Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

Abstract

The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions.T o preserve the integrity and efficiency of photosynthesis and other metabolic reactions, chloroplast enzymes need to be flexibly and appropriately controlled in response to changes in light environments. The photosynthetic electron transport chain in the chloroplast thylakoid membrane converts light energy into chemical energy, which is captured and stored in ATP and NADPH. These molecules are primarily consumed by the Calvin-Benson cycle in the stroma, but part of the reducing power is used for other metabolic reactions in chloroplasts (e.g., nitrogen and sulfur metabolism). One pathway for the reducing power is the redox cascade, mediated by ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx). In this system, FTR receives reducing power from the light-driven photosynthetic electron transport chain via ferredoxin and then donates the reducing power to Trx. A reduced form of Trx subsequently transfers reducing power to target proteins through a dithiol-disulfide exchange reaction, allowing the targets to modulate the enzymatic activities. The redox cascade via the FTR/Trx pathway provides the basis for thiol-based redox regulation in chloroplasts and ensures light-responsive control of chloroplast functions (1, 2).The FTR/Trx pathway has been considered the only pathway regulating redox reactions in chloroplasts. Information about its target proteins has also been limited to a specific set of lightactivated enzymes, such as some enzy...

NADPH Thioredoxin Reductase C and Thioredoxins Act Concertedly in Seedling Development

“…The results discussed above provide an explanation for the severe growth inhibition phenotype of the ntrctrxx and ntrc-trxf1f2 mutants and suggest the concerted action of both systems in the redox regulation of common targets in vivo, a notion further supported by the finding that NTRC interacts with well-established Trx targets (Nikkanen et al, 2016). Indeed, NTRC has been shown to participate in the regulation of enzymes such as AGPase (Michalska et al, 2009) and different enzymes of the pathway of tetrapyrrole synthesis (Richter et al, 2013;Pérez-Ruiz et al, 2014;Yoshida and Hisabori, 2016), which were identified previously as Trx regulated (Ballicora et al, 2000;Geigenberger et al, 2005;Ikegami et al, 2007). Here, we addressed this issue by analyzing FBPase, a well-established redox-regulated enzyme of the Calvin-Benson cycle.…”

“…These results are surprising, since biochemical analyses have shown that FBPase is regulated by Trxs f but not by NTRC (Yoshida and Hisabori, 2016) or Trx x (Collin et al, 2003). Thus, to analyze further the role of these thiol redox systems in the regulation of FBPase, we performed in vitro assays with the purified enzymes.…”

“…Furthermore, the Arabidopsis double mutant deficient in NTRC and FTR displays a lethal phenotype under autotrophic growth conditions (Yoshida and Hisabori, 2016), suggesting that NTRC and the Fdx/FTR/Trx systems act in concert in the redox regulation of common targets in planta. Bifluorescence complementation assays showing that NTRC interacts in vivo with well-established Trx-regulated enzymes (Nikkanen et al, 2016), further supporting this notion.…”

Thioredoxin and NADPH-Dependent Thioredoxin Reductase C Regulation of Tetrapyrrole Biosynthesis

“…Moreover, a double mutant lacking FTRb and NTRC shows a lethal phenotype on soil and is only viable on Murashige and Skoog medium supplemented with Suc. Under these conditions, the mutant displays retarded growth, a weak yellow-green pigmentation, and dramatically decreased photosynthetic activity (Yoshida and Hisabori, 2016). This phenotype resembles the TRX ms and NTRC quadruple silencing plants (Fig.…”

“…Recent research suggested that cooperative control of chloroplast functions via the ferredoxin-thioredoxin reductase (FTR)/TRX and NTRC pathways is essential for plant viability. The mutual inactivation of the catalytic subunit of FTR (FTRb) and NTRC leads to the drastic loss of photosynthetic performance and causes a lethal phenotype (Yoshida and Hisabori, 2016). Last but not least, glutaredoxins utilize the reducing power of glutathione to catalyze disulfide reductions in the presence of NADPH and glutathione reductase and operate, like TRXs, as dithiol reductants (Meyer et al, 2012).…”