In flowering plants, EMS1 (Excess Microsporocytes 1) perceives TPD1 (Tapetum Determinant 1) to specify tapeta, the last somatic cell layer nurturing pollen development. However, the signaling components downstream of EMS1 are relatively unknown. Here, we use a molecular complementation approach to investigate the downstream components in EMS1 signaling. We show that the EMS1 intracellular domain is functionally interchangeable with that of the brassinosteroid receptor BRI1 (Brassinosteroid Insensitive 1). Furthermore, expressing EMS1 together with TPD1 in the BRI1 expression domain could partially rescue bri1 phenotypes, and led to the dephosphorylation of BES1, a hallmark of active BRI1 signaling. Conversely, expressing BRI1 in the EMS1 expression domain could partially rescue ems1 phenotypes. We further show that PpEMS1 and PpTPD1 from the early land plant Physcomitrella patens could completely rescue ems1 and tpd1 phenotypes, respectively. We propose that EMS1 and BRI1 have evolved distinct extracellular domains to control different biological processes but can act via a common intracellular signaling pathway.

Endoplasmic Reticulum-Associated Degradation (ERAD) is one of the major processes in maintaining protein homeostasis. Class I α-mannosidases MNS4 and MNS5 are involved in the degradation of misfolded variants of the heavily glycosylated proteins, playing an important role for glycan-dependent ERAD in planta. MNS4 and MNS5 reportedly have functional redundancy, meaning that only the loss of both MNS4 and MNS5 shows phenotypes. However, MNS4 is a membrane-associated protein while MNS5 is a soluble protein, and both can localize to the endoplasmic reticulum (ER). Furthermore, MNS4 and MNS5 differentially demannosylate the glycoprotein substrates. Importantly, we found that their gene expression patterns are complemented rather than overlapped. This raises the question of whether they indeed work redundantly, warranting a further investigation. Here, we conducted an exhaustive genetic screen for a suppressor of the bri1-5, a brassinosteroid (BR) receptor mutant with its receptor downregulated by ERAD, and isolated sbi3, a suppressor of bri1-5 mutant named after sbi1 (suppressor of bri1). After genetic mapping together with whole-genome re-sequencing, we identified a point mutation G343E in AT1G27520 (MNS5) in sbi3. Genetic complementation experiments confirmed that sbi3 was a loss-of-function allele of MNS5. In addition, sbi3 suppressed the dwarf phenotype of bri1-235 in the proteasome-independent ERAD pathway and bri1-9 in the proteasome-dependent ERAD pathway. Importantly, sbi3 could only affect BRI1/bri1 with kinase activities such that it restored BR-sensitivities of bri1-5, bri1-9, and bri1-235 but not null bri1. Furthermore, sbi3 was less tolerant to tunicamycin and salt than the wild-type plants. Thus, our study uncovers a non-redundant function of MNS5 in the regulation of ERAD as well as plant growth and ER stress response, highlighting a need of the traditional forward genetic approach to complement the T-DNA or CRISPR-Cas9 systems on gene functional study.

Phytohormones play an important role in the adaptive evolution of terrestrial plants. Brassinosteroids (BRs) are essential hormones that regulate multiple aspects of plant growth and development in angiosperms, but the presence of BR signaling in non-seed plants such as ferns remains unknown. Here, we found that BR promotes the growth of Ceratopteris richardii, while the synthetic inhibitor PCZ inhibits the growth. Using full-length transcriptome sequencing, we identified four BRI1-like receptors. By constructing chimeric receptors, we found that the kinase domains of these four receptors could trigger BR downstream signaling. Further, the extracellular domains of two receptors were functionally interchangeable with that of BRI1. In addition, we identified a co-receptor, CtSERK1, that could phosphorylate with CtBRL2s in vitro. Together, these proved the presence of a receptor complex in Ceratopteris richardii that might perceive BR and activate downstream hormone signaling. Our results shed light on the biological and molecular mechanisms of BR signaling in ferns and the role of BR hormone signaling in the adaptive evolution of terrestrial plants.

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Summary Brassinosteroid (BR) signaling has been identified from the ligand BRs sensed by the receptor Brassinosteroid Insensitive 1 (BRI1) to the final activation of Brassinozole Resistant 1/bri1 EMS‐Suppressor 1 through a series of transduction events. Extensive studies have been conducted to characterize the role of BR signaling in various biological processes. Our previous study has shown that Excess Microsporocytes 1 (EMS1) and BRI1 control different aspects of plant growth and development via conserved intracellular signaling. Here, we reveal that another receptor, NILR1, can complement the bri1 mutant in the absence of BRs, indicating a pathway that resembles BR signaling activated by NILR1. Genetic analysis confirms the intracellular domains of NILR1, BRI1 and EMS1 have a common signal output. Furthermore, we demonstrate that NILR1 and BRI1 share the coreceptor BRI1 Associated Kinase 1 and substrate BSKs. Notably, the NILR1‐mediated downstream pathway is conserved across land plants. In summary, we provide evidence for the signaling cascade of NILR1, suggesting pan‐brassinosteroid signaling initiated by a group of distant receptor–ligand pairs in land plants.