Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification

The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT‐qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall‐forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary‐walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9‐L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.     

Functional conservation of the glycosyltransferase gene GT47A in the monocot rice

Glucuronoarabinoxylan is the major hemicellulose in grass cell walls, yet the mechanism of xylan synthesis in monocot plants is still unclear. Unraveling the genes involved in the biosynthesis of xylan in rice will be very important for the utilization of rice straw as a source of bioenergy in the future. In this report, we investigated the functional role of a rice gene homologous to Arabidopsis IRREGULAR XYLEM10 (IRX10), belonging to the glycosyl transferase (GT) gene family 47 (GT47), in the biosynthesis of xylan. The protein sequence of OsGT47A from rice exhibits a 93.49 % similarity to IRX10, which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of the GT47 glycosyl transferase family in the rice genome revealed that OsGT47A is a closely related homolog of IRX10 and IRX10L. Expression pattern analysis showed that the OsGT47A gene is highly expressed in the rice stem. Overexpression of OsGT47A in the irx10 irx10L double mutant rescued the plant growth phenotype and restored secondary wall thickness. Analysis of monosaccharides indicated that the rescued plants had levels of xylose identical to those of the wild type plants, and the fluorescence signals were restored in the complementation plants by xylan immunolocalization. The OsGT47A complementation under the native promoter of Arabidopsis IRX10L (ProIRX10L) partially rescued the double mutant, indicating that OsGT47A is functionally equivalent to IRX10L. Together, these results suggest that the IRX10 homolog OsGT47A exhibits functional conservation and is most likely involved in xylan synthesis in rice.     

Comparative analysis of GT14/GT14-like gene family in Arabidopsis, Oryza, Populus, Sorghum and Vitis

Glycosyltransferase family14 (GT14) belongs to the glycosyltransferase (GT) superfamily that plays important roles in the biosynthesis of cell walls, the most abundant source of cellulosic biomass for bioethanol production. It has been hypothesized that DUF266 proteins are a new class of GTs related to GT14. In this study, we identified 62 GT14 and 106 DUF266 genes (named GT14-like herein) in Arabidopsis, Oryza, Populus, Sorghum and Vitis. Our phylogenetic analysis separated GT14 and GT14-like genes into two distinct clades, which were further divided into eight and five groups, respectively. Similarities in protein domain, 3D structure and gene expression were uncovered between the two phylogenetic clades, supporting the hypothesis that GT14 and GT14-like genes belong to one family. Therefore, we proposed a new family name, GT14/GT14-like family that combines both subfamilies. Variation in gene expression and protein subcellular localization within the GT14-like subfamily were greater than those within the GT14 subfamily. One-half of the Arabidopsis and Populus GT14/GT14-like genes were found to be preferentially expressed in stem/xylem, indicating that they are likely involved in cell wall biosynthesis. This study provided new insights into the evolution and functional diversification of the GT14/GT14-like family genes.     

Partial functional conservation of IRX10 homologs in physcomitrella patens and Arabidopsis thaliana indicates an evolutionary step contributing to vascular formation in land plants

Background

Plant cell walls are complex multicomponent structures that have evolved to fulfil an essential function in providing strength and protection to cells. Hemicelluloses constitute a key component of the cell wall and recently a number of the genes thought to encode the enzymes required for its synthesis have been identified in Arabidopsis. The acquisition of hemicellulose synthesis capability is hypothesised to have been an important step in the evolution of higher plants.

Results

Analysis of the Physcomitrella patens genome has revealed the presence of homologs for all of the Arabidopsis glycosyltransferases including IRX9, IRX10 and IRX14 required for the synthesis of the glucuronoxylan backbone. The Physcomitrella IRX10 homolog is expressed in a variety of moss tissues which were newly formed or undergoing expansion. There is a high degree of sequence conservation between the Physcomitrella IRX10 and Arabidopsis IRX10 and IRX10-L. Despite this sequence similarity, the Physcomitrella IRX10 gene is only able to partially rescue the Arabidopsis irx10 irx10-L double mutant indicating that there has been a neo- or sub-functionalisation during the evolution of higher plants. Analysis of the monosaccharide composition of stems from the partially rescued Arabidopsis plants does not show any significant change in xylose content compared to the irx10 irx10-L double mutant. Likewise, knockout mutants of the Physcomitrella IRX10 gene do not result in any visible phenotype and there is no significant change in monosaccharide composition of the cell walls.

