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Functional analysis of MAPK phosphatase AtMKP2 Cheng, Jia 2009

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by  Jia Cheng  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  The Faculty of Graduate Studies (Botany)  UNIVERSITY OF BRITISH COUMBIA (Vancouver)  April 2009 © Jia Cheng, 2009  Abstract Plants have evolved complex signal transduction pathways to sense and respond to the fast-changing environment. Among several crucial signaling pathways, MAPK pathways are known to be involved in regulating many biological processes, including development, cytokinesis, biotic and abiotic stress signaling and hormone signaling. As negative regulators of MAP kinases (MPKs), MAPK phosphatases (AtMKPs) can reverse the activation status of AtMPKs by dephosphorylating the activation sites of AtMPKs. There are 5 putative AtMKPs in the Arabidopsis genome and previous research has shown they play an important role in MAP kinase control. AtMKP2 has been shown to be a novel regulator for ozone stress responses, but how it might be involved in other biological aspects of Arabidopsis development and growth remains unknown. In this study, I examined the biological functions of AtMKP2 in trichome development. Phenotype analysis showed that in AtMKP2-RNAi mutants, trichome density as well as trichome branching number were affected. Both proAtMKP2: GUS signal and proAtMKP2:YFP signal showed that AtMKP2 was expressed in all development stages of developing trichomes. RT-PCR showed that the expression levels of several known trichome development regulators were affected in AtMKP2RNAi plants. Genetic analysis of AtMKP2 RNAi x try and AtMKP2 RNAi x cpc double mutants showed phenotype consistent with the involvement of AtMKP2 in trichome development. To characterize the biological processes in which AtMKP2 plays a role, I also employed microarray approaches to examine the short-term transcriptional events in AtMKP2 LOF and GOF mutants. I was able to validate the microarray data of AtMKP2 LOF mutants using qRT-PCR. However, the gene expression patterns in AtMKP2 GOF mutants were not verified. This might result from the different overexpression levels of AtMKP2 in different biological replicates. Several defense-related genes showed transcriptional changes in both AtMKP2 LOF and GOF mutants, suggesting AtMKP2 might function in response to pathogen attack. In all, I studied the biological function of AtMKP2 in Arabidopsis trichome development and placed AtMKP2 within the network of known trichome development regulators. My microarray experiments provided some useful clues suggesting other biological functions of AtMKP2.  ii  Table of Contents  Abstract................................................................................................. ii Table of Contents ................................................................................ iii List of Tables ....................................................................................... vi List of Figures...................................................................................... ix Abbreviations ...................................................................................... ix Acknowledgements .............................................................................. x 1. Introduction...................................................................................... 1 1.1 Post-translational modification and protein phosphorylation...................2 1.2 MAPK signaling .............................................................................................3 1.2.1 MAPK signaling networks in mammals and yeast ...................................4 1.2.2 MAPK signaling networks in Arabidopsis ...............................................5 1.2.3 The role of MAPK signaling networks in plant development ..................6 1.3 The biological functions of MAPK phosphatases .......................................7 1.3.1 MKPs in yeast and humans.......................................................................7 1.3.2 MKPs in Arabidopsis................................................................................8 1.3.3 Previous research on AtMKP2................................................................10 1.4 Trichome development in Arabidopsis .......................................................10 1.4.1 The stages of trichome development ......................................................11 1.4.2 The genetic regulatory network that controls Arabidopsis trichome development.....................................................................................................11 1.4.3 The cellular events associated with trichome development....................12 1.5 Experiment materials to study the function of AtMKP2 .........................13 1.6 Project objectives .........................................................................................14  2. AtMKP2 is involved in trichome development............................ 15 2.1 Introduction..................................................................................................15 2.2 Material and methods..................................................................................16 2.2.1 Plant materials.........................................................................................16 2.2.2 Evaluation of trichome and root-hair number.........................................17 2.2.3 Construction of double mutants ..............................................................17 2.2.4 GUS staining analysis .............................................................................18 iii  2.2.5 AtMKP2-YFP fusion protein localization analysis ................................18 2.2.6 Nuclear DNA content measurement .......................................................19 2.2.7 RNA Isolation and RT-PCR Analysis ....................................................19 2.3 Results ...........................................................................................................21 2.3.1 AtMKP2 is involved in trichome initiation and branching.....................21 2.3.2 Gene expression pattern of AtMKP2 in developing trichomes ..............23 2.3.3 Microtubule and actin cytoskeleton structure remain unaltered in AtMKP2 LOF mutants.....................................................................................25 2.3.4 The expression patterns of trichome development-related genes is changed in AtMKP2 LOF mutants ..................................................................27 2.3.5 The endoreduplication level of AtMKP2 trichome nuclei is decreased .28 2.3.6 Genetic interaction of AtMKP2 and TRY/CPC ......................................29 2.3.7 Trichome phenotype in mutants of potential AtMKP2 substrates..........33 2.4 Discussion......................................................................................................35  3. Investigation of short-term transcriptional events in AtMKP2 LOF and GOF mutants ..................................................................... 40 3.1 Introduction..................................................................................................40 3.2 Material and methods..................................................................................40 3.2.1 Plant material and treatments..................................................................40 3.2.2 Microarray analysis.................................................................................41 3.2.2.1 RNA isolation and cDNA synthesis ................................................41 3.2.2.2 cDNA hybridization.........................................................................41 3.2.2.3 Cy3 and Cy5 labeling.......................................................................42 3.2.2.4 Image processing .............................................................................42 3.2.2.5 Data analysis ....................................................................................43 3.2.3 Quantitative Real-time PCR ...................................................................43 3.3 Results ...........................................................................................................45 3.3.1 Experimental design for the study of AtMKP2-mediated transcriptional changes.............................................................................................................45 3.3.2 AtMKP2 mediates short-term transcriptional changes ...........................46 3.3.3 Analysis of trichome development-related gene expression levels ........50 3.3.4 Verification of selected genes by Real-time PCR...................................51 3.4 Discussion......................................................................................................53  iv  4. Future directions............................................................................ 56 4.1 Yeast 2-hybrid (Y2H) screening for other possible interactors of AtMKP2 ..............................................................................................................56 4.2 Phospho-proteomics profiling using AtMKP2 mutants ...........................57 4.3 Interaction of AtMKP2 with MPK8 and MPK15 .....................................57 4.4 Conclusions...................................................................................................59  References........................................................................................... 60 Appendix............................................................................................. 70 1. Genes affected by overexpression of AtMKP2 in mature plants...............70 2. Genes affected by repression of AtMKP2 in seedlings ...............................90  v  List of Tables  Table 2. 1 RT primers for trichome development related genes..................................20 Table 2. 2 Summary of gene expression of trichome developmental regulators in AtMKP2 RNAi mutants...............................................................................................28 Table 3. 1 Primers used for qRT-PCR confirmation ...................................................44 Table 3. 2 Experimental design for microarray profiling of AtMKP2 LOF and GOF mutants.........................................................................................................................46 Table 3. 3 Transcriptional responses of genes involved in trichome development, as compared in microarray analysis and RT-PCR analysis..............................................50 Table 3. 4 Genes for QRT-PCR from AtMKP2 overexpression data..........................51 Table 3. 5 Genes for qRT-PCR from AtMKP2 RNAi data .........................................52 Table 5. 1 Genes affected by overexpression of AtMKP2 in mature plants................70 Table 5. 2 Genes affected by repression of AtMKP2 in seedlings ............................105  vi  List of Figures  Figure 1. 1 Phylogenetic tree of Arabidopsis MKPs ...................................................10 Figure 2. 1 AtMKP2 RNAi lines grown on 10μM DEX plates...................................15 Figure 2. 2 Trichome number on first and second true leaves of Arabidopsis ............21 Figure 2. 3 Root hair density of primary root ..............................................................22 Figure 2. 4 Trichome branching defect of AtMKP2 RNAi mutants............................23 Figure 2. 5 Expression pattern of AtMKP2 in developing trichomes..........................24 Figure 2. 6 Microtubule structure in AtMKP2 RNAi mutants ....................................25 Figure 2. 7 Actin cytoskeleton structure in AtMKP2 RNAi mutants ..........................26 Figure 2. 8 Endoreduplication level of AtMKP2 RNAi mutants.................................29 Figure 2. 9 AtMKP2 expression level in double mutants ............................................30 Figure 2. 10 Total trichome number on first and second true leaves of cpc and AtMKP2 RNAi x cpc double mutants .........................................................................30 Figure 2. 11 Trichome branching on first and second true leaves of cpc and AtMKP2 RNAi x cpc double mutants.........................................................................................31 Figure 2. 12 Total trichome number on first and second true leaves of try and AtMKP2 RNAi x try double mutants ..........................................................................32 Figure 2. 13 Trichome branching on first and second true leaves of try and AtMKP2 RNAi x try double mutants ..........................................................................................32 Figure 2. 14 Trichome cluster percentage on first and second true leaves of try and AtMKP2 RNAi x try double mutants ..........................................................................32 Figure 2. 15 AtMKP2 physically interact with MPK8 and MPK15 in yeast ..............33 Figure 2. 16 Total trichome number on first and second true leaves...........................34 Figure 2. 17 Trichome branching on first and second true leaves ...............................34 Figure 2. 18 Expression of trichome development related genes ................................35 Figure 2. 19 Pavement cells and stomata on true leaves of WT and AtMKP2 LOF mutants.........................................................................................................................36 Figure 2. 20 A possible model for the function of MKP2 in the regulation of trichome development.................................................................................................................38 Figure 2. 21 DAB staining of WT and AtMKP2 RNAi mutants.................................39 Figure 3. 1 Distribution of p-values from t-test ...........................................................47 Figure 3. 2 GO annotation for genes affected by over-expression of AtMKP2 ..........49  vii  Figure 3. 3 GO annotation for genes affected by repression of AtMKP2 ...................50 Figure 3. 4 qRT-PCR analysis of genes affected by AtMKP2 overexpression ...........53 Figure 3. 5 qRT-PCR confirmation of AtMKP2 RNAi line26 with different biological replicates ......................................................................................................................53  viii  Abbreviations ABA  abscisic acid  cDNA  complementary DNA  Cy3  cyanine 3 bihexanoic acid dye  Cy5  cyanine 5 bihexanoic acid dye  DAB  3,3'-diaminobenzidine  DAPI  4',6-diamidino-2-phenylindole  DEX  dexamethasone  DNA  deoxyribonucleic acid  dNTP  deoxyribonucleotide triphosphate  DTT  dithiothreitol  ERK  extracellular signal-regulated kinase  EV  empty vector  GFP  green fluorescent protein  GST  glutathione S-transferase  GUS  β-glucuronidase  MAPK  mitogen-activated protein kinase  MAPKK (or MKK)  mitogen-activated protein kinase kinase  MAPKKK  mitogen-activated protein kinase kinase kinase  MKP  mitogen-activated protein kinase phosphatase  MS  Murashige and Skoog  OMFP  3-O-methylfluorescein phosphate  PCR  polymerase chain reaction  RNA  ribonucleic acid  RNAi  RNA interference  ROS  reactive oxygen species  qRT-PCR  quantitative real-time PCR  T-DNA  transfer DNA  WT  wild type  Y2H  yeast two hybrid  YFP  yellow fluorescent protein  ix  Acknowledgements  First of all I would like to thank my supervisors Dr. Brian Ellis and Dr Jin-Gui Chen. They have provided me with many helpful suggestions and constant encouragement during my study. I also want to thank my committee members, Dr. Leonard Foster, Dr Fred Sack for their scientific advice for my project.  I wish to thank JinSuk Lee for her wonderful work and all the materials she provided for my study. I thank Dr. Jie Le and Dr. Chris Ambrose for providing seeds for my imaging studies. I also want to thank Anne Haegert for technical support of my microarray experiments, Brad Ross for technical support of ESEM experiments . I thank all lab members in Chen Lab and Ellis Lab, Dr. ShuCai Wang, Jim Guo, JunBi Wang, Hardy Hall, Dr Jun Chen, Ankit Walia, QingNing Zeng, Apurva Bhargava, Adrienne Nye and Doris Vong, for their personal support and assistance.  I want to thank all my friends and families for their endless support. I wish to thank my parents, Xiangqian Cheng and Yonghong Guo, for their understanding and encouragement. I would particularly like to thank Guang Yang, who has always been there for me and encouraged me to do my best.  Finally, I also thank the UBC Faculty of Graduate Studies for educational funding.  x  1. Introduction Plants, unlike animals, need to monitor and respond to a complex, dynamic and diverse environment without using a classical nervous system. In order to mediate the sensing process and the associated response mechanisms, plants have evolved an elaborate signal transduction system. Over the past two decades, several important signaling pathways have been identified in plants, including the highly conserved MAPK pathways which control downstream targets through catalyzing reversible phosphorylation and dephosphorylation of proteins (Jonak et al., 2002). MAPK cascades are crucial signaling cascades in all eukaryotes, including plants, where they are involved in regulating many biological processes, including development, cytokinesis, biotic and abiotic stress signaling and hormone signaling (2004; Nakagami et al., 2005).  The basic components of a MAPK cascade include a MAPKK kinase (MAPKKK), a MAPK kinase (MAPKK or MKK) and a MAP kinase (MAPK or MPK). After sensing an input signal from receptors, the upstream kinases in such a cascade activate the downstream kinases by phosphorylation of specific amino acids in the activation loop of the target protein. At the bottom of a canonical cascade, the activated MPKs are able to modify their own downstream targets by phosphorylation of serine or threonine residues, usually in the context of a S/T-P sequence motif (Irie et al., 1994; Luttrell and Luttrell, 2004).  The biological outcomes of these chains of phosphorylation reactions are determined at different levels. In addition to the specificity of a given kinase toward its potential substrates within the cascade, and the spatial and temporal pattern of distribution of both the upstream kinase and downstream substrate within plant tissues and cells, the magnitude and duration of the resulting activation must be controlled. In MAPK signaling pathways, the activation of MPKs is usually mediated by upstream MAPKKs, although one recent report demonstrates that a plant MAPKKK can directly activate a MPK, thus by-passing the usual MAPKK stage of a cascade (Seger and Krebs, 1995).  1  In contrast to the process by which MPKs are activated, their deactivation has been much less well-studied. The de-activation process was first characterized in mammalian systems, where a group of dual-specificity tyrosine phosphatases (MAPK phosphatases; MKPs) were found to be important negative regulators of MPKs (Wishart and Dixon, 1998).  In Arabidopsis, 5 MKP homologues have been identified based on amino acid sequence similarity to the corresponding functional domains in mammalian MKPs (Naoi and Hashimoto, 2004). While several mammalian MKPs have been demonstrated to be important in regulating cell differentiation, cell fate determination and disease-related processes (Dickinson and Keyse, 2006), relatively little is known about the plant MKPs and their biological functions. Therefore, I chose to study the function of a specific MKP in Arabidopsis, AtMKP2. This study is focused both on some specific biological roles of AtMKP2 as well as global approaches to explore the wider range of biological processes in which this phosphatase works.  1.1 Post-translational modification and protein phosphorylation  Proteins are essential components of all organisms and participate in most biological events in living cells. After synthesis, proteins undergo various post-translational chemical modifications in vivo, such as phosphorylation, glycosylation, biotinylation, and ubiquitination (Rucker and McGee, 1993). These modifications are essential to the biological functions of proteins. For example, some modifications are signals for the transport of proteins to certain cellular location. Some modify the activity and specificity of proteins, and some control the degradation rate of the proteins.  Among  different  kinds  of  protein  modifications,  phosphorylation  and  dephosphorylation play a significant role in a wide range of processes. Phosphorylation is the addition of a phosphate (PO4) group to a protein, usually at a serine, tyrosine, threonine or histidine residue (Rucker and McGee, 1993). Extensive studies have demonstrated that this reversible covalent modification mediates specific enzymatic and signaling events. Protein kinases, activated by specific signals, can catalyze the phosphorylation of their downstream substrates. On the other hand, 2  specific protein phosphatases act to dephosphorylate the modified substrates and thus reverse the signal.  The Arabidopsis genome encodes over 1000 protein kinases and 112 protein phosphatase catalytic subunits (Kerk et al., 2002; Wang et al., 2003). The major kinase families are receptor-like protein kinases (RLKs), calcium-dependent protein kinase (CDPK)-SNF1-related kinase (SnRK) superfamily, mitogen-activated protein kinase (MAPK) cascade members, GSK-3/Shaggy-like protein kinases and histidine protein kinases (Dornelas et al., 1998; Shiu and Bleecker, 2001; MAPKgroup, 2002; Hrabak et al., 2003). Protein phosphatases are grouped as protein phosphatases 2C (PP2C), protein serine/threonine phosphatases (ST), dual-specificity protein phosphatases (DSP), protein tyrosine phosphatases (PTP) and low-Mr protein tyrosine phosphatases (LMW-PTP) (Kerk et al., 2002). The protein kinases and phosphatases in Arabidopsis, although clearly homologous to those of other eukaryotes, have their own unique features. Studying the role of these kinases and phosphatases can help to decipher the signaling events that underpin plant development and environmental responses.  1.2 MAPK signaling  MAPK cascades are highly conserved in eukaryotes, including yeasts, plants and animals. The kinases in these cascades can be divided into three classes: MAPK kinase kinases (MAPKKK or MAP3K), MAPK kinases (MAPKK or MAP2K or MKK) and MAPK (MPK) (Marshall, 1994; Qi and Elion, 2005). In addition to being phosphorylated and activated by a wide range of different factors, the MAP3Ks can be activated by directly interacting with other proteins. Once activated, MAP3Ks turn on MAP2Ks in the cascades by phosphorylating MAP2Ks at two serine/threonine residues within a highly conserved –S/T-X3-5-S/T motif. As signal transmitters, dualspecificity kinases MAP2Ks then recognize and activate diverse groups of downstream MPKs and thereby pass the signal from MAP3K to MPK via phosphorylating MPKs at threonine and tyrosine residues within their –TXY- motif (Widmann et al., 1999). At the bottom of the MAPK signaling cascade, MPKs act  3  upon their targets by phosphorylating one or more serine and/or threonine residues within a consensus PXT/SP motif. The substrates of MPKs include transcription factors, cytoskeleton associated proteins, protein kinases and protein phosphatases which control various cellular events such as gene expression, mitosis, differentiation, and cell survival/apoptosis (Karin and Hunter, 1995; Feilner et al., 2005; Yap et al., 2005).  1.2.1 MAPK signaling networks in mammals and yeast  In mammals, thirteen MPKs have been identified and characterized. Based on their functions and structures, these MPKs are divided into three major groups: the extracellular signal-regulated protein kinases (ERKs), the p38 MPKs and the c-Jun NH2-terminal kinases (JNKs) (Cohen, 1997). Specifically, the members in ERK family share a TEY activation motif and respond to growth factors such as EGF (Morrison and Davis, 2003). In contrast, the P38 MPKs contain a TGF activation motif and are regulated by osmotic stress, endotoxins and cytokines (Kumar et al., 2003; Mikhailov et al., 2005). Finally, the JNK family members have TPY in their activation motif and are mainly involved in cell responses to stress and inflammatory cytokines (Kyriakis and Avruch, 2001; Waetzig et al., 2005).  