Phosphatidylinositol 3-phosphate-binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis.

"Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting.

Export of vacuolar manganese by AtNRAMP3 and AtNRAMP4 is required for optimal photosynthesis and growth under manganese deficiency.

Manganese (Mn) is an essential element, acting as cofactor in numerous enzymes. In particular, a Mn cluster is indispensable for the function of the oxygen-evolving complex of photosystem II. Metal transporters of the Natural Resistance-Associated Macrophage Protein (NRAMP) family have the ability to transport both iron and Mn. AtNRAMP3 and AtNRAMP4 are required for iron mobilization in germinating seeds. The results reported here show that, in adult Arabidopsis (Arabidopsis thaliana) plants, AtNRAMP3 and AtNRAMP4 have an important role in Mn homeostasis. Vacuolar Mn accumulation in mesophyll cells of rosette leaves of adult nramp3nramp4 double mutant plants was dramatically increased when compared with the wild type. This suggests that a considerable proportion of the cellular Mn pool passes through the vacuole and is retrieved in an AtNRAMP3/AtNRAMP4-dependent manner. The impaired Mn release from mesophyll vacuoles of nramp3nramp4 double mutant plants is associated with reduced growth under Mn deficiency. However, leaf AtNRAMP3 and AtNRAMP4 protein levels are unaffected by Mn supply. Under Mn deficiency, nramp3nramp4 plants contain less functional photosystem II than the wild type. These data are consistent with a shortage of Mn to produce functional photosystem II, whereas mitochondrial Mn-dependent superoxide dismutase activity is maintained under Mn deficiency in both genotypes. The results presented here suggest an important role for AtNRAMP3/AtNRAMP4-dependent Mn transit through the vacuole prior to the import into chloroplasts of mesophyll cells.

Functional characterization of NRAMP3 and NRAMP4 from the metal hyperaccumulator Thlaspi caerulescens.

The ability of metal hyperaccumulating plants to tolerate and accumulate heavy metals results from adaptations of metal homeostasis. NRAMP metal transporters were found to be highly expressed in some hyperaccumulating plant species. Here, we identified TcNRAMP3 and TcNRAMP4, the closest homologues to AtNRAMP3 and AtNRAMP4 in Thlaspi caerulescens and characterized them by expression analysis, confocal imaging and heterologous expression in yeast and Arabidopsis thaliana. TcNRAMP3 and TcNRAMP4 are expressed at higher levels than their A. thaliana homologues. When expressed in yeast TcNRAMP3 and TcNRAMP4 transport the same metals as their respective A. thaliana orthologues: iron (Fe), manganese (Mn) and cadmium (Cd) but not zinc (Zn) for NRAMP3; Fe, Mn, Cd and Zn for NRAMP4. They also localize at the vacuolar membrane in A. thaliana protoplasts. Inactivation of AtNRAMP3 and AtNRAMP4 in A. thaliana results in strong Cd and Zn hypersensitivity, which is fully rescued by TcNRAMP3 or TcNRAMP4 expression. However, metal tolerance conferred by TcNRAMP expression in nramp3nramp4 mutant does not exceed that of wild-type A. thaliana. Our data indicate that the difference between TcNRAMP3 and TcNRAMP4 and their A. thaliana orthologues does not lie in a different protein function, but probably resides in a different expression level or expression pattern.

R-type anion channel activation is an essential step for ROS-dependent innate immune response in Arabidopsis suspension cells.

Plants are constantly exposed to environmental biotic and abiotic stresses. Plants cells perceive these factors and trigger early responses followed by delayed and complex adaptation processes. Using cell suspensions of Arabidopsis thaliana (L.) as a cellular model, we investigated the role of plasma membrane anion channels in Reactive Oxygen Species (ROS) production and in cell death which occurs during non-host pathogen infection. Protoplasts derived from Arabidopsis suspension cells display two anion currents with characteristics very similar to those of the slow nitrate-permeable (S-type) and rapid sulfate-permeable (R-type) channels previously characterised in hypocotyl cells and other cell types. Using seven inhibitors, we showed that the R-type channel and ROS formation in cell cultures present similar pharmacological profiles. The efficiency of anion channel blockers to inhibit ROS production was independent of the nature of the triggering signal (osmotic stress or general elicitors of plant defence), indicating that the R-type channel represents a crossroad in the signalling pathways leading to ROS production. In a second step, we show that treatment with R-type channel blockers accelerates cell death triggered by the non-specific plant pathogen Xanthomonas campestris. Finally, we discuss the hypothesis that the R-type channel is involved in innate immune response allowing cell defence via antibacterial ROS production.

