Botrytis cinerea combines four molecular strategies to tolerate membrane-permeating plant compounds and to increase virulence.

Saponins are plant secondary metabolites comprising glycosylated triterpenoids, steroids or steroidal alkaloids with a broad spectrum of toxicity to microbial pathogens and pest organisms that contribute to basal plant defense to biotic attack. Secretion of glycosyl hydrolases that enzymatically convert saponins into less toxic products was thus far the only mechanism reported to enable fungal pathogens to colonize their saponin-containing host plant(s). We studied the mechanisms that the fungus Botrytis cinerea utilizes to be tolerant to well-characterized, structurally related saponins from tomato and Digitalis purpurea. By gene expression studies, comparative genomics, enzyme assays and testing a large panel of fungal (knockout and complemented) mutants, we unraveled four distinct cellular mechanisms that participate in the mitigation of the toxic activity of these saponins and in virulence on saponin-producing host plants. The enzymatic deglycosylation that we identified is novel and unique to this fungus-saponin combination. The other three tolerance mechanisms operate in the fungal membrane and are mediated by protein families that are widely distributed in the fungal kingdom. We present a spatial and temporal model on how these mechanisms jointly confer tolerance to saponins and discuss the repercussions of these findings for other plant pathogenic fungi, as well as human pathogens.

Root branching under high salinity requires auxin-independent modulation of LATERAL ORGAN BOUNDARY DOMAIN 16 function.

Salinity stress constrains lateral root (LR) growth and severely affects plant growth. Auxin signaling regulates LR formation, but the molecular mechanism by which salinity affects root auxin signaling and whether salt induces other pathways that regulate LR development remains unknown. In Arabidopsis thaliana, the auxin-regulated transcription factor LATERAL ORGAN BOUNDARY DOMAIN 16 (LBD16) is an essential player in LR development under control conditions. Here, we show that under high-salt conditions, an alternative pathway regulates LBD16 expression. Salt represses auxin signaling but, in parallel, activates ZINC FINGER OF ARABIDOPSIS THALIANA 6 (ZAT6), a transcriptional activator of LBD16. ZAT6 activates LBD16 expression, thus contributing to downstream cell wall remodeling and promoting LR development under high-salt conditions. Our study thus shows that the integration of auxin-dependent repressive and salt-activated auxin-independent pathways converging on LBD16 modulates root branching under high-salt conditions.

The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases.

In this work, we introduce the application of proton transfer reaction mass spectrometry (PTR-MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC-MS)-based methods, PTR-MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR-MS-based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR-Qi-ToF-MS were compared with GC-MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR-Qi-ToF-MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC-MS analysis. This study provides a first basis for the application of rapid PTR-Qi-ToF-MS detection, in combination with multi-well formats, in HTS screening methods for the selection of highly productive terpene synthases.

Effective root responses to salinity stress include maintained cell expansion and carbon allocation.

Acclimation of root growth is vital for plants to survive salt stress. Halophytes are great examples of plants that thrive even under severe salinity, but their salt tolerance mechanisms, especially those mediated by root responses, are still largely unknown. We compared root growth responses of the halophyte Schrenkiella parvula with its glycophytic relative species Arabidopsis thaliana under salt stress and performed transcriptomic analysis of S. parvula roots to identify possible gene regulatory networks underlying their physiological responses. Schrenkiella parvula roots do not avoid salt and experience less growth inhibition under salt stress. Salt-induced abscisic acid levels were higher in S. parvula roots compared with Arabidopsis. Root transcriptomic analysis of S. parvula revealed the induction of sugar transporters and genes regulating cell expansion and suberization under salt stress. 14 C-labeled carbon partitioning analyses showed that S. parvula continued allocating carbon to roots from shoots under salt stress while carbon barely allocated to Arabidopsis roots. Further physiological investigation revealed that S. parvula roots maintained root cell expansion and enhanced suberization under severe salt stress. In summary, roots of S. parvula deploy multiple physiological and developmental adjustments under salt stress to maintain growth, providing new avenues to improve salt tolerance of plants using root-specific strategies.

Terpene synthases in cucumber (Cucumis sativus) and their contribution to herbivore-induced volatile terpenoid emission.

