Intracellular Distribution of Manganese by the Trans-Golgi Network Transporter NRAMP2 Is Critical for Photosynthesis and Cellular Redox Homeostasis.

Plants require trace levels of manganese (Mn) for survival, as it is an essential cofactor in oxygen metabolism, especially O2 production via photosynthesis and the disposal of superoxide radicals. These processes occur in specialized organelles, requiring membrane-bound intracellular transporters to partition Mn between cell compartments. We identified an Arabidopsis thaliana member of the NRAMP family of divalent metal transporters, NRAMP2, which functions in the intracellular distribution of Mn. Two knockdown alleles of NRAMP2 showed decreased activity of photosystem II and increased oxidative stress under Mn-deficient conditions, yet total Mn content remained unchanged. At the subcellular level, these phenotypes were associated with a loss of Mn content in vacuoles and chloroplasts. NRAMP2 was able to rescue the mitochondrial yeast mutant mtm1∆ In plants, NRAMP2 is a resident protein of the trans-Golgi network. NRAMP2 may act indirectly on downstream organelles by building up a cytosolic pool that is used to feed target compartments. Moreover, not only does the nramp2 mutant accumulate superoxide ions, but NRAMP2 can functionally replace cytosolic superoxide dismutase in yeast, indicating that the pool of Mn displaced by NRAMP2 is required for the detoxification of reactive oxygen species.

High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions.

In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis.

The NRAMP6 metal transporter contributes to cadmium toxicity.

NRAMP (natural resistance-associated macrophage protein) homologues are evolutionarily conserved bivalent metal transporters. In Arabidopsis, AtNRAMP3 and AtNRAMP4 play a key role in iron nutrition of the germinating plantlet by remobilizing vacuolar iron stores. In the present paper we describe the molecular and physiological characterization of AtNRAMP6. AtNRAMP6 is predominantly expressed in the dry seed embryo and to a lesser extent in aerial parts. Its promoter activity is found diffusely distributed in cotyledons and hypocotyl, as well as in the vascular tissue region of leaf and flower. We show that the AtNRAMP6 transcript coexists with a partially spliced isoform in all shoot cell types tested. When expressed in yeast, AtNRAMP6, but not its misspliced derivative, increased sensitivity to cadmium without affecting cadmium content in the cell. Likewise, Arabidopsis transgenic plants overexpressing AtNRAMP6 were hypersensitive to cadmium, although plant cadmium content remained unchanged. Consistently, a null allele of AtNRAMP6, named nramp6-1, was more tolerant to cadmium toxicity, a phenotype that was reverted by expressing AtNRAMP6 in the mutant background. We used an AtNRAMP6::HA (where HA is haemagglutinin) fusion, shown to be functional in yeast, to demonstrate through immunoblot analysis of membrane fractions and immunofluorescence localization that, in yeast cells, AtNRAMP6 is targeted to a vesicular-shaped endomembrane compartment distinct from the vacuole or mitochondria. We therefore propose that AtNRAMP6 functions as an intracellular metal transporter, whose presence, when modified, is likely to affect distribution/availability of cadmium within the cell.