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Identification of thioredoxin h targets in guard cell
enriched epidermal peels using cysTMT proteomics
Tong Zhang, Ph.D.
Department of Biology
University of Florida
ROS mediate plant signaling in guard cells
Signal perception
(RLKs etc.)
Signal relay
(ROS etc.)
Cellular responses
(Protein activity;
Transcription reprogramming;
Metabolites accumulation etc.)
Plant Reponses
ABA and blue light mediated stomatal movement
Zhang et al, 2014, Plant Signal Behav.
9(11):e972845
ROS induce modifications on cysteines
Akter et al., 2015. J Exp Bot.
66, 2935-44
Redox network of chloroplast thioredoxin systems
Nikkanen et al., 2014.
Philos Trans R Soc Lond B, 369
Workflow for identification of Trx h target by cysTMT
Trx h reduces air-oxidized protein extracts
A
B
cysTMT proteomics to identify Trx h targets
How are the MS/MS spectra used to interpret Trx h triggered redox changes?
From raw MS data to Trx h target proteins
Search the raw files against protein
database using SEQUEST algorithm
Filter the high confident peptides
using 1% FDR as cutoff
Normalization of the six
quantification channels
Statistics test for redox-regulated peptides
Trx h target protein list
Grouping of the 80 identified putative Trx h targets
Novel Trx h substrates identified here point to their potential roles in guard cells.
• aspartic protease in guard cell 1 functions in ABA-mediated drought response.
• Pectin acetylesterase catalyzes the interaction between acetylated polysaccharides and
cellulose.
• a novel protein containing 2Fe-2S iron-sulfur cluster binding domain is involved in
regulation of photosynthesis activity.
Summary
• cysTMT is effective in identification and multiplex
quantification of cysteine modifications, and is
suitable in discovering potential Trx h targets.
• 46 out of 80 of putative targets identified here
have been reported as Trx h substrates previously,
demonstrating the reliability of the method.
• Interesting guard cell proteins were identified as
potential Trx h substrates.
Acknowledgements
University of Florida
Pennsylvania State University
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Dr. Sixue Chen
Dr. Alice C Harmon
Ning Zhu
Johanna M. Strul
Jacqueline D. Schneider
Dr. Mengmeng Zhu
Thermo Scientific
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Dr. Craig Dufresne