WRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown. We identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress. We identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape.

Grape production in continental climatic regions suffers from the combination of drought and cold stresses during winter. Developing a reliable system to simulate combined drought–cold stress and to determine physiological responses and regulatory mechanisms is important. Evaluating tolerance to combined stress at germplasm level is crucial to select parents for breeding grapevines. In the present study, two species, namely, Vitis amurensis and V. vinifera cv. ‘Muscat Hamburg’, were used to develop a reliable system for evaluating their tolerance to drought–cold stress. This system used tissue −cultured grapevine plants, 6% PEG solution, and gradient cooling mode to simulate drought–cold stress. V. amurensis had a significantly lower LT50 value (the temperature of 50% electrolyte leakage) than ‘Muscat Hamburg’ during simulated drought–cold stress. Thus, the former had higher tolerance than the latter to drought–cold stress based on electrolyte leakage (EL) measurements. Moreover, the chlorophyll fluorescence responses of V. amurensis and ‘Muscat Hamburg’ were also analyzed under drought–cold stress. The maximum photochemical quantum yield of PS II (Fv/Fm) exhibited a significant linear correlationship with EL. The relationship of EL with Fv/Fm in the other four genotypes of grapevines under drought–cold stress was also detected. A novel LT50 estimation model was established, and the LT50 values can be well calculated based on Fv/Fm in replacement of EL measurement. The Fv/Fm–based model exhibits good reliability for evaluating the tolerance of different grapevine genotypes to drought–cold stress.

Low temperature is one of the most important environmental factors that limits the geographical distribution and productivity of grapevine. However, the molecular mechanisms on how grapevine responds to cold stress remains to be elucidated. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play an essential role during plant development and stress responses. Although miRNAs and their targets have been identified in several Vitis species, their participation during cold accumulation in grapevine remains unknown. In this study, two small RNA libraries were generated from micropropagated ‘Muscat Hamburg’ (V. vinifera) plantlets under normal and low temperatures (4 °C). A total of 163 known miRNAs and 67 putative novel miRNAs were detected from two small RNA libraries by Solexa sequencing. Forty-four cold-inducible miRNAs were identified through differentially expressed miRNAs (DEMs) analysis; among which, 13 belonged to upregulated DEMs while 31 belonged downregulated DEMs. The expression patterns of the 13 DEMs were verified by real-time RT-PCR analysis. The prediction of the target genes for DEMs indicated that miRNA may regulate transcription factors, including AP2, SBP, MYB, bHLH, GRAS, and bZIP under cold stress. The 5’-RLM RACE were conducted to verify the cleavage site of predicted targets. Seven predicted target genes for four known and three novel vvi-miRNAs showed specific cleavage sites corresponding to their miRNA complementary sequences. The expression pattern of these seven target genes revealed negative correlation with the expression level of the corresponding vvi-miRNAs. Our results indicated that a diverse set of miRNAs in V. vinifera are cold-inducible and may play an important role in cold stress response.