The shoot apical meristem (SAM) gives rise to the aboveground organs of plants. The size of the SAM is relatively constant due to the balance between stem cell replenishment and cell recruitment into new organs. In angiosperms, the transcription factor WUSCHEL (WUS) promotes stem cell proliferation in the central zone of the SAM. WUS forms a negative feedback loop with a signaling pathway activated by CLAVATA3 (CLV3). In the periphery of the SAM, the ERECTA family receptors (ERfs) constrain WUS and CLV3 expression. Here, we show that four ligands of ERfs redundantly inhibit the expression of these two genes. Transcriptome analysis confirmed that WUS and CLV3 are the main targets of ERf signaling and uncovered new ones. Analysis of promoter reporters indicated that the WUS expression domain mostly overlaps with the CLV3 domain and does not shift along the apical-basal axis in clv3 mutants. Our three-dimensional mathematical model captured gene expression distributions at the single-cell level under various perturbed conditions. Based on our findings, CLV3 regulates cellular levels of WUS mostly through autocrine signaling, and ERfs regulate the spatial expression of WUS, preventing its encroachment into the peripheral zone.

In contrast to animals, plants generate very few organs during embryogenesis, with most of the organogenesis occurring after seed germination throughout the entire lifespan. New cells for aboveground plant organs are supplied by the shoot apical meristem (SAM) and axillary meristems. The SAM forms between cotyledons or embryonic leaves during embryogenesis. After germination, it generates the internodes, leaves and flowers of the main stem. The SAM is located at the apex of the main stem. Later in development, axillary meristems develop in the leaf axils and form branches (Wang, 2021). In all meristems, there is a small cluster of pluripotent slow-dividing stem cells in the center. Once cells are displaced from the center into the periphery, they grow and divide at a faster rate, differentiate, and ultimately are incorporated into organs. The molecular mechanisms controlling the transition of stem cells into differentiating cells of the peripheral zone are of fundamental interest to plant developmental biology. In the SAM, this transition relies on the ability of cells to communicate using a variety of extracellular signals (Demesa-Arevalo et al., 2024).

The homeobox transcription factor WUSCHEL (WUS) is essential for maintaining the SAM central zone in Arabidopsis. In the wus mutant, cells in the center differentiate prematurely, and the SAM disappears (Laux et al., 1996; Mayer et al., 1998). Ectopic and inducible expression of WUS promotes stem cell identity and increases the size of the central zone (Schoof et al., 2000; Yadav et al., 2010). Multiple signaling pathways regulate WUS expression. Cytokinins promote and position WUS expression along the apical-basal axis (Lindsay et al., 2006; Gordon et al., 2009; Chickarmane et al., 2012). A signaling pathway activated by the extracellular glycopeptide CLAVATA3 (CLV3) inhibits WUS expression. When CLV3 or its putative receptor CLAVATA1 (CLV1) are mutated, expression of WUS is increased (Clark et al., 1995, 1993; Schoof et al., 2000; Brand et al., 2000). In turn, WUS promotes the expression of CLV3, which leads to the formation of a negative feedback loop responsible for the stability of the SAM size (Schoof et al., 2000). In addition to CLV3, several other peptides encoded by the CLAVATA3-LIKE/ENDOSPERM SURROUNDING REGION (CLE) gene family, such as CLE16, CLE17 and CLE40, have been shown to regulate the size of the SAM (Dao et al., 2022; Schlegel et al., 2021).

Recently, we have demonstrated that in addition to CLV3 and CLE peptides, another signaling pathway controls WUS expression (Zhang et al., 2021). In Arabidopsis, three plasma membrane receptors, ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2, redundantly regulate the width of the vegetative SAM and promote leaf initiation in its periphery (Chen et al., 2013; Uchida et al., 2013). Collectively, these receptors are called ERECTA family receptors (ERfs). In the SAM, ERf activity is controlled by four extracellular proteins – EPFL1, EPFL2, EPFL4 and EPFL6 (herein collectively referred to as EPFLs) – that are expressed in the SAM periphery (Kosentka et al., 2019). Genetic analysis has demonstrated that ERfs and CLV3 function synergistically in controlling SAM size and organogenesis in the peripheral zone (Zhang et al., 2021; Kimura et al., 2018). The clv3 er erl1 erl2 mutant forms a gigantic meristem that cannot form leaves or internodes (Zhang et al., 2021). Our previously published data show that wus is epistatic to genes encoding ERfs (Zhang et al., 2021). Stimulation of ERf signaling with exogenous EPFL4 or EPFL6 rapidly decreases both CLV3 and WUS expression. Based on these data, we have proposed that ERfs restrict the width of the central zone in the SAM by inhibiting the expression of CLV3 and WUS in the peripheral zone (Zhang et al., 2021).

The main focus of the current investigation is the role of EPFL and CLV3 signaling pathways in SAM maintenance and organogenesis. We studied the role of EPFLs in the regulation of CLV3 and WUS expression and uncovered that the four EPFL ligands function redundantly. Using RNA sequencing (RNAseq), we analyzed gene expression changes after a brief activation of ERf signaling with EPFL6. This experiment confirmed that CLV3 and WUS are the main targets of the pathway and uncovered several new potential targets. In addition, we studied the role of CLV3 in the control of WUS expression. Whereas it is broadly accepted that CLV3 prevents WUS expression in the top layers of the meristem, our analysis indicated that CLV3 regulates the amount of WUS per cell and not its spatial expression. Finally, the role of EPFL signaling in leaf organogenesis was studied using the DORNROSCHEN (DRN; also known as ENHANCER OF SHOOT REGENERATION 1, ESR1) and DRN-LIKE (DRNL; also known as ENHANCER OF SHOOT REGENERATION 2, ESR2) markers. DRN and DRNL are transcription factors belonging to the APETALA2/Ethylene Responsive Factor (AP2/ERF) gene family. DRNL is a direct target of auxin and one of the first markers of organ initiation (Chandler et al., 2011; Comelli et al., 2016; Dai et al., 2023). Whereas DRN is normally expressed in the central zone, it is upregulated in the incipient primordia when DRNL is absent (Dai et al., 2023; Kirch et al., 2003). Our experiments indicated that although cells designated to become leaf primordia are specified in the peripheral zone, the bulging of the primordia is inefficient when ERf signaling is disrupted.

EPFL1, EPFL2, EPFL4 and EPFL6 redundantly control expression of WUS and CLV3

EPFL1, EPFL2, EPFL4 and EPFL6 redundantly restrict the size of the SAM (Kosentka et al., 2019). When EPFL4 and EPFL6 are supplied exogenously, they suppress WUS and CLV3 expression (Zhang et al., 2021). To test whether EPFL1 and EPFL2 also regulate the expression of WUS and CLV3, we analyzed the spatial expression of CLV3 and WUS in the vegetative SAM of epfl mutants using previously described H2B-GFP reporters (Zhang et al., 2021). Seedlings at 3 days post germination (3DPG) were used for all experiments. The reporter analysis showed that simultaneous knockout of either EPFL4 and EPFL6 or EPFL1 and EPFL2 had a minimal effect on the spatial expression pattern of CLV3 and WUS (Fig. 1A-D). We observed only a minute increase in the height of the WUS domain in epfl1 epfl2 (epfl1,2) and epfl4 epfl6 (epfl4,6) mutants (Fig. 1A,B) and a very slight broadening of CLV3 in the L1 layer of the epfl1,2 mutant (Fig. 1C,D). The small increase in CLV3 expression in the epfl1,2 mutant correlated with a slightly broader SAM (Fig. 1E). Next, we analyzed the expression of CLV3 and WUS in epfl1/+ epfl2 epfl4 epfl6 (epfl1/+,2,4,6) and epfl1 epfl2 epfl4 epfl6 (epfl1,2,4,6) quadruple mutant seedlings. In the seedlings heterozygous for epfl1 mutations, the sizes of CLV3 and WUS domains were slightly increased compared to those in both the wild type and double mutants (Fig. 1A-D). Again, this correlated with a subtle increase in the SAM width (Fig. 1E). In the quadruple epfl1,2,4,6 mutant, the width of WUS and CLV3 expression domains and the SAM were the most significantly increased (Fig. 1A-E). These experiments indicate that all four ligands regulate the expression of WUS and CLV3 in a mostly redundant manner.

Fig. 1.

Genes encoding EFPLs synergistically regulate the expression of CLV3 and WUS. (A,C) Representative confocal images of the SAM region of 3DPG seedlings of the indicated genotypes (wt, wild type) transformed with promoter reporters for (A) WUS (WUSp:H2B-GFP; green) or (C) CLV3 (CLV3p:H2B-GFP:CLV3t; green). All images are in the same panel are shown at the same magnification. The cell walls were stained with SR2200 (blue). (B) The width and height of the WUS expression region in seedlings of the indicated genotype, as shown by the H2B-GFP promoter reporter. n=7-20. (D) The width of CLV3 expression in the L1 and L2 layers for the indicated genotypes, as shown by the H2B-GFP promoter reporter. n=8-15. (E) Comparison of the SAM width at 3DPG for the indicated genotypes. n=13-54. Measurements in B, D and E were taken from confocal images. (F) RT-qPCR analysis of WUS and CLV3 in 3DPG seedlings of the indicated genotypes. n=3. Bars in B, D and E show the mean. Data in F are shown as the mean±s.d. Statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significance differences, with no statistically significant difference between groups marked by the same upper- or lower-case letter.

Fig. 1.

