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ScienceDirect Intracellular trafficking and PIN-mediated cell polarity during tropic responses in plants Hana Rakusova´1,2, Matya´sˇ Fendrych1 and Jirˇı´ Friml1 Subcellular trafficking and cell polarity are basic cellular processes crucial for plant development including tropisms — directional growth responses to environmental stimuli such as light or gravity. Tropisms involve auxin gradient across the stimulated organ that underlies the differential cell elongation and bending. The perception of light or gravity is followed by changes in the polar, cellular distribution of the PIN auxin transporters. Such re-specification of polar trafficking pathways is a part of the mechanism, by which plants adjust their phenotype to environmental changes. Recent genetic and biochemical studies provided the important insights into mechanisms of PIN polarization during tropisms. In this review, we summarize the present state of knowledge on dynamic PIN repolarization and its specific regulations during hypocotyl tropisms. Addresses 1 Institute of Science and Technology (IST) Austria, 3400 Klosterneuburg, Austria 2 Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Ghent University, BE-9052 Ghent, Belgium Corresponding author: Friml, Jirˇı´ ([email protected])

Current Opinion in Plant Biology 2015, 23:116–123 This review comes from a themed issue on Growth and development

phytohormone auxin [1]. Auxin induces hypocotyl cell elongation, therefore elongation of the cells on the shaded/lower side of shoots results in differential growth and bending towards the light source or against gravity vector. In the case of roots, the auxin asymmetry is the same, i.e. at the shaded or lower side of the organ, but root cells react to increased auxin levels oppositely by inhibition of elongation. Directional (polar) auxin transport seems to be the main mechanism for asymmetric auxin distribution during tropisms [2]. Directionality of auxin flow within tissues is determined by a polar localization of auxin exporters, in particular PIN-FORMED (PIN) proteins [3,4] with the assistance of auxin influx carriers such as AUXIN/LIKE AUXIN (AUX/LAX) [5] and some ATP BINDING CASSETTE B (ABCB) transporters [6]. The polar subcellular localization of PIN proteins was shown to direct auxin flow [4]. Results of the recent years support the notion that PIN polarization and resulting directional auxin flux play a crucial role in plant phototropism and gravitropism [7–9]. In this review, we focus on the operation of this system mainly in the hypocotyl, as this aerial organ has received comparably little attention in the literature.

Edited by Niko Geldner and Sigal Savaldi-Goldstein

Pin (re)polarizations during hypocotyl tropisms http://dx.doi.org/10.1016/j.pbi.2014.12.002

Phototropism

1369-5266/# 2014 Published by Elsevier Ltd.

Plants sense the intensity, direction, duration and wavelength of light (for a recent review, see [10]). Light sensing occurs in the upper hypocotyl [11] where bending also takes place. The blue-light receptors, such as PHOTOTROPIN1 (PHOT1) and PHOT2 [12], that combine blue-light sensing with kinase activity, can initiate the phototropic response with shoot bending as a consequence. One of the PHOT1 phosphorylation targets is the auxin transporter ABCB19 [13] that, in the absence of light, has been proposed to mediate the auxin flux through the vasculature. Following blue-light irradiation, auxin is redistributed from the vasculature to the epidermis in the upper part of the hypocotyl by the PHOT1-mediated phospohorylation that inhibits ABCB19 activity. Subsequently, auxin is channelled by PIN3 through the epidermis to the elongation zone. PIN3 has been also shown to play a role during phototropism in endodermal cells to mediate lateral auxin fluxes by restricting auxin to the vascular cylinder [2,8]. The expression of PIN3 in endodermal cells has an apolar localization in dark-grown hypocotyls. After

Introduction Plants have evolved sophisticated mechanisms to perceive information from their biotic and abiotic surroundings. Post-embryonic growth involving the activity of meristem tissues, and tropic growth responses are examples of processes, by which plants adapt to environmental conditions. Tropism is a directional growth of a plant in response to an environmental stimulus, such as light (phototropism) and gravity (gravitropism). Phototropism enables plants to react to changes in light direction by bending shoots towards the light source, essential for photosynthetic plants. Gravitropism is the growth reaction that orients plant development along the gravity vector. Phototropism and gravitropism involve light or gravity perception and the asymmetric distribution of the Current Opinion in Plant Biology 2015, 23:116–123

