A gibberellin-assisted study of the transcriptional and hormonal changes occurring at floral transition in peach buds (Prunus persica L. Batsch).

BACKGROUND: Flower load in peach is an important determinant of final fruit quality and is subjected to cost-effective agronomical practices, such as the thinning, to finely balance the sink-source relationships within the tree and drive the optimal amount of assimilates to the fruits. Floral transition in peach buds occurs as a result of the integration of specific environmental signals, such as light and temperature, into the endogenous pathways that induce the meristem to pass from vegetative to reproductive growth. The cross talk and integration of the different players, such as the genes and the hormones, are still partially unknown. In the present research, transcriptomics and hormone profiling were applied on bud samples at different developmental stages. A gibberellin treatment was used as a tool to identify the different phases of floral transition and characterize the bud sensitivity to gibberellins in terms of inhibition of floral transition. RESULTS: Treatments with gibberellins showed different efficacies and pointed out a timeframe of maximum inhibition of floral transition in peach buds. Contextually, APETALA1 gene expression was shown to be a reliable marker of gibberellin efficacy in controlling this process. RNA-Seq transcriptomic analyses allowed to identify specific genes dealing with ROS, cell cycle, T6P, floral induction control and other processes, which are correlated with the bud sensitivity to gibberellins and possibly involved in bud development during its transition to the reproductive stage. Transcriptomic data integrated with the quantification of the main bioactive hormones in the bud allowed to identify the main hormonal regulators of floral transition in peach, with a pivotal role played by endogenous gibberellins and cytokinins. CONCLUSIONS: The peach bud undergoes different levels of receptivity to gibberellin inhibition. The stage with maximum responsiveness corresponded to a transcriptional and hormonal crossroad, involving both flowering inhibitors and inductors. Endogenous gibberellin levels increased only at the latest developmental stage, when floral transition was already partially achieved, and the bud was less sensitive to exogenous treatments. A physiological model summarizes the main findings and suggests new research ideas to improve our knowledge about floral transition in peach.

Metabolic and Molecular Rearrangements of Sauvignon Blanc (Vitis vinifera L.) Berries in Response to Foliar Applications of Specific Dry Yeast.

Dry yeast extracts (DYE) are applied to vineyards to improve aromatic and secondary metabolic compound content and wine quality; however, systematic information on the underpinning molecular mechanisms is lacking. This work aimed to unravel, through a systematic approach, the metabolic and molecular responses of Sauvignon Blanc berries to DYE treatments. To accomplish this, DYE spraying was performed in a commercial vineyard for two consecutive years. Berries were sampled at several time points after the treatment, and grapes were analyzed for sugars, acidity, free and bound aroma precursors, amino acids, and targeted and untargeted RNA-Seq transcriptional profiles. The results obtained indicated that the DYE treatment did not interfere with the technological ripening parameters of sugars and acidity. Some aroma precursors, including cys-3MH and GSH-3MH, responsible for the typical aromatic nuances of Sauvignon Blanc, were stimulated by the treatment during both vintages. The levels of amino acids and the global RNA-seq transcriptional profiles indicated that DYE spraying upregulated ROS homeostatic and thermotolerance genes, as well as ethylene and jasmonic acid biosynthetic genes, and activated abiotic and biotic stress responses. Overall, the data suggested that the DYE reduced berry oxidative stress through the regulation of specific subsets of metabolic and hormonal pathways.

Dissecting postharvest chilling injuries in pome and stone fruit through integrated omics.

Lowering the storage temperature is an effective method to extend the postharvest and shelf life of fruits. Nevertheless, this technique often leads to physiological disorders, commonly known as chilling injuries. Apples and pears are susceptible to chilling injuries, among which superficial scald is the most economically relevant. Superficial scald is due to necrotic lesions of the first layers of hypodermis manifested through skin browning. In peaches and nectarines, chilling injuries are characterized by internal symptoms, such as mealiness. Fruits with these aesthetic or compositional/structural defects are not suitable for fresh consumption. Genetic variation is a key factor in determining fruit susceptibility to chilling injuries; however, physiological, or technical aspects such as harvest maturity and storage conditions also play a role. Multi-omics approaches have been used to provide an integrated explanation of chilling injury development. Metabolomics in pome fruits specifically targets the identification of ethylene, phenols, lipids, and oxidation products. Genomics and transcriptomics have revealed interesting connections with metabolomic datasets, pinpointing specific genes linked to cold stress, wax synthesis, farnesene metabolism, and the metabolic pathways of ascorbate and glutathione. When applied to Prunus species, these cutting-edge approaches have uncovered that the development of mealiness symptoms is linked to ethylene signaling, cell wall synthesis, lipid metabolism, cold stress genes, and increased DNA methylation levels. Emphasizing the findings from multi-omics studies, this review reports how the integration of omics datasets can provide new insights into understanding of chilling injury development. This new information is essential for successfully creating more resilient fruit varieties and developing novel postharvest strategies.

