Exploring the oxidative side of chloroplast redox regulation in Arabidopsis thaliana

Abstract

Plants are subjected to changes in light conditions, so they have a mechanism for redox regulation of photosynthetic enzymes that allows them to acclimate to different light intensity situations. The redox regulation is mainly based on the activation (reduction) of photosynthetic enzymes during the day and their inactivation (oxidation) at night. This study focuses on analyzing the mechanism of oxidative redox regulation and the role of an atypical thioredoxin (TRX) called Cdsp32 (chloroplastic drought-induced stress protein of 32 kDa), whose function is still unknown. It is thought that Cdsp32 may act together with atypical TRXs to activate 2- Cysteine-Peroxiredoxin (2cp), which allows the detoxification of hydrogen peroxide (H2O2) to water (H2O). Furthermore, a protein called NADPH TRX reductase C (NTRC) may control the activity of Cdsp32. Therefore, from previous articles, it is understood that Cdsp32 reduces and activates the 2cp protein. In addition, it is known that NTRC also reduces the 2cp. However, the function of Cdsp32 in the oxidative redox regulation and its functional relationship with 2cp and NTRC are still unknown. By confocal microscopy, it was observed that Cdsp32 is localized in the chloroplasts of mesophyll cells, and co-localized with 2cp. The levels and redox status of Cdsp32 in Arabidopsis thaliana wild-type, 2cpab and ntrc were studied by Western Blot. It was observed that 2cp and NTRC may be functionally related with Cdsp32, as the lack of these proteins is balanced by a higher amount of Cdsp32. Moreover, in mutants deficient of 2cp or NTRC, Cdsp32 is reduced in light and dark conditions, so the redox status of Cdsp32 does not change in the light-to-dark transition. In addition, the effect of Cdsp32 overexpression on the transgenic lines wt/PDEX-Cdsp32-HA and 2cpab/PDEX-Cdsp32-HA were analyzed under two types of growth conditions: with dexamethasone (DEX) and without DEX. DEX induces the overexpression of Cdsp32. Under conditions with DEX, the transgenic lines wt/PDEX-Cdsp32- HA were albino. When we analyzed the phenotypic effect, we observed that Cdsp32 overexpression induces the premature senescence. The most important objective of the study was to analyze the effect of Cdsp32 on the redox status of photosynthetic enzymes. In the darkto-light transition, the overexpression of Cdsp32 (35S-Cdsp32-cmyc) in Nicotiana benthamiana leaves does not affect the redox status of the enzymes. The redox status of enzymes was similar in control and the gene construct 35S-Cdsp32-cmyc. The photosynthetic enzymes were reduced (therefore activated) in light and oxidized (therefore inactivated) in dark. However, in the lightto-dark transition, the overexpression of Cdsp32 accelerates the reduction and delays oxidation of target enzymes (FBPase and ATPc). Overall, these results suggests that Cdsp32 does not function as an oxidant TRX.