
==== Front
J Exp Bot
J Exp Bot
exbotj
Journal of Experimental Botany
0022-0957
1460-2431
Oxford University Press UK

38717932
10.1093/jxb/erae206
erae206
Research Paper
AcademicSubjects/SCI01210
Macromolecular tool box to elucidate CLAVATA3/EMBRYO SURROUNDING REGION-RELATED–RLK binding, signaling, and downstream effects
https://orcid.org/0000-0002-8600-0671
Narasimhan Madhumitha Institute for Developmental Genetics, Heinrich Heine University, Universitätstraße 1, D-40225 Düsseldorf, North Rhine Westphalia, Germany

https://orcid.org/0009-0001-2325-9133
Jahnke Nina Institute of Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University, Universitätstraße 1, D-40225 Düsseldorf, North Rhine Westphalia, Germany

Kallert Felix Institute of Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University, Universitätstraße 1, D-40225 Düsseldorf, North Rhine Westphalia, Germany

https://orcid.org/0000-0002-2678-6246
Bahafid Elmehdi Institute for Developmental Genetics, Heinrich Heine University, Universitätstraße 1, D-40225 Düsseldorf, North Rhine Westphalia, Germany

https://orcid.org/0009-0002-2936-2104
Böhmer Franziska Institute of Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University, Universitätstraße 1, D-40225 Düsseldorf, North Rhine Westphalia, Germany

https://orcid.org/0000-0003-0115-6405
Hartmann Laura Institute of Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University, Universitätstraße 1, D-40225 Düsseldorf, North Rhine Westphalia, Germany
Institute of Macromolecular Chemistry, University Freiburg, Stefan-Meier-Straße 31, D-79104 Freiburg, Germany

https://orcid.org/0000-0002-1317-7716
Simon Rüdiger Institute for Developmental Genetics, Heinrich Heine University, Universitätstraße 1, D-40225 Düsseldorf, North Rhine Westphalia, Germany
Institute for Developmental Genetics and Cluster of Excellence in Plant Sciences, Heinrich Heine University, Universitätstraße 1, D-40225 Düsseldorf, North Rhine Westphalia, Germany

Janda Martin University of South Bohemia in České Budějovice, Czech Republic
Editor
Madhumitha Narasimhan and Nina Jahnke contributed equally to this work.

Correspondence: Laura.Hartmann@makro.uni-freiburg.de or Ruediger.Simon@hhu.de
11 9 2024
08 5 2024
08 5 2024
75 17 54385456
07 12 2023
17 4 2024
07 5 2024
15 7 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.

Abstract

Plant peptides communicate by binding to a large family of receptor-like kinases (RLKs), and they share a conserved binding mechanism, which may account for their promiscuous interaction with several RLKs. In order to understand the in vivo binding specificity of the CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptide family in Arabidopsis, we have developed a novel set of CLAVATA3 (CLV3)-based peptide tools. After carefully evaluating the CLE peptide binding characteristics, using solid phase synthesis process, we modified the CLV3 peptide and attached a fluorophore and a photoactivable side group. We observed that the labeled CLV3 shows binding specificity within the CLAVATA1 clade of RLKs while avoiding the distantly related PEP RECEPTOR clade, thus resolving the contradictory results obtained previously by many in vitro methods. Furthermore, we observed that the RLK-bound CLV3 undergoes clathrin-mediated endocytosis and is trafficked to the vacuole via ARA7 (a Rab GTPase)-labeled endosomes. Additionally, modifying CLV3 for light-controlled activation enabled spatial and temporal control over CLE signaling. Hence, our CLV3 macromolecular toolbox can be used to study rapid cell specific down-stream effects. Given the conserved binding properties, in the future our toolbox can also be used as a template to modify other CLE peptides.

A macromolecular tool box consisting of a modified CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptide with a fluorescent molecule and photoactivatable group offers reliable insights into its in vivo binding characteristics, localization, and signaling.

Chemical probe
CLV3 peptide
CLAVATA signaling
endocytosis
fluorophore labeling
photoactivation
receptor specificity
solid phase peptide synthesis
sub-cellular trafficking
Deutsche Forschungsgemeinschaft 10.13039/501100001659
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pmcIntroduction

Small secreted peptides mediate cell-to-cell communication in multicellular organisms in response to pathogens, biotic, and abiotic stimuli, and to regulate developmental processes. The CLAVATA3/EMBRYO SURROUNDING REGION (ESR)-RELATED (CLE) family of peptides is evolutionarily conserved among land plants (Furumizu et al., 2021; Whitewoods, 2021; Hirakawa, 2022). CLE peptides act over short ranges to control stem cell division and differentiation in meristems, but also over long distances between shoot and root tissues. The Arabidopsis genome comprises 32 CLE genes that encode 26 different CLE peptides (Ito et al., 2006; Hirakawa et al., 2011). CLE genes encode pre-pro-proteins containing an N-terminus signal peptide, a variable central domain that might be involved in the secretion process, and a highly conserved CLE domain close to the C-terminus. After cleavage of the signal peptide and proteolytic processing, the functional CLE domain comprises 12 or 13 amino acids that are secreted via the endoplasmic reticulum and Golgi apparatus to the apoplast. The mature CLE peptides often contain hydoxyproxiline residues that may be essential for binding and activity, which can be further modified by arabinosylation (Rojo et al., 2002; Ito et al., 2006; Kondo et al., 2006; Shinohara and Matsubayashi, 2013; Tamaki et al., 2013; Strabala et al., 2014; Xu et al., 2015; Kim et al., 2017; Roman et al., 2022).

The secreted peptide binds to the extracellular leucine-rich-repeat (LRR) domains of plasma membrane-localized receptor-like kinases (RLK), which interact with diverse co-receptors. Besides the LRR ligand binding domain, the RLKs and their co-receptors carry a transmembrane domain and a cytosolic kinase domain that transmit the intracellular signal (Shiu and Bleecker, 2001; Xi et al., 2019). The first identified CLE peptide was CLAVATA3 (CLV3), which is expressed in the stem cell domain of the shoot apical meristem (SAM) of Arabidopsis. CLV3 signals via the LRR-RLK CLAVATA1 (CLV1), which belongs to the sub-family XI of LRR-RLKs (hereafter referred to as RLKs) that further includes BARELY ANY MERISTEM1 (BAM1), -2, and -3 (DeYoung et al., 2006; Furumizu et al., 2021). The CLV3-related peptide CLE40 is expressed in non-stem cells of root and shoot apical meristems (RAM and SAM, respectively) and signals via BAM1. During SAM development, the CLV3–CLV1 and the CLE40–BAM1 modules control expression of the transcription factor WUSCHEL, which is essential for meristem maintenance (Schoof et al., 2000; Yadav et al., 2011; Schlegel et al., 2021). Overexpression or exogenous application of several different CLE peptides triggers premature differentiation of the SAM and root meristem. CLV3 and several other CLE peptides signal via the heteromeric complex of CLV2, a receptor-like protein, and the pseudo-kinase CORYNE (CRN) (Fiers et al., 2005; Müller et al., 2008; Guo et al., 2010; Nimchuk et al., 2011a; Replogle et al., 2011; Breda et al., 2019). CLV2 and CRN exit the endoplasmic reticulum together, dependent on each other, and reach the plasma membrane to form a functional CLV2/CRN receptor-like complex together. Hence, the clv2 and crn mutants have identical CLE signaling phenotypes (Müller et al., 2008; Bleckmann et al., 2010; Somssich et al., 2015).

CLV3 and several other CLE peptides can induce arrest of root meristem growth and differentiation of root stem cells. This indicates promiscuity in the interaction between CLE peptides and RLKs (Fiers et al., 2005; DeYoung et al., 2006; Ito et al., 2006; Kinoshita et al., 2007; Hazak et al., 2017; Crook et al., 2020; Narasimhan and Simon, 2022). This has been corroborated by in vitro studies on isolated receptor domains in which dissociation constants (Kd) between 0.6 nM and 14 µM were observed for CLE peptides and RLKs of subfamily XI. For example, CLE8-14 and -16 can bind BAM1 with very high affinities, but CLE9 can also bind CLV1 (Ogawa et al., 2008; Crook et al., 2020). Notably, CLV3 has been shown to bind to the RLKs CLV1, BAM1, -2, and -3, which belong to the CLV1 clade (Guo et al., 2010; Shinohara et al., 2012; Hazak et al., 2017; Crook et al., 2020; Furumizu et al., 2021).

Interestingly, some CLEs were found to interact with RLKs of different clades. CLE9, in addition to BAM1, binds also to HAESA-LIKE1 (HSL1) with lower affinity. HSL1 is an RLK that predominantly interacts with the peptide INFLORESCENCE-DEFICIENT IN ABSCISSION (IDA) and other members of the IDA-LIKE family of ligands (Qian et al., 2018; Furumizu et al., 2021; Roman et al., 2022). CLE14, in addition to BAM1, was also reported to interact with PEP RECEPTOR 2 (PEPR2), which is a receptor for the AtPep family of peptides (Gutierrez-Alanis et al., 2017). This broad interaction spectrum can be attributed to the conserved mode of binding, which is shared amongst CLEs and other peptide families. The C-terminal residues undergo close contacts with the RLK and their co-receptors (Zhang et al., 2016a, b; Li et al., 2017). The N-terminal residues would then interact only with the RLK and provide specificity for the interaction. For example, changing the first three N-terminal residues of the CLE peptide TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF or CLE41) or of CLE9 resulted in total loss of binding to their cognate RLKs. Furthermore, swapping one or more of the N-terminal residues between IDA and CLE9 resulted in swapped affinity to their cognate RLKs HSL1 and BAM1, respectively (Zhang et al., 2016a; Li et al., 2017; Roman et al., 2022). Thus, N- and C-termini form two anchoring sites for the peptide on the RLKs, with the N-terminus contributing to the specificity of interactions. Furthermore, the peptides flg22, derived from the flagellin protein, and pep1 have been shown to bind FLAGELLIN-SENSITIVE2 (FLS2) and PEPR, respectively, in a fully extended conformation, which is in stark contrast to CLE41 forming a kink-like structure in the middle while binding the RLK TDR (Zhang et al., 2016b). This indicates that peptides of different families have mechanisms that confer specificity to the RLKs of a certain clade, thus excluding unspecific RLK interactions. This brings the previously reported CLE14–PEPR2 interaction into question, particularly when PEPR clade is phylogenetically distant from the CLV1 and TDR clades, which act as CLE receptors (Gutierrez-Alanis et al., 2017; Furumizu et al., 2021). Two important questions remain unanswered: can CLE peptides interact with high affinity and induce signaling through the PEPR clade of receptors? Is there an in vivo binding affinity between CLEs and specific RLK clades, such as CLV1 and TDR sister clades, while eliminating cross-clade interaction with phylogenetically more distant RLKs?

Most of our understanding of peptide–receptor interaction comes from in silico models, in vitro studies with purified RLKs in stable conditions, or in heterologous systems (Ito et al., 2006; Ogawa et al., 2008; Shinohara et al., 2012; Zhang et al., 2016b; Gutierrez-Alanis et al., 2017; Li et al., 2017; Roman et al., 2022). However, such studies do not accurately reflect the in vivo conditions, such as receptor confirmation changes within multi-protein complexes, the chemical and physical environment, protein modifications, number of receptor molecules at the plasma membrane, and ligand diffusion kinetics (Bhattacharya et al., 2013; Kastritis and Bonvin, 2013; Chang, 2022).

