Overexpression of VaPAT1, a GRAS transcription factor from Vitis amurensis, confers abiotic stress tolerance in Arabidopsis

The plant-specific GRAS transcription factor family regulates diverse processes involved in plant growth, development and stress responses. In this study, VaPAT1, a GRAS gene from Vitis amurensis was isolated and functionally characterized. Sequence alignment and phylogenetic analysis showed that VaPAT1 has a high sequence identity to CmsGRAS and OsCIGR1, which belong to PAT1 branch of GRAS family and function in stress resistance. The transcription of VaPAT1 was markedly induced by stress-related phytohormone abscisic acid (ABA) and various abiotic stress treatments such as cold, drought and high salinity, however, it was repressed by exogenous gibberellic acid (GA) application. Overexpression of VaPAT1 increased the cold, drought and high salinity tolerance in transgenic Arabidopsis. When compared with wild type (WT) seedlings, the VaPAT1-overexpression lines accumulated higher levels of proline and soluble sugar under these stress treatments. Moreover, stress-related genes such as AtSIZ1, AtCBF1, AtATR1/MYB34, AtMYC2, AtCOR15A, AtRD29A and AtRD29B showed higher expression levels in VaPAT1 transgenic lines than in WT Arabidopsis under normal growth conditions. Together, our results indicated that VaPAT1 functions as a positive transcriptional regulator involved in grapevine abiotic stress responses

An efficient method for transgenic callus induction from Vitis amurensis petiole

Transformation is the main platform for genetic improvement and gene function studies in plants. However, the established somatic embryo transformation system for grapevines is time-consuming and has low efficiency, which limits its utilization in functional genomics research. Vitis amurensis is a wild Vitis species with remarkable cold tolerance. The lack of an efficient genetic transformation system for it has significantly hindered the functional identification of cold stress related genes in the species. Herein, an efficient method was established to produce transformed calli of V. amurensis. Segments of petioles from micro-propagated plantlets of V. amurensis exhibited better capacity to differentiate calli than leaf-discs and stem segments, and thus was chosen as target tissue for Agrobacterium-mediated transformation. Both neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (eGFP) genes were used for simultaneous selection of transgenic calli based on kanamycin resistance and eGFP fluorescence. Several parameters affecting the transformation efficiency were optimized including the concentration of kanamycin, Agrobacterium stains, bacterial densities, infection treatments and co-cultivation time. The transgenic callus lines were verified by checking the integration of NPTII gene into calli genomes, the expression of eGFP gene and the fluorescence of eGFP. Up to 20% of the petiole segments produced transformed calli after 2 months of cultivation. This efficient transformation system will facilitate the functional analysis of agronomic characteristics and related genes not only in V. amurensis but also in other grapevine species

An efficient method for transgenic callus induction from Vitis amurensis petiole.

Transformation is the main platform for genetic improvement and gene function studies in plants. However, the established somatic embryo transformation system for grapevines is time-consuming and has low efficiency, which limits its utilization in functional genomics research. Vitis amurensis is a wild Vitis species with remarkable cold tolerance. The lack of an efficient genetic transformation system for it has significantly hindered the functional identification of cold stress related genes in the species. Herein, an efficient method was established to produce transformed calli of V. amurensis. Segments of petioles from micropropagated plantlets of V. amurensis exhibited better capacity to differentiate calli than leaf-discs and stem segments, and thus was chosen as target tissue for Agrobacterium-mediated transformation. Both neomycin phosphotransferase II (NPTII) and enhanced green fluorescent protein (eGFP) genes were used for simultaneous selection of transgenic calli based on kanamycin resistance and eGFP fluorescence. Several parameters affecting the transformation efficiency were optimized including the concentration of kanamycin, Agrobacterium stains, bacterial densities, infection treatments and co-cultivation time. The transgenic callus lines were verified by checking the integration of NPTII gene into calli genomes, the expression of eGFP gene and the fluorescence of eGFP. Up to 20% of the petiole segments produced transformed calli after 2 months of cultivation. This efficient transformation system will facilitate the functional analysis of agronomic characteristics and related genes not only in V. amurensis but also in other grapevine species

