The shoot apical meristem (SAM) of higher plants comprises distinct functional zones. The central zone (CZ) is located at the meristem summit and harbors pluripotent stem cells. Stem cells undergo cell division within the CZ and give rise to descendants, which enter the peripheral zone (PZ) and become recruited into lateral organs. Stem cell daughters that are pushed underneath the CZ form rib meristem (RM). To unravel the mechanism of meristem development, it is essential to know how stem cells adopt distinct cell fates in the SAM. Here, we show that meristem patterning and floral organ primordia formation, besides auxin transport, are regulated by auxin biosynthesis mediated by two closely related genes of the TRYPTOPHAN AMINOTRANSFERASE family. In Arabidopsis SAM, TAA1 and TAR2 played a role in maintaining auxin responses and the identity of PZ cell types. In the absence of auxin biosynthesis and transport, the expression pattern of the marker genes linked to the patterning of the SAM is perturbed. Our results prove that local auxin biosynthesis, in concert with transport, controls the patterning of the SAM into the CZ, PZ and RM.

In Arabidopsis, the cell-cell signaling network controlled by the receptor-like kinase CLAVATA1 (CLV1) and its ligand CLAVATA3 (CLV3) regulates stem cell proliferation and exit in the SAM (Fletcher et al., 1999). CLV3 binds to CLV1 and initiates the signaling cascade (Ogawa et al., 2008), which restricts the WUSCHEL (WUS) expression in the organizing center (Brand et al., 2000; Mayer et al., 1998). WUS protein moves from its site of synthesis via plasmodesmata, which serves as an essential intercellular conduit for the movement of small RNAs and protein macromolecules in plants (Sevilem et al., 2013). The intercellular movement of WUS is critical to promote stem cell fate in the apical meristem (Daum et al., 2014). WUS binds to the CLV3 promoter and activates its expression (Yadav et al., 2011). Most of the genes involved in stem cell differentiation are expressed in the PZ of SAM but are repressed by WUS (Yadav et al., 2013). Thus, WUS determines the fate of stem cells in the apical meristem and serves as a central hub around which gene expression patterns are established to specify CZ, PZ and RM to form a functional SAM. Recent studies have shown that the CLV-WUS feedback is a conserved feature of seed plants. In Marchantia polymorpha CLV3-like peptide, MpCLE2 expressed outside the stem cell domain appears to regulate the proliferation of apical notch cells via the MpCLV1 signaling pathway (Hirakawa et al., 2008, 2020). The closely related MpCLE1 restricts stem cell proliferation with a related receptor (Hirakawa et al., 2019). In Physcomitrella (Physcomitrium patens), a single apical stem cell undergoes asymmetric cell division to initiate filamentous growth. Interestingly, the broadly expressed CLE peptides control stem cell identity and their division plane to promote growth (Whitewoods et al., 2018). Additionally, CLV3-like peptides expressed in the periphery of the meristem both in Arabidopsis and grasses also control the activities of stem cells in the meristem to promote growth (Hata and Kyozuka, 2021; Liu et al., 2021; Ohmori et al., 2013; Schlegel et al., 2021). These studies show that diverse sets of CLV3-like peptides can promote and inhibit stem cell activities, and thus maintain stem cell homeostasis in land plants.

In angiosperms, stem cell daughters are recruited into organ primordia at a regular interval in the PZ. Previous studies have shown that auxin, which is polarly transported by its efflux carrier PIN FORMED1 (PIN1), is required for this transition (Gälweiler et al., 1998; Reinhardt et al., 2003; Vernoux et al., 2000). By combining genetics, molecular biology and pharmacological treatments, studies have shown that auxin controls its transport by regulating the expression of PIN1 and downstream auxin signaling network genes (Adamowski and Friml, 2015). Genetic and molecular evidence collected thus far suggests that PIN1 is polarized toward the regions of high auxin concentrations, reinforcing a positive-feedback loop between PIN1 and auxin signaling network genes (Burian et al., 2022; Friml et al., 2002; Galvan-Ampudia et al., 2020). A recent study has shown that the auxin response factor MONOPTEROUS (MP), which controls the polarity of PIN1 non-cell autonomously, is expressed in the incipient primordia first; PIN1 polarization follows MP expression (Bhatia et al., 2016). However, pin1 mutant plants produce cotyledons and leaves in the vegetative phase of plant development (Guenot et al., 2012; Vernoux et al., 2000), suggesting that locally produced auxin might be involved in leaf initiation in the pin1 mutant. Previous models of phyllotactic pattern formation did not consider the role of locally produced auxin with transport in the patterning of the SAM (Smith et al., 2006). When auxin is applied externally to the SAM, it promotes auxin responses in the PZ of the SAM without affecting the fate of stem cells in the CZ, suggesting that stem cells are either insensitive to auxin or lack the cellular machinery to perceive it efficiently (Heisler et al., 2005; Reinhardt et al., 2003). An analogous mechanism is present in mosses, where auxin is synthesized in the presumptive stem cell but promotes cell growth, differentiation and auxin responses in the descendants (Eklund et al., 2015; Landberg et al., 2021). A recent study has also shown that WUS binds to auxin signaling and response pathway genes to control the auxin signaling output in the CZ (Ma et al., 2019). Interestingly, Yadav et al. (2010) observed that the position of the primordium cannot be changed by manipulation of WUS in the PZ, demonstrating that the fate of the primordia is uncoupled from the CZ identity.

Auxin is mainly synthesized from L-tryptophan (L-Trp) in land plants via the IPyA pathway (Ljung, 2013). The enzymes encoded by TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and by its close homologue TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR) catalyze the conversion of L-Trp into indole-3-pyruvic acid (IPyA) (Stepanova et al., 2008; Tao et al., 2008); subsequently, the YUCCA (YUC) monooxygenases convert IPyA to indole-3-acetic acid (IAA) (Chourey et al., 2010; Eklund et al., 2015; Gallavotti et al., 2008; Landberg et al., 2021; Phillips et al., 2011; Zhao, 2010). In Arabidopsis, single mutants in TAA1/TAR and YUC genes do not show a phenotype when grown in standard conditions owing to their high genetic redundancy (Stepanova et al., 2008; Tao et al., 2008; Yamada et al., 2009; Zhou et al., 2011). However, when higher-order mutants are generated, they display a defect in embryonic and postembryonic development that is related to auxin signaling (Cheng et al., 2006, 2007; Stepanova et al., 2008). Similarly, mutation in vanishing tassel2 (vt2), an ortholog of TAA1, dramatically affects vegetative and reproductive development in maize (Phillips et al., 2011). Among the bryophytes, M. polymorpha has the smallest tool kit for synthesizing auxin via the IPyA pathway that consists of a TAA and two YUC genes (Eklund et al., 2015). TAA1 orthologs are required for apical growth, and their transcripts are distinctly expressed in the apical region of the developing meristem or similar cell types in land plants (Eklund et al., 2015; Landberg et al., 2021; Phillips et al., 2011; Stepanova et al., 2008). Except for TAA1, it is not clear from previous studies where and when the remaining TAR genes are expressed in the Arabidopsis SAM, and whether their activity contributes to the maintenance of local auxin maxima in the SAM has yet not been explored.

A recent study implicated the role of locally produced auxin in root meristem patterning (Brumos et al., 2018). Expression studies revealed that YUC1, YUC2, YUC4 and YUC6 are active in the SAM (Cheng et al., 2006). In contrast, expression of only TAA1 is reported in a few cells in the epidermal cell layer of the shoot apex (Stepanova et al., 2008). Interestingly, Yadav et al. (2013) found that TRYPTOPHAN AMINOTRANSFERASE RELATED 2 (TAR2) is responsive to WUS doses in the shoot stem cell niche. These studies raise important questions concerning the role of local auxin biosynthesis in shoot cell types. Does IPyA synthesis occur across the SAM uniformly? Are TAA1 and TAR genes regulated spatiotemporally to control auxin responses within the SAM?

Here, we report that TAA1 and TAR2 are expressed in distinct domains in the SAM. By genetic analysis, we have uncovered a synergistic role of TAA1 and TAR2 in maintaining optimum auxin responses and PZ cell identity in the SAM. The function of TAA1 and TAR2 is masked by auxin transport. Furthermore, when the auxin transport mutant was combined with the taa1 or tar2 mutant, it revealed that TAA1 is required for stem growth, whereas TAR2 is required for floral primordia formation. In the absence of auxin transport and biosynthesis, genetic pathways involved in meristem patterning, stem cell maintenance, organ initiation and their patterning become perturbed, and this results in a complete arrest in SAM growth and development in Arabidopsis.

Auxin produced by the IPyA pathway affects the relative size of the CZ and PZ in the SAM

Previous studies have shown that the auxin produced via the IPyA pathway is required for embryo and root meristem patterning (Brumos et al., 2018; Stepanova et al., 2008). In addition, IPyA pathway genes influence the inflorescence meristem and flower development in Arabidopsis (Brumos et al., 2018; Stepanova et al., 2008). However, it is unclear how auxin synthesized by the IPyA pathway contributes to SAM maintenance in Arabidopsis. To better understand the role of TAA1 and TAR2 in shoot and flower development, and how it affects SAM growth and development, we identified the T-DNA insertion in TAA1 and TAR2 from segregating taa1−/− tar2+/− plants (Stepanova et al., 2008). To uncover their role, taa1-1 and tar2-1 single-mutant, and taa1-1 tar2-1 (hereafter they will be referred to as taa1, tar2 and taa1 tar2) double-mutant plants were grown with wild type (WT; Col-0) as a control for 4 weeks. After removing the older floral buds, SAMs were examined under the confocal microscope to study the shoot patterning and phyllotactic defects. Confocal imaging revealed no discernible impact of taa1 and tar2 on the patterning of the shoot apex (Fig. 1A-C; Fig. S1E-G). However, when we examined SAM size, we found an increase in the tar2 mutant compared with the control (Fig. 1C,O; Table S1). taa1 and tar2 single-mutant plants displayed normal inflorescence meristem (IM) architecture, and plant height was indistinguishable from WT (Fig. S1A-C and I-K), as revealed in an earlier study (Stepanova et al., 2008). In taa1 tar2 double-mutant plants, we noticed that their stem failed to elongate along the primary growth axis, and plants appeared bushier and more compact (Fig. S1L). We also discovered that the size of the shoot in the taa1 tar2 double mutant was significantly smaller than the respective single mutant and control plants (Fig. 1D,O; Table S1). The SAM appeared oval or elliptical from the top when compared with WT and respective single mutants (Fig. 1D; Fig. S1H). The taa1 tar2 double-mutant plants grew extremely slowly compared with respective single mutants and control, as reported by Stepanova et al. (2008), when grown under identical growth conditions (see Fig. 5G and Fig. S1L). When we carefully examined the SAM under a confocal microscope, we observed that, in taa1 tar2 double mutant, the SAMs occasionally looked like a pin in shape, and floral organ primordia either showed a radial patterning or failed to elongate along the proximal-distal axis (Fig. S2A,B). The flower primordium displayed a long pedicel with a semi-oval-shaped floral meristem (Fig. S2C,D), as shown by Stepanova et al. (2008). Previous studies have shown that plants deficient in auxin biosynthesis, signaling and transport do display a range of patterning defects in different stages of organ development, like the taa1 tar2 double-mutant plants (Brumos et al., 2018; Gälweiler et al., 1998; Möller and Weijers, 2009; Stepanova et al., 2008; Weijers et al., 2006; Wenzel et al., 2007). These results show that taa1-and tar2-mediated auxin biosynthesis is required to maintain the size of the shoot, floral organ growth and correct floral meristem patterning.

