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Functional analyses of somatic embryogenesis receptor-like kinase family in multiple signaling pathways in Arabidopsis

Dissertation
Author: Kai He
Abstract:
Arabidopsis SERK family contains five members: AtSERK1, AtSERK2 , AtSERK3 (BAK1) , AtSERK4 (BKK1) and AtSERK5. SERK5 might not be functional due to a natural point mutation at a highly conserved "RD" motif. All other SERKs , SERK1 to SERK4, are involved in at least four distinct signaling pathways. Previous studies indicated the function of SERK1 and SERK2 in microsporgenesis; SERK1, SERK2, SERK3 and SERK4 in BR signaling pathway; SERK1, SERK2, SERK3 and SERK4 in cell death control pathway; and SERK3 in disease resistance pathway. More importantly, the multiple SERK knockout mutant phenotypes support the essential role of SERKs in BR signaling. In our model, it is hypothesized although SERKs play redundant role to each other, some SERKs play major roles in certain pathways. SERK1 and SERK2 play major role in regulating microsporgenesis; SERK1 and SERK3 in BR signaling pathway; SERK3 and SERK4 in cell death control pathway.

vi TABLE OF CONTENTS Acknowledgement .............................................................................................. iv Table of Contents ............................................................................................... vi List of Tables ................................................................................................... viii List of Figures ................................................................................................... ix List of Abbreviations ....................................................................................... xii

Chapter I. Introduction ..................................................................................... 1 1. Brassinosteroid signal transduction pathway .................................................... 2 2. ROS in plants ................................................................................................. 12 References .......................................................................................................... 27

Chapter II. BAK1 and BKK1, Two Arabidopsis LRR Receptor-Like Protein Kinases, Regulate BR-mediated Growth and BR-Independent Cell Death Pathways ............................................................................................................................. 36 1. Summary ......................................................................................................... 37 2. Results ............................................................................................................. 37 3. Discussion ....................................................................................................... 44 4. Experimental procedures ................................................................................ 45 5. Acknowledgements ......................................................................................... 49 References ........................................................................................................... 62 Note ..................................................................................................................... 67

vii Chapter III. Arabidopsis Receptor-Like Kinases, BAK1 and BKK1, Regulate the Levels of Reactive Oxygen Species in Chloroplast......................................... 68 1. Abstract ........................................................................................................... 69 2. Introduction .................................................................................................... 69 3. Results ............................................................................................................ 71 4. Discussion ....................................................................................................... 77 5. Perspectives..................................................................................................... 80 6. Methods........................................................................................................... 81 References ........................................................................................................... 92

Chapter IV. Somatic Embryogenesis Receptor Kinase (SERK) Family Control Multiple Signing Pathways in Arabidopsis..................................................... 95 1. Abstract ........................................................................................................... 96 2. Introduction .................................................................................................... 96 3. Results ............................................................................................................ 99 4. Discussion ..................................................................................................... 106 5. Methods......................................................................................................... 109 References ......................................................................................................... 123 Note ................................................................................................................... 127

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LIST OF TABLES Chapter II Table 2.1 Genetic segregation analysis to confirm that the lethality is caused by the loss-of-function recessive mutations of both BAK1 and BKK1 ...................... 61

Chapter III Table 3.1 Nine ROS-scavenging genes are down-regulated in bak1-4 bkk1-1 ........................................................................................................................... 91

ix LIST OF FIGURES

Chapter I Figure 1.1 BL structure and representative BR mutants ................................. 20 Figure 1.2 Structure of BRI1 protein............................................................... 21 Figure 1.3 Structure of BAK1 protein ............................................................. 22 Figure 1.4 Current model for BR signaling pathway ...................................... 23 Figure 1.5 ROS are generated in chloroplast and peroxisome during photosynthesis ....................................................................................................... 24 Figure 1.6 ROS are triggered under biotic and abiotic tresses ........................ 25 Figure 1.7 ROS scavenging system ................................................................. 26

