[Show abstract][Hide abstract] ABSTRACT: Strigolactones (SLs) trigger germination of parasitic plant seeds and hyphal branching of symbiotic arbuscular mycorrhizal (AM) fungi. There is extensive structural variation in SLs and plants usually produce blends of different SLs. The structural variation among natural SLs has been shown to impact their biological activity as hyphal branching and parasitic plant seed germination stimulants. In this study, rice root exudates were fractioned by HPLC. The resulting fractions were analyzed by MRM-LC-MS to investigate the presence of SLs and tested using bioassays to assess their Striga hermonthica seed germination and Gigaspora rosea hyphal branching stimulatory activities. A substantial number of active fractions were revealed often with very different effect on seed germination and hyphal branching. Fractions containing (-)-orobanchol and ent-2'-epi-5-deoxystrigol contributed little to the induction of S. hermonthica seed germination but strongly stimulated AM fungal hyphal branching. Three SLs in one fraction, putative methoxy-5-deoxystrigol isomers, had moderate seed germination and hyphal branching inducing activity. Two fractions contained strong germination stimulants but displayed only modest hyphal branching activity. We provide evidence that these stimulants are likely SLs although no SL-representative masses could be detected using MRM-LC-MS. Our results show that seed germination and hyphal branching are induced to very different extents by the various SLs (or other stimulants) present in rice root exudates. We propose that the development of rice varieties with different SL composition is a promising strategy to reduce parasitic plant infestation while maintaining symbiosis with AM fungi.
PLoS ONE 01/2014; 9(8):e104201. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.
[Show abstract][Hide abstract] ABSTRACT: Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km value of 32µM and 51µM for GPP, respectively, upon expression in E. coli. The in planta enzymatic activity and sub-cellular localization of VoGES and LdGES were characterized in stable transformed tobacco and using transient expression in Nicotiana benthamiana. Transgenic tobacco expressing VoGES or LdGES accumulate geraniol, oxidized geraniol compounds like geranial, geranic acid and hexose conjugates of these compounds to similar levels. Geraniol emission of leaves was lower than that of flowers, which could be related to higher levels of competing geraniol-conjugating activities in leaves. GFP-fusions of the two GES proteins show that VoGES resides (as expected) predominantly in the plastids, while LdGES import into to the plastid is clearly impaired compared to that of VoGES, resulting in both cytosolic and plastidic localization. Geraniol production by VoGES and LdGES in N. benthamiana was nonetheless very similar. Expression of a truncated version of VoGES or LdGES (cytosolic targeting) resulted in the accumulation of 30% less geraniol glycosides than with the plastid targeted VoGES and LdGES, suggesting that the substrate geranyl diphosphate is readily available, both in the plastids as well as in the cytosol.The potential role of GES in the engineering of the TIA pathway in heterologous hosts is discussed.
[Show abstract][Hide abstract] ABSTRACT: Artemisia annua, which produces the anti-malaria compound artemisinin, occurs as high-artemisinin production (HAP) and low-artemisinin production (LAP) chemotypes. Understanding the basis of the difference between these chemotypes would assist breeding and optimising artemisinin biosynthesis. Here we present a systematic comparison of artemisinin biosynthesis genes that may be involved in determining the chemotype (CYP71AV1, DBR2 and ALDH1). These genes were isolated from the two chemotypes and characterized using transient expression in planta. The enzyme activity of DBR2 and ALDH1 from the two chemotypes did not differ, but structural differences in CYP71AV1 from LAP and HAP chemotypes (AMOLAP and AMOHAP, respectively) resulted in altered enzyme activity. AMOLAP displays a seven amino acids N-terminal extension compared with AMOHAP. The GFP fusion of both proteins show equal localization to the ER but AMOHAP may have reduced stability. Upon transient expression in Nicotiana benthamiana, AMOLAP displayed a higher enzyme activity than AMOHAP. However, expression in combination with the other pathway genes also resulted in a qualitatively different product profile ('chemotype'); that is, in a shift in the ratio between the unsaturated and saturated (dihydro) branch of the pathway.