Fig. 1.

TAA1 and TAR2 are required for meristem development. (A-D) Three dimensional (3D) reconstructed top view of the L1 layer of SAM from Col-0 (A), taa1-1 (B), tar2-1 (C) and taa1-1 tar2-1 double mutant (D) inflorescence meristems. (E-L) pCLV3::mGFP-ER reporter in Col-0 (E,I), taa1-1 (F,J), tar2-1 (G,K) and taa1-1 tar2-1 (H,L). (E-H) 3D top view of the SAM; (I-L) side view of the same images. Propidium iodide (PI, in red) was used to visualize the cell outline. (M) Quantification of pCLV3::mGFP-ER (CLV3-expressing cells, i.e. CZ cells) and non-expressing cells (PZ cells) in Col-0 (n=9), taa1-1 (n=9), tar2-1 (n=9) and taa1-1 tar2-1 (n=9). One-way ANOVA followed by Tukey's multiple comparisons tests; different letters represent statistically significant differences in cell number (M), in the ratio of CLV3-expressing and non-expressing cells (N), and in SAM size (O). The raw data relate to quantifying cells expressing the CLV3 reporter in epidermal cells (CZ); cells not expressing the reporter were considered PZ and are given in Table S1. SAM size measurements are given in Table S1. Scale bars: 20 µm.

Fig. 1.

TAA1 and TAR2 are required for meristem development. (A-D) Three dimensional (3D) reconstructed top view of the L1 layer of SAM from Col-0 (A), taa1-1 (B), tar2-1 (C) and taa1-1 tar2-1 double mutant (D) inflorescence meristems. (E-L) pCLV3::mGFP-ER reporter in Col-0 (E,I), taa1-1 (F,J), tar2-1 (G,K) and taa1-1 tar2-1 (H,L). (E-H) 3D top view of the SAM; (I-L) side view of the same images. Propidium iodide (PI, in red) was used to visualize the cell outline. (M) Quantification of pCLV3::mGFP-ER (CLV3-expressing cells, i.e. CZ cells) and non-expressing cells (PZ cells) in Col-0 (n=9), taa1-1 (n=9), tar2-1 (n=9) and taa1-1 tar2-1 (n=9). One-way ANOVA followed by Tukey's multiple comparisons tests; different letters represent statistically significant differences in cell number (M), in the ratio of CLV3-expressing and non-expressing cells (N), and in SAM size (O). The raw data relate to quantifying cells expressing the CLV3 reporter in epidermal cells (CZ); cells not expressing the reporter were considered PZ and are given in Table S1. SAM size measurements are given in Table S1. Scale bars: 20 µm.

After noticing that the taa1 tar2 double-mutant plants display slower growth, and their shoot meristems are smaller in size compared with the control, we asked whether it was linked to the reduced number of stem cells. A reduction in the number of stem cells in the CZ would lead to a decrease in the stem cell progenitors and an overall decrease in the rate of cell division and differentiation within the PZ, which might eventually affect the size of the SAM in the double-mutant plants. To test this hypothesis, we made crosses between taa1-1−/− tar2-1−/+ and the pCLV3::mGFP-ER line, and isolated taa1, tar2 and taa1 tar2 double-mutant plants carrying the pCLV3::mGFP-ER reporter (Fig. 1E-L). A closer examination of pCLV3::mGFP-ER activity in taa1, tar2, taa1 tar2 and WT revealed that the number of CLV3-expressing cells in taa1 tar2 double-mutant SAM was reduced (∼43; n=9) compared with WT (∼54; n=9) (Fig. 1M; Table S1), suggesting that auxin produced by the IPyA pathway affects the CZ size. To evaluate the relative size of the PZ, we counted epidermal cells outside the CLV3 expression domain in the taa1 tar2 double mutant, single mutant and control plant SAMs (Table S1). This analysis revealed that taa1 tar2 double-mutant plants have a smaller PZ than respective single mutants and control (Fig. 1M,N; Table S1). Our analysis shows that auxin produced via the IPyA pathway affects the relative number of cells both in CZ and PZ in the SAM. Thus, in the taa1 tar2 double mutant, the overall size of the shoot is reduced.

Local auxin biosynthesis in meristem provides robustness to auxin signaling

The polar transport of auxin is involved in positioning and patterning the emerging organ primordia within the SAM. To determine whether plants defective in synthesizing the IPyA can maintain the normal auxin transport and responses in the SAM, we crossed the pDR5rev::3xVenus-N7; pPIN1::PIN1-GFP line with taa1-1−/−tar2-1+/− plants. We examined the meristems after breeding the reporter to homozygosity in the taa1, tar2 and taa1 tar2 double mutant background. The DR5 promoter anchored to a reporter gene is commonly used to study auxin responses at cellular and tissue level in Arabidopsis. In taa1 and tar2 single mutants, we did not see a change in DR5 activity compared with wild type (Fig. 2A-C,E-G,Y; Table S2). In contrast, in taa1 tar2 double mutant SAM, DR5 expression was reduced in the PZ of the SAM (Fig. 2D,H,Y; Table S2). pPIN1::PIN1-GFP expression is detected throughout the meristem in the epidermal cell layer in control plants (Fig. 2I,M). In the incipient primordium (I1), high PIN1-GFP expression is detected. To investigate the role of TAA1 and TAR2 on PIN1 expression, we imaged taa1 and tar2 single mutants, and WT plant SAMs carrying pPIN1::PIN1-GFP (Fig. 2I-K,M-O). Compared with the control, we did not see a change in the PIN1-GFP expression pattern in the taa1 and tar2 single-mutant SAMs (Fig. 2I-K). However, in taa1 tar2 double mutant plants, we observed an overall decrease in the PIN1-GFP expression in the SAM. The incipient primordia showing a typical intense build-up of PIN1-GFP expression in the meristem appeared wider compared with the control (Fig. 2L,P; Fig. S3). Together with the observations of DR5 and PIN1-GFP data, our findings reveal a combined role of TAA1 and TAR2 in maintaining optimum auxin responses in the SAM. In taa1 tar2 double-mutant SAM, a decrease in the PIN1-GFP expression level was also spotted at the site of the incipient primordia. This result implies that the auxin produced by the IPyA pathway controls the PIN1 expression and thereby regulates the downstream signaling network genes.

Fig. 2.

Locally produced auxin contributes to auxin maxima formation in SAM. (A-D) Maximum projection images of Col-0, taa1-1, tar2-1 and taa1-1 tar2-1 SAM expressing pDR5rev::3xVenus-N7. (E-H) Side views of A-D, respectively. (D) The taa1-1 tar2-1 double mutant SAM lacks DR5 activity. (I-L,M-P) pPIN1::PIN1-GFP expression and PIN1-GFP localization, as visualized using the shoot apices of Col-0, taa1-1, tar2-1 and taa1-1 tar2-1 in top (I-L) and side (M-P) views. (L) Despite the auxin biosynthesis defect, taa1-1 tar2-1 double mutant plants show PIN1-GFP polarization and organogenesis. (Q-X) R2D2 in Col-0, taa1-1, tar2-1 and taa1-1 tar2-1; (Q-T) maximum projection of Col-0, taa1-1, tar2-1 and taa1-1 tar2-1; the side views are displayed in U-X. Ratiometric analysis for Q-T is shown in Fig. S4. In taa1-1 tar2-1 double mutant SAM, DII-Venus appears stable despite attenuated auxin signaling in the CZ. (Y) Quantification of auxin maxima was carried out in Col-0 (n=10), taa1-1 (n=10), tar2-1 (n=10) and taa1-1 tar2-1 (n=10) SAMs. For counting DR5-positive cells, a maximum projection in the form of a 3D image of the SAM was used. One-way ANOVA followed by Tukey's multiple comparisons tests; different letters represent the statistically significant differences in auxin maxima. Quantification of data is given in Table S2. Scale bars: 20 µm.

Fig. 2.

Locally produced auxin contributes to auxin maxima formation in SAM. (A-D) Maximum projection images of Col-0, taa1-1, tar2-1 and taa1-1 tar2-1 SAM expressing pDR5rev::3xVenus-N7. (E-H) Side views of A-D, respectively. (D) The taa1-1 tar2-1 double mutant SAM lacks DR5 activity. (I-L,M-P) pPIN1::PIN1-GFP expression and PIN1-GFP localization, as visualized using the shoot apices of Col-0, taa1-1, tar2-1 and taa1-1 tar2-1 in top (I-L) and side (M-P) views. (L) Despite the auxin biosynthesis defect, taa1-1 tar2-1 double mutant plants show PIN1-GFP polarization and organogenesis. (Q-X) R2D2 in Col-0, taa1-1, tar2-1 and taa1-1 tar2-1; (Q-T) maximum projection of Col-0, taa1-1, tar2-1 and taa1-1 tar2-1; the side views are displayed in U-X. Ratiometric analysis for Q-T is shown in Fig. S4. In taa1-1 tar2-1 double mutant SAM, DII-Venus appears stable despite attenuated auxin signaling in the CZ. (Y) Quantification of auxin maxima was carried out in Col-0 (n=10), taa1-1 (n=10), tar2-1 (n=10) and taa1-1 tar2-1 (n=10) SAMs. For counting DR5-positive cells, a maximum projection in the form of a 3D image of the SAM was used. One-way ANOVA followed by Tukey's multiple comparisons tests; different letters represent the statistically significant differences in auxin maxima. Quantification of data is given in Table S2. Scale bars: 20 µm.

