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The efficacy of calcium sprays for improving fleshy fruit resistance to abiotic/biotic stress and enhancement of fruit shelf life has increasingly been explored. However, because calcium is a powerful secondary messenger in many signaling pathways, including those driven by abscisic acid (ABA) and jasmonates, it may interfere with the biosynthesis of specialized metabolites highly important for fruit and wine quality, such as phenolic compounds. In this study, a combination of biochemical and molecular biology approaches were applied to grape cell cultures and detached grape berries, in order to investigate the effect of calcium in the modulation of enzymes involved in the biosynthesis of phenolic compounds and in cell wall organization. Concentrations up to 10 mM CaCl 2 did not affect cell growth, size or viability, but triggered modifications in total phenolics content, particularly in anthocyanin levels in grape cell suspensions. The effects of calcium applied alone or in combination with ABA or methyl jasmonate (MeJA) were visible in several branches of specialized metabolic pathways, confirming that the calcium-hormone interplay regulates the expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), dihydroflavonol reductase (DFR) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The activity of PAL and UFGT enzymes was also specifically modulated by calcium, ABA and MeJA. These results closely correlated to the modifications observed in the expression of VvAM1 and VvABCC1 encoding vacuolar anthocyanin transporters. Modulation of the expression and activity of pectin methyl esterases (PME) and polygalacturonases (PG) by calcium was also evident, confirming an important role of calcium in cell wall organization via the regulation of enzyme activity, besides its well-known role in the formation of cross links between pectin molecules. Overall, this study uncovers important biochemical mechanisms induced by calcium and stress hormones on grape berries, and highlights the need to consider the consequences of calcium treatments and stress for fruit quality., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Rhamnogalacturonan I (RG-I) is a domain of plant cell wall pectin. The rhamnogalacturonan lyase (RGL) enzyme (EC 4.2.2.23) degrades RG-I by cleaving the α-1,4 glycosidic bonds located between the l-rhamnose and d-galacturonic residues of the main chain. While RGL's biochemical mode of action is well known, its effects on plant physiology remain unclear. To investigate the role of the RGL enzyme in plants, we have expressed the Solyc11g011300 gene under a constitutive promoter (CaMV35S) in tomato cv. 'Ohio 8245' and evaluated the expression of this and other RGL genes, enzymatic activity and alterations in vegetative tissue, and tomato physiology in transformed lines compared to the positive control (plants harboring the pCAMBIA2301 vector) and the isogenic line. The highest expression levels of the Solyc11g011300, Solyc04g076630, and Solyc04g076660 genes were observed in leaves and roots and at 10 and 20 days after anthesis (DAA). Transgenic lines exhibited lower RGL activity in leaves and roots and during fruit ripening, whereas higher activity was observed at 10, 20, and 30 DAA than in the isogenic line and positive control. Both transgenic lines showed a lower number of seeds and fruits, higher root length, and less pollen germination percentage and viability. In red ripe tomatoes, transgenic fruits showed greater firmness, longer shelf life, and reduced shriveling than did the isogenic line. Additionally, a delay of one week in fruit ripening in transgenic fruits was also recorded. Altogether, our data demonstrate that the Solyc11g011300 gene participates in pollen tube germination, fruit firmness, and the fruit senescence phenomena that impact postharvest shelf life., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The effects of 15 days of storage at 12 °C and 7 °C followed by fruit ripening at 20 °C on oxidative status, antioxidant defense systems and carotenoid accumulation were studied for two successive years in mango fruits (Mangifera indica L.) cv. Cogshall. Changes in the non-enzymatic (ascorbate) and enzymatic (SOD, CAT, APX, MDHAR, DHAR and GR) antioxidant systems, as well as oxidative parameters (H 2 O 2 and MDA) and the contents of the major carotenoids were measured for three maturity stages, at harvest and after ripening following cold temperature storage. In control conditions (20 °C), ripening induced an increase in oxidation resulting in ROS production and a decrease in ascorbate content. Fruit tissue protection was activated by means of antioxidant and ascorbate regeneration enzyme systems. Carotenoid accumulated exponentially during ripening. Storage at low temperatures increased respiration crisis intensity and therefore increased oxidation in the fruit pulp. Fruit response to this increase varied according to the maturity stage, i.e., enzymatic responses in younger fruits were very low in comparison to the control, whereas second harvest fruits had a significantly higher degree of enzymatic activity to cope with the oxidative stress. Carotenoid contents decreased with low temperatures and first harvest fruits showed significantly lower values than the control, in opposition to second harvest fruits that appeared not to be affected. We also suggest that, based on a review of the literature, a link can be made between antioxidant system defense and carotenoid metabolism since ROS seems to play a central role as a stress signal in plants., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

In this study the occurrence of a number of enzymes involved in gluconeogenesis was investigated in both tomato fruits and leaves during their development and senescence and in some other fruits. The enzymes studied were phosphoenolpyruvate carboxykinase (PEPCK), pyruvate orthophosphate dikinase (PPDK) and glyoxysomal isocitrate lyase (ICL). PPDK was detected in the ripe flesh of tomato, and much smaller amounts were detected in the flesh of both peach and pepper, whereas it was not detected (not present or at very low abundance) in the other fruits which were investigated (apricot, aubergine, blackberry, blueberry, cherry, grape, plum, raspberry and red current). By contrast PEPCK was present in the flesh of all the fruits investigated. Very small amounts of ICL were detected in ripe tomato flesh. PEPCK was present in the skin, flesh, locular gel and columella of tomato fruit, and in these its abundance increased greatly during ripening. PPDK showed a similar distribution, however, its abundance did not increase during ripening. PEPCK was not detected in tomato leaves at any stage of their development or senescence. The content of PPDK g(-1) fresh weight (FW) increased in tomato leaves as they matured, however, it declined during their senescence. In tomato leaves the content of ICL g(-1) FW increased until the mid-stage of development, then decreased as the leaf matured, and then increased during the latter stages of senescence. In the flesh of tomato fruits the contents of PPDK and PEPCK g(-1) FW decreased during senescence. The results suggest that in fruits other than tomato the bulk of any gluconeogenic flux proceeds via PEPCK, whereas in tomato both PEPCK and PPDK could potentially be utilised. Further, the results indicate that the conversion of pyruvate/acetyl-CoA to malate by the glyoxylate cycle, for which ICL is necessary, is not a major pathway utilised by gluconeogenesis in fruits under normal conditions of growth. Finally, the results contribute to our understanding of the role of several enzymes in the senescence of both leaves and fruits., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

