Arabidopsis ECHIDNA plays an important role in auxin regulation of clathrin-mediated trafficking during root gravitropism

In plant cells, clathrin and its adaptor protein complexes at the plasma membrane (PM) and/or trans -Golgi network/early endosome (TGN/EE) regulate clathrin-mediated endocytosis and post-Golgi trafficking, processes crucial for responses to many environmental cues. Studies show that phytohormone auxin differentially regulates clathrin light (CLC) and heavy chain (CHC) recruitment to establish the asymmetric distribution of the auxin efflux carrier PIN-FORMED2 (PIN2), promoting root gravitropic responses. The results showed that the loss-of-function of the TGN/EE component protein ECHIDNA (ECH) in Arabidopsis resulted in defective root gravitropism and impaired PIN2 trafficking. We found that levels of membrane-associated clathrin and its adaptor protein complexes AP-1, AP-2, and the TPLATE complex at the PM and/or TGN/EE were reduced in ech mutants. Furthermore, loss of ECH function disrupted the localization of the auxin receptor TRANSMEMBRANE KINASE1 (TMK1), thereby preventing auxin-induced modulation of CLC and CHC membrane association during root gravitropism. These findings suggest that ECH is crucial for the membrane localization of clathrin and its adaptor complexes, highlighting its critical role in clathrin-mediated trafficking and auxin-responsive regulation.

Vesicle trafficking is a crucial mechanism for regulating intracellular and extracellular transport in plants. The trans -Golgi network/early endosome (TGN/EE) functions as the central hub for cargo delivery and retrieval from the cell surface and sorting into vacuoles for degradation (Dettmer et al., 2006; Viotti et al., 2010; Scheuring et al., 2011; Gendre et al., 2015; Sparks et al., 2016). The ECHIDNA (ECH) protein, an evolutionarily conserved TGN/EE component, is crucial for maintaining its structural and functional integrity (Gendre et al., 2011). ECH is required for the secretion of auxin influx carrier AUXIN RESISTANT1 (AUX1) in apical hook epidermal cells, cell wall polysaccharides such as pectin and hemicellulose in seed coat cells, and for secretory trafficking during anther and pollen development (Boutté et al., 2013; Gendre et al., 2013; McFarlane et al., 2013; Fan et al., 2014). Beyond its roles in plant growth and development, evidence indicates that ECH also participates in regulating plant immunity and stress responses (Liu et al., 2023). However, the molecular mechanisms underlying ECH-mediated vesicle trafficking remain largely unknown. Clathrin triskelia, a vesicle coat protein complex composed of clathrin heavy (CHCs) and light chains (CLCs), localizes to the plasma membrane (PM) and TGN/EE in plants (Konopka et al., 2008; Ito et al., 2012; Wang et al., 2013a; Yan et al., 2021). Clathrin-mediated endocytosis (CME) is an evolutionally conserved pathway in which clathrin is recruited by the heterotetrameric ADAPTOR PROTEIN complex 2 (AP-2, composed of AP2α, AP2β, AP2μ, and AP2σ) and the ancient heterooctameric TPLATE complex (TPC) to internalize specific PM cargo. CME represents the predominant endocytic pathway in plants (Fan et al., 2013; Kim et al., 2013; Yamaoka et al., 2013; Gadeyne et al., 2014; Zhang et al., 2015; Johnson et al., 2021; Grones et al., 2022). Studies show that clathrin-dependent post-Golgi trafficking mediated by the heterotetrameric ADAPTOR PROTEIN complex 1 (AP-1, composed of AP1γ, AP1β, AP1μ, and AP1σ) at the TGN/EE, including secretion and vacuolar trafficking, is as crucial for plant growth and development as CME (Park et al., 2013; Wang et al., 2013a; Wang et al., 2013b; Wang et al., 2014; Yan et al., 2021). These clathrin-mediated trafficking (CMT) pathways are interdependent, with coordinated regulation of clathrin and adaptor protein complex recruitment at the PM and TGN/EE to maintain balance between endocytosis and exocytosis (Yan et al., 2021). The plant hormone auxin regulates membrane-associated clathrin and adaptor protein at the PM and TGN/EE to rapidly modulate CMT, thereby influencing the distribution of PM proteins, including the PIN-FORMED (PIN) auxin efflux carriers ( Paciorek et al., 2005; Wang et al., 2013a; Wang et al., 2016 ). PIN2-mediated basipetal auxin transport is crucial for the root gravitropic response ( Abas et al., 2006; Retzer et al., 2019; Han et al., 2021; Luschnig and Friml, 2024 ). Auxin inhibits PIN2 endocytosis by differentially regulating membrane-associated CLCs and CHCs in root tip epidermal cells on the lower side of the root, leading to asymmetric PIN2 distribution, enhanced auxin accumulation on that side, and the establishment of auxin asymmetric distribution during root gravitropic growth ( Wang et al., 2016; Wang et al., 2023 ). This study aims to investigate the roles of ECH in CMT and its function in root gravitropism using genetic and cytological approaches. The results show that ECH is required for the membrane association of clathrin and its adaptor protein at the PM and TGN/EE, and that it plays a critical role in auxin-mediated regulation of CMT.

