Engineered sorbitol accumulation induces dwarfism in Japanese persimmon

A cDNA encoding sorbitol-6-phosphate dehydrogenase (S6PDH), which is a key enzyme in sorbitol biosynthesis in Rosaceae, was introduced into the Japanese persimmon (Diospyros kaki) to increase the environmental stress tolerance. Resultant transformants exhibited salt-tolerance with dwarfing phenotypes. Therefore, we studied two transgenic lines to understand the physiological mechanism of this dwarfism: lines PS1 and PS6 accumulated high and moderate levels of sorbitol, respectively. The average length of shoots was significantly shorter as compared with the wild-type in line PS1, while no such decrease was observed in line PS6. The myo-inositol and glucose 6-phosphate (G6P) contents were measured in the transgenic lines because previous work with tobacco transformed with S6PDH had suggested that growth inhibition was due to depletion of these metabolites. Although the myo-inositol content was decreased in PS1 plants, the decrease was much smaller than that observed in transgenic tobacco that accumulates sorbitol. The G6P contents were the same in PS1 plants and phenotypically normal PS6 plants. The level of indole-3-acetic acid (IAA), which affects stem elongation, in line PS1 was similar to the levels in the other lines. A decrease in gibberellin (GA) content generally induces dwarfism in plants. However, GA was not decreased in PS1 plants compared with wild-type or control plants. Therefore, we focused on sorbitol accumulation as the most remarkable feature of PS1 plants. As one possibility, the observed growth inhibition was likely caused by an osmotic imbalance between the cytosol and vacuole.     

Adjustments of the enzymatic complement for polyol biosynthesis and accumulation in diapausing cold-acclimated adults of Pyrrhocoris apterus

The capacity to accumulate winter polyols (mainly ribitol and sorbitol) during cold-acclimation in Pyrrhocoris apterus is restricted only to the adults that have previously entered diapause. The enzymatic complement involved in polyol biosynthesis was found to differ in a complex manner between diapause and non-diapause adults. Nearly 100% of glycogen phosphorylase (GPase) was present in its active form in non-diapause adults irrespective of their acclimation status. In contrast, less than 40% of GPase was present in its active form in diapause adults prior to cold-acclimation and the inactive form was rapidly activated upon transition from 5 to 0 degrees C, concomitantly with the start of rapid polyol accumulation. The flow of carbon released by activation of glycogen degradation might be routed to the pentose cycle because the activity of glucose-6-P dehydrogenase (G(6)P-DH) was significantly higher and it increased with cold-acclimation in diapause adults while it was relatively low and it decreased with cold-acclimation in non-diapause adults. Reducing equivalents in the form of NADPH, which were generated in the pentose cycle, might require re-oxidation. Such re-oxidation might be achieved during reduction of sugars to polyols. The activity of NADP(H)-dependent aldose reductase (AR) was about 20-fold higher in diapause than in non-diapause adults. Similarly, the activity of NAD(H)-dependent polyol dehydrogenase (PDH) was higher in diapause adults. In addition, we found a very high activity of an unusual enzyme, NADP(H)-dependent ketose reductase (KR), exclusively in diapause adults. KR might be involved in reduction of fructose to sorbitol. Although its affinity for fructose as a substrate was low (K(M)=0.64M), its activity was about 10-fold higher than that of PDH with fructose. Moreover, the activity of KR significantly increased with cold-acclimation while that of PDH remained unchanged. Different electrophoretic mobilities in PAGE gel suggested that KR and PDH are two different enzymes with specific requirement for NADP(H) or NAD(H), respectively, as co-factors.     

