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Comparative and Reverse Genetic Analysis of the Cytokinin Response Regulator Gene Family in Populus

Permanent Link: http://ufdc.ufl.edu/UFE0024141/00001

Material Information

Title: Comparative and Reverse Genetic Analysis of the Cytokinin Response Regulator Gene Family in Populus
Physical Description: 1 online resource (119 p.)
Language: english
Creator: Ramirez, Gustavo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: adventitious, arabidopsis, cuttings, cytokinin, microarrays, populus, ptrr13, regulators, response, roots, transgenic
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Plant Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cytokinins are important plant hormones that influence a diverse array of physiological and developmental processes such as root and shoot morphogenesis. However, little is known with respect to whether or how cytokinin action is connected to biomass distribution in any forest tree. A better understanding of the molecular mechanisms by which cytokinin signaling is connected to specific traits can provide the genetic tools to improve agronomic characteristics (e.g, stem wood production per unit root biomass). Cytokinin signaling resembles two-component systems from bacteria and yeast in which an external signal is sensed by a histidine kinase (HK) and then transferred to a response regulator (RR). Because of their ability to activate transcription and regulate protein activity, RRs have been proposed to coordinate most of physiological processes regulated by cytokinin. I identified, annotated and characterized at the transcript level 11 type-As, 11 type-Bs and 11 pseudo cytokinin response reulators (RRs) in Populus balsamifera ssp. trichocarpa (Torr. and Gray) genotype Nisqually-1. Developmental and cytokinin-responsive expression of the Populus RRs indicate that while the type-As and type-Bs are preferentially expressed in nodes, pseudo-RRs are preferentially expressed in mature leaves. Next I investigated the in vivo role of a particular Populus RR using a reverse genetic approach. Transgenic lines with ectopic expression of a constitutively active form of PtRR13 (Delta DDKPtRR13) were found to exhibit a delay in rootability during propagation. Microarray analysis in non transgenic (NT) plants evidenced a massive transcriptome remodeling during the 24 h following excision with aproximately 30% of the nuclear genes differentially regulated. During this time gene networks involved in wound and stress responses showed significant regulation while genes with potential roles in root morphogenesis were significantly regulated later during the 24 to 48 hour interval. Misregulated genes in Delta DDKPtRR13 included COV1, a negative regulator of vascularization; PDR9, an auxin transporter; two genes with sequence similarity to TINY1; and BELL1, encoding a homeodomain protein. I also observed a time point-specific influence of Delta DDKPtRR13 expression on the transcriptome at 24 h where 273 genes were differentially regulated. Results obtained show organ-preferred expression patterns of Populus RRs, suggesting possible roles for the type-As and type-Bs in development and pseudo-RRs in integration of environmental signals with plant function. I confirmed the negative role of cytokinin action in root developmental processes previously hypothesized in other plant systems, and obtained direct evidence that links a specific type RR (PtRR13) with inhibition of adventitious root formation. I propose that the inhibitory effects of PtRR13 on adventitious rooting are manifest physiologically, as reflected by transcriptome shifts, 24 h after shoot excision. This defines a discrete time frame during which cytokinin may act in adventitious root formation in vivo. Finally, putative direct and indirect targets of the constitutively active PtRR13 transcription factor imply that cross-talk between cytokinin, auxin and ethylene are important during adventitious rooting in cuttings.
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 Gustavo Ramirez.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Davis, John M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024141:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024141/00001

Material Information

Title: Comparative and Reverse Genetic Analysis of the Cytokinin Response Regulator Gene Family in Populus
Physical Description: 1 online resource (119 p.)
Language: english
Creator: Ramirez, Gustavo
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: adventitious, arabidopsis, cuttings, cytokinin, microarrays, populus, ptrr13, regulators, response, roots, transgenic
Plant Molecular and Cellular Biology -- Dissertations, Academic -- UF
Genre: Plant Molecular and Cellular Biology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Cytokinins are important plant hormones that influence a diverse array of physiological and developmental processes such as root and shoot morphogenesis. However, little is known with respect to whether or how cytokinin action is connected to biomass distribution in any forest tree. A better understanding of the molecular mechanisms by which cytokinin signaling is connected to specific traits can provide the genetic tools to improve agronomic characteristics (e.g, stem wood production per unit root biomass). Cytokinin signaling resembles two-component systems from bacteria and yeast in which an external signal is sensed by a histidine kinase (HK) and then transferred to a response regulator (RR). Because of their ability to activate transcription and regulate protein activity, RRs have been proposed to coordinate most of physiological processes regulated by cytokinin. I identified, annotated and characterized at the transcript level 11 type-As, 11 type-Bs and 11 pseudo cytokinin response reulators (RRs) in Populus balsamifera ssp. trichocarpa (Torr. and Gray) genotype Nisqually-1. Developmental and cytokinin-responsive expression of the Populus RRs indicate that while the type-As and type-Bs are preferentially expressed in nodes, pseudo-RRs are preferentially expressed in mature leaves. Next I investigated the in vivo role of a particular Populus RR using a reverse genetic approach. Transgenic lines with ectopic expression of a constitutively active form of PtRR13 (Delta DDKPtRR13) were found to exhibit a delay in rootability during propagation. Microarray analysis in non transgenic (NT) plants evidenced a massive transcriptome remodeling during the 24 h following excision with aproximately 30% of the nuclear genes differentially regulated. During this time gene networks involved in wound and stress responses showed significant regulation while genes with potential roles in root morphogenesis were significantly regulated later during the 24 to 48 hour interval. Misregulated genes in Delta DDKPtRR13 included COV1, a negative regulator of vascularization; PDR9, an auxin transporter; two genes with sequence similarity to TINY1; and BELL1, encoding a homeodomain protein. I also observed a time point-specific influence of Delta DDKPtRR13 expression on the transcriptome at 24 h where 273 genes were differentially regulated. Results obtained show organ-preferred expression patterns of Populus RRs, suggesting possible roles for the type-As and type-Bs in development and pseudo-RRs in integration of environmental signals with plant function. I confirmed the negative role of cytokinin action in root developmental processes previously hypothesized in other plant systems, and obtained direct evidence that links a specific type RR (PtRR13) with inhibition of adventitious root formation. I propose that the inhibitory effects of PtRR13 on adventitious rooting are manifest physiologically, as reflected by transcriptome shifts, 24 h after shoot excision. This defines a discrete time frame during which cytokinin may act in adventitious root formation in vivo. Finally, putative direct and indirect targets of the constitutively active PtRR13 transcription factor imply that cross-talk between cytokinin, auxin and ethylene are important during adventitious rooting in cuttings.
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 Gustavo Ramirez.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Davis, John M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-11-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024141:00001


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COMPARATIVE AND REVERSE GENETIC ANAL YSIS OF THE CYTOKININ RESPONSE REGULATOR GENE FAMILY IN Populus By GUSTAVO A. RAMIREZ CARVAJAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Gustavo A. Ramirez Carvajal 2

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To my parents and Vero 3

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ACKNOWLEDGMENTS First I would like to thank th e faculty and students of the Plant Molecular and Cellular Biology program at the University of Florida for a llowing me to be part of it. I also thank my advisor John Davis and all the pa st and present members of the fo rest genomics lab (especially Alison Morse, Chris Dervinis, Philip Bocock, and Ma tias Kirst) for their valuable support. I also would like to express gratitude to my comm ittee members (Mark Settles, Harry Klee, Tim Martin and Alice Harmon) for th eir constructive and wi se contributions. Most important I thank my family for their unconditional support and love. Finally, I thank my beloved partner and best friend through the years, Vernica. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT.....................................................................................................................................9 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW..............................................................11 Poplar as a Reference Species for Tree Biology....................................................................12 Cytokinin Action............................................................................................................... .....14 Cytokinin Signaling................................................................................................................15 Type-As...........................................................................................................................17 Type-Bs...........................................................................................................................17 Pseudo-RRs.....................................................................................................................18 Cytokinin Effects on Primary and Lateral Roots....................................................................19 Cytokinins Are Negative Regul ators of Vascular Development in Primary Roots........20 Cytokinins Are Negativ e Regulators of Lateral Root Initiation......................................21 Cytokinins and Adventitious Roots........................................................................................21 Project Objectives...................................................................................................................23 2 TRANSCRIPT PROFILES OF TH E CYTOKININ RESPONSE REGULATOR GENE FAMILY IN Populus IMPLY DIVERSE ROLES IN PLANT DEVELOPMENT...............27 Introduction................................................................................................................... ..........27 Materials and Methods...........................................................................................................30 Plant Material and Growth Conditions............................................................................30 Gene Annotation and Sequence Analysis........................................................................30 Data Analysis...................................................................................................................31 Detached-Leaves Cytokinin Experiment.........................................................................32 Semiquantitative RT-PCR...............................................................................................33 Results.....................................................................................................................................34 Cytokinin Response Regulator Gene Family in Populus ................................................34 Expression of Populus RRs.............................................................................................36 Populus Type-As and Bs Are Rapidly Upregulated by Cytokinin..................................38 Discussion...............................................................................................................................39 5

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3 THE CYTOKININ TYPE-B RESPO NSE REGULATOR PTRR13 IS A NEGATIVE REGULATOR OF ADVENTITIOUS ROOTS DEVELOPMENT IN Populus ...................56 Introduction................................................................................................................... ..........56 Materials and Methods...........................................................................................................60 Plant Material and Growth Conditions............................................................................60 Semiquantitative RT-PCR and Real-Time PCR..............................................................60 Tissue Culture Experiments............................................................................................61 Response Regulator PtRR13 Plasmid Construction and Transgenic Line Generation...61 Detached-Leaves Cytokinin Experiment.........................................................................62 Microarray Analysis........................................................................................................62 Gene Set Analysis............................................................................................................63 Results.....................................................................................................................................64 Transcriptome Analysis of Early Stages of Adventitious Root Formation.....................64 The Response Regulator PtRR13 and Its Relatives........................................................66 The Response Regulator PtRR13 Tr ansgenes and Their Effects....................................67 The Response Regulator PtRR13 Is a Nega tive Regulator of Adventitious Root Formation.....................................................................................................................6 9 Transgenic DDK PtRR13 Lines Show Altered Gene Expression during Adventitious Root Formation......................................................................................70 Discussion...............................................................................................................................73 Massive Transcriptome Remodeling Is Associated with Adventitious Rooting.............73 The Effect of PtRR13 Is Discrete....................................................................................75 Transcriptome Regulation at 24 h after Presumed Acquisition of Competence.............76 Potential Direct and Indi rect Targets of PtRR13.............................................................77 4 CONCLUSIONS.....................................................................................................................98 APPENDIX CYTOKININ BIOS YNTHETIC, DEGRADING AND SIGNALING GENES IN Populus ..............................................................................................................102 LIST OF REFERENCES.............................................................................................................104 BIOGRAPHICAL SKETCH.......................................................................................................119 6

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LIST OF TABLES Table page 2-1 Gene-specific Populus type-A and B RRs primers...............................................................53 2-2 Populus response regulators (RRs)........................................................................................54 2-3 Duplicated Populus RRs as reported by Tuskan et al. (2006)...............................................55 3-1 Primer list and gene models.............................................................................................. .....91 3-2 Gene set analysis of differentially re gulated genes in NT during early stages of adventitious root formation................................................................................................92 3-3 Differentially regulated genes between NT and DDKPtRR13 from 0 to 48 h...................94 3-4 Differentially regulated genes in DDKPtRR13 at 24 h.......................................................95 3-5 Top 10 differentially regulated genes in DDKPtRR13 at 24 h...........................................97 A-1 List of cytokinin biosyntheti c, degrading and signaling genes in Populus ........................102 7

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LIST OF FIGURES Figure page 1-1 Overview of Arabidopsis two-component asso ciated proteins............................................24 1-2 Cytokinin signaling pathway................................................................................................25 1-3 Lateral root development in Arabidopsis.............................................................................26 2-1 Populus response regulators (RRs) exhibit similar receiver domains..................................46 2-2 Populus response regulator (RR) gene structures and se quence similarity tree...................47 2-3 Amino acid alignments of the GARP DNA-binding domain................................................48 2-4 Receiver domains of Populus Arabidopsis and rice response regulators............................49 2-5 Tissue regulation of Populus response regulators................................................................50 2-6 Relative transcript abundance of Populus type As and Bs in various tissues......................51 2-7 Exogenous cytokinin induces transcript accumulation of Populus type-A and -B response regulators (RRs) in leaves...................................................................................52 3-1 Microarray data veri fication by real-time PCR....................................................................80 3-2 Differentially expressed genes during ea rly stages of adventi tious root formation.............81 3-3 Amino acid sequ ence similarity tree of Populus and Arabidopsis GARP-containing RRs (A). Transcript abundance of PtRR12 and PtRR13 (B).............................................82 3-4 Response regulator PtRR13 domain organization and screening of transgenic lines..........83 3-5 Overexpression of DDKPtRR13 stimulates stem growth in culture..................................84 3-6 Overexpression of DDKPtRR13 interferes with exog enous cytokinin induction of type-As in leaves.............................................................................................................. ..85 3-7 The response regulator PtRR13 is a negativ e regulator of adventitious root formation......86 3-8 Overall effects of DDKPtRR13 overexpression on gene expression................................87 3-9 Overexpression of DDKPtRR13 perturbs the expression of regulatory genes..................88 3-10 Arabidopsis RR1, DRE and ERF binding motifs in promoters of regulated genes across all time points......................................................................................................... .89 3-11 Hypothesized role of PtRR13 in adventitious root development.........................................90 8

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy COMPARATIVE AND REVERSE GENETIC ANAL YSIS OF THE CYTOKININ RESPONSE REGULATOR GENE FAMILY IN Populus By Gustavo A. Ramirez Carvajal May 2009 Chair: John Davis Major: Plant Molecular and Cellular Biology Cytokinins are important plant hormones that influence a diverse array of physiological and developmental processes such as root and shoot morphogenesis. Ho wever, little is known with respect to whether or how cytokinin acti on is connected to biom ass distribution in any forest tree. A better understanding of the molecu lar mechanisms by which cytokinin signaling is connected to specific traits can provide the gene tic tools to improve agronomic characteristics (e.g, stem wood production per unit root biomass). Cytokinin signaling resembles tw o-component systems from bacteria and yeast in which an external signal is sensed by a histidine kina se (HK) and then transferred to a response regulator (RR). Because of their ability to ac tivate transcription and regulate protein activity, RRs have been proposed to coordinate most of physiological processes regulated by cytokinin. I identified, annotated an d characterized at the transcript level 11 type-As, 11 type-Bs and 11 pseudo cytokinin response reulators (RRs) in Populus balsamifera ssp. trichocarpa (Torr. and Gray) genotype Nisqually-1. Developmental an d cytokinin-responsive expression of the Populus RRs indicate that while the type-As and type -Bs are preferentially expressed in nodes, pseudoRRs are preferentially expressed in mature leaves. 9

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10 Next I investigated the in vivo role of a particular Populus RR using a reverse genetic approach. Transgenic lines with ectopic expression of a constitutively active form of PtRR13 (Delta DDKPtRR13) were found to exhibit a dela y in rootability during propagation. Microarray analysis in non transgenic (NT) plants eviden ced a massive transcriptome remodeling during the 24 h following excision with aproximately 30% of the nuclear genes differentially regulated. During this time gene networks involved in wound and stress responses showed significant regulation while genes with poten tial roles in root morphogenesi s were significantly regulated later during the 24 to 48 hour interval. Misr egulated genes in Delta DDKPtRR13 included COV1 a negative regulator of vascularization; PDR9 an auxin transporter; two genes with sequence similarity to TINY1 ; and BELL1 encoding a homeodomain protein. I also observed a time point-specific influence of Delta DDKPtRR13 expression on the transcriptome at 24 h where 273 genes were differentially regulated. Results obtained show organ-pr eferred expression patterns of Populus RRs, suggesting possible roles for the type-As and type-Bs in development and pseud o-RRs in integration of environmental signals with plan t function. I confirmed the negativ e role of cytokinin action in root developmental processes previously hypothesized in other plant systems, and obtained direct evidence that links a specific type RR (PtRR13) with inhibition of adventitious root formation. I propose that the inhibitory effects of Pt RR13 on adventitious r ooting are manifest physiologically, as reflected by transcriptome sh ifts, 24 h after shoot excision. This defines a discrete time frame during which cytokinin may act in adventitious root formation in vivo Finally, putative direct and indi rect targets of the constituti vely active PtRR13 transcription factor imply that cross-talk between cytoki nin, auxin and ethylene are important during adventitious rooting in cuttings.

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CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Forests are the major terrestrial sink for atmo spheric carbon dioxide, and trees are the most prominent feature of forests. Forests cover 30% of the earths terrestrial surface, harbor substantial amounts of biodiversity, and provide humanity with benefits such as clean air and water, lumber, fiber and fuels (BOA, 1991). Poplar ( Populus sp.) trees are ecologically and economically important in the northern hemisphe re. Globally, 91% of poplars grow in natural forests, 6% in plantations and 3% in agroforest ry systems and as individual or small groups of trees outside of forests. The to tal area of natural poplar sta nds reported by the International Poplar Commission is over 70 million hectares, 97% of which occur in Canada, the Russian Federation and the United States, where they are managed predominantly for wood production (Ball et al., 2005). The recent completion of the Populus genome sequence (Tuskan et al. 2006) creates a new suite of tools for understanding the role of genetics in forested ecosystems. By having a complete genetic parts list for a forest tree, it is now feasible to link the functions of individual genes with traits that have adaptive, ecological, evolutionary or econ omic significance. A comparative approach exploits the relatively close evolutionary relationship between poplar and Arabidopsis, the current genetic reference system for plant biol ogy (both are in the Eurosi d clade of Eudicots). Comparative studies of these sp ecies enable researchers to le verage the massive amounts of genetic information that have emerged in Ar abidopsis (Jansson and Douglas, 2007), with the knowledge that while most genes are probably conserved, there are presumably some genes unique to Populus (or uniquely regulated in Populus) that create the obvious trait differences between the two taxa. Functi onal analysis in genes in Populus is now feasible given the wide assortment of genetic tools available. Successful linkage of genes with traits will help us 11

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understand how forest ecosystems function, and provi de tree breeders with candidate genes that control important characteristic s in trees such as yield, wood quality, disease resistance and environmental responsiveness. A more explicit genetic understanding of forest ecosystems provides more tools to inform science-based forest resource management and conservation practices worldwide. Poplar as a Reference Species for Tree Biology Poplars have several attributes that have le d to their emergence as a reference genus for genetics, molecular biology and physiological st udies. They can be genetically transformed, regenerated and clonally propagate d in a relatively short amount of time (Bradshaw et al., 2000). Poplars were the first forest tree s to be genetically transformed and regenerated (Fillatti et al., 1987), and Agrobacterium -mediated transformation of differe nt genotypes is now routinely performed (Tuskan et al., 2006). Unlike many other tree species that are recalcitrant to in vitro organogenesis, poplars are easily regenerated from leaves and stems, facilitating propagation and phenotypic characterization of desi rable genotypes (Bradshaw et al., 2000). Under rapid-growth conditions, poplars can grow approx imately 3 meters per year with wood and tree architecture phenotypes produced in 1-3 years in greenhouse or field environments (Tuskan et al., 2006). Flowering can be observed between 3-6 years in the field, but can be induced within 1 year using the LEAFY transgene or early flowering genotypes (H an et al., 2000). Field tests indicate that transgene expression is highly stab le in most hybrids, and somaclonal variation is thought to be minor (~0.06%) such that it does not pose ma jor constraints on functional genomic studies. Pedigree and transgenic lines can be eas ily and efficiently clonally propagated in vivo by stem cuttings; adventitious ro ot formation is usually achieved with in days and without the requirement of exogenous auxin formulations. 12

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The power of poplar as a reference plant has been enhanced by th e recent sequencing of the Populus trichocarpa genome (Tuskan et al., 2 006). For a forest species, P. trichocarpa has a relatively small haploid genome size (approximatel y 500 Mb), almost 40 times smaller than pine and only 4 times larger than Arabidopsis. Th e number of protein-coding genes has been predicted to be 45,000, a number that may decrease as annotation improves, but is comparatively similar to rice (~41,000) and only twice as larg e as Arabidopsis (~27,000) (Sterck et al., 2007). The availability of more than 200,000 ESTs from a wide range of tissues has greatly facilitated gene annotation and genome-wide transcriptome an alyses by microarray technologies (Quesada et al., 2008). Likewise, the availability of a whole genome sequen ce in poplar and other species like rice and Arabidopsis facili tates the identification of cis regulatory elements based on phylogenetic conservation and similarity in expression (Creux et al., 2008). The long generation time and outcrossing mating sy stem of poplars (i.e they are dioeceous and wind-pollinated) make the development of near-isogenic lines, which provide a powerful means for converting quantitative trait loci to Mendelian traits for precise mapping, impractical (Tuskan et al., 2006).Reverse genetics, in which a gene sequence is first obtained and then its function learned via directed alte ration in transgenic plants, provi des an alternative approach to link single genes to phenotypes (Bradshaw and St rauss, 2001). Typically, modification of gene expression is either suppressed by RNA-mediated s ilencing, or elevated us ing a strong promoter or enhancer. Since the genes used for reverse genetics are usually selected for study based on prior knowledge of gene function in other organism s, the number of independent plant lines that must be produced and maintained for phenotypic analysis is one or two orders of magnitude smaller than in forward genetic analysis. The av ailability and flexibility of different expression cassettes for reverse genetics studies in poplars and the availability of whole-genome sequence 13

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allows functional studies of vi rtually every gene in the genome. Identification of actively expressed genes with intact coding regions and defined relationships with homologs in other plant systems can lead to predic tive hypotheses regarding the functi on of those candidate genes. In this study, I coupled gene family annotation (assisted by prior knowledge of genes for cytokinin action that had previ ously been identified in Arab idopsis and rice) with wholetranscriptome analysis in poplar to identify candidate genes fo r reverse genetic manipulation. Cytokinin Action Cytokinins are plant hormones that influence diverse processes of growth and development such as cell proliferation and differentiation, va scular morphogenesis, shoot-root development, chloroplast morphogenesis, leaf senescence, and axillary bud dormancy (Gan and Amasino, 1995; Inoue et al., 2001; Fukuda, 2004; Kim et al., 2006; Mahonen et al., 2006; Tanaka et al., 2006). Cytokinins are adenine deriva tives with an aromatic or is oprenoid side chain at the N-6 position. The initial step in cytokinin biosynthesis is catalyzed by the enzyme adenosine phosphate-isopentenyltransferase (IPT). This enzy me catalyzes the prenyl ation of adenosine 5phosphate (AMP, ADP, or ATP) at the N6 position with dimethylallyl diphosphate (DMAPP). Although expression of Arabidops is IPTs indicate that cy tokinins can be locally synthesized in different parts of the plant body such as leaves, stems or seeds; roots have been shown to be major sites of cytokinin biosynthe sis (Miyawaki et al., 2004; Nordstrom et al., 2004; Takei et al., 2004). In species such as cucumber, root-borne cytokinins ha ve been shown to be translocated with the xylem flow to above ground organs mostly as trans-zeatin ribosides (Takei et al., 2001; Kuroha et al., 2002). Such root to sh oot transport is proposed to signal nitrogen and nutrient status of the soil since nitrate supplementation to roots induces rapid accumulation of cytokinins in Arabidopsis, maize and barley (Samuelson et al., 1992; Sakakibara et al., 1998; Takei et al., 2001). AtIPT3 and AtIPT5, the most abundantly expressed IPTs from Arabidopsis, 14

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are upregulated when soil nitroge n availability is high (Takei et al., 2004). However, acropetally transported cytokinins may be involved in regulatory processe s other than nutrient signaling, such as developmental events including inhibition of lateral and ectopic r oots, and promotion of lateral bud elongation (Sakakibara, 2006). Analogous to the importance of auxin gradie nts in shoots, cytokinin concentration gradients along primary roots are thought to contro l lateral and adventiti ous root initiation. By analogy to the well-known phenomenon of shoot apic al dominance, this has been called root apical dominance (Aloni et al., 2006). trans -Zeatin present in xylem sa p has been identified as a root-derived suppressor of adventitious root formation in cucumber hypocotyls (Kuroha et al., 2002). Likewise, in lateral roots, cy tokinins interfere with the initia l cell divisions le ading to root primordia organization (Laplaze et al., 2007). B ecause of their important effects on plant architecture, genetic manipulation of the cyto kinin signaling pathway ma y have profound effects in economically and ecologically important proce sses in plants like nitrogen signaling, biomass production-partitioning and root system architecture. Cytokinin Signaling The cytokinin signaling pathway resembles b acterial and yeast two-component signal transduction pathways. Recent genetic and molecu lar studies in Arabidopsis have allowed the identification of three key components of this signaling pathwa y in plants: sensor histidine kinases (HKs), histidine-cont aining phosphotransfer proteins (HPs) and response regulators (RRs) (Mok and Mok, 2001; Kakimoto, 2003; Fe rreira and Kieber, 2005) (Figure 1-1). Cytokinin responses are initiated when cytokini n binds to the HK in a conserved extracellular domain (also known as the CHASE domain), inducing autophosphorylation on a His residue within the cytoplasmic transmitter domain of the HK. The same phosphate group then gets transferred to a HP which has the ability to shut tle to the nucleus and phosphorylate RR proteins 15

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(type-As and type-Bs). While phosphorylated ty pe-Bs act as transcri ptional activators of cytokinin-regulated genes, the type-As down regul ate the cytokinin signa ling by competing with the type-Bs for phosphoryl groups (Figure1-2). Three cytokinin receptors have been found in Arabidopsis: AHK2, AHK3 and AHK4/CRE1. They show different expression patte rns; AHK4 is mainly expressed in roots, whereas AHK2 and AHK3 are present in all major organs (Nishimura et al., 2004). The ahk4 mutant allele was initially isolated in a screen for mutants that failed to form large green calli on shoot initiating media (Inoue et al ., 2001). Complementation tests in Schizosaccharomyces pombe lacking three HK (Phk1, Phk2, Phk3) showed that AHK4 functions in a cytokinindependent manner (Suzuki et al., 2001). Interestingly in vivo and in vitro studies indicate that AHK4 is a bidirectional regul ator of cytokinin signaling that can phosphorylate and dephosphorylate HP proteins (Mahonen et al., 2006). The HP proteins function as a bridge in the primary phosphotransfer between the Asp residues in the receiver domain of the hybrid sensor kinase and in the receiver domain of the RR (S uzuki et al., 2001). Upon induction by cytokinin, some HPs localize specifi cally and transiently to the nucleus indicating that members of this class of protein function as cytoplasmi c-nuclear shuttles (Hwang and Sheen, 2001). In Arabidopsis, special atte ntion has been given to the RRs since they are the final recipients of phosphoryl groups coming from the HK receptors. Because of their ability to activate transcription an d regulate protein activity, RRs have been proposed to coordinate most of physiological processes regul ated by cytokinin. Based on do main structure, RRs can be classified into three subg roups: type-As, type-Bs and pseudo-RRs. Although functional characterization of these genes in different species is still in progress preliminary results have confirmed their importance as cy tokinin signaling regulators. 16

