JIPB

Journal of Integrative Plant Biology

ROP GTPase‐mediated auxin signaling regulates pavement cell interdigitation in Arabidopsis thaliana Deshu Lin1*, Huibo Ren1 and Ying Fu2 1

Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fuzhou 350002, China, 2State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China.

Abstract In multicellular plant organs, cell shape formation depends on molecular switches to transduce developmental or environmental signals and to coordinate cell‐to‐cell communication. Plants have a specific subfamily of the Rho GTPase family, usually called Rho of Plants (ROP), which serve as a critical signal transducer involved in many cellular processes. In the last decade, important advances in the ROP‐mediated regulation of plant cell morphogenesis have been made by using Arabidopsis thaliana leaf and cotyledon pavement cells. Especially, the auxin‐ROP signaling networks have been demonstrated to control interdigitated growth of pavement

INTRODUCTION

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Keywords: Auxin; leaf pavement cell; Rho of plants GTPase Citation: Lin D, Ren H and Fu Y (2014) ROP GTPase‐mediated auxin signaling regulates pavement cell interdigitation in Arabidopsis thaliana. J Integr Plant Biol XX: XXX–XXX. doi: 10.1111/jipb.12281 Edited by: Tobias Baskin, University of Massachusetts Amherst, USA Received Jun. 24, 2014; Accepted Aug. 27, 2014 Available online on Aug. 29, 2014 at www.wileyonlinelibrary.com/ journal/jipb © 2014 Institute of Botany, Chinese Academy of Sciences

inhibition of the neighboring cell. Therefore, in addition to its physiological importance for the development of the leaf epidermis, jigsaw‐puzzle pavement cells have been taken advantage of frequently as an ideal model system to investigate cell‐to‐cell signaling communication and the coordination of cell polarity and shape formation in multicellular tissues and organs (Qiu et al. 2002; Li et al. 2003, 2013; Fu et al. 2005, 2009; Xu et al. 2011, 2014; Zhang et al. 2011; Nagawa et al. 2012; Lucas et al. 2013; Sampathkumar et al. 2014). It has long been accepted that the cytoskeleton elements, microtubules and microfilaments, play important roles in plant cell shape formation (Hepler and Palevitz 1974; Fu et al. 2002, 2005, 2009; Smith 2003; Wasteneys and Galway 2003; Smith and Oppenheimer 2005; Ivakov and Persson 2013; Lin et al. 2013). In diffusely growing cells, cortical microtubules are organized into a parallel array that is transverse to the elongation axis. This highly ordered transverse microtubule array is believed to restrict the lateral cell expansion through guiding the deposition of cellulose microfibrils (Baskin 2001, 2005; Wasteneys 2004; Smith and Oppenheimer 2005; Ehrhardt and Shaw 2006; Paradez et al. 2006; Gutierrez et al. 2009). Disturbance of microtubule organization or microtubule‐dependent processes leads to isotropic cell XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

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Cell polarity and morphogenesis are critical for plant tissue and organ development and pattern formation. To investigate how plants regulate the establishment and maintenance of cell polarity as well as cell shape formation, scientists have adopted several model systems, such as the pollen tube, trichome, and leaf and cotyledon pavement cells, and exploited them productively, as revealed by recent studies (Yang 2002; Fu et al. 2005; Gu et al. 2005; Yang 2008; Duan et al. 2010; Qin and Yang 2011; Wu et al. 2011; Grebe 2012; Nakamura et al. 2012; Gu and Nielsen 2013; Guan et al. 2013). Arabidopsis thaliana leaf and cotyledon pavement cells display interdigitated jigsaw‐puzzle appearance with lobes and indentations (Figure 1A, B). The development of pavement cells is divided into three stages (Figure 1C) (Fu et al. 2002, 2005). Stage I cells are small cells that just start to expand, with more or less regular morphology. Expending stage II cells display slightly wavy outlines, resulting from the protrusion of lobes as well as the inhibition of expansion at indentation regions. Stage III cells are cells interlocking with each other, with fully extended lobes and highly wavy cell outlines. To create such deep interdigitation, the local outgrowth in one cell is presumably coordinated with the local growth

Invited Expert Review

Deshu Lin *Correspondence: [email protected]

cells to form jigsaw‐puzzle shapes. Here, we review findings related to the discovery of this novel auxin‐signaling mechanism at the cell surface. This signaling pathway is to a large extent independent of the well‐known Transport Inhibitor Response (TIR)–Auxin Signaling F‐Box (AFB) pathway, and instead requires Auxin Binding Protein 1 (ABP1) interaction with the plasma membrane‐localized, transmembrane kinase (TMK) receptor‐like kinase to regulate ROP proteins. Once activated, ROP influences cytoskeletal organization and inhibits endocytosis of the auxin transporter PIN1. The present review focuses on ROP signaling and its self‐organizing feature allowing ROP proteins to serve as a bustling signal decoder and integrator for plant cell morphogenesis.

