Vitislactone, a non-canonical strigolactone exudated by grapevine rootstocks in response to nitrogen starvation.

Strigolactones are compounds produced by plant roots in response to nutrient deficiency, acting both as local and systemic signals to control development and nutrition. Strigolactones are exuded in the rhizosphere to positively influence interactions with beneficial microbes. LC-MS/MS analysis shows that two genetically distinct grapevine rootstocks exudate one or two non-canonical strigolactones when subjected to low nitrogen conditions. Gene expression profiles and orobanche seed germination assays confirm that the biosynthesis and exudation of non-canonical compounds is the preferred pathway. The first compound, corresponding to heliolactone or 6-epi-heliolactone, is only exuded by the rootstock showing lower shoot branching and a higher level of mycorrhization with arbuscular mycorrhizal fungi. The structure of the second compound exuded by both rootstocks was identified by NMR and LC-MS/MS analysis. It is a non-canonical strigolactone, which has never been identified in another species. This first identification of a natural compound with the potential to stimulate beneficial root-microbe interactions in grapevines opens new perspectives in viticulture.

Wild Helianthus species: A reservoir of resistance genes for sustainable pyramidal resistance to broomrape in sunflower.

Orobanche cumana Wall., sunflower broomrape, is one of the major pests for the sunflower crop. Breeding for resistant varieties in sunflower has been the most efficient method to control this parasitic weed. However, more virulent broomrape populations continuously emerge by overcoming genetic resistance. It is thus essential to identify new broomrape resistances acting at various stages of the interaction and combine them to improve resistance durability. In this study, 71 wild sunflowers and wild relatives accessions from 16 Helianthus species were screened in pots for their resistance to broomrape at the late emergence stage. From this initial screen, 18 accessions from 9 species showing resistance, were phenotyped at early stages of the interaction: the induction of broomrape seed germination by sunflower root exudates, the attachment to the host root and the development of tubercles in rhizotron assays. We showed that wild Helianthus accessions are an important source of resistance to the most virulent broomrape races, affecting various stages of the interaction: the inability to induce broomrape seed germination, the development of incompatible attachments or necrotic tubercles, and the arrest of emerged structure growth. Cytological studies of incompatible attachments showed that several cellular mechanisms were shared among resistant Helianthus species.

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.

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.

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.

Strigolactone-Like Bioactivity via Parasitic Plant Germination Bioassay.

Strigolactones are a class of plant hormones involved in shoot branching, growth of symbiotic arbuscular mycorrhizal fungi, and germination of parasitic plant seeds. Assaying new molecules or compound exhibiting strigolactone-like activities is therefore important but unfortunately time-consuming and hard to implement because of the extremely low concentrations at which they are active. Seeds of parasite plants are natural integrator of these hormones since they can perceive molecule concentrations in the picomolar to nanomolar range stimulating their germination. Here we describe a simple and inexpensive method to evaluate the activity of these molecules by scoring the germination of parasitic plant seeds upon treatment with these molecules. Up to four molecules can be assayed from a single 96-well plate by this method. A comparison of SL-like bioactivities between molecules is done by determining the EC50 and the maximum percentage of germination.

Electrochromic Properties and Electrochemical Behavior of Marennine, a Bioactive Blue-Green Pigment Produced by the Marine Diatom Haslea ostrearia.

Marennine has long been known as the unique peculiar pigment responsible for the natural greening of oysters. It is specifically produced by the marine diatom Haslea ostrearia and it is a natural blue molecule indeed promising for food industry because of the rarity of such non-toxic, blue-colored pigments. In the search for its still not defined molecular structure, investigation of the color changes with the redox state has been carried out combining different approaches. Reducing and oxidizing chemicals have been added to purified marennine solutions and a stable blue-green color has been confirmed for the oxidized state, while a yellow color corresponded to the reduced unstable state. Raman spectroscopy has been used to monitor changes in the Raman spectra corresponding to the different colored states, and cyclic voltammetry has allowed the detection of a redox system in which protons and electrons are exchanged. These findings show that marennine is a suitable stable blue pigment for use in food applications and help in the elucidation of the chromophore structure.