Plant materials and growth conditions All Arabidopsis thaliana materials used in this study were authorized and derived from the Columbia-0 (Col-0) background. The following transgenic lines and mutants were used in this study: DR5pro:GFP (Benková et al., 2003), DR5pro:NLS-3×VENUS (Rodriguez-Villalon et al., 2015), PIN2pro:PIN2-GFP (Xu and Scheres, 2005), ECHpro:ECH–YFP (Gendre et al., 2011), 35Spro:CLC1-GFP (Wang et al., 2013a), RPS5Apro:CHC2-GFP (Ortiz-Morea et al., 2016), AP1μ2pro:AP1μ2-RFP (Wang et al., 2013b), 35Spro:AP1σ2-GFP (Yan et al., 2021), AP1/2β1pro: AP1/2β1-YFP (Liu et al., 2022), AP2μpro:AP2μ-YFP (Bashline et al., 2013), AP2σpro:AP2σ-GFP (Fan et al., 2013), 35Spro:TPLATE-GFP (Van Damme et al., 2006), TMLpro:TML-YFP (Gadeyne et al., 2014), 35Spro:DRP1A-GFP (Konopka and Bednarek, 2008), DRP1Cpro:DRP1C-GFP (Konopka et al., 2008), TMK1pro:TMK1-GFP (Xu et al., 2014), PIN2pro:HUB (this study), and ech (SAIL_163_E09; Gendre et al., 2011). The PIN2pro:HUB construct was generated in 1300 vector. The 1,669-bp PIN2 promoter region was cloned into 1300 using the HindIII and KpnI restriction sites. The HUB was inserted into 1300-PIN2pro at the KpnI restriction site, generating 1300-PIN2pro-HUB vector, which was subsequently transformed into Col-0. Arabidopsis thaliana plants are transformed using the floral dip method (Clough and Bent, 1998). Supplemental Table 1 lists the primer sequences used for cloning. The ech homozygous mutant lines (Gendre et al., 2011) were isolated and identified using a PCR-based assay. Fluorescently tagged marker lines were crossed into ech homozygous lines, which were confirmed based on their mutant phenotypes, genotyping PCR (Supplementary Table 1), and fluorescence. Seeds were surface-sterilized, stratified for 3 days at 4°C in the dark, and sown on 0.5× MS medium with 1.5% (w/v) agar unless otherwise specified. Seedlings were grown vertically on plates in a climate-controlled growth room at 22°C/20°C (day/night) with a 16 h/8 h light/dark photoperiod and 80 μE s −1 m −2 light intensity. Five-day-old seedlings with healthy roots were used in this study. Chemical solutions and treatments Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich. DMSO was used to dissolve CHX (50 mM) and BFA (50 mM) for stock solutions. 2,4-D (10 mM) and IAA (10 mM) were first dissolved in a few drops of 1 M KOH and then diluted with water. ddH 2 O was used to dissolve FM4-64 (2 mmol/L; Invitrogen) for stock solution. Unless otherwise indicated, final working concentrations were 2 μM for FM4-64, 10 μM for 2,4-D, 20 μM for IAA, and 50 μM for CHX and BFA. Treatment durations are indicated in the text or figure legends. All pretreatments and treatments for subcellular localization of membrane-associated proteins were performed in liquid medium (0.5× MS basal salts, 1% sucrose, and 0.05% MES [w/v], pH 5.6–5.8). All treatments were performed as previously described (Yan et al., 2021). The resulting seedlings were used in live-cell imaging and IF analyses. Immunofluorescence analysis and live-cell confocal microscopy Immunolocalization was performed as previously described (Wang et al., 2013a; Wang et al., 2016). All primary antibodies used for immunolocalization were detected using Cy3-labeled anti-rabbit secondary antibodies (1:100 dilution; Sigma-Aldrich, USA, Catalog No. C2306). Images were captured using a confocal laser scanning microscope (Leica TCS SP5 AOBS and Leica Stellaris 5). For Cy3 imaging, the 560 nm laser line was used for excitation, and emission was detected from 570–590 nm. For GFP/VENUS and YFP live-cell imaging, the 488- and 514-nm lines of the laser were used for excitation, and emission was detected from 496–535 and 520–560 nm, respectively. For FM4-64 and RFP imaging, the 594-nm laser line was used for excitation, and emission was detected from 615–660 nm. For quantitative fluorescence measurements, confocal microscope parameters (laser, pinhole, and gain settings) were identical among different treatments and genotypes. Fluorescence intensity at the PM or intracellular compartments was quantified from digital images of root epidermal cells using ImageJ (http://rsb.info.nih.gov/ij/). Further details of the quantification methods are provided in previous studies (Wang et al., 2013a; Wang et al., 2016; Yan et al., 2021). All experiments were independently performed at least three times, and statistical significance was evaluated using two-tailed paired Student’s t -tests. Statistical analyses were performed using Microsoft Excel 2019. Live-cell total internal reflection fluorescence microscopy Vertically grown seedlings were mounted on a glass slide with 0.5× MS liquid medium and observed using a total internal reflection fluorescence (TIRF) system on an Olympus IX-83 microscope equipped with a 100× oil-immersion objective (NA 1.49; Olympus). GFP-/YFP-fused proteins were excited with the 488 nm laser line. Detailed methods are provided in previous studies (Yan et al., 2021). Quantitative reverse transcription polymerase chain reaction assays TotalRNA was extracted using a RNeasy Plant Mini Kit (Qiagen). The RT-qPCR assay was performed with gene-specific primers (Supplementary Table 2) as previously described (Wang et al., 2016). Polyclonal antibodies Polyclonal antibodies were raised in rabbits using synthesized peptides for each protein (Supplemental Table 3) together