Polar‐localized EXO70G1 regulates root development in Arabidopsis thaliana

ABSTRACT

Cellular asymmetry, which represents a fundamental characteristic of cell polarity, is prominently illustrated by the apical‐basal localization of PIN‐FORMED (PIN) auxin efflux carriers in Arabidopsis thaliana. Although the maintenance of PIN polarity at the plasma membrane (PM) relies on endomembrane trafficking, the pivotal factors responsible for recruiting PIN proteins to the PM remain largely unknown. In this study, we discover that EXO70G1 displays a polarized distribution at the PM in root cells. Acting as a putative subunit of the exocyst complex, which mediates the tethering of exocytic vesicles to the PM, EXO70G1 exhibits continuous recycling foci at the PM, and its dynamic behavior is akin to that of SEC6 and SEC8. Disruption of EXO70G1 and its homolog EXO70G2 in Arabidopsis reduces auxin accumulation and primary root length. Importantly, the recycling of PIN2 from the brefeldin A (BFA) compartment to the PM is compromised, and the abundance of PIN2 at the PM is reduced in the exo70G1 exo70G2 backgrounds. Interestingly, live‐cell imaging reveals that the polarity of EXO70G1 is established during cytokinesis, prior to that of PIN2, and is maintained throughout the subsequent phases of cell elongation and differentiation. When the lipid raft was disturbed, the accumulation of EXO70G1 at the PM decreased. Our findings highlight the crucial role of EXO70G1 in root development by providing positional cues that facilitate the recycling efficiency of PIN2 to the PM.

The putative exocyst subunit EXO70G1 exhibits apical‐basal polarity in Arabidopsis root cells; this polarity stabilizes membrane domains to facilitate targeting of the auxin transporter PIN‐FORMED2 during root development.

INTRODUCTION

Cell polarity, manifested by the asymmetric distribution of cellular components such as plasma membrane (PM)‐associated proteins, is a fundamental biological phenomenon. The establishment of this polarity critically depends on polarized intracellular trafficking. While numerous polarly localized PM proteins have been identified, the regulators orchestrating their polar trafficking remain largely unexplored, particularly in plants (Ma et al., 2025). A prime example of functional polarity in plants is the establishment of precise auxin gradients is vital for plant organogenesis (Vanneste and Friml, 2009), and coordinated auxin transport systems contribute to the formation of auxin maxima within the root tip (Benkova et al., 2003; Wisniewska et al., 2006). The PIN‐FORMED (PIN) proteins, known for their asymmetric distribution at the PM, play a pivotal role in facilitating spring‐like auxin flow in roots, alongside auxin influx carrier/like auxin resistant (AUX/LAX) proteins and ATP‐binding cassette B (ABCB) transporters (Band et al., 2014). Various PIN proteins exhibit distinct cell‐specific subcellular distribution patterns in roots, for instance, PIN1 is found on the basal membrane of stele cells, while PIN2 localizes to the apical membrane of epidermal cells and the basal membrane of cortex cells (Blilou et al., 2005; Wisniewska et al., 2006). Loss‐of‐function of PIN protein results in defects in stem cell maintenance, cell division, elongation, and abnormal gravity responses in roots (Blilou et al., 2005). Additionally, the expression of key regulators, including WUSCHEL‐RELATED HOMEOBOX 5 (WOX5), PLETHORAs (PLTs), and SCARECROW (SCR), becomes abnormal when auxin distribution is disrupted (Blilou et al., 2005).

The dynamic polarity of PIN proteins is intricately regulated by multiple mechanisms, including membrane trafficking, phosphorylation, transcriptional control, and cellular factors such as membrane lipids, the cytoskeleton, the cell wall, and cell wall–PM connection (Dhonukshe et al., 2007; Kleine‐Vehn and Friml, 2008; Armengot et al., 2016; Li et al., 2021). It is well established that endomembrane trafficking, encompassing processes of clathrin‐mediated endocytosis (CME), recycling, and trans‐cytosis, plays a crucial role in the polarity maintenance of PIN proteins at the PM (Kleine‐Vehn et al., 2011). PIN proteins at the PM enter the cytoplasm via CME, where they either degrade in the vacuole or recycle back to the PM (Yang et al., 2020). Numerous regulators have been identified to govern different aspects of intracellular trafficking. For example, Rho‐like guanosine triphosphate (GTP) binding protein 6 (ROP6) and its downstream effector ROP‐interactive crib motif‐containing protein 1 (RIC1) regulate endocytosis of PIN proteins (Chen et al., 2012). ROP3 is required for the polar localization of PINs and affects the trafficking of PIN1 and PIN3 in roots (Huang et al., 2014). ARF1A1C and RabA1b play a pivotal role in the recycling and trafficking of PIN proteins from the trans‐Golgi network/early endosome (TGN/EE) to the PM (Feraru et al., 2012; Tanaka et al., 2014). The inactivation of small GTPases results in the altered accumulation and abundance of PIN proteins at the PM. While numerous regulators involved in various aspects of intracellular trafficking have been identified, they do not exhibit obvious polarized localization at the PM. Therefore, the regulators specifically governing the polar trafficking of PIN proteins warrant further investigation.

Endomembrane trafficking involves consecutive cytological events, including vesicle budding, movement, tethering/docking, and membrane fusion. Tethering is critical for directing exocytic vesicles to their target membrane, and the exocyst complex is one of tethering complex mediating this progress in yeast, animals, and plants (He et al., 2007; He and Guo, 2009; Mei and Guo, 2019; Žárský, 2022). Comprising eight subunits (SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84), the exocyst facilitates the delivery of PIN proteins from endomembrane compartments to the PM (Drdová et al., 2013; Tan et al., 2016). Mutation in the single‐copy gene of SEC6 has been shown to lead to an accumulation of exocytic vesicles adjacent to the PM in Arabidopsis roots (Tan et al., 2016), indicating that there is an impairment in the recycling process of PIN proteins to the PM. The EXO70 family in Arabidopsis consists of 23 paralogous genes derived from three subfamilies (EXO70.1, EXO70.2, EXO70.3), exhibiting highly tissue‐ and cell‐type‐specific expression patterns (Li et al., 2010). EXO70A1 (EXO70.1 subfamily) was the first characterized member involved in PINs recycling (Drdová et al., 2013), and EXO70A3 (also EXO70.1) modulates PIN4 exocytosis to regulate root architecture (Ogura et al., 2019). Although the exocyst mediates trafficking of PIN proteins to the PM, both EXO70A1 and SEC6 localize preferentially to the lateral PM in root cells (Fendrych et al., 2013). Indeed, no EXO70 isoforms have been reported to exhibit apical‐basal polarity like PINs in these cells. Meanwhile, phylogenetic analysis reveals that the EXO70.3 subfamily—including EXO70G1 and EXO70G2—shares a common evolutionary branch with the EXO70.1 subfamily, yet its functional significance remains to be investigated (Marković et al., 2021).