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PIN-mediated cell polarity during tropism Rakusova´, Fendrych and Friml 117

unidirectional light stimulation, PIN3 gradually disappears from the outer side of endodermal cells at the illuminated hypocotyl side and gets stabilized at the inner cell side. This PIN3 asymmetry then instructs the auxin flow to the shaded side of hypocotyls as visualized by high auxin response reporter expression [8]. Consequently, auxin accumulation induces cell elongation at the shaded side, resulting in hypocotyl bending [8]. Interestingly, both the auxin gradient formation and the phototopic bending require activity of the PM H+ ATPase [14] in line with the notion that the proper regulation of apoplastic pH is crucial for polar auxin transport. The role of the PINs and in particular PIN3 in phototropism has been characterized under various light regimes and in different mutant alleles [7,8,15,16]. Despite the phototropic defects are relatively weak, confirmed pin3 knock-out mutant alleles are always clearly impaired and show progresivelly stronger defects in higher order pin mutants suggesting extensive functional redundancy. Furthermore, light-induced auxin asymmetry and phototropism can be completely inhibited by chemical inhibitors of auxin transport [2] that act by yet unclear molecular mechanism [17].

Gravitropism

In the hypocotyls, gravity is sensed by the sedimentation of amyloplasts in endodermal cells [18]. One model proposes that the gravity signal transduction starts when sedimenting amyloplasts promote the opening of mechano-sensitive ion channels, either directly or through interaction with the actin cytoskeleton. Alternatively, signal transduction may initiate after sedimentation, when amyloplast-borne ligands interact with receptors located on sensitive structures within the gravity-sensing cells (reviewed in [18]). The main player in the differential auxin transport during hypocotyl gravitropism is again PIN3, the mutant of which shows no differential auxin distribution and reduced bending response [2,7]. After gravity has been sensed by the amyloplasts sedimentation in endodermal cells, the plasma membrane (PM)-localized PIN3 relocates to the lower side of the same cells, presumably redirecting the auxin flow to the lower hypocotyl side [7]. There auxin promotes elongation resulting in upward hypocotyl bending (Figure 1). Shade avoidance

Plants communities have to compete for light and nutrients, and display typical responses to the presence of

Figure 1

(a) Light control

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(b) Shade avoidance

(c) Dark control

low R/FR

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(d) Phototropism

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PIN3 localization in Arabidopsis hypocotyls. Light-mediated and gravity-mediated PIN3 re-localization in the endodermis cells of upper hypocotyls. PIN3 is localized on the inner-lateral side in the light (a) and relocalizes to the outer-lateral domain during the shade avoidance response (b). Arrows point the outer lateral endodermal cell side. In the dark, PIN3 is apolarly localized (c). PIN3 gradually relocalizes to the inner-lateral side at the illuminated hypocotyl side after unilateral light (d) or at the upper hypocotyl side after gravity stimulation (e). Arrowheads point the outer endodermis membranes. Arrows indicate direction of light (c, d) or gravity (e) [2,7,8,24].

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neighbouring plants. One type of these behaviours is the shade avoidance [19] that, strictly speaking, is not a tropic response, because it leads to a uniform organ elongation. The phytochrome receptor signalling pathway allows plants to monitor the ratio between red (R) and far-red (FR) light that is specific for shade caused by neighbouring plants [20] and initiate shade avoidance response [21,22]. Here, the PIN3 expression is induced by the low R:FR and in the hypocotyl, PIN3 in the endodermal cells relocates from basal to outer-lateral cell sides, triggering an auxin flow into the outer cell layers of the hypocotyl. Consequently, the hypocotyl elongates, exerting the typical shade avoidance response [23]. Either chemical inhibition or genetic interference with the polar auxin transport in pin mutants restricts shade avoidance-induced elongation [24]. Relatively weak effects of the single pin mutations again imply functional redundancy within PIN family and/or involvement of other components such as ABCB auxin transporters. The connection between light-dependent responses such as phototropism and shade avoidance, in particular, the role and regulation of PIN3, is intensively studied [25] (Figure 1). In summary, PIN proteins play an important role in the regulation of auxin fluxes during tropic responses and shade avoidance. Despite the extensive functional redundancy, the main player seems to be PIN3, therefore, in the following we will focus on the PIN3 trafficking and polarity regulation in hypocotyl endodermal cells.

