Molecular Plant Volume 5 Number 6 Pages November 2012

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1 Molecular Plant Volume 5 Number 6 Pages November 2012 RESEARCH ARTICLE Endocytic Trafficking towards the Vacuole Plays a Key Role in the Auxin Receptor SCF TIR -Independent Mechanism of Lateral Root Formation in A. thaliana Patricio Pérez-Henríquez a,b, Natasha V. Raikhel c and Lorena Norambuena a,b,1 a Plant Molecular Biology Laboratory, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile b Millenium Nucleus in Plant Cell Biotechnology, Chile c Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA ABSTRACT Plants developmental plasticity plays a pivotal role in responding to environmental conditions. One of the most plastic plant organs is the root system. Different environmental stimuli such as nutrients and water deficiency may induce lateral root formation to compensate for a low level of water and/or nutrients. It has been shown that the hormone auxin tunes lateral root development and components for its signaling pathway have been identified. Using chemical biology, we discovered an Arabidopsis thaliana lateral root formation mechanism that is independent of the auxin receptor SCF TIR. The bioactive compound Sortin2 increased lateral root occurrence by acting upstream from the morphological marker of lateral root primordium formation, the mitotic activity. The compound did not display auxin activity. At the cellular level, Sortin2 accelerated endosomal trafficking, resulting in increased trafficking of plasma membrane recycling proteins to the vacuole. Sortin2 affected Late endosome/pvc/mvb trafficking and morphology. Combining Sortin2 with well-known drugs showed that endocytic trafficking of Late E/PVC/MVB towards the vacuole is pivotal for Sortin2- induced SCF TIR -independent lateral root initiation. Our results revealed a distinctive role for endosomal trafficking in the promotion of lateral root formation via a process that does not rely on the auxin receptor complex SCF TIR. Key words: lateral root; development; endocytic trafficking; Sortin2; endosomes; Arabidopsis. Introduction Plant organ development both in the aerial and below-ground parts occurs mainly post-embryonically by highly regulated spatiotemporal processes. Root plasticity dynamically integrates different stimuli such as nutrients and water as well as soil aeration and salinity (Malamy, 2005; Nibau et al., 2008). For instance, roots under nutrient deficiency are able to remodel their structure, increasing lateral roots (LR) to facilitate nutrient acquisition (Drew and Saker, 1978; Malamy and Ryan, 2001; Svistoonoff et al., 2007; Lima et al., 2010; Martín-Rejano et al., 2011; Miura et al., 2011). This strategy contributes to their spatial distribution in the soil and also increases surface area, stimulating absorption of water and nutrients from the soil. LR originate in the inner pericyle cell layer, where a root primordium is formed and finally emerges from the primary root (Casimiro et al., 2001). Specific pericycle cells are selected to be founder cells in a developmental stage called priming (Peret et al., 2009; Overvoorde et al., 2010). LR develop at defined positions, suggesting a very tight control on the selection and activation of founder cells in the primary root. LR initiation begins at the first divisions of founder cells (Peret et al., 2009). The consecutive divisions that pericycle founder 1 To whom correspondence should be addressed at Las palmeras 3425 Ñuñoa, Santiago, Chile. lnorambuena@uchile.cl, tel , fax The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: /mp/sss066, Advance Access publication 30 July 2012 Received 27 March 2012; accepted 10 May 2012

2 1196 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation cells undergo to initiate a LR primordium have been revealed by means of live imaging (De Smet et al., 2008). The hormone auxin has been shown to be a key regulator of LR formation. Exogenous application or endogenous overproduction of auxin induces LR primordium development (Klee et al., 1987; Celenza et al., 1995; Himanen et al., 2002; Dubrovsky et al., 2008). Stronger evidence of auxin-regulated LR formation comes from experiments that correlate physical features of primordium formation with the spatiotemporal distribution of auxin and its transcriptional signaling (Dubrovsky et al., 2008, 2011). Auxin is transported through the tissues by cell-to-cell transport and its effects have been related mainly to an auxin gradient concentration (Benkova et al., 2003; Scarpella et al., 2006; Sundberg and Østergaard, 2009; Shkolnik-Inbar and Bar-Zvi, 2010; Dubrovsky et al., 2011; Forestan and Varotto, 2012). Inhibitors of auxin transport cause accumulation of auxin in the root apex, arresting LR development by blocking cell division at a very early stage (Karabahlidegron et al., 1998; Reed et al., 1998; Casimiro et al., 2001). Auxin signaling activates the family of transcription factors called auxin response factors (ARF), which are responsible for the auxin-regulated gene expression of the physiological events that auxin triggers (Ulmasov et al., 1997b). ARFs are negatively regulated by nuclear proteins known as AUX/ IAA (Auxin/Indole Acetic Acid) (Ulmasov et al., 1997a; Tiwari et al., 2003, 2004). Within the cell, auxin is recognized by the auxin receptor complex SCF TIR, which induces AUX/IAA protein degradation and releases ARF inhibition, thus allowing auxin-regulated gene expression to set a particular developmental program (Gray et al., 2001). Mutants with defects in auxin homeostasis, signaling, and transport have few or no LR (Peret et al., 2009; Zhu et al., 2012). The characterization of some of LR-impaired mutants showed that auxin is required to initiate cell division in the pericycle (Celenza et al., 1995). There is evidence that transcriptional activation is required for auxin LR development (Vanneste et al., 2005). Nevertheless, it has been controversial to define auxin as the earliest and unique signal for LR formation. Recently, additional signals for LR initiation have been found (Ditengou et al., 2008; Ortíz-Castro et al., 2008; Richter et al., 2009); LR initiation was induced by mechanical stimulus, independently of the AUX/IAA and ARF protein functions (Ditengou et al., 2008). Richter et al. (2009) showed that root bending triggers LR independently of shoot auxin supply and this response was not disrupted in mutants of the auxin pathway (Richter et al., 2009). Root-tip-derived signals, calcium signaling, and plasma membrane protein relocalization appear to be important cues for LR initiation mediated by mechanical stimulus (Ditengou et al., 2008; Richter et al., 2009). The plant endomembrane system has been implicated in a variety of processes including plant development and hormone signaling, among others (Samaj et al., 2004; Surpin and Raikhel, 2004; Drakakaki et al., 2009; Grunewald and Friml, 2010). At the cellular level, endomembrane trafficking is fundamental for the intracellular relocalization of macromolecular cargo and membranes by secretion and endocytosis. In plant biosynthetic trafficking, new proteins are translocated to the endoplasmic reticulum (ER) and subsequent protein processing and targeting occur via vesicle trafficking through the secretory pathway (Bassham et al., 2008). In plant endocytic trafficking, protein components of plasma membrane (PM) and cell wall are recycled to internal endosomal compartments (Müller et al., 2007). The endocytosed components reach the early endosome/trans-golgi network (EE/TGN) (Viotti et al., 2010) from where they may either recycle back to the PM via recycling endosomes or traffic onwards via the endocytic route to later compartments such as the late endosome/prevacuolar compartment/multi-vesicular body (LE/PVC/MVB). Endocytosed components may finally reach the vacuole, where they can be degraded (Emans et al., 2002; Kleine-Vehn et al., 2008; Laxmi et al., 2008). In the context of auxin-dependent LR formation, the endomembrane system plays an important role, establishing cellular localization and polarity of the auxin transporter proteins (Geldner et al., 2001; Friml et al., 2003; Friml, 2010). Polarity of auxin transporters allows the formation of auxin gradients that are important for both LR initiation and the establishment of the primordium cell patterning (Benkova et al., 2003). Chemical biology, as a strategy, has provided instrumental tools to understand plant cellular processes and their implications at the physiological level (Hicks and Raikhel, 2009; Robert et al., 2009; Hicks and Raikhel, 2010). Bioactive chemicals that impair protein trafficking called Sortins were identified using a chemical genomic screen (Zouhar et al., 2004). Among these compounds, Sortin2 affects vacuolar protein targeting in S. cerevisiae as well as vacuole morphology in Arabidopsis thaliana (Zouhar et al., 2004; Norambuena et al., 2008). Interestingly, in A. thaliana, Sortin2 affects root development (Zouhar et al., 2004); however, how this links with the disruption of endomembrane trafficking has not yet been resolved. Here, Sortin2 was instrumental to unravel a pathway that triggers LR initiation by a mechanism independent of the auxin receptor complex SCF TIR. We provide evidence that Sortin2 induces endocytic trafficking, and thus targets plasma membrane/endosome recycling proteins towards the vacuole. A combination of chemical biology tools shows that Sortin2-LR initiation was due to the effect of Sortin2 on endocytic protein trafficking that requires the route from LE/PVC/MVB to the vacuole. The overall evidence suggests a mechanism whereby endocytic trafficking induces initiation of LR by a mechanism independent of SCF TIR. RESULTS The synthetic bioactive compound Sortin2 affects root development and the endomembrane system in plants (Zouhar et al., 2004). When A. thaliana seeds are sown with 100 µg ml 1, Sortin2 germination occurs, but root growth is strongly

3 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation 1197 inhibited (Zouhar et al., 2004). We found that inhibition of the main root growth due to Sortin2 was dose-dependent (Supplemental Figure 1). Moreover, 25 µg ml 1 Sortin2 induced adventitious roots along with the main root growth inhibition (Supplemental Figure 1). These data strongly indicate that Sortin2 affects mechanisms involved in root growth and organogenesis in A. thaliana. LR Development Occurs by an SCF TIR -Independent Mechanism To study the effect of Sortin2 on root development, 7-day-old A. thaliana seedlings were treated with the compound and root phenotypes were analyzed. After a 4-d treatment with 25 µg ml 1 Sortin2, length was 34.7 mm, 39% of the control seedlings (57.0 mm) (Figure 1A). There was no significant difference between root length with 25 and 50 µg ml 1 Sortin2. In addition, the chemical treatment increased the LR index from 0.25 in the control to 1.9 with 25 µg ml 1 Sortin2 (Figure 1B). With 25 µg ml 1 Sortin2, the number of LR was more variable; however, the mean was significantly greater than in control seedlings and seedlings treated with 50 µg ml 1 Sortin2 (Figure 1B). Sortin2-induced LR appeared normal in shape, even though 36% shorter than in control conditions (Figure 1C and 1D, and Supplemental Figure 2). Short LR was consistent with Sortin2 inhibition of main root growth (Figure 1A). LR develop alternately along the main root (De Smet et al., 2007); however, Sortin2-treated seedlings often showed two opposite LR (Figure 1D and Supplemental Figure 2). This suggests that Sortin2 alters a mechanism that determines where LR are initiated. There are several steps to LR development: founder cell specification (priming), initiation, emergence, and elongation (Casimiro et al., 2003; Peret et al., 2009; Duclercq et al., 2011). Obviously, for more emerged LR on Sortin2-treated seedlings, more initiation events were needed. However, it is also possible that the chemical treatment accelerates the emergence of already initiated primordia in the 7-day-old root seedling. To determine whether Sortin2 affects LR initiation or emergence, we utilized the well-characterized marker line CYCB1;1::GUS, which has been useful to identify the initiation step of LR (Himanen et al., 2002). This transgenic line expresses GUS under the CYCB1;1 promoter, a mitotic activity reporter which labels root primordia as well as the main root and LR meristems (Ferreira et al., 1994a, 1994b). Control Figure 1. Sortin2 Induces Lateral Root Initiation. (A C) Seven-day-old A. thaliana (Col-0) seedlings were treated with Sortin2 at 25 and 50 μg ml 1. After 96 h, roots were analyzed by measuring primary root length (A), number of lateral roots (B), and lateral root length (C). The number of analyzed seedlings is indicated in each case (n). Scattering graphs show the average value. (D) Seven-day-old CycB1;1:GUS A. thaliana line was treated with control treatment, 1 μm NAA, and 25 μg ml 1 Sortin2. After 72-h treatment, GUS staining was performed. (Low magnification) images were taken with the same magnification. (High magnification) images showing primordium close-up for each low-magnification image. (E) Emerged lateral roots and primordia were scored as CYCB1;1 positive events per plant (n = 8). SEM is shown in (E).

