Substrate Report for: Orobanchyl-acetate

Chickpea (Cicer arietinum L.) is a major pulse crop in Israel grown on about 3000 ha spread, from the Upper Galilee in the north to the North-Negev desert in the south. In the last few years, there has been a gradual increase in broomrape infestation in chickpea fields in all regions of Israel. Resistant chickpea cultivars would be simple and effective solution to control broomrape. Thus, to develop resistant cultivars we screened an ethyl methanesulfonate (EMS) mutant population of F01 variety (Kabuli type) for broomrape resistance. One of the mutant lines (CCD7M14) was found to be highly resistant to both Phelipanche aegyptiaca and Orobanche crenata. The resistance mechanism is based on the inability of the mutant to produce strigolactones (SLs)-stimulants of broomrape seed germination. LC/MS/MS analysis revealed the SLs orobanchol, orobanchyl acetate, and didehydroorobanchol in root exudates of the wild type, but no SLs could be detected in the root exudates of CCD7M14. Sequence analyses revealed a point mutation (G-to-A transition at nucleotide position 210) in the Carotenoid Cleavage Dioxygenase 7 (CCD7) gene that is responsible for the production of key enzymes in the biosynthesis of SLs. This nonsense mutation resulted in a CCD7 stop codon at position 70 of the protein. The influences of the CCD7M14 mutation on chickpea phenotype and chlorophyll, carotenoid, and anthocyanin content were characterized.

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.

Major strigolactones (SLs) produced by rice (Oryza sativa L. cv. Nipponbare) and tobacco (Nicotiana tabacum L. cv. Michinoku No. 1) were purified and their stereochemical structures were determined by comparing with optically pure synthetic standards for their NMR and CD data and retention times and mass fragmentations in ESI-LC/MS and GC-MS. SLs purified from root exudates of rice plants were orobanchol, orobanchyl acetate, and ent-2'-epi-5-deoxystrigol. In addition to these SLs, 7-oxoorobanchyl acetate and the putative three methoxy-5-deoxystrigol isomers were detected by LC-MS/MS. The production of 7-oxoorobanchyl acetate seemed to occur in the early growth stage, as it was detected only in the root exudates collected during the first week of incubation. The root exudates of tobacco contained at least 11 SLs, including solanacol, solanacyl acetate, orobanchol, ent-2'-epi-orobanchol, orobanchyl acetate, ent-2'-epi-orobanchyl acetate, 5-deoxystrigol, ent-2'-epi-5-deoxystrigol, and three isomers of putative didehydro-orobanchol whose structures remain to be clarified. Furthermore, two sorgolactone isomers but not sorgolactone were detected as minor SLs by LC-MS/MS analysis. It is intriguing to note that rice plants produced only orobanchol-type SLs, derived from ent-2'-epi-5-deoxystrigol, but both orobanchol-type and strigol-type SLs, derived from 5-deoxystrigol were detected in tobacco plants.

Chickpea (Cicer arietinum L.) is a major pulse crop in Israel grown on about 3000 ha spread, from the Upper Galilee in the north to the North-Negev desert in the south. In the last few years, there has been a gradual increase in broomrape infestation in chickpea fields in all regions of Israel. Resistant chickpea cultivars would be simple and effective solution to control broomrape. Thus, to develop resistant cultivars we screened an ethyl methanesulfonate (EMS) mutant population of F01 variety (Kabuli type) for broomrape resistance. One of the mutant lines (CCD7M14) was found to be highly resistant to both Phelipanche aegyptiaca and Orobanche crenata. The resistance mechanism is based on the inability of the mutant to produce strigolactones (SLs)-stimulants of broomrape seed germination. LC/MS/MS analysis revealed the SLs orobanchol, orobanchyl acetate, and didehydroorobanchol in root exudates of the wild type, but no SLs could be detected in the root exudates of CCD7M14. Sequence analyses revealed a point mutation (G-to-A transition at nucleotide position 210) in the Carotenoid Cleavage Dioxygenase 7 (CCD7) gene that is responsible for the production of key enzymes in the biosynthesis of SLs. This nonsense mutation resulted in a CCD7 stop codon at position 70 of the protein. The influences of the CCD7M14 mutation on chickpea phenotype and chlorophyll, carotenoid, and anthocyanin content were characterized.