[Show abstract][Hide abstract] ABSTRACT: During evolution, plants have adapted an ecological balance with their associates, competitors, predators, and pests. Keeping this balance intact is an active process during which the plant needs to respond to many dif-ferent stimuli in order to survive. For example, plants have developed an array of physiological and bio-chemical responses to phosphate deprivation. One of these responses is the production of isoprenoid-derived molecules called strigolactones. Strigolactones are used to stimulate the formation of symbiotic associations
[Show abstract][Hide abstract] ABSTRACT: The concentration of the lifesaving antimalarial compound artemisinin (AN) in cultivated Artemisia annua (A. annua) plants is relatively low, and thus research in improving the content is important. In the present study, external stress was applied to adult plants of A. annua and the effect was examined on the concentrations of AN and its immediate precursors in leaves, and these concentrations were related to densities and sizes of the glandular trichomes (GT). Plants were stress treated weekly five times by sandblasting or spraying with salicylic acid, chitosan oligosaccharide, H(2)O(2), and NaCl solutions. Contents of AN-related compounds (AN-c) were analysed in leaf samples from an upper and a lower position of the plants, and GT were quantified and measured. In lower leaves, several stress treatments had significant negative effects on concentrations of AN-c, whereas the ratios between compounds showed an increased conversion to AN. In the upper leaves, no changes were observed compared to controls. Linear relations were found between the concentrations of metabolites and the density of GT in both upper and lower leaves, and size of GT in lower leaves. Results suggested that older and younger leaves may respond differently to applied stress. A part of the plants were infected by powdery mildew, and this caused significantly different compositions of the AN-c, compared to uninfected plants. In conclusion, changes in concentrations of AN-c seemed largely to be related to changes in GT densities and sizes.
[Show abstract][Hide abstract] ABSTRACT: Highlights
► We investigate the plant health diagnosis based on VOC measurement in greenhouse. ► We define a numerical index to evaluate VOC emission from plants in greenhouse. ► The index distinguishes VOCs emitted by plants from those emitted by others. ► The VOC emission of non-stressed tomato plants in greenhouse is determined. ► The effects of physical damage on tomato plant's VOC emission are determined.
[Show abstract][Hide abstract] ABSTRACT: Jamil M, Charnikhova T, Cardoso C, Jamil T, Ueno K, Verstappen F, Asami T & Bouwmeester HJ (2011). Quantification of the relationship between strigolactones and Striga hermonthica infection in rice under varying levels of nitrogen and phosphorus. Weed Research51, 373–385.SummaryStrigolactone exudation, as well as Striga hermonthica germination and attachment, was studied under different levels of nitrogen (N) and phosphorus (P) in two cultivars of rice (IAC 165 and TN 1). Exudation of strigolactones by rice was the highest under mineral-deficient conditions, whereas increasing N and P dose reduced the amount of strigolactones in the exudates. Deficiency of P led to the highest strigolactone exudation, when compared with N or NP deficiency. Production of strigolactones differed strongly between the two cultivars. IAC 165 produced about 100-fold higher amounts than TN 1 of 2′-epi-5-deoxystrigol, orobanchol and three new strigolactones. Across all N and P treatments, a positive relationship was found between the amount of strigolactones in the exudates of both cultivars and in vitro S. hermonthica germination. These results show that the positive effect of fertiliser application in S. hermonthica control is, at least partly, because of the suppression of strigolactone production and hence of S. hermonthica germination and subsequent attachment. This warrants further research into practical application. Maintaining suitable N and P nutrient status of soil through fertiliser use might be a promising strategy to reduce damage in cereals by this notorious weed.
