Molecular cloning and characterization of two distinct caffeoyl CoA O-methyltransferases (CCoAOMTs) from the liverwort Marchantia paleacea.

Caffeoyl CoA O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to a hydroxyl moiety of caffeoyl-CoA as part of the lignin biosynthetic pathway. CCoAOMT-like proteins also catalyze to a variety of flavonoids, coumarins, and phenylpropanoids. Several CCoAOMTs that prefer flavonoids as substrates have been characterized from liverworts. Here, we cloned two CCoAOMT genes, MpalOMT2 and MpalOMT3, from the liverwort Marchantia paleacea. MpalOMT3 has a second ATG codon downstream and the truncated version that lacks 11 amino acids was named MpalOMT3-Tr. Phylogenetic analysis placed MpalOMT3 at the root of the clade with true CCoAOMTs from vascular plants and placed MpalOMT2 between the CCoAOMT and CCoAOMT-like proteins. Recombinant OMTs methylated caffeoyl CoA, phenylpropanoids, and flavonoids containing two or three vicinal hydroxyl groups. MpalOMT3 showed higher catalytic activity for phenylpropanoids than MpalOMT2, but MpalOMT2 showed more promiscuous towards eriodictyol and myricetin. The lignin content in Arabidopsis thaliana stems increased with constitutive heterologous expression of MpalOMT3-Tr, but not MpalOMT2. Subcellular localization experiments indicated that the N-terminus of MpalOMT3 probably served as a chloroplast transit peptide and inhibited its enzymatic activity. Combining the phylogenetic analysis and functional characterization, we conclude that the liverwort M. paleacea harbors true CCoAOMT and CCoAOMT-like genes.

Distribution of adenylyl cyclase/cAMP phosphodiesterase gene, CAPE, in streptophytes reproducing via motile sperm.

We recently isolated a novel adenylyl cyclase/cAMP phosphodiesterase gene from the liverwort, Marchantia polymorpha. The protein encoded by this gene has a class III adenylyl cyclase (AC) in the C-terminal domain and class I phosphodiesterase (PDE) in the N-terminal domain; therefore, we named it CAPE (COMBINED AC with PDE). CAPE protein is likely involved in spermatogenesis and sperm motility due to its tissue-specific expression pattern in M. polymorpha and the distribution of CAPE genes in streptophytes. However, little is known about the distribution of CAPE in gymnosperms that use motile sperm for fertilization, such as cycads and ginkgo. The present study aimed to isolate CAPE genes from the cycad, Cycas revoluta, the ginkgo, Ginkgo biloba, and the hornwort, Anthoceros agerestis. Sequences with high homology to CAPE were obtained from these species. Our analyses revealed that all plant taxonomic groups reproducing via motile sperm possessed CAPE, whereas those that do not produce motile sperm did not possess CAPE, with one exception in gymnosperm Cupressales. The phylogenic distribution of CAPE almost corresponds to the evolutionary history of motile sperm production and further suggests that CAPE may be involved in sexual reproduction process using motile sperm in streptophytes.

Diversified amino acid-mediated allosteric regulation of phosphoglycerate dehydrogenase for serine biosynthesis in land plants.

The phosphorylated pathway of serine biosynthesis is initiated with 3-phosphoglycerate dehydrogenase (PGDH). The liverwort Marchantia polymorpha possesses an amino acid-sensitive MpPGDH which is inhibited by l-serine and activated by five proteinogenic amino acids, while the eudicot Arabidopsis thaliana has amino acid-sensitive AtPGDH1 and AtPGDH3 as well as amino acid-insensitive AtPGDH2. In this study, we analyzed PGDH isozymes of the representative land plants: the monocot Oryza sativa (OsPGDH1-3), basal angiosperm Amborella trichopoda (AmtriPGDH1-2), and moss Physcomitrium (Physcomitrella) patens (PpPGDH1-4). We demonstrated that OsPGDH1, AmtriPGDH1, PpPGDH1, and PpPGDH3 were amino acid-sensitive, whereas OsPGDH2, OsPGDH3, AmtriPGDH2, PpPGDH2, and PpPGDH4 were either sensitive to only some of the six effector amino acids or insensitive to all effectors. This indicates that PGDH sensitivity to effectors has been diversified among isozymes and that the land plant species examined, except for M. polymorpha, possess different isozyme types in terms of regulation. Phylogenetic analysis suggested that the different sensitivities convergently evolved in the bryophyte and angiosperm lineages. Site-directed mutagenesis of AtPGDH1 revealed that Asp538 and Asn556 residues in the ACT domain are involved in allosteric regulation by the effectors. These findings provide insight into the evolution of PGDH isozymes, highlighting the functional diversification of allosteric regulation in land plants.

