Genome editing of a recalcitrant wine grape genotype by lipofectamine-mediated delivery of CRISPR/Cas9 ribonucleoproteins to protoplasts.

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.

Photosynthetic recovery in drought-rehydrated grapevines is associated with high demand from the sinks, maximizing the fruit-oriented performance.

To understand how grapevine sinks compete with each other during water stress and subsequent rehydration, carbon (C) allocation patterns in drought-rehydrated vines (REC) at the beginning of fruit ripening were compared with control vines maintained under drought (WS) or fully irrigated (WW). In the 30 days following rehydration, the quantity and distribution of newly fixed C between leaves, roots and fruits was evaluated through 13 CO2 pulse-labeling and stable isotope ratio mass spectrometry. REC plants diverted the same percentage of fixed C towards the berries as the WS plants, although the percentage was higher than that of WW plants. Net photosynthesis (measured simultaneously with root respiration in a multichamber system for analysis of gas exchange above- and below-ground) was approximately two-fold greater in REC compared to WS treatment, and comparable or even higher than in WW plants. Maximizing C assimilation and delivery in REC plants led to a significantly higher amount of newly fixed C compared to both control treatments, already 2 days after rehydration in root, and 2 days later in the berries, in line with the expression of genes responsible for sugar metabolism. In REC plants, the increase in C assimilation was able to support the requests of the sinks during fruit ripening, without affecting the reserves, as was the case in WS. These mechanisms clarify what is experienced in fruit crops, when occasional rain or irrigation events are more effective in determining sugar delivery towards fruits, rather than constant and satisfactory water availabilities.

Stress responses and epigenomic instability mark the loss of somatic embryogenesis competence in grapevine.

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.

The Role of Italy in the Use of Advanced Plant Genomic Techniques on Fruit Trees: State of the Art and Future Perspectives.

Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the 'Made in Italy' label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The 'new genomic techniques' (NGTs), renamed in Italy as 'technologies for assisted evolution' (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.

'Nebbiolo' genome assembly allows surveying the occurrence and functional implications of genomic structural variations in grapevines (Vitis vinifera L.).

BACKGROUND: 'Nebbiolo' is a grapevine cultivar typical of north-western Italy, appreciated for producing high-quality red wines. Grapevine cultivars are characterized by possessing highly heterozygous genomes, including a great incidence of genomic rearrangements larger than 50 bp, so called structural variations (SVs). Even though abundant, SVs are an under-explored source of genetic variation mainly due to methodological limitations at their detection. RESULTS: We employed a multiple platform approach to produce long-range genomic data for two different 'Nebbiolo' clones, namely: optical mapping, long-reads and linked-reads. We performed a haplotype-resolved de novo assembly for cultivar 'Nebbiolo' (clone CVT 71) and used an ab-initio strategy to annotate it. The annotated assembly enhanced our ability to detect SVs, enabling the study of genomic regions not present in the grapevines' reference genome and accounting for their functional implications. We performed variant calling analyses at three different organizational levels: i) between haplotypes of clone CVT 71 (primary assembly vs haplotigs), ii) between 'Nebbiolo' and 'Cabernet Sauvignon' assemblies and iii) between clones CVT 71 and CVT 185, representing different 'Nebbiolo' biotypes. The cumulative size of non-redundant merged SVs indicated a total of 79.6 Mbp for the first comparison and 136.1 Mbp for the second one, while no SVs were detected for the third comparison. Interestingly, SVs differentiating cultivars and haplotypes affected similar numbers of coding genes. CONCLUSIONS: Our results suggest that SVs accumulation rate and their functional implications in 'Nebbiolo' genome are highly-dependent on the organizational level under study. SVs are abundant when comparing 'Nebbiolo' to a different cultivar or the two haplotypes of the same individual, while they turned absent between the two analysed clones.

Molecular basis of differential adventitious rooting competence in poplar genotypes.