In this study, we present cytological, pharmacological, and genetic evidence demonstrating that EXO70G1 polarizes to transverse PM domains throughout the root. The trafficking of EXO70G1 to the PM is cytoskeleton‐independent, while its accumulation at the PM depends on lipid raft density. EXO70G1 and its paralog EXO70G2 facilitate PIN2 recycling to the PM redundantly in primary root elongation. Taken together, we propose that EXO70G1 serves as a spatial landmark to define or stabilize membrane domains for PIN2 recycling, rather than acting as a direct delivery machinery, thus linking its function to root development.

RESULTS

EXO70G1 predominately localizes to the transverse PM domains of the root

Consistent with the root meristem‐enriched expression of pEXO70G1::GUS (Li et al., 2010), we generated an EXO70G1‐GFP (green fluorescent protein) fusion protein under its native promoter to examine subcellular localization. Initially, we conducted a growth assay on the exo70G1 single mutant to characterize the gene function of EXO70G1 in root development. Following vertical growth on half‐strength Murashige and Skoog (1/2MS) medium for 5 or 7 d, no significant difference in root length was observed between the exo70G1 single mutant and wild‐type (WT) Arabidopsis thaliana (Col‐0). Given that the EXO70.3 subfamily contains two members sharing 39.6% protein homology (Li et al., 2010), we presumed that EXO70G1 and EXO70G2 might function redundantly. Indeed, exo70G1 exo70G2 double mutant exhibited significantly reduced root length compared to Col‐0 (Figure S1A–D). Importantly, this phenotype was fully rescued by a pEXO70G1::EXO70G1‐GFP or pEXO70G2::EXO70G2‐GFP transgene in the double mutant background (Figure S1E–H; hereafter EXO70G1‐RS or EXO70G2‐RS), demonstrating the functionality of the EXO70G1‐GFP and EXO70G2‐GFP fusion protein.

To investigate EXO70G1 expression pattern at the PM, we employed the pEXO70G1::EXO70G1‐GFP complementation line. Initial characterization in roots revealed that EXO70G1 expressed throughout all cell types within the meristematic zone, including epidermal, cortical, and endodermal layers, as well as the stele (Figure 1A). Further line scan analyses identified that the EXO70G1‐GFP co‐localized with FM4‐64 (styryl dye N‐(3‐triethylammoniumpropyl)‐4‐(6‐(4‐(diethylamino) phenyl) hexatrienyl) pyridinium dibromide) (Figure 1A, B), suggesting its apparent membrane localization. Moreover, in epidermal cells, the density of the EXO70G1‐GFP signal on the apical and basal membrane was higher than that of the lateral membrane, and the EXO70G1‐GFP appears as punctate foci on the membrane (Figure 1C). Moreover, EXO70G1‐GFP signal did not co‐localize with FM4‐64‐labeled endocytic vesicles in the cytoplasm (Figure 1C). A plasmolysis experiment was conducted to discriminate the apical or basal membrane localization of EXO70G1. The EXO70G1‐GFP signal was expressed near the propidium iodide (PI) counterstained cell wall, like a sandwich, confirming its polarized localization (Figure 1D). To further distinguish between PM and cell wall localization, we co‐stained cells with Calcofluor White (a specific cell wall dye) and observed that the EXO70G1‐GFP fluorescence signal localized to the PM, immediately adjacent to the Calcofluor White signal (Figure S2A). Furthermore, the distribution of EXO70G1 signal at the elongation zone was also polarized (Figure S2B). The findings illustrated that the distribution of EXO70G1 was asymmetric at the PM and predominantly localized at the apical‐basal PM domains.

Figure 1.

EXO70G1 polarizes at the PM

(A) Co‐localization analysis between membrane dye FM4‐64 and EXO70G1‐GFP (green fluorescent protein) in the meristem zone of Arabidopsis roots. Scale bars: 20 μm. (B) Line scan assessment between FM4‐64 and EXO70G1‐GFP in the cortex cells, white line in (A). (C) Distribution of endocytosis vesicles and EXO70G1‐GFP in epidermal cells. Scale bars: 5 μm. (D) Distribution pattern of EXO70G1‐GFP in epidermal cells following treatment with 30% sucrose for 1 h. Root meristems were stained with 10 μg/mL propidium iodide (PI). Scale bars: 5 μm.

To identify the subcellular distribution of EXO70G1 in the cytoplasm, we performed co‐localization analysis between EXO70G1‐GFP and a panel of fluorescent protein markers for distinct endomembrane compartments, including Golgi, endoplasmic reticulum (ER), and TGN, prevacuolar compartment (PVC), late endosome (LE), and EE markers, respectively (Geldner et al., 2009; Li et al., 2017). No obvious co‐localization was detected between EXO70G1‐GFP and the Golgi marker SYP32‐RFP (red fluorescent protein), the ER marker RabD2b‐RFP, the TGN marker VHAa1‐ATPase‐mRFP and the PVC marker RFP‐ARA7, the EE marker RabA1g‐RFP, and the LE marker RabG3f‐RFP (Figure S3). These results indicate that the delivery or recruitment of EXO70G1 to the PM might not be through these structures.

To examine the dynamic behavior of EXO70G1 at the PM, we conducted a fluorescence recovery after photobleaching (FRAP) experiment. Twenty percent fluorescence recovery occurred faster at apical/basal membranes (18.5 s) compared to lateral membranes (25.7 s) (Figure 2A, B), indicating that cytoplasmic EXO70G1 was preferentially transported to the apical/basal membrane instead of the lateral membrane. To determine whether brefeldin A (BFA) affects the subcellular localization of EXO70G1, we performed a BFA washout assay. When treated with 50 μM BFA, EXO70G1 was not co‐localized with FM4‐64 labeled BFA bodies, and the BFA bodies disappeared after 1/2MS wash out for 60 min, as anticipated (Figure S4A–D). Moreover, the recovery time and polarity index (the rate of fluorescence intensity of the apical membrane to that of lateral membrane) of EXO70G1 did not change following BFA treatment (Figure 2C–E). Quantification confirmed EXO70G1's BFA resistance, aligning with canonical exocyst subunit responses (Drdová et al., 2013; Fendrych et al., 2013). Next, we assessed whether the cytoskeleton influences the polarity of EXO70G1. The microfilament depolymerizer latrunculin B (LatB), and microtubule depolymerizer (oryzalin), had no influence on the recovery of EXO70G1 during FRAP (Figure 2F, G, I, J). Additionally, the polarity index of EXO70G1 under LatB and oryzalin treatment was similar to dimethyl sulfoxide (DMSO) treatment (Figure 2H, K). Thus, we conclude that EXO70G1 targeting to the PM and its polar localization are BFA insensitive and cytoskeleton‐independent.

Figure 2.

Dynamics analysis of EXO70G1 at the PM

(A, B) Recovery curves of EXO70G1 at the apical/basal membrane and lateral membrane in the meristem zone of Arabidopsis roots following photobleaching; more than 15 cells in different roots were analyzed. (C) Recovery curve of EXO70G1‐GFP following dimethyl sulfoxide (DMSO) and brefeldin A (BFA) treatment. (D, E) Recovery time and polarity index of EXO70G1‐GFP following DMSO and BFA treatment (n = 68 cells from 10 roots, 76 cells, from 12 roots, respectively). (F) Recovery curve of EXO70G1‐GFP following DMSO and latrunculin B (LatB) treatment. (G, H) Recovery time and polarity index of EXO70G1‐GFP following DMSO and LatB treatment (n = 54 cells from 9 roots, 63 cells, from 13 roots, respectively). (I) Recovery curve of EXO70G1‐GFP after DMSO and Oryzalin treatment. (J, K) Recovery time and polarity index of EXO70G1‐GFP following DMSO and Oryzalin treatment (n = 48 cells from 9 roots, 55 cells from 11 roots, respectively).

