Generation of fruit postharvest gene datasets and a novel motif analysis tool for functional studies: uncovering links between peach fruit heat treatment and cold storage responses.

MAIN CONCLUSION: A survey of developed fruit gene-specific datasets and the implementation of a novel cis-element analysis tool indicate specific transcription factors as novel regulatory actors under HT response and CI protection. Heat treatment (HT) prior to cold storage (CS) has been successfully applied to ameliorate fruit chilling injury (CI) disorders. Molecular studies have identified several HT-driven benefits and putative CI-protective molecules and mechanisms. However, bioinformatic tools and analyses able to integrate fruit-specific information are necessary to begin functional studies and breeding projects. In this work, a HT-responsive gene dataset (HTds) and four fruit expression datasets (FEds), containing gene-specific information from several species and postharvest conditions, were developed and characterized. FEds provided information about HT-responsive genes, not only validating their sensitivity to HT in different systems but also revealing most of them as CS-responsive. A special focus was given to peach heat treatment-sensitive transcriptional regulation by the development of a novel Perl motif analysis software (cisAnalyzer) and a curated plant cis-elements dataset (PASPds). cisAnalyzer is able to assess sequence motifs presence, localization, enrichment and discovery on biological sequences. Its implementation for the enrichment analysis of PASPds motifs on the promoters of HTds genes rendered particular cis-elements that indicate certain transcription factor (TF) families as responsible of fruit HT-sensitive transcription regulation. Phylogenetic and postharvest expression data of these TFs showed a functional diversity of TF families, with members able to fulfil roles under HT, CS and/or both treatments. All integrated datasets and cisAnalyzer tool were deposited in FruitGeneDB (https://www.cefobi-conicet.gov.ar/FruitGeneDB/search1.php), a new available database with a great potential for fruit gene functional studies, including the markers of HT and CS responses whose study will contribute to unravel HT-driven CI-protection and select tolerant cultivars.

Peach Fruit Development: A Comparative Proteomic Study Between Endocarp and Mesocarp at Very Early Stages Underpins the Main Differential Biochemical Processes Between These Tissues.

Peach (Prunus persica) is an important economically temperate fruit. The development follows double sigmoid curve with four phases (S1-S4). We centered our work in the early development. In addition to S1, we studied the very early stage (E) characterized by the lag zone of the exponential growing phase S1, and the second stage (S2) when the pit starts hardening. "Dixiland" peach fruit were collected at 9 (E), 29 (S1), and 53 (S2) days after flowering (DAF) and endocarp and mesocarp were separated. There was a pronounced decrease in total protein content along development in both tissues. Quantitative proteomic allowed the identification of changes in protein profiles across development and revealed the main biochemical pathways sustaining tissue differentiation. Protein metabolism was the category most represented among differentially proteins in all tissues and stages. The decrease in protein synthesis machinery observed during development would be responsible of the protein fall, rather than a proteolytic process; and reduced protein synthesis during early development would reroute cell resources to lignin biosynthesis. These changes were accompanied by net decrease in total amino acids in E1-S1 and increase in S1-S2 transitions. Amino acid profiling, showed Asn parallels this trend. Concerted changes in Asn and in enzymes involved in its metabolism reveal that increased synthesis and decreased catabolism of Asn may conduct to an Asn increase during very early development and that the ß-Cyano-Alanine synthase/ß-Cyano-Alanine hydratase could be the pathway for Asn synthesis in "Dixiland" peach fruit. Additionally, photosynthetic machinery decays during early development in mesocarp and endocarp. Proteins related to photosynthesis are found to a higher extent in mesocarp than in endocarp. We conclude mesocarpic photosynthesis is possible to occur early on the development, first providing both carbon and reductive power and latter only reductive power. Together with proteomic, histological tests and anatomical analysis help to provide information about changes and differences in cells and cell-walls in both tissues. Collectively, this work represents the first approach in building protein databases during peach fruit development focusing on endocarp and mesocarp tissues and provides novel insights into the biology of peach fruit development preceding pit hardening.

Differential lipidome remodeling during postharvest of peach varieties with different susceptibility to chilling injury.

