Green Lineage Research Articles

The DnaJ proteins DJA6 and DJA5 are essential for chloroplast iron-sulfur cluster biogenesis.

Fe-S clusters are ancient, ubiquitous and highly essential prosthetic groups for numerous fundamental processes of life. The biogenesis of Fe-S clusters is a multistep process including iron acquisition, sulfur mobilization, and cluster formation. Extensive studies have provided deep insights into the mechanism of the latter two assembly steps. However, the mechanism of iron utilization during chloroplast Fe-S cluster biogenesis is still unknown. Here we identified two Arabidopsis DnaJ proteins, DJA6 and DJA5, that can bind iron through their conserved cysteine residues and facilitate iron incorporation into Fe-S clusters by interactions with the SUF (sulfur utilization factor) apparatus through their J domain. Loss of these two proteins causes severe defects in the accumulation of chloroplast Fe-S proteins, a dysfunction of photosynthesis, and a significant intracellular iron overload. Evolutionary analyses revealed that DJA6 and DJA5 are highly conserved in photosynthetic organisms ranging from cyanobacteria to higher plants and share a strong evolutionary relationship with SUFE1, SUFC, and SUFD throughout the green lineage. Thus, our work uncovers a conserved mechanism of iron utilization for chloroplast Fe-S cluster biogenesis.

Molecular Interaction and Evolution of Jasmonate Signaling With Transport and Detoxification of Heavy Metals and Metalloids in Plants.

An increase in environmental pollution resulting from toxic heavy metals and metalloids [e.g., cadmium (Cd), arsenic (As), and lead (Pb)] causes serious health risks to humans and animals. Mitigation strategies need to be developed to reduce the accumulation of the toxic elements in plant-derived foods. Natural and genetically-engineered plants with hyper-tolerant and hyper-accumulating capacity of toxic minerals are valuable for phytoremediation. However, the molecular mechanisms of detoxification and accumulation in plants have only been demonstrated in very few plant species such as Arabidopsis and rice. Here, we review the physiological and molecular aspects of jasmonic acid and the jasmonate derivatives (JAs) in response to toxic heavy metals and metalloids. Jasmonates have been identified in, limiting the accumulation and enhancing the tolerance to the toxic elements, by coordinating the ion transport system, the activity of antioxidant enzymes, and the chelating capacity in plants. We also propose the potential involvement of Ca2+ signaling in the stress-induced production of jasmonates. Comparative transcriptomics analyses using the public datasets reveal the key gene families involved in the JA-responsive routes. Furthermore, we show that JAs may function as a fundamental phytohormone that protects plants from heavy metals and metalloids as demonstrated by the evolutionary conservation and diversity of these gene families in a large number of species of the major green plant lineages. Using ATP-Binding Cassette G (ABCG) transporter subfamily of six representative green plant species, we propose that JA transporters in Subgroup 4 of ABCGs may also have roles in heavy metal detoxification. Our paper may provide guidance toward the selection and development of suitable plant and crop species that are tolerant to toxic heavy metals and metalloids.

New Insights into the Evolution of the Electron Transfer from Cytochrome f to Photosystem I in the Green and Red Branches of Photosynthetic Eukaryotes

In cyanobacteria and most green algae of the eukaryotic green lineage, the copper-protein plastocyanin (Pc) alternatively replaces the heme-protein cytochrome c6 (Cc6) as the soluble electron carrier from cytochrome f (Cf) to photosystem I (PSI). The functional and structural equivalence of ‘green’ Pc and Cc6 has been well established, representing an example of convergent evolution of two unrelated proteins. However, plants only produce Pc, despite having evolved from green algae. On the other hand, Cc6 is the only soluble donor available in most species of the red lineage of photosynthetic organisms, which includes, among others, red algae and diatoms. Interestingly, Pc genes have been identified in oceanic diatoms, probably acquired by horizontal gene transfer from green algae. However, the mechanisms that regulate the expression of a functional Pc in diatoms are still unclear. In the green eukaryotic lineage, the transfer of electrons from Cf to PSI has been characterized in depth. The conclusion is that in the green lineage, this process involves strong electrostatic interactions between partners, which ensure a high affinity and an efficient electron transfer (ET) at the cost of limiting the turnover of the process. In the red lineage, recent kinetic and structural modeling data suggest a different strategy, based on weaker electrostatic interactions between partners, with lower affinity and less efficient ET, but favoring instead the protein exchange and the turnover of the process. Finally, in diatoms the interaction of the acquired green-type Pc with both Cf and PSI may not yet be optimized.