To map the auxin input within the SAM, we crossed the line carrying the R2D2 reporter with taa1-2−/−tar2-1+/−. In R2D2, the degradable version of the domain II of Aux/IAA is fused to n3×Venus, whereas the non-degradable version is combined with ntdTomato. The DII and mDII are expressed under the RPS5A promoter as a single transgene (Liao et al., 2015). The stability of DII-n3×Venus depends upon the availability of auxin input (Brunoud et al., 2012). Thus, the ratio of the two fluorochromes can be used to infer the relative auxin distribution across the different domains of the SAM. By following parents, taa1, tar2 and taa1 tar2 plants carrying the R2D2 reporter gene were identified. The expression pattern of R2D2 was comparable with WT in the taa1 and tar2 single mutants (Fig. 2Q-S,U-W; Fig. S4A-C). Strikingly, in the taa1 tar2 double mutant plant SAMs, the DII-n3×Venus protein was stable in the CZ (Fig. 2T,X; Fig. S4D). This finding suggests that TAA1-and TAR2-mediated auxin biosynthesis contributes to auxin signaling in both the CZ and PZ of the SAM. The ratiometric analysis of R2D2 in taa1 tar2 double-mutant SAM revealed a low stability of DII in PZ compared with CZ. This result suggests that the relative accumulation of auxin is perturbed in the double mutant across the SAM. The differences observed in the DII stability in the CZ and PZ cells in the double mutant plant SAMs suggest that TAA1 and TAR2, in concert with auxin transport, play a crucial role in maintaining optimum auxin signaling in the meristem summit.

TAA1 and TAR2 expression patterns complement each other

Our analysis of auxin output by DR5 and input using the DII degron system revealed that TAA1 and TAR2 are necessary to maintain the optimum threshold of auxin signaling in CZ and PZ of the SAM. Surprisingly, taa1 and tar2 single mutant plants do not show any phenotype in Arabidopsis. This could be due to their overlapping spatiotemporal expression pattern, and, thus, it is possible that they could complement each other. To understand the role of TAA1 and TAR2 in shoot and flower development, we examined the expression pattern of TAA1 and TAR2 by in situ hybridization in Arabidopsis SAM. The TAA1 transcript was detected in the epidermal cell layer of CZ cells in 4-week-old WT-Ler SAM (Fig. 3A). In the sense probe, we did not see a signal (Fig. 3B). The expression pattern of TAA1 was further investigated in the SAM using a translational fusion construct, where the YPet sequence was inserted upstream of the TAA1-coding sequence (pTAA1::YPet-TAA1) to make a N-terminal fusion (Brumos et al., 2018). Compared with the mRNA expression pattern reported by in situ hybridization, we found the pTAA1::YPet-TAA1 expression was slightly wide in the SAM but spatially restricted to the epidermal cell layer (Fig. 3C).

Fig. 3.

TAA1 and TAR2 transcripts complement each other in their expression pattern within the SAM. (A) TAA1 expression is detected in the epidermal cell layer of SAM. (B) A TAA1 sense probe was applied to the shoot apices. (C) Reconstructed surface projection of the L1-layer of SAM expressing pTAA1::YPet-TAA1 along with a side view. (D,E) TAR2 expression was detected in the PZ of the SAM and in emerging floral primordia (D); signal was not detected with the sense probe (E). (F) 3D reconstructed top of the epidermis and the side view of the SAM expressing pTAR2::H2B-YFP. Scale bars: 10 µm in A,B,D,E; 20 µm in C,F.

Fig. 3.

TAA1 and TAR2 transcripts complement each other in their expression pattern within the SAM. (A) TAA1 expression is detected in the epidermal cell layer of SAM. (B) A TAA1 sense probe was applied to the shoot apices. (C) Reconstructed surface projection of the L1-layer of SAM expressing pTAA1::YPet-TAA1 along with a side view. (D,E) TAR2 expression was detected in the PZ of the SAM and in emerging floral primordia (D); signal was not detected with the sense probe (E). (F) 3D reconstructed top of the epidermis and the side view of the SAM expressing pTAR2::H2B-YFP. Scale bars: 10 µm in A,B,D,E; 20 µm in C,F.

Similarly, we studied the mRNA expression pattern of TAR2 by in situ hybridization in the 4-week-old shoot apices of WT-Ler. In situ studies revealed expression of TAR2 in the PZ of SAM, where the primordium emerges within the shoot (Fig. 3D). Sense probe did not yield a signal (Fig. 3E). However, TAR2 transcript was conspicuously absent in CZ cells (Fig. 3D). To further investigate the expression of TAR2, we made a transcriptional fusion construct by amplifying the 3 kb promoter region of TAR2 above the translational start site (TSS). We placed the 3 kb promoter fragment upstream to H2B-YFP translational fusion in a modified pGreen vector. To examine the TAR2 expression in the shoot apex, several independent T1 lines were selected (n=18). Most of the lines showed activity of a TAR2 reporter-like native mRNA expression pattern, as reported by in situ studies (Fig. 3F).

To understand when and where TAA1- and TAR2-mediated auxin biosynthesis played a role in plant development, we checked the expression of TAA1 and TAR2 in embryos and seedlings using the pTAA1::YPet -TAA1 and pTAR2::H2B-YFP lines, respectively, as shown earlier by Stepanova et al. (2008). Both pTAA1::Ypet-TAA1 and pTAR2::H2B-YFP did not show expression in the globular stage (16- to 32-cell stage) (Fig. S5A,J-K). However, at the 64-cell stage, Ypet-TAA1 fluorescence was visible in the epidermal cell layer of the apical domain of the pro-embryo (Fig. S5B). Ypet-TAA1 is retained in the epidermal cell layer in the presumptive shoot apex and root stem cell niche in the heart stage (Fig. S5C) (Stepanova et al., 2008). In postembryonic development, TAA1 expression was confined to the epidermal cell layer in the SAM in 3-days after sowing (DAS) seedlings and 4-week-old SAM, respectively (Fig. S5D-F). In stage 3 and stage 4 flowers, TAA1 expression was observed in the center of the floral meristem (Fig. S5G-I) (Stepanova et al., 2008). In contrast, TAR2 promoter activity was noticed first in the heart stage in the presumptive hypocotyl region of the pro-embryo (Fig. S5L). In postembryonic development, TAR2 expression was present in the periphery of the SAM in 3-DAS seedlings (Fig. S5M,N). In flowers and IM, TAR2 expression is extended beyond the L1 cell layer in the PZ (Fig. S5O-R). These results prove that TAA1 and TAR2 show complementary expression patterns in embryonic and postembryonic development, and are regulated differentially in distinct stages of development.

Plants lacking auxin biosynthesis in the incipient organ primordia show defects in organ initiation

The TAA1 and TAR2 in situ hybridization studies revealed that their transcripts are restricted in the CZ and PZ, respectively, in Arabidopsis SAM. However, plants lacking either TAA1 or TAR2 do not show a phenotype. This suggests that a transport protein could mobilize IPyA and IAA produced in single mutants. To dissect how auxin transport mediated by PIN1 masks the function of genes involved in local auxin production in the SAM, we crossed the taa1-1−/−tar2-1+/− plants with the pin1-5 allele. As strong alleles of a pin1 mutant, such as pin1-4 and pin1-6, do not form floral primordia in the reproductive phase, despite having functional CZ, PZ and RM (Gälweiler et al., 1998; Reinhardt et al., 2003; Vernoux et al., 2000), we decided to use a pin1-5 allele – a mild allele of PIN1 in which mutant plants bear floral organs and set seeds (Sohlberg et al., 2006). Compared with the control, pin1-5 mutant plants displayed a defect in plant architecture and floral meristem patterning (Fig. 4A,B,E,F,I,J; Fig. S6A,B). To ascertain the relative contribution of TAA1 and TAR2 based on their spatiotemporal expression pattern in auxin signaling and organogenesis, we isolated pin1 taa1 and pin1 tar2 double mutant plants, respectively (Fig. 4C,D; Fig. S6C,D). In the double mutant plants, the presence of the pin1-5 allele was confirmed based on the flower organ number and morphology. To resolve the taa1-1 and tar2-1 genotype in the pin1 taa1 and pin1 tar2 double mutants, T-DNA PCR was set up for both taa1 and tar2 alleles. Unlike strong alleles, the pin1-5 allele also initiates and maintains axillary IMs (AMs), which bear the flowers (Fig. 4B,M,N; Table S3). We observed a significant decrease in the number of AMs in the pin1 tar2 double mutant compared with taa1, tar2 and pin1 single-mutant, and pin1 taa1 double-mutant plants (Fig. 4M). In pin1 taa1 and pin1 tar2 double-mutant plants, SAMs terminate into pins after producing fewer flowers than control and respective single mutant plants (Fig. 4G,H,K,L,N,O; Tables S4 and S5). The pin1-5 single mutant plants produce at least five flowers (n=18), and the flower primordia abut in the PZ-like WT (Fig. 4F,J; Table S4). The overall number of flowers and siliques is reduced in the pin1 tar2 double mutant compared with the pin1 taa1 double mutant, suggesting that auxin produced by the TAR2-mediated IPyA pathway in the PZ has more influence on the floral primordia emergence (Fig. 4D,N,O; Tables S4 and S5). In the pin1 taa1 double mutant, we found that AMs bore flowers in 35-day-old plants. In contrast, in the pin1 tar2 double mutant, AMs terminate into pin-shaped shoot apices, indicating that the floral primordia are not produced in the pin1 tar2 double mutant (Fig. 4C,D). This genetic evidence makes TAR2 more critical than TAA1 in floral primordia formation in the reproductive phase of plant development, which correlates well with its expression in the PZ of the SAM. In contrast, in pin1 taa1 double mutant plants, primary IM grew slower than in control and in the pin1 tar2 double mutant, but AMs initiated from the primary IM arrested plants (Fig. 4C; Fig. S6A-D). As the TAA1 transcript is present in the epidermal cell layer of SAM, its expression correlates with its role in promoting apical dominance and stem tissue growth (Fig. 4C,P; Fig. S6A-D and Table S6).

Fig. 4.

Expression of TAR2 in the periphery of SAM is crucial for floral primordia formation. (A-D) 35-day-old wild-type Ler (A), pin1-5 (B), pin1-5 taa1-1 (C) and pin1-5 tar2-1 (D). (E-I) 3D reconstructed top view of wild-type Ler, pin1-5, pin1-5 taa1 and pin1-5 tar2-1 SAM (E-H); side views are given in I-L. (C,D) White arrows indicate the pin-like inflorescence in pin1-5 taa1-1 and pin1-5 tar2-1 plants. (F) pin1-5 makes floral primordia in the same way as wild type (white asterisks). (M-P) Graphs showing the quantification of the axillary meristem (M), siliques numbers (O), flower numbers (N) and stem length (P). Wild-type Ler (n=18), Col-0 (n=20), taa1-1 (n=19), tar2-1 (n=20), pin1-5 (n=18), pin1-5 taa1-1 (n=18) and pin1-5 tar2-1 (n=18). For stem length, the height of the plant on the 35th day was measured. pin1, pin1 taa1 and pin1 tar2 mutant plants maintained in Ler were used for counting flowers, siliques and axillary meristem number. One-way ANOVA followed by Tukey's multiple comparisons tests, different letters represent the statistically significant differences in the axillary meristem (M), flowers (N), siliques (O) and stem length (P). Quantification data for axillary meristem, flower numbers, silique numbers and inflorescence meristem height are given in Tables S3, S4, S5 and S6. Scale bars: 3 cm in A-D; 20 µm in E-H.

