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SUMMARY: Barley produces several specialized metabolites, including five α‐, β‐, and γ‐hydroxynitrile glucosides (HNGs). In malting barley, presence of the α‐HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite‐GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock‐out (KO)‐lines, isolated from the barley FIND‐IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO‐lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG‐0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production. [ABSTRACT FROM AUTHOR]

Summary: Aluminium (Al) toxicity decreases crop production in acid soils in general, but many crops have evolved complex mechanisms to resist it. However, our current understanding of how plants cope with Al stress and perform Al resistance is still at the initial stage. In this study, the citrate transporter CcMATE35 was identified to be involved in Al stress response. The release of citrate was increased substantially in CcMATE35 over‐expression (OE) lines under Al stress, indicating enhanced Al resistance. It was demonstrated that transcription factor CcNFYB3 regulated the expression of CcMATE35, promoting the release of citrate from roots to increase Al resistance in pigeon pea. We also found that a Long noncoding RNA Targeting Citrate Synthase (CcLTCS) is involved in Al resistance in pigeon pea. Compared with controls, overexpression of CcLTCS elevated the expression level of the Citrate Synthase gene (CcCS), leading to increases in root citrate level and citrate release, which forms another module to regulate Al resistance in pigeon pea. Simultaneous overexpression of CcNFYB3 and CcLTCS further increased Al resistance. Taken together, these findings suggest that the two modules, CcNFYB3‐CcMATE35 and CcLTCS‐CcCS, jointly regulate the efflux and synthesis of citrate and may play an important role in enhancing the resistance of pigeon pea under Al stress. [ABSTRACT FROM AUTHOR]

In higher plants, the shoot apical meristem produces lateral organs in a regular spacing (phyllotaxy) and timing (plastochron). The molecular analysis of mutants associated with phyllotaxy and plastochron would increase our understanding of the mechanism of shoot architecture formation. In this study, we identified mutant mnd8ynp5 that shows an increased rate of leaf emergence and a larger number of nodes in combination with a dwarfed growth habit from an EMS-treated population of the elite barley cultivar Yangnongpi 5. Using a map-based cloning strategy, the mnd8 gene was narrowed down to a 6.7-kb genomic interval on the long arm of chromosome 5H. Sequence analysis revealed that a C to T single-nucleotide mutation occurred at the first exon (position 953) of HORVU5Hr1G118820, leading to an alanine (Ala) to valine (Val) substitution at the 318th amino acid site. Next, HORVU5Hr1G118820 was defined as the candidate gene of MND8 encoding 514 amino acids and containing two multidrug and toxic compound extrusion (MATE) domains. It is highly homologous to maize Bige1 and has a conserved function in the regulation of plant development by controlling the leaf initiation rate. Examination of modern barely varieties showed that Hap-1 was the dominant haplotype and was selected in barley breeding around the world. Collectively, our results indicated that mnd8ynp5 is a novel allele of the HORVU5Hr1G118820 gene that is possibly responsible for the shortened plastochron and many noded dwarf phenotype in barley.

Summary: More than half of the world's food is provided by cereals, as humans obtain >60% of daily calories from grains. Producing more carbohydrates is always the final target of crop cultivation. The carbohydrate partitioning pathway directly affects grain yield, but the molecular mechanisms and biological functions are poorly understood, including rice (Oryza sativa L.), one of the most important food sources. Here, we reported a prolonged grain filling duration mutant 1 (gfd1), exhibiting a long grain‐filling duration, less grain number per panicle and bigger grain size without changing grain weight. Map‐based cloning and molecular biological analyses revealed that GFD1 encoded a MATE transporter and expressed high in vascular tissues of the stem, spikelet hulls and rachilla, but low in the leaf, controlling carbohydrate partitioning in the stem and grain but not in the leaf. GFD1 protein was partially localized on the plasma membrane and in the Golgi apparatus, and was finally verified to interact with two sugar transporters, OsSWEET4 and OsSUT2. Genetic analyses showed that GFD1 might control grain‐filling duration through OsSWEET4, adjust grain size with OsSUT2 and synergistically modulate grain number per panicle with both OsSUT2 and OsSWEET4. Together, our work proved that the three transporters, which are all initially classified in the major facilitator superfamily family, could control starch storage in both the primary sink (grain) and temporary sink (stem), and affect carbohydrate partitioning in the whole plant through physical interaction, giving a new vision of sugar transporter interactome and providing a tool for rice yield improvement. [ABSTRACT FROM AUTHOR]

Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-μM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter. [Display omitted] • The substrate/H+-antiport mechanism of PfMATE was structurally analyzed by solution NMR • The equilibrium between the IF/OF conformations is dictated by Glu163 protonation • Only the IF conformation has a sufficient affinity for substrate binding • The affinity-directed transport mechanism ensures H+-coupled substrate excretion Takeuchi et al. structurally characterized the substrate/H+-antiport mechanism of the multidrug transporter PfMATE. PfMATE is an affinity-directed H+ antiporter and substrates selectively bind to its protonated IF conformation. Subsequent proton release is coupled to a structural rearrangement to the OF conformation, which mechanistically ensures H+-coupled substrate excretion. [ABSTRACT FROM AUTHOR]

