Genome-Wide Identification of MADS-Box Family Genes in Safflower (Carthamus tinctorius L.) and Functional Analysis of CtMADS24 during Flowering

Abstract

Safflower is an important economic crop with a plethora of industrial and medicinal applications around the world. The bioactive components of safflower petals are known to have pharmacological activity that promotes blood circulation and reduces blood stasis. However, fine-tuning the genetic mechanism of flower development in safflower is still required. In this study, we report the genome-wide identification of MADS-box transcription factors in safflower and the functional characterization of a putative CtMADS24 during vegetative and reproductive growth. In total, 77 members of MADS-box-encoding genes were identified from the safflower genome. The phylogenetic analysis divided CtMADS genes into two types and 15 subfamilies. Similarly, bioinformatic analysis, such as of conserved protein motifs, gene structures, and cis-regulatory elements, also revealed structural conservation of MADS-box genes in safflower. Furthermore, the differential expression pattern of CtMADS genes by RNA-seq data indicated that type II genes might play important regulatory roles in floral development. Similarly, the qRT-PCR analysis also revealed the transcript abundance of 12 CtMADS genes exhibiting tissue-specific expression in different flower organs. The nucleus-localized CtMADS24 of the AP1 subfamily was validated by transient transformation in tobacco using GFP translational fusion. Moreover, CtMADS24-overexpressed transgenic Arabidopsis exhibited early flowering and an abnormal phenotype, suggesting that CtMADS24 mediated the expression of genes involved in floral organ development. Taken together, these findings provide valuable information on the regulatory role of CtMADS24 during flower development in safflower and for the selection of important genes for future molecular breeding programs.

Keywords: MADS-box; Safflower (Carthamus tinctorius L.); expression patterns; flowering; phylogenetic analysis.

Figure 1

Phylogenetic analysis of safflower, sunflower, and Arabidopsis MADS-box family proteins. A total of 77 safflower MADS-box proteins, 23 Sunflower MADS-box proteins, and 101 Arabidopsis MADS-box proteins were included in the phylogenetic tree constructed using a maximum likelihood method. The red circles represent the MADS-box proteins in safflower.

Figure 2

Distribution and sequences of signature motifs of MADS and K-box structural domains obtained from safflower MADS-box proteins using MEME analysis. Different motifs are represented by different colors. Typical MADS and K-box structural domains extracted from MADS-box protein sequence are also shown on the right side.

Figure 3

The organization of exon–intron structures of CtMADS genes. The lines indicate introns, the red rectangles indicate exons, and the blue rectangle indicate UTRs.

Figure 4

Distribution of cis-acting regulatory elements of CtMADS genes in safflower. The different colored boxes represent the presence of specific cis-regulatory elements in the promoters of CtMADS-box genes.

Figure 5

Expression analysis of MADS-box genes in safflower. The FPKM values of the safflower MADS-box genes were log2-transformed to create the heatmap using TBtools software. The white and red colors represent the expression levels of CtMADS genes from low to high.

Figure 6

Organ-specific expression analysis of 12 CtMADS genes in safflower. (A) The genes were classified into different classes including class A, B, C, and E and four different organs (leaf, stamen, pistil, and sepal) were selected for expression analysis. Error bars represent the standard deviation n = 3. (B) The phenotype of different safflower flower organs used in the study.

Figure 7

Subcellular localization of CtMADS24 using transient expression system in tobacco leaves. The CtMADS24-GFP (green fluorescent protein) fusion construct was localized to the nucleus. The fluorescence signal was detected with a laser scanning confocal microscope. GFP indicates fluorescence of green fluorescent protein, and the red color shows the auto-fluorescence of chlorophyll. Scale bar = 50 μm.

Figure 8

Phenotypic variations and expression of key floral regulator genes in CtMADS24-overexpressed transgenic Arabidopsis. (A) The flowering phenotype of CtMADS24 transgenic Arabidopsis and WT plants. (B) Statistics for anthesis time of CtMADS24 transgenic Arabidopsis and WT plants. (C) The rosette phenotype of CtMADS24 transgenic Arabidopsis and WT plants. (D) Statistics for rosette leaves of CtMADS24 transgenic Arabidopsis and WT plants. (E) The sepal phenotype of CtMADS24 transgenic Arabidopsis and WT plants, bar = 1 cm. The red arrows pointed the internal floral organ in WT and transgenic Arabidopsis lines. (F) Legume morphology of CtMADS24 transgenic Arabidopsis and WT plants, bar = 0.5 cm. (G) Statistics for legume length of CtMADS24 transgenic Arabidopsis and WT plants. (H) The expression pattern of key genes involved in flowering development (* significant difference at p < 0.05, ** significant difference at p < 0.01).