Abstract Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar‐proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.
SUMMARY The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine ( Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene‐free grapevine plants, exploiting the lipofectamine‐mediated direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo ( V. vinifera ), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG‐mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.
Abstract Priming modulates plant stress responses before the stress appears, increasing the ability of the primed plant to endure adverse conditions and thrive. In this context, we investigated the effect of biological (i.e. arbuscular mycorrhizal fungi, AMF) agents and natural compounds (i.e. salicylic acid applied alone or combined with chitosan) against water deficit and salinity on a commercial tomato genotype (cv. Moneymaker). Effects of seed treatments on AMF colonization were evaluated, demonstrating the possibility of using them in combination. Responses to water and salt stresses were analysed on primed plants alone or in combination with the AMF inoculum in soil. Trials were conducted on potted plants by subjecting them to water deficit or salt stress. The effectiveness of chemical seed treatments, both alone and in combination with post-germination AMF inoculation, was investigated using a multidisciplinary approach that included ecophysiology, biochemistry, transcriptomics, and untargeted metabolomics. Results showed that chemical seed treatment and arbuscular mycorrhizal symbiosis modified the tomato response to water deficit and salinity triggering a remodelling of both transcriptome and metabolome, which ultimately elicited the plant antioxidant and osmoprotective machinery. The plant physiological adaptation to both stress conditions improved, confirming the success of the adopted approaches in enhancing stress tolerance.
Plants have evolved different strategies to cope with environmental stresses and, although still debated, it was observed that they can remember past stress occurrence. Anatomical and physiological adjustments have been observed in different grapevine cultivars after repeated drought exposure, however epigenetic, transcriptional and biochemical changes associated with drought-primed ecological memory have been poorly studied. This work was conceived to test whether exposure to recurring events of mild drought could prime vines to endure severe drought stress. Particularly, we investigated whether the expected improved stress tolerance of Vitis vinifera cv Nebbiolo plants subjected over years to moderate and long-lasting water stress events (WS-primed) depended on molecular memory phenomena or on resetting of stress-induced signals. To this aim, a combined multidisciplinary approach, involving eco-physiological, anatomical, biochemical and molecular analyses was adopted. First results revealed that WS-primed vines had reduced gas exchange in well-watered conditions, but at the end of WS imposition were able to maintain higher transpiration and assimilation rates with respect to unprimed plants. Moreover, WS-primed plants accumulated lower amounts of root abscisic acid and had higher content of resveratrol and viniferin, suggesting an increased antioxidant capacity that could help them in counteracting stress effects at the cellular level. WGBS analysis is ongoing to profile changes in DNA methylation landscapes in search of epigenetic signatures associated with specific transcriptome and physiological modifications. In a future perspective, the gained information will deliver a predictive framework to estimate the impact of moderately dry periods on vine performance, considering memory-associated protective effects against drought.
Abstract Somatic embryogenesis (SE) represents the most appropriate tool for next-generation breeding methods in woody plants such as grapevine (Vitis vinifera L.). However, in this species, the SE competence is strongly genotype-dependent and the molecular basis of this phenomenon is poorly understood. We explored the genetic and epigenetic basis of SE in grapevine by profiling the transcriptome, epigenome, and small RNAome of undifferentiated, embryogenic, and non-embryogenic callus tissues derived from two genotypes differing in competence for SE, Sangiovese and Cabernet Sauvignon. During the successful formation of embryonic callus, we observed the upregulation of epigenetic-related transcripts and short interfering RNAs in association with DNA hypermethylation at transposable elements in both varieties. Nevertheless, the switch to nonembryonic development matched the incomplete reinforcement of transposon silencing, and the evidence of such effect was more apparent in the recalcitrant Cabernet Sauvignon. Transcriptomic differences between the two genotypes were maximized already at early stage of culture where the recalcitrant variety expressed a broad panel of genes related to stress responses and secondary metabolism. Our data provide a different angle on the SE molecular dynamics that can be exploited to leverage SE as a biotechnological tool for fruit crop breeding.
