Molecular Characterization of an Isoflavone 2'-Hydroxylase Gene Revealed Positive Insights into Flavonoid Accumulation and Abiotic Stress Tolerance in Safflower

Abstract

Flavonoids with significant therapeutic properties play an essential role in plant growth, development, and adaptation to various environments. The biosynthetic pathway of flavonoids has long been studied in plants; however, its regulatory mechanism in safflower largely remains unclear. Here, we carried out comprehensive genome-wide identification and functional characterization of a putative cytochrome P45081E8 gene encoding an isoflavone 2'-hydroxylase from safflower. A total of 15 CtCYP81E genes were identified from the safflower genome. Phylogenetic classification and conserved topology of CtCYP81E gene structures, protein motifs, and cis-elements elucidated crucial insights into plant growth, development, and stress responses. The diverse expression pattern of CtCYP81E genes in four different flowering stages suggested important clues into the regulation of secondary metabolites. Similarly, the variable expression of CtCYP81E8 during multiple flowering stages further highlighted a strong relationship with metabolite accumulation. Furthermore, the orchestrated link between transcriptional regulation of CtCYP81E8 and flavonoid accumulation was further validated in the yellow- and red-type safflower. The spatiotemporal expression of CtCYP81E8 under methyl jasmonate, polyethylene glycol, light, and dark conditions further highlighted its likely significance in abiotic stress adaption. Moreover, the over-expressed transgenic Arabidopsis lines showed enhanced transcript abundance in OE-13 line with approximately eight-fold increased expression. The upregulation of AtCHS, AtF3'H, and AtDFR genes and the detection of several types of flavonoids in the OE-13 transgenic line also provides crucial insights into the potential role of CtCYP81E8 during flavonoid accumulation. Together, our findings shed light on the fundamental role of CtCYP81E8 encoding a putative isoflavone 2'-hydroxylase via constitutive expression during flavonoid biosynthesis.

Keywords: CYP81E8; constitutive expression; flavonoid biosynthesis; isoflavone 2′-hydroxylase; safflower; transgenic Arabidopsis.

Figure 1

The phylogenetic classification of CtCYP81E-encoding genes with other members of Arabidopsis P450s using a neighbor-joining (N-J) phylogenetic tree. MEGA-X software was used to generate the phylogeny tree. The different background colors demonstrated various clans of P450 subfamilies. The CtCYP81E-encoding genes were represented with red triangles in the phylogenetic tree.

Figure 2

The organization of gene structure, conserved motifs, and promoter analysis of CtCYP81E subfamily members (a) The organization of different components of CtCYP81E genes structure such as exon/intron organization and untranslated regions (UTRs). Untranslated regions (UTRs) were shown in blue, exons were depicted in yellow, and the total number of introns was shown in grey (b) conserved protein motifs distribution of CtCYP81E encoding proteins. The motif analysis was predicted with MEME webtool (c) promoter analysis of CtCYP81E encoding. The presence of different regulatory elements was shown with multiple colors at specific positions.

Figure 3

The illustration of the protein–protein interaction network of CtCYP81E encoding proteins. The online webserver of STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) was utilized to annotate the potential network of CtCYP81E8 using the model plant as an input. The red lines represented the presence of more than four different P450 proteins in each set of proteins. Below the Arabidopsis protein, in parenthesis, were the CtCYP81E proteins.

Figure 4

Expression analysis of CtCYP81E genes in safflower at different flowering stages. (a) the differential expression level of CtCYP81E genes using the FPKM values obtained from RNA-seq data. The expression data were illustrated as a heatmap in five different tissues. The extent of expression is shown by the color spectrum to the right, with red denoting strong expression and green denoting low expression. (b) The quantitative real-time expression variations of the CtCYP81E genes using qRT-PCR test in four flowering stages. The 18S rRNA gene was used to standardize the relative fold expression level, and the data were presented as means ± SE (n = 3).

Figure 5

Expression The reciprocal relation of CtCYP81E8 expression and accumulation of total metabolites in four different flowering stages of safflower (a) the quantitative expression analysis of CtCYP81E8 using qRT-PCR assay (b) four different flowering stages of safflower and accumulation of hydroxysafflor yellow pigment. Data were presented as means ± SE (n = 3), and the asterisks * denotes p < 0.05 and *** denotes p < 0.001.

Figure 6

The correlation analysis of CtCYP81E8 expression and flavonoid accumulation in yellow and red-type safflower varieties. Phenotype of (a) yellow-type safflower and (b) red-type safflower (c) qRT-PCR expression analysis of CtCYP81E8 and total flavonoid accumulation in four different flowering stages of yellow type safflower (d) qRT-PCR expression analysis of CtCYP81E8 and total flavonoid accumulation in four different flowering stages of red-type safflower. Data were presented as means ± SE (n = 3), and the asterisks * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001. Alpha-numeric codes denote; Y: yellow type safflower and R: red-type safflower. Y1/R1: bud flowering, Y2/R2: initial flowering, Y3/R3: full flowering, Y4/R4: Fade flowering.

Figure 7

The effects of methyl jasmonate, drought, and light and dark stress on safflower CtCYP81E8 gene expression. Six separate time periods (0, 12, 24, 36, 48, and 60 h) of relative fold expression data were displayed on graphs. Three biological replicates were analyzed to determine the variation in expression level. Data were presented as means ± SE (n = 3).

Figure 8

The relative expression analysis of CtCYP81E8 and core structural genes of flavonoid biosynthetic pathway in transgenic Arabidopsis. (a) The Phenotype of wild-type, OE-13, and OE-15 transgenic lines. (b) The expression level of CtCYP81E8 in different transgenic lines (OE1-15). (c) The expression level of key structural genes involved in the downstream regulatory pathway of flavonoid biosynthesis. Gene encoding these enzymes includes: Anthocyanidin reductase (ANR), CHS (Chalcone synthase), DFR (Dihydroflavonol 4-reductase), ANS (Anthocyanidin synthase), F3′H (flavonoid 3′-hydroxylase), FLS (Flavonol synthase), F3H (Flavonoid 3-hydroxylase), CHI (Chalcone isomerase) LAR, (Leucoanthocyanidin reductase). The relative fold expression level was normalized according to the 18S rRNA gene. Data were presented as means ± SE (n = 3), and the asterisks * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

Figure 9

HPLC-MS/MS profiling of OE-13 transgenic line and WT Arabidopsis. The quantification of metabolite in transgenic Arabidopsis and WT plants. The numbers in abscissa represent: (a) 6: eqaps denotes dihydroquercetin; (b) 3: snf denotes kaempferol; (c) 2: yps denotes naringenin; and (d) 1: cet denotes chalcone.