Fig. 4.

Expression of TAR2 in the periphery of SAM is crucial for floral primordia formation. (A-D) 35-day-old wild-type Ler (A), pin1-5 (B), pin1-5 taa1-1 (C) and pin1-5 tar2-1 (D). (E-I) 3D reconstructed top view of wild-type Ler, pin1-5, pin1-5 taa1 and pin1-5 tar2-1 SAM (E-H); side views are given in I-L. (C,D) White arrows indicate the pin-like inflorescence in pin1-5 taa1-1 and pin1-5 tar2-1 plants. (F) pin1-5 makes floral primordia in the same way as wild type (white asterisks). (M-P) Graphs showing the quantification of the axillary meristem (M), siliques numbers (O), flower numbers (N) and stem length (P). Wild-type Ler (n=18), Col-0 (n=20), taa1-1 (n=19), tar2-1 (n=20), pin1-5 (n=18), pin1-5 taa1-1 (n=18) and pin1-5 tar2-1 (n=18). For stem length, the height of the plant on the 35th day was measured. pin1, pin1 taa1 and pin1 tar2 mutant plants maintained in Ler were used for counting flowers, siliques and axillary meristem number. One-way ANOVA followed by Tukey's multiple comparisons tests, different letters represent the statistically significant differences in the axillary meristem (M), flowers (N), siliques (O) and stem length (P). Quantification data for axillary meristem, flower numbers, silique numbers and inflorescence meristem height are given in Tables S3, S4, S5 and S6. Scale bars: 3 cm in A-D; 20 µm in E-H.

To understand the requirement of IPyA in rescuing the shoot phenotype in the PZ of the SAM, we replaced H2B-YFP in the pTAR2::H2B-YFP reporter construct with the TAR2-coding sequence. The resulting pTAR2::TAR2 construct was transformed into the taa1−/− tar2+/ genetic background, and independent lines rescuing the taa1 tar2 double mutant phenotype were identified by T-DNA PCR analysis (n=6) (Fig. S7A-F). Despite the redundant role played by TAA1 and TAR2, their spatiotemporal expression pattern indicates their probable functions in stem tissue growth and lateral organ formation, respectively. Auxin transport mediated by PIN1 masks the effects of local auxin biosynthesis in taa1 and tar2 mutant backgrounds, suggesting that auxin transport and biosynthesis act in concert to promote SAM growth and development.

Abrogation in auxin biosynthesis via the IPyA pathway and transport results in the arrest of SAM development

To unravel the role of auxin transport and biosynthesis on SAM growth in vegetative development, we carried out an analysis of overall plant morphology in 15-day-old WT, taa1, tar2, pin1, pin1 taa1, pin1 tar2 and pin1 taa1 tar2 mutant plants. Leaf shape and number in taa1 and tar2 single mutant plants closely resembled the WT control (Fig. 5A-C). However, pin1, pin1 taa1 and pin1 tar2 mutant plants produced fewer leaves and perturbed plant architecture (Fig. 5D-F). The taa1 tar2 double mutant plants displayed stunted growth and small rosettes (Fig. 5G). To understand the effect of auxin transport and biosynthesis on SAM growth and architecture, we identified pin1-5 taa1 tar2 triple mutant plants. The pin1 taa1 tar2 triple mutant seedlings either did not form a rosette or produced only one rosette in the vegetative phase (Fig. 5H). Most triple mutant seedlings isolated from these crosses displayed unequally sized cotyledons and were rootless. Furthermore, when kept in soil for 2-3 weeks in the plant growth chamber, they formed a mound-shaped structure between the cotyledons (Fig. 5H, inset). We could not determine whether the heap of cells accumulated between the cotyledons was a mass of undifferentiated SAM tissue or regenerated shoot tissue (Fig. 5H, inset).

Fig. 5.

Plants lacking auxin biosynthesis and transport display defects in meristem patterning and growth. (A-H) 15-day-old Col-0 (A), taa1-1 (B), tar 2-1 (C), pin1-5 (D), pin1-5 taa1-1 (E), pin1-5 tar2-1 (F), taa1-1 tar2-1 (G) and pin1-5 taa1-1 tar2-1 (H). Shoots emerge as an undifferentiated mass of cells without lateral organs and stem tissue in pin1 taa1 tar2 triple mutant (H and inset).

Fig. 5.

Plants lacking auxin biosynthesis and transport display defects in meristem patterning and growth. (A-H) 15-day-old Col-0 (A), taa1-1 (B), tar 2-1 (C), pin1-5 (D), pin1-5 taa1-1 (E), pin1-5 tar2-1 (F), taa1-1 tar2-1 (G) and pin1-5 taa1-1 tar2-1 (H). Shoots emerge as an undifferentiated mass of cells without lateral organs and stem tissue in pin1 taa1 tar2 triple mutant (H and inset).

Auxin signaling controls the transition of stem cell progenitors into differentiating cells in the PZ

To investigate the role of auxin biosynthesis and transport in cell identity acquisition, we studied the expression pattern of marker genes linked to CZ, PZ and RM identity. CLV3 and WUS genes are usually expressed in CZ and RM cells in WT SAM, respectively (Fig. 6A,B) (Fletcher et al., 1999; Mayer et al., 1998). In pin1 taa1 tar2 triple mutant plants, expression of CLV3 mRNA was detected in the tip of the mound (Fig. 6T). A closer examination of the in situ hybridization images revealed a noticeable expansion of the CLV3 expression pattern in the epidermal and subepidermal cell layers towards the PZ in triple mutant compared with the pin1 single mutant and the taa1 tar2 double mutant (Fig. 6H,N). Interestingly, the WUS expression level is reduced dramatically in the pin1 taa1 tar2 triple mutant compared with WT, the pin1 single mutant and the taa1 tar2 double mutant SAMs, possibly owing to increased expression of CLV3 (Fig. 6A,G,M,S). To investigate whether these plants lack functional PZ or fail to form the PZ cell types without auxin signaling, we examined marker gene expression, which marks organ boundary regions and organ primordia in a functional SAM. The overall expression pattern of CUC1, ARF3, ARF4 and ARF5 is similar to that of WT in pin1 single mutant and taa1 tar2 double mutant SAMs (Fig. 6C-F,I-L,O-R) (Aida et al., 1999; Sessions et al., 1997). However, the expression level is weakened significantly in pin 1 single mutant and taa1 tar2 double mutant SAMs (Fig. 6I-L,O-R), resulting in irregular organ boundaries and defects in plant architecture. In the pin1 taa1 tar2 triple mutant, both the expression patterns and levels of CUC1, ARF3, ARF4 and ARF5 were perturbed (Fig. 6U-X), suggesting that auxin signaling has been impaired in these plants. The typical pattern of WUS expression is not maintained in the pin1 taa1 tar2 triple mutant SAM (Fig. 6A,S), indicating that auxin signaling plays a crucial role in maintaining fully functional niche in the apical meristem.

Fig. 6.

CZ, PZ and RM marker genes and their expression pattern in auxin biosynthesis and transport mutant background. (A-F) WUS, CLV3, CUC1, ARF3, ARF4 and ARF5 mRNA transcripts were detected in the inflorescence meristems of wild-type Ler using in situ hybridization. (G,I-L) pin1-5 shoot apices probed with WUS (G), CUC1 (I), ARF3 (J), ARF4 (K) and ARF5 (L). (H) A side view of a pin1-5 shoot apex showing the pCLV3::mGFP-ER reporter expression. (M-R) taa1-1 tar2-1 double mutant SAM probed for WUS, CLV3, CUC1, ARF3, ARF4 and ARF5. (S-X) Similarly, pin1-5 taa1-1 tar2-1 triple mutant SAMs were probed for WUS, CLV3, CUC1, ARF3, ARF4 and ARF5. (T) There was a strong reduction in the transcript levels of PZ and RM marker genes in pin1-5 taa1-1 tar2-1 triple mutant SAM, whereas the expression of the CZ marker CLV3 expanded in the L1 layer. Scale bars: 20 µm.

Fig. 6.

CZ, PZ and RM marker genes and their expression pattern in auxin biosynthesis and transport mutant background. (A-F) WUS, CLV3, CUC1, ARF3, ARF4 and ARF5 mRNA transcripts were detected in the inflorescence meristems of wild-type Ler using in situ hybridization. (G,I-L) pin1-5 shoot apices probed with WUS (G), CUC1 (I), ARF3 (J), ARF4 (K) and ARF5 (L). (H) A side view of a pin1-5 shoot apex showing the pCLV3::mGFP-ER reporter expression. (M-R) taa1-1 tar2-1 double mutant SAM probed for WUS, CLV3, CUC1, ARF3, ARF4 and ARF5. (S-X) Similarly, pin1-5 taa1-1 tar2-1 triple mutant SAMs were probed for WUS, CLV3, CUC1, ARF3, ARF4 and ARF5. (T) There was a strong reduction in the transcript levels of PZ and RM marker genes in pin1-5 taa1-1 tar2-1 triple mutant SAM, whereas the expression of the CZ marker CLV3 expanded in the L1 layer. Scale bars: 20 µm.

The IPyA pathway of auxin biosynthesis controls auxin responses and organogenesis

How are the auxin responses controlled by the combined action of auxin transport and biosynthesis within the meristem? This question is still unanswered. To dissect it further, we isolated pin1 taa1 and pin1 tar2 double mutant plants carrying the DR5 reporter in the respective double-mutant background. DR5 expression was examined in 30-day-old shoot apices of WT, pin1 single mutant, and pin1 taa1 and pin1 tar2 double mutant plants. In taa1 and tar2 single mutant plants, auxin responses are maintained as auxin maxima in the PZ of SAM, as in WT (Fig. 7A-C). However, when the transport of auxin is perturbed by biosynthesis (e.g. in pin1 taa1 and pin1 tar2), DR5 expression in the PZ of the SAM is completely abolished. (Fig. 7E-G; Table S8), suggesting that local auxin biosynthesis acts synergistically with auxin transport to maintain the optimum auxin responses in the PZ.

Fig. 7.

