Dependent Sorbitol Dehydrogenase Research Articles

An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields. However, this option is disallowed by the configuration of the native yeast metabolic network. To explore metabolic engineering strategies for eliminating this constraint, we studied alcoholic fermentation of sorbitol. Sorbitol cannot be fermented anaerobically by S. cerevisiae because its oxidation to pyruvate via glycolysis yields one more NADH than conversion of glucose. To enable re-oxidation of this additional NADH by alcoholic fermentation, sorbitol metabolism was studied in S. cerevisiae strains that functionally express heterologous genes for ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK). Together with the yeast non-oxidative pentose-phosphate pathway, these Calvin-cycle enzymes enable a bypass of the oxidative reaction in yeast glycolysis.ResultsConsistent with earlier reports, overproduction of the native sorbitol transporter Hxt15 and the NAD+-dependent sorbitol dehydrogenase Sor2 enabled aerobic, but not anaerobic growth of S. cerevisiae on sorbitol. In anaerobic, slow-growing chemostat cultures on glucose–sorbitol mixtures, functional expression of PRK-RuBisCO pathway genes enabled a 12-fold higher rate of sorbitol co-consumption than observed in a sorbitol-consuming reference strain. Consistent with the high Km for CO2 of the bacterial RuBisCO that was introduced in the engineered yeast strains, sorbitol consumption and increased ethanol formation depended on enrichment of the inlet gas with CO2. Prolonged chemostat cultivation on glucose–sorbitol mixtures led to loss of sorbitol co-fermentation. Whole-genome resequencing after prolonged cultivation suggested a trade-off between glucose-utilization and efficient fermentation of sorbitol via the PRK-RuBisCO pathway.ConclusionsCombination of the native sorbitol assimilation pathway of S. cerevisiae and an engineered PRK-RuBisCO pathway enabled RuBisCO-dependent, anaerobic co-fermentation of sorbitol and glucose. This study demonstrates the potential for increasing the flexibility of redox-cofactor metabolism in anaerobic S. cerevisiae cultures and, thereby, to extend substrate range and improve product yields in anaerobic yeast-based processes by enabling entry of additional electrons.

The variation of NAD+-SDH gene in mutant white-fleshed loquat

Loquat (Eriobotrya japonica Lindl.) can be divided into yellow- and white-fleshed cultivars by flesh color. However, a Dongting loquat mutant, which involved bud sport and growing white-fleshed fruit in the central region of the trunk (as wild loquat bears yellow-fleshed fruits naturally), was discovered in the preliminary study. The study cloned the coding sequence (CDS) of NAD+-dependent sorbitol dehydrogenase (NAD+-SDH) gene from the selected materials of mutant loquat, wild loquat and other nine loquat cultivars/accessions, and found that the CDS of NAD+-SDH gene from the mutant loquat, other than the rest two types of materials, had three single nucleotide polymorphisms (SNPs) loci; in addition, the amino acid encoded at variation loci changed accordingly. NAD+-SDH plays an active role in converting sorbitol into fructose in loquat cultivars. For the mutant white-fleshed loquat, the activity of NAD+-SDH rises first and then drops, the sorbitol content decreases steadily, and its fructose content is higher than that in wild loquat from coloration to maturation stage. As demonstrated by the real-time fluorescence quantification PCR analysis, the expression level of NAD+-SDH gene at maturation stage is about 5-fold lower than wild type. It may be assumed that, the three SNPs loci might lead to excessive conversion of sorbitol into fructose under the catalytic action of NAD+-SDH of white-fleshed mutant loquat at maturation stage, resulting in the increase of fructose content and reduced expression abundance of mRNA after transcription. Besides, NAD+-SDH gene may be related to flesh color and carbohydrate variation of white-fleshed mutant loquat.

Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling.

Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. Nicotinamide adenine dinucleotide+-dependent SORBITOL DEHYDROGENASE (SDH, E. C. 1.1.1.14) from Arabidopsis thaliana L. sorbitol dehydrogenase (AtSDH) is capable of oxidizing several polyols including sorbitol, ribitol, and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S) and C4 (R) polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling, and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn.) (Hemiptera: Aleyrodidae) SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S) and C4 (R).

The level of mRNA NAD-SDH is regulated through RNA splicing by sugars and phytohormones

Unspliced (L-SDH) and spliced (S-SDH) transcripts of NAD+-dependent sorbitol dehydrogenase (NAD-SDH) of strawberry (Fragaria ananassa Duch., cv. Nyoho) were investigated. Fructose, mannitol, and IAA increased S-SDH transcript level. Sorbitol decreased the level of L-SDH transcript 2.5-fold. ABA and bensyladenin (BA) decreased S-SDH thanscript level and increased L-SDH transcripts. Ratio of S-SDH to L-SDH was increased by sorbitol (2.7-fold), fructose (2.2-fold), IAA (1.5-fold), and decreased by BA (4-fold) and ABA (1.7-fold) as compared to control. These results suggest that sorbitol, fructose, and IAA stimulated splicing of pre-mRNA NAD-SDH, but BA and ABA repressed this process.

Non-climacteric ripening and sorbitol homeostasis in plum fruits

During ripening fruits undergo several physiological and biochemical modifications that influence quality-related properties, such as texture, color, aroma and taste. We studied the differences in ethylene and sugar metabolism between two genetically related Japanese plum cultivars with contrasting ripening behaviors. ‘Santa Rosa’ (SR) behaved as a typical climacteric fruit, while the bud sport mutant ‘Sweet Miriam’ (SM) displayed a non-climacteric ripening pattern.SM fruit displayed a delayed ripening that lasted 120 days longer than that of the climacteric fruit. At the full-ripe stage, both cultivars reached similar final size and weight but the non-climacteric fruits were firmer than the climacteric fruits. Fully ripe non-climacteric plum fruits, showed an accumulation of sorbitol that was 2.5 times higher than that of climacteric fruits, and the increase in sorbitol were also paralleled to an increase in sucrose catabolism. These changes were highly correlated with decreased activity and expression of NAD+-dependent sorbitol dehydrogenase and sorbitol oxidase and increased sorbitol-6-phosphate dehydrogenase activity, suggesting an enhanced sorbitol synthesis in non-climacteric fruits.

Mining and comparison of the genes encoding the key enzymes involved in sugar biosynthesis in apple, grape, and sweet orange

Fruit taste depends on the contents and types of soluble sugars and organic acids. The large-scale identification and comparison of genes involved in sugar biosynthesis in apple, grape, and sweet orange based on the genome and expressed sequence tag information have not been reported. In this study, we found that sorbitol-6-phosphate dehydrogenase (S6PDH) was one of the most important genes in sorbitol biosynthesis in apple leaf, and was decomposed by NAD+-dependent sorbitol dehydrogenase (NAD+SDH) in apple fruit. The high expression level of soluble acidic invertase (AIV) gene possibly caused the high soluble sugar content (i.e., glucose and fructose) in grape. The expression level of sucrose synthase (SUS) in grape and sweet orange was also significantly higher than that in apple. The results show that SUS was involved in the resynthesis of fructose and UDP-glucose into sucrose in grape and sweet orange. By contrast, sucrose was transported into a vacuole and decomposed into glucose and fructose by AIV. Thus, ADP-glucose pyrophosphorylase (AGP), granule-bound starch synthase (GBSS), isoamylase 1 (ISA1), and isoamylase 2 (ISA2) could be the key genes in starch biosynthesis, whereas α-amylase (AMY3), isoamylse 3 (ISA3), and pullulanase (PUL) could be the key genes in starch degradation in apple. This study provides new insight into the isolation and comparison of candidate genes involved in the sugar biosynthetic pathway among multiple plants.

