A molecular phenology scale of grape berry development

Fruit growth and development consist of a continuous succession of physical,biochemical,and physiological changes driven by a genetic program that dynamically responds to environmental cues.Establishing recognizable stages over the whole fruit lifetime represents a fundamental requirement for research and fruit crop cultivation.This is especially relevant in perennial crops like grapevine(Vitis vinifera L.)to scale the development of its fruit across genotypes and growing conditions.In this work,molecular-based information from several grape berry transcriptomic datasets was exploited to build a molecular phenology scale(MPhS)and to map the ontogenic development of the fruit.The proposed statistical pipeline consisted of an unsupervised learning procedure yielding an innovative combination of semiparametric,smoothing,and dimensionality reduction tools.The transcriptomic distance between fruit samples was precisely quantified by means of the MPhS that also enabled to highlight the complex dynamics of the transcriptional program over berry development through the calculation of the rate of variation of MPhS stages by time.The MPhS allowed the alignment of time-series fruit samples proving to be a complementary method for mapping the progression of grape berry development with higher detail compared to classic time-or phenotype-based approaches.

Dissecting the effect of soil on plant phenology and berry transcriptional plasticity in two Italian grapevine varieties (Vitis vinifera L.).

Grapevine embodies a fascinating species as regards phenotypic plasticity and genotype-per-environment interactions.The terroir,namely the set of agri-environmental factors to which a variety is subjected,can influence the phenotype at the physiological,molecular,and biochemical level,representing an important phenomenon connected to the typicality of productions.We investigated the determinants of plasticity by conducting a field-experiment where all terroir variables,except soil,were kept as constant as possible.We isolated the effect of soils collected from different areas,on phenology,physiology,and transcriptional responses of skin and flesh of a red and a white variety of great economic value:Corvina and Glera.Molecular results,together with physio-phenological parameters,suggest a specific effect of soil on grapevine plastic response,highlighting a higher transcriptional plasticity of Glera in respect to Corvina and a marked response of skin compared to flesh.Using a novel statistical approach,we identified clusters of plastic genes subjected to the specific influence of soil.These findings could represent an issue of applicative value,posing the basis for targeted agricultural practices to enhance the desired characteristics for any soil/cultivar combination,to improve vineyards management for a better resource usage and to valorize vineyards uniqueness maximizing the terroir-effect.

DNA-free genome editing in grapevine using CRISPR/Cas9 ribonucleoprotein complexes followed by protoplast regeneration

CRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding,offering enormous potential for crop improvement and food production.Although the direct delivery of Cas9-single guide RNA(sgRNA)ribonucleoprotein(RNP)complexes to grapevine(Vitis vinifera)protoplasts has been shown before,the regeneration of edited protoplasts into whole plants has not been reported.Here,we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus.As proof of concept,a single-copy green f luorescent protein reporter gene(GFP)in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts.CRISPR/Cas9 activity,guided by two independent sgRNAs,was confirmed by the loss of GFP f luorescence.The regeneration of GFP−protoplasts into whole plants was monitored throughout development,confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls.We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts,helping to address the regulatory concerns related to genetically modified plants.This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants.

Genome and transcriptome analysis of the grapevine(Vitis vinifera L.) WRKY gene family

The plant WRKY gene family represents an ancient and complex class of zinc-finger transcription factors(TFs)that are involved in the regulation of various physiological processes,such as development and senescence,and in plant response to many biotic and abiotic stresses.Despite the growing number of studies on the genomic organisation of WRKY gene family in different species,little information is available about this family in grapevine(Vitis vinifera L.).In the present study,a total number of 59 putative grapevine WRKY transcription factors(VvWRKYs)were identified based on the analysis of various genomic and proteomic grapevine databases.According to their structural and phylogentic features,the identified grapevine WRKY transcription factors were classified into three main groups.In order to shed light into their regulatory roles in growth and development as well as in response to biotic and abiotic stress in grapevine,the VvWRKYs expression profiles were examined in publicly available microarray data.Bioinformatics analysis of these data revealed distinct temporal and spatial expression patterns of VvWRKYs in various tissues,organs and developmental stages,as well as in response to biotic and abiotic stresses.To also extend our analysis to situations not covered by the arrays and to validate our results,the expression profiles of selected VvWRKYs in response to drought stress,Erysiphe necator(powdery mildew)infection,and hormone treatments(salicilic acid and ethylene),were investigated by quantitative real-time reverse transcription PCR(qRT-PCR).The present study provides a foundation for further comparative genomics and functional studies of this important class of transcriptional regulators in grapevine.

Rapid dehydration of grape berries dampens the post-ripening transcriptomic program and the metabolite profile evolution

The postharvest dehydration of grape berries allows the concentration of sugars and other solutes and promotes the synthesis of metabolites and aroma compounds unique to high-quality raisin wines such as the passito wines made in Italy.These dynamic changes are dependent on environmental parameters such as temperature and relative humidity,as well as endogenous factors such as berry morphology and genotype,but the contribution of each variable is not well understood.Here,we compared berries subjected to natural or accelerated dehydration,the latter driven by forced air flow.We followed the evolution of transcript and metabolite profiles and found that accelerated dehydration clearly dampened the natural transcriptomic and metabolomic programs of postharvest berries.We found that slow dehydration over a prolonged duration is necessary to induce gene expression and metabolite accumulation associated with the final quality traits of dehydrated berries.The accumulation of key metabolites(particularly stilbenoids)during postharvest dehydration is inhibited by rapid dehydration conditions that shorten the berry life time.