Peaches ripen and deteriorate rapidly at room temperature. Therefore, refrigeration is used to slow these processes and to extend fruit market life; however, many fruits develop chilling injury (CI) during storage at low temperature. Given that cell membranes are likely sites of the primary effects of chilling, the lipidome of six peach varieties with different susceptibility to CI was analyzed under different postharvest conditions. By using liquid chromatography coupled to mass spectrometry (LC-MS), 59 lipid species were detected, including diacyl- and triacylglycerides. The decreases in fruit firmness during postharvest ripening were accompanied by changes in the relative amount of several plastidic glycerolipid and triacylglyceride species, which may indicate their use as fuels prior to fruit senescence. In addition, levels of galactolipids were also modified in fruits stored at 0°C for short and long periods, reflecting the stabilization of plastidic membranes at low temperature. When comparing susceptible and resistant varieties, the relative abundance of certain species of the lipid classes phosphatidylethanolamine, phosphatidylcholine and digalactosyldiacylglycerol correlated with the tolerance to CI, reflecting the importance of the plasma membrane in the development of CI symptoms and allowing the identification of possible lipid markers for chilling resistance. Finally, transcriptional analysis of genes involved in galactolipid metabolism revealed candidate genes responsible for the observed changes after cold exposure. When taken together, our results highlight the importance of plastids in the postharvest physiology of fruits and provide evidence that lipid composition and metabolism have a profound influence on the cold response.

Comparative proteomic and metabolomic studies between Prunus persica genotypes resistant and susceptible to Taphrina deformans suggest a molecular basis of resistance.

The worldwide-distributed leaf peach curl disease is caused by the biotroph Taphrina deformans. To characterize the plant-fungus interaction, resistant and susceptible Prunus persica genotypes grown in the orchard were studied. Asymptomatic leaves were tested for fungal presence. In all resistant leaves analyzed the fungus was not detected. Conversely, leaves from the susceptible genotype were categorized according to the presence or absence of the pathogen. Comparative metabolomic analysis disclosed the metabolite composition associated with resistant and susceptible interactions, and of compounds involved in fungal growth inhibition such as chlorogenic acid, whose in vitro antifungal activity was verified in this work. Differential proteome studies revealed that chloroplasts are important site of plant defense responses against T. deformans. Members of the Bet-v1-like family protein differentially responded to the pathogen. Extracellular pathogenesis-related proteins, evaluated by qRT-PCR, and an enone oxidoreductase are constitutively present in leaves of resistant trees and could be related to fungal resistance. This study is a global view of the changes in the metabolome, proteome and transcripts related to plant defense in naturally infected leaves of susceptible plants during the asymptomatic stage. Additionally, it provides clues to the successful molecular mechanisms operating in resistant plants, which neither develop the disease nor harbor the pathogen.

Unravelling early events in the Taphrina deformans-Prunus persica interaction: an insight into the differential responses in resistant and susceptible genotypes.

Leaf peach curl is a devastating disease affecting leaves, flowers and fruits, caused by the dimorphic fungus Taphrina deformans. To gain insight into the mechanisms of fungus pathogenesis and plant responses, leaves of a resistant and two susceptible Prunus persica genotypes were inoculated with blastospores (yeast), and the infection was monitored during 120 h post inoculation (h.p.i.). Fungal dimorphism to the filamentous form and induction of reactive oxygen species (ROS), callose synthesis, cell death and defence compound production were observed independently of the genotype. Fungal load significantly decreased after 120 h.p.i. in the resistant genotype, while the pathogen tended to grow in the susceptible genotypes. Metabolic profiling revealed a biphasic re-programming of plant tissue in susceptible genotypes, with an initial stage co-incident with the yeast form of the fungus and a second when the hypha is developed. Transcriptional analysis of PRs and plant hormone-related genes indicated that pathogenesis-related (PR) proteins are involved in P. persica defence responses against T. deformans and that salicylic acid is induced in the resistant genotype. Conducted experiments allowed the elucidation of common and differential responses in susceptible versus resistant genotypes and thus allow us to construct a picture of early events during T. deformans infection.

Differential Metabolic Rearrangements after Cold Storage Are Correlated with Chilling Injury Resistance of Peach Fruits.