Grape Berry Responses to Sequential Flooding and Heatwave Events: A Physiological, Transcriptional, and Metabolic Overview.

Grapevine cultivation, such as the whole horticulture, is currently challenged by several factors, among which the extreme weather events occurring under the climate change scenario are the most relevant. Within this context, the present study aims at characterizing at the berry level the physiological response of Vitis vinifera cv. Sauvignon Blanc to sequential stresses simulated under a semi-controlled environment: flooding at bud-break followed by multiple summer stress (drought plus heatwave) occurring at pre-vèraison. Transcriptomic and metabolomic assessments were performed through RNASeq and NMR, respectively. A comprehensive hormone profiling was also carried out. Results pointed out a different response to the heatwave in the two situations. Flooding caused a developmental advance, determining a different physiological background in the berry, thus affecting its response to the summer stress at both transcriptional levels, with the upregulation of genes involved in oxidative stress responses, and metabolic level, with the increase in osmoprotectants, such as proline and other amino acids. In conclusion, sequential stress, including a flooding event at bud-break followed by a summer heatwave, may impact phenological development and berry ripening, with possible consequences on berry and wine quality. A berry physiological model is presented that may support the development of sustainable vineyard management solutions to improve the water use efficiency and adaptation capacity of actual viticultural systems to future scenarios.

Transcriptomic Insights on the Preventive Action of Apple (cv Granny Smith) Skin Wounding on Superficial Scald Development.

Superficial scald is a post-harvest chilling storage injury leading to browning of the surface of the susceptible cv Granny Smith apples. Wounding of skins has been reported to play a preventive role on scald development however its underlying molecular factors are unknown. We have artificially wounded the epidermal and sub-epidermal layers of apple skins consistently obtaining the prevention of superficial scald in the surroundings of the wounds during two independent vintages. Time course RNA-Seq analyses of the transcriptional changes in wounded versus unwounded skins revealed that two transcriptional waves occurred. An early wave included genes up-regulated by wounding already after 6 h, highlighting a specific transcriptional rearrangement of genes connected to the biosynthesis and signalling of JA, ethylene and ABA. A later transcriptional wave, occurring after three months of cold storage, included genes up-regulated exclusively in unwounded skins and was prevented from its occurrence in wounded skins. A significant portion of these genes was related to decay of tissues and to the senescence hormones ABA, JA and ethylene. Such changes suggest a wound-inducible reversed hormonal balance during post-harvest storage which may explain the local inhibition of scald in wounded tissues, an aspect that will need further studies for its mechanistic explanation.

Rosaceae Fruit Development, Ripening and Post-harvest: An Epigenetic Perspective.

Rosaceae is a family with an extraordinary spectrum of fruit types, including fleshy peach, apple, and strawberry that provide unique contributions to a healthy diet for consumers, and represent an excellent model for studying fruit patterning and development. In recent years, many efforts have been made to unravel regulatory mechanism underlying the hormonal, transcriptomic, proteomic and metabolomic changes occurring during Rosaceae fruit development. More recently, several studies on fleshy (tomato) and dry (Arabidopsis) fruit model have contributed to a better understanding of epigenetic mechanisms underlying important heritable crop traits, such as ripening and stress response. In this context and summing up the results obtained so far, this review aims to collect the available information on epigenetic mechanisms that may provide an additional level in gene transcription regulation, thus influencing and driving the entire Rosaceae fruit developmental process. The whole body of information suggests that Rosaceae fruit could become also a model for studying the epigenetic basis of economically important phenotypes, allowing for their more efficient exploitation in plant breeding.

The peach HECATE3-like gene FLESHY plays a double role during fruit development.

Tight control of cell/tissue identity is essential for a correct and functional organ patterning, an important component of overall fruit development and eventual maturation and ripening. Despite many investigations regarding the molecular determinants of cell identity in fruits of different species, a useful model able to depict the regulatory networks governing this relevant part of fruit development is still missing. Here we described the peach fruit as a system to link the phenotype of a slow ripening (SR) selection to an altered transcriptional regulation of genes involved in determination of mesocarp cell identity providing insight toward molecular regulation of fruit tissue formation. Morpho-anatomical observations and metabolomics analyses performed during fruit development on the reference cultivar Fantasia, compared to SR, revealed that the mesocarp of SR maintained typical immaturity traits (e.g. small cell size, high amino acid contents and reduced sucrose) throughout development, along with a strong alteration of phenylpropanoid contents, resulting in accumulation of phenylalanine and lignin. These findings suggest that the SR mesocarp is phenotypically similar to a lignifying endocarp. To test this hypothesis, the expression of genes putatively involved in determination of drupe tissues identity was assessed. Among these, the peach HEC3-like gene FLESHY showed a strongly altered expression profile consistent with pit hardening and fruit ripening, generated at a post-transcriptional level. A double function for FLESHY in channelling the phenylpropanoid pathway to either lignin or flavour/aroma is suggested, along with its possible role in triggering auxin-ethylene cross talk at the start of ripening.

