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


(tel +001 775 784 1918; fax +001 775 784 1419; email jcushman@unr.edu)


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.


Vitis vinifera or wine grape was domesticated more than 7000 years ago and continues to the present day to produce one of the world’s most important fruit crops (Arroyo-Garcia et al., 2006; This et al., 2006). Cultivation of V. vinifera and other Vitis spp. encompasses ∼8 million hectares of land worldwide, more than any other cultivated fruit (Vivier and Pretorius, 2002). Vitis vinifera cultivars grow well in temperate, semi-arid climates that can sometimes experience freezing or subfreezing temperatures each winter.

As a deciduous perennial, Vitis spp. acquire freezing tolerance in advance of annual freezes, when shoots mature into overwintering canes and buds enter dormancy, cued by chilling temperatures and/or shortened day length (Sreekantan et al., 2010). Cooled gradually, V. vinifera cultivars can tolerate sustained winter temperatures as low as −15 °C without injury, whereas wild North American and Asian species can tolerate exotherms of −35 to −40 °C (Mullins et al., 1992; Fennell, 2004). As vines exit dormancy each spring, freezing vulnerability returns quickly (Fennell, 2004). Breaking buds and newly emergent green tissues can suffer injury at just −2.5 °C (Fuller and Telli, 1999). Moreover, damage to floral primordia of primary and secondary buds can drastically reduce crop yields (Fennell, 2004).

Cold acclimation, the process whereby plants sense low temperatures and activate mechanisms to increase tolerance to chilling or freezing stress, is a complex response involving multiple biochemical pathways (Nakashima and Yamaguchi-Shinozaki, 2006; Sreenivasulu et al., 2007). Central to the early cold-response pathway is the C-repeat binding factor (CBF)/dehydration-responsive element binding (DREB) family of transcription factors, of which the overexpression of a single Arabidopsis CBF gene family member is sufficient to impart an improved stress tolerance phenotype in Arabidopsis (Stockinger et al., 1997; Gilmour et al., 1998; Jaglo-Ottosen et al., 1998; Liu et al., 1998), canola (Brassica napus) (Jaglo-Ottosen et al., 2001), tomato (Hsieh et al., 2002; Lee et al., 2003) and potato (Pino et al., 2007). In addition to cold tolerance, constitutive, ectopic expression of AtCBF2 or AtCBF3 has been shown to delay leaf senescence and extend plant longevity in Arabidopsis presumably to enable winter survival until spring for lifecycle completion (Sharabi-Schwager et al., 2010).

C-repeat binding factor/DREB genes have been identified in monocots and eudicots alike (Benedict et al., 2006; El Kayal et al., 2006; Nakashima and Yamaguchi-Shinozaki, 2006; Badawi et al., 2007; Campoli et al., 2009), many of which have been characterized to have similar or conserved functions. For example, overexpression of CBF genes from cotton (Huang et al., 2009), rice (Dubouzet et al., 2003), birch (Welling and Palva, 2008), perennial ryegrass (Zhao and Bughrara, 2008) and wine grape (V. vinifera) cv. Koshu (Takuhara et al., 2011) or wild grape (V. riparia) (Siddiqua and Nassuth, 2011) in transgenic Arabidopsis improved freezing tolerance in a manner similar to AtCBF overexpression. Ectopic expression of two Eucalyptus CBF genes in transgenic Eucalyptus plants resulted in improved cold tolerance (Navarro et al., 2011). Constitutive overexpression of AtCBF1 (AtDREB1b) in transgenic V. vinifera cv. Centennial Seedless resulted in about a 2 °C improvement in cold resistance as measured by electrolyte leakage and improved vine survival at −4 °C (Jin et al., 2009). Lastly, constitutive overexpression of a peach (Prunus persica) CBF gene in apple (Malus × domestica) not only improved cold hardiness, but also resulted in short day length–related growth cessation and leaf senescence (Wisniewski et al., 2011).

The transcription factors encoded by the first three CBF/DREB family members described for V. vinifera and V. riparia were found to regulate genes that respond to low temperature, drought stress and exogenous ABA application (Xiao et al., 2006). These three transcription factor genes (VvCBF1, VvCBF2 and VvCBF3) showed increased mRNA expression in young compared with mature vegetative tissues upon exposure to freezing and drought stresses (Xiao et al., 2006). A fourth member of the Vitis CBF/DREB1 gene family, VvCBF4, was also identified for both V. vinifera and V. riparia (Xiao et al., 2008). This transcription factor gene is unique among the Vitis CBF gene family in both its expression profile and sequence (Xiao et al., 2008). Expression of VvCBF4 mRNA was sustained for several days following induction of a 4 °C cold stress, in contrast to the transient expression of VvCBF1–3 transcripts. In addition, VvCBF4 was induced similarly in both young and mature tissues. More recently, VvCBF4 transcripts have been shown to be induced after 4 h cold (4 °C) stress in leaf, stem and flower of V. vinifera cv. Koshu (Takuhara et al., 2011). VvCBF4:green fluorescent protein (GFP) fusions localized to the nucleus, and VvCBF4 expression was shown to induce beta-glucuronidase (GUS) expression under the control of the rd29a promoter in trans when co-infiltrated in tobacco leaves (Xiao et al., 2008). CBF gene transcripts with similarly sustained cold-induction patterns have been reported in other woody perennial species, such as birch (Welling and Palva, 2008), poplar (Benedict et al., 2006) and Eucalyptus (Navarro et al., 2009). These CBF transcription factors and their target genes have been suggested to play a role in the freezing tolerance of overwintering woody perennials. Additional members of the AP2/ERF gene superfamily in Vitis vinifera display diverse expression patterns in both vegetative and reproductive tissues (Licausi et al., 2010).

In this report, the overexpression of the VvCBF4 gene is shown to impart a reduced growth phenotype as well as confer improved freezing survival in nonacclimated vines, similar to the effects of CBF overexpression in Arabidopsis and other species. Microarray transcriptional profiling of a VvCBF4 overexpressor identified 47 and 12 genes with either a >1.5-fold increase or decrease in mRNA abundance, respectively, in young, unstressed shoots. These observed changes in mRNA expression suggest that the CBF regulon is widely conserved among woody and herbaceous species and that modulations in cell wall structure, lipid metabolism, epicuticular wax formation, and various stress-responses might participate in the acquisition of freezing tolerance in wine grape.


VvCBF4 is most similar to CBFs of woody perennials

VvCBF4 was identified previously (Xiao et al., 2008) and so-named simply because of the order of its discovery. To identify its relationship with other CBF genes in Vitis, and eudicots in general, phylogenetic analyses were performed (Figure 1). Phylogenetic analysis of all four Vitis CBF genes with the full-length amino acid sequences of CBFs from other dicotyledonous plants (and a non-CBF, AP2-domain protein AtERD10 as an outgroup gene) confirmed that among the Vitis CBFs, the VvCBF4 gene product shares the greatest similarity with Arabidopsis CBFs as well as two CBF gene products from another woody species, Populus trichocarpa, PtDREB70 and PtDREB71 (Figure 1a). The amino acid sequence alignment of the full-length CBF-encoding genes of Arabidopsis, P. trichocarpa and V. vinifera (Figure 1b) showed that the VvCBF4 gene product has 100% conservation with the Arabidopsis CBF consensus sequences that flank the AP2 region (N-flanking: PKKR/PRAGRxKFxETHRP, C-flanking: DSAWR), which are required for CBF function (Canella et al., 2010). In contrast, the proteins encoded by the three other Vitis CBF genes (VvCBF1, VvCBF2 and VvCBF3) as well as those encoded by the PtDREB66 and PtDREB67 genes share a variant of the established consensus between positions 1 and 4 of the N-flanking consensus (H/KKR/NK instead of PKKR/P). However, only the three other Vitis CBF (VvCBF1, VvCBF2 and VvCBF3) gene products contain a 21–25-amino acid stretch between the DSAWR motif and the C-terminus found in neither the Arabidopsis and Populus CBF gene products nor that of VvCBF4. Additionally, the gene products of VvCBF1, VvCBF2 and VvCBF3 as well as those of the PtDREB66 and PtDREB67 genes share a variation (e.g., LWN(E)D(H/E)) in the generally conserved LWSY amino acid sequence motif at their C-terminus.

Figure 1.

 Comparative sequence analysis of C-repeat binding factor (CBF) protein family members in eudicot species. (a) Phylogenetic analysis of CBF/DREB proteins found in eudicot species. CBF proteins were identified from Arabidopsis thaliana, Vitis vinifera, Populus trichocarpa, Glycine max and Medicago truncatula– species with published draft genomes. AtERF10 is included as an outgroup root. (b) Amino acid alignment of A. thaliana, P. trichocarpa and V. vinifera CBF transcription factors. Identical and similar amino acids are coloured dark and light gray, respectively. The conserved APETALA 2 (AP2) domain and the CBF-conserved AP2 N-flanking PKKP/RAGRxKFxETRHP, AP2 C-flanking DSAWRL and LWSY motifs are labelled with boxes above the alignment. Accession numbers are listed in the Experimental Procedures section.

Quantitative real-time RT-PCR (qRT-PCR)

To quantify expression of the cauliflower mosaic virus (CaMV) 35S::VvCBF4 transgene in three independently transformed lines of Vitis rootstock ‘Freedom,’ quantitative real-time, reverse-transcriptase PCR (qRT-PCR) was performed. Whereas the ORFs of native and transgenic VvCBF4 transcript are identical, the native 3′UTR was replaced by a 35S CaMV terminator region in the 35S::VvCBF4 construct. Thus, primers were designed to detect either ‘native’ or transgenic (‘tg’) VvCBF4 transcripts exclusively from these regions (Table S1, Figure S1).

qRT-PCR experiments demonstrated that both native and tg primers were specific to the mRNAs for which they were designed. In each of the three independent transgenic lines, 9–3, 9–12 and 9–1, tg transcripts were readily detected, whereas no amplification of tg could be detected in control line 8–6 (Figure 2). VvCBF4 overexpression resulted in no observable changes in native transcript abundance. One-way ANOVA of native VvCBF4 abundance showed no significant difference in genotype (F > 0.05), which remained low regardless of the line tested (Figure 2).

Figure 2.

 Overexpression of 35S::VvCBF4 in transformed Vitis cv. ‘Freedom’ shoots. qRT-PCR of native and transgenic VvCBF4 transcripts for control (line 8–6) and three independently transformed 35S::VvCBF4-overexpressing lines (9–3, 9–12, 9–1). Wild-type VvCBF4 (wt) transcript relative abundance; transgenic VvCBF4 (tg) transcript relative abundance. Transcript abundances were normalized to an actin reference gene. Error bars indicate ± standard error (SE). Significantly different abundance was determined using the Student’s t-test with Bonferroni correction, P < 0.05; n = 3). Significant pairwise differences in tg expression are indicated with letters (a, b).

