Expression Analyses of Soybean VOZ Transcription Factors and the Role of GmVOZ1G in Drought and Salt Stress Tolerance

Abstract

Vascular plant one-zinc-finger (VOZ) transcription factor, a plant specific one-zinc-finger-type transcriptional activator, is involved in regulating numerous biological processes such as floral induction and development, defense against pathogens, and response to multiple types of abiotic stress. Six VOZ transcription factor-encoding genes (GmVOZs) have been reported to exist in the soybean (Glycine max) genome. In spite of this, little information is currently available regarding GmVOZs. In this study, GmVOZs were cloned and characterized. GmVOZ genes encode proteins possessing transcriptional activation activity in yeast cells. GmVOZ1E, GmVOZ2B, and GmVOZ2D gene products were widely dispersed in the cytosol, while GmVOZ1G was primarily located in the nucleus. GmVOZs displayed a differential expression profile under dehydration, salt, and salicylic acid (SA) stress conditions. Among them, GmVOZ1G showed a significantly induced expression in response to all stress treatments. Overexpression of GmVOZ1G in soybean hairy roots resulted in a greater tolerance to drought and salt stress. In contrast, RNA interference (RNAi) soybean hairy roots suppressing GmVOZ1G were more sensitive to both of these stresses. Under drought treatment, soybean composite plants with an overexpression of hairy roots had higher relative water content (RWC). In response to drought and salt stress, lower malondialdehyde (MDA) accumulation and higher peroxidase (POD) and superoxide dismutase (SOD) activities were observed in soybean composite seedlings with an overexpression of hairy roots. The opposite results for each physiological parameter were obtained in RNAi lines. In conclusion, GmVOZ1G positively regulates drought and salt stress tolerance in soybean hairy roots. Our results will be valuable for the functional characterization of soybean VOZ transcription factors under abiotic stress.

Keywords: VOZ transcription factor; drought and salt tolerance; expression characterization; soybean; stress response.

Figure 1

Phylogenetic analyses of vascular plant one-zinc-finger (VOZ) proteins from soybean, Arabidopsis, rice, maize, foxtail millet, and P. patens. Each cluster (I, II, III) is highlighted in a different color.

Figure 2

Analysis of exon/intron structures and cis–acting elements. (a). Exon/intron structures of soybean VOZ genes were created with GSDS. Lengths of introns and exons are shown proportionally. (b). Putative cis-acting elements in a 2.0 kb 5’ flanking region upstream from the start codon of VOZ genes in soybean.

Figure 3

Subcellular localization of soybean VOZ proteins. The recombinant plasmids of GmVOZs–GFP were transformed into Arabidopsis protoplasts. Results were visualized with confocal microscopy 18 h after transformation. Scale bars = 10 μm.

Figure 4

Transcriptional activation activity of soybean VOZ proteins in yeast cells. pGBKT7 and pGBKT7-AtVOZ1 were used as negative and positive controls, respectively.

Figure 5

Expression analysis of soybean VOZ genes at different developmental stages in specific organs. Data was extracted from the SoyBase database [28].

Figure 6

Expression profiles of soybean VOZ genes under dehydration, salt, and salicylic acid (SA) treatments. Quantitative real-time PCR (qRT-PCR) analysis of VOZ genes in soybean seedlings in response to dehydration (a), NaCl (b), and SA (c). The relative transcription levels of GmVOZ genes were normalized to the expression of tubulin. The data are presented as means ± SD of four biological replicates. The asterisks indicate statistical differences in comparison with the corresponding controls at p < 0.01 (**) and 0.01 < P < 0.05 (*), respectively.

Figure 7

Assessment of the effect of drought and salt stress on GmVOZ1G transgenic soybean hairy roots. (a). GUS detection of the transformation efficiency in soybean hairy roots. (b). qTR–PCR analysis of GmVOZ1G transcription levels in overexpression of GmVOZ1G, RNAi, and empty vector (EV) control soybean hairy roots. The expression level of tubulin was used as a quantitative control. The means of four biological replicates and the standard deviation are presented. (c). Soil water potential (SWP) was determined under normal and drought stress conditions. (d). Phenotypes of soybean composite seedlings with overexpression, RNAi, and EV control hairy roots under drought and salt stress treatments. (e−h). Relative water content (RWC), malondialdehyde (MDA) content, peroxidase (POD), and superoxide dismutase (SOD) activities in leaves of soybean composite seedlings with different transgenic hairy roots under normal and stress conditions. All values are the means and SD of three independent replicates. The asterisks indicate statistical differences in comparison with the corresponding controls at p < 0.01 (**) and 0.01 < p< 0.05 (*), respectively.