4 1198 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation seedlings had GUS-positive events along the primary root of root primordia as well as emerged LR (Figure 1D). The number of GUS-positive events increased with auxin naphthalene acetic acid (NAA; Figure 1D and 1E; Casimiro et al., 2001). Sortin2 treatment induced mitotic activity along the primary root since Sortin2-treated seedlings had more GUS-positive LR events than the control (Figure 1D and 1E). This result indicates that, under Sortin2 treatment, the initiation step of LR development was stimulated. LR formation has been explained as a result of cellular induction by auxin at the initiation step in the pericycle (Casimiro et al., 2001). Therefore, the fact that Sortin2 stimulated LR formation could be explained if this compound was acting as an auxin. To evaluate Sortin2 auxin activity, its capacity to induce AUX/IAA protein degradation was determined. Using the transgenic line HS::AXR3NT GUS that expresses the AUX/IAA family member AXR3 fused to GUS, we evaluated AXR3 stability by quantifying GUS activity (Gray et al., 2001). Treatment with NAA induced significant degradation of AXR3 (p < 0.05, Tukey s test, Figure 2A), while treatment with Sortin2 did not affect the AXR3 level (Figure 2A). This result shows that Sortin2 did not behave as an auxin signaling inducer. To evaluate the effect of Sortin2 on auxin-responsive transcriptional activity, the DR5::GUS reporter line was used. This transgenic plant expresses GUS under control of the auxin-driven promoter DR5 and therefore reports transcriptional activity induced by auxin (Ulmasov et al., 1997b). There were no significant differences in GUS protein activity between Sortin2-treated seedlings and control conditions (Figure 2B). In contrast, treatments with auxins such as indole-3-acetic acid (IAA), NAA, and 2,4-Dichlorophenoxyacetic acid (2,4-D) resulted in a fourfold induction of DR5::GUS activity compared to the control (Figure 2B). Therefore, Sortin2 was not able to induce transcriptional activity of DR5; thus, its effect on LR initiation may be auxin-independent. To analyze further the participation of auxin signaling on Sortin2 LR induction, we challenged with Sortin2 the auxin response mutant axr1-12, which carries a loss-of-function allele Figure 2. Lateral Root Development Induction by SCF TIR -Independent Mechanism. (A)Seven-day-old HS::AXR3NT:GUS seedlings were pre-incubated at 37 C for 2 h to induce protein expression. Thereafter, they were incubated with 10 μm NAA or 50 μg ml 1 Sortin2 for different periods of time. GUS activity was measured by a fluorometric assay. Asterisks show statistically significant differences between NAA by ANOVA between treatments and the control (*** p < 0.01 and * p < 0.1). (B) Seven-day-old DR5::GUS seedlings were treated for 24 h with 50 μg ml 1 Sortin2, 10 μm IAA, 1 μm NAA, or 1 μm 2,4-D and control conditions. GUS activity was quantified the same as in (A). Statistical testing was performed with Student s t-test (ns, not significant). (C) Seven-day-old A. thaliana Col-0 (WT, white bars) and axr1-12 mutant (axr1-12, gray bars) seedlings were treated with Sortin2 at 25 μg ml 1. After 72 h, the number of LR was scored (n = 36). (D) Seven-day-old A. thaliana Col-0 (WT, white bars) and the quadruple tir mutant (tir1/afb1/afb2/afb3, gray bars) seedlings were treated with 25 μg ml 1 Sortin2. After 72 h, the number of LR was evaluated (n = 21). (C) and (D) experiments were performed on liquid and solid MS media, respectively. (E) Seven-day-old A. thaliana (Col-0) seedlings were treated with concentrations of 10 μm NPA and 25 μg ml 1 Sortin2. After 72 h, LR number was scored. Statistical significance was evaluated with Student s t-test (** p < 0.05; ns, not significant).

5 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation 1199 of the SCF TIR activation factor AXR1 (Lincoln et al., 1990; Leyser et al., 1993), and the auxin receptor TIR/AFB quadruple mutant tir/afb1/afb2/afb3 (Dharmasiri et al., 2005). When axr1-12 was grown in the presence of Sortin2, it had an LR index similar to the Sortin2-treated wild-type (Figure 2C). The auxin receptor quadruple mutant tir/afb1/afb2/afb3 develops very few LR, and exogenous auxin application does not induce LR in this mutant (Dharmasiri et al., 2005). Interestingly, this quadruple mutant showed an increase in LR abundance in the presence of Sortin2, as did the wild-type (Figure 2D). Therefore, we concluded that Sortin2 induces the formation of LR and does it independently of the auxin receptor complex SCF TIR. Auxin polar transport has been defined as an important regulator of LR initiation. Interfering with auxin transport by mutations or with chemical inhibitors leads to a decrease in LR occurrence (Himanen et al., 2002; Benkova et al., 2003; Geldner et al., 2004). To test whether auxin transport is needed for the Sortin2 effect on LR, we used the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) (Casimiro et al., 2001; Moreno-Risueno et al., 2010). NPA is able to inhibit LR occurrence (Figure 2E; Casimiro et al., 2001). As Figure 2E shows, there was no difference in the LR index in treatments with Sortin2 and Sortin2 plus 10 µm NPA. Therefore, we concluded that the Sortin2 effect in LR is not inhibited by the auxin transport inhibitor NPA. Endocytic Trafficking Is Altered by Sortin2 Sortin2 was identified by its ability to alter protein trafficking (Zouhar et al., 2004). Since Sortin2 stimulates LR initiation, we further analyzed its effect on the endomembrane system at the plant cellular level. Since Sortin2 triggers secretion of the vacuole-soluble cargo protein carboxypeptidase Y (CPY) in S. cerevisiae (Zouhar et al., 2004; Norambuena et al., 2008), we tested its effect on vacuole protein trafficking in plants. By means of immunoelectron microscopy using antibodies against A. thaliana CPY (AtCPY) (Rojo et al., 2003), no gold particles could be detected in the extracellular compartment of normal hypocotyl cells (Figure 3A). By contrast, Sortin2-treated seedlings showed the presence of AtCPY in the extracellular space (Figure 3B). AtCPY, as a vacuolar protein, is synthesized on the ER and glycosylated in the Golgi apparatus (GA) (Rojo et al., 2003). Sortin2 had no effect on either ER or GA fluorescent protein marker localization, by confocal microscopy