Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3 (CLV3), produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clv3 mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here, we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clv3 mutant meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation, in which cells simultaneously express distinct fate markers, is lost in mutants. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus, all benchmarks show that clv3 mutant meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem.

The shoot apical meristem (SAM) is a structure at the growing tip of a plant that gives rise to all its aboveground tissues. The SAM is sustained by the activity of a small pool of centrally located undifferentiated stem cells in the slow-dividing central zone (CZ). Through successive divisions, their daughter cells exit from the CZ into the surrounding peripheral zone (PZ), where they may differentiate into lateral organs, such as leaves or flowers. The CZ is itself maintained via the activity of the underlying organising centre (OC) (Laux et al., 1996).

The regulatory network driving stem cell maintenance in Arabidopsis is well studied. At its core is a feedback loop between WUSCHEL (WUS), a homeobox transcription factor expressed in the OC, and CLAVATA3 (CLV3), a signalling peptide expressed in the CZ (Brand et al., 2000; Clark et al., 1995; Laux et al., 1996; Schoof et al., 2000). WUS moves from the OC to the CZ and induces stem cell identity by directly binding to the CLV3 promoter and activating its expression (Daum et al., 2014; Yadav et al., 2011). In turn, CLV3 binds receptors, such as the leucine-rich-repeat receptor-like kinase CLV1 (Clark et al., 1997) or the receptor-like proteins CLV2 (Kayes and Clark, 1998) and CORYNE (CRN) (Fletcher et al., 1999; Jeong et al., 1999; Miwa et al., 2008; Müller et al., 2008). Signalling from these ligand-receptor complexes ultimately leads to the downregulation of WUS in the OC (Miwa et al., 2008; Müller et al., 2006). Additionally, CLV3 regulates WUS at the transcriptional and post-transcriptional levels, whereas WUS activates CLV3 in a concentration-dependent manner (Plong et al., 2021). The absence of WUS activity leads to SAM arrest early in development (Laux et al., 1996), whereas the loss of the CLV genes results in a vast enlargement of the SAM (Clark et al., 1993, 1995; Kayes and Clark, 1998) through a phenomenon called fasciation. Several examples of fasciated tissues have been observed in the wild, as well as in crop plants, including beef tomatoes and the maize fasciated ear2 mutant (Taguchi-Shiobara, 2001). It is thought that fasciation is caused by stem cell overproliferation in the CZ (Brand et al., 2000; Busch et al., 2010; Dao et al., 2022; Kwon et al., 2005; Lenhard and Laux, 2003; Müller et al., 2008; Nimchuk et al., 2011; Whitewoods et al., 2018; Wu et al., 2005) or, conceivably, by lower rates of cell transit from the CZ to the PZ (Laufs et al., 1998).

Over the years, CLV3 has been the only known genetic marker for studying stem cells at the shoot apex (Müller et al., 2006; Reddy and Meyerowitz, 2005), including for analysing clv mutants. Although microarray analyses have uncovered several other genes that are enriched in the CZ (Aggarwal et al., 2010; Yadav et al., 2009), none has been extensively characterised. More recently, atomic force microscopy (AFM) experiments, which measure cellular mechanical properties, have revealed that CLV3-expressing cells are stiffer than surrounding PZ cells, and that the onset of CLV3 expression in flowers coincides with an increase in cell wall stiffness (Milani et al., 2014). Thus, although stem cell identity is both spatially and temporally associated with increased cell stiffness (Milani et al., 2014), it is unknown how these mechanical patterns are altered when stem cell regulation is perturbed.

The core stem cell regulatory network is also influenced by hormone signalling, including cytokinin and auxin (Gordon et al., 2009; Busch et al., 2010). Exogenous cytokinin treatment phenocopies the clv mutant by increasing WUS expression and decreasing CLV1 expression (Lindsay et al., 2006). Although exogenous auxin treatment induces organogenesis markers in the PZ, the CZ itself remains unaffected, displaying reduced responsivity to auxin (de Reuille et al., 2006). Whereas in the PZ, the auxin response factor MONOPTEROS (MP) activates the expression of lateral organ identity genes in an auxin-dependent manner (Berleth and Jürgens, 1993; Bhatia et al., 2016; Hardtke and Berleth, 1998; Yamaguchi et al., 2013), it also functions to repress CZ genes that are themselves activators of CLV3 expression (Luo et al., 2018; Zhao et al., 2010). Despite these advances, it remains unclear how auxin signalling is perturbed in clv mutants and, furthermore, whether this plays a direct role in generating the fasciated clv phenotype.

In this study, we use quantitative approaches to study the effects of the loss of proper stem cell regulation that reveal hitherto unobserved cellular and tissular phenotypes, such as cell size and local surface curvature in clv mutant SAM. Analytical mechanical models predict that heterogeneities in stiffness and/or growth regimes are sufficient to account for the changes in surface curvature observed in the mutant. We provide experimental support for these predictions by showing that both growth and mechanical properties display regional differences, which are likely caused by the chimeric identities of mutant meristematic cells. Finally, auxin-response quantifications in clv mutant meristems reveal a behaviour similar to undifferentiated wild-type (WT) PZ cells, rather than to stem cells.

Altered cellular and tissular properties characterise fasciated clv SAM

In order to better understand the defects associated with abnormal stem cell regulation, we first quantified the highly fasciated phenotype of the canonical clv3-2 allele (Clark et al., 1995). WT shoot meristems displayed a stereotypical dome shape, whereas clv3-2 meristems comprised a large centrally-located bulge and two or more arm-like outgrowths that elongate laterally (Fig. 1A,B; Fig. S1A-C). Further analysis revealed that the central areas were usually devoid of any discernible cellular organisation, whereas the lateral arms were often composed of cell files (Fig. S1D,E). These results were consistent across three clv1 and four clv3 alleles (Fig. S1A-C), although individual plants showed varying degrees of fasciation.

Fig. 1.

Altered cell and tissue properties characterise fasciated SAM. (A,B) Brightfield images showing dissected WT Ler (A) and clv3-2 (B) SAM. The asterisk in B shows the central outgrowth and arrows point out two linear elongations. (C-F) Brightfield images showing a dissected clv3-21 SAM with filaments (arrows) on the side of the stem and abnormally shaped flowers (arrowheads) (C), a close-up of the filaments (D) and clv3-21 abnormally-shaped flowers (E,F). (G,H) Boxplots of cell areas (G) or cell volumes (H) in the L1 (black) and L2 (grey) cell layers of WT and clv3-2 SAM. Mean±s.d. areas (G) are 23.9±5.8 µm2 (WT L1) and 24.5± 7.2 µm2 (WT L2), 18.4±5.3 µm2 (clv3 L1) and 31.2±9.8 µm2 (clv3 L2). P-values (Welch's t-test)=0.545 (ns, WT) and 4.65e-54 (***, clv3-2). n=103 (L1) and 115 (L2) cells from three WT SAM; n=400 (L1) and 244 (L2) cells from three clv3-2 SAM. Mean±s.d. volumes (H) are 136.5±40.5 µm3 (WT L1) and 148.7± 44.5 µm3 (WT L2), 129.5±40.9 µm3 (clv3 L1) and 249.1±130.7 µm3 (clv3 L2). P-values (Welch's t-test)=2.32e-9 (***, WT) and 1.53e-204 (***, clv3-2). n=1468 (L1) and 529 (L2) cells from four WT SAM; 6426 (L1) and 3428 (L2) cells from three clv3-2 SAM. (I,J) Colour maps quantifying minimal curvature in representative WT Col-0 (I) and in clv3-2 (J) SAM. The clv3-2 SAM (J) is only a small region of the entire meristem. Colourmap: warm helix (I,J). Scale bars: 30 μm (A,B,I,J); 1 mm (C); 200 μm (D); 100 μm (E,F).

Fig. 1.

Altered cell and tissue properties characterise fasciated SAM. (A,B) Brightfield images showing dissected WT Ler (A) and clv3-2 (B) SAM. The asterisk in B shows the central outgrowth and arrows point out two linear elongations. (C-F) Brightfield images showing a dissected clv3-21 SAM with filaments (arrows) on the side of the stem and abnormally shaped flowers (arrowheads) (C), a close-up of the filaments (D) and clv3-21 abnormally-shaped flowers (E,F). (G,H) Boxplots of cell areas (G) or cell volumes (H) in the L1 (black) and L2 (grey) cell layers of WT and clv3-2 SAM. Mean±s.d. areas (G) are 23.9±5.8 µm2 (WT L1) and 24.5± 7.2 µm2 (WT L2), 18.4±5.3 µm2 (clv3 L1) and 31.2±9.8 µm2 (clv3 L2). P-values (Welch's t-test)=0.545 (ns, WT) and 4.65e-54 (***, clv3-2). n=103 (L1) and 115 (L2) cells from three WT SAM; n=400 (L1) and 244 (L2) cells from three clv3-2 SAM. Mean±s.d. volumes (H) are 136.5±40.5 µm3 (WT L1) and 148.7± 44.5 µm3 (WT L2), 129.5±40.9 µm3 (clv3 L1) and 249.1±130.7 µm3 (clv3 L2). P-values (Welch's t-test)=2.32e-9 (***, WT) and 1.53e-204 (***, clv3-2). n=1468 (L1) and 529 (L2) cells from four WT SAM; 6426 (L1) and 3428 (L2) cells from three clv3-2 SAM. (I,J) Colour maps quantifying minimal curvature in representative WT Col-0 (I) and in clv3-2 (J) SAM. The clv3-2 SAM (J) is only a small region of the entire meristem. Colourmap: warm helix (I,J). Scale bars: 30 μm (A,B,I,J); 1 mm (C); 200 μm (D); 100 μm (E,F).

We also detected novel floral phenotypes. First, we frequently observed radially symmetrical, filament-like organs either growing on the flanks of the meristem or within the inflorescence itself (Fig. 1C,D). Such filaments were recently noted as aborted flowers in double mutants of clv2 or clv3 and with auxin biosynthesis mutants (John et al., 2023; Jones et al., 2020). Second, although the majority (>90%) of flowers showed previously described phenotypes of supernumerary organs (Clark et al., 1993, 1995), the rest displayed two types of opposing phenotypes. One group of flowers bore two or three sepals and petals, a single stamen and no carpels (Fig. 1C,E,F), reminiscent of the inner-whorl phenotypes of wus mutants (Laux et al., 1996), whereas a second group displayed a tubular phenotype with fused sepals (Fig. 1C). These flowers were distinctly smaller than those with supernumerary organs. These novel floral phenotypes were observed in all clv1 and clv3 alleles in our culture conditions.