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Type-As The members of this group of RRs have a receiver domain with conserved D-D-K residues characteristic of the RR gene family and a s hort C-terminus of unknown function (Sakai et al., 2000). Type-As are cytokinin primary response genes; transcripts accu mulate rapidly after cytokinin treatment without requirement for prev ious protein synthesis (Brandstatter and Kieber, 1998; Taniguchi et al., 1998; D' Agostino et al., 2000). Type-As are negative regulators of cytokinin signaling. A variety of cy tokinin response assays indicate that at least six of the ten Arabidopsis type-As act as nega tive regulators of cytokinin signaling and their overexpression results in reduced sensitivity to cytokinin in r oots and shoots (Osakabe et al., 2002; Kiba et al., 2003; To et al., 2004). No molecula r function has yet been assigne d to type-As; it has been hypothesized that they might act as on-off molecular switc hes by interacting with other proteins, including HPs and/or type-B RRs (Mizuno, 2004). Over expression of some type-As inhibit expression of an ARR6 (type-A) prom oter-luciferase reporte r gene in cultured Arabidopsis cells, suggesting that type-As have the ability to negativ ely regulate their own transcription (Hwang and Sheen, 2001) ARR4, another type-A, has been shown to interact with and stabilize the far-re d active form of phytochrome B (PhyB) (Sweere et al., 2001). Single typeA Arabidopsis mutants are indistinguishable from wild type in various cytokinin assays; double or higher order type-A mutants show increasing sensitivity to the hormone, indicating functional overlap among them (To et al., 2004). Type-Bs The type-Bs are characterized by the presence of a receiver domain with conserved D-D-K residues and a large C-terminal extension that contains a Myb-like DNAbinding region referred to as the GARP domain (Sakai et al., 1998; Imamura et al., 1999). The GARP domain is common to a class of plant-spec ific transcription factors including GOLDEN2 of maize, ARRs 17

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of Arabidopsis, and Psr1 of Chlamydomonas (Rie chmann et al., 2000). The C-terminal region is highly variable among the family members and is of ten rich in glutamine and proline, a feature usually observed in transcrip tional activators (Triezenberg, 19 95). The GARP domains of ARR1 and ARR2 bind DNA in a sequence-specific manner to the core sequence (G/A)GAT(T/C). In tobacco leaves, transient expression of ARR1 and ARR2 was able to activate transcription of a reporter gene fused to this core sequence. De leting the receiver domains of ARR1 and ARR2 increases their transactivation activity, indicatin g that the receiver domains act negatively on transcriptional activation (Sakai et al., 2000). The C-terminal domains of the type-Bs also contain potential nuclear localization signals a nd several type-Bs have been demonstrated to localize to the nucleus when fused to reporter genes (Sakai et al., 1998; Lohrmann et al., 1999; Sakai et al., 2000; Imamura et al ., 2001). In contrast to the ty pe-As, exogenous cytokinin does not affect steady state transcript levels of type-B RRs in Arab idopsis (Imamura et al., 1999; Kiba et al., 1999). Pseudo-RRs Pseudo-RRs are genes that encode proteins that resemble authentic RRs (i.e. contain a receiver-like domain). However, they have a glutamate in place of th e central aspartate (D E) in the conserved D-D-K domain that prevents their phosphorylation (Stock et al., 1989; Imamura et al., 1998; Makino et al., 2000). These prot eins also feature a CCT-motif (for C ONSTANS, C ONSTANS-LIKE and T OC1) at the C terminus that is thought to be involved in protein-protein interactions (Strayer et al., 2000; Wenke l et al., 2006). Arabidopsis and rice pseudo-RRs have been implicated in circadian controlled events such as flowering time and photomorphogenic responses (Makino et al., 2000; Murakami et al., 2004; Mizuno and Nakamichi, 2005). APRR1, an Arabidopsis pseudo -RR also referred to as TOC1 ( T IMING O F C AB EXPRESSION 1 ), is a component of the central oscilla tor of the circadian clock (Somers et al., 1998; Strayer et al., 18

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2000). Transcripts of several Arabidopsis and rice pseudo-RRs, including TOC1 and its putative ortholog in rice (OsPRR1), have been detected in leaves and exhibi t circadian regulation (Makino et al., 2000; Murakami et al., 2005). Expression of the Arabidopsis pseudo-RRs peak at different times of the day and loss-of-function muta tions result in altered circadian rhythmicity (Matsushika et al., 2000; Nakamichi et al., 2005). The extent to wh ich this subfamily is involved in cytokinin responses is an open question; however their inab ility to participate in the phosphorelay as phospho-aceptors is an indicatio n that they are not direct modulators of cytokinin responses. The comprehensive characteriza tion of the RR genes in Arabidopsis can provide valuable information a priori about structure and function of equi valent genes in related species. Populus is more phylogenetically related to Arabidopsis th an to the vast majority of other dicot taxa facilitating comparative functional genomic st udies (Jansson and Douglas, 2007). The recent completion of the genome sequencing for an economically and ecologically important tree species like Populus would facilitate the study of the effects of hormones in traits relevant to long-lived species like seconda ry growth, bud dormancy and adventitious root formation. Cytokinin Effects on Primary and Lateral Roots Cytokinins are key regulators of meristematic activity in shoots and roots where they play contrasting roles. Depletion of endogenous cyto kinins by altering the expression of cytokinin biosynthetic/degrading enzymes usually results in stunted shoots and enhanced root systems (Werner et al., 2001; Werner et al., 2003; Mahonen et al., 2006; Dello Ioio et al., 2007). Possibly the most remarkable effect of cytokinin in root growth is the regulati on of the meristem size. Analysis of the root meristem of cytokinin biosyn thetic mutants has revealed that cytokinins are positive regulators of cell differentiation at the transition zone (the boundary between the meristem and the elongation-differentiation zone). Reduction in e ndogenous cytokinin in the root 19

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meristem results in an increased population of cells with meristematic features and vice versa (Dello Ioio et al., 2007). The inhi bitory effects of cytokinins in root systems appear to vary between root types with recent stud ies indicating that late ral roots are more sensitive to cytokinin treatment than primary roots (Laplaze et al., 200 7). Such differences in response to cytokinin may reflect root-type specific interactions with other phytohormones like ethylene. Studies using different ethylene signaling mutant s and inhibitors of ethylene bi osynthesis have revealed that, while the inhibition of root elonga tion by cytokinin is ethylene-dependent in primary roots, it is ethylene-independent in latera l roots (Laplaze et al., 2007). Cytokinins Are Negative Regulators of Vascular Development in Primary Roots Cytokinins are also involve d in vascular morphogenesi s in roots. Arabidopsis AHK4/CRE1/WOL, the first cytokini n receptor identified, is requi red for proper root vascular differentiation and loss-of-function mutations of this gene result in the reduc tion of root vascular initials and in the specificati on of all vascular cell files as protoxylem (Mahonen et al., 2000; Inoue et al., 2001; Higuchi et al., 2004). Altered root vasc ular development has also been reported for mutations affecting other cytokinin receptors as well as down-stream cytokinin signaling genes including HP prot eins and type-B RRs (Hutchison et al., 2006; Ar gyros et al., 2008; Ishida et al., 2008). Quintuple loss-of-f unction HP mutants in Arabidopsis (ahp1,2,3,4,5) have similar phenotypes to th e wooden leg1 allele of AHK4/CRE1/WOL; short primary roots with reduced vascular tissue (Hutchison et al., 2006). The Arabidopsis ty pe-B triple loss-offunction mutant arr1-3 arr10-5 arr12-1 also exhibits altered root vascular development similar to wooden leg1 (Argyros et al., 2008; Ishida et al., 2008). The vascular morphogenesis developmental pathway is also regulated by AHP6, a novel pseudo-phosphotransfer protein which lacks the conserved phosphor-accepting his tidine residue found in other HP proteins. AHP6 was identified as an extragenic suppressor of wol, whose function is to inhibit cytokinin 20

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signaling during protoxylem speci fication (Mahonen et al., 2006). The common overlapping phenotypes observed in these different phosphorel ay elements suggest that cytokinin-twocomponent signaling cascade is required for nor mal vascular morphoge nesis in roots. Cytokinins Are Negative Regulators of Lateral Root Initiation In flowering plants and gymnosperms, lateral roots are derived from a group of cells in the periphery of the primary root xylem poles called peri cycle founder cells (C asimiro et al., 2001). In the early stages of lateral root formation, the pericycle founder cells de-differentiate and asymmetrically divide to give ri se to a lateral root primordium (LRP). Auxin has been shown to be crucial for LRP formation. The formation of an auxin gradient me diated by auxin efflux carriers is required for new pr imordia development (Benkova et al., 2003; Geldner et al., 2004). Genetic and molecular studies of cytokinin signal transduction and biosynthetic mutants have shown that cytokinins are nega tive regulators of lateral root formation (Werner et al., 2001; Werner et al., 2003; To et al., 2004 ; Riefler et al., 2006). Cytokinins play an inhibitory role by delaying the first cell divisions at the pericycle cells next to the xylem poles. Such delays results in a disorganized pattern of cell divisions and affects PIN-dependent auxin gradient establishment (Laplaze et al., 2007). Ectopic ex pression of IPT in the pericycle or the LRP reveals that, in the very early stages of root primordia formation, only the xylem pole cells are susceptible to the negative effect s of cytokinin; after the cells ha ve differentiated into LRP, they become cytokinin-insensitive (Laplaze et al., 2007). Cytokinins and Adventitious Roots Adventitious roots are defined as roots that originate other than from the embryo or from branches of the primary root. They can arise spon taneously on intact plants, especially at nodes of prostrate stems, or they can develop as a resp onse to damage when part of the plant has been severed from the existing root system. In woody plants, adventitious root primordia primarily 21

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arise from ray cells (close to the phloem and ca mbium), buds or leaf gaps, pericycle, or callus formed at the base of the cutting (Lovel a nd White, 1986). Adventitious root formation is regulated by endogenous as well as exogenous factors including hormones, temperature, light, sugars and phenolic compounds. Among the plant hormones, auxin appears to be the major positive regulator of de novo root formation processes. The e ffectiveness of different types of auxin to promote adventitious r oots depends on several factors su ch as uptake, transport and inactivation (degradation or conjugation). Accumula tion of endogenous auxins at the base of the stem or shoot is required for successful de novo root system formation in cuttings and explants. Inhibition of basipetal auxin transport in stems se verely reduces rooting in different species (Liu and Reid, 1992; Hausman et al., 1995; Guerre ro et al., 1999; Ludwig-Muller et al., 2005). Adventitious root formation can be seen as a two-stage process: (1) induction, where the cells from which the root primordia originate become competent to respond to the rhizogenic action of auxin, and (2) formation, where initial cell divisi ons and root primordia establishment occurs. The position of adventitious roots is thought to arise through the auxin-stimulated differentiation and elongation of cells arising from clusters of phloem parenchyma cells adjacent to vascular bundles (Lund et al., 1996; De Klerk et al., 1999). The formation of both adventitious and lateral roots are de novo processes in which new roots are form ed from pre-existing structures like stems, callus or roots. Since in both instances th e outcome is newly formed roots, there is likely to be significant overlap in the molecular mechan isms regulating these two parallel processes. Although not much is known about the molecular mechanisms governing adventitious root formation, physiological and biochemical studies in different species such as tomato, pea and poplar, have observed a decrease in cytokinin content during th e induction of rooting followed by a rise during the initial LRP cell divisions (Bollmark and Eliasson, 1986; Maldiney et al., 22

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1986; Hausman et al., 1997). The initial decrease dur ing the induction phase is concomitant with the rise of IAA and might indicate a requirement for high auxin/cytokinin ratio in the induction of rooting (Kevers et al., 1997). Direct evidence of th e inhibitory effects of cytokinin during adventitious root formation has been reported (Kuroha et al., 2002). In these experiments, the cytokinin trans-zeatin riboside was identified as a root-derived compound responsible for the suppression of adventitious root formation in cucumber hypocot yls (Kuroha et al., 2002). In recent years, the characterization of the cytokini n signaling pathway in Arabidopsis has started to give some insights into the molecular mechanisms affecting adventitious root formation. For example, several cytokinin receptors and type-B loss-of-function mutants have been reported to spontaneously grow roots in stems and hypocotyl s (Higuchi et al., 2004; Nishimura et al., 2004; Kuroha et al., 2006; Argyros et al., 2008). Similarl y, transgenic Arabidopsi s plants deficient in cytokinins show enhanced formation of adven titious roots (Werner et al., 2003). These findings reinforce the role of cytokinin as an important negative growth regul ator of all types of roots. Project Objectives Cytokinins are hypothesized to be important regulators of vegetative growth and development, and negative regulators of root development. However, no genetic evidence has been reported linking cytokinins with development in any forest tree. The main objective of this project was to characterize the cytokinin RR gene family in Populus an economically and ecologically relevant genus of forest trees, w hose genome was recently sequenced. To achieve this goal, I identified, annotated and characterized at the transcri pt level the cytokinin RRs in P.balsamifera ssp. trichocarpa (Torr. and Gray) genotype Nisqually -1. This aspect of the project is described in Chapter 2. In Chapter 3, I descri be experiments in which I used a reverse genetic approach to show that a particular Populus RR, PtRR13, plays an important role in adventitious root formation. In Chapter 4, I summarize these fi ndings and suggest avenues for future research. 23

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Ethylene receptors Cytokinin receptors CHASE HK HPt RRs PRR Ethylene receptors Cytokinin receptors CHASE HK HPt RRs PRR Figure 1-1. Overview of Arabidopsis two-comp onent associated proteins (modified from Mizuno and Nakamichi, 2005). Two-component proteins in plants include histidine kinases (HK), histidine phos phor-transfer proteins (HPt), response regulators (RR) and pseudo-response regulators (PRR). Histidin e kinase domains are indicated by rectangles, transmembrane domains by bars receiver domains by ovals. Phosphorelay relevant histidine (H), aspa rtic acid (D) and glutamic acid (E) residues are indicated. Nuclear localization signals (NLS) and GAR P, CCT, CHASE domains are shown as boxes. 24

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H P H D H P P D P GARP D P Cytokinin-regulated genes Type-As Type-Bs Histidine kinase D D GARP Cytokinin Phosphotransfer H H P H P H H D H P P D H P P D P D P GARP D P GARP D P Cytokinin-regulated genes Type-As Type-Bs Histidine kinase D D D GARP D D GARP Cytokinin Phosphotransfer Figure 1-2. Cytokinin signali ng pathway. Cytokinin binding to the membrane bound histidine kinases triggers an autophos phorylation at a histidine re sidue in kinase catalytic domain. This phosphoryl group then is transfer red to an aspartic residue located the receiver-like domain. The phosphoryl moiety is then transferred to a histidinecontaining phosphotransfer protein that shuttles to the nucleus where it phosphorylates the type-A and type-B res ponse regulators. Phosphorylated type-Bs bind to cis -elements in the promoters of cytokini n-regulated genes including the typeAs. Type-As negatively regulate cytokinin signaling by competing with type-Bs for phosphoryl groups and thus adjusting th e output of the si gnaling cascade. 25

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26 A B C D A B C D Figure 1-3. Lateral root devel opment in Arabidopsis (Nibau et al., 2008). P, pericycle; En, endodermis; Co, cortex; Epi, epidermis. (A) Early initiation: a founder xylem pole pericycle cell (dark grey) undergoes initial anticlinal cell divisions (perpendicular to the surface of the root). (B) Periclinal cell divisions (par allel to the surface of the root) begin and the lateral root primordi um (LRP) begins to grow. (C) The LRP undergoes further organized cell divisions and begins to emerge through the outer cell layers of the primary root, resulting in cell separation (asterisks). (D) The new lateral root is fully emerged and its new meristem is activated (dark grey star). It will continue to grow and elongate. At each stage, the effect of various key plant hormones is indicated. ABA, abscis ic acid; BR, brassinosteroids.

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CHAPTER 2 TRANSCRIPT PROFILES OF THE CYTOKININ RESPONSE REGULATOR GENE FAMILY IN Populus IMPLY DIVERSE ROLES IN PLANT DEVELOPMENT This Chapter has been published in New Phytologist 2008;177(1) :77-89 (PMID: 17944821) Introduction Cytokinins are plant hormones that influence diverse processes of growth and development such as cell proliferation a nd differentiation, vascular morphogenesis, shoot development, chloroplast morphogenesis, leaf senescence, and axillary bud dormancy (Cline, 1991; Gan and Amasino, 1995; Inoue et al., 2001; Mok and Mok, 2001; Fukuda, 2004; Kim et al., 2006; Mahonen et al., 2006; Tanaka et al., 2006). Cytokinins have been im plicated in the regulation of cell proliferation in the vascular cambium. In woody perennials like Populus, growth of this meristem produces secondary phloem (bark) and secondary xylem (wood), and results in stem girth increase. In additio n to the vascular cambium, stems al so contain a secondary shoot apical meristem (axillary meristem) in each node (axil of each leaf) that can generate sylleptic branches under appropriate developmental and environmental conditions. Direct cyt okinin treatments to Populus buds promote the elongation of sylleptic buds to generate new branches during the same season in which they are formed without an intervening rest peri od (Cline et al., 1997). Cytokinins have also been implicated in belo wground meristematic activ ities (Mahonen et al., 2000; Inoue et al., 2001; Werner et al., 2001). Exogenous cytokinin application can inhibit both primary root elongation and lateral root formation, resulting in reduced ro ot growth and biomass (Werner et al., 2001; Higuchi et al., 2004). Because of their importance in controlling crown architecture through the induction of sylleptic branching and thei r unquestionable effect on root and shoot growth, cytokinin signa ling genes are likely to be key elements coordinating the production and distribution of biomass in trees. 27

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The cytokinin signaling pathway resembles b acterial and yeast two-component signal transduction pathways in which an external signal is perceived by a sensor protein and transmitted to a response regulator by transf er of a phosphate group (Mizuno, 1998; West and Stock, 2001). Recent genetic and molecular studies in Arabidopsis have identified 3 key components of this signaling pathway in plants : sensor histidine kina ses (HKs), histidinecontaining phosphotransfer protei ns (HPs) and response regula tors (RRs; Mok and Mok, 2001; Kakimoto, 2003; Ferreira and Kieber, 2005). Cytoki nin responses are initiated when cytokinin binds to the HK in a conserved extracellula r domain and induces autophosphorylation on a histidine residue within the cytoplasmic transmitter domain. The phosphate group is then transferred to a HP which has the ab ility to phosphorylate RR proteins. Cytokinin RRs are key elements in this phosphorelay cascade because they modulate downstream signaling through transc riptional activation and regulat ion of protein activity. Based on their domain structure and ami no acid sequence, RRs are classified as type-As, type-Bs and pseudo-RRs. The relative abundance of type-As and Bs, 23 in Arabidopsis (Ferreira and Kieber, 2005) and 26 in rice (Ito and Kurata, 2006), indica tes they have the poten tial to coordinate many physiological processes regulated by cytokinin. The type-As have a receiver domain with conserved aspartate-aspartatelysine (D-D-K) residues and a short C-terminus of unknown function (Sakai et al., 2000). T ype-As are cytokinin primary response genes whose transcripts accumulate rapidly after cytokinin treatment w ithout the requirement for previous protein synthesis (Brandstatter and Kieber, 1998; Taniguchi et al., 1998; D' Agostino et al., 2000). Analyses of gain and loss-of-function Arabidopsis RRs have shown that type-As decrease cytokinin sensitivity and negatively regulate th eir own transcription (Hwang and Sheen, 2001; To et al., 2004). The type-Bs are characterized by the presence of a receiver domain with the 28

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conserved D-D-K residues and a large C-terminal extension. The C-terminal extension contains a Myb-like DNA-binding region referred to as a GARP domain (Imamura et al., 1998; Sakai et al., 1998) that is common to a class of plant-specif ic transcription factor s that includes maize GOLDEN2, Arabidopsis ARRs, and Chlamydomonas Psr1 (Riechmann et al., 2000). This region is highly variable and rich in glutamine and proline residues, a feat ure usually observed in transcriptional activators (Trie zenberg, 1995), and contains putati ve nuclear localization signals (Sakai et al., 1998; Lohrmann et al., 1999) that localize the RR to the nucleus when fused to reporter genes (Lohrmann et al., 1999; Sakai et al., 2000; Imamura et al., 2001). In contrast to type-As, exogenous cytokinin has not been found to alter steady state transcri pt levels of type-B RRs (Imamura et al., 1998; Kiba et al., 1999). Pseudo -RRs are genes that encode proteins that resemble authentic RRs (i.e. contain a receiver-like domain) however, they have a glutamate in place of the central aspartate in the conserved D-D-K domain that prevents phosphorylation (Stock et al., 1989; Imamura et al., 1998; Makino et al., 2000). It is thought that the evolution of ma ny gene families in Arabidopsis and Populus was influenced by 3 genome duplications. The most recent genome duplication (salicoid event) occurred in Populus between 8 and 13 Myr ago in an ance stor of the Salicaceae and affected roughly 92% of the genome (Sterc k et al., 2005) and generated nearly 8000 pairs of paralogous genes (Tuskan et al., 2006). The gene content of Populus is predicted to be 45,000, almost twice the number in Arabidopsis (Tuskan et al., 2006 ). The difference in gene number between Populus and Arabidopsis is largely due to gene fam ily expansion since the relative frequency of protein domains present in the 2 species is si milar (Tuskan et al., 2006). Families that have undergone expansion in Populus include genes involved in wood formation such as cellulose biosynthesis, flavonoid biosynthesis, and membrane transport (Tuskan et al., 2006). However, 29

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certain gene families involved in hormone homeostasis and signal transduction have not expanded. This is the case for families encoding cytokinin homeostasis-related enzymes like isopentenyl transferases and cy tokinin oxidases, where the nu mber of members is similar between Populus and Arabidopsis (Tuskan et al., 2006). The recent completion of the Populus genome sequence (Tuskan et al., 2006) significantly improves our ability to understand the structure and function of gene families involved in cytokinin signal transduction. In the present study, we identified 11 t ype-A, 11 type-B and 11 pseudo-RRs in Populus. Using microarray analysis and semiquantitative RT-PCR, we show expression data for these genes in different organs and tissues as well as cytokinin transc ript inducibility using a detached-leaf system. Materials and Methods Plant Material and Growth Conditions Experiments were conducted in a greenhouse at ambient temperature. Populus balsamifera ssp. trichocarpa (Torr. and Gray) genotype Nisqually-1 and Populus tremula x Populus alba INRA-clone No. 717-1-B4 plants were given 12 -14 hours of natural light, supplemented in the winter with artificial illumination to maintain indeterminat e growth. Rooted softwood cuttings were produced in 25 cm2 pots under mist and then transferred to 11.4 liter pots. Plants were placed in a completely randomi zed design on flood benches subirrigated once daily with a nutrient solution containing Peters Professional Blend 20-10-20 fertil izer solution (adjusted to 4 mM nitrogen). When plants reached 60-80 cm tall, they were used in experiments. Gene Annotation and Sequence Analysis RR gene discovery and annotation was perf ormed by TBLASTN searches of the JGI genome assembly v1.0 using full-length Arabidop sis response regulators as queries. A second round of TBLASTN searches was performed using candidate Populus RRs obtained in the first round of queries. Putative amino acid sequences (Ramirez-Carvajal et al ., 2008) were generated 30

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by gene-finder software GeneScan (Burge and Karlin, 1997) and compared to EST sequences obtained from the National Center for Biotechno logy Information (NCBI). Erroneous sequences generated by GeneScan were hand-edited. Af ter the release of version v1.1 of the Populus trichocarpa genome assembly, all RR sequences we re blasted (BLASTN) against the new version. No differences were identified with our predicted gene models. Sequence similarity trees were generated by aligning the receiver domains using Clus talX (1.81) default settings (Gonnet series). NEXUS output was imported into the Phylogenetic Analysis Using Parsimony software (PAUP) version 4.0 for bootstrap analysis using default settings (parsimony) and 10,000 iterations. Subcellular localization of the Populus RRs was predicted in silico using the software packages TargetP1.1 (Emanuelsson et al., 2000) and WoLF PSORT (Horton et al., 2007). Data Analysis Whole-genome microarray analyses were performed on chips containing features representing 42,364 predicted tran scriptional units from the P. trichocarpa nuclear genome. Four biological replicates of the Nis qually-1 genotype were used in the analysis of each organ. All transcriptional units were re presented by 3 60-mer probes (pro beset), designed by NimbleGen (Madison, WI) in collaboration with Oak Ridge National Laboratory an d synthesized using maskless lithography. cRNA was synthesized from to tal RNA extracted from 5 different major tissues: young leaves (LPI 4), mature leaves (LPI 5), nodes, internodes and roots. Labeling, hybridization and scanning were carried out by NimbleGen (Madison, WI) using standard procedures. Microarray expression data are available at Quesada et al. (2008). The data were analyzed using a two-step stra tegy previously outlined by Chu et al. (2002). For identification of genes expressed above background in each vege tative organ, the signal intensity detected for each probe was log2-transformed and normalized by subtracting the chip 31

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mean and dividing by the chip standard deviation. No rmalized values were contrasted to a set of 20 negative control probes ( E.coli genes, not shown). A mixed-model analysis of variance (ANOVA) was applied to each indi vidual probeset with gene as a fixed effect and probe as a random effect. Least-square means were calculated and pairwise comparisons ( t -tests) were carried out to contrast the estimated transcript le vel of each gene relative to the negative controls. P -values were adjusted for false discovery rate (FDR; Benjamini and Hochberg, 1995), with the modifications reported by Storey and Tibshirani (2003). Genes were considered expressed above background if they had a FDR below 0.05. Tissue preference of each gene was identifie d by contrasting tran script abundance among the 5 tissues ( t -test) by using a mixed linear model that included tissue type (node, internode, young and mature leaf and root) as fixed effect s and probe ID and plant as random effects. Pairwise tissue contrasts ( t -tests) were considered st atistically significant at p-values 0.05. All analyses described above were carried out usin g the statistical analysis software SAS (SAS Institute, Cary, NC) and the stat istical discovery software JMPTM. Microarray expression data were validated using real-time PCR for a set of 9 genes that showed ti ssue-specific expression (Quesada et al., 2008). Detached-Leaves Cytokinin Experiment Expanding leaves with a leaf plastochron index (LPI) of 4, 5 and 6 from the hybrid P. tremula x alba, the genotype most commonly used in functional (transgenic) studies, were harvested and treated with 1 M 6-benzylaminopurine (BAP) using 2 different means of hormone delivery. The first approach consis ted of placing leaves in a vial containing the hormone such that only the petioles were submerged in the solution (Sugiharto et al., 1992). One hour later, leaves were removed and frozen in liquid ni trogen. In the second approach, leaves were harvested and rubbed with an aqueous solution of 3% carborundum and submerged in hormone 32