In the 35S::VvCBF4 transformed line 9–12, tg transcript accumulated to concentrations 20-fold greater than native transcript levels. Another line, 9–3, expressed 35S::VvCBF4 less strongly than 9–12, with only 71% of the transgene abundance of 9–12, or 14-fold greater than native transcript. The tg mRNA abundance difference between 9–3 and 9–12 was statistically significant as determined by Student’s t-test with Bonferroni correction (P = 0.048). Transgene expression in line 9–1, however, was highly variable among biological replicates, with expression ranging between 72% and 360% of 9–12 transgene relative abundance (or between 14- and 72-fold of native transcript). Owing to this large variability, comparison of expression in 9–1 with the other transgenic lines failed to reveal any significant expression differences in contrast to 9–3 or 9–12.

VvCBF4 overexpression reduces shoot elongation

The constitutive overexpression of some CBF/DREB genes using the 35S-CaMV promoter is known to result in dwarfing effects in species such as Arabidopsis thaliana (Liu et al., 1998; Gilmour et al., 2000), Medicago sativa (Zhang et al., 2005) and Lolium perenne L. (Zhao and Bughrara, 2008). To assess the effect of 35S::VvCBF4 transgene expression on plant phenotype, the shoot elongation rate (SER) was measured at 3-day intervals in young vines over an 18-day period for control line 8–6 and the three selected 35S::VvCBF4-overexpressing lines (Figure 3a,b). By one-way anova, genotype accounted for highly significant observed differences in SER (F < 0.0001). Compared to control line 8–6, which had an average SER of 12.5 mm/day, SER was reduced in all 35S::VvCBF4-transformed vines. Line 9–3 SER was reduced slightly, although significantly, to 10.9 mm/day (P < 0.05). Lines 9–12 and 9–1 vines displayed even more dramatic reductions in SER, with rates of 7.4 and 4.5 mm/day, respectively (both were significantly different from control line 8–6, each P < 0.001) (Figure 3b).

Figure 3.

 The C-repeat binding factor (CBF)-induced dwarfing effect is dependent on relative level of VvCBF4 overexpression in 35S::VvCBF4 Vitis cv. ‘Freedom.’ (a) Image showing two representative vines each of control line 8–6 and 35S::VvCBF4 overexpressing lines 30 days after transplantation to soil. (b) Shoot elongation rate for line 8–6 (control) and 35S::VvCBF4-overexpressing lines (9–3, 9–12, 9–1). Each bar represents the mean shoot elongation rate of eight vines measured over 18 days, every third day. Error bars indicate standard error of the mean; n = 8. Growth for each 35S::VvCBF4-overexpressing line was significantly different from control line 8–6 (repeated measures anova). *P < 0.05; ***P < 0.001. (c) Average length of the maximum (Max.) internode length per plant. (d) Total increase in number of nodes (Δ nodes) after 18 days of observation for control line 8–6 and 35S::VvCBF4-overexpressing lines (n = 8). Error bars indicate standard error of the mean; n = 8. *significant difference between control (line 8–6) and VvCBF4-overexpressing lines based on the Student’s t-test with Bonferroni correction, P < 0.05; n = 8).

In lines 9–12 and 9–1, lengths of stem sections, or internodes, were also reduced greatly (Figure 3c). Over the course 18 days of growth observation, the longest internodes on individual 9–12 vines (e.g., the single longest stem section from each of eight vines, averaged) were 36.8 mm, or 66% of the lengths achieved by 8–6 vines. Similarly, line 9–1 grew to maximum lengths of 33.1 mm (59% of control). These reduced node lengths were highly significant (P < 0.01).

The two most dwarfed lines, 9–12 and 9–1, differed in the number of nodes and the attachment site of leaves produced during this 18-day period (Figure 3d). Line 9–12 vines initiated 11.6 (SD ± 2.1) leaves on average, 86% as many leaves as did 8–6, which produced an average of 13.5 leaves (SD ± 1.3). This difference was not statistically significant. In contrast, the other dwarfed line, 9–1, produced an average of only 8.0 (SD ± 1.1) leaves in the same period. Genotypic differences were significant (one-way anova by genotype, P < 0.001), and line 9–1 (and only line 9–1) differed from each of the other three lines (Bonferroni-corrected pairwise tests, P < 0.01).

Taken together with the VvCBF4 transgene expression data for each of these transformed lines, the growth data indicated that a dose–response relationship exists between VvCBF4 transcript abundance and the dwarf phenotype, with high-overexpressing line 9–12 showing a more reduced SER than the medium-overexpressing line 9–3. Line 9–1 growth reduction was the most severe, although the high variability of VvCBF4 transgene expression (Figure 2) limited the correlation with the growth phenotype in this line. Overall, these results are consistent with reports for other species in which constitutive overexpression of a CBF/DREB gene under the control of a constitutive promoter resulted in dwarf or reduced aerial biomass phenotypes (Kasuga et al., 1999; Gilmour et al., 2000; Benedict et al., 2006; Pino et al., 2007; Achard et al., 2008a; Navarro et al., 2011).

VvCBF4 overexpression increases freezing survival

As VvCBF4 transcripts were previously reported to undergo increased relative abundance during the application of 4 °C chilling in Chardonnay (Xiao et al., 2008), the effect that VvCBF4 overexpression had on freezing survival rates was investigated. Freezing stress resistance was assessed in 35S::VvCBF4 transgenic vines by whole-plant survival analysis of nonacclimated vines. The 50% lethal temperature (LT50) for control line 8–6 vines was determined experimentally to be at or near −2 °C for 24 h (data not shown), so these conditions were chosen for survival testing between control and 35S::VvCBF4 transgenic lines. Following nine replicate experiments with 10 young vines/line in each experiment, the freezing survival rate for control line 8–6 averaged only 29% (Figure 4). Survival of 35S::VvCBF4 line 9–12 was significantly higher than control, at 52% survival (P < 0.01). Survival of lines 9–1 and 9–3 were 39% and 43%, respectively, which was not significantly different from the control line (P > 0.05).

Figure 4.

 35S::VvCBF4 overexpression enhances freezing survival in transformed line 9–12. (a) Percentage survival for mock-transformed line 8–6 (control) and 35S::VvCBF4-overexpressing lines (9–3, 9–12, 9–1) following exposure to freezing at −2 °C for 24 h with 14 days recovery at 22 °C. Each bar represents the mean of nine replicate experiments with 10 individual vines used for each genotype in each experiment. Error bars indicate ± SE. The results indicated by different letters are significantly different based on the Student’s t-test with Bonferroni correction; (P < 0.01). (b) Exemplar images of freezing survival after recovery from one of the nine experimental trials.

Cold acclimation in plants is a dynamic process that results from exposure to low nonfreezing temperatures (Thomashow, 1999). Following a given cold acclimation period, electrolyte leakage assays are employed typically to assay any increased cold- or freezing-tolerance imparted to the cell by the numerous biochemical changes that occur during acclimation (Gilmour et al., 2000). Because overexpression of CBF/DREB genes might mimic the effects of cold acclimation, we performed electrolyte leakage assays on leaf discs cut from fully expanded leaves of nonacclimated control line 8–6 and VvCBF4-overexpressing vines. Line 9–3, which expressed ∼70% as much VvCBF4 mRNA as line 9–12 (Figure 2), exhibited a change in leakage only at −6 °C (Figure 5a). Line 9–12 showed a 2 °C greater resistance to electrolyte leakage than the control line with these differences appearing at −6 and −7 °C (Figure 5b). At −8 °C, line 9–12 leaf discs had begun leaking and no further differences could be observed. No significant changes between line 9–1 and control line 8–6 were observed (Figure 5c). The lack of measurably significant enhancement of survival or electrolyte leakage in line 9–1 might be related to the severity of growth retardation in this line (Figure 3) in combination with the propagation and assay conditions employed. Sterile cuttings from line 9–1 typically had shorter and fewer roots and had a lower survival rate when transplanted to soil compared with the other lines under identical propagation conditions, suggesting a lowered fitness for 9–1 vines propagated on the same time scale as faster-developing vines. Based upon freezing survival and electrolyte leakage assays, line 9–12 was selected for detailed genotypic evaluation.

Figure 5.

 35S::VvCBF4-transformed ‘Freedom’ lines exhibit less electrolyte leakage upon freezing stress. Percentage leaf-disc electrolyte leakage for line 8–6 (control) and 35S::VvCBF4-overexpressing lines (a) 9–3, (b) 9–12 and (c) 9–1 following exposure to subfreezing temperatures as indicated. Each point represents the mean electrolyte leakage of leaf discs from 11. Error bars indicate ± SE. *Significant difference between the 35S::VvCBF4-transformed line and control line 8–6 at the marked temperature (one-way anova with post hoc Student’s t-test with ordered differences test, P < 0.05; n = 11).

Global changes in gene expression in 35S::VvCBF4 overexpressor

To investigate the molecular mechanisms involved in the observed enhancement of freezing tolerance in VvCBF4 overexpressor line 9–12, differences in relative mRNA abundance changes in this line were compared with those of empty vector control line 8–6 using microarray transcript profiling. RNA was extracted from whole aerial portions of young vines of line 9–12 and control line 8–6 (four biological replicates each), which had been propagated and harvested 30 day after transplantation to soil. RNA was verified to be of high quality with no degradation using an Agilent 2100 Bioanalyzer (Figure S2). After cRNAs were hybridized to Affymetrix®Vitis GeneChip® microarrays and probeset intensities normalized by robust multiaverage (RMA), principal component analysis (PCA) of the RMA probe intensities found 99.4% of the variation among all samples to be explained by two components, p1 and p2 (Figure 6). Along these two axes, the microarray data can be seen to segregate by genotype, indicating that expression of the transgene is associated strongly with the observed phenotypic differences.

Figure 6.

Principal component analysis of microarray probeset variation. Robust Multichip Average (RMA) normalized intensities were analysed with ∼16 000 probesets on Affymetrix®Vitis GeneChip® microarrays for the RNA transcripts of 35S::VvCBF4-overexpressing line 9–12 (black) and empty vector-transformed Vitis rootstock ‘Freedom’ control line 8–6 (white). Whole aerial portions (stem + five leaves) of nonstressed young vines were used for RNA extraction, and tissue from three different vines was pooled for RNA isolation and microarray hybridization (n = 4 arrays per genotype). Principal components 1 and 2 (p1, p2) account for 99% and 0.4% of the variation among the eight microarrays.

In VvCBF4-overexpressing line 9–12, 48 probesets were identified to have 1.5-fold or greater abundance than in control line 8–6, (significance of P < 0.05) using t statistics corrected for multiple comparisons (Benjamini and Hochberg, 1995). A total of 47 unique transcripts increased in abundance in the VvCBF4 overexpressor, which included an acid phosphatase class B (XP_002273448) that was specified by two independent probesets that showed 2.0-fold (probeset ID 1621892_a_at) and 1.6-fold (probeset ID 1617422_at) increase, respectively (Table 1). Changes in increased relative expression ranged from 1.5-fold to 5.6-fold. An additional 12 transcripts exhibited significantly decreased relative abundance in line 9–12, compared with control line 8–6 (Table 1). Changes in decreased relative expression ranged from −1.5-fold to −37.1-fold. The complete list of transcripts that displayed significantly different patterns of expression is presented in Table S2.

Table 1.   Transcripts with significantly different expression in VvCBF4 overexpressor line (9–12) compared with control line (8–6)
AnnotationFold ratioadj. P-valueProbeset IDProtein IDGene ID
Increased abundance
Pectin methylesterase Inhibitor (PMEI)5.6290.0261606429_atXP_002264167LOC100251832
AAA-type ATPase domain (chaperone-like)4.7240.0041610816_atXP_002275572LOC100252698
Sulphite exporter TauE/SafE2.5320.0481609884_atXP_002267318LOC100253061
Leucine-rich repeat extensin2.4130.0261620976_atXP_002277227LOC100252056
Hypothetical protein2.3210.0451612536_s_atXP_002283137LOC100255628
Gibberellin-regulated protein (GASA5)2.2720.0491617881_atXP_002280219LOC100259439
Phytepsin aspartyl protease2.2520.0481616668_atXP_002276363LOC100266485
Lipid transfer protein (LTP3)2.1930.0491608175_atXP_002275107LOC100265591
Lactoylglutathione lyase/glyoxalase I2.1480.0481619235_atXP_002271396LOC100266132
Flavonoid 3′,5′-hydroxylase (F3′5′H)/CYP96A102.0890.0261612325_atXP_002279531LOC100266173
Serine carboxypeptidase III2.0690.051620729_atXP_002271855LOC100242723
Unknown protein2.0520.0311613368_atXP_002283053LOC100243457
Acid phosphatase class B2.0240.011621892_a_atXP_002273448LOC100246316
Xyloglucan endo-transglycosylase (XET)2.0080.0381617739_atXP_002274520LOC100232906
Rho GDP-dissociation inhibitor 21.9310.051611669_atXP_002263904LOC100260088
DUF1070 domain AGP41-like1.9270.051622530_atXP_002283086LOC100249036
Lipase GDSL1.880.0391607341_atXP_002283363LOC100242887
DNA-3-methyladenine glycosidase I1.8760.0441621976_atXP_002263612LOC100256507
Lipase GDSL1.8290.0491620618_atXP_002271851LOC100263626
Pepsin-like aspartic protease1.8080.0451611138_s_atXP_002265771LOC100246744
Glycosyl hydrolase 17-like1.7840.0311622656_atXP_002285661LOC100257244
Glycerol-3-phosphate acyltransferase 4-like1.7750.0311612479_atXP_002275348LOC100243093
BSL1-like serine/threonine phosphoesterase1.7720.0311615748_atXP_002270638LOC100249353
Pollen proteins Ole e I1.7610.0271610746_atXP_002272595LOC100265200
Embryo-specific 31.7570.0261614771_atXP_002266473LOC100242179
Lipase GDSL1.7520.0311607744_atXP_002272970LOC100249332
Zinc finger (C3HC4-type RING)1.7290.0311612240_atXP_002262825LOC100263579
Polyketide cyclase/dehydratase (MLP28-like)1.7240.0451607196_atXP_002284578LOC100262819
Pectate lyase1.7170.0481616158_atXP_002285340LOC100242302
Unknown protein1.6960.0311617384_atXP_002281002LOC100261133
Similar to thaumatin-like VVTL11.6890.0491614746_atXP_002284403LOC100259225
Exordium-like 31.6890.0481614951_atXP_002264723LOC100266367
Acid phosphatase class B1.6860.0461617433_atXP_002273448LOC100246316
DUF1070 domain AGP20-like1.6550.0191619401_atXP_002280494LOC100250031
LACERATA-like CYP4501.5640.0481621973_atXP_002275806LOC100247907
GDP-fucose protein O-fucosyltransferase1.5620.0451613171_atXP_002267185LOC100260861
Calcium-binding EF-hand-containing MSS3-like1.5540.0481612996_atXP_002266359LOC100254364
Tubulin beta-1 chain1.5360.0451607001_atXP_002275306LOC100259087
Glucose–methanol–choline(GMC) oxidoreductase1.5350.051622345_atXP_002282510LOC100266705
Unknown protein1.5290.0351610862_atXP_002274279LOC100260563
Plant Basic Secretory Protein1.5180.0311615434_atXP_002283729LOC100257403
Protein transport Sec61 subunit beta1.5070.0481622732_atXP_002276029LOC100247887
Decreased abundance
Chlorophyllase 2 (CHL2)−1.5140.0351616275_atXP_002279285LOC100252835
Unknown protein (DUF1279, thylakoid)−1.5210.0451619538_atXP_002271410LOC100258627
Galactinol synthase 4-like−1.6180.0481608907_s_atXP_002265947LOC100260266
Copper amine oxidase−1.6750.0451608650_atXP_002263349LOC100257527
ABC transporter MRP4-like−1.7030.0491617849_atXP_002265012LOC100253698
Secretory peroxidase−2.0950.0481621431_atXP_002269918LOC100257005
Chitinase class IV C (CHI4C)−2.2050.0481617192_atXP_002275516LOC100232911
Ubiquitin-protein ligase (PUB23-like)−2.2560.041606741_atXP_002267438LOC100250551
Nicotinamidase 1−2.2640.0481610169_atXP_002270896LOC100250570
CYP94 family protein−2.4910.0261609552_atXP_002278009LOC100267102
Alpha-glucan phosphorylase (PHS2-like)−3.040.0431614707_atXP_002280732LOC100251865

Validation of transcript abundance by qRT-PCR

To validate the observed transcript abundance changes obtained using the Vitis GeneChip® microarray, qRT-PCR was performed on a set of nine genes that were selected at random from among those genes exhibiting either significantly increased or decreased transcript abundance using gene-specific primer pairs (Table S1). Linear regression analysis of the log2-transformed values from the microarray analysis with those from qRT-PCR showed a goodness of fit (R2) of 0.81, confirming that the observed changes in transcript abundance were accurate (Figure 7).

Figure 7.

 Verification of microarray results by real-time qRT-PCR. Log2-transformed values of Affymetrix®Vitis GeneChip® signal intensities (x-axis) and real-time PCR expression ratios (y-axis) for 35S::VvCBF4/control of nine differentially expressed microarray probe sets (open circles). The linear regression had a goodness of fit R2 = 0.81.

Comparison of woody CBF regulons

Transcriptome changes observed because of ectopic 35S::VvCBF4 expression were compared with those identified in annual (leaf) and perennial (stem) tissues of 35S::AtCBF1 regulons in the woody species Populus (Benedict et al., 2006). The closest protein homologues within previously reported CBF regulons were compared using BLAST homology searches. Comparing 35S::VvCBF4 transgene expression with that in 35S::AtCBF1 poplar revealed 10 genes with similar trends of increased and one gene, a chitinase class IV C gene, with decreased transcript abundance (Table 2). Three additional genes shared significant expression differences between the two transgenic woody species, but displayed varying directionality of expression (Table 2). These results indicate that at least part of the CBF regulon is shared between these two diverse woody species.

Table 2. Comparison of changes in gene expression in VvCBF4-overexpressing grapevine with AtCBF1-overexpressing Poplar
In 35S::VvCBF4-grapevineIn 35S::AtCBF1-Poplar (as reported by Benedict et al., 2006) 
Vitis vinifera gene descriptionV. vinifera RefSeq IDVitis Shoot FCPoplar Leaf FCPoplar Stem FCPoplar gene ID (best At match)*Poplar gene descriptionBLAST
  1. FC, fold change.

  2. *Arabidopsis gene locus identifiers used as the sole designators for UPSC-KTH 13K Poplar microarray probes designed by Benedict et al., 2006 (Tables 1a–c).

Reported transcript changes similar in Vitis and Poplar CBF overexpressors
Exordium-like 3XP_0022647231.7−1.41.8At2g17230.1Exordium-like 53.4E-92
Glycosyl hydrolase 17-likeXP_0022856611.82.3−1.1At5g55180.2O-Glycosyl hydrolase family 172.0E-18
Calcium-binding EF-hand MSS3-likeXP_0022663591.61.01.8At5g39670.1Calcium-binding EF-hand family protein9.0E-16
Xyloglucan endo-transglycosylase (XET)XP_0022745202.01.62.0At3g23730.1Xyloglucan endo-transglucosylase/hydrolase 162.6E-87
Unknown proteinXP_0022830532.11.11.9At4g35320.1Unknown protein4.7E-30
Leucine-rich repeat extensinXP_0022772272.41.8−1.1At1g28290.1Arabinogalactan protein 31 (AGP31)9.4E-30
Rare Cold-Inducible 2B (RCI2B)XP_0022793331.41.91.9At3g05880.1Low temperature and salt responsive protein (LTI6A)5.0E-22
Lipase GDSL (1)XP_0022718511.82.31.1At5g45950.1GDSL-like Lipase/Acylhydrolase (1)2.3E-70
Lipase GDSL (2)XP_0022729701.82.31.2At1g29660.1GDSL-like Lipase/Acylhydrolase (2)1.1E-56
Lipase GDSL (3)XP_0022833631.92.81.1At1g71691.2GDSL-like Lipase/Acylhydrolase (3)6.1E-55
Chitinase class IV C (CHI4C)XP_002275516−2.2−1.2−2.0At3g54420.1Chitinase (ATEP3, ATCHITIV)1.1E-111
Reported transcript changes differ between Vitis and Poplar CBF overexpressors
Pectate lyaseXP_0022853401.7−2.0−2.1At4g24780.1Pectin lyase-like7.1E-202
Lactoylglutathione lyase/glyoxalase IXP_0022713962.1−2.51.1At1g15380.1Lactoylglutathione lyase/glyoxalase I9.0E-45
ABC transporter MRP4-likeXP_002265012−1.7−1.22.4At3g21250.2Multidrug resistance-associated protein 6 (MRP6)3.7E-297

Comparison of herbaceous CBF regulons

Next, the ectopic 35S::VvCBF4 mRNA expression profile was compared with that from various independent reports of ectopic expression of AtCBF or closely related genes including the 35S::AtCBF1, 35S::AtCBF2 35S::AtCBF3 regulons (Fowler and Thomashow, 2002; Maruyama et al., 2004, 2009; Vogel et al., 2005; Sharabi-Schwager et al., 2010) and the 35S::DDF1 regulon (Magome et al., 2008) in the herbaceous species Arabidopsis (Table 3). A total of nine and four shared genes showed similar increases or decreases, respectively, in relative transcript abundance with two genes, a pectin methylesterase inhibitor (PMEI) and a GDSL lipase being common to four and five independent studies, respectively. Another five genes were shared among different experiments, but had only partially similar transcript expression trends between 35S::VvCBF4 and the different AtCBF- or DDF1f-overexpressing Arabidopsis lines. Three genes showed consistent transcript expression trends with one or more independent experiments: a chitinase class IV gene, a thaumatin-like gene and a pepsin-like aspartic protease (Table 3). Lastly, six genes showed disparate transcript expression trends between 35S::VvCBF4 expression profiles and among the different AtCBF- or AtDDF1f-overexpressing Arabidopsis lines (Table 3). The amount of overlap in target genes observed for 35S::VvCBF4 and these various Arabidopsis 35S::AtCBF (DDF1f) transgenic lines is comparable to the degree of overlap observed when 35S::AtCBF1-driven expression in Poplar was compared with the AtCBF3 regulon in Arabidopsis, wherein 12 genes showed similar increased transcript abundance (Benedict et al., 2006). These results indicate that CBF regulons share common targets in both woody and herbaceous species.