analysis (data not shown). Therefore, the Sortin2 effect within the secretory pathway was very specific, affecting post-golgi trafficking. AtCPY reaches the vacuole by the N-terminal peptide pathway (NTPP; Rojo et al., 2003) that traffics from EE/TGN through the LE/PVC/MVB (Surpin and Raikhel, 2004). Therefore, the effect of Sortin2 on trafficking to the endosomal compartments was tested using GFP-fused protein markers on root cells of 7-day-old seedlings. The normal distribution of EE/ TGN, LE/PVC/MVB, and vacuole is shown in Figure 3. EE/TGN (VHA-a1:GFP; Dettmer et al., 2006) and LE/PVC/MVB (ARA7:GFP; Jaillais et al., 2006) are punctuate structures distributed over Figure 3. Trafficking within the Endomembrane System Is Altered by Sortin2. Seven-day-old A. thaliana Col-0 and transgenic line seedlings were treated with 25 μg ml 1 Sortin2 (B, D, F, H, J, and L) and control conditions (A, C, E, G, I, and K). (A, B) AtCPY was detected by electron microscopy of hypocotol cells of A. thaliana (Col-0) seedlings. (C L) Roots of A. thaliana transgenic lines expressing fluorescent protein marker were visualized by confocal microscopy. (C, D) EE/TGN marker, VHA-a1:GFP (E, F) LE/PVC/MVB marker, ARA7:GFP (G, H) LE/ PVC/MVB-tonoplast marker, SYP22:YFP (I, J) PM/endosome recycling marker, PIN2:GFP (K, L) PM/endosome recycling marker, BRI1:GFP. Arrows show altered phenotypes. Scale bars are indicated. the cell (Figure 3C and 3E). The marker SYP22:YFP that traffics between LE/PVC/MVB and the vacuole localized as expected in the round vacuole structures, as well as in the punctuate structures LE/PVC/MVB (Figure 3G; Robert et al., 2008). The LE/ PVC/MVB proved to be aggregated in Sortin2-treated seedlings (Figure 3F and 3H). In Sortin2 treatments, the LE/PVC/ MVB marker ARA7:GFP was aggregated in larger compartments (Figure 3F, white arrow) and there was also a more abundant GPF signal in these compartments (Figure 3F). In

6 1200 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation addition, with Sortin2 treatment, SYP22:YFP was localized on the tonoplast, but also clustered in more abundant and larger structures than normal LE/PVC/MVB (Figure 3H, white arrow). Interestingly, the EE/TGN marker, VHA-a1:GFP (Dettmer et al., 2006), was not affected by Sortin2 (Figure 3D). Overall, these results indicate that cellular component(s) affected by this compound are probably located post TGN trafficking and before protein localization in the vacuole. Secretory and endocytic pathways of the endomembrane system are crucial for protein localization within the cell. These pathways are highly connected, especially since they share compartments such as EE/TGN and LE/PVC/MVB (Viotti et al., 2010; Scheuring et al., 2011). In order to test whether Sortin2 affects endocytosis, we examined the subcellular localization of the plasma membrane marker PIN2:GFP (Xu and Scheres, 2005) that constitutively traffics back and forth from the PM to endosomes (Figure 3I; Geldner et al., 2001; Dhonukshe et al., 2007). Sortin2 triggered PIN2:GFP accumulation into the vacuole after 6 h of treatment (Figure 3J). This phenotype was due to PIN2 endocytic trafficking, since it was independent of de novo protein synthesis; cycloheximide did not inhibit the Sortin2 effect (Supplemental Figure 3C). Furthermore PIN2 vacuole accumulation due to Sortin2 was sensitive to Wortmannin (Wn) and Latrunculin B (Supplemental Figure 3E and 3G, respectively) as in the physiological PIN2 degradation pathway (Kleine-Vehn et al., 2008; Laxmi et al., 2008). Therefore, the Sortin2 effect is upstream of Wm, a drug that inhibits trafficking from LE/PVC/MVB to the vacuole (Matsuoka et al., 1995), consistently with the altered pattern of the LE/PVC/MVB marker. In addition to the cellular effect, Sortin2 inhibited the gravitropic response of the seedlings (Supplemental Figures 1 and 4B). After gravistimulation, Sortin2-treated seedlings were not able to establish the PIN2 differential degradation (Supplemental Figure 4) that leads to the bending of the tip root (Abas et al., 2006; Kleine-Vehn et al., 2008; Peer et al., 2011). After gravitropic stimulus, PIN2 level is diminished in the upper part of the root tip (Abas et al., 2006; Kleine-Vehn et al., 2008). However, upon Sortin2 treatment, both sides of the root tip showed the same PIN2 abundance (Supplemental Figure 4). The plasma membrane/endosome recycling brassinosteroid transporter BRI1 (Geldner et al., 2007), which is also targeted to the vacuole by endocytic mechanisms (Kleine-Vehn et al., 2008), was also accumulated in the vacuole due to Sortin2 (Figure 3L). This result indicates that Sortin2-induced protein vacuole accumulation was not specific to PIN2; other proteins may also be affected by the chemical treatment. To test whether Sortin2 affects protein trafficking or vacuole protein stability, the effect of Sortin2 on endocytic trafficking of the fluorescent tracer FM4-64 was analyzed (Bolte et al., 2004). Under normal conditions, FM4-64 labels endosomes and vacuoles after incubation for 60 and 120 min, respectively (Supplemental Figure 5). Interestingly, Sortin2 treatment was able to accelerate the internalization of FM4-64 to the vacuole. Upon Sortin2 treatment, it took 60 min for FM4-64 to reach the vacuoles (Supplemental Figure 5). Therefore, Sortin2 did affect the mechanism(s) of endocytic protein trafficking. Overall, the results showed that Sortin2 accelerates endocytic protein trafficking to the vacuole and most likely affects one or more cellular components of LE/PVC/MVB trafficking. Sortin2 Stimulates Trafficking from Endosomes to the Vacuole To get more insight into Sortin2-specific effects on the endocytic pathway, we analyzed Sortin2-induced trafficking to the vacuole using PIN2:GFP as the protein marker. Brefeldin A (BFA) is well known as an exocytosis inhibitor that blocks trafficking from endosomes to the PM (Robinson et al., 2008a, 2008b). With BFA treatment, plasma membrane/ endosome recycling proteins are accumulated in intracellular compartments called BFA bodies, due to endosome membrane aggregation (Nebenführ et al., 2002). BFA bodies are characterized to be one or two per root cell, with very particular size and location within the cell (Kleine-Vehn et al., 2008; Robert et al., 2010). Geldner et al. (2003) described PIN2 BFA-induced accumulation in BFA bodies