The potential of glucosidase and glucose oxidase for aroma improvement in concentrated peach puree based on volatilomics and metabolomics.

Cooked off-flavor was produced during the processing of concentrated peach puree (CPP), which led to aroma deterioration. Enzymatic treatment was beneficial in eliminating off-flavors and improving the aroma quality. Herein, the efficacy of glycosidase (AR2000), glucose oxidation (GOD), and their combination on the inhibition of off-flavors and aroma enhancement were evaluated. Compared with CPP, contents of benzaldehyde, benzyl alcohol, nonanal, and linalool increased by 198%, 1222%, 781%, and 71% after AR2000 treatment via the metabolisms of shikimate, glucose, linoleic acid, and linolenic acid, leading to the strengthening of floral and grassy. Due to the removal of 1-octen-3-one via linolenic acid metabolism, cooked off-flavor could be significantly weakened by GOD. Furthermore, Furthermore, the combination of AR2000 and GOD could not only inhibit the production of 1-octen-3-one to weaken the cooked note but also enhance grassy and floral attributes via the increase of aldehydes and alcohols.

Fine-tuning the performance of ddRAD-seq in the peach genome.

The advance of Next Generation Sequencing (NGS) technologies allows high-throughput genotyping at a reasonable cost, although, in the case of peach, this technology has been scarcely developed. To date, only a standard Genotyping by Sequencing approach (GBS), based on a single restriction with ApeKI to reduce genome complexity, has been applied in peach. In this work, we assessed the performance of the double-digest RADseq approach (ddRADseq), by testing 6 double restrictions with the restriction profile generated with ApeKI. The enzyme pair PstI/MboI retained the highest number of loci in concordance with the in silico analysis. Under this condition, the analysis of a diverse germplasm collection (191 peach genotypes) yielded 200,759,000 paired-end (2 × 250 bp) reads that allowed the identification of 113,411 SNP, 13,661 InDel and 2133 SSR. We take advantage of a wide sample set to describe technical scope of the platform. The novel platform presented here represents a useful tool for genomic-based breeding for peach.

Double NCED isozymes control ABA biosynthesis for ripening and senescent regulation in peach fruits.

During ripening, peach fruits (Prunus persica L. Batsch) rapidly progress to the senescent stage, resulting in a brief shelf life. Abscisic acid (ABA) plays an important role in regulating the ripening process, both in climacteric and non-climacteric fruits. A key enzyme for ABA biosynthesis in higher plants is 9-cis-epoxycarotenoid dioxygenase (NCED). In this study, two NCED isozymes, PpNCED1 and PpNCED5, were identified in peach fruits. While both NCED genes had similar transcriptional patterns (up-regulation) at the beginning of peach ripening, PpNCED5 showed a consistently lower expression level than PpNCED1. During the post-harvest stage, gene expression of PpNCED1 declined, while PpNCED5 expression increased relative to PpNCED1 expression. Considering the dynamic process of ABA accumulation during fruit ripening and senescence in peach, this study indicates that both NCED genes cooperatively control ABA biosynthesis in peach fruits. Moreover, spatio-temporal expression and transcriptional response to hormone and abiotic stress suggested that there is functional divergence between PpNCED1 and PpNCED5 genes in peach. A carotenoid-rich callus system was used to verify the function of PpNCED1 and PpNCED5. In the transgenic callus system, both PpNCED1 and PpNCED5 isozymes promoted ABA biosynthesis, which likely accelerated cell senescence through activating ROS signals. The results from this study provide evidence supporting an ABA biosynthetic regulation process via the two NCED genes in peach fruit, and suggest a mechanism of ABA-induced fruit ripening and senescence.

Functional analysis of PpRHM1 and PpRHM2 involved in UDP-l-rhamnose biosynthesis in Prunus persica.

UDP-l-rhamnose (UDP-Rha) is an important sugar donor for glycosylation of various cell molecules in plant. Rhamnosides are widely present in different plant tissues and play important biological roles under different developmental or environmental conditions. However, enzymes involved in UDP-Rha biosynthesis and their encoding genes have been identified in few plants, which limits the functional analysis of plant rhamnosides. Here, two UDP-Rha biosynthesis genes, named PpRHM1 (2028 bp) and PpRHM2 (2016 bp), were isolated and characterized from Prunus persica, which is rich sources of flavonol rhamnosides. Both recombinant RHM proteins can catalyze the transformation from UDP-d-glucose (UDP-Glc) to UDP-Rha, which was confirmed by LC-MS and formation of flavonol rhamnosides. Biochemical analysis showed that both recombinant RHM proteins preferred alkaline conditions in pH range of 8.0-9.0 and had optimal reaction temperature between 25 and 30 °C. PpRHM1 showed the better UDP-Glc substrate affinity with Km of 360.01 µM. Gene expression analysis showed different transcript levels of both RHMs in all plant tissues tested, indicating the involvement of rhamnosides in various tissues in plant. Such results provide better understanding of UDP-Rha biosynthesis in fruit tree and may be helpful for further investigation of various rhamnose derivatives and their biological functions.

