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The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate

  1. Xingyun Qi
  2. Akira Yoshinari
  3. Pengfei Bai
  4. Michal Maes
  5. Scott M Zeng
  6. Keiko U Torii  Is a corresponding author
  1. Howard Hughes Medical Institute and Department of Biology, University of Washington, United States
  2. Institute of Transformative Biomolecules (WPI-ITbM), Nagoya University, Japan
  3. Howard Hughes Medical Institute and Department of Molecular Biosciences, The University of Texas at Austin, United States
  4. Department of Physics, University of Washington, United States
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Cite this article as: eLife 2020;9:e58097 doi: 10.7554/eLife.58097

Abstract

Receptor endocytosis is important for signal activation, transduction, and deactivation. However, how a receptor interprets conflicting signals to adjust cellular output is not clearly understood. Using genetic, cell biological, and pharmacological approaches, we report here that ERECTA-LIKE1 (ERL1), the major receptor restricting plant stomatal differentiation, undergoes dynamic subcellular behaviors in response to different EPIDERMAL PATTERNING FACTOR (EPF) peptides. Activation of ERL1 by EPF1 induces rapid ERL1 internalization via multivesicular bodies/late endosomes to vacuolar degradation, whereas ERL1 constitutively internalizes in the absence of EPF1. The co-receptor, TOO MANY MOUTHS is essential for ERL1 internalization induced by EPF1 but not by EPFL6. The peptide antagonist, Stomagen, triggers retention of ERL1 in the endoplasmic reticulum, likely coupled with reduced endocytosis. In contrast, the dominant-negative ERL1 remained dysfunctional in ligand-induced subcellular trafficking. Our study elucidates that multiple related yet unique peptides specify cell fate by deploying the differential subcellular dynamics of a single receptor.

Introduction

Receptor-mediated endocytosis is an integral part of cellular signaling, as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. In mammalian systems, endocytosis of receptor tyrosine kinases can attenuate the signal outputs, by removing the active receptor pools from the plasma membrane, or it can specify signals at defined sites of action, such as signaling through endosomes (Sigismund et al., 2012). As a sessile organism, plants make use of a large number of receptor-like kinases (RLKs) for cell-cell, shoot-to-root, and inter-kingdom communications (Shiu and Bleecker, 2001). The RLKs with extracellular leucine-rich repeat domain, known as LRR-RLKs, comprise the largest RLK subfamily (Shiu and Bleecker, 2001), and they specify critical aspects of development, environmental response, and immunity by perceiving extrinsic signals (Torii, 2004; Macho and Zipfel, 2014). Increasing evidence shows that the subcellular localization and trafficking routes of LRR-RLKs regulate their function and activity (Ben Khaled et al., 2015). In Arabidopsis, bacterial flagellin peptide flg22 induces the heterodimer formation consisting of the LRR-RLKs FLAGELLIN SENSING2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE (BAK1)/SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 3 (Chinchilla et al., 2007). This triggers the endocytosis and degradation of the receptor complex to generate transient cellular immune signaling but also to prevent continuous signaling to the same stimulus (Robatzek et al., 2006; Beck et al., 2012). The brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE1 (BRI1) forms a complex with BAK1 (Li et al., 2002; Nam and Li, 2002; Bücherl et al., 2013). BRI1 can undergo constitutive endocytosis independent of BRs, but BRs can elevate BRI1 and BAK1 interaction and reduce the number of available BRI1-BAK1 complexes on the plasma membrane (Geldner et al., 2007; Bücherl et al., 2013; Hutten et al., 2017). CLAVATA1 (CLV1), an LRR-RLK that controls stem cell homeostasis within the shoot meristem (Clark et al., 1997), is downregulated by ligand-dependent internalization upon perception of its ligand CLV3 (Nimchuk et al., 2011). It remains a key question as to where within the cell these LRR-RLKs transduce signals and how different activation states of LRR-RLKs influence their subcellular localization.

Developmental patterning of stomata, adjustable pores on the plant epidermis for gas-exchange and transpiration, relies on intricate cell-cell communication mediated by signaling peptides and their receptors (Lau and Bergmann, 2012; Pillitteri and Torii, 2012). In Arabidopsis, secreted peptides from the EPF family, and their shared receptor LRR-RLKs, ERECTA, ERL1, and ERL2, mediate this process (Rychel et al., 2010). Amongst the plant LRR-RLKs, the ERECTA family offers a unique advantage to study how multiple signals are perceived to achieve cell fate and patterning. EPF2 and EPF1 negatively regulate stomatal development primarily through ERECTA and ERL1, respectively (Hara et al., 2007; Hara et al., 2009; Hunt and Gray, 2009). In contrast, EPF-LIKE9 (EPFL9), also known as Stomagen, promotes stomatal development by competing with EPF2 and, to some extent, with EPF1 for receptor binding (Sugano et al., 2010; Lee et al., 2015; Lin et al., 2017; Qi et al., 2017). Moreover, EPFL4/5/6, a subfamily only expressed in hypocotyls and stems, also act as ligands for the ERECTA family to inhibit stomatal formation when an LRR receptor protein, TOO MANY MOUTHS (TMM), is missing (Abrash and Bergmann, 2010; Abrash et al., 2011). Although the final phenotypic outcomes of these different EPF signaling events are well characterized, the very early step of signal transmission by the receptors remain elusive. While internalization of ERL2 was documented briefly (Ho et al., 2016), it is unknown whether it has any implications in signal transduction or in which subcellular organelle ERL2 was localized.

ERL1 regulates guard cell differentiation in an autocrine manner in addition to enforcing stomatal spacing of neighboring cells in a paracrine manner (Lee et al., 2012; Qi et al., 2017). This dual function of ERL1 can be attributed to its cell-type specific expression patterns as well as its ability to perceive different EPF/EPFL peptide ligands (Shpak et al., 2005; Lin et al., 2017). On the other hand, its sister receptor ERECTA is broadly and ubiquitously expressed in the epidermis (Horst et al., 2015). The specific expression of ERL1 in stomatal meristemoids as well as its dual function as an autocrine and paracrine signaling receptor provide a unique advantage to study how receptor subcellular dynamics translates into the eventual cell fate. Here, we combined genetic, pharmacological, and live imaging approaches to explore the initial events that occurred at ERL1 upon perception of different EPF peptides. Our study shows that EPF1 and EPFL6, the ligands activating the inhibitory stomatal signaling, trigger ERL1 endocytosis into multi-vesicular bodies/late endosomes (MVB/LEs). TMM, which can form a receptor complex with ERL1, is required for the EPF1-induced ERL1 internalization and suppression of stomatal fate but is superfluous for EFPL6-induced ERL1 internalization. Surprisingly, Stomagen interferes with the inhibitory regulation of stomatal differentiation by retaining ERL1 to the endoplasmic reticulum, similar to when endocytosis was pharmacologically blocked by Tyrphostin A23 (Tyr A23) (Santuari et al., 2011) and Endosidin 9–17 (ES9-17) (Dejonghe et al., 2019). Additionally, we extensively examined the effects of Brefeldin A (BFA) on subcellular organelle behaviors in stomatal meristemoids and established optimal conditions to observe BFA body formation in true leaves. Combined, our study reveals a mechanism by which plant cells interpret multiple signals through the subcellular localization and trafficking route of a single receptor.

Results

ERL1 undergoes endocytosis through multivesicular bodies to vacuole in stomatal meristemoids

To understand how stomatal cell fate decisions are made at the level of receptor subcellular dynamics, we first examined the localization of ERL1 (Figure 1). As reported previously (Qi et al., 2017), a functional ERL1-YFP fusion protein driven by its endogenous promoter (ERL1pro::ERL1-YFP) in erl1 seedlings marks the plasma membrane of stomatal-lineage cells, most notably late meristemoids. In addition, we detected some punctae highlighted by ERL1-YFP that co-localized with FM4-64, a styryl dye used to trace the endocytic pathways in plants (Meckel et al., 2004Figure 1A). To define the subcellular localization of ERL1-YFP, its co-localization analysis was performed with marker proteins SYP43-RFP for trans-Golgi network/early endosomes (TGN/EEs), RFP-ARA7 for MVB/LE, and SYP22-RFP for vacuoles (occasionally MVB/LE)(Ebine et al., 2011; Postma et al., 2016Figure 1B, Video 1). ERL1-YFP extensively co-localizes and moves together with RFP-ARA7 (Figure 1B,C, Video 1), whereas only 25% and 18% of ERL1-YFP-positive punctae are also labelled by SYP43-RFP and SYP22-RFP, respectively. Thus, ERL1-YFP predominantly resides on the MVB/LE. This is further confirmed by a pharmacological approach using Wortmannin (Wm), a fungal drug that can cause fusion of MVB/LEs by inhibiting phosphatidylinositol-3 (PI3) and phosphatidylinositol-4 (PI4) kinases (Foissner et al., 2016). The Wm application on Arabidopsis seedlings resulted in the formation of typical ring-like Wm bodies marked by both ERL1-YFP and RFP-ARA7 (Figure 1D; Figure 1—figure supplement 1A). These Wm bodies are much larger (average size of 0.993 ± 0.203 μm) than the endosomes (average size of 0.383 ± 0.063 μm), thus easy to recognize and quantify (Figure 1—figure supplement 1B).

Figure 1 with 1 supplement see all
ERL1-YFP has dual localization on plasma membrane and late endosomes.

(A) Representative confocal microscope images of ERL1-YFP expression in a meristemoid (yellow) co-stained with 10 μM FM4-64 (magenta). Cyan arrowheads, ERL1-YFP endosomes co-localizing with FM4-64 stains. Scale bars = 7.5 µm. (B) Representative confocal microscope images of ERL1-YFP co-localization analysis with the TGN marker SYP43-RFP (top), the MVB/LE marker RFP-ARA7 (middle), and the MVB/LE and vacuole marker SYP22-RFP (bottom) in the abaxial epidermis of developing true leaves of the 7-day-old seedlings. Merged images are shown in the third column, with enlarged images of representative meristemoids in the right column. Arrowheads point to endosomes bearing ERL1-YFP, SYP43-RFP, RFP-ARA7, and/or SYP22-RFP: cyan, single channels; green, YFP; magenta, RFP; white, co-localization . Scale bars = 10 µm. (C) Quantitative analysis of the co-localized endosomes between ERL1-YFP and the subcellular marker proteins. Percentage of the endosomes of the former protein that co-localize with the latter protein is shown as dots. Lines in the boxplot show the median value of each group, and the boxes represent from the first to third quartiles. Number of cells analyzed, n = 40 for ERL1 vs ARA7 or ARA7 vs ERL1; n = 12 for ERL1 vs SYP43 or SYP43 vs ERL1; n = 7 for ERL1 vs SYP22 or SYP22 vs ERL1. (D) ERL1-YFP and RFP-ARA7 treated with Wm. Shown are RFP-ARA7 (left column) and ERL1-YFP (middle column) in the abaxial epidermis of developing true leaves of the 7-day-old seedlings treated with mock (top row) or 30 µM Wm (bottom row). Arrowheads point to ERL1-YFP and/or RFP-ARA7 endosomes: cyan, single channels; magenta, YFP; white, co-localization. Scale bars = 10 µm.

Video 1
ERL1-YFP and RFP-Ara7.

A time-lapse movie of an Arabidopsis meristemoid co-expressing ERL1-YFP and RFP-Ara7. Images were taken every 30 s for 10 frames and presented as four frames per second.

Receptors internalized via MVB/LE are destined for degradation in lytic vacuoles (Reyes et al., 2011). Because we did not observe clear accumulation of ERL1 in a vacuole, and co-localization of SYP22-RFP and ERL1-YFP was limited to the endosomes (Figure 1B,C), we took a pharmacological approach using Concanamycin A, a specific inhibitor of vacuolar H+-ATPase that is known to reduce protein degradation in the lytic vacuole (Kleine-Vehn et al., 2008). As shown in Figure 2, Concanamycin A treatment following the wash-out of the endocytic tracer dye FM4-64 led to accumulation of strong vacuolar YFP signals surrounded by an FM4-64 stained tonoplast. Taken together, our results indicate that, within the stomatal precursor cells, ERL1 undergoes endocytic trafficking from the plasma membrane to MVB/LE and is destined to vacuolar degradation.

ERL1 is transported into vacuole.

(A) Representative confocal microscopy images of ERL1-YFP (yellow) and FM4-64 (magenta) in the abaxial epidermis of developing true leaves of the 7-day-old seedlings expressing ERL1pro::ERL1-YFP in erl1. The seedlings were treated with 2 μM FM4-64 for 30 min followed by incubation in water for 6 hr and subsequently incubated in 1 μM Concancmycin A for 5 hr. Tonoplast is highlighted by FM4-64. Scale bars = 10 µm. (B) Enlarged merged image of ERL1-YFP (yellow) and FM4-64 (tonoplast, magenta). Scale bars = 5 µm. (C) Line plots of ERL1-YFP (yellow) and FM4-64 (magenta) fluorescence on 10-pixel-thickness of arrow indicated in (B). Arrows indicate FM4-64 fluorescence staining the tonoplast.

The effects of BFA on endomembrane behaviors in stomatal meristemoids

Endocytosis is an essential process to regulate cell signaling by controlling the turnover of plasma membrane proteome. Activated plant receptor kinases can either be recycled back to the plasma membrane or are destined for endocytic degradation via MVB/LE for signal termination. BFA, a chemical inhibitor of ADP-ribosylation factor guanine-nucleotide exchange factors (ARF-GEFs) including GNOM has been widely used to block endosomal recycling and endocytosis of membrane proteins (Geldner et al., 2003). BFA treatment in roots results in formation of characteristic BFA bodies, which are conglomerates of TGN surrounded by the Golgi apparatus (Geldner et al., 2003). It has been reported, however, that BFA application to Arabidopsis leaves triggers re-absorption of Golgi membrane collapsed onto the endoplasmic reticulum (Robinson et al., 2008; Langhans et al., 2011). Thus, to study the effects of BFA on ERL1 endomembrane dynamics, we first sought to fully document the BFA effects in stomatal meristemoids. The previous reports used high concentration (e.g. 50, and 90, 180, 360 μM of BFA) (Robinson et al., 2008; Langhans et al., 2011) compared to the lower concentration (30 μM of BFA) generally used in roots.

First, we treated Arabidopsis seedlings expressing the endoplasmic reticulum marker, GFP-HDEL (Mitsuhashi et al., 2000) or the Golgi marker ST-YFP (Takagi et al., 2013) co-stained with FM4-64 with different BFA concentrations (0, 30, 90, 180 μM) (Figure 3). Low BFA concentration at 30 μM does not confer any discernable effects on the endoplasmic reticulum (GFP-HDEL; Figure 3A,B). Under this condition, BFA bodies (magenta arrows) are surrounded by ST-YFP (green arrows, Figure 3C), exhibiting characteristic membrane organization of BFA bodies reported in roots (Figure 3D). These BFA bodies are also observed at 90 μM treatment (Figure 3B,C, magenta arrows). In contrast, BFA at very high concentration, 180 μM, conferred aberrant spherical structures of the endoplasmic reticulum (Figure 3A,B), which was also previously reported (Nakano et al., 2009). Indeed, at 180 μM BFA treatment, we observed Golgi absorbed ER, which confers ring-like structure of membrane accumulating ST-YFP (Figure 3C). These results demonstrate that previously reported effects of BFA on leaves were observed only when extremely high concentration of BFA was applied.

Figure 3 with 1 supplement see all
Effects of BFA on subcellular membrane structures in the meristemoids.

(A) Representative Z-stack confocal images of an ER marker, GFP-HDEL (green) and FM4-64 (magenta) in true leaf abaxial epidermis from the 7-day-old seedlings expressing CaMV35Spro::GFP-HDEL treated with 5 µM FM4-64 alone (mock) or co-treated with 30, 90, 180 µM BFA for 1 hr. Scale bars = 20 µm. BFA treatment at low concentration (30 µM) does not alter the characteristic, mesh-like ER structure inside the meristemoids and at the edge of nuclei. In contrast, high concentration of BFA results in aberrant spherical structures in the ER. (B) Representative confocal images of an ER marker, GFP-HDEL (green) and FM4-64 (magenta) treated with BFA as described in (A). BFA treatment at low concentration (30 µM) causes the formation of BFA bodies (magenta arrows) without impacting the ER structure. In high BFA concentration confers aberrant spherical ER structure (green arrows). In addition, the FM4-64 signals in the BFA bodies disappear. Scale bars = 5 µm. (C) Representative confocal images of a Golgi marker, ST-YFP (green) and FM4-64 (magenta) in true leaf abaxial epidermis from the 7-day-old seedlings expressing CaMV35Spro::N-ST-YFP treated with 5 µM FM4-64 alone (mock) or co-treated with 30, 90, 180 µM BFA for 1 hr. Scale bars = 5 µm. In a lower and medium concentration (30 and 90 µM) of BFA, Golgi (green arrows) are surrounding BFA bodies (magenta arrows). By contrast, in higher concentration (180 µM), the Golgi marker becomes collapsed (green arrow) to ER. (D) Schematic diagrams depicting probable subcellular membrane structures in the meristemoids based on confocal microscopy.

