Update on Peptide Signaling Pathways in Vascular Differentiation

Peptide Signaling Pathways in Vascular Differentiation[OPEN]

Hiroo Fukuda,a,1,2,3 and Christian S. Hardtkeb,1

aDepartment of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, 113-0033JapanbDepartment of Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne, Switzerland

ORCID ID: 0000-0003-3203-1058 (C.S.H.).

The modern plant vascular system is a central featureof extant angiosperms and consists of xylem, phloem,and the intervening procambium (Lucas et al., 2013).During vegetative growth, the apical meristem servesas a continuous source of procambial initial cells, whichproduce the primary xylem, the primary phloem, andthe procambium. In secondary growth, vascular cam-bium, which is derived from the procambium, acts as astem cell reservoir and continues to give rise to second-ary xylem and phloem cells. The formation of the vas-cular tissue is a well-organized plant developmentalprocess, whose central step is the regulation of vascularstem cell fates. It is governed by cell-to-cell communi-cation, and symplasticmovement of signalingmoleculescontributes to vascular development. Various secretedpeptides as well as plant hormones play crucial roles incell-to-cell communication for regulating vascular de-velopment (Fukuda and Ohashi-Ito, 2019). Among thepeptides, several members of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION, or CLE, familyact at different points of key processes in vascular de-velopment (Hazak and Hardtke, 2016; Fukuda andOhashi-Ito, 2019). The CLE family genes are conservedamong land plants. In the Arabidopsis (Arabidopsisthaliana) genome, there are 32 genes encoding 27 distinctCLE peptides (Yamaguchi et al., 2016). CLE precursorproteins contain an N-terminal signal peptide and atleast one conserved C-terminal 12–14 amino acid CLEdomain, from which a mature CLE peptide is producedthrough proteolytic cleavage. The activity of someprocessed peptides is increased by modificationssuch as hydroxylation on Pro residues, and in somecases by glycosylation with three arabinose residues(Ito et al., 2006; Ohyama et al., 2009; Matsubayashi,2011; Shinohara and Matsubayashi, 2013). Duringvascular development, CLE41/CLE44, CLE45, andCLE9/CLE10 peptides function in distinct processes.In addition, other peptides such as phytosulfokine and

EPIDERMAL PATTERNING FACTOR-LIKE (EPFL)peptides are thought to contribute to the regulation ofvascular development (Ikematsu et al., 2017; Holzwartet al., 2018). Crosstalk among peptides and betweenpeptides and plant hormones is an important currenttopic in vascular development. In this update,we focus onrecent advances in our understanding of the regulation ofvascular fate with an emphasis on peptide signaling.

THE TDIF–TDR/PXY SIGNALING PATHWAY INXYLEM DIFFERENTIATION

TRACHEARY ELEMENT DIFFERENTIATION IN-HIBITORY FACTOR (TDIF) is a CLE-family peptidecomposed of 12 amino acids including two hydroxylatedPro residues (Ito et al., 2006). TDIFwasfirst isolated fromzinnia (Zinnia elegans) xylogenic culture medium as afactor inhibiting tracheary element differentiation (Itoet al., 2006). The TDIF sequence is conserved in the

1These authors contributed equally to this article.2Senior author.3Author for contact: fukuda@bs.s.u-tokyo.ac.jH.F. and C.S.H. wrote the article.[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.01259