Post CLE peptide interaction, the fates of the peptide and the RLK have not been extensively studied. In plants, several receptors have been shown to be internalized via clathrin-mediated endocytosis (CME) (Paez Valencia et al., 2016). Different RLKs and ligands have been shown to undergo CME with varying rates and subsequent endosomal trafficking. For example, pep1–PEPR complexes undergo a slower rate of CME compared with BRASSINOSTEROID-INSENSITIVE1 (BRI1). This could be attributed to the pre-formed BRI1–co-receptor complexes at the plasma membrane to receive brassinosteroids (Bucherl et al., 2013; Ortiz-Morea et al., 2016). Similarly, CLV3 peptide binding can induce rapid formation of larger multimeric aggregates of CLV1 with co-receptors at the plasma membrane. In the SAM, CLV1 is endocytosed and trafficked towards the vacuole for degradation (Nimchuk et al., 2011b; Somssich et al., 2015; Wang et al., 2023). However, the sub-cellular trafficking dynamics of CLE peptides and their fate within the cell after RLK binding are unknown.

To enable in vivo studies of CLE–receptor interactions, we developed two new tools: (i) we synthesized a functional, fluorescently labeled CLV3 peptide in order to track the spatial and temporal distribution of CLE peptides and their cognate RLKs in vivo, and (ii) we generated a photocaged CLV3 peptide that can be activated by light with high spatial and temporal control. Fluorophore-labeled pep1, flg22, and brassinosteroid hormone have been used previously to understand their cognate receptor-mediated signaling and subsequent sub-cellular dynamics (Irani et al., 2012; Ortiz-Morea et al., 2016; Jelenska et al., 2017). Based on the existing knowledge of the CLE–RLK recognition mechanism and key interacting residues (Yamaguchi et al., 2016), we modified the CLE motif of the CLV3 peptide to allow linkage of a fluorescent dye, while retaining specific binding activity to the RLKs. This was achieved by conjugation of a fluorophore to the side chain. To accomplish this, we replaced the second amino acid, threonine, previously identified as non-essential for binding in depletion assays (Fiers et al., 2006), with lysine and linked a suitable fluorophore to this position. A quantum-dot based probe for CLV3 was previously reported, but it could not be used in planta due to toxic effects (Yu et al., 2014). Our functional CLE peptide probe allows the study of in vivo binding specificity and subsequent sub-cellular trafficking.

We then used photo-caging to obtain a peptide probe that can be activated locally and rapidly using light (Mangubat-Medina and Ball, 2021). A photocage is a photolabile protecting group that can be used to block the functional groups of the peptide required for receptor binding and biological activity. The caged, non-binding peptide can then be transformed into its active form by releasing the cage group through sample illumination with a specific wavelength of light (Fig. 1). Photo-caging techniques have a very broad application, ranging from enzyme activation to release of therapeutic agents, amino acids, and other molecules in target cells (Silva et al., 2019; Y. Li et al., 2023). This has been applied to study plant hormone effects as well. Photo-caged auxin has enabled control of the auxin response at a single cell level. Photo-caging of gibberellin has enabled monitoring its real time movement and also gradual release of the active compound for a prolonged effect. Photo-caging and release of the active cytokinin form has been achieved as well (Kusaka et al., 2009; Wexler et al., 2019; J. Li et al., 2023; Sun et al., 2023). However, no photoactivatable and fluorescently labeled plant peptide has been synthesized and applied in planta so far.

Fig. 1. CLV3 sequence and schematic presentation of the modifications. (A) Introduction of a rhodamine fluorophore (green) to CLV3 to derive an active CLV3 probe followed by introduction of an N-terminal 2-nitrobenzyl photocage (red) to allow for light-activated binding to CLV1 receptor in planta. CLV1, CLAVATA1; CLV3, CLAVATA3; TAMRA, 6-carboxy-tetramethylrhodamine.

In this study, we present the development of a macromolecular tool box of CLE peptides including fluorescently labeled and photoactivatable peptides. Performing a series of in vivo tests, we demonstrate their bioactivity, specificity, and ability to be locally activated. Using these tools, we study binding specificities and subcellular dynamics in a spatially and temporally controlled manner. Overall, we demonstrate how to derive a truly functional and controllable CLE peptide to understand the recognition and binding capacity to its cognate RLKs. Furthermore, the techniques we have developed are transferrable and can be applied to other members of the CLE peptides, and possibly peptides of other families, as well.

Materials and methods

Chemicals and materials

Acetonitrile (≥99%), dichlormethane (≥99%), and acetic acid (≥99%) were purchased from Merck. Acetic anhydride (99%) was purchased from VWR. Diethyl ether (stabilized with butylated hydroxytoluene, ≥99%) was purchased from Honeywell. 1,3-Dimethylbarbituric acid and lithium chloride were purchased from Carl Roth. Piperidine (99%), tetrakis (triphenylphosphine) palladium (0), and trifluoroacetic acid (99%) were purchased from Acros Organics. N,N-Dimethylformamide (DMF; for peptide synthesis), triisopropylsilane (TIPS, 99%), N,N-diisopropylethylamine (DIPEA, ≥99%), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 99%) were purchased from Fluorochem. 6-Carboxy-tetramethylrhodamine succinimidyl ester (TAMRA-N-hydroxysuccinimide) was purchased from Carbosynth. Dichlormethane (DCM, ≥99 %) and 4,5-dimethoxy-2-nitrobenzyl (NVOC) chloroformate (NVOC-Cl, 97%) were purchased from Sigma-Aldrich. Fmoc-Hyp(tBu)-OH (99%) was purchased from BLDpharm. Fmoc-Leu-OH (98%) was purchased from Carbolution Chemicals. Boc-Arg(Pbf)-OH, Fmoc-l-Arg(Boc)2-OH, Fmoc-l-Arg(Pbf)-OH, Fmoc-l-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-l-Pro-OH, Fmoc-l-Ser(tBu)-OH, Fmoc-l-Thr(tBu)-OH, and Fmoc-l-Val-OH were purchased from Iris Biotech with purities of 98.0%. Fmoc-His(Trt)-OH (98.0%), Fmoc-Lys(Boc)-OH (98.0%), and Fmoc-Pro-OH (99.0%) were purchased from Merck. H-l-His(Trt)-2CT resin (loading 0.63 mmol g−1) was purchased from Rapp Polymer. For trifluoroacetic acid (TFA) removal, AG 1-X8 resin from Bio-Rad Laboratories was used. A Strata C18-E column (1g/6ml) was purchased from Phenomenex. Centrifugal Concentrator Vivaspin20 (1000 MWCO, 20ml) was purchased from Satorius.

Plant materials

Arabidopsis ecotype Col-0 was used as WT and generally as the background for the mutant and the transgenic lines, unless specified. crn and clv2 mutant lines have been described as crn-10 (CRISPR-Cas9-derived) (Nimchuk, 2017) and clv2-gabi (GK-686A09) (Pallakies and Simon, 2014), respectively. crn-10 was verified using dCAPS strategy which involves amplifying the gene with the oligomers 5ʹ-GTAGAAGCAGCAATGAAGCAAAGAAGAAGGTG-3ʹ and 5ʹ-GTGTAGATGATGTTGAAGTT GTGGATAAGTG-3ʹ followed by HphI digestion. bam1 single and bam1;bam2 double mutants are CRISPR-Cas9-derived (Fan et al., 2021). The transgenic lines used in the study are: p35s::ARA7-GFP (Ueda et al., 2001; Dettmer et al., 2006), XVE>>AXL2 (Adamowski et al., 2018), pBAM1:BAM1-GFP/bam1-3 (Schlegel et al., 2021), pUBQ10:VAMP711-YFP (Geldner et al., 2009), pCLC2:CLC2-GFP (ecotype: Wassilewskija) (Konopka et al., 2008), pCLV1::CLV1-2xGFP; clv1-11 (Nimchuk et al., 2011b), and RPS5:PEPR1-GFP/pepr1;pepr2 (Ortiz-Morea et al., 2016).

Macromolecule synthesis

General procedure for solid phase synthesis

All peptides were synthesized on solid phase using an automated synthesizer (Activotec P11). The standard Fmoc protocol was used. Therefore, fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids were used for the synthesis. H-l-His(Trt)-2CT resin (loading 0.63 mmol g−1) was used and the structures were synthesized by repetitive Fmoc cleavage and amide coupling. For Fmoc cleavage, the resin was treated three times with 25 vol% piperidine in DMF for 10, 15, and 20 min. Afterwards the resin was washed 10 times with DMF. For coupling, the resin was treated with a solution of 5 eq. Fmoc amino acid, 5 eq. PyBOP and 10 eq. DIPEA in DMF for 1 h. Afterwards the resin was washed 10 times with DMF. After assembly of the full sequence, the resin was washed three times with DCM. Thereafter, the modifications of the peptide described in detail below were performed. For final cleavage the resin was treated with 70 vol% TFA, 30 vol% TIPS for 1 h. The peptides were precipitated in diethyl ether, lyophilized, and then TFA removal and further purification were performed.

Alloc deprotection and TAMRA labeling

After finishing the sequence, the allyloxycarbonyl (Alloc) deprotection of lysine on the resin was performed using 10 eq. 1,3-dimethylbarbituric acid and a spatula tip of tetrakis (triphenylphosphine) palladium (0) in DCM (1 ml per 100 mg resin). First 1,3-dimethylbarbituric acid was dissolved DCM and flushed with argon for 2 min. The palladium catalyst was added and flushed with argon for 2 min. The solution was then incubated in the syringe for 45 min. Afterwards the resin was washed 10 times with DCM, 10 times with 0.2 M DIPEA in DMF, and 10 times with DMF. The whole step was repeated once. After lysine was deprotected under reductive conditions, 5-carboxy tetramethyl rhodamine (TAMRA) conjugation took place. For this purpose, 1.3 eq. TAMRA-N-hydroxysuccinimide was dissolved in 4 ml of DMF and then 10 eq. of DIPEA was added. The solution was incubated in the syringe for 16 h. The resin was then washed with DMF 10 times and then alternately washed three times with DCM, three times with methanol until the solution becomes clear.

Acetylation

Acetic anhydride (1 ml per 70 mg resin) was incubated to the syringe for 15 min two times. Afterwards the resin was washed 10 times with DMF and 10 times with DCM.

4,5-Dimethoxy-2-nitrobenzyl coupling

NVOC-Cl was dissolved in a 50:50 vol% DCM/DMF mixture (1 ml per 100 mg resin). Then 10 eq. DIPEA was added and the mixture incubated in the solid phase syringe for 16 h. Afterwards the resin was washed 10 times with DMF and 10 times with DCM.

Trifluoroacetic acid removal

TFA removal was performed with an AG 1-X8, quaternary ammonium, 100–200 mesh, acetate form resin. For 100 mg sample, 500 mg of the ion exchange resin was used. The resin was activated by washing three times with 10 ml of 1.6 M acetic acid solution, followed by three times with 10 ml of 0.16 M acetic acid solution. A 100 mg sample was dissolved in 2 mL Milli-Q water, and the solution was loaded into the resin into the syringe. The syringe was shaken for 1 h. The supernatant was recovered, and the resin was washed three times with 0.1 ml Milli-Q. The liquid phases were lyophilized to obtain the crude product as a white or pink solid (in case of TAMRA labelled peptides).

