The movement of the non-cell-autonomous transcription factor, SHORT-ROOT relies on the endomembrane system.

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Received Date : 17-Dec-2013 Revised Date : 29-Jul-2014 Accepted Date : 04-Aug-2014 Article type

: Original Article

The movement of the non-cell-autonomous transcription factor, SHORT-ROOT relies on endomembrane system.

Shuang Wu & Kimberly L. Gallagher1

Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, 19104 USA

1

Correspondence should be addressed to K.L.G ([email protected]).

Running Title: Endosomes and SHR Movement

Keywords: Intercellular protein movement, SHORT-ROOT, Cell Signaling, Plasmodesmata, SHR INTERACTING EMBRYONIC LETHAL, endosomes.

Summary Plant cells are able to convey positional and developmental information between cells through the direct transfer of transcription factors. One well-studied example of this is the SHORT-ROOT protein, which moves from the stele into the neighboring ground tissue layer to specify endodermis. While it has been shown that SHR trafficking relies on plasmodesmata (PD), and interaction with the SHR INTERACTING EMBRYONIC LETHAL (SIEL) protein, little is known about how SHR trafficking

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is controlled or how SIEL promotes the movement of SHR. Here we show that SHR can move from multiple different cell types in the root. Analysis of subcellular localization indicates that in the cytoplasm of root or leaf cells, SHR localizes to endosomes in a SIEL dependent manner. Interference of early and late endosomes disrupts intercellular movement of SHR. Our findings reveal an essential role for the plant endomembrane, independent of secretion, in the intercellular trafficking of SHR.

Introduction The intercellular movement of transcription factors is pervasive during plant development (Lee et al. 2006; Rim et al. 2011, 2). Transcription factors made by one cell in the plant can move into neighboring cells and affect the behavior and fate of the recipient cells. Mobile transcription factors regulate trichome and root hair patterning (Bouyer et al. 2008, Digiuni et al. 2008, Kurata et al. 2005, Pesch et al. 2009, Wester et al. 2009), development of the root and shoot apical meristems (RAM and SAM respectively) (Kim et al. 2009; Lucas et al. 1995; Schlereth et al. 2010; Xu et al. 2011) and the patterning and development of the mature root (Kurata et al. 2005; Cui et al. 2007; Gallagher et al. 2004; Koizumi et al. 2012a; Nakajima et al. 2001). While plant cells are capable of both exo- and endocytosis, most proteins are thought to move between cells via plasmodesmata (PD), plasma membrane lined intercellular channels that connect the ER and cytoplasm of neighboring cells and therefore provide a direct route for protein transfer (Oparka 2004).

Recent work on the KNOTTED1 (KN1) protein, which moves in the SAM of Zea mays has shown that KN1 must be unfolded in order to pass through the PD (Xu et al. 2011, Kragler et al. 1998). After KN1 passes through the PD, it interacts with the CHAPERONIN CONTAINING TCP1 SUBUNIT 8 (CCT8) protein, which as part of a type II chaperonin complex, refolds KN1. CCT8 also interacts with TRANSPARENT TESTA GLABRA1 (TTG1) and facilitates its refolding after passage through PD. Mosaic analysis has shown that CCT8 is specifically required in the recipient cells and appears not to function in promoting movement of either KN1 or TTG1 from the donor cell to the PD (Xu et al. 2011). In fact very little is known about how cytoplasmically localized proteins are targeted to PD.

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Here, using the SHORT-ROOT (SHR) transcription factor as a model, we address how mobile transcription factors access the PD. In the root of Arabidopsis thaliana, the SHR protein is expressed in stele cells, where the protein is both nuclear and cytoplasmically localized. The SHR protein moves into the neighboring cells including the endodermis, quiescent center (QC) cells and the cortical endodermal initial (founder) cells where the protein is nuclear localized (Nakajima et al. 2001). Movement of SHR is required for the cell divisions in the root apex that produce the separate endodermis and cortex cell layers and in the endodermis to specify endodermal cell fate and inhibit precocious cell divisions (Koizumi et al. 2012b; Koizumi et al. 2012a; Nakajima et al. 2001). The question of how SHR is able to move between cells, therefore, is of developmental importance.

Yeast two-hybrid assays identified an essential protein, SHR INTERACTING EMBRYONIC LETHAL (SIEL) that directly interacts with SHR and promotes SHR movement from the stele into the endodermis. SIEL is a nuclear and cytoplasmically localized protein that associates with endosomes in the cytoplasm (Koizumi et al. 2011). Localization of SIEL to endosomes requires intact microtubules; depolymerization of microtubules with oryzalin causes SIEL to aggregate around the nucleus and reduces SHR movement (Wu and Gallagher 2013). However, neither localization of SHR to endosomes, nor a role for endosomes in SHR movement has been demonstrated.

Endosomes are well-recognized intracellular sorting compartments that carry luminal cargoes from both the recycling and biosynthetic pathways. The role of endosomes in the internalization and recycling of plasma membrane receptors to control signaling is well documented, as is the transport of proteins from the trans-Golgi network via endosomes (Contento and Bassham 2012; Robinson et al. 2008). However work largely in animals has led to a more expansive view of endosome functions that are detailed in the signaling endosome hypothesis (Howe 2005; Howe 2004; Miaczynska et al. 2004; Palfy et al. 2012; Sehgal 2008). One aspect of the signaling endosome hypothesis posits that vesicular trafficking of cytoplasmically associated proteins from the plasma membrane to the nucleus (to initiate transcription) is facilitated by interaction with endosomes. For example cytosolic STAT3 has been shown to bind to endosomes and shuttle to the nucleus to activate transcription (Sehgal 2008). In


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addition, endosome can serve as platforms for protein-protein interaction; late endosomes can bring together on their surface cytoplasmically localized proteins to form functional complexes or to promote post-translational modifications that either promote or inhibit signaling (Murphy et al. 2009). Endosomal localization of receptor proteins can also enhance signaling; for example endocytosis of the brassinosteroid receptor, BR1 increases its signaling potential in A. thaliana (Geldner et al. 2007). In these examples, endosome function either as protein transporters or as platforms for the interaction of cytosolic proteins.