DNA methylation of LDOX gene contributes to the floral colour variegation in peach.

Peach is an important fruit and ornamental plant around the globe. Variegation in flowers often captures consumers' attention, and variegated plants are of high ornamental value. To determine the relationship between DNA methylation and phenotype, we obtained the first single-nucleotide resolution DNA methylation of variegation cultivars in peach through bisulfite sequencing. In this study, a similar methylation rate of 12.90 % in variegated flower buds (VF) and 11.96 % in red flower buds (RF) was determined. The methyl-CG (mCG) was the main context in both samples. We identified 503 differentially methylated regions (DMRs) in all chromosomes. These DMRs were focused on 96 genes and 156 promoters. Associated with the transcriptional and proteome analysis, 106 differently expressed genes and 52 different proteins had varying degrees of methylation. Silent genes exhibited higher methylation levels than expressed genes. The methylation state of the leucoanthocyanidin dioxygenase (LDOX) promoter in VF was higher than RF at flower stages 2 (FS2) based on bisulfite sequencing PCR (BSP) results. Moreover, further experiments showed LDOX gene expression and enzyme activity in RF was higher than VF. The DNA methylation trend consisted of the gene expression and flower colour phenotype. Several cis-acting regulatory elements on BSP sequences were involved in phytohormones, transcription factors, and light responsiveness, which could affect gene expression. The higher level of LDOX gene expression promoted synthesis of colourful anthocyanidins, which caused red spots on the petal. Together, this study identified the context and level of methylation at each site with bisulfite sequencing (BS). These results are helpful in uncovering the mechanism of variegated flower petal formation in peach.

Effects of Bacillus Subtilis CF-3 VOCs Combined with Heat Treatment on the Control of Monilinia fructicola in Peaches and Colletotrichum gloeosporioides in Litchi Fruit.

In order to study the effect of volatile organic compounds (VOCs) produced by Bacillus subtilis CF-3 combined with heat treatment on Monilinia fructicola in peach and Colletotrichum gloeosporioides in litchi fruit, fruits were treated with B. subtilis CF-3 VOCs and hot air alone or in combination. The quality indexes of peach and litchi fruit after treatment and the changes in defense-related enzymes were measured. The results showed that the B. subtilis CF-3 VOCs combined with heat treatment could significantly reduce the rot index of peach and litchi fruit, and effectively maintain firmness and soluble solids content, as well as reduce weight loss of fruits. The combined treatment effectively enhanced the activity of peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), and superoxide dismutase (SOD) than either treatment alone, and enhanced the resistance of fruit to pathogenic fungi by activating disease-resistant enzymes (phenylalanine ammonia-lyase [PAL], chitinase [CHI], ß-1, 3-glucanase [GLU]) activity. In this study, B. subtilis CF-3 VOCs combined with heat treatment maintained the quality and delayed the decline of peach and litchi fruit, providing a theoretical basis for future applications. PRACTICAL APPLICATION: The combination of B. subtilis CF-3 VOCs and heat treatment reduce the extent of M. fructicola and C. gloeosporioides. The combination maintain the quality of peach and litchi better. The combination obviously improve the activity of defense-related enzyme in fruit.

Genome-wide identification and transcriptome profiling reveal that E3 ubiquitin ligase genes relevant to ethylene, auxin and abscisic acid are differentially expressed in the fruits of melting flesh and stony hard peach varieties.

