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Rho-GTPase-regulated vesicle trafficking in plant cell polarity Xu Chen*1 and Jiˇrı´ Friml*†1 *Institute of Science and Technology Austria (IST Austria), Am Campus 1, 3400 Klosterneuburg, Austria †Mendel Centre for Plant Genomics and Proteomics, Masaryk University, CEITEC MU, CZ-625 00 Brno, Czech Republic
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Abstract ROPs (Rho of plants) belong to a large family of plant-specific Rho-like small GTPases that function as essential molecular switches to control diverse cellular processes including cytoskeleton organization, cell polarization, cytokinesis, cell differentiation and vesicle trafficking. Although the machineries of vesicle trafficking and cell polarity in plants have been individually well addressed, how ROPs co-ordinate those processes is still largely unclear. Recent progress has been made towards an understanding of the coordination of ROP signalling and trafficking of PIN (PINFORMED) transporters for the plant hormone auxin in both root and leaf pavement cells. PIN transporters constantly shuttle between the endosomal compartments and the polar plasma membrane domains, therefore the modulation of PIN-dependent auxin transport between cells is a main developmental output of ROP-regulated vesicle trafficking. The present review focuses on these cellular mechanisms, especially the integration of ROP-based vesicle trafficking and plant cell polarity.
Introduction One of the essential features of cells in the context of multicellular organisms is their ability to migrate or coordinately generate asymmetry of their cellular components, known as cell polarity [1]. Plant cells display an impressive array of polarized cell types to maintain different axes of growth, providing a framework along which they develop and adapt to environmental changes. The best characterized model of plant cell polarity is the apical–basal distribution of PIN (PINFORMED) auxin transporters, which control the direction of auxin flow at the cellular level and thus mediate auxin gradients and local maxima in different plant developmental processes [2]. PINs have been identified as endocytic cargoes undergoing a constitutive dynamic trafficking between the PM (plasma membrane) and endomembrane system [3]. Basically, vesicle trafficking initiates at the PM with the recruitment of cargoes into forming clathrin-coated vesicles that are internalized into the cell interior by a process called endocytosis [4]. In contrast, intracellular vesicles move to the PM and subsequently fuse with the acceptor PM by exocytosis [4]. PIN cargoes are associated with the endocytic/exocytic vesicles [5] and are dependent on several small GTPase regulators to determine their proper trafficking and polarization at different steps
Key words: cell polarity, Rho GTPase, Rho of plants (ROP), vesicle trafficking. Abbreviations: ABP1, AUXIN-BINDING PROTEIN1; GAP, GTPase-activating protein; GEF, guaninenucleotide-exchange factor; ICR1, interactor of constitutively active ROP1; MIDD1, microtubule depletion domain 1; PC, pavement cell; PIN, PINFORMED; PM, plasma membrane; REN1, ROP1 enhancer 1; RIC, CRIB (Cdc42/Rac-interactive binding) motif-containing ROP-interacting protein; ROP, Rho of plants. 1
Correspondence may be addressed to either author (email [email protected] or [email protected]).
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[5,6]. Therefore PINs are good molecular landmarks for the investigation of cell polarity and vesicle trafficking events. An important class of vesicle trafficking regulators are small GTPases that are a form of G-proteins that hydrolyse GTP to form GDP [7]. They function as molecular switches shuttling between the active GTP-bound state and the inactive GDP-bound state [7]. GEFs (guanine-nucleotideexchange factors) are responsible for switching on the active form of small GTPases by promoting GDP-toGTP exchange, whereas GAPs (GTPase-activating proteins) are inactivators via switching off the active form, thereby increasing the GDP-bound state [7]. In plants, the wellknown members of small GTPases are the Ras-like family consisting of Rho, Arf, Rab and Ran [8], among which Arf and Rab GTPases are the best characterized regulators in vesicle trafficking pathways. In plants, our knowledge of Arf’s role in vesicle trafficking comes from the studies of the developmentally important Arf-GEF GNOM [9], which also co-localizes with ArfGAP VAN3 (VASCULAR NETWORK DEFECTIVE 3) at clathrin foci on the PM [5]. In order to better understand the function of small GTPases, researchers mutate the specific amino acids to lock a mutant GTPase either in the inactive DN (dominant-negative) form or in the CA (constitutively active) form [10], and illustrate that both a GTP-locked form (such as Arf1-Q71L) and a GDP-locked form (such as ArfT31N) interfere with vesicle trafficking [11]. Rho GTPases, also called ROP (Rho of plants)/RAC, are plant-specific Rho-related small GTPases, which contain 11 members in Arabidopsis [8]. Recent evidence highlights the indispensable roles of ROPs in vesicle trafficking [12– 16]. In the present review, we focus on the mechanisms of Biochem. Soc. Trans. (2014) 42, 212–218; doi:10.1042/BST20130269
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ROP-regulated vesicle dynamics in plants, and summarize their functions in plant-specific cell polarity regulation.
