Interconversions of 14C-labelled Sugars in Apple Tree Tissues12

Tissue pieces excised from orchard-grown apple trees duringa growing season exhibited different and changing capabilitiesof transferring ¹⁴C-label from sucrose, fructose and sorbitolto other soluble carbohydrates. All tissues incorporated fructose¹⁴C into sucrose but only leaves incorporated significant amountsof label from sucrose into sorbitol. As seeds developed andmatured, their ability to incorporate ¹⁴C from sorbitol andfrom fructose into sucrose increased. Sorbitol and sucrose arethe major translocated photosynthetic products of apple leavesbut whereas sorbitol appears to be an end-product of synthesis,sucrose may be considered as a substrate involved more directlyin carbohydrate utilization. Key words: Malus domestica, Apple, Carbohydrate interconversions     

Genomic Structure, Sub-Cellular Localization, and Promoter Analysis of the Gene Encoding Sorbitol-6-Phosphate Dehydrogenase from Apple

Sorbitol is the primary photosynthetic product and the translocatable and storage carbohydrate in apple (Malus domestica) and other fruit tree species within the Rosaceae family. Sorbitol-6-phosphate dehydrogenase (S6PDH, EC 1.1.1.200) is the key enzyme in the biosynthesis of sorbitol. In this study, we isolated two full-length genomic sequences for S6PDH from ??Gala?? apple. The two sequences have same six exons and first two introns, but the sizes of their last three introns are different. The two sequences were mapped to the same loci on chromosome 10. Immunogold electron microscopy analysis demonstrates that the S6PDH is localized mainly in leaf cytosol and chloroplasts. We also isolated and analyzed the promoter region of S6PDH and constructed a series of promoter deletion derivatives with ??-glucuronidase (GUS) gene to identify the upstream region of the S6PDH gene required for promoter activity. The GUS activity in Agrobacterium-mediated transient transformation of tobacco leaves reveals that the -1719 region is more important for gene expression contrasting with other regions in the S6PDH promoter. The promoter region can be induced by cold, dark, and abscisic acid treatment.     

Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO<Subscript>2</Subscript> assimilation in apple leaves

Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, Greensleeves apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15–30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. ¹⁴CO₂ pulse-chase labeling at ambient CO₂ demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO₂ assimilation in response to photon flux density or intercellular CO₂ concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis. As a result, CO₂ assimilation in source leaves was sustained at both ambient CO₂ and saturating CO₂.     

ATP-Promoted Sorbitol Transport into Vacuoles Isolated from Apple Fruit

Sorbitol was transported actively into vacuoles isolated fromapple (Malus pumilla Mill, var domestica Schneid.) fruit flesh.The uptake was stimulated up to twofold by the addition of ATP,and the ATP dependent uptake showed a saturation curve as tothe substrate concentration. The optimum uptake of sorbitolwas pursued in the acidic range of pH 5 to 6. The K_m value forthe ATP dependent sorbitol uptake was about 5 mM. Sorbitol uptake was clearly inhibited by PCMB and uncouplers(CCCP and DCCD), and to a lesser extent by orthovanadate, butonly slightly by oligomycin. K⁺ stimulated sorbitol uptake.Sorbitol was converted to other sugars (glucose) only very slowlywhen transported across the tonoplast. This suggests that sorbitolis transported into vacuoles by a carrier mediated transportsystem coupled with H⁺- ATPase, localized on the tonoplast.Sucrose uptake into the vacuoles was also enhanced by ATP. (Received May 31, 1986; Accepted March 2, 1987)     

NADP+-Dependent Sorbitol Dehydrogenase Found in Apple Leaves

An NADP⁺-dependent sorbitol dehydrogenase that catalyzes sorbitoland glucose was found in apple leaves. The partially purifiedenzyme had optimum activity at pH 9.6 and a K_m value of 128mM for sorbitol. Among the polyols studied, this enzyme showedthe most activity for sorbitol. ¹This paper is contribution A-173 of the Fruit Tree ResearchStation. (Received June 4, 1984; Accepted July 31, 1984)     

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.     