Table 3.   Comparisons of changes in gene expression in VvCBF4-overexpressing grapevine with multiple C-repeat binding factor (CBF)/DREB1f-overexpressing Arabidopsis
In 35S::VvCBF4-grapevineIn CBF-overexpressing Arabidopsis 
Vitis vinifera RefSeq IDV. vinifera gene descriptionVitis CBF4CBF1* FCCBF2 FCCBF2 FCCBF3§ FCDDF1 FCArabidopsis Gene IDArabidopsis gene descriptionBLAST
  1. FC, fold change.

  2. *35S::AtCBF1 in Arabidopsis (Fowler and Thomashow, 2002).

  3. 35S::AtCBF2 (Sharabi-Schwager et al., 2010).

  4. 35S::AtCBF2 (Vogel et al., 2005).

  5. §35S::AtCBF3 (Maruyama et al., 2009).

  6. 35S::DDF1 (Magome et al., 2008).

  7. GASA, gibberellic acid-stimulated Arabidopsis.

Transcript abundance trends similar between 35S::VvCBF4-grapevine and CBFs in Arabidopsis
XP_002264167Pectin methylesterase Inhibitor (PMEI)5.6 At5g62350.1Invertase/PMEI1.9E-52 At5g62360.1Invertase/PMEI6.9E-42
 4.4  3.9 At3g47380.1Invertase/PMEI1.7E-35
XP_002280219Gibberellin-regulated protein (GASA5)2.3 3.9   At1g75750.1GASA11.6E-19
XP_002271855Serine carboxypeptidase III2.1 2.5   At2g22980.1Serine carboxypeptidase-like 132.3E-39
XP_002283363Lipase GDSL (#1)1.9 3.0   At1g29660.1GDSL-like Lipase/Acylhydrolase (#2)1.1E-56
XP_002271851Lipase GDSL (#2)1.832.411.0258.5 64.0At2g24560.1GDSL-like Lipase/Acylhydrolase (#3)1.0E-68
XP_002272970Lipase GDSL (#3)1.8 3.8 2.1 At1g29670.1GDSL-like Lipase/Acylhydrolase (#4)1.7E-59
    2.3 At4g30140.1CDEF1 | GDSL-like5.4E-42
XP_002280885Stellacyanin-like1.8   2.2 At4g27520.1Early nodulin-like 21.5E-16
XP_002285340Pectate lyase1.7 2.54.5  At3g09540.1Pectin lyase-like superfamily1.4E-39
XP_002275806LACERATA-like CYP4501.6    4.0At2g27690.1CYP94C11.0E-84
XP_002279285Chlorophyllase 2 (CHL2)−1.5 −9.8−5.1−2.8−5.3At1g19670.1Chlorophyllase 1 (CHL1)3.2E-70
XP_002263349Copper amine oxidase−1.7 −2.7   At4g12280.1Copper amine oxidase3.6E-129
XP_002269918Secretory peroxidase−2.1    −8.6At5g06730.1Peroxidase superfamily2.7E-85
XP_002278009CYP94 family protein−2.5    −5.3At5g63450.1CYP94B11.3E-118
Transcript abundance trends partially similar between 35S::VvCBF4-grapevine and CBFs in Arabidopsis
XP_002275516Chitinase class IV C (CHI4C)−2.2    −4.0At2g43590.1Chitinase family protein2.2E-91 family protein1.0E-69
XP_002284403Similar to thaumatin-like VVTL11.7   −4.2−3.2At1g75040.1Pathogenesis-related gene 51.5E-64 At4g36010.1Pathogenesis-related thaumatin1.1E-59
XP_002265771Pepsin-like aspartic protease1.8   −2.7 At5g10770.1Eukaryotic aspartyl protease family7.8E-41
   −3.6−4.6 At5g10760.1Eukaryotic aspartyl protease family5.4E-34
  5.5   At3g52500.1Eukaryotic aspartyl protease family2.1E-30
  10.03.0 3.7At3g54400.1Eukaryotic aspartyl protease family1.9E-26
XP_002265947Galactinol synthase 4-like−1.617.7 6.1 4.6At1g60470.1Galactinol synthase 45.5E-158
  3.5   At2g47180.1Galactinol synthase 13.6E-149
  −4.4 18.3 At1g56600.1Galactinol synthase 23.2E-142
 88.462.7346.550.621.1At1g09350.1Galactinol synthase 31.3E-138
XP_002284578Polyketide cyclase/dehydratase (MLP28-like)1.7   2.4−3.2At1g70850.3MLP34 | MLP-like protein 345.2E-44
     −17.1At1g70880.1Polyketide cyclase/dehydrase8.4E-34
Transcript abundance trends dissimilar between 35S::VvCBF4-grapevine and CBFs in Arabidopsis
XP_002265012ABC transporter MRP4-like−1.7   2.2 At3g62700.1Multidrug resistance-associated protein 100.0E+00
XP_002273448Acid phosphatase class B2.0 −13.3  −5.7At5g24780.1Vegetative storage protein (VSP1)8.0E-42
XP_002279531Flavonoid 3′,5′-hydroxylase CYP96A102.1   −2.4−19.7At5g52320.1CYP96A42.5E-138
XP_002280732Alpha-glucan phosphorylase (PHS2-like)−3.0   3.2 At3g29320.1Glycosyl transferase, family 350.0E+00
    3.9 At3g46970.1Alpha-glucan phosphorylase 2 (PHS2)0.0E+00
XP_002266359Calcium-binding EF-hand MSS3-like1.6   −2.2 At2g43290.1Calcium-binding EF-hand (MSS3)4.5E-76
    −2.2 At3g59440.1Calcium-binding EF-hand family3.7E-70
XP_002274520Xyloglucan endo-transglycosylase (XET)2.0   −2.5 At5g65730.1XTH61.0E-87
    −2.9 At4g30270.1XTH241.8E-79
  −2.8 −2.4 At3g44990.1XTH317.6E-54

Two-dimensional difference in gel electrophoresis (2D-DIGE) analysis

To quantify the effect of 35S::VvCBF4 transgene expression on relative protein abundance, 2D-DIGE analysis was performed. Seventy-seven spots showed significant differences (one-way anova, P ≤ 0.05) in abundance in 35S::VvCBF4 line 9–12 when compared with control line 8–6 across four biological replicates (data not shown). Of these, 29 spots, of which 17 and 12 proteins were increased or decreased in protein abundance, respectively, were identified by MALDI-TOF/TOF analysis (Table S4).


In this study, the role of a wine grape CBF transcription factor in conferring improved freezing tolerance was confirmed in the leaves of V. vinifera cv. Freedom. VvCBF4 is unique among the four known CBF/DREB genes found in grapevine, sharing only 48%, 45% and 45% homology with VvCBF1, VvCBF2 and VvCBF3, respectively (Xiao et al., 2008). In contrast, VvCBF4 shares 58% sequence homology with A. thaliana CBF1, one of four known Arabidopsis CBF/DREB1 genes. Among all genes found in the fully sequenced plant genomes, VvCBF4 gene product has the greatest amino acid similarity the Populus trichocarpa PtDREB70 and PtDREB 71 gene products (Figure 1). PtDREB70 and PtDREB 71, which were previously identified as PtCBF2 and PtCBF1 by Benedict et al. (2006), are cold-inducible CBF genes with peak mRNA expression at 9 and 6 h after cold-exposure, respectively. Previously, transient expression assays of VvCBF4 constructs in tobacco leaves confirmed that VvCBF4 was capable of inducing the transcription of reporter genes via CRT cis-elements and that Vitis CBF4:GFP localized to the nucleus (Xiao et al., 2008). These observations, combined with the long duration of cold induction (2–5 days) of its transcript, which is even longer in the cold-hardy Vitis riparia species (Xiao et al., 2008), suggested that VvCBF4 is likely to play an important role in adaptation to cold or freezing conditions. In Eucalyptus, another woody species, differences in the duration of cold induction of CBF genes between cold-hardy and cold-sensitive species have also been reported (Navarro et al., 2009).

The constitutive overexpression of VvCBF4 enhanced freezing survival (Figure 4) and reduced electrolyte leakage under freezing conditions by about 2 °C (Figure 5) in two of three VvCBF4-expressing lines tested. The observed improvements in freezing tolerance are comparable with those observed in transgenic poplar ectopically expressing AtCBF1, which showed an improvement in freezing tolerance of 1.5 °C in stems and 3 °C in leaves, as assessed by electrolyte leakage assays (Benedict et al., 2006). These results were also consistent with the improved freezing tolerance afforded by ectopic, constitutive expression of two endogenous CBF genes in transgenic Eucalyptus (Navarro et al., 2011). Overexpression of AtCBF1 (AtDREB1b) under the control of the constitutive CaMV 35S promoter in transgenic V. vinifera cv. Centennial Seedless resulted in about a 2 °C improvement in cold resistance as assessed by electrolyte leakage assays, reduced vine wilting and improved vine survival at −4 °C after 12 h (Jin et al., 2009). Ectopic expression of a VvCBF4 gene from cv. Koshu under the control of the CaMV 35S promoter conferred cold tolerance in transgenic Arabidopsis (Takuhara et al., 2011). Lastly, these results were also consistent with the 2–3 °C or 4–6 °C increase in freezing tolerance exhibited by different transgenic apple trees expressing a peach PpCBF1 gene compared with untransformed control trees as determined by electrolyte leakage assays (Wisniewski et al., 2011). Taken together, these results indicate that Vitis spp. employ CBF-mediated response regulons, in part, to modulate cold acclimation responses. If the protection against frost damage observed here for shoot tissues were pertinent to floral primordia of primary and secondary buds (Fennell, 2004), then this engineering strategy might serve to improve crop yields by reducing frost damage to fruit-bearing structures.

