Claudio Bonghi

A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype

BackgroundAlternative splicing (AS) significantly enhances transcriptome complexity. It is differentially regulated in a wide variety of cell types and plays a role in several cellular processes. Here we describe a detailed survey of alternative splicing in grape based on 124 SOLiD RNAseq analyses from different tissues, stress conditions and genotypes.ResultsWe used the RNAseq data to update the existing grape gene prediction with 2,258 new coding genes and 3,336 putative long non-coding RNAs. Several gene structures have been improved and alternative splicing was described for about 30% of the genes. A link between AS and miRNAs was shown in 139 genes where we found that AS affects the miRNA target site. A quantitative analysis of the isoforms indicated that most of the spliced genes have one major isoform and tend to simultaneously co-express a low number of isoforms, typically two, with intron retention being the most frequent alternative splicing event.ConclusionsAs described in Arabidopsis, also grape displays a marked AS tissue-specificity, while stress conditions produce splicing changes to a minor extent. Surprisingly, some distinctive splicing features were also observed between genotypes. This was further supported by the observation that the panel of Serine/Arginine-rich splicing factors show a few, but very marked differences between genotypes. The finding that a part the splicing machinery can change in closely related organisms can lead to some interesting hypotheses for evolutionary adaptation, that could be particularly relevant in the response to sudden and strong selective pressures.

Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach

Ethylene-induced abscission in leaf and fruit explants of peach involves different enzymes. In leaves abscission is accompanied by increased occurrence of cellulase forms differing in isoelectric point (pI 6.5 and 9.5). A polypeptide with a molecular mass of 51 kDa gives in a western blot a strong cross-reaction with an antibody raised against a maturation cellulase from avocado fruit. Cellulase activity is also found in abscising fruit explants but the amount is very low compared to that of the leaf explants. A northern analysis with a cellulase clone from avocado reveals the presence of two hybridizing mRNAs with a size of 2.2 kb and 1.8 kb, respectively. The steady-state level of the 2.2 kb mRNA is significantly increased by treatment with ethylene.Polygalacturonases are not detected in abscising leaves, but are strongly induced by ethylene in fruit explants. Of the three forms found, two are exopolygalacturonases while the third is an endoenzyme. Ethylene activates preferentially the endoenzyme and the basic exoenzyme but depresses the acid exopolygalacturonases. A northern analysis carried out with a cDNA coding for tomato endopolygalacturonase shows hybridization only with one endopolygalacturonase mRNA from in the fruit abscission zone. Treatment with ethylene causes an increase in the steady-state level of this mRNA. The differences in the enzyme patterns observed in fruit and leaf abscission zones and a differential enzyme induction suggest the feasibility to regulate fruit abscission in peach with the aid of antisense RNA genes.

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.

Jasmonate-induced transcriptional changes suggest a negative interference with the ripening syndrome in peach fruit

Peach (Prunus persica L. Batsch) was chosen as a model to shed light on the physiological role of jasmonates (JAs) during fruit ripening. To this aim, the effects of methyl jasmonate (MJ, 0.40 mM) and propyl dihydrojasmonate (PDJ, 0.22 mM), applied in planta at different fruit developmental stages, on the time-course of ethylene production and fruit quality traits were evaluated. MJ-induced changes in fruit transcriptome at harvest and the expression profiling of relevant JA-responsive genes were analysed in control and JA-treated fruit. Exogenously applied JAs affected the onset of ripening depending upon the fruit developmental stage, with PDJ being more active than MJ. Both compounds enhanced the transcription of allene oxide synthase (PpAOS1), the first specific enzyme in the biosynthesis of jasmonic acid, and altered the pattern of jasmonic acid accumulation. Microarray transcriptome profiling showed that MJ down-regulated some ripening-related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (PpACO1) and polygalacturonase (PG), and the transcriptional modulator IAA7. MJ also altered the expression of cell wall-related genes, namely pectate lyase (PL) and expansins (EXPs), and up-regulated several stress-related genes, including some of those involved in JA biosynthesis. Time-course expression profiles of PpACO1, PL, PG, PpExp1, and the transcription factor LIM confirmed the array results. Thus, in peach fruit, exogenous JAs led to a ripening delay due to an interference with ripening- and stress/defence-related genes, as reflected in the transcriptome of treated fruit at harvest.

