Markus Klein

ATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxification processes in plants and animals. Recent evidence shows that this class of transporters may also be involved in many other cellular processes. Because of their homology with human multidrug resistance-associated proteins (MRP), cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities. This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation. Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane. Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate. In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs. Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants. These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture.

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.

In many different plant species, genes belonging to the multidrug resistance-associated protein (MRP, ABCC) subfamily of ABC transporters have been identified. Following the discovery of vacuolar transport systems for xenobiotic or plant-produced conjugated organic anions, plant MRPs were originally proposed to be primarily involved in the vacuolar sequestration of potentially toxic metabolites. Indeed, heterologous expression of different Arabidopsis MRPs in yeast demonstrates their activity as ATP-driven pumps for structurally diverse substrates. Recent analysis of protein-protein interactions and the characterization of knockout mutants in Arabidopsis suggests that apart from transport functions plant MRPs play additional roles including the control of plant transpiration through the stomata. Here, we review and discuss the diverse functions of plant MRP-type ABC transporters and present an organ-related and developmental analysis of the expression of Arabidopsis MRPs using the publicly available full-genome chip data.

In this paper we present results on the vacuolar uptake mechanism for two flavone glucuronides present in rye mesophyll vacuoles. In contrast to barley flavone glucosides ( Klein et al. (1996 ) J. Biol. Chem. 271, 29666–29671), the flavones luteolin 7-O-diglucuronyl-4′-O-glucuronide (R1) and luteolin 7-O-diglucuronide (R2) were taken up into vacuoles isolated from rye via a directly energised mechanism. Kinetic studies suggested that the vacuolar glucuronide transport system is constitutively expressed throughout rye primary leaf development. Competition experiments argued for the existence of a plant MRP-like transporter for plant-specific and non-plant glucuronides such as β-estradiol 17-(β- d-glucuronide) (E217G). The interaction of ATP-dependent vacuolar glucuronide uptake with glutathione and its conjugates turned out to be complex: R1 transport was stimulated by dinitrobenzene-GS and reduced glutathione but was inhibited by oxidized glutathione in a concentration-dependent manner. In contrast, R2 uptake was not increased in the presence of reduced glutathione. Thus, the transport system for plant-derived glucuronides differed from the characteristic stimulation of vacuolar E217G uptake by glutathione conjugates but not by reduced glutathione ( Klein et al. (1998 ) J. Biol. Chem. 273, 262–270). Using tonoplast vesicles isolated with an artificial K+ gradient, we demonstrate for the first time for plant MRPs that the ATP-dependent uptake of R1 is membrane-potential dependent. We discuss the kinetic capacity of the ABC-type glucuronide transporter to explain net vacuolar flavone glucuronide accumulation in planta during rye primary leaf development and the possibility of an interaction of potential substrates at both the substrate binding and allosteric sites of the MRP transporter regulating the activity towards a certain substrate.

Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein (MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers.

We study a large class of reversible Markov chains with discrete state space and transition matrix $P_N$. We define the notion of a set of {\it metastable points} as a subset of the state space $\G_N$ such that (i) this set is reached from any point $x\in \G_N$ without return to x with probability at least $b_N$, while (ii) for any two point x,y in the metastable set, the probability $T^{-1}_{x,y}$ to reach y from x without return to x is smaller than $a_N^{-1}\ll b_N$. Under some additional non-degeneracy assumption, we show that in such a situation: \item{(i)} To each metastable point corresponds a metastable state, whose mean exit time can be computed precisely. \item{(ii)} To each metastable point corresponds one simple eigenvalue of $1-P_N$ which is essentially equal to the inverse mean exit time from this state. The corresponding eigenfunctions are close to the indicator function of the support of the metastable state. Moreover, these results imply very sharp uniform control of the deviation of the probability distribution of metastable exit times from the exponential distribution.

We study a class of Markov chains that describe reversible stochastic dynamics of a large class of disordered mean field models at low temperatures. Our main purpose is to give a precise relation between the metastable time scales in the problem to the properties of the rate functions of the corresponding Gibbs measures. We derive the analog of the Wentzell-Freidlin theory in this case, showing that any transition can be decomposed, with probability exponentially close to one, into a deterministic sequence of “admissible transitions”. For these admissible transitions we give upper and lower bounds on the expected transition times that differ only by a constant factor. The distributions of the rescaled transition times are shown to converge to the exponential distribution. We exemplify our results in the context of the random field Curie-Weiss model.

We study a large class of reversible Markov chains with discrete state space and transition matrix P N . We define the notion of a set of metastable points as a subset of the state space ΓN such that (i) this set is reached from any point x∈ΓN without return to x with probability at least b N , while (ii) for any two points x, y in the metastable set, the probability T − 1x ,y to reach y from x without return to x is smaller than a N − 1< b N . Under some additional non-degeneracy assumption, we show that in such a situation: (i) To each metastable point corresponds a metastable state, whose mean exit time can be computed precisely. (ii) To each metastable point corresponds one simple eigenvalue of 1 −P N which is essentially equal to the inverse mean exit time from this state. Moreover, these results imply very sharp uniform control of the deviation of the probability distribution of metastable exit times from the exponential distribution.

Warum werden Bürger Mitglieder politischer Parteien? Und warum werden einige Parteimitglieder aktiv und andere nicht? Diesen Leitfragen wird im Rahmen des General-Incentives-Modell von Seyd und Whiteley nachgegangen. Die Grundlage der empirischen Analysen bilden eine Bevölkerungsumfrage sowie eine Umfrage unter den deutschen Parteimitgliedern. Es kann gezeigt werden, dass für die Entscheidung zum Parteibeitritt bereits weiche Anreize hinreichend sein können, während die Entscheidung für aktives innerparteiliches Engagement vor allem durch harte Anreize beeinflusst wird. Da weiche Anreize eine rückläufige Verbreitung haben, können diese Befunde auch zu einer Erklärung der rückläufigen Parteimitgliederzahlen beitragen. Why do citizens become members of political parties? And why are some party members activists and others not? These guiding questions are analyzed within the context of the general incentives model proposed by Seyd and Whiteley. The basis of the empirical analyses is a survey of the German population as well as a survey of the German party members. It is shown that soft incentives can be sufficient for the decision to become a member of a political party while hard incentives are the main predictor of the decision to become active within a party. Since soft incentives had become less widespread these findings contribute to an explanation of the declining number of party members in Germany.

Warum werden Bürger Mitglieder politischer Parteien? Und warum werden einige Parteimitglieder aktiv und andere nicht? Diesen Leitfragen wird im Rahmen des General-Incentives-Modell von Seyd und Whiteley nachgegangen. Die Grundlage der empirischen Analysen bilden eine Bevölkerungsumfrage sowie eine Umfrage unter den deutschen Parteimitgliedern. Es kann gezeigt werden, dass für die Entscheidung zum Parteibeitritt bereits weiche Anreize hinreichend sein können, während die Entscheidung für aktives innerparteiliches Engagement vor allem durch harte Anreize beeinflusst wird. Da weiche Anreize eine rückläufige Verbreitung haben, können diese Befunde auch zu einer Erklärung der rückläufigen Parteimitgliederzahlen beitragen. Why do citizens become members of political parties? And why are some party members activists and others not? These guiding questions are analyzed within the context of the general incentives model proposed by Seyd and Whiteley. The basis of the empirical analyses is a survey of the German population as well as a survey of the German party members. It is shown that soft incentives can be sufficient for the decision to become a member of a political party while hard incentives are the main predictor of the decision to become active within a party. Since soft incentives had become less widespread these findings contribute to an explanation of the declining number of party members in Germany.