[Show abstract][Hide abstract] ABSTRACT: Strigolactones (SLs) are a class of phytohormones and rhizosphere signaling compounds with high structural diversity. Three enzymes, carotenoid isomerase DWARF27 and carotenoid cleavage dioxygenases CCD7 and CCD8, were previously shown to convert all-trans-β-carotene to carlactone (CL), the SL precursor. However, how CL is metabolized to SLs has remained elusive. Here, by reconstituting the SL biosynthetic pathway in Nicotiana benthamiana, we show that a rice homolog of Arabidopsis MORE AXILLARY GROWTH 1 (MAX1), encodes a cytochrome P450 CYP711 subfamily member that acts as a CL oxidase to stereoselectively convert CL into ent-2'-epi-5-deoxystrigol (B-C lactone ring formation), the presumed precursor of rice SLs. A protein encoded by a second rice MAX1 homolog then catalyzes the conversion of ent-2'-epi-5-deoxystrigol to orobanchol. We therefore report that two members of CYP711 enzymes can catalyze two distinct steps in SL biosynthesis, identifying the first enzymes involved in B-C ring closure and a subsequent structural diversification step of SLs.
Full-text · Article · Oct 2014 · Nature Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: Chicory (Cichorium intybus L) is rich in bitter sesquiterpene lactones, mainly guaianolides: lactucin, 8-deoxylactucin, lactupicrin and their 11(S),13-dihydroderivatives-compounds recognized for their antimicrobial and anti-cancer effects. In vitro plant tissue culture, and particularly Agro bacterium rhizogeries-generated hairy root (HR) cultures, have many advantages as systems for production of valuable secondary metabolites. Although chicory HRs grow better than control culture, having nearly 60 times greater fresh weight gain, they do not contain a higher content of guaianolides than wild type (wt) roots. Thus we have established in vitro system comprised of wt root and HR cultures, and wt and transformed regenerated plants of the same age, in rosette and flowering stage, in order to study the effects of transformation, organogenesis and flowering on guaianolides production. Both regeneration and flowering in vitro were spontaneous, so the results were not influenced by exogenous growth regulators. Some of the transformed clones grew better, but all flowered earlier in comparison to wt plants. Floral transition increased guaianolides content in both roots and leaves of transformed, but not of wt plants. Expression of RolC oncogene correlated with floral transition and with guaianolides accumulation. We propose A. rhizogenes transformed plants at the flowering stage as an alternative source of free guaianolides, where, in contrast to HRs, entire plants can be used for the extraction.
[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.
[Show abstract][Hide abstract] ABSTRACT: Strigolactones, plant-secreted underground signalling molecules, play an important role in agricultural ecosystems, because they mediate the interaction of crops with symbiotic AM fungi and parasitic weeds like Striga hermonthica. Cereal host plants secret these signalling molecules particularly under nutrient-deficient conditions and especially when phosphate (P) is limiting. The objective of the present study was to see the potential of P seed priming for S. hermonthica management in cereals in relation to strigolactone production. It has been demonstrated that P fertiliser application down-regulates the production of these signalling molecules in the rhizosphere, which results in lower S. hermonthica infection of cereals. The laboratory study showed maximum production of strigolactones from dry and water-soaked seeds, while seed soaking in P solution reduced their production. Similarly, maximum S. hermonthica infection was observed under control treatments with dry sowing or water soaking, while P seed soaking decreased S. hermonthica germination, emergence and dry biomass in all cereal crops. Our study shows that P seed priming resulted in lower exudation of strigolactones, which induced less S. hermonthica seeds germination and hence may lead to lower S. hermonthica infection. P-based seed priming could prove to be an effective and affordable strategy to reduce S. hermonthica infection in cereals. Further research for practical field application is needed
[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: Measurement of volatile organic compounds (VOCs) emitted by plants allows us to monitor plant health status without touching the plant. To bring this technique a step further towards a practical plant diagnosis technique for greenhouse crop production, we have defined a numerical index named "Emission index" to evaluate VOC emissions from tomato plants isolated from ambient greenhouse air using an open-bottom chamber. The emission index of a VOC is a ratio of the concentration of the VOC in the air inside the chamber to that in the ambient greenhouse air and the index should be larger than one if the VOC is emitted by the plants in the chamber. Measurement of the emission indices of representative tomato VOCs proved that non-stressed tomato plants in a greenhouse emit n-hexanal, 2-carene, beta-phellandrene, alpha-copaene, beta-caryophyllene and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT), do not emit (Z)-3-hexenol, and might absorb and/or adsorb n-hexanol. Another experiment proved that a routine crop maintenance operation, i.e. removal of old leaves from the lower part of plants and removal of side shoots, enhances the emissions of n-hexanal, (E)-2-hexenal, (Z)-3-hexenol, but does not enhance the emissions of alpha-copaene, methyl salicylate (MeSA) and TMTT. The results suggest that the measurement of emission index with the open-bottom chamber is a useful technique for monitoring VOC emissions by plants in a greenhouse under practical conditions.
No preview · Article · Oct 2012 · Biosystems Engineering
[Show abstract][Hide abstract] ABSTRACT: Meer dan altijd zichtbaar en merkbaar is, spelen hormonen een cruciale rol in de teelt van bloembollen. Het Laboratorium voor Plantenfysiologie in Wageningen en PPO Bloembollen in Lisse onderzochten welke hormonen er zoal in bloembollen voorkomen en hoe hun gehaltes door de tijd heen al dan niet veranderen. Inmiddels kunnen er 48 stoffen in één keer worden gemeten.
[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.
[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.
No preview · Article · Apr 2011 · Acta horticulturae
[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.
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: 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.
Full-text · Article · Feb 2011 · Metabolic Engineering
[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: 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.