Terpenoids play important roles in flavour, pollinator attraction and defence of plants. In cucumber (Cucumis sativus) they are important components of the herbivore-induced plant volatile blend that attracts natural enemies of herbivores. We annotated the cucumber TERPENE SYNTHASE gene (CsTPS) family and characterized their involvement in the response towards herbivores with different feeding guilds using a combined molecular and biochemical approach. Transcripts of multiple CsTPS genes were upregulated in leaves upon herbivory and the products generated by the expressed proteins match the terpenoids recorded in the volatile blend released by herbivore-damaged leaves. Spatial and temporal analysis of the promoter activity of CsTPS genes showed that cell content-feeding spider mites (Tetranychus urticae) and thrips (Frankliniella occidentalis) induced promoter activity of CsTPS9 and CsTPS19 within hours after initiation of infestation, while phloem-feeding aphids (Myzus persicae) induced CsTPS2 promoter activity. Our findings offer detailed insights into the involvement of the TPS gene family in the dynamics and fine-tuning of the emission of herbivore-induced plant volatiles in cucumber, and open a new avenue to understand molecular mechanisms that affect plant-herbivore interactions.

The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase.

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, ß-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.

Identification of the Bisabolol Synthase in the Endangered Candeia Tree (Eremanthus erythropappus (DC) McLeisch).

Candeia (Eremanthus erythropappus (DC) McLeisch, Asteraceae) is a Brazilian tree, mainly occurring in the cerrado areas. From ethnobotanical information its essential oil is known to have wound healing and nociceptive properties. These properties are ascribed to result from a sesquiterpene alcohol, (-)-α-bisabolol, which is present at high concentrations in this oil. Bisabolol is highly valued by the cosmetic industry because of its antibacterial, anti-inflammatory, skin-smoothing and wound healing properties. Over the past decades, Candeia timber has been collected at large scale for bisabolol extraction from wild reserves and the species is thereby at risk of extinction. To support the development of breeding and nursing practices that would facilitate sustainable cultivation of Candeia, we identified a terpene synthase gene, EeBOS1, that appears to control biosynthesis (-)-α-bisabolol in the plant. Expression of this gene in E. coli showed that EeBOS1 protein is capable of producing (-)-α-bisabolol from farnesyl pyrophosphate in vitro. Analysis of gene expression in different tissues from Candeia plants in different life stages showed a high correlation of EeBOS1 expression and accumulation of (-)-α-bisabolol. This work is the first step to unravel the pathway toward (-)-α-bisabolol in Candeia, and in the further study of the control of (-)-α-bisabolol production.

The Use of Metabolomics to Elucidate Resistance Markers against Damson-Hop Aphid.

Phorodon humuli (Damson-hop aphid) is one of the major pests of hops in the northern hemisphere. It causes significant yield losses and reduces hop quality and economic value. Damson-hop aphid is currently controlled with insecticides, but the number of approved pesticides is steadily decreasing. In addition, the use of insecticides almost inevitably results in the development of resistant aphid genotypes. An integrated approach to pest management in hop cultivation is therefore badly needed in order to break this cycle and to prevent the selection of strains resistant to the few remaining registered insecticides. The backbone of such an integrated strategy is the breeding of hop cultivars that are resistant to Damson-hop aphid. However, up to date mechanisms of hops resistance towards Damson-hop aphids have not yet been unraveled. In the experiments presented here, we used metabolite profiling followed by multivariate analysis and show that metabolites responsible for hop aroma and flavor (sesquiterpenes) in the cones can also be found in the leaves, long before the hop cones develop, and may play a role in resistance against aphids. In addition, aphid feeding induced a change in the metabolome of all hop genotypes particularly an increase in a number of oxidized compounds, which suggests this may be part of a resistance mechanism.

Engineering storage capacity for volatile sesquiterpenes in Nicotiana benthamiana leaves.

Plants store volatile compounds in specialized organs. The properties of these storage organs prevent precarious evaporation and protect neighbouring tissues from cytotoxicity. Metabolic engineering of plants is often carried out in tissues such as leaf mesophyll cells, which are abundant and easily accessible by engineering tools. However, these tissues are not suitable for the storage of volatile and hydrophobic compound such as sesquiterpenes and engineered volatiles are often lost into the headspace. In this study, we show that the seeds of Arabidopsis thaliana, which naturally contain lipid bodies, accumulate sesquiterpenes upon engineered expression. Subsequently, storage of volatile sesquiterpenes was achieved in Nicotiana benthamiana leaf tissue, by introducing oleosin-coated lipid bodies through metabolic engineering. Hereto, different combinations of genes encoding diacylglycerol acyltransferases (DGATs), transcription factors (WRINKL1) and oleosins (OLE1), from the oil seed-producing species castor bean (Ricinus communis) and Arabidopsis, were assessed for their suitability to promote lipid body formation. Co-expression of α-bisabolol synthase with Arabidopsis DGAT1 and WRINKL1 and OLE1 from castor bean promoted storage of α-bisabolol in N. benthamiana mesophyll tissue more than 17-fold. A clear correlation was found between neutral lipids and storage of sesquiterpenes, using synthases for α-bisabolol, (E)-ß-caryophyllene and α-barbatene. The co-localization of neutral lipids and α-bisabolol was shown using microscopy. This work demonstrates that lipid bodies can be used as intracellular storage compartment for hydrophobic sesquiterpenes, also in the vegetative parts of plants, creating the possibility to improve yields of metabolic engineering strategies in plants.

