Abstract
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO2 into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO2 to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.Electronic supplementary materialThe online version of this article (doi:10.1007/s11120-013-9964-5) contains supplementary material, which is available to authorized users.
Highlights
Improving the catalytic or regulatory properties of Rubisco to increase the rate of carbon assimilation in photosynthesis has been suggested as a strategy for boosting crop yields (Parry et al 2013)
To determine the effects of naturally-occurring or artificiallyintroduced modifications of Rubisco on carboxylation activity or the interaction with the catalytic chaperone, Rubisco activase (RCA), it is important to have a reliable method for measuring Rubisco and RCA activity
Given the central role of RCA in controlling the activation state of Rubisco, it is desirable that the assay can measure RCA activity in response to variable ratios of ADP:ATP
Summary
Improving the catalytic or regulatory properties of Rubisco to increase the rate of carbon assimilation in photosynthesis has been suggested as a strategy for boosting crop yields (Parry et al 2013). Increasing the turnover rate of Rubisco or its affinity and/or specificity for CO2 (Spreitzer and Salvucci 2002; Whitney et al 2011), preventing inactivation of Rubisco during periods of high temperature (Kurek et al 2007; Parry et al 2011; Carmo-Silva and Salvucci 2012) or improving the response time of Rubisco activation. Given the central role of RCA in controlling the activation state of Rubisco, it is desirable that the assay can measure RCA activity in response to variable ratios of ADP:ATP. The ratio of these adenine nucleotides is the major physiological factor affecting RCA activity (Robinson and Portis 1989a; Carmo-Silva and Salvucci 2013)
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