Auxin binding protein: curiouser and curiouser

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<ul><li><p>TRENDS in Plant Science Vol.6 No.12 December 2001</p><p> 1360-1385/01/$ see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)02150-1</p><p>586 ReviewReview</p><p>Candace TimpteDept Biological Sciences,University of NewOrleans, New Orleans,LA 70148, USA.e-mail:</p><p>Curiouser and curiouser! cried Alice. (As her bodygrew after swallowing a potion, she realized that sheshould give her now-distant feet a new pair of bootsfor Christmas). Alice went on planning to herself howshe would manage it. They must go by the carrier,she thought; and how funny it will seem, sendingpresents to ones own feet! And how odd thedirections will look!Through the Looking Glass (Lewis Carrol)</p><p>The plant hormone auxin (indole-3-acetic acid, orIAA) is central to diverse plant growth anddevelopmental responses. Some of the best-characterized examples are tropic growth responses(such as to gravity or light), stem elongation, lateralbranching of roots and shoots, and vasculardevelopment1. These whole-plant responses are theresult of changes at the cellular level that includeelongation, division or differentiation. However, themechanisms of auxin perception and response areunderstood poorly. Some responses are rapid andothers occur after a lag period, complicating thesituation further.</p><p>The first step in a classic hormone responsepathway is a receptor binding a hormone. Manyinvestigators have sought auxin receptors and severalgood candidates have been isolated2. However, as wellas binding auxin, the receptor must also transduce theauxin stimulus into the known responses. Collectingevidence that the auxinreceptor interaction causesdirect changes in the cell has been difficult. Theimmediate short-term auxin responses includechanges in protoplast electrophysiology, guard-cellgating and early-response-gene induction. Longer-term responses include cell elongation, cell divisionand phenotypic changes in the whole plant. The choiceof assay is the key to establishing an auxinreceptorinteraction; one must remember that more than onepathway might be activated by one receptor, anddirect cause-and-effect relations must be established.</p><p>Auxin-binding proteinTwenty years ago, an auxin-binding activity waspurified from maize coleoptiles by several groups2,3.This auxin-binding protein, ABP1, was shownby photoaffinity labeling to bind auxin4 (itscharacterization is summarized in Ref. 2). The maizeABP1 cDNA encodes a 201 amino acid protein, with a38 residue signal sequence. The unglycosylatedprotein is 20 kDa, whereas the mature protein is22 kDa, containing a high-mannose-typeoligosaccharide2. ABP1 was the first plant proteindiscovered with a C-terminal KDEL sequence, whichis an endoplasmic reticulum (ER) retention signal5.ABP1 has no hydrophobic regions. Thus, to functionas a receptor, it probably associates with amembrane-bound dockingprotein. ABP1 bears noresemblance to well-known hormone receptors fromanimal systems and does not have substantialsimilarity to any mammalian gene. Yet, ABP1 hasbeen identified from many plant species includingmaize, Arabidopsis, tobacco and radish2.</p><p>In spite of excellent research efforts, importantquestions need to be answered if ABP1 is to beestablished as the auxin receptor. First, does ABP1ligand binding have biological relevance? The auxinreceptor must bind auxin but also must evokechanges in the cell. Second, what is the structure ofthis protein, and how does this structure relate to itssignaling mechanism? Third, where does ABP1reside in the cell? Typically, a mammalian hormonebinds the target ligand at the plasma membrane,although one exception is the steroid hormonereceptor. Paradoxically, the KDEL sequence of ABP1suggests an ER, not a plasma membrane, location.Could it be elsewhere in the cell?</p><p>ABP1 is crucial for embryogenesisRecent genetic studies provide strong evidence forABP1 mediating responses leading to cell elongationand embryogenesis. Arabidopsis has a single geneencoding ABP1 (Ref. 6) and disruption of this gene isexpected to affect auxin signaling processes and toreveal ABP1s role in plant development. A knockoutplant harboring a T-DNA insertion in the first exon ofthe ABP1 gene has been identified7. Homozygousindividuals were not recovered from this plant line,strongly indicating that disruption of the ABP1 geneis lethal. About 25% of the seeds in transgenic siliqueswere white and nonviable, clear evidence ofsegregation of a lethal homozygous phenotype.</p><p>Auxin is implicated in a variety of plant developmental processes, yet themolecular mechanism of auxin response remains largely unknown. Auxinbinding protein 1 (ABP1) mediates cell expansion and might be involved in cellcycle control. Structural modeling shows that it is a -barrel dimer, with theC terminus free to interact with other proteins. We do not know where ABP1performs its receptor function. Most ABP1 is detected within the endoplasmicreticulum but the evidence indicates that it functions at the plasma membrane.ABP1 is established as a crucial component of auxin signaling, but its precisemechanism remains unclear.