Transcriptional regulatory networks controlling woolliness in peach in response to preharvest gibberellin application and cold storage

BACKGROUND: Postharvest fruit conservation relies on low temperatures and manipulations of hormone metabolism to maintain sensory properties. Peaches are susceptible to chilling injuries, such as ‘woolliness’ that is caused by juice loss leading to a ‘wooly’ fruit texture. Application of gibberellic acid at the initial stages of pit hardening impairs woolliness incidence, however the mechanisms controlling the response remain unknown. We have employed genome wide transcriptional profiling to investigate the effects of gibberellic acid application and cold storage on harvested peaches. RESULTS: Approximately half of the investigated genes exhibited significant differential expression in response to the treatments. Cellular and developmental process gene ontologies were overrepresented among the differentially regulated genes, whereas sequences in cell death and immune response categories were underrepresented. Gene set enrichment demonstrated a predominant role of cold storage in repressing the transcription of genes associated to cell wall metabolism. In contrast, genes involved in hormone responses exhibited a more complex transcriptional response, indicating an extensive network of crosstalk between hormone signaling and low temperatures. Time course transcriptional analyses demonstrate the large contribution of gene expression regulation on the biochemical changes leading to woolliness in peach. CONCLUSION: Overall, our results provide insights on the mechanisms controlling the complex phenotypes associated to postharvest textural changes in peach and suggest that hormone mediated reprogramming previous to pit hardening affects the onset of chilling injuries. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-015-0659-2) contains supplementary material, which is available to authorized users

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>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.</p> <p>Conclusions</p> <p>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.</p> <p>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).</p

Ethylene-auxin crosstalk regulates postharvest fruit ripening process in apple

The ripening of climacteric fruits, such as apple, is represented by a series of genetically programmed events orchestrated by the action of several hormones. In this study, we investigated the existence of a hormonal crosstalk between ethylene and auxin during the post-harvest ripening of three internationally known apple cultivars: 'Golden Delicious', 'Granny Smith' and 'Fuji'. The normal climacteric ripening was impaired by the exogenous application of 1-methylcyclopropene (1-MCP) that affected the production of ethylene and the physiological behaviour of specific ethylene-related quality traits, such as fruit texture and the production of volatile organic compounds. The application of 1-MCP induced, moreover, a de-novo accumulation of auxin. The RNA-Seq wide-transcriptome analysis evidenced as the competition at the level of the ethylene receptors induced a cultivar-dependent transcriptional re-programming. The DEGs annotation carried out through the KEGG database identified as most genes were assigned to the plant hormone signaling transduction category, and specifically related to auxin and ethylene. The interplay between these two hormones was further assessed through a candidate gene analysis that highlighted a specific activation of GH3 and ILL genes, encoding key steps in the process of the auxin homeostasis mechanism. Our results showed that a compromised ethylene metabolism at the onset of the climacteric ripening in apple can stimulate, in a cultivar-dependent fashion, an initial de-novo synthesis and de-conjugation of auxin as a tentative to restore a normal ripening progression

A PLENA-like gene of peach is involved in carpel formation and subsequent transformation into a fleshy fruit

MADS-box genes have been shown to play a role in the formation of fruits, both in Arabidopsis and in tomato. In peach, two C-class MADS-box genes have been isolated. Both of them are expressed during flower and mesocarp development. Here a detailed analysis of a gene that belongs to the PLENA subfamily of MADS-box genes is shown. The expression of this PLENA-like gene (PpPLENA) increases during fruit ripening, and its ectopic expression in tomato plants causes the transformation of sepals into carpel-like structures that become fleshy and ripen like real fruits. Interestingly, the transgenic berries constitutively expressing the PpPLENA gene show an accelerated ripening, as judged by the expression of genes that are important for tomato fruit ripening. It is suggested that PpPLENA might interfere with the endogenous activity of TAGL1, thereby activating the fruit ripening pathway earlier compared with wild-type tomato plants

