A phylogenomically informed five-order system for the closest relatives of land plants.

The evolution of streptophytes had a profound impact on life on Earth. They brought forth those photosynthetic eukaryotes that today dominate the macroscopic flora: the land plants (Embryophyta).1 There is convincing evidence that the unicellular/filamentous Zygnematophyceae-and not the morphologically more elaborate Coleochaetophyceae or Charophyceae-are the closest algal relatives of land plants.2-6 Despite the species richness (>4,000), wide distribution, and key evolutionary position of the zygnematophytes, their internal phylogeny remains largely unresolved.7,8 There are also putative zygnematophytes with interesting body plan modifications (e.g., filamentous growth) whose phylogenetic affiliations remain unknown. Here, we studied a filamentous green alga (strain MZCH580) from an Austrian peat bog with central or parietal chloroplasts that lack discernible pyrenoids. It represents Mougeotiopsis calospora PALLA, an enigmatic alga that was described more than 120 years ago9 but never subjected to molecular analyses. We generated transcriptomic data of M. calospora strain MZCH580 and conducted comprehensive phylogenomic analyses (326 nuclear loci) for 46 taxonomically diverse zygnematophytes. Strain MZCH580 falls in a deep-branching zygnematophycean clade together with some unicellular species and thus represents a formerly unknown zygnematophycean lineage with filamentous growth. Our well-supported phylogenomic tree lets us propose a new five-order system for the Zygnematophyceae and provides evidence for at least five independent origins of true filamentous growth in the closest algal relatives of land plants. This phylogeny provides a robust and comprehensive framework for performing comparative analyses and inferring the evolution of cellular traits and body plans in the closest relatives of land plants.

Search for evolutionary roots of land plant arabinogalactan-proteins in charophytes: presence of a rhamnogalactan-protein in Spirogyra pratensis (Zygnematophyceae).

Charophyte green algae (CGA) are assigned to be the closest relatives of land plants and therefore enlighten processes in the colonization of terrestrial habitats. For the transition from water to land, plants needed significant physiological and structural changes, as well as with regard to cell wall composition. Sequential extraction of cell walls of Nitellopsis obtusa (Charophyceae) and Spirogyra pratensis (Zygnematophyceae) offered a comparative overview on cell wall composition of late branching CGA. Because arabinogalactan-proteins (AGPs) are considered common for all land plant cell walls, we were interested in whether these special glycoproteins are present in CGA. Therefore, we investigated both species with regard to characteristic features of AGPs. In the cell wall of Nitellopsis, no hydroxyproline was present and no AGP was precipitable with the ß-glucosyl Yariv's reagent (ßGlcY). By contrast, ßGlcY precipitation of the water-soluble cell wall fraction of Spirogyra yielded a glycoprotein fraction rich in hydroxyproline, indicating the presence of AGPs. Putative AGPs in the cell walls of non-conjugating Spirogyra filaments, especially in the area of transverse walls, were detected by staining with ßGlcY. Labelling increased strongly in generative growth stages, especially during zygospore development. Investigations of the fine structure of the glycan part of ßGlcY-precipitated molecules revealed that the galactan backbone resembled that of AGPs with 1,3- 1,6- and 1,3,6-linked Galp moieties. Araf was present only in small amounts and the terminating sugars consisted predominantly of pyranosidic terminal and 1,3-linked rhamnose residues. We introduce the term 'rhamnogalactan-protein' for this special AGP-modification present in S. pratensis.

On the Edge of Dispensability, the Chloroplast ndh Genes.

