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.

Polymorphism for novel tetraglycosylated flavonols in an Eco-model crucifer, Barbarea vulgaris.

Nineteen apparent flavonoids were determined by HPLC-DAD in foliage of a chemotype (G-type) of Barbarea vulgaris , and four were isolated. Two were novel tetraglycosylated flavonols with identical glycosylation patterns, kaempferol 3-O-(2,6-di-O-ß-d-glucopyranosyl)-ß-d-glucopyranoside-7-O-α-l-rhamnopyranoside (1) and quercetin 3-O-(2,6-di-O-ß-d-glucopyranosyl)-ß-d-glucopyranoside-7-O-α-l-rhamnopyranoside (2). The identification of d/l configuration was tentatively based on susceptibility to α-l-rhamnosidase and ß-d-glucosidases. A characteristic feature of 1 and 2 was appreciable water solubility, an expected consequence of the extensive glycosylation. A less complex pair of flavonols comprised 3-O-ß-d-glucopyranoside-7-O-α-l-rhamnopyranosides of kaempferol and quercetin. Two natural chemotypes of B. vulgaris differed in levels of 1 and 2, with the P-type deficient in 1 and 2 and the insect-resistant G-type rich in 1 (ca. 3-4 µmol/g dry wt) and with moderate levels of 2 (ca. 0.3-0.8 µmol/g dry wt). However, there was only modest seasonal variation in flavonols 1 and 2, in contrast to a strong seasonal variation in insect resistance.

Barbarea vulgaris linkage map and quantitative trait loci for saponins, glucosinolates, hairiness and resistance to the herbivore Phyllotreta nemorum.

Combined genomics and metabolomics approaches were used to unravel molecular mechanisms behind interactions between winter cress (Barbarea vulgaris) and flea beetle (Phyllotreta nemorum). B. vulgaris comprises two morphologically, biochemically and cytologically deviating types, which differ in flea beetle resistance, saponin and glucosinolate profiles, as well as leaf pubescence. An F2 population generated from a cross between the two B. vulgaris types was used to construct a B. vulgaris genetic map based on 100 AFLP and 31 microsatellite markers. The map was divided into eight linkage groups. QTL (quantitative trait loci) analysis revealed a total of 15 QTL affecting eight traits, including nine QTL for four saponins, two QTL for two glucosinolates, two QTL for hairiness, and two QTL for flea beetle resistance. The two QTL for resistance towards flea beetles in B. vulgaris co-localized with QTL for the four saponins associated with resistance. Furthermore, global QTL analysis of B. vulgaris metabolites identified QTL for a number of flavonoid glycosides and additional saponins from both resistant and susceptible types. The transcriptome of the resistant B. vulgaris type was sequenced by pyrosequencing, and sequences containing microsatellites were identified. Microsatellite types in B. vulgaris were similar to Arabidopsis thaliana but different from Oryza sativa. Comparative analysis between B. vulgaris and A. thaliana revealed a remarkable degree of synteny between a large part of linkage groups 1 and 4 of B. vulgaris harboring the two QTL for flea beetle resistance and Arabidopsis chromosomes 3 and 1. Gene candidates that may underlie QTL for resistance and saponin biosynthesis are discussed.

Identification of defense compounds in Barbarea vulgaris against the herbivore Phyllotreta nemorum by an ecometabolomic approach.

Winter cress (Barbarea vulgaris) is resistant to a range of insect species. Some B. vulgaris genotypes are resistant, whereas others are susceptible, to herbivory by flea beetle larvae (Phyllotreta nemorum). Metabolites involved in resistance to herbivory by flea beetles were identified using an ecometabolomic approach. An F2 population representing the whole range from full susceptibility to full resistance to flea beetle larvae was generated by a cross between a susceptible and a resistant B. vulgaris plant. This F2 offspring was evaluated with a bioassay measuring the ability of susceptible flea beetle larvae to survive on each plant. Metabolites that correlated negatively with larvae survival were identified through correlation, cluster, and principal component analyses. Two main clusters of metabolites that correlate negatively with larvae survival were identified. Principal component analysis grouped resistant and susceptible plants as well as correlated metabolites. Known saponins, such as hederagenin cellobioside and oleanolic acid cellobioside, as well as two other saponins correlated significantly with plant resistance. This study shows the potential of metabolomics to identify bioactive compounds involved in plant defense.

Genetic differentiation between resistance phenotypes in the phytophagous flea beetle, Phyllotreta nemorum.

The flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae) is genetically polymorphic for resistance against the defences of one of its host plants, Barbarea vulgaris R.Br. (Brassicales: Brassicaceae). Whereas resistant flea beetles are able to use B. vulgaris as well as other cruciferous plants as food, non-resistant beetles cannot survive on B. vulgaris. This limitation to host plant use of non-resistant beetles could potentially lead to asymmetric gene flow and some degree of genetic isolation between the different resistance-genotypes. Therefore, we studied the extent of genetic differentiation at neutral allozyme loci between samples of flea beetles that were collected at different locations and first tested for resistance phenotype. Since earlier work has shown a weak, but significant, effect of geographical distance between the samples on their genetic differentiation, in the present study variation at the neutral allozyme loci in P. nemorum was partitioned between geographical distance and resistance-phenotype. Both sources independently contributed statistically significantly to population differentiation. Thus, there appears to be a limitation to genetic exchange between the resistant and non-resistant flea beetles when corrections are made for their geographic differentiation. This is consistent with the presence of some degree of host race formation in this flea beetle.

Glucosinolates, flea beetle resistance, and leaf pubescence as taxonomic characters in the genus Barbarea (Brassicaceae).

Glucosinolate content of leaves and roots, diversity in leaf pubescence, and resistance to two near-isogenic lines of the flea beetle Phyllotreta nemorum with or without an R-gene, were determined for 27 accessions of 7 Barbarea taxa, i.e. B. stricta, B. orthoceras, B. intermedia, B. verna, B. vulgaris var. vulgaris, the G-type of B. vulgaris var. arcuata and the P-type of B. vulgaris var. arcuata. Four variable glucosinolate biosynthetic characters were deduced. For (formally) homophenylalanine-derived glucosinolates: (1). Presence or absence of 2-hydroxylation, and if present, R- or S-configuration of 2-hydroxylation; (2). presence or absence of p-hydroxylation; and for tryptophan-derived glucosinolates: (3). presence or absence of N-methoxyglucobrassicin; and (4). presence or absence of 1,4-dimethoxyglucobrassicin. Three phenotypes of leaf-pubescence were observed; (1). glabrous to glabrate leaves; (2). glabrous to glabrate leaves with hairs along the edge; (3). pubescent leaves. The hairs were characterized as simple by scanning electron microscopy. Full resistance to a flea beetle line (ST) was found in B. vulgaris var. vulgaris and in the G-type of var. arcuata; partial resistance was found in B. verna and B. intermedia, while the remaining taxa were fully susceptible to the ST line. All investigated Barbarea taxa were susceptible to larvae from another line containing an R-gene, indicating a similar flea beetle resistance mechanism in the three resistant species. Most Barbarea taxa could be characterized by a particular combination of the investigated characters. The most aberrant was the P-type of B. vulgaris var. arcuata, and the taxonomic status of this type should be reconsidered.