MYB30 and MYB14 form a repressor-activator module with WRKY8 that controls stilbene biosynthesis in grapevine.

When exposed to pathogen infection or ultraviolet (UV) radiation, grapevine (Vitis vinifera) plants rapidly accumulate the stilbenoid resveratrol (Res) with concomitant increase of stilbene synthase (STS), the key enzyme in stilbene biosynthesis. Although a few transcription factors have been shown to regulate STSs, the molecular mechanism governing the regulation of STSs is not well elucidated. Our previous work showed that a VvMYB14-VvWRKY8 regulatory loop fine-tunes stilbene biosynthesis in grapevine through protein-protein interaction; overexpression of VvWRKY8 down-regulates VvMYB14 and VvSTS15/21; and application of exogenous Res up-regulates WRKY8 expression. Here, we identified an R2R3-MYB repressor, VvMYB30, which competes with the activator VvMYB14 for binding to the common binding sites in the VvSTS15/21 promoter. Similar to VvMYB14, VvMYB30 physically interacts with VvWRKY8 through their N-termini, forming a complex that does not bind DNA. Exposure to UV-B/C stress induces VvMYB14, VvWRKY8, and VvSTS15/21, but represses VvMYB30 in grapevine leaves. In addition, MYB30 expression is up-regulated by VvWRKY8-overexpression or exogenous Res. These findings suggest that the VvMYB14-VvWRKY8-VvMYB30 regulatory circuit allows grapevine to respond to UV stress by producing Res and prevents over-accumulation of Res to balance metabolic costs. Our work highlights the stress-mediated induction and feedback inhibition of stilbene biosynthesis through a complex regulatory network involving multiple positive and negative transcriptional regulators.

The Genome of Vitis zhejiang-adstricta Strengthens the Protection and Utilization of the Endangered Ancient Grape Endemic to China.

Vitis zhejiang-adstricta (V. zhejiang-adstricta) is one of the most important and endangered wild grapes. It is a national key protected wild, rare and endangered ancient grape endemic to China and used as a candidate material for resistance breeding owing to its excellent significant disease resistance. Here, we present a high-quality chromosome-level assembly of V. zhejiang-adstricta (IB-VB-01), comprising 506.66 Mb assembled into 19 pseudo-chromosomes. The contig N50 length is 3.91 Mb with 31,196 annotated protein-coding genes. Comparative genome and evolutionary analyses illustrated that V. zhejiang-adstricta has a specific position in the evolution of East Asian Vitis and shared a common ancestor with Vitis vinifera during the divergence of the two species about 10.42 (between 9.34 and 11.12) Mya. The expanded gene families compared with those in plants were related to disease resistance, and constructed gene families were related to plant growth and primary metabolism. With the analysis of gene family expansion and contraction, the evolution of environmental adaptability and especially the NBS-LRR gene family of V. zhejiang-adstricta was elucidated based on the pathways of resistance genes (R genes), unique genes and structural variations. The near-complete and accurate diploid V. zhejiang-adstricta reference genome obtained herein serves as an important complement to wild grape genomes and will provide valuable genomic resources for investigating the genomic architecture of V. zhejiang-adstricta as well as for improving disease resistance breeding strategies in grape.

Far-red light modulates grapevine growth by increasing leaf photosynthesis efficiency and triggering organ-specific transcriptome remodelling : Author.

