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Lectins are carbohydrate-binding proteins that occur widely among plants. Lectins of plant vegetative tissues are less well characterized than those of seeds. Previously, a protein of soybean (Glycine max [L.] Merr.) leaves was shown to possess properties similar to the seed lectin. Here we show that the N-terminal amino acid sequence of this protein shares 63% identity with the seed lectin. Immunoblot analysis indicated that the protein occurs in leaves, petioles, stems, and cotyledons of seedlings but not in seeds. These observations prompted designation of the protein as a soybean vegetative lectin (SVL). Immunohistochemical localization in leaves indicated that SVL was localized to the vacuoles of bundle-sheath and paraveinal mesophyll cells. Removal of sink tissues or exposure to atmospheric methyl jasmonate caused increased levels of SVL in leaves and cotyledons. Co-precipitation of SVL and the soybean vegetative storage protein (VSP) during purification suggested an interaction between these proteins. SVL-horseradish peroxidase conjugate bound to dot blots of VSP or SVL, and binding was inhibited by porcine stomach mucin and heparin but not simple carbohydrates. Binding between SVL and VSP and similarities in localization and regulation support a possible in vivo interaction between these proteins.

The plant growth regulator, jasmonic acid (JA) has been shown to increase the level of a glycopeptide with an apparent molecular mass of 30 000 in soybean cells grown photomixotrophically in suspension culture. JA sprayed on soybean leaves increased the level of two glycoproteins with apparent molecular masses of 28 000 and 30 000. These glycoproteins were identified on Western blots as the two subunits of the vegetative storage protein (VSP) found in the paraveinal mesophyll of depoded soybeans. The identity of the VSP bands in the Western blots was determined by using antibody raised against native VSP and antibody that was affinity purified using deglycosylated VSP. The JA-dependent increase in leaf storage proteins suggests that JA may play a role in the regulation of the amount of leaf nigrogen that partitions into VSP, possibly under stressed conditions.

Activity of pyrophosphate:fructose-6-phosphate phosphotransferase (PFP) was investigated in relation to carbohydrate metabolism and physiological growth stage in mixotrophic soybean (Glycine max Merr.) 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, UDP-glucose 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(a)) 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.

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. STARCH GRAINS in latex of Euphorbia differ in their metabolic processes and biophysical-biochemical properties from grains of other plant cells (Biesboer and Mahlberg, 1978; Spilatro and Mahlberg, 1985). The period of laticifer starch grain accumulation in relation to growth and development of the laticifer is unclear. If starch grain formation only occurs in the laticifer tip, then the total starch content should be established prior to the expansion phase of laticifer and organ growth, as for a leaf. In this

The elongated laticifer starch grains of Euphorbia pulcherrima Willd. `Eckespoint C-1 Red' were studied to evaluate compositional and structural properties on grain nonutilization by the laticifer cell. Carbohydrate components were studied specifically. Gas-liquid chromatographic analysis of acid hydrolysates of laticifer starch showed it to be a polyglucan. Solubilized laticifer starch was hydrolyzed readily by α-amylase. Amylose content was ca. 30%. Crystallinity of the starch grains by X-ray diffraction was B-type, typical of starch grains in root and tuber storage tissues. Laticifer starch grains were significantly less soluble in anhydrous dimethylsulfoxide than other B-type starch grains and were rapidly hydrolyzed by α-amylase. The array of properties of laticifer starch grains is distinctive among B-type starch grains and indicates an unusually strong molecular association within the grain. However, our results indicate that neither the starch composition nor structure in these grains prevents its...

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 was due to an up to 40-fold increase in the activity of an extrachloroplast β-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of β-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that β-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.

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 R(F) of 0.44 on 7% polyacrylamide gel electrophoresis. Exoamylase fraction, A1, has an R(F) of 0.23 on the polyacrylamide gel. Viscometric analysis showed that A1 has a slope of 0.013, which is same as that of A3, the extrachloroplastic amylase. A1, 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. A1 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 d-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 R(F) 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.

Synthesis of starch in leaves of Zea mays, an NADP-ME type C4 plant, is normally restricted to the bundle sheath cells (1). The absence of starch accumulation in maize mesophyll may be related to the distribution of starch metabolizing enzymes between the bundle sheath and mesophyll cells, although this has not been convincingly established (1–3). The stromal enzyme, ADPglucose pyrophosphorylase catalyzes the formation of ADPglucose, the glucosyl donor for starch synthase, and is believed to be the principal site of regulation for starch biosynthesis. This enzyme from all plants studied to date is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi) (14). The rate of starch synthesis is believed to be coupled to the rate of photosynthesis and to the rate of photosynthate export from the cell through the interaction of this enzyme and the stromal ratio of 3-PGA/Pi. Activity of this enzyme from whole leaf extracts of sorghum, also a NADH-ME type C4 plant, is also allosterically regulated by 3-PGA and Pi (5). However, significance of the kinetic properties of bundle sheath and mesophyll specific ADPglucose pyrophosphorylases to localization of starch synthesis in leaves of maize or other C4 plants has not been previously investigated.

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 Mg(2+), ATP, and glucose-1-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.

Starch grain morphology in laticifer amyloplasts of Euphorbia pulcherrima Willd. (poinsettia) was examined for evidence of starch metabolism in vegetative and flowering plants. Laticifer starch grains in vegetative plants were rod shaped with lengths ranging from 3 to 60 μm. Average grain size was significantly larger in stems than leaves, and in older than younger tissues. Starch grain length frequency was unimodal and approximated a normal probability distribution in stems, but was skewed positively toward smaller grains in leaves. Frequency distributions were shifted toward larger grains in older tissues. Under short-day photoperiod (flowering) conditions, round starch grains formed in latex of stems, and the average length of rod-shaped grains decreased in latex of stems and leaves. Round grains did not occur in laticifers of leaves or bracts. Round starch grains often occurred in aggregates of two or more subunits. Changes in size and shape of latex starch grains indicate that amyloplasts in fully differentiated laticifers metabolize starch. Identification of metabolically active amyloplasts in differentiated laticifers suggests that the function of these organelles may involve starch mobilization under certain physiological conditions.

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 NaCl. The native relative 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 identified 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.