Synthesis of Active Strigolactone Analogues Based on Eudesmane- and Guaiane-Type Sesquiterpene Lactones.

Strigolactones are natural products that are exuded by plants and stimulate parasitic weed germination. Their use in herbicides is limited since they are produced in small quantities, but the synthesis of bioactive analogues provides an alternative source. In this work, eleven analogues have been synthesized. Among them, nine compounds belong to a novel family named eudesmanestrigolactones. The procedure is short (3-6 steps), the starting materials are isolated on a multigram scale, and global yields are up to 8%, which significantly enhance isolated yields. In bioassay, the compounds germinated high percentages of Phelipanche ramosa, Orobanche cumana, and Orobanche crenata seeds, even at nanogram doses (100 nM). Bioactivity was stereochemistry-dependent, and it was discussed in terms of the presence and geometry of the enol ether, orientation of the butenolide, and unsaturation of ring A. The reported compounds provide a set of readily obtained allelochemicals with potential applications as preventive herbicides.

Effects of drought and N level on the interactions of the root hemiparasite Rhinanthus alectorolophus with a combination of three host species.

Increasing nitrogen deposition and more frequent drought events are likely to change plant interactions in natural grasslands. Both factors may also influence the interactions between hemiparasitic plants, regarded as keystone species in many grasslands, and their host species. We grew a combination of three suitable hosts, a grass, a forb and a legume, with and without the hemiparasite Rhinanthus alectorolophus at three levels of nitrogen (N) and two levels of water availability in a factorial design. Biomass of the hemiparasite and host community increased with N level and was reduced by drought to a similar degree. Larger plants in fertilised pots started to wilt earlier, and the presence of a hemiparasite further increased drought sensitivity. The hemiparasite strongly reduced biomass of the host community and overall productivity, and affected the competitive balance among host plants because it particularly reduced biomass of the dominant grass. These effects were the opposite of those of high N. The hemiparasite increased the root mass fraction of the hosts at all levels of N and water availability, indicating that the effect of the hemiparasite on the hosts was mainly due to loss of belowground resources. Our results indicate that hemiparasites will not always respond more strongly to increased N availability and drought than autotrophic plants, and that hemiparasites can have similarly strong effects on grassland communities as soil fertility and drought. By preferentially attacking dominant species the hemiparasites might alleviate the negative effects of nutrient enrichment on grassland diversity.

Hybrid-type strigolactone analogues derived from auxins.

BACKGROUND: Strigolactones (SLs) have a vast number of ecological implications because of the broad spectrum of their biological activities. Unfortunately, the limited availability of SLs restricts their applicability for the benefit of humanity and renders synthesis the only option for their production. However, the structural complexity of SLs impedes their economical synthesis, which is unfeasible on a large scale. Synthesis of SL analogues and mimics with a simpler structure, but with retention of bioactivity, is the solution to this problem. RESULTS: Here, we present eight new hybrid-type SL analogues derived from auxin, synthesized via coupling of auxin ester [ethyl 2-(1H-indol-3-yl)acetate] and of ethyl 2-phenylacetate with four D-rings (mono-, two di- and trimethylated). The new hybrid-type SL analogues were bioassayed to assess the germination activity of seeds of the parasitic weeds Striga hermonthica, Orobanche minor and Phelipanche ramosa using the classical method of counting germinated seeds and a colorimetric method. The bioassays revealed that analogues with a natural monomethylated D-ring had appreciable to good activity towards the three species and were the most active derivatives. By contrast, derivatives with the trimethylated D-ring showed no activity. The dimethylated derivatives (2,4-dimethyl and 3,4-dimethyl) were slightly active, especially towards P. ramosa. CONCLUSIONS: New hybrid-type analogues derived from auxins have been prepared. These analogues may be attractive as potential suicidal germination agents for parasitic weed control because of their ease of preparation and relevant bioactivity. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Small-Molecule Screens Reveal Novel Haustorium Inhibitors in the Root Parasitic Plant Triphysaria versicolor.

