Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Complex Polyploids: Origins, Genomic Composition, and Role of Introgressed Alleles.

Introgression allows polyploid species to acquire new genomic content from diploid progenitors or from other unrelated diploid or polyploid lineages, contributing to genetic diversity and facilitating adaptive allele discovery. In some cases, high levels of introgression elicit the replacement of large numbers of alleles inherited from the polyploid's ancestral species, profoundly reshaping the polyploid's genomic composition. In such complex polyploids it is often difficult to determine which taxa were the progenitor species and which taxa provided additional introgressive blocks through subsequent hybridization. Here, we use population-level genomic data to reconstruct the phylogenetic history of Betula pubescens (downy birch), a tetraploid species often assumed to be of allopolyploid origin and which is known to hybridize with at least four other birch species. This was achieved by modeling of polyploidization and introgression events under the multispecies coalescent and then using an approximate Bayesian computation (ABC) rejection algorithm to evaluate and compare competing polyploidization models. We provide evidence that B. pubescens is the outcome of an autoploid genome doubling event in the common ancestor of B. pendula and its extant sister species, B. platyphylla, that took place approximately 178,000-188,000 generations ago. Extensive hybridization with B. pendula, B. nana, and B. humilis followed in the aftermath of autopolyploidization, with the relative contribution of each of these species to the B. pubescens genome varying markedly across the species' range. Functional analysis of B. pubescens loci containing alleles introgressed from B. nana identified multiple genes involved in climate adaptation, while loci containing alleles derived from B. humilis revealed several genes involved in the regulation of meiotic stability and pollen viability in plant species.

DE-ETIOLATED1 has a role in the circadian clock of the liverwort Marchantia polymorpha.

Previous studies of plant circadian clock evolution have often relied on clock models and genes defined in Arabidopsis. These studies identified homologues with seemingly conserved function, as well as frequent gene loss. In the present study, we aimed to identify candidate clock genes in the liverwort Marchantia polymorpha using a more unbiased approach. To identify genes with circadian rhythm we sequenced the transcriptomes of gemmalings in a time series in constant light conditions. Subsequently, we performed functional studies using loss-of-function mutants and gene expression reporters. Among the genes displaying circadian rhythm, a homologue to the transcriptional co-repressor Arabidopsis DE-ETIOLATED1 showed high amplitude and morning phase. Because AtDET1 is arrhythmic and associated with the morning gene function of AtCCA1/LHY, that lack a homologue in liverworts, we functionally studied DET1 in M. polymorpha. We found that the circadian rhythm of MpDET1 expression is disrupted in loss-of-function mutants of core clock genes and putative evening-complex genes. MpDET1 knock-down in turn results in altered circadian rhythm of nyctinastic thallus movement and clock gene expression. We could not detect any effect of MpDET1 knock-down on circadian response to light, suggesting that MpDET1 has a yet unknown function in the M. polymorpha circadian clock.

Rates and patterns of molecular evolution in bryophyte genomes, with focus on complex thalloid liverworts, Marchantiopsida.

Plants commonly referred to as "bryophytes" belong to three major lineages of non-vascular plants: the liverworts, the hornworts and the mosses. They are unique among land plants in having a dominant haploid generation and a short-lived diploid sporophytic generation. The dynamics of selection acting on a haploid genome differs from those acting on a diploid genome: new mutations are directly exposed to selection. The general aim of this paper is to investigate the diversification rateof bryophytes - measured as silent site substitution rate representing neutral evolution (mutation rate) and the nonsynonymous to synonymous substitution rate ratio (dN/dS) representing selective evolution - and compare it with earlier studies on vascular plants. Results show that the silent site substitution rate is lower for liverworts as compared to angiosperms, but not as low as for gymnosperms. The selection pressure, measured as dN/dS, isnot remarkably lower for bryophytes as compared to other diploid dominant plants as would be expected by the masking hypothesis, indicating that other factors are more important than ploidy.

Class C ARFs evolved before the origin of land plants and antagonize differentiation and developmental transitions in Marchantia polymorpha.