Fungal-Type Terpene Synthases in Marchantia polymorpha Are Involved in Sesquiterpene Biosynthesis in Oil Body Cells.

The liverwort Marchantia polymorpha possesses oil bodies in idioblastic oil body cells scattered in its thallus. Oil bodies are subcellular organelles in which specific sesquiterpenes and bisbibenzyls are accumulated. Therefore, a specialized system for the biosynthesis and accumulation of these defense compounds specifically in oil bodies has been implied. A recent study on M. polymorpha genome sequencing revealed 10 genes that shared high similarities with fungal-type terpene synthases (TPSs). Eight of these fungal-type TPS-like genes in M. polymorpha (MpFTPSL1-6, -9 and -10) are located within a 376-kb stretch on chromosome 6 and share similarities of over 94% at the nucleotide level. Therefore, these genes have likely originated from recent gene duplication events. The expression of a subset of MpFTPSLs was induced under non-axenic growth on vermiculite, which increased the amounts of sesquiterpenes and number of oil bodies. The tdTomato fluorescent protein-based in-fusion reporter assay with MpFTPSL2 promoter revealed fluorescent signals specifically in oil body cells of the thallus, indicating that MpFTPSL2 functions in oil body cells. Recombinant MpFTPSL2 expression in Escherichia coli led to sesquiterpene synthesis from farnesyl pyrophosphate. Moreover, suppression of a subset of MpFTPSLs through RNA interference reduced sesquiterpene accumulation in thalli grown on vermiculite. Taken together, these results suggest that at least a subset of MpFTPSLs is involved in sesquiterpene synthesis in oil body cells.

Molecular identification of histone acetyltransferases and deacetylases in lower plant Marchantia polymorpha.

Histone is the core component of nucleosome and modification of amino acid residues on histone tails is one of the most pivotal epigenetic regulatory mechanisms. Histone acetylation or deacetylation is carried out by two groups of proteins: histone acetyltransferases (HATs) or histone deacetylases (HDACs), and has been proven to be tightly linked to regulation of gene expression in animals and vascular plants. The biological functions of HATs and HDACs in non-flowering plants remain largely unknown. We found that there are seven MpHAT genes and twelve MpHDAC genes present in the Marchantia genome, and the comprehensive protein sequence analysis of the HAT and HDAC families was introduced to investigate their potential functions. On the basis of the functional domain analysis, eight MpHATs and twelve MpHDACs contain the conserved functional domains as the defining feature of each family. Phylogenetic trees of all families of MpHATs and MpHDACs along with their homologs from different plant and green algae species were constructed to illustrate evolutionary relationship of HAT and HDAC proteins. We found both SIR2 family and RPD3/HDA1 superfamily possess lower plant-specific proteins indicating the potential unknown functions of HATs and HDACs in Marchantia and other lower plant or algae species. Subcellular localization prediction suggests that MpHATs and MpHDACs are likely functioning in various organelles. Expression analysis shows that all MpHAT and MpHDAC genes are expressed in all tissues with differences at the transcriptional level. In addition, their expression patterns were altered in response to various treatments with plant hormones and environmental stress. We concluded that all MpHATs and MpHDACs are functional proteins in Marchantia and involved in various signaling pathways. Marchantia could have developed a complex histone acetylation epigenetic mechanism to regulate growth and development, as well as responses to environment.

Molecular Diversity of Alkenal Double Bond Reductases in the Liverwort Marchantia paleacea.