as a consequence of endocytosis (Geldner et al., 2003). Figure 4A, 4C, 4E, and 4G (arrows) show PIN2 accumulated on BFA bodies. When 7-day-old seedlings were treated with 50 µm BFA along with 50 µg l 1 Sortin2 for 2 h, accumulation of PIN2:GFP in BFA bodies was inhibited (Figure 4B). In this co-treatment, BFA bodies were present, as was observed using FM4-64 (Supplemental Figure 6). Therefore, the inhibition of BFA effect was specific for protein trafficking. More interestingly, longer co-treatment BFA-Sortin2 (6 h) caused PIN2 accumulation in the vacuole, which was an indication that endocytic trafficking was active (Figure 4D, arrowheads). However, no PIN2-labeled BFA bodies were observed (Figure 4D). This result suggests that Sortin2 inhibited the BFA effect by transporting PIN2 protein from endosomes to the vacuole. To verify this hypothesis, a pretreatment with BFA for 1.5 h was performed in order to induce PIN2 BFA bodies (Figure 4E) and Sortin2 was added afterwards for 2 h (Figure 4F). In this treatment, intracellular PIN2 coexisted in BFA bodies and the vacuole (Figure 4F). Longer treatment with Sortin2 BFA after BFA bodies were formed caused PIN2 BFA body disappearance, whereas PIN2 vacuole accumulation was observed (Figure 4H). Notice that Sortin2 application once BFA bodies were formed induced PIN2 vacuole targeting in less time (4 h instead of 6 h) than when both drugs were applied concomitantly, supporting that PIN2 trafficked to the vacuole from BFA bodies in the former condition (Figure 4F and 4H). Together, these results showed that Sortin2 induced PM/ endosome recycling marker relocalization from BFA bodies to the vacuole. Sortin2-Induced SCF TIR -Independent LR Formation Relies on Endocytic Trafficking Sortin2 induced a mechanism for LR initiation as well as endocytic trafficking. To examine how these two processes

7 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation 1201 Figure 4. Sortin2 Alters Trafficking from Endosomes to Vacuole. Seven-day-old PIN2:GFP seedlings were incubated under different conditions. Treatments used 50 μg ml 1 Sortin2 and 50 μm BFA as indicated below. PIN2:GFP distribution in root cells was visualized by confocal microscopy. (A) 2.5 h with BFA. (B) Sortin2 pretreatment for 0.5 h followed by 2 h of co-treatment of BFA and Sortin2. (C) 6.5 h with BFA. (D) Sortin2 pretreatment for 0.5 h followed for 6 h of co-treatment of BFA along with Sortin2. (E) Treatment with BFA for 3.5 h. (F) BFA pretreatment for 1.5 h followed by 2 h of co-treatment of BFA and Sortin2. (G) Treatment with BFA for 5.5 h. (H) BFA pretreatment for 1.5 h followed for 4 h of co-treatment with BFA plus Sortin2. Arrows and arrowheads indicate BFA bodies and vacuole, respectively. Scale bars are indicated. are linked, co-treatments with bioactive compounds known to alter endomembrane trafficking along with Sortin2 were performed. Tyrphostin A23 (TyrA23) inhibits clathrin-mediated endocytosis from plasma membrane to EE/TGN (Ortiz-Zapater et al., 2006; Dhonukshe et al., 2007). At a concentration that efficiently inhibits endocytosis (30 µm; Dhonukshe et al., 2007), this drug inhibited LR occurrence on 7-day-old seedlings (Figure 5A). Lower concentrations such as 20 µm had no effect on LR occurrence, suggesting its effect was specifically due to endocytosis impairment (Figure 5A). Sortin2, however, was able to overcome the inhibitory effect of 30 µm TyrA23 on LR. When plants were treated with Sortin2 and 30 µm TyrA23 simultaneously, the number of LR was ~2.5 times greater than control untreated seedlings (Figure 5A). This result is likely due to the stimulation of endocytosis from PM caused by Sortin2. Inhibition of exocytosis by BFA causes agglomeration of endosomal compartments (Figure 4; Geldner et al., 2003; Grebe et al., 2003). Figure 5B shows that LR occurrence with 25 and 50 μm BFA was significantly less than in the control. Contrastingly, there was an induction of LR on seedlings treated with Sortin2 along with different concentrations of BFA (Figure 5B). Therefore, Sortin2 LR induction prevailed over the inhibitory effect of 25 and 50 μm BFA (Figure 5B). This result is consistent with the fact that Sortin2 inhibited the accumulation of PIN2, and perhaps other proteins such as PINs in BFA bodies (Figure 4B). This indicates that trafficking from BFA bodies towards the vacuole is required for the SCF TIR -independent LR initiation mechanism. The lower LR index (~2.5 times) of seedlings co-treated with Sortin2 and 50 μm BFA (Figure 4B) may be explained by the existence of a pathway for sorting PIN to the vacuole which is sensitive to 50 μm BFA (Kleine-Vehn et al., 2008). In order to evaluate whether trafficking of LE/PVC/MVB to the vacuole was required for Sortin2 LR induction, the trafficking inhibitor Wm was used. Wm inhibits trafficking to the vacuole of PM/endosome recycling proteins (Kleine-Vehn et al., 2008). Furthermore, Wm alters trafficking and morphology of LE/PVC/MVB in plants (Matsuoka et al., 1995). Figure 5C shows that neither 15 nor 33 µm Wm had an effect on LR occurrence; however, both concentrations inhibited Sortin2-LR induction. Therefore, in plants growing in normal conditions, alterations of LE/PVC/MVB and vacuole trafficking were not relevant to LR formation. However, LE/PVC/MVB-vacuole trafficking was a prerequisite for the SCF TIR -independent LR initiation mechanism induced by Sortin2 (Figure 5C). DISCUSSION LR Initiation Independent of the Auxin Receptor SCF TIR It has been established that founder cell specification and LR initiation occur in a defined region of the main root pericycle layer where auxin level is critical (De Smet et al., 2007;