Here we show that SHR associates with early, late and recycling endosomes in a SIEL dependent manner. Disruption of early and late endosomes, but not the Golgi or recycling endosomes significantly reduces SHR levels in the endodermis, indicating that endosomes promote intercellular movement of SHR. Our paper provides direct evidence for the plant endomembrane system in the intercellular movement of a transcription factor. Our collective results are consistent with a model for SHR movement in which endosomes serve as platforms for the assembly of a movement competent SHR protein complex. These results expand our understanding of endosome function and the mechanisms of intercellular protein movement in plants.

Results SHORTROOT mobility is independent of cell type. Previously we showed that movement of the SHR protein from the stele into the endodermis is facilitated by the endosome localized protein, SIEL (Koizumi et al. 2011). Loss of SIEL or mislocalization of SIEL so that it no longer associated with endosomes inhibited movement of SHR (Koizumi et al. 2011; Wu and Gallagher 2013). These results suggested that association of SIEL with endosomes is important for intercellular movement of SHR. However, there was no direct evidence showing that endosomes regulate SHR trafficking, nor localization of SHR to endosomes. When imaged by confocal microscopy, SHR-GFP appears homogenously distributed throughout the cytoplasm of stele cells (Figure S1a, b and c). As stele cells are small and embedded within the center of the root, determining the specific subcellular localization


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of SHR is difficult. To facilitate imaging of SHR, we examined the ability of SHR to move between cells of the epidermis and the subcellular localization of SHR-GFP in these cells.

When driven from the CPC promoter in a wild-type background, SHR-GFP was expressed in the stele and in the non-hair cells of the epidermis -the normal domain of CPC expression (Figure 1a, d and g) (Kurata et al. 2005). SHR-GFP fluorescence, however, was found not only in the stele and in the non-hair cells, but also in the endodermis, cortex and the hair cells (Figure 1b, e and h). Since SHR-GFP was fully nuclear localized in the endodermis, SHR-GFP must enter the cortex and the hair cells by movement from the non-hair cells in the epidermis (Gallagher et al. 2004; Gallagher and Benfey 2009). These results show that the capacity for SHR movement is not restricted to the stele.

To test whether movement of SHR between epidermal cells occurs via the same pathway as movement from the stele, we expressed a movement defective form of SHR from the CPC promoter. Structure-function analysis of SHR showed that multiple regions within SHR protein are required for its movement from the stele to the endodermis (Gallagher et al. 2004; Gallagher and Benfey 2009). Mutation of an LNELDV motif (to AAA) in the leucine heptad repeat II (LHR2) domain resulted in a reduction in the nuclear localization of SHR-GFP in the stele (Figure 1c, f and S1) and eliminated movement into the endodermis (Figure 1c and f). However independent of movement, the mutated protein was functional (could activate transcription; Gallagher and Benfey 2009). When expressed from the CPC promoter, SHR∆LNELDV-GFP was restricted to the stele and non-hair cells in the epidermis (the domain of CPC promoter activity), indicating a lack of cell-to-cell movement (Figure 1c, f and i). In addition, the SHR∆LNELDV-GFP protein showed decreased nuclear localization in both the stele and the non-hair cells of the epidermis. These results indicate that similar mechanisms regulate the subcellular localization and movement of SHR-GFP in the stele and the epidermis. Therefore examination of SHR-GFP movement in the epidermis can inform our understanding of the mechanisms of endogenous SHR movement.


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SHR is not homogenously distributed in the cytoplasm of epidermal or stele cells. The localization patterns of wild-type SHR-GFP driven in the stele from the CPC or SHR promoters were identical (Figure S1a and b); SHR-GFP was detected in the cytoplasm and nuclei of stele cells and limited to the nuclei of endodermal cells. In the epidermis, SHR-GFP was largely nuclear localized in the hair cells. In the non-hair cells, SHR-GFP showed both nuclear and cytoplasmic localization. Within the cytoplasm of the non-hair cells, SHR was not uniformly distributed. Instead the protein was observed in strands that extended from the nucleus to the plasma membrane and in punctate structures in the cytoplasm (Figure 2a). This punctate localization was not observed in epidermal cell expressing the nonmobile SHRΔLNELDV-GFP. Instead SHRΔLNELDV-GFP was uniformly distributed in the cytoplasm (Figure 2b), The localization of SHRΔLNELDV-GFP was similar to free (untagged) GFP, which has moved into the epidermis from the stele (GFP expressed from the SUCROSE-H+SYMPORTER 2 (SUC2) promoter; Figure 2c) (Truernit and Sauer 1995) or GFP directly expressed in the epidermis from the constitutive 35S promoter (Figure 2d). These results suggest that the capacity of SHR to move is associated with specific localization in the cytoplasm.