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solution for 40 minutes. Leaves were removed from the solution and frozen in liquid nitrogen. Since both methods gave similar results (data no t shown), data are repo rted from the petiole feeds. Samples from 3 biological replicates were used per treatment in all experiments, and both the petiole feed and carborundum experiments were performed twice with similar results. Semiquantitative RT-PCR RT-PCR analyses were pe rformed on RNA from P. trichocarpa genotype Nisqually-1 and Populus deltoides (Bartr. ex Marsh). Vegetative tissu es (phloem and xylem) used in the experiments reported here were harvested from 3 month old clonally propagated Nisqually-1 plants. Because greenhouse-grow n Nisqually-1 plants were not reproductively mature, catkin samples were collected from a female P. deltoides located on the University of Florida, Gainesville campus. Total RNA was isolated using a cetyltr imethylammonium bromide (CTAB) method (Chang et al., 1993). RNA samples were subjecte d to DNase treatment with RQ1 RNase-free DNase (Promega, Madison, WI, USA) and purifie d using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). One g of DNA-free RNA was used to synthesi ze first strand cDNAs using oligo-dT primers and M-MLV reverse transcriptase (Prome ga, Madison, WI, USA). Gene specific primers were designed for all Populus response regulators using th e Joint Genome Institute (JGI) assembly for P. trichocarpa and are shown in Table 21. To avoid non-specific PCR amplification, primers were designed against the most variable regions in the coding sequences, 5 UTR or 3 UTR using NetPrimer (Premier Biosoft International, Palo Alto, CA, USA). Primers were tested on genomic DNA from th e 2 species and showed equal amounts of PCRproduct of the appropriate sizes (data not shown). The detection of higher expression of PtRR9 and PtRR11 in P. deltoides catkin tissues than in P. trichocarpa vegetative tissues provided us 33

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with confidence that the amplification product was related to transcript abundance difference among tissues and not sequence differences among the 2 species. For RT-PCR, 1 l of the 20 l RT reaction was used as template. One of 2 Populus genes; actin2 or ubiquitin (UBQ; Table 2-1), was used as internal control for each PCR reaction. Each pair of gene-specific primers was assayed in a PCR reaction alone and combined with each pair of control primers using genomic DNA as template. The combination giving higher PCR product was chosen for further gene expression quantific ation. The number of PCR cycles (Figure 2-6) for each gene was determined such that the le vel of product was in the linear range of the amplification. PCR products were separated by agarose gel electrophoresis, stained with ethidium bromide, and band intensities were scaled to the band intensity of the internal control genes using Kodak 1D Image analysis software. Tissues were collected from 3 plants and 3 technical reps were performed for each ge ne-tissue combination. Differences between normalized tissue intensities were identified as st atistically significant by standard two-sample t tests ( = 0.05). Results Cytokinin Response Regulator Gene Family in Populus Arabidopsis RR genes were selected to query the Populus database because all Arabidopsis members have been identified, thei r functions and tissue specificity are being clarified, and Arabidopsis is the closest relative of Populus for which whole-genome sequence is available (Soltis et al., 1999). TBLASTN searches of the JGI Populus trichocarpa genome assembly v1.1 identified 11 type -As, 11 type-Bs and 11 putative pseudo-RRs. Putative Populus RRs were then used to re-search the JGI genom e assembly to improve the power of our gene discovery searches, however, no additiona l RRs were found. All of the identified Populus RRs 34

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share the conserved receiver domain (Figure 2-1). The Populus pseudo-RRs identified here have substitutions in the first 2 residues of the D-D-K, yielding either E-E-K or D-E-K. Since gene structure may provide clues to gene evolution, we investig ated the distribution of introns and exons by comparing gene models with ESTs. Partial and/or complete ESTs were found for 9 type-As, 9 type-Bs, and 10 pseudo-RRs (Figure 2-2). The receiver domains of typeAs and type-Bs appear to have distinct origins be cause they are encoded by a different number of exons; 5 for type-As and 3 to 4 for type-Bs. The pseudo-RRs are more heterogeneous with a variable number of exons (6 to 15) and with receiver domains encoded by 2 to 6 exons (Figure 22). Six of the pseudo-RRs contain a motif of 50 a.a. known as a CCT motif (for C ONSTANS, C ONSTANS-like and T OC1; Makino et al., 2000; Strayer et al ., 2000). The three subfamilies also coalesced into distinct groups when clustered based on receiver domain amino acid sequence (Figure 2-2). By using in silico localization methods, we found that 29 out of 33 RRs were predicted to be nuclear (Table 2-2), which is consistent with localization of e quivalent Arabidopsis subfamilies to the nucleus (Lohrmann et al., 1999; Sakai et al., 2000; Imamura et al., 2001). For 2 of the 3 type-As that were not predicted to be nuclear (PtRR5 and PtRR8), no consensus was found among the prediction programs, whereas 1 type-A (PtRR9) was predicted to be cytoplasmic. Short stretches of positively char ged amino acids (mostly Arg and Lys) in the region downstream of the receiver domain may function as nuclear localization signals in both type-As and type-Bs (Sakai et al ., 2000; Imamura et al., 2001). De letion of the short C-terminal region of the Arabidopsis type-As ARR6 and ARR7 abolish their ability to enter the nucleus (Imamura et al., 2001). Populus RRs are most likely nuclear proteins and the presence of a 35

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GARP domain (Figure 2-3) appe ars unnecessary for nuclear local ization since 8 type-As and 10 pseudo-RRs, which lack this domain, we re predicted to be nuclear. A sequence similarity tree using the conser ved receiver domains of RR gene family members in Populus Arabidopsis and rice (Figure 2-4) reve aled that a significant number of the RRs (26 in Populus 20 in Arabidopsis and 14 in rice) gr ouped in species-specific pairs. As expected, we observed a tendency of monocot RRs to group in clades separate from dicot RRs. Models for gene family evolution propose that s ister pairs of genes w ould arise as the product of chromosomal duplication events that occur in dependently in different species (Blanc and Wolfe, 2004; Van de Peer, 2004; Sterck et al., 2005). We tested this model by comparing the chromosomal distribution of the Populus RRs. Consistent with this hypothesized model, we found that 71% of the sister pa irs are located on different chro mosomes that share duplicated segments (Tuskan et al., 2006; Table 2-3). Expression of Populus RRs To define the tissue preferences of the Populus RR gene family, we generated expression data using a combination of microarray anal yses and semiquantitative RT-PCR. Transcript abundance for 8 type-As, 7 type-Bs and 10 pseudo-RRs was detected above background in the microarray experiment. Of the 8 type-As, 6 (PtRR1, PtRR2, PtRR4, PtRR5, PtRR6, and PtRR10) showed significant differences in transcript abundance among tissues ( p-value 0.05), while 2 (PtRR7 and PtRR8) showed no tissue preference (Figure 2-5A). Of the 6 with differences in transcript abundance, 5 (PtRR1, PtRR2, PtRR 5, PtRR6 and PtRR10) were preferentially expressed in nodes over young or ma ture leaves. PtRR4 was signifi cantly more abundant in roots than in young leaves, mature leaves and nodes. Thus, Populus type-As appear preferentially expressed in stem tissues (comprised of node s and internodes) over leaf tissues (young or mature). 36

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The microarray results also revealed tissue e xpression preferences for the type-Bs. Of the 7 members whose expression was significan tly higher than background (PtRR12, PtRR13, PtRR15, PtRR16, PtRR18, PtRR19 and PtRR22), 6 showed significant differences among tissues (Figure 2-5B). A more detailed examination of contrasts among tissues re vealed that 5 of the type-Bs (PtRR12, PtRR13, PtRR18, PtRR19 and Pt RR22) were significantly more abundant ( pvalue 0.05) in nodes than in mature and/or young leaves. Thus 5 of the 7 type-Bs expressed above background in the microarray analysis appear ed preferentially expressed in stem tissues. The pseudo-RRs subfamily had the most members detected above background (10 out of 11; Figure 2-5C). Seven (PtpRR3, PtpRR4, PtpRR5, PtpRR6, PtpRR7, PtpRR10 and PtpRR11) showed significant differences among tissues ( p-value 0.05), all of which exhibited higher transcript abundance in mature and/or young leaves than in roots. The sister pair PtpRR3 and PtpRR5 were the only genes for which the expre ssion in mature leaves was significantly higher than in the other 4 tissues. The sister pair PtpRR10 and PtpRR11 exhi bited significantly higher expression in nodes than roots, mature leaves or young leaves (Figure 2-5C). Overall, pseudoRRs appear more abundant in aboveground organs than in belowground organs (7 out of 10), with mature leaves being the organs of highest transcript enrichment. To obtain additional information about type-A and type-B RR tissue preference we performed semiquantitative RT-PCR analysis on ti ssues not included in the microarray analyses: outer stem (including phloem, axillary buds and pe tioles referred to as phloem), inner stems (including xylem and pith referred to as xylem ) and preand post-receptive female catkins. Two pairwise tissue comparisons ( t -tests with = 0.05) are shown: phloem vs. xylem and prereceptive catkins vs. post-receptive catkins (Figur e 2-6). We detected expression of 20 family members but not the type-Bs PtRR14 and PtRR 20. Although Arabidopsis RRs have been shown 37

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to be expressed in the vasculat ure of shoots and roots (D'Agos tino et al., 2000; To et al., 2004), differential regulation of RRs within the stem can be quantified after physi cal separation of stem tissues in Populus. Three type-As and 8 type-Bs were significantly more abundant in phloem than in xylem at = 0.05 (Figure 2-6) while only 1, the type-A PtRR5, was significantly higher in xylem than in phloem. Our results agree with pr evious findings that t ype-As and Bs are highly expressed in vasculature and reveal that type-Bs are preferenti ally expressed in outer stem tissues. Semiquantitative RT-PCR of pre-receptive a nd post-receptive catkins provided us with new information about Populus type-A and B expression in repr oductive tissues. Transcripts for 18 of the 22 type-As and Bs were detected in fl oral tissues (Figure 2-6) Seven type-As (PtRR1, PtRR2, PtRR3, PtRR5, PtRR7, PtRR8 and PtRR11) were more abundant in pre-receptive catkins and 1 type-A (PtRR4) and 1 type-B (PtRR22) we re more abundant in post-receptive catkins ( pvalue 0.05). Three type-As (PtRR3, PtRR9 and PtRR 11), whose expression were not detected in the microarray experiment, were highly abundant in pre-receptive catkins (Figure 2-6). The RT-PCR analysis of PtRR9 transc ript abundance in vegetative and reproductive tissues revealed the presence of 3 aberrant tr anscripts (approximately 360, 560 and 580bp each) that were shorter than expected (~800 bp). Populus Type-As and Bs Are Rapidly Upregulated by Cytokinin In several plant species, type-A RRs are defi ned as primary cytokinin response genes since de novo protein synthesis is not requ ired for their expression in re sponse to cytokinin treatment. Maximal transcript induction occurs, on average, within 1 hour after ap plication of exogenous cytokinin (Brandstatter and Kieber, 1998; Sakakiba ra et al., 1998; Asakura et al., 2003; Jain et al., 2006). Treatment of detached maize leaves with cytokinin rapidly induces transcript accumulation of ZmRR1 and ZmRR2, 2 type-A RRs (S akakibara et al., 1998; Sakakibara et al., 38

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1999). The effect of exogenous cy tokinin on the abundance of the 22 Populus type-As and Bs in leaves was analyzed by semiquantitative RT-PCR (Figure 2-7). The maize detached-leaf system (Sugiharto et al., 1992) was adapted for Populus and individual detached mature leaves were treated with exogenous cytokinin (1 M BAP) for 1 hour. Seven of the 11 Populus type-As (PtRR2 through PtRR7 and PtRR10) revealed tran script accumulation after cytokinin treatment when compared to the dimethyl sulfoxide (DMSO) treated control. In this group of cytokinininducible RR genes, transcripts for PtRR2, PtRR3 and PtRR6 were not detected in untreated leaves, while transcripts for PtRR4, PtRR5, PtRR7 and PtRR10 were detected even in the absence of the exogenous cytokinin. Differences in baseline levels of tr anscript abundance may reflect responses of genes to di fferent baseline levels of endoge nous cytokinin, or responses of genes to signals other than cytokinin. Because cytokinin inducibility of Populus RRs was performed only in mature leaves, family member s expressed in different organs may also be cytokinin responsive but not detected in our as say. Unexpectedly, since type-Bs have been considered non-responsive to exogenous cyt okinin (Imamura et al., 1998; Kiba et al., 1999), three Populus type-Bs (PtRR13, PtRR18 and PtRR22) show ed increased transcript levels after exogenous treatment with cytokinin. Discussion Our study identified 33 type-A, type-B and pseudo-RRs in Populus This number is approximately equivalent to the RR family size in Arabidopsis (32 genes) and rice (36 genes). The Populus genome is predicted to contain 45,000 gene s (Tuskan et al., 2006), almost twice the number in Arabidopsis (~27,000) and approximate ly the same number as rice (~41,000). Based on the JGI gene models, Populu s has more protein-coding genes than Arabidopsis, averaging 1.4 to 1.6 putative Populus homologs for each Arabidopsis gene (Tuskan et al., 2006), yet the similarity in protein domain fre quencies in the 2 genomes suggests that the grea ter gene number 39

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in Populus is largely due to gene family expans ion and not to the presence of unique Populus genes (Tuskan et al., 2006). Families that have undergone expansion in Populus include genes involved in lignocellulosic wall forma tion (cellulose synthases, 93 in Populus vs. 78 in Arabidopsis); lignin and phenylpr opanoid biosynthesis (34 in Populus vs. 18 in Arabidopsis), flavonoid biosynthesis (cha lcone synthases, 6 in Populus vs. 1 in Arabidopsis), and disease resistance (NBS coding R genes, 399 in Populus vs. ~200 in Arabidopsis; Tuskan et al., 2006). The Populus type-As, type-Bs and pseudo-RRs however, shows no expansion compared to Arabidopsis and rice. Because of the importance of cytokinin in plant growth and development, the conservation in family size observed among Populus, Arabidopsis and rice type-As, type-Bs and pseudo-RRs, may reflect selection ag ainst substantial changes in the stoichiometry of the components of this signaling cascade. Genes en coding regulatory molecules involved in signal transduction pathways, like transcription factors, tend to be dosage-dependent; changing the concentration of a regulator could change the con centration of its targets (Birchler et al., 2001). Populus type-As and type-Bs exhibit the invari ant D-D-K residues in the receiver domain originally identified in bacterial signal transducers (P arkinson and Kofoid, 1992) and are predicted to be phosphorylated at the central aspartate in a cy tokinin dependent manner. Such phosphorylation is hypothesized to modulate the DNA binding activity of the C-terminal domain in the type-Bs but the effects on type-A protei n function remain elusiv e (Miyata et al., 1998; Sakai et al., 2000). However, site-directed mutagene sis of the central aspartate to a glutamine in the Arabidopsis type-B ARR2 had no effect on DNA-binding (Hwang and Sheen, 2001), suggesting that phosphorylation is no t required for target DNA binding. Searching the Populus genome revealed the presence of atypical RRs referred to here as pseudo-RRs. The proteins encoded by these genes have the central aspartate of the invariant D40

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D-K substituted by glutamate (D E). We speculate that Populus pseudo -RRs may be components of the plant biological clock since pseudo-RRs in Arabidopsis and rice have been implicated in circadian controlled events such as flowering time and photomorphogenic responses (Makino et al., 2000; Murakami et al., 2004; Mizuno and Nakamichi, 2005). APRR1, an Arabidopsis pseudo -RR also referred to as TOC1 ( T IMING O F C AB EXPRESSION 1 ), is a component of the central oscilla tor of the circadian clock (Somers et al., 1998; Strayer et al., 2000). Transcripts of several Arabidopsis and rice pseudo-RRs, including TOC1 and its putative ortholog in rice (OsPRR1), have been detected in leaves and exhibi t circadian regulation (Makino et al., 2000; Murakami et al ., 2005). High expression of several Populus pseudo-RRs in mature leaves may indicate a role for these gene s in clock-regulated events such as stomatal opening and phenylpropanoid accumulation (Harmer et al., 2000). The classification scheme for response regul ators was recently expanded to include an additional subclass (type-Cs; Schall er et al., 2007), which have a re ceiver domain more similar to that of histidine kinases than to the type-As, Bs or pseudo-RRs. The rice and Arabidopsis genomes each encode 2 type-Cs, however, the Populus genome appears to have 15 (data not shown). It will be of interest to test the functional significance of this expansion in Populus. Our analysis of the relationship of the Populus RR gene family revealed that 78% of the genes grouped in pairs. Comparing the chromosomal distribution of the Populus RR gene family revealed that 71% of the sister pairs were located in paralogo us genome regions as defined by Tuskan et al. (2006). The high similarity in am ino acid sequence and gene structure among these sister pairs suggest their orig ination from the duplication of a common ancestor. We also found that Arabidopsis and rice RRs grouped in sister pairs. All of these results are consistent with the proposed genome duplications that Populus (Sterck et al., 2005; Tuskan et al., 2006), 41

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Arabidopsis and rice have under gone (Bowers et al., 2003). Typicall y, an individual member of a sister pair can be lost wit hout affecting fitness since th e pair has redundant functions immediately after the duplicati on event (Blanc and Wolfe, 2 004). However, gene loss or retention is not random and regul atory genes involved in signal transduction, such as RRs, tend to be dosage dependent and preferentially retain ed (Blanc and Wolfe, 2004). Intriguingly, the Populus RR sister pairs that arose during the salicoid duplication, th e type-As (4), type-Bs (5) and pseudo-RRs (4), reflect a large increase in the ge ne family but evenly distributed across the subfamilies. In Arabidopsis, functional redundancy among RR sister pairs is positively correlated with overlapping expression profile s (Mason et al., 2004; To et al ., 2004; Mason et al., 2005) and may explain the weak phenotypes exhibited by single loss-of-function mu tants (To et al., 2004). In contrast, the RR gene family in rice appears less functionally redundant since single RR mutants often exhibit str onger phenotypes (Hirose et al., 2007). Our finding that Populus RR sister pairs exhibit overlapping expression profiles suggests pote ntial functional redundancy in this gene family. High transcript abundance of Populus RRs in nodes and phloem is consistent with a role for members of this gene family in regulating me ristematic activity in the vascular cambium and axillary apical meristems. Roots also appear to be sites of active cytokinin signaling in Populus since transcripts for most of the Populus type-As and Bs (17 out of 22) were detected in this organ. Similarly, expression of 13 RRs and HKs have been detected in Arabidopsis roots (Imamura et al., 1999; D'Agostino et al., 2000; Higuc hi et al., 2004; Nishimura et al., 2004; To et al., 2004). Arabidopsis AHK4/CRE1/WOL, the firs t cytokinin receptor id entified, was found to be preferentially expressed in roots and require d for root xylem differen tiation (Mahonen et al., 2000; Inoue et al., 2001; Hi guchi et al., 2004). The cre1 mutant exhibits a reduced number of cell 42

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files within the vascular bundle because of the lack of peric linal procambial cell divisions (Mahonen et al., 2000). Because of the abundant ev idence that cytokinin has negative effects on root growth in Arabidopsis a nd tobacco, we speculate that Populus RRs negatively regulate root growth by blocking lateral root fo rmation, restricting the size of the meristem in root tips, and preventing protoxylem specification. Cytokinins have also been identified as 1 of the signaling molecules required for the formation of floral meristems (Bernier and Perilleux, 2005). In Sinapis alba flowering is inhibited when long-distance signaling is in terrupted by phloem removal and restored upon application of cytokinin to the apex (Havelange et al., 2000). Additional documented roles of cytokinin in reproductive tissue development in clude maintaining cell division within the embryo, greening of sepals, and enhancing sink strength of seeds (H erbers and Sonnewald, 1998). We detected expression of 77% of the Populus type-A and B RRs in pre-receptive and post-receptive catkins with type-As being more abundant in pre-receptive catkins. Arabidopsis cytokinin RRs and HKs have been detected in different floral ti ssues, including petals, stigmas, styles, immature siliques, the abscission zone of flowers, the junction of sepals and pedicels (Urao et al., 1998; Imamura et al ., 1999; Mason et al., 2004; Tajim a et al., 2004; Mason et al., 2005). Two Populus type-As appear to be specifically expressed during the reproductive phase of development since they were not detected in vegetative tissues or in any publicly available EST resources. Interestingly, th ese 2 genes grouped with the 2 Arabidopsis type-As (ARR16 and ARR17) whose expression is also high in reprod uctive tissues (Zimmermann et al., 2004). Such similarities in both protein sequence and tissue preference may indicate identical gene function in the 2 species. 43

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Consistent with findings in Arabidopsis, rice and maize (Brandsta tter and Kieber, 1998; Sakakibara et al., 1998; D'Agos tino et al., 2000; Asakura et al., 2003; Jain et al., 2006), transcripts of 7 Populus type-As were induced after 1 hour of cytokinin treatment. Type-As are thought to be negative regulators that medi ate a feedback mechanism by which the plant decreases its sensitivity to the hormone (To et al., 2004) and are predic ted to inhibit type-B activation by competing for phosphotransfer from upstream HP proteins (To et al., 2004). Although Arabidopsis type-Bs do not appear induced after cytokini n treatment (Imamura et al., 1999; Rashotte et al., 2003; Kiba et al., 2004; Brenne r et al., 2005), 3 Populus type-Bs (PtRR13, PtRR18 and PtRR22) showed increased transcript levels after exogenous cytokinin treatment. We speculate these results could refl ect a novel regulation of particular Populus type-Bs. In the present study we have identified the members of the Populus RR gene family. They exhibit typical features of other plant RRs such as transcript induction in response to exogenous cytokinin, the presence of a receiver domain and a GARP domain (characteristic of the type-Bs). A significant proportion of the genes in this family seem to be the product of the recent salicoid whole-genome duplication event. Most of the ty pe-As and Bs are preferentially expressed in stem tissues, while pseudo -RRs are preferentially expressed in mature leaves Unraveling the contributions of indivi dual cytokinin RRs in Populus will contribute to our understanding of the roles that cytokinin can play in perennial plant growth and de velopment. There is a growing realization that combustion of fossil fuels and other human activities, including deforestation and other changes in land use, is driving an imbala nce in the global carbon cycle (DeLucia et al., 2005). Forest trees such as Populus are seen as clean alterna tives to reducing anthropogenic carbon dioxide in the atmosphere be cause of their capacity to store large quant ities of biomass in belowground organs and as a feedstock for re newable bioenergy. A better understanding of the 44

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hormone response pathways that govern producti vity should improve opportunities for genetic enhancement of woody biomass. 45