In Saccharomyces cerevisiae, there are five MPKs. All these MPKs contain the canonical TXY motif in the activation loop. Like mammalian ERKs, yeast MPK Fus3, Kss1 and Slt2/Mpk1 contain a similar –TEY- motif (Chen and Thorner, 2007). In yeast, Fus3 mediates cellular response to pheromones (Sabbagh et al., 2001; Maleri et al., 2004), while Kss1 permits adjustment to nutrient-limiting conditions (Roberts and Fink, 1994). Slt2/Mpk1 plays an important role in cell wall repair and budding under different environmental conditions (Staleva et al., 2004). A yeast p38 MAPK homologue has been also identified (Hog1), which contains a-TGY- motif and participates in cell responses to osmotic stress (Lawrence et al., 2004). Moreover, a more divergent MPK, Smk1, which possesses a –TNY- motif, has been shown to mediate spore wall assembly during meiosis and sporulation (Krisak et al., 1994).  4  1.2.2 MAPK signaling networks in Arabidopsis  MAPK signaling pathways are involved in a lot of biological processes in Arabidopsis, including plant development, cytokinesis, biotic and abiotic stress signaling and hormone signaling. There are 20 MPKs, 10 MAP2Ks and 60 putative MAP3Ks in Arabidopsis (MAPKgroup, 2002). Based on the similarity with mammalian MAP3Ks, 60 putative Arabidopsis MAP3Ks could be divided into two subfamilies, MEKK-like protein kinase (such as . ANP1, ANP2, ANP3, YDA etc.) and Raf-like protein kinase (such as EDR1, CTR1 etc.) (MAPKgroup, 2002). Comparing with mammalian MAP2Ks’ /T-X3-S/T motif, plant MAP2Ks have the S/T-X5-S/T motif as phosphorylation site. Arabidopsis MAP2Ks can also be classified into four different groups (group A to D) (Jonak et al., 2002). All of Arabidopsis MAPKs are homologous of human extracellular signal-regulated kinases (ERK). Based on protein sequence similarity, they could be divided into four subgroups (group A-D) (MAPKgroup, 2002).  Although the diversity of Arabidopsis MPKs suggests that they might have divergence roles in signaling networks, the detailed functions of MPKs remain largely unknown. In group A MPK, MPK3 and MPK6 are known to participate in various biological processes such as plant development, biotic/abiotic response and hormone signaling (Liu and Zhang, 2004; Menke et al., 2004; Wang et al., 2008a). Group B MPK MPK4 is involved in pathogen defense machineries (Calderini et al., 1998; Bogre et al., 1999; Droillard et al., 2004), MPK12 is a negative regulator of auxin signaling(Lee et al., 2008a) and MPK13 plays a role in cell division (Bogre, Calderini et al. 1999;). All members of group C (MPK1,2,7,14) can be activated by MKK3, which is involved in pathogen defense(Doczi et al., 2007). However, not much is known about the specific roles of these MPKs in Arabidopsis. Group D MPKs contain –TDY- motif in their activation loop and they also have featured extended C-terminal region. There are no reports on functions of group D MPKs in Arabidopsis, the only published information on MPKs with –TDY- motif is in rice for OsSJMK1 and BWMK1, which are both involved in defense signaling(Cheong et al., 2003; Ning et al., 2006).  5  1.2.3 The role of MAPK signaling networks in plant development  Emerging evidences show that MAPK cascades are also tightly involved in plant growth and development. More and more studies reveal that the functions of environmental stimulated kinases are broader than short term impacts on gene expression and hormone signaling. They may serve as crucial links that integrate both environmental cues and genetic cues in controlling plant growth and development.  The MAPKKK YODA in Arabidopsis is known to function in embryo development and patterning (Lukowitz et al., 2004). The zygotes of yda mutants cannot perform the normal asymmetric division, but go through a nearly symmetrical division, resulting in equal sized apical and basal cells. The basal cells of yda mutants then undergo abnormal divisions and the suspensor growth is then abolished. So far the downstream targets in this process are still unknown.  MAPK cascades are also known to control stomata patterning in Arabidopsis. (Wang et al., 2007a; Lampard et al., 2008). Stomata are microscopic pores on shoot epidermis formed by two kidney shaped guard cells. They control water and CO2 exchanges and their patterning is tightly controlled by both environmental and genetic signals. The distribution of stomata in Arabidopsis follows the one-cell spacing rule. In loss of function alleles of YODA, MKK4/MKK5 and MPK3/MPK6, the cell fate determination is disrupted, resulting in stomata clusters (Wang et al., 2007a). Correspondingly, the activation of MKK4/MKK5 and MPK3/MPK6 suppresses stomatal cell fate determination and causes reduction of stomata number. It is further proved that MPK3/MPK6 can phosphorylate a transcription factor, SPEECHLESS (SPCH), which is an important regulator in stomata patterning (Lampard et al., 2008). MPK3 and MPK6 are also crucial factors for flower organ development. In mpk3+/mpk6-/- mutants, the integument of ovules cannot develop normally and the cell divisions are arrested at later stages. This result indicates an essential role of MPK3/6 in ovule development (Wang et al., 2008a). Moreover, research also showed MPK3/MPK6 mutants have defects in anther lobe formation and anther cell differentiation. It is indicated that MPK3/MPK6 might function with ERECTA family proteins to regulate anther cell division and differentiation (Bush and Krysan, 2007). 6  1.3 The biological functions of MAPK phosphatases  Regarding the multiple functions of MAPK signaling, proper regulatory mechanisms must be applied to these delicate cascades to achieve the optimal biological activities. The specificity of phosphorylation reactions is usually controlled by enzyme/substrate interaction and/or the mediation of scaffold proteins. The magnitude and duration of MAPK signal, on the other hand, is usually controlled by various activators and inactivators. Therefore, the negative regulators of MPKs also play essential roles in balancing the signaling cascades.  Previous research in mammalian systems first identified a group of dual specificity phosphatases as the negative regulators of MPKs. These phosphatases are named MAPK phosphatases (MKPs) and they are evolutionarily related to protein tyrosine phosphatases. Their common structural feature is a C-terminal catalytic domain containing a highly conserved signature motif HCXXXXXR and two Cdc25-like domains (Keyse and Ginsburg, 1993). Unlike tyrosine specific phosphatases, MKPs can dephosphorylate both Tyr and Ser/Thr residues. Consequently, they inactivate MPKs through dephosphorylating threonine and/or tyrosine residues within the – TXY- motif located in the activation loop of certain targets (Theodosiou and Ashworth, 2002).  1.3.1 MKPs in yeast and humans  In mammals, thirteen members of MKP family (Theodosiou and Ashworth, 2002; Farooq and Zhou, 2004) have been characterized. They have specified substrates and locate in different cellular compartments, suggesting that they are involved in sophisticated regulatory networks to regulate MPKs. Based on their substrate preference, these phosphatases can be divided into four different groups (Zhang and Dong, 2007).  Group one phosphatases consist of MKPs that can dephosphorylate ERKs, including VHR, MKP2, MKP3, MKP4, and MKP6. Studies have showed that MKP4 is involved in early development of embryo and the MKP4 deficient mouse is embryonic lethal due to a failure of labyrinth development (Christie et al., 2005). 7  Group two phosphatases have preference for JNKs and contain four members, VH5, Pac-1, MKP5 and MKP7. Several members in this group are related to innate and adaptive immune response. For instance, MKP5 is found to be an important negative regulator of innate inflammatory cytokine production (Tanoue et al., 1999; Zhang et al., 2004). Pac-1 has a possible role in regulating macrophage function (Jeffrey et al., 2006). Group three phosphatases include MKP1 and DSP2, both of which are located in nucleus and have preference for p38 MAPKs. MKP1 is involved in inflammatory and metabolic processes since MKP1-deficient mice have increased disease incidence and metabolic syndrome (Abraham and Clark, 2006). Group four contains two MKPs, VH3 and PYST2, whose substrates and functions remain unknown.  Two MKPs, Msg5 and Sdp1, have been identified in yeast. Msg5 promotes adaptation to the pheromone response by dephosphorylating the Fus3 (Andersson et al., 2004). Furthermore, another MAPK, Slt2, can phosphorylate Msg5 after the activation of the cell integrity pathway, indicating Slt2 controls the action of Msg5 via the modulation of protein-protein interactions (Andersson et al., 2004). The other MKP, Sdp1, have a very high sequence similarity to that of Msg5. It has been shown to target Slt2, regulating the phosphorylation level of this MAPK in response to heat shock. (Hahn and Thiele, 2002)  1.3.2 MKPs in Arabidopsis  In Arabidopsis, the MPK family has 20 members. While the functions of many of them remain obscure, the information obtained thus far suggests that each MPK family member is likely to be preferentially involved in specific sets of physiological functions.  In contrast to the MPKs, the Arabidopsis genome encodes only five  putative MKPs, as based on the conserved domain structure defined by the features of the mammalian MKPs (Figure. 1.1). In mammalian cells, MKPs operate as key modulators of MAPK signal transduction networks and thus help determine the outcomes of MAPK signaling in development, metabolic homeostasis and stress response (Dickinson and Keyse, 2006). Similarly, in Arabidopsis there are also evidence that MKPs play important roles in various processes.  8  AtMKP1 is shown to be involved in genotoxic resistance (Ulm et al., 2002). The mkp1 mutants are indistinguishable from wild-type under standard conditions but they are  hypersensitive  to  genotoxic  stress  treatments  (UV-C  and  methyl  methanesulphonate). In-gel MBP-kinase activity assays after both genotoxic treatments show apparent deregulation of the MAP kinase activity levels in the mkp1 mutant and the AtMKP1 over-expressing line in comparison to the wild type. These data imply that AtMKP1 can regulate MAP kinase activity in vivo, which in turn regulates the outcome of the cellular reaction and the level of genotoxic resistance. It is also reported that AtMKP1 is a CaM binding protein and the phosphatase activity of AtMKP1 can be regulated by CaM in a Ca(2+)-dependent manner (Lee et al., 2008b).  PHS1 is involved in the organization of cortical microtubules (Naoi and Hashimoto, 2004). phs1-1 mutant, in which a conserved Arg residue in the noncatalytic Nterminal region of PHS1 is exchanged with Cys, exhibits phenotypes indicative of compromised cortical microtubule functions. It is hypothesized that the corresponding region is important for MAPKs interaction, and analogous Arg substitutions severely inhibit the kinase-phosphatase association. Another mutant, phs1-3, has about 50% knock down expression level of PHS1. This mutant shows hypersensitivity to abscisic acid (ABA), which indicate a negative role of PHS1 in ABA signaling (Quettier et al., 2006).  IBR5 is shown to modulate auxin and abscisic acid responsiveness (Monroe-Augustus et al., 2003). Deficiency in IBR5 results in decreased plant height, defective vascular development, increased leaf serration, fewer lateral roots, and resistance to the auxin and abscisic acid. It has been identified that MPK12 and IBR5 are physically coupled and MPK12 is a physiological substrate of IBR5 (Lee et al., 2008a).  Not much is known about the biological function of AtDsPTP1. It has been reported that this protein can physically interact with CaM family proteins and this interaction can modulate its phosphatase activity (Gupta et al., 1998; Yoo et al., 2004).  9  1.3.3 Previous research on AtMKP2  Research in the Ellis lab revealed that another member of AtMKP family, AtMPK2 (At3g06110), can positively regulate oxidative stress tolerance and inactivate AtMPK3/6 in vitro (Lee and Ellis, 2007). Interestingly, Y2H screening of the 20 AtMPKs using AtMKP2 as bait showed strong interaction between AtMKP2 and AtMPK8 or AtMPK15, but not with MPK3 or MPK6 (Lee and Ellis, unpublished). Moreover, AtMKP2 RNAi mutants exhibit pleiotropic phenotypes, implying that AtMKP2 also has a crucial role in regulating developmental processes (Lee, Cheng and Ellis, unpublished).  Figure 1. 1 Phylogenetic tree of Arabidopsis MKPs The conserved catalytic domain of MKPs were aligned with the ClustalW method (http://www.ebi.ac.uk/clustalw/#) The graph was modified from Naoi and Hashimoto, 2004.  1.4 Trichome development in Arabidopsis  In Arabidopsis, trichome cells are large single cells with long stalks that originate from the epidermis. The cell can grow up to 500μm tall with 2-4 branches so the  10  phenotype can be easily scored. Trichome cells make a great model to study different developmental and cellular events in plants including cell differentiation, cell fate determination, cell shape control and cell polarity.  1.4.1 The stages of trichome development  During development, trichome cells go through a series of morphogenetic changes. Generally the development period can be divided into six different stages according to cell size and polarity (Schnittger and Hulskamp, 2002). During stage one and stage two, protodermal cells that are committed to trichome cell fate switch from mitotic cycles to endoreduplication cycles. After nuclear enlargement, incipient cells expand within the plane of the epidermis. After that, the growth orientation changes and the cells grow out of the leaf surface. In stage three and four, one or two successive branching events take place after the cells have finished two to three endoreduplication cycles. The first branching direction is parallel to the basal–distal leaf axis. The second branching event is perpendicular with respect to the first one. At stage five, the nucleus of trichome cells migrate to the base of the second branching point and the cells start to expand until they reach a final height of around 400-500μm. During the last stage, the trichome cells start to mature. The cell wall thickens and papillae appear on the surface of trichome. The average DNA content of mature Arabidopsis trichome cell is 32C.  1.4.2 The genetic regulatory network that controls Arabidopsis trichome development  Previous molecular and genetic analyses have established a regulatory network of both positive and negative players during trichome growth. GL2 gene, which encodes a homeodomain leucine-zipper protein, is thought to be crucial to translate the cues that determine epidermal cell fates including trichomes, root hairs and the seed coat (Koornneef et al., 1982; Rerie et al., 1994; Di Cristina et al., 1996). The expression of GL2 in trichome cells, sequentially, is tightly regulated by four positive regulators, GL1, TTG1, GL3 and EGL3 (Szymanski et al., 1998). GL1 encodes a MYB-related transcription factor and the gl1 mutants are glabrous (Koornneef et al., 1982; Oppenheimer et al., 1991). TTG1 encodes a WD40 protein with unknown function, and the ttg1 mutants are also glabrous (Walker et al., 1999). GL3 encodes a bHLH 11  transcription factor (Payne et al., 2000); EGL3 is a close homologue of GL3 (Zhang et al., 2003). The knockout of both genes produces glabrous plants. Based on genetic analysis and yeast two-hybrid data, it is believed that GL3 can bind to GL1 and TTG1 by different domains and this transcriptional-activation complex up-regulates the expression of GL2 (Payne et al., 2000; Esch et al., 2003; Zhang et al., 2003).  For the negative regulation of GL2 expression, there are a group of single-repeat MYB proteins. So far TRY, CPC, ETC1, ETC2, ETC3 and TCL1 have been demonstrated to have redundant function in different part of Arabidopsis (Wada et al., 1997; Schellmann et al., 2002; Kirik et al., 2004a; Kirik et al., 2004b; Wang et al., 2007b; Wang et al., 2008b). TRY is thought to be the major negative player in trichome fate determination as well as trichome cell patterning. The try mutant has small trichome clusters and the cluster phenotype becomes more severe when try is crossed to other mutants in this family (Schellmann et al., 2002; Kirik et al., 2004a). CPC mainly functions in root hair development but also plays a role in trichome development (Wada et al., 1997).  Three-hybrid analysis showed that TRY can  counteract the interaction between GL1 and GL3, which will affect the proper formation of transcriptional-activation complex (Esch et al., 2003).  1.4.3 The cellular events associated with trichome development  As a giant cell which undergoes several rounds of morphological and cell polarity changes, the trichome cell performs proper subcellular events to conduct this process (Hulskamp et al., 1994). The failing of any event will affect trichome cell development and cause morphological defects.  Endoreduplication is the duplication of DNA without mitosis and cytokinesis, which produces cells with a greater DNA content than 2C. In mature Arabidopsis trichome cells, the average nuclear DNA level is around 32C (Walker et al., 2000). Many genes involved in trichome development are reported to affect the endoreduplication level of trichome cells. The defect in endoreduplication might cause reduced trichome branching, while the increase in the endoreduplication level results in trichomes with more branches. For instance, mutation of GL3 reduces endoreduplication, trichome  12  branching and trichome cell size (Hulskamp et al., 1994). Mutation of TRY increases endoreduplication, trichome branching and trichome cell size (Hulskamp et al., 1994). Another factor involved in trichome development is the cytoskeleton structures. Both microtubule and actin cytoskeleton network are known to affect trichome branching and trichome cell morphology. Previous research shows that microtubule reorientation is important for trichome branching (Mathur and Chua, 2000). It is shown that microtubules reorient with respect to the longitudinal growth axis during branching event. Microtubule stabilizing drugs induced microtubule stabilization leads to new branch formation on unbranched trichome mutants. Actin cytoskeleton, on the other hand, is known to coordinate trichome expansion after branching (Mathur et al., 1999). Actin cytoskeletons form bundles parallel with the growth axis while trichome expansion. Actin disruption causes distort trichome mutants (Schwab et al., 2003).  1.5 Experiment materials to study the function of AtMKP2  The large number of MPKs (20) and small number of MKPs (5) indicate that MKPs in Arabidopsis might have multiple substrates and might serve non-redundant roles in different biological processes. this might explain why there are no available AtMKP2 knockout lines in public seed stocks (ABRC and GABI). To study the function of MKPs in a transgenic plant context, Lee built multiple dexamethasone (DEX) inducible loss of function (LOF) and gain of function (GOF) lines (Lee and Ellis, 2007).  The DEX inducible system comprises two parts, a chimeric transcription factor, GVG, and the transgene of interest linked after GAL4 promoter sequence that GVG binds to. GVG combines the DNA-binding domain of the yeast transcription factor GAL4, the transactivating domain of the herpes viral protein VP16, and the hormone binding domain of rat glucocorticoid receptor (Aoyama and Chua, 1997). The glucocorticoid receptor sequence will mediate nuclear translocation of the transcription factor upon binding to a glucocorticoid. After treating transgenic plants with synthetic glucocorticoid DEX, the DEX-GVG complex will be transported into the nucleus and activate transcription of the transgene. This system is widely used to study protein function in plants (Kim et al., 2003; Soyano et al., 2003; Lee and Ellis, 2007). 13  1. 6 Project objectives  The small number of MKPs in Arabidopsis suggests that these phosphatases might act as a point of convergence within the plant signaling networks. Although some interesting research has demonstrated that different MKPs are involved in plant hormone signaling and stress responses, however there is still much that remains unknown about the physiological functions of MKPs. Previous research in our laboratory showed that AtMKP2 can positively regulate oxidative stress tolerance and it has several potential MPK substrates (Lee and Ellis, 2007). Moreover, AtMKP2 RNAi mutants exhibit pleiotropic phenotypes, indicating that AtMKP2 is also crucial in plant development. Among other developmental impacts of MKP2 silencing, we specifically observed that AtMKP2 RNAi mutants showed a trichome developmental defect. My research goal was to study the role of AtMKP2 in plant development, as well as to explore the wider range of biological functions of AtMKP2 by using global analysis approaches.  14  2. AtMKP2 is involved in trichome development 2.1 Introduction  Since there are 5 AtMKPs, compared to 20 AtMPKs, in Arabidopsis, it is legitimate to assume that AtMKPs play multiple roles in plant growth. AtMKP2 has been shown to be a novel regulator for ozone stress response (Lee and Ellis, 2007), but how it is involved in other biological aspects of Arabidopsis development and growth remains unknown. There are no available T-DNA insertion knockout lines of AtMKP2 in the public stock collections. The loss of function (LOF) and gain of function (GOF) mutants I used for all phenotype characterization are DEX-inducible mutant lines generated previously by Jin Suk Lee. For characterization of the developmental deficiencies of AtMKP2 RNAi plants, I used at least three independent RNAi lines in which such phenotypic deficiencies have been observed (Figure. 2.1).  Figure 2. 1 AtMKP2 RNAi lines grown on 10μM DEX plates A. Five-day-old plants grown on 10 µM DEX plates. B. Two-week-old plants, scale bar shown is 5mm. 1. Empty vector line EVL1, 2. MKP2RNAi line P2iL22, 3. MKP2-RNAi line P2iL33, 4. MKP2-RNAi line P2iL21, 5. MKP2RNAi line P2iL26  Trichomes are highly differentiated epidermal cells found on the surfaces of leaves and stems, where they are thought to serve as a barrier against herbivores. In Arabidopsis, trichomes usually have two to four branches and their distribution is  15  non-random because trichome clusters are rarely observed on the leaf surface (Larkin et al., 1996). Trichome initiation and development in Arabidopsis are processes that have provided a useful model to address questions concerning cell fate specification, pattern formation and cellular differentiation.  In AtMKP2 RNAi mutants, trichome density as well as trichome branching number were affected. Both proAtMKP2:GUS signal and proAtMKP2:YFP signal showed that AtMKP2 was expressed in all development stages of developing trichomes, consistent with the idea that AtMKP2 plays a role in trichome development. RT-PCR showed that the expression levels of several important trichome development regulator genes were affected in AtMKP2-RNAi plants. Genetic analysis of AtMKP2RNAi x try and AtMKP2 RNAi x cpc double mutants further demonstrated the involvement of AtMKP2 in trichome development. To identify the downstream targets of AtMKP2 for trichome development, I obtained all available mutant and/or over-expression lines for each of the MPKs thought to interact with MKP2, and analyzed their trichome phenotype. However, none of these showed significant trichome defects. This might reflect the level of redundancy within the MPKs. Another possibility is that AtMKP2 has novel unknown substrates that function in this pathway. More research in the future would be needed to better understand this developmental pathway.  