Auxin transport: ABC Proteins Join the Club

Dr. Christian Luschnig
The cloning and characterization of transporter-like proteins advanced our knowledge in the understanding of auxin delivery in plants. AUX1 is considered to be a membrane-integrated auxin carrier. This is believed to catalyze the uptake of IAAH+ into cells. A number of other transmembrane proteins encoded by members of the PIN family has been implicated in auxin efflux. The polar auxin transport of auxin is believed to be related to asymmetrical distribution of protein carriers of auxin within the plasma membrane. Such an array, not conforming to symmetry, makes it possible for the establishment of auxin gradients facilitating cellular transport of auxin.
In the August issue of Trends in Plant Science (vol 7 : 329-332, 2002), Christian Luschnig at the Centre for Applied Genetics, University of Agricultural Sciences, Vienna, Austria, reviews the recent progress made in the field of auxin transport. He introduces the subject by pointing out that the discovery of an involvement of ATP-binding cassette (ABC) proteins in auxin transport, has been a major breakthrough in elucidating the mechanisms of auxin distribution in Arabidopsis. ABC proteins, a multimember family are membrane proteins such as MDR (multiple drug resistance), have been implicated to counteract the harmful effects of toxic chemicals and drugs by removing them from the cytosol. For example, the mutant mouse strain, mdr i.e., containing a recessive allele of MDR grows normally but is extremely susceptible to toxic synthetic elements. Describing catalytic and pharmacological property of ABC proteins, the author points out that these proteins have two transmembrane domains abbreviated to TMD1 and TMD2. The two are connected by a large hydrophilic linker region designated nucleotide binding fold (NBF), extending from the cytosol. Both TMD1 and TMD2 are required for substrate interaction.
The author describes a model according to which there is a substrate-binding site on the inner side of the membrane and a substrate-release site on the outer membrane surface. ATP hydrolysis results in the translocation of substrates from one compartment into another.
Mutation in an Arabidopsis ABC protein affects auxin homeostasis .
Approximately 100 members of the ABC protein family have been reported from the Arabidopsis genome. A recent report indicates that auxin might be just another substrate for plant ABC transporters. It was considered that AtMRP5, one of many ABC proteins, has the ability to pump organic anions. An AtMRP5 recessive mutant, generated through T-DNA insertion at this locus confirmed this belief. Compared to the wild-type, the mutant primary root length was significantly reduced concomitant with earlier emergence of lateral roots. There was a two-fold increase of auxin concentrations in the mutant Atmrp5-1. According to the author, still further confirmation is necessary
to establish the direct involvement of AtMRP1 in auxin transport. It remains to be seen whether the atmrp5-1 defect in auxin homeostasis is related to alterations in auxin transport.
Different ways of Auxin transport: According to the recent findings there are three different classes of transporters implicated in auxin transport: AUX1, PIN1 and AtMDR1. Different types of carriers are required depending upon the site of auxin delivery, specificity/affinity for substrate. For instance, the passage of negatively charged auxin- conjugates from the cytoplasm into the vacuole could be catalyzed by ATMRP5 located at the vacuolar membrane (tonoplast) . In such a scenario, the defects in root morphogenesis, apparent in the atmrp5 mutant, might result form a failure in active removal of auxin conjugates from the cytosol into the vacuole.
AtMDR1 and AtPGP may be involved in polar auxin transport: Recent studies
on characterization of genes by using an inhibitor of anion channel activity have
shown that there is a link between ABC transporters such as AtMDR1 and AtPGP (P-glycoprotein) and auxin delivery. It was further substantiated by examining AtMDR1 and AtPGP mutants produced through T–DNA insertions. Cotyledons were deformed (epinasty) and leaves were wrinkled in the mutants , being more pronounced in the double recessives, Atmdr1 Atpgp. The latter also demonstrated lack of apical dominance. Auxin bioassays showed that there is a significant reduction of polar auxin transport, significantly more pronounced in the double mutants. Moreover, the fact that AtMDR1 binds the auxin efflux inhibitor NPA, provides another link between polar auxin transport and ABC proteins.. Further proof of an association between NPA and auxin transport came from the analysis of another Arabidopsis mutant defective in BIG in which correlation was found between reduced polar auxin transport and a reduction of NPA-binding sites.
Conclusion: Recent studies in Arabidopsis have produced a large body of evidence to demonstrate that auxin transport is mediated by three different families of membrane proteins. However, to date it has not been possible to follow inter-compartmental movements of auxin. Failure to do so is mainly on account of the absence of a procedure to assay auxin transport in a quantitative fashion. Once methodology for transport assays is developed, it would help us gain in-depth knowledge about the complexity of auxin delivery system.
Auxin Transport: Its Role in Modulating Plant Growth
Center for Plant Molecular Biology
In the February, 03, issue of Current Opinion in Plant Biology (vol. 6:7-12), Ji í Friml at the Center for Plant Molecular Biology, Tübingen University, Germany, gives an overview of the current status of knowledge in auxin transport and its role in modulating growth and development in plants.
In his introductory remark, the author relates the story leading to the discovery of auxin. While Sachs proposed that some substances act as a transporting signal, Charles Darwin and his son, Francis demonstrated that unilateral light directs the course of growth of a grass coleoptile towards the source of light (phototropism) by transporting a signal. The following century witnessed the unraveling of the chemical nature of auxin which proved to be a simple molecule, namely, indole-3-acetic acid (IAA).