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Figure 1. Development of cotyledon pavement cells of Arabidopsis thaliana (A) Cotyledons of A. thaliana seedlings are used as a model for studying the interdigitation of pavement cells. (B) A confocal microscopic view of cotyledon pavement cells of A. thaliana stained with propidium iodide for visualization. Scale bar ¼ 50 mm. (C) A schematic illustration showing development of the cotyledon pavement cell. The development can be separated into three stages and is associated with cortical fine actin microfilaments (MF; F‐actin) (red patches) in the cortex, and with cortical microtubules (green lines). Arrows indicate directions of expansion. Images are reproduced from Fu et al. (2005) with permission.

expansion. A well‐ordered transverse cortical microtubule array is associated with the indentation regions of pavement cells. A. thaliana mutants with loss of function of various microtubule‐associated proteins (MAPs, such as mor1, clasp, ktn1, map18, and ric1) display disordered cortical microtubule arrays and defective pavement cell shapes with wider indentation regions than wild‐type cells (Burk et al. 2001; Whittington et al. 2001; Ambrose et al. 2007; Wang et al. 2007; Fu et al. 2009; Lin et al. 2013; Lindeboom et al. 2013; Zhang et al. 2013). Interestingly, cortical fine microfilaments required for the initiation and outgrowth of lobes are found to associate with the growing lobe tips of pavement cells where transverse well‐ ordered microtubules are absent (Frank and Smith 2002; Fu et al. 2002; Fu et al. 2005). Reduced lobe formation (sometimes complete elimination) is observed in loss of function of actin‐ related proteins, such as brk1 as well as several ARP2/3 complex subunit mutants (Li et al. 2003; Deeks et al. 2004; Frank et al. 2004; Djakovic et al. 2006). It is proposed that these fine microfilaments are important for the trafficking of exocytotic vesicles to deliver new plasma membrane and cell wall materials to the growth sites (Lee et al. 2008). Both XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

microfilaments and microtubules are highly dynamic structures and are both regulated spatiotemporally. In eukaryotic cells, a conserved regulator of these cytoskeletal elements is the family of Rho GTPase (Etienne‐ Manneville and Hall 2002; Yang 2002). In plants, a distinct subfamily of Rho has been found, usually termed ROP (Rho of Plants). ROP subfamily members serve as versatile signaling switches involved in many cellular and developmental processes and stress responses (Yang 2002; Nagawa et al. 2010; Wu et al. 2011; Li et al. 2012a, 2012b; Oda and Fukuda 2012). Upon activation by upstream signals, active ROP proteins interact with immediate effectors that relay signals to downstream cellular components and consequently induce a variety of cellular responses (Yang 2002). In the last decade, control of pavement cell interdigitation has been found to rely on ROP GTPase through their manipulation of the cytoskeleton and intracellular vesicle trafficking (Fu et al. 2005, 2009; Xu et al. 2010; Nagawa et al. 2012). Recently, the discovery of a novel auxin‐signaling mechanism mediated by ROP GTPase makes a notable progress toward solving the signaling mechanism puzzle of pavement cell morphogenesis (Xu et al. 2010, 2014). www.jipb.net

ROP Signaling Regulates Pavement Cell Interdigitation Although there are recent reviews that summarize the roles of ROP GTPases in cell polarity, vesicle trafficking, and auxin signaling (Nagawa et al. 2010; Wu et al. 2011; Xu et al. 2011; Craddock et al. 2013; Chen and Friml 2014), we still think it is well worth writing an additional specific review regarding the pavement cell, an emerging and powerful model system in plant cell biology. We hope that by discussing recent research progresses on pavement cells in A. thaliana, this review together with the latest review (Chen and Yang 2014), will refine our understanding of the ROP GTPase‐mediated cell‐surface auxin signal in the regulation of cytoskeletal organization and vesicle trafficking during pavement cell interdigitation.

