Cell and molecular biology of ATP-binding cassette proteins in plants.

ATP-binding cassette (ABC) proteins constitute a large and diverse superfamily of membrane-bound and soluble proteins, which are involved in a wide range of biological processes in all organisms from prokaryotes to eukaryotes. Genome analyses of model plants, for example, Arabidopsis and rice, have revealed that plants have more than double numbers of this family member in their genomes compared to animals and insects. In recent years, various biochemical and physiological functions of ABC proteins in plants have been reported.

Improved accumulation of ajmalicine and tetrahydroalstonine in Catharanthus cells expressing an ABC transporter.

The biosynthetic pathway of monoterpenoid indole alkaloids in Catharanthus roseus is located throughout various membranes at both the cellular and intercellular levels. ATP-binding cassette (ABC) transporters are known to export vincristine and vinblastine from human cancer cells. It has recently been shown that ABC transporters are also involved in the transport of various monoterpenoid alkaloids in Catharanthus roseus cells.

Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid.

The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane alpha-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level.

Prenylation of flavonoids by biotransformation of yeast expressing plant membrane-bound prenyltransferase SfN8DT-1.

Prenylated flavonoids are natural products that exhibit diverse biological effects and often represent the active components of various medicinal plants. This study demonstrated the production of prenylated naringenin by biotransformation using transgenic yeast expressing naringenin 8-dimethylallyltransferase, a membrane-bound enzyme, without feeding of prenyl donors. This method provides the possibility of generating prenylated flavonoids that occur rarely in nature.

Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum.

Alkaloids play a key role in plant defense mechanisms against pathogens and herbivores, but the plants themselves need to cope with their toxicity as well. The major alkaloid of the Nicotiana species, nicotine, is translocated via xylem transport from the root tissues where it is biosynthesized to the accumulation sites, the vacuoles of leaves. To unravel the molecular mechanisms behind this membrane transport, we characterized one transporter, the tobacco (Nicotiana tabacum) jasmonate-inducible alkaloid transporter 1 (Nt-JAT1), whose expression was coregulated with that of nicotine biosynthetic genes in methyl jasmonate-treated tobacco cells.

Multidrug and toxic compound extrusion-type transporters implicated in vacuolar sequestration of nicotine in tobacco roots.

Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane.

Molecular cloning and characterization of a cDNA for pterocarpan 4-dimethylallyltransferase catalyzing the key prenylation step in the biosynthesis of glyceollin, a soybean phytoalexin.

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C5 prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety.

Cross-sectional analysis of germline BRCA1 and BRCA2 mutations in Japanese patients suspected to have hereditary breast/ovarian cancer.

The prevalence of BRCA1/2 germline mutations in Japanese patients suspected to have hereditary breast/ovarian cancer was examined by a multi-institutional study, aiming at the clinical application of total sequencing analysis and validation of assay sensitivity in Japanese people using a cross-sectional approach based on genetic factors estimated from personal and family histories. One hundred and thirty-five subjects were referred to the genetic counseling clinics and enrolled in the study. Full sequencing analysis of the BRCA1/2 gene showed 28 types of deleterious mutations in 36 subjects (26.

Production of the monoterpene limonene and modulation of apoptosis-related proteins in embryonic-mouse NIH 3T3 fibroblast cells by introduction of the limonene synthase gene isolated from Japanese catnip (Schizonepeta tenuifolia).

The monoterpene D-limonene shows cancer preventative and cancer therapeutic activities in vitro and in vivo. Unlike plants, animals are unable to synthesize limonene de novo and obtain limonene through dietary sources. In the present study we established transgenic mouse embryonic NIH 3T3 fibroblast cells that produce limonene by introducing the D-limonene synthase gene obtained from Japanese catnip (Schizonepeta tenuifolia).

Plant ABC proteins--a unified nomenclature and updated inventory.

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products.

Coordination between leaf and stem traits related to leaf carbon gain and hydraulics across 32 drought-tolerant angiosperms.

We examined 15 traits in leaves and stems related to leaf C economy and water use for 32 co-existing angiosperms at ridge sites with shallow soil in the Bonin Islands. Across species, stem density was positively correlated to leaf mass per area (LMA), leaf lifespan (LLS), and total phenolics and condensed tannins per unit leaf N (N-based), and negatively correlated to leaf osmotic potential and saturated water content in leaves. LMA and LLS were negatively correlated to photosynthetic parameters, such as area-, mass-, and N-based assimilation rates.

Galactinol synthase gene of Coptis japonica is involved in berberine tolerance.

Many plant secondary metabolites show strong biological activities and are potentially also toxic to plants, while plants producing such active compounds are usually insensitive to their own metabolites, suggesting that they have species-specific detoxification mechanisms. In order to clarify the detoxification mechanism of alkaloids, we used cultured cells of Coptis japonica, which are capable of producing a yellow benzylisoquinoline alkaloid, berberine, and accumulate it in the vacuole. Unlike other plant cells that do not produce berberine, C.

Cloning and characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase of Sophora flavescens.

Prenylated flavonoids are natural compounds that often represent the active components in various medicinal plants and exhibit beneficial effects on human health. Prenylated flavonoids are hybrid products composed of a flavonoid core mainly attached to either 5-carbon (dimethylallyl) or 10-carbon (geranyl) prenyl groups derived from isoprenoid (terpenoid) metabolism, and the prenyl groups are crucial for their biological activity. Prenylation reactions in vivo are crucial coupling processes of two major metabolic pathways, the shikimate-acetate and isoprenoid pathways, in which these reactions are also known as a rate-limiting step.

Signaling from soybean roots to rhizobium: An ATP-binding cassette-type transporter mediates genistein secretion.

