David M. Pharr

Purification of NAD-Dependent Mannitol Dehydrogenase from Celery Suspension Cultures

Mannitol dehydrogenase, a mannitol:mannose 1-oxidoreductase, constitutes the first enzymatic step in the catabolism of mannitol in nonphotosynthetic tissues of celery (Apium graveolens L.). Endogenous regulation of the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contributes to stress tolerance of celery. The enzyme was purified to homogeneity from celery suspension cultures grown on D-mannitol as the carbon source. Mannitol dehydrogenase was purified 589-fold to a specific activity of 365 [mu]mol h-1 mg-1 protein with a 37% yield of enzyme activity present in the crude extract. A highly efficient and simple purification protocol was developed involving polyethylene glycol fractionation, diethylaminoethyl-anion-exchange chromatography, and NAD-agarose affinity chromatography using NAD gradient elution. Sodium dodecyl sulfate gel electrophoresis of the final preparation revealed a single 40-kD protein. The molecular mass of the native protein was determined to be approximately 43 kD, indicating that the enzyme is a monomer. Polyclonal antibodies raised against the enzyme inhibited enzymatic activity of purified mannitol dehydrogenase. Immunoblots of crude protein extracts from mannitol-grown celery cells and sink tissues of celery, celeriac, and parsley subjected to sodium dodecyl sulfate gel electrophoresis showed a single major immunoreactive 40-kD protein.

Cloning and Expression Analysis of a UDP-Galactose/Glucose Pyrophosphorylase from Melon Fruit Provides Evidence for the Major Metabolic Pathway of Galactose Metabolism in Raffinose Oligosaccharide Metabolizing Plants

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by α-galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism.

Sugar Repression of Mannitol Dehydrogenase Activity in Celery Cells.

We present evidence that the activity of the mannitol-catabolizing enzyme mannitol dehydrogenase (MTD) is repressed by sugars in cultured celery (Apium graveolens L.) cells. Furthermore, this sugar repression appears to be mediated by hexokinases (HKs) in a manner comparable to the reported sugar repression of photosynthetic genes. Glucose (Glc)-grown cell cultures expressed little MTD activity during active growth, but underwent a marked increase in MTD activity, protein, and RNA upon Glc starvation. Replenishment of Glc in the medium resulted in decreased MTD activity, protein, and RNA within 12 h. Addition of mannoheptulose, a competitive inhibitor of HK, derepressed MTD activity in Glc-grown cultures. In contrast, the addition of the sugar analog 2-deoxyglucose, which is phosphorylated by HK but not further metabolized, repressed MTD activity in mannitol-grown cultures. Collectively, these data suggest that HK and sugar phosphorylation are involved in signaling MTD repression. In vivo repression of MTD activity by galactose (Gal), which is not a substrate of HK, appeared to be an exception to this hypothesis. Further analyses, however, showed that the products of Gal catabolism, Glc and fructose, rather than Gal itself, were correlated with MTD repression.

Changes in carbohydrate and enzyme levels during the sink to source transition of leaves of Cucumis sativus L., a stachyose translocator

Abstract The sink to source transition of expanding leaves of cucumber was studied by sampling leaves sequentially from the growing stem apex toward the base of the plant. Leaf fresh weight and photosynthetic rate increased in the progression to lower nodes. Sucrose and raffinose concentrations were higher in sink leaves than in source leaves. Galactinol and stachyose concentrations increased during leaf expansion. Both alkaline and acidic α-galactosidase activity declined during leaf expansion, whereas galactinol synthase activity increased abruptly in leaves beginning in the leaf at the fourth node below the apex. This increase in activity corresponded temporally to marked changes in the oligosaccharide composition of the leaves in favor of galactosyl-saccharides, particularly stachyose. Across all leaf positions, galactinol synthase correlated positively with galactosyl-oligosaccharide concentration ( r = 0.90) and correlated negatively with sucrose concentration ( r = −0.82). The ratio of stachyose to raffinose correlated positively with the ratio of galactinol synthase to α-galactosidase ( r = 0.95). The results point to the importance of changes in enzyme levels per se as determinants of changes in soluble carbohydrate levels associated with acquisition of export capability during leaf expansion.

Regulation of Photosynthetic Carbon Metabolism in Cucumber by Light Intensity and Photosynthetic Period

The effects of photosynthetic periods and light intensity on cucumber (Cucumis sativus L.) carbon exchange rates and photoassimilate partitioning were determined in relation to the activities of galactinol synthase and sucrose-phosphate synthase. Carbon assimilation and partitioning appeared to be controlled by different mechanisms. Carbon exchange rates were influenced by total photon flux density, but were nearly constant over the entire photoperiod for given photoperiod lengths. Length of the photosynthetic periods did influence photoassimilate partitioning. Assimilate export rate was decreased by more than 60% during the latter part of the short photoperiod treatment. This decrease in export rate was associated with a sharp increase in leaf starch acccumulation rate. Results were consistent with the hypothesis that starch accumulation occurs at the expense of export under short photoperiods. Galactinol synthase activities did not appear to influence the partitioning of photoassimilates between starch and transport carbohydrates. Sucrose phosphate synthase activities correlated highly with sugar formation rates (sucrose, raffinose, stachyose + assimilate export rate, r = 0.93, alpha = 0.007). Cucumber leaf sucrose phosphate synthase fluctuated diurnally in a similar pattern to that observed in vegetative soybean plants.

Growth of callus initiated from cucumber hypocotyls on galactose and galactose-containing oligosaccharides

Abstract Initiation and growth of cucumber hypocotyl callus on galactose, glucose, sucrose, melibiose, raffinose, stachyose, and sucrose + galactose (1 : 1, w/w) were examined. Abundant callus was produced on all of the above carbon sources. However, callus grew best on stachyose or raffinose after a substantial lag period (relative to growth on sucrose). Callus growth was similar on sucrose or galactose. α-Galactosidase activity was detected in hypocotyl explants and in calli grown on raffinose, melibiose, sucrose and galactose. Regardless of carbon source, cultured cells accumulated primarily sucrose, glucose and fructose internally. Free space sugars varied depending on carbon source and provided evidence of an extracellular location for α-galactosidase and invertase from cucumber cells.

Immunolocalization of Mannitol Dehydrogenase in Celery Plants and Cells

Immunolocalization of mannitol dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme. MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles. Suspension cells that were grown in medium with mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction. Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts. The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent w ith the hypothesis that the Mtd gene may be regulated by sugar repression. Additionally, a developmental component may regulate the distribution of MTD within celery plants.

Cucumber fruit sucrose synthase isozymes

Two forms of sucrose synthase (SSI and SSII) were resolved from cucumber (Cucumis sativus) fruit pericarp and fruit peduncle tissue using DEAE-cell

Partial characterization of the galactinol forming enzyme from leaves of cucumis sativus L.

Uridine diphosphate-D-galactose: inositol galactosyltransferase, the enzyme responsible for galactinol synthesis, was purified 41-fold from leaves of Cucumis sativus L. by a combination of gel filtration and ion exchange chromatography. Mn2+ concentration influenced the pH optimum of the enzyme. At low Mn2+ concentration (0.2 mM) the optimum was at pH 7.0. At high Mn2+ concentration (7.0 mM) the optimum was pH 5.5. The enzyme was not found in lysates of chloroplasts isolated from cucumber leaves.

Subcellular localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei.

Mannitol dehydrogenase (MTD) is the first enzyme in mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. Cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown.