Conserved functions of MATE transporters and their potential to enhance plant tolerance to aluminium toxicity

Almost half of the world’s arable land has acidic pH. Aluminum salts present in acid soils dissociate to release Al³⁺ ions in the soil solution that inhibit root growth causing severe loss in crop yields. Aluminium toxicity accounts for the second highest loss in plant productivity after drought. Aluminium in high doses causes damage to the plant cell wall, cytoskeleton and DNA. One of the ways by which plants alleviate aluminium toxicity is by the exudation of citrate from the roots that chelates the free Al³⁺ and prevents its entry into the plant. In several crop plants Multidrug and Toxic Compound Extrusion (MATE) transporters regulate citrate exudation from the roots. The MATE proteins are ubiquitously present in bacteria, archaea, fungi, animals and plants. The origin and evolution of these membrane transporters in plants is not well known. Here, using protein sequence information we identify MATE transporters in major groups of land plants and their algal ancestors. Our study indicates that the MATE family members expanded in number and functionally diverse among the land plants. We also identify motifs present across the streptophyte clade and a conserved aspartate residue that might regulate citrate exudation. This study can provide leads to engineer MATE transporters to confer enhanced tolerance in acid soils.

     

Acyl‐lipid desaturase 1 primes cold acclimation response in Arabidopsis

Membrane fluidity change has long been suggested as the primary mechanism by which, plants adapt to cold stress, but the underlying molecular mechanisms are not completely established. In this study, we found that a knockout of acyl‐lipid/CoA desaturase 1 gene (ADS1; EC 1.14.99) enhances freezing tolerance after cold acclimation (CA). Fatty acid composition analysis demonstrated that 18:1 content in ads1 mutant plants was 20% lower than in wild‐type (WT) grown at 23°C. Lipidomics revealed that 34C‐species of monogalactosyl diacylglycerol (MGDG) content in ads1 mutants were 3.3–14.9% lower than in WT. Lipid positional analysis identified 10% lower 18:1 fatty acid content at the sn‐2 position of MGDG. The cytosolic calcium content in ads1 mutant plants was also approximately two‐times higher than that of WT in response to cold shock. Each of these biochemical differences between WT and ads1 mutant disappeared after CA. Subcellular localization of C‐ and N‐terminal enhanced‐fluorescence‐fusion proteins indicated that ADS1 localized exclusively to chloroplasts. These observations suggest that ADS1‐mediated alteration of chloroplast membrane fluidity is required to prime a CA response, and is the upstream event of cytosolic calcium signaling.     

Arabidopsis desaturase 2 gene is involved in the regulation of cadmium and lead resistance

Aims

It was shown previously that Arabidopsis (Arabidopsis thaliana) desaturase 2 (ADS2) cDNA was isolated and it was shown that the expression of ADS2 was organ-dependent and up-regulated by low temperature. However, little is known about the role of ADS2 gene in heavy metal resistance in plants. In this study, we showed that ADS2 gene is involved in the regulation of cadmium (Cd) and lead (Pb) resistance.

Methods

For heavy metal resistance tests, seeds were germinated and grown on 1/2 MS media supplemented with the indicated concentrations of metal ions. To quantify root length, plants were grown vertically in plates. For heavy metal treatments, two-week old wild-type seedlings grown on MS media were treated with cadmium (Cd) or lead (Pb) for 24 h, and then sampled for metal content measurement and qPCR analysis.

Results

ADS2 was strongly repressed by Cd(II), and ads2-1 mutant plants showed increased Cd(II) resistance. A lower Cd content was detected in ads2-1 plants than in wild-type plants subjected to Cd(II) treatment, which was associated with activation in expression of AtPDR8 gene, a pump excluding Cd(II) and/or Cd(II)-containing toxic compounds from the cytoplasm, suggesting that ADS2-mediated Cd(II) resistance is AtPDR8 dependent. We also found that ads2-1 plants showed increased Pb(II) sensitivity, and ADS2 was strongly repressed by hydrogen peroxide (H₂O₂) but not by Pb(II). The ads2-1 mutant showed increased sensitivity to oxidative stresses mediated by H₂O₂ and paraquat, and higher levels of H₂O₂ accumulation were observed in leaves of ads2-1 plants than those of wild-type plants when subjected to Pb(II) and H₂O₂, indicating that ADS2 mediates Pb(II) resistance indirectly by impaired ROS scavenging.