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genome of Euphyllophytes (Hirakawa and Bowman,2015) and encoded by the CLE41 and CLE44 genes inthe Arabidopsis genome (Hirakawa et al., 2008). The ac-tivity of TDIF in vascular development is also conservedamong extant Euphyllophytes (Hirakawa and Bowman,2015). TDIF RECEPTOR/PHLOEM INTERCALATEDWITH XYLEM (TDR/PXY) was demonstrated to be aTDIF receptor (Hirakawa et al., 2008). TDR/PXY belongsto Class XI LEU-RICH REPEAT RECEPTOR-LIKE KI-NASES, which consist of an extracellular LRR domain, asingle transmembrane domain for anchorage in theplasma membrane, and a cytoplasmic kinase domain(Fisher and Turner, 2007; Hirakawa et al., 2008). The ex-tracellular domain of TDR forms a twisted right-handedsuperhelical structure, comprising 22 LRRs and the Nterminus, and specifically recognizes TDIF by its innerconcave surface (Morita et al., 2016, Zhang et al., 2016a).The crystal structure of TDR ectodomains containsN-linked glycans (Zhang et al., 2016b). The PXY (TDR)family is composed of TDR (PXY), PXY-LIKE1 (PXL1),and PXL2 (Fig. 1A). PXL1 and PXL2 also participate invascular development together with TDR, because TDIFbinds the ligand binding pocket of both PXL1 and PXL2(Zhang et al., 2016b), and pxl1 and pxl2 mutations en-hance the vascular organization defects in tdr mutants(Etchells et al., 2013; Fisher and Turner, 2007). SOMATICEMBRYOGENESIS RECEPTOR KINASE (SERK) familyproteins (SERK1 and others) act as coreceptors of TDR(Fig. 1A; Zhang et al., 2016b). In the TDIF–TDR–SERK2complex, TDIFmediates interactions between the LRRs ofTDR and SERK2. Similarly, the CLE42 peptide, the closesthomolog of TDIF that also displays TDIF activity (Itoet al., 2006), promotes an interaction between PXL2 andSERK2 (Mou et al., 2017). Because SERK2 does notchange the basic structure of the TDIF–TDR complex,TDIF functions as a “glue” that joins TDR and a SERK,which may contribute to phosphorylation of a cyto-plasmic downstream factor (Zhang et al., 2016b).In plants, the TDIF–TDR signaling module acts by

inhibiting xylem differentiation from procambial cellsand promoting procambial cell proliferation, which re-sults in themaintenance of the procambial cell population(Hirakawa et al., 2008). WUSCHEL-RELATED HOME-OBOX4, which is a member of theWOX gene family, is adownstream factor of TDR and promotes procambial celldivisions (Fig. 1A; Hirakawa et al., 2010; Ji et al., 2010;Suer et al., 2011). Moreover, the SK1 (ARABIDOPSISTHALIANA SK11 [ATSK11], ATSK12, and ATSK13) andSK2 (BRASSINOSTEROID-INSENSITIVE2 [BIN2], BIN2-LIKE1 [BIL1], and BIL2) subgroups of GLYCOGENSYNTHASE KINASE3/SHAGGY-LIKE KINASE pro-teins (GSK3s) contribute redundantly to suppress xy-lem differentiation downstream of TDR (Fig. 1A).

PROMOTION OF PROCAMBIAL/CAMBIAL CELLPROLIFERATION BY TDIF–TDR

TDR is expressed preferentially in procambium andcambium (Fisher and Turner 2007; Hirakawa et al.,

2008), while CLE41 and CLE44 are expressed specifi-cally in phloem and more widely in its neighboring cellfiles, respectively (Fig. 1A; Hirakawa et al., 2008). De-fects in TDR or CLE41 cause the depletion of the pro-cambial cells (Fisher and Turner 2007; Hirakawa et al.,2008, 2010; Etchells and Turner 2010). Ectopic expres-sion of CLE41 under different promoters revealed thatexpression of CLE41 in or around procambial cells issufficient to drive vascular cell division, but localizedexpression of CLE41 in the phloem is required formaintaining properly orientated cell divisions (Etchellsand Turner 2010). Thus, phloem-synthesized TDIFregulates the orientation of procambial cell divisions ina non-cell–autonomous fashion.TDIF upregulates WOX4 expression in a TDR-

dependent manner (Hirakawa et al., 2010). Geneticanalyses showed thatWOX4 is required to promote theproliferation of procambial/cambial cells, but not forrepressing xylemdifferentiation in response to the TDIFsignal. The WOX14 transcription factor may also func-tion redundantly in Arabidopsis to modulate procam-bial cell proliferations (Etchells et al., 2013). Moreover,WOX14 may act in xylem fiber differentiation throughpromotion of gibberellin biosynthesis (Denis et al.,2017), consistent with the requirement of gibberellinfor xylem expansion (Ragni et al., 2011; Fig. 1A). Al-though WOX4 was shown to interact physically withthe HAIRY MERISTEM (HAM) family protein HAM4(Zhou et al., 2015), it is still unknown how WOX4 reg-ulates gene expression with HAM proteins.The cambium plays a central role in radial growth of