BACKGROUND: Ubiquitin ligases (E3) are the enzymes in the ubiquitin/26S proteasome pathway responsible for targeting proteins to the degradation pathway and play major roles in multiple biological activities. However, the E3 family and their functions are yet to be identified in the fruit of peach. RESULTS: In this study, genome-wide identification, classification and characterization of the E3 ligase genes within the genome of peach (Prunus persica) was carried out. In total, 765 E3 (PpE3) ligase genes were identified in the peach genome. The PpE3 ligase genes were divided into eight subfamilies according to the presence of known functional domains. The RBX subfamily was not detected in peach. The PpE3 ligase genes were not randomly distributed among the 8 chromosomes, with a greater concentration on the longer chromosomes. The primary mode of gene duplication of the PpE3 ligase genes was dispersed gene duplication (DSD). Four subgroups of the BTB subfamily never characterized before were newly identified in peach, namely BTBAND, BTBBL, BTBP and BTBAN. The expression patterns of the identified E3 ligase genes in two peach varieties that display different types of fruit softening (melting flesh, MF, and stony hard, SH) were analyzed at 4 different stages of ripening using Illumina technology. Among the 765 PpE3 ligase genes, 515 (67.3%) were expressed (FPKM > 1) in the fruit of either MF or SH during fruit ripening. In same-stage comparisons, 231 differentially expressed genes (DEGs) were identified between the two peach cultivars. The number of DEGs in each subfamily varied. Most DEGs were members of the BTB, F-box, U-box and RING subfamilies. PpE3 ligase genes predicted to be involved in ethylene, auxin, or ABA synthesis or signaling and DNA methylation were differentially regulated. Eight PpE3 ligase genes with possible roles in peach flesh texture and fruit ripening were discussed. CONCLUSIONS: The results of this study provide useful information for further understanding the functional roles of the ubiquitin ligase genes in peach. The findings also provide the first clues that E3 ligase genes may function in the regulation of peach ripening.

The Role of IP3 in NO-Enhanced Chilling Tolerance in Peach Fruit.

The role of inositol 1,4,5-trisphosphate (IP3) in nitric oxide (NO)-reduced chilling injury (CI) in peach fruit was investigated. The fruit were immersed in sodium nitroprusside (SNP) (NO donor) and neomycin (IP3 inhibitor). Results showed that chilling tolerance was enhanced upon exogenous SNP in postharvest peach fruit. Further, GABA accumulation was stimulated by SNP. The increase in protein expression and activity for enzymes in GABA biosynthesis, including glutamate decarboxylase (GAD), polyamine oxidase (PAO), and amino aldehyde dehydrogenase (AMADH), upon SNP treatment was also observed. Also, the up-regulation of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine d-aminotransferase (OAT) and the down-regulation of proline dehydrogenase (PDH) were induced by SNP treatment, thereby accelating proline production. Additionally, SNP treatment elevated protein expression and activity of alternative oxidase (AOX). The above effects induced upon SNP were partly weakened by neomycin. Therefore, IP3 mediated NO-activated GABA and proline accumulation as well as AOX, thus inducing chilling tolerance in postharvest peach fruit.

Identification of the Fungal Pathogens of Postharvest Disease on Peach Fruits and the Control Mechanisms of Bacillus subtilis JK-14.

Postharvest fungal disease is one of the significant factors that limits the storage period and marketing life of peaches, and even result in serious economic losses worldwide. Biological control using microbial antagonists has been explored as an alternative approach for the management of postharvest disease of fruits. However, there is little information available regarding to the identification the fungal pathogen species that cause the postharvest peach diseases and the potential and mechanisms of using the Bacillussubtilis JK-14 to control postharvest peach diseases. In the present study, a total of six fungal isolates were isolated from peach fruits, and the isolates of Alternaria tenuis and Botrytis cinerea exhibited the highest pathogenicity and virulence on the host of mature peaches. In the culture plates, the strain of B. subtilis JK-14 showed the significant antagonistic activity against the growth of A. tenuis and B. cinerea with the inhibitory rates of 81.32% and 83.45% at 5 days after incubation, respectively. Peach fruits treated with different formulations of B. subtilis JK-14 significantly reduced the mean disease incidences and lesion diameters of A. tenuis and B. cinerea. The greatest mean percent reduction of the disease incidences (81.99% and 71.34%) and lesion diameters (82.80% and 73.57%) of A. tenuis and B. cinerea were obtained at the concentration of 1 × 107 CFU mL-1 (colony forming unit, CFU). Treatment with the strain of B. subtilis JK-14 effectively enhanced the activity of the antioxidant enzymes-superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in A. tenuis and B. cinerea inoculated peach fruits. As such, the average activities of SOD, POD and CAT were increased by 36.56%, 17.63% and 20.35%, respectively, compared to the sterile water treatment. Our results indicate that the isolates of A. tenuis and B. cinerea are the main pathogens that cause the postharvest peach diseases, and the strain of B. subtilis JK-14 can be considered as an environmentally-safe biological control agent for the management of postharvest fruits diseases. We propose the possible mechanisms of the strain of B. subtilis JK-14 in controlling of postharvest peach diseases.