with keyhole limpet hemocyanin with an additional N-terminal Cys (Huabio). All antibodies were prepared as previously described (Wang et al., 2013a; Wang et al., 2016; Yan et al., 2021) Immunoblot analysis Total protein fractions were prepared as previously described (Abas et al., 2006; Wang et al., 2016). For immunoblot analysis, the antibodies against CLC1 and CHC were used. Coomassie brilliant blue staining served as a loading control. All primary antibodies for immunoblotting were detected using anti-rabbit secondary antibody (1:50,000; Huabio) conjugated to horseradish peroxidase, detected via a chemiluminescent enhanced substrate kit (Thermo Scientific). ACCESSION NUMBERS Sequence data are available in the Arabidopsis Genome Initiative under the following accession numbers: ECH (At1g09330), PIN2 (At5g57090), CLC1 (At2g20760), CHC1 (At3g11130), CHC2 (At3g08530), AP1μ2 (At1g60780), AP1σ2 (At2g17380), AP1/2β1 (At4g11380), AP2σ (At1g47830), AP2μ (At5g46630), TPLATE (At3g01780), TML (At5g57460), DRP1A (At5g42080), DRP1C (At1g14830), UBIQUITIN7 (At2g35635), and TMK1 (At1g66150). Defective root gravitropism in ech mutants Seedlings of the ech mutant exhibited significant gravitropic defects in roots. Figure 1a shows that the roots of five-day-old vertically grown ech seedlings deviated from the gravity vector. Quantitative analysis revealed that the gravitropic index was lower in ech mutants than in the wild type (Figure 1b). To further assess gravitropic responses, a root bending assay was performed. ech mutant roots exhibited significantly less gravitropic bending at 2, 4, 6, and 8 h after seedling reorientation than in the wild type (Figure 1c,d). Basipetal auxin transport and asymmetric auxin distribution are crucial for root gravitropism (Han et al., 2021). To determine whether auxin asymmetric distribution is disrupted in ech mutants, auxin-responsive fluorescence was examined using the DR5:GFP (green fluorescent protein) and DR5:NLS-3 × VENUS (hereafter referred to as DR5:VENUS ) reporters using confocal laser scanning microscopy. Before vertical gravistimulation, the bottom/top fluorescence ratio of DR5-GFP and DR5-VENUS in root tip epidermal cells did not differ significantly between the wild type and ech mutants (Figure 1e,f). After 2 h of vertical gravistimulation, both reporters showed significantly higher bottom/top signal ratio in wild-type roots than in the ech mutants (Figure 1g,h). Collectively, these findings demonstrate that ECH contributes to root gravitropism by regulating asymmetric auxin distribution. Impaired PIN-FORMED 2 trafficking in ech mutants Upon gravistimulation, PIN2 endocytosis was inhibited in epidermal and cortical cells on the lower side of the root, but not on the upper side, thereby generating asymmetric PIN2 and auxin distribution (Pan et al., 2009; Wang et al., 2023). To further elucidate the basis of impaired gravitropism in ech mutants, PIN2-GFP distribution was examined during the root gravitropic response. In the absence of gravistimulation, the bottom/top PIN2-GFP fluorescence ratio did not differ between wild-type and ech roots (Figure 2a). However, following 2 h of gravistimulation, wild-type roots exhibited higher PIN2-GFP signals at the bottom than at the top, with no detectable differences in ech mutants (Figure 2b). However, ech mutants exhibited a significant increase in PIN2-GFP abundance at the PM compared to the wild type (Figure 2c). Although PIN2-GFP predominantly localized at the PM, ech mutants displayed additional PIN2-GFP signal in vacuolar ring-shaped membranes, which was not observed in the wild type (Figure 2c). These findings suggest that ECH is required for establishing PIN2 asymmetric distribution and proper subcellular localization. Studies show that CME facilitates the internalization of PM-resident PIN proteins and their polar localization (Dhonukshe et al., 2007; Kitakura et al., 2011; Wang et al., 2013a). To determine whether CME is compromised in ech mutants, PIN2-GFP internalization was assessed using the vesicle trafficking inhibitor Brefeldin A (BFA), which clusters late secretory pathway compartments into BFA bodies (Geldner et al., 2003; Wang et al., 2013a), and the de novo protein synthesis inhibitor cycloheximide (CHX). After 15 min of BFA treatment in the presence of CHX, ech mutant root cells displayed significantly fewer PIN2-GFP-labeled BFA bodies than those of wild-type cells, indicating impaired CME of PIN2-GFP in ech mutants (Figure 2d). Additionally, ech mutants exhibited delayed internalization of the lipophilic PM tracer dye FM4-64, further confirming that loss of ECH function impairs CME (Supplemental Figure 1). Following internalization, PM proteins are sorted at the TGN/EE and subsequently transported to vacuoles for degradation or recycled to the PM (Lam et al., 2007; Kleine-Vehn et al., 2008). Vacuolar accumulation of internalized GFP-fused PM proteins can be visualized in light-grown seedlings after dark incubation (Kleine-Vehn et al., 2008). After 2 h of dark treatment, ech mutants exhibited significantly reduced vacuolar accumulation of PIN2-GFP compared to the wild type (Figure 2e,f). These findings suggest that ECH is necessary for the intracellular trafficking of PIN2. Links between ECHIDNA and clathrin-mediated trafficking in Arabidopsis In plants, CMT dynamically regulates the polar localization