Report. Halotropism Is a Response of Plant Roots to Avoid a Saline Environment

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1 Current Biology 23, , October 21, 2013 ª2013 Elsevier Ltd All rights reserved Halotropism Is a Response of Plant Roots to Avoid a Saline Environment Report Carlos S. Galvan-Ampudia, 1 Magdalena M. Julkowska, 1 Essam Darwish, 1,3 Jacinto Gandullo, 1 Ruud A. Korver, 1 Geraldine Brunoud, 2 Michel A. Haring, 1 Teun Munnik, 1 Teva Vernoux, 2 and Christa Testerink 1, * 1 Section of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, the Netherlands 2 Laboratoire de Reproduction et Développement des Plantes, CNRS, INRA, ENS Lyon, UCBL, Université de Lyon, Lyon, France Summary Tropisms represent fascinating examples of how plants respond to environmental signals by adapting their growth and development. Here, a novel tropism is reported, halotropism, allowing plant seedlings to reduce their exposure to salinity by circumventing a saline environment. In response to a salt gradient, Arabidopsis, tomato, and sorghum roots were found to actively prioritize growth away from salinity above following the gravity axis. Directionality of this response is established by an active redistribution of the plant hormone auxin in the root tip, which is mediated by the PIN-FORMED 2 (PIN2) auxin efflux carrier. We show that salt-induced phospholipase D activity stimulates clathrinmediated endocytosis of PIN2 at the side of the root facing the higher salt concentration. The intracellular relocalization of PIN2 allows for auxin redistribution and for the directional bending of the root away from the higher salt concentration. Our results thus identify a cellular pathway essential for the integration of environmental cues with auxin-regulated root growth that likely plays a key role in plant adaptative responses to salt stress. Results and Discussion Plant Roots Grow Away from High Salinity Salinity is one of the most devastating abiotic stresses in plants, causing major losses in crop production worldwide. Well-characterized responses to salt include the regulation of osmotic balance and ion transport [1] and, more recently, adjustment of root system architecture [2, 3]. Interestingly, salt stress has been observed to inhibit gravitropic growth of the primary root [4, 5]. Yet, it was unknown whether root bending in response to salt represented a bona fide tropism, i.e., a directional response induced by salt, or simply an alteration of the gravitropic response. We set up a vertical agarplate assay, in which Arabidopsis and tomato seedlings were challenged by exposure to diagonal NaCl gradients (Figure 1A; Figure S1D and Movie S1 available online). Roots were found to modify their direction of growth to circumvent the high salt-containing media in the gradient (Figures 1A and 1B). 3 Present address: Plant Physiology Division, Agricultural Botany Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt *Correspondence: c.s.testerink@uva.nl Quantification of the salt concentration in the media showed that roots experience a rapid increase of NaCl, raising up to only half the initial concentration of the salt-containing medium (Figure S1A). The response is concentration-dependent and is not triggered by an equal level of osmotic stress induced by mannitol or KCl (Figures 1A, 1B, and S1B), clearly distinguishing it from hydrotropism, which is growth of roots toward moisture [6]. In line with the nomenclature of other tropisms and a previous publication that hypothesized the existence of such a response [5], we call this novel negative tropic response to salt halotropism. Addition of CaCl 2 to the medium did not alter the response of roots to salt gradients (Figure S1B), showing it is not caused by lower external Ca 2+ activity [7]. Early responses to Na + disequilibrium involve the salt overly sensitive (SOS) pathway [5, 8], and accordingly, sos1 and sos2 mutants responded to lower salt concentrations than wild-type plants (Figure S1C). Moreover, plants overexpressing SOS1 showed a reduced halotropic response (Figure S1C), indicating that high internal sodium concentrations trigger the directional root growth. To investigate if the negative halotropic response could also occur in soil, we used tomato and sorghum plants to visualize and quantify their primary root growth in a soil slice system. Also in this setup, primary roots changed direction away from the site where salt was applied (Figures 1C and 1D). These results suggest that roots are constantly monitoring the direction and strength of the salt gradient and integrate these signals with the gravity stimulus