Evolutionary adaptations to the hormonal regulation of vascular tissue development

Significance

Vascular tissues are a hallmark of land plants, enabling long-distance transport and structural support. Although the interplay between auxin and cytokinin during vascular development is well characterized in few model plants, it remains unclear how conserved these regulatory networks are in other lineages. Unlike the vascular plants studied to date where cross-talk between these two phytohormones is observed during vascular tissue development, here, we demonstrate that the lycophyte Selaginella moellendorffii employs a unique task separation strategy, with auxin and cytokinin exclusively controlling cell proliferation and differentiation, respectively. This finding provides insight into how fundamental tissue patterning principles can be modified and rewired over evolution, which could be important to explain the diversification of vascular architectures found in extant plants today.

Abstract

Vascular tissues provide long-distance transport and physical support in the vascular plant lineage, providing a significant adaptive advantage. Although the cross talk between auxin and cytokinin in promoting both vascular cell proliferation and differentiation has been well studied in angiosperms such as Arabidopsis thaliana, little is known about this regulation in other vascular plant lineages. Here, we found that unlike the hormonal cross talk found in all other species under study, the lycophyte Selaginella moellendorffii shows clear task separation, with auxin driving vascular cell proliferation only and cytokinin specifically triggering cell differentiation. Using a cross-species transcriptomics approach, we found that members of the AUXIN/INDOLE-ACETIC ACID (AUX/IAA) and CYTOKININ OXIDASE (CKX) gene families exhibited divergent expression patterns in response to auxin and cytokinin treatments. Despite these regulatory differences, we show that AUX/IAA and CKX proteins are functionally conserved between Arabidopsis and Selaginella. Taken together, our findings suggest an evolutionary adaptation to the hormonal regulation of vascular tissue development in which core protein functions are conserved, but regulatory circuits diverged in lycophytes.

Multicellular land plants have shaped the Earth’s biosphere as we know it today (1) and can be divided into bryophytes (nonvascular plants) and tracheophytes (vascular plants) (2). Unlike green algae, dominant land plants use a complex vascular system to transport nutrients, hormones, and other signaling molecules throughout the plant body and store carbon (1, 3). Together with other innovations, this development of vascular tissues contributed to a significant advantage in competition, enabling vascular plants to ultimately dominate the planet. Vascular tissues consist of two specialized cell types: phloem cells, which function as conduits for sugar transport, and xylem cells, which distribute water throughout the plant (4). These pools of cells are replenished by divisions of the intervening (pro)cambium cells (5, 6). Research in the model species Arabidopsis thaliana has shown that phytohormone signaling is crucial in maintaining (pro)cambium identities and regulating its proliferation activity, leading to the establishment of functional vascular tissues (7–9). During primary growth in root meristems, both auxin and cytokinin promote procambial cell proliferation and vascular cell differentiation.

Auxin is an essential phytohormone that plays a fundamental role in developmental processes during both embryogenesis and postembryogenesis. Its signaling is initiated upon binding to the TIR1 (TRANSPORT INHIBITOR RESPONSE1)/AFB (AUXIN SIGNALING F-BOX) family of F-box receptors, which initiates the ubiquitin-mediated degradation of AUXIN/INDOLE-ACETIC ACID (AUX/IAA) transcriptional repressors (10). This degradation releases AUXIN RESPONSE FACTOR (ARF) transcription factors from inhibition, thereby activating gene expression. During vascular tissue development, BODENLOS/IAA12 disrupts the division orientation of provascular initial cells by repressing the transcriptional activities of MONOPTEROS (MP/ARF5) (11, 12). SHORT HYPOCOTYL 2 (SHY2/IAA3) acts as a negative regulator of secondary vascular formation and ectopic expression of stabilized SHY2 variants in the vascular cambium disrupts the differentiation of specialized vascular tissues (13–15). Moreover, the interplay with auxin and cytokinin also plays an essential role in vascular tissue development (5–7, 16, 17). MP/ARF5 induces expression of the MP-dependent basic HELIX-LOOP-HELIX (bHLH) transcription factor TARGET OF MONOPTEROS 5 (TMO5) (18), which forms a heterodimer complex with another bHLH TF called LONESOME HIGHWAY (LHW). This complex activates expression of LOG LONELY GUY 3 (LOG3), LOG4, and BETA GLUCOSIDASE 44 (BGLU44), thereby increasing active cytokinin levels (5, 19, 20). The TMO5/LHW complex also induces expression of the cytokinin-inducible DOF2.1 TF, promoting vascular cell proliferation (21). Additionally, cytokinin diffuses into adjacent procambium and phloem, stimulating cell division. This effect is counterbalanced by CYTOKININ OXIDASE3 (CKX3), which is regulated by SHORT ROOT (SHR) as another TMO5/LHW downstream target (16). The phytohormones auxin and cytokinin were also shown to exhibit conserved functions on vascular tissue development across diverse tracheophyte species, such as Populus trees (22) and Oryza sativa (23). Despite these anecdotal examples of a putative conserved role for auxin and cytokinin in regulating vascular proliferation and differentiation processes, it remains unclear whether this mechanism is indeed maintained throughout land plant evolution.

