REVIEW

Receptor-like protein kinase-mediated signalingin controlling root meristem homeostasis

Yafen Zhu1, Chong Hu1, Xiaoping Gou1&

1 Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, LanzhouUniversity, Lanzhou 730000, China

Received: 30 March 2020 / Accepted: 9 June 2020

Abstract Generation of the root greatly benefits higher plants living on land. Continuous root growth anddevelopment are achieved by the root apical meristem, which acts as a reservoir of stem cells. The stemcells, on the one hand, constantly renew themselves through cell division. On the other hand, theydifferentiate into functional cells to form diverse tissues of the root. The balance between the main-tenance and consumption of the root apical meristem is governed by cell-to-cell communications.Receptor-like protein kinases (RLKs), a group of signaling molecules localized on the cell surface, havebeen implicated in sensing multiple endogenous and environmental signals for plant development andstress adaptation. Over the past two decades, various RLKs and their ligands have been revealed toparticipate in regulating root meristem homeostasis. In this review, we focus on the recent studiesabout RLK-mediated signaling in regulating the maintenance and consumption of the root apicalmeristem.

Keywords Distal root meristem, Peptides, Proximal root meristem, Receptor-like protein kinases, Root apicalmeristem, Signal transduction

INTRODUCTION

Continuous new organ initiation and outgrowth inplants rely on meristems that act as reservoirs ofpluripotent stem cells. Like most of the plants, Ara-bidopsis (Arabidopsis thaliana) carries two primarymeristems, the shoot apical meristem (SAM) and theroot apical meristem (RAM), which are responsible forthe initiation of leaves, flowers, and the elongation ofthe main plant axes (Kitagawa and Jackson 2019).Besides the primary meristems, some plants also harbora secondary meristem, the vascular meristem, which isresponsible for the thickening of these axes (Fischeret al. 2019).

The RAM is a complex and dynamic structure thatharbors different cell types with divergent functions

(Fig. 1). In the core of the RAM, there is a distinct cen-tral region called the stem cell niche, which comprisesthe quiescent center (QC) and initial cells (stem cells).The quiescent cells are mitotically inactive, which inhi-bit differentiation of the surrounding cells and maintaintheir stem cell characteristics (van den Berg et al. 1997).Four types of initial cells surround the QC, named vas-cular initials, cortex/endodermal initials, epidermal/lateral root cap initials, and columella initials/columellastem cells (Sozzani and Iyer-Pascuzzi 2014). Through anasymmetric anticlinal cell division, the vascular initialsgive rise to the self-renewing stem cells and the sieveelement–procambium precursor cells. The sieve ele-ment–procambium precursor cell then gives rise to twocells by a periclinal division. The inner cell forms theprocambium, the outer cell is termed the sieve element–precursor cell. The sieve element–precursor cell even-tually divides periclinally once more to produce the

& Correspondence: gouxp@lzu.edu.cn (X. Gou)

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inner metaphloem and the outer protophloem (Ro-driguez-Villalon et al. 2014). The cortex/endodermalinitials anticlinally divide into the self-renewing stemcells and daughter cells, and the daughter cells switchtheir division planes to periclinally give rise to the innercortex and the outer endodermis. The epidermal/lateralroot cap initials and columella initials also go throughasymmetric cell division, giving rise to the self-renewingstem cells and daughter cells that finally form the epi-dermis and the root cap, respectively (Sozzani and Iyer-Pascuzzi 2014).

According to the location and function, the RAM canbe artificially classified into the proximal root meristemand the distal root meristem. The proximal root meris-tem locates above the QC, which divides continuously,and ultimately cells away from the stem cells will exitthe cell cycle and gradually differentiate to finally formthe mature area, also called the elongation/differentia-tion zone (Perilli et al. 2012). The root length is greatlyaffected by the proximal root meristem size. The distalroot meristem locates under the QC, where the col-umella stem cells differentiate into the columella cells

that can protect root tips and sense gravity (Stahl et al.2009) (Fig. 1).

These highly ordered cell arrangements and thecorresponding position signals regulating cell divisionand differentiation in the RAM suggest that cell–cellcommunication plays pivotal roles in maintaining rootmeristem homeostasis. Plant receptor-like proteinkinases (RLKs) located on the plasma membrane areoriginal sensors of numerous signals, such as phyto-hormones and small peptides, to mediate intercellularcommunications during plant growth, development, andresponse to stresses. A typical RLK comprises a uniqueextracellular domain which is thought to sense theexternal signal, a single transmembrane domain, and anintracellular kinase domain to transmit the signal intothe cytoplasm. In Arabidopsis, there are more than 610RLKs (Shiu and Bleecker 2001). A number of RLK-me-diated signaling pathways have been identified in reg-ulating plant growth, development and defenseresponse (Olsson et al. 2019). For instance, CLAVATA1(CLV1) associates with CLAVATA3 INSENSITIVERECEPTOR KINASES (CIKs) to perceive the CLV3 signal