Because it had been suggested that cell size in clv mutants differs from the WT (Laufs et al., 1998), we wondered whether we could use such changes in cellular morphologies to further characterise the mutant phenotypes described above. We quantified cell size in two ways. First, we calculated cell areas by acquiring images of meristems from WT and multiple clv1 and clv3 SAM and extracting slices near the midpoints of cells in the L1 and L2 layers, which we then segmented using the MorphoGraphX software (Barbier de Reuille et al., 2015) (Fig. S1F,G). In WT SAM, the average area was similar in both cell layers, with L1 cells at 23.9±5.8 µm2 and L2 cells at 24.5±7.2 µm2 (mean±s.d.; Fig. 1G). In contrast, clv3-2 mutants exhibited significant differences in the L1 and L2, with L2 cells on average 69% larger than L1 cells – 31.2±9.8 µm2 in the L2 versus 18.4±5.3 µm2 in the L1 (Fig. 1G). Secondly, we measured cell volumes in WT and clv mutant SAM, by generating 3D reconstructions using the MultiAngle Reconstruction and Segmentation (MARS) pipeline (Fernandez et al., 2010), which involves imaging samples from multiple angles and fusing those images to generate high-resolution images that are then segmented in 3D (Fig. S1H). Consistent with our findings for cell areas, we found that, although L1 and L2 cells had comparable volumes in the WT (136.5±40.5 µm3 and 148.7±44.6 µm3, respectively), L2 cells in clv3-2 SAM were almost twice as large as L1 cells (249.1±130.7 µm3 versus 129.5±40.9 µm3) (Fig. 1H). Similar trends were observed for cell size in other clv1 and clv3 alleles, suggesting that altered cell morphology is a general feature of these mutants (Fig. S1I).

We had noticed that meristems in clv3 mutants are less smooth than WT meristems. We quantified this by measuring curvature at the sample surface, using a method that detects the outer surface in MARS-reconstructed 3D meristems (Kiss et al., 2017). We used MorphoGraphX to calculate curvature within a 10 µm radius of every pixel on the surface. In WT plants, the meristem dome and young flowers were regions of uniformly positive curvature, separated by clearly defined boundary areas with negative curvature (Fig. 1I). However, the imaged areas of clv1 and clv3 SAM, which are distant from the regions where flowers form, displayed a much more variable surface curvature, with crests and troughs of varying shapes and depths distributed throughout (Fig. 1J). Together, our data reveal that clv mutants exhibit abnormal cellular traits and buckling defects that contrast with the stereotypical characteristics of WT meristems.

Morphoelastic models reveal how growth and stiffness contribute to tissue buckling

To elucidate a theoretical basis for the buckling defects of clv mutants, we next generated analytical models of SAM development using a growth and remodelling framework (Goriely, 2017). Specifically, we adapted a model developed to explore the biomechanical basis of morphogenesis in unrelated systems that display similar buckling behaviour to that observed in clv mutants (Almet et al., 2019; Moulton et al., 2013). We modelled the SAM as an axially growing planar elastic rod (the outer L1 layer), attached to an elastic foundation (the inner layers), and clamped at the two ends (Fig. S2). We used the theoretical model to explore how growth stretch, a proxy for tissue growth, and/or mechanical properties, a proxy for cellular stiffness, might contribute to the altered morphology of clv mutants (see supplementary Materials and Methods, Analytic models for further details).

First, we used linear stability analysis to plot the output shapes when either the growth stretch (γ) alone or the system stiffness (, the ratio of foundation and rod stiffnesses) was varied (Fig. S3A). These analyses showed that when the foundation and rod had similar mechanical properties and when growth stretch was at a low, subcritical value (γ*inf=5.82), the output shape resembled the smooth dome of a WT meristem (Fig. 2A). In contrast, when the growth stretch was raised to a near-critical point (γ*inf=8.99), the size of the dome increased, while remaining smooth. A further increase in growth stretch generated various modes of buckling (Fig. 2A) that broadly capture the curvature defects of clv mutants. Next, we investigated the role of system stiffness on SAM morphology, by varying stiffness within a background of constant growth stretch (γ*=12.23) (Fig. 2B). Although no buckling was visible for higher values of stiffness , lowering it led to the appearance of buckling in the rod (Fig. 2B), which was similar to those obtained using growth values above the critical point (Fig. 2A). Taken together, these analytical results indicate that, in a biological context, cellular growth and stiffness characteristics could influence tissue architecture of the SAM.

Fig. 2.

Growth and stiffness influence SAM shapes via growth-induced buckling. (A) Deformed shapes due to growth-induced buckling obtained for different values of critical growth rates (γ*) causing various buckling modes, with the stiffness of the underlying foundation fixed . (B) Deformed shapes due to stiffness-induced buckling obtained for different values of foundation stiffness (, , ), with the growth rate fixed (γ*=12.23). (C) The choice of growth law affects the deformed shapes. We select three different forms of growth laws for the same value of growth rate γ0: cosine, linear and exponential. (D) Deformed shape obtained as a result of growth-induced buckling in the case of cosine growth distribution. (E) Deformed shape obtained as a result of stiffness-induced buckling in the case of cosine stiffness distribution. (F) Effect of foundation stiffness on the asymmetric rod shapes obtained using a cosine growth distribution. The colour bar in D,E represents the magnitude of growth and stiffness, respectively, that was varied along the length of the rod.

Fig. 2.

Growth and stiffness influence SAM shapes via growth-induced buckling. (A) Deformed shapes due to growth-induced buckling obtained for different values of critical growth rates (γ*) causing various buckling modes, with the stiffness of the underlying foundation fixed . (B) Deformed shapes due to stiffness-induced buckling obtained for different values of foundation stiffness (, , ), with the growth rate fixed (γ*=12.23). (C) The choice of growth law affects the deformed shapes. We select three different forms of growth laws for the same value of growth rate γ0: cosine, linear and exponential. (D) Deformed shape obtained as a result of growth-induced buckling in the case of cosine growth distribution. (E) Deformed shape obtained as a result of stiffness-induced buckling in the case of cosine stiffness distribution. (F) Effect of foundation stiffness on the asymmetric rod shapes obtained using a cosine growth distribution. The colour bar in D,E represents the magnitude of growth and stiffness, respectively, that was varied along the length of the rod.

In contrast to the symmetrical shapes obtained above, clv mutant SAM in fact display highly variable buckling, such that the positions, numbers and amplitudes of folds differ within, and between, individual mutant meristems. We reasoned that this variability might result from local differences in growth, stiffness or both. To investigate the effects of such heterogeneities on buckling, we replaced constant growth stretch or system stiffness with one of three distinct functions to generate differential distributions across the rod (Fig. 2C). Indeed, all three functions generated variable buckling over the length of the rod when either growth stretch (Fig. 2D; Fig. S3B,B′) or system stiffness (Fig. 2E) was spatially distributed, with the resulting shapes often closely resembling the asymmetry and variable buckling amplitudes visible in sections through various clv mutant SAMs in the literature as well as in our experiments (Fig. S3C-G). These results suggest that local differences in growth or stiffness within the SAM could explain the phenotypes of clv mutants.

Finally, we asked whether variations in system stiffness and growth stretch could act together to generate even greater differences in buckling characteristics. To address this question, we used a cosine spatial growth stretch distribution and examined how buckling was affected when system stiffness was varied between 0.5 and 1.5. Indeed, for identical distributions of growth, rod buckling varied locally as a function of stiffness (Fig. 2F); these analyses suggest that small changes at the cellular level could alter shape locally.

Taken together, our results indicate that growth and mechanical traits are sufficient to describe the differences in meristem shape between WT and clv mutant SAM. Furthermore, local variations of these two cellular parameters could lead to the local curvature defects observed in mutant meristems.

Heterogeneous growth correlates with buckling in clv mutants

We tested predictions from the analytical model by first examining whether clv mutants display local heterogeneities in growth and, more specifically, whether these differences correlate with areas of differential curvature. We did this by dissecting WT and clv3-7 meristems, placing them on culture medium and imaging them once a day over 72 h by confocal microscopy. We then segmented the image stacks, tracked cell lineages and analysed cell size, growth and curvature over the tissue (Fig. 3A-H; Fig. S4A-C). In the WT, cells had a mean size of 22.8±5.8 µm2 at the start of the experiment and over 72 h their daughters had grown 5.5±1.3-fold (Fig. 3A-C). Of these, lineages that remained entirely within the meristem dome grew 4.9±1.1-fold. In contrast, cells at the very top of the clv3 meristem had an initial size of 17±6.2 µm2 and had grown 1.5±0.3-fold over 72 h (Fig. 3E-G). This is consistent with previous studies showing reduced proliferation in clv3-1 mutants (Laufs et al., 1998).

Fig. 3.

Regional growth heterogeneities correlate with surface buckling in clv mutants. (A-H) 3D surface reconstruction of a WT Col-0 (A-D) and a clv3-7 (E-H) SAM, expressing the pUBQ10::Lti6b-tdTomato membrane marker, at the start of the experiment (A,E) and 72 h later (B,F). Solid white line in A and B delineates the same group of cells. Segmented cells are coloured by lineage and outlines of unsegmented cells are shown. For each lineage, heatmaps for either growth fold-change over 72 h (C,G) or minimal curvature (D,H) are represented on the second time point. (I-L) Scatter plots of minimal curvature per lineage as a function of fold-growth per lineage (I,K) or of minimal curvature per lineage as a function of initial lineage surface area (J,L) in WT (I,J) and clv3-7 (K,L) SAM. Regression lines representing the best fit are shown in blue, and confidence intervals as shaded regions around them. n=64 WT lineages (I,J); n=1064 clv3 lineages (K,L). Pearson's correlation coefficients (r) are shown on the plots. P-values: P=0.18 (I); P=0.79 (J); P<0.001 (K); P<0.001 (L). Colourmap: Turbo (C,G); warm helix (D,H). Scale bars: 20 µm (A-H).

Fig. 3.

Regional growth heterogeneities correlate with surface buckling in clv mutants. (A-H) 3D surface reconstruction of a WT Col-0 (A-D) and a clv3-7 (E-H) SAM, expressing the pUBQ10::Lti6b-tdTomato membrane marker, at the start of the experiment (A,E) and 72 h later (B,F). Solid white line in A and B delineates the same group of cells. Segmented cells are coloured by lineage and outlines of unsegmented cells are shown. For each lineage, heatmaps for either growth fold-change over 72 h (C,G) or minimal curvature (D,H) are represented on the second time point. (I-L) Scatter plots of minimal curvature per lineage as a function of fold-growth per lineage (I,K) or of minimal curvature per lineage as a function of initial lineage surface area (J,L) in WT (I,J) and clv3-7 (K,L) SAM. Regression lines representing the best fit are shown in blue, and confidence intervals as shaded regions around them. n=64 WT lineages (I,J); n=1064 clv3 lineages (K,L). Pearson's correlation coefficients (r) are shown on the plots. P-values: P=0.18 (I); P=0.79 (J); P<0.001 (K); P<0.001 (L). Colourmap: Turbo (C,G); warm helix (D,H). Scale bars: 20 µm (A-H).

