MAdLand Projects

Phosphorus is a biocritical element for all forms of life, but of minute abundance in the Earth’s crust. Globally, apatite minerals of volcanic rock are the only significant source of inorganic phosphate (Pi), the assimilated form of the macronutrient. Chemical and microbial rock weathering sequesters Pi into insoluble co-precipitates with metal cations, and by strong adsorption onto abundant metal oxides and oxyhydroxides into recalcitrant mineral (foremost FeIII und Al) phases. Thus, in rocky nascent soils, prospecting the essential but immobile nutrient posed a major challenge to the first emerging land plants (embryophytes) with only simple, rhizoid-based systems for water and nutrient absorption. Rhizomatous axes of the earliest embryophytes gave rise to the root systems of tracheophytes, including the capacity of growing root tips to monitor Fe-dependent Pi availability in the growth substrate (local Pi sensing). We recently showed that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1) plays a dominant role in local Pi sensing and typifies a novel, bacterial-type cohort of Fe-oxidizing multicopper oxidases (MCOs), which are ubiquitous in the land plants and encoded by small orthogroups (1-5 genes). Intriguingly, the progenitor of the streptophytes (charophyte freshwater algae plus land plants) acquired LPR1-type MCO ferroxidase from soil-dwelling bacteria via horizontal gene transfer (HGT). At least two additional genes, coding for an ABC transporter complex involved in Fe-dependent root Pi sensing, were transferred during the same HGT episode. Arabidopsis LPR1 also affects root hair development, which is controlled by evolutionary conserved processes shared with rhizoid formation. Thus, LPR1-type MCOs offer an excellent framework for dissecting the impact of HGT on the evolution of land plants and their adaptations to dramatically altered geochemical conditions. The first goal of our project will test the predicted biochemical activity of LPR1-like MCOs as specific ferroxidases. We will express, purify, and determine kinetic parameters of recombinant LPR1-type MCOs from select seedless tracheophytes, bryophytes, streptophyte algae, and soil-dwelling bacteria, which cover the evolutionary range relevant to plant terrestrialization. To probe a possibly conserved function in local Pi sensing, we will conduct complementation analyses with select LPR1-type MCO genes in Arabidopsis lpr1 knockout lines. A second goal will test the proposition that the acquisition of LPR1-type MCO ferroxidase activity facilitated the evolution of Fe-dependent Pi sensing during plant terrestrialization. We will use the established Marchantia bryophyte model system to characterize Pi deficiency responses and to analyze by reverse genetics the function of presumed key players in Marchantia such as LPR1-type MCOs for external Pi sensing.

https://gepris.dfg.de/gepris/projekt/528056313

Phosphorus (P) like nitrogen (N), is a critical nutrient required for all life. It generally occurs in small quantities in the natural environment. Therefore, P is usually considered the "limiting" nutrient in aquatic and terrestrial ecosystems, meaning that the availability of this nutrient controls the growth and productivity of aquatic and terrestrial plants, including algae. In most land plants, symbiosis with mycorrhizal fungi, and nitrogen-fixing rhizobia mainly in legumes, greatly facilitates soil P and N uptake. The development of arbuscular mycorrhiza (AM) as well as other plant symbioses depends on the common symbiosis signaling pathway (CSSP) in host plants which recognizes the microbial partner and stimulates the expression of genes required for the symbiosis. Furthermore, AM and nodulation symbioses are controlled by the P and N status of the host via interlinked response pathways. Key components of the CSSP and the phosphate starvation response (PSR) pathway are conserved between algae and land plants. However, the function of algal orthologues of PSR and CSSP components is currently unknown. Recent work has shown that the AM symbiosis is modulated downstream of the CSSP by a network of receptor kinases including surface localized, transmembrane receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs). Our project aims to elucidate the interactions between RLK/RLCK proteins and upstream and downstream regulatory steps that are important for understanding the evolution and the molecular mechanisms of RLCKs in AM symbiosis signaling in the context of the CSSP in land plants. We plan to compare the liverwort Marchantia paleacea, which forms AM symbiosis, with the evolutionarily distant aquatic and terrestrial alga Zygnema, which belongs to the extant sister group of land plants. We expect to gain significant insight into the evolution of AM symbiosis regulation and the integration of phosphate sensing and signaling. Our project will complement other proposals within MAdLand on integrating sensing of environmental cues, downstream cellular signaling and transcriptional responses underlying complex adaptive traits for life on land.

https://gepris.dfg.de/gepris/projekt/528027896

This project aims to elucidate the functional evolution of plant calcium- and electrical signaling for stress adaptation crucial for plant success on dry land, to address three outstanding questions within the Priority Programme “MAdLand”: i) which features enabling conquest of land evolved in charophyte freshwater algae, ii) what is the succession and nature of molecular adaptations in early land plant evolution, and iii) what are the molecular evolutionary drivers of tolerance to abiotic and biotic stresses?Using three model species, Chara braunii, Marchantia polymorpha and Ceratopteris richardii, we will focus on three ion channel classes considered to provide for local signal generation and systemic signal propagation: i) Glutamate receptor like channels (GLRs), as plasma membrane entry pathways for calcium ions and mediators of calcium and electrical signal propagation, ii) calcium-activated Tandem-Pore Channel 1 (TPC1) proteins as mediators of vacuolar electrical excitability, and iii) Big-K+-channels (BKs), as the putative molecular link that connects calcium- and electrical signaling, expressed in algae and early land plants but not in angiosperms.Using genome editing and molecular genetics, together with cell biology and electrophysiology, we will functionally characterize GLR, TPC and BK channels and dissect their structure-function relationships in heterologous expression systems, in planta and in cross-complementation studies. Using this approach, we will address the following specific questions: i) which ion channels are required for calcium and electrical signaling, ii) how and approximately when did they evolve during plant evolution, iii) what are their physiological roles in different early-diverging plant groups, and what functional changes occurred after the divergence of the alga Chara, the liverwort Marchantia and the fern Ceratopteris, respectively from shared common ancestors with well-characterized angiosperm models, and iv) which structural changes in GLRs, TPC1s, and BKs provided for systemic calcium- and electrical signaling?

