Effects of abiotic stresses on sorbitol biosynthesis and metabolism in tomato (Solanum lycopersicum).

Polyols such as sorbitol and ribitol are a class of compatible solutes in plants that may play roles in tolerance to abiotic stresses. This study investigated the effects of water stress on sorbitol biosynthesis and metabolism and sorbitol and ribitol accumulation in tomato (Solanum lycopersicum L.). Water stress induced by withholding water and by using polyethylene glycol as a root incubation solution to mimic water stress, and NaCl stress were applied to wild-type (WT) and three genetically-modified lines of tomato (cv. Ailsa Craig), a control vector line TR22, and 2 sorbitol dehydrogenase (sdh) antisense lines TR45 and TR49. Sorbitol and ribitol content, as well as the enzymatic activities, protein accumulation, and gene expression patterns of the key sorbitol cycle enzymes aldose-6-phosphate reductase (A6PR), aldose reductase (AR), and sorbitol dehydrogenase (SDH), were measured in mature leaves. In response to the stresses, both sorbitol and ribitol accumulated in leaf tissue, most significantly in the sdh antisense lines. A6PR, characterised for the first time in this work, and AR both exhibited increased enzymatic activity correlated with sorbitol accumulation during the stress treatments, with SDH also increasing in WT and TR22 to metabolise sorbitol, reducing the content to control levels within 3 days after re-watering. In the sdh antisense lines, the lack of significant SDH activity resulted in the increased sorbitol and ribitol content above WT levels. The results highlighted a role for both A6PR and AR in biosynthesis of sorbitol in tomato where the high activity of both enzymes was associated with sorbitol accumulation. Although both A6PR and AR are aldo-keto reductases and use NADPH as a co-factor, the AR-specific inhibitor sorbinil inhibited AR only indicating that they are different enzymes. The determination that sorbitol, and perhaps ribitol as well, plays a role in abiotic responses in tomato provides a cornerstone for future studies examining how they impact tomato tolerance to abiotic stresses, and if their alteration could improve stress tolerance.

Comparative analyses of polyphenolic composition of Fragaria spp. color mutants.

White-fruited mutants of Fragaria vesca, and one of F. x ananassa, were studied to determine the identity and quantity of major flavonols (FVLs), flavan-3-ols (FV3Ls), hydroxycinnamic acids (HCAs), and ellagic acid (EA)-derived compounds, by using HPLC-MS. The content of 22 compounds across the major groups were used to assess the possibility of unique mutations among the mutant gentoypes. Total HCAs were lower in the white than the red cultivars of both species, except for 2 white F. vesca cultivars. Total FVLs were comparable in white fruit of both species, although a red F. x ananassa had more than a red F. vesca. Total FV3Ls were higher in red than white cultivars of both species. Total EA-derived content was generally higher in white than in red F. vesca. Principal component analysis and a combined heatmap and hierarchical cluster analysis clearly discriminated among the five white F. vesca genotypes.

Effects of Phenolic Compounds on Growth of Colletotrichum spp. In Vitro.

Colletotrichum acutatum is responsible for anthracnose fruit rot, one of the most devastating diseases in strawberry. Phenolic compounds have been described as contributors to anthracnose resistance in strawberry (Fragaria x ananassa, Duch.). Six isolates of Colletotrichum acutatum and four isolates of three other Colletotrichum species, C. gloeosporioides, C. fragariae, and C. graminicola, associated with disease symptoms were investigated in this study. The potential inhibitory effect of phenolic acids (gallic acid, caffeic acid, chlorogenic acid, ferulic acid, trans-cinnamic acid, p-coumaric acid, salicylic acid), flavonoids (catechin, quercetin, naringenin), and ellagic acid, which are naturally found in strawberry, were screened against two different spore suspension concentrations of the Colletotrichum isolates at 5, 10, 50 mM in vitro. Among the phenolic acids and flavonoids tested in this study, only trans-cinnamic acid, ferulic acid, and p-coumaric acid inhibited fungal growth. The inhibitory effects were concentration-dependent but also varied with the spore suspension concentration of the isolates. The results demonstrated that trans-cinnamic acid had the greatest inhibitory effect on all Colletotrichum spp. isolates tested.