Conclusions

ADS2 gene mediates Cd(II) and Pb(II) resistance, at least in part, through two distinct mechanisms, an AtPDR8-dependent mechanism and a ROS detoxification system-mediated mechanism, respectively.     

How a microbial drug transporter became essential for crop cultivation on acid soils: aluminium tolerance conferred by the multidrug and toxic compound extrusion (MATE) family

Background

Aluminium (Al) toxicity is a major agricultural constraint for crop cultivation on acid soils, which comprise a large portion of the world''s arable land. One of the most widely accepted mechanisms of Al tolerance in plants is based on Al-activated organic acid release into the rhizosphere, with organic acids forming stable, non-toxic complexes with Al. This mechanism has recently been validated by the isolation of bona-fide Al-tolerance genes in crop species, which encode membrane transporters that mediate Al-activated organic acid release leading to Al exclusion from root apices. In crop species such as sorghum and barley, members in the multidrug and toxic compound extrusion (MATE) family underlie Al tolerance by a mechanism based on Al-activated citrate release.

Scope and Conclusions

The study of Al tolerance in plants as conferred by MATE family members is in its infancy. Therefore, much is yet to be discovered about the functional diversity and evolutionary dynamics that led MATE proteins to acquire transport properties conducive to Al tolerance in plants. In this paper we review the major characteristics of transporters in the MATE family and will relate this knowledge to Al tolerance in plants. The MATE family is clearly extremely flexible with respect to substrate specificity, which raises the possibility that Al tolerance as encoded by MATE proteins may not be restricted to Al-activated citrate release in plant species. There are also indications that regulatory loci may be of pivotal importance to fully explore the potential for Al-tolerance improvement based on MATE genes.     

Structures of multidrug and toxic compound extrusion transporters and their mechanistic implications

Multidrug resistance poses grand challenges to the effective treatment of infectious diseases and cancers. Integral membrane proteins from the multidrug and toxic compound extrusion (MATE) family contribute to multidrug resistance by exporting a wide variety of therapeutic drugs across cell membranes. MATE proteins are conserved from bacteria to humans and can be categorized into the NorM, DinF and eukaryotic subfamilies. MATE transporters hold great appeal as potential therapeutic targets for curbing multidrug resistance, yet their transport mechanism remains elusive. During the past 5 years, X-ray structures of 4 NorM and DinF transporters have been reported and guided biochemical studies to reveal how MATE transporters extrude different drugs. Such advances, although substantial, have yet to be discussed collectively. Herein I review these structures and the unprecedented mechanistic insights that have been garnered from those structure-inspired studies, as well as lay out the outstanding questions that present exciting opportunities for future work.     

Twelve Transmembrane Helices Form the Functional Core of Mammalian MATE1 (Multidrug and Toxin Extruder 1) Protein