Modification of chrysanthemum odour and taste with chrysanthemol synthase induces strong dual resistance against cotton aphids.

Aphids are pests of chrysanthemum that employ plant volatiles to select host plants and ingest cell contents to probe host quality before engaging in prolonged feeding and reproduction. Changes in volatile and nonvolatile metabolite profiles can disrupt aphid-plant interactions and provide new methods of pest control. Chrysanthemol synthase (CHS) from Tanacetum cinerariifolium represents the first committed step in the biosynthesis of pyrethrin ester insecticides, but no biological role for the chrysanthemol product alone has yet been documented. In this study, the TcCHS gene was over-expressed in Chrysanthemum morifolium and resulted in both the emission of volatile chrysanthemol (ca. 47 pmol/h/gFW) and accumulation of a chrysanthemol glycoside derivative, identified by NMR as chrysanthemyl-6-O-malonyl-ß-D-glucopyranoside (ca. 1.1 mM), with no detrimental phenotypic effects. Dual-choice assays separately assaying these compounds in pure form and as part of the headspace and extract demonstrated independent bioactivity of both components against the cotton aphid (Aphis gossypii). Performance assays showed that the TcCHS plants significantly reduced aphid reproduction, consistent with disturbance of aphid probing activities on these plants as revealed by electropenetrogram (EPG) studies. In open-field trials, aphid population development was very strongly impaired demonstrating the robustness and high impact of the trait. The results suggest that expression of the TcCHS gene induces a dual defence system, with both repellence by chrysanthemol odour and deterrence by its nonvolatile glycoside, introducing a promising new option for engineering aphid control into plants.

Floral Volatiles in Parasitic Plants of the Orobanchaceae. Ecological and Taxonomic Implications.

The holoparasitic broomrapes, Orobanche spp. and Phelipanche spp. (Orobanchaceae), are root parasites that completely depend on a host plant for survival and reproduction. There is considerable controversy on the taxonomy of this biologically and agronomically important family. Flowers of over 25 parasitic Orobanchaceae and a number of close, parasitic and non-parasitic, relatives emitted a complex blend of volatile organic compounds (VOCs), consisting of over 130 VOCs per species. Floral VOC blend-based phylogeny supported the known taxonomy in internal taxonomic grouping of genus and eliminated the uncertainty in some taxonomical groups. Moreover, phylogenetic analysis suggested separation of the broomrapes into two main groups parasitizing annual and perennial hosts, and for the annual hosts, into weedy and non-weedy broomrapes. We conclude that floral VOCs are a significant tool in species identification and possibly even in defining new species and can help to improve controversial taxonomy in the Orobanchaceae.

Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis.

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-ß-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.

Differential activity of Striga hermonthica seed germination stimulants and Gigaspora rosea hyphal branching factors in rice and their contribution to underground communication.

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.

Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers.

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.

Characterization of two geraniol synthases from Valeriana officinalis and Lippia dulcis: similar activity but difference in subcellular localization.

Two geraniol synthases (GES), from Valeriana officinalis (VoGES) and Lippia dulcis (LdGES), were isolated and were shown to have geraniol biosynthetic activity with Km values of 32 µM and 51 µM for GPP, respectively, upon expression in Escherichia 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.

The metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin biosynthetic pathway genes is a function of CYP71AV1 type and relative gene dosage.

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.

Artemisinin production and precursor ratio in full grown Artemisia annua L. plants subjected to external stress.

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, H2O2, 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.

Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana.

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.

Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns.

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-ß-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.

Strigolactones are transported through the xylem and play a key role in shoot architectural response to phosphate deficiency in nonarbuscular mycorrhizal host Arabidopsis.

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.