A Metal Importer and Exporter Interact Differently in the Chloroplast and Cell Membrane

Metal homeostasis, which coordinates the influx and efflux of essential elements such as iron (Fe) and manganese (Mn) in chloroplasts, is essential for optimum photosynthesis, especially in metal accumulating plants. Brassica juncea (Indian mustard) is a metal-tolerant species with a strong metal accumulation capacity, making it a suitable model for studying transition metal homeostasis. In this study, we identified two efflux transporters, BjYSL6.1 and BjYSL6.4, that localize in the endomembrane system of Schizosaccharomyces pombe and interact with the chloroplast Mn influx transporter BjNRAMP4.1 at the plasma membrane and within the chloroplasts. Bimolecular fluorescence complementation and split-ubiquitin yeast two-hybrid assays confirmed specific protein-protein interactions among these transporters, as well as with the membrane-bound thioredoxin BjHCF164, a known regulator of photosynthetic electron transport. Gene expression studies revealed that BjNRAMP4.1 and BjYSL6 isoforms are inversely regulated under Fe and Mn stress conditions, with BjNRAMP4.1 being strongly induced under deficiency, whereas BjYSL6.1 and BjYSL6.4 are downregulated. These findings suggest that a coordinated network involving BjNRAMP4.1, BjYSL6s, and BjHCF164 modulates metal influx and efflux at the chloroplast and plasma membrane interfaces, thereby maintaining metal homeostasis, which is critical for photosynthetic efficiency in B. juncea.