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. 2023 Jan 7;192(1):65–76. doi: 10.1093/plphys/kiad005

BRASSINOSTEROID INSENSITIVE1 internalization can occur independent of ligand binding

Lucas Alves Neubus Claus 1,2,#, Derui Liu 3,4,#,3, Ulrich Hohmann 5,4, Nemanja Vukašinović 6,7, Roman Pleskot 8,9,5, Jing Liu 10, Alexei Schiffner 11, Yvon Jaillais 12, Guang Wu 13, Sebastian Wolf 14, Daniël Van Damme 15,16, Michael Hothorn 17, Eugenia Russinova 18,19,✉,7,6,8
PMCID: PMC10152650  EMSID: EMS170881  PMID: 36617237

Abstract

The brassinosteroid (BR) hormone and its plasma membrane (PM) receptor BR INSENSITIVE1 (BRI1) are one of the best-studied receptor–ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the PM pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. As BRs are ubiquitously distributed in the plant, the tools available to study the BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR binding-deficient Arabidopsis (Arabidopsis thaliana) mutant based on protein sequence-structure analysis and homology modeling of members of the BRI1 family. This tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We showed that despite impaired phosphorylation and ubiquitination, BR binding-deficient BRI1 internalizes similarly to the wild type form. Our data indicate that BRI1 internalization relies on different endocytic machineries. In addition, the BR binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.

Introduction

Brassinosteroids (BRs) are low abundant and ubiquitously distributed plant steroidal hormones that play an essential role in growth, development, immunity and responses to stress (Nolan et al., 2020). BR biosynthetic or signaling mutants display severe phenotypes including dwarfism, dark-green leaves, photomorphogenesis in the dark, and late flowering (Nolan et al., 2020). BRs are perceived at the cell surface by a leucine-rich repeat (LRR) receptor kinase BR INSENSITIVE1 (BRI1) (He et al., 2000; Wang et al., 2001; Kinoshita et al., 2005; Hothorn et al., 2011; She et al., 2011). BR binding triggers the dissociation of the inhibitory proteins BRI1 KINASE INHIBITOR1 (BKI1) and BRI1-ASSOCIATED KINASE1 (BAK1)-INTERACTING RECEPTOR-LIKE KINASE3 (BIR3), allowing interaction between BRI1 and its co-receptor BAK1, which is required for downstream signaling (Li et al., 2002; Wang and Chory, 2006; Hohmann et al., 2018a). BR signal is conveyed from the cell surface to the nucleus through a sequence of phosphorylation/dephosphorylation events that activate the transcription factors of the BRASSINAZOLE-RESISTANT1 (BZR1) and BRI1-EMS-SUPPRESSOR1 (BES1)/BZR2 family (Wang et al., 2001; Yin et al., 2002; Chen et., 2019). BRI1 receptor functions go beyond the canonical BR signaling becuase together with the RECEPTOR-LIKE PROTEIN44 (RLP44) and BAK1, BRI1 controls xylem cell fate independently of BRs (Holzwart et al., 2018, 2020).

An important regulatory step in BR signaling is the control of the plasma membrane (PM) pool of BRI1, which is determined by BRI1 endocytosis, recycling, and secretion (Irani et al., 2012; Luo et al., 2015). As a consequence, impaired endocytosis and receptor secretion enhance and reduce BR signaling, respectively (Irani et al., 2012; Luo et al., 2015). Several studies have focused on BRI1 dynamics upon ligand binding. Given that exogenous BRs did not change BRI1 internalization dynamics, BRI1 endocytosis was described as ligand independent (Russinova et al., 2004; Geldner et al., 2007; Luo et al., 2015). Subsequent studies on post-translation modifications (PTMs) showed that BRI1 undergoes polyubiquitination that is mediated by the plant U-box (PUB) E3 ubiquitin ligases PUB12 and PUB13 and requires BR binding (Zhou et al., 2018). BRI1 ubiquitination is a signal for BRI1 endocytosis and vacuolar sorting, because disruptions in this process by mutations in either the ubiquitination sites or the E3 ligases translate into accumulation of BRI1 in the PM and consequently increase in BR sensitivity (Martins et al., 2015; Zhou et al., 2018).

Available tools used to study the dependence of BRI1 trafficking on ligand binding are limited and rely on the depletion of the endogenous BRs using BR biosynthetic mutants or the BR biosynthesis inhibitor, brassinazole (BRZ) (Asami et al., 2000). However, the possibility cannot be excluded that treatment with BRZ might not completely deplete bioactive BRs and that the BR biosynthetic mutant might contain biologically active BR precursors. Furthermore, BR biosynthetic mutants display pleiotropic phenotypes that could lead to general changes in membrane trafficking. Therefore, the use of these tools could hamper analysis of ligand-independent BRI1 dynamics.

Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) quintuple (Q) BR binding-deficient BRI1 receptor mutant, designated as BRI1Q, that was generated using homology and structure analysis of BRI1 and its three homologs BRI1-LIKE1 (BRL1), BRL2, and BRL3 (Caño-Delgado et al., 2004). This tool revealed that BRI1 endocytosis is largely independent of BRs and despite strongly decreased phosphorylation and ubiquitination, the BRI1Q mutant held normal endocytosis rates. These results reinforce the hypothesis that BRI1 is internalized by means of different endocytic machineries. Moreover, we used the BRI1Q mutant to investigate BR-independent BRI1 functions, such as xylem cell differentiation. We showed that BRI1Q can partially complement the xylem cell fate phenotype of the bri1 null mutant, suggesting that BRI1Q can be used to study BRI1 non-canonical functions.