Auxin transport and local biosynthesis control auxin responses in the SAM. (A-F) Thirty-day-old wild-type Ler (A), taa1-1 (B), tar2-1 (C), pin1-5 (D), pin1-5 taa1-1 (E) and pin1-5 tar2-1 (F) inflorescence meristems expressing pDR5rev::3xVenus-N7. (G) DR5-positive cells were counted in pin1-5, pin1-5 taa1-1 and pin1-5 tar2-1 (Table S8). (H) Relative enrichment of the transcript levels for the various IPyA pathways genes was determined from the pCLV3::mGFP-ER (CLV3), pFIL::Ds-Red (FIL), pHMG15::H2B-YFP (HMG15) and pWUS::eGFP-ER (WUS) cell population data (Table S7). The color scheme and numbers in the heatmap scale indicate the relative enrichment of TAR and YUC transcript levels on a scale of 0-30. (I) Schematic model illustrating the TAA1 and TAR2 expression together with auxin transport in the SAM. (J) Diagrammatic model showing the distinct role of the TAA1 and TAR2 on plant architecture when combined with a transport mutant. Scale bars: 50 µm.

Fig. 7.

Auxin transport and local biosynthesis control auxin responses in the SAM. (A-F) Thirty-day-old wild-type Ler (A), taa1-1 (B), tar2-1 (C), pin1-5 (D), pin1-5 taa1-1 (E) and pin1-5 tar2-1 (F) inflorescence meristems expressing pDR5rev::3xVenus-N7. (G) DR5-positive cells were counted in pin1-5, pin1-5 taa1-1 and pin1-5 tar2-1 (Table S8). (H) Relative enrichment of the transcript levels for the various IPyA pathways genes was determined from the pCLV3::mGFP-ER (CLV3), pFIL::Ds-Red (FIL), pHMG15::H2B-YFP (HMG15) and pWUS::eGFP-ER (WUS) cell population data (Table S7). The color scheme and numbers in the heatmap scale indicate the relative enrichment of TAR and YUC transcript levels on a scale of 0-30. (I) Schematic model illustrating the TAA1 and TAR2 expression together with auxin transport in the SAM. (J) Diagrammatic model showing the distinct role of the TAA1 and TAR2 on plant architecture when combined with a transport mutant. Scale bars: 50 µm.

Previous studies have shown that L-Trp is the primary precursor of indole 3 acetic acid in plants (Ljung, 2013). TAA1 and its close homologue TAR genes convert L-Trp into IPyA (Stepanova et al., 2008; Tao et al., 2008). It has been shown in earlier studies that the YUC class of genes shows overlapping functions (Cheng et al., 2006, 2007). However, it is not clear from previous studies how IPyA pathways genes contribute to auxin responses within the meristem of higher plants. We carried out RNAseq experiments to understand the role of IPyA pathway genes in auxin responses. As described earlier by Saini et al. (2020), epidermal, CZ, PZ and RM cells were labeled by expressing a reporter gene encoding fluorescent protein under the regulatory sequences of HMG15 (HMGBD15), CLV3, FILAMENTOUS FLOWER and WUS, respectively. Distinct cell populations marked with the fluorescent reporter protein were isolated using a fluorescent-activated cell sorter from the apetala1 cauliflower1 double-mutant SAMs (Yadav et al., 2014).

To understand the role of the TAR family genes, we determined the relative transcript levels of TAA1, TAR1, TAR2, TAR3 and TAR4 from the cell population data (Fig. 7G; Table S7). The TAA1 transcript is enriched in the epidermal cell layer (Fig. 3A,C), while TAR2 is expressed in organ primordia and RM cells (Fig. 3D,F). We analyzed the enrichment of TAR genes in RNAseq data, excluding TAA1 and TAR2; the remaining TAR genes are either not expressed at all or are expressed weakly in the apical meristem (Fig. 7G). Next, we checked the expression of YUC genes. YUC1 and YUC4 are expressed in the apical meristem. The YUC1 transcript shows high expression within the epidermal cell layer, whereas YUC4 is expressed in organ primordia and RM cells in the meristem (Fig. 7H). Based on the RNAseq data, we could see a strong correlation in the expression pattern of TAA1, TAR2, YUC1 and YUC4 genes in the epidermis, organ primordia and RM cells, indicating that IPyA pathway-mediated auxin biosynthesis would result in elevated auxin responses in these cell populations.

Despite being a simple molecule, auxin is fundamental for exquisitely regulating distinct aspects of plant development. In Arabidopsis shoot, the developing organ primordia cells accumulate considerably higher amounts of auxin than the neighboring cells that do not participate in primordium development. Considering the role of PIN1-mediated polar auxin transport, mathematical modelling studies explained this apparent disparity in auxin build-up and responses in the shoot apex (de Reuille et al., 2006; Smith et al., 2006). However, previous studies could not explain why pin1 mutant plants form lateral organs in the vegetative phase. Here, we show that disruption in the local auxin biosynthesis in the SAM, mediated by TAA1 and TAR2, leads to a decline in auxin signaling in SAM. This becomes weakened further when auxin transport is abolished in the biosynthesis mutants. The striking effect of this reduction in auxin signaling was on shoot patterning. In taa1 tar2 double mutant plants, the relative size of the CZ and PZ is reduced (Fig. 1D,H,M,N).

TAA1 and TAR2 are upstream of YUC genes in the IPyA pathways of auxin biosynthesis (Cheng et al., 2006; Ljung, 2013). IPyA is an essential auxin-generating substrate for YUC genes, which are also expressed in SAM. Despite their importance in achieving auxin maxima, their overlapping expression patterns and high genetic redundancy make it challenging to interpret the precise contribution of YUC genes in auxin biosynthesis in distinct cell types of the shoot apex. This study uncoupled specific roles of TAA1 and TAR2, especially in stem growth and floral primordia formation (Fig. 4C,D). Without local auxin biosynthesis in the shoot apex, auxin responses are abolished in the PZ. Considering the low expression of CUC1, ARF3, ARF4 and ARF5 in auxin transport and biosynthesis mutant SAMs (Fig. 6G-L,M-R), our findings implicate auxin signaling in maintaining their expression level. In pin1 taa1 tar2 triple mutant SAM, auxin signaling is severely compromised (Figs 5H and 6U-X), demonstrating that auxin plays a fundamental role in meristem growth and patterning. A crucial part of IPyA pathway-mediated auxin biosynthesis has been established in recent years in the promotion of apical fate promotion in land plants (Brumos et al., 2018; Eklund et al., 2015; Landberg et al., 2021; Phillips et al., 2011). Our findings confirm that the Trp-dependent IPyA pathway contributes to auxin production in the Arabidopsis shoot.

Genetic evidence presented here shows that organ boundary patterning is affected in the taa1 tar2 double mutant; this genetic defect becomes more pronounced in the pin1 taa1 tar2 triple mutant, suggesting that auxin optima are not only crucial for organ initiation but are also required for organ patterning. Furthermore, CUC1 expression is dependent upon the auxin signaling. Expression of CUC1 gradually decreases in biosynthesis mutants (Fig. 6O). In taa1 tar2 double-mutant plants, CUC1, ARF3 and ARF4 expression is reduced in the SAM (Fig. 6O-R), which is then further reduced in the pin1 taa1 tar2 triple mutant (Fig. 6V-X).

The meristem-like growth shown by early plants came from the activity of the apical cell, which is analogous to the CZ/stem cells of angiosperms (Harrison et al., 2007; Moody, 2019, 2020; Moody et al., 2018). In non-vascular plants, the functional organization of the SAM into CZ, PZ and RM is missing; however, the components that participate in regulatory networks to control the cell division activity and planer cell growth related to angiosperms are expressed (Furumizu et al., 2021; Takahashi et al., 2021; Whitewoods et al., 2018). The apical cell present within the notch region of the M. polymorpha thallus expresses the ortholog of TAA1, MpTAA, and supports the meristematic growth (Eklund et al., 2015). In P. patens PpTARA, which is equivalent to TAA1 with regards to its expression pattern in the apical cell/stem cell region, promotes filament growth in the descendant cells (Landberg et al., 2021). By combining auxin biosynthesis and transport mutants, we discovered that, in angiosperms, intercalary growth is promoted by TAA1, which is expressed in the epidermis of CZ (Figs 3A and 4C). TAR2 expression is restricted in the periphery in Arabidopsis SAM and promotes floral primordia formation (Figs 3D and 4D). At the phenotypic level, the functions of both TAA1 and TAR2 are correlated with their expression patterns (Fig. 7J). However, their synergistic role in promoting auxin responses in the PZ remains masked by PIN1. A mutation in the TAA1 orthologue gene (vt2) alone reduces apical dominance in maize. When this was combined with a transport mutant, the resulting double-mutant plants showed severe defects in the ear length and kernel number (Phillips et al., 2011). The expression pattern of TAA1 and its orthologs in the apical cell/CZ epidermis signifies a conserved role for IPyA in promoting apical growth in land plants.

Studies have shown that auxin can activate auxin-responsive genes by canonical binding to TIR/AFB co-receptor family proteins (Dharmasiri et al., 2005; Kepinski and Leyser, 2005). Of the six canonical auxin receptor proteins encoded by the Arabidopsis genome, five are expressed uniformly in SAM (Vernoux et al., 2011), suggesting an intricate role in achieving the auxin signaling responses in the PZ cells (Prigge et al., 2020). Previous studies have shown that the transcription of ARFs, which are involved in auxin responses (e.g. ARF5), is controlled by auxin (Bhatia et al., 2016; Dharmasiri et al., 2005; Kepinski and Leyser, 2005). It is not understood whether the expression of repressive ARFs can be regulated by auxin in the SAM, e.g. ARF3 and ARF4. Our in situ studies on taa1 tar2 and pin1 taa1 tar2 shoot apices support that both locally produced auxin and transport control the expression of activating and repressing ARFs in SAM (Simonini et al., 2016).

Plant material and growth conditions

Arabidopsis thaliana Columbia-0 (Col-0) and Landsberg erecta (Ler) ecotypes were used as wild-type strains and were obtained from the Arabidopsis Biological Resource Center (ABRC, Ohio University, USA). T-DNA insertion alleles for TAA1 and TAR2 used in this study were obtained from ABRC. The pin1-5 allele is in the Ler ecotype and obtained from ABRC. R2D2 is in Col-0 and described by Liao et al. (2015), the pTAA1::TAA1-YPet line is in Col-0 and was gifted by Anna Stepanova. pRPS5A-DII-n3xVenus has been described previously (Brunoud et al., 2012). The pCLV3::mGFP-ER line was available in house. pPIN1::PIN1-GFP and pDR5rev::3xVenus-N7 have been described previously (Heisler et al., 2005). Seeds were surface sterilized with 70% ethanol, followed by 4% (v/v) sodium hypochlorite (MERCK 1.93607.1021) containing 0.02% Triton X-100 for 3 min and rinsed three times with sterile distilled water. The seeds were sown on Murashige and Skoog (MS) medium (Sigma) containing 0.8% Bacto agar (Himedia, India), 1% (w/v) sucrose and 0.1% (w/v) MES. Stratified seeds were kept in darkness at 4°C for 3 days and then transferred to plant growth chambers (Conviron, Canada; Percival Scientific, USA). For transformation, wild-type Ler and Col-0 dry seeds were sown on soilrite mix (KELTECH Energies, India), kept for vernalization at 4°C for 3 days and then placed in the growth chambers (Conviron PGC Flex, Canada) under Philips fluorescent tube lights (120 µmol light and 22°C) on a long-day cycle (16 h light and 8 h dark). The soil was prepared by mixing soilrite mix (KELTECH Energies, India), compost and perlite in a ratio of 3:1:0.5.