35S::VvCBF4 overexpressor results in dwarf phenotypes

Interestingly, the 35S::VvCBF4 expressing lines displayed a dwarf phenotype characterized by slower shoot elongation rates, shorter internodes and fewer interodes per plant in a transgene dose-dependent manner (Figure 3). This observation is well known from constitutive, ectopic expression of AtCBF1, AtCBF2 and AtCBF3 genes in Arabidopsis, which results in dwarf phenotypes with smaller leaves (Liu et al., 1998; Kasuga et al., 1999; Gilmour et al., 2004; Sharabi-Schwager et al., 2010). Although direct measurements were not made to assess changes in either cell numbers or cell size in the transgenic Vitis lines, the dwarf phenotype observed here is likely to be the result of reduced cell expansion or elongation rather than cell division as observed in EguCBF1a overexpressing Eucalyptus (Navarro et al., 2011).

In the woody species poplar, ectopic AtCBF1 expression resulted in a slowing of growth rate in plants <6 weeks of age (Benedict et al., 2006). After this age, tree growth rates returned to normal. Similarly, constitutive ectopic expression of two endogenous CBF genes in Eucalyptus resulted in reduced growth of microcuttings and leaf area, with EguCBF1a having a more pronounced effect than EguCBF1b (Navarro et al., 2011). Similarly, ectopic expression of a peach CBF gene in transgenic apple, a woody perennial species, resulted in reduced leaf size, but increased leaf dry weight, increased anthocyanin accumulation in cold-acclimated leaves, reduced shoot growth, and onset of dormancy as exhibited by terminal bud set and basipetal leaf senescence triggered by exposure to short day length (SD) or cold (4 °C) conditions (Wisniewski et al., 2011). In contrast, constitutive expression of a VvCBF4 gene from cv. Koshu in transgenic Arabidopsis apparently did not result in dwarfing, but only limited morphometric data were reported (Takuhara et al., 2011).

In wine grape, the observed reduction in growth rates observed for the 35S::VvCBF4 transgenic lines might be desirable in a production setting because it would be expected to reduce vine vigour, thereby reducing labour costs normally associated with pruning and leaf-pulling, activities customarily practiced in vineyards to improve fruit and wine quality via increased sunlight exposure of the fruit (Matus et al., 2009). CBF gene overexpression is also known to delay flowering in some instances depending upon the CBF gene family member (Gilmour et al., 2004; Sharabi-Schwager et al., 2010). Delays in flowering time in wine grape might help avoid late spring frost damage to floral primordial and improve berry yield in some areas (Fennell, 2004). However, such delays might also limit berry-ripening potential in areas with short growing seasons.

Gene expression changes in the 35S::VvCBF4 overexpressor

The 47 transcripts with significantly increased relative abundance in VvCBF4-overexpressing grape vines spanned a wide range of molecular functions (Table 1). Five transcripts encoded proteins that catalyse the polymerization or depolymerization of cell wall structural components. For example, increased expression of the proteinaceous pectin methylesterase inhibitor (PMEI; 5.6-fold; 1606429_at) might act to reduce pectin depolymerization-based cell wall loosening leading to growth inhibition consistent with a dwarf phenotype (Figure 3), although the exact in vivo role of this inhibitor remains unclear (Jolie et al., 2010). In another example, increased transcript abundance of a leucine-rich repeat extensin (LRX; 2.4-fold; 1620976_at), which are thought to reinforce and stabilize cell wall polysaccharide structure by cross-linking (Baumberger et al., 2003; Ringli, 2010), might also be consistent with dwarfing as increased activity of this enzyme would be expected to limit cell size. Increased expression of an xyloglucan endotransglycosylase (XET; 2.0-fold; 1617739_at), which can participate in cell wall strengthening and reduce cell wall extension in vitro (Maris et al., 2009), might also be in agreement with the observed dwarf phenotype. However, the exact role of this gene product is difficult to predict based on sequence information alone given the large size of this gene family and should be investigated by direct experimentation as other xyloglucan-specific enzymes have been reported to stimulate cell expansion (Maris et al., 2009). Increased abundance of two DUF1070 transcripts predicted to encode short arabinogalactosylated proteins (AGP; 1.7- and 1.9-fold; 1622530_at, 1619401_at) might also contribute to dwarfing as overexpression of an Arabidopsis arabinogalactan protein gene AtAGP18 resulted in plants with smaller rosettes and shorter stems and roots (Zhang et al., 2011). Poplar, acclimated to cold for 7 days, or overexpressing AtCBF1, also displayed increased abundance of an AGP31 gene (Table 2) (Benedict et al., 2006). The elevated mRNA expression of LRX and EXGT genes was also observed in AtCBF1-overexpressing Poplar (Table 2), and that of the PMEI was conserved in AtCBF2 and AtCBF3 overexpressing Arabidopsis (Table 3) indicative of conservation of CBF gene regulons in both woody and herbaceous species. Lastly, a gibberellin-regulated gibberellic acid-stimulated Arabidopsis (GASA) GASA5-like gene (1617881_at) increased in abundance 2.27-fold (Table 1). In Arabidopsis, GASA5 gene expression is stimulated by GA signalling and subsequent GASA5 activity negatively regulates GA-related gene expression and inflorescence stem growth, making it both GA-regulated and GA-regulating (Zhang et al., 2009). Transcripts for a related GASA1 gene have also been observed to be elevated in AtCBF2 overexpressing Arabidopsis (Table 3); however, the exact role of this gene remains unclear at this time. Additional experiments are required to determine whether the expression of the Vitis GASA5-like gene is influenced by GA and its product is involved in GA-signalling.

Another class of genes showing increased relative transcript abundance in the VvCBF4 gene overexpressing line included those involved in lipid metabolism and epicuticular wax formation or modification. A homolog of the lipid transfer protein 3 (LTP3-like) gene increased in abundance 2.2-fold (1608175_at). Although the function of this gene is unclear, various functions have been assigned to lipid transfer genes including lipid exchange between membranes or as lipid sensors or chaperones (D’Angelo et al., 2008). The Vitis LTP3-like gene is most similar to the Arabidopsis LTP3 (At5g59320) gene, which encodes a PR-14 family lipid transfer protein that is located in the apoplast and cell wall and whose transcript abundance is regulated by water deficit and ABA (Huang et al., 2008).

Three GDSL-motif lipase genes increased in abundance between 1.7- and 1.9-fold (1607744_at, 1607341_at, 1620618_at). In plants, members of the GDSL-lipase family are known to be involved in various types of lipid and acyl-group metabolism, including lipase, lysophospholipase, esterase, thioesterase and arylesterase activities (Akoh et al., 2004). GDSL lipases comprise a large gene family in land plant species, including grapevine, which contains more than 90 GDSL-lipase genes (Volokita et al., 2010). Some GDSL-lipase genes are required for proper cuticle formation. For example, in rice, the GDSL-lipase gene wilted dwarf and lethal 1 (WLD1) is required for correct cuticle formation; wld1 knockouts contain abnormal wax crystals and exhibit rapid water loss as a result of aberrant cuticle (Park et al., 2010). GDSL-lipase genes might also participate in bacterial and fungal pathogen resistance through eliciting local and systemic resistance (Kwon et al., 2009). Homologues of three GDSL-motif lipase genes also exhibited increased transcript abundance in AtCBF1 and CBF-overexpressing Poplar and Arabidopsis, respectively (Tables 2 and 3).

The relative mRNA expression of genes involved in biosynthesis of the epicuticular wax also increased, including a glycerol-3-phosphate acyltransferase 4-like gene (GPAT4; 1.8-fold; 1612479_at), a LACERATA-like CYP450 (1.6-fold; 1621973_at) and a glucose-methanol-choline (GMC) oxidoreductase (1.5-fold; 1622345_at). The best matches in Arabidopsis for each of these transcripts were elevated in A. thaliana transgenic plants overexpressing the wax-inducing WIN1/SHN1 AP2 domain-containing ERF transcription factor genes (Kannangara et al., 2007). The increased expression of epicuticular wax biosynthetic genes is consistent with the observed enhancement of epicuticular wax deposition in transgenic Eucalyptus (E. urophylla × E. grandis) overexpressing an endogenous CBF1a gene (Navarro et al., 2011). Although additional experiments are needed to determine whether and how ectopic expression of VvCBF4 might have affected cuticle formation in grapevine, transgenic vines retained a normal appearance.

A set of stress-responsive genes also showed increased transcript abundance in the VvCBF4-overexpressing line including genes encoding the RESPONSIVE TO DESICCATION 22 (VvRD22) gene (1.9-fold; 1621818_at), a polyketide cyclase/dehydratase (major latex protein 28-like) (1.7-fold; 1607196_at), thaumatin-like VvTL1 (1.7-fold; 1614746_at), stress-related basic secretory protein (1.5-fold; 1615434_at) and an unknown stress-regulated transcript (2.1-fold; 1613368_at) genes, which have each been reported previously to increase in mRNA abundance under abiotic stress conditions either in grape or in Arabidopsis (Kuwabara et al., 1999; Fowler and Thomashow, 2002; Hanana et al., 2008; Lytle et al., 2009). Transcripts coding for the DNA repair enzyme DNA-3-methyladenine glycosidase I (1.9-fold; 1621976_at) and the abiotic stress-responsive methylglyoxal detoxification enzyme lactoylglutathione lyase/glyoxalase I (2.1-fold; 1619235_at) also increased (Santerre and Britt, 1994; Yadav et al., 2008). Two other stress-regulated transcripts with increased mRNA expression with likely roles in signal transduction included a calmodulin (CaM)-like gene (2.6-fold; 1617516_at), which has also been shown to increase in expression in cold-stressed Arabidopsis (Fowler and Thomashow, 2002), and a calcium-binding EF-hand-containing MSS3-like transcript (1.6-fold; 1612996_at). Lastly, the mRNA expressing an AAA-type ATPase domain (chaperone-like) gene (1610816_at) increased in abundance 4.7-fold (Table 1). A homolog of this gene is known to respond to salt stress in Arabidopsis (Fujita et al., 2005).

