Basal Land Plants Research Articles

Conifers exhibit a characteristic inactivation of auxin to maintain tissue homeostasis.

Dynamic regulation of the concentration of the natural auxin (IAA) is essential to coordinate most of the physiological and developmental processes and responses to environmental changes. Oxidation of IAA is a major pathway to control auxin concentrations in angiosperms and, along with IAA conjugation, to respond to perturbation of IAA homeostasis. However, these regulatory mechanisms remain poorly investigated in conifers. To reduce this knowledge gap, we investigated the different contributions of the IAA inactivation pathways in conifers. MS-based quantification of IAA metabolites under steady-state conditions and after perturbation was investigated to evaluate IAA homeostasis in conifers. Putative Picea abies GH3 genes (PaGH3) were identified based on a comprehensive phylogenetic analysis including angiosperms and basal land plants. Auxin-inducible PaGH3 genes were identified by expression analysis and their IAA-conjugating activity was explored. Compared to Arabidopsis, oxidative and conjugative pathways differentially contribute to reduce IAA concentrations in conifers. We demonstrated that the oxidation pathway plays a marginal role in controlling IAA homeostasis in spruce. By contrast, an excess of IAA rapidly activates GH3-mediated irreversible conjugation pathways. Taken together, these data indicate that a diversification of IAA inactivation mechanisms evolved specifically in conifers.

The origin and evolution of the ALOG proteins, members of a plant-specific transcription factor family, in land plants.

The Arabidopsis LSH1 and Oryza G1 (ALOG) protein is a family of plant-specific transcription factors that regulate reproductive growth in angiosperms. Despite their importance in plant development, little research has been conducted on ALOG proteins in basal land plants and the processes involved in their evolution remain largely unknown. Here, we studied the molecular evolution of ALOG family proteins. We found that ALOG proteins are absent in green algae but exist in all land plants analyzed as well as in some Charophycean algae, closest relatives of land plants. Multiple sequence alignments identified the high sequence conservation of ALOG domains in divergent plant lineages. Phylogenetic analyses also identified a distinct clade of ALOG protein member of lycophytes and bryophytes, including two of Marchantia polymorpha LATERAL ORGAN SUPPRESOR (MpLOS1 and MpLOS2) with a long branch length in MpLOS2. Consistent with this, the function of MpLOS1 was replaceable by Phycomitrella patens ALOG proteins, whereas MpLOS2 failed to replace the molecular function of MpLOS1. Moreover, the rice ALOG proteins, OsTAW1 and OsG1, were not able to replace the molecular function of MpLOS1 although we previously found that the function of OsG1 was replaceable by MpLOS1. Altogether, these findings suggest that ALOG proteins emerged before the evolution of land plants and that they exhibit functional conservation and diversification during the evolution of land plants. The finding that MpLOS1 is able to complement rice ALOG mutants but not vice versa also suggest the existence of conserved and the partly divergent functions of ALOG proteins in bryophytes and angiosperms.

Evolution of Plant Hormone Response Pathways.

This review focuses on the evolution of plant hormone signaling pathways. Like the chemical nature of the hormones themselves, the signaling pathways are diverse. Therefore, we focus on a group of hormones whose primary perception mechanism involves an Skp1/Cullin/F-box-type ubiquitin ligase: auxin, jasmonic acid, gibberellic acid, and strigolactone. We begin with a comparison of the core signaling pathways of these four hormones, which have been established through studies conducted in model organisms in the Angiosperms. With the advent of next-generation sequencing and advanced tools for genetic manipulation, the door to understanding the origins of hormone signaling mechanisms in plants beyond these few model systems has opened. For example, in-depth phylogenetic analyses of hormone signaling components are now being complemented by genetic studies in early diverging land plants. Here we discuss recent investigations of how basal land plants make and sense hormones. Finally, we propose connections between the emergence of hormone signaling complexity and major developmental transitions in plant evolution.

Factors Affecting Organelle Genome Stability in Physcomitrella patens.

Organelle genomes are essential for plants; however, the mechanisms underlying the maintenance of organelle genomes are incompletely understood. Using the basal land plant Physcomitrella patens as a model, nuclear-encoded homologs of bacterial-type homologous recombination repair (HRR) factors have been shown to play an important role in the maintenance of organelle genome stability by suppressing recombination between short dispersed repeats. In this review, I summarize the factors and pathways involved in the maintenance of genome stability, as well as the repeats that cause genomic instability in organelles in P. patens, and compare them with findings in other plant species. I also discuss the relationship between HRR factors and organelle genome structure from the evolutionary standpoint.