A plethora of developmental and physiological processes in land plants is influenced by auxin, to a large extent via alterations in gene expression by AUXIN RESPONSE FACTORs (ARFs). The canonical auxin transcriptional response system is a land plant innovation, however, charophycean algae possess orthologues of at least some classes of ARF and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, suggesting that elements of the canonical land plant system existed in an ancestral alga. We reconstructed the phylogenetic relationships between streptophyte ARF and AUX/IAA genes and functionally characterized the solitary class C ARF, MpARF3, in Marchantia polymorpha. Phylogenetic analyses indicate that multiple ARF classes, including class C ARFs, existed in an ancestral alga. Loss- and gain-of-function MpARF3 alleles result in pleiotropic effects in the gametophyte, with MpARF3 inhibiting differentiation and developmental transitions in multiple stages of the life cycle. Although loss-of-function Mparf3 and Mpmir160 alleles respond to exogenous auxin treatments, strong miR-resistant MpARF3 alleles are auxin-insensitive, suggesting that class C ARFs act in a context-dependent fashion. We conclude that two modules independently evolved to regulate a pre-existing ARF transcriptional network. Whereas the auxin-TIR1-AUX/IAA pathway evolved to repress class A/B ARF activity, miR160 evolved to repress class C ARFs in a dynamic fashion.

Auxin Produced by the Indole-3-Pyruvic Acid Pathway Regulates Development and Gemmae Dormancy in the Liverwort Marchantia polymorpha.

The plant hormone auxin (indole-3-acetic acid [IAA]) has previously been suggested to regulate diverse forms of dormancy in both seed plants and liverworts. Here, we use loss- and gain-of-function alleles for auxin synthesis- and signaling-related genes, as well as pharmacological approaches, to study how auxin regulates development and dormancy in the gametophyte generation of the liverwort Marchantia polymorpha. We found that M. polymorpha possess the smallest known toolkit for the indole-3-pyruvic acid (IPyA) pathway in any land plant and that this auxin synthesis pathway mainly is active in meristematic regions of the thallus. Previously a Trp-independent auxin synthesis pathway has been suggested to produce a majority of IAA in bryophytes. Our results indicate that the Trp-dependent IPyA pathway produces IAA that is essential for proper development of the gametophyte thallus of M. polymorpha. Furthermore, we show that dormancy of gemmae is positively regulated by auxin synthesized by the IPyA pathway in the apex of the thallus. Our results indicate that auxin synthesis, transport, and signaling, in addition to its role in growth and development, have a critical role in regulation of gemmae dormancy in M. polymorpha.

A Simple Auxin Transcriptional Response System Regulates Multiple Morphogenetic Processes in the Liverwort Marchantia polymorpha.

In land plants comparative genomics has revealed that members of basal lineages share a common set of transcription factors with the derived flowering plants, despite sharing few homologous structures. The plant hormone auxin has been implicated in many facets of development in both basal and derived lineages of land plants. We functionally characterized the auxin transcriptional response machinery in the liverwort Marchantia polymorpha, a member of the basal lineage of extant land plants. All components known from flowering plant systems are present in M. polymorpha, but they exist as single orthologs: a single MpTOPLESS (TPL) corepressor, a single MpTRANSPORT inhibitor response 1 auxin receptor, single orthologs of each class of auxin response factor (ARF; MpARF1, MpARF2, MpARF3), and a single negative regulator auxin/indole-3-acetic acid (MpIAA). Phylogenetic analyses suggest this simple system is the ancestral condition for land plants. We experimentally demonstrate that these genes act in an auxin response pathway--chimeric fusions of the MpTPL corepressor with heterodimerization domains of MpARF1, MpARF2, or their negative regulator, MpIAA, generate auxin insensitive plants that lack the capacity to pattern and transition into mature stages of development. Our results indicate auxin mediated transcriptional regulation acts as a facilitator of branching, differentiation and growth, rather than acting to determine or specify tissues during the haploid stage of the M. polymorpha life cycle. We hypothesize that the ancestral role of auxin is to modulate a balance of differentiated and pluri- or totipotent cell states, whose fates are determined by interactions with combinations of unrelated transcription factors.

Early evolution of the land plant circadian clock.

While angiosperm clocks can be described as an intricate network of interlocked transcriptional feedback loops, clocks of green algae have been modelled as a loop of only two genes. To investigate the transition from a simple clock in algae to a complex one in angiosperms, we performed an inventory of circadian clock genes in bryophytes and charophytes. Additionally, we performed functional characterization of putative core clock genes in the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis. Phylogenetic construction was combined with studies of spatiotemporal expression patterns and analysis of M. polymorpha clock gene mutants. Homologues to core clock genes identified in Arabidopsis were found not only in bryophytes but also in charophytes, albeit in fewer copies. Circadian rhythms were detected for most identified genes in M. polymorpha and A. agrestis, and mutant analysis supports a role for putative clock genes in M. polymorpha. Our data are in line with a recent hypothesis that adaptation to terrestrial life occurred earlier than previously expected in the evolutionary history of charophyte algae. Both gene duplication and acquisition of new genes was important in the evolution of the plant circadian clock, but gene loss has also contributed to shaping the clock of bryophytes.

