Abstract
Stem cell homeostasis in plant shoot meristems requires tight coordiantion between stem cell proliferation and cell differentiation. In Arabidopsis, stem cells express the secreted dodecapeptide CLAVATA3 (CLV3), which signals through the leucine-rich repeat (LRR)–receptor kinase CLAVATA1 (CLV1) and related CLV1-family members to downregulate expression of the homeodomain transcription factor WUSCHEL (WUS). WUS protein moves from cells below the stem cell domain to the meristem tip and promotes stem cell identity, together with CLV3 expression, generating a negative feedback loop. How stem cell activity in the meristem centre is coordinated with organ initiation and cell differentiation at the periphery is unknown.
We show here that the CLE40 gene, encoding a secreted peptide closely related to CLV3, is expressed in the SAM in differentiating cells in a pattern complementary to that of CLV3. CLE40 promotes WUS expression via BAM1, a CLV1-family receptor, and CLE40 expression is in turn repressed in a WUS-dependent manner. Together, CLE40-BAM1-WUS establish a second negative feedback loop. We propose that stem cell homeostasis is achieved through two intertwined pathways that adjust WUS activity and incorporate information on the size of the stem cell domain, via CLV3-CLV1, and on cell differentiation via CLE40-BAM1.
Introduction
In angiosperms, the stem cell domain in shoot meristem is controlled by the directional interplay of two adjacent groups of cells. These are the central zone (CZ) at the tip of the dome-shaped meristem, comprising slowly dividing stem cells, and the underlying cells of the organising centre (OC). Upon stem cell division, daughter cells are displaced laterally into the peripheral zone (PZ), where they can enter differentiation pathways (Fletcher et al., 1999; Hall & Watt, 1989; Reddy et al., 2004; Schnablová et al., 2020; Stahl & Simon, 2005; Steeves & Sussex, 1989). Cells in the OC express the homeodomain transcription factor WUSCHEL (WUS), which moves through plasmodesmata to CZ cells to maintain stem cell fate and promote expression of the secreted signalling peptide CLAVATA3 (CLV3) (Brand et al., 2000; Daum et al., 2014; Müller et al., 2006; Schoof et al., 2000; Yadav et al., 2011). Perception of CLV3 by plasma-membrane localised receptors in the OC cells triggers a signal transduction cascade and downregulates WUS activity, thus establishing a negative feedback loop (Mayer et al., 1998; Ogawa et al., 2008; Yadav et al., 2011). Mutants of CLV3 or its receptors (see below) fail to confine WUS expression and cause stem cell proliferation, while WUS mutants cannot maintain an active stem cell population (Brand et al., 2002; Clark et al., 1993, 1995; Endrizzi et al., 1996; Laux et al., 1996; Schoof et al., 2000). WUS function in the OC is negative regulated by HAM transcription factors, and only WUS protein that moves upwards to the stem cell zone, which lacks HAM expression, can activate CLV3 expression (Han et al., 2020; Zhou et al., 2018). The CLV3-WUS interaction can serve to maintain the relative sizes of the CZ and OC, and thereby meristem growth along the apical-basal axis. However, cell loss from the PZ due to production of lateral organs requires a compensatory size increase of the stem cell domain.
The CLV3 signalling pathway, which acts along the apical-basal axis of the meristem, has been widely studied in several plant species and shown to be crucial for stem cell homeostasis in shoot and floral meristems (Somssich et al., 2016). The CLV3 peptide is perceived by a leucin-rich-repeat (LRR) receptor kinase, CLAVATA1 (CLV1), which interacts with coreceptors of the CLAVATA3 INSENSITIVE RECEPTOR KINASES (CIK) 1-4 family (Clark et al., 1997; Cui et al., 2018). CLV1 activation involves autophoshorylation, interaction with membrane-associated and cytosolic kinases and phosphatases (Blümke et al., 2021; Defalco et al., 2021). Furthermore, heterotrimeric G-proteins and MAPKs have been implicated in this signal transduction cascade in maize and Arabidopsis (Betsuyaku et al., 2011; Bommert et al., 2013; Ishida et al., 2014; Lee et al., 2019). Besides CLV1, several other receptors contribute to WUS regulation, among them RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), the CLAVATA2-CORYNE heteromer (CLV2-CRN) and BARELY ANY MERISTEM1-3 (BAM1-3) (Bleckmann et al., 2010; DeYoung & Clark, 2008; Hord et al., 2006; Jeong et al., 1999; Kinoshita et al., 2010; Müller et al., 2008). The BAM receptors share high sequence similarity with CLV1, and perform diverse functions throughout plant development. Double mutants of BAM1 and BAM2 maintain smaller shoot and floral meristems, thus displaying the opposite phenotype to mutants of CLV1 (DeYoung et al., 2006; DeYoung & Clark, 2008; Hord et al., 2006). Interestingly, ectopic expression experiments showed that CLV1 and BAM1 can perform similar functions in stem cell control (Nimchuk et al., 2015). In addition, one study showed that CLV3 could interact with CLV1 and BAM1 in cell extracts (Shinohara & Matsubayashi, 2015), although another in vitro study did not detect BAM1-CLV3 interaction at physiological levels of CLV3 (Crook et al., 2020). Furthermore, CLV1 was shown to act as a negative regulator of BAM1 expression, which was interpreted as a genetic buffering system, whereby a loss of CLV1 is compensated by upregulation of BAM1 in the meristem centre (Nimchuk, 2017; Nimchuk et al., 2015). Comparable genetic compensation models for CLE peptide signalling in stem cell homeostasis were established for other species, such as tomato and maize (Rodriguez-Leal et al., 2019).
Maintaining the overall architecture of the shoot apical meristem during the entire life cycle of the plant requires replenishment of differentiating stem cell descendants in the PZ, indicating that cell division rates and cell fate changes in both regions are closely connected (Stahl & Simon, 2005). Overall meristem size is restricted by the ERECTA-family signalling pathway, which is activated by EPIDERMAL PATTERNING FACTOR (EPF)-LIKE (EPFL) ligands from the meristem periphery and confines both CLV3 and WUS expression (Mandel et al., 2014; Shpak, 2013; Shpak et al., 2004; Torii et al., 1996; Zhang et al., 2021). In the land plant lineage, the shoot meristems of bryophytes such as the moss Physcomitrium patens appear less complex than those of angiosperms, and carry only a single apical stem cell which ensures organ initiation by continuous asymmetric cell divisions (de Keijzer et al., 2021; Harrison et al., 2009). Broadly expressed CLE peptides were here found to restrict stem cell identity, and act in division plane control (Whitewoods et al., 2018). Proliferation of the apical notch cell in the liverwort Marchantia polymorpha is promoted by MpCLE2 peptide which acts from outside the stem cell domain via the receptor MpCLV1, while cell proliferation is confined by MpCLE1 peptide through a different receptor (Hata & Kyozuka, 2021; Hirakawa et al., 2019, 2020; Takahashi et al., 2021). Thus, antagonistic control of stem cell activities through diverse CLE peptides is conserved between distantly related land plants. In the grasses, several CLEs were found to control the stem cell domain. In maize, ZmCLE7 is expressed from the meristem tip, while ZmFCP1 is expressed in the meristem periphery and its centre. Both peptides restrict stem cell fate via independent receptor signalling pathways (Liu et al., 2021; Rodriguez-Leal et al., 2019). In rice, overexpression of the CLE peptides OsFCP1 and OsFCP2 downregulates the homeobox gene OSH1 and arrests meristem function (Ohmori et al., 2013; Suzaki et al., 2008). Common for rice and maize, CLE peptide signalling restricts stem cell activities in the shoot meristem, but a stem cell promoting pathway were not been identified so far.
Importantly, how stem cell activities in the CZ and OC are coordinated to regulate organ initiation and cell differentiation in the PZ, which is crucial to maintain an active meristem, is not yet known. In maize, the CLV3-related peptide ZmFCP1 was suggested to be expressed in primordia, and convey a repressive signal on the stem cell domain (Je et al., 2016). In Arabidopsis, the most closely related peptide to CLV3 is CLE40, which was shown to act in the root meristem to restrict columella stem cell fate and regulate the expression of the WUS paralog WOX5 (Berckmans et al., 2020; Hobe et al., 2003; Pallakies & Simon, 2014; Stahl et al., 2013; Stahl & Simon, 2010). Functions of CLE40 in the SAM have not previously been described. Overexpression of CLE40 causes shoot stem cell termination, while CLE40 expression from the CLV3 promoter fully complements the shoot and floral meristem defects of clv3 mutants (Hobe et al., 2003). We therefore hypothesized that CLE40 could act in a CLV3-related pathway in shoot stem cell control.
