Acorus calamus

Ibdah, Mwafaq, Hino, Shada, Nawade, Bhagwat, Yahyaa, Mosaab, Bosamia, Tejas C. & Shaltiel-Harpaz, Liora, 2022, Identification and characterization of three nearly identical linalool / nerolidol synthase from Acorus calamus, Phytochemistry (113318) 202, pp. 1-10 : 4

2.4. Analysis of sequences of terpene synthases from A. calamus

Based on our bioinformatics analysis, we were able to identify six contigs with sequence similarity to plant TPS genes, which were designated as Acorus calamus AcTPS 1 to AcTPS6 (Supplementary Fig. S1 ). From the sequence comparison with previously characterized TPSs from other plant species, the sequences of AcTPS1 to AcTPS6 appear to contain complete open reading frames (ORFs; Supplemental Fig. S1 ). A. calamus AcTPS 1 to AcTPS6 ORF sequences encoded 462, 494, 314, 339, 759, and 785 deduced amino acids, respectively. The calculated molecular weight of proteins encoded by these AcTPS genes ranged from 36.5 to 89.9 kDa (Supplementary Table S2). Among the 6 AcTPS genes, the TargetP-2.0 software (https://services.healthtech.dtu.dk/service.ph p?TargetP-2.0) predicted a plastidic localization for the proteins AcTPS2 and AcTPS6 with the putative amino-terminal extension of 32 and 39 amino acids, respectively, upstream of the RRx8W motif. A phylogenetic tree based on protein sequence comparisons with representative TPS sequences from other plant species indicated that the AcTPS1 protein belongs to the TPS-b clad, in which the majority of angiospermous monoterpene synthases reside ( Chen et al., 2011). AcTPS1 was found to have an identity with germacrene D synthase-like from Camellia sinensis , Vitis vinifera , and V. riparia (50% of amino acids identity) ( Fig. 4 ). The AcTPS2, AcTPS3, and AcTPS4 belong to the TPS-g clade, and the BLASTP analysis of these proteins with the NCBI protein database revealed that the α -terpineol synthase-like from V. riparia and Camellia sinensis have 50%, 52%, and 48% amino acid identity with AcTPS2, AcTPS3, and AcTPS4, respectively ( Fig. 4 ). Comparisons with representative TPS sequences from other plant species indicated that AcTPS-5 belongs to the clade TPS-c. The TPS sequence most similar to AcTPS5 is ent-kaur-16-ene synthase, from Elaeis guineensis (62% of amino acids) ( Fig. 4 ). As for AcTPS6 was found to belong to the clade TPS-e clade and had the highest sequence similarity of amino acids with ent -copalyl diphosphate synthase from C. sinensis (56% of amino acids identity) ( Fig. 4 ).

Figures

Fig. 1. The most abundant terpenes and phenylpropanoids in Acorus calamus leaves and rhizomes. (A) The level of terpenes founds only in A. calamus leaves. (B) The level of terpenes founds only in A. calamus rhizomes. (C) The level of most abundant terpenes founds in A. calamus leaves and rhizomes. (D) The level of phenylpropanoids founds only in A. calamus leaves and rhizomes. All analyses were performed using ten biological replicates. Columns marked with different letters differ significantly at P <0.05 (Tukey–Kramer HSD means comparison test). All analyses were performed using five biological replicates.

Fig. 4. An unrooted neighbor-joining tree based on protein sequences of Acorus calamus AcTPSs, and selected plant TPS sequences. The tree was generated using Phylogeny Analysis MEGA X program. The resulting tree was bootstrap analyzed with 1000 replicates, with the values at the nodes reported in percentages. The subdivisions (clades) of the TPS gene family, designated TPS-a to TPS-f, are according (Chen et al., 2011). The bold black underlines indicate the AcTPS sequences identified in this study (AcTPS1 to AcTPS6). Ag: Abies grandis, Ae: Aralia elata, Am: Antirrhinum majus, Ar: Actinidia rufa1, Cb: Clarkia breweri, Cc: Clarkia concinna, Cm: Cinnamomum micranthum, Cn: Cocos nucifera, Cs: Camellia sinensis, Eg: Elaeis guineensis, Fc: Ficuc carcica, Fv: Fragaria vesca, Gh: Gossypium hirsutum, Jr: Juglans regia, Md: Malus x domestica, Mn: Morus notabilis, Nn: Nelumbo nucifera, Ob: Ocimum basilicum, Pd: Prunus dulcis, Pb: Pyrus x bretschneideri, Pg: Picea glauca, Pp: Prunus persica, Pt: Populus trichocarpa, Qs: Quercus suber, Sl: Solanum lycopersicum, Ta: Triticum aestivum, VV: Vitis vinifera, Vr: Vitis riparia.