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.

Lessons from a century of apical dominance research.

The process of apical dominance by which the apical bud/shoot tip of the plant inhibits the outgrowth of axillary buds located below has been studied for more than a century. Different approaches were used over time, with first the physiology era, the genetic era, and then the multidisciplinary era. During the physiology era, auxin was thought of as the master regulator of apical dominance acting indirectly to inhibit bud outgrowth via unknown secondary messenger(s). Potential candidates were cytokinin (CK) and abscisic acid (ABA). The genetic era with the screening of shoot branching mutants in different species revealed the existence of a novel carotenoid-derived branching inhibitor and led to the significant discovery of strigolactones (SLs) as a novel class of plant hormones. The re-discovery of the major role of sugars in apical dominance emerged from modern physiology experiments and involves ongoing work with genetic material affected in sugar signalling. As crops and natural selection rely on the emergent properties of networks such as this branching network, future work should explore the whole network, the details of which are critical but not individually sufficient to solve the 'wicked problems' of sustainable food supply and climate change.

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.

Structural and functional analyses explain Pea KAI2 receptor diversity and reveal stereoselective catalysis during signal perception.

KAI2 proteins are plant α/ß hydrolase receptors which perceive smoke-derived butenolide signals and endogenous, yet unidentified KAI2-ligands (KLs). The number of functional KAI2 receptors varies among species and KAI2 gene duplication and sub-functionalization likely plays an adaptative role by altering specificity towards different KLs. Legumes represent one of the largest families of flowering plants and contain many agronomic crops. Prior to their diversification, KAI2 underwent duplication resulting in KAI2A and KAI2B. Here we demonstrate that Pisum sativum KAI2A and KAI2B are active receptors and enzymes with divergent ligand stereoselectivity. KAI2B has a higher affinity for and hydrolyses a broader range of substrates including strigolactone-like stereoisomers. We determine the crystal structures of PsKAI2B in apo and butenolide-bound states. The biochemical, structural, and mass spectra analyses of KAI2s reveal a transient intermediate on the catalytic serine and a stable adduct on the catalytic histidine, confirming its role as a bona fide enzyme. Our work uncovers the stereoselectivity of ligand perception and catalysis by diverged KAI2 receptors and proposes adaptive sensitivity to KAR/KL and strigolactones by KAI2B.

Strigolactones (SLs) modulate the plastochron by regulating KLUH (KLU) transcript abundance in Arabidopsis.

The timing of leaf emergence at the shoot apical meristem, or plastochron, is highly regulated in plants. Among the genes known to regulate the plastochron in Arabidopsis (Arabidopsis thaliana), KLUH (KLU), orthologous to the rice (Oryza sativa) PLASTOCHRON1, encodes the cytochrome P450 CYP78A5, and is thought to act through generation of a still unknown mobile signal. As klu mutants display not only a short plastochron but also a branching phenotype reminiscent of strigolactone (SL) mutants, we investigated whether KLU/CYP78A5 is involved in SL biosynthesis. We combined a genetic approach, a parasitic plant seed germination bioassay to test klu root exudates, and analysis of transcript abundances of SL-biosynthesis genes in the Arabidopsis klu mutants. We demonstrate that KLU is not involved in the SL-biosynthesis pathway. Moreover, this work allowed us to uncover a new role for SL during Arabidopsis development in modulating plastochron via a KLU-dependent pathway. Globally our data reveal that KLU is required for plastochron-specific SL responses, a first indication of crosstalk between SL and the KLU-derived signal.

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.

Integration of the SMXL/D53 strigolactone signalling repressors in the model of shoot branching regulation in Pisum sativum.

DWARF53 (D53) in rice (Oryza sativa) and its homologs in Arabidopsis (Arabidopsis thaliana), SUPPRESSOR OF MAX2-LIKE 6 (SMXL6), SMXL7 and SMXL8, are well established negative regulators of strigolactone (SL) signalling in shoot branching regulation. Little is known of pea (Pisum sativum) homologs and whether D53 and related SMXLs are specific to SL signalling pathways. Here, we identify two allelic pea mutants, dormant3 (dor3), and demonstrate through gene mapping and sequencing that DOR3 corresponds to a homolog of D53 and SMXL6/SMXL7, designated PsSMXL7. Phenotype analysis, gene expression, protein and hormone quantification assays were performed to determine the role of PsSMXL7 in regulation of bud outgrowth and the role of PsSMXL7 and D53 in integrating SL and cytokinin (CK) responses. Like D53 and related SMXLs, we show that PsSMXL7 can be degraded by SL and induces feedback upregulation of PsSMXL7 transcript. Here we reveal a system conserved in pea and rice, whereby CK also upregulates PsSMXL7/D53 transcripts, providing a clear mechanism for SL and CK cross-talk in the regulation of branching. To further deepen our understanding of the branching network in pea, we provide evidence that SL acts via PsSMXL7 to modulate auxin content via PsAFB5, which itself regulates expression of SL biosynthesis genes. We therefore show that PsSMXL7 is key to a triple hormone network involving an auxin-SL feedback mechanism and SL-CK cross-talk.