The role of SORBITOL DEHYDROGENASE in Arabidopsis thaliana.

SORBITOL DEHYDROGENASE (SDH, EC 1.1.1.14) catalyses the interconversion of polyols and ketoses (e.g. sorbitol ⟷ fructose). Using two independent Arabidopsis thaliana (L.) Heynh. sdh knockout mutants, we show that SDH (At5g51970) plays a primary role in sorbitol metabolism as well as an unexpected role in ribitol metabolism. Sorbitol content increased in both wild-type (WT) and mutant plant leaves during drought stress, but mutants showed a dramatically different phenotype, dying even if rewatered. The lack of functional SDH in mutant plants was accompanied by accumulation of foliar sorbitol and at least 10-fold more ribitol, neither of which decreased in mutant plants after rewatering. In addition, mutant plants were uniquely sensitive to ribitol in a concentration-dependent manner, which either prevented them from completing seed germination or inhibited seedling development, effects not observed with other polyols or with ribitol-treated WT plants. Ribitol catabolism may occur solely through SDH in A. thaliana, though at only 30% the rate of that for sorbitol. The results indicate a role for SDH in metabolism of sorbitol to fructose and in ribitol conversion to ribulose in A. thaliana during recovery from drought stress.

Loss of ripening capacity of pawpaw fruit with extended cold storage.

The fruit ripening traits of pawpaw [ Asimina triloba (L.) Dunal] were examined after harvest and after cold storage at -2, 2, 4, and 6 degrees C for up to 12 weeks. Generally, fruits stored at 2-4 degrees C for 4 weeks ripened normally, but those stored at -2 degrees C did not ripen normally, those stored at 6 degrees C were overripe, and by 6-8 weeks those stored at 2-4 degrees C had a lower respiration rate and ethylene production, lower firmness, and lower pH than fruit cold-stored for 4 weeks or less. These changes, and the occasional development of brown discoloration in the pulp once the fruits were moved back to room temperature, were evidence of chilling injury by 6 weeks. After harvest and through 4 weeks of cold storage, the main volatile compounds produced by fruit were methyl and ethyl octanoates and hexanoates. Volatile production significantly increased >5-fold in fruit ripened for 72 h after harvest or after removal from up to 4 weeks of cold storage. Fruit cold-stored for 6 weeks or more produced fewer total volatiles and esters but increased levels of such off-flavor compounds as ethyl acetate, ethyl propionate, and hexanoic and decanoic acids. Alcohol acyltransferase (AAT) activity declined in cold-stored fruit but was not correlated with either total volatile production or total ester production. Alcohol dehydrogenase activity did not change during ripening after harvest or cold storage. Lipoxygenase activity was highest just after harvest or after 2 weeks of cold storage, but was low by 4 weeks. Thus, ripening of pawpaw fruit seems to be limited to 4 weeks at 2-4 degrees C with loss of ability to continue ripening and chilling injury symptoms evident at colder temperatures and after longer periods of cold storage.

Tissue-specific expression of SORBITOL DEHYDROGENASE in apple fruit during early development.

