The dynamics of embolism refilling in abscisic acid (ABA)-deficient tomato plants.

Plants are in danger of embolism formation in xylem vessels when the balance between water transport capacity and transpirational demand is compromised. To maintain this delicate balance, plants must regulate the rate of transpiration and, if necessary, restore water transport in embolized vessels. Abscisic acid (ABA) is the dominant long-distance signal responsible for plant response to stress, and it is possible that it plays a role in the embolism/refilling cycle. To test this idea, a temporal analysis of embolism and refilling dynamics, transpiration rate and starch content was performed on ABA-deficient mutant tomato plants. ABA-deficient mutants were more vulnerable to embolism formation than wild-type plants, and application of exogenous ABA had no effect on vulnerability. However, mutant plants treated with exogenous ABA had lower stomatal conductance and reduced starch content in the xylem parenchyma cells. The lower starch content could have an indirect effect on the plant's refilling activity. The results confirm that plants with high starch content (moderately stressed mutant plants) were more likely to recover from loss of water transport capacity than plants with low starch content (mutant plants with application of exogenous ABA) or plants experiencing severe water stress. This study demonstrates that ABA most likely does not play any direct role in embolism refilling, but through the modulation of carbohydrate content, it could influence the plant's capacity for refilling

New plant breeding technologies towards a more sustainable viticulture

European grapevine cultivars are highly susceptible to many pathogens that are managed through large pesticide use. Nevertheless, the European policies promote pesticide use reduction and new environmentally friendly methods for a more sustainable agriculture. In this framework, grapevine genetic improvement could benefit from New Plant Breeding Technologies. In order to reduce fungal susceptibility, we will produce knock-out plants from embryogenic calli using CRISPR/Cas9 technology. Studies in barley reported the acquisition of powdery mildew resistance by knocking out susceptibility genes belonging to the MLO (Mildew Locus O) family. In this study, our approach takes advantage from CRISPR/Cas9 technology to perform a multiple knockout of MLO genes. Among the 17 VvMLOs reported in grapevine we designed constructs to target VvMLO6 and VvMLO7. Golden Gate assembly was used to produce three different constructs (containing two guideRNAs for each gene) to knocking-out the targets singularly or by producing a double mutant. Usually, the genetic engineering techniques, mediated by A. tumefaciens, involve the insertion of exogenous selectable marker genes. These markers are required for selection of transgenic plants, but they are undesirable to be retained in commercial transgenic plants due to possible toxicity or allergenicity to humans and potential environmental hazard. To overcome these limits, we opted for a \u201cclean\u201d editing strategy developing an inducible excision system. This approach is based on a recombinase technology involving the Cre-loxP system from the P1 bacteriophage under a heat-shock inducible promoter to be activated once the editing event(s) will be confirmed. Obtainment of embryogenic calli is one of the main bottlenecks for application of CRISPR/Cas9: for two seasons, we collected inflorescences from Chardonnay, Glera, Microvine, Pinot Noir, Sangiovese cultivars and two rootstocks, 110 Richter and SO4, cultured and maintained in vitro up to embryo development and then used to perform Agrobacterium tumefaciens GV3101 mediated transformation

miRVIT: A Novel miRNA Database and Its Application to Uncover Vitis Responses to Flavescence dorée Infection

Micro(mi)RNAs play crucial roles in plant developmental processes and in defense responses to biotic and abiotic stresses. In the last years, many works on small RNAs in grapevine (Vitis spp.) were published, and several conserved and putative novel grapevine-specific miRNAs were identified. In order to reorganize the high quantity of available data, we produced “miRVIT,” the first database of all novel grapevine miRNA candidates characterized so far, and still not deposited in miRBase. To this aim, each miRNA accession was renamed, repositioned in the last version of the grapevine genome, and compared with all the novel and conserved miRNAs detected in grapevine. Conserved and novel miRNAs cataloged in miRVIT were then used for analyzing Vitis vinifera plants infected by Flavescence dorée (FD), one of the most severe phytoplasma diseases affecting grapevine. The analysis of small RNAs from healthy, recovered (plants showing spontaneous and stable remission of symptoms), and FD-infected “Barbera” grapevines showed that FD altered the expression profiles of several miRNAs, including those involved in cell development and photosynthesis, jasmonate signaling, and disease resistance response. The application of miRVIT in a biological context confirmed the effectiveness of the followed approach, especially for the identification of novel miRNA candidates in grapevine. miRVIT database is available at http://mirvit.ipsp.cnr.it.Highlights: The application of the newly produced database of grapevine novel miRNAs to the analysis of plants infected by Flavescence dorée reveals key roles of miRNAs in photosynthesis and jasmonate signaling