ROP SIGNALING REGULATION OF LEAF PAVEMENT CELL INTERDIGITATION A family of plant‐specific ROP effectors containing a CDC42/ RAC Interactive Binding (CRIB) motifs discovered in fungal Rho GTPase effectors (Burbelo et al. 1995), named ROP Interactive CRIB motif‐containing protein (RIC), has been identified (Wu et al. 2001). There are 11 annotated RIC proteins in the A. thaliana genome, and it has been reported that these proteins function in a wide range of cellular and developmental processes, such as tip growth of pollen tubes, intercalation of leaf pavement cells, and root development (Wu et al. 2001; Yang 2002; Fu et al. 2005, 2009; Gu et al. 2005; Lee et al. 2008; Nagawa et al. 2010; Chen et al. 2012; Lin et al. 2012, 2013; Choi et al. 2013). To date, two antagonistic pathways, mediated by two different RIC proteins (RIC1 and RIC4), have been shown to regulate the interdigitated growth of pavement cells. One of them is the ROP6–RIC1 pathway, which promotes well‐ordered transverse cortical microtubules in the indentation regions, and the other is the ROP2–RIC4 pathway, which promotes the accumulation of fine microfilaments in the lobe tips (Fu et al. 2005, 2009). In plant cells, well‐ordered transverse cortical microtubule arrays are usually associated with restriction of expansion in the direction of their dominant orientation. The dynamics of microtubules and their organization into different arrays are known to be regulated by microtubule self‐organization and sustained treadmilling, as well as by many microtubule‐ associated proteins (Shaw et al. 2003; Ehrhardt and Shaw 2006; Chan et al. 2009; Gardiner 2013). The discovery of the ROP6–RIC1 pathway has helped us understand how pavement cells produce a well‐ordered microtubule array in the indentation region. Fu et al. (2009) have found that the ROP6 effector RIC1 is a novel microtubule‐associated protein in pavement cells. Loss of RIC1 function leads to randomly oriented cortical microtubules and subsequently increases expansion in the indentation regions of pavement cells. However, overexpression of RIC1 increases the abundance of transverse microtubules and turns the jigsaw‐puzzle appearance of pavement cells into a cylindrical shape (Fu et al. 2005, 2009). RIC1 is known to be able to interact with several ROP proteins, including ROP1, ROP2, and ROP6. Analysis of pavement cell phenotypes as well as microtubule organization in the rop knockout mutants has demonstrated that ROP6 is the upstream activator of RIC1 for its function of ordering microtubules (Fu et al. 2009). Overexpression of ROP6 www.jipb.net

