Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

Abstract

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30 degreesC, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40 degreesC. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45 degreesC. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40 degreesC. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40 degreesC for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.

Figure 1

The effect of temperature on light activation of Rubisco. Intact cotton (A) and wheat (B) leaf tissue was irradiated with 1800 μmol photons m⁻² s⁻¹ PAR either for 15 min at 22.5°C followed by an additional 5 min at the indicated temperature (○), or for 5 min at the indicated temperature followed by a 15-min incubation at 22.5°C (•). After the 20-min irradiation period, leaf tissue was immediately homogenized to determine Rubisco activity. Each point represents the mean ± se of two replications.

Figure 2

Effect of temperature on the time course of qN of dark-adapted cotton (A) and wheat (B) leaf tissue. The time course of Chl fluorescence was measured at 25°C after leaf tissue was incubated in the dark for 10 min at 25°C followed by an additional 5 min at 25°C (•), 35°C (▪), or 40°C (▴). Data points represent the mean ± se of three replications.

Figure 3

Effect of DTT and nigericin on the time course of qN in wheat leaf tissue. Detached leaves were supplied with 3 mm DTT (○, •) (A) or 100 μm nigericin (○, •) (B) via the transpiration stream. Detached control leaves (□, ▪) were allowed to transpire in water. Subsequently, leaf tissue was incubated in the dark for 15 min at 25°C (○, □) or for 10 min at 25°C followed by 5 min at 37.5°C (•, ▪). After incubation, the time course of Chl fluorescence was measured at 25°C. Data points represent the mean ± se of two replications.

Figure 4

Effect of temperature on the F_o (•) and F_m (▪) Chl fluorescence of dark-adapted cotton (A) and wheat (B) leaf tissue. Leaf tissue was treated as described in Figure 2. Data points represent the mean ± se of three replications.

Figure 5

Effect of temperature on the distribution of activase in the soluble and insoluble fractions of extracts of wheat leaves. Detached leaf tissue was incubated for the indicated times and at the indicated temperatures and then was immediately homogenized and separated into soluble (A) and insoluble (B) fractions by centrifugation. Polypeptides in the two fractions were separated by SDS-PAGE and analyzed for the presence of activase by western-blot analysis. Lanes were loaded with extract corresponding to equal amounts of leaf area. The activase subunits are indicated by the 43-kD label.

Figure 6

Effect of temperature on the formation of high-molecular-weight aggregates of Rubisco activase in the supernatant (A) and pellet (B) fractions of wheat leaf extracts and in the pellet (C) fraction of cotton leaf extracts. Detached leaf tissue was incubated for the indicated times and at the indicated temperatures and then processed and analyzed as described in Figure 5. Lanes were loaded with extract corresponding to equal amounts of leaf area. To better visualize large-molecular-weight aggregates, the gels were overloaded with protein. The activase subunits are indicated by the 43-kD label and the predominant high-molecular-weight aggregates of activase are indicated by the arrows.

Figure 7

Effect of temperature on the form of phosphoribulokinase in the soluble leaf extracts of wheat (A) and cotton (B). Detached leaf tissue was incubated for the indicated times and at the indicated temperatures and then processed and analyzed as described in Figure 5. Lanes were loaded with extract corresponding to equal amounts of leaf area. Only the supernatant fraction is shown because there was no phosphoribulokinase visible on western blots of proteins from the pellet fraction.