The DEAD-box RNA helicase eIF4A regulates plant development and interacts with the hnRNP LIF2L1 in Physcomitrella patens

eIF4A is a RNA-stimulated ATPase and helicase. Besides its key role in regulating cap-dependent translation initiation in eukaryotes, it also performs specific functions in regulating cell cycle progression, plant growth and abiotic stress tolerance. Flowering plants encode three eIF4A paralogues, eIF4A1, eIF4A2 and eIF4A3 that share conserved sequence motifs but differ in functions. To date, however, no information is available on eIF4A in basal land plants. In this study we report that genome of the moss Physcomitrella patens encodes multiple eIF4A genes. The encoded proteins possess the highly conserved motifs characteristic of the DEAD box helicases. Spatial expression analysis shows these genes to be ubiquitously expressed in all tissue types with Pp3c6_1080V3.1 showing high expression in filamentous protonemata. Targeted deletion of conserved core motifs in Pp3c6_1080V3.1 slowed protonemata growth and resulted in dwarfing of leafy gametophores suggesting a role for Pp3c6_1080V3.1 in regulating cell division/elongation. Rapid and strong induction of Pp3c6_1080V3.1 under salt stress and slow recovery of knockout plants upon exposure to high salt further suggest Pp3c6_1080V3.1 to be involved in stress management in P. patens. Protein-protein interaction studies that show Pp3c6_1080V3.1 to interact with the Physcomitrella heterogenous ribonucleoprotein, LIF2L1, a transcriptional regulator of stress-responsive genes in Arabidopsis. The results presented in this study provide insight into evolutionary conserved functions of eIF4A and shed light on the novel link between eIF4A activities and stress mitigation pathways/RNA metabolic processes in P. patens.

Regulation of the Poly(A) Status of Mitochondrial mRNA by Poly(A)-Specific Ribonuclease Is Conserved among Land Plants.

Regulation of the stability and the quality of mitochondrial RNA is essential for the maintenance of mitochondrial and cellular functions in eukaryotes. We have previously reported that the eukaryotic poly(A)-specific ribonuclease (PARN) and the prokaryotic poly(A) polymerase encoded by AHG2 and AGS1, respectively, coordinately regulate the poly(A) status and the stability of mitochondrial mRNA in Arabidopsis. Mitochondrial function of PARN has not been reported in any other eukaryotes. To know how much this PARN-based mitochondrial mRNA regulation is conserved among plants, we studied the AHG2 and AGS1 counterparts of the liverwort, Marchantia polymorpha, a member of basal land plant lineage. We found that M. polymorpha has one ortholog each for AHG2 and AGS1, named MpAHG2 and MpAGS1, respectively. Their Citrine-fused proteins were detected in mitochondria of the liverwort. Molecular genetic analysis showed that MpAHG2 is essential and functionally interacts with MpAGS1 as observed in Arabidopsis. A recombinant MpAHG2 protein had a deadenylase activity in vitro. Overexpression of MpAGS1 and the reduced expression of MpAHG2 caused an accumulation of polyadenylated Mpcox1 mRNA. Furthermore, MpAHG2 suppressed Arabidopsis ahg2-1 mutant phenotype. These results suggest that the PARN-based mitochondrial mRNA regulatory system is conserved in land plants.

GEMMA CUP-ASSOCIATED MYB1, an Ortholog of Axillary Meristem Regulators, Is Essential in Vegetative Reproduction in Marchantiapolymorpha.

A variety of plants in diverse taxa can reproduce asexually via vegetative propagation, in which clonal propagules with a new meristem(s) are generated directly from vegetative organs. A basal land plant, Marchantia polymorpha, develops clonal propagules, gemmae, on the gametophyte thallus from the basal epidermis of a specialized receptacle, the gemma cup. Here we report an R2R3-MYB transcription factor, designated GEMMA CUP-ASSOCIATED MYB1 (GCAM1), which is an essential regulator of gemma cup development in M.polymorpha. Targeted disruption of GCAM1 conferred a complete loss of gemma cup formation and gemma generation. Ectopic overexpression of GCAM1 resulted in formation of cell clumps, suggesting a function of GCAM1 in suppression of cell differentiation. Although gemma cups are a characteristic gametophyte organ for vegetative reproduction in a taxonomically restricted group of liverwort species, phylogenetic and interspecific complementation analyses support the orthologous relationship of GCAM1 to regulatory factors of axillary meristem formation, e.g., Arabidopsis REGULATOR OF AXILLARY MERISTEMS and tomato Blind, in angiosperm sporophytes. The present findings in M.polymorpha suggest an ancient acquisition of a transcriptional regulator for production of asexual propagules in the gametophyte and the use of the regulatory factor for diverse developmental programs, including axillary meristem formation, during land plant evolution.

