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Multiple Non-Redundant Roles for Plastidic 6-Phosphogluconate Dehydrogenase (6PGDH) in Maize

Permanent Link: http://ufdc.ufl.edu/UFE0022731/00001

Material Information

Title: Multiple Non-Redundant Roles for Plastidic 6-Phosphogluconate Dehydrogenase (6PGDH) in Maize
Physical Description: 1 online resource (56 p.)
Language: english
Creator: Li, Li
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The oxidative pentose phosphate pathway (OPPP) serves multiple roles in primary metabolism. Enzymes for the oxidative section of the OPPP are found both in the cytosol and plastid. Several mutant studies have suggested that cytosolic and plastidic OPPP enzymes are redundant including 6-phosphogluconate dehydrogenase (6PGDH). 6PGDH enzymes catalyze the third non-reversible step of the oxidative section of the OPPP. Maize mutations in the cytosolic 6PGDH enzymes, pgd1 and pgd2, do not show obvious phenotypes beyond loss of enzyme activity. In this thesis, two knockout alleles of the maize Pgd3 locus were identified to investigate the role of this gene in central carbon metabolism. The pgd3 mutants disrupt plastid-localized 6PGDH activity and cause a rough endosperm (rgh) phenotype that affects both grain-fill and embryo development. Consistent with the reduced grain-fill phenotype, 13C-glucose labeling experiments during seed development suggested that pgd3 mutants disrupt carbohydrate flux for starch synthesis. PGD1, PGD2, and PGD3 are all active in both the endosperm and embryo. These data suggest that PGD3 has a non-redundant role for seed development. Moreover, homozygous pgd3 seeds can be rescued through tissue culture experiment. The addition of asparagine in the tissue culture medium increases the rescue of mutant seeds, suggesting that amino acid synthesis is limiting in pgd3 mutants. The homozygous pgd3 mutant plants show normal morphology but are slow to green and late flowering. PGD3 activity is restricted to sink tissues suggesting that the slow to green phenotype is due to disruptions in carbon metabolism during leaf expansion.
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 Li Li.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Settles, Andrew M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022731:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022731/00001

Material Information

Title: Multiple Non-Redundant Roles for Plastidic 6-Phosphogluconate Dehydrogenase (6PGDH) in Maize
Physical Description: 1 online resource (56 p.)
Language: english
Creator: Li, Li
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The oxidative pentose phosphate pathway (OPPP) serves multiple roles in primary metabolism. Enzymes for the oxidative section of the OPPP are found both in the cytosol and plastid. Several mutant studies have suggested that cytosolic and plastidic OPPP enzymes are redundant including 6-phosphogluconate dehydrogenase (6PGDH). 6PGDH enzymes catalyze the third non-reversible step of the oxidative section of the OPPP. Maize mutations in the cytosolic 6PGDH enzymes, pgd1 and pgd2, do not show obvious phenotypes beyond loss of enzyme activity. In this thesis, two knockout alleles of the maize Pgd3 locus were identified to investigate the role of this gene in central carbon metabolism. The pgd3 mutants disrupt plastid-localized 6PGDH activity and cause a rough endosperm (rgh) phenotype that affects both grain-fill and embryo development. Consistent with the reduced grain-fill phenotype, 13C-glucose labeling experiments during seed development suggested that pgd3 mutants disrupt carbohydrate flux for starch synthesis. PGD1, PGD2, and PGD3 are all active in both the endosperm and embryo. These data suggest that PGD3 has a non-redundant role for seed development. Moreover, homozygous pgd3 seeds can be rescued through tissue culture experiment. The addition of asparagine in the tissue culture medium increases the rescue of mutant seeds, suggesting that amino acid synthesis is limiting in pgd3 mutants. The homozygous pgd3 mutant plants show normal morphology but are slow to green and late flowering. PGD3 activity is restricted to sink tissues suggesting that the slow to green phenotype is due to disruptions in carbon metabolism during leaf expansion.
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 Li Li.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Settles, Andrew M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022731:00001


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fe9583532c6fa4061447ae3b977706f82e9c1352







MULTIPLE NON-REDUNDANT ROLES FOR PLASTIDIC 6-PHOSPHOGLUCONATE
DEHYDROGENASE (6PGDH) IN MAIZE






















By

LI LI

















A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE


UNIVERSITY OF FLORIDA

2008
































2008 Li Li




































To my Mom and Dad









ACKNOWLEDGMENTS

I would like to express my sincere gratitude and thanks to my adviser Dr. Mark Settles,

for introducing me to this project. His kindness, pleasant personality, and patience made my

master's study at UF very enjoyable. More importantly, he showed me the way to find great

happiness from research and supported my ideas to broaden my research experience.

I also thank Dr. Cline for all his support during my project. His insight and good advice

helped me improve on my research and thesis. I also learned a lot of other helpful information

through the happy work with him.

Meanwhile, I also want to express my thanks to the lab members of Dr. Settles, for their

assistance and time to make this project come from a proposal to reality. Dr. Tseung taught me

about enzyme activity analysis and mutant seed rescue. Dr. Spielbauer conducted much of the

13C-labeling experiments.

Other people I would like to thank are my committee members: Dr. William Gurley and

Dr. Kevin Folta. Their support and guidance helped me during my thesis research and the writing

of this document.

Likewise, special thanks go to the researchers in Technical University of Munich,

Germany. Their NMR analysis of the isotopolog pattern of starch in 13C-labeling experiments

was a critical contribution to this project.

Finally, I would like to thank my parents for their support, my thanks to them is beyond

any scope of the languages.









TABLE OF CONTENTS

page

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

LIST OF FIGURES .................................. .. .. .... ..... ..................7

A B S T R A C T ............ ................... .................. .......................... ................ .. 8

CHAPTER

1 IN TR OD U CTION .......................................................... ...................... .... 10

G general B background ................................................... ................... .......... 10
Oxidative Pentose Phosphate Pathway (OPPP) ................................... ............... 10
Subcellular Localization of OPPP Enzymes ................ ................................ ........... 12
Connections between Cytosolic and Plastidic OPPP Enzymes.................. ........... 12
Mechanism of 6-Phosphogluconate Dehydrogenase (6PGDH) Enzymes .................... 13
The Importance of the OPPP and 6PGDH to Cellular Metabolism ................................... 15
Substrates for N ucleotide Synthesis ..................................................... ...... ......... 16
Substrates for Aromatic Amino Acid Synthesis................................... ............... 17
NADPH for Fatty Acid Synthesis ............................................................................18
NADPH for Nitrate Assimilation ........ ......... ............................. 19
Starch Synthesis in M aize Kernels ........... .................... .............. .. ........... .... 20
Sugar Induction of Transporter Expression............................................................22
Q u e stio n s ............................................................................................ 2 3

2 RESULTS: MUTATIONS IN PGD3 CAUSES RGH SEED PHENOTYPES......................24

B a ck g ro u n d ................. .............. ........ ...... ...................... ................ 2 4
Co-segregation Analysis ofpgd3-umul ................................. ........ ................... 25
Identification of the pgd3-umu2 Allele............................ .......................... 25
Com plem entation Test.................................... .. ...... ..... .. ............27

3 RESULTS: PGD3 HAS MULTIPLE NON-REDUNDANT ROLES IN MAIZE.................30

B ackgrou n d ......................................................... ................... ................ 30
pgd3 Seed Phenotype and Plant Phenotype........ ........ .. ..... .. .. ................ 31
Both pgd3-umul and pgd3-umu2 Mutants Are Enzymatic Knockouts in Seeds ...................31
Differences in PGD1/PGD2 and PGD3 Activity Cannot Explain pgd3 Mutant
P h en oty p es ........................................ .... ................... .................................3 3
PGD1, PGD2 and PGD3 Can be Co-purified with Plastids............................................34









4 RESULTS: POTENTIAL NON-REDUNDANT ROLES OF PLASTIDIC 6PGDH............36

B a c k g ro u n d ................... .......................................................................... .. 3 6
Plastidic 6PGDH is Required for Normal Starch Synthesis in Seeds ..................................37
Nitrogen Assimilation May be Limited in pgd3 Mutants..................... ......................37

5 D ISC U S SIO N ..............................................................................................40

6 M A TER IA L S A N D M ETH O D S ........................................ .............................................43

G enom ic D N A E extraction ............................................................................ ....................43
PCR Assay ........................................... .......................... 43
T otal P rotein E xtraction .......... .................................................................... ..................44
P lastid Isolation ................................................................4 5
6PGDH Activity A ssay ..................................... ................ ............ .. ...... 46
Mutant Seeds Rescue ............... ................................................... ... 46
13C -lab selling E xperim ents ............................................................................ ....................47
T total N nitrogen M easurem ent........................................................................... .............. 48

L IST O F R E F E R E N C E S .............................................................................. ...........................50

B IO G R A PH IC A L SK E T C H .......................................................................... ........................56









LIST OF FIGURES


Figure page

1-1 Schematic of the oxidative pentose phosphate pathway...................................................11

1-2 Multiple levels of redundancy of the OPPP in Arabidopsis...................................... 13

1-3 6-Phosphogluconate dehydrogenase (6PGDH) convertes 6-phosphogluconate to
ribulose 5-phosphate a three-step acid-base mechanism. .............................................15

1-4 Generalized schematic of starch biosynthesis in maize kernels utilizing central
carbon metabolism..................................... .. ... ... .. ........ ........ 22

2-1 pgd3-umul co-segregates with a rough endosperm (rgh) seed phenotype........................26

2-2 pgd3-umu2, the second mutant allele ofPgd3, was identified by reverse genetic
screen ......................................................... ....................................2 7

2-3 The phenotype ofpgd3-umu2 is same as the one ofpgd3-umul.................................28

2-4 Reciprocal crosses between heterozygouspgd3-umul and pgd3-umu2 plants fail to
complement resulting in F ears segregating for the rgh mutant phenotype...................29

3-1 Phenotype of thepgd3 m utants ......... ............................................................... ... ... 32

3-2 6PGDH activity of pgd mutant and normal seeds. ................................. ..................... 33

3-3 6PGDH activity in different tissues from a wild type plant (W22). ..................................34

3-4 6PGDH activity after chloroplast isolation using total protein extract from fresh leaf
tissue as a control. ...........................................................................35

3-5 6PGDH activity after protease treatm ent ........................................ ....................... 35

4-1 Mutation in Pgd3 changes isotopolog patterns of glucose in starch from the maize
k ern els...................................................... ...................................... 3 8

4-2 The germination percentage of thepgd3 homozygous mutant seeds in the culture
rescue experim ent. ....................................................... ................. 39

6-1 Schematic of the mutant seeds rescue experiments............ ............................................47

6-2 Schematic of the 13C-labeling experiments......... .................................48









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

MULTIPLE NON-REDUNDANT ROLES FOR PLASTIDIC 6-PHOSPHOGLUCONATE
DEHYDROGENASE (6PGDH) IN MAIZE

By

Li Li

August 2008

Chair: Andrew Settles
Major: Horticultural Science

The oxidative pentose phosphate pathway (OPPP) serves multiple roles in primary

metabolism. Enzymes for the oxidative section of the OPPP are found both in the cytosol and

plastid. Several mutant studies have suggested that cytosolic and plastidic OPPP enzymes are

redundant including 6-phosphogluconate dehydrogenase (6PGDH). 6PGDH enzymes catalyze

the third non-reversible step of the oxidative section of the OPPP. Maize mutations in the

cytosolic 6PGDH enzymes, pgd] and pgd2, do not show obvious phenotypes beyond loss of

enzyme activity. In this thesis, two knockout alleles of the maize Pgd3 locus were identified to

investigate the role of this gene in central carbon metabolism. The pgd3 mutants disrupt plastid-

localized 6PGDH activity and cause a rough endosperm (rgh) phenotype that affects both grain-

fill and embryo development. Consistent with the reduced grain-fill phenotype, 13C-glucose

labeling experiments during seed development suggested thatpgd3 mutants disrupt carbohydrate

flux for starch synthesis. PGD1, PGD2, and PGD3 are all active in both the endosperm and

embryo. These data suggest that PGD3 has a non-redundant role for seed development.

Moreover, homozygous pgd3 seeds can be rescued through tissue culture experiment. The

addition of asparagine in the tissue culture medium increases the rescue of mutant seeds,









suggesting that amino acid synthesis is limiting in pgd3 mutants. The homozygous pgd3 mutant

plants show normal morphology but are slow to green and late flowering. PGD3 activity is

restricted to sink tissues suggesting that the slow to green phenotype is due to disruptions in

carbon metabolism during leaf expansion.









CHAPTER 1
INTRODUCTION

General Background

Oxidative Pentose Phosphate Pathway (OPPP)

The oxidative pentose phosphate pathway (OPPP) is a central process in plant

metabolism. The OPPP includes a series of enzymes that converts glucose 6-phosphate into a

pool of phosphorylated sugars with 3 to 7 carbons. Those sugars can be converted back into

glucose 6-phosphate allowing the pathway to undergo cycles. Figure 1-1 shows a generalized

schematic of the OPPP showing the reactions and intermediates through the pathway. The OPPP

has two major metabolic roles: providing reducing power to the cell and providing carbon

intermediates for multiple biosynthetic pathways. Both of these roles are thought to be essential

to the cell and will be discussed in greater detail in section 1.2.

The OPPP provides reductant to the cell in the form of NADPH, which is synthesized in

the oxidative section. The oxidative section includes three enzymes catalyzing three reactions.

Initially, glucose 6-phosphate dehydrogenase (G6PDH) oxidizes glucose 6-phosphate (G6P) to

phosphogluconolactone. 6-Phosphogluconolactonase then converts phosphogluconolactone to 6-

phosphogluconate very quickly. After that, 6-phosphogluconate dehydrogenase (6PGDH)

oxidatively decarboxylates 6-phosphogluconate to ribulose 5-phosphate. Importantly, all three of

the oxidative reactions are non-reversible and loss of any of these enzyme activities is expected

to disrupt the entire OPPP.

The non-oxidative section of the OPPP provides the pool of phosphorylated sugars that

are needed in a variety of metabolic pathways. The five enzymes working in the non-oxidative

section are: ribose 5-phosphate isomerase (RPI), ribulose 5-phosphate 3-epimease (RPE),

transaldolase, transketolase, and glucose 6-phosphate isomerase. These enzymes convert the









product of 6PGDH, ribulose 5-phosphate, to other sugar phosphates. The pentose phosphate

sugar pool includes: 5 carbon sugars (ribose 5-phosphate and xylulose 5-phosphate), a 7 carbon

sugar (sedoheptulose 7-phosphate), a 4 carbon sugar (erythrose 4-phosphate), a 3 carbon sugar

(triose 3-phosphate), and 6 carbon sugars (fructose 6-phosphate and glucose 6-phosphate). The

non-oxidative enzymes catalyze reversible reactions, so it is possible to synthesize all of the

phosphate sugar pool by utilizing glucose-6-phosphate and ATP (ap Rees, 1985; reviewed in

Kruger and von Schaewen, 2003).


NADPH

SP-Gluconolactone..


6-P-Gluconate
Glucose 6-P 6-P-Guconate




Fructose 6-P x :.. ,


NADPH


VU VZM:


5-P


Figure 1-1. Schematic of the oxidative pentose phosphate pathway. The number in black circle
denotes the enzyme that catalyzes each of the steps: 1. Glucose 6-phosphate
dehydrogenase; 2. 6-Phosphogluconolactonase; 3. 6-Phosphogluconate
dehydrogenase (6PGDH); 4. Ribose 5-phosphate isomerase; 5. Ribulose 5-phosphate
3-epimerase; 6. Transketolase; 7. Transaldolase; 8. Glucose 6-phosphate isomerase.


:C









Subcellular Localization of OPPP Enzymes

OPPP enzymes in animal and yeast cells are located exclusively in the cytosol. In higher

plants, the complete set of OPPP enzymes are found in the plastids (Nishimura and Beevers,

1979; Journet and Douce, 1985; Hong and Copeland, 1990).

A subset of plant OPPP enzymes are present in the cytosol, with most plant cells

containing a cytosolic oxidative branch. Genes encoding cytosolic G6PDH have been identified

in Arabidopsis, potato, tobacco, and maize (Schnarrenberger et al., 1995; von Schaewen et al.,

1995; The Arabiopsis Genome Initiative, 2000; Knight et al., 2001). Non-oxidative enzymes are

also found in the cytosol of some plant species. The global genome analysis of Arabidopsis

showed except for transaldolase and transketolase, all other non-oxidative enzymes have both

cytosolic and plastidic isozymes (The Arabidopsis Genome Initiative, 2000).

Connections between Cytosolic and Plastidic OPPP Enzymes

In at least some species, OPPP enzymes are not completely duplicated in the cytosol or

plastid, and the cytosol is likely to produce intermediates that need to be utilized in the plastid. A

number of transporters on the plastid envelope membrane connect the cytosolic and plastidic

OPPP.

In the Arabidopsis genome, six genes encode functional plastidic phosphate transporters

that can be grouped into four classes: the glucose 6-phosphate/phosphate transporters (AtGPT1

and AtGPT2), the triose-phosphate/phosphate transporter (AtTPT), the

phosphoenolpyruvate/phosphate transporters (AtPPT1 and AtPPT2), and the xylulose 5-

phosphate (Xul 5-P)/phosphate transporter (AtXPT) (reviewed in Weber, 2004).

Generally, all transporters have broad substrate specificity. For example, GPT can accept

glucose 6-phosphate (G6P), triose phosphate, 3-phosphoglyceric acid, and Xul 5-P as counter-

exchange substrates for inorganic phosphate. XPT, the most recently identified transporter in the









plastidic phosphate transporter family, can transport triose phosphates, Xul 5-P, and inorganic

phosphate (Eicks et al., 2002).

The reversible reactions in the non-oxidative section, gene redundancy and subcellular

distribution of OPPP enzymes combined with a set of transporters on the plastid membrane,

suggests that the OPPP in plants has multiple levels of redundancy (Figure 1-2).



Nittlte reduction
Sucrose Starch Glutamine synthesis

I t t
GIC-6-P ADP-GIc Fatty acid
Frx/Trx
NADPH GI c-6-P systm
Glc-6-P
6-PGIcA
6PGDH Pi GPT Pi
PGDH6PGDH
NADPH J- CO.. r -I
NADPH C Triose-P TPT Triose-P CO,
oxPPP
Rbu-5-P oxPPP

Pi W1* XPT pi Rib-5-P

Rib-5-P XIu-5-P Xlu-5-P Nucleotides
Ery-4-P



PPT Shikimic acid
pathway
Pi Pi
Cytosol iPlastid



Figure 1-2. Multiple levels of redundancy of the OPPP in Arabidopsis. The OPPP provides
reducing power and carbon skeletons for many biosynthetic pathways (shown in
pink). Adapted from Kruger and von Schaewen, 2003, pp240, Figure 2.