woody plants. In the poplar (Populus trichocarpa) ge-nome, orthologs of CLE41, TDR/PXY, and WOX4 areconserved (Etchells et al., 2015; Hirakawa and Bowman,2015; Kucukoglu et al., 2017), and PttWOX4a andPttWOX4b are specifically expressed in the cambial re-gion during vegetative growth, but not after growthcessation and during dormancy (Kucukoglu et al., 2017).A decrease in PttWOX4a/b levels achieved by RNA in-terference treatment caused severe reduction of thewidth of the vascular cambium and greatly diminishedsecondary growth, although primary growth was notaffected.Because there is no homologous sequence to Ara-

bidopsis WOX14 in poplar or spruce (Picea) genomes,WOX4 is expected to be a major player of cambialproliferation in woody plants. PtPP2::PttCLE41-PttANT::PttPXY double overexpression lines, in whichphloem-specific expression of PttCLE41 and cambium-specific expression of PXY are induced, exhibited highlyorganized vascular tissue, comparable to that of wild-type controls (Etchells et al., 2015). Because orthologsof CLE41, TDR/PXY, andWOX4 are widely distributedin angiosperm and gymnosperm tree species (Hirakawaand Bowman, 2015; Kucukoglu et al., 2017), the TDIF–TDR/PXY–WOX4 pathway is an evolutionarily con-served program for the regulation of the vascularcambium activity in wood formation. Recently twogroups identified bifacial, strongly proliferating cambialstem cells that feed both xylem and phloem production

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during secondary growth inArabidopsis (Shi et al., 2019;Smetana et al., 2019). In fact, in the cambial stem cells,TDR/PXY and WOX4 genes were actively expressed.Recently, Zhu et al. (2019) found that PtrCLE20 isexpressed in xylem cells and represses cambium ac-tivity in poplar. The molecular mechanism underlyingthe xylem-producing CLE peptide-dependent regula-tion of cambium activity should be analyzed further.

INTERACTION OF THE TDIF–TDR PATHWAYWITH PHYTOHORMONES

The role of auxin in the regulation of vascular cam-bium activity is well established and the auxin con-centration gradient peaks over the cambial region anddecreases toward the surrounding secondary tissues inhybrid aspen (Populus tremula 3 Populus tremuloidesMichx; Tuominen et al., 1997).WOX4 has been shown tobe necessary both for the auxin responsiveness of the

cambium cells and for the auxin-dependent increasein the cambium cell division activity (Suer et al.,2011). Local auxin signaling in TDR-positive stemcells stimulates cambium activity (Brackmann et al.,2018). An analysis with a highly sensitive auxin re-sponse marker revealed a moderate auxin responsein TDR-positive stem cells and a higher responsein xylem and phloem tissues. Whereas the auxin re-sponse factors (ARFs) ARF3 and ARF4 act redundantlyas general cambium activators, MONOPTEROS (MP)/ARF5 acts as an inactivator specifically in cambiumstem cells (Fig. 1A). Indeed, MP induction resulted inboth WOX4 repression and the induction of xylem-and phloem-related genes (Brackmann et al., 2018).Recently, Smetana et al. (2019) proposed that auxin-dependent WOX4 regulation occurs in xylem precursorcells with an auxin maximum, which promotes divi-sion of vascular stem cells in a non-cell–autonomousmanner. This idea should be verified from variousviewpoints.

Figure 1. Peptide-related signalingpathways that regulate proliferationof procambial/cambial cells and theirdifferentiation into xylem and phloemcells. A, Signaling pathways in hypo-cotyls and stems. B, Signaling pathwaysin the RAM. BR, brassinosteroid; CK,cytokinin; AHKs, cytokinin receptors.

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Periclinal and radial cell divisions in procambial cellsof the root apical meristem (RAM) are also regulated bytwo bHLH transcription factors, LONESOME HIGH-WAY (LHW) andTARGETOFMONOPTEROS5 (TMO5;Schlereth et al., 2010). In xylem precursor cells in theRAM, auxin induces TMO5 expression, which allowsthe formation of heterodimers between LHW andTMO5 (Fig. 1B). The heterodimers directly promotethe expression of target genes involved in the final stepof cytokinin biosynthesis, LONELYGUY3 (LOG3) andLOG4 (De Rybel et al., 2014; Ohashi-Ito et al., 2014).Therefore, cytokinin was hypothesized to act as amobile signal from the xylem to trigger divisions in theneighboring procambium cells. DOF2.1 and its closesthomologs, TMO6 and DOF6, may be key transcriptionfactors downstream of cytokinin in terms of pro-cambial cell proliferation (Miyashima et al., 2019;Smet et al., 2019). In xylem precursor cells, however,LHW–TMO5-induced HIS PHOSPHOTRANSFER PRO-TEIN6 represses cytokinin signaling to inhibit cell prolif-eration (De Rybel et al., 2014; Ohashi-Ito et al., 2014).