Vivaspin

Samples were separated from non-bound material using a Vivaspin20 Centrifugal Concentrator with 1000 MWCO. The sample was washed five times with 10 ml of Milli-Q water, five times with 10 ml of 2 M LiCl solution, and five times with 10 ml of Milli-Q water, each step using a Heraeus Megafuge 8R centrifuge at 2597 g for 15 min.

Column purification

For 4,5-dimethoxy-2-nitrobenzyl (NVOC)–CLV3–TAMRA a further purification step was performed. A Strata C18-E column from Phenomenex (1 g/6 ml) was used to separate the samples from other incorrect sequences. First the column was conditioned with 6 ml acetonitrile and equilibrated with 6 ml Milli-Q water. A 100 mg sample was dissolved in 1–2 ml of Milli-Q water and added to the column. Then the sample was washed with acetonitrile/Milli-Q gradients from 5 to 30% each 3–6 ml on the column.

Reversed-phase high performance liquid chromatography electron spray ionization mass spectrometry

Reversed-phase high performance liquid chromatography (RP-HPLC)-MS was performed on an Agilent Technologies 1260 Infinity instrument in combination with an Agilent Technologies 6120-quadrupole mass spectrometer. The instrument has a wavelength detector (VWD1 A) that measures the absorbance at 214 nm. The mass spectrometer generates ions using the electrospray method, which is used at a charge-to-mass ratio detected between 200 and 2000. The separation of the sample was performed on the MZ-AquaPerfect C18 column (3.0 × 50 mm, 3 μm) at 25 °C. As the mobile phase the following mixtures were used: A: H2O–acetonitrile (95:5 vol%) with 0.1 vol% formic acid; and B: H2O–acetonitrile (5:95 vol%) with 0.1 vol% formic acid. The flow rate was 0.4 ml min−1. A linear gradient was from 100% A–0% B to 0% A–100% B with a total measurement time of 17 min.

Ultra-high resolution mass spectrometry

Ultra-high resolution (UHR) MS measurements were performed with a Bruker UHR quadrupole time-of-flight (QTOF) maXis 4G instrument with a direct inlet via syringe pump, an electrospray ionization (ESI) source and a QTOF mass analyser.

MALDI-TOF-MS

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS spectra were recorded on an UltrafleXtreme instrument from Bruker Daltonik. The concentration of the sample was 1 mg ml−1. The ratio to the α-cyano-4-hydroxycinnamic acid matrix was 1:10.

Lyophilization

The final oligomers were lyophilized with an Alpha 1–4 LD plus instrument from Martin Christ Freeze Dryers GmbH. The drying method was set to −40 °C and 0.1 mbar

Seed sterilization and plant growth conditions

The seeds were sterilized using chlorine gas (1 h in a desiccator after mixing 50 ml of 13% w/v sodium hypochlorite with 4 ml 37% HCl) and were sown on Murashige and Skoog (MS) medium containing ½ 0.22% w/v MS salts with B5 vitamins, 1% w/v sucrose, 0.05% w/v MES and 12 g l−1 plant agar, adjusted to pH 5.7 with KOH. The seedlings for root experiments were grown in phytocabinets (poly klima; model: M4Z-TDL+rF) for 4 or 5 d vertically with continuous light at 21 °C. Later, they were transferred to soil and grown in phytochambers for 5–6 weeks under 16 h light–8 h dark to experiment on the shoot apical meristem. The light spectrum for the plant growth condition spans the wavelengths from UV to far red with very minimal irradiance of 1–5 mW m−2 nm−1 for wavelength <400 nm, and 11–170 mW m−2 nm−1 for photosynthetically active radiation in the 400–700 nm range.

Treatment and imaging conditions

Peptide treatment

The peptides—pep1 (synthesized by Davids Biotechnologie), pep1–TAMRA (obtained personally from Prof. Russinova), CLV3–TAMRA, Ac–CLV3–TAMRA, NVOC–CLV3, NVOC–CLV3–TAMRA—and the control free TAMRA were dissolved in water to make stocks and subsequently added to the MS medium to reach the indicated working concentration. CLV3p (synthesized by Davids Biotechnologie) was dissolved in peptide buffer of pH 6 (mixture of 87.7 ml of 0.2 M potassium phosphate, mono-potassium salt and 12.3 ml of 0.2 M potassium phosphate, di-potassium salt in 100 ml buffer).

Chemical treatment

Four-day-old seedlings of the XVE>>AXL2 line were transferred to MS agar plates containing 10 µM estradiol [10 mM stock dissolved in dimethyl sulfoxide (DMSO)] for 24 h AXL2 induction. For the uninduced control condition, seedlings were transferred to MS agar plates containing the solvent DMSO. Subsequent mock or peptide treatments in AXL2-induced seedlings were also made in MS agar plates containing either 10 µM estradiol or DMSO.

Brefeldin A (BFA) treatment of seedlings was done in GM liquid medium containing 50 µM BFA together with 1 µM peptide or the controls for 50 min.

Sample handling and imaging

In vivo live fluorescence microscopy was performed with the Zeiss LSM 880 and Zeiss LSM 900 confocal laser scanning microscopy systems employing C-Apochromat ×40/1.20 water objectives. For root length assays, the MS agar plates were scanned using a CanoScan 9000F with 600 dpi resolution.

For root meristem imaging, 4- or 5-day-old seedlings were incubated for the indicated time in MS medium (with or without agar) containing either mock or peptide. For photoactivation of the NVOC–CLV3–TAMRA peptide, the seedlings were mounted with MS medium containing the peptide and directly exposed to UV light (UV lamp X-Cite XYLIS/model XT720L) with DAPI Filter Set 49 (the excitation wavelength is between 300 and 395 nm, peak 365 nm). For targeting the UV radiation over a small field of cells, the field aperture at the microscope was manually controlled, and the field of view was adjusted by locating the cells through the ocular objective.

For shoot apical meristem imaging, the entire inflorescence of 5- or 6-week-old plants was submerged in water containing mock or peptide, 0.01% Tween 20 and 0.1% DMSO. The inflorescence was then cut off and mounted onto the slide over a double-sided adhesive tape. It was then dissected to expose the apical meristem. For a CLV1 localization assay, the inflorescences were treated for 30 s and imaged after 30 min. For a CLV3–TAMRA binding assay, the inflorescences were treated for 1 min, washed thrice, and imaged immediately.

For root length assays, the seeds were directly sown on MS agar plates containing mock or peptide. For the root length assays with NVOC–CLV3, 3 d after germination, one set of plates with peptides or mock were exposed to UV black light (OUSIDE) for 3 h at a distance of 25 cm while the control set were kept unexposed. The plates were covered in yellow foil during growth to filter off light under 500 nm in order to avoid NVOC cleavage before and after UV radiation. After the indicated number of days, the plates were scanned and the length of the roots was analysed. For pre-cleaving the NVOC peptide, a solution of 0.5 mg ml−1 was prepared in Milli-Q water. It was filled in a cuvette and radiated with UV black light for 3 h. The pre-radiated NVOC peptide was examined with UV spectra and RP-HPLC-MS and used for the root length assays.

Testing the degradation of the NVOC group

The samples were irradiated from a distance of 25 cm under a UV medium pressure lamp (Heraeus Noblelight) fitted with a mercury lamp that emitted light in the 250–600 nm range, or a 50 W UV black light (OUSIDE) fitted with COB LED chip that emits light in the 395–400 nm range.

Image analysis

Root length analysis and the plasma membrane and cytosolic intensity analysis were performed using Fiji ImageJ software tools (Schindelin et al., 2012). The number of SAMs with vacuolar CLV1 localization and the number of root meristem cells with plasma membrane PEPR1 were recorded by visual inspection. Pearson’s correlation coefficient analysis was performed and the cytofluorogram was made with the JaCoP plugin (Bolte and Cordelières, 2006). The plots were made using GraphPad Prism 9 and Origin.

Statistical analysis

All the statistical tests on intensity measurements were performed using GraphPad Prism 9. Statistical tests for root length analysis were performed using R version 4.3.1. ‘n’ indicates biological replicates and ‘N’ indicates technical replicates.

Results

Synthesis of a functional, fluorescently labeled CLV3 probe (CLV3–TAMRA)

Arabidopsis CLE domains share several conserved residues indicating that they interact with RLKs through a conserved mechanism (Zhang et al., 2016a, b). We chose CLV3, one of the best understood CLEs, as a test system (Fig. 1A).

Synthesis of all modified peptides was performed on an automated peptide synthesizer using well-established solid phase peptide synthesis employing fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids (Wellings and Atherton, 1997). In short, the terminal carboxy group of the amino acid is activated in situ to form an active ester that allows for coupling to a resin-bound amine group (e.g. the N-terminus of the previous amino acid, at room temperature and with high yields). Upon coupling of the amino acid, the Fmoc group is selectively cleaved by piperidine, releasing the N-terminus, which is now available for coupling of the next amino acid. After successful build-up of the desired amino acid sequence through such iterative coupling, the peptide is cleaved from the resin, typically under acidic conditions, including the cleavage of potential side chain protecting groups (Fig. 2A). Synthetic protocols for the unmodified CLV3 peptide were developed by choosing a suitable side chain protecting group strategy (Fig. 2B) (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine, tert-butyloxycarbonyl (tBu) for hydroxyproline, serine and aspartic acid, and trityl (trt) for histidine). Upon final cleavage of the peptide from the resin using acidic conditions, the fully deprotected CLV3 peptide (CLV3p) (1) was isolated in high purity (Supplementary Datasets S1–S3). Based on this protocol, a series of modified and fluorescently labeled CLV3 peptides were synthesized (Fig. 2C, D).

Fig. 2. Solid phase peptide synthesis of CLV3 using preloaded Fmoc-His(Trt)–resin and Fmoc-protecting group (PG) standard protocol. (A) Deprotection of N-terminal Fmoc-PG (a) using 25 vol% piperidine in N,N-dimethylformamide (DMF) for 5, 15, and 20 min. Coupling of Fmoc-protection amino acid (AA) (b) using 5 eq. AA, 5 eq. benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 10 eq. N,N-diisopropylethylamine (DIPEA) in DMF for 1 h. Both steps (a) and (b) were repeated to build up a sequence. (B) Selective Alloc deprotection (c) of lysine under reductive conditions with tetrakis (triphenylphosphine) palladium (0) catalyst and 10 eq. 1,3-dimethylbarbituric acid in dichlormethane (DCM) two times for 45 min. (C) Conjugation of TAMRA–N-hydroxysuccinimide (d) at free lysine by adding 10 eq. DIPEA in DMF for 16 h. Cleavage of the final structure (e) was with 70 vol% trifluoroacetic acid and 30 vol% triisopropylsilane. (D) Overview of different structures. See Supplementary Datasets S1–S3 for further details on the synthesis and analytical data of different peptide probes. Alloc, allyloxycarbonyl; Boc, tert-butyloxycarbonyl; CLV3, CLAVATA3; FITC, fluorescein isothiocyanate; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; TAMRA, 6-carboxy-tetramethylrhodamine; tBu, tert-butyloxycarbonyl; Trt, trityl.

