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Photosynthesis converts light into metabolic energy which fuels plant growth. In nature, many factors influence light availability for photosynthesis on different time scales, from shading by leaves within seconds up to seasonal changes over months. Variability of light energy supply for photosynthesis can limit a plant´s biomass accumulation. Plants have evolved multiple strategies to cope with strongly fluctuation light (FL). These range from long-term optimization of leaf morphology and physiology and levels of pigments and proteins in a process called light acclimation, to rapid changes in protein activity within seconds. Therefore, uncovering how plants deal with FL on different time scales may provide key ideas for improving crop yield. Photosynthesis is not an isolated process but tightly integrates with metabolism through mutual regulatory interactions. We thus require mechanistic understanding of how long-term light acclimation shapes both, dynamic photosynthesis and its interactions with downstream metabolism. To approach this, we analyzed the influence of growth light on i) the function of known rapid photosynthesis regulators KEA3 and VCCN1 in dynamic photosynthesis (Chapter 2-3) and ii) the interconnection of photosynthesis with photorespiration (PR; Chapter 4).
We approached topic (i) by quantifying the effect of different growth light regimes on photosynthesis and photoprotection by using kea3 and vccn1 mutants. Firstly, we found that, besides photosynthetic capacity, the activities of VCCN1 and KEA3 during a sudden high light phase also correlated with growth light intensity. This finding suggests regulation of both proteins by the capacity of downstream metabolism. Secondly, we showed that KEA3 accelerated photoprotective non-photochemical quenching (NPQ) kinetics in two ways: Directly via downregulating the lumen proton concentration and thereby de-activating pH-dependent NPQ, and indirectly via suppressing accumulation of the photoprotective pigment zeaxanthin.
For topic (ii), we analyzed the role of PR, a process which recycles a toxic byproduct of the carbon fixation reactions, in metabolic flexibility in a dynamically changing light environment. For this we employed the mutants hpr1 and ggt1 with a partial block in PR. We characterized the function of PR during light acclimation by tracking molecular and physiological changes of the two mutants. Our data, in contrast to previous reports, disprove a generally stronger physiological relevance of PR under dynamic light conditions. Additionally, the two different mutants showed pronounced and distinct metabolic changes during acclimation to a condition inducing higher photosynthetic activity. This underlines that PR cannot be regarded purely as a cyclic detoxification pathway for 2PG. Instead, PR is highly interconnected with plant metabolism, with GGT1 and HPR1 representing distinct metabolic modulators.
In summary, the presented work provides further insight into how energetic and metabolic flexibility is ensured by short-term regulators and PR during long-term light acclimation.
The emerging threat of antibiotic-resistant bacteria has become a global challenge in the last decades, leading to a rising demand for alternative treatments for bacterial infections. One approach is to target the bacterial cell envelope, making understanding its biophysical properties crucial. Specifically, bacteriophages use the bacterial envelope as an entry point to initiate infection, and they are considered important building blocks of new antibiotic strategies against drug-resistant bacteria.. Depending on the structure of the cell wall, bacteria are classified as Gram-negative and Gram-positive. Gram-negative bacteria are equipped with a complex cell envelope composed of two lipid membranes enclosing a rigid peptidoglycan layer. The synthesis machinery of the Gram-negative cell envelope is the target of antimicrobial agents, including new physical sanitizing procedures addressing the outer membrane (OM). It is therefore very important to study the biophysical properties of the Gram-negative bacterial cell envelope. The high complexity of the Gram-negative OM sets the demand for a model system in which the contribution of individual components can be evaluated separately. In this respect, giant unilamellar vesicles (GUVs) are promising membrane systems to study membrane properties while controlling parameters such as membrane composition and surrounding medium conditions.
The aim of this work was to develop methods and approaches for the preparation and characterization of a GUV-based membrane model that mimics the OM of the Gram-negative cell envelope. A major component of the OM is the lipopolysaccharide (LPS) on the outside of the OM heterobilayer. The vesicle model was designed to contain LPS in the outer leaflet and lipids in the inner leaflet. Furthermore, the interaction of the prepared LPS-GUVs with bacteriophages was tested. LPS containing GUVs were prepared by adapting the inverted emulsion technique to meet the challenging properties of LPS, namely their high self-aggregation rate in aqueous solutions. Notably, an additional emulsification step together with the adaption of solution conditions was employed to asymmetrically incorporate LPS containing long polysaccharide chains into the artificial membranes. GUV membrane asymmetry was verified with a fluorescence quenching assay. Since the necessary precautions for handling the quenching agent sodium dithionite are often underestimated and poorly described, important parameters were tested and identified to obtain a stable and reproducible assay. In the context of varied LPS incorporation, a microscopy-based technique was introduced to determine the LPS content on individual GUVs and to directly compare vesicle properties and LPS coverage. Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.
Employing LPS-GUVs we could demonstrate that a Salmonella bacteriophage bound with high specificity to its LPS receptor when presented at the GUV surface, and that the number of bound bacteriophages scaled with the amount of presented LPS receptor. In addition to binding, the bacteriophages were able to eject their DNA into the vesicle lumen. LPS-GUVs thus provide a starting platform for bottom-up approaches for the generation of more complex membranes, in which the effects of individual components on the membrane properties and the interaction with antimicrobial agents such as bacteriophages could be explored.