Next, to evaluate the intactness of different endomembrane organelles in stomatal meristemoids when treated with 30 μM BFA, we examined seven endomembrane markers, YFP-SYP32 (cis-Golgi), N-ST-YFP (trans-Golgi), GFP-SYP43 (TGN/EE), YFP-RabA1e (TGN/EE, plasma membrane), YFP-RabA5d (uncharacterized endosomes), ARA6-GFP (MVB/LE, plasma membrane) and YFP-ARA7 (MVB/LE) co-stained with FM4-64 (Geldner et al., 2009; Ebine et al., 2011; Postma et al., 2016). All marker lines show the formation of characteristic BFA bodies in meristemoids, without discernible collapse of Golgi apparatus into the endoplasmic reticulum (Figure 3—figure supplement 1). Taken together, our extensive documentation shows that 30 μM BFA confers BFA body formation just like when applied to roots. Therefore, BFA can be used to study the endocytosis of ERL1 in meristemoids.

TMM is required for the process of ERL1 endocytosis in true leaves

We wondered if the ERL1 endocytosis that we observed (Figures 1 and 2) is related to its biological signaling. A previous work has shown that ERL1 forms a heterodimer with TMM, a receptor protein, to create a pocket for the proper binding of its major ligand EPF1 (Lee et al., 2012; Lin et al., 2017). The absence of TMM results in clustered stomata (Figure 4A), indicating that TMM is required for EPF1-ERL1 signaling to enforce proper stomatal spacing (Hara et al., 2007; Lee et al., 2012). As a first step to test whether active ERL1 signaling is a prerequisite for its endocytosis, we monitored ERL1 dynamics in tmm background (Figure 4). The number of ERL1-YFP-positive endosomes per meristemoid cell was reduced in tmm (Figure 4B; p=1.58e-14, Figure 4—figure supplement 1). Fluorescent volume intensity (voxel) ratio analysis also revealed statistically-significant reduction of ERL1-YFP-positive endosomes in tmm (Figure 4D, p=0.0049) as well as significant reduction of ERL1-YFP-positive BFA bodies in 30 µM BFA-treated tmm (Figure 4D, p=4.64e-11) compared to those in wild type. To assess the role of the secretory pathway in the observed difference in ERL1-YFP BFA bodies between wild-type and tmm, the BFA treatment was further performed in the presence of protein synthesis inhibitor cycloheximide (CHX). Treatment with 50 µM CHX for 1 hr followed by either mock or 30 µM BFA led to reduction in the number of ERL1-YFP-positive BFA bodies in tmm mutant (Figure 4—figure supplement 1, p=0.0154). Thus, ERL1-YFP-positive BFA bodies are reduced in tmm mutant regardless of the presence or absence of CHX, thereby suggesting that the absence of TMM does not impact the ERL1 secretory pathway. Next, we treated the seedlings with Wm, which conferred significant reduction of ERL1-YFP-labelled Wm-bodies in tmm compared to that in wild type (Figure 4E,F, p=1.40e-5). Combined, the results suggest that TMM is critical for the endocytosis/internalization of ERL1 to MVB/LE.

Figure 4 with 3 supplements see all
ERL1 internalization requires its co-receptor TMM.

(A) Representative confocal microscopy images of PI-stained true leaf abaxial epidermis of ERL1-YFP in erl1 (top) and in erl1 tmm (bottom), the latter shows characteristic stomatal cluster phenotype (orange brackets). Scale bars = 10 µm. (B) ERL1-YFP subcellular localization in erl1 (top row) and in erl1 tmm (bottom row) in the true leaf abaxial epidermis from the 7-day-old seedlings. Right column; enlarged images. Their stomatal phenotypes are shown in (A). Arrowheads indicate endosomes. Scale bars = 10 µm. (C) Representative confocal images of ERL1-YFP in erl1 (top row) or in erl1 tmm (bottom row) of the abaxial epidermis of developing true leaves from the 7-day-old seedlings treated with mock (left column) or 30 µM BFA (right column). Arrowheads indicate BFA bodies. Scale bars = 10 µm. (D) Quantitative analysis of the volume ratio of YFP-positive endosomes and BAF bodies per cell volume (voxels) when ERL1-YFP in erl1 (yellow) or erl1 tmm (orange) are treated with mock or 30 µM BFA. Welch’s Two sample T-test was performed for pairwise comparisons. Experiments were repeated three times. Two-way ANOVA analysis: genotype (WT, tmm), p<3.85e-14; treatment (mock, BFA), p<2.2e-16. The total numbers of cells subjected to YFP signal intensity measurements are 86, 130, 66 (WT mock); 138, 94, 131 (tmm mock); 230, 336, 109 (WT BFA); 300, 393, 211 (tmm BFA). (E) Representative images of ERL1-YFP in erl1 (top row) or in erl1 tmm (bottom row) treated with mock (left column) or 25 µM Wm (right column). Inset, an enlarged image of a representative meristemoid. Arrowheads indicate Wm bodies. Scale bars = 10 µm. (F) Quantitative analysis of the number of Wm bodies per cell when ERL1-YFP in erl1 (yellow) or erl1 tmm (orange) are treated with mock or 25 µM Wm. Lines in the boxplot show the median value, and each dot represents individual data point with jitter. Welch’s Two sample T-test was performed for pairwise comparisons. Two-way ANOVA analysis: genotype (WT, tmm), p=1.597e-6; treatment (mock, Wm), p<2.2e-16. Experiments were repeated three times. the total numbers of cells counted are 74 (WT mock); 91 (tmm mock); 82 (WT Wm); 90 (tmm Wm). (G) FRAP analyses of plasma membrane ERL1-YFP in wild type (erl1) or in tmm (erl1 tmm). Shown are representative fluorescence recovery curves plotted as a function of time and fitted to Single Exponential Fitting. ERL1-YFP in erl1 (top; yellow); ERL1-YFP in erl1 tmm (bottom; orange). (H) Quantitative analysis of the half time of fluorescence recovery of plasma membrane ERL1-YFP in erl1 (yellow) and erl1 tmm (orange). Lines in the boxplot show the median value. T-test was performed for pairwise comparisons between erl1 and erl1 tmm. n = 3 for WT and n = 9 for tmm.

To rule out the possibility that TMM influences a general endocytic degradation machinery, we examined the effects of tmm on general endocytosis using FM4-64. In wild type, 92.8% (n = 20 cells) of ERL1-YFP-labelled endosomes can be stained by FM4-64. In tmm, however, FM4-64 still internalizes to endosomes whereas ERL1-YFP internalization can only be detected in 30% of the cells examined (n = 30 cells) (Figure 4—figure supplement 2A). The effects of tmm on the formation of MVB/LEs were subsequently tested (Figure 4—figure supplement 2B–E). In the cells co-expressing RFP-ARA7 and ERL1-YFP, no significant difference was observed in the numbers of RFP-ARA7-labelled endosomes and Wm bodies per meristemoid cell between wild type and tmm (Figure 4—figure supplement 2B,C). In contrast, the tmm mutation conferred reduction in ERL1-YFP-labelled Wm bodies that co-localized with RFP-ARA7 (Figure 4—figure supplement 2B–C). Thus, the results suggest that TMM mediates endocytosis of ERL1 for vacuolar sorting without influencing general endomembrane trafficking.

To further explore the role of TMM for the ERL1 receptor dynamics, we performed fluorescence recovery after photobleaching (FRAP) assays on ERL1-YFP on the plasma membrane (Figure 4—figure supplement 3), and the half time of fluorescence recovery was calculated from modeling to exponential curves (Figure 4G. H, Videos 2 and 3). In wild type, the calculated mean half-time of ERL1-YFP fluorescence recovery (t1/2) was 23.55 ± 5.55 s, whereas in tmm it was 70.89 ± 24.63 s (Figure 4G. H). The longer recovery time of ERL1-YFP in tmm could be due to reduced diffusion rate/mobility of ERL1 receptor at the plasma membrane or, alternatively, slower cycle of ERL1 receptor between the plasma membrane and the cytoplasmic fractions. The results imply that, in the absence of TMM, un-activated ERL1 receptors may not be readily targeted for the endocytic pathway and, consequently, remain stable on the plasma membrane.

Video 2
FRAP analysis of ERL1-YFP in erl1.

A time-lapse FRAP movie of ERL1-YFP in a meristemoid in erl1 mutant. 5 images were taken every 0.453 s (minimum speed) both before and during photobleaching, and then 120 frames were taken every 3 s after photobleaching. The movie was presented as five frames per second.

Video 3
FRAP analysis of ERL1-YFP in erl1 tmm.

A time-lapse FRAP movie of ERL1-YFP in a meristemoid in erl1 tmm mutant. 5 images were taken every 0.453 s (minimum speed) both before and during photobleaching, and then 120 frames were taken every 3 s after photobleaching. The movie was presented as five frames per second.

EPF1 triggers TMM-dependent ERL1 internalization

Of the 11 EPF family members, EPF1 is the major ligand for ERL1 (Lee et al., 2012). EPF1 signaling plays a negative role in stomatal development, and the induction of EPF1 peptide (iEPF1) confers arrested stomatal precursors (Figure 5AHara et al., 2007; Lee et al., 2012; Qi et al., 2017). We therefore tested whether the ERL1 internalization is ligand dependent. For this purpose, we first examined ERL1-YFP dynamics in epf1 mutants. As shown in Figure 5—figure supplement 1A, both plasma membrane and highly mobile endosomes are highlighted by ERL1-YFP in epf1. When treated with BFA or Wm, the number of ERL1-YFP-positive BFA or Wm bodies per cell are similar between wild type and epf1 (Figure 5—figure supplement 1B–E, p=0.512 and p=0.647 for BFA and Wm treatment, respectively). Therefore, although EPF1 is a primary ligand for ERL1, ERL1-YFP endocytosis is not severely affected in the absence of EPF1.

Figure 5 with 1 supplement see all
MEPF1 triggers ERL1-YFP internalization in erl1 but not in erl1 tmm.

(A) Representative confocal microscopy images of cotyledon abaxial epidermis from the 4-day-old iEPF1 seedlings treated with mock (left) or 10 µM Estradiol (right). Scale bars = 10 µm. (B) Representative confocal microscopy images of cotyledon abaxial epidermis from the 4-day-old iEPF1 in tmm seedlings treated with mock (left) or 10 µM Estradiol (right). Brackets indicate clustered stomata in both mock- and estradiol-induced samples. Scale bars = 10 µm. (C) Representative confocal microscopy images of ERL1-YFP in erl1 treated with mock (top left), 1 µM MEPF1 (top right), 2.5 µM MEPF1 (bottom left) and 5 µM MEPF1 (bottom right) are shown. Arrowheads indicate endosomes. Scale bars = 10 µm. (D) Representative confocal microscopy images of ERL1-YFP in erl1 tmm treated with mock (top left), 1 µM MEPF1 (top right), 2.5 µM MEPF1 (bottom left) and 5 µM MEPF1 (bottom right) are shown. Arrowheads indicate endosomes. Scale bars = 10 µm. (E) Quantitative analysis of the number of ERL1-YFP-positive endosomes per cell at different concentrations of MEPF1 application in erl1 shown as a violin plot. Dots, individual data points. Median values are shown as lines in the boxplot, and mean values are shown as yellow dots in the plot. Welch's two sample T-test was performed for pairwise comparisons of samples treated with the mock and different concentration of MEPF1. Number of cells analyzed, n = 79, 27, 38, 82 for treatment with mock, 1 µM, 2.5 µM, 5 µM MEPF1. (F) Quantitative analysis of the number of ERL1-YFP-positive endosomes per cell at different concentrations of MEPF1 application in erl1 tmm shown as a violin plot. Dots, individual data points. Median values are shown as lines in the boxplot, and mean values are shown as yellow dots in the plot. Welch's two sample T-test was performed for pairwise comparisons of samples treated with the mock and different concentration of MEPF1. Number of cells analyzed, n = 76, 113, 109, 114 for treatment with mock, 1 µM, 2.5 µM, and 5 µM MEPF1, respectively.

Considering the high similarity among the 11 EPF members, it is possible that the functional redundancy of other EPFs alleviates the defect of ERL1 internalization in epf1. To address this, we took advantage of the biologically-active mature EPF1 (MEPF1) peptide (Figure 5Lee et al., 2012; Qi et al., 2017). Different concentrations of MEPF1 were applied to the true leaf epidermis of 7-day-old seedlings expressing ERL1pro::ERL1-YFP. The number of ERL1-YFP-positive endosomes per cell increases as the peptide concentration increases (Pearson correlation coefficient, r = 0.56, p=2.2 e-16; Figure 5C,E), indicating that MEPF1 peptide triggers the internalization of ERL1 in a dosage-dependent manner.

In the tmm background, however, the number of ERL1-YFP-positive endosomes per cell remains low regardless of the MEPF1 dosage applied (Figure 5D,F). Thus, in the absence of TMM, ERL1-YFP endocytosis is insensitive to MEPF1 application, consistent with the genetic evidence that the tmm mutation is epistatic to induced EPF1 overexpression (iEPF1) (Hara et al., 2007; Lee et al., 2012Figure 5B). Taken together, we conclude that EPF1 peptide ligand perception triggers the internalization of ERL1 receptor in a TMM-dependent manner.

EPFL6 triggers ERL1 internalization in the absence of TMM

A previous structural analysis has shown that binding of EPF1 to the ERL1-TMM receptor complex does not lead to conformational change (Lin et al., 2017). To test if the pre-formed ERL1-TMM receptor complex is required for the internalization of ERL1, we took advantage of EPFL6, a peptide related to EPF1 with a distinct property (Abrash and Bergmann, 2010; Abrash et al., 2011Figure 6). EPFL6 is normally expressed in the internal tissues of hypocotyls and stems, but not in the stomatal-lineage cells. Unlike EPF1, ectopic EPFL6 is a potent inhibitor of stomatal development even in the tmm mutant background (Abrash and Bergmann, 2010; Abrash et al., 2011; Uchida et al., 2012Figure 6A,B). Using a similar strategy as MEPF1, we purified biologically active, predicted mature EPFL6 (MEPFL6) peptide. Indeed, the inhibition of stomatal formation by MEPFL6 is more sensitive in tmm mutant than in wild type (Figure 6—figure supplement 1). In contrast to MEPF1, MEPFL6 application induced ERL1-YFP internalization in a dosage-dependent manner regardless of the presence or absence of TMM (Figure 6C–F). The results indicate that TMM is not required for EPFL6-triggered ERL1-YFP internalization. Rather, the ERL1-YFP endocytosis accurately reflects the activity of ERL1 signaling to inhibit stomatal development (Figure 6 and Figure 6—figure supplement 1), thereby supporting the notion that distinct EPF/EPFL peptide ligands activate a sub-population of ERL1 receptor complexes to internalize through a TMM-based discriminatory mechanism.

Figure 6 with 1 supplement see all
MEPFL6 triggers ERL1-YFP internalization in both erl1 and erl1 tmm.

(A) Representative confocal microscopy images of cotyledon abaxial epidermis from the 5-day-old wild type seedlings treated with mock (left) or 5 µM MEPFL6 (right). Scale bars = 10 µm. (B) Shown are representative confocal microscopy images of cotyledon abaxial epidermis from the 5-day-old tmm seedlings treated with mock (left) or 5 µM MEPFL6 (right). Scale bar = 10 µm. (C) Representative images of ERL1-YFP in erl1 treated with mock (top left), 1 µM MEPFL6 (top right), 2.5 µM MEPFL6 (bottom left) and 5 µM MEPFL6 (bottom right) are shown. Arrowheads indicate endosomes. Scale bar = 10 µm. (D) Representative images of ERL1-YFP in erl1 tmm treated with mock (top left), 1 µM MEPFL6 (top right), 2.5 µM MEPFL6 (bottom left) and 5 µM MEPFL6 (bottom right) are shown. Arrowheads indicate endosomes. Scale bars = 10 µm. (E) Quantitative analysis of the number of ERL1-YFP-positive endosomes per cell at different concentrations of MEPFL6 application in erl1 are shown as a Violin plot. Median values are shown as lines in the boxplot, and mean values are shown as yellow dots in the plot. Dots, individual data points. Welch's two sample T-test was performed for pairwise comparisons of samples treated with the mock and different concentration of MEPFL6. Number of cells analyzed, n = 37, 28, 27, 30 for treatment with mock, 1 µM, 2.5 µM, 5 µM MEPFL6. (F) Quantitative analysis of the number of ERL1-YFP-positive endosomes per cell at different concentrations of MEPFL6 application in erl1 tmm are shown as a Violin plot. Dots, individual data points. Median values are shown as lines in the boxplot, and mean values are shown as yellow dots in the plot. Welch's two sample T-test was performed for a pairwise comparisons of samples treated with the mock and different concentration of MEPFL6. Number of cells analyzed, n = 55, 63, 48, 35 for treatment with mock, 1 µM, 2.5 µM, 5 µM MEPFL6.

An antagonistic EPFL peptide, Stomagen, elicits retention of ERL1-YFP in the endoplasmic reticulum

Stomagen promotes stomatal development by competing with other EPFs for binding to the same receptor complex, including ERL1 (Figure 7AKondo et al., 2010; Sugano et al., 2010; Lee et al., 2015; Lin et al., 2017; Qi et al., 2017). Because the activated ERL1 receptor undergoes endocytosis to MVB/LEs, we sought to address the role of Stomagen on subcellular dynamics of ERL1. For this purpose, we first applied bioactive Stomagen peptide on seedlings expressing ERL1pro::ERL1-YFP in erl1. Unlike in mock-treated samples, YFP signal was detected inside the cells (Figure 7—figure supplement 1A). We subsequently treated Stomagen peptides to ERL1-YFP in the erecta erl1 erl2 triple mutant background to remove any potential redundancy among the three ERECTA-family receptors. Strikingly, strong ERL1-YFP signals were detected in a ring-like structure surrounding the nucleus (Figure 7B), which co-localizes with the endoplasmic reticulum marker protein RFP-KDEL (Faraco et al., 2017Figure 7B).