The KNOTTED-like genes of peach (Prunus persica L. Batsch) are differentially expressed during drupe growth and the class 1 KNOPE1 contributes to mesocarp development.

The Knotted-like transcription factors (KNOX) contribute to plant organ development. The expression patterns of peach KNOX genes showed that the class 1 members act precociously (S1-S2 stages) and differentially during drupe growth. Specifically, the transcription of KNOPE1 and 6 decreased from early (cell division) to late (cell expansion) S1 sub-stages, whilst that of STMlike1, 2, KNOPE2, 2.1 ceased at early S1. The KNOPE1 role in mesocarp was further addressed by studying the mRNA localization in the pulp cells and vascular net at early and late S1. The message signal was first diffuse in parenchymatous cells and then confined to hypodermal cell layers, showing that the gene down-tuning accompanied cell expansion. As for bundles, the mRNA mainly featured in the procambium/phloem of collateral open types and subsequently in the phloem side of complex structures (converging bundles, ducts). The KNOPE1 overexpression in Arabidopsis caused fruit shortening, decrease of mesocarp cell size, diminution of vascular lignification together with the repression of the major gibberellin synthesis genes AtGA20ox1 and AtGA3ox1. Negative correlation between the expression of KNOPE1 and PpGA3ox1 was observed in four cultivars at S1, suggesting that the KNOPE1 repression of PpGA3ox1 may regulate mesocarp differentiation by acting on gibberellin homeostasis.

Grape berry ripening delay induced by a pre-véraison NAA treatment is paralleled by a shift in the expression pattern of auxin- and ethylene-related genes.

BACKGROUND: Auxins act as repressors of ripening inception in grape (véraison), while ethylene and abscisic acid (ABA) play a positive role as inducers of the syndrome. Despite the increasing amount of information made available on this topic, the complex network of interactions among these hormones remains elusive. In order to shed light on these aspects, a holistic approach was adopted to evaluate, at the transcriptomic level, the crosstalk between hormones in grape berries, whose ripening progression was delayed by applying naphtalenacetic acid (NAA) one week before véraison. RESULTS: The NAA treatment caused significant changes in the transcription rate of about 1,500 genes, indicating that auxin delayed grape berry ripening also at the transcriptional level, along with the recovery of a steady state of its intracellular concentration. Hormone indices analysis carried out with the HORMONOMETER tool suggests that biologically active concentrations of auxins were achieved throughout a homeostatic recovery. This occurred within 7 days after the treatment, during which the physiological response was mainly unspecific and due to a likely pharmacological effect of NAA. This hypothesis is strongly supported by the up-regulation of genes involved in auxin conjugation (GH3-like) and action (IAA4- and IAA31-like). A strong antagonistic effect between auxin and ethylene was also observed, along with a substantial 'synergism' between auxins and ABA, although to a lesser extent. CONCLUSIONS: This study suggests that, in presence of altered levels of auxins, the crosstalk between hormones involves diverse mechanisms, acting at both the hormone response and biosynthesis levels, creating a complex response network.

A microarray approach to identify genes involved in seed-pericarp cross-talk and development in peach.

BACKGROUND: Field observations and a few physiological studies have demonstrated that peach embryogenesis and fruit development are tightly coupled. In fact, attempts to stimulate parthenocarpic fruit development by means of external tools have failed. Moreover, physiological disturbances during early embryo development lead to seed abortion and fruitlet abscission. Later in embryo development, the interactions between seed and fruit development become less strict. As there is limited genetic and molecular information about seed-pericarp cross-talk and development in peach, a massive gene approach based on the use of the µPEACH 1.0 array platform and quantitative real time RT-PCR (qRT-PCR) was used to study this process. RESULTS: A comparative analysis of the transcription profiles conducted in seed and mesocarp (cv Fantasia) throughout different developmental stages (S1, S2, S3 and S4) evidenced that 455 genes are differentially expressed in seed and fruit. Among differentially expressed genes some were validated as markers in two subsequent years and in three different genotypes. Seed markers were a LTP1 (lipid transfer protein), a PR (pathogenesis-related) protein, a prunin and LEA (Late Embryogenesis Abundant) protein, for S1, S2, S3 and S4, respectively. Mesocarp markers were a RD22-like protein, a serin-carboxypeptidase, a senescence related protein and an Aux/IAA, for S1, S2, S3 and S4, respectively.The microarray data, analyzed by using the HORMONOMETER platform, allowed the identification of hormone-responsive genes, some of them putatively involved in seed-pericarp crosstalk. Results indicated that auxin, cytokinins, and gibberellins are good candidates, acting either directly (auxin) or indirectly as signals during early development, when the cross-talk is more active and vital for fruit set, whereas abscisic acid and ethylene may be involved later on. CONCLUSIONS: In this research, genes were identified marking different phases of seed and mesocarp development. The selected genes behaved as good seed markers, while for mesocarp their reliability appeared to be dependent upon developmental and ripening traits. Regarding the cross-talk between seed and pericarp, possible candidate signals were identified among hormones.Further investigations relying upon the availability of whole genome platforms will allow the enrichment of a marker genes repertoire and the elucidation of players other than hormones that are involved in seed-pericarp cross-talk (i.e. hormone peptides and microRNAs).

