Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco1

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°C, 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°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. 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°C. 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°C 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.

Re-Aeration following Hypoxia or Anoxia Leads to Activation of the Antioxidative Defense System in Roots of Wheat Seedlings1

The response of the ascorbate-glutathione cycle was investigated in roots of young wheat (Triticum aestivum L.) seedlings that were deprived of oxygen either by subjecting them to root hypoxia or to entire plant anoxia and then re-aerated. Although higher total levels of ascorbate and glutathione were observed under hypoxia, only the total amount of ascorbate was increased under anoxia. Under both treatments a significant increase in the reduced form of ascorbate and glutathione was found, resulting in increased reduction states. Upon the onset of re-aeration the ratios started to decline rapidly, indicating oxidative stress. Hypoxia caused higher activity of ascorbate peroxidase, whereas activities of monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase were diminished or only slightly influenced. Under anoxia, activities of ascorbate peroxidase and glutathione reductase decreased significantly to 39 and 62%, respectively. However, after re-aeration of hypoxically or anoxically pretreated roots, activity of enzymes approached the control levels. This corresponds with the restoration of the high reduction state of ascorbate and glutathione within 16 to 96 h of re-aeration, depending on the previous duration of anoxia. Apparently, anoxia followed by re-aeration more severely impairs entire plant metabolism compared with hypoxia, thus leading to decreased viability.

Identification of a Novel Isoform of the Chloroplast-Coupling Factor α-Subunit 1

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF1) α-subunit. Antibodies for the 64-kD protein also recognized the α-subunit of CF1. Both the 64-kD protein and the 61-kD CF1 α-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF1 α-subunit. The 64-kD protein accumulated in response to high irradiance (1000 μmol photons m−2 s−1) and declined in response to low irradiance (80 μmol photons m−2 s−1) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF1 α-subunit found only in monocots. Analysis of purified CF1 complexes showed that the 64-kD protein represented up to 15% of the total CF1 α-subunit.