PROTEOMIC TECHNOLOGIES FOR THE ANALYSIS OF PLANT SIGNALING PATHWAYS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1003457
• Project Start Date: Oct 1, 2014
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Biochemistry

Non Technical Summary
Our understanding of the molecular control of many biological functions and disease pathologies requires the identification of clear kinase-substrate relationships. For example, the phytohormone abscisic acid (ABA) plays important roles in plant development and responses to stressful environments. Recently, the discovery of the PYR1 (Pyrabactin Resistance 1)/PYL (PYR1-Like)/RCAR (Regulatory Component of ABA Receptor) family of ABA receptors led to the elucidation of the core ABA signaling pathway. ABA binds to the PYLs, triggering the PYLs to interact with and inactivate clade A protein phosphatase 2Cs (PP2Cs). This releases Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) from inhibition by the PP2Cs, allowing the kinases to phosphorylate downstream effectors of ABA responses.The proteomic approaches that will be performed in next five years can provide a technical platform to map any signaling pathway and contribute to our understanding of molecular mechanisms accountable for many biological events in plants. As a high throughput experiment, it has the potential of identifying direct substrates of protein kinases, which provides a basis for designing novel protein kinase inhibitors. The approach also identifies the sites of phosphorylation by the kinase, presenting structural details for molecular targeting.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	2499	1000	100%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

protein phosphorylation

plant signaling

proteomics

mass spectrometry

kinase

Goals / Objectives
In-depth understanding of plant signal transduction processes and interactions with other signaling pathways in plants is critical to effectively increase plant yield, increase food security and improve nutrition disorders, which are considered the greatest threats to public safety in the world by the World Health Organization. Global phosphorylation changes in plants in response to environmental stress have been relatively poorly characterized to date. Compared to mammalian cell culture or tissue extracts, plant samples have many more intrinsic metabolites which may interfere with proteomic analyses. A plant phosphoproteomics platform will be developed to study the plant signaling network. First the plant sample preparation will be optimized by using filter aided proteome preparation (FASP) prior to phosphorylation enrichment to remove cell wall debris and plant metabolites. A Polymer-based metal ion chromatography (PolyMAC) will be applied to enrich phosphopeptides with high efficiency. A novel mass spectrometry-based label-free quantitation method will be introduced to facilitate systematic profiling plant phosphoproteome changes with high efficiency and accuracy. Finally, proteomic strategies will be introduced to map direct kinase-substrate relationship in plants.Specific aims are:Aim 1: Establish a high throughput proteomic platform to study plant signaling.Aim 2: Develop a quantitative proteomic approach to the identification of direct substrates of plant protein kinases.Aim 3: Develop a quantitative proteomics approach to the identification of upstream direct kinases in plants.

