Characterization of Coptis japonica CjABCB2, an ATP-binding cassette protein involved in alkaloid transport.

Higher plants produce a large number of secondary metabolites. Among these are the alkaloids, a group of small nitrogen-containing molecules. Alkaloids often have strong biological activity that protects alkaloid-producing plants from herbivores, and often accumulate to high concentrations in a specific organelle of a particular organ in the producing plant. However, knowledge of the membrane transport mechanism of alkaloids is still limited. Coptis japonica, a perennial Ranunculaceous plant, produces the benzylisoquinoline alkaloid berberine. This alkaloid, though biosynthesized in root tissues, accumulates in the rhizome, suggesting translocation of the molecule via xylem. In this study, a gene encoding a ATP-binding cassette (ABC) protein of B-type, Cjabcb2, was isolated from C. japonica. Northern analysis showed that Cjabcb2 was preferentially expressed in the rhizome, which is the sink organ of berberine. Functional analysis of CjABCB2 using yeast suggested that CjABCB2 transports berberine in an inward direction. Membrane separation and in situ hybridization data indicated that CjABCB2 might be involved in translocation of berberine from the root to the rhizome by transporting berberine at the plasma membrane of cells around the xylem of the rhizome.

Immuno-PCR--a new tool for paleomicrobiology: the plague paradigm.

BACKGROUND: The cause of past plague pandemics was controversial but several research teams used PCR techniques and dental pulp as the primary material to reveal that they were caused by Yersinia pestis. However, the degradation of DNA limits the ability to detect ancient infections. METHODS: We used for the first time immuno-PCR to detect Yersinia pestis antigens; it can detect protein concentrations 70 times lower than the standard ELISA. After determining the cut-off value, we tested 34 teeth that were obtained from mass graves of plague, and compared previous PCR results with ELISA and immuno-PCR results. RESULTS: The immuno-PCR technique was the most sensitive (14 out of 34) followed by the PCR technique (10 out of 34) and ELISA (3 out of 34). The combination of these three methods identified 18 out of 34 (53%) teeth as presumably being from people with the plague. CONCLUSION: Immuno-PCR is specific (no false-positive samples were found) and more sensitive than the currently used method to detect antigens of ancient infections in dental pulp. The combination of three methods, ELISA, PCR and immuno-PCR, increased the capacity to identify ancient pathogens in dental pulp.

Brief communication: co-detection of Bartonella quintana and Yersinia pestis in an 11th-15th burial site in Bondy, France.

Historical and anthropological data suggest that skeletons excavated from an 11th to 15th century mass grave in Bondy, France, may be those of victims of the Great Plague. Using high-throughput real-time PCR investigation of the dental pulp collected from 14 teeth from five such skeletons, we detected Bartonella quintana DNA in three individuals and Yersinia pestis DNA in two individuals. DNA from five other deadly pathogens was not found. Suicide PCR genotyping confirmed Y. pestis DNA belonging to the Orientalis biotype. One individual had co-infection. These data suggest a plague epidemic in a population already infected by the body louse-transmitted B. quintana or a body louse-driven transmission of the plague that drove a medieval epidemic in inland Europe.

A common highly conserved cadmium detoxification mechanism from bacteria to humans: heavy metal tolerance conferred by the ATP-binding cassette (ABC) transporter SpHMT1 requires glutathione but not metal-chelating phytochelatin peptides.

Cadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers' yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the co-substrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO(3), As(III), As(V), CuSO(4), or HgCl(2). Abolishment of the catalytic activity by expression of SpHMT1(K623M) mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHMA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans.

AtMRP6/AtABCC6, an ATP-binding cassette transporter gene expressed during early steps of seedling development and up-regulated by cadmium in Arabidopsis thaliana.

BACKGROUND: ABC proteins constitute one of the largest families of transporters found in all living organisms. In Arabidopsis thaliana, 120 genes encoding ABC transporters have been identified. Here, the characterization of one member of the MRP subclass, AtMRP6, is described. RESULTS: This gene, located on chromosome 3, is bordered by AtMRP3 and AtMRP7. Using real-time quantitative PCR (RT-Q-PCR) and the GUS reporter gene, we found that this gene is essentially expressed during early seedling development, in the apical meristem and at initiation point of secondary roots, especially in xylem-opposite pericycle cells where lateral roots initiate. The level of expression of AtMRP6 in response to various stresses was explored and a significant up-regulation after cadmium (Cd) treatment was detected. Among the three T-DNA insertion lines available from the Salk Institute library, two knock-out mutants, Atmrp6.1 and Atmrp6.2 were invalidated for the AtMRP6 gene. In the presence of Cd, development of leaves was more affected in the mutants than wild-type plants, whereas root elongation and ramification was comparable. CONCLUSION: The position of AtMRP6 on chromosome 3, flanked by two other MRP genes, (all of which being induced by Cd) suggests that AtMRP6 is part of a cluster involved in metal tolerance, although additional functions in planta cannot be discarded.

