AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco

Abstract

In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants.

Keywords: Epigenetic regulation; ROS1 overexpression; coding regions; methylation status; promoters; salt stress; tobacco..

Fig. 1.

Confirmation of AtROS1 expression in transgenic tobacco by reverse transcription PCR analysis. The expression analysis of 26SrRNA was used as internal control. R1, internal primer 1; R2, internal primer 2; Bar, Basta selection marker-specific primer.

Fig. 2.

Salt-stress response of AtROS1 transgenic tobacco plants. (A) Morphological affect of salt stress in wild-type and transgenic lines. Fifteen-day-old seedlings of wild-type and transgenic lines grown on MS medium supplemented with different NaCl concentrations for 30 days and photographed. (B) Fresh weight of wild-type and transgenic seedlings treated with different concentrations of NaCl for 30 days. Different letters on error bar represents significant differences within treatment group, i.e. data marked ‘a’ is significantly different from data marked ‘b’ with P < 0.05.

Fig. 3.

Influence of AtROS1 overexpression on relative transcript expression of genes encoding enzymes of flavonoid biosynthetic pathway in transgenic tobacco. Relative transcript expression of genes encoding enzymes (A) CHS, (B) CHI, (C) F3H, (D) FLS, (E) DFR, and (F) ANS under normal and salt stress conditions in wild type and transgenic lines. 26SrRNA was used as internal control in the expression analyses experiment. Different letters on error bar represents significant differences within treatment group, i.e. data marked ‘a’ is significantly different from data marked ‘b’ with P < 0.05.

Fig. 4.

Influence of AtROS1 overexpression on relative transcript expression of genes encoding enzymes of antioxidative system. Relative transcript expression of genes encoding enzymes (A) GST, (B) APx, (C) GPx, and (D) GR under normal and salt stress conditions in wild-type and transgenic lines. 26SrRNA was used as internal control in the expression analyses experiment. Different letters on error bar represents significant differences within treatment group, i.e. data marked ‘a’ is significantly different from data marked ‘b’ with P < 0.05.

Fig. 5.

Total demethylase activity of wild-type and AtROS1 transgenic tobacco lines under normal and salt stress conditions. Different alphabet on error bar represents significant difference. Different letters on error bar represents significant differences within treatment group, i.e. data marked ‘a’ is significantly different from data marked ‘b’ with P < 0.05, and from data marked ‘c’ with P < 0.01.

Fig. 6.

Methylation pattern of coding regions of genes encoding enzymes of the flavonoid and antioxidative pathways by methylation-specific PCR in wild-type and transgenic tobacco lines under normal and salt stress conditions. Methylation pattern of coding regions of genes encoding enzymes. M, methylated; U, unmethylated.