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Plant Phosphoproteomics to Study SnRK2 Kinase Signaling in P lant Hormone Abscisic Acid (ABA) Pathway. Haley Clark, Liang Xue , Pengcheng Wang, Jian -Kang Zhu, and W. Andy Tao. Abstract. Materials and Methods. Results.

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Abstract

Plant Phosphoproteomicsto Study SnRK2 Kinase Signaling in Plant Hormone Abscisic Acid (ABA) Pathway

Haley Clark, Liang Xue, Pengcheng Wang, Jian-Kang Zhu, and W. Andy Tao

Abstract

Materials and Methods

Results

Abscisic Acid (ABA) is a necessary hormone produced in plants that regulates their growth, development, and reaction to environmental stresses. ABA allows the plant to protect itself from cold, drought, and salinity conditions. A major response of ABA signaling is phosphorylation of SnRK2 kinases. Reactions like these allow the plants to modify water levels, as well as, develop and grow faster or slower. To better understand this process, we use large-scale phosphoproteomics and we have identified that SnRK2/OST1 kinase, Nuclear transport factor 2 (NTF1), and phosphoribulonkinase are likely to reside in the ABA SnRK2 pathway.

Fig. 1 (a)

Fig. 1 (b)

Figure 1 (a) compares the ABA induced wild type samples compared to the wild type controlled samples. Figure 1 (b) compares ABA and control of SnKR2 kinase knock out samples.

  • KO ABA vs. Ctrl

Arabidopsis

WT ABA vs. Ctrl

Protein extraction

2,867

834

770

797

3,027

808

ABA

Ctrl

ABA

Ctrl

ABA & Ctrl

ABA & Ctrl

Chloroform/ Methanol Precipitation

Phosphopeptide confirmation and comparison from previous study (4)

“P signifies phosphate group”

Protein Digestion

8 M Urea

5 mM DTT

IAA reagent

Trypsin (1:100)

Background

An organism’s genome remains relatively stable, where as their proteome is subject to change in different cells and environmental conditions (1). For this reason, proteomics is a highly valuable area of research as it allows us to identify changes in cellular activity in the presence and absence of known and unknown environmental factors. An important change caused by environmental “cues” is phosphorylation. When signaled, a phosphate group will bind to the proteins and alter their enzymatic activities (2). The combined study of phosphorylation and proteomics is known as phosphoproteomics, and it is necessary when studying kinase pathways. Our research focuses on the downstream pathways of the SnRK2 kinase caused by the phytohormone ABA.

PolyMAC Enrichment (3)

Conclusion

Through experiments and research, we have compiled a large number of phosphopeptides that may belong in the ABA pathway. We identified 4,640 phosphopeptides in the wild type experiments, and 4,472 in the knock out group. Of the wild type, 834 phosphopeptides were found in ABA induced conditions only, and 779 in the control group. In contrast, the knock out ABA induced group contained 797 phosphopeptides, and the knock out control group yielded 808. In addition, SnRK2.6/OST1 kinase, Nuclear transport factor 2, phosphoribulokinase, and an unknown protein were identified to be phosphorylated in a previous study (4) and appeared in our WT ABA only list. These proteins plus others in the list above are very likely to be in relation to the SnRK2 and ABA pathway. Other phosphorylated peptides were both identified in wild type and knock out ABA induced versus control groups. Being phosphorylated in the absence or presence of ABA and with or without the SnRK2 gene would indicate that these proteins act independently from SnRK2.

Peptides

Solid-phase beads

PolyMAC

Mass Spectrometry Analysis

Phosphopeptides

Objectives of Research

  • Study the ABA signaling pathway and activation of SnRK2 by phosphorylation

  • Identify which proteins become phosphorylated in the activated SnRK2 downstream effects

References

(1) Roix, Jeffrey, and Tom Misteli. "Genomes, Proteomes, andDynamic Networks in the Cell Nucleus." Histochemistryand Cell Biology 118.2 (2002): 105-16. Print.

(2) Krebs, E. G., and J. A. Beavo. "PhosphorylationDephosphorylation ofEnzymes." Annual Review ofBiochemistry 48.1 (1979): 923-59. Print.

(3) Iliuk, Anton B., Victoria Martin, Bethany Alicie, Robert L. Gaehlen,and W. Andy Tao.“In-depthAnalyses of Kinase-dependent TyrosinePhosphoproteomes Based on Metal Ion-functionalized Soluble Nanopolymers.” Molecular & Cellular Proteomics9.10(2010): 2162-2172.Print.

(4) Kline, Kelli G., Gregory A. Barrett-Wilt, and Michael R. Sussman. “In plantachanges in protein phosphorylation induced by plant hormone abscisic acid.”PNAS 107.36 (2010): 15986-15991. Print

Acknowledgements: DURI Program, Purdue University ▪ Dr. W. Andy Tao, Purdue University Center for Cancer Research


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