The multidrug and toxic compound extrusion (MATE) protein family has been implicated in the transport of a diverse range of molecules, including specialized metabolites. In tobacco (Nicotiana tabacum), only a limited number of MATE transporters have been functionally characterized, and no MATE transporter has been studied in the context of flavonoid transport in this plant species so far. In the present study, we characterize two homeologous tobacco MATE genes, NtMATE21 and NtMATE22 , and demonstrate their role in flavonol transport and in plant growth and development. The expression of these two genes was reported to be up-regulated in trichomes as compared with the trichome-free leaf. The transcript levels of NtMATE21 and NtMATE22 were found to be higher in flavonol overproducing tobacco transgenic lines as compared with wild type tobacco. The two transporters were demonstrated to be localized to the plasma membrane. Genetic manipulation of NtMATE21 and NtMATE22 led to altered growth phenotypes and modulated flavonol contents in N. tabacum. The β-glucuronidase and green fluorescent protein fusion transgenic lines of promoter regions suggested that NtMATE21 and NtMATE22 are exclusively expressed in the trichome heads in the leaf tissue and petals. Moreover, in a transient transactivation assay, NtMYB12, a flavonol-specific MYB transcription factor, was found to transactivate the expression of NtMATE21 and NtMATE22 genes. Together, our results strongly suggest the involvement of NtMATE21 and NtMATE22 in flavonol transport as well as in the regulation of plant growth and development. [ABSTRACT FROM AUTHOR]

Drought is one of the most severe environmental factors limiting plant growth and crop yield, necessitating the identification of genes that enhance drought resistance for crop improvement. Through screening an ethyl methyl sulfonate-mutagenized rice mutant library, we isolated the PEG tolerance mutant 97-1 (ptm97-1), which displays enhanced resistance to osmotic and drought stress, and increased yield under drought conditions. A point mutation in OsMATE6 was identified as being associated with the drought-resistant phenotype of ptm97-1. The role of OsMATE6 in conferring drought resistance was confirmed by additional OsMATE6 knockout mutants. OsMATE6 is expressed in guard cells, shoots and roots and the OsMATE6-GFP fusion protein predominantly localizes to the plasma membrane. Our ABA efflux assays suggest that OsMATE6 functions as an ABA efflux transporter; mutant protoplasts exhibited a slower ABA release rate compared to the wild type. We hypothesize that OsMATE6 regulates ABA levels in guard cells, influencing stomatal closure and enhancing drought resistance. Notably, OsMATE6 knockout mutants demonstrated greater yields under field drought conditions compared to wild-type plants, highlighting OsMATE6 as a promising candidate for improving crop drought resistance., (© 2024 John Wiley & Sons Ltd.)

Soybean mosaic virus (SMV) is detrimental to soybean (Glycine max) breeding, seed quality, and yield worldwide. Improving the basic resistance of host plants is the most effective and economical method to reduce damage from SMV. Therefore, it is necessary to identify and clone novel SMV resistance genes. Here, we report the characterization of two soybean cultivars, DN50 and XQD, with different levels of resistance to SMV. Compared with XQD, DN50 exhibits enhanced resistance to the SMV strain SC7. By combining bulked-segregant analysis (BSA)-seq and fine-mapping, we identified a novel resistance locus, R SMV -11 , spanning an approximately 207-kb region on chromosome 11 and containing 25 annotated genes in the reference Williams 82 genome. Of these genes, we identified eleven with non-synonymous single-nucleotide polymorphisms (SNPs) or insertion-deletion mutations (InDels) in their coding regions between two parents. One gene, GmMATE68 (Glyma.11G028900), harbored a frameshift mutation. GmMATE68 encodes a multidrug and toxic compound extrusion (MATE) transporter that is expressed in all soybean tissues and is induced by SC7. Given that MATE transporter families have been reported to be linked with plant disease resistance, we suggest that GmMATE68 is responsible for SC7 resistance in DN50. Our results reveal a novel SMV-resistance locus, improving understanding of the genetics of soybean disease resistance and providing a potential new tool for marker-assisted selection breeding in soybean. [ABSTRACT FROM AUTHOR]

Soybean mosaic virus (SMV) is detrimental to soybean (Glycine max) breeding, seed quality, and yield worldwide. Improving the basic resistance of host plants is the most effective and economical method to reduce damage from SMV. Therefore, it is necessary to identify and clone novel SMV resistance genes. Here, we report the characterization of two soybean cultivars, DN50 and XQD, with different levels of resistance to SMV. Compared with XQD, DN50 exhibits enhanced resistance to the SMV strain SC7. By combining bulked-segregant analysis (BSA)-seq and fine-mapping, we identified a novel resistance locus, RSMV-11, spanning an approximately 207-kb region on chromosome 11 and containing 25 annotated genes in the reference Williams 82 genome. Of these genes, we identified eleven with non-synonymous single-nucleotide polymorphisms (SNPs) or insertion-deletion mutations (InDels) in their coding regions between two parents. One gene, GmMATE68 (Glyma.11G028900), harbored a frameshift mutation. GmMATE68 encodes a multidrug and toxic compound extrusion (MATE) transporter that is expressed in all soybean tissues and is induced by SC7. Given that MATE transporter families have been reported to be linked with plant disease resistance, we suggest that GmMATE68 is responsible for SC7 resistance in DN50. Our results reveal a novel SMV-resistance locus, improving understanding of the genetics of soybean disease resistance and providing a potential new tool for marker-assisted selection breeding in soybean.