SUPPRESSION OF XYLEM DIFFERENTIATION FROMPROCAMBIAL CELLS

TDIF signaling releases BIN2 and BIL2, but notBIL1, from TDR, and then activates these GSK3s, whichresults in the suppression of xylem differentiation(Fig. 1A; Kondo et al., 2014). Conversely, the inhibitionof GSK3s with a chemical inhibitor (De Rybel et al.,2009), bikinin, induces xylem differentiation (Kondoet al., 2014, 2015). Bikinin-dependent ectopic xylemdifferentiation is suppressed in loss-of-function mutantsfor BRI1–EMS–SUPPRESSOR1 (BES1; Kondo et al.,2016, Saito et al., 2018), a direct substrate of GSK3s inthe brassinosteroid signaling pathway (Yin et al., 2002).By contrast, constitutively active BES1 (bes1-D) pro-motes xylem differentiation (Kondo et al., 2014). Theloss-of-function mutant of BRASSINAZOLE RESIS-TANT 1 (BZR1), the closest homolog of BES1, alsoshows a bes1-like phenotype, but a weaker one (Saitoet al., 2018). Thus, BES1 and BZR1,which are suppressedby GSK3s, act redundantly in promoting xylem differ-entiation (Fig. 1A). Further analysis indicated that theGSK3s–BES1/BZR1 signal not only regulates xylem butalso phloem differentiation (Kondo et al., 2016; Saitoet al., 2018). This finding is consistent with a previousreport showing that both bes1-D and bzr1-D partiallyrescued the phenotype of the octopus mutant, in whichroot protophloem differentiation is suppressed (Anneet al., 2015), and with a role of brassinosteroid signal-ing in phloem sieve element differentiation (Kang et al.,2017). Nevertheless, whether the canonical brassinoste-roid pathway effectors also have a role in root proto-phloemdevelopment remains unclear (Kang et al., 2017).Of SK1 and SK2 GSK3s, BIL1 may have a distinct role invascular development (Fig. 1A). Han et al. (2018) sug-gested that BIL1 phosphorylates and activates MP, andthe activatedMP promotes the expression of two A-type

Arabidopsis Response Regulators (ARRs), ARR7 andARR15, which suppress cambial cell divisions.The finding that bikinin induces ectopic xylem cell

differentiation led to the establishment of a useful tissueculture system for vascular cell differentiation namedvascular cell induction culture system using Arabi-dopsis leaves (VISUAL). In VISUAL, mesophyll cells incultured cotyledons or leaf disks differentiate at a highfrequency into tracheary elements and sieve ele-ments via the procambial stage in the presence ofbikinin (Kondo et al., 2015, 2016). This system enabledthe analysis of the roles of various genes in vasculardifferentiation by using their mutants as starting ma-terials of VISUAL.Brassinosteroid signaling may interact with the TDIF–

TDR–GSK3s–BES1/BZR1 signaling pathway, becauseGSK3s and BES1/BZR1 are among its key components(Fig. 1A). Indeed, brassinosteroid promotes xylemdifferentiation (Yamamoto et al., 1997; Caño-Delgadoet al., 2004; Kubo et al., 2005), which suggests thatbrassinosteroid regulates procambial cell fates to coun-teract TDIF signaling through the reverse regulation ofGSK3s. BRASSINOSTEROID INSENSITIVE1 (BRI1) isthe major receptor for brassinosteroid and the BRI1 geneis expressed widely in plants, while its homologs, BRI1-LIKE1 (BRL1) and BRL3, are expressed predominantly invascular tissues. In particular, BRL1 expression is asso-ciated with the procambial cells in inflorescence stems(Caño-Delgado et al., 2004). In procambial cells, there-fore, the TDIF and brassinosteroid signals may be bal-anced via GSK3s to determine procambial cell identity.Because brassinosteroid biosynthesis occurs in pro-cambial cells, which is promoted by auxin and cyto-kinin (Yamamoto et al., 2001, 2007), brassinosteroidmay act as an autocrine factor in xylem differentiationfrom procambial cells.