and PM abundance of PIN2 via constitutive endocytic recycling and vacuolar trafficking for degradation (Dhonukshe et al., 2007; Kleine-Vehn et al., 2008; Kitakura et al., 2011; Wang et al., 2013a). Therefore, the potential relationship between ECH function and CMT was evaluated. Overexpression of the C-terminal fragment of CHC1 (HUB) acts as a dominant-negative form of clathrin by competing for CLC binding, thereby inhibiting endocytosis in plant cells (Dhonukshe et al., 2007; Robert et al., 2010; Kitakura et al., 2011). To investigate the role of CMT in PIN2-mediated gravitropism, the effect of HUB expression driven by the PIN2 promoter was examined. As expected, ProPIN2:HUB seedlings exhibited impaired root gravitropic response (Figure 3a,b), resembling the phenotype of ech mutant roots. Furthermore, co-localization of ECH with clathrin and AP-1 at the TGN/EE was assessed. Immunofluorescence (IF) microscopy using an affinity-purified anti-CLC1 antibody (Wang et al., 2013a) revealed partial co-localization of ECH-YFP with CLC1 at the TGN/EE (Figure 3c). Similarly, live-cell imaging showed that ECH-YFP partially colocalized with AP1μ2 tagged with red fluorescent protein (AP1μ2-RFP) in root epidermal cells (Figure 3d). These observations suggest a potential association between ECH and CMT at the TGN/EE. ECHIDNA is required for the membrane localization of clathrin-mediated trafficking components To determine whether ECH regulates the localization of core CMT components, the subcellular localization of CLC and CHC was first examined in ech mutant root cells. IF analysis using anti-CLC1 and anti-CHC antibodies revealed that loss of ECH reduced the levels of endogenous CLC1 and CHCs at the PM and TGN/EE (Figure 4a,b). Similarly, the membrane association of CLC1-GFP and CHC2-GFP was significantly reduced in the ech mutant compared to that in the wild type (Figure 4c,d). To further evaluate the effect of ECH on clathrin localization, TIRF microscopy was used to analyze the membrane association of CLC1-GFP and CHC2-GFP in wild-type and ech root epidermal cells. In accordance with the reduced clathrin abundance at the PM, the densities of foci containing CLC1-GFP and CHC2-GFP were significantly decreased in the ech mutant compared to the wild type (Figure 4e,f; small foci). The number of CLC1-GFP- and CHC2-GFP-labeled TGN/EE, which appear as larger structures upon entering the cortical TIRF imaging field, was also significantly reduced in ech mutant compared to wild-type cells (Figure 4e,f; large structure). Furthermore, internalized FM4-64 predominantly colocalized with CLC1-GFP in both genotypes (Supplemental Figure 2), indicating that loss of ECH did not alter clathrin localization at the TGN/EE. Previous results demonstrate that clathrin localization at the PM and TGN/EE requires AP-2/TPC and AP-1 recruitment, respectively (Wang et al., 2016; Yan et al., 2021). To determine whether ECH influences AP-2 localization at the PM, the subcellular localization of endogenous AP2σ/AP2μ and AP2σ-GFP/AP2μ-YFP was examined in the wild-type and ech mutant root cells by IF microscopy using affinity-purified anti-AP2σ/AP2μ-specific antibodies and live-cell imaging, respectively. PM-associated AP2σ, AP2μ, AP2σ-GFP, and AP2μ-YFP levels were reduced in the ech mutant compared to the wild type (Figure 5a–h). Similarly, PM-associated levels of the transgenically derived TPC subunits TPLATE-GFP and TML-YFP were also reduced in ech mutants (Figure 5i–l). These results were corroborated by quantitative TIRF microscopy (Figure 5m–p), which confirmed reduced levels of PM-associated AP-2 and TPC subunits in ech mutant seedlings. Beyond the AP-2 and TPC adaptor protein complexes, loss of ECH function also decreased the PM abundances of transgenically derived DYNAMIN-RELATED PROTEIN1 (DRP1) family members DRP1A-GFP and DRP1C-GFP (Supplemental Figure 3), which are crucial for CME (Konopka et al., 2008; Fujimoto et al., 2010). Furthermore, the association of fluorescent protein-tagged AP1σ2 (AP1σ2-GFP) and APμ2 (AP1μ2-RFP) with the TGN/EE was examined in wild-type and ech mutant. Compared to wild-type root cells, ech mutants exhibited reduced levels of TGN/EE-associated AP1σ2-GFP and AP1μ2-RFP (Figure 6a–d). The AP1/2β subunits, AP1/2β1 and AP1/2β2 function as shared components of the AP-1 and AP-2 complexes (Wang et al., 2016; Liu et al., 2022). The membrane association of AP1/2β1 subunit was assessed by quantitative live-cell and IF microscopy using an affinity-purified anti-AP1/2β1 antibody. In the ech mutants, PM- and TGN/EE-associated AP1/2β1-YFP and endogenous AP1/2β1 levels were reduced, relative to the wild type (Figure 6e–h). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis revealed that the reduced levels of these membrane-associated CMT components in ech mutants are not attributable to altered transcript levels (Supplemental Figure 4). Furthermore, total protein extracts showed no significant differences in CLC1 or CHC abundance between wild-type and ech mutant seedlings (Supplemental Figure 5). Collectively, these findings suggest that ECH regulates vesicle trafficking by influencing the recruitment of CMT components at the PM and TGN/EE. ECHIDNA is required for auxin regulation of membrane-associated clathrin Auxin differentially regulates membrane-associated CLCs and CHCs, which are required for auxin asymmetric distribution during gravitropism (Wang et al., 2016; Wang et al., 2023). To examine whether ECH functions are required for gravity-stimulation-induced changes in clathrin membrane abundance, we analyzed endogenous membrane-associated CLC1 and CHCs levels during the gravitropic response using IF microscopy. The vertically grown roots of wild-type and ech mutants showed comparable levels of membrane-associated CLC1 on both sides (Figure 7a–d). After 2 h of gravistimulation, the levels of PM- and TGN/EE-associated CLC1 at the lower side of wild-type roots were reduced compared with those at the top side (Figure 7e,f). No discernible difference in membrane-associated CLC1 was observed between the upper and lower sides of ech mutant roots after 2 h of gravistimulation (Figure 7g,h). Similarly, a decrease in CLC1-GFP signal at the lower side was observed after 2 h of gravistimulation in wild-type roots, whereas ech mutants showed a symmetrical distribution of CLC1-GFP between the upper and lower sides before and after gravity stimulation (Supplemental Figure 6). The bottom side signal of the PM- and TGN/EE-associated CHCs increased after 2 h of gravistimulation in wild-type roots, whereas ech mutant showed no significant difference in membrane-associated CHCs levels between the bottom and top sides, regardless of gravistimulation (Figure 7i–p). These data indicated that ECH is required for gravistimulation-induced asymmetric distribution of clathrin in root tips. To investigate whether auxin regulates clathrin association with the membranes dependent on ECH, we analyzed PM- and TGN/EE-associated CLC and CHC levels in wild-type and ech mutant root epidermal cells after a 30-min treatment with exogenous auxin 2,4-dichlorophenoxyacetic acid (2,4-D; 10 µM) and indole-3-acetic acid (IAA; 20 µM). Membrane-associated CLC1 levels decreased in wild type after treatment with 2,4-D or IAA relative to the mock controls, whereas no decrease was observed in ech mutants (Figure 8a–c). Similarly, live-cell imaging of CLC1-GFP revealed that exogenous auxin regulates the association of the CLC1 isoform with the PM and TGN/EE in wild-type but not in ech mutant root epidermal cells (Supplemental Figure 7). In contrast, PM- and TGN/EE-associated CHCs levels increased in wild-type roots after 30 min of auxin treatments (Figure 8d), consistent with previous observation (Wang et al., 2013a). Nevertheless, membrane-associated CHC levels did not differ significantly between mock- and auxin-treated ech mutant seedlings (Figure 8e,f). Together, these results showed that ECH is essential for auxin regulation of clathrin membrane association. Previously, Arabidopsis TRANSMEMBRANE KINASE1 (TMK1) was shown to mediate auxin signaling that regulates membrane-associated clathrin in root cells at the PM (Wang et al., 2023). To explore how the potential mechanism of ECH affects auxin-mediated clathrin recruitment, we analyzed TMK1-GFP localization in wild-type and ech mutant root cells. The PM intensity of TMK1-GFP in ech mutants was significantly higher than in wild-type cells. In addition to the PM, TMK1-GFP localized to vacuolar ring-shaped membranes in ech cells (Figure 8g–i), indicating that ECH is essential for TMK1 localization and function.

ECHIDNA-mediated intracellular trafficking Plant TGN functions as an EE and serves as a central hub for sorting and trafficking newly synthesized proteins and endocytic cargoes to the PM and vacuole (Dettmer et al., 2006; Viotti et al., 2010; Gendre et al., 2015). Arabidopsis ECH is essential for maintaining TGN/EE structure and function and mediates multiple vesicle trafficking pathways at the TGN/EE (Gendre et al., 2011). Substantial evidence indicates that ECH is involved in secretory trafficking from the TGN to PM (Gendre et al., 2011; Boutté et al., 2013; Gendre et al., 2013; McFarlane et al., 2013). Initially, ECH is thought not to affect the endocytic or vacuolar pathways (Gendre et al., 2011). However, studies have reported contradictory results, including defects in FM4-64 internalization and vacuolar protein sorting in ech mutants (Ravikumar et al., 2018; Ichino et al., 2020). Our results showed that loss of ECH inhibits FM4-64 internalization (Supplemental Figure 1). One potential reason for the discrepancies in FM4-64 experiments among studies is the variation in experimental methods and quantitative approaches used. Here, FM4-64 dye was first loaded into the PM of root cells at 4°C to inhibit endocytosis. After a 6-min incubation at 23°C, when endocytosis was active, the difference between the wild type and ech mutant was determined by the ratio of intracellular/PM FM4-64 signal (Supplemental Figure 1). Additionally, ech mutants shows increased levels of PIN2-GFP PM abundance, compared with wild type (Figure 2c). Given the constitutive endocytosis and recycling of PIN in plants (Dhonukshe et al., 2007; Kitakura et al., 2011), disruption of endocytosis and/or vacuolar trafficking likely causes PIN2-GFP accumulation at the PM of ech mutants. Therefore, we examined PIN2-GFP endocytosis and vacuolar trafficking using BFA and dark-treatment, respectively. ech mutants exhibit decreased PIN2-GFP accumulation in BFA bodies and vacuoles (Figure 2d,f), supporting a role for ECH in PM endocytosis and vacuolar protein trafficking. Mechanism by which ECHIDNA regulates clathrin-mediated