Flavonol‐mediated stabilization of PIN efflux complexes regulates polar auxin transport

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue‐native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo‐ and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1‐naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole‐3‐acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

[1]  A. Murphy,et al.  The Arabidopsis ATP-BINDING CASSETTE Transporter ABCB21 Regulates Auxin Levels in Cotyledons, the Root Pericycle, and Leaves , 2019, Front. Plant Sci..

[2]  A. Carruthers,et al.  Red wine and green tea flavonoids are cis-allosteric activators and competitive inhibitors of glucose transporter 1 (GLUT1)-mediated sugar uptake , 2018, The Journal of Biological Chemistry.

[3]  J. Friml,et al.  Flavonol-induced changes in PIN2 polarity and auxin transport in the Arabidopsis thaliana rol1-2 mutant require phosphatase activity , 2017, Scientific Reports.

[4]  C. Lücke,et al.  TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics , 2016, Plant Cell.

[5]  M. Ivanchenko,et al.  Master and servant: Regulation of auxin transporters by FKBPs and cyclophilins. , 2016, Plant science : an international journal of experimental plant biology.

[6]  N. Zheng,et al.  Molecular Mechanism Underlying the Plant NRT1.1 Dual-Affinity Nitrate Transporter , 2015, Front. Physiol..

[7]  R. Nitschke,et al.  Protocol: an improved and universal procedure for whole-mount immunolocalization in plants , 2015, Plant Methods.

[8]  J. Friml,et al.  PIN-Dependent Auxin Transport: Action, Regulation, and Evolution , 2015, Plant Cell.

[9]  A. Plückthun,et al.  Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry , 2014, Biology Open.

[10]  D. Baehrens,et al.  Regional Diversity and Developmental Dynamics of the AMPA-Receptor Proteome in the Mammalian Brain , 2014, Neuron.

[11]  B. Kuster,et al.  Auxin efflux by PIN-FORMED proteins is activated by two different protein kinases, D6 PROTEIN KINASE and PINOID , 2014, eLife.

[12]  R. Scott,et al.  Flavonoids and the regulation of seed size in Arabidopsis. , 2014, Biochemical Society transactions.

[13]  Klaus Palme,et al.  The iRoCS Toolbox--3D analysis of the plant root apical meristem at cellular resolution. , 2014, The Plant journal : for cell and molecular biology.

[14]  J. Payandeh,et al.  Crystal Structure of A Plant Dual-Affinity Nitrate Transporter , 2014, Nature.

[15]  Klaus Palme,et al.  A quantitative ratiometric sensor for time-resolved analysis of auxin dynamics , 2013, Scientific Reports.

[16]  C. Hocart,et al.  Alteration of flavonoid accumulation patterns in transparent testa mutants disturbs auxin transport, gravity responses, and imparts long-term effects on root and shoot architecture , 2013, Planta.

[17]  K. Moin,et al.  Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter* , 2011, The Journal of Biological Chemistry.

[18]  B. Fakler,et al.  Extending the Dynamic Range of Label-free Mass Spectrometric Quantification of Affinity Purifications* , 2011, Molecular & Cellular Proteomics.

[19]  S. Pollastri,et al.  Flavonols: old compounds for old roles. , 2011, Annals of botany.

[20]  A. Plückthun,et al.  Her2-specific multivalent adapters confer designed tropism to adenovirus for gene targeting. , 2011, Journal of molecular biology.

[21]  E. Spalding,et al.  The ER-Localized TWD1 Immunophilin Is Necessary for Localization of Multidrug Resistance-Like Proteins Required for Polar Auxin Transport in Arabidopsis Roots[OA] , 2010, Plant Cell.

[22]  H. Nam,et al.  Identification of an ABCB/P-glycoprotein-specific Inhibitor of Auxin Transport by Chemical Genomics* , 2010, The Journal of Biological Chemistry.

[23]  Fang Huang,et al.  Phosphorylation of Conserved PIN Motifs Directs Arabidopsis PIN1 Polarity and Auxin Transport[W][OA] , 2010, Plant Cell.

[24]  Filip Vandenbussche,et al.  Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana , 2010, Development.

[25]  A. Murphy,et al.  Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe. , 2009, The Plant journal : for cell and molecular biology.

[26]  Daniel R. Lewis,et al.  Measurement of auxin transport in Arabidopsis thaliana , 2009, Nature Protocols.

[27]  Suresh Chand,et al.  Interaction of PIN and PGP transport mechanisms in auxin distribution-dependent development , 2008, Development.

[28]  Diana Santelia,et al.  Modulation of P-glycoproteins by Auxin Transport Inhibitors Is Mediated by Interaction with Immunophilins* , 2008, Journal of Biological Chemistry.

[29]  S. Cutler,et al.  Arabidopsis P-glycoprotein19 participates in the inhibition of gravitropism by gravacin. , 2007, Chemistry & biology.

[30]  Klaus Palme,et al.  A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase interactors. , 2007, The Plant journal : for cell and molecular biology.

[31]  Elliot M. Meyerowitz,et al.  Antagonistic Regulation of PIN Phosphorylation by PP2A and PINOID Directs Auxin Flux , 2007, Cell.

[32]  Daniel R. Lewis,et al.  Mutations in Arabidopsis Multidrug Resistance-Like ABC Transporters Separate the Roles of Acropetal and Basipetal Auxin Transport in Lateral Root Development[W][OA] , 2007, The Plant Cell Online.

[33]  O. Kwon,et al.  Inhibition of the intestinal glucose transporter GLUT2 by flavonoids , 2007, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[34]  A. Murphy,et al.  Interactions of PIN and PGP auxin transport mechanisms. , 2007, Biochemical Society transactions.