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. A consolidated nomenclature will provide much-needed clarity and a framework for future research.

The ATP binding cassette transporter AtMRP5 modulates anion and calcium channel activities in Arabidopsis guard cells.

Stomatal guard cells control CO(2) uptake and water loss between plants and the atmosphere. Stomatal closure in response to the drought stress hormone, abscisic acid (ABA), results from anion and K(+) release from guard cells. Previous studies have shown that cytosolic Ca(2+) elevation and ABA activate S-type anion channels in the plasma membrane of guard cells, leading to stomatal closure. However, membrane-bound regulators of abscisic acid signaling and guard cell anion channels remain unknown. Here we show that the ATP binding cassette (ABC) protein AtMRP5 is localized to the plasma membrane. Mutation in the AtMRP5 ABC protein impairs abscisic acid and cytosolic Ca(2+) activation of slow (S-type) anion channels in the plasma membrane of guard cells. Interestingly, atmrp5 insertion mutant guard cells also show impairment in abscisic acid activation of Ca(2+)-permeable channel currents in the plasma membrane of guard cells. These data provide evidence that the AtMRP5 ABC transporter is a central regulator of guard cell ion channel during abscisic acid and Ca(2+) signal transduction in guard cells.

Transport of antimony salts by Arabidopsis thaliana protoplasts over-expressing the human multidrug resistance-associated protein 1 (MRP1/ABCC1).

ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of "safe-food" strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.

Heavy metal transport by AtHMA4 involves the N-terminal degenerated metal binding domain and the C-terminal His11 stretch.

The Arabidopsis thaliana AtHMA4 is a P1B-type ATPase that clusters with the Zn/Cd/Pb/Co subgroup. It has been previously shown, by heterologous expression and the study of AtHMA4 knockout or overexpressing lines in Arabidopsis , that AtHMA4 is implicated in zinc homeostasis and cadmium tolerance. Here, we report the study of the heterologous expression of AtHMA4 in the yeast Saccharomyces cerevisiae. AtHMA4 expression resulted in an increased tolerance to Zn, Cd and Pb and to a phenotypic complementation of hypersensitive mutants. In contrast, an increased sensitivity towards Co was observed. An AtHMA4::GFP fusion protein was observed in endocytic vesicles and at the yeast plasma membrane. Mutagenesis of the cysteine and glutamate residues from the N-ter degenerated heavy metal binding domain impaired the function of AtHMA4. It was also the case when the C-ter His11 stretch was deleted, giving evidence that these amino acids are essential for the AtHMA4 binding/translocation of metals.

Inventory and comparative analysis of rice and Arabidopsis ATP-binding cassette (ABC) systems.

ATP-binding cassette (ABC) proteins constitute a large superfamily found in all kingdoms of living organisms. The recent completion of two draft sequences of the rice (Oryza sativa) genome allowed us to analyze and classify its ABC proteins and to compare to those in Arabidopsis thaliana. We identified a similar number of ABC proteins in rice and Arabidopsis (121 versus 120), despite the rice genome being more than three times the size of Arabidopsis. Both Arabidopsis and rice have representative members in all seven major subfamilies of ABC ATPases (A to G) commonly found in eukaryotes. This comparative analysis allowed the detection of 29 potential orthologous sequences in Arabidopsis and rice. However, plant share with prokaryotes a specific set of ABC systems that is not detected in animals. These ABC systems might be inherited from the cyanobacterial ancestor of chloroplasts. The present work provides the first complete inventory of rice ABC proteins and an updated inventory of those proteins in Arabidopsis.

Differential sensitivity of plant and yeast MRP (ABCC)-mediated organic anion transport processes towards sulfonylureas.

The role of ATP-binding cassette (ABC) proteins such as multidrug resistance-associated proteins (MRPs) is critical in drug resistance in cancer cells and in plant detoxification processes. Due to broad substrate spectra, specific modulators of these proteins are still lacking. Sulfonylureas such as glibenclamide are used to treat non-insulin-dependent diabetes since they bind to the sulfonylurea receptor. Glibenclamide also inhibits the cystic fibrosis transmembrane conductance regulator, p-glycoprotein in animals and guard cell ion channels in plants. To investigate whether this compound is a more general blocker of ABC transporters the sensitivity of ABC-type transport processes across the vacuolar membrane of plants and yeast towards glibenclamide was evaluated. Glibenclamide inhibits the ATP-dependent uptake of beta-estradiol 17-(beta-D-glucuronide), lucifer yellow CH, and (2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein. Transport of glutathione conjugates into plant but not into yeast vacuoles was drastically reduced by glibenclamide. Thus, irrespective of the homologies between plant, yeast and animal MRP transporters, specific features of plant vacuolar MRPs with regard to sensitivity towards sulfonylureas exist. Glibenclamide could be a useful tool to trap anionic fluorescent indicator dyes in the cytosol.

The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use.