Methods for Phenotyping Shoot Branching and Testing Strigolactone Bioactivity for Shoot Branching in Arabidopsis and Pea.

Shoot branching is a highly variable trait that evolves during plant development and is influenced by environmental and endogenous cues such as hormones. In particular, strigolactones (SLs) are hormones that play a key role in the control of shoot branching. Branch primordia, axillary buds formed in the leaf axils, display differential growth depending on their position in the plant and also respond to hormone signaling. In this chapter, we will describe how to quantify the degree of shoot branching in two plant model species, Arabidopsis and pea, commonly used to decipher the control of this complex trait. We will also propose several methods to perform treatments of SL or SL analogs, to investigate their bioactivity and effect on the shoot branching patterns of plants of different genotypes.

Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling.

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P. patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors.

The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data.

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.

Validated Method for Strigolactone Quantification by Ultra High-Performance Liquid Chromatography - Electrospray Ionisation Tandem Mass Spectrometry Using Novel Deuterium Labelled Standards.

INTRODUCTION: Strigolactones (SLs) are important plant hormones. They are difficult to analyse because they occur in very small concentrations especially in comparison with other plant hormones and other substances can interfere with their detection. OBJECTIVE: To develop a procedure for the extraction, purification and quantification of SLs from plant roots. METHODOLOGY: Samples were prepared by extraction of plant root tissues with ethyl acetate. Then the extracts were further purified with silica column chromatography. The natural SLs in the final extracts were quantified using novel deuterium labelled SLs. The results of the methodology were compared with those of the procedure of Yoneyama and coworkers. RESULTS: This procedure required about 1-g root samples to detect and quantify simultaneously the SLs (orobanchyl acetate and fabacyl acetate) concentration with high reliability. CONCLUSION: A method was developed for determining endogenous fabacyl acetate and orobanchyl acetate in plant tissue based on novel deuterium labelled standards. A method of orobanchol quantification using a synthetic SL GR24 as internal standard was proposed. Copyright © 2017 John Wiley & Sons, Ltd.

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop.

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner.

Phloem Transport of the Receptor DWARF14 Protein Is Required for Full Function of Strigolactones.

The cell-to-cell transport of signaling molecules is essential for multicellular organisms to coordinate the action of their cells. Recent studies identified DWARF14 (D14) as a receptor of strigolactones (SLs), molecules that act as plant hormones and inhibit shoot branching. Here, we demonstrate that RAMOSUS3, a pea ortholog of D14, works as a graft-transmissible signal to suppress shoot branching. In addition, we show that D14 protein is contained in phloem sap and transported through the phloem to axillary buds in rice. SLs are not required for the transport of D14 protein. Disruption of D14 transport weakens the suppression of axillary bud outgrowth of rice. Taken together, we conclude that the D14 protein works as an intercellular signaling molecule to fine-tune SL function. Our findings provide evidence that the intercellular transport of a receptor can regulate the action of plant hormones.

Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 in Physcomitrella patens.

Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organism Physcomitrella patens has experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families in Physcomitrella is substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.

An histidine covalent receptor and butenolide complex mediates strigolactone perception.

Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/ß-hydrolases, and is known to hydrolyze the bond between the ABC lactone and the D ring. Here we characterized the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using new profluorescent probes with strigolactone-like bioactivity, we found that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We demonstrated the formation of a covalent RMS3-D-ring complex, essential for bioactivity, in which the D ring was attached to histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception in which the receptor performs an irreversible enzymatic reaction to generate its own ligand.

Structural modelling and transcriptional responses highlight a clade of PpKAI2-LIKE genes as candidate receptors for strigolactones in Physcomitrella patens.

