@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix dc: . @prefix skos: . vivo:departmentOrSchool "Science, Faculty of"@en, "Botany, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Cheng, Jia"@en ; dcterms:issued "2009-04-17T16:52:11Z"@en, "2009"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description "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 AtMKP2-RNAi 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."@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/7297?expand=metadata"@en ; dcterms:extent "1888572 bytes"@en ; dc:format "application/pdf"@en ; skos:note "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 ii 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 AtMKP2- RNAi 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. iii 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 iv 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 v4. 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 vi 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 vii 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 viii 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 ix 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 xAcknowledgements 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. 11. 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). 2In 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, 3specific 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, dual- specificity 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 4upon 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). 51.2.2 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). 61.2.3 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). 71.3 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). 8Group 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. 9AtMKP1 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 N- terminal 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). 10 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 11 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 12 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 13 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). 14 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. 15 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. MKP2- RNAi line P2iL22, 3. MKP2-RNAi line P2iL33, 4. MKP2-RNAi line P2iL21, 5. MKP2- RNAi 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 16 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 AtMKP2- RNAi 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 surface- sterilized 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 17 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. 18 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 X- 100, 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 AtMKP2- YFP 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. 19 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-2- phenylindole (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' 20 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 21 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. 22 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 root- hair 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 two- branched trichomes (Figure. 2.4). This result suggests that AtMKP2 also positively regulates trichome branching. 23 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 S- 2600N 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 24 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. 25 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 Green- fluorescent 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. 26 (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 27 (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, 28 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). 29 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 RT- PCR analysis (Figure 2.9). 30 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 31 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). 32 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 33 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) 34 Figure 2. 16 Total trichome number on first and second true leaves Figure 2. 17 Trichome branching on first and second true leaves 35 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. 36 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. 37 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 over- expression 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. 38 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 39 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. 40 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 over- expressing 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-week- old 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. 41 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 42 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 Anti- Fade 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. 43 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 Realtime- PCR 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 oC 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 44 samples were then calculated by the△△Ct method, using the formula 2 △CT (ACT) - △CT (GENE) (△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 45 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 down- regulated 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 over- expression 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. 46 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 up- regulated 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 fold- change 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 47 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 48 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%). 49 Figure 3. 2 GO annotation for genes affected by over-expression of AtMKP2 50 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 51 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 Other name Fold- change Additional annotation At3g22231 PCC1 -9.56 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. At2g14610 PR-1 -7.47 Expression is induced in response to a variety of pathogens. It is a useful molecular marker for the SAR response. At1g75040 PR-5 -4.27 Thaumatin-like protein involved in response to pathogens. At5g13930 CHS -4.22 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. At2g40750 WRKY54 -2.82 Induced by avirulent P. syringae in NPR1-independent manner; not induced by SA At5g25350 EBF2 2.45 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. At5g44610 MAP18 3.13 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. At4g35770 SEN1 5.51 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 Fold- change 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. 52 Genes Other name Fold- change Additional annotation At5g66680 DGL1 1.61 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. At3g13870 RHD3 1.61 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. At2g14610 PR1 1.63 Expression is induced in response to a variety of pathogens. It is a useful molecular marker for the SAR response. At4g12720 GFG1 1.76 Encodes a protein with ADP-ribose hydrolase activity. Negatively regulates EDS1-conditioned plant defense and programmed cell death. At4g17490 ATERF6 1.86 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. 53 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 up- regulated in the new sample compared to microarray data. This means the RNAi data set can be verified both technically and biologically. 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 0.1 1 10 100 1000 M KP 2 PR 1 SE N1 DG L1 ER F6 GF G1 RH D3 54 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 55 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 development- related genes by RT-PCR. 56 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 57 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 58 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 59 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. 60 References Abraham, S.M., and Clark, A.R. (2006). Dual-specificity phosphatase 1: a critical regulator of innate immune responses. 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Oligo_ID AGInumber Annotation Fold- change P- value A010068_01 At3g06110 dual specificity protein phosphatase family protein, contains Pfam profile: PF00782 dual specificity phosphatase, catalytic domain 25.66319 6.76E-05 A000993_01 At1g30250 expressed protein 8.987 0.00137 A021199_01 none 6.801222 0.00312 A203371_01 At5g59330 hypothetical protein 6.771661 0.000246 A016393_01 At5g59310 lipid transfer protein 4 mRNA, complete cds 6.67766 0.007102 A014758_01 At4g35770 senescence-associated gene 5.510784 0.00249 A011098_01 At3g24420 hydrolase, alpha/beta fold family protein, low similarity to 3-oxoadipate enol-lactone hydrolase (Pseudomonas sp. B13) 5.220583 0.000483 A016337_01 At5g59320 lipid transfer protein 3 mRNA, complete cds 4.928037 0.007373 A010731_01 At3g27690 Lhcb2 protein (Lhcb2:4) mRNA, complete cds 4.914438 0.002369 A013858_01 At4g21870 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. 