Project Methods
Aim 1: Establish a high throughput proteomic platform to study plant signaling.A comprehensive strategy based on proteomics will be introduced to study the plant signaling network. First, the plant sample preparation will be optimized by using filter aided proteome preparation (FASP) prior to phosphorylation enrichment. Compared to other approaches, including Rapigest digestion, protein precipitation-Urea digestion, and in-gel digestion, this approach can lead to exceptional phosphopeptide identification. Phosphopeptides after FASP will be enriched by Polymer-based metal ion chromatography (PolyMAC).(1, 2)The superior performance of PolyMAC in terms of capturing efficiency and specificity further improved the yield of phosphopeptides prior to LC-MS analysis.A novel label-free quantitation strategy termed Library Assisted eXtracted Ion Chromatogram (LAXIC) for plant phosphoproteomic analyses with high accuracy and consistency will be developed. The approach employs synthetic peptide libraries as the internal standard. These peptides will be prepared to have proper properties for quality control assessments and mass spectrometric measurements. In particular, peptides will be designed to elute sequentially over an entire LC gradient and to have suitable ionization efficiency and m/z values within the normally scanned mass range. Local normalization of peak intensity will be performed using Loess Procedure, a data treatment adopted from cDNA microarray data analysis (3). To monitor the diverse ion suppression effect across retention time, the local normalization factors (LNFs) are determined by internal standard pairs in individual time windows. Finally, samples will be quantified with LNFs in order to correct variance of LC-MS conditions. This quantification occurs in a small time frame centered to each target peptide. In order to evaluate the quantitation accuracy of LAXIC, different amounts of library peptides will be spiked into a complex phosphopeptide sample and then analyzed by LC-MS. Identical phosphopeptides will be quantified when the absolute amount of samples 25ug, 50ug, and 100ug lysate is used. To test whether the LAXIC technique is applicable for complex biological samples, plant samples with abscisic acid (ABA) treatments will be examined.Aim 2: Develop a quantitative proteomic approach to the identification of direct substrates of plant protein kinases. Besides classical biochemical and genetic methods, mass spectrometry-based high throughput approaches have become increasingly attractive since they are capable of sequencing proteins and localizing phosphorylation sites at the same time. Mass spectrometry-based proteomic methods have been extensively applied to kinase-substrate interaction mapping (4)and global phosphorylation profiling (5-7). Although thousands of phosphorylation events can be inspected simultaneously (8, 9), large-scale phosphoproteomics does not typically reveal direct relationships between protein kinases and their substrates.A proteomic strategy suitable for identifying kinase specificity and direct substrates in high throughput will be developed. This approach includes an in vitro kinase assay-based substrate screening and an endogenous kinase dependent phosphorylation profiling. In the in vitro kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the kinase of interest is added. Quantitative proteomics identifies the re-phosphorylated proteins as direct substrates in vitro. In parallel, the in vivo quantitative phosphoproteomics is performed in which cells are treated with or without the kinase inhibitor. Together, proteins phosphorylated in vitro overlapping with the kinase-dependent phosphoproteome in vivo represents the physiological direct substrates in high confidence. The major issue is how to reduce potential interference by endogenous kinase activities. One effective solution is to use a generic kinase inhibitor, 5′-(4-fluorosulfonylbenzoyl)adenosine (FSBA), which was widely used for covalent labeling of kinases (10, 11), kinase isolation (12), kinase activity exploration (13, 14), and more recently kinase substrate identification by Kothary and co-workers (15). However, an extra step is required to effectively remove the inhibitor before the kinase reaction, which may decrease the sensitivity. ProKALIP addresses the issue by carrying out the kinase reaction using formerly in vivo phosphorylated proteins as candidates. This step efficiently improves the sensitivity and specificity of the in vitro kinase reaction. Coupled with in vivo phosphoproteomics, KALIP can gain a high sensitivity and provide physiologically relevant substrates with high confidence.The KALIP strategy will be applied to identify direct substrates of sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s). SnRK2s are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. These results will shine new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.Aim 3: Develop a quantitative proteomics approach to the identification of upstream direct kinases in plants.There have been multiple studies on screening kinase substrates but few on upstream kinase identification. Bimolecular fluorescence complementation (BiFC)(16)has been widely used to visualize protein-protein interactions in living cells by re-association of fluorescence protein fragment fused to interacting protein pairs. It is reported that this re-association is irreversible in vivo, which can be utilized to stabilize the transient or weak interactions, such as kinase-substrate interactions. Thus, a new strategy, termed fluorescence complementation-mass spectrometry, has been devised based on BiFC and mass spectrometry to identify direct upstream kinases. The use of SnRK2s will continue as the model system to identify their upstream direct kinases. Known kinases will be selected to verify the method, and a kinases collection will be applied to study the novel kinases. Fluorescence protein Venus will be split to two fragments with N terminal (VN) or C terminal (VC). SnRK2-VN and kinase-VC will be constructed. Corresponding plamids will be co-transfected. After 48 hours expression, cells will be observed under fluorescence microscope and harvested. Cell lysates will prepared and immunoprecipitated by GFP nanotrap. Different SnRK2 mutants will be constructed and co-transfected with kinases to examine SnRK2-kinase interactions. Mass spectrometry can identify not only the SnRK2 interacting kinases but also corresponding phosphorylation changes.