Genes encoding EFPLs synergistically regulate the expression of CLV3 and WUS. (A,C) Representative confocal images of the SAM region of 3DPG seedlings of the indicated genotypes (wt, wild type) transformed with promoter reporters for (A) WUS (WUSp:H2B-GFP; green) or (C) CLV3 (CLV3p:H2B-GFP:CLV3t; green). All images are in the same panel are shown at the same magnification. The cell walls were stained with SR2200 (blue). (B) The width and height of the WUS expression region in seedlings of the indicated genotype, as shown by the H2B-GFP promoter reporter. n=7-20. (D) The width of CLV3 expression in the L1 and L2 layers for the indicated genotypes, as shown by the H2B-GFP promoter reporter. n=8-15. (E) Comparison of the SAM width at 3DPG for the indicated genotypes. n=13-54. Measurements in B, D and E were taken from confocal images. (F) RT-qPCR analysis of WUS and CLV3 in 3DPG seedlings of the indicated genotypes. n=3. Bars in B, D and E show the mean. Data in F are shown as the mean±s.d. Statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significance differences, with no statistically significant difference between groups marked by the same upper- or lower-case letter.

At the same time, there were subtleties in the contribution of individual EPFLs to the regulation of these two genes. When we analyzed the expression of WUS and CLV3 in epfl triple and quadruple mutants using reverse transcription-quantitative PCR (RT-qPCR), we observed increased expression of these two genes, and the mutant combination dictated which one was upregulated more (Fig. 1F). The experiment suggests that EPFL1 and EPFL2 might have a more significant role in the regulation of CLV3 whereas EPFL4 and EPFL6 are more important for the regulation of WUS. In addition, a comparison of epfl1,2,4,6 and er erl1 erl2 mutants identified some differences: in the epfl1,2,4,6 mutant, CLV3 and WUS were expressed in a narrower domain (Fig. 1B,D), CLV3 was expressed at a lower level (Fig. 1F) and the SAM was slightly narrower (Fig. 1E). This suggests that either ERfs can weakly regulate the SAM in a ligand-independent manner or additional EPFL ligands contribute to the regulation of the SAM structure.

Transcriptome analysis identifies several meristematic genes, including CLV3 and WUS, as the immediate targets of EPFL6

A previous study in which transient activation of ERfs with EPFL4 and EPFL6 was followed by RT-qPCR identified CLV3 and WUS as downstream targets (Zhang et al., 2021). To discover targets of ERf signaling using an unbiased approach, we performed transcriptome sequencing. As we were specifically interested in the meristematic targets, many of which are expressed at low levels, we used clv3 epfl1,2,4,6 seedlings that have very large vegetative SAMs (Zhang et al., 2021) and performed a relatively deep sequencing (>50 M reads per sample). We treated 3DPG seedlings exogenously with 10 µM EPFL6 for 3 h with and without 10 µM cycloheximide (CHX). CHX was used to test whether the regulation of gene expression by EPFL6 depends on translation. Based on principal component analysis (PCA), the obtained RNAseq data clustered according to treatment and showed a high degree of intra-treatment reproducibility (Fig. S1A). A Pearson correlation heatmap of replicates also indicated the similarity between biological replicates (Fig. S1B). In the samples treated with EPFL6 only, we observed minimal changes in gene expression compared to the mock treatment (Fig. S1A,B). In response to EPFL6 alone, eight genes were upregulated by at least 1.5-fold, or 0.585 log2 fold change (LFC), and fourteen genes were downregulated at least −0.585 LFC. All results were corrected for multiple comparisons and had a false discovery rate (FDR) of less than 0.05 (Table S1). The RNAseq data from plants treated with CHX or with CHX and EPFL6 (CHX+EPFL6) clustered together but were not as similar. As expected, global inhibition of translation by CHX led to widespread dysregulation of gene expression in the mock sample (Table S1). Downregulated and upregulated targets of EPFL6-only treatment showed no coordinated expression pattern under CHX-only treatment (Fig. 2B). Using the CHX treatment as a control, we analyzed CHX+EPFL6-induced changes and observed a reduction of gene expression for both genes downregulated and upregulated by EPFL6 alone (Table S1). This suggests that most of these genes are directly repressed by the ERf signaling pathway.

Fig. 2.

Downstream targets of EPFL6 based on RNAseq analysis. (A) A volcano plot shows changes in gene expression in 3DPG clv3 epfl1,2,4,6 seedlings after treatment with 10 µM EPFL6. Vertical dashed lines indicate an LFC cutoff of ±0.585; horizontal dashed lines mark the FDR cutoff of 0.05. Selected genes that are discussed in the manuscript are indicated. Blue indicates genes studied previously. Two groups of paralog genes are indicated by green and purple. (B) Comparison of changes in gene expression in response to 10 µM EPFL6 (versus mock treatment; EPFL6-Mock), 10 µM CHX (versus mock treatment; CHX-mock) and CHX+EPFL6 cotreatment versus only CHX treatment (CHX&EPFL6-CHX). Most genes downregulated in response to EPFL6 are also downregulated in response to CHX+EPFL6 (left panel). None of the genes upregulated in response to EPFL6 are upregulated in response to CHX+EPFL6 (right panel), suggesting that their upregulation is indirect. Genes discussed in the text are in bold. *FDR<0.05. Data are presented as the mean±s.e.m. of n=3 replicates. (C) RT-qPCR analysis of selected gene expression in 3DPG seedlings of the indicated genotypes (wt, wild type). Data are presented as the mean±s.d. of n=3. Statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significance differences, with no statistically significant difference between groups marked by the same letter.

Fig. 2.

Downstream targets of EPFL6 based on RNAseq analysis. (A) A volcano plot shows changes in gene expression in 3DPG clv3 epfl1,2,4,6 seedlings after treatment with 10 µM EPFL6. Vertical dashed lines indicate an LFC cutoff of ±0.585; horizontal dashed lines mark the FDR cutoff of 0.05. Selected genes that are discussed in the manuscript are indicated. Blue indicates genes studied previously. Two groups of paralog genes are indicated by green and purple. (B) Comparison of changes in gene expression in response to 10 µM EPFL6 (versus mock treatment; EPFL6-Mock), 10 µM CHX (versus mock treatment; CHX-mock) and CHX+EPFL6 cotreatment versus only CHX treatment (CHX&EPFL6-CHX). Most genes downregulated in response to EPFL6 are also downregulated in response to CHX+EPFL6 (left panel). None of the genes upregulated in response to EPFL6 are upregulated in response to CHX+EPFL6 (right panel), suggesting that their upregulation is indirect. Genes discussed in the text are in bold. *FDR<0.05. Data are presented as the mean±s.e.m. of n=3 replicates. (C) RT-qPCR analysis of selected gene expression in 3DPG seedlings of the indicated genotypes (wt, wild type). Data are presented as the mean±s.d. of n=3. Statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significance differences, with no statistically significant difference between groups marked by the same letter.

Consistent with our previously published data (Zhang et al., 2021), RNAseq showed ∼50% downregulation of CLV3 and WUS expression in response to EPFL6 treatment (Fig. 2A). The reduction in WUS and CLV3 expression was independent of the production of new proteins, as we observed their downregulation in samples treated simultaneously with EPFL6 and CHX (Fig. 2B). These experiments confirmed that WUS and CLV3 are direct targets of the ERf signaling pathway.

Additionally, we found that genes encoding three members of the MEI2 family of RNA-binding proteins – MEI2 C-TERMINAL RRM ONLY LIKE1 (MCT1), MCT2 and TERMINAL EAR-LIKE 2 (TEL2) – were significantly downregulated by EPFL6 treatment (Fig. 2A,B). The expression of the fourth member of this family, TEL1, was slightly reduced by CHX+EPFL6 treatment but not by EPFL6 alone (Fig. 2B). Another gene downregulated by EPFL6 treatment was that encoding the leucine-rich-repeat protein AT3G25670 (hereafter referred to as AtFEA3), one of three Arabidopsis homologs of maize FASCIATED EAR 3 (FEA3), which regulates the SAM size (Je et al., 2016). RT-qPCR analysis of er erl1 erl2 and epfl1,2,4,6 seedlings detected increased expression of MCT1, MCT2 and TEL2, which is consistent with their downregulation by ERfs (Fig. 2C). We could not detect TEL1 expression consistently in either the wild type or the mutants due to its extremely low expression, if any. There was no change in AtFEA3 expression in the mutants (Fig. 2C), suggesting that it might not be a significant target of EPFLs. Finally, of note is the downregulation of ERL1 and ERL2 expression by EPFL6 (Fig. 2B and Table S1). Previously published data show that ERf signaling negatively regulates ERL1 and ERL2 expression (Pillitteri et al., 2007).

Out of ten genes upregulated in response to EPFL6, none were upregulated when EPFL6 was applied with CHX, suggesting that upregulation of these genes is an indirect response to EPFL6. The two upregulated genes with the lowest FDR were DRN and DRNL (Fig. 2A), which encode transcription factors that regulate meristem maintenance and organ initiation (Kirch et al., 2003; Ikeda et al., 2021, 2006). However, both of these genes were downregulated by EPFL6 in the presence of CHX (Fig. 2B, right panel, CHX&EPFL6-CHX bars). In addition, EPFL6 downregulated expression of the gene encoding AT1G80580, a close paralog of DRN and DRNL (Fig. 2A,B). This result suggests that EPFLs might downregulate this gene family directly while simultaneously indirectly promoting DRN and DRNL expression. EPFL6-only treatment upregulated the expression of UNUSUAL FLORAL ORGANS (UFO) (Fig. 2A), another meristematic gene (Long and Barton, 1998). However, in CHX+EPFL6 treatment, we observed the downregulation of UFO (Fig. 2B), suggesting that EPFL6 regulates the expression of this gene in a complex manner.