INTRODUCTION: Strigolactones (SLs) are the most representative germination stimulants for seeds of root parasitic plants, and they show activity even at concentrations below 10(-10) M. The low amounts of stimulants produced by the host and their rapid degradability make it crucial to develop analytical methods with very low limits of quantification. OBJECTIVE: To develop a sensitive and validated analytical method for the simultaneous quantification of seven SLs [7-oxoorobanchyl acetate (1), solanacol (2), orobanchol (4), strigol (5), fabacyl acetate (6), orobanchyl acetate (7), and 5-deoxystrigol (8)]. METHODS: SLs were analysed using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), with (+/-)-GR24 (3) employed as internal standard (IS). Validation was based on selectivity, linearity, precision of the peak areas (repeatability and intermediate precision), detection and quantification limits, and stability. RESULTS: A simple, rapid and reliable UHPLC-MS/MS method has been validated for the routine analysis of seven SLs and has been successfully applied to quantify them in exudates and extracts from tomato roots (Solanum lycopersicum). The limits of quantifications range from 0.05 microg/L for 5-deoxystrigol to 0.96 microg/L for solanacol. CONCLUSION: The method provides a useful tool for research in all the fields related to SLs, both for studies related to their function as hormones, and signalling molecules in the rhizosphere, without sample preparation required for extracts and root exudates in less than 11 minutes.

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.

Present investigations report the presence of strigolactones (SLs) and photomodulation of their biosynthesis in sunflower seedlings (roots, cotyledons and first pair of leaves) during early phase of seedling development. Qualitative analyses and characterization by HPLC, ESI-MS and FT-IR revealed the presence of more than one type of SLs. Orobanchyl acetate was detected both in roots and leaves. Five-deoxystrigol, sorgolactone and orobanchol were exclusively detected in seedling roots. Sorgomol was detectable only in leaves. HPLC eluted fraction from seedling roots and leaves co-chromatographing with GR24 (a synthetic SL) could also bring about germination in Orobanche cernua (a weed) seeds, which are established to exhibit SL - mediated germination, thereby indicating the SL identity of the eluates using this bioassay. SLs accumulation was always more in the roots of light-grown seedlings, it being maximum at 4 d stage. Although significant activity of carotenoid cleavage dioxygenase (CCD, the enzyme critical for SL biosynthesis) was detected in 2 d old seedling roots, SLs remained undetectable in cotyledons at all stages of development and also in the roots of 2 d old light and dark-grown seedlings. Roots of light-grown seedlings showed maximum CCD activity during early (2 d) stage of development, thereby confirming photomodulation of enzyme activity. These observations indicate the migration of a probable light-sensitized signaling molecule (yet to be identified) or a SL precursor from light exposed aerial parts to the seedling roots maintained in dark. Thus, a photomodulation and migration of SL precursor/s is evident from the present work.

Faba bean yield is severely constrained in the Mediterranean region and Middle East by the parasitic weeds Orobanche crenata, O. foetida, and Phelipanche aegyptiaca. Seed germination of these weeds is triggered upon recognition of host root exudates. Only recently faba bean accessions have been identified with resistance based in low induction of parasitic seed germination, but the underlying mechanism was not identified. Strigolactones are a group of terpenoid lactones involved in the host recognition by parasitic plants. Our LC-MS/MS analysis of root exudates of the susceptible accession Prothabon detected orobanchol, orobanchyl acetate, and a novel germination stimulant. A time course analysis indicated that their concentration increased with plant age. However, low or undetectable amounts of these germination stimulants were detected in root exudates of the resistant lines Quijote and Navio at all plant ages. A time course analysis of seed germination induced by root exudates of each faba bean accession indicated important differences in the ability to stimulate parasitic germination. Results presented here show that resistance to parasitic weeds based on low strigolactone exudation does exist within faba bean germplasm. Therefore, selection for this trait is feasible in a breeding program. The remarkable fact that low induction of germination is similarly operative against O. crenata, O. foetida, and P. aegyptiaca reinforces the value of this resistance.