CDPK kinases are a unique class of calcium sensor/responders that regulate many growth and developmental processes as well as stress responses of plants. PnCDPK1 kinase from Pharbitis nil is regulated by light and contributes to seed germination, seedling growth and flower formation. Following an earlier work in which we identified the PnCDPK1 coding sequence and a 330bp long 3'UTR (untranslated region), we present for the first time the genomic organization of PnCDPK1, including intron analysis and the gene copy number designation. We completed the research by identifying the 5'-flanking region of PnCDPK1 and analyzed it in silico, which led to the discovery of several cis-regulatory elements involved in light regulation, embryogenesis and seed development. The functional analysis of P. nil CDPK showed characterization of the PnCDPK1 transcript and PnCDPK protein level during seed formation and fruit maturation. The greatest amount of PnCDPK1 mRNA was present in the last stages of seed maturation. Moreover, two PnCDPK proteins of different molecular masses were discovered during fruit development, showing various protein accumulation and activity profile. The 56kDa protein dominated in the early stages of fruit development, whereas the smaller protein (52kDa) was prominent in the latter stages., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The effects of a reduction in water supply during fruit development and postharvest fruit ripening on the oxidative status and the antioxidant defense system were studied in the mango fruit (Mangifera indica L.) cv. Cogshall. Changes in non-enzymatic (ascorbate) and enzymatic (SOD, CAT, APX, MDHAR, DHAR and GR) antioxidants, as well as oxidative parameters (H2O2 and MDA) and major carotenoids, were measured in unripe and ripe fruits from well-irrigated and non-irrigated trees. Under non-limiting water supply conditions, ripening induced oxidation as a result of the production of ROS and decreased ascorbate content. Antioxidant enzymatic systems were activated to protect fruit tissues and to regenerate the ascorbate pool. The carotenoid pool, mainly represented by β-carotene and esterified violaxanthine isomers, accumulated naturally during mango ripening. The suppression of irrigation decreased fruit size and induced accumulation of ABA and of its storage form, ABA-GE, in fruit pulp from the earliest harvest. It also increased oxidation, which was observable by the high levels of ascorbate measured at the early stages at harvest, and by the delay in the time it took to reach the pseudo constant carotene-to-xanthophyll ratio in ripe fruits. Nevertheless, differences between the irrigation treatments on the antioxidant system in ripe fruits were not significant, mainly because of the drastic changes in this system during ripening., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The presence of peroxisomes in olive (Olea europaea L.) fruits and different antioxidant enzymes occurring in this plant tissue is reported for the first time. Ultrastructural analysis showed that olive cells were characterized by the presence of large vacuoles and lipid drops. Plastids, mitochondria and peroxisomes were placed near the cell wall, showing some type of association with it. Olive fruit peroxisomes were purified by sucrose density-gradient centrifugation, and catalase, glutathione reductase and ascorbate peroxidase were found in peroxisomes. In olive fruit tissue the presence of a battery of antioxidant enzymes was demonstrated, including catalase, four superoxide dismutase isozymes (mainly an Fe-SOD plus 2 Cu,Zn-SOD and a Mn-SOD), all the enzymes of the ascorbate-glutathione cycle, reduced and oxidized glutathione, ascorbate, and four NADPH-recycling dehydrogenases. The knowledge of the full composition of antioxidants (enzymatic and non-enzymatic) in olive fruits is crucial to be able to understand the processes regulating the antioxidant composition of olive oil., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The volatile metabolite methyl jasmonate (MeJA) plays an important role in intra- and interplant communication and is involved in diverse biological processes. In this study, we report the cloning and functional characterization of a S-adenosyl-l-methionine:jasmonic acid carboxyl methyltransferase (JMT) from Fragaria vesca and Fragaria×ananassa. Biochemical assays and comprehensive transcript analyses showed that JMT has been erroneously annotated as gene fusion with a carboxyl methyltransferase (CMT) (gene15184) in the first published genome sequence of F. vesca. Recombinant FvJMT catalyzed the formation of MeJA with KM value of 22.3μM while FvCMT and the fusion protein were almost inactive. Activity of JMT with benzoic acid and salicylic acid as substrates was less than 1.5% of that with JA. Leucine at position 245, an amino acid missing in other JMT sequences is essential for activity of FvJMT. In accordance with MeJA levels, JMT transcript levels decreased steadily during strawberry fruit ripening, as did the expression levels of JA biosynthesis and regulatory genes. It appears that CMT has originated by a recent duplication of JMT and lost its enzymatic activity toward JA. In the newest version of the strawberry genome sequence (June 2014) CMT and JMT are annotated as separate genes in accordance with differential temporal and spatial expression patterns of both genes in Fragaria sp. In conclusion, MeJA, the inactive derivative of JA, is probably involved in early steps of fruit development by modulating the levels of the active plant hormone JA., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Chestnut rose (Rosa roxburghii Tratt) is a fruit crop that contains unusually high levels of l-ascorbic acid (AsA; ∼1300 mg 100g(-1) FW). To explore the mechanisms underlying AsA metabolism, we investigated the distribution and abundance of AsA during fruit development. We also analyzed gene expression patterns, enzyme activities, and content of metabolites related to AsA biosynthesis and recycling. AsA first accumulated during late fruit development and continued to accumulate during ripening, with the highest accumulation rate near fruit maturity. The redox state of AsA in fruit was also enhanced during late fruit development, while leaf and other tissues had much lower levels of AsA and the redox state of AsA was lower. In mature fruit, AsA was mainly distributed in the cytoplasm of the mesocarp. Correlation analysis suggested that the gene expression patterns, enzyme activities, and related metabolite concentrations involved in the l-galactose pathway showed relatively high correlations with the accumulation rate of AsA. The gene expression pattern and activity of dehydroascorbate reductase (DHAR, EC 1.8.5.1) correlated strongly with AsA concentration, possibly indicating the crucial role of DHAR in the accumulation of high levels of AsA in chestnut rose fruit. Over expression of DHAR in Arabidopsis significantly increased the reduced AsA content and redox state. This was more effective than over expression of the l-galactose pathway gene GDP-d-mannose-3,5-epimerase (EC 5.1.3.18). These findings will enhance understanding of the molecular mechanisms regulating accumulation of AsA in chestnut rose., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The ripening hormone, ethylene is known to initiate, modulate and co-ordinate the expression of various genes involved in the ripening process. The burst in ethylene production is the key event for the onset of ripening in climacteric fruits, including tomatoes. Therefore ethylene is held accountable for the tons of post-harvest losses due to over-ripening and subsequently resulting in fruit rotting. In the present investigation, delayed ripening tomatoes were generated by silencing three homologs of 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) gene during the course of ripening using RNAi technology. The chimeric RNAi-ACS construct designed to target ACS homologs, effectively repressed the ethylene production in tomato fruits. Fruits from such lines exhibited delayed ripening and extended shelf life for ∼45 days, with improved juice quality. The ethylene suppression brought about compositional changes in these fruits by enhancing polyamine (PA) levels. Further, decreased levels of ethylene in RNAi-ACS fruits has led to the altered levels of various ripening-specific transcripts, especially the up-regulation of PA biosynthesis and ascorbic acid (AsA) metabolism genes and down-regulation of cell wall hydrolyzing enzyme genes. These results suggest that the down-regulation of ACS homologs using RNAi can be an effective approach for obtaining delayed ripening with longer shelf life and an enhanced processing quality of tomato fruits. Also, the chimeric gene fusion can be used as an effective design for simultaneous silencing of more than one gene. These observations would be useful in better understanding of the ethylene and PA signaling during fruit ripening and molecular mechanisms underlying the interaction of these two molecules in affecting fruit quality traits., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Sucrose metabolism enzymes, including invertase (EC 3.2.1.26), sucrose synthase (SS, EC 2.4.1.13), and sucrose phosphate synthase (SPS, EC 2.4.1.14), are key factors that determine fruit sugar accumulation and composition. Sugar concentration and sugar composition in the arils of 42 litchi cultivars were determined at maturity. The cultivars were grouped into three types according to their hexose/sucrose ratio. Five cultivars of each type were selected to monitor the activities and gene expressions of enzymes related to sucrose metabolism. Pattern changes in the arils of four cultivars with different sugar concentrations and compositions were traced from around 40 d after anthesis to full maturity. Highly significant positive correlations were observed between hexose/sucrose ratios and the activities and expression levels of soluble acid invertase (SAI) and SS among the 15 cultivars tested. The increase in hexose/sucrose ratio was accompanied by enhanced acid invertase (AI) and SS activities and the expression of their genes in Feizixiao (FZX) and Heiye (HY). By contrast, no significant correlation was observed between hexose/sucrose ratio and SPS. These results indicate that the sugar composition in litchi aril depends mainly on the sucrose cleavage enzymes AI and SS and not on the sucrose synthetic enzyme SPS. The cultivar Nuomici, which had the highest sugar content among the cultivars studied, displayed significantly lower activities of cell wall acid invertase, SAI, neutral invertase, and SS and lower expression levels of SAI and SS compared with HY, the cultivar with the lowest sugar content. The inconsistent patterns of sugar accumulation and activities and expressions of sucrose metabolism enzymes suggest that these sucrose metabolism enzymes are not necessarily related to sugar accumulation., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T(0)). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T(1)) produced the highest GUS activity when treated with 150 μM Cu(2+) compared to the control (without Cu(2+)). However, Zn(2+) and Fe(2+) treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T(1) seedlings of tomato when subjected to Cu(2+) ions., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Raffinose synthase (RS, EC2.4.1.82) is one of the key enzymes that channels sucrose into the raffinose family oligosaccharides (RFOs) biosynthetic pathway. However, the gene encoding RS is poorly characterized in cucumber (Cucumis sativus L.), which is a typical RFOs-translocating plant species. Here we isolated the gene encoding RS (CsRS) from the leaves of cucumber plants. The complete cDNA of CsRS consisted of 2552 nucleotides with an open reading frame encoding a polypeptide of 784 amino acid residues. Reverse transcription-polymerase chain reaction and RNA hybridization analysis revealed that expression of CsRS was the highest in leaves followed by roots, fruits, and stems. The RS activity was up-regulated and the raffinose content was high in the leaves of transgenic tobacco with over-expression of CsRS, while both the RS activity and the raffinose content decreased in the transgenic cucumber plants with anti-sense expression of CsRS. The expression of CsRS could be induced by low temperature and exogenous phytohormone abscisic acid (ABA). In cucumber growing under low temperature stress, CsRS expression, RS activity and raffinose content increased gradually in the leaves, the fruits, the stems and the roots. The most notable increase was observed in the leaves. Similarly, the expression of CsRS was induced in cucumber leaves and fruits with 200 μM and 150 μM ABA treatments, respectively., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Based on previous results in which oxidative metabolism was suggested as a possible inducer of blossom-end rot (BER), the main questions addressed here were whether calcium deficiency is the main factor that induces BER or whether this physiological disorder a general stress-related phenomenon? Tomato plants were grown under optimal or deficient calcium concentrations. Only the application of 0.1mM calcium resulted in BER induction, although only half of the fruits grown under this treatment had this disorder. Having fruits showing or not showing BER in the same plant and treatment provided us with a powerful tool that we used to investigate whether calcium deficiency operates alongside another mechanism in the induction of BER. Whether or not this other mechanism was the one controlling BER incidence was also investigated. We performed a complete study of the oxidative metabolism in the pericarp of healthy fruits and in the healthy portion of BER-affected fruits. Calcium deficiency led to an induction of NADPH oxidase, superoxide dismutase, dehydro- and monodehydroascorbate reductase, and to an inhibition of catalase, ascorbate peroxidase and glutathione reductase, with a concomitant accumulation of hydrogen peroxide and an increase in lipid peroxidation. While the ascorbate redox state was not affected by calcium deficiency, the glutathione redox state was markedly reduced. We conclude that calcium deficiency fundamentally affected the activity of the ascorbate-glutathione enzymes, with special importance to the inhibition of GR, which lead to a reduction of the glutathione redox state. This could cause the breakdown of cellular homeostasis, the inhibition of other enzymes responsible for H(2)O(2) detoxification, and ultimately an increase of lipid peroxidation. Therefore, BER is defined here as the visual symptom of a massive lipid peroxidation event caused by the breakdown of cellular glutathione homeostasis., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The stearoyl-acyl-carrier-protein (ACP) desaturase is a plastid-localized enzyme that catalyzes the conversion of stearoyl-ACP to oleoyl-ACP and plays an important role in the determination of the properties of the majority of cellular glycerolipids. Functional characterization of the fatty acid desaturase genes and their specific promoters is a prerequisite for altering the composition of unsaturated fatty acids of palm oil by genetic engineering. In this paper, the specificity and strength of the oil palm stearoyl-ACP desaturase gene promoter (Des) was evaluated in transgenic tomato plants. Transcriptional fusions between 5' deletions of the Des promoter (Des1-4) and the β-glucuronidase (GUS) reporter gene were generated and their expression analyzed in different tissues of stably transformed tomato plants. Histochemical analysis of the Des promoter deletion series revealed that GUS gene expression was confined to the tomato fruits. No expression was detected in vegetative tissues of the transgenic plants. The highest levels of GUS activity was observed in different tissues of ripe red fruits (vascular tissue, septa, endocarp, mesocarp and columella) and in seeds, which harbored the promoter region located between -590 and +10. A comparison of the promoter-deletion constructs showed that the Des4 promoter deletion (314bp) produced a markedly low level of GUS expression in fruits and seeds. Fluorometric analysis of the GUS activity revealed a 4-fold increase in the activity of the full-length Des promoter compared to the CaMV35S promoter. RNA-hybridization analyses provided additional evidence of increased GUS expression in fruits driven by a Des fragment. Taken together, these results demonstrate the potential of the Des promoter as a tool for the genetic engineering of oil palms and other species, including dicots, in improving the quality and nutritional value of the fruits., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Cell walls of tomato fruit contain hemicellulosic mannans that may fulfill a structural role. Two populations were purified from cell walls of red ripe tomato tissue and named galactoglucomannan-glucuronoxylan I and II (GGM-GX I and II), respectively. Both polysaccharides not only consisted of mannose, glucose and galactose, indicating the presence of GGM, but also contained xylose and glucuronic acid, indicating the presence of GX. Treatment of both polysaccharides with xylanase or endo-β-mannanase showed that the GX and the GGM were associated in a complex. The composition of GGM-GX II changed slightly during tomato ripening, but both GGM-GX I and II showed no change in molecular weight, indicating that they were not hydrolyzed during ripening. Ripe tomato fruit also possess an endo-β-mannanase, an enzyme that in vitro was capable of either hydrolyzing GGM-GX I and II (endo-β-mannanase activity), or transglycosylating them in the presence of mannan oligosaccharides (mannan transglycosylase activity). The lack of evidence for hydrolysis of these potential substrates in vivo suggests either that the enzyme and potential substrates are not accessible to each other for some reason, or that the main activity of endo-β-mannanase is not hydrolysis but transglycosylation, a reaction in which polysaccharide substrates and end-products are indistinguishable. Transglycosylation would remodel rather than weaken the cell wall and allow the fruit epidermis to possibly retain flexibility and plasticity to resist cracking and infection when the fruit is ripe., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The flavor and taste of fruits are often determined by terpenes. We identified three cDNAs encoding putative terpene synthases from olive fruits of cv. Frantoio and Grignano. Heterologous expression in a bacterial system demonstrated that one of the terpene synthases, OeGES1, was an active monoterpene synthase that converted geranyl diphosphate to the monoterpene alcohol geraniol. The transcript accumulation pattern of this gene showed a peak during fruit ripening in both genotypes, indicating that the enzyme may be involved in the production of monoterpene flavor compounds in olive fruit. Although the putative terpene synthases OeTPS2 and OeTPS3 clustered with α-farnesene synthases and angiosperm monoterpene synthases, no detectable in vitro activity was found after expression in a bacterial system. Nevertheless, their transcripts sharply accumulated during fruit ripening starting from véraison., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