Results and discussion

BRI1Q cannot bind BRs

The Arabidopsis genome encodes three BRI1 homologs, designated BRL1, BRL2, and BRL3, of which BRL2 does not bind BRs (Caño-Delgado et al., 2004; Kinoshita et al., 2005). Sequence analyses of BRI1, BRL1, BRL2, and BRL3 ectodomains (Figure 1A) and examination of the crystal structure of the BRI1 ectodomain in a complex with brassinolide (BL), the most active BR (Wang et al., 2001; Hothorn et al., 2011; She et al., 2011; Figure 1, B–D), revealed five putative residues important for BR binding, three derived from changes in the BRL2 sequence [tyrosine (Y)597, Y599 and methionine (M)657] and two bulky hydrophobic residues located at a 4 Å distance from the BL molecule in BRI1 [Y642 and phenylalanine (F)681] (Figure 1, B and C). Y597, Y599, and Y642 map to the inner surface of the BRI1 island domain, forming the distal part of the BR binding pocket, whereas M657 and F681 are located in the LRR core and establish hydrophobic interactions with the aliphatic BL moiety (Figure 1, B and C). The identified residues were mutated to the corresponding ones in BRL2 either individually (BRI1Y599F and BRI1M657E) or in a combination (BRI1Y597M/Y599F/M657E) or to alanine (A) (BRI1Y597A/Y599A/M657A). Finally, a quintuple BRI1Y597M/Y599F/Y642A/M657E/F681A version was generated and designated as BRI1Q. Next, the binding kinetics of BL to the BRI1Q ectodomain was determined by grating-coupled interferometry (GCI) (Figure 2B). As controls, the ectodomains of the Arabidopsis wild type BRI1 and that of the previously characterized Arabidopsis bri1-6 mutant that carries the glycine (G) 644 to asparagine (D) missense mutation (BRI1G644D) were included (Noguchi et al., 1999; Wang et al., 2001; Kinoshita et al., 2005; Hothorn et al., 2011; Hohmann et al., 2018b). Analytical size-exclusion chromatography (SEC) and right-angle light scattering (RALS) experiments confirmed that all BRI1 variants were monodisperse, suggesting that mutations in the BR binding pocket do not affect the overall shape and oligomeric state of the BRI1 ectodomain (Supplemental Figure S1). The GCI experiments revealed that BRI1 bound BL with a dissociation constant (KD) of ∼10 nM as previously reported (Wang et al., 2001; Hohmann et al., 2018b) and BRI1Q did not bind BL, whereas the BRI1G644D mutant displayed a strongly reduced BL binding capacity with a KD of ∼11.6 μM (Figure 2B). After confirmation that BRI1Q cannot bind BL, mutated full-length BRI1 versions fused to mCitrine (mCit) were expressed in the bri1 null mutant from the native promoter and plants with similar protein expression levels of the transgenes were selected (Figure 2A; Supplemental Figure S2A). BRI1Y599F-mCit, BRI1M657E-mCit, and BRI1Y597M/Y599F/M657E-mCit partially complemented the bri1 dwarf phenotype and localized in the PM and in intracellular punctate structures, similar to the wild type BRI1 (Russinova et al., 2004; Geldner et al., 2007; Figure 2A). However, BRI1Y597A/Y599A/M657A-mCit and BRI1Q-mCit did not complement the bri1 mutant (Figure 2A). BRI1Y597A/Y599A/M657A-mCit displayed an aberrant accumulation in the vacuole, probably due to misfolding of the LRR domain caused by the mutations. Asimilar localization was reported for the artificially ubiquitinated BRI1, in which the ubiquitin was recognized before reaching the PM, likely at the trans-Golgi network/early endosome (TGN/EE) compartments, and it served as a targeting signal for vacuolar degradation (Martins et al., 2015). In contrast, BRI1Q-mCit exhibited correct BRI1 localization in the PM and in intracellular punctate structures. Hence, the absence of BRI1Q functionality corroborates the in vitro ligand binding deficiency results (Figure 2B). To further characterize the BRI1Q-mCit line, we tested the BL-induced BRI1 PTMs. It is well established that after ligand binding, BRI1 heterodimerizes with its co-receptor BAK1 and undergoes PTMs such as phosphorylation and ubiquitination (Belkhadir and Jaillais, 2015; Martins et al., 2015; Zhou et al., 2018). Moreover, BL treatment promotes the dephosphorylation of the transcription factor BES1 in a dose-dependent manner, which is frequently used as a BR signaling indicator (Yin et al., 2002). In agreement with the impaired BL binding, BRI1Q-mCit had no detectable phosphorylation or ubiquitination, did not interact with BAK1 and did not promote BES1 dephosphorylation after BL treatment (Figure 2, C and D). These findings indicate that BRI1Q is unable to perceive BRs.

Figure 1.

Figure 1

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Selection of the essential residues for BR binding in the BRI1 ectodomain. A, Sequence alignment of the wild type BRI1 ectodomain (only the region corresponding to BRI1 550–700 is shown) with that of BRL1, BRL2, and BRL3 and BRI1Q with the mutated residues marked with an asterisk. B, The BRI1 ectodomain structure in a complex with BL (PDB ID 3RJ0). C and D, The five residues selected are indicated as single-letter abbreviation with their locations. The figures (B–D) were generated with UCSF chimera where the BL molecule is shown in a balls-and-sticks representation.

Figure 2.

Figure 2

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BRI1Q cannot bind BL. A, Phenotypes (upper panel) and subcellular localizations (lower panel) of homozygous bri1 mutant transgenic plants expressing the indicated BRI1 isoform mutations grown in short-day cycle for 6 weeks. The quintuple BRI1Y597M/Y599F/Y642A/M657E/F681A mutation is designated BRI1Q. Epidermal root meristem cells of 5-day-old seedlings were imaged. Scale bars, 2 cm (upper panel) and 10 μm (lower panel). B, Binding kinetics for BL versus wild type BRI1, BRI1G644D, and BRI1Q as obtained from GCI. Sensograms with recorded data are shown with the respective fits (when applicable) and include the corresponding association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD). C, BRI1-mCitrine (mCit) and BRI1Q-mCit phosphorylation and ubiquitination state and interaction with BAK1 were tested by isolation of microsomal fractions of 5-day-old seedlings followed by immunoprecipitation (IP) and Western blot (WB) analysis with α-ubiquitin (α-Ub), α-pThreonine (α-pThr), and α-BAK1 antibodies. D, BES1 phosphorylation state assessed in 5-day-old seedlings treated with DMSO (mock), 1 nM or 10 nM BL for 1 h subjected to WB analysis with the α-BES1 antibody; α-tubulin was used as loading control. All experiments were repeated twice with similar results (C and D).

Endocytosis of BRI1 is independent of BR binding

The lack of BL binding in the BRI1Q mutant provides a powerful tool to investigate different aspects of BRI1 regulation, including endocytosis, without interference from BRs. Although initially BRI1 endocytosis had been described as ligand independent (Russinova et al., 2004; Geldner et al., 2007), later BR perception has been demonstrated to promote BRI1 ubiquitination (Zhou et al., 2018), which assists BRI1 internalization and vacuolar targeting Luo et al., 2022; Martins et al., 2015; Zhou et al., 2018). We revisited the BRI1 ligand-dependent endocytosis model using BRI1Q by evaluating the internalization of BRI1Q-mCit in root meristem epidermal cells (Figure 3, A and B). The PM pool of BRI1 is regulated by secretion, recycling, and endocytosis (Irani et al., 2012; Luo et al., 2015). To avoid interference of the newly synthesized and secreted BRI1, we analyzed 5-day-old plants expressing BRI1Q-mCit treated with 50 µM of the protein synthesis inhibitor cycloheximide (CHX) for 1.5 h. Interestingly, the PM versus cytoplasm fluorescence intensity did not significantly differ between BRI1Q-mCit and the control BRI1-mCit, both in bri1 null background (Figure 3, A and B, upper panel). In addition to the CHX treatment, we applied Brefeldin A (BFA), an inhibitor of endosomal trafficking that is widely used to visualize endocytosis (Geldner et al., 2003). In Arabidopsis roots, BFA treatment promotes the formation of BFA bodies, composed of aggregated TGN/EEs (Geldner et al., 2003; Lam et al., 2009). When combining CHX (50 µM, 1.5 h) with BFA (50 µM, 30 min), both BRI1-mCit and BRI1Q-mCit accumulated in similar size BFA bodies (Figure 3, A and B, lower panel). These results were in agreement with the measurements of the PM versus cytoplasm fluorescence intensity in BRI1Q-mCit (Figure 3, A and B, upper panel). Comparable PM versus cytoplasm fluorescence intensity ratios and BFA body sizes were also obtained when BRI1Q-mCit was introduced into the Arabidopsis Columbia-0 (Col-0) wild type (Supplemental Figure S2, B and D) to avoid artifacts in quantitative microscopy due to the strong dwarfism of the bri1 mutant (Figure 3, C and D).

