Edited by Karen G. Fleming Thiol-based redox regulation via ferredoxin-thioredoxin (Trx) reductase/Trx controls various functions in chloroplasts in response to light/dark changes. Trx is a key factor of this regulatory system, and five Trx subtypes, including 10 isoforms, have been identified as chloroplast-localized forms in Arabidopsis thaliana. These subtypes display distinct target selectivity, and, consequently, they form a complicated redox regulation network in chloroplasts. In this study, we developed a FRET-based sensor protein by combining CFP, YFP, and the N-terminal region of CP12, a redox-sensitive regulatory and Trx-targeted protein in chloroplasts. This sensor protein enabled us to monitor the redox change of chloroplast thioredoxin in vivo, and we therefore designated this protein "change in redox state of Trx" (CROST). Using CP12 isoforms, we successfully prepared two types of CROST sensors that displayed different affinities for two major chloroplast Trx isoforms (f-type and m-type). These sensor proteins helped unravel the real-time redox dynamics of Trx molecules in chloroplasts during the light/dark transition. This work was supported by MEXT-KAKENHI Grant 16H06556 (to T. H.) and the Dynamic Alliance for Open Innovation Bridging Human, Environment, and Materials. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Fig. S1.
Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first reaction in the oxidative pentose phosphate pathway. In green plant chloroplasts, G6PDH is a unique redox-regulated enzyme, since it is inactivated under the reducing conditions. This regulation is accomplished using a redox-active cysteine pair, which is conserved in plant G6PDH. The inactivation of this enzyme under conditions of light must be beneficial to prevent release of CO from the photosynthetic carbon fixation cycle. In the filamentous, heterocyst-forming, nitrogen-fixing cyanobacterium sp. PCC 7120 ( 7120), G6PDH plays a pivotal role in providing reducing power for nitrogenase, and its activity is also reported to be suppressed by reduction, though G6PDH does not conserve the critical cysteines for regulation. Based on the thorough analyses of the redox regulation mechanisms of G6PDH from 7120 and its activator protein OpcA, we found that -type thioredoxin regulates G6PDH activity by changing the redox states of OpcA. Mass spectrometric analysis and mutagenesis studies indicate that Cys and Cys of OpcA are responsible for the redox regulation property of this protein. Moreover, analyses of the redox states of OpcA showed that more than half of the OpcA is present as an oxidized form, even under conditions of light, when cells are cultured under the nitrogen-fixing conditions. This redox regulation of OpcA might be necessary to provide reducing power for nitrogenase by G6PDH in heterocysts even during the day.
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