Fig. 1 Schematic diagram ofthe Arabidopsis root meristemwith each cell typedifferentially colored. SCNstem cell niche, PM proximalroot meristem, DM distal rootmeristem

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in controlling SAM homeostasis (Clark et al. 1997;Fletcher et al. 1999; Hu et al. 2018); BRASSINOSTEROIDINSENSITIVE 1 (BRI1) recruits BRI1-ASSOCIATEDRECEPTOR KINASE 1 (BAK1) to sense brassinosteroidsignals to regulate diverse aspects of plant growth anddevelopment (Li and Chory 1997; Li et al. 2002);ERECTA and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES (SERKs) are required for perceivingEPIDERMAL PATTERNING FACTOR (EPF) family pep-tides to regulate stomata development (Shpak et al.2005; Meng et al. 2015; Lin et al. 2017); HAESA andSERKs act as the receptor complex of INFLORESCENCEDEFICIENT IN ABSCISSION (IDA) to regulate flowerorgan abscission (Jinn et al. 2000; Meng et al. 2016).

In the past years, several RLK-mediated signalingpathways have been revealed in maintaining RAMhomeostasis. For example, columella-originated CLV3/EMBRYO SURROUNDING REGION-RELATED 40 (CLE40)signal is perceived by ARABIDOPSIS CRINKLY4 (ACR4)to maintain the distal root meristem; ROOT MERISTEMGROWTH FACTOR (RGF) peptides are perceived by RGFRECEPTORS (RGFRs)/RGF1 INSENSITIVES (RGIs) tomaintain the proximal root meristem. Some of thesesignaling components act as conserved regulators inboth the RAM and SAM. In this review, we mainly focuson the advances of the RLK-mediated pathways incontrolling RAM homeostasis.

RLK-MEDIATED SIGNALING REGULATES THE DISTALROOT MERISTEM

Peptides and RLKs modulating meristem homeostasiswere first characterized in the SAM where the CLV-WUSCHEL (WUS) negative feedback loop plays anessential role (Brand et al. 2000; Schoof et al. 2000).CLV3 is a member of the CLE peptide family. The pre-propeptide of CLV3 is proteolytically cleaved into asmall secreted peptide with 12–14 amino acid residues(Cock and McCormick 2001; Ito et al. 2006). In Ara-bidopsis, there are 32 CLE members, and some of themare expressed in shoot and root apices (Jun et al. 2010).In the regulation of SAM homeostasis, the CLV3 signal istransmitted by several RLKs, including CLV1, BARELYANY MERISTEMS (BAMs), and RECEPTOR-LIKE PRO-TEIN KINASE 2 (RPK2), a receptor-like protein (RLP)CLV2 and a pseudokinase CORYNE (CRN) (Clark et al.1997; Kayes and Clark 1998; Muller et al. 2008;Kinoshita et al. 2010). Members of another group ofRLK, CIKs, function as coreceptors of these receptors tomediate CLV3 signaling, finally repressing the expres-sion of WUS, a critical homeodomain transcription fac-tor that is generated in the organizing center and moves

to the central zone of the SAM to promote stem cellactivity via a non-cell-autonomous way (Lenhard 2003;Yadav et al. 2011).

As an evolution-related structure of the SAM, theRAM also employs a similar CLE40-WUSCHEL-RELATEDHOMEOBOX5 (WOX5) signaling module to regulate thedistal root meristem (Fig. 2). CLE40 is the closesthomolog of CLV3 peptide, which is expressed in thecolumella cells. Loss of CLE40 signaling results inexpanded expression of WOX5, a homolog of WUS, anddelayed differentiation of the columella stem cells.Consistent with this, exogenous application of CLE40promotes differentiation of the columella stem cells,phenocopying that of the wox5 mutants (Stahl et al.2009). The wox5 mutants displayed enlarged QC cellswith abnormal shapes and differentiated columella stemcells, but no other abnormalities were observed in theproximal root meristem in comparison with the wildtype. Ubiquitous expression of WOX5 is sufficient toblock the differentiation of the columella stem celldaughters. Therefore, WOX5 is required for the main-tenance of the stem cell niche in the distal root meris-tem, similar to that of WUS in the SAM (Sarkar et al.2007).

As a secreted peptide, CLE40 is perceived by ACR4.The acr4 mutants, like the cle40 mutants, generateadditional columella stem cells and exhibit expandedexpression of WOX5 into the columella stem cells andvascular initials. Moreover, the acr4 mutants wereinsensitive to CLE40 treatment, and fewer columellastem cells can differentiate into the columella cellswhen compared with the wild type. CLV1, a key regu-lator in the SAM, is also involved in RAM regulation.

