Proteomic profiling of root microsomal membrane fractions reveals distinct RBOHC- and RBOHF-associated responses to cadmium in Arabidopsis

Abstract

Cadmium (Cd) is a toxic heavy metal for plants, and although its mechanisms of toxicity are well characterized, the regulatory networks underlying plant responses remain incompletely understood. This study investigated membrane-associated molecular responses linked to RBOHC and RBOHF during Cd stress through a label-free proteomics of root microsomal membrane fractions from Arabidopsis thaliana wild-type (WT), rbohC and rbohF plants. Proteomic analysis revealed pronounced genotype-dependent responses, with most Cd-responsive differentially expressed proteins (DEPs) being genotype-specific. The rbohF mutant displayed broader proteomic reprogramming than rbohC, whereas rbohC exhibited a comparatively attenuated response. Functional analyses indicated that Cd stress altered proteins associated with detoxification, ion transport, membrane trafficking, redox regulation and stress signaling. WT plants showed coordinated accumulation of glutathione S-transferases (GSTs), MATE/DTX transporters, ABC transporters and Ca²⁺-related proteins, whereas rbohC lacked GST accumulation and showed reduced abundance of the IRT1-CIPK23 module. In contrast, rbohF exhibited enhanced accumulation of several GSTs together with altered abundance of proteins associated with redox regulation, Casparian strip organization and ion homeostasis, including SBP1, CASP1 and PER64. Physiological analyses further revealed increased superoxide accumulation in rbohC roots under Cd stress, while GST activity differed among genotypes, supporting distinct antioxidant responses. Together with previous physiological evidence, these findings identify membrane-associated proteins and pathways associated with the contrasting Cd responses of rbohC and rbohF mutants. Overall, this work provides new insights into membrane-associated molecular responses linked to RBOHC and RBOHF during Cd stress and identifies candidate proteins for future functional studies on Cd tolerance.