Manganese specificity determinants in the Arabidopsis metal/H+ antiporter CAX2

In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H(+) antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca(2+) into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn(2+) transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn(2+) specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn(2+) or Ca(2+) transport, the second class showed a reduction in Ca(2+) transport and no measurable Mn(2+) transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca(2+) transport and a complete loss of Mn(2+) transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn(2+) specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H(+) antiporter, an important family of transporters found in a variety of organisms.     

Functional dependence on calcineurin by variants of the Saccharomyces cerevisiae vacuolar Ca2+/H+ exchanger Vcx1p

The Ca(2+)-dependent protein phosphatase calcineurin is an important regulator of ion transporters from many organisms, including the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger Vcx1p. In yeast and plants, cation/H(+) exchangers are important in shaping cytosolic Ca(2+) levels involved in signal transduction and providing tolerance to potentially toxic concentrations of cations such as Ca(2+), Mn(2+) and Cd(2+). Previous genetic evidence suggested Vcx1p is negatively regulated by calcineurin. By utilizing direct transport measurements into vacuolar membrane vesicles, we demonstrate that Vcx1p is a low-affinity Ca(2+) transporter and may also function in Cd(2+) transport, but cannot transport Mn(2+). Furthermore, direct Ca(2+) transport by Vcx1p is calcineurin sensitive. Using a yeast growth assay, a mutant allele of VCX1 (VCX1-S204A/L208P), termed VCX1-M1, was previously found to confer strong Mn(2+) tolerance. Here we demonstrate that this Mn(2+) tolerance is independent of the Ca(2+)/Mn(2+)-ATPase Pmr1p and results from Mn(2+)-specific vacuolar transport activity of Vcx1-M1p. This Mn(2+) transport by Vcx1-M1p is calcineurin dependent, although the localization of Vcx1-M1p to the vacuole appears to be calcineurin independent. Additionally, we demonstrate that mutation of L208P alone is enough to confer calcineurin-dependent Mn(2+) tolerance. This study demonstrates that calcineurin can positively regulate the transport of cations by VCX1-M1p.     

Managing the manganese: molecular mechanisms of manganese transport and homeostasis

Manganese (Mn) is an essential metal nutrient for plants. Recently, some of the genes responsible for transition metal transport in plants have been identified; however, only relatively recently have Mn2+ transport pathways begun to be identified at the molecular level. These include transporters responsible for Mn accumulation into the cell and release from various organelles, and for active sequestration into endomembrane compartments, particularly the vacuole and the endoplasmic reticulum. Several transporter gene families have been implicated in Mn2+ transport, including cation/H+ antiporters, natural resistance-associated macrophage protein (Nramp) transporters, zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPases. The identification of mutants with altered Mn phenotypes can allow the identification of novel components in Mn homeostasis. In addition, the characterization of Mn hyperaccumulator plants can increase our understanding of how plants can adapt to excess Mn, and ultimately allow the identification of genes that confer this stress tolerance. The identification of genes responsible for Mn2+ transport has substantially improved our understanding of plant Mn homeostasis.     

植物对重金属镉的耐受机制

镉离子（Cd^2＋）具有强植物毒性,抑制植物生长,甚至使植物死亡。由于长期的环境选择和适应进化,植物发展出耐受机制,可减轻或避免Cd^2＋的毒害。硫转运蛋白、硫还原相关酶类以及半胱氨酸、谷胱甘肽和植物螯合肽合成基因的表达受Cd^2＋调控。同时这些基因的过表达也能提高植物对Cd^2＋的耐性。植物抗氧化系统对Cd^2＋胁迫诱发的活性氧的清除作用,具转运Cd^2＋活性的质膜转运蛋白促进Cd^2＋经共质体途径向木质部运输、装载,而后随蒸腾流向地上部迁移,具转运Cd^2＋活性的液泡膜转运蛋白促进Cd^2＋进入液泡的隔离作用,都在植物对Cd^2＋的耐性中起作用。     