8 1202 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation Figure 5. Endomembrane Trafficking Impairment Alters Lateral Root Development. Seven-day-old A. thaliana (Col-0) seedlings were treated with different concentrations of Brefeldin A (BFA), Tyrphostin A23 (TyrA23), Wortmannin (Wm), and 25 μg ml 1 Sortin2 (S2). After 72 h, the number of LR was scored. White bars: treatment with trafficking-impairing drugs. Gray bars: treatments including Sortin2. Black bars: control treatment. After 72 h, number of LR was scored. Results were analyzed by Student s t-test (* p < 0.1, ** p < 0.05, *** p < 0.01, ns, not significant). Dubrovsky et al., 2008, 2011). This mechanism depends on both TIR1/AFB auxin perception and polar auxin transport (Dubrovsky et al., 2011). Genetically, we showed that the auxin receptor SCF TIR is not necessary for Sortin2-LR induction. Mutants in two different components of the SCF TIR, the quadruple mutant of the TIR1/AFB receptor and a mutant of the SCF TIR activator protein, were able to develop LR due to Sortin2. Furthermore, Sortin2 does not induce the downstream SCF TIR signaling pathway, since chemical treatment did not change AUX/IAA stability. The fact that, with Sortin2 treatment, the auxin-responsive promoter DR5 activity, which responds to the TIR1 pathway (Ulmasov et al., 1997b; Robert et al., 2010), was unaffected strongly supports that Sortin2 is not inducing the SCF TIR pathway. Therefore, Sortin2 induces LR initiation by a different molecular pathway led by a DR5-independent transcription program. The Sortin2 mechanism may occur by inhibiting the constitutive ARF transcriptional repression or by inducing an ARF-independent transcription (Figure 6A). LR development independent of TIR and ARF has been described; however, the underlying mechanism remains undetermined (Ditengou et al., 2008; Richter et al., 2009). Sortin2 may be triggering this mechanism, but this is difficult to determine without genetic tools. Therefore, Sortin2 is able to induce LR by a novel mechanism that depends on endomembrane trafficking. Whether Sortin2 mechanism is independent of auxin or auxin-dependant mechanism has to be resolved. Auxin induces endocytosis from plasma membrane to endosomes; however, there is no evidence of its effect on trafficking of later compartments. In addition to SCF TIR, auxin binding protein 1 (ABP1) is an auxin receptor that mediates auxin physiological responses (Jones et al., 1998; Chen et al., 2001; Tromas et al., 2009). We did not test whether Sortin2-induced LR requires ABP1, since no role in LR development has been reported for the ABP1-mediated pathway (Jones et al., 1998; Tromas et al., 2009; Robert et al., 2010; Xu et al., 2010). ABP1 loss of function produces defects in the growth of the main root; however, no alteration of LR occurrence is observed (Tromas et al., 2009). Inhibition of root growth or cutting the root tip may trigger LR (Torrey, 1950). However, there are examples where mutants with shorter main roots do not increase LR number (Geldner et al., 2004; Gendre et al., 2011) or even have less LR (Lucas et al., 2011). In addition, chemical root growth inhibition does not always induce LR increase (Rojas-Pierce et al., 2007; Robert et al., 2008; Rosado et al., 2011). Therefore, the effect of Sortin2 on triggering LR is unlikely to be caused by a pleiotropic effect on primary root growth. The fact that the normal alternate pattern of LR is altered by Sortin2 indicates stimulation of the LR initiation step instead of the emergence step, which is strongly supported by the increase of LR CYCB1;1 positive events. The LR alternation is established due to an inhibitory radial signal (De Smet et al., 2008). In A. thaliana, a key regulator of this process is ACR4, which encodes the membrane-localized receptor-like kinase ARABIDOPSIS CRINKLY4 (Gifford et al., 2005). The acr4 gene family loss-of-function mutants have more primordia initiated closer to one another and are often opposite (De Smet et al., 2008), resembling the effect of Sortin2. ACR4 is internalized to endosomes (Gifford et al., 2005); therefore, Sortin2 treatment could target it to the vacuole for degradation, explaining the increase of LR. However, ACR4 is also involved in LR emergence, because even though acr4 has more primordia, it has a lower density of emerged LR than

9 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation 1203 Figure 6. Lateral Root Initiation Pathways. (A) The canonical auxin receptor SCF TIR -dependent pathway is shown. SCF TIR binds auxin, inducing AUX/IAA degradation. Consequently, ARF proteins are activated to establish the gene transcriptional program leading LR initiation. In contrast, the SCF TIR -independent pathway relies on cellular endocytic trafficking. Trafficking of key proteins, towards the vacuole, triggers a signal to set the LR initiation. The activation of the gene transcriptional program may be executed by releasing the constitutive ARF inhibition. Alternatively, a signaling mechanism, independent of ARF proteins, could be induced by endocytic trafficking. Dotted lines indicate effects of Sortin2 and solid lines the auxin pathway. (B) Sortin2 impact on endocytic protein trafficking is shown. Sortin2 accelerates endocytic trafficking affecting mainly LE/PVC/MVB morphology and trafficking. Sortin2 induces a cellular pathway upstream of Wm and downstream of EE/TGN. Segmented and dotted arrows indicate the endocytic route and Sortin2-related processes, respectively. CW, cell wall; PM, plasma membrane; EE/TGN, early endosome/tgn; LE/PVC/MVB, late endosome/pvc/mvb; TyrA23, Thyrphostin A23; BFA, Brefeldin A; Wm, Wortmannin. the wild-type (De Smet et al., 2008). Therefore, if Sortin2 were affecting ACR4 function, the number of emerged LR would have diminished, contrary to what we observed. Alternatively, Sortin2-induced LR could be due to inhibition of an unidentified downstream component of the ACR4 pathway that only functions for LR radial inhibition. Sortin2, an Endocytic Trafficking Accelerator Consistently, by looking at compartment morphology combined with drugs of known effect, we found that the Sortin2 putative cellular target pathway is located downstream of the EE/TGN. Sortin2 induced trafficking towards the vacuole of the endocytic tracer FM4-64 as well as the PM/endosome recycling proteins PIN2 and BRI1. It is unlikely that this effect is due to increase of endocytosis from PM, since Sortin2 inhibited PIN2 accumulation into BFA bodies. Furthermore, PIN2 localized in BFA bodies traffics to the vacuole induced by Sortin2. The evidence indicates that Sortin2 mainly affects trafficking of LE/PVC/MVB, strongly supported by the larger size of LE/PVC/MVB observed in Sortin2 treatments. Since morphology of compartments is a consequence of dynamic trafficking back and forth, more abundant trafficking would increase the size of LE/PVC/ MVB. Larger LE/PVC/MVB are also observed by inhibiting trafficking from it to the vacuole using Wm (Jaillais et al., 2006). Furthermore, the effect of Sortin2 