https://gepris.dfg.de/gepris/projekt/440539846

The colonization of terrestrial habitats by ancestral streptophyte algae approximately 450 Mya ago, followed by the rise of land plants relied on the evolution of novel microtubule cytoskeletal arrays and was accompanied by the diversification and emergence of plant-specific microtubule-associated proteins (MAPs) to adjust cellular processes to novel conditions and interactions. One of the key innovations in plant cytoskeleton organization is the emergence of the preprophase band (PPB)/phragmoplast system. This system facilitates a switch in the mode of cell division from a cleavage-based mechanisms to an inside-out mechanism, in which new cell walls are inserted at the cell center. Functional studies in Arabidopsis thaliana identified networks of eukaryotic and plant-specific MAPs that control the formation of the PPB and phragmoplast. The origin, architecture and dynamics of these complexes, however, are still unknown. Plant-specific IQ67 DOMAIN (IQD) proteins emerged as key regulators of PPB formation and division plane control in Arabidopsis thaliana with proposed functions as hubs in microtubule-associated PPI networks. IQDs share scaffold-like properties, physically interact with known regulators of plant cell division, and are implicated in auxin-dependent control of division plane positioning. Our preliminary data indicate emergence of IQDs early during land plant evolution. We hypothesize that plant-specific IQD scaffolds facilitated the rewiring and acquisition of PPIs at the cytoskeleton to support neo-functionalization and functional specialization during plant cell division and growth regulation. In this project, we aim to identify key principles that govern the adaptations of the microtubule cytoskeleton by investigating the evolution of IQD-assembled PPI networks and their function in cell division. We will approach these aims by functional analyses of IQD functions in the liverwort Marchantia polymorpha and by comparative analyses of microtubule-binding and PPI specificities of IQDs from streptophyte algae, bryophytes, and seed plants.

https://gepris.dfg.de/gepris/projekt/528023844

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.

https://gepris.dfg.de/gepris/projekt/440540015

The evolutionary pressures that occured during terrestrialisation most propably brought about cell wall modifications to cope with the new conditions. In a first step, sequential extraction of cell walls of at least all MAdLand model organisms followed by monosaccharide analyses of these fractions will offer an overview on whole cell wall composition and the elucidation of differences over a broader phylogenetic range. Our further work will focus on streptophyte algae and bryophytes, as their ancestors were mainly involved in transition from water to land. We will isolate and characterize arabinogalactan-proteins (AGPs) and pectic polysaccharides, which are the two most complex macromolecule groups present in the plant cell wall. In the first phase, we detected AGPs in bryophytes and ferns, which contained unusual features, especially terminally linked 3-O-methylrhamnose, a monosaccharide not present in angiosperms. Within the streptophyte algae, no AGPs were detected in Chara sp. (Charophyceae), whereas cell walls of Spirogyra pratensis (Zygnematophyceae), contained AGP-like molecules with nearly complete replacement of arabinose by rhamnose. To find more evidence, we will search for AGPs in the new streptophyte algae models Zygnema circumcarinatum and Mesotaenium endlicherianum and additionally Mougeotiopsis calospora and Coleochaete scutata as well as in the liverwort Riccia fluitans and suspension cultures of the hornwort Anthoceros agrestis. Presence and nature of pectic polysaccharides in streptophyte algae and bryophytes is not finally settled. It is generally accepted that homogalacturonan has deep roots in the green lineage, but knowledge on rhamnogalacturonan-I (RG-I) and -II (RG-II) in bryophytes and streptophyte algae is mainly based on reactivity with antibodies directed against glycan motifs present in pectins. Therefore we will isolate and structurally characterize RG-I and RG-II from Chara sp., Spirogyra pratensis, Riccia fluitans and Anthoceros agrestis. Antibodies directed against AGP- and pectic glycan epitopes will be used in ELISA to complement analytical structure elucidation of the molecules. Furthermore, these antibodies will be used for immunolocalization in plant tissue which offers first hints on possible functions of these macromolecules in these organisms. Additionally, bioinformatic searches for genes of AGP protein backbones and glycosyltransferases involved in AGP and pectin biosynthesis will be performed. Thus, our work on biochemical and bioinformatic characterization of cell walls will identify the cell wall key features necessary for the conquest of land and broaden the knowledge on molecular adaptations in early land plant evolution. This will contribute to elucidate the nature of the most common ancestor of all extant land plants and of streptophyte algae and land plants.

https://gepris.dfg.de/gepris/projekt/440046237

Terrestrialization of plants was a key evolutionary process to for the transformation of the earths surface as we know it today. To thrive on land, plants needed to adapt to terrestrial conditions, which majorly differ from submerged habitats in salt or fresh water. Land plants evolved in freshwater ponds from streptophyte algae and different from algae in submerged habitats, land plants needed to develop specialised tissues and cells to achieve this conquest of land. Here, we propose a project that will contribute to the MAdLand mission to understand which molecular features enabled streptophyte algae for terrestrialization, and which characteristics allowed the last common ancestor of land plants and streptophyte algae to start this evolutionary. We investigate ROPGEFs, activators of RhoGTPases signalling, that regulate polar growth and cell differentiation. We find that ROPGEFs are Streptophyta specific and diversified in land plants. This makes ROPGEFs a great model to study how molecular features needed to evolve during the conquest of land. We will use cross-species complementation of Marchantia polymorpha with ROPGEFs of Streptophyte algae to find the evolutionary origin of this protein family. Further, we will use different cell biological techniques, like immunolocalisation, to describe these ROPGEFs in streptophyte algae. Our project will contribute to our knowledge about the nature of the last common ancestor of land plants and streptophyte algae was, and which molecular pathways adapted during the conquest of land.

https://gepris.dfg.de/gepris/projekt/528090862

Plasmodesmata (PD) control intercellular communication in plants and estabilish a direct symplasmic continuity between adjacent cells across the cell walls. For seed plants, it is well known that the PD networks within tissues are highly dynamic and undergo sophisticated structural and functional modifications in a precisely regulated manner. These changes play essential roles in the regulation of plant development, as well as in metabolic adaptations to changing environmental conditions, and in the coordinaton of cell- or non-cell autonomous pathogen defense. PD do regularly occur in non-seed plants, and they have also been found in some groups of the streptophyte algae, which are closely related to all land plants. Thus, it has been assumed that intercellular communication via PD has already had an impact on plant evolution during the conquest of land. PD connectivity might have been a prerequisite for the increasing complexity of the plant bodies allowing division of labour between spatially patterned cells, tissues, and organs. Further, PD might have been essential for the adaptation to the hazardous conditions on land, when plant tissues have had to develop coordinated mechanisms to face a multitude of abiotic and biotic stresses. This raises the central question whether PD and symplasmic network have kept the same properties and the same regulatory circuits from the very beginning on, or whether there was an "evolution of PD" in the course of land plant evolution over hundreds of million of years, allowing an increasingly complex regulation of intercellular communication?This question remains open, since there is only scarce and partly contradictory information available on the plasmodesmal networks in streptophyte algae and non-seed plants that could be compared to a bulk of findings on seed plants. This is why we want to study the evolution of plasmodesmata in the present project. Before asking molecular questions, we will gather more information on the structural adaptations that PD and PD networks have experienced in the course of evolution. Thus, we will use transmission electron microscopy and confocal laser scanning microscopy to answer, e.g., the following questions: -- are typical PD substructures, derived from the endoplasmic reticulum, already present in PD of streptophyte algae?-- are there differences in the complexity of symplasmic networks in hornworts and thalloid liverworts that reflect the distinct complexibility of their vegetative bodies?-- do the same mechanisms of PD modifications take place during "leaf" development of non- seed plants that have been found for seed plants? Although not all "leaf" organs are homologous?-- do all non-seed plants form secondary PD in already existing walls? And which mechanism(s) do they use?

