SUPPRESSOR OF MAX2 1-LIKE (SMXL) homologs are MAX2-dependent repressors of Physcomitrium patens growth.

SUPPRESSOR OF MAX2 (SMAX)1-LIKE (SMXL) proteins are a plant-specific clade of type I HSP100/Clp-ATPases. SMXL genes are present in virtually all land plant genomes. However, they have mainly been studied in angiosperms. In Arabidopsis (Arabidopsis thaliana), 3 functional SMXL subclades have been identified: SMAX1/SMXL2, SMXL345, and SMXL678. Of these, 2 subclades ensure endogenous phytohormone signal transduction. SMAX1/SMXL2 proteins are involved in KAI2 ligand (KL) signaling, while SMXL678 proteins are involved in strigolactone (SL) signaling. Many questions remain regarding the mode of action of these proteins, as well as their ancestral roles. We addressed these questions by investigating the functions of the 4 SMXL genes in the moss Physcomitrium patens. We demonstrate that PpSMXL proteins are involved in the conserved ancestral MAX2-dependent KL signaling pathway and negatively regulate growth. However, PpSMXL proteins expressed in Arabidopsis cannot replace SMAX1 or SMXL2 function in KL signaling, whereas they can functionally replace SMXL4 and SMXL5 and restore root growth. Therefore, the molecular functions of SMXL proteins are conserved, but their interaction networks are not. Moreover, the PpSMXLC/D clade positively regulates SL signal transduction in P. patens. Overall, our data reveal that SMXL proteins in moss mediate crosstalk between the SL and KL signaling pathways.

The Physcomitrium (Physcomitrella) patens PpKAI2L receptors for strigolactones and related compounds function via MAX2-dependent and -independent pathways.

In angiosperms, the α/ß hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (-)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (-)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.

Synthesis and signalling of strigolactone and KAI2-ligand signals in bryophytes.

Strigolactones (SLs), long known as butenolide rhizospheric signals, have been recognized since 2008 as a class of hormones regulating many aspects of plant development. Many authors also anticipate 'KAI2-ligand' (KL) as a novel class of phytohormones; however, this ligand remains elusive. Core genes of SL and KL pathways, first described in angiosperms, are found in all land plants and some even in green algae. This review reports current knowledge of these pathways in bryophytes. Data on the pathways mostly come from two models: the moss Physcomitrium patens and the liverwort Marchantia. Gene targeting methods have allowed functional analyses of both models. Recent work in Marchantia suggests that SLs' ancestral role was to recruit beneficial microbes as arbuscular mycorrhizal fungi. In contrast, the hormonal role of SLs observed in P. patens is probably a result of convergent evolution. Evidence for a functional KL pathway in both bryophyte models is very recent. Nevertheless, many unknowns remain and warrant a more extensive investigation of SL and KL pathways in various land plant lineages.

Phytohormone biosynthesis and signaling pathways of mosses.

KEY MESSAGE: Most known phytohormones regulate moss development. We present a comprehensive view of the synthesis and signaling pathways for the most investigated of these compounds in mosses, focusing on the model Physcomitrium patens. The last 50 years of research have shown that most of the known phytohormones are synthesized by the model moss Physcomitrium patens (formerly Physcomitrella patens) and regulate its development, in interaction with responses to biotic and abiotic stresses. Biosynthesis and signaling pathways are best described in P. patens for the three classical hormones auxins, cytokinins and abscisic acid. Furthermore, their roles in almost all steps of development, from early filament growth to gametophore development and sexual reproduction, have been the focus of much research effort over the years. Evidence of hormonal roles exist for ethylene and for CLE signaling peptides, as well as for salicylic acid, although their possible effects on development remain unclear. Production of brassinosteroids by P. patens is still debated, and modes of action for these compounds are even less known. Gibberellin biosynthesis and signaling may have been lost in P. patens, while gibberellin precursors such as ent-kaurene derivatives could be used as signals in a yet to discover pathway. As for jasmonic acid, it is not used per se as a hormone in P. patens, but its precursor OPDA appears to play a corresponding role in defense against abiotic stress. We have tried to gather a comprehensive view of the biosynthesis and signaling pathways for all these compounds in mosses, without forgetting strigolactones, the last class of plant hormones to be reported. Study of the strigolactone response in P. patens points to a novel signaling compound, the KAI2-ligand, which was likely employed as a hormone prior to land plant emergence.

