Does fungal infection increase the palatability of oilseed rape to insects?

BACKGROUND: Multiple and simultaneous attacks by pathogens and insect pests frequently occur in nature. Plants respond to biotic stresses by activating distinct defense mechanisms, but little is known about how plants cope with multiple stresses. The focus of this study was the combined interaction of fungal infection caused by Leptosphaeria maculans (synonym Plenodomus lingam) and arthropod infestation by the diamondback moth (Plutella xylostella) in oilseed rape (Brassica napus). We hypothesized that infection by the fungal pathogen L. maculans could alter oilseed rape palatability to P. xylostella-chewing caterpillars. Feeding preference tests were complemented with analyses of defense gene transcription, and levels of glucosinolates (GLSs) and volatile organic compounds (VOCs) in L. maculans-inoculated and non-inoculated (control) leaves to determine possible causes of larval choice. RESULTS: Caterpillars preferred true leaves to cotyledons, hence true leaves were used for further experiments. True leaves inoculated with L. maculans were more palatable to caterpillars over control leaves during the early stage of infection at 3 days post inoculation (dpi), but this preference disappeared in the later stages of infection at 7 dpi. In parallel, genes involved in the salicylic acid and ethylene pathways were up-regulated in L. maculans-inoculated leaves at 3 and 7 dpi; L. maculans increased the level of total aliphatic GLSs, specifically glucobrassicanapin, and decreased the level of glucoiberin at 3 dpi and altered the content of specific VOCs. A group of 55 VOCs with the highest variability between treatments was identified. CONCLUSION: We suggest that the P. xylostella preference for L. maculans-inoculated leaves in the early stage of disease development could be caused by the underlying mechanisms leading to changes in metabolic composition. Further research should pinpoint the compounds responsible for driving larval preference and evaluate whether the behavior of the adult moths, i.e. the stage that makes the first choice regarding host plant selection in field conditions, correlates with our results on larval host acceptance. © 2024 Society of Chemical Industry.

Hybridization and complex evolution of Barbarea vulgaris and related species (Brassicaceae).

Barbarea, winter-cress, is a genus of 29 species in Brassicaceae, the mustard family, which has emerged as a model for evolution of plant defence and specialised metabolites. Notably, some Barbarea species have evolved the ability to produce triterpenoid saponins as the only ones in Brassicaceae, some of which make plants resistant to important herbivores. Resistance has, however, been lost in a distinct group of plants within B. vulgaris ssp. arcuata, which is genetically strongly diverged from other B. vulgaris plants. This divergence is not reflected present in taxonomy. Thus, a phylogeny is needed to understand evolution and defence in Barbarea. Here, we analysed the nuclear ITS and the plastid matK, ndhF, rps16, and psbA-trnH DNA regions from seven out of 29 Barbarea species, 57 accessions of B. vulgaris, 10 accessions of other Barbarea species, and eight outgroup species, in addition to sequences available from GenBank. All Barbarea species formed a highly supported monophyletic group, separated from sister genera. Several clades seem to have radiated within the genus with no simple branching pattern, and discordant nuclear and plastid DNA phylogenies indicate reticulate evolution and chloroplast capture. One of the complex patterns may have resulted from chloroplast capture of a non-Nordic Barbarea species not included in the study. Two pairs of species were almost identical, B. australis and B. grayi, and B. orthoceras and B. stricta. Despite hybridization, chloroplast capture, and incongruence among the plastid and nuclear DNA data, the high level of intraspecific diversity, coupled with lineage specificity, lead us to recognize three groups of Barbarea vulgaris: G-type (glabrous) and P-type (pubescent) individuals of the current B. vulgaris ssp. arcuata as two distinct groups and the current B. vulgaris ssp. vulgaris as the third. Despite the high molecular diversity below species level, the evolutionary history of the saponin-based resistance remains unsettled due to unresolved basal branching.

Specialist Aphid Feeding Causes Local Activation of Salicylic and Jasmonic Acid Signaling in Arabidopsis Veins.