High Temperature Alters Sorbitol Metabolism in Pyrus pyrifolia Leaves and Fruit Flesh during Late Stages of Fruit Enlargement

Sorbitol is the main photosynthetic product and primary translocated carbohydrate in the Rosaceae and plays fundamental roles in plant growth, fruit quality, and osmotic stress adaptation. To investigate the effect of frequent high temperature during advanced fruit development on fruit quality of chinese sand pear [Pyrus pyrifolia (Burm. f.) Nakai], we analyzed sorbitol metabolism in mature leaves and fruit flesh of potted ‘Wonhwang’ pear trees. In mature leaves, sorbitol synthesis catalyzed by NADP+-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) was repressed, while sorbitol utilization mainly catalyzed by NAD+-dependent sorbitol dehydrogenase (NAD+-SDH) and NADP+-dependent sorbitol dehydrogenase (NADP+-SDH) was higher than that before high-temperature treatment, which resulted in decreased sorbitol accumulation. In contrast, sucrose accumulation in mature leaves was significantly enhanced in response to high temperatures. In fruit flesh, accumulation of sorbitol and sucrose was increased at the time of harvest under high temperatures. Among sorbitol metabolic enzymes, only NAD+-SDH was sensitive to high temperature in fruit flesh, and significant decrease of NAD+-SDH activity indicated that the fruit sorbitol-uptake capacity was undermined under high temperatures. Transcription analysis revealed tissue-specific responses of NAD+-SDH genes (PpSDH1, PpSDH2, and PpSDH3) to high-temperature treatment. The NAD+-SDH activity and regulation of PpSDH1 and PpSDH3 were positively correlated in mature leaves. However, the downregulation of PpSDH1 and PpSDH2 was consistent with decreased enzyme activity in the fruit flesh. With regard to sorbitol transport, two sorbitol transporter genes (PpSOT1 and PpSOT2) were isolated, and downregulation of PpSOT2 expression in mature leaves indicated that the sorbitol-loading capability decreased under high-temperature conditions because of the limited sorbitol supply. These findings suggested that sorbitol metabolism responded differently in mature leaves and fruit flesh under high temperature, and that these dissimilar responses influenced fruit quality and may play important roles in adaptation to high temperatures.

Cloning, expression, purification and assay of sorbitol dehydrogenase from "Feicheng" peach fruit (Prunus persica)

A gene encoding NAD+-dependent sorbitol dehydrogenase (SDH) in peach fruit was cloned and expressed in Escherichia coli. Recombinant SDH protein with 6×His-tagged was localized exclusively in the cytoplasmic soluble fraction of E. coli when the strains were grown for 4-5 h at 37 ºC. Highly pure protein was isolated by Ni2+-resin chromatography with 150 mM imidazole in 50 mM Tris, pH 8.0, by elution. In order to ensure that the recombinant SDH could be used for further study, the fluorescence and ultraviolet spectrum of the recombinant SDH were detected. Recombinant SDH was confirmed to be capable of oxidizing sorbitol by enzymatic activity assay. The activity of the recombinant SDH was 2.73 U mg-1min-1, which was similar with that directly extracted from peach fruits. The activities of SDH extracted from the fruits in different periods (30, 60, 90 days after flowing) were 7.75, 5.95, 3.26 U mg-1min-1, respectively.

Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed

Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD+-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH

Identification of genes possibly related to loss of quality in late-season ‘Hass’ avocados in Chile

The ‘Hass’ avocado (Persea americana Mill.) is the most important avocado variety cultivated worldwide. To reach destination markets, long-term cold storage and transit is usually needed to reduce fruit deterioration. However, low temperatures can affect fruit quality during ripening by increasing the incidence of several physiological disorders. In this work, ‘Hass’ avocados harvested in Chile in the early summer showed more incidence of physiological disorders than fruit harvested in the spring after storage for 40d at 5°C. As an initial approach to understand the types of metabolism affected during ripening after low temperature exposure, a forward suppression subtractive hybridization (SSH) cDNA library was constructed. A total of 117 differentially expressed genes encoding proteins involved in different biochemical pathways were identified. Genes related to stress response, protein synthesis and signal transduction were the most abundant. To corroborate our results with some of the previously found key differentially expressed genes, full length cDNA was obtained by rapid amplification of cDNA ends (RACE-PCR). Analysis of gene expression using real-time quantitative PCR was performed. Six avocado genes relating to cell wall, sugar and lipid metabolism were analyzed: polygalacturonase (PamPG), putative NAD+-dependent sorbitol dehydrogenase (PamSDH), stearoyl-ACP desaturase (SAD) and three putative acyl-CoA synthetase (PamACoAS1, 2, 3). The expression of all genes was up-regulated after ripening, especially in avocados harvested in the spring, except for PamSAD. These results imply that these genes could be related to the loss in quality observed in the fruit from the second harvest. Further studies should be performed to identify markers for quality loss in ‘Hass’ avocados.

Cloning of NAD-SDH cDNA from plum fruit and its expression and characterization

A full-length cDNA consisting of 1444 bp for NAD dependent sorbitol dehydrogenase (NAD-SDH) was cloned from fruit of plum (Prunus salicina var. cordata cv. Younai) by means of RT-PCR and RACE. The cDNA containing an open reading frame (ORF) of 1101 bp encoded a polypeptide of 367 amino acid residues. The maltose binding protein fusion SDH (MBP-SDH) was expressed and partially purified from Escherichia coli cells, and biochemical properties of MBP-SDH and SDH cleaved from the fusion protein by factor Xa were characterized. The MBP-SDH had the specific affinity for NAD and was able to oxidize sorbitol, xylitol, l-ribitol and mannitol but not ethyl alcohol, arabitol and other polyols. The optimum pH for the oxidation of sorbitol and the reduction of fructose was 9.0 and 7.0, respectively; the maximum reaction rate occurred when temperature increased up to 50 °C in the presence of sorbitol. The MBP-SDH with a subunit of 80 kDa appears to be a hexamer. Its molecular weight was 478.6 kDa estimated by gel filtration and 493.2 kDa estimated using native linear gradient PAGE. The K(m) values for sorbitol, NAD, fructose and NADH were 95.86 mM, 0.31 mM, 1.04 M and 0.038 mM, respectively. However, when MBP was cleaved from the fusion enzyme, the SDH exists as a homotetramer with the native molecular weight of 164.8 kDa estimated by gel filtration. The K(m) values were 111.8 mM, 0.35 mM, 1.25 M and 0.048 mM for sorbitol, NAD, fructose and NADH, respectively. The MBP-SDH and the SDH were similar with respect to their kinetic characteristics despite their difference in quaternary structures.

Ubiquitous distribution and different subcellular localization of sorbitol dehydrogenase in fruit and leaf of apple

NAD+-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), a key enzyme in sorbitol metabolism, plays an important role in regulating sink strength and determining the quality of apple fruit. Understanding the tissue and subcellular localization of NAD-SDH is helpful for understanding sorbitol metabolism in the apple. In this study, two NAD-SDH cDNA sequences were isolated from apple fruits (Malus domestica Borkh cv. Starkrimson) and named MdSDH5 and MdSDH6. Immunohistochemical analysis revealed that NAD-SDH is distributed in both the flesh and the vascular tissue of the fruit, and the vascular tissue and mesophyll tissue in the young and old leaves, indicating that it is a ubiquitous protein expressed in both sink and source organs. Immunogold electron microscopy analysis demonstrated that NAD-SDH is localized mainly in the cytoplasm and chloroplast of the fruit and leaves. The chloroplast localization of NAD-SDH was confirmed by the transient expression of MdSDH5-GFP and MdSDH6-GFP in the mesophyll protoplast of Arabidopsis. NAD-SDH was also found in electron opaque deposits of vacuoles in young and mature leaves. These data show that NAD-SDH has different subcellular localizations in fruit and leaves, indicating that it might play a different role in sorbitol metabolism in different tissues of apple.