Mechanism of 6-Phosphogluconate Dehydrogenase (6PGDH) Enzymes

One method to investigate the biological roles of the plastidic and cytosolic OPPP is to

identify OPPP mutants. 6PGDH is and ideal enzyme to target for mutant studies, because of its

non-reversible mechanism. Complete loss of the enzyme activity is lethal since a high









concentration of 6PG is toxic to eukaryotic cells, including Drosophila melanogaster (Gvozdev

et al., 1976; Hughes and Lucchesi, 1977; He et al., 2007), Saccharomyces cerevisae (Lobo and

Maitra, 1982) and Trypanosoma brucei (Hanau et al., 2004). However, plants have multicopies

of 6PGDH enzymes, and these isozymes are localized both in the cytosol and plastid. A loss of

6PGDH activity has not yet been reported in higher plants.

6PGDH( EC 1.1.1.44) converts 6-phosphogluconate (6-PG) to ribulose 5-phosphate and

CO2 by a three-step acid-base mechanism: dehydrogenation, decarboxylation and keto-enol

tautomerization (Cervellati et al., 2008). Two residues in 6PGDH assist all those three steps, one

acting as an acid and the other as a base (Figure 1-3). In Trypanosoma brucei, the catalytic

residues are Glu192 and Lysl85. When the enzyme binds to the substrate, the lysine residue is

unprotonated, and it receives a proton from the 3-hydroxyl of 6-PG to give a 3-keto intermediate.

Then this same residue lysine donates the proton to help decarboxylation and form 1,2-enediol of

ribulose 5-phosphate, which is converted to ribulose 5-phosphate (Montin et al., 2007). In this

oxidative decarboxylation reaction, NADP works as the oxidant to accept a proton from aqueous

environment to give NADPH, one of the major reductants in the cell. The release of CO2 in the

decarboxylation step makes the reaction being non-reversible.

In yeast as well as many other species, the 6PGDH monomer contains two domains, N-

terminal domain and C-terminal domain. The N-terminal a/P "co-enzyme binding" domain of

6PGDH is a NADP+ binding domain. The C-terminal domain is almost fully helical, contributing

to the dimerization (He et al., 2007). It has been shown that 6PGDH isozymes can form

heterodimers and homodimers in maize (Bailey-Serres and Nguyen, 1992).










--COOH --COOH

En --" NADP NADPH En O
H -- OH H --OH


H OH H OH
H+
H --OH H -- OH
CH,2PO3" CH2OPO-

6-phosphogluconate (6PG) 3-keto-6PG



-- COO- --COOH C
0CO

En En
NH+ --H -NH HO OH

H OH H OH
H OH H OH
CH2OPO3- CH2OPO3

Ribulose 5-phosphate (Ru5P) 1,2-enediol of Ru5P

Figure 1-3. 6-Phosphogluconate dehydrogenase (6PGDH) convertes 6-phosphogluconate to
ribulose 5-phosphate a three-step acid-base mechanism: dehydrogenation,
decarboxylation and keto-enol tautomerization, producing NADPH and CO2.

The Importance of the OPPP and 6PGDH to Cellular Metabolism

The oxidative pentose phosphate pathway (OPPP) is a major source of reducing power

and metabolic intermediates in central carbon metabolism. NADPH produced in the non-

reversible oxidative section of this pathway is the major source of reductant in nonphotosynthetic

cells. The reversible non-oxidative section of this pathway provides substrates for glycolysis and

several biosynthetic pathways, such as biosynthesis of nucleic acids, lignin, polyphenols, amino

acids.









Substrates for Nucleotide Synthesis

In plants, de novo synthesis of nucleotides requires 5- phosphoribosyl-1-pyrophosphate

(PRPP). PRPP is synthesized from ribose 5-phosphate by PRPP synthase. The OPPP provides

ribose 5-phosphate through the action of ribose 5-phosphate isomerase (RPI), which converts

ribulose 5-phosphate to ribose 5-phosphate.

The Arabidopsis radial swelling 10 (rsw]O) mutant suggests that a primary requirement

for nucleotides is in cellulose synthesis. The rsw]O mutant is mutated in a gene predicted to

encode a cytosolic ribose 5-phosphate isomerase (RPI). The root elongation in rsw]O mutants is

greatly reduced, suggesting a defect in cell wall biosynthesis. Since the orientation of

microfibrils assembled by cellulose influences the balance between longitudinal and radial

growth, the level of cellulose in rsw]O mutant was analyzed and it was lower than in wild type

(Howles et al., 2006). The mutation in RPI and the defect of cellulose synthesis in rsw]O can be

connected by UDP-glucose, theoretically. UDP-glucose is the substrate for the growing cellulose

chain (Carpita and Delmer, 1981). Uridine nucleotides, the substrate of UDP-glucose, are

products from the activity of RPI in the OPPP (reviewed in Boldt and Zrenner, 2003). It has been

shown that with exogenous uridine and UDP-glucose, the phenotype of rswlO mutant can be

suppressed. In contrast, rswl mutant, which is defective in the enzyme believed to use UDP-

glucose as substrate, cannot be rescued by exogenous uridine or UDP-glucose. Thus, the

cellulose defect in rswlO mutants is caused by the defect of nucleotides synthesis (Howles et al.,

2006).

In Arabidopsis, three nuclear genes are predicted to encode RPIs (reviewed in Kruger and

von Schaewen, 2003). Generally, there is a duplication of the pyrimidine synthesis pathway

between the cytosol and plastid, and transporters on the plastid membrane will enhance the

redundancy. Since the rsw]O mutation only changed a single amino acid residue in one of two









cytosolic RPI enzymes, those RPI isoforms should have a very different expression pattern,

which has been confirmed on the transcript level. A further support is that both the cytosolic

RPIs can complement the rsw]O phenotype when expressed behind a constitutive promoter

(Howles et al., 2006).

Substrates for Aromatic Amino Acid Synthesis

Another important cabon skeleton provided by OPPP is erythrose 4-phosphate, which is a

downstream product in the non-oxidative sugar pool. Both erythrose 4-phosphate and

phosphoenolpyruvate (PEP) are condensed and reduced to give shikimic acid. After that,

shikimic acid is condensed with another PEP to give chorismic acid, the precursor of aromatic

amino acids: phenylalanine, tryptophan, and tyrosine (reviewed in Herrmann and Weaver, 1999).

The Arabidopsis cuel mutant is the indirect evidence suggesting that the substrate defect

of the shikimate pathway will cause aromatic amino acid defect. This mutant is mutated in

AtPPT1, which transports another precursor of shikimate pathway phosphoenolpyruvate (PEP)

into plastids. The cuel mutant was originally isolated because of its defect in the light-induced

expression of the chlorophyll a/b binding protein. The mutant is unable to produce anthocyanins

and several other products that are derived from the shikimate pathway. Moreover, the reticulate

leaf phenotype of cuel can be rescued by feeding of aromatic amino acids (Streatfield et al.,

1999).

Additionally, the phenotype of cuel can be complemented by constitutive overexpression

of a heterologous PPT from cauliflower. Also, the defect in plastidic PEP import could be

bypassed by overexpression of plastid-targeted pyruvate orthosphophate dikinase. This is

because that the overexpression of pyruvate orthosphophate dikinase allows pyruvate imported

into the plastids and converted to PEP in the stroma (Voll et al., 2003).









The reason that the biosynthesis of aromatic amino acids is not severely affected in the

whole cue] mutant plant is because of the gene redundancy. As there are two PPTs in

Arabidopsis, AtPPT2 is suggested to be a more housekeeping functional in providing

chloroplasts with PEP as a precursor for the shikimate pathway, while AtPPT1 is involved in

provision of signals for correct mesophyll development (Knappe et al., 2003). Therefore, the

import of PEP is reduced but not eliminated in cue] mutant plant (Voll et al., 2003).

NADPH for Fatty Acid Synthesis

NADPH, which can be produced by the 6PGDH reaction, is the major power resource in

nonphotosynthetic tissue for maintaining the redox potential necessary to protect against

oxidative stress, especially for fatty acid biosynthesis and nitrogen assimilation.

In almost all plants, de novo fatty acid synthesis occurs in plastids. The first committed

step of fatty acid synthesis is the formation of malonyl-CoA from acetyl-CoA and bicarbonate

which is catalysed by acetyl-CoA carboxylase (Harwood, 1988). Since acetyl-CoA cannot cross

the plastid membrane, it must be generated within the plastid using precursors which are

synthesized inside the plastid or actively imported from the cytosol. The imported precursors

include glucose 6-phosphate (G6P), dihydroxyacetone phosphate, phosphoenolpyruvate (PEP),

pyruvate, malate, and acetate, and their relative rates of utilization depend on the plant species,

the tissue studied, and also the developmental stage (reviewed in Rawsthome, 2002).

The production of fatty acids requires the provision of reducing power in the form of

NADPH and NADH (Slabas and Fawcett, 1992). Those reducing equivalents are used for the

reduction of 3-ketoacyl-ACP to acyl-ACP, a reaction catalyzed by two subunits of the fatty acid

synthase complex 3-ketoacyl reductase and enoyl-ACP reductase. Photosynthesis can provide

reductants directly. In nonphotosynthetic tissues, those reductants can be generated during the

synthesis of acetyl-CoA from glucose 6-phosphate (G6P), malate or pyruvate (Smith et al., 1992;









Kang and Rawsthorne, 1996), or via the OPPP. B. napus plastids have a full complement of

glycolytic enzymes as well as OPPP oxidative reaction enzymes, so they have both

photosynthetic and heterotrophic properties. But it has been confirmed that in B. napus, OPPP

does provide a source of NADPH for fatty acid synthesis, contributing an estimated about 35%

of the total required (Schwender et al., 2003). Thus, the 6PGDH enzyme is likely to be involved

in providing reducing power for fatty acid synthesis in B. napus.

An additional evidence of the relationship between NADPH produced by OPPP and fatty

acid synthesis is found from sunflower. In sunflower embryo plastids, pyruvate utilization for

fatty acid synthesis can be stimulated by the addition of glucose 6-phosphate (G6P). In contrast,

glucose 6-phosphate (G6P) addition has no effect on the utilization of malate. Furthermore,

while addition of pyruvate stimulated the activity of the OPPP, malate suppressed its activity

(Pleite et al., 2005). This is because malate utilization can provide NADPH, NADH and acetyl-

CoA via plastidic NADP-malic enzyme and pyruvate dehydrogenase complex. Under these

conditions there would be no demand for additional NADPH from the OPPP.

Furthermore, an Arabidopsis gpt mutant shows a large reduction of the number of oil

bodies in pollen with gametogenesis defects (Niewiadomski et al., 2005). The gpt mutant is

mutated in AtGPT1, which transport glucose 6-phosphate (G6P) into plastids. Since there is a

10-fold increase in the accumulation of AtGPT1 transcripts in guard cells relative to mesophyll

cells in wild type plants (Niewiadomski et al., 2005), it is suggested that the role of glucose 6-

phosphate in lipid synthesis in Arabidopsis pollen is to provide reducing power via OPPP rather

than the precursor of acetyl-CoA.

NADPH for Nitrate Assimilation

The NADPH provided by OPPP is also important in nitrogen assimilation and glutamine

synthesis. As nitrate is reduced to nitrite in the cytosol, the uptake of nitrite into the plastids and









its subsequent reduction by nitrite reductase and glutamate synthase are potentially important

control points (Bowsher et al., 2007). Several studies have shown how electrons from the plastid-

localized OPPP go to nitrite in wheat and pea roots (Oji et al., 1985; Bowsher et al., 1992).

Generally, OPPP-generated NADPH acts as the initial reductant to generate reduced ferredoxin

via a ferredoxin- NADP oxidoreductase (FNR). Then, the reduced ferredoxin provides electrons

to the nitrite reduction process.

Moreover, it has been shown that carbohydrate flux through the plastidic OPPP can be

stimulated by feeding NO2- or glutamine to isolated chloroplasts of green pepper fruits (Thom

and Neuhaus, 1995). Finally, the treatment of nitrate induced the increase of 6PGDH activity as

well as protein and transcript level in maize root plastids (Redinbaugh and Campbell, 1998).

Leaves of C4 plants such as maize have two kinds of photosynthetic cells: the bundle

sheath cells (BSC) and the mesophyll cells (MC). The distribution of enzymes involved in

nitrogen metabolism is different in these two cell types. Nitrate reductive reaction occurs in MC

(Harel et al., 1977; Moore and Black, 1979) while the photorespiratory pathway is in BSC

(Ohnishi and Kanai, 1983). In maize, although different photosynthetic ferredoxin- NADP

oxidoreductases (FNRs) are localized in MC and BSC, respectively nonphotosynthetic

ferredoxin- NADP oxidoreductases (FNRs) are predominantly detected in MC rather than BSC

(Matsumura et al., 1999). Thus, even in photosynthetic organs, the reductant for nitrogen

assimilation is supplied, at least partially, via OPPP (Favery et al., 1998).

Starch Synthesis in Maize Kernels

In plants, the glucose 6-phosphate (G6P) is either the substrate of OPPP or the precursor

of starch biosynthesis. Glucose 6-phosphate is converted to glucose 1-phosphate by

phosphoglucomutase. Then ADP-glucose is synthesized from glucose 1-phosphate and ATP by









the action of ADP-glucose pyrophosphorylase (AGPase). Next, ADP-glucose is transferred to

the elongating starch chain by the activity of starch synthase isoforms.

The localization of ADP-glucose production is different between plant species and

tissues. In most plants or tissues, this enzyme reaction is localized in the plastids. Thus, the

import of G6P is required for starch synthesis. For example, Arabidopsis wild type pollen

contains many starch granules in plastids. However, the gpt mutant pollen only contains starch-

free plastids (Niewiadomski et al., 2005). In fact, other evidence suggests that not only G6P

uptake but also the plastidic OPPP is involved in starch synthesis.

In cereal endosperm such as maize, the AGPase is known to be largely extraplastidic

(Beckles et al., 2001). In this tissue, sucrose is the major nutrient. Thus, ADP-glucose could also

be converted from glucose 1-phosphate, the product of sucrose degradation with UDP-glucose

as the intermediate. As ADP-glucose is synthesized in cytosol, cereal endosperm has an

additional transporter to transport ADP-glucose across the plastid envelope membrane. In maize,

this transporter was identified by the brittle] mutant, which has a reduced starch content and

accumulates ADP-glucose in the cytosol (Shannon et al., 1996). Also, the amyloplasts of the

brittle] mutant do not synthesize starch from exogenously supplied ADP-glucose (Shannon et

al., 1998). A similar phenotype has been found for barley mutants carrying mutations in the

Hv.Nstl gene (Patron et al., 2004). It is suggested that ADP-glucose is exchanged with AMP by

those adenylate transporters (reviewed in Emes and Neuhaus, 1997). Thus, a model of starch

synthesis in maize kernel is presented in Figure 1-4. Although many starch biosynthetic mutants,

such as brittle and shrunken2 mutants, suggested that major ADP-glucose is synthesized in

cytosol, 13C-labeling experiment suggested that about 80% of the carbons must go to glycolysis

or the OPPP before they are incorporated into starch in maize kernels (Spielbauer et al., 2006).









Since the precursor for starch synthesis is not from the plastidic OPPP, the NADPH produced by

OPPP may be more important in explanation of this phenomenon.



/ ADP-Glucose-- ADP-Glucose STARCH

Sucrosel.... .... Hexose-P Hexose-P PGD3
Glucose
Glucose Glycolysis + 4 OPPP
Triose-P Triose-P ntose-P


PEP

PLASTID





TCA


MITOCHONDRION
CYTOSOL


Figure 1-4. Generalized schematic of starch biosynthesis in maize kernels utilizing central
carbon metabolism. The conventional flow of starch biosynthesis is: sucrose/glucose
is transported into cell and converted to ADP-glucose in cytosol; then ADP-glucose is
transported into plastid and incorporated into starch. Glycolysis, OPPP and TCA
cycle in mitochondrion contribute carbon skeleton for starch biosynthesis, too.

Sugar Induction of Transporter Expression

Although it has been shown that the products of 6PGDH, ribulose 5-phosphate and

NADPH, are very important in organisms, there was no direct phenomenon caused by 6PGDH

defect in plants until 6-amino nicotinamide (6-AN), an inhibitor of 6PGDH, is applied on

Arabidopsis (Lejay et al., 2008). 6-AN is converted in vivo to an analogue of NADP+, which is a









potent inhibitor of 6PGDH and G6PD in neural tissue (Favery et al., 1998) and restricts flux

through the OPPP (Garlick et al., 2002).

Ion transporter gene expression in the roots is up-regulated by light and sugars, such as

NRT1.1 and NRT2.1 (NO3- transporter), AMT1.3 (NH4+ transporter), SULTR1.1 (S042-

transporter) (Lejay et al., 2003). However, after applying 6-AN to Arabidopsis roots, sugar

induction at the transcript level of the transporter genes is reduced (Lejay et al., 2008). As the

sulfur and nitrogen assimilatory pathways are well coordinated, it is not surprising that the

availability of one element regulates the other pathway. Since the NADPH-dependent regulation

has been found in animals for the redox regulation of fertilization in the mouse (Urner and

Sakkas, 2005), the reducing power produced by the OPPP might be the key element in regulation

of root ion transporters.

Questions

In a word, OPPP is a central portion of carbon metabolism in plants as it serves multiple

roles, such as providing substrates and reducing power for many nutrient biosynthesis. This

pathway has a great redundancy in plants, since there are multicopies of OPPP enzymes in the

cytosol and plastid, and many transporters on the plastid membrane connect the carbohydrate

pools of the cytosol and plastid. 6PGDH, the enzyme working in the third step of OPPP, has

three copies in maize: Pgd], Pgd2, and Pgd3 (Bailey-Serres et al., 1992). In this thesis, I will

show that mutation in Pgd3 gives a visible phenotype, and this phenotype is caused by the some

roles non-redundant of PGD3.









CHAPTER 2
RESULTS: MUTATIONS IN PGD3 CAUSES RGH SEED PHENOTYPES

Background

In maize, mutants in Pgd] and Pgd2 have been previously identified (Averill et al.,

1998). A mutant in Pgd3 was identified recently via forward genetic screen from a maize

mutagenic population. Most mutagenesis experiments in maize apply maize transposons, such as

Ac/Ds (Activator and Dissociation) and MuDR/Mu (Robertson's Mutator) elements (Walbot,

2000). Mutations in Pgd3 were identified in the UniformMu transposon-tagging population

(McCarty et al., 2005). UniformMu has the MuDR/Mu elements in the W22 inbred, which create

tagged mutations at a high rate.