Populations of the Parasitic Plant Phelipanche ramosa Influence Their Seed Microbiota.

Seeds of the parasitic weed Phelipanche ramosa are well adapted to their hosts because they germinate and form haustorial structures to connect to roots in response to diverse host-derived molecular signals. P. ramosa presents different genetic groups that are preferentially adapted to certain hosts. Since there are indications that microbes play a role in the interaction especially in the early stages of the interaction, we studied the microbial diversity harbored by the parasitic seeds with respect to their host and genetic group. Twenty-six seed lots from seven cropping plots of three different hosts-oilseed rape, tobacco, and hemp-in the west of France were characterized for their bacterial and fungal communities using 16S rRNA gene and ITS (Internal transcribed spacer) sequences, respectively. First seeds were characterized genetically using twenty microsatellite markers and phenotyped for their sensibility to various germination stimulants including strigolactones and isothiocyanates. This led to the distinction of three P. ramosa groups that corresponded to their host of origin. The observed seed diversity was correlated to the host specialization and germination stimulant sensitivity within P. ramosa species. Microbial communities were both clustered by host and plot of origin. The seed core microbiota was composed of seventeen species that were also retrieved from soil and was in lower abundances for bacteria and similar abundances for fungi compared to seeds. The host-related core microbiota of parasitic seeds was limited and presumably well adapted to the interaction with its hosts. Two microbial candidates of Sphingobacterium species and Leptosphaeria maculans were especially identified in seeds from oilseed rape plots, suggesting their involvement in host recognition and specialization as well as seed fitness for P. ramosa by improving the production of isothiocyanates from glucosinolates in the rhizosphere of oilseed rape.

Contalactone, a contaminant formed during chemical synthesis of the strigolactone reference GR24 is also a strigolactone mimic.

Strigolactone (SL) plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. GR24, a synthetic SL analog, is the worldwide reference compound used in all bioassays for investigating the role of SLs in plant development and in rhizospheric interactions. In 2012, the first characterization of the SL receptor reported the detection of an unknown compound after incubation of GR24 samples with the SL receptor. We reveal here the origin of this compound (P270), which comes from a by-product formed during GR24 chemical synthesis. We present the identification of this by-product, named contalactone. A proposed chemical pathway for its formation is provided as well as an evaluation of its bioactivity on pea, Arabidopsis, root parasitic plant seeds and AM fungi, characterizing it as a SL mimic. Quality of GR24 samples can be easily checked by carrying out microscale hydrolysis in a basic aqueous medium to easily detect P270 as indicator of the presence of the contalactone impurity. In all cases, before being used for bioassays, GR24 must be careful purified by preparative HPLC.

CYP707As are effectors of karrikin and strigolactone signalling pathways in Arabidopsis thaliana and parasitic plants.

Karrikins stimulate Arabidopsis thaliana germination, whereas parasitic weeds of the Orobanchaceae family have evolved to respond to host-exuded compounds such as strigolactones, dehydrocostus lactone, and 2-phenylethyl isothiocyanate. In Phelipanche ramosa, strigolactone-induced germination was shown to require one of the CYP707A proteins involved in abscisic acid catabolism. Here, germination and gene expression were analysed to investigate the role of CYP707As in germination of both parasitic plants and Arabidopsis upon perception of germination stimulants, after using pharmacological inhibitors and Arabidopsis mutants disrupting germination signals. CYP707A genes were up-regulated upon treatment with effective germination stimulants in both parasitic plants and Arabidopsis. Obligate parasitic plants exhibited both intensified up-regulation of CYP707A genes and increased sensitivity to the CYP707A inhibitor abscinazole-E2B, whereas Arabidopsis cyp707a mutants still positively responded to germination stimulation. In Arabidopsis, CYP707A regulation required the canonical karrikin signalling pathway KAI2/MAX2/SMAX1 and the transcription factor WRKY33. Finally, CYP707As and WRKY33 also modulated Arabidopsis root architecture in response to the synthetic strigolactone rac-GR24, and wrky33-1 exhibited a shoot hyperbranched phenotype. This study suggests that the lack of host-independent germination in obligate parasites is associated with an exacerbated CYP707A induction and that CYP707As and WRKY33 are new players involved in a variety of strigolactone/karrikin responses.