The polypeptides encoded by the chloroplast ndh genes and some nuclear genes form the thylakoid NADH dehydrogenase (Ndh) complex, homologous to the mitochondrial complex I. Except for Charophyceae (algae related to higher plants) and a few Prasinophyceae, all eukaryotic algae lack ndh genes. Among vascular plants, the ndh genes are absent in epiphytic and in some species scattered among different genera, families, and orders. The recent identification of many plants lacking plastid ndh genes allows comparison on phylogenetic trees and functional investigations of the ndh genes. The ndh genes protect Angiosperms under various terrestrial stresses, maintaining efficient photosynthesis. On the edge of dispensability, ndh genes provide a test for the natural selection of photosynthesis-related genes in evolution. Variable evolutionary environments place Angiosperms without ndh genes at risk of extinction and, probably, most extant ones may have lost ndh genes recently. Therefore, they are evolutionary endpoints in phylogenetic trees. The low number of sequenced plastid DNA and the long lifespan of some Gymnosperms lacking ndh genes challenge models about the role of ndh genes protecting against stress and promoting leaf senescence. Additional DNA sequencing in Gymnosperms and investigations into the molecular mechanisms of their response to stress will provide a unified model of the evolutionary and functional consequences of the lack of ndh genes.

Hemicellulose-remodelling transglycanase activities from charophytes: towards the evolution of the land-plant cell wall.

Transglycanases remodel cell-wall polymers, having a critical impact on many physiological processes. Unlike xyloglucan endotransglucosylase (XET) activity, widely studied in land plants, very little is known about charophyte wall-modifying enzymes - information that would promote our understanding of the 'primordial' wall, revealing how the wall matrix is remodelled in the closest living algal relatives of land plants, and what changed during terrestrialisation. We conducted various in-vitro assays for wall-remodelling transglycosylases, monitoring either (a) polysaccharide-to-[3 H]oligosaccharide transglycosylation or (b) non-radioactive oligosaccharide-to-oligosaccharide transglycosylation. We screened a wide collection of enzyme extracts from charophytes (and early-diverging land plants for comparison) and discovered several homo- and hetero-transglycanase activities. In contrast to most land plants, charophytes possess high trans-ß-1,4-mannanase activity, suggesting that land plants' algal ancestors prioritised mannan remodelling. Trans-ß-1,4-xylanase activity was also found, most abundantly in Chara, Nitella and Klebsormidium. Exo-acting transglycosidase activities (trans-ß-1,4-xylosidase and trans-ß-1,4-mannosidase) were also detected. In addition, charophytes exhibited homo- and hetero-trans-ß-glucanase activities (XET, mixed-linkage glucan [MLG]:xyloglucan endotransglucosylase and cellulose:xyloglucan endotransglucosylase) despite the paucity or lack of land-plant-like xyloglucan and MLG as potential donor substrates in their cell walls. However, trans-α-xylosidase activity (which remodels xyloglucan in angiosperms) was absent in charophytes and early-diverging land plants. Transglycanase action was also found in situ, acting on endogenous algal polysaccharides as donor substrates and fluorescent xyloglucan oligosaccharides as acceptor substrates. We conclude that trans-ß-mannanase and trans-ß-xylanase activities are present and thus may play key roles in charophyte walls (most of which possess little or no xyloglucan and MLG, but often contain abundant ß-mannans and ß-xylans), comparable to the roles of XET in xyloglucan-rich land plants.

A fossil record of land plant origins from charophyte algae.

Molecular time trees indicating that embryophytes originated around 500 million years ago (Ma) during the Cambrian are at odds with the record of fossil plants, which first appear in the mid-Silurian almost 80 million years later. This time gap has been attributed to a missing fossil plant record, but that attribution belies the case for fossil spores. Here, we describe a Tremadocian (Early Ordovician, about 480 Ma) assemblage with elements of both Cambrian and younger embryophyte spores that provides a new level of evolutionary continuity between embryophytes and their algal ancestors. This finding suggests that the molecular phylogenetic signal retains a latent evolutionary history of the acquisition of the embryophytic developmental genome, a history that perhaps began during Ediacaran-Cambrian time but was not completed until the mid-Silurian (about 430 Ma).

Multi-genomic analysis of the cation diffusion facilitator transporters from algae.

