Cyrille Forestier

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.

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.

The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement

In the present study, we investigated a new member of the ABC transporter superfamily of Arabidopsis thaliana, AtMRP5. AtMRP5 encodes a 167 kDa protein and exhibits low glutathione conjugate and glucuronide conjugate transport activity. Promotor‐β‐glucuronidase fusion constructs showed that AtMRP5 is expressed mainly in the vascular bundle and in the epidermis, especially guard cells. Using reverse genetics, we identified a plant with a T‐DNA insertion in AtMRP5 (mrp5‐1). mrp5‐1 exhibited decreased root growth and increased lateral root formation. Auxin levels in the roots of mrp5‐1 plants were increased. This observation may indicate that AtMRP5 works as an auxin conjugate transporter or that mutant plants are affected in ion uptake, which may lead to changes in auxin concentrations. Experiments on epidermal strips showed that in contrast to wild type, the sulfonylurea glibenclamide had no effect on stomatal opening in mrp5‐1 plants. This result strongly suggests that AtMRP5 may also function as an ion channel regulator.

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 [1–3] , 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.

The ATP Binding Cassette Transporter AtMRP5 Modulates Anion and Calcium Channel Activities in Arabidopsis Guard Cells

Stomatal guard cells control CO2 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 Ca2+ 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 Ca2+ 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 Ca2+-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 Ca2+ signal transduction in guard cells.

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

BackgroundABC 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.ResultsThis 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.ConclusionThe 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.

Pharmacological properties of slow anion currents in intact guard cells of Arabidopsis. Application of the discontinuous single-electrode voltage-clamp to different species

Abstract More electrophysiological studies have been carried out on guard cells than on any other cell type of vascular plants. The characterization of their ion channels has been achieved using mainly the whole-cell patch-clamp technique applied to guard-cell protoplasts. The aim of this study was to obtain recordings of ion channel currents in intact guard cells and especially of slow anion channels of Arabidopsis thaliana, a species of fundamental genetic interest. Application of the discontinuous single-electrode voltage-clamp technique enabled the first characterization of K+ currents in Commelina communis and of slow anion currents in C. communis and A. thaliana in intact guard cells to be made. Inward K+ channels from A. thaliana were inhibited by external application of tetraethylammonium (TEA) or Ca2+. In the presence of K+ channel blockers, slow anion channel currents were elicited in almost all guard cells tested and were confirmed by the application of anion channel blockers. In A. thaliana, only anthracene-9 carboxylic acid was able to inhibit slow anion currents, to promote stomatal opening in the dark and to reverse the effect of 25 µM abscisic acid under light. Use of a single microelectrode and preservation of cell integrity make this technique well suited for the study of ion channel regulation in species that have guard cell protoplasts with which it is difficult to form good seals.

Modification by protons of frog skeletal muscle KATP channels: effects on ion conduction and nucleotide inhibition.

1. The molecular mechanisms underlying pH regulation of skeletal muscle ATP‐sensitive K+ (KATP) channels were studied using the patch clamp technique in the inside‐out configuration. Two effects of intracellular protons were studied in detail: the decrease in magnitude of single‐channel currents and the increase in open probability (Po) of nucleotide‐inhibited channels. 2. The pH dependence of inward unit currents under different ionic conditions was in poor agreement with either a direct block of the pore by protons or an indirect proton‐induced conformational change, but was compatible with the protonation of surface charges located near the cytoplasmic entrance of the pore. This latter electrostatic mechanism was modelled using Gouy‐Chapman‐Stern theory, which predicted the data accurately with a surface charge density of about 0.1 negative elementary charges per square nanometre and a pK (pH value for 50% effect) value for protonation of these charges of 6.25. The same mechanism, i.e. neutralization of negative surface charges by cation binding, could also account for the previously reported reduction of inward unit currents by Mg2+. 3. Intracellular alkalization did not affect Po of the KATP channels. Acidification increased Po. In the presence of 0.1 mM ATP (no Mg2+), the channel activation vs. pH relationship could be fitted with a sigmoid curve with a Hill coefficient slightly above 2 and a pK value of 6. This latter value was dependent on the ATP concentration, decreasing from 6.3 in 30 microM ATP to 5.3 in 1 microM ATP. 4. Conversely, the channel inhibition vs. ATP concentration curve was shifted to the right when the pH was lowered. At pH 7.1, the ATP concentration causing half‐maximal inhibition was about 10 microM. At pH 5.4, it was about 400 microM. The Hill coefficient values remained slightly below 2. Similar effects were observed when ADP was used as the inhibitory nucleotide. 5. These results confirm that a reciprocal competitive link exists between proton and nucleotide binding sites. Quantitatively, they are in full agreement with a steady‐state model of a KATP channel possessing four identical protonation sites (microscopic pK, 6) allosterically connected to the channel open state and two identical nucleotide sites (microscopic ATP dissociation constant, approximately 30 microM) connected to the closed state.

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.