In the WT, a gradient of differential growth was observed from the centre to the periphery of the meristem, whereas in the mutant, groups of cells with varying growth rates were seemingly scattered across the enlarged meristem (Fig. 3C,G). To determine whether this observation was relevant in the context of buckling in clv mutant meristems, we then measured curvature at the surface of WT and mutant samples, determined the mean value per cell (Fig. 3D,H; Fig. S4C) and tested correlations between cell size, growth rate and local curvature (Fig. 3I-L; Fig. S4D,E). WT meristems showed no significant correlations between any parameters [Pearson's correlation coefficients (r) between: growth and curvature, r=−0.17, P=0.18; size and curvature, r=−0.035, P=0.79; growth and size, r=−0.23, P=0.07] (Fig. 3I,J; Fig. S4D). clv3 mutants, however, displayed significant moderate or strong positive correlations between all parameters (Pearson's correlation coefficients between: growth and curvature, r=0.59, P<0.001; size and curvature, r=0.41, P<0.001; growth and size, r=0.36, P<0.001) (Fig. 3K,L; Fig. S4E). To account for differences in numbers of cell lineages between genotypes, we confirmed that significant correlations also exist in randomly selected subsets of mutant lineages equal in size to the WT (Fig. S4F). These analyses show that in clv3 mutants, both cell size and growth rate are variable across the tissue and are linked to local buckling characteristics.

Loss of CLV activity is associated with reduced and variable epidermal cell stiffness

In addition to growth heterogeneity, our analytical models predicted that buckling is also dependent on stiffness changes in the SAM. We had previously shown that CLV3-expressing CZ cells are stiffer than PZ cells (Milani et al., 2014). Because the clv mutant phenotype is thought to be caused by stem cell overproliferation, we predicted that cells in clv mutant meristems might bear higher values of stiffness. To test this, we probed epidermal cells in WT, clv1 and clv3 SAM using AFM, as previously published (Milani et al., 2014; Beauzamy et al., 2015) (Fig. S5A-D). The resultant force-displacement curves at each measured point from individual AFM scans were analysed to determine the apparent Young's modulus up to a depth of 100 nm, and these values were subsequently corrected to remove artefacts caused by local slope (Fig. 4A-D). The data were either pooled and plotted as density curves (Fig. 4E,F) or analysed by individual scans as box plots (Fig. 4G,H). When compared with the appropriate WT accession, we found that both clv3-2 (Fig. 4E,G) and clv1-8 cells (Fig. 4F,G) were significantly softer, with apparent Young's moduli that were 15-20% lower than WT cells (Fig. 4G; Tables S1, S2). Similar trends were observed in other clv alleles (Fig. S5E; Table S1).

Fig. 4.

Loss of clv is associated with reduced and heterogenous stiffness. (A-D) Maps showing apparent Young's moduli derived from atomic force microscope scans of representative Ler (A), Col-0 (B), clv3-2 (C) and clv1-8 (D) SAM. (E,F) Density plots of apparent Young's moduli from all probed WT (Ler) and clv3-2 (E), and WT (Col-0) and clv1-8 (F) samples. (G,H) Box plots of either mean values (G) or standard deviation values (H) of apparent Young's moduli measurements of scans from all probed samples. Black dots show values of different samples probed by a single scan, whereas coloured dots represent values from samples bearing multiple non-overlapping scans, with each colour identifying an individual sample. Overall mean values derived from individual mean apparent Young's moduli (G): Ler, 12.2±1.2 MPa; clv3-2, 9.7±0.7; Col-0, 10.58±0.4; and clv1-8, 8.94±0.5. Overall mean values derived from individual standard deviations (H): Ler, 1.7±0.3 MPa; clv3-2, 1.9±0.4; Col-0, 1.2±0.2; clv1-8, 1.9±0.3. P-values (Welch's t-test): WT–clv3, 6.65e-8 (***) and WT–clv1, 1.85e-7 (***) (G); WT–clv3, 3.4e-2 (*) and WT–clv1, 7.1e-5 (***) (H). n (non-overlapping scans/independent meristems): Ler, 18/11; Col-0, 8/2; clv3-2, 34/8; and clv1-8, 16/1.

Fig. 4.

Loss of clv is associated with reduced and heterogenous stiffness. (A-D) Maps showing apparent Young's moduli derived from atomic force microscope scans of representative Ler (A), Col-0 (B), clv3-2 (C) and clv1-8 (D) SAM. (E,F) Density plots of apparent Young's moduli from all probed WT (Ler) and clv3-2 (E), and WT (Col-0) and clv1-8 (F) samples. (G,H) Box plots of either mean values (G) or standard deviation values (H) of apparent Young's moduli measurements of scans from all probed samples. Black dots show values of different samples probed by a single scan, whereas coloured dots represent values from samples bearing multiple non-overlapping scans, with each colour identifying an individual sample. Overall mean values derived from individual mean apparent Young's moduli (G): Ler, 12.2±1.2 MPa; clv3-2, 9.7±0.7; Col-0, 10.58±0.4; and clv1-8, 8.94±0.5. Overall mean values derived from individual standard deviations (H): Ler, 1.7±0.3 MPa; clv3-2, 1.9±0.4; Col-0, 1.2±0.2; clv1-8, 1.9±0.3. P-values (Welch's t-test): WT–clv3, 6.65e-8 (***) and WT–clv1, 1.85e-7 (***) (G); WT–clv3, 3.4e-2 (*) and WT–clv1, 7.1e-5 (***) (H). n (non-overlapping scans/independent meristems): Ler, 18/11; Col-0, 8/2; clv3-2, 34/8; and clv1-8, 16/1.

While these results show that clv mutant SAM bear different physical characteristics to WT cells, they do not reveal whether they also display greater heterogeneity. To test this, we plotted standard deviation values for each scanned region by genotype (Fig. 4H) and found that clv mutant samples indeed display a significantly greater variability in values of apparent Young's moduli when compared to the WT. This altered regime is also visible in the density plots (Fig. 4E,F), where mutant distributions are notably broader than in the WT. Furthermore, scatter plots of the dataset show that mean and standard deviation are not correlated (Fig. S5F-I). Consistent with our analytical models, these results suggest that altered local stiffness and increased heterogeneity could indeed contribute to tissue buckling in clv mutants.

Genetic domain separation is lost in clv SAM

We reasoned that the local heterogeneities in growth and stiffness described above might be caused by underlying variations in genetic identity. To examine this, we first retested the expression patterns of OC and CZ markers using mRNA in situ hybridisation assays in WT and different clv3 alleles. We used WUS to examine OC identity (Laux et al., 1996; Mayer et al., 1998), whereas for the CZ we tested several genes previously identified as enriched in CLV3-expressing cells (Yadav et al., 2009) and retained the APUM10 gene, which encodes a member of the Puf family, with a conserved Pumillio homology domain (Tam et al., 2010). APUM10 was observed in a few centrally located cells of the L1 and L2 in WT SAM (Fig. 5A), and WUS in an underlying group of cells (Fig. 5B). In clv3 mutants, we detected both APUM10 and WUS in broad domains occupying almost the entire meristem, with APUM10 expressed principally in the L2 layer (Fig. 5C,C′) and WUS in deeper layers (Fig. 5D,D′). However, both genes were expressed in a discontinuous manner, with no detectable expression in scattered groups of cells. An inspection of published expression data in clv mutants revealed that such patchy expression was frequently visible, but had gone unremarked (Brand et al., 2000; Kinoshita et al., 2015; Schuster et al., 2014).

Fig. 5.

Gene expression is heterogenous in clv3 SAM. (A-F′) Pattern of mRNA localisation for APUM10 (A,C,C′), WUS (B,D,D′), and MP (E-F′) in WT Ler (A,B,E) or clv3-2 (C,C′,D,D′,F,F′) SAM. C′, D′ and F′ are magnified views of the boxed regions in C, D and F, respectively. (G-L′) Pairwise localisation of APUM10 (G,H,H′,J,K,K′), WUS (G′,I,I′), and MP (J′,L,L′) transcripts on alternating sections of meristems from individual WT Ler (G,G′,J,J′) or clv3-2 (H-I′,K-L′) plants. Arrowheads (H′,I′,K′,L′) indicate zones where only one of the two transcripts is detected, asterisks (K′,L′) indicate regions where neither is detected, and arrows indicate areas where both are detected. H′, I′, K′ and L′ are magnified views of the boxed regions shown in H, I, K and L, respectively. Section thickness: 10 μm. Scale bars: 50 µm (A-C,E,F,G,G′,H′,I′,J,J′,K′,L′); 100 µm (D,H,I,K,L); 25 µm (C′,D′,F′).

Fig. 5.

Gene expression is heterogenous in clv3 SAM. (A-F′) Pattern of mRNA localisation for APUM10 (A,C,C′), WUS (B,D,D′), and MP (E-F′) in WT Ler (A,B,E) or clv3-2 (C,C′,D,D′,F,F′) SAM. C′, D′ and F′ are magnified views of the boxed regions in C, D and F, respectively. (G-L′) Pairwise localisation of APUM10 (G,H,H′,J,K,K′), WUS (G′,I,I′), and MP (J′,L,L′) transcripts on alternating sections of meristems from individual WT Ler (G,G′,J,J′) or clv3-2 (H-I′,K-L′) plants. Arrowheads (H′,I′,K′,L′) indicate zones where only one of the two transcripts is detected, asterisks (K′,L′) indicate regions where neither is detected, and arrows indicate areas where both are detected. H′, I′, K′ and L′ are magnified views of the boxed regions shown in H, I, K and L, respectively. Section thickness: 10 μm. Scale bars: 50 µm (A-C,E,F,G,G′,H′,I′,J,J′,K′,L′); 100 µm (D,H,I,K,L); 25 µm (C′,D′,F′).

One explanation for why some patches of cells in clv3 mutants expressed neither CZ nor OC identity was that they might instead bear other identities. To test this, we localised mRNA for the MP gene (Hardtke and Berleth, 1998), which is expressed mainly in the WT PZ (Zhao et al., 2010) (Fig. 5E). In clv mutant SAM, the PZ is thought to be restricted to areas at the base of the enlarged dome, in the region in which flowers form. However, we found that in clv3 mutants, MP is expressed in a broad and patchy manner (Fig. 5F,F′), similar to the APUM10 and WUS patterns. Taken together, our results suggest that identities of the three meristematic zones are present in overlapping groups of cells in clv mutant apices.

We thought it possible that normal domain organisation might exist at a more local level. In order to investigate the positions of the OC and PZ markers relative to the CZ marker within clv mutant SAM, we performed pairwise in situ hybridisations using alternating sections from individual fixed meristems, with one probe used on even-numbered sections, and a second on odd-numbered sections. In WT meristems, the APUM10, WUS and MP expression domains were never detected in similar regions within adjacent sections (Fig. 5G,G′,J,J′). However in clv3 mutants, APUM10 and WUS expression overlapped across the entire SAM (Fig. 5H-I′; Fig. S6), except for a few areas where APUM10 was observed but WUS was not (Fig. 5H′,I′) and rare areas where WUS was expressed without APUM10. Similarly, in clv3 mutants APUM10 and MP were expressed in overlapping patterns, except for a few areas where MP was expressed without APUM10 (Fig. 5K-L′; Fig. S7) and no instances where APUM10 was expressed without MP. We also observed a few cell patches devoid of any signal.