Triterpenoid saponins, synthesized via the mevalonic acid (MVA) pathway in the cytoplasm, provide protection against pathogens and pests in plants and health benefits for humans. However, the mechanisms by which triterpenoid saponins are transported between cellular compartments remain uncharacterized. Here, we characterize a tonoplast localized multidrug and toxic compound extrusion transporter, GmMATE100 (encoded by Glyma.18G143700 ), from soybean ( Glycine max L.). GmMATE100 is co-expressed with soyasaponin biosynthetic genes, and its expression was induced by MeJA treatment, which also led to soyasaponin accumulation in soybean roots. GmMATE100 efficiently transports multiple type-B soyasaponins as well as type-A soyasaponins with low affinity from the cytosol to the vacuole in a yeast system. The GmMATE100 loss-of-function mutant showed a significant decrease in type-A and type-B soyasaponin contents in soybean roots. This study not only characterized the first soybean triterpenoid saponin transporter but also provided new knowledge for the rational engineering of soyasaponin content and composition in soybean plants to modulate their levels within crop environments.

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter. [ABSTRACT FROM AUTHOR]

Summary: Iron (Fe) is an essential element, its transport is regulated by the cell redox balance. In seeds, Fe enters the embryo as Fe2+ and is stored in vacuoles as Fe3+. Through its ferric reduction activity, ascorbate plays a major role in Fe redox state and therefore Fe transport within the seed.We searched for ascorbate membrane transporters responsible for controlling Fe reduction by screening the yeast ferric reductase‐deficient fre1 strain and isolated AtDTX25, a member of the Multidrug And Toxic compound Extrusion (MATE) family.AtDTX25 was shown to mediate ascorbate efflux when expressed in yeast and Xenopus oocytes, in a pH‐dependent manner. Inplanta, AtDTX25 is highly expressed during germination and encodes a vacuolar membrane protein. Isolated vacuoles from AtDTX25‐1 knockout mutant contained less ascorbate and more Fe than wild‐type (WT), and mutant seedlings were highly sensitive to Fe deficiency. Iron imaging further showed that the remobilisation of Fe from vacuoles was highly impaired in mutant seedlings.Taken together, our results established AtDTX25 as a vacuolar ascorbate transporter, required during germination to promote the reduction of the pool of stored Fe3+ and its remobilisation to feed the developing seedling. [ABSTRACT FROM AUTHOR]

Nitrogen (N) as well as Phosphorus (P) are key nutrients determining crop productivity. Legumes have developed strategies to overcome nutrient limitation by, for example, forming a symbiotic relationship with N-fixing rhizobia and the release of P-mobilizing exudates and are thus able to grow without supply of N or P fertilizers. The legume-rhizobial symbiosis starts with root release of isoflavonoids that act as signaling molecules perceived by compatible bacteria. Subsequently, bacteria release nod factors, which induce signaling cascades allowing the formation of functional N-fixing nodules. We report here the identification and functional characterization of a plasma membrane-localized MATE-type transporter (LaMATE2) involved in the release of genistein from white lupin roots. The LaMATE2 expression in the root is upregulated under N deficiency as well as low phosphate availability, two nutritional deficiencies that induce the release of this isoflavonoid. LaMATE2 silencing reduced genistein efflux and even more the formation of symbiotic nodules, supporting the crucial role of LaMATE2 in isoflavonoid release and nodulation. Furthermore, silencing of LaMATE2 limited the P-solubilization activity of lupin root exudates. Transport assays in yeast vesicles demonstrated that LaMATE2 acts as a proton-driven isoflavonoid transporter.

As a tetracycline antibiotic, the enrichment of doxycycline in soil seriously endangers agricultural cultivation and food security. Salvianolic acid B (SAB), as a natural product, has high antioxidant activity. We applied SAB and doxycycline (DOX) externally to Salvia miltiorrhiza seedlings, and measured the activities of total superoxide dismutase, peroxidase and catalase, as well as the accumulation of malondialdehyde, active oxygen and glutathione. It was found that SAB can participate in the DOX stress release of S. miltiorrhiza through the antioxidant system. We also analyzed the evolutionary relationship of multidrug and toxic compound extrusion (MATE) family proteins related to antibiotic transport in 50 representative evolutionary genomes, and revealed the potential binding ability of MATE and DOX through macromolecular docking. Finally, based on transcriptome and RT-qPCR analysis, we identified a novel gene SmMATE1 , and found that it has a co-expression pattern with SmRAS and SmCYP98A14. Transgenic in tobacco, yeast and hairy root of S. miltiorrhiza revealed the important role of this new gene and SAB in synergistically releasing DOX stress damage, which provided a reference for natural product to alleviate the stress of antibiotics on plants, and also provided a theoretical basis for the development of new soil antibiotic sustained-release agents. [Display omitted] • SmMATE1 involved in tetracycline transport was found in Salvia miltiorrhiza. • Evolution analysis speculates the transport capacity of MATE to doxycycline. • MATEs and salvianolic acid B biosynthesis genes are involved in doxycycline relief. • Transgenic analysis demonstrated the relieve function of DOX toxicity of SmMATE1. • Salvianolic acid B as a potential remediation agent for soil antibiotic pollution. [ABSTRACT FROM AUTHOR]