Initial applications of transposon tagging in maize relied on correlating the inheritance of

a plant phenotype with a band on a DNA hybridization blot (Walbot, 2000). Several techniques

have been developed for amplifying and sequencing genomic DNA flanking to transposon

insertions. A specific band can be amplified by PCR primers specific to transposons TIRs with a

gene specific primer, priming from the flanking genomic DNA. Since this specific band indicates

a specific insertion, it can be determined whether this transposon insertion segregates with a

phenotype.

UniformMu is a high-copy transposon population, and the numerous Mu elements create

challenges for the molecular analysis of the tagged mutations. MuTAIL PCR was developed to

identify transposon insertion sites in genetic backgrounds with high-copy transposons (Settles et

al., 2004). By conducting the optimized PCR with the Mu-specific primer and a series of

arbitrary primers, a collection of genomic sequences flanking to the transposon insertions can be

obtained. Informatic analysis showed that only a small fraction of the flanking sequences have a

significant similarity to maize repetitive sequences. Also, those sequences are matched to the









TIGR Zea mays Gene Index (ZMGI) to get the annotations of the loci disrupted by those novel

insertions. Moreover, by matching to assembled genomic islands (MAGI,

http://www.plantgenomics.iasate.edu/maize/), we can design locus-specific primers as the co-

dominant markers for those novel insertions. Thus, it is possible to know whether those novel

insertions may be the cause of the mutant phenotype by co-segregation analysis. For example, a

mutant allele pgd3-umul has been shown to co-segregate with a rough endosperm (rgh)

phenotype (Settles et al., 2007).

Co-segregation Analysis of pgd3-umul

The co-segregation between pgd3-umul and a rgh phenotype was analyzed by PCR with

co-dominant markers, and this analysis has been extended up to 323 meiotic products. All PCR

results showed that, with the gene specific left and right primer, the wild type allele can be

amplified from both heterozygous and wild type seeds, and cannot be amplified from the

homozygous mutants. However, the mutant allele pgd3-umul can only be amplified when there

is a rgh phenotype allele by conducting PCR with a gene specific primer and a TIR primer

(Figure 2-1). Thus, pgd3-umul is tightly linked with the rgh phenotype, and the linkage is less

than 0.31cM.

Identification of the pgd3-umu2 Allele

An additional mutant allele was identified from the UniformMu population by a reverse

genetic screen (Figure 2-2). DNA was extracted from the remaining independent rgh mutants

from the UniformMu transposon-tagging population. The Pgd3 gene-specific primer and the TIR

primer were used to screen for mutants that can give a positive amplification. Those PCR

products which had different size with the product of pgd3-umul amplification were sequenced

to confirm the insertion. The insertion site of the second allele, named as "pgd3-umu2", is 308 bp









TIR
006-


,' Right


pgd3 FS

Pgd3 Ger


T


98%


Left
rgh/+


Left/Right



TIR/Right


C


Right
+/+


0+/+ rghlrgh


Left/Right




TTR/Right


Figure 2.1. pgd3-umul co-segregates with a rough endosperm (rgh) seed phenotype. A)
Schematic of the pgd3-umul insertion site. The identity between pgd3 FST and Pgd3
gene sequence is 98%. The left and right primers are specific to genomic region of the
insertion site as marked by green arrows. The TIR primer is specific to transposon
sequence. B-C)pgd3-umul co-segregates with a rough endosperm (rgh) seed
phenotype. Left/Right primer pair can amplify normal alleles. TIR/Right primer pair
can amplify the insertion site. PCR was completed with genomic DNA extracted from
homozygous normal, heterozygous and homozygous rgh mutant kernels.

3' further from the one of pgd3-umul (Figure 2-2). The homozygous pgd3-umu2 seeds give the

same rgh phenotype as pgd3-umul and the heterozygous pgd3-umu2 plants also exhibit a rgh


ne


&*mass*


ci"9
re ^









segregating ear (Figure 2-3). As pgd3-umul, co-segregation analysis showed that pgd3-umu2 is

linked with the rgh phenotype.


L.. fi`


PCR result


Grid of rgh mutants













B




Pgd3 Gene -


C
(c .


Screen with the primer
pair (TIR/Right) that
can amplify pgd3-umul


TIR TIR
4= 006


Pool
deconvolution
and
sequencing
the PCR
product


TIR TIR
4W= m"


Left Right


Figure 2.2. pgd3-umu2, the second mutant allele ofPgd3, was identified by reverse genetic
screen. A) Schematic of the reverse genetic screen procedure. Genomic DNA was
extracted from the remaining rgh mutants to make pools. B) Schematic of the pgd3-
umu2 insertion site, which is 308bp 3' further from the one of the pgd3-umul.

Complementation Test

Reciprocal crosses between the heterozygous plants of pgd3-umul and pgd3-umu2 have

been done in greenhouse and field. Before the crosses, both parents were genotyped using PCR

markers. Also, they were self-pollinated to confirm the transmission of the mutation. All

reciprocal crosses of two alleles failed to complement the rgh phenotype (Figure 2-4). Thus,

mutation in Pgd3 causes a rgh phenotype.










pgd3-umull+


pgd3-umu2/+



B pgd3-umu 1 pgd3-umu2


Figure 2-3. The phenotype ofpgd3-umu2 is same as the one ofpgd3-umul. A) Both
heterozygouspgd3 ears segregate for rgh mutant seeds. B) The longitudinal hand
section of the homozygous pgd3 mutant shows that they have same rgh phenotype.







pgd3-umull+


X pgd3-umu2
I


pgd3-umu21+ X pgd3-umul
I


Figure 2-4. Reciprocal crosses between heterozygouspgd3-umul and pgd3-umu2 plants fail to
complement resulting in Fl ears segregating for the rgh mutant phenotype (shown in
the circle).









CHAPTER 3
RESULTS: PGD3 HAS MULTIPLE NON-REDUNDANT ROLES IN MAIZE

Background

In maize, there are three copies of 6PGDH: PGD1, PGD2 and PGD3. The cytosolic loci,

Pgdl and Pgd2, are located on long arm of chromosome 6 and the long arm of chromosome 3,

respectively (Stuber and Goodman, 1984). The gene products, PGD1 and PGD2, respectively,

form homodimeric and heterdimeric isozymes that can be visible as separate bands by activity

staining after native gel electrophoresis (Bailey-Serres et al., 1992). Cytosolic activity is present

in extracts from roots and leaves, but the double-null homozygous mutant (pgdl; pgd2) has no

detectable cytosolic isozymes in those tissues (Averill et al., 1998). However, there is about

30% of wild type activity in the pgdl; pgd2 double mutant seedling roots. Also, the double-null

homozygous mutant has no visible phenotype and is still reproductively viable, so the wild type

levels of cytosolic 6PGDH are not required for development (Averill et al., 1998).

The plastidic 6PGDH is more difficult to purify because of the degradation and

contamination from the cytosolic activity. Krepinsky et al. 2001 have separated the plastidic

6PGDH from the cytosolic 6PGDH from spinach leaves by anion-exchange chromatography and

have sequenced the digested peptide, showing that the chloroplast 6PGDH had a blocked N-

terminus (Krepinsky et al., 2001).

In maize, the predicted Pgd3 locus has been assembled from ZMGI and MAGI

sequences. The predicted PGD3 protein contains a short N-terminal extension, which is absent in

PGD1 and PGD2 sequences and is predicted to be a chloroplast targeting signal. In addition, the

predicted PGD3 protein is more similar to the spinach chloroplast-localized 6PGDH than to the

maize PGD1 and PGD2 (Settles et al., 2007). Finally, the residual activity in homozygous









pgdl;pgd2 double mutants is 30% of wild type and confined to the plastid (Averill et al., 1998).

Thus, PGD3 is predicted to be plastid-localized 6PGDH.

pgd3 Seed Phenotype and Plant Phenotype

Although pgdl; pgd2 homozygous mutants do not have any visible phenotype, the pgd3

mutant seeds show a rough endosperm (rgh) seed mutant phenotype (Figure 3-1). The

longitudial hand sections of mature mutant kernels showed both reduced grain-fill as well as the

failure of embryo development (Figure 2-3). The homozygous mutant seeds could not germinate

under normal conditions, such as in soil or wet paper towels. However, a small fraction of those

homozygous pgd3 mutant seeds could be rescued by growth in culture. Those homozygous

mutant plants show normal plant morphology except for a pale green leaf phenotype and a

reduced growth speed (Figure 3-1).

Both pgd3-umul and pgd3-umu2 Mutants Are Enzymatic Knockouts in Seeds

After activity staining, there are two bands on the native gel loaded with the whole

protein extract of wild type seeds: one slower migrating band, and one faster migrating wide

band, which in fact contains several closely migrating bands (Figure 3-2). The faster migrating

band indicates cytosolic 6PGDH activity, homodimers or heterodimers (Bailey-Serres et al.,

1992). It is missing in the pgdl; pgd2 double homozygous mutant seed protein extract. Also, this

faster migrating band showed a reduced level in pgd] and pgd2 single mutant seeds. The slower

migrating band is PGD3 or the plastidic 6PGDH activity. Although both cytosolic and plastidic

6PGDH are active in wild type endosperm and embryo, the enzyme activity assay showed that

the plastidic activity is lost in both pgd3-umul and pgd3-umu2 homozygous mutant seeds. Thus,

both pgd3 mutant alleles are enzymatic knockouts in maize seeds.







A Normal


Normal

EI1 11


Mutant


Homozygous mutant ear
Ma ANw fl-


Figure 3-1. Phenotype ofthepgd3 mutants. A) Mature normal andpgd3 kernels. The top row
shows the abgerminal side of the kernels, and the bottom row shows the germinal side
of the kernels. B) Normal andpgd3 mutant plants. C) Homozygouspgd3 mutant ear.


Mutant










pgdl; pgd3- pgd3- W22 W22
pgd1 pgd2 W22
pgd2 umul umu2 22 Endo. Emb.



SM O N PGD3
*nw A PGD1/
PGD2

Figure 3-2. 6PGDH activity of pgd mutant and normal seeds. The fast migrating band on this
native PAGE gel indicates PGD1/PGD2 isozyme activities. The slow migrating band
on the native PAGE gel indicates PGD3 activity. Thus, bothpgd3-umul andpgd3-
umu2 are enzymatic nulls. The PGD1/PGD2 isozymes are active inpgd3 mutants
indicating that the cytosolic activity is not sufficient to complement the loss of
plastidic 6PGDH.

Differences in PGD1/PGD2 and PGD3 Activity Cannot Explain pgd3 Mutant Phenotypes

The enzyme activity assay from wild type plant showed that almost all tissues show a

great cytosolic activity in vitro. However, PGD3 activity level showed tissue differences (Figure

3-3). Protein extract from mature leaves has a relatively very low level of PGD3 activity, but

almost all other non-photosynthetic tissues have a great PGD3 activity, including seeds,

immature leaves, and roots. Interesting, although there is a substantial PGD3 activity in the

inflorescence tissues, loss of PGD3 activity in those tissues did not affect the function and

maturation of inflorescence in pgd3 mutant plants. In fact, both the male and female gametes of

the homozygous mutant are fertile, and homozygous mutant ears can be obtained (Figure 3-1).

Another interesting thing is that the most obvious pgd3 phenotype is in tissues with the lowest

PGD3 activity, suggesting that the pale green leaf phenotype is caused by loss of PGD3 activity

in sink tissues.








Mature Immature
Seed Root Adult White Stem Tassel Immature
Seed Root Stem Tassel
Leaf Leaf Ear


|PGD3

MIU EU5PGD11
PGD2



Figure 3-3. 6PGDH activity in different tissues from a wild type plant (W22). The fast migrating
band on this native PAGE gel indicates PGD1/PGD2 isozyme activities. The slow
migrating band on the native PAGE gel indicates PGD3 activity. It is interesting that
most obvious pgd3 phenotype in plant level (pale green leaves) is in tissues with
lowest PGD3 activity (mature adult leaf).
PGD1, PGD2 and PGD3 Can be Co-purified with Plastids
PGD1 and PGD2 are cytosolic and form isoenzyme dimers (Bailey-Serres et al., 1992),

and PGD3 is predicted to be plastidic-localized (Settles et al., 2007). Thus, PGD3 activity is

expected to co-purify with intact plastids. Indeed, PGD3 activity is found in the intact plastid

fraction of isolated chloroplasts and etioplasts from W22 seedling leaves. However,

PGD1/PGD2 also co-purify with plastids, and all three enzymes also co-purify with the stroma

subfraction and membrane subfraction (Figure 3-4). One possibility is that PGD1/PGD2 may

stick on the plastid envelope.The protease thermolysin was used to digest proteins on the outside

of the purified plastids, but this treatment did not alter the activity of any of the 6PGDH enzymes

(data not shown). To test the digestion efficiency of proteases, protease K, thermolysin, and

trypsin were applied on the total protein extract from leaf tissue. The results showed that even

the concentration of protease is increased to 20-fold of the typical treatment, the remaining

PGD1/PGD2 activity is still detectable, but PGD3 activity is completely gone (Figure 3-5). Thus







PGD1/PGD2 isozymes are highly resistant to proteases, and the PGD1/PGD2 activity that
purifies with isolated plastids was not been conclusively shown to be cytosolic.

Protein Super- Intact SMemb-
Extract natant Plastid rane

I!-m PGD3
4 'UIIb PGD1/
PGD2

Figure 3-4. 6PGDH activity after chloroplast isolation using total protein extract from fresh leaf
tissue as a control. The fast migrating band on this native PAGE gel indicates
PGD1/PGD2 isozyme activities. The slow migrating band on the native PAGE gel
indicates PGD3 activity. The first two lanes are control sample, total protein extract,
from leaf tissue. The second two samples are supernatant after the gradient centrifuge
in plastid isolation experiment. The third two samples are intact plastid layer after the
gradient centrifuge. The forth two samples are stroma subfraction after breaking the
intact plastid extract. The final two samples are membrane subfraction after breaking
the intact plastid extract. Thus, both PGD1/PGD2 and PGD3 are active in the plastid
extracts.

Protease K Trypsin
I I I I
Un 1X 5X 10X 20X 20X 10X 5X 1X Un
PGD3
_PGGD1/
W" W O W"MWNoPGD2

Figure 3-5. 6PGDH activity after protease treatment. The total protein extract from seedling leaf
was treated with proteases in 1-fold, 5-fold, 10-fold, 20-fold concentration at 250C for
25min, then loaded on the native PAGE gel for enzyme activity assay. The cytosolic
activity (PGD1/PGD2) is still detectable after treated with 500ug/ml protease. 1-fold:
25ug/ml.









CHAPTER 4
RESULTS: POTENTIAL NON-REDUNDANT ROLES OF PLASTIDIC 6PGDH

Background

It has been shown that the plastidic OPPP is related to starch biosynthesis in maize kernel

by 13C-labelling experiments (Spielbauer et al., 2006). Generally, cereal seeds utilize exogenous

glucose or sucrose to synthesize starch. The exogenous hexose is converted to ADP-glucose by

AGPase in cytosol, after that ADP-glucose is transported into the plastid and is incorporated into

starch. Although it has been suggested that most of ADP-glucose for starch synthesis in cereal

endosperm is from the cytosol, hexose in plastids can be converted to ADP-glucose as well.

Also, both hexose and triose in the cytosol can be transported into the plastid, becoming a part of

OPPP or starch biosynthesis. It has been shown that after providing 13C-labeled glucose, only

20% of 13C -labeled glucose goes into starch directly. It means that about 80% of carbons go

through glycolysis or the OPPP before they are incorporated into starch in maize (Spielbauer et

al., 2006). This suggests that carbohydrate fluxes are robust in maize endosperm. Also, those

fluxes are very stable as the distribution of 13C-labeled glucose in synthesized starch had a very

similar pattern in wild type maize kernels as well as many starch synthesis defect mutants, such

brittle2 and shrunken2 mutants (Spielbauer et al., 2006).

The plastidic OPPP also plays a very important role in plants. It has been suggested that

the restriction of the plastidic OPPP in roots affects the sugar induction of nitrogen and sulfur

transporter expression. (Lejay et al., 2008) Also, in non-photosynthesis tissues, the NAPDH

produced by the plastidic OPPP is a major resource of reducing power for the ferredoxin

regulation system, a major regulation system in many biosynthesis pathways. For example, in

nitrogen assimilation and glutamine synthesis, although nitrate is reduced to nitrite in the cytosol,









nitrite needs to be reduced in plastid by ferredoxin system and nitrite reductase (Bowsher et al.,

1992).

Plastidic 6PGDH is Required for Normal Starch Synthesis in Seeds

Since 13C fully labeled glucose is supplied to cultured wild type kernels, if such glucose

is converted into starch without going through any other metabolic flux, the carbon in the

glucose digested from synthesized starch in kernels should be all 13C carbons, so the percentage

of the 111111 isotopolog glucose would be 100%. However, the isotopolog pattern of starch in

wild type kernels showed that about 15% glucose is incorporated into starch directly (Figure 4-

1). About 65% glucose is converted to triose prior to starch synthesis, indicated by the 111000

and 000111 type glucose. Also, about 10% glucose needs to be converted to pentose, then goes

into starch, indicated by the 110000 and 001111 type glucose (Figure 4-1).

However, the isotopolog pattern in pgd3 homozygous mutant kernels is very different.

About 30% glucose is directly incorporated into starch, shown by the percentage of the 111111

type glucose, which is 2-fold of the percentage in wild type seeds. The percentage of triose

incorporation (111000 and 000111 type glucose) is reduced, but the percentage of pentose

incorporation did not have a significant change. Thus, the metabolic fluxes of normal starch

synthesis are altered significantly by mutations in Pgd3.

Nitrogen Assimilation May be Limited in pgd3 Mutants

As the plastidic OPPP is suggested to be related to nitrogen assimilation and glutamine

synthesis, it is hypothesized that exogenous nutrients may help pgd3 mutant seed germination in

the embryo rescue experiments. Since the germination percentage of the pgd3 seeds in culture

rescue experiments are very low, it is important to find an assimilated nitrogen source that can be

absorbed by kernels and does not impair tissue culture growth. Asparagine is common nutrient in

endosperm culture medium, so this nutrient was added into the MS medium. Asparagine rescue









experiment suggests that exogenous asparagine gives a 2-fold increase in pgd3 mutant

germination (Figure 4-2). Also, this nutrient accelerated germination to some extent.

In addition, total nitrogen and carbon content inpgd3 homozygous mutants and wild type

plants were measured with a CN analyzer to investigate whether pgd3 mutants were able to

uptake nitrate. The homozygous pgd3 mutants are very sick and hard to survive and only one

comparison between mutant and wild type was possible. The mutants in this single experiment

had about 8% nitrogen while normal sibling plants had about 5% nitrogen. These data gives

suggestive evidence thatpgd3 mutants are not significantly impaired in nitrate transport.