One of the challenges in modifying small peptide probes with fluorescent labels is the decrease or even loss of their biological activity, e.g. due to a change in polarity, capping of essential amino acid residues, or sterical shielding of peptide sites that are required for interaction with the receptor (Boaro et al., 2020). Previous studies showed that the C-terminal residues interact with the receptor–co-receptor interface in a conserved manner, and that the N-terminal residue, either R or H, undergoes specific interactions with the receptor. The first and third residues are vital for recognizing and anchoring the CLE peptide to the RLK. The second residue is less conserved and can be modified without strongly compromising peptide activity (Song et al., 2012; Zhang et al., 2016a; Li et al., 2017). Therefore, the threonine at position 2 in CLV3p was replaced by lysine, giving CLV3-(Lys) (2) (Fig. 2D). The amino side chain of the lysine amino acid allows for the introduction of another orthogonal protecting group, allyloxycarbonyl (Alloc), which can be cleaved under reductive conditions on a solid support (Fig. 2B) (Wojcik et al., 2012). Therefore, the N-terminal amino acid arginine was changed from Fmoc to a Boc protecting group as during reductive deprotection of the lysine side chain partial deprotection of the N-terminal Fmoc protecting groups was observed. Selective release of Alloc allowed for quantitative and site-selective introduction of a fluorophore, here either TAMRA or fluorescein isothiocyanate (FITC), giving the two fluorescently labeled CLV3 probes CLV3–TAMRA (3) (Fig. 2C, D) and CLV3-FITC (4) (Fig. 2D). TAMRA and fluorescein were chosen as they are both commonly used in fluorescence microscopy, but differ in their excitation and emission spectra (λex,max=550 nm and λem,max=580 nm for TAMRA and λex,max=500 nm and λem,max=520 nm for FITC) (Tung, 2004).

In order to assess the binding characteristics of CLV3–TAMRA as a peptide probe, we synthesized a series of CLV3 peptides that are acetylated at the N-terminus, Ac–CLV3 (5), Ac–CLV3-(Lys) (6), and Ac–CLV3–TAMRA (7) (Fig. 2D), as negative controls. Instead of the Boc-protected arginine, its Fmoc-protected variant was used. Fmoc was cleaved off under basic conditions while every other protection group was stable under these conditions and capped with acetic anhydride to receive the acetylated N-terminus.

Testing the bioactivity of CLV3–TAMRA

Bioactivity of the modified peptides was tested using a root length assay. Exogenous application of the synthesized native CLV3 peptide (CLV3p) elicits a premature differentiation of the root meristem, leading to a short root phenotype, at CLV3p concentrations of 10 nM or higher (Hazak et al., 2017; Blümke et al., 2021). We compared root lengths of seedlings grown on medium with different concentrations of CLV3p or the modified peptides (Fig. 3; Supplementary Fig. S1). Seedlings showed a strong reduction in root length in response to CLV3p at 100 nM or lower (Blümke et al., 2021) (Fig. 3; Supplementary Fig. S1A); CLV3–TAMRA was ineffective at 100 nM, but triggered root length reduction at 1 µM concentration (Fig. 3; Supplementary Fig. S1A). This reduced activity is likely due to the steric hindrance to peptide binding caused by TAMRA. For comparison, we found that CLV3-(Lys) was bioactive at the much lower concentration of 200 nM (Supplementary Fig. S1B). Neither Ac–CLV3–TAMRA, nor free TAMRA fluorophore, nor CLV3-FITC elicited any response in our assays (Fig. 3; Supplementary Fig. S1A, C, D). Signalling of CLV3p during root development depends on the CLV2/CRN-receptor heteromer (Fiers et al., 2005; Miwa et al., 2008). We used one of the mutants of the CLV2/CRN heteromeric complex, crn, to thwart the CLV3 signaling pathway. The mutant seedlings did not respond to the tested peptides, indicating that the CLV3–TAMRA peptide acts through the canonical CLV3 signaling pathway, although with a lower efficacy than the unmodified CLV3p (Fig. 3; Supplementary Fig. S1A). Lack of CLV3–FITC biological activity could be due to structural differences between the fluorophores: TAMRA carries tertiary amines in the xanthene core and is unable to form hydrogen bonds, while FITC contains hydroxyl groups that could interact with the peptide backbone, potentially altering its conformation and preventing binding to the receptor protein. In summary, modifying the second amino acid residue of the CLE domain to attach the fluorophore is an effective strategy in creating a bioactive CLE peptide probe. TAMRA, as a fluorescent agent for CLE peptide probes, can retain their bioactivity, although with a significantly diminished efficiency.

Fig. 3. Bioactivity of CLV3–TAMRA. (A) Violin plot representing root length analyses of Col-0 and crn after 7 d treatment with mock, CLV3p, CLV3–TAMRA and Ac–CLV3–TAMRA. n≥ 43 roots for each condition; N=3. The lines represent the median and the quartiles. Two-way ANOVA with interaction, F-test; P<2 × 10−16. Statistical grouping was calculated by Tukey’s HSD test (α=0.001). Groups sharing the same letter are not significantly different. (B) Images of seedlings of Col-0 and crn after 10 d treatment. Scale bar: 1 cm. CLV3, CLAVATA3; TAMRA, 6-carboxy-tetramethylrhodamine.

Sub-cellular localization and trafficking of CLV3–TAMRA

We tested if CLV3–TAMRA is recognized by the RLKs at the plasma membrane, and how it interacts with the endocytic sub-cellular trafficking machinery. When added to Arabidopsis root or shoot meristems, CLV3–TAMRA localized to the plasma membrane in all layers of the meristems within 3 min after addition (Fig. 4A, B; Supplementary Fig. S2A, S2B). The negative control peptide Ac–CLV3–TAMRA showed a very weak signal at the plasma membrane, possibly due to unspecific binding to other plasma membrane proteins (Fig. 4A, B).

Fig. 4. Sub-cellular localization and trafficking of CLV3–TAMRA. (A–G) Representative confocal images of root meristem epidermal cells (A, C–G) or shoot apical meristem (SAM) cells (B) after 1 µM treatment of the peptide CLV3–TAMRA, or the controls Ac–CLV3–TAMRA or free TAMRA fluorophore. Lines used: Col-0 (A, B); pBAM1:BAM1-GFP/bam1-3 (C); XVE>> AUXILIN-LIKE2 (D); p35s:ARA7-GFP (E–G). (A) Plasma membrane localization after 15 min treatment. n=6; N=2. The inset on the right shows the same images with increased brightness. (B) Plasma membrane localization in SAM cells after 1 min treatment and washing. N≥5. The inset on the right shows the same images with increased brightness. (C) Vacuolar accumulation after continuous treatment overtime. n≥6; N=2. (D) Clathrin-mediated endocytosis and vacuolar trafficking after 30 min treatment. The seedlings either had induction of AUXILIN-LIKE 2 overexpression under estradiol treatment or no induction under mock treatment. n≥6; N=2. (E) Localization of the peptide in late endosome after 4–6 min treatment. n=4; N=2. (F) Localization of peptide and controls in late endosome 10–15 min post 5 min treatment. n≥5; N=2. Pearson’s correlation coefficient (r) of co-localization between ARA7–GFP and peptide and controls was calculated in three cells for each condition: CLV3–TAMRA versus ARA7–GFP: r=0.791, 0.711, and 0.664; Ac–CLV3–TAMRA versus ARA7–GFP: r=−0.027, −0.111, and 0.07; TAMRA-CO versus ARA7–GFP: r=0.041, 0.032, and −0.192. (Eʹ) Cytofluorogram of one measurement from each condition is shown. (G) Localization of peptide and controls in Brefeldin A (BFA) bodies containing late endosomes after 50 min co-treatment with BFA. n≥5; N=3. Yellow arrows indicate the CLV3–TAMRA co-localized with late endosomes marked by ARA7–GFP. White arrows indicate the BFA bodies containing late endosomes, and the peptide or the controls. Note: in (D–F), Ac–CLV3–TAMRA and free TAMRA were imaged at higher laser intensity and gain than CLV3–TAMRA to observe their sub-cellular localization. Scale bar: (A, C, D, F, G) 10 µm; (B) 15 µm; (E) 3 µm. CLV3, CLAVATA3; GFP, green fluorescent protein; TAMRA, 6-carboxy-tetramethylrhodamine.

During the course of 40 min treatment, CLV3–TAMRA accumulated increasingly in the vacuole, which is surrounded by the tonoplast (Fig. 4C; Supplementary Fig. S2C). We first tested if the accumulating vacuolar signal is due to endocytosed CLV3–TAMRA peptide, or free TAMRA fluorophore that resulted from CLV3–TAMRA instability or degradation. We analysed CLV3–TAMRA from our stock solution via RP-HPLC after 1 and 2 years’ storage, and saw no degradation of the fluorophore, suggesting that the CLV3–TAMRA peptide remained intact and did not undergo dissociation into free fluorophore during storage (Supplementary Dataset S4). We concluded that CLV3–TAMRA is functional and reaches the vacuole through the endocytic sub-cellular trafficking pathway.

We then examined the CLE peptide sub-cellular trafficking process. In animal cells, after endocytosis, ligand-bound receptors enter the early endosome for sorting. The membrane bound receptors are primarily recycled and return to the plasma membrane, but ligands are mostly delivered to the late endosome and then degraded (French and Lauffenburger, 1997; Lakadamyali et al., 2006; Solinger and Spang, 2022). We first examined if CLV3–TAMRA undergoes CME from the plasma membrane using AUXILIN-LIKE2 overexpression, which strongly hinders CME (Adamowski et al., 2018). After 60 min of CLV3–TAMRA treatment, CLV3–TAMRA accumulated in the vacuole. However, estradiol inducible overexpression of AUXILIN-LIKE2 strongly diminished the vacuolar signal compared with mock treatment, showing that CLV3–TAMRA is subject to CME (Fig. 4D). The inactive Ac–CLV3–TAMRA and free TAMRA fluorophore also reached the vacuole (Supplementary Fig. S2C), although to a lesser extent. CME inhibition interfered with uptake and vacuolar localization of Ac–CLV3–TAMRA (Fig. 4D), indicating that it might still bind plasma membrane localized RLKs with reduced affinity, possibly via the conserved C-terminal interaction. Free TAMRA fluorophore was neither bound at the plasma membrane nor trafficked via endosomes; therefore, it likely entered cells via diffusion (Supplementary Fig. S2D).

We then investigated the endocytic route to the vacuole. We found that CLV3–TAMRA co-localized with a few late endosomes labeled with ARA7 (a Rab GTPase) (Lee et al., 2004) rapidly within 4–6 min (Fig. 4E). After 15 min, CLV3–TAMRA reached almost all the ARA7-labeled late endosome compartments and aggregated into Brefeldin A (BFA) bodies after BFA treatment (Geldner et al., 2009; Narasimhan et al., 2021) (Fig. 4F, Fʹ, G). However, we only occasionally observed localization of CLV3–TAMRA in early endosomes, marked by CLC2–GFP (Supplementary Fig. S2E, Eʹ; the control for ARA7–GFP and CLC2–GFP bleed-through into the TAMRA channel is shown in Supplementary Fig. S2F). This is in concordance with flg22 and pep1 ligands that are also predominantly localized in late endosomes en route to the vacuole, and in contrast to animal cells where the ligand–receptor sorting occurs in early endosomes (Ortiz-Morea et al., 2016; Jelenska et al., 2017). However, neither free TAMRA nor Ac–CLV3–TAMRA co-localized with late endosomes (Fig. 4F, Fʹ), although a weak Ac–CLV3–TAMRA signal was observed in BFA bodies (Fig. 4G). We conclude that CLV3–Tamra is endocytosed via CME and effectively trafficked along the endosomal pathway. When and where the separation of endocytosed peptide ligands from the RLKs occurs in plant cells remains to be solved.

