Genome-Wide Analysis, Classification, Evolution, and Expression Analysis of the Cytochrome P450 93 Family in Land Plants

<div><p>Cytochrome P450 93 family (CYP93) belonging to the cytochrome P450 superfamily plays important roles in diverse plant processes. However, no previous studies have investigated the evolution and expression of the members of this family. In this study, we performed comprehensive genome-wide analysis to identify CYP93 genes in 60 green plants. In all, 214 CYP93 proteins were identified; they were specifically found in flowering plants and could be classified into ten subfamilies—CYP93A–K, with the last two being identified first. CYP93A is the ancestor that was derived in flowering plants, and the remaining showed lineage-specific distribution—CYP93B and CYP93C are present in dicots; CYP93F is distributed only in Poaceae; CYP93G and CYP93J are monocot-specific; CYP93E is unique to legumes; CYP93H and CYP93K are only found in <i>Aquilegia coerulea</i>, and CYP93D is Brassicaceae-specific. Each subfamily generally has conserved gene numbers, structures, and characteristics, indicating functional conservation during evolution. Synonymous nucleotide substitution (<i>d</i>_N/<i>d</i>_S) analysis showed that CYP93 genes are under strong negative selection. Comparative expression analyses of CYP93 genes in dicots and monocots revealed that they are preferentially expressed in the roots and tend to be induced by biotic and/or abiotic stresses, in accordance with their well-known functions in plant secondary biosynthesis.</p></div

Expression profiles of plant CYP93 genes in response to abiotic stresses.

<p>(A) Expression profiles of <i>AtCYP93</i> and representative P450 genes in response to abiotic stresses. (B) Expression profiles of eight probe sets representing eight soybean CYP93 genes based on four microarray datasets of abiotic stresses. (C) Expression profiles of rice CYP93 genes based on four microarray datasets of abiotic stresses. Color bar at the base represents log2 expression values.</p

Sequence logos of the multiple alignments of the 214 CYP93 proteins in plants.

<p>The sequence logos of plant CYP93 proteins based on amino acid alignment using MAFFT are shown. The logos were generated using Weblogo. The bit score indicates the information content for each position in the sequence. The height of the letter designating the amino acid residue at each position represents the degree of conservation. The key conserved motifs are underlined; the red lines indicate the less conserved regions; the black ones, the P450 motifs; and the blue ones, the substrate recognition sites (SRSs). The white triangles indicate the conserved intron insertion location of plant CYP93 genes; the numbers within the triangles indicate the splicing phase of the intron (0 refers to phase 0). The red and black dots indicate the conserved amino acid insertion or deletion sites, respectively, in a given subfamily and/or clade; the number below each dot indicates the corresponding subfamily, i.e., B indicates the CYP93B subfamily.</p

Phylogenetic relationships of the 60 species investigated in the present study.

<p>Phylogenetic relationships (branch lengths are arbitrary) among these species have been described previously (<a href="http://www.phytozome.net/" target="_blank">http://www.phytozome.net/</a>). The total number of cytochrome P450 93 (CYP93) proteins identified in each genome is indicated on the right.</p

Expression profiles of CYP93 homologous genes in <i>Arabidopsis</i>, soybean, rice, and maize.

<p>(A) Expression profiles of the <i>AtCYP93D1</i> gene in <i>Arabidopsis</i>. (B) and (C) expression profiles of <i>GmCYP93</i> genes in soybean expression dataset1[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165020#pone.0165020.ref046" target="_blank">46</a>] and dataset2[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165020#pone.0165020.ref047" target="_blank">47</a>]. (D) and (E) expression profiles of <i>OsCYP93</i> genes in rice expression dataset1 (GSE14304) and dataset2 (GSE19024)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165020#pone.0165020.ref031" target="_blank">31</a>]. (F) expression profiles of <i>ZmCYP93</i> genes in maize[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165020#pone.0165020.ref048" target="_blank">48</a>]. Color bar at the base represents log2 expression values.</p

Expression profiles of <i>GmCYP93</i> genes in response to biotic stresses.

<p>(A) Expression profiles of GmCYP93 genes after infection with root-knot nematode (GSE33410). (B) Expression profiles of GmCYP93 genes after infection with <i>Phytophthora sojae</i> (GSE9687). (C) Expression profiles of GmCYP93 genes after aphid infestation (GSE35427). Color bar at the base represents log2 expression values.</p

Architecture of conserved protein motifs in the eight subgroups of the plant CYP93 family.

<p>The sequence logos of the P450 transmembrane, I-helix, K-helix, PERF, and heme-binding motifs based on the amino acid alignments are shown. The bit score indicates the information content for each position in the sequence. A−K indicate subfamilies CYP93A−CYP93K.</p

Evolution and expression analyses of the MADS-box gene family in <i>Brassica napus</i>

<div><p>MADS-box transcription factors are important for plant growth and development, and hundreds of MADS-box genes have been functionally characterized in plants. However, less is known about the functions of these genes in the economically important allopolyploid oil crop, <i>Brassica napus</i>. We identified 307 potential MADS-box genes (<i>BnMADSs</i>) in the <i>B</i>. <i>napus</i> genome and categorized them into type I (M_α, M_β, and M_γ) and type II (MADS DNA-binding domain, intervening domain, keratin-like domain, and C-terminal domain [MIKC]^c and MIKC*) based on phylogeny, protein motif structure, and exon-intron organization. We identified one conserved intron pattern in the MADS-box domain and seven conserved intron patterns in the K-box domain of the MIKC^c genes that were previously ignored and may be associated with function. Chromosome distribution and synteny analysis revealed that hybridization between <i>Brassica rapa</i> and <i>Brassica oleracea</i>, segmental duplication, and homologous exchange (HE) in <i>B</i>. <i>napus</i> were the main <i>BnMADSs</i> expansion mechanisms. Promoter cis-element analyses indicated that <i>BnMADSs</i> may respond to various stressors (drought, heat, hormones) and light. Expression analyses showed that homologous genes in a given subfamily or sister pair are highly conserved, indicating widespread functional conservation and redundancy. Analyses of <i>BnMADSs</i> provide a basis for understanding their functional roles in plant development.</p></div

MADS-box domain of MADSs-box genes in the <i>Brassica napus</i> (<i>BnMADSs</i>) genome.

<p>A multiple alignment analysis was performed using the MAFFT program. The sequence logos are based on the alignments of all type I (M_α, M_β, and M_γ) and type II (MIKC) <i>B</i>. <i>napus</i> MADS-box domains. Bit scores indicate the information content for each position in the sequence. Black and grey dots indicate 100%- and 90%-conserved residues, respectively.</p

Schematic diagram of intron distribution patterns within the K-box of proteins translated from type II <i>BnMADSs</i>.

<p>Alignment of the K-box domains is representative of 7 intron patterns, designated A to G. Intron locations are indicated by white triangles, and the number within each triangle indicates the splicing phases: 0 refers to phase 0; 1 to phase 1; and 2 to phase 2. The number of <i>BnMADSs</i> within each pattern is presented on the left. The correlation between intron distribution patterns and phylogenetic subfamilies is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200762#pone.0200762.g002" target="_blank">Fig 2</a>.</p