BACKGROUND: Growing evidence demonstrates that the synergistic interaction of far-red light with shorter wavelength lights could evidently improve the photosynthesis efficiency of multiple species. However, whether/how far-red light affects sink organs and consequently modulates the source‒sink relationships are largely unknown. RESULTS: Here, equal intensities of white and far-red lights were added to natural light for grape plantlets to investigate the effects of far-red light supplementation on grapevine growth and carbon assimilate allocation, as well as to reveal the underlying mechanisms, through physiological and transcriptomic analysis. The results showed that additional far-red light increased stem length and carbohydrate contents in multiple organs and decreased leaf area, specific leaf weight and dry weight of leaves in comparison with their counterparts grown under white light. Compared to white light, the maximum net photosynthetic rate of the leaves was increased by 31.72% by far-red light supplementation, indicating that far-red light indeed elevated the photosynthesis efficiency of grapes. Transcriptome analysis revealed that leaves were most responsive to far-red light, followed by sink organs, including stems and roots. Genes related to light signaling and carbon metabolites were tightly correlated with variations in the aforementioned physiological traits. In particular, VvLHCB1 is involved in light harvesting and restoring the balance of photosystem I and photosystem II excitation, and VvCOP1 and VvPIF3, which regulate light signal transduction, were upregulated under far-red conditions. In addition, the transcript abundances of the sugar transporter-encoding genes VvSWEET1 and VvSWEET3 and the carbon metabolite-encoding genes VvG6PD, VvSUS7 and VvPGAM varied in line with the change in sugar content. CONCLUSIONS: This study showed that far-red light synergistically functioning with white light has a beneficial effect on grape photosystem activity and is able to differentially affect the growth of sink organs, providing evidence for the possible addition of far-red light to the wavelength range of photosynthetically active radiation (PAR).

Advances in understanding cold tolerance in grapevine.

Grapevine (Vitis ssp.) is a deciduous perennial fruit crop, and the canes and buds of grapevine should withstand low temperatures (LTs) annually during winter. However, the widely cultivated Vitis vinifera is cold-sensitive and cannot survive the severe winter in regions with extremely LTs, such as viticulture regions in northern China. By contrast, a few wild Vitis species like V. amurensis and V. riparia exhibit excellent freezing tolerance. However, the mechanisms underlying grapevine cold tolerance remain largely unknown. In recent years, much progress has been made in elucidating the mechanisms, owing to the advances in sequencing and molecular biotechnology. Assembly of grapevine genomes together with resequencing and transcriptome data enable researchers to conduct genomic and transcriptomic analyses in various grapevine genotypes and populations to explore genetic variations involved in cold tolerance. In addition, a number of pivotal genes have been identified and functionally characterized. In this review, we summarize recent major advances in physiological and molecular analyses of cold tolerance in grapevine and put forward questions in this field. We also discuss the strategies for improving the tolerance of grapevine to cold stress. Understanding grapevine cold tolerance will facilitate the development of grapevines for adaption to global climate change.

A chromosome-level genome of Syringa oblata provides new insights into chromosome formation in Oleaceae and evolutionary history of lilacs.

Lilacs (Syringa L.), a group of well-known ornamental and aromatic woody plants, have long been used for gardening, essential oils and medicine purposes in East Asia and Europe. The lack of knowledge about the complete genome of Syringa not only hampers effort to better understand its evolutionary history, but also prevents genome-based functional gene mining that can help in the variety improvement and medicine development. Here, a chromosome-level genome of Syringa oblata is presented, which has a size of 1.12 Gb including 53 944 protein coding genes. Synteny analysis revealed that a recent duplication event and parallel evolution of two subgenomes formed the current karyotype. Evolutionary analysis, transcriptomics and metabolic profiling showed that segment and tandem duplications contributed to scent formation in the woody aromatic species. Moreover, phylogenetic analysis indicated that S. oblata shared a common ancestor with Osmanthus fragrans and Olea europaea approximately 27.61 million years ago (Mya). Biogeographic reconstruction based on a resequenced data set of 26 species suggested that Syringa originated in the northern part of East Asia during the Miocene (approximately 14.73 Mya) and that the five Syringa groups initially formed before the Late Miocene (approximately 9.97 Mya). Furthermore, multidirectional dispersals accompanied by gene introgression among Syringa species from Northern China during the Miocene were detected by biogeographic reconstruction. Taken together, the results showed that complex gene introgression, which occurred during speciation history, greatly contributed to Syringa diversity.