Here, we show that the expression level of WUS in the OC is subject to feedback regulation from the PZ, which is mediated by the secreted peptide CLE40. In the shoot meristem, CLE40 is expressed in a complementary pattern to CLV3, and excluded from the CZ and OC. In cle40 loss of function mutants, WUS expression is reduced, and shoot meristems remain small and flat, indicating that CLE40 signalling is required to maintain WUS expression in the OC. Ectopic expression of WUS represses CLE40 expression, while in wus loss-of-function mutants CLE40 is expressed in the meristem centre, indicating that CLE40, in contrast to CLV3, is subject to negative feedback regulation by WUS. CLE40 likely acts as an autocrine signal that is perceived by BAM1 in a domain flanking the OC.
Based on our findings, we propose a new model for the regulation of the stem cell domain in the shoot meristem in which signals and information from both, the CZ and the PZ are integrated through two interconnected negative feedback loops that sculpt the dome-shaped shoot meristems of angiosperms.
Results
CLE40 signalling promotes IFM growth from the peripheral zone
Previous studies showed that CLE40 expression from the CLV3 promoter can fully complement a clv3-2 mutant, indicating that CLE40 can substitute CLV3 function in the shoot meristem to control stem cell homeostasis, if expressed from the stem cell domain. Furthermore, while all other CLE genes in Arabidopsis lack introns, the CLE40 and CLV3 genes carry two introns at very similar positions (Hobe et al., 2003). Phylogenetic analysis revealed that CLV3 and CLE40 locate in the same cluster together with CLV3 orthologues from rice, maize and tomato (Goad et al., 2017) (Fig. 1A).
(A) The amino acid (AA) sequences of the mature CLV3 and CLE40 peptides differ in four AAs (differences marked in red). (B) Col-0 inflorescence at 6 WAG with flowers. (B’) IFM at 6 WAG, maximum intensity projection (MIP) of a z-stack taken by confocal microscopy. (C) clv3-9 inflorescence at 6 WAG (Ć) MIP of a clv3-9 IFM at 6 WAG. (D) Inflorescence of cle40-2 at 6 WAG (D’) MIP of a cle40-2 IFM. (E) Box and whisker plot of IFM sizes of Col-0 (N=59), clv3-9 (N=22), and cle40-2 (N=27) plants.
Scale bars: 10mm (B, C, D), 50µm (B’, C’, D’), Statistical groups were assigned after calculating p-values by ANOVA and Turkey’s multiple comparison test (differential grouping from p ≤ 0.01). Yellow dotted lines in B’ to D’ enclose the IFM, red line in the inset meristem in E indicates the area that was used for the quantifications in E.
Mutations in CLE40 were previously found to affect distal stem cell maintenance in the root meristem, revealing that a CLV3 related signalling pathway also operates in the root stem cell niche. To uncover a potential role of CLE40 in shoot development, we analysed seedling and flower development, and inflorescence meristem (IFM) sizes of the wild type Col-0, and clv3-9 and cle40-2 loss-of-function mutants. At 4 weeks after germination (WAG), leaves of clv3-9 mutants remained shorter than those of Col-0 or cle40-2 (Fig1-SupplFig.1). After floral induction, the inflorescences of clv3-9 mutants were compact with many more flowers than the wild type, while cle40-2 mutant inflorescences appeared smaller than the control (Fig. 1B-D). To first investigate effects on meristem development in detail, longitudinal optical sections through the inflorescence meristem (IFM) at 6 WAG were obtained by confocal microscopy and meristem areas were analysed (Fig. 1B-E). In clv3-9 mutants, meristem areas increased to approx. 450% of wild type (Col-0) levels, while shoot meristems from 4 independent cle40 mutant alleles in a Col-0 background (cle40-2, cle40-cr1, cle40-cr2, cle40-cr3) reached only up to 65% of wild type (Fig. 1E, Fig1-SupplFig.2C) (Yamaguchi et al., 2017). Next, we used carpel number as a rough proxy for flower meristem (FM) size, which was 2±0.0 (N=290) in Col-0 and cle40-2 (N=290) but 3.7±0.4 (N=340) in clv3-9 (Fig1-SupplFig.3). Hence, we concluded that CLE40 mainly promotes IFM growth, whereas CLV3 serves to restrict both IFM and FM sizes.
We next analysed the precise CLE40 expression pattern using a transcriptional reporter line, CLE40:Venus-H2B (Wink, 2013). We first concentrated on the IFMs and FMs. CLE40 is expressed in IFMs and in FMs, starting at P5 to P6 onwards (Fig. 2A-C). We found stronger expression in the PZ than in the CZ, and no expression in young primordia. Using MorphoGraphX software, we extracted the fluorescence signal originating from the outermost cell layer (L1) of the IFM, and noted reduced CLE40 expression in the CZ (Fig. 2B). Optical longitudinal sections through the IFM showed that CLE40 is not expressed in the CZ, and only occasionally in the OC region (Fig. 2C). Expression of CLE40 changed dynamically during development: expression was concentrated in the IFM, but lacking at sites of primordia initiation (P0 to P4/5, Fig. 2C). In older primordia from P5/6 onwards, CLE40 expression is detectable from the centre of the young FM and expands towards the FM periphery. In the FMs, CLE40 is lacking in young sepal primordia (P6), but starts to be expressed on the adaxial sides of petals at P7 (Fig. 2A, P1-P7).
(A) MIP of an inflorescence at 5 WAG expressing the transcriptional reporter CLE40:Venus-H2B//Col-0 showing CLE40 expression in the IFM, older primordia and sepals (N=23). (B) The L1 projection shows high expression in the epidermis of the periphery of the IFM and only weak expression in the CZ. (C) Longitudinal section through the IFM shows expression of CLE40 in the periphery, but lack of expression in the CZ. (P1 –P6) Longitudinal section through primordia show no CLE40 expression in young primordia (P1-P4), but in the centre of older primordia (P5-P6). (D) The MIP of the double reporter line of CLE40 and CLV3 (CLE40:Venus-H2B;CLV3:NLS-3xmCherry//Col-0) shows CLV3 expression in the CZ surrounded by CLE40 expression in the periphery (N=12). (E-E’’’) The L1 projection shows CLV3 (E’) expression in the centre of the IFM and CLE40 (E’’) expression in a distinct complementary pattern in the periphery of the IFM. (F) The longitudinal section through the centre of the IFM shows CLV3 expression in the CZ while CLE40 (F’) is mostly expressed in the surrounding cells. (F’’) CLE40 and CLV3 are expressed in complementary patterns.
Dashed blue lines indicate magnified areas, dashed white and orange lines indicate planes of optical sections, dashed yellow line in B marks CZ and in F’’ the OC. Scale bars: 50µm (A, D), 20µm (B, C, E, E’’’, F’’), 10µm (P0 to P6), MIP = Maximum intensity projection, PI = Propidium iodide, L1 = visualisation of layer 1 only, P1 to P7 = primordia at consecutive stages.
To compare the CLE40 pattern with that of CLV3, we introgressed a CLV3:NLS-3xmCherry transcriptional reporter into the CLE40:Venus-H2B background. CLV3 and CLE40 are expressed in almost mutually exclusive domains of the IFM, with CLV3 in the CZ surrounded by CLE40 expressing cells (Fig. 2D-F’’). In the deeper region of the IFM, where the OC is located, both CLV3 and CLE40 are not expressed (Fig. 2F).
We noted that CLE40 is downregulated where WUS is expressed, or where WUS protein localises, such as the OC and CZ. Furthermore, CLE40 is also lacking in very early flower primordia and in incipient organs.
