A complete ferredoxin/thioredoxin system regulates fundamental processes in amyloplasts

Abstract

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 ferredoxin/Trx 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 ferredoxin-Trx 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.

Fig. 1.

Trx m was detected in amyloplasts isolated from wheat starchy endosperm by using antibodies against spinach chloroplast Trx m. Lane 1, amyloplast extract, wheat endosperm; lane 2, chloroplast extract, spinach leaves. Immunoblot was prepared as before by using 20 μg of protein per lane (33).

Fig. 2.

Regulation of amyloplast enzymes by the ferredoxin/Trx system.

Fig. 3.

Trx as a regulatory link between photosynthesis and metabolic processes in amyloplasts.