The NSF 2010 Arabidopsis Project





Project Participant
Project Abstract
Project Gene List
Project Signal List
MAPK Cascades
MAPK Cascades Resources
MAPK Cascades Publications
MAPK Cascades Clones


Project Participant

Jen Sheen Massachusetts General Hospital PI
Organize the project and develop most of the experimental and bioinformatics tools

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Project Abstract


     The goal of this project is to build a detailed molecular regulatory network centered on six MAPKKs (MKK1, 2, 4, 5, 7, 9) that play essential roles in linking ten upstream MAPKKKs (MTKs), six downstream MAPKs (MPKs), 12 transcription factors (TFs), and thousands of target genes in response to 12 signals that control Arabidopsis growth and development. Numerous studies have indicated that plant MAPK cascades are important for controlling diverse stress and defense responses and various developmental processes. The project will determine how specific MPKs and their immediate upstream regulators, MKKs and MTKs are integrated into the plant-signaling network connecting upstream signals and downstream transcription factors (TFs) and target genes in Arabidopsis.

      In the first phase of the functional genomic analysis of Arabidopsis MAPK cascade signaling, 20 MPK, 10 MKK and 60 putative MTK genes have been cloned and analyzed in Arabidopsis mesophyll protoplasts to establish MAPK cascade functions in the H2O2, flg22, and ethylene signaling pathways. The information has established the core of a MAPK cascade-signaling network including 10 MTKs, 6 MKKs and 6 MPKs that will serve as the foundation to launch new genome-wide studies linking 12 dynamic and overlapping signal transduction pathways. The 34 genes and 12 signals are listed on the MAPK Cascade Project website This project combines global gene expression profiling and bioinformatics tools for individual MKKs and TFs with gain-of-function and loss-of-function mutant analyses to construct overlapping MAPK signaling cascades linked directly to key TFs and a large number of target genes in response to stress, elicitors, and hormonal signals.

      The experimental design is based on the novel Arabidopsis protoplast technology combined with microarray and bioinformatics analysis in WT and mutants. The transient nature of the protoplast system enables direct and dynamic functional analyses of MAPK cascades and TFs at an unprecedented high throughput rate and at relatively low cost. The proposed experimental approaches are especially powerful in unraveling the regulation of a large number of genes that are difficult to tackle by traditional genetic and biochemical approaches due to redundancy, lethality or low levels of expression. Our proposal aims to elucidate the functional roles of MAPK signaling cascades in stress, defense and development by combining cell-based assays with genomic and genetic tools and whole plant analyses. The unique advantage of the integrated approach is to carry out whole plant studies guided by hypotheses established using simplified cell and molecular assays scaled up to the genome level. The elucidation and manipulation of MAPK cascades in plants will reveal fundamentally important signaling processes.

      This project integrates broad experience, resources and information on stress, defense, and plant hormone signaling and gene regulation to facilitate comprehensive and molecular understanding and integration of complex but evolutionarily conserved signaling networks in Arabidopsis and other plant species.  The project will provide excellent and unique training opportunities for graduate and undergraduate students, postdoctoral fellows and women/minority in a multidisciplinary academic environment, especially in the application of cutting-edge functional genomics and bioinformatics. Information, tools, protocols, materials, lectures, services and publications generated from this project will continue to be freely available to the plant community via a comprehensive web-accessible MAPK cascade and plant signal transduction database (MAPKDB) and TAIR, PlantsP and ABRC.


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Project Gene List

Genes AGI
MKK1 At4g26070
MKK2 At4g29810
MKK4 At1g51660
MKK5 At3g21220
MKK7 At1g18350
MKK9 At1g73500
MPK2 At1g59580
MPK3 At3g45640
MPK4 At4g01370
MPK5 At4g11330
MPK6 At2g43790
MPK11 At1g01560
MEKK1 At4g08500
MEKK2 At4g08480
MEKK3 At4g08470
ANP1 At1g09000
ANP2 At1g54960
ANP3 At3g06030
MTK-a At1g53570
MTK-g At5g66850
MTK18 At1g05100
CTR1 At5g03730
EIN3 At3g20770
ABF4 At3g19290
ICE1 At3g26744
RGA1 At2g01570
ARF1 At1g59750
ARF7 At5g20730
ERF5 AT5g47230
ERF6 AT4g17490
WKY6 At1g62300
WKY29 At4g23550
WKY30 At5g24110
WKY53 At4g23810

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Project Signal List

Arabidopsis leaves and mesophyll protoplasts respond similarly to diverse signals. Signals:
1. H: H2O2 (200 μM for protoplasts or 1 mM for whole plants, 15 min for MAPK activation, and 1h for early gene expression analysis)
2. UV: UV (using a UV lamp or in a UV Stratalinker/Stratagene with 120 J/m2 for MAPK activation and 1 h for early gene expression analysis)
3. W: Wounding (pressing leaves with a pair of metal forceps for 5sec for MAPK activation and wait for 1 h for early gene expression analysis)
4. T: Touch (brushing leaves for 10sec for MAPK activation and 30 min for early gene expression analysis, and twice a day for long-term growth effect)
5. C: Cold (4oC, 20 min for MAPK activation and 1h for early gene expression analysis)
6. F22: Flg22 (a 22 amino-acid peptide from bacterial flagellin, 1 μM, 10 min for MAPK activation and 1h for early gene expression analysis)
7. Hrp: HrpZ (the bacterial harpin protein elicitor, 1 μM, 10 min MAPK activation and 1h for early gene expression analysis)
8. NPP: NPP1 (a fungal cell wall protein elicitor as a GST-NPP1 fusion purified from E. coli, 20 nM, 10 min for MAPK activation and 1h for early gene expression analysis)
9. E: Ethylene (15min 5 ppm ethylene or 1h 10 μM ACC for MAPK activation, and 1h for early gene expression analysis)
10. A: Auxin (1 μM NAA, 20 min for MAPK activation and 1h for early gene expression analysis)
11. G: Gibberellin (1 μM GA3, 15 min for MAPK activation and 1h for early gene expression analysis and seedling expansion)
12. ABA: Abscisic Acid (10 μM ABA, 1 h for early gene expression analysis)

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