The transcription factor TaMYB31 regulates the benzoxazinoid biosynthetic pathway in wheat

Abstract

Benzoxazinoids are specialized metabolites that are highly abundant in staple crops, such as maize and wheat. Although their biosynthesis has been studied for several decades, the regulatory mechanisms of the benzoxazinoid pathway remain unknown. Here, we report that the wheat transcription factor MYB31 functions as a regulator of benzoxazinoid biosynthesis genes. A transcriptomic analysis of tetraploid wheat (Triticum turgidum) tissue revealed the up-regulation of two TtMYB31 homoeologous genes upon aphid and caterpillar feeding. TaMYB31 gene silencing in the hexaploid wheat Triticum aestivum significantly reduced benzoxazinoid metabolite levels and led to susceptibility to herbivores. Thus, aphid progeny production, caterpillar body weight gain, and spider mite oviposition significantly increased in TaMYB31-silenced plants. A comprehensive transcriptomic analysis of hexaploid wheat revealed that the TaMYB31 gene is co-expressed with the target benzoxazinoid-encoded Bx genes under several biotic and environmental conditions. Therefore, we analyzed the effect of abiotic stresses on benzoxazinoid levels and discovered a strong accumulation of these compounds in the leaves. The results of a dual fluorescence assay indicated that TaMYB31 binds to the Bx1 and Bx4 gene promoters, thereby activating the transcription of genes involved in the benzoxazinoid pathway. Our finding is the first report of the transcriptional regulation mechanism of the benzoxazinoid pathway in wheat.

Keywords: Rhopalosiphum padi aphid; Spodoptera littoralis caterpillar; Tetranychus urticae spider mite; Triticum aestivum; Abiotic stress; Triticum turgidum; biotic stress; herbivores; specialized metabolites.

Fig. 1.

Effects of TaMYB31 gene silencing on benzoxazinoid content. RNA and metabolites were extracted from the second leaves of wheat plants at 14 dpi BSMV-VIGS containing the TaMYB31 gene. (A) Expression of TaMYB31 by qRT–PCR using two reference genes Glyceraldehyde-3-phosphate dehydrogenase (GAPC) and Actin (ACT-1) (n=4). (B) Left panel, BXD metabolite levels presented as mg g^–1 FW. Right panel, BXD metabolite levels in peak area from chromatogram (n=10–11). The comparison was made using Student’s t-test relative to BSMV::empty vector, P<0.05. Asterisks indicate a significant difference. In this figure, a single independent biological experiment is presented.

Fig. 2.

Gene silencing of TaMYB31 affects insect herbivory susceptibility. Insect bioassay was performed on BSMV-VIGS-inoculated plants. (A) Expression of TaMYB31 by qRT–PCR using two reference genes Glyceraldehyde-3-phosphate dehydrogenase (GAPC) and Actin (ACT-1) (n=5). Data are presented as means from a single independent biological experiment ±SE. Asterisks indicate a significant difference, Student’s t-test P<0.05. (B) Aphid (Rhopalosiphum padi) progeny production after 4 d (n=5); caterpillar (Spodoptera littoralis) weight after 3 d (n=10); two-spotted spider mite (Tetranychus urticae) oviposition after 3 d (n=12).

Fig. 3.

TaMYB31 activates promoters of genes from the benzoxazinoid pathway. TaMYB31 protein and Bx gene promoter interactions were evaluated using a dual fluorescence system based on transient expression. Background promoter activity was assayed by infiltration of the same empty vector for each gene without a TaMYB31 cassette. Promoter sequences of the following genes were analyzed: IGPS3 (TraesCS7B02G423900), Bx1 (TraesCS7B02G294800), Bx3 (TraesCS5B02G007200), Bx4a (TraesCS5A02G008800), Bx4b (TraesCS5B02G007100), Bx8/9 (TraesCS7B02G016800), and TaKCS1 (KU737579). Shown are means of the GFP/RFP reporters ratio ±SE. Asterisks indicate a significant difference by Student’s t-test: *P<0.05; ***P<0.001 (n=5). In this figure, a single independent biological experiment is presented.

Fig. 4.

Environmental stresses induce BXD levels in wheat leaves. (A) BXD profile of plants grown under drought conditions. (B) BXD profile of plants affected by polyethylene glycol (PEG) treatment. (C) BXD levels of plants grown under either low temperature or salt stress conditions. Asterisks indicate a significant difference, P<0.05 by Student’s t-test (n=7–8). ND, not detected.