CLE9/CLE10 PERCEPTION IN XYLEM FORMATION

CLE9 and CLE10, which encode the same CLE pep-tide, are preferentially expressed in vascular cells ofroots (Fig. 1B; Kondo et al., 2011). cle9mutants and cle9cle10 double mutants exhibit a phenotype of increasedmetaxylem cell file numbers, which results from en-hanced periclinal cell division of xylem precursor cellsin the RAM (Qian et al., 2018). The receptor kinaseHAESA-LIKE1 (HSL1) is a CLE9/10 receptor that reg-ulates stomatal lineage cell division, while BARELYANY MERISTEM (BAM) class receptor kinases areCLE9/10 receptors that regulate periclinal cell divisionof xylem precursor cells (Fig. 1B; Qian et al., 2018). BothHSL1 and BAM1 bind to CLE9/10, but only HSL1 re-cruits SERKs as coreceptors in the presence of CLE9/10,suggesting different signaling modes for these receptorsystems (Hazak et al., 2017; Qian et al., 2018). Geneticanalysis indicated that BAM1, BAM2, and BAM3 areinvolved in the action of CLE9/10, but BAM1 is mostimportant among them. Their genes are expressed invascular cells, with BAM1 being expressed in xylem

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and phloem precursor cells in roots, consistent with thefunction of CLE9/10 in xylem precursor cells.

Application of CLE9/10 peptide suppressed proto-xylem vessel formation in Arabidopsis roots (Kondoet al., 2011). An early event caused by exogenousCLE9/10 peptide was the reduced expression of type-AARR genes such asARR5 andARR6, which are negativeregulators of cytokinin signaling (Fig. 1B). Consistently,an arr10 arr12 doublemutant, which is a combination ofmutations of B-type ARRs, showed CLE9/10-resistancein terms of protoxylem vessel suppression, and displayedectopic protoxylem vessel formation (Kondo et al., 2011).Therefore, CLE9/10 signaling may control xylem differ-entiation through a cytokinin signaling pathway. How-ever, because cle9 cle10 double mutants did not enhanceprotoxylem vessel differentiation, other CLE peptide(s)may also be involved redundantly in this process.

PHYTOSULFOKINE PEPTIDE PERCEPTION INPROCAMBIAL CELL FATE

Phytosulfokines (PSKs) are sulfated pentapeptidesthat are perceived by PHYTOSULFOKINE RECEPTOR-1 (PSKR1) and PSKR2 (Matsubayashi et al., 2002). PSKstabilizes the PSKR island domain for recruitment of aSERK (Wang et al., 2015). A recent study revealed asmall RECEPTOR-LIKE PROTEIN44 (RLP44)-mediatedinteraction between PSKRs and BRI1 that regulatesprocambial cell fate in roots (Fig. 1B; Holzwart et al.,2018). In the rlp44 loss-of-function mutant, super-numerary metaxylem-like cells are formed frequentlyoutside the primary xylem axis in the position ofthe procambium. This rlp44 phenotype can be res-cued by application of PSK peptide. Moreover, pskr1pskr2 double mutants displayed an rlp44-like xylemphenotype. Likewise, bri1 mutants show ectopic xy-lem in the procambial position, while brassinosteroid-deficient mutants do not exhibit an rlp44-like xylemphenotype. Therefore, PSK-PSKR signaling acts tomaintain procambial cell identity and RLP44 mediatesthe interaction between PSKR1 and BRI1. These re-sults may provide a model for the integration ofmultiple signaling pathways at the plasma membraneby shifting associations of receptors in multiproteincomplexes in response to different ligands (Holzwartet al., 2018). Because RLP44 and most PSK genes areexpressed preferentially in xylem/phloem and pro-cambium of roots, respectively, this signaling may oc-cur in a noncell autonomous fashion.

ERECTA AND RELATED RECEPTORS COOPERATEWITH TDR/PXY FAMILY RECEPTORS

The LEU-RICH REPEAT RECEPTOR-LIKE KINASEencoded by the ERECTA (ER) gene is part of a smallgene family together with ER-LIKE1 (ERL1) and ERL2.In er erl1 stems, intervening cambial cells are decreasedand phloem cells are frequently located adjacent to

xylem cells (Uchida and Tasaka, 2013). This also oc-curs in tdr (Fisher and Turner, 2007; Hirakawa et al.,2008), suggesting that ER and ERL1, like TDR, actby suppressing xylem differentiation and promotingcambial cell activity. By contrast, er erl1 hypocotylsdisplay a remarkable expansion of the xylem and anincrease in xylem fiber cells (Ragni et al., 2011; Ikematsuet al., 2017).