Overall, we found that CLV3–TAMRA endocytosis and sub-cellular trafficking dynamics are very rapid, and that the trafficking route is concordant with other plant signaling peptides.

Binding specificity of CLV3–TAMRA to receptor-like kinases

Studies in heterologous systems reported that CLV3 binds to the RLKs CLV1 and BAM1, although reports on binding affinities vary significantly between studies (Ogawa et al., 2008; Guo et al., 2010; Hazak et al., 2017; Crook et al., 2020). With our probes in hand, we here tested the interaction of CLV3–TAMRA with CLV1 and BAM1 in vivo.

After CLV3 binding, CLV1 in the SAM undergoes endocytosis and is targeted to the vacuole. The CLV1 population at the plasma membrane is strongly reduced over time (Nimchuk et al., 2011b; Wang et al., 2023). We observed that CLV3–TAMRA, but not Ac–CLV3–TAMRA, induced CLV1 endocytosis and vacuolar targeting (Fig. 5A, Aʹ). However, CLV3 binding does not trigger endocytic loss of the BAM1 plasma membrane pool or increased vacuolar trafficking of BAM1 (Supplementary Fig. S3A). This difference could be due to a high rate of recycling of BAM1, or alternatively due to a smaller fraction of BAM1 being involved in active signaling that further undergo degradation (Sorkin and von Zastrow, 2009; Bucherl et al., 2013). Therefore, to test for interaction with BAM1, we used a transgenic Arabidopsis line that overexpressed BAM1–GFP in the root meristem (Supplementary Fig. S3B). Compared with wild-type root meristems, a significantly higher amount of CLV3–TAMRA bound to the plasma membrane of BAM1–GFP (OX) root meristems (Fig. 5B; Supplementary Fig. S3C, Cʹ; the control for GFP bleed-through in the TAMRA channel is shown in Supplementary Fig. S3D). Plasma membrane binding of Ac–CLV3–TAMRA, on the other hand, was generally very low although slightly increased in the BAM1–GFP (OX) roots (Supplementary Fig. S3C, Cʹ). In contrast, we observed a general decrease in plasma membrane bound CLV3–TAMRA in bam1 and bam1;bam2 mutants compared with WT, which is particularly obvious in the stele layers. It is to be noted that this decrease could be attributed to an increased endocytic rate in the mutants as indicated by strong cytosolic signal (Supplementary Fig. S3E, Eʹ). We then incubated root meristems of BAM1–GFP (OX) line with CLV3p and CLV3–TAMRA to test if both compete for the same binding sites on the plasma membrane (Fig. 5C, Cʹ). With increasing concentration of CLV3p in the medium, the amount of CLV3–TAMRA fluorescence on the plasma membrane decreased, showing that CLV3p can compete with CLV3–TAMRA binding to the same site. This demonstrates that CLV3–TAMRA has strong affinity to both CLV1 and BAM1 in vivo, in contrast to results obtained from in vitro studies.

Fig. 5. Binding specificity of CLV3–TAMRA to RLKs. (A–F) Representative confocal images of shoot apical meristem (SAM) (A) or root meristem epidermal cells (B–E) after respective treatments. Lines used: pCLV1:CLV1-2X-GFP/clv1-11 (A); Col-0 and pBAM1:BAM1-GFP/bam1-3 (B, E); pBAM1:BAM1-GFP/bam1-3 (C); Col-0 and pRPS5A:PEPR1-GFPpepr1;pepr2 (D); pRPS5A:PEPR1-GFP/pepr1;pepr2 (F). (A) Top, CLV1 localization after 30 min of treatment with either mock or 100 nM CLV3. N≥5. Bottom, CLV1 localization after 30 min of treatment with 1 µM of either Ac–CLV3–TAMRA or CLV3–TAMRA. N≥9. The insets showcase the plasma membrane (white arrow) or vacuolar localized CLV1 (blue arrow) in a cell. Stacked bar graph represents the relative number of SAMs with plasma membrane and vacuolar localized CLV1. (B) CLV3–TAMRA plasma membrane signal after 1 µM, 30 min treatment in Col-0 or BAM1–GFP overexpression line. n≥6; N=3. (Bʹ) Violin plot representing CLV3–TAMRA mean fluorescence intensity of the plasma membrane from 13–39 cells/root. Two-sided t-test. For Col-0>BAM1–GFP, P<0.0001 (****). (C) CLV3–TAMRA plasma membrane signal after 30 min treatment with CLV3p/CLV3–TAMRA combination: from left to right, 0 µM/1 µM, 20 µM/1 µM, 40 µM/1 µM. n≥7; N=2. (Cʹ) Violin plots representing the mean fluorescence intensity of the plasma membrane (top) or the intracellular region (bottom) from 5–17 cells/root. (D) pep1–TAMRA and CLV3–TAMRA plasma membrane signal after 200 nM, 1 min treatment in Col-0 and PEPR1–GFP overexpression line. n=6; N=1. (Dʹ) Violin plot representing TAMRA mean fluorescence intensity of the plasma membrane from 14–31 cells/root. Two-sided t-test. For pep1–TAM/PEPR–GFP>Col-0, P<0.0001 (****); CLV3–TAM/PEPR–GFP>Col-0, was not significant. (E) pep1–TAMRA plasma membrane signal after 200 nM, 1 min treatment in Col-0 or BAM1–GFP overexpression line. n≥6; N=1. (Eʹ) Violin plot representing pep1–TAMRA mean fluorescence intensity of the plasma membrane from 10–25 cells/root. Two-sided t-test. BAM1–GFP>Col-0 was not significant. (F) PEPR1 plasma membrane localization 1 h after 30 s pulse with mock, 100 nM pep1–TAMRA or 1 µM CLV3–TAMRA. Grey, violet, and blue arrowheads indicate an example cell with strong, weak, and no plasma membrane PEPR, respectively. (Fʹ) Stacked bar graph representing relative number of cells with strong, weak, or no PEPR at the plasma membrane. n=5; N=2. At least 55 cells were analysed per root. Error bars indicate mean ±SD. The lines in the violin plots indicate the median and the quartiles. Scale bar: (A, B, E, F) 15 µm; (C, D) 10 µm. BAM1, BARELY ANY MERISTEM1; CLV1, CLAVATA1; CLV3, CLAVATA3; GFP, green fluorescent protein; n.s., not significant; PEPR, PEP RECEPTOR; PM, plasma membrane; RLK, receptor-like kinase; TAMRA, 6-carboxy-tetramethylrhodamine.

Because CLV3 has a strong affinity to CLV1 clade RLKs, we tested for its cross-clade interaction with PEPR1, an RLK of a phylogenetically distant clade (Furumizu et al., 2021). pep1 and pep1–TAMRA bound with strong affinity and induced endocytosis of its plasma membrane localized cognate receptor PEPR1–GFP, causing a significant down-regulation of the plasma membrane PEPR population through vacuolar targeting (Ortiz-Morea et al., 2016) (Fig. 5D–F, Dʹ–Fʹ; Supplementary Fig. S3F, Fʹ). However, CLV3–TAMRA did not exhibit an increased binding in a PEPR1 overexpression transgenic line (Fig. 5D, Dʹ); moreover, neither CLV3p nor CLV3–TAMRA induced PEPR endocytosis from the plasma membrane (Fig. 5F, Fʹ; Supplementary Fig. S3F, Fʹ). Similarly, pep1–TAMRA, which exhibited an increased binding in PEPR1 overexpression line (Fig. 5D, Dʹ), failed to show increased binding affinity to BAM1 overexpression (Fig. 5E, Eʹ). We conclude from our in vivo studies with modified CLV3 peptides that interactions of peptides are limited to a specific RLK family, pep1 to the PEPR clade and CLV3 to the CLV1 clade, contradicting earlier reports of cross-clade promiscuity in CLE peptide interactions (Gutierrez-Alanis et al., 2017; Qian et al., 2018; Furumizu et al., 2021).

Spatial and temporal control of CLE signaling

CLV3–TAMRA is a functional bioactive probe that, when externally applied, will rapidly reach all tissue layers and elicit a response. In order to achieve a controlled activity of the peptide in the targeted tissue or at a specific time point, we used nitroveratryloxycarbonyl (NVOC) as photo-cleavable protecting group, or photocage for the N-terminus of the CLV3 peptide with a maximum absorption for cleavage at 365 nm (Kneuttinger, 2022) (Fig. 6A). Linking the NVOC group to the N-terminus generates an inactive CLV3 probe; biological activity is then reinstated upon light-triggered cleavage of the NVOC group. As before, the TAMRA fluorophore was linked to the AA2 position, giving fluorescently labeled, photoactivatable NVOC–CLV3–TAMRA (8) (Fig. 6B).

Fig. 6. Synthesis and activation of photocaged peptide by UV radiation. (A) General scheme for peptide probe activation through 365 nm light-induced cleavage of N-terminal NVOC photocage. (B) Solid phase peptide synthesis of NVOC–CLV3–TAMRA using preloaded Fmoc-His (Trt)-resin and Fmoc-protecting group (PG) standard protocol. Deprotection of N-terminal Fmoc-PG (a) using 25 vol% piperidine in N,N-dimethylformamide (DMF) for 5, 15, and 20 min. Coupling photocage (b) using 5 eq. NVOC-Cl and 10 eq N,N-diisopropylethylamine (DIPEA) in DMF/dichlormethane (DCM) 1:1 for 4 h. Selective Alloc deprotection (c) of lysin under reductive conditions with tetrakis (triphenylphosphine) palladium (0) catalyst and 10 eq. dimethylbarbituric acid in DCM two times for 45 min. Conjugation of TAMRA–N-hydroxysuccinimide (d) at free lysine by adding 10 eq. DIPEA in DMF for 16 h. Cleavage of the final structure (e) with 70 vol% trifluoroacetic acid and 30 vol% TIPS. (C) Section of RP-HPLC chromatograms in which NVOC–CLV3 was irradiated with an UV medium-pressure lamp including wavelength of 365 nm for a total duration of 150 min. After 150 min, a complete degradation of the NVOC–CLV3 signal was observed, indicating a complete cleavage of the NVOC-protecting group. See Supplementary Dataset S5 for full spectra. (D) Violin plot representing root length analysis of 7-day-old Col-0 and clv2 seedlings 7 d treatment with mock, 200 nM CLV3p, 200 nM NVOC–CLV3 or pre-cleaved 200 nM NVOC–CLV3. The lines represent the median and the quartiles. n>9 roots; N=1. Two-way ANOVA with interaction, F-test, P<9.59 × 10−12. Statistical grouping was calculated by Tukey’s HSD test (α=0.001). Groups sharing the same letter are not significantly different. (E–H) Representative confocal images of root meristem epidermal cells after treatment with 1 µM (E, G, H) or 5 µM (F) NVOC–CLV3–TAMRA. Line used: pBAM1:BAM1-GFP/bam1-3. (E) Plasma membrane binding of NVOC–CLV3–TAMRA before and after photoactivation by UV exposure of root tip for 30 s. Top, image of the epidermal layer. Bottom, image of the root center with all layers visible. The inset shows the zoomed-in cells with CLV3–TAMRA in the apoplast around the cells (yellow arrows) or at the plasma membrane (blue arrows). (Eʹ) Violin plot representing the CLV3–TAMRA mean fluorescence intensity of the plasma membrane from 12–26 cells/root. n=4; N=6. Two-sided t-test. For After>Before, P<0.0001 (****). (F) Vacuolar localization of photoactivated NVOC–CLV3–TAMRA 60 min after 15 s UV exposure. n=5; N=2. (G) Late endosome localization of photoactivated NVOC–CLV3–TAMRA 20 min after 15 s UV exposure. n=2; N=3. (H) Targeted photoactivation of NVOC–CLV3–TAMRA after 3 min UV exposure of specific cells (marked by dotted circle) through controlled field aperture. Plasma membrane binding of NVOC–CLV3–TAMRA before and after photoactivation in BAM1–GFP expressing line is shown. BAM1–GFP shows mild bleaching due to UV exposure. Scale bar: (E, F, H) 20 µm; (G) 10 µm. BAM1, BARELY ANY MERISTEM1; CLV3, CLAVATA3; GFP, green fluorescent protein; NVOC, 4,5-dimethoxy-2-nitrobenzyl; TAMRA, 6-carboxy-tetramethylrhodamine.

