Plant Physiology Online: Exploring the Cellul...

HOME :: CHAPTER 19 :: Essay 19.1

PREVIOUS :: NEXT

Essay 19.1

Exploring the Cellular Basis of Polar Auxin Transport

Angus Murphy, Department of Horticulture and Landscape Architecture, Purdue University

May, 2006

Plant form is dependent on the establishment of polarity: growth takes place in apical regions of roots and shoots in response to basic developmental programming which is then modulated by environmental cues. Plants can also undergo tropic growth in order to adapt to changes in light, orientation, or surface contact. However, even when tropic responses alter the direction of growth, the overall polarity of the plant remains intact.

Biochemical and physiological evidence suggests that the polarity of plant growth is regulated at the cellular level and involves components of the cytoskeleton, plasma membrane, and cell wall. Plant cells must therefore possess mechanisms to asymmetrically direct proteins to specific cell surfaces. These mechanisms appear to be regulated by developmental and environmental cues.

Auxin, or indole acetic acid (IAA), is an essential, multifunctional plant hormone that influences virtually every aspect of plant growth and development. Although auxin-dependent growth is evident in all plant tissues, it is synthesized primarily in apical regions of the shoot and is then transported in a polar fashion to other sites. When auxin reaches the root apex, it is redistributed away from the root tip through cortical and epidermal tissues (Lomax, 1995). In tropic growth, auxin is diverted laterally to one side of the plant stem or root; therefore, As a result, the cells in that portion of the stem or root below the point of redistribution elongate. The result is bending toward light, gravitational pull, or a potential point of attachment.

Auxin is taken up into cells by diffusion augmented by a proton co-transporter (Swarup et al., 2001), but can only exit from cells via basally-localized efflux carriers (reviewed in Muday and DeLong, 2001). Mutants deficient in auxin transport generally display aberrant morphology. Auxin is thus thought to maintain cellular polarity and, as a result, its own asymmetric transport mechanism. The genes that encode the auxin efflux carriers have been identified and are generally referred to as PIN genes, for the pin-formed phenotype resulting from mutations of these genes (reviewed in Palme and Gälweiler, 1999). Biochemical evidence suggests that the PIN proteins effectively transport auxin only when functionally associated with other proteins. A number of proteins that appear to modulate PIN-associated auxin transport have recently been identified (reviewed in Muday and Murphy, 2002). For a more detailed explanation of auxin function and transport, please refer to Ch 19.

An asymmetric targeting mechanism for transport proteins

Recent cellular localization studies have shown that PIN1 cycles between the plasma membrane and an internal compartment in membrane vesicles associated with actin cytoskeletal fibers (Steinmann et al., 1999; Geldner et al., 2001). The role of actin in this process may be to provide "tracks" for vesicle movement and to fix the efflux carriers in a specific location after delivery to the membrane surface. When chemical agents are used to disrupt cytoskeletal tracking, auxin transport inhibitors prevent relocalization of PIN proteins on the plasma membrane. This suggests that the proteins that bind auxin transport inhibitors may provide a bridge between the efflux carriers and the actin network used to transport and localize these complexes. Rapid vesicular cycling is now thought to redistribute carriers to a new site when auxin transport polarity is changed by environmental stimuli, such as light or gravity. Therefore, direct analysis of the proteins that bind auxin efflux inhibitors, and examination of endogenous molecules, such as flavonoids, that may regulate auxin efflux in vivo is crucial to understanding how the PIN cycling apparatus functions.

There is a striking similarity between the cycling of PIN transporters and the mechanism that mediates the movement of glucose transporters to the plasma membrane in mammalian insulin-responsive tissues (Muday and Murphy, 2002). In those tissues, when blood glucose levels rise, an insulin-induced signaling cascade causes endomembrane vesicles containing the GLUT4 glucose transporter to be dispatched asymmetrically to the plasma membrane (reviewed in Baumann and Saltiel, 2001; Simpson et al., 2001). Changes in protein phosphorylation states activates some components of GLUT4 secretory vesicles (GSVs) and deactivates anchoring components that normally repress movement. The net result is relocation of transporters from internal compartments to docking sites on the plasma membrane. A diagrammatic comparison of the mammalian GLUT4 system with the proposed PIN cycling system in Arabidopsis is shown in Figure 1.