Fig. 2 RLK-mediated pathway regulates distal root meristemhomeostasis. CLE40 generated from the differentiated columellacells and diffused into the columella stem cells is recognized byACR4 and CLV1 to restrict the expression of WOX5 in thequiescent center. PP2A-3 is phosphorylated by ACR4 and can alsodephosphorylate ACR4 to facilitate the membrane localization ofACR4. Arrows indicate positive interactions; barred arrowsindicate repressive interactions

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CLV1 and ACR4 exhibit overlapping expression domainsin the distal root meristem and co-localize on plas-modesmata. The clv1 mutants generate more columellastem cell layers than the wild type, which resembles theacr4 mutants. Interestingly, the clv1 mutants were moresensitive to CLE40 treatment. Further studies revealedthat CLV1 and ACR4 can form heterodimers to mediatethe CLE40 signal. But at the same time, ACR4 itself canform homodimers to more efficiently transduce theCLE40 signal. When CLV1 is mutated, CLE40 canupregulate the expression of ACR4, and ACR4 can formmore homodimers to more effectively transduce theCLE40 signal, resulting in hypersensitivity to CLE40 inthe clv1 mutants (Stahl et al. 2013) (Fig. 2).

ACR4 can form homomeric complexes and also formheteromeric complexes with CLV1 on plasmodesmata inthe columella stem cells, leading to a hypothesis thatACR4-mediated signaling in the RAM restricts cell-to-cell movement of the QC-expressed WOX5 or anotherstemness factor (Stahl et al. 2013). The recent resultsdemonstrated that WOX5 protein is mainly localized inthe QC cells, which is not changed in the cle40, acr4, andclv1 mutants. In addition, WOX5 fused to a 2 9 GFP tagto prevent its movement can completely complementthe columella stem cell defects of wox5 mutants.Therefore, WOX5 is not the proposed mobile factor,which is different from that of WUS in the SAM. It ispossible that additional mobile factors, whose activitiesor expression can be regulated by CLE40-ACR4-WOX5signaling in the QC, act non-cell-autonomously to con-trol the differentiation of the columella stem cells(Berckmans et al. 2020).

Some other factors are also involved in the CLE40-ACR4-WOX5 pathway to control columella stem cell fate.PP2A, a phosphatase, has been uncovered to be a targetof and can be phosphorylated by the receptor ACR4. Onthe other hand, PP2A can also dephosphorylate ACR4,functioning as a positive regulator to facilitate themembrane localization of ACR4. Similar to acr4, thepp2a-3 mutants displayed increased columella stem celllayers. In addition, the pp2a-3 mutants did not act asadditive to the acr4 mutants, indicating that PP2A-3 andACR4 act in a common pathway to regulate columellastem cell differentiation (Yue et al. 2016) (Fig. 2).Besides CLV1, RPK2, another regulator in the SAM, alsofunctions redundantly with RPK1 to control root stemcell niche homeostasis (Racolta et al. 2018).

The CLE40-ACR4-WOX5 pathway in the distal rootmeristem is similar to the CLV3-CLV-WUS pathway inthe SAM. Many reports indicated that a ligand-bindingRLK usually requires a coreceptor RLK possessing arelatively short extracellular domain to mediate signaltransduction (Gou and Li 2020). Up to date, however, no

coreceptor RLK has been identified to function togetherwith ACR4 to regulate the root distal meristem. Previousstudies demonstrated that CIKs function as coreceptorsof CLV1 to regulate the homeostasis of the SAM (Huet al. 2018). It was also showed that CIKs are requiredto sense multiple CLE signals, and are involved in antherwall cell specification (Cui et al. 2018; Hu et al. 2018).Whether CIKs also act as conserved coreceptors of ACR4in the RAM is worthy of further investigation. Further-more, future work needs to reveal the downstreamsignaling components of the CLE40-ACR4-WOX5 path-way, such as the transcription factors that directly reg-ulate the expression of WOX5.

RLK-MEDIATED SIGNALING PROMOTESTHE DIFFERENTIATION OF THE PROXIMAL ROOTMERISTEM

During dissecting the CLV signaling pathway in the SAM,researchers found that the overexpression of CLV3,CLE40 or CLE19 as well as application of synthetic14-amino acid peptides containing the conserved CLEdomain resulted in a premature loss of the proximalroot meristem, which leads to a short-root phenotype(Casamitjana-Martinez et al. 2003; Hobe et al. 2003;Fiers et al. 2005). Besides CLV3, CLE40 and CLE19, allgroup A and B CLE peptides can consume the proximalroot meristem to inhibit root growth, resulting in asimilar short-root phenotype (Ito et al. 2006; Betsuyakuet al. 2011). To explore the components of CLE peptide-mediated signaling in roots, two suppressors in thebackground with overexpressed CLE19 were identifiedthrough genetic screening, which were designated sup-pressor of LIGAND-LIKE PROTEIN1 (LLP1) 1 and 2 (sol1and sol2) (Casamitjana-Martinez et al. 2003). SOL1encodes a putative Zn2?-carboxypeptidase involved inC-terminal processing of CLE19, which is essential forCLE19 activity (Tamaki et al. 2013). SOL2 encodes apseudokinase also named CRN, which is required forCLV3 signaling in the SAM and CLE signaling in theproximal root meristem (Miwa et al. 2008; Muller et al.2008). CLV2, an interacting cooperator of CRN in theSAM, is also involved in the perception of CLE peptidesin the root (Fiers et al. 2005). Loss function of RPK2,another regulator in the SAM, suppresses the short-rootphenotype caused by CLV3 application, indicating thatRPK2 is also required for CLE signal transduction in theroot (Kinoshita et al. 2010).