NAD(P)H-glutamate dehydrogenase (GDH, EC 1.4.1.3) contributes to the control of glutamate homeostasis in all living organisms. In bacteria and animals, GDH is a homohexamer allosterically regulated, whereas in plants NADH-GDH (EC 1.4.1.2) is also found as heterohexamer of α- and β-subunits, but its regulation remains undefined. In tomato (Solanum lycopersicum), GDH activity increases during the fruit ripening along with the content of free glutamate, the most abundant amino acid of ripe fruit involved in conferring the genuine tomato flavour. In this work, novel Slgdh-NAD genes were identified in the recently deciphered tomato genome: three encoding the α-subunit (Slgdh-NAD;A1-3) and one additional gene encoding the β-subunit of GDH (Slgdh-NAD;B1) isolated from a genomic library. These genes are located in different chromosomes. Slgdh-NAD;A1-3 show conserved structures, whereas Slgdh-NAD;B1 includes a novel 5'-untranslated exon. Slgdh-NAD;A1-3 transcripts were detected in all tomato tissues examined, showing the highest levels in mature green fruits, contrasting with Slgdh-NAD;B1 transcripts which were detected mainly in roots or in mature fruits when treated with glutamate, NaCl or salicylic acid. Analyses of GDH activity and protein distribution in different tissues of the Micro-Tom cultivar showed that only the active homohexamer of GDH β-subunits was detected in roots while heterohexamers of GDH α- and β-subunits were found in fruits. These results indicate that GDH β-subunit could modulate the heteromeric isoforms of GDH in response to the environment and physiology of the tomato fruit. This information is relevant to manipulate glutamate contents in tomato fruits genetically., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The growth characteristics of some plums and their component parts have been previously studied, as have some aspects of their developmental anatomy and composition. However, little is known about either their metabolism or about the interactions between the metabolism of their component parts. In this study we investigated these aspects in the Japanese plum Ozark Premier. Throughout fruit and seed development, changes in sugar and organic acid contents, protein composition and abundance of selected enzymes were determined. In the stone, there was a transient accumulation of vegetative storage proteins. These were subsequently mobilized and this coincided with the onset of the lignification of the stone and the start of storage protein accumulation in the seed. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was present in the seeds, even though they lacked chlorophyll, and its presence may be related to limited gas exchange. In the flesh of some fruits, phosphoenolpyruvate carboxykinase (PEPCK) and NADP malic enzyme (NADP-ME) are thought to function in the dissimilation of malate and/or citrate during ripening. However, PEPCK and NADP-ME were present in plum flesh for most of its development, although there was no net dissimilation of malate until the latter stages of ripening. There is an interaction between the developing seed and endocarp with respect to the utilization of imported sugars and amino acids. An hypothesis is presented to account for the presence of PEPCK and NADP-ME enzyme in plum flesh when there was no net dissimilation of organic acids., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The aim of this study was to obtain new insights into the mechanisms that regulate endogenous abscisic acid (ABA) levels by β-glucosidase genes during the development of watermelons (Citrullus lanatus) and under drought stress conditions. In total, five cDNAs from watermelons were cloned by using reverse transcription-PCR (RT-PCR). They included three cDNAs (ClBG1, ClBG2 and ClBG3) homologous to those that encode β-glucosidase l that hydrolyzes the ABA glucose ester (ABA-GE) to release active ABA, ClNCED4, which encodes 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthesis, and ClCYP707A1, encoding ABA 8'-hydroxylase. A BLAST homology search revealed that the sequences of cDNAs and the deduced amino acids of these genes showed a high degree of homology to comparable molecules of other plant species. During fruit development and ripening, the expressions of ClBG1, ClNCED4 and ClCYP707A1 were relatively low at an early stage, increased rapidly along with fruit ripening, and reached the highest levels at 27 days after full bloom (DAFB) at the harvest stage. This trend was consistent with the accumulation of ABA. The ClBG2 gene on the other hand was highly expressed at 5 DAFB, and then decreased gradually with fruit development. Unlike ClBG1 and ClBG2, the expression of ClBG3 was low at an early stage; its expression peak occurred at 15 DAFB and then declined to the lowest point. When watermelon seedlings were subjected to drought stress, expressions of ClBG1 and ClCYP707A1 were significantly down-regulated, while expressions of ClBG2 and ClNCED4 were up-regulated in the roots, stems and leaves. The expression of ClBG3 was down-regulated in root tissue, but was up-regulated in stems and leaves. In conclusion, endogenous ABA content was modulated by a dynamic balance between biosynthesis and catabolism regulated by ClNCED4, ClCYP707A1 and ClBGs during development and under drought stress condition. It seems likely that β-glucosidase genes are important for this regulation process., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Citrus fruit is widely consumed and provides ascorbate for human health. The ascorbate content in pulp is generally higher in orange (Citrus sinensis Osb.) than in Satsuma mandarin (Citrus unshiu Marc.). However, what contributes to such difference is still unknown. In the present study, ascorbate accumulation, expression profiles of genes involved in L-galactose pathway and activity changes of enzymes related with L-ascorbic acid (AA) oxidation and recycling were investigated during fruit development and ripening in fruit pulp of Satsuma mandarin and orange. As fruit ripens, total ascorbate (T-ASC) or AA content increased in mandarin whereas fluctuated on a relatively high level in orange. Concentrations of T-ASC or AA in pulp of orange were over 1.5-fold higher than that in pulp of Satsuma mandarin during fruit ripening. Further analysis showed that each transcript of four genes (encoding GDP-D-mannose-3',5'-epimerase, GDP-L-galactose-pyrophosphatase, L-galactose dehydrogenase and L-galactono-1,4-lactone dehydrogenase respectively) in orange was almost on a higher level and the activities of oxidation enzymes (ascorbate oxidase and ascorbate peroxidase) were lower during fruit ripening as compared with Satsuma mandarin. As ascorbate pool size is decided by the combination of biosynthesis, oxidation and recycling, therefore, higher expression of four genes along with lower activity of oxidation enzymes should contribute at least partially to the higher ASC accumulation in orange pulp., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Vacuolar H(+)-translocating inorganic pyrophosphatase (VHP, EC 3.6.1.1) is an electrogenic proton pump, which is related to growth as well as abiotic stress tolerance in plants. In this study, a VHP gene MdVHP1 was isolated from apple. The alignment of nucleotide and amino acid sequences showed that it encoded a type I VHP protein. It expressed in vegetative and reproductive organs, and its expression was induced by salt, PEG-mediated osmotic stress, cold and heat in apple in vitro shoot cultures. MdVHP1 expression showed a similar pattern in different apple tissues, but different change dynamics in response to abiotic stresses, compared with MdVHP2 (another MdVHP gene in apple). MdVHP1 overexpression enhanced tolerance to salt, PEG-mimic drought, cold and heat in transgenic apple calluses, which was related to an increased accumulation of proline and decreased MDA content compared with control calluses. In addition, MdVHP1 overexpression confers improved tolerance to salt and drought in transgenic tomato, along with an increased ion accumulation, high RWC and low solute potential compared with wild type. These results indicate that MdVHP1 is an important regulator for plant tolerance to abiotic stresses by modulating internal stores of ions and solutes., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