Peptide synthesis was performed following previously discussed solid phase peptide protocols. Coupling of NVOC was performed on solid phase using NVOC chloride and coupling to the N-terminus of the terminal amino acid after Fmoc release. After installation of the photocage, the TAMRA fluorophore was coupled as previously described. After cleavage from the resin the crude peptide was purified by chromatography to give NVOC–CLV3–TAMRA with <90% relative purity (Supplementary Datasets S1, S3). First, release of the NVOC group upon light irradiation in solution was evaluated. Therefore, the NVOC–CLV3 peptide was exposed to UV light at 365 nm and analysed by RP-HPLC-MS measurements at different time points. After 150 min, full deprotection was observed. Importantly, no decomposition or fragmentation of the deprotected peptide was found (Fig. 6C; Supplementary Dataset S5).

We first tested the efficiency of NVOC cleavage from the peptide and the bioactivity of the NVOC-cleaved CLV3. We performed root length assays on whole seedlings that were grown in medium containing NVOC–CLV3 or CLV3p or mock, that were either (i) irradiated with UV for 120 min or (ii) not irradiated. UV irradiation of plates with NVOC–CLV3 resulted in shorter roots than controls, equivalent to CLV3p-treated roots. This indicated that the NVOC group was cleaved off, thereby releasing CLV3, which inhibited meristem development. clv2 mutants were resistant to NVOC–CLV3 with or without irradiation, indicating that the short roots were indeed due to activation of the photo-caged CLV3 peptide and signaling, and not a direct consequence of UV irradiation (Fig. 6D). Roots protected from UV exposure developed similar to mock-treated negative controls, showing that the NVOC group remained attached, which rendered the peptide inactive (Fig. 6D). Additionally, the roots grown in medium with pre-cleaved peptide (NVOC+CLV3) had shorter roots, proving that photoactivation of the photocaged peptide by UV is a reproducible technique and that NVOC in the medium does not interfere with the bioactivity of the peptide or plant development.

We then tested if the photocaged peptide can be rapidly photoactivated in the target tissue. To visualize peptide uncaging and perception, we photoactivated the NVOC–CLV3–TAMRA peptide by non-targeted, whole meristem UV irradiation at 385 nm, which is close to the maximum absorption wavelength for NVOC cleavage (Schaper et al., 2009). NVOC–CLV3–TAMRA predominantly localized to the apoplastic space before photoactivation due to the blocked N-terminus. After 30 s of UV irradiation using the UV lamp at the confocal microscope, we observed a rapid plasma membrane localization of the deprotected CLV3–TAMRA (Fig. 6E, top, Eʹ). Notably, CLV3–TAMRA localized to the epidermal layer of the root (Fig. 6E, bottom). Twenty minutes after photoactivation, we observed CLV3–TAMRA in endosomal compartments, and subsequently also in vacuoles (Fig. 6F, G). We then performed targeted irradiation and peptide activation in a few epidermal cells using the UV lamp or the laser. We observed localized plasma membrane binding of the deprotected CLV3–TAMRA peptide in and around the irradiated area (Fig. 6F; Supplementary Fig. S4A). It is to be noted that focusing UV radiation over target cells manually through the field aperture is not as precise as laser-based activation, and hence there is a slightly broader dispersion of energy and photoactivation. On the other hand, a 405 nm laser is not as energetically efficient as high energy UV lamp radiation.

We have successfully created photoactivatable CLE peptide tools, NVOC–CLV3 and NVOC–CLV3–TAMRA, that can be activated simply by very short exposure to UV radiation in target cells or tissue in vivo. The released bioactive peptide can be used to study rapid down-stream signaling responses or cell/tissue specific effects.

Discussion

Synthetic chemistry has aided in generating ligands to dissect ligand–receptor pairing, receptor activation, and down-stream signaling. Such chemical tools have also been used to label phytohormones and peptides to probe their interaction with receptor kinases and subsequent signaling (Sharma and Russinova, 2018). In this study we have synthesized a set of CLV3 probes with different modifications to study and decipher diverse aspects of CLE–RLK signaling.

Advantages of studies with fluorophore labeling

Following the identification of CLE molecules, synthesis of the active CLE peptides enabled rapid elucidation of their effects and downstream signaling effectors through in vivo and in vitro bioassays (Fiers et al., 2005; Ito et al., 2006; Stahl et al., 2009; Qian et al., 2018; Takahashi et al., 2018; Breda et al., 2019; Blümke et al., 2021; Breiden et al., 2021). Synthetic approaches have also provided a faster alternative to mutagenesis in creating peptide variants. Replacing individual residues or a combination of residues, and swapping of residues between peptides have facilitated breakthroughs in dissecting CLE affinities, specificities, and recognition mechanism for individual RLKs (Ito et al., 2006; Song et al., 2012; Zhang et al., 2016a; Li et al., 2017; Roman et al., 2022). However, most of those studies were performed in vitro. An alternative assay that allowed differentiation of affinities for RLKs was photoaffinity labeling, and binding of CLE9 and CLV3 peptides to several RLKs was resolved using this technique (Ogawa et al., 2008; Shinohara and Matsubayashi, 2013, 2015). However, photoaffinity labeling is an expensive technique, requires elaborate protocols, and does not spatially resolve the ligand–receptor interactions within a tissue (Sharma and Russinova, 2018). We emphasize here the advantages of fluorescently labeled peptides, which exhibit non-covalent reversible binding and native endocytic sub-cellular trafficking interactions.

Challenges in CLE modification to study interaction specificity

Attaching a fluorophore while maintaining biological activity is challenging, since CLE peptides are only 12 or 13 amino acids long, and modification of residues can change recognition and binding to cognate RLKs. Ogawa et al. (2008) had previously synthesized modified CLV3 peptides replacing L10 and T2, which still effectively bound the CLV1 ectodomain. Based on these in vitro experiments, we modified the second CLV3 residue to lysine (T2K) and added the fluorophore TAMRA. Nevertheless, the second residue variation between CLE41 and -42 is enough to significantly alter their binding affinity to TDR (Zhang et al., 2016a). Therefore, it is crucial to test both the bioactivity and specificity of the synthesized peptide tools. We observed that the peptide was indeed bioactive but showed reduced activity, which is likely caused by the attached TAMRA fluorophore (Fig 3; Supplementary Fig. S1). However, the peptide showed no significant decrease in affinity to CLV1 clade receptors, supporting the notion that CLV3–TAMRA peptide is functional. Furthermore, we utilized the probe to test for cross-clade interaction to the phylogenetically distant PEPR clade. We observed no binding affinity of CLV3–TAMRA to PEPR1. Importantly, pep1–TAMRA also showed no affinity to BAM1, a member of the CLV1 clade (Fig. 5, Supplementary Fig. S3). This demonstrates that, in vivo, there is minimal cross-interaction of peptides from distinct families with different RLK clades. Gutierrez-Alanis et al. (2017) previously reported that CLE14 interacts with PEPR2, based on a bimolecular fluorescence complementation (BiFC) assay of CLE14–nYFP with PEPR2–cYFP. However, CLE peptides undergo extensive processing, which involves not only the removal of the N-terminal signal peptide, but also proteolytic cleavage of the active CLE peptide from a larger precursor. Any C- or N-terminal fusions, such as nYFP in this case study, would not remain linked to the bioactive CLE peptide. CLE peptide interactions with receptors can therefore not be reliably detected using BiFC assays, and the fluorescence detected in the aforementioned study is likely due to constitutive interaction of the proteolytically released nYFP with PEPR2–cYFP (Fiers et al., 2006; Ni and Clark, 2006; Tamaki et al., 2013).

Terminal labeling to form NVOC–CLE peptides

Previous studies illustrated the importance of terminal residues for peptide function. Addition of an arginine group to the C-terminus significantly reduced CLE41 activity and its binding to TDR (Ito et al., 2006; Zhang et al., 2016a). Similarly, unprocessed CLE19 proprotein with an extra C-terminal arginine exhibited diminished activity in vivo (Tamaki et al., 2013). Alanine scanning experiments by Song et al. (2012) demonstrated that the N-terminal arginine residue of CLV3 is vital for its function, and addition of an extra tyrosine residue to the N-terminus of CLV3 or CLE45 rendered these peptides inactive (Hazak et al., 2017). Interestingly, we observed that a simple acetylation of the N-terminus was enough to deactivate CLV3 (Fig. 2). Based on this finding, we were able to manipulate the peptide N-terminus by attaching a photocleavable group that temporarily deactivates CLV3 and can be cleaved off efficiently with light at 365–380 nm (Fig. 6). This enables the tracking of rapid or time-sensitive cell biological effects, and elucidation of tissue specific effects with spatial and temporal control. For example, rapid changes in sub-cellular localization of a protein of interest within seconds after CLE perception can be observed microscopically in vivo. Alternatively, the peptide can be activated in target cells, and subsequent non-cell autonomous effects can be followed in adjacent cells or distant tissues.

The rapid NVOC cleavage observed under the microscope (Fig. 6E), in comparison with in vitro experiments (Fig. 6C), can be attributed to several factors. Firstly, differences in the light sources employed, such as lasers versus UV lamps, can lead to variations in energy output, potentially influencing the rate of cleavage. It is also crucial to note that the fluorescence observed under the microscope after 30 s does not necessarily indicate complete NVOC protection of group cleavage. In contrast, in vitro experiments involving spectral methods (e.g. absorbance and RP-HPLC spectra) offer a more quantitative assessment of cleavage completeness. The microscopic method confirms the occurrence of cleavage but does not provide the same level of quantitative information.

