MAdLand Projects

The evolution from green algae to land plants involved a dramatic change of body plan. This transition required that simple 1-D algal filaments advanced to 2-D and eventually 3-D growth. The formation of the new body plans became possible through the evolution of branching mechanisms, including profound changes in the mode of cell division. The suggested research is a comparative study aiming at the discovery and analysis of branching and cell division genes. In the zygnematophycean alga Mougeotia filament branching is inducible through a simple terrestrialization system, for which the algal filaments are transferred to solid (e.g. agar) surfaces. Based on this system the cell biology of Mougeotia terrestrialization will be studied using live-cell microscopy and TEM. Next, branching and cell division will be analyzed by a transcriptomic study. Orthologues of genes revealed in this way will be knocked out in the liverwort Marchantia. Phenotypic analyses of the obtained Marchantia plants can reveal how algal genes and proteins evolved to allow for the formation of a bryophyte body plan. This evolutionary comparative approach will reveal how the evolution of cell division and branching enabled the formation of the land plants.

As AGPs are considered common for all angiosperms cell walls, they seem to be essential for life on land. They are involved in different processes like tolerance to drought stress or to other abiotic and biotic stress situations and are therefore molecular candidates important for the drastic habitat change during terrestrialisation. Furthermore, involvement of AGPs during embryogenesis and in plant-microbe interactions have been shown for many land plant species and implicates that AGPs might also play a role in evolution of these processes.
Our work will focus on isolation and structural characterization of arabinogalactan-proteins (AGPs) from the different fern, bryophyte and charophyte model organisms. Monoclonal antibodies directed against arabinogalactan glycan motifs will be used to further elucidate fine structure and to detect AGPs in plant tissue by immunolocalization. The work will be complemented by bioinformatic search for genes of AGP protein backbones and glycosyltransferases responsible for AGP biosynthesis (collaborations see below). In spore-producing land plants, we already detected special structural features probably connected with divergent functions. In charophytic algae, our first investigations revealed fundamental differences with regard to AGPs between Spirogyra (Zygnematophyceae) and Chara (Charophyceae) which support the proposal of a sister-group relationship between land plants and the Zygnematophyceae. Search for molecular ancestors of AGPs in charophytes will enlighten molecular adaptions of these glycoproteins with regard to plant terrestrialisation.

The invention of intercellular communication networks was a crucial step in plant evolution that enabled i) coordinated strategies to face the hazardous biotic and abiotic stresses occurring during the conquest of land and that allowed ii) the development of increasingly complex plant bodies with division of labour between spatially patterned, cooperative tissues and organs.
Plasmodesmata (PD) are complex cytoplasmic channels that interconnect the cells of multicellular plant organisms and mediate the symplasmic exchange of (macro)molecules. In seed plants, the PD networks are highly dynamic and undergo drastic functional and structural changes controlling developmental processes, metabolic acclimatisation and pathogen responses. Yet, information on PD networks in non-seed plants and streptophyte algae is scarce and often inconsistent.
PD-like cell connections have evolved multiple times independently in several algal lineages. Within the streptophytes, homology of PD-like structures in the ZCC grade algae and land plant PD is hypothesised, although there are still uncertainties pertaining to a uniform PD architecture and a common mode of PD formation. The project aims to elucidate whether charophyte PD already include a central ER component and are regularly formed during cytokinesis with the help of the ER.
Postcytokinetic formation of secondary PD in pre-existing cell walls is an appropriate mode to adjust PD numbers (and transport capacities) to changing requirements and has evolved (independently?) at least in some lycophyte lineages and spermatophytes. Whether secondary PD formation also occurs in other land plant lineages is unclear and will systematically be investigated in this project. In order to trace back the structural PD development, PD numbers will be determined by transmission electron microscopy in young and old tissues of the MAdLand model plant species - preferentially in leaf(-like) organs, but also in thalloid bryophyte gametophytes with different tissue complexity, and in hornwort sporophytes. We will also investigate, whether the modification of simple PD into highly branched forms, which also has functional implications, occurs in all land plant taxa.
With these data at hand, basic differences in PD structure and origin can be detected among the streptophyte lineages, the molecular basis of which will be addressed in the second funding period. By using refined proteomic data sets of seed plant PD proteins, presence or absence of orthologues will be investigated in the model species, focussing e.g. on ER-shaping reticulon proteins, membrane tether proteins, or cell wall modelling enzymes.