40.0000
35.0000 pgd3 mutant
0) = W22
0 30.0000

0)
-0 20 0000

W. 15.0000


50000 1 Ti-

0.0000 I

N3C SQ S, S 1 "



Different isotopolog glucose

Figure 4-1. Mutation in Pgd3 changes isotopolog patterns of glucose in starch from the maize
kernels. The percentage of 111111 isotopolog type glucose inpgd3 mutant is 2-fold
of the one in wild type, indicating that the percentage of direct incorporation of
glucose into starch inpgd3 mutant is greatly increased.













w 2.5/

S2
OC
:P Co 1.5





0-
MS MS+Asn
Culture Medium Type

Figure 4-2. The germination percentage ofthepgd3 homozygous mutant seeds in the culture
rescue experiment. The addition of the asparagine monohydrates in medium helped
germination as the germination percentage in MS+Asn medium is 2-fold higher than
the one in MS medium.









CHAPTER 5
DISCUSSION

The characteristics of 6PGDH enzyme have been studied extensively in Drosophila

melanogaster, Saccharomyces cerevisa, Ovis aries and Trypanosoma brucei. Although the

crystal structure of yeast Gndl protein suggested that there are three domains in 6PGDH (He et

al., 2007), the third domain (C-terminal tail) can be included into the alpha domain structure.

Thus, all 6PGDH enzymes are separated into two domains. The C-terminal is an all-alpha

domain, and the NAD binding domain forms a Rossman fold.

The predicted PGD3 protein shows many similarities to other 6PGDH enzymes, as the

BLAST search of this sequence on the NCBI webpage gives hit on NAD-binding domain and C-

terminal domain. The insertion inpgd3-umul andpgd3-umu2 alleles occurred at 1151 and 1459

bp 3' further of the predicted ATG site, respectively. Thus, the mutation is likely disrupted the

C-terminal domain of 6PGDH, causing the loss of activity.

It is very hard to eliminate cytosolic contamination when purifying the plastidic enzyme

from spinach, so the evidence to confirm purification of plastidic 6PGDH is based on sequence

analysis. However, although the localization of PGD3 is predicted to be plastidic (Settles et al.,

2007), the N-terminal signal in PGD3 is a very weak evidence since it is very short compared to

the plastidic 6PGDH from spinach. Thus, it is not as clear that PGD3 is plastid-localized.

As maize has three copies of 6PGDH, it has been suggested that the complete loss of

PGD1/PGD2 can be complemented by the PGD3 activity (Averill et al., 1998). The enzyme

activity assay suggested that both PGD1/PGD2 and PGD3 were highly active in endosperm and

embryo. However, the visible phenotype ofpgd3 mutants suggested that the PGD3 protein has a

non-redundant role in maize kernels. Aspgd3 seeds show a rgh phenotype with a greatly reduced

grain-fill, it is reasonable to hypothesize that mutation in Pgd3 affects starch biosynthesis in









maize kernels. In maize, brittle2 and shrunken2 mutants are starch biosynthesis defect mutants

that mutated in cytosolic AGPase, which converts glucose 6-phosphate to ADP-glucose in the

cytosol. Thus, those two mutants should reduce the incorporation of glucose from cytosolic

carbon flux. However, the 13C-labelling experiments showed that the carbon fluxes pattern in

those two mutants did not have a significant change (Spielbauer et al., 2006). It has shown that

the metabolic fluxes inpgd3 mutant seeds are significantly different from those in wild type

seeds, as the percentage of directly incorporation is doubled. Thus, PGD3 is required for normal

starch biosynthesis. Then, there are two possible models for starch biosynthesis in maize kernels.

Both two models required exogenous hexose to go through OPPP cycles before starch

incorporation. After OPPP cycles, hexose may be converted into ADP-glucose in the plastid to

synthesize starch, requiring plastidic AGPase. Alternatively, hexose may be transported out of

the plastid, converted to ADP-glucose in the cytosol, and ADP-glucose is transported back to

plastid. The reason why the plastidic OPPP is required is still unknown. There are several

possibilities. The ADP-glucose transporter might be affected by Pgd3 mutation, as the sequence

analysis of this transporter suggested that it is a target of ferredoxin regulation, related to

reducing power provided by OPPP. Also, OPPP provides substrates for nucleotide biosynthesis,

such as AMP, ATP. AMP is the exchanger for ADP-glucose transporter. ATP is a substrate for

synthesizing ADP-glucose.

Although the plastidic 6PGDH is essential in maize seeds, it is interesting that both

female and male gametes of homozygous pgd3 mutant plants are fertile. Thus, the cytosolic

6PGDH might complement the mutation of Pgd3 in some tissues. Also, 6PGDH activity assays

of various plant tissues suggests that both cytosolic and plastidic activity maintain a high level in

almost all non-photosynthetic tissues, including roots and developing leaves. The homozygous









pgd3 plants give a visible phenotype, suggesting that the plastidic 6PGDH has a non-redundant

role at the whole plant level. However, the study of the whole plant level is limited, as the

homozygous mutant plants exhibit very poor growth and very few can survive. Only 1-5% of the

homozygous mutants can germinate by embryo rescue experiments on the basic MS medium.

Interestingly, exogenous asparagine helped mutant seed germination suggesting thatpgd3

mutants have a defect in amino acid biosynthesis. There are several possible explanations for this

phenomenon. First, plastidic OPPP provides NADPH, which is required for nitrite reductase to

convert nitrate into ammonia in plastids. Second, is has been shown that inhibition of 6PGDH

activity will suppress the sugar induction of the nitrate transporter expression. A mutation in

Pgd3 may limit nitrate uptake. However, it is still not clear that whether the amino acid

biosynthesis defect is the primary defect of Pgd3 mutation, as there are many difficulties in

comparing the metabolites inpgd3 plants to wild type plants. First, all mutants are come from the

culture medium and are transplanted to the soil, but the wild type plants are germinated directly

from soil. Also, the mutants grow much slower than wild type plants, so it is very difficult to

make the mutant and wild type plants in the same development level for sampling. A possible

resolution would be to transplant both mutants and wild type plants to hydroponic growth. In

such case, environment effects will be reduced. Also, it would provide the possibility to give

different nutrient sources to know the key nutrient for complementing or worsening thepgd3

phenotype.









CHAPTER 6
MATERIALS AND METHODS

Genomic DNA Extraction

Fresh leaf tissues were ground with urea extraction buffer, or frozen leaf tissues were

ground with liquid nitrogen, and then urea extraction buffer was added in a g F.W. : mL ratio of

1:2.5. The urea extraction buffer was made from 168 g urea, 25 mL of 5M NaC1, 20 mL of 1M

Tris-HCl pH 8, 16 mL of 0.5M EDTA pH 8, 20 mL of sarkosine and 190 mL of H20. The

mixture was transferred to a 2 mL-microfuge tube. An equal volume of phenol:chloroform

:Isoamylalcohol (25:24:1) was added to the tubes and mixed well by gently shaking the tubes for

15 min. The tubes were centrifuged at room temperature for 10 min at 1600 Xg. The upper

aqueous phase was collected in a new tube. The DNA was precipitated from the solution by

adding 0.1 volume of 3 M sodium acetate pH 7.0 and 0.7 volume of isopropanol. After 10 min of

incubation on ice the tubes were centrifuged for 15 min at 1600 Xg. The DNA pellet was washed

with 70% ethanol and air dried. The DNA was dissolved in TE (10 mM Tris-HCl pH 8, 1 mM

EDTA pH 8). Each DNA sample was diluted to approximately100 ng/[tL in water and 1 [tL were

added to a 20 [tL PCR assay reaction mixture.

PCR Assay

The wild type allele was amplified with gene specific primer that is 5' to the transposon

insertion (02S-2018L1: 5'-GGTTAATGTCGACAAGAAGGTGCTG-3') and 3' to the

transposon insertion (02S-2018R1: 5'-CCCTTCTCATACCAACCAATTCCTC-3'). The mutant

allele was amplified with one gene specific primer and TIR8 primer. The TIR8 primer was

composed of the TIR8.1 (5'-CGCCTCCATTTCGTCGAATCCCCTS-3'), TIR8.2 (5'-

CGCCTCCATTTCGTCGAATCCSCTT-3'), TIR8.3 (5'-

SGCCTCCATTTCGTCGAATCCCKT-3') and TIR8.4 (5'-









CGCCTCCATTTCGTCGAATCACCTC-3') primers mixed in a 2:4:4:1 ratio, respectively. The

PCR amplification was carried out in a volume of 20 [L containing 100 ng of template DNA, 1.0

uL of 40 mM MgC12, luL of 100%DMSO, 2.0 tL of 10X PCR buffer, 2.0 uL of 2 mM each

dNTP, 25 pmole of primer and 0.3 uL 50 Unit Taq DNA Polymerase. Thermocycling conditions

were generally 940C for 1 min, 600C for 1 min, 720C for 1 min for 40 cycles. The same DNA

extraction and PCR assay were used for co-segregation analysis, reverse genetic screen ofpgd3-

umu2, and genotyping plants.

Total Protein Extraction

Seeds for enzyme activity were harvest 16 days after pollination (DAP). Roots and

immature white leaves were harvested from seedling 1 week after germination. Tassel and

immature ear were harvested from mature plant before flowering. Mature adult leaves were

harvested from mature plant after flowering. Leaves for all fresh tissues were harvested into

liquid nitrogen and stored at -80 oC. The extraction procedures were carried out at 40C. Frozen

tissues were ground with liquid nitrogen and then added extraction buffer (100 mM Tris-HCl pH

7.5, 30 mM 1,4-Dithiothreitol (DTT), 15% (v/v) glycerol) in the mg/ul ratio of 1:1 for seeds and

mg/ul ratio of 1:2 for other tissues. Then the mixtures were centrifuged for 20 min at 1600 Xg.

The upper aqueous phase was collected in a new tube, and stored at -800C. For protease

treatment, fresh extract was distributed at 50 uL per centrifuge tube, and added protease to make

the final protease concentration to be 25 uL/mL for 1-fold digestion. The stock of protease K and

trypsin was made in 20 mM HEPES pH 8 up to 2 mg/mL. Thermolysin was made at 2 mg/mL in

import buffer (50 mM HEPES/KOH pH 8, 0.33 M sorbitol) with addition of 10 mM CaC12. After

incubation on ice for 45 min or at 250C for 25 min, protease K was terminated with an equal

volume of 4 mM PMSF (phenyl methyl sulfonyl fluoride). PMSF stock solution is made freshly

up to 100 mM in ethanol, and used soon after preparation. Trypsin was terminated with equal









volume of 4 mM PMSF and 0.2 mg/mL soybean trypsin inhibitor. Thermolysin was terminated

with 100 mM EDTA.

Plastid Isolation

Seeds were planted in growth chamber either in dark to obtain etioplastids or in light to

obtain chloroplasts. The temperature of the growth chamber is 30C at day and 20C at night, so

seeds germinated very fast. All procedures were carried out at 40C. Percoll gradients were

prepared freshly before the isolation experiment. To make percoll gradients, 1 mg glutathione

was mixed with 17.5 2X GR-buffer by vortexing, mixed with 17.5 mL Percoll by inversion, spin

at 48000 Xg for 40 min, and then stored in 4C. Seedling leaves were harvested 6 days after

planting, starting at the basal meristem, and then cut into 0.5 to 1 cm sections. Approximately 30

g leaf tissues were ground in 200 mL GR-buffer (50 mM HEPES/KOH pH 7.5, 0.33 M sorbitol,

2 mM EDTA, 5 mM Na-ascorbate, 0.1% BSA) with a Polytro w/PTA35/2M probe set at medium

power. After filtering slurry through one layer of Miracloth, the collected samples were spin at

1800 Xg for 3 min, and supernatant was removed. The pellet was resuspended by swirling in 5

mL GR-buffer, and loaded on the performed Percoll gradients. After gradient centrifugation at

1800 Xg for 15 min, intact plastids were in bottom band, and other bands were removed

carefully with a wide bore pipet. The intact plastid band were diluted about 1:4 with import

buffer (50 mM HEPES/KOH pH 8, 0.33 M sorbitol), invert gently for mixing, and then

centrifuged at 1500 Xg for 6 min. The pellet was resuspend at 1 mg chlorophyll/mL in import

buffer, and stored at -800C. For breaking intact plastids, the pellet was resuspended at 1 mg

chlorophyll/mL in HKM buffer (10 mM HEPES/KOH pH 8, 10 mM MgC12), incubated on ice

for 5 min, and added an equal volume of 2X import buffer. Then the lysates were centrifuged at

42000 Xg for 30 min. The supernatant is the membrane fraction, and the stroma pellet was

resuspended in import buffer at same volume of membrane fraction. For protease thermolysin









treatment, intact chloroplasts were mixed with import buffer at 100 ug chlorophyll/ 0.5 mL

import buffer, and then incubated with 25 uL thermolysin (2 mg/mL in import buffer, 10 mM

CaC12) at 40C for 45 min or at 250C for 25 min. The digestion was terminated by adding 100 uL

50 mM EDTA-import buffer. The plastids were repurified on 35% Percoll-5 mM EDTA-import

buffer, and then resuspended in 1 mL HKM buffer for subfraction.

6PGDH Activity Assay

The protein samples were loaded onto a native polyacrylamide gel and electrophoresed at

20-25 mA and 4C for 2.5 hr. 6PGDH activity was revealed by incubating gels at room

temperature for 30 min to 1 hr, in the dark, in 6PGDH activity stain (0.1 mg/mL NADP 0.1

mg/mL nitro blue tetrazolium, 0.1 mg/mL phenazine methosulfate, 0.5 mg/mL 6-

phosphogluconate, 100 mM Tris-HCl pH 7.5). Activity stain solutions were made just before

using. Gels were stored in water overnight prior to drying.

Mutant Seeds Rescue

The medium for embryo rescue was made with MS and 3% sucrose and sterilized for 30

min, then stored at 40C. The addition of 0.2% asparagine monohydrates was made before

sterilization. Both mutant and normal seeds were harvested 21 days after pollination from

heterozygous ears. The freshly harvested seeds were sterilized with 70% ethanol for 2 min,

followed by 20% (v/v) bleach for 15 min. Next, the seeds were washed by sterilized water for

several times. The pericarp was carefully cut at the endosperm and embryo axis to get the

immature embryo with some endosperm tissues, which was then incubated on the sterilized

medium in a growth chamber (30C at day and 20C at night).











Mutant
pgd31+ ear


21 Mutant
DAP seeds

20% Bleach Culture on
sterilization MS medium,
Normal
seeds
Normal.,






Figure 6-1. Schematic of the mutant seeds rescue experiments. Both mutant and normal seeds
were selected from heterozygous ear 21 days after pollination. After sterilization and
then removing pericarp, those seeds were cultured on MS medium.

13C-labelling Experiments

Heterozygous ears were harvested 8-10 day after pollination and kernels were cultured as

previously described in Spielbauer et al., 2006. For labeling, kernel blocks were transferred onto

fresh culture media containing 77.4 g/L glucose and 2.6 g/L [U-13C 12] glucose. Kernels were

harvested after 7 days, frozen in liquid nitrogen and stored at -800C and transported to Germany

for starch analysis. The measurement of the percentage of isotopolog glucose in starch by NMR

spectroscopy was described in Spielbauer et al., 2006. Different isotopolog glucose was marked

as 6 digit number in order like Figure 6-2.












OH OH
OH OOH
-)H +"H OH Starch

OH OH
3% 97% Hydrolysis
[U-13C6]- unlabeled Sterile culture of maize kernels
glucose glucose
13C-NM R:
Glucose isotopolog
distribution


B
OH OH OH OH OH
5 OOH OOH OOH OOH 0oOH
4 OH OH OH OH OH
o 0 0 0 0
OH OH OH OH OH
001000 110000 000111 111000 111111

1 = 13C, = 12C, X = 13C or 12C


Figure 6-2. Schematic of the 13C-labeling experiments. A) Procedure of the 13C-labeling
experiment. B) Different isotopolog glucose in starch is marked as 6 digit number in
order: 0 stands for 12C and 1 stands for 13C.

Total Nitrogen Measurement

Homozygous mutant plants were obtained from the embryo rescue experiments. Wild type

seeds were planted at the same time when mutants were transplanted into the greenhouse. The

top and second leaves were harvested from those plants. Starting from 2 cm further from the tip,

a 10 cm long rectangle of leaf tissue was cut and dried in 65C for 3 days. Then the dried leaf

tissues were ground to powder. The weights of the fresh and dry tissue were recorded. About 5









mg dry powder was rolled in tin cups for the carbon and nitrogen analysis in CN analyzer. Apple

standard sample in range from 3 mg to 6 mg was used for calibration in this measurement.









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BIOGRAPHICAL SKETCH

Li Li was born in Changsha, Hunan province, China in 1982. She finished her primary

school and middle school in her hometown Yueyang, Hunan province. After graduating from

Yueyang No. 1 High School in 1999, she attended Wuhan University in Hubei province. In July,

2003, she received her Bachelor of Science degree in biology. In January 2006, Li enrolled in the

Horticultural Sciences Department at the University of Florida to pursue graduate education. She

received her master degree from the University of Florida in the summer of 2008. She plans to

pursue a career dedicated to biology research.