Strigolactones are plant signaling molecules that induce germination of parasitic plant seeds, initiate host plant - arbuscular mycorrhizal fungus symbiosis and act as plant hormones controlling shoot branching and root architecture. To date four unique strigolactones (e.g., orobanchol, didehydroorobanchol isomers 1 and 2 and the aromatic strigolactone solanacol) have been reported in the root exudates and extracts of tomato (Solanum lycopersicum). Here we report on the presence of several additional strigolactones in tomato root exudates and extracts, orobanchyl acetate, two 7-hydroxyorobanchol isomers, 7-oxoorobanchol and two additional didehydroorobanchol isomers and discuss their possible biological relevance.

Major strigolactones (SLs) produced by rice (Oryza sativa L. cv. Nipponbare) and tobacco (Nicotiana tabacum L. cv. Michinoku No. 1) were purified and their stereochemical structures were determined by comparing with optically pure synthetic standards for their NMR and CD data and retention times and mass fragmentations in ESI-LC/MS and GC-MS. SLs purified from root exudates of rice plants were orobanchol, orobanchyl acetate, and ent-2'-epi-5-deoxystrigol. In addition to these SLs, 7-oxoorobanchyl acetate and the putative three methoxy-5-deoxystrigol isomers were detected by LC-MS/MS. The production of 7-oxoorobanchyl acetate seemed to occur in the early growth stage, as it was detected only in the root exudates collected during the first week of incubation. The root exudates of tobacco contained at least 11 SLs, including solanacol, solanacyl acetate, orobanchol, ent-2'-epi-orobanchol, orobanchyl acetate, ent-2'-epi-orobanchyl acetate, 5-deoxystrigol, ent-2'-epi-5-deoxystrigol, and three isomers of putative didehydro-orobanchol whose structures remain to be clarified. Furthermore, two sorgolactone isomers but not sorgolactone were detected as minor SLs by LC-MS/MS analysis. It is intriguing to note that rice plants produced only orobanchol-type SLs, derived from ent-2'-epi-5-deoxystrigol, but both orobanchol-type and strigol-type SLs, derived from 5-deoxystrigol were detected in tobacco plants.

The biosynthesis of the recently identified novel class of plant hormones, strigolactones, is up-regulated upon phosphate deficiency in many plant species. It is generally accepted that the evolutionary origin of strigolactone up-regulation is their function as a rhizosphere signal that stimulates hyphal branching of arbuscular mycorrhizal fungi. In this work, we demonstrate that this induction is conserved in Arabidopsis (Arabidopsis thaliana), although Arabidopsis is not a host for arbuscular mycorrhizal fungi. We demonstrate that the increase in strigolactone production contributes to the changes in shoot architecture observed in response to phosphate deficiency. Using high-performance liquid chromatography, column chromatography, and multiple reaction monitoring-liquid chromatography-tandem mass spectrometry analysis, we identified two strigolactones (orobanchol and orobanchyl acetate) in Arabidopsis and have evidence of the presence of a third (5-deoxystrigol). We show that at least one of them (orobanchol) is strongly reduced in the putative strigolactone biosynthetic mutants more axillary growth1 (max1) and max4 but not in the signal transduction mutant max2. Orobanchol was also detected in xylem sap and up-regulated under phosphate deficiency, which is consistent with the idea that root-derived strigolactones are transported to the shoot, where they regulate branching. Moreover, two additional putative strigolactone-like compounds were detected in xylem sap, one of which was not detected in root exudates. Together, these results show that xylem-transported strigolactones contribute to the regulation of shoot architectural response to phosphate-limiting conditions.