Biofilms are heterogeneous structures made of microorganisms embedded in a self-secreted extracellular matrix. Recently, biofilms have been studied as sustainable living materials with a focus on the tuning of their mechanical properties. One way of doing so is to use metal ions. In particular biofilms have been shown to stiffen in presence of some metal cations and to soften in presence of others. However, the specificity and the determinants of those interactions vary between species. While Escherichia coli is a widely studied model organism, little is known concerning the response of its biofilms to metal ions. In this work, we aimed at tuning the mechanics of E. coli biofilms by acting on the interplay between matrix composition and metal cations. To do so, we worked with E. coli strains producing a matrix composed of curli amyloid fibres or phosphoethanolamine-cellulose (pEtN-cellulose) fibres or both. The viscoelastic behaviour of the resulting biofilms was investigated with rheology after incubation with one of the following metal ion solutions: FeCl3, AlCl3, ZnCl2 and CaCl2 or ultrapure water. We observed that the strain producing both fibres stiffen by a factor of two when exposed to the trivalent metal cations Al(III) and Fe(III) while no such response is observed for the bivalent cations Zn(II) and Ca(II). Strains producing only one matrix component did not show any stiffening in response to either cation, but even a small softening. In order to investigate further the contribution of each matrix component to the mechanical properties, we introduced additional bacterial strains producing curli fibres in combination with non-modified cellulose, non-modified cellulose only or neither component. We measured biofilms produced by those different strains with rheology and without any solution. Since rheology does not preserve the architecture of the matrix, we compared those results to the mechanical properties of biofilms probed with the non-destructive microindentation. The microindentation results showed that biofilm stiffness is mainly determined by the presence of curli amyloid fibres in the matrix. However, this clear distinction between biofilm matrices containing or not containing curli is absent from the rheology results, i.e. following partial destruction of the matrix architecture. In addition, rheology also indicated a negative impact of curli on biofilm yield stress and flow stress. This suggests that curli fibres are more brittle and therefore more affected by the mechanical treatments. Finally, to examine the molecular interactions between the biofilms and the metal cations, we used Attenuated total reflectance - Fourier transform infrared spectroscopy (ATR-FTIR) to study the three E.coli strains producing a matrix composed of curli amyloid fibres, pEtN-cellulose fibres or both. We measured biofilms produced by those strains in presence of each of the aforementioned metal cation solutions or ultrapure water. We showed that the three strains cannot be distinguished based on their FTIR spectra and that metal cations seem to have a non-specific effect on bacterial membranes in absence of pEtN-cellulose. We subsequently conducted similar experiments on purified curli or pEtN-cellulose fibres. The spectra of the pEtN-cellulose fibres revealed a non-valence-specific interaction between metal cations and the phosphate of the pEtN-modification. Altogether, these results demonstrate that the mechanical properties of E. coli biofilms can be tuned via incubation with metal ions. While the mechanism involving curli fibres remains to be determined, metal cations seem to adsorb onto pEtN-cellulose and this is not valence-specific. This work also underlines the importance of matrix architecture to biofilm mechanics and emphasises the specificity of each matrix composition.
The light reactions of photosynthesis are carried out by a series of multiprotein complexes embedded in thylakoid membranes. Among them, photosystem I (PSI), acting as plastocyanin-ferderoxin oxidoreductase, catalyzes the final reaction. Together with light-harvesting antenna I, PSI forms a high-molecular-weight supercomplex of ~600 kDa, consisting of eighteen subunits and nearly two hundred co-factors. Assembly of the various components into a functional thylakoid membrane complex requires precise coordination, which is provided by the assembly machinery. Although this includes a small number of proteins (PSI assembly factors) that have been shown to play a role in the formation of PSI, the process as a whole, as well as the intricacy of its members, remains largely unexplored.
In the present work, two approaches were used to find candidate PSI assembly factors. First, EnsembleNet was used to select proteins thought to be functionally related to known PSI assembly factors in Arabidopsis thaliana (approach I), and second, co-immunoprecipitation (Co-IP) of tagged PSI assembly factors in Nicotiana tabacum was performed (approach II).
Here, the novel PSI assembly factors designated CO-EXPRESSED WITH PSI ASSEMBLY 1 (CEPA1) and Ycf4-INTERACTING PROTEIN 1 (Y4IP1) were identified. A. thaliana null mutants for CEPA1 and Y4IP1 showed a growth phenotype and pale leaves compared with the wild type. Biophysical experiments using pulse amplitude modulation (PAM) revealed insufficient electron transport on the PSII acceptor side. Biochemical analyses revealed that both CEPA1 and Y4IP1 are specifically involved in PSI accumulation in A. thaliana at the post-translational level but are not essential. Consistent with their roles as factors in the assembly of a thylakoid membrane protein complex, the two proteins localize to thylakoid membranes. Remarkably, cepa1 y4ip1 double mutants exhibited lethal phenotypes in early developmental stages under photoautotrophic growth. Finally, co-IP and native gel experiments supported a possible role for CEPA1 and Y4IP1 in mediating PSI assembly in conjunction with other PSI assembly factors (e.g., PPD1- and PSA3-CEPA1 and Ycf4-Y4IP1). The fact that CEPA1 and Y4IP1 are found exclusively in green algae and higher plants suggests eukaryote-specific functions. Although the specific mechanisms need further investigation, CEPA1 and Y4IP1 are two novel assembly factors that contribute to PSI formation.
Biostimulant SuperFifty based molecular priming to increase plant strength and stress tolerance
(2023)
The development of seeds in angiosperms starts with a complex process of double fertilization, involving the fusion of the maternal egg cell and central cell with two paternal sperm cells. This gives rise to the embryo and the nourishing endosperm, which are then enclosed by the seed coat, derived from the maternal integuments. The growth of the seed coat in Arabidopsis thaliana (Arabidopsis) is actively inhibited before fertilization by epigenetic regulators known as Polycomb Group (PcG) proteins. These proteins deposit a repressive histone mark called H3K27me3, which must be removed to enable seed coat formation. In this thesis, I explored the mechanism of removal of H3K27me3 marks from the integument cells following fertilization, which allows for seed coat formation. We hypothesized that this removal should be primarily facilitated by histone demethylases from the JMJ family and potentially influenced by the plant hormones Brassinosteroids (BRs). This hypothesis was supported by the expression patterns of the JMJ protein REF6 and of BR related genes, which are specifically expressed in the integuments and in the seed coat. Moreover, mutations in both these pathways lead to developmental defects, such as reduced ovule viability and delayed seed coat growth. Our research provides evidence suggesting that BR signalling is likely involved in recruiting JMJ-type histone demethylases to target loci responsible for seed coat growth. Moreover, we have discovered an additional pathway through which BRs regulate seed coat development, independent of their influence on H3K27me3 marks. This finding emphasizes the diverse roles of BRs in coordinating seed development, extending beyond their well-known involvement in plant growth and development. Furthermore, I explored the role of another epigenetic mark, DNA methylation, in fertilization-independent (or autonomous) seed formation in Arabidopsis. For this, we utilized epigenetic Recombinant Inbred Lines (epiRILs) and thus identified an epigenetic Quantitative Trait Locus (epiQTL) on chromosome II, potentially responsible for the larger autonomous seed size observed in DNA methylation mutants. Overall, this thesis significantly enhances our comprehension of the intricate relationship between epigenetic modifications, hormonal signaling, and plant reproductive processes. It offers valuable insights into the genetic mechanisms governing both sexual and asexual seed formation, while also presenting potential avenues for the engineer of advantageous traits in agricultural crops.
