Angela Rasori

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

A large-scale transcriptome analysis has been conducted using μPEACH1.0 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 °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.

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

BackgroundAuxins 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.ResultsThe 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.ConclusionsThis 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

BackgroundField 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.ResultsA 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.ConclusionsIn 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).

Differential expression of two lipid transfer protein genes in reproductive organs of peach (Prunus persica L. Batsch)

Abstract Two cDNA clones (named Pp-LTP1 and Pp-LTP2 ) corresponding to different lipid transfer protein (LTP) genes have been isolated from peach ( Prunus persica L. Batsch) epicarp and ovary, respectively. Sequence analysis revealed that the two fragments share 54% identity at nucleotide level and show common features of plant LTP genes, such as conserved cysteine residues and lipid-binding motifs. Phylogenetic analysis grouped Pp-LTP1 and Pp-LTP2 in two distinct clusters, the former with most of LTP genes sequenced in the Rosaceae family, the latter only with one almond LTP. Genomic Southern data indicated that a small LTP gene family is present in peach. Pp-LTP1 and Pp-LTP2 have been used as gene-specific probes to describe expression in flowers and fruits throughout development. In petals, sepals and stamen only Pp-LTP1 was expressed whereas transcripts of Pp-LTP2 strongly accumulated in non-pollinated and pollinated ovary with a decreasing trend in the period of four weeks after pollination. In fruits, a dramatic accumulation of Pp-LTP1 mRNA was detected in epicarp at all stages of fruit development and, with the exception of the early growth stage, no Pp-LTP1 transcripts have been detected in mesocarp. When Pp-LTP2 was used as a probe in the same fruit tissues, a faint hybridisation signal was observed only in epicarp of fruitlets collected at an early growth stage. Infection with Monilia induced only a slight increase of Pp-LTP1 transcript in epicarp of pre-climacteric and climacteric fruits. These results support the hypothesis of multiple roles played by LTPs and, considering that LTPs have been recognised as the major allergen of peach, indicate that Pp-LTP1 could be related to the allergenicity of peach.

Characterization of a major latex protein (MLP) gene down-regulated by ethylene during peach fruitlet abscission☆

We report the isolation of a new peach gene, Pp-MLP1 , that shows significant similarity to a family of fruit- and flower-specific genes, designated as major latex protein (MLP) homologues. Transcript of Pp-MLP1 highly accumulated in cells of fruit pedicel, similar to lacticifers, adjacent to the abscission zone (non-abscission zone) and, to a lesser extent, in epicotyls, stems and roots, while no accumulation was detected in leaves. In contrast to the MLP homologues isolated so far, the Pp-MLP1 transcript was detected during fruit cells expansion, though its expression appeared unrelated to fruit ripening. Propylene treatment caused a decrease in mRNA accumulation of Pp-MLP1 in all tested tissues. The function of Pp-MLP1, as with all previously described MLP homologues, is unknown. MLPs are associated with fruit and flower development in addition to plant pathogenesis responses. Expression in tissues associated with abscission would be consistent with a role in implementing this aspect of floral development or possibly protective responses to plant pathogens which may infect post-abscission wounds. In addition, the high similarity between proteins encoding by Pp-MLP1 and Csf2 , an MLP gene associated with the early development of cucumber fruit, could suggest an alternativ ed evelopmental role such as cell and tissue expansion. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Functional analysis of peach ACC oxidase promoters in transgenic tomato and in ripening peach fruit

Abstract To investigate the transcriptional regulation of two 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) genes of peach, chimeric fusions between β-glucuronidase (GUS) reporter gene, and Pp-ACO1 and Pp-ACO2 promoters have been constructed and introduced in tomato (cv Microtom). Pp-ACO1 promoter is able to induce in transgenic tomato plants the same pattern of expression observed in peach. In fact, Pp-ACO1–GUS activity was localized in leaf blade, ovary, leaf and fruit abscission zones and pericarp, and it is up-regulated by propylene and wounding. A gradient of transcript accumulation has been observed in ripening fruit tissues: in tomato it decreased moving from the inner to the outer pericarp, while in peach the opposite occurred. This different behavior could be related to the fruit type (berry vs. drupe). In order to identify cis-acting element involved in ethylene induction of Pp-ACO1, portions of its promoter fused with the GUS gene have been constructed and used in peach fruit transient activity assay. The deletion analysis has shown that a region located between −716 and −346 bp, containing an ethylene-responsive element (ERE), is responsible for the higher stimulation by the gas. In addition, two auxin-responsive elements (AUXre), probably responsible for the auxin suppression of the propylene induction of Pp-ACO1 gene expression, are present upstream from EREs. Pp-ACO2 promoter is able to drive the expression in vascular bundles of immature and ripe fruit, senescent leaf blade, and in fruit and leaf abscission zones. In peach, Pp-ACO2 mRNA is detected only in immature fruit, epicotyl and root of seedling. This discrepancy might be imputed to a lesser stability and translation of Pp-ACO2 mRNA in comparison to that of chimeric one.

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.

Gene expression analysis of peach fruit at different growth stages and with different susceptibility to Monilinia laxa

Monilinia brown rot is one of the most important diseases of peach (Prunus persica L. Batsch). In this study, the susceptibility to Monilinia rot of peach fruit during ripening was analysed weekly by assessing infected fruits upon artificial inoculation. Fruit drastically reduced their susceptibility to Monilinia rot along with ripening, becoming resistant in correspondence to pit hardening (a two-week period). Susceptibility increases again thereafter. With the aim to identify genes possibly correlated with the variation of brown rot susceptibility, a microarray based-transcriptome analysis was undertaken to compare the expression of genes between susceptible fruit (two weeks before the pit hardening stage) and resistant fruit (at the pit hardening stage). This approach pointed out that genes involved in defence and primary and secondary metabolism, in particular some phenylpropanoid and flavonoid related genes, are differentially expressed in susceptible and resistant fruit.Considering that several aromatic compounds with antifungal properties are known to accumulate during endocarp lignification, the expression levels of genes encoding key enzymes of the phenylpropanoid and jasmonate pathways was quantified by real time RT-PCR in the peel of both susceptible and resistant fruit. Results show that during the two-week time between the susceptible and resistant fruit stages the expression of several genes involved in the synthesis of phenylpropanoid and jasmonate compounds drastically changes, supporting a role for these metabolites in the fruit response to Monilinia.

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.