In this study, we observed that the hormonal control of vascular tissue development in the lycophyte Selaginella moellendorffii diverged from the complex auxin-cytokinin cross talk found in all other lineages under study. In Selaginella, auxin uniquely regulates cell proliferation, while cytokinin specifically triggers vascular cell differentiation. We further show that diverged responses of AUX/IAA and CKX family members to these phytohormones likely lay at the basis of this task separation, despite their conserved protein functions during vascular tissue development. As such, our work suggests that changes to the regulatory logic likely drove the evolutionary divergence of hormonal control during vascular tissue development across vascular plants.

Results

Selaginella Has Diverged Hormonal Functions during Vascular Tissue Development.

In tracheophytes, the presence of vascular tissues is a defining feature and its development is largely determined by the interplay between the phytohormones auxin and cytokinin. To explore the potential conservation of this interaction, we first investigated vascular tissue development in response to exogenous auxin and cytokinin treatments in a representative species from the dicot, monocot, fern, and lycophyte clades (Fig. 1A). In the root of Arabidopsis thaliana, a 2-d auxin (1 µM 1-naphthaleneacetic acid (NAA)) treatment promoted cell proliferation by increasing the total cell numbers in primary vascular tissues (Fig. 1 B and F). Additionally, we observed that auxin treatment promoted the differentiation of xylem cells visualized by the presence of secondary cell wall accumulations (Fig. 1 B and G). A similar effect was observed for a 2-d cytokinin (1 µM 6-benzylaminopurine (BAP)) treatment in the root of Arabidopsis (Fig. 1 B, F, and G). This cross-talk in function between auxin and cytokinin signaling on vascular cell proliferation and differentiation (as typed by secondary cell wall accumulation) was also observed in the roots of the monocot Triticum aestivum (Fig. 1 C, F, and G) and the fern Azolla filiculoides (Fig. 1 D, F, and G). In contrast, when analyzing these responses in Selaginella moellendorffii as a lycophyte model species (Fig. 1A) (24–26), we observed that a 7-d auxin treatment (1 µM NAA) in Selaginella root promoted cell proliferation near the root tip (observed about 0.2 cm from root tip) (Fig. 1 E–G), but inhibited differentiation into specialized vascular cells compared to the mock treated control (observed about 0.5 cm from root tip) (SI Appendix, Fig. S1 A and B). To determine the identity of the auxin-induced proliferating cells, we first stimulated proliferation with auxin treatment (1 µM NAA) and then transferred the roots to mock medium to allow differentiation to occur (SI Appendix, Fig. S1C). One day after auxin removal, the proliferating cells initiated specialized vascular cell differentiation (SI Appendix, Fig. S1C). Seven days after auxin removal, well-developed vascular tissues ultimately formed in the root center (SI Appendix, Fig. S1 C–E); suggesting that normal differentiation took place after auxin removal, but in an enlarged vascular area compared to the mock control. Moreover, a cytokinin treatment (1 µM BAP) applied to the root tip significantly promoted vascular cell differentiation without changing the total amount of vascular tissue cell numbers on a cross-section compared to a mock treated control (Fig. 1 E–G and SI Appendix, Fig. S1 A and B) and reduced root meristem size (SI Appendix, Fig. S1 F and G) as shown in the longitudinal sections.

Fig. 1.