Progress 10/01/14 to 09/30/19

Outputs
Target Audience:Biochemical researchers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has supported one graudate student and one undergraduate student through summer research. In addition, the Department of Biochemistry at Purdue University has implemented IDP for graduate students and postdoctoral scholars. It provides a planning process that identifies annual progress, professional development needs, and career objectivesfor individual trainees. The IDP serves as a communication tool between trainees and their mentors and offers a platformfor broader discussion. During the funding period for this grant, we identify short-term goals as a clearer sense of expectations and milestones along the way to achieving specific objectives. In doing so, graduate students and postdoctoral scholars have a process that assists in developing and achieving long-term career goals. How have the results been disseminated to communities of interest? The project has allowed us to establish as a premier lab in the field of plant signaling. Using state-of-the-art high throughput mass spetrometry, we havegenerated high quality data and novel techiques, such as databases and reagents. Besides several obvious approaches such as publications and conference presentations, we have alsodisseminated information quickly through networking with relevant research groups who critically need the technology.? What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1.The perception and relay of cell-wall signals are critical for plants to regulate growth and stress responses, but the underlying mechanisms are poorly understood. Through collaboration between Tao and Zhu groups, we found that the cell-wall leucine-rich repeat extensins (LRX) 3/4/5 are critical for plant salt tolerance inArabidopsisThe LRXs physically associate with the rapid alkalinization factor (RALF) peptides RALF22/23, which in turn interact with the plasma membrane-localized receptor-like protein kinase FERONIA (FER). Thelrx345triple mutant as well asfermutant plants display retarded growth and salt hypersensitivity, which are mimicked by overexpression ofRALF22/23Salt stress promotes S1P protease-dependent release of mature RALF22 peptides. Treatment of roots with mature RALF22/23 peptides or salt stress causes the internalization of FER. Our results suggest that the LRXs, RALFs, and FER function as a module to transduce cell-wall signals to regulate plant growth and salt stress tolerance. 2.The capability to maintain cell wall integrity is critical for plants to adapt to unfavorable conditions. l-Arabinose (Ara) is a constituent of several cell wall polysaccharides and many cell wall-localised glycoproteins, but so far the contribution of Ara metabolism to abiotic stress tolerance is still poorly understood. We discovered that mutations in the MUR4 (also known as HSR8) gene, which is required for the biosynthesis of UDP-Arap in Arabidopsis, led to reduced root elongation under high concentrations of NaCl, KCl, NaNO3, or KNO3. The short root phenotype of the mur4/hsr8 mutants under high salinity is rescued by exogenous Ara or gum arabic, a commercial product of arabinogalactan proteins (AGPs) from Acacia senegal. Mutation of the MUR4 gene led to abnormal cell-cell adhesion under salt stress. MUR4 forms either a homodimer or heterodimers with its isoforms. Analysis of the higher order mutants of MUR4 with its three paralogues, MURL, DUR, MEE25, reveals that the paralogues of MUR4 also contribute to the biosynthesis of UDP-Ara and are critical for root elongation. Taken together, our work revealed the importance of the Ara metabolism in salt stress tolerance and also provides new insights into the enzymes involved in the UDP-Ara biosynthesis in plants.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang C, Arrington J, Ratliff AC, Chen J, Horton HE, Nie Y, Yue F, Hrycyna CA, Tao WA, Kuang S. Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice. J Biol Chem. 2019 Sep 13;294(37):13718-13728.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhao C, Zayed O, Zeng F, Liu C, Zhang L, Zhu P, Hsu CC, Tuncil YE, Tao WA, Carpita NC, Zhu JK. Arabinose biosynthesis is critical for salt stress tolerance in Arabidopsis. New Phytol. 2019 Oct;224(1):274-290.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Arrington J, Xue L, Wang WH, Geahlen RL, Tao WA. Identification of the Direct Substrates of the ABL Kinase via Kinase Assay Linked Phosphoproteomics with Multiple Drug Treatments. J Proteome Res. 2019 Apr 5;18(4):1679-1690.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhao C, Zayed O, Yu Z, Jiang W, Zhu P, Hsu CC, Zhang L, Tao WA, Lozano-Durÿ¿ÿ¡n R, Zhu JK. Leucine-rich repeat extensin proteins regulate plant salt tolerance in Arabidopsis. Proc Natl Acad Sci U S A. 2018 Dec 18;115(51):13123-13128.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Biochemists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The Department of Biochemistry at Purdue University has implemented IDP for graduate students and postdoctoral scholars. Itprovides a planning process that identifies annual progress, professional development needs, and career objectives forindividual trainees. The IDP serves as a communication tool between trainees and their mentors and offers a platform forbroader discussion.During the funding period for this grant, we identify short-term goals as a clearer sense of expectations and milestones alongthe way to achieving specific objectives. In doing so, graduate students and postdoctoral scholars have a process that assistsin developing and achieving long-term career goals. How have the results been disseminated to communities of interest?The project has generated high quality data and novel techiques, such as databases and reagents, centered around the stateof-the-art mass spectrometry. Besides several obvious approaches such as publications and conference presentations, wewill disseminate information quickly through networking with relevant research groups who critically need the technology. Purdue University has a strong research team that involves the measurement of basic signaling pathways in human, animals,and plants in response to physiological stimuli, developmental state, and genetic modifications. Proteomics data will greatlycomplement information obtained in molecular biology, thus provide key insights for biochemical mechanisms. What do you plan to do during the next reporting period to accomplish the goals?We will further develop the techniques to improve their sensitivity and specificity, and at the same time apply them to a variety of plant biology projects.