4.161562 0.000564 A020085_01 At5g37940 At5g38000 NADP-dependent oxidoreductase, putative, similar to probable NADP- dependent oxidoreductase (zeta- crystallin homolog) P1 (SP|Q39172)(gi:886428), Arabidopsis thaliana 4.05971 0.043012 A014791_01 At4g27450 expressed protein, similar to auxin down-regulated protein ARG10 (Vigna radiata) GI:2970051, wali7 (aluminum-induced protein) (Triticum aestivum) GI:451193 4.002576 0.001604 A007306_01 At2g05100 At2g05070 Lhcb2 protein (Lhcb2.3) mRNA, complete cds 3.917433 0.000847 A203309_01 At5g50335 Expressed protein 3.91707 0.002185 A020603_01 At5g06530 ABC transporter family protein 3.871633 0.004138 A001370_01 At1g27020 expressed protein 3.815838 0.001167 A203464_01 At2g05070 At2g05100 Lhcb2 protein (Lhcb2.2) mRNA, complete cds 3.779338 0.002173 A024527_01 At1g75750 GA-responsive GAST1 protein homolog regulated by BR and GA 3.667072 0.000236 72 Oligo_ID AGInumber Annotation Fold- change P- value antagonistically. Possibly involved in cell elongation based on expression data A005727_01 At3g47340 asparagine synthetase 1 (glutamine- hydrolyzing) / 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} 3.626826 6.67E-05 A015883_01 At5g14130 At5g14120 peroxidase, putative, identical to peroxidase ATP20a (Arabidopsis thaliana) gi|1546694|emb|CAA67338 3.611591 0.001803 A021505_01 At5g47330 palmitoyl protein thioesterase family protein, 3.582128 0.002161 A016891_01 At5g22430 expressed protein 3.488863 0.011968 A001095_01 At1g62480 vacuolar calcium-binding protein- related, contains weak similarity to vacuolar calcium binding protein (Raphanus sativus) gi|9049359|dbj|BAA99394 3.470357 0.000219 A008440_01 At2g31980 cysteine proteinase inhibitor-related, contains similarity to extracellular insoluble cystatin GI:2204077 from (Daucus carota) 3.438672 0.000902 A012747_01 At3g15450 At3g15460 expressed protein, similar to auxin down-regulated protein ARG10 (Vigna radiata) GI:2970051, wali7 (aluminum-induced protein) (Triticum aestivum) GI:451193 3.415517 0.001577 A025459_01 At2g02010 glutamate decarboxylase, putative, strong similarity to glutamate decarboxylase isozyme 3 (Nicotiana tabacum) GI:13752462 3.413869 0.001927 A019919_01 At1g27030 expressed protein 3.313548 0.000222 A200108_01 At4g25100 Fe-superoxide dismutase 3.234958 0.000954 A020778_01 At3g08940 Lhcb4.2 protein (Lhcb4.2) mRNA, complete cds 3.203188 0.001084 A005379_01 none 3.193668 0.001661 A016319_01 At5g44610 DREPP plasma membrane polypeptide-related, contains Pfam profile: PF05558 DREPP plasma membrane polypeptide 3.127811 0.040966 A021504_01 At5g36130 cytochrome P450 family, simialr to taxane 13-alpha-hydroxylase (Taxus cuspidata) GI:17148242; contains Pfam profile PF00067: Cytochrome P450 3.116403 0.005355 A020286_01 At3g23560 MATE efflux family protein, similar to ripening regulated protein DDTFR18 (Lycopersicon esculentum) GI:12231296; contains Pfam profile: PF01554 uncharacterized membrane protein family 3.088999 0.002407 A018671_01 At5g17950 hypothetical protein 3.081181 0.035608 A020560_01 At3g47340 asparagine synthetase 1 (glutamine- 3.030386 0.012564 73 Oligo_ID AGInumber Annotation Fold- change P- value 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.) A011800_01 At3g47470 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. 2.993971 1.51E-05 A014936_01 At4g36410 ubiquitin-conjugating enzyme 17 (UBC17) mRNA, complete cds 2.962985 0.002648 A005366_01 At5g20250 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 2.941709 0.00274 A024680_01 At5g54270 At5g54280 Lhcb3 protein is a component of the main lightharvesting chlorophyll a/b- protein complex of Photosystem II (LHC II). 2.919609 0.00015 A200017_01 At1g28330 dormancy-associated protein (DRM1) mRNA, complete cds 2.900423 0.00096 A021034_01 At4g27440 light-dependent NADPH:protochlorophyllide oxidoreductase B 2.896053 1.94E-05 A020748_01 At2g05540 glycine-rich protein 2.889876 0.047875 A008707_01 At2g45180 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 2.781147 0.000231 A201667_01 At3g15356 legume lectin family protein, contains Pfam domain, PF00139: Legume lectins beta domain 2.773161 0.001872 A024643_01 At3g15356 legume lectin family protein, contains Pfam domain, PF00139: Legume lectins beta domain 2.754956 0.000785 A015071_01 At4g25100 Fe-superoxide dismutase 2.738357 0.003598 A002224_01 At1g78460 SOUL heme-binding family protein, weak similarity to SOUL protein (Mus musculus) GI:4886906; contains Pfam profile PF04832: SOUL heme- binding protein 2.736347 0.00082 A019670_01 none 2.713866 0.002161 A012331_01 At3g16240 At3g16250 delta tonoplast integral protein. functions as water channel and highly expressed in flower, shoot, and stem. protein localized to vacuolar membrane. 2.689999 0.002897 74 Oligo_ID AGInumber Annotation Fold- change P- value A200156_01 At5g28770 bZIP protein BZO2H3 mRNA, partial cds 2.677186 0.022797 A007535_01 At2g15960 expressed protein 2.673482 0.000447 A009135_01 At3g59930 At5g33355 expressed protein 2.668988 0.025331 A020704_01 At1g02205 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 2.667085 0.001684 A000381_01 At1g01600 member of CYP86A 2.578533 0.007992 A012865_01 At4g19420 pectinacetylesterase family protein, contains Pfam profile: PF03283 pectinacetylesterase 2.576198 0.002272 A007154_01 At2g04570 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 2.567906 0.009702 A012754_01 At3g13750 Arabidopsis thaliana mRNA for putative beta-galactosidase (BGAL1 gene). 2.555611 0.024521 A009890_01 At3g30775 osmotic stress-induced proline dehydrogenase (pro1) mRNA, 2.551062 0.003258 A008891_01 At3g60290 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 2.546272 0.010541 A016886_01 At5g24490 At5g24500 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 2.513831 0.000484 A202311_01 At4g17245 zinc finger (C3HC4-type RING finger) family protein, contains Pfam profile: PF00097 zinc finger, C3HC4 type (RING finger) 2.506057 0.000777 A007362_01 At2g33830 dormancy/auxin associated family protein, contains Pfam profile: PF05564 dormancy/auxin associated protein 2.493506 0.000171 A020688_01 At2g20870 cell wall protein precursor, putative, identical to Putative cell wall protein precursor (Swiss-Prot:P47925) (Arabidopsis thaliana); weak similarity to mu-protocadherin 2.490368 0.023493 75 Oligo_ID AGInumber Annotation Fold- change P- value (GI:7861967) (Rattus norvegicus) A019843_01 At2g38310 expressed protein, low similarity to early flowering protein 1 (Asparagus officinalis) GI:1572683, SP|P80889 Ribonuclease 1 (EC 3.1.-.-) {Panax ginseng} 2.490248 0.002756 A200012_01 At1g12080 expressed protein 2.482652 0.001695 A016820_01 At5g63530 Farnesylated protein that binds metals. 2.477417 0.01464 A006874_01 At2g40970 myb family transcription factor, contains Pfam profile: PF00249 myb- like DNA-binding domain 2.464852 0.036251 A006076_01 At2g40330 Bet v I allergen family protein, contains Pfam profile PF00407: Pathogenesis-related protein Bet v I family 2.46232 0.02964 A004704_01 At1g28330 dormancy-associated protein (DRM1) mRNA, complete cds 2.457608 8.36E-05 A011815_01 At3g05900 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) 2.453253 0.019225 A202864_01 At5g25350 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. 2.45238 0.000939 A007964_01 At2g06850 endoxyloglucan transferase (EXGT- A1) gene 2.446044 0.000364 A023934_01 At1g06350 fatty acid desaturase family protein, similar to delta 9 acyl-lipid desaturase (ADS1) GI:2970034 from (Arabidopsis thaliana) 2.441986 0.023405 A010370_01 At3g12440 extensin family protein, contains similarity to Swiss-Prot:Q38913 extensin 1 precursor (AtExt1) (AtExt4) (Arabidopsis thaliana) 2.419394 0.012674 A017271_01 At5g07550 member of Oleosin-like protein family 2.41797 0.00017 A005592_01 At5g25610 responsive to dehydration 22 (RD22) mediated by ABA 2.396124 0.000556 A015984_01 At5g40450 expressed protein 2.394075 0.000135 A006181_01 At2g14900 gibberellin-regulated family protein, similar to SP|P46690 Gibberellin- regulated protein 4 precursor {Arabidopsis thaliana} GASA4; contains Pfam profile PF02704: Gibberellin regulated protein 2.391966 0.003858 A203500_01 At4g21650 subtilase family protein, contains Pfam domain, PF00082: Subtilase family; contains Pfam domain, PF02225: protease associated (PA) domain 2.318953 0.001695 76 Oligo_ID AGInumber Annotation Fold- change P- value A006021_01 none 2.314774 0.004384 A021605_01 At2g26120 glycine-rich protein, 2.299743 0.001746 A202197_01 none 2.276257 0.015558 A022137_01 At3g48720 transferase family protein, similar to hypersensitivity-related hsr201 protein - Nicotiana tabacum,PIR2:T03274; contains Pfam transferase family domain PF00248 2.262015 0.019013 A024470_01 At1g51400 photosystem II 5 kD protein, 100% identical to GI:4836947 (F5D21.10) 2.261651 0.001784 A024649_01 At1g29680 At1g29670 expressed protein 2.255592 0.00064 A002045_01 At1g74670 gibberellin-responsive protein, putative, similar to SP|P46690 Gibberellin-regulated protein 4 precursor {Arabidopsis thaliana} GASA4; contains Pfam profile PF02704: Gibberellin regulated protein 2.232983 0.002409 A024378_01 At4g04630 expressed protein, contains Pfam profile PF04520: Protein of unknown function, DUF584 2.226941 0.003069 A003990_01 At1g74870 expressed protein, contains similarity to hypothetical proteins 2.216655 0.019728 A010009_01 At3g51910 heat shock transcription factor family protein, contains Pfam profile: PF00447 HSF-type DNA-binding domain 2.214072 0.026084 A021515_01 At5g09490 40S ribosomal protein S15 (RPS15B), ribosomal protein S15 - Arabidopsis thaliana, EMBL:Z23161 2.210122 0.048967 A006824_01 At2g33810 putative transcription factor that binds DNA and may directly regulate AP1, involved in regulation of flowering 2.209626 0.001809 A019305_01 At5g37300 expressed protein 2.203243 0.010707 A007422_01 At2g48020 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 2.188867 0.009809 A024664_01 At3g10450 serine carboxypeptidase S10 family protein, similar to glucose acyltransferase GB:AAD01263 (Solanum berthaultii); also similar to serine carboxypeptidase I GB:P37890 (Oryza sativa) 2.182121 0.018903 A005629_01 At5g21170 5'-AMP-activated protein kinase beta-2 subunit, putative, similar to Swiss-Prot:Q9QZH4 5'-AMP- activated protein kinase, beta-2 subunit (AMPK beta-2 chain) (Rattus norvegicus) 2.180966 0.005202 77 Oligo_ID AGInumber Annotation Fold- change P- value A025164_01 At3g23550 MATE efflux family protein, similar to ripening regulated protein DDTFR18 (Lycopersicon esculentum) GI:12231296; contains Pfam profile: PF01554 uncharacterized membrane protein family 2.177566 0.025718 A021615_01 none 2.176194 0.000704 A002988_01 At1g04220 At1g04210 beta-ketoacyl-CoA synthase, putative, Strong similarity to beta- keto-Coa synthase gb|U37088 from Simmondsia chinensis, GI:4091810 2.172909 0.011086 A025898_01 At1g78830 curculin-like (mannose-binding) lectin family protein, similar to S glycoprotein (Brassica rapa) GI:2351186; contains Pfam profile PF01453: Lectin (probable mannose binding) 2.171303 0.003993 A012008_01 At3g02710 nuclear associated protein-related / NAP-related, similar to Nuclear associated protein (NAP) (NYD- SP19) (Swiss-Prot:Q8WYA6) (Homo sapiens) 2.171094 0.042299 A014768_01 none 2.158403 0.020627 A001679_01 At1g55330 arabinogalactan-protein (AGP21) 2.154897 0.001069 A021052_01 At2g34430 Photosystem II type I chlorophyll a/b-binding protein 2.145318 0.006586 A023904_01 At1g49130 zinc finger (B-box type) family protein, contains similarity to zinc finger protein GI:3618318 from (Oryza sativa) 2.138207 0.014893 A003745_01 At3g29770 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 2.133221 0.028744 A005969_01 At1g61520 PSI type III chlorophyll a/b-binding protein (Lhca3*1) 2.130999 0.004365 A003475_01 At1g80920 J8 mRNA, nuclear gene encoding plastid protein, complete 2.119444 0.000416 A203242_01 At5g40730 arabinogalactan-protein (AGP24) 2.119073 0.015532 A024617_01 At1g49240 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 2.115946 0.003498 A003291_01 At1g58290 2.112983 0.000237 A016210_01 At5g14780 NAD-dependent formate dehydrogenase 1B (FDH1B) mRNA, 2.112535 0.001473 A021481_01 At5g23370 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 2.10653 0.009315 78 Oligo_ID AGInumber Annotation Fold- change P- value PF02893: GRAM domain A019439_01 At2g37430 zinc finger (C2H2 type) family protein (ZAT11), contains Pfam domain, PF00096: Zinc finger, C2H2 type 2.102014 0.010609 A007604_01 At2g04795 Expressed protein 2.098601 0.002497 A014175_01 At4g33970 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 2.096639 0.001719 A005989_01 At1g29910 At1g29930 At1g29920 member of Chlorophyll a/b-binding protein family 2.095632 0.012656 A020902_01 At1g68530 member of Strong similarity to beta- keto-Coa synthase family 2.085214 0.004439 A021237_01 At3g55240 expressed protein 2.080588 0.008297 A009579_01 At3g56950 At3g56940 small basic membrane integral family protein, contains similarity to small basic membrane integral protein ZmSIP2-1 (GI:13447817) (Zea mays) 2.078797 0.008324 A202455_01 At4g31290 ChaC-like family protein, contains Pfam profile: PF04752 ChaC-like protein 2.070445 0.006895 A005741_01 At2g34420 Photosystem II type I chlorophyll a/b-binding protein 2.070295 0.000265 A200460_01 At1g19510 myb family transcription factor, contains PFAM profile: PF00249 myb-like DNA binding domain 2.063297 0.012819 A202671_01 At5g19190 expressed protein, predicted protein, Arabidopsis thaliana 2.06156 0.006473 A012626_01 At3g22160 VQ motif-containing protein, contains PF05678: VQ motif 2.059864 0.003617 A000828_01 At1g80180 expressed protein 2.053053 0.001978 A006657_01 At2g16850 plasma membrane intrinsic protein, putative, very strong similarity to plasma membrane intrinsic protein (SIMIP) (Arabidopsis thaliana) GI:2306917 2.050502 0.022481 A020400_01 At3g15630 expressed protein 2.048221 0.001173 A015182_01 At4g13830 DnaJ-like protein (J20) mRNA, complete cds; nuclear gene 2.040772 0.00319 A011875_01 At3g57690 arabinogalactan-protein, putative (AGP23), similar to arabinogalactan protein (Arabidopsis thaliana) gi|10880503|gb|AAG24281 2.036752 0.00919 A010748_01 At3g58910 F-box family protein, contains F-box domain Pfam:PF00646 2.030622 0.002078 A008578_01 At2g38400 alanine--glyoxylate aminotransferase, putative / beta- alanine-pyruvate aminotransferase, putative / AGT, putative, similar to SP|Q64565 Alanine--glyoxylate aminotransferase 2, mitochondrial 2.029291 0.001145 79 Oligo_ID AGInumber Annotation Fold- change P- value precursor (EC 2.6.1.44) (AGT 2) (Beta-alanine-pyruvate aminotrans A018838_01 At5g64920 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. 2.02389 0.000504 A021811_01 At1g80920 At1g80910 J8 mRNA, nuclear gene encoding plastid protein, complete 2.014919 0.005877 A016445_01 At5g64750 AP2 domain-containing transcription factor, putative, contains similarity to transcription factor 2.012769 0.004196 A202546_01 At5g04310 pectate lyase family protein, similar to pectate lyase GP:14531296 from (Fragaria x ananassa) 2.012254 0.034286 A020760_01 At4g32930 At4g32940 expressed protein, predicted protein, Caenorhabditis elegans, gb:Z70780 2.012174 0.001988 A003507_01 At1g12080 expressed protein 2.010814 0.013008 A002276_01 none 2.010058 0.010156 A025135_01 At5g49730 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 2.000484 0.005866 A008472_01 At2g37040 encodes a protein similar to phenylalanine ammonia-lyase -2.00046 0.000614 A022194_01 At3g55190 esterase/lipase/thioesterase family protein, similar to monoglyceride lipase from (Homo sapiens) GI:14594904, (Mus musculus) GI:2632162; contains Interpro entry IPR000379 -2.00112 0.007028 A025177_01 At3g13470 chaperonin, putative, similar SWISS- 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 -2.00543 0.012066 A024162_01 none -2.00669 0.000971 A020001_01 At1g55490 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 -2.00908 0.002888 A017188_01 At5g01900 member of WRKY Transcription Factor; Group III -2.00984 0.031323 A014515_01 At4g04330 expressed protein -2.01 0.00278 80 Oligo_ID AGInumber Annotation Fold- change P- value A200947_01 At1g64900 cytochrome P450 (CYP89A2) mRNA, complete cds -2.01187 0.000753 A005197_01 At1g27350 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 -2.02227 0.00025 A008080_01 At2g27660 DC1 domain-containing protein, contains Pfam profile PF03107: DC1 domain -2.02487 0.009601 A023474_01 At2g29120 member of Putative ligand-gated ion channel subunit family -2.02979 0.002438 A021656_01 At2g07880 hypothetical protein -2.03148 0.046524 A022469_01 At4g10500 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 -2.05042 0.004005 A006046_01 At2g43510 Encodes putative trypsin inbitor protein which may function in defense against herbivory. -2.05616 0.00794 A020806_01 At2g30860 Encodes glutathione transferase belonging to the phi class of GSTs. Naming convention according to Wagner et al. (2002). -2.05808 0.000798 A200051_01 At2g32160 expressed protein -2.0596 0.006234 A015892_01 At5g58070 lipocalin, putative, similar to temperature stress-induced lipocalin (Triticum aestivum) GI:18650668 -2.07427 0.000533 A011678_01 At3g54960 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 -2.07574 0.028395 A003138_01 At1g78570 At1g78580 NAD-dependent epimerase/dehydratase family protein, similar to dTDP-glucose 4,6- dehydratase from Aneurinibacillus thermoaerophilus GI:16357461, RmlB from Leptospira borgpetersenii GI:4234803; contains Pfam profile PF01370 NAD dependent epimerase/dehydrata -2.0839 0.002802 A020763_01 At1g10960 ferredoxin, chloroplast, putative, strong similarity to FERREDOXIN PRECURSOR GB:P16972 (SP|P16972) from (Arabidopsis thaliana) -2.08469 2.58E-05 A020462_01 At4g39950 Belongs to cytochrome P450 and is involved in tryptophan metabolism. Converts Trp to indo-3- acetaldoxinme (IAOx), a precursor to IAA and indole glucosinolates. -2.09598 5.20E-05 A022417_01 At4g24920 protein transport protein SEC61 -2.09687 4.48E-06 81 Oligo_ID AGInumber Annotation Fold- change P- value gamma subunit, putative, similar to Swiss-Prot:Q19967 protein transport protein SEC61 gamma subunit (Caenorhabditis elegans) A017938_01 At5g06320 NDR1/HIN1-like