To test whether SHR associates with structures within the cytoplasm of stele cells, we used fluorescence recovery after photo-bleaching (FRAP; Figure S2), which can be used to measure the intracellular dynamics of SHR mobility. If SHR-GFP is freely distributed in the cytosol, then there should be rapid and full recovery of fluorescence following photo-bleaching in the cytoplasm. However, if there are distinct populations of the SHR-GFP protein within the cytoplasm, for example subpopulations that are associated with components of the endomembrane or the cytoskeleton, these will show reduced mobility compared to free cytosolic SHR-GFP (Sprague and McNally 2005). To test what fraction of SHR is mobile, SHR-GFP fluorescence was bleached within a region of the cytoplasm, and then the recovery was monitored (Figure S2a). In the cytoplasm of both stele and epidermal cells, approximately 75% of SHR-GFP displayed free movement and migrated into the bleached area in less than one second (Figure S2c). However in both the epidermis and stele, 25% of SHR-GFP was in a less mobile fraction that was unable to rapidly diffuse back into the bleached region of the cytoplasm (Figure S2c). This less mobile fraction presumably represents the population


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of SHR-GFP that is associated with cytoplasmic components that prevent rapid and free diffusion. As a control, the mobile fraction of SHR-GFP in the nuclei of endodermal cells, where SHR forms a complex with SCR, MAGPIE, JACKDAW and DNA (Gallagher et al. 2004;Sozzani et al. 2010; Welch et al. 2007) (Figure S2b and c) was also measured. In nuclei, only 30% of SHR-GFP was in a mobile fraction. In contrast, the mobile fraction of free GFP in the stele or the epidermis was over 90%. These results suggest that a significant fraction of SHR is not free within the cytosol of stele or epidermal cells, but instead associates with cytoplasmic components that limit its free diffusion.

SHR localizes to the endomembrane. To determine what cytoplasmic components SHR-GFP interacts with, we examined SHR localization in leaf protoplasts, which are often used as a single cell system to examine gene expression or protein localization (for a recent example see Carluccio et al. 2014). SHR-GFP consistently localized to the nucleus and cytoplasm of protoplasts and was found in punctate structures throughout the cytoplasm (Figure 2e). In contrast, SHRΔLNELDV-GFP showed no specific localization in the cytoplasm, which is consistent with the localization of the SHRΔLNELDV-GFP protein the stele and epidermis (Figure 2f). Consistent with the kinetics of FRAP, free GFP showed no punctate structures in the cytoplasm of protoplasts (Figure 2 g and h), suggesting association to vesicles is specific to the SHR protein. Incubation of the SHR-GFP expressing protoplasts with the lipophilic dye, FM4-64, revealed overlap between the SHR-GFP signal and FM4-64. Based upon the time frame for FM4-64 incubation, these results suggest that SHR localizes to endosomes (Figure 2 I and j).

Since FM4-64 is not specific to endosomes, we examined SHR-GFP together with the wave markers, mCherry tagged proteins that localize to specific endomembrane compartments (Figure 3 and Figure S3) (Geldner et al. 2009 and updated localization data found at http://www.unil.ch/dbmv/page52928_en.html). The highest degree of overlap with SHR-GFP was detected for Rab2Fa (7R, a marker for late endosomes; Figure 3a, b and e) and the VTI12 protein (13R, a marker for early endosomes; Figure 3c, d and e). SHR-GFP also co-localized with recycling endosomes (RabA5d; 24R; Figure 3e and Figure S3e) and post-golgi endosomes (RabD1; 25R; Figure


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3e and Figure S3f). However, we found very little co-localization between SHR-GFP and Golgi markers, which included Got1p homolog (18R) and SYP32 (22R) (Figure 3e and Figure S3a-d). Likewise we found no co-localization between free GFP and any of the endomembrane markers. Nor was significant overlap seen in pixel-shifted controls. These results suggest that movement competent SHR associates largely with endosomes, particularly early and late endosomes.

As shown in Figure 2a, SHR-GFP localized non-uniformly in the cytoplasm of non-hair cells. In order to determine if SHR-GFP localized to endosomes in these cells, CPC:SHR-GFP was crossed into the wave marker lines (Geldner et al. 2009) and co-localization was imaged in intact root tissue. As was the case with protoplast assays, a sub-population of the SHR protein colocalized with markers of the early, late and recycling endosomes (Figure 4). We saw very little co-localization of SHR-GFP with Golgi specific markers (e.g. 22R, SYP32; Figure 4c). However SHR-GFP was often near the Golgi markers (Figure S3 c and d). These results indicate that a subpopulation of the SHR protein localizes to endosomes in intact root tissues.

SHR-GFP moves with markers of the early and late endosome. To determine if co-localization of SHR with markers of the endomembrane is indicative of SHR binding to these compartments, we examined the dynamics of co-localization in living cells. Since a high proportion of the SHR-GFP signal localized to early and late endosomes, we chose the 7R (late endosomes) and 13R (early endosomes) markers for analysis (Figure 5 and S4). The mCherry and the SHR-GFP markers were simultaneously observed for 90 s. Many of the 7R or 13R tagged endosomes showed significant and directional movement; however there was no obvious preference for movement towards the plasma membrane. Throughout the time of observation, the SHR-GFP signal either partially or entirely overlapped with the endosomes markers (7R and 13R). By focusing on individual vesicles, we saw matched movement between SHR-GFP and the endosomal markers, indicating that SHR-GFP is bound to the endosome (Figure 5a and c). By compressing the time series images along the x axis, the identical trafficking pathway of SHR-GFP and the mCherry markers is more easily discerned (Figure


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5b and d). This result indicates that co-localization between SHR and endosomes is not merely a transient intersection of the two fluorescent signals, but instead bone fide interaction.