Figure 1   Targeting of the PIN auxin efflux carrier in Arabidopsis resembles the human IRAP/GLUT4 mechanism. In human insulin responsive tissues, a vesicular cycling mechanism mediates the asymmetric localization of the GLUT4 glucose transporter on the plasma membrane. Recent studies of the localization of PIN auxin efflux carriers and protein interactions with auxin transport inhibitors suggest that a similar mechanism may regulate auxin transport in Arabidopsis. Sequence homologies and analogous functions of many of the protein components of the two systems further suggest parallel mechanisms. An external signal (hormone binding) triggers a phosphatidylinositol/Protein kinase C (Pinoid) phosphorylation cascade that activates asymmetric vesicular trafficking by: 1) causing dissociation of either an FKB506 binding protein (Twisted Dwarf) or Cyclophilin 5; 2) relocation of an inhibitory Grp ARF-GEF protein (GNOM) from an endomembrane compartment to PIP3–enriched plasma membranes; 3) recruitment of a dynamin GTPase (ADL1a) and a secretory adaptor (adaptin) complex; 4) phosphorylation of both IRAP (AtAPM1) and the Vamp2 v-snare adaptor protein. Vesicles then traffic on cytoskeletal fibers to a Munc18c (Keule) plasma membrane docking site where Vamp2 interacts with the t-snare Syntaxin 4 (Knolle) to initiate vesicle fusion. Membrane transport proteins are then recycled by endocytosis into early endosomes for redistribution to plasma membranes or, if processed sequentially by a KEXP-APP complex and PM-localized IRAP (AtAPM1), are routed to late endosomes for subsequent proteolytic degradation. For comparison, Arabidopsis proteins implicated in auxin transport are listed adjacent to their mammalian orthologs in the columns at the right of the figure. PKC, protein kinase C; AKT, protein kinase B/AKT; RCN1, root curling in NPA-1 PP2a; Vamp2, vesicle associated membrane protein 2 (v-SNARE2); IRAP, insulin responsive aminopeptidase; AtAPM1, Arabidopsis thaliana microsomal aminopeptidase; GRP1 ARF-GEF, general receptor for phosphoinositides ADP ribosylation factor-guanine nucleotide exchange factor; Munc18c, mammalian homolog of C. elegans unc18c; FKB506BP, FKB506-binding immunophilin; TWD, Twisted Dwarf; APP1, mammalian aminopeptidase P 1; AtAPP1, Arabidopsis APP1; AtKEXP, Arabidopsis KEXP neutral metallopeptidase. (Click image to enlarge.)

Many of the components of the mammalian GLUT4 inducible vesicle secretion mechanism have orthologs in Arabidopsis, a number of which have been directly or indirectly implicated in the regulation of auxin transport and/or the asymmetric distribution of the PIN1 protein. For example, mutations in kinase and phosphatase genes homologous to their mammalian GSV counterparts results in growth defects, altered auxin transport, and altered sensitivity to auxin transport inhibitors (Bennett et al, 1995; Christensen et al., 2000; Benjamins et al., 2001; Reru?re et al., 1999, Rashotte et al., 2001). Other Arabidopsis proteins known to associate with the PIN proteins or to be implicated in auxin transport are also homologs of important components of the GLUT4 cycling mechanism (reviewed in Muday and Murphy, 2002). One of the most important of these may be the apparent Arabidopsis counterpart of the mammalian Insulin Responsive Aminopeptidase (IRAP), which is essential for mammalian GLUT4 cycling. IRAP and its Arabidopsis homolog, AtAPM1, have a high degree of sequence similarity, have similar membrane orientations and enzymatic activities, and undergo unique processing of their carboxy-terminal domains when secreted to the plasma membrane (Murphy et al., 2000; 2002). Recently, we have shown that treatment of Arabidopsis seedlings with IRAP inhibitors results in delocalization of PIN1 from the plasma membrane and strong localization of AtAPM1 to the basal ends of auxin-conducting cells. Natural flavonoid inhibitors of AtAPM1 have been found to alter PIN1 localization as well.