Alkenal double bond reductases (DBRs), capable of catalyzing the NADPH-dependent reduction of the α,ß-unsaturated double bond, play key roles in the detoxication of alkenal carbonyls. Here, the isolation and characterization of two DBRs encoded by the liverwort species Marchantia paleacea are described. The two DBRs share a relatively low similarity, and phylogenetic analysis indicated that MpMDBRL is more closely related to microbial DBRs than to other plant DBRs, while MpDBR shares common ancestry with typical plant DBRs. Both DBR proteins exhibited hydrogenation ability towards hydroxycinnamyl aldehydes; however, their temperature optimums were strikingly different. MpMDBRL demonstrated slightly weaker catalytic efficiency compared to MpDBR, and the structural models of their active binding sites to the substrate may provide a parsimonious explanation. Furthermore, both DBRs significantly responded to phytohormone treatment. In conclusion, M. paleacea produces two distinct types of functional DBRs, both of which participate in the protection against environmental stress in liverwort. The presence of a microbial type of DBR in a plant is herein reported for the first time.

Structural and biochemical characterization of the plant type III polyketide synthases of the liverwort Marchantia paleacea.

Chalcone synthases (CHSs) of the type III polyketide synthases (PKSs), catalyze the formation of a tetraketide intermediate from a CoA-tethered starter and malonyl-CoA but use different cyclization mechanisms to produce distinct chemical scaffolds. Herein, we characterized CHS and CHS-like enzymes (designated MpCHS and MpCHSL1, 2 and 3) from Marchantia paleacea and determined the crystal structure of MpCHSL1. MpCHS catalyzed a Claisen condensation to form chalcone, while MpCHSLs catalyzed the formation of lactonized α-pyrones in vitro. Based on the structural, mutational and in vitro biochemical analyses, we established that MpCHSL1 is structurally and functionally closer to prototype CHS than stilbene synthase, and characterized the structural basis for the functional diversity of the type III PKSs. A chalcone-forming mutant of MpCHSL1 was build directed by the structural information. These findings pave the way for future studies to elucidate the functional diversity of type III PKSs in liverwort.

RAB GTPases in the Basal Land Plant Marchantia polymorpha.

The RAB GTPase is an evolutionarily conserved machinery component of membrane trafficking, which is the fundamental system for cell viability and higher order biological functions. The composition of RAB GTPases in each organism is closely related to the complexity and organization of the membrane trafficking pathway, which has been developed uniquely to realize the organism-specific membrane trafficking system. Comparative genomics has suggested that terrestrialization and/or multicellularization were associated with the expansion of membrane trafficking pathways in green plants, which has yet to be validated in basal land plant lineages. To obtain insight into the diversification of membrane trafficking systems in green plants, we analyzed RAB GTPases encoded in the genome of the liverwort Marchantia polymorpha in a comprehensive manner. We isolated all genes for RAB GTPases in Marchantia and analyzed their expression patterns and subcellular localizations in thallus cells. While a majority of MpRAB GTPases exhibited a ubiquitous expression pattern, specific exceptions were also observed; MpRAB2b, which contains a sequence similar to an intraflagellar transport protein at the C-terminal region; and MpRAB23, which has been secondarily lost in angiosperms, were specifically expressed in the male reproductive organ. MpRAB21, which is another RAB GTPase whose homolog is absent in Arabidopsis, exhibited endosomal localization with RAB5 members in Marchantia. These results suggest that Marchantia possesses unique membrane trafficking pathways involving a unique repertoire of RAB GTPases.

Isolation and functional characterization of hydroxycinnamoyltransferases from the liverworts Plagiochasma appendiculatum and Marchantia paleacea.

Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT, EC: 2.3.1.133) is a key metabolic entry point for the synthesis of monolignols in vascular plants; however, little is known about HCT in liverworts. Here, the isolation and characterization of HCTs encoded by the two liverwort species, Plagiochasma appendiculatum and Marchantia paleacea, are described. The sequences of the two enzymes harbor features typical of BAHD family members, except for the presence of a stretch of >100 residues that are not represented in higher plant HCTs. When truncated versions of both genes, which were constructed to clarify the significance of these extra residues, were investigated, it became apparent that the full-length and the truncated gene products shared similar catalytic activity and recognized the same substrates in vitro. They also functioned equivalently in vivo either when transiently expressed in tobacco to cause a higher total production of CGA (5-CQA) and 4-CQA or stably expressed in liverworts to accumulate the lignin-like contents. A structural model of MpHCT suggests that its active site bind to its substrate similar to that of Arabidopsis thaliana HCT. While truncated forms of HCT were deposited in the nucleocytoplasm, the full-length versions occurred exclusively in the cytoplasm. The conclusion is that liverworts produce bona fide HCTs that represent a point of departure in studying the evolution of lignin synthesis in plants.

Functional Characterization of a Hydroxyacid/Alcohol Hydroxycinnamoyl Transferase Produced by the Liverwort Marchantia emarginata.

The aerial organs of most terrestrial plants are covered by a hydrophobic protective cuticle. The main constituent of the cuticle is the lipid polyester cutin, which is composed of aliphatic and aromatic domains. The aliphatic component is a polyester between fatty acid/alcohol and hydroxycinnamoyl acid. The BAHD/HxxxD family enzymes are central to the synthesis of these polyesters. The nature of this class of enzymes in bryophytes has not been explored to date. Here, a gene encoding a fatty ω-hydroxyacid/fatty alcohol hydroxycinnamoyl transferase (HFT) has been isolated from the liverwort Marchantia emarginata and has been functionally characterized. Experiments based on recombinant protein showed that the enzyme uses ω-hydroxy fatty acids or primary alcohols as its acyl acceptor and various hydroxycinnamoyl-CoAs-preferentially feruloyl-CoA and caffeoyl-CoA-as acyl donors at least in vitro. The transient expression of a MeHFT-GFP fusion transgene in the Nicotiana benthamiana leaf demonstrated that MeHFT is directed to the cytoplasm, suggesting that the feruloylation of cutin monomers takes place there.

The isolation and functional characterization of three liverwort genes encoding cinnamate 4-hydroxylase.

The plant phenylpropanoid pathway is responsible for the synthesis of a wide variety of secondary metabolites. The second step in phenylpropanoid synthesis is carried out by the cytochrome P450 monooxygenase enzyme cinnamate 4-hydroxylase (C4H), which catalyzes the p-hydroxylation of trans-cinnamic acid to p-coumarate. Genes encoding C4H have been characterized in many vascular plant species, but as yet not in any bryophyte species. Here, a survey of the transcriptome sequences of four liverwort species was able to identify eight putative C4Hs. The three liverwort C4H genes taken forward for isolation and functional characterization were harbored by Plagiochasma appendiculatum (PaC4H) and Marchantia paleacea (MpC4H1 and MpC4H2). When the genes were heterologously expressed in yeast culture, an assay of enzyme activity indicated that PaC4H and MpC4H1 had a higher level of activity than MpC4H2. The favored substrate (trans-cinnamic acid) of all three liverwort C4Hs was the same as that of higher plant C4Hs. The co-expression of PaC4H in yeast cells harboring PaPAL (a P. appendiculatum ene encoding phenylalanine ammonia lyase) allowed the conversion of L-phenylalanine to p-coumaric acid. Furthermore, the expression level of PaC4H was enhanced after treatment with abiotic stress inducers UV irradiation or salicylic acid in the thallus of P. appendiculatum. The likelihood is that high activity C4Hs evolved in the liverworts and have remained highly conserved across the plant kingdom.

An adenylyl cyclase with a phosphodiesterase domain in basal plants with a motile sperm system.

Adenylyl cyclase (AC), which produces the signalling molecule cAMP, has numerous important cellular functions in diverse organisms from prokaryotes to eukaryotes. Here we report the identification and characterization of an AC gene from the liverwort Marchantia polymorpha. The encoded protein has both a C-terminal AC catalytic domain similar to those of class III ACs and an N-terminal cyclic nucleotide phosphodiesterase (PDE) domain that degrades cyclic nucleotides, thus we designated the gene MpCAPE (COMBINED AC with PDE). Biochemical analyses of recombinant proteins showed that MpCAPE has both AC and PDE activities. In MpCAPE-promoter-GUS lines, GUS activity was specifically detected in the male sexual organ, the antheridium, suggesting MpCAPE and thus cAMP signalling may be involved in the male reproductive process. CAPE orthologues are distributed only in basal land plants and charophytes that use motile sperm as the male gamete. CAPE is a subclass of class III AC and may be important in male organ and cell development in basal plants.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

Biochemical characterization of allene oxide synthases from the liverwort Marchantia polymorpha and green microalgae Klebsormidium flaccidum provides insight into the evolutionary divergence of the plant CYP74 family.