PIN3 trafficking for polarization during tropisms The adaptation of growth to changing environmental conditions with complex developmental reprogramming often involves resetting of the developmental fate and polarity of cells within differentiated tissues [26]. Although some components of the signalling pathways activated by light or gravity are known, the downstream processes leading to regulation of PIN localization and auxin redistribution are still largely elusive [7,8]. The changes in polar PIN localization involve several processes, notably secretion [27], constitutive endocytic recycling [28], and transcytosis [29]. This subcellular dynamics enable rapid changes in PIN polarity that are essential for the fast redirections of auxin fluxes in response to stimuli, such as light or gravity. Endocytic recycling

PM-localized PIN proteins are constitutively internalized by clathrin-mediated endocytosis [30] and recycled back to the PM [28]. In plants, the coat protein clathrindependent trafficking is essential for the polar PIN distribution and plays a role during gravitropism of roots [9,31]. Clathrin function is required for the rapid PIN3 internalization following a gravity stimulus in roots [9] to Current Opinion in Plant Biology 2015, 23:116–123

possibly create an intracellular PIN3 pool to be redirected to the preferred cell side (Figure 2). Both PIN internalization and recycling back to the PM depend on the activity of ADP-ribosylation factor (ARF) GTPases, guanine-nucleotide exchange factors (GEFs) [32–36] and their counter-partners ARF GTPase-activating proteins (GAPs) [37]. The ARF GEF GNOM is involved in PIN3 trafficking following photo- and gravitropic stimulation in roots and hypocotyls [7–9]. Notably, the chemical inhibition of the GNOM function interferes specifically with the gravity-induced or light-induced destabilization of PIN3 at the outer PM [7,8]. This observation suggests that GNOM may play an additional role on the PM of hypocotyl endodermal cells, apart from its described recycling role at the Golgi apparatus. Other ARF GEFs and ARF GAPs have been also shown to regulate PIN trafficking [35,37]. The early endosomal components, the ARF GEF BFA-visualized endocytic trafficking defective1 (BEN1), BEN2 and syntaxinbinding protein 1-like 1 (SEC1-/mammalian uncoordinated-18 (MUNC18) [38] proteins are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic re-polarization, and, consequently, for auxin response gradient formation and auxin-related developmental processes [36]. GTP exchange factors on the ADP-ribosylation factors GNOM-LIKE1 (GNL1) regulate trafficking at the Golgi apparatus and trans-Golgi network/early endosome (TGN/EE) [39]. BFA-visualized exocytic trafficking defective1 (BEX1), a member of the ARF1 gene family [40], localizes to the TGN/EE and Golgi apparatus, acts synergistically with the ARF GEF BEN1/HOPM Interactor 7 (MIN7) and is important for PIN recycling to the PM [41]. BEX5 encodes a Ras genes from rat brainA1b (RabA1b), a member of the large RabA GTPase class, localizes to the TGN/EE compartment, participates in PIN trafficking processes distinct from GNL1, presumably by regulating trafficking from the TGN/EE to the PM [42]. PIN protein recycling to the PM and secretion is compromised in the mutant of the Interactor of constitutive active ROP 1 (ICR1) [43] and in the mutant of the exocyst complex component EXO70A1 [44], highlighting the role of the secretory pathway for PIN recycling. The roles of these proteins in PIN1 and PIN2 trafficking have been described, but a possible role during tropic responses and in PIN3 trafficking has still to be assessed. Role of cytoskeleton in tropic responses

