NADPH supply and mannitol biosynthesis. Characterization, cloning, and regulation of the non-reversible glyceraldehyde-3-phosphate dehydrogenase in celery leaves.

Mannitol, a sugar alcohol, is a major primary photosynthetic product in celery (Apium graveolens L. cv Giant Pascal). We report here on purification, characterization, and cDNA cloning of cytosolic non-reversible glyceraldehyde-3-P dehydrogenase (nr-G3PDH, EC 1.2.1. 9), the apparent key contributor of the NADPH required for mannitol biosynthesis in celery leaves. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified nr-G3PDH showed a molecular mass of 53 kD. A 1,734-bp full-length cDNA clone (accession no. AF196292) encoding nr-G3PDH was identified using polymerase chain reaction and rapid amplification of cDNA ends techniques. The cDNA clone has an open reading frame of 1,491 bp encoding 496 amino acid residues with a calculated molecular weight of 53,172. K(m) values for the celery nr-G3PDH were low (6.8 microM for NADP(+) and 29 microM for D-glyceraldehyde-3-P). NADPH, 3-phosphoglycerate, and ATP were competitive inhibitors, and cytosolic levels of these three metabolites (as determined by nonaqueous fractionation) were all above the concentrations necessary to inhibit activity in vitro, suggesting that nr-G3PDH may be regulated through feedback inhibition by one or more metabolites. We also determined a tight association between activities of nr-G3PDH and mannose-6-P reductase and mRNA expression levels in response to both leaf development and salt treatment. Collectively, our data clearly show metabolic, developmental, and environmental regulation of nr-G3PDH, and also suggest that the supply of NADPH necessary for mannitol biosynthesis is under tight metabolic control.

Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery.

Compared with other primary photosynthetic products (e.g. sucrose and starch), little is known about sugar alcohol metabolism, its regulation, and the manner in which it is integrated with other pathways. Mannose-6-phosphate reductase (M6PR) is a key enzyme that is involved in mannitol biosynthesis in celery (Apium graveolens L.). The M6PR gene was cloned from a leaf cDNA library, and clonal authenticity was established by assays of M6PR activity, western blots, and comparisons of the deduced amino acid sequence with a celery M6PR tryptic digestion product. Recombinant M6PR, purified from Escherichia coli, had specific activity, molecular mass, and kinetic characteristics indistinguishable from those of authentic celery M6PR. Sequence analyses showed M6PR to be a member of the aldo-keto reductase superfamily, which includes both animal and plant enzymes. The greatest sequence similarity was with aldose-6-phosphate reductase (EC 1.1.1.200), a key enzyme in sorbitol synthesis in Rosaceae. Developmental studies showed M6PR to be limited to green tissues and to be under tight transcriptional regulation during leaf initiation, expansion, and maturation. These data confirmed a close relationship between the development of photosynthetic capacity, mannitol synthesis, and M6PR activity.

Gas Exchange and Carbon Partitioning in the Leaves of Celery (Apium graveolens L.) at Various Levels of Root Zone Salinity.

Both mannitol and sucrose (Suc) are primary photosynthetic products in celery (Apium graveolens L.). In other biological systems mannitol has been shown to serve as a compatible solute or osmoprotectant involved in stress tolerance. Although mannitol, like Suc, is translocated and serves as a reserve carbohydrate in celery, its role in stress tolerance has yet to be resolved. Mature celery plants exposed to low (25 mM NaCl), intermediate (100 mM NaCl), and high (300 mM NaCl) salinities displayed substantial salt tolerance. Shoot fresh weight was increased at low NaCl concentrations when compared with controls, and growth continued, although at slower rates, even after prolonged exposure to high salinities. Gas-exchange analyses showed that low NaCl levels had little or no effect on photosynthetic carbon assimilation (A), but at intermediate levels decreases in stomatal conductance limited A, and at the highest NaCl levels carboxylation capacity (as measured by analyses of the CO2 assimilation response to changing internal CO2 partial pressures) and electron transport (as indicated by fluorescence measurements) were the apparent prevailing limits to A. Increasing salinities up to 300 mM, however, increased mannitol accumulation and decreased Suc and starch pools in leaf tissues, e.g. the ratio of mannitol to Suc increased almost 10-fold. These changes were due in part to shifts in photosynthetic carbon partitioning (as measured by 14C labeling) from Suc into mannitol. Salt treatments increased the activity of mannose-6-phosphate reductase (M6PR), a key enzyme in mannitol biosynthesis, 6-fold in young leaves and 2-fold in fully expanded, mature leaves, but increases in M6PR protein were not apparent in the older leaves. Mannitol biosynthetic capacity (as measured by labeling rates) was maintained despite salt treatment, and relative partitioning into mannitol consequently increased despite decreased photosynthetic capacity. The results support a suggested role for mannitol accumulation in adaptation to and tolerance of salinity stress.