The musculoskeletal system provides support and enables movement to the body, and its deterioration is a crucial aspect of age-related functional decline. Mesenchymal stromal cells (MSCs) play an important role in musculoskeletal homeostasis due to their broad differentiation potentials and their ability to support osteogenic and myogenic tissue maintenance and regeneration. In the bone, MSCs differentiate either into osteochondrogenic progenitors to form osteocytes and chondrocytes, or increasingly with age into adipogenic progenitors which give rise to bone-resident adipocytes. In skeletal muscle, during healthy regeneration MSCs provide regulatory signals that activate local, tissue-specific stem cells, known as satellite cells, which regenerate contractile myofibres. This process involves a significant cross-talk to immune cells stemming from both lymphoid and myeloid lineages. During ageing, muscle-resident MSCs undergo increased adipogenic lineage commitment, causing niche changes that contribute to fatty infiltration in muscles. These shifts in cell populations in bone lead to the loss of osteogenic cells and subsequently osteoporosis, or in muscle to impaired regeneration and to the development of sarcopenia. However, the signals that drive transition of MSCs into their respective cellular fates remain elusive.
This thesis aims to elucidate the transcriptional shifts modulating cell states and cell types in musculoskeletal MSC fate determination. Single-cell RNA-sequencing (scRNA-seq) was used to characterise cell type-specific transcript regulation. State-of-the-art bioinformatics tools were combined with different analytical platforms that include both droplet-based scRNA-seq for large heterogeneous populations, and microfluidics-based scRNA-seq to assess small, rare subpopulations. For each platform, distinct computational pipelines were established including filtering steps to exclude low-quality cells, and data visualisation was performed by dimensionality reduction. Downstream analysis included clustering, cell type annotation, and differential gene expression to investigate transcriptional states in defined cell types during ageing and injury in the muscle and bone. Finally, a novel tool to assess publication activities in defined areas of research for the identified marker genes was developed.
The results in the bone indicate that ageing MSCs increasingly commit towards an adipogenic fate at the expense of osteogenic specialisation. The data also suggests that significant cell population shifts of MSC-type fibro-adipogenic progenitors during muscle ageing underlie the pathologies observed in homeostatic and post-injury regenerative conditions. High-throughput visualisation of publication activity for candidate genes enabled more effective biological evaluation of scRNA-seq data. These results expose critical age-related changes in the stem cell niches of skeletal muscle and bone, highlight their respective sensitivity to nutrition and pathology, and elucidate novel factors that modulate stem cell-based regeneration. Targeting these processes might improve musculoskeletal health in the context of ageing and prevent the negative effects of pathological lineage determination.
In times of ongoing biodiversity loss, understanding how communities are structured and what mechanisms and local adaptations underlie the patterns we observe in nature is crucial for predicting how future ecological and anthropogenic changes might affect local and regional biodiversity. Aquatic zooplankton are a group of primary consumers that represent a critical link in the food chain, providing nutrients for the entire food web. Thus, understanding the adaptability and structure of zooplankton communities is essential. In this work, the genetic basis for the different temperature adaptations of two seasonally shifted (i.e., temperature-dependent) occurring freshwater rotifers of a formerly cryptic species complex (Brachionus calyciflorus) was investigated to understand the overall genetic diversity and evolutionary scenario for putative adaptations to different temperature regimes. Furthermore, this work aimed to clarify to what extent the different temperature adaptations may represent a niche partitioning process thus enabling co-existence. The findings were then embedded in a metacommunity context to understand how zooplankton communities assemble in a kettle hole metacommunity located in the northeastern German "Uckermark" and which underlying processes contribute to the biodiversity patterns we observe. Using a combined approach of newly generated mitochondrial resources (genomes/cds) and the analysis of a candidate gene (Heat Shock Protein 40kDa) for temperature adaptation, I showed that the global representatives of B. calyciflorus s.s.. are genetically more similar than B. fernandoi (average pairwise nucleotide diversity: 0.079 intraspecific vs. 0.257 interspecific) indicating that both species carry different standing genetic variation. In addition to differential expression in the thermotolerant B. calyciflorus s.s. and thermosensitive B. fernandoi, the HSP 40kDa also showed structural variation with eleven fixed and six positively selected sites, some of which are located in functional areas of the protein. The estimated divergence time of ~ 25-29 Myr combined with the fixed sites and a prevalence of ancestral amino acids in B. calyciflorus s.s. indicate that B. calyciflorus s.s. remained in the ancestral niche, while B. fernandoi partitioned into a new niche. The comparison of mitochondrial and nuclear markers (HPS 40kDa, ITS1, COI) revealed a hybridisation event between the two species. However, as hybridisation between the two species is rare, it can be concluded that the temporally isolated niches (i.e., seasonal-shifted occurrence) they inhabit based on their different temperature preferences most likely represent a pre-zygotic isolation mechanism that allows sympatric occurrence while maintaining species boundaries. To determine the processes underlying zooplankton community assembly, a zooplankton metacommunity comprising 24 kettle holes was sampled over a two-year period. Active (i.e., water samples) and dormant communities (i.e., dormant eggs hatched from sediment) were identified using a two-fragment DNA metabarcoding approach (COI and 18S). Species richness and diversity as well as community composition were analysed considering spatial, temporal and environmental parameters. The analysis revealed that environmental filtering based on parameters such as pH, size and location of the habitat patch (i.e., kettle hole) and surrounding field crops largely determined zooplankton community composition (explained variance: Bray-Curtis dissimilarities: 10.5%; Jaccard dissimilarities: 12.9%), indicating that adaptation to a particular habitat is a key feature of zooplankton species in this system. While the spatial configuration of the kettle holes played a minor role (explained variance: Bray-Curtis dissimilarities: 2.8% and Jaccard dissimilarities: 5.5%), the individual kettle hole sites had a significant influence on the community composition. This suggests monopolisation/priority effects (i.e., dormant communities) of certain species in individual kettle holes. As environmental filtering is the dominating process structuring zooplankton communities, this system could be significantly influenced by future land-use change, pollution and climate change.