Insulin signaling is a key component of vesicle targeting in the GLUT4 localization system. For vesicle mediated targeting of IAA transport proteins to be truly parallel to the GLUT4 model, it is necessary to ask what signal(s) might control the localization of auxin transport proteins. The simplest possibility is that auxin acts as the signal to stimulate its own transport. Auxin has been reported to stimulate IAA transport (Rayle et al., 1969) and is generally thought to be required for the establishment of both embryonic polarity (Geldner et al., 2000) and auxin transport pathways themselves ( reviewed by Berleth and Sachs, 2001).

The focus of the research in my lab is to dissect the interactions of the potential components of the PIN vesicular cycling apparatus in Arabidopsis. We are currently analyzing the localization of PIN proteins in mutants lacking various components of the vesicular cycling mechanism in order to better understand the asymmetric targeting of membrane proteins and polar growth in plants. We are complementing the localization studies with biochemical assays of protein-protein interactions. It is my hope that these experiments will help us determine the applicability of the GLUT4 cycling model to plant growth and development.

References

Assaad, F. F., Huet, Y., Mayer, U., and Jurgens, G., (2001) The cytokinesis gene KEULE encodes a Sec1 protein that binds the syntaxin KNOLLE. J Cell Biol 152, 531-543.

Baumann, C. A., and Saltiel, A.,R. (2001) Spatial compartmentalization of signal transduction in insulin action. Bioessays 23, 215-222.

Benjamins, R., Quint, A., Weijers, D., Hooykaas, P., and Offringa R., (2001) The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. Development 128, 4057-4067.

Bennett, S.,R.,M., Alvarez, J., Bossinger, G., and Smyth, D.R. (1995) Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant J 8, 505-520.

Berleth, T., and Sachs, T. (2001) Plant morphogenesis: long-distance coordination and local patterning. Curr Opin Plant Biol 4, 57-62.

Christensen, S. K., Dagenais, N., Chory, J. and, Weigel D., (2000) Regulation of auxin response by the protein kinase PINOID. Cell 100, 469-478.

Deru?re, J., Jackson K., Garbers, C., Soll, D., and Delong, A. (1999) The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo. Plant J 20, 389-399.

Geldner, N., Friml, J., Stierhof Y. D., Jurgens G., and Palme K. (2001) Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413, 425-428.

Geldner, N., Hamann, T., and Jurgens, G. (2000) Is there a role for auxin in early embryogenesis? Plant Growth Reg 32, 187-191.

Lomax, T. L., Muday, G. K., and Rubery, P. H. (1995) Auxin transport. In Plant Hormones and Their Role in Plant Growth Development, 2nd ed., P. J. Davies, ed., Kluwer, Dordrecht, Netherlands, pp. 509–530.

Muday, G. K., and DeLong, A. (2001) Polar auxin transport: controlling where and how much. Trends Plant Sci 6, 535-542.

Muday, G. K., and Murphy, A. S. (2002) An emerging model of auxin transport regulation. Plant Cell 14:293-299.

Murphy, A., Hoogner, K., Peer, W. and Taiz, L. (2002) Identification, purification, and molecular cloning of N-1-naphthylphthalamic acid-binidng plasma membrane-associated aminopeptidases from Arabidopsis. Plant Physiol. 128: 935-950.

Murphy, A., Peer, W. A. and Taiz, L. (2000). Regulation of auxin transport by aminopeptidases and endogenous flavonoids. Planta 211, 315-324.

Palme, K., and G?lweiler, G., (1999) PIN-pointing the molecular basis of auxin transport. Curr Op in Plant Biol 2, 375-381.

Rashotte, A. M., DeLong, A. and Muday, G. K. (2001) Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth. Plant Cell 13, 1683-1697.

Rayle, D. L., Ouitrakul, R., and Hertel, R., (1969) Effect of auxins on the auxin transport system in coleoptiles. Planta 87, 49-53.

Simpson, F., Whitehead, J. P. and James, D. E., (2001) GLUT4--at the cross roads between membrane trafficking and signal transduction. Traffic 2, 2-11.

Steinmann, T., Geldner, N., Grebe, M., Mangold, S., Jackson, C.L., Paris, S., Galweiler, L., Palme, K., and Jurgens G. (1999) Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF. Science 286, 316-318.

Swarup, R., Friml, J., Marchant, A., Ljung, K., Sandberg, G., Palme, K., and Bennett, M., (2001) Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes Dev 15, 2648-2653.

HOME :: CHAPTER 19 :: Essay 19.1