CLE40 expression is repressed by WUS activity
To further analyse the regulation of CLE40 expression, we introduced the CLE40 transcriptional reporter into the clv3-9 mutant background (Fig. 3A-B, Fig3-SupplFig.1). In clv3-9 mutants, WUS is no longer repressed by the CLV signalling pathway, and the CZ of the meristem increases in size as described previously (Clark et al., 1995). In the clv3-9 mutant meristems, both CLV3 and WUS promoter activity is now found in an expanded domain (Fig3-SupplFig.1). CLE40 is not expressed in the tip and centre of the IFM but is rather confined to the peripheral domain, where neither CLV3 nor WUS are expressed (Fig. 3B’, Fig3-SupplFig.1B’). To further explore the expression dynamics of CLE40 in connection with regulation of stem cell fate and WUS, we misexpressed WUS from the CLV3 promoter and introgressed it into plants carrying the CLE40:Venus-H2B construct. Since WUS activates the CLV3 promoter, CLV3:WUS misexpression triggers a positive feedback loop. This results in a continuous enlargement of the CZ (Brand et al., 2002). Young seedlings carrying the CLV3:WUS transgene at 10 DAG displayed a drastically enlarged SAM, compared to wild type seedlings of the same age (Fig. 3C-D’). Wild type seedlings at this stage express CLE40 in older leaf primordia and in deeper regions of the vegetative SAM (Fig. 3E-E’). The CLV3:WUS transgenic seedlings do not initiate lateral organs from the expanded meristem, and CLE40 expression is confined to the cotyledons (Fig. 3F-F’). CLE40 is also lacking in the deeper regions of the vegetative SAM (Fig. 3F’). Thus. we conclude that either WUS itself, or a WUS-dependent regulatory pathway represses CLE40 gene expression.
(A) MIP of CLE40 expression (CLE40:Venus-H2B//Col-0) at 5 WAG, (A’) Optical section through the centre of the IFM (indicated by orange line in (A)) reveals no CLE40 expression in the CZ and in the centre of the meristem. Cells in the L2 layer also show less CLE40 expression. High CLE40 expression is found in the PZ (N=23). (B) MIP of CLE40 expression in a clv3-9 mutant (CLE40:Venus-H2B//clv3-9) shows expression only in the PZ of the meristem, in FMs and in sepals (N=6). (B’) Optical section through the IFM depicts no CLE40 expression at the tip and the centre of the meristem. CLE40 expression is only detected in cells at the flanks of the IFM and in sepals. (C) Arabidopsis seedling at 10 DAG. (D) Seedling expressing WUS from the CLV3 promoter, 10 DAG. (D’) Magnification of seedling in (D). The meristem fasciates without forming flowers. (E) L1 projection, vegetative seedling with CLE40 expression in the PZ and in leaf primordia starting from LP4, at 10 DAG (N=5). (E’) Optical section of (E) with CLE40 expression primordia and rib meristem or periphery. (F) MIP of fasciated meristem as in (D). CLE40 expression can only be found in the cotyledons (C1 and C2) next to the meristem (N=5). (F’) Optical section shows CLE40 expression only in the epidermis of cotyledons. (G and G’) MIP (G) and optical section (G’) of CLE40 expression (CLE40:Venus-H2B//Ler) in a wild type (L.er) background at 5WAG shows no signal in the CZ or OC. CLE40 is confined to the PZ and the centre of older flower primordia, and to sepals (N=8). (H and H’) MIP of CLE40 in a wus-7 background shows expression through the entire IFM and in the centre of flower primordia. The optical section (H’) reveals that CLE40 is also expressed in the CZ as well as in the OC of the IFM (N= 12).
Dashed orange lines indicate the planes of optical sections, dashed blue lines in E and F enclose the meristem, the dashed white line in É marks the CZ. Scale bars: 50µm (A, B, G, H), 20µm (A’, B’, E, E’, F, F’, G’, H’), 1mm (C, D), 500µm (D’), MIP = Maximum intensity projection, PI = propidium iodide, L1 = layer 1 projection, C = cotyledon, LP = leaf primordium
We next determined if CLE40 repression in the CZ can be alleviated in mutants with reduced WUS activity. Since wus loss-of-function mutants fail to maintain an active CZ and shoot meristem, we used the hypomorphic wus-7 allele (Graf et al., 2010; Ma et al., 2019). wus-7 mutants are developmentally delayed. Furthermore, wus-7 mutants generate an IFM, but the FMs give rise to sterile flowers that lack inner organs (Fig3-SupplFig.2). We introgressed the CLE40 reporter into wus-7, and found that at 5WAG, all wus-7 mutants expressed CLE40 in both the CZ and the OC of the IFM (Fig. 3G-H’, Fig3-SupplFig.2). Similar to wild type, CLE40 is only weakly expressed in the young primordia of wus-7. Therefore, we conclude that a WUS-dependent pathway downregulates CLE40 in the centre of the IFM during normal development.
CLE40 signals through BAM1
Given that CLV1 and BAM1 perform partially redundant functions to perceive CLV3 in shoot and floral meristems, we asked if these receptors also contribute in a CLE40 signalling pathway. We therefore generated the translational reporter lines CLV1:CLV1-GFP and BAM1:BAM1-GFP, and analysed their expression patterns in detail. We observed dynamic changes of CLV1 expression during the different stages of flower primordia initiation. CLV1:CLV1-GFP is continuously expressed in deeper regions of the IFM comprising the OC, and in the meristem periphery where new FMs are initiating (Fig. 4A). CLV1 is expressed strongly in cells of the L1 and L2 of incipient organ primordia (P-1, P0), and only in L2 at P1. P2 and P3 show only very faint expression in the L1, but in stages from P4 to P6, CLV1 expression expands from the L3 into the L2 and L1 (Fig. 4, P1-P6).
(A) MIP of CLV1 under its endogenous promoter (CLV1:CLV1-GFP//Col-0) at 5 WAG shows CLV1 expression in the OC of the meristems, IFM and FMs, in incipient organ primordia (P-1 to P1) and in sepals (N=15). (B) In the L1 projection CLV1 expression is detected in cells of incipient organs. (C) Optical section through the IFM shows CLV1 expression in the OC and in P0. (P-1-P6) CLV1 expression is detected in incipient organ primordia in L1 and L2 (-P1, P0), in the L2 of P1, and in the OC of the IFM and FMs from P4 to P6.
Dashed white and blue lines indicate the planes of optical sections, yellow dashed line in (A) and (B) mark incipient organ primordia (P-1 to P1), yellow lines (P-1 to P6) indicate the IFM region, white lines mark the primordium. Scale bars: 50µm (A), 20µm (B, C), 10µm (P1 to P6), MIP = maximum intensity projection, PI = propidium iodide, L1 = layer 1, P = primordium
The translational BAM1:BAM1-GFP reporter is expressed in the IFM, the FMs and in floral organs (Fig. 5A). In the IFM, expression is found throughout the L1 layer of the meristem, and, at an elevated level, in L2 and L3 cells of the PZ, but not in the meristem centre around the OC, where CLV1 expression is detected (Fig. 5B,C, compare to Fig. 4C). BAM1 is less expressed in the deeper regions of primordia from P6 onwards (Fig. 5C). BAM1 transcription was reported to be upregulated in the meristem centre in the absence of CLV3 or CLV1 signalling (Nimchuk, 2017). Using our translational BAM1 reporter in the clv1-20 mutant background, we confirmed that BAM1 is expressed in the meristem centre, similar to the pattern of CLV1 in the wild type, and that BAM1 is upregulated in the L1 of the meristem. Importantly, in a clv1-20 background BAM1 is absent in the peripheral region of the IFM and the L2 (Fig. 5D-F).
(A) MIP of BAM1 under its endogenous promoter (BAM1:BAM1-GFP//bam1-3) at 5 WAG. BAM1 expression is detected nearly throughout the entire inflorescence (IFM, FM, sepals) with weak expression in the CZ of IFM and FMs (N=15). (B) The L1 projection of the IFM shows ubiquitous expression of BAM1. (C) Optical section through the IFM shows elevated BAM1 expression in the flanks (yellow arrows) and a lack of BAM1 expression in the OC. (P1 - P6) BAM1 expression is found in all primordia cells. (D) MIP of BAM1 in a clv1-20 mutant (BAM1:BAM1-GFP//bam1-3;clv1-20). BAM1 expression is detected in most parts of the inflorescence, especially in the centre of the IFM and FMs (N=9). (E) Cross section (XY) through of the IFM (from D) shows BAM1 expression in a clv1-20 mutant in the CZ (IFM and FMs) and in the L1/L2. (F) Optical section through the meristem (from D) shows BAM1 expression in the OC and in the L1, while no BAM1 expression is detected in the PZ (yellow arrows).
