GRAPERIPE · The role of ethylene and of ERF gene regulators in the grapevine berry ripening

The role of ethylene and of ERF gene regulators in the grapevine berry ripening

The aims of this project were to understand the role of ethylene, a volatile plant hormone, in fruit ripening of non climacteric species, in particular grapevine, and to characterize the role in planta of the ethylene responsive factor VvERF045, a berry specific transcription factor highly expressed during ripening. Traditionally fruits are classified as climacteric, when an increase in respiration and a burst of ethylene occur during ripening, unlike when these processes are absent or extremely limited they are considered non-climacteric. Grapevine is one of the most economically important crops in the world and the fruit is consumed either fresh and dried or, once fermented, as wine and distilled beverages. Almost half of the entire world vineyard is located in Europe. Fruit ripening is a complex developmental process whereby the fruit undergoes dramatic changes that promote seed dispersal and that greatly impact the final fruit quality. Climate change is nowadays negatively affecting European viticulture especially in the regions facing the Mediterranean basin where the high temperatures are causing accelerated ripening, excessive sugar content and drought stress. The objective of this study, namely dissecting the ethylene-mediated molecular processes behind grapevine berry ripening, is then of great relevance for the viticulture and enology sectors but also for our understanding of fruit ripening in climacteric and non climacteric fruits. - To address the role of ethylene in fruit ripening during the project we worked with grapevine and melon. In grapevine we were able to measure, for the first time, ethylene emitted by Pinot noir grapes attached to the vine by using highly sophisticated and sensitive instruments. The measurement in ten different cultivated varieties highlighted that indeed at the beginning of berry ripening there is ethylene emission but the level is very low, difficult to detect, and comparable among the tested varieties. A step further in this research was taken by treating Pinot noir grapes around color change in the field with 1-MCP, a chemical which blocks ethylene activity. Samples were then collected at different time points after treatment and several biochemical parameters, such as color, acidity, sugar and polyphenol content were determined to understand the impact of blocking the ethylene signal. Global gene expression analysis was also carried out on the same samples. Preliminary analysis of the data indicates a specific but important role of ethylene in the grape ripening process. Melon is a nice model to study climacteric and non climacteric ripening because it comprises varieties which display the two different ripening behaviors. In collaboration with the group of B. Pico at the Universitat Politècnica of Valencia we studied the variability in ripening behavior and sugar accumulation in a collection of 175 melon accessions of different origin. The same samples were also investigated at the genetic level to score the DNA sequence variability in 53 candidate genes involved in sugar accumulation and fruit ripening processes. The association of the phenotypic variability to the genetic variability has allowed to identify several candidate genes likely involved in the melon ripening phenotypic diversity. The results were published in a scientific article [LEIDA C. et al., 2015. Variability of candidate genes and genetic association for sugar accumulation and climacteric behavior in melon (Cucumis melo L.). BMC Genetics 16:28. DOI :10.1186/s12863-015-0183-2]. - To address the role of the grapevine ethylene responsive factor VvERF045 we analyzed grapevine transgenic lines grown in vitro that over-express this transcription factor. Sequence and phylogenetic analysis of the amino acid sequence of VvERF045 demonstrated that it is a close relative to the SHINE genes from Arabidopsis and tomato known to be involved in the regulation of wax biosynthesis and cuticle morphology in these species. A broad phenotypic and molecular characterization of the in vitro grown lines was then undertaken to understand the role of VvERF045 in planta. We measured the emission of ethylene and volatile organic compounds (VOCs), evaluated plant morphology (leaf area, color, insertion angle, root shape) and leaf surface via microscope analysis and looked at the effect of the transgene over-expression on the transcriptome analysis of one of the lines. The results demonstrated that the effect of VvERF045 on the phenotype was related to the level of transgene expression: high-expressing lines showed stunted growth, discolored and smaller leaves, and a lower level of chlorophylls and carotenoids. The transcriptomic analysis revealed 573 differentially expressed genes in the transgenic line compared to wild type plants. The main result, supported by optical and cryo-electron microscopy as well as gene expression analysis, was the observation that VvERF045 plays a major role in leaf epidermis patterning by acting on waxes and cuticle. Our data also suggested that VvERF045 affects polyphenol secondary metabolism and induces a reaction resembling a plant immune response with modulation of receptor like-kinases and pathogen related genes. These results have been summarized in an article submitted to Journal of Experimental Botany (LEIDA C. et al. “The role of the grapevine berry-specific ethylene responsive factor VvERF045.”) The results of this study have several impacts: - Lead Users: the immediate users of the research results will be other scientists working in the field of fruit ripening, grapevine molecular biology, ethylene role. - Outcome: 2 scientific publications in high ranking journals, 1 Master thesis - Exploitation: The transgenic grapevine plants characterized in this project have shown an altered morphology and structure of the leaf epicuticular waxes. This phenotype can be interesting for future exploitation because these plants might be more resilient to biotic and abiotic stresses (e.g. drought because of reduced water transpiration). - Dissemination: the results of the project have been presented at six international and national meetings Apart the scientific community, other target groups interested in the results stemmed from the project could be the communities of grapevine breeders and grapevine growers (treatments with 1-MCP to control ripening). Contacts: Carmen Leida (carmen.leida@gmail.com) Claudio Moser (claudio.moser@fmach.it)

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