The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply

Zinc ions are required to maintain the biological activity of numerous proteins. However, when mislocalized or accumulated in excess, Zn(2+) ions are toxic because of adventitious binding to proteins and displacement of other metal ions, among them Fe(2+), from their binding sites. Heterologous expression of a previously uncharacterized Arabidopsis thaliana metal tolerance protein, MTP3, in the zrc1 cot1 mutant of budding yeast restores tolerance to, and cellular accumulation of, zinc and cobalt. An MTP3-GFP fusion protein localizes to the vacuolar membrane when expressed in Arabidopsis. Ectopic over-expression of MTP3 increases Zn accumulation in both roots and rosette leaves of A. thaliana, and enhances Zn tolerance. Exposure of wild-type plants to high but non-toxic concentrations of Zn or Co, or Fe deficiency, strongly induce MTP3 expression specifically in epidermal and cortex cells of the root hair zone. Silencing of MTP3 by RNA interference causes Zn hypersensitivity and enhances Zn accumulation in above-ground organs of soil-grown plants and of seedlings exposed to excess Zn or to Fe deficiency. Our data indicate that, in wild-type A. thaliana, the AtMTP3 protein contributes to basic cellular Zn tolerance and controls Zn partitioning, particularly under conditions of high rates of Zn influx into the root symplasm.     

Arabidopsis thaliana MTP1 is a Zn transporter in the vacuolar membrane which mediates Zn detoxification and drives leaf Zn accumulation

The Arabidopsis thaliana metal tolerance protein 1 (MTP1) of the cation diffusion facilitator family of membrane transport proteins can mediate the detoxification of Zn in Arabidopsis and yeast. Xenopus laevis oocytes expressing AtMTP1 accumulate more Zn than oocytes expressing the AtMTP1(D94A) mutant or water-injected oocytes. An AtMTP1-GFP fusion protein localizes to the vacuolar membrane in root and leaf cells. The analysis of Arabidopsis transformed with a promoter-GUS construct suggests that AtMTP1 is not produced throughout the plant, but primarily in the subpopulation of dividing, differentiating and expanding cells. RNA interference-mediated silencing of AtMTP1 causes Zn hypersensitivity and a reduction in Zn concentrations in vegetative plant tissues.     

Nicotiana tabacum Tsip1-interacting ferredoxin 1 affects biotic and abiotic stress resistance

Tsip1, a Zn finger protein that was isolated as a direct interactor with tobacco stress-induced 1 (Tsi1), plays an important role in both biotic and abiotic stress signaling. To further understand Tsip1 function, we searched for more Tsip1-interacting proteins by yeast two-hybrid screening using a tobacco cDNA library. Screening identified a new Tsip1-interacting protein, Nicotiana tabacum Tsip1-interacting ferredoxin 1 (NtTfd1), and binding specificity was confirmed both in vitro and in vivo. The four repeats of a cysteine-rich motif (CXXCXGXG) of Tsip1 proved important for binding to NtTfd1. Virus-induced gene silencing of NtTfd1, Tsip1, and NtTfd1/Tsip1 rendered plants more susceptible to salinity stress compared with TRV2 control plants. NtTfd1- and Tsip1-silenced tobacco plants were more susceptible to infection by Cucumber mosaic virus compared with control plants. These results suggest that NtTfd1 might be involved in the regulation of biotic and abiotic stresses in chloroplasts by interaction with Tsip1.     

A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance

The metal tolerance of metal hyper-accumulating plants is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the Zn hyper-accumulating plant Arabidopsis halleri ssp. halleri, we carried out a functional screening of an A. halleri cDNA library in the yeast Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. The screening revealed four A. halleri defensin genes (AhPDFs), which induced Zn but not cadmium (Cd) tolerance in yeast. The expression of AhPDF1.1 under the control of the 35S promoter in A. thaliana made the transgenic plants more tolerant to Zn than wild-type plants, but did not change the tolerance to Cd, copper (Cu), cobalt (Co), iron (Fe) or sodium (Na). Thus, AhPDF1.1 is able to confer Zn tolerance both to yeast and plants. In A. halleri, defensins are constitutively accumulated at a higher level in shoots than in A. thaliana. A. halleri defensin pools are Zn-responsive, both at the mRNA and protein levels. In A. thaliana, some but not all defensin genes are induced by ZnCl2 treatment, and these genes are not induced by NaCl treatment. Defensins, found in a very large number of organisms, are known to be involved in the innate immune system but have never been found to play any role in metal physiology. Our results support the proposition that defensins could be involved in Zn tolerance in A. halleri, and that a role for plant defensins in metal physiology should be considered.     