Peach Carboxylesterase PpCXE1 Is Associated with Catabolism of Volatile Esters.

Peach fruit volatile acetate esters impact consumer sensory preference and contribute to defense against biotic stresses. Previous studies showed that alcohol acyltransferase (AAT) family PpAAT1 is correlated with volatile ester formation in peach fruits. However, fruits also contain carboxylesterase (CXE) enzymes that hydrolyze esters. The functions of this family with regard to volatile ester content has not been explored. Here, we observed that content of acetate ester was negatively correlated with expression of PpCXE1. Recombinant PpCXE1 protein exhibited hydrolytic activity toward acetate esters present in peach fruit. Kinetic analysis showed that PpCXE1 showed the highest catalytic activity toward E-2-hexenyl acetate. Subcellular localization demonstrated that PpCXE1 is present in the cytoplasm. Transient expression in peach fruit and stable overexpression in tomato fruit resulted in significant reduction of volatile esters in vivo. Taken together, the results indicate that PpCXE1 expression is associated with catabolism of volatile acetate esters in peach fruit.

Cofactor Specificity Switch on Peach Glucitol Dehydrogenase.

Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than that for NADP+. Herein, we combined structural and kinetic data to reverse the cofactor specificity of this enzyme. Using site-saturation mutagenesis, we obtained the D216A mutant, which uses both NAD+ and NADP+, although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1, respectively). This mutant was used as a template to introduce further mutations by site-directed mutagenesis, using information from the fruit fly NADP-dependent GolDHase. The D216A/V217R/D218S triple mutant displayed a 2-fold higher catalytic efficiency with NADP+ than with NAD+. Overall, our results indicate that the triple mutant has the potential to be used for metabolic and cellular engineering and for cofactor recycling in industrial processes.

Carotenoid cleavage in chromoplasts of white and yellow-fleshed peach varieties.

BACKGROUND: In peach fruit, carotenoid accumulation in the mesocarp causes the difference between yellow and white genotypes. The latter are generally characterized by a peculiar and more intense aroma, because of higher release of volatiles deriving from dioxygenase-catalysed breakdown of the tetraterpene skeleton. The rate of carotenoid oxidation was investigated in peach (Prunus persica L.) fruits harvested at various stages of development. Two couples of white and yellow-fleshed isogenic varieties and an ancestral white-fleshed genotype were analysed, which had previously shown to differ in Carotenoid Cleavage Dioxygenase 4 allelic composition resulting in various combinations of putatively active/inactive proteins. RESULTS: Carotenoid bleaching activity was localized in the insoluble fraction of fruit flesh chromoplasts. Higher rates of trans-ß-apo-8'-carotenal than ß-carotene bleaching suggest that the first cleavage reaction is the rate-limiting step. Consistently, HPLC analysis did not show the appearance of coloured intermediates in reaction mixtures. High levels of substrate breakdown were found during the initial phases of fruit development in all genotypes examined, whereas significant differences were evident during the second exponential growth phase and ripening onset. Also, the ratio of carotene versus carotenale utilization varied significantly. CONCLUSION: Pattern comparison among activity levels measured in vitro on chromoplast enriched fractions suggests that cleavage enzyme(s) other than Carotenoid Cleavage Dioxygenase 4 play a significant role in carotenoid breakdown during fruit development and ripening. © 2018 Society of Chemical Industry.

The effect of auxin and strigolactone on ATP/ADP isopentenyltransferase expression and the regulation of apical dominance in peach.

KEY MESSAGE: We confirmed the roles of auxin, CK, and strigolactones in apical dominance in peach and established a model of plant hormonal control of apical dominance in peach. Auxin, cytokinin, and strigolactone play important roles in apical dominance. In this study, we analyzed the effect of auxin and strigolactone on the expression of ATP/ADP isopentenyltransferase (IPT) genes (key cytokinin biosynthesis genes) and the regulation of apical dominance in peach. After decapitation, the expression levels of PpIPT1, PpIPT3, and PpIPT5a in nodal stems sharply increased. This observation is consistent with the changes in tZ-type and iP-type cytokinin levels in nodal stems and axillary buds observed after treatment; these changes are required to promote the outgrowth of axillary buds in peach. These results suggest that ATP/ADP PpIPT genes in nodal stems are key genes for cytokinin biosynthesis, as they promote the outgrowth of axillary buds. We also found that auxin and strigolactone inhibited the outgrowth of axillary buds. After decapitation, IAA treatment inhibited the expression of ATP/ADP PpIPTs in nodal stems to impede the increase in cytokinin levels. By contrast, after GR24 (GR24 strigolactone) treatment, the expression of ATP/ADP IPT genes and cytokinin levels still increased markedly, but the rate of increase in gene expression was markedly lower than that observed after decapitation in the absence of IAA (indole-3-acetic acid) treatment. In addition, GR24 inhibited basipetal auxin transport at the nodes (by limiting the expression of PpPIN1a in nodal stems), thereby inhibiting ATP/ADP PpIPT expression in nodal stems. Therefore, strigolactone inhibits the outgrowth of axillary buds in peach only when terminal buds are present.