Weed Research 02/2011; 51(4):373 - 385. · 2.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, the role of the recently identified class of phytohormones, strigolactones, in shaping root architecture was addressed. Primary root lengths of strigolactone-deficient and -insensitive Arabidopsis (Arabidopsis thaliana) plants were shorter than those of wild-type plants. This was accompanied by a reduction in meristem cell number, which could be rescued by application of the synthetic strigolactone analog GR24 in all genotypes except in the strigolactone-insensitive mutant. Upon GR24 treatment, cells in the transition zone showed a gradual increase in cell length, resulting in a vague transition point and an increase in transition zone size. PIN1/3/7-green fluorescent protein intensities in provascular tissue of the primary root tip were decreased, whereas PIN3-green fluorescent protein intensity in the columella was not affected. During phosphate-sufficient conditions, GR24 application to the roots suppressed lateral root primordial development and lateral root forming potential, leading to a reduction in lateral root density. Moreover, auxin levels in leaf tissue were reduced. When auxin levels were increased by exogenous application of naphthylacetic acid, GR24 application had a stimulatory effect on lateral root development instead. Similarly, under phosphate-limiting conditions, endogenous strigolactones present in wild-type plants stimulated a more rapid outgrowth of lateral root primordia when compared with strigolactone-deficient mutants. These results suggest that strigolactones are able to modulate local auxin levels and that the net result of strigolactone action is dependent on the auxin status of the plant. We postulate that the tightly balanced auxin-strigolactone interaction is the basis for the mechanism of the regulation of the plants' root-to-shoot ratio.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize (Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MIC<46 μM). To evaluate the possibility of enhancing fungal resistance in maize by overexpressing geranic acid, we generated transgenic plants with the geraniol synthase gene cloned from Lippia dulcis under the control of a ubiquitin promoter. The volatile and non-volatile metabolite profiles of leaves from transgenic and control lines were compared. The headspaces collected from intact seedlings of transgenic and control plants were not significantly different, although detached leaves of transgenic plants emitted 5-fold more geranyl acetate compared to control plants. Non-targeted LC-MS profiling and LC-MS-MS identification of extracts from maize leaves revealed that the major significantly different non-volatile compounds were 2 geranic acid derivatives, a geraniol dihexose and 4 different types of hydroxyl-geranic acid-hexoses. A geranic acid glycoside was the most abundant, and identified by NMR as geranoyl-6-O-malonyl-β-d-glucopyranoside with an average concentration of 45μM. Fungal bioassays with C. graminicola and F. graminearum did not reveal an effect of these changes in secondary metabolite composition on plant resistance to either fungus. The results demonstrate that metabolic engineering of geraniol into geranic acid can rely on the existing default pathway, but branching glycosylation pathways must be controlled to achieve accumulation of the aglycones.
[Show abstract][Hide abstract] ABSTRACT: The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.
PLoS ONE 01/2011; 6(8):e23255. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Strigolactone exudation, as well as Striga hermonthica germination and attachment, was studied under different levels of nitrogen (N) and phosphorus (P) in two cultivars of rice (IAC 165 and TN 1). Exudation of strigolactones by rice was the highest under mineral-deficient conditions, whereas increasing N and P dose reduced the amount of strigolactones in the exudates. Deficiency of P led to the highest strigolactone exudation, when compared with N or NP deficiency. Production of strigolactones differed strongly between the two cultivars. IAC 165 produced about 100-fold higher amounts than TN 1 of 2′-epi-5-deoxystrigol, orobanchol and three new strigolactones. Across all N and P treatments, a positive relationship was found between the amount of strigolactones in the exudates of both cultivars and in vitro S. hermonthica germination. These results show that the positive effect of fertiliser application in S. hermonthica control is, at least partly, because of the suppression of strigolactone production and hence of S. hermonthica germination and subsequent attachment. This warrants further research into practical application. Maintaining suitable N and P nutrient status of soil through fertiliser use might be a promising strategy to reduce damage in cereals by this notorious weed.