Conclusions

The fact that the Physcomitrella IRX10 (PpGT47A) protein can partially complement an Arabidopsis irx10 irx10-L double mutant suggests that it shares some function with the Arabidopsis proteins, but the lack of a phenotype in knockout lines shows that the function is not required for growth or development under normal conditions in Physcomitrella. In contrast, the Arabidopsis irx10 and irx10 irx10-L mutants have strong phenotypes indicating an important function in growth and development. We conclude that the evolution of vascular plants has been associated with a significant change or adaptation in the function of the IRX10 gene family.     

Focus Issue on Plant Cell Walls: The Arabidopsis Family GT43 Glycosyltransferases Form Two Functionally Nonredundant Groups Essential for the Elongation of Glucuronoxylan Backbone

There exist four members of family GT43 glycosyltransferases in the Arabidopsis (Arabidopsis thaliana) genome, and mutations of two of them, IRX9 and IRX14, have previously been shown to cause a defect in glucuronoxylan (GX) biosynthesis. However, it is currently unknown whether IRX9 and IRX14 perform the same biochemical function and whether the other two GT43 members are also involved in GX biosynthesis. In this report, we performed comprehensive genetic analysis of the functional roles of the four Arabidopsis GT43 members in GX biosynthesis. The I9H (IRX9 homolog) and I14H (IRX14 homolog) genes were shown to be specifically expressed in cells undergoing secondary wall thickening, and their encoded proteins were targeted to the Golgi, where GX is synthesized. Overexpression of I9H but not IRX14 or I14H rescued the GX defects conferred by the irx9 mutation, whereas overexpression of I14H but not IRX9 or I9H complemented the GX defects caused by the irx14 mutation. Double mutant analyses revealed that I9H functioned redundantly with IRX9 and that I14H was redundant with IRX14 in their functions. In addition, double mutations of IRX9 and IRX14 were shown to cause a loss of secondary wall thickening in fibers and a much more severe reduction in GX amount than their single mutants. Together, these results provide genetic evidence demonstrating that all four Arabidopsis GT43 members are involved in GX biosynthesis and suggest that they form two functionally nonredundant groups essential for the normal elongation of GX backbone.Secondary walls constitute the bulk of cellulosic biomass produced by vascular plants. Cellulosic biomass in the form of fibers and wood is an important raw material for a myriad of industrial uses, such as timber, pulping, papermaking, and textiles. Due to the dwindling of nonrenewable fossil fuels and the detrimental effects of burning fossil fuels on the global environment, there has been an urgent call to develop alternative renewable energy sources, and the lignocellulosic biomass from plants is considered to be an attractive renewable source for biofuel production (Somerville, 2006). However, lignocellulosic biomass is recalcitrant to the enzymatic conversion of cellulose into sugars, because cellulose is embedded in a complex mixture of polysaccharides and lignin polymers that block the accessibility of degrading enzymes. It has been shown that reduction of lignin and xylan by chemical or enzymatic treatment or by the transgenic approach reduces the recalcitrance of the lignocellulosic biomass to saccharification (Chen and Dixon, 2007; Himmel et al., 2007; Lee et al., 2009a). Therefore, a complete understanding of how individual components of lignocellulosic biomass are biosynthesized will potentially allow us to design novel strategies for genetic modification of cell wall composition and, hence, reduction in biomass recalcitrance to biofuel production.Xylan is the main hemicellulose that cross-links with cellulose in the secondary walls of dicot plants (Carpita and McCann, 2000). It is made of a linear backbone of β-(1,4)-linked xylosyl residues, about 10% of which are attached with side chains of single residues of glucuronic acid (GlcA) and/or 4-O-methylglucuronic acid (MeGlcA) via α-(1,2)-linkages. The backbone xylosyl residues may also be substituted with the arabinosyl group and acetylated. Based on the nature of the side chains, xylan is generally grouped as (methyl)glucuronoxylan (GX), which is the main hemicellulose in dicots, and arabinoxylan and glucuronoarabinoxylan, which are the most abundant hemicelluloses in grass cell walls (Ebringerová and Heinze, 2000). In addition to the xylosyl backbone, the reducing end of xylan from birch (Betula verrucosa), spruce (Picea abies), Arabidopsis (Arabidopsis thaliana), and poplar (Populus alba × Populus tremula) contains a unique tetrasaccharide sequence β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (Shimizu et al., 1976; Johansson and Samuelson, 1977; Andersson et al., 1983; Peña et al., 2007; Lee et al., 2009a).The biosynthesis of xylan requires multiple glycosyltransferases and other modifying enzymes. Early biochemical studies revealed the activities of xylosyltransferases, glucuronosyltransferases, arabinosyltransferases, methyltransferases, and acetyltransferases that are likely involved in the biosynthesis of xylan (Baydoun et al., 1983, 1989; Kuroyama and Tsumuraya, 2001; Gregory et al., 2002; Porchia et al., 2002; Urahara et al., 2004; Zeng et al., 2008). However, none of the genes corresponding to these xylan biosynthetic enzymes have been identified. Recent molecular and genetic studies in Arabidopsis and poplar have led to the identification of a number of glycosyltransferases that are essential for GX biosynthesis. Among them, several members of the families GT47 and GT8 from Arabidopsis (FRA8, F8H, IRX8, and PARVUS) and poplar (GT47C, GT8D, and GT8E/8F) are implicated in the biosynthesis of the GX reducing end sequence (Aspeborg et al., 2005; Brown et al., 2005, 2007; Zhong et al., 2005; Zhou et al., 2006, 2007; Lee et al., 2007b, 2009b, 2009c; Peña et al., 2007; Persson et al., 2007). These glycosyltransferase genes are specifically expressed in vessels and fibers, and their encoded proteins are targeted to Golgi, where GX is synthesized, except for PARVUS and GT8E/8F, which are predominantly located in the endoplasmic reticulum (Lee et al., 2007b, 2009c). Mutations of the Arabidopsis FRA8, IRX8, and PARVUS genes all led to a near loss of the reducing end tetrasaccharide sequence and a reduction in GX amount (Brown et al., 2007; Lee et al., 2007b; Peña et al., 2007), indicating their essential roles in the biosynthesis of the GX reducing end sequence, although their exact enzymatic activities are still unknown.The genetic studies have also identified roles of two members of family GT43 glycosyltransferases, IRX9 and IRX14, from Arabidopsis and GT43B from poplar in the biosynthesis of the GX xylosyl backbone (Brown et al., 2007; Peña et al., 2007; Zhou et al., 2007). The expression of IRX9 has been shown to be associated with cells undergoing secondary wall biosynthesis, and its encoded protein is targeted to the Golgi. Mutation of the IRX9 gene causes a drastic reduction in xylan xylosyltransferase activity (Brown et al., 2007; Lee et al., 2007a) and concomitantly a substantial decrease in the GX chain length and GX amount (Peña et al., 2007). Mutation of IRX14 was shown to result in a reduction in the GX level and the xylosyltransferase activity (Brown et al., 2007). In addition, two functionally redundant glycosyltransferases, IRX10 and IRX10-like, which belong to family GT47, were also demonstrated to be required for the normal GX level and xylan xylosyltransferase activity, suggesting their involvement in the biosynthesis of the GX xylosyl backbone (Brown et al., 2009; Wu et al., 2009).In this report, we performed comprehensive molecular and genetic studies of the roles of all members of the Arabidopsis family GT43 glycosyltransferases in GX biosynthesis. We show that, like IRX9, the other three GT43 members, I9H (IRX9 homolog), IRX14, and I14H (IRX14 homolog), are expressed in secondary wall-containing cells and that their encoded proteins are targeted to the Golgi. We have found that the GX defects in the irx9 mutant can be rescued by overexpression of I9H but not IRX14 and I14H. Similarly, overexpression of I14H but not IRX9 and I9H is able to complement the GX defects caused by the irx14 mutation. Furthermore, genetic analysis of an array of double mutants revealed redundant and nonredundant roles of GT43 members in GX biosynthesis. Our findings demonstrate that the Arabidopsis family GT43 glycosyltransferases form two functionally nonredundant groups essential for the normal elongation of GX backbone.     

