Enhancing tonoplast Cd/H antiport activity increases Cd,Zn, and Mn tolerance,and impacts root/shoot Cd partitioning in <Emphasis Type="Italic">Nicotiana tabacum </Emphasis>L.

Sequestration mechanisms that prevent high concentrations of free metal ions from persisting in metabolically active compartments of cells are thought to be central in tolerance of plants to high levels of divalent cation metals. Expression of AtCAX2 or AtCAX4, which encode divalent cation/proton antiporters, in Nicotiana tabacum cv. KY14 results in enhanced Cd- and Zn-selective transport into root tonoplast vesicles, and enhanced Cd accumulation in roots of plants exposed to moderate, 0.02 μM Cd in solution culture (Korenkov et al. in Planta 225:403–411, 2007). Here we investigated effects of expressing AtCAX2 and AtCAX4 in the same lines on tolerance to growth with near-incipient toxicity levels of Cd, Zn and Mn. Less growth inhibition (higher tolerance) to all three metals was observed in 35S::AtCAX2 and FS3::AtCAX4 expressing plants. Consistent with the tolerance observed for Cd was the finding that while root tonoplast vesicle proton pump activities of control and FS3AtCAX4 expressing plants grown in 3 μM Cd were similarly reduced, and vesicle proton leak was enhanced, root tonoplast vesicle antiporter activity of these plants remained elevated above that in controls. We suggest that CAX antiporters, unlike tonoplast proton pump and membrane integrity, are not negatively impacted by high Cd, and that supplementation of tonoplast with AtCAX compensates somewhat for reduced tonoplast proton pump and proton leak, and thereby results in sufficient vacuolar Cd sequestration to provide higher tolerance. Results are consistent with the view that CAX2 and CAX4 antiporters of tonoplast play a role in tolerance to high, toxic levels of Cd, Zn, and Mn in tobacco.     

植物液泡膜阳离子/H+反向转运蛋白结构和功能研究进展

阳离子转运蛋白在调节细胞质阳离子浓度过程中发挥关键作用。液泡是一个储存多种离子的重要细胞器,阳离子 (Ca2+)/H+反向转运蛋白CAXs定位在液泡膜上,主要参与Ca2+向液泡的转运,也参与其他阳离子的转运。近年来,植物中分离鉴定了多个CAX基因,植物CAXs主要有4个功能域：NRR通过自抑制机制调节Ca2+转运活性,CaD和C功能域分别赋予CAXs的Ca2+和Mn2+专一性转运活性,D功能域可调节细胞质pH。拟南芥AtCAXs参与植物的生长发育和胁迫适应过程,AtCAX3主要在盐胁迫下转运Ca2+,At     

Transgenic tobacco co-expressing flavodoxin and betaine aldehyde dehydrogenase confers cadmium tolerance through boosting antioxidant capacity

Excessive heavy metal (HM) levels in soil have become a source of concern due to their adverse effects on human health and the agriculture industry. Soil contamination by HMs leads to an accumulation of reactive oxygen species (ROSs) within the plant cell and disruption of photosynthesis-related proteins. The response of tobacco lines overexpressing flavodoxin (Fld) and betaine aldehyde dehydrogenase (BADH) to cadmium (Cd) toxicity was investigated in this study. PCR results demonstrated the expected amplicon length of each gene in the transgenic lines. Absolute qRT-PCR demonstrates a single copy of T-DNA integration into each transgenic line. Relative qRT-PCR confirmed overexpression of Fld and BADH in transgenic lines. The maximum quantum yield of photosystem II (Fv/Fm) was measured under Cd toxicity stress and revealed that transgenic lines had a higher Fv/Fm than wild-type (WT) plants. Accumulation of proline, glycine betaine (GB), and higher activity of antioxidant enzymes alongside lower levels of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) was indicative of a robust antioxidant system in transgenic plants. Therefore, performing a loop in reducing the ROS produced in the photosynthesis electron transport chain and stimulating the ROS scavenger enzyme activity improved the plant tolerance to Cd stress.

     

Metallothioneins BcMT1 and BcMT2 from Brassica campestris enhance tolerance to cadmium and copper and decrease production of reactive oxygen species in Arabidopsis thaliana

Aims

Metallothioneins are cysteine-rich, metal-binding proteins, but their exact functions are not fully understood. In this study, we isolated two metallothionein genes, BcMT1 and BcMT2 from Brassica campestris to increase our understanding of metal tolerance mechanisms in Brassica plants.