Figure 7 with 3 supplements see all
Stomagen application confers accumulation of ERL1 in endoplasmic reticulum.

(A) Representative confocal microscopy images of cotyledon abaxial epidermis from the 5-day-old wild type seedlings (top) or tmm seedlings (bottom) treated with mock (left) or 5 µM Stomagen (right). Scale bars = 10 µm. (B) Representative confocal microscopy images of ERL1-YFP (left column) and co-localization analysis with the endoplasmic reticulum marker RFP-KDEL (middle column) in the abaxial epidermis of cotyledons of the 5-day-old erecta (er) erl1 erl2 seedlings treated with mock (top row) or 5 µM Stomagen (bottom row). Right, merged images with the line slicing along which quantification analysis of the YFP intensity (green) and RFP intensity (magenta) was done; graphs are shown on the right, with two middle peaks (pointed by arrowheads) showing signals from the endoplasmic reticulum and two big peaks on both sides showing signals of the plasma membrane. Scale bars = 10 µm. (C) Representative confocal microscopy images of ERL1-YFP (left) in the abaxial epidermis of cotyledons of the 5-day-old erecta erl1 erl2 tmm seedlings stained with the endoplasmic reticulum dye Rhodamine (second left column). The merged image is shown in the third left column. Quantification analysis of the YFP intensity (green) and RFP intensity (magenta) along the line drawn in the right image is shown as a graph on the right, with two middle peaks (pointed by arrowheads) showing signals from the endoplasmic reticulum and two big peaks on both sides showing signals of the plasma membrane. Scale bars = 10 µm. (D) Immunoblot analysis of 3-day-old ERL1-FLAG erecta erl1 erl2 seedlings treated with mock or 5 µM Stomagen for 2 days and then digested without or with Endo-H. Top panel shows the ERL1-FLAG detected by α-FLAG. Lower panel shows the loading control of Tubulin detected by α-Tubulin. Black arrow, ERL1 band without digestion, blue and cyan arrows, ERL1 cut with Endo-H digestion. (E) Representative confocal microscopy images of abaxial epidermis of true leaves from ERL1-YFP in erecta erl1 erl2 seedlings treated with mock (top left) or 50 µM Tyr A23 (top right); mock (bottom left) or 100 µM ES9-17 (bottom right). Arrow indicates the ring-like structure, characteristics of endoplasmic reticulum localization, detected after treatment with Tyr A23 or ES9-17. Scale bars = 10 µm.

Next, to reveal a consequence of inactive ERL1 receptor on its subcellular dynamics, we applied Stomagen peptide on tmm seedlings expressing ERL1pro::ERL1-YFP and carefully reexamined the signal. Very faint ring-like structures were highlighted by ERL1-YFP in both mock and Stomagen-treated meristemoids (Figure 7—figure supplement 1A). This was enhanced in the erecta erl1 erl2 tmm quadruple mutant (Figure 7C). These ERL1-YFP signals co-localized with Rhodamine B hexyl esters, a dye that stains the endoplasmic reticulum (Hawes et al., 2018), suggesting that the absence of TMM intensifies accumulation of ERL1 in the endoplasmic reticulum (Figure 7C). To address the specificity of Stomagen effects on ERL1 subcellular behaviors, we applied Stomagen peptide to Arabidopsis seedlings expressing BRI1pro::BRI1-GFP, which is endogenously expressed in cotyledon and leaf epidermis (Friedrichsen et al., 2000). Strong BRI1-GFP signals are detected at the plasma membrane of stomatal meristemoids, guard cells, as well as pavement cells (Figure 7—figure supplement 2). Application of Stomagen peptide did not influence plasma membrane localization of BRI1-GFP (Figure 7—figure supplement 2), thus the endoplasmic reticulum accumulation of ERL1-YFP upon Stomagen perception is not a universal phenomenon to LRR-RLKs.

To biochemically characterize the effects of Stomagen application and tmm mutation on ERL1 accumulation in the endoplasmic reticulum, we further performed endoglycosidase H (Endo-H) enzymatic sensitivity assays. Endo-H cleaves N-glycans of proteins specifically in the endoplasmic reticulum, including LRR-RLKs (Jin et al., 2007; Nekrasov et al., 2009), but not the remodeled glycan chains of proteins transported to the Golgi or further. To detect slight molecular mass changes, proteins from erecta erl1 erl2 triple mutant seedlings rescued by ERL1pro::ERL1-FLAG were subjected to Endo-H treatment (see Methods). Under normal conditions, ERL1-FLAG is detected as a single band on immunoblots (Figure 7D, black arrow). The Endo-H digestion resulted in a faster mobility of ERL1-FLAG protein with at least three different sizes, suggestive of heterogeneous glycans (Figure 7D, blue and cyan arrows). In contrast, ERL1-FLAG protein from Stomagen-treated seedlings was hypersensitive to Endo-H and cleaved completely (Figure 7D, light arrow). Likewise, the tmm mutation enhanced the Endo-H sensitivity of ERL1 (Figure 7—figure supplement 1B), suggesting an increase in endoplasmic reticulum retention.

Because exogenous application of Stomagen blocks the activation of ERECTA-family signaling (Lee et al., 2015) and results in stomatal clustering (Figure 7A), we sought to address if insufficient internalization of ERL1 from the plasma membrane triggers its stalling in the endoplasmic reticulum. For this purpose, we first treated erecta erl1 erl2 seedlings expressing ERL1-YFP with Tyr A23, an inhibitor that has been widely used to block clathrin-mediated endocytosis in plant cells (Banbury et al., 2003; Santuari et al., 2011). Indeed, the Tyr A23 treatment enhanced ERL1-YFP signals in the endoplasmic reticulum (Figure 7E, pink arrow), whereas in mock ERL1-YFP was only detected on the plasma membrane and endosomes.

A recent report showed that Tyr A23 functions as a protonophore, which inadvertently blocks endocytosis through cytoplasmic acidification (Dejonghe et al., 2016). Chemical screening and subsequent derivatization identified ES9-17 as a specific inhibitor of clathrin-mediated endocytosis without the side effects of cytoplasmic acidification (Dejonghe et al., 2019). We sought to test the effects of ES9-17 on ERL1-YFP subcellular localization to rule out the possibility that retention of ERL1-YFP in the endoplasmic reticulum is due to cellular acidification. ES9-17 previously has been applied only to root cells (Dejonghe et al., 2019). We first optimized the treatment condition for developing seedling shoots (see Methods). At 100 μM, ES9-17 inhibited the internalization of FM4-64 dye in epidermal pavement cells and stomatal-lineage cells, just like 50 μM ES9-17 in root cells (Figure 7—figure supplement 3A,B). Under this condition, ES9-17 treatment caused the accumulation of ERL1-YFP in the endoplasmic reticulum, just like the Tyr A23 treatment (Figure 7E). Taken together, our cell biological, pharmacological, and biochemical analyses reveal that inefficient endocytosis due to perception of an antagonistic peptide, Stomagen, as well as loss of co-receptor TMM, causes retention of ERL1-YFP in the endoplasmic reticulum.

Cytoplasmic domain of ERL1 is required for proper subcellular trafficking behavior

Removal of the entire cytoplasmic domain from ERECTA-family RLKs is known to confer strong dominant-negative effects both in aboveground organ growth and in stomatal patterning (Shpak et al., 2003; Lee et al., 2012). The dominant-negative ERL1ΔK can directly bind its ligand EPF1 through the extracellular LRR domain. However, it is unable to signal and, consequently, confers paired and clustered stomata, thereby phenocopying epf1 mutant (Figure 8A,BLee et al., 2012).

Figure 8 with 1 supplement see all
Dominant-negative form of ERL1 is compromised in subcellular trafficking.

(A) Diagram of the full-length ERL1 protein (top) and the dominant-negative ERL1 protein lacking the cytoplasmic domain (bottom). (B) Representative confocal microscopy images of cotyledon abaxial epidermis from 4-day-old seedlings of wild type, epf1, ERL1-YFP erl1 and ERL1ΔK-CFP erl1, stained by PI. Orange brackets indicate the paired stomata in epf1 and ERL1ΔK -CFP in erl1. Scale bars = 10 µm. (C) Representative confocal microscopy images of ERL1ΔK-CFP treated with mock (top left for BFA treatment), 30 µM BFA (top right), mock (bottom left for Wm treatment) and 25 µM Wm (bottom right). Inset, enlarged image of a representative meristemoid. Arrowheads indicate BFA bodies. Scale bars = 10 µm. (D) Representative confocal microscopy images of an abaxial true leaf epidermis from seedlings expressing ERL1-YFP in erecta erl1 erl2 (top) and ERL1ΔK-CFP in erl1 (bottom) treated with mock (left) or 5 μM MEPF1. Scale bars = 5 µm. (E) Quantitative analysis of the number of ERL1-YFP-positive or ERL1ΔK-CFP-positive endosomes per cell in mock or upon 5 μM MEPF1 peptide application. Dots, individual data points with a jitter (0.2). A box plot is overlaid to each violin plot, with a median shown as a line. T-test was performed for pairwise comparisons of samples treated with the mock and MEPF1. Two-way ANOVA analysis: genotype (ERL1-YFP, ERL1ΔK-CFP), p=7.081e-12; treatment (mock, MEPF1), p=0.001498. Tukey's HSD, genotype: treatment, ERL1-YFP mock: ERL1-YFP MEPF1 treatment, p=0.00045, ERL1ΔK-CFP mock: ERL1ΔK-CFP EPF1 treatment, p=0.9998. Number of cells analyzed, n = 88 (ERL1-YFP, mock), 92 (ERL1-YFP, MEPF1), 50 (ERL1ΔK-CFP, mock), 59 (ERL1ΔK-CFP, MEPF1). (F) Representative confocal microscopy images of abaxial epidermis of true leaves from ERL1-YFP expressed in erecta erl1 erl2 seedlings treated with mock or 5 µM Stomagen peptide.). Arrow indicates the ring-like structure, characteristics of endoplasmic reticulum localization. Scale bars = 5 µm. (G) Representative confocal microscopy images of abaxial epidermis of true leaves from ERL1ΔK-CFP in erl1 seedlings treated with mock or 5 µM Stomagen peptide. Scale bars = 5 µm.

To gain further insight into the mechanism behind the complex subcellular dynamics of ERL1 upon different EPF/EPFL peptide perception, we examined the subcellular behaviors of the dominant-negative ERL1, ERL1ΔK-CFP driven by the endogenous ERL1 promoter (Figure 8). Strong CFP signal is detected on the plasma membrane of stomatal precursor cells, but only very few mobile punctae can be seen in the cytoplasm (Figure 8C). BFA treatment results in 86% cells possessing an average of 2 ERL1ΔK-CFP-labelled BFA bodies (Figure 8C, Figure 8—figure supplement 1A). However, the BFA sensitivity of ERL1ΔK was also observed in the presence of CHX in a statistically-indistinguishable manner from those in tmm mutant (Figure 4—figure supplement 1, p=0.412), supporting that the dominant-negative form of ERL1 may confer reduced endocytosis/internalization. Moreover, ERL1ΔK-CFP exhibits insensitivity to Wm treatment, with only 18% cells showing few Wm bodies highlighted by ERL1ΔK-CFP (Figure 8—figure supplement 1A). The reduced endocytosis of ERL1ΔK-CFP is not due to defects in the general endocytosis process, as FM4-64 can still internalize in the ERL1ΔK-CFP-positive cells on the transgenic seedling epidermis, like it does in cells with the full-length ERL1 (Figure 8—figure supplement 1B).

Next, we examined the subcellular behaviors of ERL1ΔK-CFP upon perception of EPF1 and Stomagen peptides. As shown in Figure 8D and E, application of 5 μM MEPF1 did not increase endocytosis of ERL1ΔK-CFP (p=0.9972), whereas the same treatment triggered the endocytosis of control ERL1-YFP (p=0.0002) (Figure 8D,E). Thus, the dominant-negative ERL1 is insensitive to EPF1 peptide ligand. Together with the case of ERL1-YFP in tmm mutant (Figure 5), the results suggest that the internalization of ERL1 by EPF1 peptide reflects the active signaling events.

To address whether ERL1ΔK-CFP behaves similarly to signaling-incompetent full-length ERL1-YFP (i.e. in tmm erecta erl1 erl2 background), we next applied Stomagen peptide. To our surprise, unlike ERL1-YFP (Figure 8F), ERL1ΔK-CFP failed to accumulate in the endoplasmic reticulum and remained insensitive to Stomagen peptide (Figure 8G). Combined, these results indicate that the dominant-negative ERL1ΔK is dysfunctional in ligand-induced subcellular trafficking and suggest that the cytoplasmic domain of ERL1 is critical for its proper subcellular dynamics.

Discussion

In this study, we revealed that ERL1 endocytosis accurately reflects EPF signal perception based on three pieces of evidence (Figure 9): first, both EPF1 and EPFL6 peptides trigger ERL1 endocytosis. Second, in the absence of the co-receptor TMM, ERL1 endocytosis is compromised and becomes insensitive to EPF1 application. Third, the cytoplasmic domain of ERL1 is required for ERL1 endocytosis. EPF1 and EPFL6 peptide application increased ERL1 population in endosomes in a dosage-dependent manner (Figures 5 and 6). Our pharmacological application of Wortmannin, BFA, BFA with CHX and Concanamycin A (Figures 23, Figure 4—figure supplement 1), indicate that ERL1 is constitutively recycled whereas the receptor activation triggers endocytosis for vacuolar sorting via MVB/LE. In this aspect, the subcellular dynamics of ERL1 resembles that of FLS2, which is also constitutively recycled but rapidly removed from the cell surface upon flg22 perception (Robatzek et al., 2006; Smith et al., 2014). Unlike FLS2, however, a vast majority of ERL1-YFP signal remained at the plasma membrane even after treatment of 5 µM MEPF1 (Figure 5). These differences could be attributed to the roles of FLS2 and ERL1 in immunity vs. development, respectively. FLS2 mediates acute pathogen-induced defense response, whereas ERL1 likely detects endogenous peptides to influence slower processes of cell division and differentiation. A recent study showed, however, that defects in the clathrin-mediated FLS2 endocytosis impair only a subset of FLS2-mediated immune responses (Mbengue et al., 2016). Thus, the precise contributions of endocytosis and cellular response remain open questions.

Schematic model of ERL1 subcellular dynamics triggered by diverse EPF peptides with different biological activities.

ERL1 (light green) is constitutively recycling and follows BFA-sensitive endosomal pathway (Receptor Recycling). EPF1 (orange) and EPFL6 (pink) peptide ligands both activate ERL1 to inhibit stomatal differentiation, trigger ERL1 trafficking via Wm-sensitive MVB/LE to the vacuole (Signal Activation). EPF1-triggered ERL1 trafficking requires the presence of TMM (gray). In contrast, EPFL6 triggers ERL1 trafficking in TMM-independent manner. Stomagen (dark green), which blocks ERL1 signaling, causes stalling of ERL1 in endoplasmic reticulum (ER) (Signal Inhibition). The dominant-negative ERL1ΔK is overwhelmingly plasma-membrane localized and does not undergo normal subcellular trafficking (Dominant Negative). The site of action of each inhibitor/enzyme is indicated in red.

It has been shown that EPF1, but not EPFL6, requires TMM for the inhibition of stomatal development (Hara et al., 2007; Abrash and Bergmann, 2010). Likewise, structural analyses of the EPF-ERECTA family complexes showed that EPF1, but not EPFL6, requires TMM for binding to the ectodomain of ERECTA family receptors (Lin et al., 2017). Here, we demonstrate that TMM is required for endocytosis triggered by EPF1, but not by EPFL6 (Figures 4, 5, 6 and 9). Thus, at least two populations of ERL1 receptor complexes must be present on the plasma membrane, with and without TMM. The BFA treatment on tmm mutants (Figure 4, Figure 4—figure supplement 1) revealed that in the absence of TMM, ERL1-YFP internalization is compromised but not absent. This is most likely due to the presence of EPF-LIKE peptide (e.g. EPFL4/5/6) family members that do not require TMM. The FRAP analysis indeed detected the different mobility of these two ERL1 compositions on the plasma membrane (Figure 4). Multiple compositions of receptor complexes have also been reported in CLV3 signaling, where CLV1 homomers, CLV2/CORYNE (CRN) heterodimers and CLV1/CLV2/CRN multimers co-exist on the plasma membrane (Somssich et al., 2015). However, only the microdomain-localized CLV1/CLV2/CRN multimers can perceive the sole ligand CLV3. In the case of BRI1 and FLS2, pre-formed BRI1-BAK1 complex was detected regardless of BRs whereas FLS2 forms FLS2-BAK1 complex upon flg22 application (Bücherl et al., 2013; Somssich et al., 2015). These receptor complexes are spatially separated, even though BRI1 and FLS2 share the same co-receptor BAK1 (Bücherl et al., 2013; Somssich et al., 2015; Bücherl et al., 2017; Hutten et al., 2017). On the contrary, both populations of ERL1 complexes are ‘functional’ and ligand-inducible, as they can perceive EPF1 or EPFL6, respectively (Figures 5 and 6). It is possible that the distinct ERL1 receptor complexes reside in different microdomains on the plasma membrane and undergo different trafficking routes upon the correlated ligand perception. EPF1 triggers ERL1 association with BAK1 (Meng et al., 2015). Examining spatiotemporal subcellular dynamics of ERL1 together with TMM and BAK1 at a super resolution scale may reveal the contribution of each receptor complex for specific signal perception and transduction.