Diverged hormonal functions during vascular tissue development. (A) Phylogenetic relationships of selected land plants. (B) Cross-section through the root of Arabidopsis thaliana (Col-0). 4-d-old plants were transferred to either mock (Left), 1 μM NAA (Middle), or 1 μM BAP (Right) plates for 2 d before analysis. (C) Cross-section through the root of Triticum aestivum. 2-d-old plants were transferred to either mock (Upper), 1 μM NAA (Middle), or 1 μM BAP (Lower) plates for 2 d before analysis. (D) Cross-section through the root of Azolla filiculoides. Plants were transferred to either mock (Left), 1 μM NAA (Middle), or 1 μM BAP (Right) plates for 7 d before analysis. (E) Cross-section (~0.2 cm from root tip) through the root of Selaginella moellendorffii. 14-d-old plants were transferred to either mock (Left), 1 μM NAA (Middle), or 1 μM BAP (Right) plates for 7 d before analysis. The blue circle indicated the vascular tissues. (F and G) Quantification of the number of vascular tissue cells and differentiated xylem cells in panels B–E. The red asterisk in B–E indicates the differentiated xylem cell. Ph: Phloem, Xy: Xylem. Black scale bars: 20 μm. Count data in F and G were modelld with a generalized mixed model (P-values were adjusted using Holm–Bonferroni correction; *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant).

Taken together, our findings suggest that both auxin and cytokinin signaling can promote root vascular cell proliferation and differentiation in Arabidopsis (dicot), Triticum (monocot), and Azolla (fern) roots. However, in Selaginella (lycophyte), these hormones operate through independent pathways with auxin inducing vascular cell proliferation only, while cytokinin specifically initiates vascular cell differentiation. This divergence highlights a unique regulatory mechanism in lycophyte vascular tissue development.

Characterizing Auxin and Cytokinin Regulation in Selaginella Vascular Tissue Development.

As Selaginella exhibits a unique hormonal task separation, we next analyzed the response of vascular tissue development to auxin and cytokinin in more detail. Vascular tissue formation in the Selaginella root can be separated into several characteristic growth phases (SI Appendix, Fig. S2). A first stage can be characterized by cytoplasmic dense and rather equal sized cells above the apical initial cell(s) (Stage 1). Subsequently, cells in the center of the root become more specialized in size and shape and the first patterning is observable (Stage 2). Next, the first differentiated xylem and phloem cells are observed (Stage 3). Finally, mature phloem and xylem were present in the center of the Selaginella root (Stage 4) (SI Appendix, Fig. S2) (24).

After building on our characterization of vascular tissue development in Selaginella, we next investigated the modalities of how auxin treatments affect vascular tissue development in this model system. First, we applied different concentrations of 2,4-D (2,4-Dichlorophenoxyacetic acid) to sustain high auxin level in the roots, enabling us to investigate the functions of auxin in root vascular tissue development (27–29). Auxin treatments (0.1 µM 2,4-D and 1 µM 2,4-D) in mature root (Stage 4) vascular tissues had no effect on the number of specialized vascular tissues (phloem and xylem) or total vascular tissue cells count (SI Appendix, Fig. S3 A and B). By contrast, an auxin treatment in root tips (Stage 3) significantly promoted cell proliferation in a dose-dependent manner, without inducing specialized vascular tissue differentiation (SI Appendix, Fig. S3 C–E). This effect was observed for different types of auxin (NAA, indole-3-acetic acid (IAA), or 2,4-D) albeit with different strengths (SI Appendix, Fig. S3 F–H). In line with expectations, blocking polar auxin transport using NPA (N-1-naphthylphthalamic acid) reduced the number of vascular cells (SI Appendix, Fig. S3 F–H). Subsequently, we conducted a similar analysis to elucidate the precise role of cytokinin signaling pathways in regulating cell differentiation within the vascular tissues of Selaginella root systems. We first applied different concentrations of cytokinin (0.1 µM BAP and 1 µM BAP) to mature root (Stage 4) vascular tissues and root tips (Stage 2) and found that a 7-d cytokinin treatment had no effect on the number of specialized vascular tissues (phloem and xylem) or the total number of vascular cells (SI Appendix, Fig. S4 A and B). However, a 1 µM BAP treatment in root tips (Stage 2) is sufficient to induce vascular tissue differentiation (Fig. 1 E–G) while this is not the case for lower cytokinin concentrations (SI Appendix, Fig. S4 C and D). Furthermore, the cytokinin treatment was able to counteract the auxin-induced cell proliferation effect and enhance vascular cell differentiation in the presence of auxin (SI Appendix, Fig. S4 E–I). These findings suggest that both auxin and cytokinin operate in vascular stem cells in Selaginella but have distinct functions. More specifically, cytokinin signaling acts antagonistically to auxin to suppress vascular cell proliferation, and drive vascular cell differentiation.