Potential contribution of strigolactones in regulating scion growth and branching in grafted grapevine in response to nitrogen availability.

In grafted plants, rootstocks assure the mineral nutrition of the scion and modify its development. In this study, we show that two grapevine rootstock genotypes have different shoot branching architectures when cultivated as cuttings and that this trait is transmitted to the scion when grafted. Shoot branching plasticity in response to nitrogen supply was also studied. As strigolactones are known to have a role in the regulation of shoot development in response to nutrient availability, their involvement in the control of scion architecture by the rootstock was investigated. Functional characterization of putative grapevine strigolactone biosynthetic genes in Arabidopsis mutants or grapevine cell suspensions showed similar functions to those of Arabidopsis. Both rootstocks produced strigolactone-like compounds; the quantity produced in response to nitrogen treatments differed between the two rootstock genotypes and correlated with the expression of putative strigolactone biosynthetic genes. Exudation of strigolactone-like compounds by both rootstocks was closely related to the developmental pattern of the scion in grafted plants. These results suggest that differential regulation of strigolactone biosynthesis in response to nitrogen availability may contribute to the control of scion development conferred by each rootstock genotype.

Haustorium initiation in the obligate parasitic plant Phelipanche ramosa involves a host-exudated cytokinin signal.

The heterotrophic lifestyle of parasitic plants relies on the development of the haustorium, a specific infectious organ required for attachment to host roots. While haustorium development is initiated upon chemodetection of host-derived molecules in hemiparasitic plants, the induction of haustorium formation remains largely unknown in holoparasitic species such as Phelipanche ramosa. This work demonstrates that the root exudates of the host plant Brassica napus contain allelochemicals displaying haustorium-inducing activity on P. ramosa germinating seeds, which increases the parasite aggressiveness. A de novo assembled transcriptome and microarray approach with P. ramosa during early haustorium formation upon treatment with B. napus root exudates allowed the identification of differentially expressed genes involved in hormone signaling. Bioassays using exogenous cytokinins and the specific cytokinin receptor inhibitor PI-55 showed that cytokinins induced haustorium formation and increased parasite aggressiveness. Root exudates triggered the expression of cytokinin-responsive genes during early haustorium development in germinated seeds, and bio-guided UPLC-ESI(+)-/MS/MS analysis showed that these exudates contain a cytokinin with dihydrozeatin characteristics. These results suggest that cytokinins constitutively exudated from host roots play a major role in haustorium formation and aggressiveness in P. ramosa.

Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel.

Seed dormancy release of the obligate root parasitic plant, Phelipanche ramosa, requires a minimum 4-day conditioning period followed by stimulation by host-derived germination stimulants, such as strigolactones. Germination is then mediated by germination stimulant-dependent activation of PrCYP707A1, an abscisic acid catabolic gene. The molecular mechanisms occurring during the conditioning period that silence PrCYP707A1 expression and regulate germination stimulant response are almost unknown. Here, global DNA methylation quantification associated with pharmacological approaches and cytosine methylation analysis of the PrCYP707A1 promoter were used to investigate the modulation and possible role of DNA methylation during the conditioning period and in the PrCYP707A1 response to GR24, a synthetic strigolactone analogue. Active global DNA demethylation occurs during the conditioning period and is required for PrCYP707A1 activation by GR24 and for subsequent seed germination. Treatment with 5-azacytidine, a DNA-hypomethylating molecule, reduces the length of the conditioning period. Conversely, hydroxyurea, a hypermethylating agent, inhibits PrCYP707A1 expression and seed germination. Methylated DNA immunoprecipitation followed by PCR experiments and bisulfite sequencing revealed that DNA demethylation particularly impacts a 78-nucleotide sequence in the PrCYP707A1 promoter. The results here demonstrate that the DNA methylation status during the conditioning period plays a crucial role independently of abscisic acid in the regulation of P. ramosa seed germination by controlling the strigolactone-dependent expression of PrCYP707A1.

Marennine, promising blue pigments from a widespread Haslea diatom species complex.

