Abstract
The first step of the benzoxazinoid (BX) synthesis pathway is catalyzed by an enzyme with indole-3-glycerol phosphate lyase activity encoded by 3 genes, Bx1, TSA and Igl. A gene highly homologous to maize and wheat Bx1 has been identified in rye. The goal of the study was to analyze the gene and to experimentally verify its role in the rye BX biosynthesis pathway as a rye ortholog of the Bx1 gene. Expression of the gene showed peak values 3 days after imbibition (dai) and at 21 dai it was undetectable. Changes of the BX content in leaves were highly correlated with the expression pattern until 21 dai. In plants older than 21 dai despite the undetectable expression of the analyzed gene there was still low accumulation of BXs. Function of the gene was verified by correlating its native expression and virus-induced silencing with BX accumulation. Barley stripe mosaic virus (BSMV)-based vectors were used to induce transcriptional (TGS) and posttranscriptional (PTGS) silencing of the analyzed gene. Both strategies (PTGS and TGS) significantly reduced the transcript level of the analyzed gene, and this was highly correlated with lowered BX content. Inoculation with virus-based vectors specifically induced expression of the analyzed gene, indicating up-regulation by biotic stressors. This is the first report of using the BSMV-based system for functional analysis of rye gene. The findings prove that the analyzed gene is a rye ortholog of the Bx1 gene. Its expression is developmentally regulated and is strongly induced by biotic stress. Stable accumulation of BXs in plants older than 21 dai associated with undetectable expression of ScBx1 indicates that the function of the ScBx1 in the BX biosynthesis is redundant with another gene. We anticipate that the unknown gene is a putative ortholog of the Igl, which still remains to be identified in rye.
Highlights
Benzoxazinoids (BXs) are a group of defense-related molecules accumulated as glycosylated precursors in grasses and sporadically in a few dicot species [1]
It is worthwhile to point that the benzoxazineless1 mutation was originally used to identify and to clone Bx1, the gene itself and the encoded protein (BX1) are not the only ones responsible for synthesis of indole, which is the substrate for the BX biosynthesis pathway
Conversion of indole-3-glycerol phosphate to indole is catalyzed by enzymes encoded by at least three different genes: Bx1, tryptophan synthase alpha (TSA) and Igl
Summary
Benzoxazinoids (BXs) are a group of defense-related molecules accumulated as glycosylated precursors in grasses and sporadically in a few dicot species [1]. The first reports published in the 1950s identified the BX as components of rye resistance against Fusarium [2] and maize resistance to the European corn borer Ostrinia nubilalis [3]. Further studies confirmed the diverse roles of BX in innate immunity against pests and fungal diseases in maize [4, 5], wheat [6] and rye [7]. Nutritional investigations revealed that the BXs themselves or the BX-derived compounds found in rye or wheat food products show potentially health-promoting activities [11]. The diverse spectrum of biological functions as well as the possible nutritional values caused that the BXs became a widely studied group of secondary metabolites in plants
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