A molecular phenology scale of grape berry development

SPL is a plant-specific transcription factor family. Many researchers reported that SPL members targeted by miR156s could play crucial roles in the modulation of plant growth and development. Although there are similar reports on grapes, till now little is known about grape berry development and ripening. To gain more insight into how grape miR156s (Vv-miR156s) modulated the above given processes of grape berries by mediating their target gene Vv-SPLs, here we identified the precise sequences of Vv-miR156s in 'Giant Rose' grape berries, predicted their potential targets, and revealed that the matching degree of various Vv-miR156: Vv-SPL pairs exhibited some discrepancy, implying the divergence of their interaction. Subsequently, we also discovered similar motifs such as ABRE, CGTCA and ERE, which are more specific to berry development and ripening, within the promoters of both Vv-MIR156s and Vv-SPLs. With berry development and ripening, meanwhile, Vv-miR156a, b/c/d, e and f/g/i exhibited an overall increasing expression trend, while their targets showed opposite trends at the corresponding stages. Additionally, exogenous ABA and NAA application promoted or curbed the expression of Vv-miR156s to some extent, before grape berry ripening stage. The cleavage products, sites and frequencies of Vv-miR156a, b/c/d, e, f/g/i and their respective targets (Vv-SPL2, 9, 10, 16) during grape berry development and ripening process were validated by our developed PPM-RACE and modified RLM-RACE together with qRT-PCR, which demonstrated that Vv-miR156s can be involved in the modulation of grape berry development and ripening process by mediating the expression of Vv-SPL2, 9, 10, 16. Our findings lay an important foundation for further recognizing their functions in grape berries, and enrich the knowledge of the regulatory mechanism of miRNA-mediated grape berry development and ripening.

The ethylene signalling pathway has never been fully described in grapes. Regarded as a non-climacteric fruit, grape berry seems to ripen independently to ethylene, however 1-methylcyclopropene (1-MCP), a specific inhibitor of ethylene receptors has been shown to alter berry ripening processes. Here, we report profiles of transcript abundance of various receptors and transcription factors, associated with ethylene signalling, throughout berry development. Transcript abundance of ortholog VvETR2 gene showed a transient peak at the inception of ripening in 'Cabernet Sauvignon' berries coinciding with an internal ethylene peak, prior to colour changes. The transcripts of other orthologs such as VvRTE1 and VvEIN4 steadily increased over the berry development, while VvERS1 ortholog transcripts exhibited a peak of accumulation only when the berries were fully coloured. Finally, mRNAs of two transcription factors, VvEIN3 and VvMADS4, showed strong accumulation during the late phase of berry ripening. We also observed inflections of mRNA accumulation after incubating berry clusters with ethylene and 1-MCP (inhibitor of ethylene action). The main effect was observed with VvEIN3 transcripts that showed a significant up-regulation after incubation with 1-MCP. Furthermore, other transcript levels (VvETR2 and VvCTR1) were also increased by exogenous ethylene, once the colour change was initiated ( i.e. 10 to 11 weeks after bloom). Some studies have already indicated that non-climacteric fruits shared signalling pathways with climacteric fruits. However, most differences between these ripening classifications remain poorly described at the genetic/molecular level. This data set will contribute to a better understanding on potential involvements of ethylene signalling in a non-climacteric fruit such as grape berry.

In plants, hexokinase (HXK, EC 2.7.1.1) involved in hexose phosphorylation, plays an important role in sugar sensing and signaling. In this study, we found that at Phase I of grape berry development, lower hexose (glucose or fructose) levels were concomitant with higher HXK activities and protein levels. After the onset of ripening, we demonstrated a drastic reduction in HXK activity and protein levels accompanied by a rising hexose level. Therefore, our results revealed that HXK activity and protein levels had an inverse relationship with the endogenous glucose or fructose levels during grape berry development. A 51 kDa HXK protein band was detected throughout grape berry development. In addition, HXK located in the vacuoles, cytoplasm, nucleus, proplastid, chloroplast, and mitochondrion of the berry flesh cells. During grape berry development, HXK transcriptional level changed slightly, while cell wall invertase (CWINV) and sucrose synthase (SuSy) expression was enhanced after véraison stage. Intriguingly, when sliced grape berries were incubated in different glucose solutions, CWINV and SuSy expression was repressed by glucose, and the intensity of repression depended on glucose concentration and incubation time. After sliced, grape berries were treated with different glucose analogs, CWINV and SuSy expression analyses revealed that phosphorylation of hexoses by hexokinase was an essential component in the glucose-dependent CWINV and SuSy expression. In the meantime, mannoheptulose, a specific inhibitor of hexokinase, blocked the repression induced by glucose on CWINV and SuSy expression. It suggested that HXK played a major role in regulating CWINV and SuSy expression during grape berry development.