苹果S6PDH基因克隆与原核表达

6-磷酸山梨醇脱氢酶(sorbitol-6-phosphate dehydrogenase,S6PDH)是蔷薇科植物中合成山梨醇的重要酶。以苹果叶片为材料,利用RT-PCR法克隆到S6PDHcDNA全长,将其与大肠杆菌表达载体pET-32a( )构建原核表达载体pET-S并转化大肠杆菌BL21,经IPTG诱导表达后,SDS-PAGE检测结果表明该基因表达了1个约54kD的蛋白,为进一步研究目的蛋白的结构和功能提供了试验基础。     

蔷薇科植物中山梨醇代谢酶的研究进展

蔷薇科植物中,与山梨醇代谢相关的酶主要有：6-磷酸山梨醇脱氢酶、山梨醇脱氢酶和山梨醇氧化酶。综述了近些年来国内外关于这几种酶的研究进展,涉及的内容有：酶的性质与作用、酶的活性变化与转录的关系,及其在生物技术方面的研究成果,并对今后的研究工作进行了展望。     

Transgenically enhanced sorbitol synthesis facilitates phloem-boron mobility in rice

The tolerance of crops to a shortage of boron (B) in the soil varies markedly among species. This variation in tolerance is due, in part, to a species ability to form phloem mobile B-sugar-alcohol complexes (such as B-mannitol or B-sorbitol) which enhance the remobilization of B within the plant. Species lacking the capacity to form B-sugar alcohol complexes are intolerant of even short-term deficits in soil B supply. Here we have genetically engineered rice ( Oryza sativa L.) cultivar Taipei 309 (TP309) with the sorbitol-6-phosphate dehydrogenase (S6PDH) gene, a key enzyme for sorbitol production, and determined the effect of this transformation on the physiology of B remobilization. Sorbitol was detected in the S6PDH transgenic plants as well as in vector-transformed plants and wild-type (TP 309) plants, although the concentration of sorbitol in the S6PDH transgenic plants was significantly enhanced. Remobilization of B from mature leaves to flag leaves correlated with increased levels of sorbitol. The presence of sorbitol and detection of B remobilization in the wild-type and vector-transformed plants suggests that rice utilizes an unknown pathway for sorbitol synthesis and may partly explain the relative insensitivity of rice to B deficits when compared to other graminaceous crops.     

Molecular evidence of sorbitol dehydrogenase in tomato, a non-Rosaceae plant

The enzyme NAD-dependent sorbitol dehydrogenase (SDH) is well characterized in the Rosaceae family of fruit trees, which synthesizes sorbitol as a translocatable photosynthate. Expressed sequence tags of SDH-like sequences have also been generated from various non-Rosaceae species that do not synthesize sorbitol as a primary photosynthetic product, but the physiological roles of the encoded proteins in non-Rosaceae plants are unknown. Therefore, we isolated an SDH-like cDNA (SDL) from tomato (Lycopersicon esculentum Mill.). Genomic Southern blot analysis suggested that SDL exists in the tomato genome as a single-copy gene. Northern blot analysis showed that SDL is ubiquitously expressed in tomato plants. Recombinant SDL protein was produced and purified for enzymatic characterization. SDL catalyzed the interconversion of sorbitol and fructose with NAD (H). SDL showed highest activity for sorbitol among the several substrates tested. SDL showed no activity with NADP+. Thus, SDL was identified as a SDH, although the Km values and substrate specificity of SDL were significantly different from those of SDH purified from the Japanese pear (Pyrus pyrifolia), a Rosaceae fruit tree. In addition, tomato was transformed with antisense SDL to evaluate the contribution of SDL to SDH activity in tomato. The transformation decreased SDH activity to approximately 50% on average. Taken together, these results provide molecular evidence of SDH in tomato, and SDL was renamed LeSDH.     

Effect of drought on sorbitol and sucrose metabolism in sinks and sources of peach