Metal transport processes are relatively poorly understood in algae in comparison to higher plants and other eukaryotes. A screen of genomes from 33 taxonomically diverse algal species was conducted to identify members of the Cation Diffusion Facilitator (CDF) family of metal ion transporter. All algal genomes contained at least one CDF gene with four species having >10 CDF genes (median of 5 genes per genome), further confirming that this is a ubiquitous gene family. Phylogenetic analysis suggested a CDF gene organisation of five groups, which includes Zn-CDF, Fe/Zn-CDF and Mn-CDF groups, consistent with previous phylogenetic analyses, and two functionally undefined groups. One of these undefined groups was algal specific although excluded chlorophyte and rhodophyte sequences. The majority of sequences (22 out of 26 sequences) from this group had a putative ion binding site motif within transmembrane domain 2 and 5 that was distinct from other CDF proteins, such that alanine or serine replaced the conserved histidine residue. The phylogenetic grouping was supported by sequence cluster analysis. Yeast heterologous expression of CDF proteins from Chlamydomonas reinhardtii indicated Zn2+ and Co2+ transport function by CrMTP1, and Mn2+ transport function by CrMTP2, CrMTP3 and CrMTP4, which validated the phylogenetic prediction. However, the Mn-CDF protein CrMTP3 was also able to provide zinc and cobalt tolerance to the Zn- and Co-sensitive zrc1 cot1 yeast strain. There is wide diversity of CDF transporters within the algae lineage, and some of these genes may be attractive targets for future applications of metal content engineering in plants or microorganisms.

An Ancient COI1-Independent Function for Reactive Electrophilic Oxylipins in Thermotolerance.

The jasmonate signaling pathway regulates development, growth, and defense responses in plants. Studies in the model eudicot, Arabidopsis thaliana, have identified the bioactive hormone (jasmonoyl-isoleucine [JA-Ile]) and its Coronatine Insensitive 1 (COI1)/Jasmonate-ZIM Domain (JAZ) co-receptor. In bryophytes, a conserved signaling pathway regulates similar responses but uses a different ligand, the JA-Ile precursor dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), to activate a conserved co-receptor. Jasmonate responses independent of JA-Ile and COI1, thought to be mediated by the cyclopentenone OPDA, have also been suggested, but experimental limitations in Arabidopsis have hindered attempts to uncouple OPDA and JA-Ile biosynthesis. Thus, a clear understanding of this pathway remains elusive. Here, we address the role of cyclopentenones in COI1-independent responses using the bryophyte Marchantia polymorpha, which is unable to synthesize JA-Ile but does accumulate OPDA and dn-OPDA. We demonstrate that OPDA and dn-OPDA activate a COI1-independent pathway that regulates plant thermotolerance genes, and consequently, treatment with these oxylipins protects plants against heat stress. Furthermore, we identify that these molecules signal through their electrophilic properties. By performing comparative analyses between M. polymorpha and two evolutionary distant species, A. thaliana and the charophyte alga Klebsormidium nitens, we demonstrate that this pathway is conserved in streptophyte plants and pre-dates the evolutionary appearance of the COI1-dependent jasmonate pathway, which later co-opted the pre-existing dn-OPDA as its ligand. Taken together, our data indicate that cyclopentenone-regulated COI1-independent signaling is an ancient conserved pathway, whose ancestral role was to protect plants against heat stress. This pathway was likely crucial for plants' successful land colonization and will be critical for adaption to current climate warming.

Plant Evolution: Assembling Land Plants.

Traditional evolutionary scenarios posit that land plants emerged from land plant-like relatives, the charophytes. New phylogenies suggest a closer affinity to simpler pond scum relatives, and evidence the gradual assembly of the land plant genome, revealing a phenotypic simplification from the complex ancestors envisaged by traditional scenarios.

Evolution of the Auxin Response Factors from charophyte ancestors.

Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most 'ancient' ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex.