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.     

烟草DREBP转录因子结合DRE元件的关键氨基酸

从烟草品种本塞母氏中分离出2条DREB类转录因子基因,分别命名为NbDREB1和 NbDREB2.根据测序结果推导出的氨基酸序列分析显示,NbDREB1和NbDREB2都具有典型的AP2/EREBP转录因子家族EREBP亚族A类特征.酵母单杂交结果显示,它们都不具有激活功能.连接pGADT7反式激活载体形成融合基因表达结果显示,NbDREB1能与DRE顺式作用元件结合,NbDREB2则不能.比较NbDREB1和NbDREB2的AP2区,发现两者的第2和49位氨基酸残基不同.对NbDREB2的第2位氨基酸残基N点突变为Y,NbDREB2也显示出与DRE顺式元件结合的活性,表明烟草DREB转录因子的AP2区第2位氨基酸残基Y是识别及结合DRE顺式作用元件必需的氨基酸残基.     

The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae

To avoid metal toxicity, organisms have evolved mechanisms including efflux of metal ions from cells and sequestration into internal cellular compartments. Members of the ubiquitous cation diffusion facilitator (CDF) family are known to play an important role in these processes. Overexpression of the plant CDF family member metal tolerance protein 1 (MTP1) from the Ni/Zn hyperaccumulator Thlaspi goesingense (TgMTP1), in the Saccharomyces cerevisiaeDelta zinc resistance conferring (zrc)1Delta cobalt transporter (cot)1 double mutant, suppressed the Zn sensitivity of this strain. T. goesingense was found to contain several allelic variants of TgMTP1, all of which confer similar resistance to Zn in Deltazrc1Deltacot1. Similarly, MTP1 from various hyperaccumulator and non-accumulator species also confer similar resistance to Zn. Deltazrc1Deltacot1 lacks the ability to accumulate Zn in the vacuole and has lower accumulation of Zn after either long- or short-term Zn exposure. Expression of TgMTP1 in Deltazrc1Deltacot1 leads to further lowering of Zn accumulation and an increase in Zn efflux from the cells. Expression of TgMTP1 in a V-type ATPase-deficient S. cerevisiae strain also confers increased Zn resistance. In vivo and in vitro immunological staining of hemagglutinin (HA)-tagged TgMTP1::HA reveals the protein to be localized in both the S. cerevisiae vacuolar and plasma membranes. Taken together, these data are consistent with MTP1 functioning to enhance plasma membrane Zn efflux, acting to confer Zn resistance independent of the vacuole in S. cerevisiae. Transient expression in Arabidopsis thaliana protoplasts also reveals that TgMTP1::green fluorescent protein (GFP) is localized at the plasma membrane, suggesting that TgMTP1 may also enhance Zn efflux in plants.     

Combined expression of the Arabidopsis metallothionein MT2b and the heavy metal transporting ATPase HMA4 enhances cadmium tolerance and the root to shoot translocation of cadmium and zinc in tobacco

We expressed the AtMt2b and AtHMA4 genes under the 35S cauliflower mosaic virus promoter simultaneously in Nicotiana tabacum (SR1), using leaf disc transformation. A single AtMT2b tobacco T2 line was used for re-transformation with AtHMA4 to obtain the double transformant. Cadmium (Cd) and zinc (Zn) tolerance, uptake and translocation were measured in the double transformant, and compared to untransformed (‘wild type’) tobacco and single gene transformants. The double transformant exhibited enhanced Cd-tolerance, enhanced Cd and Zn root to shoot transport, but unaltered Zn tolerance and Cd and Zn uptake, compared with wild type.The single transformant lines did not show significant phenotypes. Our results suggest that the phenotypes of the double transformant are due to synergistic interaction between the transgenes. Except for Cd tolerance, the phenotypes were moderate for Cd and Zn root to shoot transport, which may be due to use of the 35S promotor, resulting in incorrect tissue-specificity.     