Dashed white and orange lines indicate longitudinal sections; yellow lines (P1 to P6) indicate the IFM region, white lines (P1 to P6) mark the primordium, yellow arrows indicate high (C) or no (F) BAM1 expression in the PZ. Scale bars: 50µm (A, D), 20µm (B, C, E, F), 10µm (P1 to P6), MIP = maximum intensity projection, PI = propidium iodide, L1 = layer 1, P = primordium
In longitudinal and optical cross sections through the IFM, we found that complementarity of CLE40 and CLV3 is reflected in the complementary expression patterns of BAM1 and CLV1 (Fig. 6A-D’). Therefore, we conclude that expression patterns of CLV1 and BAM1 are mostly complementary in the meristem itself and during primordia development. When comparing CLE40 and BAM1 expression patterns, we found a strong overlap in the peripheral zone of the meristem, during incipient primordia formation, in older primordia, and in L3 cells surrounding the OC (Fig. 6A’B’, Fig6-SupplFig. 1). Similarly, CLV3 and CLV1 are confined to the CZ and OC, respectively.
(A and A’) Longitudinal and cross sections of CLE40 (CLE40:Venus-H2B//Col-0) through the IFM show CLE40 expression in the PZ while no CLE40 expression is detected in the CZ or the OC (dashed yellow line). (B and B’) In optical sections of BAM1 (BAM1:BAM1-GFP//bam1-3) through the IFM elevated BAM1 expression in the PZ and in young primordia can be detected, while low expression is found in the CZ and no expression is observed in the OC (dashed yellow line). (C and C’) Optical and cross section of CLV3 through the IFM (CLV3:NLS-3xmCherry//Col-0) show CLV3 expression in the CZ (dashed yellow line). (D and D’) The native expression of CLV1 (CLV1:CLV1-GFP//Col-0) in an optical and cross section through the IFM is depicted in the OC (dashed yellow line) and in cells of the L1 and L2 close to emerging primordia. (E) Box and whisker plot of the IFM area size of Col-0 (N=82), various single (clv3-9 (N=22), cle40-2 (N=42), clv1-101 (N=32), bam1-3 (N=68)) and double mutants (cle40-2;clv1-101 (N=37), cle40-2;bam1-3 (N=25) and bam1-3;clv1-101 (N=36)) at 6 WAG. Scale bars: 20µm (A – D’), yellow dashed lines indicate the OC (in A’, B’, D’) or the CZ (C’), Statistical groups were assigned after calculating p-values by ANOVA and Turkey’s multiple comparison test (differential grouping from p ≤ 0.01). Red line in the inset meristem in (E) indicates the area that was used for the quantifications in (E).
To analyse if CLE40-dependent signalling requires CLV1 or BAM1, we measured the sizes of IFMs in the respective single and double mutants (Fig. 6E, Fig6-SupplFig. 2). While cle40-2 mutant IFMs reached 65% of the wild type size, clv1-101 plants develop IFMs that were 140% wild type size, whereas bam1-3;clv1-101 double mutant meristems reached 450% wild type size, similar to those of clv3-9 mutants. This supports the notion that BAM1 can partially compensate for CLV1 function in the CLV3 signalling pathway when expressed in the meristem centre (Fig. 5F) (Nimchuk et al., 2015). The relationship between CLV1 and BAM1 is not symmetrical, since CLV1 is expressed in a wildtypic pattern in bam1-3 mutants (Fig8-SupplFig. 4). Meristem sizes of bam1-3 mutants reached 70% of the wild type, and double mutants of cle40-2;bam1-3 did not differ significantly. However, double mutants of cle40-2;clv1-101 developed like the clv1-101 single mutant, indicating an epistatic relationship. Importantly, both clv1-101 and bam1-3 mutants lack BAM1 function in the meristem periphery (Fig. 5F), where also CLE40 is highly expressed, which could explain the observed epistatic relationships of cle40-2 with both clv1-101 and bam1-3. Similar genetic relationships for CLV3, CLE40, CLV1 and BAM1 were noticed when analysing carpel number as a proxy for FM sizes. We also noted that generation of larger IFMs and FMs in different mutants was negatively correlated with leaf size, which we cannot explain so far (Fig1-SupplFig.1).
We hypothesize that CLE40 signals from the meristem periphery via BAM1 to promote meristem growth. Next, we aimed to determine if the commonalities between cle40-2 and bam1-3 mutants extend beyond their effects on meristem size.
A CLE40 and BAM1 signalling pathway promotes WUS expression in the meristem periphery
We next analysed the number of WUS-expressing cells in wild type and mutant meristems using a WUS:NLS-GFP transcriptional reporter. Compared to wild type, the WUS expression domain was laterally strongly expanded in both clv3-9 and clv1-101. Interestingly, WUS signal extended also into the L1 layer of clv1-101, albeit in a patchy pattern (Fig. 7A-C’,F). Also noteworthy is that BAM1 was expressed at a higher level in the L1 layer of clv1 mutants. cle40-2 mutants showed a reduction in the number of WUS expressing cells down to approx. 50% wild-type levels (Fig. 7D-D’,F). Importantly, WUS remained expressed in the centre of the meristem, but was there found in a narrow domain. In bam1-3 mutants, the WUS domain was similarly reduced as in cle40-2, and WUS expression focussed in the meristem centre (Fig. 7E,E’,F). In contrast, both clv3-9 and clv1-101 mutants express WUS in a laterally expanded domain (Fig. 7B’,C’).
(A – E’) MIP and optical section of inflorescences at 5WAG expressing the transcriptional reporter WUS:NLS-GFP in a (A and A’) Col-0, (B and B’) clv1-101, (C and C’) clv3-9, (D and D’) cle40-2 and (E and E’) bam1-3 background. In (A) wild type plants the WUS domain is smaller compared to the expanded WUS domain in (B) clv1-101 and (C) clv3-9 mutants. The WUS domain of (D) cle40-2 and (E) bam1-3 mutants is decreased compared to wild type plants. Optical sections of (B’) clv1-101 and (C’) clv3-9 mutants expand along the basal-apical axis while the meristem shape of (D’) cle40-2 and (E’) bam1-3 mutants are flatter compared to (A’) wild type plants,. (F) Box and whisker plot shows the number of WUS-expressing cells in the OC of IFMs of Col-0 (N=9), cle40-2 (N=9), bam1-3 (N=9), clv1-101 (N=8), and clv3-9 (N=5). (G) After 5 WAG bam1-3 (N=11) and cle40-2 (N=11) mutants have flatter meristems than wild type plants (decreased σ value compared to Col-0 (N=11)), while clv1-101 (N=9) and clv3-9 (N=6) mutants increase in their IFM height showing a higher σ value.
Scale bars: 50µm (A – E), 20µm (A’-J), Statistical groups and stars were assigned after calculating p-values by ANOVA and Turkey’s multiple comparison test (differential grouping from p ≤ 0.01). WAG = weeks after germination, yellow numbers = WUS expressing cells in the CZ, σ value = height/width of IFMs
To integrate our finding that CLE40 expression is repressed by WUS activity with the observation that WUS, in turn, is promoted by CLE40 signalling, we hypothesize that the CLE40-BAM1-WUS interaction establishes a new negative feedback loop. The CLE40-BAM1-WUS negative feedback loop acts in the meristem periphery, while the CLV3-CLV1-WUS negative feedback loop acts in the meristem centre along the apical-basal axis. Both pathways act in parallel during development to regulate the size of the WUS expression domain in the meristem, possibly by perceiving input signals from two different regions, the CZ and the PZ, of the meristem.
We then asked how the two signalling pathways, converge on the regulation of WUS expression, control meristem growth and development. So far, we showed that both CLV3-CLV1 and CLE40-BAM1 signalling control meristem size, but in an antagonistic manner. However, we noticed that the different mutations in peptides and receptors affected distinct aspects of meristem shape. We therefore analysed meristem shape by measuring meristem height (the apical-basal axis) at its centre, and meristem diameter (the radial axis) at the base in longitudinal sections. The ratio of height to width then gives a shape parameter “σ” (from the greek word σχήμα = shape). In young inflorescence meristems at 4-5 WAG, when inflorescence stems were approximately 5-8 cm long, meristems of cle40-2 and bam1-3 mutants were slightly reduced in width, and strongly reduced in height, resulting in reduced σ in comparison to Col-0 (Fig. 7G, Fig7-SupplFig. 1A). Meristems of clv1-101 and clv3-9 mutants were similar in width to wild type, but strongly increased in height, giving high σ values (Fig. 7, Fig7-SupplFig. 1A-C). This indicates that CLV3-CLV1 signalling mostly restricts meristem growth along the apical-basal axis, while CLE40-BAM1 signalling promotes meristem growth along both axes.
