Comparative transcript profiling of a peach and its nectarine mutant at harvest reveals differences in gene expression related to storability

Gene expression at harvest was compared for two stone fruit cultivars, a peach and its near-isogenic nectarine mutant, using two microarray platforms, μPEACH1.0 and ChillPeach. Together, both platforms covered over 6,000 genes out of which 417 were differentially expressed between the fruits of the two cultivars at a p value of 0.05. A total of 47 genes in nectarine and 60 genes in peach were at least twofold higher relative to each other. Nectarine had much better storage characteristics than peach and could be stored for over 5 weeks at 5 °C without storage disorders. In an attempt to determine whether gene expression at harvest could give an indication of storage potential, the expression analysis of the two cultivars was compared to that of two genotypes with different sensitivities to chilling injury. Principal component analysis of gene expression results across four fruit types differing in chilling sensitivity resulted in 41 genes whose expression levels separated the fruits according to sensitivity to storage disorders, suggesting that the genes have a role in cold response adaptation.     

DNA marker-assisted evaluation of fruit firmness at harvest and post-harvest fruit softening in a diverse apple germplasm

Several different genes have been proposed as responsible for fruit texture variability at harvest and/or after storage. We have analysed 127 apple cultivars for allelic composition in two key genes that are directly involved in the ethylene biosynthesis pathway, Md-ACS1 and Md-ACO1, and two other genes that are involved in cell wall degradation, Md-Exp7 and Md-PG1. Firmness was measured with a penetrometer at harvest and after 6 or 12 weeks (early- and late-maturing cultivars, respectively) of cold storage. Maturation time was positively correlated with firmness at harvest and negatively correlated with fruit softening rate (difference between firmness at harvest and after storage, divided by number of weeks in storage). Polyploid cultivars showed significantly higher firmness at harvest compared to diploids. Alleles previously described as responsible for good texture were associated with significantly lower softening for Md-ACS1 and Md-PG1, but the opposite was noted for Md-EXP7. Results were nonsignificant for Md-ACO1. Allele frequencies were very uneven in all four loci, with the three most common multi-locus configurations accounting for 64 % of the entire material. The predictive power of these genes was calculated with a partial least squares discriminant analysis, and these accounted for 15 % of the observed variation in initial firmness and 18 % for softening rate. Inclusion of maturation time, storage time (i.e. 6 or 12 weeks) and initial firmness into the model however increased the predictability of softening rate to 38 %. Dividing the material in modern (released after 1960) and old cultivars did not change the outcome of our analyses.     

<Emphasis Type="Italic">PpVIN2</Emphasis>, an acid invertase gene family member,is sensitive to chilling temperature and affects sucrose metabolism in postharvest peach fruit

We previously reported that expression and activity of acid invertases (AI) are increased in peach fruit under chilling stress. In order to determine which AI genes respond to chilling stress, seven AI genes, two vacuolar invertases (VINs) and five cell wall-bound invertases (CWINs), were identified and cloned. The predicted amino acid sequences of the genes contain conserved sites characteristic of plant AIs such as NDPNG/A, the sucrose-binding site, and MWECV/P, a cysteine catalytic motif. Using gene-specific primers, the expression of each gene was measured in ‘Baifeng’ and ‘Yulu’ peach fruits stored at 0, 5, 10 and 20 °C. Of the seven genes, expression of PpVIN2 was the most affected by chilling stress; the largest increases were in fruit stored at 5 °C, up to 17-fold in ‘Baifeng’ fruit, and up to 280-fold in ‘Yulu’ fruit. Overall, VIN activity was much higher than CWIN activity in stored peach fruit. In both cultivars reducing sugar content increased significantly and sucrose content decreased gradually during storage at 5 °C relative to other temperatures, and was accompanied by severe chilling injury symptoms. Thus, PpVIN2 appears to be induced by chilling and may play an important role in sucrose metabolism in peach fruit subjected to cold storage.     

Deciphering the metabolic pathways influencing heat and cold responses during post‐harvest physiology of peach fruit

Peaches are highly perishable and deteriorate quickly at ambient temperature. Cold storage is commonly used to prevent fruit decay; however, it affects fruit quality causing physiological disorders collectively termed ‘chilling injury’ (CI). To prevent or ameliorate CI, heat treatment is often applied prior to cold storage. In the present work, metabolic profiling was performed to determine the metabolic dynamics associated with the induction of acquired CI tolerance in response to heat shock. ‘Dixiland’ peach fruits exposed to 39 °C, cold stored, or after a combined treatment of heat and cold, were compared with fruits ripening at 20 °C. Dramatic changes in the levels of compatible solutes such as galactinol and raffinose were observed, while amino acid precursors of the phenylpropanoid pathway were also modified due to the stress treatments, as was the polyamine putrescine. The observed responses towards temperature stress in peaches are composed of both common and specific response mechanisms to heat and cold, but also of more general adaptive responses that confer strategic advantages in adverse conditions such as biotic stresses. The identification of such key metabolites, which prime the fruit to cope with different stress situations, will likely greatly accelerate the design and the improvement of plant breeding programs.     