Aphids, the phloem sap feeders, probe into leaf tissues and activate a complex network of plant defense responses. Phytohormonal signaling plays a major role in this network; however, the dynamics of the signal spreading is yet to be clarified. Despite the growing knowledge about transcriptomic changes upon infestation, results often differ due to sampling, varying strongly between the tissues collected at the single feeding site, individual leaves, pooled infested leaves, or whole plant rosettes. This study focuses on activation of salicylic acid (SA) and jasmonic acid (JA) signals in Arabidopsis leaves during infestation by cabbage aphid (Brevicoryne brassicae) in high spatio-temporal resolution. We used genetically encoded fluorescent biosensors, histochemistry, and quantitative reverse transcription-PCR to precisely map activation of distinct branches of phytohormonal signaling. We found a rapid induction of SA and JA signaling markers in cells surrounding stylet puncture, colocalizing with callose deposition. For both PR1 and JAZ10, we detected activation at 24 h postinfestation (hpi), increasing and spreading along the veins until 72 hpi and, to a lesser extent, within the epidermal pavement cells. The SA signaling wave appeared in parallel with JA-associated signaling and continued to increase in time. Our results first show a local activation of SA- and JA-related responses after stylet penetration of Arabidopsis leaves and bring a detailed insight into the spatio-temporal complexity of plant defense activation during specialist aphid attack.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Induction of a Compensatory Photosynthetic Response Mechanism in Tomato Leaves upon Short Time Feeding by the Chewing Insect Spodoptera exigua.

In addition to direct tissue consumption, herbivory may affect other important plant processes. Here, we evaluated the effects of short-time leaf feeding by Spodoptera exigua larvae on the photosynthetic efficiency of tomato plants, using chlorophyll a fluorescence imaging analysis. After 15 min of feeding, the light used for photochemistry at photosystem II (PSII) (ΦPSII), and the regulated heat loss at PSII (ΦNPQ) decreased locally at the feeding zones, accompanied by increased non-regulated energy losses (ΦNO) that indicated increased singlet oxygen (1O2) formation. In contrast, in zones neighboring the feeding zones and in the rest of the leaf, ΦPSII increased due to a decreased ΦNPQ. This suggests that leaf areas not directly affected by herbivory compensate for the photosynthetic losses by increasing the fraction of open PSII reaction centers (qp) and the efficiency of these centers (Fv'/Fm'), because of decreased non-photochemical quenching (NPQ). This compensatory reaction mechanism may be signaled by singlet oxygen formed at the feeding zone. PSII functionality at the feeding zones began to balance with the rest of the leaf 3 h after feeding, in parallel with decreased compensatory responses. Thus, 3 h after feeding, PSII efficiency at the whole-leaf level was the same as before feeding, indicating that the plant managed to overcome the feeding effects with no or minor photosynthetic costs.

Glucosinolate profiles and phylogeny in Barbarea compared to other tribe Cardamineae (Brassicaceae) and Reseda (Resedaceae), based on a library of ion trap HPLC-MS/MS data of reference desulfoglucosinolates.

A library of ion trap MS2 spectra and HPLC retention times reported here allowed distinction in plants of at least 70 known glucosinolates (GSLs) and some additional proposed GSLs. We determined GSL profiles of selected members of the tribe Cardamineae (Brassicaceae) as well as Reseda (Resedaceae) used as outgroup in evolutionary studies. We included several accessions of each species and a range of organs, and paid attention to minor peaks and GSLs not detected. In this way, we obtained GSL profiles of Barbarea australis, Barbarea grayi, Planodes virginica selected for its apparent intermediacy between Barbarea and the remaining tribe and family, and Rorippa sylvestris and Nasturtium officinale, for which the presence of acyl derivatives of GSLs was previously untested. We also screened Armoracia rusticana, with a remarkably diverse GSL profile, the emerging model species Cardamine hirsuta, for which we discovered a GSL polymorphism, and Reseda luteola and Reseda odorata. The potential for aliphatic GSL biosynthesis in Barbarea vulgaris was of interest, and we subjected P-type and G-type B. vulgaris to several induction regimes in an attempt to induce aliphatic GSL. However, aliphatic GSLs were not detected in any of the B. vulgaris types. We characterized the investigated chemotypes phylogenetically, based on nuclear rDNA internal transcribed spacer (ITS) sequences, in order to understand their relation to the species B. vulgaris in general, and found them to be representative of the species as it occurs in Europe, as far as documented in available ITS-sequence repositories. In short, we provide GSL profiles of a wide variety of tribe Cardamineae plants and conclude aliphatic GSLs to be absent or below our limit of detection in two major evolutionary lines of B. vulgaris. Concerning analytical chemistry, we conclude that availability of authentic reference compounds or reference materials is critical for reliable GSL analysis and characterize two publicly available reference materials: seeds of P. virginica and N. officinale.