Together, these results show that clv3 meristems comprise a heterogeneous group of cells with mixed genetic identities, rather than a homogeneous population of stem cells. Furthermore, we observed similar expression patterns in SAM from other alleles of clv1 and clv3 (Fig. S8), suggesting that chimeric cell identities are a general feature of fasciated clv mutant meristems.

Exogenous auxin elicits a strong response in clv3 meristems

Because cells in fasciated clv3 SAM displayed traits not usually associated with stem cells, such as chimeric cell identities and reduced stiffness, we wished to better understand whether they were also functionally different from stem cells. We chose auxin response as a functional readout for the following reasons. First, auxin-associated genes are differentially expressed in WT and clv3 mutants (John et al., 2023). Second, a key aspect of organogenesis at the shoot apex is the presence of a CZ that is refractive to auxin signalling (de Reuille et al., 2006; Douady and Couder, 1996; Galvan-Ampudia et al., 2020; Vernoux et al., 2011), suggesting that auxin insensitivity is an indicator of stem cell identity. Third, MP, a polarity regulator of the auxin efflux transporter PIN1, which itself promotes auxin accumulation and drives organogenesis (Bhatia et al., 2016), is misregulated in clv mutants (Fig. 5E,F).

In order to test how clv mutants respond to auxin, we dissected shoot apices from WT, clv1 and clv3 plants carrying the auxin signalling reporter, pDR5rev::GFPer, and treated them with exogenous auxin (Galvan-Ampudia et al., 2020), while monitoring expression every 24 h. As is well understood, in 100% of WT samples before treatment, GFP was localised in small groups of cells corresponding to the positions of floral anlagen, accompanied by low expression in the geometric centre of the SAM (Fig. 6A). After 24 h, we observed high levels of GFP throughout the periphery of the SAM and in internal layers, whereas expression levels at the centre remained low (Fig. 6B). clv mutant meristems differed from the WT in that, before auxin treatment, we detected only very basal levels – several thousand-fold lower than in the WT – of GFP (Fig. 6C), whereas 24 h after treatment, a clear response was visible in all samples (Fig. 6D). More specifically, a minority of meristems (1 out of 11 clv3 SAM) showed high levels of GFP in both outer and inner layers (Fig. S9A,B), whereas in all other samples (10 out of 11 clv3, and 5 out of 5 clv1) expression levels were variable and patchy in outer layers, and high in inner layers (Fig. 6D).

Fig. 6.

clv3 meristematic cells respond strongly to exogenous auxin treatment. (A-D) Projections of confocal stacks showing the effect of IAA on WT Col-0 (A,B) and clv3-2/clv3-21 (C,D) SAM expressing the pUBQ10::Lti6b-tdTomato and pDR5::GFPer reporters. Samples are shown either before (A,C) or 25 h after (B,D) a 5-h IAA treatment. Orthogonal views of the SAM are shown alongside each panel. n=12 WT SAM (five independent experiments), n=11 clv3 SAM (four independent experiments). (E-L) Quantification of response to IAA treatment observed in representative SAM from WT Col-0 (E-H) or clv3-2/clv3-21 heterozygotes (I-L) carrying the pUBQ10::Lti6b-tdTomato and pDR5::GFPer reporters. Views of the SAM surface before (E,I) and after (F,J) treatment, showing segmented cells coloured by lineage and unsegmented cells as grey outlines. Numbers in E represent the phyllotactic order of flowers from oldest to youngest. Heatmaps show DR5 signal per cell presented as a concentration (G,H,K,L) with a common colour scale (G). (M) Violin plot reflecting distributions of DR5 fold-change per cell lineage between clv3 SAM and putative WT CZ and PZ (shown in E-L). ***P<0.001 (Welch's t-test). n=12 cells for WT CZ; n=300 cells for WT PZ; n=713 cells for clv3. Colourmap: Turbo (G,H,K,L). Scale bars: 50 µm (main views in A-D); 20 µm (orthogonal sections in A-D,F-H,J,L); 10 μm (E,I,K).

Fig. 6.

clv3 meristematic cells respond strongly to exogenous auxin treatment. (A-D) Projections of confocal stacks showing the effect of IAA on WT Col-0 (A,B) and clv3-2/clv3-21 (C,D) SAM expressing the pUBQ10::Lti6b-tdTomato and pDR5::GFPer reporters. Samples are shown either before (A,C) or 25 h after (B,D) a 5-h IAA treatment. Orthogonal views of the SAM are shown alongside each panel. n=12 WT SAM (five independent experiments), n=11 clv3 SAM (four independent experiments). (E-L) Quantification of response to IAA treatment observed in representative SAM from WT Col-0 (E-H) or clv3-2/clv3-21 heterozygotes (I-L) carrying the pUBQ10::Lti6b-tdTomato and pDR5::GFPer reporters. Views of the SAM surface before (E,I) and after (F,J) treatment, showing segmented cells coloured by lineage and unsegmented cells as grey outlines. Numbers in E represent the phyllotactic order of flowers from oldest to youngest. Heatmaps show DR5 signal per cell presented as a concentration (G,H,K,L) with a common colour scale (G). (M) Violin plot reflecting distributions of DR5 fold-change per cell lineage between clv3 SAM and putative WT CZ and PZ (shown in E-L). ***P<0.001 (Welch's t-test). n=12 cells for WT CZ; n=300 cells for WT PZ; n=713 cells for clv3. Colourmap: Turbo (G,H,K,L). Scale bars: 50 µm (main views in A-D); 20 µm (orthogonal sections in A-D,F-H,J,L); 10 μm (E,I,K).

We next quantified this response by segmenting image stacks of WT (Fig. 6E,F) or mutant (Fig. 6I,J) meristems before and after auxin treatment, and extracted average fluorescence values per cell (Fig. 6G,H,K,L). For each cell, we then calculated the ratio of expression between the two time points, normalised this across the sample, and compared values in the mutant with values for cells in either the geometric centre or the periphery of WT meristems. A histogram of cell signal intensities shows that, before auxin treatment, 22% of WT cells and 100% of mutant cells were in a low-expressing category of signal intensities under an arbitrary threshold set to 1/10th of the highest value (Fig. S9C). After treatment with auxin, only 6.4% of WT and 5.9% of mutant cells remained in this category, suggesting that the clv mutant meristem responded to exogenous auxin in a similar manner to the WT. Furthermore, plots of normalised intensity change per lineage show that cells from the putative WT CZ and WT PZ, as well as from the clv3 mutant, had significantly different distributions from each other, although qualitatively the mutant resembled the PZ more than the CZ (Fig. 6M). Similar results are obtained for clv1 mutants carrying the DR5 reporter (Fig. S9D,E). Taken together, these results show that cells in clv mutant SAM are capable of responding to auxin signalling, an output more associated with the WT PZ, where organ specification occurs.

The development of fasciated meristems due to the loss of CLV activity in Arabidopsis shoot apical stem cells has generally been interpreted as being caused by a massive increase in stem cell numbers (Brand et al., 2002; Busch et al., 2010; Dao et al., 2022; Kwon et al., 2005; Lenhard and Laux, 2003; Müller et al., 2006; Nimchuk et al., 2011; Whitewoods et al., 2018; Wu et al., 2005). Direct experimental evidence for this comes principally from a CLV3 transcriptional reporter, which is expressed throughout the enlarged meristem of clv mutants (Brand et al., 2002; Reddy and Meyerowitz, 2005). However, because WUS protein is known to directly activate CLV3 transcription (Yadav et al., 2011), and because WUS is expressed throughout clv mutant meristems (Schoof et al., 2000), it is not surprising that CLV3 reporter expression is broad in the clv mutant. We suggest that the CLV3 reporter might thus not be an appropriate marker to study cell identity in clv mutant meristems. Furthermore, the use of the CLV3 promoter for various misexpression studies of the role of stem cells in meristem maintenance might also be inappropriate.

An alternate way of deciphering regional identities is by characterising cellular properties, which we have done using recently developed approaches to precisely quantify cell volumes, areas and surface curvatures. Although we show that epidermal and subepidermal cell sizes in clv mutant meristems are different from the WT, the effect of such changes on meristem structure is difficult to estimate. One possibility is that they generate a higher frequency of cell boundary overlaps between the L1 and L2 layers, which could generate a mechanically fragile structure, akin to a wall built with small bricks in one layer and much larger bricks in the next.

Our analytical model attempts to explain meristem morphology through a mechanical framework, in contrast to various other modelling studies that have shed light on different aspects of meristem shape and function, such as the role of auxin in phyllotactic patterning or homeostasis of the CLV-WUS feedback loop (Banwarth-Kuhn et al., 2022; Battogtokh and Tyson, 2022; Chickarmane et al., 2012; Fujita et al., 2011; Galvan-Ampudia et al., 2020; Gruel et al., 2016; Heisler et al., 2005; Jönsson et al., 2005, 2006; Klawe et al., 2020; Michael et al., 2023; Plong et al., 2021). Our results suggest that the specific buckling-like patterns displayed by clv mutants can be explained by a combination of local heterogeneities in growth rates and in mechanical properties. Indeed, we show that mutant tissues are 15-20% less stiff than the WT and display a wider range of stiffnesses. We had expected clv mutant cells to be stiffer than WT cells, because CLV3-expressing WT CZ cells are stiffer than PZ cells (Milani et al., 2014) and because the prevailing view in the literature is that cells in clv mutant SAM bear stem cell identity. Our findings indicate that this view might be incorrect.

A wavy surface can arise from buckling, with the epidermis growing more than subepidermal layers, or from differences along the epidermis, with accelerated growth in bumps or growth constriction in the valleys. The results presented here cannot fully distinguish between these scenarios. However, a recent study revealed that cellular turgor pressure is anticorrelated to the size and number of neighbours of a given cell, but is correlated to its growth rate, such that smaller cells with fewer neighbours display higher pressure and higher growth (Long et al., 2020). In this context, our results showing a large epidermal-to-subepidermal cell size differential suggest that subepidermal cells in clv mutants are less stiff and grow less than the significantly smaller epidermal cells, which is consistent with predictions from our analytical model, that buckling occurs when inner layers are less stiff than the L1. A further experimental validation of this would require techniques to measure stiffness or pressure in internal layers. In the WT, although a growth rate differential is observed between the centre and the periphery, buckling would not necessarily occur if the intrinsic growth rate of the epidermis falls below the threshold predicted by the model and determined by growth and stiffness of the different layers.

That tissue-level shape robustness emerges from cell growth and cell size variability averaged through space and time has been shown in multiple contexts (Hervieux et al., 2016; Hong et al., 2016; Kamimoto et al., 2016; Tsugawa et al., 2017). Consistent with previous studies (Laufs et al., 1998), we find that clv mutants grow at a much lower rate than the WT, indicating that the enlarged meristem of mature plants is likely to be a result of the amplification of early changes through time, rather than due to increased proliferation later in development. Evidence for such a scenario is provided by Reddy and Meyerowitz (2005), who showed that the immediate effect of the loss of CLV3 is an increase in proliferation in the meristem. A recent study has shown that impairing cellulose synthesis decreases both cell proliferation and cell stiffness, leading to smaller SAM, and that in the context of a clv1 mutant, it results in a dramatic reduction of the fasciated phenotype (Sampathkumar et al., 2019). As we now show that clv mutants themselves have slower growth and reduced stiffness, it is not evident how a further reduction of both parameters could result in an amelioration of the clv mutant phenotype. A recent study shows that cell expansion in parts of the meristem can be accounted for by stress-dependent orientation of cell division planes (Banwarth-Kuhn et al., 2022). Thus, the early effects of lowered cellulose synthase activity in reducing tensile stress may prevent the clv mutant phenotype from developing in plants with impaired cellulose synthase activity. Furthermore, an important distinction is that our study focuses on local buckling, rather than on global fasciation.