</p><p>Auxin binding protein: curiouser andcuriouserCandace Timpte</p></li><li><p>Addition of a transgenic, functional copy of ABP1rescued the embryonic-lethal phenotype, suggestingthat normal embryo development requires at leastone copy of ABP1.</p><p>Examination of the nonviable embryos revealedthat ABP1 is required early in plant development:embryos arrested after the globular stage. Newlyformed cross walls between cells were wronglyoriented and cells failed to elongate, leading toembryo death7. These results provide direct evidencethat ABP1 plays a crucial role in embryonicmorphogenesis. Whether the role is in cell elongation,embryo polarity establishment or individual cellpolarity could not be determined. To address thepolarity versus elongation issue, antisensesuppression was used to create an ABP1 loss-of-function mutation in the BY-2 tobacco cell line. Theresults enabled the two types of expansion commonlyobserved in cultured plant cells to be differentiated8.Auxin-induced elongation to increase cell volumebeyond that of the divided cell was abolished in thesetransgenic cells7. However, cell expansion to replacecell volume following division was not affected in theABP1-antisense lines. Thus, elongation growth is thecrucial auxin response in cultured cells and failure toelongate is the probable cause of embryo lethality inthe transgenic knockout plants.</p><p>ABP1 mediates cell expansionThe complementary approach, overproducing ABP1,confirms the role of ABP1 in auxin-mediated cellexpansion. Tobacco was transformed with ABP1under the control of an inducible promoter9. In controlplants, auxin only induced growth at the leaf tips,whereas, in overproducing transgenic plants, itinduced growth throughout the leaf. Regions that arenot normally auxin responsive acquired induciblegrowth that was strictly dependent on the presenceof auxin; a structurally similar inactive auxin did notstimulate growth. Thus, overproducing ABP1extended the range of auxin sensitivity in matureleaf tissue9. A meticulous analysis of individual cellsfrom ABP1-overproducing plants reveals thatauxin-inducible cell expansion is a component ofthis growth10. The abundance of ABP1 in each cellcorrelates with the extent of auxin-induced cellexpansion and with cell size in transgenic plants.</p><p>Evidence from cultured cells supports a role forABP1 in cell expansion. Cultured maize cellsoverproducing ABP1 expanded in an auxin-dependent fashion and were greater in volume thancontrol cells9. Antisense-suppressed ABP1 tobaccoBY-2 cells had undetectable levels of ABP1 proteinand lacked auxin-induced cell expansion whencompared with wild-type cells10.</p><p>Auxin-induced cell division might involve ABP1Auxin-mediated growth might also have a divisioncomponent. Cells from ABP1-overproducing tobaccoleaves were examined for nuclear division stage10.The proportion of nuclei in G2 stage was double thatof the wild type. By sequential analysis of cells indeveloping leaves, cell expansion was found toprecede the G2 advance in the cycle. The prematureG2 advance is probably an indirect effect of theincreased cell volume of transgenic plants10.</p><p>A conditional ABP1 knockout mutation has been constructed by producing a transgenic ABP1 antibody in the tobacco BY2 cell line(C. Perrot-Rechenmann, pers. commun.). Thistransgenic antibody presumably binds ABP1in planta and limits its activity within the cell.These knockout cells showed no significant change incell volume but arrested at the G1 phase of the cellcycle. Thus, ABP1 might play a crucial role in theregulation G1 and G2/M phases of the cell cycle.</p><p>Although the conclusions from these twotransgenic studies differ, the results indicate acrucial role for ABP1 in plant cells. Furthermore,either knocking out or overproducing ABP1 providescrucial evidence that ABP1 mediates perception ofauxin in cultured cells and that disruption of thissignal causes changes in the cell cycle.</p><p>ABP1 triggers a plasma membrane electrical responseHyperpolarization of the cell membrane occurswithin minutes after applying biologically activeauxin, providing a convenient assay for evaluatingauxin response at the outer face of the plasmamembrane. ABP1 has been implicated in thisresponse in many studies11. Synthetic peptidescorresponding to the C terminus of ABP1 were testedin the hyperpolarization assay12,13 (Table 1). PeptidePz152-163 is a maize-derived sequence. Two othersare tobacco-derived peptides: the first, Nt-C15, ismost similar to the wild-type sequence whereas thesecond, Nt-C12, lacks three conserved residues.The maize and Nt-C15 peptides all inducehyperpolarization, much as auxin does when appliedto tobacco protoplasts. The truncated Nt-C12 peptidefails to induce the hyperpolarization response. Thisstudy confirms previous results that exogenouspeptides derived from ABP1 can elicit an electricalresponse. These results confirm that the homologoussystem is more efficient than the heterologoussystem, because peptides and membranes werederived from the same species13.