Comparison of glucosinolate diversity in the crucifer tribe Cardamineae and the remaining order Brassicales highlights repetitive evolutionary loss and gain of biosynthetic steps.

We review glucosinolate (GSL) diversity and analyze phylogeny in the crucifer tribe Cardamineae as well as selected species from Brassicaceae (tribe Brassiceae) and Resedaceae. Some GSLs occur widely, while there is a scattered distribution of many less common GSLs, tentatively sorted into three classes: ancient, intermediate and more recently evolved. The number of conclusively identified GSLs in the tribe (53 GSLs) constitute 60% of all GSLs known with certainty from any plant (89 GSLs) and apparently unique GSLs in the tribe constitute 10 of those GSLs conclusively identified (19%). Intraspecific, qualitative GSL polymorphism is known from at least four species in the tribe. The most ancient GSL biosynthesis in Brassicales probably involved biosynthesis from Phe, Val, Leu, Ile and possibly Trp, and hydroxylation at the ß-position. From a broad comparison of families in Brassicales and tribes in Brassicaceae, we estimate that a common ancestor of the tribe Cardamineae and the family Brassicaceae exhibited GSL biosynthesis from Phe, Val, Ile, Leu, possibly Tyr, Trp and homoPhe (ancient GSLs), as well as homologs of Met and possibly homoIle (intermediate age GSLs). From the comparison of phylogeny and GSL diversity, we also suggest that hydroxylation and subsequent methylation of indole GSLs and usual modifications of Met-derived GSLs (formation of sulfinyls, sulfonyls and alkenyls) occur due to conserved biochemical mechanisms and was present in a common ancestor of the family. Apparent loss of homologs of Met as biosynthetic precursors was deduced in the entire genus Barbarea and was frequent in Cardamine (e.g. C. pratensis, C. diphylla, C. concatenata, possibly C. amara). The loss was often associated with appearance of significant levels of unique or rare GSLs as well as recapitulation of ancient types of GSLs. Biosynthetic traits interpreted as de novo evolution included hydroxylation at rare positions, acylation at the thioglucose and use of dihomoIle and possibly homoIle as biosynthetic precursors. Biochemical aspects of the deduced evolution are discussed and testable hypotheses proposed. Biosyntheses from Val, Leu, Ile, Phe, Trp, homoPhe and homologs of Met are increasingly well understood, while GSL biosynthesis from mono- and dihomoIle is poorly understood. Overall, interpretation of known diversity suggests that evolution of GSL biosynthesis often seems to recapitulate ancient biosynthesis. In contrast, unprecedented GSL biosynthetic innovation seems to be rare.

Root-Associated Entomopathogenic Fungi Modulate Their Host Plant's Photosystem II Photochemistry and Response to Herbivorous Insects.