Two cotton fiber‐associated glycosyltransferases,GhGT43A1 and GhGT43C1, function in hemicellulose glucuronoxylan biosynthesis during plant development

Xylan is the major hemicellulosic constituent in dicot secondary cell walls. Cell wall composition of cotton fiber changes dynamically throughout development. Not only the amounts but also the molecular sizes of the hemicellulosic polysaccharides show substantial changes during cotton fiber development. However, none of the genes encoding glycosyltransferases (GTs) responsible for synthesizing xylan have been isolated and characterized in cotton fiber. In this study, we applied a bioinformatics approach and identified two putative GTs from cotton, designated GhGT43A1 and GhGT43C1, which belong to the CAZy GT43 family and are closely related to Arabidopsis IRX9 and IRX14, respectively. We show that GhGT43A1 is highly and preferentially expressed in 15 and 20 days post‐anthesis (dpa) cotton fiber, whereas GhGT43C1 is ubiquitously expressed in most organs, with especially high expression in 15 dpa fiber and hypocotyl. Complementation analysis demonstrates that GhG43A1 and GhGT43C1 are orthologs of Arabidopsis IRX9 and IRX14, respectively. Furthermore, we show that overexpression of GhGT43A1 or GhGT43C1 in Arabidopsis results in increased xylan content. We also show that overexpression of GhGT43A1 or GhGT43C1 leads to more cellulose deposition. These findings suggest that GhGT43A1 and GhGT43C1 likely participate in xylan synthesis during fiber development.     