The ATG4 protease integrates redox and stress signals to regulate autophagy.

Autophagy is a highly conserved degradative pathway that ensures cellular homeostasis through the removal of damaged or useless intracellular components including proteins, membranes, or even entire organelles. A main hallmark of autophagy is the biogenesis of autophagosomes, double-membrane vesicles that engulf and transport to the vacuole the material to be degraded and recycled. The formation of autophagosomes responds to integrated signals produced as a consequence of metabolic reactions or different types of stress and is mediated by the coordinated action of core autophagy-related (ATG) proteins. ATG4 is a key Cys-protease with a dual function in both ATG8 lipidation and free ATG8 recycling whose balance is crucial for proper biogenesis of the autophagosome. ATG4 is conserved in the green lineage, and its regulation by different post-translational modifications has been reported in the model systems Chlamydomonas reinhardtii and Arabidopsis. In this review, we discuss the major role of ATG4 in the integration of stress and redox signals that regulate autophagy in algae and plants.

Widespread polycistronic gene expression in green algae

Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: Chlamydomonas reinhardtii and Chromochloris zofingiensis Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.

Arabidopsis Mitochondrial Transcription Termination Factor mTERF2 Promotes Splicing of Group IIB Introns.

Plastid gene expression (PGE) is essential for chloroplast biogenesis and function and, hence, for plant development. However, many aspects of PGE remain obscure due to the complexity of the process. A hallmark of nuclear-organellar coordination of gene expression is the emergence of nucleus-encoded protein families, including nucleic-acid binding proteins, during the evolution of the green plant lineage. One of these is the mitochondrial transcription termination factor (mTERF) family, the members of which regulate various steps in gene expression in chloroplasts and/or mitochondria. Here, we describe the molecular function of the chloroplast-localized mTERF2 in Arabidopsis thaliana. The complete loss of mTERF2 function results in embryo lethality, whereas directed, microRNA (amiR)-mediated knockdown of MTERF2 is associated with perturbed plant development and reduced chlorophyll content. Moreover, photosynthesis is impaired in amiR-mterf2 plants, as indicated by reduced levels of photosystem subunits, although the levels of the corresponding messenger RNAs are not affected. RNA immunoprecipitation followed by RNA sequencing (RIP-Seq) experiments, combined with whole-genome RNA-Seq, RNA gel-blot, and quantitative RT-PCR analyses, revealed that mTERF2 is required for the splicing of the group IIB introns of ycf3 (intron 1) and rps12.

Transcriptional profiling reveals conserved and species-specific plant defense responses during the interaction of Physcomitrium patens with Botrytis cinerea.

Evolutionary conserved defense mechanisms present in extant bryophytes and angiosperms, as well as moss-specific defenses are part of the immune response of Physcomitrium patens. Bryophytes and tracheophytes are descendants of early land plants that evolved adaptation mechanisms to cope with different kinds of terrestrial stresses, including drought, variations in temperature and UV radiation, as well as defense mechanisms against microorganisms present in the air and soil. Although great advances have been made on pathogen perception and subsequent defense activation in angiosperms, limited information is available in bryophytes. In this study, a transcriptomic approach uncovered the molecular mechanisms underlying the defense response of the bryophyte Physcomitrium patens (previously Physcomitrella patens) against the important plant pathogen Botrytis cinerea. A total of 3.072 differentially expressed genes were significantly affected during B. cinerea infection, including genes encoding proteins with known functions in angiosperm immunity and involved in pathogen perception, signaling, transcription, hormonal signaling, metabolic pathways such as shikimate and phenylpropanoid, and proteins with diverse role in defense against biotic stress. Similarly as in other plants, B. cinerea infection leads to downregulation of genes involved in photosynthesis and cell cycle progression. These results highlight the existence of evolutionary conserved defense responses to pathogens throughout the green plant lineage, suggesting that they were probably present in the common ancestors of land plants. Moreover, several genes acquired by horizontal transfer from prokaryotes and fungi, and a high number of P. patens-specific orphan genes were differentially expressed during B. cinerea infection, suggesting that they are important players in the moss immune response.