Our data expand the current model of shoot meristem homeostasis by taking into account that stem cells are lost from the OC during organ initiation in the PZ (Fig. 8). CLV3 signals from the CZ via CLV1 in the meristem centre to confine WUS expression to the OC. The diffusion of WUS protein along the apical-basal axis towards the meristem tip establishes the CZ and activates CLV3 expression as a feedback signal. During plant growth, rapid cell division activity and organ initiation requires the replenishment of PZ cells from the CZ, which can be mediated by increased WUS activity. We now propose that the PZ generates CLE40 as a short range or autocrine signal that acts through BAM1 in the meristem periphery. Since BAM1 and WUS expression do not overlap, we postulate the generation of a diffusible factor that relies on CLE40-BAM1, and acts from the PZ to promote WUS expression. WUS, in turn, represses CLE40 expression from the OC, thus establishing a second negative feedback regulation. Together, the two intertwined pathways serve to adjust WUS activity in the OC and incorporate information on the actual size of the stem cell domain, via CLV3-CLV1, and the growth requirements from the PZ via CLE40-BAM1.
(A and B) Schematic representation of two negative feedback loops in the IFM of Arabidopsis thaliana. CLV3 in the CZ binds to the LRR receptor CLV1 to activate a downstream signalling cascade which leads to the repression of the transcription factor WUS. In a negative feedback loop WUS protein moves to the stem cells to activate CLV3 gene expression. In the PZ of the IFM a second negative feedback loop controls meristem growth by CLE40 and its receptor BAM1. CLE40 binds to BAM1 in an autocrine manner, leading to the activation of a downstream signal “X” which promotes WUS activity. WUS protein in turn represses the expression of the CLE40 gene.
CZ = central zone, PZ = peripheral zone, arrows indicate a promoting effect and the blocked line indicates a repressing signal
Discussion
Shoot meristems are the centres of growth and organ production throughout the life of a plant. Meristems fulfil two main tasks, which are the maintenance of a non-differentiating stem cell pool, and the assignment of stem cell daughters to lateral organ primordia and differentiation pathways (Hall & Watt, 1989). Shoot meristem homeostasis requires extensive communication between the CZ, the OC and the PZ. The discovery of CLV3 as a signalling peptide, which is secreted exclusively from stem cells in the CZ, and its interaction with WUS in a negative feedback loop was fundamental for our understanding of such communication pathways (Fletcher, 2020). Here, we analysed the function of CLE40 in shoot development of Arabidopsis, and found that WUS expression in the OC is under positive control from the PZ due to the activity of a CLE40-BAM1 signalling pathway. IFM size is reduced in cle40 mutants, indicating that CLE40 signalling promotes meristem size. Importantly, CLE40 is expressed in the PZ, in late stage FMs and in differentiating organs. A common denominator for the complex and dynamic expression pattern is that CLE40 expression is confined to meristematic tissues, but not in organ founder sites or in regions with high WUS activity, such as the OC and the CZ. Both misexpression of WUS in the CLV3 domain (Fig. 3F), studies of clv3 mutants with expanded stem cell domains (Fig. 3B, Fig3-SupplFig.1) and analysis of wus mutants (Fig. 3, Fig3-SupplFig.2) underpinned the notion that CLE40 expression, in contrast to CLV3, is negatively controlled in a WUS-dependent manner. Furthermore, we found that the number of WUS expressing cells in cle40 mutant IFMs is strongly reduced, indicating that CLE40 exerts its positive effects on IFM size by expanding the WUS expression domain.
So far, the antagonistic effects of Arabidopsis CLV3 and CLE40 on meristem size can only be compared to the antagonistic functions of MpCLE1 and MpCLE2 on the gametophytic meristems of M. polymorpha, which signal through two distinct receptors, MpTDR and MpCLV1, respectively (Hata & Kyozuka, 2021). By the complementation of clv3 mutants through expression of CLE40 from the CLV3 promoter it was shown previously that CLE40 and CLV3 are able to activate the same downstream receptors (Hobe et al., 2003). Our detailed analysis of candidate receptor expression patterns showed that CLV3 and CLV1 are expressed in partially overlapping domains in the meristem centre, while CLE40 and BAM1 are confined to the meristem periphery. Like cle40 mutants, bam1 mutant IFMs are smaller and maintain a smaller WUS expression domain, supporting the notion that CLE40 and BAM1 comprise a signalling unit that increases meristem size by promoting WUS expression. The antagonistic functions of the CLV3-CLV1 and CLE40-BAM1 pathways in the regulation of WUS are reflected in their complementary expression patterns. There is cross-regulation between these two signalling pathways at two levels: (1) WUS has been previously shown to promote CLV3 levels in the CZ, and we here show that WUS represses (directly or indirectly) CLE40 expression in the OC and in the CZ (Fig. 3B, Fig3-SupplFig.1); (2) CLV1 represses BAM1 expression in the OC, and thereby restricts BAM1 to the meristem periphery (Fig. 5, Fig. 6). In clv1 mutants, BAM1 shifts from the meristem periphery to the OC, and the WUS domain laterally expands in the meristem centre (Fig. 5F, Fig. 7B’). Furthermore, BAM1 expression increases also in the L1, which could cause the observed irregular expression of WUS in the outermost cell layer of clv1 mutants. The role of BAM1 in the OC is not entirely clear: despite the high sequence similarity between CLV1 and BAM1, the expression of BAM1 in the OC is not sufficient to compensate for the loss of CLV1 (Fig.5D-F, Nimchuk et al., 2015). In the OC, BAM1 appears to restrict WUS expression to some extent, since clv1;bam1 double mutants reveal a drastically expanded IFM (DeYoung & Clark, 2008). However, it is possible that BAM1 in the absence of CLV1 executes a dual function: to repress WUS in response to CLV3 in the OC as a substitute for CLV1, and simultaneously to promote WUS expression in the L1 in response to CLE40.
The expression domains of CLE40 and its receptor BAM1 largely coincide, suggesting that CLE40 acts as an autocrine signal. Similarly, protophloem sieve element differentiation in roots is inhibited by CLE45, which acts as an autocrine signal via BAM3 (Kang & Hardtke, 2016). Since WUS is not expressed in the same cells as BAM1, we have to postulate a non-cell autonomous signal X that is generated in the peripheral zone due to CLE40-BAM1 signalling, and diffuses towards the meristem centre to promote WUS expression (Hohm et al., 2010). As a result, CLE40-BAM1 signalling from the PZ will provide the necessary feedback signal that stimulates stem cell activity and thereby serves to replenish cells in the meristem for the initiation of new organs. The CLV3-CLV1 signalling pathway then adopts the role of a necessary feedback signal that avoids an excessive stem cell production.
The two intertwined, antagonistically acting signalling pathways that we described here allow us to better understand the regulation of shoot meristem growth, development and shape. The previous model, which focussed mainly on the interaction of the CZ and the OC via the CLV3-CLV1-WUS negative feedback regulation, lacked any direct regulatory contribution from the PZ. EPFL peptides were shown to be expressed in the periphery and to restrict both CLV3 and WUS expression via ER (Zhang et al., 2021). However, EPFL peptide expression is not reported to be feedback regulated from the OC or CZ, and the main function of the EPFL-ER pathway is therefore to restrict overall meristem size (Zhang et al., 2021). The second negative feedback loop controlled by CLE40, which we uncovered here, enables the meristem to fine-tune stem cell activities in response to fluctuating requirements for new cells during organ initiation. Due to the combined activities of CLV3 and CLE40, the OC (with WUS as a key player) can now record and compute information from both, the CZ and PZ. Weaker CLV3 signalling, indicating a reduction in the size of the CZ, induces preferential growth of the meristem along the apical-basal axis (increasing σ), while weaker CLE40 signals, reporting a smaller PZ, would decrease σ and flatten meristem shape. It will be intriguing to investigate if different levels of CLV3 and CLE40 also contribute to the shape changes that are observed during early vegetative development, or upon floral transition in Arabidopsis.