A genomic investigation of the ripening regulation in peach fruit

The aim of this work is the study of the gene regulation of peach ripening with a genomic approach. The microarray platform μPEACH 1.0 and Real Time PCR experiments have been used in order to find out genes involved in peach ripening (219 genes up-regulated and 188 down-regulated during the transition from stage S3II to stage S4I) and to study the effect of two hormones (auxin and ethylene) and 1-MCP (1-Methylcyclopropene), an inhibitor of ethylene receptors, during ripening. This approach has confirmed that ethylene can affect the expression of many genes and has confirmed its basic role in the regulation of ripening of peach fruit (102 genes induced and 80 repressed by treatment with ethylene associated with the S3II-S4I transition) and, more generally, of climacteric fruit (Alba et al., 2005). Also it has been possible to show that auxin is actively involved in the ripening of peaches (43 and 48 genes induced and repressed, respectively, by the NAA treatment and modulated by the transition from pre-climacteric to climacteric stage) but with a role independent from ethylene. Indeed, many genes involved in the biosynthesis, transport, perception and signaling of auxin had increased expression in the mesocarp during ripening. Moreover, there is an important cross-talk between auxin and ethylene, with genes in the auxin domain regulated by ethylene, as ctg3721 encoding a PIN auxin efflux facilitator, and genes in the ethylene domain regulated by auxin, as ACS1 (1-aminocyclopropane-1-carboxylate synthase). The microarray analyses carried out with 1-MCP treated fruit revealed that this chemical modified the expression of 121 genes. Besides inducing ethylene-, auxin- and ripening-repressed genes and repressing ethylene-, auxin- and ripening-induced genes, also genes either induced or repressed by ripening, auxin and 1-MCP were discovered. Thus, blocking ethylene perception with 1-MCP can induce ripening-related effects through a not-yet identified mechanism. Some of the genes identified as “ripening-related” and “belonging to either auxin or ethylene domains” were mapped with the bin mapping technique. In particular, the Prunus reference map constructed with an almond (Prunus dulcis) x peach (Prunus persica) F2 population (Howad et al., 2005) has been used, namely, the TxE population (Texas, almond x Earlygold, peach), available in the laboratory of prof. Pere Arus (IRTA, Barcelona, Spain). Some of these genes are transcription factors, whose expression parallel peach ripening. A functional analysis of these TF genes is a possible way to study the genetic regulation involved in peach ripening. Thus an analysis of the promoters of a gene, named PpIAA57, coding for an Aux/IAA protein and of a gene, named PpbZIP298, coding for a bZIP transcription factor has been carried out. These genes present a high transcript abundance in ripe fruit and they are up-regulated by the S3II-S4I transition. Nonetheless, their expression is not influenced by treatments with either ethylene or auxin. These promoter sequences have been evaluated with a bioinformatic analysis to discover the presence of cis-elements. Progressive deletions of the PpbZIP298 and PpIAA57 promoters, fused with the GUS reporter gene, were used for tobacco stable transformation and for agroinfiltration experiments performed on peach fruit. Unexpectedly, the strength of the two promoters resulted to be very weak, despite the length of the used fragments. Anyway, a possible putative regulatory region has been identified in the 5' UTR of PpbZIP298.Lo scopo di questo lavoro è lo studio della regolazione genica durante la maturazione della pesca mediante un approccio di tipo genomico. La piattaforma μPEACH 1.0 ed esperimenti di Real Time PCR sono stati utilizzati per scoprire geni coinvolti nella maturazione della pesca (219 geni indotti e 188 repressi durante la transizione dallo stadio S3II allo stadio S4I) e per studiare l’effetto di due ormoni (auxina ed etilene) e di un inibitore dei recettori dell’etilene, 1-MCP (1-Metilciclopropene) durante la maturazione. Questo approccio ha confermato che l’etilene controlla l’espressione di molti geni e ha avvalorato il suo ruolo fondamentale nella regolazione della maturazione della pesca (102 geni indotti e 80 repressi dal trattamento con etilene associato alla transizione S3II-S4I) e, più in generale, dei frutti climaterici (Alba et al., 2005). Inoltre è stato possibile dimostrare che l’auxina è coinvolta attivamente nella maturazione delle pesche (43 e 48 geni rispettivamente indotti e repressi dal trattamento con NAA e modulati dalla