to optimize their growth direction. We hypothesize the response to be relevant for growth of plants in natural conditions, such as salinized Australian soils, in which salt diffuses from deep layers [9]. Negative halotropism could be specific to salt-sensitive species, as in the halophyte Bassia indica an opposite, positive growth response of roots toward high salinity has been reported, although specificity for other ions was not tested [10]. Halotropism Depends on Redistribution of Auxin in the Root Tropisms enable plants to optimize their growth in response to their environment. Active redistribution of the phytohormone auxin through polar transport is an essential process in establishing tropisms. In the case of gravistimulated roots (e.g., rotated 90 from the gravity axis), transient accumulation of auxin at the lower side of the root inhibits cell elongation, resulting in the bending of the root tip toward gravity [11 13]. To test whether halotropism is regulated through differential auxin accumulation, we used DII-VENUS, a synthetic fusion protein rapidly degraded in response to auxin [12]. This reporter revealed increased auxin levels at the side of the root opposite to the higher salinity (Figure 2A), notably in the outer tissues (epidermis and cortex), as early as 2 hr (Figures 2C and S2A). Consistently, changes in expression of the auxin-induced transcription synthetic marker, DR5::N7- VENUS [14] were detected after 6 hr in salt gradients (Figures 2B and 2C), indicating higher auxin signaling at epidermis and lateral root cap opposite to the high salt. Disruption of auxin distribution (Figure S2B) by application of a low concentration

2 Plant Roots Avoid Salinity 2045 A Figure 1. Halotropism Is a Directional Response of Roots to Salinity (A) Seedlings exposed to diagonal gradients exhibit a halotropic response (white) followed by re-establishment of gravitropic growth (orange arrowhead). The scale bar represents 1 cm. (B) Quantification of percentage of roots in angle categories of 10 degrees after 24 hr, with number of roots measured. Blue bars represent halotropism. Only root tips positioned cm above the control-salt or control-mannitol interface were analyzed to minimize activation of hydrotropism (see [6]). Statistical comparisons by multivariate ANOVA (post hoc Tukey honestly significant difference [HSD] p < 0.01) among the treatments are denoted to indicate no significant differences (same letter) or significant differences (different letters). (C and D) Tomato (C) and sorghum (D) seedlings were exposed to a salt gradient in soil (arrow). Tomato roots were highlighted in white to aid visibility. Error bars depict the SE. Asterisk denotes significant differences (Student s t test). See also Figure S1. B C D

3 Current Biology Vol 23 No A B C D E F Figure 2. Halotropism Is Mediated by Asymmetric Auxin Distribution (A and B) DII-VENUS (yellow) (A) and DR5::N7-VENUS (B) growing in a salt gradient (white arrow). Seedlings were directly imaged on the gradient as described in Supplemental Experimental Procedures. (C) Quantification of fluorescence intensity of DII-VENUS and DR5::N7-VENUS during halotropism. Quantification of DR5::N7-VENUS was performed by selecting a region of interest corresponding to the lateral root cap and epidermis cell layer for eight roots per time point in four independent biological experiments, as described in [13]. Error bars depict the SE. Asterisk indicates statistical differences (Student s t test) between left and right. a.u., arbitrary units. (D and E) External auxin application inhibits halotropism. After 5 days of germination in control conditions, DII-VENUS seedlings were transferred to mock (D) or 10 nm IAA plates (E), after which the salt gradient was established. (F) Quantification of root angles after 24 hr gradient, as in Figure 1B. See also Figure S2. of 3-indoleacetic acid (IAA) largely abolished the halotropic response (Figures 2D 2F), indicating that the asymmetrical distribution of auxin in the root is required for halotropism. Salinity Triggers Clathrin-Mediated Endocytosis of PIN2 PIN-FORMED auxin efflux carriers (PINs) are major regulators of polar auxin transport [15 17]. Cell-specific polar localization of PINs has an essential role during development by generating local auxin maxima and minima throughout the plant body [17, 18]. During gravitropism, asymmetrical auxin distribution is modulated by the auxin efflux carrier proteins PIN2 and PIN3 and the auxin influx carrier AUX1 [11, 19 21]. Upon gravistimulation, PIN2 is targeted for ubiquitin-dependent degradation in the epidermis cell layer at the