Grapevine VlbZIP30 improves drought resistance by directly activating VvNAC17 and promoting lignin biosynthesis through the regulation of three peroxidase genes

Drought stress severely affects grapevine quality and yield,and recent reports have revealed that lignin plays an important role in protection from drought stress.Since little is known about lignin-mediated drought resistance in grapevine,we investigated its significance.Herein,we show that VlbZIP30 mediates drought resistance by activating the expression of lignin biosynthetic genes and increasing lignin deposition.Transgenic grapevine plants overexpressing VlbZIP30 exhibited lignin deposition(mainly G and S monomers)in the stem secondary xylem under control conditions,which resulted from the upregulated expression of VvPRX4 and VvPRX72.Overexpression of VlbZIP30 improves drought tolerance,characterized by a reduction in the water loss rate,maintenance of an effective photosynthesis rate,and increased lignin content(mainly G monomer)in leaves under drought conditions.Electrophoretic mobility shift assay,luciferase reporter assays,and chromatin immunoprecipitation-qPCR assays indicated that VlbZIP30 directly binds to the G-box cis-element in the promoters of lignin biosynthetic(VvPRX N1)and drought-responsive(VvNAC17)genes to regulate their expression.In summary,we report a novel VlbZIP30-mediated mechanism linking lignification and drought tolerance in grapevine.The results of this study may be of value for the development of molecular breeding strategies to produce drought-resistant fruit crops.

Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

The CRISPR(clustered regularly interspaced short palindromic repeats)-associated protein 9(Cas9)system is a powerful tool for targeted genome editing,with applications that include plant biotechnology and functional genomics research.However,the specificity of Cas9 targeting is poorly investigated in many plant species,including fruit trees.To assess the off-target mutation rate in grapevine(Vitis vinifera),we performed whole-genome sequencing(WGS)of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type(WT)plants.In total,we identified between 202,008 and 272,397 single nucleotide polymorphisms(SNPs)and between 26,391 and 55,414 insertions/deletions(indels)in the seven Cas9-edited grapevine plants compared with the three WT plants.Subsequently,3272 potential off-target sites were selected for further analysis.Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing.In addition,we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome(PN40024)but no true off-target mutations.In conclusion,we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

VlbZIP30 of grapevine functions in dehydration tolerance via the abscisic acid core signaling pathway

Drought stress limits the growth and development of grapevines,thereby reducing productivity,but the mechanisms by which grapevines respond to drought stress remain largely uncharacterized.Here,we characterized a group A bZIP gene from“Kyoho”grapevine,VlbZIP30,which was shown to be induced by abscisic acid(ABA)and dehydration stress.Overexpression of VlbZIP30 in transgenic Arabidopsis thaliana enhanced dehydration tolerance.Transcriptome analysis revealed that a major proportion of ABA-responsive and/or drought-responsive genes are transcriptionally regulated by VlbZIP30 during ABA or mannitol treatment at the cotyledon greening stage.We identified an A.thaliana G-box motif(CACGTG)and a potential grapevine G-box motif(MCACGTGK)in the promoters of the 39 selected A.thaliana genes upregulated in the transgenic plants and in the 35 grapevine homologs,respectively.Subsequently,using two grapevine-related databases,we found that 74%(23/31)and 84%(21/25)of the detected grapevine genes were significantly upregulated by ABA and drought stress,respectively,suggesting that these genes are involved in ABA or dehydration stress and may be regulated by VlbZIP30 in grapevine.We propose that VlbZIP30 functions as a positive regulator of dehydration-responsive signaling in the ABA core signaling pathway.