Sphingolipids are ubiquitous metabolites in eukaryotic cells. Across kingdoms, they are essential membrane components enriched in the plasma membrane, as well as signaling molecules. In plants, they are involved in several processes such as maintenance of plasma membrane integrity and microdomain formation, cell growth and division, polar secretion, and programmed cell death signaling. The precise functions of sphingolipids have been challenging to study in vascular plant models due to non-viable and pleiotropic mutant phenotypes, complex organ structure, and difficulties with sphingolipid extraction and detection. The model bryophytes Marchantia polymorpha and Physcomitrium patens have simpler developmental patterning that may help overcome some of these issues. Moreover, a more phylodiverse study of land plant species is necessary for understanding the acquisition of sphingolipids as a major membrane lipid component of plant cells, and may offer clues as to what the most conserved functions of sphingolipids are in the land plant lineage. Preliminary work by our group revealed a unique sphingolipid profile for Physcomitrium, and diversification of gene families associated with biosynthesis of the central building block of sphingolipids, ceramides. We will now carry out comprehensive sphingolipid profiling of Marchantia, as well as the algae Spirogyra pratensis and Mesotaenium endlicherianum, for a broader perspective of the establishment of characteristic plant sphingolipid building blocks. We will also use Physcomitrium and Marchantia to study the ancestral functions of sphingolipids in land plant. We will focus on the analysis of (1) a sphingolipid desaturase family that our work has suggested is specific to bryophytes and microalgae, (2) the ceramide synthase family as key enzymes in sphingolipid biosynthesis, and (3) ceramide glucosyltransferases, which catalyze the simplest modification of the ceramide backbone. We will further use these systems to test how sphingolipids contribute to tolerance of abiotic and biotic stresses associated with terrestrial life.

Regulation of gene expression is crucial for all developmental decisions as well as adaptations to changing environmental conditions. In contrast to prokaryotes, eukaryotic organisms have evolved complex regulatory pathways that control gene expression at the post-transcriptional level through small RNAs. One specific class of small RNAs are microRNAs (miRNAs) which bind to reverse complementary sequences within their target RNAs to trigger their cleavage or translational inhibition. These miRNAs are processed from specific RNA precursors by DICER-LIKE (DCL) proteins and miRNAs exist already in chlorophytes such as Chlamydomonas reinhardtii, but miRNA biogenesis and the miRNA repertoire differs from that of land plants. In the moss P. patens we identified an autoregulatory loop involving the DCL protein essential for miRNA processing suggesting a feedback control of the miRNA biogenesis pathway. The specific DCL autoregulation has evolved in early land plants and we provide evidence that it is essential for adaptation to elevated salt concentrations. In this project, we intend to unravel the impact of the autoregulation on a genome-wide molecular scale. Furthermore, using a comparative approach we aim to introduce the feedback control into the liverwort Marchantia polymorpha which harbors the miRNA pathway, but lacks the autoregulation and is less tolerant to salt compared to P. patens. By this, we will elucidate whether the invention of the autoregulation presents one of the crucial molecular adaptations required for the water to land transition of the green lineage.