In peach (Prunus persica [L.] Batsch.), sorbitol and sucrose are the two main forms of photosynthetic and translocated carbon and may have different functions depending on the organ of utilization and its developmental stage. The role and interaction of sorbitol and sucrose metabolism was studied in mature leaves (source) and shoot tips (sinks) of ‘Nemaguard’ peach under drought stress. Plants were irrigated daily at rates of 100, 67, and 33% of evapotranspiration (ET). The relative elongation rate (RER) of growing shoots was measured daily. In mature leaves, water potential (Ψ_w), osmotic potential (Ψ_s), sorbitol‐6‐phosphate dehydrogenase (S6PDH, EC 1.1.1.200), and sucrose‐phosphate synthase (SPS, EC 2.4.1.14) activities were measured weekly. Measurements of Ψ_s, sorbitol dehydrogenase (SDH, 1.1.1.14), sucrose synthase (SS, EC 2.4.1.13), acid invertase (AI, EC 3.2.1.26), and neutral invertase (NI, EC 3.2.1.27) activities were taken weekly in shoot tips. Drought stress reduced RER and Ψ_w of plants in proportion to water supply. Osmotic adjustment was detected by the second week of treatment in mature leaves and by the third week in shoot tips. Both SDH and S6PDH activities were reduced by drought stress within 4 days of treatment and positively correlated with overall Ψ_w levels. However, only SDH activity was correlated with Ψ_s. Among the sucrose enzymes, only SS was affected by drought, being reduced after 3 weeks. Sorbitol accumulation in both mature leaves and shoot tips of stressed plants was observed starting from the second week of treatment and reached up to 80% of total solutes involved in osmotic adjustment. Sucrose content was up to 8‐fold lower than sorbitol content and accumulated only occasionally. We conclude that a loss of SDH activity in sinks leads to osmotic adjustment via sorbitol accumulation in peach. We propose an adaptive role of sorbitol metabolism versus a maintenance role of sucrose metabolism in peach under drought stress.     

干旱胁迫下平邑甜茶各器官中山梨醇的累积

检测聚乙二醇6000(PEG6000)模拟干旱下的水培苗、自然干旱下的盆栽苗和离体叶片自然失水后平邑甜茶不同器官中山梨醇含量和相关代谢酶活性的结果表明,轻度和中度干旱胁迫下的平邑甜茶叶中山梨醇含量、6-磷酸山梨醇脱氢酶(S6PDH)和6-磷酸山梨醇磷酸酶(SorPP)活性以及韧皮部中山梨醇含量显著增加;根和韧皮部中山梨醇含量平行上升,山梨醇脱氢酶(SDH)活性随之下降。     

Effect of high temperature on the metabolic processes affecting sorbitol synthesis in the silverleaf whitefly,Bemisia argentifolii

Whiteflies accumulate the polyhydric alcohol, sorbitol, when exposed to temperatures greater than about 30 degrees C. Feeding experiments using artificial diets containing labeled sucrose showed that more of the label was incorporated into whitefly bodies and less was excreted in the honeydew when feeding was conducted at 41 compared with 25 degrees C. Analysis of the components of the honeydew showed that more of the excreted label was in glucose and fructose and less in trehalulose at 41 degrees C than at 25 degrees C. A similar effect of temperature on honeydew composition occurred for whiteflies feeding on cotton leaves. Measurement of the activities of glycolytic, pentose-phosphate and polyol pathway enzymes at 30 and 42 degrees C showed that NADPH-dependent ketose reductase/sorbitol dehydrogenase (NADPH-KR/SDH), sucrase, glucokinase and glucose-6-phosphate dehydrogenase activities were stimulated to a greater extent at 42 degrees C than trehalulose synthase and fructokinase. NAD(+)-sorbitol dehydrogenase (NAD(+)-SDH) activity was inhibited at 42 degrees C. We propose that high temperature alters metabolic activity in a way that increases the availability of fructose and stimulates pentose-phosphate pathway activity, providing both the substrate and coenzyme for sorbitol synthesis. High temperature also increases the activity of NADPH-KR/SDH, the enzyme in whiteflies that synthesizes sorbitol, but inhibits the activity of NAD(+)-SDH, the enzyme that degrades sorbitol.     

Fructose supports glycogen accumulation,heterocysts differentiation,N2 fixation and growth of the isolated cyanobiont Anabaena azollae

Cells of the cyanobiont Anabaena azollae isolated from the water fern Azolla filiculoides were found to take up and utilize fructose in the light for mixotrophic growth. Fructose was favored by the cyanobiont as a substrate over sucrose and glucose. Cell growth in the presence of 8 mM fructose led to glycogen accumulation in the cells which approached 20% of the cell dry weight within 2 to 3 days, followed by reduction of glycogen content during the fourth day. Glucose-6-phosphate dehydrogenase activity was increased 5–6-fold in the fructose grown cells from the third day of growth onwards. The frequency of heterocysts in fructose-grown cells increased from 6 to 18%, and acetylene reduction by nitrogenase was increased 3-fold in the presence of fructose as compared with control cells, with maximum values observed between the third and fifth day of mixotrophic growth. Fructose-supported growth yielded a 2–4-fold increase in cell dry weight over controls.It is suggested that fructose-supported development and growth of the cyanobiont in batch cultures may resemble its mixotrophic growth and development in situ in the leaf cavity of the host fern Azolla.Abbreviation G6PDH glucose-6-phosphate dehydrogenase     

Aldose reductase and sorbitol dehydrogenase distribution in rat kidney.

