The Vitis vinifera C-repeat binding protein 4 (VvCBF4) transcriptional factor enhances freezing tolerance in wine grape.

Chilling and freezing can reduce significantly vine survival and fruit set in Vitis vinifera wine grape. To overcome such production losses, a recently identified grapevine C-repeat binding factor (CBF) gene, VvCBF4, was overexpressed in grape vine cv. 'Freedom' and found to improve freezing survival and reduced freezing-induced electrolyte leakage by up to 2 °C in non-cold-acclimated vines. In addition, overexpression of this transgene caused a reduced growth phenotype similar to that observed for CBF overexpression in Arabidopsis and other species. Both freezing tolerance and reduced growth phenotypes were manifested in a transgene dose-dependent manner. To understand the mechanistic basis of VvCBF4 transgene action, one transgenic line (9-12) was genotyped using microarray-based mRNA expression profiling. Forty-seven and 12 genes were identified in unstressed transgenic shoots with either a >1.5-fold increase or decrease in mRNA abundance, respectively. Comparison of mRNA changes with characterized CBF regulons in woody and herbaceous species revealed partial overlaps, suggesting that CBF-mediated cold acclimation responses are widely conserved. Putative VvCBF4-regulon targets included genes with functions in cell wall structure, lipid metabolism, epicuticular wax formation and stress-responses suggesting that the observed cold tolerance and dwarf phenotypes are the result of a complex network of diverse functional determinants.

CBF4 is a unique member of the CBF transcription factor family of Vitis vinifera and Vitis riparia.

The CBF/DREB1 transcription factors control an important pathway for increased freezing and drought tolerance in plants. We report here the isolation of one CBF/DREB1-like gene, CBF4, from both freezing-tolerant wild grape (Vitis riparia) and freezing-sensitive cultivated grape (Vitis vinifera). The deduced protein in V. riparia is 99% identical to the corresponding protein in V. vinifera; 45-48% to three other Vitis CBF proteins reported earlier and 57% to AtCBF1, and contains CBF-specific amino acid motifs. Agroinfiltration experiments in tobacco leaves revealed that VrCBF4 activates expression from reporter genes driven by a CRT-containing promoter. Expression of the endogenous Vitis CBF4 genes was low at ambient temperature, but enhanced upon exposure to low temperature (4 degrees C). Uncommon for CBF genes, this expression was maintained for several days. No significant difference in expression pattern was observed between V. riparia and V. vinifera. Vitis CBF4 was expressed in both young and mature tissue, in contrast to the previously described Vitis CBF1, 2 and 3. Together, these results suggest that CBF4 represents a second type of CBF in grape that might be more important for the over-wintering of grape plants.

Transcript abundance profiles reveal larger and more complex responses of grapevine to chilling compared to osmotic and salinity stress.

Cabernet Sauvignon grapevines were exposed to sudden chilling (5 degrees C), water deficit (PEG), and an iso-osmotic salinity (120 mM NaCl and 12 mM CaCl(2)) for 1, 4, 8, and 24 h. Stomatal conductance and stem water potentials were significantly reduced after stress application. Microarray analysis of transcript abundance in shoot tips detected no significant differences in transcript abundance between salinity and PEG before 24 h. Chilling stress relates to changes in membrane structure, and transcript abundance patterns were predicted to reflect this. Forty-three percent of transcripts affected by stress vs control for 1 through 8 h were affected only by chilling. The functional categories most affected by stress included metabolism, protein metabolism, and signal transduction. Osmotic stress affected more protein synthesis and cell cycle transcripts, whereas chilling affected more calcium signaling transcripts, indicating that chilling has more complex calcium signaling. Stress affected many hormone (ABA, ethylene, and jasmonate) and transcription factor transcripts. The concentrations and transporter transcripts of several anions increased with time, including nitrate, sulfate, and phosphate. The transcript abundance changes in this short-term study were largely the same as a gradually applied long-term salinity and water-deficit study (Cramer et al. Funct Integr Genomics 7:111-134, 2007), but the reverse was not true, indicating a larger and more complex response in the acclimation process of a gradual long-term stress.

Water and salinity stress in grapevines: early and late changes in transcript and metabolite profiles.

Grapes are grown in semiarid environments, where drought and salinity are common problems. Microarray transcript profiling, quantitative reverse transcription-PCR, and metabolite profiling were used to define genes and metabolic pathways in Vitis vinifera cv. Cabernet Sauvignon with shared and divergent responses to a gradually applied and long-term (16 days) water-deficit stress and equivalent salinity stress. In this first-of-a-kind study, distinct differences between water deficit and salinity were revealed. Water deficit caused more rapid and greater inhibition of shoot growth than did salinity at equivalent stem water potentials. One of the earliest responses to water deficit was an increase in the transcript abundance of RuBisCo activase (day 4), but this increase occurred much later in salt-stressed plants (day 12). As water deficit progressed, a greater number of affected transcripts were involved in metabolism, transport, and the biogenesis of cellular components than did salinity. Salinity affected a higher percentage of transcripts involved in transcription, protein synthesis, and protein fate than did water deficit. Metabolite profiling revealed that there were higher concentrations of glucose, malate, and proline in water-deficit-treated plants as compared to salinized plants. The metabolite differences were linked to differences in transcript abundance of many genes involved in energy metabolism and nitrogen assimilation, particularly photosynthesis, gluconeogenesis, and photorespiration. Water-deficit-treated plants appear to have a higher demand than salinized plants to adjust osmotically, detoxify free radicals (reactive oxygen species), and cope with photoinhibition.

Proteomic analysis reveals differences between Vitis vinifera L. cv. Chardonnay and cv. Cabernet Sauvignon and their responses to water deficit and salinity.

The impact of water deficit and salt stress on two important wine grape cultivars, Chardonnay and Cabernet Sauvignon, was investigated. Plants were exposed to increasing salinity and water deficit stress over a 16 d time period. Measurements of stem water potentials, and shoot and leaf lengths indicated that Chardonnay was more tolerant to these stresses than Cabernet Sauvignon. Shoot tips were harvested every 8 d for proteomic analysis using a trichloroacetic acid/acetone extraction protocol and two-dimensional gel electrophoresis. Proteins were stained with Coomassie Brilliant Blue, quantified, and then 191 unique proteins were identified using matrix-assisted laser desorption ionization time of flight/time of flight mass spectrometry. Peptide sequences were matched against both the NCBI nr and TIGR Vitis expressed sequence tag (EST) databases that had been implemented with all public Vitis sequences. Approximately 44% of the protein isoforms could be identified. Analysis of variance indicated that varietal difference was the main source of protein expression variation (40%). In stressed plants, reduction of the amount of proteins involved with photosynthesis, protein synthesis, and protein destination was correlated with the inhibition of shoot elongation. Many of the proteins up-regulated in Chardonnay were of unclassified or of unknown function, whereas proteins specifically up-regulated in Cabernet Sauvignon were involved in protein metabolism.