Figure 3.

Figure 3

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BRI1Q endocytosis is independent of ligand binding. A and C, Representative confocal images of epidermal root meristem cells of 5-day-old seedlings expressing BRI1-mCitrine (mCit) or BRI1Q-mCit treated with CHX (50 µM) for 1.5 h or pretreated with CHX for 1 h, followed by treatment for 30 min with CHX and BFA (50 μM). Scale bar, 10 µm. B and D, PM versus intracellular BRI1-mCit fluorescence intensity and BFA body size. For each line, 15 cells from at least 5 seedlings were measured. Box plots show the first and third quartiles, split by the medians (lines), with whiskers extending 1.5-fold interquartile range beyond the box, and dots as outliers. Statistical analysis was performed using Mann Whitney test. E and F, Time series analysis of meristem epidermal cells of 5-day-old seedling expressing BRI1-mCit or BRI1Q-mCit after heat induction (1 h at 37°C). Images were taken with a vertical confocal microscope with a 12 min interval between the frames. Scale bar, 10 µm. PM fluorescence intensity of the same cells was quantified for all time points and the signal peak was set as 1. Box plots show the first and third quartiles, split by the medians (lines), with whiskers extending 1.5-fold interquartile range beyond the box, and dots as outliers. Asterisks indicate the cells that were quantified. Four roots and 3–5 cells were measured per genotype. Asterisks indicate statistically significant differences, *P<0.05, **P<0.01, ***P<0.001, based on two-way analysis of variance (ANOVA) and post hoc Sidak’s multiple comparisons test. All experiments were repeated twice with similar results (A–F).

We next assessed the recycling dynamics of BRI1Q-mCit in bri1, by performing BFA washout experiments after treatment with CHX (50 µM) for 1 h, followed by a combined application of CHX and BFA (50 µM) for 30 min. Epidermal cells of root meristem were imaged at 0, 30, 60, 90, and 120 min after the BFA washout in the presence of CHX. By quantifying the percentage of epidermal cells with BFA bodies, we did not observe differences between the BRI1-mCit and BRI1Q-mCit (Supplemental Figure S3, A and B).

Besides recycling, BRI1 is also subjected to vacuole degradation (Martins et al., 2015; Zhou et al., 2018). To evaluate the vacuolar targeting of BRI1Q, we transferred BRI1Q-mCit/Col-0 and BRI1-mCit/Col-0 plants to dark conditions, which suppress vacuolar lytic activity and allow detection of the vacuolar pH-resistant mCit fluorescent fusion proteins (Tamura et al., 2003). By measuring the fluorescence intensity of BRI1Q-mCit and BRI1-mCit in the vacuole, we observed that the vacuolar accumulation of BRI1Q-mCit is slightly reduced when compared to that of BRI1-mCit (Supplemental Figure S3, C and D).

Because BRI1Q and BRI1 were endocytosed, recycled, and degraded with similar dynamics, we hypothesized that they share endomembrane trafficking routes. Indeed, as previously reported for BRI1 (Irani et al., 2012), BRI1Q co-localized with endomembrane markers labeling TGN/EEs, late endosomes/multivesicular bodies (MVBs), and Golgi compartments but not with the autophagy marker ATG8e (Supplemental Figure S4). Moreover, by taking advantage of the bioactive fluorescently labeled BR precursor Alexa Fluor 647-Castasterone (AFCS), which specifically marks the endocytic route of BRI1 (Irani et al., 2012), we showed that AFCS co-localized with BRI1Q-mCit in the wild type Arabidopsis (Supplemental Figure S5), suggesting that the two receptors are internalized through similar routes.

Despite the widespread use of CHX and BFA treatments to analyze endocytosis in plant cells, it cannot be excluded that the chemical treatments might cause pleiotropic effects (Oksvold et al., 2012; Smith et al., 2014). To circumvent this problem, we expressed BRI1-mCit and BRI1Q-mCit under the control of the heat shock-inducible promoter (pHS) in the Col-0 background and studied the protein internalization during the recovery phase after a heat treatment at 37°C for 1 h, which did not affect the endocytic rates of FM4-64 in wild type seedlings (Supplemental Figure S6). First, we selected transgenic lines expressing pHS::BRI1-mCit and pHS::BRI1Q-mCit with similar expression levels following the heat induction (37°C) for 1 h (Supplemental Figure S2C). Taking advantage of a vertical confocal microscope setup equipped with the TipTracker software (von Wangenheim et al., 2017) that allows the monitoring of growing root tips over time, we observed that BRI1Q-mCit reached the PM a little later than the BRI1-mCit (Figure 3, E and F). However, after a signal intensity peak in the PM, between 87 and 99 min, the internalization rate of BRI1-mCit and BRI1Q-mCit was very similar (Figure 3, E and F). Thus, our findings further confirm previous reports (Russinova et al., 2004; Geldner et al., 2007; Irani et al., 2012) that the BRI1 internalization is largely independent of BR binding and, consequently, of receptor activation. After ligand binding and interaction with BAK1, BRI1 is phosphorylated and ubiquitinated, both essential for receptor internalization (Martins et al., 2015; Zhou et al., 2018). However, although BRI1Q displayed reduced levels of both PTMs, it still had a normal endocytosis. These results reinforce the hypothesis that BRI1 is internalized using different mechanisms. For instance, the BRI1 internalization has been demonstrated to partially depend on both the classical clathrin Adaptor Protein 2 (AP-2) complex that binds to a canonical YXXΦ endocytic motif in BRI1 (Liu et al., 2020) and on ubiquitin recognition machinery, because endocytosis of the ubiquitin-deficient BRI125KR-mCit or BRI1-mCit in the pub12 pub13 double mutant was not completely abolished (Martins et al., 2015; Zhou et al., 2018). Similar observations were reported for the borate exporter BOR1, in which AP-2-dependent and AP-2-independent internalization had been activated by low and high borate concentration, respectively (Yoshinari et al., 2019). Equally in mammals, the well-studied epidermal growth factor receptor (EGFR) is also internalized via different endocytic mechanisms, including canonical ligand-dependent clathrin-mediated endocytosis and clathrin-independent endocytosis, which both depend on the ligand concentration (Zhou and Sakurai, 2022), and a ligand-independent internalization where EGFR endocytosis is induced by stress conditions and does not require kinase activity or ubiquitination (Metz et al., 2021).