RISAP is a TGN-associated RAC5 effector regulating membrane traffic during polar cell growth in tobacco.

RAC/ROP GTPases coordinate actin dynamics and membrane traffic during polar plant cell expansion. In tobacco (Nicotiana tabacum), pollen tube tip growth is controlled by the RAC/ROP GTPase RAC5, which specifically accumulates at the apical plasma membrane. Here, we describe the functional characterization of RISAP, a RAC5 effector identified by yeast (Saccharomyces cerevisiae) two-hybrid screening. RISAP belongs to a family of putative myosin receptors containing a domain of unknown function 593 (DUF593) and binds via its DUF593 to the globular tail domain of a tobacco pollen tube myosin XI. It also interacts with F-actin and is associated with a subapical trans-Golgi network (TGN) compartment, whose cytoplasmic position at the pollen tube tip is maintained by the actin cytoskeleton. In this TGN compartment, apical secretion and endocytic membrane recycling pathways required for tip growth appear to converge. RISAP overexpression interferes with apical membrane traffic and blocks tip growth. RAC5 constitutively binds to the N terminus of RISAP and interacts in an activation-dependent manner with the C-terminal half of this protein. In pollen tubes, interaction between RAC5 and RISAP is detectable at the subapical TGN compartment. We present a model of RISAP regulation and function that integrates all these findings.

In vivo Rac/Rop localization as well as interaction with RhoGAP and RhoGDI in tobacco pollen tubes: analysis by low-level expression of fluorescent fusion proteins and bimolecular fluorescence complementation.

Polarized Rac/Rop GTPase signaling plays a key role in polar cell growth, which is essential for plant morphogenesis. The molecular and cellular mechanisms responsible for the polarization of Rac/Rop signaling during polar cell growth are only partially understood. Mutant variants of Rac/Rop GTPases lacking specific functions are important tools to investigate these mechanisms, and have been employed to develop a model suggesting that RhoGAP (GTPase activating protein) and RhoGDI (Guanine Nucleotide Dissociation Inhibitor) mediated recycling of Rac/Rop GTPases maintains apical polarization of Rac/Rop activity in pollen tubes, which elongate by 'tip growth' (an extreme form of polar cell growth). Despite the importance of these mutant variants for Rac/Rop functional characterization, their distinct intracellular distributions have not been thoroughly comparatively and quantitatively analyzed. Furthermore, support for the proposed RhoGAP and RhoGDI functions in apical polarization of Rac/Rop activity based on the analysis of in vivo interactions between these proteins and Rac/Rop GTPases has been missing. Here, extensive fluorescent protein tagging and bimolecular fluorescence complementation (BiFC) analyses are described of the intracellular distributions of wild type and mutant variants of the tobacco pollen tube Rac/Rop GTPase Nt-Rac5, as well as of interactions of these Nt-Rac5 variants with RhoGAP and RhoGDI proteins, in normally growing transiently transformed pollen tubes. Presented results substantially enhance our understanding of apical dynamics of pollen tube Rac/Rop signaling proteins, confirm previously proposed RhoGAP and RhoGDI functions in Rac/Rop polarization and provide important technical insights facilitating future in vivo protein localization and BiFC experiments in pollen tubes.

Homologues of the Arabidopsis thaliana SHI/STY/LRP1 genes control auxin biosynthesis and affect growth and development in the moss Physcomitrella patens.