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1 MULTIPLE NON-REDUNDANT ROLES FOR PLASTIDIC 6-PHOSPHOGLUCONATE DEHYDROGENASE (6PGDH) IN MAIZE By LI LI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Li Li

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3 To my Mom and Dad

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4 ACKNOWLEDGMENTS I would like to express my sincere gratitude an d thanks to my adviser Dr. Mark Settles, for introducing me to this project. His kindne ss, pleasant personality, and patience made my masters study at UF very enjoyable. More im portantly, he showed me the way to find great happiness from research and supported my ideas to broaden my research experience. I also thank Dr. Cline for al l his support during my project His insight and good advice helped me improve on my research and thesis. I also learned a lot of other helpful information through the happy work with him. Meanwhile, I also want to express my thanks to the lab members of Dr. Settles, for their assistance and time to make this project come from a proposal to reality. Dr. Tseung taught me about enzyme activity analysis and mutant seed rescue. Dr. Spielbauer conducted much of the 13C-labeling experiments. Other people I would like to thank are my committee members: Dr. William Gurley and Dr. Kevin Folta. Their support and gu idance helped me during my th esis research and the writing of this document. Likewise, special thanks go to the researchers in Technical University of Munich, Germany. Their NMR analysis of the isotopolog pattern of starch in 13C-labeling experiments was a critical contribution to this project. Finally, I would like to thank my parents for their support, my thanks to them is beyond any scope of the languages.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4 LIST OF FIGURES .........................................................................................................................7 ABSTRACT ...................................................................................................................... ...............8 CHAP TER 1 INTRODUCTION .................................................................................................................. 10 General Background ...............................................................................................................10 Oxidative Pentose Phosphate Pathway (OPPP) ..............................................................10 Subcellular Localizatio n of OPPP Enzym es ................................................................... 12 Connections between Cytosolic and Plastidic OP PP Enzymes .......................................12 Mechanism of 6-Phosphogluconate Dehydrogenase (6PGDH) Enzym es ......................13 The Importance of the OPPP and 6PGDH to Cellular Metabolism .......................................15 Substrates for Nucleotide Synthesis ................................................................................16 Substrates for Aromatic Amino Acid Synthesis .............................................................. 17 NADPH for Fatty Acid Synthesis ................................................................................... 18 NADPH for Nitrate Assimilation .................................................................................... 19 Starch Synthesis in Maize Kernels .................................................................................. 20 Sugar Induction of Tran sporter Expression .....................................................................22 Questions ................................................................................................................................23 2 RESULTS: MUTATIONS IN PGD3 C AUSES RGH SEED PHENOTYPES ...................... 24 Background .................................................................................................................... .........24 Co-segregation Analysis of pgd3-umu1 .................................................................................25 Identification of the pgd3-umu2 Allele ...................................................................................25 Complementation Test .......................................................................................................... ..27 3 RESULTS: PGD3 HAS MULTIPLE NONRE DUNDANT ROLES IN MAIZE ................. 30 Background .................................................................................................................... .........30 pgd3 Seed Phenotype and Plant Phenotype ............................................................................31 Both pgd3-umu1 and pgd3-umu2 Mutants Are E nzymatic Knockouts in Seeds ...................31 Differences in PGD1/PGD2 and PGD3 Activity Cannot Explain pgd3 Mutant Phenotypes .................................................................................................................... ......33 PGD1, PGD2 and PGD3 Can be Co-purified with Plastids ...................................................34

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6 4 RESULTS: POTENTIAL NON-REDUNDANT ROLES OF PLASTIDI C 6PGDH ............36 Background .................................................................................................................... .........36 Plastidic 6PGDH is Required for Norm al Starch Synthesis in Seeds .................................... 37 Nitrogen Assimilation May be Limited in pgd3 Mutants ....................................................... 37 5 DISCUSSION .................................................................................................................... .....40 6 MATERIALS AND METHODS ...........................................................................................43 Genomic DNA Extraction ...................................................................................................... 43 PCR Assay ..............................................................................................................................43 Total Protein Extraction ..........................................................................................................44 Plastid Isolation ............................................................................................................. .........45 6PGDH Activity Assay .......................................................................................................... .46 Mutant Seeds Rescue ..............................................................................................................46 13C-labelling Experiments ......................................................................................................47 Total Nitrogen Measurement ..................................................................................................48 LIST OF REFERENCES ...............................................................................................................50 BIOGRAPHICAL SKETCH .........................................................................................................56

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7 LIST OF FIGURES Figure page 1-1 Schematic of the oxidative pentose phosphate pathway.. .................................................. 11 1-2 Multiple levels of redundancy of the OPPP in Arabidopsis.. ............................................ 13 1-3 6-Phosphogluconate dehydrogenase (6 PGDH) convertes 6-phosphogluconate to ribulose 5-phosphate a three-st ep acid-base m echanism. .................................................. 15 1-4 Generalized schematic of starch b iosynthesis in maize kernels utilizing central carbon metabolism.. ........................................................................................................... 22 2-1 pgd3-umu1 co-segregates with a rough endosperm ( rgh) seed phenotype.. ......................26 2-2 pgd3-umu2, the second mutant allele of Pgd3, was identified by reverse genetic screen. ....................................................................................................................... .........27 2-3 The phenotype of pgd3-umu2 is sam e as the one of pgd3-umu1 ......................................28 2-4 Reciprocal crosses between heterozygous pgd3-umu1 and pgd3-umu2 pla nts fail to complement resulting in F1 ears segregating for the rgh mutant phenotype. ....................29 3-1 Phenotype of the pgd3 mutants. .........................................................................................32 3-2 6PGDH activity of pgd mutant and norm al seeds. ............................................................ 33 3-3 6PGDH activity in different tissu es from a wild type plant (W22). .................................. 34 3-4 6PGDH activity after chloroplast isolation using total protein extr act from fresh leaf tissue as a control. .......................................................................................................... ....35 3-5 6PGDH activity after protease treatm ent. .......................................................................... 35 4-1 Mutation in Pgd3 changes isotopolog patterns of gl ucose in starch from the maize kernels.. ..................................................................................................................... .........38 4-2 The germination percentage of the pgd3 hom ozygous mutant seeds in the culture rescue experiment. ............................................................................................................ .39 6-1 Schematic of the mutant seeds rescue experiments.. ......................................................... 47 6-2 Schematic of the 13C-labeling experiments........................................................................ 48

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8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science MULTIPLE NON-REDUNDANT ROLES FOR PLASTIDIC 6-PHOSPHOGLUCONATE DEHYDROGENASE (6PGDH) IN MAIZE By Li Li August 2008 Chair: Andrew Settles Major: Horticultural Science The oxidative pentose phosphate pathway (O PPP) serves multiple roles in primary metabolism. Enzymes for the oxidative section of the OPPP are found both in the cytosol and plastid. Several mutant studies have suggested that cytosolic and plastidic OPPP enzymes are redundant including 6-phosphogluconate dehydroge nase (6PGDH). 6PGDH enzymes catalyze the third non-reversible step of the oxidative section of the OPPP. Maize mutations in the cytosolic 6PGDH enzymes, pgd1 and pgd2, do not show obvious phenotypes beyond loss of enzyme activity. In this thesis, two knockout alleles of the maize Pgd3 locus were identified to investigate the role of this gene in central carbon metabolism. The pgd3 mutants disrupt plastidlocalized 6PGDH activity and cause a rough endosperm ( rgh ) phenotype that affects both grainfill and embryo development. Consistent with the reduced grain-fill phenotype, 13C-glucose labeling experiments during seed development suggested that pgd3 mutants disrupt carbohydrate flux for starch synthesis. PGD1, PGD2, and PG D3 are all active in both the endosperm and embryo. These data suggest that PGD3 has a non-redundant ro le for seed development. Moreover, homozygous pgd3 seeds can be rescued through tissue culture experiment. The addition of asparagine in the ti ssue culture medium increases the rescue of mutant seeds,

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9 suggesting that amino acid synthesis is limiting in pgd3 mutants. The homozygous pgd3 mutant plants show normal morphology but are slow to green and late flower ing. PGD3 activity is restricted to sink tissues suggesting that the sl ow to green phenotype is due to disruptions in carbon metabolism during leaf expansion.

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10 CHAPTER 1 INTRODUCTION General Background Oxidative Pentose Phosphate Pathway (OPPP) The oxidative pentose phosphate pathway (OPPP) is a central process in plant m etabolism. The OPPP includes a series of enzy mes that converts glucose 6-phosphate into a pool of phosphorylated sugars with 3 to 7 carb ons. Those sugars can be converted back into glucose 6-phosphate allowing the pathway to undergo cycles. Figure 1-1 shows a generalized schematic of the OPPP showing the reactions and intermediates through the pathway. The OPPP has two major metabolic roles: providing redu cing power to the cell and providing carbon intermediates for multiple biosynthetic pathways. Both of these roles are thought to be essential to the cell and will be discussed in greater detail in section 1.2. The OPPP provides reductant to the cell in the form of NADPH, which is synthesized in the oxidative section. The oxidative section incl udes three enzymes catalyzing three reactions. Initially, glucose 6-phosphate dehydrogenase (G6PDH) oxidizes glucose 6-phosphate (G6P) to phosphogluconolactone. 6-Phosphogluconolactonase th en converts phosphogluconolactone to 6phosphogluconate very quickly. After that 6-phosphogluconate dehydrogenase (6PGDH) oxidatively decarboxylates 6-phosphogluconate to ri bulose 5-phosphate. Importantly, all three of the oxidative reactions are non-reve rsible and loss of any of these enzyme activities is expected to disrupt the entire OPPP. The non-oxidative section of the OPPP provide s the pool of phosphorylated sugars that are needed in a variety of metabolic pathways The five enzymes working in the non-oxidative section are: ribose 5-phosphate isomerase (RP I), ribulose 5-phosphat e 3-epimease (RPE), transaldolase, transketolase, and glucose 6-phosphate isomeras e. These enzymes convert the

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11 product of 6PGDH, ribulose 5-phosphate, to other sugar phosphates. The pentose phosphate sugar pool includes: 5 carbon sugars (ribose 5phosphate and xylulose 5-phosphate), a 7 carbon sugar (sedoheptulose 7-phosphate), a 4 carbon s ugar (erythrose 4-phosph ate), a 3 carbon sugar (triose 3-phosphate), and 6 carbon sugars (fructose 6-phosphate and glucose 6-phosphate). The non-oxidative enzymes catalyze reversible reactions so it is possible to synthesize all of the phosphate sugar pool by utilizing glucose-6-p hosphate and ATP (ap Rees, 1985; reviewed in Kruger and von Schaewen, 2003). Figure 1-1. Schematic of the oxidative pentose p hosphate pathway. The number in black circle denotes the enzyme that catalyzes eac h of the steps: 1. Glucose 6-phosphate dehydrogenase; 2. 6-Phosphogluconol actonase; 3. 6-Phosphogluconate dehydrogenase (6PGDH); 4. Ribose 5-phosphate isomerase; 5. Ribulose 5-phosphate 3-epimerase; 6. Transketolase; 7. Transal dolase; 8. Glucose 6-phosphate isomerase.

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12 Subcellular Localization of OPPP Enzymes OPPP enzym es in animal and yeast cells are lo cated exclusively in the cytosol. In higher plants, the complete set of OPPP enzymes are found in the plastids (Nishimura and Beevers, 1979; Journet and Douce, 1985; Hong and Copeland, 1990). A subset of plant OPPP enzymes are present in the cytosol, with most plant cells containing a cytosolic oxidative branch. Genes encoding cytosolic G6PDH have been identified in Arabidopsis, potato, tobacco, and maize (Sc hnarrenberger et al., 1995; von Schaewen et al., 1995; The Arabiopsis Genome Initiative, 2000; Kn ight et al., 2001). Non-oxidative enzymes are also found in the cytosol of some plant species. The global ge nome analysis of Arabidopsis showed except for transaldolas e and transketolase, all other non-oxidative enzymes have both cytosolic and plastidic isozymes (The Arabidopsis Genome Initiative, 2000). Connections between Cytosolic and Plastidic OPPP Enzymes In at leas t some species, OPPP enzymes are not completely duplicated in the cytosol or plastid, and the cytosol is likely to produce intermediates that need to be utilized in the plastid. A number of transporters on the plastid envelope membrane conn ect the cytosolic and plastidic OPPP. In the Arabidopsis genome, six genes encode functional plastidic phosphate transporters that can be grouped into four classes: the glucose 6-phosphate/ phosphate transporters (AtGPT1 and AtGPT2), the triose-phosphate/ phosphate transporter (AtTPT), the phosphoenolpyruvate/phosphate transporters (AtPPT1 and AtPPT2), and the xylulose 5phosphate (Xul 5-P)/phosphate transporte r (AtXPT) (reviewed in Weber, 2004). Generally, all transporters have broad subs trate specificity. For example, GPT can accept glucose 6-phosphate (G6P), triose phosphate, 3-phosphoglyceric acid, and Xul 5-P as counterexchange substrates for inorganic phosphate. XPT, the most recently identif ied transporter in the

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13 plastidic phosphate transporter family, can trans port triose phosphates, Xul 5-P, and inorganic phosphate (Eicks et al., 2002). The reversible reactions in the non-oxidative section, gene redundancy and subcellular distribution of OPPP enzymes combined with a se t of transporters on the plastid membrane, suggests that the OPPP in plants has multip le levels of redundancy (Figure 1-2). Figure 1-2. Multiple levels of redundancy of the OPPP in Arabidopsis. The OPPP provides reducing power and carbon skeletons for many biosynthetic pathways (shown in pink). Adapted from Kruger a nd von Schaewen, 2003, pp240, Figure 2. Mechanism of 6-Phosphogluconate Dehydrogenase (6PGDH) Enz ymes One method to investigate the biological roles of the plastidic and cytosolic OPPP is to identify OPPP mutants. 6PGDH is and ideal enzyme to target for mutant studies, because of its non-reversible mechanism. Complete loss of the enzyme activity is lethal since a high

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14 concentration of 6PG is toxic to eukaryotic cells, including Drosophila melanogaster (Gvozdev et al., 1976; Hughes and Lucchesi, 1977; He et al., 2007), Saccharomyces cerevisae (Lobo and Maitra, 1982) and Trypanosoma brucei (Hanau et al., 2004). However, plants have multicopies of 6PGDH enzymes, and these isozymes are locali zed both in the cytosol and plastid. A loss of 6PGDH activity has not yet been reported in higher plants. 6PGDH( EC 1.1.1.44) converts 6-phosphogluconate (6-PG) to ribulos e 5-phosphate and CO2 by a three-step acid-base mechanism: dehydrogenation, decarboxylation and ketoenol tautomerization (Cervellati et al., 2008). Two residues in 6PGDH assist all those three steps, one acting as an acid and the other as a base (Figure 1-3). In Trypanosoma brucei the catalytic residues are Glu192 and Lys185. When the enzyme bi nds to the substrate, the lysine residue is unprotonated, and it receives a proton from the 3-hydr oxyl of 6-PG to give a 3-keto intermediate. Then this same residue lysine donates the proton to help decarboxylation a nd form 1,2-enediol of ribulose 5-phosphate, which is converted to ribulose 5-phosphate ( Montin et al., 2007). In this oxidative decarboxylatio n reaction, NADP works as the oxidant to accept a proton from aqueous environment to give NADPH, one of the major reductants in the cel l. The release of CO2 in the decarboxylation step makes the r eaction being non-reversible. In yeast as well as many other species, the 6PGDH monomer contains two domains, Nterminal domain and C-terminal domain. The N-terminal / "co-enzyme binding" domain of 6PGDH is a NADP+ binding domain The C-terminal domain is almost fully helical, contributing to the dimerization (He et al ., 2007). It has been shown that 6PGDH isozymes can form heterodimers and homodimers in maize (Bailey-Serres and Nguyen, 1992).

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15 Figure 1-3. 6-Phosphogluconate dehydrogenase (6PGDH) convertes 6-phosphogluconate to ribulose 5-phosphate a three-step ac id-base mechanism: dehydrogenation, decarboxylation and ketoenol tautom erization, producing NADPH and CO2. The Importance of the OPPP and 6PGDH to Cellular Metabolism The oxidative pentose phosphate pathway (O PPP) is a m ajor source of reducing power and metabolic intermediates in central car bon metabolism. NADPH produced in the nonreversible oxidative section of this pathway is the major source of reductant in nonphotosynthetic cells. The reversible non-oxidative section of this pathway provides substrates for glycolysis and several biosynthetic pathways, su ch as biosynthesis of nucleic acids, lignin, pol yphenols, amino acids.

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16 Substrates for Nucleotide Synthesis In plants, de novo synthesis of nucleotides requir es 5phosphoribos yl-1-pyrophosphate (PRPP). PRPP is synthesized from ribose 5-ph osphate by PRPP synthase. The OPPP provides ribose 5-phosphate through the action of ribose 5-phosphate isom erase (RPI), which converts ribulose 5-phosphate to ribose 5-phosphate. The Arabidopsis radial swelling 10 (rsw10 ) mutant suggests that a primary requirement for nucleotides is in cellulose synthesis. The rsw10 mutant is mutated in a gene predicted to encode a cytosolic ribose 5-phosphate isomerase (RPI). The root elongation in rsw10 mutants is greatly reduced, suggesting a defect in cell wall biosynthesis. Sin ce the orientation of microfibrils assembled by cellulose influences the balance between l ongitudinal and radial growth, the level of cellulose in rsw10 mutant was analyzed and it was lower than in wild type (Howles et al., 2006). The mutation in RPI a nd the defect of cellulose synthesis in rsw10 can be connected by UDP-glucose, theoretically. UDP-gluco se is the substrate fo r the growing cellulose chain (Carpita and Delmer, 1981). Uridine nucleo tides, the substrate of UDP-glucose, are products from the activity of RPI in the OPPP (revi ewed in Boldt and Zrenner, 2003). It has been shown that with exogenous uridine and UDPglucose, the phenotype of rsw10 mutant can be suppressed. In contrast, rsw1 mutant, which is defective in the enzyme believed to use UDP glucose as substrate, cannot be rescued by exogenous uridine or UDPglucose. Thus, the cellulose defect in rsw10 mutants is caused by the defect of nucleotides synthesis (Howles et al., 2006). In Arabidopsis, three nuclear genes are predic ted to encode RPIs (reviewed in Kruger and von Schaewen, 2003). Generally, ther e is a duplication of the pyrimidine synthesis pathway between the cytosol and plastid, and transporte rs on the plastid membrane will enhance the redundancy. Since the rsw10 mutation only changed a single am ino acid residue in one of two

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17 cytosolic RPI enzymes, those RPI isoforms sh ould have a very different expression pattern, which has been confirmed on the transcript level. A further support is that both the cytosolic RPIs can complement the rsw10 phenotype when expressed behind a constitutive promoter (Howles et al., 2006). Substrates for Aromatic Amino Acid Synthesis Another im portant cabon skeleton provided by OPPP is erythrose 4-phosphate, which is a downstream product in the non-oxidative suga r pool. Both erythrose 4-phosphate and phosphoenolpyruvate (PEP) are condensed and redu ced to give shikimic acid. After that, shikimic acid is condensed with another PEP to give chorismic acid, the precursor of aromatic amino acids: phenylalanine, tryptophan, and tyrosine (reviewed in Herrmann and Weaver, 1999). The Arabidopsis cue1 mutant is the indirect evidence suggesting that the substrate defect of the shikimate pathway will cause aromatic amino acid defect. This mutant is mutated in AtPPT1, which transports another precursor of shikimate pathway phosphoenolpyruvate (PEP) into plastids. The cue1 mutant was originally isolated beca use of its defect in the light-induced expression of the chlorophyll a/b binding protein. The mutant is un able to produce anthocyanins and several other products that are derived from the shikimate pathway. Moreover, the reticulate leaf phenotype of cue1 can be rescued by feedi ng of aromatic amino ac ids (Streatfield et al., 1999). Additionally, the phenotype of cue1 can be complemented by constitutive overexpression of a heterologous PPT from cauliflower. Also, the defect in plastidic PEP import could be bypassed by overexpression of pl astid-targeted pyruvate orthosphophate dikinase. This is because that the overexpression of pyruvate or thosphophate dikinase allows pyruvate imported into the plastids and converted to PE P in the stroma (Voll et al., 2003).