As one exocyst complex subunit, we further analyzed the co‐localization between EXO70G1 and the exocyst complex subunits SEC6/8 in epidermal cells. Along the apical/basal membrane, EXO70G1 co‐localized with the exocyst subunit SEC6/8 at the PM (Figures 3A, S5A). However, the GFP signals of SEC6/8 were focused on the outer lateral membrane, which contributed to the polarity index of SEC6/8 being lower than that of EXO70G1 (Figures 3B, S5B), and polarity index of the inner and outer lateral membrane differed in SEC6/8 (Figures 3B, S5B). To determine if EXO70G1 function depends on the exocyst complex, we quantified co‐localization and dynamics of EXO70G1 and SEC6/8 using variable angle total internal reflection microscope (VEAM) in epidermal cells at the elongation zone. We observed that EXO70G1 and SEC6/8 showed overlapping trajectories (Figures 3C, D, S5C, D) with similar velocities during time‐lapse imaging (Figures 3E, S5E). The co‐localization levels between EXO70G1 and SEC6/8 were 40.4% and 34.3%, respectively (Figures 3F, S5F). The findings above indicate that SEC6/8 is enriched at lateral PM domains, while EXO70G1 polarizes at the apical/basal PM. Despite differential abundance at the PM, EXO70G1 and SEC6/8 maintain co‐localization with similar dynamics, supporting functional integration of EXO70G1 within the exocyst complex.

Figure 3.

Co‐localization of EXO70G1 and SEC8 in epidermal cells of Arabidopsis roots

(A) Co‐localization analysis between EXO70G1‐tdTomato and SEC8‐GFP in epidermal cells of the meristem zone. Scale bars: 5 μm. (B) The polarity index of EXO70G1‐tdTomato and SEC8‐GFP. AP indicates the apical PM, while ILP and OLP denote the inner and outer lateral PM, respectively. (C) Co‐localization analysis between EXO70G1‐tdTomato and SEC8‐GFP in epidermal cells of the elongation zone when observed using variable angle total internal reflection microscope (VAEM). Scale bars: 10 μm. (D) A kymographic representation of EXO70G1‐tdTomato and SEC8‐GFP co‐localization in exocyst foci. The horizontal and vertical arrowheads represent 22 μm and 4 s, respectively. (E) Velocity of the EXO70G1 and SEC8 foci in epidermal cells. (F) Quantification (%) of the EXO70G1 and SEC8 co‐localization in Arabidopsis root epidermal cells. Pairs denoted in (C) were examined. Green, red, and yellow colors represent GFP only, monomeric tdTomato only, and co‐localization, respectively.

EXO70G1 and EXO70G2 redundantly promote root growth

The transgene line of EXO70G1‐RS rescued the root elongation defect in exo70G1 exo70G2 mutants (Figure S1E, F). Additionally, p35S::EXO70G1 overexpression lines (5‐9 and 9‐8) showed significantly increased root length compared to Col‐0 (Figures 4A, B, S6). These results collectively demonstrate EXO70G1's essential function in root growth regulation. Since cell division and elongation contribute to root growth (Perilli et al., 2012; Di Mambro and Sabatini, 2018), we examined the cell length and number in the roots of the exo70G1 exo70G2 double mutant. Compared to Col‐0, both cell length and cell number in the meristem zone were reduced in the double mutant (Figure 4C–E). To further characterize the change of cell number in the meristem zone, the cell division marker pCYCB1;1::GUS was introduced into the exo70G1 exo70G2 double mutant background. Consistently, the β‐glucuronidase (GUS) signal was reduced in the exo70G1 exo70G2 double mutant (Figure 4F). No difference was observed in the size of the elongation zone and epidermal cell length in the mature zone (Figure 4G, H). In addition, SCR expression was reduced in the exo70G1 exo70G2 double mutant, whereas PLT expression showed no significant difference between Col‐0 and the double mutant (Aida et al., 2004; Figure S7). These findings demonstrated that EXO70G1 and EXO70G2 redundantly promote cell division of the meristem zone in root growth.

Figure 4.

EXO70G1 and EXO70G2 promote primary root elongation

(A) Seedlings of Col‐0, exo70G1 exo70G2, p35S::EXO70G1 9‐8, and EXO70G1‐RS 3‐1 grown on half‐strength Murashige and Skoog (1/2MS) vertically for 5 d. Scale bars: 1 cm. (B) Quantitation of root length of 5‐d‐old seedlings (n = 27, 26, 28, 32, respectively). (C) Meristem zones of 5‐d‐old Col‐0 and exo70G1 exo70G2. Root meristems were stained in 10 μg/mL propidium iodide. Scale bars: 20 μm. (D, E) Root meristem length and cell number in the meristem zone (n = 35, 38, respectively). (F) β‐glucuronidase (GUS) staining illustrates the expression pattern of pCYCB1;1::GUS in Col‐0 and exo70G1 exo70G2. Scale bars: 20 μm. (G, H) Length of elongation zone and epidermal cell length of 5‐d‐old Col‐0 (n = 56 cells from 16 roots) and exo70G1 exo70G2 (n = 63 cells from 17 roots). Data represent mean ± SD, ** and *** represent means that differ at P < 0.01 and 0.001, respectively (Student's t‐test).

EXO70G1 co‐localizes with PIN2 at the PM

Given that the polar localization of EXO70G1 strongly overlapped with that of PIN proteins and that PIN2 is ubiquitously expressed and polar‐localized in epidermal cells of Arabidopsis roots, we introduced EXO70G1‐tdTomato into PIN2‐GFP to examine their co‐localization. Line scan analysis showed that EXO70G1‐tdTomato and PIN2‐GFP co‐localized in the root epidermal cells, and the peaks of the GFP signal and the tdTomato signal overlapped (Figure 5A, B). Both EXO70G1‐tdTomato and PIN2‐GFP were observed as punctate foci at the PM, and their puncta strongly co‐localized (Figure 5C–F). Moreover, EXO70G1 was co‐expressed with PIN2 at the basal membrane in the cortex cells (Figure 5G–I). To assess the details of EXO70G1 and PIN2 at the PM, Z‐axis stacking scans revealed significant overlap between distinct PIN2 clusters and EXO70G1 signals on the apical membrane (Figure 5J, K). These results revealed that the distribution pattern of EXO70G1 at the PM was highly similar to that of PIN2.

Figure 5.

