Characterization of genes involved in micronutrients and toxic metals detoxification in Brassica napus by genome-wide cDNA library screening.

Stresses caused by deficiency/excess of mineral nutrients or of pollution of toxic metals have already become a primary factor in limiting crop production worldwide. Genes involved in minerals and toxic metals accumulation/tolerance could be potential candidates for improving crop plants with enhanced nutritional efficiency and environmental adaptability. In this study, we first generated a high-quality yeast expression cDNA library of Brassica napus (Westar), and 46 genes mediating excess micronutrients and toxic metals detoxification were screened using the yeast genetic complementation system, including 11, 5, 6, 14, 6, and 5 genes involved in cadmium (Cd), zinc (Zn), iron (Fe), manganese (Mn), boron (B), and copper (Cu) tolerance, respectively. Characterization of genes mediating excess ions stress resistance in this study is beneficial for us to further understand ions homeostasis in B. napus.

Cloning and Functional Characterization of SpZIP2.

Zinc (Zn)-regulated and iron (Fe)-regulated transporter-like proteins (ZIP) are key players involved in the accumulation of cadmium (Cd) and Zn in plants. Sedum plumbizincicola X.H. Guo et S.B. Zhou ex L.H. Wu (S. plumbizincicola) is a Crassulaceae Cd/Zn hyperaccumulator found in China, but the role of ZIPs in S. plumbizincicola remains largely unexplored. Here, we identified 12 members of ZIP family genes by transcriptome analysis in S. plumbizincicola and cloned the SpZIP2 gene with functional analysis. The expression of SpZIP2 in roots was higher than that in the shoots, and Cd stress significantly decreased its expression in the roots but increased its expression in leaves. Protein sequence characteristics and structural analysis showed that the content of alanine and leucine residues in the SpZIP2 sequence was higher than other residues, and several serine, threonine and tyrosine sites can be phosphorylated. Transmembrane domain analysis showed that SpZIP2 has the classic eight transmembrane regions. The evolutionary analysis found that SpZIP2 is closely related to OsZIP2, followed by AtZIP11, OsZIP1 and AtZIP2. Sequence alignment showed that most of the conserved sequences among these members were located in the transmembrane regions. A further metal sensitivity assay using yeast mutant Δyap1 showed that the expression of SpZIP2 increased the sensitivity of the transformants to Cd but failed to change the resistance to Zn. The subsequent ion content determination showed that the expression of SpZIP2 increased the accumulation of Cd in yeast. Subcellular localization showed that SpZIP2 was localized to membrane systems, including the plasma membrane and endoplasmic reticulum. The above results indicate that ZIP member SpZIP2 participates in the uptake and accumulation of Cd into cells and might contribute to Cd hyperaccumulation in S. plumbizincicola.

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.

Comparative understanding of metal hyperaccumulation in plants: a mini-review.

Hyperaccumulator plants are ideal models for investigating the regulatory mechanisms of plant metal homeostasis and environmental adaptation due to their notable traits of metal accumulation and tolerance. These traits may benefit either the biofortification of essential mineral nutrients or the phytoremediation of nonessential toxic metals. A common mechanism by which elevated expression of key genes involved in metal transport or chelation contributes to hyperaccumulation and hypertolerance was proposed mainly from studies examining two Brassicaceae hyperaccumulators, namely Arabidopsis halleri and Noccaea caerulescens (formerly Thlaspi caerulescens). Meanwhile, recent findings regarding systems outside the Brassicaceae hyperaccumulators indicated that functional enhancement of key genes might represent a strategy evolved by hyperaccumulator plants. This review provides a brief outline of metal hyperaccumulation in plants and highlights commonalities and differences among various hyperaccumulators.

OsProT1 and OsProT3 Function to Mediate Proline- and γ-aminobutyric acid-specific Transport in Yeast and are Differentially Expressed in Rice (Oryza sativa L.).

BACKGROUND: Proline (Pro) and γ-aminobutyric acid (GABA) play important roles in plant development and stress tolerance. However, the molecular components responsible for the transport of these molecules in rice remain largely unknown. RESULTS: Here we identified OsProT1 and OsProT3 as functional transporters for Pro and GABA. Transient expression of eGFP-OsProTs in plant protoplasts revealed that both OsProT1 and OsProT3 are localized to the plasma membrane. Ectopic expression in a yeast mutant demonstrated that both OsProT1 and OsProT3 specifically mediate transport of Pro and GABA with affinity for Pro in the low affinity range. qRT-PCR analyses suggested that OsProT1 was preferentially expressed in leaf sheathes during vegetative growth, while OsProT3 exhibited relatively high expression levels in several tissues, including nodes, panicles and roots. Interestingly, both OsProT1 and OsProT3 were induced by cadmium stress in rice shoots. CONCLUSIONS: Our results suggested that plasma membrane-localized OsProT1 and OsProT3 efficiently transport Pro and GABA when ectopically expressed in yeast and appear to be involved in various physiological processes, including adaption to cadmium stress in rice plants.

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.

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.

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.

Vacuolar sequestration capacity and long-distance metal transport in plants.

The vacuole is a pivotal organelle functioning in storage of metabolites, mineral nutrients, and toxicants in higher plants. Accumulating evidence indicates that in addition to its storage role, the vacuole contributes essentially to long-distance transport of metals, through the modulation of Vacuolar sequestration capacity (VSC) which is shown to be primarily controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them. Plants adapt to their environments by dynamic regulation of VSC for specific metals and hence targeting metals to specific tissues. Study of VSC provides not only a new angle to understand the long-distance root-to-shoot transport of minerals in plants, but also an efficient way to biofortify essential mineral nutrients or to phytoremediate non-essential metal pollution. The current review will focus on the most recent proceedings on the interaction mechanisms between VSC regulation and long-distance metal transport.

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.