Methods

Semi-quantitative RT-PCR was used to analyze expression of the two BcMTs genes. BcMT1 and BcMT2 were ectopically expressed in Arabidopsis thaliana. Quantitative real-time RT-PCR and GUS-staining method were used to select transgenic Arabidopsis plants. Cd and Cu concentrations were analyzed by flame atomic absorption spectrometry. Histochemical detection of H₂O₂ and O₂ ^?? were conducted by 3,3-diaminobenzidine and nitroblue tetrazoliu-staining methods.

Results

BcMT1 is expressed predominantly in roots, whereas BcMT2 is expressed mainly in leaves of B. campestris. Expression of BcMT1 was induced by both Cd and Cu, but expression of BcMT2 was enhanced only by Cd. Ectopic expression of BcMT1 and BcMT2 in Arabidopsis thaliana enhanced the tolerance to Cd and Cu and increased the Cu concentration in the shoots of the transgenic plants. Transgenic Arabidopsis accumulated less reactive oxygen species (ROS) than wild-type plants.

Conclusions

BcMT1 and BcMT2 increased Cd and Cu tolerance in transgenic Arabidopsis, and decreased production of Cd- and Cu-induced ROS, thereby protecting plants from oxidative damage.     

Effects of root and foliar applications of exogenous NO on alleviating cadmium toxicity in lettuce seedlings

The effects of sodium nitroprusside (SNP, a donor of NO) on cadmium (Cd) toxicity in lettuce seedlings were studied. SNP was added into hydroponic systems or sprayed directly on the leaves of plants grown with and without Cd. Excess supply of Cd (100 μM) caused growth inhibition, dramatically increased Cd accumulation in both leaves and roots, and inhibited the absorption of Ca, Mg, Fe and Cu. Excess Cd also decreased activities of superoxide dismutase peroxidase and catalase in leaves and roots, and increased the accumulation of superoxide anion (O ₂ ^·? ), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). Root or foliar applications of exogenous NO alleviated Cd-induced growth suppression, especially root application of 250 μM SNP and foliar addition of 500 μM SNP. Addition of SNP promoted the chlorophyll synthesis suggesting that the photosynthesis was up-regulated. Exogenous NO increased Cd-decreased activities of antioxidant enzymes and markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation. Moreover, the absorption of Ca, Mg, Fe and Cu was increased, indicating that exogenous NO stimulated H⁺-ATPase activity to promote sequestration or uptake of ions. In addition, exogenous NO also inhibited Cd transfer from roots to shoots, which may indicate that Cd retention in roots induced by NO plays a significant role in Cd tolerance in lettuce seedlings. These data suggest that under Cd stress, exogenous NO improves photosynthesis by increasing chlorophyll synthesis, protects lettuce seedlings against oxidative damage by scavenging ROS, helps to maintain the uptake of nutrient elements, and inhibits Cd transferred to shoots effectively.     

Root-selective expression of AtCAX4 and AtCAX2 results in reduced lamina cadmium in field-grown Nicotiana tabacum L.

To assess the impact of enhanced root vacuole cadmium (Cd) sequestration on leaf Cd accumulation under a low Cd dose, as generally occurs in agriculture, leaf Cd accumulation was examined in field-grown tobacco plants expressing genes encoding the high-capacity-Cd, tonoplast-localized, divalent cation/H antiporters AtCAX4 and AtCAX2 (AtCAX, Arabidopsis cation exchanger). It has been shown previously that root tonoplast vesicles isolated from plants expressing these genes, directed by root-selective promoters, show enhanced Cd transport activity, and young plants show enhanced root Cd accumulation when grown in solution culture containing 0.02 µ m Cd, a moderate Cd dose. In this article, we present results which show that the lower leaves of mature plants expressing AtCAX2 or AtCAX4 , under the control of two different root-selective promoters, accumulate 15%–25% less lamina Cd than control plants when grown in the field (3 years, three different collection methods). Reciprocal grafting experiments of AtCAX2 shoots onto control roots (and vice versa), grown in solution culture with 0.005 µ m Cd, indicated that the root controls Cd translocation and accumulation in the shoot in control and AtCAX2 and AtCAX4 tobacco plants exposed to low Cd concentration. The results are consistent with a model in which supplementation of Cd/H antiporter activity in root cell tonoplasts enhances root Cd sequestration, resulting in decreased translocation of Cd to the shoot of field-grown plants. These results suggest that human Cd intake from food and tobacco use could be reduced via the enhancement of root vacuolar sequestration of this pollutant.     