Adaptive strategies to study other peptides

Several in vivo and in vitro studies (detailed in ‘Introduction’), including this, have investigated the binding specificity of CLV3 and CLE41 to LRR-RLKs of sub-family XI. However, the in vivo interaction and recognition mechanisms of most other CLE peptides remain unexplored. Owing to the conserved architecture of the CLE domain amongst all CLE peptides, fluorophore labeling and photocaging strategies from this study (Figs 2, 6) could be transferred to investigate binding characteristics, short-term effects, turnover or localization.

There are several plant peptide families with multiple members that could potentially interact with one or more members of a sub-clade of RLKs. Modelling and crystal structure analysis of the interaction complex will give insights into ligand binding pockets and the conserved recognition mechanism that may be shared between the members of the family. Subsequently, targeted amino acid modifications and fluorophore labeling will enable confirmation of in vivo interaction and expand our understanding of their sub-cellular dynamics.

Conclusion

We have developed a macromolecular tool box of CLE peptides with fluorescent labeling and photocaging. After testing the bioactivity of these modified peptides, we have demonstrated that photocaged peptides can be regionally activated in plant tissues and be used in vivo to study their sub-cellular properties. Using these tools, we studied CLE–RLK binding specificities, clarifying the contradictory reports on CLE–RLK interactions and further substantiating the existence of highly selective mechanisms that discriminate between different peptide families. We have, for the first time, followed the in vivo endocytic and post-endocytic trafficking dynamics of a CLE peptide and RLKs. Our methodology for CLE peptide modification creates a functional and controllable peptide without compromising the recognition and binding capacity to its corresponding RLKs. Furthermore, based on our approach and developed synthetic strategy, additional CLE peptides can be synthesized to elucidate the signaling pathways mediated by CLE–RLK interactions.

Supplementary data

The following supplementary data are available at JXB online.

Fig. S1. Bioactivity of CLV3–TAMRA (related to Fig. 3).

Fig. S2. Sub-cellular localization and trafficking of CLV3–TAMRA (related to Fig. 4).

Fig. S3. Binding specificity of CLV3–TAMRA to RLKs.

Fig. S4. Activation of photocaged peptide by 405 nm laser irradiation.

Dataset S1. RP-HPLC-ESI-MS for CLV3 derivatives.

Dataset S2. MALDI-TOF-MS for CLV3 derivatives.

Dataset S3. HR-ESI for CLV3 derivatives.

Dataset S4. Storing stability test of CLV3–TAMRA.

Dataset S5. Absorption measurements and RP-HPLC-MS analysis after irradiation of NVOC–CLV3.

erae206_suppl_Supplementary_Figures_S1-S4_Datasets_S1-S5

Acknowledgements

We would like to thank Prof. Eugenia Russinova for sharing resources. We would like to acknowledge the Center for Advanced Imaging (CAi) at Heinrich-Heine-University Düsseldorf for providing access to the Zeiss LSM 880 Airyscan Fast.

Author contributions

MN, NJ, RS, and LH: conceptualization; MN, NJ, and EB: methology; MN and NJ: validation; MN and NJ: formal analysis; MN, NJ, EB, FK, and FB: investigation; RS and LH: resources; MN and NJ: writing—original draft; MN, NJ, RS, and LH: writing—review and editing; MN and NJ: visualization; LH and RS: supervision and funding acquisition. LH was responsible for synthetic chemistry and molecular characterization, and RS was responsible for plant biology.

Conflict of interest

The authors declare no competing interests

Funding

MN and NJ were supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through grants within the collaborative research center (CRC) 1208, B04 and A11 awarded to RS and LH, respectively. LSM 880 Airyscan Fast funded by DFG-INST 208/746-1. LSM900 was funded through the ERC Synergy grant, Sympore.

Data availability

The original raw data files of microscopy and phenotypic analysis and the corresponding files from data analysis are available upon request.
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References

