• Energy Research

Stomatal density and distribution are under the control of small cysteine-rich peptides called epidermal patterning factors (EPFs), highly conserved in a wide range of higher plants. In Arabidopsis and cereals, three members of this family play a key role in the formation of stomata: EPF1, EPF2 and EPFL9. EPF2 and EPF1 prevent stomata formation while EPFL9 is a positive regulator, acting antagonistically to the other two. In this work, CRISPR/Cas9 technology was applied to investigate the role of VvEPFL9-1 gene in grapevine via knockout (KO) mutants. Gene transfer experiments were carried out co-culturing embryogenic calli of a table grape and a rootstock with Agrobacterium tumefaciens carrying the binary vector with a specific sgRNA. Edited lines were phenotypically characterized under well-watered and water-stressed conditions using a full suite of physiological parameters. Vvepfl-1 KO plants showed a reduction in stomatal density compared to the wild type (WT) followed by an improved water-use efficiency both under optimal and water stress conditions. In addition, ad-hoc experiments, carried out to test micrografted combinations of KO and WT lines, showed a moderate gain in water-use efficiency when micrografts were formed by an edited scion or rootstock. Our results highlight the possibility to optimize water-use efficiency by manipulating stomatal density via VvEPFL9-1 knockout and suggests stomatal anatomical features as a promising target for climate change resilient grapevine varieties.

Epidermal Patterning Factor Like 9 (EPFL9), also known as STOMAGEN, is a cysteine-rich peptide that induces stomata formation in vascular plants, acting antagonistically to other epidermal patterning factors (EPF1, EPF2). In grapevine there are two EPFL9 genes, EPFL9-1 and EPFL9-2 sharing 82% identity at protein level in the mature functional C-terminal domain. In this study, CRISPR/Cas9 system was applied to functionally characterize VvEPFL9-1 in ‘Sugraone’, a highly transformable genotype. A set of plants, regenerated after gene transfer in embryogenic calli via Agrobacterium tumefaciens, were selected for evaluation. For many lines, the editing profile in the target site displayed a range of mutations mainly causing frameshift in the coding sequence or affecting the second cysteine residue. The analysis of stomata density revealed that in edited plants the number of stomata was significantly reduced compared to control, demonstrating for the first time the role of EPFL9 in a perennial fruit crop. Three edited lines were then assessed for growth, photosynthesis, stomatal conductance, and water use efficiency in experiments carried out at different environmental conditions. Intrinsic water-use efficiency was improved in edited lines compared to control, indicating possible advantages in reducing stomatal density under future environmental drier scenarios. Our results show the potential of manipulating stomatal density for optimizing grapevine adaptation under changing climate conditions.

Abstract Climate change and rapid adaption of invasive pathogens pose a constant pressure on the fruit industry to develop improved varieties. Aiming to accelerate the development of better-adapted cultivars, new breeding techniques have emerged as a promising alternative to meet the demand of a growing global population. Accelerated breeding, cisgenesis, and CRISPR/Cas genome editing hold significant potential for crop trait improvement and have proven to be useful in several plant species. This review focuses on the successful application of these technologies in fruit trees to confer pathogen resistance and tolerance to abiotic stress and improve quality traits. In addition, we review the optimization and diversification of CRISPR/Cas genome editing tools applied to fruit trees, such as multiplexing, CRISPR/Cas-mediated base editing and site-specific recombination systems. Advances in protoplast regeneration and delivery techniques, including the use of nanoparticles and viral-derived replicons, are described for the obtention of exogenous DNA-free fruit tree species. The regulatory landscape and broader social acceptability for cisgenesis and CRISPR/Cas genome editing are also discussed. Altogether, this review provides an overview of the versatility of applications for fruit crop improvement, as well as current challenges that deserve attention for further optimization and potential implementation of new breeding techniques.

The necessity to adapt to climate change is even stronger for grapevine than for other crops, because grape berry composition—a key determinant of fruit and wine quality, typicity and market value— highly depends on “terroir” (complete natural environment), on vintage (annual climate variability), and on their interactions. In the same time, there is a strong demand to reduce the use of pesticides. Thus, the equation that breeders and grape growers must solve has three entries that cannot be dissociated: adaptation to climate change, reduction of pesticides, and maintenance of wine typicity. Although vineyard management may cope to some extent to the short–medium-term effects of climate change, genetic improvement is necessary to provide long-term sustainable solutions to these problems. Most vineyards over the world are planted using vines that harbor two grafted plants’ genomes. Although this makes the range of interactions (scion-atmosphere, rootstock-soil, scion-rootstock) more complex, it also opens up wider possibilities for the genetic improvement of either or both the grafted genotypes. Positive aspects related to grapevine breeding are as follows: (a) a wide genetic diversity of rootstocks and scions that has not been thoroughly explored yet; (b) progress in sequencing technologies that allows high-throughput sequencing of entire genomes, faster mapping of targeted traits and easier determination of genetic relationships; (c) progress in new breeding technologies that potentially permit precise modifications on resident genes; (d) automation of phenotyping that allows faster and more complete monitoring of many traits on relatively large plant populations; (e) functional characterization of an increasing number of genes involved in the control of development, berry metabolism, disease resistance, and adaptation to environment. Difficulties involve: (a) the perennial nature and the large size of the plant that makes field testing long and demanding in manpower; (b) the low efficiency of transformation, regeneration and small size of breeding populations; (c) the complexity of the adaptive traits and the need to define more clearly future ideotypes; (d) the lack of shared and integrative platforms allowing a complete appraisal of the genotype-phenotype-environmental links; (e) legal, market and consumer acceptance of new genotypes. The present chapter provides an overview of suitable strategies and challenges linked to the adaptation of viticulture to a changing environment.