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P tRR1 1 -----------------------------------------------------------------------M SSNSIASNRWMSEKMDGFD-------PSPNSNSDNEEEG VHVLA VDDSLVDRKVIERLLK ISS CKP tRR2 1 -----------------------------------------------------------------------M GSNSIVSNRWMSEKMNCLD-------LSPNSNSDNEEEE VHVLA VDDSFVDRKVIERLLK ISS CKP tRR10 1 -----------------------------------------------------------------------M ATAGEILRRSLTEEVG----------FSKGSVSGSEE-LHVLA VDDSFVDRKVIERLLK ISS CKP tRR9 1 -----------------------------------------------------------------------M AGSSSSS-------------------PSMGFDFDEKLHV L A V EAVDDGLIDRK A IERLLINSE Y KP tRR11 1 -----------------------------------------------------------------------M ASSSSSL-------------------PSMEFDFDEKPHV L ---AVDDSLIDRKVIERLL INS T CRP tRR8 1 -----------------------------------------------------------------------M VGG----------------------------AYTEEPHV L ---AVDDSLVDRKLVERLLK NSS CKP tRR4 1 -----------------------------------------------------------------------M ATLATLAT----------------------------ETQF HVLA VDDSLIDRKLIERLLK TSSY QP tRR5 1 --------------------------------------------------------------------MAVE M ALATTMAT----------------------------ETQF HVLA VDDCLIDRKLIERLLK TSSY QP tRR6 1 --------------------------------------------------------------------MAVE M ALATTMAT----------------------------ETQF HVLA VDDSMIDRKLIERLLK TSSY QP tRR7 1 --------------------------------------------------------------------------MAITGDS----------------------------LSQF HVLA VDDSLIDRKLIERLLK TSSY QP tRR3 1 -------------------------------------------------------------------MEIMESGAVDTQHQEEEKHQQQKEEEGEEKHKQQRDQEGEEEEKHF HVLA VDDSFIDRKLLERLLK VSSY QP tRR12 1 -----------------------------------------------------------------------M MNLANCKGSMSTATSGG-------VWKASDGASDQFPAG LRVLVVDDD P T CLV ILEKMLR T C R Y EP tRR13 1 -----------------------------------------------------------------------M LNLGYCKGSMSTASSG--------------GVSDQFPAG LRVLVVDDD P T CLV ILEKMLR T C L Y EP tRR14 1 -----------------------------------------------------------------------M ENN-----GFSSP------------------RNDSFPAG LRVLVVDDD P T WLKILEKMLK K CSYEP tRR21 1 -----------------------------------------------------------------------M EN------GFSSP------------------RNDSFPAG LRVLVVDDD P T WLKILEKMLK R CSYEP tRR15 1 -----------------------------------------------------------------------M AALQRVAPSLGTSASTYGSC---KGAGADVIVSDQFPAG LRVLVVDDD I T CLRLLEKMLR R C L Y HP tRR16 1 -----------------------------------------------------------------------M AALQRVASSLSTSASSYGSC---KGAGADLMFSDQFPAG LRVLVVDDD I T CLRLLEKML CR C L Y NP tRR18 1 -----------------------------------------------------------------------M AVDDQRGGNSVNE--------------------KKFPVG MRILA VDDDPICLKVLE N LLR K C Q Y EP tRR19 1 -----------------------------------------------------------------------M AVDDQRGGNSVNE--------------------KKFPVG MRVLA VDDDPICLKVLE N LLR K C Q Y EP tRR22 1 -----------------------------------------------------------------------M TVEQGIGDSNID----------------------QFPIG MRVLA VDDDP T CLL LLE T LLR R C Q Y NP tRR17 1 -----------------------------------------------------------------------M ASTQSSNALS------------------------SSLPK IHILIVDDD S T SLS VVSAT LK TVSY KD P tRR20 1 -----------------------------------------------------------------------M ASSQSSNIIQN-----------------------SSLLK IKILVVDDD S T SLS IVSA MLK T CSYKE P tpRR1 1 -----------------------------------------------------------------------M TSEQFLSVEVTKN------------------NASSFAEG VRILVVE S D P T CLRIV S KMLQAFG Y EP tpRR2 1 -----------------------------------------------------------------------M TSERVLSVEVTEN------------------NAGSFPIG VRILVVE S D P T CLRIV S KMLQAFG Y EP tpRR3 1 -------------------MGVVVVSSG---EELEVKTGSETEEEKQSKEETESETGEVKRKR-------------KKK-EGEGS------------DNGLVRWERFLPRMV LRVLLVE A D D S T R Q IIAA LLR K CSYRP tpRR5 1 -------------------MGEVVISSG---EELEVRSKSEREEEKQRKQSKE-ETGEVKKKK-------------KKKKEGEGL------------NDGLVRWDGFLPRMV LRVLLVE A D D S T R Q IIAA LLR K CSYRP tpRR4 1 ---MLTEGEYINKHSNLSYYPISIVASKDYFDLLNQKTKPKHTIEIKGKENPPGEEDIFRGSRGS-YLRMGE V VVSSSSEEVEGMAVELETEKKDIGSSEVVRWEKFLPRMV L S VLLVE A D D S T R Q IIAA LLR K CSYRP tpRR6 1 ---MLTR-----HLNQPVANGEPPFDRPS--DLLNQIKKPKLSQLKKLKKILQTRRYFQRFCGLSSCLRMGK V VLSSSSEEAGGMVVELETEKKDIGSSEVVRWEKFLPKMV LRVLLVE A D D S T R Q IIVA LLR K C G Y RP tpRR7 1 MNKNASIDQRVAEQNHIVEDEQKEIRDGIMGEGQELSEEDESQINEDGKYMNDKGMELLQVQN------DAQAVIQSQQQQSQGP---------------LVIWERFLPLRS LKVLLVEND D S T RHVVSA LLR N C G Y EP tpRR8 1 ---MLSMNNGFAEQNHIVEDEQKKIRDGIMGEDQELSEEGESQINEDEKDVNDKGMESLQVLT------DAQ V VIQSQHQQSQGP---------------LVHWERFLPRRS LKVLLVEND D S T RHVVSA LLR N C G Y EP tpRR10 1 -----------------------------------------------------------------------M VCTTNDLS-----------------------AWKDFPKG L S VLLLDED N S SAAEI KSK L EALD Y IP tpRR11 1 -----------------------------------------------------------------------M VCTTNDLS-----------------------AWKDFPKG LRVLLLDED SMSAAE I KSK L EAMD Y IP tpRR9 1 -------------------------------------MEGEVDEQNLSG----------------------H M KESGNGKSVGGGG----------------AGDGFVDRSK VRILLC DND AKSSQE V FT LLLK CSYQP tRR1 ------VTA V DS G WRAL K LL GLLD--EEDKSSSSSSSSS--AGFDVLK VDLIITD YCMP G MTG Y E LL KKI KEST---------------------TF REIPVVIMSSE NVVAR I D RCL EE GAE DFIVKPVKL S DVK R165 P tRR2 -------E T A V DS G WGAL K LL GLLDDDEEDKSSSSSSSSSSSAGFEGLK VDLIITD YCMP G MTG Y E LL KKI KESS---------------------SF REIPVVIMSSE NVMAR I D RCL EE GAE EFIA KPVKLS DVK R169 P tRR10 ------VTV V ES G SRAL QY L GLDG----EKSS---------VGFNDLK INLIMTD YSMP G MTG Y E LL KKI KESS---------------------AF REIPVVIMSSE NILAR I D RCL EE GAE EYILKPVKL S DVK R151 P tRR9 ------VTT AENKKK AI EY L GLAD---GHHTNH------------DLK VNLIITD YCM RGMTG Y E LL KRI KESP---------------------TM K GDT VVVVSSE NIP T R I KG CMEE GAQ EFLLKPL Q L SG V TK L 143 P tRR11 ------VTT AEN G KRAL EY L GLAD---GQHPSHSQ----------DLK VNMIITD YSMP G MTG Y E LL KRI KESP---------------------TM KEIPVVVVSSE NIP T R I NQ CMEG GAQ EFLLKPL Q L S D ATK L 142 P tRR8 ------VTT AEN G LRAL EY L GLGD---EKRTSLEDN---------VSK VNLIITD YCMP G MTG Y E LL KKI KESS--------------------M L KEIPVVIMSSE NIP T R I N KCL EE GAQM FMLKPLK QSDV VK L 134 P tRR4 ------VTA V DS G SKAL EF L GLHGD-DEQRDSNP-SSVSPDHHHQHIE INMIITD YCMP G MTG Y D LL KKI KESK---------------------YF KDIPVVIMSSE NVP S R I N RCL EE GAE EFF LKPV Q L S DV NK L 147 P tRR5 ------VTA V DS G SKAL EF L GLNGE-NELRDSKP-ASVSPDPYHQHIE INMIITD YCMP G MTG Y D LL KKI KESK---------------------YF KDIPVVIMSSE NVP S R I N RCL KE GAE EFF LKPV Q L S DV NK L 151 P tRR6 ------VTA V DS G SKAL KF L GLHEE-DDHSNPDTVPSVSPN-DHREVE VNLIITD YCMP G MTG Y D LL KKV KESS---------------------SL RDIPVVIMSSE NVP S R I T RCL EE GAE EFF LKPVRL A DL NR L 151 P tRR7 ------VTTVDS G SKAL KF L GLQE--DEQSNPDT-PYVSPN-NHQEME VNLIITD YCMP G MTG Y D LL KKV KESS---------------------SL R N IPVVIMSSE NVP S R I T RCL EE GAE EFF LKPVRL S DL NR L 143 P tRR3 ------VTF V DS G DKAL EY L GLLDS-IDNVNATSSSSSSQSPQQEGMK VNLIMTD YCMP G MSG Y D LL KRV KGS----------------------YW KDVPVVVMSSE NIP S R I RM CLEE GAE EFLLKPL Q L S DV EK179 P tRR12 ------VTKCNR A EIAL S LLR ENKN----------------------GY DIVISDV H MP D M D G I F K LL EQI G-----------------------M SSS LPIVVF SAD NNV S A M LGWL Y KGA AL YLMKPI VKNDVK N L 146 P tRR13 ------VTKCNR A EIAL S LLR ENRN----------------------GY DIVISDV H MP D M D G F K LL ELI G-----------------------L EMDLPVIMMSAD DGKNV V M K G V T HGA C DYLIKPIRI EA LKN I 138 P tRR14 ------VTT CGL A RDAL N LLR ERKG----------------------GY DIVISDV Y MP D M D G F K LL EQV G-----------------------L EMDLPVIMMS V D GET S R V M K G V Q HGA C DYLLKPIRM K ELR N I 129 P tRR21 ------VTT CGL A RDAL N LLR ERKG----------------------GY DIVISDV Y MP D M D G F K LL EHV G-----------------------L EMDLPVIMMS V D GET S R V M K G V Q HGA C DYLLKPIRM K ELR N I 128 P tRR15 ------VTT CSQ A TAAL K LLR ERKG----------------------CF DVVLSDV H MP D M D G F K LL ELV G-----------------------L EMDLPVIMMSAD GRT S A V M R G I S HGA C DYLIKPIR EEELK N I 149 P tRR16 ------VTT CSQ A TAAL K LLR ERKG----------------------CF DVVLSDV H MP D M D G F K LL ELV G-----------------------L EMDLPVIMMSAD GRT S A V M R G I R HGA C DYLIKPIR EEELK N I 149 P tRR18 ------VTT TNQ A VTAL E MLR ENRN----------------------KY DLVISDV N MP D M D G F K LL ELV G-----------------------L EMDLPVIMLSS HGDKEF V Y K G V T HGA V DYLLKPVRM E ELK N I 132 P tRR19 ------VTT TNQ A VTAL E MLR ENRN----------------------KY DLVISDV N MP D M D G F K LL ELV G-----------------------L EMDLPVIMLSS HGDKEF V F K G I T HGA V DYLLKPVRL E ELK N I 132 P tRR22 ------VTT TSQ A ITAL R MLR ENKN----------------------KF DLVISDV H MP D M D G F K LL ELV G-----------------------L EMDLPVIMLSA NGDPKL V M K G I T HGA CY YLLKPVRI E ELK T I 130 P tRR17 NRNLELI V V TV KKPFDAL S ILR LKKG----------------------LF DLVVSDL H MP E M N G -MEL QKQ V E------------------------EEFK LPVIIMSSD ESKNV I S R S L EG GAAF YIVKPANKVDLK N V 136 P tRR20 NRKLELF V V TV KNPFDAL ST LRLKKG----------------------LF DLVVTDL H MP E M N G -MEL QQQ V D------------------------EEFK LPVIIMSSD DSEKV I L R T L EG GAAF YIVKPI NKDDLK N V 137 P tpRR1 ------VTT ATR A TDAL H ILR EKED----------------------E INLILI E THLP D M DQ-Y E II ETV RA----------------------L SSLPIVVF SAD NNE S A M LG CLY KGA AL YLMKPI IKNDVK N L 135 P tpRR2 ------VTT ATR A TDAL R ILR EKED----------------------E INLILI E TRLP D M NQ-Y E IL ETL GE----------------------L EMDLPVIMMSAD DGKNV V M K G V T HGA C DYLIKPIRI EA LKN I 134 P tpRR3 ------V A TV SD G LKA WEILK ERPH----------------------N IDLILTEV D LP S VSG Y A LL TLI MEHE--------------------I C K N IPVIMMSS QDSIKT V Y KCM L RGA A DYLVKPIR KNELR N L 179 P tpRR5 ------V V SV PD G LKA WEILK GRPH----------------------G IDLILTEV D LP S I ----------------------------------I C K N IPVIMMSS QDSIS T V Y KCM L RGA A DYLVKPLR KNELR N L 166 P tpRR4 ------V AA V PD G LMA WET LKGGPH----------------------N IDLILTEV E LP L ISG Y A LL TLV TEHA--------------------V C K N IPVIMMSS QDSIS M V L KCM L KGA A DFLIKPVR KNELR N L 223 P tpRR6 ------VSA V PD G LMA WET LKERPH----------------------S IDLILTEV E LP L ISG Y AFL ALV MEHD--------------------V C K N IPVIMMSS HDSIS V V L KCM L KGS A DFLVKPVR KNELR N L 217 P tpRR7 ------VTA V SN G LQA WKVL QDLTN----------------------H IDLVLTEV A MP C LSG -IGLL SKI MSHK---------------------TC R N IPVIMMSS HDSMNV V F KCL S KGA V DFLVKPIR KNELK I L 206 P tpRR8 -------A T A V AN G LQA WKLL QDLTN----------------------H IDLVLTEV A MP C LSG -IGLL SNI MSHK---------------------TC R N IPVIMMSS HDSMNV V F RCL S KGA V DFLVKPIR KNELK I L 203 P tpRR10 ------V Y T FCNENE AL LA I SNEPG----------------------SFH V A I V EVISFSSNPT F QYL VNV VKPAATMMNIVQESKQEKHTNLTWN L LYDL NA M KRI SDFID S EQAFNAGGSVQSES LKPVKDSI V SM L 154 P tpRR11 ------V Y T FCNETE AL SA I SNEPG----------------------SFH V A I V EVS M SNS S RSF KFL --------------ETSK------------DLP T I KKV SDFMDPEQAFNAGGSVQSKS LKPVKDSV V SM L 127 P tpRR9 ------VTSVRS A RQV I DA L NAEGP----------------------E IDIILSEV D IP MTKG -MKML KYI MRDK---------------------DL R R IPVIMTVHCVHT S E L LGLAEK NILNY DFDPV ASDPSDAN 152 P tRR1 1 -----------------------------------------------------------------------M SSNSIASNRWMSEKMDGFD-------PSPNSNSDNEEEG VHVLA VDDSLVDRKVIERLLK ISS CKP tRR2 1 -----------------------------------------------------------------------M GSNSIVSNRWMSEKMNCLD-------LSPNSNSDNEEEE VHVLA VDDSFVDRKVIERLLK ISS CKP tRR10 1 -----------------------------------------------------------------------M ATAGEILRRSLTEEVG----------FSKGSVSGSEE-LHVLA VDDSFVDRKVIERLLK ISS CKP tRR9 1 -----------------------------------------------------------------------M AGSSSSS-------------------PSMGFDFDEKLHV L A V EAVDDGLIDRK A IERLLINSE Y KP tRR11 1 -----------------------------------------------------------------------M ASSSSSL-------------------PSMEFDFDEKPHV L ---AVDDSLIDRKVIERLL INS T CRP tRR8 1 -----------------------------------------------------------------------M VGG----------------------------AYTEEPHV L ---AVDDSLVDRKLVERLLK NSS CKP tRR4 1 -----------------------------------------------------------------------M ATLATLAT----------------------------ETQF HVLA VDDSLIDRKLIERLLK TSSY QP tRR5 1 --------------------------------------------------------------------MAVE M ALATTMAT----------------------------ETQF HVLA VDDCLIDRKLIERLLK TSSY QP tRR6 1 --------------------------------------------------------------------MAVE M ALATTMAT----------------------------ETQF HVLA VDDSMIDRKLIERLLK TSSY QP tRR7 1 --------------------------------------------------------------------------MAITGDS----------------------------LSQF HVLA VDDSLIDRKLIERLLK TSSY QP tRR3 1 -------------------------------------------------------------------MEIMESGAVDTQHQEEEKHQQQKEEEGEEKHKQQRDQEGEEEEKHF HVLA VDDSFIDRKLLERLLK VSSY QP tRR12 1 -----------------------------------------------------------------------M MNLANCKGSMSTATSGG-------VWKASDGASDQFPAG LRVLVVDDD P T CLV ILEKMLR T C R Y EP tRR13 1 -----------------------------------------------------------------------M LNLGYCKGSMSTASSG--------------GVSDQFPAG LRVLVVDDD P T CLV ILEKMLR T C L Y EP tRR14 1 -----------------------------------------------------------------------M ENN-----GFSSP------------------RNDSFPAG LRVLVVDDD P T WLKILEKMLK K CSYEP tRR21 1 -----------------------------------------------------------------------M EN------GFSSP------------------RNDSFPAG LRVLVVDDD P T WLKILEKMLK R CSYEP tRR15 1 -----------------------------------------------------------------------M AALQRVAPSLGTSASTYGSC---KGAGADVIVSDQFPAG LRVLVVDDD I T CLRLLEKMLR R C L Y HP tRR16 1 -----------------------------------------------------------------------M AALQRVASSLSTSASSYGSC---KGAGADLMFSDQFPAG LRVLVVDDD I T CLRLLEKML CR C L Y NP tRR18 1 -----------------------------------------------------------------------M AVDDQRGGNSVNE--------------------KKFPVG MRILA VDDDPICLKVLE N LLR K C Q Y EP tRR19 1 -----------------------------------------------------------------------M AVDDQRGGNSVNE--------------------KKFPVG MRVLA VDDDPICLKVLE N LLR K C Q Y EP tRR22 1 -----------------------------------------------------------------------M TVEQGIGDSNID----------------------QFPIG MRVLA VDDDP T CLL LLE T LLR R C Q Y NP tRR17 1 -----------------------------------------------------------------------M ASTQSSNALS------------------------SSLPK IHILIVDDD S T SLS VVSAT LK TVSY KD P tRR20 1 -----------------------------------------------------------------------M ASSQSSNIIQN-----------------------SSLLK IKILVVDDD S T SLS IVSA MLK T CSYKE P tpRR1 1 -----------------------------------------------------------------------M TSEQFLSVEVTKN------------------NASSFAEG VRILVVE S D P T CLRIV S KMLQAFG Y EP tpRR2 1 -----------------------------------------------------------------------M TSERVLSVEVTEN------------------NAGSFPIG VRILVVE S D P T CLRIV S KMLQAFG Y EP tpRR3 1 -------------------MGVVVVSSG---EELEVKTGSETEEEKQSKEETESETGEVKRKR-------------KKK-EGEGS------------DNGLVRWERFLPRMV LRVLLVE A D D S T R Q IIAA LLR K CSYRP tpRR5 1 -------------------MGEVVISSG---EELEVRSKSEREEEKQRKQSKE-ETGEVKKKK-------------KKKKEGEGL------------NDGLVRWDGFLPRMV LRVLLVE A D D S T R Q IIAA LLR K CSYRP tpRR4 1 ---MLTEGEYINKHSNLSYYPISIVASKDYFDLLNQKTKPKHTIEIKGKENPPGEEDIFRGSRGS-YLRMGE V VVSSSSEEVEGMAVELETEKKDIGSSEVVRWEKFLPRMV L S VLLVE A D D S T R Q IIAA LLR K CSYRP tpRR6 1 ---MLTR-----HLNQPVANGEPPFDRPS--DLLNQIKKPKLSQLKKLKKILQTRRYFQRFCGLSSCLRMGK V VLSSSSEEAGGMVVELETEKKDIGSSEVVRWEKFLPKMV LRVLLVE A D D S T R Q IIVA LLR K C G Y RP tpRR7 1 MNKNASIDQRVAEQNHIVEDEQKEIRDGIMGEGQELSEEDESQINEDGKYMNDKGMELLQVQN------DAQAVIQSQQQQSQGP---------------LVIWERFLPLRS LKVLLVEND D S T RHVVSA LLR N C G Y EP tpRR8 1 ---MLSMNNGFAEQNHIVEDEQKKIRDGIMGEDQELSEEGESQINEDEKDVNDKGMESLQVLT------DAQ V VIQSQHQQSQGP---------------LVHWERFLPRRS LKVLLVEND D S T RHVVSA LLR N C G Y EP tpRR10 1 -----------------------------------------------------------------------M VCTTNDLS-----------------------AWKDFPKG L S VLLLDED N S SAAEI KSK L EALD Y IP tpRR11 1 -----------------------------------------------------------------------M VCTTNDLS-----------------------AWKDFPKG LRVLLLDED SMSAAE I KSK L EAMD Y IP tpRR9 1 -------------------------------------MEGEVDEQNLSG----------------------H M KESGNGKSVGGGG----------------AGDGFVDRSK VRILLC DND AKSSQE V FT LLLK CSYQP tRR1 ------VTA V DS G WRAL K LL GLLD--EEDKSSSSSSSSS--AGFDVLK VDLIITD YCMP G MTG Y E LL KKI KEST---------------------TF REIPVVIMSSE NVVAR I D RCL EE GAE DFIVKPVKL S DVK R165 P tRR2 -------E T A V DS G WGAL K LL GLLDDDEEDKSSSSSSSSSSSAGFEGLK VDLIITD YCMP G MTG Y E LL KKI KESS---------------------SF REIPVVIMSSE NVMAR I D RCL EE GAE EFIA KPVKLS DVK R169 P tRR10 ------VTV V ES G SRAL QY L GLDG----EKSS---------VGFNDLK INLIMTD YSMP G MTG Y E LL KKI KESS---------------------AF REIPVVIMSSE NILAR I D RCL EE GAE EYILKPVKL S DVK R151 P tRR9 ------VTT AENKKK AI EY L GLAD---GHHTNH------------DLK VNLIITD YCM RGMTG Y E LL KRI KESP---------------------TM K GDT VVVVSSE NIP T R I KG CMEE GAQ EFLLKPL Q L SG V TK L 143 P tRR11 ------VTT AEN G KRAL EY L GLAD---GQHPSHSQ----------DLK VNMIITD YSMP G MTG Y E LL KRI KESP---------------------TM KEIPVVVVSSE NIP T R I NQ CMEG GAQ EFLLKPL Q L S D ATK L 142 P tRR8 ------VTT AEN G LRAL EY L GLGD---EKRTSLEDN---------VSK VNLIITD YCMP G MTG Y E LL KKI KESS--------------------M L KEIPVVIMSSE NIP T R I N KCL EE GAQM FMLKPLK QSDV VK L 134 P tRR4 ------VTA V DS G SKAL EF L GLHGD-DEQRDSNP-SSVSPDHHHQHIE INMIITD YCMP G MTG Y D LL KKI KESK---------------------YF KDIPVVIMSSE NVP S R I N RCL EE GAE EFF LKPV Q L S DV NK L 147 P tRR5 ------VTA V DS G SKAL EF L GLNGE-NELRDSKP-ASVSPDPYHQHIE INMIITD YCMP G MTG Y D LL KKI KESK---------------------YF KDIPVVIMSSE NVP S R I N RCL KE GAE EFF LKPV Q L S DV NK L 151 P tRR6 ------VTA V DS G SKAL KF L GLHEE-DDHSNPDTVPSVSPN-DHREVE VNLIITD YCMP G MTG Y D LL KKV KESS---------------------SL RDIPVVIMSSE NVP S R I T RCL EE GAE EFF LKPVRL A DL NR L 151 P tRR7 ------VTTVDS G SKAL KF L GLQE--DEQSNPDT-PYVSPN-NHQEME VNLIITD YCMP G MTG Y D LL KKV KESS---------------------SL R N IPVVIMSSE NVP S R I T RCL EE GAE EFF LKPVRL S DL NR L 143 P tRR3 ------VTF V DS G DKAL EY L GLLDS-IDNVNATSSSSSSQSPQQEGMK VNLIMTD YCMP G MSG Y D LL KRV KGS----------------------YW KDVPVVVMSSE NIP S R I RM CLEE GAE EFLLKPL Q L S DV EK179 P tRR12 ------VTKCNR A EIAL S LLR ENKN----------------------GY DIVISDV H MP D M D G I F K LL EQI G-----------------------M SSS LPIVVF SAD NNV S A M LGWL Y KGA AL YLMKPI VKNDVK N L 146 P tRR13 ------VTKCNR A EIAL S LLR ENRN----------------------GY DIVISDV H MP D M D G F K LL ELI G-----------------------L EMDLPVIMMSAD DGKNV V M K G V T HGA C DYLIKPIRI EA LKN I 138 P tRR14 ------VTT CGL A RDAL N LLR ERKG----------------------GY DIVISDV Y MP D M D G F K LL EQV G-----------------------L EMDLPVIMMS V D GET S R V M K G V Q HGA C DYLLKPIRM K ELR N I 129 P tRR21 ------VTT CGL A RDAL N LLR ERKG----------------------GY DIVISDV Y MP D M D G F K LL EHV G-----------------------L EMDLPVIMMS V D GET S R V M K G V Q HGA C DYLLKPIRM K ELR N I 128 P tRR15 ------VTT CSQ A TAAL K LLR ERKG----------------------CF DVVLSDV H MP D M D G F K LL ELV G-----------------------L EMDLPVIMMSAD GRT S A V M R G I S HGA C DYLIKPIR EEELK N I 149 P tRR16 ------VTT CSQ A TAAL K LLR ERKG----------------------CF DVVLSDV H MP D M D G F K LL ELV G-----------------------L EMDLPVIMMSAD GRT S A V M R G I R HGA C DYLIKPIR EEELK N I 149 P tRR18 ------VTT TNQ A VTAL E MLR ENRN----------------------KY DLVISDV N MP D M D G F K LL ELV G-----------------------L EMDLPVIMLSS HGDKEF V Y K G V T HGA V DYLLKPVRM E ELK N I 132 P tRR19 ------VTT TNQ A VTAL E MLR ENRN----------------------KY DLVISDV N MP D M D G F K LL ELV G-----------------------L EMDLPVIMLSS HGDKEF V F K G I T HGA V DYLLKPVRL E ELK N I 132 P tRR22 ------VTT TSQ A ITAL R MLR ENKN----------------------KF DLVISDV H MP D M D G F K LL ELV G-----------------------L EMDLPVIMLSA NGDPKL V M K G I T HGA CY YLLKPVRI E ELK T I 130 P tRR17 NRNLELI V V TV KKPFDAL S ILR LKKG----------------------LF DLVVSDL H MP E M N G -MEL QKQ V E------------------------EEFK LPVIIMSSD ESKNV I S R S L EG GAAF YIVKPANKVDLK N V 136 P tRR20 NRKLELF V V TV KNPFDAL ST LRLKKG----------------------LF DLVVTDL H MP E M N G -MEL QQQ V D------------------------EEFK LPVIIMSSD DSEKV I L R T L EG GAAF YIVKPI NKDDLK N V 137 P tpRR1 ------VTT ATR A TDAL H ILR EKED----------------------E INLILI E THLP D M DQ-Y E II ETV RA----------------------L SSLPIVVF SAD NNE S A M LG CLY KGA AL YLMKPI IKNDVK N L 135 P tpRR2 ------VTT ATR A TDAL R ILR EKED----------------------E INLILI E TRLP D M NQ-Y E IL ETL GE----------------------L EMDLPVIMMSAD DGKNV V M K G V T HGA C DYLIKPIRI EA LKN I 134 P tpRR3 ------V A TV SD G LKA WEILK ERPH----------------------N IDLILTEV D LP S VSG Y A LL TLI MEHE--------------------I C K N IPVIMMSS QDSIKT V Y KCM L RGA A DYLVKPIR KNELR N L 179 P tpRR5 ------V V SV PD G LKA WEILK GRPH----------------------G IDLILTEV D LP S I ----------------------------------I C K N IPVIMMSS QDSIS T V Y KCM L RGA A DYLVKPLR KNELR N L 166 P tpRR4 ------V AA V PD G LMA WET LKGGPH----------------------N IDLILTEV E LP L ISG Y A LL TLV TEHA--------------------V C K N IPVIMMSS QDSIS M V L KCM L KGA A DFLIKPVR KNELR N L 223 P tpRR6 ------VSA V PD G LMA WET LKERPH----------------------S IDLILTEV E LP L ISG Y AFL ALV MEHD--------------------V C K N IPVIMMSS HDSIS V V L KCM L KGS A DFLVKPVR KNELR N L 217 P tpRR7 ------VTA V SN G LQA WKVL QDLTN----------------------H IDLVLTEV A MP C LSG -IGLL SKI MSHK---------------------TC R N IPVIMMSS HDSMNV V F KCL S KGA V DFLVKPIR KNELK I L 206 P tpRR8 -------A T A V AN G LQA WKLL QDLTN----------------------H IDLVLTEV A MP C LSG -IGLL SNI MSHK---------------------TC R N IPVIMMSS HDSMNV V F RCL S KGA V DFLVKPIR KNELK I L 203 P tpRR10 ------V Y T FCNENE AL LA I SNEPG----------------------SFH V A I V EVISFSSNPT F QYL VNV VKPAATMMNIVQESKQEKHTNLTWN L LYDL NA M KRI SDFID S EQAFNAGGSVQSES LKPVKDSI V SM L 154 P tpRR11 ------V Y T FCNETE AL SA I SNEPG----------------------SFH V A I V EVS M SNS S RSF KFL --------------ETSK------------DLP T I KKV SDFMDPEQAFNAGGSVQSKS LKPVKDSV V SM L 127 P tpRR9 ------VTSVRS A RQV I DA L NAEGP----------------------E IDIILSEV D IP MTKG -MKML KYI MRDK---------------------DL R R IPVIMTVHCVHT S E L LGLAEK NILNY DFDPV ASDPSDAN 152 Figure 2-1. Populus response regulators (RRs) exhibit similar receiver domains. Amino acid sequences of the receiver domains of the type-As and Bs (denoted as PtRR) and pseudo-RRS (denoted as PtpRR) were aligned using ClustalX and shaded using the BoxShade 3.21 program. D/E-D/E-K residues are marked with asterisks and the receiver domain is underlined. Structurally similar residues present in 50% or more of the sequences are highlighted in bl ack (identical) a nd gray (similar). 46