Masks Start to Drop: Suppressor of MAX2 1-Like Proteins Reveal Their Many Faces.

Although the main players of the strigolactone (SL) signaling pathway have been characterized genetically, how they regulate plant development is still poorly understood. Of central importance are the SUPPRESSOR OF MAX2 1-LIKE (SMXL) proteins that belong to a family of eight members in Arabidopsis thaliana, of which one subclade is involved in SL signaling and another one in the pathway of the chemically related karrikins. Through proteasomal degradation of these SMXLs, triggered by either DWARF14 (D14) or KARRIKIN INSENSITIVE2 (KAI2), several physiological processes are controlled, such as, among others, shoot and root architecture, seed germination, and seedling photomorphogenesis. Yet another clade has been shown to be involved in vascular development, independently of the D14 and KAI2 actions and not relying on proteasomal degradation. Despite their role in several aspects of plant development, the exact molecular mechanisms by which SMXLs regulate them are not completely unraveled. To fill the major knowledge gap in understanding D14 and KAI2 signaling, SMXLs are intensively studied, making it challenging to combine all the insights into a coherent characterization of these important proteins. To this end, this review provides an in-depth exploration of the recent data regarding their physiological function, evolution, structure, and molecular mechanism. In addition, we propose a selection of future perspectives, focusing on the apparent localization of SMXLs in subnuclear speckles, as observed in transient expression assays, which we couple to recent advances in the field of biomolecular condensates and liquid-liquid phase separation.

Expansion of the Strigolactone Profluorescent Probes Repertory: The Right Probe for the Right Application.

Strigolactones (SLs) are intriguing phytohormones that not only regulate plant development and architecture but also interact with other organisms in the rhizosphere as root parasitic plants (Striga, Orobanche, and Phelipanche) and arbuscular mycorrhizal fungi. Starting with a pioneering work in 2003 for the isolation and identification of the SL receptor in parasitic weeds, fluorescence labeling of analogs has proven a major strategy to gain knowledge in SL perception and signaling. Here, we present novel chemical tools for understanding the SL perception based on the enzymatic properties of SL receptors. We designed different profluorescent SL Guillaume Clavé (GC) probes and performed structure-activity relationship studies on pea, Arabidopsis thaliana, and Physcomitrium (formerly Physcomitrella) patens. The binding of the GC probes to PsD14/RMS3, AtD14, and OsD14 proteins was tested. We demonstrated that coumarin-based profluorescent probes were highly bioactive and well-adapted to dissect the enzymatic properties of SL receptors in pea and a resorufin profluorescent probe in moss, contrary to the commercially available fluorescein profluorescent probe, Yoshimulactone Green (YLG). These probes offer novel opportunities for the studies of SL in various plants.

Methods for Medium-Scale Study of Biological Effects of Strigolactone-Like Molecules on the Moss Physcomitrium (Physcomitrella) patens.

As a bryophyte and model plant, the moss Physcomitrium (Physcomitrella) patens (P. patens) is particularly well adapted to hormone evolution studies. Gene targeting through homologous recombination or CRISPR-Cas9 system, genome sequencing, and numerous transcriptomic datasets has allowed for molecular genetics studies and much progress in Evo-Devo knowledge. As to strigolactones, like for other hormones, both phenotypical and transcriptional responses can be studied, in both WT and mutant plants. However, as in any plant species, medium- to large-scale phenotype characterization is necessary, owing to the general high phenotypic variability. Therefore, many biological replicates are required. This may translate to large amount of the investigated compounds, particularly expensive (or difficult to synthesize) in the case of strigolactones. These issues prompted us to improve existing methods to limit the use of scarce/expensive compounds, as well as to simplify subsequent measures/sampling of P. patens. We hence scaled up well-tried experiments, in order to increment the number of tested genotypes in one given experiment.In this chapter, we will describe three methods we set up to study the response to strigolactones and related compounds in P. patens.