Summary: Manihot esculenta (cassava) is a root crop originating from South America that is a major staple in the tropics, including in marginal environments. This study focused on South American and African germplasm and investigated the genetic architecture of hydrogen cyanide (HCN), a major component of root quality. HCN, representing total cyanogenic glucosides, is a plant defense component against herbivory but is also toxic for human consumption. We genotyped 3354 landraces and modern breeding lines originating from 26 Brazilian states and 1389 individuals were phenotypically characterized across multi‐year trials for HCN. All plant material was subjected to high‐density genotyping using genotyping by sequencing. We performed genome‐wide association mapping to characterize the genetic architecture and gene mapping of HCN. Field experiments revealed strong broad‐ and narrow‐sense trait heritability (0.82 and 0.41, respectively). Two major loci were identified, encoding for an ATPase and a MATE protein, and contributing up to 7 and 30% of the HCN concentration in roots, respectively. We developed diagnostic markers for breeding applications, validated trait architecture consistency in African germplasm and investigated further evidence for the domestication of sweet and bitter cassava. Fine genomic characterization revealed: (i) the major role played by vacuolar transporters in regulating HCN content; (ii) the co‐domestication of sweet and bitter cassava major alleles are dependent upon geographical zone; and (iii) the major loci allele for high HCN in M. esculenta Crantz seems to originate from its ancestor, M. esculenta subsp. flabellifolia. Taken together, these findings expand our insights into cyanogenic glucosides in cassava roots and its glycosylated derivatives in plants. [ABSTRACT FROM AUTHOR]

Key message: This review summarizes the recent updates in the area of transporters of plant secondary metabolites, including their applied aspects in metabolic engineering of economically important secondary metabolites. Plants have evolved biosynthetic pathways to produce structurally diverse secondary metabolites, which serve distinct functions, including defense against pathogens and herbivory, thereby playing a pivotal role in plant ecological interactions. These compounds often display interesting bioactivities and, therefore, have been used as repositories of natural drugs and phytoceuticals for humans. At an elevated level, plant secondary metabolites could be cytotoxic to the plant cell itself; therefore, plants have developed sophisticated mechanisms to sequester these compounds to prevent cytotoxicity. Many of these valuable natural compounds and their precursors are biosynthesized and accumulated at diverse subcellular locations, and few are even transported to sink organs via long-distance transport, implying the involvement of compartmentalization via intra- and intercellular transport mechanisms. The transporter proteins belonging to different families of transporters, especially ATP binding cassette (ABC) and multidrug and toxic compound extrusion (MATE) have been implicated in membrane-mediated transport of certain plant secondary metabolites. Despite increasing reports on the characterization of transporter proteins and their genes, our knowledge about the transporters of several medicinally and economically important plant secondary metabolites is still enigmatic. A comprehensive understanding of the molecular mechanisms underlying the whole route of secondary metabolite transportome, in addition to the biosynthetic pathways, will aid in systematic and targeted metabolic engineering of high-value secondary metabolites. The present review embodies a comprehensive update on the progress made in the elucidation of transporters of secondary metabolites in view of basic and applied aspects of their transport mechanism. [ABSTRACT FROM AUTHOR]

Multidrug and toxic compound extrusion (MATE) transporters mediate excretion of xenobiotics and toxic metabolites, thereby conferring multidrug resistance in bacterial pathogens and cancer cells. Structural information on the alternate conformational states and knowledge of the detailed mechanism ofMATE transport are of great importance for drug development. However, the structures of MATE transporters are only known in V-shaped outward-facing conformations. Here, we present the crystal structure of a MATE transporter from Pyrococcus furiosus (PfMATE) in the long-soughtafter inward-facing state, which was obtained after crystallization in the presence of native lipids. Transition from the outward-facing state to the inward-facing state involves rigid body movements of transmembrane helices (TMs) 2-6 and 8-12 to form an inverted V, facilitated by a loose binding of TM1 and TM7 to their respective bundles and their conformational flexibility. The inward-facing structure of PfMATE in combinationwith the outward-facing one supports an alternating access mechanism for the MATE family transporters. [ABSTRACT FROM AUTHOR]

The response to inclination in plants is an attractive and extensively studied biological process. The most commonly held theory proposes a differential growth in stem tissue due to unequal auxin redistribution. Further evidence proposed that flavonoids act as molecular regulators of auxin distribution or flux. It is well known that flavonoids affect auxin distribution, but how intracellular concentration is controlled during the gravitropic response in woody species is still unknown. The MATE family has been widely studied, however the molecular basis of flavonoids transport is still poorly understood. Here, we identified and characterized a full-length cDNA from radiate pine encoding a putative MATE protein. Transcript abundance analysis showed that PrMATE1 is expressed in a spatial and temporal manner in inclined stems. Additionally, PrMATE1 fused to GFP is mainly localized in the vacuolar membrane. A 3D protein model showed 12 transmembrane helices and an open cavity. The protein–ligand interaction was evaluated; favourable binding affinity energies were obtained and suggested epicatechin 3′-O-glucoside as the best putative ligand. In silico mutagenesis analysis was used to identify five residues as important to protein–ligand interaction. The data provide a dynamic view of interaction between PrMATE1 and their putative ligands at the molecular scale. [ABSTRACT FROM AUTHOR]

