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The Arabidopsis (Arabidopsis thaliana) Heavy Metal Associated3 (AtHMA3) protein belongs to the P 1B-2 subgroup of the P-type ATPase family, which is involved in heavy metal transport. In a previous study, we have shown, using heterologous expression in the yeast Saccharomyces cerevisiae, that in the presence of toxic metals, AtHMA3 was able to phenotypically complement the cadmium/lead (Cd/Pb)-hypersensitive strain ycf1 but not the zinc (Zn)-hypersensitive strain zrc1. In this study, we demonstrate that AtHMA3 in planta is located in the vacuolar membrane, with a high expression level in guard cells, hydathodes, vascular tissues, and the root apex. Confocal imaging in the presence of the Zn/Cd fluorescent probe BTC-5N revealed that AtHMA3 participates in the vacuolar storage of Cd. A T-DNA insertional mutant was found more sensitive to Zn and Cd. Conversely, ectopic overexpression of AtHMA3 improved plant tolerance to Cd, cobalt, Pb, and Zn; Cd accumulation increased by about 2-to 3-fold in plants overexpressing AtHMA3 compared with wild-type plants. Thus, AtHMA3 likely plays a role in the detoxification of biological (Zn) and nonbiological (Cd, cobalt, and Pb) heavy metals by participating in their vacuolar sequestration, an original function for a P 1B-2 ATPase in a multicellular eukaryote.

AtHMA4 is an Arabidopsis thaliana P1B-ATPase which transports Zn and Cd. Here, we demonstrate that AtHMA4 is localized at the plasma membrane and expressed in tissues surrounding the root vascular vessels. The ectopic overexpression of AtHMA4 improved the root growth in the presence of toxic concentrations of Zn, Cd and Co. A null mutant exhibited a lower translocation of Zn and Cd from the roots to shoot. In contrast, the AtHMA4 overexpressing lines displayed an increase in the zinc and cadmium shoot content. Altogether, these results strongly indicate that AtHMA4 plays a role in metal loading in the xylem.

Nitric oxide (NO) functions as a cell-signaling molecule in plants. In particular, a role for NO in the regulation of iron homeostasis and in the plant response to toxic metals has been proposed. Here, we investigated the synthesis and the role of NO in plants exposed to cadmium (Cd 2+ ), a nonessential and toxic metal. We demonstrate that Cd 2+ induces NO synthesis in roots and leaves of Arabidopsis (Arabidopsis thaliana) seedlings. This production, which is sensitive to NO synthase inhibitors, does not involve nitrate reductase and AtNOA1 but requires IRT1, encoding a major plasma membrane transporter for iron but also Cd 2+ . By analyzing the incidence of NO scavenging or inhibition of its synthesis during Cd 2+ treatment, we demonstrated that NO contributes to Cd 2+ -triggered inhibition of root growth. To understand the mechanisms underlying this process, a microarray analysis was performed in order to identify NO-modulated root genes up-and down-regulated during Cd 2+ treatment. Forty-three genes were identified encoding proteins related to iron homeostasis, proteolysis, nitrogen assimilation/metabolism, and root growth. These genes include IRT1. Investigation of the metal and ion contents in Cd 2+ -treated roots in which NO synthesis was impaired indicates that IRT1 up-regulation by NO was consistently correlated to NO's ability to promote Cd 2+ accumulation in roots. This analysis also highlights that NO is responsible for Cd 2+ -induced inhibition of root Ca 2+ accumulation. Taken together, our results suggest that NO contributes to Cd 2+ toxicity by favoring Cd 2+ versus Ca 2+ uptake and by initiating a cellular pathway resembling those activated upon iron deprivation.

Although ions play important roles in the cell and chloroplast metabolism, little is known about ion transport across the chloroplast envelope. Using a proteomic approach specifically targeted to the Arabidopsis chloroplast envelope, we have identified HMA1, which belongs to the metal-transporting P 1B -type ATPases family. HMA1 is mainly expressed in green tissues, and we validated its chloroplast envelope localization. Yeast expression experiments demonstrated that HMA1 is involved in copper homeostasis and that deletion of its N-terminal His-domain partially affects the metal transport. Characterization of hma1 Arabidopsis mutants revealed a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase activity. No effect was observed on the plastocyanin content in these lines. The hma1 insertional mutants grew like WT plants in standard condition but presented a photosensitivity phenotype under high light. Finally, direct biochemical ATPase assays performed on purified chloroplast envelope membranes showed that the ATPase activity of HMA1 is specifically stimulated by copper. Our results demonstrate that HMA1 offers an additional way to the previously characterized chloroplast envelope Cu-ATPase PAA1 to import copper in the chloroplast.Chloroplasts contain a large variety of ions among which metal ions such as copper, iron, manganese, and zinc that are essential for their development and function. Copper is an essential redox cofactor required for a wide variety of processes, including photosynthetic electron transfer reactions (plastocyanin) and detoxification of superoxide radicals (Cu/Zn-superoxide dismutase, SOD) 4 (1). Other metal ions are cofactors for several enzymatic reactions: zinc is associated with chloroplast SOD, methionine synthase, carbonic anhydrase; manganese is required for oxygen evolution in photosynthesis; whereas iron is a cofactor of iron SOD and is found in iron-sulfur clusters of cytochrome b 6 f complex, ferredoxin, photosystem I (PSI), and photosystem II (PSII) (2). All these metals are essential micronutrients but are toxic when present in excess (3). To maintain the concentration of metals within physiological limits, cells possess mechanisms that control the uptake, accumulation, trafficking, and also detoxification of metal ions. Little is known about metal transport into chloroplasts. Until now, the sole chloroplast proteins demonstrated as being involved in metal ions transport are PAA1 (4) and very recently PAA2 (5), two P 1B -type ATPases. PAA1, localized into the chloroplast envelope, supplies copper to the chloroplast, whereas PAA2, localized into the thylakoid membrane, delivers copper to the thylakoid lumen. One important result derived from this work is the fact that the disruption of the PAA1 gene does not fully abolish the import of copper into the chloroplast. From this recent observation, Abdel-Ghany and coworkers (5) concluded that an as yet unidentified and additional way to PAA1 must exist to import copper into the chloroplast....