Reconfiguration of the metabolome is a key component involved in the acclimation to cold in plants; however, few studies have been devoted to the analysis of the overall metabolite changes after cold storage of fruits prior to consumption. Here, metabolite profiling of six peach varieties with differential susceptibility to develop mealiness, a chilling-injury (CI) symptom, was performed. According to metabolic content at harvest; after cold treatment; and after ripening, either following cold treatment or not; peach fruits clustered in distinct groups, depending on harvest-time, cold treatment, and ripening state. Both common and distinct metabolic responses among the six varieties were found; common changes including dramatic galactinol and raffinose rise; GABA, Asp, and Phe increase; and 2-oxo-glutarate and succinate decrease. Raffinose content after long cold treatment quantitatively correlated to the degree of mealiness resistance of the different peach varieties; and thus, raffinose emerges as a candidate biomarker of this CI disorder. Xylose increase after cold treatment was found only in the susceptible genotypes, indicating a particular cell wall reconfiguration of these varieties while being cold-stored. Overall, results indicate that peach fruit differential metabolic rearrangements due to cold treatment, rather than differential metabolic priming before cold, are better related with CI resistance. The plasticity of peach fruit metabolism renders it possible to induce a diverse metabolite array after cold, which is successful, in some genotypes, to avoid CI.

Cell wall-related genes studies on peach cultivars with differential susceptibility to woolliness: looking for candidates as indicators of chilling tolerance.

KEY MESSAGE: The results obtained indicate that a ß-xylosidase gene may act as good indicator of chilling tolerance and provide new insights into the complex issue of peach fruit woolliness. The storage of peaches at low temperatures for prolonged periods can induce a form of chilling injury (CI) called woolliness, characterized by a lack of juiciness and a mealy texture. As this disorder has been associated with abnormal cell wall dismantling, the levels of 12 transcripts encoding proteins involved in cell wall metabolism were analysed in cultivars with contrasting susceptibility to this disorder selected from five melting flesh peach cultivars. The resistant ('Springlady') and susceptible ('Flordaking') cultivars displayed differences in the level of expression of some of the selected genes during fruit softening and in woolly versus non-woolly fruits. From these genes, the level of expression of PpXyl, which encodes for a putative ß-xylosidase, was the one that presented the highest correlation (negative) with the susceptibility to woolliness. PpXyl expression was also analysed in a cultivar ('Rojo 2') with intermediate susceptibility to woolliness, reinforcing the conclusion about the correlation of PpXyl expression to the presence of woolliness symptom. Moreover, the level of expression of PpXyl correlated to protein level detected by Western blot. Analyses of the promoter region of the PpXyl gene (1637 bp) isolated from the three cultivars showed no differences suggesting that cis-elements from other regions of the genome and/or trans elements could be responsible of the differential PpXyl expression patterns. Overall, the results obtained indicate that PpXyl may act as a good indicator of woolliness tolerance and that the regulation of expression of this gene in different cultivars does not depend on sequences upstream the coding sequence.

Metabolic profiling of a range of peach fruit varieties reveals high metabolic diversity and commonalities and differences during ripening.

Peach (Prunus persica) fruits from different varieties display differential organoleptic and nutritional properties, characteristics related to their chemical composition. Here, chemical biodiversity of peach fruits from fifteen varieties, at harvest and after post-harvest ripening, was explored by gas chromatography-mass spectrometry. Metabolic profiling revealed that metabolites involved in organoleptic properties (sugars, organic and amino acids), stress tolerance (raffinose, galactinol, maltitol), and with nutritional properties (amino, caffeoylquinic and dehydroascorbic acids) displayed variety-dependent levels. Peach varieties clustered into four groups: two groups of early-harvest varieties with higher amino acid levels; two groups of mid- and late-harvest varieties with higher maltose levels. Further separation was mostly dependent on organic acids/raffinose levels. Variety-dependent and independent metabolic changes associated with ripening were detected; which contribute to chemical diversity or can be used as ripening markers, respectively. The great variety-dependent diversity in the content of metabolites that define fruit quality reinforces metabolomics usage as a tool to assist fruit quality improvement in peach.

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.

Transcriptomic profiling during the post-harvest of heat-treated Dixiland Prunus persica fruits: common and distinct response to heat and cold.