The plant hormone auxin plays fundamental roles in vascular plants. Although exogenous auxin also stimulates developmental transitions and growth in non-vascular plants, the effects of manipulating endogenous auxin levels have thus far not been reported. Here, we have altered the levels and sites of auxin production and accumulation in the moss Physcomitrella patens by changing the expression level of homologues of the Arabidopsis SHI/STY family proteins, which are positive regulators of auxin biosynthesis genes. Constitutive expression of PpSHI1 resulted in elevated auxin levels, increased and ectopic expression of the auxin response reporter GmGH3pro:GUS, and in an increased caulonema/chloronema ratio, an effect also induced by exogenous auxin application. In addition, we observed premature ageing and necrosis in cells ectopically expressing PpSHI1. Knockout of either of the two PpSHI genes resulted in reduced auxin levels and auxin biosynthesis rates in leafy shoots, reduced internode elongation, delayed ageing, a decreased caulonema/chloronema ratio and an increased number of axillary hairs, which constitute potential auxin biosynthesis sites. Some of the identified auxin functions appear to be analogous in vascular and non-vascular plants. Furthermore, the spatiotemporal expression of the PpSHI genes and GmGH3pro:GUS strongly overlap, suggesting that local auxin biosynthesis is important for the regulation of auxin peak formation in non-vascular plants.

The Arabidopsis thaliana STYLISH1 protein acts as a transcriptional activator regulating auxin biosynthesis.

The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

Physcomitrium patens PpRIC, an ancestral CRIB-domain ROP effector, inhibits auxin-induced differentiation of apical initial cells.

RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain-containing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hormone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remarkably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in mediating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity.

The Arabidopsis thaliana transcriptional activator STYLISH1 regulates genes affecting stamen development, cell expansion and timing of flowering.

SHORT-INTERNODES/STYLISH (SHI/STY)-family proteins redundantly regulate development of lateral organs in Arabidopsis thaliana. We have previously shown that STY1 interacts with the promoter of the auxin biosynthesis gene YUCCA (YUC)4 and activates transcription of the genes YUC4, YUC8 and OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF (ORA)59 independently of protein translation. STY1 also affects auxin levels and auxin biosynthesis rates. Here we show that STY1 induces the transcription of 16 additional genes independently of protein translation. Several of these genes are tightly co-expressed with SHI/STY-family genes and/or down-regulated in SHI/STY-family multiple mutant lines, suggesting them to be regulated by SHI/STY proteins during plant development. The majority of the identified genes encode transcription factors or cell expansion-related enzymes and functional studies suggest their involvement in stamen and leaf development or flowering time regulation.

Expression of Arabidopsis SHORT INTERNODES/STYLISH family genes in auxin biosynthesis zones of aerial organs is dependent on a GCC box-like regulatory element.

Auxin/indole-3-acetic acid (IAA) biosynthesis in Arabidopsis (Arabidopsis thaliana) plays a major role in growth responses to developmental and genetic signals as well as to environmental stimuli. Knowledge of its regulation, however, remains rudimentary, and few proteins acting as transcriptional modulators of auxin biosynthesis have been identified. We have previously shown that alteration in the expression level of the SHORT INTERNODES/STYLISH (SHI/STY) family member STY1 affects IAA biosynthesis rates and IAA levels and that STY1 acts as a transcriptional activator of genes encoding auxin biosynthesis enzymes. Here, we have analyzed the upstream regulation of SHI/STY family members to gain further insight into transcriptional regulation of auxin biosynthesis. We attempted to modulate the normal expression pattern of STY1 by mutating a putative regulatory element, a GCC box, located in the proximal promoter region and conserved in most SHI/STY genes in Arabidopsis. Mutations in the GCC box abolish expression in aerial organs of the adult plant. We also show that induction of the transcriptional activator DORNRÖSCHEN-LIKE (DRNL) activates the transcription of STY1 and other SHI/STY family members and that this activation is dependent on a functional GCC box. Additionally, STY1 expression in the strong drnl-2 mutant or the drn drnl-1 puchi-1 triple mutant, carrying knockdown mutations in both DRNL and its close paralogue DRN as well as one of their closest homologs, PUCHI, was significantly reduced, suggesting that DRNL regulates STY1 during normal plant development and that several other genes might have redundant functions.

PIF-independent regulation of growth by an evening complex in the liverwort Marchantia polymorpha.