Pharmacological interference of endosomes limits intercellular movement of SHR. If SHR localizes to endosome, and endosomes participate in the intercellular movement of SHR, then disrupting endosomes should impair movement of SHR. To test this, we took advantage of two different assays that we developed to assess SHR trafficking. In the first assay, we used a semidominant, synthetic form of CALLOSE SYNTHASE 3 that in response to treatment with estradiol, produces PD localized callose in stele cells (from the WOODENLEG/CYTOKININ RESPONSE 1 promoter). As a consequence of the increased callose, movement through PD is blocked and the SHR-GFP signal in the endodermis is considerably reduced (Wu and Gallagher 2013; Vaten et al. 2011). However, upon removal from estradiol, SHR-GFP moves again through PD into the endodermis. This system allows us to decrease the SHR-GFP signal in endodermis and then monitor its recovery in the absence of estradiol, but in the presence of pharmacological inhibitors of different cellular components (Wu and Gallagher 2013).

Here we examined the recovery of the SHR-GFP signal in the endodermis of roots treated with 20 M Wortmannin (Wort), 1 M Concanamycin A (ConcA) or 10 M Brefeldin A (BFA). Wort is an inhibitor of phosphatidylinositol-3 kinase, which is essential for the formation of internal vesicles. In the presence of Wort, plant cells fail to endocytose FM4-64 dye. Wort also targets the prevacuolar compartments and late endosomes, inducing homotypic fusion, but has no reported effect on the Golgi apparatus or on early endosomes (once formed) (Reichardt et al. 2007; Wang et al. 2009). Concanamycin A (ConcA) is an antibiotic that binds to the V-ATPase c subunits to inhibit proton transport. ConcA blocks both the trafficking of newly synthesized proteins to the plasma membrane and the transport of FM4-64 from the TGN/EE to the vacuole (Dettmer et al.2006). BFA is a widely used inhibitor that targets GTP-exchange factors (GEFs) and therefore inhibits protein secretion in green tissues (Richter et al. 2007).


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To verify that Wort, ConcA and BFA affect endosomal pathways, we examined the waveline markers in roots treated with these drugs (Figure S5). Consistent with previous reports, 24Y (RabA5d, recycling endosomes) was affected by 10µM BFA treatment (Geldner et al. 2009). None of the other markers showed significant changes in response to 10µM BFA treatment. 20µM Wort treatment caused extensive aggregation of late endosomes (ARA7, 2Y and RabF2a, 7Y) and recycling endosomes (RabA5d, 24Y). Treatment of roots with, 1µM ConcA lead to considerable endosomal aggregation in almost all lines we tested (early, late and recycling endosomes). These results show that Wort, BFA and ConcA are effective inhibitors of partially overlapping endosomal compartments in root cells.

Having shown that Wort, BFA and ConcA are effective inhibitors of endosomes in the A. thaliana root, we tested how they affect SHR movement. Since the stele is the sole source of SHRGFP in the endodermis, the endodermal to stele (E:S) ratio of GFP fluorescence reflects SHR-GFP movement. Prior to treatment with estradiol, the average E:S ratio for all seedlings was 1.36. (i.e. endodermal fluorescence is 1.36 times higher than the stele). After estradiol treatment, the ratio dropped to 0.48 and then increased in the control roots to 1.01 after 6 hr on estradiol free medium. When recovery was assessed in the presence of Wort or ConcA, the SHR-GFP ratio increased to 0.54 and 0.65 respectively (11% and 32% of the recovery in control roots; Figure S6a and b). In contrast, treatment of roots with 10µM BFA had no significant effect on recovery (in the presence of BFA, the E:S ratio of SHR-GFP was 1.08 after 6 hr recovery). These results suggest that perturbation of endocytotic pathways disrupts SHR movement; however disruption of the recycling endosome appears to have no effect on SHR movement.

To confirm the result of the recovery assay, we quantified FRAP of SHR-GFP in the root endodermis. In these assays, the SHR-GFP signal in the endodermis was bleached using a 488 nm laser. Recovery of SHR-GFP in the endodermis was then monitored over 120 min. In this timeframe, we observed a 60% recovery in the level of SHR-GFP in the endodermis. Roots treated with 10µM BFA showed a 55% recovery over 120 min, indicating that BFA does not inhibit SHR movement


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(Figure 6a and b). In the presence of Wort or ConcA, recovery of SHR-GFP was only 26% and 25% respectively of the prebleached levels (i.e. around half of the controls). Similar results were seen in roots expressing the CPC:SHR-GFP transgene. 120 min after being bleached in root-hair epidermal cells, SHR-GFP levels increased to 85% of the prebleached levels. CPC:SHR-GFP expressing roots treated with Wort or ConcA recovered to 41% and 49% respectively of the prebreached level (again about one-half of the control roots; Figure S6c-d).

To further analyze how the inhibitors affect SHR, the localization of SHR-GFP was examined in protoplasted cells treated with 20µM Wort, 1µM ConcA or 10µM BFA (Figure 7a-d). 10µM BFA did not dramatically change the subcellular localization of SHR-GFP (Figure 7b). In contrast, 20µM Wort led to partial aggregation of SHR-GFP (Figure 7c) and 1µM ConcA caused reduction of endosomal localization of the SHR-GFP signal (Figure 7d). In the Wort treated roots, RabF2a (7R) associated endosomes often formed ring-like structures (Figure 7e). SHR-GFP often clustered near the RabF2a positive vesicles or was surrounded by doughnut-like structures positive for RabF2a-mCherry (Figure 7f and g). This localization is different from the colocalization pattern in the control (Figure 7h). These results show that the endomembrane inhibitors that have the most effect on movement of SHR-GFP also most significantly effect the subcellular localization of the SHR protein. These results reinforce the assertion that localization of SHR to endosomes promotes the intercellular movement of SHR.