The x-ray structure of the prototypic MATE family member, NorM from Vibrio cholerae, reveals a protein fold composed of 12 transmembrane helices (TMHs), confirming hydropathy analyses of the majority of (prokaryotic and plant) MATE transporters. However, the mammalian MATEs are generally predicted to have a 13(th) TMH and an extracellular C terminus. Here we affirm this prediction, showing that the C termini of epitope-tagged, full-length human, rabbit, and mouse MATE1 were accessible to antibodies from the extracellular face of the membrane. Truncation of these proteins at or near the predicted junction between the 13(th) TMH and the long cytoplasmic loop that precedes it resulted in proteins that (i) trafficked to the membrane and (ii) interacted with antibodies only after permeabilization of the plasma membrane. CHO cells expressing rbMate1 truncated at residue Gly-545 supported levels of pH-sensitive transport similar to that of cells expressing the full-length protein. Although the high transport rate of the Gly-545 truncation mutant was associated with higher levels of membrane expression (than full-length MATE1), suggesting the 13(th) TMH may influence substrate translocation, the selectivity profile of the mutant indicated that TMH13 has little impact on ligand binding. We conclude that the functional core of MATE1 consists of 12 (not 13) TMHs. Therefore, we used the x-ray structure of NorM to develop a homology model of the first 12 TMHs of MATE1. The model proved to be stable in molecular dynamic simulations and agreed with topology evident from preliminary cysteine scanning of intracellular versus extracellular loops.     

A gain-of-function mutation in the Arabidopsis pleiotropic drug resistance transporter PDR9 confers resistance to auxinic herbicides

Arabidopsis (Arabidopsis thaliana) contains 15 genes encoding members of the pleiotropic drug resistance (PDR) family of ATP-binding cassette transporters. These proteins have been speculated to be involved in the detoxification of xenobiotics, however, little experimental support of this hypothesis has been obtained to date. Here we report our characterization of the Arabidopsis PDR9 gene. We isolated a semidominant, gain-of-function mutant, designated pdr9-1, that exhibits increased tolerance to the auxinic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Reciprocally, loss-of-function mutations in PDR9 confer 2,4-D hypersensitivity. This altered auxin sensitivity defect of pdr9 mutants is specific for 2,4-D and closely related compounds as these mutants respond normally to the endogenous auxins indole-3-acetic acid and indole-butyric acid. We demonstrate that 2,4-D, but not indole-3-acetic acid transport is affected by mutations in pdr9, suggesting that the PDR9 transporter specifically effluxes 2,4-D out of plant cells without affecting endogenous auxin transport. The semidominant pdr9-1 mutation affects an extremely highly conserved domain present in all known plant PDR transporters. The single amino acid change results in increased PDR9 abundance and provides a novel approach for elucidating the function of plant PDR proteins.     

Genome-wide identification and expression analysis of MATE gene family in citrus fruit (Citrus clementina)

Multidrug and toxic compound extrusion (MATE) proteins are a class of secondary active multidrug transporters. In plants, this family has significantly expanded and is involved in numerous plant physiological processes. Although MATE proteins have been identified in an increasing number of species, the understanding about this family in citrus remains unclear. In this study, a total of 69 MATE transporters were identified in the citrus genome (Citrus clementina) and classified into four groups by phylogenetic analysis. Tandem and segmental duplication events were the main causes of the citrus MATE family expansion. RNA-seq and qRT-PCR analyses were performed during citrus fruit development. The results indicated that CitMATE genes showed specific expression profiles in citrus peels and flesh at different developmental stages. Combined with the variations of flavonoids and citrate levels in citrus fruit, we suggested that CitMATE43 and CitMATE66 may be involved in the transport process of flavonoids and citrate in citrus fruit, respectively. In addition, two flavonoids positive regulators, CitERF32 and CitERF33, both directly bind to and activated the CitMATE43 promoter. Our results provide comprehensive information on citrus MATE genes and valuable understanding for the flavonoids and citrate metabolism in citrus fruit.     

Functional characterization of testis-specific rodent multidrug and toxic compound extrusion 2, a class III MATE-type polyspecific H+/organic cation exporter

Mammalian multidrug and toxic compound extrusion (MATE) proteins are classified into three subfamilies: classes I, II, and III. We previously showed that two of these families act as polyspecific H(+)-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney (Otsuka et al. Proc Natl Acad Sci USA 102: 17923-17928, 2005; Omote et al. Trends Pharmacol Sci 27: 587-593, 2006). Rodent MATE2 proteins are class III MATE transporters, the molecular nature, as well as transport properties, of which remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). On expression in human embryonic kidney (HEK)-293 cells, mMATE2 localized to the intracellular organelles and plasma membrane. mMATE2 mediated pH-dependent TEA transport with substrate specificity similar to, but distinct from, that of mMATE1, which prefers N-methylnicotinamide and guanidine as substrates. mMATE2 expressed in insect cells was solubilized and reconstituted with bacterial H(+)-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone but unaffected by valinomycin in the presence of K(+). Immunologic techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus mMATE2 appears to act as a polyspecific H(+)/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporters of organic cations.     