Impacts
What was accomplished under these goals? Protein phosphorylation is one of the most important and widespread molecular regulatory mechanisms that controls almost all aspects of cellular functions in animals and plants. Here, we introduce a novel chemically functionalized Reverse Phase PhosphoProtein Array (RP3A) to capture and measure phosphoproteomes. RP3A uses polyamidoamine (PAMAM) dendrimer immobilized with Ti(IV) ions to functionalize nitrocellulose membrane, facilitating specific chelation of phosphoproteins from complex protein samples on the array. Globular, water-soluble Ti(IV)-dendrimer allows the RP3A surface to be highly accessible to phosphoproteins multi-dimensionally and the captured phosphoproteins were subsequently detected using the same validated antibodies as in regular reverse-phase protein arrays. The novel chemical strategy demonstrated superior specificity (1:10,000), high sensitivity (fg level), and good quantitative nature (R2=0.99) for measuring phosphoproteins. We further applied quantitative phosphoproteomics followed by RP3A to validate the phosphorylation status of a panel of phosphoproteins in response to environmental stresses in Arabidopsis.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhang C, Du X, Tang K, Yang Z, Pan L, Zhu P, Luo J, Jiang Y, Zhang H, Wan H, Wang X, Wu F, Tao WA, He XJ, Zhang H, Bressan RA, Du J, Zhu JK. Arabidopsis AGDP1 links H3K9me2 to DNA methylation in heterochromatin. Nat Commun. 2018 Oct 31;9(1):4547.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhang Y, Zhao C, Li L, Hsu CC, Zhu JK, Iliuk A, Tao WA. High-Throughput Phosphorylation Screening and Validation through Ti(IV)-Nanopolymer Functionalized Reverse Phase Phosphoprotein Array. Anal Chem. 2018 Sep 4;90(17):10263-10270.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Hsu CC, Zhu Y, Arrington JV, Paez JS, Wang P, Zhu P, Chen IH, Zhu JK, Tao WA. Universal Plant Phosphoproteomics Workflow and Its Application to Tomato Signaling in Response to Cold Stress. Mol Cell Proteomics. 2018 Oct;17(10):2068-2080.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhao Y, Zhang Z, Gao J, Wang P, Hu T, Wang Z, Hou YJ, Wan Y, Liu W, Xie S, Lu T, Xue L, Liu Y, Macho AP, Tao WA, Bressan RA, Zhu JK. Arabidopsis Duodecuple Mutant of PYL ABA Receptors Reveals PYL Repression of ABA-Independent SnRK2 Activity. Cell Rep. 2018 Jun 12;23(11):3340-3351.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang P, Zhao Y, Li Z, Hsu CC, Liu X, Fu L, Hou YJ, Du Y, Xie S, Zhang C, Gao J, Cao M, Huang X, Zhu Y, Tang K, Wang X, Tao WA, Xiong Y, Zhu JK. Reciprocal Regulation of the TOR Kinase and ABA Receptor Balances Plant Growth and Stress Response. Mol Cell.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhu Y, Wang B, Tang K, Hsu CC, Xie S, Du H, Yang Y, Tao WA, Zhu JK. An Arabidopsis Nucleoporin NUP85 modulates plant responses to ABA and salt stress. PLoS Genet. 2017 Dec 12;13(12):e1007124.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Department of Biochemistry at Purdue University has implemented IDP for graduate students and postdoctoral scholars. It provides a planning process that identifies annual progress, professional development needs, and career objectives for individual trainees. The IDP serves as a communication tool between trainees and their mentors and offers a platform for broader discussion. During the funding period for this grant, we identify short-term goals as a clearer sense of expectations and milestones along the way to achieving specific objectives. In doing so, graduate students and postdoctoral scholars have a process that assists in developing and achieving long-term career goals. How have the results been disseminated to communities of interest?The project has generated high quality data and novel techiques, such as databases and reagents, centered around the state-of-the-art mass spectrometry. Besides several obvious approaches such as publications and conference presentations, we will disseminate information quickly through networking with relevant research groups who critically need the technology. Purdue University has a strong research team that involves the measurement of basic signaling pathways in human, animals, and plants in response tophysiological stimuli, developmental state, and genetic modifications. Proteomics data will greatly complement information obtained in molecular biology, thus provide key insights for biochemicalmechanisms. What do you plan to do during the next reporting period to accomplish the goals?We will further optimize the technology and expand the technology to other kinases and other plants.