On the one hand, this result supports the idea that ERand ERL1 suppress xylem differentiation but appear tosuppress cambial activity. On the other hand, the rela-tive xylem expansion in the Landsberg er genotype wasfound to be due to early cessation of phloem formationand reduced cambial activity (Sankar et al., 2014). Thedifference between stems and hypocotyls in er erl1maybe explained by the tissue-specific gene regulation ofERL1 and ERL2: In the er pxy pxl1 pxyl2 genetic back-ground, ERL1 and ERL2 gene expression is reducedgreatly in stems, while their expression is strikinglyenhanced in hypocotyls (Wang, et al., 2019). Thesefindings clearly indicate a tight interaction betweenPXY family genes and ER family genes (Fig. 1A). In-deed, the sextuple mutant, er erl1 erl2 pxy pxl1 pxyl2,shows a complete suppression of secondary growth ofhypocotyls, supporting the view that both TDR and ERfamily genes are crucial factors of secondary growth(Wang, et al., 2019).

Both ER and ERL1 are expressed widely in the epi-dermis, phloem, and xylem (Uchida and Tasaka, 2013),but the vascular defect in er erl1 stems is rescued byphloem-specific activity of ER (Uchida and Tasaka,2013), consistent with a proposed role of ER in phloemproliferation (Sankar et al., 2014). Therefore, ER ERL1-dependent suppression of xylem precursor cell divisionshould occur in a non-cell–autonomousmanner (Fig. 1A;Tameshige et al., 2017). ER and ERL1 also function inpreventing xylem fiber differentiation, which is medi-ated by the NAC SECONDARY WALL THICKENINGPROMOTING FACTORs in hypocotyls (Ikematsu et al.,2017). Although BREVIPEDICELLUS is required for thehypocotyl to gain competency to respond to gibberellinand trigger fiber differentiation in both the wild-typeand er erl1, it is still unknown whether the ER/ERL1pathway is associated with the gibberellin pathway.

Ligands for ER and EFR1 that function in xylemdevelopment have not been identified. However, it isknown that EPIDERMAL PATTERNING FACTOR1(EPF1), EPF2, and EPFL9 peptides function as ligandsfor ER and ERL1 in stomata development (Han andTorii, 2016). In stem elongation, EPFL4 and EPFL6,which are produced in epidermal cells, act as ligandsfor phloem-located ER (Abrash et al., 2011; Uchidaet al., 2012). In hypocotyls, EPFL4 and EPFL6 areexpressed in xylem parenchyma cells and differenti-ating xylem cells (Wang et al., 2019). However, thereis presently no evidence that EPFL4 and EPFL6function as ligands for regulating hypocotyl second-ary growth. In the Arabidopsis genome, there are 11EPF/EPFL family members (Hara et al., 2009). It isplausible that some EPF/EPFL family members may

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function as ligands for ER and ERL1 to suppress xy-lem development in stems and hypocotyls. TOOMANY MOUTHS (TMM) and SERK family proteinsfunction as coreceptors in ER signaling (Han andTorii, 2016). Therefore, a specific combination of ER/ERL1, EPF/EPFL peptides, and coreceptors, such asTMM and SERK family proteins, may function in vas-cular development in stems and hypocotyls.

PHLOEM-RELATED CLE PEPTIDE SIGNALS

An interesting aspect emerging from the analysis ofpxy/tdr and related mutants is that they do not losetheir capacity to produce proper xylem and phloemtissues, as judged from morphological and molecular-genetic criteria (Fisher and Turner, 2007; Hirakawaet al., 2008). This even holds true for higher order (sex-tuple) mutants that largely remove redundancy (Wanget al., 2019). Thus, the main role of the PXY/TDR–CLE41/44/TDIF system is likely the organization ofvascular patterning, which is, for instance, evident in theregular spacing of phloem strands during hypocotylexpansion (Sankar et al., 2014).An easier, more accessible system to study a possibly

autonomous role of CLE peptide signaling in vasculardevelopment is the root tip with its simple diarch vas-cular organization. Here, two protophloem poles flanka central xylem axis and allow continuous observationof development along a spatio-temporal gradient. More-over, protophloemexpresses keyplayers ormarkers, suchas ALTERED PHLOEM DEVELOPMENT (APL; Bonkeet al., 2003) or COTYLEDON VASCULAR PATTERN2(CVP2; Carland and Nelson, 2004; Rodriguez-Villalonet al., 2015), which are also expressed in secondaryphloem development (Lehmann and Hardtke, 2016),indicating that the principal findings in one contextlikely apply to the other.