However, after internalization, BRI1 and BRI1Q pursued the same trafficking routes. Interestingly and in contrast to BRI1, the immune receptors, FLAGELLIN SENSING2 (FLS2) and PEP RECEPTOR1 (PEPR1), follow distinct trafficking pathways depending on their activation state. Inactive FLS2 and PEPR1 constitutively recycle between PM and TGN/EEs in a BFA-sensitive manner, while after activation, FLS2 and PEPR are internalized in MVBs that are insensitive to BFA (Beck et al., 2012; Mbengue et al., 2016; Ortiz-Morea et al., 2016).

Taken together, our results show that BRI1 internalization is not abolished in the BR binding-deficient mutant but it remains to be established which signals or conditions trigger endocytosis of the inactive receptor.

BRI1Q can partially complement the xylem phenotype of bri1

In addition to its primary role in perceiving BRs, recent studies suggest that BRI1 might also have non-canonical functions in sensing cell wall integrity. After disturbance of the cell wall integrity by the inhibition of the pectin de-methyl esterification, BRI1 is recruited together with the RLP44 and BAK1 to activate a BR signaling for a compensatory feedback loop to remodel the cell wall (Wolf et al., 2012). Besides cell wall integrity monitoring, RLP44 is also implicated in controlling xylem cell fate through the phytosulfokine signaling (Holzwart et al., 2018). Interestingly, BRI1 and BAK1 are also necessary to regulate the vasculature cell fate, but independently of BRs, because BR biosynthesis mutants have no ectopic xylem in the procambial position present in the rlp44 and bri1 mutants (Holzwart et al., 2018, 2020). To test whether BRI1Q retains its non-BRs receptor functions, we examined whether BRI1Q could still interact with RLP44 (Holzwart et al., 2018). RLP44-RFP was transiently co-expressed with either BRI1-GFP or BRI1Q-GFP in Nicotiana benthamiana leaves and co-immunoprecipitation (Co-IP) assay revealed that RLP44-RFP was co-purified with both BRI1-GFP and BRI1Q-GFP but not with the negative control, indicating that BRI1Q, like the wild type BRI1, can form a complex with RLP44 (Figure 4A). Furthermore, confocal analysis of N. benthamiana leaves transiently expressing BRI1-GFP, BRI1Q-GFP, and RLP44-RFP shows that both BRI1 and BRI1Q co-localize with RLP44 in dynamic punctate structures (Figure 4B). The intracellular punctate structures containing RLP44-RFP and BRI1-GFP or BRI1Q-GFP are probably endosomes, because BRI1 is a bona fide endosomal PM cargo (Russinova et al., 2004; Geldner et al., 2007). Moreover, RLP44 had already been shown to localize in endosomal structures in Arabidopsis roots (Wolf et al., 2014). Finally, we tested whether BRI1Q-mCit could recover the BR-independent xylem cell fate phenotype of the bri1 null mutants (Holzwart et al., 2018, 2020). Indeed, BRI1Q-mCit could partially complement the ectopic number of xylem cells present in the bri1 mutant (Figure 4). Collectively, our result provides evidence that BRI1Q could still be active in BR-independent pathways.

Figure 4.

Figure 4

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BRI1Q partially recovers bri1 xylem cell fate phenotype. A, Co-IP of RLP44-RFP transiently co-expressed with BRI1-GFP, BRI1Q-GFP, or free GFP (negative control) in Nicotiana benthamiana leaf epidermal cells. Proteins were extracted (input) and immunoprecipitated (IP) by means of magnetic GFP beads. The immunoblots were done with α-GFP and α-RFP antibodies. B, Co-localization of RLP44-RFP with BRI1-GFP or BRI1Q-GFP in subcortical discrete punctate structures in Nicotiana benthamiana leaf epidermal cells. Insets show enlarged images. Scale bars, 10 µm. C, Seven-day-old roots stained with Basic Fuchsin for the visualization of xylem cells. Scale bars, 10 µm. D, Frequency quantification of roots with the indicated number of metaxylem cells. Asterisks indicate statistically significant differences, *P<0.05, ***P<0.001, Chi-square test. n = 20–57 as indicated. All experiments were repeated twice with similar results (C and D).

In conclusion, analyses of the crystal structure of the ectodomain of BR receptors and homology modeling allowed us to create a BR binding-deficient BRI1 mutant, BRI1Q. Characterization of BRI1Q showed that it displays a clear bri1-like phenotype and is unable to respond to exogenous BRs. By means of BRI1Q as a tool to study BRI1 endocytosis and in agreement with previous observations (Russinova et al., 2004; Geldner et al., 2007; Irani et al., 2012), we conclude that BRI1 internalization can occur without BR binding. Moreover, the BR binding-deficient BRI1 mutant might provide opportunities to discover additional non-canonical ligand-independent BRI1 functions in vasculature development and other processes (Holzwart et al., 2018, 2020; Graeff et al., 2020).

Materials and methods

Plant materials, growth conditions, and treatments

The experimental model used in this study was Arabidopsis (Arabidopsis thaliana (L.) Heynh). The wild type used was accession Columbia-0 (Col-0). Dual-marker lines were generated by crossing pBRI1::BRI1-mCit/Col-0 or pBRI1::BRI1Q-mCit/Col-0 with plants expressing pVHAa1::VHAa1-RFP/Col-0 (Dettmer et al., 2006), pUBQ10::MEMB12-mCherry/Col-0 (Gelgner et al., 2009), pARA7::ARA-RFP/Col-0 (Geldner et al., 2009), and pUBQ10::mCherry-ATG8e/Col-0 (Zhao et al., 2022).

For phenotypic analysis, plants were grown in soil in a growth chamber at 22°C, 58% relative humidity, and a 16-h light/8-h dark photoperiod for 6 weeks. The Arabidopsis seeds were surface-sterilized with chlorine gas, and then placed on plates with half-strength Murashige and Skoog medium (½MS) containing 0.5% (w/v) sucrose, 0.8% (w/v) agar, and 2.5 mM methyl ester sulfonate at pH 5.7. After vernalization for 2 days at 4°C, the plates were moved to the growth chamber under a 16-h/8-h light/dark cycle. Nicotiana benthamiana plants were grown in a greenhouse under a normal 14-h light/10-h dark regime at 25°C. For the microsomal protein preparation, plants were grown for 6 days on plates. For the BRI1 internalization assay and the BRI1 transcript analysis, plants were grown for 5 or 7 days on plates. The vacuolar targeting of BRI1 was evaluated in plants grown for 5 days on plates and then transferred to dark for 10 h. MG-132 (10 mM stock in dimethylsulfoxide [DMSO]), BFA (50 mM stock in DMSO), FM4-64 (2 mM stock in water), and CHX (50 mM stock in DMSO) were used at the concentrations indicated in the figure legends.