Transcriptome profiling of ripening nectarine (Prunus persica L. Batsch) fruit treated with 1-MCP.

A large-scale transcriptome analysis has been conducted using microPEACH1.0 microarray on nectarine (Prunus persica L. Batsch) fruit treated with 1-methylcyclopropene (1-MCP). 1-MCP maintained flesh firmness but did not block ethylene biosynthesis. Compared with samples at harvest, only nine genes appeared to be differentially expressed when fruit were sampled immediately after treatment, while a total of 90 targets were up- or down-regulated in untreated fruit. The effect of 1-MCP was confirmed by a direct comparison of transcript profiles in treated and untreated fruit after 24 h of incubation with 106 targets differentially expressed. About 30% of these targets correspond to genes involved in primary metabolism and response processes related to ethylene, auxin, and other hormones. In treated fruit, altered transcript accumulation was detected for some genes with a role in ripening-related events such as softening, colour development, and sugar metabolism. A rapid decrease in flesh firmness and an increase in ethylene production were observed in treated fruit maintained for 48 h in air at 20 degrees C after the end of the incubation period. Microarray comparison of this sample with untreated fruit 24 h after harvest revealed that about 45% of the genes affected by 1-MCP at the end of the incubation period changed their expression during the following 48 h in air. Among these genes, an ethylene receptor (ETR2) and three ethylene-responsive factors (ERF) were present, together with other transcription factors and ethylene-dependent genes involved in quality parameter changes.

Characterization of two putative ethylene receptor genes expressed during peach fruit development and abscission.

Two peach genes homologous to the Arabidopsis ethylene receptor genes ETR1 and ERS1, named Pp-ETR1 and Pp-ERS1 respectively, have been isolated and characterized. Pp-ETR1 and Pp-ERS1 are conserved in terms of exon numbers and intron positions, although the first and fifth introns of Pp-ETR1 have an unusual length. In addition, two putative polyadenylation sites, that may cause an incomplete splicing at the 3' terminus, are present in the fifth intron. A motif of 28 nt, which shows high homology with ethylene responsive elements found in promoters of genes up-regulated by ethylene, is present in the promoter region of Pp-ERS1. Expression analysis, carried out by quantitative RT-PCR, was performed during fruit development and ripening, and leaf and fruitlet abscission. The level of Pp-ETR1 transcripts remained unchanged in all the tissues and developmental stages examined, whereas Pp-ERS1 mRNA abundance increased in ripening mesocarp, in leaf and fruitlet activated abscission zones, and following propylene application. 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, did not affect Pp-ETR1 transcription, while it down-regulated Pp-ERS1. A rise in ethylene evolution, accompanied by an increase of Pp-ERS1 transcript accumulation occurred within 24 h from the end of 1-MCP treatment. These results indicate that Pp-ERS1 might play a role in abscission and ripening.

Characterization and expression of two members of the peach 1-aminocyclopropane-1-carboxylate oxidase gene family.

The characterization and expression of PP-ACO1 and PP-ACO2, two members of the peach 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) gene family, are reported. PP-ACO1 is organized in 4 exons interrupted by 3 introns, whereas PP-ACO2 has only 2 of the 3 introns present in PP-ACO1. Comparison of the deduced amino acid sequences of PP-ACO1 and PP-ACO2 reveals a 77.7% identity. PP-ACO1 and PP-ACO2 show highest degree of similarity with petunia (PH-ACO3; 84.1%) and apple (85.4%) ACO genes, respectively. PP-ACO1 is expressed in flowers, fruitlet abscission zones, mesocarp and in young fully expanded leaves. PP-ACO1 transcript accumulation strongly increases during fruitlet abscission, in ripe mesocarp and senescing leaves, and is enhanced by propylene. PP-ACO2 mRNA accumulation is detected in fruits only during early development and is unaffected by propylene treatment. Both ACO genes are expressed in epicotyl and roots of growing seedlings, although a stronger accumulation of PP-ACO2 mRNA is observed.