MAIN CONCLUSION: Allene oxide synthases (AOSs) were isolated from liverworts and charophytes. These AOSs exhibited enzymatic properties similar to those of angiosperms but formed a distinct phylogenetic clade. Allene oxide synthase (AOS) and hydroperoxide lyase (HPL) mediate the formation of precursors of jasmonates and carbon-six volatiles, respectively. AOS and HPL utilize fatty acid hydroperoxides and belong to the plant cytochrome P450 74 (CYP74) family that mediates plant defense against herbivores, pathogens, or abiotic stresses. Although members of the CYP74 family have been reported in mosses and other species, the evolution and function of multiple CYP74 genes in plants remain elusive. Here, we show that the liverwort Marchantia polymorpha belongs to a basal group in the evolution of land plants; has two closely related proteins (59% identity), MpAOS1 and MpAOS2, that are similar to moss PpAOS1 (49 and 47% identity, respectively); and exhibits AOS activity but not HPL activity. We also found that the green microalgae Klebsormidium flaccidum, consist of multicellular and non-branching filaments, contains an enzyme, KfAOS, that is similar to PpAOS1 (37% identity), and converts 13-hydroperoxide of linolenic acid to 12-oxo-phytodienoic acid in a coupled reaction with allene oxide cyclase. Phylogenetic analysis showed two evolutionarily distinct clusters. One cluster comprised AOS and HPL from charophytic algae, liverworts, and mosses, including MpAOSs and KfAOS. The other cluster was formed by angiosperm CYP74. Our results suggest that plant CYP74 enzymes with AOS, HPL, and divinyl ether synthase activities have arisen multiple times and in the two different clades, which occurred prior to the divergence of the flowering plant lineage.

Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha.

12-Oxo-phytodienoic acid (OPDA) is an intermediate in jasmonic acid (JA) biosynthesis. OPDA exerts JA-dependent and JA-independent biological effects; therefore, it is considered a signaling molecule in flowering plants. OPDA is induced by bacterial infection and wounding and inhibits growth in the moss Physcomitrella patens. The functions of OPDA and allene oxide cyclase (AOC) in the liverwort Marchantia polymorpha were explored, which represents the most basal lineage of extant land plants. The analysis of OPDA showed that it is present in M. polymorpha and is increased by wounding. OPDA has been suggested to be involved in the response to environmental stresses. Moreover, OPDA showed growth inhibitory activity in M. polymorpha. Nonetheless JA in M. polymorpha was not found in this study. AOC synthesizes OPDA from an unstable allene oxide. A database search of the M. polymorpha genome identified only a putative gene encoding allene oxide cyclase (MpAOC). Recombinant MpAOC showed AOC activity similar to that in flowering plants. MpAOC was localized to chloroplasts, as in flowering plants. Expression of MpAOC was induced by wounding and OPDA treatment, and positive feedback regulation of OPDA was demonstrated in M. polymorpha. Overexpression of MpAOC increased the endogenous OPDA level and suppressed growth in M. polymorpha. These results indicate the role of OPDA as a signaling molecule regulating growth and the response to wounding in the liverwort M. polymorpha.

Biosynthetic routes of hydroxylated carotenoids (xanthophylls) in Marchantia polymorpha, and production of novel and rare xanthophylls through pathway engineering in Escherichia coli.