Striga gesnerioides is a root parasitic weed of economic significance to cowpea (Vigna unguiculata) crops in Western Africa. Seeds of the parasite germinate in response to cowpea root exudates. Germination stimulants for the seeds were isolated from the hydroponic culture filtrate of cowpea, and their structures were unambiguously determined as (-)-(3aR,4R,8bR,2'R)-ent-2'-epi-orobanchol and (+)-(3aR,4R,8bR,2'R)-ent-2'-epi-orobanchyl acetate, on the basis of mass, CD, and (1)H NMR spectra; optical rotatory power; and chromatographic behavior on HPLC. The alcohol was first isolated and identified from the cowpea root exudates, and the acetate may be the same compound that had been previously isolated from the exudates and designated as alectrol. Identity of the stimulants produced by cowpea to those produced by red clover (Trifolium pratense) was confirmed.

White lupin (Lupinus albus L.), a non-host plant for arbuscular mycorrhizal (AM) fungi in the typically mycotrophic family Fabaceae, has been investigated for root metabolites that inhibit hyphal development in AM fungi. Four known pyranoisoflavones, licoisoflavone B (1), sophoraisoflavone A (2), alpinumisoflavone (3) and 3'-hydroxy-4'-O-methylalpinumisoflavone (4), together with three previously unknown pyranoisoflavones, lupindipyranoisoflavone A (5), 10'-hydroxylicoisoflavone B (6) and 10'-hydroxysophoraisoflavone A (7) were isolated from the root exudates of white lupin as an inhibitor of germ tube growth in the AM fungus Gigaspora margarita. Pyranoisoflavones 1, 2 and 3 strongly inhibited germ tube growth at 0.63, 1.25 and 0.63 microg/disc, respectively. The remaining compounds 4-7 were either moderate or weak inhibitors that inhibited germ tube growth at concentrations higher than 10 microg/disc. Licoisoflavone B (1) and sophoraisoflavone A (2) completely inhibited hyphal branching induced by a lupin strigolactone, orobanchyl acetate, in G. margarita at 0.16 and 0.63 microg/disc, respectively.

Germination stimulants for root parasitic plants produced by flax (Linum usitatissimum L.) were purified and characterized. The root exudate of flax contained at least 8 active fractions, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography mass spectrometry (GC-MS) analyses suggested that there were 6 strigolactones. Two of them were identified as orobanchol and orobanchyl acetate by comparing NMR and GC-MS and LC-MS/MS data with those of synthetic standards. One of the two novel strigolactones was purified and determined as 7-oxoorobanchyl acetate [((3aS,4S,8bS,E)-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2,7-dioxo-3,3a,4,5,6,7,8,8b-octahydro-2H-indeno[1,2-b]furan-4-yl acetate) by 1D and 2D NMR spectroscopic, and ESI- and EI-MS spectrometric analyses. The other one was also purified and identified as 7-oxoorobanchol. The remaining two compounds could not been characterized due to their scarcity.

Both root parasitic plants and arbuscular mycorrhizal (AM) fungi take advantage of strigolactones, released from plant roots as signal molecules in the initial communication with host plants, in order to commence parasitism and mutualism, respectively. In this study, strigolactones in root exudates from 12 Fabaceae plants, including hydroponically grown white lupin (Lupinus albus), a nonhost of AM fungi, were characterized by comparing retention times of germination stimulants on reverse-phase high-performance liquid chromatography (HPLC) with those of standards and by using tandem mass spectrometry (LC/MS/MS). All the plant species examined were found to exude known strigolactones, such as orobanchol, orobanchyl acetate, and 5-deoxystrigol, suggesting that these strigolactones are widely distributed in the Fabaceae. It should be noted that even the nonmycotrophic L. albus exuded orobanchol, orobanchyl acetate, 5-deoxystrigol, and novel germination stimulants. By contrast to the mycotrophic Fabaceae plant Trifolium pratense, in which phosphorus deficiency promoted strigolactone exudation, neither phosphorus nor nitrogen deficiency increased exudation of these strigolactones in L. albus. Therefore, the regulation of strigolactone production and/or exudation seems to be closely related to the nutrient acquisition strategy of the plants.