an ebb-and-flow flood bench system with a daily supply of Peters Professional 20-10-20 water-soluble fertilizer diluted to a final concentration of 4 mM nitroge n. Plants were grown to a height of 60-100 cm prior to experimentation. Arabidopsis plant material was prepared as described by Huang (2006). Briefly, Arabidopsis thaliana (Col-0) seeds were sterilized and soaked in water for 4-5 d at 4 C in the

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59 dark. Seeds were sown on half-strength Murashige and Skoog (MS) medium, pH 5.8 (GIBCOBRL, U.S.A.) solidified with 0.16% (w/v ) phytagel. The typical sugar supplement for enhanced plant growth was omitted. Plantlets were cultured under cycles of 12 h light/dark at 25 C. All plant material was immediately frozen in liquid nitrogen after harv esting, and stored at 80 C prior to RNA extraction. Sequence Alignments and Similarity Trees Predicted amino acid sequences were aligned using CLUSTALW (http://clustalw.genome.jp) to construct simila rity trees using the TR EEVIEW program (Page, 1996). PAUP (Swofford, 1993) was used for boot strap analysis with 100,000 iterations. RNA Extraction Total RNA was extracted from poplar and pine leaves using standard methods (Chang, 1993), DNase-treated and purified on RNAeasy QIAGEN columns (Valencia, CA). Total RNA was extracted from Arabidopsis plants and rice embryos using the RNeasy Plant Mini Kit (QIAGEN) and DNase treated (DNA-free Kit, Am bion, Austin, TX) accord ing to manufacturers instructions. Quantitative RT-PCR in Poplar and Pine Poplar and pine cDNA was synt hesized from purified RNA (5 g) using a mixture of 500 ng oligo-dT, 100 ng random primers, and M-ML V-RT as per manufacturers instructions (Invitrogen, Carlsbad, CA), with the exception th at the DTT was excluded. Gene expression was analyzed using the SYBR Green kit (Stratagen e, La Jolla, CA) and Mx3000P thermo-cycler (Stratagene) as per manufactur ers instructions. Briefly, 1 l of synthesized cDNA and 0.15 l of a 0.25 M solution of each primer were used for each 50 l RT-qPCR reaction. Primers were designed using NetPrimer (Premier Biosoft In ternational, Palo Alto, CA) software and synthesized by Invitrogen. Primer sequences are as follows: PtVIN1 forward, 5-

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60 GCCCTGGTTCCACCTATAGTT-3; reverse, 5-TCCTGGCCTCAAAATTTGC-3 (yields 216 bp product); PtVIN2 forward, 5-CCCAGCTATAGTCCTGCCC-3; reverse, 5CGCATCTTCGTCTTCTTGTG-3 (yields 189 bp product); PtVIN3 forward, 5ATACACTCCCTTGCTAGACAACC-3; revers e, 5-GCTTGGAAAATGACTCTGTAGGTC3(yields 197 bp product); PtaedaVIN1 forward, 5-TGATTCCCGACCGCTGG-3; reverse, 5ATTAGCCTCCGACTTCACCC-3 (yields 185 bp product); PtaedaVIN2 forward, 5GGGCTGCGGTATGATTATGG-3; reverse, 5 -CTGGAGCAGCACATTCTCG-3 (yields 261 bp product). Each real-time PCR reaction was perf ormed in triplicate (t echnical replicates) on four individual plants (biological replicates) in poplar and six individual plants in pine and carried out for 40 cycles with annealing, extension, and melting temperatures of 55 C, 72 C, and 95 C, respectively. Melting curves were generate d to check the specificity of the amplified fragments. In the case of PtVIN2 an extension temperature of 79 C with the fluorescence reading taken at the end of the run was used to correct for a spurious primer-dimer amplicon. Changes (n-fold) in gene expression relati ve to the geometric mean (Vandesompele et al. 2002) of three control genes encoding ac tin, ubiquitin and ubiquitin_L (Brunner et al. 2004) were determined using the program DART-PCRv1.0 (Peirson et al. 2003). Quantitative RT-PCR in Arabidopsis Real time quantitative RT-PCR in Arabidopsis was conducted as described by Huang (2006). Briefly, 200 ng of RNA were used in 25 l reactions with Taq-Man one-step RT-PCR Master Mix reagents (Applied Biosystems, Fo ster City, CA) accord ing to manufacturers instructions.

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61 Sugar Treatments for Poplar, Arabidopsis Pine and Rice Mature leaves (LPI 12) from four greenhouse grown poplars (biological replicates, one leaf per plant, n=4) were covered in aluminum foil fo r three to five days. Leaves were then excised and taken to a dark room where aluminum foil wa s removed. Leaves were lightly rubbed with a carborundum suspension to remove the cuticle and subsequently cut in half (lengthwise). The two leaf halves were placed in 1 % (w/v) mannitol (negative c ontrol) and 1 % (w/v) glucose, respectively, and incubated in the dark at room temperature on a shaker. After a 16 h incubation period, leaves were frozen in li quid nitrogen and RNA extracted. Sugar treatments in Arabidopsis were conducted on two week ol d plants that were placed in the dark for three days, after which they we re transferred to 1/2 strength liquid MS minimal media supplemented with 1 % (w/v) mannitol or glucose and incubated in the dark at room temperature on a shaker. After a 16 h in cubation period, plants were harvested. Greenhouse grown Pinus taeda branches containing fully mature needles were wrapped in aluminum foil for five days. Branches were th en excised and taken to a dark room where aluminum foil was removed. Needles were remove d and then lightly rubbed with a carborundum suspension to remove the cuticle. Needles from a single branch were then divided in half between solutions of 1 % (w/v) mannitol and 1 % (w/v) glucose, respectively, and incubated in the dark at room temperature on a shaker. After a 16 h incubation period, needles were frozen in liquid nitrogen and RNA extracted. Needles were used from six different plants (biological replicates, n=6). Light-Dark Treatments for Poplar, Pine and Arabidopsis Mature leaves (LPI 12) from four greenhous e grown poplars (biological replicates, n=4) were covered in aluminum foil for five days. After the fifth day, half of the leaves were uncovered and exposed to sunlight for approxima tely 5 h. At noon, wrapped and exposed leaves

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62 were harvested and RNA extracted. Greenhouse grown Pinus taeda branches containing fully mature needles from six plants (biological repl icates, n=6) were wrapped in aluminum foil for five days. After the fifth day, half of the bran ches were uncovered and exposed to sunlight for approximately 5 h. At noon, needles from the wrapped and exposed branches were harvested and the RNA extracted. Treatments in Arabidopsis were conducted on two week old plants that were placed in the dark for three days, after which ha lf were exposed to light for approximately 5 h before plants were harvested and RNA extracted. Results Predicting Gene Orthology Using Protein Sequence Similarity The hypothesis that the r eciprocal regulation of VIN first identified in the maize VIN s Ivr1 ( ZmIVR1 ) and Ivr2 ( ZmIVR2 ),(Xu et al. 1996) is conserved not only in monocots, but also in eudicots was tested. To my knowledge, a ll plants studied to date contain two VIN s, with the exception of poplar, which contains three. Poplars PtVIN1 is thought to be a very recent innovation specific to poplar si nce it has no introns and likel y arose through a processed transcript of PtVIN2 inserting into the genome in trans (Bocock et al. 2007). I predict that the poplar VIN s PtVIN2 and -3 (not PtVIN1 ) are the likely othologs of ZmIVR1 and -2 Arabiodopsis and rice both contain only two VIN s and are referred to here as AtvacIVR1 AtvacIVR2 OsVIN1 and OsVIN2 respectively (Haouazine-Takvorian et al. 1997; Ji et al. 2005). Nucleotide and protein sequences were obtained from the various databases (Joint Genome Institute, poplar; NCBI, Arabidopsis and maize; TIGR, rice and maize). I then translated the nucleotide sequences and constructed amino acid a lignments between the four species (data not shown). I discovered that ZmIVR2 (accession # CAD91358) as it exists in NCBI, is missing information on the N-terminal end. In order to obtain more complete sequence, I searched the TIGR Maize database. I found that ZmIVR2 corresponds to TIGR contig AZM5_84630.

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63 Subsequent analysis of the contig in GENESC AN (http://genes.mit.edu/GENSCAN.html) (Burge and Karlin, 1997) allowed me to identify an a dditional 42 amino acids on the N-terminal end. Based on amino acid alignments with the other VIN s, I estimate that there are still roughly 140 amino acids missing from the N-te rminal end (data not shown). To obtain VIN sequences for pine, I searched for Pinus taeda ESTs on NCBI using AtvacIVR1 and -2 as queries. The ESTs were then aligned and assembled using the Sequencher program (Gene Codes, Ann Arbor, MI). This techni que resulted in two, dis tinct contigs referred to hereafter as PtaedaVIN1 and PtaedaVIN2 The lack of a third contig resulting from the assembled ESTs indicates that pine, like the other plant species mentioned above, encodes only two VIN s. The nucleotide sequences were translat ed into amino acid sequences and protein alignments revealed that PtaedaVIN1 and -2 are missing roughly 130 and 270 amino acids from their N-terminal ends, resp ectively (data not shown). The amino acid sequences were then analyzed in an attempt to identify the vacuolar orthologs between species. As can be seen in Table 3-1, OsVIN1 and ZmIVR1 appear to be orthologs as they are more simila r to each other (82 % similarity) than they are to their family members within each species. This pattern is repeated between OsVIN2 and ZmIVR2 (88 % similarity) indicating that these two genes are al so orthologous, based on sequence similarity. In the case of poplar, Arabidopsis and pine, orthology cannot be determined. In Arabidopsis and pine, the VINs are most similar to their family member within the species rather than their putative orthologs in other specie s. In contrast, the poplar VINs in comparison to each other are less similar than compared to any other VIN (Table 3-1). These alignments were then used to generate a bootstrapped protein similarity tree (Figure 3-1). As expected from percent similarity, OsVI N1 and ZmIVR1 cluster together as do OsVIN2

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64 and MzIVR2. However, the poplar, Arabidopsis and pine VINs tend to cluster within each species rather than with their orthologs (Figure 3-1). Using th is data, it was predicted that ZmIVR1 and OsVIN1 are orthologs whose transcri pts are sugar repressed while ZmIVR2 and OsVIN2 encode sugar induced transcri pts. It is not straightforwar d to predict orthology between poplar, Arabidopsis and pine using this approach due to the lack of obvious pairwise clustering of protein sequences among species. A Chromosome Duplication Event is Responsible for the Two-Member VIN Family in Poplar and Arabidopsis Large scale chromosomal or genome duplications are responsible for the growth of many gene families (Sankoff, 2001). Chromosomal segm ents arising from genome duplication events often retain a conserved linear order of loci. Conservation of loci order on a chromosomal segment is not only evidence of gene family expansion through large scale chromosomal duplication, but it also can serve as another me thod to be used in predicting orthology, and therefore function. Figure 3-2A de picts the locations of several putative large scale or genomic chromosomal duplications overlayed on an evol utionary tree outlining the development of poplar, Arabidopsis maize, pine and rice. In a previous work, I examined the chromosomal segments containing the VIN s in poplar and Arabidopsis and identified neighbori ng open reading frames (ORF) that were used to establish colinearity between the species (Bocock et al. 2007). In summary, I showed that poplar and Arabidopsis VIN s lie within conserved, colinear ch romosomal segments (Figure 3-2B) (Bocock et al. 2007). These data indicate that a chromo somal duplication event is responsible for the development of the two-member VIN family in poplar and Arabidopsis. Conservation of colinearity between the two species indicates that the duplication event must have occurred prior to poplar and Arabidopsis divergence, likely in duplication event or (Figure 3-2A).

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65 I hypothesized that the duplication ev ent giving rise to the two member VIN family occurred prior to the eudicot and monocot dive rgence, which would explain the conserved two member VIN family size in maize and rice. To address this questi on, I examined the chromosomal segments containing the VIN s in maize and rice to iden tify neighboring ORFs that could be used to establish colinearity between maize, rice and the eudictos. Unfortunately, I was unsuccessful in identifying neighboring ORFs in maize as the contigs containing ZmIVR1 and -2 were not large enough to contai n any ORFs other than the VIN s. In rice, I was able to identify numerous ORFs upstream and downstream of OsVIN1 and -2 and compare these to the eudicots. No evidence of colinearity wa s found between rice and the eudicots. This finding does not support the hypothesis, however it also does not n ecessarily contradict the hypothesis as it is quite common for small insertions, deletions and rearrangements to occur subsequent to genome duplication events (Sankoff, 2001). If the VIN s truly arose from the event, then there have been approximately 221-300+ million years (Blanc and Wo lfe, 2004) for rearrangements to occur and obscure the duplication event. Sugar Response Demonstrates Cons ervation of Gene Function in VIN s Based on amino acid sequence similarity, I hypothesized that OsVIN1 and ZmIVR1 are orthologs and likely retain the same transcript repression to exogenous sugar. Similarly, I predicted OsVIN2 and ZmIVR1 to be orthologous and retain the same transcript induction to exogenous sugar. In poplar and Arabidopsis the link is a bit more c onfusing. I was able to use conservation of colinearity to establish chromosoma l duplication as the like ly originator of the two VIN s, however I was unable to us e it to estab lish orthology. I next examined the response of the VIN s to exogenous sugar treatment to address the above questions. Leaves from greenhouse-grown popl ars were wrapped in foil for approximately three to five days in order to clear the leaves of any carbohydrate reserves. The leaves were then

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66 excised, treated with a suspension of carborundu m to remove the cuticle, and incubated overnight in solutions of either 1 % mannitol (t o serve as an osmotic and wounding control) or glucose in the dark. Transcripts for PtVIN1,2 and -3 were then assayed. I found that, like ZmIVR1 transcripts for PtVIN2 were repressed in the pres ence of sugar (Figure 3-4A). Transcripts for PtVIN3 were induced in the presen ce of sugar (data not shown). PtVIN1 the intronless poplar invertase disc ussed earlier showed no sugar re sponse at all. Transcripts for PtVIN1 were also found to be two orders of magnitude less abundant than its presumed progenitor, PtVIN2 (data not shown). I speculate that PtVIN1 lost at least some of the regulatory features of PtVIN2 when it re-inserted into the genome. Huang (2006) examined Arabidopsis plants for this sugar resp onse. Whole plants grown on sugar free media were transferred to the dark for approximately three days to clear the carbohydrate reserves. The plants were then transf erred to 1 % mannitol or glucose in the dark. After incubation in the solutes, transcripts for AtvacIVR1 and -2 were assayed. Similar to ZmIVR1 and PtVIN2 transcripts for AtvacIVR2 were found to be repressed (Figure 3-4A). AtvacIVR1 was found to be induced under th ese conditions (data not shown). In a similar fashion, Huang (unpublished data ) treated rice embryos with 3 % sucrose, which in poplar, Arabidopsis and maize incurs the same VIN response as glucose (data not shown). OsVIN1 transcripts were found to be do wn-regulated as expected from OsVIN1 s similarity to ZmIVR1 (Figure 3-4A). Reciprocal Response in Light Exogenous sugar treatments serve as an in vitro assay to mimic photosynthesis conditions in vivo by recreating the high sugar environments associated w ith active photosynthesis. I hypothesized that the VIN s repressed by sugar would also be repressed by light, while the sugar induced VIN s would be induced by light. Leaves of greenhouse grown poplar were covered for