Recalcitrant adventitious root (AR) development is a major hurdle in propagating commercially important woody plants. Although significant progress has been made to identify genes involved in subsequent steps of AR development, the molecular basis of differences in apparent recalcitrance to form AR between easy-to-root and difficult-to-root genotypes remains unknown. To address this, we generated cambium tissue-specific transcriptomic data from stem cuttings of hybrid aspen, T89 (difficult-to-root) and hybrid poplar OP42 (easy-to-root), and used transgenic approaches to verify the role of several transcription factors in the control of adventitious rooting. Increased peroxidase activity was positively correlated with better rooting. We found differentially expressed genes encoding reactive oxygen species scavenging proteins to be enriched in OP42 compared with T89. A greater number of differentially expressed transcription factors in cambium cells of OP42 compared with T89 was revealed by a more intense transcriptional reprograming in the former. PtMYC2, a potential negative regulator, was less expressed in OP42 compared with T89. Using transgenic approaches, we demonstrated that PttARF17.1 and PttMYC2.1 negatively regulate adventitious rooting. Our results provide insights into the molecular basis of genotypic differences in AR and implicate differential expression of the master regulator MYC2 as a critical player in this process.

Somatic embryogenesis is an effective strategy for dissecting chimerism phenomena in Vitis vinifera cv Nebbiolo.

KEY MESSAGE: The tendency of somatic embryogenesis to regenerate plants only from the L1 layer, associated with the spread of chimerism in grapevine, must be carefully considered in the framework of biotechnological improvement programmes. Grapevine is an important fruit crop with a high economic value linked to traditional genotypes that have been multiplied for centuries by vegetative propagation. In this way, somatic variations that can spontaneously occur within the shoot apical meristem are fixed in the whole plant and represent a source of intra-varietal variability. Previously identified inconsistencies in the allelic calls of single nucleotide variants (SNVs) suggested that the Vitis vinifera 'Nebbiolo' CVT185 clone is a potential periclinal chimera. We adopted the somatic embryogenesis technique to separate the two genotypes putatively associated with the L1 and L2 layers of CVT185 into different somaclones. Despite the recalcitrance of 'Nebbiolo' to the embryogenic process, 58 somaclones were regenerated and SNV genotyping assays attested that the genotype of all them differed from that of the mother plant and was only attributable to L1. The results confirmed that L2 has low or no competence for differentiating somatic embryos. After one year in the greenhouse, the somaclones showed no phenotypic alterations in comparison with the mother plant; however further analyses are needed to identify potential endogenous sources of variation. The tendency of somatic embryogenesis to regenerate plants only from L1 must be carefully considered in the framework of biotechnological improvement programmes in this species.

Secondary Metabolism and Defense Responses Are Differently Regulated in Two Grapevine Cultivars during Ripening.

Vitis vinifera 'Nebbiolo' is one of the most important wine grape cultivars used to produce prestigious high-quality wines known throughout the world, such as Barolo and Barbaresco. 'Nebbiolo' is a distinctive genotype characterized by medium/high vigor, long vegetative and ripening cycles, and limited berry skin color rich in 3'-hydroxylated anthocyanins. To investigate the molecular basis of these characteristics, 'Nebbiolo' berries collected at three different stages of ripening (berry pea size, véraison, and harvest) were compared with V. vinifera 'Barbera' berries, which are rich in 3',5'-hydroxylated anthocyanins, using transcriptomic and analytical approaches. In two consecutive seasons, the two genotypes confirmed their characteristic anthocyanin profiles associated with a different modulation of their transcriptomes during ripening. Secondary metabolism and response to stress were the functional categories that most differentially changed between 'Nebbiolo' and 'Barbera'. The profile rich in 3'-hydroxylated anthocyanins of 'Nebbiolo' was likely linked to a transcriptional downregulation of key genes of anthocyanin biosynthesis. In addition, at berry pea size, the defense metabolism was more active in 'Nebbiolo' than 'Barbera' in absence of biotic attacks. Accordingly, several pathogenesis-related proteins, WRKY transcription factors, and stilbene synthase genes were overexpressed in 'Nebbiolo', suggesting an interesting specific regulation of defense pathways in this genotype that deserves to be further explored.

VvPIP2;4N aquaporin involvement in controlling leaf hydraulic capacitance and resistance in grapevine.