EXO70G1 co‐localizes with PIN2 at the PM

(A) Co‐localization analysis between EXO70G1‐tdTomato and PIN2‐GFP in epidermal cells of Arabidopsis roots. Scale bars: 5 μm. (B) Line scan analysis between EXO70G1‐tdTomato and PIN2‐GFP in epidermal cells, indicated by the white line in (A). (C) Co‐localization analysis between EXO70G1 and PIN2 with Pearson coefficient. (D) Co‐localization analysis between EXO70G1‐tdTomato and PIN2‐GFP at the apical membrane of epidermal cells in Arabidopsis roots. Scale bars: 5 μm. (E) Line scan analysis of EXO70G1‐tdTomato and PIN2‐GFP in (D). (F) Co‐localization analysis between EXO70G1 and PIN2 with Mander's coefficient. (G) Co‐localization analysis between EXO70G1‐tdTomato and PIN2‐GFP at the basal membrane of cortex cells in Arabidopsis roots. End, endodermis cells. Cor, cortex cells. Scale bars: 10 μm. (H) Line scan analysis of EXO70G1‐tdTomato and PIN2‐GFP in cortex cells. (I) Line scan analysis of EXO70G1‐tdTomato and PIN2‐GFP in the basal membrane of cortex cells. (J) Vertical view of EXO70G1‐tdTomato and PIN2‐GFP from Z‐stack analysis. Scale bars: 1 μm. (K) Line scan analysis of EXO70G1‐tdTomato and PIN2‐GFP in (J), depicted as a white line.

Although EXO70G1‐tdTomato and PIN2‐GFP are co‐localized to the apical/basal membranes, their distribution patterns during cell division exhibit notable differences. After cytokinesis, EXO70G1 displays a clear apical‐basal polarity (Figure S8A, B), while PIN2 remained non‐polarized in daughter cells (Figure S8A, C). To further confirm the temporal relationship between EXO70G1 and PIN2 in the polarity establishment, we performed time‐lapse imaging to monitor the dynamic behaviors of both proteins throughout cytokinesis. At the early stage of cytokinesis, EXO70G1‐tdTomato was found to localize at the FM4‐64 labeled cell plate (Figure S9). Then, the EXO70G1‐tdTomato signal appears at the onset of cytokinesis at the 3.0 min time point, slightly later than that of PIN2 at the 1.5 min time point (Figure S10A, B). It is noteworthy that the EXO70G1‐tdTomato persisted at both apical and basal membranes after initial appearance and was maintained throughout the post‐cytokinetic period (Figures S8, S10). This finding contrasts with PIN2, which exhibits a temporary loss of polarity during cytokinesis (Figures S8, S10). Our observations are consistent with those reported in the article (Glanc et al., 2018). These results strongly suggest that the polarity establishment of EXO70G1 precedes that of PIN2.

EXO70G1 and EXO70G2 function redundantly in regulating abundance of PIN2 at the PM

PIN proteins are responsible for auxin accumulation in root tips, which is crucial for root growth and development (Benkova et al., 2003). First, we detected auxin homeostasis using a pDR5rev::GFP reporter to confirm whether auxin accumulation was altered, the relative fluorescence intensity was decreased in the root tips of the exo70G1 exo70G2 double mutant compared to Col‐0 (Figure 6A, B). Auxin accumulation in the root tips is partly dependent on the polar transport system mediated by PIN proteins (Blilou et al., 2005). Since PIN2 is widely expressed in epidermal cells, we selected PIN2 as a representative marker to verify the regulation of PIN proteins by EXO70G1. We analyzed the expression pattern of PIN2‐GFP in the exo70G1 exo70G2 double mutant and identified that the PIN2 fluorescence intensity of the apical membrane in epidermal cells decreased in the double mutant compared to the WT (Figure 6C–E). However, the lateral membrane intensity was unchanged, and the polarity index was altered in the exo70G1 exo70G2 double mutant because of decreased apical membrane intensity (Figure 6F, G). The fluorescence intensity of the basal membrane in cortex cells was also decreased in the exo70G1 exo70G2 double mutant (Figure 6H). Because PIN proteins aggregate at PM‐like clusters, we evaluated whether the mutation of EXO70G1 and EXO70G2 impact PIN2 clustering. As anticipated, the density of the PIN2 cluster was reduced in the exo70G1 exo70G2 double mutant (Figure 6I, J). Then we concluded that mutations in EXO70G1 and EXO70G2 influence the abundance and clustering of PIN2 at the PM.

Figure 6.

Decreased PIN2‐GFP abundance at the PM in exo70G1 exo70G2 double mutants

(A) Expression pattern of pDR5rev:GFP in Col‐0 and exo70G1 exo70G2 cultured on 1/2MS vertically for 5 d. Scale bars: 20 μm. (B) Quantitation of relative fluorescence intensity of pDR5rev:GFP in Col‐0 (n = 25) and exo70G1 exo70G2 (n = 28). (C, D) Expression pattern of PIN2‐GFP in Col‐0 and exo70G1 exo70G2 background at the surface plane and the median section. Epi, epidermal cells. Cor, cortex cells. Fire color scale represents the fluorescence intensity of PIN2‐GFP. Scale bars: 10 μm. (E–G) Quantitative analysis of the individual PIN2‐GFP in Col‐0 (n = 123 cells from 17 roots) and exo70G1 exo70G2 (n = 140 cells from 22 roots), with respect to relative fluorescence intensity of PIN2‐GFP at the apical membrane (E), lateral membrane (F) of epidermal cells, the polarity index (G). (H) Relative fluorescence intensity of PIN2‐GFP at the basal membrane of cortex cells in Col‐0 and exo70G1 exo70G2. (I) Distribution of PIN2‐GFP cluster in Col‐0 and exo70G1 exo70G2 backgrounds. Scale bars: 3 μm. (J) Number of PIN2‐GFP clusters in Col‐0 (n = 57 cells from 6 roots) and exo70G1 exo70G2 (n = 61 cells from 7 roots). Data represent mean ± SD, ** and * represent means that differ at P < 0.01 and 0.05, respectively (Student's t‐test).

As polar localization maintenance of PIN proteins at the PM depends on CME, recycling, and degradation in the vacuole (Dhonukshe et al., 2007; Kleine‐Vehn and Friml, 2008; Yang et al., 2020), we predicted that EXO70G1 might regulate PIN2 trafficking from the cytoplasm toward the PM. FRAP analysis demonstrated that the recovery time of PIN2 was delayed in the exo70G1 exo70G2 double mutant (Figure 7A–C), implying the critical role of EXO70G1 and EXO70G2 in the trafficking of PIN2. The BFA washout experiment was investigated to clarify our assumption. When treated with BFA for 1 h, the PIN2‐GFP‐labeled BFA bodies per cell were significantly higher in the exo70G1 exo70G2 double mutant compared to Col‐0 (Figure 7D, E). Following BFA treatment for 1 h and BFA washout for 30 to 90 min, PIN2‐GFP‐labeled BFA bodies were not recycled back to the PM in exo70G1 exo70G2. The percentage of cells retaining BFA bodies was much higher than Col‐0 (Figure 7D, F). Then, we utilized the membrane dye FM4‐64 to explore the role of EXO70G1 and EXO70G2 on endocytosis. However, no significant difference was observed in the number of vesicles per cell or in fluorescence intensity between Col‐0 and the exo70G1 exo70G2 double mutant (Figure S11), suggesting that EXO70G1 and EXO70G2 might not influence endocytosis. Next, we examined whether EXO70G1 and EXO70G2 are involved in the degradation of PIN2 in the vacuole. The distribution of PIN2 in the exo70G1 exo70G2 double mutant was similar to the WT after 4 h of dark treatment (Figure S12), indicating the degradation of PIN2 in the vacuole was not impacted. To confirm whether PIN2 was the cargo of EXO70G1‐mediated trafficking, a luciferase (LUC) split and complementary experiment was conducted and no direct interaction was identified between PIN2 and EXO70G1, indicating that the regulation of PIN2 trafficking by EXO70G1 and EXO70G2 occurs indirectly. The findings presented above suggest that EXO70G1, together with EXO70G2, play a significant role in regulating the abundance of PIN2 at the PM by enhancing the recycling efficiency of PIN2.

