GhRCD1 promotes cotton tolerance to cadmium by regulating the GhbHLH12-GhMYB44-GhHMA1 transcriptional cascade

Heavy metal pollution poses a significant risk to human health and wreaks havoc on agricultural productivity. Phytoremediation, a plant-based, environmentally benign, and cost-effective method, is employed to remove heavy metals from contaminated soil, particularly in agricultural or heavy metal-sensitive lands. However, the phytoremediation capacity of various plant species and germplasm resources display significant genetic diversity, and the mechanisms underlying these differences remain hitherto obscure. Given its potential benefits, genetic improvement of plants is essential for enhancing their uptake of heavy metals, tolerance to harmful levels, as well as overall growth and development in contaminated soil. In this study, we uncover a molecular cascade that regulates cadmium (Cd²⁺) tolerance in cotton, involving GhRCD1, GhbHLH12, GhMYB44, and GhHMA1. We identified a Cd²⁺-sensitive cotton T-DNA insertion mutant with disrupted GhRCD1 expression. Genetic knockout of GhRCD1 by CRISPR/Cas9 technology resulted in reduced Cd²⁺ tolerance in cotton seedlings, while GhRCD1 overexpression enhanced Cd²⁺ tolerance. Through molecular interaction studies, we demonstrated that, in response to Cd²⁺ presence, GhRCD1 directly interacts with GhbHLH12. This interaction activates GhMYB44, which subsequently activates a heavy metal transporter, GhHMA1, by directly binding to a G-box cis-element in its promoter. These findings provide critical insights into a novel GhRCD1-GhbHLH12-GhMYB44-GhHMA1 regulatory module responsible for Cd²⁺ tolerance in cotton. Furthermore, our study paves the way for the development of elite Cd²⁺-tolerant cultivars by elucidating the molecular mechanisms governing the genetic control of Cd²⁺ tolerance in cotton.