In contrast to the relatively large numbers of genes with elevated transcript abundance, only twelve genes showed a significant decrease in mRNA abundance in the VvCBF4 overexpressor. Most notable was a BAG-domain (BCL-2-associated athanogene) gene that showed a 37-fold decrease in relative transcript abundance (Table 1). This nuclear localized, calmodulin-binding domain containing gene is most similar to AtBAG6, which when overexpressed induces programmed cell death in transformed yeast and Arabidopsis (Kang et al., 2006). AtBAG6 is also thought to coordinate stress-induced hormone signalling and might play a role in limiting pathogen colonization (Doukhanina et al., 2006). Additional experiments will be necessary to determine the exact role of this gene in Vitis.

Lack of correlation between mRNA and protein expression

Interestingly, none of the proteins identified as being significantly overexpressed in the VvCBF4-overexpressing line 9–12 (Table S4) matched the differentially expressed transcripts described here. This result is in contrast to the correlation observed between transcript and protein abundance changes described for heat-stressed, 35S::DREB2C overexpressing Arabidopsis plants, wherein a total of 10 different proteins showed significant changes in protein abundance (six and four proteins showed increased or decreased abundance, respectively) compared with wild-type plants (Lee et al., 2009). However, no significant differences were apparent if the transgenic plants were not heat-treated. One possible explanation for these different results is that heat stress might be necessary for transcription factor activation as described for DREB2A, which was activated by dehydration, high salinity, ABA or cold treatments, presumably by post-translational modification (Liu et al., 1998). Indeed, phosphorylation of a stress-inducible DREB2A transcription factor from Pennisetum glaucum was shown to prevent DNA binding (Agarwal et al., 2007). However, such post-translational modifications are not always necessary as in the case of DREB1A-related transcription factors (Liu et al., 1998). In another example, comparison of nontransgenic and transgenic potato expressing 35S::AtDREB1A using 2D-DIGE showed increased expression of only patatin, a major storage protein and decreased expression of lipoxygenase and starch synthase (Nakamura et al., 2010). Thus, there is precedent for ectopic expression of CBF-family transgenes having a relatively small effect on the proteome.

One possible explanation for the lack of correlation between transcript and protein abundance is that VvCBF4 might require post-translational modification for activation. Additional experiments would be required to test this hypothesis. Studies in various plant species, including wine grape berries, have shown that there are only moderate-to-weak correlations (r = 0.50 or less) between mRNA and protein depending on the study (Watson et al., 2003; Gion et al., 2005; Liu et al., 2006; Faurobert et al., 2007; Grimplet et al., 2009b). Additional possible explanations for this weak correlation include the observation that mRNA abundance changes are typically presented as fold changes and not in absolute terms, so this might favour the reporting of low-abundance transcripts. Also, alternatively spliced transcripts might not be detected by microarray or qRT-PCR analytical methods; however, such events might result in changes in relative protein expression.


In conclusion, ectopic expression of the VvCBF4 resulted in improved freezing survival in nonacclimated vines to a degree comparable with that observed for CBF overexpression in Arabidopsis and other woody species within Eucalyptus, Malus, Populus and Vinifera genera. The gene expression profiling results presented here clearly show that wine grape possesses an evolutionarily conserved CBF regulon that is widely conserved among cold-adapted herbaceous species as well as other woody species. Improvement in freezing tolerance should be useful in reducing late spring frost damage to floral primordia of primary and secondary buds in Vitis. The observed dwarf phenotype might be advantageous to improve berry ripening by reducing canopy vigour and allowing more sun exposure to developing berries. However, to avoid possible undesirable effects of dwarfing, such as reductions in sink tissue (i.e., berries) production typically associated with constitutive expression of CBF transcription factors, future experiments should focus on the development of transgenic vines expressing this or other VvCBF gene family members under the control of abiotic stress-inducible promoters. VvCBF4-overexpressing lines might also be predicted to be more tolerant to salinity and water deficit stress. Additional experiments are in progress to verify these possibilities.

Experimental procedures

VvCBF4 sequence analysis

Phylogenetic analysis, based on minimum evolution, was conducted on the full-length protein sequences of CBF gene family members from eudicots for which complete genome drafts are available. The polypeptides aligned included four CBF genes from Arabidopsis: AtCBF1 (NP_567721.1), AtCBF2 (NP_567719.1), AtCBF3 (NP_567720.1), AtCBF4 (NP_200012.1); four CBFs from Glycine max: GmCBF1 (AAQ02703.1), GmCBF2 (ACB45077.1), GmCBF3 (ACA63936.1) and GmDREB7 (ABQ42206.1); six CBFs from Populus trichocarpa: PtDREB66 (XP_002313656.1), PtDREB67 (XP_002328068.1), PtDREB68 (XP_002299565.1), PtDREB69 (XP_002298067.1), PtDREB70 (XP_002318846.1) and PtDREB71 (XP_002321877.1); four CBF genes from Medicago truncatula: MtCBF1 (ABX80062.1), MtDREB1A (ABG75914.1), MtCBF2 (ABX80063.1) and MtDREB (ABB72792.1); and four CBF genes from Vitis vinifera: VvCBF1 (AAR28673.1), VvCBF2 (AAR28677.1), VvCBF3 (XP_002267961.1) and VvCBF4 (XP_002280097.1). The Arabidopsis AP2-AE ERF10 (NP_171876.1) was included as a non-CBF/DREB rooting outlier. Alignments were generated in the MacVector (ver. 12.0; MacVector, Inc., Cary, NC) software suite, using ClustalW alignment. The phylogeny tree was constructed using neighbour-joining tree building, with Poisson-corrected distances, 1000 bootstrap replicates and postbuild outlier rooting.

CBF4 overexpression construct

The full-length VvCBF4 open reading frame (ORF, GenBank accession: DQ497624) was PCR-amplified from genomic DNA using the forward primer 5′-CACCATGAATACTACTTCTCCACCATATTCC-3′ and reverse primer 5′- CTAAATAGAGTAACTCCATAATGACATGTC-3′. The PCR product was cloned into pENTR/D-TOPO and then into the pH2GW7 GATEWAY-type expression vector using LR clonase to recombine the VvCBF4 ORF between sites attr1 and attr2, downstream of the CaMV 35S constitutive promoter. Empty vector control line (e.g., ‘8–6’) vectors were constructed by the removal of the GATEWAY cassette from pH2GW7 with ApaI/SstI digestion, blunt-end formation via T4 polymerase 3′ exonuclease activity, and re-ligation with T4 ligase (see Figure S1 for details). Both the VvCBF4 expression vector and the empty vector control were verified by DNA sequencing and restriction digestion (Tattersall, 2006). Expression constructs were then introduced into Agrobacterium tumefaciens strain EHA105 by electroporation.

Vitis vinifera cv. Freedom transformation

Grape vine transformation was performed by the Ralph M. Parsons Plant Transformation Facility, University of California, Davis, CA. Briefly, immature anthers from the Vitis hybrid rootstock cv. ‘Freedom’ were used as a source of tissue for embryogenic callus. The embryogenic callus was inoculated with an overnight suspension of Agrobacterium tumefaciens containing the expression cassettes outlined earlier and adjusted to an O.D.600 of 0.075. Callus tissue was plated onto a sterile No. 1 Whatman filter paper, which was placed on top of coculture medium consisting of Woody Plant Media (WPM) (Lloyd and McCown, 1981) supplemented with 10 mg/L picloram, 2 mg/L thidiazuron (TDZ), 500 mg/L activated charcoal, 1000 mg/L casein and 200 μm acetosyringone. Agrobacterium was added dropwise to the callus until thoroughly moistened. After 15 min, the callus was blotted dry using a second piece of sterile No. 1 Whatman filter paper and incubated in the dark at 23 °C. After 48–72 h, the callus was transferred to WPM supplemented with 10 mg/L picloram, 2 mg/L thidiazuron (TDZ), 500 mg/L activated charcoal, 1000 mg/L casein, 400 mg/L carbenicillin, 250 mg/L cefotaxime and 25 mg/L hygromycin sulphate. Callus was incubated in the dark at 26 °C and transferred to fresh medium every 21–28 days. Hygromycin-resistant embryogenic callus colonies formed after 3–4 months. These colonies were harvested and transferred to germination medium consisting of WPM medium supplemented with 20 g/L sucrose, 500 mg/L activated charcoal, 1000 mg/L casein, 150 mg/L timentin, 0.5 mg/L benzylaminopurine (BAP) and 0.1 mg/L naphthalene acetic acid (NAA). Elongating embryos were transferred to rooting media consisting of one-half strength Murashige and Skoog minimal organics medium (Murashige and Skoog, 1962) supplemented with 15 g/L sucrose, 0.01 mg/L NAA, 150 mg/L timentin and 25 mg/L hygromycin. Rooted plantlets were acclimated to soil in a growth chamber under 16 h photoperiod at 26 °C and then transferred to greenhouse for further development.

Vine propagation

Green cuttings (2–3 internodes in length) from well-established woody vines were sterilized with 50% (v/v) household bleach and 0.02% (v/v) Triton X-100 for 30 s and placed in autoclaved, sterile 77 × 77 × 97 mm (W × L × H) Magenta GA-7 boxes (Magenta Corp., Chicago, IL) containing 80 mL of 0.8% (w/v) plant tissue culture agar (#A111; Phytotechnology Laboratories, Shawnee Mission, KS) with Murashige and Skoog modified basal medium w/Gamborg vitamins (#M404; Phytotechnology Laboratories), 1.5% sucrose, pH 5.6–5.7 and 300 μm indoleacetic acid (IAA) to promote rooting (Murashige and Skoog, 1962; Gamborg et al., 1968). Vines were allowed to develop roots in a Percival Scientific growth chamber (Model # CU-32L; Perry, IA) under 18 h of fluorescent light (200 μmol/m2 s−1 PPFD) at 25 °C and 6 h darkness at 20 °C. Upon development of root primordia (∼14 days), individual vines were transferred to a second Magenta box containing media prepared as above, except without IAA, to promote normal plant growth for about 10 days. Upon establishment of normal growth, cuttings from these vines were further propagated in Magenta boxes (one cutting/box) for subsequent use in stress phenotyping assays, electrolyte leakage assays, mRNA and protein expression assays and growth phenotyping. These cuttings were handled identically as the ‘mother vines’ (omitting the bleaching step, as they were already sterile) and were allowed ∼24 days to develop roots in Magenta boxes, followed by transplantation to 4′ square plastic pots (Part # TSD4; McConkey Co., Garden Grove, CA) containing Metromix® 200 soil (Sun Gro Horticulture, Bellevue, WA). Vines were allowed an additional 2 weeks to adapt to soil and low humidity before abiotic stress assays were performed. Acclimation was accomplished by placing the newly potted vines in a tray covered with a clear plastic dome to maintain high humidity. After 5 days, a dome having a 50% vented surface area replaced the full dome. After an additional 5 days, the vented dome was removed completely. Twice during the 2-week time period, vines were given ¼ strength modified Hoagland’s solution with full-strength iron and micronutrients (600 μm KNO3, 400 μm Ca(NO3)2·H2O, 200 μm NH4H2PO4, 100 μm MgSO4·7H2O, 280 μm Fe-EDTA, 50 μm KCl, 25 μm H3BO3, 2 μm MnSO4·H2O, 2 μm ZnSO4·7H2O, 0.5 μm CuSO4·5H2O, and 0.5 μm H2MoO4) (Hoagland and Arnon, 1950).