Model-assisted analysis for tuning anthocyanin composition in grape berries.

Anthocyanin composition is responsible for the red colour of grape berries and wines, and contributes to their organoleptic quality. However, anthocyanin biosynthesis is under genetic, developmental and environmental regulation, making its targeted fine-tuning challenging. We constructed a mechanistic model to simulate the dynamics of anthocyanin composition throughout grape ripening in Vitis vinifera, employing a consensus anthocyanin biosynthesis pathway. The model was calibrated and validated using six datasets from eight cultivars and 37 growth conditions. Tuning the transformation and degradation parameters allowed us to accurately simulate the accumulation process of each individual anthocyanin under different environmental conditions. The model parameters were robust across environments for each genotype. The coefficients of determination (R2) for the simulated versus observed values for the six datasets ranged from 0.92 to 0.99, while the relative root mean square errors (RRMSEs) were between 16.8 and 42.1 %. The leave-one-out cross-validation for three datasets showed R2 values of 0.99, 0.96 and 0.91, and RRMSE values of 28.8, 32.9 and 26.4 %, respectively, suggesting a high prediction quality of the model. Model analysis showed that the anthocyanin profiles of diverse genotypes are relatively stable in response to parameter perturbations. Virtual experiments further suggested that targeted anthocyanin profiles may be reached by manipulating a minimum of three parameters, in a genotype-dependent manner. This model presents a promising methodology for characterizing the temporal progression of anthocyanin composition, while also offering a logical foundation for bioengineering endeavours focused on precisely adjusting the anthocyanin composition of grapes.

The genome of Shanputao (Vitis amurensis) provides a new insight into cold tolerance of grapevine.

Vitis amurensis (Shanputao) is the most cold tolerant Vitis species and so is of great interest to grape breeders and producers in areas with low winter temperatures. Here, we report its high-quality, chromosome-level genome assembly based on a combination of sequence data from Illumina and PacBio platforms, BioNano optical mapping and high-throughput chromosome conformation Capture (Hi-C) mapping. The 604.56-Mb genome contains 32 885 protein-coding genes. Shanputao was found to share a common ancestor with PN40024 (V. vinifera) approximately 2.17-2.91 million years ago, and gene expansion observed in Shanputao might contribute to the enhancement of cold tolerance. Transcriptome analysis revealed 17 genes involved in cold signal transduction, suggesting that there was a different response mechanism to chilling temperature and freezing conditions. Furthermore, a genome-wide association study uncovered a phosphoglycerate kinase gene that may contribute to the freezing resistance of buds in the winter. The Shanputao genome sequence not only represents a valuable resource for grape breeders, but also is important for clarifying the molecular mechanisms involved in cold tolerance.

Hormone biosynthesis and metabolism members of 2OGD superfamily are involved in berry development and respond to MeJA and ABA treatment of Vitis vinifera L.