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67 three to five days to clear the leaves of any star ch reserves. Half of the foil covered leaves were then uncovered and exposed to light. At noon on the same day, the covered and light exposed leaves were harvested and tr anscripts associated with PtVIN2 and -3 were assayed. Similarly, Arabidopsis plants grown on strength MS media lack ing sugars were placed in the dark for three to five days. Half of the plants were then moved to the li ght and allowed to photosynthesize for approximately 5 h. The plants were then harvested and transcripts for AtvacIVR1 and -2 were assayed. As expected, light was found to repress transcripts for PtVIN2 and AtvacIVR2 while inducing transcripts for PtVIN3 and AtvacIVR1 (Figure 3-3). Discussion In this study, I utilized newly released sequence information to establish orthology between VIN s in poplar, Arabidopsis maize, pine and rice. I was ab le to establish clear orthology between ZmIVR1 and OsVIN1 as well as ZmIVR2 and OsVIN2 using available nucleotide data, however establishing orthology between the other species proved more difficult. Through an examination of gene colinearity on chromosomal segments encoding the VIN loci, I was able to establish that a chromoso mal duplication gave rise to the two-member VIN family in poplar and Arabidopsis I was unable to establish cons ervation of colinearity between the eudicots and rice, however a lack of sequence information fr om maize made it impossible to compare the two monocots. The fact that the ma ize and rice genomes both appear to encode only two VIN s makes it likely that the chromosome d uplication event giving rise to the two VIN s in the eudicots occurred prior to the eudicot-monocot divergence in the event. Convergent evolution in the monocots could se rve as an alternate explanation for the appearance of the twomember VIN family in the monocots. This can only be ruled out by identify ing a conservation of colinearity on the VIN chromosomal regions between rice and another monocot, such as maize or sorghum, as more genomic sequence becomes av ailable. The grasses retain considerable

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68 conservation of colinearity (Bennetzen and Ma, 2003) so it is not unreasonable to expect that the predicted colinear regions will be identified. It will be more difficult to test colinearity between the e udicot lines and those of the monocot lines due to the extent of time and e volutionary change sin ce their divergence. The conservation of function described in this pa per, however does go a long way in establishing orthology between the monocot and eudicot VIN s. While the conservati on of reciprocal sugar response cannot rule out the explanation of c onvergent evolution, the likelihood that only two VIN family members would be encoded by such a di verse array of plant genomes and also retain the same functions for such a long evolutiona ry time frame makes the convergent evolution hypothesis extremely unlikely. This work also describes the identificati on of two EST contigs from the gymnosperm, Pinus taeda, that are highly similar to previously described VIN s in other species. I name these two contigs PtaedaVIN1 and PtaedaVIN2 These two putative VIN s had good EST coverage for nearly the entire coding region. As I was unable to find any ESTs that assembled into a third VIN contig, I believe that PtaedaVIN1 and -2 are either the only two VIN s encoded by the Pinus taeda genome, or, at the very least, the only two significantly expressed VIN s in this species. In order to identify conservation of VIN function between the gymnosperms and angiosperms, I conducted the all the same sugar and light /dark experiments as done for poplar and Arabidopsis I was unable to identify the same reciprocal response that was seen in poplar and Arabdidopsis It is unclear whether this is because the P. taeda VIN s do not have the reciprocal regulation feature as do the angiosperms, or that conditions tested did not effectivel y reveal these responses. The possibility remains that additional experi mentation could define a differential sugar responsiveness in pine under somewhat different conditions.

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69 In this study, I show that the reciprocal sugar regulation of VIN s first identified in maize (Xu et al. 1996) is conserved in at least three eudicot species. S ugar treatments were conducted on poplar and Arabidopsis and demonstrate the reciprocal re sponse in these two species. I also demonstrate that sugar repressed OsVIN1 transcripts, as predicted based on its sequence similarity to the sugar-repressed ZmIVR1 A search of the literatu re also revealed that TIV1 one of tomatos two vacuolar invert ases is also repressed after tr eatment with glucose (Godt and Roitsch, 1997a). Collectively, these data help to es tablish this sugar respon se as highly conserved throughout plants. Poplar and Arabidopsis plants were subjected to a ligh t/dark experiment intended to mimic the exogenous sugar treatment experiments. The pur pose of this is two-fol d. First, demonstrating a commonality between exogenous suga r applications and light treat ments helps to establish the sugar-induced VIN s as feast genes. Feast genes are those that are induced in response to sugars and are thought to play roles in storage proces ses and carbon utilization. These genes would also be up-regulated in actively photos ynthesizing cells (Koch, 1996). This helps us to place these sugar up-regulated genes in a more general cont ext regarding their role in plant growth and development. The second purpose of establishi ng the commonality of transcript response between the light/dark treatment and the exogenous sugar treatmen t would be the ease of the assay. For plants that are less amenable to experimental manipulation than poplar and Arabidopsis the light/dark treatment provides anot her tool for testing the reciprocal VIN response. Advances in genome sequencing are rapidly in creasing available genomic sequence. This abundance of data is allowing new types of quest ions to be asked as well as application of functional data to the larger story of plant evolution. In this study of the VIN gene family, I have

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70 added to our knowledge of the response of VIN s to sugar and light treatments. I have tied this into a larger, evolutionary stor y and discussed how this gene family has developed with the evolution of plants. Hopefully this work has ach ieved its goal of aiding our understanding of how plants process and utilize carbohydrate s obtained through photosynthesis.

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71 Table 3-1. Percent similarity of predicted VINs from poplar, Arabidopsis rice and maize. Protein names are in bold with the co rresponding gene model names below. PtVIN2PtVIN3AtvacIVR1AtvacIVR2OsVIN1OsVIN2MzIVR1MzIVR2PtaedaVIN1PtaedaVIN2 PtVIN2697776776972767474 estExt_fgenesh4_pg.C_LG_III0902 PtVIN36971716771707170 estExt_Genewise1_v1.C_LG_XV2841 AtvacIVR185706769717371 At1g62660 AtvacIVR2 707069727371 At1g12240 OsVIN1 7782767473 Os04g45290.1 OsVIN2 74887069 Os02g01590 MzIVR1 767375 P49175 MzIVR2 6970 AZM5_84630 PtaedaVIN1 89 PtaedaVIN2

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72 AtvacIVR2 AtvacIVR1 PtVIN2 PtVIN3 OsVIN1 OsVIN2 ZmIVR1 ZmIVR2 PtaedaVIN2 PtaedaVIN1100 100 100 100 98 86 91 Figure 3-1. Protein similarity tree of VINs. Bootstrap values are reported as a percentage of 100,000 repetitions. Branch lengths denote protein similarity with the exception of ZmIVR2, PtaedaVIN1 and -2 where the firs t ~140-230 amino acids have not yet been sequenced.

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73 A Poplar Arabidopsis Maize Rice Pine ? B 4 Kb no apical meristem (NAM) family protein PtVIN3 Multicopperoxidase Trehalose-6-phosphate phosphatase no apical meristem(NAM) family proteinAtvacINV1disease resistance protein (CC-NBS-LRR class) 3-oxoacyl-[acylcarrier-protein] synthaseIII flavin-containing monooxygenase family protein flavin-containing monooxygenase family protein disease resistance protein (CC-NBS-LRR class) no apical meristem(NAM) family proteinAtvacINV2 Thylakoid lumenalproteinrelated no apical meristem(NAM) family protein flavin-containing monooxygenase family protein PtVIN2 Multicopperoxidase Trehalose-6-phosphate phosphatase 45 Kb 45 Kb 3-oxoacyl-[acylcarrier-protein] synthaseIII 32 Kb Thylakoid lumenalproteinrelated Figure 3-2. A) Phylogenetic repr esentation of relevant plant speci es. Stars represent large scale duplication events as proposed by Blanc a nd Wolfe (2004), Van de Peer (2004), and Sterck et al. (2005). Greek letters deno te duplication events as described by Bowers et al. (2003). There is some uncertain ty as to the location of the event relative to the divergence of the gymnosperms and angios perms, this is denoted by ?. B) Microcolinearity between poplar and Arabidopsis VIN s. Boxes depict genes. Poplar VIN s are checkered, and Arabidopsis VIN s are solid, all other me mbers of a particular gene family are depicted with matching patterns. Labels appear over the first appearance of a certain gene family member. Panel B adapted from Bocock et al. (2007).

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74 A PtVIN20 20 40 60 80 100 120 MannitolGlucose% mRNA Actin 0% OsVIN1 3% Suc Rice embryos AtvacIVR20 20 40 60 80 100 120 MannitolGlucose% mRNA Figure 3-3. Conser vation of specific VIN isoform transcript repres sion under sugar treatments across taxa. A) PtVIN2, AtvacIVR2 and OsVIN1 transcripts are repressed by sugar. Bars represent the mean level of transcri pt; error bars denote SEM; n=4 and n=3 for PtVIN2 and AtvacIVR2 respectively. Rice panel c ontributed by L-F Huang. Suc, sucrose.

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75 A PtVIN30 20 40 60 80 100 120 DarkLight% mRNA PtVIN20 20 40 60 80 100 120 DarkLight% mRNA B AtvacIVR20 20 40 60 80 100 120 DarkLight% mRNA AtvacIVR10 20 40 60 80 100 120 DarkLight% mRNA Figure 3-4. Conservation of reciprocal regulation of VIN transcript under dark and light treatments across taxa. A) Poplars PtVIN2 is repressed by light while PtVIN3 is induced by light. Bars represent the mean level of transcript; error bars denote standard error (SEM); n=3. B) Arabidopsis AtvacIVR2 is repressed by light while AtvacIVR1 is induced by light. Bars represent th e mean level of transcript; error bars denote SEM; n=3. Panel B cont ributed by L-F Huang (2006).

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76 CHAPTER 4 OVEREXPRESSION OF YEAST INVERTASE IN POPLAR Introduction Plants fix carbon and obtain energy through phot osynthesis whereby sun light is harnessed and used to reduce and fix carbon obtained from CO2. As photosynthesis does not occur in all plant cells, carbohydrates synthesized in th e photosynthesizing source cells must be transported to the non-photosynt hesizing sink cells. The prim ary products of photosynthesis are starch and sucrose. While starch serves pr imarily as a means of storage, sucrose plays a central role in the transport of photoassimilates throughout the plant. Sucrose, as the primary form of sugar transpor t in most plants, acts as a major player in plant growth and development. Sucrose is a non -reducing disaccharide ma de of a molecule of glucose covalently bonded to a molecule of fructose, and is found in multiple compartments within the cell. In source cells, sucrose is synthesi zed in the cytosol, can be stored in the vacuole for later use, can travel to ne ighboring cells through plasmodesmat a, and eventually enters the phloem via plasmodesmata or from the surroundi ng apoplast depending on whether the plant is a symplastic or apoplastic loader of phloem (Turgeon, 1989; Grusak et al. 1996). The composition of sucrose from two hexoses means sucrose plays roles not only as a transport molecule, but also in osmotic main tenance and sugar signaling. The cleavage of a single sucrose molecule in solution results in two molecules of hexoses thereby doubling the osmotic potential of the solution. By cleaving sucrose and compartmentalizing the resulting hexoses in various cellular compartments, osmo tic gradients are created providing the basis not only for short-distance transport from cell to cell but also for long-distan ce transport from organ to organ via the phloem as first proposed by Mnch (1930). Sucrose and its component hexoses

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77 provide sugar signals triggering numerous biological pathways playing roles in cell division, expansion, differentiation and maturation (Koch, 2004). The enzymes that cleave sucrose are infl uential players of plant metabolism and development. Invertase (EC 3.2.1.26) is one of only two enzymes able to cleave sucrose. Invertase, also known as -fructofuranosidase, hydrolyzes su crose into two hexoses, glucose and fructose, in an irreversible reaction. Invertase is found in multiple cellular compartments and can be divided into three sub-families base on cellu lar localization. Invertas es localized to the vacuole and apoplast are also know n as acid invertases based upon their pH optimum for activity (Haouazine-Takvorian et al. 1997; Sherson et al. 2003). The third sub-family of invertase is localized to the cytosol and ar e also known as neutral/alkalin e invertases due to their pH optimum (Chen and Black, 1992; Van den Ende and Van Laere, 1995). Several groups in the past have utilized tran sgenic forms of plants to study the role of invertase in sucrose transloca tion. Invertase derived from yeas t has been overexpressed in the apoplast in Arabidopsis (Von Schaewen et al. 1990), potato (Heineke et al. 1992), tobacco (Sonnewald et al. 1991) and tomato (Dickinson et al. 1991). Overexpression in this compartment resulted in numerous growth defects summarized by stunted growth, inhibition of photosynthesis, accumulation of leaf starch, and necrotic lesion s on leaves followed by overall yellowing of the leaf (Von Schaewen et al. 1990; Dickinson et al. 1991; Sonnewald et al. 1991; Heineke et al. 1992). Sonnewald et al. (1991) also overexpresse d yeast derived invertase in the vacuolar and cytosolic compartments of tobacco resulting in similar phenotypes to the apoplastic overexpressing tobacco plants. Here I describe the overexpr ession of yeast invertase in Populus a deciduous, perennial tree species. While the previously mentioned wo rk has done much to es tablish the role of