2.2 Material and methods  2.2.1 Plant materials  For seedlings used for phenotypic and RT-PCR analyses, seeds were surfacesterilized by 20% commercial bleach and grown on Murashige and Skoog Basal Salts with minimal organics (Plant cell culture tested, Sigma) and 1% (w/v) sucrose, solidified with 0.7% (w/v) agar (Sigma). Seeds were stratified at 4 °C dark condition for 24 hours before incubation at 22 °C under 16-h photo-period constant white light for seed germination and seedling growth. Wild-type Arabidopsis (ecotype "Columbia-0") and four AtMKP2 RNAi lines were used in trichome phenotype analysis. For phenotype observation and DAPI staining, plants were sterilized and 16  either sown on MS medium with 10µM DEX for 10 days or on MS medium without DEX for 2 days and then treated with 10 µM DEX every 3 days until 10 days. Plants under both treatments showed same trends. For RT-PCR, plants were grown on MS medium for 10 days, treated by 10 µM for 24 hours. Aerial parts were frozen in liquid nitrogen and then store in -80oC. For double mutant analysis, wild-type Arabidopsis (ecotype "WS") was used as control for AtMKP2 x cpc, wild-type Arabidopsis (ecotype "Columbia-0") was used as control for AtMKP2 x try. For GUS staining, plants were sterilized and sown on MS medium for 8 days.  2.2.2 Evaluation of trichome and root-hair number  Adaxial surface of first and second leaves were used for scoring trichome number. Leaves were mounted on glass slides and observed under a dissecting microscope. More than 10 plants for each line were taken for analysis. Root hair density was determined from at least 10 one-week-old seedlings from each line, as previously described (Lee and Schiefelbein, 2002). An epidermal cell was scored as a root-hair cell if any protrusion was visible. For scanning electron microscopy, fresh plant tissues were mounted on a stub and scanned by Hitachi S-2600N scanning electron microscope.  2.2.3 Construction of double mutants  The try and cpc mutants were provided by Chen Lab in Dept. of Botany, UBC. The try mutant, try_29760, is in the Columbia-0 (Col-0) ecotype background (Esch et al., 2003). The cpc mutant is in the WS ecotype background (Wada et al., 1997). The TUB6-GFP line (Ueda et al., 2003) was provided by the Wasteneys Lab and the ABD2-GFP line (Sheahan et al., 2004) was provided by the Sack Lab. Two AtMKP2 RNAi lines, line 22 and line 26 were each crossed with try, cpc, TUB6-GFP and ABD2-GFP. In F1 progeny, seedlings showed try or cpc phenotype were selected on 50 µg/ml hygromycin plates. The hygromycin resistant lines were then transferred to soil to get F2 seeds. The F2 progeny were selected on 50 µg/ml hygromycin plate to get homozygous double mutant lines. These lines were then used for RT-PCR analysis and phenotype observation.  17  2.2.4 GUS staining analysis  The AtMKP2 promoter: GUS construct was generated using a DNA fragment corresponding to the region 410 bp upstream of AtMKP2’s ATG start codon, and this construct was transformed into Arabidopsis by J.S. Lee in the Ellis Lab. Homozygous T3 lines were then analyzed using the histochemical GUS assay. The histochemical staining and fixation method for GUS activity was performed as described (Jefferson et al., 1987; Malamy and Benfey, 1997). Transgenic lines were grown on ½ MS plates and 8-day-old plants with the first pair of true leaves forming were collected. Plants were treated with heptane for 5 minutes and then air-dried for 5minutes. Then leaf tissues were incubated in GUS staining solution (0.5 mg/ml X-gluc, 0.1% Triton X100, 0.25 mM K4Fe(CN)6·3H2O, 0.25 mM K3Fe(CN)6, 50 mM sodium phosphate buffer, pH 7.0) for 4 hours at 37 °C. After removing staining solution, tissue samples were incubated for 15 minutes at 57 °C in 20% methanol containing 0.24 N HCl, replaced by 60% ethanol containing 7% (w/v) NaOH for 15 minutes at room temperature. Afterwards, tissues were incubated in series rehydration solution for 20 minutes each in 40%, 20%, and 10% (v/v)  ethanol at room temperature. After  rehydration, tissues were stored in 5% v/v ethanol, 20% v/v glycerol solution. GUS activity was observed under a light microscope equipped with a digital camera.  2.2.5 AtMKP2-YFP fusion protein localization analysis  The AtMKP2 promoter:AtMKP2-YFP fusion protein construct was generated and transformed into Arabidopsis by Lee in Ellis Lab. Constructs expressing AtMKP2YFP fusion protein were prepared using the GatewayTM system (Invitrogen). To generate the ProAtMKP2:AtMKP2:YFP construct, a 1.5-kb AtMKP2 genomic fragment upstream of the open reading frame was amplified, and the amplified fragment was introduced into the vector pGWB40 (Research Institute of Molecular Genetics). Transgenic Arabidopsis seedlings expressing ProAtMKP2:AtMKP2:YFP fusion construct were grown in MS medium for 8 days and fresh leaf tissues were used for confocal microscopy analysis.  18  2.2.6 Nuclear DNA content measurement  First and second true leaves of 2-week-old plants were used. Trichomes were isolated from these leaves to reduce the impact of background fluorescence; the trichome isolation method was modified from Zhang (Zhang and Oppenheimer, 2004). Leaf tissues were incubated in PBST (phosphate-buffered saline containing 0.05% [v/v] Triton X-100, pH 7.2) supplemented with 100mM EGTA at room temperature for 24 hours. Trichomes were removed by gentle rubbing using a paintbrush, after which all the liquid in the Petri dish was transferred by pipetting to centrifuge tubes and centrifuged gently at 2000rpm for 2 minutes. The supernatant was removed and the trichome pellet was washed another 2 times by PBST, after which the washed trichomes were resuspended in the desired volume of PBST. For 4’, 6-diamidino-2phenylindole (DAPI) staining, the washed trichomes were stained with 10µg/ml DAPI for 10 min, washed three times for 10 minutes with PBST and mounted in water under glass coverslips for microscopy. All pictures were taken at the same magnification and images were processed for signal strength by using ImageJ software. Nuclear contents were calculated based on the fluorescence signal from nuclear area, after subtraction of the background value.  2.2.7 RNA Isolation and RT-PCR Analysis  Ten-day-old plants grown on ½ MS plates were treated with 10μM DEX for 24hrs and then the aerial parts were harvested. Total RNA samples were prepared using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration of RNA was determined by NanoDropTM 1000 Spectrophotometer. Reverse transcription was performed using a First-strand cDNA Synthesis Kit (Amersham Biosciences), and same volume of resulting RT reaction products from selected samples were used as template for RT-PCR analysis. The primers used for RT-PCR are presented in Table 2.1.  Primer Name  Primer Sequence  ACT8_F  5'-ATTAAGGTCGTGGCA-3'  ACT8_R  5'-TCCGAGTTTGAAGAGGCTAC-3'  MKP2_F  5'-CGCGGATCCGCGATGGAGAAAGTGGTTGATCTCTTCG-3'  19  Primer Name  Primer Sequence  MKP2_R  5'-CCGGAATTCCGGAAGCAATCATGCATTACCTTGGATG-3'  MPK3_F  5'-CCAAGAAGCCATAGCACTCA-3'  MPK3_R  5'-AGCCATTCGGATGGTTATTG-3'  MPK6_F  5'-ACCACCACCAACCTCAAAAG-3'  MPK6_R  5'-CCTCCAGGAGCTTCTGTCAT-3'  MPK8_F  5'-TAATAATAATAATCACGAACAACCCATTTTCAATTC-3'  MPK8_R  5'-GAAATATGGATCAGCTAGTGCATCTTCA-3'  MPK15_F  5'-ATTATTATCAGCCTCTAAATCAATGGGTGGTG-3'  MPK15_R  5'-GTAGGGGCATCATTAAAAGATACACGAGCT-3'  GL1_F  5'-GCCACACCTTCTTCTTGTCA-3'  GL1_R  5'-ATCGTCGTCATGAACCCATA-3'  GL2_F  5'-AGATGAGCAGCGAGAACTCA-3'  GL2_R  5'-TCTGATCTGATCGGTGGTGT-3'  GL3_F  5'-CAACAGATTCTAGGCGACGA-3'  GL3_R  5'-CCTCCAGTGATTCTTTCGGT-3'  EGL3_F  5'-CAACCAGGAGTGTTGGAGTG-3'  EGL3_R  5'-CTACCGGAAGCTGAGGATTC-3'  TTG_F  5'-TCTCTCCTTCGAGCATCCTT-3'  TTG_R  5'-GCTGTTGTTGAGAACCGAGA-3'  TRY_F  5'-CCTCTTCTTCTTCTTGTTCGC-3'  TRY_R  5'-AGAGTCATGGAGGGCGATT-3'  CPC_F  5'-TGGGAAGCTGTGAAGATGTC-3'  CPC_R  5'-AGTCTCTTCGTCTGTTGGCA-3'  ETC1_F  5'-GTGAGCAGTCTTGAGTGGGA-3'  ETC1_R  5'-GTTGGCCATCAACGTAATTG-3'  ETC2_F  5'-GTGAGTAGCATCGAATGGGA-3'  ETC2_R  5'-AAGACGTCGTCGTTTGTGAG-3'  TCL1_F  5'-GTGAGTAGCATCGAATGGGA-3'  TCL1_R  5'-AAGACGTCGTCGTTTGTGAG-3'  AN_F  5'-TGAGACGGTGCCGTGGTATGG-3'  AN_R  5'-GTTGCCTACTGGTGGATTCC-3'  ZWI_F  5'-TTTCGATGCCGAGTCGTCTTCTC-3'  ZWI_R  5'-CTATATATTCCTCATTTCCGGGATCAGAT-3'  STI_F  5'-ATGTCAGGTTCGAGAGTTTCGG-3'  STI_R  5'-CTACTTCCGGTTCTTCTCAAAGTA T-3'  Table 2. 1 RT primers for trichome development related genes  20  2.3 Results  2.3.1 AtMKP2 is involved in trichome initiation and branching  T-DNA insertion lines for AtMKP2 were not available in the public seed stocks; therefore, we used multiple RNAi lines in this study. To assess the function of AtMKP2 in trichome development, we used four AtMKP2-RNAi lines with different repression levels to check trichome growth condition on the first and second true leaves of 10-day-old seedlings. All RNAi lines showed significantly lower numbers of trichomes compared to wild-type, and the most severely affected line showed a 70% reduction (Figure 2.2). This result indicates that AtMKP2 positively regulates trichome initiation.  Since it has been reported that several transcription factors (such as GL1, GL2, GL3 and CPC) could be recruited both during trichome initiation and root hair initiation (Masucci et al., 1996; Wada et al., 1997), I hypothesized that AtMKP2 might also play a role in root hair initiation; however, we did not observe a significant change in root hair density in AtMKP2 LOF lines (Figure. 2.3) which, together with the trichome data, indicates that AtMKP2 plays a specific role in trichome regulation.  Figure 2. 2 Trichome number on first and second true leaves of Arabidopsis Four different RNAi lines were used for phenotypic analysis. Plants were grown on ½ MS media with 10µM DEX for 10 days and the first and second leaves were scored. To reduce the variation caused by leaf size, mutant leaves that had similar size as control were scored.  21  Figure 2. 3 Root hair density of primary root Root hair density was determined from at least 10 one-week-old seedlings from each line, as previously described (Lee and Schiefelbein, 2002). An epidermal cell was scored as a roothair cell if any protrusion was visible.  Trichome branching is another crucial feature of trichome development. The branching number and the morphology of trichome stalks are both tightly regulated phenotypic traits controlled by a series of genes related to nuclear endoreduplication, Golgi-related transportation, and microtubule and actin cytoskeleton organization (Mathur and Chua, 2000; Walker et al., 2000; Schwab et al., 2003; Ishida et al., 2008). Trichomes on wild-type Arabidopsis leaves usually have three branches, although two- to four-branched trichomes can also sometimes be observed. As observed by scanning electron microscopy, no obvious alterations in trichome stalk morphology were observed when comparing wild-type and AtMKP2 RNAi lines (Figure 2.4); however, I found obvious defects in trichome branching in AtMKP2 RNAi lines. Compared with the wild-type, RNAi lines had an increased percentage of twobranched trichomes (Figure. 2.4). This result suggests that AtMKP2 also positively regulates trichome branching.  22  Figure 2. 4 Trichome branching defect of AtMKP2 RNAi mutants Four different RNAi lines were used for phenotypic analysis. Plants were grown on ½ MS media with 10µM DEX for 10 days and the first and second leaves were scored. For scanning electron microscopy, fresh plant tissues were mounted on a stub and scanned by Hitachi S2600N scanning electron microscope. Scale bar shown is 50µm.  2.3.2 Gene expression pattern of AtMKP2 in developing trichomes  Since AtMKP2 is involved in trichome development, I tested whether the expression pattern of AtMKP2 was associated in some way with trichomes. To address this, I first used transgenic lines expressing beta-glucuronidases (GUS) driven by the AtMKP2 promoter. The GUS staining pattern showed that the AtMKP2 promoter was ubiquitously activated in leaf tissues, although at a low level. However, an enhanced GUS signal was detected in developing and mature trichomes (Figure 2.5A-C). To study  the  subcellular  localization  of  AtMKP2  protein,  I  examined  proAtMKP2:AtMKP2-YFP lines. Similar expression pattern were observed by  23  confocal microscopy (Figure 2.5D-G). These results confirm that AtMKP2 is expressed through all stages of trichome development.  Figure 2. 5 Expression pattern of AtMKP2 in developing trichomes Transgenic  Arabidopsis  seedlings  expressing  ProAtMKP2:  GUS  and  ProAtMKP2:AtMKP2:YFP fusion construct were grown in MS medium for 8 days and fresh leaf tissues were used for analysis. Figure A, B, C show proAtMKP2: GUS signal in trichomes of different development stages. The arrowheads are pointing the trichomes expressing GUS signal. Figure D, E, F show proAtMKP2:AtMKP2-YFP signal in different development stages of trichomes. The arrows are pointing the trichome cells expressing YFP signal. Figure G is WT YFP signal under same condition; the yellow signal of cell wall is auto-fluorescence. Scare bar shown is 10µm.  24  2.3.3 Microtubule and actin cytoskeleton structure remain unaltered in AtMKP2 LOF mutants  Trichome cells have large cell bodies and a distinctive cell shape. To maintain the normal developmental path and cell shape of trichome cells, it is important to have a normal microtubule and actin cytoskeleton structure. Previous studies showed that the microtubule organization center (MTOC) plays an important role in controlling trichome branching. The disruption of microtubule distribution around the branch position of trichomes resulted in trichomes forming abnormal numbers of branches (Mathur and Chua, 2000). To visualize microtubule structure in vivo, I crossed Greenfluorescent protein–α-tubulin 6 fusion protein (TUB6-GFP)-expressing lines (Ueda et al., 2003) with the AtMKP2 RNAi mutant lines. No obvious difference in microtubule distribution was observed in the resulting progeny in comparison to control (Figure 2.6), suggesting that AtMKP2 does not regulate trichome branching by controlling microtubule cytoskeleton structure.  Figure 2. 6 Microtubule structure in AtMKP2 RNAi mutants (A) And (B) show trichomes from GFP:TUB6 plants. The arrowheads are pointing at the microtubule bundle structure in trichome cells.  25  (C) and (D) show trichomes from AtMKP2 RNAi X GFP:TUB6-expressing plant. The arrowheads are pointing at the microtubule bundle structure in trichome cells. The scale bar is 10µm.  The actin-based cytoskeleton is also important for trichomes to maintain their branch shape and reach final maturation. Mutants with actin cytoskeleton defects usually exhibit curving and bulging trichomes (Schwab et al., 2003). To examine the actin layout in AtMKP2 RNAi mutants, I crossed AtMKP2 RNAi lines with ABD2-GFP lines. ABD2-GFP is a fusion protein between green fluorescent protein (GFP) and the second actin-binding domain (fABD2) of Arabidopsis fimbrin, AtFIM1 (Sheahan et al., 2004). When the progeny of these crosses were examined, I could detect no obvious differences in actin distribution between control and mutant lines (Figure 2.7). This result is consistent with the morphological features of AtMKP2 RNAi mutant trichomes, since the branches and cell bodies do not show any distorted or twisted shape.  Figure 2. 7 Actin cytoskeleton structure in AtMKP2 RNAi mutants  26  (A)and(B) show trichomes from GFP:ABD2 plants. The arrowheads are pointing at the actin structure in trichome cells. (C)and(D) show trichomes from RNAi X GFP:ABD2-expressing plants. The arrowheads are pointing at the actin structure in trichome cells. The scale bar shown is 10µm.  2.3.4 The expression patterns of trichome development-related genes is changed in AtMKP2 LOF mutants  In Arabidopsis, trichome development is regulated by series of positive and negative regulators. Some of the positive regulators, such as GL1, GL3, EGL1, TTG1 and negative regulators, such as TRY, CPC, ETC1, ETC2, TCL1, have been demonstrated to work together in controlling GL2 expression and, in turn, affecting trichome development (Wada et al., 1997; Szymanski et al., 1998; Schellmann et al., 2002; Kirik et al., 2004b). There are also genes such as AN, STI and ZWI, which are not closely related with the transcription factor network, but are still functioning in trichome branching (Oppenheimer et al., 1997; Folkers et al., 2002; Ilgenfritz et al., 2003). To investigate whether AtMKP2 regulates trichome development via a specific group of regulators, I employed RT-PCR to check the expression level of these trichome development-related genes in AtMKP2 RNAi background. I found that the major positive-regulators GL1, EGL1 and GL2 were down-regulated when MKP2 was suppressed, whereas the critical negative-regulator TRY was up-regulated (Table 2.2). In contrast to the behavior of genes belonging to the regulatory complex of GL1 and TRY, expression of genes thought to be specifically involved in trichome branching regulation, such as AN, STI and ZWI, were unaltered in the AtMKP2 RNAi background. This suggests that AtMKP2 may regulate trichome development via a regulatory complex containing TRY and GL1. There are also a slight up-regulation of TTG1, and down-regulation of ETC1, ETC2 and TCL1 observed in these experiments. ETC1 and ETC2 are functionally redundant with TRY and CPC, although the double mutants of etc1/etc2 show no trichome phenotype, indicating they are not as crucial to this process as TRY or CPC (Kirik et al., 2004b). The tcl1 mutants develop ectopic trichomes on their inflorescence stem but have normal leaf trichome numbers (Wang et al., 2007b). Therefore, the reduced expression of ETC1, ETC2 and TCL1 would not be predicted to affect the trichome number on the first and second true leaves. Overall,  27  the pattern of expression of trichome regulatory genes in AtMKP2 RNAi lines showed a reduced expression of positive regulators and an increased expression of negative regulators.  Table 2. 2 Summary of gene expression of trichome developmental regulators in AtMKP2 RNAi mutants ↓,↑, and – indicate increase, decrease and no change, respectively. Ten day old wild-type and AtMKP2 RNAi line 26 were used.  2.3.5 The endoreduplication level of AtMKP2 trichome nuclei is decreased  TRY is a canonical negative regulator in trichome development. It has been reported that the try mutant has an increased endoreduplication level (Hulskamp et al., 1994). To further support our RT-PCR findings, and to test the hypothesis that AtMKP2 functions in the same regulatory pathway with TRY, I examined the nuclear DNA content in trichomes from the AtMKP2 LOF lines. A decrease in endoreduplication would be consistent with the model that AtMKP2 functions together with TRY in trichome development. As shown in Figure 2.8, trichomes from AtMKP2 RNAi lines showed 21%~31% lower nuclei DNA content compared to WT trichomes (Figure 2.8).  28  Figure 2. 8 Endoreduplication level of AtMKP2 RNAi mutants First and second true leaves of 2-week-old plants were used. Trichomes were isolated from these leaves to reduce the impact of background fluorescence. The isolated trichomes were stained with DAPI and mounted in water under glass coverslips for microscopy. All pictures were taken at the same magnification and images were processed for signal strength by using ImageJ software. Nuclear contents were calculated based on the fluorescence signal from nuclear area, after subtraction of the background value.  2.3.6 Genetic interaction of AtMKP2 and TRY/CPC  Although the RT-PCR analysis of AtMKP2 RNAi lines showed that GL1, GL2, and EGL3 were down-regulated and TRY and TTG1 were up-regulated, genetic analysis is needed to confirm AtMKP2’s involvement in this network. Since the LOF mutants of positive regulators of trichome initiation are usually glabrous, it was not feasible to observe the phenotype of double mutants involving this class of regulators. However, both try and cpc have increased overall trichome numbers and clustered trichomes, which can be easily scored (Hulskamp et al., 1994; Wada et al., 1997). I therefore crossed AtMKP2-RNAi line 22 and line 26 into the try and cpc mutant backgrounds. The repression of AtMKP2 in the resulting double mutants was confirmed by RTPCR analysis (Figure 2.9).  29  Figure 2. 9 AtMKP2 expression level in double mutants Plants were grown on ½ MS for 10 days and then treated with 10µM DEX for 24 hours before harvesting. Same amount of cDNA from each sample was used for RT-PCR.  The AtMKP2 RNAi X cpc double mutants showed increased numbers of trichomes similar to the cpc single mutants (Figure 2.10). This indicates that, for the regulation of trichome number, CPC and AtMKP2 are likely in the same pathway and CPC likely acts downstream of AtMKP2. However, AtMKP2-RNAi X cpc double mutants still showed trichome branching defects, indicating that AtMKP2 also functions in a CPC-independent signaling pathway that controls trichome branching (Figure 2.11).  Figure 2. 10 Total trichome number on first and second true leaves of cpc and AtMKP2 RNAi x cpc double mutants  30  Figure 2. 11 Trichome branching on first and second true leaves of cpc and AtMKP2 RNAi x cpc double mutants  Two AtMKP2 RNAi lines were crossed with cpc mutants. Plants were grown on ½ MS media with 10µM DEX for 10 days and the first and second leaves were scored. To reduce the variation caused by leaf size, mutant leaves that had similar size as control were scored.  AtMKP2 RNAi X try double mutants showed increased trichome number, but that number is still significantly lower than that in WT (Figure 2.12). This means that, for the regulation of trichome number, TRY is not sufficient to rescue the reduced trichome number observed in AtMKP2-RNAi lines. AtMKP2 might therefore be controlling multiple other factors that act in parallel with TRY. Nevertheless, since the double mutant still partly rescued the reduced trichome number, AtMKP2 might be one of the upstream regulators of TRY. AtMKP2-RNAi X try double mutants showed more 3- and 4-branched trichomes compared to the AtMKP2 RNAi single mutant (Figure 2.13), which indicates that TRY is one of the major downstream targets of AtMKP2 in trichome branching event. Since TRY is also involved in trichome patterning, I examined the cluster frequency in try single mutants and AtMKP2 X try double mutants. The double mutants did not show any significant differences compared to try mutants, indicating that AtMKP2 does not affect trichome patterning (Figure 2.14).  31  Figure 2. 12 Total trichome number on first and second true leaves of try and AtMKP2 RNAi x try double mutants  Figure 2. 13 Trichome branching on first and second true leaves of try and AtMKP2 RNAi x try double mutants  Figure 2. 14 Trichome cluster percentage on first and second true leaves of try and AtMKP2 RNAi x try double mutants  32  Two AtMKP2 RNAi lines were crossed with try mutants. Plants were grown on ½ MS media with 10µM DEX for 10 days and the first and second leaves were scored. To reduce the variation caused by leaf size, mutant leaves that had similar size as control were scored.  2.3.7 Trichome phenotype in mutants of potential AtMKP2 substrates  The direct substrates for AtMKP2 are likely AtMPKs. Previous studies showed that AtMKP2 can dephosphorylate MPK3 and MPK6 (Lee and Ellis, 2007), but AtMKP2 also interacted with MPK8 and MPK15 in a yeast 2-hybrid assay (Figure 2.15) (Lee and Ellis, unpublished). I thus hypothesized that one or more of these MPKs could be downstream targets of AtMKP2 in trichome development. To test this idea, I examined the trichome phenotype of MPK3, MPK6, and MPK8 KO lines, MPK8/15 double RNAi lines, and MPK8 and MPK15 over-expression lines. However, I did not notice obvious trichome defects in any of these knockout and over-expression transgenic plants (Figure 2.16, 2.17). Furthermore, no significant change was observed for the gene expression level of any of the transcription factors which had shown altered expression in the AtMKP2 LOF lines (Figure 2.18). These results suggest that AtMKP2 could be working through other, non-MPK-downstream targets to control trichome development.  Figure 2. 15 AtMKP2 physically interact with MPK8 and MPK15 in yeast (Lee and Ellis, unpublished)  33  Figure 2. 16 Total trichome number on first and second true leaves  Figure 2. 17 Trichome branching on first and second true leaves  34  Figure 2. 