Solanales, Juss. ex Bercht. & J.Presl, 1820

Uda, Kouji, Edashige, Yumika, Nishimura, Rie, Shikano, Yuuna, Matsui, Tohru, Radkov, Atanas D. & Moe, Luke A., 2020, Distribution and evolution of the serine / aspartate racemase family in plants, Phytochemistry (112164) 169, pp. 1-8 : 4

2.3. Evolution of the AspR gene in order Solanales

To verify our hypothesis that Solanum lycopersicum AspR gene has evolved from SerR by gene duplication and amino acid substitutions, the gene loci of the SerR and AspR genes were compared among seven plants of the order Solanales and Coffea arabica , order Gentianales , as an outgroup ( Fig. 2 ). Each plant genome sequence obtained from GenBank contained one to three SerR homologous genes. These SerR homologous genes were separated into two types: one with HPN residues at positions 150 to 152 and one with PSS residues at the same positions, and we assumed the former to be a SerR and the latter to be an AspR according to the prediction of enzyme activity mentioned above (triple serine loop region).

The Nicotiana tabacum ( Solanales , Solanaceae ) and Coffea arabica (Gentianales) genomes have only one SerR gene. The chromosomal gene arrangement around the SerR gene is shown as gene arrangement (B) in Fig. 2a . The Ipomoea trifida ( Solanales , Convolvulaceae ) genome lacks the NOB1 gene compared to type (B) and has type (A) gene arrangement ( Fig. 2a ). Gene arrangement (C) in which the SerR and AspR genes are arranged in tandem, is found in Solanum verrucosum , S olanum tuberosum and Capsicum baccatum . Moreover, Solanum pennellii and Solanum lycopersicum genomes contain one more tandem duplicated AspR gene and have type (D) gene arrangement ( Fig. 2a ). The phylogenetic tree constructed from the nucleotide sequences of SerR and AspR genes ( Fig. 2b ), and the comparison between gene arrangements (A to D) have revealed the following findings. Gene arrangement (B), which has one SerR gene, is considered to be an ancestral arrangement type in Solanales species since type (B) is conserved in the outgroup species of Coffea arabica (Gentianales) . Hence, the lack of the NOB1 gene in arrangement (A) is considered to have occurred after the divergence of Solanaceae and the Convolvulaceae . It became clear that the AspR gene was obtained by tandem duplication of the SerR gene and subsequent amino acid substitution. We thought that this event occurred for the first time in the common ancestor species of genus Capsicum and genus Solanum without genus Nicotiana . Moreover, the SerR and AspR genes were also arranged in tandem in the Manihot esculenta genome, suggesting that the Manihot esculenta AspR was obtained by a similar evolutionary process. The second AspR gene was obtained by tandem duplication of the AspR gene after the divergence of the genus Solanum . Considering the phylogenetic tree, the second tandem duplication event occurred in the ancestral species of four Solanum species, and then ancestral species of Solanum verrucosum and Solanum tuberosum lost one AspR gene. These findings indicate that Solanum lycopersicum AspRs (AspR1 and AspR2) genes have evolved from SerR by gene tandem duplication and amino acid substitutions. The hypothesis that AspR evolved from SerR would be supported by that plant SerRs isolated in this study and many animal SerRs showed weak but distinct racemase activity toward aspartate ( Ito et al., 2016; Uda et al., 2016). We isolated and characterized only the AspR1 gene from Solanum lycopersicum and called it AspR in this study. The Solanum lycopersicum AspR1 and AspR2 have 97% amino acid sequence identity and the expression level of the AspR2 gene was too low to isolate as described below.

Figures

Fig. 2. Chromosomal gene arrangement around SerR homologous genes (a) and phylogenetic tree based on the nucleotide sequence of SerR homologous genes (b) from Solanales species. (a) The arrows indicate the direction of transcription. The genome sequences were obtained from GenBank and the accession numbers are as follows: CP025656, Ipomoea trifida; NW_015850497, Nicotiana tabacum; CM008450, Capsicum baccatum; NC_015445, Solanum lycopersicum; NC_028644, Solanum pennellii; NW_006239070, Solanum tuberosum; FYAA01000154, Solanum verrucosum; NC_039913, Coffea arabica. Each SerR homologous gene was predicted as AspR or SerR based on the amino acid sequence of the triple serine loop region. AHL5, AT-hook motif nuclear-localized protein 5; FTIP1, FT-interacting protein 1; NOB1, RNA-binding protein NOB1; LOR, LURP-one-related. (b) The nucleotide sequence of SerR homologous genes was obtained from genome sequences. The ML tree was constructed using the MEGA 7 (Kumar et al., 2016) and the ML bootstrap values are shown at the branching point. The symbols (A) to (D) before sequence name correspond to the gene arrangement shown in (a).