Mannose-6-Phosphate Reductase, a Key Enzyme in Photoassimilate Partitioning, Is Abundant and Located in the Cytosol of Photosynthetically Active Cells of Celery (Apium graveolens L.) Source Leaves.

Mannitol, a major photosynthetic product and transport carbohydrate in many plants, accounts for approximately 50% of the carbon fixed by celery (Apium graveolens L.) leaves. Previous subfractionation studies of celery leaves indicated that the enzymes for mannitol synthesis were located in the cytosol, but these data are inconsistent with that published for the sites of sugar alcohol synthesis in other families and taxa, including apple (Malus) and a brown alga (Fucus). Using antibodies to a key synthetic enzyme, NADPH-dependent mannose-6-phosphate reductase (M6PR), and immunocytochemical techniques, we have resolved both the inter-cellular and intracellular sites of mannitol synthesis. In leaves, M6PR was found only in cells containing ribulose-1,5-bisphosphate carboxylase/oxygenase. M6PR was almost exclusively cytosolic in these cells, with the nucleus being the only organelle to show labeling. The key step in transport carbohydrate biosynthesis that is catalyzed by M6PR displays no apparent preferential association with vascular tissues or with the bundle sheath. These results show that M6PR and, thus, mannitol synthesis are closely associated with the distribution of photosynthetic carbon metabolism in celery leaves. The principal role of M6PR is, therefore, in the assimilation of carbon being exported from the chloroplast, and it seems unlikely that this enzyme plays even an indirect role in phloem loading of mannitol.

Mannitol Synthesis in Higher Plants : Evidence for the Role and Characterization of a NADPH-Dependent Mannose 6-Phosphate Reductase.

Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase (14)C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent K(m) (M6P) was 15.8 millimolar, but the apparent K(m) (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg(2+), Ca(2+), Zn(2+), and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.

[(14) C]-Assimilate translocation in the light and dark in celery (Apium graveokns) leaves of different ages.

The 2 major photosynthetic products and translocated carbohydrates in celery (Apium graveolens L.) are sucrose and the sugar alcohol, mannitol. Sucrose is produced and utilized in leaves of all ages. Mannitol, however, is synthesized primarily in mature leaves, utilized in young leaves and stored in all leaves. Here we show that mannitol export was lower from young, expanding leaves than from older leaves. After a 10 min pulse of (14) CO(2) and a 2 h chase in the light or dark there was more radioactivity in sucrose than in mannitol in petiole tissues from leaves of all ages. However, after a chase of 15 h in the dark or 6 h in the light followed by 9 h in the dark, mannitol was the predominant [(14) C]-labeled carbohydrate remaining in all leaf and petiole tissues. Thus, newly synthesized sucrose was apparently exported at a faster rate than mannitol and more mannitol was partitioned into vacuolar storage pools than was sucrose. It also appears that in the light both sucrose and mannitol were exported, but in the dark, once sucrose pools were depleted, mannitol remained as the predominant substance translocated. Both mannitol and sucrose were unloaded into petiole storage parenchyma tissue, but sucrose was hydrolyzed prior to storage.

Biosynthesis of Sucrose and Mannitol as a Function of Leaf Age in Celery (Apium graveolens L.).

In celery (Apium graveolens L.), the two major translocated carbohydrates are sucrose and the acyclic polyol mannitol. Their metabolism, however, is different and their specific functions are uncertain. To compare their roles in carbon partitioning and sink-source transitions, developmental changes in (14)CO(2) labeling, pool sizes, and key enzyme activities in leaf tissues were examined. The proportion of label in mannitol increased dramatically with leaf maturation whereas that in sucrose remained fairly constant. Mannitol content, however, was high in all leaves and sucrose content increased as leaves developed. Activities of mannose-6-P reductase, cytoplasmic and chloroplastic fructose-1,6-bisphosphatases, sucrose phosphate synthase, and sucrose synthase increased with leaf maturation and decreased as leaves senesced. Ribulose bisphosphate carboxylase and nonreversible glyceraldehyde-3-P dehydrogenase activities rose as leaves developed but did not decrease. Thus, sucrose is produced in all photosynthetically active leaves whereas mannitol is synthesized primarily in mature leaves and stored in all leaves. Onset of sucrose export in celery may result from sucrose accumulation in expanding leaves, but mannitol export is clearly unrelated to mannitol concentration. Mannitol export, however, appears to coincide with increased mannitol biosynthesis. Although mannitol and sucrose arise from a common precursor in celery, subsequent metabolism and transport must be regulated separately.