Despite CLE signaling that regulates the proximalroot meristem was identified many years ago, theunderlying mechanisms, for example, how CLEs affectroot length, remained to be elucidated. In the recent

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years, great progress has been made in uncovering CLEsignaling that suppresses protophloem differentiation toregulate the proximal root meristem (Anne and Hardtke2018). The COTYLEDON VASCULAR PATTERN 2 (CVP2)report line showing specific expression in the develop-ing sieve elements was used to monitor CLE peptideeffects in more detail. The results showed that CLEpeptide treatments specifically suppressed protophloemdifferentiation in 24 h before the proximal root meris-tem was greatly affected, indicating that the developingphloem is a crucial site of action for CLE peptides(Hazak et al. 2017). BREVIS RADIX (BRX) was identifiedas another critical regulator of protophloem develop-ment. The brx mutants displayed a short-root pheno-type, which was caused by discontinued protophloem(Mouchel et al. 2004). Using brx as the background,several genetic suppressors of protophloem develop-ment in the proximal root meristem were identified,including bam3, octopus (ops) and membrane-associatedkinase regulator5 (makr5) (Depuydt et al. 2013; Rodri-guez-Villalon et al. 2014; Kang and Hardtke 2016)(Fig. 3A).

BAM3 encodes an RLK, a closely related homolog ofCLV1. Loss function of BAM3 perfectly suppressed thedefects of perturbed protophloem development as wellas root growth in the brx mutants. In addition, the bam3mutants displayed specific resistance to the applicationof CLE45 peptide that can suppress protophloem dif-ferentiation and promote proximal root meristem con-sumption in the wild type. In the brx mutants, CLE45-mediated signaling was activated, thus leading to dis-continued protophloem and reduced proximal rootmeristem. Therefore, a paradigm that CLE45 is per-ceived by BAM3 to suppress protophloem differentia-tion in regulating the proximal root meristem has been

established (Hazak et al. 2017). As mentioned before,CLV2 and CRN are required for CLE peptide perception,which is probably due to that CLV2 and CRN interactwith BAM3 and enhance the expression of BAM3 in thedeveloping protophloem (Hazak et al. 2017). RPK2 isspecifically expressed in the phloem pole pericycle andcompanion cells, but not in the protophloem to perceiveCLE45 peptide, which restricts protophloem sieve ele-ment (PSE) identity to the PSE position (Gujas et al.2020) (Fig. 3A). In addition, RPK2 also forms hetero-meric complexes with BAM1, a homolog of BAM3, whichare involved in CLE peptide-dependent proximal rootmeristem shrinkage (Shimizu et al. 2015). It is possiblethat RPK2 also interacts with BAM3 or CLV2/CRN forsensing the CLE45 signal to suppress phloem identity.Most recently, it was reported that the bri1 brl1 brl3triple mutants display defective protophloem differen-tiation and are slightly hypersensitive to CLE45 treat-ment, which can be rescued by bam3 mutation orphloem-specific BRI1 expression, indicating that theCLE45-BAM3 signaling in regulating protophloem dif-ferentiation is also modulated by brassinosteroid per-ception (Kang et al. 2017; Graeff et al. 2020).

OPS encodes a polarly localized and plasma mem-brane-associated protein of unknown biochemicalfunction, which was found to be a key regulator ofprotophloem development. The ops mutants displayedperturbed protophloem differentiation and small prox-imal root meristem size, phenotypes resembling the brxmutants (Truernit et al. 2012). Enhancing OPS expres-sion level or increasing OPS activity can greatly rescuethe short root of brx mutants as well as the inhibitoryeffects of CLE45 treatment on root length (Rodriguez-Villalon et al. 2014). Further studies revealed that OPSdirectly disturbs the interaction between CLV2/CRN and

Fig. 3 RLK-mediatedpathways regulate proximalroot meristem homeostasis.A CLE signaling networkssuppress protophloemdifferentiation. B The RGFs-RGFRs/RGIs and PSK/PSY1-PSKRs/PSY1R pathwaysregulate the maintenance ofthe proximal root meristem.Arrows indicate positiveinteractions; barred arrowsindicate repressiveinteractions

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BAM3 to dampen the perception of CLE45 (Breda et al.2019) (Fig. 3A). Both OPS and BRX are plasma mem-brane-associated proteins, but they display shootwardand rootward polar localization, respectively. Despitethat the activated form of OPS acted as a suppressor ofbrx, and ops displayed a phenotype similar to brx, theops brx double mutants exhibited an additive phenotypeof ops and brx, indicating that OPS and BRX act in par-allel pathways to regulate protophloem differentiation(Breda et al. 2017).