It has been known that methyl jasmonate (MeJA) interacts with ethylene to elicit resistance. In green mature tomato fruits (Lycopersicon esculentum cv. Lichun), 0.02mM MeJA increased the activity of 1-aminocyclopropane-1-carboxylate oxidase (ACO), and consequently influenced the last step of ethylene biosynthesis. Fruits treated with a combination of 0.02 MeJA and 0.02 α-aminoisobutyric acid (AIB, a competitive inhibitor of ACO) exhibited a lower ethylene production comparing to that by 0.02mM MeJA alone. The increased activities of defense enzymes and subsequent control of disease incidence caused by Botrytis cinerea with 0.2mM MeJA treatment was impaired by AIB as well. A close relationship (P<0.05) was found between the activity alterations of ACO and that of chitinase (CHI) and β-1,3-glucanase (GLU). In addition, this study further detected the changes of gene expressions and enzyme kinetics of ACO to different concentrations of MeJA. LeACO1 was found the principal member from the ACO gene family to respond to MeJA. Accumulation of LeACO1/3/4 transcripts followed the concentration pattern of MeJA treatments, where the largest elevations were reached by 0.2mM. For kinetic analysis, K(m) values of ACO stepped up during the experiment and reached the maximums at 0.2mM MeJA with ascending concentrations of treatments. V(max) exhibited a gradual increase from 3h to 24h, and the largest induction appeared with 1.0mM MeJA. The results suggested that ACO is involved in MeJA-induced resistance in tomato, and the concentration influence of MeJA on ACO was attributable to the variation of gene transcripts and enzymatic properties., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The aim of this study was to investigate the connection between heat-induced ethylene signal changes and enhanced disease resistance. Heat enhanced ripening and elevated MaACO1 expression in naturally ripened bananas (NRB), while it delayed ripening and reduced MaACO1expression in the ethephon-treated bananas (ETB). However, in both cases, heat reduced lesion sizes infected by Colletotrichum musae. This indicates that heat-induced disease resistance in bananas was independent of ripening rate. The expression of MaERS1 gene was inhibited by heat treatment in both NRB and ETB, implying that heat as a physical signal could be sensed by banana fruits through the inhibition of ethylene receptor gene expression. The intensity of MaERF1 transcript signals was elevated in heated bananas, suggesting that the enhanced accumulation of MaERF1 transcript following heat treatment could play an important role in activation of the defense system. In ETB, inhibition of JA biosynthesis by application of IBU down-regulated the expression of MaERF and significantly weakened disease resistance, suggesting involvement of endogenous JA in induction of the gene expression, which was reconfirmed by the fact that exposure to exogenous MeJA following the combination of heat plus IBU treatment restored part of the gene expression. On the other hand, in NRB, application of IBU elevated level of MaERF1 expression at 24h and enhanced disease resistance, suggesting that, when banana was not exposed to ethephon, the expression of MaERF1 gene was not JA dependent, which was verified by the fact that MeJA application did not enhance MaERF1 gene expression. In conclusion, heat-induced disease resistance in harvested bananas could involve down-regulation of MaERS1 expression and up-regulation of MaERF1 expression and JA pathway could be involved in heat activation of the defense system in bananas exposed to ethephon., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The limiting steps of ethylene-dependent (methylsulfanyl)alkanoate ester biosynthesis have been investigated in this study, using closely related Actinidia chinensis genotypes and the commercial cultivar 'Hort16A'. Quantification of methylsulfanyl-compounds from the headspace of ethylene-producing kiwifruits revealed little variation in their volatile composition but remarkable differences in the magnitude of the fruit volatile levels. To test whether the variations in fruit volatile levels can be correlated with the genotype-specific apparent catalytic efficiency, the initial slope of the substrate response curve (V'(Max)K(M)(-1) where V'(Max) is the apparent V(Max) in a crude extract) was evaluated for total alcohol acyltransferase (EC 2.3.1.84) activity. The V'(Max)K(M)(-1) values of different (methylsulfanyl)alkyl-CoAs were in a similar range for most genotypes, which suggests substrate availability as the limiting factor for (methylsulfanyl)alkanoate ester synthesis in these kiwifruit. Furthermore, gene expression analysis of acyltransferase expressed sequence tags points towards the action of multiple isozymes for (methylsulfanyl)alkanoate ester synthesis, emphasizing the central role of substrate levels on final ester concentrations. Volatile levels of the potential precursor methional were increased in ethylene-producing A. chinensis kiwifruit and a close connection between (methylsulfanyl)alkanoate ester formation and ethylene synthesis in plants is proposed. Finally, a possible biosynthetic pathway is presented., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Polyamines are involved in crucial plant physiological events, but their roles in fruit development remain unclear. We generated transgenic tomato plants that show a 1.5- to 2-fold increase in polyamine content by over-expressing the spermidine synthase gene, which encodes a key enzyme for polyamine biosynthesis. Pericarp-columella and placental tissue from transgenic tomato fruits were subjected to (1)H-nuclear magnetic resonance (NMR) for untargeted metabolic profiling and high-performance liquid chromatography-diode array detection for carotenoid profiling to determine the effects of high levels of polyamine accumulation on tomato fruit metabolism. A principal component analysis of the quantitative (1)H NMR data from immature green to red ripe fruit showed a clear discrimination between developmental stages, especially during ripening. Quantification of 37 metabolites in pericarp-columella and 41 metabolites in placenta tissues revealed distinct metabolic profiles between the wild type and transgenic lines, particularly at the late ripening stages. Notably, the transgenic tomato fruits also showed an increase in carotenoid accumulation, especially in lycopene (1.3- to 2.2-fold), and increased ethylene production (1.2- to 1.6-fold) compared to wild-type fruits. Genes responsible for lycopene biosynthesis, including phytoene synthase, phytoene desaturase, and deoxy-d-xylulose 5-phosphate synthase, were significantly up-regulated in ripe transgenic fruits, whereas genes involved in lycopene degradation, including lycopene-epsilon cyclase and lycopene beta cyclase, were down-regulated in the transgenic fruits compared to the wild type. These results suggest that a high level of accumulation of polyamines in the tomato regulates the steady-state level of transcription of genes responsible for the lycopene metabolic pathway, which results in a higher accumulation of lycopene in the fruit., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