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78 invertase in carbon allocation and partitioning, it is stil l unknown what roles invertase may play in a deciduous, perennial plant. The deciduous, perennial nature of poplar requires numerous cycles of sugar movement and carbon sequestration in storage organs such as the root and stem in the winter, followed by remobilization of these stored carbon compounds the following spring. It is also thought that poplar may employ a unique mode of sucrose movement into the phloem of source leaves analogous to that of poplars closely related cousin, willow (both members of Salicaceae ) (Turgeon and Medville, 1998). A reverse ge netic approach was utilized here to directly manipulate invertase levels in three su bcellular compartments, so that the effects of ectopic invertase expression c ould be assessed in poplar. Materials and Methods Plant Material, Transgenes is, and Growth Conditions Hybrid poplar clone, INRA 717-1-B4 ( P. tremula x P. alba ) was placed into sterile culture prior to Agrobacterium -mediated transformation (Leple et al. 1992). Individual clones from independent lines were clonally propagated as softwood cuttings under mist, transferred to 8 L pots and grown to a height of 60-100 cm prior to experimentation in a fanand pad-cooled greenhouse with natural light augmented with full spectrum fluorescent lighting during the winter to give a day length of 15 h (Lawrence et al. 1997). Greenhouse temperatures ranged from 20-35 C. At noon, the light intensity in the greenhouse averaged 500-700 E/m2/min PAR, which is one-half the light inte nsity outside the greenhouse. Plan ts were grown on an ebb-andflow flood bench system with a da ily supply of Peters Professional 20-10-20 water-soluble fertilizer diluted to a final c oncentration of 4 mM nitrogen. Vector Construction The cell wall targeted SUC2 construct ( CwSUC2 ) was obtained by a generous donation from Uwe Sonnewald (Institut fr Biologie de r Universitt Erlangen-Nrnberg, Erlangen,

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79 Germany). The cytosolic targeted SUC2 construct ( CytSUC2 ) was PCR amplified without the Nterminal signal peptide from the CwSUC2 construct using the primer sequences: CytSUC2 forward, 5-CACCATGACAAACGAAACTAGCGATAG-3; CytSUC2 reverse, 5CAGGTAACTGGGGTCGGGAGAA-3. The vacuolar targeted SUC2 construct ( VacSUC2 ) was designed by adding a vacuolar targ eting domain from tobacco chitinase to the 3 end of the CwSUC2 construct in two PCR steps. The fi rst step used the primer sequences: CwSUC2 forward, 5-CACCATGGATGTTCACAAGGAAGTTA-3; VacSUC2 reverse_step_1, 5GTCCAACAAACCATTACCTTTT ACTTCCCTTACTTGGAACTTGTC AAT-3. The second step used the CwSUC2 forward primer again, and the primer VacSUC2 reverse_step_2, 5TCACATCGTATCTACCAAGTCCAACAAACCA TTACCTTTTACTTCC-3. Cycling parameters for all PCR reactions were 95 C for 5 min to activate DNA polymerase, then 35 cycles of 95 C for 30 s, 60 C for 30 s and 72 C for 2 min, followed by a final step of 72 C for 5 min. The above PCR amplicons were cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA) and then sub-cloned into Gate way destination vectors by LR recombination reaction for expression in E. coli and Agrobacterium The Gateway destination vectors were kindly donated by G. Tuskan (Oak Ridge National Laboratory, Oak Ridge, TN). Construction of Similarity Trees Amino acid sequences were aligned using CLUSTALW (http://clustalw.genome.jp ) to construct similarity trees us ing the TREEVIEW program (Pag e, 1996). The PAUP (Swofford, 1993) program was used for bootstra p analysis with 1,000 iterations. Isolation of RNA, Generation of cDNA and Real-Time PCR Assay Total RNA was extracted using standard methods (Chang, 1993), DNase-treated and purified in RNAeasy QIAGEN columns (V alencia, CA). Purified RNA (5 g) was used to

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80 synthesize cDNA using a mixture of 500 ng olig o-dT, 100 ng random hexamer primers, and MMLV-RT as per manufacturers instructions (In vitrogen), with the excep tion that the DTT was excluded. Gene expression was analyzed using the SYBR Green kit (Stratagene, La Jolla, CA) and Mx3000P thermo-cycler (Stratagene) as per manuf acturers instructions. Briefly, each reaction was run in triplicate and contained 1 l of synthesized cDNA along with 0.15 l of each 0.25 M primer in a final reaction volume of 50 l. Primers were designed using NetPrimer (Premier Biosoft International) software and synthesized by Invitrogen. Prim er sequences were as follows: SUC2 forward, 5-TTTGAGTTGGTTTACGCTG-3; SUC2 reverse, 5TATTTTACTTCCCTTACTTGG-3 (428 bp produc t). Cycling parameters were 95 C for 10 min to activate DNA polymerase, then 40 cycles of 95 C for 30 s, 46 C for 30 s and 72 C for 1 min. Melting curves were generated to check the specificity of the amplified fragments. Changes (n-fold) in gene expression relative to the geometric mean (Vandesompele et al. 2002) of three control genes encoding actin, ub iquitin and ubiquitin_L (Brunner et al. 2004) were determined using the program DART-PCRv1.0 (Peirson et al. 2003). Genomic DNA Isolation and PCR Amplification To confirm transgene presence, genomic DNA was isolated from poplar leaves using the Plant DNAeasy Kit (Qiagen) as per manufacturers instructions. Approxi mately 25 ng of DNA was used as template for PCR. Transg ene presence was confirmed by PCR using nptII -specific primers with the following sequences: fo rward, 5-ATCCATCATGGCTGATGCAATGCG-3; reverse, 5-CCATGATATTCGGC AAGCAGGCAT-3 (253 bp of TDNA insertion). Cycling parameters were 30 cycles of 94 C for 1 min, 58 C for 1 min and 72 C for 1 min. Amplicons were then separated on 1 % (w/v) agarose ge ls and stained with ethidium bromide.

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81 Metabolic Profiling: Extraction, Separation and Identification For metabolic profiling by GC-MS, three biolog ical replicates for each of the selected transgenic lines ( CwSUC2-2 -14 CytSUC2-18 -50 and VacSUC2-20 -30 ) and a non-transgenic (NT) control line were randomized in the greenhouse and grown to a final height of 1 m prior to the harvesting and pooling of LPI 10 -12 leaf tissues. Metabolites we re analyzed in pooled leaf tissues using GC-MS where chromatogram peaks were evaluated via thei r retention times and mass spectra as described in Morse et al. (2007). Construction and Experime ntal Design of Grafts Plants were grown under normal conditions unti l approximately 1 m in height and were then used as the source for rootstock and scion buds. A bud from scion material was attached to the rootstock with Parafilm approximately 5 cm above the soil surface. The lateral shoots that emerged from the rootstock after grafting were re moved to facilitate grow th of the scion. After the grafts took (approximately 2 weeks), Paraf ilm was removed as well as all plant material above the graft and leaves below the graft inducing the scion bud to break. The scion was maintained as the only growing stem on the plan t and measurements were taken regularly during growth. Grafting combinations were conducted such that each combination had three replicates (in some cases, one replicate was lost; these ar e noted). Grafting combinations are as follows (scion/rootstock): CwSUC2-2 /NT (n=2), CwSUC2-2 / CwSUC2-2 (n=2), CwSUC2-14 /NT, CwSUC2-14 / CwSUC2-14 (n=2), VacSUC2-20 /NT, VacSUC2-20 / VacSUC2-20 (n=2), VacSUC230 /NT (n=2), VacSUC2-30 / VacSUC2-30 NT/ CwSUC2-2 NT/ CwSUC2-14 NT/ VacSUC2-20 NT/ VacSUC2-30 NT/NT. Plants were randomized in the greenhouse and grown to a final height of approximately 1 m.

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82 Measurement of Photosynthesis and Respiration Photosynthesis parameters were measured on a ma ture leaf (LPI 12) from each plant of the grafted plants described previ ously. Light saturated net photos ynthesis was measured with a portable photosynthesis system (Li-6400, Li-Cor, Lincoln, NE) equipped with a red/blue LED light source under the following chamber conditions: chamber [CO2] 380 mol mol-1, photosynthetic photon flux density 2000 mol m-2 s-1, leaf temperature 18 to 22 C, vapor pressure deficit < 1.5 kPa, air flow rate 500 mol s-1. Measurements were recorded when the sum of the coefficients of variation for [CO2], [H2O], and flow rate for a 30 s running period dropped below 0.3 %. Immediately following the light-satur ated net photosynthesis measurement, a net photosynthesis versus leaf internal CO2 concentration (A-Ci) curve was generated by taking measurements at nine additional levels of reference [CO2] ranging from 50 mol mol-1 to 1800 mol mol-1, with all other chamber c onditions remaining constant. The parameters for maximum rate of carboxylat ion of Rubisco (Vcmax) and maximum rate of electron transport (Jmax) were derived from the A-Ci curves with a program (Photosyn As sistant, Dundee Sc ientific, Dundee, UK) that fits a model proposed by Farquhar et al. (1980) and modified by von Caemmerer and Farquhar (1981), Sharkey (1985), Harley and Sharkey (1991) and Harley et al. (1992). Parameters were adjusted to a standard temperature of 20 C using the methods described in Walcroft et al. (1997). Statistical differences were assessed by ANOVA using the GLM procedure of the SAS Version 8.0 statistical so ftware package (SAS Institute, Cary, NC). To measure foliage respiration, four biological replicates for each of the transgenic lines CytSUC2-18 and -50 and a NT control line were randomized in the greenhouse and grown to a final height of approximately 1 m on an ebb-and -flow flood bench system with a daily supply of Peters Professional 20-10-20 water soluble fe rtilizer diluted to a fina l concentration of 10 mM

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83 nitrogen. At noon, plants were moved to a dark growth chamber maintained at 35 C while respiration measurements were conducted. Measurements were re corded on both a sink (LPI 5) and source leaf (LPI 12) from each plant. Respiration was measured with the same photosynthesis system used for photosynthesis m easurements. The photosynthesis leaf chamber conditions were as follows: chamber [CO2] 380 mol mol-1, leaf temperature 34 to 36 C, air flow rate 200 mol s-1. Measurements were recorded when the sum of the coefficients of variation for [CO2], [H2O], and flow rate for a 30 s running period dropped below 0.3 %. Statistical differences were s ubjected to ANOVA using the Mixed procedure of the SAS Version 8.0 statistical software package (SAS Institute). Protein Extraction Poplar leaves were frozen and ground in liq uid nitrogen. Total protein was extracted by sonicating 300 mg ground tissue for 30 s in 1 ml of cold extraction buffer containing 50 mM Hepes-KOH, pH 7.4, 2 mM EDTA, 2 mM EG TA, 5 mM DTT, 100 M PMSF and 0.3 % DIECA. Supernatant was collected after centri fugation at 3,220 g for 20 min at 4 C and is referred to as the soluble fraction. The pellet wa s then washed four times with 5 ml extraction buffer and resuspended in 2.5 ml extraction buffer containing 1 M NaCl and incubated overnight at 4 C. Supernatant was collected after centri fugation at 3,220 g for 20 min at 4 C and is referred to as the insoluble fract ion. Soluble and insoluble fractions were desalted with extraction buffer using a 30 kD cutoff Centricon (Millipore, Billerica, MA) in order to concentrate protein, remove the NaCl from the insoluble fraction a nd remove the invertase inhibitor from both fractions. Proteins were quantif ied using a BCA Protein Assay Kit (Pierce, Bonn, Germany) as per manufacturers instructions for the microplate procedure.

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84 Total Invertase Activity Assay Total protein extracts were assayed for total invertase activity using a modification of the method of Arnold (1965). Briefly, pr otein extracts (25 l of solubl e or insoluble fractions) were added to 155 l of 60 mM phosphate-citrate buffe r, pH 4.5. Assays were brought up to 30 C and at time zero, 20 l of 1 M sucrose was added. Reactions were stopped at 30 min by adding 600 l of a modified Sumners r eagent containing 1 % (w/v) 3,5 di nitrosalicylic acid, 0.05 % (w/v) Na2SO3, 1 % (w/v) NaOH and 0.2 % (v/v ) phenol. Samples were then incubated at 95 C for 10 min and absorbance read at 540 nm. Detection of Invertase Activity in Native Polyacrylamide Gels SUC2 activity was detected using a native i nvertase activity gel assay (Gabriel and Wang, 1969). Briefly, protein extracts in 100 mM Tris-phosphate, pH 6.7, 0.1 % (w/v) bromophenol blue, 10 % (v/v) glycerol, and 0.1 % (v/v) Triton X-100 were loaded on a 10 % (w/v) polyacrylamide gel. The gel and running buffe rs were 100 mM Tris-phosphate, pH 6.7. After running overnight at 40 V and 4 C, gels were incubated in a 100 mM sucrose, 100 mM NaOAc, pH 5.0 solution for 30 min at 30 C. After a quick ri nse in distilled water, gels were developed by incubating in a 95 C solution of 500 mM NaOH containing 0.1 % (w/v) 2,3,5triphenyltetrazolium chloride, giving rise to re d bands at positions of invertase activity. Sugar and Starch Determination For soluble sugar (glucose, fructose and su crose) determinations, ground, frozen tissue samples were extracted using double distilled water and clarified via the Carrez method to remove proteins. Glucose, fructose and sucros e were calculated from the spectrophotometric measurement of NADPH producti on using a D-glucose/D-fructo se sugar assay kit as per manufacturers instruct ions (Boehringer Mannheim, Germany) Starch was solubilized from

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85 ground, frozen samples using dimethylsulfoxide and hydrochloric acid and assayed using a Boehringer Mannheim starch assay kit as per manufacturers instructions. Results and Discussion Construction of Overexpressing Yeast Invertase Vectors To further define the role of invertase in the different cellular compartments (apoplast, cytosol and vacuole) in poplar, I created three constructs designed to target and express the yeast invertase gene, SUC2, in those compartments via Agrobacterium -mediated transformation (Figure 4-1). A plasmid cont aining a cell wall targeted SUC2 was received through a generous donation from Uwe Sonnewald (Institut fr Bi ologie der Universitt Erlangen-Nrnberg, Erlangen, Germany). This construct contains a fusion of SUC2 (nucleotides 64-1765) (Taussig and Carlson, 1983) with the signal peptide of potato proteinase i nhibitor II (nucleotides 1-230) (Keil et al. 1986). The gene fusion was PCR amplified and recombined into an overexpression vector using the Gateway (Invitrogen) cloni ng system and is hereafter referred to as CwSUC2 In order to create a vacuolar targeted SUC2 gene fusion, the vacuolar targeting domain from tobacco chitinase (Neuhaus et al. 1991) was fused to the C-terminal end of the CwSUC2 construct and is hereaf ter referred to as VacSUC2 To direct accumulation of SUC2 in the cytosol, the coding region of SUC2 was PCR amplified without any signal peptide from the original CwSUC2 construct and subsequently recombined into the overexpression vector and is hereafter referred to as CytSUC2 All constructs were driven by two, identical constitutive CaMV 35S promoters linked in tandem and terminated with the OCS terminator (Figure 4-1). Yeast invertase was used in these co nstructs for three reasons: first, SUC2 is known to be active over a broad pH range (Goldstein and La mpen, 1975), which is important considering the pH differences between the acidic vacuole and apopl ast as compared to the more neutral cytosol. Secondly, this invertase is not inhibited by the en dogenous invertase inhibitors present in plants

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86 (Greiner et al. 1998; Greiner et al. 1999; Link et al. 2004; Huang et al. 2007). Lastly, SUC2 has been expressed in a variet y of plants to date including Arabidopsis (Von Schaewen et al. 1990), potato (Heineke et al. 1992), tobacco (Von Schaewen et al. 1990; Sonnewald et al. 1991; Ding et al. 1993; Sonnewald et al. 1993; Tomlinson et al. 2004) and tomato (Dickinson et al. 1991), with sign ificant results. The two major sub-families of plant invertases are the acid and neutral/alkaline invertases. The acid invertases are thought to have evolved fr om respiratory eukaryotes and aerobic bacteria (Sturm and Chrispeels, 1990a) while the neutra l/alkaline invertases are thought to have originated from cyanobacteria (Vargas et al. 2003). SUC2 coming from yeast, a eukaryote, indicates that SUC2 is more closely related to the plant acid invertase sub-family than the neutral/alkaline sub-family. The acidic pH optim um for activity of SUC2 also supports this conclusion. To further address this, amino aci d alignments of SUC2 with endogenous poplar invertases were examined (Figure 4-2A). Additionally, a protein similarity tree was constructed using full length sequences of invertases included from Arabidopsis carrot, corn, poplar, potato, tomato, various micro-organisms, as well as leva nase and inulinase (related enzymes provided as outliers) (Figure 4-2B). These data indicate that although SUC2 may be evolutionarily more closely related to the acid invertases than the neutral/alkaline invertas es, amino acid sequences have diverged such that SUC2 se quence is as different from the aci d invertases as it is from the neutral/alkaline invertases (Figure 2B). This lack of sequence sim ilarity from the plant invertases is likely the reason behind the lack of SUC2 inhibition from the endogenous plant invertase inhibitors. Expression of SUC2 in Poplar In order to verify the constitutive nature of the double 35S ( 2x35S ) promoter in poplar, I analyzed expression of the 2x35S::SUC2 construct in three different organs with RT-qPCR.