MAKR5 works downstream of BAM3 and acts as apositive regulator of CLE45–BAM3 signaling, which isopposite to its prototypical homolog, BRI1 KINASEINHIBITOR 1 (BKI1), a negative regulator of brassinos-teroid signaling (Wang and Chory 2006). Loss of MAKR5did not show any apparent morphological phenotypesand partially suppressed brx phenotypes. The mark5mutants showed reduced sensitivity to low concentra-tion of CLE45. CLE45 application triggered a strongaccumulation of MAKR5 in developing protophloem andincreased plasma membrane association of MAKR5(Kang and Hardtke 2016) (Fig. 3A).

Recently, it was reported that the cik2 3 4 triplemutants showed resistance to all of those CLE peptidesthat can inhibit root growth, indicating that CIKs areessential for CLE peptide sensing in the proximal rootmeristem (Hu et al. 2018). By screening EMS-mutage-nized seeds and T-DNA insertion lines of leucine-richrepeat (LRR)-RLKs, mutants resistant to CLE25, orCLE26 treatment were obtained and CIK2/CLE RESIS-TANT KINASE (CLERK) was finally identified indepen-dently (Anne et al. 2018; Ren et al. 2019). CIK2/CLERKis mainly expressed in the protophloem and metaph-loem, and required for sensing root-active CLE peptides,including CLE25, CLE26, and CLE45. These CLE pep-tides are also expressed in the protophloem (Depuydtet al. 2013; Anne et al. 2018; Ren et al. 2019). Inter-estingly, unlike BAM3 and CLV2/CRN, loss of CIK2/CLERK cannot suppress the brx phenotype, however, canweakly rescue the ops phenotype (Anne et al. 2018). Inaddition, the extracellular domains of CIK2/CLERK andBAM3 did not interact with each other. These resultssuggested that CIK2/CLERK may act in parallel path-ways with BAM3 and CLV2/CRN in sensing CLE signalto suppress protophloem differentiation (Anne et al.2018). However, another report showed that CLERK caninteract with CLV2 in vivo, and clerk was able to par-tially rescue the ops phenotype, which suggested thatCIK2/CLERK and CLV2 form a complex, and act in theOPS pathway (Ren et al. 2019).

CLE signaling in controlling protophloem differenti-ation and proximal root meristem maintenance iscomplex. Many receptors, including BAM3, CLV2/CRN,

RPK2, and CIK2/CLERK, are involved in the perceptionof root-active CLE peptides (Depuydt et al. 2013; Hazaket al. 2017; Anne et al. 2018; Ren et al. 2019; Gujas et al2020) (Fig. 3A). However, these receptor-mediated sig-naling pathways are far from well-characterized. Forinstance, how these RLK-mediated signals are trans-duced into the nucleus is largely unknown. Previousstudies indicated that CLE25, CLE26, and CLE45 wereexpressed in the protophloem to regulate its develop-ment (Depuydt et al. 2013; Anne et al. 2018; Ren et al.2019). In addition, the bam3 mutants were still sensi-tive to exogenous application of CLE25 and CLE26(Depuydt et al. 2013). These results suggest that otherreceptors, probably the homologs of BAM3, may berequired to perceive these CLE peptides in regulatingprotophloem development.

Moreover, whether these receptors act in the samepathway or in parallel pathways is still controversial.From the perspective of expression pattern, BAM3 andCIK2/CLERK are specifically expressed in the developingprotophloem. Both the bam3 and cik2/clerk mutantsshowed resistance to CLE45, but bam3 could greatlyrescue the short-root phenotype of brx and ops whilecik2/clerk could not. Hence, it was considered thatCIK2/CLERK acts in a parallel pathway with BAM3 toregulate protophloem differentiation and root growth(Anne et al. 2018). However, it is worthy of noting thatCIK2/CLERK belongs to the CIK subfamily which con-tains six members and these CIKs showed high redun-dancy in limiting SAM size by acting as coreceptors ofCLV1 (Hu et al. 2018). Whether high-order cik mutantscan suppress the short-root phenotype of brx and opsremains to be elucidated. Previous results showed thatCLV2/CRN, RPK2, and CIK2/CLERK cannot directly bindCLE peptides alone (Shinohara and Matsubayashi 2015;Anne et al. 2018). On the other hand, interactions ofCLV2/CRN–BAM3, RPK2–BAM1 and CIK2/CLERK–CRN/RPK2 were previously revealed (Shimizu et al. 2015;Hazak et al. 2017; Hu et al. 2018). It is therefore rea-sonable to propose that all these receptors can form acomplex in sensing CLE signals to suppress pro-tophloem differentiation and root growth.

