Source: UNIVERSITY OF MICHIGAN submitted to NRP
PROTEIN PHOSPHORYLATION AND POLY(ADP-RIBOSYL)ATION IN COTTON DROUGHT STRESS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1031672
Grant No.
2020-67013-41537
Cumulative Award Amt.
$395,455.09
Proposal No.
2023-11175
Multistate No.
(N/A)
Project Start Date
Sep 1, 2023
Project End Date
May 31, 2025
Grant Year
2024
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
UNIVERSITY OF MICHIGAN
(N/A)
ANN ARBOR,MI 48109
Performing Department
(N/A)
Non Technical Summary
Cotton sustains one of the world's largest industries (textiles) and serves as a significant source of fiber, feed and oil products. With increasingly dwindling water supplies, water deficit is the major abiotic stress limiting cotton productivity. However, the genetic and molecular basis of cotton drought stress responses remains largely unknown. Recent advances in cotton genome sequencing and functional genomics make it possible to understand cotton drought stress at the genome and proteome level and to leverage genetic resources for improving cotton drought tolerance by molecular breeding and genetic engineering. The proposed research is based on our recent findings which reveal a novel phosphorelay cascade and protein poly(ADP-ribosyl)ation (PARylation) in cotton drought stress. We have identified a key transcription factor that is phosphorylated by an evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade and PARylated by poly(ADP-ribose) polymerases (PARPs) in regulating cotton drought responses in a phytohormone abscisic acid (ABA)-independent manner. The goal of this application is to understand the mechanisms and the intertwined crosstalk of these two key post-translational modifications, protein phosphorylation and PARylation, in orchestrating cotton drought stress responses, and genetically improve cotton drought tolerance by rewiring this unique transcriptional regulon. The proposed objectives represent a systems approach to decipher genetic and biochemical mechanisms controlling an important trait in the complex genome of an essential crop and establish platforms to generate drought tolerant cotton cultivars. The proposal is submitted in response to Physiology of Agricultural Plants (A1152).
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20317101080100%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1710 - Upland cotton;

Field Of Science
1080 - Genetics;
Goals / Objectives
Cotton sustains one of the world's largest industries (textiles) and serves as a significant source of fiber, feed and oil products. With increasingly dwindling water supplies, water deficit is the major abiotic stress limiting cotton productivity. However, the genetic and molecular basis of cotton drought stress responses remains largely unknown. Recent advances in cotton genome sequencing and functional genomics make it possible to understand cotton drought stress at the genome and proteome level and to leverage genetic resources for improving cotton drought tolerance by molecular breeding and genetic engineering. The proposed research is based on our recent findings that reveal a novel phosphorelay cascade and protein poly(ADP-ribosyl)ation (PARylation) in cotton drought stress. We have identified a key transcription factor that is phosphorylated by an evolutionarily conserved mitogen-activated protein kinase (MAPK) cascade and PARylated by poly(ADP-ribose) polymerases (PARPs) in regulating cotton drought responses in a phytohormone abscisic acid (ABA)-independent manner. The goal of this application is to understand the mechanisms and the intertwined crosstalk of these two key post-translational modifications, protein phosphorylation and PARylation, in orchestrating cotton drought stress responses, and genetically improve cotton drought tolerance by rewiring this unique transcriptional regulon. The proposed objectives represent a systems approach to decipher genetic and biochemical mechanisms controlling an important trait in the complex genome of an essential crop and establish platforms to generate drought tolerant cotton cultivars.
Project Methods
In this cycle, we mainly focused on Protein ADP-ribosylation in cotton drought stress. The process is mediated by ADP-ribosyltransferases (ARTs), which transfer single ADP-ribose (MAR, mono-ADP-ribose) or multiple ADP-ribose (PAR, poly-ADP-ribose) from nicotinamide adenine dinucleotide (NAD+) to acceptor proteins, termed mono(ADP-ribosyl)ation (MARylation) or poly(ADP-ribosyl)ation (PARylation), respectively. The PARylated or/and MARylated proteins could be immunoprecipitated by macrodomain affinity resin (MD-resin), whereas MARylated proteins are only immunoprecipitated by PARP14m3 resin. Therefore, we can identify ADP-ribosylated proteins in cotton response to drought stress using MD-resin and PARP14m3 resin coupled with label-free quantitative proteomics. We collected 14-day-old cotton seedlings grown in hydroponic medium with or without 5% PEG (a reagent mimicking drought stress) treatment for 12 hrs. Total protein extracts were incubated with MD-resin and PARP14m3 resin at 4 ? for 3-4 hrs. Mock and PEG treatment samples includes three independent repeats, respectively. The immunoprecipitated cotton PARylated and MARylated proteins are identified using mass spectrometry-based label-free quantitative proteomics.As a results, we identified total 1330 candidates (include three PARPs, Table 1) for PARylated or MARylated proteins, and we also identified total 2231 candidates only for MARylated proteins. For PARylated or/and MARylated proteins, 22 candidates show increased ADP-ribosylation, whereas 325 candidates show decreased ADP-ribosylation upon PEG treatment. For only MARylated proteins, 416 candidates show increased MARylation, whereas 14 candidates show decreased MARylation upon PEG treatment . In addition, there are 942 overlapped candidates identified by MD-resin and PARP14m3 resin, which means that 388 among 1330 candidates are likely PARylated proteins.Among the PARylated and/or MARylated candidates, a subset of candidates belonging to RNA-binding proteins (RBP) were identified response to drought stress. Importantly, their homologs in mammalian involved in forming stress granules response to different biotic and abiotic stresses including H2O2, heat, temperature, and pathogens. Also, mammalian system research show that RNA-proteins function as ADP-ribosylation substrates to regulate various cellular processes. These information give us more rationale to focus on the function of RBPs in cotton response to the drought stress. We have made the VIGS constructs of these RBPs to knockdown them in cotton using our previously established VIGS-based gene silence system. We are in the process of screening the growth and drought phenotypes to focus on certain genes that involved in regulating drought stress. We will also study the molecular mechanisms of these RBPs in regulating drought stress through PARylation and/or MARylation.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience: Our target audiences are researchers and scientists with an aim to advance knowledge in cotton biology and cotton stress tolerance; farmers and producers with an aim to improve the production and value of cotton; students and graduates with an aim to train the next generation of scientists. agricultural professionals and leaders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training for graduate students: Suji Ye, Ph.D candidate, Jane Champagne, M.S. candidate,Molecular, Cellular, and DevelopmentalBiology,University of Michigan Training for undergraduate students: Corinna Chen, Saarim Iqbal, Tiancheng Liu, Biology Program,University of Michigan How have the results been disseminated to communities of interest?Brendon Mormile gave a talk about our cotton research in the retreat ofMolecular, Cellular, and DevelopmentalBiology at theUniversity of Michigan. What do you plan to do during the next reporting period to accomplish the goals? Among the PARylated and/or MARylated candidates, a subset of candidates belonging to RNA-binding proteins (RBP) were identified in response to drought stress. Importantly, their homologs in mammals are involved in forming stress granules in response to different biotic and abiotic stresses, including H2O2, heat, temperature, and pathogens.Also, mammalian system research shows that RNA-proteins function as ADP-ribosylation substrates to regulate various cellular processes. This information gives us more rationale to focus on the function of RBPs in cotton response to drought stress. We have made the VIGS constructs of these RBPs to knock them down in cotton using our previously established VIGS-based gene silence system. We are in the process of screening the growth and drought phenotypes to focus on certain genes that are involved in regulating drought stress. We will also study the molecular mechanisms of these RBPs in regulating drought stress through PARylation and/or MARylation.