Polarized ROPs in plants The PM lipid bilayer, peppered with numerous proteins, appears to be a fundamental platform for vesicle trafficking. ROPs are incorporated in the PM via S-acylation and prenylation modification [17]. As central regulators of cell polarity, ROPs are found to be polarized on the PM in different cell types. For example, ROP1 is preferentially localized to the apical area of pollen tube PMs [18,19]; ROP4 is enriched in the apical and basal PM of the division zone of root and at places of root hair initiation [20]; ROP6 is distributed in different sides of later root cells according to different growth stage and asymmetric cell division [21]. Thus asymmetrically distributed ROP proteins have potentially to act as cell polarity determinants in multiple developmental contexts. Besides, the upstream regulators of ROPs such as ROP-GEF12C (C-terminally truncated ROP-GEF12), ROP-GAP1, REN1 (ROP1 enhancer 1, encoding a ROPGAP), and downstream effectors such as RIC1 [CRIB (Cdc42/Rac-interactive binding) motif-containing ROPinteracting protein 1], RIC3, RIC4, RIP1/ICR1 (interactor of constitutively active ROP1) also polarly target to the apical PM of the growing pollen tube [19,22–26]. Unlike in mammals and yeast, where several Rho-GTPases are visualized to stay in vesicular compartments [27], in plants ROPs have not been found to be distributed in endosomes or to undergo an endocytic/exocytic trafficking, suggesting that ROPs require their upstream or downstream signalling to indirectly influence the vesicle trafficking pathway.
ROP-regulated vesicle trafficking for polarized tip growth of the pollen tube and root hair As a single cell system, a pollen tube extends and elongates in an extreme form of polarized growth, thus this model system facilitates our understanding of the core mechanisms behind cell polarity. It was generally accepted that pollen tube growth occurs by accumulation of secretory vesicles in the apical region, which is dependent on endocytosis of vesicles to retrieve the excess cell membrane and wall molecules, and exocytosis of vesicles to accumulate at the extreme apical growing area [28]. At the molecular level, rapid pollen tube growth is accompanied by a continuous increase in ROP1 activity, and the highest activated ROP1 expression is at the extreme tip region [18,19]. Tip-localized activated ROP1 captures and promotes the exocytic vesicles to be targeted to the PM of the pollen tube apex [18]. Subsequently, a Ca2 + gradient that is higher close to the apical region, but lower in the subapical region is established [29]. Therefore activated ROP1 at the tip site is a major contributing factor in finetuning of the spatial distribution of exocytic vesicles and Ca2 + -dependent pollen tube elongation.
Consistent with ROP1 overexpression, genetic manipulation of ROP1 downstream regulators such as overexpression of RIC3, RIC4, ICR1 or deficiency in REN1 all cause depolarized and swollen pollen tubes [18,22,26,29,30]. The activity of ROP-GAPs such as ROP-GAP1 and REN1 is also highest at the extreme apical region of the pollen tube in turn to restrict the lateral propagation of apical ROP1 activity [19]. In particular, REN1 is distributed in exocytic vesicles, indicating the direct interaction of ROP signalling and vesicle trafficking. The modules of global inhibition-based balance of ROP1 activity accompanied by exocytosis regulation provide an efficient manner for rapid tip growth (Figure 1). Moreover, the above mechanisms of polarized tip growth are supported by another similar tip growing system: root hair [20]. Interestingly, mathematical modelling in root hair shows that auxin spontaneously leads to localized patches of activated ROP and the correct patterning of ROP controls appropriate root hair position and initiation [31].