Light is the essential energy source for plants to drive photosynthesis. In nature, light availability is highly variable and often fluctuates on very short time scales. As a result, plants developed mechanisms to cope with these fluctuations. Understanding how to improve light use efficiency in natural fluctuating light (FL) conditions is a major target for agronomy.
In the first project, we identified an Arabidopsis thaliana plant that showed reduced levels of rapidly inducible non-photochemical quenching (NPQ). This plant was devoid of any T-DNA insertion. Using a mapping-by-sequencing approach, we successfully located the causal genomic region near the end of chromosome 4. Through variant investigations in that region, we identified a deletion of about 20 kb encompassing 9 genes. By complementation analysis, we confirmed that one of the deleted genes, VTC2, is the causal gene responsible for the low NPQ. Loss of VTC2 decreased NPQ particularly in old leaves, with young leaves being only slightly affected. Additionally, ascorbate levels were almost abolished in old leaves, likely causing the NPQ decrease by reducing the activity of the xanthophyll cycle. Although ascorbate levels in younger leaves were reduced compared to wild-type plants, they remained at a comparably higher level. This difference may be due to the VTC2 paralog VTC5, which is expressed at a higher level in young leaves than in old ones.
Plants require the PROTON GRADIENT REGULATION 5 (PGR5) protein for survival in FL. pgr5 mutants die because they fail to increase the luminal proton concentration in response to high light (HL) phases. A rapid elevation in ∆pH is needed to slow down electron transport through the Cytochrome b6 f complex (photosynthetic control). In FL, such lack of control in the pgr5 mutants results in photosystem I (PSI) overreduction, reactive oxygen species (ROS) production, and cell death. Decreases in photosystem II (PSII) activity introduced by crossing pgr5 with PSII deficient mutants
rescued the lethality of pgr5 in FL. PGR5 was suggested to act as part of the ferredoxin-plastoquinone reductase (FQR), involved in cyclic electron transfer around PSI. However, the proposed molecular role of PGR5 remains highly debated. To learn more about PGR5 function, we performed a forward genetic screen in Arabidopsis thaliana to identify EMS-induced suppressor mutants surviving longer when grown in FL compared to pgr5 mutants (referred to as ”suppressor of pgr5 lethality in fluctuating light”, splf ). 11 different candidate genes were identified in a total of 22 splf plants.
Mutants of seven of these genes in the pgr5 background showed low Fv/Fm values when grown in non-fluctuating low light (LL). Five of these 4genes were previously reported to have a role in PSII biogenesis or function. Two others, RPH1 and a DEAD/DEAH box helicase (AT3G02060), have not been linked to PSII function before. Three of splf candidate genes link to primary metabolism, fructose-2,6-bisphosphatase (F2KP ), udp-glucose pyrophosphorylase 1 (UGP1 ) and ferredoxin-dependent glutamate synthase (Fd-GOGAT ). They are characterized by the fact that they survive longer in FL than pgr5 mutants but do not procede beyond the early vegetative
phase and then die.
Heat stress (HS) is one of the major abiotic stresses which adversely affects the survival and growth of plants due to their sessile nature. To combat the detrimental effects of HS and develop thermotolerance, plants have evolved several defense mechanisms. Thermomemory is one such molecular mechanism whereby plants that have been acclimated (or primed/P) by a moderate HS can respond more efficiently and continue their growth after exposure to a severe or lethal HS (called triggering/T), while unprimed plants cannot survive. Thermomemory is known to be regulated by several transcription factors (TFs), epigenetic changes, chromatin remodellers, post-transcriptional changes and it also involves protein stability control and primary metabolism adjustment. Recent research has suggested that the shoot apical meristem (SAM) in Arabidopsis thaliana has a distinct transcriptional thermomemory which is possibly regulated by eight TFs called HEAT SHOCK FACTORS (HSFs). The main objective of this PhD thesis is to investigate the role of HSFA7b (one of the eight HSFs), in regulating thermomemory at the SAM by identifying the molecular networks it regulates. HSFA7a, a close homolog of HSFA7b, is also one of the eight HSFs that are involved in regulating thermomemory at the SAM. Thermomemory was found to be defective in the hsfa7b and hsfa7a hsfa7b mutants; the percentage survival of these seedlings was significantly lower than in wild-type (WT) seedlings after the priming and triggering (PT) treatment. Transcriptome and ChIP analyses were performed to identify the molecular networks controlled by HSFA7b and its close homolog HSFA7a, in regulating thermomemory at the SAM. The chromatin regulator SPLAYED (SYD) was found to be regulated by both HSFA7a and HSFA7b at the SAM during thermomemory. SYD is directly involved in SAM maintenance by directly regulating WUSCHEL (WUS), a master regulator of stem cell maintenance. WUS expression was down-regulated at the SAM of PT treated hsfa7a/b mutants compared to WT-Col-0 seedlings. HSFA7a and HSFA7b also jointly regulate the expression of orphan gene QUA QUINE STARCH (QQS) during thermomemory. Starch accumulation negatively correlates with QQS expression and this trend was observed in WT plants in response to thermopriming. The remobilization of starch was affected in the hsfa7a/b mutants compared to WT plants during the recovery period after T treatment. These findings indicate that defects in SAM maintenance and starch remobilization could possibly contribute to the reduced thermomemory in the hsfa7a/b mutants. Moreover, transcriptome and ChIP analysis indicate that ethylene signaling genes are directly regulated by HSFA7b during thermomemory. Transcriptome analysis of the HSFA7b-IOE line indicates that HSFA7b positively regulates the expression of HEAT STRESS ASSOCIATED 32 (HSA32), an important thermomemory gene, and HSFA7b strongly suppresses the expression of the reactive oxygen species (ROS) responsive REDOX RESPONSIVE TRANSCRIPTION FACTOR 1 (RRTF1) gene, which is also a repressed target of SYD. In Arabidopsis, the HSFA7b transcript undergoes alternative splicing at high temperatures to form two splice variants: one correctly/constitutively spliced variant which is functional and codes for the HSFA7b protein and one intron retained splice variant. Higher accumulation of the functional HSFA7b splice variant was found at the SAM compared to other tissues. Moreover, accumulation of the functional splice variant was higher in P and PT plants compared to control plants, whereas higher levels of the intron retained splice variant is found in plants subjected directly to the T treatment. The intron retained HSFA7b splice variant is degraded by the non-sense mediated decay (NMD) pathway as a means of regulating transcript level essential for protein synthesis at high temperatures. Importantly, HSFA7b protein accumulation was observed in plants subjected to PT treatment that survive and continue growth, but not in plants subjected directly to T treatment that do not survive, indicating that constitutive/ correct splicing of the HSFA7b transcript is a component of thermomemory. Taken together, these findings suggest that HSFA7a and HSFA7b jointly regulate SAM maintenance via the chromatin remodeller SYD and starch remobilization via QQS. In addition to them, HSFA7b also regulates the expression of ethylene signaling genes, heat responsive genes and the ROS responsive RRTF1. Furthermore, constitutive/correct splicing in the HSFA7b transcript is also an essential component of thermomemory.