Site-Directed Mutagenesis of IRX9, IRX9L and IRX14 Proteins Involved in Xylan Biosynthesis: Glycosyltransferase Activity Is Not Required for IRX9 Function in Arabidopsis

Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic activity.     

A molecular phylogenomic analysis of the ILR1-like family of IAA amidohydrolase genes

The ILR1-like family of hydrolase genes was initially isolated in Arabidopsis thaliana and is thought to help regulate levels of free indole-3-acetic-acid.We have investigated how this family has evolved in dicotyledon, monocotyledon and gymnosperm species by employing the GenBank and TIGR databases to retrieve orthologous genes. The relationships among these sequences were assessed employing phylogenomic analyses to examine molecular evolution and phylogeny. The members of the ILR1-like family analysed were ILL1, ILL2, ILL3, ILL6, ILR1 and IAR3. Present evidence suggests that IAR3 has undergone the least evolution and is most conserved. This conclusion is based on IAR3 having the largest number of total interspecific orthologues, orthologous species and unique orthologues. Although less conserved than IAR3, DNA and protein sequence analyses of ILL1 and ILR1 suggest high conservation. Based on this conservation, IAR3, ILL1 and ILR1 may have had major roles in the physiological evolution of 'higher' plants. ILL3 is least conserved, with the fewest orthologous species and orthologues. The monocotyledonous orthologues for most family-members examined have evolved into two separate molecular clades from dicotyledons, indicating active evolutionary change. The monocotyledon clades are: (a) those possessing a putative endoplasmic reticulum localizing signal; and (b) those that are putative cytoplasmic hydrolases. IAR3, ILL1 and ILL6 are all highly orthologous to a gene in the gymnosperm Pinus taeda, indicating an ancient enzymatic activity. No orthologues could be detected in Chlamydomonas, moss and fern databases.     

Evolution of Plant Mitochondrial Intron-Encoded Maturases: Frequent Lineage-Specific Loss and Recurrent Intracellular Transfer to the Nucleus

Among land plants, mitochondrial and plastid group II introns occasionally encode proteins called maturases that are important for splicing. Angiosperm nuclear genomes also encode maturases that are targeted to the organelles, but it is not known whether nucleus-encoded maturases exist in other land plant lineages. To examine the evolutionary diversity and history of this essential gene family, we searched for maturase homologs in recently sequenced nuclear and mitochondrial genomes from diverse land plants. We found that maturase content in mitochondrial genomes is highly lineage specific, such that orthologous maturases are rarely shared among major land plant groups. The presence of numerous mitochondrial pseudogenes in the mitochondrial genomes of several species implies that the sporadic maturase distribution is due to frequent inactivation and eventual loss over time. We also identified multiple maturase paralogs in the nuclear genomes of the lycophyte Selaginella moellendorffii, the moss Physcomitrella patens, and the representative angiosperm Vitis vinifera. Phylogenetic analyses of organelle- and nucleus-encoded maturases revealed that the nuclear maturase genes in angiosperms, lycophytes, and mosses arose by multiple shared and independent transfers of mitochondrial paralogs to the nuclear genome during land plant evolution. These findings indicate that plant mitochondrial maturases have experienced a surprisingly dynamic history due to a complex interaction of multiple evolutionary forces that affect the rates of maturase gain, retention, and loss.     

Characterization of IRX10 and IRX10-like reveals an essential role in glucuronoxylan biosynthesis in Arabidopsis

Xylan, the major hemicellulosic polysaccharide in Arabidopsis secondary cell walls, requires a number of glycosyltransferases (GT) to catalyse formation of the various glycosidic linkages found in the polymer. In this study, we characterized IRX10 and IRX10-like ( IRX10-L ), two highly homologous genes encoding members of the glycosyltransferase family 47 (GT47). T-DNA insertions in IRX10 gave a mild irregular xylem (irx) phenotype consistent with a minor defect in secondary cell-wall synthesis, whereas plants containing mutations in IRX10-L showed no change. However, irx10 irx10-L double mutant plants showed a much more severe irx and whole-plant phenotype, suggesting considerable functional redundancy between these two genes. Detailed biochemical analysis of the irx10 irx10-L double mutant showed a large reduction of xylan in the secondary cell walls, consistent with a specific defect in xylan biosynthesis. Furthermore, the irx10 irx10-L mutant retains the unique oligosaccharide found at the reducing end of Arabidopsis xylan, but shows a severe reduction in β(1,4) xylosyltransferase activity. These characteristics are similar to those of irx9 and irx14 , mutants that are believed to be defective in xylan chain elongation, and suggests that IRX10 and IRX10-L also play a role in elongation of the xylan backbone.     