ROP-regulated vesicle trafficking for auxin distribution in root and leaf pavement cells The plant root determines the capacity of a sessile organism to acquire nutrients and water, and its bending in response to gravity stimuli provides a means to spatially arrange plants in their environment. The root patterning processes, including control of stem cell differentiation status [32], are dependent on a local auxin maximum in the centre of the root meristem [33]. Polarized distribution of several PIN proteins [34] is sufficient to set up an auxin maximum within roots [33]. The polar delivery of PIN cargoes requires the involvement of ROP-based vesicle trafficking [12,14]. Although ROPs are not polarly localized in primary root cells, ROP6 and its regulators SPIKE1 [belonging to the conserved DHR2 (Dock homology region 2)-Dock family of ROP-GEF] and RIC1 all regulate clathrin-dependent endocytosis, and thus influence PIN-mediated directional auxin flow during root gravitropism [12,13] (Figure 2). It seems that, in root cells, only ROP6, ROP11 and ROP9 execute the responsibility of vesicle trafficking regulation, representing the diverse functions among different ROP homologues. Apart from the endotytosis modulation, ICR1, which is a ROP1 interactor, has been identified as an PIN exocytosis regulator. ICR1 protein is polarized in lateral root founder cells and in embryos; the icr1 mutant displays a swollen and collapsed root tip correlating with a diminished local auxin maximum [15,16]. Similar to yeast Cdc42, which is the best characterized Rho GTPase functioning in the endocytic machinery [15] and which interacts directly with the exocyst complex subunit SEC3 [36], Arabidopsis ICR1 also binds SEC3A [33]. The regulation of the exocyst complex might be the underlying mechanism for reduced recycling of PINs to the PM and PIN polarization defects in icr1 mutant [16]. Thus the interaction of ICR1 and SEC3A provides the link between ROP signalling and exocytosis processes (Figure 2). C The
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Figure 1 ROPs-regulated vesicle trafficking in polarized tip growth of pollen tubes In the growing pollen tube, the tip growth is tightly controlled by the activity of ROPs, RICs, REN1, ROP-GEF12C and ICR1 which accumulate more in the apical region, but less in the subapical region (marked by red), as well as cytoskeleton-directed exocytic vesicles (more at the apical region and less in the subapical region) and Ca2 + oscillation (higher close to the apical region and lower in the subapical region).
Studies of PIN proteins also revealed a novel plantspecific mechanism for endocytosis regulation by auxin, which decreases clathrin recruitment to the PM via a presumably extracellular auxin receptor, ABP1 (AUXINBINDING PROTEIN1) [37]. ROP2 and ROP6 are activated by ABP1-dependent auxin signalling within 30 s in leaf PCs (pavement cells) [38], hinting at the possible involvement of ROPs in ABP1-mediated inhibition of endocytosis. Indeed, the endocytic morphology of double mutants between abp1knockdown lines and rop6 or ric1 show defects comparable with those of rop6 or ric1 single mutants, consistent with a notion that ROP6/RIC1 signalling acts downstream of ABP1 to inhibit endocytosis in roots [12]. The regulatory module of auxin–ABP1–ROP6/RIC1–PIN in endocytosis in roots provides a mechanism for feedback regulation of auxin transport by ROP-mediated vesicle trafficking [12] (Figure 2). Auxin is produced and has a role in nearly every plant organ and tissue, including PCs of the leaf epidermis, and the ROP signalling module provides a common mechanism for both root and leaf cell patterning. Hence the similar machinery is found in the co-ordinated interdigitation of leaf PCs. Following auxin stimulation, activated ROP2 inhibits PIN1 endocytosis in an ABP1-dependent manner, in turn allowing more PIN1 to be polarly localized in at the lobe apex [14,38]. Additionally, PIN1 endocytosis is preferentially inhibited in lobes, but not in indentations [14]. Therefore PIN1-drived differential auxin efflux in lobes and indentations defines interdigitated PC patterning (Figure 2). C The
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ROP-regulated cytoskeleton arrangement in vesicle trafficking and polarity control So far, members of the Rho GTPase family are considered primarily to control the organization of the cytoskeleton to act on downstream events. In plants, ROPs rely on their interacting protein RICs targeting Arp2/3 (actin-related protein 2/3) to control actin assembly [10,38,39]. A number of studies have demonstrated that the defects of pollen tube or PC expansion by genetic manipulation of ROPs and their effectors are the consequences of disorganized actin [10,14,18,30,38,39]. In growing pollen tubes, RIC4 promotes assembly of actin to the tip apical region, allowing the apical accumulation of exocytic vesicles; by contrast, RIC3 disassembles actin to realize the feedback regulation of ROP1 activity [18]. In leaf PCs, activated ROP2 promotes RIC4dependent assembly of actin for lobe outgrowth, whereas ROP6 promotes RIC1-dependent microtubule ordering for outgrowth suppression, which in turn suppresses ROP2 activity [10,38]. Moreover, the study of pollen tubes shows that clathrin-dependent endocytosis occurs preferentially in the apex of pollen tubes, which coincides with the area of actin accumulation [40]. All of these scenarios are tightly connected with actin cytoskeleton arrangement. Interestingly, those cytoskeleton-involving mutually antagonistic regulations are often seen in different ROP-based signalling mechanisms (Figure 3). Compared with ROP-controlled actin dynamics, the ROP machinery upstream of microtubules is relatively less
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Figure 2 Models of ROP-regulated vesicle trafficking and auxin signalling in root and leaf PCs In root cells, SPIKE1, ROP6 and RIC1 attenuate PIN1 and PIN2 endocytosis to co-ordinate PIN-dependent auxin transport, which is activated by ABP1-dependent auxin signalling. ROP1 interacts with ICR1 and recruits ICR1 and SEC3A to regulate PIN2 exocytosis. In leaf PCs, lobe and indention formation is controlled by ABP1-triggered Rho-GTPase pathways: ROP2-RIC4 signalling promotes lobe outgrowth (more auxin efflux) via actin assembly and ROP6-RIC1 signalling suppresses lobe outgrowth but triggers indention formation (less auxin efflux) via microtubule organization. Only in lobe, exocytosis and in turn polarization of PIN1 is enhanced by ROP2-RIC4 signalling.
