Fern-tastic discoveries: CLAVATA3 and WOX signaling pathways in fern gametophyte development

Abstract

Developmental processes and responses to environmental stimuli are governed by plant-specific signals, such as peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family (Cock & McCormick, 2001). They are involved in processes like stomata closure, vascular development, and meristem homeostasis (reviewed in Fletcher, 2020). The CLE signaling pathway is one such pathway; it maintains the stem cell population in the shoot apical meristem in Arabidopsis. The homeobox transcription factor WUSCHEL (WUS) is expressed in cells of the meristem organizing center, then moves to stem cells in the outer layers of the meristem, where it activates the expression of the CLE peptide CLV3 (Carles & Fletcher, 2003). The mature peptide is secreted and binds to the extracellular leucine-rich repeats of receptor-like kinase CLV1, triggering a signal cascade that represses WUS expression in the organizing center. A similar pathway also regulates shoot meristem homeostasis of other plants (e.g., Je et al., 2016; Suzaki et al., 2008).

CLE peptides are found across all land plant lineages, but they are absent in algae, suggesting that CLEs likely evolved in the last common ancestor of land plants (Whitewoods, 2021). However, while bryophyte genomes have both WUS and CLE genes, the genes function in separate processes (Hirakawa et al., 2020). Therefore, the prevalent hypothesis is that WOX-CLE signaling evolved after the divergence of bryophytes (Whitewoods, 2021). Kelley Renninger, first author of the highlighted publication and then a PhD student in Chi-Lien Cheng's group at the University of Iowa, decided to address the question of how and when WOX transcription factors were integrated into CLE-receptor signaling pathways during the evolution of land plants.

As ferns represent an evolutionary intermediate between bryophytes and flowering plants, the authors decided to use Ceratopteris richardii, a homosporous fern, for their study. Its mature sporophyte produces haploid spores, which germinate and grow into multicellular hermaphrodite or male gametophytes. Male gametophytes produce multiple antheridia that produce sperm. The hermaphrodite gametophyte develops one multicellular meristem, called the marginal meristem, and next to the meristem notch, the egg-bearing archegonia initiate (Figure 1a) (Geng et al., 2022). The Ceratopteris genome has five WOX genes (Nardmann & Werr, 2012) and CLE peptides, but the sequences encoding CLE peptides and their functions are unknown.

Based on BLASTp searches with the CLEs of Arabidopsis and those of the ferns Azolla filiculoides and Salvinia cucullata, the authors identified 11 loci in the Ceratopteris genome encoding CLEs. The C-terminal CLE motif represents the mature peptide. The CLE motif of CrCLV3 was highly similar to CLV3 from Arabidopsis, only differing at one residue. The authors used RT-qPCR to show that CrCLV3 was expressed in gametophytes when the marginal meristem was present, and that expression decreased at 14 h post fertilization, correlating with a loss of meristematic activity. RNA in situ hybridization showed that CLV3 was expressed in the marginal meristem in hermaphrodite gametophytes and in developing antheridia of both hermaphrodite and male gametophytes.

Hermaphrodite gametophytes of CrCLV3 knockdown lines contained fewer cells, were delayed in growth, and produced antheridia and archegonia less frequently, suggesting that CrCLV3 promotes cell proliferation in hermaphrodites and has a positive role in both antheridia and archegonia formation. Treatment with synthetic CrCLV3 peptide resulted in an increase in cell number and wider marginal meristems, while simultaneously inhibiting the production of antheridia and archegonia. The authors think this inhibition might be caused by meristem expansion, so that undifferentiated cells are not able to produce differentiated reproductive organs. When plants were pulse-dosed with CrCLV3 peptide during early growth, the peptide concentration that promoted cell proliferation also promoted antheridium development, supporting the idea that both the meristem and antheridia are promoted by the peptide. Exposure to CrCLV3p for 6 days or longer, within the first 8 days post-plating (dpp) spores, even induced the formation of a second marginal meristem (Figure 1b). The authors used aniline blue staining, which detects transient callose deposition marking frequently dividing marginal meristem cells. This showed an increase in the number of cells exhibiting marginal meristem identity, likely caused by CrCLV3 promoting cell division in this region.

The authors then asked if WOX genes played a role in this regulation in Ceratopteris. CrWOXA and CrWOXB were expressed in the gametophyte meristem, and gametophytes treated with CrCLV3p for 13 days showed significantly weaker expression of several WOX genes, including CrWOX13A, CrWOXA, and CrWOXB. However, when peptide treatment was limited to gametophytes at 8–10 days post-plating (dpp), only CrWOXA had weaker expression. CrCLV3p treatment also downregulated CrCLV3 expression in older stages of gametophyte development, suggesting that Ceratopteris gametophytes have a feedback mechanism to control the levels of CrCLV3p, but at earlier stages, cell proliferation at the meristem is not regulated by CrWOXA (Figure 1c).

The study provides the first evidence for WOX-CLE signaling in a seed-free plant, placing the evolution of this regulatory relationship earlier in land plant evolution. In Physcomitrella patens and Marchantia polymorpha, no influence of CLEs on WOX function and vice versa was found (Hirakawa et al., 2020; Sakakibara et al., 2014). As both of those species have gametophyte-dominant life cycles, it was hypothesized that WOX-CLE signaling was a derived trait restricted to the sporophyte generation in vascular plants. In Ceratopteris, a connection between the CLE and WOX genes was observed in the gametophyte generation. These findings support that a free-living gametophyte body can also utilize WOX-CLE signaling.