ERL1 is retained in the endoplasmic reticulum when treated with exogenous Stomagen. This ERL1 subcellular behavior is associated with its own biological contexts, given that BRI1, an LRR-RK with unrelated function, does not change its plasma membrane localization upon Stomagen application (Figure 7—figure supplement 2). Extensive studies support that the steady state of a protein in its subcellular compartment is interdependent on the anterograde and retrograde trafficking routes (Brandizzi and Barlowe, 2013). For example, a secretory protein is often retained in the endoplasmic reticulum when the downstream secretion pathway is compromised (Zheng et al., 2005). Blocking the endoplasmic reticulum-to-Golgi retrograde trafficking will accelerate protein transport to the cell surface (Fossati et al., 2014). It is thus possible that Stomagen binding prevents the ERL1 endocytosis and the plasma membrane-accumulated ERL1 interferes with the normal transport of incoming ERL1 from the endoplasmic reticulum. Two additional pieces of evidence support this hypothesis. First, when endocytosis is blocked by Tyr A23 (Banbury et al., 2003) or ES7-19, the improved, specific inhibitor of clathrin heavy chain (Dejonghe et al., 2019), strong ERL1 signals become evident in the endoplasmic reticulum (Figure 7E). Second, in leaves of the tmm mutant, EPF1-triggered ERL1 endocytosis is compromised, ERL1 also accumulates in the endoplasmic reticulum (Figure 7C and Figure 7—figure supplement 1).

Stomagen-triggered ERL1 accumulation in endoplasmic reticulum may be highlighting the role of the endoplasmic reticulum-plasma membrane contact sites as a direct communication link between the two compartments (Carrasco and Meyer, 2011). The VAP-RELATED SUPPRESSOR OF TMM (VST) family plasma membrane proteins that interact with integral endoplasmic reticulum proteins, have been reported to facilitate ERECTA family-mediated signaling in stomatal development (Ho et al., 2016). Hence, Stomagen perception by the ERL1-TMM complex on the plasma membrane may directly influence signaling via the contact sites and therefore affect the transport of ERL1 to the cell surface. The endoplasmic reticulum accumulation of ERL1-YFP is notable in the erecta erl1 erl2 mutant background, that is when functional ERECTA and ERL2 genes are missing. Because Stomagen interacts with ERECTA at the early stage of stomatal initiation (Lee et al., 2015), the absence of ERECTA likely confers sensitized, exaggerated response of ERL1-YFP to Stomagen. Alternatively, a dysregulated cell-cell signaling in the erecta erl1 erl2 background may abrogate the buffering of exogenously applied Stomagen peptide.

Posttranslational modifications, such as phosphorylation and ubiquitination, of the receptors have emerged as key regulatory mechanisms of receptor subcellular dynamics in FLS2 and BRI1 (Robatzek et al., 2006; Lu et al., 2011; Martins et al., 2015; Zhou et al., 2018). Specific posttranslational modifications of ERL1 are yet unknown, but perception of different EPF/EPFL peptides may trigger unique phosphorylation codes that influence subsequent trafficking of ERL1. The dominant-negative ERL1 fails to respond to either MEPF1 or Stomagen peptides (Figure 8, Figure 8—figure supplement 1). Clearly, the behaviors of ERL1ΔK-CFP is different from the full-length ERL1-YFP. The accumulation of ERL1 in the endoplasmic reticulum occurs when ERL1-YFP becomes signaling incompetent- (i) by Stomagen peptide perception; (ii) due to the absence of co-receptor and redundant receptors, TMM, ERECTA, and ERL2; or (iii) when ERL1 endocytosis is pharmacologically blocked (Figure 7). Thus, subcellular dynamics of the full-length ERL1 is highly tuned and coordinated. Such coordination is lost by removal of the cytoplasmic domain, which includes juxtamembrane domain, kinase domain, and C-terminal tail region (Figure 8A). It is highly plausible that ERL1ΔK-CFP can no longer be recognized as the original endocytosis cargo by adaptor proteins. For instance, the endocytosis of BRI1 is mediated by direct association with Clathrin adaptor complex AP2 (Di Rubbo et al., 2013). Future studies may reveal the molecular basis of how ERL1 interacts with specific cargo receptors.

Previous work reported that BFA effect (BFA body formation) is different in root vs. shoot tissues (Robinson et al., 2008; Langhans et al., 2011). Here, we extensively documented the effects of BFA on subcellular behaviors using eight endomembrane organelle markers together with endocytic tracer dye (Figure 3, Figure 3—figure supplement 1). Our extensive analyses show that with appropriate concentration and treatment condition, BFA body formation can be recapitulated in leaf cells, just like in root cells. The results could facilitate future studies on recycling and endocytosis of membrane proteins in intact leaves. Taken together, our work revealed the mechanism by which multiple peptide ligands with distinct activities, EPF1, EPFL6, and Stomagen, fine-tune stomatal patterning at the level of the subcellular dynamics of a single receptor, ERL1. Successful development of visible functional peptide ligands and identification of the immediate biochemical events by the ERECTA-family perceiving different EPF peptides will help elucidate the exact roles of receptor trafficking and signaling specifying developmental patterning in plants.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (Arabidopsis thaliana)erecta
(er-105)
Shpak et al., 2005 PMID:16002616See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)erl1-2Shpak et al., 2005 PMID:16002616See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)erl2-1Shpak et al., 2005 PMID:16002616See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)epf1-1Hara et al., 2007
PMID:17639078
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)tmm-KOHara et al., 2007
PMID:17639078
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ERL1pro::ERL1-YFP in erl1-2Lee et al., 2012
PMID:22241782
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ERL1pro::ERL1-YFP in erl1-2 tmmThis studySee Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ERL1pro::ERL1-YFP in erl1-2 epf1This studySee Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ERL1pro::ERL1-FLAG in erl1-2Lee et al., 2012
PMID:22241782
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ERL1pro::ERL1-FLAG in erecta erl1-2 erl2-1Lee et al., 2012
PMID:22241782
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ERL1pro::ERL1ΔK-CFP in erl1-2Lee et al., 2012
PMID:22241782
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)MUTEpro::ERL1-YFP in er-105 erl1-2 erl2-1Qi et al., 2017
PMID:28266915
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)iEPF1Qi et al., 2017
PMID:28266915
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)BRI1pro::BRI1-GFPFriedrichsen et al., 2000
PMID:10938344
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)UBQ10pro::YFP-SYP32Geldner et al., 2009
PMID:19309456
Wave22YSee Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)UBQ10pro::YFP-ARA7Geldner et al., 2009
PMID:19309456
Wave2YSee Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)UBQ10pro::YFP-RabA5dGeldner et al., 2009
PMID:19309456
Wave24YSee Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)UBQ10pro::YFP-RabA1eGeldner et al., 2009
PMID:19309456
Wave34YSee Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)SYP43pro::GFP-SYP43Uemura et al., 2012
PMID:22307646
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)SYP22pro:: SYP22- mRFPEbine et al., 2011Postma et al., 2016,PMID:21666683, 26765243See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ARA7pro::mRFP-ARA7Ebine et al., 2011; Postma et al., 2016,PMID:21666683,26765243See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)SYP43pro:: SYP43- mRFPEbine et al., 2011Postma et al., 2016,PMID:21666683,26765243See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ARA6pro::ARA6-GFPEbine et al., 2011
PMID:21666683
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)ST-RFPFaraco et al., 2017
PMID:28636930
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)KDEL-RFPFaraco et al., 2017
PMID:28636930
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)CaMV35S::N-ST-YFPTakagi et al., 2013
PMID:24280388
See Materials and method, section 1
Genetic reagent (Arabidopsis thaliana)CaMV35S::GFP-HDELMitsuhashi et al., 2000
PMID:11100771
See Materials and method, section 1
Chemical compound, drugBrefeldin A (BFA)Cayman ChemicalCat. #: 11861
Lot #: 0533646–12
See Materials and method, section 3
Chemical compound, drugConcanamycin AAdipogen Life SceincesCat. #: BVT-0237-C025
Lot #: B1105151
See Materials and method, section 3
Chemical compound, drugBrefeldin A (BFA)SigmaCat. #: B7651See Materials and method, section 3
Chemical compound, drugWortmannin (Wm)SigmaCat. #: W1628See Materials and method, section 3
Chemical compound, drugCycloheximide (CHX)SigmaCat. #: C4859See Materials and method, section 3
Chemical compound, drugRhodamine BSigmaCat. #: R6626See Materials and method, section 3
Chemical compound, drugTyrphostin (Tyr A23)SigmaCat. #: T7165See Materials and method, section 3
Chemical compound, drugES9-17Dejonghe et al., 2019 PMID:31011214See Materials and method, section 3
Peptide, recombinant proteinMEPF1Lee et al., 2012
PMID:22241782
pJSL11See Materials and method, section 2
Peptide, recombinant proteinMEPFL6This workpJSL79See Materials and method,section 2
Peptide, recombinant proteinSTOMAGENLee et al., 2015
PMID:26083750
See Materials and method, Section 2
AntibodyMouse monoclonal anti-FLAG M2SigmaCat. #: F-3165
Lot #: 065K6236
WB (1:5000 dilution)
AntibodyHorseradish peroxidase-conjugated goat anti-mouse IgGGE HealthcareCat. #: NA931VS
Lot #: 9708060
WB (1:50,000 dilution)
AntibodyMouse anti-tubulinMillipore SigmaCat. #: MABT205
Lot #: 2999783
WB (1:5000 dilution)
Software, algorithmR (ver 3.4.1)Ritz et al., 2015
PMID:26717316
See Materials and method, section 2 and section 7
Software, algorithmLeica LAS AFhttp://www.leica-microsystems.comSee Materials and method, section 5
Software, algorithmFijihttps://imagej.net/FijiSee Materials and method, section 5
Software, algorithmImaris 8.1BitplaneSee Materials and method, section 5
Software, algorithmFrapBotKohze et al., 2017 www.frapbot.kohze.comSee Materials and method, section 6
OtherFM4-64Invitrogen/Thermo Fisher ScientificCat. #: T13320See Materials and method, section 3
OtherEndo-HNEBCat. #: P0703SSee Materials and method, section 4

Plant materials and growth conditions

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The Arabidopsis accession Columbia (Col) was used as wild type. The following mutants and reporter transgenic plant lines were reported previously: erecta (er-105) (Shpak et al., 2005); erl1-2 (Shpak et al., 2005); erl2-1 (Shpak et al., 2005); epf1-1 (Hara et al., 2007); tmm-KO (Hara et al., 2007); ERL1pro::ERL1-YFP in erl1-2, ERL1pro:: ERL1-FLAG in erl1-2 and erecta erl1-2 erl2-1, and ERL1pro::ERL1ΔKinase in erl1-2 (Lee et al., 2012); MUTEpro::ERL1-YFP in er-105 erl1-2 erl2-1 and iEPF1 lines (Qi et al., 2017); BRI1pro::BRI1-GFP (Friedrichsen et al., 2000), UBQ10pro::YFP-SYP32 (Wave22Y), UBQ10pro::YFP-RabA5d (Wave24Y), UBQ10pro::YFP-RabA1e (Wave34Y), and UBQ10pro::YFP-ARA7 (Wave2Y) (Geldner et al., 2009); SYP43pro::GFP-SYP43 (Uemura et al., 2012), ARA6pro::ARA6-GFP (Ebine et al., 2011), ARA7pro::mRFP-ARA7, SYP22pro::mRFP-SYP22, and SYP43pro::mRFP-SYP43 (Ebine et al., 2011; Postma et al., 2016) are a gift from Prof. Takashi Ueda (NIBB, Japan); ST-RFP and KDEL-RFP constructs (Faraco et al., 2017) are from Prof. Gian Pietro Di Sansebastiano (Univ. of Salento, Italy); 35Spro::GFP-HDEL (Mitsuhashi et al., 2000) and CavM35S::ST-YFP (Takagi et al., 2013) are from Prof. Ikuko Hara-Nishimura (Konan University, Japan); Reporter lines were introduced into respective mutant backgrounds by genetic crosses or by Agrobacterium-mediated floral-dipping transformation, and genotypes were confirmed by PCR. Seedlings and plants were grown as described previously (Lee et al., 2012). For a list of PCR-based genotyping primer sequence, see Table S1.

Recombinant peptide production

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For recombinant peptide production of EPFL6, a coding sequence covering a predicted mature EPFL6 (MEPFL6) peptide was cloned into pBADgIII vector to make a fusion construct with 6xHis-tag (pJSL79: generated by Dr. Jin Suk Lee). Expression, purification, and refolding of predicted mature EPF1 (MEPF1) or EPFL6 (MEPFL6) peptides were performed as described previously (Lee et al., 2012), except for the following. His-tagged MEPF1 or MEPFL6 was affinity purified on 5 ml His-Trap HP column (GE Healthcare) using NGC Chromatography System (Bio-Rad). Inclusion bodies from 1.0 L of E. coli were solubilized in guanidine hydrochloride (Gdn-HCl) buffer (6.0 M Gdn-HCl, 500 mM NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol, 50 mM Tris, pH 8.0) and loaded onto the column and washed with 10 column volumes (50 mL) of Wash Buffer (8.0 M urea, 500 mM NaCl, 30 mM imidazole, 1 mM β-mercaptoethanol, 50 mM Tris, pH 8.0) at a flow rate of 3.00 ml/min, and MEPF1 or MEPFL6 peptides were eluted with a 0–100% gradient of Wash to Elution Buffer (8.0 M urea, 500 mM NaCl, 500 mM imidazole, 1 mM β-mercaptoethanol, 50 mM Tris, pH 8.0) over 10 column volumes at 3.00 mL/min prior to refolding. The quality of refolded peptide was analyzed by HPLC (Walters DataPrep 300), its bioactivity was confirmed using Arabidopsis seedlings, and bioassay on Arabidopsis seedlings were performed as described previously (Lee et al., 2012; Lee et al., 2015). Peptide treatments were performed as described previously (Lee et al., 2015). For a control experiment of Stomagen treatment on ERL1ΔK-CFP, ERL1-YFP driven by MUTE promoter in erecta erl1 erl2 (Qi et al., 2017) was used due to the germination issues of ERL1-YFP driven by its own promoter. For the dose-response analysis of EPFL6, the R-package ‘drc’ (Ritz et al., 2015) was used to fit the binding curve to the generalized log logistic distribution (Uchida et al., 2018).

Pharmacological treatment

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Surface-sterilized seeds were sown on half-strength MS media containing 1% (w/v) sucrose and 1% (w/v) agar for 7 days in growth chamber. To synchronize germination, plates were placed in the dark at 4°C for 2 days. Plants were grown vertically in a 16 hr/8 hr light/dark cycle (40 μE m−2 s−1). Cotyledons were removed by scissors before drug treatment. For Concanamycin A treatment, plants were soaked in Milli-Q ultrapure-water (Merck Millipore) containing 2 µM FM4-64 (Thermo Fisher Scientific) with vacuum and then incubated in ultrapure water without FM4-64 for 6 hr to washout excessive dye. Then plants were transferred into ultrapure water containing 1 µM concanamycin A (AdipoGen Life Sciences, CA, USA) for 5 hr.

BFA (Sigma: Cat No. B7651) and Wortmannin (Sigma: Cat No. W1628) were dissolved as 10 mM stock using ethanol and DMSO, respectively. For BFA treatment, cotyledons of 7-day-old seedlings were removed, and the rest of the seedlings were immersed into either mock (0.3% of ethanol), or 30 µM BFA solution, vacuumed for 1 min, and immersed for 30 min before imaging. For a series of BFA concentration treatment experiments analyze its effect on endomembrane systems, whole plants were soaked in ultrapure water containing 5 µM FM4-64 and 30, 90, or 180 µM BFA (Cayman Chemical, MI, USA; Stock solution: 50 mM in DMSO) or equivalent concentration of solvent DMSO with vacuum.

For Wortmannin treatment, seedlings were treated with 25 µM Wortmannin in 0.25% DMSO. 0.25% DMSO solution was used as a mock condition. For MEPF1 and MEPFL6 treatment, purified peptide solution was diluted to 5 µM using liquid ½ MS media. Cotyledons of 7-day-old seedlings were removed, and the rest of the seedlings were immersed into the above solutions, vacuumed for 1 min, and immersed for 10 min before imaging. The same procedure was done for Stomagen treatment except that the seedlings were immersed into the solution for 1 hr. For co-treatment of cycloheximide (CHX: Sigma, C4859) and BFA, 7-day-old seedlings, with cotyledons moved, were immersed into 50 µM CHX for 1 hr followed by either mock (0.3% of ethanol), or 30 µM BFA solution, vacuumed for 1 min, and immersed for 30 min before imaging.

For Tyr A23 (Sigma: Cat No. T7165) treatment, Tyr A23 was dissolved as 50 mM stock using DMSO. 5-day-old seedlings were immersed into either mock (0.1% of DMSO) or 50 µM Tyr A23 solution, vacuumed for 1 min, and immersed for 1 hr before imaging.

ES9-17 was generously provided by Dr. Eugenia Russinova (VIB, Gent), and ES9-17 treatment was done as described in Dejonghe et al., 2019. Briefly, ES9-17 was dissolved as 50 mM stock using DMSO. For ES9-17 and FM 4–64 treatment on true leaves, cotyledons of 7-day-old seedlings were removed, and the rest of the seedlings were immersed into either mock (1/2 MS medium with 0.4% of DMSO) or 100 µM ES9-17 solution (1/2 MS medium with 50 µM ES9-17) for 30 min followed by 5 µM FM 4–64 (Thermo Fisher, T13320) staining for 30 min before imaging. For ES9-17 and FM 4–64 treatment in roots, 3-day-old seedlings were immersed into either mock (1/2 MS medium with 0.4% of DMSO), or 50 µM ES9-17 solution (1/2 MS medium with 100 µM ES9-17) for 30 min, followed by FM 4–64 (5 µM) staining for 30 min before imaging.