https://gepris.dfg.de/gepris/projekt/440525456

Sphingolipids are essential lipids that are ubiquitous among eukaryotes. Plant sphingolipids are involved in many processes, including maintenance of plasma membrane integrity and microdomain formation, cell growth and division, polar secretion, and programmed cell death signaling. They have primarily been investigated in Arabidopsis thaliana, for which an extensive genetic toolkit has been available for decades. The precise functions of sphingolipids have been challenging to study in Arabidopsis, however, due to non-viable or pleiotropic mutant phenotypes, complex organ structure, and difficulties with sphingolipid extraction and detection. Genome sequences and tools for genome editing are now available for a wide variety of species, offering a better understanding of metabolic and functional diversity, and enabling study of evolutionary history and ancestral functions. The bryophytes Marchantia polymorpha and Physcomitrella patens are early-diverged land plants and relatively new model organisms suitable for this purpose. Preliminary work by my group revealed a unique sphingolipid profile for Physcomitrella, 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 Zygnematophyceaen algae Spirogyra pratensis and Mougeotia scalaris, for a broader perspective of the establishment of characteristic plant sphingolipid building blocks. Next, we will use Physcomitrella and Marchantia to study the ancestral functions of sphingolipids in the land plant lineage. 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. Physcomitrella and Marchantia will be particularly useful for the study of sphingolipid functions given their relatively simple morphology, which will facilitate observation of traits known to be affected by sphingolipid metabolism such as cell growth, division, and differentiation. Further, we will use these systems to test how sphingolipids contribute to tolerance of abiotic and biotic stresses associated with terrestrial life.

https://gepris.dfg.de/gepris/projekt/440232164

In nature, plants live in association with a large variety of microbes, which are beneficial, neutral, or detrimental. Given that the mode of plant-microbe interactions is presumed to be very different in terrestrial and aquatic environments, molecular innovations or adaptations of immunity-related pathways probably have 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 angiosperms. Thus, a molecular dissection of the immune system of bryophytes and streptophyte algae is needed to puzzle out the adaptation processes to the terrestrial environment. Salicylic acid (SA) is a major defence-related phytohormone, which primarily induces resistance against biotrophic and hemi-biotrophic pathogens in angiosperms. SA is also present in a range of bryophytes and streptophyte algae. However, SA function in defence responses and SA pathway components in bryophytes and streptophyte algae remain uncharacterized. Notably, NPR gene is absent in the sequenced genomes of streptophyte algae, and thus plants probably have acquired the canonical SA receptor NPR during terrestrialization. In this project, we aim to unravel the evolution of SA function and signalling. We will approach this aim by firstly describing SA responses at transcriptome and proteome levels in plants either having or lacking the canonical embryophyte SA receptor NPR, which include three bryophytes and six streptophyte algae. Secondary, the evolution of the SA perception mechanism will be investigated at the molecular level by characterising NPR in bryophytes and by exploring novel SA receptors.

Trehalose 6-phosphate (Tre6P) is a low abundance metabolite that plays an important role in flowering plants in regulating sugar metabolism, in developmental decision making, and in maintaining optimal sucrose levels. Tre6P is made by Tre6P synthase (TPS) and dephosphorylated by Tre6P phosphatase (TPP) into trehalose. Both, TPS and TPP proteins are present in all chloroplastida and have diversified in land plants, suggesting a key role of Tre6P during land plant evolution. Importantly, catalytically inactive TPS proteins are already found in streptophyte algae and bryophytes suggesting a function specifically for Tre6P signalling in these organisms. This is further supported by Tre6P-related genes responding transcriptionally in decapitated Physcomitrium patens shoots and Tre6P synthesis mutants in P. patens being unable to produce sporophytes. In Marchantia polymorpha TPS and TPP genes are highly expressed in reproductive structures suggesting a conserved role of Tre6P in reproduction in bryophytes. A TPP has been identified in interacting with a central regulator of the arbuscular mycorrhiza symbiosis program. However, a direct connection of Tre6P in mycorrhiza symbiosis is yet to be established. The presence of Tre6P synthesis and degradation genes in green algae and bryophytes, especially the occurrence of catalytically inactive forms, suggests that there is a function for Tre6P signalling which might have contributed to the evolution of land plants. The goal of this project is to uncover the role of Tre6P in sugar metabolism and developmental processes in both streptophyte algae and bryophytes by these three distinct objectives: 1) Investigating the role of Tre6P in regulating metabolism in streptophyte algae, 2) Identifying the function of Tre6P in regulating metabolism and development in bryophytes, and 3) Investigating the role of Tre6P in arbuscular mycorrhiza symbiosis in liverworts. The methods used will include growing different algae and bryophytes under varying light and carbon regimes to examine if Tre6P levels are responsive to changes in sugar availability and if changes in Tre6P levels result in a reprogramming of metabolism similar to what is observed in angiosperms. By genetic manipulation, Tre6P levels will be altered in bryophytes which will be combined with detailed metabolic and phenotypic analyses. This will help us to elucidate whether Tre6P is linked to nutrition in order to provide trehalose to plants as a storage and transport sugar, and/or regulates metabolic and developmental responses. Overall, the aim of this project is to determine if Tre6P is a signalling molecule that plays a crucial role in regulating metabolism and development in streptophytes and if it contributed to the adaptation of plants to life on land.

https://gepris.dfg.de/gepris/projekt/527912383

Evolutionary impact of small RNA-dependent gene expression in bryophytes during the molecular adaptation for life on land We will perform comparative analyses of small RNA-dependent regulatory pathways in two bryophyte species, the moss Physcomitrium patens and the liverwort Marchantia polymorpha. The project involves functional studies on specific DICER-LIKE dependent small RNA (sRNA) pathways and the characterisation of their targets. Analyses of a specific P. patens PpDCL1a mutant that lacks an autoregulatory feedback loop revealed an essential contribution of this control of miRNA biogenesis in the adaptation to salt. Transcriptome analyses in the mutant allowed the identification of genes that contribute to adaptive processes required for the water-to-land transition. Alterations in the expression levels of two of these genes encoding a flotillin-type membrane protein (PpFLOT) acting in vesicle transport and signalling and a MYB transcription factor (PpMYB), respectively, led to altered growth and impaired sensitivity to salt as well as to phytohormones. The underlying molecular mechanisms causing these phenotypic abnormalities will be explored. Further, we generated mutant lines for all four known M. polymorpha DCL genes and detected severe abnormalities of these mutant lines with respect to development, phytohormone response and salt sensitivity. The Mpdcl mutants present a valuable tool for the dissection of all sRNA pathways in M. polymorpha and their comparison with already analysed sRNA pathways in P. patens. We will use the Mpdcl lines for genome-wide sRNA and mRNA transcriptome profiling to elucidate the specific sRNA repertoire and sRNA-mediated gene control in the liverwort M. polymorpha. Based on our initial analyses of the sRNA-dependent genes in P. patens and the preliminary work on Mpdcl mutant lines we expect to address several key aspects of the MAdLand priority program including the evolution of phytohormone-related signalling and associated control of gene expression, the increase in body plan complexity as well as the evolution of regulatory mechanisms related to the adaptation to new environments.