Impacts
What was accomplished under these goals? Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. However, mass spectrometry-based strategies for studying the plant phosphoproteome still need improvement to be able to handle the high complexity and dynamic range of plant proteome. Here, we developed a novel sample preparation approach for profiling the plant phosphoproteome. This advanced workflow significantly improved phosphopeptide identifications by 2.5-fold, which enabled deep insight into the plant phosphoproteome. This protocol has been further applied to study the phosphorylation events involved in tomato cold tolerance mechanisms. Cold stress is one of the major abiotic stresses that affects plant growth and productivity in the subtropical area. Using our newly developed approach, we profiled phosphoproteomic changes under cold stress of two tomato species (N135 Green Gage and Atacames) with distinct cold tolerance phenotypes. In total, we identified more than 5,000 phosphoproteins and 14,000 class I phosphorylation sites from tomato leaves, which is to our knowledge the largest tomato phosphoproteomic resource. Based on the identification of kinases involved in cold tolerant signaling, we found distinct kinase-substrate events in two tomatoes under a cold environment. In particular, the activation of SnRK2s and their direct substrates may assist N135 Green Gage tomatoes in surviving long-term cold stress. Taken together, this advanced approach and deep phosphoproteomic analysis revealed a global view of tomato cold-induced signaling mechanisms.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yan J, Wang P, Wang B, Hsu CC, Tang K, Zhang H, Hou YJ, Zhao Y, Wang Q, Zhao C, Zhu X, Tao WA, Li J, Zhu JK (2017). The SnRK2 kinases modulate miRNA accumulation in Arabidopsis. PLoS Genet. 13(4):e1006753.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hsu CC, Xue L, Arrington JV, Wang P, Paez Paez JS, Zhou Y, Zhu JK, Tao WA (2017). Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry. J Am Soc Mass Spectrom. 28(6):1127-1135.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Chen IH, Xue L, Hsu CC, Paez JS, Pan L, Andaluz H, Wendt MK, Iliuk AB, Zhu JK, Tao WA (2017). Phosphoproteins in extracellular vesicles as candidate markers for breast cancer. Proc Natl Acad Sci U S A. 114(12):3175-3180.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Li G, Zhang X, Tian H, Choi YE, Tao WA, Xu JR (2017). MST50 is involved in multiple MAP kinase signaling pathways in Magnaporthe oryzae. Environ Microbiol. 19(5):1959-1974.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Zeng L, Shin WH, Zhu X, Park SH, Park C, Tao WA*, Kihara D* (2017). Discovery of Nicotinamide Adenine Dinucleotide Binding Proteins in the Escherichia coli Proteome Using a Combined Energetic- and Structural-Bioinformatics-Based Approach. J Proteome Res. 16(2):470-480.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Arrington JV, Hsu CC, Tao WA (2017). Kinase Assay-Linked Phosphoproteomics: Discovery of Direct Kinase Substrates. Methods Enzymol. 586:453-471.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang C, Wang M, Arrington J, Shan T, Yue F, Nie Y, Tao WA, Kuang S (2017). Ascl2 inhibits myogenesis by antagonizing the transcriptional activity of myogenic regulatory factors. Development. 144(2):235-247.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Duan CG, Wang X, Xie S, Pan L, Miki D, Tang K, Hsu CC, Lei M, Zhong Y, Hou YJ, Wang Z, Zhang Z, Mangrauthia SK, Xu H, Zhang H, Dilkes B, Tao WA, Zhu JK (2017). A pair of transposon-derived proteins function in a histone acetyltransferase complex for active DNA demethylation. Cell Res. 27(2):226-240.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Our understanding of the molecular control of many biological functions and disease pathologies requires the identification of clear kinase-substrate relationships. For example, the phytohormone abscisic acid (ABA) plays important roles in plant development and responses to stressful environments. Recently, the discovery of the PYR1 (Pyrabactin Resistance 1)/PYL (PYR1-Like)/RCAR (Regulatory Component of ABA Receptor) family of ABA receptors led to the elucidation of the core ABA signaling pathway. ABA binds to the PYLs, triggering the PYLs to interact with and inactivate clade A protein phosphatase 2Cs (PP2Cs). This releases Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) from inhibition by the PP2Cs, allowing the kinases to phosphorylate downstream effectors of ABA responses. The proteomic approaches that will be performed during the five-year periodcan provide a technical platform to map any signaling pathway and contribute to our understanding of molecular mechanisms accountable for many biological events in plants. As a high throughput experiment, it has the potential of identifying direct substrates of protein kinases, which provides a basis for designing novel protein kinase inhibitors. The approach also identifies the sites of phosphorylation by the kinase, presenting structural details for molecular targeting. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the accomplishment of the aims, this interdisciplinary project has led to the advancement of modern bioanalytical capabilities and providing unique opportunities to train postdoctoral, graduate and undergraduate students. Our research group has continuously strived for integration of research and education through multiple programs, projects, activities at Purdue University. How have the results been disseminated to communities of interest?High quality data and novel techiques, such as databases and reagents, centered around the state-of-the-art mass spectrometry, have been developed for this important field. Besides several obvious approaches such as publications and conference presentations, we will disseminate information quickly through networking with relevant research groups who critically need the technology. Purdue University has a strong research team that involves the measurement of basic signaling pathways in human, animals, and plants in response to physiological stimuli, developmental state, and genetic modifications. Proteomics data will greatly complement information obtained in molecular biology, thus provide key insights for biochemical mechanisms. What do you plan to do during the next reporting period to accomplish the goals?We are currently working on a large-scale experiment to dissect more than 20 kinases in plant signaling that are not characterized and their direct substrates are not known.