Chapter II Figure 2.1 BKK1 plays a redundant role with BAK1 in suppressing bri1-5 when overexpressed ................................................................................. 50 Figure 2.2 A bak1-4 bkk1-1 double null mutant shows a seedling lethal phenotype at an early developmental stage .............................................................. 51 Figure 2.3 BKK1 interacts with BRI1 and mediates BR signal transduction . 52 Figure 2.4 BAK1 and BKK1 are also involved in BR-independent cell death signaling pathway .......................................................................................... 54 Figure 2.5 A proposed model indicating that BAK1 and BKK1 positively regulate BR signaling pathway, and negatively regulate a spontaneous cell death pathway .......................................................................................... 56

x Figure 2.6 Partial sequence alignment of BAK1, BKK1/SERK4, and SERK5… ............................................................................................................................. 57 Figure 2.7 Shoot apical meristems (SAMs) of wild-type (Col-0) and bak1-4 bkk1-4 seedlings revealed by Scanning Electron Microscopy (SEM) ....... 58 Figure 2.8 bak1-3 bkk1-1 show early senescence and seedling lethality phenotype ........................................................................................................ 59 Figure 2.9 In bak1-4 bkk1-1 double mutant, BR signaling pathway is partially blocked ........................................................................................................ 60

Chapter III Figure 3.1 bak1 bkk1 double mutants are lethal .............................................. 84 Figure 3.2 Cell death in bak1-3 bkk1-1 weak mutant is environment-dependent ............................................................................................................................. 85 Figure 3.3 ROS product and cell death effect is accumulated in bak1-3 bkk1-1 ............................................................................................................................. 86 Figure 3.4 Light triggers cell death in bak1-4 bkk1-1 ..................................... 87 Figure 3.5 ROS accumulated in chloroplast due to down-regulation of ROS-savaging genes in bak1-4 bkk1-1 .................................................................. 88 Figure 3.6 bak1-3 bkk1-1 shows accelerated leaf senescence ......................... 89 Figure 3.7 Current model for BAK1/BKK1-controlling pathways. ................. 90

Chapter IV Figure 4.1 SERK proetin sequcne allignment ............................................... 114

xi Figure 4.2 Overexpression of SERK genes rescues cell death phenotype in bak1 bkk1 ...................................................................................................... 115 Figure 4.3 Expression patterns of SERK genes ............................................ 116 Figure 4.4 SERK genes play redundant roles in regulating BR signaling pathway ...................................................................................................... 117 Figure 4.5 SERK gene T-DNA insertion mutants ......................................... 118 Figure 4.6 Double mutants serk1 serk2 and serk1 serk3 are sterile .............. 119 Figure 4.7 Phenotypes of SERK mutants....................................................... 120 Figure 4.8 serk1 serk3 is insensitive to BL treatment. .................................. 121 Figure 4.7 SERKs regulate multiple singling pathways................................ 122

xii LIST OF ABBREVIATIONS

2D-DFE: two-dimensional difference gel electrophoresis ABA: abscisic acid ACC: 1-Aminocyclopropane-1-carboxylic acid ACS: ACC synthase AOX: alternative oxidase APN1: ARABIDOPSIS NPK1-LIKE PROTEIN KINASE1 APX: ascorbate peroxidase BAK1: BRI1 ASSOCIATED RECEPTOR KINASE 1 BES1: BRI1-EMS-SUPPRESSOR1 BFA: brefeldin A BKI1: BRI1 KINASE INHIBITOR1 BKK1; BAK1-LIKE 1 BIM: BES1-INTERACTING MYC-LIKE PROTEIN BIN2: BRASSINOSTEROID INSENSITIVE2 BL: brassinolide BPCS: biotin-tagged photoaffinity CS BR: brassinosteroid BR6ox1: BR-6-oxidase1 BRI1: BRASSINOSTEROID INSENSITIVE 1 BRL1: BRI1-LIKE 1 BRS1: BRI1 SUPPRESSOR 1 BRZ: brassinazole BSK: BR-SIGNALING KINASE BSU1: BRI1 SUPPRESSOR1 BZR1: BRASSINAZOLE-RESISTANT1 CaMV 35S: Cauliflower mosaic virus 35S CAT: catalase CDC: CELL DIVISION CYCLE

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CFP: cyan fluorescent protein cGMP: cyclic guanosine monophosphate CPD: CONSTITUTIVE PHOTOMORPHOGENESIS ANDDWARFISM CS: castasterone CT: cathasterone DAB: 3, 3’-diaminobenzidine DAG: day after germination DET2: DE-ETIOLATED 2 DHA: dehydroascorbate DHAR: DHA reductase DWF4: DWARF4 EDS1: ENHANCED DISEASE SUSCEPTIBILITY 1 EFR: EF-Tu RECEPTOR EF-Tu: elongation factor thermo unstable ER: endoplasmic reticulum FLS2: FLAGELLIN SENSITIVE2 GA: gibberellins GC: guanylyl cyclase GC-MS: gas chromatography-mass spectrometry GFP: green fluorescent protein GPX: glutathione peroxidase GR: glutathione reductase GSH: glutathione GSK3: glycogen synthase kinase-3-like protein GST: glutathione S-transferase, GTP: guanosine-5'-triphosphate GUS: β-glucuronidase