Vector construction and plant transformation

The BRI1-coding region without the stop codon was cloned into pMD19-T (simple) (Takara Biotechnology) to generate pMD19-BRI1 that was used as a template to generate the mutants BRI1Y599F, BRI1M657E, BRI1Y597M/Y599F/M657E, BRI1Y597A/Y599A/M657A, and BRI1Y597M/Y599F/Y642A/M657E/F681A by overlapping polymerase chain reaction (PCR) and subcloned into pDONR221 to generate pDONRP1P2-BRI1 (with mutations). The primers used to generate the BRI1 mutations are listed in Supplemental Table S1. The destination vectors were generated by recombining pK7m34GW, pB7m34GW, pDONRP4P1r-pBRI1, pDONRP4P1r-pHS (Del Mar Marquès-Bueno et al., 2016), pDONRP4P1r-pBRI1, pDONRP4P1r-pHS, pDONR221-BRI1, and pDONRP2rP3-mCit (Martins et al., 2015). The resulting constructs were transformed into the heterozygous bri1 null mutant (GABI_134E10) (Jaillais et al., 2011) or into Col-0 plants by floral dip. For transient expression in N. benthamiana leaves, pDONR221-BRI1 and pDONR221-BRI1Q were recombined in pK7FWG2 that contained the 35S promoter and C-terminal green fluorescent protein (GFP). The Gateway technology (Invitrogen) was used for cloning.

Western blot analysis and IP

For the BRI1 expression assay, 5-day-old seedlings were homogenized in liquid nitrogen. Total proteins were extracted with a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% (w/v) sodium dodecyl sulfate (SDS), 100 mM dithiothreitol, and ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail cOmplete (Roche). For blocking and antibody dilutions, 3% (w/v) bovine serum albumin (BSA) powder in 0.2% (v/v) Tris-buffered saline-containing Tween-20 was used. For the microsomal fraction isolation, 6-day-old seedlings treated with 50 µM MG-132 for 5 h were ground in liquid nitrogen and resuspended in ice-cold sucrose buffer (100 mM Tris [pH 7.5], 810 mM sucrose, 5% [v/v] glycerol, 10 mM EDTA [pH 8.0], 10 mM ethyleneglycoltetraacetic acid [pH 8.0], 5 mM KCl, protease inhibitor [Sigma-Aldrich], and phosphatase inhibitor [Sigma-Aldrich]). The homogenate was transferred to polyvinyl polypyrrolidone pellets, mixed, and left to rest for 5 min. Samples were centrifuged for 5 min at 600×g at 4°C. The supernatant was collected. The extraction was repeated for two more times. The supernatant was filtered through a Miracloth mesh. The same amount of water was added to the clear supernatant and centrifuged at 21,000×g for 2 h at 4°C to pellet microsomes (Abas and Luschnig, 2010). The pellet was resuspended in IP buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 0.1% [w/v] SDS, protease inhibitor, and phosphatase inhibitor). IPs were carried out on solubilized microsomal proteins with GFP-Trap-MA (Chromotek) according to the manufacturer’s protocol. For protein detection, the following antibodies were used: monoclonal α-GFP horseradish peroxidase-coupled (1/5,000; Miltenyi Biotech), monoclonal α-tubulin (1/10,000; Sigma-Aldrich), α-ubiquitin (Ub) P4D1 (1/2,500; Millipore), α-pThr (1/2,000; Cell Signaling), αBES1 (Yin et al., 2002) (1/4,000), and α-BAK1 (1/5,000; custom-made by Eurogentec). Uncropped blots are shown in Supplemental Figure S7.

Xylem staining

Seven-day-old Arabidopsis seedlings were stained with Basic Fuchsin as described (Ursache et al., 2018).

AFCS uptake assays

AFCS uptake assay was performed as previously described (Irani et al., 2014) with modifications. Six-day-old seedlings grown on solid ½MS were transferred to 200 µl of liquid ½MS on a piece of parafilm placed in a Petri plate, humidified with wet laboratory wipes for 10 min. The medium was replaced with ½MS supplemented with 30 µM AFCS for 40 min (pulse). Seedlings were washed six times and chased for 2 min on ½MS followed by imaging.

For confocal microscopy and image analysis of BRI1 localization and BFA washout, Arabidopsis seedlings were imaged with an Olympus FluoView1000 confocal laser scanning microscope equipped with UPLSAPO 60×/1.2 n.a. water-corrected immersion objective at digital zoom 2 and UPLSAPO 40×/1.3 n.a. oil-corrected immersion objective at digital zoom 1, respectively. The excitation/emission wavelengths were 514 nm/530–600 nm for BRI1-mCit, with laser intensity of 40% and gain 667. For BRI1 internalization, BFA treatment, and FM4-64 uptake, a Leica SP8X confocal microscope was used with a HC PL 584 APO CS2 40×/1.1 n.a. water-corrected immersion objective at digital zoom 5, 3, and 5, respectively. The excitation/emission wavelengths were 514 nm/530–600 nm for BRI1-mCit and 514 nm/600–700 nm for FM4-64 with white light laser (WLL) intensity of 70% and gain 150. For BRI1 co-localization, Leica SP8X confocal microscope was used with a HC PL APO CS2 63×/1.20 water-corrected immersion objective at digital zoom 2.5. The excitation/emission wavelengths were 514 nm/530–600 nm for BRI1-mCit and 594 nm/600–650 nm for red fluorescent protein (RFP) and mCherry with WLL intensity of 70% and gain 110. For the BRI1 internalization and FM4-64 uptake, the membrane of individual cells was selected using the brush tool of ImageJ with a size of 5 pixels as well as using the polygon selection tool to mark the intracellular space. The average intensity of the top 5% highest pixels for both the PM and the intracellular space was used to obtain a ratio between PM and intracellular fluorescence. The BFA body size and percentage of epidermal cells with BFA bodies were calculated as previously described (Luo et al., 2015).

For AFCS uptake, epidermal cells of the root meristematic zone were imaged using laser scanning confocal microscope a Leica SP8X equipped with an HC PL APO CS2 63×/1.20 water-corrected objective with 4× digital zoom. The excitation wavelength was 633 nm by WLL. Emission was detected at 650–700 nm by Leica hybrid detectors with WLL intensity of 70% and gain 180.