An engineered sorbitol cycle alters sugar composition, not growth, in transformed tobacco

Many efforts have been made to engineer stress tolerance by accumulating polyols. Transformants that accumulate polyols often show growth inhibition, because polyols are synthesized as a dead-end product in plants that do not naturally accumulate polyols. Here, we show a novel strategy in which a sorbitol cycle was engineered by introducing apple cDNA encoding NAD-dependent sorbitol dehydrogenase (SDH) in addition to sorbitol-6-phosphate dehydrogenase (S6PDH). Tobacco plants transformed only with S6PDH showed growth inhibition, and very few transformants were obtained. In contrast, many transgenic plants with both S6PDH and SDH were easily obtained, and their growth was normal despite their accumulation of sorbitol. Interestingly, the engineered sorbitol cycle enhanced the accumulation of sucrose instead of fructose that was expected to be increased. Sucrose, rather than fructose, was also increased in the immature fruit of tomato plants transformed with an antisense fructokinase gene in which the phosphorylation of fructose was inhibited. A common phenomenon was observed in the metabolic engineering of two different pathways, showing the presence of homeostatic regulation of fructose levels.     

Phloem Mobility of Boron is Species Dependent: Evidence for Phloem Mobility in Sorbitol-rich Species

Boron is generally considered to be phloem immobile or to haveonly limited phloem mobility in higher plants. Evidence suggests,however, that B may be mobile in some species within thePyrus,Malus andPrunusgenera. These genera utilize sorbitol as a primarytranslocated photosynthate and it has been clearly demonstratedthat B forms stable complexes with sorbitolin vitro.In the researchpresented here we demonstrate, further, that B is freely phloemmobile inPyrus, MalusandPrunusspecies and suggest that thisis mediated by the formation and transport of B-sorbitol complexes. The pattern of B distribution within shoot organs and the translocationof foliar-applied, isotopically-enriched¹⁰B was studied in sixtree species. Results demonstrate that in species in which sorbitolis a major sugar (sorbitol-rich), B is freely mobile while inspecies that produce little or no sorbitol (sorbitol-poor) Bis largely immobile. The sorbitol-rich species used here werealmond [Prunus amygdalusB. syn.P. dulcis(Mill.)], apple (MalusdomesticaB.) and nectarine (Prunus persicaL. B. var.nectarinaM.),sorbitol-poor species included fig (Ficus caricaL.), pistachio(Pistacia veraL.) and walnut (Juglans regiaL.). In sorbitol-richspecies foliar applied¹⁰B was transported from the treated leavesto adjacent fruit and specifically to the fruit tissues (hull,shell or kernel) that developed during the experimental period.Whereas, foliar-applied¹⁰B was rapidly translocated out of leaves,only a small percentage of the¹¹B present in the leaf at thetime of foliar application was retranslocated. In sorbitol-richspecies, B concentrations differed only slightly between oldand young leaves while fruit tissue had significantly greaterB concentrations than leaves. In contrast, sorbitol-poor specieshad significantly higher B concentrations in older leaves thanyoung leaves while fruit tissue had the lowest B concentration.This occurred irrespective the source of plant B (soil, solutionor foliar-applied). In a subsequent experiment the growth ofapple trees in solutions free of applied B was maintained solelyby foliar applications of B to mature leaves. These resultsindicate that B is mobile in species that produce significantamounts of sorbitol. We propose that the mobility of B in thesespecies is mediated by the formation of B-sorbitol complexes. Almond; Prunus amygdalus ; apple; Malus domestica; nectarine; Prunus persica; fig; Ficus carica; pistachio; Pistacia vera; walnut; Juglans regia; boron; phloem mobility; deficiency; toxicity; inductively coupled plasma-mass; spectrometer     

Manipulation of in Vivo Sorbitol Production Alters Boron Uptake and Transport in Tobacco