Selaginella Shows Diverged Transcriptional Responses to Auxin and Cytokinin.

To uncover how auxin and cytokinin signaling might regulate vascular tissue development at the molecular level in the root of Selaginella and Arabidopsis treated with these hormones, we performed bulk RNA sequencing (RNA-seq). First, we analyzed the whole-genome transcriptional changes in both Selaginella and Arabidopsis root tips after 2-h treatments with mock, auxin (10 µM NAA) or cytokinin (10 µM BAP) (Fig. 2 A and B, SI Appendix, Fig. S5 A and B, and Dataset S1). Moreover, given that an auxin treatment in Selaginella is able to induce a population of undifferentiated vascular cells, which subsequently differentiate upon removal of auxin (SI Appendix, Fig. S1 C–E), this offers a unique system to observe de novo establishment of vascular cells. We thus decided to take advantage of this effect to further narrow down potential regulators. In this second set-up, we conducted a time-series RNA-seq experiment following NAA removal at 0, 3, 6, 12, and 24 h (SI Appendix, Fig. S5C and Dataset S1). By integrating data from both experimental conditions, we identified two distinct gene subsets regulating vascular tissue development in Selaginella roots: one promoting cell proliferation and another driving differentiation (SI Appendix, Fig. S5 D and E). Considering that the diverged functions of auxin and cytokinin signaling in root vascular tissues of Arabidopsis and Selaginella are likely to be tightly connected to distinct transcriptional profiles, we hypothesized that the orthologous regulators should be differently regulated in our bulk RNA-seq datasets. Among the orthologous gene families differentially regulated between Arabidopsis and Selaginella (Dataset S1), members of the AUX/IAA and CKX gene families (SI Appendix, Fig. S5 F and S6 A and B) stood out as being highly regulated upon auxin and cytokinin treatment. Given their divergent role in auxin and cytokinin signaling in regulating vascular tissue development between Arabidopsis and Selaginella, we considered these as logical targets for deeper analysis. To further investigate this hypothesis, we first validated whether these genes are expressed in vascular cells at the correct developmental window. For the Arabidopsis orthologs, several selected genes have already been reported to be expressed in vascular tissues, including AtIAA1 (30), AtIAA3/SHY2 (14), AtIAA5 (31), and AtIAA19 (32) from the AUX/IAA gene family, as well as AtCKX1 (33) and AtCKX3 (16) from the CKX gene family. In order to validate the expression patterns for the Selaginella orthologs, we used hybridization chain reaction RNA fluorescence in situ hybridization (HCR RNA-FISH) (34) and designed probes for SmIAA1, SmIAA3, and SmCKX1. We found that both SmIAA1 and SmIAA3 were expressed in the young vascular tissues of the root apical meristem (Fig. 2C). In contrast, the expression of SmCKX1 in the root apical meristem appeared in later stages of vascular tissue development (Fig. 2C) in a mutually exclusive pattern compared to the SmIAA genes. In summary, our results show distinct transcriptomics responses in AUX/IAA and CKX gene families upon auxin and cytokinin treatment in roots of Arabidopsis and Selaginella, suggesting lineage-specific evolutionary adaptations in vascular tissue development.

Fig. 2.

Diverged transcriptional responses to auxin and cytokinin in Selaginella. (A and B) Overview of the RNA-seq experimental design. In Selaginella moellendorffii, 14-d-old plants were transferred for 2 h on mock, 10 μM NAA or 10 μM BAP plates, and the root tips (~0.5 cm) were collected for each sample (A). In Arabidopsis thaliana, 4-d-old plants were transferred for 2 h on mock, 10 μM NAA or 10 μM BAP plates, and the root tips (~0.5 cm) were collected for each sample (B). (C) Whole-mount HCR RNA-FISH of SmIAA1, SmIAA3, and SmCKX1 in the root tip of Selaginella moellendorffii. Note that the negative control (including the hairpin but no probes) is valid for experiments with both probes. The white arrow indicates the vascular tissues. White scale bars (for panels C and D): 20 μm and yellow scale bars (for panels A and B): 1 mm.