Sorbitol, the primary photosynthate and translocated carbohydrate in apple (Malusxdomestica Borkh.), is converted to fructose by sorbitol dehydrogenase (SDH; EC 1.1.1.14) which is active in apple fruit throughout development. In the apple genome, nine SDH genes have been isolated and their sequences characterized, but their individual expression patterns during apple fruit set and development have not been determined. The objective of this work was to ascertain if SDH genes are differentially expressed and how their patterns of expression may relate to SDH activity in apple seed and cortex during early fruit development. Seed SDH activity was found to be much higher than cortex SDH activity per mg and g fresh weight (FW), and seed SDH activity contributed significantly to whole fruit SDH activity during weeks 2-5 after bloom. Five of the nine SDH genes present in the apple genome were expressed in apple fruit. Two SDH genes, SDH1 and SDH3, were expressed in both seed and cortex tissues. SDH2 expression was limited to cortex, while SDH6 and SDH9 were expressed in seed tissues only. SDH isomeric proteins of different pI values were detected in apple fruit. SDH isomers with pI values of 4.2, 4.8, 5.5, and 6.3 were found in seeds, and SDH isomers with pI values of 5.5, 6.3, 7.3, and 8.3 were found in cortex. The present work is the first to show that SDH is highly active in apple seed and that SDH genes are differentially expressed in seed and cortex during early development.

Interaction with and effects on the profile of proteins of Botrytis cinerea by C6 aldehydes.

The natural volatile compounds cis-3-hexenal (c-3-H) and trans-2-hexenal (t-2-H) have significant antifungal activity with potential for use as postharvest fumigants of fruits and vegetables. However, the nature of their interaction with fungi and impact on fungal growth at the molecular level are largely unknown. The sites of interaction of these six carbon (C6) aldehydes with Botrytis cinerea, a common pathogen of many plant species, was characterized using 3H-labeled c-3-H and t-2-H. Radiolabeled C6 aldehydes were produced with lipoxygenase and hydroperoxide lyase extracts using [9,10,12,13,15,16-3H6]linolenic acid as a substrate. Following exposure of B. cinerea cultures to radiolabeled C6 aldehydes, radiolabel was recovered in protein-enriched but not lipid-enriched fractions. Radiolabel was incorporated at higher levels (6-fold per milligram of fresh weight and 4-fold per microgram of protein) into conidia than mycelia. About 95% of the radiolabeled aldehyde recovered in the protein fraction was from the surface of the fungal tissue, while 5% was from protein in internal tissue (cell wall, membrane, and cytosol). Exposure to t-2-H at both 5.4 and 85.6 micromol affected the protein profile of B. cinerea, changing the intensity of over one-third of all proteins. Both up-regulation and down-regulation of specific proteins were observed by two-dimensional gel electrophoresis, indicating a clear effect of t-2-H on changes in the protein profile of B. cinerea. This is the first evidence that fungal proteins are targets of the volatile C6 aldehydes and that sublethal levels of the aldehydes cause changes in the protein profile of a fungus.

Biosynthesis of trans-2-hexenal in response to wounding in strawberry fruit.

Wounded strawberry fruit produces a diverse group of volatile compounds including aldehydes, alcohols, and esters derived from the lipoxygenase (LOX) and hydroperoxide lyase (HPL) pathways. Because the wound volatiles may play an important role in plant-fungal interaction, the goal of this study was to develop a greater understanding about the biosynthesis of the major wound volatile, trans-2-hexenal (t-2-H), produced by strawberry fruit upon wounding. To that end, composition and quantity of total and free fatty acids of control and wounded strawberry fruit were analyzed. In addition, activities of the key enzymes, LOX and HPL, and production of C6 aldehydes were determined. Intact strawberry fruit did not produce detectable t-2-H which is derived from alpha-linolenic acid (18:3). However, in response to wounding by bruising, strawberry fruit emitted t-2-H and its precursor cis-3-hexenal (c-3-H). The level of total lipid 18:3 in the fruit increased 2-fold in response to wounding, whereas free 18:3 declined slightly ( approximately 30%). At 10 min following wounding, fruit exhibited a 25% increase in LOX activity, which leads to the production of 13-hydroperoxyoctadecatrienoic acid (13-HPOT) from 18:3. The activity of HPL, which catalyzes formation of cis-3-hexenal from 13-HPOT, increased 2-fold by 10 min after wounding. Thus, during a 15 min period after wounding, free 18:3 substrate availability and the activity of two key enzymes, LOX and HPL, changed in a manner consistent with increased c-3-H and t-2-H biosynthesis.