Root parasitic weeds in Orobanchaceae pose a tremendous threat to agriculture worldwide. We used an in vitro assay to screen libraries of small molecules for those capable of inhibiting or enhancing haustorium development in the parasitic plant Triphysaria versicolor. Several redox-modifying molecules and one structural analog of 2,6-dimethoxybenzoquine (DMBQ) inhibited haustorium development in the presence of the haustorium-inducing factor DMBQ, some of these without apparent growth inhibition to the root. Triphysaria seedlings were able to acclimate to some of these redox inhibitors. Transcript levels of four early-stage haustorium genes were differentially influenced by inhibitors. These novel haustorium inhibitors highlight the importance of redox cycling for haustorium development and suggest the potential of controlling parasitic weeds by interrupting early-stage redox-signaling pathways.

Triazolide strigolactone mimics as potent selective germinators of parasitic plant Phelipanche ramosa.

BACKGROUND: Strigolactones are a unique class of plant metabolites which serve as a rhizosphere signal for parasitic plants and evocate their seed germination. The expansion of these parasitic weeds in the food crop fields urgently calls for their increased control and depletion. Simple strigolactone analogues able to stimulate seed germination of these parasitic plants may represent an efficient control measure through the induction of suicidal germination. RESULTS: Triazolide-type strigolactone mimics were easily synthesized in three steps from commercially available materials. These derivatives induced effectively seed germination of Phelipanche ramosa with EC50 as low as 5.2 × 10-10 M. These mimics did not induce seed germination of Striga hermonthica even at high concentration (≥1 × 10-5 M). CONCLUSIONS: Simple and stable strigolactone mimics with selective activity against Phelipanche ramosa were synthesized. © 2019 Society of Chemical Industry.

Recent advances in the synthesis of analogues of phytohormones strigolactones with ring-closing metathesis as a key step.

In this paper, we synthesized and evaluated the biological activity of structural analogues of natural strigolactones in which the butenolide D-ring has been replaced with a γ-lactam. The key step to obtain the α,ß-unsaturated-γ-lactam was an RCM on suitably substituted amides. Strigolactones (SLs) are plant hormones with various developmental functions. As soil signaling chemicals, they are required for establishing beneficial mycorrhizal plant/fungus symbiosis. Beside these auxinic roles, recently SLs have been successfully investigated as antitumoral agents. Peculiar to the SL perception system is the enzymatic activity of the hormone receptor. SARs data have shown that the presence of the butenolide D-ring is crucial to retain the biological activity. The substitution of the butenolide with a lactam might shed light on the mechanism of perception. In the following, a dedicated in silico study suggested the binding modes of the synthesized compounds to the receptor of SLs in plants.

New Strigolactone Mimics as Exogenous Signals for Rhizosphere Organisms.

The importance of strigolactones in plant biology prompted us to synthesize simplified strigolactone mimics effective as exogenous signals for rhizosphere organisms. New strigolactone mimics easily derived from simple and available starting materials in significant amounts were prepared and fully characterized. These compounds contain an aromatic or heterocyclic ring, usually present in various bioactive molecules, connected by an ether link to a furan-2-one moiety. The new synthesized strigolactone mimics were confirmed to be active on plant pathogenic fungi and parasitic weed seeds.

Mechanism of glyphosate control of Phelipanche aegyptiaca.

MAIN CONCLUSION: Despite its total reliance on its host plant, the holoparasite Phelipanche aegyptiaca suffers from a deficiency of aromatic amino acids upon exposure to glyphosate. The herbicide glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the biosynthesis of aromatic amino acids. However, the functionality of the EPSPS pathway in the obligate root holoparasite Phelipanche aegyptiaca is not straightforward because of the parasite's total dependence on the host plant. Despite the importance of glyphosate as a means of controlling P. aegyptiaca, the mechanism of action of the herbicide in this parasite is not clearly understood. We characterized glyphosate control of P. aegyptiaca by using a glyphosate-resistant tomato (GRT) genotype as the host plant and evaluating the activity of EPSPS and the levels of free aromatic amino acids in the parasite. The viability of the parasite's tissues deteriorated within the first 40 h after treatment (HAT) with glyphosate. In parallel, shikimate accumulation in the parasite was first detected at 24 HAT and increased over time. However, shikimate levels in the GRT host did not increase, indicating that the host was indeed glyphosate tolerant. Free phenylalanine and tyrosine levels decreased by 48 HAT in the parasite, indicating a deficiency of aromatic amino acids. The use of GRT as the host enabled us to observe, in an in situ experimental system, both endogenous EPSPS inhibition and a deficiency of aromatic amino acids in the parasite. We thus provided evidence for the presence of an active EPSPS and aromatic amino acid biosynthesis pathway in P. aegyptiaca and pinpointed this pathway as the target of glyphosate action in this parasite.

