Overexpression of the tomato nuclear-cytoplasmic shuttling bZIP transcription factor VSF-1 in Arabidopsis retards plant development under mannitol-stressed conditions

VASCULAR SPECIFICITY FACTOR 1 (VSF-1) is a basic leucine zipper transcription factor identified in tomato (Solanum lycopersicum L.). VSF-1 regulates vascular-specific gene expression and is homologous to an Arabidopsis thaliana mechanical stress regulator, VIP1, but physiological roles for VSF-1 remain unclear. Here, we demonstrate that VSF-1 shuttles between the nucleus and the cytoplasm in response to hypo-osmotic stress. In Arabidopsis plants overexpressing the VSF-1-GFP fusion protein, VSF-1-GFP was mainly detected in the cytoplasm under unstressed conditions but in the nucleus under hypo-osmotically stressed conditions. VSF-1 contains three serine residues within HXRXXS motifs, which can serve as its phosphorylation and 14-3-3 protein-binding sites. In a transient gene expression system in Nicotiana benthamiana leaves, GFP-fused VSF-1 variants where those serine residues were replaced with alanine exhibited nuclear accumulation even under unstressed conditions. GFP-fused VSF-1 variants lacking those HXRXXS motifs also exhibited such nuclear accumulation. The VSF-1 variants lacking those HXRXXS motifs failed to interact with 14-3-3 proteins in a yeast two-hybrid system. These findings suggest that the nuclear accumulation of VSF-1 is triggered by hypo-osmotic stress through its dissociation from 14-3-3 proteins, similar to that of VIP1. The Arabidopsis VSF-1-GFP-overexpressing lines exhibited retarded germination and growth in the presence of mannitol, which can induce hyper-osmotic stress and repress nuclear accumulation of VSF-1. These results are consistent with phenotypes from VIP1-GFPoverexpressing lines in a previous study, indicating a conserved role for VIP1 and VSF-1 in regulating osmotic stress responses.