A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance

The Arabidopsis AtMTP family of genes encode proteins of the cation diffusion facilitator (CDF) family, with several members having roles in metal tolerances. Four of the 11 proteins in the family form a distinct cluster on a phylogenetic tree and are closely related to ShMTP8, a CDF identified in the tropical legume Stylosanthes hamata that is implicated in the transport of Mn(2+) into the vacuole as a tolerance mechanism. Of these four genes, AtMTP11 was the most highly expressed member of the Arabidopsis subgroup. When AtMTP11 was expressed in Saccharomyces cerevisiae, it conferred Mn(2+) tolerance and transported Mn(2+) by a proton-antiport mechanism. A mutant of Arabidopsis with a disrupted AtMTP11 gene (mtp11) was found to have increased sensitivity to Mn(2+) but not to Cu(2+) or Zn(2+). At a non-toxic but sufficient Mn(2+) supply (basal), the mutant accumulated more Mn(2+) than the wild type, but did not show any obvious deleterious effects on growth. When grown with Mn(2+) supplies that ranged from basal to toxic, the mutant accumulated Mn(2+) concentrations in shoots similar to those in wild-type plants, despite showing symptoms of Mn(2+) toxicity. AtMTP11 fused to green fluorescent protein co-localized with a reporter specific for pre-vacuolar compartments. These findings provide evidence for Mn(2+)-specific transport activity by AtMTP11, and implicate the pre-vacuolar compartments in both Mn(2+) tolerance and Mn(2+) homeostasis mechanisms of Arabidopsis.     

Emerging mechanisms for heavy metal transport in plants

Heavy metal ions such as Cu(2+), Zn(2+), Mn(2+), Fe(2+), Ni(2+) and Co(2+) are essential micronutrients for plant metabolism but when present in excess, these, and non-essential metals such as Cd(2+), Hg(2+) and Pb(2+), can become extremely toxic. Thus mechanisms must exist to satisfy the requirements of cellular metabolism but also to protect cells from toxic effects. The mechanisms deployed in the acquisition of essential heavy metal micronutrients have not been clearly defined although a number of genes have now been identified which encode potential transporters. This review concentrates on three classes of membrane transporters that have been implicated in the transport of heavy metals in a variety of organisms and could serve such a role in plants: the heavy metal (CPx-type) ATPases, the natural resistance-associated macrophage protein (Nramp) family and members of the cation diffusion facilitator (CDF) family. We aim to give an overview of the main features of these transporters in plants in terms of structure, function and regulation drawing on information from studies in a wide variety of organisms.     

Arsenic induced oxidative stress in plants

Arsenic is a highly toxic metalloid for all forms of life including plants. Arsenic enters in the plants through phosphate transporters as a phosphate analogue or through aquaglycoporins. Uptake of arsenic in plant tissues adversely affects the plant metabolism and leads to various physiological and structural disorders. Photosynthetic apparatus, cell division machinery, energy production, and redox status are the major section of plant system that are badly affected by As (V). Similarly As (III) can react with thiol (-SH) groups of enzymes and inhibits various metabolic processes. Arsenic is also known to induce oxidative stress directly by generating reactive oxygen species (ROS) during conversion of its valence forms or indirectly by inactivating antioxidant molecules through binding with their -SH groups. As-mediated oxidative stress causes cellular, molecular and physiological disturbances in various plant species. Activation of enzymatic antioxidants namely, superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), Glutathione s-transferase, glutathione peroxidase (GPX) as well as non antioxidant compounds such as, ascorbate, glutathione, carotenoids are reported to neutralize arsenic mediated oxidative stress. Understanding of biochemistry of arsenic toxicity would be beneficial for the development of arsenic tolerant crops and other economically important plants.     

Transporters: the molecular drivers of arsenic stress tolerance in plants

Arsenic (As), the toxic metalloid, is taken up by plant roots and transported to different parts of the plant through transporters of the essential elements due to the structural analogy. The analogy of arsenate (AsV) with phosphate enables As (V) to enter plant through phosphate transporter, while, arsenite (AsIII) which is analogous to silicic acid, is taken up by plants through aquaporins. After the uptake, the different forms of As are translocated to shoot via xylem, imposing toxicity to plants that affect their growth and yield, however this depends on the effective concentration of free As anion at particular cellular organelle /site. To this end, the role of transporters becomes crucial as the central and prime regulator of As movement throughout the plant and in various cellular compartments. It is essential to understand the precise roles of different transporters involved in As uptake and transportation to avoid As accumulation and stress in plant. Therefore, this review discusses the transporters namely, phosphate transporters, nodulin 26-like intrinsic proteins, plasma membrane intrinsic proteins, tonoplast intrinsic proteins, C-type ATP binding cassette transporters, arsenical resistance 3 transporter, inositol transporters, multidrug and toxic compound extrusion transporters, and natural resistance-associated macrophage protein transporters, which are involved in As uptake, sequestration, translocation and efflux in plants, with an emphasis on As stress tolerance through the regulation of expression of the different transporters.