Haustorial Hairs Are Specialized Root Hairs That Support Parasitism in the Facultative Parasitic Plant Phtheirospermum japonicum.

A haustorium is the unique organ that invades host tissues and establishes vascular connections. Haustorium formation is a key event in parasitism, but its underlying molecular basis is largely unknown. Here, we use Phtheirospermum japonicum, a facultative root parasite in the Orobanchaceae, as a model parasitic plant. We performed a forward genetic screen to identify mutants with altered haustorial morphologies. The development of the haustorium in P. japonicum is induced by host-derived compounds such as 2,6-dimethoxy-p-benzoquinone. After receiving the signal, the parasite root starts to swell to develop a haustorium, and haustorial hairs proliferate to densely cover the haustorium surface. We isolated mutants that show defects in haustorial hair formation and named them haustorial hair defective (hhd) mutants. The hhd mutants are also defective in root hair formation, indicating that haustorial hair formation is controlled by the root hair development program. The internal structures of the haustoria in the hhd mutants are similar to those of the wild type, indicating that the haustorial hairs are not essential for host invasion. However, all the hhd mutants form fewer haustoria than the wild type upon infection of the host roots. The number of haustoria is restored when the host and parasite roots are forced to grow closely together, suggesting that the haustorial hairs play a role in stabilizing the host-parasite association. Thus, our study provides genetic evidence for the regulation and function of haustorial hairs in the parasitic plant.

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.

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway.

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.

Effects of ozone on species composition in an upland grassland.

Northern hemispheric background concentrations of ozone are increasing, but few studies have assessed the ecological significance of these changes for grasslands of high conservation value under field conditions. We carried out a 3-year field experiment in which ozone was released at a controlled rate over three experimental transects to produce concentration gradients over the field site, an upland mesotrophic grassland located in the UK. We measured individual species biomass in an annual hay cut in plots receiving ambient ozone, and ambient ozone elevated by mean concentrations of approximately 4 ppb and 10 ppb in the growing seasons of 2008 and 2009. There was a significant negative effect of ozone exposure on herb biomass, but not total grass or legume biomass, in 2008 and 2009. Within the herb fraction, ozone exposure significantly decreased the biomass of Ranunculus species and that of the hemi-parasitic species Rhinanthus minor. Multivariate analysis of species composition, taking into account spatial variation in soil conditions and ozone exposure, showed no significant ozone effect on the grass component. In contrast, by 2009, ozone had become the dominant factor influencing species composition within the combined herb and legume component. Our results suggest that elevated ozone concentrations may be a significant barrier to achieving increased species diversity in managed grasslands.

Strigolactones as germination stimulants for root parasitic plants.

Witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.) are the two most devastating root parasitic plants belonging to the family Orobanchaceae and are causing enormous crop losses throughout the world. Seeds of these root parasites will not germinate unless they are exposed to chemical stimuli, 'germination stimulants' produced by and released from plant roots. Most of the germination stimulants identified so far are strigolactones (SLs), which also function as host recognition signals for arbuscular mycorrhizal fungi and a novel class of plant hormones inhibiting shoot branching. In this review, we focus on SLs as germination stimulants for root parasitic plants. In addition, we discuss how quantitative and qualitative differences in SL exudation among sorghum cultivars influence their susceptibility to Striga.

Susceptibility of Phelipanche and Orobanche species to AAL-toxin.

Fusarium and Alternaria spp. are phytopathogenic fungi which are known to be virulent on broomrapes and to produce sphinganine-analog mycotoxins (SAMs). AAL-toxin is a SAM produced by Alternaria alternata which causes the inhibition of sphinganine N-acyltransferase, a key enzyme in sphingolipid biosynthesis, leading to accumulation of sphingoid bases. These long chain bases (LCBs) are determinant in the occurrence of programmed cell death (PCD) in susceptible plants. We showed that broomrapes are sensitive to AAL-toxin, which is not common plant behavior, and that AAL-toxin triggers cell death at the apex of the radicle as well as LCB accumulation and DNA laddering. We also demonstrated that three Lag1 homologs, encoding components of sphinganine N-acyltransferase in yeast, are present in the Orobanche cumana genome and two of them are mutated leading to an enhanced susceptibility to AAL-toxin. We therefore propose a model for the molecular mechanism governing broomrape susceptibility to the fungus Alternaria alternata.