upper side of the root [11]. Exposure to salt gradients induced internalization of PIN2-GFP at the side of the root facing the higher salt concentration, but not on the other side of the root (Figures 3A 3C), whereas mannitol did not affect PIN2 subcellular localization (Figures S3A S3E), suggesting that NaCl, but not osmotic stress, triggers the response. Salt treatment also reduced PIN2 apical localization in root epidermal cells (Figure S3E), possibly as a result of the increased rate of endocytosis. No effect of salt treatment was found on subcellular location or internalization of the PIN1, AUX1, or PIN3 proteins (Figures S3H and S3I) or the plasma membrane (PM)-adenosine triphosphatase (ATPase) PMA2 [22](Figure S3G), showing that internalization of PIN2 in response to salt is a specific event, rather than a general response of membrane proteins. A marked difference with gravitropism is that during halotropism, the directional effect on root growth appears to be

4 Plant Roots Avoid Salinity 2047 A B C D F G J E H I K L Figure 3. Clathrin-Mediated Endocytosis of PIN2 Is Required for Halotropism (A and B) PIN2-GFP (cyan) imaged in roots growing in diagonal salt gradients (white arrow) after 0 (A) or 6 hr (B). White dashed boxes are magnified sections. Ep, epidermis; Co, cortex. Arrowheads indicate PIN2 internalization. Clear PIN2 internalization on the high-salt exposed side and low or absent PIN2 internalization at the other side of the root was a consistent pattern in 30 imaged roots. (C) Quantification of PIN2-GFP pixel intensity. Statistical differences determined by Student s t test between 0 hr and 6 hr (left/right) are indicated by an asterisk (n = 60 cells). (D and E) CLC-mCherry (magenta) imaged at 5 min (D) and 60 min after exposure to 100 mm NaCl (E). Arrowheads indicate clathrin recruitment to PM. (F I) Roots pretreated for 30 min with mock (F and G) or 30 mm tyrphostin A23 (H and I), followed by 100 mm NaCl (F and G) or 30 mm tyrphostin A23 with 100 mm NaCl (H and I). Clathrin recruitment to PM is indicated by arrowheads and PIN2 internalization by arrows. (J and K) Quantification of data presented in (D) (I). CLC-CHERRY or PIN2-VENUS pixel intensity (n = 50 epidermal cells of ten roots). Asterisk indicates significant differences from control (Student s t test) and + indicates significant difference between Tyr23 and mock treatment. (L) Quantification of root angles after growing 24 hr on salt gradient, as in Figure 1B, showing a reduced halotropic response of abp1-5 and PIN2 Y505A mutants, and after tyrphostin treatment. Statistical comparisons by multivariate ANOVA (post hoc Tukey HSD p < 0.05). Error bars depict the SE. See also Figure S3. mediated by modulation of PIN2 endocytosis rather than degradation, as no reduction in overall PIN2 signal was observed (Figures S3J and S3K), whereas expression of PIN3 and PIN1 was even slightly induced upon salt treatment (Figures S3H and S3I). Both PIN2 internalization and recycling to the membrane could be increased, similar to the saltinduced enhanced cycling found for aquaporins [23]. Previous reports have shown that transfer to higher salt (>150 mm), which induces plasmolysis, causes degradation of PIN2 [5]. However, such a response is not triggered at the mm NaCl the roots experience in our gradient setup (Figures S1A and S3J). Transfer to 100 mm salt even inhibited the differential PIN2 degradation upon gravitropical challenge [11] (Figures S3L S3N). The capacity to block PIN2 degradation in response

5 Current Biology Vol 23 No A B C D E F G H I J K L M Figure 4. PIN2 Internalization Is Reduced upon Salt Treatment in the pldz2 Mutant (A D) Col-0 (A and B) or pldz2 mutant (C and D) expressing PIN2-GFP and FM4-64 staining. (E and F) Quantification of data presented in (A) (D). Error bars depict the SE (n = 40 epidermal cells of eight roots). (G L) PIN2-GFP and FM4-64 costaining in Col-0 (G and H) or pldz2 mutant (I and J) after NaCl treatment, with quantification of the data (K and L). Arrowheads indicate PIN2-GFP internalization. Statistical difference determined by Student s t test (p < 0.01) between the lines are denoted with asterisk. Error bars depict the SE (n = 40 epidermal cells of eight roots). (M) Proposed model for the molecular and cellular mechanism regulating halotropism. Salt stress induces PLDz2-dependent recruitment of clathrin to the PM and PIN2 internalization and recycling (thick red arrows) at one side of the root, causing differential auxin accumulation (green) and bending of the root away from the salt source. TGN/EE, trans-golgi network/early endosomes. See also Figure S4.