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18 The reason that the biosynthesis of aromatic am ino acids is not severely affected in the whole cue1 mutant plant is because of the gene redundancy. As there are two PPTs in Arabidopsis, AtPPT2 is suggested to be a more housekeeping functional in providing chloroplasts with PEP as a precursor for the shikimate pathway, while AtPPT1 is involved in provision of signals for correct mesophyll deve lopment (Knappe et al., 2003). Therefore, the import of PEP is reduced but not eliminated in cue1 mutant plant (Voll et al., 2003). NADPH for Fatty Acid Synthesis NADPH, which can be produced by the 6PGDH reaction, is the major power resource in nonphotosynthetic tissue for maintaining the re dox potential necessary to protect against oxidative stress, especially for fatty acid biosynthesis and nitrogen assimilation. In almost all plants, de novo fatty acid synthesis occurs in plastids. The first committed step of fatty acid synthesis is the formation of malonyl-CoA from ac etyl-CoA and bicarbonate which is catalysed by acetyl-CoA carboxylase (Harwood, 1988). Since acetyl-CoA cannot cross the plastid membrane, it must be generated w ithin the plastid using precursors which are synthesized inside the plastid or actively im ported from the cytosol. The imported precursors include glucose 6-phosphate (G6P), dihydroxyacetone phosphate, phosphoenolpyruvate (PEP), pyruvate, malate, and acetate, and their relative rates of utilization depend on the plant species, the tissue studied, and also the developmen tal stage (reviewed in Rawsthorne, 2002). The production of fatty acids requires the prov ision of reducing power in the form of NADPH and NADH (Slabas and Fawcett, 1992). Those reducing e quivalents are used for the reduction of 3-ketoacyl-ACP to acyl-ACP, a reac tion catalyzed by two subunits of the fatty acid synthase complex 3-ketoacyl reductase and e noyl-ACP reductase. Photosynthesis can provide reductants directly. In nonphotosynt hetic tissues, those reductants can be generated during the synthesis of acetyl-CoA from glucose 6-phosphate (G 6P), malate or pyruvate (Smith et al., 1992;

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19 Kang and Rawsthorne, 1996), or via the OPPP. B. napus plastids have a full complement of glycolytic enzymes as well as OPPP oxidativ e reaction enzymes, so they have both photosynthetic and hete rotrophic properties. But it has been confirmed that in B. napus OPPP does provide a source of NADPH for fatty acid s ynthesis, contributing an estimated about 35% of the total required (Schwender et al., 2003). Thus, the 6PGDH enzyme is likely to be involved in providing reducing power for fatty acid synthesis in B. napus. An additional evidence of the relationship between NADPH produced by OPPP and fatty acid synthesis is found from sunflower. In sunf lower embryo plastids, pyruvate utilization for fatty acid synthesis can be stimulated by the ad dition of glucose 6-phosphat e (G6P). In contrast, glucose 6-phosphate (G6P) additi on has no effect on the utiliza tion of malate. Furthermore, while addition of pyruvate stimul ated the activity of the OPPP, malate suppressed its activity (Pleite et al., 2005). This is because malate utilization can provide NADPH, NADH and acetylCoA via plastidic NADP-malic enzyme and pyruvate dehydrogenase complex. Under these conditions there would be no demand for additional NADPH from the OPPP. Furthermore, an Arabidopsis gpt mutant shows a large redu ction of the number of oil bodies in pollen with gametogenesis de fects (Niewiadomski et al., 2005). The gpt mutant is mutated in AtGPT1, which transport glucose 6-phosphate (G6P) into plastids. Since there is a 10-fold increase in the accumulation of AtGPT1 tr anscripts in guard cells relative to mesophyll cells in wild type plants (Niewiadomski et al., 2005), it is suggested that the role of glucose 6phosphate in lipid synthesis in Ar abidopsis pollen is to provide reducing power via OPPP rather than the precursor of acetyl-CoA. NADPH for Nitrate Assimilation The NADPH provided by OPPP is also im portant in nitrogen assimilation and glutamine synthesis. As nitrate is reduced to nitrite in the cytosol, the uptake of nitrite into the plastids and

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20 its subsequent reduction by nitrite reductase and glutamate syntha se are potentially important control points (Bowsher et al., 2007 ). Several studies have shown how electrons from the plastidlocalized OPPP go to nitrite in wheat and pea roots (Oji et al., 1985; Bowsher et al., 1992). Generally, OPPP-generated NADPH acts as the init ial reductant to generate reduced ferredoxin via a ferredoxinNADP oxidoreduc tase (FNR). Then, the reduced ferredoxin provides electrons to the nitrite re duction process. Moreover, it has been shown that carbohydrate flux through the plastidic OPPP can be stimulated by feeding NO2or glutamine to isolated chloropl asts of green pe pper fruits (Thom and Neuhaus, 1995). Finally, the treatment of nitrat e induced the increase of 6PGDH activity as well as protein and transcript level in maize root plastids (Redinbaugh and Campbell, 1998). Leaves of C4 plants such as maize have two kinds of photosynthetic cells: the bundle sheath cells (BSC) and the mesophyll cells (M C). The distribution of enzymes involved in nitrogen metabolism is different in these two cel l types. Nitrate reductive reaction occurs in MC (Harel et al., 1977; Moore and Black, 1979) while the photorespi ratory pathway is in BSC (Ohnishi and Kanai, 1983). In maize, althou gh different photosynthetic ferredoxinNADP oxidoreductases (FNRs) are localized in MC and BSC, respectively nonphotosynthetic ferredoxinNADP oxidoreductases (F NRs) are predominantly detected in MC rather than BSC (Matsumura et al., 1999). Thus, even in phot osynthetic organs, the re ductant for nitrogen assimilation is supplied, at least par tially, via OPPP (Favery et al., 1998). Starch Synthesis in Maize Kernels In plants, the glucose 6-phosphate (G6P) is eith er the substrate of OPPP or the precursor of starch biosynthesis. Glucose 6-phosphate is converted to glucose 1-phosphate by phosphoglucom utase. Then ADP-glucose is synthesized from glucose 1-phosphate and ATP by

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21 the action of ADP-glucose pyropho sphorylase (AGPase). Next, ADP-g lucose is transferred to the elongating starch chain by the activ ity of starch synthase isoforms. The localization of ADP-glucose production is different between plant species and tissues. In most plants or tissues, this enzyme reaction is localized in the plastids. Thus, the import of G6P is required for starch synthesis. For example, Arabidopsis wild type pollen contains many starch granules in plastids. However, the gpt mutant pollen only contains starchfree plastids (Niewiadomski et al., 2005). In f act, other evidence sugges ts that not only G6P uptake but also the plastidic OPPP is involved in starch synthesis. In cereal endosperm such as maize, the AGP ase is known to be largely extraplastidic (Beckles et al., 2001). In this tissu e, sucrose is the major nutrient. Thus, ADP-glucose could also be converted from glucose 1-phosphate, the pr oduct of sucrose degradation with UDPglucose as the intermediate. As ADP-glucose is synt hesized in cytosol, cereal endosperm has an additional transporter to transport ADP-glucose acr oss the plastid envelope membrane. In maize, this transporter was identified by the brittle1 mutant, which has a reduced starch content and accumulates ADP-glucose in the cytosol (Shannon et al., 1996). Also, the amyloplasts of the brittle1 mutant do not synthesize starch from exogenously supplied ADP-glucose (Shannon et al., 1998). A similar phenotype has been found for barley mutants carrying mutations in the Hv.Nst1 gene (Patron et al., 2004) It is suggested that ADP-glucose is exchanged with AMP by those adenylate transporters (reviewed in Em es and Neuhaus, 1997). Thus, a model of starch synthesis in maize kernel is presented in Figur e 1-4. Although many starch biosynthetic mutants, such as brittle2 and shrunken2 mutants, suggested that major ADP-glucose is synthesized in cytosol, 13C-labeling experiment suggested that about 80% of the carbons mu st go to glycolysis or the OPPP before they are incorporated into starch in maize kernels (Spielbauer et al., 2006).

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22 Since the precursor for starch synthesis is not from the plas tidic OPPP, the NADPH produced by OPPP may be more important in explanation of this phenomenon. Figure 1-4. Generalized schematic of starch biosynthesis in maize kernels utilizing central carbon metabolism. The conventional flow of starch biosynthesis is: sucrose/glucose is transported into cell and converted to ADP -glucose in cytosol; then ADP-glucose is transported into plastid and incorporated into starch. Glycolysis, OPPP and TCA cycle in mitochondrion contribute carbon sk eleton for starch biosynthesis, too. Sugar Induction of Transporter Expression Although it has been shown that the produc ts of 6PGDH, ribulose 5-phosphate and NADPH, are very im portant in organisms, there was no direct phenomenon caused by 6PGDH defect in plants until 6-amino nicotinamide (6AN), an inhibitor of 6PGDH, is applied on Arabidopsis ( Lejay et al., 2008 ) 6-AN is converted in vivo to an analogue of NADP+, which is a

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23 potent inhibitor of 6PGDH and G6PD in neural tissue (Favery et al., 1998) and restricts flux through the OPPP (Garlick et al., 2002). Ion transporter gene expression in the roots is up-regulated by light and sugars, such as NRT1.1 and NRT2.1 (NO3 transporter), AMT1.3 (NH4 + transporter), SULTR1.1 (SO4 2transporter) (Lejay et al., 2003). However, afte r applying 6-AN to Arabidopsis roots, sugar induction at the transcript level of the transporter genes is redu ced (Lejay et al., 2008). As the sulfur and nitrogen assimilatory pathways ar e well coordinated, it is not surprising that the availability of one element re gulates the other pathway. Sin ce the NADPH-dependent regulation has been found in animals for the redox regulati on of fertilization in the mouse (Urner and Sakkas, 2005), the reducing power produced by the OPPP might be the key element in regulation of root ion transporters. Questions In a word, OPPP is a central portion of carbon me tabolism in plants as it serves multiple roles, such as providing substrates and reduc ing power for many nutrien t biosynthesis. This pathway has a great redundancy in plants, since there are multicopies of OPPP enzymes in the cytosol and plastid, and many transporters on the plastid membrane connect the carbohydrate pools of the cytosol and plastid. 6PGDH, the enzy me working in the third step of OPPP, has three copies in maize: Pgd1, Pgd2, and Pgd3 (Bailey-Serres et al., 1992). In this thesis, I will show that mutation in Pgd3 gives a visible phenotype, and this phenotype is caused by the some roles non-redundant of PGD3.

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24 CHAPTER 2 RESULTS: MUTATIONS IN PGD3 CAUSES RGH SEED PHENOTYPES Background In m aize, mutants in Pgd1 and Pgd2 have been previously id entified (Averill et al., 1998). A mutant in Pgd3 was identified recently via forward genetic screen from a maize mutagenic population. Most mutage nesis experiments in maize apply maize transposons, such as Ac / Ds ( Activator and Dissociation ) and MuDR / Mu ( Robertsons Mutator ) elements (Walbot, 2000). Mutations in Pgd3 were identified in the UniformMu transposon-tagging population (McCarty et al., 2005). UniformMu has the MuDR / Mu elements in the W22 inbred, which create tagged mutations at a high rate. Initial applications of transposon tagging in maize relied on correlati ng the inheritance of a plant phenotype with a band on a DNA hybridizat ion blot (Walbot, 2000). Several techniques have been developed for amplifying and seque ncing genomic DNA fla nking to transposon insertions. A specific band can be amplified by P CR primers specific to transposons TIRs with a gene specific primer, priming from the flanking genomic DNA. Since this specific band indicates a specific insertion, it can be determined whether this tran sposon insertion segregates with a phenotype. UniformMu is a high-copy trans poson population, and the numerous Mu elements create challenges for the molecular analysis of the ta gged mutations. MuTAIL PCR was developed to identify transposon insertion site s in genetic backgrounds with hi gh-copy transposons (Settles et al., 2004). By conducting the optimized PCR with the Mu-specific primer and a series of arbitrary primers, a collection of genomic sequences flanking to the transposon insertions can be obtained. Informatic analysis s howed that only a small fraction of the flanking sequences have a significant similarity to maize repetitive sequen ces. Also, those sequences are matched to the

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25 TIGR Zea mays Gene Index (ZMGI) to get the annotations of the loci disrupted by those novel insertions. Moreover, by matching to assembled genomic islands (MAGI, http://www.plantgenomics.iasate.edu/maize/), we can design locus-specific primers as the codominant markers for those novel insertions. T hus, it is possible to know whether those novel insertions may be the cause of the mutant phenotype by co-segre gation analysis. For example, a mutant allele pgd3-umu1 has been shown to co-segregate with a rough endosperm ( rgh) phenotype (Settles et al., 2007). Co-segregation Analysis of pgd3-u mu1 The co-segregation between pgd3-umu1 and a rgh phenotype was analyzed by PCR with co-dominant markers, and this analysis has b een extended up to 323 meiotic products. All PCR results showed that, with the gene specific left and right primer, the wild type allele can be amplified from both heterozygous and wild type seeds, and cannot be amplified from the homozygous mutants. However, the mutant allele pgd3-umu1 can only be amplified when there is a rgh phenotype allele by conduc ting PCR with a gene specific primer and a TIR primer (Figure 2-1). Thus, pgd3-umu1 is tightly linked with the rgh phenotype, and the linkage is less than 0.31cM. Identification of the pgd3-umu2 Allele An additional mutant allele was identifie d from the UniformMu population by a reverse genetic screen (Figure 2-2). DNA was extr acted from the remaining independent rgh mutants from the UniformMu transposon-tagging population. The Pgd3 gene-specific primer and the TIR primer were used to screen for mutants th at can give a positive am plification. Those PCR products which had different size with the product of pgd3-umu1 amplification were sequenced to confirm the insertion. The insertion site of the second allele, named as pgd3-umu2, is 308 bp

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26 Figure 2.1. pgd3-umu1 co-segregates with a rough endosperm ( rgh) seed phenotype. A) Schematic of the pgd3umu1 insertion site. The identity between pgd3 FST and Pgd3 gene sequence is 98%. The left and right prim ers are specific to genomic region of the insertion site as marked by green arrows. The TIR primer is specific to transposon sequence. B-C) pgd3umu1 co-segregates with a rough endosperm ( rgh ) seed phenotype. Left/Right primer pair can amplif y normal alleles. TIR/Right primer pair can amplify the insertion site. PCR was completed with genomic DNA extracted from homozygous normal, heterozygous and homozygous rgh mutant kernels. 3 further from the one of pgd3-umu1 (Figure 2-2). The homozygous pgd3-umu2 seeds give the same rgh phenotype as pgd3-umu1 and the heterozygous pgd3-umu2 plants also exhibit a rgh

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27 segregating ear (Figure 2-3). As pgd3-umu1, co-segregation analysis showed that pgd3-umu2 is linked with the rgh phenotype. Figure 2.2. pgd3-umu2, the second mutant allele of Pgd3, was identified by reverse genetic screen. A) Schematic of the reverse genetic screen procedure. Genomic DNA was extracted from the remaining rgh mutants to make pools. B) Schematic of the pgd3umu2 insertion site, which is 308bp 3 further from the one of the pgd3-umu1. Complementation Test Reciprocal crosses betw een the heterozygous plants of pgd3-umu1 and pgd3-umu2 have been done in greenhouse and field. Before the crosses, both pare nts were genotyped using PCR markers. Also, they were self-pollinated to confirm the transmission of the mutation. All reciprocal crosses of two alleles failed to complement the rgh phenotype (Figure 2-4). Thus, mutation in Pgd3 causes a rgh phenotype.

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28 Figure 2-3. The phenotype of pgd3-umu2 is same as the one of pgd3-umu1 A) Both heterozygous pgd3 ears segregate for rgh mutant seeds. B) The longitudinal hand section of the homozygous pgd3 mutant shows that they have same rgh phenotype.

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29 Figure 2-4. Reciprocal cr osses between heterozygous pgd3-umu1 and pgd3-umu2 plants fail to complement resulting in F1 ears segregating for the rgh mutant phenotype (shown in the circle).

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30 CHAPTER 3 RESULTS: PGD3 HAS MULTIPLE NON-RE DUNDANT ROLES IN MAIZE Background In m aize, there are three copies of 6PGDH: PGD1, PGD2 and PGD3. The cytosolic loci, Pgdl and Pgd2, are located on long arm of chromosome 6 and the long arm of chromosome 3, respectively (Stuber and Goodman, 1984). The gene products, PGD1 and PGD2, respectively, form homodimeric and heterdimeric isozymes that can be visible as separate bands by activity staining after native gel electrophor esis (Bailey-Serres et al., 1992). Cytosolic activity is present in extracts from roots and leaves, but the double-null homozygous mutant ( pgd1; pgd2) has no detectable cytosolic isozymes in those tissues (A verill et al., 1998). However, there is about 30% of wild type activity in the pgd1; pgd2 double mutant seedling roots. Also, the double-null homozygous mutant has no visible ph enotype and is still reproductive ly viable, so the wild type levels of cytosolic 6PGDH are not requi red for development (Averill et al., 1998). The plastidic 6PGDH is more difficult to purify because of the degradation and contamination from the cytosolic activity. Krep insky et al. 2001 have separated the plastidic 6PGDH from the cytosolic 6PGDH from spinach leaves by anion-exchange chromatography and have sequenced the digested peptide, showi ng that the chloroplas t 6PGDH had a blocked Nterminus (Krepinsky et al., 2001). In maize, the predicted Pgd3 locus has been assembled from ZMGI and MAGI sequences. The predicted PGD3 protein contains a short N-terminal extension, which is absent in PGD1 and PGD2 sequences and is predicted to be a chloroplast targeting signal. In addition, the predicted PGD3 protein is more similar to the spinach chloroplast-localized 6PGDH than to the maize PGD1 and PGD2 (Settles et al., 2007). Finally, the residual ac tivity in homozygous

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31 pgd1;pgd2 double mutants is 30% of wild type and conf ined to the plastid (A verill et al., 1998). Thus, PGD3 is predicted to be plastid-localized 6PGDH. pgd3 Seed Phenotype and Plant Phenotype Although pgd1; pgd2 homozygous mutants do not have any visible phenotype, the pgd3 mutant seeds show a rough endosperm ( rg h ) seed mutant phenot ype (Figure 3-1). The longitudial hand sections of mature mutant kern els showed both reduced grain-fill as well as the failure of embryo development (Figure 2-3). Th e homozygous mutant seeds could not germinate under normal conditions, such as in soil or wet pa per towels. However, a small fraction of those homozygous pgd3 mutant seeds could be rescued by growth in culture. Those homozygous mutant plants show normal plant morphology ex cept for a pale green leaf phenotype and a reduced growth speed (Figure 3-1). Both pgd3-umu1 and pgd3-umu2 Mutants Are Enz ymatic Knockouts in Seeds After activity staining, there are two bands on the native gel load ed with the whole protein extract of wild type seeds: one slower migrating band, and one faster migrating wide band, which in fact contains seve ral closely migrating bands (Fi gure 3-2). The faster migrating band indicates cytosolic 6PGDH activity, homodimers or heterodimers (Bailey-Serres et al., 1992). It is missing in the pgd1; pgd2 double homozygous mutant seed protein extract. Also, this faster migrating band showed a reduced level in pgd1 and pgd2 single mutant seeds. The slower migrating band is PGD3 or the plastidic 6PGDH activity. Although both cy tosolic and plastidic 6PGDH are active in wild type endosperm and embryo, the enzyme activity assay showed that the plastidic activity is lost in both pgd3-umu1 and pgd3-umu2 homozygous mutant seeds. Thus, both pgd3 mutant alleles are enzymatic knockouts in maize seeds.