The Arabidopsis thaliana AtHMA3 protein belongs to the P 1B -adenosine triphosphatase (ATPase) transporter family, involved in heavy metal transport. Functional expression of AtHMA3 phenotypically complements the Cd/Pb-hypersensitive yeast strain v vycf1, but not the Zn-hypersensitive mutant v vzrc1. AtHMA3-complemented v vycf1 cells accumulate the same amount of cadmium as YCF1-complemented v vycf1 cells or wild-type cells, suggesting that AtHMA3 carries out an intracellular sequestration of Cd. A mutant of AtHMA3 altered in the P-ATPase phosphorylation domain did not complement v vycf1, suggesting that metal transport rather than chelation is involved. The fusion protein AtHMA3: :green £uorescent protein (GFP) is localized at the vacuole, consistent with a role in the in£ux of cadmium into the vacuolar compartment. In A. thaliana, the mRNA of AtHMA3 was detected mainly in roots, old rosette leaves and cauline leaves. The expression levels were not a¡ected by cadmium or zinc treatments.

Plants produce cytokinin (CK) hormones for controlling key developmental processes like source/sink distribution, cell division or programmed cell-death. Some plant pathogens have been shown to produce CKs but the function of this mimicry production by non-tumor inducing pathogens, has yet to be established. Here we identify a gene required for CK biosynthesis, CKS1, in the rice blast fungus Magnaporthe oryzae. The fungal-secreted CKs are likely perceived by the plant during infection since the transcriptional regulation of rice CK-responsive genes is altered in plants infected by the mutants in which CKS1 gene was deleted. Although cks1 mutants showed normal in vitro growth and development, they were severely affected for in planta growth and virulence. Moreover, we showed that the cks1 mutant triggered enhanced induction of plant defenses as manifested by an elevated oxidative burst and expression of defense-related markers. In addition, the contents of sugars and key amino acids for fungal growth were altered in and around the infection site by the cks1 mutant in a different manner than by the control strain. These results suggest that fungal-derived CKs are key effectors required for dampening host defenses and affecting sugar and amino acid distribution in and around the infection site.

The role of salicylic acid (SA) and jasmonic acid (JA) signaling in resistance to root pathogens has been poorly documented. We assessed the contribution of SA and JA to basal and partial resistance of Arabidopsis to the biotrophic clubroot agent Plasmodiophora brassicae. SA and JA levels as well as the expression of the SA-responsive genes PR2 and PR5 and the JA-responsive genes ARGAH2 and THI2.1 were monitored in infected roots of the accessions Col-0 (susceptible) and Bur-0 (partially resistant). SA signaling was activated in Bur-0 but not in Col-0. The JA pathway was weakly activated in Bur-0 but was strongly induced in Col-0. The contribution of both pathways to clubroot resistance was then assessed using exogenous phytohormone application and mutants affected in SA or JA signaling. Exogenous SA treatment decreased clubroot symptoms in the two Arabidopsis accessions, whereas JA treatment reduced clubroot symptoms only in Col-0. The cpr5-2 mutant, in which SA responses are constitutively induced, was more resistant to clubroot than the corresponding wild type, and the JA signaling-deficient mutant jar1 was more susceptible. Finally, we showed that the JA-mediated induction of NATA1 drove N(δ)-acetylornithine biosynthesis in infected Col-0 roots. The 35S::NATA1 and nata1 lines displayed reduced or enhanced clubroot symptoms, respectively, thus suggesting that in Col-0 this pathway was involved in the JA-mediated basal clubroot resistance. Overall, our data support the idea that, depending on the Arabidopsis accession, both SA and JA signaling can play a role in partial inhibition of clubroot development in compatible interactions with P. brassicae.