The escalating food demand and loss to herbivores has led to increasing interest in using resistance-inducing microbes for pest control. Here, we evaluated whether root-inoculation with fungi that are otherwise known as entomopathogens improves tomato (Solanum lycopersicum) leaflets' reaction to herbivory by Spodoptera exigua (beet armyworm) larvae using chlorophyll fluorescence imaging. Plants were inoculated with Metarhizium brunneum or Beauveria bassiana, and photosystem II reactions were evaluated before and after larval feeding. Before herbivory, the fraction of absorbed light energy used for photochemistry (ΦPSII) was lower in M. brunneum-inoculated than in control plants, but not in B. bassiana-inoculated plants. After herbivory, however, ΦPSII increased in the fungal-inoculated plants compared with that before herbivory, similar to the reaction of control plants. At the same time, the fraction of energy dissipated as heat (ΦNPQ) decreased in the inoculated plants, resulting in an increased fraction of nonregulated energy loss (ΦNO) in M. brunneum. This indicates an increased singlet oxygen (1O2) formation not detected in B. bassiana-inoculated plants, showing that the two entomopathogenic fungi differentially modulate the leaflets' response to herbivory. Overall, our results show that M. brunneum inoculation had a negative effect on the photosynthetic efficiency before herbivory, while B. bassiana inoculation had no significant effect. However, S. exigua leaf biting activated the same compensatory PSII response mechanism in tomato plants of both fungal-inoculated treatments as in control plants.

Induction and Priming of Plant Defense by Root-Associated Insect-Pathogenic Fungi.

Plants evolved in close contact with a myriad of microorganisms, some of which formed associations with their roots, benefitting from carbohydrates and other plant resources. In exchange, they evolved to influence important plant functions, e.g. defense against insect herbivores and other antagonists. Here, we test whether a fungus, Metarhizium brunneum, which is mostly known as an insect pathogen, can also associate with plant roots and contribute to above-ground plant defense. Cauliflower (Brassica oleracea var. botrytis) seeds were sown together with M. brunneum-inoculated rice grains, and the resulting plants subjected to leaf herbivory by the specialist Plutella xylostella. Activity of myrosinases, the enzymes activating glucosinolates, was measured before and after herbivory; larval consumption and plant weight at the end of experiments. Metarhizium brunneum clearly established in the plant roots, and after herbivory myrosinase activity was substantially higher in M. brunneum-treated plants than in controls; before herbivory, M. brunneum-treated and control plants did not differ. Leaf consumption was slightly lower in the M. brunneum-treated plants whereas total biomass and allocation to above- or below-ground parts was not affected by the Metarhizium treatment. Thus, M. brunneum associates with roots and primes the plant for a stronger or faster increase in myrosinase activity upon herbivory. Consistent with this, myrosinase function has been suggested to be rate-limiting for induction of the glucosinolate-myrosinase defense system. Our results show that M. brunneum, in addition to being an insect pathogen, can associate with plant roots and prime plant defense.

The cytochrome P450 CYP72A552 is key to production of hederagenin-based saponins that mediate plant defense against herbivores.

Plants continuously evolve new defense compounds. One class of such compounds is triterpenoid saponins. A few species in the Barbarea genus produce saponins as the only ones in the large crucifer family. However, the molecular mechanism behind saponin biosynthesis and their role in plant defense remains unclear. We used pathway reconstitution in planta, enzymatic production of saponins in vitro, insect feeding assays, and bioinformatics to identify a missing gene involved in saponin biosynthesis and saponin-based herbivore defense. A tandem repeat of eight CYP72A cytochromes P450 colocalise with a quantitative trait locus (QTL) for saponin accumulation and flea beetle resistance in Barbarea vulgaris. We found that CYP72A552 oxidises oleanolic acid at position C-23 to hederagenin. In vitro-produced hederagenin monoglucosides reduced larval feeding by up to 90% and caused 75% larval mortality of the major crucifer pest diamondback moth and the tobacco hornworm. Sequence analysis indicated that CYP72A552 evolved through gene duplication and has been under strong selection pressure. In conclusion, CYP72A552 has evolved to catalyse the formation of hederagenin-based saponins that mediate plant defense against herbivores. Our study highlights the evolution of chemical novelties by gene duplication and selection for enzyme innovations, and the importance of chemical modification in plant defense evolution.

The genome sequence of Barbarea vulgaris facilitates the study of ecological biochemistry.