The plant vacuole is a cellular compartment that is essential to plant development and growth. Often plant vacuoles accumulate specialized metabolites, also called secondary metabolites, which constitute functionally and chemically diverse compounds that exert in planta many essential functions and improve the plant's fitness. These metabolites provide, for example, chemical defense against herbivorous and pathogens or chemical attractants (color and fragrance) to attract pollinators. The chemical composition of the vacuole is dynamic, and is altered during development and as a response to environmental changes. To some extent these alterations rely on vacuolar transporters, which import and export compounds into and out of the vacuole, respectively. During the past decade, significant progress was made in the identification and functional characterization of the transporters implicated in many aspects of plant specialized metabolism. Still, deciphering the molecular players underlying such processes remains a challenge for the future. In this review, we present a comprehensive summary of the most recent achievements in this field. [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR) in bacterial pathogens has become a severe threat to public health. Membrane transporters of the multidrug and toxic compound extrusion (MATE) family contribute critically to MDR, making them promising drug targets. Despite recent advances, structures in different conformations and the mechanistic details of their antiport cycle are still elusive. Here we studied NorM_PS, a representative MATE transporter from Pseudomonas stutzeri , using biochemical assays in combination with hydrogen/deuterium exchange-mass spectrometry. Our results confirm that the antiport is proton dependent and electroneutral with a stoichiometry of two protons per one doubly positively charged substrate. We investigated the conformational dynamics upon substrate binding, and our hydrogen/deuterium exchange-mass spectrometry analysis revealed an occlusion in the proposed binding site as well as a closure of the cytoplasmic cavity and formation of a periplasmic cavity. Together with the results of selected variants (D38N, D373N and Q376A), we propose a six-step rocker-switch model for NorM_PS, which also increases our understanding of related MATE transporters and may help to fight the burden of MDR. [ABSTRACT FROM AUTHOR]

Proanthocyanidin and anthocyanin comprise the most important phenolic components in strawberry fruits. Biosynthesis of these products has been extensively studied, but the transportation and trafficking processes are far from clear. Members of multidrug and toxin extrusion (MATE) transporters are imparted in the secondary metabolites transportation. In this study, we identified 53 MATE family members in the diploid Fragaria vesca genome. Based on homologous strategies, we obtained a potential AtTT12 orthologue in the octoploid strawberry, nominated as FaTT12-1 . It could be detected in both vegetative and reproductive organs, but mainly in young tissues including young leaves and green fruits. Diverse cis -regulatory elements were recognized in the FaTT12-1 promoter. Blue light irradiation and abscisic acid treatment did not impact the expression of FaTT12-1 . But red light irradiation can significantly elevate the transcription. Perturbation of FaTT12-1 via virus induced gene silencing (VIGS) in strawberry fruits did not give obvious visual changes. Fruit anthocyanin remained but proanthocyanidins were affected. In conclusion, we provide evidences that FaTT12-1 is a member of MATE family, specifically involved in proanthocyanidin accumulation in strawberry fruits. [ABSTRACT FROM AUTHOR]

Anthocyanins are a subclass of flavonoids that are synthesized in the endoplasmic reticulum and then transported to the vacuole in plants. Multidrug and toxic compound extrusion transporters (MATE) is a family of membrane transporters that transport ions and secondary metabolites, such as anthocyanins, in plants. Although various studies on MATE transporters have been carried out on different plant species, this is the first comprehensive report to mine the Daucus carota genome to identify the MATE gene family. Our study identified 45 DcMATEs through genome-wide analysis and detected five segmental and six tandem duplications from the genome. The chromosome distribution, phylogenetic analysis, and cis-regulatory elements revealed the structural diversity and numerous functions associated with the DcMATEs. In addition, we analyzed RNA-seq data obtained from the European Nucleotide Archive to screen for the expression of DcMATEs involved in anthocyanin biosynthesis. Among the identified DcMATEs , DcMATE21 correlated with anthocyanin content in the different D. carota varieties. In addition, the expression of DcMATE21 and anthocyanin biosynthesis genes was correlated under abscisic acid, methyl jasmonate, sodium nitroprusside, salicylic acid, and phenylalanine treatments, which were substantiated by anthocyanin accumulation in the in vitro cultures. Further molecular membrane dynamics of DcMATE21 with anthocyanin (cyanidin-3-glucoside) identified the binding pocket, showing extensive H-bond interactions with 10 crucial amino acids present in the transmembrane helix of 7, 8, and 10 of DcMATE21. The current investigation, using RNA-seq, in vitro cultures, and molecular dynamics studies revealed the involvement of DcMATE21 in anthocyanin accumulation in vitro cultures of D. carota. [Display omitted] • We identified 45 DcMATEs containing domain genes in D. carota. • The involvement of DcMATE21 in anthocyanin accumulation was revealed in vitro cultures. • Correlation of DcMATE21 and anthocyanin genes observed under abiotic stress. • The membrane simulation and dynamics of DcMATE21 with cyanidin were unveiled. [ABSTRACT FROM AUTHOR]