Arabinogalactan-Proteins from the Liverwort Marchantia polymorpha L., a Member of a Basal Land Plant Lineage, Are Structurally Different to Those of Angiosperms.

The thalloid liverwort Marchantia polymorpha as a member of a basal land plant lineage has to cope with the challenge of terrestrial life. Obviously, the plant cell wall has been strongly involved in the outstanding evolutionary process of water-to-land-transition. AGPs are signaling glycoproteins of the cell wall, which seem to be ubiquitous in seed plants and might play a role in adaption to abiotic and biotic stress situations. Therefore, we investigated the cell wall composition of Marchantia polymorpha with special focus on structural characterization of arabinogalactan-proteins. The Marchantia AGP shows typical features known from seed plant AGPs like precipitation with β-glucosyl-Yariv’s reagent, a protein moiety with hydroxyproline and a carbohydrate part with 1,3,6-linked galactose and terminal arabinose residues. On the other hand, striking differences to AGPs of angiosperms are the occurrence of terminal 3-O-methyl-rhamnose and a highly branched galactan lacking appreciable amounts of 1,6-linked galactose. Binding of different AGP-antibodies (JIM13, KM1, LM2, LM6, LM14, LM26, and MAC207) to Marchantia AGP was investigated and confirmed structural differences between liverwort and angiosperm AGP, possibly due to deviating functions of these signaling molecules in the different taxonomic groups.

The RopGEF KARAPPO Is Essential for the Initiation of Vegetative Reproduction in Marchantia polymorpha.

Many plants can reproduce vegetatively, producing clonal progeny from vegetative cells; however, little is known about the molecular mechanisms underlying this process. Liverwort (Marchantia polymorpha), a basal land plant, propagates asexually via gemmae, which are clonal plantlets formed in gemma cups on the dorsal side of the vegetative thallus [1]. The initial stage of gemma development involves elongation and asymmetric divisions of a specific type of epidermal cell, called a gemma initial, which forms on the floor of the gemma cup [2, 3]. To investigate the regulatory mechanism underlying gemma development, we focused on two allelic mutants in which no gemma initial formed; these mutants were named karappo, meaning "empty." We used whole-genome sequencing of both mutants and molecular genetic analysis to identify the causal gene, KARAPPO (KAR), which encodes a ROP guanine nucleotide exchange factor (RopGEF) carrying a plant-specific ROP nucleotide exchanger (PRONE) catalytic domain. Invitro GEF assays showed that the full-length KAR protein and the PRONE domain have significant GEF activity toward MpROP, the only ROP GTPase in M.polymorpha. Moreover, genetic complementation experiments showed a significant role for the N- and C-terminal variable regions in gemma development. Our investigation demonstrates an essential role for KAR/RopGEF in the initiation of plantlet development from a differentiated cell, which may involve cell-polarity formation and subsequent asymmetric cell division via activation of ROP signaling, implying a similar developmental mechanism in vegetative reproduction of various land plants.

BLADE-ON-PETIOLE genes are not involved in the transition from protonema to gametophore in the moss Physcomitrella patens.

The timing of the transition between developmental phases is a critical determinant of plant form. In the moss Physcomitrella patens, the transition from protonema to gametophore is a particularly important step as it results in a change from two-dimensional to three-dimensional growth of the plant body. It is well known that this transition is promoted by cytokinin (CK), however, the underlying mechanisms are poorly understood. Previously, it was reported that P. patens orthologs of BLADE-ON-PETIOLE (BOP) genes (PpBOPs) work downstream of CK to promote the transition to gametophore. To further understand the role of PpBOPs in the control of this transition, we performed functional analyses of PpBOP genes. We simultaneously disrupted the function of all three PpBOP genes in P. patens using CRISPR technology, however, no abnormal phenotypes were observed in the triple mutant during either the gametophytic or the sporophytic growth stages. CK treatment did not alter the phase change in the triple mutant. We conclude that PpBOP genes are unnecessary in the control of P. patens development under normal conditions. We propose that BOP genes are not involved in the control of developmental processes in bryophytes and other basal land plants, but may function in physiological processes such as in the defense response.