Jasmonate-Related MYC Transcription Factors Are Functionally Conserved in Marchantia polymorpha.

The lipid-derived phytohormone jasmonoyl-isoleucine regulates plant immunity, growth and development in vascular plants by activating genome-wide transcriptional reprogramming. In Arabidopsis (Arabidopsis thaliana), this process is largely orchestrated by the master regulator MYC2 and related transcription factors (TFs). However, the TFs activating this pathway in basal plant lineages are currently unknown. We report the functional conservation of MYC-related TFs between the eudicot Arabidopsis and the liverwort Marchantia polymorpha, a plant belonging to an early diverging lineage of land plants. Phylogenetic analysis suggests that MYC function first appeared in charophycean algae and therefore predates the evolutionary appearance of any other jasmonate pathway component. M. polymorpha possesses two functionally interchangeable MYC genes, one in females and one in males. Similar to AtMYC2, MpMYCs showed nuclear localization, interaction with JASMONATE-ZIM-DOMAIN PROTEIN repressors, and regulation by light. Phenotypic and molecular characterization of loss- and gain-of-function mutants demonstrated that MpMYCs are necessary and sufficient for activating the jasmonate pathway in M. polymorpha, but unlike their Arabidopsis orthologs, do not regulate fertility. Therefore, despite 450 million years of independent evolution, MYCs are functionally conserved between bryophytes and eudicots. Genetic conservation in an early diverging lineage suggests that MYC function existed in the common ancestor of land plants and evolved from a preexisting MYC function in charophycean algae.

Identification, classification, and evolution of putative xylosyltransferases from algae.

Xylosyltransferases (XylTs) play key roles in the biosynthesis of many different polysaccharides. These enzymes transfer D-xylose from UDP-xylose to substrate acceptors. In this study, we identified 30 XylTs from primary endosymbionts (green algae, red algae, and glaucophytes) and secondary or higher endosymbionts (brown algae, diatoms, Eustigmatophyceae, Pelagophyceae, and Cryptophyta). We performed comparative phylogenetic studies on key XylT subfamilies, and investigated the functional divergence of genes using RNA-Seq. Of the 30 XylTs, one ß-1,4-XylT IRX14-related, one ß-1,4 XylT IRX10L-related, and one xyloglucan 6-XylT 1-related gene were identified in the Charophyta, showing strong similarities to their land plant descendants. This implied the ancient occurrence of xylan and xyloglucan biosynthetic machineries in Charophyta. The other 27 XylTs were identified as UDP-D-xylose: L-fucose-α-1,3-D-XylT (FucXylT) type that specifically transferred D-xylose to fucose. We propose that FucXylTs originated from the last eukaryotic common ancestor, rather than being plant specific, because they are also distributed in Choanoflagellatea and Echinodermata. Considering the evidence from many aspects, we hypothesize that the FucXylTs likely participated in fucoidan biosynthesis in brown algae. We provide the first insights into the evolutionary history and functional divergence of FucXylT in algal biology.

Recurrent acquisition of cytosine methyltransferases into eukaryotic retrotransposons.

Transposable elements are in a constant arms race with the silencing mechanisms of their host genomes. One silencing mechanism commonly used by many eukaryotes is dependent on cytosine methylation, a covalent modification of DNA deposited by C5 cytosine methyltransferases (DNMTs). Here, we report how two distantly related eukaryotic lineages, dinoflagellates and charophytes, have independently incorporated DNMTs into the coding regions of distinct retrotransposon classes. Concomitantly, we show that dinoflagellates of the genus Symbiodinium have evolved cytosine methylation patterns unlike any other eukaryote, with most of the genome methylated at CG dinucleotides. Finally, we demonstrate the ability of retrotransposon DNMTs to methylate CGs de novo, suggesting that retrotransposons could self-methylate retrotranscribed DNA. Together, this is an example of how retrotransposons incorporate host-derived genes involved in DNA methylation. In some cases, this event could have implications for the composition and regulation of the host epigenomic environment.