Xylem was imaged with Leica SP8X confocal microscope equipped with a HC PL APO CS2 63×/1.20 n.a. water-corrected immersion objective. The excitation/emission wavelengths used were 561 nm/600–650 nm for Basic Fuchsin staining with WLL intensity of 70% and gain 180. The BRI1 heat shock lines were analyzed under a vertical ZEISS LSM900 microscope equipped with a Plan-Apochromat M27 20×/0.8 n.a. objective. The excitation/emission wavelengths were 514 nm/530–600 nm for BRI1-mCit with laser intensity of 10% and gain 660. The root tip was tracked over time with the TipTracker software (von Wangenheim et al., 2017). Quantification was obtained by measuring the PM signal of the same cell over time and normalized by the time point with the highest PM signal. Because the heat shock promoter generated a patchy expression pattern, quantification cells with similar fluorescence intensity were selected. Image processing and quantification were performed with the Fiji software package.

GCI

A Creoptix WAVE system (Creoptix AG, Switzerland) was used for the GCI binding assays. Experiments were done on a 4PCH WAVE GCI chip (long polycarboxylate surface; Creoptix AG). After a borate buffer conditioning (100 mM sodium borate, pH 9.0, 1 M NaCl; XanTec Bioanalytics, Germany), streptavidin was immobilized through a standard amine coupling protocol, followed by passivation of the surface (0.5% BSA [Roche] in 10 mM sodium acetate, pH 5.0), and final quenching with 1 M ethanolamine, pH 8.0 for 7 min (XanTec Bioanalytics). The LRR ectodomains of wild type BRI1 and the respective mutants were biotinylated and coupled to the streptavidin-coated chip. For the BL binding experiments, BL was injected in a 1:2 dilution series, starting from 3 µM, in 20 mM citrate, pH 5.0, 250 mM NaCl at 25°C. Blank injections were used for double referencing and a DMSO calibration curve for bulk correction. All analyses and corrections were done with the Creoptix WAVE control software, with a one-to-one binding model with bulk correction used to fit all experiments.

Analytical SEC

Analytical SEC experiments were carried out on a Superdex 200 increase 10/300 column (GE Healthcare), preequilibrated in 20 mM sodium citrate, pH 5.0, 250 mM NaCl. Two hundred microgram of protein, injected in a 100-µl volume, was loaded onto the column, and elution at 0.75 ml min−1 was monitored by ultraviolet absorbance at λ = 280 nm. Peak fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE).

RALS

BRI1 ectodomains (residues 1–788 with a C-terminal Avi-tag as well as a Tobacco Etch Virus (TEV) protease cleavable TwinStrep—9x His tag) were expressed and purified as described previously (Hohmann et al., 2018a) and analyzed by SEC paired with a RALS and a refractive index detector, using an OMNISEC RESOLVE/REVEAL system. The calibration of the instrument was carried out with a BSA standard (Thermo Scientific). In a 50 µl volume, 100 µg of protein was separated on a Superdex 200 increase column (GE Healthcare) in 20 mM sodium citrate, pH 5.0, 250 mM NaCl at a column temperature of 35°C and a 0.7 ml min−1. Data were analyzed using the OMNISEC software (v10.41).

Homology modeling and structure visualization

BRIQ ectodomain structure was modeled using the Modeller 9.18 program (Šali and Blundell, 1993) with the BRI1 ectodomain (Protein Data Bank (PDB) ID 3RJ0) as a template. The structures of wild type BRI1 and BRI1Q ectodomains (Figure 1) were visualized by UCSF Chimera (Pettersen et al., 2021). Multiple sequence alignment was prepared using the Jalview program (Waterhouse et al., 2009).

Reverse transcription quantitative PCR

Seven-day-old seedlings in liquid ½ MS medium were transferred to 37°C for 1 h and let to recover at room temperature for another 1 h. Total RNA was extracted with the RNeasy kit (Qiagen). cDNA was synthesized from RNA with the qScript complementary (c)DNA Supermix (Quantabio). Reverse transcription quantitative PCR was conducted with SYBR green I Master kit (Roche) on a LightCycler 480 (Roche). The mCitrine expression was normalized to that of ACTIN2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The cycling conditions were as follows: preincubation at 95°C for 10 min; 45 amplification cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s; melting curve at 95°C for 1 s and 65°C for 1 s, followed by cooling at 40°C for 10 s.

Quantification and statistical analysis

The data were subjected to statistical analysis using GraphPad Prism (https://www.graphpad.com/scientific-software/prism/) and Excel software. Comparisons between two groups were made with t-tests. Comparisons between three or more groups were made with Mann Whitney tests or two-way ANOVAs with subsequent post hoc Sidak’s multiple comparisons tests. Comparisons between discrete groups were made using Chi-square tests. The measurements are shown as box plots displaying the first and third quartiles and split by medians (center lines), with whiskers extending to 1.5-fold the interquartile range from the 25th and 75th percentiles.

Accession numbers

Sequence data from this article can be found in the GenBank/European Molecular Biology Laboratory (EMBL) data libraries under accession numbers: At4g39400 (BRI1), At1g55610 (BRL1), At2g01950 (BRL2), At3g13380 (BRL3), At4g33430 (BAK1), At3g49750 (RLP44), At1g19350 (BES1), At1g50010 (TUBULIN), At4g19640 (ARA7), At2g28520 (VHAa1), At5g50440 (MEMB12), and At2g45170 (ATG8e).

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1 . Purity and oligomeric state of wild type and mutant BRI1 ectodomains.

Supplemental Figure S2 . BRI1-mCitrine expression in different transgenic lines.

Supplemental Figure S3 . BRI1Q displays normal recycling but reduced vacuole trafficking.

Supplemental Figure S4 . Endocytic routes of BRI1 and BRI1Q in Arabidopsis root epidermal meristem cells.

Supplemental Figure S5 . BRI1and BRI1Q colocalize.

Supplemental Figure S6 . FM4-64 uptake after heat treatment.

Supplemental Figure S7 . Source blots.

Supplemental Table S1 . List of oligonucleotides used in this study.

Supplementary Material

kiad005_Supplementary_Data
Click here for additional data file. (1.2MB, pdf)

Acknowledgments

We thank Yanhai Yin (Iowa State University, Ames, USA), Grégory Vert (CNRS/Université de Toulouse, France), Cyril Zipfel (University of Zurich, Switzerland), and Yasin Dagdas (Gregor Mendel Institute, Austria) for providing the anti-BES1 antibody, the pBRI1::BRI1-mCit/bri1 Arabidopsis transgenic line, pDONRP4P1r-pBRI1, pDONRP1P2-BRI1, and pDONRP2rP3-mCit plasmids, information for making the anti-BAK1 antibody, and the pUBQ10::mCherry-ATG8e/Col-0 line, respectively, and Martine De Cock for help in preparing the manuscript.

Contributor Information

Lucas Alves Neubus Claus, Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.