For Rhodamine B (Sigma: Cat No. R6626) hexyl ester treatment, Rhodamine B hexyl ester was dissolved as 16 mM stock using DMSO. 5-day-old seedlings were immersed into either mock (1% of DMSO) or 160 µM Rhodamine B hexyl ester solution for 30 min before imaging.

Protein extraction, enzymatic assay (Endo-H), and protein gel immunoblot analysis

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For Endo-H (NEB: Cat No. P0703S) assays, erecta erl1 erl2 seedlings with functional ERL1pro::ERL1-FLAG were grown on ½ MS media plates for 3 days and then transferred to ½ MS liquid media with either Tris-HCl buffer (pH 8.8) or 5 µM Stomagen peptide in a 24-well cluster plate at room temperature for one day before being pooled for harvest. Plant materials were ground in liquid nitrogen, and then extracted with buffer (100 mM Tris-HCl pH 8.8, 150 mM NaCl, 1 mM EDTA, 20% glycerol, 20 mM NaF, 2 mM Na3VO4, 1 mM PMSF, 1% Triton X-100, 1 tablet per 50 ml extraction buffer of cOmplete proteinase inhibitor cocktail, Roche). The extracts were briefly sonicated at 4°C and centrifuged at 4000 r.p.m. for 10 min at 4°C to remove cell debris. The supernatant was then ultracentrifuged at 100,000 g for 30 min at 4°C. Total protein concentration was determined using a Bradford assay (Bio-Rad: Cat No. 5000006) before adjustment. The solution was incubated with Dynabeads Protein G (Invitrogen: Cat No. 10004D) conjugated with mouse monoclonal anti-FLAG M2 (Sigma: Cat No. F-3165) for 2 hr with slow rotation at 4°C, followed by washing with TBS with 0.1% Tween 20. The immunoprecipitates were eluted with 2x SDS sample buffer (100 mM Tris-HCl at pH 6.8, 4% SDS, 0.02% Bromophenol Blue, 20% glycerol, 2% 2-mercaptoethanol, 1% proteinase inhibitor cocktail) by boiling for 10 min. Each immunoprecipitate was then separated into two aliquots, treated with either water or Endo-H for 10 min at 37°C. Immunoblot analysis was performed using mouse monoclonal anti-FLAG M2 (Sigma: Cat No. F-3165; 1:5,000) antibody as primary antibody, and horseradish peroxidase-conjugated goat anti-mouse IgG (GE Healthcare: Cat No. NA931VS; 1:50,000) as secondary antibody. For loading control, immunoblot was performed using mouse anti-tubulin (Millipore Sigma: Cat No. MABT205; 1:5000). The protein blots were visualized using Chemiluminescence assay kit (Thermo Scientific: Cat No. 34095).

Confocal microscopy and image analysis

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Confocal microscopy images were taken on the Leica SP5X-WLL inverted confocal microscope (Solms, Germany). Time-lapse imaging of ERL1-YFP true leaves was prepared as described previously (Peterson and Torii, 2012). For internalization imaging of ERL1-YFP, ERL1ΔK-CFP and all other membrane organelle markers, maximum intensity projection of Z-stack images (0.33 μm step) covering the entire meristemoids were generated and subjected to analysis. The imaging was done with a 63x/1.2 W Corr lens on Leica SP5X. A 514 nm laser was used to excite YFP and emission window of 518–600 nm was used to collect YFP signal. A 458 nm laser was used to excite CFP and emission window of 470–510 nm was used to collect CFP signal. For the multicolor images of YFP and RFP, true leaves of 7-day-old transgenic seedlings were observed with a 63x/1.2 W Corr lens on Leica SP5X-WLL (Leica). 514 nm laser was used to excite YFP and 555 nm laser was used to excite RFP and FM4-64. Emission filter was set as 518 nm-550nm for YFP and 573–630 for RFP and FM4-64. Each experiment was repeated at least three times, each with multiple seedlings. Leica TCS-SP8 gSTED (Leica) was used for assessing the effects of Concanamycin A and high concentration of BFA on membrane organelle. For these purposes, images were taken using 93x glycerol immersion objective lens and 488/500–530 nm, 514/518–550 nm, 561/600–650 nm for GFP, YFP, and RFP signals, respectively with Time Gating of 0.3–0.6 nm to eliminate chloroplast autofluorescence. The Leica LAS AF software (http://www.leica-microsystems.com), Fiji ( https://imagej.net/Fiji), and Imaris 8.1 (Bitplane) were used for post-acquisition image processing. Quantitative analysis of BFA signal intensity was performed using the Voxel counter plugin in Fiji briefly as the following. An extensive series of Z-stack confocal images covering the entire meristemoids were converted to 8-bit. Cut-off threshold was set from 35 to 200, which effectively removed objects with non-specific signals. An ROI was defined to cover the entire inside volume of each cell. Threshold sum (BFA bodies) and ROI sum (cell volume) for each ROI were subsequently measured. For Wm bodies quantification, z-stack confocal microscopy images were taken on the Leica SP5x inverted confocal microscope. The maximum projection of z-stack images was then generated by FIJI, and the number of detectable endosomes per cell or the number of Wm bodies (diameter >0.5 µm) were counted and recorded.

Fluorescence recovery after photobleaching (FRAP) analysis

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The FRAP experiments were conducted on ERL1-YFP using a 63x/1.2 W Corr lens on the Leica SP5X confocal microscope by photobleaching ~10% of the plasma membrane with 100% 405 nm laser power. 514 nm laser was used to excite YFP and emission window of 518–600 was used to collect YFP signal. Recovery of fluorescence was monitored in the photobleached plasma membrane for 6 min with 3 s intervals. A non-photobleached region was monitored meanwhile as an internal control. Average intensities of the region of interest were quantified with Leica LAS AF software. The exported data were analyzed and modeled by using the R-based FrapBot software (www.frapbot.kohze.com) (Kohze et al., 2017) with some modification to run on the local lab computer. The FRAP recovery curves were fitted to a single-parameter exponential model to determine the half time.

Data plots and statistics

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Graphs were generated using R (ver 3.4.1) using ggplot2. For box plots and violin plots, individual data points are plotted as dot plots, and for large sample numbers the dot plots were jittered with a position of 0.2. Welch’s Two Sample T-Test and Person’s correlation were analyzed using R. For specific codes, see Supplementary Source Code.

References

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Decision letter

  1. Jürgen Kleine-Vehn
    Reviewing Editor; University of Natural Resources and Life Sciences, Austria
  2. Christian S Hardtke
    Senior Editor; University of Lausanne, Switzerland

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This work provides a better understanding of ligand-dependent control of intracellular receptor distribution. We are very pleased to publish this paper at eLife because it outlines how vesicle trafficking contributes to signal integration during stomata differentiation in Arabidopsis.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate" for consideration by eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. The following individual involved in review of your submission has agreed to reveal their identity: Grégory Vert (Reviewer #2).

While the reviewers showed interest in your cell biological analysis, they also discussed several shortcomings, which precludes its publication at eLife in its present form. On one hand, they ask you to improve the quantification and image quality throughout your manuscript. On the other hand, they request additional experiments, including the generation of new lines (e.g. kinase dead ERL1), to further support your conclusion. It is policy of eLife to avoid lengthy re-review processes with uncertain outcomes. Therefore, we decided to reject the paper at this stage. Please find the specific comments below.

Reviewer #1:

The authors use genetics, microscopy and pharmacological treatments to determine sub cellular dynamics of the receptor ERL1 upon perceiving distinct ligands in the stomatal lineage of Arabidopsis. Through pharmacological experiments they show that the receptor ligand pair goes to the MVB and the vacuole upon endocytosis and can distinguish activated receptor routes towards degradation from general receptor endocytosis. Furthermore, the authors propose an attractive hypothesis where the coreceptor TMM modulates internalization of ERL1 in a ligand-specific manner. The experiments are well controlled, described in detail both in terms of methodology and sample size and the paper is written clearly and with an easy to follow structure. I have several concerns that I would like to see addressed and a couple of suggestions that would help clarity and visualization purposes.

I would like to stress that I am not a cell biologist and not 100% familiar with the limitations of the pharmacological assays.

A) Major concerns:

1) The data clearly shows that upon ligand perception the ERL1 gets internalized, send to MVB and likely degraded in the vacuole. While it is extremely interesting that these dynamics get modulated by co-receptor and the specific ligand, to me the mechanistic proof that internalization is relevant is somewhat lacking. Particularly when considering that very little of the PM-localized receptor gets internalized (unlike for FSL2, which the authors also discuss). If endocytosis and MVB mediated degradation is relevant then I would expect a patterning and clustering phenotype upon treatment with an endocytosis inhibitor (ES9-17 or Tyr A23) or even with Wm that blocks MVBs. Even though these experiments are tricky and probably must be done in a pulsed set-up, I think the manuscript would profit from phenotypic analysis of the leaf epidermis upon blocking the aforementioned processes (particularly endocytosis).

2) I am wondering why the authors focused on mEPF1 only since they are having mEPF2 available and Lee et al., 2012 showed that mEPF2 also binds to ERL1. Since the two peptides have not quite the same genetic role (one seems to inhibit the stomatal lineage at earlier stages than the other) it would be interesting to see if mEPF2 treatments induce the same internalization and sub-cellular dynamics and whether EPF2-ERL1 internalization is TMM dependent, too.

3) I feel somewhat uneasy about the fact that the authors use BFA treatment to infer anything about "general recycling" of the receptor. In my understanding BFA bodies are stalled TGN vesicles, which can go either to the MVB-vacuole pathway or get recycled to the PM. Therefore the BFA treatments and resulting BFA bodies rather indicate general internalization/endocytosis rather than recycling. To show recycling the authors would need to perform BFA washout experiments, but this is likely not possible since so much PM signal persists. I would like to ask the authors to change recycling for endocytosis/internalization throughout the manuscript.

Along the same lines, Figure 7 shows the receptor recycling as something isolated on the upper right corner. In my opinion this also goes through the TGN and should be drawn accordingly. Everything gets internalized through endocytosis and form TGN/EE. Then they either cycle back to the PM (recycling) or go to LE/MVB to be targeted to the vacuole. Please change the model accordingly and indicate which steps are inhibited by BFA, Wm, and ES9-17 and Tyr A23 in Figure 7.

B) Concerns in Figures:

4) General comments regarding figures:

- I would like to suggest that for all the quantifications of cells with bodies (eg. Figure 1C,E; Figure 2F, Figure 3E, F etc.) to label the y-axis with "% (or n) of YFP-positive bodies/endosomes/etc.".

- In addition, I would appreciate that for all plots the jitter of the data is indicated much like for the violin plots. It is currently missing in Figure 1C, 1E, 1G, Figure 2G.

- Finally, all bar charts should be box plots or violin plots (Figure 3G; Figure 4 – Figure supplement 1 B and C)

5) Figure 1:

I don't quite get what the difference between ERL1 vs. SYP22 is compared to SYP22 vs. ERL1. Please elaborate in the legend.

6) Figure 2:

In panel A it is not clear that the leftmost π stainings are different images than the ones showing YFP channels. Please split phenotype and YFP pictures into panel A and panel B, respectively.

For the FRAP experiments, I wonder why there seems to be more recovery albeit slower recovery in tmm. Is this something that was observed in all 9 replicates? If yes, please quantify and discuss this data, too. If not, I would suggest to use a different representative fluorescence recovery curve for tmm. And please also elaborate on why WT vs. tmm was so unbalanced in terms of replicates (2G, 3 vs. 9 respectively).

7) Figure 3.

In Figure 3E, it is really hard to see the FM4-64 internalization. Maybe use a more focussed and enlarged field of view?

I think that Figure 3F and Figure 3G should be combined or at the very least should be both transformed into violin plots with jitter so that they can be compared. It would also be much easier to read if it indicated in the figure and not just the legend that one is ERL1-YFP bodies and the other is ERL1deltaK-YFP bodies.

Please indicate the n for all treatments in 3G in the figure legend and also show as jitter on a violin or box plot. Otherwise it is really hard to judge the quality of this data.

8) Figure 5.

Please specify exact p values in Figure 5E and 5F as you did in Figure 4E and 4F.

9) Figure 6.

In 6B and 6C it is really hard to see the lines indicating where the fluorescence intensity is measured.

In 6D I would suggest to choose other colors than black dark gray and light gray. Or bigger difference between dark grey and light grey.

C) Concerns in text:

10) subsection “ERL1 is internalized through multivesicular bodies to vacuolar pathway in stomatal meristemoids”.

What do you mean with differentiating meristemoids? Are not all meristemoids differentiating?

11) Subsection “TMM is required for the process of ERL1 endocytosis in true leaves”.

I would appreciate if these quantifications would be included as box plots with jitter in the Figure 2—figure supplement 2 so that we can see the n, the variation in the data etc.

12) Subsection “An antagonistic EPFL peptide, Stomagen, elicits retention of ERL1-YFP in the endoplasmic reticulum”.

For the Stomagen experiments the authors use the er erl1 erl2 triple mutant to remove potential redundancy. I am a little worried that for some of the EPF1 and EPF6l experiments the results would look different in the triple mutant as well. I would appreciate the authors discussing this issue here or in the Discussion section.

13) Subsection “An antagonistic EPFL peptide, Stomagen, elicits retention of ERL1-YFP in the endoplasmic reticulum”.

I think that the data does not warrant such a strong conclusion. The ER accumulation is seen independent of TMM but might be stronger in tmm background. But for this statement to be valid the authors have to compare fluorescence intensity in wt vs. tmm background.

14) Subsection “Pharmacological treatment”.

I am a bit worried by the immensely different incubation times for MEPF1 / MEPFL6 compared to Stomagen (10minutes vs. 1hour). Would we also see ER retention if MEPF1 was incubated for 1h, too? Please show that this is not the case.

Reviewer #2:

The manuscript by Qi et al., reports on the characterization of the dynamics of the ERECTA-LIKE1 receptor and its dependence on the building of a functional receptor complex. It is a very interesting manuscript that provides a detailed analysis of ERL1 trafficking upon ligand binding or activation. Below are listed the most important points that need to be addressed by authors to improve the quality and readability of their contribution.

1) The confocal microscopy images require some more work to fully convince the reader :

- Figure 2A, rather than quantifying the ratio of cells showing ERL1 positive endosomes in WT and tmm, it would be more informative to quantify the number of ERL1-positive endosomes per cells in WT and tmm. To do so, z-stacks across the whole cell and max projection must be performed to avoid bias from uneven distribution of endosomes between, cell surface and middle section.

This comment applies to other figures. For example, the authors quantified the number of endosomes in a given confocal section in subsequent figures (Figure 5A) but must provide quantification per cell using z-stacks and max projection.

- Quantification of % of cells showing BFA or Wm bodies is not appropriate. Please also quantify BFA body number and size per cell. This is a better readout of endocytic defects.

- Some of the images would deserve higher magnification or enlarged panels to better visualize endosomes, lack of endosomes, or BFA/Wm bodies.

2) BFA has been widely characterized and used in Arabidopsis roots where it yields bodies containing aggregated TGN surrounded by Golgi. In aerial parts, BFA is known to trigger the collapsing of Golgi into the ER. Whether it also yields endosomal defects in stomatal meristemoids must be documented or tested if undocumented. The images shown the effect of BFA being very zoomed out, it is hard to evaluate the effect of BFA.

3) BFA leads, according to the authors, to the aggregation of TGN indicating that proteins trafficking through the TGN will end up (recycled or not) in BFA bodies. The fact that ERL1 is observed in BFA bodies in tmm upon CHX is therefore puzzling. One would anticipate a decrease (or even absence) of ERL1 if internalization is compromised. A better quantification of BFA bodies (BFA body number/cell and size; see 2)) is required to better characterize possible defects in ERL1 internalization. If the result stands, the authors will have to provide some explanations in the Discussion section. Regardless, a more direct investigation of ERL1 internalization kinetics in WT or tmm background by VAEM imaging would GREATLY strengthen the authors conclusions. I am not sure the authors have access to VAEM, but if so, I strongly encourage them to try.

4) The FRAP experiment raises some questions. First, the recovery of ERL1-YFP in erl1 is almost absent in my opinion. There is hardly any recovery compared to T0 (slope is flat). As such, it is hard to interpret and one may come up with a very different conclusion where ERL1 is largely immobile at the cell surface, and that it is more mobile in tmm. The authors should (i) show the prebleach intensity on their plot and (ii) image faster after photobleaching to catch the initial recovery phase better.

5) The internalization of ERL1 and targeting to MVB suggests that ERL1 is routed to the vacuole for degradation, which the authors have not shown. The authors must therefore assess ERL1 degradation by western blot if ERL1 is detectable in leaf extracts and/or by confocal imaging inhibiting the lytic activity of the vacuole (dark conditions of concanamycin A).

6) The ER retention of ERL1 upon stomagen application is puzzling yet very interesting. The authors must back up their observations by monitoring the endogenous ERL1 protein by immunofluorescence (or alternatively an ERL1-HA or FLAG if the authors do not have an ERL1 antibody) to ascertain that the YFP fusion protein, although active, represents the behavior of genuine ERL1 for ER retention. Second, the authors must address whether this is specific or ERL1 by testing the influence of stomagen on other secreted proteins (PM proteins) that woumd be expressed (e,dogenously or artificially) in stomata.

