NtMYB4 and NtCHS1 Are Critical Factors in the Regulation of Flavonoid Biosynthesis and Are Involved in Salinity Responsiveness

Abstract

High levels of salinity induce serious oxidative damage in plants. Flavonoids, as antioxidants, have important roles in reactive oxygen species (ROS) scavenging. In the present study, the tobacco R2R3 MYB type repressor, NtMYB4, was isolated and characterized. The expression of NtMYB4 was suppressed by salinity. Overexpression of NtMYB4 reduced the salt tolerance in transgenic tobacco plants. NtMYB4 repressed the promoter activity of NtCHS1 and negatively regulated its expression. Rutin accumulation was significantly decreased in NtMYB4 overexpressing transgenic plants and NtCHS1 RNAi silenced transgenic plants. Moreover, high H₂O₂ and ${\text{O}}_2^{-}$ contents were detected in both types of rutin-reduced transgenic plants under high salt stress. In addition, exogenous rutin supplementation effectively scavenged ROS (H₂O₂ and ${\text{O}}_2^{-}$ ) and improved the salt tolerance of the rutin-reduced transgenic plants. In contrast, NtCHS1 overexpressing plants had increased rutin accumulation, lower H₂O₂ and ${\text{O}}_2^{-}$ contents, and higher tolerance to salinity. These results suggested that tobacco NtMYB4 acts as a salinity response repressor and negatively regulates NtCHS1 expression, which results in the reduced flavonoid accumulation and weakened ROS-scavenging ability under salt stress.

Keywords: NtCHS1; NtMYB4; ROS level; flavonoid pathway; salt stress.

FIGURE 1

NtMYB4 is a nuclear localized transcription repressor in tobacco. (A) Subcellular localization analysis of NtMYB4. An NtMYB4-GFP construct was transformed into leaves of N. benthamiana and examined by confocal laser scanning microscopy. A confocal micrograph is shown at the left (green fluorescent protein, GFP), the corresponding differential interference contrast (bright) image is in the middle, and the merged image is at the right. Bars, 20 μm. (B) Phylogenetic tree based on the amino acid sequences of NtMYB4 and 10 other R2R3 MYB subgroup 4 members. The NtMYB4 isolated in this study is highlighted by a black dot. Accession numbers for MYB subgroup 4 sequences are: AmMYB308 (JQ0960), AmMYB330 (P81395), AtMYB4 (AY519615), AtMYB7 (AEC06531), AtMYB32 (NP_195225), EgMYB1 (CAE09058), HvMYB1 (P20026), TaMYB4 (AAT37167), ZmMYB31 (CAJ42202), and ZmMYB42 (CAJ42204). (C) The expression pattern of NtMYB4 in tobacco root, stem, leaf flower and bud was detected by qRT-PCR. Expression data were detected in two representative growth stages of common tobacco, including the seedling (S) and mature (M) stages. Organs sampled included leaf (L), stem (S), root (R), flower (F). Asterisks in (C) indicate differences between tissues (^∗P < 0.05, ^∗∗P < 0.01). (D) UAS/GAL4-based transcriptional repression assay of NtMYB4 in N. benthamiana leaves. NtMYB4 repressed the 35S-driven GUS activity. Pairwise combinations of constructs as indicated were co-infiltrated into tobacco leaves and stained for GUS activity. The left leaf marked GAL4-BD is 35S-driven GAL4 DNA binding domain (BD) as control; the right leaf marked NtMYB4+GAL4-BD was 35S-driven full-length NtMYB4 fused to the Gal4-BD domain.

FIGURE 2

Overexpression of NtMYB4 significantly reduces salt tolerance. The relative expression levels were normalized to the expression of the Tob103 gene (internal control). The relative expression of NtMYB4 in leaves of WT plants was set to 1. (A) The expression of NtMYB4 in leaves of WT plants was significantly suppressed by the addition of 200 mM NaCl in the medium. Asterisks in (A) indicate significant difference (^∗∗P < 0.01) between control condition and salt treatment. (B) The expression level of NtMYB4 in leaves was significantly increased in overexpressing transgenic tobacco compared to the WT. (C,D) The seed germination of WT and three NtMYB4 overexpressing transgenic tobacco lines under 200 mM NaCl. (E,F) The root elongation of WT and three NtMYB4 overexpressing transgenic tobacco lines under 200 mM NaCl. Data are expressed as the mean ± SD as determined from three independent biological replicates. Values in (F) that are significantly different between treatments and among lines within each treatment are marked with ^∗∗P < 0.01 and ^∗P < 0.05.