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32 Figure 3-1. Phenotype of the pgd3 mutants. A) Mature normal and pgd3 kernels. The top row shows the abgerminal side of the kernels, and the bottom row shows the germinal side of the kernels. B) Normal and pgd3 mutant plants. C) Homozygous pgd3 mutant ear.

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33 Figure 3-2. 6PGDH activity of pgd mutant and normal seeds. The fast migrating band on this native PAGE gel indicates PGD1/PGD2 isozym e activities. The sl ow migrating band on the native PAGE gel indicates PGD3 activity. Thus, both pgd3-umu1 and pgd3umu2 are enzymatic nulls. The PGD1/PGD2 isozymes are active in pgd3 mutants indicating that the cytosolic activity is not sufficient to complement the loss of plastidic 6PGDH. Differences in PGD1/PGD2 and P GD3 Activity Cannot Explain pgd3 Mutant Phenotypes The enzyme activity assay from wild type pl ant showed that almost all tissues show a great cytosolic activity in vitro. However, PGD3 activity level s howed tissue differences (Figure 3-3). Protein extract from mature leaves has a relatively very low level of PGD3 activity, but almost all other non-photosynthetic tissues ha ve a great PGD3 activity, including seeds, immature leaves, and roots. Interesting, althou gh there is a substantial PGD3 activity in the inflorescence tissues, loss of PGD3 activity in those tissues did not a ffect the function and maturation of inflorescence in pgd3 mutant plants. In fact, both the male and female gametes of the homozygous mutant are fertile, and homozygous mutant ears can be obtained (Figure 3-1). Another interesting thing is that the most obvious pgd3 phenotype is in tissues with the lowest PGD3 activity, suggesting that the pale green l eaf phenotype is caused by loss of PGD3 activity in sink tissues.

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34 Figure 3-3. 6PGDH activity in diffe rent tissues from a wild type plant (W22). The fast migrating band on this native PAGE gel indicates P GD1/PGD2 isozyme activities. The slow migrating band on the native PAGE gel indicates PGD3 activity. It is interesting that most obvious pgd3 phenotype in plant level (pale gr een leaves) is in tissues with lowest PGD3 activity (mature adult leaf). PGD1, PGD2 and PGD3 Can be Co-purified with Plastids PGD1 and PGD2 are cytosolic and form isoe nzyme dimers (Bailey-Serres et al., 1992), and PGD3 is predicted to be plastidic-localized (Settles et al., 2007). Thus, PGD3 activity is expected to co-purify with intact plastids. Ind eed, PGD3 activity is found in the intact plastid fraction of isolated chloroplasts and etio plasts from W22 seedling leaves. However, PGD1/PGD2 also co-purify with plastids, and all three enzymes also co-purify with the stroma subfraction and membrane subfraction (Figure 3-4). One possibility is that PGD1/PGD2 may stick on the plastid envelope.The protease thermolysin was used to digest proteins on the outside of the purified plastids, but this treatment did not alter the activity of any of the 6PGDH enzymes (data not shown). To test the digestion efficien cy of proteases, protease K, thermolysin, and trypsin were applied on the total protein extract from leaf tissue. The results showed that even the concentration of protease is increased to 20fold of the typical treatment, the remaining PGD1/PGD2 activity is stil l detectable, but PGD3 activity is completely gone (Figure 3-5). Thus

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35 PGD1/PGD2 isozymes are highly resistant to proteases, and the PGD1/PGD2 acitivity that purifies with isolated plastids was not b een conclusively shown to be cytosolic. Figure 3-4. 6PGDH activity after ch loroplast isolation using total protein extract from fresh leaf tissue as a control. The fast migrating band on this native PAGE gel indicates PGD1/PGD2 isozyme activities. The slow migrating band on the native PAGE gel indicates PGD3 activity. The first two lanes are control sample, total protein extract, from leaf tissue. The second two samples are supernatant after the gradient centrifuge in plastid isolation experiment. The third tw o samples are intact plastid layer after the gradient centrifuge. The forth two samples are stroma subfraction after breaking the intact plastid extract. The final two sample s are membrane subfraction after breaking the intact plastid extract. Thus, both PGD1/PGD2 and PGD3 are active in the plastid extracts. Figure 3-5. 6PGDH activity after pr otease treatment. The total protein extract from seedling leaf was treated with proteases in 1-fold, 5-fold, 10-fold, 20-fold concentration at 25C for 25min, then loaded on the native PAGE gel for enzyme activity assay. The cytosolic activity (PGD1/PGD2) is still de tectable after treated with 500ug/ml protease. 1-fold: 25ug/ml.

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36 CHAPTER 4 RESULTS: POTENTIAL NON-REDUNDANT ROLES OF PLASTIDIC 6PGDH Background It has been shown that the plas tidic OPPP is related to starch biosynthesis in m aize kernel by 13C-labelling experiments (Spielbauer et al., 2006). Generally, cereal seeds utilize exogenous glucose or sucrose to synthesize starch. The exogenous hexose is converted to ADP-glucose by AGPase in cytosol, after that ADP-glucose is trans ported into the plastid and is incorporated into starch. Although it has been suggested that most of ADP-glucose for starch synthesis in cereal endosperm is from the cytosol, hexose in plas tids can be converted to ADP-glucose as well. Also, both hexose and triose in the cytosol can be transported into the plastid, becoming a part of OPPP or starch biosynthesis. It ha s been shown that after providing 13C-labeled glucose, only 20% of 13C -labeled glucose goes into starch directly. It means th at about 80% of carbons go through glycolysis or the OPPP befo re they are incorporated into starch in maize (Spielbauer et al., 2006). This suggests that carbohydrate fluxes are robust in maize endosperm. Also, those fluxes are very stable as the distribution of 13C-labeled glucose in synthesized starch had a very similar pattern in wild type maize kernels as well as many starch synthesis defect mutants, such brittle2 and shrunken2 mutants (Spielbauer et al., 2006). The plastidic OPPP also plays a very important role in plants. It has been suggested that the restriction of the plastidic OPPP in roots a ffects the sugar induction of nitrogen and sulfur transporter expression. (Lejay et al., 2008) Also, in non-phot osynthesis tissues, the NAPDH produced by the plastidic OPPP is a major resource of reducing power for the ferredoxin regulation system, a major regulation system in many biosynthesis pathways. For example, in nitrogen assimilation and glutamine synthesis, although nitrate is redu ced to nitrite in the cytosol,

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37 nitrite needs to be reduced in plastid by ferredoxin system and nitrite redu ctase (Bowsher et al., 1992). Plastidic 6PGDH is Required for No rmal Starch Synthesis in Seeds Since 13C fully labeled glucose is supplied to cultured wild type kernels, if such glucose is converted into starch w ithout going through any other metabolic flux, the carbon in the glucose digested from synthesized starch in kernels should be all 13C carbons, so the percentage of the 111111 isotopolog glucose would be 100%. However, the isotopolog pattern of starch in wild type kernels showed that about 15% glucose is incorporated into starch directly (Figure 41). About 65% glucose is convert ed to triose prior to starch synthesis, indicated by the 111000 and 000111 type glucose. Also, abou t 10% glucose needs to be converted to pentose, then goes into starch, indicated by the 110000 a nd 001111 type glucose (Figure 4-1). However, the isotopolog pattern in pgd3 homozygous mutant kernels is very different. About 30% glucose is directly incorporated into starch, show n by the percentage of the 111111 type glucose, which is 2-fold of the percentage in wild type seeds. The percentage of triose incorporation (111000 and 000111 type glucose) is reduced, but the percentage of pentose incorporation did not have a significant change Thus, the metabolic fluxes of normal starch synthesis are altered signigicantly by mutations in Pgd3. Nitrogen Assimilation May be Limited in pgd3 Mutants As the plastidic OPPP is suggested to be re lated to nitrogen assimilation and glutamine synthesis, it is hypothesized that exogenous nutrients may help pgd3 mutant seed germination in the embryo rescue experiments. Since the germination pe rcentage of the pgd3 seeds in culture rescue experiments are very low, it is important to find an assimilated nitrogen source that can be absorbed by kernels and does not impair tissue cu lture growth. Asparagine is common nutrient in endosperm culture medium, so this nutrient was added into the MS medium. Asparagine rescue

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38 experiment suggests that exogenous as paragine gives a 2-fold increase in pgd3 mutant germination (Figure 4-2). Als o, this nutrient accelerated germination to some extent. In addition, total nitroge n and carbon content in pgd3 homozygous mutants and wild type plants were measured with a CN analyzer to investigate whether pgd3 mutants were able to uptake nitrate. The homozygous pgd3 mutants are very sick and hard to survive and only one comparison between mutant and wild type was possible. The mutants in th is single experiment had about 8% nitrogen while normal sibling pl ants had about 5% nitrogen. These data gives suggestive evidence that pgd3 mutants are not significantly impaired in nitrate transport. Figure 4-1. Mutation in Pgd3 changes isotopolog patterns of gl ucose in starch from the maize kernels. The percentage of 111111 isotopolog type glucose in pgd3 mutant is 2-fold of the one in wild type, i ndicating that the percentage of direct incorporation of glucose into starch in pgd3 mutant is greatly increased.

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39 Figure 4-2. The germination percentage of the pgd3 homozygous mutant seeds in the culture rescue experiment. The addition of the as paragine monohydrates in medium helped germination as the germination percentage in MS+Asn medium is 2-fold higher than the one in MS medium.

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40 CHAPTER 5 DISCUSSION The characteris tics of 6PGDH enzyme have been studied extensively in Drosophila melanogaster Saccharomyces cerevisa Ovis aries and Trypanosoma brucei Although the crystal structure of yeast Gnd1 protein suggested that there are three dom ains in 6PGDH (He et al., 2007), the third domain (C-termi nal tail) can be included into the alpha domain structure. Thus, all 6PGDH enzymes are separated into two domains. The C-terminal is an all-alpha domain, and the NAD binding domain forms a Rossman fold. The predicted PGD3 protein shows many simila rities to other 6PGD H enzymes, as the BLAST search of this sequence on the NCBI webpage gives hit on NAD-binding domain and Cterminal domain. The insertion in pgd3-umu1 and pgd3-umu2 alleles occured at 1151 and 1459 bp 3 further of the predicted ATG site, respectively. Thus, the mutation is likely disrupted the C-terminal domain of 6PGDH, causing the loss of activity. It is very hard to eliminate cytosolic contamination when purifying the plastidic enzyme from spinach, so the evidence to confirm purif ication of plastidic 6PGDH is based on sequence analysis. However, although the loca lization of PGD3 is predicted to be plastidic (Settles et al., 2007), the N-terminal signal in PGD3 is a very we ak evidence since it is very short compared to the plastidic 6PGDH from spinach. Thus, it is not as clear that PGD3 is plastid-localized. As maize has three copies of 6PGDH, it has been suggested that the complete loss of PGD1/PGD2 can be complemented by the PGD3 activity (Averill et al., 1998). The enzyme activity assay suggested that bo th PGD1/PGD2 and PGD3 were highly active in endosperm and embryo. However, the visible phenotype of pgd3 mutants suggested that the PGD3 protein has a non-redundant role in maize kernels. As pgd3 seeds show a rgh phenotype with a greatly reduced grain-fill, it is reasonable to hypothesize that mutation in Pgd3 affects starch biosynthesis in

PAGE 41

41 maize kernels. In maize, brittle2 and shrunken2 mutants are starch biosynthesis defect mutants that mutated in cytosolic AGPase, which converts glucose 6-phosphate to ADP-glucose in the cytosol. Thus, those two muta nts should reduce the in corporation of glucose from cytosolic carbon flux. However, the 13C-labelling experiments showed th at the carbon fluxes pattern in those two mutants did not have a significant cha nge (Spielbauer et al., 20 06). It has shown that the metabolic fluxes in pgd3 mutant seeds are significantly diffe rent from those in wild type seeds, as the percentage of di rectly incorporation is doubled. T hus, PGD3 is required for normal starch biosynthesis. Then, there are two possible m odels for starch biosynthesis in maize kernels. Both two models required exogenous hexose to go through OPPP cycles before starch incorporation. After OPPP cycles, hexose may be converted into ADP-glucose in the plastid to synthesize starch, requiring plasti dic AGPase. Alternatively, hexose may be transported out of the plastid, converted to ADP-glucose in the cyto sol, and ADP-glucose is transported back to plastid. The reason why the pl astidic OPPP is required is still unknown. There are several possibilities. The ADP-glucose tr ansporter might be affected by Pgd3 mutation, as the sequence analysis of this transporter s uggested that it is a target of ferredoxin regulation, related to reducing power provided by OPPP. Also, OPPP provides substrates for nucleotide biosynthesis, such as AMP, ATP. AMP is the exchanger for ADP -glucose transporter. ATP is a substrate for synthesizing ADP-glucose. Although the plastidic 6PGDH is essential in maize seeds, it is in teresting that both female and male gametes of homozygous pgd3 mutant plants are fer tile. Thus, the cytosolic 6PGDH might complement the mutation of Pgd3 in some tissues. Also, 6PGDH activity assays of various plant tissues suggests that both cytosolic and plastidic activity maintain a high level in almost all non-photosynthetic tissues, includ ing roots and developi ng leaves. The homozygous

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42 pgd3 plants give a visible phenotype, suggesti ng that the plastidic 6PGDH has a non-redundant role at the whole plant level. However, the study of the whole plant level is limited, as the homozygous mutant plants exhibit very poor growth and very few can survive. Only 1~5% of the homozygous mutants can germinate by embryo resc ue experiments on the basic MS medium. Interestingly, exogenous asparagine helped mutant seed germination suggesting that pgd3 mutants have a defect in amino acid biosynthesis. There are several possible explanations for this phenomenon. First, plastidic OPPP provides NADPH, which is required for nitrite reductase to convert nitrate into ammonia in plastids. Sec ond, is has been shown th at inhibition of 6PGDH activity will suppress the sugar induction of the nitr ate transporter expression. A mutation in Pgd3 may limit nitrate uptake. However, it is still not clear that whether the amino acid biosynthesis defect is the primary defect of Pgd3 mutation, as there are many difficulties in comparing the metabolites in pgd3 plants to wild type plants. Firs t, all mutants are come from the culture medium and are transplanted to the soil, bu t the wild type plants are germinated directly from soil. Also, the mutants grow much slower than wild type plants, so it is very difficult to make the mutant and wild type plants in the same development level for sampling. A possible resolution would be to transplant both mutants and wild type plants to hydroponic growth. In such case, environment effects will be reduced. Also, it would provide the possibility to give different nutrient sources to know the key nut rient for complementing or worsening the pgd3 phenotype.

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43 CHAPTER 6 MATERIALS AND METHODS Genomic DNA Extraction Fresh leaf tissues were ground with urea extraction buffer, or frozen leaf tissues were ground with liquid nitrogen, and th en urea extraction buffer wa s adde d in a g F.W. : mL ratio of 1:2.5. The urea extraction buffer was made from 168 g urea, 25 mL of 5M NaCl, 20 mL of 1M TrisHCl pH 8, 16 mL of 0.5M EDTA pH 8, 20 mL of sarkosin e and 190 mL of H2O. The mixture was transferred to a 2 mL-microfuge tube. An equal volume of phenol:chloroform :Isoamylalcohol ( 25:24:1) was a dded to the tubes and mixed well by gently shaking the tubes for 15 min. The tubes were centrifuged at room temperature for 10 mi n at 1600 Xg. The upper aqueous phase was collected in a new tube. The DNA was precipitated from the solution by adding 0.1 volume of 3 M sodium acetate pH 7.0 and 0.7 volume of isopropanol. After 10 min of incubation on ice the tubes were centrifuged fo r 15 min at 1600 Xg. The DNA pellet was washed with 70% ethanol and air dried. The DNA was disso lved in TE (10 mM TrisHCl pH 8, 1 mM EDTA pH 8). Each DNA sample was diluted to approximately100 ng/L in water and 1 L were added to a 20 L PCR assay reaction mixture. PCR Assay The wild typ e allele was amplified with gene specific primer that is 5 to the transposon insertion (02S-2018L1: 5-GGTTAATGTC GACAAGAAGGTGCTG-3) and 3 to the transposon insertion (02S-2018R1: 5-CCCTTC TCATACCAACCAATTCCTC-3). The mutant allele was amplified with one gene specific pr imer and TIR8 primer. The TIR8 primer was composed of the TIR8.1 (5-CGCCTCCATTTCGTCGAATCCCCTS3), TIR8.2 (5CGCCTCCATTTCGTCGAATCCS CTT-3), TIR8.3 (5SGCCTCCATTTCGTCGAATCCCKT-3) and TIR8.4 (5-

