CsCYT75B1, a Citrus CYTOCHROME P450 Gene, Is Involved in Accumulation of Antioxidant Flavonoids and Induces Drought Tolerance in Transgenic Arabidopsis

Abstract

CYTOCHROME P450s genes are a large gene family in the plant kingdom. Our earlier transcriptome data revealed that a CYTOCHROME P450 gene of Citrus sinensis (CsCYT75B1) was associated with flavonoid metabolism and was highly induced after drought stress. Here, we characterized the function of CsCYT75B1 in drought tolerance by overexpressing it in Arabidopsis thaliana. Our results demonstrated that the overexpression of the CsCYT75B1 gene significantly enhanced the total flavonoid contents with increased antioxidant activity in transgenic Arabidopsis. The gene expression results showed that several genes that are responsible for the biosynthesis of antioxidant flavonoids were induced by 2-12 fold in transgenic Arabidopsis lines. After 14 days of drought stress, all transgenic lines displayed an enhanced tolerance to drought stress along with accumulating antioxidant flavonoids with lower superoxide radicals and reactive oxygen species (ROS) than wild type plants. In addition, drought-stressed transgenic lines possessed higher antioxidant enzymatic activities than wild type transgenic lines. Moreover, the stressed transgenic lines had significantly lower levels of electrolytic leakage than wild type transgenic lines. These results demonstrate that the CsCYT75B1 gene of sweet orange functions in the metabolism of antioxidant flavonoid and contributes to drought tolerance by elevating ROS scavenging activities.

Keywords: antioxidant enzymatic activity; antioxidant flavonoids; citrus; drought stress; transgenic Arabidopsis.

Figure 1

Phylogenetic analysis and gene expression pattern of the CsCYT75B1 (CYT) gene. (A) Phylogenetic analysis of CsCYT75B1 gene with its homologous genes from other plant species. (B) Gene expression pattern of CsCYT75B1 in overexpressed (OX) Arabidopsis lines. Wild type (WT), and empty vector (EV) plants. A Students t-test was used to compare CYT-OX and WT at ** p < 0.01. Each value is a mean of three replicates.

Figure 2

Drought stress effects on transgenic and wild type plants. (A) Roots of overexpressed lines, empty vector, and wild type plants after 14 days of polyethylene glycol (PEG-6000) stress. (B) Before drought stress (DS), overexpressed (OX), and wild type (WT) plants. (C) OX and WT plants after 14 days of drought stress. (D) Root length of WT and empty vector (EV) plants compared to the transgenic lines under PEG stress. A Students t-test was used to compare the CYT-OX and WT plants at ** p < 0.01. Each value is a mean of three replicates.

Figure 3

The gene expressions, total flavonoid contents and antioxidant activities of wild type and overexpressed lines without stress: (A) Total flavonoid contents, (B) antioxidant activity, (C) antioxidant capacity, (D) AT5G08640 flavonol synthase (FLS), (E) AT5G13930 TT4, (F) AT3G55120 TT5, and (G) AT3G51240 TT6. (WT) wild type and (EV) empty vector. A Student’s t-test was used to compare the CYT-OX and WT plants at * p < 0.05 and ** p < 0.01. Each value is a mean of three replicates.

Figure 3

The gene expressions, total flavonoid contents and antioxidant activities of wild type and overexpressed lines without stress: (A) Total flavonoid contents, (B) antioxidant activity, (C) antioxidant capacity, (D) AT5G08640 flavonol synthase (FLS), (E) AT5G13930 TT4, (F) AT3G55120 TT5, and (G) AT3G51240 TT6. (WT) wild type and (EV) empty vector. A Student’s t-test was used to compare the CYT-OX and WT plants at * p < 0.05 and ** p < 0.01. Each value is a mean of three replicates.

Figure 4

The q-PCR relative expression data of 6 genes under drought stress: (A) AT5G13930 TT4, (B) AT3G55120 TT5, (C) AT3G51240 TT6, (D) AT5G08640 FLS, (E) AT3G13610 F6′H1, and (F) AT4G22880 TT18. Gene IDs were taken from Arabidopsis genome website TAIR (https://www.arabidopsis.org/). FLS: flavonol synthase. 14DDS: after 14 days of drought stress. WT: wild type. EV: empty vector. A Students t-test was used to compare the CYT-OX and WT plants at * p < 0.05 and ** p < 0.01. Each value is a mean of three replicates.

Figure 5

Various metabolic and antioxidant-related parameters. (A) Total phenolic contents (TPCs). (B) Total flavonoid contents (TFCs), (C) Total anthocyanin contents (TACs). (D) Antioxidant activity (E) Antioxidant capacity. 14DDS: after 14 days of drought stress. WT: wild type. EV: empty vector. A Students t-test was used to compare the CYT-OX and WT plants at * p < 0.05 and ** p < 0.01. Each value is a mean of three replicates.

Figure 6

Antioxidant enzymatic activities under drought stress. (A) Superoxide dismutase (SOD), (B) Peroxidase (POD), and (C) Catalase (CAT). 14DDS: after 14 days of drought stress. WT: wild type. EV: empty vector. A Students t-test was used to compare the CYT-OX and WT plants at * p < 0.05 and ** p < 0.01. Each value is a mean of three replicates.

Figure 7

Drought stress effects on transgenic and wild type plants. (A) Trypan blue staining effect on OX and WT plants. (B) Nitro-blue tetrazolium (NBT) staining effect on OX and WT plants. (C) Hydrogen peroxide (H₂O₂ content). (D) Malondialdehyde (MDA). (E) Electrolytic leakage (EL). (F) Superoxide radicals production rate (O₂⁻). (G) Reactive oxygen species (ROS). (H) Chlorophyll a (I) Chlorophyll b. 14DDS: after 14 days of drought stress. WT: wild type. EV: empty vector. OX: overexpressed lines. A Students t-test was used to compare the CYT-OX and WT plants at * p < 0.05 and ** p < 0.01. Each value is a mean of three replicates.

Figure 7

Drought stress effects on transgenic and wild type plants. (A) Trypan blue staining effect on OX and WT plants. (B) Nitro-blue tetrazolium (NBT) staining effect on OX and WT plants. (C) Hydrogen peroxide (H₂O₂ content). (D) Malondialdehyde (MDA). (E) Electrolytic leakage (EL). (F) Superoxide radicals production rate (O₂⁻). (G) Reactive oxygen species (ROS). (H) Chlorophyll a (I) Chlorophyll b. 14DDS: after 14 days of drought stress. WT: wild type. EV: empty vector. OX: overexpressed lines. A Students t-test was used to compare the CYT-OX and WT plants at * p < 0.05 and ** p < 0.01. Each value is a mean of three replicates.