Iron status modulates cadmium tolerance: mechanistic insights from MsbHLH60 -mediated regulation of the FIT/IRT1 module in alfalfa.

Abstract

Basic helix-loop-helix (bHLH) transcription factors play pivotal roles in plant adaptation to metallic elements, yet the mechanisms by which leguminous bHLHs regulate iron (Fe) homeostasis and cadmium (Cd) stress adaptation remain inadequately understood. MsbHLH60 and its promoter was cloned from alfalfa. The expression of MsbHLH60 under iron deficiency (-Fe), Cd, and Cd accompanied by Fe deficiency (Cd(-Fe)) was analyzed by qRT-PCR and GUS assay. MsbHLH60 was subcellular localized. Overexpressing MsbHLH60 Arabidopsis was generated and subjected to phenotypic and physiological assays under -Fe, Cd and Cd(-Fe) stress. The interaction was studied through Y2H, Y1H and transient co-expression. MsbHLH60 belongs to bHLH transcription factor family and was localized to nucleus and cytoplasm. MsbHLH60 was strongly expressed in the young tissues of alfalfa, and was significantly upregulated in roots by -Fe or Cd stress, especially in the stele. However, MsbHLH60 was not induced by Cd(-Fe). Overexpression of MsbHLH60 enhanced Arabidopsis tolerance to -Fe and Cd stress. MsbHLH60 mediated Cd tolerance was notably attenuated by -Fe. MsbHLH60 could interact with MsFIT and enhanced the activate effect on MsIRT1 expression. The results suggested that MsbHLH60 might serve as an auxiliary regulator in the Fe regulation network and might orchestrate plant adaptive response to -Fe and Cd by modulating metal transport and distribution and enhancing antioxidant capacity. This study provides novel insights into the intersecting mechanisms of nutrient homeostasis and metal stress tolerance in plants, laying a theoretical foundation and genetic resources for crop improvement.