Properties of Sorbitol Dehydrogenase in Strawberry Fruit and Enhancement of the Activity by Fructose and Auxin

Strawberry, belonging to the Rosaceae family, translocates sucrose as the main carbohydrate to fruit, although many Rosaceae fruit trees use sorbitol as translocated sugar; however, we have found that strawberry has genes encoding sorbitol-metabolizing enzymes. In this study, the property of NAD+-dependent sorbitol dehydrogenase (NAD-SDH) and the effect of sugars and phytohormones on NAD-SDH activity and its mRNA accumulation were investigated to elucidate sorbitol metabolism and its regulation. The Km values of NAD-SDH for substrates of fructose and sorbitol were 78.7 and 7.3 mM, respectively, similar to those of maize, which synthesizes sorbitol by the reduction of fructose by NAD-SDH. This result suggested that NAD-SDH in strawberry fruit can catalyze the reduction of fructose, but the activity is not enough to accumulate sorbitol in fruit. Therefore, we investigated the effect of sugars and phytohormones on NAD-SDH activity and the transcript level by adding various sugars and phytohormones to sliced fruit discs. Tissues incubated in 100 mM fructose or sorbitol stimulated NAD-SDH activity by about 2.5-fold compared with the control. Sucrose also stimulated NAD-SDH activity, but the increase was not as high as sorbitol or fructose. Of the phytohormones treated, 100 μM indole-3-acetic acid (IAA) had a marked stimulatory effect on NAD-SDH activity, but the other phytohormones (abscisic acid, gibberellic acid and 6-benzyladenine) had no stimulative effect. The mRNA transcript level in all sugar and phytohormone treatments showed no marked increase compared with the control. Thus, the kinetics and induction profiles by phytohormones of NAD-SDH showed that sorbitol metabolism in strawberry fruit was different from that in apple and Japanese pear fruits.

Presence and expression of NAD+-dependent sorbitol dehydrogenase and sorbitol-6-phosphate dehydrogenase genes in strawberry

SummaryThe sorbitol metabolic pathway in strawberry (Fragaria ananassa Duch. var. ‘Nyoho’) is unclear, despite strawberry belonging to the family Rosaceae. Therefore, the presence or absence of genes for NAD+-dependent sorbitol dehydrogenase (NAD-SDH) and sorbitol-6-phosphate dehydrogenase (S6PDH) in strawberry, and their levels of expression related to growth were investigated. A full-length cDNA encoding the NAD-SDH gene in strawberry (FaSDH) was obtained using rapid amplification of cDNA ends (RACE). The full-length clone consisted of 1,399 bp and contained a 1,083 bp open reading frame encoding 361 amino acids with a calculated molecular mass of 38.8 kDa, and a predicted isoelectric point (pI) of 6.08. This nucleotide sequence showed 79 – 85% similarity with the NAD-SDH genes of other plants. Its amino acid sequence contained both zinc- and NAD-binding sites, confirming that this sequence belonged to the NAD-SDH gene family. Southern blotting suggested that the genomic copy number of FaSDH was less than in apple. The expression of FaSDH transcripts in strawberry fruit was higher at the young and mature stages than at other stages. The transcript level in mature leaves was higher than in the shoot tip, and in folded and young leaves. NAD-SDH activity was detected in strawberry fruits and leaves and the activity in fruit was comparable to that in other fruits. Reverse transcriptase (RT)-PCR using specific primers designed based on the S6PDH gene of Fragaria vesca, showed a clear single band in leaves and fruits of strawberry. Sequence analysis showed this RT-PCR fragment, named FaS6PDH, to have high homology to the S6PDH gene sequence of other plants, suggesting that the S6PDH gene exists in the strawberry genome and that its mRNA is expressed in leaves and fruits. However, neither the protein nor its enzyme activity were detected in leaves or fruit.