Figure 7.

EXO70G1 and EXO70G2 promote the recycling of PIN2

(A) Photobleaching and recovery of PIN2‐GFP in Col‐0 and exo70G1 exo70G2 backgrounds. Scale bar: 10 μm. (B) Relative fluorescence intensity recovery curves of PIN2‐GFP in Col‐0 and exo70G1 exo70G2 following fluorescence recovery after photobleaching (FRAP) analysis. (C) The time to obtain 50% of a maximum recovery after photobleaching in Col‐0 and exo70G1 exo70G2; more than 10 cells in different roots were analyzed per line. (D) BFA bodies in Col‐0 and exo70G1 exo70G2 background following treatment with 50 μmol/L BFA for 1 h, and 1/2MS wash out for 30 min, 60 min, and 90 min after 50 μmol/L BFA treatment for 1 h, respectively. Scale bars: 10 μm. (E) Numbers of BFA bodies in Col‐0 and exo70G1 exo70G2 in (D). (F) Percentage of cells retaining BFA bodies in Col‐0 and exo70G1 exo70G2; more than 10 cells in 13–16 roots were analyzed per line. Data represent mean ± SD, ** and * represent means that differ at P < 0.01 and 0.05, respectively (Student's t‐test).

The organization of lipid raft influences EXO70G1 abundance at the PM

Previous studies indicate that EXO70A1 provides a positional signal for Casparian strip localization via its interaction with phosphoinositides (Kalmbach et al., 2017), then we propose the question whether EXO70G1 also acts as a molecular signpost for recycling of PIN2 to the PM. Additionally, exogenous salicylic acid (SA) application increases lipid nanodomain size while concurrently decreasing their density, and methyl‐β‐cyclodextrin (mβcd) acts as a sterol extraction inhibitor to prevent lipid raft disassembly (Huang et al., 2019; Ke et al., 2021). To investigate the lipid nanodomain's effect on EXO70G1 localization, we analyzed EXO70G1‐GFP expression pattern following treatment with SA, mβcd, or SA + mβcd. The results showed that the polarity index of EXO70G1 remained unchanged; however, apical and basal membrane fluorescence intensity of EXO70G1 decreased (Figure 8A–C). Meanwhile, SA treatment decreases the abundance of PIN2 at the PM and inhibits root growth (Tan et al., 2020; Ke et al., 2021). Next, we examined the expression pattern of PIN2‐GFP in WT and the exo70G1 exo70G2 double mutant under SA treatment. The results showed that SA treatment reduced PIN2 fluorescence intensity at the apical membrane relative to mock controls, with no additional reduction in exo70G1 exo70G2 mutants (Figure S13). Correspondingly, the double mutant did not exhibit the inhibition of primary root elongation observed in WT plants on the 1/2MS supplemented with SA (Figure S14). We propose that under normal conditions, EXO70G1 and EXO70G2 enhance the recycling efficiency of PIN2. However, SA treatment alters lipid raft distribution (Ke et al., 2021), which reduces EXO70G1 PM abundance and consequently diminishes PIN2 accumulation at the PM. Taken together, these findings suggest that the function of EXO70G1 depends on lipid raft or nanodomains at the PM, and that it regulates PIN2 trafficking partially through providing a positional cue at the apical‐basal membranes.

Figure 8.

Salicylic acid (SA) inhibits abundance of EXO70G1 at the PM

(A) EXO70G1‐GFP were treated with SA (100 mmol/L, 24 h), mβcd (10 mmol/L, 24 h), SA plus mβcd, GFP signal (green) was visualized. Scale bars: 5 μm. (B–D) Quantitative analysis of the individual EXO70G1‐GFP, with respect to relative fluorescence intensity of EXO70G1‐GFP at the apical membrane (B), lateral membrane (C), and polarity index (D), more than eight cells in 15–18 roots were analyzed per treatment. Data represent mean ± SD, ** and * represent means that differ at P < 0.01 and 0.05, respectively (Student's t‐test).

Next, we investigated whether EXO70G1—beyond facilitating PIN2 delivery to the PM—participates in broader membrane trafficking processes targeting apical/basal membranes. We crossed GFP‐PIP2;1 (plasma membrane intrinsic protein 2;1) with the exo70G1 exo70G2 double mutant, fluorescence intensity analysis revealed that PIP2;1 signal at the PM was no significant difference between double mutant and WT (Figure S15). Given that PIP2;1 is a constitutive cycling protein (Li et al., 2011), which is similar to BAK1‐interacting receptor‐like kinase (BIR1), while mutation in EXO70A1 contributes disturbed BIR1 recycling (Drdová et al., 2013), thus, we speculate that conserved EXO70A function in housekeeping exocytotic processes. In exo70G1 exo70G2 double mutant, function of EXO70A1 was normal, thus the accumulation of GFP‐PIP2;1 at the PM was unaffected. Our data further indicate that different EXO70 isoforms recognize distinct cargo sets, and that EXO70G1's polar localization enables specialized membrane trafficking. Previous studies established that the primary defect in pin2 mutants is loss of gravitropism. However, we observed no comparable gravitropic impairment in exo70G1 exo70G2 mutants. We hypothesize that this phenotypic resilience stems from the retention of ~70% PIN2‐GFP at the PM in exo70G1 exo70G2 relative to Col‐0—a residual level potentially sufficient to sustain gravity sensing. Thus, we propose that EXO70G1 might coordinate accessory factors mediating PIN2 delivery to the PM.

DISCUSSION

EXO70G1‐marked polar PM domains differ from other EXO70 isoforms

The exocyst complex subunit EXO70 exhibits extensive gene duplication in plants. Based on protein homology, the 23 Arabidopsis EXO70 paralogs cluster into three clades and eight subclades (Li et al., 2010). These isoforms display tissue‐ and cell type‐specific expression patterns (Li et al., 2010), and their PM microdomain distributions show significant heterogeneity even within individual cells (Marković et al., 2021). For example, the representative EXO70A1 in the EXO70.1 subfamily was abundant at the outer lateral membrane (Fendrych et al., 2013; Marković et al., 2021), while EXO70B2 in the EXO70.2 subfamily was evenly distributed at the PM (Marković et al., 2021). However, the details of subfamily EXO70.3 remain unclear. Here, we found that, unlike other isoforms, EXO70G1 was predominantly localized to the apical and basal membranes. Despite distinct polar localization relative to EXO70A1 and other exocyst subunits, EXO70G1 exhibits overlapping distribution domains with these components within the PM. This spatial coexistence suggests that EXO70A1 and EXO70G1 may mark distinct exocytosis‐active destinations for cargo‐specific trafficking. When EXO70A1 was mutated, the distribution pattern of EXO70.1 and EXO70.2 isoforms was altered (Marković et al., 2021), indicating that EXO70A1 is functionally pivotal among EXO70 isoforms, with other paralogs partially dependent on its activity. The effect of EXO70A1 on EXO70G1 polarity is worthy of further investigation. Interestingly, the mutation of EXO70G1 resulted in a reduced distribution of EXO70A1‐GFP at both the apical and basal membranes. This finding may suggest that the polarity established by EXO70G1 plays a crucial role in targeting of EXO70A1 to the apical‐basal domains, and loss‐of‐function of EXO70G1/G2 itself might affect the stability of the exocyst complex.