In nature, plants often encounter biotic and abiotic stresses, which can cause reduced crop yield and quality, and threaten the nutrition of a growing human population. As heat stress (HS) is one of the main abiotic stresses, and is projected to increase due to global warming, it is necessary to better understand how plants respond and survive under HS. In Arabidopsis thaliana, plants can survive under severe HS if primed by a non-lethal HS, a process called acquisition of thermotolerance. This primed stated can be maintained for several days, and the ability of plants to maintain the primed state is called maintenance of acquired thermotolerance (mATT) or HS memory. According to current research, two Heat shock factors (HSFs) HSFA2 and HSFA3 are known to account for the majority of mATT capability, and there are other HSFs e.g. HSFA1b and HSFA6b in HSF complexes containing HSFA2 and/or HSFA3, however, the roles of these HSFs in HS memory is not clearly understood. Moreover, the mechanism of these HSFs in regulating HS memory is unclear, whether transcriptional machinery e.g. the Mediator complex contributes to transcriptional memory. This work investigates the role of HSFs and Mediator subunits in HS memory in A. thaliana. For the role of HSFs, the interaction between HSFA1b and HSFA2 during HS memory phase was confirmed by in vivo co- immunoprecipitation (Co-IP). HSFA1b, HSFA2, HSFA3 and HSFA6b targeted HS memory-related genes according to DNA affinity purification sequencing (DAP-seq) data, and targets of HSFA1b were confirmed in vivo by chromatin immunoprecipitation qPCR (ChIP-qPCR). The mutant of hsfa6b showed an HS memory deficiency phenotype in mATT survival assay. These data confirmed the role for HSFA2 and HSFA3 in HS memory, and suggest that HSFA1b and HSFA6b also function in HS memory. The Mediator complex functions as an RNA Polymerase II (RNA Pol II) co-regulator, and includes Head, Middle, Tail and Kinase modules. Both MED23 and MED32 belong to the Tail module, and they have a positive role in HS memory. MED23 interacted with HSFA3, as determined by yeast two hybrid (Y2H) and in vivo Co-IP assays. The med23 mutant showed a decreased HS memory phenotype, reduced expression of Type I (sustained expression) memory genes following HS, and reduced accumulation of the memory-associated Tri-methylation of histone H3 lysine 4 (H3K4me3)histone modification at HS memory-related gene loci after HS. MED23 was recruited to HS-inducible memory and non-memory genes after HS, as determined by ChIP-qPCR. The med32
mutant showed a reduced HS memory phenotype, decreased expression of Type I and Type II (hyper-induction) memory genes, and lower accumulation of H3K4me3 at memory gene lociafter HS. However, MED32 did not show interaction with any tested HSF in Y2H or in vivo Co-IP. MED32 regulated the recruitment of RNA Pol II at HS-inducible genes after HS, but was not itself recruited to HS memory genes after HS. These results provided more evidence
that the Mediator subunits MED23 and MED32 regulate HS memory on transcriptional and epigenetic levels. In general, this work provides a better insight into the molecular mechanism of how HSFs and Mediator subunits regulate HS memory in plants and will provide new perspectives to breed crops with improved thermotolerance.