Hydraulic capacitance (C) in a plant tissue buffers the xylem tension, storing and releasing water and has been highlighted in recent years as an important factor that affects water relations such as drought tolerance and embolism formation. Aquaporins (AQPs) are well known to control leaf hydraulic resistance (Rh) but their role in the control of C is unknown. Here, we assess Rh and C on detached grapevines wild-type (WT) (cv. Brachetto) leaves and over-expressing the aquaporin gene VvPIP2;4N (OE). For this purpose, we developed a new method inspired from the pressure-volume curve technique and the rehydration-kinetic-method, which allowed us to monitor the dynamics of dehydration and rehydration in the same leaf. The recovery after dehydration was measured in dark, light non-transpirative conditions, light-transpirative conditions and light-transpirative condition adding abscisic acid. Pressurizing to dehydrate leaves in the OE line, the recorded Rh and C were respectively lower and higher than those in the WT. The same results were obtained in the dark recovery by rehydration treatment. In the presence of light, either when leaves transpired or not (by depressing vapor pressure deficit), the described effects disappeared. The change in Rh and C did not affect the kinetics of desiccation of detached leaves in dark in air, in OE plants compared to WT ones. Our study highlighted that both Rh and C were influenced by the constitutive over-expression of VvPIP2;4N. The effect of AQPs on C is reported here for the first time and may involve a modulation of cell reflection coefficient.

Gene expression in vessel-associated cells upon xylem embolism repair in Vitis vinifera L. petioles.

In this work, the involvement of vessel-associated cells in embolism recovery was investigated by studying leaf petiole hydraulics and expression profiles of aquaporins and genes related to sugar metabolism. Two different stress treatments were imposed onto grapevines to induce xylem embolism: one involved a pressure collar applied to the stems, while the other consisted of water deprivation (drought). Embolism formation and repair were monitored during stress application and release (recovery). At the same time, stomatal conductance (g(s)), leaf water potential (Ψ(leaf)) and leaf abscisic acid (ABA) concentration were measured. For each treatment, gene transcript levels were assessed on vessel-associated cells (isolated from leaf petioles by laser microdissection technique) and whole petioles. Both treatments induced severe xylem embolism formation and drops in g s and Ψ (leaf) at a lesser degree and with faster recovery in the case of application of the pressure collar. Leaf ABA concentration only increased upon drought and subsequent recovery. Transcripts linked to sugar mobilisation (encoding a ß-amylase and a glucose-6-P transporter) were over-expressed upon stress or recovery, both in vessel-associated cells and whole petioles. However, two aquaporin genes (VvPIP2;1 and VvPIP2;4N) were activated upon stress or recovery only in vessel-associated cells, suggesting a specific effect on embolism refilling. Furthermore, the latter gene was only activated upon drought and subsequent recovery, suggesting that either severe water stress or ABA is required for its regulation.

Auxin is a central player in the hormone cross-talks that control adventitious rooting.

Vegetative propagation of economically important woody, horticultural and agricultural species rely on an efficient adventitious root (AR) formation. The formation of ARs is a complex genetic trait regulated by the interaction of environmental and endogenous factors among which the phytohormone auxin plays an essential role. This article summarizes the current knowledge related to the intricate network through which auxin controls adventitious rooting. How auxin and recently identified auxin-related compounds affect AR formation in different plant species is discussed. Particular attention is addressed to illustrate how auxin has a central role in the hormone cross-talk leading to AR development. In parallel, we describe the molecular players involved in the control of auxin homeostasis, transport and signaling, for a better understanding of the auxin action during adventitious rooting.

The AINTEGUMENTA LIKE1 homeotic transcription factor PtAIL1 controls the formation of adventitious root primordia in poplar.

Adventitious rooting is an essential but sometimes rate-limiting step in the clonal multiplication of elite tree germplasm, because the ability to form roots declines rapidly with age in mature adult plant tissues. In spite of the importance of adventitious rooting, the mechanism behind this developmental process remains poorly understood. We have described the transcriptional profiles that are associated with the developmental stages of adventitious root formation in the model tree poplar (Populus trichocarpa). Transcriptome analyses indicate a highly specific temporal induction of the AINTEGUMENTA LIKE1 (PtAIL1) transcription factor of the AP2 family during adventitious root formation. Transgenic poplar samples that overexpressed PtAIL1 were able to grow an increased number of adventitious roots, whereas RNA interference mediated the down-expression of PtAIL1 expression, which led to a delay in adventitious root formation. Microarray analysis showed that the expression of 15 genes, including the transcription factors AGAMOUS-Like6 and MYB36, was overexpressed in the stem tissues that generated root primordia in PtAIL1-overexpressing plants, whereas their expression was reduced in the RNA interference lines. These results demonstrate that PtAIL1 is a positive regulator of poplar rooting that acts early in the development of adventitious roots.