clear [18,25,30,39]. Two recent studies uncover regulatory mechanisms on how ROPs organize microtubule arrangement. One finding shows that the conserved microtubulesevering protein katanin (KTN1) interacts physically with RIC1 and acts downstream of ROP6/RIC1 signalling to promote severing of microtubules from branched nucleation sites [41]. The other exciting finding identifies a new Rho-
based regulatory mechanism in the cell wall: GTP-bound ROP11 accumulates at microtubule filaments of xylem cells to recruit the plant-specific microtubule-binding protein MIDD1 (microtubule depletion domain 1), thus inducing local disassembly of microtubules for a specific secondary cell wall patterning in xylem vessel cells [42]. Notably, a mutual inhibitory relationship has been observed in this C The
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Figure 3 Mutual inhibition and feedback events in ROP signalling pathways as shown in different plant tissues: pollen tube, leaf PC, xylem cell and root The mutual inhibition and feedback events are highlighted by purple barrier bars.
ROP-based cell wall formation: the activated ROP11 recruits MIDD1 to the PM, while at the same time ROP11 can be recruited along cortical microtubules by MIDD1 to eliminate active ROP11 from the PM [42] (Figure 3). Interestingly, otherwise polarly localized PIN proteins are depolarized in cell-wall-free protoplasts and PIN polarity is altered in cellwall-deficient mutants [43], hinting at a possible connection between ROPs, microtubules and PIN polarity cues in the cell wall. However, the significance of the cytoskeleton in diverse vesicle trafficking processes is still much less clear in plants than in other eukaryotes. F-actin (filamentous actin) is C The
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described to be essential in vesicle trafficking of mammalian cells, including facilitating vesicle budding from the PM, scission of coated vesicles and fusion with early endosomes [44]. Whereas the molecular details of the link between actin and vesicle trafficking in plants lag behind, initial observations show that chemical disruption of the actin cytoskeleton interferes with constitutive PIN recycling to the PM [45] and block of actin dynamics inhibits both recycling and endocytosis [46]. On the other hand, the involvement of microtubules in vesicle trafficking in plant cells has not been demonstrated directly, with the exception of processes associated with cell division [47]. Thus, given the
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importance of ROPs in both cytoskeleton arrangement and vesicle trafficking signalling, it is reasonable to expect new insights into the relationship between these two basic cellular processes.
Outlook and perspective Vesicle trafficking in plant cells is emerging as a crucial machinery not only to shuffle cargoes between different cellular destinations, but also for regulating cell-surface presence and thus activity of PM-bound proteins [48–50]. In recent years, a number of new molecular players contributing to vesicle trafficking pathways have been identified in plants, and researchers have begun to integrate endocytosis, vacuolar trafficking and exocytosis processes with the function of developmental regulators in organ formation, polarity establishment and gradient-based patterning [6,9,48,49]. In particular, the studies of ROP proteins and their upstream regulators and downstream effectors help us to gain insights into the underlying molecular mechanisms of vesicle trafficking pathways. The investigation based on several model systems such as pollen tubes, leaf PCs and primary roots have revealed that ROPs and ROP effectors/regulators not only are crucial evolutionarily conserved components of vesicle trafficking pathways, but also mediate plant-specific ‘plug-ins’ into these processes such as regulation of endocytosis and cell polarity by the plant-specific signal auxin. In the coming years, it will be interesting to uncover the unknown roles of ROPs downstream of other developmentally and physiologically important signalling pathways and identify new eventually plant-specific ROP regulators. Especially, establishment of the precise connections among ROP-regulated cytoskeleton arrangement, vesicle trafficking and polarity cues is the next challenging question to answer.