18 Expression of trichome development related genes  2.4 Discussion  The RNAi mutants of AtMKP2 showed pleiotropic phenotypes. To confirm that the trichome phenotype was not a secondary effect of general cell growth, I checked the trichome phenotype in four different RNAi lines with different growth rates. In all these mutants, the trichome defect phenotype was consistent. Since AtMKP2 can dephosphorylate MPK3 and MKP6 (Lee and Ellis, 2007), and both kinases are involved in stomata patterning (Wang et al., 2007a), I also checked the growth of other cell types in the epidermis to confirm that the trichome was the only cell type that was affected by repression of AtMKP2. This analysis showed that the pavement cells and guard cells of AtMKP2-RNAi mutants appeared normal in comparison to the wild type (Figure 2.19), and the root hair initiation in AtMKP2-RNAi lines was not affected by knockdown of this gene. These results demonstrate that AtMKP2 is specifically involved in trichome development in the epidermis.  35  Figure 2. 19 Pavement cells and stomata on true leaves of WT and AtMKP2 LOF mutants, Scale bar shown is 20µm  Each step of trichome development and morphogenesis is tightly controlled by different genes, and genetic lesions affecting different steps cause distinct morphological defects. Cell fate determination can be regulated by several negative and positive regulators, most of which are transcription factors (Oppenheimer et al., 1991; Szymanski et al., 1998; Esch et al., 2003). The up-regulation of negative regulators and down-regulation of positive regulators can cause reduced trichome density and trichome branching number. The development of trichome cells is also regulated by other factors, including genes involved in endoreduplication, trichome branching, cell expansion and cell death (Schnittger and Hulskamp, 2002; Hulskamp, 2004). In order to identify which genes AtMKP2 might work with, I applied different techniques to check the trichome defects in AtMKP2 RNAi mutants. The branching defect indicated the involvement of cell growth regulators. Therefore, I checked the expression of branching-related genes, including AN, STI, ZWI, but these remained unchanged in AtMKP2 LOF mutants. In order to examine the cytoskeleton structure in AtMKP2 mutants, I checked the double mutants of AtMKP2 RNAi X TUB6-GFP and AtMKP2 RNAi X ABD2-GFP during various trichome developmental stages, and again, no obvious defects were observed. I then checked the expression of different players in the trichome development regulatory network. Analysis of their expression by RT-PCR showed that several important factors had altered expression levels. Furthermore, measurement of the nuclear DNA content, and double mutant analysis of AtMKP2 RNAi X try/cpc confirmed that AtMKP2 functions in the same signaling pathway with TRY and CPC.  36  Since AtMKP2 is a dual-specificity phosphatase which mainly interacts with MAPKs, the possibility for AtMKP2 to directly dephosphorylate any transcription factor would seem to be low. It seems more likely that there are other protein targets between AtMKP2 and the trichome development regulators. The known potential substrates of AtMKP2 are the most probable downstream targets that would establish such a link. AtMKP2 can dephosphorylate MPK3 and MPK6 in vitro and in vivo (Lee and Ellis, 2007), while in a yeast two-hybrid assay that screened MPK interactors with AtMKP2, both MPK8 and MPK15 showed strong interaction with this phosphatase (Lee and Ellis, not published). Therefore, I examined the trichome phenotype of MPK3, MPK6, MPK8 KO lines and MPK8/15 RNAi lines; I also checked the phenotype of overexpression lines of MPK8 and MPK15. However, none of them showed trichome development defects compared to wild type.  One explanation for this result is that MPKs in Arabidopsis may be functionally redundant. Previous research showed that MPK3 and MPK6 are functionally redundant in stomatal development (Wang et al., 2007a), so it might be helpful to examine the double mutant of MPK3 and MPK6 for its trichome phenotype. For MPK8 and MPK15 mutants, we obtained homozygous KO MPK8 plants but were unable to recover homozygous KO lines of MPK15. The MPK8/15 RNAi line has a limited knock-down level of both MPK8 and MPK15, but the repression of the genes may not be strong enough to induce any trichome defects. It would be useful to get a strong KO MPK8/15 line for future research. It is also possible that MKP2 has other MPK substrates that are not identified yet, since the screening of interactors in the yeast system may not represent the true physiological condition in plants.  It is also possible that these four MAP kinases are not crucial players in trichome development pathway. This is an indication that AtMKP2 might have other unknown substrates that do not belong to the MPK family. Examples in mammalian system have shown that some atypical DUSPs can function through non-MPK substrates. For example, DUSP3 was found to dephosphorylate STAT1 (signal transducer and activator of transcription) 1 and STAT5 (Najarro et al., 2001). DUSP12 was identified as a binding partner of glucokinase and had been shown to dephosphorylate glucokinase in vitro (Munoz-Alonso et al., 2000). Therefore, it will be necessary to look for other interactors of AtMKP2 using global approaches in the future. 37  Figure 2.20 shows the possible position of AtMKP2 in trichome development regulation network based on my experimental results. From the RT-PCR results, it seems that AtMKP2 is able to regulate the expression levels of several transcription factors. The double mutant analysis shows that AtMKP2 is upstream of both TRY and CPC in the established trichome development network. The regulation of gene expression levels should be performed by downstream targets of AtMKP2, which have not yet been identified. There are few reports describing the direct mediators that control the expression of these trichome development regulators. It also remains unknown whether these transcription factors can be phosphorylated and dephosphorylated; or if such post-transcriptional modification will affect their activities and binding affinities. More research would be needed to shed light on these questions.  Figure 2. 20 A possible model for the function of MKP2 in the regulation of trichome development  Another interesting topic to pursue in the future is whether AtMKP2 might be mediating the ROS levels in plant cells, which in turn could affect trichome development. Trichomes in Arabidopsis are known to contain a glutathione (GSH) pool, indicating that hey may play a physiological role in ROS scavenging processes (Gutierrez-Alcala et al., 2000). In some mutants which show an accumulation of ROS, the trichome numbers on leaves are also increased (Rius et al., 2008). Previous research has shown that AtMKP2 may be a positive regulator of oxidative stress responses, since AtMKP2-RNAi mutants are hypersensitive to ozone treatment. Since  38  ROS also act as important signals in plant development, it is possible that AtMKP2 works as a modulator between ROS signaling and trichome development signaling. As a preliminary exploration of this idea, I examined endogenous H2O2 levels in AtMKP2 RNAi mutants by using DAB staining (Love et al., 2005). Interestingly, the AtMKP2 RNAi mutants appeared to have a higher endogenous level of H2O2 (Figure 2.21), which suggests that AtMKP2 could normally act as a negative regulator of ROS accumulation. This would be consistent with the earlier observation that AtMKP2-RNAi plants accumulate more ROS when challenged with ozone, and display earlier symptoms of oxidation-induced cell death. However, more experiments will be needed to define any possible relationship between ROS signaling and trichome development.  Figure 2. 21 DAB staining of WT and AtMKP2 RNAi mutants A, D are WT, B, C, E are AtMKP2 RNAi lines Plants were grown on ½ MS media for 10 days and treated with 10µM DEX for 24 hours. The leaves were then stained with DAB to observe the H2O2 level in vivo.  39  3. Investigation of short-term transcriptional events in AtMKP2 LOF and GOF mutants  3.1 Introduction  AtMKP2-RNAi plants display severe developmental defects, especially at the seedling stage, whereas AtMKP2-over-expression does not appear to cause any morphological defects (Cheng and Lee, unpublished results); however, the molecular events underlying such phenotypic perturbations are unknown. Therefore, it will be informative to analyze the transcript profiles in plants that either lack, or over-express, AtMKP2. This strategy has the potential to identify genes whose transcription is directly or indirectly dependent on AtMKP2 function, and thus provide additional insight into the biological processes that rely on MKP2. Therefore, AtMKP2 overexpressing plants were grown to different growth stages (seedlings and 3-week-old plants). AtMKP2-RNAi plants were grown to seedling stages. The extracted RNA was used for transcriptome analysis, using 30k-element long oligo Arabidopsis microarrays labelled by dendrimers.  3.2 Material and methods  3.2.1 Plant material and treatments  For the production of seedlings used for microarray and RT-PCR analyses, seeds were surface-sterilized with 20% commercial bleach and grown on Murashige and Skoog Basal Salts with minimal organics (Plant cell culture tested, Sigma) and 1% (w/v) sucrose, solidified with 0.7% (w/v) agar (Sigma). Seeds were stratified at 4 °C dark condition for 24 hours before incubation at 22 °C under 16-h photo-period constant white light for seed germination and seedling growth. For mature plants, one-weekold seedlings were transferred to soil and grown further under environmentally controlled conditions (22 °C under a 16-h photo-period) for 3 weeks. At the appropriate time point, silencing of AtMKP2 was induced by spraying 10µM dexamethasone (DEX), 0.025%(V/V) Silwet 77 solution.  40  3.2.2 Microarray analysis  3.2.2.1 RNA isolation and cDNA synthesis  After the chosen period of DEX treatment, plant materials were frozen in liquid nitrogen and stored at –80oC until further processing. Total RNA samples were prepared using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration of RNA was determined with a NanoDropTM 1000 Spectrophotometer and 5µg total mRNA was used for each slide. Total RNA solution was mixed with 1µl RT primer (Array 3DNA kits, Genisphere) and nuclease-free water to reach a final volume of 11µl. The mixture was then heated to 80oC for ten minutes and immediately transferred to ice for 2-3 minutes. The RNA-RT primer mix was next combined with 4 µl 5X SuperScript II first strand buffer, 1µl dNTP, 2µl 0.1M DTT, 1µl Superase-In and 1µl Superscript II enzyme. After gently mixing the solution, it was incubated at 42oC for 2 hours. After incubation, the reaction was stopped with 3.5µl cDNA synthesis stop solution (0.5M NaOH/50mM EDTA), incubated for 10 minutes at 65oC, and neutralized by adding 5µl 1M Tris-HCl pH7.5. The cDNA preparations from control and mutant tissues were then combined into 1 tube. The original tubes were rinsed with 73µl TE buffer (10mM Tris, pH 8.0, 1mM EDTA). Then the rinsing solution was combined with the total cDNA solution. The cDNA mixture was then concentrated with a Microcon YM-30 centrifugal filter device according to manufacturer’s instruction and eluted with nuclease-free water to achieve a volume of 40µl.  3.2.2.2 cDNA hybridization  The analysis made use of microarray slides printed at the Jack Bell Array Centre, Vancouver, with the Arabidopsis Genome Oligo Set Version 1.0 (26,090 70mer oligonucleotides). For pre-hybridization, the slides were gently shaken for 45 minutes at 48oC in pre-hybridization buffer (5X SSC, 0.1% SDS, 0.2% BSA) in a Coplin jar, rinsed twice in water, dipped 5 times in isopropanol and dried by 2 minutes centrifugation in 50ml Falcon tubes at 2000rpm (benchtop clinical centrifuge). At the same time, thawed and resuspended the 2X Hybridization Buffer by heating to 65 oC for 7 minutes. After preparation, mixed 40µl hybridization buffer with 40µl 41  concentrated cDNA, incubated the hybridization mix at 80 oC for 10 minutes and loaded the mix to the microarray slides covered with a glass cover slip, incubated for 18 hours in dark at 50 oC. After hybridization, the cover slips were removed and the slides were washed for 15 minutes with 2X SCC, 0.2% SDS at 50 oC, and then with agitation at room temperature for 15 minutes with 2X SCC, 15 minutes with 0.2X SCC. After washing, the slides were incubated for 2 minutes in 95% ethanol and dried in a 50ml Falcon tube by 2 minute centrifugation at 1000 rpm.  3.2.2.3 Cy3 and Cy5 labeling 2X Hybridization Buffer was prepared by heating to 55 oC for 10 minutes. 3DNA Array 350 Capture Reagent was prepared in dark at room temperature for 20 minutes and then incubated at 55 oC for 10minutes. The reagent was vortexed before use. The 3DNA Hybridization Mix was made with 40µl 2X Hybridization Buffer, 0.4µl AntiFade Reagent, 2.5µl 3DNA Capture Reagent #1, 2.5µl 3DNA Capture Reagent #2, 0.5µl Cy3 labeled GFP, 0.5µl Cy5 labeled GFP and 33.6µl nuclease free water. The hybridization mix was incubated first at 75 oC for 10 minutes and then loaded to the slides at 53 oC in dark for 3.5 hours. After hybridization, the slides were washed for 15 minutes with 2X SCC, 0.2% SDS at 65 oC, and then with agitation at room temperature for 15 minutes with 2X SCC, 15 minutes with 0.2X SCC. After washing, the slides were dried in a 50ml Falcon tube by 2 minute centrifugation at 1000 rpm. The slides were kept in dark until scanned.  3.2.2.4 Image processing  Hybridized microarrays were scanned with GenePix 4200AL (Axon Instruments) using PMT settings of 440 for Cy5 and 420 for Cy3. The spots were then identified and the intensity of labeling were quantified using the ImaGene software (BioDiscovery). Spot grids were auto-adjusted three times and then manually aligned. Raw spot and background intensities were saved as text files.  42  3.2.2.5 Data analysis  The data analysis was carried out using custom R scripts, with the help of Hardy Hall in the Ellis Lab. The mean background of each slide was calculated for each subgrid of a channel. This mean value was then subtracted from the signal obtained for each spot in the subgrid. The background-corrected intensities were then normalized using LOWESS normalization. Lowess normalization merges two-color data, applying a smoothing adjustment that removes spurious variation that can be caused by Cy3/Cy5 dye bias. A one-way ANOVA was then performed on the normalized data. After filtering, genes that showed a ‘q’ values smaller than 0.09 were selected for RealtimePCR confirmation.  3.2.3 Quantitative Real-time PCR  Total RNA samples were prepared from new plant samples using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration of RNA was determined by NanoDropTM 1000 Spectrophotometer. Reverse transcription was performed using a First-strand cDNA Synthesis Kit (Amersham Biosciences) with 2µg total mRNA. The cDNA products were diluted by a factor of ten in water for real-time PCR analysis. The primers were designed using GenScript software (https://www.genscript.com/ssl-bin/app/primer) to amplify a 200-250bp unique region of each selected gene. The primers used for RT-PCR are presented in Table 3.1.  The real-time PCR reactions were performed using QuantiTect SYBR Green PCR Master Mix (Qiagen) in a total volume of 20µl. ACT8 was used as a control for different samples and each gene had three technical replicates. The real-time PCR reactions were run in a DNA Engine Opticon2 (Bio-Rad) with the following program: 95 oC for 15 minutes, followed by 40 cycles of amplification (94 oC for 15 seconds, 60 o  C for 30 seconds, 68 oC for 45 seconds, and a fluorescence reading). After a final  elongation step of 5 minutes at 68 oC, a melting curve was performed from 60 to 90 oC to check the specificity of the primers. After amplification, data was quantified by Opticon Monitor 3 (MJ research). The baseline was subtracted between cycle 3 to 10 and the threshold was set to 0.005. The ratios of transcript abundance in different  43  samples were then calculated by the △△Ct method, using the formula 2 (GENE)  △CT  (ACT) - △CT  (△CT = the difference in threshold cycle observed between mutant and control  samples).  Primer Name  Primer Sequence  ACT8_F  5'-GAGACATCGTTTCCATGACG-3'  ACT8_R  5'-TTTCAAACCTGCTCCTCCTT-3'  MKP2_F  5'-ACTTGTAAGGAGCCGACGAC-3'  MKP2_R  5'-GAAGACACTTGCCGGAATTT-3'  PCC1_F  5'-GCAGTGGAGACAAACTCCAA-3'  PCC1_R  5'-GTTTGGGCAACGACTTCTG-3'  PR-1_F  5'-GATGTGCCAAAGTGAGGTGT-3'  PR-1_R  5'-TCCTGCATATGATGCTCCTT-3'  PR-5_F  5'-TGGCGGTCTAAGATGTAACG-3'  PR-5_R  5'-AGACACAGCCTGCGTATTTG-3'  CHS_F  5'-GGCCTCATCTCCAAGAACAT-3'  CHS_R  5'-GTCGCCCTCATCTTCTCTTC-3'  WRKY54_F  5'-TAGACGCAGGCATGGTTAAG-3'  WRKY54_R  5'-ATCGTTGTCGATGAAACCAA-3'  EBF2_F  5'-AAACGGAGTGACAGATGCAG-3'  EBF2_R  5'-GCTTGCGTTTGTGATGTTCT-3'  MAP18_F  5'-GGCTAAACCGGTGGAGGT-3'  MAP18_R  5'-AGTTTCAGCCACCGGAAC-3'  SEN1_F  5'-GAGTCGGATCAGGAATGGTT-3'  SEN1_R  5'-CTCATTCTCTGTCCAAGCGA-3'  DGL1_F  5'-GTGGCCAAGTCCCATAAAGT-3'  DGL1_R  5'-TGATTATTGAGCTGCCTTGG-3'  RHD3_F  5'-TAGACGGGAAGGAGAATTGG-3'  RHD3 _R  5'-TCCTCCTTTCCAGTCCAAAC-3'  GFG1_F  5'-GGATGCCGATCCAAGAGTAT-3'  GFG1 _R  5'-GAGAGAAGCAGAGGCTTGGT-3'  ERF6_F  5'-CGGTGGTTGAGAAAGTGCTA-3'  ERF6_R  5'-CAAGCTGACCCAAACAGAAA-3'  Table 3. 1 Primers used for qRT-PCR confirmation  44  3.3 Results  3.3.1 Experimental design for the study of AtMKP2-mediated transcriptional changes  To test the transcriptional changes caused by repression or over expression of AtMKP2, I employed DEX-inducible AtMKP2 RNAi (line26) and 35S::AtMKP2 (line10) plants for global transcriptional profiling. Since both constructs were placed in vector pTA7002, transgenic plants transformed with the empty pTA7002 vector were used as control. In order to capture the direct affect of AtMKP2 on gene expression, and reduce the noise from secondary signaling pathways triggered by the early transcriptional events associated with up- or down-regulation of AtMKP2, I examined the changes in AtMKP2 expression level in both lines at 6 hours, 12 hours and 24 hours after DEX induction. For the AtMKP2 over-expression line, MKP2 transcripts were already highly up-regulated after 6 hours, so I used 6 hour DEX treatment for this line. For the AtMKP2 RNAi line, MKP2 expression was downregulated about 50% after either 12 hours and 24 hours treatment in comparison to controls. Since a comparison of the transcriptome after 12 hours and 24 hours might help me to distinguish primary and secondary responses, I conducted microarray analysis at both time points.  Another factor that I considered was the growth stage of the plants. AtMKP2 overexpression lines showed no obvious phenotype at either the seedling or mature plant stages; therefore, to get a clue as to what biological processes AtMKP2 would be involved in, I chose plants at both growth stages to check for altered gene expression patterns. AtMKP2 RNAi lines, on the other hand, showed a severe growth phenotype at the seedling stage, but no obvious defect at more mature stages, so I chose AtMKP2 RNAi seedlings for transcriptome analysis. The detailed experimental design is shown in Table 3.2.  45  Table 3. 2 Experimental design for microarray profiling of AtMKP2 LOF and GOF mutants  3.3.2 AtMKP2 mediates short-term transcriptional changes  After background subtraction and normalization of the microarray data from the MKP2-over-expression (OX) samples, I found that AtMKP2 expression was upregulated 12.3 fold and 25.7 fold in seedlings and mature samples, respectively. In the MKP2-RNAi samples, however, AtMKP2 expression was only slightly suppressed (down-regulated 1.2 fold and 1.07 fold in 12hr DEX-treated and 24hr DEX-treated samples, respectively). The data were analyzed by Student’s t-test and a series of graphs were developed based on the distribution of the resulting p-values. These revealed that the OX AtMKP2 seedlings sample set and the 12 hour DEX-treated AtMKP2-RNAi sample set did not show significant changes in transcript profiles. In the OX AtMKP2 mature plant sample, 6.8% of the genes showed significant changes, while in the 24hr DEX-treated AtMKP2-RNAi sample, 3.8% of the genes showed significant changes in signal.  I then generated a list of genes by filtering the resulting data according to the following standard: For OX AtMKP2 mature plant sample, the p-value should be <0.05 and the observed fold-change should be >2-fold. For the 24hr DEX treatment AtMKP2 sample, the p-value should be smaller than 0.05 and the observed foldchange should be more than 1.6-fold. Based on these cut-offs, 203 genes had significant changes in AtMKP2 RNAi lines (162 genes were up-regulated and 41 genes were down-regulated). Somewhat more (311) genes had significant changes in  46  the OX AtMKP2 lines, with 139 genes up-regulated and 172 genes down-regulated (Appendix Table 5.1, 5.2).  Figure 3. 1 Distribution of p-values from t-test  To functionally classify the genes affected by changes in AtMKP2 expression, I used functional categorization based on the gene ontology classes described on the TAIR website (http://www.arabidopsis.org/tools/bulk/go/index.jsp). The ontology classes for genes affected by overexpression of AtMKP2, are graphically displayed in Figure 3.2, and for the genes affected by knock down of AtMKP2, as the classes are shown in Figure 3.3. The cellular component ontology of genes affected by overexpression of AtMKP2 appeared to contain a large proportion of chloroplast (14.118%), plastid (7.582%) and plasma membrane (5.621%) assignments. The biological process  47  ontology indicates that 13.6% of OX AtMKP2-affected genes are involved in response to stress and 12.133% are involved in response to biotic and abiotic stimulus. There are also 5.733% proteins involved in protein metabolism. For molecular functions of affected genes, the top 3 functions are protein binding (8.122%), transferase activity (8.122%) and kinase activity (6.599%).  The cellular component ontology of genes affected by repression of AtMKP2 revealed that 10.73% of proteins are predicted to be located in the chloroplast, 8.369% of proteins on or in the plasma membrane and 4.936% in the nucleus. The biological process ontology of the genes is has a similar trend as the overexpression data set, with 10.407% related to stress responses, 6.335% related to response to biotic and abiotic stimulus, and 5.656% related to protein metabolism. For molecular functions of affected genes, the top three predicted functions are hydrolase activity (10.432%), transporter activity (7.554%) and transferase activity (7.194%).  48  Figure 3. 2 GO annotation for genes affected by over-expression of AtMKP2  49  Figure 3. 3 GO annotation for genes affected by repression of AtMKP2  3.3.3 Analysis of trichome development-related gene expression levels  My phenotypic analyses have shown that AtMKP2 is involved in the trichome developmental process and my RT-PCR data also confirmed transcriptional changes of some regulators in trichome development. When I examined the AtMKP2 RNAi microarray data for trichome development-related genes, and compared these data with my RT-PCR data, I found that most of the genes showed similar trends in both analyses, except for CPC. This result further indicates that the data set of microarray is reproducible.  Table 3. 3 Transcriptional responses of genes involved in trichome development, as compared in microarray analysis and RT-PCR analysis  50  3.3.4 Verification of selected genes by Real-time PCR  Since microarray data are unavoidably affected by noise, it is necessary to validate the results obtained in this approach. For this purpose, I chose two sets of genes from over-expression of AtMKP2 mature plants and AtMKP2 RNAi 24 hour DEX treated seedlings, focusing on genes that had shown the most marked changes.  