Plasmid construct, generation of transgenic lines and RNA sequencing

To make pTAR2:H2B-YFP transcriptional fusion, the TAR2 promoter was PCR amplified using WT-Ler genomic DNA as a template and cloned into pENTER/D/TOPO. A gateway LR-reaction was set up with a modified pGreen0229 destination vector (Yadav et al., 2010). pTAR2::H2B-YFP construct was introduced in the WT-Ler to generate the reporter line. The TAR2-coding sequence was PCR amplified and cloned into pENTER/D/TOPO. We made a new vector for the rescue experiment because of the in planta BASTA resistance of taa1 T-DNA. To overcome this challenge, we assembled pTAR2::TAR2 cassettes in the pMDC32 vector. First, we replaced the 35S promoter with pTAR2 inserts in SbfI and AscI restriction sites in the pMDC32 vector. SbfI and AscI sites were introduced into pTAR2 by PCR (Table S9). Second, a pENTR clone with TAR2 CDS was used to set up a LR reaction with pTAR2 pMDC32. The pTAR2::TAR2 pMDC32 construct was transformed in taa1-1−/− tar2-1+/− and pCLV3::mGFP-ER reporter line using the floral dip method. Seeds were collected and transgenics were selected on hygromycin (50 μg/ml). Over 100 plants were selected on hygromycin and genotyped for taa1-1 and tar2-1 T-DNA insertion in the T1 generation. In total, six independent lines were identified for taa1 tar2 double mutants that looked like WT.

pCLV3::mGFP-ER, pFIL::Ds-Red, pHMG15::H2B-YFP and pWUS::eGFP-ER reporter lines were used previously by Yadav et al. (2014) for cell-sorting purposes. Promoter reporters are maintained in ap1-1 cal1-1 double mutant background for cell-sorting purposes from the shoot apices of Arabidopsis. Cell-sorting, RNA isolation, library preparations and RNAseq data analysis were performed as described previously by Saini et al. (2020). The raw data sequence files have been deposited in NCBI SRA under BioProject PRJNA529066.

In situ hybridization

Non-radioactive in situ hybridization was performed according to http://www.its.caltech.edu/~plantlab/html/protocols.html. From the Arabidopsis wild-type cDNA library, coding regions of CLV3, WUS, TAA1, TAR2, CUC1, ARF3, ARF4 and ARF5 were amplified and cloned into a pENTER/D/TOPO vector (Table S9). Clones were sequence verified. To synthesize the full-length antisense and sense probe, the template was PCR amplified with the help of respective primer pairs (Table S9). One of the strands of the PCR product carries a T7 promoter sequence, which was used for in vitro sense and antisense probe synthesis (Yadav et al., 2010).

Confocal laser scanning microscopy of SAM

Plants were grown in 16 h light/8 h dark conditions for confocal imaging. Once the bolting was induced, shoots were plucked with fine forceps and placed in boxes containing solidified agar (1.5%). The shoots were dissected gently by removing the older flower buds under a stereomicroscope. To visualize the cell outline, shoot apices were stained with 10 μg/ml propidium iodide (Invitrogen). The SAMs were scanned under the upright confocal microscope (Leica SP8, Germany). Images of 1024×1024 pixels were taken using a 63× long-distance water dipping lens with a step size of 1.5 µm. The emission and excitation spectra were collected according to the tagged fluorescence reporter. YFP, Venus and YPet were excited at 515 nm wavelength with argon laser lines at 9-10% laser power, and the emission spectra were collected between 524 and540 nm by adjusting the variable band pass filter. GFP was excited at 488 nm, and emission spectra were collected at 500-530 nm. PI was excited using a 561 nm laser line, and emission spectra were filtered with a 600-650 nm adjustable bandpass filter. Transgenic lines established based on the inflorescence meristem expression pattern were used for embryo and seedling imaging. Siliques of appropriate age with immature seeds were dissected on a glass slide in distilled water for embryo imaging. Ovules were stripped from the ovary wall and placed into FM4-64 (Invitrogen) on a microscope slide. Embryos were collected with the help of insulin syringes under a dissecting scope, and a coverslip was placed over the isolated embryos and sealed with nail polish. Imaging was carried out immediately under a 63× oil immersion objective in Leica SP8 upright confocal microscope as described above.

For seedling shoot apex imaging, seeds were surface sterilized with bleach and kept in the dark at 4°C for 3 days after putting them on MS media plates. Plates were held vertically in the growth chamber for 3 days at 22°C. We chose germinated seedlings before their cotyledons opened and transferred them to magenta boxes containing 1.5% solidified agar. Seedlings were pushed into the pre-created holes in the agar, leaving their top outside. The SAM was exposed by gently removing one cotyledon with the help of fine tweezers (5TI, Dumont, Switzerland) and oriented vertically to visualize under the upright confocal nose piece. Propidium iodide (10 µg/ml, Invitrogen) drops were put on the top of the dissected seedlings and kept for 30-40 s to visualize the cell outline. Autoclaved distilled water was poured over the seedlings to submerge them. Z-stacks were captured using a Leica SP8 upright confocal microscope equipped with a long-distance water dipping lens (63× objective). The rest of the confocal microscope settings were followed as described above.

Genetic analysis

T-DNA insertion mutants of wei8-1 tar2-1 (CS16413), tar2-1 (CS16404) and an EMS allele of pin1-5 (CS69067) were obtained from thee ABRC. To confirm the insertion, genomic DNA was first isolated using a modified CTAB method (Murray and Thompson, 1980). The pCLV3::mGFP-ER reporter line was created in the Ler background. It was used to set up crosses with taa1-1−/− tar2+/− (in Col-0) to evaluate the relative size of CZ. In F2, plants carrying pCLV3::mGFP-ER reporter in taa1-1, tar2−/− and taa1-1−/− tar2+/− were identified in Col-0 from the segregating populations by genotyping. After removing the floral buds under the fluorescence microscope, reporter activity was examined in the SAM. Plants were followed until the F4 to F5 generation to make the pCLV3::mGFP-ER reporter homozygous. A similar strategy was used to cross the R2D2 transgene.

To generate the double mutants of pin1-5 taa1-1 and pin1-5 tar2-1, and triple mutant of pin1-5 taa1-1 tar2-1, pin1-5 was crossed with the taa1−/− tar2+/− line. In F2, we identified pin1-5 taa1-1, pin1-5 tar2-1 and pin1-5 taa1-1 tar2-1. The fecundity of pin1-5 taa1-1 and pin1-5 tar2-1 double-mutant plants was extremely poor, even though we were able to collect seeds and use them to further analyze the double mutant phenotype in subsequent generations.

To obtain the pDR5::3xVenus-N7 reporter in taa1, tar2 and taa1 tar2 mutant background, taa1−/− tar2+/− Col-0 plants were crossed with the pPIN1::PIN1-GFP; pDR5::3xVenus-N7 Ler line. After raising the F1 generation, Ler-looking F2 plants, carrying either the pPIN1::PIN1-GFP or pDR5::3xVenus-N7 marker, were selected for taa1, tar2 and taa1−/− tar2+/− mutation by T-DNA PCR. To make the reporter homozygotes in the respective mutants, plants were selected for each reporter under the fluorescence microscope and, in parallel, were tested on selection media for the respective transgene. After multiple selection rounds, plants carrying pDR5::3xVenus-N7 and pPIN1::PIN1-GFP transgenes were identified in taa1, tar2 and taa1−/− tar2+/− mutants that had mixed background with a Landsberg (erecta mutant) Ler phenotype. pDR5::3xVenus-N7 taa1 (Ler) and pDR5::3xVenus-N7 tar2 (Ler) plants were used to set up a cross with pin1-5 (Ler). After raising F1 plants, in F2, pin1 taa1 and pin1 tar2 plants carrying the pDR5::3xVenus-N7 reporter were identified using fluorescence microscopy. Again, they were followed in F3 and F4 for segregation from the DR5 reporter. Double-mutant plants (taa1 pin1 and tar2 pin1) that did not show segregation from the pDR5::3xVenus-N7 reporter were used for confocal microscopy; cells showing fluorescence signal were counted to ascertain the auxin responses (Fig. 7D-F). Double mutant plants (taa1 pin1 and tar2 pin1) that did not show segregation for pDR5::3xVenus-N7 reporter were used for confocal microscopy, and cells showing fluorescence signal were counted to ascertain the auxin responses (Fig. 7D-F).

For counting the flowers, siliques, axillary IM and stem length, 35-day-old plants were used. Siliques were counted using only primary inflorescence meristem, whereas open flowers were counted on the 35th day using primary and axillary inflorescence meristem in WT-Ler, pin1, pin1 taa1 and pin1 tar2. For stem length, the height of the plant on 35th day was considered for measurement.

SAM size measurement

For SAM size measurements, confocal image stacks were obtained with a 0.35 μm step size from 4-week-old plants and analyzed using Morphograph X, as described previously de Reuille et al. (2015). The original image in TIF format was loaded into stack1 of MorphographX (Fig. S8). To remove the noise from the images, we applied a Gaussian Blur filter with a 0.3-pixel radius (Fig. S8). Signal edges were identified using an edge-detection process with a threshold value ranging from 10,000 to 15,000. The surface mess was created using Marching Cubes Surface with a 2.5 μm cube size (Fig. S8). After trimming the bottom, the visualized mess was subdivided two to three times (enough to extract the outer contour). The mess was stretched three or four times with a Smooth mess function with a pass value of 20. A signal was projected on the mess with 2 μm and 6 μm top and bottom values, respectively. This 2.5D image was used for seeding and segmentation (Fig. S8).

The first seed was used to mark the SAM boundaries, excluding the emerging primordia. Primordia were identified through a careful assessment of a 2.5D image by looking at the relative positions of the older primordia and the dip in curvature where primordia abuts from the meristem. The second seed was placed adjacent to the first seed to restrict the propagation of the first seed outside the meristem area (Fig. S8). The watershed segmentation algorithm was used for the segmentation process (Fig. S8). As the segmented area was user-defined and included only two propagated seeds, the segmentation quality was not an issue. The segmented area was converted into a heatmap to obtain the actual values (Fig. S8). A similar process was followed for all confocal image stacks to measure the size of SAM in wild-type and respective mutant plants.

