Analysis of celery (Apium graveolens) mannitol dehydrogenase (Mtd) promoter regulation in Arabidopsis suggests roles for MTD in key environmental and metabolic responses.

Of the growing list of promising genes for plant improvement, some of the most versatile appear to be those involved in sugar alcohol metabolism. Mannitol, one of the best characterized sugar alcohols, is a significant photosynthetic product in many higher plants. The roles of mannitol as both a metabolite and an osmoprotectant in celery (Apium graveolens) are well documented. However, there is growing evidence that 'metabolites' can also have key roles in other environmental and developmental responses in plants. For instance, in addition to its other properties, mannitol is an antioxidant and may have significant roles in plant-pathogen interactions. The mannitol catabolic enzyme mannitol dehydrogenase (MTD) is a prime modulator of mannitol accumulation in plants. Because the complex regulation of MTD is central to the balanced integration of mannitol metabolism in celery, its study is crucial in clarifying the physiological role(s) of mannitol metabolism in environmental and metabolic responses. In this study we used transformed Arabidopsis to analyze the multiple environmental and metabolic responses of the Mtd promoter. Our data show that all previously described changes in Mtd RNA accumulation in celery cells mirrored changes in Mtd transcription in Arabidopsis. These include up-regulation by salicylic acid, hexokinase-mediated sugar down-regulation, and down-regulation by salt, osmotic stress and ABA. In contrast, the massive up-regulation of Mtd expression in the vascular tissues of salt-stressed Arabidopsis roots suggests a possible role for MTD in mannitol translocation and unloading and its interrelation with sugar metabolism.

Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense.

Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungus Alternaria alternata in the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.

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.

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.

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 with 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.

Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3.

Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant. A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue mannitol concentration are inversely related. MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.

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 on the enzyme activity in response to environmental cues is critical in modulating tissue concentration of mannitol, which, importantly, contribute 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 mumol 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 dodecylsulfate 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 immuno-reactive 40-kD protein.

Mannitol Metabolism in Celery Stressed by Excess Macronutrients.

The effect of excess macronutrients in the root environment on mannitol and sucrose metabolism was investigated in celery (Apium graveolens L. var dulce [Mill.] Pers.). Plant growth was inhibited progressively as macronutrient concentration in the media, as measured by electrical conductivity (E.C.), increased from 1.0 to 11.9 decisiemens m-1. Plants grown for 35 d at higher E.C. had a lower water content but similar dry weight in their roots, leaves, and petioles compared to plants grown at lower E.C. Macronutrient concentrations of leaves, roots, and petioles were not affected by the imposed stress, indicating that the macronutrient stress resulted in a water-deficit stress response rather than a salt-specific response. Mannitol accumulated in sink tissues and was accompanied by a drastic decrease in activity of mannitol-1-oxidoreductase. Sucrose concentration and activities of sucrose-metabolizing enzymes in sink tissues were not affected by the macronutrient stress. Mature leaves exhibited increased concentrations of both mannitol and sucrose, together with increased activity of mannose-6-phosphate reductase and sucrose phosphate synthase, in response to macronutrient stress. Thus, mannitol accumulation in osmotically stressed celery is regulated by diminished catabolism in sink tissues and increased capacity for mannitol biosynthesis in source leaves.

Effect of Different Carbon Sources on Relative Growth Rate, Internal Carbohydrates, and Mannitol 1-Oxidoreductase Activity in Celery Suspension Cultures.

Little information exists concerning the biochemical route of mannitol catabolism in higher plant cells. In this study, the role of a recently discovered mannitol 1-oxidoreductase (MDH) in mannitol catabolism was investigated. Suspension cultures of celery (Apium graveolens L. var dulce [Mill.] Pers.) were successfully grown on nutrient media with either mannitol, mannose, or sucrose as the sole carbon source. Cell cultures grown on any of the three carbon sources did not differ in relative growth rate, as measured by packed cell volume, but differed drastically in internal carbohydrate concentration. Mannitol-grown cells contained high concentrations of mannitol and extremely low concentrations of sucrose, fructose, glucose, and mannose. Sucrose-grown cells had high concentrations of sucrose early in the growth cycle and contained a substantial hexose pool. Mannose-grown cells had a high mannose concentration early in the cycle, which decreased during the growth cycle, whereas their internal sucrose concentrations remained relatively constant during the entire growth cycle. Celery suspension cultures on all three carbon substrates contained an NAD-dependent MDH. Throughout the growth cycle, MDH activity was 2- to 4-fold higher in mannitol-grown cells compared with sucrose- or mannose-grown cells, which did not contain detectable levels of mannitol, indicating that MDH functions pre-dominantly in an oxidative capacity in situ. The MDH activity observed in celery cells was 3-fold higher than the minimum amount required to account for the observed rate of mannitol utilization from the media. Cultures transferred from mannitol to mannose underwent a decrease in MDH activity over a period of days, and transfer from mannose to mannitol resulted in an increase in MDH activity. These data provide strong evidence that MDH plays an important role in mannitol utilization in celery suspension cultures.