protein 3 (NHL3) mRNA, complete cds -2.09753 0.002908 A014477_01 At4g03450 ankyrin repeat family protein, contains ankyrin repeats, Pfam domain PF00023 -2.10199 0.010262 A021217_01 At1g75830 plant defensin-fusion protein, putative (PDF1.1), identical to SP|P30224 Cysteine-rich antifungal protein 1 precursor (AFP1) (Anther- specific protein S18 homolog) {Arabidopsis thaliana} -2.12447 0.002085 A011673_01 At3g09440 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} -2.1268 0.000202 A003569_01 At1g04980 thioredoxin family protein, similar to SP|Q63081 Protein disulfide isomerase A6 precursor (EC 5.3.4.1) {Rattus norvegicus}; contains Pfam profile PF00085: Thioredoxin -2.13514 0.012799 A021700_01 none -2.14046 0.002069 A013804_01 At4g12480 pEARLI 1 mRNA, complete cds -2.1569 0.001325 A009611_01 At3g08590 2,3-biphosphoglycerate-independent phosphoglycerate mutase, putative / phosphoglyceromutase, putative, strong similarity to SP|Q42908 2,3- bisphosphoglycerate-independent phosphoglycerate mutase (EC 5.4.2.1) (Phosphoglyceromutase) {Mesembryanthemum crystal -2.1584 0.000477 A024307_01 At1g21270 cytoplasmic serine/threonine protein kinase induced by salicylic acid -2.1598 0.026634 A025929_01 At3g49120 Encodes a peroxidase. -2.1655 0.009557 A024557_01 At1g27730 Related to Cys2/His2-type zinc- finger 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 -2.16974 6.71E-07 A025089_01 At3g25490 wall-associated kinase, putative, similar to wall-associated kinase 4 GB:CAA08793 from (Arabidopsis thaliana) -2.17798 0.00697 A005695_01 At1g65800 At1g65790 S-receptor protein kinase, putative, similar to PIR|T05180|T05180 S- receptor kinase ARK3 precursor - (Arabidopsis thaliana) -2.17808 0.005176 A014416_01 At4g20110 vacuolar sorting receptor, putative, similar to BP-80 vacuolar sorting receptor (Pisum sativum) -2.18067 0.001094 82 Oligo_ID AGInumber Annotation Fold- change P- value GI:1737222; identical to vacuolar sorting receptor-like protein (GI:2827665) (Arabidopsis thaliana) A200075_01 At3g17609 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 -2.18466 0.00142 A202426_01 At4g27657 Expressed protein -2.1849 0.012824 A202676_01 At5g19250 expressed protein -2.20053 0.000834 A021247_01 At3g46080 zinc finger (C2H2 type) family protein, contains zinc finger, C2H2 type, domain, PROSITE:PS00028 -2.20111 0.001111 A202924_01 At5g26690 heavy-metal-associated domain- containing protein, low similarity to farnesylated protein GMFP5 (Glycine max)(GI:4097571); contains Pfam profile PF00403: Heavy-metal- associated domain -2.20264 0.026079 A006717_01 At2g39030 GCN5-related N-acetyltransferase (GNAT) family protein, similar to SP|Q9SMB8 Tyramine N- feruloyltransferase 4/11 (EC 2.3.1.110) (Hydroxycinnamoyl- CoA: tyramine N- hydroxycinnamoyltransferase) {Nicotiana tabacum}; contains Pfam profile PF00583: acetyltrans -2.20293 0.027243 A025877_01 At5g56010 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. -2.2057 0.015645 A202287_01 At4g16146 Expressed protein -2.2302 0.000558 A020011_01 At2g46440 At2g46430 member of Cyclic nucleotide gated channel family -2.23143 0.00249 A002695_01 At1g56340 calreticulin (Crt1) mRNA, complete cds -2.23289 0.00423 A006761_01 At2g31880 At2g31890 leucine-rich repeat transmembrane protein kinase, putative -2.23346 0.002023 A018927_01 At5g24210 lipase class 3 family protein, contains Pfam profile PF01764: Lipase -2.23674 0.002801 A202909_01 At5g26170 member of WRKY Transcription Factor; Group II-c -2.25902 0.015372 A003835_01 At1g23050 hydroxyproline-rich glycoprotein family protein, contains proline-rich extensin domains, INTERPRO:IPR002965 -2.25965 0.0008 A025828_01 At1g66970 glycerophosphoryl diester phosphodiesterase family protein, contains Pfam PF03009 : Glycerophosphoryl diester -2.25968 0.01211 83 Oligo_ID AGInumber Annotation Fold- change P- value phosphodiesterase family A022269_01 At5g10380 zinc finger (C3HC4-type RING finger) family protein, contains Pfam profile: PF00097 zinc finger, C3HC4 type (RING finger) -2.26019 0.020234 A025696_01 At5g27060 disease resistance family protein, contains leucine rich-repeat (LRR) domains Pfam:PF00560, INTERPRO:IPR001611; similar to Hcr2-0B (Lycopersicon esculentum) gi|3894387|gb|AAC78593 -2.26542 0.028663 A203310_01 At5g50460 protein transport protein SEC61 gamma subunit, putative, similar to Swiss-Prot:Q19967 protein transport protein SEC61 gamma subunit (Caenorhabditis elegans) -2.27047 0.000586 A003204_01 At1g60050 nodulin-related, low similarity to MtN21 (Medicago truncatula) GI:2598575; contains Pfam profile PF00892: Integral membrane protein -2.27348 7.22E-05 A011710_01 At3g11340 UDP-glucoronosyl/UDP-glucosyl transferase family protein, contains Pfam profile: PF00201 UDP- glucoronosyl and UDP-glucosyl transferase -2.27802 0.006924 A018891_01 At5g20630 germin-like protein (GLP3b) mRNA, complete cds -2.27986 8.30E-07 A012346_01 At3g11090 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 -2.28563 0.014451 A011432_01 At3g25010 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 -2.29977 0.031314 A202718_01 At5g19875 Expressed protein -2.29995 0.024395 A022222_01 none -2.30165 0.010236 A021488_01 At5g52760 heavy-metal-associated domain- containing protein, contains Pfam profile PF00403: Heavy-metal- associated domain -2.31133 0.042237 A011050_01 At3g13950 expressed protein -2.3125 0.003953 A024349_01 At1g72900 At1g72910 disease resistance protein (TIR-NBS class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. -2.31323 0.003471 A008966_01 At3g47480 calcium-binding EF hand family protein, contains INTERPRO:IPR002048 calcium- binding EF-hand domain -2.31521 0.003723 A003378_01 At1g71100 ribose 5-phosphate isomerase- -2.32891 0.007076 84 Oligo_ID AGInumber Annotation Fold- change P- value related, similar to ribose-5- phosphate isomerase GI:18654317 from (Spinacia oleracea) A024141_01 At2g32680 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 -2.33548 0.037811 A002917_01 At1g68620 expressed protein, similar to PrMC3 (Pinus radiata) GI:5487873 -2.33999 0.029859 A008693_01 At2g25110 MIR domain-containing protein, similar to SP|Q99470 Stromal cell- derived factor 2 precursor (SDF-2) {Homo sapiens}; contains Pfam profile PF02815: MIR domain -2.34017 0.008408 A203264_01 At5g44575 expressed protein, -2.34122 0.009311 A024841_01 At3g61220 short-chain dehydrogenase/reductase (SDR) family protein, similar to carbonyl reductase GI:1049108 from (Mus musculus) -2.34403 0.038645 A007808_01 At2g21150 XAP5 family protein, contains Pfam profile: PF04921 XAP5 protein -2.34649 0.013879 A001618_01 At1g24145 expressed protein, -2.3554 3.18E-05 A025932_01 At1g21250 cell wall-associated kinase, may funtion as a signaling receptor of extracellular matrix component. -2.37209 0.020056 A021286_01 At4g12470 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 -2.3901 0.000479 A019418_01 At5g52940 hypothetical protein, contains Pfam profile PF03478: Protein of unknown function (DUF295) -2.41895 0.00178 A019642_01 At5g48880 peroxisomal 3-keto-acyl-CoA thiolase 2 precursor (PKT2) -2.42875 0.002278 A025079_01 At3g24900 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 -2.44017 0.000108 A019812_01 At3g51860 member of Low affinity calcium antiporter CAX2 family -2.44755 0.008316 A202674_01 At5g19230 expressed protein -2.45625 0.003891 A011469_01 At3g09940 monodehydroascorbate reductase, putative, similar to monodehydroascorbate reductase (NADH) GB:JU0182 (Cucumis sativus) -2.45627 0.003434 85 Oligo_ID AGInumber Annotation Fold- change P- value A016950_01 At5g61790 calnexin 1 (CNX1), identical to calnexin homolog 1, Arabidopsis thaliana, EMBL:AT08315 (SP|P29402) -2.46243 0.001209 A202381_01 At4g21830 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 -2.4916 0.000869 A022573_01 At4g05520 calcium-binding EF hand family protein, similar to EH-domain containing protein 1 from {Mus musculus} SP|Q9WVK4, {Homo sapiens} SP|Q9H4M9, receptor- mediated endocytosis 1 from (Caenorhabditis elegans) GI:13487775, GI:13487777, GI:13487779; contains INTER -2.49936 0.002586 A200552_01 At1g27330 expressed protein, similar to EST gb|AA650671 and gb|T20610 -2.54324 0.000574 A001038_01 At1g07050 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} -2.54419 0.004796 A201621_01 At3g11010 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 -2.56987 0.006999 A009087_01 At3g45140 Chloroplast lipoxygenase required for wound-induced jasmonic acid accumulation in Arabidopsis. -2.57021 0.001107 A003718_01 At1g28480 glutaredoxin family protein, contains INTERPRO Domain IPR002109, Glutaredoxin (thioltransferase) -2.57961 0.010168 A003555_01 At1g13930 expressed protein, weakly similar to drought-induced protein SDi-6 (PIR:S71562) common sunflower (fragment) -2.58475 0.00011 A006621_01 At2g18660 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; -2.59141 0.003253 A020903_01 At3g51240 Encodes flavanone 3-hydroxylase that is coordinately expressed with chalcone synthase and chalcone -2.60733 0.018062 86 Oligo_ID AGInumber Annotation Fold- change P- value isomerases. Regulates flavonoid biosynthesis. A012143_01 At3g25760 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 -2.60868 0.000634 A011295_01 At3g25882 NPR1/NIM1-interacting protein 2 (NIMIN-2), identical to cDNA NIMIN- 2 protein (nimin-2 gene)GI:12057155 -2.61296 0.039208 A203406_01 At5g64810 member of WRKY Transcription Factor; Group II-c -2.63625 0.014983 A202038_01 At4g00165 protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 -2.6551 0.010557 A009474_01 At3g55605 mitochondrial glycoprotein family protein / MAM33 family protein, low similarity to SUAPRGA1 (Emericella nidulans) GI:6562379; contains Pfam profile PF02330: Mitochondrial glycoprotein -2.65856 0.003917 A200110_01 At4g27410 Encodes a gene induced in response to dessication. -2.66146 0.01606 A019716_01 At5g08640 flavonol synthase -2.68662 0.024778 A002996_01 At1g56120 leucine-rich repeat family protein / protein kinase family protein, contains Pfam domains PF00560: Leucine Rich Repeat and PF00069: Protein kinase domain -2.6908 0.004171 A020317_01 At4g27410 Encodes a gene induced in response to dessication. -2.70695 0.000525 A016234_01 none -2.71935 0.004212 A012973_01 At4g23160 protein kinase family protein, contains Pfam domain PF00069: Protein kinase domain -2.74037 0.001375 A022890_01 At3g14620 putative cytochrome P450 -2.76056 0.000761 A017511_01 At5g55410 protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, contains Pfam profile: PF00234 protease inhibitor/seed storage/LTP family -2.7669 0.00298 A014949_01 none -2.78296 0.006606 A020779_01 At5g42530 expressed protein -2.80039 0.000586 A013669_01 At4g24190 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 -2.80694 0.002269 A007195_01 At2g40750 member of WRKY Transcription -2.81802 0.007894 87 Oligo_ID AGInumber Annotation Fold- change P- value Factor; Group III A002960_01 At1g72950 disease resistance protein (TIR-NBS class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. -2.8341 0.0023 A024555_01 At1g72910 disease resistance protein (TIR-NBS class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. -2.8545 0.002785 A201020_01 At1g72930 Toll/interleukin-1 receptor-like protein (TIR) mRNA, -2.85655 0.000128 A021221_01 At2g42530 cold-responsive protein / cold- regulated protein (cor15b), nearly identical to cold-regulated gene cor15b (Arabidopsis thaliana) GI:456016; contains Pfam profile PF02987: Late embryogenesis abundant protein -2.86205 0.004607 A021287_01 At4g12490 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 -2.87164 0.002353 A021021_01 At5g42020 At5g28540 mRNA for luminal binding protein (BiP), complete cds -2.9015 0.003341 A003085_01 At1g13520 expressed protein, -2.90203 0.006586 A202675_01 At5g19240 expressed protein -2.92858 0.012285 A001169_01 At1g21750 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 -2.93843 0.000793 A023779_01 At1g35710 leucine-rich repeat transmembrane protein kinase, putative, similar to many predicted protein kinases -2.96621 0.005251 A006275_01 At2g17040 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 -3.00293 0.001569 A014503_01 At4g04020 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 -3.00734 0.004865 A011053_01 At3g21560 UDP-glucosyltransferase, putative, similar to UDP-glucose:sinapate glucosyltransferase GI:9794913 from (Brassica napus) -3.0094 0.006203 88 Oligo_ID AGInumber Annotation Fold- change P- value A006596_01 At2g29350 senescence-associated gene SAG13 encoding a short-chain alcohol dehydrogenase -3.0109 0.001693 A025121_01 At3g25020 disease resistance family protein, contains leucine rich-repeat (LRR) domains Pfam:PF00560, INTERPRO:IPR001611; similar to Hcr2-0B (Lycopersicon esculentum) gi|3894387|gb|AAC78593 -3.02315 0.002726 A005242_01 At5g60900 A.thaliana receptor-like protein kinase mRNA, complete cds -3.07172 0.018309 A025947_01 At1g72900 disease resistance protein (TIR-NBS class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. -3.0977 0.00413 A003156_01 At1g16670 protein kinase family protein, contains protein kinase domain, Pfam:PF00069; similar to receptor- like serine/threonine kinase GI:2465923 from (Arabidopsis thaliana) -3.12034 0.001583 A025675_01 At1g77510 protein disulfide isomerase, putative, similar to protein disulfide isomerase precursor GB:P29828 GI:4704766 (Medicago sativa); Pfam HMM hit: PF00085 Thioredoxins -3.16763 0.004126 A200102_01 At4g23140 AF224706 Arabidopsis thaliana receptor-like protein kinase 5 (RLK5) mRNA, complete cds. Naming convention from Chen et al 2003 (PMID 14756307) -3.18849 0.002287 A014788_01 At4g21840 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 -3.20711 0.004976 A203678_01 At1g47400 expressed protein -3.24819 0.00525 A025250_01 At3g44870 At3g44860 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) -3.28392 0.000505 A021263_01 At4g23170 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) -3.2995 0.002999 A200095_01 At4g14400 ankyrin repeat family protein, contains ankyrin repeats, Pfam domain PF00023 -3.33064 0.000357 89 Oligo_ID AGInumber Annotation Fold- change P- value A011676_01 At3g57260 beta 1,3-glucanase -3.35805 0.028809 A203819_01 At3g24982 leucine-rich repeat family protein, 5' fragment, contains leucine rich- repeat domains Pfam:PF00560, INTERPRO:IPR001611 (19 copies); contains similarity to GB:AAD13301 from (Lycopersicon esculentum) -3.39185 0.00903 A020068_01 At3g22600 protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 -3.41095 6.70E-05 A022595_01 none -3.46854 6.88E-05 A007548_01 At2g41090 calmodulin-like calcium-binding protein, 22 kDa (CaBP-22), identical to SP|P30187 22 kDa calmodulin- like calcium-binding protein (CABP- 22) (Arabidopsis thaliana) -3.53543 0.007599 A003390_01 At1g13470 expressed protein, -3.63767 0.003112 A003203_01 At1g31580 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. -3.63907 1.40E-05 A006150_01 At2g43570 chitinase, putative, similar to chitinase class IV GI:722272 from (Brassica napus) -3.71296 0.010541 A021288_01 At4g12500 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 -3.75219 0.001305 A003202_01 At1g72120 proton-dependent oligopeptide transport (POT) family protein, contains Pfam profile: PF00854 POT family -3.83116 9.49E-05 A026021_01 At3g24954 leucine-rich repeat family protein, contains leucine rich-repeat domains Pfam:PF00560, INTERPRO:IPR001611 -3.89325 0.001051 A017966_01 At5g05270 chalcone-flavanone isomerase family protein, contains very low similarity to chalcone-flavonone isomerase (chalcone isomerase), GI:1705761 from Vitis vinifera; contains Pfam profile PF02431: Chalcone- flavanone isomerase -3.98125 0.001904 A021449_01 At5g44430 At2g26010 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) -3.98226 0.00544 90 Oligo_ID AGInumber Annotation Fold- change P- value gi|609322|gb|AAA69541 A021448_01 At5g44420 Encodes an ethylene- and jasmonate-responsive plant defensin. mRNA levels are not responsive to salicylic acid treatment. -3.99666 0.00951 A003191_01 At1g67020 hypothetical protein -4.01746 0.000154 A021629_01 At2g32210 expressed protein -4.02988 0.039931 A017513_01 At5g13930 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. -4.22062 6.00E-05 A001044_01 At1g75040 Thaumatin-like protein involved in response to pathogens. -4.26921 0.000155 A021056_01 At2g25510 expressed protein -4.43211 8.93E-05 A007820_01 At2g14560 expressed protein, contains Pfam profile PF04525: Protein of unknown function (DUF567) -4.45681 0.002661 A016636_01 At5g55450 protease inhibitor/seed storage/lipid transfer protein (LTP) family protein, contains Pfam protease inhibitor/seed storage/LTP family domain PF00234 -4.45768 0.000347 A200954_01 At1g65490 expressed protein -4.49258 0.000513 A025249_01 At3g44860 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) -4.51435 0.003957 A017046_01 At5g24530 oxidoreductase, 2OG-Fe(II) oxygenase family protein, similar to flavanone 3-hydroxylase (Persea americana)(GI:727410); contains PF03171 2OG-Fe(II) oxygenase superfamily domain -4.69776 0.000411 A023721_01 At1g14880 expressed protein, similar to PGPS/D12 (Petunia x hybrida) GI:4105794; contains Pfam profile PF04749: Protein of unknown function, DUF614 -4.73978 3.55E-05 A024206_01 At5g03210 expressed protein, -4.81544 0.002804 A016672_01 At5g54610 ankyrin repeat family protein, contains Pfam domain, PF00023: Ankyrin repeat -4.904 0.003625 A021604_01 At2g26020 At5g44420 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 -5.25852 0.004764 A020040_01 At1g76960 expressed protein -5.32756 0.00122 91 Oligo_ID AGInumber Annotation Fold- change P- value A021603_01 At2g26010 At5g44430 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 -5.47525 0.003609 A021371_01 none -6.28952 3.48E-05 A201730_01 At3g22235 expressed protein, -6.89457 0.000422 A018293_01 At5g10760 aspartyl protease family protein, contains Pfam domain, PF00026: eukaryotic aspartyl protease -7.01546 0.000496 A009255_01 At3g22240 expressed protein -7.05691 0.001599 A008772_01 At2g24850 Encodes a tyrosine aminotransferase that is responsive to treatment with jasmonic acid. -7.3268 0.000378 A022124_01 At2g14610 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. -7.47025 0.001426 A019613_01 At5g03350 legume lectin family protein, contains Pfam domain, PF00139: Legume lectins beta domain -7.99115 0.00128 A021039_01 At3g22231 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. -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 AGInumber Annotation Fold- change P- value A006796_01 At2g43590 chitinase, putative, similar to basic endochitinase CHB4 precursor SP:Q06209 from (Brassica napus) 5.063292 0.00029 92 Oligo_ID AGInumber Annotation Fold- change P- value A000300_01 none 3.031555 0.003103 A200571_01 At1g29290 expressed protein 2.87105 0.018244 A017276_01 At5g04050 maturase-related, contains similarity to maturase proteins from several species 2.867738 0.000814 A010001_01 none 2.781295 0.007069 A016657_01 At5g04460 expressed protein 2.779685 0.003246 A202024_01 At3g61035 cytochrome P450 family protein, similar to Cytochrome P450 76C2 (SP:O64637) (Arabidopsis thaliana) 2.617455 0.006205 A005474_01 none 2.562339 0.021362 A025973_01 At3g23810 adenosylhomocysteinase, putative / S- adenosyl-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}; 2.456892 0.005543 A000402_01 At1g23730 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 2.440065 0.010017 A203431_01 At1g07930 elongation factor 1-alpha / EF-1-alpha, identical to GB:CAA34456 from (Arabidopsis thaliana) (Plant Mol. Biol. 14 (1), 107-110 (1990)) 2.436207 0.004928 A010916_01 At3g24530 AAA-type ATPase family protein / ankyrin repeat family protein, contains Pfam profiles: PF00023 ankyrin repeat, PF00004 ATPase family associated with various cellular activities (AAA) 2.431236 0.012326 A025145_01 none 2.420466 0.00569 A200897_01 At1g61110 no apical meristem (NAM) family protein, contains Pfam PF02365: No apical meristem (NAM) domain; similar to NAM protein GI:1279639 from (Petunia hybrida) 2.379829 0.004145 A203432_01 At1g07940 elongation factor 1-alpha / EF-1-alpha, identical to GB:CAA34456 from (Arabidopsis thaliana) (Plant Mol. Biol. 14 (1), 107-110 (1990)) 2.322377 0.014531 A024959_01 At4g20830 FAD-binding domain-containing protein, similar to SP|P30986 reticuline oxidase precursor (Berberine-bridge- forming enzyme) (BBE) (Tetrahydroprotoberberine synthase) (Eschscholzia californica); contains PF01565 FAD binding domain 2.294219 0.003866 A025831_01 none 2.290884 0.023058 A025914_01 At5g12250 Encodes a beta-tubulin. Expression of TUB6 has been shown to decrease in response to cold treatment. 2.270937 0.003733 A025177_01 At3g13470 chaperonin, putative, similar SWISS- 2.248077 0.005873 93 Oligo_ID AGInumber Annotation Fold- change P- value 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 A012399_01 At3g12860 nucleolar protein Nop56, putative, similar to XNop56 protein (Xenopus laevis) GI:14799394; contains Pfam profile PF01798: Putative snoRNA binding domain 2.246667 0.001873 A025390_01 At2g42100 actin, putative, very strong similarity to SP|P53496 Actin 11 {Arabidopsis thaliana}, SP|P53493 Actin 3 {Arabidopsis thaliana}; contains Pfam profile PF00022: Actin 2.243983 0.000983 A025091_01 At3g16410 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 2.236121 0.005406 A203454_01 At1g54270 member of eIF4A - eukaryotic initiation factor 4A 2.21423 0.001458 A001980_01 At1g68570 proton-dependent oligopeptide transport (POT) family protein, contains Pfam profile: PF00854 POT family 2.197063 0.009004 A203514_01 At5g25754 expressed protein 2.178511 0.027639 A016907_01 At5g23580 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+ 2.163045 0.015737 A004779_01 none 2.162315 0.009126 A025008_01 At4g14030 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 2.124777 0.002309 A202031_01 At3g62800 double-stranded RNA-binding domain (DsRBD)-containing protein, weak similarity to SP|P19525 Interferon- induced, double-stranded RNA- activated protein kinase (EC 2.7.1.-) {Homo sapiens}; contains Pfam profile PF00035: Double-stranded RNA binding motif 2.115281 0.042284 A025898_01 At1g78830 curculin-like (mannose-binding) lectin family protein, similar to S glycoprotein 2.105369 0.000531 94 Oligo_ID AGInumber Annotation Fold- change P- value (Brassica rapa) GI:2351186; contains Pfam profile PF01453: Lectin (probable mannose binding) A025647_01 At3g06650 ATP-citrate synthase, putative / ATP- citrate (pro-S-)-lyase, putative / citrate cleavage enzyme, putative, strong similarity to ATP:citrate lyase (Capsicum annuum) GI:13160653; contains Pfam profiles PF00549: CoA- ligase, PF02629: CoA binding domain 2.093446 0.002804 A025927_01 At5g43780 sulfate adenylyltransferase 4 / ATP- sulfurylase 4 (APS4), identical to ATP sulfurylase precursor (APS4) (Arabidopsis thaliana) GI:4633131 2.087402 0.011534 A025919_01 At3g09260 Encodes beta-glucosydase.The major constituyent of ER bodies. One of the most abundant protein in Arabidopsis seedlings 2.075209 0.018045 A025923_01 At4g25860 oxysterol-binding family protein, contains Pfam profile PF01237: Oxysterol-binding protein 2.072077 0.038795 A024818_01 At3g48980 expressed protein 2.053454 0.019061 A025265_01 At5g08690 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 2.053347 0.021909 A025883_01 At4g20890 tubulin 9 2.051906 0.016096 A016665_01 At5g04600 RNA recognition motif (RRM)- containing protein, contains InterPro entry IPR000504: RNA-binding region RNP-1 (RNA recognition motif) (RRM) 2.050125 0.000502 A010975_01 At3g47540 chitinase, putative, similar to basic endochitinase CHB4 precursor SP:Q06209 from (Brassica napus) 2.029563 0.021921 A025862_01 At1g79920 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) 2.02203 0.045686 A013064_01 none 2.001979 0.007849 A021479_01 At5g23230 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 1.990825 0.041866 A002998_01 At1g50320 encodes a prokaryotic thioredoxin 1.989429 0.000109 A025217_01 At5g39030 protein kinase family protein, contains protein kinase domain, Pfam:PF00069 1.984426 0.024115 A025264_01 At5g08680 ATP synthase beta chain, 1.983095 0.014453 95 Oligo_ID AGInumber Annotation Fold- change P- value 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 A025269_01 At2g47510 fumarase (FUM1) mRNA, complete cds 1.968462 0.018863 A008440_01 At2g31980 cysteine proteinase inhibitor-related, contains similarity to extracellular insoluble cystatin GI:2204077 from (Daucus carota) 1.960682 0.002357 A011379_01 At3g51540 expressed protein, mucin 5AC, Homo sapiens, PIR:S53363 1.952314 0.003314 A009179_01 At3g23110 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 1.952196 4.42E-05 A016864_01 none 1.943734 0.004502 A025890_01 At1g64190 6-phosphogluconate dehydrogenase family protein, contains Pfam profiles: PF00393 6-phosphogluconate dehydrogenase C-terminal domain, PF03446 NAD binding domain of 6- phosphogluconate 1.935999 0.010439 A025812_01 none 1.929456 0.008721 A018948_01 At5g22260 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. 1.928706 0.000698 A203871_01 At3g42628 At2g42600 phosphoenolpyruvate carboxylase- related / PEP carboxylase-related, identical to phosphoenolpyruvate carboxylase (Arabidopsis thaliana) GP:26800701 over first 45 residues 1.925643 0.00329 A021667_01 At4g37870 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 1.912497 0.033507 A203504_01 At5g17000 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 1.903355 0.006874 A025130_01 At5g37600 encodes a glutamate ammonia lyase 1.890038 0.007683 96 Oligo_ID AGInumber Annotation Fold- change P- value A021336_01 At4g16930 disease resistance protein (TIR-NBS- LRR class), putative, domain signature TIR-NBS exists, suggestive of a disease resistance protein. 1.883945 0.007788 A003841_01 At1g17810 At1g17820 beta-tonoplast intrinsic protein (beta- TIP) mRNA, complete 1.863806 0.00181 A025459_01 At2g02010 glutamate decarboxylase, putative, strong similarity to glutamate decarboxylase isozyme 3 (Nicotiana tabacum) GI:13752462 1.857434 0.031381 A202318_01 At4g17490 ERF-6 mRNA for extracellular signal- regulated factor, 1.857432 0.022163 A016834_01 At5g10130 pollen Ole e 1 allergen and extensin family protein, contains similarity to pollen specific protein C13 precursor (Zea mays) SWISS-PROT:P33050 1.844709 0.001503 A013100_01 At4g13030 expressed protein, 1.840163 0.001641 A023935_01 At1g60030 xanthine/uracil permease family protein, contains Pfam profile: PF00860 permease family 1.833715 0.029677 A008617_01 At2g43620 chitinase, putative, similar to basic endochitinase CHB4 precursor SP:Q06209 from (Brassica napus) 1.828802 0.005146 A020112_01 At5g49280 hydroxyproline-rich glycoprotein family protein, contains proline-rich extensin domains, INTERPRO:IPR002965 1.821542 0.00615 A022490_01 At4g39340 hypothetical protein, 1.815836 0.023038 A004128_01 At1g33440 proton-dependent oligopeptide transport (POT) family protein, contains Pfam profile: PF00854 POT family 1.815406 0.008861 A023717_01 At1g63580 protein kinase-related 1.806556 0.036571 A021604_01 At2g26020 At5g44420 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 1.801706 0.037772 A025877_01 At5g56010 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. 1.801212 0.004283 A025809_01 At1g79930 encodes high molecular weight heat shock protein 70 not a HSP90 homolog, mRNA is constitutively expressed but transiently induced after heat shock 1.799541 0.006979 A019613_01 At5g03350 legume lectin family protein, contains Pfam domain, PF00139: Legume lectins beta domain 1.797542 0.003797 A020236_01 At5g17650 glycine/proline-rich protein, glycine/proline-rich protein GPRP - Arabidopsis thaliana, EMBL:X84315 1.796825 0.007912 97 Oligo_ID AGInumber Annotation Fold- change P- value A024885_01 At4g30920 At4g30910 cytosol aminopeptidase family protein, contains Pfam profiles: PF00883 cytosol aminopeptidase family catalytic domain, PF02789: cytosol aminopeptidase family N-terminal domain 1.793812 0.010239 A009736_01 At3g13440 expressed protein 1.788905 0.026247 A012386_01 At3g57810 OTU-like cysteine protease family protein, contains Pfam profile PF02338: OTU-like cysteine protease 1.787808 0.015215 A025793_01 At5g23020 methylthioalkymalate synthase-like. Also known as 2-isopropylmalate synthase (IMS2). 1.762533 0.006585 A013250_01 At4g12720 MutT/nudix family protein, similar to SP|P53370 Nucleoside diphosphate- linked moiety X motif 6 {Homo sapiens}; contains Pfam profile PF00293: NUDIX domain 1.759799 0.002869 A025875_01 At5g20000 26S proteasome AAA-ATPase subunit RPT6a (RPT6a) mRNA, 1.757893 0.00234 A010263_01 At3g29385 hypothetical protein 1.755584 0.029672 A025608_01 At1g33590 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 1.753953 0.025289 A025780_01 At3g13772 endomembrane protein 70, putative, TM4 family; 1.749513 0.016213 A017939_01 At5g44570 hypothetical protein, 1.74777 0.001341 A203995_01 At5g11170 At5g11200 DEAD/DEAH box helicase, putative (RH15), DEAD BOX RNA helicase RH15, Arabidopsis thaliana, EMBL:ATH010466 1.747099 0.019963 A203915_01 At4g04394 hypothetical protein 1.746493 0.003365 A023420_01 At2g29950 expressed protein, ; expression supported by MPSS 1.744765 0.004313 A203428_01 At1g06130 hydroxyacylglutathione hydrolase, putative / glyoxalase II, putative, similar to glyoxalase II isozyme GB:AAC49865 GI:2570338 from (Arabidopsis thaliana) 1.741517 0.030069 A000046_01 At1g48030 dihydrolipoamide dehydrogenase 1, mitochondrial / lipoamide dehydrogenase 1 (MTLPD1), identical to GB:AAF34795 (gi:12704696) from (Arabidopsis thaliana) 1.738782 0.00105 A024799_01 At3g44320 Nitrilase (nitrile aminohydrolase ,EC 3.5.5.1) catalyzes the hydrolysis of indole-3-acetonitrile (IAN) to indole-3- acetic acid (IAA). It is the only one of the four Arabidopsis nitrilases whose mRNA levels are strongly induced when plants experience sulp 1.737973 0.005221 A025455_01 At2g25520 phosphate translocator-related, low similarity to SP|P52178 Triose phosphate/phosphate translocator, 1.735082 0.029244 98 Oligo_ID AGInumber Annotation Fold- change P- value non-green plastid, chloroplast precursor (CTPT) {Brassica oleracea}, phosphoenolpyruvate/phosphate translocator precursor (Mesembryanthemum crystallinum) A025306_01 At1g31180 3-isopropylmalate dehydrogenase, chloroplast, putative, strong similarity to SP|P29102 3-isopropylmalate dehydrogenase, chloroplast precursor {Brassica napus}; EST gb|F14478 comes from this gene 1.734823 0.013313 A017954_01 At5g52730 heavy-metal-associated domain- containing protein, contains Pfam profile PF00403: Heavy-metal- associated domain 1.726847 0.013327 A000805_01 At1g27670 expressed protein 1.724531 0.007481 A007343_01 At2g17310 Encodes an F-Box protein that regulates a novel induced defense response independent of both salicylic acid and systemic acquired resistance 1.721426 0.009558 A025936_01 At1g12920 Encodes a eukaryotic release factor one homolog. 1.71993 0.005523 A014977_01 none 1.716831 0.005622 A024657_01 At2g17360 40S ribosomal protein S4 (RPS4A), contains ribosomal protein S4 signature from residues 8 to 22 1.715471 0.003309 A000416_01 At1g04040 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 1.71517 0.008459 A025051_01 At5g11880 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 1.709329 0.015159 A021710_01 At1g24851 At1g25025 At1g25112 At1g25180 At1g24938 hypothetical protein 1.708569 0.006628 A005569_01 none 1.708143 0.010565 A007101_01 At2g18350 zinc finger homeobox family protein / ZF-HD homeobox family protein 1.707773 0.018524 A024946_01 At4g11030 long-chain-fatty-acid--CoA ligase, putative / long-chain acyl-CoA synthetase, putative, similar to acyl- CoA synthetase (MF7P) gi:1617270 from Brassica napus 1.699735 0.024879 A007820_01 At2g14560 expressed protein, contains Pfam profile PF04525: Protein of unknown 1.694084 0.015208 99 Oligo_ID AGInumber Annotation Fold- change P- value function (DUF567) A003835_01 At1g23050 hydroxyproline-rich glycoprotein family protein, contains proline-rich extensin domains, INTERPRO:IPR002965 1.693878 0.010962 A019956_01 At1g75030 encodes a PR5-like protein 1.692549 0.040509 A006156_01 At2g39050 hydroxyproline-rich glycoprotein family protein, contains QXW lectin repeat domain, Pfam:PF00652 1.684324 0.044262 A024827_01 At3g51440 strictosidine synthase family protein, similar to hemomucin (Drosophila melanogaster)(GI:1280434), strictosidine synthase (Rauvolfia serpentina)(SP|P15324); contains strictosidine synthase domain PF03088 1.679627 0.036676 A003511_01 At1g11520 pliceosome associated protein-related, contains similarity to spliceosome associated protein SAP 145 GI:1173904 from (Homo sapiens) 1.678608 0.002176 A204094_01 At5g46490 disease resistance protein (TIR-NBS class), putative, domain signature TIR- NBS exists, suggestive of a disease resistance protein. 1.674529 0.011985 A020804_01 At1g52070 jacalin lectin family protein, similar to myrosinase-binding protein homolog (Arabidopsis thaliana) GI:2997767; contains Pfam profile PF01419 jacalin- like lectin domain 1.673328 0.018419 A019782_01 At1g52150 Member of the class III HD-ZIP protein family. Contains homeodomain and leucine zipper domain. Involved in vascular development. 1.672707 0.007802 A014058_01 At4g15840 expressed protein 1.671294 0.009019 A001553_01 At1g30580 expressed protein 1.668573 0.004077 A016359_01 At5g09440 phosphate-responsive protein, putative, similar to phi-1 (phosphate- induced gene) (Nicotiana tabacum) GI:3759184; contains Pfam profile PF04674: Phosphate-induced protein 1 conserved region 1.667751 0.049063 A203477_01 At3g25520 Encodes ribosomal protein L5 that binds to 5S ribosomal RNA and in involed in its export from the nucleus to the cytoplasm. 1.667167 0.009788 A017943_01 At5g11500 expressed protein, contains Pfam profile PF05670: Domain of unknown function (DUF814) 1.666249 0.008861 A203503_01 At5g16990 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 1.666197 0.004311 A012849_01 At3g42050 vacuolar ATP synthase subunit H family protein, identical to probable vacuolar ATP synthase subunit H (EC 1.665175 0.015717 100 Oligo_ID AGInumber Annotation Fold- change P- value 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 A005501_01 At5g24930 zinc finger (B-box type) family protein, similar to CONSTANS-like protein 1 GI:4091804 from (Malus x domestica) 1.658399 0.026954 A021648_01 At2g20530 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 1.657427 0.002404 A023022_01 At5g22940 exostosin family protein, contains Pfam profile: PF03016 exostosin family 1.656745 0.027754 A018783_01 At5g59880 actin-depolymerizing factor 3 (ADF3), identical to SP|Q9ZSK4 Actin- depolymerizing factor 3 (ADF 3) (AtADF3) {Arabidopsis thaliana} 1.656204 0.022043 A007548_01 At2g41090 calmodulin-like calcium-binding protein, 22 kDa (CaBP-22), identical to SP|P30187 22 kDa calmodulin-like calcium-binding protein (CABP-22) (Arabidopsis thaliana) 1.655201 0.001308 A023484_01 At2g07090 expressed protein 1.654567 0.005433 A024795_01 At5g26751 encodes a SHAGGY-related kinase involved in meristem organization. 1.650442 0.03108 A016398_01 At5g59360 expressed protein, predicted protein, Arabidopsis thaliana; expression supported by MPSS 1.649516 0.002924 A025819_01 At5g27640 AF285834 Arabidopsis thaliana eukaryotic initiation factor 3B1 subunit (TIF3B1) mRNA, complete cds 1.64778 0.005772 A203264_01 At5g44575 expressed protein, 1.647464 0.027859 A000483_01 At1g78110 expressed protein, 1.647021 0.014334 A203953_01 At4g08876 pyrophosphate--fructose-6-phosphate 1-phosphotransferase-related / pyrophosphate-dependent 6- phosphofructose-1-kinase-related, contains weak similarity to pyrophosphate-fructose 6-phosphate 1-phosphotransferase beta-subunit gi|169540|gb|AAA63452 1.646872 0.020391 A017989_01 At5g43770 At5g43780 proline-rich family protein, contains proline-rich extensin domains, INTERPRO:IPR002965 1.644252 0.025104 A025873_01 none 1.644118 0.025627 A201057_01 At1g77122 expressed protein, 1.644054 0.008336 A005401_01 At5g20020 Encodes a small soluble GTP-binding protein. Likely to be involved in nuclear translocation of proteins. May also be involved in cell cycle progression. 1.644026 0.007421 A000765_01 At1g32540 Encodes a protein with 3 plant-specific zinc finger domains that acts as a positive regulator of cell death. 