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87 Plants representing three separate transgenic events were tested in order to average out any line specific effects. I found that the transgene driven under the double 35S promoter is expressed at highest levels in leaf (Figure 4-3). Expression levels of th e transgene in root and stem are approximately 50 % of the level of expression of that in leaf (Figur e 4-3). RNA from leaf material was extracted and transgene expression leve ls analyzed in order to identify the highest expressing lines of each cellular compartment dir ected construct (Figure 4-4). The two highest expressing lines from each compartment were then selected for further experimental analysis. Yeast Invertase Active in Cytosol To test that the accumulation of SUC2 transcripts translate to increases in invertase activity, total protein was extrac ted and fractionated according to solubility from leaf. The insoluble fraction yielded no detectable increase in total invertase activity in any of the transgenic lines examined (dat a not shown). In the soluble fr action, as much as two fold increases of total invertase activity were detected in the CytSUC2-18 line (Figure 4-5A), but, unexpectedly, not in any of the VacSUC2 or CwSUC2 constructs (data not shown). This indicates that the SUC2 transcript accumulation seen in the cell wall and vacuolar targeted constructs either did not translate into incr eased activity or that the increase occurred in the wrong cellular compartment. One explanation for the lack of total invertase activity increases in the CwSUC2 and VacSUC2 transgenic lines could be th at the endogenous plant invert ases were down-regulated in order to compensate for the exogenous increase s in invertase activity. To address this, an invertase assay was employed that does not detect plant invertase activit y, but can detect SUC2 activity (Sonnewald et al. 1991). Total protein extracts from the insoluble and soluble fractions were separated by electrophoresis on a non-denatu ring polyacrylamide gel and then assayed for activity (see Materials and Methods ). Activity was detected only in the soluble fr action of lines

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88 CytSUC2-18 and -50 (Figure 4-5B). This indicates that the total soluble invertase activity increases seen in Figure 4-5A are a result of the expression of ectopic SUC2 This also indicates that the lack of activity seen in the cell wall a nd vacuolar constructs is due to the lack of an active form of SUC2 in the targeted compartmen t and not necessarily due to down-regulation of plant invertase activity. Metabolic Profiling Reveals Alterations in CwSUC2 and VacSUC2 Transgenic Lines To determine whether SUC2 expression altered endogenous leve ls of sugars and any of the metabolic pathways they feed, leaf tissue from three biological replicates from each of the selected transgenic lines and NT controls we re subjected to metabol ite analysis using gas chromatography-mass spectrometry (GC-MS) with electron impact ionization. As was expected due to lack of detectable i nvertase activity alterations, the CwSUC2 and VacSUC2 transgenic lines showed no significant change s in the levels of sucrose, gl ucose or fructose. Surprisingly, 12 metabolites were found to be significantly altered in at least one of those transgenic lines (Table 4-1). For three of those metabolites, the exact stru ctures could not be determined and are referred to by their retention index followed by their key mass/charge ( m/z ) ratios. Many of the other nine metabolites can be found in, or are associated with, both the glyoxylate and TCA cycles. This would indicate that even though I could not detect an increase in invertas e activity, metabolic shifts were still occurring in th ese transgenic poplars. If the glyoxylate cycle is indeed stimulated in the CwSUC2 and VacSUC2 transgenic lines, this could account for my inability to detect shifts in glucose and fructose as one end product of the glyoxylate cycle is gl ucose. As the glyoxylate cycle occurs in specialized peroxisomes calle d glyoxysomes, it is possible that the metabolic consequences of SUC2 expression are restricted to cellular compartments that represent a small proportion of the total metabolite pool measured in these studies. One possi ble way to test this

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89 hypothesis would be to isolate pe roxisomes and evaluate metabol ic shifts in these specific compartments. Metabolic Profiling Reveals Altera tions in Sugar Accumulation in CytSUC2 Transgenic Lines As would be expected from the increased invertase activity that accumulated in the CytSUC2 transgenic lines (Figure 4-5), metabolite anal ysis revealed alterati ons in the levels of sucrose as well as its hydrolysis products: glucose and fructose (T able 4-2). The transgenic line containing the greatest increas es of invertase activity ( CytSUC2-18 ) also expressed the greatest reduction in sucrose levels by nearly 30 %. CytSUC2-50 also revealed reduced levels of sucrose (20 % reduction), however this was not found to be a statistically significant shift and could be explained by the lower levels of invertase activity evident in line -50 compared to line -18 (Figure 4-5). One would expect that increases in invertase activity would result not only in lower levels of sucrose, but also in increas ed levels of glucose and fructo se since these compounds would be produced by sucrose hydrolysis. Surpri singly, this was not the case for the CytSUC2 lines where analyses of both -18 and -50 revealed reductions in glucose a nd fructose levels (Table 4-2). Interestingly, the greatest decrease s in hexose abundan ce occurred in line -50 which had the lowest invertase activity of the two lines. This may indicate that sucros e levels are directly related to alterations in invert ase activity, whereas observed he xose reductions are indirectly linked to invertase activity. Also of note are the 50 % reductions in malic and glyceric acid levels in the two CytSUC2 lines (Table 4-2). One possible explanation for the reductions in hexoses as well as glyceric and malic acids may lie in increased re spiration rates. To test this hypothesis, CytSUC2-18 and -50 plants were propagated and grown along with NT control pl ants with a high nutrient fertilizer in a

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90 greenhouse. Plants were then moved to a warm (35 C) growth chamber and kept in the dark while leaf respiration rates we re measured using a portable p hotosynthesis system from both source (LPI 12) and sink (LPI 5) leaves. Re spiration rates remained unchanged between the CytSUC2 lines and the NT control plants (data not shown). Whole-Plant Phenotypes Are Not Apparent Phenotypes of clonally propagated SUC2 and NT plants were compared at whole-plant, organ and molecular-levels (Table 4-3). I det ected no differences in propagation efficiency, growth rate, plant architecture or plant size when grown under controlled greenhouse conditions (data not shown). One strategy to increase whole-plant differen ces was to graft transgenic material onto non-transgenic. This al lows me to specifically target SUC2 expression to the root and/or crown of the tree thereby increasing the sink of the roots wh ile not affecting the ability of the source organs from photosynt hesizing and exporting sucrose. CwSUC2 and VacSUC2 lines (scion) were grafted onto NT rootstock as well as NT plants (scion) onto CwSUC2 and VacSUC2 rootstock. Control grafts were also performed fo r all lines (transgenic on to itself, and NT onto itself) and whole plant phenotypes including growth rates, plant ar chitecture and plant size were measured as well as photosynthetic rates. In all cases, the transgene was found to have no detectable effect relative to the NT co ntrol (data not shown). Unfortunately the CytSUC2 plants were excluded from this experiment due to prob lems in propagation that were unrelated to the transgenic construct per se Concluding Remarks My results indicate that it was possible to successfully transform poplar with the yeast invertase gene, SUC2 In contrast to the many other plant sp ecies transformed with this gene (see Introduction) I saw no whole-plant altering phenotype s. This is likely due to the relatively low level of induction as compared to what was s een in other plant species In the transformant

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91 containing the highest SUC2 activity ( CytSUC2-18 ), I saw approximately a two-fold induction in total activity (Figure 4-5A). In contrast, tob acco and potato transgenics expressing the same SUC2 gene saw as much as 50and 100-fold increases in activity, respectively (Sonnewald et al. 1991; Heineke et al. 1992). Even with the modest increases in invertase activity seen in poplar, a subtle metabolic phenotype could be detected in transgenic lines CytSUC2-18 and -50 While the reduction in sucrose is to be exp ected, the reduction in glucose and fructose is counter-intuitive. Increased respiration could be one explanation of the decrease in hexoses I observed. This hypothesis would also be supported by the more dr amatic reductions observed in malic acid. Respiration was measured, but no significant ch anges were detected. This may simply be because respiration was not altered significan tly enough to be measurable. Alternatively, respiration may not have been altered at all, in which case the metabolic shifts observed may have resulted from some other metabolic pathway. The SUC2 constructs targeted to the cell wall and vacuole pose a conundrum. While transcripts were detected for the CwSUC2 and VacSUC2 transgenes, invertase activity was not detected either in the total activity assay or in the native gel assay. This would seemingly indicate that the transcript simp ly did not result in detectable levels of mature, active protein. However, a metabolic phenotype was evident in th ese transgenics. Since no increase in total activity was found, one potential explanation for th is metabolic phenotype may reside in altered workloads of endogenous invertase to exogenous yeast invertase. The increase in activity resulting from the transgene may have led to a compensatory down-regul ation of the plants endogenous invertase activity. Mini mal changes to sugar levels would thus have been likely. The lack of detectable activity in the native ge l makes this a tenuous hypothesis, however. This hypothesis could only be correct if the yeast invertase underwent so me sort of post-translational

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92 modification in the cells secretory system rende ring the yeast invertase in active in the native gel assay, much like the undetectable plant invertases. If this were true, the in vivo increases in activity must be quite small. Collectively, my data indicate that the transgenic poplar lines did give rise to metabolic phenotypes, but that thes e were not straightforward to interpret. Also, these transgenics did not show detectable w hole-plant phenotypes pot entially due to the relatively subtle increases of SUC2 dependent inve rtase activity.

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93 SP SUC2 Vacuolar targeted invertase Chitinase VTD 35S 35S ATG TAG SP SUC2 Vacuolar targeted invertase Chitinase VTD 35S 35S ATG TAG SP SUC2 Cell wall targeted invertase 35S 35SATG TAG SP SUC2 Cell wall targeted invertase 35S 35SATG TAG SUC2 Cytosolic targeted invertase 35S 35S ATG TAG SUC2 Cytosolic targeted invertase 35S 35S ATG TAG Figure 4-1. Schematic representation of yeast invertase overexpressing constructs targeted to the cytosol, cell wall and vacuole. Block arrows represent 35S promoter, solid black box represents SUC2 coding region, brick filled box represents the signal peptide (SP) take n from potato proteinase inhibitor II, cross-hatched box represents the vacuolar targeting domain (VTD) taken from tobacco chitinase.

