Identification of Isoflavonoid Biosynthesis-Related R2R3-MYB Transcription Factors in Callerya speciosa (Champ. ex Benth.) Schot Using Transcriptome-Based Gene Coexpression Analysis

Abstract

The R2R3-MYB family is one of the largest plant transcription factor (TF) families playing vital roles in defense, plant growth, and secondary metabolism biosynthesis. Although this gene family has been studied in many species, isoflavonoid biosynthesis-related R2R3-MYB TFs in Callerya speciosa (Champ. ex Benth.) Schot, a traditional Chinese medicinal herb, are poorly understood. Here, a total of 101 R2R3-MYB TFs were identified from C. speciosa transcriptome dataset. 25 clades divided into five functional groups were clustered based on the sequence similarity and phylogenetic tree. Conserved motifs and domain distribution, expression patterns, and coexpression networks were also employed to identify the potential R2R3-MYB TFs in the regulation of isoflavonoid biosynthesis. In silico evaluation showed that the deduced R2R3-CsMYB proteins contain highly conserved R2R3 repeat domain at the N-terminal region, that is the signature motif of R2R3-type MYB TFs. Eight potential TFs (CsMYB17, CsMYB36, CsMYB41, CsMYB44, CsMYB45, CsMYB46, CsMYB72, and CsMYB81) had high degrees of coexpression with four key isoflavonoid biosynthetic genes (CsIFS, CsCHS7, CsHID-1, and CsCHI3), in which CsMYB36 as a potential regulator possessed the highest degree. HPLC analysis showed that formononetin and maackiain contents were significantly increased during the development of tuberous roots, which might be controlled by both related R2R3-CsMYBs and structural genes involved in the isoflavonoid biosynthesis pathway. The transcriptome data were further validated by reverse transcription real-time PCR (RT-qPCR) analysis, and similar expression profiles between TFs and key structural genes were identified. This study was the first step toward the understanding of the R2R3-MYB TFs regulating isoflavonoid biosynthesis in C. speciosa. The results will provide information for further functional analysis and quality improvement through genetic manipulation of these potential R2R3-CsMYB genes in C. speciosa.

Figure 1

Phylogenetic tree and classification of R2R3-MYB genes among 245 sequences. The neighbor-joining (NJ) tree was constructed using the sequences of 101 R2R3-MYB from C. speciosa, 126 from Arabidopsis, and 18 from other various species. The English letters with Arabic numbers outside the large red circle indicate the name of each clade. The amino acid sequences of 101 R2R3-CsMYBs were aligned in MEGA 6.0, and the phylogenetic tree was constructed by the NJ method with 1,000 bootstrap replicates. Bootstrap values > 50 are indicated on the nodes. Different clades are marked with different background colors.

Figure 2

Phylogenetic relationships, motif, and domain compositions of R2R3-MYB genes in C. speciosa. The amino acid sequences of 101 R2R3-CsMYBs were aligned in MEGA 6.0, and the phylogenetic tree was constructed by the NJ method with 1,000 bootstrap replicates. Bootstrap values > 50 is indicated on the nodes. Different categories were marked with different background colors. Motifs of R2R3-MYB proteins were analyzed by the MEME web server, and conserved domains were identified by the Pfam 30.0 database, respectively. Each motif/domain is represented by a number on the colored box. The length can be calculated using the scale at the bottom.

Figure 3

Logo sequences of R2 and R3 repeat of R2R3-MYB subfamily members in C. speciosa. The logo sequences of motifs 3, 1, and 2 together constitute the R2 and R3 repeats. The position of each residue and the width of the motif are represented by the Arabic numerals under the colored capital letters, respectively. The overall height of each stack represents the conservation of the sequence at that position. The capital letters indicate greater than 50% conservation of amino acids among the MYB domains. Each color of the letters represents a different type of amino acid residue.

Figure 4

The heat map for R2R3-MYB expression profiles from the transcriptome data at different developmental stages of tuberous roots. Color scale represents log₂ (FPKM+1) expression values. Changes in the expression level are indicated by a change in color; from blue to red indicates an expression level from low to high.

Figure 5

Simplified scheme and a heat map of the expression of key isoflavonoid biosynthetic genes in C. speciosa. (a) Isoflavonoid biosynthesis pathway. Enzyme names are indicated for each step. 4CL: 4-coumarate-CoA ligase; CHS: chalcone synthase; CHI: chalcone isomerase; IFS: isoflavone synthase; HID: 2-hydroxyisoflavanone dehydratase; HI4′OMT: isoflavone 4′-O-methyltransferase; VR: vestitone reductase. (b) Heat map of the expression profiles of key isoflavonoid biosynthetic genes. Color scale represents log₂ (FPKM+1) expression values. Changes in expression level are indicated by a change in color; from blue to red indicates an expression level from low to high. (c) Coexpression networks between key isoflavonoid biosynthetic genes and R2R3-MYBs related to secondary metabolism. The blue purple oval nodes indicate the key isoflavonoid biosynthetic genes, and the orange arrow nodes indicate the R2R3-MYBs. The gray solid lines indicate positive coexpression, and the red dash lines indicate negative coexpression. (d) The content of formononetin and maackiain at four developmental stages (6, 12, 18, and 30 MAG) of tuberous roots. All data shown reflect the mean of three biological replicates (n = 3). Means with different letters in each sample represent a significant difference at 0.05 level.

Figure 6

Validation of the transcriptomic data with RT-qPCR. (a) Comparison of the expression levels between RT-qPCR and FPKM values identified by transcriptome. (b) Correlation plot of the RT-qPCR (2^−ΔΔCq) and FPKM values. The R² value represents the correlation between the qPCR and RNA-seq results. Values are the means ± SD of three biological replicates.