The genus Barbarea has emerged as a model for evolution and ecology of plant defense compounds, due to its unusual glucosinolate profile and production of saponins, unique to the Brassicaceae. One species, B. vulgaris, includes two 'types', G-type and P-type that differ in trichome density, and their glucosinolate and saponin profiles. A key difference is the stereochemistry of hydroxylation of their common phenethylglucosinolate backbone, leading to epimeric glucobarbarins. Here we report a draft genome sequence of the G-type, and re-sequencing of the P-type for comparison. This enables us to identify candidate genes underlying glucosinolate diversity, trichome density, and study the genetics of biochemical variation for glucosinolate and saponins. B. vulgaris is resistant to the diamondback moth, and may be exploited for "dead-end" trap cropping where glucosinolates stimulate oviposition and saponins deter larvae to the extent that they die. The B. vulgaris genome will promote the study of mechanisms in ecological biochemistry to benefit crop resistance breeding.

Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana.

The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces ß-amyrin and α-amyrin, and is preferentially expressed in resistant plants; ß-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs).

Multiple hydroxyphenethyl glucosinolate isomers and their tandem mass spectrometric distinction in a geographically structured polymorphism in the crucifer Barbarea vulgaris.

Two distinct glucosinolate (GSL) chemotypes (P and G-types) of Barbarea vulgaris (Brassicaceae) were known from southern Scandinavia, but whether the types were consistent in a wider geographic area was not known. Populations (26) from Eastern and Central Europe were analyzed for GSLs in order to investigate whether the two types were consistent in this area. Most (21) could be attributed to one of the previously described GSL profile types, the P-type (13 populations) and the G-type (8 populations), based on differences in the stereochemistry of 2-hydroxylation, presence or absence of phenolic glucobarbarin derivatives, and qualitative differences in indole GSL decoration (tested for a subset of 8+6 populations only). The distinction agreed with previous molecular genetic analysis of the same individuals. Geographically, the P-type typically occurred in Eastern Europe while the G-type mainly occurred in Central Europe. Of the remaining five populations, minor deviations were observed in some individuals from two populations genetically assigned to the G-type, and a hybrid population from Finland contained an additional dihydroxyphenethyl GSL isomer attributed to a combinatorial effect of P-type and G-type genes. Major exceptions to the typical GSL profiles were observed in two populations: (1) A G-type population from Slovenia deviated by a high frequency of a known variant in glucobarbarin biosynthesis ('NAS form') co-occurring with usual G-type individuals. (2) A population from Caucasus exhibited a highly deviating GSL profile dominated by p-hydroxyphenethyl GSL that was insignificant in other accessions, as well as two GSLs investigated by NMR, m-hydroxyphenethylGSL and a partially identified m,p disubstituted hydroxy-methoxy derivative of phenethylGSL. Tandem HPLC-MS of seven NMR-identified desulfoGSLs was carried out and interpreted for increased certainty in peak identification and as a tool for partial structure elucidation. The distinct, geographically separated chemotypes and rare variants are discussed in relation to future taxonomic revision and the genetics and ecology of GSLs in B. vulgaris.

Interactive impacts of a herbivore and a pathogen on two resistance types of Barbarea vulgaris (Brassicaceae).

It is well known that pathogens and arthropod herbivores attacking the same host plant may affect each other. Little is known, however, about their combined impact on plant fitness, which may differ from simple additive expectations. In a 2-year common garden field experiment, we tested whether the pathogen Albugo sp. (white blister rust) and the herbivorous flea beetle Phyllotreta nemorum affected each other's performance on two resistance types (G-type and P-type) of the crucifer Barbarea vulgaris ssp. arcuata, and whether biomass, reproduction and survival of the plants were affected by interactive impacts of the antagonists. Most of the insect-resistant G-plants were severely affected by white rust, which reduced biomass and reproductive potential compared to the controls. However, when also exposed to flea beetles, biomass loss was mitigated in G-plants, even though apparent disease symptoms were not reduced. Most of the insect-susceptible P-plants were resistant to white rust; however, the number of flea beetle mines tended to increase in plants also exposed to Albugo, and biomass at the last harvest was slightly lower in the combined treatment. Thus, interactive impacts of the herbivore and pathogen differed between the two resistance types, with an antagonistic combined impact in G-plants, which lasted surprisingly long, and a slight synergistic impact in P-plants.