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produces similar highly ordered transverse microtubules as that induced by RIC1 overexpression. Pavement cells with overexpressing ROP6 also lose the intercalating jigsaw‐puzzle appearance. Recently, through a genetic screen from mutants generated by ethyl methane sulfonate mutation in the ROP6 overexpression background, the microtubule‐severing protein, katanin (KTN1, the p60 subunit) is identified as a downstream component of the ROP6–RIC1 signaling pathway (Lin et al. 2013). Lin and colleagues have shown that RIC1 physically interacts with KTN1 and promotes microtubule‐ severing activity of KTN1 in vitro. They find also that RIC1 and KTN1 at least partially co‐localize in pavement cells, and overexpression of RIC1 promotes the KTN1‐mediated detachment of branched microtubules (Nakamura et al. 2010; Lin et al. 2013). Furthermore, a point mutation in KTN1 induces a randomly oriented cortical microtubule network in the pavement cells of wild type as well as of ROP6‐ overexpression or RIC1‐overexpression lines. The restriction of lateral cell expansion by overexpressing either ROP6 or RIC1 is also released by loss of KTN1 function. Taken together, these findings reveal a novel signaling pathway mediated by ROP6–RIC1–KTN1, which regulates the organization of cortical microtubule arrays and pavement cell morphogenesis. ROP2 and ROP4 are two ROP proteins with 97% amino acid identity and are functionally redundant in regulation of pavement cell shape formation (Fu et al. 2005). Different from ROP6, loss of ROP2 or ROP4 function leads to narrower indentation regions and shallower lobes phenotypes compared to wild‐type pavement cells. Expression of a constitutively active ROP2 (CA‐rop2) protein causes isotropic expansion and results in cubical pavement cells, while in contrast expression of ROP2 dominant‐negative (DN‐rop2) gives rise to plants with fewer lobes and reduced lobe outgrowth (Fu et al. 2002, 2005). Further investigation demonstrates that ROP2 preferentially localizes to the lobe tips of pavement cells and interacts with its effector RIC4 to promote the assembly of fine cortical microfilaments, correlated with the outgrowth of pavement cell lobes (Fu et al. 2002, 2005). However, RIC4 itself does not directly bind to microfilaments. It is likely that the ROP2–RIC4 pathway targets to one or more actin‐binding proteins to regulate the organization and dynamics of microfilaments. Although the ROP6–RIC1 and the ROP2–RIC4 pathways regulate the organization of cortical microtubules and microfilaments, respectively, these two pathways are required to interact for proper pavement cell morphogenesis and interdigitation between neighboring cells. Indeed, details of the interaction between these pathways have been uncovered. Active ROP2 at the lobe tips inhibits RIC1 association with microtubules and prevents the formation of a well‐ordered microtubule array in the lobes. Furthermore, ROP6, which is preferentially localized to the indentation regions of pavement cells, activates RIC1 to promote the formation of parallel arrays of microtubules, which not only restricts the lateral expansion of the pavement cell but also suppresses ROP2 activity in the indentation regions (Fu et al. 2005, 2009). Thus, the outgrowth‐ promoting ROP2–RIC4 pathway in the lobe tips and the outgrowth‐inhibiting ROP6–RIC1 pathway in the indentation regions antagonize each other and consequently cause XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

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interdigitations between adjacent pavement cells and lead to the jigsaw‐puzzle shape of pavement cells.

AUXIN ACTIVATES ROP SIGNALING TO REGULATE PAVEMENT CELL INTERDIGITATION As molecular switches, coordinative ROP proteins relay signals to microtubules and microfilaments to develop puzzle‐like pavement cells with interlocking lobes and indentations. However, the upstream developmental signal that triggers ROP signaling had been unclear until recent work established a novel auxin‐dependent signaling pathway (Xu et al. 2010). The hormone, auxin, is involved in nearly every aspect of plant growth and development. A common mechanism for auxin participating in so many developmental processes is through the regulation of gene expression mediated by Transport Inhibitor Response (TIR)/Auxin Signaling F‐Box (AFB) proteins, which target the transcriptional repressors AUX/IAA for degradation (Parry and Estelle 2006; Wang and Estelle 2014). In addition to the TIR1–AFB‐mediated nuclear pathway, recent studies by Xu and colleagues have established a novel auxin signaling pathway involving the Auxin Binding Protein 1 (ABP1)‐Transmembrane Kinase 1 (TMK1) auxin‐sensing complex that regulates pavement cell morphogenesis through activation of ROP proteins (Xu et al. 2010). Xu and colleagues found that application of auxin significantly increases pavement cell interdigitation in wild‐type pavement cells (Xu et al. 2010). They then analyzed pavement cell phenotypes of the yucca quadruple mutant, which is deficient in auxin biosynthesis (Zhao et al. 2001). The yucca pavement cells have reduced lobes compared to wild type, which could be rescued by addition of auxin (Xu et al. 2010). These data suggest a role of auxin for control of pavement cell interdigitation; however, exogenous auxin could not rescue the reduced lobe phenotype of the ROP2RNAi rop4‐1 plant (Xu et al. 2010), indicating that ROP2 and ROP4 are required for auxin activation of pavement cell interdigitation. Further investigation indeed detects a rapid increase of ROP2 activity by as low as 1 nM 1‐naphthaleneacetic acid treatment within a period as short as 30 s. Furthermore, auxin treatments also increase the amount of active ROP6 in a dose‐dependent manner (Xu et al. 2010). These findings link auxin to ROP‐ dependent pavement cell morphogenesis, but also raise an interesting question: how does auxin activate two antagonistic ROP pathways to coordinate pavement cell interdigitation?