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94 A PtVIN2 1 WQRTAFHFQ P EEN WMNDPNG P LY YKG--W YHFFYQYNP HAA VW G D I V WGHA V S K DLI H W PtCIN1 1 VHRTGYHFQ P PRH WINDPNA P MY YKG--L YH L FYQYNP KGA VW G N I V WAH SV S K DLI N W PtNIN2 1 -------------AA NDP GDK M PLNY--DQV F VRD F V P S----A LAFLLR G -E GE I VK PtNIN10 1 -------------AAM D TSADALNY---NQV F VRD F V P T----G LACLMKEPP E PE I VR S_cerSUC2 1 --------P NKG WMNDPNG LW Y DEKDAK WH L YFQYNP NDT VW GTP L F WGHA T SDDL TN W PtVIN2 58 LH L PL AM VADK W YDKN G V WTGS AT ILP DGKIVMLYTGSTN-E S VQ V Q N LAYPADHD D PL L PtCIN1 58 ES L EP AI YPSK W FDNY G C WSGS AT VLP NGEPVIFYTGIVDKNNSQ I Q N YA V PANLS D PY L PtNIN2 39 NF L LH AL QLQS W EKTVDC YS PGQG LMP --------------A S FK V RTVP LD DNNL E EV L PtNIN10 40 NF L LKT L HLQGLEKRVDN FT LGEG VLP --------------A S FK V ---LY D SDLEKET L S_cerSUC2 52 EDQPI AI APK--RNDS G A FSGS MV V DYN-------------N T SGFF N DT ID PRQRCVA I PtVIN2 117 LK W VKY SG NPV L V P PPGIGAKDFRDPTTA W KTSE G K W R III GSKIN K T G IALV Y DTEDFI PtCIN1 118 RE W VKPDDNPI V N P DANVNGSAFRDPTTA W WA D G-H W R ILI GSRRKHR G VA Y L Y RS K DFK PtNIN2 85 DPDFGE SA IGR V A P ----------------VD S G L WWIIL L R AYG K LTGD Y ALQ ER --PtNIN10 83 LVDFGA SA IGR V A P ----------------VD S G F WWIIL L R SYI K RTRD Y ALL DR --S_cerSUC2 97 WT Y NTPESEEQYIS--------------Y S LD G G YT F TEYQ K NPVLA A NSTQ F R D PKVF PtVIN2 177 N Y ELLSGI L HGVP KT G MW E CV D F YPV S KTGQNGL D T S VNGPQ V KH V IKTSLD DDRH DYY PtCIN1 177 K W VKAKHP L H S VQG T G MW E C PD F YPV S LSGENGL D P S V M GQN V KH V LKVSLDM TRY EYY PtNIN2 125 ------VD V Q TG I K LI L NL CL ADGF D MFP S LLVT DGS C M IDRRMG I HGH P LEI QALF Y S PtNIN10 123 ------PE V QN G M K LI L KL CL SDGF DT FP T LLCA DG CS M IDRRMG I YGY P IEI QALF YF S_cerSUC2 142 W Y EPSQKW I M TA A KS QD Y KIEI Y SS D DLK S WKLES A FANEGF L GYQYEC P G L IEVPT E QD PtVIN2 236 AL G TY ADK V GKWY P DNPE ID VGIGIRYDYGIFYA S KT F Y D Q SK GRR V LWG W IG E SDSEVA PtCIN1 236 T M G TY DKKKDKYF P DEGL VD GWAGLRLDYGNFYA S KT F F D P S TNRR I LWG W AN E SDDPQK PtNIN2 178 ALRS SRE ML VVNDGSKNL V RAINNRLS A LSFHIREYY W V D MR KI NE I Y R YKTE E Y S TEAT PtNIN10 176 ALR CAKQ ML KPELDGKEF IE RIEKRIT A LSYHIQ T YY W L D F T Q L NN I Y R YKTE E Y S HTAV S_cerSUC2 202 PS KSY WV M FISIN P GAPAGGSFNQYFV G SFNGTHFEA F DNQ SRV VDFG K D Y YALQ T FFNT PtVIN2 296 D VKK G W A SLQG IP RT V VLDTKTGS N L L QWP V EE V ESLRLKSKNF NN IE V KAGSAV P LE L D PtCIN1 296 DK DK G W A GIQL IP RK V WLDP SGK Q L L QWP V AE L EKLRGHNVQLS N QM L DQGNHVEVK V I PtNIN2 238 N K FN--IYPEQ IP SW L MDWI P EEGGY L IGN L QPA H MDFRFFTLG N LWS V VSSLGT P KQNE PtNIN10 236 N K FN--VIPES IP DW V FDFM P LRGGY L IGN V SPA R MDFRWFLVG N CVA I LSSLVT P AQAT S_cerSUC2 262 D PTY G S A LGIAWASNWEYSAFVPT N PWRSS M SL VR KFSLNTEYQA N PETELINLKAEP I L PtVIN2 356 G AT QL DIVAE FEL D----RKAIERTAESN V EFSCSTNG G ASH R GA LG PFGLLVLADDD L T PtCIN1 355 TAA Q ADVDVT F SFSSLDKAEPFDPKWAKLDA L DVC A QK G SKDPGG LG PFGLL T LA S EN L E PtNIN2 296 AV L NL IESKWD DL VG-----------NMP L K I CYP A LESEDW R I I T G SDPKN T PW S YHNG PtNIN10 294 AI MD L VEERWE DL IG-----------EMP L K I TYP A LE G HEW R L V T G FDPKN T RW S YHNG S_cerSUC2 322 N I S N AGPWSR F ATNT-------TLTKANSYN V DLSNST G TLEFE LV YAVNTTQTI S KS V F PtVIN2 412 EYT PVYFFV AK G NNGS L KTFFCTDQ S RSSVAND V R KE I Y GS Y VPVLE G EK L S----V R IL PtCIN1 415 EFT PVFF R V FK AA DK H KVLLCSDA RS SS LG KELYKPSFAGFVDVDL T D K K L S----L RS L PtNIN2 345 G SW P TLL W QFTL A CM KM DRMELAQ K AIA LAE KR L QV D H W PE Y YDTR SGK F I G----KQ S R PtNIN10 343 G SW PM LL WL LS AA CI KV GRPQIAK R AIE LAE QR L S KD G W PE Y YDGK TGR Y V G----KQAR S_cerSUC2 375 A DLS LWF KGLEDPEEY L RMGFEVSA S SFFL D RGNS K VK F VKENPYF T N R MSVNNQPF KS E

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95 PtVIN2 468 VD H S IIES FA QG GR TC I TSRVYP T RA IY GSA R LF L FN NAT -EAGV TS S L KIWN M NSAFIR PtCIN1 471 ID H S VVES FG AG GRI A I SSRVYP TI A VFE KA H LY V FN NGS -ETI T VKN L NAWS M NTPVMN PtNIN2 401 L YQ TW TVAGF L TS KVLLE NPEKA SLL F WD EDYDL L EFCVC-GL NTS GRKRCSR V AARSQI PtNIN10 399 KYQ TW SIAGY L V AKMMVE NPSNLL MI SL E ED K KSARSRL T -RS NSTS F-----------S_cerSUC2 435 N D L SY YKV YGL LDQN ILE LYFNDGD VV STNTYFMTTG NA LGSV N M TT G V DNLFYIDKFQV PtVIN2 527 PYSNEQQ PtCIN1 530 VPVKS-PtNIN2 460 LV----PtNIN10 ------S_cerSUC2 495 REVK--B tomatoTIV1 tomatoTIV1 AT4G09510 AT4G09510 AT1G35580 AT1G35580 PtNIN12 PtNIN12 PtNIN8 PtNIN8 AT1G22650 AT1G22650 AtcwINV1 AtcwINV1 AT3G06500 AT3G06500 Inulinase Inulinase carrotInv carrotInv Dc4 Dc4 PtVIN3 PtVIN3 AT1G72000 AT1G72000 AtcwINV6 AtcwINV6 maizecwIVR4 maizecwIVR4 AT4G34860 AT4G34860 PtNIN9 PtNIN9 PtNIN11 PtNIN11 maizecwIVR2 maizecwIVR2 maizecwIVR3 maizecwIVR3 PtCIN5 PtCIN5 PtCIN4 PtCIN4 PtCIN2 PtCIN2 PtCIN1 PtCIN1 PtCIN3 PtCIN3 maizecwIVR1 maizecwIVR1 tomatoLin8 tomatoLin8 tomatoLin7 tomatoLin7 carrotInv carrotInv Dc3 Dc3 carrotInv carrotInv Dc2 Dc2 carrotInv carrotInv Dc1 Dc1 AtcwINV3 AtcwINV3 AtcwINV5 AtcwINV5 tomatoLin5 tomatoLin5 potatoIVRGE potatoIVRGE tomatoLin6 tomatoLin6 S cerevisiaeSUC2 S cerevisiaeSUC2100 100 100 100 100 100 93 93 100 100 100 100 I I III III II II IV IV Figure 4-2. S. cerevisiae SUC2 in relationship to other plant invertases. A ) Amino acid alignment of SUC2 and se lect poplar invertases. Black shading corresponds to identical residues, grey shading corr esponds to similar residues. Alignment produced with CLUSTALW and fi gure produced with BOXSHADE. B) Amino acid similarity tree using full length sequences. Depicts phylogenetic relationship of S. cerevisiae SUC2 (in bold) with selected invertases from Arabidopsis carrot, corn, poplar, potato and tomato. Invertases from various micro-organisms are also included along with the closely related enzymes, levanase and inulinase, provided as outli ers. Clades I-IV represent vacuolar, cell wall, -neutral/alkaline and -neutral/alkaline invertases, respectively. and designations of the neutral/alkalin e invertases represent two distinct clades as described by Bocock et al. (2007). Bootstrap values of the major clades are depicted and reported as a percentage of 1,000 repetitions. Branch lengths denote protein similarity.

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96 Double 35S driven SUC2 expression0 20 40 60 80 100 120 140 rootstemleafOrganRelative transcript (%) Figure 4-3. Relativ e expression of double 35S driven SUC2 in root, stem and leaf. Bars represent the mean level of SUC2 transcript across three separate transgenic events; error bars denote standard error, n=3.

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97 A Relative expression of CytSUC20 20 40 60 80 100 120 18501622125172831126936NT19 Transgenic lineRelative transcript (%) B Relative expression of CwSUC20 20 40 60 80 100 120 140 21425262811310NT Transgenic lineRelative transcript (%) C Relative expression of VacSUC2-20 0 20 40 60 80 100 120 140 160 3020291521382736242534NT7 Transgenic lineRelative transcript (%) Figure 4-4. Relative expression of SUC2 transcript in overexpre ssing transgenic events in leaf. A) Cytosolic targeted construc t. B) Cell wall targeted construct. C) Vacuolar targeted construct. In all panels, bars represent mean level of SUC2 transcript; error bars denote sta ndard error when available, n=3.

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98 A Total invertase activity (soluble fraction)0 20 40 60 80 100 120 140 NTCyt-18Cyt50 Transgenic lineActivity (mol suc hydrolyzed min-1 mg protein-1) B Cyt18-1 Cyt18-2 Cyt50-1 Cyt50-2 NT NT Cyt18-1 Cyt18-2 Cyt50-1 Cyt50-2 NT NT ISS Cyt18-1 Cyt18-2 Cyt50-1 Cyt50-2 NT NT Cyt18-1 Cyt18-2 Cyt50-1 Cyt50-2 NT NT ISS Figure 4-5. CytSUC2 transgenic plants di splay increased invertase activity due to presence of transgene. A) Soluble prot eins were extracted from a source leaf (LPI 12) and assayed for invertase acti vity. Bars represent mean invertase activity, error bars denote standard error, n=2. B) Pr otein extracts (20 g total protein each lane) were separated us ing non-denaturing gel electrophoretic conditions and assayed for activity.Top panel: No invertase activity is detected in NT plants or in the insol uble fraction (IS). Activity can be seen (red bands) in the soluble fraction (S) of CytSUC2 lines -18 and -50 in both biological reps. Bottom panel: Coomassi e stain of gel to show loading.

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99 Table 4-1. Metabolites altered in CwSUC2 and VacSUC2 overexpressing plants expressed as a ratio of g/g FW (glucose equivalents) of transgenic/NT controls. VacSUC2-20/NTVacSUC2-30/NTCwSUC2-2/NTCwSUC2-14/NT 5 Carbon-sugar alcohol 1.08 1.18*1.17*1.18Iditol 1.11 1.29*1.21* 1.16 Glucoside (m/z 434) 1.08 1.551.211.37 16.25-273 (Unknown compo und) 0.740.56 0.520.45alpha-keto-Glutaric acid 1.111.27 1.63*2.43* Maleic acid 1.131.25 1.42* 1.33 Citric acid 1.211.25 1.39* 0.79 10.79-184 (Unknown compo und) 1.08 0.58*0.35*0.28* Fumaric acid 1.432.48 1.58* 1.32 Serine 0.880.870.89 0.69* Carbamoyl-phosphate 0.97 0.620.35*0.35* 15.7-221 327 (Unknown compo und)0.5*0.62*0.51*0.42* *=significant at 0.05 =significant at 0.10

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100 Table 4-2. Metabolites altered in CytSUC2 overexpressing plants e xpressed as a ratio of g/g FW (sorbitol equivalents) of transgenic/NT controls. CytSUC2-18/NTCytSUC2-50/NT Fructose0.83*0.80* Sucrose0.71* 0.82 Myoinositol0.68* 0.96 Glucose 0.75 0.70Galactose0.650.61Glyceric acid0.520.57Malic acid0.520.52*=significant at 0.05 =significant at 0.10

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101 Table 4-3. Summary of phenot ypic experiments performed. Trait TestedPlants TestedSignificant Changes FoundPCR verification of transgeneCyt, Cw, VacCyt, Cw, Vac Transcriptional changesCyt, Cw, VacCyt, Cw, Vac Invertase activityCyt, Cw, VacCyt Sugars (fructose, glucose, sucrose) (root, stem, leaf)Cyt, Cw, VacCyt (leaf) MetabolitesCyt, Cw, VacCyt, Cw, Vac RootabilityCyt, Cw, Vacno Growth ratesCyt, Cw, Vacno Starch (leaf, root and stem)Cyt, Cw, Vacno RespirationCytno Growth rates after graftingCw, Vacno Photosynthetic rates after graftingCw, Vacno

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102 CHAPTER 5 CONCLUSIONS As a first step in examining the role inve rtase plays in carbon allocation and partitioning, the invertase family was identified in Populus trichocarpa a woody, perennial tree species whose genome was recently sequenced (Tuskan et al. 2006) (Chapter 2). The identification of eight acid invertase genes; three of which belong to th e vacuolar targeted group ( PtVIN1-3 ), and five of which belong to th e cell wall targeted group ( PtCIN1-5 ) is described. Similarly, I report the identification of 16 neutra l/alkaline invertase genes ( PtNIN1-16 ). Expression analyses using whole genome microarrays and RT-PCR revealed evid ence for expression of all invertase family members. Evidence is also reported for expres sion of a novel intronless vacuolar invertase ( PtVIN1 ), which apparently arose from a processed PtVIN2 transcript that re-inserted into the genome. An examination of the microsyntenic regions surrounding the p oplar invertase genes revealed extensive colinearity with Arabidopsis invertases, indicati ng strong evolutionary conservation in the development of the invertase family between the two species. To further determine if the evolutionary conservation of the invertase fa mily, demonstrated by conserved colinearity, extended to the functional level, vac uolar invertases were se lected as a case study (Chapter 3). Vacuolar invertases have a well documented reciprocal response to sugar treatment in which one invertase is up-regulated while the other is down-regulated af ter sugar treatment in maize (Xu et al. 1996). The conserved, colinear struct ure of the chromosomal regions containing these vacuolar invertase genes in Arabidopsis and poplar did not extend to maize and rice. However, the reciprocal response was f ound to occur in all f our plant species. One explanation for this is that th e function of these genes has be en conserved throughout evolution. An alternative hypothesis is that th is reciprocal response is an exam ple of parallel evolution that arose independently in the different plant lin eages. Finally, invertase activity in poplar was

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103 manipulated with transgenesis by ectopically expressing yeast invertase in three cellular compartments (apoplast, cytosol and vacuole) (Cha pter 4). This doubled inve rtase activity in the cytosol, but did not alte r invertase activity in other cellular compartments. Subtle shifts in carbon partitioning within the cell were observed, but no detectable alterations in carbon allocation between tissues. Specific invertas e genes identified in Chapter 2 we re also targeted for repression via RNAi (Appendix A). The overall objective of determining contribu tions by invertase to carbon allocation and partitioning in a woody perennial was met in the following ways: Poplar contains three vacuolar invertases, one of which is a novel, intronless gene found only in poplar. Poplar contains five cell wall invertases. Poplar contains 16 neutral/alkal ine invertases that clearly di vide into two, structurally distinct groups and represents a significan t expansion in gene number over that of Arabidopsis and rice. Poplar and Arabidopsis acid invertases share substantial similarities in their chromosomal arrangement indicating that chromosoma l duplication can explain much of the development of the invertase family. Poplar and Arabidopsis vacuolar orthologs identified thr ough shared colin earity both retain a conserved, reciprocal response to exogenous sugar treatment that is also found in the monocots, maize and rice. Invertase activity in poplar can be increased through the introduc tion of exogenous yeast invertase. An increase in invertase activity shifts car bon partitioning within the cell. This research has opened up seve ral new questions that would be valuable to be addressed in future research. The first question is to addres s how invertase fits into the hypothesized lack of active phloem loading model proposed by Turge on and Medville (1998) for poplar and similar species. One possibility could be that the expans ion of the neutral/alkalin e invertase sub-family