Testosterone is considered to be released from Leydig cells via passive diffusion because of its hydrophobicity; however, the exact mechanism underlying testosterone secretion and the transporter involved are both unknown. Multidrug and toxic compound extrusion (MATE) transporters are predominantly found in the kidneys and liver and are thought to function in the elimination of metabolic organic cations during the final step of excretion in the kidney. In contrast, mMATE2 has been shown to be predominantly expressed in testicular Leydig cells. Although the physiological function of mMATE2 in Leydig cells is unknown, we hypothesized that mMATE2 acts as a testosterone exporter and is responsible for the secretion of testosterone from Leydig cells. Therefore, in the present study, we investigated the involvement of the MATE transporter in testosterone secretion from pig Leydig cells. Immunohistochemical analysis with anti-pig MATE2 antiserum indicated that the MATE transporter is present in pig Leydig cells. Additionally, treatment with the MATE inhibitors cimetidine and pyrimethamine reduced the testosterone secretion from pig Leydig cells but increased the intracellular testosterone levels. Estradiol release and intracellular estradiol level induced by human chorionic gonadotropin (hCG) further increased with cimetidine treatment. These results indicated that testosterone produced by hCG treatment is secreted from Leydig cells via the MATE transporter; however, in the presence of cimetidine or pyrimethamine, this MATE transporter-mediated secretion was inhibited, resulting in increased intracellular testosterone levels and estradiol production in Leydig cells. Thus, the MATE transporter may be responsible for testosterone secretion from Leydig cells. [ABSTRACT FROM AUTHOR]

The multidrug and toxin extrusion ( MATE) transporter family comprises 70 members in the Medicago truncatula genome, and they play seemingly important, yet mostly uncharacterized, physiological functions. Here, we employed bioinformatics and molecular genetics to identify and characterize MATE transporters involved in citric acid export, Al3+ tolerance and Fe translocation. Mt MATE69 is a citric acid transporter induced by Fe-deficiency. Overexpression of Mt MATE69 in hairy roots altered Fe homeostasis and hormone levels under Fe-deficient or Fe-oversupplied conditions. Mt MATE66 is a plasma membrane citric acid transporter primarily expressed in root epidermal cells. The mtmate66 mutant had less root growth than the wild type under Al3+ stress, and seedlings were chlorotic under Fe-deficient conditions. Overexpression of Mt MATE66 rendered hairy roots more tolerant to Al3+ toxicity. Mt MATE55 is involved in seedling development and iron homeostasis, as well as hormone signaling. The mtmate55 mutant had delayed development and chlorotic leaves in mature plants. Both knock-out and overexpression mutants of Mt MATE55 showed altered Fe accumulation and abnormal hormone levels compared with the wild type. We demonstrate that the zinc-finger transcription factor Mt STOP is essentially required for Mt MATE66 expression and plant resistance to H+ and Al3+ toxicity. The proper expression of two previously characterized MATE flavonoid transporters Mt MATE1 and Mt MATE2 also depends on several transcription factors. This study reveals not only functional diversity of MATE transporters and regulatory mechanisms in legumes against H+ and Al3+ stresses, but also casts light on their role in metal nutrition and hormone signaling under various stresses. [ABSTRACT FROM AUTHOR]

The multidrug and toxic compound extrusion (MATE) transporters mediate the coupled exchange of organic substrates and monovalent cations have been recently implicated in various plant biological activities. In this work, we isolated a dominant mutant from an Arabidopsis activation-tagging mutant pool. This mutant exhibits pleiotropic phenotype including early flowering, dwarf and bushy architecture, minified lateral organs and early leaf senescence, and is therefore designated early leaf senescence 1-Dominaint ( els1-D ). Genotyping assays showed that els1-D is a gain-of-function mutant of a novel MATE transporter gene, ELS1 , which encodes a close homolog of the previously reported ADP1, BCD1 and DTX50. Further investigations revealed that the overexpression of ELS1 reduces iron content in els1-D , and the accelerated senescence of the detached els1-D leaves can be recovered by exogenous iron supply. In addition, we also found that ELS1 is an iron responsive gene. Based on these findings, we proposed that ELS1 is related to leaf senescence and iron homeostasis in Arabidopsis . [ABSTRACT FROM AUTHOR]

Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was over-expressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters. [ABSTRACT FROM AUTHOR]

Forward genetics approaches have helped elucidate the anthocyanin biosynthetic pathway in plants. Here, we used the Arabidopsis banyuls ( ban) mutant, which accumulates anthocyanins, instead of colorless proanthocyanidin precursors, in immature seeds. In contrast to standard screens for mutants lacking anthocyanins in leaves/stems, we mutagenized ban plants and screened for mutants showing differences in pigmentation of immature seeds. The pale banyuls1 ( pab1) mutation caused reduced anthocyanin pigmentation in immature seeds compared with ban. Immature pab1 ban seeds contained less anthocyanins and flavonols than ban, but showed normal expression of anthocyanin biosynthetic genes. In contrast to pab1, introduction of a flavonol-less mutation into ban did not produce paler immature seeds. Map-based cloning showed that two independent pab1 alleles disrupted the MATE-type transporter gene FFT/DTX35. Complementation of pab1 with FFT confirmed that mutation in FFT causes the pab1 phenotype. During development, FFT promoter activity was detected in the seed-coat layers that accumulate flavonoids. Anthocyanins accumulate in the vacuole and FFT fused to GFP mainly localized in the vacuolar membrane. Heterologous expression of grapevine MATE-type anthocyanin transporter gene partially complemented the pab1 phenotype. These results suggest that FFT acts at the vacuolar membrane in anthocyanin accumulation in the Arabidopsis seed coat, and that our screening strategy can reveal anthocyanin-related genes that have not been found by standard screening. [ABSTRACT FROM AUTHOR]

