Mechanism of light regulation of Rubisco: A specific role for the larger Rubisco activase isoform involving reductive activation by thioredoxin-f

Abstract

Rubisco activase is a nuclear-encoded chloroplast protein that is required for the light activation of ribulose 1,5-bisphosphate carboxylase͞oxygenase (Rubisco) in vivo. In most plants examined to date, there are two isoforms of Rubisco activase arising from alternative splicing that differ only at the carboxyl terminus. Here we demonstrate with recombinant proteins that in Arabidopsis the larger isoform has a unique role in the regulation of Rubisco activity. At physiological ratios of ADP͞ATP, the 46-kDa isoform has minimal ATP hydrolysis and Rubisco activation activity in comparison with the 43-kDa isoform. Analysis of a series of carboxyl-terminal deletion and Ala substitution mutants of the 46-kDa isoform revealed that the presence of Cys residues at positions 411 and 392 were essential to preserve a low ATP hydrolysis and Rubisco activation activity in the presence of ADP. Consequently, incubation of the 46-kDa isoform with DTT and thioredoxin-f increased both activities, whereas incubations with DTT alone or with thioredoxin-m were ineffective. Thioredoxin-f and DTT had no effect on the 43-kDa isoform. However, premixing both isoforms before conducting a reduction and oxidation cycle demonstrated that the activity of both isoforms could be regulated. Reduction and oxidation also modulated the activity of native activase proteins isolated from either Arabidopsis or spinach, but not tobacco, which only has the smaller isoform. These findings suggest that in plants containing both isoforms, Rubisco activase regulates the activity of Rubisco in response to light-induced changes in both the ADP͞ATP ratio and the redox potential via thioredoxin-f.

Drought stress and carbon assimilation in a warming climate: Reversible and irreversible impacts

“…These two residues can be reduced via the thioredoxin system (Portis, 2003). The α-isoform of Rubisco activase can be reversibly inactivated by two mechanisms: It may be inactivated/reactivated by oxidation/reduction of the two cysteine residues near the C-terminus via the thioredoxin system (Zhang and Portis, 1999;Portis, 2003) and may additionally be inactivated by direct effects of the moderately elevated temperature (Keown and Pearce, 2014) as schematically shown in Fig. 3 (processes 5 and 6).…”

“…The shorter β-isoform missing the two cysteine residues is also sensitive to moderately elevated temperature, but is not regulated by redox reactions mediated by the thioredoxin system. Only the shorter β-isoform of Rubisco activase was detected in tobacco, while the longer α-isoform with the C-terminal extension containing two cysteine residues was absent (Zhang et al, 1999;Carmo-Silva and Salvucci, 2013). It must be borne in mind that no regulation via the thioredoxin system is possible when the α-isoform is absent (Carmo-Silva and Salvucci, 2013).…”

Analysis of Alternatively Spliced Rice Transcripts Using Microarray Data

“…Alternative splicing has been shown to modulate protein function in plants. For example, alternative splicing of the Arabidopsis Rubisco activase transcripts affects the activity of the resulting proteins in regulating Rubisco in vitro [6,36]. Function-changing alternative splicing has also been observed in rice.…”

“…Alternative splicing generates different forms of mature messenger RNA (mRNA) from the same genetic locus and contributes to expanding diversity of gene function in most eukaryotes [24,34]. For example, relative to each other, different splice forms may have altered mRNA stability or translational efficiency or encode proteins with modified localization or function [6,19,36]. Significantly, around 15% of disease-causing mutations in human genes are point mutations in the vicinity of mRNA splice junctions, supporting the hypothesis that alternative splicing can have major effects on gene function [10,18].…”

Tansley Review No. 112

“…Chloroplastic target enzymes are reduced by Td f , Td m , or both (Buchanan, 1991). The list of enzymes subject to this regulation is ever-growing : recent evidence suggests that Td functions to activate acetyl-CoA carboxylase, which catalyses the first committed step in the synthesis of fatty acids (Sasaki et al, 1997), and also the regulatory protein Rubisco activase (Zhang & Portis, 1999 Fig. 4.…”