Aromatic Glucosinolate Biosynthesis Pathway in Barbarea vulgaris and its Response to Plutella xylostella Infestation

Abstract

The inducibility of the glucosinolate resistance mechanism is an energy-saving strategy for plants, but whether induction would still be triggered by glucosinolate-tolerant Plutella xylostella (diamondback moth, DBM) after a plant had evolved a new resistance mechanism (e.g., saponins in Barbara vulgaris) was unknown. In B. vulgaris, aromatic glucosinolates derived from homo-phenylalanine are the dominant glucosinolates, but their biosynthesis pathway was unclear. In this study, we used G-type (pest-resistant) and P-type (pest-susceptible) B. vulgaris to compare glucosinolate levels and the expression profiles of their biosynthesis genes before and after infestation by DBM larvae. Two different stereoisomers of hydroxylated aromatic glucosinolates are dominant in G- and P-type B. vulgaris, respectively, and are induced by DBM. The transcripts of genes in the glucosinolate biosynthesis pathway and their corresponding transcription factors were identified from an Illumina dataset of G- and P-type B. vulgaris. Many genes involved or potentially involved in glucosinolate biosynthesis were induced in both plant types. The expression patterns of six DBM induced genes were validated by quantitative PCR (qPCR), while six long-fragment genes were validated by molecular cloning. The core structure biosynthetic genes showed high sequence similarities between the two genotypes. In contrast, the sequence identity of two apparent side chain modification genes, the SHO gene in the G-type and the RHO in P-type plants, showed only 77.50% identity in coding DNA sequences and 65.48% identity in deduced amino acid sequences. The homology to GS-OH in Arabidopsis, DBM induction of the transcript and a series of qPCR and glucosinolate analyses of G-type, P-type and F1 plants indicated that these genes control the production of S and R isomers of 2-hydroxy-2-phenylethyl glucosinolate. These glucosinolates were significantly induced by P. xylostella larvae in both the susceptiple P-type and the resistant G-type, even though saponins are the main DBM-resistance causing metabolites in G-type plants. Indol-3-ylmethylglucosinolate was induced in the G-type only. These data will aid our understanding of the biosynthesis and induction of aromatic glucosinolates at the molecular level and also increase our knowledge of the complex mechanisms underpinning defense induction in plants.

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

“…QTLs were detected for both G-type glucobarbarin ( S -configuration) and P-type epi-glucobarbarin ( R -configuration) on different linkage groups, clearly showing that different genes are involved. This is supported by recent transcriptomics analyses suggesting two related but quite diverged genes are responsible for the hydroxylations21. QTLs for G-type saponins have also been identified, together with genes involved in their biosynthesis17.…”

“…2, alignments in Supplementary Fig. 11); the gene encodes an enzyme that is 82 % identical to CYP79F1 and has been named CYP79F6 by the P450 nomenclature committee21. The gene is highly expressed and induced by diamondback moth infestation as expected for a gene responsible for biosynthesis of phenethylglucosinolate and derivatives such as glucobarbarins21.…”

“…11); the gene encodes an enzyme that is 82 % identical to CYP79F1 and has been named CYP79F6 by the P450 nomenclature committee21. The gene is highly expressed and induced by diamondback moth infestation as expected for a gene responsible for biosynthesis of phenethylglucosinolate and derivatives such as glucobarbarins21. If CYP79F6 is responsible for the committed biosynthetic step to phenethylglucosinolate and glucobarbarins21, the apparent lack of methionine derived glucosinolates would seem to be due to a changed substrate specificity37 of CYP79F6.…”

“…Additional hydroxylation in the P-type leads to other P-type specific glucosinolates and hydrolysis products2. The biosynthetic pathway of glucobarbarins was recently proposed21. In general the P-type deviates markedly from the G-type and other investigated Barbarea species19, and is for this reason regarded as an ‘innovative’ evolutionary lineage with respect to specialized metabolites, including a number of rare and even unique glucosinolates and saponins101517.…”

Biosynthesis and bioactivity of glucosinolates and their production in plant in vitro cultures

“…There are three different types of glucosinolates depending on the amino acid nature: aliphatic glucosinolates derived from leucine, valine, methionine, isoleucine and alanine; indole glucosinolates derived from tryptophan and aromatic glucosinolates derived from phenylalanine or tyrosine. The biosynthetic pathway of aliphatic and indole glucosinolates is well known, however the biosynthetic pathway of aromatic glucosinolates remains largely unknown (Liu et al 2016). …”