Derui Liu, Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.

Ulrich Hohmann, Structural Plant Biology Laboratory, Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland.

Nemanja Vukašinović, Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.

Roman Pleskot, Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.

Jing Liu, College of Life Sciences, Shaanxi Normal University, Xi’an, 710062 Shaanxi, China.

Alexei Schiffner, Center for Plant Molecular Biology (ZMBP), University of Tübingen, 72076 Tübingen, Germany.

Yvon Jaillais, Laboratoire Reproduction et Développement des Plantes (RDP), Ecole Normale Supérieure de Lyon, Centre National de la Recherche Scientifique (CNRS), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Lyon, 69342 Lyon, France.

Guang Wu, College of Life Sciences, Shaanxi Normal University, Xi’an, 710062 Shaanxi, China.

Sebastian Wolf, Center for Plant Molecular Biology (ZMBP), University of Tübingen, 72076 Tübingen, Germany.

Daniël Van Damme, Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.

Michael Hothorn, Structural Plant Biology Laboratory, Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland.

Eugenia Russinova, Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.

Funding

This work was supported by Ghent University Special Research Fund Grant (BOF15/24J/048 to E.R.), the Research Foundation-Flanders (project G008416N and G0E5718N to E.R. and a postdoctoral fellowship 12R7819N to N.V.), the European Research Council (ERC Co T-Rex grant 682436 to D.V.D.), the Swiss National Science Foundation (project number 31003A_176237 to M.H.), the Howard Hughes Medical Institute (to M.H.) and the German Research Foundation (DFG) (project WO 1660/6-2 to S.W.).