Developmental Changes in Photosynthetic Gas Exchange in the Polyol-Synthesizing Species, Apium graveolens L. (Celery).

Developmental changes in photosynthetic gas exchange were investigated in the mannitol synthesizing plant celery (Apium graveolens L. ;Giant Pascal'). Greenhouse-grown plants had unusually high photosynthetic rates for a C(3) plant, but consistent with field productivity data reported elsewhere for this plant. In most respects, celery exhibited typical C(3) photosynthetic characteristics; light saturation occurred at 600 micromoles photons per square meter per second, with a broad temperature optimum, peaking at 26 degrees C. At 2% O(2), photosynthesis was enhanced 15 to 25% compared to rates at 21% O(2). However, celery had low CO(2) compensation points, averaging 7 to 20 microliters per liter throughout the canopy. Conventional mechanisms for concentrating CO(2) were not detectable.

A pathway for photosynthetic carbon flow to mannitol in celery leaves : activity and localization of key enzymes.

In the polyol producing plant, celery (Apium graveolens L.), mannitol is a major photosynthetic product and a form in which carbohydrate is translocated. Measurements of whole leaf extracts of celery indicated substantial activity of the following enzymes: mannose-6-P reductase, mannose-6-P isomerase, mannitol-1-P phosphatase, and nonreversible glyceraldehyde-3-P dehydrogenase. The activities of these enzymes were either undetectable or very low in the nonpolyol producing plants, Secale cereale L. (rye) and Vigna mungo (L.) Hepper (black gram).Mesophyll protoplasts were enzymically isolated from celery leaves, broken with a Yeda press and the intracellular localization of the above enzymes for mannitol synthesis studied following differential and/or sucrose density gradient centrifugation of the protoplast extract. These data suggested the enzymes involved in mannitol synthesis are exclusively localized in the cytoplasm. Ninety-five to 100% of the activity of these enzymes, along with the cytoplasmic marker enzyme phosphoenolpyruvate carboxylase, was found in the cytosolic fraction.We propose the pathway of photosynthetic carbon flow from triose-P to mannitol in celery occurs via fructose-6-P, mannose-6-P, and mannitol-1-P; these final reactions being catalyzed by the cytoplasmic enzymes, mannose-6-P isomerase, NADPH-dependent mannose-6-P reductase, and mannitol-1-P phosphatase, respectively. The requirement for NADPH may be met via the cytoplasmically located NADP-linked nonreversible glyceraldehyde-3-P dehydrogenase.

Sorbitol metabolism and sink-source interconversions in developing apple leaves.

In apple (Malus domestica Borkh.) sorbitol is the primary product of photosynthesis, the major translocated form of carbon, and a common fruit constituent and storage compound. Previous work on sorbitol metabolism has revealed a NADPH-dependent aldose 6-phosphate reductase (A6PR) in green tissues, and a NAD-dependent sorbitol dehydrogenase in nongreen tissues. Results here show a decrease in sorbitol dehydrogenase activity and an increase in A6PR activity as leaves developing in the spring undergo the transition from sink to source. Sorbitol dehydrogenase activity reached a minimum as A6PR peaked. These changes were related to increases in leaf carbohydrate levels, especially sorbitol, and to increases in rates of net photosynthesis. Studies conducted in the autumn on senescing leaves also showed changes in enzyme activites, leaf carbohydrate levels, and photosynthesis. At this time, however, sorbitol dehydrogenase increased in specific activity, whereas A6PR activity, leaf carbohydrates, and photosynthetic rates all decreased substantially. Other experiments showed differences in the ability of young and mature leaves to metabolize sorbitol and in the distribution of sorbitol enzymes in leaves at transitional developmental stages. The results suggest that sorbitol metabolism in apple is tightly controlled and may be related to mechanisms regulating partitioning or source and sink activity.

Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves.