Although the roles of many root-active CLE peptideswere thought to be involved in root meristem regula-tion, genetic analysis of high-order mutants of these CLEgenes is still strongly demanding due to their small genesize and extremely high redundancy. CRISPR/Cas9-me-diated gene editing is a powerful approach to overcomethese limits and was successfully utilized to generate allcle single mutants (Yamaguchi et al. 2017). However, nodefects of the protophloem and root meristem werereported in these single mutants. Since CLE25, CLE26,and CLE45 are expressed in the developing protophloem

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(Depuydt et al. 2013; Anne et al. 2018; Ren et al. 2019),generation of high-order cle mutants may take thechance to clarify this problem. Intriguingly, the CLEpeptide–resistance phenotype of both the bam3 and crnmutants can be rescued by protophloem-specificexpression of BAM3 and CRN (Hazak et al. 2017), raisinga hypothesis that differentiated protophloem may pro-duce stemness factors to maintain the proximal rootmeristem non-cell-autonomously. Figuring out thisquestion will be a challenge to understand the molec-ular mechanisms of CLE signaling in regulating theproximal root meristem.

RLK-MEDIATED SIGNALING REGULATESTHE MAINTENANCE OF THE PROXIMAL ROOTMERISTEM

Phytosulfokine (PSK) is a secreted 5-amino acid tyr-osine-sulfated peptide that was initially identified as aproliferation promoting factor in low-density plant cellsuspension culture (Matsubayashi and Sakagami 1996).In Arabidopsis, there are five PSK genes that encode thesame conserved pentapeptide. Application of PSK pro-moted root growth by regulating the proximal rootmeristem size and cell expansion in the elongation zone(Kutschmar et al. 2009). PSK was recognized by thereceptors PSKR1 and PSKR2 (Matsubayashi et al. 2002;Amano et al. 2007). Crystal structures revealed that twosulfate moieties of PSK interact directly with PSKR,which stabilizes the island domain of PSKR for therecruitment of the coreceptor SERKs. In line with this,both the pskr1 single and serk1/? 2 3 triple mutants hadshortened roots and were much less sensitive to PSKthan the wildtype plants (Wang et al. 2015). PSY1, an18-amino acid tyrosine-sulfated glycopeptide, wasidentified in Arabidopsis cell suspension culture. Similarto PSK, PSY1 significantly promoted cellular prolifera-tion and expansion. PSY1 was perceived by PSKR1,PSKR2 and PSY1R. The pskr1 pskr2 psy1r triple mutantshad reduced RAM size and root length (Amano et al.2007). A recent study showed that PSY1R and SERKsinteracted with and transphosphorylated each other,suggesting that SERKs may act as the coreceptor ofPSY1R, but more genetic and structural evidence isneeded (Oehlenschlæger et al. 2017) (Fig. 3B).

Both PSK and PSY1 are tyrosine-sulfated peptides,suggesting that tyrosine sulfation modification is criticalfor plant development. An Arabidopsis tyrosylproteinsulfotransferase (TPST) was purified from microsomalfractions that can specifically catalyze tyrosine sulfationof PSY1 precursor peptide (Komori et al. 2009). Anothergroup identified AQC1 that encodes the same TPST

independently by utilizing combinational approaches ofmap-based cloning and thermal asymmetric interlaced-PCR (TAIL-PCR) (Zhou et al. 2010). The expression ofTPST was positively regulated by auxin. The tpstmutants displayed defective maintenance of the rootstem cell niche, decreased proximal root meristem, andstunted root growth due to impaired auxin-inducedexpression of transcription factor genes PLETHORAS(PLTs) (Komori et al. 2009; Zhou et al. 2010). PLTsencode AP2 class transcription factors, which areessential for QC specification and stem cell activity in adosage-dependent manner. High levels of PLT activityare required for stem cell identity; lower levels promotecell division in the meristematic zone; and furtherreduced levels allow cells to differentiate (Aida et al.2004; Galinha et al. 2007). The plt1 2 double mutantsexhibited abnormal QC identity, extremely reducedproximal root meristem size and root growth as well asdefective distal cell division patterns (Aida et al. 2004)(Fig. 3B).

As the tpst mutants displayed severely reducedproximal root meristem size and short roots, whichwere not rescued by exogenously applied PSK and PSY1peptides, indicating that there existed undiscoveredtyrosine-sulfated peptide(s) regulating root meristemactivity and maintenance of the stem cell niche in Ara-bidopsis (Matsuzaki et al. 2010). Through bioinformaticsanalysis, members of a candidate polypeptide familycontaining tyrosine–sulfation motifs were identified,which were later named ROOT MERISTEM GROWTHFACTORS (RGFs) (Matsuzaki et al. 2010). RGF1 isexpressed in the QC and columella stem cells; RGF2 andRGF3 show stronger activity and are expressed in thecentral columella cells. However, RGF1, RGF2, and RGF3proteins diffuse into the meristematic region. Overex-pression of RGF1 or application of synthesized sulfatedform of RGF1 significantly increased the proximal rootmeristem size of the wildtype plants. Synthesized sul-fated RGF1 also greatly restored the defects of theproximal root meristem of the tpst mutants as well asthe expression level of PLTs, suggesting that TPST cat-alyzes the sulfation of RGFs to maintain the root stemcell niche. The RGF family contains nine members. Thergf1 2 3 mutants showed a short-root phenotype char-acterized by reduced proximal root meristem size aswell as decreased expression level of PLTs. Thus, a TPST-RGFs-PLTs pathway regulating root meristem activitywas revealed (Fig. 3B). In the tpst mutants, both PLT1-GFP and PLT2-GFP signals were dramatically reduced inthe meristematic zone, but quantitative RT-PCR andin situ hybridization analyses revealed that the tran-scription level of PLT2 was decreased and the expres-sion of PLT1 is comparable to that of the wild type,