Recent evidence that some species can retranslocate boron as complexes with sugar alcohols in the phloem suggests a possible mechanism for enhancing boron efficiency. We investigated the relationship between sugar alcohol (sorbitol) content, boron uptake and distribution, and translocation of foliar-applied, isotopically enriched ¹⁰B in three lines of tobacco (Nicotiana tabacum) plants differing in sorbitol production. In tobacco line S11, transformed with sorbitol-6-phosphate dehydrogenase, the production of sorbitol was accompanied by an increase in the concentration of boron in plant tissues and an increased uptake of boron compared with either tobacco line A4, transformed with antisense orientation of sorbitol-6-phosphate dehydrogenase, or wild-type tobacco (line SR1, zero-sorbitol producer). Foliar application of ¹⁰B to mature leaves was translocated to the meristematic tissues only in line S11. These results demonstrate that the concentration of the boron-complexing sugar alcohol in the plant tissue has a significant effect on boron uptake and distribution in plants, whereas the translocation of the foliar-applied ¹⁰B from the mature leaves to the meristematic tissues verifies that boron is mobile in sorbitol-producing plants (S11) as we reported previously. This suggests that selection or transgenic generation of cultivars with an increased sugar alcohol content can result in increased boron uptake, with no apparent negative effects on short-term growth.     

Tobacco Transformants Bearing Antisense Suppressor of Proline Dehydrogenase Gene, Are Characterized by Higher Proline Content and Cytoplasm Osmotic Pressure

The antisense suppressor was constructed for proline dehydrogenase gene (PDH; a fragment of PDH from Arabidopsis in antisense orientation and under the control of 35S promoter of cauliflower mosaic virus, CMV). In Nicotiana tabacum SR1 tobacco transformants bearing antisense suppressor for PDH, the proline content and the cytoplasm osmotic pressure were increased. The proline content in these transformants varied, whereas cytoplasm osmotic pressure was stable, which seems to reflect complicated relationships between these characteristics of the plant cell.     

Characterization of the Permeability of Excised Apple Tissue 2

Sorbitol uptake from a bathing solution into the compartmentedspace and into the diffusible or apparent free space of excisedparenchyma tissue from apple fruit (Pyrus malus L. cv. GoldenDelicious) was investigated. Uptake into the two cell compartmentswas measured after washing of ^l4C-loaded tissue for 1 h withan osmoticum-free bathing solution. Compartmental analysis showedthat this treatment released sorbitol taken up into the cytoplasmof the cell, which was considered to be part of the apparentfree space. Uptake of sorbitol into the apparent free space was dependenton the osmotic concentration of the incubation medium. Usingmannitol up to 200 mM, uptake decreased by 60%, and increasedagain above 600 mM mannitol, the external concentration whereturgor was eliminated. Uptake in the compartmented space wasabout 3 times lower and was hardly affected by the externalosmotic concentration. PCMBS inhibited sorbitol transport intothe apparent free space by 25% at 100 mM mannitol, but at 600mM the inhibitor had no effect. The results indicate that sorbitoltransport across the plasma membrane is possibly facilitatedby a turgor-sensitive carrier. Uptake of ^l4C-sorbitol into thefreely diffusible space of tissue discs also increased by 200%after storage of unripe fruit for 70 d. This increase in agedtissue did not occur when uptake was measured at 4C or in thepresence of 200 mM PEG. Enhanced uptake was concomitant withan increased release of endogenous sugars from aged tissue. It would appear that the effect of a hypotonic bathing solutionon the permeability of excised apple tissue is related to structuralchanges, such as stretching of the plasma membrane. This effect,which becomes more marked as unripe fruit ages, is probablybrought about by turgor-driven relaxation of the tissue. Itmay increase non-specific leakage of sugars but could also bea factor affecting carrier-mediated transport of sorbitol atthe plasma membrane. Key words: Apple, sugar transport, sorbitol, plasma membrane, apoplast     

