MicroRNA-Mediated Post-Transcriptional Regulation of Enzymes Involved in Herbicide Resistance in Echinochloa oryzicola (Vasinger) Vasinger

Abstract

Herbicide resistance is an emerging phytosanitary threat, causing serious yield and economic losses. Although this phenomenon has been widely studied, only recently has the role of epigenetic factors in its occurrence been considered. In the present study, we analyzed the microRNA-mediated regulation in Echinochloa oryzicola (Vasinger) Vasinger (late-watergrass) of the expression of cytochromes P450, glutathione S-transferase (GST), and eIF4B, all of which are enzymes involved in profoxydim (AURA^®) detoxification. Before and after profoxydim application, the expression profiles of microRNAs (miRNAs) were selected for their ability to target the genes considered, and their targets were assessed by means of RT-qPCR. Susceptible and resistant biotypes showed different responses to this herbicide. After profoxydim application, in resistant biotypes, osa-miR2099-5p, ath-miR396b, osa-miR395f, osa-miR396a-5p, osa-miR166a-5p, osa-miR166d-5p, gra-miR8759, and gma-miR396f were not triggered, allowing the expression of CYP81A, GSTF1, and eIF4B genes and the herbicide's detoxification. Meanwhile, the transcription of ata-miR166c-5p, ath-miR847, osa-miR5538, and gra-miR7487c was triggered, down-regulating CYP71AK2, CYP72A254, CYP72A122, and EcGST expression. In susceptible biotypes, the herbicide stimulated ata-miR166c-5p, ath-miR847, osa-miR5538, gra-miR7487c, osa-miR166a-5p, and gra-miR8759, down-regulating their respective target genes (CYP72A122, CYP71AK2, EcGST, CYP72A254, CYP81A12, and eIF4B). A better understanding of the role of miRNA-mediated epigenetic regulation in herbicide resistance will be useful in planning more targeted and sustainable methods for controlling this phytosanitary threat.

Keywords: CYP450; CYP81A; GST; eIF4B; late-watergrass; microRNA; profoxydim resistance.

Figure 1

Expression levels of the miRNAs studied (red) and the mRNA of their target genes (blue) in susceptible (SUS) and resistant (RES1 and RES2) biotypes of Echinochloa oryzicola before (BT) and after (AT) profoxydim treatment. Mean and standard error values are indicated as bar plots and error bars, respectively. The expressions of genes and miRNAs were normalized with those of the respective internal references (b-Actin and non-coding small nuclear RNA U6). (A) CYP81A12 and osa-miR166a-5p; (B) CYP81A21 and osa-miR166d-5p; (C) CYP81A22 and ath-miR396b-5p; (D) CYP81A24 and osa-miR2099-5p; (E) CYP81A6 and osa-miR395f; (F) CYP81A63 and osa-miR396a-5p. t-test: *: p < 0.05.

Figure 2

Expression levels of the miRNAs studied (red) and the mRNA of their target genes (blue) in susceptible (SUS) and resistant (RES1 and RES2) biotypes of Echinochloa oryzicola before (BT) and after (AT) profoxydim treatment. Mean and standard error values are indicated as bar plots and error bars, respectively. The expressions of genes and miRNAs were normalized with those of the respective internal references (b-Actin and non-coding small nuclear RNA U6). (A) CYP71AK2 and ath-miR847; (B) CYP72A122 and ata-miR166c-5p; (C) CYP72A254 and gra-miR7487c; (D) EcGST and osa-miR5538; (E) GSTF1 and gma-miR396f; (F) eIF4B and gra-miR8759. t-test: *: p < 0.05.

Figure 3

Radar chart of the expression profiles of genes (blue line) and miRNAs (red line) in the SUS biotype (A), in the RES1 biotype (B), and in the RES2 biotype (C) after profoxydim treatment. ata—Aegilops tauschii Coss.; ath—Arabidopsis thaliana (L.) Heynh; gma—Glycine max (L.) Merr.; gra—Gossypium raimondii Ulbr.; osa—Oryza sativa L.