The land plant cell, from root to leave tissue, houses dozens of plastids. The contrary is true for the majority of algae that are monoplastidic and house a single plastid per cell and nucleus. Polyplastidy is the norm for land plants, but an exception among algae. The transition from mono- to polyplastidy occurred a few times independently in evolution and appears to enable macroscopic phenotypes. The steps that allow escaping the monoplastidic bottleneck remain unexplored, but are essentially linked to the de-synchronization of the cell cycle and plastid division. The land plant ancestor was monoplastidic, as are extant streptophyte algae sister to the land plant ancestor. Traces of monoplastidy are evident in bryophytes such as Marchantia. This common liverwort undergoes a switch from poly- to monoplastidy during sporogenesis, such that the released sporocytes carry only one plastid. This, we speculate, is associated with a check-point in which inheritance of healthy plastids is guaranteed, also because meiosis only commences upon successful plastid division. We plan on exploring the genetics and mechanisms behind reduction and increase of plastid number per cell in the bryophyte.
In collaboration, the Zachgo and Bräutigam laboratories (University of Osnabrück and University of Bielefeld, respectively) have recently received funding to generate a high resolution RNA-Seq database of Marchantia’s gene expression. This includes the sequencing of the RNA profile of three different stages (both 2n and 1n) of sporophytes. Prof Zachgo, who we are closely collaborating with on this project, and Prof. Bräutigam have both agreed to providing us with early access to the generated RNA-Seq atlas, which we will screen for expression changes (in relation to the global expression pattern of Marchantia that will be calculated from the newly generated data) in the relevant gene families. Regarding the latter we will focus on everything that has been described to be associated with plastid division, both of the outer and inner division ring (e.g. FtsZ, DRP5B, MinD and E, PARC6, etc.; [see also Osteryoung and Pyke 2014], and the regular cell-cycle. The expression pattern of the cell-cycle genes is important for comparison with the expression data we will collect on certain mutant lines (e.g. DRP5, see task 3) and sampled sporocytes treated with ampicillin to manipulate and study the role of the remnant murein layer.
In addition, we plan on generating an independent but complementary set of RNASeq data on freshly released sporocytes [Shimamura 2016]. Spore release and germination will be induced by changes in the day/night cycle, moisture and light quality [Nakazato et al. 1999; Shimamura 2016]. In all cases we will perform our analysis on a combination of biological and technical triplicates. We have long-standing experience in the analysis of RNA-Seq data from a variety of different species and with varying biological conditions tested [Wägele et al. 2011; Woehle et al. 2011, Gould et al. 2013, de Vries et al. 2015; Rauch et al. 2017; II]. We will use an existing pipeline implemented by us, which is based on the analysis of Trinity-assembled datasets [Haas et al. 2013] and using edgeR as the backbone analysis tool [McCarthy et al. 2012]. Data generated on the released spores will be compared to that of spores from the fern Ceratopteris richardii [Bushart et al. 2013] in order to compare their patterns and identify both similarities, as well as differences in the genes relevant for especially plastid division, its regulation and the hosts cell cycle.
Among the genes of special interest are in particular those of the FtsZ family, MinD and MinE, the Dynamin-related protein 5 (DRP5), and – see also WP2 – those of the murein layer biosynthesis pathway. The minicell genes minD and MinE are of cyanobacterial origin and mediate the positioning of the FtsZ-based ring on the inner plastid membrane [Fujiwara et al., 2008; Osteryoung and Pyke, 2014]. Marchantia polymorpha encodes homologs of MinD (Mapoly0042s0122) and MinE (Mapoly0021s0017) in the nuclear, but not plastid genome. Intriguingly, it appears that all species that are polyplastidic, no longer encode these genes in the plastid itself [V]). How their expression and use is regulated during the switch between mono- and polyplastidy in Marchantia, is one question this proposal aims to tackle.

Charophytes are submerged macrophytes belonging to the Streptophyta, a clade which also include all land plants. Together with the Zygnematophyceae and the Coleochaetophycae, Charophyceae form the ZCC clade, from which the common ancestor of land plants evolved. Charophytes are the most complex streptophyte algae, which exhibit features thought to be mandatory for successful colonization of the terrestrial habitat (e.g. functional rhizoids, gametangia envelopes, cortication). Hence, they represent an important stepping stone worth being investigated in detail in order to understand the mechanisms of terrestrialization. The drastic decline in availability of inorganic carbon (Ci) was probably one of the most striking challenge during terrestrialization. Passive diffusion of CO2 is unable to meet the photosynthetic demand of complex aquatic eukaryotic photoautotrophs at the recent low atmospheric pCO2 conditions. Therefore, eukaryotic algae usually employ sophisticated carbon-concentrating mechanisms (CCMs) to enrich CO2 in the vicinity of Rubisco. However, the existence and function of a CCM in Charophytes is uncertain. Moreover, s strong interference between pH and ion acclimation and Ci acquisition is anticipated.
In preliminary experiments we established cultivation systems for Charophytes under defined conditions. Selected species are able to grow under a wide range of pCO2, salinity and pH levels. For the project we will concentrate on Chara braunii, because its available well-annotated genome sequence provides a solid base for the planned molecular work. Searches in the C. braunii genome resulted in the detection of many genes that potentially encode proteins involved in the CCM of eukaryotic model algae such as Chlamydomonas reinhardtii.
The initial aim of this proposal is to unravel whether or not C. braunii performs a CCM. For this purpose, we will compare the CO2 affinities of intact C. braunii and its carboxylating enzyme Rubisco. The alga will be cultivated under different CO2 and pH levels to investigate if it can respond to different Ci levels by changing the photosynthetic CO2 affinity. The expression of selected genes putatively involved in Chara CCM will be analyzed by qPCR. Finally, the interaction of Ci and ion acclimation will be investigated in C. braunii after cultivation at different salinities. Collectively, we aim to find out if Charophytes perform a CCM, which genes may be involved in this process, and, how far it is interacting to different ionic relations in the surrounding waters.