https://gepris.dfg.de/gepris/projekt/440035902

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. We will pay particular attention to the plastid division apparatus and involved proteins (MinD, MinE, FtsZ1-3, Drp5), and the peptidoglycan layer (and its related mur genes). We plan on examining the mono- to polyplastiy transition (monoplastidic bottleneck) by the interplay of the cell cycle and the plastid division apparatus. The release from the monoplastidic constrain was a critical step in evolving higher plants and the different types of plastids (pro-, chromo-, amyloplast, etc.) that embryophytes can mobilize. Marchantia offers a great model system, both because of its evident transition between poly- and monoplastidy as part of its life cycle (a unique window into organelle evolution and plastid inheritance), as well as the ease with which both the nuclear and plastid genomes can be manipulated.

https://gepris.dfg.de/gepris/projekt/440043394

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. In this proposal, 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. The proposal 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.

https://gepris.dfg.de/gepris/projekt/440411476

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 thesurrounding waters.

https://gepris.dfg.de/gepris/projekt/439896201

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 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.

https://gepris.dfg.de/gepris/projekt/440415732

The cells of most green algae and land plants are surrounded by a primary cell wall, which is a load bearing yet extendable matrix composed of various polysaccharides and protects algae from environmental stress such as wate scarcity. A major fraction of these cell wall polysaccharides are hemicelluloses, which can be enzymatically cut and re-connected to another hemicellulose molecules nearby. Such reactions are catalysed by cell wall-bound transglycanases, which occur abundantly in both the Zygnematophyceae and their sister lineage, the land plants. Interestingly, transglycanases are less abundant in early diverging Charophytes (e.g. Klebsormidiophyceae) and Chlorophyta. Hemicellulose transglycosylation is considered to play a key role in the cell wall metabolism of uprightly growing land plants, however, its functions in algae are elusive, even though gene families encoding the responsible enzymes strongly expanded in land-conquering algae, suggesting that they have played important roles in preparing algae and their cell walls for terrestrialization. Moreover, the major substrate of transglycanses and most abundant hemicellulose in land plants – xyloglucan – evolved in Zygnematophyceae. Our recent data indicate that these algae can secrete xyloglucan and other hemicelluloses in surprisingly large amounts into the environment and – vice versa – recruit external polysaccharides back into their cell walls via transglycosylation. We believe that this unexplored hemicellulose secretion and recruitment of polysaccharides helped algae to produce biocrusts that most likely served as their primary habitats during terrestrialization and can be still found worldwide in harsh environments. In order to explore the role of polysaccharide secretion and recruitment, we will devise a series of cultivation experiments and analyses the external polysaccharide footprint of Zygnema and Mesotaenium grown on different substates by state-of-the-art glycobiology techniques. We will compare the secretion pattern with the basal land plant Marchantia, which is known to release xyloglucan from rhizoids. This will be complemented by evaluation compositional and ultrastructural consequences for the cell wall and by tracking polysaccharide secretion and recruitment on a cellular level using new in vivo click chemistry-based imaging approaches. Tracking will consider cross-species exchange of polysaccharides. Finally, transcriptomic changes of relevant carbohydrate-active enzymes will be monitored in response to different external polysaccharides and desiccation stress. We anticipate that our in-depth investigation of polysaccharide remodelling and recruitment will provide novel insights into the role of the cell wall during terrestrialization.

https://gepris.dfg.de/gepris/projekt/528114108

Plants differentiate specialized cell types, tissues, and organs that are fundamental to their photosynthetic lifestyle and for thriving in their respective ecological niches. All differentiated cells ultimately descend from stem cells, undifferentiated, pluripotent progenitors. During ontogenesis, small groups of stem cells are maintained at certain positions, the so-called stem cell niches. Such niches exist in plants within the root, shoot and vascular meristems. In these stem cell niches, a precisely balanced set of regulatory factors ensures that the undifferentiated, pluripotent state is maintained, while cells at the periphery are able to enter specific differentiation pathways. To control cell differentiation status, plants possess multiple mechanisms that include specialized transcription factors and microRNAs. Our key hypothesis is that the mechanisms underlying the complex land plant morphologies evolved in specific members of the charophytes, and that by characterization of specific regulators’ molecular functions it is possible to track the evolutionary process. Therefore, this project follows the hypothesis that the emergence of these regulatory mechanisms was of central importance in early land plant evolution. We will focus on the Chara braunii model but compare to insight obtained in other algae and land plant models such as Arabidopsis and Physcomitrium. In Chara braunii, nodal central cells are candidates for pluripotent cells, which will be characterized at single-cell level. We will then investigate the role of certain regulators in controlling cellular and chloroplast differentiation and follow the regulatory responses to selected abiotic stresses. While the focus is on charophytes, the systematic comparison with data from mosses and ferns will advance, based on publicly available datasets and in particular through collaborations within the SPP. In addition, we will provide bioinformatics expertise for the analysis of gene expression datasets and RNA:RNA interactions and Chara braunii material and cultures, thereby strengthening the know-how of the consortium as a whole.

https://gepris.dfg.de/gepris/projekt/440274755

Compared to aquatic habitats, the light conditions on land can be very different, suggesting that plant terrestrialisation required adaptations in photosensory receptors and downstream signalling pathways. Phytochromes (PHYs) are an important class of photoreceptors in plants. In seed plants, ferns, and mosses, independent gene duplications events resulted in small PHY gene families. Functional diversification of PHYs in seed plants into red and far-red light sensing PHYs is well known. However, due to the independent evolution of the PHY diversity in seed plants, ferns, and mosses, functional diversification of PHYs in ferns and mosses cannot be predicted based on studies on seed plant PHYs. Investigating the functional diversification of PHYs is an important goal of the proposed project Work in the frame of the first funding period of MAdLand has shown that the three clades of PHYs in the moss Physcomitrium patens functionally diversified into clades with red and far-red light sensing PHYs, respectively, and into a clade that has dual-specificity and acts in red and far-red light. In the proposed project, we want to investigate functional diversification of PHYs in the fern Ceratopteris richardii using PHY knock-out or knock-down mutants. In addition, we also want to compare the characteristics of red and far-red light induced responses mediated by the different PHYs in Physcomitrium and Ceratopteris to identify similarities and differences that could point to common or lineage-specific mechanisms of light signalling. In contrast to ferns and mosses, liverworts contain a single PHY that is active in red and far-red light. By cross-species complementation studies with the liverwort Marchantia polymorpha and the moss Physcomitrium patens, we then want to identify amino acid residues or motifs that are important for functional diversification of PHYs. Finally, we found that two key components of light signalling in land plants possibly have been acquired in the last common ancestor of land plants and therefore could be innovations in light signalling that facilitated plant terrestrialisation. In the last part of the project, we want to explore this idea.