In all assays, 10µM BFA had no significant effect on movement of SHR-GFP suggesting that the recycling endosome is not required for SHR movement. To further address this finding, we examined movement of SHR-GFP in roots defective for SORTING NEXIN 1 (AtSNX1). AtSNX1 defines a BFA sensitive endosomal pathway for auxin-carrier recycling. To test whether the AtSNX1 pathway is involved in SHR movement, we examined the CPC:SHR-GFP in the snx1-1 background (Figure 8d). Since SHR-GFP moves from non-hair cells to hair cells in roots, we used the ratio of fluorescence intensity between non-hair and hair cell to evaluate SHR movement. While the variation in the fluorescence ratio was larger in snx1-1, we did not detect a significant difference in movement


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of SHR-GFP between snx1-1 and WT (Figure 8e). Consistent with this finding, we found minimal colocalization between SHR-GFP and SNX1-mRFP (Figure 8f). Therefore, the AtSNX1 mediated endosomal pathway is not essential for SHR trafficking

Association to endosomes and intercellular movement of SHR rely on SIEL. Previous results from our lab have shown that SIEL facilitates the intercellular movement of SHR. Likewise mislocalization of SIEL via disruption of microtubules, inhibits SHR movement. To determine whether SIEL promotes the localization of SHR to endosomes, SHR-GFP was examined in protoplasts made from siel3 mutant seedlings (strong hypomorphs, which have significantly reduced SHR movement; Figure 8c and e), the association of SHR with endosomes was drastically reduced (Figure 8a and b). Collectively these data indicate that both SHR localization and intercellular movement of SHR are dependent upon SIEL and a functional endocytic pathway.

Discussion SHR is one of many transcription factors that can move between cells in A. thaliana (Lee et al. 2006; Rim et al. 2011). Recently we showed that SHR movement occurs via PD, and that careful regulation of movement is important both for activating and inhibiting asymmetric cell divisions in the endodermis (Koizumi et al. 2012a; Koizumi et al. 2012b; Vaten et al. 2011). Structure function analysis of SHR has shown that sequences within the SHR protein are important for intercellular movement and that defects in subcellular localization inhibit SHR movement (Cui et al. 2007;, Gallagher and Benfey 2009). Previous results by Sena et al. (2004) suggested that only stele cell were able to support SHR movement. However here we show that the epidermis is also able to support SHR movement; when expressed in non-hair cells, SHR-GFP was detected also in hair cells indicating cell-to-cell movement. In addition the subcellular localization and movement of SHR in the epidermis was inhibited by the LNELDV mutation that blocks normal localization and movement from stele cells as well. Collectively these results suggest that the factors and pathways that promote SHR movement are more widely expressed in the root than previously thought.


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Recently, we showed that the movement of SHR is promoted through interaction with an essential protein, SIEL that associates with endosomes and interacts with multiple non-cellautonomous proteins (Koizumi et al. 2012a). Here we show that the SHR protein localizes to early, late and recycling endosomes in a SIEL dependent manner. Treatment of roots with BFA, Wort or ConcA showed that early and late endosome functions are required for intercellular movement of SHR, but surprising not recycling endosomes. An inability of SHR to move between cells was strictly associated with a loss of endosomal localization, indicating that endosomes promote signaling functions in plants by facilitating the movement of proteins between cells. Analogous trafficking and signaling functions (Howe 2005; Howe 2004; Miaczynska et al. 2004; Palfy et al. 2012; Sehgal 2008) have been proposed for endosomes in animals.

The endocytic pathway in animals is composed of a series of interrelated compartments including the early, late and recycling endosomes that efficiently recycle membrane components back to the plasma membrane or luminal cargos to various endomembrane compartments including the lysosome for degradation. In plants the early endosome and the trans-Golgi network are not distinct compartments (Contento and Bassham 2012), neither are the late endosome and prevacuolar compartment. Lysosomes are rare in plants; instead proteins are targeted to the vacuole for degradation. In animals, one of the most recognized functions of endosomes is the internalization of plasma membrane receptors and the consequential down-regulation of signaling. Roles for endosomes in the attenuation of signaling in A. thaliana have also been demonstrated, for example through the uptake of the FLAGELLIN-SENSING2 (FLS2) receptor, which is then targeted for degradation (Robatzek et al. 2006). In A. thaliana, endosome mediated recycling also plays critical roles in membrane polarity and in signaling. This has been shown for both PIN1 and PIN2 proteins, which use distinct but related recycling mechanisms for asymmetrical localization in the plasma membrane (Geldner et al. 2003; Jaillais et al. 2006; Pan et al. 2009). The AtSXN1 pathway for PIN recycling is not essential for SHR movement.


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Over the past several years, new roles for endosomes have been proposed, which suggest that endosomes serve as platforms to prevent or enhance protein-protein interactions and hence signaling. For example, Taelman et al. (2010) have shown that WNT signaling is enhanced by the sequestration of cytosolic GSK3 to multivesicular endosomes, which prevents the phosphorylation and subsequent down-regulation of GSK3 targets. In contrast, interaction with early endosomes by SMAD2 promotes protein-protein interactions that result in the phosphorylation and movement of SMAD2 to the nucleus. The activation of SMAD2 begins with the internalization of the activated TGFβ receptor. The early endosome then serves as a platform for the association SARA, which recruits SMAD2. SMAD2 can then be phophorylated by activated TGFβ and carried to the nucleus where it activates gene expression. The early endosome therefore provides two functions in SMAD2 signaling; it acts as a stage for the assembly of the SMAD2 protein complex and subsequent posttranslational modification, and as a directional carrier to facilitate nuclear localization.