Light environments have long been known to influence grape (Vitis vinifera L.) berry development and biosynthesis of phenolic compounds, and ultimately affect wine quality. Here, the accumulation and compositional changes of hydroxycinnamic acids (HCAs) and flavonoids, as well as global gene expression were analyzed in Cabernet Sauvignon grape berries under sunlight exposure treatments at different phenological stages. Sunlight exposure did not consistently affect the accumulation of berry skin flavan-3-ol or anthocyanin among different seasons due to climatic variations, but increased HCA content significantly at véraison and harvest, and enhanced flavonol accumulation dramatically with its timing and severity degree trend. As in sunlight exposed berries, a highly significant correlation was observed between the expression of genes coding phenylalanine ammonia-lyase, 4-coumarate: CoA ligase, flavanone 3-hydroxylase and flavonol synthase family members and corresponding metabolite accumulation in the phenolic biosynthesis pathway, which may positively or negatively be regulated by MYB, bHLH, WRKY, AP2/EREBP, C2C2, NAC, and C2H2 transcription factors (TFs). Furthermore, some candidate genes required for auxin, ethylene and abscisic acid signal transductions were also identified which are probably involved in berry development and flavonoid biosynthesis in response to enhanced sunlight irradiation. Taken together, this study provides a valuable overview of the light-induced phenolic metabolism and transcriptome changes, especially the dynamic responses of TFs and signaling components of phytohormones, and contributes to the further understanding of sunlight-responsive phenolic biosynthesis regulation in grape berries.

Some of the most important physiological and biochemical processes that occur during the development and maturation of de Chaunac grape berries have been investigated. The studies included monitoring changes in the pH, in total and individual major acids, cations, sugars, anthocyanins and their biosynthetic enzymes. Brix, pH, K+, sugars and anthocyanins showed a sharp rise during and immediately after veraison. There was a concomitant rise in the activity of anthocyanin biosynthetic enzymes. Total acidity and malic acid content increased during the first four weeks and declined thereafter, reaching a steady level approximately five weeks later. The concentration of phosphoric acid showed a rise during the maturation process, while tartaric acid levels declined continuously. On the per gram basis, the levels of Na+ showed no consistent changes, while Ca++, Cu++, Mg++, and Mn++ showed sharp declines during the expansion period of the berries and steady, low levels during the maturation process. The data suggest that the majority of metabolic events in the berries come to a steady state approximately eight weeks after veraison under the climatic conditions of the northeastern United States.

Although the effects of girdling on grape berry development have been widely studied, the underlying mechanisms are poorly understood, especially at the molecular level. This study investigated the effect of trunk girdling on grape (Vitis L.) berry maturation. Girdling was performed on 5-year-old ‘Summer Black’ grapevines at early veraison, and transcriptional and physiologic analyses were performed. Trunk girdling promoted sugar accumulation and color development in berries and accelerated berry ripening by 25 days. Genes related to sucrose cleavage and polysaccharide degradation were upregulated at the transcriptional level, which was associated with increased monosaccharide accumulation and berry softening. Anthocyanin biosynthesis and accumulation were also enhanced by trunk girdling through the upregulation of anthocyanin biosynthesis genes including phenylalanine ammonia-lyase and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The increased expression of two VvUFGT genes was accompanied by the upregulation of VvMYBA2 under girdling. The upregulation of genes involved in ethylene biosynthesis and hormone (abscisic acid and brassinosteroid) responses and downregulation of genes involved in indoleacetic acid biosynthesis and response may have also promoted berry ripening in the girdling group. A total of 120 differentially expressed transcription factor genes from 29 gene families including MYB, ERF, and MYB-related were identified in the girdling group, which may participate in the regulation of berry development and ripening. These results provide molecular-level insight into the positive effects of trunk girdling on berry development in grapes.