Image analysis

The CLV3-expressing and non-expressing cells were counted in the L1 layer from the reconstructed top view of the SAM, as described by Yadav et al. (2010). First, a 3D volume stack of confocal images was obtained using the Leica Application Suite X (version 3.5.7.23225). This 3D image was loaded and opened in FIJI software directly, and all the primordia were marked. Cells were manually counted by labelling in the FIJI software cell counter tool. The same protocol was followed for quantification in mutant and wild-type SAM.

Virtual ratio images for the R2D2 reporter were produced in Leica Application Suite X (version 3.5.7.23225) software using the ‘Calcium Imaging Calculator’. Briefly, stacks were projected with maximum intensity, and background ROI was selected to determine the noise signal intensity at each pixel. After subtracting the background noise from both channels, a ratio image was created by calculating the ratio between the signal intensities of each pixel.

pPIN1::PIN1-GFP LUT images were produced in Fiji. Maximum intensity projection images were converted to LUT after setting the same thresholds for all images. DR5 maxima were counted in 3D projected images of SAMs from wild-type and mutant plants.

For preparing the final figures, 3D volume images and side views were taken from the Leica Application Suite X (version, 3.5.7.23225). Images were loaded into Fiji and cropped to an appropriate size to place them on the white canvas in Adobe Photoshop 2023.

Statistical analysis

All bar graphs were generated using GraphPad Prism 6 Software. Two-tailed t-tests or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests were carried out for statistical analysis using GraphPad Prism 6 software.

We thank Anna Stepanova for the pTAA1::YPet-TAA1 seed lines and Dolf Weijers for the R2D2 line. We thank Venu Reddy for sharing pCLV3::mGFP-ER lines and stimulating feedback on the manuscript. Imaging and sorting were performed at the confocal microscopy and FACS core facility at IISER Mohali. The IISER Mohali core grant funds support both confocal imaging and FACS facility.

Author contributions

Conceptualization: R.K.Y.; Methodology: S.Y., H.K., M.M., S.K.Sahu, R.K.Y.; Software: S.K.Sahu; Validation: S.Y., H.K., S.K.Singh; Formal analysis: S.Y., H.K., M.M., S.K.Sahu, S.K.Singh, R.K.Y.; Investigation: S.Y., H.K., M.M., S.K.Singh, R.K.Y.; Resources: S.Y., H.K., M.M., S.K.Singh, R.K.Y.; Data curation: S.Y., H.K., M.M., S.K.Sahu, S.K.Singh, R.K.Y.; Writing - original draft: S.Y., H.K., M.M., S.K.Sahu, S.K.Singh, R.K.Y.; Writing - review & editing: S.Y., M.M., S.K.Sahu, S.K.Singh, R.K.Y.; Visualization: H.K., S.K.Sahu, R.K.Y.; Supervision: R.K.Y.; Project administration: R.K.Y.; Funding acquisition: R.K.Y.

Funding

This work was supported by the Department of Biotechnology, Ministry of Science and Technology, India (BT/PR15352/BPA/118/159/2015), and by a IISER Mohali core grant sanctioned to R.K.Y. R.K.Y. is a recipient of an Innovative Young Biotechnologist Award and Ramalingaswami fellowship, and acknowledges grants received from the Department of Biotechnology, Ministry of Science and Technology, India.

Data availability

The raw data sequence files have been deposited in NCBI SRA under BioProject PRJNA529066.