[Show abstract][Hide abstract] ABSTRACT: This paper describes a method to alert growers of the presence of a pathogen infection in their greenhouse based on the detection of pathogen-induced emissions of volatile organic compounds (VOCs) from plants. Greenhouse-grown plants were inoculated with spores of a fungus to learn more about this concept. The specific objective of the present study was to determine whether VOCs are detectable after inoculation, and if so, to determine the time course of the concentrations of these compounds. To achieve this objective, we inoculated 60 greenhouse-grown tomato plants (Lycopersicon esculentum) with an aqueous suspension of Botrytis cinerea spores. Upon inoculation, the greenhouse air was sampled semi-continuously with a one hour time interval until 72 hours after inoculation (HAI). The samples were transferred to the laboratory and analysed using gas chromatography - mass spectrometry. Ten leaves were randomly selected to monitor the visible symptoms of infection. The severity of these visual symptoms was assessed at 0, 24, 48, and 72 HAI. Results demonstrated no detection of C6-compounds, and an almost constant concentration of all monoterpenes, most sesquiterpenes, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. However, the concentration of methyl salicylate increased 10-fold and 3-fold at 32 and 34 HAI respectively. At 24 HAI, 10% of the selected leaves showed mild symptoms while 20% of the selected leaves showed mild symptoms at 48 HAI. These results indicate that methyl salicylate might alert a grower of the presence of a B. cinerea infection of tomato plants at greenhouse scale. Further research is required to confirm these findings.
[Show abstract][Hide abstract] ABSTRACT: The strigolactones are internal and rhizosphere signalling molecules in plants that are biosynthesised through carotenoid cleavage. They are secreted by host roots into the rhizosphere where they signal host-presence to the symbiotic arbuscular mycrorrhizal (AM) fungi and the parasitic plants of the Orobanche, Phelipanche and Striga genera. The seeds of these parasitic plants germinate after perceiving these signalling molecules. After attachment to the host root, the parasite negatively affects the host plant by withdrawing water, nutrients and assimilates through a direct connection with the host xylem. In many areas of the world these parasites are a threat to agriculture but so far very limited success has been achieved to minimize losses due to these parasitic weeds. Considering the carotenoid origin of the strigolactones, in the present study we investigated the possibilities to reduce strigolactone production in the roots of plants by blocking carotenoid biosynthesis using carotenoid inhibitors. Hereto the carotenoid inhibitors fluridone, norflurazon, clomazone and amitrole were applied to rice either through irrigation or through foliar spray. Irrigation application of all carotenoid inhibitors and spray application of amitrole significantly decreased strigolactone production, Striga hermonthica germination and Striga infection, also in concentrations too low to affect growth and development of the host plant. Hence, we demonstrate that the application of carotenoid inhibitors to plants can affect S. hermonthica germination and attachment indirectly by reducing the strigolactone concentration in the rhizosphere. This finding is useful for further studies on the relevance of the strigolactones in rhizosphere signalling. Since these inhibitors are available and accessible, they may represent an efficient technology for farmers, including poor subsistence farmers in the African continent, to control these harmful parasitic weeds.
Archives of Biochemistry and Biophysics 12/2010; 504(1):123-31. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SUMMARY: *Strigolactones are considered a novel class of plant hormones that, in addition to their endogenous signalling function, are exuded into the rhizosphere acting as a signal to stimulate hyphal branching of arbuscular mycorrhizal (AM) fungi and germination of root parasitic plant seeds. Considering the importance of the strigolactones and their biosynthetic origin (from carotenoids), we investigated the relationship with the plant hormone abscisic acid (ABA). *Strigolactone production and ABA content in the presence of specific inhibitors of oxidative carotenoid cleavage enzymes and in several tomato ABA-deficient mutants were analysed by LC-MS/MS. In addition, the expression of two genes involved in strigolactone biosynthesis was studied. *The carotenoid cleavage dioxygenase (CCD) inhibitor D2 reduced strigolactone but not ABA content of roots. However, in abamineSG-treated plants, an inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED), and the ABA mutants notabilis, sitiens and flacca, ABA and strigolactones were greatly reduced. The reduction in strigolactone production correlated with the downregulation of LeCCD7 and LeCCD8 genes in all three mutants. *The results show a correlation between ABA levels and strigolactone production, and suggest a role for ABA in the regulation of strigolactone biosynthesis.
