Figure 3. Generation and characterization of constitutively nuclear-localized variants of VIP1.

(A) Schematic representations of the VIP1 variants. Numbers indicate amino acid positions of full-length VIP1. Lowercase letters (a–f) correspond to those in the panels B–D. NLS: nuclear localization signal; NES: nuclear export signal. (B) Subcellular localization of the VIP1 variants in onion cells. The indicated forms of VIP1 (a–f) were expressed as GFP-fused proteins in onion epidermal cells. For each construct, more than 10 cells were observed, and a representative image is shown. Scale bars  = 100 µm. (C) Subcellular localization of the VIP1 variants in Arabidopsis. Roots of the transgenic Arabidopsis plants expressing GFP-fused VIP1 variants (forms a, d, e and f) were observed without being treated by a hypotonic solution. For each genotype, more than 10 plants were used for observation, and a representative image is shown. Scale bars  = 100 µm. (D) Expression of the VIP1 variants retards the cotyledon growth under a mannitol-stressed condition. The transgenic plants expressing the GFP-fused VIP1 variants (35S-VIP1ΔN80-GFP, for example) were grown in the presence of 0 (Control) or 200 mM mannitol for 10 days, and their cotyledon lengths were measured. vip1, which lacks the 3′ region of the VIP1 transcript because of a T-DNA insertion in the genomic region of VIP1 [13], is shown as control. Values are means ± SD. (n = 15–20). *: P<0.05; **: P<0.01 vs. the wild type in Student's t-test. (E) Quantitative RT-PCR analyses of expression of the genes with promoters containing AGCTGT/G in the transgenic plants expressing the VIP1 variants. The transgenic plants were grown in the presence of 0 (Control) or 200 mM mannitol (+ Mannitol) for 18 days, and sampled for RNA extraction and cDNA synthesis. Relative expression levels were calculated by the comparative cycle threshold (C_T) method using UBQ5 as an internal control gene. Data are means of three biological replicates. Error bars indicate SD.