Adamowski M , NarasimhanM, KaniaU, GlancM, De JaegerG, FrimlJ. 2018. A functional study of AUXILIN-LIKE1 and 2, two putative clathrin uncoating factors in Arabidopsis. The Plant Cell 30 , 700–716.29511054
Bhattacharya A , KimYC, MittalJ. 2013. Protein-protein interactions in a crowded environment. Biophysical Reviews 5 , 99–108.28510161
Bleckmann A , Weidtkamp-PetersS, SeidelCA, SimonR. 2010. Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane. Plant Physiology 152 , 166–176.19933383
Blümke P , SchlegelJ, Gonzalez-FerrerC, BecherS, PintoKG, MonaghanJ, SimonR. 2021. Receptor-like cytoplasmic kinase MAZZA mediates developmental processes with CLAVATA1 family receptors in Arabidopsis. Journal of Experimental Botany 72 , 4853–4870.33909893
Boaro A , AgeitosL, TorresM, BartoloniFH, de la Fuente-NunezC. 2020. Light-emitting probes for labeling peptides. Cell Reports Physical Science 1 , 100257.34396352
Bolte S , CordelièresFP. 2006. A guided tour into subcellular colocalization analysis in light microscopy. Journal of Microscopy 224 , 213–232.17210054
Breda AS , HazakO, SchultzP, AnneP, GraeffM, SimonR, HardtkeCS. 2019. A cellular insulator against CLE45 peptide signaling. Current Biology 29 , 2501–2508.e3.31327718
Breiden M , OlssonV, BlümkeP, SchlegelJ, Gustavo-PintoK, DietrichP, ButenkoMA, SimonR. 2021. The cell fate controlling CLE40 peptide requires CNGCs to trigger highly localized Ca2+ transients in Arabidopsis thaliana root meristems. Plant and Cell Physiology 62 , 1290–1301.34059877
Bucherl CA , van EsseGW, KruisA, LuchtenbergJ, WestphalAH, AkerJ, van HoekA, AlbrechtC, BorstJW, de VriesSC. 2013. Visualization of BRI1 and BAK1 (SERK3) membrane receptor heterooligomers during brassinosteroid signaling. Plant Physiology 162 , 1911–1925.23796795
Chang Z. 2022. Some random thoughts on the life of protein molecules in living cells. Biochemical and Biophysical Research Communications 633 , 33–38.36344157
Crook AD , WilloughbyAC, HazakO, et al . 2020. BAM1/2 receptor kinase signaling drives CLE peptide-mediated formative cell divisions in Arabidopsis roots. Proceedings of the National Academy of Sciences, USA 117 , 32750–32756.
Dettmer J , Hong-HermesdorfA, StierhofY-D, SchumacherK. 2006. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. The Plant Cell 18 , 715–730.16461582
DeYoung BJ , BickleKL, SchrageKJ, MuskettP, PatelK, ClarkSE. 2006. The CLAVATA1-related BAM1, BAM2 and BAM3 receptor kinase-like proteins are required for meristem function in Arabidopsis. The Plant Journal 45 , 1–16.16367950
Fan P , AguilarE, BradaiM, et al . 2021. The receptor-like kinases BAM1 and BAM2 are required for root xylem patterning. Proceedings of the National Academy of Sciences, USA 118 , e2022547118.
Fiers M , GolemiecE, van der SchorsR, van der GeestL, LiKW, StiekemaWJ, LiuC-M. 2006. The CLAVATA3/ESR motif of CLAVATA3 is functionally independent from the nonconserved flanking sequences. Plant Physiology 141 , 1284–1292.16751438
Fiers M , GolemiecE, XuJ, van der GeestL, HeidstraR, StiekemaW, LiuCM. 2005. The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway. The Plant Cell 17 , 2542–2553.16055633
French AR , LauffenburgerDA. 1997. Controlling receptor/ligand trafficking: effects of cellular and molecular properties on endosomal sorting. Annals of Biomedical Engineering 25 , 690–707.9236981
Furumizu C , KrabberødAK, HammerstadM, AllingRM, WildhagenM, SawaS, AalenRB. 2021. The sequenced genomes of nonflowering land plants reveal the innovative evolutionary history of peptide signaling. The Plant Cell 33 , 2915–2934.34240188
Geldner N , Dénervaud-TendonV, HymanDL, MayerU, StierhofYD, ChoryJ. 2009. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. The Plant Journal 59 , 169–178.19309456
Guo Y , HanL, HymesM, DenverR, ClarkSE. 2010. CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification. The Plant Journal 63 , 889–900.20626648
Gutierrez-Alanis D , Yong-VillalobosL, Jimenez-SandovalP, Alatorre-CobosF, Oropeza-AburtoA, Mora-MaciasJ, Sanchez-RodriguezF, Cruz-RamirezA, Herrera-EstrellaL. 2017. Phosphate starvation-dependent iron mobilization induces CLE14 expression to trigger root meristem differentiation through CLV2/PEPR2 signaling. Developmental Cell 41 , 555–570.e3.28586647
Hazak O , BrandtB, CattaneoP, SantiagoJ, Rodriguez-VillalonA, HothornM, HardtkeCS. 2017. Perception of root-active CLE peptides requires CORYNE function in the phloem vasculature. EMBO Reports 18 , 1367–1381.28607033
Hirakawa Y. 2022. Evolution of meristem zonation by CLE gene duplication in land plants. Nature Plants 8 , 735–740.35854003
Hirakawa Y , KondoY, FukudaH. 2011. Establishment and maintenance of vascular cell communities through local signaling. Current Opinion in Plant Biology 14 , 17–23.20934371
Irani NG , Di RubboS, MylleE, et al . 2012. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nature Chemical Biology 8 , 583–589.22561410
Ito Y , NakanomyoI, MotoseH, IwamotoK, SawaS, DohmaeN, FukudaH. 2006. Dodeca-CLE peptides as suppressors of plant stem cell differentiation. Science 313 , 842–845.16902140
Jelenska J , DavernSM, StandaertRF, MirzadehS, GreenbergJT. 2017. Flagellin peptide flg22 gains access to long-distance trafficking in Arabidopsis via its receptor, FLS2. Journal of Experimental Botany 68 , 1769–1783.28521013
Kastritis PL , BonvinAM. 2013. On the binding affinity of macromolecular interactions: daring to ask why proteins interact. Journal of the Royal Society Interface 10 , 20120835.23235262
Kim HJ , WuCY, YuHM, SheenJ, LeeH. 2017. Dual CLAVATA3 peptides in Arabidopsis shoot stem cell signaling. Journal of Plant Biology 60 , 506–512.30310351
Kinoshita A , NakamuraY, SasakiE, KyozukaJ, FukudaH, SawaS. 2007. Gain-of-function phenotypes of chemically synthetic CLAVATA3/ESR-related (CLE) peptides in Arabidopsis thaliana and Oryza sativa. Plant and Cell Physiology 48 , 1821–1825.17991631
Kneuttinger AC. 2022. A guide to designing photocontrol in proteins: methods, strategies and applications. Biological Chemistry 403 , 573–613.35355495
Kondo T , SawaS, KinoshitaA, MizunoS, KakimotoT, FukudaH, SakagamiY. 2006. A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis. Science 313 , 845–848.16902141
Konopka CA , BackuesSK, BednarekSY. 2008. Dynamics of Arabidopsis dynamin-related protein 1C and a clathrin light chain at the plasma membrane. The Plant Cell 20 , 1363–1380.18502847
Kusaka N , MaischJ, NickP, HayashiK, NozakiH. 2009. Manipulation of intracellular auxin in a single cell by light with esterase-resistant caged auxins. ChemBioChem 10 , 2195–2202.19637145
Lakadamyali M , RustMJ, ZhuangX. 2006. Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell 124 , 997–1009.16530046
Lee GJ , SohnEJ, LeeMH, HwangI. 2004. The Arabidopsis rab5 homologs rha1 and ara7 localize to the prevacuolar compartment. Plant and Cell Physiology 45 , 1211–1220.15509844
Li J , LiangP, GaoL, LuH, DongY, ZhangJ. 2023. o-Nitrobenzyl-based caged exo-16,17-dihydro-gibberellin A5-13-acetate for photocontrolled release of plant growth regulators. Journal of Agricultural and Food Chemistry 71 , 16533–16541.37878916
Li Y , WangM, WangF, LuS, ChenX. 2023. Recent progress in studies of photocages. Smart Molecules 1 , e20220003.
Li Z , ChakrabortyS, XuG. 2017. Differential CLE peptide perception by plant receptors implicated from structural and functional analyses of TDIF-TDR interactions. PLoS One 12 , e0175317.28384649
Mangubat-Medina AE , BallZT. 2021. Triggering biological processes: methods and applications of photocaged peptides and proteins. Chemical Society Reviews 50 , 10403–10421.34320043
Miwa H , BetsuyakuS, IwamotoK, KinoshitaA, FukudaH, SawaS. 2008. The receptor-like kinase SOL2 mediates CLE signaling in Arabidopsis. Plant and Cell Physiology 49 , 1752–1757.18854335
Müller R , BleckmannA, SimonR. 2008. The receptor kinase CORYNE of Arabidopsis transmits the stem cell-limiting signal CLAVATA3 independently of CLAVATA1. The Plant Cell 20 , 934–946.18381924
Narasimhan M , GalleiM, TanS, et al . 2021. Systematic analysis of specific and nonspecific auxin effects on endocytosis and trafficking. Plant Physiology 186 , 1122–1142.33734402
Narasimhan M , SimonR. 2022. Spatial range, temporal span, and promiscuity of CLE-RLK signaling. Frontiers in Plant Science 13 , 906087.36092449
Ni J , ClarkSE. 2006. Evidence for functional conservation, sufficiency, and proteolytic processing of the CLAVATA3 CLE domain. Plant Physiology 140 , 726–733.16407446
Nimchuk ZL. 2017. CLAVATA1 controls distinct signaling outputs that buffer shoot stem cell proliferation through a two-step transcriptional compensation loop. PLoS Genetics 13 , e1006681.28355208
Nimchuk ZL , TarrPT, MeyerowitzEM. 2011a. An evolutionarily conserved pseudokinase mediates stem cell production in plants. The Plant Cell 23 , 851–854.21398569
Nimchuk ZL , TarrPT, OhnoC, QuX, MeyerowitzEM. 2011b. Plant stem cell signaling involves ligand-dependent trafficking of the CLAVATA1 receptor kinase. Current Biology 21 , 345–352.21333538
Ogawa M , ShinoharaH, SakagamiY, MatsubayashiY. 2008. Arabidopsis CLV3 peptide directly binds CLV1 ectodomain. Science 319 , 294.18202283
Ortiz-Morea FA , SavatinDV, DejongheW, et al . 2016. Danger-associated peptide signaling in Arabidopsis requires clathrin. Proceedings of the National Academy of Sciences, USA 113 , 11028–11033.
Paez Valencia J , GoodmanK, OteguiMS. 2016. Endocytosis and endosomal trafficking in plants. Annual Review of Plant Biology 67 , 309–335.
Pallakies H , SimonR. 2014. The CLE40 and CRN/CLV2 signaling pathways antagonistically control root meristem growth in Arabidopsis. Molecular Plant 7 , 1619–1636.25178283
Qian P , SongW, YokooT, MinobeA, WangG, IshidaT, SawaS, ChaiJ, KakimotoT. 2018. The CLE9/10 secretory peptide regulates stomatal and vascular development through distinct receptors. Nature Plants 4 , 1071–1081.30518839
Replogle A , WangJ, BleckmannA, HusseyRS, BaumTJ, SawaS, DavisEL, WangX, SimonR, MitchumMG. 2011. Nematode CLE signaling in Arabidopsis requires CLAVATA2 and CORYNE. The Plant Journal 65 , 430–440.21265896
Rojo E , SharmaVK, KovalevaV, RaikhelNV, FletcherJC. 2002. CLV3 is localized to the extracellular space, where it activates the Arabidopsis CLAVATA stem cell signaling pathway. The Plant Cell 14 , 969–977.12034890
Roman AO , Jimenez-SandovalP, AugustinS, BroyartC, HothornLA, SantiagoJ. 2022. HSL1 and BAM1/2 impact epidermal cell development by sensing distinct signaling peptides. Nature Communications 13 , 876.
Schaper K , EtinskiM, FleigT. 2009. Theoretical investigation of the excited states of 2-nitrobenzyl and 4,5-methylendioxy-2-nitrobenzyl caging groups. Photochemistry and Photobiology 85 , 1075–1081.19508640
Schindelin J , Arganda-CarrerasI, FriseE, et al . 2012. Fiji: an open-source platform for biological-image analysis. Nature Methods 9 , 676–682.22743772
Schlegel J , DenayG, WinkR, PintoKG, StahlY, SchmidJ, BlümkeP, SimonRG. 2021. Control of Arabidopsis shoot stem cell homeostasis by two antagonistic CLE peptide signalling pathways. eLife 10 , e70934.34643181
Schoof H , LenhardM, HaeckerA, MayerKF, JürgensG, LauxT. 2000. The stem cell population of Arabidopsis shoot meristems in maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100 , 635–644.10761929
Sharma I , RussinovaE. 2018. Probing plant receptor kinase functions with labeled ligands. Plant and Cell Physiology 59 , 1520–1527.29726983
Shinohara H , MatsubayashiY. 2013. Chemical synthesis of Arabidopsis CLV3 glycopeptide reveals the impact of hydroxyproline arabinosylation on peptide conformation and activity. Plant and Cell Physiology 54 , 369–374.23256149
Shinohara H , MatsubayashiY. 2015. Reevaluation of the CLV3-receptor interaction in the shoot apical meristem: dissection of the CLV3 signaling pathway from a direct ligand-binding point of view. The Plant Journal 82 , 328–336.25754504
Shinohara H , MoriyamaY, OhyamaK, MatsubayashiY. 2012. Biochemical mapping of a ligand-binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR-RKs. The Plant Journal 70 , 845–854.22321211
Shiu SH , BleeckerAB. 2001. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proceedings of the National Academy of Sciences, USA 98 , 10763–10768.
Silva JM , SilvaE, ReisRL. 2019. Light-triggered release of photocaged therapeutics - Where are we now? Journal of Controlled Release 298 , 154–176.30742854
Solinger JA , SpangA. 2022. Sorting of cargo in the tubular endosomal network. Bioessays 44 , e2200158.36344475
Somssich M , MaQ, Weidtkamp-PetersS, StahlY, FelekyanS, BleckmannA, SeidelCA, SimonR. 2015. Real-time dynamics of peptide ligand-dependent receptor complex formation in planta. Science Signaling 8 , ra76.26243190
Song XF , YuDL, XuTT, RenSC, GuoP, LiuCM. 2012. Contributions of individual amino acid residues to the endogenous CLV3 function in shoot apical meristem maintenance in Arabidopsis. Molecular Plant 5 , 515–523.22259020
Sorkin A , von ZastrowM. 2009. Endocytosis and signalling: intertwining molecular networks. Nature Reviews Molecular Cell Biology 10 , 609–622.19696798
Stahl Y , WinkRH, IngramGC, SimonR. 2009. A signaling module controlling the stem cell niche in Arabidopsis root meristems. Current Biology 19 , 909–914.19398337
Strabala TJ , PhillipsL, WestM, StanbraL. 2014. Bioinformatic and phylogenetic analysis of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) and the CLE-LIKE signal peptide genes in the Pinophyta. BMC Plant Biology 14 , 47.24529101
Sun X , LiuL, ChengL. 2023. Photo- and tetrazine-responsive modulation of trans-zeatin. The Journal of Organic Chemistry 88 , 2921–2930.36763518
Takahashi F , SuzukiT, OsakabeY, BetsuyakuS, KondoY, DohmaeN, FukudaH, Yamaguchi-ShinozakiK, ShinozakiK. 2018. A small peptide modulates stomatal control via abscisic acid in long-distance signalling. Nature 556 , 235–238.29618812
Tamaki T , BetsuyakuS, FujiwaraM, FukaoY, FukudaH, SawaS. 2013. SUPPRESSOR OF LLP1 1-mediated C-terminal processing is critical for CLE19 peptide activity. The Plant Journal 76 , 970–981.24118638
Tung CH. 2004. Fluorescent peptide probes for in vivo diagnostic imaging. Biopolymers 76 , 391–403.15389488
Ueda T , YamaguchiM, UchimiyaH, NakanoA. 2001. Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. The EMBO Journal 20 , 4730–4741.11532937
Wang J , JiangQ, PleskotR, et al . 2023. TPLATE complex-dependent endocytosis attenuates CLAVATA1 signaling for shoot apical meristem maintenance. EMBO Reports 24 , e54709.37458257
Wellings DA , AthertonE. 1997. Standard Fmoc protocols. Methods in Enzymology 289 , 44–67.9353717
Wexler S , SchayekH, RajendarK, TalI, ShaniE, MerozY, DobrovetskyR, WeinstainR. 2019. Characterizing gibberellin flow in planta using photocaged gibberellins. Chemical Science 10 , 1500–1505.30809367
Whitewoods CD. 2021. Evolution of CLE peptide signalling. Seminars in Cell & Developmental Biology 109 , 12–19.32444290
Wojcik F , MoscaS, HartmannL. 2012. Solid-phase synthesis of asymmetrically branched sequence-defined poly/oligo (amidoamines). The Journal of Organic Chemistry 77 , 4226–4234.22486248
Xi L , WuXN, GilbertM, SchulzeWX. 2019. Classification and interactions of LRR receptors and Co-receptors within the Arabidopsis plasma membrane – an overview. Frontiers in Plant Science 10 , 472.31057579
Xu C , LiberatoreKL, MacAlisterCA, et al . 2015. A cascade of arabinosyltransferases controls shoot meristem size in tomato. Nature Genetics 47 , 784–792.26005869
Yadav RK , PeralesM, GruelJ, GirkeT, JonssonH, ReddyGV. 2011. WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex. Genes & Development 25 , 2025–2030.21979915
Yamaguchi YL , IshidaT, SawaS. 2016. CLE peptides and their signaling pathways in plant development. Journal of Experimental Botany 67 , 4813–4826.27229733
Yu G , TanY, HeX, QinY, LiangJ. 2014. CLAVATA3 dodecapeptide modified CdTe nanoparticles: a biocompatible quantum dot probe for in vivo labeling of plant stem cells. PLoS One 9 , e89241.24586624
Zhang H , HanZ, SongW, ChaiJ. 2016a. Structural insight into recognition of plant peptide hormones by receptors. Molecular Plant 9 , 1454–1463.27743937
Zhang H , LinX, HanZ, QuLJ, ChaiJ. 2016b. Crystal structure of PXY-TDIF complex reveals a conserved recognition mechanism among CLE peptide-receptor pairs. Cell Research 26 , 543–555.27055373
