|Do Carmo Silva, Anaelisabete|
Submitted to: Photosynthesis Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 18, 2011
Publication Date: September 1, 2011
Citation: Do Carmo Silva, A., Salvucci, M.E., 2011. The activity of Rubisco's molecular chaperone, Rubisco activase, in leaf extracts. Photosynthesis Research. 108:143-155. Interpretive Summary: In the process of photosynthesis, plants convert sunlight into chemical energy. The energy produced by photosynthesis is then used to synthesize sugars and other foodstuffs. Previous research from this research group identified an enzyme called Rubisco activase as a major regulator of photosynthesis, particularly under heat stress. In the past, measuring the activity of Rubisco activase required purifying the enzyme for leaves or isolating the gene and expressing the gene product in bacterium. In this manuscript, we describe for the first time a method for measuring the activity of Rubisco activase directly in leaf extracts without the need for purifying the enzyme. By greatly simplifying measurement of the activity of this key photosynthetic enzyme, this method will allow researchers to investigate the properties of this enzyme in a variety of plants. These investigations include research designed to improve the efficiency of photosynthesis under heat stress, by increasing the temperature optimum of Rubisco activase.
Technical Abstract: Rubisco frequently undergoes unproductive interactions with its sugar-phosphate substrate that stabilize active sites in an inactive conformation. Restoring catalytic competence to these sites requires the “molecular chiropractic” activity of Rubisco activase (activase). To make the study of activase more routine and physiologically relevant, an assay was devised for measuring activase activity in leaf extracts based on the ATP-dependent activation of inactive Rubisco. Control experiments with an Arabidopsis activase-deficient mutant confirmed that the rate of Rubisco activation was dependent on the concentration of activase in the extracts. Activase catalyzed Rubisco activation at rates equivalent to 9-14% catalytic sites per min in desalted extracts of Arabidopsis, camelina, tobacco, cotton and wheat. Higher rates were observed in a transgenic line of Arabidopsis that expresses only the ß-isoform of activase, whereas no activity was detected in a line that expresses only the a-isoform. Activase activity was also low or undetectable in rice, maize and Chlamydomonas, revealing differences in the stability of the enzyme in different species. These differences are discussed in terms of the ability of activase subunits to remain associated or to reassociate into active oligomers when the stromal milieu is diluted by extraction. Finally, the temperature response of activase activity in leaf extracts differed for Arabidopsis, camelina, tobacco and cotton, corresponding to the respective temperature responses of photosynthesis for each species. These results confirmed the exceptional thermal lability of activase at physiological ratios of activase to Rubisco.