NAD-dependent Sorbitol Dehydrogenase Research Articles

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii.

Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized d-sorbitol as its carbon source, among the four polyols examined (d-galactitol, d-mannitol, d-sorbitol and d-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153 and Lys157 as catalytic active site residues and existed as an ∼67-kDa dimer in size-exclusion chromatography. PmSDH converted d-sorbitol to d-fructose using nicotinamide adenine dinucleotide (NAD+) as a cofactor and, vice versa, d-fructose to d-sorbitol using nicotinamide adenine dinucleotide reduced (NADH) as a cofactor. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4-25°C. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

High Carbon Dioxide Treatment Modulates Sugar Metabolism and Maintains the Quality of Fresh-Cut Pear Fruit.

The purpose of this study is to explore the effect of 10% carbon dioxide (CO2) on the fruit quality and sugar metabolism of fresh-cut pear during storage. The results indicated that carbon dioxide treatment maintained fruit quality by delaying the decline of firmness and promoting the accumulation of total soluble solids (TSS). Moreover, carbon dioxide enhanced activities of sucrose synthase (SS), and sucrose phosphate synthase (SPS). The activities of amylase, acid invertase (AI), neutral invertase (NI), SS-cleavage, fructokinase (FK), hexokinase (HK), sorbitol oxidase (SOX), NAD-dependent sorbitol dehydrogenase (NAD-SDH), and NADP-SDH in CO2-treated fruit were inhibited. Expression levels of key genes were found to correspond with the related enzyme activities. As a result, the accumulation of glucose, fructose, sorbitol, and sucrose were accelerated by CO2, which were 12.58%, 13.86%, 24.7%, and 13.9% higher than those of the control at the end of storage, respectively. The results showed that CO2 could maintain the quality of fresh-cut pears by regulating the conversion of various sugar components to enhance soluble sugars content.

WHITISH-FLESH COLOR MUTATION IN LOQUAT INVOLVES FRUCTOSE BIOSYNTHESIS AND S-LOCUS

Loquat (Eriobotrya japonica Lindl.) produces yellowish-flesh and whitish-flesh fruits. A bud mutation with whitish-flesh fruits originating from the yellowish-flesh cultivar ‘Dongting’ was discovered. The white mutant and yellow loquats were collected and used in this study. Two polymorphic bands were obtained with the primers UBC813/535 and OPH01/1719, discriminating between wild and mutant genomes. The deduced amino acids of the two sequences were highly correlated with NAD-dependent sorbitol dehydrogenase (NAD+-SDH) in E. japonica and F-box proteins, S ribonuclease (S-RNase) in P. pyrifolia. Three SNP variances at nucleotide 32, 370 and 461 in the coding sequence of the NAD-SDH gene, were observed in whitish fruit mutants, and the amino acids of the three alleles were also different. In addition, real-time quantitative PCR showed that NAD-SDH and some other carotenogenic genes were considerably suppressed in ripe mutant fruits compared to those in wild fruits. These results suggest that flesh color of loquat might be controlled by at least two loci referring to NAD+-SDH and S-locus, in addition to expression levels of carotenogenic genes. The NAD-SDH might be an upstream gene involved in I²-carotene accumulation in yellowish-flesh fruit and it may be important for flesh color and fructose biosynthesis.

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.

Carbohydrate metabolism and its possible roles in endodormancy transition in Japanese pear

In order to elucidate the possible roles of carbohydrate in Japanese pear (Pyrus pyrifolia, ‘Kosui’) during endodormancy, seasonal changes of carbohydrate concentrations and the activities of sorbitol- and sucrose-metabolizing enzymes were investigated in the lateral flower buds and current shoots. ‘Kosui’ flower buds were released from endodormancy between mid- and late December. During this period, a decrease in the shoot starch concentration and an increase of soluble sugars in both the flower buds and shoots were observed, concomitant with the onset of acid invertase and sucrose synthase activity in flower buds. Following these changes, a notable accumulation of sorbitol was recorded in xylem sap in late-December, accompanied by considerable expression of the sorbitol transporter gene (PpSOT2) in shoots. After that, in mid- to late January, flower buds exhibited increased activity of NAD-dependent sorbitol dehydrogenase simultaneous to an increase of sorbitol transporter gene (PpSOT3) expression. We, thus, assume that pear flower buds regulate endodormancy by modulating the systems for sorbitol- and sucrose-catabolism depending on the dormancy stage. We also discuss the possible inhibitory effect of sorbitol on flower bud growth during endodormancy.