The grapevine root-specific aquaporin VvPIP2;4N controls root hydraulic conductance and leaf gas exchange under well-watered conditions but not under water stress.

We functionally characterized the grape (Vitis vinifera) VvPIP2;4N (for Plasma membrane Intrinsic Protein) aquaporin gene. Expression of VvPIP2;4N in Xenopus laevis oocytes increased their swelling rate 54-fold. Northern blot and quantitative reverse transcription-polymerase chain reaction analyses showed that VvPIP2;4N is the most expressed PIP2 gene in root. In situ hybridization confirmed root localization in the cortical parenchyma and close to the endodermis. We then constitutively overexpressed VvPIP2;4N in grape 'Brachetto', and in the resulting transgenic plants we analyzed (1) the expression of endogenous and transgenic VvPIP2;4N and of four other aquaporins, (2) whole-plant, root, and leaf ecophysiological parameters, and (3) leaf abscisic acid content. Expression of transgenic VvPIP2;4N inhibited neither the expression of the endogenous gene nor that of other PIP aquaporins in both root and leaf. Under well-watered conditions, transgenic plants showed higher stomatal conductance, gas exchange, and shoot growth. The expression level of VvPIP2;4N (endogenous + transgene) was inversely correlated to root hydraulic resistance. The leaf component of total plant hydraulic resistance was low and unaffected by overexpression of VvPIP2;4N. Upon water stress, the overexpression of VvPIP2;4N induced a surge in leaf abscisic acid content and a decrease in stomatal conductance and leaf gas exchange. Our results show that aquaporin-mediated modifications of root hydraulics play a substantial role in the regulation of water flow in well-watered grapevine plants, while they have a minor role upon drought, probably because other signals, such as abscisic acid, take over the control of water flow.

Recovery from water stress affects grape leaf petiole transcriptome.

Fast and efficient recovery from water stress is a key determinant of plant adaptation to changing meteorological conditions modulating transpiration, i.e. air temperature and humidity. We analysed transcriptomic responses during rehydration after water stress in grapevine leaf petioles, where embolism formation and repair commonly take place, and where metabolic changes related to embolism recovery are expected to be particularly important. We compared gene expression of recovering plants with irrigated controls, upon high and low transpiration conditions, using cDNA microarrays. In parallel, we assessed the daily dynamics of water relations, embolism formation and repair, and leaf abscisic acid concentration. In recovering plants, the most affected gene categories were secondary metabolism, including genes linked to flavonoid biosynthesis; sugar metabolism and transport, and several aquaporin genes. The physiological dynamics of recovery were lower and the number of differentially expressed probes was much lower upon low transpiration than found in actively transpiring grapevines, suggesting the existence of a more intense metabolic reorganization upon high transpiration conditions and of a signal eliciting these responses. In plants recovering under high transpiration, abscisic acid concentrations significantly increased, and, in parallel, transcripts linked to abscisic acid metabolism and signalling (ABA-8'-hydroxylase, serine-threonine kinases, RD22 proteins) were upregulated; a trend that was not observed upon low transpiration. Our results show that recovery from water stress elicits complex transcriptomic responses in grapevine. The increase observed in abscisic acid cellular levels could represent a signal triggering the activation of responses to rehydration after stress.

Isolation of a gene encoding for a class III peroxidase in female flower of Corylus avellana L.

