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

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

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

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

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

Phenotypic T Cell Exhaustion in a Murine Model of Bacterial Infection in the Setting of Pre-Existing Malignancy

<div><p>While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8⁺ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with <i>Listeria monocytogenes</i>, and antigen-specific CD8⁺ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8⁺ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8⁺ T cells and total CD4⁺ and CD8⁺ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8⁺ T cells in the cancer group. Taken together, these data suggest that cancer's immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8⁺ T cell functionality and differentiation.</p></div

Effect of impaired intestinal lipid transport on serum lipoproteins.

<p>Triglyceride (A) and cholesterol (B) in serum were measured before and 24 hours after induction of pneumonia in the same mice. Sepsis decreased both triglyceride and cholesterol after induction of pneumonia in WT mice (C, D, n = 16–18/group, p<0.0001 for both). Inhibiting chylomicron assembly also resulted in lower triglyceride and cholesterol at baseline (n = 17–18/group, p<0.0001 for both). In contrast, sepsis did not alter triglyceride levels in <i>Mttp-IKO</i> mice (n = 11–17/group, p>0.05). Additionally, sepsis increased cholesterol levels in <i>Mttp-IKO</i> mice after induction of pneumonia (n = 11–17/group, p<0.0001). Sepsis virtually eliminated triglyceride from large, VLDL and LDL size lipoproteins in WT mice (note fractions 10 and 25, respectively). By contrast, Mttp-IKO mice exhibited virtually no triglyceride in lipoprotein particles, as expected. Sepsis resulted in an increase in HDL cholesterol content in <i>Mttp-IKO</i> mice (fractions 38–40). Serum bile acids (E) were similar between WT and <i>Mttp-IKO</i> mice (n = 7–8/group, p>0.05).</p

Serum cytokine suggests Th2 skewing in EtOH-fed septic relative to water-fed septic animals.

<p>A) Chronic alcohol ingestion induced elevated baseline levels of serum IL1β over water-feeding in sham animals (906.5±187.7 vs 1883±444.6, p = 0.01). B) Alcohol ingestion increased baseline serum IL-4 concentration in sham animals (19322±473.2 vs 26584±2516, p = 0.02), with a concurrent strong trend toward increase in septic animals (19629±523 vs 24433±1686, p = 0.05). C) Chronic alcohol ingestion increased baseline serum IL12 concentration in sham animals (1073±421.5 vs 35.9±3.1, p = 0.04). D) Alcohol ingestion increased baseline serum TNF concentration in sham animals (1093±79.9 vs 823.8±34.7, p = 0.005). E) Serum IL-6 was increased in alcohol sepsis over water sepsis (58697±27081 vs 896.7±356, p = 0.02). F) Serum IL-10 concentration was increased is alcohol sepsis over water sepsis (10057±4412 vs 979.7±896.2, p = 0.01). n = 8/group.</p