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United States Department of Agriculture

Agricultural Research Service

Research Project: Commercialization of New Industrial Crop Germplasm and Cropping Systems Title: Activation of Rubisco by activase in leaf extracts

Authors
item Do Carmo Silva, Anaelisabete
item Salvucci, Michael

Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 14, 2010
Publication Date: N/A

Technical Abstract: The activation of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) is catalyzed by a chaperone called Rubisco activase. Rubisco activation is an early target of moderate heat stress, due largely to the acute sensitivity of Rubisco activase to thermal inactivation. In the past, assay of Rubisco activase activity required purified Rubisco and Rubisco activase. To address the need to measure Rubisco activase activity in leaf extracts, we modified the in vitro assay based on the ATP-dependent reactivation of a dead-end complex of Rubisco. This complex was produced in leaf extracts by decarbamylating the endogenous Rubisco and then binding the substrate, RuBP, to the inactive enzyme. Reactivation was measured by the ability to restore catalytic competence to the inactive Rubisco complex. Control experiments with the Rubisco activase-deficient rca mutant of Arabidopsis thaliana verified that reactivation of Rubisco was dependent on the presence of Rubisco activase in the extract. In extracts of wild-type Arabidopsis, Rubisco activase catalyzed Rubisco activation at rates equivalent to 12-15% catalytic sites activated per min. The ability to measure Rubisco activase activity in extracts allowed us to compare the relative temperature response and thermal stability of Rubisco activase from several species including Arabidopsis thaliana, Camelina sativa and Nicotiana tabacum. Thus, the assay developed for leaf extracts provides a fundamental tool for studying the temperature response of Rubisco activase activity at physiological ratios of Rubisco: Rubisco activase.

Last Modified: 12/22/2014