Partial purification and characterization of mannitol: mannose 1-oxidoreductase from celeriac (Apium graveolens var. rapaceum) roots.

A mannitol:mannose 1-oxidoreductase was isolated from celeriac (Apium graveolens var. rapaceum) root tips by fractionation with (NH4)2SO4, followed by chromatography on a Fractogel DEAE column and then concentration with (NH4)2SO4. This newly discovered mannitol dehydrogenase catalyzes the NAD-dependent oxidation of mannitol to mannose, not mannitol to fructose. The sugar product of the enzyme reaction was identified by three independent HPLC systems and by an enzymatically linked system as being mannose and not fructose or glucose. Normal Michaelis--Menten kinetics were exhibited for both mannitol and NAD with Km values of 72 and 0.26 mM, respectively, at pH 9.0. The Vmax was 40.14 mumol/h/mg protein for mannitol synthesis and 0.8 mumol/h/mg protein for mannose synthesis at pH 9.0. In the polyol oxidizing reaction, the enzyme was very specific for mannitol with a low rate of oxidation of sorbitol. In the reverse reaction, the enzyme was specific for mannose. The enzyme was strongly inhibited by NADH and sensitive to alterations of NAD/NADH ratio. The enzyme is of physiological importance in that it is mainly localized in root tips (sink tissue) where it functions to convert mannitol into hexoses which are utilized to support root growth. Product determination and kinetic characterization were carried out on an enzyme preparation with a specific activity (SA) of 30.44 mumol/h/mg protein. Subsequently, the enzyme was further purified to a SA of 201 mumol/h/mg protein using an NAD affinity column. This paper apparently represents the first evidence of the existence of a mannitol:mannose 1-oxidoreductase and also the first evidence of the presence of a mannitol dehydrogenase in vascular plants.

CO(2)-Enhanced Yield and Foliar Deformation among Tomato Genotypes in Elevated CO(2) Environments.

Yield increases observed among eight genotypes of tomato (Lycopersicon esculentum Mill.) grown at ambient CO(2) (about 350) or 1000 microliters per liter CO(2) were not due to carbon exchange rate increases. Yield varied among genotypes while carbon exchange rate did not. Yield increases were due to a change in partitioning from root to fruit. Tomatoes grown with CO(2) enrichment exhibited nonepinastic foliar deformation similar to nutrient deficiency symptoms. Foliar deformation varied among genotypes, increased throughout the season, and became most severe at elevated CO(2). Foliar deformation was positively related to fruit yield. Foliage from the lower canopy was sampled throughout the growing season and analysed for starch, K, P, Ca, Mg, Fe, and Mn concentrations. Foliar K and Mn concentrations were the only elements correlated with deformation severity. Foliar K decreased while deformation increased. In another study, foliage of half the plants of one genotype received foliar applications of 7 millimolar KH(2)PO(4). Untreated foliage showed significantly greater deformation than treated foliage. Reduced foliar K concentration may cause CO(2)-enhanced foliar deformation. Reduced K may occur following decreased nutrient uptake resulting from reduced root mass due to the change in partitioning from root to fruit.

Role of sucrose phosphate synthase in sucrose biosynthesis in ripening bananas and its relationship to the respiratory climacteric.

During ripening of bananas (Musa spp. [AAA group, Cavendish subgroup]), there is a massive conversion of starch to sucrose. Also during ripening there is a rise in respiration known as the respiratory climacteric. In this study changes in carbohydrate content, activities of starch and sucrose metabolizing enzymes, and respiration were measured to assess their potential interrelationships. Sucrose phosphate synthase activity increased dramatically during the first 4 days after initiation of ripening by ethylene treatment. Starch concentration decreased and sucrose concentration increased during this time period. Developmental changes in sucrose phosphate synthase activity were measured with limiting substrate (plus Pi) and saturating substrate concentrations. Activities were not parallel under the two assay conditions, providing tentative evidence that kinetically different forms of the enzyme may exist at different stages of ripening. Sucrose accumulation rate was most highly correlated with sucrose phosphate synthase activity assayed with limiting substrate concentrations (plus Pi). The cumulative amount of CO(2) respired during ripening was positively correlated with sugar accumulation (R(2) = 0.97). From this linear regression it was calculated that a constant 0.605 millimoles of CO(2) was evolved per mole of sucrose formed throughout ripening. Using this quantity, the percentage of the total respiratory ATP produced which was required for the conversion of starch to sucrose was calculated assuming different models for carbon export from the amyloplast. The results suggest that sucrose biosynthesis during ripening constitutes a significant sink for respiratory ATP.

