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Induction of haustorium development by sphaeropsidones in radicles of the parasitic weeds Striga and Orobanche. A structure-activity relationship study. Monica Fernández-Aparicio, Marco Masi, Lucia Maddau, Alessio Cimmino, Marco Evidente, Diego Rubiales, and Antonio Evidente J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01910 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 7, 2016
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Journal of Agricultural and Food Chemistry 1
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Induction of haustorium development by sphaeropsidones in radicles of the
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parasitic weeds Striga and Orobanche. A structure-activity relationship study.
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Mónica Fernández-Aparicio,† Marco Masi,‡ Lucia Maddau,§ Alessio Cimmino,‡ Marco Evidente,‡
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Diego Rubiales,┴ and Antonio Evidente*,‡
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†
INRA UMR1347, Agroécologie, BP 86510, F-21000 Dijon, France
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‡
Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario
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Monte Sant’Angelo, Via Cintia 4, 80126, Napoli, Italy.
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§
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Sassari, Viale Italia 39, 07100, Sassari, Italy
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┴Institute
Dipartimento di Agraria, Sezione di Patologia Vegetale ed Entomologia, Università degli Studi di
for Sustainable Agriculture, CSIC, Apdo. 4084, 14080 Córdoba, Spain
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Journal of Agricultural and Food Chemistry 2
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ABSTRACT: Crop attack by parasitic weeds such as Striga and Orobanche occurs through
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developmental processes triggered by host chemodetection. Seeds of those weed species remain
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dormant in the soil until germination is triggered by host root exudates. The development of
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haustorium, a parasitic plant organ that invades the host to withdraw its nutrients, is also initiated in
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Orobanchaceae by host molecular cues. The induction of haustorium development by exogenous
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signals has previously been reported for Striga but not for Orobanche species. In this work we
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demonstrate that sphaeropsidone and epi-sphaeropsidone, two phytotoxic cyclohexene oxides
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isolated from the fungus Diplodia cupressi, a causal agent of cypress canker, induce haustorium
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development in radicles of the parasitic weeds Striga hermonthica, Orobanche crenata and O.
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cumana. This is the first report of chemical stimulation of haustorium development in radicles of
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Orobanche in the absence of host. In addition SAR studies were carried out by testing the
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haustorium-inducing activity of the natural cyclohexene oxides, seven already known and four new
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hemisynthetic derivatives, in O. cumana, O. crenata and S. hermonthica, to find a molecular
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specificity model required for haustorium induction. The results suggested that the haustorium-
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inducing activity is due to the possibility to convert the natural sphaeropsidone and natural and
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hemisynthetic derivatives in the corresponding 4-methoxyquinone, and that the stereochemistry at
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C-5 also seems to affect this activity.
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KEYWORDS: Striga hermonthica, Orobanche crenata, Orobanche cumana, cyclohexene oxides,
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attachment organ, haustorium-inducing factor,
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ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry 3
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INTRODUCTION
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Root parasitic plants have evolved to exploit another plant root system to fulfil their water and
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nutrient requirements. Two main types of parasitic plants can be recognized regarding their
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photosynthetic status: hemiparasitic plants perform some degree of photosynthesis but are
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dependent on the host mainly for water and inorganic nutrients while holoparasitic plants lack of
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photosynthetic machinery and in the nutrient diversion from the host they include all their required
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reduced carbon. Hemiparasitic plants are also classified in obligated or facultative parasites
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depending on their ability to develop an autotrophic way of life in the absence of a suitable host.1
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Plants from up to 28 dicotyledonous families use the plant-parasitic strategy to obtain competitive
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advantage from neighbouring host plants.2 In all of them, the core feature of their parasitic strategy
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is a specialized host-invasive organ called haustorium that develops sequential functions of host
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attachment, penetration and connection with host vascular tissues.3,4 At least in one family of
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parasitic plants, the Orobanchaceae, the haustorium development is initiated by host-derived
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metabolites.5 Some members of this family i.e. the hemiparasitic plants Striga spp. and the
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holoparasitic plants Orobanche spp. are weeds to which despite their high negative economic
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impact in agriculture there is not effective and or practicable control and thus the development of
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innovative control strategies is urgent.