The author describes the ‘chemiosmotic hypothesis’. The model assumes that the auxin transport is a cell-to-cell movement and specific auxin-influx and -efflux carriers are involved in the process. This movement takes place in one direction, and is termed ‘polar’ transport system. The asymmetric positioning of the efflux carrier at a particular location of the cell was considered to determine the direction of auxin flux. The identification and characterization of candidate proteins for auxin influx (AUXIN1 [AUX1]/LIKE-AUX1 [LAX] family) and efflux (PIN-FORMED [PIN] family) carriers provide a number of indirect evidence that these proteins are involved in auxin transport. The PIN genes-encoded proteins were detected in different proportions in various cell types, so far matching with the known direction of auxin influx.
Is auxin to be regarded a ‘morphogen’? The author poses the question whether or not auxin is to be considered a ‘morphogen’ – a substance which is implicated in developmental patterning. In order for a chemical to be called a morphogen, it must fulfill three criteria: (a) ability to form a stable concentration gradient, (b) directing concerned cells to respond to the stimulus without the participation of a signal relay, and (c) the degree of response proportional to the concentration of the morphogen. Auxin partly meets these criteria. For instance, the roots of both Scots pine and Arabidopsis show an auxin gradient. Furthermore, the root meristem of Arabidopsis shows the maximum levels of auxin concentration in the root columella initial cells, with its levels falling off in the surrounding differentiated tissues, constituting epidermis, cortex, endodermis, stele and the lateral root cap. Such an observation establishes a close correlation between the pattern of root development and the auxin gradient, which reaches its maximum in the columella initials. In spite of the fulfillment of most of the criteria, no positive conclusion whether to regard auxin as a morphogen can be drawn. One of the compelling reasons against assigning auxin to a morphogen class of chemicals is that its downstream signaling is so far not enough characterized to establish the direct effect of auxin on cell fate decisions.
How does the auxin gradient arise? The PIN4 protein regulates the auxin efflux in the root. Recent study has shown that the PIN4-gene encoded protein occurs asymmetrically in cells and that in pin4 mutants the DR5 (synthetic promotor strongly responsive to auxin) activity is disrupted. These findings indicate that the auxin-gradient is actively maintained by the PIN4-dependent efflux-driven auxin transport. The author proposed that auxin itself can in turn influence PIN4 position and activity implying that auxin gradient is stabilized by a feedback loop.
Tropisms, transport and traffic The author describes recently characterized members of a ATP-binding cassette (ABC) protein family. They seem to be involved in auxin transport. Two important proteins of this family, detected in Arabidopsis thaliana, are AtMDR1 (multidrug resistance 1 ) and P-glycoprotein1 (AtPGP1). The evidence that they are involved in auxin transport comes from a recent study in which it was shown that the mutants and double mutants of Atmdr and Atpgp1 show a reduced rate of auxin transport. Furthermore, the two sides of the bent hypocotyl of an Arabidopsis seedling, subjected to gravitational force, showed differential staining by DR5:GUS, the curved side showing more blue color than the rest of the tissue. The results of the above experiment indicated that the gravitational force induces accumulation of higher levels of auxin on the more elongated side and that this unequal distribution of auxin results in the curvature of the hypocotyl.
By means of an illustration the author describes active cell-to-cell polar transport from both the apex to the base (basipetal transport) as well as lateral auxin transport, initiating asymmetric growth. A recent study has shown that PIN3 is involved in the tropic movement of auxin. In the absence of gravistimula- tion, the protein encoded by this gene is evenly distributed at the plasma membranes of the columella cells, which are below a few quiescent cells in the middle of the root apex. Two minutes after gravistimula- tion, PIN3-encoded protein is relocated at the new bottom of the cells. It takes up the auxin-export function at the newly created bottom which is now the lower side of the root. However, it is still to be demonstrated that lateral auxin redistribution is impaired in pin3 mutants.
Stimulus perception- and growth response centers: In roots these two centers are located at a distance from each other. The root cap contains ‘stimulus perception center’, while in the elongation zone the growth response center occurs. Results of some recent experiments indicate that in the newly formed root cap, there is already lateral auxin redistribution. From then on, translocation of auxin is basipetal and takes place in an auxin efflux- and influx-dependent manner. It is also presumed that the uptake of auxin into the lateral root cap and epidermis is mediated by AUX1. PIN2 participates in this process by directing auxin transport towards the elongation zone.
How is gravity perceived? The author discusses how auxin transport is activated and regulated by a stimulus of gravity. It is presumed that the sedimentation of statoliths, (= starch-containing organelles) in the root cap and shoot endodermis help them in the perception of gravity and that PIN3 protein mediates connect gravity perception and auxin redistribution. The author speculates that following gravity stimulation, there is an AUX1-dependent accumulation of a temporary pool of auxin, utilized for its asymmetric distribution. Interestingly, the auxin influx component AUX1 also shows strong subcellular dynamics in the columella cells.
Mechanism enabling the rapid subcellular relocation of PIN3: Recent experiments in this area have demonstrated that PIN1 and PIN3 proteins move around in a continuous cycle between the plasma membrane and the endosome along the actin cytoskeleton. Membrane vesicles are involved in their movement. PIN3′s involvement in gravitropic bending is provided by pin3 mutants. Disruption of the movement of PIN3 by means of the vesicle-traficking inhibitor brefeldin A or actin depolymerization, leads to gravitropism defects in the treated seedlings. The author mentions about the two endodermis mutants the sgr2 and sgr4in which gravity perception is impaired, implicating that their normal alleles SGR2 and SGR4 mediate between membrane traffic, vacuole organization and shoot gravitropism.
Conclusion: Although an impressive progress has been made explaining the mode of action of auxin, there is a still a lot of ground to cover towards advancing our understanding of its molecular mechanism. Establishment of the connection between perception and protein relocation as well as the downstream signaling of auxin distribution are some of the areas in which the future research should be directed. Study of pattern formation in plants and animals will help researchers reach an in-depth understanding and discover common grounds, underlying the vital processes of development in plant and animal kingdoms.

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