Given that EXO70G1 exhibits a bundle‐shaped distribution pattern—similar to and overlapping with PIN2 clustering in Z‐axis stacks—we propose that the polarity regulatory mechanisms of EXO70G1 may parallel those of PIN proteins. Cellular requirements, including cell wall and PM components, play a vital role in the clustering of PIN proteins at the PM (Li et al., 2021). It is intriguing to further investigate the distribution change of EXO70G1 when phosphoinositide lipids were altered. Moreover, the exocyst complex is responsible for the trafficking of cell wall components like cellulose (Zhu et al., 2018), and the PIN2 cluster was decreased in the cellulose synthase (cesa) mutant (Li et al., 2021). Therefore, it might be interesting to investigate whether cellulose synthase complexes (CESA) regulate EXO70 localization. In endomembrane trafficking, small GTPases modulate endocytosis and recycling‐exocytosis of PIN proteins (Chen et al., 2012; Feraru et al., 2012; Tanaka et al., 2014). Interestingly, the EXO70 is a downstream effector of GTPases in yeast and animals (Inoue et al., 2003; He et al., 2007; He and Guo, 2009), however, their relationship is unclear in plants. Considering that EXO70G1 was highly dynamic at the PM, we are also curious about the question whether the targeting or trafficking of EXO70G1 to the PM was regulated by small GTPases. Taken together, the upstream regulators in EXO70G1 homeostasis maintenance on the PM are worthy of further investigation.

The EXO70 family is highly divergent and partially redundant

In response to their spatial–temporal specificity, the gene functions of EXO70 are characteristic and complex. For instance, mutation in EXO70A1 leads to developmental defects in branching, root elongation, and fertility (Synek et al., 2006). However, only EXO70A2 could restore development defects of exo70A1 (Marković et al., 2021). EXO70C1 and EXO70C2 show high transcription levels in mature pollen, where they redundantly regulate tip growth (Li et al., 2010; Synek et al., 2017). In plant immunity, EXO70B1 interacts with EXO70B2 to maintain homeostasis of FLAGELLIN‐SENSITIVE 2 (FLS2) at the PM (Wang et al., 2020). Notably, none of the EXO70 isoforms could substitute the function of EXO70B1 during senescence (Marković et al., 2021). In this study, we found that neither exo70G1 nor exo70G2 exhibits a discernible phenotype, whereas both EXO70G1‐RS and EXO70G2‐RS line successfully rescue the impaired root elongation phenotype of the double mutant. Besides that, EXO70G1 polar localize in different root cell types, and EXO70G2‐GFP is evenly distributed at the PM, with occasional cytoplasmic puncta (Figure S16). This broader spatial distribution compared to EXO70G1 suggests a more widespread role for EXO70G2 in membrane trafficking. The phylogenetic analysis of EXO70 proteins across different species indicates that these two paralogs share a common ancestor and form a clade that is evolutionarily distant from other EXO70 members (Li et al., 2010). Based on these results, we propose that the polar‐localized EXO70G1 plays a predominant role in root elongation and functions partially redundantly with EXO70G2. In addition, the exo70G1 exo70G2 double mutant exhibited cell division defects within the meristem zone. Consequently, we tracked the expression pattern of EXO70G1 during cytokinesis and observed its localization at the cell plate, while it has been reported that only the first phase of initial vesicle fusion is delayed in exo70A1 mutants during both early cytokinesis and cell plate maturation (Fendrych et al., 2010). Considering the functional redundancy between EXO70 isoforms, we believe that EXO70A1 plays a dominant role in cell plate formation. Interestingly, in our study, EXO70A1 and EXO70G1 were co‐expressed at the apical and basal membranes, and the triple mutant of exo70A1 exo70G1 exo70G2 exhibited substantial defects in root development. Therefore, the results further demonstrate the functional redundancy between EXO70.1 and EXO70.3 isoforms. Our findings support the conclusion that the gene function of the EXO70 family is highly divergent and partially redundant.

As one subunit of the exocyst complex, EXO70 could function dependently or independently of the other subunits. For targeting of the CESAs to the PM, EXO70A1 cooperates with other exocyst subunits to function as an integrated complex (Zhu et al., 2018; Jiang et al., 2024). For the recruitment of PATL3 to the PM and the cell plate, EXO70A1 is independent of its role as an exocyst subunit (Wu et al., 2017). The functional conservation of the exocyst complex in tethering secretion vesicles to the target membrane is well‐documented (He and Guo, 2009; Vukasinovic and Žárský, 2016; Gingras et al., 2022). SEC6 and EXO84b subunits co‐localized with EXO70A1 at PM sites of active exocytosis (Fendrych et al., 2013). Our results show EXO70G1 co‐localizes with SEC6/SEC8 at the apical/basal PM domains. Its BFA resistance profile resembles that of EXO70A1, SEC6, SEC8, and EXO84b (Drdová et al., 2013; Fendrych et al., 2013), suggesting functional dependence on core exocyst subunits. However, EXO70G1 was insensitive to prolonged actin disruption, differing from EXO84b (Fendrych et al., 2013). The reason might be that the recovery time of EXO84b was tested at the lateral PM of elongating root epidermal cells. Based on PINs recycling regulation, we propose that EXO70 function depends on the exocyst complex. For instance, mutations in EXO70A1 and SEC6 impair PIN proteins recycling during root development (Drdová et al., 2013; Tan et al., 2016), and EXO70A3 modulates PIN4 dynamics to influence root architecture (Ogura et al., 2019). EXO70G1 polarizes to the PM and regulates PIN2 recycling redundantly with EXO70G2. The expression patterns of EXO70 isoforms are characteristic and cell‐type specific, whereas SEC6 expression is broader. Therefore, the precise regulation of PINs trafficking may be achieved through the interactions of specific EXO70 isoforms with other exocyst subunits. The hypothesis that distinct EXO70 isoforms associate with other subunits to mediate specialized functions was proposed and verified (Drdová et al., 2013; Marković et al., 2020). Intriguingly, the two SEC15 exocyst subunit isoforms, SEC15a and SEC15b, exhibit distinct distribution patterns; SEC15b localizes to the outer lateral membrane, whereas SEC15a is polarized at apical and basal membranes (Batystova et al., 2022). This differential accumulation resembles patterns observed for EXO70A1 and EXO70G1. Therefore, EXO70G1 may cooperate with polarized subunits to facilitate the delivery of other polar cargos. It is interesting to investigate their effects on PINs trafficking when two polarly distributed subunits were mutated. This suggests that the specificity of pairing between individual EXO70 isoforms and their cognate subunits may be far greater than previously appreciated.

