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.

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.

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.

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.

DROUGHT STRESS AND INBREEDING DEPRESSION IN LYCHNIS FLOS-CUCULI (CARYOPHYLLACEAE).

Interactions between drought stress and inbreeding depression were studied in Lychnis flos-cuculi. Four inbreeding levels (F = 0, 0.25, 0.50 and 0.75), and three watering treatments were used. Performance was scored for germination rate and proportion, survival, plant size, proportion of plants flowering, flowering date, stem height, number of flowers, flower size, anther weight, fruiting proportion and number of capsules. Multiplicative fitness values were estimated from these traits. Inbreeding affected most of the traits studied, and a severe inbreeding depression was found for the combined fitness estimates. The higher inbreeding depression found here relative to the same family groups in a former experiment may reflect greater dominance and suppression in the present experiment at higher density.