Sucrose Phosphate Synthase and Acid Invertase as Determinants of Sucrose Concentration in Developing Muskmelon (Cucumis melo L.) Fruits.

Fruits of orange-fleshed and green-fleshed muskmelon (Cucumis melo L.) were harvested at different times throughout development to evaluate changes in metabolism which lead to sucrose accumulation, and to determine the basis of differences in fruit sucrose accumulation among genotypes. Concentrations of sucrose, raffinose saccharides, hexoses and starch, as well as activities of the sucrose metabolizing enzymes sucrose phosphate synthase (SPS) (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), and acid and neutral invertases (EC 3.2.1.26) were measured. Sucrose synthase and neutral invertase activities were relatively low (1.7 +/- 0.3 micromole per hour per gram fresh weight and 2.2 +/- 0.2, respectively) and changed little throughout fruit development. Acid invertase activity decreased during fruit development, (from as high as 40 micromoles per hour per gram fresh weight) in unripe fruit, to undetectable activity in mature, ripened fruits, while SPS activity in the fruit increased (from 7 micromoles per hour per gram fresh weight) to as high as 32 micromoles per hour per gram fresh weight. Genotypes which accumulated different amounts of sucrose had similar acid invertase activity but differed in SPS activity. Our results indicate that both acid invertase and SPS are determinants of sucrose accumulation in melon fruit. However, the decline in acid invertase appears to be a normal function of fruit maturation, and is not the primary factor which determines sucrose accumulation. Rather, the capacity for sucrose synthesis, reflected in the activity of SPS, appears to determine sucrose accumulation, which is an important component of fruit quality.

Effect of Restricted Root Growth on Carbohydrate Metabolism and Whole Plant Growth of Cucumis sativus L.

The effects of varied rooting volumes on root growth and source leaf carbohydrate metabolism were studied in greenhouse-grown cucumber (Cucumis sativus L cv Calypso) plants. Plants were grown for 7 weeks in container volumes that ranged from 0.4 to 5.9 liters. Plants grown in the smaller containers exhibited less leaf expansion, lower root and shoot weight, and fewer lateral stems than plants grown in the 5.9 liter containers. Shoot/root ratio was not altered by the container volume, suggesting coordination of root and shoot growth due to rooting volume. Source leaf carbon exchange rates, assimilate export rates, and starch accumulation rates for plants grown in 0.4 liter containers were approximately one-half or less in comparison to those for plants grown in 5.9 liter containers. Starch concentrations per unit leaf area were maintained at high levels in source leaves of plants grown in 0.4 liter containers over the entire day/night cycle. Lower extractable galactinol synthase activities and higher galactinol concentrations occurred in leaves of plants grown in 0.4 liter container volumes. The reduced sink demand, induced by restricted root growth, may have led to increased starch concentrations and to a reduction in stachyose biosynthesis in cucumber source leaves.

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.

Galactinol synthase activity and soluble sugars in developing seeds of four soybean genotypes.

Galactinol synthase (UDP-galactose:inositol galactosyltransferase) is the first unique enzyme in the biosynthetic pathway of raffinose saccharides. Its role as a regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean (Glycine max L. Merrill) seeds was examined. Galactinol synthase activity and concentrations of sucrose, stachyose, and raffinose were compared during seed development between two genotypes that were high and two genotypes that were low in mature seed raffinose saccharide concentration. In all genotypes, sucrose concentration increased as seed development progressed, but in both low raffinose saccharide genotypes, greater increases in sucrose concentration were observed late in seed development. Sucrose to stachyose ratios in mature seeds were 2.3-fold greater in low raffinose saccharide genotypes than in the high raffinose saccharide genotypes. During seed development, higher levels of galactinol synthase activity were observed in the high raffinose saccharide genotypes than in the low raffinose saccharide genotypes. A common linear relationship for all four soybean genotypes was shown to exist between galactinol formed estimated from galactinol synthase activity data and the concentration of galactose present in raffinose saccharides. Results of this study implied that galactinol synthase is an important regulator of carbon partitioning between sucrose and raffinose saccharides in developing soybean seeds.

Leaf Carbohydrate Status and Enzymes of Translocate Synthesis in Fruiting and Vegetative Plants of Cucumis sativus L.