Insights on Na+ binding and conformational dynamics in multidrug and toxic compound extrusion transporter NorM

MATE (multidrug and toxic compound extrusion) transporter proteins mediate metabolite transport in plants and multidrug resistance in bacteria and mammals. MATE transporter NorM from Vibrio cholerae is an antiporter that is driven by Na+ gradient to extrude the substrates. To understand the molecular mechanism of Na+‐substrate exchange, molecular dynamics simulation was performed to study conformational changes of both wild‐type and mutant NorM with and without cation bindings. Our results show that NorM is able to bind two Na⁺ ions simultaneously, one to each of the carboxylic groups of E255 and D371 in the binding pocket. Furthermore, this di‐Na⁺ binding state is likely more efficient for conformational changes of NorM_VC toward the inward‐facing conformation than single‐Na⁺ binding state. The observation of two Na⁺ binding sites of NorM_VC is consistent with the previous study that two sites for ion binding (denoted as Na1/Na2 sites) are found in the transporter LeuT and BetP, another two secondary transporters. Taken together, our findings shed light on the structure rearrangements of NorM on Na⁺ binding and enrich our knowledge of the transport mechanism of secondary transporters. Proteins 2014; 82:240–249. © 2013 Wiley Periodicals, Inc.     

OsRAR1 and OsSGT1 physically interact and function in rice basal disease resistance

The RAR1 and SGT1 proteins function synergistically or antagonistically in plant innate immune responses. Here, we show that the rice orthologs OsRAR1 and OsSGT1 physically interact in vivo and in yeast. They displayed conserved roles in Arabidopsis disease resistance through ectopic expression in the Arabidopsis rar1 and sgt1 mutants. Overexpression of OsRar1 and OsSGT1 in rice significantly increased basal resistance to a virulent bacterial blight Xanthomonas oryzae pv. oryzae PXO99 but not to another virulent strain DY89031, suggesting race-specific-like basal resistance conferred by OsRar1 and OsSGT1. OsRar1-OE and OsSGT1-OE plants also enhanced resistance to all four virulent blast fungal Magnaporthe oryzae races. Overexpression of the OsSGT1-green fluorescent protein (GFP) fusion most likely caused a dominant negative phenotype which led to race-specific-like basal resistance. Transgenic plants overexpressing OsSGT1-GFP show enhanced resistance to DY89031 but decreased resistance to PXO99, implying that OsSGT1 might be the target of a component required for DY89031 virulence or OsSGT1-GFP might stabilize weak resistance proteins against DY89031. Consistent with the hypothesis of the dominant negative regulation, we observed the reduced sensitivity to auxin of OsSGT1-GFP plants compared with the wild-type ones, and the curling-root phenotype in OsSGT1-OE plants. These results collectively suggest that OsRar1 and OsSGT1 might be differentially required for rice basal disease resistance. Our current study also provides new insight into the roles of OsSGT1 in basal disease resistance.     

植物蔗糖转运蛋白表达的调控因素与分子机制

植物光合作用的产物主要以蔗糖的形式在植物体内进行从源到库的运输.蔗糖转运蛋白是此过程的重要参与者,其表达和调控与植物中光合作用产物的分配紧密关联,从而调控着植物的生长发育、结果结实、抗逆抗病等性状.蔗糖转运蛋白的表达受到植物发育时期、外界环境条件及激素的影响.蔗糖转运蛋白的调控机制有转录因子的调节、基因内部序列调控、蛋...     