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Figure 2-2. Populus response regulator (RR) gene structur es and sequence similarity tree. Exonintron distribution was defined using Gene Scan software (Burge and Karlin, 1997) and expressed sequence tags (ESTs) availa ble at Genebank. Solid black boxes denote the conserved receiver domain, gray boxes the DNA-binding domain, white boxes nonconserved coding regions and diagonal cr osshatching the CCT motif. Horizontal lines denote introns. RRs with EST support ar e indicated by an asterisk. The unrooted sequence similarity tree was generated by aligning the receiver domain of all genes using ClustalX. The output nexus file was imported into PAUP 4.0 to generate the displayed bootstrap tree (n = 10 000 iterations). 47

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P tRR12 KRR---KD E EEEA--EE R DDT S ALKKPRVVWSVELHQ Q FVA AVNQLGID S-----KAVPKKILELMNVPGLTRENVASHLQKYRLYLRRL P tRR13 KRR---KD E EEEA--DE R DDT S TLKKPRVVWSVELHQ Q FVA AV H QLGID------KAVPKKILELMNVPGLTRENVASHLQKYRLYLRRL P tRR15 KKRSIAKD E DDA E --LE N DDP S ASKKPRVVWSVELHQ Q FVS AVN H LGID------KAVPKRILELMNVPGLTRENVASHLQKFRLYLKRL P tRR16 KKRNIAKE E DDN E --LE I DDP S ASKKPRVVWSVELHQ Q FVS AVNQLGID ------KAVPKRILELMNVPGLTRENVASHLQKFRLYLKRL P tRR14 I KKRKHIE SKHDE--KD TGD SISTKKA RVVWSVDLHQKFV K AVNQIGF D ------K VGPKKILDMMNVP W LTRENVASHLQKYRLYL S RL P tRR21 I KKRKD I E SKHDE--KD IGD N T SAKKA RVVWSVELHQKFV K AVNQIGF D ------K VGPKKILDLMNVP R LTRENVASHLQKYRLYL S RL P tRR18 DQD-D E D E E E G ED-GDD N E VSGNQKKPRVVWSVDLHQKFV A AVNQMGLD ------KAVPKKILDLMNV D GLTRENVASHLQKFRLYLKRL P tRR19 K QD-EE E E G E G ED-GND N EESGNQKKPRVVWSVELHQKFV S AVNQLGLD ------KAVPKKILDLMNV D GLTRENVASHLQKFRLYLKRL P tRR22 DQNGD E D E DHDEDEDHE N EDP T TQKKPRVVWSVELH R KFVA AVNQLGVD ------KAVPKKILDLMNV EKLTRENVASHLQKYR H YLKRI P tpRR1 RKELEE T D N DDED----ND NL T VLKKPE LVWTN ELHNRFLQ AI RILGVD------GA H PKKIL QHMNVS GLK KENVS SHLQKYRL S LKRE P tpRR2 RKELEE TNNDDED----NNNL T VPKKR KLVWTN ELHNRFLQ AI RILGIDDLIINAGA H PKKIL QHMNVPGLK KENVS SHLQKYRLYLKR E P tRR17 K NGRKE ERKSTKD -DQE V D SQPASKKPKVVWT NSLHNRFLL ALN H IGLD------KAVPKRILE C M S V R GLSRENIASHLQKYRIFLKKV P tRR20 K D R INKNRKRTK E -DQE V D SQLAPKKPKVVWT NSLHS RFLQ AIN H IGLD------KAVPKRILE F M S VPGLSRENVASHLQKYRIFLKKV Figure 2-3. Amino acid alignments of the GARP DNA-binding domain. Amino acids sequences of all type-Bs plus two pseudo-RRs were aligned using ClustalX and shaded using BoxShade 3.21 program. Structurally similar residues present in 50% or more of the sequences are highlighted in black and gray. 48

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Figure 2-4. Receiver domains of Populus Arabidopsis and rice res ponse regulators (RRs) are highly conserved. The unrooted amino acid sequence similarity tree was generated by aligning the conserved D-D-K domain of Populus (balck), Arabidopsis (red) and rice (blue) using ClustalX. Type As, Bs and pseudo-RRs group separately and are labeled accordingly. The output nexus file was imported into PAUP 4.0 to generate the displayed bootstrap tree (n = 10 000 iterations). 49

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A B C A B C Figure 2-5. Tissue regulation of Populus response regulator (RR) type As (A), type Bs (B) and pseudo-RRs (C). Microarray analyses were performed on five tissues; young leaves (YL), mature leaves (ML), nodes (N), inter nodes (IN) and roots (R). The y-axis is the least square mean (LSM) value of each ge ne after adjusting by adding 0.8 units to avoid presenting negative values Same-gene tissues marked with different letters are significantly different ( p = 0.05). Genes whose expre ssion was not significantly higher than background at a false discovery rate of 5% in a ll tissues tested are labeled nondetectable (N.D.). 50

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Figure 2-6. Relative tran script abundance of Populus type As and Bs in various tissues. Semiquantitative RT-PCR was carried out using RNA samples isolated from P. trichocarpa phloem and xylem, and from P. deltoides prereceptive and postreceptive catkins. Transcript intensity signals for each gene were divided by the internal control (actin2 or ubiquitin) and visualized using TreeView version 1.60. Two-sample t -tests were performed for phloem vs xylem (lef t heatmap) and prereceptive catkins (pre. catkins) vs postreceptive cat kins (post. Catkins) (ri ght heatmap). Significant differences at = 0.05 are denoted by an asterisk Genes whose transcripts are not detected are not shown. Intensity denotes fold change. 51

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Figure 2-7. Exogenous cytokinin in duces transcript accumulation of Populus type-A and -B response regulators (RRs) in leav es. Detached mature leaves of Populus tremula Populus alba were treated for 1 h with 1 m BAP (+) or 0.1% DMSO (). Actin2 or UBQ was used as internal control fo r each RT-PCR reaction. The number of PCR cycles used in the amplification of each RR was the same as in Figure 2-4 except for PtRR3, PtRR6, PtRR13 and PtRR19, for wh ich the numbers were 33, 45, 40 and 38, respectively. 52

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Table 2-1. Gene-specific Populus type-A and B RRs primers Name5' primer 3' primer PtRR1CCACCCTTAATTGTAAACTGGATATGAGCTCATCATCCTCGTTATCATATCC PtRR2ATGGGTTCTAACAGTGTTGTTTCGTCTCCTTCGCCTTCCTCTGC PtRR3ATGGAGATCATGGAATCAGGT CCTAAAATGAAAGGATTGGCTGTTGTATAG PtRR4ATGGCTACTTTGGCTACTCTG GCTTGGAAGTGGTCTTATCCCAATAG PtRR5ATGGCTGTGGAAATGGCTC CAATGATGGCTTGGAAGTTGTCTGA PtRR6CTCTTCTATATTTTTGCTCTTTACAGGTGGCTAGAGTCACAATCACAACCACAA PtRR7ATGGCAATAACAGGGGATTC GTGCAGCAGCCATCATCACAG PtRR8ATGGTGGGGGGAGCATATAC ATTGAGATGTAATTTGATGAACTGCAGAAGTTAA PtRR9TTGAAGCTGTTGATGATGGCTTGGGGGAAGATGAACATGCAGAAAA PtRR10ATGGCAACGGCCGGCGAGA CAAAGAGGCCTAAATTGCAAACCAGAGACTAA PtRR11TTGATGATAGTTTGATAGATCGCAAAGGAATAATTAATCGATATTACAAGGGGGC PtRR12ATTGCTTCTCTTGGAGATCAAAGTTCCGCTCT TTTATCAG CCCACA AGAGG PtRR13GTTCGGAAGAGAAAGAACGAG GGG TTTT GGAAAGTTGCC TTTGA PtRR14-1CCTTGTCTAAATGGTTCGTTTGGTGTCACTCACCTCTGGTACCTGCATT PtRR14-2GTCTCCCTATCTGTCTCTGTCCA TG GAAGCCATGAC AGTGATCGAAAGAGAC PtRR15TATTTGAAGAGATTAAGTGGGGTTGCCAAGGTTCCTATGGTGCTAAGTCAA PtRR16CTCAACAAGGTGGGATTTCTAATACCATCCCAATATTACAACAATTGCC PtRR17CCTGGTGGCTCATCAAGAAAATC TGGAAAGCCCTAGACATGAGAGATACCAC PtRR18TAAGTAGTGGAGGAAACCAGCAAGGACTCTATCCATG TTTCAGCATCC PtRR19ACCTCAAAAGACTAAGTTGTGGGGCCTTGTCTAAATGGTTCGTTTGGT PtRR20-1CGTATCTTCCTAAAAAAGGTTGCAGGGAAATTACAATCAGCAGCAACA PtRR20-2GGATTTATTCACAGCATAGCTTGTGG AGAAAACGTTGCCAGCCACTTA PtRR21AGAGATATTGAAATTCTCGAAGGAATATTCTCTAATAGATCAAAGCTTG TTTATATGA PtRR22AGTGATAACAAAGACAGAAACAGCTCGGGGCTAATGGTCTAAGTCACTTCCAACC Actin2CCCATTGAGCACGGTATTGT TACGACCACTGGCATACAGG UBQGTTGATTTTTGCT GGGAAGC GATCTTGGCCTTCACGTTGT 53

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Table 2-2. Populus response regulators (RRs) Name Predicted size (amino acids) Type Predicted localization TargetP1.1/ WoLF PSORT /ProtComp Chromosome location Linkage group (nucleotide position) Gene model PtRR1257ANucleus LG_VIII(15159506-15158278)grail3.0113000201 PtRR2248ANucleus LG VIII(13279009-13277609) eugene3.00081821 PtRR3243ANucleus LG_II(5724266-5721514)eugene3.00020757 PtRR4193ANucleus(membrane bound) LG_III(17348335-17347029)eugene3.00031671 PtRR5203ANucleus/Plasma membrane LG_I(2060121-2061214) eugene3.00010260 PtRR6235ANucleus LG_VI(2665007-2666550)eugene3.00060364 PtRR7227ANucleus LG_XVI(2308339-2309809)eugene3.00160317 PtRR8143ANucleus/Cytoplasm LG_XIX(7831782-7832640)eugene3.00190596 PtRR9146ACytoplasm LG_XIII(12808568-12810101)eugene3.00131279 PtRR11151ANucleus LG_XIX(10741174-10743356)eugene3.00190915 PtRR10222ANucleus LG_XV(4993461-4991698)eugene3.00150492 PtRR12678BNucleus scaffold_77(542536-538989)eugene3.00770034 PtRR13673BNucleus LG_X(109067-112814) eugene3.00100010 PtRR14576BNucleus LG_VIII(12181811-12178061)eugene3.00081689 PtRR15624BNucleus LG_VIII(8931205-8935830)eugene3.00081269 PtRR16663BNucleus LG_X(10954136-10949431)fgenesh4_pg.C_LG_X000965 PtRR171045BNucleus LG_XII(13244168-13247591)eugene3.00121175 PtRR18691BNucleus LG_VI(11563959-11566041)gw1.VI.371.1 PtRR19685BNucleus LG_XVIII(11094572-11092394)gw1.XVIII.3323.1 PtRR20871BNucleus LG_XV(9898648-9893242)eugene3.00151142 PtRR21545BNucleus LG_X(6378193-6374174)gw1.X.5015.1 PtRR22661BNucleus LG_XVIII ( 6075507-6079079)fgenesh4_pg.C_LG_XVIII000471 PtpRR1458 Pseudo Nucleus LG_II (11584449-11587070)fgenesh4_pg.C_LG_II001405 PtpRR2880 Pseudo Nucleus/Cytoplasm LG_XIV (2012304-2003513)eugene3.00140231 PtpRR3495 Pseudo Nucleus LG_XV(134835-139313) eugene3.00150024 PtpRR5682 Pseudo Nucleus LG_XII(3179833-3184499)estExt_fgenesh4_pg.C_LG_XIV0468 PtpRR4680 Pseudo Nucleus LG_XIV (4270858-4278021)gw1.XII.1231.1 PtpRR6588 Pseudo Nucleus LG_II (14066193-114071399)fgenesh4_pg.C_LG_II001656 PtpRR7753 Pseudo Nucleus LG_VIII(2475953-2483643)estExt_fgenesh4_pm.C_LG_VIII0151 PtpRR8828 Pseudo Nucleus LG_X(18604749-18611132)gw1.X.2468.1 PtpRR9554 Pseudo Nucleus scaffold_129(470213-475672)fgenesh4_pg.C_scaffold_129000038 PtpRR10428 Pseudo Nucleus scaffold_118(468759-473575)estExt_fgenesh4_pg.C_1180049 PtpRR11471 Pseudo Nucleus scaffold_29(1744763-1749474)gw1.29.358.1 54

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55 Table 2-3. Duplicated Populus RRs as reported by Tuskan et al. (2006) P airGene type Gene 1Chromosome Gene 2ChromosomeDuplication 1APtRR1LGVIIIPtRR2LGVIIIPutative tandem* 2APtRR4LGIIIPtRR5LGIRecent 3APtRR6LGVIPtRR7LGXVIRecent 4APtRR9LGXIIIPtRR11LGXIXRecent 5BPtRR12Scaffold 77PtRR13LGXPutative recent* 6BPtRR14LGVIIIPtRR21LGXRecent 7BPtRR15LGVIIIPtRR16LGXRecent 8BPtRR17LGXIIPtRR20LGXVRecent 9BPtRR18LGVIPtRR19LGXVIIIRecent 10 Pseudo PptRR1LGIIPptRR2LGXIVRecent 11 Pseudo PptRR3LGXVPptRR5LGXIIPutative ancient* 12 Pseudo PptRR4LGXIVPptRR6LGIIRecent 13 Pseudo PptRR7LGVIIIPptRR8LGXRecent 14 Pseudo PptRR10Scaffold 118PptRR11Scaffold 29Putative recent* ( ) Suspected duplicated genes not reported by Tuskan et al. (2006).

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CHAPTER 3 THE CYTOKININ TYPE-B RESPONSE REGULATOR PTRR13 IS A NEGATIVE REGULATOR OF ADVENTITIOUS ROOTS DEVELOPMENT IN Populus This chapter will be submitted to a plant physiology-related journal for publication Introduction Adventitious rooting is an ecologically and economically important developmental process. Populus and other perennial species adapted to riparian ecosy stems naturally propagate clonally when stems or branches detached by a natural disturbance are carried downstream and lodge in a moist environment conducive to r ooting. For many species therefore, clonal propagation via adventitious root formation is a natural complement to sexual propagation by seeds. The stimulation of adventitious rooting to facilitate clonal propaga tion is a cornerstone of the ornamental horticulture and forest products industries. Treatment of cuttings with synthetic auxins has been used for more than 70 years to induce and accelerate r ooting in hard-to-root species (Thimann and Went, 1934; Kevers et al., 1997). Genetic improvement programs for forest trees including Eucalyptus spp., Populus spp. and Pinus radiata are almost exclusively based on the production of rooted cuttings for breeding and deployment in operational plantations (Davis and Becwar, 2007). In woody plants, adventitious root primordia primarily arise from stem ray cells (close to the phloem and cambium), buds or leaf gaps, or fr om callus formed at the base of the cutting (Lovel and White, 1986). Adventitious root formati on can be seen as a three-stage process: (1) activation, where the cells origin ating the root primordia become competent to respond to the rhizogenic action of auxin, (2) induction, comprisi ng the initial cell divisi ons and establishment of root primordia and (3) outgrowth, where the primordium initiates growth and emerges from the stem. Ectopic formation of roots is regu lated by both endogenous (e.g. hormones, sugars, phenolic compounds) and exogenous (e.g. temperatur e, light) factors (Eliasson, 1978; Davies 56

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and Hartmann, 1988; Kevers et al., 1997; De Kl erk et al., 1999). Among the plant hormones, auxin and cytokinin play antagonist ic roles in this process (De Klerk et al., 1997). Quantification of the endogenous levels of auxin and cytokinins in the basal part of cuttings from diverse species such as Populus tomato and Phaseolus aureus, reveal that the concentrations of these two hormones follow opposite patterns during th e initial 48 h of rooting; when auxin concentrations increase, cytokinin concentratio ns rapidly decrease (B lakesley et al., 1985; Bollmark and Eliasson, 1986; Maldiney et al., 1986; Hausman et al., 1997). It is well established that basipetal transpor t and accumulation of auxin at the base of cuttings precedes adventitious root fo rmation in a variety of species including Populus (Liu and Reid, 1992; Hausman et al., 1995; Guerrero et al., 1999). Endogenous auxin synthesized in shoot tips is important during adventitous rooting sin ce removal of the shoot apex decreases both the level of endogenous auxin in the basal portion of a cutting and the number of adventitious roots produced (Nordstrom and Eliasson, 1991). Elevated a uxin concentrations give rise to new root primordia by activating the differentiation and elongation of phloem parenchyma cells adjacent to vascular bundles in the stem (Lund et al., 1996; De Klerk et al., 1999) In contrast, exogenous applications of cytokinins to cuttings strongly inhibit root fo rmation (De Klerk et al., 1999). trans -zeatin riboside (ZR), a natura lly occurring cytokinin in cu cumber root xylem sap, was identified as a main suppressor of adventiti ous root formation in hypocotyls (Kuroha et al., 2002). Histological studies during ro ot primordia formation indicate that cytokinins inhibit the differentiation of primordia at an early stage in development (Bo llmark and Eliasson, 1986). It is well established that auxi n:cytokinin ratios cont rol organogenesis in culture; low ratios promote shoot formation and high ratios stimulate root formation (Skoog and Miller, 1957). Likewise, the interaction between these two hormones is cr itical for the induction and formation of 57

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adventitious roots in cuttings; an elevation of endogenous auxin typically occurs simultaneously with a reduction in the level of endogenous cytokinins in the i nductive phase of rooting in many types of cuttings (Kevers et al., 1997). Cytokinin signaling resembles tw o-component systems first id entified in bacteria and yeast. In this signaling pathway, an extracellular cue is sensed by a plasma membrane localized histidine kinase (HK) which tran sfers the signal to a response re gulator (RR) in the form of a phosphoryl group (Mizuno, 1998; West and Stock, 2001). These signaling pathways in plants include a third component, a his tidine phosphor-transfer protein, wh ich function as a carrier of the phosphoryl group from the HK to the RR (Mok and Mok, 2001; Kakimoto, 2003; Ferreira and Kieber, 2005). The RRs are the final step of this phospho-relay and have been traditionally divided into two groups; the type -As and the type-Bs. The type-As are small proteins consisting of a receiver domain with cons erved D-D-K residues. The type-Bs are more complex proteins that contain, in addition to the receiver domain, a GARP DNA-binding motif that resembles a domain originally found in the mammalian oncoprotein c-Myb (Imamura et al., 1998; Sakai et al., 1998). Forward genetic experiments carried out to define the function of RRs in Arabidopsis suggest functional redundancy am ong family members since phe notypes are not obvious unless mutant lines contain null alleles from several loci (To et al., 2004; Argyros et al., 2008; Ishida et al., 2008). Because of their potential functions as transcriptional activat ors, type-Bs are thought to represent key elements orchestrating tran scriptome changes triggered by cytokinin. In Populus we previously identified 22 genes exhibiti ng typical features of plant RRs (RamirezCarvajal et al., 2008). Although the physiological and anat omical processes associated with adventitious rooting have been described for a variety of species including Eucalyptus, pine and apple (De Klerk et 58

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al., 1999; Fett-Neto et al., 2001), the molecula r mechanisms involved in determining the competence of cells to generate adventitious ro ots as well as the development of adventitious roots per se, are not well defined. Reverse gene tic studies in Arabidopsis and tobacco have provided compelling evidence linking cytokinin signaling to adventiti ous root formation. Arabidopsis mutants lacking the cytokinin HK receptors AHK2, AHK3 and AH4 exhibit enhanced adventitious root gr owth (Higuchi et al., 2004; Ni shimura et al., 2004). Similar phenotypes have been observed in cytokinin deficient tobacco plants where overexpression of the cytokinin degrading enzyme cytokinin oxidase (AtCKX) results in ab undant adventitious roots (Werner et al., 2001; Werner et al., 2003). Similarly, type-Bs have also been associated with inhibition of the root morphogenesis proces ses. The Arabidopsis triple loss-of-function mutant arr1 arr10 arr12 shows almost complete insensitiv ity to exogenously applied cytokinin and spontaneously produces adventitious root s in hypocotyls (Argyros et al., 2008). Recent advancements in transcript analyses, includ ing microarrays and high throughput sequencing, have allowed more comprehensive studies about gene expression duri ng adventitious root formation in economically important species such as Populus and pine (Kohler et al., 2003; Brinker et al., 2004). However, thes e studies examined relatively sm all set of genes or targeted late stages of root formation (samples were ta ken several days to mont hs after shoot excision) when many of the cytokinin-se nsitive stages may be over. As part of a comprehensive analysis of the primary molecular mechanisms orchestrating de novo adventitious root formation in Populus cuttings, we report transcriptome remodeling during very early stages in nontransgenic (NT) plants using Populus whole-genome microarrays. We show massive transcriptome remodeling during th e 24 h involving almost half of the nuclear genes of Populus To link cytokinin signaling effectors (i.e. response regulators) with root 59

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formation processes, these microarray results were contrasted with whole-genome expression data acquired from transgenic lines exhibiting de layed rooting as a result of ectopic expression of a constitutive active form of PtRR13, referred here as DDKPtRR13. Perturbation of rooting due to alteration of cytokinin si gnaling through the expression of DDKPtRR13 becomes physiologically relevant at 24 h where a plethora of signaling, developmental and metabolic networks are differentially regulat ed in the transgenics. Promoter analyses of differentially regulated genes across all time points between NT and DDKPtRR13 lines reveal putative direct and indirect targets of PtRR13 such as a negati ve regulator of vascular ization COV1, an auxin efflux transporter PDR9, two AP2/ERF proteins similar to TINY1 and a homeodomain protein involved in ovule identity BELL1. Materials and Methods Plant Material and Growth Conditions Experiments were conducted in a greenhouse at ambient temperature between the months of September and December in Gainesville, Florida. Populus balsamifera ssp. trichocarpa (Torr. and Gray) genotype Nisqually-1 and Populus tremula x Populus alba INRA-clone No. 717-1-B4 plants were given 12-14 h of natura l light, supplemented in the wint er with artificial illumination to maintain indeterminate growth. Plants were grown in 11.4 liter pots on flood benches subirrigated once daily with a nutrient soluti on containing Peters Prof essional Blend 20-10-20 fertilizer solution (adjusted to 4 mM nitrogen). When plants reached 60-80 cm tall, single cuttings per plant were collected, planted in 25 cm2 pots and placed under mist until harvested. Semiquantitative RT-PCR and Real-Time PCR Total RNA was isolated using a cetyltr imethylammonium bromide (CTAB) method (Chang et al., 1993). RNA samples were subjecte d to DNase treatment with RQ1 RNase-free DNase (Promega, Madison, WI, USA) and purifie d using RNeasy Mini Kit (Qiagen, Valencia, 60

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CA, USA). One g of DNA-free RNA was used to synthesi ze first strand cDNAs using oligo-dT primers and M-MLV reverse transcriptase (Prome ga, Madison, WI, USA). Gene specific primers were designed using the Joint Genome Institute (JGI) assembly for P. trichocarpa v1.1 and are shown in supplementary Table 3-1. To avoid non-specific PCR amplification, primers were designed against the most variable regions in the coding sequences using NetPrimer (Premier Biosoft International, Palo Alto, CA, USA). One l of the 20 l RT reaction was used as template in both semiquantitative and real-tim e PCR reactions. For the semiquantitative RTPCR, PCR products were separated by agarose gel electrophoresis, st ained with ethidium bromide, and band intensities scaled to the band intensity of the actin2 internal control gene using Kodak 1D Image analysis software. For th e real-time PCR experiments, gene expression was quantified using the SYBR Green kit (Strat agene, La Jolla, CA) and Mx3000P thermocycler (Stratagene) as per manufacturers inst ructions. The obtained expression values were normalized using the mean expression of the two controls actin2 and ubiquitin. Tissue Culture Experiments Stems were harvested from ~60 cm tall plan ts grown as described. After removing all leaves, petioles and shoots; stems were steriliz ed by four consecutive washes of 70% ethanol; 30% concentrated bleach; and two washes with 100% deionized water. Stems were cut into 0.5 cm sections and placed on MS-agar plates (0.443 % MS, 0.01 % Myo-inositol, 3% sucrose, 0.65% phytoagar) containing the indicat ed cytokinin:auxin concentra tion. Plates were placed in a Percival growth chamber under12h light-dark cycles at a constant temperature of 25oC and 75% relative humidity. Response Regulator PtRR13 Plasmid Construc tion and Transgenic Line Generation cDNAs for the full-length (F LPtRR13), DDK truncation ( DDKPtRR13) and RNAi fragment were cloned into the TOPO entry vector (Invitrogen USA, Carlsbad, CA). Cloned 61

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sequences were transferred to the destinati on vectors using Gateway technology (Invitrogen USA, Carlsbad, CA) with the FLPtRR13 and DDKPtRR13 cDNA sequences cloned into pZKY1 Overexpress and the RNAi sequence into pZKY2 Direct. Vector s were kindly provided by G Tuskan (Oak Ridge National Laboratory, Oak Ridge, TN). Populus tremula x Populus alba INRA-clone No. 717-1-B4 was transformed via an Agrobacterium -mediated protocol developed by Han et al. (2000). Twenty independent transf ormations (lines) per co nstruct were obtained and explants grown on kanamycin selection medi a. Transgenic lines were screened using PtRR13 specific primers and primers directed against the 35S promoter and octopine synthase inverted repeats (for full-length and DDK truncated lines) or the PIV2 intron (for RNAi lines). Detached-Leaves Cytokinin Experiment Expanding leaves with a leaf plastochron index (LPI) of 4, 5 and 6 from the hybrid P. tremula x P. alba INRA-clone No. 717-1-B4 and the transgenic lines DDKPtRR13-16 and DDKPtRR13-20, were harvested an d treated with 0, 0.1, or 1 M 6-benzylaminopurine (BAP) by submerging only the petioles in a vial containing the hormone (Sugiharto et al., 1992). One hour later, leaves were remove d and frozen in liquid nitrogen. Total RNA extractions and RT reactions were performed as explained above. A to tal of three biological re plicates (three single leaves) per treatment were used for all lines. Microarray Analysis For these analyses, 14 cm tall apical cuttings we re collected from 60 cm tall mother plants. Cuttings were sown in 25 cm2 pots and placed on a mist bench. Samples were collected at the indicated time points and consisted of a 5 mm sec tion measured up from the base of the cutting (one sample per cutting). Total RNA was extrac ted with the RNeasy mini kit (Qiagen USA) and DNase treated in-column with the RNase-Free DNase set (Qiagen USA). Double-stranded cDNA was synthesized using SuperScript Double St rand cDNA Synthesis Kit (Invitrogen USA, 62