Regulation of PIN2 by EXO70G1 depending on lipid raft organization at the PM

During exocyst complex‐mediated exocytosis, the C‐terminal region of EXO70 can interact with negatively charged phosphoinositides to define the active sites for vesicle tethering at the PM (He and Guo, 2009; Synek et al., 2021). Therefore, the recognition of target membrane is important for cargo trafficking. For the precise localization of the Casparian strip, EXO70A1 could provide a transient positional signal (Kalmbach et al., 2017). Then, how do EXO70G1 and EXO70G2 regulate recycling efficiency of PIN2 and its abundance at the PM? The regulatory mechanisms of polarized protein trafficking primarily include: (i) protein sorting at the TGN/EE, (ii) cytoskeleton‐dependent vesicle transport, and (iii) tethering of exocytic vesicles to the target membranes (Gao and Chao, 2022). Subcellular localization analyses revealed that EXO70G1 co‐localizes with PIN2 at the PM but shows no significant co‐localization in the cytoplasm. Furthermore, unlike PIN2, EXO70G1 trafficking to the PM is insensitive to BFA and LatB treatments. These findings indicate that EXO70G1 does not participate in BFA‐sensitive sorting or actin‐dependent vesicle transport of PIN2.

As established, the aggregation of sterol at the PM forms a specialized microdomain; the asymmetric distribution of proteins at the PM is not only dependent on polarized trafficking mechanisms but also on microdomain compartmentalization (Ott, 2017). Salicylic acid treatment induces the nanodomain compartmentalization (Ke et al., 2021). Under SA treatment, the abundance of EXO70G1 at the PM decreased, the accumulation of PIN2 at the PM was impaired in the exo70G1 exo70G2 double mutant. This suggests that EXO70G1's regulation of PIN2 depends on the lipid raft clustering. On the other hand, lipids recruit PIN proteins to the nanodomain through direct binding (Li et al., 2021), and alterations in phosphoinositide lipids at the PM affect the PIN clustering density (Li et al., 2021). Thus, phosphoinositide lipids serve as a binding platform for PM localized proteins and a recognition site for vesicle trafficking. We propose that EXO70G1 may function in transporting components required for the formation of these microdomains. Besides that, the polarized trafficking mechanism of PIN2 is a complex and actively debated topic, with multiple regulatory factors implicated but with unclear mechanistic details. For instance, guanine nucleotide exchange factor for ARF GTPase (GNOM), a key regulator of PIN polarity, localizes to TGN/EE and the PM but lacks obvious polarized distribution at the PM, leaving its role in PIN polarized trafficking enigmatic (Geldner et al., 2003). Therefore, we hypothesize that apical‐basally localized EXO70G1 acts as a landmark for microdomains to guide PIN2, rather than directly mediating its transport to apical or basal membranes, whereas its contribution to microdomain formation remains an open question for further exploration.

MATERIALS AND METHODS

Plant materials and culture conditions

All mutants used in this article were established in a Columbia‐0 (Col‐0) background. T‐DNA insertion lines exo70G1 (SALK_019409) and exo70G2 (SALK_097393C) were obtained from the Nottingham Arabidopsis Stock Centre. The following published materials were used: the endomembrane compartment markers (Geldner et al., 2009), prevacuole compartment markers (Li et al., 2017), pin2 mutant, and transgenic lines expressing pDR5rev:GFP, pSCR::GFP (Tian et al., 2014), GFP‐PIP2;1 (Li et al., 2011).

Seeds were surface‐sterilized using 75% ethanol containing 0.04% Triton X‐100, followed by two washes with 70% ethanol. Seedlings were grown on 1/2MS medium (MS medium with Gamborg's vitamins, Sigma, USA) supplemented with 10 g/L sucrose, 0.5 g/L 2‐(N‐morpholine)‐ethanesulphonic acid (MES, Sigma, USA), and 8 g/L agar, pH 5.8) under continuous light (20 μmol photons/m²/s) at 22°C ± 1°C.

Generation of constructs and transgenic lines

The 2,064 bp coding sequence (CDS) of EXO70G1 was inserted into the pCAMBIA‐3301H plant expression vector under the control of the CAMV 35S promoter to produce the p35S::EXO70G1 transgene. For the pEXO70G1::EXO70G1‐GFP construct, a 2,061 bp EXO70G1 CDS fragment and its 1,998 bp native promoter were ligated into the pGFPGUSplus vector (Vickers et al., 2007). For the pEXO70G2::EXO70G2‐GFP construct, a 1,980 bp EXO70G2 CDS fragment and its 835 bp native promoter were ligated into the pGFPGUSplus vector (Vickers et al., 2007). The construction of pEXO70G1::EXO70G1‐GFP and pEXO70G2::EXO70G2‐GFP was transformed into the exo70G1 exo70G2 double mutant to establish transgenic “RS” lines. To generate pEXO70G1::EXO70G1‐tdTomato, the GFP cassette in pGFPGUSplus was replaced with tdTomato to create the p35S::tdTomato vector. Subsequently, the EXO70G1 promoter and CDS were cloned into p35S::tdTomato vector using a Hieff Clone^@ Plus One Step Cloning Kit (10911ES62). The pEXO70G1::EXO70G1‐tdTomato was transformed into Col‐0 and PIN2‐GFP backgrounds respectively via the floral dip method (Clough and Bent, 1998). Homozygous exo70G1 exo70G2 mutants and its hybrid lines with pDR5rev::GFP, pSCR::GFP, PLT1‐GFP, PLT2‐GFP, PIN2‐GFP lines were genotyped by polymerase chain reaction using primers designed via the SIGnAL T‐DNA verification tool (http://signal.salk.edu/isects.html). All primers used are listed in Table S1.

Root length and meristem size measurement

For the root length measurements, 5–7‐d old seedlings were grown vertically on 1/2MS medium. Seedlings were imaged with a digital scanner, and root lengths were quantified using ImageJ software (https://imagej.net/). Meristem length, meristematic cell number, and epidermal cell length were analyzed as previously described (Di Mambro and Sabatini, 2018).

Confocal microscopy and image quantification

For live‐cell imaging, seedlings were vertically grown in Petri dishes on 1/2MS medium for 5 d. Imaging was performed using either: (i) a spinning‐disk confocal system (Andor dragonfly 505) mounted on a Lecia DMi8 microscope, equipped with a 100 × 1.47‐mumerical aperture (NA) UPlanSApo oil objective (Lecia), and two Sona 2.0B‐11 sCOMS cameras (Andor); or (ii) a Lecia TCS SP8 confocal microscope based on a DMi8 platform, equipped with a 100 × 1.40 NA objective and two HyD GaAsP spectral detectors (Lecia).