Changes in the Activity and Gene Expression of Sorbitol- and Sucrose-related Enzymes with Leaf Development of 'La France' Pear

The activity and gene expression of sorbitol-6-phosphate dehydrogenase (S6PDH), NAD+-dependent sorbitol dehydrogenase (NAD-SDH), sucrose phosphate synthase (SPS), sucrose synthase (SS) and soluble acid invertase (S-AIV), in developing ‘La France’ pear leaves in relation to the metabolism of sorbitol and sucrose as translocated sugars, were investigated. The levels of activity and mRNA of S6PDH were significantly higher in mature leaves than in folded and young ones. S6PDH activity was more than ten times higher than SPS activity. SPS activity remained at an almost constant level throughout the developmental stages; it was undetectable except in the very young leaves although its mRNA was present. NAD-SDH and SS activities were higher in young leaves than in mature ones; the levels of mRNAs, however, exhibited an inverse patterns to that of activities. The levels of both activity and mRNA of S-AIV were significantly higher in folded and young leaves than mature ones. The distinctly higher expression of S6PDH than that of SPS in mature leaves suggests that the former is more important than the latter in relation to CO2 assimilation (source activity). Thus, the most important change in the transition from sink to source of the pear leaf is the increasing expression of S6PDH gene with leaf maturation.

Changes in the Activity and Gene Expression of Sorbitol- and Sucrose-related Enzymes Associated with Development of 'La France' Pear fruit

To clarify the roles of the five sorbitol- and sucrose-related enzymes important to the development of ‘La France’ pear fruit, we traced the fluctuations in the activities and mRNA levels of these enzymes. Such an approach of simultaneously assaying five sugar-metabolizing enzymes is a first in pear fruit and the Rosaceae family. Both the activity and the mRNA level of NAD+-dependent sorbitol dehydrogenase (NAD-SDH) were high in young fruit, but decreased during fruit enlargement and rose again with fruit maturation. Sucrose synthase (SS) showed nearly the same fluctuation pattern as did NAD-SDH. The changes in both NAD-SDH and SS activities, based on fresh weight, were proportional to that of the relative growth rate (RGR) of the fruit. This suggests that they function closely in supplying hexose from the unloaded sugars in the phloem for active growth. Two isogenes of soluble acid invertase (S-AIV), S-AIV1 and S-AIV2, were present in pear fruit; the former was expressed highly in young fruit, whereas the latter was expressed both in young and mature fruits. The fluctuation pattern of the activity for sorbitol-6-phosphate dehydrogenase (S6PDH) was similar to that of NAD-SDH. However, the role of S6PDH in fruit is unclear. Although sucrose phosphate synthase (SPS) activity was higher in young fruit, it decreased and remained low with fruit maturation. This may be attributed to the finding that ‘La France’ pear fruit accumulates hexose but not sucrose. These results showed that NAD-SDH, SS, and S-AIV are of prime importance in supplying sugars for the development and accumulation of hexose necessary for pear fruit enlargement.

Sorbitol- and Other Sugar-induced Expressions of the NAD+-dependent Sorbitol Dehydrogenase Gene in Japanese Pear Fruit

