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A growing number of processes throughout biology are regulated by redox via thiol-disulfide exchange. This mechanism is particularly widespread in plants, where almost 200 proteins have been linked to thioredoxin (Trx), a widely distributed small regulatory disulfide protein. The current study extends regulation by Trx to amyloplasts, organelles prevalent in heterotrophic plant tissues that, among other biosynthetic activities, catalyze the synthesis and storage of copious amounts of starch. Using proteomics and immunological methods, we identified the components of the ferredoxin͞Trx system (ferredoxin, ferredoxin-Trx reductase, and Trx), originally described for chloroplasts, in amyloplasts isolated from wheat starchy endosperm. Ferredoxin is reduced not by light, as in chloroplasts, but by metabolically generated NADPH via ferredoxin-NADP reductase. However, once reduced, ferredoxin appears to act as established for chloroplasts, i.e., via ferredoxinTrx reductase and a Trx (m-type). A proteomics approach in combination with affinity chromatography and a fluorescent thiol probe led to the identification of 42 potential Trx target proteins, 13 not previously recognized, including a major membrane transporter (Brittle-1 or ADP-glucose transporter). The proteins function in a range of processes in addition to starch metabolism: biosynthesis of lipids, amino acids, and nucleotides; protein folding; and several miscellaneous reactions. The results suggest a mechanism whereby light is initially recognized as a thiol signal in chloroplasts, then as a sugar during transit to the sink, where it is converted again to a thiol signal. In this way, amyloplast reactions in the grain can be coordinated with photosynthesis taking place in leaves.redox regulation ͉ target proteins ͉ ferredoxin-thioredoxin reductase O ur understanding of the function of the regulatory disulfide protein thioredoxin (Trx) has increased dramatically in the past few years, with the advent of new methodologies. Recent approaches make it possible to identify proteins linked to Trx by combining proteomics with affinity chromatography (1) or thiol probes (2-4). These capabilities have defined an extended role of Trx in chloroplasts (1, 5) and helped elucidate its function in other plant systems: mitochondria (6), seeds (2,3,7,8), and seedlings (4, 9). Currently almost 200 proteins appear to be linked to Trx in plants (10). One major organelle that has been neglected, however, is the amyloplast, a plastid of heterotrophic tissues that performs a wide range of biosynthetic reactions, including the synthesis and storage of abundant quantities of starch.To help fill this gap, we have applied proteomic and immunological approaches to investigate the occurrence and function of Trx in amyloplasts. We now report evidence that amyloplasts isolated from wheat starchy endosperm resemble chloroplasts in containing a complete ferredoxin͞Trx system composed of ferredoxin, ferredoxin-Trx reductase (FTR), and Trx (m-type). Application of affinity chromatography and fl...

A combined two-dimensional gel electrophoresis-mass spectrometry approach was utilized to identify over 250 proteins of wheat (Triticum aestivum L., cv. Butte 86) starchy endosperm that participate in 13 biochemical processes: ATP interconversion reactions, carbohydrate metabolism, cell division, cytoskeleton, lipid metabolism, nitrogen metabolism, protein synthesis/assembly, protein turnover, signal transduction, protein storage, stress/defense, transcription/translation, and transport. Endosperm protein populations were compared at early (10 days post-anthesis, dpa) and late (36 dpa) stages of grain development. Analysis of protein number and spot volume revealed that carbohydrate metabolism, transcription/translation, and protein synthesis/assembly were the principal endosperm functions at 10 dpa followed by nitrogen metabolism, protein turnover, cytoskeleton, cell division, signal transduction, and lipid metabolism. Carbohydrate metabolism and protein synthesis/assembly were also major functions at 36 dpa, but stress/defense and storage were predominant. The results provide insight into biochemical events taking place during wheat grain development and highlight the value of proteomics in characterizing complex biochemical processes. Further, the proteome maps will facilitate future studies addressing the effects of genetic and environmental factors on the development and quality of wheat grain.

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Application of a thiol-speci¢c probe, monobromobimane, with proteomics and enzyme assays led to the identi¢ca-tion of 23 thioredoxin targets in the starchy endosperm of mature wheat seeds (Triticum aestivum cv. Butte), almost all containing at least two conserved cysteines. The identi¢ed targets, 12 not known to be thioredoxin-linked, function in a spectrum of processes : metabolism (12 targets), protein storage (three), oxidative stress (three), protein degradation (two), protein assembly/folding (one) and unknown reactions (two). In addition to formulating metabolic pathways functional in the endosperm, the results suggest that thioredoxin acts in redox regulation throughout the life cycle of the seed. ß