PIN phosphorylation is sufficient to mediate PIN polarity and direct auxin transport

The plant hormone auxin plays a crucial role in regulating plant development and plant architecture. The directional auxin distribution within tissues depends on PIN transporters that are polarly localized on the plasma membrane. The PIN polarity and the resulting auxin flow directionality are mediated by the antagonistic actions of PINOID kinase and protein phosphatase 2A. However, the contribution of the PIN phosphorylation to the polar PIN sorting is still unclear. Here, we identified an evolutionarily conserved phosphorylation site within the central hydrophilic loop of PIN proteins that is important for the apical and basal polar PIN localizations. Inactivation of the phosphorylation site in PIN1(Ala) resulted in a predominantly basal targeting and increased the auxin flow to the root tip. In contrast, the outcome of the phosphomimic PIN1(Asp) manipulation was a constitutive, PINOID-independent apical targeting of PIN1 and an increased auxin flow in the opposite direction. Furthermore, the PIN1(Asp) functionally replaced PIN2 in its endogenous expression domain, revealing that the phosphorylation-dependent polarity regulation contributes to functional diversification within the PIN family. Our data suggest that PINOID-independent PIN phosphorylation at one single site is adequate to change the PIN polarity and, consequently, to redirect auxin fluxes between cells and provide the conceptual possibility and means to manipulate auxin-dependent plant development and architecture.

[1]  R. Offringa,et al.  PINOID-Mediated Signaling Involves Calcium-Binding Proteins , 2003, Plant Physiology.

[2]  P. Cole,et al.  Peptide and protein phosphorylation by protein tyrosine kinase Csk: insights into specificity and mechanism. , 1998, Biochemistry.

[3]  Masahiko Hibi,et al.  c-Jun Can Recruit JNK to Phosphorylate Dimerization Partners via Specific Docking Interactions , 1996, Cell.

[4]  Ben Scheres,et al.  Polar PIN Localization Directs Auxin Flow in Plants , 2006, Science.

[5]  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.

[6]  G. Hagen,et al.  Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. , 1997, The Plant cell.

[7]  Yunde Zhao,et al.  NPY genes and AGC kinases define two key steps in auxin-mediated organogenesis in Arabidopsis , 2008, Proceedings of the National Academy of Sciences.

[8]  Michael Sauer,et al.  Efflux-dependent auxin gradients establish the apical–basal axis of Arabidopsis , 2003, Nature.

[9]  Albert J R Heck,et al.  Quantitative Phosphoproteomics of Early Elicitor Signaling in Arabidopsis*S , 2007, Molecular & Cellular Proteomics.

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

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

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

[13]  M. Cobb,et al.  Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. , 2001, Endocrine reviews.

[14]  O. Leyser,et al.  Plant Development: Auxin in Loops , 2005, Current Biology.

[15]  Joanne Chory,et al.  Rapid Synthesis of Auxin via a New Tryptophan-Dependent Pathway Is Required for Shade Avoidance in Plants , 2008, Cell.

[16]  Keithanne Mockaitis,et al.  Auxin receptors and plant development: a new signaling paradigm. , 2008, Annual review of cell and developmental biology.

[17]  Hong-Wei Zhou,et al.  Disparate Roles for the Regulatory A Subunit Isoforms in Arabidopsis Protein Phosphatase 2A , 2004, The Plant Cell Online.

[18]  C. Schwechheimer,et al.  The polarly localized D6 PROTEIN KINASE is required for efficient auxin transport in Arabidopsis thaliana , 2009, Development.

[19]  K. Feldmann,et al.  Arabidopsis AUX1 Gene: A Permease-Like Regulator of Root Gravitropism , 1996, Science.

[20]  G. Fink,et al.  EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. , 1998, Genes & development.

[21]  Jiří Friml,et al.  Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism , 2006, Nature Cell Biology.

[22]  P. Robles,et al.  A regulated auxin minimum is required for seed dispersal in Arabidopsis , 2009, Nature.

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

[24]  J. Friml,et al.  Spatiotemporal asymmetric auxin distribution: a means to coordinate plant development , 2006, Cellular and Molecular Life Sciences CMLS.

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

[26]  Yunde Zhao.  The role of local biosynthesis of auxin and cytokinin in plant development. , 2008, Current opinion in plant biology.

[27]  Yidong Liu,et al.  Phosphorylation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase by MPK6, a Stress-Responsive Mitogen-Activated Protein Kinase, Induces Ethylene Biosynthesis in Arabidopsisw⃞ , 2004, The Plant Cell Online.

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

[29]  G. Muday,et al.  PINOID Kinase Regulates Root Gravitropism through Modulation of PIN2-Dependent Basipetal Auxin Transport in Arabidopsis1[W][OA] , 2009, Plant Physiology.

[30]  J. Knoblich.  Pins for spines , 2005, Nature Cell Biology.

[31]  Klaus Palme,et al.  AtPIN4 Mediates Sink-Driven Auxin Gradients and Root Patterning in Arabidopsis , 2002, Cell.

[32]  Jan Petrásek,et al.  Auxin transport routes in plant development , 2009, Development.

[33]  Gerrit T. S. Beemster,et al.  Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal , 2005, Nature Cell Biology.

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

[35]  M. Sauer,et al.  Immunocytochemical techniques for whole-mount in situ protein localization in plants , 2006, Nature Protocols.

[36]  Anna N. Stepanova,et al.  TAA1-Mediated Auxin Biosynthesis Is Essential for Hormone Crosstalk and Plant Development , 2008, Cell.

[37]  J. Friml,et al.  Automated whole mount localisation techniques for plant seedlings. , 2003, The Plant journal : for cell and molecular biology.

[38]  Jing Zhang,et al.  Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter , 2009, Nature.

[39]  L. Dolan,et al.  Morphometric analysis of root shape. , 2004, The New phytologist.

[40]  Michael Sauer,et al.  Molecular and cellular aspects of auxin-transport-mediated development. , 2007, Trends in plant science.

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

[42]  J. Chory,et al.  Regulation of Auxin Response by the Protein Kinase PINOID , 2000, Cell.

[43]  R. Offringa,et al.  Regulation of auxin transport polarity by AGC kinases. , 2008, Current opinion in plant biology.

[44]  B. Frantz,et al.  p38 map kinase substrate specificity differs greatly for protein and peptide substrates. , 2000, Archives of biochemistry and biophysics.

[45]  A. Levine,et al.  Structure of the MDM2 Oncoprotein Bound to the p53 Tumor Suppressor Transactivation Domain , 1996, Science.

[46]  D. Schachtman,et al.  High-Affinity Auxin Transport by the AUX1 Influx Carrier Protein , 2006, Current Biology.

[47]  Steffen Vanneste,et al.  Auxin: A Trigger for Change in Plant Development , 2009, Cell.

[48]  M. Strnad,et al.  Isolation of novel indole-3-acetic acid conjugates by immunoaffinity extraction. , 2009, Talanta.