Many shoot-expressed CLE peptides are encoded in the genomes of maize, rice and barley, which could act analogously to CLV3 and CLE40 of Arabidopsis. It is tempting to speculate that in grasses, a CLE40-like, stem cell promoting signalling pathway is more active than a CLV3-like, stem cell restricting pathway. This could contribute to the typical shape of cereal SAMs, which are, compared to the dome-shaped SAM of dicotyledonous plants, extended along the apical-basal axis.
Material and Methods
All chemicals used for the experiments are listed in Tab. 1.
Chemicals and substances used in this study.
Plant material and growth conditions
All wild type Arabidopsis thaliana (L.) Heynh. plants used in this study are ecotype Columbia-0 (Col-0), except for wus-7 mutants which are in Landsberg erecta (L.er.) background. Details about Arabidopsis thaliana plants carrying mutations in the following alleles: bam1-3, cle40-2, cle40-cr1, cle40-cr2, cle40-cr3, clv1-101, clv3-9 and wus-7 are described in Tab. 2. All mutants are in Col-0 background and are assumed to be null-mutants, except for wus-7 mutants. cle40 mutants (cle40-2, cle40-cr1, cle40-cr2, cle40-cr3) have either a stop codon, a T-DNA insertion or deletion in or before the crucial CLE box domain (Fig1-SupplFig.2B’). clv3-9 mutants were generated in 2003 by the lab of R. Simon. clv3-9 mutants were created by EMS resulting in a W62STOP mutation before the critical CLE domain region. bam1-3 and clv1-101 mutants have been described as null mutants before (DeYoung et al., 2006; Kinoshita et al., 2010), while clv1-20 is a weak allele which contains a insertion within the 5’-UTR of CLV1 and results in a reduced mRNA level (Durbak & Tax, 2011). wus-7 is a weak allele and mutants were described in previous publications (Graf et al., 2010). Double mutants were obtained by crossing the single mutant plants until both mutations were proven to be homozygous for both alleles. Genotyping of the plants was performed either by PCR or dCAPS method with the primers and restrictions enzymes listed in Tab. 3. Before sowing, seeds were either sterilized for 10min in an ethanol solution (80% v/v ethanol, 1,3% w/v sodium hypochloride, 0,02% w/v SDS) or for 1h in a desiccator in a chloric gas atmosphere (50mL of 13% w/v sodium hypochlorite with 1mL 37% HCL). Afterwards, seeds were stratified for 48h at 4°C in darkness. Seeds on soil were then cultivated in phytochambers under long day (LD) conditions (16h light/ 8h dark) at 21°C. For selection of seeds or imaging of vegetative meristems seeds were sowed on ½ Murashige & Skoog (MS) media (1% w/v sucrose, 0.22% w/v MS salts + B5 vitamins, 0.05% w/v MES, 12g/L plant agar, adjusted to pH 5.7 with KOH) in squared petri dishes. Seeds in petri dishes were kept in phytocabinets under continuous light conditions at 21°C and 60% humidity.
Mutants analysed in this study.
Primers and methods used for genotyping.
Cloning of reporter lines
CLV1 (CLV1:CLV1-GFP), BAM1 (BAM1:BAM1-GFP) and CLV3 (CLV3:NLS-3xmCherry) reporter lines were cloned using the GreenGate method (Lampropoulos et al., 2013). Entry and destination plasmids are listed in Tab. 4 and Tab. 5. Promoter and coding sequences were PCR amplified from genomic Col-0 DNA which was extracted from rosette leaves of Col-0 plants. Primers used for amplification of promoters and coding sequences can be found in Tab. 6 with the specific overhangs used for the GreenGate cloning system. Coding sequences were amplified without the stop codon to allow transcription of fluorophores at the C-terminus. BsaI restriction sites were removed by site-directed mutagenesis using the “QuickChange II Kit” following the manufacturer’s instructions (Agilent Technologies). Plasmid DNA amplification was performed by heat-shock transformation into Escherichia coli DH5α cells (10min on ice, 1min at 42°C, 1min on ice, 1h shaking at 37°C), which were subsequently plated on selective LB medium (1% w/v tryptone, 0.5% w/v yeast extract, 0.5% w/v NaCl) and cultivated overnight at 37°C. All entry and destination plasmids were validated by restriction digest and Sanger sequencing.
Entry vectors used for cloning.
Destination vectors used to generate transgenic A.thaliana reporter lines.
Primers used for cloning the entry vectors.
Generation of stable A. thaliana lines
Generation of stable Arabidopsis thaliana lines was done by using the floral dip method (Clough & Bent, 1998).
Translational CLV1 (CLV1:CLV1-GFP) and transcriptional CLV3 (CLV3:NLS-3xmCherry) reporter carry the BASTA plant resistance cassette. T1 seeds were sown on soil and sprayed with Basta ® (120mg/mL) after 5 and 10 DAG. Seeds of ∼10 independent Basta-resistant lines were harvested. The translational BAM1 (BAM1:BAM1-GFP) reporter line carries a D-Alanin resistance cassette and T1 seeds were sown on ½ MS media containing 3-4mM D-Alanin. T2 seeds were then selected on ½ MS media supplied with either 3-4mM D-Alanin or 10µg/mL of DL-phosphinothricin (PPT) as a BASTA alternative. Only plants from lines showing about ∼75% viability were kept and cultivated under normal plant conditions (21°C, LD). Last, T3 seeds were plated on ½ MS media supplied with 3-4mM D-Alanin or PPT again and plant lines showing 100% viability were kept as homozygous lines. The CLV3:NLS-3xmCherry and CLV1:CLV1-GFP constructs were transformed into Col-0 wild type plants and after a stable T3 line was achieved, plants carrying the CLV1:CLV1-GFP construct were crossed into bam1-3, cle40-2, clv3-9 and clv1-101 mutants until a homozygous mutant background was reached. BAM1:BAM1-GFP lines were floral dipped into bam1-3 mutants and subsequently crossed into the clv1-20 mutant background which rescued the extremely fasciated meristem phenotype of bam1-3;clv1-20 double mutants (Fig. 5D-F). BAM1:BAM1-GFP//bam1-3 plants were also crossed into cle40-2 and clv3-9 mutants until a homozygous mutant background was achieved. The CLE40:Venus-H2B reporter line was created and described in Wink, 2013 and the WUS:NLS-GFP;CLV3:NLS-mCherry reporter line was a gift from the Lohmann lab (Wink, 2013). CLE40:Venus-H2B reporter line was crossed into homozygous clv3-9 and heterozygous wus-7 mutants. Homozygous clv3-9 mutants were detected by its obvious phenotype and were brought into a stable F3 generation. Homozygous wus-7 mutants were genotyped. Seeds were kept in the F2 generation, since homozygous wus-7 plants do not develop seeds. The CLE40:Venus-H2B reporter line was also crossed with the CLV3:NLS-3xmCherry reporter line and was brought into a stable F3 generation. To generate the CLE40:Venus-H2B//CLV3:WUS line, plants carrying the CLE40:Venus-H2B line were floral dipped with the CLV3:WUS construct. T1 seeds were sown on 10µg/mL of DL-phosphinothricin (PPT) and the viable seedlings were imaged. WUS:NLS-GFP/CLV3:NLS-mCherry//Col-0 reporter line was crossed into clv3-9, cle40-2, clv1-101 and bam1-3 mutants until a stable homozygous F3 generation was reached respectively. Detailed information of all used A. thaliana lines can be found in Tab. 7.
Arabidopsis lines that were analysed in this study.