</p><p>TRENDS in Plant Science Vol.6 No.12 December 2001</p><p></p><p>587ReviewReview</p><p>Table 1. C-terminal sequences tested for hyperpolarizationPeptide Sequence Hyperpolarization?</p><p>Consensus WDE.C......KEDL Not known</p><p>Maize ABP1 WDEDCFEAA..KDEL Yes</p><p>Nicotiana tabacum ABP1 WDEECYQTTSWKDEL Yes</p><p>Pz152163 .DEDCFEAA..KDEL Yes</p><p>Nt-C15 WDEECYQTTSWKDEL Yes</p><p>Nt-C12 ..ECYQTTSW.KDEL No</p><p>Mutation targets WDEECYQTTSWKDELa No</p><p>Deletion WDEECYQTTSW.... NoaText in bold indicates target residues for mutation.</p></li><li><p>The C-terminal charged residues in ABP1 weremutagenized and the entire protein was tested in thehyperpolarization assay14. The charged residues weremutated to the cognate amine residues, singly andpaired, and a KDEL deletion mutant protein wasconstructed (Table 1). The KDEL deletion evoked thesame hyperpolarization response as the wild type whenapplied to cells. None of the charge-substituted mutantproteins evoked a hyperpolarization response14. Thus,the substitution of charged residues causes ABP1 tofail to interact with the plasma membrane proteinthat affects hyperpolarization, implicating achargecharge interaction between the proteins.Alternatively, the mutated ABP1 might simply misfoldand fail to interact with the plasma membrane protein.</p><p>The electrical response of plant cells was affected byantibodies directed against ABP1. Several monoclonalantibodies induce hyperpolarization in tobacco cellprotoplasts and act as auxin agonists13. Three othermonoclonal antibodies act as antagonists and blockauxin action, either by recognizing the auxin-bindingsite as the epitope13 or by immobilizing ABP1 in a non-functional conformation. Similarly, antibodies affectedorchid cell stomatal opening. Both the D16 monoclonalantibody, raised against the putative auxin bindingsite of ABP1 (Ref. 15), and a monoclonal antibodyagainst an ABP1 peptide, induced stomatal openingand acidification, similar to the effects of auxin16. Amonoclonal antibody that targets the C terminus ofABP1 and the peptide Pz152-163 stimulated stomatalclosure and increased pH, similar to the mode ofabscisic acid. A Pz152-163 peptide lacking the KDELsequence had no effect. This result is curious becauseother data suggests that the KDEL is not requiredfor hyperpolarization stimulation. However, smallchanges in a peptide can cause great changes inpeptide structure. These immunological resultsindicate that ABP1 transduces the auxin signal tothe plasma membrane to effect hyperpolarization,perhaps by interacting with another protein.</p><p>The amount of ABP1 might be tightly regulatedin the cell. As the evidence above indicates,increased production of ABP1 enhances auxinsensitivity9,10,17,18. Examination at the molecular level</p><p>reveals that transgenic overexpression of wild-typeABP1 generated a ~100-fold increase in expression byRNA blotting but only a 30% increase in detectableprotein by immunoblotting18. In antisense transgenicplants, maximal inhibition of ABP1 protein wasmerely 50%, indicating that complete inhibition ofABP1 might be detrimental to the plant18.</p><p>Structure of ABP1The three-dimensional structure of ABP1 could giveclues about the mechanism of signaling or potentialproteinprotein interactions. The first model for theauxin-binding site of ABP1 was based on thestructure and interaction of 45 different auxinanalogs19. This model proposed a planar, indole ring-binding platform, a charged carboxylic acid-bindingsite and a hydrophobic transition region. Byphotoaffinity labeling with azido IAA (Ref. 20) andimmunology21, two regions were implicated in auxinbinding. Structure mapping studies using a panel ofmonoclonal antibodies further defined the identityof residues forming the auxin-binding platform andthe carboxylic acid-binding site13.</p><p>-Barrel dimerComparisons of amino acid sequences show that thereare several highly conserved residues between auxinbinding proteins in monocots and dicots2,22. Anaugmented model has been proposed based on theseand additional comparisons with the cupin and vicilinsuperfamily of proteins23. The structural basis of thismodel relies on conserved residues corresponding to-barrel turn anchors in the germin protein structure24.The proposed structure is a -barrel homodimer,containing -sheets and no -helix, consistent withcircular dichroism spectra25, and resembles thepseudodimer symmetry of a vicilin monomer23.Recently, ABP1 was crystallized, and X-ray diffractionanalysis to 1.9 resolution shows two glycosylatedhomodimers in asymmetric units26. These crystalstructure data are consistent with a -barrel (Fig. 1).This level of resolution cannot confirm the auxin-binding site. A conserved region might be analogous tothe metal-binding site of oxalate oxidase23 and thusindicate that ABP1 has some unknown enzymefunction. This speculation is intriguing, because noenzymatic activity has been reported for ABP1.</p><p>Mobile C-terminusExperi...</p></li></ul>


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