Sesquiterpene biosynthesis in a leafy liverwort Radula lindenbergiana Gottsche ex C. Hartm.

Liverworts (Marchantiophyta) are among the earliest diverging lineages of extant land plants. Among their unique features, most liverworts contain membrane-bound oil bodies, organelles that accumulate diverse secondary metabolites, especially terpenoids. In contrast to the rich information on liverwort terpenoid chemistry, little is known about their biosynthesis. Recently, terpenoid biosynthesis was studied in a model thalloid species Marchantiapolymorpha, in which sesquiterpenes and monoterpenes are biosynthesized by a new type of terpene synthases termed microbial terpene synthase-like (MTPSL) proteins. Here we study terpenoid biosynthesis in a leafy liverwort Radula lindenbergiana. Vegetative plants of R.lindenbergiana were found to contain a mixture of sesquiterpenes, with (E,E)-α-farnesene/ß-curcumene and (Z)-ß-bisabolene being the most abundant constituents. From the analysis of the R. lindenbergiana transcriptome, five full-length MTPSL genes were identified. They were designated RlMTPSL1-5, respectively. Recombinant RlMTPSL proteins were produced in Escherichia coli and tested for sesquiterpene synthase activities using farnesyl diphosphate (FPP) as substrate. All except RlMTPSL5 were demonstrated to catalyze the formation of different sesquiterpenes. RlMTPSL1 produced multiple sesquiterpenes with eremophilene and an unidentified sesquiterpene as major products. The major products of RlMTPSL2 and RlMTPSL3 were ß-elemene and an unidentified sesquiterpene, respectively. RlMTPSL4 was also a multi-product sesquiterpene synthase with an unidentified sesquiterpene being the major product. Homology-based structural modeling was performed to understand the structural basis underlying different product profiles of the RlMTPSLs proteins. Most of the sesquiterpene products of the four active RlMTPSLs were also detected in R. lindenbergiana plants. Expression levels of the four RlMTPSL genes encoding active enzymes in vegetative plants were compared. In phylogenetic analysis, RlMTPSL genes were found to cluster together, indicating lineage-specific expansion of MTPSL genes in lineages leading to R.lindenbergiana and M. polymorpha. This study strengthens evidence for the contribution of MTPSL genes to terpenoid biosynthesis in liverworts.

Organellomic data sets confirm a cryptic consensus on (unrooted) land-plant relationships and provide new insights into bryophyte molecular evolution.

PREMISE: Phylogenetic trees of bryophytes provide important evolutionary context for land plants. However, published inferences of overall embryophyte relationships vary considerably. We performed phylogenomic analyses of bryophytes and relatives using both mitochondrial and plastid gene sets, and investigated bryophyte plastome evolution. METHODS: We employed diverse likelihood-based analyses to infer large-scale bryophyte phylogeny for mitochondrial and plastid data sets. We tested for changes in purifying selection in plastid genes of a mycoheterotrophic liverwort (Aneura mirabilis) and a putatively mycoheterotrophic moss (Buxbaumia), and compared 15 bryophyte plastomes for major structural rearrangements. RESULTS: Overall land-plant relationships conflict across analyses, generally weakly. However, an underlying (unrooted) four-taxon tree is consistent across most analyses and published studies. Despite gene coverage patchiness, relationships within mosses, liverworts, and hornworts are largely congruent with previous studies, with plastid results generally better supported. Exclusion of RNA edit sites restores cases of unexpected non-monophyly to monophyly for Takakia and two hornwort genera. Relaxed purifying selection affects multiple plastid genes in mycoheterotrophic Aneura but not Buxbaumia. Plastid genome structure is nearly invariant across bryophytes, but the tufA locus, presumed lost in embryophytes, is unexpectedly retained in several mosses. CONCLUSIONS: A common unrooted tree underlies embryophyte phylogeny, [(liverworts, mosses), (hornworts, vascular plants)]; rooting inconsistency across studies likely reflects substantial distance to algal outgroups. Analyses combining genomic and transcriptomic data may be misled locally for heavily RNA-edited taxa. The Buxbaumia plastome lacks hallmarks of relaxed selection found in mycoheterotrophic Aneura. Autotrophic bryophyte plastomes, including Buxbaumia, hardly vary in overall structure.