The idea that the endomembrane promotes intercellular movement of proteins between plant cells is supported not only by analysis of SHR, but also by the association of virally encoded mobile proteins with the ER and endosomes (Haupt et al. 2005; Ju et al. 2005; Lewis and Lazarowitz 2010; Wu et al. 2011). For example the triple gene block (TGB) proteins from Potato mop-top virus (PMTV) interact both with the ER and early endosomes. Mutations in the TGB3 that blocked localization to the ER also blocked association with PD (Haupt et al. 2005). The movement of Cabbage leaf curl virus Movement Protein (CaLCuV MP), and the Tobamovirus Tobacco mosaic virus Movement Protein (TMV MP30K) are both dependent upon interaction with the SYNTAGOMIN A protein, which localizes to endosomes. Knockdown of SYTA decreased the formation of endosomes and the cell-to-cell spread of both TMV MP30K and CaLCuV MP suggesting that endosome recycling promotes the transport of the viral movement proteins to PD (Lewis and Lazarowitz 2010).


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Since PD are integrated within the plant endomembrane (bound by the plasma membrane and spanned by the ER-derived desmotubule), it is not surprising that endocytic pathways would promote PD mediated movement (Oparka 2005). Recently Liu et al. (2012) showed that FLOWERING LOCUS T (FT) movement is dependent upon the ER localized, FT INTERACTING PROTEIN (FTIP). The authors suggest that localization of FTIP to the ER allows FT to interact with the desmotubule. The FT-FTIP complex then either passes through the desmotuble or undergoes lateral diffusion in the ER membrane to pass through the PD into the neighboring cell.

It is not clear how localization to early and late endosomes promotes SHR movement. One possibility is that endosomes, via interaction with SIEL facilitate the movement of SHR to the plasma membrane where SHR then diffuses to PD (Figure S7). However the lack of an effect on SHR movement in roots treated with BFA, which targets the recycling endosome argues against this. In animal cells the late endosome can perform some of the functions of a recycling endosome and move bi-directionally between the nucleus and the plasma membrane. Defects in bi-directional movement cause an aggregation of late endosomes in a perinuclear localization (Lebrand et al. 2002; Loubery et al. 2008) that is similar to SIEL localization when microtubules are disrupted with oryzalin. In animals, multi-vesicular (late) endosomes can also fuse with the plasma membrane (Figure S7, scenario 1), thus SHR could hitch-hike on vesicles destined for the PM. To our knowledge, fusion of the late endosome with the plasma membrane has not been shown in plant cells, making this scenario less likely than other possible set-ups. However Jaillais and Gaude (2007) suggest that the LE/PVC is involved in the recycling of PIN1 back to the plasma membrane so late endosomes may have a role in membrane recycling.

An alternative and perhaps more likely explanation for how SHR movement is promoted by endosomes is that endosomes provide a stable platform (a meeting place akin the scenario with SARA and SMAD2) for the association of SHR with SIEL and perhaps other proteins that render it competent to move between cells, or proteins that directly facilitate transport (Figure S7, numbers 2 and 3). Support for this hypothesis comes from the finding that 10 µM BFA, and therefore disruption


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of the RabA5d positive recycling endosome has no effect on SHR movement. Likewise previous studies have shown that disruption of actin, which is thought to mediate endosome movement in plants, only mildly decreases SHR movement (Wu and Gallagher 2013). Further studies are required to determine whether endosomes provide a direct trafficking function that promotes SHR movement or whether endosomes serve primarily as a stable platform for protein-protein interactions that facilitate intercellular trafficking of SHR. Since SIEL interacts with multiple non-cell-autonomous transcription factors this could be a widespread pathway for protein movement.

Experimental procedures Plant material and growth conditions: Arabidopsis thaliana ecotype Col-0 was used as the wildtype. Plants were germinated and grown vertically on 1.0 x Murashige & Skoog (MS) medium (Caisson Laboratories, North Logan, UT) containing 0.05% w/v MES (pH 5.7), 1.0% w/v sucrose and 1.0% granulated agar (Difco Laboratories, Detroit, MI) in a growth chamber at 22°C under a 16 h light/8 h dark cycle. Plants were used 5 days after plating unless otherwise stated. All endosomal marker lines (waveline markers) were purchased from The Nottingham Arabidopsis Stock Centre (NASC). The CPC:SHR-GFP line was crossed into each individual waveline. Homozygous seedlings expressing both the SHR–GFP and the mCherry tagged endosomal markers were selected based upon fluorescence.

Treatment with inhibitors: Stock solutions of 10mM 17–β–estradiol (Sigma, St. Louis, MO), 20 mM Wortmannin (Wort), 1mM Concanamycin A (ConcA) and 10mM Brefeldin A all in dimethylsulfoxide (DMSO) were prepared and stored at −20°C. Plates containing the specified inhibitors were prepared by adding the appropriate stock solution to liquified ½ MS agar. Controls received the same amount of DMSO. The callose induction and recovery studies were performed as described previously (Wu and Gallagher 2013). For treatment of protoplasts, the inhibitors were added into the incubation solution and the mixture was kept at room temperature for 3-4 hr before imaging.