IAA and CKX Proteins Are Functionally Conserved in Selaginella and Arabidopsis.

Given the vascular expression of these auxin- and cytokinin-related genes, we next sought to investigate their evolutionary relationships by constructing a phylogenetic tree of AUX/IAA and CKX gene family members in Selaginella and Arabidopsis. The phylogenetic analysis of the AUX/IAA gene family revealed that Selaginella orthologs cluster together and separately from the orthologs in Arabidopsis. In contrast, the CKX gene family members from Selaginella and Arabidopsis showed intermixing in the phylogenetic tree (SI Appendix, Figs. S6B and S7B). However, a strong conservation at the level of the protein sequence was observed in several protein domains for both species (SI Appendix, Figs. S6A and S7A). These results suggested that the biochemical functions of the AUX/IAA and CKX orthologs are likely conserved, despite the observed differential responses to auxin and cytokinin at the transcript level.

Based on the protein sequence conservation presented in AUX/IAA and CKX family members from Selaginella and Arabidopsis, we next explored whether the protein functions themselves are also conserved. Given the AUX/IAA family is highly redundant, few phenotypes are observed in loss-of-function mutants. Indeed, our understanding of the AUX/IAA family in Arabidopsis mostly relies on gain of function mutants (35, 36). A noticeable exception is the shy2-31 loss-of-function mutant causing lateral root hydropatterning defects (37). We thus next analyzed the role of AtIAA3/SHY2 in vascular tissue development using a gain-of-function (shy2-2) and loss-of-function (shy2-31) mutant. shy2-2 exhibited fewer vascular cells and delayed secondary cell wall deposition in primary xylem compared to the Col-0 control (SI Appendix, Fig. S8 A and B). In contrast, shy2-31 showed no difference from Col-0 (SI Appendix, Fig. S8 A and B). Given functional redundancy among AUX/IAA proteins during vascular tissue development in Arabidopsis (SI Appendix, Fig. S8 A and B) (35), we then generated Arabidopsis lines ectopically overexpressing AtIAA1, AtIAA5, and AtIAA19 under the ubiquitously expressed UBI10 promoter (pUBI10::AtIAA1-GFP, pUBI10::AtIAA5-GFP, pUBI10::AtIAA19-GFP in Col-0). Similar to the shy2-2 mutant, overexpression of these AtAUX/IAA proteins inhibited vascular cell proliferation and secondary cell wall deposition in the primary xylem (Fig. 3 A and B and SI Appendix, Fig. S8 A–F). Subsequently, we examined the AUX/IAA proteins from Selaginella (pUBI10::SmIAA1-GFP and pUBI10::SmIAA4-GFP in Col-0). Although a diverged hormonal regulatory pattern was found for SmIAA1 and SmIAA4 (SI Appendix, Fig. S6 and Dataset S1), we observed that the produced phenotypes were identical to those of AtAUX/IAA members, exhibiting similar impairments in vascular cell proliferation and primary xylem secondary cell wall deposition (Fig. 3 C and D and SI Appendix, Fig. S8 G and H).

Fig. 3.

Aux/IAA and CKX orthologs are functionally conserved. (A) Cross-section through 6-d-old Col-0 and pUBI10::AtIAA1-GFP roots. (B) Quantification of the number of vascular tissue cells and differentiated xylem cells in panel A. (C) Cross-section through 6-d-old Col-0 and pUBI10::SmIAA1-GFP roots. (D) Quantification of the number of vascular tissue cells and differentiated xylem cells in panel C. (E) Cross-section through 6-d-old Col-0 and pUBI10::AtCKX7-GFP roots. (F) Quantification of the number of vascular tissue cells and differentiated xylem cells in panel E. (G) Cross-section through 6-d-old Col-0 and pUBI10::SmCKX1-GFP roots. (H) Quantification of the number of vascular tissue cells and differentiated xylem cells in panel G. The red asterisk indicates the differentiated xylem cell. Black scale bars: 20 μm. Count data were modeled with a generalized mixed model (P-values were adjusted using Holm–Bonferroni correction).