Kingdom-wide analysis of the evolution of the plant type III polyketide synthase superfamily.

The emergence of type III polyketide synthases (PKSs) was a prerequisite for the conquest of land by the green lineage. Within the PKS superfamily, chalcone synthases (CHSs) provide the entry point reaction to the flavonoid pathway, while LESS ADHESIVE POLLEN 5 and 6 (LAP5/6) provide constituents of the outer exine pollen wall. To study the deep evolutionary history of this key family, we conducted phylogenomic synteny network and phylogenetic analyses of whole-genome data from 126 species spanning the green lineage including Arabidopsis thaliana, tomato (Solanum lycopersicum), and maize (Zea mays). This study thereby combined study of genomic location and context with changes in gene sequences. We found that the two major clades, CHS and LAP5/6 homologs, evolved early by a segmental duplication event prior to the divergence of Bryophytes and Tracheophytes. We propose that the macroevolution of the type III PKS superfamily is governed by whole-genome duplications and triplications. The combined phylogenetic and synteny analyses in this study provide insights into changes in the genomic location and context that are retained for a longer time scale with more recent functional divergence captured by gene sequence alterations.

Uncharted routes: exploring the relevance of auxin movement via plasmodesmata.

ABSTRACTAuxin is an endogenous small molecule with an incredibly large impact on growth and development in plants. Movement of auxin between cells, due to its negative charge at most physiological pHs, strongly relies on families of active transporters. These proteins import auxin from the extracellular space or export it into the same. Mutations in these components have profound impacts on biological processes. Another transport route available to auxin, once the substance is inside the cell, are plasmodesmata connections. These small channels connect the cytoplasms of neighbouring plant cells and enable flow between them. Interestingly, the biological significance of this latter mode of transport is only recently starting to emerge with examples from roots, hypocotyls and leaves. The existence of two transport systems provides opportunities for reciprocal cross-regulation. Indeed, auxin levels influence proteins controlling plasmodesmata permeability, while cell–cell communication affects auxin biosynthesis and transport. In an evolutionary context, transporter driven cell–cell auxin movement and plasmodesmata seem to have evolved around the same time in the green lineage. This highlights a co-existence from early on and a likely functional specificity of the systems. Exploring more situations where auxin movement via plasmodesmata has relevance for plant growth and development, and clarifying the regulation of such transport, will be key aspects in coming years.This article has an associated Future Leader to Watch interview with the author of the paper.

A Reference Genome from the Symbiotic Hydrozoan, Hydra viridissima.

Various Hydra species have been employed as model organisms since the 18th century. Introduction of transgenic and knock-down technologies made them ideal experimental systems for studying cellular and molecular mechanisms involved in regeneration, body-axis formation, senescence, symbiosis, and holobiosis. In order to provide an important reference for genetic studies, the Hydra magnipapillata genome (species name has been changed to H. vulgaris) was sequenced a decade ago (Chapman et al., 2010) and the updated genome assembly, Hydra 2.0, was made available by the National Human Genome Research Institute in 2017. While H. vulgaris belongs to the non-symbiotic brown hydra lineage, the green hydra, Hydra viridissima, harbors algal symbionts and belongs to an early diverging clade that separated from the common ancestor of brown and green hydra lineages at least 100 million years ago (Schwentner and Bosch 2015; Khalturin et al., 2019). While interspecific interactions between H. viridissima and endosymbiotic unicellular green algae of the genus Chlorella have been a subject of interest for decades, genomic information about green hydras was nonexistent. Here we report a draft 280-Mbp genome assembly for Hydra viridissima strain A99, with a scaffold N50 of 1.1 Mbp. The H. viridissima genome contains an estimated 21,476 protein-coding genes. Comparative analysis of Pfam domains and orthologous proteins highlights characteristic features of H. viridissima, such as diversification of innate immunity genes that are important for host-symbiont interactions. Thus, the H. viridissima assembly provides an important hydrozoan genome reference that will facilitate symbiosis research and better comparisons of metazoan genome architectures.