Carbon dioxide uptake and water release through stomata, controlling the opening and closure of stomatal pore size in the leaf surface, is critical for optimal plant performance. Stomatal movements are regulated by multiple signalling pathways involving guard cell ion channels. Using reverse genetics, we recently isolated a T-DNA insertion mutant for the Arabidopsis ABC-transporter AtMRP5 (mrp5-1). Guard cells from mrp5-1 mutant plants were found to be insensitive to the sulfonylurea compound glibenclamide, which in the wild type induces stomatal opening in the dark. Here, we report that the knockout in AtMRP5 affects several signalling pathways controlling stomatal movements. Stomatal apertures of mrp5-1 and wild-type Ws-2 were identical in the dark. In contrast, opening of stomata of mrp5-1 plants was reduced in the light. In the light, stomatal closure of mrp5-1 was insensitive to external calcium and abscisic acid, a phytohormone responsible for stomatal closure during drought stress. In contrast to Ws-2, the phytohormone auxin could not stimulate stomatal opening in the mutant in darkness. All stomatal phenotypes were complemented in transgenic mrp5-1 plants transformed with a cauliflower mosaic virus (CaMV) 35S-AtMRP5 construct. Both whole-plant and single-leaf gas exchange measurements demonstrated a reduced transpiration rate of mrp5-1 in the light. Excised leaves of mutant plants exhibited reduced water loss, and water uptake was strongly decreased at the whole-plant level. Finally, if plants were not watered, mrp5-1 plants survived much longer due to reduced water use. Analysis of CO2 uptake and transpiration showed that mrp5-1 plants have increased water use efficiency. Mutant plants overexpressing AtMRP5 under the control of the CaMV 35S promoter again exhibited wild-type characteristics. These results demonstrate that multidrug resistance-associated proteins (MRPs) are important components of guard cell functioning.

Engineering tolerance and accumulation of lead and cadmium in transgenic plants.

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.

Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica.

Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica, a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter (Cjmdr1) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1-injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome.

Heavy metal toxicity: cadmium permeates through calcium channels and disturbs the plant water status.

Because plant wilting has been described as a consequence of cadmium (Cd2+) toxicity, we investigate Cd2+ effects on plant water losses, gas exchanges and stomatal behaviour in Arabidopsis thaliana L. Effects of 1-week Cd2+ application in hydroponic condition (CdCl2 10-100 micro m) were analyzed. A 10- micro m Cd2+ concentration had no significant effect on the plant-water relationship and carbon assimilation. At higher Cd2+ concentrations, a Cd2+ -dependent decrease in leaf conductance and CO2 uptake was observed despite the photosynthetic apparatus appeared not to be affected as probed by fluorescence measurements. In epidermal strip bioassays, nanomolar Cd2+ concentrations reduced stomatal opening under light in A. thaliana, Vicia faba and Commelina communis. Application of 5 micro m ABA limited the root-to-shoot translocation of cadmium. However, the Cd2+-induced stomatal closure was likely ABA-independent, since a 5-day treatment with 50 micro m Cd2+ did not affect the plant relative water content. Additionally, a similar Cd2+-induced stomatal closure was observed in the ABA insensitive mutant abi1-1. Interestingly, this mutant displayed a higher transpiration rate than the wild type but did not accumulate more Cd2+, arguing that Cd2+ uptake is not dependent only on the transpiration flow. Application of putative calcium channels inhibitors suppressed the inhibitory effect of Cd2+ in epidermal strip experiments, suggesting that Cd2+ could enter the guard cell through calcium channels. Patch-clamp studies with V. faba guard cell protoplasts showed that plasma membrane K+ channels were insensitive to external Cd2+ application whereas Ca2+ channels were found permeable to Cd2+. In conclusion, we propose that Cd2+ affects guard cell regulation in an ABA-independent manner by entering the cytosol via Ca2+ channels.

Multifunctionality of plant ABC transporters--more than just detoxifiers.

The ABC-transporter superfamily is one of the largest protein families, and members can be found in bacteria, fungi, plants and animals. The first reports on plant ABC transporters showed that they are implicated in detoxification processes. The recent completion of the genomic sequencing of Arabidopsis thaliana (L.) Heynh. [Arabidopsis Genome Initiative (2000) Nature 408:796-815] showed that Arabidopsis contains more than 100 ABC-type proteins; 53 genes code for so-called full-size transporters, which are large proteins of about 150 kDa consisting of two hydrophobic and two hydrophilic domains. The large number of genes in the MDR/MRP and PDR5-like sub-clusters and the strong sequence homology found in many cases suggest functional redundancy. One reason for the high number of genes can be attributed to the duplication of large segments of Arabidopsis chromosomes. Recent results indicate that the function of this protein family is not restricted to detoxification processes. Plant ABC transporters have been demonstrated to participate in chlorophyll biosynthesis, formation of Fe/S clusters, stomatal movement, and probably ion fluxes; hence they may play a central role in plant growth and developmental processes.