CAX1 suppresses Cd‐induced generation of reactive oxygen species in Arabidopsis halleri

The molecular analysis of metal hyperaccumulation in species such as Arabidopsis halleri offers the chance to gain insights into metal homeostasis and into the evolution of adaptation to extreme habitats. A prerequisite of metal hyperaccumulation is metal hypertolerance. Genetic analysis of a backcross population derived from Arabidopsis lyrata × A. halleri crosses revealed three quantitative trait loci for Cd hypertolerance. A candidate gene for Cdtol2 is AhCAX1, encoding a vacuolar Ca²⁺/H⁺ antiporter. We developed a method for the transformation of vegetatively propagated A. halleri plants and generated AhCAX1‐silenced lines. Upon Cd²⁺ exposure, several‐fold higher accumulation of reactive oxygen species (ROS) was detectable in roots of AhCAX1‐silenced plants. In accordance with the dependence of Cdtol2 on external Ca²⁺ concentration, this phenotype was exclusively observed in low Ca²⁺ conditions. The effects of external Ca²⁺ on Cd accumulation cannot explain the phenotype as they were not influenced by the genotype. Our data strongly support the hypothesis that higher expression of CAX1 in A. halleri relative to other Arabidopsis species represents a Cd hypertolerance factor. We propose a function of AhCAX1 in preventing a positive feedback loop of Cd‐elicited ROS production triggering further Ca²⁺‐dependent ROS accumulation.     

Regulation Effects of Na+/H+ Antiporter (NHX1) on Nicotiana tabacum Stressed with Metals of Different Valences

Na⁺/H⁺ antiporter (NHX1) was reported to be induced by NaCl and conferred salt tolerance to transgenic plants overexpressing NHX1 gene. Cadmium (Cd) resistance and Cd accumulation in tobacco were also closely correlated with the expression of NHX1 gene. However, it was still unclear the regulation effects of NHX1 gene on plants under other metal stresses with different valence states. The present study focuses on the resistance of NHX1-overexpressing tobacco plants to Li (I), Pb (II), Cr (III), or Cr (VI) stress and elucidates the potential mechanisms involved in this process. SseNHX1 gene obtained by shuffling using SsNHX1 from Suaeda salsa and SeNHX1 from Salicornia europaea as sequence templates in our previous work, conferred higher NaCl and Cd tolerance to tobacco than SsNHX1 and SeNHX1. The overexpression of SseNHX1 gene in tobacco was used to explore the relationship between metal stresses that differed in valence states and NHX1 gene expression in plants. Our results showed that the content of metal ions in vacuole and the translocation factor from root to leaf were gradually reduced in NHX1-overexpressing tobacco under different metal stresses with the metal valence states increased from monovalent to hexavalent. Comparing with non-transgenic tobacco plants, NHX1 transgenic plants showed higher photosynthesis and transpiration rates when exposed to Li (I), Pb (II), or Cr (III) stress. Decreased ROS and MDA content and increased antioxidant enzyme activities were also found in transgenic tobacco roots than in non-transgenic roots under Li (I), Pb (II), or Cr (III) exposure.

     

Increased calcium in carrots by expression of an Arabidopsis H+/Ca2+ transporter

We demonstrate that carrots expressing the Arabidopsis H⁺/Ca²⁺ transporter CAX1 (Cation Exchanger 1) contained up to 50% more calcium (Ca) than plants transformed with control vectors. The CAX1-expressing carrots were fertile, and robust plant growth was seen in the majority of the transgenic plants. CAX1-expressing carrots were crossed to a commercial carrot variety to confirm that the increased Ca accumulation was mediated by CAX1-expression, and the increased Ca content was clearly correlated with the transgene. This study suggests that modulation of ion transporters could be an important means of increasing the Ca content of agriculturally important crops. To our knowledge, this study represents the first attempts to use biotechnology to increase the Ca content of an agriculturally important crop.     

Expression of arabidopsis CAX2 in tobacco. Altered metal accumulation and increased manganese tolerance

Metal transport from the cytosol to the vacuole is thought to be an important component of ion tolerance and of a plant's potential for use in phytoremediation. The Arabidopsis antiporter CAX2 (calcium exchanger 2) may be a key mediator of this process. CAX2 expression in yeast suppressed both Ca(2+) and Mn(2+) growth defects. A peptide-specific antibody to the antiporter reacted with a 39-kD protein from plant vacuolar membranes. Tobacco (Nicotiana tabacum) plants expressing CAX2 accumulated more Ca(2+), Cd(2+), and Mn(2+) and were more tolerant to elevated Mn(2+) levels. Expression of CAX2 in tobacco increased Cd(2+) and Mn(2+) transport in isolated root tonoplast vesicles. These results suggest that CAX2 has a broad substrate range and modulation of this transporter may be an important component of future strategies to improve plant ion tolerance.     

Inhibition of DNA demethylation enhances plant tolerance to cadmium toxicity by improving iron nutrition

Although the alteration of DNA methylation due to abiotic stresses, such as exposure to the toxic metal cadmium (Cd), has been often observed in plants, little is known about whether such epigenetic changes are linked to the ability of plants to adapt to stress. Herein, we report a close linkage between DNA methylation and the adaptational responses in Arabidopsis plants under Cd stress. Exposure to Cd significantly inhibited the expression of three DNA demethylase genes ROS1/DML2/DML3 (RDD) and elevated DNA methylation at the genome-wide level in Col-0 roots. Furthermore, the profile of DNA methylation in Cd-exposed Col-0 roots was similar to that in the roots of rdd triple mutants, which lack RDD, indicating that Cd-induced DNA methylation is associated with the inhibition of RDD. Interestingly, the elevation in DNA methylation in rdd conferred a higher tolerance against Cd stress and improved cellular Fe nutrition in the root tissues. In addition, lowering the Fe supply abolished improved Cd tolerance due to the lack of RDD in rdd. Together, these data suggest that the inhibition of RDD-mediated DNA demethylation in the roots by Cd would in turn enhance plant tolerance to Cd stress by improving Fe nutrition through a feedback mechanism.     

Expression of an Arabidopsis Ca/H antiporter CAX1 variant in petunia enhances cadmium tolerance and accumulation

Phytoremediation is a cost-effective and minimally invasive technology to cleanse soils contaminated with heavy metals. However, few plant species are suitable for phytoremediation of metals such as cadmium (Cd). Genetic engineering offers a powerful tool to generate plants that can hyperaccumulate Cd. An Arabidopsis CAX1 mutant (CAXcd), which confers enhanced Cd transport in yeast, was ectopically expressed in petunia to evaluate whether the CAXcd expression would enhance Cd tolerance and accumulation in planta. The CAXcd-expressing petunia plants showed significantly greater Cd tolerance and accumulation than the controls. After being treated with either 50 or 100 μM CdCl₂ for 6 weeks, the CAXcd-expressing plants showed more vigorous growth compared with controls, and the transgenic plants accumulated significantly more Cd (up to 2.5-fold) than controls. Moreover, the accumulation of Cd did not affect the development and morphology of the CAXcd-expressing petunia plants until the flowering and ultimately the maturing of seeds. Therefore, petunia has the potential to serve as a model species for developing herbaceous, ornamental plants for phytoremediation.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.     

Enhanced Cd<Superscript>2+</Superscript>-selective root-tonoplast-transport in tobaccos expressing Arabidopsis cation exchangers

Several Arabidopsis CAtion eXchangers (CAXs) encode tonoplast-localized transporters that appear to be major contributors to vacuolar accumulation/sequestration of cadmium (Cd(2+)), an undesirable pollutant ion that occurs in man largely as a result of dietary consumption of aerial tissues of food plants. But, ion-selectivity of individual CAX transporter types remains largely unknown. Here, we transformed Nicotiana tabacum with several CAX genes driven by the Cauliflower Mosaic Virus (CaMV) 35S promoter and monitored divalent cation transport in root-tonoplast vesicles from these plants in order to select particular CAX genes directing high Cd(2+) antiporter activity in root tonoplast. Comparison of seven different CAX genes indicated that all transported Cd(2+), Ca(2+), Zn(2+), and Mn(2+) to varying degrees, but that CAX4 and CAX2 had high Cd(2+) transport and selectivity in tonoplast vesicles. CAX4 driven by the CaMV 35S and FS3 [figwort mosaic virus (FMV)] promoters increased the magnitude and initial rate of Cd(2+)/H(+) exchange in root-tonoplast vesicles. Ion selectivity of transport in root-tonoplast vesicles isolated from FS3::CAX4-expressing plant lines having a range of gene expression was Cd(2+)>Zn(2+)>Ca(2+)>Mn(2+) and the ratios of maximal Cd(2+) (and Zn(2+)) versus maximal Ca(2+) and Mn(2+) transport were correlated with the levels of CAX4 expression. Root Cd accumulation in high CAX4 and CAX2 expressing lines was increased in seedlings grown with 0.02 muM Cd. These observations are consistent with a model in which expression of an Arabidopsis-gene-encoded, Cd(2+)-efficient antiporter in host plant roots results in greater root vacuole Cd(2+) transport activity, increased root Cd accumulation, and a shift in overall root tonoplast ion transport selectivity towards higher Cd(2+) selectivity. Results support a model in which certain CAX antiporters are somewhat more selective for particular divalent cations.