GsIRT3: a zinc-iron transporter gene from Glycine soja, enhancing alkali stress tolerance in Arabidopsis thaliana.

ZIP family genes are known to play important roles in the transport of divalent metal ions such as zinc and iron. However, their roles in alkali stress have not been well characterized so far. In this study, a ZIP (Zrt, Irt-like Protein) cDNA was isolated from wild soybean (Glycine soja) by reverse transcription PCR (RT-PCR) and named as GsIRT3. GsIRT3 displayed differential gene expression levels at different time points under alkali treatment in both roots and leaves. Heterologous expression of GsIRT3 in yeast (pYES2-GsIRT3) conferred enhanced tolerance to both iron- and zinc-deficiency stress conditions. In addition, GsIRT3 overexpression lines (OX) of Arabidopsis thaliana were created by floral dip method for functional characterization of GsIRT3 under stress treatments. The results displayed that GsIRT3 OX lines performed better under alkali stress than wild-type (WT) plants in terms of higher root lengths and fresh biomass. Physiological indicator assays showed that OX lines appeared with higher chlorophyll, lower malondialdehyde (MDA), and H2O2 contents than WT plants under alkali stress. Further, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activities increased in OX lines under alkali stress. Superoxide radical accumulation was further evaluated by nitro-blue tetrazolium (NBT) staining, where WT plants exhibited more intense staining than the overexpression (OX) lines, indicating higher superoxide levels in WT. To further verify the role of GsIRT3 in stress mechanisms, expression levels of stress responsive marker genes (RD29A, COR15, KIN1, and H+-ATPase) were determined by qRT-PCR (Quantitative Real‑time PCR) analysis and all marker genes showed high transcript expression in OX lines after stress application compared to WT. Finally, functional characterization of GsIRT3 overexpression in soybean displayed better hairy root growth and increased fresh weight in OX lines compared to the soybean K599 line. These results clearly suggest the positive roles of GsIRT3 in alkali stress tolerance mechanisms.