When seedlings of barley (Hordeum vulgare L.) were transferred from a natural light/dark cycle into darkness, (1-->3,1-->4)-beta- D-glucan endohydrolase (EC 3.2.1.73) activity in leaf extracts increased 3- to 4-fold after 2 days. Activity decreased to normal levels within a day if the light/dark cycle was restored. Although there are two (1-->3,1-->4)-beta-D-glucan endohydrolase isoenzymes in barley, the increased enzyme activity in dark-grown seedlings was attributable entirely to increases in isoenzyme EI. Northern hybridization analyses confirmed that mRNA transcripts encoding (1-->3,1-->4)-beta-D-glucan endohydrolase isoenzyme EI accumulated in the leaves of dark-incubated seedlings; no isoenzyme EII mRNA was detected. Activity of beta-D-glucan glucohydrolases also increased 10-fold after 2 days of dark treatment. The latter, broad-specificity enzymes release glucose from (1-->3,1-->4)-beta-D-glucans and from beta-D-oligoglucosides released by (1-->3,1-->4)-beta-D-glucan endohydrolases. Consistent with the activity patterns of these enzymes, the (1-->3,1-->4)-beta-D-glucan content of leaf cell walls decreased by about 30% when barley seedlings were transferred into darkness. Soluble sugars in the leaves decreased by about 60% during the same period. Because no measurable leaf elongation was detected during the various light/dark treatments, the enzymes were unlikely to be participating in wall loosening and cell elongation. Instead, the results suggest that cell wall (1-->3,1-->4)-beta-D-glucans can be re-mobilized in the non-elongating, dark-incubated leaves and the glucose so generated could serve as an energy source under conditions of sugar depletion.

Intact chloroplasts were isolated from mature pea (Pisum sativum L.) leaves in order to study the degradation of several stromal proteins in organello. Changes in the abundances of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), glutamine synthetase (EC 6.3.1.2) and ferredoxin-dependent glutamine:a-ketoglutarate aminotransferase (glutamate synthase; EC 1.4.7.1) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie-staining of the gels or immunoblotting using speci®c antibodies for the dierent enzymes. Degradation of several stromal proteins was strongly stimulated when intact chloroplasts were incubated in the light in the presence of dithiothreitol. Since free radicals may arti®cially accumulate in the chloroplast under such conditions and interfere with the stability of stromal proteins, the general relevance of these processes remains questionable. In the absence of light, proteolysis proceeded slowly in isolated chloroplasts and was not stimulated by dithiothreitol. Inhibition by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or excess zinc ions as well as the requirement for divalent cations suggested that a zinccontaining metalloprotease participated in this process. Furthermore, light-independent degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase was enhanced in chloroplasts isolated from leaves in which senescence was accelerated by nitrogen starvation. Our results indicate that light-independent stromal protein degradation in intact chloroplasts may be analogous to proteolysis that occurs in intact leaves during senescence.

A (1-->3,1-->4)-beta-D-glucan endohydrolase [(1-->3,1-->4)-beta-glucanase, EC 3.2.1.73] was detected in wheat (Triticum aestivum L.) leaves by Western analyses and activity measurements. This enzyme is able to degrade the (1-->3,1-->4)-beta-glucans present in the cell walls of cereals and other grass species. In wheat, enzyme levels clearly increased during leaf development, reaching maximum values at full expansion and then decreasing upon leaf ageing. To test whether the abundance of (1-->3,1-->4)-beta-glucanase might be controlled by the carbohydrate status, environmental and nutritional conditions capable of altering the leaf soluble sugar contents were used. Both the activity and enzyme protein levels rapidly and markedly increased when mature leaves were depleted of sugars (e.g. during extended dark periods), whereas elevated carbohydrate contents (e.g. following continuous illumination, glucose supply in the dark or nitrogen deficiency during a light/dark cycle) caused a rapid decrease in (1-->3,1-->4)-beta-glucanase abundance or prevented its accumulation in the leaves. The physiological significance of (1-->3,1-->4)-beta-glucanase accumulation under sugar depletion remains to be elucidated.

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