BACKGROUND: Hormones play an indispensable role during fruit ripening, nine clades in 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily are responsible for the hormone biosynthesis and metabolism, but less information is known about them. RESULTS: A total of 163 Vv2OGD superfamily members were identified from grape genome, which were mainly expanded by local (tandem and proximal) duplication. Phylogenetic analysis of 2OGD members in grape and Arabidopsis indicates 37 members in Vv2OGD superfamily are related to hormone biosynthesis and metabolism process (Vv2OGD-H), which could be divided into 9 clades, gibberellin (GA) 3-oxidase (GA3ox), GA 20-oxidase (GA20ox), carbon-19 GA 2-oxidase (C19-GA2ox), carbon-20 GA 2-oxidase (C20-GA2ox), 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), dioxygenase for auxin oxidation (DAO), lateral branching oxidoreductas (LBO), downy mildew resistant 6 and DMR6-like oxygenase (DMR6/DLO) and jasmonate-induced oxygenase (JOX). Sixteen of these 37 Vv2OGD-Hs are expressed in grape berry, in which the expression patterns of VvGA2oxs, VvDAOs and VvJOXs shows a correlation with the change patterns of GAs, indole-3-acetic acid (IAA) and jasmonates (JAs), indicating the involvement of these genes in grape berry development by regulating corresponding hormones. Twelve Vv2OGD-Hs respond to methyl JA (MeJA) treatment, of which eight may lead to the inhibition of the ripening process by the crosstalk of JAs-salicylic acids (SAs), JAs-GAs and JAs-JAs, while seven Vv2OGD-Hs respond to ABA treatment may be responsible for the promotion of ripening process by the interplay of abscisic acid (ABA)-strigolactones (SLs), ABA-SAs, ABA-GAs, ABA-JAs. Especially, VvLBO1 reach an expression peak near véraison and up-regulate about four times after ABA treatment, which implies SLs and ABA-SLs crosstalk may be related to the onset of berry ripening in grape. CONCLUSIONS: This study provides valuable clues and new insights for the mechanism research of Vv2OGD-Hs in hormones regulation during the grape berry development.

Overexpression of grape ABA receptor gene VaPYL4 enhances tolerance to multiple abiotic stresses in Arabidopsis.

BACKGROUND: Abscisic acid (ABA) plays a crucial role in abiotic stress responses. The pyrabactin resistance (PYR)/PYR-like (PYL)/regulatory component of ABA receptor (RCAR) proteins that have been characterized as ABA receptors function as the core components in ABA signaling pathway. However, the functions of grape PYL genes in response to different abiotic stresses, particularly cold stress, remain less studied. RESULTS: In this study, we investigated the expression profiles of grape PYL genes upon cold treatment and isolated the VaPYL4 gene from Vitis amurensis, a cold-hardy grape species. Overexpression of VaPYL4 gene in grape calli and Arabidopsis resulted in enhanced cold tolerance. Moreover, plant resistance to drought and salt stress was also improved by overexpressing VaPYL4 in Arabidopsis. More importantly, we evaluated the contribution of VaPYL4 to plant growth and development after the treatment with cold, salt and drought stress simultaneously. The transgenic plants showed higher survival rates, earlier flowering phenotype, and heavier fresh weight of seedlings and siliques when compared with wild-type plants. Physiological analyses showed that transgenic plants had much lower content of malondialdehyde (MDA) and higher peroxidase (POD) activity. Stress-responsive genes such as RD29A (Responsive to desiccation 29A), COR15A (Cold responsive 15A) and KIN2 (Kinase 2) were also significantly up-regulated in VaPYL4-overexpressing Arabidopsis plants. CONCLUSIONS: Our results show that overexpression of VaPYL4 could improve plant performance upon different abiotic stresses, which therefore provides a useful strategy for engineering future crops to deal with adverse environments.

GRAS-domain transcription factor PAT1 regulates jasmonic acid biosynthesis in grape cold stress response.

Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis.

Grapevine U-Box E3 Ubiquitin Ligase VlPUB38 Negatively Regulates Fruit Ripening by Facilitating Abscisic-Aldehyde Oxidase Degradation.

The plant U-box E3 ubiquitin ligase-mediated ubiquitin/26S proteasome degradation system plays a key role in plant growth and development. Previously identified as a member of the grape PUB gene family, PUB38 was shown to participate in the berry-ripening progress. Here, we demonstrate that the E3 ligase VlPUB38 mediates abscisic acid (ABA) synthesis via 26S proteasome degradation and its involvement in regulating fruit-ripening processes. Strawberry-overexpressing VlPUB38 lines displayed obvious inhibition of mature phenotype, and this was rescued by exogenous ABA treatment and MG132. Post-ABA treatment, expression levels of ABA response-related genes in VlPUB38-overexpressed Arabidopsis significantly exceeded controls. Strawberry and Arabidopsis ectopic expression assays suggest that VlPUB38 negatively regulates fruit ripening in an ABA-dependent manner. Moreover, VlPUB38 has ubiquitin ligase activity, which depends on the U-box-conserved domain. VlPUB38 interacts with abscisic-aldehyde oxidase (VlAAO), targeting VlAAO proteolysis via the 26S proteasome system. These results indicate that VlPUB38 negatively regulates grape fruit ripening by mediating the degradation of key factor VlAAO in the ABA synthesis pathway.