Natural metabolites for parasitic weed management.

Compounds of natural origin, such as phytotoxins produced by fungi or natural amino acids, could be used in parasitic weed management strategies by interfering with the early growth stages of the parasites. These metabolites could inhibit seed germination or germ tube elongation, so preventing attachment to the host plant, or, conversely, stimulate seed germination in the absence of the host, contributing to a reduction in the parasite seed bank. Some of the fungal metabolites assayed were very active even at very low concentrations, such as some macrocyclic trichothecenes, which at 0.1 microM strongly suppressed the germination of Orobanche ramosa L. seeds. Interesting results were also obtained with some novel toxins, such as phyllostictine A, highly active in reducing germ tube elongation and seed germination both of O. ramosa and of Cuscuta campestris Yuncker. Among the amino acids tested, methionine and arginine were particularly interesting, as they were able to suppress seed germination at concentrations lower than 1 mM. Some of the fungal metabolites tested were also able to stimulate the germination of O. ramosa seeds. The major findings in this research field are described and discussed.

Recognition of root exudates by seeds of broomrape (Orobanche and Phelipanche) species.

BACKGROUND AND AIMS: The long co-existence of broomrapes and their hosts within the same environment has culminated in a strong adaptation and effective parasitism. As a first step of specialization in the parasitic process, seed receptors of parasitic plant species vary in their ability to recognize compounds released by their hosts. This work aims to investigate potential patterns for the reception requirements needed to activate germination within Orobanche and Phelipanche species. METHODS: Induction of the germination of seeds of nine Orobanche and Pheliphanche species by root exudates of 41 plant species was studied and subjected to biplot multivariate analysis. KEY RESULTS: A high level of specialization in root exudate recognition was found in Orobanche densiflora, O. gracilis and O. hederae, which germinated almost exclusively in contact with root exudates from the plants they infect in nature. At the opposite extreme, Phelipanche aegyptiaca, P. ramosa and O. minor were highly generalist, germinating when in contact with the root exudates of most plant species. Orobanche crenata, O. cumana and O. foetida showed intermediate behaviour. CONCLUSIONS: A universal germination stimulant for all broomrape species has not being identified to date. The synthetic stimulant GR24 is active against most of the weedy broomrape species, but fails with the non-weedy species tested in this study and with the very recent weedy species O. foetida. In addition, germination behaviour of broomrape species depends on the crop plant tested. Weedy broomrapes with a broad host spectrum respond better to the different exudates released by a wide range of crops and wild species than do non-weedy broomrapes, which have a narrow host spectrum and are more restricted to their host range. Root exudates of many plant species were active in stimulating germination of seeds of Orobanche and Phelipanche species for which they are not described as hosts, representing interesting examples of potential trap crops.

Pscroph, a parasitic plant EST database enriched for parasite associated transcripts.

BACKGROUND: Parasitic plants in the Orobanchaceae develop invasive root haustoria upon contact with host roots or root factors. The development of haustoria can be visually monitored and is rapid, highly synchronous, and strongly dependent on host factor exposure; therefore it provides a tractable system for studying chemical communications between roots of different plants. DESCRIPTION: Triphysaria is a facultative parasitic plant that initiates haustorium development within minutes after contact with host plant roots, root exudates, or purified haustorium-inducing phenolics. In order to identify genes associated with host root identification and early haustorium development, we sequenced suppression subtractive libraries (SSH) enriched for transcripts regulated in Triphysaria roots within five hours of exposure to Arabidopsis roots or the purified haustorium-inducing factor 2,6 dimethoxybenzoquinone. The sequences of over nine thousand ESTs from three SSH libraries and their subsequent assemblies are available at the Pscroph database http://pscroph.ucdavis.edu. The web site also provides BLAST functions and allows keyword searches of functional annotations. CONCLUSION: Libraries prepared from Triphysaria roots treated with host roots or haustorium inducing factors were enriched for transcripts predicted to function in stress responses, electron transport or protein metabolism. In addition to parasitic plant investigations, the Pscroph database provides a useful resource for investigations in rhizosphere interactions, chemical signaling between organisms, and plant development and evolution.