Consequences of combined herbivore feeding and pathogen infection for fitness of Barbarea vulgaris plants.

Plants are often attacked by pathogens and insects. Their combined impact on plant performance and fitness depends on complicated three-way interactions and the plant's ability to compensate for resource losses. Here, we evaluate the response of Barbarea vulgaris, a wild crucifer, to combined attack by an oomycete Albugo sp., a plant pathogen causing white rust, and a flea beetle, Phyllotreta nemorum. Plants from two B. vulgaris types that differ in resistance to P. nemorum were exposed to Albugo and P. nemorum alone and in combination and then monitored for pathogen infection, herbivore damage, defence compounds, nutritional quality, biomass and seed production. Albugo developed infections in the insect-resistant plants, whereas insect-susceptible plants were scarcely infected. Concentrations of Albugo DNA were higher in plants also exposed to herbivory; similarly, flea beetle larvae caused more damage on Albugo-infected plants. Concentrations of saponins and glucosinolates strongly increased when the plants were exposed to P. nemorum and when the insect-susceptible plants were exposed to Albugo, and some of these compounds increased even more in the combined treatment. The biomass of young insect-susceptible plants was lower following exposure to flea beetles, and the number of leaves of both plant types was negatively affected by combined exposure. After flowering, however, adult plants produced similar numbers of viable seeds, irrespective of treatment. Our findings support the concept that pathogens and herbivores can affect each other's performance on a host plant and that the plant reacts by inducing specific and general defences. However, plants may be able to compensate for biomass loss from single and combined attacks over time.

Different geographical distributions of two chemotypes of Barbarea vulgaris that differ in resistance to insects and a pathogen.

The interactions of plants with herbivores and pathogens have been suggested to drive the evolution of resistances in plants and in some cases new lineages and taxa. However, such divergence may require reproductive isolation, e.g., in allopatry. In the crucifer Barbarea vulgaris, some plants are resistant to the flea beetle Phyllotreta nemorum, due to production of specific saponins, whereas others are susceptible. Resistant and susceptible plants additionally differ in resistance to the pathogen Albugo candida, content of glucosinolates, and leaf pubescence, and they are genetically strongly divergent and partially reproductively incompatible. This suggests that at some point they were separated for a considerable length of time. Previously, the insect susceptible P-type had been described only from Denmark, Sweden, and Estonia, whereas the resistant G-type is widely distributed in Western Europe. Here, we tested whether the two plant types have divergent geographical distributions and maintain their distinct trait associations throughout their range. The insect-susceptible type was found in Russia, the Baltics, and parts of Fennoscandia, but not in Central Europe. In contrast, the insect resistant type was found from Finland and westwards. Their different trait associations were consistent within the two ranges. We therefore suggest that the two plant types diverged in allopatry at some time in the past, and evolved different resistances in response to local antagonists. The two plant types probably maintain their distinctness due to a hybridization barrier. Thus, the present distributions of the two types may be shaped by both historical processes and current differential biotic selection.

Phylogeny of an Albugo sp. infecting Barbarea vulgaris in Denmark and its frequency of symptom development in natural populations of two evolutionary divergent plant types.