MAIN CONCLUSION: MpBHY codes for a carotene ß-ring 3(,3')-hydroxylase responsible for both zeaxanthin and lutein biosynthesis in liverwort. MpCYP97C functions as an ε-ring hydroxylase (zeinoxanthin 3'-hydroxylase) to produce lutein in liverwort. Xanthophylls are oxygenated or hydroxylated carotenes that are most abundant in the light-harvesting complexes of plants. The plant-type xanthophylls consist of α-xanthophyll (lutein) and ß-xanthophylls (zeaxanthin, antheraxanthin, violaxanthin and neoxanthin). The α-xanthophyll and ß-xanthophylls are derived from α-carotene and ß-carotene by carotene hydroxylase activities, respectively. ß-Ring 3,3'-hydroxylase that mediates the route of zeaxanthin from ß-carotene via ß-cryptoxanthin is present in higher plants and is encoded by the BHY (BCH) gene. On the other hand, CYP97A (or BHY) and CYP97C genes are responsible for ß-ring 3-hydroxylation and ε-ring 3'-hydroxylation, respectively, in routes from α-carotene to lutein. To elucidate the evolution of the biosynthetic routes of such hydroxylated carotenoids from carotenes in land plants, we identified and functionally analyzed carotenoid hydroxylase genes of liverwort Marchantia polymorpha L. Three genes homologous to higher plants, BHY, CYP97A, and CYP97C, were isolated and named MpBHY, MpCYP97A, and MpCYP97C, respectively. MpBHY was found to code for ß-ring hydroxylase, which is responsible for both routes starting from ß-carotene and α-carotene. MpCYP97C functioned as an ε-ring hydroxylase not for α-carotene but for zeinoxanthin, while MpCYP97A showed no hydroxylation activity for ß-carotene or α-carotene. These findings suggest the original functions of the hydroxylation enzymes of carotenes in land plants, which are thought to diversify in higher plants. In addition, we generated recombinant Escherichia coli cells, which produced rare and novel carotenoids such as α-echinenone and 4-ketozeinoxanthin, through pathway engineering using bacterial carotenogenic genes that include crtW, in addition to the liverwort MpLCYb, MpLCYe and MpBHY genes.

Arachidonic acid-dependent carbon-eight volatile synthesis from wounded liverwort (Marchantia polymorpha).

Eight-carbon (C8) volatiles, such as 1-octen-3-ol, octan-3-one, and octan-3-ol, are ubiquitously found among fungi and bryophytes. In this study, it was found that the thalli of the common liverwort Marchantia polymorpha, a model plant species, emitted high amounts of C8 volatiles mainly consisting of (R)-1-octen-3-ol and octan-3-one upon mechanical wounding. The induction of emission took place within 40min. In intact thalli, 1-octen-3-yl acetate was the predominant C8 volatile while tissue disruption resulted in conversion of the acetate to 1-octen-3-ol. This conversion was carried out by an esterase showing stereospecificity to (R)-1-octen-3-yl acetate. From the transgenic line of M. polymorpha (des6(KO)) lacking arachidonic acid and eicosapentaenoic acid, formation of C8 volatiles was only minimally observed, which indicated that arachidonic and/or eicosapentaenoic acids were essential to form C8 volatiles in M. polymorpha. When des6(KO) thalli were exposed to the vapor of 1-octen-3-ol, they absorbed the alcohol and converted it into 1-octen-3-yl acetate and octan-3-one. Therefore, this implied that 1-octen-3-ol was the primary C8 product formed from arachidonic acid, and further metabolism involving acetylation and oxidoreduction occurred to diversify the C8 products. Octan-3-one was only minimally formed from completely disrupted thalli, while it was formed as the most abundant product in partially disrupted thalli. Therefore, it is assumed that the remaining intact tissues were involved in the conversion of 1-octen-3-ol to octan-3-one in the partially disrupted thalli. The conversion was partly promoted by addition of NAD(P)H into the completely disrupted tissues, suggesting an NAD(P)H-dependent oxidoreductase was involved in the conversion.

Carotenoid analysis of a liverwort Marchantia polymorpha and functional identification of its lycopene ß- and ε-cyclase genes.