The algae-to-plant transition and associated conquest of the continental realm was a complex process, enabled by evolutionary innovation of biochemical pathways. Amongst these are changes in the biosynthesis of terpenoids and other secondary metabolites that differ between algae and plants, and which may have been recorded in the rock record. Tracing the incipient terrestrialization process—i.e. its timing, mode, and extent—is of paramount importance for understanding larger Earth system changes across the terminal Neoproterozoic and early Paleozoic. Enhanced rates of carbon burial, changes in the marine redox structure, nutrient-modulated primary productivity and the explosive radiation of animals may all have been indirectly affected as a consequence of the conquest of land by the earliest plants. Tracing the oldest plants using the fossil record is complicated due to generally poor preservation in terrestrial environments, whilst early spores are exceedingly rare. Fossil lipids can offer a solution. Yet the molecular remnants of early-branching plants in earliest Paleozoic sedimentary deposits have thus far received insufficient attention, which is likely partially due to the fact that our knowledge of molecular signatures of these early phylogenetic branches spanning the algae-to-plant transition (i.e. charophytes, liverworts and bryophytes) is still sparse. Here I propose a dual approach, in which modern biomass of said species (grown under regular conditions as well as under induced drought-stress) will be ‘artificially aged’ using pyrolysis and catalytic hydrogenation in order to obtain a better understanding of characteristic early plant biomarkers, whilst sedimentary rocks from three terrigenous-influenced sequences from Australia, spanning the Cambrian and Ordovician will be systematically studied in order to find any traces of the algae-to-plant transition. The outcomes of this study not only carry the potential to reveal evolutionary changes in terpenoid biosynthesis across the algae-to-plant transition, but may also shed more light on the incipient colonization of the terrestrial realm, thereby placing constraints on triggers, facilitators and global consequences.

Cell-to-cell signaling for the conquest of land
The first land plant arose from within the predominantly freshwater, green algal division Charophyta, approximately 500 million years ago [1]. Compared to freshwater, the dry terrestrial environment presented new challenges, including abiotic stresses such as drought and heat. In order to thrive on land, plants would have benefited from signaling pathways to facilitate the transmission of signals between cells in the plant body and to enable coordinated responses to environmental changes. A prerequisite for the evolution of cellular excitability and electrical communication are voltage-gated cation channels that modulate ion fluxes into and out of the cell in response to changes in membrane potential. However, despite the importance of calcium and electrical signals for cell-to-cell communication in multicellular plants, there is currently little knowledge of how membrane-delimited signalling networks evolved. In this proposal, we aim to address this through in-depth examination of key membrane-bound proteins in calcium and electrical signalling in early-diverging plant models.

Plants possess multiple mechanisms, including small RNAs (sRNAs), to control gene expression for proper cell differentiation and organ formation and to cope with abiotic stresses such as alterations in irradiation, drought or nutrient limitations. Some sRNAs, especially in the class of regulatory microRNAs (miRNAs), are widely conserved throughout the plant kingdom suggesting that their evolution might have been critical for the colonization of land. Up to now, there is no information about the miRNA population in the evolutionarily oldest members of the Phragmoplastophyta, i.e. the Charophyceae and they were not addressed during the genome and transcriptome analyses of Chara braunii. Therefore, our project follows the hypothesis that miRNAs have been important in early land plant evolution, for developing the capability for the colonization of land as well as to deal with biotic and abiotic stress factors. We investigate the miRNA population in Chara in different cell populations under various conditions, aim at characterizing the components for RNA interference and identify the regulatory targets of selected miRNAs. Our project work will be complemented by studies on selected transcription factors, which are likely of interest with regard to miRNAs and the specific evolutionary adaptations to live on land. Our primary focus is on Charophytes, while the systematic comparison to data from mosses and ferns is pursued on the basis of publicly available datasets and through collaborations within the SPP. In addition, we contribute with our bioinformatics expertise in analyzing RNA:RNA interactions and expression datasets.

