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

Abstract

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 pH 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 RT-PCR, and named as GsIRT3. GsIRT3 displayed differential gene expression levels at different time points under alkali treatment in both roots and leaves. When expressed in yeast cells, the recombinant yeast pYES2-GsIRT3 was highly tolerant to iron deficiency stress and zinc deficiency stress. In addition, GsIRT3 overexpression lines of Arabidopsis thaliana were created by floral dip method for functional characterization of GsIRT3 under stress treatments. The results displayed that OX lines performed better under high pH stress than WT plants in terms of higher root lengths and fresh biomass. Physiological indicators assays showed that OX lines appeared with higher chlorophyll, low MDA, and H₂O₂ contents than WT plants under alkali stress. Further, CAT, POD and SOD activities increased in OX lines under alkali stress. The superoxide radicals were further assessed by NBT staining in which WT plants stained deep compared to OX lines. To further verify the role of GsIRT3 in stress mechanisms, expression levels of stress responsive marker genes (RD29A, COR15, KIN1, and H⁺ATP) were determined by qRT-PCR analysis and all marker genes showed high transcript expression in OX lines after stress application compared to WT. In last, functional characterization of GsIRT3 overexpression in soybean displayed better hairy root growth and increased fresh weight in OX lines compared to soybean WT (K599) line.These results clearly suggests the positive roles of GsIRT3 in pH stress tolerance mechanisms.