Carotenoid biosynthesis in bryophytes has yet to be clarified. The liverwort Marchantia polymorpha L. is known to be an early land plant and is an emerging bryophyte model. In order to gain insight into the evolution of carotenoid biosynthesis in plants, we studied carotenoid biosynthesis in this liverwort. As is the case in higher plants, liverwort thalli contain lutein and ß-carotene, as major carotenoids, as well as zeaxanthin, antheraxanthin, violaxanthin and 9'-cis-neoxanthin. Based on liverwort expressed sequence tag (EST)/cDNA and genome sequences, we isolated two cyclase genes encoding lycopene ß-cyclase (LCYb) and lycopene ε-cyclase (LCYe), which were involved in the synthesis of ß-carotene and α-carotene. These enzymes were phylogenetically positioned between corresponding proteins of a green alga (Chlorophyta) and higher plants. Functional analysis of the two genes was performed using a heterologous Escherichia coli expression system, in which the Pantoea ananatis lycopene biosynthesis genes were co-expressed. The results indicated liverwort LCYb activity for the synthesis of ß-carotene from lycopene, which was the same as that of higher plants. On the other hand, liverwort LCYe was able to form two ε-rings from lycopene to ε-carotene via δ-carotene, which was different from the Arabidopsis LCYe enzyme which generates only one ε-ring from lycopene.

Essential role of the E3 ubiquitin ligase nopperabo1 in schizogenous intercellular space formation in the liverwort Marchantia polymorpha.

The vast majority of land plants develop gas-exchange tissues with intercellular spaces (ICSs) connected directly to the air. Although the developmental processes of ICS have been described in detail at the morphological and ultrastructural level in diverse land plants, little is known about the molecular mechanism responsible for ICS formation. The liverwort Marchantia polymorpha develops a multilayered tissue with a large ICS (air chamber), whose formation is initiated at selected positions of epidermal cells. We isolated a mutant of M. polymorpha showing impaired air-chamber formation, nopperabo1 (nop1), from T-DNA-tagged lines. In nop1 plants, no ICS was formed; consequently, a single-layered epidermis developed on the dorsal side of the thallus. The causal gene NOP1 encodes a Plant U-box (PUB) E3 ubiquitin ligase carrying tandem ARMADILLO (ARM) repeats in the C terminus. An in vitro ubiquitination assay indicated that the NOP1 protein possesses E3 ubiquitin ligase activity in a U-box-dependent manner. Confocal microscopy and biochemical analysis showed that NOP1 was localized to the plasma membrane. Our investigation demonstrated the essential role of the PUB-ARM-type ubiquitin ligase in ICS formation in M. polymorpha, which sheds light on the molecular mechanism of schizogenous ICS formation in land plants.

Composition and physiological function of the chloroplast NADH dehydrogenase-like complex in Marchantia polymorpha.

The chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport and chloro-respiration and consists of five sub-omplexes, which in angiosperms further associate with photosystem I (PSI) to form a super-complex. In Marchantia polymorpha, 11 plastid-encoded subunits and all the nuclear-encoded subunits of the A, B, membrane and ferredoxin-binding sub-complexes are conserved. However, it is unlikely that the genome of this liverwort encodes Lhca5 and Lhca6, both of which mediate NDH-PSI super-complex formation. It is also unlikely that the subunits of the lumen sub-complex, PnsL1-L4, are encoded by the genome. Consistent with this in silico prediction, the results of blue-native gel electrophoresis showed that NDH subunits were detected in a protein complex with lower molecular mass in Marchantia than the NDH-PSI super-complex in Arabidopsis. Using the plastid transformation technique, we knocked out the ndhB gene in Marchantia. Although the wild-type genome copies were completely segregated out, the ΔndhB lines grew like the wild-type photoautotrophically. A post-illumination transient increase in chlorophyll fluorescence, which reflects NDH activity in vivo in angiosperms, was absent in the thalli of the ΔndhB lines. In ruptured chloroplasts, antimycin A-insensitive, and ferredoxin-dependent plastoquinone reduction was impaired, suggesting that chloroplast NDH mediates similar electron transport in Marchantia and Arabidopsis, despite its possible difference in structure. As in angiosperms, linear electron transport was not strongly affected in the ΔndhB lines. However, the plastoquinone pool was slightly more reduced at low light intensity, suggesting that chloroplast NDH functions in redox balancing of the inter system, especially under low light conditions.