WOX Going On: CLE Peptides in Plant Development

Abstract

The development of plant tissues requires cell-cell communication facilitated by chemical and peptide hormones, including small signaling peptides in the CLAVATA3/ EMBRYO SURROUND REGION (CLE) family. The paradigmatic CLE signaling peptide CLAVATA3 regulates the size of the shoot apical meristem and the expression of the stem cell-promoting WUSCHEL transcription factor through an unknown mechanism. This review discusses recent advances in CLE signaling showing that CLE pathways are conserved in bryophytes, CLE peptides in Arabidopsis thaliana regulate stem cell identity and cell division in root tissues, and connections to auxin biosynthesis in regulating flower and leaf development. These advances shed light on potential WUSCHEL-family independent aspects of CLE signaling and the overlap between CLE and auxin signaling.

Introduction

The development of complex plant tissues requires extensive cell-cell communication networks driven by a diversity of ligands including hormones, gasses, and genetically encoded peptides. In Arabidopsis thaliana there are 32 genes in the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family of signaling peptides, representing one of the largest families of peptide ligands [1, 2]. Variable numbers of CLEs are found in all species studied and have conserved and divergent roles in regulating development. CLE peptides, like the founding member CLAVATA3 (CLV3p) in A. thaliana, are processed from larger propeptides into a 12–13 amino acid active form which can be post-translationally modified through hydroxyprolination and arabinosylation [3,4]. Apoplastic CLE peptides (CLEp) primarily bind to and signal through leucine-rich repeat receptor-like kinases (LRR-RLK) from the CLAVATA1 (CLV1) and BARELY ANY MERISTEM (BAM) subclade, of which there are variable members across species. CLE peptides from the TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) subfamily signal via a trio of CLV-related TDIF receptors (TDR, also known as PHLOEM INTERCALATED WITH XYLEM (PXY)) [5,6]. Other LRR-containing receptors such as RECEPTOR PROTEIN KINASE2 (RPK2) and CLAVATA2 (CLV2) do not appear to directly bind CLV3p but have important roles in CLE signaling [3, 7]. These diverse primary receptors require CLAVATA3 INSENSITIVE RECEPTOR KINASEs (CIK) family co-receptors for function and signaling [7–11]. The signaling pathways downstream of CLV/BAM receptors remain unclear, but studies have suggested roles for MAP kinase and G-protein modules as potential downstream effectors, depending on the plant species [12, 13].

CLV3 signaling is required for the organization of the shoot apical meristem (SAM). SAMs in clv3 mutants become disorganized with the disruption of anticlinal stem cell division planes, and enlarged with the expansion of the expression domain of WUSCHEL (WUS), a homeobox transcription factor that promotes CLV3 expression and stem cell maintenance [14–16]. WUS binds to the promoter of CLV3, and along with other transcription factors and hormones, forms a negative feedback loop that maintains stem cell homeostasis [17–21]. Additionally, complex and species-specific transcriptional cross regulation of CLE and/or CLV/BAM genes contributes to meristem stem cell homeostasis by modulating CLE signaling strength [22, 23]. The CLV3-WUS paradigm remains foundational to our current understanding of CLE signaling in plants. Indeed, CLE signaling intersects with WUS ortholog WUSCHEL-RELATED HOMEOBOX (WOX) function in other tissues. TDIF signaling regulates division plane specification in the meristematic vascular cambium and promotes WOX4/14 expression [5, 6]. CLE40 regulates the division and differentiation of root apical meristem (RAM) stem cells in opposition to WOX5 [24]. Despite this, recent work exploring the evolutionary foundations of CLE signaling, CLE pathway functions in root development, and new aspects of CLE signaling in the SAM suggest that CLEs play broader roles in development, and in some cases independent of WOX genes.