HR: hypersensitive response IP: immunoprecipitation KAPP: kinase-associated protein phosphatase KD: kinase domain

xiv LRR: leucine-rich repeat LSD1: LESION SIMULATING DISEASE 1 MAPK: mitogen-activated protein kinase MAPKK: mitogen-activated protein kinase kinase MAPKKK: mitogen-activated protein kinase kinase kinase MBP: maltose binding protein MDA: monodehydroascorbate MDAR: MDA reductase mRNA: messenger RNA NADP: nicotinamide adenine dinucleotide phosphate NahG: nd6 salicylate hydroxylase gene PAD: PHYTOALEXIN DEFICIENT PAMP: pathogen-associated molecular pattern PCD: programmed cell death PCR: polymerase chain reaction PR: pathogen-related PrxR: peroxiredoxin PS: photosystem PTI: PAMP-triggered immunity RLK: receptor-like kinase ROS: reactive oxygen species ROT3: ROTUNDFOLIA3 RT: reverse transcription SA: salicylic acid SAM: shoot apical meristem SEM: scanning electron microscopy SERK: SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE SOC: SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 SOD: superoxide dismutase T-DNA: transferred DNA TE: teasterone

xv TRIP1: TGF-β receptor interacting protein 1 tRNA: transfer RNA TTL: TRANSTHYRETIN-LIKE UDP: uridine diphosphate UGT: UDP-glycosyltransferase enzyme UGGT: UDP-glucose:glycoprotein glucosyltransferase

1

Chapter I

Introduction

2 1 Brassinosteroid signal transduction pathway 1.1 BRs are a new class of plant hormones In 1970, Mitchell et al. reported unknown compounds extracted from rape (brassica napus L.) pollen could stimulate plant growth; and the compounds were named as brassins (Mitchell et al., 1970). It was not clear what the nature of brassins was and how brassins promoted plant growth. After the first brassin, brassinolide (BL) (Figure 1.1) was purified and the structure of BL was determined by X-ray analysis subsequently (Grove et al., 1979), researchers were able to analyze the roles of brassins in regulating plant growth and development. To date, more than 60 branssins have been identified and they are collectedly named as brassinosteroids (BRs) (Fujioka et al., 2003). The essential roles of BRs have been demonstrated by severe phenotypes of numerous mutants in either BR biosynthesis pathway or BR signaling pathway (Figure 1.1) (Li et al., 1996; Clouse et al., 1996; Szekeres et al., 1996). Lately, the identification of the BR receptor BRI1 (BRASSINOTEROID INSENSITIVE 1) (Li et al., 1997) in Arabidopsis not only dramatically accelerated the research of BR signal transduction, but also indisputably supported BRs as a new class of plant hormones, together with other well-known plant phytohormone classes including auxins, gibberellins (GAs), abscisic acid (ABA), cytokines and ethylene. This section will focus on the progress in studies on BR signaling pathway, including the processes from ligand binding, receptor activation, downstream signaling and induction of BR response genes. 1.2 BRI1 is the ligand-binding receptor of BRs 1.2.1 BRI1 protein structure

3 Arabidopsis BRI1 belongs to a protein kinase family called leucine-rich repeat receptor-like kinase (LRR-RLK) family with at least 223 members (Shiu et al., 2001). BRI1 consists of an extracellular domain, a single-pass transmembrane domain and a cytoplasmic kinase domain (Figure 1.2). As a typical LRR-RLK, BRI1 has twenty-five leucine-rich repeats in the extracellular domain. LRRs form paralleled β-sheets connected by α-helix, proving conformational structure for protein-protein interaction (Bella et al., 2008). Although the LRRs in BRI1 suggest BRI1 might interact with other proteins in extracellular space, there is still no direct evidence showing BRI1 has any extracellular protein interactors. LRR21 and LRR22 is separated by a 70-amino acid island. In the cytosol, there is a Thr/Ser kinase domain, containing 11 conserved subdomains. Activation of BRI1 kinase domain triggered by perception of BR signals plays a central role in initiating BR signaling cascade. 1.2.2 BRs bind to BRI1 Null BRI1 mutants show complete insensitivity to the BL treatment (Clouse et al., 1996), implicating the essential function of BRI1 in BR perception. By using biotin- tagged photoaffinity BL precursor castasterone, BPCS, it was demonstrated that a 94 amino acid region including the 70 amino acid island domain and its flanking LRR22 within the BRI1 extracellular domain is responsible for the direct BR binding (Kinoshita et al., 2005). Thus, BRI1 has been confirmed as a ligand-binding receptor in BR signaling. 1.2.3 Activation of BRI1 The kinase activity of BRI1 is negatively regulated by its cytoplasmic C-terminal tail. Deletion of a 41 amino acid fragment at the C-terminal enhanced BRI1 kinase activity, as was revealed by its capability to suppress BR biosynthesis mutant det2 (Wang