Out of Water: The Origin and Early Diversification of Plant R-Genes.

During plant-pathogen interactions, plants use intracellular proteins with nucleotide-binding site and Leu-rich repeat (NBS-LRR) domains to detect pathogens. NBS-LRR proteins represent a major class of plant disease resistance genes (R-genes). Whereas R-genes have been well characterized in angiosperms, little is known about their origin and early diversification. Here, we perform comprehensive evolutionary analyses of R-genes in plants and report the identification of R-genes in basal-branching streptophytes, including charophytes, liverworts, and mosses. Phylogenetic analyses suggest that plant R-genes originated in charophytes and R-proteins diversified into TIR-NBS-LRR proteins and non-TIR-NBS-LRR proteins in charophytes. Moreover, we show that plant R-proteins evolved in a modular fashion through frequent gain or loss of protein domains. Most of the R-genes in basal-branching streptophytes underwent adaptive evolution, indicating an ancient involvement of R-genes in plant-pathogen interactions. Our findings provide novel insights into the origin and evolution of R-genes and the mechanisms underlying colonization of terrestrial environments by plants.

Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis.

Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-ß-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-ß-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-ß-xylan synthase activity, and 1,4-ß-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-ß-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls.

Enhanced Desiccation Tolerance in Mature Cultures of the Streptophytic Green Alga Zygnema circumcarinatum Revealed by Transcriptomics.

Desiccation tolerance is commonly regarded as one of the key features for the colonization of terrestrial habitats by green algae and the evolution of land plants. Extensive studies, focused mostly on physiology, have been carried out assessing the desiccation tolerance and resilience of the streptophytic genera Klebsormidium and Zygnema. Here we present transcriptomic analyses of Zygnema circumcarinatum exposed to desiccation stress. Cultures of Z. circumcarinatum grown in liquid medium or on agar plates were desiccated at ∼86% relative air humidity until the effective quantum yield of PSII [Y(II)] ceased. In general, the response to dehydration was much more pronounced in Z. circumcarinatum cultured in liquid medium for 1 month compared with filaments grown on agar plates for 7 and 12 months. Culture on solid medium enables the alga to acclimate to dehydration much better and an increase in desiccation tolerance was clearly correlated to increased culture age. Moreover, gene expression analysis revealed that photosynthesis was strongly repressed upon desiccation treatment in the liquid culture while only minor effects were detected in filaments cultured on agar plates for 7 months. Otherwise, both samples showed induction of stress protection mechanisms such as reactive oxygen species scavenging (early light-induced proteins, glutathione metabolism) and DNA repair as well as the expression of chaperones and aquaporins. Additionally, Z. circumcarinatum cultured in liquid medium upregulated sucrose-synthesizing enzymes and strongly induced membrane modifications in response to desiccation stress. These results corroborate the previously described hardening and associated desiccation tolerance in Zygnema in response to seasonal fluctuations in water availability.

Primitive Auxin Response without TIR1 and Aux/IAA in the Charophyte Alga Klebsormidium nitens.

The phytohormone auxin regulates many aspects of growth and development in land plants, but the origin and evolution of auxin signaling and response mechanisms remain largely unknown. Indeed, it remains to be investigated whether auxin-related pathways diverged before the emergence of land plants. To address this knowledge deficit, we analyzed auxin responses in the charophyte alga Klebsormidium nitens NIES-2285, whose ancestor diverged from a green algal ancestor during the evolution of land plants. This strain is the same as Klebsormidium flaccidum NIES-2285, for which the draft genome was sequenced in 2014, and was taxonomically reclassified as K. nitens This genome sequence revealed genes involved in auxin responses. Furthermore, the auxin indole-3-acetic acid (IAA) was detected in cultures of K. nitens, but K. nitens lacks the central regulators of the canonical auxin-signaling pathway found in land plants. Exogenous IAA inhibited cell division and cell elongation in K. nitens Inhibitors of auxin biosynthesis and of polar auxin transport also inhibited cell division and elongation. Moreover, exogenous IAA rapidly induced expression of a LATERAL ORGAN BOUNDARIES-DOMAIN transcription factor. These results suggest that K. nitens has acquired the part of the auxin system that regulates transcription and cell growth without the requirement for the central players that govern auxin signaling in land plants.

Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.

The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell.

The Evolutionary Origin of a Terrestrial Flora.

Life on Earth as we know it would not be possible without the evolution of plants, and without the transition of plants to live on land. Land plants (also known as embryophytes) are a monophyletic lineage embedded within the green algae. Green algae as a whole are among the oldest eukaryotic lineages documented in the fossil record, and are well over a billion years old, while land plants are about 450-500 million years old. Much of green algal diversification took place before the origin of land plants, and the land plants are unambiguously members of a strictly freshwater lineage, the charophyte green algae. Contrary to single-gene and morphological analyses, genome-scale phylogenetic analyses indicate the sister taxon of land plants to be the Zygnematophyceae, a group of mostly unbranched filamentous or single-celled organisms. Indeed, several charophyte green algae have historically been used as model systems for certain problems, but often without a recognition of the specific phylogenetic relationships among land plants and (other) charophyte green algae. Insight into the phylogenetic and genomic properties of charophyte green algae opens up new opportunities to study key properties of land plants in closely related model. This review will outline the transition from single-celled algae to modern-day land plants, and will highlight the bright promise studying the charophyte green algae holds for better understanding plant evolution.

Phytochrome diversity in green plants and the origin of canonical plant phytochromes.

Phytochromes are red/far-red photoreceptors that play essential roles in diverse plant morphogenetic and physiological responses to light. Despite their functional significance, phytochrome diversity and evolution across photosynthetic eukaryotes remain poorly understood. Using newly available transcriptomic and genomic data we show that canonical plant phytochromes originated in a common ancestor of streptophytes (charophyte algae and land plants). Phytochromes in charophyte algae are structurally diverse, including canonical and non-canonical forms, whereas in land plants, phytochrome structure is highly conserved. Liverworts, hornworts and Selaginella apparently possess a single phytochrome, whereas independent gene duplications occurred within mosses, lycopods, ferns and seed plants, leading to diverse phytochrome families in these clades. Surprisingly, the phytochrome portions of algal and land plant neochromes, a chimera of phytochrome and phototropin, appear to share a common origin. Our results reveal novel phytochrome clades and establish the basis for understanding phytochrome functional evolution in land plants and their algal relatives.

Glycosyltransferase family 43 is also found in early eukaryotes and has three subfamilies in Charophycean green algae.

The glycosyltransferase family 43 (GT43) has been suggested to be involved in the synthesis of xylans in plant cell walls and proteoglycans in animals. Very recently GT43 family was also found in Charophycean green algae (CGA), the closest relatives of extant land plants. Here we present evidence that non-plant and non-animal early eukaryotes such as fungi, Haptophyceae, Choanoflagellida, Ichthyosporea and Haptophyceae also have GT43-like genes, which are phylogenetically close to animal GT43 genes. By mining RNA sequencing data (RNA-Seq) of selected plants, we showed that CGA have evolved three major groups of GT43 genes, one orthologous to IRX14 (IRREGULAR XYLEM14), one orthologous to IRX9/IRX9L and the third one ancestral to all land plant GT43 genes. We confirmed that land plant GT43 has two major clades A and B, while in angiosperms, clade A further evolved into three subclades and the expression and motif pattern of A3 (containing IRX9) are fairly different from the other two clades likely due to rapid evolution. Our in-depth sequence analysis contributed to our overall understanding of the early evolution of GT43 family and could serve as an example for the study of other plant cell wall-related enzyme families.