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Arabidopsis Protoplast Transient Expression: Protoplasts were isolated from 3-week-old plants grown under normal light conditions. To remove the epidermal cell layer, we utilized a previously reported tape-Arabidopsis sandwich method (Wu et al. 2009). The mesophyll cells were digested by an enzyme solution containing 1.5% Cellulase R-10 and 0.4% Macerozyme R-10 (Yakult Pharmaceutical, Tokyo, Japan) for 30 min-1 h. The transfection was conducted as described by (Yoo et al. 2007). The freshly isolated protoplasts were incubated with 20 to 30 μg of plasmid DNA, and then mixed with an equal volume of a solution of 40% PEG (MW 4000; Fluka, Ronkonkoma, NY) with 0.1 M CaCl2 and 0.2 M mannitol. The mixture was kept at room temperature for 5-8 min and then washed in W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, and 2 mM MES, pH 5.7) followed by incubation overnight in the dark. Protoplasts were imaged 16 to 20 h after transfection.

Microscopy and imaging: For confocal microscopy roots were stained with propidium iodide (0.01 μg/ml in water) for 1 min and then imaged using a Leica TCS SL microscope with the appropriate filter sets for visualizing GFP and propidium iodide. Confocal images were captured using GFP/mRFP filter sets. FRAP of SHR-GFP was performed using a Leica TCS SL confocal. Seedlings expressing SHR-GFP were removed from solid media and then incubated in liquid ½ MS medium on a slide at room temperature. Before imaging, the inhibitors were added into the liquid ½ MS medium (10uM BFA, 20uM Wort or 1uM ConcA). The SHR-GFP signal was bleached in either endodermal cells or root-hair epidermal cells with 5 iterations at full laser power, which lead to around 70-80% reduction in fluorescence. Multiple cells in each cell layer were bleached at the same time for each root. To acquire recovery images, laser power was set to 20% to avoid bleaching. The recovery images of the bleached areas were captured every 40 min to reduce the possibility of additional bleaching from frequent scanning. Several methods can be used to facilitate the identification of the same region of interest during the time-course imaging. First, the X, Y and Z position of the initial images and bleaching steps are noted. Second the cells expressing the fluorescent protein often form recognizable patterns that can be used as landmarks to identify the


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same region of interest. Thirdly, a brief Z-stack series of sections with short interval (several µm) around the region of interest were captured. In this way, the same region should be included in the series of images and can be re-tracked later for analysis. Only cells with clear nuclear localized signal in the endodermis from the same focal plane were used for calculation. The fluorescence intensity ratio (endodermis/stele) before bleach, after bleach and after recovery was determined using ImageJ. The time 0 post-bleach fluorescence was set to 0 and all measurements were normalized to this point by subtracting the time 0 post-bleach fluorescence signal from all of the pre-bleach and the recovery values. The percent recovery was calculated using the normalized values. For analysis of intracellular FRAP, a frame size of 125X125 pixel with a scanning speed of 800 Hz and bidirectional scanning was used to accelerate the scanning rate for each frame. The protoplasts were imaged on a Zeiss 710 confocal using GFP/mRFP filter sets. For co-localization imaging, GFP emission was captured using a 505-530 nm filter and mCherry fluorescence was selected with a 590-620 nm filter. The quantification of colocalization was performed as described before (Fendrych et al. 2013). As the vesicles in protoplasts were well dispersed within the cell, colocalization can be easily determined by evaluating each labeled spot by eye. All vesicles detected in both green and red channels were counted and the percentage of GFP only, mCherry only and colocalization were calculated respectively. The time-course observation was performed as described before (Ambrose et al. 2013).

Plasmid Construction and Transformation: The full-length SHR and SHRΔLNELDV were cloned into pDONR221 using standard Gateway protocols (Invitrogen/ Life Technologies). The entry clones were recombined with other entry clones (pCPC in pDONR P4-P1R and GFP in pDONR P2R-P3) into dpGreen BarT (Lee et al. 2006) using standard Gateway protocols (Invitrogen/Life Technologies). The binary vectors were introduced into Agrobacterium strain GV3101-pSoup-pMP and transformed into Arabidopsis using the floral dip method. To make the vectors for protoplast transfection, pDONR221 containing SHR and SHRΔLNELDV were recombined into pBlueScript1510 (pK7GWF2 and pK7FWG2 cassette to pBLUESCRIPT KS plasmid) (Pomeranz et al. 2010).The recombined plasmids were then extracted using Qiagen Endofree Maxi kit (12362).


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Acknowledgements N. Geldner provided assisitance and advice with the wave lines. J Ugochukwu provided technical support. C-M Lee and S. Price provided comments on the manuscript. A. Stout manages the confocal facility. S. Wu is supported by an NSF grant, 1243945 awarded to K.L. Gallagher. The authors declare no competing interests.

Short legends for Supporting Information Supplemental Figure 1. Subcellular localization of wild-type and mutant SHR-GFP. Supplemental Figure 2. SHR is not uniformly mobile in the cytoplasm of stele cells. Supplemental Figure 3. Co-localization of SHR-GFP with markers of the endomembrane. Supplemental Figure 4. SHR-GFP tagged endosomes move within the cell. Supplemental Figure 5. Effects of drug treatments on endosome morphology. Supplemental Figure 6. Wortmanin and Concanamycin A inhibit the recovery of SHR-GFP movement. Supplemental Figure 7. Schematic diagram illustrating possible pathways for SHR movement.

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Figure Legends Figure 1. SHR can move not only from the stele into the endodermis, but also from non-hair cells to hair cells in the root epidermis. (a-f) Confocal cross-sections through the middle of A.thaliana roots expressing (a and d) cell autonomous CPC:erGFP; (b and e) mobile CPC:SHR:GFP and (c and f) immobile CPC:SHRΔLNELD-GFP.