EPFL2 is expressed in the boundaries between the SAM and forming primordia (Kosentka et al., 2019). To test whether EPFL2 can alter the expression of genes identified by transcriptomics from the boundary, we created epfl1,2,4,6 transgenic plants with inducible EPFL2 expression (epfl1,2,4,6T). We used the pOp/LhGR system that allows tissue-specific expression of a gene of choice in response to dexamethasone (DEX) (Samalova et al., 2005). The construct was created in such a way that in response to DEX, expression of EPFL2 and H2B-GFP was induced in tissues where the EPFL2 promoter was active (Fig. S2). Without induction, H2B-GFP could not be detected by confocal microscopy, but after 7 h of induction, H2B-GFP was clearly visible in the boundary zone of the SAM (Fig. 3A). Interestingly, RT-qPCR indicated that both H2B-GFP and EPFL2 were expressed in transgenic plants without induction (Fig. 3B,C). After induction, their expression increased slightly. This suggests that our construct led to a leaky, unspecific expression at very low levels throughout the plant. This expression did not noticeably alter the phenotype of the epfl1,2,4,6 mutant (data not shown). In response to DEX, expression of GFP, and presumably of EPFL2, was strongly activated in cells where the EPFL2 promoter functions (Fig. 3A). Because this happens in very few cells out of many, RT-qPCR barely detected any change. Expression of CLV3, MCT1, MCT2 and TEL2 genes in transgenic seedlings without induction (epfl1,2,4,6T-mock) was similar to their expression in untransformed epfl1,2,4,6 seedlings (Figs 1FD, 2CD and 3D). When DEX was used to induce EPFL2 expression for 7 h in the boundary of the SAM, expression of CLV3, MCT1 and MCT2 decreased (Fig. 3D). TEL2 might be a specific target of EPFL4 and EPFL6. Unexpectedly, very low broad expression of EPFL2 reduced the expression of WUS in epfl1,2,4,6T seedings without induction to levels that were only slightly above the wild-type levels (Fig. 3E). Induction of EPFL2 expression in the boundary did not significantly lower WUS expression. In summary, this experiment confirmed that CLV3, MCT1 and MCT2 are endogenous targets of EPFL2.

Fig. 3.

Induction of EPFL2 in the SAM boundary leads to decreased expression of CLV3, MCT1 and MCT2. (A) Confocal images of 3DPG epfl1,2,4,6T seedlings following mock treatment or DEX treatment for 7 h. The seedlings express H2B-GFP (green) under the EPFL2 promoter in response to DEX. Both images were acquired with the same magnification, and the same settings were used for confocal microscopy. The cell walls were stained with SR2200 (blue). Images are representative of ten seedlings. (B-E) RT-qPCR analysis of (B) EPFL2; (C) H2B-GFP; (D) CLV3, MCT1, MCT2 and TEL2; and (E) WUS expression in (B,D,E) 3DPG wild-type (wt) seedlings, (B,C,E) 3DPG epfl1,2,4,6 seedlings and (B-E) 3DPG epfl1,2,4,6T seedlings expressing inducible EPFL2 and H2B-GFP under the EPFL2 promoter, with either mock treatment or DEX treatment as indicated. Data are presented as the mean±s.d. of n=3. Statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significance differences, with no statistically significant difference between groups marked by the same letter. *P<0.05 in C.

Fig. 3.

Induction of EPFL2 in the SAM boundary leads to decreased expression of CLV3, MCT1 and MCT2. (A) Confocal images of 3DPG epfl1,2,4,6T seedlings following mock treatment or DEX treatment for 7 h. The seedlings express H2B-GFP (green) under the EPFL2 promoter in response to DEX. Both images were acquired with the same magnification, and the same settings were used for confocal microscopy. The cell walls were stained with SR2200 (blue). Images are representative of ten seedlings. (B-E) RT-qPCR analysis of (B) EPFL2; (C) H2B-GFP; (D) CLV3, MCT1, MCT2 and TEL2; and (E) WUS expression in (B,D,E) 3DPG wild-type (wt) seedlings, (B,C,E) 3DPG epfl1,2,4,6 seedlings and (B-E) 3DPG epfl1,2,4,6T seedlings expressing inducible EPFL2 and H2B-GFP under the EPFL2 promoter, with either mock treatment or DEX treatment as indicated. Data are presented as the mean±s.d. of n=3. Statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significance differences, with no statistically significant difference between groups marked by the same letter. *P<0.05 in C.

The expression patterns of DRN and DRNL are altered in the epfl1,2,4,6 mutant

ERf signaling plays an important role in the initiation of cotyledons and leaves. However, the molecular mechanism is unknown (Chen et al., 2013; DeGennaro et al., 2022). Based on the role the AP2/ERF family plays in cotyledon and leaf initiation in a variety of species (Chandler et al., 2007; Capua and Eshed, 2017; Kusnandar et al., 2021) and on our RNAseq data, we hypothesized that ERfs might regulate organogenesis through control of DRN and/or DRNL expression. To test this hypothesis, we compared the expression of their H2B-GFP reporters in the wild type and epfl mutants. We used a 4.9 kb sequence upstream of the start codon and a 1.4 kb sequence downstream of the stop codon to analyze DRN expression. These regulatory regions have been reported to reflect the DRN expression similarly to RNA in situ hybridization (Kirch et al., 2003; Luo et al., 2018). To analyze DRNL expression, we used a 4.3 kb region upstream of the start codon as a promoter. The expression pattern of this regulatory region has also been tested previously and is consistent with RNA in situ hybridization (Luo et al., 2018). Based on published data, DRN is expressed in the young leaf primordia and the L1 and L2 layers (called the tunica) of the vegetative SAM (Kirch et al., 2003). DRNL is expressed in leaf and flower primordia (Ikeda et al., 2006; Nag et al., 2007). During flower development, DRNL is expressed in the primordia founder cells before the formation of auxin maxima and in the outer periphery of the future auxin peak (Luo et al., 2018; Chandler and Werr, 2014).

In agreement with published data, we observed expression of the DRN H2B-GFP reporter (DRNp:H2B-GFP) in the tunica of the wild-type SAM (Fig. 4A). In the L1 layer, the reporter was expressed broadly. In the L2 layer and deeper tissues, expression was narrow and correlated with the formation of leaf primordia. We observed a similar pattern in epfl1,2 and epfl1/+,2,4,6 seedlings, with the exception that, because the SAM in these mutants is slightly broader, DRN was expressed in a wider area of the L1 layer (Fig. 4B), and the correlation of DRN expression in L2 and L3 layers with forming organ primordia was more obvious. Even though the SAM of the epfl1,2,4,6 mutant forms very few primordia, DRN was found to be expressed very broadly in the L2 and L3 layers of this mutant. Because DRN is induced by auxin (Cole et al., 2009), and in the absence of ERf/EPFL signaling, auxin is present at higher levels in the SAM (DeGennaro et al., 2022), it is not clear whether the broader DRN expression observed in the SAM is related to altered auxin levels or whether ERf signaling directly downregulates this gene.

Fig. 4.

Broader expression of DRN and DRNL in the SAM of epfl mutants. (A,C) Representative confocal images of the SAM region of 3DPG seedlings of the indicated genotypes (wt, wild type) expressing a promoter reporter construct for (A) DRN (DRNp:H2B-GFP:DRNt; green) or (C) DRNL (DRNLp:H2B-GFP; green). White arrows in A indicate the induction of DRN in incipient leaf primordia. All images in the same panel are shown at the same magnification. The cell walls were stained with SR2200 (blue). Images are representative of 20 seedlings. (B) The average width of DRN expression in the L1 layer of the SAM was measured on the confocal images. Bars indicate the mean; n=4-11 seedlings. Statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significant differences.

Fig. 4.

Broader expression of DRN and DRNL in the SAM of epfl mutants. (A,C) Representative confocal images of the SAM region of 3DPG seedlings of the indicated genotypes (wt, wild type) expressing a promoter reporter construct for (A) DRN (DRNp:H2B-GFP:DRNt; green) or (C) DRNL (DRNLp:H2B-GFP; green). White arrows in A indicate the induction of DRN in incipient leaf primordia. All images in the same panel are shown at the same magnification. The cell walls were stained with SR2200 (blue). Images are representative of 20 seedlings. (B) The average width of DRN expression in the L1 layer of the SAM was measured on the confocal images. Bars indicate the mean; n=4-11 seedlings. Statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significant differences.

In the wild type, DRNL was expressed in the narrow strip of primordia founder cells (Fig. 4C). The epfl1,2,4,6 mutant forms leaf primordia very inefficiently (Kosentka et al., 2019). Unexpectedly, we observed efficient expression of DRNL in the SAM of this mutant, suggesting that in the mutant founder cells of leaf primordia are specified. This result indicates that ERf signaling promotes the subsequent step of leaf primordia outgrowth. In addition, we observed broader DRNL expression. Whereas the width of the DRNL expression in the wild type was 2-3 cells, in the epfl1,2,4,6 mutant, it was in the range of 4-7 cells. In the mutant, DRNL was also expressed in the L1 layer of the central zone. The DRNL promoter region contains auxin-responsive elements (Comelli et al., 2016). The broader expression of DRNL in the epfl1,2,4,6 mutant could be either a direct consequence of the altered ERf signaling or due to changes in auxin accumulation.

In summary, analysis of DRN and DRNL reporters in epfl mutants demonstrated a broader expression of these genes. This result is consistent with the downregulation of these genes we observed upon CHX+EPFL6 treatment. However, this change could also result from increased auxin accumulation in the SAM of plants with mutation of genes encoding ERfs or EPFLs. DRN and DRNL have been linked with the induction of leaf initiation (Chandler et al., 2007). Our data suggest that our original hypothesis that ERfs promote leaf initiation through induction of DRN and DRNL was incorrect. Whereas increased expression of DRN and DRNL in the SAM of the epfl1,2,4,6 mutant might alter some aspects of meristem maintenance, it is unlikely to inhibit leaf initiation. We speculate that ERfs do not specify the primordia founder cells but promote the outgrowth of demarcated leaf primordia.