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104 in poplar may contribute to the movement of photoassimilates through the mesophyll cells and into the phloem. A second question that arises from this research deals with the significance and development of the intronless PtVIN1 It is unknown when in popl ars evolutionary history PtVIN1 arose. When this question is addressed, it may also help us to understand the function of this intronless gene. The third question deals with the activity of th e cell wall invertase sub-family members. It has recently been reported that tw o of the six cell wall invertases in Arabidopsis may not be invertases, but rather fructan exohydrolases (FEHs) (De Coninck et al. 2005). FEH protein sequences are nearly identical to those of demonstrated acid i nvertase proteins, but FEH does not use sucrose as a substrate (De Coninck et al. 2005). It is likely that at least one poplar cell wall invertase is in fact an FEH, but analysis of recombinant cell wall invertase activity was outside the scope of research presented here. The fourth question is why the poplars ectopical ly expressing yeast invertase lack wholeplant phenotypes. Several groups overexpressed yeast derived invertase in the apoplast of Arabidopsis (Von Schaewen et al. 1990), potato (Heineke et al. 1992), tobacco (Sonnewald et al. 1991) and tomato (Dickinson et al. 1991). Sonnewald et al. (1991) also overexpressed yeastderived invertase in the vacuolar and cytoso lic compartments of tobacco. In all cases, overexpression resulted in numerous growth defect s that included stunted growth, inhibition of photosynthesis, accumulation of leaf starch and formation of necr otic lesions on leaves followed by overall yellowing of the leaf (Von Schaewen et al. 1990; Dickinson et al. 1991; Sonnewald et al. 1991; Heineke et al. 1992). The lack of phenotypes might be due to the relatively modest induction of activity (two-f old) as compared to these other pl ants. This may have resulted from

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105 inadvertent selection and propagation of the mo st vigorous transgenic plants, and hence those least markedly altered. Fifth, and finally, valuable work could be pe rformed on poplar plants with a reduction in invertase activity. As describe d in Appendix A, transgenic poplars were produced encoding constructs designed to repress specific endoge nous invertase genes through the use of RNAi. These plants have been confirmed to express the transgene, howeve r they have not been screened for reduced expression of the targeted invertase gene or even a reduction in overall invertase activity. The phenotype of these lines is normal and thus the cost of intensive phenotypic screening must be weighed against the likely benefit of the approach. The purpose of this research was to investig ate invertase contributi ons to carbon allocation and partitioning in a woody perennial. To achieve this goal, the invertase family was identified and annotated using the sequenced genome of Populus trichocarpa Several novel features were identified in the poplar invertase family. These include a three-member vacuolar invertase subfamily, an expressed, intronless v acuolar invertase as well as an expansion of the neutral/alkaline invertase family relative to that of Arabidopsis and rice. The response of two poplar vacuolar invertases ( PtVIN2 and -3 ) to exogenous sugar treatments was examined. A reciprocal regulation of transcript first documented in maize was also found to occur, similar to that of Arabidopsis and rice. A reverse genetic appr oach was also employed by ectopi c expression of yeast invertase in transgenic poplar resulting in a subtle sh ift in carbon partitioning as shown by metabolic analyses. In conjunction, RNAi was used to target endogenou s poplar invertases for downregulation.

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106 APPENDIX A REPRESSION OF INVERTASE IN POPULUS Introduction The cell wall and vacuolar invertases have been repressed in several plant species, including tomato (Ohyama et al. 1995), potato (Zrenner et al. 1996), and carrot (Tang et al. 1999). The results from these experiments were more varied than those of the invertase overexpressors, although the theme of increased sucrose content accompanied by a decrease in hexoses pervaded. In tomato these alterations in sugar accumulations appeared in both the fruit and leaves. In addition to altered sugar levels, the fruit also had elev ated rates of ethylene evolution. In carrot, growth was shown to be alte red at very early stages of development. The transgenic, cotyledon-stage embr yos were still masses of cells while the control plants had already developed two to three leaves and one primary root. Ho wever, when these plantlets were grown on media supplemented with hexoses, their growth returned to normal. After maturation and transfer to soil, the plan ts expressing the cell wall anti-s ense cDNA had a significantly increased shoot to root ratio and accumulated mo re sucrose and starch than the controls. The vacuolar anti-sense plants had increased numbers of leaves, while tap root size was slightly decreased. Here I describe the design of vectors aime d at endogenous invertase repression via RNAi in Populus a deciduous, perennial tree species. While the previously mentioned work has done much to establish the role of invertase in carbon allocation and pa rtitioning, it is still unknown what roles invertase may play in a deciduous, perennial plant system. The deciduous, perennial nature of poplar requires numerous cycles of sugar movement and carbon sequestration in storage organs such as the root and stem in th e winter, followed by remobilization of these stored carbon compounds the following spring. It is al so thought that poplar may employ a unique

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107 mode of sucrose movement into the phloem of source leaves analogous to that of poplars closely related cousin, willow (both members of Salicaceae ) (Turgeon and Medville, 1998). These added levels of complexity that can be se en by utilizing the model tree, poplar, may offer us new insights into the function of invertase. Materials and Methods Plant Material, Transgenes is and Growth Conditions Hybrid poplar clone, INRA 717-1-B4 ( P. tremula x P. alba ) was placed into sterile culture prior to Agrobacterium -mediated transformation (Leple et al. 1992) on media supplemented with glucose and fructose. Indivi dual clones from independent lines were clonally propagated as softwood cuttings under mist, transferred to 8 L pots and grown to a height of 60-100 cm prior to experimentation in a fanand pad-cooled greenhouse with natu ral light augmented with full spectrum fluorescent lighting during the winter to give a day length of 15 h (Lawrence et al. 1997). Greenhouse temperatures ranged from 20 -35 C. At noon, the light intensity in the greenhouse averaged 500-700 E/m2/min PAR, which is one-half th e light intensity outside the greenhouse. Plants were grown on an ebb-and-flow flood bench system with a daily supply of Peters Professional 20-10-20 water-soluble fer tilizer diluted to a final concentration of 4 mM nitrogen. Vector Construction The pCAPT binary vectors for RNAi was constructed as described by Filichkin et al. (2007). Briefly, the vector was constructed us ing the pART27 backbone (Gleave, 1992). The GATEWAY Conversion System (Invitrogen) was used to incorporate the proper recombination sites and genes for positive and negativ e selection. This consists of an attR recombination site flanking a ccdB gene and a chloramphenicol resistance ge ne. The vector contains spectinomycin and kanamycin ( NPTII ) resistance genes for selection in bacteria and plants, respectively.

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108 Approximately 200 bp target fragments of acid i nvertase genes from poplar (primer sequences listed in Table A-1) were PCR amplif ied using primers with tails encoding attB recombination sites. GATEWAY entry clones were created using BP Clonase (Invitrogen) mediated recombination. The entry clones were finally clon ed into the pCAPT binary vector using LR Clonase (Invitrogen) acco rding to manufacturers instructions All constructs were sequenced. Genomic DNA Isolation and PCR Amplification To clone target gene sequences and transg ene presence, genomic DNA was isolated from poplar shoot tip tissue. Approximately 250 mg of tissue was ground in liquid nitrogen and added to a buffer containing 0.3 M sucrose, 10 mM Tris (pH 7.9), 1 mM EDTA, and 4 mg/mL diethyldithiocarbamic acid. Samples were spun ( 20,800 rcf) and pellets resuspended in a buffer containing 100 mM Tris (pH 7.9), 500 mM NaCl, 20 mM EDTA, 1 % SDS, 0.1 % 2mercaptoethanol, and 100 g/ml proteinase K. Samp les were incubated at 65 C for 1 h, spun (20,800 rcf) and supernatant extrac ted with chloroform. Isopropanol was used to precipitate the DNA at room temperature, spun (20,800 rcf), and resuspended in Tris-EDTA buffer. One l RNase A was added to DNA sample and incubated at 37 C for 30 min. The chloroform extraction was repeated, followed by ethanol precip itation in presence of sodium acetate at -80 C for 1 h. Samples were spun for 10 min (20,800 rc f), air dried and resuspended in Tris-EDTA buffer. Approximately 25 ng of genomic DNA was used as template for PCR. Cycling parameters were 95 C for 5 min to activate DNA polymerase, then 35 cycles of 95 C for 30 s, annealing temperature for 30 s and 72 C for 1 min followed by a final step at 72 C for 5 min. The annealing temperature was set to five degr ees below the melting temperature for the primer pair (Tm listed in Table A-1). Amplicons were then separated on 1 % (w/v) agarose gels and stained with ethidium bromide. Transg ene presence was confirmed by PCR using nptII -specific

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109 primers with the following sequences: fo rward, 5-ATCCATCATGGCTGATGCAATGCG-3; reverse, 5-CCATGATATTCGGC AAGCAGGCAT-3 (253 bp of TDNA insertion). Cycling parameters were 30 cycles of 94 C for 1 min, 58 C for 1 min and 72 C for 1 min. Amplicons were then separated on 1 % (w/v) agarose ge ls and stained with ethidium bromide. Results Construction of RNAi Vector To repress endogenous poplar acid invertase genes, I used a binary vector, pCAPT, graciously donated by Stephen DiFazio (West Vi rginia University, Morgantown, WV) (Figure A-1). The pCAPT vector is designed for the cloni ng and insertion of a target sequence. A single GATEWAY cassette (Invitrogen) is located upstream of an inverted repeat of the octapine synthase ( OCS ) terminator. Transcription is controll ed by two CaMV 35S promoters linked in tandem. This transcript results in a hair pin formed by the inverted repeat of the OCS terminator. Transitive silencing of the inserted target ge ne fragment then occurs via RNAi (Filichkin et al. 2007). Insertion of Target Gene Sequence The acid invertase gene family was selected to be used for RNAi directed gene silencing. Using annotated sequences described in Chapter 2, nucleotide alignments were constructed using the eight acid invertases in poplar. Two gene sp ecific primer pairs per gene were designed such that a ~200 bp fragment from both the N-terminal and C-terminal ends of the gene could be PCR amplified from genomicDNA (Figure A-2; Table A-1). These fragments were then cloned into the pCAPT binary vector which was then sequenced to verify insertion in the proper orientation. The pCAPT binary containing the target gene insert was then grown in Agrobactierium and used to transform poplar trees as out lined in the Materials and Met hods. Shoot tips from regenerated transgenic poplars were used as a source of genomic DNA to screen for the presence of the

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110 transgene using primers directed against the NPTII reporter gene as outlin ed in the Materials and Methods. Table A-2 contains a complete list of all pCAPT vectors and regenerated transgenic poplars that were successfully made. Further Work The next step in this project is to screen the approximately 30 separate transgenic events per construct in order to verify the effectiven ess of the RNAi vector and rank the transgenic plants according to target gene transcript levels. Transcripts fro m the other acid invertase family members then need to be quantified to check fo r the specificity of th e RNAi mediated gene silencing as well as to screen for compensatory increases in related gene transcripts in case of functional redundancy. Total invert ase activity should then be assayed to verify that a successfully silenced invertase gene results in decreased invertase ac tivity. Phenotyping could then be done where one would expect increases in available sucrose, decreases in available hexoses, as well as whole-plant phenotypes such as alterations in growth rates.

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111 RB 35S gfp R1 R2 OCS -R PIV2 OCS -F nos5NPTIInos3 LB Gene Fragment Figure A-1. Schematic represen tation of pCAPT vector (16041 bp) used for RNAi silencing. R1 and R2, attR recombination sites flanking a ccdB gene and a chloramphenicol resistance gene from the GATEWAY vector conversion cassette (Invitrogen). RB and LB, right and left T-DNA borders, respect ively; 35S, double CaMV 35S promoter linked in tandem; gfp fragment of GFP coding sequence; PIV2 potato intron; OCS-R and OCS-F octopine synthase terminator frag ments in reverse (R) and forward (F) orientations, respectively; NPTII neomycin phosphotransferase II; gene fragment, location of targeted invertase sequences Diagram not to scale, adapted from (Filichkin et al. 2007).

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112 Spacer ~200 base pair oligoOCSOCS PtCIN2 ~3.6kb 5construct 3construct 35S 35S Spacer ~200 base pair oligoOCSOCS 35S 35S Figure A-2. Schematic representation of RNAi directed constructs. Endogenous PtCIN2 demonstrates strategy of RNAi construct design. PtCIN2 exons are represented by black boxes and introns by black lines. The 5 and 3 ~200 bp-oligos to be targeted by RNAi constructs are marked by black lin es below their respective locations. The RNAi constructs containing the ~200 bp-oligos are depicted below the PtCIN2 schematic. Grey block arrows represent 35S promoter, solid black box represents endogenous ~200bp-oligo to be targeted by RNAi machinery, dotted block arrows represent OCS terminator oriented in oppos ing directions and separated by a short potato intron (grey box) in order to form ha irpin in vivo. Figure not drawn to scale.