     

Manganese-excess induces oxidative stress,lowers the pool of antioxidants and elevates activities of key antioxidative enzymes in rice seedlings

Manganese (Mn) is an essential element for plant growth but in excess, specially in acidic soils, it can become phytotoxic. In order to investigate whether oxidative stress is associated with the expression of Mn toxicity during early seedling establishment of rice plants, we examined the changes in the level of reactive oxygen species (ROS), oxidative stress induced an alteration in the level of non-enzymic antioxidants and activities of antioxidative enzymes in rice seedlings grown in sand cultures containing 3 and 6 mM MnCl₂. Mn treatment inhibited growth of rice seedlings, the metal increasingly accumulated in roots and shoots and caused damage to membranes. Mn treated plants showed increased generation of superoxide anion (O₂ ^.−), elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) and decline in protein thiol. The level of nonprotein thiol, however, increased due to Mn treatment. A decline in contents of reduced ascorbate (AsA) and glutathione (GSH) as well as decline in ratios of their reduced to oxidize forms was observed in Mn-treated seedlings. The activities of antioxidative enzymes superoxide dismutase (SOD) and its isoforms Mn SOD, Cu/Zn SOD, Fe SOD as well as guaiacol peroxidase (GPX) increased in the seedlings due to Mn treatment however, catalase (CAT) activity increased in 10 days old seedlings but it declined by 20 days under Mn treatment. The enzymes of Halliwell-Asada cycle, ascorbate peroxidase (APX) monodehydoascorbate reductase (MDHAR), dehyroascorbate reductase (DHAR) and glutathione reductase (GR) increased significantly in Mn treated seedlings over controls. Results suggest that in rice seedlings excess Mn induces oxidative stress, imbalances the levels of antioxidants and the antioxidative enzymes SOD, GPX, APX and GR appear to play an important role in scavenging ROS and withstanding oxidative stress induced by Mn.     

Biotechnological approach of improving plant salt tolerance using antioxidants as markers

Salt stress causes multifarious adverse effects in plants. Of them, production of reactive oxygen species (ROS) is a common phenomenon. These ROS are highly reactive because they can interact with a number of cellular molecules and metabolites thereby leading to a number of destructive processes causing cellular damage. Plants possess to a variable extent antioxidant metabolites, enzymes and non-enzymes, that have the ability to detoxify ROS. In the present review, the emphasis of discussion has been on understanding the role of different antioxidants in plants defense against oxidative stress caused by salt stress. The role of different antioxidants as potential selection criteria for improving plant salt tolerance has been critically discussed. With the advances in molecular biology and availability of advanced genetic tools considerable progress has been made in the past two decades in improving salt-induced oxidative stress tolerance in plants by developing transgenic lines with altered levels of antioxidants of different crops. The potential of this approach in counteracting stress-induced oxidative stress has been discussed at length in this review.     

Identification of the N-terminal region of TjZNT2, a Zrt/Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity

The Zrt/Irt-like protein (ZIP) family of transporter proteins is involved in the uptake of essential metal elements in plants. Two homologous ZIP genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that share high amino acid sequence similarity except at the N-terminus and the cytoplasmic loop between transmembrane domains III and IV, and that have been shown to be Zn(2+) and Mn(2+) transporters, respectively. To identify the region that determines the ion selectivity of these transporters, we constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed for the Zn(2+) uptake of yeast cells expressing them. As a result, the extracellular N-terminal ends were identified as regions involved in Zn(2+) selectivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its N-terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking the N-terminal extension was shown to possess Zn(2+) uptake activity. This suggests that the extended N-terminal region inhibits Zn(2+) transport by TjZNT2. Further studies showed that it is the first 25 amino acid region of the N-terminus that is important for the inhibition of Zn(2+) transport. Furthermore, the N-terminal truncated TjZNT2 lacked Mn(2+) uptake activity. These findings suggest that the N-terminal region is a novel substrate selector in the ZIP family of transporters.     

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.     

