The role of NADPH oxidases in regulating leaf gas exchange and ion homeostasis in Arabidopsis plants under cadmium stress

NADPH oxidase, an enzyme associated with the plasma membrane, constitutes one of the main sources of reactive oxygen species (ROS) which regulate different developmental and adaptive responses in plants. In this work, the involvement of NADPH oxidases in the regulation of photosynthesis and cell ionic homeostasis in response to short cadmium exposure was compared between wild type (WT) and three RBOHs (Respiratory Burst Oxidase Homologues) Arabidopsis mutants (AtrbohC, AtrbohD, and AtrbohF). Plants were grown under hydroponic conditions and supplemented with 50 µM CdCl₂ for 24 h. Cadmium treatment differentially affected photosynthesis, stomatal conductance, transpiration, and antioxidative responses in WT and Atrbohs mutants. The loss of function of RBOH isoforms resulted in higher Cd²⁺ influx, mainly in the elongation zone of roots, which was more evident in AtrbohD and AtrbohF mutants. In the mature zone, the highest Cd²⁺ influx was observed in rbohC mutant. The lack of functional RBOH isoforms also resulted in altered patterns of net K⁺ transport across cellular membranes, both in the root epidermis and leaf mesophyll. The analysis of expression of metal transporters by qPCR demonstrated that a loss of functional RBOH isoforms has altered transcript levels for metal NRAMP3, NRAMP6 and IRT1 and the K⁺ transporters outward-rectifying K⁺ efflux GORK channel, while RBOHD specifically regulated transcripts for high-affinity K⁺ transporters KUP8 and HAK5, and IRT1 and RBOHD and F regulated the transcription factors TGA3 and TGA10. It is concluded that RBOH-dependent H₂O₂ regulation of ion homeostasis and Cd is a highly complex process involving multilevel regulation from transpirational water flow to transcriptional and posttranslational modifications of K/metals transporters.