The water-to-land transition of green organisms was associated with different challenges, including vastly altered light conditions in terrestrial compared to aquatic habitats. This change of the light environment might have been a driver of evolutionary diversity, leading to specific light signalling networks in different species. Phytochromes are red/far-red light receptors in plants. Together with a group of transcription factors, the PHYTOCHROME INTERACTING FACTORs (PIFs), phytochromes regulate growth and development of plants. Independent gene duplication events resulted in small phytochrome and PIF gene families in seed plants and bryophytes, and it has been hypothesised that the resulting phytochrome and PIF diversity is an advantage for survival in diverse and rapidly changing light environments. Preliminary work shows that the mechanism of phytochrome and PIF-mediated light signalling is different in Arabidopsis and Physcomitrium. Here, we want to identify the molecular mechanism, by which phytochromes regulate PIF-dependent gene expression in Physcomitrium, and define the specific function of different phytochromes and PIFs for responses of Physcomitrium to changes in the environment.

Upon the transition from aquatic to terrestrial life, green organisms were challenged by vastly altered ambient light conditions, including an increase in the intensity of visible and UV light, an increase in the fluctuation of light intensity, a change in the light spectrum and eventually also canopy shade. Moreover, the newly evolved plant complexity and sessile life style necessitated that light perception be interconnected with endogenous developmental programs to allow light-adapted growth and development. We will therefore study light signaling and light responses in the moss Physcomitrium patens as well as in a charophyte alga. We will focus in particular on the functions of the COP1/SPA E3 ubiquitin ligase, the transcription factors HY5 and CONSTANS-LIKE (PpCOL) and their control by cryptochrome photoreceptors. To this end, we will analyze cop1 and spa knock-out mutants of Physcomitrium for growth and development as well as for photoprotection mechanisms protecting against stress from excess light. Because COP1 is also found in animals, while SPA proteins are specific to the green lineage, these experiments will also address the important question, when and why during evolution the activity of COP1 came under the control of SPA proteins and light. A molecular and functional analysis of the putative ubiquitination targets of COP1/SPA (PpHY5, PpCOL) will address the mechanism of molecular and developmental adaptation to terrestrial life and define conservations and distinctions between light signaling in angiosperms and early land plants. At last, our studies will be extended to a charophyte alga to examine whether innovations in light response were already present in the charophyte lineage to facilitate terrestrialization.

Land plants (embryophytes) have coevolved with a large variety of microbes since their colonization of land more than 450 million years ago, and it has been hypothesized that mutualistic symbiotic interactions with fungi facilitated plant terrestrialization. Given that most microbes are not beneficial to plants and because most plants are resistant to the majority of potentially pathogenic microbes, the establishment of a sophisticated immune system probably also played a crucial role in plant terrestrialization. Extensive studies have revealed conserved and unique molecular mechanisms underlying plant-microbe interactions across different plant species; however, most insights gleaned to date have been limited to seed plants. Thus, we still lack knowledge of basic components and molecular mechanisms controlling plant-microbe interactions that would have assisted plant terrestrialization. A molecular dissection of the immune system of early diverging land plant lineages and streptophyte algae is needed to puzzle out the adaptation processes. In seed plants, cell-surface sensors recognizing microbe-derived or plant endogenous molecules play central roles in various plant-microbe interactions, which could be beneficial, neutral, or detrimental. Therefore, it is reasonable to hypothesize that acquisition and diversification of the sensors and their downstream signaling networks played a key role during plant terrestrialization. Homologs of characterized cell-surface sensors can be partly found in genomes of the liverwort Marchantia polymorpha and the charophyte alga Chara braunii. However, we know very little about their functions in plant-microbe interactions in bryophytes and streptophyte algae. In this project, we aim at unraveling the evolution of cell-surface sensor networks involved in plant-microbe interactions. We will approach this aim by primarily describing the network components in the liverwort M. polymorpha, capitalizing the materials and tools that we have developed for plant-microbe interaction studies of M. polymorpha. We will investigate the roles of M. polymorpha cell-surface sensors in plant-microbe interactions by using the established disruptant mutant lines and the isolated pathogenic microbes. As a next step, we will take recent interactomics, phosphoproteomics, and transcriptomics approaches to reveal the immune sensor networks in M. polymorpha. Furthermore, we will investigate the functions of sensor homologues in streptophyte algae using a trans-species complementation approach. These studies should provide insights to origin and evolution of the cell-surface immune sensor networks in plants.