Funding This work was supported by the European Research Council [project ERC-2011-StG-20101109-PSDP], Central European Institute of Technology (CEITEC) [grant number CZ.1.05/1.1.00/02.0068], European Social Fund [grant number CZ.1.07/2.3.00/20.0043] and ˇ [grant number GA13-406375]. the Czech Science Foundation (CACR)
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Received 6 December 2013 doi:10.1042/BST20130269
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Rho-GTPase-regulated vesicle trafficking in plant cell polarity Xu Chen*1 and Jiˇrı´ Friml*†1 *Institute of Science and Technology Austria (IST Austria), Am Campus 1, 3400 Klosterneuburg, Austria †Mendel Centre for Plant Genomics and Proteomics, Masaryk University, CEITEC MU, CZ-625 00 Brno, Czech Republic
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Abstract ROPs (Rho of plants) belong to a large family of plant-specific Rho-like small GTPases that function as essential molecular switches to control diverse cellular processes including cytoskeleton organization, cell polarization, cytokinesis, cell differentiation and vesicle trafficking. Although the machineries of vesicle trafficking and cell polarity in plants have been individually well addressed, how ROPs co-ordinate those processes is still largely unclear. Recent progress has been made towards an understanding of the coordination of ROP signalling and trafficking of PIN (PINFORMED) transporters for the plant hormone auxin in both root and leaf pavement cells. PIN transporters constantly shuttle between the endosomal compartments and the polar plasma membrane domains, therefore the modulation of PIN-dependent auxin transport between cells is a main developmental output of ROP-regulated vesicle trafficking. The present review focuses on these cellular mechanisms, especially the integration of ROP-based vesicle trafficking and plant cell polarity.
Introduction One of the essential features of cells in the context of multicellular organisms is their ability to migrate or coordinately generate asymmetry of their cellular components, known as cell polarity [1]. Plant cells display an impressive array of polarized cell types to maintain different axes of growth, providing a framework along which they develop and adapt to environmental changes. The best characterized model of plant cell polarity is the apical–basal distribution of PIN (PINFORMED) auxin transporters, which control the direction of auxin flow at the cellular level and thus mediate auxin gradients and local maxima in different plant developmental processes [2]. PINs have been identified as endocytic cargoes undergoing a constitutive dynamic trafficking between the PM (plasma membrane) and endomembrane system [3]. Basically, vesicle trafficking initiates at the PM with the recruitment of cargoes into forming clathrin-coated vesicles that are internalized into the cell interior by a process called endocytosis [4]. In contrast, intracellular vesicles move to the PM and subsequently fuse with the acceptor PM by exocytosis [4]. PIN cargoes are associated with the endocytic/exocytic vesicles [5] and are dependent on several small GTPase regulators to determine their proper trafficking and polarization at different steps
Key words: cell polarity, Rho GTPase, Rho of plants (ROP), vesicle trafficking. Abbreviations: ABP1, AUXIN-BINDING PROTEIN1; GAP, GTPase-activating protein; GEF, guaninenucleotide-exchange factor; ICR1, interactor of constitutively active ROP1; MIDD1, microtubule depletion domain 1; PC, pavement cell; PIN, PINFORMED; PM, plasma membrane; REN1, ROP1 enhancer 1; RIC, CRIB (Cdc42/Rac-interactive binding) motif-containing ROP-interacting protein; ROP, Rho of plants. 1
Correspondence may be addressed to either author (email [email protected] or [email protected]).
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[5,6]. Therefore PINs are good molecular landmarks for the investigation of cell polarity and vesicle trafficking events. An important class of vesicle trafficking regulators are small GTPases that are a form of G-proteins that hydrolyse GTP to form GDP [7]. They function as molecular switches shuttling between the active GTP-bound state and the inactive GDP-bound state [7]. GEFs (guanine-nucleotideexchange factors) are responsible for switching on the active form of small GTPases by promoting GDP-toGTP exchange, whereas GAPs (GTPase-activating proteins) are inactivators via switching off the active form, thereby increasing the GDP-bound state [7]. In plants, the wellknown members of small GTPases are the Ras-like family consisting of Rho, Arf, Rab and Ran [8], among which Arf and Rab GTPases are the best characterized regulators in vesicle trafficking pathways. In plants, our knowledge of Arf’s role in vesicle trafficking comes from the studies of the developmentally important Arf-GEF GNOM [9], which also co-localizes with ArfGAP VAN3 (VASCULAR NETWORK DEFECTIVE 3) at clathrin foci on the PM [5]. In order to better understand the function of small GTPases, researchers mutate the specific amino acids to lock a mutant GTPase either in the inactive DN (dominant-negative) form or in the CA (constitutively active) form [10], and illustrate that both a GTP-locked form (such as Arf1-Q71L) and a GDP-locked form (such as ArfT31N) interfere with vesicle trafficking [11]. Rho GTPases, also called ROP (Rho of plants)/RAC, are plant-specific Rho-related small GTPases, which contain 11 members in Arabidopsis [8]. Recent evidence highlights the indispensable roles of ROPs in vesicle trafficking [12– 16]. In the present review, we focus on the mechanisms of Biochem. Soc. Trans. (2014) 42, 212–218; doi:10.1042/BST20130269
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ROP-regulated vesicle dynamics in plants, and summarize their functions in plant-specific cell polarity regulation.
