Poster: Proteomics

Abs # 972: Unraveling thioredoxin-linked metabolic processes of cereal starchy endosperm using proteomics

Presenter:	Buchanan, Bob B, view@nature.berkeley.edu
Authors	Buchanan, Bob B (A)   Wong, Joshua H (A)   Balmer, Yves  (A)   Tanaka, Charlene K (B)   Vensel, William H (B)   Hurkman, William J (B)
Affiliations:	(A): Department of Plant Biology, University of California-Berkeley (B): USDA, Western Regional Research Center-Albany

Aside from the hydrolysis of starch and protein, the metabolic processes operative in the endosperm of germinating cereal grain—a biochemically active, genetically inactive tissue—are not well understood. Many of the proteins oxidized to the disulfide (S-S) state during grain maturation and drying are reduced to the sulfhydryl (SH) state upon germination. There is a growing body of evidence that thioredoxin h, reduced by NADPH via NADP-thioredoxin reductase, is a key participant in this process. To gain further insight into its regulatory role, we have used a fluorescent thiol-specific probe, monobromobimane (mBBr), coupled with a proteomics approach, to identify soluble thioredoxin-linked proteins of wheat endosperm (Triticum aestivum, L., cv. Butte ) isolated 40 days postanthesis (Yano, H. et al. 2001 PNAS 98:4794-4799). KCl soluble proteins were extracted from the endosperm, reacted with mBBr and separated by 2D-PAGE. The mBBr-labeled proteins were identified by comparison to an extensive 2D map of KCl soluble endosperm proteins that was constructed by mass spectroscopy (MS) and tandem MS. This approach has led to the identification of approximately 25 proteins linked to thioredoxin. These proteins, some previously unrecognized as thioredoxin targets, are members of three metabolic processes—starch breakdown, protein breakdown and oxidative stress response. The identification of most of these proteins was confirmed in parallel experiments based on an affinity column procedure in which a thioredoxin mutated in the active site traps targeted proteins covalently via heterodisulfide bridge formation (Balmer, Y. et al. 2003 PNAS 100:370-375). The results suggest that thioredoxin functions broadly in the regulation of metabolic processes of wheat endosperm.