Fluorescence markers were excited at 488 nm (GFP), or 561 nm (mCherry/tdTomato/FM4‐64), with emission filtered through bandpass filters (521/38 nm for GFP, 594/43 nm for mCherry and tdTomato). Co‐localization analysis of EXO70G1 with PIN2 was performed using the Coloc 2 plugin in Fiji. To ensure measurement robustness, both Pearson's and Mander's coefficients were calculated. For 3D reconstruction of EXO70G1‐tdTomato and PIN2‐GFP, z‐stacks were acquired at 0.2 μm intervals using spinning‐disk confocal microscopy (SDCM). The apical‐to‐lateral PM fluorescence ratio—defined as mean apical PM intensity divided by mean lateral PM intensity—was quantified for PIN2‐GFP and EXO70G1‐GFP. This ratio was computed using a modified ImageJ script: Polarity Index Calculator (https://github.com/totti0223/polarity_index_calculator; Yoshinari et al., 2021).

For FRAP experiments, photobleaching was performed using a 405‐nm laser line at 100% power. PIN2‐GFP imaging was conducted with SDCM. EXO70G1‐GFP imaging under DMSO control and drug treatments was performed on a Lecia TCS SP8 system. Time‐lapse imaging of PIN2‐GFP was acquired at 60 s intervals for 35 frames. For EXO70G1‐GFP imaging under DMSO, PM time‐lapse sequences were captured at 3 s intervals for 44 frames. Relative fluorescence intensity and half recovery time (1/2 t) were quantified using the FRAP Profiler v2 plugin in Fiji (https://worms.zoology.wisc.edu/research/microscopy/#frap).

For the imaging of EXO70G1‐tdTomato/SEC6‐GFP and EXO70G1‐tdTomato /GFP‐SEC8, time‐lapse images were at the cytoplasm of the root elongation zone using a TIRF illuminator, with a 100 ms interval for 200 frames. Velocity analysis of EXO70G1‐tdTomato, SEC6‐GFP, and GFP‐SEC8 in cells was performed using kymographs. Kymographs were generated by tracking the trajectories of EXO70G1‐tdTomato particles, SEC6‐GFP particles, or GFP‐SEC8 particles. The motility rate of individual particles was calculated as the reciprocal of the slope of their trajectories on the kymographs. For EXO70G1‐tdTomato/SEC6‐GFP and EXO70G1‐tdTomato/GFP‐SEC8 pairs, co‐localization analysis was conducted by plotting relative fluorescence intensity profiles. Peaks of SEC6‐GFP or GFP‐SEC8 fluorescence coinciding with peaks of EXO70G1‐tdTomato fluorescence were considered co‐localized. The number of overlapping peaks and the number of peaks that did not overlap with SEC6, SEC8, or EXO70G1 were counted.

β‐glucuronidase staining

β‐glucuronidase staining was performed as previously described (Friml et al., 2003).

Chemical treatment

For BFA treatment, 5‐d‐old seedlings were cultured in liquid MS medium containing 50 μmol/L BFA for 1 h. For BFA washout experiment, 5‐d‐old seedlings were pre‐treated in liquid MS medium with 50 μmol/L BFA for 1 h and then transferred into liquid MS medium (without BFA) for 30–90 min. To quantify BFA bodies, at least 150 epidermal cells were counted from a minimum 15 seedlings per treatment replicate. Retention of BFA bodies was quantified by calculating the ratio of cells containing residual BFA bodies to the total number of cells analyzed per seedling image. For LatB treatment, 5‐d‐old EXO70G1‐GFP seedlings were treated with either 10 μmol/L LatB or 0.3% DMSO (vehicle control) in liquid MS medium for 1 h.

For FM4‐64 uptake analysis, 5‐d‐old seedlings were incubated in liquid MS medium containing 5 μmol/L FM4‐64 for 15 min.

Salicylic acid treatment was performed as previously described (Huang et al., 2019; Ke et al., 2021).

Generative AI usage statement

During the preparation of this work, the authors utilized Deepseek (https://www.deepseek.com/) for the purpose of revising grammatical issues. After using this tool/service, the authors carefully reviewed and revised the content where necessary, and take full responsibility for the final version of the publication.

Accession numbers

Genic DNA sequences can be obtained from www.arabidopsis.org, using the formal synonyms indicated in parentheses: EXO70A1 (AT5G03540), EXO70G1 (AT4G31540), EXO70G2 (AT1G51640), PIN2 (AT5G57090), SCR (AT3G54220), PLT1 (AT3G20840), PLT2 (AT1G51190), SEC6 (AT1G71820), EXO70A3 (AT5G52350), SYP32 (AT3G24350), RabD2b (AT5G47200), ARA7 (AT4G19640), and VHAa1‐ATPase (AT2G28520), RabA1g (AT3G15060), and RabG3f (AT3G18820).

CONFLICTS OF INTEREST

The authors declare no competing interests.

AUTHOR CONTRIBUTIONS

J.L. and Z.L. performed most of the research and J.L. drafted the manuscript. L.G. and S.Z. carried out grafting experiments and drug treatment experiments, X.L., S.G., and S.J. performed some expression and phenotype analyses. Z.L., L.G., and L.M. carried out microscopy analysis. Q.L., G.D., and D.C. revised the manuscript. S.L. designed the experiments, supervised the study, and revised the manuscript. All authors read and approved of its content.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70053/suppinfo

Figure S1. Phenotype analysis of EXO70G1‐RS and EXO70G2‐RS transgenetic line

Figure S2. Polarized localization of EXO70G1 in epidermal cells of the meristem and elongation zone of Arabidopsis roots

Figure S3. Subcellular localization of EXO70G1‐GFP in Arabidopsis roots

Figure S4. EXO70G1 is insensitive to treatment with BFA and LatB

Figure S5. Co‐localization of EXO70G1 and SEC6 in epidermal cells of Arabidopsis roots

Figure S6. Root elongation analysis of p35S::EXO70G1

Figure S7. Expression pattern of SCR and PLTs in Col‐0 and exo70G1 exo70G2

Figure S8. Subcellular co‐localization of EXO70G1 and PIN2 in Arabidopsis roots

Figure S9. EXO70G1‐GFP localization during cell plate assembly

Figure S10. Dynamic co‐localization of EXO70G1 and PIN2 during cell plate formation

Figure S11. EXO70G1 and EXO70G2 are not involved in endocytosis

Figure S12. EXO70G1 and EXO70G2 are not involved in degradation of PIN2 in vacuoles

Figure S13. PIN2 membrane abundance is SA‐insensitive in exo70G1 exo70G2 mutants

Figure S14. The exo70G1 exo70G2 double mutant is insensitive to SA treatment

Figure S15. Distribution of GFP‐PIP2;1 in Arabidopsis root epidermis

Figure S16. Distribution of EXO70G2‐GFP in Arabidopsis roots

Movie S1. Dynamic co‐localization of EXO70G1 and SEC8 in epidermal cells of the elongation zone

Movie S2. Dynamic co‐localization of EXO70G1 and SEC6 in epidermal cells of the elongation zone

Movie S3. Dynamic co‐localization of EXO70G1 and PIN2 during cell plate formation

Biographies

Li, J. , Liu, Z. , Gong, L. , Zhao, S. , Lu, Q. , Gao, S. , Jiang, S. , Liu, X. , Ma, L. , Duan, G., et al. (2026). Polar‐localized EXO70G1 regulates root development in Arabidopsis thaliana . J. Integr. Plant Biol. 68: 96–112.