on CPY trafficking through the secretory pathway may be explained by its effect on LE/PVC/MVB trafficking. Alteration in LE/PVC/MVB trafficking causes mistargeting of the CPY receptor VPS10 in yeast, triggering secretion of CPY (Burda et al., 2002). Mislocalization of the AtCPY pathway receptor causes secretion of soluble cargos in plants (dasilva et al., 2005). Furthermore, the increase in membrane trafficking to the vacuole produced by Sortin2 correlates with the increase of the vacuole membrane in Sortin2-treated seedlings (Zouhar et al., 2004). Therefore, the Sortin2 effects on trafficking are consistent with perturbation of the secretory and endocytic pathways. Regarding its biological value, Sortin2 is a novel and distinct bioactive compound that activates a cellular and a physiological process, while the other abundant discovered drugs block or inhibit such processes. This compound is also very specific, affecting a restrictive set of cellular compartments. The power of using such a biological modulator was shown by our discovery of a mechanism for LR formation and its dependency on protein trafficking. Sortin2 induces a physiologically relevant

10 1204 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation trafficking route for environmental stimuli such as darkness and gravity changes (Kleine-Vehn et al., 2008; Laxmi et al., 2008). These attributes project Sortin2 as a powerful tool for further research at the cellular and molecular levels, as widely used drugs such as BFA, Latrunculin B, Wm, among others. LR Regulators and Endocytic Trafficking The PIN auxin transporters are responsible for auxin gradients, and thus for multiple plant responses (Friml et al., 2003). With Sortin2 treatment, PIN2 and perhaps other members of the PIN family as well as other proteins which follow the same pathway are internalized to the vacuole. Sortin2-PIN internalization may explain inhibition of root growth and impairment of gravitropism, because PIN loss-of-function mutants show these phenotypes (Chen et al., 1998; Friml et al., 2002; Benkova et al., 2003). Sortin2-induced LR could be a consequence of higher internalization of PIN, which affects PIN abundance at the plasma membrane. However, BFA treatment which captures PIN inside the cell inhibited LR occurrence in a dose-dependent manner, consistent with Geldner et al. (2001). Interestingly, inhibition of trafficking to the vacuole with Wm blocked Sortin2-induced LR. This strongly suggests that trafficking towards the vacuole is crucial for Sortin2 to induce LR initiation. Whether PIN2 or another member of the family needs to be internalized remains unknown. PIN1, the auxin transporter in pericycle cells is probably a suitable candidate. Recently, Ditengou et al. (2008) described PIN1 subcellular relocalization in the protoxylem as an earlier event for LR primordia by mechanical stimulation (Ditengou et al., 2008). PIN1 internalization is also seen where the new LR will develop; however, its localization and trafficking to vacuole has not been determined (Ditengou et al., 2008). However, the hormone cytokinins induce endocytic trafficking of PIN1 to vacuole which inhibits LR initiation (Laplaze et al., 2007; Marhavý et al., 2011). Therefore, regulation of LR would have differential regulation by molecular components. Our results showed that the effect of Sortin2 on endocytic trafficking is tightly linked to its effects on LR initiation. We propose that Sortin2 stimulation of LR initiation is based on the protein relocalization mediated by endocytic endomembrane trafficking (Figure 6). Endosomes are important sites for signaling initiated by receptors in the PM that traffic to intracellular compartments in animals, yeast, and plant cells (Irani and Russinova, 2009; Sadowski et al., 2009; Sallese et al., 2009). Regulators of developmental processes are located in different endosome populations where they may interact with different partners to convey a given signal in animals (Schenck et al., 2008; Sadowski et al., 2009). Interestingly, in signaling for the hormone brassinosteroid in plants, in addition to the localized plasma membrane, an endosomal pool of its receptor is essential for signaling (Geldner et al., 2007; Robert et al., 2008). The identity of the proteins that re-localize and their endosomal interactors in SCF TIR -independent signaling in regulating root organogenesis remain unknown, although PIN proteins are well suited to participate. These unidentified proteins may localize in PM and/or endosomal compartments. Alternatively, they may traffic within endosomes and to different destinations to signal. They may transduce signaling in endosomes by associating with certain partners proteins generating a kinase cascade as well as transcription factors trafficking to nuclei as described for endosome signaling in yeast and animals (Hayes et al., 2002; von Zastrow and Sorkin, 2007; Puria et al., 2008; Schenck et al., 2008; Dobrowolski and De Robertis, 2011). The plant body plan is the result of integrating intrinsic developmental programs with exogenous stimuli. For the plant endogenous LR developmental program, a mechanism has been established that includes oscillation of auxin concentration as well as the recently described gene transcription oscillation (De Smet et al., 2007; Moreno-Risueno et al., 2010). The role of Sortin2-induced SCF TIR -independent LR development in plant physiology should be evaluated further, since it may be an additional plant strategy to adapt to constantly changing environmental conditions. An auxin-independent LR development induced by mechanical stimulus has being described (Ditengou et al., 2008; Richter et al., 2009). Furthermore, bacterial quorum-sensing molecules have also been shown to induce LR through an auxin-independent mechanism (Ortíz-Castro et al., 2008). However, the dependency of these auxin-independent processes on protein trafficking has not been reported. It is possible that Sortin2-activated mechanism works in conditions where cells have to respond quickly to certain stimuli. Certain stimulus may activate a pathway mediated by signal molecules that traffic to endosome compartments to execute the signaling. These signaling molecules may interact with protein at endosomal compartments leading LR occurrence. Indeed, animals and yeasts use endosomal trafficking as