In diatoms, the main photosynthetic pigments are chlorophylls a and c, fucoxanthin, diadinoxanthin and diatoxanthin. The marine pennate diatom Haslea ostrearia has long been known for producing, in addition to these generic pigments, a water-soluble blue pigment, marennine. This pigment, responsible for the greening of oysters in western France, presents different biological activities: allelopathic, antioxidant, antibacterial, antiviral, and growth-inhibiting. A method to extract and purify marennine has been developed, but its chemical structure could hitherto not be resolved. For decades, H. ostrearia was the only organism known to produce marennine, and can be found worldwide. Our knowledge about H. ostrearia-like diatom biodiversity has recently been extended with the discovery of several new species of blue diatoms, the recently described H. karadagensis, H. silbo sp. inedit. and H. provincialis sp. inedit. These blue diatoms produce different marennine-like pigments, which belong to the same chemical family and present similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms thus present a commercial potential for aquaculture, cosmetics, food and health industries.

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.

Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction.

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa.

A high-throughput seed germination assay for root parasitic plants.

BACKGROUND: Some root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries. Compared with most of the other weeds, parasitic weeds are difficult to control by conventional methods because of their life style. The main difficulties that currently limit the development of successful control methods are the ability of the parasite to produce a tremendous number of tiny seeds that may remain viable in the soil for more than 15 years. Seed germination requires induction by stimulants present in root exudates of host plants. Researches performed on these minute seeds are until now tedious and time-consuming because germination rate is usually evaluated in Petri-dish by counting germinated seeds under a binocular microscope. RESULTS: We developed an easy and fast method for germination rate determination based on a standardized 96-well plate test coupled with spectrophotometric reading of tetrazolium salt (MTT) reduction. We adapted the Mosmann's protocol for cell cultures to germinating seeds and determined the conditions of seed stimulation and germination, MTT staining and formazan salt solubilization required to obtain a linear relationship between absorbance and germination rate. Dose-response analyses were presented as applications of interest for assessing half maximal effective or inhibitory concentrations of germination stimulants (strigolactones) or inhibitors (ABA), respectively, using four parameter logistic curves. CONCLUSION: The developed MTT system is simple and accurate. It yields reproducible results for germination bioassays of parasitic plant seeds. This method is adapted to high-throughput screenings of allelochemicals (stimulants, inhibitors) or biological extracts on parasitic plant seed germination, and strengthens the investigations of distinctive features of parasitic plant germination.

PrCYP707A1, an ABA catabolic gene, is a key component of Phelipanche ramosa seed germination in response to the strigolactone analogue GR24.

After a conditioning period, seed dormancy in obligate root parasitic plants is released by a chemical stimulus secreted by the roots of host plants. Using Phelipanche ramosa as the model, experiments conducted in this study showed that seeds require a conditioning period of at least 4 d to be receptive to the synthetic germination stimulant GR24. A cDNA-AFLP procedure on seeds revealed 58 transcript-derived fragments (TDFs) whose expression pattern changed upon GR24 treatment. Among the isolated TDFs, two up-regulated sequences corresponded to an abscisic acid (ABA) catabolic gene, PrCYP707A1, encoding an ABA 8'-hydroxylase. Using the rapid amplification of cDNA ends method, two full-length cDNAs, PrCYP707A1 and PrCYP707A2, were isolated from seeds. Both genes were always expressed at low levels during conditioning during which an initial decline in ABA levels was recorded. GR24 application after conditioning triggered a strong up-regulation of PrCYP707A1 during the first 18 h, followed by an 8-fold decrease in ABA levels detectable 3 d after treatment. In situ hybridization experiments on GR24-treated seeds revealed a specific PrCYP707A1 mRNA accumulation in the cells located between the embryo and the micropyle. Abz-E2B, a specific inhibitor of CYP707A enzymes, significantly impeded seed germination, proving to be a non-competitive antagonist of GR24 with reversible inhibitory activity. These results demonstrate that P. ramosa seed dormancy release relies on ABA catabolism mediated by the GR24-dependent activation of PrCYP707A1. In addition, in situ hybridization corroborates the putative location of cells receptive to the germination stimulants in seeds.

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.