Adamowski
,
M.
and
Friml
,
J.
(
2015
).
PIN-dependent auxin transport: action, regulation, and evolution
.
Plant Cell
27
,
20
-
32
.
Aida
,
M.
,
Ishida
,
T.
and
Tasaka
,
M.
(
1999
).
Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: Interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes
.
Development
126
,
1563
-
1570
.
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 (1979)
289
,
617
-
619
.
Brumos
,
J.
,
Robles
,
L. M.
,
Yun
,
J.
,
Vu
,
T. C.
,
Jackson
,
S.
,
Alonso
,
J. M.
and
Stepanova
,
A. N.
(
2018
).
Local auxin biosynthesis is a key regulator of plant development
.
Dev. Cell
47
,
306
-
318.e5
.
Brunoud
,
G.
,
Wells
,
D. M.
,
Oliva
,
M.
,
Larrieu
,
A.
,
Mirabet
,
V.
,
Burrow
,
A. H.
,
Beeckman
,
T.
,
Kepinski
,
S.
,
Traas
,
J.
,
Bennett
,
M. J.
et al. 
(
2012
).
A novel sensor to map auxin response and distribution at high spatio-temporal resolution
.
Nature
482
,
103
-
106
.
Burian
,
A.
,
Paszkiewicz
,
G.
,
Nguyen
,
K. T.
,
Meda
,
S.
,
Raczyńska-Szajgin
,
M.
and
Timmermans
,
M. C. P.
(
2022
).
Specification of leaf dorsiventrality via a prepatterned binary readout of a uniform auxin input
.
Nat. Plants
8
,
269
-
280
.
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
.
Cheng
,
Y.
,
Dai
,
X.
and
Zhao
,
Y.
(
2007
).
Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis
.
Plant Cell
19
,
2430
-
2439
.
Chourey
,
P. S.
,
Li
,
Q.-B.
and
Kumar
,
D.
(
2010
).
Sugar-hormone cross-talk in seed development: Two redundant pathways of IAA biosynthesis are regulated differentially in the invertase-deficient miniature1 (mn1) seed mutant in maize
.
Mol. Plant
3
,
1026
-
1036
.
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. USA
103
,
1627
-
1632
.
de Reuille
,
P. B.
,
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
.
Dharmasiri
,
N.
,
Dharmasiri
,
S.
and
Estelle
,
M.
(
2005
).
The F-box protein TIR1 is an auxin receptor
.
Nature
435
,
441
-
445
.
Eklund
,
D. M.
,
Ishizaki
,
K.
,
Flores-Sandoval
,
E.
,
Kikuchi
,
S.
,
Takebayashi
,
Y.
,
Tsukamoto
,
S.
,
Hirakawa
,
Y.
,
Nonomura
,
M.
,
Kato
,
H.
,
Kouno
,
M.
et al. 
(
2015
).
Auxin produced by the indole-3-pyruvic acid pathway regulates development and gemmae dormancy in the liverwort marchantia polymorpha
.
Plant Cell
27
,
1650
-
1669
.
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 (1979)
283
,
1911
-
1914
.
Friml
,
J.
,
Benková
,
E.
,
Blilou
,
I.
,
Wisniewska
,
J.
,
Hamann
,
T.
,
Ljung
,
K.
,
Woody
,
S.
,
Sandberg
,
G.
,
Scheres
,
B.
,
Jürgens
,
G.
et al. 
(
2002
).
AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis
.
Cell
108
,
661
-
673
.
Furumizu
,
C.
,
Krabberød
,
A. K.
,
Hammerstad
,
M.
,
Alling
,
R. M.
,
Wildhagen
,
M.
,
Sawa
,
S.
and
Aalen
,
R. B.
(
2021
).
The sequenced genomes of nonflowering land plants reveal the innovative evolutionary history of peptide signaling
.
Plant Cell
33
,
2915
-
2934
.
Gallavotti
,
A.
,
Barazesh
,
S.
,
Malcomber
,
S.
,
Hall
,
D.
,
Jackson
,
D.
,
Schmidt
,
R. J.
and
Mcsteen
,
P.
(
2008
).
sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize
.
Proc. Natl. Acad. Sci. USA
105
,
15196
-
15201
.
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
.
Gälweiler
,
L.
,
Guan
,
C.
,
Müller
,
A.
,
Wisman
,
E.
,
Mendgen
,
K.
,
Yephremov
,
A.
and
Palme
,
K.
(
1998
).
Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue
.
Science (1979)
282
,
2226
-
2230
.
Guenot
,
B.
,
Bayer
,
E.
,
Kierzkowski
,
D.
,
Smith
,
R. S.
,
Mandel
,
T.
,
Zádníková
,
P.
,
Benková
,
E.
and
Kuhlemeier
,
C.
(
2012
).
Pin1-independent leaf initiation in arabidopsis
.
Plant Physiol.
159
,
1501
-
1510
.
Harrison
,
C. J.
,
Rezvani
,
M.
and
Langdale
,
J. A.
(
2007
).
Growth from two transient apical initials in the meristem of Selaginella kraussiana
.
Development
134
,
881
-
889
.
Hata
,
Y.
and
Kyozuka
,
J.
(
2021
).
Fundamental mechanisms of the stem cell regulation in land plants: lesson from shoot apical cells in bryophytes
.
Plant Mol. Biol.
107
,
213
-
225
.
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
.
Hirakawa
,
Y.
,
Shinohara
,
H.
,
Kondo
,
Y.
,
Inoue
,
A.
,
Nakanomyo
,
I.
,
Ogawa
,
M.
,
Sawa
,
S.
,
Ohashi-Ito
,
K.
,
Matsubayashi
,
Y.
and
Fukuda
,
H.
(
2008
).
Non-cell-autonomous control of vascular stem cell fate by a CLE peptide/receptor system
.
Proc. Natl. Acad. Sci. USA
105
,
15208
-
15213
.
Hirakawa
,
Y.
,
Uchida
,
N.
,
Yamaguchi
,
Y. L.
,
Tabata
,
R.
,
Ishida
,
S.
,
Ishizaki
,
K.
,
Nishihama
,
R.
,
Kohchi
,
T.
,
Sawa
,
S.
and
Bowman
,
J. L.
(
2019
).
Control of proliferation in the haploid meristem by CLE peptide signaling in marchantia polymorpha
.
PLoS Genet.
15
,
e1007997
.
Hirakawa
,
Y.
,
Fujimoto
,
T.
,
Ishida
,
S.
,
Uchida
,
N.
,
Sawa
,
S.
,
Kiyosue
,
T.
,
Ishizaki
,
K.
,
Nishihama
,
R.
,
Kohchi
,
T.
and
Bowman
,
J. L.
(
2020
).
Induction of Multichotomous branching by CLAVATA peptide in Marchantia polymorpha
.
Curr. Biol.
30
,
3833
-
3840.e4
.
Kepinski
,
S.
and
Leyser
,
O.
(
2005
).
The Arabidopsis F-box protein TIR1 is an auxin receptor
.
Nature
435
,
446
-
451
.
Landberg
,
K.
,
Šimura
,
J.
,
Ljung
,
K.
,
Sundberg
,
E.
and
Thelander
,
M.
(
2021
).
Studies of moss reproductive development indicate that auxin biosynthesis in apical stem cells may constitute an ancestral function for focal growth control
.
New Phytol.
229
,
845
-
860
.
Liao
,
C.-Y.
,
Smet
,
W.
,
Brunoud
,
G.
,
Yoshida
,
S.
,
Vernoux
,
T.
and
Weijers
,
D.
(
2015
).
Reporters for sensitive and quantitative measurement of auxin response
.
Nat. Methods
12
,
207
-
210
.
Liu
,
L.
,
Gallagher
,
J.
,
Arevalo
,
E. D.
,
Chen
,
R.
,
Skopelitis
,
T.
,
Wu
,
Q.
,
Bartlett
,
M.
and
Jackson
,
D.
(
2021
).
Enhancing grain-yield-related traits by CRISPR–Cas9 promoter editing of maize CLE genes
.
Nat. Plants
7
,
287
-
294
.
Ljung
,
K.
(
2013
).
Auxin metabolism and homeostasis during plant development
.
Development (Camb.)
140
,
943
-
950
.
Ma
,
Y.
,
Miotk
,
A.
,
Šutiković
,
Z.
,
Ermakova
,
O.
,
Wenzl
,
C.
,
Medzihradszky
,
A.
,
Gaillochet
,
C.
,
Forner
,
J.
,
Utan
,
G.
,
Brackmann
,
K.
et al. 
(
2019
).
WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis
.
Nat. Commun.
10
,
5093
.
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
.
Möller
,
B.
and
Weijers
,
D.
(
2009
).
Auxin control of embryo patterning
.
Cold Spring Harb. Perspect. Biol.
5
,
a001545
.
Moody
,
L. A.
(
2019
).
The 2D to 3D growth transition in the moss Physcomitrella patens
.
Curr. Opin. Plant Biol.
47
,
88
-
95
.
Moody
,
L. A.
(
2020
).
Three-dimensional growth: a developmental innovation that facilitated plant terrestrialization
.
J. Plant Res.
133
,
283
-
290
.
Moody
,
L. A.
,
Kelly
,
S.
,
Rabbinowitsch
,
E.
and
Langdale
,
J. A.
(
2018
).
Genetic Regulation of the 2D to 3D Growth Transition in the Moss Physcomitrella patens
.
Curr. Biol.
28
,
473
-
478.e5
.
Murray
,
M. G.
and
Thompson
,
W. F.
(
1980
).
Rapid isolation of high molecular weight plant DNA
.
Nucleic Acids Res.
8
,
4321
-
4326
.
Ogawa
,
M.
,
Shinohara
,
H.
,
Sakagami
,
Y.
and
Matsubayash
,
Y.
(
2008
).
Arabidopsis CLV3 peptide directly binds CLV1 ectodomain
.
Science (1979)
319
,
294
.
Ohmori
,
Y.
,
Tanaka
,
W.
,
Kojima
,
M.
,
Sakakibara
,
H.
and
Hirano
,
H.-Y.
(
2013
).
WUSCHEL-RELATED HOMEOBOX4 Is involved in meristem maintenance and is negatively regulated by the CLE gene FCP1 in rice
.
Plant Cell
25
,
229
-
241
.
Phillips
,
K. A.
,
Skirpan
,
A. L.
,
Liu
,
X.
,
Christensen
,
A.
,
Slewinski
,
T. L.
,
Hudson
,
C.
,
Barazesh
,
S.
,
Cohen
,
J. D.
,
Malcomber
,
S.
and
McSteen
,
P.
(
2011
).
vanishing tassel2 Encodes a Grass-Specific tryptophan aminotransferase required for vegetative and reproductive development in maize
.
Plant Cell
23
,
550
-
566
.
Prigge
,
M. J.
,
Platre
,
M.
,
Kadakia
,
N.
,
Zhang
,
Y.
,
Greenham
,
K.
,
Szutu
,
W.
,
Pandey
,
B. K.
,
Bhosale
,
R. A.
,
Bennett
,
M. J.
,
Busch
,
W.
et al. 
(
2020
).
Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions
.
eLife
9
,
e54740
.
Reinhardt
,
D.
,
Pesce
,
E.-R.
,
Stieger
,
P.
,
Mandel
,
T.
,
Baltensperger
,
K.
,
Bennett
,
M.
,
Traas
,
J.
,
Friml
,
J.
and
Kuhlemeier
,
C.
(
2003
).
Regulation of phyllotaxis by polar auxin transport
.
Nature
426
,
255
-
260
.
Saini
,
P.
,
Bhatia
,
S.
,
Mahajan
,
M.
,
Kaushik
,
A.
,
Sahu
,
S. K.
,
Kumar
,
A.
,
Satbhai
,
S. B.
,
Patel
,
M. K.
,
Saxena
,
S.
,
Chaurasia
,
O. P.
et al. 
(
2020
).
ELONGATED HYPOCOTYL5 negatively regulates DECREASE WAX BIOSYNTHESIS to increase survival during UV-B stress
.
Plant Physiol.
184
,
2091
-
2106
.
Schlegel
,
J.
,
Denay
,
G.
,
Wink
,
R.
,
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
.
Sessions
,
A.
,
Nemhauser
,
J. L.
,
McCall
,
A.
,
Roe
,
J. L.
,
Feldmann
,
K. A.
and
Zambryski
,
P. C.
(
1997
).
ETTIN patterns the Arabidopsis floral meristem and reproductive organs
.
Development
124
,
4481
-
4491
.
Sevilem
,
I.
,
Miyashima
,
S.
and
Helariutta
,
Y.
(
2013
).
Cell-to-cell communication via plasmodesmata in vascular plants
.
Cell Adh. Migr.
7
,
27
-
32
.
Simonini
,
S.
,
Deb
,
J.
,
Moubayidin
,
L.
,
Stephenson
,
P.
,
Valluru
,
M.
,
Freire-Rios
,
A.
,
Sorefan
,
K.
,
Weijers
,
D.
,
Friml
,
J.
and
Østergaard
,
L.
(
2016
).
A noncanonical auxin-sensing mechanism is required for organ morphogenesis in arabidopsis
.
Genes Dev.
30
,
2286
-
2296
.
Smith
,
R. S.
,
Guyomarc'h
,
S.
,
Mandel
,
T.
,
Reinhardt
,
D.
,
Kuhlemeier
,
C.
and
Prusinkiewicz
,
P.
(
2006
).
A plausible model of phyllotaxis
.
Proc. Natl Acad. Sci. USA
103
,
1301
-
1306
.
Sohlberg
,
J. J.
,
Myrenås
,
M.
,
Kuusk
,
S.
,
Lagercrantz
,
U.
,
Kowalczyk
,
M.
,
Sandberg
,
G.
and
Sundberg
,
E.
(
2006
).
STY1 regulates auxin homeostasis and affects apical-basal patterning of the Arabidopsis gynoecium
.
Plant J.
47
,
112
-
123
.
Stepanova
,
A. N.
,
Robertson-Hoyt
,
J.
,
Yun
,
J.
,
Benavente
,
L. M.
,
Xie
,
D.-Y.
,
Doležal
,
K.
,
Schlereth
,
A.
,
Jürgens
,
G.
and
Alonso
,
J. M.
(
2008
).
TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development
.
Cell
133
,
177
-
191
.
Takahashi
,
G.
,
Betsuyaku
,
S.
,
Okuzumi
,
N.
,
Kiyosue
,
T.
and
Hirakawa
,
Y.
(
2021
).
An evolutionarily conserved coreceptor gene is essential for CLAVATA signaling in Marchantia polymorpha
.
Front. Plant Sci.
12
,
657548
.
Tao
,
Y.
,
Ferrer
,
J.-L.
,
Ljung
,
K.
,
Pojer
,
F.
,
Hong
,
F.
,
Long
,
J. A.
,
Li
,
L.
,
Moreno
,
J. E.
,
Bowman
,
M. E.
,
Ivans
,
L. J.
et al. 
(
2008
).
Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants
.
Cell
133
,
164
-
176
.
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
.
Weijers
,
D.
,
Schlereth
,
A.
,
Ehrismann
,
J. S.
,
Schwank
,
G.
,
Kientz
,
M.
and
Jürgens
,
G.
(
2006
).
Auxin triggers transient local signaling for cell specification in arabidopsis embryogenesis
.
Dev. Cell
10
,
265
-
270
.
Wenzel
,
C. L.
,
Schuetz
,
M.
,
Yu
,
Q.
and
Mattsson
,
J.
(
2007
).
Dynamics of MONOPTEROS and PIN-FORMED1 expression during leaf vein pattern formation in Arabidopsis thaliana
.
Plant J.
49
,
387
-
398
.
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
.
Yadav
,
R. K.
,
Tavakkoli
,
M.
and
Reddy
,
G. V.
(
2010
).
WUSCHEL mediates stem cell homeostasis by regulating stem cell number and patterns of cell division and differentiation of stem cell progenitors
.
Development
137
,
3581
-
3589
.
Yadav
,
R. K.
,
Perales
,
M.
,
Gruel
,
J.
,
Girke
,
T.
,
Jönsson
,
H.
and
Venugopala Reddy
,
G.
(
2011
).
WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex
.
Genes Dev.
25
,
2025
-
2030
.
Yadav
,
R. K.
,
Perales
,
M.
,
Gruel
,
J.
,
Ohno
,
C.
,
Heisler
,
M.
,
Girke
,
T.
,
Jönsson
,
H.
and
Venugopala Reddy
,
G.
(
2013
).
Plant stem cell maintenance involves direct transcriptional repression of differentiation program
.
Mol. Syst. Biol.
9
,
654
.
Yadav
,
R. K.
,
Tavakkoli
,
M.
,
Xie
,
M.
,
Girke
,
T.
and
Venugopala Reddy
,
G.
(
2014
).
A high-resolution gene expression map of the arabidopsis shoot meristem stem cell niche
.
Development (Camb.)
141
,
2735
-
2744
.
Yamada
,
M.
,
Greenham
,
K.
,
Prigge
,
M. J.
,
Jensen
,
P. J.
and
Estelle
,
M.
(
2009
).
The TRANSPORT INHIBITOR RESPONSE2 Gene is required for auxin synthesis and diverse aspects of plant development
.
Plant Physiol.
151
,
168
-
179
.
Zhao
,
Y.
(
2010
).
Auxin biosynthesis and its role in plant development
.
Annu. Rev. Plant Biol.
61
,
49
-
64
.
Zhou
,
Z.-Y.
,
Zhang
,
C.-G.
,
Wu
,
L.
,
Zhang
,
C.-G.
,
Chai
,
J.
,
Wang
,
M.
,
Jha
,
A.
,
Jia
,
P.-F.
,
Cui
,
S.-J.
,
Yang
,
M.
et al. 
(
2011
).
Functional characterization of the CKRC1/TAA1 gene and dissection of hormonal actions in the Arabidopsis root
.
Plant J.
66
,
516
-
527
.

Competing interests

The authors declare no competing or financial interests.