Philip A. Rea

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.

Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters.

Arsenic is an extremely toxic metalloid causing serious health problems. In Southeast Asia, aquifers providing drinking and agricultural water for tens of millions of people are contaminated with arsenic. To reduce nutritional arsenic intake through the consumption of contaminated plants, identification of the mechanisms for arsenic accumulation and detoxification in plants is a prerequisite. Phytochelatins (PCs) are glutathione-derived peptides that chelate heavy metals and metalloids such as arsenic, thereby functioning as the first step in their detoxification. Plant vacuoles act as final detoxification stores for heavy metals and arsenic. The essential PC–metal(loid) transporters that sequester toxic metal(loid)s in plant vacuoles have long been sought but remain unidentified in plants. Here we show that in the absence of two ABCC-type transporters, AtABCC1 and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic and arsenic-based herbicides. Heterologous expression of these ABCC transporters in phytochelatin-producing Saccharomyces cerevisiae enhanced arsenic tolerance and accumulation. Furthermore, membrane vesicles isolated from these yeasts exhibited a pronounced arsenite [As(III)]–PC2 transport activity. Vacuoles isolated from atabcc1 atabcc2 double knockout plants exhibited a very low residual As(III)–PC2 transport activity, and interestingly, less PC was produced in mutant plants when exposed to arsenic. Overexpression of AtPCS1 and AtABCC1 resulted in plants exhibiting increased arsenic tolerance. Our findings demonstrate that AtABCC1 and AtABCC2 are the long-sought and major vacuolar PC transporters. Modulation of vacuolar PC transporters in other plants may allow engineering of plants suited either for phytoremediation or reduced accumulation of arsenic in edible organs.

Mechanism of Heavy Metal Ion Activation of Phytochelatin (PC) Synthase BLOCKED THIOLS ARE SUFFICIENT FOR PC SYNTHASE-CATALYZED TRANSPEPTIDATION OF GLUTATHIONE AND RELATED THIOL PEPTIDES

The dependence of phytochelatin synthase (γ-glutamylcysteine dipeptidyltranspeptidase (PCS), EC 2.3.2.15) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana(AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd2+- and Zn2+-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g.Cd·GS2 or Zn·GS2) and free glutathione act as γ-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both γ-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzymes requirement for glutathione-like peptides containing blocked thiol groups for activity.

AtMRP2, an Arabidopsis ATP Binding Cassette Transporter Able to Transport Glutathione S-Conjugates and Chlorophyll Catabolites: Functional Comparisons with AtMRP1

Three ATP binding cassette (ABC) transporter–like activities directed toward large amphipathic organic anions have recently been identified on the vacuolar membrane of plant cells. These are the Mg-ATP–energized, vanadate-inhibitable vacuolar accumulation of glutathione S-conjugates (GS conjugates), chlorophyll catabolites, and bile acids, respectively. Although each of these activities previously had been assigned to distinct pumps in native plant membranes, we describe here the molecular cloning, physical mapping, and heterologous expression of a gene, AtMRP2, from Arabidopsis thaliana that encodes a multispecific ABC transporter competent in the transport of both GS conjugates and chlorophyll catabolites. Unlike its isoform, AtMRP1, which transports the model Brassica napus chlorophyll catabolite transporter substrate Bn-NCC-1 at low efficiency, heterologously expressed AtMRP2 has the facility for simultaneous high-efficiency parallel transport of GS conjugates and Bn-NCC-1. The properties of AtMRP2 therefore establish a basis for the manipulation of two previously identified plant ABC transporter activities and provide an explanation for how the comparable transporter in native plant membranes would be systematically mistaken for two distinct transporters. These findings are discussed with respect to the functional organization of AtMRP2, the inability of AtMRP2 and AtMRP1 to transport the model bile acid transporter substrate taurocholate (despite the pronounced sensitivity of both to direct inhibition by this agent), the differential patterns of expression of their genes in the intact plant, and the high capacity of AtMRP2 for the transport of glutathionated herbicides and anthocyanins.

Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream

Vacuolar-type H(+)-translocating inorganic pyrophosphatases have long been considered to be restricted to plants and to a few species of phototrophic bacteria. However, in recent investigations, these pyrophosphatases have been found in organisms as disparate as thermophilic Archaea and parasitic protists, and have resulted in the definition of a novel subclass in plants themselves. Among the many evolutionary and practical implications of these findings is the possibility that this research will spawn new approaches to the treatment of several prolific and debilitating parasite-mediated infections.

The protein storage vacuole: a unique compound organelle

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.

The GS-X Pump in Plant, Yeast, and Animal Cells: Structure, Function, and Gene Expression

This review addresses the recent molecular identification of several members of the glutathione S-conjugate (GS-X) pump family, a new class of ATP-binding cassette (ABC) transporters responsible for the elimination and/or sequestration of pharmacologically and agronomically important compounds in mammalian, yeast and plant cells. The molecular structure and function of GS-X pumps encoded by MRP, cMOAT, YCF1. and AtMRP genes, have been conserved throughout molecular evolution. The physiologic function of GS-X pumps is closely related with cellular detoxification, oxidative stress, inflammation, and cancer drug resistance. Coordinated expression of GS-X pump genes, e.g., MRP1 and YCF1, and γ-glutamylcystaine synthetase, a rate-limiting enzyme of cellular glutathione (GSH) biosynthesis, has been frequently observed.

Vacuolar H+-translocating pyrophosphatases: a new category of ion translocase

The membrane surrounding the central vacuole of plant cells contains an H(+)-translocating ATPase (H(+)-ATPase) and an H(+)-translocating inorganic pyrophosphatase (H(+)-PPase). Both enzymes are abundant and ubiquitous in plants but the H(+)-PPase is unusual in its exclusive use of inorganic pyrophosphate (PPi) as an energy source. The lack of sequence identity between the vacuolar H(+)-PPase and any other characterized ion pump implies a different evolutionary origin for this translocase. The existence of the vacuolar H(+)-PPase, in conjunction with increasing recognition of PPi as a key metabolite in plant systems, necessitates reconsideration of ATP as the primary energy source for membrane transport in plant cells.

Enhanced multispecificity of arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2.

Recent investigations have established that Arabidopsis thaliana contains a family of genes encoding ATP-binding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family. So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes. Here we show that Arabidopsis MRP2 (AtMRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast. Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17β-estradiol 17-(β-d-glucuronide) (E217βG) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, nottrans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each others transportvia distinct but coupled binding sites. The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2. The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione. Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E217βG uptake by AtMRP2. Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E217βG uptake, neither competes with DNP-GS for uptake nor is subject to E217βG-promoted uptake. A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2.

Weeds, Worms, and More. Papain's Long-Lost Cousin, Phytochelatin Synthase

This Update is concerned with the mechanism of synthesis of heavy metal-binding thiol peptides, phytochelatins (PCs), by the enzyme PC synthase (EC 2.3.2.15). The bulk of the considerations in this review centers on what has been learned recently of the fundamental mechanics of PC synthesis, the domain organization and phylogenetic distribution of PC synthases, and PC synthase-like enzymes, and what this tells us about the chemistry underlying and the enzyme residues necessary for PC synthesis. It was decided to prepare a review of this type rather than aim at a more comprehensive treatment of heavy metal homeostasis and detoxification in plants for two reasons. The first is that there are already several contemporary reviews dealing with the more global aspects of plant heavy metal physiology. Excellent examples are Cobbett (2000), Clemens (2001), and Cobbett and Goldsbrough (2002). Readers who have not already read these are encouraged to do so. The second reason is that some of the most fascinating and unexpected developments for our understanding in this area of late derive from investigations of the catalytic mechanism and distribution of PC synthases, facets of this field of research that have yet to be reviewed in detail.