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suggesting that PLT abundance is regulated at bothtranscriptional and posttranscriptional levels. RGF1treatment expanded the expression domain of PLT2protein, but the accumulated PLT2 transcripts were stillrestricted in the stem cells, suggesting that RGF signal-ing may stabilize PLT proteins by posttranscriptionalregulation (Matsuzaki et al. 2010; Zhou et al. 2010).Recently, the posttranslational regulation of PLTs by RGFsignaling has been proved. Transcriptomic analysesidentified a downstream transcription factor, RGF1-INDUCIBLE TRANSCRIPTION FACTOR 1 (RITF1), whichwas upregulated by RGF1 treatment. RGF1 treatmentresulted in enhanced O2- accumulation in the meris-tematic zone, thus enhancing the stability of PLT2 pro-tein to increase the proximal root meristem size.Consistent with this, the ritf1 mutants displayedreduced proximal root meristem size and RGF1-inducedO2- accumulation, suggesting that RITF1 has a crucialrole in mediating RGF1 signaling (Yamada et al. 2020)(Fig. 3B).

The receptors of RGFs, which were named RGFRECEPTORS (RGFRs)/RGF1 INSENSITIVES (RGIs), wereindependently identified by three groups using pho-toaffinity labeling, reverse genetics and structural biol-ogy, respectively (Ou et al. 2016; Shinohara et al. 2016;Song et al. 2016). The rgi quintuple mutant generated ashort root with a small proximal root meristem size andwas insensitive to RGF1 treatment, which was similar tothe tpst and plt1 2 mutants (Ou et al. 2016). Expressionof PLT1 and PLT2 was dramatically reduced in the rgi/rgfr mutants and ectopic expression of PLT2 driven byan RGI2 promoter can greatly rescue the root meristemdefects of the rgi mutants (Ou et al. 2016; Shinoharaet al. 2016). Structural evidence showed that the sulfategroup of RGF1 was specifically recognized by the Arg-x-Gly-Gly (RxGG) motif of RGFRs/RGIs, and the binding ofRGF1 to RGFRs induced heterodimerization betweenRGFRs and SERKs. Consistent with this, the serk multi-ple mutants exhibited smaller proximal root meristem,shorter roots and reduced sensitivity to RGF1 treat-ment. These results indicate that SERKs act as core-ceptors of RGFRs/RGIs to mediate RGF signaling inregulating root meristem homeostasis (Song et al.2016). In addition, RGFs can rapidly induce phospho-rylation and ubiquitination of RGFRs/RGIs, thus tofinally turnover RGF signaling after it is activated (Ouet al. 2016). This process can be reversed by ubiquitin-specific proteases UBP12 and UBP13 that de-ubiquiti-nate RGFRs/RGIs to maintain their stability. The ubp1213 double mutants displayed a short-root phenotypewith reduced root meristem cell numbers, and werecompletely insensitive to exogenously applied RGF1 (Anet al. 2018) (Fig. 3B).

Although great progress of the RGF-RGFR/RGI-SERK-PLT pathway has been achieved, the downstream sig-naling components remain to be elucidated. The mito-gen-activated protein kinase (MAPK) cascade has beenfound to act downstream of SERKs in several biologicalprocesses including stomata patterning, plant immuneresponse, floral organ abscission and embryo develop-ment (Meng et al. 2015, 2016; Ma et al. 2016; Li et al.2019). Whether a MAPK cascade is also involved in RGF-mediated pathway needs to be deciphered in futurestudies. Furthermore, the previous data showed thatPLTs were transcriptionally regulated by RGF signaling(Matsuzaki et al. 2010; Ou et al. 2016). However, atranscription factor post-translationally modified byRGF signaling through a possible MAPK cascade todirectly regulate the expression of PLTs has not yet beenidentified.

RLK-MEDIATED SIGNALING REGULATES ROOTRESPONSES TO ENVIRONMENTAL FACTORS

Root development is coordinated by developmental andenvironmental cues, which relies on cell-to-cell com-munications mediated by peptides and receptors. CLE14was shown to act as a major effector in the Pi starvationresponse. Pi starvation up-regulated the expression ofseveral CLE members, especially CLE14, which in turnled to meristem exhaustion and a short root (Gutierrez-Alanis et al. 2017). CLV2 and CRN were reported tofunction in response to CLE-triggered meristem con-sumption (Fiers et al. 2005). Interestingly, both clv2 andclv2 sol2 showed reduced proximal root meristem androot length during Pi starvation, suggesting other oradditional receptors are required for Pi starvation-in-duced CLE perception. Indeed, PEP1 RECEPTOR 2(PEPR2), a receptor of PEP1 regulating immuneresponse (Yamaguchi et al. 2010), is involved in Pistarvation-triggered RAM differentiation (Gutierrez-Alanis et al. 2017). The clv2 pepr2 seedlings showed along root with a well-organized RAM under Pi starvationconditions. Plants with down-regulated expression ofCLE14 by artificial microRNA did not show any differ-ence with regard to RAM differentiation compared withthe wild type in Pi starvation, suggesting that generedundancy or genetic compensatory effect may exist inCLE gene family. Generation of higher-order cle mutantswill further elucidate CLE signaling in response to Pistarvation.

