Abstract
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.
Highlights
This sensor protein enabled us to monitor the redox change of chloroplast thioredoxin in vivo, and we designated this protein “change in redox state of Trx” (CROST)
Detection of Trx activity in vivo is important for understanding the states of the redox regulation system and various Trx-regulated enzymes in chloroplasts such as ATP synthase [2], four enzymes involved the Calvin–Benson cycle [3], the malate valve [4], glucose 6-phosphate dehydrogenase for the oxidative pentose phosphate pathway [4, 5], and the enzymes involved in chlorophyll biosynthesis [6]
CP12 of A. thaliana (CP12-2) has two pairs of cysteines (Cys75-Cys84 and Cys117-Cys126) that form disulfide bonds under oxidizing conditions, and this protein is considered the target of Trx-f in vivo [24]
Summary
The thioredoxin (Trx) redox state sensor protein can visualize Trx activities in the light/dark response in chloroplasts. 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 designated this protein “change in redox state of Trx” (CROST). Genome sequencing of the model plant Arabidopsis thaliana revealed that 10 Trx isoforms, classified into five subtypes (f, m, x, y, and z), are localized in chloroplasts These five subtypes of Trx exhibit different target selectivities and form a complicate redox network, these Trx isoforms maintain a common structural motif named Trx-fold [7]. We succeeded in conferring Trx specificity to the sensor protein using CP12 from a different origin
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