Regulation of WUS expression by CLV3 signaling

In our previous work, we observed expression of WUS directly under the L2 layer in the wild-type vegetative SAM (Zhang et al., 2021). This contradicts the widely used description of WUS expression in the deeper layers of the SAM, only partially overlapping with CLV3 expression (Truskina and Vernoux, 2018; Uchida and Torii, 2019; Fuchs and Lohmann, 2020; Han et al., 2020a; Hirakawa, 2021; Lopes et al., 2021; Shimotohno, 2022; Ince and Sugimoto, 2023), based on early studies of this gene (Schoof et al., 2000; Brand et al., 2000). To determine the WUS expression domain, we performed further analysis using promoter reporters for WUS and CLV3.

Identification of cell layers on two-dimensional images of the SAM can be misleading because the meristem is often sectioned at an oblique angle (Fig. S3A). During the analysis of WUS expression, we realized that unless we examined a three-dimensional (3D) image, we often erroneously detected WUS in deeper tissue layers than it was actually expressed (Fig. S3B,C). Thus, we carefully analyzed z-stacks of WUS and CLV3 H2B-GFP reporter expression in the wild type, the er erl1 erl2 mutant and the clv3 mutant. In all images and in all seedlings, WUS was expressed in the third cell layer from the top at a constant distance from the surface of the SAM (Fig. 5D-G). In the clv3 mutant, we never detected a shift of WUS upward, only a slight expansion of WUS downward. In the wild type, WUS was mostly expressed in two cell layers, whereas in the clv3 mutant, WUS was sometimes expressed in three or four layers (Fig. 5D,E). In the clv3 er erl1 erl2 mutant, WUS was primarily expressed in layers three and four from the top, with only occasional expression in deeper layers (Fig. 6). Interestingly, WUS expression was discontinuous in this mutant, although all cells, based on CLV3 expression, were a part of the central zone (Fig. 6). In the clv3 mutant, er erl1 erl2 mutant and clv3 er erl1 erl2 mutant, we observed much broader expression of WUS along the radial axis (Figs 5D,E and 6; Zhang et al., 2021). Thus, CLV3 and ERf signaling mainly regulate WUS expression along the radial axis of the SAM and not the apical-basal axis.

Fig. 5.

CLV3 regulates the level of WUS expression but not its apical-basal pattern. (A-F) Confocal images of the SAM in 3DPG seedlings of the indicated genotypes (wt, wild type) expressing a promoter reporter construct for (A-C) CLV3 (CLV3p:H2B-GFP:CLV3t; green) or (D-F) WUS (WUSp:H2B-GFP; green). The cell walls were stained with SR2200 (blue). All images are shown at the same magnification. (G) The distance of the WUS domain from the top of the SAM in 3DPG seedlings of the indicated genotypes, measured using the H2B-GFP reporter as shown in D-F. n=4-8. (H) RT-qPCR of CLV3 and WUS in 3DPG seedlings of the indicated genotypes. n=15. (I,J) The number of cells expressing (I) WUS or (J) CLV3 in 3DPG seedlings of the indicated genotypes, as assessed using the promoter reporters shown in A-F. WUS, n=16-26; CLV3, n=12-22. (K) The number of cells expressing WUS or CLV3 in the L1 and L3 layers of the SAM of wild-type and er erl1 erl2 seedlings, determined as described in I and J. n=12-21. Bars in G and I-K indicate the mean. Data in H are presented as the mean±s.d. For H-K, statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significance differences, with no statistically significant difference between groups marked by the same upper- or lower-case letter. In I-K, horizontal lines represent the mean and points represent individual seedlings.

Fig. 5.

CLV3 regulates the level of WUS expression but not its apical-basal pattern. (A-F) Confocal images of the SAM in 3DPG seedlings of the indicated genotypes (wt, wild type) expressing a promoter reporter construct for (A-C) CLV3 (CLV3p:H2B-GFP:CLV3t; green) or (D-F) WUS (WUSp:H2B-GFP; green). The cell walls were stained with SR2200 (blue). All images are shown at the same magnification. (G) The distance of the WUS domain from the top of the SAM in 3DPG seedlings of the indicated genotypes, measured using the H2B-GFP reporter as shown in D-F. n=4-8. (H) RT-qPCR of CLV3 and WUS in 3DPG seedlings of the indicated genotypes. n=15. (I,J) The number of cells expressing (I) WUS or (J) CLV3 in 3DPG seedlings of the indicated genotypes, as assessed using the promoter reporters shown in A-F. WUS, n=16-26; CLV3, n=12-22. (K) The number of cells expressing WUS or CLV3 in the L1 and L3 layers of the SAM of wild-type and er erl1 erl2 seedlings, determined as described in I and J. n=12-21. Bars in G and I-K indicate the mean. Data in H are presented as the mean±s.d. For H-K, statistical differences were detected using a one-way ANOVA followed by a Tukey post-hoc test with P<0.05; letters denote statistically significance differences, with no statistically significant difference between groups marked by the same upper- or lower-case letter. In I-K, horizontal lines represent the mean and points represent individual seedlings.

Fig. 6.

In the clv3 er erl1 erl2 mutant, cells that express WUS also express CLV3. (A-C) Representative confocal images of the SAM region of 3DPG clv3 er erl1 erl2 seedlings transformed with a promoter reporter construct for either (A) CLV3 (CLV3p:H2B-GFP:CLV3t; green) or (B,C) WUS (WUSp:H2B-GFP; green). All images are shown at the same magnification. The cell walls were stained with SR2200 (blue). Images are representative of ten seedlings for A and 20 for B and C.

Fig. 6.

In the clv3 er erl1 erl2 mutant, cells that express WUS also express CLV3. (A-C) Representative confocal images of the SAM region of 3DPG clv3 er erl1 erl2 seedlings transformed with a promoter reporter construct for either (A) CLV3 (CLV3p:H2B-GFP:CLV3t; green) or (B,C) WUS (WUSp:H2B-GFP; green). All images are shown at the same magnification. The cell walls were stained with SR2200 (blue). Images are representative of ten seedlings for A and 20 for B and C.

In the vegetative SAM, CLV3 is expressed in the top 4-5 layers, and the depth of its expression is not altered in clv3 and er erl1 erl2 mutants (Fig. 5A-C) (Zhang et al., 2021). Thus, in the wild type, expression of CLV3 and WUS strongly overlaps. All cells that express WUS also express CLV3. This means that CLV3 should inhibit WUS expression primarily through autocrine signaling, with WUS-expressing cells secreting CLV3 and the same cells sensing it. Paracrine signaling with CLV3 being secreted by L1 and L2 cells and sensed by L3 cells might also contribute to the regulation of WUS expression, but we speculate that the diffusion of CLV3 from the above layers will probably provide only a small fraction of the available ligand.

To understand how ERfs and CLV3 regulate WUS expression, we estimated the amount of WUS per cell. The z-stacks were used to calculate the number of cells expressing WUS in the wild type and the mutants (Fig. 5I; Movies 1, 2 and 3). Compared to the wild type, there were approximately six and five times more WUS-expressing cells in the clv3 mutant and er erl1 erl2 mutant, respectively. RT-qPCR was used to determine the difference in WUS expression (Fig. 5H). After the difference in the number of WUS-positive cells was taken into account, the RT-qPCR data indicated that individual cells in the clv3 mutant expressed ∼22 times more WUS. In contrast, the amount of WUS per cell was not significantly changed in the er erl1 erl2 mutant (Table 1). This result indicates that the function of CLV3 is to regulate the levels of WUS in cells of the central zone. On the other hand, ERfs restrict the WUS expression domain in the SAM periphery but do not control the cellular levels of WUS in the central zone. In addition, we observed a considerable variance in the number of WUS-positive cells in individual meristems of both mutants (Fig. 5I), suggesting that both CLV3 and ERfs are necessary for the SAM size stability.

Table 1.

Comparison of CLV3 and WUS expression

Comparison of CLV3 and WUS expression
Comparison of CLV3 and WUS expression

The clv3 mutant used (clv3-9) has a point mutation that results in a premature stop codon (W62STOP), but it still produces CLV3 mRNA. Compared to the wild type, there were ∼9.7 and 3.5 times more CLV3-expressing cells in the clv3 mutant and er erl1 erl2 mutant, respectively (Fig. 5J). Surprisingly, the dramatic ∼22-fold increase in WUS in the clv3 mutant led only to a relatively modest ∼2.1-fold increase in CLV3 (Table 1). One possibility is that the premature stop codon decreases CLV3 mRNA stability. However, it has previously been proposed that at high concentrations, WUS can inhibit CLV3 expression (Perales et al., 2016). Whereas individual meristematic cells in the er erl1 erl2 mutant did not have increased WUS expression, they accumulated approximately five times more CLV3 (Table 1), suggesting that ERfs regulate CLV3 expression in individual cells independently of WUS.