Characterization of Chromatin Accessibility and Gene Expression upon Cold Stress Reveals that the RAV1 Transcription Factor Functions in Cold Response in Vitis Amurensis.

Cold tolerance is regulated by a variety of transcription factors (TFs) and their target genes. Except for the well-characterized C-repeat binding factors (CBFs)-dependent transcriptional cascade, the mechanisms of cold tolerance mediated by other transcriptional regulatory networks are still largely unknown. Here, we used the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq to identify cold responsive TFs in Vitis amurensis, a grape species with high cold hardiness. Nine TFs, including CBF4, RAV1 and ERF104, were identified after cold treatment. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) analysis revealed that these TFs may regulate cold response through different pathways. As a prime candidate TF, overexpression of VaRAV1 in grape cells improved its cold tolerance. The transgenic cells exhibited low electrolyte leakage and malondialdehyde content and high peroxidase activity. Moreover, the TF gene TCP8 and a gene involving in homogalacturonan biosynthesis were found to be regulated by VaRAV1, suggesting that the contribution of VaRAV1 to cold tolerance may be achieved by enhancing the stability of cell membrane and regulating the expression of target genes involved in plant cell wall composition. Our work provides novel insights into plant response to cold stress and demonstrates the utility of ATAC-seq and RNA-seq for the rapid identification of TFs in response to cold stress in grapevine. VaRAV1 may play an important role in adaption to cold stress.

Metabolite analysis reveals distinct spatio-temporal accumulation of anthocyanins in two teinturier variants of cv. 'Gamay' grapevines (Vitis vinifera L.).

MAIN CONCLUSION: White-fleshed grape cv. 'Gamay' and its two teinturier variants presented distinct spatial-temporal accumulation of anthocyanins, with uncoupled accumulation of sugars and anthocyanins in 'Gamay Fréaux'. In most red grape cultivars, anthocyanins accumulate exclusively in the berry skin, while 'teinturier' cultivars also accumulate anthocyanins in the pulp. Here, we investigated the teinturier cvs. 'Gamay de Bouze' and 'Gamay Fréaux' (two somatic variants of the white-fleshed cv. 'Gamay') through metabolic and transcript analysis to clarify whether these two somatic variants have the same anthocyanin accumulation pattern in the skin and pulp, and whether primary metabolites are also affected. The skin of the three cultivars and the pulp of 'Gamay de Bouze' begun to accumulate anthocyanins at the onset of berry ripening. However, the pulp of 'Gamay Fréaux' exhibited a distinct anthocyanin accumulation pattern, starting as early as fruit set with very low level of sugars. The highest level of anthocyanins was found in 'Gamay Fréaux' skin, followed by 'Gamay de Bouze' and 'Gamay'. Consistently, the transcript abundance of genes involved in anthocyanin biosynthesis were in line with the anthocyanin levels in the three cultivars. Despite no evident differences in pulp sugar content, the concentration of glucose and fructose in the skin of 'Gamay Fréaux' was only half of those in the skin of 'Gamay' and 'Gamay de Bouze' throughout all berry ripening, suggesting an uncoupled accumulation of sugars and anthocyanins in 'Gamay Fréaux'. The study provides a comprehensive view of metabolic consequences in grape somatic variants and the three almost isogenic genotypes can serve as ideal reagents to further uncover the mechanisms underlying the linkage between sugar and anthocyanin accumulation.

Transcriptomic analysis of grapevine Dof transcription factor gene family in response to cold stress and functional analyses of the VaDof17d gene.

