Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions

Abstract

Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication-a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family 1 are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms 2-7 . Polar PIN localization determines the direction of intercellular auxin flow 8 , yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosisdependent mechanism of PIN polarity generation and analyse its developmental implications. Realtime PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.The plant signalling molecule auxin acts as a versatile trigger in many aspects of plant development and mediates different cellular responses on the basis of its graded distribution between cells. Establishment and maintenance of these auxin gradients requires local auxin biosynthesis 9,10 and directional cell-to-cell transport that depends on PIN auxin transporters 11 . PINs have a polar plasma membrane localization that determines the direction of intercellular auxin flow 8 . Thus, the mechanisms underlying PIN polarity belong to centralCorrespondence and requests for materials should be addressed to P.D. (E-mail: P.B.Dhonukshe@uu.nl) or J.F. (E-mail: jiri.friml@psb.ugent.be). * These authors contributed equally to this work.Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Reprints and permissions information is available at www.nature.com/reprints. are important components of polar PIN localization. However, it remains unresolved how PIN polarity is initially generated. In mammalian epithelia, segregation of membrane proteins into apical and basolateral plasma membrane domains is mainly achieved by polar exocytosis of newly synthesized proteins, or by non-polar exocytosis followed by endocytosis and polarized recycling 19 . Here we demonstrate an endocytosis-dependent mechanism for PIN polarity generation, and its import...

Embryogenesis: Pattern Formation from a Single Cell

“…Consistent with this interpretation, EMB30/GN mutations were shown to interfere with the coordinated polar localization of PIN1 proteins (Geldner et al, 2003) ( Figure 8B) (for a detailed review, see Kleine-Vehn and Friml, 2008). The role of endocytic recycling in establishing polar localization of PIN proteins has been further elaborated by manipulation of the Arabidopsis Rab5 GTPase pathway (Dhonukshe et al, 2008). The critical role of the Rab5 GTPase pathway in endocytosis has been well characterized in mammalian systems and two homologues of Rab5 in Arabidopsis, ARA7 and RHA1, have been shown to localize to the endosome (Ueda et al, 2004).…”

“…The critical role of the Rab5 GTPase pathway in endocytosis has been well characterized in mammalian systems and two homologues of Rab5 in Arabidopsis, ARA7 and RHA1, have been shown to localize to the endosome (Ueda et al, 2004). Although single ara7 or rha1 mutants show no obvious phenotypic defects and the double ara7 rha1 mutant is gametophytic lethal, an inducible dominant negative version of ARA7 has been used to demonstrate a requirement for Rab5-mediated endocytosis in the polarized distribution of PIN1 and PIN2 proteins (Dhonukshe et al, 2008).…”

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response inArabidopsis

“…In addition to a potential role facilitating outer lateral targeting of some membrane transporters, CRK5 could also play a role in recycling proteins from the outer lateral membranes and thereby contribute to their targeting to the BFA-sensitive membrane recycling pathway. As polar localization of PIN proteins appears to be mediated though nonpolar secretion followed by their internalization and recycling-dependent polarization (Dhonukshe et al, 2008), current models propose that sequential phosphorylation of hydrophilic T-loops of PINs by AGCs and possibly other protein kinases (Zhang et al, 2010) directs their stabilization in basal membranes and subsequent apical transcytosis (Rademacher and Offringa, 2012;Löfke et al, 2013). As most evidence supporting these models is derived from the analysis of polar localization of PIN1 and PIN2 in vertically grown and gravistimulated roots, in this work, we examined in detail how inactivation of CRK5 affects the root gravitropic response.…”

“…PINs are probably secreted to membranes in an apolar fashion and then their basal localization is established through membranerecycling controlled by the Rab5-GTPase ARA7, VPS9A (vacuolar protein sorting), and brefeldin A (BFA)-sensitive ADP-ribosylation-GDP/GTP-exchange factor GNOM proteins (Dhonukshe et al, 2008). Brefeldin inhibition of GNOM-dependent basal targeting stimulates PIN accumulation in so-called brefeldin compartments and leads ultimately to their relocalization to apical membranes .…”

The AP-3 adaptor complex is required for vacuolar function in Arabidopsis

“…As an approach to identify new regulators of protein trafficking pathways we carried out a fluorescence imaging-based forward genetic screen for mutants affecting the subcellular trafficking of the PIN1 auxin efflux carrier [14], which undergoes elaborate subcellular dynamics including secretion [15], clathrin-mediated endocytosis [16], polar recycling [17,18] and trafficking to the vacuole [19,20]. By using an ethyl methanesulfonate (EMS)-mutagenized PIN1 pro :PIN1-GFP population we originally identified three independent pat mutant loci from about 1 500 M1 families; among them pat2-1 is defective in the putative β adaptin of the AP-3 complex [10].…”