trafficking Studies show an ECH/YIP/RABH secretory regulatory module at the TGN in Arabidopsis (Gendre et al., 2013; Baral et al., 2024). However, the molecular mechanisms by which ECH regulates vesicle trafficking in plants remain unclear. The transport pathway that depends on clathrin, adaptor proteins, and accessory proteins to form clathrin-coated vesicles is evolutionarily conserved (McMahon and Boucrot, 2011; Paez Valencia et al., 2016; Kaksonen and Roux, 2018; Dahhan et al., 2022). ech mutant cells exhibits no significant changes in clathrin subcellular localization at the TGN/EE (Boutté et al., 2013; Supplemental Figure 2). ECH loss-of-function significantly reduces CLC and CHC abundance at the TGN/EE (Figure 4), and RT-qPCR and immunoblot analyses indicate that this reduction results from decreased recruitment rather than changes in protein levels (Supplemental Figure 4; Supplemental Figure 5). Similarly, TGN/EE-associated levels of the AP-1 subunits, required for clathrin recruitment to the TGN/EE (Yan et al., 2021), are reduced in ech mutants (Figure 6). Studies show that defects in AP-1-dependent post-Golgi trafficking reduce the association of clathrin/AP-2/TPC/DRP1 with the PM, subsequently decreasing the internalization of PM cargo proteins through CME (Yan et al., 2021). All of the above CME core components exhibit reduced PM localization in ech mutants (Figure 5; Supplemental Figure 3), consistent with endocytosis defects in ech mutant root cells (Figure 2d; Supplemental Figure 1). Our results showed that loss-of-function of ECH significantly reduced the recruitment of clathrin, AP-1, AP-2, TPC, and DRP1 to the TGN/EE and PM. Based on ECH and CLC1/AP1μ2 partially colocalized at the TGN/EE (Figure 3c,d), ECH likely acts directly on clathrin/AP-1 to regulate their TGN/EE recruitment. Although the absence of ECH function does not prevent FM4-64 from reaching the TGN/EE (Supplemental Figure 2), the reduced TGN/EE localization of clathrin/AP-1 in ech mutants may still result from structural disruption of the TGN/EE (Gendre et al., 2011). Further investigation is required to elucidate the molecular mechanisms by which ECH regulates membrane-associated clathrin and CMT function in plants. Role of ECHIDNA in plant tropism In plants, tropism is an adaptive mechanism of directional growth in response to specific environmental cues, including gravity or light, with PIN-mediated polar auxin transport playing a critical role in tropic growth ( Ding et al., 2011; Wan et al., 2012; Han et al., 2021 ). Root gravitropism relies on the asymmetric auxin distribution generated by basipetal auxin transport in response to gravistimulation, with PIN2 playing a crucial role in this process ( Rashotte et al., 2000; Abas et al., 2006; Rahman et al., 2010 ). Hypocotyl phototropism allows seedlings to capture maximum light for photosynthesis, which depends on the establishment of asymmetric auxin distribution mediated by PIN3 ( Ding et al., 2011; Willige et al., 2013 ). Studies show that clathrin-mediated membrane trafficking is essential for PIN2 PM abundance and PIN3 polar localization, during gravitropism and phototropism, respectively ( Zhang et al., 2017; Wang et al., 2023 ). Root gravitropism defects are found in Arabidopsis ECH loss-of-function mutants ( Figure 1 ), similar to the clathrin light chain mutant clc2 clc3 ( Wang et al., 2013a ) and in transgenic lines expressing a dominant-negative form of CHC1 ( HUB ) under the PIN2 promoter ( Figure 3a,b ). Further studies show that clathrin adjusts its membrane abundance in the root tip in response to gravity stimuli, directing the formation of an asymmetric PIN2 distribution, which is impaired in ech mutants ( Figure 2; Figure 7; Supplemental Figure 6 ). Studies show that auxin regulates its polar transport by differentially modulating the PM and TGN/EE localization of CLC and CHC, thereby influencing CME to determine the PM abundance of PIN proteins ( Wang et al., 2013a; Wang et al., 2016 ). Live-cell and IF microscopy analyses reveal that exogenous auxin treatment rapidly decreases CLC membrane localization while enhancing CHC. The differential auxin regulation of CLC and CHC membrane association is abolished in ech mutant roots ( Figure 8; Supplemental Figure 7 ). Plant TMK1 interact with the auxin receptor ABP1 (AUXIN BINDING PROTEIN1) at the PM and transduces auxin signals to activate the ROP GTPases ROP2 and ROP6, thereby regulating CME ( Xu et al., 2014; Wang et al., 2023 ). In ech mutant, TMK1-GFP mislocalizes to vacuolar ring-shaped membranes, accompanied by a significant increase in PM-associated TMK1-GFP fluorescence signal intensity relative to wild-type root epidermal cells ( Figure 8g–i ), indicating that ECH is essential for TMK1-mediated auxin signaling to regulate membrane-associated clathrin dynamics. Hypocotyl phototropism, which depends on CMT regulation of PIN3 localization, may have defects in ech mutants. Although PIN3 subcellular localization in the apical hook of ech mutants does not show significant alterations, unlike the auxin influx carrier AUX1 ( Boutté et al., 2013 ), whether PIN3 exhibits normal lateral redistribution during phototropic bending of the hypocotyl remains unclear. These findings suggest that ECH is essential for CMT modulation by auxin and for regulating auxin distribution through properly localized PINs