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.

Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron.

Iron (Fe) is necessary for all living cells, but its bioavailability is often limited. Fe deficiency limits agriculture in many areas and affects over a billion human beings worldwide. In mammals, NRAMP2/DMT1/DCT1 was identified as a major pathway for Fe acquisition and recycling. In plants, AtNRAMP3 and AtNRAMP4 are induced under Fe deficiency. The similitude of AtNRAMP3 and AtNRAMP4 expression patterns and their common targeting to the vacuole, together with the lack of obvious phenotype in nramp3-1 and nramp4-1 single knockout mutants, suggested a functional redundancy. Indeed, the germination of nramp3 nramp4 double mutants is arrested under low Fe nutrition and fully rescued by high Fe supply. Mutant seeds have wild type Fe content, but fail to retrieve Fe from the vacuolar globoids. Our work thus identifies for the first time the vacuole as an essential compartment for Fe storage in seeds. Our data indicate that mobilization of vacuolar Fe stores by AtNRAMP3 and AtNRAMP4 is crucial to support Arabidopsis early development until efficient systems for Fe acquisition from the soil take over.

Distinct pH regulation of slow and rapid anion channels at the plasma membrane of Arabidopsis thaliana hypocotyl cells.

Variations in both intracellular and extracellular pH are known to be involved in a wealth of physiological responses. Using the patch-clamp technique on Arabidopsis hypocotyl cells, it is shown that rapid-type and slow-type anion channels at the plasma membrane are both regulated by pH via distinct mechanisms. Modifications of pH modulate the voltage-dependent gating of the rapid channel. While intracellular alkalinization facilitates channel activation by shifting the voltage gate towards negative potentials, extracellular alkalinization shifts the activation threshold to more positive potentials, away from physiological resting membrane potentials. By contrast, pH modulates slow anion channel activity in a voltage-independent manner. Intracellular acidification and extracellular alkalinization increase slow anion channel currents. The possible role of these distinct modulations in physiological processes involving anion efflux and modulation of extracellular and/or intracellular pH, such as elicitor and ABA signalling, are discussed.

AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency.

Metal homeostasis is critical for the survival of living organisms, and metal transporters play central roles in maintaining metal homeostasis in the living cells. We have investigated the function of a metal transporter of the NRAMP family, AtNRAMP3, in Arabidopsis thaliana. A previous study showed that AtNRAMP3 expression is upregulated by iron (Fe) starvation and that AtNRAMP3 protein can transport Fe. In the present study, we used AtNRAMP3 promoter beta-glucoronidase (GUS) fusions to show that AtNRAMP3 is expressed in the vascular bundles of roots, stems, and leaves under Fe-sufficient conditions. This suggests a function in long-distance metal transport within the plant. Under Fe-starvation conditions, the GUS activity driven by the AtNRAMP3 promoter is upregulated without any change in the expression pattern. We analyze the impact of AtNRAMP3 disruption and overexpression on metal accumulation in plants. Under Fe-sufficient conditions, AtNRAMP3 overexpression or disruption does not lead to any change in the plant metal content. Upon Fe starvation, AtNRAMP3 disruption leads to increased accumulation of manganese (Mn) and zinc (Zn) in the roots, whereas AtNRAMP3 overexpression downregulates Mn accumulation. In addition, overexpression of AtNRAMP3 downregulates the expression of the primary Fe uptake transporter IRT1 and of the root ferric chelate reductase FRO2. Expression of AtNRAMP3::GFP fusion protein in onion cells or Arabidopsis protoplasts shows that AtNRAMP3 protein localizes to the vacuolar membrane. To account for the results presented, we propose that AtNRAMP3 influences metal accumulation and IRT1 and FRO2 gene expression by mobilizing vacuolar metal pools to the cytosol.