Cold storage is extensively used to slow the rapid deterioration of peach (Prunus persica L. Batsch) fruit after harvest. However, peach fruit subjected to long periods of cold storage develop chilling injury (CI) symptoms. Post-harvest heat treatment (HT) of peach fruit prior to cold storage is effective in reducing some CI symptoms, maintaining fruit quality, preventing softening and controlling post-harvest diseases. To identify the molecular changes induced by HT, which may be associated to CI protection, the differential transcriptome of peach fruit subjected to HT was characterized by the differential display technique. A total of 127 differentially expressed unigenes (DEUs), with a presence-absence pattern, were identified comparing peach fruit ripening at 20°C with those exposed to a 39°C-HT for 3 days. The 127 DEUs were divided into four expression profile clusters, among which the heat-induced (47%) and heat-repressed (36%) groups resulted the most represented, including genes with unknown function, or involved in protein modification, transcription or RNA metabolism. Considering the CI-protection induced by HT, 23-heat-responsive genes were selected and analyzed during and after short-term cold storage of peach fruit. More than 90% of the genes selected resulted modified by cold, from which nearly 60% followed the same and nearly 40% opposite response to heat and cold. Moreover, by using available Arabidopsis microarray data, it was found that nearly 70% of the peach-heat responsive genes also respond to cold in Arabidopsis, either following the same trend or showing an opposite response. Overall, the high number of common responsive genes to heat and cold identified in the present work indicates that HT of peach fruit after harvest induces a cold response involving complex cellular processes; identifying genes that are involved in the better preparation of peach fruit for cold-storage and unraveling the basis for the CI protection induced by HT.

Heat treatment of peach fruit: modifications in the extracellular compartment and identification of novel extracellular proteins.

Ripening of peach (Prunus persica L. Batsch) fruit is accompanied by dramatic cell wall changes that lead to softening. Post-harvest heat treatment is effective in delaying softening and preventing some chilling injury symptoms that this fruit exhibits after storage at low temperatures. In the present work, the levels of twelve transcripts encoding proteins involved in cell wall metabolism, as well as the differential extracellular proteome, were examined after a post-harvest heat treatment (HT; 39 °C for 3 days) of "Dixiland" peach fruit. A typical softening behaviour, in correlation with an increase in 1-aminocyclopropane-1-carboxylic acid oxidase-1 (PpACO1), was observed for peach maintained at 20 °C for 3 days (R3). Six transcripts encoding proteins involved in cell wall metabolism significantly increased in R3 with respect to peach at harvest, while six showed no modification or even decreased. In contrast, after HT, fruit maintained their firmness, exhibiting low PpACO1 level and significant lower levels of the twelve cell wall-modifying genes than in R3. Differential proteomic analysis of apoplastic proteins during softening and after HT revealed a significant decrease of DUF642 proteins after HT; as well as an increase of glyceraldehyde-3-phosphate dehydrogenase (GAPC) after softening. The presence of GAPC in the peach extracellular matrix was further confirmed by in situ immunolocalization and transient expression in tomato fruit. Though further studies are required to establish the function of DUF642 and GAPC in the apoplast, this study contributes to a deeper understanding of the events during peach softening and after HT with a focus on this key compartment.

Metabolic profiling during peach fruit development and ripening reveals the metabolic networks that underpin each developmental stage.

Fruit from rosaceous species collectively display a great variety of flavors and textures as well as a generally high content of nutritionally beneficial metabolites. However, relatively little analysis of metabolic networks in rosaceous fruit has been reported. Among rosaceous species, peach (Prunus persica) has stone fruits composed of a juicy mesocarp and lignified endocarp. Here, peach mesocarp metabolic networks were studied across development using metabolomics and analysis of key regulatory enzymes. Principal component analysis of peach metabolic composition revealed clear metabolic shifts from early through late development stages and subsequently during postharvest ripening. Early developmental stages were characterized by a substantial decrease in protein abundance and high levels of bioactive polyphenols and amino acids, which are substrates for the phenylpropanoid and lignin pathways during stone hardening. Sucrose levels showed a large increase during development, reflecting translocation from the leaf, while the importance of galactinol and raffinose is also inferred. Our study further suggests that posttranscriptional mechanisms are key for metabolic regulation at early stages. In contrast to early developmental stages, a decrease in amino acid levels is coupled to an induction of transcripts encoding amino acid and organic acid catabolic enzymes during ripening. These data are consistent with the mobilization of amino acids to support respiration. In addition, sucrose cycling, suggested by the parallel increase of transcripts encoding sucrose degradative and synthetic enzymes, appears to operate during postharvest ripening. When taken together, these data highlight singular metabolic programs for peach development and may allow the identification of key factors related to agronomic traits of this important crop species.

