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.

Three mutations repurpose a plant karrikin receptor to a strigolactone receptor.

Uncovering the basis of small-molecule hormone receptors' evolution is paramount to a complete understanding of how protein structure drives function. In plants, hormone receptors for strigolactones are well suited to evolutionary inquiries because closely related homologs have different ligand preferences. More importantly, because of facile plant transgenic systems, receptors can be swapped and quickly assessed functionally in vivo. Here, we show that only three mutations are required to turn the nonstrigolactone receptor, KAI2, into a receptor that recognizes the plant hormone strigolactone. This modified receptor still retains its native function to perceive KAI2 ligands. Our directed evolution studies indicate that only a few keystone mutations are required to increase receptor promiscuity of KAI2, which may have implications for strigolactone receptor evolution in parasitic plants.

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.

A structural homologue of the plant receptor D14 mediates responses to strigolactones in the fungal phytopathogen Cryphonectria parasitica.

Strigolactones (SLs) are plant hormones and important signalling molecules required to promote arbuscular mycorrhizal (AM) symbiosis. While in plants an α/ß-hydrolase, DWARF14 (D14), was shown to act as a receptor that binds and cleaves SLs, the fungal receptor for SLs is unknown. Since AM fungi are currently not genetically tractable, in this study, we used the fungal pathogen Cryphonectria parasitica, for which gene deletion protocols exist, as a model, as we have previously shown that it responds to SLs. By means of computational, biochemical and genetic analyses, we identified a D14 structural homologue, CpD14. Molecular homology modelling and docking support the prediction that CpD14 interacts with and hydrolyses SLs. The recombinant CpD14 protein shows α/ß hydrolytic activity in vitro against the SLs synthetic analogue GR24; its enzymatic activity requires an intact Ser/His/Asp catalytic triad. CpD14 expression in the d14-1 loss-of-function Arabidopsis thaliana line did not rescue the plant mutant phenotype. However, gene inactivation by knockout homologous recombination reduced fungal sensitivity to SLs. These results indicate that CpD14 is involved in SLs responses in C. parasitica and strengthen the role of SLs as multifunctional molecules acting in plant-microbe interactions.

Noncanonical Strigolactone Analogues Highlight Selectivity for Stimulating Germination in Two Phelipanche ramosa Populations.

Strigolactones (SLs) are plant hormones exuded in the rhizosphere with a signaling role for the development of arbuscular mycorrhizal (AM) fungi and as stimulants of seed germination of the parasitic weeds Orobanche, Phelipanche, and Striga, the most threatening weeds of major crops worldwide. Phelipanche ramosa is present mainly on rape, hemp, and tobacco in France. P. ramosa 2a preferentially attacks hemp, while P. ramosa 1 attacks rapeseed. The recently isolated cannalactone (14) from hemp root exudates has been characterized as a noncanonical SL that selectively stimulates the germination of P. ramosa 2a seeds in comparison with P. ramosa 1. In the present work, (-)-solanacol (5), a canonical orobanchol-type SL exuded by tobacco and tomato, was established to possess a remarkable selective germination stimulant activity for P. ramosa 2a seeds. Two cannalactone analogues, named (±)-SdL19 and (±)-SdL118, have been synthesized. They have an unsaturated acyclic carbon chain with a tertiary hydroxy group and a methyl or a cyclopropyl group instead of a cyclohexane A-ring, respectively. (±)-SdL analogues are able to selectively stimulate P. ramosa 2a, revealing that these minimal structural elements are key for this selective bioactivity. In addition, (±)-SdL19 is able to inhibit shoot branching in Pisum sativum and Arabidopsis thaliana and induces hyphal branching in the AM fungus Rhizophagus irregularis, like SLs.

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 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.

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.

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.

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.

Strigolactones stimulate internode elongation independently of gibberellins.

Strigolactone (SL) mutants in diverse species show reduced stature in addition to their extensive branching. Here, we show that this dwarfism in pea (Pisum sativum) is not attributable to the strong branching of the mutants. The continuous supply of the synthetic SL GR24 via the root system using hydroponics can restore internode length of the SL-deficient rms1 mutant but not of the SL-response rms4 mutant, indicating that SLs stimulate internode elongation via RMS4. Cytological analysis of internode epidermal cells indicates that SLs control cell number but not cell length, suggesting that SL may affect stem elongation by stimulating cell division. Consequently, SLs can repress (in axillary buds) or promote (in the stem) cell division in a tissue-dependent manner. Because gibberellins (GAs) increase internode length by affecting both cell division and cell length, we tested if SLs stimulate internode elongation by affecting GA metabolism or signaling. Genetic analyses using SL-deficient and GA-deficient or DELLA-deficient double mutants, together with molecular and physiological approaches, suggest that SLs act independently from GAs to stimulate internode elongation.

Antagonistic action of strigolactone and cytokinin in bud outgrowth control.