Impacts
What was accomplished under these goals? In this cycle, we mainly focused on Protein ADP-ribosylation in cotton drought stress. The process is mediated by ADP-ribosyltransferases (ARTs), which transfer single ADP-ribose (MAR, mono-ADP-ribose) or multiple ADP-ribose (PAR, poly-ADP-ribose) from nicotinamide adenine dinucleotide (NAD+) to acceptor proteins, termed mono(ADP-ribosyl)ation (MARylation) or poly(ADP-ribosyl)ation (PARylation), respectively. The PARylated or/and MARylated proteins could be immunoprecipitated by macrodomain affinity resin (MD-resin), whereas MARylated proteins are only immunoprecipitated by PARP14m3 resin. Therefore, we can identify ADP-ribosylated proteins in cotton response to drought stress using MD-resin and PARP14m3 resin coupled with label-free quantitative proteomics. We collected 14-day-old cotton seedlings grown in a hydroponic medium with or without 5% PEG (a reagent mimicking drought stress) treatment for 12 hrs. Total protein extracts were incubated with MD-resin and PARP14m3 resin at 4 ? for 3-4 hrs. Mock and PEG treatment samples include three independent repeats, respectively. The immunoprecipitated PARylated and MARylated cotton proteins are identified using mass spectrometry-based label-free quantitative proteomics. As a result, we identified a total of 1330 candidates (including three PARPs, Table 1) for PARylated or MARylated proteins, and we also identified a total of 2231 candidates only for MARylated proteins. For PARylated or/and MARylated proteins, 22 candidates show increased ADP-ribosylation, whereas 325 candidates show decreased ADP-ribosylation upon PEG treatment. For only MARylated proteins, 416 candidates show increased MARylation, whereas 14 candidates show decreased MARylation upon PEG treatment. In addition, there are 942 overlapped candidates identified by MD-resin and PARP14m3 resin, which means that 388 among 1330 candidates are likely PARylated proteins.

Publications

  • Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Wang, P., He, P. (2024) The symphony of maize signaling quartet defending against gray leaf spot. Stress Biology 4 (1): 18.
  • Type: Other Journal Articles Status: Published Year Published: 2024 Citation: The antagonistic role of an E3 ligase pair in regulating NLR-mediated autoimmunity and fungal pathogen resistance. Cell Host & Microbe