ROOT-ACTIVE CLE PEPTIDES ARE PERCEIVED INTHE PHLOEM

The root protophloem is also a system where a keyquestion can be answered: Is CLE peptide signalingrequired to form functional phloem? Early hints thatCLE peptides could play a role in root developmentcame from the observation that several CLE genes areexpressed in the root (Jun et al., 2010), and that treat-ments with many chemically synthesized CLE peptidessuppress root growth (Fiers et al., 2005; Ito et al., 2006;Kinoshita et al., 2007). Such so-called “root-active” CLEpeptides were also employed to identify componentsthat are necessary for CLE peptide perception in the root,and notably identified the RLP CLV2 and its inter-acting partner, the pseudokinase SUPPRESSOR OFOVEREXPRESSION OF LLP1-2/CORYNE (SOL2/CRN; Jeong et al., 1999; Miwa et al., 2008; Müller et al.,2008; Meng and Feldman, 2010).

Both clv2 and crn mutants display substantial resis-tance to root growth inhibition by a range of root-activeCLE peptides, and both the CLV2 and CRN genes areexpressed at low levels throughout the root (Hazaket al., 2017). However, recently it was shown that re-stricted expression of CRN in the developing proto-phloem is sufficient to fully recover CLE perception in acrn null mutant background (Hazak et al., 2017). Thismatched the observation that root-active CLE peptidesa priori suppress protophloem sieve element differen-tiation (Rodriguez-Villalon et al., 2014; Hazak et al.,2017), which is essential for root growth (Furuta et al.,2014; Rodriguez-Villalon et al., 2014). Given that inter-actions between CLE peptides and their cognate re-ceptors are rather specific (Hohmann et al., 2017), therequirement of the CLV2jCRN heteromer for percep-tion of root-active CLE peptides likely reflects a moregeneral rate-limiting role in receptor complex activity.For example, it has been suggested that CLV2jCRNstabilizes the expression of BAM3, the cognate CLE45receptor in the root (Kang and Hardtke, 2016; Hazaket al., 2017).

SUPPRESSION OF CLE PEPTIDE SIGNALINGPERMITS ROOT PROTOPHLOEM SIEVEELEMENT DIFFERENTIATION

CLE45 is one of the few CLE peptides that isexpressed in the developing protophloem sieve ele-ments, the others being the related CLE25 and CLE26peptides (Rodriguez-Villalon et al., 2014; Anne et al.,2018; Ren et al., 2019). Recently, it was suggested thatCLE25 is required for protophloem sieve element for-mation, although a cle25 mutant did not display a rootgrowth phenotype and only showed a marginal delayin sieve element elongation (Ren et al., 2019). While thismight reflect redundancy with other CLE peptides,expression of a supposedly dominant negative mutantversion of CLE25 (a G6T exchange in the peptide) abol-ished protophloem sieve element differentiation as wellas one of the formative divisions in the phloem lineage(Ren et al., 2019).Interestingly, such divisions can also be suppressed

by mild auxin antagonist treatment (Rodriguez-Villalonet al., 2014) and appear to be nonessential. However, itoften remains unclear which of the two successive peri-clinal formative divisions in the phloem lineage is af-fected, and it might verywell turn out that one of them isessential for sieve element formation. Nonetheless, theCLE25G6T phenotype strongly resembles the so-calledDisturbed Protophloem Syndrome observed in back-grounds with elevated CLE45 signaling (Rodriguez-Villalon et al., 2014; Anne and Hardtke, 2018) and isconsistent with simple dominant (rather than dominantnegative) action of the CLE25G6T version, as observed forother CLE peptides in other contexts (Czyzewicz et al.,2015), including CLE45G6T in protophloem formation(Rodriguez-Villalon et al., 2014; Czyzewicz et al., 2015).This interpretation is also consistentwith observations in

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pertinent receptor mutants. For example, neither crnloss-of-function mutants, nor CLE-RESISTANT RECEP-TOR KINASE (clerk) loss-of-function mutants, which areboth strongly resistant to CLE25, CLE26, and CLE45application, display any root growth or protophloemsieve element differentiation phenotypes (Hazak et al.,2017; Anne et al., 2018). The same applies to the crn clerkdouble mutant (Anne et al., 2018).