Mechanical and growth heterogeneities in clv mutants may be caused by underlying perturbations in genetic patterns. Variable and patchy expression of CZ and OC markers are in fact visible in the literature (Brand et al., 2000; Reddy and Meyerowitz, 2005; Schlegel et al., 2021; Schoof et al., 2000), although few studies have remarked upon, or interpreted, these data. It is unclear precisely how this pattern arises in the mutant and how it behaves over time. Furthermore, chimeric identities might exist in regions of fate transit within WT meristems, for example at the CZ-PZ or the PZ-flower boundaries. In this context, it would be informative to look at the expression of a related negative feedback loop that functions specifically in the PZ (Schlegel et al., 2021).

In the absence of external stimuli, cells in clv mutant meristems clearly do not undergo differentiation, like WT CZ cells. However, we show that cells in clv mutant meristems are more akin to WT PZ cells in their capacity to respond to auxin signalling, perhaps due, at least in part, to their heterogeneous identities. Bhatia et al. (2016) show that for organogenesis to occur, spatial differences in MP are a necessary condition. This is indeed the case in clv mutants, where MP expression is both broad and patchy, suggesting that improper auxin distributions or signalling contribute to the clv mutant phenotype. It is thus unclear why organogenesis is affected in clv mutant plants. One possibility is that CLV signalling might regulate auxin activity directly, such as in the moss (Nemec-Venza et al., 2022). Alternatively, because auxin accumulation in space and time is crucial for organogenesis (Galvan-Ampudia et al., 2020; Reddy and Meyerowitz, 2005; Reinhardt et al., 2000; Vernoux et al., 2000), it is possible that the abnormal shapes of clv mutant SAM do not allow proper auxin patterns to be generated over most of the meristem. In this model, an initial change in the geometry of clv mutant SAM would lead to perturbations in auxin fluxes, thereby inhibiting the production of lateral organs, which are a major auxin source (Shi et al., 2018). The presence of fewer primordia during early development (e.g. embryogenesis or the vegetative phase) could generate even lower auxin levels in the SAM and lead to an increase in size (Shi et al., 2018). In support of this, the previously unreported floral phenotypes we reveal are reminiscent of certain auxin pathway components (Cheng et al., 2006). Furthermore, it was recently shown that clv mutant SAM are sensitised to auxin levels (John et al., 2023; Jones et al., 2020). A detailed analysis of auxin fluxes and auxin signalling in clv mutant meristems will be necessary to understand the role of auxin in fasciation.

Taken together, all examined parameters in this study show that cells in clv mutant SAM are different from WT CZ cells, and that they do not fit the current idea of typical stem cells. We suggest that stem cell identity needs to account for genetic, mechanical and functional parameters. Reporters for diverse cellular properties, as well as detailed real-time analyses, will help elucidate how chimeric identities arise and what their effects are on plant architecture.

Plant material and culture conditions

The following plant lines were used in this study: Ler and Col-0 as wild types, clv3-2 (Clark et al., 1995), clv3-7 (Fletcher et al., 1999), clv3-9 (Nimchuk et al., 2015), clv1-8 (Medford et al., 1992), clv1-11 (Diévart et al., 2003), pCLV3::GFPer (Reddy and Meyerowitz, 2005), pDR5rev::GFPer (Friml et al., 2003), pDR5::3xVENUS-N7 (Vernoux et al., 2011) and pUBQ10::LTI6b-TdTomato (Shapiro et al., 2015).

Seeds were germinated on soil and placed in short-day conditions (8 h light, 21°C, 50-60% humidity and 16 h dark, 19°C, 50-60% humidity) for 10 days. Seedlings were then transplanted into individual pots and placed back in the short-day conditions. Two weeks after transplantation, plants were transferred from short-day to long-day conditions (16 h light period, 21°C, 60% humidity and 8 h dark period, 19°C, 60% humidity) until flowering, when they were used for further experiments, as described below. Light sources were LED fixtures (Valoya, C75, spectrum NS12), with an intensity of 150 μmol m−2 s−1.

CRISPR mutagenesis of CLV3

Generation of clv mutants was carried out using a multiplexing CRISPR system (Stuttmann et al., 2021) to create the clv3-21 allele in Col-0 plants carrying a pUBQ10::LTI6b-tdTomato reporter construct. Two guide RNAs targeting CLV3 (5′-gATCTCACTCAAGCTCATGC-3′ and 5′-TCAAGGACTTTCCAACCGCA-3′) were designed using Benchling software (www.benchling.com) and placed under the control of the U6 promoter in the pDGE333 and pDGE334 ‘level 0’ shuttle (Stuttmann et al., 2021). These sgRNA constructs were subsequently assembled in the CAS9-carrying pDGE347 binary vector, along with the FAST-red marker (fluorescence-accumulating seed technology) (Shimada et al., 2010) and the bialophos resistance gene (BAR) that confers resistance to glufosinate ammonium (Basta) for positive selection of transformants. T1 generation seeds that were positive for FAST-red were germinated on plates containing Basta and target genes from putative transformants were sequenced by Sanger sequencing, screened for clv-like phenotypes in the T2 and T3 generations and resequenced to generate independent homozygous lines. The clv3-21 allele bears an insertion of an adenine after the second base pair of exon 2, thus leading to a frameshift at residue 24 of the CLV3 protein and the creation of a stop codon at residue 46, 23 amino acids before the start of the CLE domain.

Plant dissection and preparation for confocal microscopy

Plants were grown as described above and dissected 2-5 days after bolting. An ∼1 cm region of primary or secondary meristem was placed in apex culture medium (ACM): ½ MS medium, 1% [w/v] sucrose, pH adjusted to 5.8 with 1 M KOH, 0.8% [w/v] agarose, supplemented with 1× vitamins [1000× filtered stock: 5 g myo-inositol, 0.05 g nicotinic acid, 640.05 g pyridoxine hydrochloride (B6), 0.5 g thiamine hydrochloride (B1), 0.1 g glycine, H2O to 50 ml] and cytokinins [6-benzylaminopurine (BAP); 125-175 nM final]. Older flower buds were removed until the shoot meristem was sufficiently exposed for experimentation. Where necessary, dissected samples were placed in a growth cabinet under long-day conditions until treated.

Brightfield photos

Brightfield photographs of plants were taken with a binocular Nikon SMZ18 equipped with an objective Nikon SHR Plan Apo 1× WD:60 and an ORCA-Flash4.OLT camera.

Confocal imaging

Imaging was carried out on a Leica TCS SP8 (DM6000 CS) upright confocal laser scanning microscope, equipped with a 25× water dipping lens (Leica HC FLUOTAR L 25×/0.95 W VISIR) and a Leica HyD hybrid detector. Either plants expressed the pUBQ10::LTI6b-TdTomato fluorescent plasma membrane marker, or FM4-64 (Thermo Fisher Scientific, T13320) was used to mark the plasma membrane as previously described (Fernandez et al., 2010). FM4-64 was excited with a 488 nm laser diode and detected at 600-640 nm. GFP was excited at 488 nm and detected at 500-520 nm. VENUS was excited at 514 nm and detected at 520-535 nm. tdTomato was excited at either 514 or 552 nm and detected at 560-600 nm.

Image analysis

Fiji (Schindelin et al., 2012) was used to generate 2D projections from 3D confocal stacks (3D viewer plugin) and to generate orthogonal slices.

The MARS pipeline (Fernandez et al., 2010) was used to generate 3D reconstructions of confocal stacks. For this, shoot meristems were imaged from multiple angles by means of a custom-made device to tilt and/or rotate the sample by 15-20° between acquisitions. After fusion using MARS, the external contours of the sample were detected using the Level Set Method (Kiss et al., 2017) and this contour was then used to segment the 3D reconstructed sample using a watershed segmentation algorithm. Cells were then extracted from the segmented image for the L1 and L2 tissue layers and the volume of each cell was calculated.

MorphoGraphX (Barbier de Reuille et al., 2015; Kiss et al., 2017) was used to measure cell size, growth and curvature for individual image stacks. For cell size, we determined the image slice within the L1 and L2 layers that was orthogonal to the axis of acquisition, and which contained the midline of the group of cells near the centre of the image (in order to avoid cells that had been imaged from the side). We then segmented and extracted cell areas. For growth, we first identified the tissue surface, generated the mesh, projected signal from the first cell layer (usually 5.5-6 μm), watershed-segmented the mesh and determined cell lineages over two or more time points. Minimal curvature was calculated within a 10 μm radius for each point in the image and, for segmented tissues, was then normalised to the size of each cell. R was used to plot the data and run statistical tests.

Statistical analysis

Plots and statistical analyses were carried out using R studio version 2023.03.0 (https://posit.co/) using the ggplot2 package (Wickham, 2016). Welch's t-tests were used to compare the mean of two independent groups: cell areas and cell volumes in different cell layers, and the apparent Young's modulus in WT and mutant SAM. When we use boxplots, the boxes extend from the first to the third quartile and the whiskers from 10% to 90% of the values, the solid black line represents the median of the distribution. Throughout the paper, average values are mean±s.d. On scatter plots, the regression line represents the best linear fit of the data, along with its 95% confidence interval. Pearson's correlation coefficients and the related P-values were calculated using ggplot2.

Analytical models

We used a morphoelastic framework, which involves a multiplicative decomposition of the deformation gradient into growth tensor, describing changes in mass or density, and an elastic tensor, accounting for the elastic material response, commonly used in the literature (Goriely and Ben Amar, 2005; Moulton et al., 2013). The outer L1 layer of cells in the SAM is modelled as a planar elastic rod attached to an elastic foundation that represents the inner layers (Almet et al., 2019; Moulton et al., 2013) (Fig. S2). The elastic rod grows axially in the x-direction, which generates compressive stresses due to clamped boundary conditions at the two rod ends. These stresses lead to instability and buckling that result in the formation of folded structures, which closely resemble the shapes obtained in our experimental data. We used the model to explore differences in the growth rates and stiffness in the L1 and L2 layers of the various groups in our study (see supplementary Materials and Methods, Analytical models for further details).

