Rubisco activation by wheat Rubisco activase isoform 2β is insensitive to inhibition by ADP

Abstract

Rubisco activase (Rca) is a catalytic chaperone that remodels the active site, promotes the release of inhibitors and restores catalytic competence to Rubisco. Rca activity and its consequent effect on Rubisco activation and photosynthesis are modulated by changes to the chloroplast environment induced by fluctuations in light levels that reach the leaf, including redox status and adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio. The Triticum aestivum (wheat) genome encodes for three Rca protein isoforms: 1β (42.7 kDa), 2β (42.2 kDa) and 2α (46.0 kDa). The regulatory properties of these isoforms were characterised by measuring rates of Rubisco activation and ATP hydrolysis by purified recombinant Rca proteins in the presence of physiological ADP/ATP ratios. ATP hydrolysis by all three isoforms was sensitive to inhibition by increasing amounts of ADP in the assay. In contrast, Rubisco activation activity of Rca 2β was insensitive to ADP inhibition, while Rca 1β and 2α were inhibited. Two double and one quadruple site-directed mutants were designed to elucidate if differences in the amino acid sequences between Rca 1β and 2β could explain the differences in ADP sensitivity. Changing two amino acids in Rca 2β to the corresponding residues in 1β (T358K & Q362E) resulted in significant inhibition of Rubisco activation in presence of ADP. The results show that the wheat Rca isoforms differ in their regulatory properties and that amino acid changes in the C domain influence ADP sensitivity. Advances in the understanding of Rubisco regulation will aid efforts to improve the efficiency of photosynthetic CO2 assimilation.

An isoleucine residue acts as a thermal and regulatory switch in wheat Rubisco activase

“…To test the hypothesis that M159I might affect the regulation as well as the thermotolerance of Rca, the rates of ATP hydrolysis and Rubisco activation by Rca2b-M159I were determined at 25°C in the presence of increasing ratios of ADP:ATP. Rubisco activation by Rca2b was previously shown to be insensitive to inhibition by ADP, in contrast to Rca1b, which shows inhibition of both ATP hydrolysis and Rubisco activation as the ADP:ATP ratio increases (Perdomo et al, 2019). Rubisco activation by Rca2b-M159I was inhibited to a greater extent than ATP hydrolysis in the presence of increasing ADP:ATP ratios ( Table 2), indicating that this amino acid substitution causes Rubisco activation by Rca to become more thermotolerant, but also more sensitive to inhibition by ADP.…”

“…The apparent increase in Rca efficiency at 40°C for Rca2b and Rca2b-M159I/K161N resulted from a more marked decrease in ATP hydrolysis than Rubisco activation activity at this temperature ( Figure 2). The efficiency of Rca2b was also increased in the presence of ADP (Figure 3b) through the inhibition of ATP hydrolysis, with no effect on Rubisco activation (Perdomo et al, 2019). Rca2b-M159I and Rca2b-M159I/K161N maintained efficiency independently of ADP presence, because both ATP hydrolysis and Rubisco activation activities were inhibited (Rca2b-M159I; Table 2) or very little affected (Rca2b-M159I/K161N; Perdomo et al, 2019) as ADP:ATP ratios increased.…”

“…The efficiency of Rca2b was also increased in the presence of ADP (Figure 3b) through the inhibition of ATP hydrolysis, with no effect on Rubisco activation (Perdomo et al, 2019). Rca2b-M159I and Rca2b-M159I/K161N maintained efficiency independently of ADP presence, because both ATP hydrolysis and Rubisco activation activities were inhibited (Rca2b-M159I; Table 2) or very little affected (Rca2b-M159I/K161N; Perdomo et al, 2019) as ADP:ATP ratios increased. The response of Rca2b-M159I to ADP contrasts with that observed for Rca2b and shows that the single mutation significantly affects both the regulatory properties and the temperature optimum of Rubisco activation by Rca.…”

“…To test the hypothesis that replacing the wheat Rca2b methionine at position 159 with an isoleucine increases the temperature optimum of Rca activity, the single mutant Rca2b-M159I was generated by site-directed mutagenesis. A double mutant of wheat Rca2b with two residues changed to the corresponding amino acids in Rca1b, including the residue change at position 159, was recently described (Rca2b-M159I/K161N; Perdomo et al, 2019). The mutation M159I was also present, whereas K161N was absent, in two recently characterized mutant proteins of wheat Rca containing 8-and 11-residue changes and characterized by enhanced thermal stability (Scafaro et al, 2019b).…”

“…Wheat Rca isoforms have also been shown to differ in their response to increasing ADP:ATP ratios (Perdomo et al, 2019;Scafaro et al, 2019a), which are likely to change in wheat at high temperatures through reduced photosystem-II efficiency and increased cyclic electron flow (Sharkey, 2005;Haque et al, 2014). Combined, these observations suggest that Rca thermotolerance and ADP sensitivity might be co-regulated.…”