Impacts
What was accomplished under these goals? Phosphorylation, as one of critical regulation mechanisms in plants, remains underrepresented compared to massive phosphoproteomics data in other organisms such as mammalian systems. We focus on the SNF1-related protein kinase 2 (SnRK2), an important regulator which links abscisic acid (ABA) signaling with plant stress responses under osmotic stresses such as drought and high salinity. With the core components of ABA signaling that have been identified, our groups have attempted to identify the direct substrates of SnRK2 activated in response to ABA stimulus. Identification the downstream substrates of SnRK2 not only facilitates dissecting the signaling transduction of ABA-dependent pathways but also shedding light on improving the plant survival under osmotic stresses. Here, we report recent development of stable isotope labeled protein kinase assay linked-phosphoproteomics (siKALIP) by using phosphoproteomes as substrate candidates. The subsequent 18O-ATP labeled kinase reaction significantly simplified phosphoproteome complexity for LC-MS analyses while achieving better sensitivity and specificity. This approach was applied to identify SnRK2 substrates in response to ABA activation. A total of 778 18O-phosphopeptides representing 614 phosphoproteins were idenitfied as the candidate ABA responsive substrates of SnRK2. Besides the reported basophilic phosphorylation motifs of SnRK2 were enriched from the candidate substrates, we have also found a new phosphorylation motif in which the glycine residue at -1 position of the phosphorylation sites is significantly enriched on SnRK2 substrates. The substrate interaction networks reveal that a variety of biological processes are activated through SnRK2 under ABA stimulus, suggesting the diverse functions of SnRK2 in regulating plant stress responses.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Iliuk A, Li L, Melesse M, Hall MC, Tao WA. Multiplexed Imaging of Protein Phosphorylation on Membranes Based on Ti(IV) Functionalized Nanopolymers. Chembiochem. 2016 May 17;17(10):900-3. doi: 10.1002/cbic.201600068. PubMed PMID:27037847; PubMed Central PMCID: PMC4870103.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Xue L, Arrington JV, Tao WA. Identification of Direct Kinase Substrates via Kinase Assay-Linked Phosphoproteomics. Methods Mol Biol. 2016;1355:263-73. doi:10.1007/978-1-4939-3049-4_18. PubMed PMID:26584932.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Duan CG, Wang X, Tang K, Zhang H, Mangrauthia SK, Lei M, Hsu CC, Hou YJ, Wang C, Li Y, Tao WA, Zhu JK. MET18 Connects the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Active DNA Demethylation in Arabidopsis. PLoS Genet. 2015 Oct 22;11(10):e1005559. doi:10.1371/journal.pgen.1005559. PubMed PMID: 26492035;PubMed Central PMCID: PMC4619598.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang L, Yang L, Pan L, Kadasala NR, Xue L, Schuster RJ, Parker LL, Wei A, Tao WA. Time-Resolved Proteomic Visualization of Dendrimer Cellular Entry and Trafficking. J Am Chem Soc. 2015 Oct 14;137(40):12772-5. doi: 10.1021/jacs.5b07875. PubMed PMID: 26425924; PubMed Central PMCID: PMC4766814.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Plant scientists andbiochemists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through the accomplishment of the aims, this interdisciplinary project has led to the advancement of modern bioanalytical capabilities and providing unique opportunities to train postdoctoral,graduate and undergraduate students. Our research group has continuously strived for integration of research and education through multiple programs, projects, activities at Purdue University. How have the results been disseminated to communities of interest?High quality data and novel techiques, such as databases and reagents, centered around the state-of-the-art mass spectrometry, have been developedfor this important field. Besides several obvious approaches such as publications and conference presentations, we will disseminate information quickly through networking with relevant research groups who critically need the technology. Purdue University has a strong research team that involves the measurement of basic signaling pathways in human, animals, and plants in response to physiological stimuli, developmental state, and genetic modifications. Proteomics data will greatly complement information obtained in molecular biology, thus provide key insights for biochemical mechanisms. What do you plan to do during the next reporting period to accomplish the goals?We plan to further develop the technology and strategy to dissect signaling pathways critical to plant stress response. Furthermore, we are applying the approach to different biological systems in which specific kinase-substrate relationship can be mapped.