Wine viticulture, being firmly linked to the vine-terroir relationship, has always encountered significant bottlenecks to genetic innovation. Nonetheless, the development of new breeding strategies leading to the selection of stress resilient genotypes is urgent, especially in viticulture, where it would allow reducing the use of chemical treatments adopted to control fungal diseases. Genome editing represents an extremely promising breeding technique. Unfortunately, the well-known recalcitrance of several wine grape cultivars to in vitro regeneration strongly limits the exploitation of this approach, which to our knowledge has so far been developed on table grape genotypes with high regeneration potential. By targeting the phytoene desaturase gene as visual editing proof, we developed a genome editing and regeneration protocol to produce transgene-free grapevine plants exploiting the lipofectamine–mediated delivery of CRISPR–Cas9 ribonucleoproteins into protoplasts. We regenerated edited grapevines of Vitis vinifera'Nebbiolo', a cultivar extremely recalcitrant to in vitro regeneration and at the basis of outstanding quality wines, such as 'Barolo' and 'Barbaresco'. Successful editing was confirmed by a combination of approaches: HRM, Sanger and amplicon deep sequencing, phenotype visualization. We then exploited the method to silence two micro(mi)RNAs involved in biotic stress responses: vv-miR482, which is conserved in different species, and the grapevine-specific vv-miR3623. Since NBS-LRR disease-resistance genes are the targets of those miRNAs, the objective is to regenerate vines with a broad-spectrum level of plant tolerance/resistance to different pathogens. The developed strategy could be extended to other important wine grape varieties and recalcitrant woody species.
Shelf life is an important quality trait for many fruit, including tomatoes. We report that enrichment of anthocyanin, a natural pigment, in tomatoes can significantly extend shelf life. Processes late in ripening are suppressed by anthocyanin accumulation, and susceptibility to Botrytis cinerea, one of the most important postharvest pathogens, is reduced in purple tomato fruit. We show that reduced susceptibility to B. cinerea is dependent specifically on the accumulation of anthocyanins, which alter the spreading of the ROS burst during infection. The increased antioxidant capacity of purple fruit likely slows the processes of overripening. Enhancing the levels of natural antioxidants in tomato provides a novel strategy for extending shelf life by genetic engineering or conventional breeding.
Abstract Despite microbe-based products for grapevine protection and growth improvement are already available, a few of them contain microbes directly isolated from vine tissues. For this reason, a collection of endophytic bacterial isolates obtained directly from grapevine woody tissues has been used for producing an ad-hoc inoculum. The selected bacterial isolates were tested in biocontrol assays against some of the main grapevine pathogens ( e.g. , Botrytis cinerea , Guignardia bidwellii , Neofusicoccum parvum ) and the best performing ones were screened for plant growth promoting (PGP)-traits ( e.g. , phosphorous solubilization, indole-acetic acid and siderophore production). Before being planted, rooted cuttings were inoculated with two different synthetic communities: the first one was an ad-hoc developed microbial community (SynCom), whereas the second was a commercial consortium formed by arbuscular mycorrhizal fungi (AMF) and a rhizosphere bacterial strain (AMF+B). Physiological parameters were monitored to evaluate effects on plant performances, and samples for biochemical and molecular analyses were collected. Integration of physiological data with metabolite profiling and transcriptome sequencing highlighted that the SynCom treatment shaped the plant growth-defence trade-off, by regulating photosynthesis and diverting energy sources towards the activation of defence metabolic pathways. On the other hand, the AMF+B treatment led to a more balanced growth-defence trade-off, though a mild activation of defence mechanisms was also observed in these plants. Our findings suggest that an experimental approach considering both the features of associated microbes and their impacts on plant growth and defence could shed light on the “dark-side effects” of SynCom application, thereby enabling their exploitation with a refined awareness.
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