Report PIN Polarity Maintenance by the Cell Wall in Arabidopsis (2011)

Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech,INRA Centre de Versailles-Grignon, 78026 Versailles Cedex,FranceSummaryA central question in developmental biology concerns themechanism of generation and maintenance of cell polarity,because these processes are essential for many cellularfunctions and multicellular development [1]. In plants, cellpolarityhasanadditionalroleinmediatingdirectionaltrans-port of the plant hormone auxin that is crucial for multipledevelopmental processes [2–4]. In addition, plant cellshave a complex extracellular matrix, the cell wall [5, 6],whose role in regulating cellular processes, including cellpolarity,isunexplored.Wehavefoundthatpolardistributionof PIN auxin transporters [7] in plant cells is maintained byconnections between polar domains at the plasmamembrane and the cell wall. Genetic and pharmacologicalinterference with cellulose, the major component of thecell wall, or mechanical interference with the cell walldisrupts these connections and leads to increased lateraldiffusion and loss of polar distribution of PIN transportersfor the phytohormone auxin. Our results reveal a plant-speciﬁc mechanism for cell polarity maintenance andprovide a conceptual framework for modulating cell polarityand plant development via endogenous and environmentalmanipulations of the cellulose-based extracellular matrix.Results and DiscussionsForward Genetic Screen for PIN Polarity RegulatorsCell polarization is an essential feature for many cellprocesses, and the establishment and maintenance of cellpolarity require intracellular vesicle trafﬁcking, cytoskeletonmodiﬁcation, and signaling events. The regulation of cellpolarity in plant and animal cells is fundamentally different[8,9]: (1) plants appear to have higher complexity of polar traf-ﬁcking pathways [10], (2) their cells generally lack the tightjunctions that separate the polar domains in animal epithelialcells [11], and, very importantly, (3) plant cells have a cellwall, a crucial cellular component that provides structuralintegrity to plant tissues and regulates cellular growth andform [12, 13].To identify components of the mechanism for cell polarityestablishment and maintenance in plants, we used as polaritymarkersPINtransportersfortheplanthormoneauxin[7].Polardistribution of PIN proteins at the plasma membrane deter-mines directionality of auxin ﬂow [14] and thus contributes toregulation of multiple aspects of plant development [4]. Apical(upper, shootward cell side) polar localization of PIN2 in rootepidermal cells is required for asymmetric auxin translocationfollowing gravistimulation and thus for gravitropic root growth[15, 16]. In contrast, the transgenic PIN2::PIN1-HA into pin2mutant background has apically localized PIN2 replaced bythebasally(lower,rootwardside)localizedPIN1protein(whichnormally localizes at the basal side of stele cells), and, asa consequence, this line does not rescue the agravitropicroot growth of pin2 mutant [14]. We designed a screen forregulators of PIN polarity with a rationale that, in lines in whichthe basal PIN1 localization is defective, a presence of PIN1 atthe upper side will at least partially replace the endogenousfunction of PIN2 and could, to some extent, restore the gravi-tropic root growth (Figure 1A). This rationale is supported byprevious observations that weaker polarity mutants oftenshow defects preferentially in the basal targeting, leading toectopic apical localization of basal cargos [17]. This strategyenabled us to screen for polarity mutants macroscopically,based on gravitropic root growth, and also to detect weakerpolarity mutants, because the polarity of PIN1-HA is moresensitive to perturbations than is the polarity of endogenousPIN proteins [17]. The strategy can also circumvent the ex-pected risk of embryo or seedling lethality of stronger polaritymutants. Hence, we screened for M2 EMS-mutagenizedPIN2::PIN1-HA;pin2 seedlings that positively responded totwo successive gravistimulation stimuli, and these candidateswere tested in the M3 generation by immunolocalizations forchanges in the polarity of PIN1-HA localization. From 2600M1 families, we identiﬁed four regulator of PIN polarity (repp)Arabidopsis mutants that showed positive gravitropicresponse and changed localization of PIN1-HA in epidermalcells.Asexpected,thereppmutantsdonotshowpronounceddefects in polarity of endogenous PIN proteins and thus cangenerate all basic cell types correctly (data not shown). Herewe present novel insights into the regulation of PIN polarityby analysis of the repp3 mutant (PIN2::PIN1-HA;pin2background).repp3 Is a PIN Polarity MutantIn contrast to predominantly basal and nonpolar PIN1-HAlocalization in PIN2::PIN1-HA;pin2 epidermal cells, roots ofrepp3haddefectsinbasallocalizationofPIN1-HA,asrevealedby anti-PIN1 and anti-HA immunolocalization experiments.These included partial recovery of PIN2-like localization atthe apical side of epidermal cells (Figures 1B and 1C; seealso Figure S1A available online). Accordingly, repp3 rootswere able to generate an asymmetric distribution of auxinresponse at the lower side of gravistimulated roots, as visual-ized using the DR5rev::GFP auxin response reporter (Fig-ure 1D; see also Figure S1B), and, despite a slightly wavyroot growth, showed positive gravitropic response (Figures