FIGURE 3

Supplementation with rutin increases salt tolerance of tobacco. (A) Rutin accumulation was significantly decreased in the leaves of NtMYB4 overexpressing transgenic tobacco lines compared to WT. DW, dry weight. Asterisks in (A) indicate significant difference (^∗∗P < 0.01) between NtMYB4-OE and WT. (B,C) The root length of WT and three NtMYB4 overexpressing transgenic lines under 200 mM NaCl with or without 100 μM rutin. Data are expressed as the mean ± SD as determined from three independent biological replicates. Values in (C) that are significantly different between treatments and among lines within each treatment are marked with ^∗∗P < 0.01 and ^∗P < 0.05.

FIGURE 4

Rutin effectively scavenges the ROS levels under salt stress. (A) DAB or NBT staining of WT plants under control (CK) and salt (NaCl) treatment with or without rutin supplementation. (B,C) H₂O₂ or ${\text{O}}_2^{-}$ content of WT plants and NtMYB4 overexpressing transgenic lines under control (CK) and salt (NaCl) treatment with or without rutin supplementation, measured by visible spectrophotometry. FW, fresh weight. Data are expressed as the mean ± SD as determined from three independent biological replicates. Values in (B,C) that are significantly different between treatments and among lines within each treatment are marked with ^∗∗P < 0.01 and ^∗P < 0.05.

FIGURE 5

NtMYB4 negatively regulates the expression of flavonoid pathway genes. (A) The expression level of structural genes in NtMYB4 overexpressing transgenic plants were significant inhibited compared to WT. The relative expression of structural genes for the WT control were set to 1. (B) Dual luciferase (LUC) assays of NtMYB4 (effector) and the promoters fused to pGreen luciferase plasmid (reporter). Empty vector was used as the effector in the control assay. The promoters of PAL, C4H, 4CL, CHI, CHS1, FLS, and ANS genes were used in dual luciferase assays (n = 3). Data are expressed as the mean ± SD as determined from three independent biological replicates. Asterisks indicate that the value is significantly different from that of the control (^∗P < 0.05, ^∗∗P < 0.01).

FIGURE 6

NtCHS1 RNAi silenced transgenic plants show a similar phenotype to NtMYB4 overexpressing transgenic plants in salt stress. (A) The expression level of NtCHS1 decreased in RNAi silenced T₀ transgenic tobacco leaves compared to WT. Asterisks in (A) indicate remarkable difference (^∗∗P < 0.01) between NtCHS1 RNAi transgenic lines and WT. (B) Rutin content decreased in NtCHS1 RNAi silenced transgenic tobacco lines compared to WT. DW, dry weight. Asterisks in (B) indicate remarkable difference (^∗P < 0.05) between NtCHS1 RNAi transgenic lines and WT. (C,D) Root length of WT and NtCHS1 RNAi transgenic plants treated with salt stress with or without exogenous rutin. Values in (D) that are significantly different between treatments and among lines within each treatment are marked with ^∗∗P < 0.01 and ^∗P < 0.05. (E,F) H₂O₂ or ${\text{O}}_2^{-}$ content of WT plants and NtCHS1 RNAi silenced transgenic lines under control (CK) and salt treatment (NaCl) with or without rutin supplementation, measured by visible spectrophotometry. FW, fresh weight. Data are expressed as the mean ± SD as determined from three independent biological replicates. Values in (E,F) that are significantly different between treatments and among lines within each treatment are marked with ^∗∗P < 0.01 and ^∗P < 0.05.

FIGURE 7

NtCHS1 overexpressing transgenic plants significantly increase salt tolerance. (A) The expression level of NtCHS1 in leaves was significantly increased in overexpressing T₀ transgenic tobacco leaves compared to WT. Asterisks in (A) indicate remarkable difference (^∗∗P < 0.01) between NtCHS1 overexpressing transgenic lines and WT. (B) Rutin content increased in the leaves of typical NtCHS1 overexpressing transgenic tobacco lines compared to WT. Asterisks in (B) indicate remarkable difference (^∗P < 0.05) between NtCHS1 overexpressing transgenic lines and WT. (C,D) Root length of WT and NtCHS1 overexpressing transgenic plants (CO-3 line) treated with salt stress. Values in (D) that are significantly different between treatments and among lines within each treatment are marked with ^∗∗P < 0.01 and ^∗P < 0.05. (E,F) H₂O₂ or ${\text{O}}_2^{-}$ content of WT plants and NtCHS1 overexpressing transgenic lines under control (CK) and salt treatment (NaCl), measured by visible spectrophotometry. FW, fresh weight. Data are expressed as the mean ± SD as determined from three independent biological replicates. Values in (E,F) that are significantly different between treatments and among lines within each treatment are marked with ^∗∗P < 0.01 and ^∗P < 0.05.