Metabolism of phenolic natural compounds at the threshold to life on land
Phenolic metabolism is crucial for the plants’ molecular adaptation to land with respect to cell wall-reinforcing compounds, reduction of water loss by transpiration and adaptation to abiotic (e.g. UV) and biotic (e.g. pathogens, herbivores) stresses. Genomes of model organisms at the threshold to life on land are the basis of our project within the MAdLand SPP. The annotated model organism genomes give interesting information about genes present in the organism, but may also contain wrong annotations. On the basis of our biochemical expertise and interest in phenolic metabolism we will evaluate the catalytic properties of enzymes encoded by (tentatively) annotated genes that are important in phenolic metabolism. Annotated genes (with emphasis on PAL, C4H, 4CL and HCTs) from the model organism genomes will be expressed in suitable host systems and the proteins tested for their catalytic properties. This will include basic biochemical characterization with special emphasis on the range of accepted substrates and catalytic efficiencies. Increasing adaptation to the life on land might be accompanied by a change and an increase in enzyme specificity and efficiency. Also alternative pathways to the phenolic metabolism known from higher plants are considered. Comparative investigations will be mainly performed with an aquatic plant (Chara braunii) and the land-inhabiting hornwort Anthoceros agrestis as well as the liverwort Marchantia polymorpha and the moss Physcomitrium patens.

Altered light, temperature, and water conditions were most likely among the greatest challenges plants had to cope with during the step from water to land. Due to very different capacities of water and air, maximum ambient temperatures on land usually exceed those in water. Consequently, being able to cope with elevated temperatures was likely an important innovation for the successful colonization of land by plants. For Arabidopsis thaliana (At) several (eco-)physiological and molecular mechanisms are known to contribute to thermomorphogenesis e.g. hypocotyl and petiole elongation induced by PIF4 mediated auxin and brassinosteriod pathways. Phytochromes, and possibly other photoreceptors like cryptochromes and UVR8 , serve also as thermosensors and control PIF4 and possibly other PIFs. Additional temperature-sensitive regulators inhibiting PIF4 include the DET1-COP1-HY5 signaling cascade known from photomorphogenesis and ELF3. Hardly anything has been reported about thermomorphogenic responses in bryophytes. It is unknown whether or not bryophytes change their growth behavior to acclimate to elevated ambient temperatures. We analyzed growth patterns of Marchantia polymorpha (Mp) and Physcomitrium patens (Pp) in different ambient temperatures, and asked how this compares to what is known from thermomorphogenesis in At. We found that a variety of growth-related phenotypes are significantly affected by temperature in both Pp and Mp. We aim to investigate how the ability to acclimate plant growth to elevated temperatures has evolved in early land plants.

The alternation of generations of plants was coined middle of the 19th century by Hofmeister and describes the haplodiplontic life cycle of all land plants, with two multicellular phases: the haploid gametophyte and the diploid sporophyte. Early in the 20th century Bower hypothesized that this peculiar life cycle evolved from the haplontic life cycle of streptophyte algae (sister to land plants) via intercalation of mitosis before meiosis occurs, at the zygotic stage. Flowering plants, and with them the prime plant model, Arabidopsis, possess a drastically reduced gametophytic generation that is difficult to assess experimentally. Moreover, flowering plants have secondarily lost motile (flagellated) spermatozoids and replaced it by pollen. We want to study the role of the male germ line (spermatozoids) for the alternation of generations. For that, we are employing bryophyte model organisms that possess motile spermatozoids, and have easily tractable gametophytic and sporophytic generations. It is based on our preliminary work that has unrooted several candidate genes involved in the male germ line, and addresses two questions of relevance for MAdLand: “How did embryogenesis and the alternation of generations evolve?” and “Which features enabling conquest of land evolved in charophyte freshwater algae?”. The project involves comparative studies of bryophytes, charophytes and seed plants and is expected to further our understanding of the evolution of the plant male germ line and the alternation of generations.

Copper Zinc Superoxide Dismutase (CuZnSOD) proteins appeared at the time of the Great Oxidation Event around 2.4 billion years ago. In plants three compartment specific proteins are present, of which the cytosolic form arose first as it is the only form found in the freshwater algae Chara braunii and the liverwort Marchantia polymorpha. We found that the cytosolic CuZnSOD of Arabidopsis, AtCSD1, is essential for embryogenesis and posses two functions: on the one hand as an antioxidant enzyme and on the other as a nuclear transcriptional regulator interfering with stress related gene loci. The presence of CuZnSOD in freshwater algae might represent an early molecular adaptation to enable the transition to land. We aim at revealing the role of CSD1 during embryogenesis, the regulation of abiotic stress tolerance and phenylpropanoid metabolism. We focus on M. polymorpha and P. patens to unravel the role of the cytosolic CSD1 during plant terrestrialization.

