Abstract
Thiol-based redox regulation ensures light-responsive control of chloroplast functions. Light-derived signal is transferred in the form of reducing power from the photosynthetic electron transport chain to several redox-sensitive target proteins. Two types of protein, ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx), are well recognized as the mediators of reducing power. However, it remains unclear which step in a series of redox-relay reactions is the critical bottleneck for determining the rate of target protein reduction. To address this, the redox behaviors of FTR, Trx, and target proteins were extensively characterized in vitro and in vivo. The FTR/Trx redox cascade was reconstituted in vitro using recombinant proteins from Arabidopsis. On the basis of this assay, we found that the FTR catalytic subunit and f-type Trx are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and Rubisco activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the Trx family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Both of these biochemical and physiological findings suggest that reducing power transfer from Trx to its target proteins is a rate-limiting step for chloroplast redox regulation, conferring distinct light-responsive redox behaviors on each of the targets.
Highlights
Thiol-based redox regulation is a post-translational mechanism for the control of enzymatic activity through modification of redox-active Cys residues on the target protein
The Arabidopsis ferredoxin-thioredoxin reductase (FTR)/Trx redox cascade was reconstituted as previously described [17] with modifications; in this study, all proteins were incubated at an equimolar concentration (2 μM each)
The in vitro and in vivo studies carried out here led to a consistent conclusion; reducing power transfer from Trx to its target proteins is a critical bottleneck for determining the rate of target protein reduction
Summary
Thiol-based redox regulation is a post-translational mechanism for the control of enzymatic activity through modification of redox-active Cys residues on the target protein (e.g., formation/cleavage of disulfide bonds). A small protein, thioredoxin (Trx), serves as a key factor in redox regulation. Trx has a highly conserved amino acid sequence of WCGPC at its active site, allowing it to reduce disulfide bonds of its target proteins through the dithiol-disulfide exchange reaction. The redox regulation system is unique in terms of linking to the excitation of photosynthetic electron transport and, thereby, light. A part of the reducing power generated from photochemical reactions is transmitted from the photosynthetic electron transport chain to the ferredoxin-thioredoxin reductase (FTR)/Trx redox cascade [2,3,4].
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