In summary, current data suggest that CLE peptidesignaling is not required per se for sieve element for-mation in the Arabidopsis root. On the contrary, itseems that suppression of autocrine CLE peptide sig-naling is a prerequisite for proper protophloem sieveelement differentiation (Breda et al., 2019). This doesnot, however, preclude a role in other aspects of rootdevelopment, for example formative divisions or radialorganization. For instance, CLE9 and CLE10 redun-dantly appear to affect formative xylem divisions in theroot, through their interaction with the BAM1 andBAM2 receptors (Qian et al., 2018). Such mechanismsmay also exist for phloem formation.

CLE PEPTIDE SIGNALING AS RAPID MEDIATORSOF STRESS-INDUCED SINK SHUTDOWN

Beyond purely developmental aspects, CLE peptidesmay also transmit environmental inputs, and these tworoles might not be mutually exclusive in the case ofindividual peptides. Indeed, CLE25 is a prime examplein this context. In addition to its expression in the pro-tophloem, CLE25 expression is induced by droughtstress and acts as a long-distance signal that is trans-ported through the vasculature to convey this physio-logical state to the shoot system (Takahashi et al., 2018).Coincidentally, such induction should also suppressprotophloem differentiation and thereby root growth(Hazak et al., 2017; Anne and Hardtke, 2018), whichcould be advantageous in drought conditions. Yet an-other example is the root-active CLE peptide, CLE14,which is induced upon phosphate starvation and trig-gers a breakdown of root meristem maintenance androot growth (Gutierrez-Alanis et al., 2017). Because thisresponse requires the CLV2jCRN module, it appearsthat it is achieved through a shutdown of protophloemformation (Meng and Feldman, 2010; Gutierrez-Alaniset al., 2017; Hazak et al., 2017).

In summary, beyond any positive developmentalroles in phloem development that largely remain tobe discovered, root-active CLE peptides might bemainly involved in the sensing of adverse environ-mental conditions. From this data, it seems that theirup-regulation in response to such adverse conditionssuppresses protophloem differentiation and therebyhalts root growth. From a physiological perspective,such a mechanismwould be a very efficient and rapidway to stop growth and eliminate a metabolic sinkthat finds itself in suboptimal conditions. Becausephloem transport is essentially auto-regulated, thiswould “automatically” redirect phloem sap to actively

growing roots. Thus, CLE peptide regulation mayprimarily serve to fine-tune and optimize root systemexploration of the wildly heterogenous soil matrix,which could represent a substantial adaptive advantage.

CONCLUDING REMARKS

In summary, peptide signaling pathways havebeen increasingly implicated in vascular develop-ment over recent years (Fig. 1 summarizes peptideand hormone signaling pathways discussed in thisupdate). From this data, it appears clear that variouspeptides and plant hormones not only function asligands in vascular cell proliferation, but also in bothxylem and phloem cell type fate regulation, and bothin a cell-autonomous or non-cell–autonomous man-ner. In addition, the emerging picture is becomingconsiderably complex because the exact impact of agiven signaling pathway seems to depend on thedevelopmental stage or the organ. For example, theTDIF–TDR signaling pathway apparently acts inthe cambium and actively proliferating procambiumof hypocotyls and stems, but not in the RAM. More-over, the picture is complicated by the observationthat at least in the RAM context, CLE45–BAM3 sig-naling has to be suppressed to permit phloem dif-ferentiation, although CLE peptide signaling mightbe required to initiate proper phloem formation.From the genetic analyses, it is also apparent that thecomplex network composed of multisignaling path-ways makes the regulation of procambial/cambialcell identity and proliferation robust. Similar redun-dancy might exist at the level of differentiation of

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vascular tissues, and future efforts could focus onuncovering such redundancy through genetic as wellas biochemical/cell biological investigations.Received October 9, 2019; accepted November 17, 2019; published December 3,2019.

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