A biological trade-off situation denotes the dependence between traits whereby an increase in the value of one of the traits leads to a decrease in the value of at least one of the others. Understanding trade-offs in cellular systems is relevant to understanding the limits and constraints to tuning desired phenotypes. Therefore, it is mainly the case for rates (i.e. fluxes) of biochemical reactions that shape not only molecular traits, like metabolite concentrations but also determine physiological traits, like growth. Intracellular fluxes are the final phenotype from transcriptional and (post)translational regulation. Quantifying intracellular fluxes provides insights into cellular physiology under particular growth conditions and can be used to characterize the metabolic activity of different pathways. However, estimating fluxes from labelling experiments is labour-intensive; therefore, developing approaches to accurately and precisely predict intracellular fluxes is essential. This thesis addresses two main problems: (i) identifying flux trade-offs and (ii) predicting accurate and precise reaction flux at a genome-scale level. To this end, the concept of an absolute flux trade-off is defined, and a constraint-based approach, termed FluTO, was developed to identify absolute flux trade-offs. FluTO is cast as a mixed integer programming approach applied to genome-scale metabolic models of E. coli, S. cerevisiae, and A. thaliana, imposing realistic constraints on growth and nutrient uptake.. The findings showed that trade-offs are not only species-specific but also specific to carbon sources. In addition, we found that different models of a single species have a different number of reactions in trade-offs. We also showed that absolute flux trade-offs depend on the biomass reaction used to model the growth of A. thaliana under different carbon and nitrogen conditions. Findings reflect the strong relation between nitrogen, carbon, and sulphur metabolisms in the leaves of C3 plants. The concept of relative trade-offs was introduced to further study trade-offs in metabolic networks. A constraint-based approach, FluTOr, was proposed to identify reactions whose fluxes are in relative trade-off concerning an optimized fitness-related cellular task, like growth. FluTOr was employed to find the relative flux trade-offsin the genome-scale metabolic networks of E. coli, S. cerevisiae, and A. thaliana. The results showed that in contrast to the A. thaliana model, the relative trade-offs in the two microorganisms depend on the carbon source, reflecting the differences in the underlying metabolic network. Furthermore, applying FluTOr also showed that reactions that participated in relative trade-offs were implicated in cofactor biosynthesis in the two microorganisms. Prediction of reaction fluxes in the constraint-based metabolic framework is usually performed by parsimonious flux balance analysis (pFBA), employing the principle of efficient usage of protein resources. However, we argued that principles related to the coordination of flux values, neglected in previous studies, provide other means to predict intracellular fluxes. To this end, we designed a constraint-based approach, termed complex-balanced FBA (cbFBA), to predict steady-state flux distributions that maximize the number of balanced complexes in a flux distribution, whereby multi-reaction dependencies are maximized. The comparative analysis showed a better agreement of the flux distributions resulting from cbFBA compared to pFBA with experimentally measured fluxes from 17 E. coli strains and 26 S. cerevisiae knock-out mutants. The results also showed that the predictions from cbFBA are more precise than those from pFBA since cbFBA results in a smaller space of alternative solutions than pFBA.
Pichia pastoris (syn. Komagataella phaffi) is a distinguished expression system widely used in industrial production processes. Recent molecular research has focused on numerous approaches to increase recombinant protein yield in P. pastoris. For example, the design of expression vectors and synthetic genetic elements, gene copy number optimization, or co-expression of helper proteins
(transcription factors, chaperones, etc.). However, high clonal variability of transformants and low screening throughput have hampered significant success.
To enhance screening capacities, display-based methodologies inherit the potential for efficient isolation of producer clones via fluorescence-activated cell sorting (FACS). Therefore, this study focused on developing a novel clone selection method that is based on the non-covalent attachment of Fab fragments on the P. pastoris cell surface to be applicable for FACS.
Initially, a P. pastoris display system was developed, which is a prerequisite for the surface capture of secreted Fabs. A Design of Experiments approach was applied to analyze the influence of various genetic elements on antibody fragment display. The combined P. pastoris formaldehyde dehydrogenase promoter (PFLD1), Saccharomyces cerevisiae invertase 2 signal peptide (ScSUC2), - agglutinin (ScSAG1) anchor protein, and the ARS of Kluyveromyces lactis (panARS) conferred highest display levels.
Subsequently, eight single-chain variable fragments (scFv) specific for the constant part of the Fab heavy or light chain were individually displayed in P. pastoris. Among the tested scFvs, the anti-human CH1 IgG domain scFv allowed the most efficient Fab capture detected by flow cytometry.
Irrespective of the Fab sequence, exogenously added as well as simultaneously secreted Fabs were successfully captured on the cell surface. Furthermore, Fab secretion capacities were shown to correlate to the level of surface-bound Fabs as demonstrated for characterized producer clones.
Flow-sorted clones presenting high amounts of Fabs showed an increase in median Fab titers (factor of 21 to 49) compared to unsorted clones when screened in deep-well plates. For selected candidates, improved functional Fab yields of sorted cells vs. unsorted cells were confirmed in an upscaled shake flask production. Since the scFv capture matrix was encoded on an episomal plasmid with inherently unstable autonomously replicating sequences (ARS), efficient plasmid curing was observed after removing the selective pressure. Hence, sorted clones could be immediately used for production without the need to modify the expression host or vector. The resulting switchable display/secretion system provides a streamlined approach for the isolation of Fab producers and subsequent Fab production.
Potato FLC-like and SVP-like proteins jointly control growth and distinct developmental processes
(2023)
Based on worldwide consumption, Solanum tuberosum L. (potato) is the most important non-grain food crop. Potato has two ways of stable propagation: sexually via flowering and vegetatively via tuberization. Remarkably, these two developmental processes are controlled by similar molecular regulators and mechanisms. Given that FLC and SVP genes act as key flowering regulators in the model species Arabidopsis and in various other crop species, this study aimed at identifying FLC and SVP homologs in potato and investigating their roles in the regulation of plant development, with a particular focus on flowering and tuberization. Our analysis demonstrated that there are five FLC-like and three SVP like proteins encoded in the potato genome. The expression profiles of StFLCs and StSVPs throughout potato development and the detected interactions between their proteins indicate tissue specificity of the individual genes and distinct roles of a variety of putative protein complexes. In particular, we discovered that StFLC-D, as well as StFLC-B, StSVP-A, and StSVP-B play a complex role in the regulation of flowering time, as not only increased but also decreased levels of their transcripts promote earlier flowering. Most importantly, StFLC-D has a marked impact on tuberization under non-inductive conditions and susceptibility to temperature-induced tuber malformation, also known as second growth. Plants with decreased levels of StFLC-D demonstrated a strong ability to produce tubers under long days and appeared to be insensitive to temperature-induced second growth. Lastly, our data also suggests that StFLCs and StSVPs may be involved in the nitrogen-dependent regulation of potato development. Taken together, this study highlights the functional importance of StFLC and StSVP genes in the regulation of distinct developmental processes in potato.