Next, we investigated whether, in clv3 and er erl1 erl2 mutants, there was a comparable increase in the number of cells expressing WUS and CLV3. In the wild type, there were 1.9 times more cells expressing CLV3 than cells expressing WUS (Table 1). This is consistent with the fact that CLV3 is expressed in almost all WUS-expressing cells plus tunica cells. In the clv3 mutant, the ratio of CLV3 cells to WUS cells increased from 1.9 to 3.0 due to faster cell proliferation of the tunica cells. This proliferation leads to the convex shape of the clv3 SAM. We were surprised that in the er erl1 erl2 mutant, the ratio of CLV3 cells to WUS cells decreased from 1.9 to 1.4. To understand the cause of this decrease, the expression of both genes was compared in individual cell layers of the wild type and the er erl1 erl2 mutant (Fig. 5K). In the mutant, CLV3 expression expanded very broadly in the L1 layer. However, it did not spread as widely in the internal tissues as WUS expression. This finding has several implications. First, it suggests a complex tissue-specific pattern in which CLV3 expression is regulated and that ERf signaling plays an especially strong role in the inhibition of CLV3 expression in the L1 layer. An additional mechanism might restrict CLV3 expression in the internal tissues of the meristem periphery. Second, uneven expansion of CLV3 and WUS domains in the er erl1erl2 mutant indicates that the changes in their expression are not due to the overall expansion of the central zone but are due to a particular mechanism by which ERfs regulate them.

A mechanistic model for 3D expression patterning in the SAM

Previous mathematical models for apical-basal patterning of gene expression in the SAM have either assumed or produced antiparallel gradients of WUS and CLV3 expression with minimal overlap (Chickarmane et al., 2012; Hohm et al., 2010; Liu et al., 2020), which contradicts our 3D high-resolution imaging data (Fig. 5A-F; Movies 1-3). To test whether our current understanding of the regulatory network involving WUS, CLV3 and ERf signaling is sufficient to explain the up-to-date expression data, we built a 3D mathematical model that describes both the steady-state geometry of the SAM in terms of cell location and the expression regulation of WUS and CLV3. In this reaction-diffusion model, cells in the SAM were represented as 326 points in a 3D half dome. For gene regulation that occurs in cells, we considered the canonical negative feedback loop between WUS and CLV3 (Brand et al., 2000); the negative regulation of both WUS and CLV3 by EPFLs, whose expression zones were restricted to the peripheral areas of the SAM (Kosentka et al., 2019; Zhang et al., 2021); and the negative regulation of CLV3 by HAM (HAIRY MERISTEM protein family) signal from the meristem rib (Fig. 7A) (Han et al., 2020b). For WUS and CLV3, mRNA and protein levels were modeled separately. The model also considered movements of WUS, CLV3 and EPFLs between neighboring cells. Finally, we considered the inhibition of CLV3 by high concentrations of WUS (Perales et al., 2016) (Fig. 7A, dashed line). We restricted the WUS expression to L3 and lower layers and the HAM signal to the 6th layer from the epidermis and below to account for other spatial factors (e.g. cytokinin receptor) not described in the model (Fig. 7B).

Fig. 7.

A mathematical model of SAM patterning. (A) General gene regulatory network describing transcriptional regulation in the SAM. For CLV3 and WUS, both mRNA and protein are explicitly described, whereas only proteins were explicitly described for EPFLs and HAM. (B) Assumptions of maximum spatial distributions of regulatory factors in the absence of the regulations shown in A. Dots show the positions of simulated cells. Small dots indicate the absence of the factor from the regulatory network in A. Each length unit represents approximately 0.025 μm. (C-E) Model simulations under experimental conditions included in this study. The colors of the balls, which represent cells within the SAM, show normalized expression levels of the indicated factors (see Materials and Methods). CLV3 mRNA in the clv3 mutant (asterisk) is nonfunctional and is not translated. Because ERF mutants cannot perceive EPFL signals, in D the loss of ERF function (erf mutants) is represented as loss of EPFL expression. (F-H) Additional model conditions for predictions of the roles of specific transcriptional regulations. The specific transcriptional regulation perturbed in each panel is indicated by a red X.

Fig. 7.

A mathematical model of SAM patterning. (A) General gene regulatory network describing transcriptional regulation in the SAM. For CLV3 and WUS, both mRNA and protein are explicitly described, whereas only proteins were explicitly described for EPFLs and HAM. (B) Assumptions of maximum spatial distributions of regulatory factors in the absence of the regulations shown in A. Dots show the positions of simulated cells. Small dots indicate the absence of the factor from the regulatory network in A. Each length unit represents approximately 0.025 μm. (C-E) Model simulations under experimental conditions included in this study. The colors of the balls, which represent cells within the SAM, show normalized expression levels of the indicated factors (see Materials and Methods). CLV3 mRNA in the clv3 mutant (asterisk) is nonfunctional and is not translated. Because ERF mutants cannot perceive EPFL signals, in D the loss of ERF function (erf mutants) is represented as loss of EPFL expression. (F-H) Additional model conditions for predictions of the roles of specific transcriptional regulations. The specific transcriptional regulation perturbed in each panel is indicated by a red X.

We fitted the model to our high-resolution experimental data using biologically plausible parameter values. The model reproduced the CLV3 expression region encompassing the WUS expression region under the wild-type condition (Fig. 7C). This substantial overlap was also observed with the er erl1 erl2 mutant (erf mutant; the removal of EPFL signal) and the clv3 mutant (note that in the latter case, the CLV3 mRNA is produced but nonfunctional). In addition to the CLV3-WUS overlap, the absence of EPFL signal resulted in an expansion of the WUS expression region, but the level of expression in WUS mRNA-containing cells was unchanged (Fig. 7D). In contrast, the absence of functional CLV3 gave rise to both expansion of expression region and single-cell upregulation of WUS (Fig. 7E). The expression of CLV3 in both mutants was expanded in the SAM, but its single-cell upregulation was much less prominent compared to that of WUS (Fig. 7D,E).

We found that the loss of CLV3 inhibition by high concentration of WUS resulted in both expansion and single-cell upregulation of CLV3 expression (compare Fig. 7F and C). We next asked whether the inhibition of both CLV3 and WUS by EPFLs is required for correct SAM patterning. The removal of CLV3 inhibition by EPFLs resulted in upregulation of CLV3 in single cells and expansion of CLV3 expression (Fig. 7G), whereas the removal of WUS inhibition by EPFLs resulted in expansion of the expression regions of both CLV3 and WUS (Fig. 7H). The slight decrease in WUS expression was due to CLV3 upregulation (Fig. S4). This suggests that the two regulations by EPFLs are required for SAM patterning. Taken together, our experimentally inspired 3D model reproduced both key single-cell expression changes upon perturbations and distributions of gene expressions that were not captured by previous models simultaneously (Zhou et al., 2018; Liu et al., 2020). The model also suggests new mutant phenotypes that can be tested in future experiments.

The role of ERf/EPFL signaling in regulation of CLV3 and WUS expression

ERfs are important negative regulators of SAM size, functioning through suppression of CLV3 and WUS expression (Chen et al., 2013; Uchida et al., 2013, 2012b; Mandel et al., 2014; Zhang et al., 2021). The analysis of transcriptome changes after brief activation of ERf signaling confirmed that these two genes are the core targets of the pathway in the SAM. A mechanistic model for 3D expression patterning in the SAM indicated that regulation of both genes is necessary for the correct SAM patterning. The three ERfs redundantly control the width of the vegetative SAM and are particularly important during embryogenesis when the meristematic domain is defined (Chen et al., 2013; Uchida et al., 2012b). This is in contrast to CLV3 signaling, which controls both the width and the height of the SAM and functions in maintaining the meristem at a relatively constant size throughout the life of a plant (Clark et al., 1995). Consistent with their different roles in SAM establishment and maintenance, ERfs and CLV3 play distinct roles in the regulation of WUS. ERfs regulate the width of the WUS domain, suppressing its expression in the periphery of the SAM. Thus, ERf signaling contributes to the patterning of the SAM, defining different zones. In contrast, CLV3 regulates the cellular concentration of WUS in the central zone, defining its size.

The second function of ERfs is to reduce CLV3 cellular levels and prevent its expression in the periphery of the SAM, especially the L1 layer. ERfs regulate CLV3 independently of WUS, since in the central zone of the er erl1 erl2 mutant, the CLV3 cellular levels increase without an increase of WUS cellular levels, and knockout of ERf signaling in the wus background promotes CLV3 expression (Kimura et al., 2018). In early land plants, CLV regulates auxin and cytokinin signaling and does it independently of WUS homeobox-containing (WOX) genes (Fouracre and Harrison, 2022), as WUS-lineage WOX genes first appeared only in ferns (Nardmann and Werr, 2012). In liverwort, Marchantia polymorpha, CLAVATA signaling regulates meristematic cells independently of its only WOX gene (Hirakawa et al., 2020). We speculate that in angiosperms, CLV3 signaling might have other targets besides WUS, and suppression of CLV3 by ERfs might be related to these other functions.

