|Hurkman Ii, William|
Submitted to: Phytochemistry
Publication Type: Other
Publication Acceptance Date: 5/12/2004
Publication Date: 6/1/2004
Citation: Wong, J.H., Cai, N., Balmer, Y., Tanaka, C.K., Vensel, W.H., Hurkman, W.J., Buchanan, B.B. 2004. Thioredoxin targets of developing wheat seeds identified by complementary proteomics approaches.. Phytochemistry. 65:1629-1640 Interpretive Summary: Proteins constitute a large percentage of the plant cell and are fundamental to all biological processes. Protein function is often facilitated through interactions with other proteins. Thioredoxin is a protein with a catalytically active disulfide group that is known to interact with many other proteins (target proteins) and, in this way, regulates a range of cellular activities. The role of thioredoxin in the early life of wheat seeds is not clear. To help fill this gap, we used two methods, fluorescence gel electrophoresis and affinity column procedures, to identify thioredoxin targets in developing wheat seeds. This analysis led to the identification of 68 targets, including 40 not previously described in seeds. Based on target functions, thioredoxin regulates a number of processes in wheat seeds, including carbohydrate metabolism, cell division, protein assembly, nitrogen metabolism, and stress response. Knowledge of the regulation of cellular processes is essential for improving complex crop traits such as productivity and quality.
Technical Abstract: The role of thioredoxin in wheat starchy endosperm was investigated utilizing two proteomic approaches. Thioredoxin targets were isolated from total KCl-soluble extracts of endosperm and flour and separated by 2-DE following (1) reduction of the extract by the NADP/thioredoxin system and labeling the newly generated sulfhydryl (SH) groups with monobromobimane (mBBr), and, in parallel, (2) trapping covalently interacting proteins on an affinity column prepared with mutant thioredoxin h in which one of the active site cysteines was replaced by serine. The two procedures were complementary: of the total targets, one-third were observed with both procedures and one-third were unique to each. Altogether 68 potential targets were identified; almost all containing conserved cysteines. In addition to confirming known interacting proteins, we identified 40 potential thioredoxin targets not previously described in seeds. A comparison of the results obtained with young endosperm (isolated 10 days after flowering) to those with mature endosperm (isolated 36 days after flowering) revealed a unique set of proteins functional in processes characteristic of each developmental stage. Flour contained 36 thioredoxin targets, most of which have been found in the isolated developing endosperm.