The regulation of NAD+-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14) by sugar was investigated by using sliced tissues of japanese pear (Pyrus serotina Nakai cv. Kousui) fruit in order to determine its role in the mechanism of sugar accumulation in fruit tissue. The results of the activities and steady-state levels of the protein and mRNA indicate that NAD-SDH in japanese pear fruit is among the sugar-inducible genes. By preincubating the sliced tissues for 16 hours in a medium without sugar, NAD-SDH activity declined and reached a stable level that was maintained for up to 40 hours. The washing procedure also reduced the sugar concentration in the apoplast and cytosol of the sliced tissues to low concentrations and enabled them to be manipulated by exogenous applications of carbohydrate solutions. Incubation of tissues in 50 or 100 mm sorbitol for 8 hours led to enhanced expression of the NAD-SDH gene as determined by increased mRNA and protein levels and enhanced enzyme activity. The presence of 100 mm glucose, sucrose, or mannitol also gave significant stimulation on the levels of activity, protein, and mRNA of NAD-SDH compared with those of control tissues bathed in media in which the osmotic potential had been adjusted to that of the sugar solutions by adding polyethylene glycol. However, fructose was ineffective in stimulating NAD-SDH activities and the level of the protein was not enhanced but the level of mRNA was increased. Therefore, it is suggested that NAD-SDH gene transcription is enhanced by each sugar investigated, and fructose appears to be unique as it also influences NAD-SDH at a post-transcriptional level.

Partial cloning and expression analysis of genes encoding NAD +-dependent sorbitol dehydrogenase in pear bud during flower bud formation

Partial fragments of five NAD +-dependent sorbitol dehydrogenase (NAD-SDH) genes were isolated from the developing buds of Japanese pear ( Pyrus pyrifolia cv. Kosui) by RT-PCR (tentatively named PpySDH1-5). The derived amino acid sequences of PpySDH1-5 have 94 to 98% homology with NAD-SDH genes from apple. PpySDH1-4 were clearly distinguished from PpySDH5 based on primary structure, which showed 88–95% identity with each other, but only 69–71% identity with PpySDH5. Shading of limbs for 20 days increased NAD-SDH enzymatic activity in the buds and correspondingly increased the expression detected by PpySDH1. On the other hand, shoot bending increased NAD-SDH activity in lateral buds, and also increased the expression detected by PpySDH1. The expression of PpySDH5 was barely detected in any of these tissues. These results suggest that PpySDH1-5 encodes the isoforms of NAD-SDH protein which can be categorized into at least two groups and that PpySDH5 may not be requisite to bud growth, though some of the other NAD-SDH gene(s) may be. The increases of NAD-SDH activity, both by shading and shoot bending, are suspected to be regulated at the transcriptional level.

Purification and characterization of a NAD +-dependent sorbitol dehydrogenase from Japanese pear fruit

NAD +-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein. The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer. The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E. coli harboring an apple NAD-SDH cDNA. The K m values for sorbitol and fructose were 96.4±8.60 and 4239±33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction. Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested. These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s). Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine. However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.

Gene Expression of NAD+-Dependent Sorbitol Dehydrogenase and NADP+-Dependent Sorbitol-6-Phosphate Dehydrogenase During Development of Loquat(Eriobotrya japonica Lindl.) Fruit.

The aim of this study is to clarify the roles of NAD+-dependent sorbitol dehydrogenase (NAD-SDH) and NADP+-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) in fruit development, and the regulatory mechanism (s) underlying their expression during development using loquat as the study material. The cDNA of NAD-SDH, cloned from loquat fruit, consisted of 1, 572 bp and contained an open reading frame of 1, 023 bp capable of encoding a protein of 371 amino acids. The deduced amino acid sequence has 97.8% identity to that of an apple fruit. The activity of NAD-SDH is a function of fruit development, i.e. the increase in protein of this enzyme synchronized consistently with that of activity showing no posttranslational modification throughout the developmental stages. Furthermore, NAD-SDH activity correlated with the mRNA levels, indicating that the key regulatory step of the activity is at the transcriptional level. The increase in NAD-SDH with fruit development plays a dominantly important role during fruit maturation and sugar accumulation. The trend of S6PDH activity during fruit development paralleled that of NAD-SDH activity. It correlated with protein and mRNA levels revealing that the regulation of the activity is mainly at the transcriptional level like NAD-SDH. However, the role of S6PDH in fruit is not yet clear.