Mitochondria and plastids are organelles with an endosymbiotic origin. During evolution, many genes are lost from the organellar genomes and get integrated in the nuclear genome, in what is known as intracellular/endosymbiotic gene transfer (IGT/EGT). IGT has been reproduced experimentally in Nicotiana tabacum at a gene transfer rate (GTR) of 1 event in 5 million cells, but, despite its centrality to eukaryotic evolution, there are no genetic factors known to influence the frequency of IGT in higher eukaryotes. The focus of this work was to determine the role of different DNA repair pathways of double strand break repair (DSBR) in the integration step of organellar DNA in the nuclear genome during IGT. Here, a CRISPR/Cas9 mutagenesis strategy was implemented in N. tabacum, with the aim of generating mutants in nuclear genes without expected visible phenotypes. This strategy led to the generation of a collection of independent mutants in the LIG4 (necessary for non-homologous end joining, NHEJ) and POLQ genes (necessary for microhomology mediated end joining, MMEJ). Targeting of other DSBR genes (KU70, KU80, RPA1C) generated mutants with unexpectedly strong developmental phenotypes.. These factors have telomeric roles, hinting towards a possible relationship between telomere length, and strength of developmental disruption upon loss of telomere structure in plants. The mutants were made in a genetic background encoding a plastid-encoded IGT reporter, that confers kanamycin resistance upon transfer to the nucleus. Through large scale independent experiments, increased IGT from the chloroplast to the nucleus was observed in lig4 mutants, as well as lines encoding a POLQ gene with a defective polymerase domain (polqΔPol). This shows that NHEJ or MMEJ have a double-sided relationship with IGT: while transferred genes may integrate using either pathway, the presence of both pathways suppresses IGT in wild-type somatic cells, thus demonstrating for the first time the extent on which nuclear genes control IGT frequency in plants. The IGT frequency increases in the mutants are likely mediated by increased availability of double strand breaks for integration. Additionally, kinetic analysis reveals that gene transfer (GT) events accumulate linearly as a function of time spent under antibiotic selection in the experiment, demonstrating that, contrary to what was previously thought, there is no such thing as a single GTR in somatic IGT experiments. Furthermore, IGT in tissue culture experiments appears to be the result of a "race against the clock" for integration in the nuclear genome, that starts when the organellar DNA arrives to the nucleus granting transient antibiotic resistance. GT events and escapes of kanamycin selection may be two possible outcomes from this race: those instances where the organellar DNA gets to integrate are recovered as GT events, and in those cases where timely integration fails, antibiotic resistance cannot be sustained, and end up considered as escapes. In the mutants, increased opportunities for integration in the nuclear genome change the overall ratio between IGT and escape events. The resources generated here are promising starting points for future research: (1) the mutant collection, for the further study of processes that depend on DNA repair in plants (2) the collection of GT lines obtained from these experiments, for the study of the effect of DSBR pathways over integration patterns and stability of transferred genes and (3) the developed CRISPR/Cas9 workflow for mutant generation, to make N. tabacum meet its potential as an attractive model for answering complex biological questions.
The G protein-coupled estrogen receptor (GPER1) is acknowledged as an important mediator of estrogen signaling. Given the ubiquitous expression of GPER1, it is likely that the receptor plays a role in a variety of malignancies, not only in the classic hormonally regulated tissues (e.g., breast, ovary, and prostate), but also in the colon. As colorectal cancer (CRC) is the third most common cancer in both men and women worldwide and environmental factors and dietary habits are important risk factors, it is increasingly recognized that natural and synthetic hormones and their associated receptors might play a role in CRC. Through oral consumption, environmental contaminants with endocrine activity are in contact with the gastrointestinal mucosa, where they might exert their toxic effects. Although GPER1 has been shown to be engaged in physiological and pathophysiological processes, its role in CRC remains poorly understood. Thus, pro- as well as anti-tumorigenic effects are described in the literature. This thesis has uncovered novel roles of GPER1 in mediating major CRC-associated phenotypes in transformed and non-transformed colon cell lines. Exposure to the estrogens 17β-estradiol (E2), bisphenol-A (BPA) and diethylstilbestrol (DES) but also the androgen dihydrotestosterone (DHT) resulted in GPER1-dependent induction of supernumerary centrosomes, whole chromosomal instability (w-CIN) and aneuploidy. Indeed, both knockdown and inhibition of GPER1 attenuated the generation of (xeno)hormone-driven supernumerary centrosomes and karyotype instability. Mechanistically, (xeno)hormone-induced centrosome amplification was associated with transient multipolar mitosis and the generation of so called anaphase “lagging” chromosomes. The results of this thesis propose a GPER1/PKA/AKAP9-pathway in regulating centrosome numbers in colorectal cancer cells and the involvement of the centriolar protein centrin. Remarkably, exposure to (xeno)hormones resulted in atypical enlargement and unexpected phosphorylation of the centriole marker centrin in interphase. These findings provide a novel role for GPER1 in key CRC-prone lesions and shed light on underlying mechanisms that involve GPER1 function in the colon. Elucidating to what extent centrosomal proteins are involved in the GPER1-mediated aneugenic effect will be an important task for future studies. The present study was intended to lay a first foundation to understand the molecular basis and potential risk factors of CRC which might help to reduce the use of laboratory animals. Since numerous animal experiments are conducted in biomedical research, the development of alternative methods is indispensable. The Federal Institute for Risk Assessment (BfR) as the German Center for the Protection of Laboratory Animals (Bf3R) addresses this issue by uncovering underlying mechanisms leading to colorectal cancer as necessary prerequisite in order to develop alternative methods.
Plant metabolism serves as the primary mechanism for converting assimilated carbon into essential compounds crucial for plant growth and ultimately, crop yield. This renders it a focal point of research with significant implications. Despite notable strides in comprehending the genetic principles underpinning metabolism and yield, there remains a dearth of knowledge regarding the genetic factors responsible for trait variation under varying environmental conditions. Given the burgeoning global population and the advancing challenges posed by climate change, unraveling the intricacies of metabolic and yield responses to water scarcity became increasingly important in safeguarding food security.
Our research group has recently started to work on the genetic resources of legume species. To this end, the study presented here investigates the metabolic diversity across five different legume species at a tissue level, identifying species-specific biosynthesis of alkaloids as well as iso-/flavonoids with diverse functional groups, namely prenylation, phenylacylation as well as methoxylation, to create a resource for follow up studies investigation the metabolic diversity in natural diverse populations of legume species.