4 et al., 2005a). Phosphorylation at S/T residues at C-terminal is believed to play a positive role in BRI1 activation; mutations of certain S/T residues to D in distal C-terminal domain, mimicking a phosphorylation effect, dramatically increased BRI1 kinase activity, suggesting the mechanism of inhibition of BRI1 activity by the C-terminal domain and mechanism of BRI1 activation by phosphorylation in the C-terminal domain (Wang et al., 2005a). 1.2.4 Guanylyl cyclase activity of BRI1 Near the C-terminal region, BRI1 contains a guanylyl cyclase (GC) domain. This GC activity of BRI1 was confirmed by an experiment using the recombinant BRI1 GC domain containing 114 amino acids can catalyze GTP to cGMP (Kwezi et al., 2007). Since cGMP acts as a secondary messenger in multiple signaling pathways such as stresses and hormones, it suggests cGMP may play a role in BR signaling. 1.2.5 BRI1 endocytosis BRI1 was observed to be endocytosed from plasma membrane into endosomes (Russinova et al., 2004). The endocytosis of BRI1, however, is independent of BR treatment or BR deficiency. Brefeldin A (BFA), a protein transport inhibitor, treatment resulted in an accumulation of BRI1 in endosomes. The BR signaling, however, was not blocked, indicating endosomal BRI1 is active (Geldner et al., 2007). A mutated BRI1 protein, bri1-9, is retained in endoplasmic reticulum (ER), leading to a typical BR mutant phenotype (Jin et al., 2007). With the help of a UGGT (UDP-glucose:glycoprotein glucosyltransferase), bri1-9 protein was relocated to plasma membrane, which suppressed the bri1 mutant phenotype. This result implicated BRI1 can initiate BR signaling in

5 endosomes but not in ER (Jin et al., 2007). However, the reason of BRI1 endocytosis is still unknown. 1.3 BRI1-interactors 1.3.1 BAK1 1.3.1.1 Identification and structure of BAK1 BAK1 (BRI1 ASSOCIATED RECEPTOR KINASE 1) was identified as a BRI1 kinase domain interactor by a yeast-two hybrid screen (Nam et al., 2002) and a bri1-5, a BRI1 weak mutant, genetic suppressor by an activation tagging screen (Li et al., 2002). Like BRI1, BAK1 is also a LRR-RLK, containing an extracellular domain with only five LRRs, a transmembrane domain and a cytoplasmic Thr/Ser kinase domain (Figure 1.3). Adjacent to the signal peptide, there is a leucine-rich domain called leucine zipper with the pattern Lx6Lx6Lx6L. Leucine zipper motif contains leucine residues at every seven amino acid residues, providing the conformational structure to form protein-protein interaction through α-helix (Landschulz et al., 1988). SERK1, a BAK1 paralog, interacts with another SERK1 molecule to form a homodimer. The homodimerization of SERK1 is reduced when the extracellular leucine zipper is deleted, indicating leucine zipper is essential for homodimerization. Following the leucine zipper motif, there are five LRRs. There is a unique proline-rich region between five LRRs and the transmembrane domain. Proline-rich motif creates flexibility to extracellular domain during the signal perception. BAK1 protein has a typical Thr/Ser protein kinase domain, containing 11 characteristic subdomains. 1.3.1.2 BAK1 interacts with BRI1 to regulate BR signaling