NorM from Neisseria gonorrhoeae (NorM-NG) belongs to the multidrug and toxic compound extrusion (MATE) family of membrane-transport proteins, which can extrude cytotoxic chemicals across cell membranes and confer multidrug resistance. Here, the structure determination of NorM-NG is described, which had been hampered by low resolution (∼4 Å), data anisotropy and pseudo-merohedral twinning. The crystal structure was solved using molecular replacement and was corroborated by conducting a difference Fourier analysis. The NorM-NG structure displays an extracellular-facing conformation, similar to that of NorM-NG bound to a crystallization chaperone. The approaches taken to determine the NorM-NG structure and the lessons learned from this study are discussed, which may be useful for analyzing X-ray diffraction data with similar shortcomings. [ABSTRACT FROM AUTHOR]

The growth of higher plants is under complex regulation to ensure the elaboration of developmental programmes under a changing environment. To dissect these regulatory circuits, we carried out genetic screens for Arabidopsis abnormal shoot (abs) mutants with altered shoot development. Here, we report the isolation of two dominant mutants, abs3-1D and abs4-1D, through activation tagging. Both mutants showed a 'bushy' loss of apical dominance phenotype. ABS3 and ABS4 code for two closely related putative Multidrug and Toxic Compound Extrusion (MATE) family of efflux transporters, respectively. ABS3 and ABS4, as well as two related MATE genes, ABS3-Like1 (ABS3L1) and ABS3L2, showed diverse tissue expression profiles but their gene products all localized to the late endosome/pre-vacuole (LE/PVC) compartment. The over-expression of these four genes individually led to the inhibition of hypocotyl cell elongation in the light. On the other hand, the quadruple knockout mutant (mateq) showed the opposite phenotype of an enhanced hypocotyl cell elongation in the light. Hypocotyl cell elongation and de-etiolation processes in the dark were also affected by the mutations of these genes. Exogenously applied sucrose attenuated the inhibition of hypocotyl elongation caused by abs3-1D and abs4-1D in the dark, and enhanced the hypocotyl elongation of mateq under prolonged dark treatment. We determined that ABS3 genetically interacts with the photoreceptor gene PHYTOCHROME B (PHYB). Our results demonstrate that ABS3 and related MATE family transporters are potential negative regulators of hypocotyl cell elongation and support a functional link between the endomembrane system, particularly the LE/PVC, and the regulation of plant cell elongation. [ABSTRACT FROM AUTHOR]

Highlights: [•] Multidrug transporters cause failure of antibacterial and cancer chemotherapy. [•] Inhibition of these pumps is a target for therapeutic intervention. [•] Molecular explanations for multidrug resistance (MDR) pump promiscuity are required. [•] Structures of these pumps are enabling insights into their mechanisms. [•] Development of inhibitors using these structural advances is a genuine possibility. [Copyright &y& Elsevier]

Heterologous expression of membrane proteins in Escherichia coli often requires optimization to overcome problems with toxicity of the recombinant protein to the host cell. A number of Gateway-based destination vectors were constructed to investigate expression of membrane proteins using a high-throughput approach. These vectors were tested using putative drug transporter proteins from the multidrug and toxic compound extrusion (MATE) family and the resistance-nodulation-cell division superfamily encoded by the human pathogen Acinetobacter baumannii. Active transport of antibiotics and antiseptics mediated by efflux proteins contributes to the high level of multidrug resistance observed in A. baumannii. Substrates for 4 of the 5 putative efflux proteins investigated were identified using the expression vectors designed in this study. Additionally, a Gateway-based suicide vector was designed for construction of specific A. baumannii insertion disruption mutants. This knockout cloning strategy was tested and shown to be successful in inactivating AbeM4, a putative MATE family protein. Therefore, we have shown that the Gateway-based vectors constructed in this study are versatile tools that can be used for manipulation and characterization of membrane proteins. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

FLOWER FLAVONOID TRANSPORTER (FFT) encodes a multidrug and toxin efflux family transporter in Arabidopsis thaliana. FFT (AtDTX35) is highly transcribed in floral tissues, the transcript being localized to epidermal guard cells, including those of the anthers, stigma, siliques and nectaries. Mutant analysis demonstrates that the absence of FFT transcript affects flavonoid levels in the plant and that the altered flavonoid metabolism has wide-ranging consequences. Root growth, seed development and germination, and pollen development, release and viability are all affected. Spectrometry of mutant versus wild-type flowers shows altered levels of a glycosylated flavonol whereas anthocyanin seems unlikely to be the substrate as previously speculated. Thus, as well as adding FFT to the incompletely described flavonoid transport network, it is found that correct reproductive development in Arabidopsis is perturbed when this particular transporter is missing. [ABSTRACT FROM PUBLISHER]

The Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY 5 gene ( EDS5) is required for salicylic acid (SA) synthesis in pathogen-challenged plants. SA and EDS5 have an important role in the Arabidopsis RCY1 gene-conferred resistance against the yellow strain of Cucumber mosaic virus [CMV(Y)], a Bromoviridae, and HRT-conferred resistance against the Tombusviridae, Turnip crinkle virus (TCV). EDS5 expression and SA accumulation are induced in response to CMV(Y) inoculation in the RCY1-bearing ecotype C24. To further discern the involvement of EDS5 in Arabidopsis defence against viruses, we overexpressed the EDS5 transcript from the constitutively expressed Cauliflower mosaic virus 35S gene promoter in ecotype C24. In comparison to the non-transgenic control, the basal level of salicylic acid (SA) was twofold higher in the 35S:EDS5 plant. Furthermore, viral spread and the size of the hypersensitive response associated necrotic local lesions (NLL) were more highly restricted in CMV(Y)-inoculated 35S:EDS5 than in the non-transgenic plant. The heightened restriction of CMV(Y) spread was paralleled by more rapid induction of the pathogenesis-related gene, PR-1, in the CMV(Y)-inoculated 35S:EDS5 plant. The 35S:EDS5 plant also had heightened resistance to the virulent CMV strain, CMV(B2), and TCV. These results suggest that, in addition to R gene-mediated gene-for-gene resistance , EDS5 is also important for basal resistance to viruses. However, while expression of the Pseudomonas putida nahG gene, which encodes the SA-degrading salicylate hydroxylase, completely suppressed 35S:EDS5-conferred resistance against CMV(Y) and TCV, it only partially compromised resistance against CMV(B2), indicating that SA-dependent and -independent mechanisms are associated with 35S:EDS5-conferred resistance against viruses. [ABSTRACT FROM AUTHOR]

Bacteria use a number of mechanisms to defend themselves from antimicrobial drugs. One important defense strategy is the ability to export drugs by multidrug transporters. One class of multidrug transporter, the so-called multidrug and toxic compound extrusion (MATE) transporters, extrude a variety of antibiotic compounds from the bacterial cytoplasm. These MATE transporters are driven by a Na+, H+, or combined Na+/H+ gradient, and act as antiporters to drive a conformational change in the transporter from the outward to the inward-facing conformation. In the inward-facing conformation, a chemical compound (drug) binds to the protein, resulting in a switch to the opposite conformation, thereby extruding the drug. Using molecular dynamics simulations, we now report the structural basis for Na+ and H+ binding in the dual ion coupled MATE transporter ClbM from Escherichia coli , which is connected to colibactin-induced genotoxicity, yielding novel insights into the ion/drug translocation mechanism of this bacterial transporter. Unlabelled Image • MATE transporters expel drugs from the bacterial cytoplasm. • Drug extrusion is coupled to a sodium and proton gradient in ClbM. • Simulations clarify the location of the proton and sodium binding site. • Mechanistic implications of ion binding are proposed. [ABSTRACT FROM AUTHOR]

Ionic aluminum (Al) in acidic soils, comprising approximately 50% of arable land globally, is highly toxic to most plant species. Populus grow naturally in acidic soils and tolerate high concentrations of Al. Multidrug and toxic compound extrusion (MATE) family genes in plants are involved in responses to Al tolerance. To date, however, the functional roles of the MATE genes in Populus remain unclear. In the present study, 71 putative MATE transporters were predicted in the genome of Populus trichocarpa. The chromosome distribution, phylogenetic relationships, and expression level analysis revealed that four candidate MATE genes belonging to subgroup IIIc might contribute to high Al tolerance in poplar. Further, the expression levels of two subgroup IIIc members, PtrMATE1 and PtrMATE2, were induced by Al stress. Transient expression in onion epidermal cells showed that PtrMATE1 was localized to the plasma membrane. Overexpression of PtrMATE1 increased Al-induced secretion of citrate from the root apex of transgenic plants. Al-induced inhibition of root growths were alleviated in both PtrMATE1 overexpression lines in Populus and in Arabidopsis compared with wild-type plants. In addition, PtrMATE1 expression was induced at 12 h after exposure to Al stress whereas PtrMATE2 expression was induced at 24 h, indicating that these proteins coordinately function in response to Al stress in poplar. Taken together, these results provide important insights into the molecular mechanisms involved in Al tolerance in poplar., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

Soybean seeds are a rich source of phenolic compounds, especially isoflavonoids, which are important nutraceuticals. Our study using 14 wild- and 16 cultivated-soybean accessions shows that seeds from cultivated soybeans generally contain lower total antioxidants compared to their wild counterparts, likely an unintended consequence of domestication or human selection. Using a recombinant inbred population resulting from a wild and a cultivated soybean parent and a bin map approach, we have identified an overlapping genomic region containing major quantitative trait loci (QTLs) that regulate the seed contents of total antioxidants, phenolics, and flavonoids. The QTL for seed antioxidant content contains 14 annotated genes based on the Williams 82 reference genome (Gmax1.01). None of these genes encodes functions that are related to the phenylpropanoid pathway of soybean. However, we found three putative Multidrug And Toxic Compound Extrusion (MATE) transporter genes within this QTL and one adjacent to it (GmMATE1-4). Moreover, we have identified non-synonymous changes between GmMATE1 and GmMATE2, and that GmMATE3 encodes an antisense transcript that expresses in pods. Whether the polymorphisms in GmMATE proteins are major determinants of the antioxidant contents, or whether the antisense transcripts of GmMATE3 play important regulatory roles, awaits further functional investigations.