The oomycete Albugo candida has long been considered a broad spectrum generalist pathogen, but recent studies suggest that it is diverged into several more specialized species in addition to the generalist Albugo candida sensu stricto. Whereas these species cause the disease white blister rust in many crucifer plants, asymptomatic endophytic infections may be important in the epidemiology of others. One of the plant species attacked by Albugo sp. is the wild crucifer Barbarea vulgaris ssp. arcuata, which is diverged into two phytochemically and genetically different types with different geographical distributions in Europe. These were previously shown to differ strongly in propensity to develop white rust upon controlled infections in the greenhouse. Here, we analyse the phylogenetic relatedness of this local Albugo sp. field isolate to other species and lines of Albugo spp., including others collected on B. vulgaris. We further ask whether the difference in incidence of white rust between the two types of B. vulgaris are also expressed in natural populations. Phylogenetically, the local Albugo sp. field isolate clustered tightly together with previously analysed samples from B. vulgaris, supporting that the Albugo sp. infecting B. vulgaris may indeed be an independent specialized species. White blister rust and Albugo DNA was only detected in two populations of the plant type that frequently develops symptoms upon controlled inoculations. The lack of white rust and Albugo sp. DNA in the other plant type may be due to either resistance, preventing infection, or asymptomatic infection of other tissues than leaves, which we analysed.

UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance.

Triterpenoid saponins are bioactive metabolites that have evolved recurrently in plants, presumably for defense. Their biosynthesis is poorly understood, as is the relationship between bioactivity and structure. Barbarea vulgaris is the only crucifer known to produce saponins. Hederagenin and oleanolic acid cellobioside make some B. vulgaris plants resistant to important insect pests, while other, susceptible plants produce different saponins. Resistance could be caused by glucosylation of the sapogenins. We identified four family 1 glycosyltransferases (UGTs) that catalyze 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. Among these, UGT73C10 and UGT73C11 show highest activity, substrate specificity and regiospecificity, and are under positive selection, while UGT73C12 and UGT73C13 show lower substrate specificity and regiospecificity and are under purifying selection. The expression of UGT73C10 and UGT73C11 in different B. vulgaris organs correlates with saponin abundance. Monoglucosylated hederagenin and oleanolic acid were produced in vitro and tested for effects on P. nemorum. 3-O-ß-d-Glc hederagenin strongly deterred feeding, while 3-O-ß-d-Glc oleanolic acid only had a minor effect, showing that hydroxylation of C23 is important for resistance to this herbivore. The closest homolog in Arabidopsis thaliana, UGT73C5, only showed weak activity toward sapogenins. This indicates that UGT73C10 and UGT73C11 have neofunctionalized to specifically glucosylate sapogenins at the C3 position and demonstrates that C3 monoglucosylation activates resistance. As the UGTs from both the resistant and susceptible types of B. vulgaris glucosylate sapogenins and are not located in the known quantitative trait loci for resistance, the difference between the susceptible and resistant plant types is determined at an earlier stage in saponin biosynthesis.

The variability of processes involved in transgene dispersal-case studies from Brassica and related genera.