6 Overexpression of BAK1 suppressesd the dwarfed phenotype of a weak bri1-5 allele but not a null bri1-4 allele (Li et al., 2002), suggesting the role of BAK1 in mediating BR signaling is dependent on a functional BRI1. The in vivo interaction between BRI1 and BAK1 was demonstrated by the dominant negative phenotype resulted from the overexpression of a kinase dead form of BAK1 (mBAK1) in bri1-5 (Li et al., 2002); and the co-immunoprecipitation result showing BRI1 interacted with BAK1 in vivo (Li et al., 2002; Nam et al., 2002). BAK1 and BRI1 can also phosphorylate each other in vitro and in vivo. Further analyses indicated the interaction between BRI1 and BAK1 was dramatically enhanced by BL treatment and the phosphorylation levels of BRI1 and BAK1 were also stimulated by BL (Wang et al., 2005b). Since a BAK1 single mutant did not show the dwarfed phenotype as severe as bri1 null mutant does (Li et al., 2004; He et al., 2007), it was assumed that there are BAK1 homologous genes playing redundant roles with BAK1 in BR signaling. 1.3.1.3 BKK1, the closest paralog of BAK1, plays a redundant role with BAK1 in BR signaling BKK1 (BAK1-LIKE 1), also known as SERK4, was proven to function in the BR signaling pathway in a way similar to BAK1 (He et al., 2007). BKK1 is the closest paralog of BAK1, sharing 82% amino acid identity. BKK1 also interacts with BRI1 in vivo and the interaction is stimulated by BL. In addition, the kinase activity of BKK1 also can be regulated by BL. Although bak1 and bkk1 single mutants do not obviously show a typical bri1 mutant phenotype, bak1 bkk1 double mutant exhibits a de-etiolation phenotype with opened cotyledons when grown in dark, a typical BR mutant response, further suggesting the roles of BAK1 and BKK1 in BR signaling.

7 1.3.1.4 The interplays of BR signaling and other pathways regulated by BAK1 and its paralogs 1.3.1.4.1 bak1 bkk1 double null mutant is lethal Besides showing some bri1 mutant phenotype, bak1 bkk1 double mutant also exhibits a spontaneous cell death phenotype that is not observed in any other BR mutants (He et al., 2007), including BR deficient and BR signaling mutants. The cell death phenotype of bak1 bkk1 double mutant is not observed until 5 days after germination (DAG). Lesions on the cotyledons of the bak1 bkk1 start emerging around 7 DAG. Accompanied with the cell death are the accumulation of ROS, deposit of callose, and up-regulation of defense-related genes. Introduction of NahG gene partially suppresses bak1 bkk1 cell death phenotype, suggesting the salicylic acid (SA) signaling pathway is partially related to the cell death in the double mutant (He et al., 2007). 1.3.1.4.2 The cell death in bak1 bkk1 double mutant is independent of BR signaling pathway The spontaneous cell death phenotype seen in bak1 bkk1 double mutant is opposite to BR mutant phenotype that usually shows dark-green leaves, delayed senescence, and prolonged life span. The up-regulation of defense-related genes, such as PR1, PR2, PR5, ACS2, and ACS6, observed in bak1-4 bkk1-1 double mutant was not seen or showed opposite expression patterns in bri1-4 mutant (He et al., 2007). These observations implicate the BAK1/BKK1-controlling cell death is BR signaling-independent. bak1 single mutant developed a runaway cell death (RCD) phenotype upon bacterial or fungal pathogen infection, whereas the BL treatment cannot rescue the disease phenotype, also

8 supporting that the BAK1-controlling cell death pathway is BL-independent (Kemmerling et al., 2007). 1.3.1.4.3 BAK1 is involved in an FLS2-mediated innate immunity response pathway The recognition of pathogen-associated molecular patterns (PAMPs) by corresponding cell surface receptors initiates PAMP-triggered immunity (PTI). FLS2 functions as the receptor of bacterial flagellin. A 22-amino acid peptide conserved in flagellin, flg22, is sufficient to trigger PTI through FLS2-mediated pathway (Gomez- Gomez et al., 2000). Recently, two groups reported simultaneously that BAK1 was involved in FLS2-mediated plant defense pathway. BAK1 interacted with FLS2 in vivo upon flg22 treatment and bak1-4 showed reduced sensitivity to the flg22 treatment (Chinchilla et al., 2007; Heese et al., 2007). 1.3.2 BKI1 Through a yeast-two hybrid screen, BKI1 was identified as an interactor of BRI1 kinase domain (KD) via BKI1’s C-terminal region (Wang et al., 2006). Overexpression of BKI1 resulted in a dwarfed phenotype similar to a bri1 mutant and showed reduced accumulation of phosphorylated BES1, a BR downstream signaling transcription factor. In absence of BRs, BKI1 interacts with BRI1 in vivo; whereas, BKI1 is rapidly disassociated from BRI1 complex upon BL treatment. The BL-dependent release of BKI1 from BRI1 is thought to be an essential process to activate BRI1, which allows BAK1 to associate with BRI1. Transphosphorylation between BRI1 and BAK1 kinase domains activates their downstream substrates, triggering intracellular BR signaling. 1.3.3 BSKs