This study investigates whether, and how, polyamines (PAs) are involved in mature fruit abscission of olive (Olea europaea L.). Physiological abscission was studied in relation to the activation of the abscission zone (AZ), located between fruit and peduncle, from two olive cultivars where the breakstrength profiles and the scanning electron micrographs illustrated differences in the abscission program, under natural conditions, of mature fruit. The localization and activities of diamine oxidase (DAO), polyamine oxidase (PAO) and PA biosynthetic enzymes, together with PA content were investigated in the fruit AZ during development and abscission. The activities of arginine decarboxylase and S-adenosyl-l-methionine decarboxylase in the fruit AZ were significantly increased and decreased, respectively, by mature fruit abscission, in good agreement with the rise in free putrescine (Put), and content in uncommon PAs there, such as homospermidine and cadaverine, while no significant differences in free spermidine (Spd) and spermine (Spm) contents were detected. By contrast, an abscission-induced decrease was noted in the contents of insoluble conjugated Put, Spd and Spm. The maximum activity of PAO coincided with the maximum content of Spd and Spm, and it was localized mainly in parenchyma cells of pith, while DAO was present mainly in parenchyma cells of pith and cortex as well as at the base of the vascular tissue. These results suggest a clear correlation between the PA distribution and mature fruit abscission. The regulation of PA metabolism is discussed in relation to mature fruit abscission., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Abscisic acid (ABA) plays a key role in various aspects of plant growth and development, including adaptation to environmental stress and fruit maturation in sweet cherry fruit. In higher plants, the level of ABA is determined by synthesis and catabolism. In order to gain insight into ABA synthesis and catabolism in sweet cherry fruit during maturation and under stress conditions, four cDNAs of PacCYP707A1 -PacCYP707A4 for 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA, and one cDNA of PacNCED1 for 9-cis-epoxycarotenoid dioxygenase, a key enzyme in the ABA biosynthetic pathway, were isolated from sweet cherry fruit (Prunus avium L.). The timing and pattern of PacNCED1 expression was coincident with that of ABA accumulation, which was correlated to maturation of sweet cherry fruit. All four PacCYP707As were expressed at varying intensities throughout fruit development and appeared to play overlapping roles in ABA catabolism throughout sweet cherry fruit development. The application of ABA enhanced the expression of PacCYP707A1 -PacCYP707A3 as well as PacNCED1, but downregulated the PacCYP707A4 transcript level. Expressions of PacCYP707A1, PacCYP707A3 and PacNCED1 were strongly increased by water stress. No significant differences in PacCYP707A2 and PacCYP707A4 expression were observed between dehydrated and control fruits. The results suggest that endogenous ABA content is modulated by a dynamic balance between biosynthesis and catabolism, which are regulated by PacNCED1 and PacCYP707As transcripts, respectively, during fruit maturation and under stress conditions., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