BACKGROUND, AIM, AND SCOPE: We strive to predict consequences of genetically modified plants (GMPs) being cultivated openly in the environment, as human and animal health, biodiversity, agricultural practise and farmers' economy could be affected. Therefore, it is unfortunate that the risk assessment of GMPs is burdened by uncertainty. One of the reasons for the uncertainty is that the GMPs are interacting with the ecosystems at the release site thereby creating variability. This variability, e.g. in gene flow, makes consequence analysis difficult. The review illustrates the great uncertainty of results from gene-flow analysis. MAIN FEATURES: Many independent experiments were performed on the individual processes in gene flow. The results comprise information both from laboratory, growth chambers and field trials, and they were generated using molecular or phenotypic markers and analysis of fitness parameters. Monitoring of the extent of spontaneous introgression in natural populations was also performed. Modelling was used as an additional tool to identify key parameters in gene flow. RESULTS: The GM plant may affect the environment directly or indirectly by dispersal of the transgene. Magnitude of the transgene dispersal will depend on the GM crop, the agricultural practise and the environment of the release site. From case-to-case these three factors provide a variability that is reflected in widely different likelihoods of transgene dispersal and fitness of introgressed plants. In the present review, this is illustrated through a bunch of examples mostly from our own research on oilseed rape, Brassica napus. In the Brassica cases, the variability affected all five main steps in the process of gene dispersal. The modelling performed suggests that in Brassica, differences in fitness among plant genome classes could be a dominant factor in the establishment and survival of introgressed populations. DISCUSSION: Up to now, experimental analyses have mainly focused on studying the many individual processes of gene flow. This can be criticised, as these experiments are normally carried out in widely different environments and with different genotypes, and thus providing bits and pieces difficult to assemble. Only few gene-flow studies have been performed in natural populations and over several plant generations, though this could give a more coherent and holistic view. CONCLUSION: The variability inherent in the processes of gene flow in Brassica is apparent and remedies are wished for. One possibility is to expose the study species to additional experiments and monitoring, but this is costly and will likely not cover all possible scenarios. Another remedy is modelling gene flow. Modelling is a valuable tool in identifying key factors in the gene-flow process for which more knowledge is needed, and identifying parameters and processes which are relatively insensitive to change and therefore require less attention in future collections of data. But the interdependence between models and experimental data is extensive, as models depend on experimental data for their development or testing. RECOMMENDATIONS: More and more transgenic varieties are being grown worldwide harbouring genes that might potentially affect the environment (e.g. drought tolerance, salt tolerance, disease tolerance, pharmaceutical genes). This calls for a thorough risk assessment. However, in Brassica, the limited and uncertain knowledge on gene flow is an obstacle to this. Modelling of gene flow should be optimised, and modelling outputs verified in targeted field studies and at the landscape level. Last but not least, it is important to remember that transgene flow in itself is not necessarily a thread, but it is the consequences of gene flow that may jeopardise the ecosystems and the agricultural production. This emphasises the importance of consequence analysis of genetically modified plants.

Survival and flowering of hybrids between cultivated and wild carrots (Daucus carota) in Danish grasslands.

Many crop species are able to hybridize with related weedy or wild relatives, which could lead to transfer of cultivar genes, and among them transgenes, into wild populations. It is not clear, however, whether the hybrids and their descendants are able to survive and reproduce in natural habitats, as inherited cultivar traits may be maladaptive under such conditions. To test this, we produced hybrid (F(1)) seeds by controlled crosses between wild [see text for formula] and cultivated carrots (Daucus carota ssp. carota and ssp. sativa, respectively) and sowed them into three Danish grasslands of different age, in parallel with seeds of wild carrots. Replicate plots were sown in fall and spring. Survival and flowering of the emerging plants were monitored for the following three years. Both hybrid and wild carrots survived and flowered in highest frequency at a recently disturbed site, and much less at two older sites. Hybrids emerged in higher proportions than wild carrots in the first year and survived to similar or slightly lower frequencies at the end of the experiment. Hybrids flowered as frequently or slightly less frequently than wild plants, and developed fewer and smaller umbels. Despite a somewhat lower reproductive potential compared to wild carrots, first generation hybrids between cultivated and wild carrots are likely to survive and produce offspring in natural grasslands in Denmark. This, together with other studies, suggests that cultivar genes may transfer relatively easily into wild carrot populations.

Frequency-dependent fitness of hybrids between oilseed rape (Brassica napus) and weedy B. rapa (Brassicaceae).

Fitness of interspecific hybrids is sometimes high relative to their parents, despite the conventional belief that they are mostly unfit. F(1) hybrids between oilseed rape (Brassica napus) and weedy B. rapa can be significantly more fit than their weedy parents under some conditions; however, under other conditions they are less fit. To understand the reasons, we measured the seed production of B. napus, B. rapa, and different generations of hybrid plants at three different densities and in mixtures of different frequencies (including pure stands). Brassica napus, B. rapa, and backcross plants (F(1) ♀ × B. rapa) produced many more seeds per plant in pure plots than in mixtures and more seeds in plots when each was present at high frequency. The opposite was true for F(1) plants that produced many more seeds than B. rapa in mixtures, but fewer in pure stands. Both vegetative and reproductive interactions may be responsible for these effects. Our results show that the fitness of both parents and hybrids is strongly frequency-dependent and that the likelihood of introgression of genes between the species thus may depend on the numbers and densities of parents and their various hybrid offspring in the population.