The activity of ERfs is regulated by small extracellular cysteine-rich proteins from the EPF/EPFL family. In Arabidopsis, this family consists of eleven genes that form four clades (Takata et al., 2013). The function of two clades is linked with the formation of stomata (Richardson and Torii, 2013). The other two clades regulate the SAM (Kosentka et al., 2019). There are differences in the expression and overall function of these two last clades. One clade, consisting of EPFL4, EPFL5 and EPFL6 (EPFL4/5/6), promotes elongation of aboveground organs. EPFL4 and EPFL6 are expressed in the endodermis and regulate the elongation of internodes and pedicels (Abrash et al., 2011; Uchida et al., 2012a). All three genes redundantly promote the elongation of stamen filaments (He et al., 2023; Negoro et al., 2023; Huang et al., 2014). Another clade consists of EPFL1, EPFL2 and EPFL3 (EPFL1/2/3). EPFL2 regulates ovule initiation, elongation of leaf teeth and growth of cotyledons, and it is often expressed in organ boundaries (Tameshige et al., 2016; Kawamoto et al., 2020; Fujihara et al., 2021). EPFL4/5/6 and EPFL1/2/3 differ not only in function but also in structure. All EPF/EPFLs are made of a loop and a scaffold (Ohki et al., 2011). The loop structure is important for ligand function and might define whether it is an agonist or antagonist. EPFL9 is an antagonist; it competes with EPF2 for the same receptors but does not activate the downstream mitogen-activated protein kinase cascade (Lee et al., 2015). Swapping the loops between EPF2 and EPFL9 reverses their function (Ohki et al., 2011). The sequence and length of loops differ significantly between EPFL1/2/3 and EPFL4/5/6 clades. EPFL1, EPFL2, EPFL4 and EPFL6 redundantly regulate SAM width, leaf initiation and internode elongation (Kosentka et al., 2019). But do they regulate the same set of genes? Previously, we have demonstrated that EPFL4 and EPFL6 inhibit the expression of WUS and CLV3 (Zhang et al., 2021). Our current work shows that EPFL1 and EPFL2 also regulate the expression of these two genes. In summary, although the two clades of EPFL ligands differ structurally, their function in the SAM is very similar.

Previously, we have observed that whereas the SAM of the clv3 er erl1 erl2 mutant forms very few organs, the SAM of the clv3 epfl1,2,4,6 mutant forms some leaves and flowers (Zhang et al., 2021). Our current comparison of WUS and CLV3 expression in er erl1 erl2 and epfl1,2,4,6 mutants detected some small but statistically significant differences. This finding suggests that either ERfs regulate the SAM in a ligand-independent manner or other EPFLs also regulate the SAM structure.

ERf/EPFL signaling does not designate cells for leaf primordia but promotes primordia outgrowth

ERf signaling promotes the initiation of cotyledons and leaves and regulates phyllotaxis (Chen et al., 2013; DeGennaro et al., 2022). The hormone auxin induces initiation of aboveground organs. However, in the absence of ERf signaling, auxin cannot initiate leaves and cotyledons efficiently (DeGennaro et al., 2022). Previously, we have proposed that auxin and ERfs have common downstream targets (DeGennaro et al., 2022). In Arabidopsis, two AP2/ERF transcription factors, DRN and DRNL, promote cotyledon and leaf initiation (Chandler et al., 2007). During organogenesis, DRNL is expressed in incipient organ primordia before the formation of auxin response maxima, and it functions synergistically with auxin and PID (Chandler et al., 2011). DRNL in complex with transcription factor MONOPTEROS (MP) inhibits cytokinin accumulation in forming primordia (Dai et al., 2023). Our RNAseq data indicated that in the absence of CHX, a brief activation of ERf signaling promotes DRN and DRNL expression; however, in the presence of CHX, it downregulates DRN and DRNL expression. To understand the role of ERf signaling in the regulation of these two genes, we analyzed their expression in the SAM. This analysis indicated that DRN and DRNL are expressed more broadly in the epfl1,2,4,6 mutant than in the wild type, which is consistent with the downregulation of DRN and DRNL by EPFL6 in the presence of CHX. Overall, these data indicate that cells designated to become leaf primordia are specified, but some other requirements for leaf primordia outgrowth still must be met.

Transcriptomic analysis identified the MEI2 family of RNA-binding proteins as additional putative targets. Nine MEI2 family genes in Arabidopsis form two clades (Anderson et al., 2004). We found that EPFL6 inhibits the expression of three genes belonging to the same clade: MCT1, MCT2 and TEL2. Based on in situ hybridization, all three of these genes are expressed in the central zone of the SAM (Anderson et al., 2004; Yadav et al., 2009). In maize, rice and moss, these genes inhibit leaf initiation and control phyllotaxy (Veit et al., 1998; Xiong et al., 2006; Kawakatsu et al., 2006; Vivancos et al., 2012), but their meristematic function in Arabidopsis is unknown. It is tempting to speculate that ERfs regulate organogenesis by inhibiting MEI2 family gene expression. However, analysis of the function of these genes function in Arabidopsis is necessary before any definitive conclusion can be made.

CLV3 controls WUS cellular levels but not the apical-basal position of its expression domain

In Arabidopsis, the WUS-CLV3 negative feedback loop is central to the stability of the SAM size (Han et al., 2020a). WUS promotes the identity and proliferation of stem cells. CLV3 inhibits the expression of WUS to prevent stem cell overproliferation. Because WUS positively regulates CLV3 expression, CLV3 signaling can decrease WUS expression only to a certain extent and can never completely shut it down. The prevailing model asserts that the function of CLV3 is not only to regulate cellular levels of WUS but also to position the WUS domain in the deeper tissues of the SAM. This model was proposed during early investigations of the CLV3 and WUS feedback loop. Comparisons of WUS expression in the wild type and the clv3 mutant by in situ hybridization have been interpreted as showing that WUS is expressed in deep layers of the SAM and that WUS expression moves upward directly under the L2 cell layer when CLV3 is absent (Schoof et al., 2000; Brand et al., 2000). However, multiple published in situ hybridization images show that in the wild type, WUS is expressed directly under the L2 layer in both the vegetative and inflorescence SAM (Luo et al., 2018; Lenhard and Laux, 2003; Mayer et al., 1998; Hu et al., 2018). In addition, the original manuscript by Schoof and colleagues states that in embryos, WUS is expressed directly under the L2 layer in the wild type, and its expression domain does not move upward in the clv3 mutant (Schoof et al., 2000). An examination of in situ hybridization images of WUS expression in the manuscript by Brand and colleagues shows WUS expression directly under the L2 layer in the wild type (Brand et al., 2000). In wild-type meristems, the presence of WUS mRNA directly under the L2 layer has been observed for pWUS:GFP:WUS, pWUS:WUSlinker-GFP and pWUS:2×Venus-NLS:tWUS constructs (Yadav et al., 2011; Daum et al., 2014; Gruel et al., 2016; Wenzl and Lohmann, 2023). Despite all this evidence, the assumption that CLV3 regulates WUS expression along the apical-basal axis has not been explicitly challenged. All recent reviews show only a partial overlap of CLV3 and WUS expression, with the majority of WUS expression occurring in the cells that do not express CLV3 (Truskina and Vernoux, 2018; Uchida and Torii, 2019; Fuchs and Lohmann, 2020; Han et al., 2020a; Hirakawa, 2021; Lopes et al., 2021; Shimotohno, 2022; Ince and Sugimoto, 2023).

The correct detection of WUS expression in in situ hybridization images depends on precise vertical sectioning exactly through the center of the SAM. If the section is made at an oblique angle, the WUS expression will be perceived to be deeper than it is. We analyzed WUS expression using the WUSp:H2B-GFP promoter reporter construct. This construct contains a 4.5 kb regulatory sequence that has been used previously and that includes all regulatory elements necessary for expression in the SAM (Yadav et al., 2009; Bäurle and Laux, 2005; Zhang et al., 2017). When we examined two-dimensional images, we realized that they provide an inconsistent pattern of WUS expression and are challenging to interpret. However, analysis of z-stacks firmly placed WUS expression in the third and fourth cell layers of the SAM in the wild type and in any mutant that we observed. There was no shift of WUS expression apically in the clv3 mutant. If there was any expansion of WUS expression along the apical-basal axis, it was basally into the fifth layer in some clv3 mutant and epfl mutant seedlings. The expression of WUS is induced by cytokinins, which are produced in the L1 layer of the SAM but are perceived only underneath the tunica (Lindsay et al., 2006; Gordon et al., 2009; Chickarmane et al., 2012). Diffusion of cytokinins from the L1 layer tethers WUS expression to a specific distance from the apex; there is no apparent need for additional regulation. Our data indicate that CLV3 does not define the WUS expression domain but controls the concentration of WUS in the central zone.

Based on published in situ hybridization images, expression of CLV3 varies and can be detected in either three or four top layers of the SAM (Brand et al., 2000; Fletcher et al., 1999; Lenhard and Laux, 2003; Reddy and Meyerowitz, 2005). This inconsistency could be due to differences in the SAM sectioning (through the middle or at an angle); differences between vegetative, inflorescence and floral meristems; or variable growth conditions. Recent findings suggest that the depth of CLV3 expression is regulated by temperature, with higher temperatures inhibiting CLV3 expression in the deeper tissues (Wenzl and Lohmann, 2023). Our data indicate that in the vegetative SAM at 21°C, CLV3 is expressed in the four top cell layers in the wild type and in the clv3, er erl1 erl2, epfl and clv3 er erl1 erl2 mutants, and that CLV3 expression strongly overlaps with WUS. Thus, in the vegetative SAM, CLV3 should regulate WUS cellular levels through autocrine signaling, which is typical for CLE peptides (Narasimhan and Simon, 2022). Modeling predicted that the overlap of CLV3 and WUS expression removes the necessity for HAM influence close to the epidermis in the SAM. Consistently, we observed that CLV3 expression in the L1 layer and deeper tissues is controlled differently. Further research on the mechanisms controlling CLV3 expression should provide deeper insights into the molecular processes that oversee the size of the SAM and impact the overall plant architecture.