New Phytologist 07/2010; 187(2):343-54. · 6.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cucumber plants (Cucumis sativus L.) respond to spider-mite (Tetranychus urticae) damage with the release of specific volatiles that are exploited by predatory mites, the natural enemies of the spider mites, to locate their prey. The production of volatiles also can be induced by exposing plants to the plant hormone jasmonic acid. We analyzed volatile emissions from 15 cucumber accessions upon herbivory by spider mites and upon exposure to jasmonic acid using gas chromatography-mass spectrometry. Upon induction, cucumber plants emitted over 24 different compounds, and the blend of induced volatiles consisted predominantly of terpenoids. The total amount of volatiles was higher in plants treated with jasmonic acid than in those infested with spider mites, with (E)-4,8-dimethyl-1,3,7-nonatriene, (E,E)-alpha-farnesene, and (E)-beta-ocimene as the most abundant compounds in all accessions in both treatments. Significant variation among the accessions was found for the 24 major volatile compounds. The accessions differed strongly in total amount of volatiles emitted, and displayed very different odor profiles. Principal component analysis performed on the relative quantities of particular compounds within the blend revealed clusters of highly correlated volatiles, which is suggestive of common metabolic pathways. A number of cucumber accessions also were tested for their attractiveness to Phytoseiulus persimilis, a specialist predator of spider mites. Differences in the attraction of predatory mites by the various accessions correlated to differences in the individual chemical profiles of these accessions. The presence of genetic variation in induced plant volatile emission in cucumber shows that it is possible to breed for cucumber varieties that are more attractive to predatory mites and other biological control agents.
Journal of Chemical Ecology 04/2010; 36(5):500-12. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper describes a model to calculate the concentrations of (Z)-3-hexenol, α-pinene, α-terpinene, β-caryophyllene, and methyl salicylate in a greenhouse on the basis of their source and sink behaviour. The model was used to determine whether these volatile organic compounds (VOCs) can be used to indicate Botrytis cinerea infection in a large-scale tomato production greenhouse with a volume of 5 × 104 m3 containing 2.2 × 104 plants. Seven experiments were done to parameterise the model for these VOCs. Based on model predictions, the B. cinerea-induced increase in concentration of methyl salicylate is detectable in a large-scale tomato production greenhouse when: (a) windows are fully opened, and (b) the increase continues for at least 1 h, and (c) 5% of the plants are infected. The B. cinerea-induced increase in concentration of methyl salicylate is also detectable when: (a) windows are closed, and (b) the increase continues for at least 6 h, and (c) 5% of the plants are infected. The B. cinerea-induced increase in concentration of (Z)-3-hexenol is detectable under all conditions studied. However, it is expected that besides infected plants, many additional sources of (Z)-3-hexenol exist including plant debris and nearby field crops especially upon harvest or stress. The B. cinerea-induced increases in concentration of α-pinene, α-terpinene and β-caryophyllene are probably undetectable in a large-scale tomato production greenhouse. Therefore, it is recommended to focus on the detection of methyl salicylate to indicate B. cinerea infections in large-scale tomato production greenhouses.
[Show abstract][Hide abstract] ABSTRACT: A novel approach to support the inspection of greenhouse crops is based on the measurement of volatile organic compounds emitted by unhealthy plants. This approach has attracted some serious interest over the last decade. In pursuit of this interest, we performed several experiments at the laboratory-scale to pinpoint marker volatiles that can be used to indicate certain health problems. In addition to these laboratory experiments, pilot and model studies were performed in order to verify the validity of these marker volatiles under real-world conditions. This paper provides an overview of results and gives an outlook on the use of plant volatiles for plant health monitoring.