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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The intercellular localization of enzymes involved in starch metabolism and the kinetic properties of ADPglucose pyrophosphorylase were studied in mesophyll protoplasts and bundle sheath strands separated by cellulase digestion of Zea mays L. leaves. Activities of starch synthase, branching enzyme, and ADPglucose pyrophosphorylase were higher in the bundle sheath, whereas the degradative enzymes, starch phosphorylase, and amylase were more evenly distributed and slightly higher in the mesophyll. ADPglucose pyrophosphorylase partially purified from the mesophyll and bundle sheath showed similar apparent affinities for Mg2", ATP, and glucose-i-phosphate. The pH optimum of the bundle sheath enzyme (7.0-7.8) was lower than that of the mesophyll enzyme (7.8-8.2). The bundle sheath enzyme showed greater activation by 3-phosphoglycerate than did the mesophyll enzyme, and also showed somewhat higher apparent affinity for 3-phosphoglycerate and lower apparent affinity for the inhibitor, orthophosphate. The observed activities of starch metabolism pathway enzymes and the allosteric properties of the ADPglucose pyrophosphorylases appear to favor the synthesis of starch in the bundle sheath while restricting it in the mesophyll.

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Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Meff.) suspension cells. In the presence of exogenous sugars, log phase growth occurred and the cells displayed mixotrophic metabolism. During this stage, photosynthetic oxygen evolution was depressed and sugars were assimilated from the medium. Upon depletion of medium sugar, oxygen evolution and chlorophyll content increased, and cells entered stationary phase. Activities of various enzymes of glycolysis and sucrose metabolism, including PFP, sucrose synthase, fructokinase, glucokinase, UDPglucose pyrophosphorylase, and fructose-1,6-bisphosphatase, changed as the cells went from log to stationary phases of growth. The largest change occurred in the activity of PFP, which was three-fold higher in log phase cells. PFP activity increased in cells grown on media initially containing sucrose, glucose, or fructose and began to decline when sugar in the medium was depleted. Western blots probed with antibody specific to the -subunit of potato PFP revealed a single 56 kilodalton immunoreactive band that changed in intensity during the growth cycle in association with changes in total PFP activity. The level of fructose-2,6-bisphosphate, an activator of the soybean PFP, increased during the first 24 hours after cell transfer and returned to the stationary phase level prior to the increase in PFP activity. Throughout the growth cycle, the calculated in vivo cytosolic concentration of fructose-2,6-bisphosphate exceeded by more than two orders of magnitude the previously reported activation coefficient (K.) for soybean PFP. These results indicate that metabolism of exogenously supplied sugars by these cells involves a PFP-dependent step that is not coupled directly to sucrose utilization. Activity of this pathway appears to be controlled by changes in the level of PFP, rather than changes in the total cytosolic level of fructose-2,6-bisphosphate.Recently, the roles of PFP3 (EC 2.7.1.90) and PFK (EC 2.7.1.11) in pathways of glycolysis and sucrose metabolism have been intensely studied (3-5, 7, 8, 16, 28, 29). Both enzymes catalyze the phosphorylation of fru-6-P to fru-1,6-bisP, although only the reaction catalyzed by PFP is freely reversible (29).

Levels of several polypeptides in addition to the vegetative storage protein (VSP) increase in soybean leaves following depodding. Two of these polypeptides interact specifically with antibodies raised against the seed lectins of Phaseolus vulgaris and soybean. The two polypeptides, which had apparent molecular masses of 29,000 daltons and 33,000 daltons, were present in the sink-deprived plants but not in control podded plants and were the subunit polypeptides of a glycoprotein designated lectin-related protein (LRP). Soybean LRP was purified to near homogeneity by a combination of ammonium sulfate precipitation and gel filtration. Dialysis of the resuspended ammonium sulfate precipitate caused LRP to reprecipitate, and LRP was soluble only in the presence of molar NaCI. The native relafive molecular mass of LRP was 119,000 daltons, a size consistent with a tetrameric organization of the two polypeptides. LRP precipitated during dialysis in association with a 28,000 dalton polypeptide. The protein coprecipitating with LRP was idenfified as the dimer of the 28,000 dalton subunit of VSP, one of three native isomeric forms of VSP occurring in leaves of depodded plants. Although the specific association between LRP and VSP was intriguing, an in vivo interaction between LRP and VSP was doubtful. LRP was shown to be immunologically similar to soybean agglutinin but did not have detectable hemagglutinating activity. LRP also was shown to be made up of polypeptides distinct from soybean agglutinin.

Laticifer starch accumulation was compared to laticifer growth for developing leaves of Euphorbia pulcherrima Willd. (poinsettia). Measurements of the laticifer‐specific triterpenol, cycloartenol, in latex and in whole leaf extracts were used to calculate the total latex volume in leaves of different developmental stages. Latex volume and starch concentration in the latex were used to determine total laticifer starch and to compare laticifer growth and starch synthesis. Young leaves contained the highest latex and laticifer starch contents on dry wt and leaf area bases. In older expanding leaves, laticifer growth produced an increase in total latex volume accompanied by an increase in total laticifer starch. Laticifer growth and starch accumulation stopped upon cessation of leaf expansion. Starch concentration was similar in latex from all leaves, but differed between plant organs. Thus, laticifer starch accumulation correlated with laticifer growth, but mobilization of the starch out of the laticifer was not observed in old or senescent leaves. This is evidence that laticifer starch grains function within the laticifer independently of degradation or export to other cell types.