The role of cytoskeleton in the distinct stages of the gravitropic response is a subject of intensive research [45–47]. Depolymerization of actin microfilaments reduces the hypocotyl growth rates, but after a prolonged time leads to gravitropic overbending of hypocotyls. www.sciencedirect.com

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Figure 2

actin

(a)

auxin flow PIN3-mediated auxin flow

PIN3 PID

BFA

PINOID kinase GNOM

clathrin GNOM

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TyrA23

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Current Opinion in Plant Biology

Intracellular PIN3 repolarization regulates auxin flow during gravitropism of hypocotyls. (a) Intracellular trafficking pathways for PIN3 polar distribution during gravitropic response. PIN3 undergoes dynamic translocation between different cell sides. Inhibition of clathrin-mediated endocytosis or the GNOM-dependent outer-PM trafficking prevents the PIN3 relocation. PINOID (PID)-dependent phosphorylation of PIN3 may affect its targeting to the inner-lateral cell side. Following gravistimulation, PIN3 relocalizes rapidly to the bottom side of hypocotyl endodermis cells and, thus, redirects the auxin flow towards the lower side of the hypocotyl. (b) PIN3-mediated asymmetric auxin response during hypocotyl gravitropism as visualized by the DR5::GUS auxin response reporter. The arrows indicate the direction of auxin flux. BFA (brefeldin A); TyrA23 (Tyrphostin A23).

Therefore, actin might serve as a negative regulator of gravitropism, possibly by preventing a too fast sedimentation of statoliths [48,49]. In contrast, in roots, the actin cytoskeleton is required for PIN3 recycling during gravitropism [2] and for endomembrane cycling of PIN proteins in general [28]. Previous data indicate that the microtubule (MT) cytoskeleton is involved in the hypocotyl gravitropic response. Blue-light illumination initiates rapid MT redirection from transversal to longitudinal orientation on the lighted side, probably contributing to phototropic bending [50] since longitudinally oriented cortical array will likely inhibit cell elongation through deposition of longitudinal cellulose microfibrils. As auxin accumulates on the shaded side during phototropism and at the lower www.sciencedirect.com

side during gravitropism, here we would expect stimulation of transversal MT orientation to induce elongation also in case of both gravitropism and phototropism. But recent data showed that auxin application causes a very rapid re-orientation of MT from transversal to longitudinal [51]. On the other hand, auxin was reported to correlate with transversal MT orientation in light grown hypocotyls [52], but both the phototropic and gravitropic hypocotyl experiments are performed using etiolated hypocotyls. Therefore the role of auxin in the MT orientation during tropisms remains unclear, but evidently light and auxin signalling can converge on the regulation of MT arrangement to control cell elongation. Taken together, actin and the MT cytoskeleton are important components at different stages of tropic Current Opinion in Plant Biology 2015, 23:116–123

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responses. The MT cytoskeleton is predominantly involved in hypocotyl responses to changing light and gravity by their effect on cells elongation, whereas the actin cytoskeleton would be additionally required for gravity sensing and PIN3 re-polarization. PIN3 transcytosis during tropisms

Similar to other PM proteins also PINs are targeted to the vacuole to be degraded [53]. The vacuolar targeting of PIN2, which is enhanced in root epidermal cells during gravity responses presumably maintains the asymmetric auxin distribution at the later stages of the response [54,55], but vacuolar trafficking of PIN3 does not seem to be influenced during the root gravity response [9]. Also PIN3 relocation after gravi-stimulation in hypocotyls does not seem to require degradation processes [7]. Therefore it seems that the mechanism of gravity-induced PIN3 repolarization relies on relocation of the existing PIN3 pool. Relocation-like mechanisms, generally termed transcytosis, combine endocytic recycling and recruitment into distinct polar targeting pathways [55]. Light or gravity modulate multiple steps of the PIN3 trafficking, leading to PIN3 transcytosis that is physiologically important for redirection of auxin fluxes during tropic responses [7–9]. PIN proteins have to re-localize fast in response to environmental changes. The relevant trafficking machineries must be targeted by so far largely elusive regulations downstream of external stimuli, such as light/shade or gravity. Phosphorylation might be one of the key mechanisms that regulate the directional trafficking of PIN proteins during the tropic responses. PIN3 polarity regulation by phosphorylation