9 By using two-dimensional difference gel electrophoresis (2D DIGE), BSK1 and BSK2 were identified as early BR-regulated proteins (Tang et al., 2008). BSK1 and BSK2 belong to a receptor-like cytoplasmic kinase subfamily RLCK-XII. Co- immunoprecipitation assay showed the interaction of BRI1 and BSK1. Furthermore, BSK1 Ser-230 was phosphorylated by BRI1 in vitro, suggesting BSK1 is a substrate of BRI1. Lost-of-function of BSK3, a paralog of BSK1 and BSK2, showed reduced BR sensitivity; overexpression of BSK3 partially suppressed bri1 null mutant but not bin2 mutant, indicating BSKs function as downstream components of BRI1 but upstream components of BIN2. However, no evidence has proved BIN2 is the direct substrate of BSKs; and whether and how BR signaling is transduced from BSKs to BIN2 is still not understood. 1.3.4 TTL Transthyretin-Like (TTL) protein was identified as a BRI1-interacting protein through a yeast-two hybrid screen by using BRI1 kinase domain as bait (Nam et al., 2004). N-terminus of TTL is involved in the interaction with BRI1. Overexpression of TTL resulted in a growth inhibition, whereas the ttl mutant showed opposite growth- promoting phenotype. Although TTL overexpression line and ttl mutant show some different phenotype compared to wildtype, more evidence is needed to prove TTL is involved in BRI1-mediated BR signaling. 1.3.5 TRIP-1 In mammals, TRIP-1 is involved in TGP-β signaling, acting as the substrate of TGF-β type II receptor kinase. Arabidopsis TRIP-1 can be phosphorylated by BRI1 on three sites, Thr-14, Thr-89 and Thr-197/Ser-198 (Ehsan et al., 2005). Co-

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immunoprecipitation array revealed TRIP-1 had interaction with BRI1 in vivo. However, like TTL, there is no evidence showing TRIP-1 connects BR receptor BRI1 with BR downstream components BIN2 or BSU1; and the function of TRIP-1 in BR signaling pathway is not confirmed in plants. 1.4 BR downstream signaling BZR1 and BES1 are two BR downstream transcription factors, positively regulated by BR signaling. Through DNA binding domain, BZR1 directly interacts with the CGT(T/G)G sequence of the promoters of BR feedback-regulated biosynthesis genes, such as DWF4, CPD, ROT3 and BR6OX, acting as a repressor (Wang et al., 2002; He et al., 2005). As a paralog of BZR1, BES1 binds to BIM1, a basic helix-loop-helix transcription factor, and its homologs, BIM2 and BIM3. The BES1/BIM complex binds to the E box (CANNTG) of the promoters of BR-induced genes (Yin et al., 2005). BZR1 and BES1 are regulated by a glycogen synthase kinase-3 (GSK)-like kinase named BIN2 (BRASSINOSTEROID-INSENSITIVE 2) in a post-translational level (Yin et al., 2002; He et al., 2002). BIN2 is negatively regulated by BR signaling; and bin2-1, a BIN2 dominant mutant, shows a bri1-like dwarfed phenotype and blocked feedback regulation of BR biosynthesis genes (Mathur et al., 1998). BIN2 phosphorylates BZR1 and BES1 and the phosphorylated BZR1 and BES1 are unstable and are likely recognized by E3 ligase then degraded through an E3-mediated ubiquitin-dependent protein degradation pathway. Phosphorylated BZR1 also binds to phosphopeptide-binding proteins, 14-3-3, leading to accumulation in the cytoplasm and inhibition of BZR1 function (Gampala et al., 2007). Playing an opposite role with BIN2, a Thr/Ser phosphatase, BSU1,