Carbon partitioning in the leaves of Cucumis sativus L., a stachyose translocating plant, was influenced by the presence or absence of a single growing fruit on the plant. Fruit growth was very rapid with rates of fresh weight gain as high as 3.3 grams per hour. Fruit growth was highly competitive with vegetative growth as indicated by lower fresh weights of leaf blades, petioles, stem internodes and root systems on plants bearing a single growing fruit compared to plants not bearing a fruit. Carbon exchange rates, starch accumulation rates and carbon export rates were higher in leaves of plants bearing a fruit. Dry weight loss from leaves was higher at night from fruiting plants, and morning starch levels were consistently lower in leaves of fruiting than in leaves of vegetative plants indicating rapid starch mobilization at night from the leaves of fruiting plants. Galactinol, the galactosyl donor for stachyose biosynthesis, was present in the leaves of fruit-bearing plants at consistently lower concentration than in leaves of vegetative plants. Galactinol synthase, and sucrose phosphate synthase activities were not different on a per gram fresh weight basis in leaves from the two plant types; however, stachyose synthase activity was twice as high in leaves from fruiting plants. Thus, the lower galactinol pools may be associated with an activation of the terminal step in stachyose biosynthesis in leaves in response to the high sink demand of a growing cucumber fruit.

Carbohydrate Changes during Maturation of Cucumber Fruit : Implications for Sugar Metabolism and Transport.

Changes in the carbohydrate profiles in the mesocarp, endocarp, and seeds of maturing cucumber (Cucumis sativus, L.) fruit were analyzed. Fruit maturity was measured by a decrease in endocarp pH, which was found to correlate with a loss in peel chlorophyll and an increase in citric acid content. Concentrations of glucose and fructose (8.6-10.3 milligrams per gram fresh weight, respectively) were found to be higher than the concentration of sucrose (0.3 milligrams per gram fresh weight) in both mesocarp and endocarp tissue. Neither raffinose nor stachyose were found in these tissues. The levels of glucose and fructose in seeds decreased during development, but sucrose, raffinose, and stachyose accumulated during the late stages of maturation. Both raffinose and stachyose were found in the seeds of six lines of Cucumis sativus L. This accumulation of raffinose saccharides coincided with an increase in galactinol synthase activity in the seeds. Funiculi from maturing fruit were found to be high in sucrose concentration (4.8 milligrams per gram fresh weight) but devoid of both raffinose and stachyose. The results indicated that sucrose is the transport sugar from the peduncle to seed, and that raffinose saccharide accumulation in the seed is the result of in situ biosynthesis and not from direct vascular transport of these oligosaccharides into the seeds.

Carbon dioxide effects on ethanol production, pyruvate decarboxylase, and alcohol dehydrogenase activities in anaerobic sweet potato roots.

The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O(2) and N(2) gases disappeared quickly and were replaced by CO(2). There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO(2) environment, followed by submergence and a N(2) environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO(2)-treated and 100% N(2)-treated roots. CO(2) atmospheres also resulted in higher pyruvate decarboxylase activity than did N(2) atmospheres. Concentrations of CO(2) were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO(2) concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase.

A Potential Pathway for Galactose Metabolism in Cucumis sativus L., A Stachyose Transporting Species.

Conversion of [(14)C]galactose (Gal) 1-P, UDP-[(14)C]Gal, or UDP-[(14)C]glucose to [(14)C]sucrose was observed when cell-free homogenates of cucumber (Cucumis sativus L.) fruit peduncles were incubated with individual (14)C-labeled substrates, appropriate cofactors, and fructose. The sucrose product was labeled only in the glucose moiety. Conversion of [(14)C]Gal-1-P to [(14)C]sucrose was catalyzed by extracts of peduncles from all other stachyose transporting species tested, as well as green bean (a sucrose transporter) but was not catalyzed by peduncle extracts from three other sucrose transporting species. In cucumber, the ability of extracts to form [(14)C]sucrose from [(14)C]Gal-1-P was greater when peduncles were harvested from growing fruit than from unpollinated ovaries. [(14)C]Sucrose formation from [(14)C]Gal-1-P was inhibited by Mg . PPi, Mg . UDP, UMP, and sucrose. alpha-Galactosidase, galactokinase, UDP-gal pyrophosphorylase, UDP-Gal-4'-epimerase, UDP-glucose pyrophosphorylase, and sucrose synthase activities were detected in peduncle extracts. Neither sucrose phosphate synthetase nor hexose-1-P uridyltransferase were detected. Peduncle tissue contained a small pool of free galactose. These results suggest a potential pathway for the metabolism of galactose moieties hydrolyzed from stachyose, the major sugar transported by cucumber plants.