The Roots of CLE Signaling-

CLE signaling pathways regulating cell proliferation and stem cell organization are conserved in the two studied bryophyte lineages, suggesting that CLE signaling was present in the last common ancestor of all land plants and evolved prior to and independently of WUS gene function [25–27]. CLE signaling through CLV1 receptors in the moss Physcomitrella patens is required for cell plane orientation of specific cell divisions that organize three-dimensional apical growth [25]. Loss of function mutations in the liverwort Marchantia polymorpha CLE (MpCLE1) cause misoriented divisions in the apical meristem cell division plane and overexpression of MpCLE1 shortens the proliferative region of the apical notch (analogous to a SAM) [26]. Conversely treatments with MpCLE2p causes an accumulation of apical stem cells that disrupt the dichotomous branching pattern of this liverwort. Instead, “multichotomous” branching occurs from the additional apical stem cells, with more than two growth axes developing from the same apical notch. [27]. Taken together, these results from two different bryophyte lineages demonstrate the conservation of CLE regulation of stem cells. This work also suggests that CLE signaling in apical stem cell regulation predates the co-option of WOX genes into such networks, as mutations in P. patens and M. polymorpha WOX genes do not appear to impact CLE processes and the WUS clade of WOX proteins linked to CLE signaling is an innovation of ferns and seed plants. [25, 27–33].

CLE Signaling in Roots-

Root growth in A. thaliana is maintained by the RAM composed of a niche of stem cells maintaining a population of transit amplifying cells for all root tissues which then differentiate and elongate. Stem cells or root initials surround the quiescent center (QC), a mitotically inactive group of stem cells that organize the root initials and can replace them following damage (Fig 1A) [34, 35]. Plants grown in media containing chemically synthesized representatives of many non-TDIF CLE peptides display inhibited root elongation and reduced RAM proliferation, superficially paralleling the effects of CLV3p on SAM proliferation [36, 37]. Despite this, CLE signaling plays a more complex and cell specific role in root development. CLE40p specifically promotes the differentiation of the columella cells making up the tip of the root cap from the columella stem cells (CSCs) and promotes divisions of the cells in the distal root meristem. (Fig 1B) [38]. WOX5 is expressed in the quiescent center (QC), and like WUS is a mobile protein [24, 39]. CLE40 signaling through CLV1 was proposed to limit CSC maintenance by inhibiting WOX5 movement into the CSC [40, 41]. However, recent work suggests that CLE40 signaling does not regulate general cell-cell transport, or specifically WOX5 movement, and WOX5 does not require movement into the CSC for function [42], contrasting with WUS in SAMs. CLE40p treatment does not immediately down-regulate WOX5 expression but instead causes QC division and downregulation of QC identity markers [42]. The authors suggest that CLE40p represses QC identity, with effects on columella differentiation and WOX5 expression being a secondary consequence [42]. Interestingly, clv1 mutants are hypersensitive to CLE40p, even though they share the cle40 mutant phenotype suggesting that other receptors may be playing a role with CLV1 in CLE40 signaling [40]. Indeed, bam1/2 mutants are resistant to CLE40p effects on the QC (unpublished data).

Figure 1:

CLEs regulate specific cell populations of the RAM

The RAM is composed of many different cell types displayed in (A) (From left to right, Lateral Root Cap (LRC), Epidermis, Columella, Cortex, Cortex/Endodermal Initial (CEI), Endodermis (En), Pericycle, Protophloem lineage cells, Procambium, and Xylem lineage cells). CLEs and their receptors regulate aspects of root development; (B) shows cell types regulated by CLE/Receptor combinations.