The genetic basis of zinc tolerance in the metallophyte Arabidopsis halleri ssp. halleri (Brassicaceae): an analysis of quantitative trait loci

The species Arabidopsis halleri, an emerging model for the study of heavy metal tolerance and accumulation in plants, has evolved a high level of constitutive zinc tolerance. Mapping of quantitative trait loci (QTL) was used to investigate the genetic architecture of zinc tolerance in this species. A first-generation backcross progeny of A. halleri ssp. halleri from a highly contaminated industrial site and its nontolerant relative A. lyrata ssp. petraea was produced and used for QTL mapping of zinc tolerance. A genetic map covering most of the A. halleri genome was constructed using 85 markers. Among these markers, 65 were anchored in A. thaliana and revealed high synteny with other Arabidopsis genomes. Three QTL of comparable magnitude on three different linkage groups were identified. At all QTL positions zinc tolerance was enhanced by A. halleri alleles, indicating directional selection for higher zinc tolerance in this species. The two-LOD support intervals associated with these QTL cover 24, 4, and 13 cM. The importance of each of these three regions is emphasized by their colocalization with HMA4, MTP1-A, and MTP1-B, respectively, three genes well known to be involved in metal homeostasis and tolerance in plants.     

Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.     

Phylogenetic fragrance patterns in Nicotiana sections Alatae and Suaveolentes

We analyzed floral volatiles from eight tobacco species (Nicotiana; Solanaceae) including newly discovered Brazilian taxa (Nicotiana mutabilis and "Rastroensis") in section Alatae. Eighty-four compounds were found, including mono- and sesquiterpenoids, nitrogenous compounds, benzenoid and aliphatic alcohols, aldehydes and esters. Floral scent from recent accessions of Nicotiana alata, Nicotiana bonariensis and Nicotiana langsdorffii differed from previously published data, suggesting intraspecific variation in scent composition at the level of biosynthetic class. Newly discovered taxa in Alatae, like their relatives, emit large amounts of 1,8-cineole and smaller amounts of monoterpenes on a nocturnal rhythm, constituting a chemical synapomorphy for this lineage. Fragrance data from three species of Nicotiana sect. Suaveolentes, the sister group of Alatae, (two Australian species: N. cavicola, N. ingulba; one African species: N. africana), were compared to previously reported data from their close relative, N. suaveolens. Like N. suaveolens, N. cavicola and N. ingulba emit fragrances dominated by benzenoids and phenylpropanoids, whereas the flowers of N. africana lacked a distinct floral scent and instead emitted only small amounts of an aliphatic methyl ester from foliage. Interestingly, this ester also is emitted from foliage of N. longiflora and N. plumbaginifolia (both in section Alatae s.l.), which share a common ancestor with N. africana. This result, combined with the synapomorphic pattern of 1,8 cineole emission in Alatae s.s., suggests that phylogenetic signal explains a major component of fragrance composition among tobacco species in sections Alatae and Suaveolentes. At the intraspecific level, interpopulational scent variation is widespread in sect. Alatae, and may reflect edaphic specialization, introgression, local pollinator shifts, genetic drift or artificial selection in cultivation. Further studies with genetically and geographically well-defined populations are needed to distinguish between these possibilities.     

Investigation of the basis for Ni tolerance conferred by the expression of TjZnt1 and TjZnt2 in yeast strains.

Introduction of ZIP family transporter gene homologues TjZnt1 and TjZnt2 (metal ion transporters) into yeast strains conferred increased Ni(II) tolerance in that species. The action of ZIP family transporter homologues, however, could not explain the Ni resistance of yeast strains transformed with TjZnt1 and TjZnt2. To elucidate the mechanism of Ni tolerance conferred by TjZnt1 and TjZnt2 in yeast strains, we made a series of investigations based upon three hypotheses, including (1) cellular Ni efflux, (2) exclusion of Ni due to competitive uptake of other metals, and (3) Ni binding to histidine-rich domains (chelation). The critical Ni tolerance level of TjZnt2 expressing yeast strains was 1.4mM, whereas, the TjZnt1 expressing yeast strains were tolerant of Ni concentrations as high as 2.0mM. The TjZnt1 expressing yeast strain had significantly lower Ni content and significantly higher Zn content than the control and TjZnt2 expressing yeast strain. Effects of the deletion of histidine-rich domain HRD1 or HRD2, or deletion of the region from HRD1 to HRD2, resulted in the same or slightly less Ni(II) tolerance in the TjZnt1 expressing yeast strain. These data indicate that Ni tolerance of the TjZnt2 expressing yeast strain is not correlated with binding to HRDs (Hypothesis 3). Ni tolerance of TjZnt1 expressing yeast strain was, however, partially correlated with Zn influx, which suppressed Ni influx, therefore Ni influx (Hypothesis 1) and competitive inhibition of Ni influx by other metals (Hypothesis 2), remain viable hypotheses which will be subject to further testing.     

Six active phage-type RNA polymerase genes in Nicotiana tabacum

In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identified six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.