Genes At3g22231  Other name PCC1  Foldchange -9.56  At2g14610  PR-1  -7.47  At1g75040 At5g13930  PR-5 CHS  -4.27 -4.22  At2g40750  WRKY54  -2.82  At5g25350  EBF2  2.45  At5g44610  MAP18  3.13  At4g35770  SEN1  5.51  Additional annotation Encodes a member of a novel six-member Arabidopsis gene family. Expression of PCC1 is regulated by the circadian clock and is also up-regulated in response to both virulent and avirulent strains of Pseudomonas syringae pv. tomato. Expression is induced in response to a variety of pathogens. It is a useful molecular marker for the SAR response. Thaumatin-like protein involved in response to pathogens. Participates in the biosynthesis pathway of all flavonoids. Metabolism of defense and communication. Transcriptionally regulated by light. Required for the accumulation of anthocyanins in leaves and stems. Induced by avirulent P. syringae in NPR1-independent manner; not induced by SA Arabidopsis thaliana EIN3-binding F-box protein 2 (EBF2) mRNA. Part of the SCF complex, it is located in the nucleus and is involved in the ethylene-response pathway. Microtubule-associated protein 18. RNAi and overexpression experiments suggest that the gene is not involved in cell division but might be consequential for cell shape of epidermal and cortical cells. The MAP18 protein binds to cortical microtubules and inhibits tubulin polymerization. SENESCENCE ASSOCIATED GENE 1; strongly induced by phosphate starvation. Transcripts are differentially regulated at the level of mRNA stability at different times of day. SEN1 mRNAs are targets of a mRNA degradation pathway mediated by the "downstream instability determinant" gene (DST).  Table 3. 4 Genes for QRT-PCR from AtMKP2 overexpression data Genes  Other name  Foldchange  Additional annotation  At4g35770  SEN1  -1.71  Senescence-associated gene that is strongly induced by phosphate starvation. Transcripts are differentially regulated at the level of mRNA stability at different times of day. mRNAs are targets of the mRNA degradation pathway mediated by the downstream (DST) instability determinant.  51  Genes  Other name  Foldchange  Additional annotation  At5g66680  DGL1  1.61  At3g13870  RHD3  1.61  At2g14610  PR1  1.63  At4g12720  GFG1  1.76  At4g17490  ATERF6  1.86  Encodes a protein ortholog of human SOT48 or yeast WBP1, an essential protein subunit of the oligosaccharyltransferase (OST) complex, which is responsible for the transfer in the ER of the N-linked glycan precursor onto Asn residues of candidate proteins. Required for regulated cell expansion and normal root hair development. Encodes an evolutionarily conserved protein with putative GTP-binding motifs that is implicated in the control of vesicle trafficking between the endoplasmic reticulum and the Golgi compartments. Expression is induced in response to a variety of pathogens. It is a useful molecular marker for the SAR response. Encodes a protein with ADP-ribose hydrolase activity. Negatively regulates EDS1-conditioned plant defense and programmed cell death. Encodes a member of the ERF (ethylene response factor) subfamily B-3 of ERF/AP2 transcription factor family (ATERF-6).  Table 3. 5 Genes for qRT-PCR from AtMKP2 RNAi data  For qRT-PCR analysis of the AtMKP2 overexpression lines, I used both the original plant material as well as newly prepared plant material. Initially, I used a different biological replicate to validate the selected genes. However, only EBF2 and MAP18 showed similar trend as in the microarray data set. This was unexpected, and to check if my microarray experiment might have had technical problems, I next used the original plant tissues to re-do the qRT-PCR analysis. In this analysis, most of the genes chosen for validation except MAP18, showed similar transcriptional changes as had been found earlier in the microarray dataset. Therefore, the microarray results could be technically replicated. I observed that the plant tissue used for the original sample set had a higher level of AtMKP2 expression than did my new biological replicate (Figure 3.4). This different level of expression of AtMKP2 may have resulted in a very different response from the downstream genes.  52  Figure 3. 4 qRT-PCR analysis of genes affected by AtMKP2 overexpression Left graph shows qRT-PCR result with different biological replicate. Right graph shows qRT-PCR confirmation with original plants used for microarray.  For AtMKP2 RNAi seedlings (24 hour DEX treatment), I prepared a new biological replicate for qRT-PCR validation. The transcriptional changes observed in the RNAi plants were less dramatic than in the overexpression mutants, and most of the genes showed changes within a 1.5-2-fold range. All the genes showed the same trend in the qRT-PCR analysis as in the microarray analysis, but PR1 was more highly upregulated in the new sample compared to microarray data. This means the RNAi data set can be verified both technically and biologically. 1000  100  10  RH D3  GF G1  F6 ER  DG L1  N1 SE  1 PR  MK  0.1  P2  1  Figure 3. 5 qRT-PCR confirmation of AtMKP2 RNAi line26 with different biological replicates  3.4 Discussion  MAPK cascades play vital and complex roles in plant growth and development. The crosstalk with other signaling pathways increases the difficulty of assigning specific  53  biological functions to a single component of any MAPK cascade. Furthermore, the pleiotropic phenotype of AtMKP2 RNAi mutants and the relative normal phenotype of AtMKP2 overexpression lines provides limited insight into the biological function of AtMKP2, based on visual phenotype analysis. Therefore, in this chapter, I employed microarray techniques to capture the genome-wide transcriptional changes in AtMKP2 RNAi and overexpression mutants. The resulting data set might provide some valuable information on the biological processes in which AtMKP2 is involved.  For the four sets of the microarray experiment I conducted, AtMKP2 RNAi seedlings (12 hour DEX treated) and AtMKP2 overexpression seedlings (6 hour DEX treated) did not show significant transcriptome changes. Since the RNAi machinery requires a significant amount of time to bring down the expression level of the target gene, it is possible that at the 12-hour time point, the cellular signaling network had not yet been affected by the knock-down of AtMKP2. In addition, as a phosphatase, it is less likely for AtMKP2 to directly regulate transcriptional changes, compared to a transcription factor. Since AtMKP2 OX seedlings displayed no significant growth phenotype, it appeared that the overexpression of AtMKP2 did not affect the early stages of plant development too much. However, since the overexpression construct was driven by a strong 35S promoter, it was not necessarily being expressed in the appropriate tissue at the correct time. It also remained unknown whether the strong expression of the gene was resulting in accumulation of increased amounts of functional phosphatase in the cells of the seedlings.  For the more mature AtMKP2 overexpression plants, the data were quite interesting. Many genes related to biotic and abiotic stresses had their expression altered. For example, a lot of pathogen response-related genes, including PCC1, PR-1, PR-5, CHS, WRKY, were all down-regulated, indicating that AtMKP2 might play a negative role in pathogen defense; however, when I validated these data with different biological replicates, I found that all of these pathogenesis-related genes were up-regulated. This difference might be caused by different overexpression level of AtMKP2 in these samples. The tissues used for microarray showed about 100-fold up-regulation and the tissues used for qRT-PCR showed around 10-fold up-regulation. More experiments would be needed to test whether AtMKP2 overexpression mutants and wild type plants respond differently to pathogen infection. Of the other candidate genes in the 54  qRT-PCR verification set, EBF2 (EIN3-binding F-box protein 2) showed consistent up-regulation. EBF2 is part of the SCF complex; it is located in the nucleus and is involved in the ethylene-response pathway (Konishi and Yanagisawa, 2008).  In AtMKP2 RNAi seedling samples (24 hour DEX treated), the transcriptional changes were less dramatic than in over-expression lines. This made it hard to distinguish in which primary biological event(s) AtMKP2 might be involved. Since the expression level of AtMKP2 was only about 1.07 fold reduced from WT levels, the resulting transcriptional changes might be less pronounced. Nevertheless, when I chose the most affected genes for qRT-PCR confirmation, all the genes tested showed the same trend as in the microarray experiment. Interestingly, the PR-1 gene showed over 100 fold up-regulation in this confirmation experiment. PR-1 gene expression is induced in response to a variety of pathogens and it is also a useful molecular marker for the SAR response (Uknes et al., 1992). Since the expression of PR-1 was also altered in AtMKP2 overexpression plants, based on my microarray data, this may indicate that AtMKP2 is involved in pathogen-related responses. That would be consistent with previous reports that Arabidopsis MAPK, MPK6, as well as the tobacco ortholog, SIPK, help to regulate plant defense responses (Zhang and Liu, 2001; Menke et al., 2004), and the fact that MKP2 is capable of de-phosphorylating, and thus de-activating MPK6 (Lee and Ellis, 2008).  Finally, in addition to this global expression profiling related to MKP2 function, I was able to specifically confirm the expression responses of the trichome developmentrelated genes by RT-PCR.  55  4. Future directions  Through use of transgenic plants and microarray transcriptome profiling, I have demonstrated that AtMKP2 is a positive regulator in trichome development and likely involved in other important biological roles such as pathogen-related response. However, much remains unknown regarding the downstream targets and the regulators of this phosphatase. In this chapter, I wish to propose some interesting directions that could be followed up in future research.  4.1 Yeast 2-hybrid (Y2H) screening for other possible interactors of AtMKP2  The yeast 2-hybrid technique is a useful tool to study protein-protein interactions. Information on proteins that physically interact with AtMKP2 might provide useful clues about the regulatory machinery and the biological function of AtMKP2. To identify interacting partners, yeast 2-hybrid screening can be conducted using MKP2 as bait to screen against a full cDNA library of prey clones.  In Chapter 2, I was not able to identify the exact downstream targets of AtMKP2 in the trichome development signaling pathway. Previous Ellis lab member, JinSuk Lee, screened MKP2 against all 20 Arabidopsis MPKs using Y2H screening and she identified MPK8 and MPK15 as specific interactors of AtMKP2. She also proved that MPK2 can dephosphorylate MPK3 and MPK6 in vivo; however, analysis of the trichome phenotypes of available MPK3, MPK6, MPK8 and MPK15 mutant lines did not support the idea that each plays an obligatory role in trichome development. It is possible that sufficient functional redundancy exists among these four MPKs to mask any single mutant phenotypic deficiencies. To test this possibility, we would need to examine multiple mutant combinations.  It is also possible that non-MPK proteins could be involved in this process, rather than MPKs. Y2H screening of a seedling cDNA library could help to resolve this question, if it detected new candidate proteins associated with AtMKP2. It is noteworthy that previous research showed that although AtMKP2 is a positive regulator in ozone 56  responses, the levels of AtMKP2 transcripts do not change markedly in ozone-treated wild-type plants or ozone-treated mpk3 and mpk6 mutant plants. Interestingly, in vitro phosphatase assays demonstrated that the association of either MPK3 or MPK6 can enhance the catalytic activity of AtMKP2, and this effect was not related to MAPK activity (Lee and Ellis, 2007). These results indicate that the activity of AtMKP2 might be regulated by binding to its respective substrates. Y2H screening could allow us to identify other potential regulators of AtMKP2, improving our knowledge of the regulation of MKPs in Arabidopsis.  4.2 Phospho-proteomics profiling using AtMKP2 mutants  Although yeast 2-hybrid screening might find potential substrates or regulators of AtMKP2, it does not provide details of cell signaling events in vivo. Quantitative phospho-proteomics profiling, on the other hand, enables us to examine the dynamics of global protein phosphorylation of different mutants or under different conditions. Mass spectrometry is a powerful tool for identification and global profiling of protein phosphorylation. This assay might allow us to find direct targets of MKP2. In principle, the direct targets of AtMKP2 will become more highly phosphorylated in AtMKP2 RNAi plants and less phosphorylated in AtMKP2 overexpression plants. We are now collaborating with the Foster Lab to conduct a phospho-peptide profiling comparison of 10-day-old AtMKP2 RNAi mutants and WT plants. Besides the direct mass spectrometry (MS) approach, we are also trying to visualize the global protein phosphorylation changes in AtMKP2 mutants and WT on 2D-gel format. By using Pro-Q Diamond staining, which stains phosphoproteins specifically, we can look for significant changes in the abundance of individual phosphoprotein spots when comparing WT and MPK2 mutant protein profiles on 2D-gels.  4.3 Interaction of AtMKP2 with MPK8 and MPK15  MPK8 and MPK15 are two Group D MPKs that share 79% amino acid sequence identity. To date, there are no published reports concerning the functions of group D MPKs in Arabidopsis. Both of them were identified from the yeast 2-hybrid assay as interactors of AtMKP2; however, these interactions took place within the context of  57  the yeast nucleus. To further study the relation of AtMKP2 and MPK8/15, it is necessary to confirm the physical interaction by pull-down assays, both in vitro and in vivo. For in vitro studies, one might need to link AtMKP2 and AtMPK8/15 with different affinity tags (e.g. GST tag, 6xhis tag ) and then express them in E. coli. After purification, the fusion proteins would be used in appropriate combinations for in vitro pull-down assays. After pull-down, the recovered proteins can be probed on western blots with an antibody directed against the tag. To further confirm that AtMKP2 interacts with AtMPK8/15 in vivo, one would need to make AtMKP2 and MPK8/15 with appropriate tags and express them in N. benthamiana or Arabidopsis leaves by agroinfiltration. Then one can extract total protein from the transgenic plant leaves and conduct the in vivo pull-down assay.  Phosphatase activity assay will also be needed to detect the phosphatase/ substrate relationship of AtMKP2 and MPK8/15. To do so, one would first need to identifying one or more upstream CA-MKK clones capable of phosphorylating recombinant AtMPK8 and AtMPK15 in vitro. Then it will be possible to conduct in vitro MAPK dephosphorylation assays by incubating different concentrations of GST-AtMKP2 with purified phospho-AtMPK8 and phospho-AtMPK15 and checking the phosphorylation status of AtMPK8 and AtMPK15 by immunoblot analysis, using anti-pERK1/2 antibody. It will also be interesting to test whether the presence of recombinant MPK8 and 15 has any effect on the catalytic function of MKP2 in its activity against 3-O-methyl fluorescein phosphate (OMFP), since recombinant MPK3 and MPK6 have both been shown to stimulate the activity of MKP2 in vitro (Lee and Ellis 2007)  It was reported that two rice MPKs, which had a same –TDY- motif as Group D Arabidopsis MPKs, are both involved in defense signaling(Cheong et al., 2003; Ning et al., 2006). Therefore it might be worth trying to check the response of MPK8 and MPK15 mutants towards different pathogens. Interestingly, the microarray data of AtMKP2 mutants also showed significant changes for some defense related genes, indicating AtMKP2 is involved in defense response. These clues can be used as a  58  starting point to investigate the biological role of AtMKP2 and MPK8/15 in defense response.  4.4 Conclusions  The work presented in this thesis contains two main parts. The first part focuses on detailed examination of the biological function of AtMKP2 in Arabidopsis trichome development. The second part generated essential background knowledge concerning the global processes that AtMKP2 might be involved in. The data I obtained place AtMKP2 within the network of known trichome development regulators. By pursuing the additional experiments proposed in this chapter, we will have exciting opportunities to further elucidate the connections between the MAPK signaling network and the trichome development regulatory network. The microarray data I generated make it possible to have a more global view of the transcriptome changes taking place in AtMKP2 mutants. This information will provide useful leads for future research on other biological functions of AtMKP2, such as responses to biotic stresses, including pathogen attack.  59  References Abraham, S.M., and Clark, A.R. (2006). 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Genes affected by overexpression of AtMKP2 in mature plants  OX AtMKP2 microarray gene list (mature plant) with 2 fold changes and 0.05 p value cut.  Oligo_ID  AGI number  A010068_01  At3g06110  A000993_01 A021199_01 A203371_01 A016393_01  At1g30250 none At5g59330 At5g59310  A014758_01 A011098_01  At4g35770 At3g24420  A016337_01  At5g59320  A010731_01  At3g27690  A013858_01  At4g21870  A020085_01  At5g37940 At5g38000  A014791_01  At4g27450  A007306_01  At2g05100 At2g05070 At5g50335 At5g06530 At1g27020 At2g05070 At2g05100 At1g75750  A203309_01 A020603_01 A001370_01 A203464_01 A024527_01  Annotation dual specificity protein phosphatase family protein, contains Pfam profile: PF00782 dual specificity phosphatase, catalytic domain expressed protein hypothetical protein lipid transfer protein 4 mRNA, complete cds senescence-associated gene hydrolase, alpha/beta fold family protein, low similarity to 3-oxoadipate enol-lactone hydrolase (Pseudomonas sp. B13) lipid transfer protein 3 mRNA, complete cds Lhcb2 protein (Lhcb2:4) mRNA, complete cds 26.5 kDa class P-related heat shock protein (HSP26.5-P), contains Pfam profile: PF00011 Hsp20/alpha crystallin family: identified in Scharf, K-D., et al,Cell Stress & Chaperones (2001) 6: 225-237. NADP-dependent oxidoreductase, putative, similar to probable NADPdependent oxidoreductase (zetacrystallin homolog) P1 (SP|Q39172)(gi:886428), Arabidopsis thaliana expressed protein, similar to auxin down-regulated protein ARG10 (Vigna radiata) GI:2970051, wali7 (aluminum-induced protein) (Triticum aestivum) GI:451193 Lhcb2 protein (Lhcb2.3) mRNA, complete cds Expressed protein ABC transporter family protein expressed protein Lhcb2 protein (Lhcb2.2) mRNA, complete cds GA-responsive GAST1 protein homolog regulated by BR and GA  FoldPchange value 25.66319  6.76E-05  8.987 6.801222 6.771661 6.67766  0.00137 0.00312 0.000246 0.007102  5.510784 5.220583  0.00249 0.000483  4.928037  0.007373  4.914438  0.002369  4.161562  0.000564  4.05971  0.043012  4.002576  0.001604  3.917433  0.000847  3.91707 3.871633 3.815838 3.779338  0.002185 0.004138 0.001167 0.002173  3.667072  0.000236  71  Oligo_ID  AGI number  A005727_01  At3g47340  A015883_01  At5g14130 At5g14120  A021505_01  At5g47330  A016891_01 A001095_01  At5g22430 At1g62480  A008440_01  At2g31980  A012747_01  At3g15450 At3g15460  A025459_01  At2g02010  A019919_01 A200108_01 A020778_01  At1g27030 At4g25100 At3g08940  A005379_01 A016319_01  none At5g44610  A021504_01  At5g36130  A020286_01  At3g23560  A018671_01 A020560_01  At5g17950 At3g47340  Annotation antagonistically. Possibly involved in cell elongation based on expression data asparagine synthetase 1 (glutaminehydrolyzing) / glutamine-dependent asparagine synthetase 1 (ASN1), identical to SP|P49078 Asparagine synthetase (glutamine-hydrolyzing) (EC 6.3.5.4) (Glutamine- dependent asparagine synthetase) {Arabidopsis thaliana} peroxidase, putative, identical to peroxidase ATP20a (Arabidopsis thaliana) gi|1546694|emb|CAA67338 palmitoyl protein thioesterase family protein, expressed protein vacuolar calcium-binding proteinrelated, contains weak similarity to vacuolar calcium binding protein (Raphanus sativus) gi|9049359|dbj|BAA99394 cysteine proteinase inhibitor-related, contains similarity to extracellular insoluble cystatin GI:2204077 from (Daucus carota) expressed protein, similar to auxin down-regulated protein ARG10 (Vigna radiata) GI:2970051, wali7 (aluminum-induced protein) (Triticum aestivum) GI:451193 glutamate decarboxylase, putative, strong similarity to glutamate decarboxylase isozyme 3 (Nicotiana tabacum) GI:13752462 expressed protein Fe-superoxide dismutase Lhcb4.2 protein (Lhcb4.2) mRNA, complete cds DREPP plasma membrane polypeptide-related, contains Pfam profile: PF05558 DREPP plasma membrane polypeptide cytochrome P450 family, simialr to taxane 13-alpha-hydroxylase (Taxus cuspidata) GI:17148242; contains Pfam profile PF00067: Cytochrome P450 MATE efflux family protein, similar to ripening regulated protein DDTFR18 (Lycopersicon esculentum) GI:12231296; contains Pfam profile: PF01554 uncharacterized membrane protein family hypothetical protein asparagine synthetase 1 (glutamine-  FoldPchange value  3.626826  6.67E-05  3.611591  0.001803  3.582128  0.002161  3.488863 3.470357  0.011968 0.000219  3.438672  0.000902  3.415517  0.001577  3.413869  0.001927  3.313548 3.234958 3.203188  0.000222 0.000954 0.001084  3.193668 3.127811  0.001661 0.040966  3.116403  0.005355  3.088999  0.002407  3.081181 3.030386  0.035608 0.012564  72  Oligo_ID  AGI number  A011800_01  At3g47470  A014936_01  At4g36410  A005366_01  At5g20250  A024680_01  At5g54270 At5g54280  A200017_01  At1g28330  A021034_01  At4g27440  A020748_01 A008707_01  At2g05540 At2g45180  A201667_01  At3g15356  A024643_01  At3g15356  A015071_01 A002224_01  At4g25100 At1g78460  A019670_01 A012331_01  none At3g16240 At3g16250  Annotation hydrolyzing) / glutamine-dependent asparagine synthetase 1 (ASN1), identical to SP|P49078 Asparagine synthetase (glutamine-hydrolyzing) (EC 6.3.5.4) (Glutamine- dependent asparagine synthetase) (Love et al.) Encodes a chlorophyll a/b-binding protein that is more similar to the PSI Cab proteins than the PSII cab proteins. The predicted protein is about 20 amino acids shorter than most known Cab proteins. ubiquitin-conjugating enzyme 17 (UBC17) mRNA, complete cds encodes a member of glycosyl hydrolase family 36. Expression is induced within 3 hours of dark treatment, in senescing leaves and treatment with exogenous photosynthesis inthibitor. Induction of gene expression was suppressed in excised leaves supplied wi Lhcb3 protein is a component of the main lightharvesting chlorophyll a/bprotein complex of Photosystem II (LHC II). dormancy-associated protein (DRM1) mRNA, complete cds light-dependent NADPH:protochlorophyllide oxidoreductase B glycine-rich protein protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, similar to 14 kDa polypeptide (Catharanthus roseus) GI:407410; contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 legume lectin family protein, contains Pfam domain, PF00139: Legume lectins beta domain legume lectin family protein, contains Pfam domain, PF00139: Legume lectins beta domain Fe-superoxide dismutase SOUL heme-binding family protein, weak similarity to SOUL protein (Mus musculus) GI:4886906; contains Pfam profile PF04832: SOUL hemebinding protein delta tonoplast integral protein. functions as water channel and highly expressed in flower, shoot, and stem. protein localized to vacuolar membrane.  