Arabidopsis roots contain two ground tissue layers, the endodermis and cortex, which are derived from formative divisions in cortex-endodermal initial (CEI) cells post embryonically. CEI divisions are promoted by transcription of CYCLIND6;1 by the SHORTROOT (SHR) and SCARECROW (SCR) transcription factor dimer [43]. Recent work has demonstrated that CLE16, and likely other root expressed CLEs, signal via BAM1/2 receptors to promote CEI divisions (Figure 1B) [44]. BAM1/2 are required for CEI divisions and are defective in CYCLIND6;1 CEI expression, but not SHR or SCR expression [44, 45]. Correspondingly, CLE16p treatment promotes CYCLIND6;1 and stimulates ectopic asymmetric ground tissue divisions in a SHR-dependent manner. As such, although CLE16p and CLE40p treatment reduce root meristem size, both signaling pathways also promote divisions in distinct stem cell subtypes [42, 44, 46]. Root xylem and phloem development requires asymmetric cell divisions, and CLE9/10p inhibit the asymmetric division of meristematic protoxylem cells that produce additional metaxylem via BAM1/2 (Figure 1B) [47]. CLE25p and CLE45p regulate phloem development through pathways that require CLV2 and CLV2/BAM3, respectively (Figure 1B) [8, 9, 48–50]. In the case of CLE45, this function is independent of STERILITY-REGULATING KINASE MEMBER1/2 (SKM1/2) receptors implicated in CLE45-mediated pollen tube growth promotion [51,52], but involves RPK2[53]. When CLE25 was expressed with modifications that alter key CLE domain residues previously suggested to create a dominant negative form of the peptide, it was found to inhibit one of the two cell fate-determining asymmetric divisions of the protophloem [9]. Previous work has highlighted discrepancies in the ability of these modifications to render a true dominant negative response [54], and this inhibition of protophloem-lineage divisions has also been found in mutants with elevated CLE45 signaling. These complexities, as well as the roles of CLE signaling in phloem differentiation have been recently reviewed [49].

CLE signaling in the root intersects with phytohormones such as cytokinin (the highlight of a previous review [55]) and increasingly with auxin signaling in controlling vascular development and QC function. TDIF signaling regulates auxin signaling by enhancing phosphorylation of AUXIN RESPONSE FACTOR7 (ARF7) to promote lateral root development [56]. BAM3 regulates phloem development downstream of BREVIS RADIX (BRX), a member of a small family of plant-specific proteins. BRX inhibits polar auxin transport to promote the steep gradient of auxin concentration that facilitates continuous phloem differentiation [51]. bam3 mutations can rescue brx phloem defects, and treatment with CLE45 mimics brx phenotypes [58, 59]. Future studies are needed to explore the molecular nature of the genetic interactions between CLE45/BAM3 signaling and the inhibition of auxin transport by BRX.

QC identity and inhibition of QC division requires high auxin concentrations supported by the PLETHORA transcription factors [60]. WOX5 promotes auxin biosynthesis, providing a mechanism for its control over QC function and the non-cell-autonomous effects on columella differentiation [61]. Separately, the timing of CYCLIND6;1 expression and therefore CEI division has also been proposed to be controlled by auxin [62], although CLE16p does not affect the DR5 auxin reporter and no known auxin mutants have CEI defects [44]. The links between CLE40 or CLE16 and auxin, if any, have yet to be directly explored.

WOX up with auxin in shoots?

CLE-mediated stem cell regulation is critical for SAM maintenance, allowing for the continuation of SAM activity. Lateral organs such as leaves and flowers emerge from the peripheral zone on flanks of the SAM, and continued renewal of the peripheral zone by the stem cells at the center of the SAM is required to sustain organ production. These stem cells known as the central zone overlie the mitotically quiescent organizing center that expresses WUS (Fig 2A). Recent work has shown that CLV2, and its obligate cytoplasmic pseudokinase partner CORYNE (CRN), are required for floral primordia outgrowth in ambient and cooler temperatures (Fig 2B). In these environmental conditions, the apices of clv2 and crn mutants stall, temporarily ceasing floral primordia production. Auxin is a well-known promotor of floral primordia production and auxin function is diminished in clv2/crn SAMs in cooler temperatures, most likely at the level of auxin biosynthesis regulation. At warmer temperatures floral primordia production is restored to clv2/crn mutants, due to upregulation of auxin biosynthesis by the thermomorphogenesis response. Consistently, this effect can be mimicked in cold by mutating the thermomorphogenesis repressor EARLY FLOWERING3 (ELF3) in crn mutants [11]. Collectively CLV2/CRN and ELF3 pathways confer robustness to floral primordia production across thermal environments. WUS has been suggested to modulate auxin responsiveness in shoots [63]. Although enlarged clv2/crn inflorescence meristems have expanded WUS expression, WUS expression does not expand into terminated primordia. Termination is only seen in clv2/crn mutants and not in clv1 or clv3, mutants with stronger SAM phenotypes and expanded WUS expression, suggesting that CLV2 regulation of primordia outgrowth may be WUS-independent [11].