Marchantia polymorpha, a New Model Plant for Autophagy Studies.

Autophagy is a catabolic process for bulk and selective degradation of cytoplasmic components in the vacuole/lysosome. In Saccharomyces cerevisiae, ATG genes were identified as essential genes for autophagy, and most ATG genes are highly conserved among eukaryotes, including plants. Although reverse genetic analyses have revealed that autophagy is involved in responses to abiotic and biotic stresses in land plants, our knowledge of its molecular mechanism remains limited. This limitation is partly because of the multiplication of some ATG genes, including ATG8, in widely used model plants such as Arabidopsis thaliana, which adds complexity to functional studies. Furthermore, due to limited information on the composition and functions of the ATG genes in basal land plants and charophytes, it remains unclear whether multiplication of ATG genes is associated with neofunctionalization of these genes. To gain insight into the diversification of ATG genes during plant evolution, we compared the composition of ATG genes in plants with a special focus on a liverwort and two charophytes, which have not previously been analyzed. Our results showed that the liverwort Marchantia polymorpha and the charophytes Klebsormidium nitens and Chara braunii harbor fundamental sets of ATG genes with low redundancy compared with those of A. thaliana and the moss Physcomitrella patens, suggesting that multiplication of ATG genes occurred during land plant evolution. We also attempted to establish an experimental system for analyzing autophagy in M. polymorpha. We generated transgenic plants expressing fluorescently tagged MpATG8 to observe its dynamics in M. polymorpha and produced autophagy-defective mutants by genome editing using the CRISPR/Cas9 system. These tools allowed us to demonstrate that MpATG8 is transported into the vacuole in an MpATG2-, MpATG5-, and MpATG7-dependent manner, suggesting that fluorescently tagged MpATG8 can be used as an autophagosome marker in M. polymorpha. M. polymorpha can provide a powerful system for studying the mechanisms and evolution of autophagy in plants.

Genome-wide analysis of MpBHLH12, a IIIf basic helix-loop-helix transcription factor of Marchantia polymorpha

The evolution of plants on land required adaptation to UV radiation and dry environments, and involved the appearance and/or rewiring of genetic connections, known as gene regulatory networks (GRNs), which consist of one or more transcription factors (TFs). The liverwort, Marchantia polymorpha, is a basal land plant, with a recently sequenced genome. The number of genes encoding basic helix-loop-helix (bHLH) family members is considerably higher in M. polymorpha than in charophyte green algae, suggesting the contribution of bHLH proteins to the evolution of GRNs associated with the adaptation of plants to land. Although an understanding of the evolutionary aspects of GRNs is fundamental for elucidating the mechanisms of environmental adaptation, the evolution of GRNs that led to land adaptation in plants remains poorly understood. In this study, we isolated a single gene encoding a IIIf bHLH TF from M. polymorpha, MpBHLH12. Transgenic M. polymorpha constitutively overexpressing MpBHLH12 showed smaller and fewer gemma cups than wild type, suggesting that MpBHLH12 is involved in the regulation of morphological development. Transcriptomic analysis of MpBHLH12 overexpressor (MpBHLH12ox) lines revealed an overlap with the GRN of MpMYB14, which regulates the biosynthesis of anthocyanins and phenolic compounds. However, MpBHLH12ox did not show anthocyanin accumulation. Results of the transient reporter assay suggest that MpBHLH12 could function in repression rather than activation. Our findings suggest that although the IIIf bHLH MpBHLH12 shows highest amino acid similarity with IIIf bHLH clade and is involved in developmental process and partly biosynthesis of phenolic compounds in M. polymorpha like Arabidopsis IIIf bHLH, the GRN involving MpBHLH12 would be distinct one from those of the IIIf bHLH TFs of seed plants.

Reproductive Induction is a Far-Red High Irradiance Response that is Mediated by Phytochrome and PHYTOCHROME INTERACTING FACTOR in Marchantia polymorpha.