This chapter describes the role of plant hormones during the development of the nonclimacteric grape berry. Advances in analytical methods have enabled a better understanding of hormone accumulation although the role of some hormones, for example ethylene, during berry development, remains unclear. Even though the biosynthesis pathways for a number of hormones have been elucidated in grapevine, our understanding of some, for example, the auxin biosynthesis pathway, is still embryonic in grapes. Recent work regarding the breakdown of ABA and auxins has demonstrated the importance of catabolism in determining the final hormone profile during development. Much attention has been focused on the role of hormones during ripening due to the importance of the ripening process to the relevant grape industries. Brassinosteroids have now been added to the list of hormones that can advance ripening. High throughput techniques for analysing changes in gene expression, such as microarray analysis and deep sequencing, are now being used to assess the role of hormones during berry development. These techniques are ideally suited to analyse the effects of exogenous hormones on gene transcript levels, and the information gained will provide a global view of hormonal control at the transcriptome level. It is much more difficult to obtain an overall picture of the changes in protein levels as a response to hormone treatment. However, proteomic studies have demonstrated changes in the levels of a number of proteins, and some of these changes reflect changes in gene transcript levels. This chapter is a review of the current state of knowledge regarding the hormonal control of berry development (excluding polyamines which are described in Chapter 7). A portion of this work has been referred to in a previous review [1].

Most of the carbon found in fruits at harvest is imported by the phloem. Imported carbon provide the material needed for the accumulation of sugars, organic acids, secondary compounds, in addition to the material needed for the synthesis of cell walls. The accumulation of sugars during fruit development influences not only sweetness but also various parameters controlling fruit composition (fruit “quality”). The accumulation of organic acids and sugar in grape berry flesh cells is a key process for berry development and ripening. The present review presents an update of the research on grape berry development, anatomical structure, sugar and acid metabolism, sugar transporters, and regulatory factors.

BackgroundCytokinins are known to play an important role in fruit set and early fruit growth, but their involvement in later stages of fruit development is less well understood. Recent reports of greatly increased cytokinin concentrations in the flesh of ripening kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang & A.R. Ferguson) and grapes (Vitis vinifera L.) have suggested that these hormones are implicated in the control of ripening-related processes.ResultsA similar pattern of isopentenyladenine (iP) accumulation was observed in the ripening fruit of several grapevine cultivars, strawberry (Fragaria ananassa Duch.) and tomato (Solanum lycopersicum Mill.), suggesting a common, ripening-related role for this cytokinin. Significant differences in maximal iP concentrations between grapevine cultivars and between fruit species might reflect varying degrees of relevance or functional adaptations of this hormone in the ripening process. Grapevine orthologues of five Arabidopsis (Arabidopsis thaliana L.) gene families involved in cytokinin metabolism and signalling were identified and analysed for their expression in developing grape berries and a range of other grapevine tissues. Members of each gene family were characterised by distinct expression profiles during berry development and in different grapevine organs, suggesting a complex regulation of cellular cytokinin activities throughout the plant. The post-veraison-specific expression of a set of biosynthesis, activation, perception and signalling genes together with a lack of expression of degradation-related genes during the ripening phase were indicative of a local control of berry iP concentrations leading to the observed accumulation of iP in ripening grapes.ConclusionsThe transcriptional analysis of grapevine genes involved in cytokinin production, degradation and response has provided a possible explanation for the ripening-associated accumulation of iP in grapes and other fruit. The pre- and post-veraison-specific expression of different members from each of five gene families suggests a highly complex and finely-tuned regulation of cytokinin concentrations and response to different cytokinin species at particular stages of fruit development. The same complexity and specialisation is also reflected in the distinct expression profiles of cytokinin-related genes in other grapevine organs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0611-5) contains supplementary material, which is available to authorized users.

Grapevine fruit development is a dynamic process that can be divided into three stages: formation (I), lag (II), and ripening (III), in which physiological and biochemical changes occur, leading to cell differentiation and accumulation of different solutes. These stages can be positively or negatively affected by multiple environmental factors. During the last decade, efforts have been made to understand berry development from a global perspective. Special attention has been paid to transcriptional and metabolic networks associated with the control of grape berry development, and how external factors affect the ripening process. In this review, we focus on the integration of global approaches, including proteomics, metabolomics, and especially transcriptomics, to understand grape berry development. Several aspects will be considered, including seed development and the production of seedless fruits; veraison, at which anthocyanin accumulation begins in the berry skin of colored varieties; and hormonal regulation of berry development and signaling throughout ripening, focusing on the transcriptional regulation of hormone receptors, protein kinases, and genes related to secondary messenger sensing. Finally, berry responses to different environmental factors, including abiotic (temperature, water-related stress and UV-B radiation) and biotic (fungi and viruses) stresses, and how they can significantly modify both, development and composition of vine fruit, will be discussed. Until now, advances have been made due to the application of Omics tools at different molecular levels. However, the potential of these technologies should not be limited to the study of single-level questions; instead, data obtained by these platforms should be integrated to unravel the molecular aspects of grapevine development. Therefore, the current challenge is the generation of new tools that integrate large-scale data to assess new questions in this field, and to support agronomical practices.