Freezing stress assays

For freezing stress assays, groups of vines (= 10) from the empty vector control line (8–6) and three 35S::VvCBF4 overexpressor lines (9–1, 9–3 and 9–12) were placed in a prechilled Percival Scientific growth chamber (model # AR75L) for 24 h at −2 °C. The built-in humidifier was turned off and water drained from the reservoir to avoid ice build-up and damage to the growth chamber. Twenty kilograms of frozen ice packs and a small fan were added to the growth chamber to maintain maximum temperature equilibrium throughout the chamber during stress application. Plant position within the growth chamber was randomized. Assays began and ended at 10 am, with 18 h light (200 μmol/m2/s light) beginning at 6 am and 6 h dark beginning at 10 pm. Vines were watered 2 days poststress and allowed to recover under normal growth conditions (25 °C day/20 °C night temperature) under the same light conditions as above for 2 weeks, at which point survival was scored by the presence of new growth. Freezing stress assays were performed on multiple groups of vines, as described, and Student’s t-test corrected for multiple comparisons was used to identify statistically significant genotypic differences in freezing tolerance (adjusted P-value <0.05).

Electrolyte leakage assay

The freezing tolerance of the three transgenic V. vinifera cv. ‘Freedom’ lines overexpressing VvCBF4 and one control line was also assayed by leaf electrolyte leakage. Transgenic V. vinifera lines 9–1, 9–3, 9–12, and the empty vector control line, 8–6, were grown in pots for 6 weeks (150–200 μmol/m2/s PPFD, 16 h light at 25 °C/8 h dark at 20 °C) without any cold acclimation. Identical-sized leaf discs (6 mm diameter) were punched from individual fully expanded leaves and were equilibrated in 200 μL of Nanopure water (Millipore, Inc., Bedford, MA) in 1.5-mL microfuge tubes for 1 h. For freezing treatment, we manually adjusted a Neslab RTE-4 (Thermo Fisher Scientific, Inc., Waltham, MA) refrigerated circulating antifreeze bath to achieve 1 °C/30 min decrement from 0 to −16 °C. Tubes were floated in the refrigerated water bath and exposed to the freezing conditions. At each 1 °C/30-min interval, tubes were removed and gently thawed at 4 °C for 18 h. Leaf discs and incubation solutions were transferred to another tube containing 4 mL of Nanopure water. After shaking overnight, conductivity was measured using an Orion 4Star Portable Conductivity Meter with the Orion 013010MD Conductivity Cell (Thermo Fisher Scientific, Inc.). Samples were autoclaved for 20 min at 121 °C, and the conductivity was re-measured. The level of electrolyte leakage induced by freezing was determined as the percentage of conductivity before autoclaving versus conductivity because of total leakage after autoclaving. Student’s t-test corrected for multiple comparisons was used to identify significant genotypic differences in electrolyte leakage (adjusted P-value <0.05).

Growth phenotyping

The shoot elongation rate of 2-week-old soil-rooted plantlets of VvCBF4 overexpressor lines and empty vector control lines (n = 8) was measured to identify possible alterations (reductions) in growth. The shoots were marked with acrylic nail polish just below the first above-soil internode, and all internodal lengths were measured using dial callipers (Mitutoyo America Corp., model no. 505-675-66, Aurora, IL) every third day. Total shoot length and SER were determined over 21 days. Student’s t-test corrected for multiple comparisons was used to identify significant genotypic differences in growth rates and internode length (adjusted P-value <0.05).

RNA extraction

Entire aerial portions (stems and young leaves) of grapevines that had been growing in soil for 2 weeks were collected in 50-mL conical tubes and immediately frozen in liquid nitrogen. Total RNA was isolated using a three-step process. First, total RNA was extracted and applied to an RNeasy Midi column (Qiagen, Inc., Valencia, CA) with 2% polyethylene glycol (PEG, mw = 20 000; Sigma Aldrich, Inc., St. Louis, MO) added to the RLT buffer, followed by on-column DNase digestion. The resultant RNA was then subjected to phenol:chloroform:isoamyl–alcohol (25:24:1) extraction (Sambrook and Russell, 2001). Lastly, the RNA extract was further purified using an RNeasy mini column (Qiagen, Inc.) with 2% PEG (Tattersall et al., 2005). Total RNA was quantified and 260/280 ratios determined using a NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.). RNA integrity was confirmed by electrophoresis on formaldehyde-containing 1.5% agarose gels.

Quantitative real-time (reverse transcription) PCR (qRT-PCR)

cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Inc., Foster City, CA) according to manufacturer’s instructions, using uniform 2 μg RNA per reaction volume reverse transcription reactions. Gene-specific primers for qRT-PCRs were selected using the Primer-BLAST tool at NCBI (http://www.ncbi.nlm.nih.gov/tools/primer-blast) using RefSeq V. vinifera transcripts as input, screened against all other V. vinifera RefSeq sequences, and the following Primer3 (Rozen and Skaletsky, 2000) settings: Tm range 58–60 °C, product size = 50–150 bp, primer size = 13–25 nt, max poly-X = 3, and G/C content = 30%–80%. Primer pair selection against a GC clamp, such that no more than two of the last five 3′ nucleotides were G or C, was conducted per qRT-PCR instrument recommendations. qRT-PCRs were prepared using Fast SYBR® Green Master Mix and performed using the ABI PRISM® 7500 Sequence Detection System (Applied Biosystems, Inc.) using four biological replicate samples and normalized to an endogenous actin 7 control gene (NCBI locus ID, LOC 100232968) (Reid et al., 2006). Gene-specific primer pairs and products used are summarized in Table S1. Relative quantitation of qRT-PCR outputs was performed using the Pfaffl method, an elaborated ΔΔCt method that employs observed primer efficiencies (rather than assuming 2−ΔΔCt) when comparing different primer pairs/products (Pfaffl, 2001).

Microarray-based mRNA expression profiling

For microarray experiments, RNA was re-quantified using RiboGreen® fluorescent nucleic acid stain (Invitrogen Life Technologies, Inc., Carlsbad, CA) and read using a Labsystems Fluoroskan Ascent fluorescence plate reader (Thermo Fisher Scientific, Inc.). Sample RNA integrity was determined with an Agilent 2100 Bioanalyzer microfluidics station (Agilent Technologies, Inc., Santa Clara, CA). Total RNA (300 ng) was loaded onto a capillary electrophoretic column to determine major (rRNA) band sizes and quality (see Figure S2). Complementary DNA (cDNA) was generated from mRNA using a GeneChip® T7-Oligo(dT) Promoter Primer Kit containing the T7 polymerase promoter sequence and oligo(dT) priming region (5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3′) (Affymetrix®, Santa Clara, CA) and SuperScript™ II Reverse Transcriptase (Invitrogen Life Technologies, Inc.) according to the manufacturer’s instructions (Affymetrix, 2009). Biotinylated complementary RNAs (cRNAs) were synthesized in vitro from four biological replicates using T7 RNA polymerase in the presence of biotin-labelled UTP/CTP, then purified, fragmented and hybridized to GeneChip® 16K Vitis vinifera (Grape) Genome Arrays ver. 1.0 (Affymetrix®). The hybridized arrays were washed and stained with streptavidin–phycoerythrin and biotinylated antistreptavidin antibody using an Affymetrix® 3000 7G Scanner, Hybridization, and Fluidics System. Scanner and image data were collected and processed on a GeneChip® Workstation using Affymetrix® GCOS software.

The images of all arrays were examined, and no obvious scratches or spatial variations were observed. The ‘present’ call rates were also consistent across the eight arrays, ranging from 66% to 70% (mean rate = 69%). Raw hybridization intensity values were processed by robust multiarray average (RMA) (Irizarry et al., 2003), using the R package affy (Gautier et al., 2004). Specifically, expression values were extracted from raw CEL files by first applying the RMA model of probe-specific correction to PM (perfect match) probes. Corrected probe values were then normalized via quantile normalization, and a median value was computed for the PM probeset. Resulting RMA expression values were log2-transformed. Distributions of the RMA-expression values of all arrays (four biological replicates) were visualized by two-dimensional PCA.

A t-test was performed for each RMA-PM probeset to determine which genes were differentially expressed between the transgenic line (9–12) and empty vector control-transformed (8–6) genotypes. Multiple testing correction was performed on the t statistic of each probeset to minimize false discovery rate (Benjamini and Hochberg, 1995). Genes that were differentially expressed (±1.5-fold) at adjusted P-value <0.05 were identified and assigned functional annotation derived from VitisNet (Grimplet et al., 2009a) and the PlexDB microarray annotations http://www.plexdb.org/modules/PD_probeset/annotation.php?genechip=Grape (Wise et al., 2007) and presented in Table 1. The complete list of significantly differentially expressed genes is presented in Table S2. In this table, the light red shading indicates increased abundance, light green shading indicates decreased abundance, and genes not significant by false discovery were shaded in light gray. Microarray data were deposited in the NCBI GEO database under series GSE29948 viewable at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE29948.

Genes identified by microarray analysis that showed significantly increased or decreased transcript abundance in the 35S::VvCBF4 overexpressor were compared by sequence homology against other CBF/DREB1f regulon genes from previous studies as listed in Tables 2 and 3. The translated protein sequences of the genes or transcripts associated with each increased/decreased probeset were compared using basic local alignment search tool (BLAST), with the NCBI BLAST+ software (Camacho et al., 2009) using the command line BLASTP algorithm, word size 3, open penalty 11, extend penalty 1, window size 40 and maximum e-value cut-off 1 × 10−15.