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dephosphorylates BZR1 and BES1 and stabilizes both transcription factors (Mora-Garcia et al., 2004). 1.5 Current model for BR signaling BRs are perceived by two plasma membrane-localized LRR-RLKs, BRI1, and BAK1. BRI1 functions as a ligand-binding receptor; whereas BAK1 acts as a co-receptor. BR binding to BRI1 releases BKI1, a BRI1 kinase domain-binding protein, from BRI1 complex and the conformational change subsequently leads to BRI1 autophosphorylation. The phosphorylation activates BRI1 kinase domain that interacts with BAK1 kinase domain to form BRI1-BAK1 heterodimmer complex. Transphosphorylation between BRI1 and BAK1 initiates BR signaling cascade. BIN2 phosphorylates two BR-regulating transcription factors, BZR1 and BES1. Phosphorylated BZR1 and BES1 are degraded through E3-mediated ubiquitin-dependent protein degradation pathway. A phosphatase, BSU1, dephosphorylates BZR1 and BES1, stabilizes both transcription factors and maintains their normal functions. BZR1 acts as a repressor in regulating BR biosynthesis genes; and BES1 promotes the expression of BR-induced genes. Although a variety of proteins have been identified to interact with BRI1, such as TTL, TRIP-1 and BSKs, there is still no evidence to show any BRI1-interacitng proteins connecting BRI1/BAK1 with BIN2 or BUS1. (Figure 1.4) 1.6 Perspectives Identification of a secreted serine carboxypeptidase, BRS1, in the BR signaling pathway suggested a proteolytic protein modification proceeding BR binding to BRI1 may be necessary (Li et al., 2001). One possible function of BRS1 is to degrade a steroid- binding protein MSBP1 and releases free BRs that can be used to trigger BR signaling. It

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is equally possible that BRS1 is to degrade a BRI1-binding protein occupying the BR- binding site in order to make BR perception possible (Figure 1.4). Nevertheless, these hypotheses need to be further tested in the future. One of the major questions in BR signal transduction is what the relationship between BRI1 and BAK1 is. As a co-receptor of BRs, BAK1 does not seem to interact with BRs directly. This raises a question that whether BAK1 functions as an essential regulator in BR signaling or alternatively BAK1 only serves as an enhancer of BRI1 kinase activity. Wang et al. reported even in the bak1 bkk1 double mutant, BRI1 was still active and BR signaling pathway was intact. The authors proposed that although BAK1 can enhance BR singling pathway by tansphosphorylating BRI1, it is not essential for BR signaling pathway. Nevertheless, given the fact that BAK1 has three additional paralogs besides BKK1, and they might play redundant roles with BAK1 in BR signaling as well, it prompts us to investigate the functions of other BAK1 paralogs in BR signaling. Although a number of proteins were identified as BRI1 substrates or BRI1- interactors, such as TTL, TRIP-1 and BSKs, the immediate upstream regulator of BIN2, however, is not identified yet. The development of new approaches, such as 2D-DFE which was recently used to successfully identify BSKs (Tang et al., 2008), in BR signaling research provide alternative opportunities to discover new regulatory components, filling gaps of the BR signaling pathway. 2 ROS in plants ROS (reactive oxygen species), which include superoxide radical, hydrogen peroxide and singlet oxygen, are ubiquitous molecules produced as a consequence of normal cellular metabolisms. In green plants, ROS are continuously produced as

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byproduct of several physiological processes, such as photorespiration and photosynthesis (Foyer et al., 1994). In addition, ROS also act as signaling molecules produced during biotic and abiotic tresses, regulating plant responses to various environmental challenges (Elstner et al., 1991; Malan et al., 1990; Prasad et al., 1994; Tsugane et al., 1999). 2.1 Generation of ROS 2.1.1 ROS are byproducts of normal physiological processes ROS are produced continuously during photosynthesis, a plant unique and essential physiological process. In chloroplasts, H 2 O loses an electron and is oxidized to O 2 in photosystem II (PSII). Through electron transport, the PSII complex passes electrons to photosystem I (PSI), where the electrons are used to oxidize O 2 to O 2._ . O 2._ is unstable and is rapidly catalyzed by superoxide dismutase to form H 2 O 2 . PSI also transfers electrons to NADP + to generate NADPH and eventually produce glycolate. Glycolate is relocated to peroxisome where it is oxidized by glycolate oxidase to produce H 2 O 2 . (Figure 1.5) In mammals, respiration is the major resource of ROS that are produced in the mitochondria. However, the contribution of mitochondria to ROS generation is relatively low in plants due to two reasons: one is the production of ROS in chloroplasts is relative high in green plants (Purvis et al., 1997); the second reason is the presence of alternative oxidase (AOX) in plant mitochondria, catalyzing the reduction of O 2 generated during electron transport (Wagner et al., 1995; Maxwell et al., 1999). 2.1.2 ROS are triggered by stresses