https://gepris.dfg.de/gepris/projekt/440515704

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 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 and in the charophyte alga Mesotaenium endlicherianum, thereby focusing on photoprotection towards high light stress, photomorphogenesis and chloroplast development. We will study two transcription factors that interact with the COP1/SPA E3 ubiquitin ligase in P. patens and M. endlicherianum in order to identify innovations in the regulation of chloroplast development, photomorphogenesis, photoprotection and light-regulated gene expression networks that may have predated plant terrestrialization.

https://gepris.dfg.de/gepris/projekt/440235374

Drought is a prime stressor that plants had to overcome in their conquer of land. Most land plants are able to adapt to drought on the cellular level. Furthermore, they are able to produce desiccation tolerant cells and tissues as part of their reproductive cycle such as seeds, pollen and spores. However, many streptophyte algae are also displaying high stress resilience. While it is likely that drought resistance evolved several times independently, it is also likely that these independent origins of desiccation tolerance are underpinned by similar strategies and the co-option of existing regulatory programs for resilience. Common to both drought resistance and desiccation tolerance is the accumulation of neutral lipids, foremost triacylglycerol (TAG), in cytosolic lipid droplets (LDs) also referred to as oil bodies, lipid bodies or oleosomes. Several protein families are known to localize to LDs and two of them caleosin (CLO) and lipid droplet associated protein (LDAP) are associated with drought stress responses and possibly evolved this function in streptophyte algae. The goal of the research project is to find out if the accumulation of neutral lipids in LDs is a universal drought stress and desiccation response in the clade of Streptophyta by investigating the five species Mesotaenium endlicherianum, Zygnema circumcarinatum, Physcomitrium patens, Marchantia polymorpha and Azolla filiculoides. Furthermore, we want to collect evidence that LD-associated proteins that are upregulated during drought are important to cope with this stress, and evolved this function already in streptophyte algae. Overall, this data would support the hypothesis that the accumulation of LDs and its associated proteins during drought was one of the prerequisites for the conquer of land. The key objectives are to collect evidence for the following hypotheses: 1. The remodeling of the lipidome and the accumulation of neutral lipids in LDs is a universal stress response. We will monitor the abundance of LDs by microcopy and perform lipidome analyses in drought-treated vegetative tissues of the five species. 2. The proteins CLO and LDAP are universally upregulated under desiccation and drought stress, respectively. We will test the transcript of all respective genes by qPCR in all five species and isolate LDs from drought stressed tissues to analyze these by proteomics. 3. CLO and LDAP are important during drought stress and/or in spores in P. patens. We will knock out the three CLO and the single LDAP gene by CRISPR/Cas9 and analyze the phenotypes in respect to LD number and size, drought resistance and the performance of the spores. 4. CLO and LDAP have kept a similar function throughout evolution from streptophyte algae to land plants. We will complement the P. patens mutant lines that have displayed phenotypes with homolog genes from M. endlicherianum.

https://gepris.dfg.de/gepris/projekt/528045595

MLO proteins are integral membrane proteins that operate as calcium channels and that are specific to photosynthetic eukaryotes. In land plants, they are typically present in multiple sequence-diversified isoforms per species and contribute to essential physiological processes such as root thigmomorphogenesis and gravitropism, fertilization, as well as pathogen defense. Exocyst is an evolutionarily conserved multisubunit protein complex found in all eukaryotes. Different from other taxa, in land plants the EXO70 exocyst subunit is present in multiple sequence-diversified isoforms. We recently discovered that Arabidopsis thaliana MLOs and EXO70s form a functional module that involves physical association of these proteins and that likely governs focal secretion, e.g. of cell wall-related cargo. We hypothesize that key features of the MLO-EXO70 functional module evolved in the course of plant terrestrialization. We, therefore, propose analyzing the co-evolution and molecular adaptation of the MLO-EXO70 module in Chara braunii (Charophyceae) and Mesotaenium endlicherianum (Zygnematophyceae), which as an extant representatives of the so-called ZCC grade/clade mark the organismal transition from water to land. The C. braunii and M. endlicherianum genomes code for MLO-like proteins (seven and three, respectively) that in part markedly differ from their canonical land plant counterparts regarding size and membrane topology. Further, these genomes encode two prototypical EXO70 subunits each. Using this set of proteins derived from sister species of land plants, we will assess the recently discovered calcium channel activity of MLOs, their well-established ability to associate with the cytoplasmic calcium sensor calmodulin, and their isoform-preferential interaction with specific EXO70 family members. We further intend to explore the expression patterns of MLO and EXO70 genes in the various parts of the complex C. braunii thallus in different conditions and to examine the natural genetic variation of these genes within the species C. braunii and, more broadly, within the Charophyceae. Together, these studies will reveal a global picture regarding the presumed co-evolution and molecular adaptation of the MLO-EXO70 functional module in the course of transition of life from water to land.

https://gepris.dfg.de/gepris/projekt/527875163

Plants changing from an aquatic to a terrestrial lifestyle encounter various biotic (pathogens, herbivores) and abiotic (UV irradiation, loss of water) stresses which can be overcome with help of phenolic compounds that have deterring or antibiotic as well as UV-absorbing properties or are incorporated into lipophilic barriers. The basis for the biosynthesis of phenolic compounds is the phenylpropanoid pathway which forms an activated hydroxycinnamic acid from L-phenylalanine or L-tyrosine by the action of three enzymes: phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate CoA-ligase (4CL). 4-Coumaroyl-CoA then is further metabolized to monolignols and hydroxycinnamic acid esters/amides under participation of hydroxycinnamoyltransferases (HCT), cytochrome P450 monooxygenases (CYP98) and caffeoylshikimate esterases (CSE). All these enzymes are well known in seed plants, but our knowledge on them in early diverged land plants or algae is much more limited. In the first funding period, our main focus was on the above-mentioned genes and enzymes in extant members of the earliest land plants using the bryophytes Anthoceros agrestis, Marchantia polymorpha and Physcomitrium patens as model organisms. Chara braunii as green alga of the Characeae was also investigated to some extent. Gene sequences from these plants for the enzymes of interest were selected, heterologously expressed and biochemically characterized. Generally, our results showed that annotations in the bryophyte databases are mostly correct and the biochemical characteristics are similar to those of the respective enzymes from seed plants. This was different for Chara braunii. Here, firstly genes can not be identified as easily using seed plant sequences as baits, and secondly, the annotations often are not correct or confusing. This shows us, that it is important to concentrate on the algal model systems to follow the evolution of genes/enzymes of the phenylpropanoid pathway as organisms where this evolution might have occurred. Besides Chara braunii, we will therefore concentrate on another algal model organism, Mesotaenium endlicherianum (Zygnematophyceae), to look there for the presence of genes/enzymes of phenolic metabolism and to characterize the encoded enzymes. First inspections of Chara braunii and Mesotaenium endlicherianum genome databases have shown that the identification of our target genes is more tedious and alternative biosynthetic pathways must be taken into consideration.