Protein extraction

Whole aerial portions of young vines grown in soil for 3 weeks (control line 8–6 plant height of ∼16 cm; 35S::VvCBF4, line 9–12 plant height of ∼10 cm) were collected and frozen immediately in liquid nitrogen and stored at −80 °C until further use. Four biological replicates from each treatment were individually ground in liquid nitrogen with a mortar and pestle, followed by extraction of total proteins with a phenol-based protocol optimized for grape (Vincent et al., 2006), based upon protocols previously developed for recalcitrant plant tissues (Hurkman and Tanaka, 1987; Saravanan and Rose, 2004). From each of the eight samples, 5 g of frozen, ground tissue was added to 10 mL of protein extraction buffer (0.5 m Tris–HCl pH 7.5, 0.7 m sucrose, 50 mm EDTA, 0.1 mm KCl, 2 mm PMSF and 2% (v/v) β-mercaptoethanol in water) containing 1 Complete™ Protease Inhibitor Cocktail Tablet per 10 mL of buffer (Roche Applied Science, Inc., Indianapolis, IN) in a 50-mL BD Falcon™ tube and vortexed for 30 s followed by a 10-min incubation at 4 °C. Next, 10 mL of Tris-saturated phenol (pH 7.9) was added to the mixture vortexed for 30 s, followed by a 30-min incubation at 4 °C with inversion of the samples every 10 min. Samples were then centrifuged at 3650 g and 4 °C for 30 min in a Beckman Allegra™ 6R centrifuge (Beckman Coulter Inc., Brea, CA). The upper phenol phase was then removed to a new 50-mL BD Falcon™ tube and an equal volume of fresh protein extraction buffer added. This was followed by vortexing for 30 s and incubation for 30 min at 4 °C with inversion of the samples every 10 min. Samples were again centrifuged at 3650 g and 4 °C for 30 min, and the phenol phase for each sample was again removed to a new 50-mL BD Falcon™ tube. To precipitate proteins, five volumes of cold (−20 °C) methanolic ammonium acetate (0.1 m ammonium acetate in methanol) were added to each Falcon tube, followed by placement of samples for 3 h at −20 °C with inversion every 10 min. Next, the 50-mL BD Falcon™ tubes were spun at 3650 g at 4 °C for 30 min using the Beckman Allegra™ centrifuge to pellet the proteins. Following centrifugation, the supernatants were discarded and 5 mL cold (−20 °C) methanol was added to each tube to wash the pellet. The samples were vortexed for 30 s and placed for 1 h at −20 °C with inversion every 10 min, followed by centrifugation at 3650 g at 4 °C for 30 min. The supernatant was discarded, and three additional wash, vortex and centrifugation steps were performed in the same manner as the methanol wash, all with ice-cold (−20 °C) acetone. Following the final acetone wash, wet pellets were transferred to 2-mL Eppendorf tubes and placed on ice until 2D-DIGE analysis.

2D-DIGE analysis

The acetone-wetted protein pellets were centrifuged at 13 000 g at 0 °C for 10 min and washed an additional two times with acetone/water (4:1) before being chilled to −20 °C. The final pellets were allowed to dry in open tubes for 10 min, including 1 min of drying at 3000 g in a Speed Vac System (Thermo Fisher Scientific Inc.). Individual dried pellets were resuspended in 200 μL DIGE Reaction Buffer (7 m urea, 2 m thiourea, 4% CHAPS, 30 mm Tris, pH 8.74). The tubes were vortexed frequently a minimum of 10 times and then sonicated for a total of 10 min using 30 s pulses in a water bath sonicator (model no. FS30; Fisher Scientific, Pittsburgh, PA) over a period of 2 h. Samples were then centrifuged at 13 000 g at 22 °C for 10 min. The supernatant from each sample was removed to a clean 1.7-mL microfuge tube and assayed for protein concentration using an EZQ™ Protein Quantification Kit with ovalbumin as the standard (Bio-Rad Laboratories, Inc., Hercules, CA). Samples were stored overnight at −20 °C.

Following thawing on ice, additional aliquots of DIGE reaction buffer were added to each sample to bring the final protein concentration to 1.33 mg/mL. Sixty microlitres of each sample (80 μg protein) was then pipetted into a 0.5-mL microfuge tube. Each of three Cy-dyes (e.g., Cy2, Cy3, Cy5; GE Healthcare, Inc., Piscataway, NJ), which were solutions of 5 nmol dye in 5 μL dimethylformamide (DMF), were diluted 5 × (1 μL Cy dye in 4 μL DMF) prior to use. Samples were then subjected to a random dye-swap scheme for normalization of differences in Cy-dye fluorescence intensity (Table S3). A 1.15-μL aliquot of Cy3 and Cy5 was added to each sample. For an internal control, 30 μL of resuspended protein from each sample was mixed in a single tube and 4.6 μL Cy2 was added. At this point, each of the eight sample tubes contained approximately 80 μL total protein and 230 pmoles total Cy dyes (Cy3 and Cy5), whereas the pooled sample control tube contained 331 μg protein and 920 pmol Cy2. The tubes were quickly vortexed and then placed on ice, centrifuged briefly, then placed back on ice. The ice bucket was then covered with aluminium foil and stored in a dark cabinet for 30 min for Cy dye/protein binding. At the end of the 30 min incubation, 2 μL of 10 mm aqueous lysine was added to each tube. The tubes were vortexed and then put back on ice in the dark for an additional 10 min. Four 1.7-mL microfuge tubes were numbered gel 1–4, corresponding to the gel numbers given in Table S3 The contents of the incubated protein/Cy dye mixes were then transferred to the labelled ‘gel’ tubes, completed by washing each 0.5-mL tube with 50 μL 2 × DIGE Reaction buffer (7 m urea, 2 m thiourea, 4% CHAPS). To each of the ‘gel’ tubes, the following were added: 60 μL pooled sample, 137 μL 2 × DIGE reaction buffer, 4 μL 0.1% bromophenol blue, 2.35 μL carrier ampholytes pI = 3–10 (Bio-Rad Laboratories, Inc.), and 47.0 μL of 10.5 m DTT solution. The mixtures were then centrifuged at 13 000 g and 22 °C for 10 min. From each of the four gel tube mixtures, 450 μL of supernatant was applied to a 24-cm 4–7 immobilized pH gradient (IPG) strip (GE Healthcare, Inc.). The strip was rehydrated passively overnight (for 22 h) at 20 °C. Isoelectric focusing (IEF) was performed as follows: active rehydration at 50 V for 4 h, a linear increase to 200 V in 1 h, a linear increase to 500 V in 1 h, a linear increase to 1000 V in 1 h, a linear increase to 10 000 V in 2 1/2 h, and maintenance of steady voltage from 10 000 V to 70 000 Vh (Volt-hours) were conducted (∼7½ h). IPG strips were removed and stored at −80 °C until the second dimension was run.

IEF strips were thawed at room temperature (10 min) followed by equilibration in equilibration base buffer (6 m urea, 30% v/v glycerol, 2 m Tris–HCl pH 8.8, and 2% w/v SDS) containing 1% w/v DTT for 20 min to reduce proteins, and then 2.5% w/v iodoacetamide for 20 min to alkylate proteins. The strips were then loaded onto 12.0% (v/v) 26 × 20 × 0.1 cm polyacrylamide gels (Jule Inc., Milford, CT). The gels had been treated with Bind-Silane (γ-methacryloxypropyltrimethoxysilane) and had reference markers attached on the short plate. Electrophoresis was performed using a Protean® Plus Dodeca Cell (Bio-Rad Laboratories, Inc.) with standard Tris–glycine–SDS buffer (25 mm Tris-base, 0.5 m glycine, 0.1% v/v SDS) under the following conditions: 40 V for 2 h followed by 100 V for 24 h at 10 °C.

Prior to imaging, the larger glass plate from each of the gel cassettes was removed. A Typhoon Trio™ Imaging System (part no. 63-0055-87; GE Healthcare, Inc.) was used for image acquisition using the blue (488 nm) laser. Immediately following imaging, each gel was placed in destain solution (7% v/v acetic acid, 10% v/v methanol) and shaken gently for 72 h. Images captured with the Typhoon were analysed using DeCyder™ 2D Software ver. 7.0 (GE Healthcare, Inc.) for protein quantification. Sample labelling and CyDye swapping schema are summarized in Table S3.

Protein identification

For spot picking and protein identification, two gels (replicates #1 and #4) were selected for Sypro® Ruby staining. The destain solution was removed from the gels, and ∼110 mL of Sypro® Ruby Staining solution (Invitrogen, Inc., Carlsbad, CA) in ∼960 mL of fresh destain solution was added. Gels were gently shaken for 48 h followed by washing once with fresh destain solution and once with water. Spot excision was performed using the EXQuest™ spot cutter (Bio-Rad Laboratories, Inc.) followed by trypsin digestion according to the protocol developed by Rosenfeld et al. (Rosenfeld et al., 1992) using the Investigator™ ProPrep™ protein digestion kit (Genomic Solutions, Ann Arbor, MI). The trypsin-digested fragments were analysed using an ABI 4700 Proteomics Analyzer™ (Applied Biosystems) matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) mass spectrometer (MS). A 0.5-mL aliquot of matrix solution with 5 mg/mL alpha-cyano-4-hydroxycinnamic acid (CHCA) (Sigma-Aldrich, Inc.) and 10 mm ammonium phosphate (Sigma-Aldrich, Inc.) in 0.2% formic acid was cospotted with 0.5 mL of sample (Zhu and Papayannopoulos, 2003). The data were acquired in reflector mode from a mass range of 700–4000 Da, and 1200 laser shots were averaged for each mass spectrum. Each sample was internally calibrated if both the 842.51 and 2211.10 Da ions from trypsin autolysis were present. The eight most intense ions from the MS analysis not present on the exclusion list were subjected to MS/MS analysis. To this end, the mass range was from 70 to precursor ion size with a precursor window of 1–3 Da using an average of 2500 laser shots for each spectrum. The resulting mass data were then used to search the NCBI nr database (ver. 10_09_2009; 9 694 989 sequences) and the contigs from Vitis Gene Index (ver. 18_9_2009, 23 493 sequences) using automated MASCOT V.2.1 software (http://www.matrixscience.com). Peptide tolerance was 20 ppm, one missed cleavage was allowed and MS/MS tolerance was 0.8 Da. The possibility of matching multiple translated isoforms was examined by manual analysis of peptides present within the sequences. All MS analyses were performed in cooperation with the Nevada Proteomic Center at the University of Nevada, Reno. The complete list of differentially expressed proteins is presented in Table S4.


This work was supported by funding from the National Science Foundation NSF (DBI-0217653) and the University of Nevada Agricultural Experiment Station (to GRC and JCC). The authors thank David Tricoli and Kim Carney of the Ralph M. Parsons Plant Transformation Facility, Davis, CA for performing the Vitis transformations. The authors thank Craig Osborn of the Nevada Genomic Center for performing microarray services, Rebecca Woolsey, Kathy Schegg and David Quilici of the Nevada Proteomics Center for support and for performing MS analyses, and Rebecca Albion and Kitty Spreeman for invaluable technical support. We thank Mary Ann Cushman for her critical reading of the manuscript. This publication was also made possible by NIH Grant Number P20 RR-016464 from the INBRE Program of the National Center for Research Resources through its support of the Nevada Genomics, Proteomics and Bioinformatics Centers.