Effect of ethylene and 1-MCP treatments on strawberry fruit ripening.

BACKGROUND: Strawberry is a soft fruit, considered as non-climacteric, being auxins the main hormones that regulate the ripening process. The role of ethylene in strawberry ripening is currently unclear and several studies have considered a revision of the possible role of this hormone. RESULTS: Strawberry fruit were harvested at the white stage and treated with ethephon, an ethylene-releasing reagent, or 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. The effects of the treatments on fruit quality parameters and on the activity of enzymes related to anthocyanin synthesis and cell wall degradation were evaluated. Some aspects of ripening were accelerated (anthocyanin accumulation, total sugar content and increment of phenylalanine ammonia-lyase (PAL; EC 4.3.1.24) and beta-galactosidase (EC 3.2.1.23) activities), while others were repressed (chlorophyll levels and increment of endo-1,4-beta-glucanase (EC 3.2.1.4) and beta-xylosidase (EC 3.2.1.37) activities) or unchanged (reducing sugar content, pH, titratable acidity and alpha-L-arabinofuranosidase (EC 3.2.1.55) activity) by ethylene. 1-MCP treatment caused the opposite effect. However, its effects were more pronounced, particularly in anthocyanin accumulation, phenolics, PAL and polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activities. CONCLUSION: These observations probably indicate that strawberry produces low levels of ethylene that are sufficient to regulate some ripening aspects.

Involvement of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in response to oxidative stress.

Glyceraldehyde-3-phosphate dehydrogenases catalyze key steps in energy and reducing power partitioning in cells of higher plants. Because non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (NP-Ga3PDHase) is involved in the production of reductive power (NADPH) in the cytosol, its behavior under oxidative stress conditions was analyzed. The specific activity of the enzyme was found to increase up to 2-fold after oxidative conditions imposed by methylviologen in wheat and maize seedlings. Under moderate oxidant concentration, lack of mRNA induction was observed. The increase in specific activity would thus be a consequence of a significant stability of NP-Ga3PDHase. Our results suggest that the enzyme could be modified by oxidation of cysteine residues, but formation of disulfide bridges is dependent on levels of divalent cations and 14-3-3 proteins. The latter differential effect could be critical to relatively maintain energy and reductant levels in the cytoplasm of plant cells under oxidative stress.

ß-Xylosidase in strawberry fruit: Isolation of a full-length gene and analysis of its expression and enzymatic activity in cultivars with contrasting firmness.

Strawberry is a non-climateric fleshy fruit, which softens quickly and has short post-harvest life. Ripening is associated with an increment of pectin solubility and a reduction of the content of hemicelluloses. In this work, we have cloned the full-length cDNA encoding a ß-xylosidase (FaXyl1) from Fragaria×ananassa and we have characterized its expression in two strawberry cultivars with contrasting fruit firmness. The analysis of the predicted protein showed that FaXyl1 is closely related to other ß-xylosidases from higher plants. The recombinant protein obtained by over-expressing FaXyl1 in Escherichia coli had ß-xylosidase activity against the artificial substrate p-nitrophenyl ß-d-xilopyranoside. Differently from other bifunctional xylosidases, no α-l-arabinofuranosidase activity was detected in the recombinant enzyme. The expression of FaXyl1 gene was analyzed by northern-blot in Camarosa and Toyonaka strawberry cultivars, and compared with the corresponding protein data obtained by Western-blot and with the ß-xylosidase activity during ripening. The softest cultivar (Toyonaka) showed an early accumulation of FaXyl1 transcript and a higher expression of the corresponding protein during ripening, which correlates with a higher ß-xylosidase activity in all ripening stages analyzed.