Confocal imaging of IFMs
To image IFMs in vivo, plants were grown under LD (16h light/ 8h dark) conditions and inflorescences were cut off at 5 or 6 WAG. Inflorescences were stuck on double sided adhesive tape on an objective slide and dissected until only the meristem and primordia from P0 to maximum P10 were visible. Next, inflorescences were stained with either propidium iodide (PI 5mM) or 4′,6-Diamidin-2-phenylindol (DAPI 1µg/mL) for 2 to 5min. Inflorescences were then washed three times with water and subsequently covered with water and a cover slide and placed under the microscope. Imaging was performed with a Zeiss LSM780 or LSM880 using a W Plan-Apochromat 40x/1.2 objective. Laser excitation, emission detection range and detector information for fluorophores and staining can be found in Tab. 8. All IFMs were imaged from the top taking XY images along the Z axis, resulting in a Z-stack through the inflorescence. The vegetative meristems were imaged as described for IFMs. Live imaging of the reporter lines in A. thaliana plants was performed by dissecting primary inflorescences (except for clv3-9 mutants) at 5 WAG under LD conditions. For imaging of the reporter lines in the mutant backgrounds of clv3-9 secondary IFMs were dissected, since the primary meristems are highly fasciated. Vegetative meristems were cultivated in continuous light conditions at 21°C on ½ MS media plates and were imaged at 10 DAG. For each reporter line at least 3 independent experiments were performed and at least 5 IFMs were imaged.
Microscopy settings used for imaging.
Phenotyping of CLV mutants
For meristem measurements (area size, width and height) primary and secondary IFMs of wild type (Col-0) and mutant plants (cle40-2, cle40-cr1-3, bam1-3, cle40-2;bam1-3, clv1-101) were dissected at 6 WAG under LD conditions. For clv3-9 and clv1-101;bam1-3 only secondary IFMs were imaged and analyzed, due to the highly fasciated primary meristems. Optical sections of the Z-stacks were performed through the middle of the meristem starting in the centre of primordia P5 and ending in the centre of primordia P4. Based on the optical sections (XZ), meristem height and area size were measured as indicated in Fig6-SupplFig. 2. IFM sizes from Fig. 1E are also used in Fig. 6E for Col-0, cle40-2 and clv3-9 plants.
Same procedure was used to count the cells expressing WUS in different mutant backgrounds (Fig. 7A-E). Optical sections of IFMs at 5 WAG were performed from P4 to P5 and only nuclei within the meristem area were counted and plotted. For analyses of carpel numbers, the oldest 10 - 15 siliques per plant at 5 WAG were used. Each carpel was counted as one, independent of its size. N number depicts number of siliques. Leaf measurements were performed at 4 WAG and four leaves of each plant were measured and plotted. Data was obtained from at least 3 independent experiments.
Data analysis
For visualization of images the open-source software ImageJ v 1.53c (Schneider et al., 2012) was used. All images were adjusted in “Brightness and Contrast”. IFMs in Fig. 7 were imaged with identical microscopy settings (except for clv3-9 mutants) and were all changed in “Brightness and Contrast” with the same parameters to ensure comparability. clv3-9 mutants were imaged with a higher laser power since meristems are highly fasciated. MIPs were created by using the “Z-Projection“ function and optical sections were performed with the “Reslice…” function resulting in the XZ view of the image. Meristem width, height and area size were measured with the “Straight line” for width and height and the “Polygon selection” for area size. The shape parameter σ was calculated by the quotient of height and width from each IFM. For L1 visualization the open-source software MorphoGraphX (https://www.mpipz.mpg.de/MorphoGraphX/) was used that was developed by Richard Smith. 2½ D images were created by following the steps in the MrophoGraphX manual (de Reuille et al., 2015). After both channels (PI and fluorophore signal) were projected to the created mesh, both images were merged using ImageJ v 1.53c.
For all statistical analyses, GraphPad Prism v8.0.0.224 was used. Statistical groups were assigned after calculating p-values by ANOVA and Turkey’s or Dunett’s multiple comparison test (differential grouping from p ≤ 0.01) as indicated under each figure. Same letters indicate no statistical differences. All plasmid maps and cloning strategies were created and planned using the software VectorNTI®.
Author contributions
J.S., G.D., Y.S. and R.S. designed and planned the experiments. J.S. performed experiments and data analysis, besides counting carpels (Fig1-SupplFig.3), which was performed by J. Schmidt. G.D., K.G.P. and J.S. generated stable Arabidopsis lines. Y.S. provided material. G.D., P.B. and J.S. performed the cloning. J.S. and R.S wrote the manuscript with input from all authors.
Declaration of Interests
The authors declare no competing interests.
Supplementary Figures
(A) Wild type (Col-0) and different single and double mutants (clv3-9, cle40-2, clv1-101, clv1-101;cle40-2, bam1-3, bam1-3;cle40-2, bam1-3;clv1-101) at 4 WAG. (B) Leaf lengths were measured and plotted. Wild type (Col-0 N=47), cle40-2 (N=32) and bam1-3;cle40-2 (N=29) mutant plants do not show a significant difference in leaf length to each other. While bam1-3 (N=32) mutants exhibit in average significantly longer leaves than wild type plants, the single mutants clv3-9 (N=33) and clv1-101 (N=33) and the double mutants clv1-101;cle40-2 (N=32) and bam1-3;clv1-101 (N=45) show significantly shorter leaves. Statistical groups were assigned after calculating p-values by ANOVA and Turkey’s multiple comparison test (differential grouping from p ≤ 0.01).
(A) Wild type A. thaliana plants (Col-0) and cle40 mutants (cle40-2, cle40-cr1, cle40-cr2, cle40-cr3) at 6 WAG. All plants show a similar height ranging from 17.5cm to 21.2cm and do not have an obvious plant phenotype. (B) Schematic representation of the CLE40 gene, consisting of three exons (green arrows) and two introns. Exon 3 carries the crucial CLE box (dashed red line). (B’) Schematic representation of all four cle40 mutations. All four lines have mutations in or before the CLE box domain in Exon 3. cle40-2 mutants were created by transposon mutagenesis resulting in a stop codon in front of the CLE box (Stahl et al., 2009). cle40-cr1, cle40-cr2 and cle40-cr3 mutants were created using the CRISPR-Cas9 method (Yamaguchi et al., 2017). cle40-cr1 has an 11bp insertion inside the CLE box domain while cle40-cr2 and cle40-cr3 have a deletion of -34bp and -28bp within the CLE box. (C) At 6 WAG, IFMs of wild type (Col-0 N=16) and cle40 mutant plants were dissected and the area of each meristem was imaged and measured. All four cle40 mutants show significantly reduced IFM sizes compared to Col-0 plants (cle40-2 N=17, cle40-cr1 N=24, cle40-cr2 N=20, cle40-cr3 N=19). Statistical stars were assigned after calculating p-values by ANOVA and Dunett’s multiple comparison test (differential grouping from p ≤ 0.01).
(A) Carpels of Arabidopsis thaliana plants at 6 WAG in wild type (Col-0) or different mutant backgrounds: clv3-9, cle40-2, clv1-20, clv1-20;cle40-2, bam1-3, bam1-3;cle40-2, bam1-3;clv1-101. (B) Carpel number was counted and plotted. Wild type (Col-0 N=290), cle40-2 (N=300), bam1-3 (N=300) and bam1-3;cle40-2 (N=280) mutant plants always develop tow carpels, while clv3-9 (N=340) plants exhibits 3 to 5 carpels and clv1-20 (N=350) and clv1-20;cle40-2 (N=320) mutants show in average 2 to 3 carpels. The double mutant bam1-3;clv1-20 (N=280) develops 6 carpels in average. N number depicts number of siliques. Statistical groups were assigned after calculating p-values by ANOVA and Turkey’s multiple comparison test (differential grouping from p ≤ 0.01).
(A) MIP of CLV3 and WUS expression (CLV3:NLS-mCherry;WUS:NLS-GFP//clv3-9) in a clv3-9 mutant IFM (N=5). CLV3 expression is detected at the tip of the meristem, while WUS expression is predominantly found in young primordia surrounding the meristem. (A’) Optical section through the IFM shows an extended expression domain of CLV3 in the CZ and WUS expressing cells in OC of the IFM. (B) MIP of a clv3-9 mutant IFM expressing CLE40:Venus-H2B. CLE40 is expressed in the PZ of the IFM, in flower primordia and in mature sepals (N=6). (B’) Optical section through the IFM shows CLE40 expression in the outer layers of the PZ while it is lacking in the CZ and OC, where CLV3 and WUS are expressed.