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Carlsbad, CA) with oligo-dT primers following the manufacturers protocol except that the synthesis step was extended to 16 h. Cy-3 labeling and hybridization steps were performed by NimbleGen using their standard procedures. A custom-designed microarray platform was used comprising single 60-mer probes designed agai nst 55,793 annotated gene models from the sequenced genome of P. trichocarpa Details about probe design and selection are from D. Drost et al., (Drost and Kirst persona l communication; manuscript in re view). A total of 40 microarray chips were used in these experime nts: 40 chips = 2 genotypes (NT and DDK) x 4 time points (0, 6, 24 and 48 h) x 5 biological replications. Signal intensities were log2 transformed and quantile normalized (Bolstad et al., 2003). Normalized signals we re analyzed in SAS 9.1 (SAS Institute, Cary, NC) using a mixed model analysis of variance (ANOVA) with genotype and genotype by time interactions as fixed effects, and biological replication as random effects. Microarray expression data was verified for a set of genes by real-time PCR obtaining similar gene expression patterns with bot h analysis methods (Figure 3-1). Gene Set Analysis Gene set analysis was performed using the ove r-representation analysis (ORA) application of the ErmineJ software (Version 2.1.16) which uses the binomial approximation to the hypergeometric distribution to find gene-set en richment. The correspond ing GO annotations of the Populus genes were obtained by que rying the GO annotation t ool of the Arabidopsis Information Resource (TAIR) with the closest Arabidopsis hit for each Populus gene. GO annotations were downloaded from the Gene Ontology project website. Only biological processes-related GO categories were considered in the analysis. Genes with contrasts significant at a FDR < 0.5% were used for gene-set enri chment analysis. Gene-sets were determined significant at a FDR < 10%. The range of ge ne set sizes used was between 5 and 500. 63

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Results Transcriptome Analysis of Early Stage s of Adventitious Root Formation P.tremula x P.alba softwood cuttings form adventiti ous roots within 10 to 14 days, depending on season, with no exogenous auxin treat ment required (unpublished observations). The lack of a requirement for auxin treatment may be due to high rates of auxin synthesis and/or transport in the actively growing s hoots. Observations in apple and Populus microcuttings reveal cell divisions as early as 48 h after auxin exposure and the pres ence of organized meristemoids by 96 h (De Klerk et al., 1995; Wu, 2004). Because early physiological and biochemical evidence indicates that there are significant changes in endogenous hormone pools, including ethylene, auxin and cytokinin, during the first 48 h after excision (M aldiney et al., 1986; Selby et al., 1992; Hausman, 1993; De Kler k et al., 1997), we predicted changes in gene expression during this time frame would be of significant magnitude and provi de valuable information about the early and still unknown molecular networks regulated du ring adventitious root formation. To analyze transcriptome changes du ring adventitious root formation in Populus basal stem sections of approximately 5 mm of length, where adventitious root s originate in softwood cuttings, were harvested at 0, 6, 24 and 48 h after excision. RNA extracted from these samples was used for microarray analyses with a cu stom designed NimbleGen array containing 55,793 Populus gene models (Drost and Kirst, personal communication). Genes significantly regulated between adjacent time points (0h-6h, 6h-24h and 24h-48h) were identified by contrasting their normalized, log2-transformed intensity estimates. As s een in the Venn diagram (Figure 3-2A), 15,134 genes (27%) were differentially regulated between 0 and 6 h; 20,111 (36%) between 6 and 24 h, and 2,474 (4%) between 24 and 48 h at a FDR of 5%. Such high numbers of differentially regulated genes are consistent with a massive tran scriptome remodeling after root system removal. Due to the magnitude of the tr anscriptome change, we analyzed the expression 64

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data following a gene-set approach in which genes are grouped based on gene ontology (GO) terms prior to analysis. By changing the focus from individual genes to sets of functionally related genes, gene set analysis enables the understanding of cellu lar processes as a network of functionally related components (Dopazo, 2006). I us ed the over-representation analysis (ORA) application of the software tool ErmineJ (Lee et al., 2005) and identi fied 163, 198 and 56 gene sets showing enrichment at the 0h-6h, 6h-24h, a nd 24h-48h contrasts, respectively (genes with contrasts significant at a FDR < 0.5% were used for gene-set enrichment analysis, gene-sets were determined significant at a FDR < 10%). The top 40 gene set GO categories from each contrast were grouped into arbitrary higher order categories (Table 3-2). Differences in the GO categories enriched at each contrast suggest that different biological processes are regulated at the different samp ling intervals. During the initial 6 h following excision, a significant enrichment of categories related to wounding, ethyle ne and abiotic stress responses was identified (Table 3-2). The ethyl ene regulated gene set included genes encoding the ethylene biosynthetic enzymes ACC synthase and ACC oxidase (data not shown). Gene sets related to response and defense ag ainst biotic factors were prim arily enriched in the 24 to 48 hour interval, implying a temporal separation in the regulation of biotic and abiotic defense responses. The time interval between 6 and 24 h re vealed a significant enrichment in gene set categories related to transcription and translation, suggesting that most of the transcriptional and translational reprogramming preceding root formati on occurs in this time frame. Gene sets involved in development also showed enrichment differences between time intervals, several categories related to embryonic and anatomical stru cture formation were significant, particularly between 6 and 24 h. Categorie s potentially related to de novo root formation, such as root morphogenesis and cell proliferation, showed up as enriched in the 24 to 48h interval, indicating 65

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the initiation of root formation. Among hormone si gnaling genes, the cytokinin type-A RRs were significantly upregulated dur ing this same time interval. I an alyzed the expression of individual Populus cytokinin responsive type-As during advent itious rooting and s howed that, during the first 24 h, transcript abundance significantly drops, reaching a minimum between 6 and 24 h, then begins to increase toward original leve ls (Figure 3-2B). Because type-As are cytokinin primary response genes, differences in transcri pt abundance over time may imply variation in endogenous cytokinin levels. Whether this variat ion is relevant for rooting, or not, requires further experiments. Transcriptome changes duri ng the first 48 h after shoot excision reveals differential regulation of gene networks with potential roles in wound re sponses, transcriptional regulation, hormone signaling and adve ntitious root morphogenesis. The Response Regulator PtRR13 and Its Relatives Since cytokinins are negative regulators of r oot growth, the mis-expression and/or misregulation of RR genes and protei ns may provide insights into the molecular mechanisms by which this hormone inhibits root developmental processes in cuttings. Type-B RRs, because of their DNA-binding activity, represent good ca ndidates for modulating the downstream transcription of root formation factors. Three Arabidopsis type-Bs ARR1, ARR10 and ARR12, are known to play essential roles in cytokinin signaling and are presumed negative regulators of adventitious root formation (Ar gyros et al., 2008; Ishida et al., 2008). Of these, ARR1 has been extensively studied and is thus a good candidate for comparative genomic studies. A sequence similarity tree of Populus and Arabidopsis type-Bs reve als that the GARP domains of Populus PtRR12 and Populus PtRR13 are closely related to those of ARR1 and ARR2 from Arabidopsis (Figure 3-3A). To gain additional evidence about evolutionary relatedne ss between these 2 pairs of RRs, I searched for synteny in adjacent chromosomal regions using the Plant Genome Duplication Database (Tang et al., 2008). Interestingly, ARR1 and PtRR13 are located in a 66

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significant syntenic block containing 16 anchors ( E -value 0.0; data not shown). However, no significant colinearity was dete cted between PtRR13 and ARR2. To obtain additional evidence of orthology between ARR1 and PtRR13, I investig ated the tissue preference of these genes. While transcripts for both ARR1 and ARR2 have b een detected in vegetative tissues (Tajima et al., 2004), ARR2 appears more abundantly expre ssed in reproductive tis sues (Lohrmann et al., 2001; Tajima et al., 2004). I explored the tissue preferences of PtRR12 and PtRR13 by measuring transcript abundance in vegetative and reproductive tissues us ing semi-quantitative RT-PCR and gene specific primers (Table 3-1). Transcript abundance data from eight tissues (roots, phloem, xylem, young stems, leaves, shoo t tips, and preand post-receptive catkins) indicated that PtRR13 expressi on is relatively high in roots and post-receptive catkins, intermediate in phloem, young stems and pre-rece ptive catkins, and low in xylem, leaves and shoot tips. Overall PtRR12 transcript abundance, however, was lower than for PtRR13 with the exception of relatively higher expression in postreceptive catkins (Figure 3-3B). The evidence presented here, including amino acid sequence si milarity, chromosomal gene order, and tissue preference, represents preliminary evidence for an orthological relati onship between ARR1 and PtRR13. The Response Regulator PtRR13 Transgenes and Their Effects PtRR13 displays the typical domain organiza tion of type-Bs: an N-terminal receiver domain with the conserved D-D-K residues, a GARP domain, and a long C-terminal transactivation domain (Figure 3-4A ). To acquire knowledge about the in vivo roles of PtRR13, I generated three different constr ucts to direct gain or loss of PtRR13 function. Two different overexpression constructs were engineered: one for production of full-length PtRR13 (FLPtRR13) and a second for production of a tr uncated version in whic h the receiver domain was deleted ( DDKPtRR13). Since the receive r domain of type-Bs is proposed to inhibit the 67

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DNA binding activity of the GARP domain in the ab sence of the phosphoryl residue associated with cytokinin signaling (Sakai et al., 2000; Sakai et al., 2001), engineering a construct in which this domain is missing is predicted to create a constitutively active version of PtRR13. The lossof-function approach consisted of generating a RNAi construct containing ~200 bp of the last exon of PtRR13. Transgenic P. tremula x alba plants were generated fo r all three constructs via Agrobacterium -mediated transformation. Transgene transcript abunda nce for the FLPtRR13 and DDKPtRR13 lines was assayed by semiquantitativ e RT-PCR and the two lines with highest expression for each construct were chosen fo r further phenotyping (FLPtRR13-1 and FLPtRR1314 for the full length and DDKPtRR13-16 and DDKPtRR13-20 for the truncated; Figure 34B). The two RNAi lines with the greatest reductions in endogenous PtRR13 expression compared to the nontransgenic controls were also selected for further phenotyping (lines RNAi45 and RNAi57 with 73% and 62% reduc tions, respectively ; Figure 3-4C). I obtained evidence that the DDKPtRR13 plant lines had an altered cytokinin response. First, I cultured stem explants under six di fferent auxin:cytokinin ratios (0:0, 10:0, 100:0, 0:10, 10:10 and 10:100 M IBA: M zeatin, respectively; results shown in Figure 3-5). Second, I measured type-A transcript abundance in detached leaves treated with two concentrations of cytokinin (0.1 and 1 M of BAP, results shown in Figure 3-6). In both experiments, altered responses to exogenous cytokinins in the DDKPtRR13 lines were observed. In the stem explant culture experiment, I observe d stem growth enhancement in the absence of exogenous cytokinins. In the detached leaf experiment, I observed decreased induction of type-A transcript accumulation, implying a disruption of the cytokinin primary response. I also identified a perturbati on in adventitious rooting duri ng propagation. I measured total root length of apical cuttings 10 days after excision and found that the two DDKPtRR13 lines 68

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had significantly lower total root lengths than the nontransgenic line (36.3 5.2 and 27.3.5 for DDKPtRR13-16 and DDKPtRR13-20 respectively, vs. 84.2 10.5 for the NT; Figure 3-7A). I did not observe any significant differences between the FLPtRR13, RNAi, and NT lines. The absence of phenotypes for the FLPtRR13 lines wa s not entirely unexpected since the ectopically expressed PtRR13 would not be active (phosphor ylated) until activati on of the cytokinin signaling pathway. The lack of phenotypes in the RNAi lines could be a consequence of incomplete gene silencing since the line with the highest PtRR13 reduction still retained 27% of the endogenous transcript levels. This residua l amount of PtRR13 may be sufficient for normal functioning of the cytokinin signaling pathway. Alternativel y, PtRR13 may be functionally redundant with another RR. Based on th e presence of root phenotypes for the DDKPtRR13 lines, my subsequent analyses focused on these lines. The Response Regulator PtRR13 Is a Negative Re gulator of Adventitious Root Formation The differences in total root length observed above were a consequence of decreased numbers of roots in the DDKPtRR13 lines (Figure 3-7B). The control NT line formed, on average, 11.66 ( 0.84 SE) roots per cutting, whereas the DDKPtRR13-16 and DDKPtRR1320 lines formed 4.92 ( 0.65 SE) and 6.07 ( 0.99 SE ) roots per cutting, respectively. However, no significant differences were observed among the NT and DDKPtRR13 lines when aboveground leaves and stems were measured (Figure 3-7C), demonstrating that the differences observed in rooting were not driv en by differences in shoot biom ass. The fewer number of roots formed in the DDKPtRR13 lines (Figure 3-7D-E) was typically accompanied by callus formation at the base of the cutting adjacent to th e wound site (Figure 3-7F). This proliferation of undifferentiated cells phenocopies explants grown in callus i nduction media. Interestingly, cytokinins have been shown to activate the transcription of CYCD3 cyclins, positive cell cycle regulators whose overexpression causes ectopic cell divisions in leaves an d other organs (Riou69

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Khamlichi et al., 1999). The resu lts obtained in these experime nts represent evidence of a negative role of DDKPtRR13 in adventiti ous root formation. Transgenic DDK PtRR13 Lines Show Altered Gene Ex pression during Adventitious Root Formation To gain insights into the molecular mechanisms altered early during adventitious root formation in a constitutively active version of PtRR13, whole-transcri ptome monitoring data were generated to detect shifts in transcript abundance in stems after shoot excision. Line DDKPtRR13-20 was arbitrarily selected since both DDKPtRR13-16 and DDKPtRR13-20 lines displayed identical phenotype s. Expression data for line DDKPtRR13-20 were contrasted with NT and analyzed for genes significantly re gulated across all time poi nts (transgene effect) and genes significantly regulated at each time point (transgene by time interactions). From these analyses, a total of 11 genes were differentially expressed across all time points at a 10% FDR and a 2 fold regulation threshold (Figure 3-8). Th e transgene by time interaction analysis revealed no significant gene regulation at 6 and 48 h (above and beyond that incited by the transgene alone), however there was a significa nt transcriptional re sponse at 24 h. Using stringent criteria for significance (1% FDR and 2 fold regulation threshold), 273 genes were significantly regulated at 24 h (Figure 3-8). Of the 11 genes differentially regulated acr oss all time points, 5 were upregulated (including the transgene DDKPtRR13-20) and 6 were downr egulated (Table 3-3). The upregulated and downregulated genes showed similar expressi on patterns within each group, indicating potential coregulati on (Figure 3-9A). Expression of the endogenous PtRR13 in NT was assayed by real-time quantitative RT-PCR conf irming the pattern observed in the microarray experiments (Figure 3-9B). The two ge nes most significantly upregulated in DDKPtRR13 were COV1 ( CO NTINUOUS V ASCULAR RING 1 ), a negative regulator of vasculature formation in 70

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stems (Parker et al., 2003) and PDR9 ( P LEIOTROPIC D RUG R ESISTANCE TRANSPORTER 9 ; Ito and Gray, 2006) encoding a protein for auxin efflux. The other genes upregulated by DDKPtRR13 were FIM2 ( FIM BRIN-LIKE 2 ) encoding an actin-binding protein, and MSL10 ( M ECHANOSENSITIVE CHANNEL OF S MALL CONDUCTANCEL IKE 10 ) encoding an ion channel protein. The genes downregulated in DDKPtRR13 (or upregulated genes in NT) included two TINY-like transcription factor s, a cytochrome p450 CYP94B3, a caffeic acid Omethyltransferase family 2 protein, a metal-nicotinamide transporter YSL7 ( Y ELLOW S TRIPEL IKE 7 ), and a homeodomain protein required for ovule identity, BELL1 ( BEL1 ). Of the 273 genes differentially regulated at 24 h (Table 3-4), 160 (59%) had significantly greater expression in DDKPtRR13-16 than the NT, while the remaining 113 (41%) were more abundantly expressed in NT. From these 273 genes, the top 10 most signif icantly regulated were involved in diverse biological processes such as hormone homeostasis and signaling, transcriptional regulation, de fense responses, signal transduction, and amino acid transport (Table 3-5). Arabidopsis ARR1 has been shown to bind DNA in a sequence specific manner to the semi-palindromic motif AGATC (Sakai et al., 20 01). This motif has been detected in the promoters of several Arabidopsis type-As and in severa l cytokinin-regulated genes (Rashotte et al., 2003). I manually searched for this ARR1 mo tif in 1.5 Kb regions upstream of the starting methionine of the 10 genes (less the DDKPtRR13 transgene) regula ted across all time points and observed that the COV1 promoter had the highest numbe r of AGATC motifs present 5 times in a 374 bp segment approximately 680 bp ups tream of the start methionine (the average number of this motif occurrences in 100 randomly selected Populus promoters was 2.9 per 1.5 Kb, Figure 3-10). The number of ARR1 motifs ra nged from 1 to 4 for the promoters of the 71

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remaining 9 genes. Because Arabidopsis TINY has been demonstrated to bind dehydration responsive element (DRE) with a core seque nce of A/GCCGAC, as well as the ethylene responsive element (ERE) with a core sequen ce of AGCCCGCC (Sun et al., 2008), I manually searched for these motifs in the promoters of genes regulated across a ll time points with the intent to find potential targets of the 2 TINY-like transcription f actors. However, no statistically significant enrichment was detected. To determin e if there was significant enrichment of any particular cis -element(s) in the genes differentia lly regulated at 24 h, I adapted the overrepresentation computational analysis described by Nemhauser et al. (2004) to Populus Although no significant enrichment of AGATC was found in the genes upregulated in DDKPtRR13-16, I did detect enrichme nt of a different element, OSE2ROOTNODULE (CTCTT; Fehlberg et al., 2005), that is found in promoters of genes activated during root nodule formation (Z-score = 3.44 and p-value < 0.001, data not shown). For the genes downregulated in DDKPtRR13-16, I detected enrichment of the root-hair specific cis -element RHERPATEXPA7 (KCACGW; Z -score = 2.37 and p-value = 0.009; Kim et al., 2 006). Interestingly, cytokinin receptors are required to activate cell divisi ons during nodule organogenesis (Murray et al., 2007), implying potential biological significance of these promoter motifs. I summarize the predicted role of PtRR13 duri ng adventitious rooti ng in Figure 3-11. In the presence of cytokinin, phosphoryl groups ar e transferred through a cytokinin phosphorelay signaling pathway from the HK receptors to the RRs, including PtRR13. Phosphorylated PtRR13 (the active form) directly activates the transcription of COV1 a negative regulator of vascularization, by interacting with cis -elements present in the COV1 promoter. Activation of PtRR13 by phosphorylation would interfere with th e establishment of an auxin gradient by stimulating transcription of the auxin efflux pump PDR9 and by inhibiting the expression of 72

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stress-inducible TINY-like transcription factors that have po tential roles in ethylene-dependent adventitious root formation. Discussion Adventitious root formation in cuttings is an economically important developmental process that has been exploited for decades in the agricultural industry. It is a complex process because it encompasses signaling networks involve d in physiological res ponses to mechanical injury, wound repair as well as organ devel opment. Although transcript abundance has been measured for some genes dur ing the rooting process in Populus and Pinus only a subset of the transcriptome was monitored, and at relatively late stages of advent itious root formation (several days after removal; Kohler et al., 2003; Brinker et al., 2004). Considering that significant changes in endogenous hormones such as auxin, et hylene and cytokinin occu r within hours after shoot excision (Hausman et al., 1997; Kevers et al., 1997), we targeted th e entire transcriptome at early time points. To our knowledge, th is study represents the first whole-genome transcriptome analysis performed during the very early stages of adventitious root formation and without altering the natural hormone balan ce by the application of exogenous auxins. Massive Transcriptome Remodeling Is A ssociated with Adventitious Rooting Our results indicate that transcriptome re programming following shoot excision is of massive proportions; 27% and 36% of the Populus nuclear genes were differentially regulated between 0 and 6 h and between 6 and 24 h respec tively. Networks regulated during the initial 48 h after excision included genes related to very br oad processes such as hormone homeostasis and signaling, primary metabolism, gene expression and development. Hormone categories included ethylene, auxin and cytokinin; all known major players in regu lating rooting processes. Among them, ethylene-related genes were the most si gnificantly regulated in the initial 6 h, placing ethylene at the very beginning of the adve ntitious rooting activ ation-induction phase. 73

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Interestingly, during the same time interval, a si gnificant enrichment of gene sets involved in wounding and abiotic stress responses was observe d. Ethylene is a central regulator of plant defense responses to biotic and abiotic st resses including wounding and pathogen infection (O'Donnell et al., 1996; Van Loon et al., 2006). Aminocyclopropane-1-carboxylate (ACC) synthase, the rate-limiting enzyme in ethylene biosynthesis, is induced in tomato plants upon wounding (Kende, 1993). In our experiment, the i nduction of ethylene-related genes, including the biosynthetic enzymes ACC synthase and ACC oxidase, during the initial 6 h is in agreement with the role of ethylene as an important signaling molecule for wounding and defense responses. However, ethylene can also regulate developmental processes including root growth and formation. At low concentrations ethylene in directly promotes lateral root formation by stimulating auxin biosynthesis and transport (Stepanova et al., 2007; Ivanchenko et al., 2008). Ethylene insensitive tomato and pe tunia plants produce fewer advent itious roots than wild type plants (Clark et al., 1999). Therefore, in Populus cuttings, ethylene may be important for regulating not only wound responses but also fo r priming adventitious root formation. In cuttings, cytokinin levels are significnatly reduced around the rooting zone, reaching a minimum between 24 and 48 h (Blakesley et al., 1985; Bollmark and El iasson, 1986; Maldiney et al., 1986; Hausman et al., 1997). St udies on the stages of root formation in apple microcuttings reveal that exogenous applications of cytokini ns between 24 and 72 h st rongly inhibit rooting (De Klerk et al., 1995). This period of high cytokinin sensitivity co incides with the interval in which endogenous cytokinin content is transiently low (Bollmark et al., 1988). The pattern of the type-As transcript abundance after cutting app ears correlated with cy tokinin levels and sensitivity in that they decrea se during the first 24 h followed by an increase between 24 and 48 h. Although, the significant decreas e in type-As transcript abunda nce may reflect depletion of 74

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root-born cytokinins caused by de tachment from the root system t, it may indicate changes in cytokinin sensitivity in the root founder cells at th is last interval. Interestingly, during the same interval I detected an enrichme nt of genes involved in root morphogenesis and cell proliferation. Co-regulation of the type-As, cytokinin negative regulators and genes involv ed in root formation may be developmentally relevant considering the negative role that cytokinin exerts on de novo root formation. The Effect of PtRR13 Is Discrete Ectopic expression of full-length PtRR13 protei n did not lead to de tectable phenotypes in Populus a result consistent with the predicted inhib itory role of the DDK domain on the activity of type-B proteins (Sakai et al., 2001). Overexpression of PtRR13 without the DDK domain ( DDKPtRR13), on the other hand, re sulted in a highly specific phenotype characterized by a delay in adventitious rooting a nd aberrant callus grow th at the wound site. Similar results have been obtained in Arabidopsis, where only tran sgenic lines expressing ARR1 without the DDK domain (ARR1 DDK) showed visible phenotypes including reduced plant growth, disordered cellular proliferation aro und the shoot apex and ectopic shoots on cotyledons (Sakai et al., 2001). Differences in the pleiotropic effects of the Populus DDKPtRR13 (discrete phenotype) and Arabidopsis ARR1 DDK (diverse phenotypes) may reflect the functional dive rgence of these two related genes, the higher intrinsic plasticity of Populus to adjust to aberrant gene expression, or the differences in the transformation/regenera tion procedures that facilitate the recovery of lines with stronger alleles with the Arabidopsis floral dip method compared to the Populus culture based regeneration met hod. Regardless of the reason for the discrete phenotype in Populus it illuminated a potential role for a specifi c RR in a distinct developmental process. Other cytokinin signaling genes have been asso ciated with adventiti ous root inhibition; Arabidopsis plants lacking cytokinin receptors develop numerous adventitious roots (Kuroha et 75

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al., 2002; Werner et al., 2003; Hi guchi et al., 2004; Nishimura et al., 2004). Other cytokinin mutants with enhanced ectopic root formation include the Arabidopsis cytokinin insensitive triple mutant arr1 arr10 arr12 that spontaneously grows advent itious roots in the hypocotyl (Argyros et al., 2008). Surprisingl y, I did not detect significant differences for transcript abundance of type-As throughout the time c ourse experiments between the NT and DDKPtRR13 lines, indicating normal transcri ptional regulation of type-As in the DDKPtRR13 during this time interval. Transcriptome Regulation at 24 h after Presumed Acquisition of Competence. Concomitant with the de lay in rooting of the DDKPtRR13 lines, important changes in gene expression were observed compared to the NT. Transcriptome contrasts over time revealed small changes in gene expression across all time points but significan t differences specifically at 24 h. Adventitious rooting has been shown to progress in a stepwise manner with different physiological/developmental stages that can be identified (De Klerk et al., 1999). In vitro rooting experiments of apple microcuttings reveal that during the first 24 h there is a lag period in which cuttings show low to no sensitivity to exogenous auxin and cytokinin. However, as time progresses, cells become increasingly sensitive to these hormones, and any alteration in the auxin:cytokinin ratios strongly affects rooting (De Klerk et al., 1995). The absence of visible phenotypes in the DDKPtRR13 lines and the significant e ffects on gene expression specific to 24 h indicates that transgene effects may depe nd on the acquisition of competence by the root founder cells to initiate organ development. The ability of DDKPtRR13 cuttings to develop normal adventitious root systems, albeit more slow ly than the NT line, may imply that there are multiple routes to generate adventitious roots this might be expected for adaptive traits. 76