Hazelnut is a monoecious species characterized by mid-winter blooming and sporophytic incompatibility. The molecular mechanisms at the basis of the female flower development and of the pollen-stigma interaction are little known, although pollination in this species is a critical factor to ensure good yield. Differential display technique was used to study genes expressed during the female flower development, comparing styles before emergence from the bud and styles at full bloom. The full-length cDNA clone, designated CavPrx (Corylus avellana peroxidase) and isolated in mature styles, was characterized as a sequence encoding for a 330 amino acids protein, containing all the conserved features of class III peroxidases. CavPrx resulted expressed only in styles, with a peak in mature styles pollinated with compatible pollen. Class III peroxidases are expressed in several different plant tissue types and are involved in a broad spectrum of physiological processes. Until now, four peroxidases expressed in the stigma were identified in Arabidopsis thaliana and Senecio squalidus: they were assumed to be possibly involved in pollen-pistil interaction, pollen tube penetration/growth and/or in defence against pathogens. CavPrx is the first gene for a floral peroxidase isolated in hazelnut and its expression pattern suggests a possible role in the pollination process.

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.

Somatic Embryogenesis as a Tool for Studying Grapevine-Virus Interaction.

More than 80 viral species, many of which are not associated with a clear disease or symptomatology, can infect grapevine. The study of grapevine-virus interactions in recent years is playing an increasingly important role and these studies have shown that the molecular and physiological responses to a virus greatly vary depending on the viral strains, the presence of multiple viral infections, the grapevine genotype, and the environment. Moreover, due to the characteristics of the grapevine cultivation and its vegetative propagation, it is very difficult to find healthy plants in vineyards to use them as control in the experiments. Starting from these considerations, in order to investigate the plant-virus interaction in an unbiased way, it is important to set up an experimental system able to control as much of these variables as possible. The protocol here proposed provides the overcome some of these factors by: (i) the production of healthy plants by somatic embryogenesis; (ii) the virus transmission using in vitro micrografting, and (iii) the transfer of in vitro plants to ex-vitro conditions for the analysis of interest.

Unlocking grapevine in vitro regeneration: Issues and perspectives for genetic improvement and functional genomic studies.

In vitro plant regeneration is a pivotal process in genetic engineering to obtain large numbers of transgenic, cisgenic and gene edited plants in the frame of functional gene or genetic improvement studies. However, several issues emerge as regeneration is not universally possible across the plant kingdom and many variables must be considered. In grapevine (Vitis spp.), as in other woody and fruit tree species, the regeneration process is impaired by a recalcitrance that depends on numerous factors such as genotype and explant-dependent responses. This is one of the major obstacles in developing gene editing approaches and functional genome studies in grapevine and it is therefore crucial to understand how to achieve efficient regeneration across different genotypes. Further issues that emerge in regeneration need to be addressed, such as somaclonal mutations which do not allow the regeneration of individuals identical to the original mother plant, an essential factor for commercial use of the improved grapevines obtained through the New Breeding Techniques. Over the years, the evolution of protocols to achieve plant regeneration has relied mainly on optimizing protocols for genotypes of interest whilst nowadays with new genomic data available there is an emerging opportunity to have a clearer picture of its molecular regulation. The goal of this review is to discuss the latest information available about different aspects of grapevine in vitro regeneration, to address the main factors that can impair the efficiency of the plant regeneration process and cause post-regeneration problems and to propose strategies for investigating and solving them.

Grapevine virome and production of healthy plants by somatic embryogenesis.

Grapevine (Vitis spp.) is a widespread fruit tree hosting many viral entities that interact with the plant modifying its responses to the environment. The production of virus-free plants is becoming increasingly crucial for the use of grapevine as a model species in different studies. Using high-throughput RNA sequencing, the viromes of seven mother plants grown in a germplasm collection vineyard were sequenced. In addition to the viruses and viroids already detected in grapevine, we identified 13 putative new mycoviruses. The different spread among grapevine tissues collected in vineyard, greenhouse and in vitro conditions suggested a clear distinction between viruses/viroids and mycoviruses that can successfully be exploited for their identification. Mycoviruses were absent in in vitro cultures, while plant viruses and viroids were particularly accumulated in these plantlets. Somatic embryogenesis applied to the seven mother plants was effective in the elimination of the complete virome, including mycoviruses. However, different sanitization efficiencies for viroids and grapevine pinot gris virus were observed among genotypes. The absence of mycoviruses in in vitro plantlets, associated with the absence of all viral entities in somaclones, suggested that this regeneration technique is also effective to eradicate endophytic/epiphytic fungi, resulting in gnotobiotic or pseudo-gnotobiotic plants.