Acyl-CoA oxidase 1 is involved in γ-decalactone release from peach (Prunus persica) fruit.

KEY MESSAGE: γ-Decalactone accumulation in peach mesocarp was highly correlated with ACX enzyme activity and natural PpACX1 content. Adding the purified recombinant PpACX1 induced γ-decalactone biosynthesis in cultured mesocarp discs in vitro. Previous gene expression studies have implied that acyl coenzyme A oxidase (ACX) is related to lactones synthesis, the characteristic aroma compounds of peach. Here, we analysed the correlation between γ-decalactone content and ACX enzyme activity in mesocarp of five different types of fully ripe peach varieties. Furthermore, 'Hu Jing Mi Lu' ('HJ') and 'Feng Hua Yu Lu' ('YL'), which have strong aroma among them, at four ripening stages were selected to study the role of ACX in lactone biosynthesis. The result showed that γ-decalactone was the most abundant lactone compound. γ-Decalactone accumulation was highly correlated with ACX enzyme activity. Mass spectrometry (MS) showed that PpACX1 was the most abundant PpACX protein in fully ripe mesocarp of cv. 'HJ'. To further elucidate the function of the PpACX1 protein, the PpACX1 gene was heterologously expressed in a bacterial system and characterized in vitro. MS identification gave the molecular weight of the recombinant PpACX1 as 94.44 kDa and the coverage rate of the peptide segments was 47.3%. In cultured mesocarp discs in vitro, adding the purified recombinant PpACX1 and C16-CoA substrate induced the expected γ-decalactone biosynthesis. Using a sandwich ELISA based on mixed mono- and polyclonal antibodies against recombinant PpACX1, PpACX1 content in mesocarp was found to be highly correlated with γ-decalactone accumulation in mesocarp of five fully ripe varieties and four ripening stages of 'HJ' and 'YL'. This study revealed the vital function of PpACX1 in γ-decalactone biosynthesis in peach fruit.

Peach leaf curl disease shifts sugar metabolism in severely infected leaves from source to sink.

Peach leaf curl is a disease that affects the leaves of peach trees, and in severe cases all of the leaf can be similarly affected. This study investigated some effects of this disease on the metabolism of peach leaves in which all parts of the leaf were infected. These diseased leaves contained very little chlorophyll and performed little or no photosynthesis. Compared to uninfected leaves, diseased leaves possessed higher contents of fructose and especially glucose, but lowered contents of sucrose, sorbitol and especially starch. The activities of soluble acid invertase, neutral invertase, sorbitol dehydrogenase and sucrose synthase were all higher in diseased leaves, whereas, those of aldose-6-phosphate reductase and sucrose phosphate synthase were lower. The activities of hexokinase and fructokinase were little changed. In addition, immunblots showed that the contents of Rubisco and ADP-glucose phosphorylase were reduced in diseased leaves, whereas, the content of phosphoenolpyruvate carboxylase was increased. The results show that certain aspects of the metabolism of diseased leaves are similar to immature sink leaves. That is photosynthetic function is reduced, the leaf imports rather than exports sugars, and the contents of non-structural carbohydrates and enzymes involved in their metabolism are similar to sink leaves. Further, the effects of peach leaf curl on the metabolism of peach leaves are comparable to the effects of some other diseases on the metabolism of photosynthetic organs of other plant species.

Identification and Expression Analysis of Polygalacturonase Family Members during Peach Fruit Softening.