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Potential Direct and Indirect Targets of PtRR13 I identified 10 genes that were mis-regulated across all time points, some of which could be direct transcriptional ta rgets of native PtRR13. The COV1 gene, encoding an integral membrane protein that negatively regulates vascular tissu e differentiation in stems (Parker et al., 2003), exhibited a 4.4 fold upregulation in DDKPtRR13. In Arabidops is, mutations in COV1 cause disruptions in the pattering of differentiated vascular tissue in the stem and results in a continuous ring-like pattern of xylem and phloem with very littl e interfascicular tissue and the loss of defined vascular bundles (Parker et al., 2003). The phenotypes of these mutants are independent of auxin and have b een attributed to the action of an unknown inhibitory element. Because the establishment of vascular continu ity between the root primordia and the stem vascular tissue must occur in order for the nasc ent root to efficiently access nutrients and water coming from the plant, I hypothesi ze that direct activation of COV1 expression by DDKPtRR13 interferes with the early establishm ent of vascular connections between the new root and the stem, thus limiting adventitious root initiation. Another upregulated gene in the DDKPtRR13 mutant was PDR9 a member of the ATPbinding cassette transporter (ABC) family. Arabidopsis PDR9 is exclusively expressed in roots and the protein is predicted to act as a 2,4-dichlorophenoxyacetic acid efflux pump (Ito and Gray, 2006). Recent studies by Delker et al. (2008) have found that PDR9 functions as a genetic suppressor of the SCF complex mutant tir1-1 that is able to restore wi ld type root growth when supplied with auxins. In lateral roots, the establishmen t of an auxin gradient mediated by auxin transporters (PIN proteins) is required for proper root primordi a formation (Benkova et al., 2003; Geldner et al., 2004). Cytokinin accu mulation in the root founder cells results in disorganized patterns of cell divisions by pert urbing the expression of PIN genes and thus disrupting polar auxin transport (Laplaze et al., 2007). Upregulation of the auxin efflux pump PDR9 in 77

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DDKPtRR13 lines may interfere with the establishm ent of an auxin gradient required for root primordia initiation and organization. Because cytokinins are inhibitors of rooting processes, the identific ation of downregulated genes across all time points in DDKPtRR13 may provide clues about specific cell processes repressed by this hormone during de novo root formation. I identified two TINY-like genes that were significantly lower in the transgenics. TI NY belongs to the DREB (dehydration-responsive element binding protein) subfamily of the AP2/ERF transcription factor gene superfamily. Several AP2/ERF proteins including PLETH ORA1 and 2, and BABY BOOM (BBM) are upregulated during the formati on of adventitious roots in Medicago truncatula cultures (Imin et al., 2007). Among ERF/AP2 family members, the DR EB and ERF (ethylene-responsive element binding factor) subfamilies are of particular interest because they participate in responses to biotic and abiotic responses. Genes belonging to the DREB subfamily play major roles in responses to abiotic stresses like cold and drought while member s of the ERF subfamily play roles in defense responses to pathogen in fection and wounding (Shinozaki and YamaguchiShinozaki, 2000; Lorenzo et al., 2003). Recent studies indicating that some DREB family members, such as TINY and BnDREBIII, can activat e the transcription of genes involved in both biotic and abiotic stresses, represent evidence fo r a broader role of DREBs in stress responses (Liu et al., 2006; Sun et al., 2008) Wounding of tissues is frequen tly required for the formation of adventitious roots (De Kler k et al., 1999). However, how w ounding signaling is integrated into the developmental processes underlying ad ventitious root formation remains elusive. Although the interactions between auxin-cytokinin and auxin-et hylene during root formation have drawn most of the research attention, not much is known about the interactions between cytokinin and ethylene (Laplaze et al., 2007; Ru zicka et al., 2007; Stepanova et al., 2007; 78

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Ivanchenko et al., 2008). Our findings that two TINY-like genes are downregulated in DDKPtRR13 lines represents a po tential point for crosstalk be tween cytokinin and ethylene signaling during adventitious r oot formation and would require further research. The molecular mechanism by which stem/leaf tissu es are transformed in to root cells during adventitious root formation is still unclear. In cutt ings, the formation of adventitious roots requires that stem-localized meristematic cells are reprogrammed to form root primordia instead of stem tissues (i.e. phloem or xylem). During this process, the establishment of cell identities in the initial divi ding mass of cells would be critical for a successful root organogenesis. In Medicago truncatula and Arabibopsis, the WUSHEL-type homeobox genes also known as WOX5 play a role in root apical meristem formation during both embryonic and post embryonic root formation, however the role of other homeodomain proteins can not be discarded (Haecker et al., 2004; Xu et al., 2006 ; Imin et al., 2007). BELL1, is a homedomain transcription factor involved in the establishment of ovule cell iden tity in Arabidopsis (Reiser et al., 1995). However, its transcript s are not only found in reproductiv e tissues, but also in leaves and roots (Reiser et al., 1995).The observation that BELL1 is downregulated in the DDKPtRR13 lines indicates a potentia l novel role of this gene in root cell fate specification. 79

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PtIAA50 20 40 60 80 100 1200h6h24h48hRelative transcript abundance PtPIN20 20 40 60 80 100 1200h6h24h48hRelative transcript abundance PtIAA80 20 40 60 80 100 1200h6h24h48hRelative transcript abundance PtIAA20 20 40 60 80 100 1200h6h24h48hRelative transcript abundance PtIAA60 20 40 60 80 100 1200h6h24h48hRelative transcript abundance PtIAA30 20 40 60 80 100 1200h6h24h48hRelative transcript abundance PtIAA70 20 40 60 80 100 1200h6h24h48hRelative transcript abundance PtPIN30 20 40 60 80 100 1200h6h24h48hRelative transcript abundance PtIAA10 20 40 60 80 100 1200h6h24h48hRelative transcript abundance Figure 3-1. Microarray data ve rification by real-time PCR. RNA samples used for microarray analysis were used in real-time PCR analyses. Expression data from both analyses was normalized and is presented relative to the time point with the highest expression for a given gene. White bars represent microa rray data and black bars represent realtime PCR data. Error bars represent SE where n = 5. 80

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A 7,053 11,809 445666 504 6,777 859 24-48h 0-6h6-24h 7,053 11,809 445666 504 6,777 859 24-48h 0-6h6-24h B 7 8 9 10 11 12 13 14 15 160h6h24h48hTranscirpt abundance (log2) PtRR10 PtRR2 PtRR3 PtRR4 PtRR5 PtRR7 Figure 3-2. Differentially expre ssed genes during early stages of adventitious root formation. Microarray expression data fo r four time points; 0, 6, 24 and 48 h after shoot excision were contrasted. Differentially regulated ge nes at a FRD < 5% are shown in the Venn diagram (A). Microarray e xpression data for a group of cytokinin responsive type-As was plotted for four time points in NT (B). Error bars represent SE where n = 5. 81

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A PtpRR1* PtpRR2* PtRR17 PtRR20 ARR13 ARR21 ARR22 ARR10 PtRR22 ARR12 ARR18 PtRR18 PtRR19 ARR20 ARR19 PtRR12 PtRR13 ARR1 ARR2 PtRR15 PtRR16 ARR14 ARR11 PtRR14 PtRR21 96 82 97 70 59 56 71 72 73 63 PtpRR1* PtpRR2* PtRR17 PtRR20 ARR13 ARR21 ARR22 ARR10 PtRR22 ARR12 ARR18 PtRR18 PtRR19 ARR20 ARR19 PtRR12 PtRR13 ARR1 ARR2 PtRR15 PtRR16 ARR14 ARR11 PtRR14 PtRR21 PtpRR1* PtpRR2* PtRR17 PtRR20 ARR13 ARR21 ARR22 ARR10 PtRR22 ARR12 ARR18 PtRR18 PtRR19 ARR20 ARR19 PtRR12 PtRR13 ARR1 ARR2 PtRR15 PtRR16 ARR14 ARR11 PtRR14 PtRR21 96 82 97 70 59 56 71 72 73 63 Young stems Post. catkins 55PtRR12 38PtRR13 ActinRoots Xylem Phloem Leaves Pre. catkins Shoot tips Cycles0.950.070.390.300.080.150.431 0.61000.35000.181Young stems Post. catkins 55PtRR12 38PtRR13 ActinRoots Xylem Phloem Leaves Pre. catkins Shoot tips Cycles0.950.070.390.300.080.150.431 0.61000.35000.181B Figure 3-3. Amino acid sequence similarity tree of Populus and Arabidopsis GARP-containing RRs (A). The unrooted sequence similarity tree was generate d by aligning the GARP domain of the Populus and Arabidopsis RRs using Cl ustalX. The output nexus file was imported into PAUP 4.0 to generate the displayed bootstrap tree (n=10 000 iterations). Populus pseudo-RRs are marked with an aste risk. Transcript abundance of PtRR12 and PtRR13 (B). Semiquantitativ e RT-PCR was carried out using RNA samples isolated form P. trichocarpa (roots, phloem, xylem, shoot tips, leaves and immature stems) and from P. deltoides (preand post-receptive catkins). Transcript abundance for each tissue was normalized to the level of the c ontrol actin and is presented relative to the tissu e with the highest expression. 82

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A Transactivation domain D D K GARP1 138 186 268 673 DDK FL RNAiPtRR13 Transactivation domain D D K GARP1 138 186 268 673 Transactivation domain D D K GARP1 138 186 268 673 DDK FL RNAiPtRR13 Actin PtRR13 NT 1141620 FL DDK Lines MW Actin PtRR13 NT 1141620 FL DDK Lines MW B C 0 0.2 0.4 0.6 0.8 1 1.2NT4557Relative transcript abundance Figure 3-4. Response regulator Pt RR13 domain organization and scre ening of transgenic lines. Domain organization of PtRR13 was deduced by amino acid sequence similarity with Arabidopsis RRs (Ramirez-Carv ajal et al., 2008). The thr ee coding regions used for constructs included the fu ll-length protein (FL), a tr uncated version missing the receiver domain ( DDK), and a short region (~50 a.a.) in the last exon (RNAi). (A). Transgenic line screening (B-C). Tr ansgenic lines carrying the FL and DDK constructs were screened for transgene expression by semiquantitative RT-PCR and compared to the non transgenic control NT (B). RNA extracted from shoots was reverse-transcribed and used in a PCR reaction (30 cycles) using PtRR13 genespecific primers. Endogenous PtRR13 transc ript reduction in RNAi lines (C) was measured by real-time PCR. RNA extracte d from shoots was reversed-transcribed and used as template in real-time PCR reactions. Fluorescence intensities were normalized to the intensity of the actin c ontrol and are presented relative to the NT. Error bars represen t SE where n = 3. 83

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01 0Zeatin ( M) IBA ( M)% fresh weight increase 0 200 400 600 800 1000 1200 1400010100% fresh weight increase 0 50 100 150 200 250010100 NT DDKPtRR13 01 0Zeatin ( M) IBA ( M)% fresh weight increase 0 200 400 600 800 1000 1200 1400010100% fresh weight increase 0 50 100 150 200 250010100 NT DDKPtRR13 Figure 3-5. Overexpression of DDKPtRR13 stimulates stem grow th in culture. Stem sections were grown on culture under six different IBA:trans -zeatin ratios for 60 days. The percentage of fresh weight increase was de termined by subtracting the weight of the stem sections at day 0 from the weight at day 60, and then divided by the weight at day 0. Error bars represent SE where n = 16. 84

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PttRR100 50 100 150 200 250 300 350 400NT DDK16 DDK20Relative fluorescence PtRR20 50 100 150 200 250 300 350 400 450 500NT DDK16 DDK20Relative fluorescence Control 0.1 M BAP 1 M BAP PtRR50 100 200 300 400 500 600 700NT DDK16 DDK20Relative fluorescence PtRR70 50 100 150 200 250 300 350 400NT DDK16 DDK20Relative fluorescence PtRR40 20 40 60 80 100 120 140 160 180NT DDK16 DDK20Relatice fluorescence PtRR30 50 100 150 200 250 300NT DDK16 DDK20Relative fluorescence Figure 3-6. Overexpression of DDKPtRR13 interferes with exogenous cytokinin induction of type-As in leaves. Detached mature leaves of non transgenic, and DDKPtRR13 lines 16 and 20 ( DDK16 and DDK20 respectively) were treated for 1 hour with 0.1 M BAP (grey), 1 M BAP (hatched) or .01% DMSO (white). Transcript abundance for each type-A was measured by real-time PCR using gene specific primers. Fluorescence intensities for each individua l gene were divided by the intensities obtained for actin. Error bars represent SE where n = 3. 85

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0 20 40 60 80 100 120 140NT45571411620Total root length (mm) RNAi FL DDK 0 20 40 60 80 100 120 140NT45571411620Total root length (mm) RNAi RNAi FL FL DDK DDKA B C 0 2 4 6 8 10 12 14NT DDK20 DDK16Average root number per cutting 0 0.2 0.4 0.6 0.8NT DDK20 DDK16Above-ground dry weight (g) D E F DDK16 NT D E F DDK16 NT DDK16 NT Figure 3-7. PtRR13 is a negative regulator of adventitious root formation. Total root length was measured in 14 cm/10 day old cuttings fo r two independent transgenic lines per construct (FL, DDK and RNAi) and in NT (A). The DDKPtRR13 lines exhibit reduced number of roots compared to the NT (B), and the phenotype is specific to roots (C). Overview of the rooting phenotypes (D). Phloroglucinol-HCl stained stem sections at rooting sites (E). Abnormal callus formation at the base of the cutting in DDK lines (F). The rooting experiments de scribed here were repeated a minimum of five times to confirm the phenotypes Error bars represent SE where n = 15. 86

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Upregulated Downregulated0h6h24h48h 4 6 6 4 6 4 6 113 160 4 6 Upregulated Downregulated0h6h24h48h 0h6h24h48h 4 6 6 4 6 4 6 113 160 4 6 Figure 3-8. Overall effects of DDKPtRR13 overexpression on ge ne expression. 11 genes were differentially regulated between NT and DDKPtRR13 across all time points (blue bars) at a 10% FDR and 2 fold regulation th reshold; 4 were upregulated while 6 were downregulated in DDKPtRR13. 273 genes were differentially regulated between NT and DDKPtRR13 at 24 h at a 1% FDR and 2 fold regulation threshold (yellow bars); 160 were upregulated wh ile113 were downregulated in DDKPtRR13. 87

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A 7 8 9 10 11 12 13 14 15 160h6h24h48hTranscript abundance (log2) PtRR13 PDR9 COV1 TINY-like 1 TINY-like 2 BELL1 7 8 9 10 11 12 13 14 15 160h6h24h48hTranscript abundance (log2)NT DDK 7 8 9 10 11 12 13 14 15 160h6h24h48hTranscript abundance (log2) PtRR13 PDR9 COV1 TINY-like 1 TINY-like 2 BELL1 7 8 9 10 11 12 13 14 15 160h6h24h48hTranscript abundance (log2)NT DDK B 0 20 40 60 80 100 1200h6h24h48hRelative transcript abundance Figure 3-9. Overexpression DDKPtRR13 perturbs the expression of regulatory genes. Time course microarray expression data for regulat ory genes whose expres sion is altered in DDKPtRR13 across all time points using a 10% FDR and 2 fold regulation threshold. In DDK solid diamonds represent en dogenous and transgene PtRR13 expression (A). Endogenous PtRR13 expressi on was assayed by real-time PCR using gene specific primers and is presented relative to the time point with highest expression (B). Error bars represent SE where n = 5. 88

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FIM2 PDR9 COV MSL10 ATG -500 -1000 -1500 ARR1 AGATCT (+/-) CYP94B3 Tiny-like 1 O -methyl transferase TINY-like 2 YSL7 BELL1 DRE A/GCCGAC (+/-) ERF AGCCGCC (+/-) Up-regulated Down-regulatedFIM2 PDR9 COV MSL10 ATG -500 -1000 -1500 ARR1 AGATCT (+/-) CYP94B3 Tiny-like 1 O -methyl transferase TINY-like 2 YSL7 BELL1 DRE A/GCCGAC (+/-) ERF AGCCGCC (+/-) FIM2 PDR9 COV MSL10 ATG -500 -1000 -1500 ATG -500 -1000 -1500 ARR1 AGATCT (+/-) ARR1 AGATCT (+/-) CYP94B3 Tiny-like 1 O -methyl transferase TINY-like 2 YSL7 BELL1 DRE A/GCCGAC (+/-) DRE A/GCCGAC (+/-) ERF AGCCGCC (+/-) Up-regulated Down-regulated Figure 3-10. Arabidopsis RR1, DRE and ERF bi nding motifs in promoters of regulated genes across all time points. 1.5 kb up-stream of the translati on starting site (ATG) where manually searched for each of the three motifs shown. 89

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90 PDR9 Auxin Cytokinin PtRR13 COV1 Ethylene TINY-like FC MCC Auxin Cytokinin Root primordium formation Root primordium formation PDR9 Auxin Cytokinin PtRR13 COV1 Ethylene TINY-like FC FC MCC Auxin Cytokinin Auxin Cytokinin Root primordium formation Root primordium formation Figure 3-11. Hypothesized role of PtRR13 in adventitious root developmen t. Adventitious root formation requires that a small group of stem xylem pole cells or root founder cells (FC) become competent to form a root me ristematic cell cluster (MCC). During this time I predict that active PtRR13 (phosphorylated) plays a negative role by activating transcription of COV1 a negative regulator of vascul arization. At the same time PtRR13 also potentially pertur bs root primordia formation by interfering with auxin gradient establishment by stimulating tr anscription of the auxin efflux pump PDR9 and also by inhibiting stre ss-ethylene inducible TINY-lik e transcription factors.

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91 Table 3-1. Primer list and gene models Gene 5' primer 3' primer Poplar gene mode l RT-PCR PtRR12GTTCGCAAGAGAAAGAATG GAAGTCAAA TTTCC AAAACCAAACGTTTAAeugene3.00 770034 PtRR13GTTCGGAAGAGAAAGAACGAG GGGTTTTGG AAAGTTGCCTTTGA eugene3.00 100010 PTRR13 Constructs FL ATGTTGAATCTCGGTTACTGTAAAGGATCGGG TTTTGG AAAGTTGCCTTTGA DDKGTTCGGAAGAGAAAGAACGAG GGGTTTTGG AAAGTTGCCTTTGA RNAi ATGAGATGCAACGAGCCATGTG GGGTTTTGG AAAGTTGCCTTTGA Quantitative RT-PCR PtRR13GTGAATTTAGTAGTGTCTCTCTTTTGTTTAGCAT AAAGGCAGGTCCTAAGCATC eugene3.00100010 PtPIN2TTACGAAGGGTCTAATTTGTCTTGCACTGCAAACAGAGCAACAAATCTGestExt_Genewise1_v1.C_LG_XVI1213 PtPIN3GCTATATTTCAGTCGAAAATACATATCTAAGTTGGTGGAGATAAAATGGAATGACgw1.X.6584.1 PtIAA1ACCCTTTCGTGAACCTCTTCTTTC GCATATTGTTGACTGCTACGCTGACestExt_Genewise1_v1.C_LG_II1635 PtIAA2TCTGATGTCTCCACCACTACTTGG GCAATTCAGTAGCCTTCAGATTTAAACestExt_fgenesh4_pm.C_LG_II0495 PtIAA3CCTCTAGCTCTTCATCATCTTTCG ATGCCTTCCATGTACACCTTGAC grail3.0050017401 PtIAA5ACCCTTTCGTGAACCTCTTCTTTC GCATATTGTTGACTGCTACGCTGACestExt_Genewise1_v1.C_LG_II1635 PtIAA6AAATGGCAACAGCTACCGTGTTAG ATCAGCAGGAAGGTGGTTCTTGTC eugene3.00 100709 PtIAA7TTGAGGTAAAACGTAATCTCATCATGGTTCTTCTCCCTTCGAGTCTTCTC gw1.V.1085.1 PtIAA8AAACTTCA TTTCATTCAAGATCGTTGGATGACACAGTGCTAGAATTTTCCCestExt_fgenesh4_pg.C_LG_XIII0196

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Table 3-2. Gene set analysis of differentially regulated genes in NT during early stages of adventitious root formation. Microarray expr ession data from four time points (0, 6, 24 and 48 h) after shoot excision were contrasted between 0h and 6h, 6h and 24h, and 24 and 48h. Genes with contrasts significant at a FDR < 5% were used for gene-set enrichment analysis. Gene-sets were dete rmined significant at a FDR < 10%. Only the most significant top 40 ge ne ontology (GO) categories were included in this table 0h-6h6h-24h24h-48h GO categor y IDGenesFDRFDRFDR Hormone and signal transduction Ethylene-related genes U.D.C.1351.98E-11.. Hormone metabolic process GO:0042445255.11E-04.. Auxin-related genes U.D.C.2582.63E-04.. Cytokinin response regulators typeA U.D.C.11..2.49E-04 Regulation of kinase activit y GO:00435496..5.65E-02 Small GTPase mediated signal transduction GO:000726422..2.72E-02 Stress responses Indole glucosinolate metabolic process GO:004234360.. Regulation of response to extracellular stimulus GO:003210460.. Response to osmotic stress GO:0006970671.04E-05.. Response to salt stress GO:0009651474.16E-04.. Response to toxin GO:0009636372.52E-04.. Glycosinolate metabolic process GO:001975773.79E-04.. Response to abiotic stimulus GO:00096284112.77E-06.. Toxin metabolic process GO:0009404281.57E-04.. Wound-induced genes U.D.C.2218.05E-19.8.30E-03 Immune response GO:00069551037.83E-06.1.65E-03 Immune system process GO:00023761146.11E-07.3.43E-03 Innate immune response GO:00450871012.94E-05.1.66E-03 Plant-type hypersensitive response GO:0009626404.16E-06.3.87E-03 Programmed cell death GO:0012501481.67E-05.4.50E-03 Death GO:0016265602.18E-04.1.33E-02 Defense response to fungus GO:005083222.3.73E-04. Defense response to fungus, incompatible interactionGO:000981717.3.32E-044.18E-02 Response to fungus GO:000962055..1.89E-03 Defense response, incompatible interaction GO:000981456..5.73E-02 Response to biotic stimulus GO:0009607219..7.72E-03 Response to other organis m GO:0051707208..3.88E-03 Defense response GO:0006952345..7.17E-02 Transcription and translation Regulation of transcription, DNA-dependent GO:00063552721.41E-04.. Regulation of RNA metabolic process GO:00512522757.48E-05.. Ribonucleoprotein complex biogenesis and assembl y GO:002261387.1.59E-10. Translation GO:0006412235.3.46E-27. Translational elongation GO:000641411.5.06E-04. RNA metabolic process GO:0016070218.6.30E-04. RNA processing GO:0006396125.5.48E-04. Gene expression GO:0010467404.4.82E-27 Ribonucleotide biosynthetic process GO:000926015.3.98E-04. ncRNA processing GO:003447024.2.81E-04. Nucleotide biosynthetic process GO:000916533.8.10E-07. Nuclear mRNA splicing, via spliceosome GO:000039814.1.88E-042.09E-02 RNA splicing, via transesterification reactions GO:000037515.4.10E-042.61E-02 92

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93 Table 3-2. Continued 0h-6h6h-24h24h-48h GO category IDGenesFDRFDRFDR Development Organelle organization and biogenesis GO:00069962792.13E-044.35E-14 Cytoskeleton organization and biogenesis GO:0007010962.30E-114.59E-06. Reproductive process GO:00224144377.91E-051.95E-04. Reproduction GO:00000034377.60E-051.87E-04. Reproductive developmental process GO:0003006249.5.38E-07. Ribosome biogenesis GO:004225452.5.20E-11. Actin cytoskeleton organization and biogenesis GO:003003626.2.05E-04. Embryonic development GO:0009790167.5.66E-07. Embryonic development ending in seed dormanc y GO:0009793160.6.06E-07. Seed development GO:0048316178.1.47E-06. Plant-type secondary cell wall biogenesis GO:00098349.5.18E-04. Cell wall organization and biogenesis GO:000704773.4.99E-04. Cellular component organization and biogenesis GO:0016043431.2.61E-16. Anatomical structure development GO:0048856418.2.98E-04. External encapsulating structure organization and biogenesisGO:004522975.7.99E-04. Regulation of mitotic cell cycle GO:00073466..6.00E-02 Root epidermal cell differentiation GO:00100535..3.11E-03 Root morphogenesis GO:001001518..5.15E-02 Cell proliferation GO:000828311..5.08E-02 Cell wall biogenesis GO:004254613..7.06E-02 Metabolis m Phenylpropanoid biosynthetic process GO:0009699895.06E-04.. Photosynthesis GO:0015979347.72E-04.. N-terminal protein amino acid modification GO:00313651592.85E-04.. Lipoprotein metabolic process GO:00421571646.70E-04.. Amino acid transport GO:0006865347.53E-04.. Protein amino acid acylation GO:00435431603.42E-04.. Secondary metabolic process GO:00197482261.74E-08.. Aromatic compound metabolic process GO:00067251901.26E-03.. Carbohydrate metabolic process GO:00059753852.62E-071.37E-05. Cytoskeleton-dependent intracellular transport GO:0030705383.60E-101.11E-042.67E-02 Biopolymer biosynthetic process GO:0043284329.3.29E-23. Cellular macromolecule biosynthetic process GO:0034645417.1.62E-20. Chlorophyll biosynthetic process GO:00159959.5.04E-04. Pigment biosynthetic process GO:004614829.2.57E-04. Nucleotide metabolic process GO:005508684.6.05E-07. Nucleotide and nucleic acid metabolic process GO:0006139462.4.09E-11. Nucleoside phosphate metabolic process GO:000675363.1.45E-06. Glycerol metabolic process GO:000607129..7.25E-02 Trehalose biosynthetic process GO:00059926..6.70E-03 Lignan metabolic process GO:00098065..4.00E-02 Methionine metabolic process GO:000655510..3.94E-02 Sulfur amino acid metabolic process GO:000009620..6.68E-02 Polyol metabolic process GO:001975135..5.39E-02 Cadmium ion transport GO:00156915..3.85E-02 Disaccharide biosynthetic process GO:004635113..1.48E-02 Pectin biosynthetic process GO:00454897..1.20E-02 Alkene biosynthetic process GO:00434506..6.19E-02 Other processes Pollen-pistil interaction GO:00098751495.55E-12.. Pollination GO:00098561756.39E-11.. Recognition or rejection of self pollen GO:00485441396.13E-14.6.68E-02 Multi-organism process GO:00517043896.97E-11.2.42E-03 Microtubule-based movement GO:0007018344.36E-089.54E-061.40E-02 Microtubule-based process GO:0007017591.39E-11 1.21E-02 Protein targeting to mitochondrion GO:00066266.05.82E-02 Syncytium formation GO:00069499..3.31E-03 ID represents GO category identification number, U.D.C stands for user defined category.