Peach fruit ripening and quality in relation to picking time, and hypoxic and high CO2 short-term postharvest treatments

Peach fruits (Prunus persica L. Batsch, cv Springcrest) were harvested at two ripening stages (flesh firmness of 60 N, first harvest, and 45 N, second harvest) and maintained at 20°C in air (control) or for 24 and 48 h in streams of ultra low (<1%) oxygen (ULO) or high (30%) CO2 concentration and then transferred to air for up to 8 days. The decline in flesh firmness was strongly reduced by ULO and CO2 treatments in fruits of both harvests, although the effect was stronger in fruits picked earlier in which ethylene biosynthesis remained at the basal level. In fruits of the second harvest, endo β-1,4-glucanase (EGase) activity was lower in ULO- and CO2-treated fruits than in control fruits at the end of the 24 h treatment and the following two days in air. Acetaldehyde (AA) gradually accumulated in control fruit and the highest concentrations were detected during late ripening. Both treatments induced a strong accumulation of AA but, with the exception of the 24 and 48 h CO2 treatments performed on fruits of the second harvest, a decrease in AA content was observed when the fruits were transferred to air. A slight increase in ethanol (EtOH) was found throughout the ripening process in control fruits; ULO and CO2 strongly stimulated EtOH production. When fruits were transferred to air, EtOH concentration declined rapidly. Alcohol dehydrogenase (ADH) activity significantly increased in control fruit only in the late stages of ripening. Greater ADH activity was found throughout the experimental period in fruits of the first harvest treated for 24 h in ULO and CO2, whereas, at day 8, control and treated fruits of the second harvest showed similar ADH activity values. Hypoxic and, to a lesser extent, CO2-enriched atmospheres stimulated Adh gene expression.

Cell enlargement and cell separation during peach fruit development

The increase in cell size and the activities of cell wall lytic enzymes during fruit growth supported by the mesocarp cell enlargement have been studied in peach of the freestone cultivar Redhaven. Four growth stages (S1-S4) in peach fruit were observed. Cell enlargement and formation of intercellular spaces in the mesocarp were directly correlated with the increase in fruit diameter. Cellulase and exopolygalacturonase showed the highest activity during the first stage of fast fruit growth (S1), while the endopolygalacturonase activity increased during the last growth stage (S4), when the fruit reached its final size and ripening started.

Cell wall hydrolases and amylase in kiwifruit softening

Abstract The activities of amylase, β-galactosidase (β-GAL), polygalacturonase (PG), and endo-1,4-β-glucanase (EG), and ethylene evolution, were measured during ripening in kiwifruit ( Actinidia deliciosa (A.Chev.) (C.F. Liang et A.R. Ferguson var deliciosa, cv. Hayward). Fruit, harvested at firmness values (FV) of 65 N and a soluble solid (SS) content of 9%, were maintained in air at 20 °C for 40 days, until they reached the edible stage. Fruit firmness decreased throughout the experimental period and the lowest FV (about 5 N) was reached after 38 days of storage. SS content increased rapidly to about 13% within 15 days, then the rate of accumulation slowed and the SS content of 14.1% was reached after 33 days. Ethylene climacteric was a late event occurring at a FV of 10 N. The highest amylase activity was measured at harvest. During storage it declined, although a slight rise was detected at 33 days. β-GAL activity was very low at the beginning of storage and increased throughout the experimental period, while PG activity was detected only after the fruit FV was below 10 N. EG activity decreased within the first three days of storage, then increased and peaked 15 days after harvest. Later, EG activity remained low but increased again at the end of the storage period. Application of propylene (500 ppm) to fruit that had softened to 30 N in air stimulated fruit softening and EG activity, and induced the accumulation of an EG-related peptide: the other enzymes appeared not to be affected by the gas.

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).

New approaches to Prunus transcriptome analysis

The recent sequencing of the complete genome of the peach offers new opportunities for further transcriptomic studies in Prunus species in the called post-genomics era. First works on transcriptome analysis in Prunus species started in the early 2000s with the development of ESTs (expressed sequence tags) and the analysis of several candidate genes. Later, new strategies of massive analysis (high throughput) of transcriptomes have been applied, producing larger amounts of data in terms of expression of a large number of genes in a single experiment. One of these systems is massive transcriptome analysis using cDNA biochips (microarrays) to analyze thousands of genes by hybridization of mRNA labelled with fluorescence. However, the recent emergence of a massive sequencing methodology (“deep-sequencing”) of the transcriptome (RNA-Seq), based on lowering the costs of DNA (in this cases complementary, cDNA) sequencing, could be more suitable than the application of microarrays. Recent papers have described the tremendous power of this technology, both in terms of profiling coverage and quantitative accuracy in transcriptomic studies. Now this technology is being applied to plant species, including Prunus. In this work, we analyze the potential in using this RNA-Seq technology in the study of Prunus transcriptomes and the development of genomic tools. In addition, the strengths and limitations of RNA-Seq relative to microarray profiling have been discussed.