Specific features and assembly of the plant mitochondrial complex I revealed by cryo-EM

Mitochondria are the powerhouses of eukaryotic cells and the site of essential metabolic reactions. Complex I or NADH:ubiquinone oxidoreductase is the main entry site for electrons into the mitochondrial respiratory chain and constitutes the largest of the respiratory complexes. Its structure and composition vary across eukaryote species. However, high resolution structures are available only for one group of eukaryotes, opisthokonts. In plants, only biochemical studies were carried out, already hinting at the peculiar composition of complex I in the green lineage. Here, we report several cryo-electron microscopy structures of the plant mitochondrial complex I. We describe the structure and composition of the plant respiratory complex I, including the ancestral mitochondrial domain composed of the carbonic anhydrase. We show that the carbonic anhydrase is a heterotrimeric complex with only one conserved active site. This domain is crucial for the overall stability of complex I as well as a peculiar lipid complex composed of cardiolipin and phosphatidylinositols. Moreover, we also describe the structure of one of the plant-specific complex I assembly intermediates, lacking the whole PD module, in presence of the maturation factor GLDH. GLDH prevents the binding of the plant specific P1 protein, responsible for the linkage of the PP to the PD module.

The Transfer of the Ferredoxin Gene From the Chloroplast to the Nuclear Genome Is Ancient Within the Paraphyletic Genus Thalassiosira.

Ferredoxins are iron–sulfur proteins essential for a wide range of organisms because they are an electron transfer mediator involved in multiple metabolic pathways. In phytoplankton, these proteins are active in the mature chloroplasts, but the petF gene, encoding for ferredoxin, has been found either to be in the chloroplast genome or transferred to the nuclear genome as observed in the green algae and higher plant lineage. We experimentally determined the location of the petF gene in 12 strains of Thalassiosira covering three species using DNA sequencing and qPCR assays. The results showed that petF gene is located in the nuclear genome of all confirmed Thalassiosira oceanica strains (CCMP0999, 1001, 1005, and 1006) tested. In contrast, all Thalassiosira pseudonana (CCMP1012, 1013, 1014, and 1335) and Thalassiosira weissflogii (CCMP1010, 1049, and 1052) strains studied retained the gene in the chloroplast genome, as generally observed for Bacillariophyceae. Our evolutionary analyses further extend the dataset on the localization of the petF gene in the Thalassiosirales. The realization that the petF gene is nuclear-encoded in the Skeletonema genus allowed us to trace the petF gene transfer back to a single event that occurred within the paraphyletic genus Thalassiosira. Phylogenetic analyses revealed the need to reassess the taxonomic assignment of the Thalassiosira strain CCMP1616, since the genes used in our study did not cluster within the T. oceanica lineage. Our results suggest that this strains’ diversification occurred prior to the ferredoxin gene transfer event. The functional transfer of petF genes provides insight into the evolutionary processes leading to chloroplast genome reduction and suggests ecological adaptation as a driving force for such chloroplast to nuclear gene transfer.

Dissecting cholesterol and phytosterol biosynthesis via mutants and inhibitors.

Plants stand out among eukaryotes due to the large variety of sterols and sterol derivatives that they can produce. These metabolites not only serve as critical determinants of membrane structures, but also act as signaling molecules, as growth-regulating hormones, or as modulators of enzyme activities. Therefore, it is critical to understand the wiring of the biosynthetic pathways by which plants generate these distinct sterols, to allow their manipulation and to dissect their precise physiological roles. Here, we review the complexity and variation of the biosynthetic routes of the most abundant phytosterols and cholesterol in the green lineage and how different enzymes in these pathways are conserved and diverged from humans, yeast, and even bacteria. Many enzymatic steps show a deep evolutionary conservation, while others are executed by completely different enzymes. This has important implications for the use and specificity of available human and yeast sterol biosynthesis inhibitors in plants, and argues for the development of plant-tailored inhibitors of sterol biosynthesis.