Aldose-6-phosphate reductase (alditol 6-phosphate:NADP 1-oxidoreductase) was isolated and characterized from mature apple leaves (Malus domestica cv. Starkrimson). The enzyme was purified 79-fold. The enzyme catalyzed the following reversible reaction: d-glucose 6-phosphate + NADPH + H(+) right arrow over left arrow d-sorbitol 6-phosphate + NADP(+). No activity was detected when NAD(+) was substituted for NADP(+) or when NADH was substituted for NADPH. The enzyme reduced d-galactose 6-phosphate at a higher rate than d-glucose 6-phosphate. d-Mannose 6-phosphate and 2-deoxy-d-glucose 6-phosphate were reduced at low rates. d-Glucose 1-phosphate, d-fructose 6-phosphate, d-ribose 5-phosphate, d-glucose, and sorbitol did not serve as substrates. The pH optimum for both d-sorbitol 6-phosphate oxidation and d-glucose 6-phosphate reduction was 9.5. The K(m) values for d-sorbitol 6-phosphate oxidation and d-glucose 6-phosphate reduction were 3.9 and 20 millimolar, respectively. AgNO(3) (0.1 millimolar) and p-chloromercuribenzoate (1.0 millimolar) completely inhibited the enzyme.Aldose-6-phosphate reductase activity was also detected in mature leaves from Golden Delicious and Antonovka apples (Malus domestica), Conference and Bartlett pears (Pyrus communis), Redhaven peach (Prunus persica), and Perfection apricot (Prunus armeniaca). This suggests that the enzyme has a wide distribution and plays an important role in sorbitol synthesis.

Detection and characterization of sorbitol dehydrogenase from apple callus tissue.

Sorbitol dehydrogenase (l-iditol:NAD(+) oxidoreductase, EC 1.1.1.14) has been detected and characterized from apple (Malus domestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, l-arabitol, ribitol, and l-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitol, myo-inositol, d-arabitol, erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of d-fructose or l-sorbose. No detectable activity was observed with d-tagatose. NADPH could partially substitute for NADH.Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCl buffer (pH 9), or in 2-amino-2-methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. The K(m) for sorbitol and xylitol oxidation were 86 millimolar and 37 millimolar, respectively. The K(m) for fructose reduction was 1.5 molar.Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p-chloromercuribenzoate, and cysteine. ZnSO(4) (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity.

beta-d-Glucan of Avena Coleoptile Cell Walls.

A specific glucanase was used to liberate a noncellulosic beta-d-glucan from isolated cell walls of Avena sativa coleoptile tissue. Cell walls of this tissue contain as much as 7 to 9 mg of glucan/100 mg of dry wall. Because of the specific action pattern of the enzyme, a linkage sequence of.. 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc.. is indicated and the predominance of trisaccharide and tetrasaccharide as hydrolytic products suggests a rather regular repeating pattern in the polysaccharide. The trisaccharide and the tetrasaccharide are tentatively identified as 3-O-beta-cellobiosyl-d-glucose and 3-O-beta-cellotriosyl-d-glucose, respectively. Recovery of these oligosaccharides following glucanase treatment of native wall material was feasible only after wall-bound glucosidases were inactivated. In the absence of enzyme inactivation the released fragments were recovered as glucose. The beta-d-glucan was not extracted from walls by either hot water or protease treatment.Cell walls prepared from auxin-treated Avena coleoptile segments yielded less glucan than did segments incubated in buffer suggesting an auxin effect on the quantity of this wall component. No IAA-induced change in the ratio of the trisaccharide and tetrasaccharide could be detected, suggesting no shift in the 1,3 to 1,4 linkage ratio. While the enzyme acts directly on the beta-d-glucan, no elongation response was apparent when Avena sections were treated with the purified glucanase. The presence of the glucan was not associated with any wound response which could be attributed to the preparation of coleoptile segments. The relationship of glucan metabolism to auxin growth responses is discussed.

Turgor-dependent Changes in Avena Coleoptile Cell Wall Composition.

The effects of reduced turgor pressure on growth, as measured by cell elongation, and on auxin-mediated changes in cell walls, as measured by analyses of wall composition, were examined using Avena coleoptile segments. Although moderate (1-4 bar) decreases in turgor resulted in a progressive decline in growth proportional to the decrease in turgor, the major auxin-induced change in wall composition, a decrease in noncellulosic wall glucose, was unaffected. Severe (5-8 bar) decreases, however, did inhibit this auxin effect on the wall, and with turgor decreases of 9 bars or more this auxin effect was no longer apparent. The results show that turgor pressure is required for this auxin-mediated wall modification and also that this modification of wall glucose occurs at turgor pressures less than those required for wall extension. Changes in other wall components were generally unaffected by altering turgor pressure.