Genome-wide identification and expression analysis of the lipoxygenase gene family during peach fruit ripening under different postharvest treatments

In plants, lipoxygenase (LOX), facilitated by the LOX family genes is closely related to fruit ripening and senescence, but research on LOX in peach fruit is limited. To study the roles of LOX family genes in fruit ripening during storage, a comprehensive overview of the LOX gene family in peach is presented, including their phylogenetic relationships, gene structures and subcellular localizations. Additionally, the fruit quality, including fruit firmness, ethylene production and soluble solids content under different storage conditions, were assessed. Finally, 12 peach genes that encode LOX proteins have been identified, and comparisons of the PpaLOX gene expression levels under different postharvest treatments in peach fruit suggest that PpaLOX2.1, PpaLOX7.1, PpaLOX7.2, and especially PpaLOX2.2, may be required in peach fruit ripening during storage. The results will be useful to further analyze the functions of the LOX family of genes in plants.     

Postharvest temperature influences volatile lactone production via regulation of acyl-CoA oxidases in peach fruit

The biosynthesis of volatile compounds in plants is affected by environmental conditions. Lactones are considered to be peach‐like aroma volatiles; however, no enzymes or genes associated with their biosynthesis have been characterized. White‐fleshed (cv. Hujingmilu) and yellow‐fleshed (cv. Jinxiu) melting peach (Prunus persica L. Batsch) fruit were used as materials in two successive seasons and responses measured to four different temperature treatments. Five major lactones accumulated during postharvest peach fruit ripening at 20 °C. Peach fruit at 5 °C, which induces chilling injury (CI), had the lowest lactone content during subsequent shelf life after removal, while 0 °C and a low‐temperature conditioning (LTC) treatment alleviated development of CI and maintained significantly higher lactone contents. Expression of PpACX1 and activity of acyl‐CoA oxidase (ACX) with C16‐CoA tended to increase during postharvest ripening both at 20 °C and during shelf life after removal from cold storage when no CI was developed. There was a positive correlation between ACX and lactones in peach fruit postharvest. Changes in lactone production in response to temperatures are suggested to be a consequence of altered expression of PpACX1 and long‐chain ACX activity.     

Regulation of the expression of lipoxygenase genes in <Emphasis Type="Italic">Prunus persica</Emphasis> fruit ripening

Three genes of the lipoxygenase (LOX) family in peach (Prunus persica var. compressa cv. Ruipan 4) were cloned, and their expression patterns during fruit ripening were analyzed using real-time quantitative PCR. All of the three peach LOX genes had been expressed during fruit ripening; however, their expression patterns were significantly different. During the normal ripening of peach fruits, the expression levels of PpLox1, PpLox2 and PpLox3 increased in varying degrees accompanying upsurge of ethylene evolution. After treated by methyl jasmonic acid (MeJA), the peak of ethylene releasing occurred in advance, and the declining rate of fruit hardness was accelerated, the expression level of the three peach LOX genes in fruits markedly enhanced at the early stage of storage, but significantly decreased at the late storage stage. So, it could be suggested that all three LOXs relate to fruit ripening; however, their functions might be different. PpLox1 expression increase along with the upsurge of ethylene evolution in both control and MeJA-treated peach fruits suggested that PpLox1 probably played a major role in the peach fruit ripening. Expression peak of PpLox2 appeared at the 1 DAH (days after harvest) in both control and MeJA-treated peach fruits, while obvious changes in ethylene evolution and fruit hardness was not observed, which suggested that the rise of PpLox2 expression can be induced by certain stimulation related to ripening, such as harvesting stress and MeJA treatment. The expression of PpLox3 kept a lower level in the natural ripening fruits, whereas raced up at the early stage of storage in the fruits treated with MeJA, which indicated that PpLox3 was expressed inductively and had minor roles during the normal ripening of peach fruits, but when encountered with external stimulation, its expression level would rapidly enhance and accelerate the ripening of peach fruit.     

Application of Genomic and Quantitative Genetic Tools to Identify Candidate Resistance Genes for Brown Rot Resistance in Peach

The availability of a complete peach genome assembly and three different peach genome sequences created by our group provide new opportunities for application of genomic data and can improve the power of the classical Quantitative Trait Loci (QTL) approaches to identify candidate genes for peach disease resistance. Brown rot caused by Monilinia spp., is the most important fungal disease of stone fruits worldwide. Improved levels of peach fruit rot resistance have been identified in some cultivars and advanced selections developed in the UC Davis and USDA breeding programs. Whole genome sequencing of the Pop-DF parents lead to discovery of high-quality SNP markers for QTL genome scanning in this experimental population. Pop-DF created by crossing a brown rot moderately resistant cultivar ‘Dr. Davis’ and a brown rot resistant introgression line, ‘F8,1–42’, derived from an initial almond × peach interspecific hybrid, was evaluated for brown rot resistance in fruit of harvest maturity over three seasons. Using the SNP linkage map of Pop-DF and phenotypic data collected with inoculated fruit, a genome scan for QTL identified several SNP markers associated with brown rot resistance. Two of these QTLs were placed on linkage group 1, covering a large (physical) region on chromosome 1. The genome scan for QTL and SNP effects predicted several candidate genes associated with disease resistance responses in other host-pathogen systems. Two potential candidate genes, ppa011763m and ppa026453m, may be the genes primarily responsible for M. fructicola recognition in peach, activating both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) responses. Our results provide a foundation for further genetic dissection, marker assisted breeding for brown rot resistance, and development of peach cultivars resistant to brown rot.     