Land plants have evolved a series of photoreceptors to precisely perceive environmental information. Among these, phytochromes are the sole photoreceptors for red light (R) and far-red light (FR), and play pivotal roles in modulating various developmental processes. Most extant land plants possess multiple phytochromes that probably evolved from a single phytochrome in the common ancestor of land plants. However, the ancestral phytochrome signaling mechanism remains unknown due to a paucity of knowledge regarding phytochrome functions in basal land plants. It has recently been reported that Mpphy, a single phytochrome in the liverwort Marchantia polymorpha, regulates typical photoreversible responses collectively classified as low fluence response (LFR). Here, we show that Mpphy also regulates the gametangiophore formation analogous to the mode of action of the far-red high irradiance response (FR-HIR) in angiosperms. Our phenotypic analyses using mutant plants obtained by CRISPR/Cas9-based genome editing revealed that MpFHY1, an ortholog of FAR-RED ELONGATED HYPOCOTYL1, as well as Mpphy is critical for the FR-HIR signaling in M. polymorpha. In addition, knockout of MpPIF, a single PHYTOCHROME INTERACTING FACTOR gene in M. polymorpha, completely abolished the FR-HIR-dependent gametangiophore formation, while overexpression of MpPIF accelerated the response. FR-HIR-dependent transcriptional regulation was also disrupted in the Mppif mutant. Our findings suggest that plants had already acquired the FR-HIR signaling mediated by phytochrome and PIF at a very early stage during the course of land plant evolution, and that a single phytochrome in the common ancestor of land plants could mediate both LFR and FR-HIR.

Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha.

Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.

Stepwise evolution of supercomplex formation with photosystem I is required for stabilization of chloroplast NADH dehydrogenase-like complex: Lhca5-dependent supercomplex formation in Physcomitrella patens.

In angiosperms, such as Arabidopsis and barley, the chloroplast NADH dehydrogenase-like (NDH) complex associates with two copies of photosystem I (PSI) supercomplex to form an NDH-PSI supercomplex for the stabilization of the NDH complex. Two linker proteins, Lhca5 and Lhca6, are members of the light-harvesting complex I (LHCI) family and mediate this supercomplex formation. The liverwort Marchantia polymorpha has branched from the basal land plant lineage and has neither Lhca5 nor Lhca6. Consequently, the NDH complex does not form a supercomplex with PSI in this plant. The Lhca6 gene does not seem to exist also in the moss Physcomitrella patens (Physcomitrella). Conversely, the Lhca5 gene has been found in Physcomitrella, although experimental evidence is still lacking for its contribution to NDH-PSI supercomplex formation as a linker. Here, we biochemically characterized the Lhca5 knock-out mutant (lhca5) in Physcomitrella. The NDH-PSI supercomplex observed in wild-type Physcomitrella was absent in the lhca5 mutant. Lhca5 protein was detected in this NDH-PSI supercomplex. Some PSI and NDH subunits were co-immunoprecipitated with Lhca5-HA. These results indicate that the Physcomitrella gene is the functional ortholog of Lhca5 reported in Arabidopsis. Between Physcomitrella and Arabidopsis, the stromal loop region is highly conserved in Lhca5 proteins but not in other LHCI members. We found that Lhca5 contributed to the stable accumulation of the NDH complex, but part of the NDH complex was still sensitive to high light intensity, even in the wild-type. We considered that angiosperms acquired another linker protein, Lhca6, to further stabilize the NDH complex.

ANGUSTIFOLIA contributes to the regulation of three-dimensional morphogenesis in the liverwort Marchantia polymorpha.

Arabidopsis thaliana mutants deficient in ANGUSTIFOLIA (AN) exhibit several phenotypes at the sporophyte stage, such as narrow and thicker leaves, trichomes with two branches, and twisted fruits. It is thought that these phenotypes are caused by abnormal arrangement of cortical microtubules (MTs). AN homologs are present in the genomes of diverse land plants, including the basal land plant Marchantia polymorpha, and their molecular functions have been shown to be evolutionarily conserved in terms of the ability to complement the A. thaliana an-1 mutation. However, the roles of ANs in bryophytes, the life cycle of which includes a dominant haploid gametophyte generation, remain unknown. Here, we have examined the roles of AN homologs in the model bryophyte M. polymorpha (MpAN). Mpan knockout mutants showed abnormal twisted thalli and suppressed thallus growth along the growth axis. Under weak blue light conditions, elongated thallus growth was observed in wild-type plants, whereas it was suppressed in the mutants. Moreover, disordered cortical MT orientations were observed. Our findings suggest that MpAN contributes to three-dimensional morphogenesis by regulating cortical MT arrangement in the gametophytes of bryophytes.