We then investigated the functions of Arabidopsis CKX proteins. Fitting with previous studies showing that CKX family members play a crucial role in vascular tissue development in Arabidopsis (16, 38–40), we found that the ckx7 mutants impaired the xylem differentiation (SI Appendix, Fig. S9 A and B). Conversely, ectopic overexpression of AtCKX7 suppressed vascular cell proliferation and significantly triggered xylem differentiation (Fig. 3 E and F) (40). Similar to these results, ectopic overexpression of SmCKX1 (pUBI10::SmCKX1-GFP) and SmCKX2 (pUBI10::SmCKX2-GFP) in Arabidopsis, mirrored AtCKX7 function, limiting vascular cell proliferation and promoting xylem differentiation (Fig. 3 G and H and SI Appendix, Fig. S9 C and D), even though these two orthologs exhibited a different regulatory profile (SI Appendix, Fig. S6 and Dataset S1). These results suggest a functional conservation of AUX/IAA and CKX proteins between Arabidopsis and Selaginella, despite different regulation in response to auxin and cytokinin treatments at the transcript level.

Discussion

Although abundant morphological variations of vascular tissues exist across species, the differentiated vascular tissues (xylem and phloem cell types) are functionally conserved for long-distance transport of organic and inorganic compounds (41–43). Based on our results in model species of dicots, monocots, and ferns, it is likely to assume that in most plant species, auxin and cytokinin act cooperatively in a complex cross-talk to regulate both cell proliferation and differentiation during vascular development in the root (4, 9, 44–46). As an interesting divergence from this general rule, the tasks of auxin and cytokinin are clearly separated in Selaginella as lycophyte model species with auxin uniquely promoting cell proliferation, while cytokinin specifically driving differentiation of vascular cell types (Fig. 4). Moreover, despite the functional conservation of AUX/IAA and CKX proteins in controlling vascular development as downstream regulators in these hormone signaling pathways (9, 16, 35, 40), their expression and regulatory patterns diverge between Arabidopsis and Selaginella (Fig. 4). Taken together, these findings suggest that evolutionary innovations in vascular tissue development might have arisen through regulatory rewiring of core hormone signaling pathways, while their developmental roles at the protein level remained highly conserved. As such, the regulatory plasticity observed between Selaginella and Arabidopsis appears to be mediated by lineage-specific promoter architectures, suggesting evolutionary tinkering with transcriptional control mechanisms while preserving core protein functions. These findings hint toward an evolutionary strategy in which conserved developmental pathways can achieve species-specific modulation through cis-regulatory diversification (47–49). It would thus be interesting to further explore auxin- and cytokinin-related binding motifs across vascular plants through in silico analyses in order to uncover additional transcriptional networks that modulate developmental pathways.

Fig. 4.

Distinct evolutionary adaptations to hormonal regulation of vascular development. Schematic overview of the task separation in Selaginella where the phytohormones auxin and cytokinin have distinct functions (Left) versus the hormonal cross-talk between these hormones in other vascular plant species (Right).

Materials and Methods

Plant Material and Growth.

Arabidopsis thaliana and Triticum aestivum plants were grown either on 1/2 MS medium supplemented with 1% sucrose and 0.8% plant agar under continuous light until sampling, or in soil under long-day conditions (16 h light/8 h dark) for seed production. Azolla filiculoides plants were grown on liquid media as described in ref. 50 under continuous light until sampling. Selaginella moellendorffii plants were grown on 1/2 MS supplemented with 1% sucrose, 0.01% myo-inositol, 0.05% MES, and 0.8% plant agar under continuous light until sampling. Chemical treatments were added as a mixture to the media and included 2,4 D (0.1 µM/1 µM, Sigma, D-7299, diluted in DMSO), IAA (1 µM, Duchefa, I0901, diluted in DMSO), NAA (1 µM/10 µM, Duchefa, N0903, diluted in DMSO), NPA (10 µM, Duchefa, N0926, diluted in DMSO), or BAP (1 µM/10 µM, Duchefa, B0904, diluted in DMSO). For treatment, plants were initially grown on mock plates or in liquid media before being transferred to plates or liquid media containing the respective chemicals. The mutants and transgenic lines (T1 or T2 segregating homozygous populations) of Arabidopsis thaliana used in this study are listed in SI Appendix, Table S1. Genes used in this study are listed in SI Appendix, Table S5.