Legume plants have a unique ability to fix atmospheric nitrogen via symbiosis with rhizobia. For the establishment of symbiosis, legume plants secrete signaling molecules such as flavonoids from root tissues, leading to the attraction of rhizobia and the induction of rhizobial nod genes. Genistein and daidzein are found in soybean root exudates and function as signal molecules in soybean-Bradyrhizobium japonicum chemical communication.

Plants utilize isoprene emission as a thermotolerance mechanism.

Isoprene is a volatile compound emitted from leaves of many plant species in large quantities, which has an impact on atmospheric chemistry due to its massive global emission rate (5 x 10(14) carbon g year(-1)) and its high reactivity with the OH radical, resulting in an increase in the half-life of methane. Isoprene emission is strongly induced by the increase in isoprene synthase activity in plastids at high temperature in the day time, which is regulated at its gene expression level in leaves, while the physiological meaning of isoprene emission for plants has not been clearly demonstrated. In this study, we have functionally overexpressed Populus alba isoprene synthase in Arabidopsis to observe isoprene emission from transgenic plants.

Accumulation and membrane transport of plant alkaloids.

Among a large number of plant secondary metabolites, alkaloids comprise one of the most important groups due to their strong and divergent biological activities, and some are applied for clinical use. Alkaloids are often highly accumulated in particular organs of medicinal plants, which are called the 'medicinal part', whereas it is known that some alkaloids are translocated from source organs to such sink organs. The movement of biosynthetic intermediates from specific cells to other types of cells in tissue, and further detailed movement within the organelles in a cell is also suggested.

Involvement of a soybean ATP-binding cassette-type transporter in the secretion of genistein, a signal flavonoid in legume-Rhizobium symbiosis.

Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells.

Involvement of FpTRP26, a thioredoxin-related protein, in oxalic acid-resistance of the brown-rot fungus Fomitopsis palustris.

Brown-rot fungus Fomitopsis palustris grows vigorously at high concentrations of oxalic acid (OA), which is fungal metabolite during wood decay. We isolated a cDNA FpTRP26 from F. palustris by functional screening of yeast transformants with cDNAs grown on plates containing OA.

Human MDR1 and MRP1 recognize berberine as their transport substrate.

To examine whether human ATP-binding cassette (ABC) transporters play a role in the detoxification of plant alkaloid berberine, we investigated berberine transport using multidrug resistance protein1 (MDR1) and multidrug resistance-associated protein1 (MRP1). Cells expressing MDR1 or MRP1 accumulated less berberine. Berberine accumulation depended on the cellular ATP level, and was reversed by typical inhibitors of MDR1, suggesting that human MDR1 and MRP1 directly efflux berberine as their substrate.

Genome-wide analysis of ATP-binding cassette (ABC) proteins in a model legume plant, Lotus japonicus: comparison with Arabidopsis ABC protein family.

ATP-binding cassette (ABC) proteins constitute a large family in plants with more than 120 members each in Arabidopsis and rice, and have various functions including the transport of auxin and alkaloid, as well as the regulation of stomata movement. In this report, we carried out genome-wide analysis of ABC protein genes in a model legume plant, Lotus japonicus. For analysis of the Lotus genome sequence, we devised a new method 'domain-based clustering analysis', where domain structures like the nucleotide-binding domain (NBD) and transmembrane domain (TMD), instead of full-length amino acid sequences, are used to compare phylogenetically each other.

Bowman-birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast.

Cultured Coptis japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells.

Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica.

Selected cultured Coptis japonica cells produce a large amount of the benzylisoquinoline alkaloid berberine. Previous studies have suggested that berberine productivity is controlled at the transcript level of biosynthetic genes. We have identified a regulator of transcription in berberine biosynthesis using functional genomics with a transient RNA interference (RNAi) and overexpression of the candidate gene.

Influence of ascorbic acid on bonding of peroxide-affected dentin and 4-META/MMA-TBB resin.

The purpose of this study was to evaluate the tensile bond strength (TBS) to peroxide-exposed dentin. Furthermore, the effect of ascorbic acid (AA) on the bond strength of peroxide-exposed dentin was investigated. Extracted bovine dentin was exposed to 10% carbamide peroxide, 30% hydrogen peroxide, or distilled water for 30 min, then treated with 10% AA (0, 30, 90, and 180 min), and conditioned with 10% citric acid/3% ferric chloride.

Heterologous expression of a mammalian ABC transporter in plant and its application to phytoremediation.

Mammalian ATP-binding cassette (ABC) transporters involved in the multidrug-resistance of cancer cells can efflux cytotoxic compounds that show a wide variety of chemical structures and biological activities. Human multidrug resistance-associated protein (hMRP1) is one of the most intensively studied ABC transporters and many substrates have been identified, including both organic and inorganic compounds. In an attempt at novel 'transport engineering' using hMRP1 as a molecular pump, we established transgenic tobacco plants that showed clear resistance to cadmium and daunorubicin, although they were not resistant to etoposide, another known substrate of hMRP1.

Functional characterization of OsPPT1, which encodes p-hydroxybenzoate polyprenyltransferase involved in ubiquinone biosynthesis in Oryza sativa.

Prenylation of the aromatic intermediate p-hydroxybenzoate (PHB) is a critical step in ubiquinone (UQ) biosynthesis. The enzyme that catalyzes this prenylation reaction is p-hydroxybenzoate polyprenyltransferase (PPT), which substitutes an aromatic proton at the m-position of PHB with a prenyl chain provided by polyprenyl diphosphate synthase. The rice genome contains three PPT candidates that share significant similarity with the yeast PPT (COQ2 gene), and the rice gene showing the highest similarity to COQ2 was isolated by reverse transcription-PCR and designated OsPPT1a.