ABP1‐TMK1 IS THE AUXIN‐SENSING COMPLEX THAT ACTIVATES ROP SIGNALING Fast activation of ROP2 and ROP6 by auxin implicates a rapid cytosolic response mechanism that is distinct from TIR1/AFB‐ mediated regulation of gene expression. ABP1 has been shown to perceive extracellular auxin and orchestrate non‐ transcriptional auxin signaling at the cell periphery, and ABP1 XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

has been implicated in a large number of physiological processes, such as embryogenesis, expansion, and root development (Jones et al. 1998; Ruegger et al. 1998; Chen et al. 2001; David et al. 2007; Braun et al. 2008; Tromas et al. 2009; Sauer and Kleine‐Vehn 2011; Scherer 2011; Shi and Yang 2011). An ABP1 knockout mutant in A. thaliana fails to undergo expansion in the embryo, leading to early embryo lethality (Chen et al. 2001). Xu and colleagues have demonstrated that a weak allele of ABP1 (abp1‐5), containing a point mutation in the auxin‐binding pocket, has significantly reduced pavement cell interdigitation, and application of auxin activates neither ROP2 nor ROP6 in abp1‐5 protoplasts (Xu et al. 2010). Additionally, subcellular localization of GFP‐RIC4 demonstrates that GFP‐RIC4 is primarily localized to the plasma membrane of pavement cells in wild type by means of its preferential interaction with the active form of ROP2. However, in the yucca quadruple mutant, ROP2RNAi rop4‐1, and abp1‐5, the plasma membrane localization of GFP‐RIC4 is significantly reduced, reflecting decreased ROP2 activity at the plasma membrane. Furthermore, RIC1 association with cortical microtubules is greatly reduced in yucca and abp1‐5 pavement cells (Xu et al. 2010). Insofar as RIC1’s association with microtubules requiring active ROP6 (Fu et al. 2009; Xu et al. 2010), less microtubule‐ associated RIC1 represents a decrease of ROP6 activity in yucca and abp1‐5 pavement cells. Taken together, ABP1‐dependent auxin perception appears to be required for the rapid activation of both ROP2 and ROP6 pathways at the plasma membrane of pavement cells. How does ABP1 transmit the auxin signal at the cell surface to regulate cytoplasmic responses? A family of TMK of the receptor‐like kinase class with four members in the A. thaliana genome has been discovered (Chang et al. 1992; Dai et al. 2013). Pavement cells of the tmk1234 quadruple mutant have more severe defects of interdigitation than those observed in abp1‐5 mutant (Xu et al. 2014). Moreover, auxin‐ mediated activation of both ROP2 and ROP6 is significantly inhibited in tmk1234, and the localization of GFP‐RIC4 at the plasma membrane as well as the association of GFP‐RIC1 with microtubules are both reduced, indicating reduced ROP2 and ROP6 activity in tmk1234, just as observed in abp1‐5. Given that both TMK1 and ABP1 are critical for the activation of plasma membrane‐localized ROP2 and ROP6 in pavement cells, TMK1 and ABP1 may function in the same complex to perceive and transmit auxin signaling. Xu and colleagues further demonstrate that TMK1 interacts with ABP1 at the cell surface in an auxin dose‐dependent manner, and that the interaction is required for the auxin‐ mediated activation of ROP GTPase signaling in the control of pavement cell interdigitation (Baumann 2014; Xu et al. 2014). Furthermore, abp1‐5 not only greatly reduces the sensitivity to auxin in the activation of ROP2 or ROP6 but also abolishes the formation of the ABP1‐TMK1 complex in response to auxin (Xu et al. 2014). ABP1 associates with the extracellular domain of TMK1 at the cell surface. Collectively, the TMK1 receptor‐like kinase forms an auxin‐ sensing complex with ABP1 at the cell surface to activate ROP signaling for regulation of pavement cell interdigitation (Figure 2). www.jipb.net