Biosynthesis of methyl (E)-cinnamate in the liverwort Conocephalum salebrosum and evolution of cinnamic acid methyltransferase.

Methyl (E)-cinnamate is a specialized metabolite that occurs in a variety of land plants. In flowering plants, it is synthesized by cinnamic acid methyltransferase (CAMT) that belongs to the SABATH family. While rarely reported in bryophytes, methyl (E)-cinnamate is produced by some liverworts of the Conocephalum conicum complex, including C. salebrosum. In axenically grown thalli of C. salebrosum, methyl (E)-cinnamate was detected as the dominant compound. To characterize its biosynthesis, six full-length SABATH genes, which were designated CsSABATH1-6, were cloned from C. salebrosum. These six genes showed different levels of expression in the thalli of C. salebrosum. Next, CsSABATH1-6 were expressed in Escherichia coli to produce recombinant proteins, which were tested for methyltransferase activity with cinnamic acid and a few related compounds as substrates. Among the six SABATH proteins, CsSABATH6 exhibited the highest level of activity with cinnamic acid. It was renamed CsCAMT. The apparent Km value of CsCAMT using (E)-cinnamic acid as substrate was determined to be 50.5 µM. In contrast, CsSABATH4 was demonstrated to function as salicylic acid methyltransferase and was renamed CsSAMT. Interestingly, the CsCAMT gene from a sabinene-dominant chemotype of C. salebrosum is identical to that of the methyl (E)-cinnamate-dominant chemotype. Structure models for CsCAMT, CsSAMT and one flowering plant CAMT (ObCCMT1) in complex with (E)-cinnamic acid and salicylic acid were built, which provided structural explanations to substrate specificity of these three enzymes. In phylogenetic analysis, CsCAMT and ObCCMT1 were in different clades, implying that methyl (E)-cinnamate biosynthesis in bryophytes and flowering plants originated through convergent evolution.

Microbial-type terpene synthase genes occur widely in nonseed land plants, but not in seed plants.

The vast abundance of terpene natural products in nature is due to enzymes known as terpene synthases (TPSs) that convert acyclic prenyl diphosphate precursors into a multitude of cyclic and acyclic carbon skeletons. Yet the evolution of TPSs is not well understood at higher levels of classification. Microbial TPSs from bacteria and fungi are only distantly related to typical plant TPSs, whereas genes similar to microbial TPS genes have been recently identified in the lycophyte Selaginella moellendorffii The goal of this study was to investigate the distribution, evolution, and biochemical functions of microbial terpene synthase-like (MTPSL) genes in other plants. By analyzing the transcriptomes of 1,103 plant species ranging from green algae to flowering plants, putative MTPSL genes were identified predominantly from nonseed plants, including liverworts, mosses, hornworts, lycophytes, and monilophytes. Directed searching for MTPSL genes in the sequenced genomes of a wide range of seed plants confirmed their general absence in this group. Among themselves, MTPSL proteins from nonseed plants form four major groups, with two of these more closely related to bacterial TPSs and the other two to fungal TPSs. Two of the four groups contain a canonical aspartate-rich "DDxxD" motif. The third group has a "DDxxxD" motif, and the fourth group has only the first two "DD" conserved in this motif. Upon heterologous expression, representative members from each of the four groups displayed diverse catalytic functions as monoterpene and sesquiterpene synthases, suggesting these are important for terpene formation in nonseed plants.

World checklist of hornworts and liverworts.

A working checklist of accepted taxa worldwide is vital in achieving the goal of developing an online flora of all known plants by 2020 as part of the Global Strategy for Plant Conservation. We here present the first-ever worldwide checklist for liverworts (Marchantiophyta) and hornworts (Anthocerotophyta) that includes 7486 species in 398 genera representing 92 families from the two phyla. The checklist has far reaching implications and applications, including providing a valuable tool for taxonomists and systematists, analyzing phytogeographic and diversity patterns, aiding in the assessment of floristic and taxonomic knowledge, and identifying geographical gaps in our understanding of the global liverwort and hornwort flora. The checklist is derived from a working data set centralizing nomenclature, taxonomy and geography on a global scale. Prior to this effort a lack of centralization has been a major impediment for the study and analysis of species richness, conservation and systematic research at both regional and global scales. The success of this checklist, initiated in 2008, has been underpinned by its community approach involving taxonomic specialists working towards a consensus on taxonomy, nomenclature and distribution.

Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate.

We present a complete generic-level phylogeny of the complex thalloid liverworts, a lineage that includes the model system Marchantia polymorpha. The complex thalloids are remarkable for their slow rate of molecular evolution and for being the only extant plant lineage to differentiate gas exchange tissues in the gametophyte generation. We estimated the divergence times and analyzed the evolutionary trends of morphological traits, including air chambers, rhizoids and specialized reproductive structures. A multilocus dataset was analyzed using maximum likelihood and Bayesian approaches. Relative rates were estimated using local clocks. Our phylogeny cements the early branching in complex thalloids. Marchantia is supported in one of the earliest divergent lineages. The rate of evolution in organellar loci is slower than for other liverwort lineages, except for two annual lineages. Most genera diverged in the Cretaceous. Marchantia polymorpha diversified in the Late Miocene, giving a minimum age estimate for the evolution of its sex chromosomes. The complex thalloid ancestor, excluding Blasiales, is reconstructed as a plant with a carpocephalum, with filament-less air chambers opening via compound pores, and without pegged rhizoids. Our comprehensive study of the group provides a temporal framework for the analysis of the evolution of critical traits essential for plants during land colonization.

The resurrection of Neohattoria Kamim. (Jubulaceae, Marchantiophyta): a six decade systematic conflict resolved through a molecular perspective.

The systematic placement of Frullaniaherzogii has been contentious since its description six decades ago. Over the years it has been interpreted as either a member of the genus Frullania or segregated into its own genus, Neohattoria, due to morphological similarities with both Frullania and Jubula. Here we provide molecular evidence that supports the recognition of the genus Neohattoria and its inclusion within the Jubulaceae, together with Jubula and Nipponolejeunea. Jubulaceae are placed sister to Lejeuneaceae rather than to the monogeneric Frullaniaceae.

Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns.

Ferns are well known for their shade-dwelling habits. Their ability to thrive under low-light conditions has been linked to the evolution of a novel chimeric photoreceptor--neochrome--that fuses red-sensing phytochrome and blue-sensing phototropin modules into a single gene, thereby optimizing phototropic responses. Despite being implicated in facilitating the diversification of modern ferns, the origin of neochrome has remained a mystery. We present evidence for neochrome in hornworts (a bryophyte lineage) and demonstrate that ferns acquired neochrome from hornworts via horizontal gene transfer (HGT). Fern neochromes are nested within hornwort neochromes in our large-scale phylogenetic reconstructions of phototropin and phytochrome gene families. Divergence date estimates further support the HGT hypothesis, with fern and hornwort neochromes diverging 179 Mya, long after the split between the two plant lineages (at least 400 Mya). By analyzing the draft genome of the hornwort Anthoceros punctatus, we also discovered a previously unidentified phototropin gene that likely represents the ancestral lineage of the neochrome phototropin module. Thus, a neochrome originating in hornworts was transferred horizontally to ferns, where it may have played a significant role in the diversification of modern ferns.

The deepest divergences in land plants inferred from phylogenomic evidence.

Phylogenetic relationships among the four major lineages of land plants (liverworts, mosses, hornworts, and vascular plants) remain vigorously contested; their resolution is essential to our understanding of the origin and early evolution of land plants. We analyzed three different complementary data sets: a multigene supermatrix, a genomic structural character matrix, and a chloroplast genome sequence matrix, using maximum likelihood, maximum parsimony, and compatibility methods. Analyses of all three data sets strongly supported liverworts as the sister to all other land plants, and analyses of the multigene and chloroplast genome matrices provided moderate to strong support for hornworts as the sister to vascular plants. These results highlight the important roles of liverworts and hornworts in two major events of plant evolution: the water-to-land transition and the change from a haploid gametophyte generation-dominant life cycle in bryophytes to a diploid sporophyte generation-dominant life cycle in vascular plants. This study also demonstrates the importance of using a multifaceted approach to resolve difficult nodes in the tree of life. In particular, it is shown here that densely sampled taxon trees built with multiple genes provide an indispensable test of taxon-sparse trees inferred from genome sequences.