https://gepris.dfg.de/gepris/projekt/439529174

Evolutionary emergence of land plants (the backbone of the terrestrial biosphere) traces back to a singular event in which freshwater algae conquered land, a process referred to as terrestrialisation. This transition to land required a plethora of molecular adaptations that allowed the ancestor of plants to survive under the harsh environmental conditions of the new habitat. One of the most important requirements was the physiological adaptation of the chloroplast that is especially susceptible to environmental stressors. Key element of this process was the coordination of gene expression between cell nucleus and organelle in order to guarantee proper build-up and maintenance of the photosynthetic machinery. This research proposal focusses on the evolution of the plastid transcription machinery and its multiple novel protein components proposing it as crucial evolutionary innovation during and for terrestrialization. The function and regulation of the eubacterial plastid RNA polymerase (PEP) inherited from the cyanobacteria-like progenitor was supplemented with additional PEP associated proteins (PAPs) and novel sigma factors and further put under the control of a nuclear encoded phage-type RNA polymerase (NEP). Most of these protein factors evolved in streptophyte algae and a complete set of the corresponding genes appeared for the first time in Chara braunii. The project combines phylogenetic, molecular-genetic and biochemical approaches. It investigates the precise phylogenetic appearance of these components, their evolutionary adaptations within various streptophyte model organisms (including a dual nucleo-plastid localization for bi-partite gene regulation) and the structural evolution of the PEP complex.

https://gepris.dfg.de/gepris/projekt/527848546

The colonization of land by descendants of freshwater algae was a watershed moment in earths’ history. Plants encountered many environmental challenges during the conquest of the land, such as desiccation, high intensity light, UV-radiation, lack of nutrient availability, mechanical damage, and pathogen infections. In consequence, a consecutive adaptation of metabolism and morphology took place during evolution of ancestors from early Streptophyte algae to todays seed plants. Among these versatile adjustments of land plant metabolism to terrestrial environments, changes in biosynthesis, distribution and homeostasis of lipids represent crucial steps to cope with stressful life. Fatty acid transport function of FAX (fatty acid export) proteins in the inner envelope membrane of chloroplasts has been shown to be crucial for cellular lipid homeostasis under normal and stress conditions such as cold acclimation in the model organisms Arabidopsis thaliana (seed plant) and Chlamydomonas reinhardtii (green micro alga). However, nothing is known about FAX proteins in the organisms relevant for first steps in plant terrestrialization. In order to close this gap and evaluate FAX protein evolution towards adjustment of metabolism to life on land, we aim to pinpoint molecular adaptation of plastid FAX proteins in bryophytes, i.e. in Physcomitrium patens and Marchantia polymorpha. With the emergence of the prominent apo-lipoprotein α-helix bundle at the N-terminus of plastid FAX that is conserved throughout evolution of monophyletic Phragmoplastophyta, we could already identify a crucial molecular element. Interestingly, the FAX apo domain is predicted to bridge the inner and outer envelope membrane of chloroplasts for fatty acid and lipid transport. Moreover, this domain is likely to mediate chloroplast-mitochondrium contacts for lipid exchange at phosphate starvation stress. Within the research project, we now aim to identify exact membrane topology, formation of hetero-oligomeric complexes as well as in planta function of apoFAX and FAX proteins in Physcomitrium patens and Marchantia polymorpha. In the spotlight is the role in adaptation of lipid metabolism in response to cold stress and mineral deficiency, namely phosphate and potassium starvation.

https://gepris.dfg.de/gepris/projekt/528100298

The amplitude between the minimum and the maximum ambient temperature a plant has to endure during its life cycle is much larger in terrestrial than in aquatic environments. As such, especially elevated ambient temperatures are among the most prominent abiotic factors plants had to cope with to successfully colonize land. It is known that the phenotypic plasticity in eudicot land plants allows them to adjust shoot architecture to acclimate and adapt to elevated temperatures. How and when this mechanism, thermomorphogenesis, has originated is unknown. Likewise, we do not know whether bryophytes, whose body plans is probably very similar to that of the first terrestrial plants, perform thermomorphogenesis. In this project, we will therefore characterize the response of bryophytes (Physcomitrella patens and Marchantia polymorpha) to elevated temperatures on both the morphological as well as the physiological level, including elucidation of temperature sensitive signaling cascades. In addition to comparative analyses we will also follow an unbiased approach, primarily using the liverwort Marchantia polymorpha. To identify signaling and response modules, we will generate high density transcriptomic and phosphoproteomic datasets and use them to construct gene regulatory networks based on machine learning approaches. As such, we aim to combine morphological and molecular data in this project to better understand how bryophytes cope with elevated ambient temperatures. With this knowledge. we might then be in a position to design experiments that directly address the emergence of such mechanisms and their role during the terrestrialization of land by plants.

https://gepris.dfg.de/gepris/projekt/440536505

Approximately 500 million years ago, plant life conquered a barren terrestrial habitat. All plant life thriving on the surface of Earth today is derived from that singular event. Based on fossil, genomic and trait data that has become available during the first MAdLand funding period, we will perform state-of-the-art phylogenomics and ancestral state reconstruction. Thus, we aim to infer the evolution of plant key traits before and after the transition to land. These analyzes are expected to much increase our understanding of the nature and timeline of evolutionary adaptive events to a drastically different environment, allowing plants to live on land. The project aims at unravelling the enigmatic nature of the most recent common ancestor of plants.

https://gepris.dfg.de/gepris/projekt/527529748

The colonization of the land by plants was a key event in evolution and has transformed the planet. In their subaerial and terrestrial habitats, streptophyte algae and early land plants encountered multiple abiotic stresses and evolved adaptative mechanisms. Despite considerable progress, we are only in the beginning of understanding the cell biology of streptohyte algae. Peroxisomes are important cell organelles not only for fatty acid β-oxidation and photorespiration, but also for ROS detoxification and homeostasis, hormone biosynthesis, and abiotic stress tolerance. We want to identify key innovations of peroxisomes that were required for plant terrestrialization. We first addressed (i) to what extent peroxisome functions known from land plants are conserved in streptophyte algae and (ii) in which order they evolved. Using the protein landscape of Arabidopsis peroxisomes, we analyzed the conservation of matrix proteins and peroxisome localization at genome scale in streptophytes in comparison to chlorophytes. Accordingly, several peroxisome functions of land plants are conserved in chlorophytes. More advanced peroxisome functions, however, apparently evolved either after the divergence of Mesostigmatophyceae (e.g., fully established photorespiration), after that of Klebsormidiophyceae (e.g., NADPH production), or after the branching of Charophyceae (e.g., benzaldehyde biosynthesis). Furthermore, our data newly indicate that the peculiar, peroxisome-specific mechanism of “piggy-back import” may have been crucial to allow the simultaneous re-direction of neighboring pathway enzymes into peroxisomes. In this proposed research project, we want to extend our bioinformatic analyses and include further genomes of streptophyte algae and also transcriptome data (WP1). To identify new functions of streptophyte peroxisomes, we want to predict the entire matrix proteome of peroxisomes for 10 selected genomes of streptophytes in comparison to chlorophytes. Peroxisome targeting of proteins from selected streptophytes shall be verified experimentally (WP2). We will focus on (i) predicted peroxisomal proteins with non-canonical PTS and (ii) proteins of specific metabolic pathways and from extant species that are located close to the predicted point of divergence in subcellular compartmentalization. The genes will be cloned from streptophytic model algae and expressed as fluorescent fusions, first in the well-established system of Arabidopsis and for selected proteins also in the new model alga of Zygnematophyceae, Spirogyra pratensis. To allow comprehensive analyses of subcellular targeting in Spirogyra, we will create a set of transient expression vectors for the MadLand2 community. In WP3 we will investigate whether specific peroxisomal matrix enzymes of streptophytes can indeed import a neighboring pathway enzyme that lacks any PTS into peroxisomes by experimental analyses of protein-protein interactions and protein import by piggy-back mechanism.