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Figure 2. Model for regulation of pavement cell interdigitation (A) A model for coordination of the two ROP signaling pathways that regulate lobes outgrowth and necks expansion through affecting action or cortical microtubule organization, respectively. This model includes known components (Auxin, ABP1, TMK1, ROP2, ROP6, PIN1, RIC4, RIC1, KTN1), and their interactions at the cell surface‐ and ROP‐based auxin signaling network underlying pavement cell morphogenesis. (B) A model for ROP‐based auxin signaling in the control of interdigitated growth of pavement cells through inter‐ and intracellular coordination of the ROP2 and ROP6 pathways. Interdigitated growth of pavement cell is regulated by an auxin‐dependent self‐organizing mechanism. In this mechanism, the extracellular auxin is sensed by the ABP1/TMK1 complex, and then activated two counteracting ROP2 and ROP6 pathways, respectively, to control cell‐to‐cell coordination of lobing and indenting in two adjacent cells. The ROP2–RIC4 pathway promotes lobe outgrowth via a fine cortical actin network, while the ROP6–RIC1–KTN1 pathway suppresses indentation outgrowth via well‐organized cortical microtubules. Localized extracellular auxin, which is generated by self‐activation via the auxin–ROP2–PIN1–auxin feedback loop and self‐maintenance via the antagonizing ROP6 pathway.

ROP2‐PIN1 FEEDBACK IN THE AUXIN‐ SIGNALING MECHANISM Auxin as a morphogen modulates plant growth and development by means of its asymmetrical distribution. The relevant auxin gradients are established by polar auxin transport mediated by auxin transporters (Benkova et al. 2003; Vanneste and Friml 2009). Observations indicate that the efflux carries, mainly the pin formed (PIN) proteins, are polarly localized to the plasma membrane for directing auxin flow. Auxin inhibits endocytosis of PIN proteins in the A. thaliana root by interrupting clathrin recruitment to the plasma membrane (Paciorek et al. 2005; Robert et al. 2010; Kitakura et al. 2011). Conversely, ABP1 is reported to be a positive regulator of endocytosis by recruiting clathrin to the plasma membrane (Robert et al. 2010). Interestingly, recent data have that implicated ABP1 functions in the TIR–AFB pathway regulating gene transcription (Tromas et al. 2013; Paque et al. 2014), but its mode of action in this respect remains elusive. ROP proteins play essential roles in generation of cell polarity and in the configuration of microfilaments; therefore, it was proposed that ROP may feedback‐regulate auxin signaling through manipulating the polar localization of PIN proteins. Recently, PIN1, which is preferentially localized to the www.jipb.net

lobe tips at the plasma membrane, has been found to be critical for pavement cell interdigitation (Xu et al. 2010; Nagawa et al. 2012). A PIN1 knockout mutant (pin1‐1) has a defect in pavement cell interdigitation that is similar to ROP2RNAi rop4‐1 cells (Fu et al. 2005; Xu et al. 2010). ROP2 activity is reduced in the plasma membrane of pin1‐1 cells, as indicated by the decrease of the plasma membrane‐localized GFP‐RIC4, compared to wild‐type cells (Xu et al. 2010). Furthermore, application of auxin fails to rescue the lobing defects in pin1‐1. These findings suggest that PIN1 is also involved in the ROP‐ mediated auxin signaling during pavement cell interdigitation. The auxin‐dependent local activation of ROP2 at the lobe tips inhibits PIN1 endocytosis into the endosomal compartments containing ARA7, leading to higher levels of PIN1 accumulation at the tip of the lobes (Xu et al. 2010; Nagawa et al. 2012). PIN1 internalization in the lobe regions is inhibited by the stabilization of the cortical actin microfilaments through the ROP2 effector protein RIC4, while local auxin exported outside by PIN1 to the cell wall activates ROP2 at the plasma membrane to form a positive feedback loop for the polar PIN1 distribution during pavement cell morphogenesis (Xu et al. 2010; Nagawa et al. 2012). That the auxin–ROP2 pathway induces accumulation of PIN1 at the lobe tips also helps to elucidate the coordination between lobes and neighboring XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

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indentation regions governed by antagonistic ROP2 and ROP6, which are simultaneously activated by localized extracellular auxin at opposing sides of the cell wall (Figure 2) (Xu et al. 2010).