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113 Table A-1. Primer sequences used for ~200 bp target sequence cloning. Primer NamePrimer Sequence Tm (C) PtCIN1 NT forwardCACCATGGGCTATGCCACACAC61 PtCIN1 NT reverseCGTTGATCCAGTGCCTTGGAGG66 PtCIN1 CT forwardCACCGAGAGTTTTGGAGC53 PtCIN1 CT reverseAACATTCATGACAGGCGT51 PtCIN2 NT forwardCACCATGGCTATATGCCAAACAC61 PtCIN2 NT reverseGTTGATCCAGTTCCTAGGAGGCT61 PtCIN2 CT forwardCACCGAGAGTTTTGGAGC53 PtCIN2 CT reverseTTCATTGCGTGGATTCTCTC56 PtCIN3 NT forwardCACCATGGCTTTGTT AAAG TTTCTC62 PtCIN3 NT reverseGTTGATCCAGTTCTTAGGAGGCTG61 PtCIN3 CT forwardCACCGAAAG TTTTGGAG48 PtCIN3 CT reverseGACATTCATCACAGGCAC48 PtCIN4 NT forwardCACCTTCTTGGTTGGTTTATGC59 PtCIN4 NT reverseGGTTCTATATGACTGCTTTTCTTGC59 PtCIN4 CT forwardCACCGAGAGTTTTGGTGGG58 PtCIN4 CT reverseTTGATTTGGGC TTTGTTCATG59 PtCIN5 NT forwardCACCGGAGATATCTGTTATTTGG58 PtCIN5 NT reverseATTCATCCAGTTTTTAGGAGGTTG59 PtCIN5 CT forwardCACCTAGTTGAGAGTTTTGGTGGT60 PtCIN5 CT reverseCTAAAGGTGTGGCTTCCTTCG59 PtVIN2 NT forwardCACCCTTCCAGTTTCCAATTCCTTA65 PtVIN2 NT reverseAACAAAGTCTCGGG TTTCGC61 PtVIN2 CT forwardCACCCGTTGCTGAG TTTGAGTTA62 PtVIN2 CT reverseTGAGGCTGCCATTATTTCCTTTG63 PtVIN3 NT forwardCACCCCCACCATACACTCCCTTG67 PtVIN3 NT reverseTGGTGATACCCCTTGAGCCACTCC68 PtVIN3 CT forwardCACCAGTTTTGCTCAAGGAG56 PtVIN3 CT reverseTTGGTCAAATAGGAAAGGATGG59

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114 Table A-2. Summary of RNAi constructs made. NT and CT, N-terminal and C-terminal constructs, respectively; X denotes posse ssion of the RNAi construct (pCAPT) and transgenic plant expressing the construct. GenepCAPTTransgenic Plant PtCIN1 NTXX CTX PtCIN2 NTX CTXX PtCIN3 NTX CTXX PtCIN4 NTX CTX PtCIN5 NTXX CTXX PtVIN2 NTX PtVIN3 NTXX CTXX

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115 APPENDIX B REGULATION OF INVERTASE: A SUI TE OF TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL MECHANISMS This appendix contains a revi ew that resulted from a co llaboration between Dr. Li-Fen Huang, Dr. Karen Koch and myself. It was published in Functional Plant Biology While I took part in the construction and editi ng of the entire manuscript, some parts I did not write. I did not write the PPV compartmentalizati on, differential suga r regulation, or RNA turnover sections. I also originate the idea for the first figure. All other parts are my original work. Abstract Recent evidence indicates that several mechanis ms can alter invertase activity and, thus, affect sucrose metabolism and resource allocation in plants. One of these mechanisms is the compartmentalization of at least some vacuolar i nvertases in precursor protease vesicles (PPV), where their retention could control timing of de livery to vacuoles and hence activity. PPV are small, ER-derived bodies that sequester a subset of vacuolar-bound proteins (such as invertases and protease precursors) releas ing them to acid vacuoles in response to developmental or environmental signals. Another ne wly-identified effector of invertases is wa ll-associated kinase 2 (WAK2), which can regulate a sp ecific vacuolar invertase in Arabidopsis ( AtvacINV1 ) and alter root growth when osmolyte supplies ar e limiting. WAKs are ideally positioned to sense changes in the interface between the cell wall and plasma membrane (such as turgor), because the N -terminus of each WAK extends into the cell wa ll matrix (where a pectin association is hypothesised) and the C -terminus has a cytoplasmic serine/t hreonine kinase domain (signaling). Still other avenues of invert ase control are provided by a diverse group of kinases and phosphatases, consistent with input from multiple sensing systems for sugars, pathogens, ABA and other hormones. Mechanisms of regulation may also vary for the contrasting sugar responses of different acid invertase tran scripts. Some degree of hexokina se involvement and distinctive

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116 kinetics have been observed for the sugar-rep ressed invertases, but not for the more common, sugar-induced forms examined thus far. An a dditional means of regulation for invertase gene expression lies in the multiple DST (Down STream) elements of the 3untranslated region for the most rapidly repressed invertases. Similar sequences were initially identified in small auxinup RNAs (SAUR) where they mediate rapid mRNA turnover. Finally, the invertase inhibitors, cell walland vacuolar inhib itors of fructosidase (CIF and VIF, respectively) are indistinguishable by sequence alone from pectin methylesterase inhibito rs (PMEI); however, recent evidence suggests binding specificity may be determined by flexibility of a short, N terminal region. These recently characterized processes increase the suite of regulatory mechanisms by which invertase and, thus, sucr ose metabolism and resource partitioning can be altered in plants. Introduction The capacity to use sucrose is essential to most plant cells, because this sugar is typically the long-distance transport form for car bohydrates. Invertase (EC 3.2.1.26) irreversibly hydrolyses sucrose into glucose and fructose, and is thus positioned to play a central role in both carbon metabolism and sugar signaling. Glucose and fr uctose are hexoses that serve as an energy source for respiration, provide meta bolites for synthetic processes, generate osmotic pressure in growing tissues and function as metabolic signals affecting the expressi on of many downstream genes (Koch, 2004). Given the importance of sucros e cleavage and the diverse roles for products of invertase activity, it is not surprising that multiple avenue s have developed for the regulation of the invertases. Several invertase isozymes have been identi fied from plant species as either soluble (readily extractable from cytosol or vacuole), or insolubl e (bound to cell wall components). Vacuolar and cell wall invertases show optimal activity at an acidi c pH and thus are also called

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117 acid invertases. The mature acid invertases are glycosylated and lo cated in the cellular compartment their name implies (e.g. cell wall or vacuolar). Invertase is regulated at transcriptional a nd post-translational levels by many different factors including hormones, suga rs, pathogens, oxygen availability and proteinaceous inhibitors (Zeng et al. 1999; Long et al. 2002; Link et al. 2004; Trouverie et al. 2004; Voegele et al. 2006). However, important new methods of contro l have also been iden tified by recent work on the invertase path of sucrose use. These are reviewed here and incl ude protein trafficking, signaling cascades, transcript tu rnover and inhibitor binding. Compartmentalization of Vacuolar Invertases in Precursor Protease Vesicles (PPV) A new level of regulation has been revealed for vacuolar invertase activity by recent evidence for their localization in a class of minute vesicles calle d precursor prot ease vesicles (PPV) (Rojo et al. 2003). These organelles su rround the large, central va cuole, as well as the smaller, protein-storage vacuoles (C hrispeels and Herman, 2000; Hayashi et al. 2001; Rojo et al. 2003). The PPV are best known for their roles as storage sites for precur sor proteases that are later released into vacuoles, where the low pH activates maturation of the protease precursors (Chrispeels and Herman, 2000; Hayashi et al. 2001; Schmid et al. 2001). These vesicles are plant-specific ER bodies, formed from dila ted cisternae of the ER and surrounded with ribosomes (Chrispeels and Herman, 2000; Hayashi et al. 2001). PPVcontain a distinct population of proteins that incl ude protein disulfide isomerase (PDI), binding protein [(BiP) HSP-70] (Schmid et al. 2001), PYK10 -glucosidase (Matsushima et al. 2003) and precursor proteins for diverse cysteine proteases such as responsive to dehydrat ion-21 (RD21) (Hayashi et al. 2001), cysteine endopeptidase [C ysEP (from castor bean)] (Schmid et al. 2001) and vacuolar processing enzyme gamma (VPE ) (Chrispeels and Herman, 2000; Rojo et al. 2003).

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118 Presence of vacuolar invertases in PPV is s upported by localization of at least one vacuolar invertase to these compartments in Arabidopsis (Rojo et al. 2003). Collectively, the PPV contents, including the vacuolar i nvertase(s), are released into th e vacuolar lumen by a fusion of these organelles with the tonoplast. The process is distinct from the autophagic engulfment of other vesicles by the vacuole (Chr ispeels and Herman, 2000; Hayashi et al. 2001). When the PPV contents enter acidic vacuoles, the acid invertase moves into a low-pH, high-sucrose environment optimal for its activity. Previous evidence indicates that before v acuolar fusion, PPV are not acidic enough to support appreciable acid invertas e activity. A low pH would al so activate maturation of the VPE cysteine protease (Hayashi et al. 2001; Schmid et al. 2001) and, thus, rapidly degrade the invertase. The AtvacINV2 vacuolar i nvertase is indeed a target of VPE after this protease matures in the acidic central vacuole (Rojo et al. 2003). However, the rate of proteolytic degradation of invertase by VPE is modest in the v acuole and probably a result of dilution in the larger compartment. It is feasible that co-retention of both the VPE protease and vacuolar invertase in PPV could be a mechanism of retain ing their activities in re serve for later purposes. Once the PPV fuses with the central vacuole, ac tion of the maturing prot ease on the invertase would limit the duration of any incr eases in sucrose-cleaving activity that arose from the release of PPV contents into the acidic v acuole. Such pulse-type mechanisms of regulation facilitate fine control, and are evident for some of the sucrose synthases (Hardin et al. 2003), and for increments of starvation-induced autophagy (Rose et al. 2006). The impact of vacuolar invert ase retention by PPV could vary considerably. The portion of total invertase in these organelles at a ny given time could be significant under some circumstances and limited in others (Rojo et al. 2003). It is also unclear whether all vacuolar

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119 invertases follow a PPV path to acid vacuoles and whether this might vary with conditions or plant development. In Arabidopsis one or both vacuolar invertas es may be involved, because the proteins are virtua lly identical (Rojo et al. 2003; Carter et al. 2004). Still another factor is likely to be the timing and extent of PPV fusions with acid vacuoles. This can occur during senescence (Schmid et al. 2001; Rojo et al. 2003), salt stress (Hayashi et al. 2001) and possibly during diverse, pre-senescent phas es of development (Rojo et al. 2003). Recent evidence has indicated PPV may contribute to pathogen responses (Rojo et al. 2004) and aspects of seed development such as seed coat formation (Nakaune et al. 2005), seed protei n properties (Gruis et al. 2004) and remobilization of seed structures includ ing the megagametophyte (He and Kermode, 2003), endosperm (Schmid et al. 2001) and nucellar tissue (Greenwood et al. 2005). Wall-Associated Kinase (WAK) Recent work has shown that a wall-associate d kinase (WAK) can play a role in the regulation of vacuolar invertas e thus establishing a cross-compartmental link between WAK and vacuolar invertase(s) (Kohorn et al. 2006b).AT-DNA null allele of WAK2 reduced vacuolar invertase activity in Arabidopsis roots by 62% and decreased leve ls of a specific vacuolar invertase transcript ( AtvacINV1 ) by over 50%. The wak2 null allele also reduced plant growth under low nutrient conditions. Unlike effects on v acuolar invertase, the cell wall isoforms were unaltered by the wak2 null allele, indicating either that other WAKs may regulate cell wall invertases, or that WAK-based signals mediate only vacuolar forms (Kohorn et al. 2006b). This work further supports the role of vacuolar invertases in resource partitioning as well as turgor maintenance and cell wall expansion (Koch, 2004). Wall-associated kinases proteins are hypothesized to se rve as sensors of cellular turgor, the extent of cell wall loosening, and/or the degree of cell expansion (Zhang et al. 2005; Kohorn et al. 2006b). WAKs are ideally positioned to do so, because they span the plasma membrane

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120 (Figure B-1). Each of the five WAK genes en codes a transmembrane pr otein with an active cytoplasmic serine/threonine kinase domain on the Cterminus and a distinctive extracellular domain on the Nterminus. The extracellular domain is similar to the vertebrate epidermal growth factor-like domain. Eviden ce indicates this extracellula r domain may be bound to pectin in the cell wall, which would provide a link to the extracellular matrix (He et al. 1999; Wagner and Kohorn, 2001; Decreux and Messiaen, 2005; Kohorn et al. 2006a). This physical tie between the extracellular matrix and the cytoplasm could provide an anchor, or reference point enabling the WAK to play a role in sensing or transmitting information on the status of the cell wall relative to the plasma membrane. Such info rmation could be invaluable to adjustment of cell expansion or turgor. Different members of the WAK family ar e expressed at organ junctions, in shoot and root apical meristem s, in expanding leaves, and in response to environmental stimuli such as wounding and pathogen attack (Wagne r and Kohorn, 2001). Antisense reduction of WAK1 protein levels also establ ished the necessity of these gene s for expansion of leaf cells (cellular division was unaffected) (Lally et al. 2001; Wagner and Kohorn, 2001). The WAK family appears to be part of a la rger, 22-member WAK-like (WAKL) family in Arabidopsis and is widespread in the plant kingdo m (Verica and He, 2002). Protein gel blots usingWAK1 antibodies indicate related prot eins in pea, tobacco and maize (He et al. 1996; Gens et al. 2000). WAKL expressed sequence tags have also be en identified in tomato, soybean and rice (Verica and He, 2002), but a functional relationship between WAK and WAKL has yet to be shown. Other Kinases Affecting Invertases Invertase can be activated by mycorrhizati on, pathogen infection and wounding, as well as various hormones (Sturm and Chrispeels, 1990a; Benhamou et al. 1991; Ehness et al. 1997; Hall and Williams, 2000; Blee and Anderson, 2002; Pan et al. 2005). Diverse kinases appear to

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121 regulate activity of both cell wall and vacuolar invertases in many of these instances (Ehness et al. 1997; Pan et al. 2005). The serine/threonine kinase inhibitor, stau rosporine, prevents the accumulation of invertase transcripts that typi cally occurs during defence responses (Ehness et al. 1997). In contrast, the same inhibitor, and also two other serine/threon ine kinase inhibitors (K252a and H7), increase the extent to whic h ABA induces both vacuolar and cell wall invertases (Pan et al. 2005). However, the tyrosine protein kinase inhibitor, quercetin, strongly suppresses the ABA induction of acid invertases (Pan et al. 2005) indicating that multiple types of kinases with differing modes of regulation are involved in modulati ng invertase activity. Phosphatases have also been implicated in the regulation of invertases (Ehness et al. 1997; Pan et al. 2005). Pan et al. (2005) demonstrated that expressi on and activity of both vacuolar and cell wall invertases increased in respons e to ABA and acid phosphatase. These data, in conjunction with the kinase inhi bitor data, indicate that reve rsible protein phosphorylation is playing some role in the ABA signaling network (Pan et al. 2005). The phosphatase inhibitor, endothall, also induces cell wall invertase tr anscripts when added to cell suspensions of Chenopodium rubrum (Ehness et al. 1997). This work on invertase is consistent with other ongoing analyses of both ABA and defe nse-signaling cascades (Roitsch et al. 2003; Roitsch and Gonzalez, 2004). Differential Sugar Regulation of Invertases Despite the widespread use of invertase expr ession as a sugar res ponse marker in yeast (Ahuatzi et al. 2004; Kig et al. 2005), the regulation of these gene s has been difficult to dissect in plants. Not only are there many more indi vidual invertase genes (Tymowska-Lalanne and Kreis, 1998a; Sturm, 1999; Sherson et al. 2003), but they are also di fferentially regulated (Xu et al. 1996; Tymowska-Lalanne and Kreis, 1998a; Hu ang, 2006), and sugar response mechanisms vary (Roitsch et al. 1995; Xiao et al. 2000; Huang, 2006). All plant species studied to date have