CLE peptides are also responsive to nitrogen(N) starvation. CLE1, CLE3, CLE4, and CLE7 wereinduced by N deficiency, which in turn repressed lateralroot primordium emergence and outgrowth in a CLV1-

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dependent manner. Overexpression of these CLEs orapplication of these synthetic CLEs mimicked the Ndeficiency-inhibited lateral root development pheno-type. However, the cle mutants displaying normal lateralroot development under low N conditions are notavailable for further investigation (Araya et al. 2014).Members of another peptide family, the C-TERMINALLYENCODED PEPTIDES (CEPs), were also induced in Ndeficiency (Tabata et al. 2014). CEPs were first identi-fied by using an in silico approach combined with liquidchromatography/mass spectrometry. Overexpressed orexogenously applied CEP1 significantly inhibited rootgrowth with a small proximal root meristem size as wellas reduced lateral root elongation (Ohyama et al. 2008).Plants grown in the medium with unevenly distributedN induced the expression of CEPs in the root on the Ndeficiency side. These N deficiency-induced CEPs in theroot were translocated to the shoot where they werethen recognized by CEP RECEPTOR 1 (CEPR1) andCEPR2, two LRR type RLKs, to induce the expression ofnon-secreted peptides, including CEP DOWNSTREAM1(CEPD1), CEPD2, and CEPD-LIKE 2 (CEPDL2). Theseshoot-derived CEPD1, CEPD2, and CEPDL2 act as thelong-distance signals transported to the root to upreg-ulate the expression of high-affinity nitrate transportergenes; thereby, controlling primary root elongation andlateral root growth (Tabata et al. 2014; Ohkubo et al.2017; Ota et al. 2020). However, the receptors in theroot to sense CEPD1, CEPD2 and CEPDL2 are stillunknown.

In addition to nutrient deficiency, peptide–receptormodules are also involved in response to dehydrationstress. The expression of CLE25 was significantlyupregulated in the root under dehydration stress.However, the root-derived CLE25 peptide was translo-cated to the leaves, where it was recognized by BAMs toenhance abscisic acid biosynthesis, thus inducingstomatal closure and thereby resistance to dehydrationstress (Takahashi et al. 2018).

CONCLUDING REMARKS AND FUTUREPERSPECTIVES

Despite the extensive and growing knowledge on theunderstanding of RLK-mediated signaling in regulatingroot meristem homeostasis, the components of thesepathways are largely unknown, especially those trans-ducing signals in the cytoplasm. Recent advances insingle-cell RNA sequencing technology provideunprecedented opportunities to identify these unknowncomponents that direct stem cell differentiation at thesingle-cell level (Zhang et al. 2019). Combination with

cell-type specific marker-based cell sorting technologymay help us to uncover more and more factors in theroot meristem regulation network (Li et al. 2016).Recently, a method mapping proteome-wide targets ofprotein kinases has been reported for identifying puta-tive substrates for a given kinase, which thus could be avaluable strategy for further studies on the mechanismsof RLK-mediated signaling that usually works throughphosphorylation (Wang et al. 2020).

The SAM and RAM are responsible for the morpho-genesis of aboveground and underground tissues ofplants, respectively. In the process of crop domestica-tion, many SAM mutants that remodel plant architec-tures, thereby increasing fruit size and seed numbers,have been selected (Xu et al. 2015; Je et al. 2016;Kitagawa and Jackson 2019). Root system is responsivefor the uptake of nutrients and water in plants. There isevidence to show that root meristem mutants, applica-tion of some peptides or environmental signals cansignificantly remodel plant root architectures, thusaffecting water and nutrient use efficiency (Araya et al.2014; Czyzewicz et al. 2015; Gutierrez-Alanis et al.2017). However, RAM mutants possibly improvingunderground architectures have not been well noticed.Understanding ligand-RLK receptor-mediated signaltransduction processes in regulating the root meristemat a quantitative and predictive level with the help ofmodern gene editing approaches may enable efficientcontrol of crop production for sustainable agriculture.

Acknowledgements This work was supported by National Nat-ural Science Foundation of China (31770312 and 31970339), andFundamental Research Funds for the Central Universities (lzu-jbky-2019-ct04 and lzujbky-2020-kb05).

Compliance with ethical standards

Conflict of interest The authors state that there is no conflict ofinterest.

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