Ethylene positively regulates cold tolerance in grapevine by modulating the expression of ETHYLENE RESPONSE FACTOR 057

Ethylene (ET) is a gaseous plant hormone that plays essential roles in biotic and abiotic stress responses in plants. However, the role of ET in cold tolerance varies in different species. This study revealed that low temperature promotes the release of ET in grapevine. The treatment of exogenous 1-aminocyclopropane-1-carboxylate increased the cold tolerance of grapevine. By contrast, the application of the ET biosynthesis inhibitor aminoethoxyvinylglycine reduced the cold tolerance of grapevine. This finding suggested that ET positively affected cold stress responses in grapevine. The expression of VaERF057, an ET signaling downstream gene, was strongly induced by low temperature. The overexpression of VaERF057 also enhanced the cold tolerance of Arabidopsis. Under cold treatment, malondialdehyde content was lower and superoxide dismutase, peroxidase, and catalase activities were higher in transgenic lines than in wild-type plants. RNA-Seq results showed that 32 stress-related genes, such as CBF1-3, were upregulated in VaERF057-overexpressing transgenic line. Yeast one-hybrid results further demonstrated that VaERF057 specifically binds to GCC-box and DRE motifs. Thus, VaERF057 may directly regulate the expression of its target stress-responsive genes by interacting with a GCC-box or a DRE element. Our work confirmed that ET positively regulates cold tolerance in grapevine by modulating the expression of VaERF057

Expression of Vitis amurensis NAC26 in Arabidopsis enhances drought tolerance by modulating jasmonic acid synthesis

The growth and fruit quality of grapevines are widely affected by abnormal climatic conditions such as water deficits, but many of the precise mechanisms by which grapevines respond to drought stress are still largely unknown. Here, we report that VaNAC26, a member of the NAC transcription factor family, was upregulated dramatically during cold, drought and salinity treatments in Vitis amurensis, a cold and drought-hardy wild Vitis species. Heterologous overexpression of VaNAC26 enhanced drought and salt tolerance in transgenic Arabidopsis. Higher activities of antioxidant enzymes and lower concentrations of H2O2 and O-2(-) were found in VaNAC26-OE lines than in wild type plants under drought stress. These results indicated that scavenging by reactive oxygen species (ROS) was enhanced by VaNAC26 in transgenic lines. Microarray-based transcriptome analysis revealed that genes related to jasmonic acid (JA) synthesis and signaling were upregulated in VaNAC26-OE lines under both normal and drought conditions. VaNAC26 showed a specific binding ability on the NAC recognition sequence (NACRS) motif, which broadly exists in the promoter regions of upregulated genes in transgenic lines. Endogenous JA content significantly increased in the VaNAC26-OE lines 2 and 3. Our data suggest that VaNAC26 responds to abiotic stresses and may enhance drought tolerance by transcriptional regulation of JA synthesis in Arabidopsis

The effect of kanamycin on callus formation from petiole segments.

<p>A: Calli generated from petiole segments on medium without kanamycin for 30 days; B-E: Petiole segments on medium containing 5, 10, 15 and 20 mg/L kanamycin, respectively; F: The calculated differentiation proportion of petiole segments under different kanamycin concentrations. Data are the mean values ± SE of three biological replicates. * and ** indicate significant differences between control and treatments at <i>P</i><0.05 or <i>P</i><0.01 (Student’s t-test), respectively.</p

Optimizing parameters for <i>Agrobacterium</i>-mediated petiole segment transformation in <i>V</i>. <i>amurensis</i>.

<p>A: The transformation efficiency of petiole segments on different <i>Agrobacterium</i> strains (EHA105, GV3101 and LBA4404). B: The effect of bacterial concentration (OD₆₀₀ = 0.5, 1.0, 1.5 and 2.0) during transformation. C: The effect of infection time (4, 8, 12 and 16 min) on transformation. D: The effect of co-cultivation times (1, 2, 3 and 4 days) on transformation. All of the data are the mean values ± SE of three biological replicates. Lower and upper case letters indicate significant differences between treatments at <i>P</i><0.05 or <i>P</i><0.01 (Student’s t-test), respectively.</p