Reversible phosphorylation of PIN proteins by the serine/ threonine protein kinase PINOID (PID) and protein phosphatase 2A (PP2A) is important for targeting PIN proteins to the apical or basal PMs. PID loss-of-function promotes basal whereas gain-of-function apical PIN localization suggesting that phosphorylated PINs go apical and dephosphorylated preferentially basal [56–60]. The AGC3 Ser/Thr protein kinase subfamily comprising of PID, WAG1 and WAG2 is also involved in the transduction of stimuli during phototropism and gravitropism. PID-mediated phosphorylation regulates recruitment of PIN proteins into GNOM-dependent transcytosis pathway [8,9,61], thereby providing mechanism for the PIN3 relocation in response to light [8] and gravity [7]. The recently described D6 PROTEIN KINASE (D6K) subfamily of AGCVIII kinases also directly phosphorylates PIN proteins and regulates auxin transport during tropism, independently from PID [62]. The D6K-mediated PIN3 phosphorylation promotes auxin transport in the hypocotyls during the phototropic bending response by regulating PIN activity. Interestingly, the PIN3 polarizes correctly after phototropic stimulation in d6pk Current Opinion in Plant Biology 2015, 23:116–123

mutants, but the reduced PIN3 phosphorylation leads to inactivity of PIN3, which in turn impairs auxin redistribution and phototropism [16]. Thus PIN3 auxin transport activity regulated by D6K [62,63] represents an additional layer of regulation during tropic responses, in adition to the control of PIN3 polar localization. Obviously, both these mechanisms are required to achieve the creation of auxin maxima and subsequent tropic responses.

Conclusion The regulation of polar PIN localization and resulting auxin fluxes during tropisms are subject of intensive research. Here, we summarize recent insights into the cellular mechanism of the PIN3 auxin transporter polarization in response to environmental stimulations of Arabidopsis hypocotyls. Somewhere in the complex mechanism of light sensing, statolith sedimentation and downstream PIN3 trafficking, the directional information from the environment needs to get translated into aligned direction of auxin flow. In other words, the exact mechanism for the translation of the light and gravity perception into the directional PIN3 transcytosis is still very much unclear. Whether this occurs at the level of the sorting endosomes, where PIN3 is recruited to the polar recycling pathway, at the level of vesicle fusion with the bottom PM, or whether all the polar recycling trafficking routes are rearranged in response to light stimulation or statolith sedimentation, remains a fascinating question for the future research. These questions can be addressed by identification of new components and mechanisms involved in tropic bending or polar sorting and trafficking. PIN3 relocates after both light or gravity stimulation and its new cellular polarity corresponds with the presumed auxin flow redirection. Furthermore, the mutants defective in PIN3 or its relocation do not establish asymmetric auxin distribution and have tropism defects. These are suggestive observations but they do not provide a direct evidence that PIN3 relocation is the main mechanism translating the gravity or light stimuli into directional auxin flow for tropic bending. The ultimate proof would require a PIN3 protein version that still transports auxin, is correctly localized but is not relocated after light or gravity stimulation. Regulation of the PIN3 polarity in hypocotyl endodermal cell is a good model to study cell polarity regulations in general, because the PIN3 localization is inner lateral under light conditions [2], apolar in the dark [8], outer lateral in the shade [24] and switches to the inner-lateral PM side after gravity or light stimulations [7,8] thus providing many opportunities to study different polar pathways and their response to different signals. www.sciencedirect.com

PIN-mediated cell polarity during tropism Rakusova´, Fendrych and Friml 121

Another interesting but largely unexplored issue is how the tropic response is terminated and overbending prevented. It remains entirely unclear, which mechanism underlies this precisely timed process.

Acknowledgements We thank Martine De Cock for help in preparing the manuscript. This work was supported by the European Research Council (project ERC-2011-StG20101109-PSDP); the Agency for Innovation by Science and Technology (IWT) (predoctoral fellowship to H.R.); and the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement No. 291734 is gratefully acknowledged by M.F.

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