Previous studies in the liverwort Marchantia polymorpha have shown that the putative evening complex (EC) genes LUX ARRHYTHMO (LUX) and ELF4-LIKE (EFL) have a function in the liverwort circadian clock. Here, we studied the growth phenotypes of MpLUX and MpEFL loss-of-function mutants, to establish if PHYTOCHROME-INTERACTING FACTOR (PIF) and auxin act downstream of the M. polymorpha EC in a growth-related pathway similar to the one described for the flowering plant Arabidopsis. We examined growth rates and cell properties of loss-of-function mutants, analyzed protein-protein interactions and performed gene expression studies using reporter genes. Obtained data indicate that an EC can form in M. polymorpha and that this EC regulates growth of the thallus. Altered auxin levels in Mplux mutants could explain some of the phenotypes related to an increased thallus surface area. However, because MpPIF is not regulated by the EC, and because Mppif mutants do not show reduced growth, the growth phenotype of EC-mutants is likely not mediated via MpPIF. In Arabidopsis, the circadian clock regulates elongation growth via PIF and auxin, but this is likely not an evolutionarily conserved growth mechanism in land plants. Previous inventories of orthologs to Arabidopsis clock genes in various plant lineages showed that there is high levels of structural differences between clocks of different plant lineages. Here, we conclude that there is also variation in the output pathways used by the different plant clocks to control growth and development.

H2A ubiquitination is essential for Polycomb Repressive Complex 1-mediated gene regulation in Marchantia polymorpha.

BACKGROUND: Polycomb repressive complex 1 (PRC1) and PRC2 are chromatin regulators maintaining transcriptional repression. The deposition of H3 lysine 27 tri-methylation (H3K27me3) by PRC2 is known to be required for transcriptional repression, whereas the contribution of H2A ubiquitination (H2Aub) in the Polycomb repressive system remains unclear in plants. RESULTS: We directly test the requirement of H2Aub for gene regulation in Marchantia polymorpha by generating point mutations in H2A that prevent ubiquitination by PRC1. These mutants show reduced H3K27me3 levels on the same target sites as mutants defective in PRC1 subunits MpBMI1 and the homolog MpBMI1L, revealing that PRC1-catalyzed H2Aub is essential for Polycomb system function. Furthermore, by comparing transcriptome data between mutants in MpH2A and MpBMI1/1L, we demonstrate that H2Aub contributes to the PRC1-mediated transcriptional level of genes and transposable elements. CONCLUSION: Together, our data demonstrates that H2Aub plays a direct role in H3K27me3 deposition and is required for PRC1-mediated transcriptional changes in both genes and transposable elements in Marchantia.

Physcomitrella patens: a model to investigate the role of RAC/ROP GTPase signalling in tip growth.

Polarized cell expansion plays an important role in plant morphogenesis. Tip growth is a dramatic form of this process, which is widely used as a model to study its regulation by RAC/ROP GTPase signalling. During the dominant haploid phase of its life cycle, the moss Physcomitrella patens contains different types of cells that expand by tip growth. Physcomitrella is a highly attractive experimental system because its genome has been sequenced, and transgene integration by homologous recombination occurs in this plant at frequencies allowing effective gene targeting. Furthermore, together with the vascular spikemoss Selaginella moellendorffii, whose genome has also been sequenced, the non-vascular moss Physcomitrella provides an evolutionary link between green algae and angiosperms. BLAST searches established that the Physcomitrella and Selaginella genomes encode not only putative RAC/ROP GTPases, but also homologues of all known regulators of polarized RAC/ROP signalling, as well as of key effectors acting in signalling cascades downstream of RAC/ROP activity. Nucleotide sequence relationships within seven different families of Physcomitrella, Selaginella, Arabidopsis thaliana and Nicotiana tabacum (tobacco) genes with distinct functions in RAC/ROP signalling were characterized based on extensive maximum likelihood and Neighbor-Joining analyses. The results of these analyses are interpreted in the light of current knowledge concerning expression patterns and molecular functions of RAC/ROP signalling proteins in angiosperms. A key aim of this study is to facilitate the use of Physcomitrella as a model to investigate the molecular control of tip growth in plants.

Nyctinastic thallus movement in the liverwort Marchantia polymorpha is regulated by a circadian clock.

The circadian clock coordinates an organism's growth, development and physiology with environmental factors. One illuminating example is the rhythmic growth of hypocotyls and cotyledons in Arabidopsis thaliana. Such daily oscillations in leaf position are often referred to as sleep movements or nyctinasty. Here, we report that plantlets of the liverwort Marchantia polymorpha show analogous rhythmic movements of thallus lobes, and that the circadian clock controls this rhythm, with auxin a likely output pathway affecting these movements. The mechanisms of this circadian clock are partly conserved as compared to angiosperms, with homologs to the core clock genes PRR, RVE and TOC1 forming a core transcriptional feedback loop also in M. polymorpha.