CDNA sequences with the Pnl DTPS1 genes have been deposited in
CDNA sequences in the Pnl DTPS1 genes have already been deposited within the GeneBank database below the accession numbers OK245418 to OK245421. two.3. Sequence-Based Analysis Predicts That Both Monofunctional and Bifunctional Diterpene synthases Are Involved in the Biosynthesis of Diterpene Resin Acids in Calabrian Pine The deduced amino acid sequences from the four full-length cDNAs isolated from Calabrian pine (see above) have been located to include very conserved and characteristic regions of plant DTPSs (Figure two). Initially, a putative transit peptide, ranging from 33 (Pnl DTPS1) to 68 aa (Pnl DTPS4) in length, probably needed for the import from the mature DTPS proteins into plastids. Secondly, DTPS active-site signature motifs (Figure 2): Pnl DTPS1 was identified to include each class-II and class-I motifs, suggesting its nature of appropriate bi-I/II DTPS, like the already-known bifunctional DTPSs involved in DRAs biosynthesis in conifers, namely the isopimaradiene synthase-type (ISO) and levopimaradiene/abietadiene synthase-type (LAS) enzymes from grand fir (A. grandis) and balsam fir (A. balsamea) [16,29], Norway spruce (Picea abies) and Sitka spruce (P. sitchensis) [24,30], loblolly pine (P. taeda), PI3KC2β Purity & Documentation lodgepole pine (P. contorta), and jack pine (P. banksiana) [22,31]. All the 3 remaining putative DTPS isolated from Calabrian pine, instead, were discovered to contain only the class-I signature motifs, plus incomplete versions of the class-II 1, lacking D residues identified to become crucial for class-II catalysis [32] either within the middle (Pnl DTPS3) or in thePlants 2021, ten,7 offirst and last positions (Pnl DTPS2 and Pnl DTPS4). Despite the fact that representing putative monofunctional DTPSs, the three sequences only showed 33 to 34 protein sequence identity for the conifer monofunctional class II ent-copalyl diphosphates synthases and class I ent-kaurene synthases involved in GA metabolism (data not shown), suggesting their roles in specialized, as opposed to common, metabolism. A phylogenetic analysis including the four deduced amino acid sequences from Calabrian pine and all of the pine DTPSs identified in the NCBI database (Figure three), allowed us to locate the isolated predicted proteins within the 4 phylogenetic groups in which the Pinus members with the TPS-d3 clade can be divided [20].Figure 3. Phylogenetic tree in the deduced amino acid sequences of 13 diterpene synthases (DTPSs) identified in diverse Pinus species (Table S1) along with the four DTPSs from Calabrian pine isolated in the present study (red squares). The ent-kaurene synthase from Physcomitrella patens (Pt TPS-entKS, ErbB3/HER3 Purity & Documentation BAF61135) was utilised to root the tree. Branches marked with dots represent bootstrap assistance much more than 80 (1000 repetitions). The four phylogenetic groups identified inside the pine members of your d3 clade of terpene synthases are indicated by square brackets.Depending on the sequence relatedness using the previously characterized pine DTPSs, it was feasible to predict the potential functions of three out of four DTPSs isolated from Calabrian pine. Pnl DTPS1 was found to cluster in group 1 with the other five bi-I/II-DTPSs, displaying 989 aa sequence identity with the four of them which happen to be functionally characterized so far, namely Pc DTPS LAS1, Pc DTPS LAS2, Pb DTPS LAS1 and Pt DTPS LAS1. Of these, the 3 bifunctional DTPSs from P. banksiana and P. contorta were shown to make the diterpene alcohol 13-hydroxy-8 (14)-abietene [22]. This unstable allylic alcohol can undergo dehydration, resulting inside the for.