AGC1 kinases are polarly localized kinases with an important role in various polarity-requiring developmental processes in higher plants. Although the phospho-regulation of the activation of PIN auxin transporters is best studied, we have published and unpublished evidence that AGC1 kinases also regulate PIN- and auxin-independent processes, e.g. in pollen development or during root hair growth. We postulate that AGC1 kinases function similarly to aPKC in polarity processes in animals. In the green lineage, AGC1 kinases appear first in liverworts and mosses and by examining their function in Marchantia polymorpha and Physcomitrium patens the proposed research will shed light on the first AGC1 kinase-regulated polarity processes during the adaptation to land. We further hypothesize that AGC1 kinases regulate PIN proteins in these species and we will investigate the AGC1-PIN interaction at the biochemical and cell biological level. An intriguing finding of our analyses is also that liverworts and mosses do not contain AGC3 kinases that can alter PIN polarity in higher plants. We thus hypothesize that PIN polarity cannot be regulated in liverworts and mosses or, more likely, that other players, e.g. AGC1 kinases, may have a role in PIN polarity regulation. We will perform experiments dedicated to understanding PIN polarity regulation in mosses and liverworts to understand the molecular adaptation of auxin transport during evolution.

During the evolution of chloroplasts from a cyanobacterial endosymbiont, ancient prokaryotic protein targeting machineries were adapted and combined with novel targeting mechanisms to facilitate protein transport in chloroplasts. Current data indicate that the chloroplast SRP system, that mediates the co- and post-translational transport of proteins to the thylakoid membrane, underwent striking adaptations during land plant evolution. In the proposed project we aim to analyse the molecular details of post- and cotranslational thylakoid membrane protein transport mechanisms in Physcomitrium patens. These data will reveal insight into the evolutionary driving forces that triggered these drastic molecular adaptations during land plant evolution.

Biophysical carbon concentrating mechanisms (CCMs) operating at the single-cell level have evolved independently in some eukaryotic algae and a single lineage of land plants, the hornworts. An essential component for an efficient biophysical CCM is a pyrenoid, which represents a specialized compartment inside chloroplasts that mainly comprise the CO2-fixing enzyme RuBisCO. Hornworts with pyrenoids fix significantly more carbon than their relatives without pyrenoids. Given the repeated gains and losses of pyrenoids in hornworts during the last 50 million years, we may assume that their assembly is potentially controlled by a few master regulators of eco-evolutionary relevance. In a joint effort, we will combine comparative -omics with reverse genetics tools to study the genetics, function, and molecular basis of pyrenoid-based CCM in hornwort plastids under different environmental conditions. Guided by ultrastructure-based monitoring of the pyrenoid assembly in hornworts, we aim to identify the genetic toolkit of biophysical CCM in hornworts through two interdependent approaches: First, we aim to predict candidate CCM components in silico though a set of homology searches that compare the hornwort gene set with algal CCM component. Second, we employ an exploratory gene and protein (co)expression profiling of isolated plastids collected under low vs. high CO2 concentrations and under flooding. A strength of our experimental design is that we contrast up to three pairs of pyrenoid bearing and pyrenoid lacking hornwort species. This exploratory analysis is necessary because there is no guarantee that CCM induction and biophysical function of pyrenoids relies only on a set of homologous genes in hornworts and algae. Finally, we will investigate pyrenoid functionality under various environmental conditions. Specifically, we aim to conduct localization and functional validation analyses for a core set of genes discovered in our CCM gene prediction approaches. These experiments are possible through our recent advances to establish Anthoceros agrestis and other hornwort species as a tractable model system. Together, our collaborative project will not only allow a comparison of the mechanisms of pyrenoid assembly between algae and hornworts, but also reveal general principles and species-specific innovations in the evolution of carbon-concentrating plastids. Above that, focusing on and understanding the basis of land plant CCM instead of only the algal form could eventually contribute to efficiently engineer pyrenoid assembly and boost photosynthetic efficiency of crops.