Atomic force microscopy and data analysis

Meristems were dissected to remove all flower buds older than stage 3 the day before AFM observations and placed in ACM in 60 mm plastic Petri dishes. Acquisitions were carried out on either the dome of WT plants or on lateral outgrowths of clv mutant meristems. Samples were probed in distilled water at room temperature on a stand-alone JPK Nanowizard III microscope equipped with a CellHesion module (100 μm range Z-piezo) using the Quantitative Imaging mode (QI). We used a silica spherical tip with a radius of 400 nm (Special Development SD-sphere-NCH, Nanosensors) mounted on a silicon cantilever with a nominal force constant of 42 N m−1. Scan size was generally a square field of 45×45 μm with pixel size of ∼500 nm. An applied force trigger of 1 μN was used in order to indent only the cell wall (Milani et al., 2011; Tvergaard and Needleman, 2018), with a ramp size of 2 μm, an approach speed of 10 μm s−1 and a retraction speed of 100 μm s−1. Apparent Young's modulus was obtained by fitting the first 100-150 nm of the force displacement curve with a Hertz model and subsequently corrected for the effect of slope [see supplementary Materials and Methods, Atomic force microscopy (AFM) and data analysis for further details].

RNA in situ hybridisations

RNA in situ hybridisations on sections were performed according to published protocols (Long et al., 1996). Meristems were dissected just after bolting, fixed in FAA (formaldehyde 3.7% [v/v], ethanol 50% [v/v], acetic acid 5% [v/v], H2O to final volume), washed, dehydrated and embedded in paraplast. Embedded samples were cut (10 μm thick) and attached to pre-coated glass slides (Superfrost Plus Gold, Fisher Scientific). Antisense probes were made from PCR products using cDNA from inflorescences as a template, except for CLV3, which was amplified using genomic DNA (Table S3). Those PCR products were transcribed into RNA and then labelled using digoxigenin (DIG)-UTP. All probes were filtered on columns (CHROMA SPIN-30 columns, Clontech) to remove remaining nucleotides. Immunodetection was performed using an anti-DIG antibody coupled to alkaline phosphatase (Anti-Digoxygenin-AP, Fab fragments, Roche, 1:3000), the activity of which was detected by the chromogenic method using NBT/BCIP (Roche). Sections were finally washed with water and observed under a Zeiss Imager M2 microscope equipped with an AxioCam Mrc camera, and 10× or 20× objectives in differential interference contrast (DIC) mode.

Hormone treatments

Auxin treatments were performed by immersing dissected SAM in 1 or 2 mM Indole-3-acetic acid (IAA) solution (Sigma-Aldrich) for 5 h (Galvan-Ampudia et al., 2020). DR5 reporter expression was monitored in the samples by confocal microscopy ∼24 h post-treatment. Treatments were carried out on clv1-8 (in the Col-0 accession, as in Fig. S9D,E), clv3-2 plants (in the Ler accession, as in Fig. S9A,B), or in F1 plants of a cross between clv3-2 and clv3-21 (mixed Col-0 and Ler background, as in Fig. 6C,D,I-L).

We thank V. Mirabet and N. Dubrulle for help with preliminary AFM measurements, A. Kiss for help with 3D segmentations and surface curvature detection, Zofia Haftek-Terreau and G. Gilbert for help with in situ hybridisations, and Aurélie Chauvet for help with molecular biology. We thank the PLATIM/LyMIC imaging facility of the SFR Biosciences (UMS3444/CNRS, US8/Inserm, ENS de Lyon, UCBL1). We thank A. Lacroix, J. Berger, P. Bolland, H. Leyral, and I. Desbouchages for assistance with plant growth and logistics. This article is based on L.R.-L.’s PhD thesis (Rambaud-Lavigne, 2018).

Author contributions

Conceptualization: L.R.-L., A.B., P.D.; Methodology: A.C., S.B., N.G., A.B.; Software: A.C., N.G.; Validation: L.R.-L., V.B., P.D.; Formal analysis: L.R.-L., A.C., Q.L., P.D.; Investigation: L.R.-L., S.B., V.B., P.D.; Data curation: L.R.-L., P.D.; Writing - original draft: L.R.-L., A.C., N.G., P.D.; Writing - review & editing: L.R.-L., A.C., S.B., N.G., A.B., P.D.; Visualization: L.R.-L., A.C., Q.L., N.G., P.D.; Supervision: N.G., A.B., P.D.; Funding acquisition: L.R.-L., N.G., A.B., P.D.

Funding

This work was supported by a PhD fellowship from the École Normale Supérieure de Lyon to L.R.-L., and a Science and Engineering Research Board grant (SERB-POWER) to N.G.

Data availability

All datasets and scripts are available at Rambaud-Lavigne (2024): https://doi.org/10.57745/N6QM41.

The people behind the papers

This article has an associated ‘The people behind the papers’ interview with some of the authors.