Reviewer #3:

This manuscript by Qi et al. investigated how the ERL1 receptor interprets different signal inputs and how this affects its subcellular dynamics. Overall, the manuscript is rather descriptive and fails to provide convincing mechanistic insight, leaving many loose ends. In addition, the text needs to be streamlined with respect to terminology (MVBs, endosomes, BFA-bodies, etc) and what is actually shown on the figures to avoid mis- or over-interpretation. Furthermore, the conclusions need to be more accurately phrased.

1) Regarding the statement that "TMM is required for ERL1 endocytosis", I would phrase this differently. TMM is required to form an active signaling complex, and in the absence of such an active signaling complex, no endocytosis occurs (as is also the case upon Stomagen treatment or when the kinase domain is removed). How do the authors explain the Stomagen-mediated accumulation of ERL1 in the endoplasmic reticulum? This must be a different pool of ERL1 distinct from the Stomagen-bound ERL1. Do the authors assume a regulatory mechanism whereby Stomagen-bound ERL1 signals to control the production and ER retention of new ERL1? In this context, I would conclude that it is not inefficient ERL1 endocytosis that leads to retention in the ER, but an inactive receptor that stays on the membrane which leads to no new secretion. Does the ERL1Δkinase also accumulate in the ER?

2) With the current data, it cannot be concluded that kinase activity is required for ERL1 internalization. There could be internalization motifs in the kinase domain. To conclusively demonstrate this, an ERL1 kinase dead variant needs to be analyzed with respect to internalization. In the absence of TMM, un-activated ERL1 receptors are not readily targeted for endocytosis and remain stable at the membrane. What happens with ERL1Δkinase endocytosis in the presence of ligand?

3) The authors use different markers to reveal the localization of ERL1-YFP. However, a typical experiment to study endocytosis is the co-localization with FM4-64. Figure 2—figure supplement 2A and Figure 3E provide some idea, but the individual endosomes should be tracked. BFA bodies and MVBs are a proxy to study this, but then the phrasing in the text should be accordingly.

4) TMM is required for endocytosis of ERL1, and not recycling. BFA blocks the secretion of ERL1. One would expect that if there is less endocytosis, there is also less ERL1 in BFA bodies. However, based on the data in Figure 2 this is not the case. Also, Figure 2B shows an increased ERL1-YFP intensity upon BFA treatment in tmm, so both the number of cells with BFA bodies and the signal intensity would be meaningful to report. I would rephrase the conclusion in subsection “TMM is required for the process of ERL1 endocytosis in true leaves”: the absence of tmm does not impact the ERL1 secretory pathway, as reflected in the presence of ERL1-YFP positive BFA bodies.

5) The authors invoke that distinct EPF/EPFL peptide ligands activate a sub-population of ERL1 receptor complex to internalize through a TMM-based discriminatory system. This seems independent of distinct expression domains of these peptides as purified EPFL6 does not have the same effect as EPF1. The authors suggest distinct localization in nanodomains, but do not investigate this. It would be valuable to see FRAP analyses on ERL1-YFP +/1 EPF1 or EPFL6 in erl1 and erl1 tmm.

6) Throughout the manuscript, MVBs should be used when describing the "large dots" on the figures. I am not sure if the resolution allows to see endosomes. Are Figure 4E-F and Figure 5E-F really showing number of endosomes or should this be MVBs?

7) The authors write "TMM is required for endocytic sorting of ERL1 to MVBs, which is a hallmark for receptor degradation". I guess that given the fact that in tmm ERL1-YFP signal increases in the ER, it is rather difficult to investigate this statement using e.g. Western Blot.

8) Given the recent knowledge on Tyr A23, I would only include the more specific ES9-17. Is this effect on ES9-17 specific for ERL1-YFP, or are other receptor proteins also accumulating in the ER?

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate" for consideration by eLife. Your article has been reviewed by Christian Hardtke as the Senior Editor, Jürgen Kleine-Vehn as the Reviewing Editor, and two reviewers. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Your manuscript reveals remarkable insights into peptide ligands and how they modulate the intracellular distribution of their receptors. We invite you to resubmit your paper for final acceptance, considering the following main suggestions of the reviewers:

I) The reviewers ask you to clarify several issues, such as statistical evaluation and quantifications, which may not require novel experiments.

II) During their discussion, the reviewers defined a better characterization and integration of the ERL1Δkinase line, as an essential part of the revision, and one that unfortunately requires additional experimentation.

III) The reviewers judge that some experiments aimed at a better integration of your data into developmental context would substantially strengthen the impact of your manuscript. On the other hand, they do not believe that such an insight is essential for acceptance of the manuscript.

Please, see the detailed comments of the reviewers below.

Reviewer #1:

This manuscript by Qi et al., investigated how the ERL1 receptor interprets different signal inputs and how this affects its subcellular dynamics. There are several novel and interesting insights, but still some loose ends remain. The authors have addressed several of the comments raised previously and improved the manuscript.

Reviewer #2:

The authors undertook a remarkable effort to improve the cell biological aspects of this manuscript and I do appreciate the new experiments and careful quantification of the BFA experiments and analysis of new marker lines with expert Dr. Akira Yoshinari. In addition, the ConcavA treatments and the clear visualization of ERL1 in vacuoles adds significantly to this manuscript. However, I would like to stress two of my previous concerns again.

1) Previous point 1:

From a mechanistic/genetic perspective, the functional relevance of ERL1 endocytosis and targeting to MVB/LE and vacuole is still missing. I do completely understand that a phenotypic assay is not doable due to long-time cytotoxic effects of BFA or Wm. I also agree that strong genetic support can only come through analysis of how ERL1 is modified (phosphorylation etc) and how these modifications affect endocytosis, but this is clearly out of scope of this article. Nevertheless, in Qi et al., 2017, the authors show that the EPF1-ERL1 module directly represses MUTE in an autocrine manner. When EPF1 expression is induced, a clear decrease in MUTE-YFP fluorescence can be observed already 15hours post induction. I wonder whether induction of EPF1 together with mock or toxin treatment (Wm?) in the first couple of hours could reveal differences in how efficiently MUTE-YFP is downregulated. This could give at least somewhat functional support to the hypothesis that the endocytosis and targeting to the vacuole is required for efficient downregulation of key targets by the EFP1-ERL1 module.

2) Previous point 12:

Even though the authors stated in their response that they elaborated on ER ERL1 and ERL2 redundancy in the discussion, I cannot find new information or statements regarding the fact that the Stomagen-induced ER-retainment of ERL1 is much stronger in the triple erecta-erl1-erl2 background. I am aware that this is a subtle difference but as the authors confirm an important one. So please elaborate on this in the results (line 298ff) and clearly mention that Stomagen has been shown to interact initially with ER before acting through ERL1 and ERL2 at later stages, which is why a triple mutant is needed here and ER retainment in the single erl1 is much weaker.

https://doi.org/10.7554/eLife.58097.sa1

Author response

We took the constructive criticisms from the three Peer Reviewers very seriously, and performed the following experiments to fully address their concerns.

i) Concanamycin A treatment to seedlings expressing ERL1-YFP: The results demonstrate that ERL1-YFP is destined to a vacuole (see Revised Figure 2).

ii) Stomagen application to BRI1pro::BRI1-GFP seedlings: BRI1-GFP is endongenously expressed in leaf epidermis, includingstomatal meristemoids. The results show that Stomagen peptide does not influence subcellular localization of BRI1, thereby supporting the notion that the effects of Stomagen on ERL1 is specific (see Revised Figure 8–figure supplement 2).

iii) Extensive documentation of BFA body formation in stomatal meristemoids: We used eight different endomembrane organelle markers with an endocytic tracer dye FM4-64 to fully document the behavior of endomembrane system upon BFA treatment in meristemoids. Our results clearly demonstrate that, at low concentration (30 microM), BFA body formation occurs just like in root cells (See Revised Figure 3 and Figure 3–figure supplement 1).

Furthermore, we re-analyzed BFA and Wm treatment data as suggested by the Reviewers, and provided enlarged, high-resolution confocal images as much as possible. By analyzing the number of endosomes/BFA/Wm bodies per cell as well as the volume ratio, we were able to support our original conclusion that TMM is critical for the internalization of EPF1-perceived ERL1. Please see our point-by-point response for specifics.

Reviewer #1:

[…] A) Major concerns:

1) The data clearly shows that upon ligand perception the ERL1 gets internalized, send to MVB and likely degraded in the vacuole. While it is extremely interesting that these dynamics get modulated by co-receptor and the specific ligand, to me the mechanistic proof that internalization is relevant is somewhat lacking. Particularly when considering that very little of the PM-localized receptor gets internalized (unlike for FSL2, which the authors also discuss). If endocytosis and MVB mediated degradation is relevant then I would expect a patterning and clustering phenotype upon treatment with an endocytosis inhibitor (ES9-17 or Tyr A23) or even with Wm that blocks MVBs. Even though these experiments are tricky and probably must be done in a pulsed set-up, I think the manuscript would profit from phenotypic analysis of the leaf epidermis upon blocking the aforementioned processes (particularly endocytosis).

We thank reviewer 1 for finding our work extremely interesting. We agree with reviewer 1 that it would be ideal if we could use endocytosis inhibitors to block ERL1 endocytosis and then characterize the resulting stomatal pattering. Unfortunately, extended treatment of these inhibitors for several days to observe stomatal development is not possible due to high cytotoxicity of these inhibitors. All of our results clearly show the subcellular dynamics of ERL1 upon perceiving its ligands with different functions (EPF1, Stomagen, and EPFL6). Furthermore, the dominant-negative receptor that cannot signal is compromised in internalization. In the future, identification of receptor modification which influence internalization (e.g. phosphorylation, ubiquitination) may fully answer reviewer 1's question, which are out-of-scope of this manuscript.

2) I am wondering why the authors focused on mEPF1 only since they are having mEPF2 available and Lee et al., 2012 showed that mEPF2 also binds to ERL1. Since the two peptides have not quite the same genetic role (one seems to inhibit the stomatal lineage at earlier stages than the other) it would be interesting to see if mEPF2 treatments induce the same internalization and sub-cellular dynamics and whether EPF2-ERL1 internalization is TMM dependent, too.

We have previously reported that mEPF1 and ERL1 are both expressed in stomatal meristemoids and participate in autocrine signaling to restrict stomatal differentiation potential (Qi et al., 2017), in addition to their well-established role in enforcing the stomatal one-cell spacing rule (Hara et al., 2007). The clear expression pattern of ERL1 in the meristemoids and the known consequence of autocrine signaling make mEPF1-ERL1 a unique and powerful system to address how signal perception (activation vs inactivation) triggers receptor subcellular dynamics. While mEPF2 can also be perceived by ERL1, its major receptor, ERECTA is ubiquitously expressed at very high level (Lee et al., 2012), and we predict that this will interfere with the proper interpretation. In the revised manuscript, we emphasized our rationale in the Introduction.

3) I feel somewhat uneasy about the fact that the authors use BFA treatment to infer anything about "general recycling" of the receptor. In my understanding BFA bodies are stalled TGN vesicles, which can go either to the MVB-vacuole pathway or get recycled to the PM. Therefore the BFA treatments and resulting BFA bodies rather indicate general internalization/endocytosis rather than recycling. To show recycling the authors would need to perform BFA washout experiments, but this is likely not possible since so much PM signal persists. I would like to ask the authors to change recycling for endocytosis/internalization throughout the manuscript.

Along the same lines, Figure 7 shows the receptor recycling as something isolated on the upper right corner. In my opinion this also goes through the TGN and should be drawn accordingly. Everything gets internalized through endocytosis and form TGN/EE. Then they either cycle back to the PM (recycling) or go to LE/MVB to be targeted to the vacuole. Please change the model accordingly and indicate which steps are inhibited by BFA, Wm, and ES9-17 and Tyr A23 in Figure 7.

We truly thank reviewer 1 for raising an important point. We agree that BFA treatment affects not only recycling but also internalization/endocytosis. As reviewer 1 mentions, "BFA washout" time course experiments is generally performed to measure the rate of recycling from BFA body to Plasma membrane. Given that our results indicate that the main effects of tmm mutation is reduced internalization/endocytosis, we do not believe that the additional BFA washout experment is necessary. In response to reviewer 1, we have changed recycling to recycling and endocytosis/internalization throughout the manuscript. Likewise, we have revised the model figure to correctly reflect the literature and our inhibitor results (now Figure 9).

During the revision process, we extensively discussed how to address the questions raised by the reviewers regarding our BFA treatment experiments. We invited an expert, Dr. Akira Yoshinari, who studies subcellular dynamics of polarly-localized membrane transporters. Through collaboration, we performed a series of BFA-treatment experiments in the context of stomatal meristemoids using a variety of subcellular membrane markers (revised Figure 3 and Figure 3—figure supplement 1; see our specific response to reviewer 2). Furthermore, we performed a pharmacological treatment using Concanamycin A, a specific inhibitor of vacuolar H+-ATPase that is known to reduce protein degradation in the lytic vacuole (e.g. Kleine-Vehn et al., 2008). We now demonstrate that ERL1 indeed accumulates in a vacuole (see revised Figure 2). In summary, all the additional experiments we performed support our original findings that ERL1, upon activated by EPF peptides, is subjected to endocytosis/internalization destined to vacuole and the process gets compromised in the absence of TMM.

B) Concerns in Figures:

4) General comments regarding figures:

- I would like to suggest that for all the quantifications of cells with bodies (eg. Figure 1C,E; Figure 2F, Figure 3E, F etc.) to label the y-axis with "% (or n) of YFP-positive bodies/endosomes/etc.".

We have modified the y-axis labels and re-analyzed our data to plot the number of

YFP positive bodies/endosomes/etc per cell (see revised Figure 4F, Figure 6—figure supplement 1B, C). For a quantitative analysis of BFA bodies, as also requested by reviewer 2, we have re-analyzed the voxel YFP intensity within the cell (representing endosomes and BFA bodies) per voxel YFP intensity of cell periphery (plasma membrane) and plotted the data to reveal subtle reduction in tmm mutant background (see revised Figure 4D). This analysis indeed revealed a reduction in the BFA body signals by the loss-of-function mutation in TMM. We additionally reanalyzed Wm treatment and plotted boxplots as number of Wm bodies per cell (see revised Figure 4F, Figure 4 —figure supplement 2D).

- In addition, I would appreciate that for all plots the jitter of the data is indicated much like for the violin plots. It is currently missing in Figure 1C, 1E, 1G, Figure 2G.

Provided for all box plot graphs (see revised Figure 1C, Figure 4D, 4F, Figure 6—figure supplement 1B, C).

- Finally, all bar charts should be box plots or violin plots (Figure 3G; Figure 4 – Figure supplement 1 B and C)

Done. The original Figure 3G is a percentage of all cell counted. We removed the chart and just indicated the percentage in the text.

5) Figure 1:

I don't quite get what the difference between ERL1 vs. SYP22 is compared to SYP22 vs. ERL1. Please elaborate in the legend.

We thank reviewer 1 for asking for the clarification.

ERL1 vs. SYP43 indicates how many ERL1-YFP endosomes co-localize with SYP43-RFP. Co-localized endosomes are in white arrowheads.

SYP43 vs. ERL1 indicates how many SYP43-RFP endosomes co-localize with ERL1-YFP endosomes. Co-localized endosomes are in white arrowheads.

The % colocalization are not identical because some endosomes only accumulate ERL1-YFP whereas some other endosomes only accumulate SYP43-RFP. This disparity is most evident when comparing SYP22-RFP and ERL1-YFP, because almost all endosomes accumulates YFP signals alone, whereas nearly all of the RFP endosomes co-localize with YFP signals. To be clear to readers, we now describe this in the figure legend. Thank you.

6) Figure 2:

In panel A it is not clear that the leftmost π stainings are different images than the ones showing YFP channels. Please split phenotype and YFP pictures into panel A and panel B, respectively.

Reviewer 1 is correct. Our intention in the original Figure was to show the stomatal cluster phenotype of tmm. To be unambiguous, we revised the figure to split the figure panels (now Figure 4A and B).

For the FRAP experiments, I wonder why there seems to be more recovery albeit slower recovery in tmm. Is this something that was observed in all 9 replicates? If yes, please quantify and discuss this data, too. If not, I would suggest to use a different representative fluorescence recovery curve for tmm. And please also elaborate on why WT vs. tmm was so unbalanced in terms of replicates (2G, 3 vs. 9 respectively).

We have used FRAP Bot program (www.frapbot.kohze.com) to normalize and fit our fluorescence recovery curve to a single-parameter exponential model. The fitted curve, but not the original fluorescence intensity changes, are provided in the graphs (revised Figure 4G). As far as our understanding, recovery curve is a standard way to determine the half time, and similar curve fitting has been done by many others in the field (e.g. Martinière et al., 2012; Zhang et al., 2016). We performed 10 independent FRAP experiments, and the FRAP Bot program chose the experiment that passed their criteria (we thought it would be better for the algorithm to perform unbiased curve fitting, rather than we choose the 'representative' data). To be clear, we indicated this in our revised Method section. Furthermore, in response to a similar critique from reviewer 2, we now provide a graph of observed florescence intensity change in the representative FRAP experiment (Figure 4—figure supplement 3 in the revision) as well as confocal images throughout a representative FRAP experiment (Video 2 and Video 3).

7) Figure 3.

In Figure 3E, it is really hard to see the FM4-64 internalization. Maybe use a more focussed and enlarged field of view?