part of signaling under certain conditions of growth factor induction and low nutrient availability (Puria et al., 2008; Schenck et al., 2008). There are examples of protein transducing signaling at endosomes by associating with certain partners; proteins generating kinase cascade like as well of transcription factors trafficking to nucleus (Puria et al., 2008; Schenck et al., 2008). Therefore, it is possible that plants have at least two different mechanisms for LR remodeling TIR-independent and TIR-dependent which are activated differentially depending upon the stimulus plants perceive. Remodeling root structure is a pivotal strategy to improve water and nutrient capturing; therefore, understanding the mechanism and discovering of key molecular players would be invaluable. METHODS Plant Material and Plant Growth Conditions The A. thaliana transgenic lines used in this study were previously described: VHA-a1:GFP (Dettmer et al., 2006), Ara7:GFP (Jaillais et al., 2006), SYP22:YFP (Robert et al., 2008), PIN2:GFP (Xu and Scheres, 2005), BRI1:GFP (Geldner et al., 2007), CYCB1;1::GUS (Ferreira et al., 1994a, 1994b), HS::AXR3NT-GUS

11 Pérez-Henríquez et al. Endocytic Trafficking on Lateral Root Formation 1205 (Gray et al., 2001), DR5::GUS (Ulmasov et al., 1997b), axr1-12 (Leyser et al., 1993; Lincoln et al., 1990), and the quadruple mutant tir/afb1/afb2/afb3 (Dharmasiri et al., 2005). Sterilized and stratified seeds of A. thaliana were sown in normal Murashige and Skoog (MS) culture media containing 0.44% MS (Phyto Tecnology Laboratories TM ) medium with 2% sucrose (Merck), 0.05%MES (ph 5.7), 0.01% myo-inositol, and 0.7% phytoagar (Phyto Technology Laboratories TM ). Seedlings were grown vertically in a culture chamber at 23 C in 16 h light/8 h dark photoperiod. Chemical Treatments Long-term (7 d) and short-term chemical treatments were done in solid and liquid MS culture media, respectively. Sortin2 (stock 20 mg ml 1 in DMSO, ChemBridge San Diego, CA) was added to the media using 1%DMSO as final solvent concentration in order to have a homogenous solution. Chemical treatments were performed supplementing MS culture media with cycloheximide (stock 50 mm in DMSO), Wortmannin (stock 30 mm in DMSO), Latrunculin B (stock 30 mm in DMSO), Brefeldin A (stock 5 mg ml 1, in ethanol), and Tyrphostin A23 (stock 30 mm in DMSO). Control treatments contained all the solvents at the concentration included in the corresponding chemical treatment. Auxin treatments were performed using 10 µm IAA (stock 100 mm), 1 µm 2,4D (stock 100 µm), and 1 µm NAA (stock 100 µm). For FM4-64 staining, seedlings were incubated at 4 C for 10 min with 5 µm FM4-64. Internalization of FM4-64 was allowed by returning seedlings to 25 C. Root and Lateral Root Scoring Phenotypes Roots were visualized using a bright-field compound microscope and a MVX10 MacroView microscope. Root images were obtained with a Leica MVX TV1XC camera. LR were quantified under a bright-field microscope. Measurements were performed using the software Macnification (Orbicule 2008). The lateral index is the number of LR per cm of main root. Reporter Lines Activity Histochemical β-glucuronidase (GUS) activity was evaluated following the protocol described in Norambuena et al. (2009). Seedlings were incubated with the substrate X-Gluc at 1 mg ml 1 (5-bromo-4-chloro-3-indolyl β-d-glucuronide cyclohexamine salt) in staining solution (100 mm NaHPO 4 buffer, ph 7.2; 10 mm EDTA, ph 8; 0.1%Triton X-100; 2 mm Potassium Ferrocyanide; 2 mm Potassium Ferricyanide), in darkness at 37 C for 48 h. The reaction was stopped by adding 70% ethanol. Quantification of GUS activity was performed with a fluorometric assay by incubating protein extracts with MUG (4-methylumbelliferyl-β-D-glucoronide) as substrate and measuring the fluorescence of MU (4-methyl-umbelliferone) from the hydrolysis of MUG. Protein extracts were obtained from about 15 7-day-old seedlings (30 mg of tissue). After chemical treatments, seedlings were homogenized with extraction buffer (50 mm phosphate buffer, ph 7.1; 1 mm EDTA; 0.1% Triton X-100; 0.1% SDS, and 5 mm DTT). After centrifugation at g, the supernatant was collected. GUS activity was carried with 1 mm MUG in extraction buffer and stopped with 0.2 M Na 2 CO 3. Fluorescence of the reaction product was measured with the microtitre plate fluorometer BioTek Synergy2. AXR3 stability was evaluated using the transgenic line HS::AXR3NT GUS which expresses AXR3 GUS under a heat shock promoter (Gray et al., 2001). Seven-day-old HS::AXR3NT GUS seedlings were incubated at 37 C to induce transgene expression. After 20 min at 22 C, seedlings were incubated with Sortin2, NAA, or control conditions for 20, 30, 60, and 80 min. Adding cold 70% acetone stopped treatments. After washing with water, seedlings were frozen with liquid nitrogen. GUS activity was quantified by the fluorometric assay. Microscopy Immunoelectron microscopy was performed as described in Sanderfoot et al. (1998). A. thaliana seeds were sown in Sortin2 and control MS culture media. After 7 d, immunogold-labeling was performed. Sections were incubated with purified antibodies raised against AtCPY (Rojo et al., 2003). Subcellular localization of fluorescent marker lines was performed using a confocal microscope. Imaging was performed in a Zeiss LSM 510 confocal microscope using the 488, 514, or 543-nm excitation filter and , , and LP560 emission filters for GFP, YFP, and FM4-64, respectively. Images were processed with Macnification (Orbicule 2008) and Imagej 1.40 (Wayne Rasband, NIH, USA). Gravitropic Assay Seven-day-old vertically grown seedlings were transferred to treatment plates. Seedlings were gravistimulated by turning plates 90 degrees. Seedlings were allowed to grow for 4 d and the bending angle was measured as described in Norambuena et al. (2009). PIN2:GFP differential degradation was evaluated after 3 h of the gravitropic stimulus. The GFP pattern in root tips was imaged by confocal microscopy and a false color code was applied using Imagej 1.40 (Wayne Rasband, NIH, USA). SUPPLEMENTARY DATA Supplementary Data are available at Molecular Plant Online. FUNDING This work was supported by Fondo Nacional de Desarrollo Tecnológico FONDECYT ( to P.P. and L.N.); Plant Cell Biotechnology-Millenium Nucleus (P F to P.P. and L.N.), and National Science Foundation (MCB to N.V.R.).