Cytokinin (CK) has long been implicated as a promoter of bud outgrowth in plants, but exactly how this is achieved in coordination with other plant hormones is unclear. The recent discovery of strigolactones (SLs) as the long-sought branch-inhibiting hormone allowed us to test how CK and SL coordinately regulate bud outgrowth in pea (Pisum sativum). We found that SL-deficient plants are more sensitive to stimulation of bud growth by low concentrations of locally applied CK than wild-type plants. Furthermore, in contrast with SL mutant plants, buds of wild-type plants are almost completely resistant to stimulation by CK supplied to the vasculature. Regardless of whether the exogenous hormones were supplied locally or to the xylem stream, SL and CK acted antagonistically on bud outgrowth. These data suggest that SLs do not affect the delivery of CK to axillary buds and vice versa. Rather, these data combined with dose-response experiments suggest that SLs and CK can act directly in buds to control their outgrowth. These hormones may converge at a common point in the bud outgrowth regulatory pathway. The expression of pea BRANCHED1, a TCP transcription factor expressed strongly in buds and thought to act downstream of SLs in shoot branching, is regulated by CK and SL without a requirement for protein synthesis and in a manner that correlates with observed bud growth responses.

The pea TCP transcription factor PsBRC1 acts downstream of Strigolactones to control shoot branching.

The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated. The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application. The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes. The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine. The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth. However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth. In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant. Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1. This phenotype indicates a localized regulation of branching at these nodes specific to pea.

Structure-activity relationship studies of strigolactone-related molecules for branching inhibition in garden pea: molecule design for shoot branching.

Initially known for their role in the rhizosphere in stimulating the seed germination of parasitic weeds such as the Striga and Orobanche species, and later as host recognition signals for arbuscular mycorrhizal fungi, strigolactones (SLs) were recently rediscovered as a new class of plant hormones involved in the control of shoot branching in plants. Herein, we report the synthesis of new SL analogs and, to our knowledge, the first study of SL structure-activity relationships for their hormonal activity in garden pea (Pisum sativum). Comparisons with their action for the germination of broomrape (Phelipanche ramosa) are also presented. The pea rms1 SL-deficient mutant was used in a SL bioassay based on axillary bud length after direct SL application on the bud. This assay was compared with an assay where SLs were fed via the roots using hydroponics and with a molecular assay in which transcript levels of BRANCHED1, the pea homolog of the maize TEOSINTE BRANCHED1 gene were quantified in axillary buds only 6 h after application of SLs. We have demonstrated that the presence of a Michael acceptor and a methylbutenolide or dimethylbutenolide motif in the same molecule is essential. It was established that the more active analog 23 with a dimethylbutenolide as the D-ring could be used to control the plant architecture without strongly favoring the germination of P. ramosa seeds. Bold numerals refer to numbers of compounds.

The ecologically relevant genetics of plant-plant interactions.

Interactions among plants have been long recognized as a major force driving plant community dynamics and crop yield. Surprisingly, our knowledge of the ecological genetics associated with variation of plant-plant interactions remains limited. In this opinion article by scientists from complementary disciplines, the international PLANTCOM network identified four timely questions to foster a better understanding of the mechanisms mediating plant assemblages. We propose that by identifying the key relationships among phenotypic traits involved in plant-plant interactions and the underlying adaptive genetic and molecular pathways, while considering environmental fluctuations at diverse spatial and time scales, we can improve predictions of genotype-by-genotype-by-environment interactions and modeling of productive and stable plant assemblages in wild habitats and crop fields.

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.

Synthesis of Profluorescent Strigolactone Probes for Biochemical Studies.

In this chapter, we will describe a method we set up to synthesize two profluorescent strigolactone (SL) mimic probes (GC240 and GC242) and the optimized protocols developed to study the enzymatic properties of various strigolactone receptors. The Arabidopsis AtD14 SL receptor is used here as a model for this purpose.

A Phelipanche ramosa KAI2 protein perceives strigolactones and isothiocyanates enzymatically.

Phelipanche ramosa is an obligate root-parasitic weed that threatens major crops in central Europe. In order to germinate, it must perceive various structurally divergent host-exuded signals, including isothiocyanates (ITCs) and strigolactones (SLs). However, the receptors involved are still uncharacterized. Here, we identify five putative SL receptors in P. ramosa and show that PrKAI2d3 is involved in the stimulation of seed germination. We demonstrate the high plasticity of PrKAI2d3, which allows it to interact with different chemicals, including ITCs. The SL perception mechanism of PrKAI2d3 is similar to that of endogenous SLs in non-parasitic plants. We provide evidence that PrKAI2d3 enzymatic activity confers hypersensitivity to SLs. Additionally, we demonstrate that methylbutenolide-OH binds PrKAI2d3 and stimulates P. ramosa germination with bioactivity comparable to that of ITCs. This study demonstrates that P. ramosa has extended its signal perception system during evolution, a fact that should be considered for the development of specific and efficient biocontrol methods.