Enhanced metal tolerance correlates with heterotypic variation in SpMTL, a metallothionein-like protein from the hyperaccumulator Sedum plumbizincicola.

Mechanistic insight into metal hyperaccumulation is largely restricted to Brassicaceae plants; therefore, it is of great importance to obtain corresponding knowledge from system outside the Brassicaceae. Here, we constructed and screened a cDNA library of the Cd/Zn hyperaccumulator Sedum plumbizincicola and identified a novel metallothionein-like protein encoding gene SpMTL. SpMTL showed functional similarity to other known MT proteins and also to its orthologues from non-hyperaccumulators. However, three additional cysteine residues were observed in SpMTL and appeared to be hyperaccumulator specific. Removal of these three residues significantly decreased its ability to tolerate Cd and the stoichiometry of Cd against SpMTL (molar ratio of Cd/SpMTL) to a level comparable to those of Cd/SaMTL and Cd/SeMTL in the corresponding non-hyperaccumulating relatives. SpMTL expressed in S. plumbizincicola roots at a much higher level than those of its orthologues in the non-hyperaccumulator roots. Interestingly, a positive correlation was observed between transcript levels of SpMTL in roots and Cd accumulation in leaves. Taking these results together, we propose that elevated transcript levels and heterotypic variation in protein sequences of SpMTL might contribute to the trait of Cd hyperaccumulation and hypertolerance in S. plumbizincicola.

The Arabidopsis ethylene/jasmonic acid-NRT signaling module coordinates nitrate reallocation and the trade-off between growth and environmental adaptation.

Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.

Arabidopsis histone methylase CAU1/PRMT5/SKB1 acts as an epigenetic suppressor of the calcium signaling gene CAS to mediate stomatal closure in response to extracellular calcium.

Elevations in extracellular calcium ([Ca(2+)]o) are known to stimulate cytosolic calcium ([Ca(2+)]cyt) oscillations to close stomata. However, the underlying mechanisms regulating this process remain largely to be determined. Here, through the functional characterization of the calcium underaccumulation mutant cau1, we report that the epigenetic regulation of CAS, a putative Ca(2+) binding protein proposed to be an external Ca(2+) sensor, is involved in this process. cau1 mutant plants display increased drought tolerance and stomatal closure. A mutation in CAU1 significantly increased the expression level of the calcium signaling gene CAS, and functional disruption of CAS abolished the enhanced drought tolerance and stomatal [Ca(2+)]o signaling in cau1. Map-based cloning revealed that CAU1 encodes the H4R3sme2 (for histone H4 Arg 3 with symmetric dimethylation)-type histone methylase protein arginine methytransferase5/Shk1 binding protein1. Chromatin immunoprecipitation assays showed that CAU1 binds to the CAS promoter and modulates the H4R3sme2-type histone methylation of the CAS chromatin. When exposed to elevated [Ca(2+)]o, the protein levels of CAU1 decreased and less CAU1 bound to the CAS promoter. In addition, the methylation level of H4R3sme2 decreased in the CAS chromatin. Together, these data suggest that in response to increases in [Ca(2+)]o, fewer CAU1 protein molecules bind to the CAS promoter, leading to decreased H4R3sme2 methylation and consequent derepression of the expression of CAS to mediate stomatal closure and drought tolerance.

Arabidopsis NRT1.5 is another essential component in the regulation of nitrate reallocation and stress tolerance.

Nitrate reallocation to plant roots occurs frequently under adverse conditions and was recently characterized to be actively regulated by Nitrate Transporter1.8 (NRT1.8) in Arabidopsis (Arabidopsis thaliana) and implicated as a common response to stresses. However, the underlying mechanisms remain largely to be determined. In this study, characterization of NRT1.5, a xylem nitrate-loading transporter, showed that the mRNA level of NRT1.5 is down-regulated by salt, drought, and cadmium treatments. Functional disruption of NRT1.5 enhanced tolerance to salt, drought, and cadmium stresses. Further analyses showed that nitrate, as well as Na(+) and Cd(2+) levels, were significantly increased in nrt1.5 roots. Important genes including Na(+)/H(+) exchanger1, Salt overly sensitive1, Pyrroline-5-carboxylate synthase1, Responsive to desiccation29A, Phytochelatin synthase1, and NRT1.8 in stress response pathways are steadily up-regulated in nrt1.5 mutant plants. Interestingly, altered accumulation of metabolites, including proline and malondialdehyde, was also observed in nrt1.5 plants. These data suggest that NRT1.5 is involved in nitrate allocation to roots and the consequent tolerance to several stresses, in a mechanism probably shared with NRT1.8.

Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis.

Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.

Galactosylation of rhamnogalacturonan-II for cell wall pectin biosynthesis is critical for root apoplastic iron reallocation in Arabidopsis.