S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site

We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803-808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42kDa, thereby facilitating its aggregation. Two-dimensional SDS-polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.

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.

Sugars are under light control during bud burst in Rosa sp.

Bud burst in certain species is conditioned by the luminous environment. With roses, the requirement for light is absolute, and darkness totally inhibits bud burst. Few studies have looked into understanding the action of light on the physiological bud burst processes. Here, we show the impact of light on certain components of glucidic metabolism during bud burst. Measurements were taken on decapitated plants of Rosa hybrida L. 'Radrazz' exposed either to darkness, white, blue or R light. Results show that a mobilization of bud and the carrying stem sucrose reserves only takes place in light and accompanies the bud burst. Furthermore, the activity of the RhVI vacuolar acid invertase which contributes to the breakdown of sucrose in the buds, as well as the transcription of the RhVI gene, is reduced in darkness, although it is strongly stimulated by light. The same analysis concerning the RhNAD-SDH gene, coding an NAD-dependent sorbitol dehydrogenase, shows, on the contrary, a strong induction of its transcription in darkness that could reflect the use of survival mechanisms in this condition.

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.

Roles of gibberellins in increasing sink demand in Japanese pear fruit during rapid fruit growth

Our previous work demonstrated that exogenous gibberellins (GAs) applications during rapid fruit growth significantly increases sink demand and results in a larger fruit in Japanese pear. In an attempt to unravel the mechanism of increased sink demand by applied GAs, the histology, cell wall components of the flesh, and carbon accumulation in the fruit were assessed for Japanese pear (Pyrus pyrifolia, cultivar ‘Kousui’), as were the activities of sucrose- and sorbitol-cleaving enzymes. Our results show that most vascular tissues occurred in core tissue with very little vascular tissue in the flesh. Application of a mixture of GA3 + GA4 in lanolin paste significantly increased the amount of ethanol-insoluble solids, e.g., total pectins, hemicellulose, and cellulose in the cell walls. There was a significantly increased sink demand (assessed by 13C accumulation in the fruit) by the applied GAs, and this increased sink strength was closely related to increased activities of cell wall-bound invertase in the core, neutral invertase and NAD-dependent sorbitol dehydrogenase in the flesh during rapid fruit growth. As well, concentrations of sorbitol and sucrose in the flesh were decreased by GA application, while glucose concentration increased. Most importantly, the fact that sink activity can be increased by GA application implies that endogenous GAs are likely to be important modulators for sugar metabolism. Hence, selecting for genotypes with elevated GA production in the growing fruit and increased activities of key enzymes for sugar metabolism could result in increased fruit size.

Expression and cloning of the full-length cDNA for sorbitol-6-phosphate dehydrogenase and NAD-dependent sorbitol dehydrogenase from pear ( Pyrus pyrifolia N.)

Sorbitol is the primary product of photosynthesis and the major translocation form of photosynthate in the Rosaceae family, which includes pear plants. Therefore, the biosynthesis and breakdown of sorbitol is an important step in the carbohydrate metabolism of pear. In sorbitol metabolism, sorbitol-6-phosphate dehydrogenase (S6PDH) and NAD-sorbitol dehydrogenase (NAD-SDH) play key roles in biosynthesis and breakdown of sorbitol, respectively. We isolated full-length cDNAs encoding S6PDH and NAD-SDH from pear ( Pyrus pyrifolia N. cv Niitaka). PyS6PDH cDNA encodes a polypeptide consisting of 311 amino acid residues. PyNAD-SDH cDNA encodes a 371 amino acid polypeptide. The PyS6PDH gene was expressed at high levels in leaves at 30 day after full bloom (DAFB), but its expression was decreased later. Conversely, the transcripts of the PyNAD- SDH gene were detected at low levels in young leaves, but increased with age. These genes may be regulated by the transcriptional level.

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.

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.

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.

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.

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 K m 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.