Suppression of Acid Invertase Activity by Antisense RNA Modifies the Sugar Composition of Tomato Fruit

cDNA for an acid invertase (EC 3.2.1.26 [EC] ) of tomato (Lycopersiconesculentum Mill.) fruit was introduced into tomato plants underthe control of the cauliflower mosaic virus 35S promoter inthe antisense orientation. The antisense gene effectively suppressedthe invertase activity in soluble and cell wall fractions fromripening fruits. The sucrose content of fruits of the transformantswas markedly increased, while the hexose content was reduced.These results indicate that acid invertase is one of main determinantsof the sugar composition of tomato fruit. The invertase activityin the cell wall fraction of the leaf tissues of the transformantswas not suppressed to the same extent as that in the solublefraction. Wounding of the control leaf tissues induced invertaseactivity in both soluble and cell wall fractions. The inductionof activity in the soluble fraction was suppressed by the antisensegene, while that in the cell wall fraction was unaffected. Thesefindings suggest that mRNA for some other invertase, in particular,the mRNA for a cell wall-bound invertase, was present in leaves. ¹Present address: Plant Breeding and Genetics Research Laboratory,Japan Tobacco Inc., 700 Higashibara, Toyoda, Iwata, Shizuoka,438 Japan. ²Present address: National Institute of Agrobiological Resources,Kannondai, Tsukuba, Ibaraki, 305 Japan.     

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.     

Changes in soluble proteins in potato leaves infected with Phytophthora infestans

A new protein was observed in the electrophoretic gel band ofleaves of cultivar susceptible to Phytophthora infestans, whenthe DNA fraction of a resistant hybrid was applied to the leaves.The Rf value of this band coincided with that of the hybrid6 hr after die inoculation of a race of P. infestans to whichit was resistant. ¹Present adress: Laboratory of Plant Pathology, College of Agriculture.Kyoto University, Kyoto, 606) Japan. ²His former family name was Nakao. (Received December 19, 1975; )     

Modifications of Translocation Rate in Studies of Apple Leaf Efficiency

M.7 apple rootstocks were used during the peak period of shootextension for comparisons of dry-matter production per unitleaf area between intact plants and others which had been partiallydefoliated. Dry-matter increment per unit leaf area over a 16-dayinterval was some 70 per cent higher in partially defoliatedplants than in controls. ¹⁴CO₂ was supplied to designated leaves of comparable age andposition. Sample discs were taken from the ‘fed’leaves at intervals up to 9 days from supplying ¹⁴CO₂. Translocationrates were estimated by comparison with leaves on a third setof plants whose petioles were steamed to prevent translocationimmediately on removal of the ¹⁴CO₂ feeding chambers. Translocationrates in partially defoliated plants were enhanced some 30 percent compared with controls. It is suggested that features of the plant outside the studiedleaves may have contributed to the overall efficiency of assimilateproduction and utilization. Malus sylvestris L., apple, dry matter production, leaf efficiency, defoliation, translocation, assimilate distribution, sorbitol, sucrose     

Purification and Properties of NADP-Dependent Sorbitol-6-Phosphate Dehydrogenase from Apple Seedlings

NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) waspurified from apple (Malus domestica) seedlings by a purificationprocedure that included two fractionations by affinity chromatography.The purified enzyme was a homogeneous protein that migratedas a single polypeptide chain with an apparent relative massof 36,000 during SDS-polyacrylamide gel electrophoresis andthe native enzyme was a homodimer of the polypeptide. The maximumvelocity of the reduction of glucose-6-phosphate (G6P) was muchhigher than that of the oxidation of sorbitol-6-phosphate (S6P)and the enzyme had high G6P-reducing activity over the pH rangefrom 7 to 11 even though the oxidation of S6P proceeded veryslowly at neutral pH. These results are consistent with thehypothesis that S6PDH plays a major role in the biosynthesisof sorbitol in vivo. The reduction of G6P to S6P was inhibitedby the addition of nucleotide di- or triphosphates. ATP, thestrongest inhibitor, and ADP inhibited the reduction of G6Pin a competitive manner with respect to NADPH and the K_i valueswere 0.18 mM for ATP and 0.30 mM for ADP. (Received March 24, 1992; Accepted May 25, 1993)     

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.