Distinct physiological responses of two rice cultivars subjected to iron toxicity under field conditions

Iron toxicity is recognised as the most widely distributed nutritional disorder in lowland and irrigated rice, derived from the excessive amounts of ferrous ions generated by the reduction of iron oxides in flooded soils. Rice cultivars with variable degrees of tolerance to iron toxicity have been developed, and cultural practices such as water management and fertilisation can be used to reduce its negative impact. However, because of the complex nature of iron toxicity, few physiological data concerning tolerance mechanisms to excess iron in field conditions are available. To analyse the physiological responses of rice to iron excess in field conditions, two rice cultivars with distinct tolerance to iron toxicity [BR‐IRGA 409 (susceptible) and IRGA 420 (tolerant)] were grown in two areas, with a well‐established history of iron toxicity (in Camaquã, RS, Brazil) and without iron toxicity (in Cachoeirinha, RS, Brazil). Plants from the susceptible cultivar grown in the iron‐toxic site showed lower levels of chlorophylls and soluble proteins (together with higher carbonyl levels) indicating photooxidative and oxidative damage. The toxic effects observed were because of the accumulation of high levels of iron and not because of any indirectly induced shoot deficiency of other nutrients. Higher activities of antioxidative enzymes were also observed in leaves of plants from the susceptible cultivar only in the iron‐toxic site, probably as a result of oxidative stress rather than because of specific involvement in a tolerance mechanism. There was no difference between cultivars in iron accumulation in the symplastic and apoplastic space of leaves, with both cultivars accumulating 85–90% of total leaf iron in the symplast. However, susceptible plants accumulated higher levels of iron in low‐molecular‐mass fractions than tolerant plants. The accumulation of iron in the low‐molecular‐mass fraction probably has a direct influence on iron toxicity, and the adaptive strategy of tolerant plants may rely on their capacity to buffer the iron amounts present in the low mass fraction, a new parameter to be considered when evaluating tolerance to iron excess in field‐cultivated rice plants.     

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.     

Transporters of ligands for essential metal ions in plants

Essential metals are required for healthy plant growth but can be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalization. However, very little metal in plants is thought to exist as free ions. A number of small, organic molecules have been implicated in metal ion homeostasis as metal ion ligands to facilitate uptake and transport of metal ions with low solubility and also as chelators implicated in sequestration for metal tolerance and storage. Ligands for a number of essential metals have been identified and proteins involved in the transport of these ligands and of metal-ligand complexes have been characterized. Here we review recent advances in understanding the role of mugineic acid, nicotianamine, organic acids (citrate and malate), histidine and phytate as ligands for iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and nickel (Ni) in plants, and the proteins identified as their transporters.     

高等植物Na+吸收、转运及细胞内Na+稳态平衡研究进展

盐胁迫是影响农业生产的重要环境因素之一。本文对植物Na 吸收的机制和途径、Na 在植物体内的长距离转运以及细胞内Na 稳态平衡的研究进展进行了概述。参与植物Na 吸收与转运的蛋白和通道可能包括HKT、LCT1、AKT和NSCC等。其中,HKT是植物体内普遍存在的一类转运蛋白,能够介导Na 的吸收,其结构中的带电氨基酸残基对于其离子选择性有着非常明显的影响。LCT1是从小麦中发现的一类能够介导低亲和性阳离子吸收的蛋白,然而在典型的土壤Ca2 浓度下LCT1并不能发挥吸收Na 的功能。AKT家族的成员在高盐环境下可能也参与了Na 的吸收。目前虽然还没有克隆到编码NSCC蛋白的基因,但是NSCC作为植物吸收Na 的主要途径的观点已被广泛接受。SOS1和HKT参与了Na 在根部与植株地上部的长距离转运过程,它们在木质部和韧皮部的Na 装载和卸载中发挥重要作用,从而影响植物的抗盐性。另外,由质膜Na /H 逆向转运蛋白SOS1、蛋白激酶SOS2以及Ca2 结合蛋白SOS3组成的SOS复合体对细胞的Na 稳态具有重要的调节作用,单子叶和双子叶植物之间的这种调节机制在结构和功能上具有保守性。SOS复合体与其它位于质膜或液泡膜上的Na /H 逆向转运蛋白以及H 泵一起调节着细胞的Na 稳态。     

锰毒及植物耐性机理研究进展

综述了近些年国内外关于锰毒及植物耐锰机理的研究成果,并指出了存在的问题和发展前景。锰毒是酸性土壤上限制作物产量的重要因子,国内外针对锰毒及植物耐受机制进行了相关研究,但进展较为缓慢。锰对植物的毒害效应体现在不同的细胞组织及生理生化水平上,不同植物耐受锰的机理也存在差异性,但大都集中在有机酸的螯合解毒、内部积累、外部排斥及氧化等方面。某些锰胁迫所诱导的基因也被筛选出来,并且部分生物学功能得以鉴定。此外,锰与其他营养元素间的协同或拮抗作用也得以阐述,伴随锰超富积植物-商陆在中国的发现,对锰毒及植物耐性机理的深入研究和探讨,将会对植物修复技术的开展产生理论和实践意义。