Dashed orange line indicates the planes of optical sections; Scale bars: 50µm (C, D), 10µm (C’, D’), MIP = maximum intensity projection, PI = propidium iodide
(A) L.er. wild type plant at 5 WAG shows normal plant growth, while wus-7 mutants at 5 WAG are delayed in their development (dashed white line). (B) wus-7 mutant at 8 WAG. wus-7 mutants develop IFMs but give rise to sterile flowers that lack inner organs. (C and D) MIP of wus-7 IFMs at 5 WAG expressing CLE40:Venus-H2B. CLE40 expression is detected through the entire meristem and in the centre of primordia (N=12). (D’ and D’) Optical sections through the meristem show CLE40 expression in an extended pattern in the PZ and the OC.
Dashed white line in B encloses homozygous wus-7 mutants, dashed orange line indicates the planes of optical sections; Scale bars: 20mm (A, B), 20µm (C-D’)
Optical sections through an IFM and its developing primordia P1 to P6 expressing either (A) CLE40 (CLE40:Venus-H2B) (N=23) or (B) BAM1 (BAM1:BAM1-GFP) (N=15). In the IFM, CLE40 and BAM1 expression patterns overlap in the PZ, while both genes are lacking in the OC. No CLE40 expression is detected in young primordia in P1 to P3. From P4 on a faint signal in CZ of the primordia express CLE40. Its expression expands in P5 and can be found in almost all cells of P6. BAM1 is expressed ubiquitously in all primordia from P2 to P6.
Yellow lines (P1 to P6) indicate the IFM region, white lines (P1 to P6) mark the primordium, Scale bar: 10µm, P = primordium
IFMs of (A) Col-0, (B) clv3-9, (C) cle40-2, (D) clv1-101, (E) clv1-101;cle40-2, (F) bam1-3, (G) bam1-3,cle40-2 and (H) bam1-3;clv1-101 were imaged after 6 WAG. Z-stacks were taken from the top of the IFMs with a confocal microscope. A MIP and an optical section from P4 to P5 was performed for each meristem.
The yellow dashed line depicts the area of the meristem that was measured and the dashed red line indicates the height of the meristems. Scale bar: 20µm
(A) The width of Col-0 (N=11), cle40-2 (N=10), bam1-3 (N=11), clv1-101 (N=9) and clv3-9 (N=5) mutants at 5 WAG does not significantly differ from each other. The average width lays between 85 to 100µm. Wild type plants are in average 90µm wide while clv3-9 mutants depict the widest meristem average of 100µm. Only bam1-3 mutants have with an average of 79µm a significantly smaller meristem wide compared to wild type plants. (B) The height of cle40-2 (∼22µm) and bam1-3 (∼23µm) mutants is significantly shorter after 5 compared to wild type plants (∼28µm). In contrast, clv1-101 and clv3-9 have significantly higher meristems than Col-0, cle40-2 and bam1-3 mutants. (C) The σ-value represents the shape of the meristem and is defined by the quotient of height and width. cle40-2 and bam1-3 mutants have a significantly smaller σ-value compared to wild type plants, resulting in flatter meristems. clv1-101 and clv3-9 have with an average of 0.55 a significantly higher σ-value and thus have more dome-shaped meristems.
Green line in the inset meristem in (A) indicates the width that was used for the quantifications in (A); magenta line in the inset meristem in (B) indicates the height that was used for the quantifications in (B); green and magenta line in the inset meristem in (C) indicates the width and height that was used for the quantifications in (C),
(A) Optical sections of through IFMs show the expression patterns of CLV3 (N=8), WUS (N=8), BAM1 (N=9) and CLV1 (N=5) in a clv1 mutant. Compared to wild type plants, the expression of CLV3 and WUS is expanded and WUS is found in a patchy pattern in the L1. BAM1 expression shifts to the CZ and is found in an elevated expression in the L1. (B and C) Schematic representation of two intertwined negative feedback loops in the IFM of a clv1-101 mutant. The lack of CLV1 leads to a shift of BAM1 expression to the OC and to an elevated expression in the L1. In the L3, BAM1 can partly substitute for CLV1 and thus CLV3 can act via BAM1 in order to repress WUS activity. The elevated expression of BAM1 in the L1 overlaps in very few cells with CLE40 expression in the periphery and leads to a weak activation of the downstream signal “X” that promotes WUS activity. Since, WUS expression is only partly repressed by the CLV3-BAM1 signalling pathway, the WUS domain is extended and leads to an increase in stem cells (expanded CLV3 expression). WUS is now also detected in the L1 of the meristem, together with BAM1 expression. Scale bars: 20µm (A), CZ =central zone, L1 =layer 1
(A) Optical sections of through IFMs show the expression patterns of CLV3 (N=5), WUS (N=5), CLE40 (N=6), BAM1 (N=5) and CLV1 (N=5) in a clv3-9 mutant. Compared to wild type plants, the meristem is highly increased in its size along the apical-basal axis and the expression of CLV3 and WUS is expanded in the CZ and OC. CLE40 expression is limited to the outer layers of the meristemś periphery and excluded from the CZ and OC, while BAM1 expression shifts towards the inner layers of the PZ. CLV1 expression is found at the tip and not in the centre of the fasciated meristem. (B and C) Schematic representation of two intertwined negative feedback loops in the IFM of a clv3-9 mutant. The lack of CLV3 leads to a fasciated meristem with increased number of stem cells and thus an expanded CZ and a decreased PZ. Since no CLV3 peptide is available, CLV1 is not activated and expression of CLV1 shifts from the OC to the tip of the CZ, where it represses BAM1 expression. BAM1 is expressed in the inner layers of the PZ, while CLE40 expression is found in the outer layers of the PZ since it is repressed by the expanded WUS domain in the centre of the meristem. Thus only very few cells express both, BAM1 and CLE40 and hence, nearly no WUS promoting factor “X” is produced and the CLV3-CLV1 signaling pathway does not repress WUS activity. Scale bars: 20µm (A), CZ = central zone, PZ = peripheral zone
(A) Optical sections of through IFMs show the expression patterns of CLV3 (N=9), WUS (N=9), BAM1 (N=7) and CLV1 (N=9) in a cle40-2 mutant. CLV3 expression is similar to wild type plants, in the CZ. WUS expression is found in the OC, but in less cells than in Col-0 plants. BAM1 expression appears to be broader compared to wild type plants, while CLV1 expression seems to be decreased in its intensity. (B and C) Schematic representation of two intertwined negative feedback loops in the IFM of a cle40-2 mutant. In cle40-2 mutants, CLV1 expression seems to be decreased and leads to a broader BAM1 expression compared to wild type plants. Since expression of BAM1 is now also found in the CZ, CLV3 is able to bind CLV1 and BAM1 in the OC and CZ (respectively), leading to a double repression signalling cascade from the centre of the meristem. In the PZ, the downstream signaling cascade of BAM1 is not activated through CLE40 and thus the WUS promoting factor “X” is not being expressed and the WUS domain is confined to the centre of the OC.
Scale bars: 20µm (A), CZ = central zone, PZ = peripheral zone
(A) Optical sections of through IFMs show the expression patterns of CLV3 (N=9), WUS (N=9), BAM1 (N=15) and CLV1 (N=7) in a bam1-3 mutant. CLV3 expression is similar to wild type plants, at the tip of the meristem in a cone shaped domain. WUS expression is found in the OC, but in less cells than in Col-0 plants. CLV1 expression seems to increased in its intensity compared to wild type plants. (B and C) Schematic representation of two intertwined negative feedback loops in the IFM of a bam1-3 mutant. In bam1-3 mutants, CLV1 expression appears to be increased. Since BAM1 is lacking in the periphery, the WUS promoting diffusion factor “X” is not being produced and thus WUS expression is decreased and confined to the centre of the OC, similar to cle40-2 plants. With the loss of BAM1, the main receptor for CLE40 is missing, and thus CLE40 peptide now might signal through CLV1 leading to a stronger repression of WUS from the centre of the meristem.
Scale bars: 20µm (A), CZ = central zone, PZ = peripheral zone
Acknowledgements
This study was funded by DFG through iGrad-Plant (IRTG 2466), CRC 1208 and CEPLAS (EXC 2048). We thank Cornelia Gieseler, Silke Winters, and Carin Theres for technical support and Yasuka L. Yamaguchi (Sawa lab), Anne Pfeiffer (Lohmann lab) and Rene Wink (Simon lab) for sharing Arabidopsis seeds. We also thank Vicky Howe for proof reading the manuscript, the Center for Advanced imaging (CAi) at HHU for microscopy support and Aleksandra Sapala for support with MorphoGraphX.
References