FoldPchange value  2.993971  1.51E-05  2.962985  0.002648  2.941709  0.00274  2.919609  0.00015  2.900423  0.00096  2.896053  1.94E-05  2.889876 2.781147  0.047875 0.000231  2.773161  0.001872  2.754956  0.000785  2.738357 2.736347  0.003598 0.00082  2.713866 2.689999  0.002161 0.002897  73  Oligo_ID  AGI number  A200156_01  At5g28770  A007535_01 A009135_01 A020704_01  At2g15960 At3g59930 At5g33355 At1g02205  A000381_01 A012865_01  At1g01600 At4g19420  A007154_01  At2g04570  A012754_01  At3g13750  A009890_01  At3g30775  A008891_01  At3g60290  A016886_01  At5g24490 At5g24500  A202311_01  At4g17245  A007362_01  At2g33830  A020688_01  At2g20870  Annotation  FoldPchange value  bZIP protein BZO2H3 mRNA, partial cds expressed protein expressed protein  2.677186  0.022797  2.673482 2.668988  0.000447 0.025331  Expression of the CER1 gene associated with production of stem epicuticular wax and pollen fertility. Biochemical studies showed that cer1 mutants are blocked in the conversion of stem wax C30 aldehydes to C29 alkanes, and they also lack the secondary alc member of CYP86A pectinacetylesterase family protein, contains Pfam profile: PF03283 pectinacetylesterase GDSL-motif lipase/hydrolase family protein, similar to family II lipase EXL3 (GI:15054386), EXL1 (GI:15054382), EXL2 (GI:15054384) (Arabidopsis thaliana); contains Pfam profile PF00657: Lipase/Acylhydrolase with GDSL-like motif Arabidopsis thaliana mRNA for putative beta-galactosidase (BGAL1 gene). osmotic stress-induced proline dehydrogenase (pro1) mRNA, oxidoreductase, 2OG-Fe(II) oxygenase family protein, similar to flavonol synthase 1 (SP|Q96330), gibberellin 20-oxidase (GI:9791186); contains PF03171 2OG-Fe(II) oxygenase superfamily domain 30S ribosomal protein, putative, similar to SP|P19954 Plastid-specific 30S ribosomal protein 1, chloroplast precursor (CS-S5) (CS5) (S22) (Ribosomal protein 1) (PSRP-1) {Spinacia oleracea}; contains Pfam profile PF02482: Sigma 54 modulation protein / S30E zinc finger (C3HC4-type RING finger) family protein, contains Pfam profile: PF00097 zinc finger, C3HC4 type (RING finger) dormancy/auxin associated family protein, contains Pfam profile: PF05564 dormancy/auxin associated protein cell wall protein precursor, putative, identical to Putative cell wall protein precursor (Swiss-Prot:P47925) (Arabidopsis thaliana); weak similarity to mu-protocadherin  2.667085  0.001684  2.578533 2.576198  0.007992 0.002272  2.567906  0.009702  2.555611  0.024521  2.551062  0.003258  2.546272  0.010541  2.513831  0.000484  2.506057  0.000777  2.493506  0.000171  2.490368  0.023493  74  Oligo_ID  AGI number  A019843_01  At2g38310  A200012_01 A016820_01  At1g12080 At5g63530  A006874_01  At2g40970  A006076_01  At2g40330  A004704_01  At1g28330  A011815_01  At3g05900  A202864_01  At5g25350  A007964_01  At2g06850  A023934_01  At1g06350  A010370_01  At3g12440  A017271_01  At5g07550  A005592_01  At5g25610  A015984_01 A006181_01  At5g40450 At2g14900  A203500_01  At4g21650  Annotation (GI:7861967) (Rattus norvegicus) expressed protein, low similarity to early flowering protein 1 (Asparagus officinalis) GI:1572683, SP|P80889 Ribonuclease 1 (EC 3.1.-.-) {Panax ginseng} expressed protein Farnesylated protein that binds metals. myb family transcription factor, contains Pfam profile: PF00249 myblike DNA-binding domain Bet v I allergen family protein, contains Pfam profile PF00407: Pathogenesis-related protein Bet v I family dormancy-associated protein (DRM1) mRNA, complete cds neurofilament protein-related, similar to NF-180 (GI:632549) (Petromyzon marinus) similar to Neurofilament triplet H protein (200 kDa neurofilament protein) (Neurofilament heavy polypeptide) (NF-H) (Swiss-Prot:P12036) (Homo sapiens) Arabidopsis thaliana EIN3-binding Fbox protein 2 (EBF2) mRNA. Part of the SCF complex, it is located in the nucleus and is involved in the ethylene-response pathway. endoxyloglucan transferase (EXGTA1) gene fatty acid desaturase family protein, similar to delta 9 acyl-lipid desaturase (ADS1) GI:2970034 from (Arabidopsis thaliana) extensin family protein, contains similarity to Swiss-Prot:Q38913 extensin 1 precursor (AtExt1) (AtExt4) (Arabidopsis thaliana) member of Oleosin-like protein family responsive to dehydration 22 (RD22) mediated by ABA expressed protein gibberellin-regulated family protein, similar to SP|P46690 Gibberellinregulated protein 4 precursor {Arabidopsis thaliana} GASA4; contains Pfam profile PF02704: Gibberellin regulated protein subtilase family protein, contains Pfam domain, PF00082: Subtilase family; contains Pfam domain, PF02225: protease associated (PA) domain  FoldPchange value 2.490248  0.002756  2.482652 2.477417  0.001695 0.01464  2.464852  0.036251  2.46232  0.02964  2.457608  8.36E-05  2.453253  0.019225  2.45238  0.000939  2.446044  0.000364  2.441986  0.023405  2.419394  0.012674  2.41797  0.00017  2.396124  0.000556  2.394075 2.391966  0.000135 0.003858  2.318953  0.001695  75  Oligo_ID  AGI number  A006021_01 A021605_01 A202197_01 A022137_01  none At2g26120 none At3g48720  A024470_01  At1g51400  A024649_01 A002045_01  At1g29680 At1g29670 At1g74670  A024378_01  At4g04630  A003990_01  At1g74870  A010009_01  At3g51910  A021515_01  At5g09490  A006824_01  At2g33810  A019305_01 A007422_01  At5g37300 At2g48020  A024664_01  At3g10450  A005629_01  At5g21170  Annotation glycine-rich protein, transferase family protein, similar to hypersensitivity-related hsr201 protein - Nicotiana tabacum,PIR2:T03274; contains Pfam transferase family domain PF00248 photosystem II 5 kD protein, 100% identical to GI:4836947 (F5D21.10) expressed protein gibberellin-responsive protein, putative, similar to SP|P46690 Gibberellin-regulated protein 4 precursor {Arabidopsis thaliana} GASA4; contains Pfam profile PF02704: Gibberellin regulated protein expressed protein, contains Pfam profile PF04520: Protein of unknown function, DUF584 expressed protein, contains similarity to hypothetical proteins heat shock transcription factor family protein, contains Pfam profile: PF00447 HSF-type DNA-binding domain 40S ribosomal protein S15 (RPS15B), ribosomal protein S15 Arabidopsis thaliana, EMBL:Z23161 putative transcription factor that binds DNA and may directly regulate AP1, involved in regulation of flowering expressed protein sugar transporter, putative, similar to ERD6 protein {Arabidopsis thaliana} GI:3123712, sugar-porter family proteins 1 and 2 (Arabidopsis thaliana) GI:14585699, GI:14585701; contains Pfam profile PF00083: major facilitator superfamily protein serine carboxypeptidase S10 family protein, similar to glucose acyltransferase GB:AAD01263 (Solanum berthaultii); also similar to serine carboxypeptidase I GB:P37890 (Oryza sativa) 5'-AMP-activated protein kinase beta-2 subunit, putative, similar to Swiss-Prot:Q9QZH4 5'-AMPactivated protein kinase, beta-2 subunit (AMPK beta-2 chain) (Rattus norvegicus)  FoldPchange value 2.314774 2.299743 2.276257 2.262015  0.004384 0.001746 0.015558 0.019013  2.261651  0.001784  2.255592  0.00064  2.232983  0.002409  2.226941  0.003069  2.216655  0.019728  2.214072  0.026084  2.210122  0.048967  2.209626  0.001809  2.203243 2.188867  0.010707 0.009809  2.182121  0.018903  2.180966  0.005202  76  Oligo_ID  AGI number  A025164_01  At3g23550  A021615_01 A002988_01  none At1g04220 At1g04210  A025898_01  At1g78830  A012008_01  At3g02710  A014768_01 A001679_01 A021052_01  none At1g55330 At2g34430  A023904_01  At1g49130  A003745_01  At3g29770  A005969_01  At1g61520  A003475_01  At1g80920  A203242_01 A024617_01  At5g40730 At1g49240  A003291_01 A016210_01  At1g58290 At5g14780  A021481_01  At5g23370  Annotation MATE efflux family protein, similar to ripening regulated protein DDTFR18 (Lycopersicon esculentum) GI:12231296; contains Pfam profile: PF01554 uncharacterized membrane protein family beta-ketoacyl-CoA synthase, putative, Strong similarity to betaketo-Coa synthase gb|U37088 from Simmondsia chinensis, GI:4091810 curculin-like (mannose-binding) lectin family protein, similar to S glycoprotein (Brassica rapa) GI:2351186; contains Pfam profile PF01453: Lectin (probable mannose binding) nuclear associated protein-related / NAP-related, similar to Nuclear associated protein (NAP) (NYDSP19) (Swiss-Prot:Q8WYA6) (Homo sapiens) arabinogalactan-protein (AGP21) Photosystem II type I chlorophyll a/b-binding protein zinc finger (B-box type) family protein, contains similarity to zinc finger protein GI:3618318 from (Oryza sativa) hydrolase, alpha/beta fold family protein, similar to SP|Q40708 PIR7A protein {Oryza sativa}, polyneuridine aldehyde esterase (Rauvolfia serpentina) GI:6651393; contains Pfam profile: PF00561 alpha/beta hydrolase fold PSI type III chlorophyll a/b-binding protein (Lhca3*1) J8 mRNA, nuclear gene encoding plastid protein, complete arabinogalactan-protein (AGP24) actin 8 (ACT8), identical to SP|Q96293 Actin 8 {Arabidopsis thaliana}; nearly identical to SP|Q96292 Actin 2 (Arabidopsis thaliana) GI:1669387, and to At3g18780 NAD-dependent formate dehydrogenase 1B (FDH1B) mRNA, GRAM domain-containing protein / ABA-responsive protein-related, contains similarity to ABA-responsive protein in barley (GI:4103635) (Hordeum vulgare) (J. Exp. Bot. 50, 727-728 (1999); contains Pfam  FoldPchange value 2.177566  0.025718  2.176194 2.172909  0.000704 0.011086  2.171303  0.003993  2.171094  0.042299  2.158403 2.154897 2.145318  0.020627 0.001069 0.006586  2.138207  0.014893  2.133221  0.028744  2.130999  0.004365  2.119444  0.000416  2.119073 2.115946  0.015532 0.003498  2.112983 2.112535  0.000237 0.001473  2.10653  0.009315  77  Oligo_ID  AGI number  A019439_01  At2g37430  A007604_01 A014175_01  At2g04795 At4g33970  A005989_01  At1g29910 At1g29930 At1g29920 At1g68530  A020902_01 A021237_01 A009579_01  At3g55240 At3g56950 At3g56940  A202455_01  At4g31290  A005741_01  At2g34420  A200460_01  At1g19510  A202671_01  At5g19190  A012626_01  At3g22160  A000828_01 A006657_01  At1g80180 At2g16850  A020400_01 A015182_01  At3g15630 At4g13830  A011875_01  At3g57690  A010748_01  At3g58910  A008578_01  At2g38400  Annotation PF02893: GRAM domain zinc finger (C2H2 type) family protein (ZAT11), contains Pfam domain, PF00096: Zinc finger, C2H2 type Expressed protein leucine-rich repeat family protein / extensin family protein, similar to extensin-like protein (Lycopersicon esculentum) gi|5917664|gb|AAD55979; contains leucine-rich repeats, Pfam:PF00560; contains proline rich extensin domains, INTERPRO:IPR002965 member of Chlorophyll a/b-binding protein family member of Strong similarity to betaketo-Coa synthase family expressed protein small basic membrane integral family protein, contains similarity to small basic membrane integral protein ZmSIP2-1 (GI:13447817) (Zea mays) ChaC-like family protein, contains Pfam profile: PF04752 ChaC-like protein Photosystem II type I chlorophyll a/b-binding protein myb family transcription factor, contains PFAM profile: PF00249 myb-like DNA binding domain expressed protein, predicted protein, Arabidopsis thaliana VQ motif-containing protein, contains PF05678: VQ motif expressed protein plasma membrane intrinsic protein, putative, very strong similarity to plasma membrane intrinsic protein (SIMIP) (Arabidopsis thaliana) GI:2306917 expressed protein DnaJ-like protein (J20) mRNA, complete cds; nuclear gene arabinogalactan-protein, putative (AGP23), similar to arabinogalactan protein (Arabidopsis thaliana) gi|10880503|gb|AAG24281 F-box family protein, contains F-box domain Pfam:PF00646 alanine--glyoxylate aminotransferase, putative / betaalanine-pyruvate aminotransferase, putative / AGT, putative, similar to SP|Q64565 Alanine--glyoxylate aminotransferase 2, mitochondrial  FoldPchange value 2.102014  0.010609  2.098601 2.096639  0.002497 0.001719  2.095632  0.012656  2.085214  0.004439  2.080588 2.078797  0.008297 0.008324  2.070445  0.006895  2.070295  0.000265  2.063297  0.012819  2.06156  0.006473  2.059864  0.003617  2.053053 2.050502  0.001978 0.022481  2.048221 2.040772  0.001173 0.00319  2.036752  0.00919  2.030622  0.002078  2.029291  0.001145  78  Oligo_ID  AGI number  A018838_01  At5g64920  A021811_01 A016445_01  At1g80920 At1g80910 At5g64750  A202546_01  At5g04310  A020760_01 A003507_01 A002276_01 A025135_01  At4g32930 At4g32940 At1g12080 none At5g49730  A008472_01  At2g37040  A022194_01  At3g55190  A025177_01  At3g13470  A024162_01 A020001_01  none At1g55490  A017188_01  At5g01900  A014515_01  At4g04330  Annotation precursor (EC 2.6.1.44) (AGT 2) (Beta-alanine-pyruvate aminotrans Encodes a RING-H2 protein that interacts with the RING finger domain of COP1. CIP8 exhibits a strong interaction with the E2 ubiquitin conjugating enzyme AtUBC8 through its N-terminal domain and promotes ubiquitination in an E2-dependent fashion in vitro. J8 mRNA, nuclear gene encoding plastid protein, complete AP2 domain-containing transcription factor, putative, contains similarity to transcription factor pectate lyase family protein, similar to pectate lyase GP:14531296 from (Fragaria x ananassa) expressed protein, predicted protein, Caenorhabditis elegans, gb:Z70780 expressed protein ferric reductase-like transmembrane component family protein, similar to ferric-chelate reductase (FRO1) (Pisum sativum) GI:15341529; contains Pfam profile PF01794: Ferric reductase like transmembrane componenent encodes a protein similar to phenylalanine ammonia-lyase esterase/lipase/thioesterase family protein, similar to monoglyceride lipase from (Homo sapiens) GI:14594904, (Mus musculus) GI:2632162; contains Interpro entry IPR000379 chaperonin, putative, similar SWISSPROT:P21240- RuBisCO subunit binding-protein beta subunit, chloroplast precursor (60 kDa chaperonin beta subunit, CPN-60 beta) (Arabidopsis thaliana); contains Pfam:PF00118 domain, TCP-1/cpn60 chaperonin family encode a chloroplast chaperonin 60beta (Cpn60beta), a homologue of bacterial GroEL. Mutants in this gene develops lesions on its leaves, expresses systemic acquired resistance (SAR) and develops accelerated cell death to heat shock stress. The protein has member of WRKY Transcription Factor; Group III expressed protein  FoldPchange value 2.02389  0.000504  2.014919  0.005877  2.012769  0.004196  2.012254  0.034286  2.012174  0.001988  2.010814 2.010058 2.000484  0.013008 0.010156 0.005866  -2.00046  0.000614  -2.00112  0.007028  -2.00543  0.012066  -2.00669 -2.00908  0.000971 0.002888  -2.00984  0.031323  -2.01  0.00278  79  Oligo_ID  AGI number  A200947_01  At1g64900  A005197_01  At1g27350  A008080_01  At2g27660  A023474_01  At2g29120  A021656_01 A022469_01  At2g07880 At4g10500  A006046_01  At2g43510  A020806_01  At2g30860  A200051_01 A015892_01  At2g32160 At5g58070  A011678_01  At3g54960  A003138_01  At1g78570 At1g78580  A020763_01  At1g10960  A020462_01  At4g39950  A022417_01  At4g24920  Annotation cytochrome P450 (CYP89A2) mRNA, complete cds expressed protein, contains 1 transmembrane domain; similar to ribosome associated membrane protein RAMP4 GI:4585827 (Rattus norvegicus); similar to ESTs gb|T20610 and gb|AA586199 DC1 domain-containing protein, contains Pfam profile PF03107: DC1 domain member of Putative ligand-gated ion channel subunit family hypothetical protein oxidoreductase, 2OG-Fe(II) oxygenase family protein, similar to hyoscyamine 6 beta-hydroxylase (Atropa belladona)(GI:4996123); contains PF03171 2OG-Fe(II) oxygenase superfamily domain Encodes putative trypsin inbitor protein which may function in defense against herbivory. Encodes glutathione transferase belonging to the phi class of GSTs. Naming convention according to Wagner et al. (2002). expressed protein lipocalin, putative, similar to temperature stress-induced lipocalin (Triticum aestivum) GI:18650668 thioredoxin family protein, similar to protein disulfide isomerase GI:5902592 from (Volvox carteri f. nagariensis), GI:2708314 from Chlamydomonas reinhardtii; contains Pfam profile: PF00085 Thioredoxin NAD-dependent epimerase/dehydratase family protein, similar to dTDP-glucose 4,6dehydratase from Aneurinibacillus thermoaerophilus GI:16357461, RmlB from Leptospira borgpetersenii GI:4234803; contains Pfam profile PF01370 NAD dependent epimerase/dehydrata ferredoxin, chloroplast, putative, strong similarity to FERREDOXIN PRECURSOR GB:P16972 (SP|P16972) from (Arabidopsis thaliana) Belongs to cytochrome P450 and is involved in tryptophan metabolism. Converts Trp to indo-3acetaldoxinme (IAOx), a precursor to IAA and indole glucosinolates. protein transport protein SEC61  FoldPchange value -2.01187  0.000753  -2.02227  0.00025  -2.02487  0.009601  -2.02979  0.002438  -2.03148 -2.05042  0.046524 0.004005  -2.05616  0.00794  -2.05808  0.000798  -2.0596 -2.07427  0.006234 0.000533  -2.07574  0.028395  -2.0839  0.002802  -2.08469  2.58E-05  -2.09598  5.20E-05  -2.09687  4.48E-06  80  Oligo_ID  AGI number  A017938_01  At5g06320  A014477_01  At4g03450  A021217_01  At1g75830  A011673_01  At3g09440  A003569_01  At1g04980  A021700_01 A013804_01 A009611_01  none At4g12480 At3g08590  A024307_01  At1g21270  A025929_01 A024557_01  At3g49120 At1g27730  A025089_01  At3g25490  A005695_01  At1g65800 At1g65790  A014416_01  At4g20110  Annotation gamma subunit, putative, similar to Swiss-Prot:Q19967 protein transport protein SEC61 gamma subunit (Caenorhabditis elegans) NDR1/HIN1-like protein 3 (NHL3) mRNA, complete cds ankyrin repeat family protein, contains ankyrin repeats, Pfam domain PF00023 plant defensin-fusion protein, putative (PDF1.1), identical to SP|P30224 Cysteine-rich antifungal protein 1 precursor (AFP1) (Antherspecific protein S18 homolog) {Arabidopsis thaliana} heat shock cognate 70 kDa protein 3 (HSC70-3) (HSP70-3), identical to SP|O65719 Heat shock cognate 70 kDa protein 3 (Hsc70.3) {Arabidopsis thaliana} thioredoxin family protein, similar to SP|Q63081 Protein disulfide isomerase A6 precursor (EC 5.3.4.1) {Rattus norvegicus}; contains Pfam profile PF00085: Thioredoxin pEARLI 1 mRNA, complete cds 2,3-biphosphoglycerate-independent phosphoglycerate mutase, putative / phosphoglyceromutase, putative, strong similarity to SP|Q42908 2,3bisphosphoglycerate-independent phosphoglycerate mutase (EC 5.4.2.1) (Phosphoglyceromutase) {Mesembryanthemum crystal cytoplasmic serine/threonine protein kinase induced by salicylic acid Encodes a peroxidase. Related to Cys2/His2-type zincfinger proteins found in higher plants.Compensated for a subset of calcineurin deficiency in yeast.Salt tolerance produced by ZAT10 appeared to be partially dependent on ENA1/PMR2, a P-type ATPase required for Li+ and Na+ ef wall-associated kinase, putative, similar to wall-associated kinase 4 GB:CAA08793 from (Arabidopsis thaliana) S-receptor protein kinase, putative, similar to PIR|T05180|T05180 Sreceptor kinase ARK3 precursor (Arabidopsis thaliana) vacuolar sorting receptor, putative, similar to BP-80 vacuolar sorting receptor (Pisum sativum)  FoldPchange value  -2.09753  0.002908  -2.10199  0.010262  -2.12447  0.002085  -2.1268  0.000202  -2.13514  0.012799  -2.14046 -2.1569 -2.1584  0.002069 0.001325 0.000477  -2.1598  0.026634  -2.1655 -2.16974  0.009557 6.71E-07  -2.17798  0.00697  -2.17808  0.005176  -2.18067  0.001094  81  Oligo_ID  AGI number  A200075_01  At3g17609  A202426_01 A202676_01 A021247_01  At4g27657 At5g19250 At3g46080  A202924_01  At5g26690  A006717_01  At2g39030  A025877_01  At5g56010  A202287_01 A020011_01  At4g16146 At2g46440 At2g46430 At1g56340  A002695_01 A006761_01 A018927_01  At2g31880 At2g31890 At5g24210  A202909_01  At5g26170  A003835_01  At1g23050  A025828_01  At1g66970  Annotation GI:1737222; identical to vacuolar sorting receptor-like protein (GI:2827665) (Arabidopsis thaliana) bZIP transcription factor family protein / HY5-like protein (HYH), nearly identical to HY5-like protein (Arabidopsis thaliana) GI:18042111; similar to TGACG-motif binding factor GI:2934884 from (Glycine max); contains Pfam profile: PF00170 bZIP transcript Expressed protein expressed protein zinc finger (C2H2 type) family protein, contains zinc finger, C2H2 type, domain, PROSITE:PS00028 heavy-metal-associated domaincontaining protein, low similarity to farnesylated protein GMFP5 (Glycine max)(GI:4097571); contains Pfam profile PF00403: Heavy-metalassociated domain GCN5-related N-acetyltransferase (GNAT) family protein, similar to SP|Q9SMB8 Tyramine Nferuloyltransferase 4/11 (EC 2.3.1.110) (Hydroxycinnamoyl- CoA: tyramine Nhydroxycinnamoyltransferase) {Nicotiana tabacum}; contains Pfam profile PF00583: acetyltrans a member of heat shock protein 90 (HSP90) gene family. Expressed in all tissues and abundant in root apical meristem, pollen and tapetum. Expresssion is NOT heat-induced but induced by IAA and NaCl. Expressed protein member of Cyclic nucleotide gated channel family calreticulin (Crt1) mRNA, complete cds leucine-rich repeat transmembrane protein kinase, putative lipase class 3 family protein, contains Pfam profile PF01764: Lipase member of WRKY Transcription Factor; Group II-c hydroxyproline-rich glycoprotein family protein, contains proline-rich extensin domains, INTERPRO:IPR002965 glycerophosphoryl diester phosphodiesterase family protein, contains Pfam PF03009 : Glycerophosphoryl diester  FoldPchange value  -2.18466  0.00142  -2.1849 -2.20053 -2.20111  0.012824 0.000834 0.001111  -2.20264  0.026079  -2.20293  0.027243  -2.2057  0.015645  -2.2302 -2.23143  0.000558 0.00249  -2.23289  0.00423  -2.23346  0.002023  -2.23674  0.002801  -2.25902  0.015372  -2.25965  0.0008  -2.25968  0.01211  82  Oligo_ID  AGI number  A022269_01  At5g10380  A025696_01  At5g27060  A203310_01  At5g50460  A003204_01  At1g60050  A011710_01  At3g11340  A018891_01  At5g20630  A012346_01  At3g11090  A011432_01  At3g25010  A202718_01 A022222_01 A021488_01  At5g19875 none At5g52760  A011050_01 A024349_01  At3g13950 At1g72900 At1g72910  A008966_01  At3g47480  A003378_01  At1g71100  Annotation phosphodiesterase family zinc finger (C3HC4-type RING finger) family protein, contains Pfam profile: PF00097 zinc finger, C3HC4 type (RING finger) disease resistance family protein, contains leucine rich-repeat (LRR) domains Pfam:PF00560, INTERPRO:IPR001611; similar to Hcr2-0B (Lycopersicon esculentum) gi|3894387|gb|AAC78593 protein transport protein SEC61 gamma subunit, putative, similar to Swiss-Prot:Q19967 protein transport protein SEC61 gamma subunit (Caenorhabditis elegans) nodulin-related, low similarity to MtN21 (Medicago truncatula) GI:2598575; contains Pfam profile PF00892: Integral membrane protein UDP-glucoronosyl/UDP-glucosyl transferase family protein, contains Pfam profile: PF00201 UDPglucoronosyl and UDP-glucosyl transferase germin-like protein (GLP3b) mRNA, complete cds LOB domain family protein / lateral organ boundaries domain family protein (LBD21), identical to SP|Q9SRL8 Putative LOB domain protein 21 {Arabidopsis thaliana}; similar to lateral organ boundaries (LOB) domain-containing proteins from Arabidopsis thalian disease resistance family protein, contains leucine rich-repeat (LRR) domains (23 copies) Pfam:PF00560, INTERPRO:IPR001611; similar to Hcr2-5D (Lycopersicon esculentum) gi|3894393|gb|AAC78596 Expressed protein heavy-metal-associated domaincontaining protein, contains Pfam profile PF00403: Heavy-metalassociated domain expressed protein disease resistance protein (TIR-NBS class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. calcium-binding EF hand family protein, contains INTERPRO:IPR002048 calciumbinding EF-hand domain ribose 5-phosphate isomerase-  FoldPchange value -2.26019  0.020234  -2.26542  0.028663  -2.27047  0.000586  -2.27348  7.22E-05  -2.27802  0.006924  -2.27986  8.30E-07  -2.28563  0.014451  -2.29977  0.031314  -2.29995 -2.30165 -2.31133  0.024395 0.010236 0.042237  -2.3125 -2.31323  0.003953 0.003471  -2.31521  0.003723  -2.32891  0.007076  83  Oligo_ID  AGI number  A024141_01  At2g32680  A002917_01  At1g68620  A008693_01  At2g25110  A203264_01 A024841_01  At5g44575 At3g61220  A007808_01  At2g21150  A001618_01 A025932_01  At1g24145 At1g21250  A021286_01  At4g12470  A019418_01  At5g52940  A019642_01  At5g48880  A025079_01  At3g24900  A019812_01  At3g51860  A202674_01 A011469_01  At5g19230 At3g09940  Annotation related, similar to ribose-5phosphate isomerase GI:18654317 from (Spinacia oleracea) disease resistance family protein, contains leucine rich-repeat (LRR) domains Pfam:PF00560, INTERPRO:IPR001611; similar to Cf-2.2 (Lycopersicon pimpinellifolium) gi|1184077|gb|AAC15780 expressed protein, similar to PrMC3 (Pinus radiata) GI:5487873 MIR domain-containing protein, similar to SP|Q99470 Stromal cellderived factor 2 precursor (SDF-2) {Homo sapiens}; contains Pfam profile PF02815: MIR domain expressed protein, short-chain dehydrogenase/reductase (SDR) family protein, similar to carbonyl reductase GI:1049108 from (Mus musculus) XAP5 family protein, contains Pfam profile: PF04921 XAP5 protein expressed protein, cell wall-associated kinase, may funtion as a signaling receptor of extracellular matrix component. protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, similar to pEARLI 1 (Accession No. L43080): an Arabidopsis member of a conserved gene family (PGF95099), Plant Physiol. 