Loss of CG Methylation in Marchantia polymorpha Causes Disorganization of Cell Division and Reveals Unique DNA Methylation Regulatory Mechanisms of Non-CG Methylation.

DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, the M. polymorpha ortholog of the METHYLTRANSFERASE 1 (MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmet mutants using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9) system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that MpMET is required for maintaining differentiated cellular identities in the gametophyte. Even though numerous TEs were up-regulated, non-CG methylation was generally highly increased at TEs in the Mpmet mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha. Methylation of CCG sites in M. polymorpha does not depend on MET1, unlike in A. thaliana and Physcomitrella patens. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.

Light-regulated PAS-containing histidine kinases delay gametophore formation in the moss Physcomitrella patens.

Two-component systems (TCSs) are signal transduction mechanisms for responding to various environmental stimuli. In angiosperms, TCSs involved in phytohormone signaling have been intensively studied, whereas there are only a few reports on TCSs in basal land plants. The moss Physcomitrella patens possesses several histidine kinases (HKs) that are lacking in seed plant genomes. Here, we studied two of these unique HKs, PAS-histidine kinase 1 (PHK1) and its paralog PHK2, both of which have PAS (Per-Arnt-Sim) domains, which are known to show versatile functions such as sensing light or molecular oxygen. We found homologs of PHK1 and PHK2 only in early diverged clades such as bryophytes and lycophytes, but not in seed plants. The PAS sequences of PHK1 and PHK2 are more similar to a subset of bacterial PAS sequences than to any angiosperm PAS sequences. Gene disruption lines that lack either PHK1 or PHK2 or both formed gametophores earlier than the wild-type, and consistently, more caulonema side branches were induced in response to light in the disruption lines. Therefore, PHK1 and PHK2 delay the timing of gametophore development, probably by suppressing light-induced caulonema branching. This study provides new insights into the evolution of TCSs in plants.

Phosphoproteomic profiling reveals ABA-responsive phosphosignaling pathways in Physcomitrella patens.

Abscisic acid (ABA) and its signaling system are important for land plants to survive in terrestrial conditions. Here, we took a phosphoproteomic approach to elucidate the ABA signaling network in Physcomitrella patens, a model species of basal land plants. Our phosphoproteomic analysis detected 4630 phosphopeptides from wild-type P.patens and two ABA-responsive mutants, a disruptant of group-A type-2C protein phosphatase (PP2C; ppabi1a/b) and AR7, a defective mutant in ARK, identified as an upstream regulator of SnRK2. Quantitative analysis detected 143 ABA-responsive phosphopeptides in P.patens. The analysis indicated that SnRK2-mediated phosphorylation and target motifs were partially conserved in bryophytes. Our data demonstrate that the PpSnRK2B and AREB/ABF-type transcription factors are phosphorylated invivo in response to ABA under the control of ARK. On the other hand, our data also revealed the following: (i) the entire ABA-responsive phosphoproteome in P.patens is quite diverse; (ii) P.patens PP2C affects additional pathways other than the known ABA signaling pathway; and (iii) ARK is mainly involved in ABA signaling. Taken together, we propose that the core ABA signaling pathway is essential in all land plants; however, some ABA-responsive phosphosignaling uniquely developed in bryophytes during the evolutionary process.

Origin and evolution of the nuclear auxin response system.

The small signaling molecule auxin controls numerous developmental processes in land plants, acting mostly by regulating gene expression. Auxin response proteins are represented by large families of diverse functions, but neither their origin nor their evolution is understood. Here, we use a deep phylogenomics approach to reconstruct both the origin and the evolutionary trajectory of all nuclear auxin response protein families. We found that, while all subdomains are ancient, a complete auxin response mechanism is limited to land plants. Functional phylogenomics predicts defined steps in the evolution of response system properties, and comparative transcriptomics across six ancient lineages revealed how these innovations shaped a sophisticated response mechanism. Genetic analysis in a basal land plant revealed unexpected contributions of ancient non-canonical proteins in auxin response as well as auxin-unrelated function of core transcription factors. Our study provides a functional evolutionary framework for understanding diverse functions of the auxin signal.