https://gepris.dfg.de/gepris/projekt/528117925

Copper Zinc Superoxide Dismutase (CuZnSOD) proteins appeared around the time of the Great Oxidation Event around 2.4 billion years ago. In higher plants three compartment specific proteins are present, namely a cytosolic, plastidic and peroxisomal protein, of which the cytosolic form appears to have evolved first since it is already present in the freshwater algea Chara braunii. We found that the cytosolic form in Arabidopsis is essential for embryogenesis and that the CSD1 protein has two functions: on the one hand as an antioxidant enzyme and on the other as a nuclear transcriptional regulator. The appearance of CuZnSOD in freshwater algae indicate that it might represent an early molecular adaptation to enable the transition to land. Here we aim at revealing the role of CuZnSOD during embryogenesis and the regulation of abiotic stress tolerance in early land plants by modulating phenylpropanoid metabolism. We focus on Marchantia polymorpha and Physcomitrella patens to unravel the dual role of the cytosolic CuZnSOD during plant terrestrialization.

https://gepris.dfg.de/gepris/projekt/440296213

The evolution of a machinery that transports proteins across the two membranes separating the stroma of the organelle from the cytosol was crucial for the transition of a cyanobacterium endosymbiont to a plastid within a heterotrophic host. This machinery comprises a supercomplex of the translocon on the outer envelope of chloroplasts (TOC) and the translocon on the inner envelope of chloroplasts (TIC). This chloroplast translocon underwent significant changes during the evolution of streptophytes. Our objective is to understand whether changes in the preprotein import process were temporally associated with an increase in import efficiency along the evolutionary trajectory of land plant evolution. To accomplish this, we will evaluate import efficiency through in-vitro pre-protein translocation assays along the evolutionary lineage from green algae to land plants, by examining Streptophyta algae from KCC and ZCC clades, as well as bryophytes. Furthermore, by utilizing stalled-substrates and X-linking MS as well as computational modelling, we aim to identify proteins factors that enhanced the import efficiency during the evolution of land plants. Our in-vitro studies will be complemented with a top-down approach using in-situ cryo-electron tomography to answer the crucial question of how translocon complexes are organized structurally and spatially within the cellular context in selected streptophyta algae. Using this multi-scale integrative approach, we ultimately aim to establishing a correlation between import efficiency, translocon composition, structure and cellular organization. This will pinpoint the sequence of molecular adaptions that have taken place to increase chloroplast translocon efficiency, a pivotal feature in the evolution of charophyte freshwater algae, leading to their successful conquest of land.

https://gepris.dfg.de/gepris/projekt/527846412

Thylakoid membrane biogenesis requires the coordinated transport of nucleus- and plastid-encoded membrane proteins. The chloroplast signal recognition particle (SRP) system plays an important role in the post- and cotranslational transport of a subset of these proteins. Previous research has shown that these transport mechanisms underwent striking molecular adaptations with the evolution of streptophyte algae and land plants. In the proposed project we aim to get further insight into the molecular changes of the SRP system during land plant evolution and to reveal the underlying evolutionary driving forces. We will particularly analyze (i) the function of the cpSRP proteins (cpSRP43, cpSRP54) and the SRP RNA, (ii) the regulation of SRP-dependent transport and (iii) the docking mechanism of ribosomes to the Alb3 insertase in the moss Physcomitrium patens and the liverwort Marchantia polymorpha. To this end, we will combine genetic, cell biological, biochemical, and structural approaches.

https://gepris.dfg.de/gepris/projekt/440370941

Animal and plant cells establish polar regions on their plasma membranes to control cell division and cell differentiation. In animals, the regulation of polar PAR proteins is well understood and an important model for the study of numerous differentiation processes. There are no PAR orthologues in plants but we have evidence that so-called AGC1 kinases, such as D6PK or PAX from Arabidopsis thaliana, are needed for the formation of various polarity processes in higher plants. AGC1 kinases appear for the first time in mosses and liverworts, and we therefore postulate that they play a role in important evolutionary changes in the transition to terrestrial life. A very well understood function of the AGC1 kinases is the activation of the PIN auxin transporters, which localize - independently of AGC1 kinases - in the plasma membrane. In higher plants, the polarity of the PIN proteins is regulated by so-called AGC3 kinases, which, however, are absent from early land plants. We therefore want to investigate whether PIN proteins from early land plants can already respond to AGC3 signaling and whether AGC1 kinases may play the role of AGC3 kinases in early land plants. With these studies, we will be able to make an important contribution to the understanding of molecular adaptations in early land plants.

https://gepris.dfg.de/gepris/projekt/440524757

Pyrenoids for biophysical carbon concentration (CCM) are present in most extant Streptophyte algae and in a distinct group of land plants, the hornworts. Preliminary data show that i) hornwort CCM is not an adaptation to atmospheric carbon fluctuations and ii) a constitutive mechanism, in which iii) hyperoxia upregulates CCM-like genes. These findings suggest that hornwort CCM is a relic of terrestrialization and an adaptation to flooding-desiccation cycles, allowing land-borne organisms like hornworts to thrive on land by keeping photosynthetic ability at optimal levels through an equilibrated oxygen:carbon supply. This hypothesis moves into light streptophytic algae CCM and prompts for comparative molecular and ultrastructural research of Strepthophyte algae and hornworts. Molecular components and functional features of CCM and pyrenoids of these enigmatic plant groups remain to be thoroughly investigated. Here, we will investigate the evolutionary trajectory of CCM and its contribution to terrestrialization from Chlorophyte through Streptophyte algae to hornworts. We hypothesize that the highly dynamic and inducible Chlorophyte green algal CCM has evolved into a constitutive form along the transition to land, which may have been advantageous by enabling increased photosynthetic rates under optimally permissive periods of the day. The evolutionary step towards more constitutive CCM may have occurred already in Streptophyte algae. To investigate this scenario, we study how inducibility of CCM has evolved by comparatively recording CO2 consumption, plastid ultrastructural changes, and associated transcriptomic and proteomic responses in the Chlorophyte green alga Chlamydomonas, the three Streptophyte algae Zygnema, Mesotaenium and Mougeotiopsis, and the two hornwort species Anthoceros agrestis and A. fusiformis. Furthermore, we will characterize and functionally validate candidate CCM genes using immunoprecipitation, CRISPR/Cas9 editing, and artificial microRNAs. This line of research on the CCM biology of Streptophyte algae and hornworts and its role along terrestrialization will answer how molecular adaptations contributed to organisms thriving under periodic flooding and drought, which is associated with highly fluctuating oxygen and carbon concentrations. Thus, our research program will shed new light onto the series of the succession and nature of molecular adaptations during early land plant evolution by highlighting the evolutionary-genetic plasticity with which plants, Streptophyte algae and hornworts in particular, cope with adverse environmental conditions.