Polarized ROPs in plants The PM lipid bilayer, peppered with numerous proteins, appears to be a fundamental platform for vesicle trafficking. ROPs are incorporated in the PM via S-acylation and prenylation modification [17]. As central regulators of cell polarity, ROPs are found to be polarized on the PM in different cell types. For example, ROP1 is preferentially localized to the apical area of pollen tube PMs [18,19]; ROP4 is enriched in the apical and basal PM of the division zone of root and at places of root hair initiation [20]; ROP6 is distributed in different sides of later root cells according to different growth stage and asymmetric cell division [21]. Thus asymmetrically distributed ROP proteins have potentially to act as cell polarity determinants in multiple developmental contexts. Besides, the upstream regulators of ROPs such as ROP-GEF12C (C-terminally truncated ROP-GEF12), ROP-GAP1, REN1 (ROP1 enhancer 1, encoding a ROPGAP), and downstream effectors such as RIC1 [CRIB (Cdc42/Rac-interactive binding) motif-containing ROPinteracting protein 1], RIC3, RIC4, RIP1/ICR1 (interactor of constitutively active ROP1) also polarly target to the apical PM of the growing pollen tube [19,22–26]. Unlike in mammals and yeast, where several Rho-GTPases are visualized to stay in vesicular compartments [27], in plants ROPs have not been found to be distributed in endosomes or to undergo an endocytic/exocytic trafficking, suggesting that ROPs require their upstream or downstream signalling to indirectly influence the vesicle trafficking pathway.
ROP-regulated vesicle trafficking for polarized tip growth of the pollen tube and root hair As a single cell system, a pollen tube extends and elongates in an extreme form of polarized growth, thus this model system facilitates our understanding of the core mechanisms behind cell polarity. It was generally accepted that pollen tube growth occurs by accumulation of secretory vesicles in the apical region, which is dependent on endocytosis of vesicles to retrieve the excess cell membrane and wall molecules, and exocytosis of vesicles to accumulate at the extreme apical growing area [28]. At the molecular level, rapid pollen tube growth is accompanied by a continuous increase in ROP1 activity, and the highest activated ROP1 expression is at the extreme tip region [18,19]. Tip-localized activated ROP1 captures and promotes the exocytic vesicles to be targeted to the PM of the pollen tube apex [18]. Subsequently, a Ca2 + gradient that is higher close to the apical region, but lower in the subapical region is established [29]. Therefore activated ROP1 at the tip site is a major contributing factor in finetuning of the spatial distribution of exocytic vesicles and Ca2 + -dependent pollen tube elongation.
Consistent with ROP1 overexpression, genetic manipulation of ROP1 downstream regulators such as overexpression of RIC3, RIC4, ICR1 or deficiency in REN1 all cause depolarized and swollen pollen tubes [18,22,26,29,30]. The activity of ROP-GAPs such as ROP-GAP1 and REN1 is also highest at the extreme apical region of the pollen tube in turn to restrict the lateral propagation of apical ROP1 activity [19]. In particular, REN1 is distributed in exocytic vesicles, indicating the direct interaction of ROP signalling and vesicle trafficking. The modules of global inhibition-based balance of ROP1 activity accompanied by exocytosis regulation provide an efficient manner for rapid tip growth (Figure 1). Moreover, the above mechanisms of polarized tip growth are supported by another similar tip growing system: root hair [20]. Interestingly, mathematical modelling in root hair shows that auxin spontaneously leads to localized patches of activated ROP and the correct patterning of ROP controls appropriate root hair position and initiation [31].
