Amylolytic enzymes of Arabidopsis leaf tissue were partially purified and characterized. Endoamylase, starch phosphorylase, D-enzyme (transglycosylase), and possibly exoamylase were found in the chloroplasts. Endoamylase, fraction A2, found only in the chloroplast, was resolved from the exoamylases by chromatography on a Mono Q column and migrated with an RF of 0.44 on 7% polyacrylamide gel electrophoresis. Exoamylase fraction, Al, has an RF of 0.23 on the polyacrylamide gel. Viscometric analysis showed that Al has a slope of 0.013, which is same as that of A3, the extrachloroplastic amylase. Al, however, can be distinguished from A3 by having much higher amylolytic activity in succinate buffer than acetate buffer, and having much less reactivity with amylose. Al probably is also localized in the chloroplast, and contributes to the 30 to 40% higher amylolytic activity of the chloroplast preparation in succinate than acetate buffer at pH 6.0. The high activity of n-enzyme compared to the amylolytic activity in the chloroplast suggests that transglycosylation probably has an important role during starch degradation in Arabidopsis leaf. Extrachloroplastic amylase, A3, has an RF of 0.55 on 7% electrophoretic gel and constitutes 80% of the total leaf amylolytic activity. The results of substrate specificity studies, action pattern and viscometric analyses indicate that the extrachloroplastic amylases are exolytic.

Three classes of mutants of Arabidopsis thaliana (L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants wgs due to an up to 40-fold increase in the activity of an extrachloroplast j8-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of (3-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that fi-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.One of the few experimental treatments of higher plants that may result in an increase in the rate of photosynthetic CO2 fixation is to remove some of the leaves from plants at the stage during which seeds are developing (1, 2). In many species, this causes an increase in the rate of photosynthetic CO2 fixation in the remaining leaves. The mechanisms that regulate the altered rate of photosynthesis are not known, but they have been suggested to be either a hormonal signal produced by the developing seeds or a metabolic effect of the altered carbohydrate pools that occur in the leaf cells because of an imbalance between synthesis and export of carbohydrate. To investigate this phenomenon, we have previously identified three classes of mutants of Arabidopsis that either lack leaf starch (3, 4), contain reduced levels of starch (5), or have elevated levels of starch. These mutants have substantial alterations in leaf carbohydrate metabolism that may mimic some of the effects of experimentally induced changes in the ratio of carbohydrate-exporting tissue to carbohydrateimporting tissue. However, by contrast with invasive methods, the specificity and stability of the genetic differences between the mutants and the wild type facilitates a detailed analysis of the secondary metabolic responses that are directly associated with carbohydrate metabolism.A previous analysis of a mutant that was unable to synthesize starch because of a lack of chloroplast phosphoglucomutase activity (3) indicated that it had increased leaf sugar content and altered photosynthesis and dark respiration rates, which were secondary effects of an inability to synthesize starch. In a further analysis of the secondary effects of this and related mutations on other aspects of leaf metabolism, we have identified a major effect on the leaf 3-amylase (EC 3.2.1.2) activity in these mutants. Elucidation of the mechanisms responsible for this effect may provide fresh insights into the regulation of l...

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