during tropic growth. However, the molecular mechanisms by which ECH mediates auxin regulation of clathrin and its accessory factors at the PM and TGN/EE remain to be elucidated. Recently, auxin has been shown to promote its efflux by directly activating TMK1 at the cell surface, leading to PIN1 and PIN2 phosphorylation ( Huang et al., 2025; Rodriguez et al., 2025 ). Therefore, the localization of TMK1 to affect the PM abundance of PIN2 may present another potential mechanism by which ECH regulates plant root gravitropism. ACKNOWLEDGMENTS We are grateful to Rishikesh P. Bhalerao for sharing published research materials that were important for this study. FUNDING This work was supported by a grant to X.Y. from the Shaoxing Science and Technology-driven Agriculture Special Program (No. 2024A12007); grants to C.W. from the National Natural Science Foundation of China (No. 32470744); grants to M.X. from the Natural Science Foundation of Zhejiang Province (No. LQ23C160005); grants to Y.W. from the Gansu Provincial Science and Technology Department (No. 24JRRE005) and Gansu Provincial Education Department (Higher Education Innovation Fund, No. 2024A-115). AUTHOR CONTRIBUTIONS M.X., Y.W., J.W., J.S., C.W., and X.Y. conceived the study and designed the experiments. M.X., Y.S., Y.W., C.Z., L.W., and X.Y. carried out the experiments. M.X., Y.S., C.Z., L.W., and X.Y. analyzed the data. M.X., J.S., C.W., and X.Y. wrote the article. CONFLICT OF INTEREST STATEMENT The authors declare no conflict of interest. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. FIGURE LEGENDS Figure 1. Loss of ECH function reduces root gravitropism. (a) Five-day-old vertically grown seedlings in wild type (WT) and ech mutants. (b) Quantitative analysis of the gravitropic index ( n = 24 roots). (c and d) Root bending degree in 5-d-old WT and ech seedlings after being gravistimulated with a 90° rotation and grown on solid half-strength MS medium for 0, 2, 4, 6, and 8 h thereafter ( n = 32-36 roots). (e-h) The expression analysis of DR5:GFP and DR5:VENUS for auxin distribution at the both sides of vertically grown roots (e and f) and 2-h gravistimulated roots (g and h) in WT and ech in the left panels. The right panels show the quantitative data for the relative intensity of the GFP signals at the both sides of the roots in the left panels ( n = 22-27 roots). Arrows indicate the gravity vector. Data are shown as means ± SD. *** P < 0.0001 (Student’s t -test; compared to the WT). Bars = 5 mm (a and d) or 50 μm (e to h). Figure 2. Impaired PIN2 dynamics in ech . (a and b) Live‐cell imaging of PIN2‐GFP distribution in the top and bottom sides of roots grown vertically for 0 (a) or 2 h (b) in wild type (WT) and ech in the left panels. The right panels show the quantification of the relative bottom/top GFP signal ratio ( n = 25-32 roots). (c) Confocal microscopy imaging of PIN2-GFP in 5‐d‐old WT and ech seedlings treated with CHX for 45 min in the left panels. The right panel shows the quantitative data for the relative intensity of the GFP signals at the PM in the left panels ( n = 137-155 cells from 10-12 roots each). (d) Confocal microscopy imaging of PIN2-GFP in 5‐d‐old WT and ech seedlings pretreated with CHX for 30 min followed by washout with CHX and BFA for 15 min in the left panels. The right panel shows the average number of PIN2-GFP-labeled BFA bodies ( n = 124-149 cells from 11-14 roots each). (e and f) Visualization of vacuolar accumulation of PIN2-GFP in dark-treated WT and ech mutant seedlings. The right panel shows the relative intensity of PIN2-GFP in vacuoles for each cell ( n = 115-130 cells from 12-15 roots each). Arrows indicate the gravity vector. White arrowheads and red arrowheads indicate the PIN2-GFP-labeled BFA bodies/vacuoles and mislocalization of PIN2-GFP, respectively. Data are shown as means ± SD. *** P < 0.0001 (Student’s t -test; compared to the WT). Bars = 50 μm (a and b) or 10 μm (c to f). Figure 3. Correlation analysis between ECH and clathrin-mediated trafficking. (a) Five-day-old vertically grown seedlings in wild type (WT) and ProPIN2-HUB transgenic line. (b) Quantitative analysis of the gravitropic index ( n = 30 roots). (c) Co-localization of ECH-YFP with Anti-CLC1. (d) Co-localization of ECH-YFP with AP1μ2-RFP. Bar = 10 μm. Co-localization was quantified based on their linear Pearson’s correlation coefficient ( r p ) and nonlinear Spearman’s rank correlation coefficient ( r s ), which are indicated in the images. A value of r = 1.0 indicates complete co-localization of two fluorescent signals. Data are shown as means ± SD. *** P < 0.0001 (Student’s t -test; compared to the WT). Bar = 5 mm (a) or 10 μm (c and d). Figure 4. Levels of membrane-associated clathrin are reduced in ech mutant root cells. (a and b) Immunofluorescence microscopy of CLC1 (a) and CHCs (b) in wild type (WT) and ech in the top panels. The bottom panels show the quantitative analysis of the relative intensities of CLC1 and CHCs at the PM and TGN/EE ( n = 76-93 cells from 10-12 roots each). (c and d) Confocal microscopy imaging of CLC1-GFP (c) and CHC2-GFP (d) in WT and ech in the top panels. The bottom panels show the quantitative analysis of the relative intensities of CLC1-GFP and CHC2-GFP at the PM and TGN/EE ( n = 194-223 cells from 18-20 roots each). (e and f) Total internal reflection fluorescence microscopy analysis