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(g-i) Confocal sections of the root epidermis expressing (g) CPC:erGFP; (h) CPC:SHR:GFP and (i) CPC:SHRΔLNELDV-GFP. “E” marks the endodermis; “C” marks the cortex; “NH” marks non-hair cells and “H” marks hair cells throughout. Bars = 50 µm.

Figure 2. Mobile SHR localizes to the endomembrane. (a-b) Root epidermal cells expressing (a) SHR-GFP but not (b) SHR ΔLNELDV-GFP (labeled SHR∆L) show association of SHR with vesicles. White arrows point to punctate structures. (c and d) Root epidermal cells expressing free GFP shown by (c) pSUC2:GFP and (d) 35S:GFP. (e and f) Protoplasted cells expressing (e) SHR-GFP but not (f) SHR ΔLNELDV-GFP (labeled SHR∆L) show associations of SHR with vesicles. Both (e) and (f) are Z-stack projections created from optical sections through a single protoplast. “N” indicates the nucleus. Note the reduced nuclear localization of SHR∆L in both the root epidermis (b) and protoplast (f). (g and h) Protoplasted cells expressing free GFP shown by (g) pSUC2:GFP and (h) 35S:GFP. (i and j) are single confocal optical sections through protoplasts showing SHR-GFP and FM4-64 (after 50 min incubation) (i) at the surface and (j) in a median section. Yellow arrows indicate areas of colocalization. Bars = 10 µm.

Figure 3. SHR co-localizes with endosomal markers. (a-d) All images in the left column are protoplasts expressing SHR-GFP. The middle column is the mCherry tagged endomembrane marker and the column to the right is the overlay of the SHR-GFP and mCherry signals. Yellow arrows point to areas of co-localization. For the middle column: (a and b) 7R, Rabf2a-mCherry (as labeled); a marker of the late endosome “LE” . (c and d) Shows 13R, VTI12-mCherry a marker of the early endosome “EE”. Bars = 10 µm. (e) Quantification (%) of the SHR-GFP and endosomal marker colocalizations in protoplasted cells. Green, red, and yellow colors represent percentage of vesicles with GFP only, mCherry only, and their colocalization, respectively. For pixel shifted controls, colocalization with 7R, dropped from 14.1% to 0.7%; 13R dropped from 15.6% to 0.5%; 24R from 7.1% to 0.5% and 25R from 8.7% to 0.6%.


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Figure 4. SHR-GFP localizes to endosomes in intact root tissues. (a-e) Confocal images of root epidermal cells expressing both SHR-GFP (left column) and mCherry tagged waveline markers (middle column of each panel). Co-localization is indicated by yellow arrows and a lack of colocalization is indicated by white arrows. (a) 7R, Rabf2a-mCherry, marks late endosomes; (b) 13R, VTI12- mCherry marks trans-Golgi network/early endosomes; (c) 22R, SYP32 mCherry marks Golgi; (d) 24R, RabA5d- mCherry marks the endosome/recycling endosome and (e) 25R, RabD1 mCherry marks the post-Golgi/endosomes.

Figure 5. SHR-GFP co-migrates with endosomal markers in both time and space. (a) SHR-GFP with 7R (Rabf2a-mCherry, late endosome). (b) The 90 s compression of the time series images in (a) along the x axis, showing the identical trafficking pattern between SHR and the late endosome. (c) SHR-GFP with 13R (VTI12-mCherry, early endosome). (d) The 90 s compression of the time series images in (c) along x axis, showing the identical trafficking pattern between SHR and the early endosome.

Figure 6. Wort and ConcA, but not BFA inhibit intercellular movement of SHR. (a) Confocal images showing SHR-GFP recovery in photo-bleached root endodermal cell over a 120 min time course. The bleached cell files were marked by the asterisk. (b) Quantitative measurements of SHR-GFP recovery (numbers on the y-axis are percent x 100) under the conditions indicated (For each measurement, n=3 replicates, 9 roots and 97-144 cells). Bars = 50 µm.


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Figure 7. Pharmacological inhibition of endosomes affects subcellular localization of SHR. (a and b) Wild-type leaf protoplasts expressing SHR-GFP. The protoplasts are treated with (a) DMSO (b) 10µM BFA. (c and d) Leaf protoplasts expressing both SHR-GFP and endosomal markers are treated with (c, e and f) 20µM Wort or (d) 1µM ConcA for 3 hr. (e) Ring-like structure of 7R (RabF2a-mCherry) associated endosomes detected in Wort treated protoplasts. (f) SHR-GFP signal often clustered near the RabF2a positive vesicles. Compare to untreated control protoplasts in which SHR-GFP often co-localizes with RabF2a (in g).

Figure 8. Localization of SHR to endosomes relies on SIEL. (a) A series of optical sections showing SHR-GFP through the z–axis of protoplasts made from siel3 mutant leaves transformed with SHR-GFP. The last micrograph is a 3D projection of the Z-stack. (b) Quantification of SHR-GFP associated vesicles in the protoplasts from WT and siel3. The quantifiation is based on 32 protoplasts of WT and 10 protoplasts of siel3. Two-tailed Student's t-test is used to evaluate the difference (p = 2.54767E-12). (c-e) SHR-GFP movement between epidermal cells in WT, siel3 and snx1-1 roots. The fluorescence intensity ratio between hair (H) and non-hair (NH) cells was used to evaluate movement. The quantifiation is based on 42-75 cells from five roots for each genotype. A significant reduction of H:NH ratio is seen in siel3 compared to WT (p = 0.0024) while this is not seen in snx1-1. (f) SHR-GFP has a low co-localization with SNX1-mRFP. Green, red, and yellow colors represent percentage of vesicles with GFP only, mRFP only, and their colocalization, respectively.


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