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In vitro screenings of host-derived factors that initiate developmental processes in parasitic
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plants are essential in breeding programs for parasitic plant resistance.6 Striga haustorium can be
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monitored in vitro by exposing Striga seedlings to inducing factors. Upon detection of haustorium-
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inducing factors the Striga seedling initiates haustorium development and undergoes quick
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morphological changes that prepare it for its first step in the host invasion process: attachment to
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host root. Long haustorial hairs with functions of adhesion and attachment develop from elongation
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of epidermal cells surrounding the haustorium apex.4 Despite Striga and Orobanche haustoria have
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a common evolutionary origin,7 fundamental differences exist at their host-preattached haustorial ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry 4
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stage. Morphologically, Orobanche haustorium is by far less defined. It lacks of haustorial hairs but
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rather it develops short secretory papillae with function of the adhesion surface.8 Another
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fundamental difference with Striga is that in Orobanche species, the chemistry of haustorium
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initiation is completely unknown. Several authors have suggested that Orobanche species do not
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require host chemical factors for haustorium development, which would make Orobanche the
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notable exception in the Orobanchaceae.8-10 In consequence, in vitro assays of haustorium-inducing
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factors or haustorium-inducing root exudates have been unfeasible in Orobanche, hampering the
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screening for low-inducers genotypes in crop germplasm collections.
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Several phenols, flavonoids and quinones have been identified as factors inducing both
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terminal and lateral haustoria in obligated hemiparasitic plants i.e. Striga hermonthica and
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facultative hemiparasitic plants i.e. Triphysaria versicolor.4,13-15 The Atsatt’ hypothesis16 that
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parasitic plants evolved to use defense metabolites as host recognition signals is supported by the
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facts that many haustorium-inducing factors are structurally similar to phytotoxins produced by
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allelopathic plants. There is a similar gene expression in parasitic roots when they are exposed to
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host contact or allelochemicals.5,16 The haustorium development in hemiparasitic Orobanchaceae is
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under redox control mediated by cycling between the reduced and oxidized states of the haustorial-
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inducing compounds.17 Similar models in the quinone-mediated actions of both, phytotoxic
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allelopathy and haustorium initiation are proposed.5,10,18
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Some fungal metabolites are able to interfere with weed parasitism either by phytotoxic
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action on germination or growth inhibition or by triggering developmental programs in a suicidal
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fashion i.e. in the absence of a host. These new compounds constitute alternative candidate
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ingredients for the development of new herbicides. Parasitic weeds that can be targeted include
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species of high economic important members of Orobanche, Phelipanche and Striga genera.11,12,19-
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Sphaeropsidone and epi-sphaeropsidone (1 and 2, Fig. 1), are two cyclohexene oxides
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isolated together several sphaeropsidins23 as the main phytotoxins produced from Diplodia ACS Paragon Plus Environment
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cupressi, the causal agent of cypress (Cupressus sempervirens L.) canker in the Mediterranean
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basin.24 Successively, two chlorinated cyclohexenenones, close related to 1 and 2 and named
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chlorosphaeropsidone and epichloropsphaeropsidone (3 and 4, Fig. 1), were isolated from the same
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fungal culture filtrates.25 They have phytotoxic activity causing necrosis and wilting in Quercus
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species and in tomato and antifungal activity by inhibiting mycelial growth in species of
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Phytophthora.26
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In a previous study, among fungal and plant phytotoxins, sphaeropsidones were identified as
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metabolites able to induce morphological changes in the Orobanche radicle that resemble the
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attachment organ.11 Due to the nuanced morphology of Orobanche pre-attached haustorium and to
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the fact that no chemicals with haustorium-inducing activity have been previously identified for any
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Orobanche species, we have included in the present work S. hermonthica as a living marker for
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haustorium induction, due to its very distinct haustorium morphology. The present manuscript
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confirms the haustorium-inducing nature of sphaeropsidone activity and that it acts across the
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parasitic weed genera Orobanche and Striga. Haustorium-inducing assays of sphaeropsidones were
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performed in O. cumana, O. crenata and S. hermonthica. In addition, structure-activity
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relationships were carried out assaying two natural analogues (3 and 4), seven already known and
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four new sphaeropsidones derivatives26 prepared by chemical transformation of 1 and 2.
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MATERIALS AND METHODS
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General Experimental Procedures. IR spectra were recorded as deposit glass film on a
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5700 FT-IR spectrometer (Thermo Electron Corp. Nicolet, Madison, WI, USA) and UV spectra
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were measured in MeCN on a V-530 spectrophotometer (Jasco, Tokyo, Japan); 1H and
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spectra were recorded at 400/100 or 500/125 MHz in CDCl3 on Bruker spectrometers (Karlsruhe,
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Germany). The same solvent was used as internal standard. Carbon multiplicities were determined
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by DEPT spectra.27 ESI spectra were recorded on a 6230 TOF LC/MS instrument (Agilent
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Technologies, Milan, Italy). Analytical and preparative TLC were performed on silica gel ACS Paragon Plus Environment
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C NMR
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(Kieselgel 60, F254, 0.25 and 0.5 mm respectively) plates (Merck, Darmstadt, Germany). The spots
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were visualized by exposure to UV radiation (253), or by spraying first with 10% H2SO4 in MeOH
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and then with 5% phosphomolybdic acid in EtOH, followed by heating at 110 °C for 10 min.
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Column chromatography was performed using silica gel (Kieselgel 60, 0.063−0.200 mm) (Merck).
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Fungal Strains. The D. cupressi strain used in this study was purchased from
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Centraalbureau voor Schimmelcultures of Baarn (Netherland). Pure culture was maintained on
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potato dextrose agar (PDA) (Fluka, Sigma-Aldrich Chemic GmbH, Buchs, Switzerland) and stored
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at 4 °C in the collection of Dipartimento di Agraria, Università di Sassari, Italy, as 261.85 CBS. The
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fungus was grown as previously reported.26
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Extraction and Isolation of Sphaeropsidones. One-week-old colonies of the fungus on
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PDA in 9 cm Petri dish were cut off and blended for 30 s with 100 mL of sterile distilled water to
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prepare the inoculum for liquid fermentation. Each 2 L Erlenmeyer flasks containing 400 mL of
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modified Czapek medium supplemented with 2% corn meal (pH 5.7) was seeded with 5 mL of the
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mycelial suspension and then incubated at 25 °C for 4 weeks in darkness. The culture filtrates (15
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L) were acidified and extracted exhaustively with EtOAc as previously reported.24 The organic
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extract, obtained as a brown-red oil (9.2 g), was chromatographed on a silica gel column (110 × 5
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cm) eluted with 3 L of CHCl3/i-PrOH (9:1, v/v), affording nine groups of homogeneous fractions.