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As signaling molecules, ROS are rapidly generated in response to various stresses: biotic stresses (attacks by other organisms, such as bacteria, fungi and virus) and abiotic tresses (environmental challenges, such as high/low temperature, drought, wounding and high light etc.). 2.1.2.1 Biotic stresses In plants, recognitions of PAMPs by corresponding receptors triggered innate immunity responses. In Arabidopsis, cell surface RLKs were identified as pathogen receptors, such as FLS2, a receptor of bacterial flagellin (Gomez-Gomez et al., 2000); and EFR, a receptor of bacterial PAMP EF-Tu (Zipfel et al., 2006). The activation of disease resistance response pathways lead to enhanced activity of plasma membrane localized enzyme NADPH-oxidase that utilizes NADP + to produce O 2- (peroxide radicals) in apoplast (Sagi et al., 2001). Since O 2- is a strong oxidizer and highly toxic to pathogens, plants are able to protect themselves from biological invasion by using ROS as weapons. O 2- is unstable and is rapidly dismutated into H 2 O 2 that can diffuse into the cell. This rapid accumulation of ROS is called oxidative burst (Apostol et al., 1989). The highly accumulated H 2 O 2 in the cell ultimately cause programmed cell death (PCD) (Bolwell et al., 1999; Dangl et al., 2001), known as hypersensitive response (HR) (Wohlgemuth et al., 2002). By this strategy, on one hand, plants use ROS to kill invading pathogens; on the other hand, ROS work as signaling molecules to trigger HR, sacrificing infected areas to protect surrounding tissues and limit pathogen movement and spreading (Figure 1.6a). In some cases, if a plant fails to control HR in a limited area, the cell death will be spread out to the whole tissue, known as runaway cell death (RCD). 2.1.2.2 Abiotic stresses

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In abiotic stresses, intracellular ROS level is also enhanced. ROS are mainly produced in chloroplasts and mitochondria in the electron transport. In contrary to biotic stresses, the increased ROS levels are negatively regulated by ROS scavengers that are involved in the removal of ROS in cytosol and specific organelles. ROS-scavenging pathway is up-regulated by ROS signaling, suggesting a feedback regulation of ROS signaling in abiotic stresses. Cell death can also be triggered if the ROS-scavenging system fails to detoxify excessively accumulated ROS. (Figure 1.6b) 2.2 ROS-scavenging pathways As strong oxidizers, ROS are toxic to plant cell. Therefore, there must be a system continuously removing ROS to maintain a steady state redox (reduction/oxidation) homeostasis in a plant cell. Different mechanisms contribute to ROS detoxification, including antioxidants, such as glutathione and ascorbate, and ROS-scavenging enzymes. In Arabidopsis, five major groups of ROS-scavenging enzymes are involved in ROS metabolism, including SOD, CAT, APX, GPX and PrxR. (Figure 1.7) 2.2.1 SOD (superoxide dismutase) SOD catalyzes superoxide, which is highly toxic and unstable, into hydrogen peroxide (H 2 O 2 ). H 2 O 2 is more stable and steadily diffuses membranes and can be further reduced by other mechanisms. No antioxidants are needed in this process. 2.2.2 CAT (catalase) CAT reduces H 2 O 2 into H 2 O and O 2 . There are no antioxidants involved in CAT- mediated H 2 O 2 removal. 2.2.3 APX (ascorbate peroxidase)

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Abstract: Arabidopsis SERK family contains five members: AtSERK1, AtSERK2 , AtSERK3 (BAK1) , AtSERK4 (BKK1) and AtSERK5. SERK5 might not be functional due to a natural point mutation at a highly conserved "RD" motif. All other SERKs , SERK1 to SERK4, are involved in at least four distinct signaling pathways. Previous studies indicated the function of SERK1 and SERK2 in microsporgenesis; SERK1, SERK2, SERK3 and SERK4 in BR signaling pathway; SERK1, SERK2, SERK3 and SERK4 in cell death control pathway; and SERK3 in disease resistance pathway. More importantly, the multiple SERK knockout mutant phenotypes support the essential role of SERKs in BR signaling. In our model, it is hypothesized although SERKs play redundant role to each other, some SERKs play major roles in certain pathways. SERK1 and SERK2 play major role in regulating microsporgenesis; SERK1 and SERK3 in BR signaling pathway; SERK3 and SERK4 in cell death control pathway.