ROP-regulated vesicle trafficking for auxin distribution in root and leaf pavement cells The plant root determines the capacity of a sessile organism to acquire nutrients and water, and its bending in response to gravity stimuli provides a means to spatially arrange plants in their environment. The root patterning processes, including control of stem cell differentiation status [32], are dependent on a local auxin maximum in the centre of the root meristem [33]. Polarized distribution of several PIN proteins [34] is sufficient to set up an auxin maximum within roots [33]. The polar delivery of PIN cargoes requires the involvement of ROP-based vesicle trafficking [12,14]. Although ROPs are not polarly localized in primary root cells, ROP6 and its regulators SPIKE1 [belonging to the conserved DHR2 (Dock homology region 2)-Dock family of ROP-GEF] and RIC1 all regulate clathrin-dependent endocytosis, and thus influence PIN-mediated directional auxin flow during root gravitropism [12,13] (Figure 2). It seems that, in root cells, only ROP6, ROP11 and ROP9 execute the responsibility of vesicle trafficking regulation, representing the diverse functions among different ROP homologues. Apart from the endotytosis modulation, ICR1, which is a ROP1 interactor, has been identified as an PIN exocytosis regulator. ICR1 protein is polarized in lateral root founder cells and in embryos; the icr1 mutant displays a swollen and collapsed root tip correlating with a diminished local auxin maximum [15,16]. Similar to yeast Cdc42, which is the best characterized Rho GTPase functioning in the endocytic machinery [15] and which interacts directly with the exocyst complex subunit SEC3 [36], Arabidopsis ICR1 also binds SEC3A [33]. The regulation of the exocyst complex might be the underlying mechanism for reduced recycling of PINs to the PM and PIN polarization defects in icr1 mutant [16]. Thus the interaction of ICR1 and SEC3A provides the link between ROP signalling and exocytosis processes (Figure 2). C The
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Figure 1 ROPs-regulated vesicle trafficking in polarized tip growth of pollen tubes In the growing pollen tube, the tip growth is tightly controlled by the activity of ROPs, RICs, REN1, ROP-GEF12C and ICR1 which accumulate more in the apical region, but less in the subapical region (marked by red), as well as cytoskeleton-directed exocytic vesicles (more at the apical region and less in the subapical region) and Ca2 + oscillation (higher close to the apical region and lower in the subapical region).
Studies of PIN proteins also revealed a novel plantspecific mechanism for endocytosis regulation by auxin, which decreases clathrin recruitment to the PM via a presumably extracellular auxin receptor, ABP1 (AUXINBINDING PROTEIN1) [37]. ROP2 and ROP6 are activated by ABP1-dependent auxin signalling within 30 s in leaf PCs (pavement cells) [38], hinting at the possible involvement of ROPs in ABP1-mediated inhibition of endocytosis. Indeed, the endocytic morphology of double mutants between abp1knockdown lines and rop6 or ric1 show defects comparable with those of rop6 or ric1 single mutants, consistent with a notion that ROP6/RIC1 signalling acts downstream of ABP1 to inhibit endocytosis in roots [12]. The regulatory module of auxin–ABP1–ROP6/RIC1–PIN in endocytosis in roots provides a mechanism for feedback regulation of auxin transport by ROP-mediated vesicle trafficking [12] (Figure 2). Auxin is produced and has a role in nearly every plant organ and tissue, including PCs of the leaf epidermis, and the ROP signalling module provides a common mechanism for both root and leaf cell patterning. Hence the similar machinery is found in the co-ordinated interdigitation of leaf PCs. Following auxin stimulation, activated ROP2 inhibits PIN1 endocytosis in an ABP1-dependent manner, in turn allowing more PIN1 to be polarly localized in at the lobe apex [14,38]. Additionally, PIN1 endocytosis is preferentially inhibited in lobes, but not in indentations [14]. Therefore PIN1-drived differential auxin efflux in lobes and indentations defines interdigitated PC patterning (Figure 2). C The
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ROP-regulated cytoskeleton arrangement in vesicle trafficking and polarity control So far, members of the Rho GTPase family are considered primarily to control the organization of the cytoskeleton to act on downstream events. In plants, ROPs rely on their interacting protein RICs targeting Arp2/3 (actin-related protein 2/3) to control actin assembly [10,38,39]. A number of studies have demonstrated that the defects of pollen tube or PC expansion by genetic manipulation of ROPs and their effectors are the consequences of disorganized actin [10,14,18,30,38,39]. In growing pollen tubes, RIC4 promotes assembly of actin to the tip apical region, allowing the apical accumulation of exocytic vesicles; by contrast, RIC3 disassembles actin to realize the feedback regulation of ROP1 activity [18]. In leaf PCs, activated ROP2 promotes RIC4dependent assembly of actin for lobe outgrowth, whereas ROP6 promotes RIC1-dependent microtubule ordering for outgrowth suppression, which in turn suppresses ROP2 activity [10,38]. Moreover, the study of pollen tubes shows that clathrin-dependent endocytosis occurs preferentially in the apex of pollen tubes, which coincides with the area of actin accumulation [40]. All of these scenarios are tightly connected with actin cytoskeleton arrangement. Interestingly, those cytoskeleton-involving mutually antagonistic regulations are often seen in different ROP-based signalling mechanisms (Figure 3). Compared with ROP-controlled actin dynamics, the ROP machinery upstream of microtubules is relatively less
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Figure 2 Models of ROP-regulated vesicle trafficking and auxin signalling in root and leaf PCs In root cells, SPIKE1, ROP6 and RIC1 attenuate PIN1 and PIN2 endocytosis to co-ordinate PIN-dependent auxin transport, which is activated by ABP1-dependent auxin signalling. ROP1 interacts with ICR1 and recruits ICR1 and SEC3A to regulate PIN2 exocytosis. In leaf PCs, lobe and indention formation is controlled by ABP1-triggered Rho-GTPase pathways: ROP2-RIC4 signalling promotes lobe outgrowth (more auxin efflux) via actin assembly and ROP6-RIC1 signalling suppresses lobe outgrowth but triggers indention formation (less auxin efflux) via microtubule organization. Only in lobe, exocytosis and in turn polarization of PIN1 is enhanced by ROP2-RIC4 signalling.
