Phytoalexins of the crucifer Barbarea vulgaris: Structural profile and correlation with glucosinolate turnover.

Phytoalexins are antimicrobial plant metabolites elicited by microbial attack or abiotic stress. We investigated phytoalexin profiles after foliar abiotic elicitation in the crucifer Barbarea vulgaris and interactions with the glucosinolate-myrosinase system. The treatment for abiotic elicitation was a foliar spray with CuCl2 solution, a usual eliciting agent, and three independent experiments were carried out. Two genotypes of B. vulgaris (G-type and P-type) accumulated the same three major phytoalexins in rosette leaves after treatment: phenyl-containing nasturlexin D and indole-containing cyclonasturlexin and cyclobrassinin. Phytoalexin levels were investigated daily by UHPLC-QToF MS and tended to differ among plant types and individual phytoalexins. In roots, phytoalexins were low or not detected. In treated leaves, typical total phytoalexin levels were in the range 1-10 nmol/g fresh wt. during three days after treatment while typical total glucosinolate (GSL) levels were three orders of magnitude higher. Levels of some minor GSLs responded to the treatment: phenethylGSL (PE) and 4-substituted indole GSLs. Levels of PE, a suggested nasturlexin D precursor, were lower in treated plants than controls. Another suggested precursor GSL, 3-hydroxyPE, was not detected, suggesting PE hydrolysis to be a key biosynthetic step. Levels of 4-substituted indole GSLs differed markedly between treated and control plants in most experiments, but not in a consistent way. The dominant GSLs, glucobarbarins, are not believed to be phytoalexin precursors. We observed statistically significant linear correlations between total major phytoalexins and the glucobarbarin products barbarin and resedine, suggesting that GSL turnover for phytoalexin biosynthesis was unspecific. In contrast, we did not find correlations between total major phytoalexins and raphanusamic acid or total glucobarbarins and barbarin. In conclusion, two groups of phytoalexins were detected in B. vulgaris, apparently derived from the GSLs PE and indol-3-ylmethylGSL. Phytoalexin biosynthesis was accompanied by depletion of the precursor PE and by turnover of major non-precursor GSLs to resedine. This work paves the way for identifying and characterizing genes and enzymes in the biosyntheses of phytoalexins and resedine.

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.

Expression patterns, molecular markers and genetic diversity of insect-susceptible and resistant Barbarea genotypes by comparative transcriptome analysis.

BACKGROUND: Barbarea vulgaris contains two genotypes: the glabrous type (G-type), which confers resistance to the diamondback moth (DBM) and other insect pests, and the pubescent type (P-type), which is susceptible to the DBM. Herein, the transcriptomes of P-type B. vulgaris before and after DBM infestation were subjected to Illumina (Solexa) pyrosequencing and comparative analysis. RESULTS: 5.0 gigabase pairs of clean nucleotides were generated. Non-redundant unigenes (33,721) were assembled and 94.1 % of them were annotated. Compared with our previous G-type transcriptome, the expression patterns of many insect responsive genes, including those related to secondary metabolism, phytohormones and transcription factors, which were significantly induced by DBM in G-type plants, were less sensitive to DBM infestation in P-type plants. The genes of the triterpenoid saponin pathway were identified in both G- and P-type plants. The upstream genes of the pathway showed similar expression patterns between the two genotypes. However, gene expression for two downstream enzymes, the glucosyl transferase (UGT73C11) and an oxidosqualene cyclase (OSC), were significantly upregulated in the P-type compared with the G-type plant. The homologous genes from P- and G-type plants were detected by BLAST unigenes with a cutoff level E-value < e(-10). 12,980 gene families containing 26,793 P-type and 36,944 G-type unigenes were shared by the two types of B. vulgaris. 38,397 single nucleotide polymorphisms (SNPs) were found in 9,452 orthologous genes between the P- and G-type plants. We also detected 5,105 simple sequence repeats (SSRs) in the B. vulgaris transcriptome, comprising mono-nucleotide-repeats (2,477; 48.5 %) and triple-nucleotide-repeats (1,590; 31.1 %). Of these, 1,657 SSRs displayed polymorphisms between the P- and G-type. Consequently, 913 SSR primer pairs were designed with a resolution of more than two nucleotides. We randomly chose 30 SSRs to detect the genetic diversity of 32 Barbarea germplasms. The distance tree showed that these accessions were clearly divided into groups, with the G-type grouping with available Western and Central European B. vulgaris accessions in contrast to the P-type accession, B. stricta and B. verna. CONCLUSIONS: These data represent useful information for pest-resistance gene mining and for the investigation of the molecular basis of plant-pest interactions.