Isolation of intact and pure chloroplasts from leaves of Arabidopsis thaliana plants acclimated to low irradiance for studies on Rubisco regulation

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), the most abundant protein in the biosphere catalyzes photosynthetic carbon dioxide fixation and photorespiratory carbon oxidation. In land plants and green algae the enzyme is an oligomeric protein composed of eight large and eight small subunits (LSs and SSs, respectively). A lot of experimental data exist indicating that Rubisco is highly regulated in response to short term fluctuations in the environment. The mechanisms involved in Rubisco regulation may occur at the level of enzymatic activity, as exemplified by the loss of the Rubisco activity which has been reported in several plant species under drought and proposed to be due to the increase in the amount of inhibitors tightly bound to the catalytic site [1,2]. Alternatively, the amount of Rubisco protein may be regulated, by changes in relative rates of synthesis and degradation of both subunits. These processes may involve mRNA accumulation [3], translational elongation [4] as well as a wealth of posttranslational phenomena including the assembly of Rubisco holocomplex [4,5] and aggregation/degradation of individual subunits [6]. As far as posttranslational phenomena are concerned these are, specifically, reactive oxygen species (ROS) generated during the exposure of plants to various stress conditions [7–9] that may drive, at least in some instances Rubisco oxidative modifications which may lead, in their turn, to a massive degradation and/or aggregation of Rubisco subunits and a loss enzymatic activity as well. In vitro oxidative treatment of purified Rubisco leads to conformational changes in LS, followed by an increase in its susceptibility to proteolysis, the disassembly of the holoenzyme and finally also SS becomes more vulnerable to proteolysis [10]. It thus may be expected that Rubisco degradation events caused by stress-related oxidative modifications occuring in vivo may start by LS degradation as well. Indeed, it was found that LS oxidative modifications (disulfide cross-linking as well as non-disulfide polymerization) and a translocation of aggregated LS molecules towards membranes, both triggered in vivo by subjecting Chlamydomonas reinhardtii to saline stress are accompanied by an extensive LS degradation [11]. A similar scenario, involving aggregation, membranetranslocatation and degradation of LS molecules took place under cupric ions treatment of higher plants (wheat, Spirodela oligorrhiza) and algae (Chlamydomonas reinhardtii and C. moewusii) as well [6]. Most probably preexisting proteases are involved as the susceptibility of oxidatively modified LS towards proteolysis correlates positively with the scale of oxidative modifications introduced to LS by stress treatment but not with possible oxidative modifications of proteases engaged [12]. Alternatively, in some instances stress-related aggregation and/or translocation towards membranes of LS molecules are not accompanied by degradation events [13,14]. Thus from the results available today it appears that regulation of both LS and Rubisco holocomplex triggered by short-term environmental fluctuations may operate through a variety of combinations of both preand posttranslational events. The application of new experimental models for the study of Rubisco regulation poses a challenge for future research. It is interesting in this context that a dramatic decrease in Rubisco holocomplex content was found to happen in response to shading of attached tobacco leaves without a concomitant drop in LS mRNA level [15] and one likely mechanism is through posttranslational regulation, Abstract