Phylogenetic analysis of plant multi-domain SEC14-like phosphatidylinositol transfer proteins and structure\u2013function properties of PATELLIN2

Key messageSEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae. SEC14/CRAL-TRIO and GOLD domains govern membrane binding specificity.SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs) provide cues for membrane identity by exchanging lipophilic substrates, ultimately governing membrane signaling. Flowering plant SEC14L-PITPs often have modular structure and are associated with cell division, development, and stress responses. Yet, structure–function relationships for biochemical–cellular interactions of SEC14L-PITPs are rather enigmatic. Here, we evaluate the phylogenetic relationships of the SEC14L-PITP superfamily in the green lineage. Compared to green algae, land plants have an extended set of SEC14L-PITPs with increasingly complex modular structure. SEC14-GOLD PITPs, present in land plants but not Chara, diverged to three functional subgroups, represented by the six PATELLIN (PATL) proteins in Arabidopsis. Based on the example of Arabidopsis PATL2, we dissect the functional domains for in vitro binding to phosphoinositides and liposomes and for plant cell membrane association. While the SEC14 domain and its CRAL-TRIO-N-terminal extension serve general membrane attachment of the protein, the C-terminal GOLD domain directs it to the plasma membrane by recognizing specific phosphoinositides. We discuss that the different domains of SEC14L-PITPs integrate developmental and environmental signals to control SEC14L-PITP-mediated membrane identity, important to initiate dynamic membrane events.

Independent recurrent evolution of MICRORNA genes converging onto similar non-canonical organisation across green plant lineages is driven by local and segmental duplication events in species, family and lineages

The relationship between evolutionary history, organisation and transcriptional regulation of genes are intrinsically linked. These have been well studied in canonically organised protein-coding genes but not of MIRNAs. In the present study, we investigated the non-canonical arrangement of MIRNAs across taxonomic boundaries from algae to angiosperms employing a combination of genome organization, phylogeny and synteny. We retrieved the complete dataset of MIRNA from twenty-five species to identify and classify based on organisational patterns. The median size of cluster was between 2-5 kb and between 1-20 % of all MIRNAs are organized in head-to-head (with bidirectional promoter), head-to-tail (tandem), and overlapping manner. Although majority of the clusters are composed of MIRNA homologs, 25% of all clusters comprises of non-homologous genes with a potential of generating functional and regulatory complexity. A comparison of phylogeny and organizational patterns revealed that multiple independent events, some of which are species-specific, and some ancient, in different lineages, are responsible for non-canonical organization. Detailed investigation of MIR395 family across the plants revealed a complex origin of non-canonical arrangement through ancient and recent, segmental and local duplications; analysis of MIR399 family revealed major expansion occurred prior to monocot-dicot split, with few lineage-specific events. Evolution of “convergent” organization pattern of non-canonical arrangement originating from independent loci through recurrent event highlights our poor understanding of evolutionary process of MIRNA genes. The present investigation thus paves way for comparative functional genomics to understand the role of non-canonical organization on transcriptional regulation and regulatory diversity in MIRNA gene families.

Characterization of In Vivo Function(s) of Members of the Plant Mitochondrial Carrier Family

Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.

Lipidomic Analysis Reveals the Importance of GIPCs in Arabidopsis Leaf Extracellular Vesicles

Plant extracellular vesicles (EVs) are membrane-enclosed nanoparticles that play diverse roles in plant development and response. Recently, impressive progress has been made in the isolation and identification of the proteins and RNAs carried in plant EVs; however, the analysis of EV lipid compositions remains rudimentary. Here, we performed lipidomic analysis of Arabidopsis rosette leaf EVs, revealing a high abundance of certain groups of lipids, in particular sphingolipids, in the EVs. Remarkably, the EV sphingolipids are composed of nearly pure glycosylinositolphosphoceramides (GIPCs), which are green lineage abundant and negatively charged. We further showed that the Arabidopsis TETRASPANIN 8 (TET8) knockout mutant has a lower amount of cellular GIPCs and secrets fewer EVs, companied with impaired reactive oxygen species (ROS) burst toward stresses. Exogenous application of GIPCs promoted the secretion of EVs and ROS burst in both the WT and tet8 mutant. The characteristic enrichment of sphingolipid GIPCs provides valuable insights into the biogenesis and function of plant EVs.

Chloroplast mRNAs are 3' polyuridylylated in the Green Alga Pithophora roettleri (Cladophorales).