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122 two vacuolar invertases (Haouazine-Takvorian et al. 1997), with several cell wall invertases. Fully-sequenced genomes indicate six putative cell wall invertases in Arabidopsis (TymowskaLalanne and Kreis, 1998a; Sherson et al. 2003) and nine in rice (Ji et al. 2005). Additional complexity has been introduced by recent evidence that two of the putative cell wall invertase sequences may encode fructan exohydrolases (a closely-related enzyme) (De Coninck et al. 2005). Similar invertase family structures are also evident in poplar, maize, potato and tomato (Fridman and Zamir, 2003; Huang, 2006); P. N. Bocock, unpubl. data). The majority of these invertas es are sugar-induced rather th an repressed (Roitsch, 1999; Roitsch and Ehness, 2000), which is consistent with their roles in carbon use v. carbon acquisition by plants (Koch, 1996; Rolland et al. 2002; Koch, 2004). Both vacuolar and cell wall invertases enhance use of carbohydr ate resources by cleaving imported sucrose at sites of growth or storage (Winter and Huber, 2000; Koch, 2004) These invertases can also amplify sugar signals to other genes that respond to ch anges in hexose availability (Koch, 2004). The identities of sugars that induce invertas es remain unclear, however. Evidence thus far indicates limited involvement of the classi c hexokinase-mediated sensing system in upregulation of invertases by sugars. Neither overexpression of the key hexokinase gene, AtHXK1 nor its antisense reduction reportedly affects expression of a sugar-induced AtcwINV1 ( fruct1 ) gene for cell wall invertase in Arabidopsis (Xiao et al. 2000). Non-hexokinase mechanisms of sugar sensing may play more wide spread roles in sugar-induced ge ne expression than previously recognized (Purcell et al. 1998; Rolland et al. 2002; Sinha et al. 2002; Lalonde et al. 2004; Halford, 2006). Although signaling systems differ between plants and yeast, the hexokinasebased sensing in yeast is largel y linked to sugar repression rath er than induction (Johnston, 1999; Palomino et al. 2005; Kim et al. 2006), and a balance betwee n the two is hypothesized to

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123 mediate responses (Ronen and Bots tein, 2006). In plants, mechanisms of sugar sensing have thus far been studied mainly by following respons es of genes down-regulated by glucose (e.g. photosynthesis, seed germination) (Smeekens, 2000; Rolland et al. 2002; Rolland and Sheen, 2005). Another consideration is th at genes known to be sucroserather than hexose-responsive are often expressed in or n ear the vascular system (Rook et al. 1998), as are many invertases (Andersen et al. 2002; Wachter et al. 2003). Some acid invertase genes are sugar-repr essed. This is less common, but occurs consistently in all species examined thus far. The singular repression of one vacuolar invertase by sugars is conserved across gene families in maize (Xu et al. 1996), tomato (Godt and Roitsch, 1997b), rice (Huang, 2006) and poplar (P. N. Bocock, unpubl. data). At least some degree of involvement has been indicated for th e hexokinase-mediated sensing system in this repression. Contributions from this mechanism are supported by data from metabolizeable and non-metabolizeable glucose analogues tested in Arabidopsis for their capacity to repress the AtvacINV2 vacuolar invertase (Huang, 2006). Another f eature of invertase re pression by sugars is its rapid down-regulation, with responses ev ident in minutes rather than hours (Huang, 2006). Still another factor may be the demonstrated compartmentalization of the sugar-repressed AtvacINV2 invertase protein in the precursor protease vesicles [see section on PPV (Rojo et al. 2003)], implying a possible relationship between the PPV and the observed sugar responses. Finally, additional insights into the sugar repr ession of some invertases may lie in their respective roles. Demands for osmotic constituents (two hexoses from one sucrose) may well dominate sucrose partitioning in response to specific developm ental and/or stress signals. Expression of at least one v acuolar invertase under these conditions could be highly advantageous, and need not be viewed as separa te from its role in sucrose import. Sucrose

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124 entering cells is often cleaved first in the vacuole, thus giving vacuolar in vertase a prominent role in sucrose partitioning (Koch, 2004). Theoretically, e xpression of a starvation-tolerant invertase could confer an import priority for certain cel ls or tissues under stress, as well as favor immediate allocation of incoming resources to os motic constituents. A similar scenario could hold for key aspects of development that rely heavily on osmotic constituents for cellular expansion. Data thus far are consistent with contributions by both the sugar-induced and repressed vacuolar invertases to import-based osmotic support of expansion sinks. Examples include root elongation in Arabidopsis (Stessman, 2004; Huang, 2006; Kohorn et al. 2006b; Sergeeva et al. 2006), petiole growth in sugar beet (Gonzalez et al. 2005), expansion of ovaries and silks in maize (Andersen et al. 2002), enlargement of Agrobacterium -induced galls (Wachter et al. 2003) and growth of diverse fruits and ve getables such as tomatoes, carrot roots and newly-forming potato tubers (Koch and Zeng, 2002). RNA Turn Over and DST At least one mechanism of posttranscriptional regulat ion of invertase is also indicated for the sugar-repressed vacuolar invertases. In both rice and Arabidopsis the 3 untranslated regions of the sugar-repressed genes OsVIN1 and AtvacINV2 carry apparent downstream (DST) elements implicated in rapid turnover of plant mRNA s (Huang, 2006). The DST are highly conserved and can mediate sequence-specific decay of short-lived mRNA in vivo (Newman et al. 1993; Sullivan and Green, 1996). The DST are especially not able for their role in rapid destabilization of small auxin-up RNAs (SAUR) (Newman et al. 1993; Gil and Green, 1996; Johnson et al. 2000; Feldbrugge et al. 2002). Additional evidence is also consistent w ith altered mRNA longe vity for the sugarrepressed form of vacuolar i nvertase. Although glucose rapidly decreases mRNA levels of the AtvacINV2 vacuolar invertase in vivo (within 30 min), the promot er alone shows a contrasting

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125 induction by sugars (Huang, 2006). When transcri ption is blocked by cordycepin, however, a glucose-enhanced rate of decay is evident for AtvacINV2 mRNA (Huang, 2006). Glucose-based destabilization of mR NAs thus seems a likely c ontributor to the rapid re pression observed for the AtvacINV2 vacuolar invertase (Huang, 2006). Invertase Inhibitors Cell wall and vacuolar invertase activity can be regulated by a family of proteinaceous inhibitors known as cell wall in hibitor of fructosidase (CIF ) and vacuolar inhibitor of fructosidase (VIF), or collect ively as C/VIF reviewed by Ra usch and Greiner (2004). Although CIF are cell wall invertase inhibitors, they are broadly active against bot h cell wall and vacuolar invertases. In contrast, VI F inhibition is specific to vacuolar invertases. Neither of the inhibitors affect fungal invertases indicating a minimal ro le for these interactions in pathogen defense (Greiner et al. 1998; Greiner et al. 1999; Link et al. 2004). The C/VIF related protein family is not highly conserved, and in Arabidopsis sequence identities range from roughly 20 to 40% for ~14 family members (Rausch and Greiner, 2004). Th e C/VIF related protein family also contains pectin methylesterase inhibitors (PMEI). PM EI are indistinguishable from the C/VIF by sequence alone and retain nearly identic al structures to the C/VIF (Giovane et al. 2004; Hothorn et al. 2004a; Hothorn et al. 2004b; Di Matteo et al. 2005). Important implications arise from the capacity of these related proteinaceous inhibitors to distinguish between their targets. Arabidopsis has only eight putative ac id invertases (six cell wall and two vacuolar), but more than 60 pectin methylesterase (PME)related genes based on sequence similarity (Micheli, 2001; Sherson et al. 2003; Rausch and Greiner, 2004; De Coninck et al. 2005). X-ray crystollagraphy has revealed that CIF is conforma tionally stable over a broad pH and temperature range, however, the invertase /inhibitor complex is only stable at an acidic pH. This indicates that binding is determined not by conformational cha nges, but rather by pH-

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126 induced changes at the interface of th e invertase/inhibitor complex (Hothorn et al. 2004a; Hothorn and Scheffzek, 2006). In contrast, the highl y similar structure of the PMEI was found to undergo large structural rearrangements (Figur e B-2) under these same conditions suggesting PMEI uses a different mode of binding than does the CIF (Hothorn et al. 2004b). PMEI contains a flexible -hairpin that is important both in dimer formation and in binding of PME (Hothorn et al. 2004b). Chimeral domain swap experiments of the -hairpin domain between PMEI and CIF indicate that this domain of PM EI is necessary and sufficient for activity against PME; however, the corresponding -hairpin in NtCIF is not sufficien t for invertase inhibition (Hothorn et al. 2004b). The ~28 amino acid residues encoding this -hairpin may be key in distinguishing these two classes of i nhibitors (Hothorn et al. 2004b). Analysis of PMEI and CIF crystal structures not only clarifies interactions between these inhibitors and their targ ets, but also aids our understanding of how distinct functions can arise for proteins sharing very similar st ructures and ancestry. It is worth noting, however, that the crystallographic analyses were performed on th e cell wall invertase inhi bitor and thus do not explain the apparently narrower s ubstrate specificity of the vacuol ar invertase inhibitor. These data indicate that the VIF may use a different mode of action than the CIF. It is also of interest that two proteins in Arabidopsis previously annotated based on sequence similarity as cell wall invertases (AtcwINV3&6), may actually be fructan exohydrolase s (FEH).FEH protein sequences are nearly identical to those of demonstrated ac id invertase family members, yet the recombinant enzymes are completely inactive against sucrose (De Coninck et al. 2005). It has yet to be determined if the C/VIF family can distingu ish between the FEH and the known invertase and PME substrates, and given the level of sequence di versity within the inhibitor family it seems likely that biochemical and structural analyses will be required to re solve these questions.

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127 Summary Compartmentalization of vacuolar invertas e in PPV introduces a new dimension to regulation of invertases in vivo (Rojo et al. 2003; Koch, 2004). The PPV sequester at least some of the vacuolar invertase(s) (Rojo et al. 2003) and other proteins (esp ecially protease precursors) (Chrispeels and Herman, 2000; Hayashi et al. 2001) for later release into acid vacuoles. It is currently unclear whether these invertases are active inside PPV before vesicle fusion with acidic, sucrose-containing vacuoles. Wall-associated kinases can regula te vacuolar invertases (Kohorn et al. 2006b). At least one of them (WAK2) regulates a specific vacuolar invertase (A tvacINV1) that predominates in Arabidopsis roots. IfWAK2 is dysfunctional, vacuolar invertase activity drops to less than 50% in roots, and growth is impaired under low-os molyte conditions. The WAK kinases are ideally positioned to serve as status sensors for the interface between the plasma membrane and cell wall, because each WAK has an Nterminus in the extracellular matrix (possibly associated with pectin), and a Cterminal serine/threonine kinase domain in the cytoplasm (si gnaling capacity). Diverse kinases and phosphatases have been im plicated in the regulation of invertases, which is consistent with the ra nge of signaling networks that a ffect them. These extend from sugar and ABA sensing, to interactions with pathogens and symbionts. Contrasting responses to sugars within the invertase gene family may also be mediated by different mechanisms. Some degree of hexokinase involvement and distinctive kinetics have been observed for the sugar-repressed invert ases (Huang, 2006), but not for the more common, sugar-induced forms examined thus far (Xiao et al. 2000). DST elements implicated in mRNA turnover also lie in the 3 untranslated region of the most rapidly repressed invertases Their sequences resemble those in SAUR where they mediate mRNA destabilization.

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128 The invertase inhibitors, CIF and VIF shar e high sequence similarity with PMEI, but evidence from crystal structures and chimeral prot eins suggests that bind ing specificity may be determined by flexibility of a short, Nterminal region. Acknowledgements We thank the National Science Foundation, Me tabolic Biochemistry Program, GrantAward No. MCB-0080282; the Department of Energy,Office of Science, Office of Biological and Environmental Research, Grant Award No. DE-AC05OR22725; and the University of Florida Experiment Station for funding.

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129 Cell wall Extracellular domain Transmembrane domain Nucleus PPVs Plasma membrane VPE Vacuole Vacuolar invertase STK domain WAK Cell wall Extracellular domain Transmembrane domain Nucleus PPVs Plasma membrane VPE Vacuole Vacuolar invertase STK domain WAK Figure B-1. Recent additions to mechanisms cont rolling invertases include the wall associated kinases (WAK), precursor pr otease vessicles (PPV), and vacuolar processing enzymes (VPE). At least one WAK (WAK2) can regulate activity of a specific vacuolar invertase ( AtvacINV1 ) in Arabidopsis The WAK are ideally positioned to detect alterations in the interface between the cell wall and plasma membrane (e.g. turgor or expansion), because their extrac ellular terminus resides in the cell wall matrix (possibly anchored in pectin) and their cytoplasmic tail includes a kinase domain. This kinase can apparently tri gger cascades regulatin g transcription of vacuolar invertase and other effectors of its activity. Also, at least some vacuolar invertase(s) reach the vacuole through PPV, where they can be sequestered for extended periods. Timing of activity may be determined by when the enzyme is released to an acidic, sucrose-storing envi ronment. The PPV also releases protease precursors such as the vacuolar processing enzyme [VPE ( )], into the acidic vacuole which then act upon vacuolar invertases ( ) to limit the duration of their activity.

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130 PMEI Open conformation Closed conformation CIF X X PMEI Open conformation Closed conformation CIF X X X X Figure B-2. Protein inhibitors of cell wall invertases (CIF, cell wall inhibitor of fructosidase) share strong sequence similarity with inhibi tors of pectin methylesterase (PMEI, pectin methylesterase inhibito r), but crystal structures i ndicate important differences in flexibility. The top panel de picts CIF. The oval denotes an -hairpin thought responsible for binding specifi city of CIF and PMEI. The -hairpin in CIF is rigid at all pHs and temperatures tested. The botto m panel shows PMEI in three different conformations that demonstrate the flexibility of the PMEI -hairpin. (Hothorn et al. 2004a; Hothorn et al. 2004b; Hothorn and Scheffzek, 2006). Images were constructed by Protein E xplorer (Martz, 2002).

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149 BIOGRAPHICAL SKETCH Philip Bocock was born May 20, 1979 in Wilmingt on, Delaware. He was raised in Austin, Texas where he attended Westw ood High School. He graduated high school in 1997 and entered Texas A&M University in September of the same year. He earned his Bachelor of Science in biochemistry and genetics accompanied with a minor in forest science in May of 2001. In the fall of 2001, Philip began work on a PhD in the Plant Mo lecular and Cellular Biology Program at the University of Florida. At the University of Florida, Philip entered the laboratory of Dr. John Davis and began c onducting research on carbon allocation and partitioning using the model tree, Populus Under the tutelage of Dr. John Davis, Philip identified the members of the poplar invertase ge ne family and subsequently employed a variety of techniques to seek to understand the role this enzymatic family plays in carbon allocation and partitioning in poplar. The work detailed in th is dissertation was entirely conducted in the laboratory of Dr. John Davis.