Changes occurring in the cell walls of grape berries during ripening may contribute to berry softening. Pectic substances are mainly responsible for cell wall cohesion in association with calcium ions. We measured the activity of the pectin hydrolyzing enzyme, polygalacturonase, during the development of Sauvignon blanc and Semillon grape berries. Although absent during herbaceous growth, polygalacturonase activity increased during ripening. Levels were higher in Semillon than in Sauvignon blanc ripe berries. No significant differences were found between skin and flesh polygalacturonase activity in ripe Sauvignon blanc grape berries. During ripening, the calcium content of Sauvignon blanc grape berries decreased. The calcium content of the skin was higher than in the flesh of ripe Semillon and Sauvignon blanc berries. Levels were higher in Sauvignon blanc than in Semillon whole ripe berries. In all cases and in both cultivars, polygalacturonase activity and calcium content were diametrically opposed. Moreover, polygalacturonase activity in ripe berries was inhibited, in vitro, by 1 mM or more of calcium ion. Polygalacturonase activity seems to increase with the degree of maturity and with the decrease of calcium content.

Hormones play an important role during the development and ripening of grape berry. Unlike the case of ethylene in climacteric fruits, several different hormones are believed to sequentially accumulate at specific times during berry developmental stages to promote different physiological processes. To dissect this complex hormonal interaction system in a recalcitrant tissue containing several interfering compounds including sugar and phenolic compounds, an extraction protocol and an LC-MS-based analytical method that includes three hormone families have been adapted. Using this technique, we optimized a method to simultaneously detect and quantify cytokinin, auxin, and abscisic acid-related analytes in grape berries across the developmental stages and between tissues. Resulting quantifications of the analytes are consistent with the overall trend of the ABA, auxin, and cytokinin dynamics in grape and reveal new patterns not previously reported in this plant. Evolving evidence of coordinated action of several hormones during the critical phases of berry development (cell division, cell expansion, berry ripening) suggests a need to further integrate other plant growth regulator families to provide a more comprehensive picture.

Expression of three expansin genes during development and maturation of Kyoho grape berries

A large increase in hexose accumulation is typical of grape berry ripening. To analyse sugar transport processes during grape berry ( Vitis vinifera L. cv Ugni Blanc) development, a cDNA encoding for a sucrose transporter, designated VvSUT1 was cloned. Sucrose transport activity of the VvSUT1 protein was demonstrated by heterologous expression in yeast. A study of gene expression along berry development showed that VvSUT1 transcripts are present at all stages but accumulate with the onset of ripening. This work shows that sucrose transport at the plasma membrane of grape berry cells may play a role in the accumulation of sugars during maturation.

Grape berry development and ripening are under complex regulation by the nutrients, hormones, and environment cues sensed by the berry. However, the biochemical and molecular mechanisms underlying these types of regulation are poorly understood. A simplified but realistic model system that enables fruit growth conditions to be modulated easily will facilitate the deciphering of these mechanisms. Here, an in vitro culture system of intact detached grape berries was developed by coupling the production of greenhouse fruiting-cuttings and in vitro organ culture techniques. (13)C and (15)N labelling experiments showed that this system enables the intact detached berries actively to absorb and utilize carbon and nitrogen from the culture medium. It was further used to study the effects of sugars on anthocyanin accumulation. A sucrose concentration >2% could induce anthocyanin synthesis in the absence of additional exogenous abscisic acid. The higher the sucrose concentration, the earlier was the induction of anthocyanin accumulation. Glucose, fructose, and sucrose increased anthocyanin accumulation, with glucose and fructose being more effective than sucrose. This increase was not due to an increase in its precursor level, since the phenylalanine content was decreased by a high sugar supply. Instead, genome-wide transcriptome analysis suggests that the sugar-induced enhancement of anthocyanin accumulation results from altered expression of regulatory and structural genes (especially UDP-glucose:anthocyanidin 3-O-glucosyltransferase), together with massive reprogramming in signalling transduction pathways. This in vitro system may serve to study the response of berry composition to nutrient factors and hormones, and their interaction with environmental factors (e.g. light and temperature), which can all be finely tuned and controlled.