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Table 3-3. Differentially re gulated genes between NT and DDKPtRR13 from 0 to 48 h. Transgene effects on gene expression during 48 h following shoot excision were determined by contrasting expression estimates of the DDKPtRR13 lines with NT excluding time effects. Contrasts with a FD R < 10% and 2 fold regulation were called significant and are disp layed. Annotation of Populus gene models was carry out by identifying putative orthogues from Gene Ba nk databases (closest hit). Positive fold changes indicate upregulation in DDKPtRR13 and vice versa Description Populus gene model Closest hit Fold re g ulation p -value DDKPtRR13 estExt_Genewise1_v1.C_LG_X3573At3g 16857.111.007.0E-16 Membrane protein COV1 estExt_Genewise1_v1.C_570139At2g20120.14.413.0E-11 Pleiotropic drug resistance transporter PDR9 fgenesh4_pm.C_LG_X000603At3G53480.13.928.0E-06 Actin binding protein FIM2 estExt_fgenesh4_pm.C_LG_XI0146At4g26700.12.453.2E-07 Mechanosensitive ion channel MSL10 gw1.28.2.1 At5g 12080.12.302.5E-07 ERF/AP2 transcription factor TINY-like 1 fgenesh4_pm.C_scaffold_41000071At1g71450.1-4.448.2E-08 Cytochrome p450 CYP94B3 fgenesh4_pg.C_LG_XII000950At3g48520.1-3.587.7E-07 Caffeic acid O -methyltransferase fgenesh4_pg.C_LG_XIX000854At4g35160.1-2.482.1E-06 ERF/AP2 transcription factor TINY-like 2 gw1.XIX.1847.1 At1g71450.1-2.431.3E-05 Metal-nicotianamine transporter YSL7 fgenesh4_pg.C_LG_IV000291At1g65730.1-2.281.7E-05 Homeodomain protein BELL1 gw1.III.246.1 At5g 41410.1-2.081.1E-05 94

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Table 3-4. Differentially regulated genes in DDKPtRR13 at 24 h. Microarray expression data from NT and DDKPtRR13 were contrasted at 24 h after shoot excision. Differentially regulated genes at a 1% FDR and 2-fold threshold were distributed in arbitrary categories. Genes of unknown function or involved in metabolism were excluded. Positive fold changes indicate upregulation in DDKPtRR13 and vice versa Description Poplar gene model Closest hi t Fold change p -value Hormone-relate d IAA conjugating enzyme gw1.III.363.1 At4g27260.17.911.6E-07 Gibberellin 3 beta-hydroxylase eugene3.00011087 At1g15550.13.202.8E-05 Auxin-inducible SAUR gene gw1.IV.886.1 At4g38840.13.122.5E-05 Auxin-responsive proteinIAA16 eugene3.00100709 At3g04730.12.668.6E-06 b HLH ethylene-responsive protein gw1.XI.2349.1 At1g61660.12.372.7E-05 Auxin-responsive protein IAA18 grail3.0052008101 At3g16500.12.311.0E-04 ACC synthase ACS6 fgenesh4_pg.C_scaffold_2423000001At4g11280.1-5.571.0E-04 Transcription Transcription factor BEE3 eugene3.01230094 At1g73830.17.482.0E-05 ERF/AP2 transcription factor TINY2 gw1.XVIII.1182.1 At5g11590.15.231.2E-05 Transcription factor NAM (no apical meristem) fgenesh4_pg.C_LG_II000536At2g43000.14.882.8E-05 Transcription factor scarecrow-like 14 fgenesh4_pm.C_LG_I000675At1g07530.13.004.3E-05 DNA binding bHLH OBP3-RESPONSIVE GENE 2 gw1.VI.1267.1 At3g56970.12.781.0E-04 Putative Myb transcription factor (MYB55) gw1.II.3432.1 At4g01680.12.761.0E-04 Transcription factor (Phytochrome interacting factor 7 eugene3.00140120 At4g00050.12.441.0E-04 Putative Myb transcription factor (MYB55). gw1.XIV.2062.1 At4g01680.12.242.7E-05 DNA-binding protein/Cyclin-like F-box fgenesh4_pg.C_LG_V001665At4g02210.12.221.0E-04 Related to ARR1 eugene3.00180621 At2g03500.12.222.5E-05 Myb family transcription facto r eugene3.01480029 At5g06800.12.191.0E-04 R2R3-MYB family transcription factor MYB98 gw1.57.124.1 At4g18770.12.081.5E-05 ERF/AP2 transcription factor TINY-like 2 gw1.XIX.1847.1 At1g71450.1-9.881.5E-07 ERF/AP2 transcription factor TINY-like 1 fgenesh4_pm.C_scaffold_41000071At1g71450.1-6.671.0E-04 Myb transcription factor (MYB102) fgenesh4_pg.C_LG_XI001038At4g21440.1-6.391.0E-04 Transcription factor ATGRF5 gw1.XIX.1261.1 At3g13960.1-5.162.0E-07 Transcriptional regulator eugene3.22790002 ZP_00275252.1-4.901.0E-04 Predicted transcriptional regulator grail3.17523000101 ZP_00217860.1-3.831.0E-04 Myb transcription factor (MYB85) fgenesh4_pg.C_LG_XII001270At4g22680.1-3.671.5E-05 Hypothetical transcription facto r eugene3.02950002 At4g17310.1-2.291.0E-04 Signal transduction Leucine-rich repeat transmembrane protein kinase eugene3.00020250 At4g36180.16.401.0E-04 Receptor-like serine/threonine protein kinase ARK3estExt_Genewise1_v1.C_LG_VII0812At3g594105.751.0E-04 Putative serine/threonine protein kinase estExt_fgenesh4_pm.C_LG_V0213At4g02630.14.301.0E-04 Leucine-rich repeat transmembrane protein kinaseestExt_fgenesh4_pm.C_LG_XIX0116At3g03770.14.251.0E-04 Receptor protein kinase estExt_Genewise1_v1.C_LG_II3264At5g49660.13.781.0E-04 Calmodulin binding protein IQD33 estExt_fgenesh4_pg.C_LG_XIV0997At5g35670.13.722.3E-05 Ras-related GTP binding protein grail3.0046007401 At4g18800.13.401.0E-04 Leucine-rich repeat transmembrane protein kinasefgenesh4_pg.C_scaffold_1637000002NoHits3.211.0E-04 Protein kinase activit y gw1.XI.259.1 At4g21380.12.211.0E-04 Calcium-binding EF hand family protein fgenesh4_pg.C_LG_I002960At1g53210.12.161.0E-04 Calmodulin binding protein IQD33 grail3.0018026401 At4g23060.1-8.561.0E-04 Leucine-rich repeat transmembrane protein kinase gw1.I.1353.1 At1g07650.1-5.471.0E-04 Receptor-protein kinase-like protein FERONI A eugene3.185900001 At3g51550.1-4.961.0E-04 Receptor-protein kinase-like protein FERONI A eugene3.02770007 At3g51550.1-3.231.0E-04 p21-activated protein kinase eugene3.133980001 ACB38299-2.671.0E-04 Mytogen-activated protein kinase kinase MEKK1fgenesh4_pg.C_LG_VIII001413At1g04210.1-2.311.8E-05 Biotic and abiotic stresses Putative heat shock protein mDj10 gw1.1487.1.1 At5g37380.16.521.0E-04 Disease resistance protein (CC-NBS-LRR class) gw1.223.27.1 At3g50950.16.292.6E-05 Disease resistance protein (TIR-NBS-LRR class) gw1.15886.1.1 At5g36930.15.564.8E-05 Disease resistance protein (NBS-LRR class) grail3.0218000801 At4g27220.15.111.5E-05 Heat shock protein gw1.VI.2232.1 At3g52490.14.771.7E-05 Disease resistance lipoxygenase grail3.0308000501 At1g55020.14.714.3E-05 Senescence/dehydration-associated protein eugene3.01210064 At3g21600.14.501.0E-04 95

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96 Table 3-4. Continued Description Poplar gene model Closest hitFold change p -value Regulator of expression of PR genes CPR5 gw1.VII.2128.1 At5g64930.14.104.9E-05 Chloroplast-localized heat shock protei n gw1.I.502.1 At1g52560.13.911.0E-04 Disease resistance protein-like eugene3.01520050 At5g49290.13.602.7E-05 Disease resistance protein (TIR-NBS-LRR class) eugene3.07870005 At5g36930.12.721.0E-04 Disease resistance protein RPM1 (CC-NBS-LRR cla s eugene3.02660003 At3g07040.12.711.0E-04 Disease resistance protein (TIR-NBS-LRR class) gw1.XI.1626.1 At1g27170.12.541.0E-04 Disease resistance protei n eugene3.02350018 At1g74190.12.031.0E-04 Supressor of AvrBST-elicited resistance gw1.166.165.1 At4g22300.1-8.231.0E-04 Abscisic acid stress ripening-like protein. grail3.0002027201 gi16588758-6.271.0E-04 Disease resistance protein (TIR-NBS-LRR class) eugene3.00190623 At5g17680.1-2.951.0E-04 Proteosome Polyubiquitin UBQ10/senescence-associated protei n gw1.I.9457.1 At4g05320.14.471.0E-04 Putative UBQ ligase ATL1B gw1.X.3524.1 At1g20823.14.122.3E-05 UBQ ligase Armadillo/beta-catenin repeat family pro t eugene3.00051454 At1g71020.13.621.6E-05 Stress-related E3 ligase eugene3.00700057 At2g30580.13.121.0E-04 Ubiquitin-conjugating enzyme UBC18 estExt_fgenesh4_pg.C_LG_V1456At5g42990.12.171.0E-04 F-box protein-relate d fgenesh4_pg.C_LG_VIII000291At1g64290.12.091.8E-05 F-box protein-relate d eugene3.00013027 At1g64290.1-4.821.0E-04 Development Membrane protein COV estExt_Genewise1_v1.C_570139At2g20120.110.723.3E-09 Auxin-independent growth promoter estExt_fgenesh4_pm.C_1650007At3g30300.15.294.8E-05 Cyclin D2 gw1.II.3852.1 At2g22490.14.231.0E-04 Tubulin beta-6 chain grail3.0018029802 At5g12250.13.664.5E-05 Extensin-like protei n fgenesh4_pm.C_LG_X000304At3g22800.1-6.191.0E-04 Expansin-like B2 precursor EXLB2 fgenesh4_pg.C_LG_III001855At4g30380.1-4.881.0E-04 Involved in de novo shoot organogenesis Hookless1fgenesh4_pg.C_LG_I002046At2g30090.1-4.821.4E-05 Extensin-like protei n fgenesh4_pg.C_LG_XIV000653gi791146-3.511.0E-04 Tobaco extensin precurso r eugene3.13060001 g|119714-3.111.0E-04 Transport MATE efflux family protein fgenesh4_pg.C_LG_XI000017At3g21690.18.011.5E-05 Putative amino acid transporte r grail3.0090011402 At3g10600.17.117.5E-06 Amino acid transport protein eugene3.00080799 At5g15240.16.471.3E-06 Clathrin assembly protein-related eugene3.01250060 At1g33340.15.532.5E-08 Putative multidrug efflux protein eugene3.41 560001 At5g38030.14.022.1E-05 Mechanosensitive ion channel MSL10 gw1.28.2.1 At5g12080.13.161.0E-04 Permease PIGMENT DEFECTIVE EMBRYO 135 gw1.II.3007.1 At2g26510.13.151.0E-04 Antiporter estExt_Genewise1_v1.C_LG_V4674At5g65380.1-5.611.0E-04 Lysine and histidine transporte r grail3.0002076401 At1g47670.1-5.417.1E-06 Metal ion transporter NRAMP1 fgenesh4_pg.C_LG_V000999At1g80830.1-5.221.0E-04 Magnesium transporter (MRS2-3) eugene3.00012466 At3g19640.1-4.021.0E-04 Lysine and histidine transporte r gw1.IV.1395.1 At1g47670.1-3.471.0E-04 Putative aquaporin eugene3.00070779 At2g36830.1-3.321.0E-04 Dual-affinity nitrate transporter NRT1.1 eugene3.00030833 At1g12110.1-2.361.0E-04 RNA processing Double-stranded RNA binding protein DRB3 grail3.0088005301 At3g26932.16.104.4E-05 Mitochondrial transcription termination factor gw1.XVI.2283.1 At2g36000.14.501.4E-05 Mitochondrial transcription termination factor eugene3.00160413 At5g06810.14.321.0E-04 Puputative replication protein A1 grail3.0145000401 At2g06510.14.234.6E-05 RNA and export factor binding protei n estExt_fgenesh4_kg.C_LG_X0079At5g02530.13.871.0E-04 Translation releasing factor RF-2 gw1.VIII.2786.1 At5g36170.13.701.0E-04 Topoisomerase VIA eugene3.00 012639 At3g13170.13.653.4E-05 RNA binding grail3.0049011401 At2g39260.12.871.0E-04 Nuclear mRNA splicing fgenesh4_pm.C_LG_XIX000095At1g54385.12.311.0E-04 Electron transport Cytochrome p450 famil y eugene3.00640122 At4g37360.16.621.0E-04 Cytochrome p450 eugene3.17460001 At1g75130.13.714.4E-06 Cytochrome p450 CYP87A2 eugene3.00041437 At1g12740.12.113.3E-06 Cytochrome p450 CYP94B3 fgenesh4_pg.C_LG_XII000950At3g48520.1-20.722.1E-08 Cytochrome p450 family CYP71B17 gw1.I.4645.1 At3g26160.1-4.041.0E-04

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Table 3-5. Top 10 different ially regulated genes in DDKPtRR13 at 24 h. Microarray expression data from NT and DDKPtRR13 were contrasted at 24 h after shoot excision. Top 10 differentially regulated gene s at a 1% FDR and 2-fold threshold are displayed. Name Description Fold regulation p -value MATE efflux family protein Multi antimicrobial extrusion protein 8.011.5E-05 GH3-8 IAA conjugating enzyme 7.911.6E-07 Transcription factor BEE3 Brassinosteroid signaling 7.482.0E-05 CAT7 Cationic amino acid transporte r 7.117.5E-06 CYP81D2 Cytochrome p450 6.621.0E-04 IQD33 Calmodulin binding protein -8.561.0E-04 SOBER1 Carboxylesterase involved in defense response-8.231.0E-04 Myb transcription factor MYB102Involved in wounding and osmotic stress response-6.391.0E-04 Abscisic acid stress ripening-like proteinmRNA regulated during peach fruit developmen t -6.271.0E-04 Extensin-like protein Structural constituent of cell wall -6.191.0E-04 97

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CHAPTER 4 CONCLUSIONS The overall goal of this project was to charac terize the response regulator gene family in Populus and investigate the roles of these genes in Populus physiology and development. Here I reported the identification of 33 RRs in Populus : 11 type-As, 11 type-Bs and 11 pseudo-RRs. This number is comparable to the size of Arabidopsis and rice RR gene family (32 and 33 genes respectively). I hypothe size that because of the importa nce of cytokinins in plant growth and development, the conser vation in family size observed among Populus, Arabidopsis and rice type-As, type-Bs and pseudo -RRs, reflects selection against substantial changes in the stoichiometry of the components of cytokinin signaling cascades. Genes encoding regulatory molecules involved in signal transduction pathways including transcription factors, tend to be dosage-dependent; changing the con centration of a regulator could change the concentration of its targets (Birchler et al., 2001). Type-C re sponse regulators have b een recently described (Schaller et al., 2007) and preliminary data in dicates that this subfamily has significantly expanded in Populus with approximately 15 members compared to only two found in Arabidopsis and rice. Since the characterization of this family was beyond the scope of this project, it will be of particular interest to test the functional significance of this expansion in Populus Amino acid sequence analyses revealed that 78% of the Populus RRs grouped in sister pairs and were characterized by overlapping expression profiles. Comparisons of the chromosomal distribution of these sister pairs revealed that 71% of them are located in duplicated genome regions and likely represent paralogues of ancestral RRs (Tuskan et al., 2006). The high degree of sequence similarity (some pairs are 91% identical at the amino acid level), the overlapping expression patterns, and the putative shared ancestry between RR sister 98

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pairs implies functional redundancy in the Populus RR subfamilies and might be a reason for failing to detect visible phenotypes in the PtRR13 RNAi lines. The RR gene family in Arabidopsis is highly redundant and single loss-of-function mutants are usually indistinguishable from wild type plants (To et al., 2004, Argyros et al., 2008). Therefore, the implementation of single loss-offunctions approaches to study gene function of the RR gene family in Populus may not be feasible. The expression analysis detailed in Chapter 2 indicates that transcripts for most of the type-As and type-Bs were detected in vegetative and reproductive tissues while pseudo-RRs were preferentially expressed in mature leaves Widespread expression of response regulators throughout the plant implies a role for cytoki nin signaling in diverse aspects of plant development and physiology. The preferential expression of pseudo-RR in mature leaves is consistent with an involvement of this subfamily in light signaling, as an Arabidopsis pseudoRR, APRR1/TOC1, is a component of the central os cillator of the circadia n clock (Somers et al., 1998). The expression analyses he re presented were conducted on major plant organs; additional expression analysis at the tissue and cell type level would complement this work. This study shows for he first time that th e transcriptome reprogramming following shoot excision is of massive proportions; 27% and 36% of the Populus nuclear genes were differentially regulated between 0 and 6 h and be tween 6 and 24 h respectively. Analysis of these expression data following a gene -set approach instead of a single-gene approach revealed valuable information regarding functional gene networks regulated duri ng the early stages of adventitious root formation. Networks regulated during the initial 48 h after excision included genes related to very broad processes like hormone homeostasis and signaling, primary metabolism, gene expression and development. Gene networks involved in root morphogenesis 99

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and cell division were enriched passed the initial 24 h indicating that initiation of the rooting process in Populus cuttings is a relatively early re sponse. Future work on functionally characterizing these genes will provide insi ghts into the molecular events surrounding adventitious rooting. By using a reverse genetics approach I have shown that the response regulator type-B PtRR13 interferes with a dventitious root formation in cut tings and represents confirmatory evidence of the negative ro le of cytokinins in root developmen t. I have also demonstrated that the delay in rooting phenotype asso ciated with overexpression of the constitutively active version of PtRR13 is associated with significant change s in gene expression durin g the initial 48 h after shoot excision and that at 24 h the root founde r cells appear competent to respond to the cytokinin signaling pathway. These results represent a molecular complement to the physiological studies from previous decades show ing that cytokinin application to cuttings between 24 and 72 h after excision strongly inhibits rooting in mi crocuttings (De Klerk et al., 1995). If other genes involved in cytokinin sign aling exert effects similar to PtRR13, then it would appear that cytokinin signaling may play an important role as a negative regulator of carbon partitioning to roots in forested ecosystems. I finally conclude that the nega tive effects of cytokinin during de novo root formation through the activity of PtRR13 ope rate in part by cross-talk with auxin and ethylene/wounding signaling through its eff ects on expression of PDR9 and TINY-like genes, and also by affecting the expression of genes of potential importance fo r the development of the new root primordial, such as COV1 and BELL1 The presence of multiple cis -regulatory motifs in the promoter of COV1 make this gene a candidate to be under th e direct regulation of PtRR13, however testing 100

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this hypothesis will require further in vivo and in vitro PtRR13/ DDKPtRR13 promoter binding experiments. 101

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APPENDIX CYTOKININ BIOSYNTHETIC, DEGRADING AND SIGNALING GENES IN Populus Table A-1. List of cytoki nin biosynthetic, degradi ng and signaling genes in Populus C ytokinin-related genesPoplar gene model Putative function Isopentenyl transferases PtIPT1 gw1.VIII.574.1 p utative tRNA isopentenyl transferase / similar to tRNA isopentenyl transferase PtIPT2 gw1.XIV.2884.1 tRNA isopentenyl transferase -like protein / tRNA isopentenyl transferase PtIPT3 fgenesh4_pg.C_LG_VIII000257 tRNA isopentenyltransferase -like protein / tRNA isopentenyltransferase PtIPT4 gw1.VIII.295.1 tRNA isopentenyltransferase -like protein / tRNA isopentenyltransferase PtIPT5 gw1.X.185.1 p utative tRNA isopentenyl transferase / similar to tRNA isopentenyl transferase PtIPT6 gw1.X.4133.1 tRNA isopentenyltransferase -like protein / tRNA isopentenyltransferase PtIPT7 gw1.IV.412.1 tRNA isopentenyltransferase -like protein / tRNA isopentenyltransferase PtIPT8 gw1.IX.724.1 p utative tRNA isopentenylpyrophosphate transferase Cytokinin oxidases PtCKox1 gw1.III.1989.1 p utative cytokinin oxidase PtCKox2 eugene3.02200015 p utative cytokinin oxidase PtCKox3 gw1.VI.2253.1 p utative cytokinin oxidase PtCKox4 eugene3.00051394 cytokinin oxidase PtCKox5 gw1.XVI.1482.1 p utative cytokinin oxidase PtCKox6 eugene3.00051393 cytokinin oxidase PtCKox7 eugene3.00060432 p utative cytokinin oxidase PtCKox8 gw1.VII.2678.1 cytokinin oxidase PtCKox9 fgenesh4_pg.C_LG_II000281 cytokinin oxidase PtCKox10 gw1.VI.977.1 cytokinin oxidase Histidine Kinases PtHK5 gw1.XIV.3720.1 histidine kinase-like protei n PtHK3 gw1.I.7746.1 p utative sensory transduction histidine kinase PtHK4 eugene3.00031406 histidine kinase-like protei n PtHK2 gw1.VIII.2924.1 p utative histidine kinase PtHK1 gw1.X.6321.1 p utative histidine kinase Type-A RRs PtRR2 gw1.VIII.329.1 response regulator 3 PtRR5 estExt_Genewise1_v1.C_LG_I7064 p utative two-component response regulator 3 protei n PtRR3 gw1.II.42.1 responce reactor 4 PtRR7 gw1.XVI.1327.1 responce reactor 4 PtRR10 grail3.0005005001 response reactor 2 (ATRR2) PtRR4 gw1.III.113.1 responce reactor 4 PtRR9 gw1.XIII.278.1 response regulator-like protein / res ponse regulator 4 (ARR4) PtRR6 fgenesh4_pg.C_LG_VI000330 responce reactor 4 PtRR1 gw1.VIII.35.1 response regulator 3 PtRR11 gw1.XIX.2514.1 response regulator-like protein / response regulator 4 (ARR4) PtRR8 gw1.XIX.1648.1 response regulator-like protein / response regulator 4 (ARR4) Type-B RRs PtRR14 gw1.VIII.612.1 F12A21.15 / hypothetical protei n PtRR13 estExt_Genewise1_v1.C_LG_X3573ARR1 protei n PtRR21 gw1.X.5015.1 F12A21.15 / hypothetical protei n PtRR19 gw1.XVIII.3323.1 p utative two-component response regulator protein / similarity to RegA PtRR18 gw1.VI.371.1 p utative two-component response regulator protein / similarity to RegA PtRR16 gw1.X.6388.1 p utative two-component response regulator protein PtRR12 estExt_Genewise1_v1.C_770060 p utative two-component response regulator protein / similarity to RegA PtRR22 estExt_fgenesh4_pg.C_LG_XVIII0466 p utative two-component response regulator protein / similarity to RegA PtRR15 gw1.VIII.1097.1 p utative two-component response regulator protein PtRR20 eugene3.00151142 p utative protein / ARR2 protein Arabidopsis thaliana PtRR17 eugene3.00121175 ARR1 protei n Other RRs (Type-Cs?) PtRR23 eugene3.28400003 response regulators consisting of a CheY-like receiver domain PtRR26 eugene3.30140001 response regulators consisting of a CheY-like receiver domain PtRR24 eugene3.31330001 response regulators consisting of a CheY-like receiver domain PtRR25 eugene3.56540001 response regulators consisting of a CheY-like receiver domain PtRRC3 gw1.II.248.1 p utative response regulator protein (receiver component) PtRRC1 eugene3.00031425 p utative response regulator protein (receiver component) PtRRC7 fgenesh4_pg.C_LG_III001494 p utative response regulator protein (receiver component) PtRRC2 gw1.XIX.712.1 p utative response regulator protein (receiver component) PtRRC9 fgenesh4_pg.C_LG_I000495 p utative response regulator protein (receiver component) PtRRC11 fgenesh4_pg.C_LG_III001446 p utative response regulator protein (receiver component) PtRRC8 fgenesh4_pm.C_LG_IX000289 p utative response regulator protein (receiver component) PtRRC5 fgenesh4_pg.C_LG_III001495 p utative response regulator protein (receiver component) 102

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Table A-1. Continued Cytokinin-related genesPoplar gene model Putative function P seudo -RRs PtpRR7 estExt_fgenesh4_pm.C_LG_VIII0151 p utative protein / ABI3-interacting protei n PtpRR3 eugene3.00150024 p utative protein / contains similarity to two-component response regulator protei n PtpRR4 gw1.XII.1231.1 p utative protein / contains similarity to two-component response regulator protei n PtpRR8 gw1.X.2468.1 p utative protein / ABI3-interacting protei n PtpRR10 estExt_fgenesh4_pg.C_1180049 p utative protein / predicted protei n PtpRR9 fgenesh4_pg.C_scaffold_129000038 p seudo-response regulator 1 PtpRR5 estExt_fgenesh4_pg.C_LG_XIV0468 p utative protein / contains similarity to two-component response regulator protei n PtpRR6 fgenesh4_pg.C_LG_II001656 p utative protein / contains similarity to two-component response regulator protei n PtpRR1 fgenesh4_pg.C_LG_II001405 similar to putative two-component response regulator protei n PtpRR11 gw1.29.358.1 p utative protein / predicted protei n PtpRR2 eugene3.00140231 p utative protein / contains similarity to two-component response regulator protei n Phos p hotransfer p roteins PtHpt6 estExt_fgenesh4_pm.C_700061 two-component phosphorelay mediato r PtHpt3 eugene3.00081871 two-component phosphorelay mediato r PtHpt2 estExt_fgenesh4_pg.C_LG_X0275two-component phosphorelay mediato r PtHpt9 eugene3.00440046 HPt phosphotransmitte r PtHpt14 fgenesh4_pg.C_LG_XIV000773 p utative AHP2 PtHpt7 estExt_fgenesh4_pm.C_LG_VI0337 p utative AHP2 PtHpt11 fgenesh4_pg.C_LG_IX000220 two-component phosphorelay mediato r PtHpt1 estExt_Genewise1_v1.C_LG_XIII1173two-component phosphorelay mediato r PtHpt5 estExt_fgenesh4_pg.C_LG_XVI1041 p utative AHP2 PtHpt10 fgenesh4_pg.C_LG_I003334 p utative two-component phosphorelay mediator PtHpt12 fgenesh4_pg.C_LG_VI001633 p utative two-component phosphorelay mediator PtHpt4 estExt_fgenesh4_pg.C_LG_I1468 HPt phosphotransmitte r PtHpt13 fgenesh4_pg.C_LG_X002318 two-component phosphorelay mediato r PtHpt15 fgenesh4_pg.C_scaffold_697000003 p utative two-component phosphorelay mediator PtHpt8 eugene3.00180254 p utative two-component phosphorelay mediator PtHpt16 gw1.I.396.1 p utative two-component phosphorelay mediator 103

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119 BIOGRAPHICAL SKETCH Gustavo A Ramirez Carvajal was born in Cali, Colombia. Upon graduating from high school in 1993 he started his undergraduate st udies in agronomical engineering at the Universidad Nacional de Colombia in Palmira. During his studies toward the B.S. degree, he spent one semester working on a program designed to substitute illegal crops in the Colombian Amazon Forest. For his undergraduate thesis project he studied drought tolerance in soybean at the Colombian Center for Agronomic Crops. He graduated with the B.S. in 1998 and moved to the United States where he worked for the orna mental plant industry for several years. On August 2003, he joined the lab of Dr. John Davi s, where he studied cytokinin signaling in Populus