Impacts
What was accomplished under these goals? Osmotic stress associated with drought, salinity and other abiotic stresses is a major factor that limits plant productivity worldwide. Under osmotic stress sucrose nonfermenting 1 (SNF1)-related subfamily 2 proteins (SnRK2s) are promptly activated and snrk2.1/2/3/4/5/6/7/8/9/10 decuple mutant of Arabidopsis shows osmotic stress-hypersensitive phenotype. We report here a comprehensive profiling of SnRK2s substrates through quantitative phosphoproteomics using wild type and snrk2.1/2/3/4/5/6/7/8/9/10 decuple mutant seedlings under osmotic stress. Among 14,050 unique phosphopeptides identified in the experiment, representing 5,001 phosphoproteins in Arabidopsis, 205 phosphoproteins were identified as putative substrates of osmotic stress-activated SnRK2s. These putative substrates have roles in various cellular processes such as metabolism, mRNA decay and splicing, vesicle trafficking, and protein translation. We further verified nitrate reductase as SnRK2s substrate in vitro and in vivo through genetic and biochemical analyses. Nitrate reductase phosphorylation by SnRK2s is required for its degradation under osmotic stress.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Pan L, Wang L, Hsu CC, Zhang J, Iliuk A, Tao WA (2015). Sensitive measurement of total protein phosphorylation level in complex protein samples. Analyst. 140(10):3390-6.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Lang Z, Lei M, Wang X, Tang K, Miki D, Zhang H, Mangrauthia SK, Liu W, Nie W, Ma G, Yan J, Duan CG, Hsu CC, Wang C, Tao WA, Gong Z, Zhu JK (2015). The Methyl-CpG-Binding Protein MBD7 Facilitates Active DNA Demethylation to Limit DNA Hyper-Methylation and Transcriptional Gene Silencing. Mol Cell. 57(6):971-83.