Thank you for the suggestion. As also requested by reviewer 2, we now provide enlarged field of view throughout our confocal microscopy image figures.

I think that Figure 3F and and Figure 3G should be combined or at the very least should be both transformed into violin plots with jitter so that they can be compared. It would also be much easier to read if it indicated in the figure and not just the legend that one is ERL1-YFP bodies and the other is ERL1deltaK-YFP bodies.

Please indicate the n for all treatments in 3G in the figure legend and also show as jitter on a violin or box plot. Otherwise it is really hard to judge the quality of this data.

As suggested, we removed the Figure 3G, and instead, indicated the percentage of cells with endosome bodies within the manuscript.

8) Figure 5.

Please specify exact p values in 5E and 5F as you did in 4E and 4F.

We have performed Welch’s Two-Sample T-Test and the exact p values for pairwise comparison are specified (now revised Figures 6E, 6F, Figure 7E, and 7F).

9) Figure 6.

In 6B and 6C it is really hard to see the lines indicating where the fluorescence intensity is measured.

As suggested, we made the lines thicker (Figure 8 in the revision). Thank you.

In 6D I would suggest to choose other colors than black dark gray and light gray. Or bigger difference between dark grey and light grey.

Done (gray arrows blue and cyan now).

C) Concerns in text:

10) subsection “ERL1 is internalized through multivesicular bodies to vacuolar pathway in stomatal meristemoids”.

What do you mean with differentiating meristemoids? Are not all meristemoids differentiating?

Text changed to late meristemoids.

11) Subsection “TMM is required for the process of ERL1 endocytosis in true leaves”.

I would appreciate if these quantifications would be included as box plots with jitter in the Figure 2—figure supplement 2 so that we can see the n, the variation in the data etc.

Thanks for the suggestion. As also requested by other two Reviewers, we have plotted the number of YFP+ BFA/Wm bodies per cell (per ERL1-YFP expressing meristmoid) and replotted as box plots with jitter (now Figure 4—figure supplement 2 in the revision).

12) Subsection “An antagonistic EPFL peptide, Stomagen, elicits retention of ERL1-YFP in the endoplasmic reticulum”.

For the Stomagen experiments the authors use the er erl1 erl2 triple mutant to remove potential redundancy. I am a little worried that for some of the EPF1 and EPF6l experiments the results would look different in the triple mutant as well. I would appreciate the authors discussing this issue here or in the Discussion section.

We appreciate reviewer 1 for commenting on this, as we agree this is a valid point. Our group has extensively studied a redundancy among three ERECTA family genes and their specific interaction with TMM as well as different EPF peptides (e.g. Shpak, 2005; Hara, 2009; Lee, 2012; Lee, 2015); often these subtle yet important differences are ignored by the scientific community. Through these studies, we have found that whereas EPF1 has rather effects to activate ERL1, Stomagen acts stepwise throughout stomatal lineages, earlier with ERECTA, and ERL1 and ERL2 at a later stage). This complicates the interpretation. We elaborated this in the Discussion section.

13) Subsection “An antagonistic EPFL peptide, Stomagen, elicits retention of ERL1-YFP in the endoplasmic reticulum”.

I think that the data does not warrant such a strong conclusion. The ER accumulation is seen independent of TMM but might be stronger in tmm background. But for this statement to be valid the authors have to compare fluorescence intensity in wt vs. tmm background.

We have changed the sentence to "suggesting that the absence of TMM intensifies the accumulation of ERL1 in the endoplasmic reticulum".

14) Subsection “Pharmacological treatment”.

I am a bit worried by the immensely different incubation times for MEPF1 / MEPFL6 compared to Stomagen (10minutes vs. 1hour). Would we also see ER retention if MEPF1 was incubated for 1hour, too? Please show that this is not the case.

Due to very low amount of bioactive MEPF1 peptide we currently have, we did not perform additional time course experiments. Having said that, using very robust cheically inducible EPF1 overexpression system (which we used previously to robustly trigger EPF1 overexpression in time-controlled manner; Lee et al., 2012, Qi et al., 2017). We performed time course overexpression (which occurs with in less than 1 hour) up to 8 hours, and we did not observe any ERL1-YFP signal in the ER. Thus, it is highly unlikely that the different subcellular localization pattern of ERL1-YFP by MEPF1 or Stomagen application is owing to 50 min difference. If reviewer 1 request, we would be happy to provide the data as Supplemental Figure.

Reviewer #2:

The manuscript by Qi et al., reports on the characterization of the dynamics of the ERECTA-LIKE1 receptor and its dependence on the building of a functional receptor complex. It is a very interesting manuscript that provides a detailed analysis of ERL1 trafficking upon ligand binding or activation. Below are listed the most important points that need to be addressed by authors to improve the quality and readability of their contribution.

We thank reviewer 2 for finding our manuscript very interesting.

1) The confocal microscopy images require some more work to fully convince the reader :

- Figure 2A, rather than quantifying the ratio of cells showing ERL1 positive endosomes in WT and tmm, it would be more informative to quantify the number of ERL1-positive endosomes per cells in WT and tmm. To do so, z-stacks across the whole cell and max projection must be performed to avoid bias from uneven distribution of endosomes between, cell surface and middle section.

This comment applies to other figures. For example, the authors quantified the number of endosomes in a given confocal section in subsequent figures (Figure 5A) but must provide quantification per cell using z-stacks and max projection.

We sincerely appreciate reviewer 2's expert suggestions. We would like to emphasize that all of our quantitative analyses of ERL1-YFP endocytosis were performed by using maximum intensity projection of Z-stack images covering the entire meristmoids, not using single-plane images. We clarified this in the Materials and methods section.

- Quantification of % of cells showing BFA or Wm bodies is not appropriate. Please also quantify BFA body number and size per cell. This is a better readout of endocytic defects.

As suggested by reviewer 2 and all other reviewers, we re-analyzed our images and quantified BFA or Wm body number per cell (revised Figure 4D, F, Figure 4 —figure supplement1D, E, Figure 6—figure supplement 1B, C) and plotted as boxplots with individual data points as jitter.

For comparing the BFA-body formation between WT vs. tmm, in order to see subtle differences, we additionally performed quantification of the voxel signal intensity of inside of the cell (corresponding to endosomes/BFA bodies) per plasma membrane. This analysis was indeed very informative, as we were able to show a subtle yet statistically different voxel signal intensity ratio between WT and tmm (revised Figure 4D). It is well known that BFA strongly inhibits recycling, but also interferes with endocytosis/internalization. The subtle yet significant reduction in BFA bodies in tmm supports our original conclusion that ERL1 internalization requires its co-receptor TMM. Thank you for the great suggestion!

- Some of the images would deserve higher magnification or enlarged panels to better visualize endosomes, lack of endosomes, or BFA/Wm bodies.

As suggested, we now provide enlarged panels throughout (e.g. revised Figure 1A, 1D, Figure 4E, Figure 5D).

2) BFA has been widely characterized and used in Arabidopsis roots where it yields bodies containing aggregated TGN surrounded by Golgi. In aerial parts, BFA is known to trigger the collapsing of Golgi into the ER. Whether it also yields endosomal defects in stomatal meristemoids must be documented or tested if undocumented.

We thank reviewer 2 for bringing up the previous knowledge to our attention.

Indeed, Robinson et al., (2006) and Langhans et al., (2011) reported that BFA triggers re-absorption of Golgi membrane to the ER in leaves. These authors used rather high concentrations of BFA (50 µM for the former report and 90, 180, and 360 µM for the latter report) when observing the collapse of Golgi to ER. By contrast, we used low BFA concentration (30 µM), a condition generally used to block recycling/endocytosis in roots.

Since we did not find previous literatures that study membrane organelle dynamics in BFA-treated stomatal meristemoids, we took this revision process as an opportunity for the extensive documentations. For this purpose, we collaborated with Dr. Akira Yoshinari, an expert in subcellular dynamics of membrane transporters in plants (Dr. Yoshinari is the second author of our revised manuscript).

We collected eight different membrane organelle markers: GFP-HDEL (ER); ST-

YFP (Golgi); YFP-SYP32 (cis-Golgi); GFP-SYP43 (TGN/EE); YFP-RabA1e (TGN, PM); YFP-RabA5d (uncharacterized endosomes); ARA6-GFP (MVB/late endosomes, PM); YFP-ARA7 (MVB/late endosomes), and co-stained with the endocytosis tracer FM4-64, which initially stains plasma membrane.

First, we treated GFP-HDEL/FM4-64 and ST-YFP/FM4-64 with BFA at different concentrations (0, 30, 90, 180 µM). Treatment of low BFA concentration at 30 µM (which we used throughout our study) does not confer any discernable effects on ER (GFP-HEDL, revised Figure 3A, B). Under this condition, BFA bodies are surrounded by Golgi marker ST-YFP (Figure 3C), just as expected for the BFA treatment in roots (Figure 3D) (Langhans et al., 2011). In contrast, treatment of BFA at very high concentration, 180 µM, conferred aberrant spherical structure of ER (Figure 3A, B), which was also previously reported (Nakano et al., 2009). Moreover, we observed Golgi absorbed ER, which confers ring-like structure of membrane accumulating ST-YFP (Figure 3C). In short, we recapitulated the previous observations of collapsing of Golgi into the ER only at extremely high BFA concentration at 180 µM.

Next, we investigated the intactness of different endomenbrane organelles in stomatal meristemoids when treated in 30 µM BFA. For this purpose, seven additional endomembrane markers were carefully examined (YFP-SYP32, N-STYFP, GFP-SYP43, YFP-RabA1e, YFP-RabA5d, ARA6-GFP and YFP-ARA7 costained with FM4-64. All marker lines show the formation of characteristic BFA bodies in the meristemoids, without any collapse of Golgi into ER (Figure 3—figure supplement 1).

Taken together, we conclude that BFA treatment at 30 µM, in our hands, confers the formation of characteristic BFA bodies in the stomatal meristemoids in leaf, and thereby supporting the validity of our BFA treatment to study ERL1 subcellular dynamics. We strongly believe that our extensive documentation will serve as a guide to the field, and we thank reviewer 2 again for the excellent advice.

The images shown the effect of BFA being very zoomed out, it is hard to evaluate the effect of BFA.

As also requested by reviewers 1 and 3, we now provide enlarged field of view throughout our confocal microscopy image figures whenever possible.

3) BFA leads, according to the authors, to the aggregation of TGN indicating that proteins trafficking through the TGN will end up (recycled or not) in BFA bodies. The fact that ERL1 is observed in BFA bodies in tmm upon CHX is therefore puzzling. One would anticipate a decrease (or even absence) of ERL1 if internalization is compromised. A better quantification of BFA bodies (BFA body number/cell and size; see 2)) is required to better characterize possible defects in ERL1 internalization. If the result stands, the authors will have to provide some explanations in the Discussion section. Regardless, a more direct investigation of ERL1 internalization kinetics in WT or tmm background by VAEM imaging would GREATLY strengthen the authors conclusions. I am not sure the authors have access to VAEM, but if so, I strongly encourage them to try.

We have re-analyzed the effects of BFA on ERL1-YFP in WT vs. tmm backgrounds by quantifying the ratio of YFP+ signals within the cell (voxel) per YFP+ signals at plasma membrane using our maximal intensity projection of Z-stack images (formerly Figure 2C, now Figure 4D). The analysis revealed a statistically significant reduction of ERL1-YFP in BFA bodies in tmm mutant, but it is not completely absent. These results suggest that in the absence of TMM, ERL1-YFP internalization is compromised but not absent. This is most likely due to the presence of EPF-LIKE peptide family members that do not require TMM (e.g. EPFL4 and EPFL6). We discussed this point in the Discussion. We agree that VAEM (or TIRF) microscopy would be an interesting next step to study receptor internalization dynamics in the future.

4) The FRAP experiment raises some questions. First, the recovery of ERL1-YFP in erl1 is almost absent in my opinion. There is hardly any recovery compared to T0 (slope is flat). As such, it is hard to interpret and one may come up with a very different conclusion where ERL1 is largely immobile at the cell surface, and that it is more mobile in tmm. The authors should (i) show the prebleach intensity on their plot and (ii) image faster after photobleaching to catch the initial recovery phase better.

We have used FRAP Bot program (www.frapbot.kohze.com) to normalize and fit our fluorescence recovery curve to a single-parameter exponential model. The fitted curve, but not the original fluorescence intensity changes, are provided in the original graphs. As far as our understanding, recovery curve is a standard way to determine the half time, and similar curve fitting has been done by many others in the field (e.g. Martinière et al., 2012; Zhang et al., 2016).

As requested by reviewer 2, we are providing both a graph of observed prebleach florescence intensity and intensity recovery in representative FRAP experiments (Figure 4—figure supplement 3 in the revision) as well as confocal images throughout a representative FRAP experiment (Video 2 and Video 3).

5) The internalization of ERL1 and targeting to MVB suggests that ERL1 is routed to the vacuole for degradation, which the authors have not shown. The authors must therefore assess ERL1 degradation by western blot if ERL1 is detectable in leaf extracts and/or by confocal imaging inhibiting the lytic activity of the vacuole (dark conditions of concanamycin A).

We thank reviewer 2 for the excellent suggestion. With Dr. Yoshinari, we performed Concancmycin A treatment of ERL1-YFP seedlings that were pretreated with FM4-64 following the published protocols (e.g. Kleine-Vehn et al., 2008). Indeed, we observed ERL1-YFP accumulation in the vacuole of stomatal meristemoids (see revised Figure 2). The data support our original conclusion that ERL1, upon activated by EPF peptides, is subjected to internalization and targeted to MVP and destined to the vacuole.

6) The ER retention of ERL1 upon stomagen application is puzzling yet very interesting. The authors must back up their observations by monitoring the endogenous ERL1 protein by immunofluorescence (or alternatively an ERL1-HA or FLAG if the authors do not have an ERL1 antibody) to ascertain that the YFP fusion protein, although active, represents the behavior of genuine ERL1 for ER retention. Second, the authors must address whether this is specific or ERL1 by testing the influence of stomagen on other secreted proteins (PM proteins) that woumd be expressed (e,dogenously or artificially) in stomata.

We do not have endogenous antibody that specifically detect ERL1, and immunostain of epidermal cells have been unsuccessful in our hands. Our Stomagen treatment experiments have been performed multiple times, not just by Confocal microscopy observations but also using biochemical Endo-H treatment.

As suggested by reviewer 2, we treated Stomagen peptide on Arabidopsis seedlings expressing BRI1-GFP driven by its endogenous promoter (BRI1pro::BRI1-GFP). As reported previously (Friedrichsen et al., 2000), BRI1 is ubiquitously expressed in the epidermis, including meristemoids and stomata (see Figure 8—figure supplement 3). The application of Stomagen did not change plasma-membrane localization of BRI1. We did not detect BRI1-GFP in the ER, indicating that the influence of Stomagen on the ER retention is not universal to LRR-RLK.

Reviewer #3:

This manuscript by Qi et al. investigated how the ERL1 receptor interprets different signal inputs and how this affects its subcellular dynamics. Overall, the manuscript is rather descriptive and fails to provide convincing mechanistic insight, leaving many loose ends. In addition, the text needs to be streamlined with respect to terminology (MVBs, endosomes, BFA-bodies, etc) and what is actually shown on the figures to avoid mis- or over-interpretation. Furthermore, the conclusions need to be more accurately phrased.

We sincerely appreciate reviewer 3's expert comments. Our work reports extensive documentation of receptor subcellular localization upon perceiving signaling ligands with different activities as well as when the receptor cannot signal, using the system in which one cell type (stomatal meristemoids) respond to multiple different, and conflicting signals. Thus, we believe our finding add new insight and useful knowledge (together with extensive study of BFA treatment in the meristemoids now presented in Figure 3) to the field. We agree with reviewer 3, and we took our best effort to accurately phrase the observations and results in the revision.

1) Regarding the statement that "TMM is required for ERL1 endocytosis", I would phrase this differently. TMM is required to form an active signaling complex, and in the absence of such an active signaling complex, no endocytosis occurs (as is also the case upon Stomagen treatment or when the kinase domain is removed). How do the authors explain the Stomagen-mediated accumulation of ERL1 in the endoplasmic reticulum? This must be a different pool of ERL1 distinct from the Stomagen-bound ERL1. Do the authors assume a regulatory mechanism whereby Stomagen-bound ERL1 signals to control the production and ER retention of new ERL1? In this context, I would conclude that it is not inefficient ERL1 endocytosis that leads to retention in the ER, but an inactive receptor that stays on the membrane which leads to no new secretion. Does the ERL1Δkinase also accumulate in the ER?

We agree with reviewer 3 that TMM is required to form an active signaling complex, and in the absence of the active signaling complex endocytosis of the receptor gets compromised. We have revised our text throughout the manuscript to be clear. As for the effects of Stomagen on ERL1 accumulation in the ER, together with the pharmacological studies using TyrA23 and ES9-17, suggests that reduced signal activation by Stomagen will result in stalling of ERL1 in the ER. We wish to study the exact subcellular mechanism of the observed stalling in the future.

2) With the current data, it cannot be concluded that kinase activity is required for ERL1 internalization. There could be internalization motifs in the kinase domain. To conclusively demonstrate this, an ERL1 kinase dead variant needs to be analyzed with respect to internalization. In the absence of TMM, un-activated ERL1 receptors are not readily targeted for endocytosis and remain stable at the membrane. What happens with ERL1Δkinase endocytosis in the presence of ligand?