Phytochrome gene diversity

The structures and functions of the phytochrome apoprotein genes (the PHY genes), their diversity across the plant kingdom, and their evolution are central concerns in the study of red-light sensing in plants. We summarize here recent advances in two areas relating to these topics: (1) the characteristics of the PHY gene family in Arabidopsis thaliana, the higher plant species for which the most extensive information on these genes is available, and (2) the similarity relationships, phylogeny, and evolutionary implications of PHY gene sequences and partial sequences which have been described from various plants. Together, these two areas of study, one directed at understanding in detail the phytochromes present in a single species and the other directed at a much broader understanding of PHY gene relatedness and distribution, are producing an increasingly clear picture of the diversity and evolution of plant red-light photoreceptors. Moreover, they suggest that the complexity of the phytochrome family has increased as land plants have evolved novel morphologies.     

A novel bioinformatics approach identifies candidate genes for the synthesis and feruloylation of arabinoxylan

Arabinoxylans (AXs) are major components of graminaceous plant cell walls, including those in the grain and straw of economically important cereals. Despite some recent advances in identifying the genes encoding biosynthetic enzymes for a number of other plant cell wall polysaccharides, the genes encoding enzymes of the final stages of AX synthesis have not been identified. We have therefore adopted a novel bioinformatics approach based on estimation of differential expression of orthologous genes between taxonomic divisions of species. Over 3 million public domain cereal and dicot expressed sequence tags were mapped onto the complete sets of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) genes, respectively. It was assumed that genes in cereals involved in AX biosynthesis would be expressed at high levels and that their orthologs in dicotyledonous plants would be expressed at much lower levels. Considering all rice genes encoding putative glycosyl transferases (GTs) predicted to be integral membrane proteins, genes in the GT43, GT47, and GT61 families emerged as much the strongest candidates. When the search was widened to all other rice or Arabidopsis genes predicted to encode integral membrane proteins, cereal genes in Pfam family PF02458 emerged as candidates for the feruloylation of AX. Our analysis, known activities, and recent findings elsewhere are most consistent with genes in the GT43 families encoding beta-1,4-xylan synthases, genes in the GT47 family encoding xylan alpha-1,2- or alpha-1,3-arabinosyl transferases, and genes in the GT61 family encoding feruloyl-AX beta-1,2-xylosyl transferases.     

Evolution and diversity of the 2–oxoglutarate‐dependent dioxygenase superfamily in plants

The 2–oxoglutarate‐dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation/hydroxylation reactions. Despite their biochemical significance in metabolism, a systematic analysis of plant 2OGDs remains to be accomplished. We present a phylogenetic classification of 479 2OGDs in six plant models, ranging from green algae to angiosperms. These were classified into three classes – DOXA, DOXB and DOXC – based on amino acid sequence similarity. The DOXA class includes plant homologs of Escherichia coli AlkB, which is a prototype of 2OGD involved in the oxidative demethylation of alkylated nucleic acids and histones. The DOXB class is conserved across all plant taxa and is involved in proline 4–hydroxylation in cell wall protein synthesis. The DOXC class is involved in specialized metabolism of various phytochemicals, including phytohormones and flavonoids. The vast majority of 2OGDs from land plants were classified into the DOXC class, but only seven from Chlamydomonas, suggesting that this class has diversified during land plant evolution. Phylogenetic analysis assigned DOXC‐class 2OGDs to 57 phylogenetic clades. 2OGD genes involved in gibberellin biosynthesis were conserved among vascular plants, and those involved in flavonoid and ethylene biosynthesis were shared among seed plants. Several angiosperm‐specific clades were found to be involved in various lineage‐specific specialized metabolisms, but 31 of the 57 DOXC‐class clades were only found in a single species. Therefore, the evolution and diversification of DOXC‐class 2OGDs is partly responsible for the diversity and complexity of specialized metabolites in land plants.