of PM-associated foci of CLC1-GFP (e) and CHC2-GFP (f) in WT and ech in the top panels. The bottom panels show the quantitative analysis of the relative densities of PM-associated foci ( n = 69-82 cells from 21-25 roots each). Arrowheads show CLC1-GFP- and CHC2-GFP-labeled TGN/EE. Data are shown as means ± SD. *** P < 0.0001 (Student’s t -test; compared to the WT). Bars = 10 μm. Figure 5. Loss of PM-associated AP-2 and TPC in ech . (a-d) Immunofluorescence analysis of the AP2σ (a and b) and AP2μ (c and d) PM association in wild type (WT) and ech . (b and d) Relative fluorescent signal intensities of AP2σ and AP2μ at the PM ( n = 73-85 cells from 9-12 roots each). (e-l) Live-cell imaging analysis of the AP2σ-GFP (e and f), AP2μ-YFP (g and h), TPLATE-GFP (i and j), and TML-YFP (k and l) PM association in wild type (WT) and ech . (f, h, j, and l) Relative fluorescent signal intensities of AP2σ-GFP, AP2μ-YFP, TPLATE-GFP, and TML-YFP at the PM ( n = 292-384 cells from 18-21 roots each). (m-p) TIRF microscopy analysis of PM-associated foci of AP2μ-YFP (m and n) and TML-YFP (o and p) in WT and ech . (n and p) Relative densities of PM-associated foci of AP2μ-YFP and TML-YFP ( n = 54-69 cells from 16-22 roots each). Data are shown as means ± SD. *** P < 0.0001 (Student’s t -test; compared to the WT). Bars = 10 μm. Figure 6. Loss of membrane-associated AP-1 in ech mutants. (a-f) Live-cell imaging analysis of the AP1σ2-GFP (a and b), AP1μ2-RFP (c and d), and AP1/2β1-YFP (e and f) membrane-association in wild type (WT) and ech . (b, d, and f) Relative fluorescent signal intensities of AP1σ2-GFP, AP1μ2-RFP, and AP1/2β1-YFP at the TGN/EE and/or PM ( n = 131-189 cells from 14-17 roots each). (g and h) Immunofluorescence analysis of the AP1/2β1 PM and TGN/EE association in WT and ech . (h) Relative fluorescent signal intensities of AP1/2β1 at the TGN/EE and PM ( n = 76-81 cells from 9-10 roots each). Data are shown as means ± SD. *** P < 0.0001 (Student’s t -test; compared to the WT). Bars = 10 μm. Figure 7. Impaired asymmetric distribution of membrane‐associated CLC1 and CHCs during the gravitropic response in ech . (a-h) Immunofluorescence analysis of membrane‐associated CLC1 at the top and bottom sides of vertically grown (a-d) and 2-h gravistimulated (e-h) wild type (WT) and ech mutant roots. (b, d, f, and h) Relative intensity of membrane-associated CLC1 at both sides ( n = 186-216 cells from 21-24 roots each). (i-p) Immunofluorescence analysis of membrane‐associated CHCs at the top and bottom sides of vertically grown (i-l) and 2-h gravistimulated (m-p) WT and ech mutant roots. (j, l, n, and p) Relative intensity of membrane-associated CHCs at both sides ( n = 153-190 cells from 18-20 roots each). Arrows indicate the gravity vector. Data are shown as means ± SD. *** P < 0.0001 (Student’s t -test; compared to the WT). Bars = 15 μm. Figure 8. ECH is required for TMK1-mediated auxin differential regulation of CLC1 and CHCs association with the membranes. (a and b) Immunofluorescence microscopy of membrane‐associated CLC1 in wild type (WT) and ech mutant root cells. (c) Relative fluorescent signal intensities of CLC1 at the PM and TGN/EE ( n = 122-163 cells from 14-17 roots each). (d and e) Immunofluorescence microscopy of membrane‐associated CHCs in WT and ech mutant root cells. (f) Relative fluorescent signal intensities of CHCs at the PM and TGN/EE ( n = 105-132 cells from 13-15 roots each). (g and h) Confocal microscopy imaging of the subcellular distribution of TMK1-GFP in WT and ech mutant root cells. The white dotted box indicates the area that is 2×magnified on the right panel. (i) Relative fluorescent signal intensities of TMK1-GFP at the PM ( n = 145-180 cells from 17-19 roots each). Treatments were performed with DMSO (mock), 2,4-D (10 μM), and IAA (20 μM) for 30 min in the 5-day-old seedlings. Arrowheads show mislocalization of TMK1-GFP. Data are shown as means ± SD. *** P < 0.0001 (Student’s t -test; compared to the WT). Bars = 10 μm. Supplementary data Supplementary Figure 1. Loss of ECH function impairs FM4-64 internalization in root cells. Supplementary Figure 2. Co-localization analysis of internalized FM4-64 and CLC1-GFP at the TGN/EE in ech . Supplementary Figure 3. Loss of membrane-associated DRP1A-GFP and DRP1C-GFP in ech . Supplementary Figure 4. Transcript levels of core components of clathrin-mediated trafficking machinery in ech . Supplementary Figure 5. CLC1 and CHC protein levels in ech . Supplementary Figure 6. Impaired asymmetric distribution of membrane-associated CLC1-GFP during the gravitropic response in ech . Supplementary Figure 7. Auxin - dependent regulation of CLC1-GFP membrane association requires ECH function. Supplementary Table 1. PCR primer sequences for genotyping and cloning. Supplementary Table 2. RT-qPCR primer sequences. Supplementary Table 3. Antibody information.

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Authors Metrics & Citations Metrics Article Usage 306views 52downloads Citations Download citation Mei Xu, Yonghua Shao, Yutong Wang, et al. Arabidopsis ECHIDNA plays an important role in auxin regulation of clathrin-mediated trafficking during root gravitropism. Authorea. 11 November 2025. DOI: https://doi.org/10.22541/au.176286338.86740787/v1 DOI: https://doi.org/10.22541/au.176286338.86740787/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.