Some MIKC-type MADS-domain proteins constitute tetrameric transcription factors termed floral quartet-like complexes (FQCs). Recent evidence suggests that MIKC-type proteins that are able to constitute FQCs originated during the transition of plants to land. We hypothesize that the evolutionary origin of FQCs facilitated the evolution of morphological complexity and thus plant terrestrialization. The goal of our project is to clarify the structural prerequisites and functional consequences of FQC origin during the transition of plants to land. Preliminary evidence suggests that the duplication of an exon in the stem group of MIKCC-type genes (a sublineage of MIKC-type genes) provided the ability to constitute FQCs. We will test this hypothesis by studying dimerization and FQC formation of representatives of MIKC-type proteins of all major groups of charophytes, and of all MIKC-type genes (MIKC*-type and MIKCC-type genes) of two bryophyte model species, Marchantia polymorpha and Physcomitrium patens. We will also test mutant versions of the proteins that should have gained or lost the ability to constitute FQCs. Moreover, we will reconstruct ancestral sequences along the path of early MIKC-type gene evolution and test the respective proteins concerning FQC formation. To determine the functional consequences of FQC origin during early land plant evolution we will use M. polymorpha as model system, since it has only a single MIKCC-type gene. We will generate mutant versions of the respective protein that are either able or not to constitute FQCs. Analyzing the phenotypes of a knockout mutant and transgenic plants that express FQC-competent or incompetent versions will allow us to differentiate as to which functions of the M. polymorpha MIKCC-type protein depend on FQC formation, and which do not.

We have recently shown that homeodomain transcription factors of the Late Meristem Identity 1 (LMI1) / Reduced Complexity (RCO) type participate in a novel growth regulating pathway that underlies evolutionary diversification of leaf form in seed plants. Together with Prof. Sabine Zachgo, we have also obtained preliminary evidence that an LMI1 type gene regulates growth in the liverwort Marchantia polymorpha. These findings lead to the hypothesis that at least some aspects of this pathway may operate in liverworts and therefore may represent deeply conserved functions that were important for early land plant evolution. To test this hypothesis, we propose to characterize the function of LMI1 type genes in the model Marchantia polymorpha using genetics, advanced imaging and computational modelling. We also propose to investigate functions of this transcription factor in the model moss Physcomitrium patens which will help pinpoint if aspects of LMI1 type gene function in growth regulation were conserved in early land plants. Finally, through targeted transgenic experiments with related sequences from green algae, we will aim to investigate whether structural features of the protein necessary for growth regulation were already present before plants colonized land and therefore may have enabled this colonization.

The final size and body architecture of plants is controlled by the tight regulation of cell proliferation and differentiation processes. As sessile organisms, land plants can sense and respond to altered environmental stimuli via adaptation of their growth. Recently, we demonstrated that the bHLH transcription factor MpTCP1 controls meristematic cell proliferation during vegetative development in the basal land plant Marchantia polymorpha. MpTCP1 proteins bind redox-dependently to regulatory DNA motifs and thereby likely sense redox-changes. Moreover, MpTCP1 controls a downstream network involved in redox-processes and affects the formation of aminochromes, novel and putatively protective plant pigments. We will analyze the regulatory function of MpTCP1 and of TCP homologs from evolutionary informative MAdLand species. Thereby, our project aims to investigate the impact of redox-regulation on cell division and differentiation processes and their contribution to increase plant boy and secondary metabolite complexity during land plant evolution.

Land plants evolved from streptophyte algae and became the dominant players of the terrestrial biome. How this occurred is one of the classical questions in the field of plant evolution—and one of the questions that is at the heart of the efforts outlined in the framework of MAdLand. Hardwired into the molecular biology of land plants is a mix of signatures of their earliest steps on land as well as derived characters. Inferring the biology that allowed for the earliest steps of plants on land, hence, requires a comparative approach of land plants and their algal relatives (Fig. 1). One major hurdle in the conquest of land by plants was overcoming the specific stressors of the terrestrial habitat—including drought, high irradiance and rapid temperature shifts. One of the looming questions in the field hence is what the molecular toolkit for stress response has looked like in the earliest land plants (recently reviewed in de Vries & Archibald, 2018). This has so far been undertaken using a comparative genomics-based approach. A largely unexplored aspect are the deep evolutionary roots of the characteristic biochemistry of land plants. This specific biochemistry was—and still is—a major factor in the ability of land plants to adequately respond to the stressors that the terrestrial habitat poses and, hence, one of the likely facilitators of plant terrestrialization. One major source for a range of secondary metabolites with diverse functions in stress response are the carotenoids deriving from the terpenoid pathway: through oxidative cleavage a series of carotenoid-derived metabolites, termed apocarotenoids, emerge from the carotenoid pathway. These apocarotenoids include classical denominators of the stress response of land plants such as the famous phytohormone abscisic acid but also emerging key players in plant physiology. Here, I will build upon previous insights garnered through my work (de Vries et al. 2018) and focus on this key source of plant metabolites. I will test the hypothesis that streptophyte algae and land plants share the production of a core set of apocarotenoid metabolites that act as signals in stress response.

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