https://gepris.dfg.de/gepris/projekt/440370236

The overarching goal of our projects in both phases of MAdLand is to investigate the role of MIKC-type MADS-domain transcription factors (MTFs) during the transition of plants to land. MTFs constitute tetrameric proteins termed floral quartet-like complexes (FQCs) that control major developmental processes in land plants. The high combinatorial potential and cooperative DNA binding of FQCs enables the control of target gene activity in an especially efficient and specific way. This feature of FQCs is probably causally linked to the evolutionary origin of complex novelties in land plants and thus is of great biological interest. MTFs originated in the stem group of extant streptophytes, comprising all land plants and their closest relatives (charophyte algae). During the first phase of MAdLand we demonstrated that in the stem group of extant land plants important changes within the protein-protein interaction domain of MTFs occurred. These changes followed a gene duplication that led to two subfamilies of MTFs, termed MIKCC- and MIKC*-type proteins. MIKCC- and MIKC*-type proteins are major constituents of two largely independent gene regulatory networks that control important aspects of sporophyte and gametophyte development in land plants, respectively. In the second phase of MAdLand we want to study the biological relevance of FQC formation of the only MIKCC-type protein MpMADS2 in the liverwort M. polymorpha, reflecting the functional gain achieved by the ability of FQC formation during the transition of plants to land. We will do that by studying MpMADS2 mutants unable of FQC formation that we generated during the first phase. In addition, we want to determine the impact of FQC formation on genome-wide DNA-binding of MTFs for selected charophyte species. This will also provide useful insight into potential target genes of MIKC-type proteins in charophytes, for which no such data are available so far. Moreover, we want to complement our findings about the evolution of the protein-protein interaction domain by investigating major evolutionary changes within the DNA-binding MADS-domain of MTFs. Preliminary data suggest that ancestral MTFs that existed prior to the transition of plants to land, had a more promiscuous mode of DNA-binding than MIKCC- and MIKC*-type proteins. In our project we want to determine the mutations in the MADS-domain of MTFs that brought about these changes in DNA-recognition, and the consequences for the activity of those genes that are controlled by the different types of MTFs.

https://gepris.dfg.de/gepris/projekt/440291575

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 Physchomitrella 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.

https://gepris.dfg.de/gepris/projekt/440144334

One of the most remarkable challenges mastered by plants was the water-to-land-transition (plant terrestrialisation) that occurred more than 500 million years ago. This change in habitat required molecular adaptations to cope with an array of new stresses. Plant terrestrialisation transformed Earth’s atmosphere and soil cover, priming Earth for life as we know it. The Phragmoplastophyta comprise three lineages of streptophyte algae and the land plants (Embryophyta). Evolving from within the streptophytes, the earliest land plants gained features such as stomata as well as the cuticle and made use of fungal symbioses to gain access to nutrients. These traits are thought to have been instrumental for the habitat transition of plant life. Initially morphologically simple plants evolved a complexity that allowed them to conquer ever more habitats. In MAdLand, we use a comparative and functional evolutionary approach, encompassing streptophyte algae and non-seed plants as models, to study the genetic mechanisms underpinning the dramatic environmental adaptation to conditions on land and the evolution of plant complexity. During the first funding period of MAdLand, we already pinpointed important genetic mechanisms in adaptive evolution of plant morphology, physiology, biochemistry, cell biology and biotic interactions – and identified the ancestry of processes from which the diversity of land plants evolved. Now, in the second period, we will scrutinise these genetic mechanisms in light of streptophyte diversification with the aid of the new resources now established. These data will be used to infer properties of the most recent common ancestors of all land plants as well as those shared by land plants and their algal relatives. In MAdLand, we will address key questions in plant evolution: Which features enabling the conquest of land evolved in streptophyte algae? What is the nature of the most recent common ancestor of (i) all extant land plants and (ii) of land plants and algae? What is the succession and nature of molecular adaptations in early land plant evolution? How did embryogenesis and the alternation of generations evolve? How did organismic interaction of plants with fungi and bacteria evolve? What are the molecular evolutionary drivers of tolerance to abiotic and biotic stresses? Using a broad suite of biological methods and cross-discipline knowledge ranging from phylogenetic, molecular, physiological, genetic and cell biological approaches to the study of organismic interaction and biodiversity, we will study the following representatives of major non-seed plant lineages: (a) emerging model system Chara braunii (Charopyhceae), Spirogyra pratensis, Mesotaenium endlicherianum, and Zygnema circumcarinatum (Zygnematophyceae), Anthoceros agrestis (hornwort), Riccia fluitans (liverwort) Ceratopteris richardii, and Azolla filiculoides (ferns); the (b) established model systems Marchantia polymorpha (liverwort) and Physcomitrium patens (moss).

https://gepris.dfg.de/gepris/projekt/440231723

TCP transcription factors (TFs) are known to exert crucial regulatory functions in angiosperm development. Recently, we detected that the Marchantia polymorpha TF MpTCP1 exhibits a growth regulatory function and also governs the formation of novel plant pigments, which might mediate protective functions against abiotic stress. Plant terrestrialization likely started from a freshwater environment, where ancestors of land plants experienced water scarcity. The first land plants were thus facing novel abiotic stressors such as drought and high light intensities. To investigate adaptive molecular mechanisms enabling the conquest of land, the emerging model organism Riccia fluitans is ideally suited, as this amphibious liverwort can strive in aquatic as well as terrestrial habitats. Given its striking developmental plasticity, this liverwort can adapt thallus development to different habitats. Desiccation of the R. fluitans water form causes the formation of typical land plant structures: rhizoids as anchoring structures, air pores for gas exchange and a repellent surface. We will advance the tool box for R. fluitans and analyze the RfTCP1 activities by generating and investigating CRISPR/Cas9-induced mutants. The R. fluitans water and land forms also differ in their meristematic activities, thereby mediating thallus growth differences. The redox-sensitive MpTCP1 protein activity controls a complex downstream redox network, affecting also growth. We aim to investigate the impact of redox processes on meristematic activities and the formation of adaptive land plant features and in changing, challenging environments.

https://gepris.dfg.de/gepris/projekt/440539914


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