Metal tolerance protein family members are involved in Mn homeostasis through internal compartmentation and exocytosis in Brassica napus

Mineral elements, including manganese (Mn), are indispensable for plant growth and development. However, plants cultivated on acidic soils are vulnerable to Mn²⁺ toxicity due to a prevalently phytotoxic level of Mn²⁺. Metal tolerance proteins (MTPs) were demonstrated to be essential for metal homeostasis and tolerance in different plant species. In this study, we present the functional characteristics of BnMTP8 and BnMTP9 from Brassica napus. The expression level of BnMTP8 was most prominent in rapeseed roots and was only markedly up-regulated in shoots by excess Mn²⁺. In contrast, BnMTP9 was most abundant in shoots. Expression of BnMTP8 and BnMTP9 in Saccharomyces cerevisiae compensated the Mn²⁺-hypersensitivity of Δpmr1 by increasing metal sequestration and efflux, respectively. In addition, heteroexpression of BnMTP8 restored Mn²⁺ tolerance of Arabidopsis mtp8 mutants and increased Mn²⁺ accumulation in tissues and leaf vacuoles. Expression of BnMTP9 in Arabidopsis mtp11 mutants restored their growth and reduced Mn²⁺ concentrations in mesophyll protoplasts and tissues upon elevated levels of Mn²⁺. Transient expression of GFP-fusion constructs in protoplasts showed that BnMTP8 and BnMTP9 localize to pre-vacuolar compartments and the trans-Golgi network, respectively. However, high extracellular Mn²⁺ in Arabidopsis root cells triggered BnMTP8-GFP relocation to the tonoplast and BnMTP9-GFP relocation to the plasma membrane, suggesting that these two proteins are involved in vacuolar Mn²⁺ sequestration and cytosolic Mn²⁺ export, respectively. Taken together, these findings indicate that BnMTP8 and BnMTP9 load Mn²⁺ into vesicles for subsequent delivery to the vacuole or secretion into extracellular spaces, respectively, thereby cooperating to maintain Mn²⁺ homeostasis in the roots and shoots of rapeseed.