Neonicotinoid pesticides poorly interact with human drug transporters

The interactions of six neonicotinoid pesticides and one neonicotinoid metabolite with drug transporters have been characterized in vitro. Acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid and its metabolite thiacloprid amide, and thiamethoxam, each used at 100 µM, did not impair activity of the efflux pumps P‐glycoprotein, multidrug resistance‐associated proteins, and breast cancer resistance protein. They also did not inhibit that of the uptake transporters OATP1B1, OATP1B3, OAT4, and MATE1, whereas that of OATP2B1, OAT1, and MATE2‐K was affected by only one of the seven neonicotinoids. Activity of OCT1 was moderately stimulated (up to 1.5‐fold) by several neonicotinoids. By contrast, that of OAT3 and OCT2 was inhibited by most (OAT3), if not all (OCT2), neonicotinoids, with IC₅₀ values in the 20 to 60 µM range for thiacloprid, likely not relevant to environmental exposure. Thiacloprid was moreover not transported by OAT3 and OCT2. Overall, these data suggest that neonicotinoid pesticides rather poorly interact with drug transporter activities.     

Principles of Alternating Access in Multidrug and Toxin Extrusion (MATE) Transporters

The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.     

紫花苜蓿MsMATE1基因克隆及其抵御铁胁迫功能的鉴定

多药和有毒化合物外排转运蛋白MATE能转运金属、激素、次生代谢物等多种底物,因而在植物的生长发育中发挥重要作用。本研究基于紫花苜蓿(Medicago sativa L.)基因组数据和缺铁胁迫的转录组数据,克隆获得紫花苜蓿MsMATE1及pMsMATE1启动子(GenBank登录号分别为MN547958和MT505313)。MsMATE1基因长1470 bp,编码489个氨基酸。系统进化分析表明,紫花苜蓿MsMATE1蛋白属于mate家族,与蒺藜苜蓿MtMATE(XP013453190.1)亲缘关系最近。生物信息学分析表明,MsMATE1具有典型的MATE_like超家族结构域,属于H+势驱动的真核亚类;MsMATE1为跨膜蛋白,二级结构的主要构成元件是α螺旋。pMsMATE1启动子长1598 bp,序列分析显示内含多个植物激素和逆境胁迫响应元件。MsMATE1在紫花苜蓿幼苗的根茎叶中都有表达,茎中的表达量最高。高铁和缺铁胁迫下,MsMATE1基因在紫花苜蓿幼苗各部位的表达显著上调,茎中上调最明显。高铁和缺铁胁迫,转MsMATE1基因烟草的3种抗氧化酶活性、叶绿素和可溶性蛋白含量显著增加,丙二醛含量显著降低。以上结果表明,MsMATE1在烟草中的异源表达提高了植物抵御高铁和缺铁胁迫的能力。MsMATE1可以作为应用基因工程方法改良植物铁胁迫耐受的重要候选基因,本研究为深入了解MsMATE1蛋白在植物响应铁胁迫中的分子机制奠定基础,MsMATE1基因应对其他金属胁迫或环境胁迫的功能有待于进一步鉴定。     

From organelle to organ: ZRIZI MATE-Type transporter is an organelle transporter that enhances organ initiation

Plant architecture is a predictable but flexible trait. The timing and position of organ initiation from the shoot apical meristem (SAM) contribute to the final plant form. While much progress has been made recently in understanding how the site of leaf initiation is determined, the mechanism underlying the temporal interval between leaf primordia is still largely unknown. The Arabidopsis ZRIZI (ZRZ) gene belongs to a large gene family encoding multidrug and toxic compound extrusion (MATE) transporters. Unique among plant MATE transporters identified so far, ZRZ is localized to the membrane of a small organelle, possibly the mitochondria. Plants overexpressing ZRZ in initiating leaves are short, produce leaves much faster than wild-type plants and show enhanced growth of axillary buds. These results suggest that ZRZ is involved in communicating a leaf-borne signal that determines the rate of organ initiation.