Figure 2:

CLE control of the SAM and lateral shoot organs

The inflorescence meristem is a SAM that produces flowers from the peripheral zone, and the ability of the central zone to maintain the peripheral zone is regulated by the organizing center (A). Although many LRR-RLK mutants have enlarged SAMs and seem to act in the CLV3 pathway, only CLV1 and to a lesser degree the BAMs likely directly perceive CLV3 in the organizing center (B). The epidermis of cotyledons is composed of pavement cells and stomata (C). bam1/2/3 mutants have impaired leaf shape and vascular development (Scale bar .5cm) (D). Meristemoids are stomatal lineage stem cells that can produce stomata guard cells as well as additional pavement cells; CLE9/10 control stomatal spacing divisions of the meristemoids (E). The inner layers of leaves are composed of a variety of cell types, including the vascular bundles containing vascular cambium, regulated by TDIF/TDR signaling (F).

The links between CLE signaling and auxin in shoot organs also extend to leaf development (Fig 2C). The transcriptional co-activator BLADE-ON-PETIOLE1 (BOP1) as well as auxin promote expression of CLE5 and CLE6, and these CLEs are inhibited by the ASYMMETRIC LEAVES2 (AS2) transcription factor. BOP1 and AS2 regulate critical aspects of leaf polarity, symmetry, and vascularization. cle5/6 mutants have subtle leaf shape phenotypes, indicating that they may have a role in auxin-controlled leaf development [64]. bam1/2 and bam1/2/3 mutants have strong defects in leaf symmetry as well as vascular density, processes known to be regulated by auxin (Fig 2D) [65, 66]. CLE9/10 inhibits the spacing divisions of the stomatal lineage ground cells, although this is closely linked to cytokinin signaling without a clear connection to auxin (Fig 2E,F) [47, 67]. How CLE signaling impacts auxin function in leaf development, and if this is direct, remains outstanding. However, the phosphorylation of ARF5 and ARF7 downstream of TDIF/TDR signaling to dissociate these ARFs from inhibitor IAA proteins may be a guide [49, 56, 68].

Conclusion

The CLE peptide signaling module is highly conserved and regulates aspects of apical stem cell development in all land plants studied to date. In the SAM of angiosperms, the foundational CLV3 signaling pathway is closely linked with the function of the WUS transcription factor, control the expression of WUS are unknown. Nevertheless, as new roles for CLE signaling are discovered, parallel functions of WUS orthologs do emerge. CLE16p signaling through BAM receptors activates CEI division, and WOX7 is a WUS relative that is localized specifically in the CEIs of young roots [44, 69]. However, wox7 mutants lack CEI phenotypes, making a speculative role for WOX7 in the CLE16/BAM signaling pathway unclear [63]. WOX1/3/5 redundantly control leaf growth and auxin biosynthesis in leaves, which may intersect with control of leaf development by CLE5/6 or the BAM receptors [70]. But in these CLE signaling pathways, either these CLEs control development independently of WOX transcription factors like the CLE40 pathway controlling QC homeostasis or CLV2 function in floral primordia development [11, 42], or no direct links have been found. Additionally, CLE peptides are known to contribute to physiological and biotic interactions, but little data exists to tie these processes to WOX function [51,71]. Although CLE signaling interactions with WOX genes may have evolved with the WUS-clade in the euphyllophytes, the persistence of connections to auxin signaling could be an ancestral characteristic CLE signaling [32]. For example, in M. polymorpha, the apical stem cell marker used to denote the stem cell population regulated by MpCLE2p is a YUCCA auxin biosynthesis gene, of the same family that is positive regulated by CLV2 signaling in floral outgrowth [29]. The overlap of CLE peptides, auxin signaling, and WOX activity is an area of active exploration that may answer long-outstanding questions about CLE signaling.