Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Complete chloroplast genome features and phylogenetic analysis of Abies ernestii var. salouenensis (Bordères and Gaussen) W. C. Cheng and L. K. Fu from southwest China.

Abies ernestii var. salouenensis (Bordères & Gaussen) W. C. Cheng & L. K. Fu is endemic to southwest China, including the southeastern Tibetan Plateau and the northwestern Yunnan Province. The taxonomic relationships between A. ernestii var. salouenensis and two other closely related fir species (A. chensiensis Tiegh. and A. ernestii Rehd.) still need to be determined. Here, we report for the first time the whole chloroplast genome of A. ernestii var. salouenensis. Its genome is 121,759 bp long and is characterized by a circular structure with 68 peptide-encoding genes, 16 tRNAs, six ORFs, and four rRNAs. We also identified 70 microsatellite repeat sequences and 14 tandem repeat sequences in the chloroplast genome of A. ernestii var. salouenensis. Comparative genome analysis indicated considerable variation in ycf1 and ycf2. Phylogenetic analysis supported the monophyly of A. ernestii var. salouenensis, A. chensiensis Tiegh., and A. ernestii Rehd. The relationships among them should be surveyed using more samples at the species level. This study will facilitate taxonomic studies and the development of suitable chloroplast markers for fir species.

The complete chloroplast genome of Abies ernestii Rehder (Pinaceae) and its phylogenetic implications.

Abies ernestii Rehder is endemic to the montane regions of Southwest China. Till now, phylogenetic relationships between A. ernestii and other closely related species remain unclear. In this study, we first characterized the complete chloroplast (cp) genome of A. ernestii. The whole cp genome was 121,841 bp in size, including one hundred and thirteen genes. Results of comparative cp genome revealed that only ycf1 and ycf2 was characterized by a considerable variation. Our phylogenetic analyses supported the monophyly of the genus Abies and revealed a clear separation between A. ernestii and A. chensiensis Tiegh. This study highlights the significance of using cp genomes to examine species boundaries among closely related fir species.

Characterization of the complete plastid genome of Abies forrestii (Pinaceae) from southwest China.

Abies forrestii is endemic to southwest China and ecologically important as a major component of the cold temperate forests. This study was the first report complete chloroplast (cp) genome of A. forrestii. The complete chloroplast genome was 120,022 bp in size. In total, 114 genes were identified, including 68 peptide-encoding genes, 35 tRNA genes, four rRNA genes, six open reading frames and one pseudogene. Thirteen genes contain introns. In phylogenetic analysis, A. forrestii was found to be closely related with A. nukiangensis, A. fanjingshanensis and A. delavayi subsp. fansipanensis. Our study will provide potential genetic resources for further evolutionary studies of this ecologically important species.

[Research advances in methods of cyclezation mechanism of sesquiterpenes].

Terpenes are the largest group of natural products and contain the widest assortment of structural types. Terpene cyclization is also the most complex reaction found in nature. For a long time, terpenoids with diverse structures have attracted natural product chemists to explore their biosynthesis mechanism. Such a large number of terpene skeletons are catalyzed by enzymes called terpene synthase. Sesquiterpene synthase is a kind of terpene synthase, which can catalyze the cyclization of linear precursor farnesyl pyrophosphate(FPP) to sesquiterpene skeletons. Sesquiterpene synthase cyclize a single precursor FPP into many sesquiterpene skeletons. With the continuous discovery of sesquiterpene synthase, the cyclization mechanism of sesquiterpene synthase has been studied deeply. In recent years, with the development and improvement of isotope labeling of substrate FPP and structural analysis of sesquiterpene synthase, the structure and cyclization mechanism of sesquiterpene synthase have been studied more systematically and accurately. In this review, we reviewed the progress of the research methods on the mechanism of sesquiterpene cyclization by substrate isotope labeling and protein structure, as well as the summary and prospect of sesquiterpene synthase research.

Next-generation sequencing yields the complete chloroplast genome of Abies yuanbaoshanensis, an endangered species from South China.

Abies yuanbaoshanensis is critically endangered and restricted in the Yuanbao Mountain of China, with no more than 900 surviving individuals. Here, we reported the complete chloroplast (cp) genome of A. yuanbaoshanensis. The complete chloroplast genome is 121,795 bp in size. In total, 114 genes were identified, including 68 peptide-encoding genes, 35 tRNA genes, 4 rRNA genes, 6 open reading frames, and 1 pseudogene. Thirteen genes contain introns. In phylogenetic analysis, both the ML and BI analyses supported the monophyly of the genus Abies. Our study will provide potential genetic resources for further conservation and evolutionary studies of this highly endangered species.

Next-generation sequencing yields the complete chloroplast genome of Abies kawakamii.

Abies kawakamii is endemic to the island of Taiwan and has been listed as a threatened species in the Red List. In present study, we reported the complete chloroplast genome of A. kawakamii. The chloroplast genome is 121,290 bp in size. It was composed of 114 genes and they were 68 peptide-encoding genes, 35 tRNA genes, four rRNA genes, six open reading frames and one pseudogene. Loss of ndh genes was identified in the genome of A. kawakamii. Inverted repeat sequences include trnS-psaM-ycf12-trnG and trnG-ycf12-psaM-trnS were recognized in 52-kb inversion points. The phylogenetic analysis confirms that the Abies species are strongly supported as monophyletic. The complete plastome of A. kawakamii will provide potential genetic resources for further conservation and management strategies.