MAIN CONCLUSION: Dof genes enhance cold tolerance in grapevine and VaDof17d is tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides. DNA-binding with one finger (Dof) proteins comprise a large family that plays important roles in the regulation of abiotic stresses. No in-depth analysis of Dof genes has been performed in the grapevine. In this study, we analyzed a total of 25 putative Dof genes in grapevine at genomic and transcriptomic levels, compiled expression profiles of 11 selected VaDof genes under cold stress and studied the potential function of the VaDof17d gene in grapevine calli. The 25 Dof proteins can be classified into four phylogenetic groups. RNA-seq and qRT-PCR results demonstrated that a total of 11 VaDof genes responded to cold stress. Comparative mRNA sequencing of 35S::VaDof17d grape calli showed that VaDof17d was tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides (RFOs), as observed by the up-regulation of galactinol synthase (GolS) and raffinose synthase genes. We found that the Dof17d-ED (CRISPR/Cas9-mediated mutagenesis of Dof17d-ED) mutant had low cold tolerance with a decreased RFOs level during cold stress. These results formed the fundamental knowledge for further analysis of the biological roles of Dof genes in the grapevine's adaption to cold stresses.

The ethylene response factor VaERF092 from Amur grape regulates the transcription factor VaWRKY33, improving cold tolerance.

Cold stress is a major limiting factor in grape (Vitis) productivity. In this study, we characterized a cold-responsive ethylene response factor (ERF) transcription factor, VaERF092, from Amur grape (Vitis amurensis). VaERF092 expression was induced by both low temperatures and the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC), but was suppressed by treatment with the ethylene inhibitor aminoethoxyvinylglycine (AVG) under cold conditions. Ectopic expression of VaERF092 in Arabidopsis thaliana enhanced cold tolerance. Co-expression network analysis of V. vinifera genes indicated that WRKY33 might be a downstream target of VaERF092. This hypothesis was supported by the fact that VaWRKY33 was expressed temporally after VaERF092 expression and could also be induced by cold and ACC, and inhibited by AVG. Yeast one-hybrid, transient ß-glucuronidase (GUS) and dual-luciferase reporter assays provided evidence for an interaction between VaERF092 and a GCC-box element in the VaWRKY33 promoter. In addition, heterologous overexpression of VaWRKY33 in A. thaliana resulted in enhanced cold tolerance. VaERF092- and VaWRKY33 overexpressing grape calli showed lower low-temperature exothermic values than the empty vector (EV) calli, indicating enhanced tolerance to cold. Together, these results indicated that VaERF092 regulates VaWRKY33 through binding to its promoter GCC-box, leading to enhanced cold stress tolerance.

Transcriptomic Analysis of the Grapevine LEA Gene Family in Response to Osmotic and Cold Stress Reveals a Key Role for VamDHN3.

Late embryogenesis abundant (LEA) proteins comprise a large family that plays important roles in the regulation of abiotic stress, however, no in-depth analysis of LEA genes has been performed in grapevine to date. In this study, we analyzed a total of 52 putative LEA genes in grapevine at the genomic and transcriptomic level, compiled expression profiles of four selected (V. amurensis) VamLEA genes under cold and osmotic stresses, and studied the potential function of the V. amurensis DEHYDRIN3 (VamDHN3) gene in grapevine callus. The 52 LEA proteins were classified into seven phylogenetic groups. RNA-seq and quantitative real-time PCR results demonstrated that a total of 16 and 23 VamLEA genes were upregulated under cold and osmotic stresses, respectively. In addition, overexpression of VamDHN3 enhanced the stability of the cell membrane in grapevine callus, suggesting that VamDHN3 is involved in osmotic regulation. These results provide fundamental knowledge for the further analysis of the biological roles of grapevine LEA genes in adaption to abiotic stress.

Knockout of VvCCD8 gene in grapevine affects shoot branching.