transizione da stadio pre-climaterico a quello climaterico) ma con un ruolo indipendente dall’etilene. Infatti, molti geni coinvolti nella biosintesi, trasporto, percezione e trasduzione del segnale dell’auxina presentano un incremento di espressione nel mesocarpo durante la maturazione. Inoltre, è presente un importante “cross-talk” tra auxina ed etilene, con geni appartenenti al dominio dell’auxina regolati dall’etilene, come nel caso del ctg3721 codificante un facilitatore di efflusso di auxina di tipo PIN e geni appartenenti al dominio dell’etilene regolati dall’auxina, come ACS1 (acido 1-amminociclopropan-1-carbossilico sintasi). Gli esperimenti di microarray condotti su frutti trattati con 1-MCP hanno rivelato che questo composto chimico modifica l’espressione di 121 geni. Oltre a indurre geni repressi dall’etilene, dall’auxina e dalla maturazione e reprimere geni indotti dall’etilene, dall’auxina e dalla maturazione, è stato scoperto che l’1-MCP può indurre o reprimere geni che sono regolati nello stesso modo dalla maturazione e dall’auxina. Quindi il blocco della percezione dell’etilene con 1-MCP è in grado di indurre degli effetti legati alla maturazione attraverso un meccanismo non ancora identificato. Alcuni dei geni identificati come “legati alla maturazione” e “appartenenti sia al dominio dell’auxina che a quello dell’etilene” sono stati mappati mediante la tecnica del “bin mapping”. In particolare, è stata utilizzata la mappa di riferimento di Prunus costruita incrociando mandorlo (Prunus dulcis) con pesco (Prunus persica) con lo scopo di ottenere una popolazione F2 (Howad et al., 2005) chiamata popolazione TxE (Texas, mandorlo x Earlygold, pesco), disponibile presso il laboratorio del prof. Pere Arus (IRTA, Barcellona, Spagna). Alcuni di questi geni sono dei fattori di trascrizione, la cui espressione aumenta durante la maturazione della pesca. Un’analisi funzionale di questi fattori di trascrizione è un possibile modo per studiare la regolazione genica coinvolta nella maturazione della pesca. Quindi è stata condotta un’analisi dei promotori di un gene codificante una proteina di tipo Aux/IAA chiamato PpIAA57 e di un gene codificante per un fattore di trascrizione di tipo bZIP chiamato PpbZIP298. Questi geni presentano un’elevata abbondanza di trascritti nei frutti maturi ed inoltre sono regolati positivamente durante la transizione S3II-S4I. Tuttavia, la loro espressione non è influenzata dai trattamenti con etilene o auxina. Le sequenze di questi due promotori sono state analizzate mediante un’analisi di tipo bioinformatico per scoprire la presenza di cis-element. Delezioni progressive dei promotori dei due geni PpbZIP298 e PpIAA57, fuse con il gene reporter GUS, sono state utilizzate per la trasformazione permanente di piante di tabacco e per esperimenti di agroinfiltrazione condotti su pesche. Inaspettatamente, la forza dei due promotori sembra essere molto debole a discapito della lunghezza dei frammenti utilizzati. Comunque, una possibile putativa regione regolativa è stata identificata nel 5' UTR del gene PpbZIP298

The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches

Ethylene has long been regarded as the main regulator of ripening in climacteric fruits. The characterization of a few tomato mutants, unable to produce climacteric ethylene and to ripen their fruits even following treatments with exogenous ethylene, has shown that other factors also play an important role in the control of climacteric fruit ripening. In climacteric peach and tomato fruits it has been shown that, concomitant with ethylene production, increases in the amount of auxin can also be measured. In this work a genomic approach has been used in order to understand if such an auxin increase is functional to an independent role played by the hormone during ripening of the climacteric peach fruits. Besides the already known indirect activity on ripening due to its up-regulation of climacteric ethylene synthesis, it has been possible to show that auxin plays a role of its own during ripening of peaches. In fact, the hormone has shown the ability to regulate the expression of a number of different genes. Moreover, many genes involved in biosynthesis and transport and, in particular, the signalling (receptors, Auxin Response Factors and Aux/IAA) of auxin had increased expression in the mesocarp during ripening, thus strengthening the idea that this hormone is actively involved in the ripening of peaches. This study has also demonstrated the existence of an important cross-talk between auxin and ethylene, with genes in the auxin domain regulated by ethylene and genes in the ethylene domain regulated by auxi