MAIN CONCLUSION: A set of PpKAI2 - LIKE paralogs that may encode strigolactone receptors in Physcomitrella patens were identified through evolutionary, structural, and transcriptional analyses, suggesting that strigolactone perception may have evolved independently in basal land plants in a similar manner as spermatophytes. Carotenoid-derived compounds known as strigolactones are a new class of plant hormones that modulate development and interactions with parasitic plants and arbuscular mycorrhizal fungi. The strigolactone receptor protein DWARF14 (D14) belongs to the α/ß hydrolase family. D14 is closely related to KARRIKIN INSENSITIVE2 (KAI2), a receptor of smoke-derived germination stimulants called karrikins. Strigolactone and karrikin structures share a butenolide ring that is necessary for bioactivity. Charophyte algae and basal land plants produce strigolactones that influence their development. However phylogenetic studies suggest that D14 is absent from algae, moss, and liverwort genomes, raising the question of how these basal plants perceive strigolactones. Strigolactone perception during seed germination putatively evolved in parasitic plants through gene duplication and neofunctionalization of KAI2 paralogs. The moss Physcomitrella patens shows an increase in KAI2 gene copy number, similar to parasitic plants. In this study we investigated whether P. patens KAI2-LIKE (PpKAI2L) genes may contribute to strigolactone perception. Based on phylogenetic analyses and homology modelling, we predict that a clade of PpKAI2L proteins have enlarged ligand-binding cavities, similar to D14. We observed that some PpKAI2L genes have transcriptional responses to the synthetic strigolactone GR24 racemate or its enantiomers. These responses were influenced by light and dark conditions. Moreover, (+)-GR24 seems to be the active enantiomer that induces the transcriptional responses of PpKAI2L genes. We hypothesize that members of specific PpKAI2L clades are candidate strigolactone receptors in moss.

Strigolactone biosynthesis and signaling in plant development.

Strigolactones (SLs), first identified for their role in parasitic and symbiotic interactions in the rhizosphere, constitute the most recently discovered group of plant hormones. They are best known for their role in shoot branching but, more recently, roles for SLs in other aspects of plant development have emerged. In the last five years, insights into the SL biosynthetic pathway have also been revealed and several key components of the SL signaling pathway have been identified. Here, and in the accompanying poster, we summarize our current understanding of the SL pathway and discuss how this pathway regulates plant development.

Strigolactones inhibit caulonema elongation and cell division in the moss Physcomitrella patens.

In vascular plants, strigolactones (SLs) are known for their hormonal role and for their role as signal molecules in the rhizosphere. SLs are also produced by the moss Physcomitrella patens, in which they act as signaling factors for controlling filament extension and possibly interaction with neighboring individuals. To gain a better understanding of SL action at the cellular level, we investigated the effect of exogenously added molecules (SLs or analogs) in moss growth media. We used the previously characterized Ppccd8 mutant that is deficient in SL synthesis and showed that SLs affect moss protonema extension by reducing caulonema cell elongation and mainly cell division rate, both in light and dark conditions. Based on this effect, we set up bioassays to examine chemical structure requirements for SL activity in moss. The results suggest that compounds GR24, GR5, and 5-deoxystrigol are active in moss (as in pea), while other analogs that are highly active in the control of pea branching show little activity in moss. Interestingly, the karrikinolide KAR1, which shares molecular features with SLs, did not have any effect on filament growth, even though the moss genome contains several genes homologous to KAI2 (encoding the KAR1 receptor) and no canonical homologue to D14 (encoding the SL receptor). Further studies should investigate whether SL signaling pathways have been conserved during land plant evolution.

Multiple pathways regulate shoot branching.

Shoot branching patterns result from the spatio-temporal regulation of axillary bud outgrowth. Numerous endogenous, developmental and environmental factors are integrated at the bud and plant levels to determine numbers of growing shoots. Multiple pathways that converge to common integrators are most probably involved. We propose several pathways involving not only the classical hormones auxin, cytokinins and strigolactones, but also other signals with a strong influence on shoot branching such as gibberellins, sugars or molecular actors of plant phase transition. We also deal with recent findings about the molecular mechanisms and the pathway involved in the response to shade as an example of an environmental signal controlling branching. We propose the TEOSINTE BRANCHED1, CYCLOIDEA, PCF transcription factor TB1/BRC1 and the polar auxin transport stream in the stem as possible integrators of these pathways. We finally discuss how modeling can help to represent this highly dynamic system by articulating knowledges and hypothesis and calculating the phenotype properties they imply.

New strigolactone analogs as plant hormones with low activities in the rhizosphere.

Strigolactones (SLs) are known not only as plant hormones, but also as rhizosphere signals for establishing symbiotic and parasitic interactions. The design of new specific SL analogs is a challenging goal in understanding the basic plant biology and is also useful to control plant architectures without favoring the development of parasitic plants. Two different molecules (23 (3'-methyl-GR24), 31 (thia-3'-methyl-debranone-like molecule)) already described, and a new one (AR36), for which the synthesis is presented, are biologically compared with the well-known GR24 and the recently identified CISA-1. These different structures emphasize the wide range of parts attached to the D-ring for the bioactivity as a plant hormone. These new compounds possess a common dimethylbutenolide motif but their structure varies in the ABC part of the molecules: 23 has the same ABC part as GR24, while 31 and AR36 carry, respectively, an aromatic ring and an acyclic carbon chain. Detailed information is given for the bioactivity of such derivatives in strigolactone synthesis or in perception mutant plants (pea rms1 and rms4, Arabidopsis max2 and, max4) for different hormonal functions along with their action in the rhizosphere on arbuscular mycorrhizal hyphal growth and parasitic weed germination.