Cloning and Characterization of the H Subunit of a Magnesium Chelatase Gene (PpCHLH) in Peach

It has been suggested that the phytohormone abscisic acid (ABA) plays an important role in the ripening of climatic fruit, although relevant genetic/molecular evidence is lacking. In the present study, a peach gene homologous to the putative Arabidopsis ABA receptor gene ABAR/CHLH, named PpCHLH, was isolated and characterized. PpCHLH is expressed ubiquitously as a single-copy gene in peach. Using tobacco rattle virus-induced gene silencing (VIGS), the PpCHLH gene was silenced in both peach leaves and fruit. The silenced PpCHLH gene affected leaf stomatal movement and delayed fruit ripening significantly. Although exogenously applied ABA promoted the ripening of the wild-type fruits, it could not rescue the RNAi chimeric fruit ripening. Collectively, these results demonstrate that PpCHLH plays a critical role in peach fruit ripening, and suggest that ABA might function as an important signal in the regulation of climacteric fruit development.     

Effect of post-harvest heat treatment on proteome change of peach fruit during ripening

The extracted proteins from the heat-treated peach fruit (dipped in hot water at 48°C for 10min and then stored at room temperature (20°C-25°C) for up to 6 days) were used for proteomic analysis in order to understand the response of post-harvest peach fruit to heat treatment during ripening stage at proteomic level. After two dimensional gels electrophoresis (2-DE) was conducted, more than 600 protein spots were detected. Among them, 35 differently expressed spots (P<0.05) were selected to be excised and analyzed using MALDI-TOF/TOF, and finally 30 protein spots were confidently identified according to NCBI database. The results demonstrated that among the thirty protein spots expressed particularly induced by heat treatment, 43% were related to stress response, 17% to cell structure, 13% to protein fate, 7% to glycolytic pathway, 3% to ripening and senescence and 17% to unclassified. All of them are involved in the regulation of peach fruit development and ripening. All these indicated that the self-defense capability of peach fruit was improved by heat treatment. The study will enable future detailed investigation of gene expression and function linked with peach fruit ripening.     

Sources of variation in firmness and ester content of ''Cox'' apples stored in 2% oxygen

The contribution of various factors to variation in the quality of 'Cox' apples after storage in 2% oxygen at 3.3°C was investigated. Within one season variation in the firmness of fruits from different orchards could be largely accounted for by correlations with firmness at harvest and the position of the fruit on the climacteric at the time of harvest. However, different relationships were found between these variables in different seasons. The butyl and hexyl acetate contents of fruit were influenced by season and source of fruit, but little affected by maturity at harvest. Ester content was inversely correlated with fruit firmness after storage. Other factors which were expected to influence the ripening of fruit during storage were found to be unrelated to firmness and ester content. These included the respiration rate of fruit during storage, the resistance of the fruit to gaseous diffusion and its specific gravity. Carbon dioxide production was nearly constant at about 2.3 ml kg^-1h^-1 across seasons, sources and harvest dates. Specific gravity seemed to be particularly affected by season, whereas diffusive resistance decreased with maturity.     

Preconditioning treatment induces chilling tolerance in zucchini fruit improving different physiological mechanisms against cold injury

Zucchini fruit is susceptible to develop chilling injuries (CI) when stored at low temperature. In this study, the effects of a preconditioning treatment during cold storage and its relation with the physiological response to chilling tolerance have been investigated. The commercial variety Sinatra, whose fruit are very sensitive to cold storage, has been used. After harvest, fruit were kept at 4°C or preconditioned during 48 h at 15°C before cold storage. Weight loss, electrolyte leakage and lipid peroxidation were lower in preconditioned at the end of storage time, and CI index was significantly reduced in preconditioned compared to control fruit. The preconditioning treatment improved the energy status of the fruit increasing the pool of ATP, and maintaining the energy charge. The preconditioned fruit improved their antioxidant status with lower H₂O₂ content and induction of ascorbate peroxidase (APX) and catalase (CAT) activities. A reduction in putrescine was detected in preconditioned fruit along with a lower expression of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) and a rise in activity of diamine oxidase (DAO). The concentrations of glutamate and γ‐aminobutyrate (GABA) were lower during preconditioning, while that of proline was higher. In summary, preconditioning treatment induces chilling tolerance in zucchini fruit triggering a defence‐response against oxidative stress and increasing ATP pool and proline content.