The sorbitol pathway catalyzes the conversion of glucose to fructose via the intermediate sorbitol. It consists of aldose reductase (AR) and sorbitol dehydrogenase (SDH). In adult (44 day) kidney zones, AR was highest in the outer medulla. In substructures AR was highest in distal convoluted tubule. The AR was greatest in newborn and 8-day zones of developing rat kidney. Acute alloxan diabetes was associated with decreased AR in small arteries, but not glomeruli. The SDH was lowest in outer medulla. It was most active in glomeruli and distal convoluted tubules. The diabetic state leads to no change of SDH in arteries but an increase in glomeruli. SDH increased with development. This study demonstrates AR and SDH in substructures of the kidney. The pathway is present in developing kidney. In diabetes the enzymatic changes would tend to decrease accumulation of sorbitol.     

Carbohydrate availability modifies sorbitol dehydrogenase activity of apple fruit

Complete defoliation of the stem or spur and girdling the phloem (D/G) subtending a single apple ( Malus domestica Borkh.) fruit at 70 or more days after bloom resulted in reduction of fruit growth. Extractable sorbitol dehydrogenase (SDH) (enzyme code, 1.1.1.14) activity and sorbitol and starch content of the cortex tissue of fruit receiving D/G treatment subsequently declined. Co-extraction of control and D/G cortex tissues yielded expected levels of SDH activity, indicating that the loss of extractable SDH activity in D/G fruit was not due to presence of an inhibitor or proteolytic activity. Incubating cortex sections from D/G fruit in a buffered 200 m M sorbitol or glucose solution increased extractable SDH activity, and incubating cortex sections from control fruit in the sorbitol solution maintained the activity. However, neither 200 m M fructose or 27 m M PEG, the latter with the same osmotic potential as the sorbitol solution, affected extractable SDH activity of D/G fruit. The results indicate that carbohydrate availability may affect extractable SDH activity of apple fruit, and that specific carbohydrates such as sorbitol and glucose may be signals for modulating this activity.     

Altered activity of the P2 isoform of plastidic glucose 6-phosphate dehydrogenase in tobacco (Nicotiana tabacum cv. Samsun) causes changes in carbohydrate metabolism and response to oxidative stress in leaves

Expression of one specific isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauliflower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, reflecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 micro m MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.     

水稻质体葡萄糖-6-磷酸脱氢酶基因的克隆与表达研究

戊糖磷酸途径是高等植物中重要的代谢途径,主要生理功能是产生NADPH以及供核酸代谢的磷酸戊糖。葡萄糖-6-磷酸脱氢酶（G6PDH）是戊糖磷酸途径的关键酶,广泛存在于高等植物细胞的细胞质和质体中。木研究首次从水稻（Oryza sativa L.）幼苗中分离了核编码的质体G6PDH基因OsG6PDH2,序列分析表明OsG6PDH2编码一个具有588个氨基酸残基的多肽,等电点为8．5,分子量66kDa。OsG6PDH2的N端有1个70个氨基酸的信号肽,推测的裂解位点为Gly55和Val56,表明OsG6PDH2编码产物可能定位于质体。多序列比较的结果表明OsG6PDH2与拟南芥、烟草、马铃薯质体G6PDH的一致性分别达81％、87％、83％。进化关系说明水稻OsG6PDH2与拟南芥（AtG6PDH3）、马铃薯（StG6PDH1）处于高等植物P2型质体G6PDH分支上,暗示了OsG6PDH2可能是一个P2型的质体蛋白。Matinspector程序分析表明,OsG6PDH2在起始密码子上游含有一个bZIP转录因子识别位点、一个ABA应答元件、一个CRT／DRE元件和1个W-box元件。半定量RT-PCR分析表明,OsG6PDH2在水稻根、茎、叶和幼穗组织中都呈低丰度组成型表达,在根部表达较高,在水稻幼苗中的表达显著受暗处理的诱导。将OsG6PDH2的完整开放阅读框构建到大肠杆菌表达载体pET30a（＋）中,pET30a（＋）-OsG6PDH2在大肠杆菌中得到了有效表达。酶活性测定证明,OsG6PDH2的编码产物具有葡萄糖-6-磷酸脱氢酶的功能。     