A cryoprotective chitinase (BChi14) was isolated and purified from the mesocarp of CO(2)-treated cherimoya fruit (Annona cherimola Mill.) stored at chilling temperature by anion exchange and chromatofocusing chromatography. This hydrolase was characterized as an endochitinase with a M(r) of 14.31 kDa and a pI of 8.26, belonging to the family 19 of glycosyl hydrolases (GH19). While it was stable over a wide pH range and active in a broad acidic pH range, it had an optimum pH of 7.0. Its optimum temperature was low, 35 degrees C, and it retained about 30% of its maximum activity at 5 degrees C. Moreover, BChi14 was relatively heat unstable and its activity was progressively lost at temperatures above 50 degrees C. Kinetic studies revealed many similarities with other plant endochitinases. However, BChi14 had high k(cat) (6.93 s(-1)) value for the fluorogenic substrate 4-MU-(GlcNAc)(3), reflecting its great catalytic efficiency. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k(cat) at 37 and 5 degrees C, and a low E(a) (11.32 kJ mol(-1)). In vitro functional studies indicated that BChi14 had no effect on the inhibition of Botrytis cinerea hyphal growth and no antifreeze activity, as shown by the thermal hysteresis analysis using differential scanning calorimetry. However, the purified endochitinase showed very strong cryoprotective activity against freeze-thaw inactivation of lactate dehydrogenase. The PD(50) was 12.5 times higher than that of the cryoprotective protein BSA, and 2 or 3 orders of magnitude greater than sucrose, comparable with that of most cryoactive plant dehydrins. These results, together with the consolidated microstructure and the integrity of CO(2)-treated mesocarp tissue, indicate that BChi14 is functionally implicated in the mechanisms underlying chilling tolerance activated by high CO(2) concentrations., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Sugar accumulation, the key process determining fruit quality, is controlled by both the translocation of sugars and their metabolism in developing fruits. Sugar composition in watermelon, as in all cucurbit fruits, includes sucrose, fructose and glucose. The proportions of these three sugars are determined primarily by three enzyme families: invertases, sucrose synthases (SuSys) and sucrose phosphate synthases (SPSs). The goal of the present research was to explore the process of sugar metabolism in watermelon fruits. Crosses between the domestic watermelon (Citrullus lanatus) and three wild species provided a wide germplasm to explore genetic variability in sugar composition and metabolism. This survey demonstrated great genetic variability in sugar content and in the proportions of sucrose, glucose and fructose in mature fruits. Genotypes accumulating high and low percentage of sucrose provided an experimental system to study sugar metabolism in developing fruits. Insoluble invertase activity was high and constant throughout fruit development in control lines and in genotypes accumulating low levels of sucrose, while in genotypes accumulating high levels of sucrose, activity declined sharply 4 weeks after pollination. Soluble acid invertase activity was significantly lower in genotypes accumulating high levels of sucrose than in low-sucrose-accumulating genotypes. Conversely, activities of SuSy and SPS were higher in the high-sucrose-accumulating genotypes. The present results establish that, within the genus Citrullus, there are genotypes that accumulate a high percentage of sucrose in the fruit, while others accumulate high percentages of glucose and fructose. The significant negative correlation between insoluble invertase activity and fruit sucrose level suggests that sucrose accumulation is affected by both phloem unloading and sugar metabolism., ((c) 2009 Elsevier GmbH. All rights reserved.)

Subtilisin-like serine proteases (EC 3.4.21) consist of a widespread family of enzymes that is involved in various processes including in plants. The full-length cDNA (CpSUB1) and the corresponding genomic DNA for papaya subtilase have been obtained using rapid amplification of cDNA ends (RACEs) and PCR primer walking techniques, respectively. The cDNA clone contains an open reading frame of 2316bp encoding 772 amino acids with a calculated molecular mass of 82.6kDa and an isoelectric point (pI) of 8.97. The CpSUB1 gene is composed of nine exons and eight introns. The amino acid sequence encoded by CpSUB1 shared high identity (>60%) with the amino acid sequence of other plant subtilisin-like proteases. Sequence analysis of CpSUB1 revealed the presence of a possible signal peptide (25 amino acid residues) and an NH(2)-terminal prosequence (88 amino acid residues). In addition, papaya subtilase possesses the characteristic subtilisin catalytic triad amino acids namely Asp, His and Ser, together with the substrate-binding site, Asn. DNA hybridization analysis showed that subtilase gene exists as a single copy in the papaya genome. RNA hybridization analyses showed that expression of the subtilase transcripts was only detected in mesocarp but not in non-fruit tissues. Gene expression in fruit tissues reached the highest level during the ripening stage at which the fruits undergo dramatic softening process. Subsequently, pro-subtilase ( approximately 80kDa) was expressed as recombinant pro-enzyme ( approximately 97kDa), which was used to generate antiserum against papaya subtilase, anti-sub. Protein gel blot analysis using anti-sub towards total protein extracted from all ripening stages revealed that a protein with a molecular mass of approximately 70kDa reacted with the antiserum. Hence both RNA hybridization and protein gel blot analyses confirmed the presence of subtilase during papaya fruit ripening, pointing to its possible involvement in this important process.

Endo-beta-mannanase is thought to play a role in tomato fruit ripening by participating in the degradation of cell walls. Its spatial and temporal expression during ripening was examined, as was the relationship between its activity and softening of the fruit using a large number of tomato lines, and by suppression of transcription of the endo-beta-mannanase (LeMan4a) gene. Immunolocalization studies showed that the enzyme is expressed in the fruit cell wall at all ripening stages, but it is not active during the initial green stage; this is not due to the presence of inhibitors of its activity, nor due to changes in its mRNA sequence. Transient expression in onion epidermal cells of endo-beta-mannanase transcripts fused to green fluorescent protein resulted in the expressed enzyme being localized to the cell walls. Transgenic tomato plants expressing a GUS gene attached to the LeMan4a promoter showed that this occurs initially during ripening in the skin and outer pericarp of the fruit, and later in the skin and throughout the pericarp. Fruit firmness and activity of endo-beta-mannanase were not strongly correlated during ripening of many lines of tomato. Several plants of cv. Micro-Tom were transformed using RNA interference (mRNAi) and antisense RNA strategies to suppress transcription of the LeMan4a gene. When endo-beta-mannanase activity was much reduced in the transgenic fruits, their firmness was higher compared to those of control fruits at the turning and orange-color stages, but at the red-ripe stage firmness was similar between the two fruit types. We suggest that while the enzyme does participate in fruit ripening it alone is not sufficient to cause hydrolysis of the cell walls which results in their weakening; it likely plays a cooperative role with other known wall-modifying enzymes, and/or is involved in cell wall rearrangement.

Ripening and senescence are generally controlled by ethylene in climacteric fruits like peaches, and the ripening process of grape, a non-climacteric fruit, may have some relationship to abscisic acid (ABA) function. In order to better understand the role of ABA in ripening and senescence of these two types of fruits, we cloned the 9-cis-epoxycarotenoid dioxygenase (NCED) gene that encodes a key enzyme in ABA biosynthesis from peaches and grapes using an RT-PCR approach. The NCED gene fragments were cloned from peaches (PpNCED1and PpNCED2, each 740bp) and grapes (VVNCED1, 741bp) using degenerate primers designed based on the conserved amino acids sequence of NCEDs in other plants. PpNCED1 showed 78.54% homology with PpNCED2, 74.90% homology with VVNCED1, and both showed high homology to NCEDs from other plants. The expression patterns of PpNCED1 and VVNCED1 were very similar. Both were highly expressed at the beginning of ripening when ABA content becomes high. The maximum ABA preceded ethylene production in peach fruit. ABA in the grape gradually increased from the beginning of ripening and reached the highest level at 20d before the harvest stage. However, ethylene remained at low levels during the entire process of fruit development, including ripening and senescence. ABA content, and ripening and softening of both types of fruits, were promoted or delayed by exogenous ABA or Fluridone (or NDGA) treatment. The roles of ABA and ethylene in the later ripening of fruit are complex. Based on results obtained in this study, we concluded that PpNCED1 and VVNCED1 initiate ABA biosynthesis at the beginning of fruit ripening, and that ABA accumulation might play a key role in the regulation of ripeness and senescence of both peach and grape fruits.