[35]  Michael Sauer,et al.  Interactions among PIN-FORMED and P-Glycoprotein Auxin Transporters in Arabidopsis[W] , 2007, The Plant Cell Online.

[36]  C. Hawes,et al.  Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants , 2006, Nature Protocols.

[37]  Klaus Palme,et al.  Auxin in action: signalling, transport and the control of plant growth and development , 2006, Nature Reviews Molecular Cell Biology.

[38]  Zerihun Tadele,et al.  PIN Proteins Perform a Rate-Limiting Function in Cellular Auxin Efflux , 2006, Science.

[39]  C. Pikaard,et al.  Gateway-compatible vectors for plant functional genomics and proteomics. , 2006, The Plant journal : for cell and molecular biology.

[40]  Ping Wu,et al.  A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. , 2005, Plant & cell physiology.

[41]  Célia Baroux,et al.  Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1. , 2005, The Plant journal : for cell and molecular biology.

[42]  David A. Morris,et al.  Auxin inhibits endocytosis and promotes its own efflux from cells , 2005, Nature.

[43]  I. Blilou,et al.  The PIN auxin efflux facilitators: evolutionary and functional perspectives. , 2005, Trends in plant science.

[44]  B. Scheres,et al.  Dissection of Arabidopsis ADP-RIBOSYLATION FACTOR 1 Function in Epidermal Cell Polarityw⃞ , 2005, The Plant Cell Online.

[45]  Klaus Palme,et al.  The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots , 2005, Nature.

[46]  Klaus Palme,et al.  A PINOID-Dependent Binary Switch in Apical-Basal PIN Polar Targeting Directs Auxin Efflux , 2004, Science.

[47]  G. Jürgens,et al.  Local, Efflux-Dependent Auxin Gradients as a Common Module for Plant Organ Formation , 2003, Cell.

[48]  M. Evans,et al.  Gravity-regulated differential auxin transport from columella to lateral root cap cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Wilhelm Gruissem,et al.  Cell Cycle-regulated Gene Expression inArabidopsis * , 2002, The Journal of Biological Chemistry.

[50]  C. Kuhlemeier.  Faculty Opinions recommendation of The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. , 2001 .

[51]  A. Murphy,et al.  Multidrug Resistance–like Genes of Arabidopsis Required for Auxin Transport and Auxin-Mediated Development Article, publication date, and citation information can be found at www.aspb.org/cgi/doi/10.1105/tpc.010350. , 2001, The Plant Cell Online.

[52]  D. Weijers,et al.  The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. , 2001, Development.

[53]  N. Zahniser,et al.  Chronic and acute regulation of Na+/Cl- -dependent neurotransmitter transporters: drugs, substrates, presynaptic receptors, and signaling systems. , 2001, Pharmacology & therapeutics.

[54]  D. Saslowsky,et al.  Localization of flavonoid enzymes in Arabidopsis roots. , 2001, The Plant journal : for cell and molecular biology.

[55]  G. Muday,et al.  Genetic and Chemical Reductions in Protein Phosphatase Activity Alter Auxin Transport, Gravity Response, and Lateral Root Growth , 2001, The Plant Cell Online.

[56]  A. Murphy,et al.  Flavonoids act as negative regulators of auxin transport in vivo in arabidopsis. , 2001, Plant physiology.

[57]  A. Murphy,et al.  Flavonoid accumulation patterns of transparent testa mutants of arabidopsis. , 2001, Plant physiology.

[58]  D. Inzé,et al.  Auxin Transport Promotes Arabidopsis Lateral Root Initiation , 2001, Plant Cell.

[59]  E. Middleton,et al.  The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. , 2000, Pharmacological reviews.

[60]  M. Mann,et al.  Use of Mass Spectrometry to Study Signaling Pathways , 2000, Science's STKE.

[61]  C. Kuhlemeier,et al.  Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs , 2000, Plant Cell.

[62]  Klaus Palme,et al.  AtPIN2 defines a locus of Arabidopsis for root gravitropism control , 1998, The EMBO journal.

[63]  W. Michalke,et al.  Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites , 1992, Planta.

[64]  C. Bell,et al.  Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation. , 1991, The Plant cell.

[65]  P. Rubery,et al.  Naturally Occurring Auxin Transport Regulators , 1988, Science.

[66]  R. Hertel,et al.  Auxin transport in membrane vesicles from Cucurbita pepo L. , 1983, Planta.

[67]  G. F. Katekar,et al.  Auxin Transport Inhibitors: IV. EVIDENCE OF A COMMON MODE OF ACTION FOR A PROPOSED CLASS OF AUXIN TRANSPORT INHIBITORS: THE PHYTOTROPINS. , 1980, Plant physiology.

[68]  H. Söding,et al.  Über die Wirkungsweise von Phthalsäuremono-α-Naphthylamid (PNA) auf das Wachstum der Haferkoleoptile , 1958, Planta.

[69]  K. Palme,et al.  A PLA-iRoCS Pipeline for the Localization of Protein-Protein Interactions In Situ. , 2018, Methods in molecular biology.

[70]  J. Adamec,et al.  ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis. , 2009, The Plant journal : for cell and molecular biology.

[71]  H. Schägger,et al.  Blue native PAGE , 2006, Nature Protocols.

[72]  R. Hertel,et al.  Auxintransport und Schwerkraft , 2004, Naturwissenschaften.

[73]  P. Rubery.  Phytotropins: receptors and endogenous ligands. , 1990, Symposia of the Society for Experimental Biology.