6 Plant Roots Avoid Salinity 2049 to salt during a gravitropic response might represent a mechanism by which halotropism can temporarily block the capacity of roots to respond to gravity and allow for an optimal avoidance response to salinity. Clathrin-mediated endocytosis of PIN proteins plays an essential role in the establishment and maintenance of cell polarity [24, 25] and responses to environmental cues [21, 26]. Visualization of Arabidopsis clathrin light chain (CLC) subcellular localization in the root epidermis revealed an increase in PM localization after 60 min of NaCl treatment (Figures 3D, 3E, and 3J). Accordingly, Tyrphostin23, an inhibitor of clathrin assembly, repressed the salt-induced recruitment of CLC (Figures 3F, 3H, and 3J) and blocked salt-induced PIN2 internalization (Figures 3G, 3I, and 3K). Addition of tyrphostin during salt gradients markedly reduced halotropism. Likewise, both the abp1-5 mutant, which is resistant to inhibition of PIN protein endocytosis by auxin [27], and the PIN2 Y505A mutant, which has been suggested to have reduced clathrin-mediated endocytosis [24], exhibited significantly impaired halotropism (Figure 3L). Together, these results show a genetic requirement for clathrin-mediated endocytosis of PIN2 during the halotropic response. The Phospholipase PLDz2 Regulates PIN2 Trafficking during Halotropism Phosphatidic acid (PA) is a phospholipid that functions in recruiting proteins to cellular membranes in plant stress and developmental responses, including salinity [28]. In a quantitative proteomics screen, we recently identified several clathrin machinery proteins to bind PA and to be specifically enriched in the membrane fraction upon salt stress [29], suggesting a role for PA in the salt-enhanced recruitment of clathrin to the plasma membrane. Phospholipase D (PLD) activity represents one of the major pathways that generates PA in response to abiotic stresses [28, 30 32]. In yeast and animals, PLD has been implicated as essential mediator of membrane trafficking and clathrin-mediated endocytosis [33]. In Arabidopsis, the phox homology/pleckstrin homology domain containing PLD homolog AtPLDz2 has been shown to regulate gravitropic and hydrotropic responses [34, 35], and its expression is increased by salinity [4] and osmotic stress [35]. Consistent with earlier reports [34], the pldz2 mutant was not affected in PIN2 subcellular localization or uptake of the endocytic marker FM4-64 under normal growth conditions [31] (Figures 4A 4F). Upon treatment with salt, a markedly reduced internalization of both PIN2 and aberrant uptake of the lipophilic FM4-64 dye were observed (Figures 4G 4L). The mutant also showed a reduced halotropic response (Figures S4G and S4H), suggesting that pldz2 might be a common regulator of root tropisms [34, 35]. In addition, treatment with FIPI (5-Fluoro-2-indolyl des-chlorohalopemide), a potent inhibitor of animal PLDs [36], blocked clathrin membrane localization, even under control conditions, and affected PIN2 localization in wild-type roots (Figures S4A, S4B, and S4F). Moreover, both salt-induced PM localization of clathrin and internalization of PIN2 were disrupted (Figures S4C S4F). These results suggest that PLD function is essential for the increase in clathrin-mediated endocytosis of PIN2 during salt stress. A Cellular and Physiological Model for Root Halotropism Here, we report a new type of tropism, termed halotropism, that allows plants to escape from local salinity by simply growing away. We provide evidence for salt-induced phospholipase D activity controlling clathrin recruitment to the membrane to trigger internalization of PIN2 at the site of the root facing the high salt (Figure 4M). This in turn results in redistribution of auxin in the root tip, causing the root to bend away from high salt concentrations. Given the key role for PIN2 in gravitropism [11] and its recent implication in the adaptation of root growth in response to the light environment [37], our results further establish the regulation of PIN2 trafficking as a central target of pathways that allow for the integration of environmental cues during plant growth and development. Supplemental Information Supplemental Information includes Supplemental Experimental Procedures, four figures, and one movie and can be found with this article online at Acknowledgments We thank Dorus Gadella, Erik Manders, and Ronald Breedijk for confocal microscopy assistance; Ludek Tikovsky for help in the greenhouse; and Song Chen and Pilar Morera Margarit for technical assistance. We are grateful to Karin Schumacher (PMA2-GFP), Takashi Aoyama (pldz2 mutant), Jian-Kang Zhu (sos mutants), Remko Offringa (PIN2-GFP and eir1-1 PIN2- VENUS), Jiri Friml (DR5::N7-VENUS; abp1-5 and PIN2 Y505A ), and Eva Benkova (PIN3-GFP) for published and unpublished materials. We thank Remko Offringa for helpful discussions and comments and the Nottingham Arabidopsis Stock Centre (NASC) for seed stocks. 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