Depolymerization of cell wall xyloglucan has been proposed to be involved in tomato fruit softening, along with the xyloglucan modifying enzymes. Xyloglucan endotransglucosylase/hydrolases (XTHs: EC 2.4.1.207 and/or EC 3.2.1.151) have been proposed to have a dual role integrating newly secreted xyloglucan chains into an existing wall-bound xyloglucan, or restructuring the existing cell wall material by catalyzing transglucosylation between previously wall-bound xyloglucan molecules. Here, 10 tomato (Solanum lycopersicum) SlXTHs were studied and grouped into three phylogenetic groups to determine which members of each family were expressed during fruit growth and fruit ripening, and the ways in which the expression of different SlXTHs contributed to the total XET and XEH activities. Our results showed that all of the SlXTHs studied were expressed during fruit growth and ripening, and that the expression of all the SlXTHs in Group 1 was clearly related to fruit growth, as were SlXTH12 in Group 2 and SlXTH6 in Group 3-B. Only the expression of SlXTH5 and SlXTH8 from Group 3-A was clearly associated with fruit ripening, although all 10 of the different SlXTHs were expressed at the red ripe stage. Both total XET and XEH activities were higher during fruit growth, and decreased during fruit ripening. Ethylene production during tomato fruit growth was low and experienced a significant increase during fruit ripening, which was not correlated either with SlXTH expression or with XET and XEH activities. We suggest that the role of XTH during fruit development could be related to the maintenance of the structural integrity of the cell wall, and the decrease in XTHs expression, and the subsequent decrease in activity during ripening may contribute to fruit softening, with this process being regulated through different XTH genes.

This study focuses on how the length of exposure to chilling temperature and atmosphere storage conditions regulate the hydrolytic activity and expression of chitinase (PR-Q) and 1,3-beta-glucanase (PR-2) isoenzymes in cherimoyas (Annona cherimola Mill.). Storage at 6 degrees C modified the expression of constitutive isoenzymes and induced the appearance of novel acidic chitinases, AChi26 and AChi24, at the onset of the storage period, and of a basic chitinase, BChi33, after prolonged storage. The induction of this basic isoenzyme was concomitant with the accumulation of basic constitutive 1,3-beta-glucanases. These low-temperature-induced chitinases modified the growth inhibition in vitro of Botrytis cinerea. Short-term high CO(2) treatment activated a coordinated response of acidic chitinases and 1,3-beta-glucanases after prolonged storage at chilling temperature. Moreover, the high in vitro cryoprotective activity of CO(2)-treated protein extracts was associated with the induction of two low molecular mass isoenzymes, AGlu19 and BChi14. Thus, exposure to high concentrations of CO(2) modified the response of fruit to low temperature, inducing the synthesis of cryoprotectant proteins such as specific pathogenesis-related isoenzymes that could be functionally associated with an increase in chilling tolerance in vivo.

The ripening-specific genes MA-ACS1 (Musa acuminata ACC synthase1) and MA-ACO1 (M. acuminata ACC oxidase 1) are regulated in response to a wide variety of factors. Here, we have studied the differential transcript accumulation pattern and protein levels of MA-ACS1 and MA-ACO1 genes in response to ethylene, auxin, wounding and low temperature in preclimacteric banana fruit. We have shown that exogenous application of ethylene and auxin induced the expression of MA-ACS1, while MA-ACO1 showed marginal expression following ethylene treatment in preclimacteric stage. Auxin did not induce MA-ACO1 expression. Thus, auxin-treated banana fruits showed lower ethylene production rate as compared to ethylene-treated fruits. Conversely, wounding and cold treatment down-regulated the expression of both the genes and thus inhibited ethylene production. Furthermore, we have detected a GCC-box putative ethylene-responsive element (ERE)- and an auxin-responsive element (ARE)-specific DNA-binding activity in the banana pulp and studied the ethylene and auxin responsive characteristics of the GCC-box and ARE (TGTCTC) containing synthetic promoter fragments. In addition, we have detected an enhanced ethylene production rate and expression level of MA-ACS1 and MA-ACO1 genes along with a strong GCC-box-specific DNA-binding activity following exposure to constant dark period for 8d at the preclimacteric stage. Together, our study provides interesting information about the regulation of expression of MA-ACS1 and MA-ACO1 genes in response to various factors during ripening in banana fruit, which may have physiological relevance concerning ethylene biosynthesis during post-harvest conditions.

The decrease of strawberry (Fragariaxananassa Duch.) fruit firmness observed during ripening is partly attributed to pectolytic enzymes: polygalacturonases, pectate lyases and pectin methylesterases (PMEs). In this study, PME activity and pectin content and esterification degree were measured in cell walls from ripening fruits. Small green, large green, white, turning, red and over-ripe fruits from the Elsanta cultivar were analyzed. Using the 2F4 antibody directed against the calcium-induced egg box conformation of pectin, we show that calcium-bound acidic pectin was nearly absent from green and white fruits, but increased abruptly at the turning stage, while the total pectin content decreased only slightly as maturation proceeded. Isoelectrofocalisation performed on wall protein extracts revealed the expression of at least six different basic PME isoforms. Maximum PME activity was detected in green fruits and steadily decreased to reach a minimum in senescent fruits. The preliminary role of PMEs and subsequent pectin degradation by pectolytic enzymes is discussed.

The subcellular distribution and activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) were studied in developing peach (Prunus persica L. Batsch cv. Zaoyu) fruit. Fruit tissues were separated by differential centrifugation at 15,000g into plastidic and cytosolic fractions. There was no serious loss of enzyme activity (or activation) during the preparation of fractions. G6PDH activity was found in both the plastidic and cytosolic compartments. Moreover, DTT had no effect on the plastidic G6PDH activities, that is, the redox regulatory mechanism did not play an important role in the peach fleshy tissue. Results from the immunogold electron-microscope localization revealed that G6PDH isoenzymes were mainly present in the cytosol, the secondary wall and plastids (chloroplasts and chromoplasts), but scarcely found in the starch granules or the cell wall. In addition to a decrease in fruit firmness, the G6PDH activity in the cytotolic and plastidic fractions increased, and anthocyanin started to accumulate during fruit maturation. These results suggest that G6PDH, by providing precursors for metabolic processes, might be associated with the red coloration that occurs in peach fruit.

Soluble acid invertase (S-AIV; EC 3.2.1.26) in Japanese pear fruit has an important role in accumulating hexoses during fruit enlargement and regulates the sucrose-to-hexose ratio in the vacuole. Full-length cDNA of PsS-AIV1 and PsS-AIV2 isoforms were cloned from Japanese pear fruit and their amino acid sequences share 40% identity; PsS-AIV1 was confirmed to code S-AIV isozyme purified previously. The roles of PsS-AIV1 and PsS-AIV2 genes throughout fruit development and in sugar composition were investigated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using specific primers of their transcripts. PsS-AIV1 transcript had a maximum level at 34 days after full bloom (DAFB) and decreased rapidly during fruit development; PsS-AIV2 transcript increased gradually during fruit growth from 34 DAFB, had its maximum level at 79 DAFB and remained high until 107 DAFB at active fruit enlargement. The activity of S-AIV was highest at 34 DAFB, decreased during fruit growth until 66 DAFB, remained almost the same during early fruit enlargement until 79 DAFB and then decreased again. Soluble sugars fructose and glucose began accumulating predominantly during fruit enlargement from 66 DAFB; sucrose began increasing rapidly during fruit maturation from 121 DAFB. High expression of PsS-AIV1 transcript and high enzyme activity in the young fruit stage seems to have an important role in supplying a lot of substrate for energy needed for cell division and growth by hydrolyzing sucrose to hexoses. Increasing PsS-AIV2 expression during fruit enlargement may lead to rapid cell expansion through increased osmotic pressure by accumulation of a large amount of hexose in the vacuole.

We isolated from strawberry (Fragariae x ananassa Duch) a genomic clone of a beta-1,3-glucanase gene, designated as FaBG2-2. In addition, a related cDNA clone, designated as FaBG2-3, was also isolated. FaBG2-2 and FaBG2-3 are similar in their coding regions, except that FaBG2-2 does not appear to contain a signal peptide coding sequence. The 5' and 3' flanking regions of FaBG2-2 and FaBG2-3 are differentt. Using real-time PCR, the expression patterns of FaBG2-3 and a previously isolated beta-1,3-glucanase gene, FaBG2-1, in strawberry plants infected with Colletotrichum fragariae or Colletotrichum acutatum were analyzed at different time points post-infection. The results showed that expressions of both genes in the leaves of infected plants were induced by the two fungi, but the level of induction was several fold greater with C. fragariae. Comparison of the expression levels of the two genes revealed that the level of FaBG2-3 expression was several hundred to over a thousand fold higher than that of FaBG2-1. Furthermore, the expression levels of the two genes in the leaf, fruit, crown and root of uninfected strawberry plants were analyzed.