1.643781 0.011302 A025943_01 At4g29510 protein arginine N-methyltransferase, 1.641287 0.004661 101 Oligo_ID AGInumber Annotation Fold- change P- value putative, similar to protein arginine N- methyltransferase 1-variant 2 (Homo sapiens) GI:7453575 A006051_01 none 1.637279 0.00396 A025774_01 At3g16440 myrosinase-binding protein-like protein (AtMLP-300B) mRNA, 1.636876 0.000699 A019710_01 At5g23900 60S ribosomal protein L13 (RPL13D) 1.63578 0.001983 A002651_01 At1g70410 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 1.630584 0.010789 A006874_01 At2g40970 myb family transcription factor, contains Pfam profile: PF00249 myb- like DNA-binding domain 1.630558 0.002117 A020370_01 At5g39570 expressed protein 1.629537 0.016998 A003682_01 At1g19960 At2g32140 expressed protein 1.627494 0.022893 A022124_01 At2g14610 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. 1.627241 0.015205 A014152_01 At4g33110 At4g33120 coclaurine N-methyltransferase, putative, similar to coclaurine N- methyltransferase (Coptis japonica) GI:16754879; contains Pfam profile PF02353: Cyclopropane-fatty-acyl- phospholipid synthase 1.625865 0.042384 A019598_01 At4g30340 diacylglycerol kinase family protein, contains INTERPRO domain, IPR001206, DAG-kinase catalytic domain 1.622854 0.02578 A006912_01 At2g44660 ALG6, ALG8 glycosyltransferase family protein, similar to SP|P40351 Dolichyl pyrophosphate Glc1Man9GlcNAc2 alpha-1,3- glucosyltransferase (EC 2.4.1.-) (Dolichyl-P-Glc:Glc1Man9GlcNAc2- PP-dolichyl glucosyltransferase) {Saccharomyces cerevisiae}; contains Pfa 1.622794 0.014809 A014630_01 At4g18970 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 1.617291 0.026476 A004358_01 none 1.614826 0.001596 A201425_01 At2g33110 member of VAMP72 Gene Family 1.611919 0.022338 A005267_01 At3g13870 required for regulated cell expansion 1.611365 0.041743 102 Oligo_ID AGInumber Annotation Fold- change P- value and normal root hair development. Encodes an evolutionarily conserved protein with putative GTP-binding motifs A025917_01 At5g66680 dolichyl-diphosphooligosaccharide- protein glycosyltransferase 48kDa subunit family protein, similar to SP|Q05052 Dolichyl- diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit precursor (EC 2.4.1.119) (Oligosaccharyl transferase 48 kDa subu 1.610636 0.004829 A202739_01 At5g20190 expressed protein 1.607791 0.010188 A018226_01 At5g13470 expressed protein 1.605946 0.037361 A019398_01 At5g04430 KH domain-containing protein NOVA, putative, astrocytic NOVA-like RNA- binding protein, Homo sapiens, U70477 1.604741 0.042326 A015939_01 At5g43280 enoyl-CoA hydratase/isomerase family protein, similar to Delta 3,5-delta2,4- dienoyl-CoA isomerase, mitochondrial (ECH1) from Rattus norvegicus (SP|Q62651), from Homo sapiens (SP|Q13011); contains Pfam profile PF00378 enoyl-CoA hydratase/isomerase family p 1.603507 0.002344 A200427_01 At1g17600 disease resistance protein (TIR-NBS- LRR class), putative, domain signature TIR-NBS-LRR exists, suggestive of a disease resistance protein. 1.602327 0.007189 A021288_01 At4g12500 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 1.601351 0.001033 A002496_01 At1g27680 glucose-1-phosphate adenylyltransferase large subunit 2 (APL2) / ADP-glucose pyrophosphorylase, identical to SP|P55230 1.600311 0.010172 A201984_01 At3g49870 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 1.600107 0.005326 A001299_01 At1g07590 pentatricopeptide (PPR) repeat- containing protein, low similarity to DNA-binding protein (Triticum aestivum) GI:6958202; contains Pfam profile PF01535: PPR repeat -1.60121 0.00723 A006169_01 At2g24270 NADP-dependent glyceraldehyde-3- phosphate dehydrogenase, putative, -1.60491 0.024465 103 Oligo_ID AGInumber Annotation Fold- change P- value similar to NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NON-phosphorylating glyceraldehyde 3-phosphate; glyceraldehyde-3-phosphate dehydrogenase (NADP+)) (Nicotiana plumbaginif A008055_01 At2g02320 F-box family protein / SKP1 interacting partner 3-related, contains similarity to SKP1 interacting partner 3 GI:10716951 from (Arabidopsis thaliana) -1.60589 0.023207 A202682_01 At5g19330 armadillo/beta-catenin repeat family protein / BTB/POZ domain-containing protein, contains armadillo/beta- catenin-like repeats, Pfam:PF00514 and a BTB/POZ domain, Pfam:PF00651 -1.60765 0.031529 A014643_01 At4g30190 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 -1.61565 0.023611 A200929_01 At1g63500 protein kinase-related, low similarity to protein kinase (Arabidopsis thaliana); contains Pfam profile: PF00069 Eukaryotic protein kinase domain -1.61827 0.028315 A020681_01 At5g44260 zinc finger (CCCH-type) family protein, contains Pfam domain, PF00642: Zinc finger C-x8-C-x5-C-x3-H type (and similar) -1.61955 0.00543 A201923_01 none -1.62336 0.03948 A200371_01 At1g12850 phosphoglycerate/bisphosphoglycerate mutase family protein, similar to XY4 protein (Silene vulgaris) GI:21386788; contains Pfam profile PF00300: phosphoglycerate mutase family -1.62426 0.006835 A025115_01 At3g20090 member of CYP705A -1.62489 0.002132 A013216_01 At4g30900 expressed protein -1.62993 0.007533 A009135_01 At3g59930 At5g33355 expressed protein -1.63004 0.016443 A204090_01 At5g43370 phosphate transporter -1.6308 0.017952 A026001_01 At1g66270 At1g66280 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) -1.63194 0.000151 A024664_01 At3g10450 serine carboxypeptidase S10 family protein, similar to glucose acyltransferase GB:AAD01263 (Solanum berthaultii); also similar to serine carboxypeptidase I GB:P37890 (Oryza sativa) -1.63269 0.000381 104 Oligo_ID AGInumber Annotation Fold- change P- value A009579_01 At3g56950 At3g56940 small basic membrane integral family protein, contains similarity to small basic membrane integral protein ZmSIP2-1 (GI:13447817) (Zea mays) -1.63316 0.020918 A010681_01 At3g27050 expressed protein -1.63317 0.006386 A015866_01 At5g22920 zinc finger (C3HC4-type RING finger) family protein, contains Pfam profiles:PF05495 CHY zinc finger, PF00097 zinc finger, C3HC4 type (RING finger) -1.64252 0.004764 A025989_01 At5g43350 mRNA for inorganic phosphate transporter, complete cds -1.64539 0.000949 A024326_01 At5g56870 beta-galactosidase, putative / lactase, putative, similar to beta-galactosidase precursor GI:3869280 from (Carica papaya) -1.64742 0.001317 A024517_01 At5g47450 major intrinsic family protein / MIP family protein, contains Pfam profile: MIP PF00230 -1.64783 0.01628 A005187_01 At2g40940 encodes a protein with 67% identity to ETR1 and is involved in ethylene perception -1.64822 0.00687 A204083_01 At5g38430 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} -1.6568 0.011125 A007314_01 At2g28910 Arabidopsis thaliana CAX-interacting protein 4 mRNA, complete cds. -1.65955 0.029121 A024298_01 At4g30270 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. -1.66475 0.010705 A021209_01 none -1.68647 0.016502 A023187_01 none -1.69195 0.006629 A020560_01 At3g47340 asparagine synthetase 1 (glutamine- hydrolyzing) / 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} -1.70367 0.032581 A014758_01 At4g35770 senescence-associated gene -1.71305 0.00535 A008605_01 At2g22990 sinapoylglucose:malate sinapoyltransferase. Catalyzes the formation of sinapoylmalate from sinapoylglucose. Mutants accumulate excess sinapoylglucose. -1.72241 0.012196 A200323_01 At1g09690 60S ribosomal protein L21 (RPL21C), Similar to ribosomal protein L21 (gb|L38826). ESTs gb|AA395597,gb|ATTS5197 come -1.74447 0.005907 105 Oligo_ID AGInumber Annotation Fold- change P- value from this gene A015102_01 At4g33010 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 -1.7515 0.021089 A019041_01 At5g14320 30S ribosomal protein S13, chloroplast (CS13), ribosomal protein S13 precursor, chloroplast Arabidopsis thaliana, PIR:S59594; identical to cDNA ribosomal protein S13 GI:1515106 -1.77785 0.003462 A204096_01 At5g50565 At5g50665 hypothetical protein -1.78855 0.003433 A025934_01 At4g37070 patatin, putative, similar to patatin-like latex allergen (Hevea brasiliensis)(PMID:10589016); contains patatin domain PF01734 -1.8075 0.028943 A200268_01 At1g06640 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 -1.80891 0.014988 A025368_01 At1g52030 At1g52040 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. -1.8092 0.025463 A012647_01 At3g16400 myrosinase-binding protein-like protein (AtMLP-470) mRNA, -1.83774 4.21E-05 A014789_01 At4g23670 major latex protein-related / MLP- related, low similarity to major latex protein {Papaver somniferum}(GI:294060) contains Pfam profile PF00407: Pathogenesis- related protein Bet v I family -1.954 0.039754 A018064_01 At5g50920 ClpC mRNA, nuclear gene encoding chloroplast protein, -1.96097 0.021691 A015071_01 At4g25100 Fe-superoxide dismutase -2.25264 0.006585 Table 5. 2 Genes affected by repression of AtMKP2 in seedlings"@en ; edm:hasType "Thesis/Dissertation"@en ; vivo:dateIssued "2009-05"@en ; edm:isShownAt "10.14288/1.0067145"@en ; dcterms:language "eng"@en ; ns0:degreeDiscipline "Botany"@en ; edm:provider "Vancouver : University of British Columbia Library"@en ; dcterms:publisher "University of British Columbia"@en ; dcterms:rights "Attribution-NonCommercial-NoDerivatives 4.0 International"@en ; ns0:rightsURI "http://creativecommons.org/licenses/by-nc-nd/4.0/"@en ; ns0:scholarLevel "Graduate"@en ; dcterms:title "Functional analysis of MAPK phosphatase AtMKP2"@en ; dcterms:type "Text"@en ; ns0:identifierURI "http://hdl.handle.net/2429/7297"@en .