Characterization of mannitol metabolism in the mangrove red algaCaloglossa leprieurii (Montagne) J.Agardh

A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK _m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD⁺. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK _m value of 1.2 mM and a high requirement of Mg²⁺ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK _m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD⁺. For the non-specific HK, only theK _m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase     

The polyol pathway in the bovine oviduct

The oviducts likely provide optimized micro‐environments for the final maturation of gametes, fertilization, and early embryo development. Hexoses, including glucose, fructose, and sorbitol, are involved in these critical reproductive events. Monosaccharide production is controlled, in part, by the polyol pathway and requires two enzymes: an aldose reductase (AR) that reduces glucose into sorbitol, followed by its oxidation into fructose by sorbitol dehydrogenase (SDH). We analyzed the expression of AR and SDH in the isthmus and ampulla of the bovine oviduct at the proliferative, mid‐luteal, and late‐luteal phases of the estrous cycle by quantitative PCR and immunoblots. Immunochemistry and an assay of SDH activity were also performed. The quantity of hexoses in whole sections of isthmus and ampulla were determined by liquid chromatography coupled to mass spectrometry. In sum, AR expression was restricted to the isthmus, while SDH was mostly expressed in the isthmic–ampullary junction and the ampulla, specifically concentrated in the luminal epithelium of the oviduct. The estrous cycle had no impact on protein expression of AR and SDH. Instead, the levels of AR and SDH expression were associated with higher ratios of sorbitol to fructose in the isthmus (1.6) than in the ampulla (4.1; P = 0.005). These results are discussed in light of physiological events occurring in the oviduct. Mol. Reprod. Dev. 79: 603–612, 2012. © 2012 Wiley Periodicals, Inc.     

Carbon tetrachloride and the sorbitol pathway in the diabetic mouse

1. Sorbitol dehydrogenase activity and the hepatic and serum concentrations of sorbitol, glucose and fructose were quantified in diabetic mice. 2. Blood glucose concentrations were increased over 300% by diabetes and were decreased toward normal after insulin-treatment. 3. Hepatic sorbitol concentrations ranged from 7-15 mumol/g and were highest in uncontrolled diabetic mice. 4. Hepatic concentrations of fructose and sorbitol were not affected by insulin administration. 5. Challenge with carbon tetrachloride (25 microliters/kg i.p.) did not alter concentrations of glucose, sorbitol or fructose in blood or liver. 6. Sorbitol dehydrogenase activity in blood was increased similarly in normal, diabetic and insulin-treated diabetic mice after CCl4 administration. 7. The data indicate that sorbitol did not accumulate in diabetic mice, and that induction of diabetes did not increase the susceptibility of mice to CCl4 hepatotoxicity as occurs in rats.     

Biochemical characteristics of erythrocyte sorbitol dehydrogenase from selected mammals

The erythrocyte sorbitol dehydrogenase (EC 1.1.1.14) activity, regarding its action on sorbitol oxidation to fructose, was studied in 19 species of mammals, showing a striking variability, with high activity in rodents. Enzyme activity was studied against other polyols, namely xylitol, inositol, manitol and dulcitol. Most animals showed activity against all the polyols studied, but hamster and red deer only presented activity on sorbitol and xylitol. Michaelis-Menten constant determinations for sorbitol were performed, and it was observed that animals which presented high activity had a high Km. pH curves were obtained from 8 animals, with an optimum pH ranging from pH 8.0 to pH 10.0; four of the animals presented an optimum pH at 8.5.