Comparative Mutant Analysis of Arabidopsis ABCC-Type ABC Transporters: AtMRP2 Contributes to Detoxification, Vacuolar Organic Anion Transport and Chlorophyll Degradation

Abstract

The enormous metabolic plasticity of plants allows detoxification of many harmful compounds that are generated during biosynthetic processes or are present as biotic or abiotic toxins in their environment. Derivatives of toxic compounds such as glutathione conjugates are moved into the central vacuole via ATP-binding cassette (ABC)-type transporters of the multidrug resistance-associated protein (MRP) subfamily. The Arabidopsis genome contains 15 AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) cluster together in one of two major phylogenetic clades. We isolated T-DNA knockout alleles in all four highly homologous AtMRP genes of this clade and subjected them to physiological analysis to assess the function of each AtMRP of this group. None of the single atmrp mutants displayed visible phenotypes under control conditions. In spite of the fact that AtMRP1 and AtMRP2 had been described as efficient ATP-dependent organic anion transporters in heterologous expression experiments, the contribution of three of the AtMRP genes (1, 11 and 12) to detoxification is marginal. Only knockouts in AtMRP2 exhibited a reduced sensitivity towards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides. AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophyll degradation since ethylene-treated rosettes of atmrp2 showed reduced senescence, and AtMRP2 expression is induced during senescence. This suggests that AtMRP2 is involved in vacuolar transport of chlorophyll catabolites. Vacuolar uptake studies demonstrated that transport of typical MRP substrates was reduced in atmrp2. We conclude that within clade I, only AtMRP2 contributes significantly to overall organic anion pump activity in vivo.

Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis

“…The Arabidopsis AtABCC1 and especially AtABCC2 mediate the transport of structurally diverse metabolites, such as phytochelatins, folates, and conjugates of chlorophyll catabolite and xenobiotics (Liu et al, 2001;Frelet-Barrand et al, 2008;Raichaudhuri et al, 2009;Song et al, 2010). We expressed AtABCC2 in yeast and observed a distinct MgATP-dependent ABA-GE transport activity of isolated membrane vesicles (Fig.…”

“…The simultaneous addition of ABC transporter and V-ATPase inhibitors inhibited the ABA-GE uptake below the levels observed for these compounds individually. Orthovanadate and bafilomycin A1 were used at concentrations shown to completely inhibit corresponding enzymatic activity in tonoplast preparations (Frelet-Barrand et al, 2008;Zhao and Dixon, 2009). The presence of the preexisting proton gradients in isolated vacuoles explains why the combination of bafilomycin A1 with NH 4 Cl decreased the ABA-GE uptake more than bafilomycin A1 alone.…”

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

“…Both proteins transport GS conjugates (23,26), which are formed by the attack of the GS thiolate anion to an electrophilic and potentially toxic substrate. Furthermore, AtABCC2 has been shown to transport chlorophyll catabolites, which are potentially toxic, because they can absorb light and produce harmful reactive oxygen species (43). This versatile and important role in plant detoxification requires that AtABCC1 and AtABCC2 are expressed in all tissues in a constitutive way.…”

Update on the biochemistry of chlorophyll breakdown

“…After expression in yeast, two Arabidopsis transporters, ABCC2 and ABCC3, were shown to be capable of transporting a NCC from canola (Lu et al 1998;Tommasini et al 1998). However, an Arabidopsis ABCC2 mutant was only marginally affected in senescence (Frelet-Barrand et al 2008), pointing to a possible functional redundancy of different transporters.…”