Aggarwal
,
P.
,
Yadav
,
R. K.
and
Reddy
,
G. V.
(
2010
).
Identification of novel markers for stem-cell niche of Arabidopsis shoot apex
.
Gene Expr. Patterns
10
,
259
-
264
.
Almet
,
A. A.
,
Byrne
,
H. M.
,
Maini
,
P. K.
and
Moulton
,
D. E.
(
2019
).
Post-buckling behaviour of a growing elastic rod
.
J. Math. Biol.
78
,
777
-
814
.
Banwarth-Kuhn
,
M.
,
Rodriguez
,
K.
,
Michael
,
C.
,
Ta
,
C.-K.
,
Plong
,
A.
,
Bourgain-Chang
,
E.
,
Nematbakhsh
,
A.
,
Chen
,
W.
,
Roy-Chowdhury
,
A.
,
Reddy
,
G. V.
et al.
(
2022
).
Combined computational modeling and experimental analysis integrating chemical and mechanical signals suggests possible mechanism of shoot meristem maintenance
.
PLoS Comput. Biol.
18
,
e1010199
.
Barbier de Reuille
,
P.
,
Routier-Kierzkowska
,
A.-L.
,
Kierzkowski
,
D.
,
Bassel
,
G. W.
,
Schüpbach
,
T.
,
Tauriello
,
G.
,
Bajpai
,
N.
,
Strauss
,
S.
,
Weber
,
A.
,
Kiss
,
A.
et al.
(
2015
).
MorphoGraphX: A platform for quantifying morphogenesis in 4D
.
eLife
4
,
05864
.
Battogtokh
,
D.
and
Tyson
,
J. J.
(
2022
).
Nucleation of stem cell domains in a bistable activator-inhibitor model of the shoot apical meristem
.
Chaos Woodbury N
32
,
093117
.
Beauzamy
,
L.
,
Louveaux
,
M.
,
Hamant
,
O.
and
Boudaoud
,
A.
(
2015
).
Mechanically, the shoot apical meristem of arabidopsis behaves like a shell inflated by a pressure of about 1 MPa
.
Front. Plant Sci.
6
,
1038
.
Berleth
,
T.
and
Jürgens
,
G.
(
1993
).
The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo
.
Development
118
,
575
-
587
.
Bhatia
,
N.
,
Bozorg
,
B.
,
Larsson
,
A.
,
Ohno
,
C.
,
Jönsson
,
H.
and
Heisler
,
M. G.
(
2016
).
Auxin acts through MONOPTEROS to regulate plant cell polarity and pattern phyllotaxis
.
Curr. Biol.
26
,
3202
-
3208
.
Brand
,
U.
,
Fletcher
,
J. C.
,
Hobe
,
M.
,
Meyerowitz
,
E. M.
and
Simon
,
R.
(
2000
).
Dependence of stem cell fate in arabidopsis on a feedback loop regulated by CLV3 activity
.
Science
289
,
617
-
619
.
Brand
,
U.
,
Grünewald
,
M.
,
Hobe
,
M.
and
Simon
,
R.
(
2002
).
Regulation of CLV3 expression by two homeobox genes in arabidopsis
.
Plant Physiol.
129
,
565
-
575
.
Busch
,
W.
,
Miotk
,
A.
,
Ariel
,
F. D.
,
Zhao
,
Z.
,
Forner
,
J.
,
Daum
,
G.
,
Suzaki
,
T.
,
Schuster
,
C.
,
Schultheiss
,
S. J.
,
Leibfried
,
A.
et al.
(
2010
).
Transcriptional control of a plant stem cell Niche
.
Dev. Cell
18
,
841
-
853
.
Cheng
,
Y.
,
Dai
,
X.
and
Zhao
,
Y.
(
2006
).
Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis
.
Genes Dev.
20
,
1790
-
1799
.
Chickarmane
,
V. S.
,
Gordon
,
S. P.
,
Tarr
,
P. T.
,
Heisler
,
M. G.
and
Meyerowitz
,
E. M.
(
2012
).
Cytokinin signaling as a positional cue for patterning the apical–basal axis of the growing Arabidopsis shoot meristem
.
Proc. Natl. Acad. Sci. USA
109
,
4002
-
4007
.
Clark
,
S. E.
,
Running
,
M. P.
and
Meyerowitz
,
E. M.
(
1993
).
CLAVATA1, a regulator of meristem and flower development in Arabidopsis
.
Development
119
,
397
-
418
.
Clark
,
S. E.
,
Running
,
M. P.
and
Meyerowitz
,
E. M.
(
1995
).
CLAVATA3 is a specific regulator of shoot and floral meristem development affecting the same processes as CLAVATA1
.
Development
121
,
2057
-
2067
.
Clark
,
S. E.
,
Williams
,
R. W.
and
Meyerowitz
,
E. M.
(
1997
).
The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis
.
Cell
89
,
575
-
585
.
Dao
,
T. Q.
,
Weksler
,
N.
,
Liu
,
H. M.-H.
,
Leiboff
,
S.
and
Fletcher
,
J. C.
(
2022
).
Interactive CLV3, CLE16 and CLE17 signaling mediates stem cell homeostasis in the Arabidopsis shoot apical meristem
.
Development
149
,
dev200787
.
Daum
,
G.
,
Medzihradszky
,
A.
,
Suzaki
,
T.
and
Lohmann
,
J. U.
(
2014
).
A mechanistic framework for noncell autonomous stem cell induction in Arabidopsis
.
Proc. Natl. Acad. Sci. USA
111
,
14619
-
14624
.
de Reuille
,
P. B.
,
Bohn-Courseau
,
I.
,
Ljung
,
K.
,
Morin
,
H.
,
Carraro
,
N.
,
Godin
,
C.
and
Traas
,
J.
(
2006
).
Computer simulations reveal properties of the cell-cell signaling network at the shoot apex in Arabidopsis
.
Proc. Natl. Acad. Sci. U. S. A.
103
,
1627
-
1632
.
Diévart
,
A.
,
Dalal
,
M.
,
Tax
,
F. E.
,
Lacey
,
A. D.
,
Huttly
,
A.
,
Li
,
J.
and
Clark
,
S. E.
(
2003
).
CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development
.
Plant Cell
15
,
1198
-
1211
.
Douady
,
S.
and
Couder
,
Y.
(
1996
).
Phyllotaxis as a Dynamical Self Organizing Process Part II: the spontaneous formation of a periodicity and the coexistence of spiral and whorled patterns
.
J. Theor. Biol.
178
,
275
-
294
.
Fernandez
,
R.
,
Das
,
P.
,
Mirabet
,
V.
,
Moscardi
,
E.
,
Traas
,
J.
,
Verdeil
,
J.-L.
,
Malandain
,
G.
and
Godin
,
C.
(
2010
).
Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution
.
Nat. Methods
7
,
547
-
553
.
Fletcher
,
J. C.
,
Brand
,
U.
,
Running
,
M. P.
,
Simon
,
R.
and
Meyerowitz
,
E. M.
(
1999
).
Signaling of cell fate decisions by CLAVATA3 in arabidopsis shoot meristems
.
Science
283
,
1911
-
1914
.
Friml
,
J.
,
Vieten
,
A.
,
Sauer
,
M.
,
Weijers
,
D.
,
Schwarz
,
H.
,
Hamann
,
T.
,
Offringa
,
R.
and
Jürgens
,
G.
(
2003
).
Efflux-dependent auxin gradients establish the apical–basal axis of Arabidopsis
.
Nature
426
,
147
-
153
.
Fujita
,
M.
,
Himmelspach
,
R.
,
Hocart
,
C. H.
,
Williamson
,
R. E.
,
Mansfield
,
S. D.
and
Wasteneys
,
G. O.
(
2011
).
Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis
.
Plant J.
66
,
915
-
928
.
Galvan-Ampudia
,
C. S.
,
Cerutti
,
G.
,
Legrand
,
J.
,
Brunoud
,
G.
,
Martin-Arevalillo
,
R.
,
Azais
,
R.
,
Bayle
,
V.
,
Moussu
,
S.
,
Wenzl
,
C.
,
Jaillais
,
Y.
et al.
(
2020
).
Temporal integration of auxin information for the regulation of patterning
.
eLife
9
,
e55832
.
Gordon
,
S. P.
,
Chickarmane
,
V. S.
,
Ohno
,
C.
and
Meyerowitz
,
E. M.
(
2009
).
Multiple feedback loops through cytokinin signaling control stem cell number within the Arabidopsis shoot meristem
.
Proc. Natl. Acad. Sci. USA
106
,
16529
-
16534
.
Goriely
,
A.
(
2017
).
The Mathematics and Mechanics of Biological Growth
.
New York, NY
:
Springer New York
.
Goriely
,
A.
and
Ben Amar
,
M.
(
2005
).
Differential growth and instability in elastic shells
.
Phys. Rev. Lett.
94
,
198103
.
Gruel
,
J.
,
Landrein
,
B.
,
Tarr
,
P.
,
Schuster
,
C.
,
Refahi
,
Y.
,
Sampathkumar
,
A.
,
Hamant
,
O.
,
Meyerowitz
,
E. M.
and
Jönsson
,
H.
(
2016
).
An epidermis-driven mechanism positions and scales stem cell niches in plants
.
Sci. Adv.
2
,
e1500989
.
Hardtke
,
C. S.
and
Berleth
,
T.
(
1998
).
The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development
.
EMBO J.
17
,
1405
-
1411
.
Heisler
,
M. G.
,
Ohno
,
C.
,
Das
,
P.
,
Sieber
,
P.
,
Reddy
,
G. V.
,
Long
,
J. A.
and
Meyerowitz
,
E. M.
(
2005
).
Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the arabidopsis inflorescence meristem
.
Curr. Biol.
15
,
1899
-
1911
.
Hervieux
,
N.
,
Dumond
,
M.
,
Sapala
,
A.
,
Routier-Kierzkowska
,
A.-L.
,
Kierzkowski
,
D.
,
Roeder
,
A. H. K.
,
Smith
,
R. S.
,
Boudaoud
,
A.
and
Hamant
,
O.
(
2016
).
A mechanical feedback restricts sepal growth and shape in Arabidopsis
.
Curr. Biol.
26
,
1019
-
1028
.
Hong
,
L.
,
Dumond
,
M.
,
Tsugawa
,
S.
,
Sapala
,
A.
,
Routier-Kierzkowska
,
A.-L.
,
Zhou
,
Y.
,
Chen
,
C.
,
Kiss
,
A.
,
Zhu
,
M.
,
Hamant
,
O.
et al.
(
2016
).
Variable cell growth yields reproducible organ development through spatiotemporal averaging
.
Dev. Cell
38
,
15
-
32
.
Jeong
,
S.
,
Trotochaud
,
A. E.
and
Clark
,
S. E.
(
1999
).
The arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase
.
Plant Cell
11
,
1925
-
1933
.
John
,
A.
,
Smith
,
E. S.
,
Jones
,
D. S.
,
Soyars
,
C. L.
and
Nimchuk
,
Z. L.
(
2023
).
A network of CLAVATA receptors buffers auxin-dependent meristem maintenance
.
Nat. Plants
9
,
1306
-
1317
.
Jones
,
D. S.
,
John
,
A.
,
VanDerMolen
,
K. R.
and
Nimchuk
,
Z. L.
(
2020
).
CLAVATA signaling ensures reproductive development in plants across thermal environments
.
Curr. Biol.
31
,
220
-
227.e5
.
Jönsson
,
H.
,
Heisler
,
M.
,
Reddy
,
G. V.
,
Agrawal
,
V.
,
Gor
,
V.
,
Shapiro
,
B. E.
,
Mjolsness
,
E.
and
Meyerowitz
,
E. M.
(
2005
).
Modeling the organization of the WUSCHEL expression domain in the shoot apical meristem
.
Bioinformatics
21
Suppl. 1
,
i232
-
i240
.
Jönsson
,
H.
,
Heisler
,
M. G.
,
Shapiro
,
B. E.
,
Meyerowitz
,
E. M.
and
Mjolsness
,
E.
(
2006
).
An auxin-driven polarized transport model for phyllotaxis
.
Proc. Natl. Acad. Sci. USA
103
,
1633
-
1638
.
Kamimoto
,
K.
,
Kaneko
,
K.
,
Kok
,
C. Y.-Y.
,
Okada
,
H.
,
Miyajima
,
A.
and
Itoh
,
T.
(
2016
).
Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling
.
eLife
5
,
e15034
.
Kayes
,
J. M.
and
Clark
,
S. E.
(
1998
).
CLAVATA2, a regulator of meristem and organ development in Arabidopsis
.
Development
125
,
3843
-
3851
.
Kinoshita
,
A.
,
Seo
,
M.
,
Kamiya
,
Y.
and
Sawa
,
S.
(
2015
).
Mystery in genetics: PUB4 gives a clue to the complex mechanism of CLV signaling pathway in the shoot apical meristem
.
Plant Signal. Behav.
10
,
e1028707
.
Kiss
,
A.
,
Moreau
,
T.
,
Mirabet
,
V.
,
Calugaru
,
C. I.
,
Boudaoud
,
A.
and
Das
,
P.
(
2017
).
Segmentation of 3D images of plant tissues at multiple scales using the level set method
.
Plant Methods
13
,
114
.
Klawe
,
F. Z.
,
Stiehl
,
T.
,
Bastian
,
P.
,
Gaillochet
,
C.
,
Lohmann
,
J. U.
and
Marciniak-Czochra
,
A.
(
2020
).
Mathematical modeling of plant cell fate transitions controlled by hormonal signals
.
PLoS Comput. Biol.
16
,
e1007523
.
Kwon
,
C. S.
,
Chen
,
C.
and
Wagner
,
D.
(
2005
).
WUSCHEL is a primary target for transcriptional regulation by SPLAYED in dynamic control of stem cell fate in Arabidopsis
.
Genes Dev.
19
,
992
-
1003
.
Laufs
,
P.
,
Grandjean
,
O.
,
Jonak
,
C.
,
Kiêu
,
K.
and
Traas
,
J.
(
1998
).
Cellular parameters of the shoot apical meristem in Arabidopsis
.
Plant Cell Online
10
,
1375
-
1389
.
Laux
,
T.
,
Mayer
,
K. F. X.
,
Berger
,
J.
and
Jurgens
,
G.
(
1996
).
The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis
.
Development
122
,
87
-
96
.
Lenhard
,
M.
and
Laux
,
T.
(
2003
).
Stem cell homeostasis in the Arabidopsis shoot meristem is regulated by intercellular movement of CLAVATA3 and its sequestration by CLAVATA1
.
Development
130
,
3163
-
3173
.
Lindsay
,
D. L.
,
Sawhney
,
V. K.
and
Bonham-Smith
,
P. C.
(
2006
).
Cytokinin-induced changes in CLAVATA1 and WUSCHEL expression temporally coincide with altered floral development in Arabidopsis
.
Plant Sci.
170
,
1111
-
1117
.
Long
,
J. A.
,
Moan
,
E. I.
,
Medford
,
J. I.
and
Barton
,
M. K.
(
1996
).
A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis
.
Nature
379
,
66
-
69
.
Long
,
Y.
,
Cheddadi
,
I.
,
Mosca
,
G.
,
Mirabet
,
V.
,
Dumond
,
M.
,
Kiss
,
A.
,
Traas
,
J.
,
Godin
,
C.
and
Boudaoud
,
A.
(
2020
).
Cellular heterogeneity in pressure and growth emerges from tissue topology and geometry
.
Curr. Biol.
30
,
1504
-
1516.e8
.
Luo
,
L.
,
Zeng
,
J.
,
Wu
,
H.
,
Tian
,
Z.
and
Zhao
,
Z.
(
2018
).
A molecular framework for auxin-controlled homeostasis of shoot stem cells in Arabidopsis
.
Mol. Plant
11
,
899
-
913
.
Mayer
,
K. F. X.
,
Schoof
,
H.
,
Haecker
,
A.
,
Lenhard
,
M.
,
Jürgens
,
G.
and
Laux
,
T.
(
1998
).
Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem
.
Cell
95
,
805
-
815
.
Medford
,
J. I.
,
Behringer
,
F. J.
,
Callos
,
J. D.
and
Feldmann
,
K. A.
(
1992
).
Normal and abnormal development in the Arabidopsis vegetative shoot apex
.
Plant Cell
4
,
631
-
643
.
Michael
,
C.
,
Banwarth-Kuhn
,
M.
,
Rodriguez
,
K.
,
Ta
,
C.-K.
,
Roy-Chowdhury
,
A.
,
Chen
,
W.
,
Venugopala Reddy
,
G.
and
Alber
,
M.
(
2023
).
Role of turgor-pressure induced boundary tension in the maintenance of the shoot apical meristem of Arabidopsis thaliana
.
J. R. Soc. Interface
20
,
20230173
.
Milani
,
P.
,
Gholamirad
,
M.
,
Traas
,
J.
,
Arnéodo
,
A.
,
Boudaoud
,
A.
,
Argoul
,
F.
and
Hamant
,
O.
(
2011
).
In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy: measuring wall stiffness in meristems with AFM
.
Plant J.
67
,
1116
-
1123
.
Milani
,
P.
,
Mirabet
,
V.
,
Cellier
,
C.
,
Rozier
,
F.
,
Hamant
,
O.
,
Das
,
P.
and
Boudaoud
,
A.
(
2014
).
Matching patterns of gene expression to mechanical stiffness at cell resolution through quantitative tandem epifluorescence and nanoindentation
.
Plant Physiol.
165
,
1399
-
1408
.
Miwa
,
H.
,
Betsuyaku
,
S.
,
Iwamoto
,
K.
,
Kinoshita
,
A.
,
Fukuda
,
H.
and
Sawa
,
S.
(
2008
).
The receptor-like kinase SOL2 mediates CLE signaling in arabidopsis
.
Plant Cell Physiol.
49
,
1752
-
1757
.
Moulton
,
D. E.
,
Lessinnes
,
T.
and
Goriely
,
A.
(
2013
).
Morphoelastic rods. Part I: a single growing elastic rod
.
J. Mech. Phys. Solids
61
,
398
-
427
.
Müller
,
R.
,
Borghi
,
L.
,
Kwiatkowska
,
D.
,
Laufs
,
P.
and
Simon
,
R.
(
2006
).
Dynamic and compensatory responses of arabidopsis shoot and floral meristems to CLV3 signaling
.
Plant Cell
18
,
1188
-
1198
.
Müller
,
R.
,
Bleckmann
,
A.
and
Simon
,
R.
(
2008
).
The receptor kinase CORYNE of Arabidopsis transmits the stem cell–limiting signal CLAVATA3 independently of CLAVATA1
.
Plant Cell Online
20
,
934
-
946
.
Nemec-Venza
,
Z.
,
Madden
,
C.
,
Stewart
,
A.
,
Liu
,
W.
,
Novák
,
O.
,
Pěnčík
,
A.
,
Cuming
,
A. C.
,
Kamisugi
,
Y.
and
Harrison
,
C. J.
(
2022
).
CLAVATA modulates auxin homeostasis and transport to regulate stem cell identity and plant shape in a moss
.
New Phytol.
234
,
149
-
163
.
Nimchuk
,
Z. L.
,
Tarr
,
P. T.
,
Ohno
,
C.
,
Qu
,
X.
and
Meyerowitz
,
E. M.
(
2011
).
Plant Stem Cell Signaling Involves Ligand-Dependent Trafficking of the CLAVATA1 Receptor Kinase
.
Curr. Biol.
21
,
345
-
352
.
Nimchuk
,
Z. L.
,
Zhou
,
Y.
,
Tarr
,
P. T.
,
Peterson
,
B. A.
and
Meyerowitz
,
E. M.
(
2015
).
Plant stem cell maintenance by transcriptional cross-regulation of related receptor kinases
.
Development
142
,
1043
-
1049
.
Plong
,
A.
,
Rodriguez
,
K.
,
Alber
,
M.
,
Chen
,
W.
and
Reddy
,
G. V.
(
2021
).
CLAVATA3 mediated simultaneous control of transcriptional and post-translational processes provides robustness to the WUSCHEL gradient
.
Nat. Commun.
12
,
6361
.
Rambaud-Lavigne
,
L. E. S.
(
2018
).
The genetics and mechanics of stem cells at the Arabidopsis shoot apex
. PhD thesis, University of Lyon, Lyon, France. theses.hal.science/tel-02366972.
Rambaud-Lavigne
,
L.
(
2024
).
Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants
.
Recherche Data Gouv
.
Reddy
,
G. V.
and
Meyerowitz
,
E. M.
(
2005
).
Stem-cell homeostasis and growth dynamics can be uncoupled in the arabidopsis shoot apex
.
Science
310
,
663
-
667
.
Reinhardt
,
D.
,
Mandel
,
T.
and
Kuhlemeier
,
C.
(
2000
).
Auxin regulates the initiation and radial position of plant lateral organs
.
Plant Cell
12
,
507
-
518
.
Sampathkumar
,
A.
,
Peaucelle
,
A.
,
Fujita
,
M.
,
Schuster
,
C.
,
Persson
,
S.
,
Wasteneys
,
G. O.
and
Meyerowitz
,
E. M.
(
2019
).
Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth
.
Development
146
,
dev.179036
.
Schindelin
,
J.
,
Arganda-Carreras
,
I.
,
Frise
,
E.
,
Kaynig
,
V.
,
Longair
,
M.
,
Pietzsch
,
T.
,
Preibisch
,
S.
,
Rueden
,
C.
,
Saalfeld
,
S.
,
Schmid
,
B.
et al.
(
2012
).
Fiji: an open-source platform for biological-image analysis
.
Nat. Methods
9
,
676
-
682
.
Schlegel
,
J.
,
Denay
,
G.
,
Wink
,
R. H.
,
Pinto
,
K. G.
,
Stahl
,
Y.
,
Schmid
,
J.
,
Blümke
,
P.
and
Simon
,
R. G. W.
(
2021
).
Control of Arabidopsis shoot stem cell homeostasis by two antagonistic CLE peptide signalling pathways
.
eLife
10
,
e70934
.
Schoof
,
H.
,
Lenhard
,
M.
,
Haecker
,
A.
,
Mayer
,
K. F. X.
,
Jürgens
,
G.
and
Laux
,
T.
(
2000
).
The stem cell population of arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes
.
Cell
100
,
635
-
644
.
Schuster
,
C.
,
Gaillochet
,
C.
,
Medzihradszky
,
A.
,
Busch
,
W.
,
Daum
,
G.
,
Krebs
,
M.
,
Kehle
,
A.
and
Lohmann
,
J. U.
(
2014
).
A regulatory framework for shoot stem cell control integrating metabolic, transcriptional, and phytohormone signals
.
Dev. Cell
28
,
438
-
449
.
Shapiro
,
B. E.
,
Tobin
,
C.
,
Mjolsness
,
E.
and
Meyerowitz
,
E. M.
(
2015
).
Analysis of cell division patterns in the Arabidopsis shoot apical meristem
.
Proc. Natl. Acad. Sci. USA
112
,
4815
-
4820
.
Shi
,
B.
,
Guo
,
X.
,
Wang
,
Y.
,
Xiong
,
Y.
,
Wang
,
J.
,
Hayashi
,
K.-I.
,
Lei
,
J.
,
Zhang
,
L.
and
Jiao
,
Y.
(
2018
).
Feedback from lateral organs controls shoot apical meristem growth by modulating auxin transport
.
Dev. Cell
44
,
204
-
216.e6
.
Shimada
,
T. L.
,
Shimada
,
T.
and
Hara-Nishimura
,
I.
(
2010
).
A rapid and non-destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana
.
Plant J.
61
,
519
-
528
.
Stuttmann
,
J.
,
Barthel
,
K.
,
Martin
,
P.
,
Ordon
,
J.
,
Erickson
,
J. L.
,
Herr
,
R.
,
Ferik
,
F.
,
Kretschmer
,
C.
,
Berner
,
T.
,
Keilwagen
,
J.
et al.
(
2021
).
Highly efficient multiplex editing: one-shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants
.
Plant J.
106
,
8
-
22
.
Taguchi-Shiobara
,
F.
,
Yuan
,
Z.
,
Hake
,
S.
and
Jackson
,
D.
(
2001
).
The fasciated ear2 gene encodes a leucine-rich repeat receptor-like protein that regulates shoot meristem proliferation in maize
.
Genes Dev.
15
,
2755
-
2766
.
Tam
,
P. P. C.
,
Barrette-Ng
,
I. H.
,
Simon
,
D. M.
,
Tam
,
M. W. C.
,
Ang
,
A. L.
and
Muench
,
D. G.
(
2010
).
The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization
.
BMC Plant Biol.
10
,
44
.
Tsugawa
,
S.
,
Hervieux
,
N.
,
Kierzkowski
,
D.
,
Routier-Kierzkowska
,
A.-L.
,
Sapala
,
A.
,
Hamant
,
O.
,
Smith
,
R. S.
,
Roeder
,
A. H. K.
,
Boudaoud
,
A.
and
Li
,
C.-B.
(
2017
).
Clones of cells switch from reduction to enhancement of size variability in Arabidopsis sepals
.
Development
144
,
4398
-
4405
.
Tvergaard
,
V.
and
Needleman
,
A.
(
2018
).
Effect of properties and turgor pressure on the indentation response of plant cells
.
J. Appl. Mech.
85
,
061007
.
Vernoux
,
T.
,
Kronenberger
,
J.
,
Grandjean
,
O.
,
Laufs
,
P.
and
Traas
,
J.
(
2000
).
PIN-FORMED 1 regulates cell fate at the periphery of the shoot apical meristem
.
Development
127
,
5157
-
5165
.
Vernoux
,
T.
,
Brunoud
,
G.
,
Farcot
,
E.
,
Morin
,
V.
,
Van den Daele
,
H.
,
Legrand
,
J.
,
Oliva
,
M.
,
Das
,
P.
,
Larrieu
,
A.
,
Wells
,
D.
et al.
(
2011
).
The auxin signalling network translates dynamic input into robust patterning at the shoot apex
.
Mol. Syst. Biol.
7
,
508
.
Whitewoods
,
C. D.
,
Cammarata
,
J.
,
Nemec Venza
,
Z.
,
Sang
,
S.
,
Crook
,
A. D.
,
Aoyama
,
T.
,
Wang
,
X. Y.
,
Waller
,
M.
,
Kamisugi
,
Y.
,
Cuming
,
A. C.
et al.
(
2018
).
CLAVATA was a genetic novelty for the morphological innovation of 3D growth in land plants
.
Curr. Biol.
28
,
2365
-
2376.e5
.
Wickham
,
H.
(
2016
).
Ggplot2: Elegant Graphics for Data Analysis
, 2nd edn.
Springer International Publishing
.
Wu
,
X.
,
Dabi
,
T.
and
Weigel
,
D.
(
2005
).
Requirement of homeobox gene STIMPY/WOX9 for arabidopsis meristem growth and maintenance
.
Curr. Biol.
15
,
436
-
440
.
Yadav
,
R. K.
,
Girke
,
T.
,
Pasala
,
S.
,
Xie
,
M.
and
Reddy
,
G. V.
(
2009
).
Gene expression map of the Arabidopsis shoot apical meristem stem cell niche
.
Proc. Natl. Acad. Sci. USA
106
,
4941
-
4946
.
Yadav
,
R. K.
,
Perales
,
M.
,
Gruel
,
J.
,
Girke
,
T.
,
Jönsson
,
H.
and
Reddy
,
G. V.
(
2011
).
WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex
.
Genes Dev.
25
,
2025
-
2030
.
Yamaguchi
,
N.
,
Wu
,
M.-F.
,
Winter
,
C. M.
,
Berns
,
M. C.
,
Nole-Wilson
,
S.
,
Yamaguchi
,
A.
,
Coupland
,
G.
,
Krizek
,
B. A.
and
Wagner
,
D.
(
2013
).
A molecular framework for auxin-mediated initiation of flower primordia
.
Dev. Cell
24
,
271
-
282
.
Zhao
,
Z.
,
Andersen
,
S. U.
,
Ljung
,
K.
,
Dolezal
,
K.
,
Miotk
,
A.
,
Schultheiss
,
S. J.
and
Lohmann
,
J. U.
(
2010
).
Hormonal control of the shoot stem-cell niche
.
Nature
465
,
1089
-
1092
.

Competing interests

The authors declare no competing or financial interests.

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