clear [18,25,30,39]. Two recent studies uncover regulatory mechanisms on how ROPs organize microtubule arrangement. One finding shows that the conserved microtubulesevering protein katanin (KTN1) interacts physically with RIC1 and acts downstream of ROP6/RIC1 signalling to promote severing of microtubules from branched nucleation sites [41]. The other exciting finding identifies a new Rho-
based regulatory mechanism in the cell wall: GTP-bound ROP11 accumulates at microtubule filaments of xylem cells to recruit the plant-specific microtubule-binding protein MIDD1 (microtubule depletion domain 1), thus inducing local disassembly of microtubules for a specific secondary cell wall patterning in xylem vessel cells [42]. Notably, a mutual inhibitory relationship has been observed in this C The
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Figure 3 Mutual inhibition and feedback events in ROP signalling pathways as shown in different plant tissues: pollen tube, leaf PC, xylem cell and root The mutual inhibition and feedback events are highlighted by purple barrier bars.
ROP-based cell wall formation: the activated ROP11 recruits MIDD1 to the PM, while at the same time ROP11 can be recruited along cortical microtubules by MIDD1 to eliminate active ROP11 from the PM [42] (Figure 3). Interestingly, otherwise polarly localized PIN proteins are depolarized in cell-wall-free protoplasts and PIN polarity is altered in cellwall-deficient mutants [43], hinting at a possible connection between ROPs, microtubules and PIN polarity cues in the cell wall. However, the significance of the cytoskeleton in diverse vesicle trafficking processes is still much less clear in plants than in other eukaryotes. F-actin (filamentous actin) is C The
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described to be essential in vesicle trafficking of mammalian cells, including facilitating vesicle budding from the PM, scission of coated vesicles and fusion with early endosomes [44]. Whereas the molecular details of the link between actin and vesicle trafficking in plants lag behind, initial observations show that chemical disruption of the actin cytoskeleton interferes with constitutive PIN recycling to the PM [45] and block of actin dynamics inhibits both recycling and endocytosis [46]. On the other hand, the involvement of microtubules in vesicle trafficking in plant cells has not been demonstrated directly, with the exception of processes associated with cell division [47]. Thus, given the
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importance of ROPs in both cytoskeleton arrangement and vesicle trafficking signalling, it is reasonable to expect new insights into the relationship between these two basic cellular processes.
Outlook and perspective Vesicle trafficking in plant cells is emerging as a crucial machinery not only to shuffle cargoes between different cellular destinations, but also for regulating cell-surface presence and thus activity of PM-bound proteins [48–50]. In recent years, a number of new molecular players contributing to vesicle trafficking pathways have been identified in plants, and researchers have begun to integrate endocytosis, vacuolar trafficking and exocytosis processes with the function of developmental regulators in organ formation, polarity establishment and gradient-based patterning [6,9,48,49]. In particular, the studies of ROP proteins and their upstream regulators and downstream effectors help us to gain insights into the underlying molecular mechanisms of vesicle trafficking pathways. The investigation based on several model systems such as pollen tubes, leaf PCs and primary roots have revealed that ROPs and ROP effectors/regulators not only are crucial evolutionarily conserved components of vesicle trafficking pathways, but also mediate plant-specific ‘plug-ins’ into these processes such as regulation of endocytosis and cell polarity by the plant-specific signal auxin. In the coming years, it will be interesting to uncover the unknown roles of ROPs downstream of other developmentally and physiologically important signalling pathways and identify new eventually plant-specific ROP regulators. Especially, establishment of the precise connections among ROP-regulated cytoskeleton arrangement, vesicle trafficking and polarity cues is the next challenging question to answer.
Funding This work was supported by the European Research Council [project ERC-2011-StG-20101109-PSDP], Central European Institute of Technology (CEITEC) [grant number CZ.1.05/1.1.00/02.0068], European Social Fund [grant number CZ.1.07/2.3.00/20.0043] and ˇ [grant number GA13-406375]. the Czech Science Foundation (CACR)
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Received 6 December 2013 doi:10.1042/BST20130269