The complete chloroplast genome sequence of Abies chensiensis (Pinaceae).

Abies chensiensis is listed as a threatened species in the Red List and categorized as key protected wild plants in China. Here, we determined the complete chloroplast genome of A. chensiensis using the Illumina MiSeq platform. The genome was 121,795 bp in length, comprising a large single copy (LSC) region of 67,160 bp, a small single copy (SSC) region of 54,107 bp, and two inverted repeat regions (IRa and IRb) of 264 bp each. It was composed of 114 genes, including 68 peptide-encoding genes, 35 transfer RNAs (tRNAs), four ribosomal RNAs (rRNAs), six open reading frames and one pseudogene. Phylogenetic analysis revealed that A. chensiensis was most closely related to A. beshanzuensis, with high bootstrap values. The present research will provide potential genetic resources for further conservation and management strategies.

The complete chloroplast genome sequence of Abies beshanzuensis, a highly endangered fir species from south China.

Abies beshanzuensis is critically endangered and endemic to Zhengjiang province of China, with only three surviving individuals. In present study, we reported the complete chloroplast (cp) genome of Abies beshanzuensis. The complete chloroplast genome size is 121,399 bp. In total, 114genes were identified, including 68 peptide-encoding genes, 35 tRNA genes, four rRNA genes, six open reading frames and one pseudogene. Loss of ndh genes was also identified in the genome of A. beshanzuensis like other genomes in the family Pinaceae. Thirteen genes contain one (11 genes) or two (rps12 and ycf3 genes) introns. Inverted repeat sequences located in 42-kb inversion points (1186 bp) include trnS-psaM-ycf12-trnG genes. In phylogenetic analysis, the tree confirms that the four Abies species are strongly supported as monophyletic. The complete plastome of A. beshanzuensis will provide potential genetic resources for further conservation and evolutionary studies of this highly endangered species.

Elevation Shift in Abies Mill. (Pinaceae) of Subtropical and Temperate China and Vietnam-Corroborative Evidence from Cytoplasmic DNA and Ecological Niche Modeling.

The "elevational shift" scenario has been proposed as a model to explain the response of cold-adapted organisms to Quaternary climatic fluctuations in Europe and North America. However, the elevational shift model has not been well-explored in eastern Asia, which is more topographically complex than the other Northern Hemisphere biogeographic regions. Here, we evaluated the role of elevational shift in the closely related firs, or Abies Mill., of subtropical and temperate China. These firs are typical alpine trees with sensitivity to climate change. We tested the elevational shift hypothesis in firs of China using phylogeographic methods and ecological niche models. Our phylogeographic analyses comprised mitochondrial and chloroplast polymorphisms surveyed across 479 individuals from 43 populations representing 11 species. M1 of the 11 mitotypes and C1 of the 25 chlorotypes were inferred as the ancestral haplotype, and they had the widest distribution. The results of our phylogeographic survey revealed multiple centers of genetic diversity in distinct geographic regions and no latitudinal trend. Moreover, our results showed range expansions for seven taxa during the last glacial (64.9-18.2 or 32.5-9.1 kya), and this was consistent with the Quaternary fossil record of Abies in China. Taken together, our findings support a historical biogeographic pattern in firs of glacial expansions, probably through corridors at lower elevation, and interglacial fragmentations, through isolation at higher elevation peaks. Therefore, Abies in China probably undergoes elevational shift in response to climate change. Facing the forecasting global warming, the risk of several critically endangered firs was further enhanced as these species would have little escape space in situ to higher altitudes. According to our ENMs, we proposed an ex situ conservation strategy in the southern Hengduan Mountains region of south western China.

Phylogenetic relationships, possible ancient hybridization, and biogeographic history of Abies (Pinaceae) based on data from nuclear, plastid, and mitochondrial genomes.

Abies, the second largest genus of Pinaceae, consists of approximately 48 species occurring in the north temperate region. Previous molecular phylogenetic studies improved our understanding of relationships within the genus, but were limited by relying on only DNA sequence data from single genome and low taxonomic sampling. Here we use DNA data from three genomes (sequences of internal transcribed spacer of nrITS, three chloroplast DNA intergenic spacers, and two mitochondrial intergenic spacers) from 42 species to elucidate species relationships and construct the biogeographic history of Abies. We further estimated the divergence times of intercontinental disjunction using a relaxed molecular clock calibrated with three macro-fossils. Our phylogenetic analyses recovered six robust clades largely consistent with previous classifications of sections. A sister relationship between the eastern Asian and Europe-Mediterranean clades was highly supported. The monophyly of section Balsamea, disjunct in Far East and western North America, is supported by the nrITS data but not by the cpDNA data. Discordance on placement of section Balsamea between the paternally inherited cpDNA and maternally inherited mtDNA trees was also observed. The data suggested that ancient hybridization was likely involved in the origin of sect. Balsamea. Results from biogeographic analyses and divergence time estimation suggested an origin and early diversification of Abies in an area of high latitude around the Pacific during the Eocene. The present disjunction in eastern Asia and Europe-Mediterranean area of Abies was likely the result of southward migration and isolation by the Turgai Strait in the Late Eocene. An 'out-of-America' migration, for the origin of an eastern Asian and western North American disjunct species pairs in section Amabilis was supported. The results suggested a western North American origin of the section with subsequent dispersal across the Bering Land Bridge (BLB) to Japan during the Middle Miocene.