Figure 1.
Location and structure of the VaICE1,2 genes.
(A) Gene structure and locus. Chromosomal localization of VaICE1,2 genes were predicted in V. vinifera cv. Pinot Noir clone P40024 genome. VaICE1 comprises four exons spanning 2.7 kb with a predicted 1,551 bp ORF, mapping to Chromosome 1 and position 21694886–21700313. VaICE2 contains four exons spanning 4.2 kb and has a predicted 1,617 bp ORF, which is mapped to Chromosome 14 and position 24967920–24976387. Nucleotide numbers are indicated above the gene structure. (B) Schematic protein structures of VaICE1 and VaICE2 with the N-terminal domain containing a S-rich motif and the C-terminal domain represented by helix-loop-helix (HLH), ICE-specific domain, Zipper region (ZIP), and ACT_UUR_ACR-like (ACT) domain. The putative nuclear localization signals (NLS) are indicated as green boxes. Codon numbers are indicated above the protein structures. (C) The predicted tertiary structures of the bHLH-ZIP domains of the (a) VaICE1 and (b) VaICE2 proteins evaluated by the SWISS-MODEL sever.
Figure 2.
Phylogenetic tree, alignment and expression profiles of VaICE1,2.
(A) Phylogenetic tree based on the deduced amino acid sequences of ICE from a range of plant species. A neighbor-joining tree, with the following predicted ICE protein sequences, with GenBank accession numbers listed in parentheses: V. amurensis (VaICE1, AGP04217; VaICE2, AGP04218; VaICE14, ADY17816), V. vinifera (VvICE1, AFI49627; VvICE1a, AGQ03810; VvICE1b, AGQ03811), V. riparia (VrICE1, AGG34704; VrICE2, AIA58705; VrICE3, AIA58706; VrICE4, AIA58707), Arabidopsis thaliana (AtICE1, NP_189309; AtICE2, NP_172746), Brassica napus (BnICE1, AEL33687), Brassica rapa subsp. Chinensis (BrICE1, ACB70963), Capsella bursa-pastoris (CbICE1, AAS79350), Camellia sinensis (CsICE, ACT90640), Eucalyptus camaldulensis (EcICE1,ADY68776), Eucalyptus globules (EgICE1, AEF33833), Eutrema salsugineum (EsICE, ACT68317), Glycine max (GmICE1, ACJ39211), Hordeum vulgare (HvICE2, ABA25896), Malus x domestica (MdbHLH1, ABS50251), Oryza sativa (OsICE1, Os11g0523700; OsICE2, Os01g0928000), Populus suaveolens (PsICE1, ABF48720), Populus trichocarpa (PtrICE1, ABN58427), Raphanus sativus (RsICE1, ADY68771), Triticum aestivum (TaICE41, ACB69501; TaICE87, ACB69502), Zea mays (ZmICE2, ACG46593), was produced by ClustalX 2.0 alignment followed by tree construction using MEGA 5.0 with 100 bootstrap tests. The branch support values are indicated. The length of the scale bar corresponds to 5 substitutions per site. (B) Comparison of ICE amino acid sequences from V. amurensis (VaICE1,2) and Arabidopsis thaliana (AtICE1,2). Deduced amino acid sequences were aligned using ClustalW. Sequences and accession numbers are shown for the following: Vitis amurensis (VaICE1, KC815984; VaICE2, KC815985) and A. thaliana (AtICE1, AAP14668; AtICE2, AAO63441). Residues in black and gray regions indicate identical and similar residues, respectively, between isoforms. Four predicted domains are labeled: a S-rich motif, a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) region, ICE-specific domain [38] and a ACT-UUR-ACR-like domain. The red triangles indicate the position of the mutation isolated by Chinnusamy et al. [32] and Kanaoka et al. [54], and the residue targeted for sumoylation by SIZ1[53]. Green triangle indicates E-box/N-box specificity site [69], blue asterisks indicate core residues for DNA binding sites [70], and red diamonds indicate dimerization interface/polypeptide binding sites [70]. (C) Expression profiles of VaICE1 and VaICE2 during a cold stress time-course experiment. Semi-quantitative RT-PCR was used to determine VaICE1 and VaICE2 transcript levels in cold treated grapevine leaves at indicted times. Grapevine GAPDH was used as a loading control.
Figure 3.
Nuclear localization and transcriptional activation assay of VaICE1,2.
(A) Confocal imaging of the VaICE1 and VaICE2 transiently expressed in onion epidermal cells as GFP fusion proteins, driven by the 35S promoter. GFP alone was used as a control. Left hand panels show dark field images of green fluorescence, middle panels show the morphology of the cells in bright field and the right hand panels show the merged images. (B) Fusion proteins of pGBKT7-VaICE1, pGBKT7-VaICE2, pCL1 (positive control) and pGBKT7 (negative control) were expressed in the yeast strain AH109. Transformants were incubated on SD/Trp- and SD/Trp-/His-/Ade- to assess their growth and tested for β-galactosidase activity.
Figure 4.
Freezing tolerance evaluation of 35S::VaICE1,2 transgenic Arabidopsis plants.
(A) A schematic map of the T-DNA region of 35S::VaICE1,2 fusion constructs employed for Arabidopsis transformation. RB, right border; LB, left borders; CaMV35S, Cauliflower mosaic virus 35S promoter; OCS, octopine synthetase terminator; NOS, nopaline synthase promoter; NPTII, Kanamycin resistance gene; NOS-T, nopaline synthase terminator. (B) RT-PCR was used to assess the transcript abundance of VaICE1 or VaICE2 in the transgenic Arabidopsis plants. Atactin2 was used as a reference control (C) 3-week-old plants were treated at −6°C for 8 h and then transferred back to normal conditions for recovery. Photographs were taken after 7 d of recovery. (D) Survival rates of plants exposed to −6°C. Average survival rates and standard errors were calculated using the results of three separate experiments with 50 seedlings per line for each freezing stress.
Figure 5.
Effect of VaICE1, 2 expression in Arabidopsis on levels of (A) electrolyte leakage, (B) proline and (C) malondialdehyde (MDA).
Three-week-old Arabidopsis plants of vector-carrying control, VaICE1 L2, and VaICE2 L6 were grown at 0°C for the time indicated. Leaves were collected to assess electrolyte leakage and free proline and MDA. Each value is the mean ± SD of three replicates in case of electrolyte leakage, proline and MDA content (6 seedlings each) for the indicated time points.
Figure 6.
Effect of VaICE1,2 overexpression in Arabidopsis on transcript levels of genes involved in the cold stress pathway.
qRT-PCR analysis using leaves from control Arabidopsis L1, 35S::VaICE1 L2 and 35S::VaICE2 L6 plants. Three-week-old plants were grown at 0°C for the time indicated. The tested genes were AtICE1, AtICE2, AtCBF1, AtCBF2, AtCBF3, AtCOR15A, AtCOR47, ATKIN1 and AtRD29A. Atactin2 was used as a reference control. Each value is the mean of three replicates.