Following this, the second study investigates the genetic architecture of drought-induced changes in a global common bean population. Here, a plethora of quantitative trait loci (QTL) associated with various traits are identified by performing genome-wide association studies (GWAS), including for lipid signaling. On this site, overexpression of candidates highlighted the induction of several oxylipins reported to be pivotal in coping with harsh environmental conditions such as water scarcity.
Diverging from the common bean and GWAS, the following study focuses on identifying drought-related QTL in tomato using a bi-parental breeding population. This descriptive study highlights novel multi-omic QTL, including metabolism, photosynthesis as well as fruit setting, some of which are uniquely assigned under drought. Compared to conventional approaches using the bi-parental IL population, the study presented improves the resolution by assessing further backcrossed ILs, named sub-ILs.
In the final study, a photosynthetic gene, namely a PetM subunit of the cytochrome b6f complex encoding gene, involved in electron flow is characterized in an horticultural important crop. While several advances have been made in model organisms, this study highlights the transition of this fundamental knowledge to horticultural important crops, such as tomato, and investigates its function under differing light conditions. Overall, the presented thesis combines different strategies in unveiling the genetic components in multi-omic traits under drought using conventional breeding populations as well as a diverse global population. To this end, it allows a comparison of either approach and highlights their strengths and weaknesses.
Starch is an essential biopolymer produced by plants. Starch can be made inside source tissue (such as leaves) and sink tissue (such as fruits and tubers). Nevertheless, understanding how starch metabolism is regulated in source and sink tissues is fundamental for improving crop production.
Despite recent advances in the understanding of starch and its metabolism, there is still a knowledge gap in the source and sink metabolism. Therefore, this study aimed to summarize the state of the art regarding starch structure and metabolism inside plants. In addition, this study aimed to elucidate the regulation of starch metabolism in the source tissue using the leaves of a model organism, Arabidopsis thaliana, and the sink tissue of oil palm (Elaeis guineensis) fruit as a commercial crop.
The research regarding the source tissue will focus on the effect of the blockage of starch degradation on the starch parameter in leaves, especially in those of A. thaliana, which lack both disproportionating enzyme 2 (DPE2) and plastidial glucan phosphorylase 1 (PHS1) (dpe2/phs1). The additional elimination of phosphoglucan water dikinase (PWD), starch excess 4 (SEX4), isoamylase 3 (ISA3), and disproportionating enzyme 1 (DPE1) in the dpe2/phs1 mutant background demonstrates the alteration of starch granule number per chloroplast. This study provides insights into the control mechanism of granule number regulation in the chloroplast.
The research regarding the sink tissue will emphasize the relationship between starch metabolism and the lipid metabolism pathway in oil palm fruits. This study was conducted to observe the alteration of starch parameters, metabolite abundance, and gene expression during oil palm fruit development with different oil yields. This study shows that starch and sucrose can be used as biomarkers for oil yield in oil palms. In addition, it is revealed that the enzyme isoforms related to starch metabolism influence the oil production in oil palm fruit.
Overall, this thesis presents novel information regarding starch metabolism in the source tissue of A.thaliana and the sink tissue of E.guineensis. The results shown in this thesis can be applied to many applications, such as modifying the starch parameter in other plants for specific needs.
The African weakly electric fish genus Campylomormyrus includes 15 described species mostly native to the Congo River and its tributaries. They are considered sympatric species, because their distribution area overlaps. These species generate species-specific electric organ discharges (EODs) varying in waveform characteristics, including duration, polarity, and phase number. They exhibit also pronounced divergence in their snout, i.e. the length, thickness, and curvature. The diversifications in these two phenotypical traits (EOD and snout) have been proposed as key factors promoting adaptive radiation in Campylomormyrus. The role of EODs as a pre-zygotic isolation mechanism driving sympatric speciation by promoting assortative mating has been examined using behavioral, genetical, and histological approaches. However, the evolutionary effects of the snout morphology and its link to species divergence have not been closely examined. Hence, the main objective of this study is to investigate the effect of snout morphology diversification and its correlated EOD to better understand their sympatric speciation and evolutionary drivers. Moreover, I aim to utilize the intragenus and intergenus hybrids of Campylomormyrus to better understand trait divergence as well as underlying molecular/genetic mechanisms involved in the radiation scenario. To this end, I utilized three different approaches: feeding behavior analysis, diet assessment, and geometric morphometrics analysis. I performed feeding behavior experiments to evaluate the concept of the phenotype-environment correlation by testing whether Campylomormyrus species show substrate preferences. The behavioral experiments showed that the short snout species exhibits preference to sandy substrate, the long snout species prefers a stone substrate, and the species with intermediate snout size does not exhibit any substrate preference. The experiments suggest that the diverse feeding apparatus in the genus Campylomormyrus may have evolved in adaptation to their microhabitats. I also performed diet assessments of sympatric Campylomormyrus species and a sister genus species (Gnathonemus petersii) with markedly different snout morphologies and EOD using NGS-based DNA metabarcoding of their stomach contents. The diet of each species was documented showing that aquatic insects such as dipterans, coleopterans and trichopterans represent the major diet component. The results showed also that all species are able to exploit diverse food niches in their habitats. However, comparing the diet overlap indices showed that different snout morphologies and the associated divergence in the EOD translated into different prey spectra. These results further support the idea that the EOD could be a ‘magic trait’ triggering both adaptation and reproductive isolation. Geometric morphometrics method was also used to compare the phenotypical shape traits of the F1 intragenus (Campylomormyrus) and intergenus (Campylomormyrus species and Gnathonemus petersii) hybrids relative to their parents. The hybrids of these species were well separated based on the morphological traits, however the hybrid phenotypic traits were closer to the short-snouted species. In addition, the likelihood that the short snout expressed in the hybrids increases with increasing the genetic distance of the parental species. The results confirmed that additive effects produce intermediate phenotypes in F1-hybrids. It seems, therefore, that morphological shape traits in hybrids, unlike the physiological traits, were not expressed straightforward.