The phenylpropanoid pathway yields a variety of phenolics that are closely associated with fruit qualities in addition to structural and defense-related functions. However, very little has been reported concerning its metabolism in fruit. This experiment was designed to assess changes of eleven phenolic acids in grape berry (Vitis vinifera L. cv. Cabernet Sauvignon) and explore both the activities and amounts of three key enzymes--phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate:coenzyme A ligase (4CL)--catalyzing the biosynthesis of these compounds during berry development. Finally, the subcellular localizations of the enzymes within berry tissues were also investigated using immuno-gold electron microscopic technique. The results indicated that the contents of gallic, protocatechuic, gentisic and caffeic acid all changed drastically during berry development, while other compounds containing p-hydroxybenzoic, vanillic, syringic, chlorogenic, p-coumaric, ferulic and sinapic acid varied only slightly. Activities of PAL, C4H and 4CL showed similar pattern changes with two accumulated peaks throughout berry development. In addition, their activities all showed a highly positive correlation with the total contents of phenolic acids, whereas the immunoblotting analysis showed that changes in enzyme activities were independent of the enzyme amounts. Results from the subcellular-localization study revealed that PAL was mainly present in the cell walls, secondarily thickened walls, and the parenchyma cells of the berry mesocarp cells, C4H was found primarily in the chloroplast (plastid) and nucleus and 4CL predominantly in the secondarily thickened walls and the parenchyma cells of mesocarp vascular tissue.

Most previous efforts to isolate genes that are expressed during fruit development have focused on fruit ripening. As a result, information is lacking on fruit genes that are specifically expressed at early developmental stages. Using a cDNA subtraction technique, we isolated fruit-specific genes that are expressed during the cell expansion phase of tomato (Lycopersicon esculentum Mill) fruit development. One of the isolated cDNAs, LeODD, is transiently expressed 15 days after flowering in a nearly fruit-specific manner during the initial period of cell expansion. Southern blot analysis indicated that LeODD is encoded by a single gene. LeODD is homologous to 2-oxoglutarate-dependent dioxygenase genes, and the key amino acid residues in the binding sites for ferrous iron and 2-oxoglutarate are completely conserved. The amino acid sequence identity between LeODD and other 2-oxoglutarate-dependent dioxygenases is relatively low, suggesting that LeODD is a novel enzyme of this family. Another of the isolated cDNAs, LeGLO2, is also highly expressed at 15 days after flowering. LeGLO2 is thought to be a novel glycolate oxidase isoform that functions in fruit. 2-Oxoglutarate, the cosubstrate of LeODD, could be supplied by a LeGLO2-mediated glycolate pathway in immature fruit. The coordinate expression of LeODD and LeGLO2 may play a role in the biosynthesis of a metabolite, such as a plant hormone or secondary metabolite, that is required during the initial period of the cell expansion phase of fruit development.

In the apple variety 'Usterapfel', there are two known genotypes, which differ in malic acid content. One hundred days after full bloom, low-acid fruit (LA-fruit) contained 125 micromolg(-1) dry matter (DW) of malate, while the high-acid genotype (HA-fruit) reached levels up to 627 micromolg(-1) DW. There was no difference in the catalytic activity of enzymes involved in malate metabolism, such as PEPcarboxylase, malate dehydrogenase, and NADP malic enzyme. After [14C]glucose incorporation into the excised tissue of either genotype, the organic acid fraction was labeled to approximately the same extent. Furthermore, uptake of [14C]malate was significantly lower in excised tissue of LA-fruit. These findings suggest that low malate content in LA-fruit is the result of a restricted ability to accumulate malate in apple parenchyma cells. The different ability to accumulate malate had a pronounced effect on overall carbon partitioning. However, the rate of respiration and the rate of malate synthesis was similar in both genotypes. In HA-fruit, the glycolytic flux through pyruvate kinase was increased to compensate for the carbon that accumulated in the vacuole as malate. Since malate storage in the LA-fruit was restricted, it was more easily available for gluconeogenesis, and was correlated with a three-times higher activity of PEPcarboxykinase. LA-fruit showed higher concentrations of ATP, which stimulated Glc6P and fructose-6-phosphate formation. The elevated hexosephosphate content led to an enhanced partitioning of carbon into starch (+40%), hemicellulose (+104%), and sucrose (+40%) in more mature fruit. The activation of carbohydrate synthesis resulted in a significant drop in glucose-1-phosphate (Glc1P). To meet the increased demand for Glc1P, the activities of neutral and acid invertase, hexokinase, and phosphoglucomutase were higher in LA-fruit. Glucose was a more versatile substrate for this metabolic route than was fructose. It was also evident that glycolytic flux in apple was dependent on glucose level, and that the reaction catalysed by phosphoglucomutase contributed to the regulation of carbon partitioning between malate and carbohydrate polymers.

Pepper is a vegetable of importance in human nutrition. Currently, one of the most interesting properties of natural products is their antioxidant content. In this work, the purification and characterisation of peroxisomes from fruits of a higher plant was carried out, and their antioxidative enzymatic and non-enzymatic content was investigated. Green and red pepper fruits (Capsicum annuum L., type Lamuyo) were used in this study. The analysis by electron microscopy showed that peroxisomes from both types of fruits contained crystalline cores which varied in shape and size, and the presence of chloroplasts and chromoplasts in green and red pepper fruits, respectively, was confirmed. Peroxisomes were purified by differential and sucrose density-gradient centrifugations. In the peroxisomal fractions, the activity of the photorespiration, beta-oxidation and glyoxylate cycle enzymes, and the ROS-related enzymes catalase, superoxide dismutase, xanthine oxidase, glutathione reductase and NADP(+)-dehydrogenases, was determined. Most enzymes studied had higher specific activity and protein content in green than in red fruits. By native PAGE and western blot analysis, the localisation of a Mn-SOD in fruit peroxisomes was demonstrated. The ascorbate and glutathione levels were also determined in crude extracts and in peroxisomes purified from both green and red peppers. The total ascorbate content (200-220 mg per 100 g FW) was similar in crude extracts from the two types of fruits, but higher in peroxisomes from red peppers. The glutathione concentration was 2-fold greater in green pepper crude extracts than in red fruits, whereas peroxisomes from both tissues showed similar values. The presence in pepper peroxisomes of different antioxidative enzymes and their corresponding metabolites implies that these organelles might be an important pool of antioxidants in fruit cells, where these enzymes could also act as modulators of signal molecules (O2*-, H202) during fruit maturation.

Pectinmethylesterase (PME, EC 3.2.1.11) and polygalacturonase (PG, EC 3.2.1.15) are known to operate in tandem to degrade methylesterified polyuronides. In this study, PGs purified from tomato and avocado fruit were compared in terms of their capacity to hydrolyze water-soluble polyuronides from avocado before and following enzymic or chemical de-esterification. When assayed using polygalacturonic acid or polyuronides from avocado fruit, the activity of PG from tomato fruit was 3-4 times higher than that from avocado fruit. High molecular mass, low methylesterified (33%) water-soluble polyuronides (WSP) from pre-ripe avocado fruit (day 0) were partially depolymerized upon incubation with purified avocado and tomato PGs. In contrast, middle molecular mass, highly methylesterified (74%) WSP from day 2 fruit were largely resistant to the action of both PGs. PME or weak alkali treatment of highly methylesterified WSP decreased the methylesterification values to 11 and 4.5%, respectively. Treatment of de-esterified WSP with either avocado or tomato PGs caused extensive molecular mass downshifts, paralleling those observed during avocado fruit ripening. Although PME and PG are found in many fruits, the pattern of depolymerization of native polyuronides indicates that the degree of cooperativity between these enzymes in vivo differs dramatically among fruits. The contribution of PME to patterns of polyuronide depolymerization observed during ripening compared with physically compromised fruit tissues is discussed.