Species within the green algal order Cladophorales have an unconventional plastome structure where individual coding regions or small numbers of genes occur as linear single-stranded DNAs folded into hairpin structures. Another group of photosynthetic organisms with an equivalently reduced chloroplast genome are the peridinin dinoflagellates of the Alveolata eukaryotic lineage whose plastomes are mini-circles carrying one or a few genes required for photosynthesis. One unusual aspect of the Alveolata is the polyuridylylation of mRNA 3' ends among peridinin dinoflagellates and the chromerid algae. This study was conducted to understand if an unconventional highly reduced plastome structure co-occurs with unconventional RNA processing. To address this, the 5' and 3' mRNA termini of the known chloroplast genes of Pithophora roettleri (order Cladophorales) were analyzed for evidence of post-transcriptional processing. Circular Reverse Transcriptase PCR (cRT-PCR) followed by deep sequencing of the amplicons was used to analyze 5' and 3' mRNA termini. Evidence of several processing events were collected, most notably the 3' termini of six of the eight genes were polyuridylylated, which has not been reported for any lineage outside of the Alveolata. Other processing events include poly(A) and heteropolymeric 3' additions, 5' primary transcript start sites, as well as the presence of circularized RNAs. Five other species representing other green algal lineages were also tested and poly(U) additions appear to be limited to the order Cladophorales. These results demonstrate that chloroplast mRNA polyuridylylation is not the sole provenance of photosynthetic alveolates and may have convergently evolved in two distinct photosynthetic lineages.

Characterization of the diversification of phospholipid:diacylglycerol acyltransferases in the green lineage.

Triacylglycerols have important physiological roles in photosynthetic organisms, and are widely used as food, feed and industrial materials in our daily life. Phospholipid:diacylglycerol acyltransferase (PDAT) is the pivotal enzyme catalyzing the acyl-CoA-independent biosynthesis of triacylglycerols, which is unique in plants, algae and fungi, but not in animals, and has essential functions in plant and algal growth, development and stress responses. Currently, this enzyme has yet to be examined in an evolutionary context at the level of the green lineage. Some fundamental questions remain unanswered, such as how PDATs evolved in photosynthetic organisms and whether the evolution of terrestrial plant PDATs from a lineage of charophyte green algae diverges in enzyme function. As such, we used molecular evolutionary analysis and biochemical assays to address these questions. Our results indicated that PDAT underwent divergent evolution in the green lineage: PDATs exist in a wide range of plants and algae, but not in cyanobacteria. Although PDATs exhibit the conservation of several features, phylogenetic and selection-pressure analyses revealed that overall they evolved to be highly divergent, driven by different selection constraints. Positive selection, as one major driving force, may have resulted in enzymes with a higher functional importance in land plants than green algae. Further structural and mutagenesis analyses demonstrated that some amino acid sites under positive selection are critically important to PDAT structure and function, and may be central in lecithin:cholesterol acyltransferase family enzymes in general.

Phylogenomics reveals convergent evolution of red-violet coloration in land plants and the origins of the anthocyanin biosynthetic pathway

The flavonoids, one of the largest classes of plant secondary metabolites, are found in lineages that span the land plant phylogeny and play important roles in stress responses and as pigments. Perhaps the most well-studied flavonoids are the anthocyanins that have human health benefits and help plants attract pollinators, regulate hormone production, and confer resistance to abiotic and biotic stresses. The canonical biochemical pathway responsible for the production of these pigments is well-characterized for flowering plants yet its conservation across deep divergences in land plants remains debated and poorly understood. Many early land plants such as mosses, liverworts, and ferns produce flavonoid pigments, but their biosynthetic origins and homologies to the anthocyanin pathway remain uncertain. We conducted phylogenetic analyses using full genome sequences representing nearly all major green plant lineages to reconstruct the evolutionary history of the anthocyanin biosynthetic pathway then test the hypothesis that genes in this pathway are present in early land plants. We found that the entire pathway was not intact until the most recent common ancestor of seed plants and that orthologs of many downstream enzymes are absent from seedless plants including mosses, liverworts, and ferns. Our results also highlight the utility of phylogenetic inference, as compared to pairwise sequence similarity, in orthology assessment within large gene families that have complex duplication-loss histories. We suggest that the production of red-violet flavonoid pigments widespread in seedless plants, including the 3-deoxyanthocyanins, requires the activity of novel, as-yet discovered enzymes, and represents convergent evolution of red-violet coloration across land plants.