109 (4), 1497 (1995); contains Pfam protease inhibitor/se hypothetical protein, contains Pfam profile PF03478: Protein of unknown function (DUF295) peroxisomal 3-keto-acyl-CoA thiolase 2 precursor (PKT2) disease resistance family protein / LRR family protein, contains leucine rich-repeat domains Pfam:PF00560, INTERPRO:IPR001611; similar to Cf-2.2 (Lycopersicon pimpinellifolium) gi|1184077|gb|AAC15780 member of Low affinity calcium antiporter CAX2 family expressed protein monodehydroascorbate reductase, putative, similar to monodehydroascorbate reductase (NADH) GB:JU0182 (Cucumis sativus)  FoldPchange value  -2.33548  0.037811  -2.33999  0.029859  -2.34017  0.008408  -2.34122 -2.34403  0.009311 0.038645  -2.34649  0.013879  -2.3554 -2.37209  3.18E-05 0.020056  -2.3901  0.000479  -2.41895  0.00178  -2.42875  0.002278  -2.44017  0.000108  -2.44755  0.008316  -2.45625 -2.45627  0.003891 0.003434  84  Oligo_ID  AGI number  A016950_01  At5g61790  A202381_01  At4g21830  A022573_01  At4g05520  A200552_01  At1g27330  A001038_01  At1g07050  A201621_01  At3g11010  A009087_01  At3g45140  A003718_01  At1g28480  A003555_01  At1g13930  A006621_01  At2g18660  A020903_01  At3g51240  Annotation calnexin 1 (CNX1), identical to calnexin homolog 1, Arabidopsis thaliana, EMBL:AT08315 (SP|P29402) methionine sulfoxide reductase domain-containing protein / SeIR domain-containing protein, low similarity to pilin-like transcription factor (Homo sapiens) GI:5059062, SP|P14930 Peptide methionine sulfoxide reductase msrA/msrB (EC 1.8.4.6) {Neisseria gono calcium-binding EF hand family protein, similar to EH-domain containing protein 1 from {Mus musculus} SP|Q9WVK4, {Homo sapiens} SP|Q9H4M9, receptormediated endocytosis 1 from (Caenorhabditis elegans) GI:13487775, GI:13487777, GI:13487779; contains INTER expressed protein, similar to EST gb|AA650671 and gb|T20610 CONSTANS-like protein-related, contains similarity to photoperiod sensitivity quantitative trait locus (Hd1) GI:11094203 from (Oryza sativa); similar to Zinc finger protein constans-like 15 (SP:Q9FHH8) {Arabidopsis thaliana} disease resistance family protein / LRR family protein, contains leucine rich-repeat domains Pfam:PF00560, INTERPRO:IPR001611; similar to disease resistance protein (Lycopersicon esculentum) gi|3894383|gb|AAC78591 Chloroplast lipoxygenase required for wound-induced jasmonic acid accumulation in Arabidopsis. glutaredoxin family protein, contains INTERPRO Domain IPR002109, Glutaredoxin (thioltransferase) expressed protein, weakly similar to drought-induced protein SDi-6 (PIR:S71562) common sunflower (fragment) expansin family protein (EXPR3), identical to Expansin-related protein 3 precursor (Ath-ExpGamma-1.2) (Swiss-Prot:Q9ZV52) (Arabidopsis thaliana); contains Prosite PS00092: N-6 Adenine-specific DNA methylases signature; Encodes flavanone 3-hydroxylase that is coordinately expressed with chalcone synthase and chalcone  FoldPchange value -2.46243  0.001209  -2.4916  0.000869  -2.49936  0.002586  -2.54324  0.000574  -2.54419  0.004796  -2.56987  0.006999  -2.57021  0.001107  -2.57961  0.010168  -2.58475  0.00011  -2.59141  0.003253  -2.60733  0.018062  85  Oligo_ID  AGI number  A012143_01  At3g25760  A011295_01  At3g25882  A203406_01  At5g64810  A202038_01  At4g00165  A009474_01  At3g55605  A200110_01  At4g27410  A019716_01 A002996_01  At5g08640 At1g56120  A020317_01  At4g27410  A016234_01 A012973_01  none At4g23160  A022890_01 A017511_01  At3g14620 At5g55410  A014949_01 A020779_01 A013669_01  none At5g42530 At4g24190  A007195_01  At2g40750  Annotation isomerases. Regulates flavonoid biosynthesis. early-responsive to dehydration stress protein (ERD12), nearly identical to early-responsive to dehydration (ERD12) protein (GI:15320414); similar to allene oxide cyclase GI:8977961 from (Lycopersicon esculentum); identical to cDNA ERD12 partial cds GI:15 NPR1/NIM1-interacting protein 2 (NIMIN-2), identical to cDNA NIMIN2 protein (nimin-2 gene)GI:12057155 member of WRKY Transcription Factor; Group II-c protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 mitochondrial glycoprotein family protein / MAM33 family protein, low similarity to SUAPRGA1 (Emericella nidulans) GI:6562379; contains Pfam profile PF02330: Mitochondrial glycoprotein Encodes a gene induced in response to dessication. flavonol synthase leucine-rich repeat family protein / protein kinase family protein, contains Pfam domains PF00560: Leucine Rich Repeat and PF00069: Protein kinase domain Encodes a gene induced in response to dessication. protein kinase family protein, contains Pfam domain PF00069: Protein kinase domain putative cytochrome P450 protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, contains Pfam profile: PF00234 protease inhibitor/seed storage/LTP family expressed protein encodes an ortholog of GRP94, an ER-resident HSP90-like protein and is involved in regulation of meristem size and organization. Single and double mutant analyses suggest that SHD may be required for the correct folding and/or complex formation of CLV pro member of WRKY Transcription  FoldPchange value -2.60868  0.000634  -2.61296  0.039208  -2.63625  0.014983  -2.6551  0.010557  -2.65856  0.003917  -2.66146  0.01606  -2.68662 -2.6908  0.024778 0.004171  -2.70695  0.000525  -2.71935 -2.74037  0.004212 0.001375  -2.76056 -2.7669  0.000761 0.00298  -2.78296 -2.80039 -2.80694  0.006606 0.000586 0.002269  -2.81802  0.007894  86  Oligo_ID  AGI number  A002960_01  At1g72950  A024555_01  At1g72910  A201020_01  At1g72930  A021221_01  At2g42530  A021287_01  At4g12490  A021021_01 A003085_01 A202675_01 A001169_01  At5g42020 At5g28540 At1g13520 At5g19240 At1g21750  A023779_01  At1g35710  A006275_01  At2g17040  A014503_01  At4g04020  A011053_01  At3g21560  Annotation Factor; Group III disease resistance protein (TIR-NBS class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. disease resistance protein (TIR-NBS class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. Toll/interleukin-1 receptor-like protein (TIR) mRNA, cold-responsive protein / coldregulated protein (cor15b), nearly identical to cold-regulated gene cor15b (Arabidopsis thaliana) GI:456016; contains Pfam profile PF02987: Late embryogenesis abundant protein protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, similar to pEARLI 1 (Accession No. L43080): an Arabidopsis member of a conserved gene family (PGF95099), Plant Physiol. 109 (4), 1497 (1995); contains Pfam protease inhibitor/se mRNA for luminal binding protein (BiP), complete cds expressed protein, expressed protein protein disulfide isomerase, putative, similar to SP|P29828 Protein disulfide isomerase precursor (PDI) (EC 5.3.4.1) {Medicago sativa}; isoform contains non-consensus GA donor splice site at intron 9 leucine-rich repeat transmembrane protein kinase, putative, similar to many predicted protein kinases no apical meristem (NAM) family protein, contains Pfam PF02365: No apical meristem (NAM) domain; similar to petunia NAM (X92205) and A. thaliana sequences ATAF1 (X74755) and ATAF2 (X74756); probable DNA-binding protein plastid-lipid associated protein PAP, putative / fibrillin, putative, strong similarity to plastid-lipid associated proteins PAP1 GI:14248554, PAP2 GI:14248556 from (Brassica rapa), fibrillin (Brassica napus) GI:4139097; contains Pfam profile PF04755: PAP UDP-glucosyltransferase, putative, similar to UDP-glucose:sinapate glucosyltransferase GI:9794913 from (Brassica napus)  FoldPchange value -2.8341  0.0023  -2.8545  0.002785  -2.85655  0.000128  -2.86205  0.004607  -2.87164  0.002353  -2.9015  0.003341  -2.90203 -2.92858 -2.93843  0.006586 0.012285 0.000793  -2.96621  0.005251  -3.00293  0.001569  -3.00734  0.004865  -3.0094  0.006203  87  Oligo_ID  AGI number  A006596_01  At2g29350  A025121_01  At3g25020  A005242_01  At5g60900  A025947_01  At1g72900  A003156_01  At1g16670  A025675_01  At1g77510  A200102_01  At4g23140  A014788_01  At4g21840  A203678_01 A025250_01  At1g47400 At3g44870 At3g44860  A021263_01  At4g23170  A200095_01  At4g14400  Annotation senescence-associated gene SAG13 encoding a short-chain alcohol dehydrogenase disease resistance family protein, contains leucine rich-repeat (LRR) domains Pfam:PF00560, INTERPRO:IPR001611; similar to Hcr2-0B (Lycopersicon esculentum) gi|3894387|gb|AAC78593 A.thaliana receptor-like protein kinase mRNA, complete cds disease resistance protein (TIR-NBS class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. protein kinase family protein, contains protein kinase domain, Pfam:PF00069; similar to receptorlike serine/threonine kinase GI:2465923 from (Arabidopsis thaliana) protein disulfide isomerase, putative, similar to protein disulfide isomerase precursor GB:P29828 GI:4704766 (Medicago sativa); Pfam HMM hit: PF00085 Thioredoxins AF224706 Arabidopsis thaliana receptor-like protein kinase 5 (RLK5) mRNA, complete cds. Naming convention from Chen et al 2003 (PMID 14756307) methionine sulfoxide reductase domain-containing protein / SelR domain-containing protein, weak similarity to pilin-like transcription factor (Homo sapiens) GI:5059062, SP|P14930 Peptide methionine sulfoxide reductase msrA/msrB (EC 1.8.4.6) {Neisseria gon expressed protein S-adenosyl-L-methionine:carboxyl methyltransferase family protein, similar to defense-related protein cjs1 (Brassica carinata)(GI:14009292)(Mol Plant Pathol (2001) 2(3):159-169) protein kinase family protein, contains Pfam PF01657: Domain of unknown function; similar to receptor-like protein kinase 5 (GI:13506747) {Arabidopsis thaliana}; similar to receptor-like protein kinase 4 (GI:13506745) (Arabidopsis thaliana) ankyrin repeat family protein, contains ankyrin repeats, Pfam domain PF00023  FoldPchange value -3.0109  0.001693  -3.02315  0.002726  -3.07172  0.018309  -3.0977  0.00413  -3.12034  0.001583  -3.16763  0.004126  -3.18849  0.002287  -3.20711  0.004976  -3.24819 -3.28392  0.00525 0.000505  -3.2995  0.002999  -3.33064  0.000357  88  Oligo_ID  AGI number  A011676_01 A203819_01  At3g57260 At3g24982  A020068_01  At3g22600  A022595_01 A007548_01  none At2g41090  A003390_01 A003203_01  At1g13470 At1g31580  A006150_01  At2g43570  A021288_01  At4g12500  A003202_01  At1g72120  A026021_01  At3g24954  A017966_01  At5g05270  A021449_01  At5g44430 At2g26010  Annotation beta 1,3-glucanase leucine-rich repeat family protein, 5' fragment, contains leucine richrepeat domains Pfam:PF00560, INTERPRO:IPR001611 (19 copies); contains similarity to GB:AAD13301 from (Lycopersicon esculentum) protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 calmodulin-like calcium-binding protein, 22 kDa (CaBP-22), identical to SP|P30187 22 kDa calmodulinlike calcium-binding protein (CABP22) (Arabidopsis thaliana) expressed protein, Encodes cell wall protein. ECS1 is not a Xcc750 resistance gene, but the genetic data indicate that ECS1 is linked to a locus influencing resistance to Xcc750. chitinase, putative, similar to chitinase class IV GI:722272 from (Brassica napus) protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, similar to pEARLI 1 (Accession No. L43080): an Arabidopsis member of a conserved gene family (PGF95099), Plant Physiol. 109 (4), 1497 (1995); contains Pfam protease inhibitor/se proton-dependent oligopeptide transport (POT) family protein, contains Pfam profile: PF00854 POT family leucine-rich repeat family protein, contains leucine rich-repeat domains Pfam:PF00560, INTERPRO:IPR001611 chalcone-flavanone isomerase family protein, contains very low similarity to chalcone-flavonone isomerase (chalcone isomerase), GI:1705761 from Vitis vinifera; contains Pfam profile PF02431: Chalconeflavanone isomerase plant defensin-fusion protein, putative (PDF1.2c), plant defensin protein family member, personal communication, Bart Thomma (Bart.Thomma@agr.kuleuven.ac.be); similar to antifungal protein 1 preprotein (Raphanus sativus)  FoldPchange value -3.35805 -3.39185  0.028809 0.00903  -3.41095  6.70E-05  -3.46854 -3.53543  6.88E-05 0.007599  -3.63767 -3.63907  0.003112 1.40E-05  -3.71296  0.010541  -3.75219  0.001305  -3.83116  9.49E-05  -3.89325  0.001051  -3.98125  0.001904  -3.98226  0.00544  89  Oligo_ID  AGI number  A021448_01  At5g44420  A003191_01 A021629_01 A017513_01  At1g67020 At2g32210 At5g13930  A001044_01  At1g75040  A021056_01 A007820_01  At2g25510 At2g14560  A016636_01  At5g55450  A200954_01 A025249_01  At1g65490 At3g44860  A017046_01  At5g24530  A023721_01  At1g14880  A024206_01 A016672_01  At5g03210 At5g54610  A021604_01  At2g26020 At5g44420  A020040_01  At1g76960  Annotation gi|609322|gb|AAA69541 Encodes an ethylene- and jasmonate-responsive plant defensin. mRNA levels are not responsive to salicylic acid treatment. hypothetical protein expressed protein Participates in the biosynthesis pathway of all flavonoids. Metabolism of defense and communication. Trancriptionally regulated by light. Required for the accumulation of purple anthocyanins in leaves and stems. Thaumatin-like protein involved in response to pathogens. expressed protein expressed protein, contains Pfam profile PF04525: Protein of unknown function (DUF567) protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 expressed protein S-adenosyl-L-methionine:carboxyl methyltransferase family protein, similar to defense-related protein cjs1 (Brassica carinata)(GI:14009292)(Mol Plant Pathol (2001) 2(3):159-169) oxidoreductase, 2OG-Fe(II) oxygenase family protein, similar to flavanone 3-hydroxylase (Persea americana)(GI:727410); contains PF03171 2OG-Fe(II) oxygenase superfamily domain expressed protein, similar to PGPS/D12 (Petunia x hybrida) GI:4105794; contains Pfam profile PF04749: Protein of unknown function, DUF614 expressed protein, ankyrin repeat family protein, contains Pfam domain, PF00023: Ankyrin repeat plant defensin-fusion protein, putative (PDF1.2b), plant defensin protein family member, personal communication, Bart Thomma (Bart.Thomma@agr.kuleuven.ac.be); similar to antifungal protein 1 preprotein (Raphanus sativus) gi|609322|gb|AAA69541 expressed protein  FoldPchange value -3.99666  0.00951  -4.01746 -4.02988 -4.22062  0.000154 0.039931 6.00E-05  -4.26921  0.000155  -4.43211 -4.45681  8.93E-05 0.002661  -4.45768  0.000347  -4.49258 -4.51435  0.000513 0.003957  -4.69776  0.000411  -4.73978  3.55E-05  -4.81544 -4.904  0.002804 0.003625  -5.25852  0.004764  -5.32756  0.00122  90  Oligo_ID  AGI number  A021603_01  At2g26010 At5g44430  A021371_01 A201730_01 A018293_01  none At3g22235 At5g10760  A009255_01 A008772_01  At3g22240 At2g24850  A022124_01  At2g14610  A019613_01  At5g03350  A021039_01  At3g22231  Annotation plant defensin-fusion protein, putative (PDF1.3), plant defensin protein family member, personal communication, Bart Thomma (Bart.Thomma@agr.kuleuven.ac.be); similar to antifungal protein 1 preprotein (Raphanus sativus) gi|609322|gb|AAA69541 expressed protein, aspartyl protease family protein, contains Pfam domain, PF00026: eukaryotic aspartyl protease expressed protein Encodes a tyrosine aminotransferase that is responsive to treatment with jasmonic acid. PR1 gene expression is induced in response to a variety of pathogens. It is a useful molecular marker for the SAR response. Though the Genbank record for the cDNA associated to this gene is called 'PR1-like', the sequence actually corresponds to PR1. legume lectin family protein, contains Pfam domain, PF00139: Legume lectins beta domain Encodes a member of a novel 6 member Arabidopsis gene family. Expression of PCC1 is regulated by the circadian clock and is upregulated in response to both virulent and avirulent strains of Pseudomonas syringae pv. tomato.  FoldPchange value -5.47525  0.003609  -6.28952 -6.89457 -7.01546  3.48E-05 0.000422 0.000496  -7.05691 -7.3268  0.001599 0.000378  -7.47025  0.001426  -7.99115  0.00128  -9.56147  2.62E-05  Table 5. 1 Genes affected by overexpression of AtMKP2 in mature plants  2. Genes affected by repression of AtMKP2 in seedlings  AtMKP2 RNAi microarray gene list (24hr DEX treatment) with 1.6 fold changes and 0.05 p value cut.  Oligo_ID  AGI number  A006796_01  At2g43590  Annotation chitinase, putative, similar to basic endochitinase CHB4 precursor SP:Q06209 from (Brassica napus)  FoldPchange value 5.063292  0.00029  91  Oligo_ID  AGI number  A000300_01 A200571_01 A017276_01  none At1g29290 At5g04050  A010001_01 A016657_01 A202024_01  none At5g04460 At3g61035  A005474_01 A025973_01  none At3g23810  A000402_01  At1g23730  A203431_01  At1g07930  A010916_01  At3g24530  A025145_01 A200897_01  none At1g61110  A203432_01  At1g07940  A024959_01  At4g20830  A025831_01 A025914_01  none At5g12250  A025177_01  At3g13470  Annotation expressed protein maturase-related, contains similarity to maturase proteins from several species expressed protein cytochrome P450 family protein, similar to Cytochrome P450 76C2 (SP:O64637) (Arabidopsis thaliana) adenosylhomocysteinase, putative / Sadenosyl-L-homocysteine hydrolase, putative / AdoHcyase, putative, strong similarity to |P50248|SAHH_TOBAC Adenosylhomocysteinase (EC 3.3.1.1) (S-adenosyl-L-homocysteine hydrolase) (AdoHcyase) {Nicotiana sylvestris}; carbonic anhydrase, putative / carbonate dehydratase, putative, similar to SP|P27140 Carbonic anhydrase, chloroplast precursor (EC 4.2.1.1) (Carbonate dehydratase) {Arabidopsis thaliana}; contains Pfam profile PF00484: Carbonic anhydrase elongation factor 1-alpha / EF-1-alpha, identical to GB:CAA34456 from (Arabidopsis thaliana) (Plant Mol. Biol. 14 (1), 107-110 (1990)) AAA-type ATPase family protein / ankyrin repeat family protein, contains Pfam profiles: PF00023 ankyrin repeat, PF00004 ATPase family associated with various cellular activities (AAA) no apical meristem (NAM) family protein, contains Pfam PF02365: No apical meristem (NAM) domain; similar to NAM protein GI:1279639 from (Petunia hybrida) elongation factor 1-alpha / EF-1-alpha, identical to GB:CAA34456 from (Arabidopsis thaliana) (Plant Mol. Biol. 14 (1), 107-110 (1990)) FAD-binding domain-containing protein, similar to SP|P30986 reticuline oxidase precursor (Berberine-bridgeforming enzyme) (BBE) (Tetrahydroprotoberberine synthase) (Eschscholzia californica); contains PF01565 FAD binding domain Encodes a beta-tubulin. Expression of TUB6 has been shown to decrease in response to cold treatment. chaperonin, putative, similar SWISS-  FoldPchange value 3.031555 2.87105 2.867738  0.003103 0.018244 0.000814  2.781295 2.779685 2.617455  0.007069 0.003246 0.006205  2.562339 2.456892  0.021362 0.005543  2.440065  0.010017  2.436207  0.004928  2.431236  0.012326  2.420466 2.379829  0.00569 0.004145  2.322377  0.014531  2.294219  0.003866  2.290884 2.270937  0.023058 0.003733  2.248077  0.005873  92  Oligo_ID  AGI number  A012399_01  At3g12860  A025390_01  At2g42100  A025091_01  At3g16410  A203454_01  At1g54270  A001980_01  At1g68570  A203514_01 A016907_01  At5g25754 At5g23580  A004779_01 A025008_01  none At4g14030  A202031_01  At3g62800  A025898_01  At1g78830  Annotation PROT:P21240- RuBisCO subunit binding-protein beta subunit, chloroplast precursor (60 kDa chaperonin beta subunit, CPN-60 beta) (Arabidopsis thaliana); contains Pfam:PF00118 domain, TCP-1/cpn60 chaperonin family nucleolar protein Nop56, putative, similar to XNop56 protein (Xenopus laevis) GI:14799394; contains Pfam profile PF01798: Putative snoRNA binding domain actin, putative, very strong similarity to SP|P53496 Actin 11 {Arabidopsis thaliana}, SP|P53493 Actin 3 {Arabidopsis thaliana}; contains Pfam profile PF00022: Actin jacalin lectin family protein, similar to myrosinase-binding protein homolog (Arabidopsis thaliana) GI:2997767, epithiospecifier (Arabidopsis thaliana) GI:16118845; contains Pfam profiles PF01419 jacalin-like lectin family, PF01344 Kelch motif member of eIF4A - eukaryotic initiation factor 4A proton-dependent oligopeptide transport (POT) family protein, contains Pfam profile: PF00854 POT family expressed protein unique family of enzymes containing a single polypeptide chain with a kinase domain at the amino terminus and a putative calcium-binding EF hands structure at the carboxyl terminus; recombinant protein is fully active and induced by Ca2+ selenium-binding protein, putative, contains Pfam profile PF05694: 56kDa selenium binding protein (SBP56); identical to Putative selenium-binding protein (Swiss-Prot:O23264) (Arabidopsis thaliana); similar to selenium binding protein (GI:15485232) (Arabi double-stranded RNA-binding domain (DsRBD)-containing protein, weak similarity to SP|P19525 Interferoninduced, double-stranded RNAactivated protein kinase (EC 2.7.1.-) {Homo sapiens}; contains Pfam profile PF00035: Double-stranded RNA binding motif curculin-like (mannose-binding) lectin family protein, similar to S glycoprotein  FoldPchange value  2.246667  0.001873  2.243983  0.000983  2.236121  0.005406  2.21423  0.001458  2.197063  0.009004  2.178511 2.163045  0.027639 0.015737  2.162315 2.124777  0.009126 0.002309  2.115281  0.042284  2.105369  0.000531  93  Oligo_ID  AGI number  A025647_01  At3g06650  A025927_01  At5g43780  A025919_01  At3g09260  A025923_01  At4g25860  A024818_01 A025265_01  At3g48980 At5g08690  A025883_01 A016665_01  At4g20890 At5g04600  A010975_01  At3g47540  A025862_01  At1g79920  A013064_01 A021479_01  none At5g23230  A002998_01 A025217_01  At1g50320 At5g39030  A025264_01  At5g08680  Annotation (Brassica rapa) GI:2351186; contains Pfam profile PF01453: Lectin (probable mannose binding) ATP-citrate synthase, putative / ATPcitrate (pro-S-)-lyase, putative / citrate cleavage enzyme, putative, strong similarity to ATP:citrate lyase (Capsicum annuum) GI:13160653; contains Pfam profiles PF00549: CoAligase, PF02629: CoA binding domain sulfate adenylyltransferase 4 / ATPsulfurylase 4 (APS4), identical to ATP sulfurylase precursor (APS4) (Arabidopsis thaliana) GI:4633131 Encodes beta-glucosydase.The major constituyent of ER bodies. One of the most abundant protein in Arabidopsis seedlings oxysterol-binding family protein, contains Pfam profile PF01237: Oxysterol-binding protein expressed protein ATP synthase beta chain 2, mitochondrial, identical to SP|P83484 ATP synthase beta chain 2, mitochondrial precursor (EC 3.6.3.14) {Arabidopsis thaliana}; strong similarity to SP|P17614 ATP synthase beta chain, mitochondrial precursor (EC 3.6.3.14) {Nicoti tubulin 9 RNA recognition motif (RRM)containing protein, contains InterPro entry IPR000504: RNA-binding region RNP-1 (RNA recognition motif) (RRM) chitinase, putative, similar to basic endochitinase CHB4 precursor SP:Q06209 from (Brassica napus) heat shock protein 70, putative / HSP70, putative, contains Pfam profile: PF00012 Heat shock hsp70 proteins; similar to heat-shock proteins GB:CAA94389, GB:AAD55461 (Arabidopsis thaliana) isochorismatase hydrolase family protein, low similarity to SP|P45743 Isochorismatase (EC 3.3.2.1) (2,3 dihydro-2,3 dihydroxybenzoate synthase) (Superoxide-inducible protein 1) (SOI1) {Bacillus subtilis}; contains Pfam profile PF00857: isochorismatase fam encodes a prokaryotic thioredoxin protein kinase family protein, contains protein kinase domain, Pfam:PF00069 ATP synthase beta chain,  FoldPchange value  2.093446  0.002804  2.087402  0.011534  2.075209  0.018045  2.072077  0.038795  2.053454 2.053347  0.019061 0.021909  2.051906 2.050125  0.016096 0.000502  2.029563  0.021921  2.02203  0.045686  2.001979 1.990825  0.007849 0.041866  1.989429 1.984426  0.000109 0.024115  1.983095  0.014453  94  Oligo_ID  AGI number  A025269_01  At2g47510  A008440_01  At2g31980  A011379_01  At3g51540  A009179_01  At3g23110  A016864_01 A025890_01  none At1g64190  A025812_01 A018948_01  none At5g22260  A203871_01  At3g42628 At2g42600  A021667_01  At4g37870  A203504_01  At5g17000  A025130_01  At5g37600  Annotation mitochondrial, putative, strong similarity to SP|P83483 ATP synthase beta chain 1, mitochondrial precursor (EC 3.6.3.14) {Arabidopsis thaliana}, SP|P17614 ATP synthase beta chain, mitochondrial precursor (EC 3.6.3.14) {Nicotiana p fumarase (FUM1) mRNA, complete cds cysteine proteinase inhibitor-related, contains similarity to extracellular insoluble cystatin GI:2204077 from (Daucus carota) expressed protein, mucin 5AC, Homo sapiens, PIR:S53363 disease resistance family protein, contains leucine rich-repeat (LRR) domains Pfam:PF00560, INTERPRO:IPR001611; similar to Cf2.2 (Lycopersicon pimpinellifolium) gi|1184077|gb|AAC15780 6-phosphogluconate dehydrogenase family protein, contains Pfam profiles: PF00393 6-phosphogluconate dehydrogenase C-terminal domain, PF03446 NAD binding domain of 6phosphogluconate Sporophytic factor controlling anther and pollen development. Mutants fail to make functional pollen;pollen degeneration occurs after microspore release and the tapetum also appears abnormally vacuolated. Similar to PHD-finger motif transcription factors. phosphoenolpyruvate carboxylaserelated / PEP