References

  1. Abas L, Luschnig C (2010) Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation. Anal Biochem 401(2): 217–227 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asami T, Min YK, Nagata N, Yamagishi K, Takatsuto S, Fujioka S, Murofushi N, Yamaguchi I, Yoshida S (2000) Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor. Plant Physiol 123(1): 93–99 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beck M, Zhou J, Faulkner C, MacLean D, Robatzek S (2012) Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting. Plant Cell 24(10): 4205–4219 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belkhadir Y, Jaillais Y (2015) The molecular circuitry of brassinosteroid signaling. New Phytol 206(2): 522–540 [DOI] [PubMed] [Google Scholar]
  5. Caño-Delgado A, Yin Y, Yu C, Vefeados D, Mora-García S, Cheng JC, Nam KH, Li J, Chory J (2004) BRL1 And BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development 131(21): 5341–5351 [DOI] [PubMed] [Google Scholar]
  6. Chen W, Lv M, Wang Y, Wang P-A, Cui Y, Li M, Wang R, Gou X, Li J (2019) BES1 is activated by EMS1-TPD1-SERK1/2-mediated signaling to control tapetum development in Arabidopsis thaliana. Nature Communications 10(1): 1065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Del Mar Marquès-Bueno M, Morao AK, Cayrel A, Platre MP, Barberon M, Caillieux E, Colot V, Jaillais Y, Roudier F, Vert G (2016) A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis. Plant J 85(2): 320–333 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dettmer J, Hong-Hermesdorf A, Stierhof Y-D, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18(3): 715–730 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P, Delbarre A, Ueda T, Nakano A, Jürgens G (2003) The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112(2): 219–230 [DOI] [PubMed] [Google Scholar]
  10. Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof Y-D, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59(1): 169–178 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geldner N, Hyman DL, Wang X, Schumacher K, Chory J (2007) Endosomal signaling of plant steroid receptor kinase BRI1. Genes Dev 21(13): 1598–1602 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graeff M, Rana S, Marhava P, Moret B, Hardtke CS (2020) Local and systemic effects of brassinosteroid perception in developing phloem. Curr Biol 30(9): 1626–1638.e3 [DOI] [PubMed] [Google Scholar]
  13. He Z, Wang Z-Y, Li J, Zhu Q, Lamb C, Ronald P, Chory J (2000) Perception of brassinosteroids by the extracellular domain of the receptor kinase BRI1. Science 288(5475): 2360–2363 [DOI] [PubMed] [Google Scholar]
  14. Hohmann U, Nicolet J, Moretti A, Hothorn LA, Hothorn M (2018. a) The SERK3 elongated allele defines a role for BIR ectodomains in brassinosteroid signalling. Nat Plants 4(6): 345–351 [DOI] [PubMed] [Google Scholar]
  15. Hohmann U, Santiago J, Nicolet J, Olsson V, Spiga FM, Hothorn LA, Butenko MA, Hothorn M (2018b) Mechanistic basis for the activation of plant membrane receptor kinases by SERK-family coreceptors. Proc Natl Acad Sci 115(13): 3488–3493 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holzwart E, Huerta AI, Glöckner N, Garnelo Gómez B, Wanke F, Augustin S, Askani JC, Schürholz A-K, Harter K, Wolf S (2018) BRI1 controls vascular cell fate in the Arabidopsis root through RLP44 and phytosulfokine signaling. Proc Natl Acad Sci U S A 115(46): 11838–11843 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holzwart E, Wanke F, Glöckner N, Höfte H, Harter K, Wolf S (2020) A mutant allele uncouples the brassinosteroid-dependent and independent functions of BRASSINOSTEROID INSENSITIVE1. Plant Physiol 182(1): 669–678 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hothorn M, Belkhadir Y, Dreux M, Dabi T, Noel JP, Wilson IA, Chory J (2011) Structural basis of steroid hormone perception by the receptor kinase BRI1. Nature 474(7352): 467–471 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Irani NG, Di Rubbo S, Mylle E, Van den Begin J, Schneider-Pizoń J, Hniliková J, Šíša M, Buyst D, Vilarrasa-Blasi J, Szatmári A-M, et al. (2012) Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nat Chem Biol 8(6): 583–589 [DOI] [PubMed] [Google Scholar]
  20. Irani NG, Di Rubbo S, Russinova E (2014) In vivo imaging of brassinosteroid endocytosis in Arabidopsis. Methods Mol Biol 1209: 107–117 [DOI] [PubMed] [Google Scholar]
  21. Jaillais Y, Belkhadir Y, Balsemão-Pires E, Dangl JL, Chory J (2011) Extracellular leucine-rich repeats as a platform for receptor/coreceptor complex formation. Proc Natl Acad Sci U S A 108(20): 8503–8507 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kinoshita T, Caño-Delgado A, Seto H, Hiranuma S, Fujioka S, Yoshida S, Chory J (2005) Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. Nature 433(7022): 167–171 [DOI] [PubMed] [Google Scholar]
  23. Lam SK, Cai Y, Tse YC, Wang J, Law AHY, Pimpl P, Chan HYE, Xia J, Jiang L (2009) BFA-induced compartments from the Golgi apparatus and trans-Golgi network/early endosome are distinct in plant cells. Plant J 60(5): 865–881 [DOI] [PubMed] [Google Scholar]
  24. Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC (2002) BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110(2): 213–222 [DOI] [PubMed] [Google Scholar]
  25. Liu D, Kumar R, Claus LAN, Johnson AJ, Siao W, Vanhoutte I, Wang P, Bender KW, Yperman K, Martins S, et al. (2020) Endocytosis of BRASSINOSTEROID INSENSITIVE1 is partly driven by a canonical Tyr-based motif. Plant Cell 32(11): 3598–3612 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Luo Y, Scholl S, Doering A, Zhang Y, Irani NG, Di Rubbo S, Neumetzler L, Krishnamoorthy P, Van Houtte I, Mylle E, et al. (2015) V-ATPase activity in the TGN/EE is required for exocytosis and recycling in Arabidopsis. Nat Plants 1(7): 15094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Luo Y, Takagi J, Claus LAN, Zhang C, Yasuda S, Hasegawa Y, Yamaguchi J, Shan L, Russinova E, Sato T (2022) Deubiquitinating enzymes UBP12 and UBP13 stabilize the brassinosteroid receptor BRI1. EMBO reports 23(4) [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martins S, Dohmann EMN, Cayrel A, Johnson A, Fischer W, Pojer F, Satiat-Jeunemaître B, Jaillais Y, Chory J, Geldner N, et al. (2015) Internalization and vacuolar targeting of the brassinosteroid hormone receptor BRI1 are regulated by ubiquitination. Nat Commun 6(1): 6151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mbengue M, Bourdais G, Gervasi F, Beck M, Zhou J, Spallek T, Bartels S, Boller T, Ueda T, Kuhn H, et al. (2016) Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases. Proc Natl Acad Sci U S A 113(39): 11034–11039 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Metz C, Oyanadel C, Jung J, Retamal C, Cancino J, Barra J, Venegas J, Du G, Soza A, González A (2021) Phosphatidic acid-PKA signaling regulates p38 and ERK1/2 functions in ligand-independent EGFR endocytosis. Traffic 22(10): 345–361 [DOI] [PubMed] [Google Scholar]
  31. Noguchi T, Fujioka S, Choe S, Takatsuto S, Yoshida S, Yuan H, Feldmann KA, Tax FE (1999) Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids. Plant Physiol 121(3): 743–752 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nolan TM, Vukašinović N, Liu D, Russinova E, Yin Y (2020) Brassinosteroids: multidimensional regulators of plant growth, development, and stress responses. Plant Cell 32(2): 298–318 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Oksvold MP, Pedersen NM, Forfang L, Smeland EB (2012) Effect of cycloheximide on epidermal growth factor receptor trafficking and signaling. FEBS Lett 586(20): 3575–3581 [DOI] [PubMed] [Google Scholar]
  34. Ortiz-Morea FA, Savatin DV, Dejonghe W, Kumar R, Luo Y, Adamowski M, Van den Begin J, Dressano K, Pereira de Oliveira G, Zhao X, et al. (2016) Danger-associated peptide signaling in Arabidopsis requires clathrin. Proc Natl Acad Sci U S A 113(39): 11028–11033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE (2021) UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci 30(1): 70–82 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Russinova E, Borst J-W, Kwaaitaal M, Caño-Delgado A, Yin Y, Chory J, de Vries SC (2004) Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1). Plant Cell 16(12): 3216–3229 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Šali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234(3): 779–815 [DOI] [PubMed] [Google Scholar]
  38. She J, Han Z, Kim TW, Wang J, Cheng W, Chang J, Shi S, Wang J, Yang M, Wang Z-Y, et al. (2011) Structural insight into brassinosteroid perception by BRI1. Nature 474(7352): 472–476 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith JM, Salamango DJ, Leslie ME, Collins CA, Heese A (2014) Sensitivity to Flg22 is modulated by ligand-induced degradation and de novo synthesis of the endogenous flagellin-receptor FLAGELLIN-SENSING2. Plant Physiol 164(1): 440–454 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tamura K, Shimada T, Ono E, Tanaka Y, Nagatani A, Higashi SI, Watanabe M, Nishimura M, Hara-Nishimura I (2003) Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. Plant J 35(4): 545–555 [DOI] [PubMed] [Google Scholar]
  41. Ursache R, Andersen TG, Marhavý P, Geldner N (2018) A protocol for combining fluorescent proteins with histological stains for diverse cell wall components. Plant J 93(2): 399–412 [DOI] [PubMed] [Google Scholar]
  42. von Wangenheim D, Hauschild R, Fendrych M, Barone V, Benková E, Friml J (2017) Live tracking of moving samples in confocal microscopy for vertically grown roots. Elife (6): e26792 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang X, Chory J (2006) Brassinosteroids regulate dissociation of BKI1, a negative regulator of BRI1 signaling, from the plasma membrane. Science 313(5790): 1118–1122 [DOI] [PubMed] [Google Scholar]
  44. Wang Z-Y, Seto H, Fujioka S, Yoshida S, Chory J (2001) BRI1 Is a critical component of a plasma-membrane receptor for plant steroids. Nature 410(6826): 380–383 [DOI] [PubMed] [Google Scholar]
  45. Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009). Jalview version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25(9): 1189–1191 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wolf S, Mravec J, Greiner S, Mouille G, Höfte H (2012) Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling. Curr Biol 22(18): 1732–1737 [DOI] [PubMed] [Google Scholar]
  47. Wolf S, van der Does D, Ladwig F, Sticht C, Kolbeck A, Schürholz A-K, Augustin S, Keinath N, Rausch T, Greiner S, et al. (2014) A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling. Proc Natl Acad Sci U S A 111(42): 15261–15266 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yin Y, Wang Z-Y, Mora-Garcia S, Li J, Yoshida S, Asami T, Chory J (2002) BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109(2): 181–191 [DOI] [PubMed] [Google Scholar]
  49. Yoshinari A, Hosokawa T, Amano T, Beier MP, Kunied T, Shimada T, Hara-Nishimura I, Naito S, Takano J (2019) Polar localization of the borate exporter BOR1 requires AP2-dependent endocytosis. Plant Physiol 179(4): 1569–1580 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhao J, Bui MT, Ma J, Künzl F, Picchianti L, De La Concepcion JC, Chen Y, Petsangouraki S, Mohseni A, García-Leon M. et al. (2022) Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole. J Cell Biol 221(12): 444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhou J, Liu D, Wang P, Ma X, Lin W, Chen S, Mishev K, Lu D, Kumar R, Vanhoutte I, et al. (2018) Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination. Proc Natl Acad Sci U S A 115(8): E1906–E1915 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhou Y, Sakurai H (2022) New trend in ligand-induced EGFR trafficking: a dual-mode clathrin-mediated endocytosis model. J Proteomics 255 (3): 104503 [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

kiad005_Supplementary_Data
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