OUTLOOK AND PERSPECTIVES Polarity establishment and maintenance are fundamental processes in eukaryotic cells, which require precise regulation and plasticity. Using the pavement cell as a model system, the complex signaling mechanisms involving auxin, ROP proteins, cytoskeleton, and vesicle trafficking become clearer and clearer (Nagawa et al. 2010; Baumann 2014; Chen and Friml 2014). Interestingly, the auxin‐activated ROP6–RIC1 pathway also has been demonstrated to play a critical role in the regulation of PIN2 subcellular distribution in roots, possibly by stabilizing microfilaments (Lin et al. 2012), which indicates that ROP signaling to the actin cytoskeleton is a common regulatory mechanism for PIN trafficking and feedback regulation of auxin signaling in various tissues and organs. Nevertheless, the ROP‐mediated polar localization of PIN proteins is not only dependent on the cytoskeleton. Another family of ROP effectors in A. thaliana named ICR or RIP (Interactor of Constitutive Active ROP or ROP‐Interactive Partner), comprising five members of coiled‐coil domain‐ containing proteins, likely provide scaffolds to mediate ROP signaling (Lavy et al. 2007; Li et al. 2008; Hazak et al. 2010). In the A. thaliana root, ICR1 or RIP1 binds to AtSEC3A and acts as a scaffold protein linking ROP to the exocyst and regulates PIN protein distribution through its promotion of PIN recycling (Hazak et al. 2010). The icr1 mutant has small and cubical pavement cells. Overexpression of ICR1 results in defective pavement cells with the loss of the intercalating jigsaw‐puzzle appearance, similar to the appearance of plants overexpressing CA‐ROP6 (Lavy et al. 2007). Therefore, ROP proteins may affect auxin distribution through the coordination of multiple pathways in the control of the trafficking and polar localization of PIN proteins. To date, auxin activation of ROP proteins has been documented, and the auxin‐sensing TMK1‐ABP1 complex has been uncovered (Xu et al. 2010, 2014). However, there are still gaps of our understanding of steps between perception of the auxin signal and activation of the ROP protein, which need to be investigated in the future. Besides auxin, other hormones may also participate in the regulation of pavement cell interdigitation. Recently, Li and colleagues have found that cytokinin signaling acts upstream of ROP to suppress the interdigitation of pavement cells (Li et al. 2013), possibly through affecting auxin signaling, but the underlying mechanism remains a mystery. ROP GTPase activity is precisely regulated by three major classes of regulators, guanine nucleotide dissociation inhibitor (GDI), GTPase‐activating protein (GAP), and guanine nucleotide exchange factor (GEF). In the future, it will be interesting to investigate the positive and negative regulators of ROP activity in the auxin signaling pathways for regulation of pavement cell interdigitation. In the A. thaliana genome, GEF proteins that interact with ROP proteins fall into two types: the first, with just one member, named spike (Qiu et al. 2002; Basu et al. 2008; Zhang XXX 2014 | Volume XXXX | Issue XXXX | XXX-XX

et al. 2010), contains a homolog of single Dock homology region; the other is a plant specific family with 14 members, termed ROP‐GEF, containing a conserved plant specific ROP nucleotide exchange (PRONE) domain (Berken et al. 2005; Gu et al. 2006). Knockout of spike causes severe defects in leaf epidermis, such as reduced or even eliminated pavement cell interdigitation, reduced trichome branches, and reduced cell‐ cell adhesion (Qiu et al. 2002; Basu et al. 2008; Zhang et al. 2010). Spike is proposed to activate ROP signaling as a GEF, and to influence actin polymerization via WAVE and ARP 2/3 complexes (Qiu et al. 2002; Basu et al. 2008; Zhang et al. 2010). Lin and colleagues demonstrate that spike is required for the auxin‐induced ROP6 activation and is involved in the ROP6–RIC1‐mediated regulation of PIN2 polar distribution to the plasma membrane in root cells (Lin et al. 2012). Several pollen specific‐expressed ROP‐GEF proteins have been shown to physically interact with a pollen‐specific expressed receptor‐like kinase and may function in tip growth of the pollen tube (Kaothien et al. 2005; Zhang and McCormick 2007; Chang et al. 2013). However, the roles of the 14 members of the PRONE domain‐containing ROP‐GEF family in the control of pavement cell shape remain uncharacterized. Plant ROP‐GEF proteins may function together with receptor‐like kinases to form a plant‐specific signaling module to perceive specific extracellular signals and activate ROP proteins to regulate multiple downstream cellular responses. Future work should determine the role of ROP‐GEF family members, GDI proteins, and GAP proteins during pavement cell interdigitation and the interaction between these regulators and candidates receptor‐like kinases.

ACKNOWLEDGEMENTS This work is supported by startup funds to D. L. from Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University. Y. F. is supported by Natural Science Foundation of China (31361140354).

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