Molecular Cloning.

Protein-coding sequences from Arabidopsis thaliana and Selaginella moellendorffii root cDNA were amplified using primers listed in SI Appendix, Table S2. All transgenic constructs were assembled using Modular GreenGate technology (51). If protein coding sequences contain BSA sites, they were replaced as described in ref. 52. SI Appendix, Table S3 lists all modules and constructs used in this study, while SI Appendix, Table S4 details the module assembly strategies. For plant transformation, Agrobacterium tumefaciens GV3101 was used, and Arabidopsis thaliana plants were transformed via the floral dip method. Heterozygous T2 or homozygous T3 lines were used for subsequent analyses.

Histology Staining.

Root samples were collected from the root junction region (0.5 to 1 cm) for plastic embedding. Plastic embedding and cross-sectioning were performed according to the protocol described in ref. 53, using Technovit 7100 (Heraeus Kulzer, 64709003). Sections (5 to 8 µm thick) were stained with 0.1% toluidine blue (Sigma, T3260) and imaged using an Olympus BX51 microscope equipped with a digital imaging system.

RNA-FISH and Confocal Microscopy.

HCR RNA-FISH in the root of Selaginella moellendorffii is conducted as mentioned in refs. 24, 34, and 54. Gene probes were designed and synthesized by Molecular Instruments (www.molecularinstruments.com). Leica Stellaris 5 LIAchroic upright confocal was used for acquiring the confocal images with specific settings. Fluorophores were imaged using 633-nm excitation and 650 to 755 nm detection, while Calcofluor White was imaged using 405-nm excitation and 410 to 525 nm detection.

RNA Sequence and Gene Cluster Trend Analysis.

Paired-end 150 bp RNA sequencing was performed by BGI. Raw data processing and analysis were conducted using the Galaxy platform (https://usegalaxy.be/) (55). Sequence adapters were trimmed using Trimmomatic (default parameters), followed by quality assessment with FastQC. Reads were aligned to the TAIR10 reference genome (for Arabidopsis thaliana) and Genome Selaginella moellendorffii v1.0 using Salmon (quant mode, default parameters). Transcript abundance estimation and read counting were performed with tximport, and differential gene expression analysis was conducted using DESeq2 (Dataset S1). Gene cluster trend analysis was done using Mfuzz package (v2.68.0) with time-course gene expression matrix as input (Dataset S1).

Statistical Analyses.

All statistical analyses, data quantification, and visualization were performed in R (v4.0.3). Boxplots were generated using ggplot2 (v3.4.3). Count data were analyzed by fitting generalized linear (mixed) models with Poisson (stats package), quasi-Poisson (stats package), or negative binomial (stats package) distributions. Model selection was based on residual diagnostics performed using the DHARMa package (v0.4.6) with simulation-based approaches. Statistical inferences were drawn using the emmeans package (v1.5.5-1), with P-values from pairwise comparisons adjusted via the Holm–Bonferroni method. Significant differences (α = 0.05) were visualized using compact letter displays generated with the multcomp package (v1.4-16). Complete datasets and statistical test results are provided in Dataset S2.

Cell Number Quantification and Image analyses.

Cell number quantification was performed using Fiji (v2.3.0; https://fiji.sc/) (56). For each genotype, we analyzed 5 to 20 root cross-sections obtained from independent plants. While 2 to 3 independent biological replicates were performed, data from a single representative experiment are shown here.

Phylogenetic and Protein Alignment Analysis.

Representative subfamily members of the CKX or IAA gene families from Arabidopsis thaliana or Selaginella moellendorffii were used as query sequences for BLASTP searches (E-value < 1 × 10⁻⁵) against protein databases of multiple species. Retrieved orthologs were aligned using MAFFT, with sequences lacking a complete domain excluded. Candidate orthologs were then realigned to ensure only sequences with intact domains were retained. For phylogenetic analysis, the sequences were aligned using IQ-TREE, followed by Maximum Likelihood (ML) tree reconstruction with 1,000 bootstrap replicates to assess branch support.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Data, Materials, and Software Availability

Raw RNA-seq data can be accessed at NCBI with GEO number GSE301807 (57). All other data are included in the article and/or supporting information.