carboxylase-related, identical to phosphoenolpyruvate carboxylase (Arabidopsis thaliana) GP:26800701 over first 45 residues phosphoenolpyruvate carboxykinase (ATP), putative / PEP carboxykinase, putative / PEPCK, putative, similar to phosphoenolpyruvate carboxykinase (Lycopersicon esculentum) GI:16950587, SP|Q9SLZ0 Phosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49) (PEP car NADP-dependent oxidoreductase, putative, strong similarity to probable NADP-dependent oxidoreductase (zeta-crystallin homolog) P1 (SP|Q39172)(gi:886428) and P2 (SP|Q39173)(gi:886430), Arabidopsis thaliana encodes a glutamate ammonia lyase  FoldPchange value  1.968462  0.018863  1.960682  0.002357  1.952314  0.003314  1.952196  4.42E-05  1.943734 1.935999  0.004502 0.010439  1.929456 1.928706  0.008721 0.000698  1.925643  0.00329  1.912497  0.033507  1.903355  0.006874  1.890038  0.007683  95  Oligo_ID  AGI number  A021336_01  At4g16930  A003841_01 A025459_01  At1g17810 At1g17820 At2g02010  A202318_01  At4g17490  A016834_01  At5g10130  A013100_01 A023935_01  At4g13030 At1g60030  A008617_01  At2g43620  A020112_01  At5g49280  A022490_01 A004128_01  At4g39340 At1g33440  A023717_01 A021604_01  At1g63580 At2g26020 At5g44420  A025877_01  At5g56010  A025809_01  At1g79930  A019613_01  At5g03350  A020236_01  At5g17650  Annotation disease resistance protein (TIR-NBSLRR class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. beta-tonoplast intrinsic protein (betaTIP) mRNA, complete glutamate decarboxylase, putative, strong similarity to glutamate decarboxylase isozyme 3 (Nicotiana tabacum) GI:13752462 ERF-6 mRNA for extracellular signalregulated factor, pollen Ole e 1 allergen and extensin family protein, contains similarity to pollen specific protein C13 precursor (Zea mays) SWISS-PROT:P33050 expressed protein, xanthine/uracil permease family protein, contains Pfam profile: PF00860 permease family chitinase, putative, similar to basic endochitinase CHB4 precursor SP:Q06209 from (Brassica napus) hydroxyproline-rich glycoprotein family protein, contains proline-rich extensin domains, INTERPRO:IPR002965 hypothetical protein, proton-dependent oligopeptide transport (POT) family protein, contains Pfam profile: PF00854 POT family protein kinase-related plant defensin-fusion protein, putative (PDF1.2b), plant defensin protein family member, personal communication, Bart Thomma (Bart.Thomma@agr.kuleuven.ac.be); similar to antifungal protein 1 preprotein (Raphanus sativus) gi|609322|gb|AAA69541 a member of heat shock protein 90 (HSP90) gene family. Expressed in all tissues and abundant in root apical meristem, pollen and tapetum. Expresssion is NOT heat-induced but induced by IAA and NaCl. encodes high molecular weight heat shock protein 70 not a HSP90 homolog, mRNA is constitutively expressed but transiently induced after heat shock legume lectin family protein, contains Pfam domain, PF00139: Legume lectins beta domain glycine/proline-rich protein, glycine/proline-rich protein GPRP Arabidopsis thaliana, EMBL:X84315  FoldPchange value 1.883945  0.007788  1.863806  0.00181  1.857434  0.031381  1.857432  0.022163  1.844709  0.001503  1.840163 1.833715  0.001641 0.029677  1.828802  0.005146  1.821542  0.00615  1.815836 1.815406  0.023038 0.008861  1.806556 1.801706  0.036571 0.037772  1.801212  0.004283  1.799541  0.006979  1.797542  0.003797  1.796825  0.007912  96  Oligo_ID  AGI number  A024885_01  At4g30920 At4g30910  A009736_01 A012386_01  At3g13440 At3g57810  A025793_01  At5g23020  A013250_01  At4g12720  A025875_01  At5g20000  A010263_01 A025608_01  At3g29385 At1g33590  A025780_01  At3g13772  A017939_01 A203995_01  At5g44570 At5g11170 At5g11200  A203915_01 A023420_01  At4g04394 At2g29950  A203428_01  At1g06130  A000046_01  At1g48030  A024799_01  At3g44320  A025455_01  At2g25520  Annotation cytosol aminopeptidase family protein, contains Pfam profiles: PF00883 cytosol aminopeptidase family catalytic domain, PF02789: cytosol aminopeptidase family N-terminal domain expressed protein OTU-like cysteine protease family protein, contains Pfam profile PF02338: OTU-like cysteine protease methylthioalkymalate synthase-like. Also known as 2-isopropylmalate synthase (IMS2). MutT/nudix family protein, similar to SP|P53370 Nucleoside diphosphatelinked moiety X motif 6 {Homo sapiens}; contains Pfam profile PF00293: NUDIX domain 26S proteasome AAA-ATPase subunit RPT6a (RPT6a) mRNA, hypothetical protein disease resistance protein-related / LRR protein-related, contains leucine rich-repeat domains Pfam:PF00560, INTERPRO:IPR001611; similar to Hcr2-5D (Lycopersicon esculentum) gi|3894393|gb|AAC78596 endomembrane protein 70, putative, TM4 family; hypothetical protein, DEAD/DEAH box helicase, putative (RH15), DEAD BOX RNA helicase RH15, Arabidopsis thaliana, EMBL:ATH010466 hypothetical protein expressed protein, ; expression supported by MPSS hydroxyacylglutathione hydrolase, putative / glyoxalase II, putative, similar to glyoxalase II isozyme GB:AAC49865 GI:2570338 from (Arabidopsis thaliana) dihydrolipoamide dehydrogenase 1, mitochondrial / lipoamide dehydrogenase 1 (MTLPD1), identical to GB:AAF34795 (gi:12704696) from (Arabidopsis thaliana) Nitrilase (nitrile aminohydrolase ,EC 3.5.5.1) catalyzes the hydrolysis of indole-3-acetonitrile (IAN) to indole-3acetic acid (IAA). It is the only one of the four Arabidopsis nitrilases whose mRNA levels are strongly induced when plants experience sulp phosphate translocator-related, low similarity to SP|P52178 Triose phosphate/phosphate translocator,  FoldPchange value 1.793812  0.010239  1.788905 1.787808  0.026247 0.015215  1.762533  0.006585  1.759799  0.002869  1.757893  0.00234  1.755584 1.753953  0.029672 0.025289  1.749513  0.016213  1.74777 1.747099  0.001341 0.019963  1.746493 1.744765  0.003365 0.004313  1.741517  0.030069  1.738782  0.00105  1.737973  0.005221  1.735082  0.029244  97  Oligo_ID  AGI number  A025306_01  At1g31180  A017954_01  At5g52730  A000805_01 A007343_01  At1g27670 At2g17310  A025936_01  At1g12920  A014977_01 A024657_01  none At2g17360  A000416_01  At1g04040  A025051_01  At5g11880  A021710_01  A005569_01 A007101_01  At1g24851 At1g25025 At1g25112 At1g25180 At1g24938 none At2g18350  A024946_01  At4g11030  A007820_01  At2g14560  Annotation non-green plastid, chloroplast precursor (CTPT) {Brassica oleracea}, phosphoenolpyruvate/phosphate translocator precursor (Mesembryanthemum crystallinum) 3-isopropylmalate dehydrogenase, chloroplast, putative, strong similarity to SP|P29102 3-isopropylmalate dehydrogenase, chloroplast precursor {Brassica napus}; EST gb|F14478 comes from this gene heavy-metal-associated domaincontaining protein, contains Pfam profile PF00403: Heavy-metalassociated domain expressed protein Encodes an F-Box protein that regulates a novel induced defense response independent of both salicylic acid and systemic acquired resistance Encodes a eukaryotic release factor one homolog. 40S ribosomal protein S4 (RPS4A), contains ribosomal protein S4 signature from residues 8 to 22 acid phosphatase class B family protein, similar to SP|P15490 STEM 28 kDa glycoprotein precursor (Vegetative storage protein A) {Glycine max}, acid phosphatase (Glycine max) GI:3341443; contains Pfam profile PF03767: HAD superfamily (subfamily IIIB) phosp diaminopimelate decarboxylase, putative / DAP carboxylase, putative, similar to diaminopimelate decarboxylase (Arabidopsis thaliana) GI:6562332; contains Pfam profiles PF02784: Pyridoxal-dependent decarboxylase pyridoxal binding domain, PF00278: Pyridoxal hypothetical protein  zinc finger homeobox family protein / ZF-HD homeobox family protein long-chain-fatty-acid--CoA ligase, putative / long-chain acyl-CoA synthetase, putative, similar to acylCoA synthetase (MF7P) gi:1617270 from Brassica napus expressed protein, contains Pfam profile PF04525: Protein of unknown  FoldPchange value  1.734823  0.013313  1.726847  0.013327  1.724531 1.721426  0.007481 0.009558  1.71993  0.005523  1.716831 1.715471  0.005622 0.003309  1.71517  0.008459  1.709329  0.015159  1.708569  0.006628  1.708143 1.707773  0.010565 0.018524  1.699735  0.024879  1.694084  0.015208  98  Oligo_ID  AGI number  A003835_01  At1g23050  A019956_01 A006156_01  At1g75030 At2g39050  A024827_01  At3g51440  A003511_01  At1g11520  A204094_01  At5g46490  A020804_01  At1g52070  A019782_01  At1g52150  A014058_01 A001553_01 A016359_01  At4g15840 At1g30580 At5g09440  A203477_01  At3g25520  A017943_01  At5g11500  A203503_01  At5g16990  A012849_01  At3g42050  Annotation function (DUF567) hydroxyproline-rich glycoprotein family protein, contains proline-rich extensin domains, INTERPRO:IPR002965 encodes a PR5-like protein hydroxyproline-rich glycoprotein family protein, contains QXW lectin repeat domain, Pfam:PF00652 strictosidine synthase family protein, similar to hemomucin (Drosophila melanogaster)(GI:1280434), strictosidine synthase (Rauvolfia serpentina)(SP|P15324); contains strictosidine synthase domain PF03088 pliceosome associated protein-related, contains similarity to spliceosome associated protein SAP 145 GI:1173904 from (Homo sapiens) disease resistance protein (TIR-NBS class), putative, domain signature TIRNBS exists, suggestive of a disease resistance protein. jacalin lectin family protein, similar to myrosinase-binding protein homolog (Arabidopsis thaliana) GI:2997767; contains Pfam profile PF01419 jacalinlike lectin domain Member of the class III HD-ZIP protein family. Contains homeodomain and leucine zipper domain. Involved in vascular development. expressed protein expressed protein phosphate-responsive protein, putative, similar to phi-1 (phosphateinduced gene) (Nicotiana tabacum) GI:3759184; contains Pfam profile PF04674: Phosphate-induced protein 1 conserved region Encodes ribosomal protein L5 that binds to 5S ribosomal RNA and in involed in its export from the nucleus to the cytoplasm. expressed protein, contains Pfam profile PF05670: Domain of unknown function (DUF814) NADP-dependent oxidoreductase, putative, strong similarity to probable NADP-dependent oxidoreductase (zeta-crystallin homolog) P1 (SP|Q39172)(gi:886428) and P2 (SP|Q39173)(gi:886430), Arabidopsis thaliana vacuolar ATP synthase subunit H family protein, identical to probable vacuolar ATP synthase subunit H (EC  FoldPchange value 1.693878  0.010962  1.692549 1.684324  0.040509 0.044262  1.679627  0.036676  1.678608  0.002176  1.674529  0.011985  1.673328  0.018419  1.672707  0.007802  1.671294 1.668573 1.667751  0.009019 0.004077 0.049063  1.667167  0.009788  1.666249  0.008861  1.666197  0.004311  1.665175  0.015717  99  Oligo_ID  AGI number  A005501_01  At5g24930  A021648_01  At2g20530  A023022_01  At5g22940  A018783_01  At5g59880  A007548_01  At2g41090  A023484_01 A024795_01  At2g07090 At5g26751  A016398_01  At5g59360  A025819_01  At5g27640  A203264_01 A000483_01 A203953_01  At5g44575 At1g78110 At4g08876  A017989_01  At5g43770 At5g43780  A025873_01 A201057_01 A005401_01  none At1g77122 At5g20020  A000765_01  At1g32540  A025943_01  At4g29510  Annotation 3.6.3.14)(V-ATPase H subunit) (Vacuolar proton pump H subunit) (Vacuolar proton pump subunit SFD) SP:Q9LX65 from (Arabidopsis thaliana); contains Pfa zinc finger (B-box type) family protein, similar to CONSTANS-like protein 1 GI:4091804 from (Malus x domestica) prohibitin, putative, similar to SP|P24142 Prohibitin (B-cell receptor associated protein 32) (BAP 32) {Rattus norvegicus}; contains Pfam profile PF01145: SPFH domain / Band 7 family exostosin family protein, contains Pfam profile: PF03016 exostosin family actin-depolymerizing factor 3 (ADF3), identical to SP|Q9ZSK4 Actindepolymerizing factor 3 (ADF 3) (AtADF3) {Arabidopsis thaliana} calmodulin-like calcium-binding protein, 22 kDa (CaBP-22), identical to SP|P30187 22 kDa calmodulin-like calcium-binding protein (CABP-22) (Arabidopsis thaliana) expressed protein encodes a SHAGGY-related kinase involved in meristem organization. expressed protein, predicted protein, Arabidopsis thaliana; expression supported by MPSS AF285834 Arabidopsis thaliana eukaryotic initiation factor 3B1 subunit (TIF3B1) mRNA, complete cds expressed protein, expressed protein, pyrophosphate--fructose-6-phosphate 1-phosphotransferase-related / pyrophosphate-dependent 6phosphofructose-1-kinase-related, contains weak similarity to pyrophosphate-fructose 6-phosphate 1-phosphotransferase beta-subunit gi|169540|gb|AAA63452 proline-rich family protein, contains proline-rich extensin domains, INTERPRO:IPR002965 expressed protein, Encodes a small soluble GTP-binding protein. Likely to be involved in nuclear translocation of proteins. May also be involved in cell cycle progression. Encodes a protein with 3 plant-specific zinc finger domains that acts as a positive regulator of cell death. protein arginine N-methyltransferase,  FoldPchange value  1.658399  0.026954  1.657427  0.002404  1.656745  0.027754  1.656204  0.022043  1.655201  0.001308  1.654567 1.650442  0.005433 0.03108  1.649516  0.002924  1.64778  0.005772  1.647464 1.647021 1.646872  0.027859 0.014334 0.020391  1.644252  0.025104  1.644118 1.644054 1.644026  0.025627 0.008336 0.007421  1.643781  0.011302  1.641287  0.004661  100  Oligo_ID  AGI number  Annotation  FoldPchange value  putative, similar to protein arginine Nmethyltransferase 1-variant 2 (Homo sapiens) GI:7453575 A006051_01 A025774_01  none At3g16440  A019710_01 A002651_01  At5g23900 At1g70410  A006874_01  At2g40970  A020370_01 A003682_01  At5g39570 At1g19960 At2g32140 At2g14610  A022124_01  A014152_01  At4g33110 At4g33120  A019598_01  At4g30340  A006912_01  At2g44660  A014630_01  At4g18970  A004358_01 A201425_01 A005267_01  none At2g33110 At3g13870  myrosinase-binding protein-like protein (AtMLP-300B) mRNA, 60S ribosomal protein L13 (RPL13D) carbonic anhydrase, putative / carbonate dehydratase, putative, similar to SP|P42737 Carbonic anhydrase 2 (EC 4.2.1.1) (Carbonate dehydratase 2) {Arabidopsis thaliana}; contains Pfam profile PF00484: Carbonic anhydrase myb family transcription factor, contains Pfam profile: PF00249 myblike DNA-binding domain expressed protein expressed protein PR1 gene expression is induced in response to a variety of pathogens. It is a useful molecular marker for the SAR response. Though the Genbank record for the cDNA associated to this gene is called 'PR-1-like', the sequence actually corresponds to PR1. coclaurine N-methyltransferase, putative, similar to coclaurine Nmethyltransferase (Coptis japonica) GI:16754879; contains Pfam profile PF02353: Cyclopropane-fatty-acylphospholipid synthase diacylglycerol kinase family protein, contains INTERPRO domain, IPR001206, DAG-kinase catalytic domain ALG6, ALG8 glycosyltransferase family protein, similar to SP|P40351 Dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3glucosyltransferase (EC 2.4.1.-) (Dolichyl-P-Glc:Glc1Man9GlcNAc2PP-dolichyl glucosyltransferase) {Saccharomyces cerevisiae}; contains Pfa GDSL-motif lipase/hydrolase family protein, similar to family II lipases EXL3 GI:15054386, EXL1 GI:15054382, EXL2 GI:15054384 from (Arabidopsis thaliana); contains Pfam profile PF00657: GDSL-like Lipase/Acylhydrolase member of VAMP72 Gene Family required for regulated cell expansion  1.637279 1.636876  0.00396 0.000699  1.63578 1.630584  0.001983 0.010789  1.630558  0.002117  1.629537 1.627494  0.016998 0.022893  1.627241  0.015205  1.625865  0.042384  1.622854  0.02578  1.622794  0.014809  1.617291  0.026476  1.614826 1.611919 1.611365  0.001596 0.022338 0.041743  101  Oligo_ID  AGI number  A025917_01  At5g66680  A202739_01 A018226_01 A019398_01  At5g20190 At5g13470 At5g04430  A015939_01  At5g43280  A200427_01  At1g17600  A021288_01  At4g12500  A002496_01  At1g27680  A201984_01  At3g49870  A001299_01  At1g07590  A006169_01  At2g24270  Annotation and normal root hair development. Encodes an evolutionarily conserved protein with putative GTP-binding motifs dolichyl-diphosphooligosaccharideprotein glycosyltransferase 48kDa subunit family protein, similar to SP|Q05052 Dolichyldiphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit precursor (EC 2.4.1.119) (Oligosaccharyl transferase 48 kDa subu expressed protein expressed protein KH domain-containing protein NOVA, putative, astrocytic NOVA-like RNAbinding protein, Homo sapiens, U70477 enoyl-CoA hydratase/isomerase family protein, similar to Delta 3,5-delta2,4dienoyl-CoA isomerase, mitochondrial (ECH1) from Rattus norvegicus (SP|Q62651), from Homo sapiens (SP|Q13011); contains Pfam profile PF00378 enoyl-CoA hydratase/isomerase family p disease resistance protein (TIR-NBSLRR class), putative, domain signature TIR-NBS-LRR exists, suggestive of a disease resistance protein. protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, similar to pEARLI 1 (Accession No. L43080): an Arabidopsis member of a conserved gene family (PGF95-099), Plant Physiol. 109 (4), 1497 (1995); contains Pfam protease inhibitor/se glucose-1-phosphate adenylyltransferase large subunit 2 (APL2) / ADP-glucose pyrophosphorylase, identical to SP|P55230 ADP-ribosylation factor, putative, similar to ADP-ribosylation factor-like protein 1 (SP:P40616) (Homo sapiens); ARF3 ADP-RIBOSYLATION FACTOR,GP:453191 Arabidopsis thaliana; contains domain PF00025: ADP-ribosylation factor family pentatricopeptide (PPR) repeatcontaining protein, low similarity to DNA-binding protein (Triticum aestivum) GI:6958202; contains Pfam profile PF01535: PPR repeat NADP-dependent glyceraldehyde-3phosphate dehydrogenase, putative,  FoldPchange value  1.610636  0.004829  1.607791 1.605946 1.604741  0.010188 0.037361 0.042326  1.603507  0.002344  1.602327  0.007189  1.601351  0.001033  1.600311  0.010172  1.600107  0.005326  -1.60121  0.00723  -1.60491  0.024465  102  Oligo_ID  AGI number  A008055_01  At2g02320  A202682_01  At5g19330  A014643_01  At4g30190  A200929_01  At1g63500  A020681_01  At5g44260  A201923_01 A200371_01  none At1g12850  A025115_01 A013216_01 A009135_01  At3g20090 At4g30900 At3g59930 At5g33355 At5g43370 At1g66270 At1g66280  A204090_01 A026001_01  A024664_01  At3g10450  Annotation similar to NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NON-phosphorylating glyceraldehyde 3-phosphate; glyceraldehyde-3-phosphate dehydrogenase (NADP+)) (Nicotiana plumbaginif F-box family protein / SKP1 interacting partner 3-related, contains similarity to SKP1 interacting partner 3 GI:10716951 from (Arabidopsis thaliana) armadillo/beta-catenin repeat family protein / BTB/POZ domain-containing protein, contains armadillo/betacatenin-like repeats, Pfam:PF00514 and a BTB/POZ domain, Pfam:PF00651 ATPase 2, plasma membrane-type, putative / proton pump 2, putative / proton-exporting ATPase, putative, strong similarity to SP|P19456 ATPase 2, plasma membrane-type (EC 3.6.3.6) (Proton pump 2) {Arabidopsis thaliana}; contains InterPro accession IPR00175 protein kinase-related, low similarity to protein kinase (Arabidopsis thaliana); contains Pfam profile: PF00069 Eukaryotic protein kinase domain zinc finger (CCCH-type) family protein, contains Pfam domain, PF00642: Zinc finger C-x8-C-x5-C-x3-H type (and similar) phosphoglycerate/bisphosphoglycerate mutase family protein, similar to XY4 protein (Silene vulgaris) GI:21386788; contains Pfam profile PF00300: phosphoglycerate mutase family member of CYP705A expressed protein expressed protein phosphate transporter beta-glucosidase (PSR3.2), nearly identical to GI:2286069 from (Arabidopsis thaliana) (Plant Mol. Biol. 34 (1), 57-68 (1997)); similar to thioglucoside glucohydrolase (GI:984052) (Arabidopsis thaliana) serine carboxypeptidase S10 family protein, similar to glucose acyltransferase GB:AAD01263 (Solanum berthaultii); also similar to serine carboxypeptidase I GB:P37890 (Oryza sativa)  FoldPchange value  -1.60589  0.023207  -1.60765  0.031529  -1.61565  0.023611  -1.61827  0.028315  -1.61955  0.00543  -1.62336 -1.62426  0.03948 0.006835  -1.62489 -1.62993 -1.63004  0.002132 0.007533 0.016443  -1.6308 -1.63194  0.017952 0.000151  -1.63269  0.000381  103  Oligo_ID  AGI number  A009579_01  At3g56950 At3g56940  A010681_01 A015866_01  At3g27050 At5g22920  A025989_01  At5g43350  A024326_01  At5g56870  A024517_01  At5g47450  A005187_01  At2g40940  A204083_01  At5g38430  A007314_01  At2g28910  A024298_01  At4g30270  A021209_01 A023187_01 A020560_01  none none At3g47340  A014758_01 A008605_01  At4g35770 At2g22990  A200323_01  At1g09690  Annotation small basic membrane integral family protein, contains similarity to small basic membrane integral protein ZmSIP2-1 (GI:13447817) (Zea mays) expressed protein zinc finger (C3HC4-type RING finger) family protein, contains Pfam profiles:PF05495 CHY zinc finger, PF00097 zinc finger, C3HC4 type (RING finger) mRNA for inorganic phosphate transporter, complete cds beta-galactosidase, putative / lactase, putative, similar to beta-galactosidase precursor GI:3869280 from (Carica papaya) major intrinsic family protein / MIP family protein, contains Pfam profile: MIP PF00230 encodes a protein with 67% identity to ETR1 and is involved in ethylene perception ribulose bisphosphate carboxylase small chain 1B / RuBisCO small subunit 1B (RBCS-1B) (ATS1B), identical to SP|P10796 Ribulose bisphosphate carboxylase small chain 1B, chloroplast precursor (EC 4.1.1.39) (RuBisCO small subunit 1B) {Arabidopsis thaliana} Arabidopsis thaliana CAX-interacting protein 4 mRNA, complete cds. encodes a protein similar to endo xyloglucan transferase in sequence. It is also very similar to BRU1 in soybean, which is involved in brassinosteroid response.  asparagine synthetase 1 (glutaminehydrolyzing) / glutamine-dependent asparagine synthetase 1 (ASN1), identical to SP|P49078 Asparagine synthetase (glutamine-hydrolyzing) (EC 6.3.5.4) (Glutamine- dependent asparagine synthetase) {Arabidopsis thaliana} senescence-associated gene sinapoylglucose:malate sinapoyltransferase. Catalyzes the formation of sinapoylmalate from sinapoylglucose. Mutants accumulate excess sinapoylglucose. 60S ribosomal protein L21 (RPL21C), Similar to ribosomal protein L21 (gb|L38826). ESTs gb|AA395597,gb|ATTS5197 come  FoldPchange value -1.63316  0.020918  -1.63317 -1.64252  0.006386 0.004764  -1.64539  0.000949  -1.64742  0.001317  -1.64783  0.01628  -1.64822  0.00687  -1.6568  0.011125  -1.65955  0.029121  -1.66475  0.010705  -1.68647 -1.69195 -1.70367  0.016502 0.006629 0.032581  -1.71305 -1.72241  0.00535 0.012196  -1.74447  0.005907  104  Oligo_ID  AGI number  A015102_01  At4g33010  A019041_01  At5g14320  A204096_01 A025934_01  At5g50565 At5g50665 At4g37070  A200268_01  At1g06640  A025368_01  At1g52030 At1g52040  A012647_01  At3g16400  A014789_01  At4g23670  A018064_01  At5g50920  A015071_01  At4g25100  Annotation from this gene glycine dehydrogenase (decarboxylating), putative / glycine decarboxylase, putative / glycine cleavage system P-protein, putative, strong similarity to SP|P49361 Glycine dehydrogenase (decarboxylating) A, mitochondrial precursor (EC 1.4.4.2) {Flaveria pri 30S ribosomal protein S13, chloroplast (CS13), ribosomal protein S13 precursor, chloroplast Arabidopsis thaliana, PIR:S59594; identical to cDNA ribosomal protein S13 GI:1515106 hypothetical protein patatin, putative, similar to patatin-like latex allergen (Hevea brasiliensis)(PMID:10589016); contains patatin domain PF01734 2-oxoglutarate-dependent dioxygenase, putative, similar to 2A6 (GI:599622) and tomato ethylene synthesis regulatory protein E8 (SP|P10967); contains Pfam profile: PF00671 Iron/Ascorbate oxidoreductase family Similar to myrosinase binding proteins which may be involved in metabolizing glucosinolates and forming defense compounds to protect against herbivory. Also similar to lectins and other agglutinating factorsl. Expressed only in flowers. myrosinase-binding protein-like protein (AtMLP-470) mRNA, major latex protein-related / MLPrelated, low similarity to major latex protein {Papaver somniferum}(GI:294060) contains Pfam profile PF00407: Pathogenesisrelated protein Bet v I family ClpC mRNA, nuclear gene encoding chloroplast protein, Fe-superoxide dismutase  FoldPchange value -1.7515  0.021089  -1.77785  0.003462  -1.78855  0.003433  -1.8075  0.028943  -1.80891  0.014988  -1.8092  0.025463  -1.83774  4.21E-05  -1.954  0.039754  -1.96097  0.021691  -2.25264  0.006585  Table 5. 2 Genes affected by repression of AtMKP2 in seedlings  105  

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