Plant materials and growth conditions

The Arabidopsis thaliana ecotype Columbia was used as the wild type. The following mutants have been described previously: er-105 erl1-2 erl2-1 (er erl1 erl2; Shpak et al., 2004); epfl1-1 epfl2-1 (abbreviated here as epfl1,2), cll2-1 chal-2/epfl4 epfl6 (abbreviated here as epfl4,6), epfl2,4,6 and epfl1,2,4,6 (Kosentka et al., 2019); clv3-9 (Nimchuk et al., 2015); and clv3 epfl1,2,4,6 (Zhang et al., 2021). All mutants are in the Columbia background. Seedlings were grown on modified Murashige and Skoog medium (Research Products International) plates supplemented with 1% (w/v) sucrose. Plates were stratified for 2 days at 4°C and then moved to a growth room with the following conditions: 18 h light/6 h dark cycle and 21°C. The generation of the wild-type plants expressing WUS promoter reporter (WUSp:H2B-GFP:35St; pESH746) and CLV3 promoter reporter (CLV3p:H2B-GFP:CLV3t; pESH747) constructs was described previously (Zhang et al., 2021). These constructs were transformed into clv3; epfl1,2; epfl4,6; epfl1/+,2,4,6; and clv3 er erl1 erl2 plants.

The DRN promoter reporter construct (DRNp:H2B-eGFP:DRNterm) was generated by fusing the 4.86 kb sequence upstream of the DRN start codon with sequence encoding H2B-eGFP followed by a 1.38 kb sequence downstream of the DRN stop codon (Kirch et al., 2003; Luo et al., 2018). The H2B-eGFP sequence was fused to the downstream sequence using overlapping PCR and inserted into the binary vector pPZP222 (Hajdukiewicz et al., 1994) between BamHI and SalI sites. The template for amplifying the H2B-EGFP sequence was a plasmid from the Z. Nimchuk lab (UNC Chapel Hill, USA). The H2B-eGFP:DRNterm plasmid was used as a vector to introduce the 4.86 kb DRN promoter sequence using BamHI, and the plasmid was named pAMO102b. The DRNL promoter reporter construct (DRNLp:H2B-eGFP:35sT) was generated by fusing the 4.3 kb sequence upstream of the DRNL start codon to H2B-eGFP:35sTerm (Luo et al., 2018). Using overlapping PCR, H2B-eGFP was linked to the CaMV 35S terminator and inserted into the binary vector pPZP222 between BamHI and SalI sites. This plasmid was used as a vector to introduce the 4.3 kb DRNL promoter between KpnI and BamHI sites, and the construct was named pAMP109. Both constructs were confirmed by Sanger sequencing.

The following construct was generated to produce dexamethasone–inducible EPFL2 expressed in its endogenous domain (Fig. S2). The 2.58 kb promoter of EPFL2 was amplified using pPZK412 (Kosentka et al., 2019) and fused with GR-LhG4:T35S amplified from pBIN-LhGR-N (Samalova et al., 2005) via overlapping PCR. This DNA fragment was introduced into the binary vector pPZP222 between KpnI and SbfI sites, and the plasmid was named pAMO112. The next step involved eight PCR reactions. PCR1 amplified H2B-GFP:35S terminator (T35S). PCR2 amplified the omega translational enhancer (Ω) and 35S minimal promoter (35S Min) using plasmid pH-TOP (Samalova et al., 2005) as a template. PCR1 and PCR2 products were overlapped to generate a PCR3 fragment. PCR4 used pH-TOP as a template to generate a fragment containing pPOP6 followed by 35S Min and Ω. PCR5 amplified the EPFL2 coding sequence with introns using the pPZK412 vector as a template. PCR6 amplified the NOS terminator (TNOS) using pBIN-LhGR-N as a template. PCR7 was an overlapping PCR that fused DNA fragments created by PCR4, PCR5 and PCR6. Finally, PCR8 was an overlapping PCR that fused the PCR3 and PCR7 DNA fragments. It generated a DNA fragment containing T35s:H2B-GFP: Ω:35SMin:pOp6:35SMin: Ω:gEPFL2: TNOS. The DNA segment generated by PCR8 was inserted into the SbfI site of pAMO112. Orientation was confirmed via restriction digest and Sanger sequencing. The sequencing identified additional SbfI sites between the two T35S. The final construct was named pAMO113 and contained: proEPFL2:GR-LhG4:T35S___T35S:H2B-GFP:Ω:35sMin_pOp6_35sMin:Ω:gEPFL2:TNOS.

The generated constructs were transformed into an Agrobacterium tumefaciens strain GV3101/pMP90 by electroporation and introduced into wild-type (Columbia ecotype); epfl1,2; and epfl1/+,2,4,6 plants by the floral dip method (Clough and Bent, 1998).

To induce EPFL2, an epfl1,2,4,6 mutant expressing pAMO113 was grown on modified Murashige and Skoog medium (MS-0) plates for 5 days (3DPG). Then, 15 seedlings per biological replicate were transferred to 2 ml of liquid MS-0 containing 10 µM DEX (Thermo Scientific Chemicals) or an equivalent amount of DMSO (mock treatment). The 50 mM stock solution of DEX was prepared using DMSO. 24-well culture plates with the samples were kept on a rocker in a growth room for 7 h, and then seedlings were preserved in liquid nitrogen.

Due to the infertility of the epfl1,2,4,6 mutant, we isolated it from the progeny of epfl1/+,2,4,6 plants. A small piece of root was cut from a seedling and placed into the PCR mix. PCR was performed using the Phire Plant Direct PCR Master Mix kit (Thermo Fisher Scientific). The rest of the seedling was preserved in 4% paraformaldehyde for microscopy. A three-primer PCR reaction with EPFL1.74, EPFL1.436.rev and 3dspm (Table S2) was performed to genotype for epfl1. The mutant band was ∼200 bp, and the wild-type band 387 bp.

RNAseq library construction, sequencing and analysis of differential gene expression

For the RNAseq sample collection, seedlings were grown as described previously (Zhang et al., 2021). In brief, the clv3 epfl1,2,4,6 seedlings were grown on modified Murashige and Skoog medium plates for 5 days (3DPG). Four treatment conditions were used: mock, 10 μM EPFL6, 10 μM cycloheximide (CHX; Alfa Aesar), and a combination of 10 μM EPFL6 and 10 μM cycloheximide. Purification of the EPFL6 peptide was described previously (Lin et al., 2017). EPLF6 peptides were diluted in 10 mM Bis-Tris and 100 nM NaCl, pH 6.0 (treatment buffer). For treatment, 10 seedlings per biological replicate were transferred into 1 ml of liquid Murashige and Skoog medium. Seedlings in liquid medium were treated with 8.7 μl of the 1.15 mM EPFL6 solution or for mock with an equal volume of the treatment buffer. For CHX treatments, seedlings in a liquid medium were pretreated with 10 μM CHX for 10 min before EPFL6 or the mock treatment was added. The aboveground portion of the seedlings was collected 3 h after adding EPFL6 or the mock treatment buffer and flash frozen in liquid nitrogen. Three biological replicates were performed for each treatment. Total RNA was isolated using the Spectrum Plant RNA Isolation Kit (Sigma-Aldrich).

RNA quality was measured using 4200 TapeStation (Agilent); all samples had an RNA integrity number (RIN) score greater than 7.5. Paired-end cDNA libraries were constructed using the TruSeq mRNA kit from Illumina. The libraries were sequenced on a NovaSeq S4 flow cell in paired-end mode and with 150 base pair reads at the Oklahoma Medical Research Foundation. Raw read quality was assessed with FastQC v0.11.5 (Babraham Bioinformatics). Raw reads were aligned to the TAIR10.1 genome and Araport11 annotation (TAIR genome and Araport11 citation) using STAR-2.7.6a (Dobin et al., 2013), with default parameters except for the following: - alignIntronMax 1000. Mapping quality was assessed with RSeQC v4.0.0 (Wang et al., 2012). Reads were counted using subread featureCounts v2.0.1 (Liao et al., 2013) in paired-end mode. Reads were imported into R (v3.6.3). Genes not expressed in all three replicates of at least one sample were removed. Samples were inspected for batch effect by PCA, and no batch effect was found. The filtered reads were then normalized, and differential gene expression was assessed using DESeq2 v1.26.0 (Love et al., 2014) using a two-factorial design. The resulting P-values were corrected for multiple comparisons using FDR, and the resulting log2 fold changes were shrunk using ashr2.2 (Stephens, 2017).

RT-qPCR analysis

Total RNA was isolated from the tissues of 3DPG seedlings using the Spectrum Plant RNA Isolation Kit (Sigma-Aldrich). The RNA was treated with RNase-free RQ1 DNase (Promega). First-strand complementary cDNA was synthesized with LunaScript RT SuperMix Kits (New England Biolabs). Quantitative PCR was performed with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). Each experiment contained three technical replicates of three biological replicates. Cycling conditions were as follows: 30 s at 95°C; then 40 repeats of 10 s at 95°C, 10 s at 52°C for ACTIN2, 55°C for WUS and GFP, 53°C for CLV3, 56.7°C for MCT1, 50°C for MCT2 and EPFL2, and 56.1°C for TEL2, and 15 s at 68°C, followed by the melt-curve analysis. For AtFEA3, two-step PCR was performed. Cycling conditions were as follows: 30 s at 95°C; then 40 repeats of 10 s at 95°C, 30 s at 68°C, followed by the melt-curve analysis. qPCR for ACTIN2, CLV3, EPFL2 and GFP was performed in 10 µl with 4 µl of 10× diluted cDNA, whereas WUS, AtFEA3, MCT1, MCT2 and TEL2 were performed in 20 µl with 8 µl of 10× diluted cDNA reaction. All primers used in this study are shown in Table S2. The fold difference in gene expression was calculated using relative quantification by the 2−ΔΔCT algorithm (Livak and Schmittgen, 2001) ACTIN2 was used as an internal control.