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44 CGCCTCCATTTCGTCGAATCACCTC-3) primers mixe d in a 2:4:4:1 ratio, respectively. The PCR amplification was carri ed out in a volume of 20 L containing 100 ng of template DNA, 1.0 uL of 40 mM MgCl2, 1uL of 100%DMSO, 2.0 L of 10X PCR buffer, 2.0 uL of 2 mM each dNTP, 25 pmole of primer and 0.3 uL 50 Unit Taq DNA Polymerase. Thermocycling conditions were generally 94C for 1 min, 60C for 1 min, 72C for 1 min for 40 cycles. The same DNA extraction and PCR assay were used for co-seg regation analysis, revers e genetic screen of pgd3umu2, and genotyping plants. Total Protein Extraction Seeds for enzym e activity were harvest 16 days after pollination (DAP). Roots and immature white leaves were harvested from seedling 1 week after germination. Tassel and immature ear were harvested from mature plan t before flowering. Matu re adult leaves were harvested from mature plant afte r flowering. Leaves for all fresh tissues were harvested into liquid nitrogen and stored at 80 C. The extraction procedures were carried out at 4C. Frozen tissues were ground with liquid ni trogen and then added extracti on buffer (100 mM Tris-HCl pH 7.5, 30 mM 1,4-Dithiothreitol (DTT), 15% (v/v) glycerol) in the mg/ul ratio of 1:1 for seeds and mg/ul ratio of 1:2 for other tissues. Then the mixtures were centrifuged for 20 min at 1600 Xg. The upper aqueous phase was collected in a new tube, and stored at -80C. For protease treatment, fresh extract was dist ributed at 50 uL per centrifuge tube, and added protease to make the final protease concentration to be 25 uL/mL for 1-fold digesti on. The stock of protease K and trypsin was made in 20 mM HEPES pH 8 up to 2 mg/mL. Thermolysin was made at 2 mg/mL in import buffer (50 mM HEPES/KOH pH 8, 0.33 M sorbitol) with addi tion of 10 mM CaCl2. After incubation on ice for 45 min or at 25C for 25 min, protease K was terminated with an equal volume of 4 mM PMSF (phenyl me thyl sulfonyl fluoride). PMSF st ock solution is made freshly up to 100 mM in ethanol, and used soon after pr eparation. Trypsin was terminated with equal

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45 volume of 4 mM PMSF and 0.2 mg/mL soybean trypsin inhibitor. Thermolysin was terminated with 100 mM EDTA. Plastid Isolation Seeds were planted in growth ch am ber either in dark to obtain etioplastids or in light to obtain chloroplasts. The temperature of the growth chamber is 30 C at day and 20C at night, so seeds germinated very fast. All procedures were carried out at 4C. Percoll gradients were prepared freshly before the isolation experiment To make percoll gradients, 1 mg glutathione was mixed with 17.5 2X GR-buffer by vortexing, mi xed with 17.5 mL Percol l by inversion, spin at 48000 Xg for 40 min, and then stored in 4C. Seedling leaves were ha rvested 6 days after planting, starting at the basal meri stem, and then cut into 0.5 to 1 cm sections. Approximately 30 g leaf tissues were ground in 200 mL GRbuffer (50 mM HEPES/KOH pH 7.5, 0.33 M sorbitol, 2 mM EDTA, 5 mM Na-ascorbate, 0.1% BSA) with a Polytro w/PT A35/2M probe set at medium power. After filtering slurry thr ough one layer of Miracloth, the co llected samples were spin at 1800 Xg for 3 min, and supernatant was removed. The pellet was resuspended by swirling in 5 mL GR-buffer, and loaded on the performed Percoll gradients. Af ter gradient centrifugation at 1800 Xg for 15 min, intact plastids were in bottom band, and other bands were removed carefully with a wide bore pipet. The intact plastid band were diluted about 1:4 with import buffer (50 mM HEPES/KOH pH 8, 0.33 M sorbitol) invert gently for mixing, and then centrifuged at 1500 Xg for 6 min. The pellet was resuspend at 1 mg chlorophyll/mL in import buffer, and stored at -80C. For breaking intact plastids, the pellet was resuspended at 1 mg chlorophyll/mL in HKM buffer (10 mM HEPES/KOH pH 8, 10 mM MgCl2), incubated on ice for 5 min, and added an equal volume of 2X import buffer. Then the lysates were centrifuged at 42000 Xg for 30 min. The supernatant is the membrane fraction, and the stroma pellet was resuspended in import buffer at same volume of membrane fraction. For protease thermolysin

PAGE 46

46 treatment, intact chloroplasts were mixed with import buffer at 100 ug chlorophyll/ 0.5 mL import buffer, and then incubated with 25 uL thermolysin (2 mg/mL in import buffer, 10 mM CaCl2) at 4C for 45 min or at 25C for 25 min. The digestion was terminated by adding 100 uL 50 mM EDTA-import buffer. The plastids were repurified on 35% Percoll-5 mM EDTA-import buffer, and then resuspended in 1 mL HKM buffer for subfraction. 6PGDH Activity Assay The protein sam ples were loaded onto a na tive polyacrylamide gel and electrophoresed at 20~25 mA and 4C for 2.5 hr. 6PGDH activity wa s revealed by incubating gels at room temperature for 30 min to 1 hr, in the da rk, in 6PGDH activity stain (0.1 mg/mL NADP+, 0.1 mg/mL nitro blue tetrazolium, 0.1 mg/m L phenazine methosulfate, 0.5 mg/mL 6phosphogluconate, 100 mM Tris-HCl pH 7.5). Activity stain solutions were made just before using. Gels were stored in wa ter overnight prior to drying. Mutant Seeds Rescue The m edium for embryo rescue was made with MS and 3% sucrose and sterilized for 30 min, then stored at 4C. The addition of 0.2% asparagine monohydrates was made before sterilization. Both mutant and normal seeds were harvested 21 days after pollination from heterozygous ears. The freshly harvested seeds were sterilize d with 70% ethanol for 2 min, followed by 20% (v/v) bleach for 15 min. Next, the seeds were washed by sterilized water for several times. The pericarp was carefully cut at the endosperm and embryo axis to get the immature embryo with some endosperm tissues, which was then incubated on the sterilized medium in a growth chamber (30 C at day and 20C at night).

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47 Figure 6-1. Schematic of the mutant seeds resc ue experiments. Both mutant and normal seeds were selected from heterozygous ear 21 days after pollination. Af ter sterilization and then removing pericarp, those seed s were cultured on MS medium. 13C-labelling Experiments Heterozygous ears were harvested 8 day afte r pollination and kernel s were cultured as previously described in Spielbau er et al., 2006. For labeling, kernel blocks were transferred onto fresh culture media containing 77.4 g/L glucose and 2.6 g/L [U-13C 12] glucose. Kernels were harvested after 7 days, frozen in liquid nitrogen and stored at -80C and transported to Germany for starch analysis. The measur ement of the percentage of is otopolog glucose in starch by NMR spectroscopy was described in Spielbauer et al., 2006. Different isotopol og glucose was marked as 6 digit number in order like Figure 6-2.

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48 Figure 6-2. Schematic of the 13C-labeling experiments. A) Procedure of the 13C-labeling experiment. B) Different isotopolog glucose in starch is ma rked as 6 digit number in order: 0 stands for 12C and 1 stands for 13C. Total Nitrogen Measurement Hom ozygous mutant plants were obtained from the embryo rescue experiments. Wild type seeds were planted at the same time when muta nts were transplanted into the greenhouse. The top and second leaves were harvested from those plants. Starting from 2 cm further from the tip, a 10 cm long rectangle of leaf tissue was cut and dried in 65C for 3 days Then the dried leaf tissues were ground to powder. The weights of the fresh and dry tissue were recorded. About 5

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49 mg dry powder was rolled in tin cups for the car bon and nitrogen analysis in CN analyzer. Apple standard sample in range from 3 mg to 6 mg was used for calibration in this measurement.

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50 LIST OF REFERENCES Anderson LE (1971) Chloroplast and cytoplasmic enzym es. 3. Pea leaf ribose 5-phosphate isomerases. Biochim Bi ophys Acta 14;235(1):245-9 ap Rees T (1985) The organisation of gl ycolysis and the oxidative pentose phosphate pathway in plants. Encyclopaedia of Plant Physiol ogy, NS, vol 18. Edited by Douce R, Day DA. Berlin: Springer 391-417 Averill RH, Bailley-Serres J, Kruger NJ (1998) Co-operation between cytosolic and plastidic oxidative pentose phosphate pathways re vealed by 6-phosphogluc onate dehydrogenasedeficient genotypes of maize. Plant J 14:449-457 Bailey-Serres J, Nguyen MT (1992) Purification and characterization of cytosolic 6phosphogluconate dehydrogenase isozymes from maize. Plant Phys iol 100(3):1580-1583 Bailey-Serres J, Tom J, Freeling M (1992) Expression and distribution of cytosolic 6phosphogluconate dehydrogenase isozymes in maize. Biochem Genet 30(5-6):233-46 Beckles DM, Smith AM, ap Rees T (2001) A cytosolic ADP-gl ucose pyrophosphorylase is a feature of graminaceous endosperms, but not of other starch-storing organs. Plant Physiol 125(2):818-27 Boldt R, Zrenner R (2003) Purine and pyrimidine biosynthesis in higher plants. Plant Physiol 117(3):297-304 Bowsher CG, Boulton EL, Rose J, Nayagam S, Emes MJ (1992) Reductant for glutamate synthase is generated by th e oxidative pentose phosphate pathway in non-photosynthetic root plastids. Plant J 2:893-898 Bowsher CG, Lacey AE, Hanke GT, Clarks on DT, Saker LR, Stulen I, Emes MJ (2007) The effect of Glc6P uptake and its subsequent oxidation within pea root plastids on nitrite reduction and glutamate synthe sis. J Exp Bot 58(5):1109-18 Caillau M, Paul Quick W (2005) New insights into plant tr ansaldolase. Plant J 43(1):1-16 Carpita NC, Delmer DP (1981) Concentration and metabo lic turnover of UDP-glucose in developing cotton fibers J Biol Chem 256:308-315 Cervellati C, Li L, Andi B, Guariento A, Dallocchio F, Cook PF (2008) Proper orientation of the nicotinam ide ring of NADP is important for the precatalytic conformational change in the 6-phosphogluconate dehydrogenase. Biochemistry 47:1862-70 Eicks M, Maurino V, Knappe S, Flu gge UI, Fischer K (2002) The plastidic pentose phosphate translocator represen ts a link between the cytosoli c and the plastidic pentose phosphate pathways in plants. Plant Physiol 128:512-522

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52 Kang F, Rawsthorne S (1996) Metabolism of glucose-6-phos phate and utilization of multiple metabolites for fatty acid synthesis by plasti ds from developing oilseed rape embryos. Planta 199:321-327 Knappe S, Lo ttgert T, Schneider A, Voll L, Flu gge UI, Fischer K (2003) Characterization of two functional phosphoenolpyruvate/ phosphate translocator (PPT) genes in Arabidopsis AtPPT1 may be involved in the provision of signals for correct mesophyll development. Plant J 36:411-424 Knight JS, Emes MJ, Debnam PM (2001) Isolation and charac terisation of a full-length genomic clone encoding a plastidic glucose 6-phosphate dehydrogenase from Nicotiana tabacum. Planta 212:499-507 Krepinsky K, Plaumann M, Martin W, Schnarrenberger C (2001) Purification and cloning of chloroplast 6-phosphogluc onate dehydrogenase from sp inach. Cyanobacterial genes for chloroplast and cytosolic isoenzymes en coded in eukaryotic chromosomes. Eur J Biochem 268(9):2678-86 Kruger NJ, von Schaewen A (2003) The oxidative pentose phosphate pathway: structure and organisation. Curr Opin Plant Biol 6:236 Lejay L, Gansel X, Cerezo M, Tillard P, Mu ller C, Krapp A, von Wiren N, Daniel-Vedele F, Gojon A (2003) Regulation of root ion tran sporters by photosynthesis: functional importance and relation with hexokinase. Plant Cell 15:2218-2232 Lejay L, Wirth J, Pervent M, Cross JM, Tillard P, Gojon A (2008) Oxidative pentose phosphate pathway-dependent sugar sensing as a mechanism for regul ation of root ion transporters by photosynthesis Plant Physiol 146(4):2036-53 Li L, Zhang L, Cook PF (2006) Role of the S128, H186, a nd N187 triad in substrate binding and decarboxylation in the sheep liver 6-phosphogluconate dehydrogenase reaction. Biochemistry 24;45(42):12680-6 Lobo Z, Maitra PK (1982) Pentose phosphate pathway mutants of yeast. Mol Gen Genet 185(2):367-8 Matsumura T, Kimata-Ariga Y, Sakakibara H, Sugiyama T, Murata H, Takao T, Shimonishi Y, Hase T (1999) Complementary DNA cloni ng and characterization of ferredoxin localized in bundle-sheath cells of maize leaves. Plant Physiol 119(2):481-8 McCarty DR, Settles AM, Suzuki M, Tan BC Latshaw S, Porch T, Robin K, Baier J, Avigne W, Lai J, Messing J, Koch KE, Hannah LC (2005) Steady-state transposon mutagenesis in inbred ma ize. Plant J 44(1):52-61 Montin K, Cervellati C, Dallocchio F, Hanau S (2007) Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenase. FEBS J 274(24):6426-35

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53 Moore R, Black CC (1979) Nitrogen assimilation pathways in leaf mesophyll and bundle sheath cells of C4 photosynthesis plant formulated from comparative studies with Digitaria sanguinalis (L.) Scop. Pl ant Physiol 64:309-313 Niewiadomski P, Knappe S, Geimer S, Fische r K, Schulz B, Unte US, Rosso MG, Ache P, Flgge UI, Schneider A (2005) The Arabidopsis plastidic glucose 6phosphate/phosphate translocator GPT1 is esse ntial for pollen matura tion and embryo sac development. Plant Cell 17(3):760-75 Nishimura M, Beevers H (1979) Subcellular distributi on of gluconeogenic enzymes in germinating castor bean endos perm. Plant Physiol 64:31-37 Ohnishi J, Kanai R (1983) Differentiation of photorespira tory activity between mesophyll and bundle sheath cells of C4 plants. I. Glycin e oxidation by mitochondria. Plant Cell Physiol 24:1411-1420 Oji Y, Watanabe M, Wakiuchi N, Okamoto S (1985) Nitrite reduction in barley-root plastids: dependence on NADPH coupled with gluc ose-6-phosphate and 6-phosphogluconate dehydrogenases, and possible involvement of an electron carrie r and a diaphorase. Planta 165:85-90 Patron NJ, Greber B, Fahy BF, Laurie DA, Parker ML, Denyer K (2004) The lys5 mutations of barley reveal the nature and importance of plastidial ADP -Glc transporters for starch synthesis in cereal endospe rm. Plant Physiol 135:2088-2097 Pleite R, Pike MJ, Garcs R, Martnez-Force E, Rawsthorne S (2005) The sources of carbon and reducing power for fatty aci d synthesis in the heterotrophic plastid s of developing sunflower (Helianthus annuus L.) embryos. J Exp Bot 56(415):1297-303 Rawsthorne S (2002) Carbon flux and fatty acid synthesi s in plants. Prog Lipid Res 41:182-96 Redinbaugh MG, Campbell WH (1998) Nitrate regulation of the oxidative pentose phosphate pathway in maize (Zea mays L.) root plastids: induction of 6-phosphogluconate dehydrogenase activity, protein and transc ript levels. Plan t Science 134:129-140 Schnarrenberger C, Flechner A, Martin W (1995) Enzymatic evidence for a complete oxidative pentose phosphate pathway in chlor oplasts and an incomplete pathway in the cytosol of spinach leav es. Plant Physiol 108:609-614 Schwender J, Shachar-Hill Y, Ohlrogge JB (2003) A flux model of glycolysis and the oxidative pentose phosphate pathway in developing Brassica napus embryos. J Biol Chem 278:29442-29453

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54 Settles AM, Holding DR, Tan BC, Latshaw SP Liu J, Suzuki M, Li L, O'Brien BA, Fajardo DS, Wroclawska E, Tseung CW, Lai J, Hunter CT 3rd, Avigne WT, Baier J, Messing J, Hannah LC, Koch KE, Becraft PW, Larkins BA, McCarty DR (2007) Sequence-indexed mutations in maize us ing the UniformMu transposon-tagging population. BMC Genomics 9;8:116 Settles AM, Latshaw S, McCarty DR (2004) Molecular analysis of high-copy insertion sites in maize. Nucleic Acids Res 32(6):e54 Shannon JC, Pien FM, Cao H, Liu KC (1998) Brittle-1, an adenylat e translocator, facilitates transfer of extraplastidial synthesized ADP-glucose into amyloplasts of maize endosperms. Plant Physiol 117(4):1235-52 Shannon JC, Pien FM, Liu KC (1996) Nucleotides and nucleoti de sugars in developing maize endosperms (Synthesis of ADP-glucose in brittle 1). Plant Physiol 110:835-843 Slabas AR, Fawcett T (1992) The biochemistry and mo lecular biology of plant lipid biosynthesis. Plant Mol Biol 19:161-191 Smith RG, Gauthier DA, Dennis DT, Turpin DH (1992) Malateand pyruvate-dependent fatty acid synthesis in leuc oplasts from developing casto r endosperm. Plant Physiol 98:1233-1238 Spielbauer G, Margl L, Hannah LC, Rmisch W, Ettenhuber C, Bacher A, Gierl A, Eisenreich W, Genschel U (2006) Robustness of central carbohydrate metabolism in developing maize kernels. P hytochemistry 67(14):1460-75 Streatfield SJ, Weber A, Kinsman EA, Husler RE, Li J, Post-Beittenmiller D, Kaiser WM, Pyke KA, Flgge UI, Chory J (1999) The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisad e cell development, and plastid-dependent nuclear gene expression. Plant Cell 11(9):1609-22 Stuber CW, Goodman MM (1984) Inheritance, intracellular lo calization, and genetic variation of 6-phosphogluconate dehydrogenase isoz ymes in maize. Maydica 29:453-471 Thom E, Neuhaus HE (1995) Oxidation of imported or endogenous carbohydrates by isolated chloroplasts from green pepper fr uits. Plant Physiol 109(4):1421-1426 Urner F, Sakkas D (2005) Involvement of the pentos e phosphate pathway and redox regulation in fertilization in the mouse. Mol Reprod Dev 70(4):494-503 Voll L, Husler RE, Hecker R, Weber A, We issenbck G, Fiene G, Waffenschmidt S, Flgge UI (2003) The phenotype of the Arabidopsis cue1 mutant is not simply caused by a general restriction of the shikimate path way. Plant J 36(3):301-17 von Schaewen A, Langenkamper G, Gr aeve K, Wenderoth I, Scheibe R (1995) Molecular characterisation of the plastidic glucose6-phosphate dehydrogena se from potato in comparison to its cytosolic counterpart. Plant Physiol 109:1327-1335

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56 BIOGRAPHICAL SKETCH Li Li was born in Changsha, Hunan provin ce, China in 1982. She finished her prim ary school and middle school in her hometown Yuey ang, Hunan province. After graduating from Yueyang No.1 High School in 1999, she attended Wuha n University in Hubei province. In July, 2003, she received her Bachelor of Science degree in biology. In January 2006, Li enrolled in the Horticultural Sciences Department at the Univers ity of Florida to pursue graduate education. She received her master degree from the University of Florida in the summer of 2008. She plans to pursue a career dedicated to biology research.