BACKGROUND: Shoot branching is an important trait of plants that allows them to adapt to environment changes. Strigolactones (SLs) are newly identified plant hormones that inhibit shoot branching in plants. The SL biosynthesis genes CCD7 (carotenoid cleavage dioxygenase 7) and CCD8 have been found to regulate branching in several herbaceous plants by taking advantage of their loss-of-function mutants. However, the role for CCD7 and CCD8 in shoot branching control in grapevine is still unknown due to the lack of corresponding mutants. RESULTS: Here we employed the CRISPR/Cas9 system to edit the VvCCD7 and VvCCD8 genes in the grape hybrid 41B. The 41B embryogenic cells can easily be transformed and used for regeneration of the corresponding transformed plants. Sequencing analysis revealed that gene editing has been used successfully to target both VvCCD genes in 41B embryogenic cells. After regeneration, six 41B plantlets were identified as transgenic plants carrying the CCD8-sgRNA expression cassette. Among these, four plants showed mutation in the target region and were selected as ccd8 mutants. These ccd8 mutants showed increased shoot branching compared to the corresponding wild-type plants. In addition, no off-target mutation was detected in the tested mutants at predicted off-target sites. CONCLUSIONS: Our results underline the key role of VvCCD8 in the control of grapevine shoot branching.

The grapevine R2R3-type MYB transcription factor VdMYB1 positively regulates defense responses by activating the stilbene synthase gene 2 (VdSTS2).

BACKGROUND: Resveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens and abiotic stresses are well-established, yet little is known about the molecular mechanisms regulating stilbene biosynthesis in plant defense progress. RESULTS: Here, we cloned and identified the Chinese wild grape (Vitis davidii) R2R3-MYB transcription factor VdMYB1, which activates defense responses against invading pathogen. VdMYB1 transcripts were significantly upregulated after inoculation with the grapevine powdery mildew fungus Erysiphe necator (Schw.) Burr. Transient expression analysis using onion epidermal cells and Arabidopsis thaliana protoplasts showed that VdMYB1 was localized in the nucleus. Yeast one-hybrid assays revealed that VdMYB1 acts as a transcriptional activator. Grapevine leaves transiently overexpressing VdMYB1 showed a lower number of fungal conidiophores compared with wild-type leaves. Overexpression of VdMYB1 in grapevine leaves did not alter the expression of genes in salicylic acid- and jasmonate-dependent pathways, but affected the expression of stilbene synthase (STS) genes, key regulators of flavonoid metabolism. Results of electrophoretic mobility shift assays and in vivo transcriptional activation assays showed that VdMYB1 binds to the MYB binding site (MYBBS) in the STS2 gene promoter, thus activating STS2 transcription. In heterologous expression assays using tobacco leaves, VdMYB1 activated STS2 gene expression and increased the accumulation of resveratrol. CONCLUSIONS: Our study showed that VdMYB1 activates STS2 gene expression to positively regulate defense responses, and increases the content of resveratrol in leaves.

Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9.

KEY MESSAGE: The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene (PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.

Genome-wide identification and expression analysis of HKT transcription factor under salt stress in nine plant species.

High-affinity K+ (HKT) gene family is regulated the transport of Na+ and maintain the balance between Na+ and K+ in the process of plant growth, development and response to abiotic stress. Despite this fact, systemic and comprehensive studies on HKT in multiply plants remains unknown. A total of 29 HKT genes distributed on nine species were identified. Phylogenetic tree analysis results indicated that HKT genes were divided into five homology subfamilies. Combining structural analysis with protein contains five highly conservative motifs, HKT family has similar gene structures and special gene characteristics. Finally, the expression patterns of HKT showed two different dramatic changes in different organs and tissues under different salt stress in multiply plants. This study has many implications for research into the comparative genomics analysis of HTK gene family, which revealed regulation mechanism of HKT genes, is valuable for understanding development and response to abiotic stress in plant.