Microscopy

For microscopy, we used the T3 or T4 generations of transgenic plants that were homozygous for the insert, except when the WUS reporter was analyzed in T2 epfl1,2 and epfl4,6 seedlings. 3-day-old seedlings were fixed with 4% paraformaldehyde for 1.5 h. The fixed samples were washed three times for 5 min in phosphate buffer (PBS) and cleared with ClearSee (Kurihara et al., 2015) for 3 days at room temperature on a rocker. The cell wall was stained with Renaissance 2200 [0.1% (v/v) in ClearSee] (Musielak et al., 2015) for 1-2 days. For better imaging, one cotyledon was removed under a stereo microscope. A Leica SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) with 40×/1.10 water objective was used at the Advanced Microscopy and Imaging Center, University of Tennessee, Knoxville, TN, USA. An argon laser with 488 nm emission was used for the excitation of EGFP, and images were collected using a HyD ‘Hybrid’ Super Sensitivity SP Detector with the emission range of 493-550 nm. SCRI Renaissance 2200 (SR2200) dye was excited with a diode 405 nm ‘UV’ laser, and images were collected by using PMT SP Detector with the emission 415-470 nm. EGFP and SR2200 fluorescence emission was collected with HyD ‘Hybrid’ Super Sensitivity SP Detector (Leica Microsystems) and PMT SP Detector (Leica Microsystems). Z-stacks were created via sequential line scanning. Quantitative image measurements were performed using the Fiji image processing software (Schindelin et al., 2012). Two-dimensional slices from the center of the SAM were chosen based on analysis of z-stacks to determine the width and height of reporter expression. The spot detection tool of IMARIS software (Oxford Instruments) was used to calculate the number of cells in Fig. 5I-K and Table 1. Nuclei were detected on the basis of EGFP signal, and estimated nuclei diameter values were used for background subtraction.

Construction of the mathematical model

We assumed that there is a two-fold symmetry of the SAM. We used 326 points in a quarter ball (half dome) with a radius of 400 length units to represent cells in half of the SAM. The number of cells was estimated from Chen et al. (2013). In the 3D cell network model, the EPFL ligands are synthesized in two peripheral regions represented by two ‘corner’ regions. We assumed that EPFLs diffuses broadly in the SAM and inhibit the expression of both WUS and CLV3 through binding to their receptors, which were assumed to be always expressed in each cell (Kosentka et al., 2019; Zhang et al., 2021). Because CLV3 is a diffusive peptide, and WUS is a transcription factor capable of moving between cells (Daum et al., 2014; Lenhard and Laux, 2003; Yadav et al., 2011), we assumed that these molecules are diffusible in the model. Our model includes WUS-CLV3 negative feedback and its lateral regulator, the EPFLs. In addition, the model describes a HAIRY MERISTEM (HAM) signal that originates from the rib zone and inhibits CLV3 expression in the organizing center (Zhou et al., 2018). The distribution of HAM expression is likely established by other signals not considered in the model (Han et al., 2020b). It has been shown that a high concentration of WUS can cause CLV3 downregulation, forming a biphasic regulation of CLV3 by WUS (Perales et al., 2016; Shimotohno and Scheres, 2019), and this regulation is described in our model. The model also includes a CLV3-independent positive feedback involving WUS. This feedback can be supported by a WUS-cytokinin mutual activation loop: it has previously been shown that cytokinin activates WUS expression (Gordon et al., 2009; Chickarmane et al., 2012; Wang et al., 2017), whereas WUS derepresses cytokinin signal by inhibiting type A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes, which act as inhibitors of cytokinin (To et al., 2004; Leibfried et al., 2005; Shimotohno and Scheres, 2019). This positive feedback loop might also be supported by other factors (Yadav et al., 2013). Based on these assumptions, the dynamics of six interacting species representing concentrations of regulatory molecules are described with nonlinear ordinary differential equations (ODEs) in each cell (point) of the model (additional spatial constraints are shown in Fig. 7B):
Here, state variables Wr, Wp, Cr, Cp, L and H represent the concentrations (or strengths) of WUS mRNA, WUS protein, CLV3 mRNA, CLV3 protein, EPFLs and HAM, respectively. A full list of parameter descriptions and their numerical values is available in Table S3. In the ODEs, kw is the production rate constant of WUS protein; bW is the degradation rate constant of WUS protein; KWC is the threshold of inhibition of WUS by CLV3. nWC is the cooperativity of inhibition of WUS by CLV3; DW is the rate constant of passive diffusion-like transport of molecule WUS protein; kc is the production rate constant of CLV3 protein. bC is the degradation rate constant of CLV3 protein. DC is the rate constant of passive diffusion-like transport of molecule CLV3 protein. k0WW is the production rate constant of WUS mRNA. k0WL is the proportion of WUS mRNA production rate controlled by EPFLs. KWL is the threshold of WUS inhibition by EPFLs. nWL represents the cooperativity of regulation of WUS by EPFLs. k0WC is the proportion of WUS mRNA production rate controlled by CLV3. KWW is the activation threshold of WUS autoactivation. nWW is the cooperativity of WUS self-regulation. is the degradation rate constant of WUS mRNA. gc is the production rate constant of CLV3 mRNA. KCL1 is the threshold of CLV3 inhibition by EPFL. nCL1 is the cooperativity of regulation of CLV3 by EPFLs. KCW2 is the threshold of CLV3 activation by WUS. nCW2 is the cooperativity of regulation of CLV3 by WUS. apc is the constant representing the inversed strength of CLV3 inhibition by WUS. KCW3 is the threshold of CLV3 inhibition by WUS. nCW3 represents the cooperativity of negative regulation of CLV3 by WUS. KCH1 is the threshold of CLV3 activation by HAM. nCH1 represents the cooperativity of regulation of CLV3 by HAM. is the degradation rate constant of CLV3 mRNA. kL is the production rate constant of EPFL. bL is the degradation rate constant of EPFL protein. DL is the passive diffusion rate constant of EPFL proteins. kH is the production rate constant of HAM. bH is the degradation rate constant of HAM protein; Δ is the Laplace operator describing gradients of concentrations, which govern passive diffusion-like transport; ΔWp, ΔCp, ΔL have a unit of concentration per unit area. DW, DC, DL were adjusted by multiplying with a scaling factor l/l, where l represents the distance between the centers of the two cells (Delile et al., 2017); and neighboring cells are defined as cells that are located within a radius of 100 length units (∼10 μm). We neglected the subcellular geometry of the cells, their contact areas and the influence of mechanics in this study (the effective contact area for WUS transport cannot be directly inferred from total contact area of plasma membrane). The movements of EPFLs, CLV3 and WUS are responsible for the intercellular communication in the model. We used a Hill function to describe nonlinearity in the gene regulation. Previous models of the SAM and other complex systems have used similar nonlinear functions (Fujita et al., 2011; Nikolaev et al., 2007; Ye et al., 2019; Gruel et al., 2018; Liu et al., 2020). aC is a constant used to perturb the negative feedback regulation. aw0 is a constant used to perturb the auto-positive feedback regulation. Because the absolute concentrations of these molecules have not been measured experimentally, we used an arbitrary unit (a.u.) to describe concentration (or strength) of each molecule. We used a no-flux boundary condition for the model, as in other published SAM models (Zhou et al., 2018; Liu et al., 2020).

We fitted the parameters to known patterning phenotypes of the SAM under normal and genetically perturbed conditions. The erf mutant was represented by setting the EPFL production rate to 0. The clv3 mutant was represented by setting the CLV3 protein production rate to 0. Each regulation-specific perturbation was modeled by setting the inhibition threshold to large number (1000). Because only qualitative information is available from the experimental data, we performed the fitting manually. To perform a simulation for a SAM system, we solved the system of ODEs numerically using the Tellurium package (Choi et al., 2018). The initial concentrations for all variables were set to zero. For all our analyses, steady-state solutions (at time unit 98) were used to determine the patterning of the SAM. For visualization of gene expression levels, expression values were normalized to [0, 1] by dividing each value by the maximum level of the molecule across all conditions.

We thank Jaydeep Kolape for training and technical assistance with confocal microscopy at Advanced Microscopy and Imaging Center, University of Tennessee, Knoxville. We thank Guangzhong Lin and Jijie Chai for sharing with us EPFL6 protein, and Ian Moore for sharing with us GR-LhG4:T35S, pBIN-LhGR-N and pH-TOP plasmids.

Author contributions

Conceptualization: M.U., Z.L., L.Z., T.H., E.D.S.; Methodology: M.U., R.A.U.C., Z.L., A.M.O., D.D., L.Z.; Software: R.A.U.C., Z.L., T.H.; Validation: M.U., R.A.U.C., Z.L.; Formal analysis: M.U., R.A.U.C., Z.L., T.H., E.D.S.; Investigation: M.U., R.A.U.C., Z.L., A.M.O., D.D., L.Z., B.S.H., T.H., E.D.S.; Resources: A.M.O., D.D., L.Z.; Data curation: M.U., R.A.U.C.; Writing - original draft: M.U., R.A.U.C., Z.L., T.H., E.D.S.; Writing - review & editing: M.U., R.A.U.C., Z.L., A.M.O., D.D., L.Z., B.S.H., T.H., E.D.S.; Visualization: M.U., R.A.U.C., Z.L., D.D., T.H., E.D.S.; Supervision: T.H., E.D.S.; Project administration: T.H., E.D.S.; Funding acquisition: T.H., E.D.S.

Funding

This work was funded by the National Science Foundation (IOS–2016756 to E.D.S. and 2243562 to T.H.) and by the National Institutes of Health (R35GM149531 to T.H.). Deposited in PMC for release after 12 months.

Data availability

Code for reproducing the modeling result can be found at https://github.com/ZiyiiiLiu/SAM-model-3D. RNAseq data have been deposited in Gene Expression Omnibus under the accession GSE243494.

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Competing interests

The authors declare no competing or financial interests.

Supplementary information