Fructose Content and the Activity of Fructose-related Enzymes in the Fruit of Eating-quality Peach Cultivars and Native-type Peach Cultivars

Significant differences in fructose content were detected among different cultivars of the peach (Prunus persica (L.) Batsch). To investigate the factors that regulate fructose concentrations in peach fruit, we measured the activities of fructose-related enzymes in two eating-quality peaches, ‘Akatsuki’ and ‘Kawanakajima hakuto’, and two native-type peaches, ‘Nagano yaseito Early’ and ‘Noto zairaito No. 2’. Fructose contents in the fruit of the eating-quality peach cultivars, measured from 3 months after flowering to the ripe stage, ranged from 7 to 12 mg · g-1 FW, whereas those of the native-type peach cultivars were less than 1 mg · g-1 FW. There were no clear differences between the two groups with respect to fructokinase, fructose-6-phosphatase, and phosphoglucose isomerase (PGI) enzyme activities although they were low in ‘Noto zairaito No. 2’. In all cultivars, the activity of PGI was much higher than that of the other enzymes. This finding suggests that isomerization by PGI is not the rate-limiting step in the synthesis of fructose. The concentration of fructose-6-phosphate (Fru6P) in the fruit did not differ between the two groups. These results suggest that the differences in fructose content in different peach cultivars are related to different capacities for fructose production rather than to differences in the ability to convert fructose to Fru6P and then to glucose-6-phosphate. NAD-dependent sorbitol dehydrogenase (SDH) activity was found to be higher in the eating-quality peach cultivars than in the native-type peach cultivars during all fruit development stages tested in this study. It is therefore likely that SDH, which contributes to the production of fructose from sorbitol, is responsible for the regulation of fructose concentrations in peach fruit.

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.

Patterns of Sorbitol Metabolism and Availability during Apple Fruit Set

Acquiring sufficient carbohydrate is essential for successful apple fruit set. Sorbitol may be the dominant carbohydrate imported by growing fruit, and the rate of sorbitol accumulation may be a function of NAD-dependent sorbitol dehydrogenase (SDH; EC 1.1.1.14) activity. Prior work indicated that SDH activity from whole fruit (seeds plus cortex) increased for 2 or 3 weeks after initiation of fruit growth and then declined through 5 weeks. Using SDH activity assays, an SDH-specific antibody, and SDH-specific probes in Northern analyses, it is evident that SDH is expressed and is active in both apple seed and cortex tissue during the first few weeks of fruit growth. On a per unit protein basis, SDH activity in seeds increased by the pattern described above while that in fruit was generally lower and constant. During this same period of time, the sorbitol content of the expressed sap of apple shoots was analyzed. The sorbitol concentration was 50- to 100-fold higher than the sucrose concentration. The concentrations of both carbohydrates changed in parallel to the change in SDH activity of whole fruit and seeds. The lowest SDH activity and sap sorbitol levels preceded and/or coincided with the beginning of the natural fruit drop (or June drop) period.

Trophic control of bud break in peach (Prunus persica) trees: a possible role of hexoses.

Vegetative buds of peach (Prunus persica L. Batsch.) trees act as strong sinks and their bud break capacity can be profoundly affected by carbohydrate availability during the rest period (November-February). Analysis of xylem sap revealed seasonal changes in concentrations of sorbitol and hexoses (glucose and fructose). Sorbitol concentrations decreased and hexose concentrations increased with increasing bud break capacity. Sucrose concentration in xylem sap increased significantly but remained low. To clarify their respective roles in the early events of bud break, carbohydrate concentrations and uptake rates, and activities of NAD-dependent sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX) and cell wall invertase (CWI) were determined in meristematic tissues, cushion tissues and stem segments. Only CWI activity increased in meristematic tissues shortly before bud break. In buds displaying high bud break capacity (during January and February), concentrations of sorbitol and sucrose in meristematic tissues were almost unchanged, paralleling their low rates of uptake and utilization by meristematic tissues, and indicating that sorbitol and sucrose play a negligible role in the bud break process. Hexose concentrations in meristematic tissues and glucose imported by meristematic tissues correlated positively with bud break capacity, suggesting that hexoses are involved in the early events of bud break. These findings were confirmed by data for buds that were unable to break because they had been collected from trees deprived of cold. We therefore conclude that hexoses are of greater importance than sorbitol or sucrose in the early events of bud break in peach trees.