Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform

Abstract

The light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo requires the presence of Rubisco activase, a nuclear-encoded chloroplast protein that consists of two isoforms arising from alternative splicing in most plants. We examined the function of each isoform by characterizing Rubisco activation in transgenic Arabidopsis plants that express only one or both isoforms, as compared with the wild type. In plants expressing only the shorter isoform, Rubisco activity was as high as in the wild type under saturating light, but the activity was not down-regulated at intensities limiting for photosynthesis. In contrast, in plants expressing only the longer isoform, Rubisco activity was down-regulated at limiting light, but the activity was slightly lower and increased much more slowly at saturating light intensities as compared with the wild type. Light regulation of Rubisco similar to that in the wild-type plants was observed in the progeny of a genetic cross of these two transformants in which both isoforms were again present. When the capacity to redox regulate the activity of the larger activase isoform was eliminated by replacement of the critical cysteine residues in the carboxyl-terminal extension unique to this isoform, Rubisco activity in saturating light was similar to the wild type, but the ability of the larger isoform to down-regulate Rubisco activity at limiting light intensities in transgenic plants was almost abolished. These results indicate that the light modulation of Rubisco under limiting light is mainly due to the ability to regulate the activity of Rubisco activase by redox changes in the stroma.

Figure 1

Activase expression in leaves of the wild type (Wt), rca mutant (rca), and rca transgenics expressing the 43-kDa (43) or 46-kDa (46) isoforms. A typical result is shown in which each lane contained 2 μg of total soluble protein. The figure is a composite created by eliminating intervening lanes on the original immunoblot and the addition of the “rca” lane from a different immunoblot obtained under identical conditions. Immunoblot quantitation by densitometry with recombinant standards indicated that expression in the wild type was 110 mg⋅m⁻² at nearly a 1:1 ratio (51% vs. 49%). Expression as compared with 100% for the wild type averaged 71% ± 12% in the 43-kDa plants and 51% ± 8% in the 46-kDa plants (n ≥ 4).

Figure 2

Dependence of the CO₂ assimilation rate on the intracellular CO₂ concentration (Ci) in the wild type (●), rca mutant (⋄), and rca transformants expressing either the 43-kDa (▵) or 46-kDa (□) isoform of activase. The calculated initial slopes were as follows: WT, 0.061; 43-kDa, 0.054; 46-kDa, 0.040; and rca, 0.01.

Figure 3

Time course of Rubisco activation during a transition from high light to low light for 1 h and a return to high light in various plants. The plants examined are the wild type (A), the rca mutant (B), 43-kDa isoform transformant of the rca mutant (C), 46-kDa isoform transformant of the rca mutant (D), progeny of a cross between the 43- and 46-kDa transformants (E), 46-kDa isoform transformant with a C411A substitution (F), and 46-kDa isoform transformant with C411Aand C392A substitutions (G). Data are the means of two or three replicates.