Apoplastic iron (Fe) in roots represents an essential Fe storage pool. Reallocation of apoplastic Fe is of great importance to plants experiencing Fe deprivation, but how this reallocation process is regulated remains elusive, likely because of the highly complex cell wall structure and the limited knowledge about cell wall biosynthesis and modulation. Here, we present genetic and biochemical evidence to demonstrate that the Cdi-mediated galactosylation of rhamnogalacturonan-II (RG-II) is required for apoplastic Fe reallocation. Cdi is expressed in roots and up-regulated in response to Fe deficiency. It encodes a putative glycosyltransferase localized to the Golgi apparatus. Biochemical and mass spectrometry assays showed that Cdi catalyzes the transfer of GDP-L-galactose to the terminus of side chain A on RG-II. Disruption of Cdi essentially decreased RG-II dimerization and hence disrupted cell wall formation, as well as the reallocation of apoplastic Fe from roots to shoots. Further transcriptomic, Fourier transform infrared spectroscopy, and Fe desorption kinetic analyses coincidently suggested that Cdi mediates apoplastic Fe reallocation through extensive modulation of cell wall components and consequently the Fe adsorption capacity of the cell wall. Our study provides direct evidence demonstrating a link between cell wall biosynthesis and apoplastic Fe reallocation, thus indicating that the structure of the cell wall is important for efficient usage of the cell wall Fe pool.

A defensin-like protein drives cadmium efflux and allocation in rice.

Pollution by heavy metals limits the area of land available for cultivation of food crops. A potential solution to this problem might lie in the molecular breeding of food crops for phytoremediation that accumulate toxic metals in straw while producing safe and nutritious grains. Here, we identify a rice quantitative trait locus we name cadmium (Cd) accumulation in leaf 1 (CAL1), which encodes a defensin-like protein. CAL1 is expressed preferentially in root exodermis and xylem parenchyma cells. We provide evidence that CAL1 acts by chelating Cd in the cytosol and facilitating Cd secretion to extracellular spaces, hence lowering cytosolic Cd concentration while driving long-distance Cd transport via xylem vessels. CAL1 does not appear to affect Cd accumulation in rice grains or the accumulation of other essential metals, thus providing an efficient molecular tool to breed dual-function rice varieties that produce safe grains while remediating paddy soils.

Tonoplast-localized nitrate uptake transporters involved in vacuolar nitrate efflux and reallocation in Arabidopsis.

A great proportion of nitrate taken up by plants is stored in vacuoles. Vacuolar nitrate accumulation and release is of great importance to nitrate reallocation and efficient utilization. However, how plants mediate nitrate efflux from vacuoles to cytoplasm is largely unknown. The current study identified NPF5.11, NPF5.12 and NPF5.16 as vacuolar nitrate efflux transporters in Arabidopsis. Histochemical analysis showed that NPF5.11, NPF5.12 and NPF5.16 were expressed preferentially in root pericycle cells and xylem parenchyma cells, and further analysis showed that these proteins were tonoplast-localized. Functional characterization using cRNA-injected Xenopus laevis oocytes showed that NPF5.11, NPF5.12 and NPF5.16 were low-affinity, pH-dependent nitrate uptake transporters. In npf5.11 npf5.12 npf5.16 triple mutant lines, more root-fed 15NO3- was translocated to shoots compared to the wild type control. In the NPF5.12 overexpression lines, proportionally less nitrate was maintained in roots. These data together suggested that NPF5.11, NPF5.12 and NPF5.16 might function to uptake nitrate from vacuoles into cytosol, thus serving as important players to modulate nitrate allocation between roots and shoots.

Arabidopsis NRT1.5 Mediates the Suppression of Nitrate Starvation-Induced Leaf Senescence by Modulating Foliar Potassium Level.

Nitrogen deficiency induces leaf senescence. However, whether or how nitrate might affect this process remains to be investigated. Here, we report an interesting finding that nitrate-instead of nitrogen-starvation induced early leaf senescence in nrt1.5 mutant, and present genetic and physiological data demonstrating that nitrate starvation-induced leaf senescence is suppressed by NRT1.5. NRT1.5 suppresses the senescence process dependent on its function from roots, but not the nitrate transport function. Further analyses using nrt1.5 single and nia1 nia2 nrt1.5-4 triple mutant showed a negative correlation between nitrate concentration and senescence rate in leaves. Moreover, when exposed to nitrate starvation, foliar potassium level decreased in nrt1.5, but adding potassium could essentially restore the early leaf senescence phenotype of nrt1.5 plants. Nitrate starvation also downregulated the expression of HAK5, RAP2.11, and ANN1 in nrt1.5 roots, and appeared to alter potassium level in xylem sap from nrt1.5. These data suggest that NRT1.5 likely perceives nitrate starvation-derived signals to prevent leaf senescence by facilitating foliar potassium accumulation.