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 Plant Reponses Signal perception (RLKs etc.) Signal relay (ROS etc.) Cellular responses (Protein activity; Transcription reprogramming; Metabolites accumulation etc.)

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., J Exp Bot. 66,

Redox network of chloroplast thioredoxin systems Nikkanen et al., 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 Dr. Sixue Chen Dr. Alice C Harmon Ning Zhu Johanna M. Strul Jacqueline D. Schneider University of Florida Dr. Mengmeng Zhu Pennsylvania State University Thermo Scientific Dr. Craig Dufresne