We fully concur with reviewer 3. We have no intention to claim that the kinase activity is required for the internalization. We are fully aware of the fact that removal of the entire cytoplasmic domain will remove potential binding interface with endocytosis adaptor proteins, including those in AP complex or TPLATE complex. Our intention here is to show that a dominant-negative receptor, which is known to severely interfere with it signal transduction pathway (Shpak et al., 2003; Lee et al., 2012), is largely compromised in internalization. We agree that to examine whether the kinase activity affect internalization, kinase-dead variant should be used. We revised our text throughout to clarify this point. We have previously shown that dominant negative ERL1DKinase override EPF1 peptide application (i.e. EPF1 peptide has no effects on epidermis of ERL1DKinase; Lee et al., 2012). Thus, we predict that ERL1DKinase would not be strongly influenced by the peptide application.

3) The authors use different markers to reveal the localization of ERL1-YFP. However, a typical experiment to study endocytosis is the co-localization with FM4-64. Figure 2—figure supplement 2A and Figure 3E provide some idea, but the individual endosomes should be tracked. BFA bodies and MVBs are a proxy to study this, but then the phrasing in the text should be accordingly.

This is a great suggestion. We observed co-localized FM4-64 endosomes and ERL1-YFP signals (now provided in revised Figure 1A), which indeed serves as a starting point to investigate the subcellular dynamics of ERL1. It is evident from our Concanamycin A treatment (revised Figure 2) that FM4-64 signals are eventually detected to a tonoplast and ERL1-YFP targeted into a vacuole. Having said that, in our hands it was not trivial to track the exact snapshots of ERL1 subcellular loalizations in a time-resolved fashion by simply following FM4-64 signals in different endomembrane organelles (e.g. early endosomes, late endosomes, etc). We strongly believe that the use of well-studied endomembrane organelle markers combined with pharmacological analysis provide robust and reliable results. These markers and inhibitors are widely used in the field.

4) TMM is required for endocytosis of ERL1, and not recycling. BFA blocks the secretion of ERL1. One would expect that if there is less endocytosis, there is also less ERL1 in BFA bodies. However, based on the data in Figure 2 this is not the case. Also, Figure 2B shows an increased ERL1-YFP intensity upon BFA treatment in tmm, so both the number of cells with BFA bodies and the signal intensity would be meaningful to report. I would rephrase the conclusion in subsection “TMM is required for the process of ERL1 endocytosis in true leaves”: the absence of tmm does not impact the ERL1 secretory pathway, as reflected in the presence of ERL1-YFP positive BFA bodies.

Both reviewer 1 and reviewer 2 also commented on this. As our response to other reviewers, we have re-analyzed the effects of BFA on ERL1-YFP in WT vs. tmm backgrounds by quantifying the ratio of YFP+ signals within the cell (voxel) per YFP+ signals at plasma membrane using our maximal intensity projection of Z-stack images (formerly Figure 2C, now Figure 4D). The analysis revealed a statistically significant reduction of ERL1-YFP in BFA bodies in tmm mutant. As reviewer 3 thoughtful points out, we revised our Results section based on the new analysis. Thank you.

5) The authors invoke that distinct EPF/EPFL peptide ligands activate a sub-population of ERL1 receptor complex to internalize through a TMM-based discriminatory system. This seems independent of distinct expression domains of these peptides as purified EPFL6 does not have the same effect as EPF1. The authors suggest distinct localization in nanodomains, but do not investigate this. It would be valuable to see FRAP analyses on ERL1-YFP +/1 EPF1 or EPFL6 in erl1 and erl1 tmm.

This is a very interesting point, and certainly it is our future direction to pursue specific nanodomain formation, using FRAP and elaborated microscopy, such as VAEM (as encouraged by reviewer 2).

6) Throughout the manuscript, MVBs should be used when describing the "large dots" on the figures. I am not sure if the resolution allows to see endosomes. Are Figure 4E-F and Figure 5E-F really showing number of endosomes or should this be MVBs?

As suggested, we have modified to MVP/LE, which is also part of late endosome.

7) The authors write "TMM is required for endocytic sorting of ERL1 to MVBs, which is a hallmark for receptor degradation". I guess that given the fact that in tmm ERL1-YFP signal increases in the ER, it is rather difficult to investigate this statement using e.g. Western Blot.

We agree that our original statement might have been too strong. The sentence was removed.

As requested by reviewer 2, we have done pharmacological treatment of ERL1-

YFP seedlings with Concanamycin A, which specifically inhibits vacuolar H+ATPase and thus reduce protein degradation in the lytic vacuole. We followed the published protocols (e.g. Kleine-Vehn et al., 2008) and pre-treated with FM464 to track a tonoplast. Our result show that ERL1 indeed accumulates in a vacuole in the presence of Concanamycin A (see revised Figure 2), indicating that ERL1 is targeted to a vacuole. We discussed receptor degradation in the context of our new results.

8) Given the recent knowledge on Tyr A23, I would only include the more specific ES9-17. Is this effect on ES9-17 specific for ERL1-YFP, or are other receptor proteins also accumulating in the ER?

ES9-17 is very new, and as far as we believe we are one of the very first to use this inhibitor in the context of shoot stomatal meristemoids. Although TyrA23 inhibits endocytosis is due to acidification, thereby non-specific manner (Dejonghe, 2016), it has been widely used to block internalization.

Indeed, since the publication of Dejonghe paper, it is still used to study endocytosis of LRR-RLKs (e.g. Song et al., 2019). While we agree with reviewer 3 that ES9-17's effects on endocytosis is more specific and thus clean, we strongly believe that presenting our data that both TyrA23 and ES9-17 triggers the ER accumulation of ERL1-YFP would strengthen our conclusion. To address the specificity to ERL1-YFP, we think that testing Stomage application to other LRR-RLK expressed endogenously in the leaf epidermis would be the better control. To this end, as also suggested by reviewer 2, we examined the effects of Stomagen peptide on the subcellular localization of BRI1 using a published

BRI1pro::BRI1-GFP reporter line (Friedrichsen et al., 2000). The Stomagen application did not change plasma-membrane localization of BRI1, thus the influence of Stomagen is likely specific to ERL1-YFP. Please see Figure 8—figure supplement 3 or our revision.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Your manuscript reveals remarkable insights into peptide ligands and how they modulate the intracellular distribution of their receptors. We invite you to resubmit your paper for final acceptance, considering the following main suggestions of the reviewers:

We truly thank for the positive and encouraging comments.

I) The reviewers ask you to clarify several issues, such as statistical evaluation and quantifications, which may not require novel experiments.

As requested by reviewer 1, we have in addition performed two-way ANOVA for analysis for those data with two parameters, treatment and genotype (Figure 4D, 4F, Figure 4—figure supplement 1, Figure 4—figure supplement 2D, Figure 5-Figure supplement 1B, 1C in the revision). Accordingly, the additional R scripts for the analysis are provided as Source code.

II) During their discussion, the reviewers defined a better characterization and integration of the ERL1Δkinase line, as an essential part of the revision, and one that unfortunately requires additional experimentation.

We agree that testing how ERL1ΔK responds to different ligands is very important to complete our story. As you might hopefully recognize, however, this has been extremely challenging for us. The Torii laboratory at the University of Texas at Austin has been under lock down due to COVID-19 surge in the US, notably terrible in the Southern states including Texas. Even worse, we ran out of bioactive recombinant MEPF1 peptide.

After UT-Austin gave us permission to conduct time-sensitive experiments in July, my postdoc, Dr. Pengfei Bai, who is now a co-author, worked day and night to express, purify, refold, and test bioactivities of their recombinant MEPF1 peptide. The bioactive peptide was then shipped to Japan, where Dr. Akira Yoshinari performed the requested experiments.

As you can see in revised Figure 8, neither MEPF1 nor Stomagen peptide application triggered subcellular localization changes of the dominant-negative ERL1ΔK. ERL1ΔK remained predominantly at the plasma membrane upon MEPF1 or Stomagen application. These new findings suggest that ERL1ΔK is dysfunctional in ligand-induced subcellular trafficking. Because ERL1ΔK lacks the entire cytoplasmic kinase domain, including juxtamembrane domain, kinase domain, and C-terminal tail region, we predict that proper interaction site with endocytosis cargo adaptors may have been lost in ERL1ΔK. While identifying specific cargo adaptors for ERL1 is a future study that is out-of-scope of this manuscript, our additional experiments highlights a highly-tuned, complex actions of antagonistic peptides on ERL1 subcellular dynamics is a highly tuned, coordinated process. We have revised our main text to include these new findings (subsection “Cytoplasmic domain of ERL1 is required for proper subcellular trafficking behavior”, Discussion section).

III) The reviewers judge that some experiments aimed at a better integration of your data into developmental context would substantially strengthen the impact of your manuscript. On the other hand, they do not believe that such an insight is essential for acceptance of the manuscript.

We appreciate the editors' advice. Please see our detailed response to reviewer 2 regarding this point.

Please, see the detailed comments of the reviewers below.

Reviewer #2:

The authors undertook a remarkable effort to improve the cell biological aspects of this manuscript and I do appreciate the new experiments and careful quantification of the BFA experiments and analysis of new marker lines with expert Dr. Akira Yoshinari. In addition, the ConcavA treatments and the clear visualization of ERL1 in vacuoles adds significantly to this manuscript. However, I would like to stress two of my previous concerns again.

We truly thank reviewer 2 for commending our "remarkable effort to improve the cell biological aspects of this manuscript".

1) Previous point 1:

From a mechanistic/genetic perspective, the functional relevance of ERL1 endocytosis and targeting to MVB/LE and vacuole is still missing. I do completely understand that a phenotypic assay is not doable due to long-time cytotoxic effects of BFA or Wm. I also agree that strong genetic support can only come through analysis of how ERL1 is modified (phosphorylation etc) and how these modifications affect endocytosis, but this is clearly out of scope of this article. Nevertheless, in Qi et al., 2017, the authors show that the EPF1-ERL1 module directly represses MUTE in an autocrine manner. When EPF1 expression is induced, a clear decrease in MUTE-YFP fluorescence can be observed already 15hours post induction. I wonder whether induction of EPF1 together with mock or toxin treatment (Wm?) in the first couple of hours could reveal differences in how efficiently MUTE-YFP is downregulated. This could give at least somewhat functional support to the hypothesis that the endocytosis and targeting to the vacuole is required for efficient downregulation of key targets by the EFP1-ERL1 module.

We appreciate reviewer 2's comments. As we all know, chemical inhibitors of membrane trafficking, such as BFA, TyrA23, and Concanamycin A, are a very powerful tool to delineate subcellular dynamics of membrane proteins of interest, whether receptor kinases or transporters. However, there are major problems with the use of these chemical inhibitors for extended period of time to study developmental outcomes.

First and foremost, these inhibitors are not specific to one plasma membrane protein of interest, but rather affect many proteins. It is well known that BFA, for instance, affects recycling of PIN auxin efflux carriers (e.g. Kleine-Vehn et al., 2008), which also plays a role in stomatal patterning (e.g. Le et al., 2014). ES9-17 blocks endocytosis of not only ERL1, but also PIN2 (Iwatate et al., 2020), BRI1 (Dejonghe et al., 2019), and likely many other membrane proteins. Wm could somehow block endocytosis (Emans et al., 2002; Dettmer et al., 2006) in addition to vacuolar sorting. It is therefore impossible to conclude that a developmental phenotype caused by these inhibitors are due to trafficking defects of a single membrane protein. Second, as reviewer 2 acknowledges, these inhibitors possess high cellular toxicity. Extended treatment of seedlings with these chemicals will impact seedling growth and cell proliferation, making it difficult, if not impossible, to characterize stomatal development phenotype. We appreciate that reviewer 2 suggested using a reporter MUTE-YFP line as a quicker marker (~15 hours). However, in our experience, addition of these chemicals for only a few hours would impact the health of the seedlings, thus the interpretation of these experiments would be very tricky.

Our manuscript reports unique, subcellular dynamics of a single receptor (ERL1) by different peptide ligands with distinct biological activities, thereby suggesting the intricate, fine-tuning of developmental patterning by multiple peptide signals. Future analysis, such as identifying critical post-translational modifications of ERL1 and characterizing their impacts on receptor subcellular behavior, will provide a full picture of how intricate regulation of receptor dynamics directly specifies stomatal patterning.

2) Previous point 12:

Even though the authors stated in their response that they elaborated on ER ERL1 and ERL2 redundancy in the discussion, I cannot find new information or statements regarding the fact that the Stomagen-induced ER-retainment of ERL1 is much stronger in the triple erecta-erl1-erl2 background. I am aware that this is a subtle difference but as the authors confirm an important one. So please elaborate on this in the results (line 298ff) and clearly mention that Stomagen has been shown to interact initially with ER before acting through ERL1 and ERL2 at later stages, which is why a triple mutant is needed here and ER retainment in the single erl1 is much weaker.

We fully agree with reviewer 2 that the Endoplasmic Reticulum retention of ERL1-YFP is notable in the erecta erl1 erl2 mutant background, i.e. when functional ERECTA and ERL2 genes are missing. As reviewer 2 astutely points out, we also think that the lack of ERECTA, the major receptor for the early stage of stomatal initiation, confers sensitized, exaggerated response of ERL1-YFP to Stomagen. This is because ERL1 is the only receptor of the family to perceive Stomagen. In addition, it is known that the lack of ERECTA triggers excessive entry divisions of stomatal cell lineages and clustering of meristemoids, where ERL1 is expressed (e.g. Shpak et al., 2005, Lee et al., 2012, and Qi et al., 2017). The dysregulated cell-cell signaling in the erecta erl1 erl2 background likely abrogate the 'buffering' of exogenously applied Stomagen peptide.

In response to reviewer 2, we have elaborated on the main text, stating the following sentences. We sincerely thank reviewer 2 for the keen, thoughtful suggestion.

"The endoplasmic reticulum retention of ERL1-YFP is notable in the erecta erl1 erl2 mutant background, i.e. when functional ERECTA and ERL2 genes are missing. The signals in erl1 ("WT") are much weaker. Because Stomagen interacts with ERECTA at the early stage of stomatal initiation, the absence of ERECTA likely confers sensitized, exaggerated response of ERL1-YFP to Stomagen. Alternatively, a dysregulated cell-cell signaling in the erecta erl1 erl2 background may abrogate the buffering of exogenously applied Stomagen peptide." (Discussion section.)

https://doi.org/10.7554/eLife.58097.sa2

Article and author information

Author details

  1. Xingyun Qi

    Howard Hughes Medical Institute and Department of Biology, University of Washington, Seattle, United States
    Present address
    Department of Biology, Rutgers University, Camden, United States
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1261-1362
  2. Akira Yoshinari

    Institute of Transformative Biomolecules (WPI-ITbM), Nagoya University, Aichi, Japan
    Contribution
    Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7083-5674
  3. Pengfei Bai

    Howard Hughes Medical Institute and Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Contribution
    Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2281-2881
  4. Michal Maes

    Howard Hughes Medical Institute and Department of Biology, University of Washington, Seattle, United States
    Present address
    Institute of Systems Biology, Seattle, United States
    Contribution
    Resources, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7406-3982
  5. Scott M Zeng

    1. Howard Hughes Medical Institute and Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    2. Department of Physics, University of Washington, Seattle, United States
    Contribution
    Software, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3146-8207
  6. Keiko U Torii

    1. Howard Hughes Medical Institute and Department of Biology, University of Washington, Seattle, United States
    2. Institute of Transformative Biomolecules (WPI-ITbM), Nagoya University, Aichi, Japan
    3. Howard Hughes Medical Institute and Department of Molecular Biosciences, The University of Texas at Austin, Austin, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    ktorii@utexas.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6168-427X

Funding

Howard Hughes Medical Institute (TORII)

  • Keiko U Torii

Gordon and Betty Moore Foundation (GBMF3035)

  • Keiko U Torii

University of Texas at Austin (Johnson & Johnson Centennial Chair of Plant Cell Biology)

  • Keiko U Torii

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Prof. Takashi Ueda for GFP-SYP43, ARA6-GFP, RFP-ARA7, SYP43-RFP, and SYP22-RFP lines; Prof. Gian Pietro Di Sansebastiano for ST-RFP and KDEL-RFP constructs; Prof. Hugo Zheng for N-ST-YFP and N-YFP-HDEL constructs; Prof. Ikuko Hara-Nishimura for GFP-HDEL and ST-YFP lines; Prof. Joanne Chory for BRI1pro::BRI1-GFP line; Dr. Jin Suk Lee for pBAD::MEPFL6-6xHis plasmid (pJSL79); Geovanny Zarceno and Alex Hofstetter for technical assistance of peptide purification; Dr. Ayami Nakagawa for assistance in bioassays; Prof. Jenny Russinova for providing ES9-17 and insightful suggestions on experimental designs; Drs. Naoyuki Uchida, Ayami Nakagawa, Arvid Herrmann and Soon-Ki Han for commenting on the manuscript. A part of this work was conducted at Nagoya University Live Imaging Center supported by Japan Advanced Plant Science Research Network. This work was supported by the Gordon and Betty Moore Foundation (GBMF3035) to K.U.T., who is a Howard Hughes Medical Institute Investigator. K.U.T. acknowledges the generous support from The University of Texas at Austin as the Johnson and Johnson Centennial Chair.

Senior Editor

  1. Christian S Hardtke, University of Lausanne, Switzerland

Reviewing Editor

  1. Jürgen Kleine-Vehn, University of Natural Resources and Life Sciences, Austria

Publication history

  1. Received: April 21, 2020
  2. Accepted: August 14, 2020
  3. Accepted Manuscript published: August 14, 2020 (version 1)
  4. Version of Record published: September 3, 2020 (version 2)

Copyright

© 2020, Qi et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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