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The residues (3.6 g) of fractions 4-7 were combined and further purified by a silica gel column (90
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× 4 cm) eluted with 2.5 L of CHCl3/i-PrOH (9:1, v/v), yielding six groups of homogeneous
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fractions. The residue of fraction 3 was crystallized from EtOAc/n-hexane (1:5, v/v), yielding
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sphaeropsidone, 1 (Rf 0.40, 2.3 g, 153.3 mg/L) (Figure 1) as white needles. The mother liquors were
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further purified by column chromatography on silica gel (75 × 3 cm) eluted with 1 L of petroleum
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ether-Me2CO (7:3, v/v), affording epi-sphaeropsidone, 2 (Rf 0.53, 725 mg, 48.3 mg/L) (Figure 1) as
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a homogeneous oil. Chlorosphaeropsidone, 3, and epichlorosphaeropsidone, 4 (Figure 1) were
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purified from the same culture filtrate and the purification procedures for these compounds have
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been described previously.25 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry 7
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Preparation of Sphaeropsidones Derivatives (5−11). The 5-O-acetyl, 5, 2,4,5-
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triacetylanisole, 6, 1,4-dione, 7, bromohydrin, 8, 1,4-diol, 9, and 2,4-dihydroxycyclohexanone, 10,
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derivatives of sphaeropsidone, 1 (Scheme 1) as well as the 5-O-acetylderivative, 11, of epi-
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sphaeropsidone, 2 (Scheme 2) were prepared according to the procedures previously reported.26
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5-O-p-bromobenzoyl Ester of Sphaeropsidone (12). Sphaeropsidone, 1, (5 mg) was
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dissolved in CH3CN (0.5 mL) and DMAP (4-dimethylaminopyridine) (10 mg) and p-bromobenzoyl
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chloride (10 mg) were added. The reaction mixture was stirred at room temperature for 4 h, and
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then evaporated under reduced pressure. The residue (10.0 mg) was purified by TLC on silica gel
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eluted with CHCl3/i-PrOH (95:5, v/v) giving derivatives 12 (3.65 mg, Rf 0.78) (Scheme 1), as
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uncolored oil. 12 had: UV λmax (log ε) 250 (4.07) nm; IR νmax 1728, 1670, 1618, 1590 cm-1; 1H
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NMR, δ: 8.01 (d, J = 8.7 Hz, H-2′ and H-6′), 7.63 (d, J = 8.7 Hz, H-3′ and H-5′), 6.19 (d, J = 2.5
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Hz, H-5), 5.35 (br s, H-3), 3.87 (dd, J = 4.3 and 2.5, H-6), 3.71 (s, OMe), 3.54 (dd, J = 4.3 and 1.7,
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H-1); ESIMS (+) m/z 364 [M+2+Na]+, 362 [M+Na]+, 342 [M+2+H]+, 340 [M+H]+.
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5-O-5′-azidopentanoyl Ester of Sphaeropsidone (14). To compound 1 (5.0 mg) dissolved
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in anhydrous CH2Cl2 (4.5 mL) and pyridine (100 mL) were added DCC (N,N′-
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dicyclohexylcarbodiimide) (5 mg) and 5-azidopentanoic acid (20 µL). The reaction was left at 0 °C
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for 1 h and then at room temperature for 2 h. The reaction was stopped by evaporation under N2.
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The residue (10 mg) was purified by TLC on silica gel eluted with CHCl3/i-PrOH (95:5, v/v) giving
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derivatives 14 (7.90 mg, Rf 0.74) (Scheme 1), as uncolored oil. 14 had: UV λmax (log ε) 253 (3.51)
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nm; IR νmax 2100, 1746, 1672, 1618 cm-1; 1H NMR, 5.97 (d, J =2.9 Hz, H-5), 5.30 (br s, H-3), 3.76
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(dd, J = 4.0 and 2.9, H-6), 3.70 (s, OMe), 3.50 (dd, J = 4.0 and 1.4, H-1), 3.32 (t, J = 6.5 Hz, CH2-
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5′), 2.53 (t, J = 7.3 Hz, CH2-2′), 1.83 – 1.76 (m, CH2-3′), 1.71 – 1.64 (m, CH2-4′); ESIMS (+) m/z
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304 [M+Na]+, 282 [M+H]+.
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NaBH4 Reduction of epi-Sphaeropsidone (13). To epi-Sphaeropsidone, 2, (5 mg),
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dissolved in MeOH (15 mL), was added NaBH4 (5 mg) under stirring at room temperature for 30
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min. The mixture was neutralized with 0.1M HCl, extracted with CH2Cl2 (3 × 30 mL), and dried ACS Paragon Plus Environment
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(Na2SO4). The oily residue was purified by preparative TLC, using CHCl3-i-PrOH (9:1) for elution,
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to give the derivative 13 as white needles (3.45 mg, Rf 0.37) (Scheme 2). 13 had: UV λmax (log ε)
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