Polygalacturonase (PG) is an important hydrolytic enzyme involved in pectin degradation during fruit softening. However, the roles of PG family members in fruit softening remain unclear. We identified 45 PpPG genes in the peach genome which are clustered into six subclasses. PpPGs consist of four to nine exons and three to eight introns, and the exon/intron structure is basically conserved in all but subclass E. Only 16 PpPG genes were expressed in ripening fruit, and their expression profiles were analyzed during storage in two peach cultivars with different softening characteristics. Eight PGs (PpPG1, -10, -12, -13, -15, -23, -21, and -22) in fast-softening "Qian Jian Bai" (QJB) fruit and three PGs (PpPG15, -21, and -22) in slow-softening "Qin Wang" (QW) fruit exhibited softening-associated patterns; which also were affected by ethylene treatment. Our results suggest that the different softening characters in QW and QJB fruit is related to the amount of PG members. While keeping relatively lower levels during QW fruit softening, the expression of six PGs (PpPG1, -10, -12, -11, -14, and -35) rapidly induced by ethylene. PpPG24, -25 and -38 may not be involved in softening of peach fruit.

The Impact of Maturity Stage on Cell Membrane Integrity and Enzymatic Browning Reactions in High Pressure Processed Peaches (Prunus persica).

Fruit maturity is an important factor associated with final product quality, and it may have an effect on the level of browning in peaches that are high pressure processed (HPP). Peaches from three different maturities, as determined by firmness (M1 = 50-55 N, M2 = 35-40 N, and M3 = 15-20 N), were subjected to pressure levels at 0.1, 200, and 400 MPa for 10 min. The damage from HPP treatment results in loss of fruit integrity and the development of browning during storage. Increasing pressure levels of HPP treatment resulted in greater damage, particularly in the more mature peaches, as determined by shifts in transverse relaxation time (T2) of the vacuolar component and by light microscopy. The discoloration of peach slices of different maturities processed at the same pressure was comparable, indicating that the effect of pressure level is greater than that of maturity in the development of browning.

Phosphoenolpyruvate carboxykinase, pyruvate orthophosphate dikinase and isocitrate lyase in both tomato fruits and leaves, and in the flesh of peach and some other fruits.

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.

Potential role of reactive oxygen species and antioxidant genes in the regulation of peach fruit development and ripening.

The roles of reactive oxygen species (ROS) as both toxic by-products and as signaling molecules have been reported in fruit development and ripening. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) play important roles in balancing the induction and removal of ROS in plants, and are respectively encoded by families of closely homologous genes. In the present study, we investigated the roles of ROS and the above-mentioned antioxidant genes during the development and ripening of peach fruit. The experimental results indicated that O2(-) and H2O2 acted as potential signaling molecules in the middle stage of fruit development, and only H2O2 might function as a main toxic molecule to stimulate lipid peroxidation and oxidative stress in the late stage of fruit ripening. PpaCu/Zn-SODs were the most abundant members in the PpaSOD gene family and they expressed steadily in peach fruit development and ripening. Low temperature (4 °C) postponed and suppressed the climacteric peaks of respiration and ethylene, significantly enhanced the activities of CAT and GPX, and up-regulated the expression of PpaCAT1 and PpaGPX6 in the late stage of fruit ripening. PpaCAT1 and PpaGPX6 were two key genes in alleviating oxidative stress in the late stage of fruit ripening.

Two ω-3 FADs Are Associated with Peach Fruit Volatile Formation.

Aroma-related volatiles, together with sugars and acids, play an important role in determining fruit flavor quality. Characteristic volatiles of peach fruit are mainly derived from fatty acids such as linoleic acid (18:2) and linolenic acid (18:3). In the present study, six genes encoding fatty acid desaturases (FAD) were cloned, including two ω-6 FAD genes (PpFAD2, PpFAD6) and four ω-3 FAD genes (PpFAD3-1, PpFAD3-2, PpFAD7 and PpFAD8). Heterologous expression of peach FADs in tobacco plants showed that PpFAD3-1, and PpFAD3-2 significantly reduced contents of 18:2, and accumulated significant higher levels of 18:3. In the case of volatiles, transgenic plants produced lower concentrations of hexanal and higher levels of (E)-2-hexenal. Consequently, the ratio of the (E)-2-hexenal and hexanal was about 5- and 3-fold higher than that of wild type (WT) in PpFAD3-1 and PpFAD3-2 transformants, respectively. No significant changes in volatile profiles were observed in transgenic plants overexpressing the four other peach FAD genes. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that ripe fruit had high PpFAD3-1 and low PpFAD3-2 transcript levels. In contrast, high PpFAD3-2 and low PpFAD3-1 transcript levels were observed in young fruit. These results indicate a temporal regulation of these two ω-3 FADs during development and ripening, influencing peach fruit volatile formation.