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Biochemical and cellular characterization of filamin binding proteins in cross striated muscle
(2006)
Besiedlung naturnaher Wälder unterschiedlicher Isolation durch Tiergruppen verschiedener Mobilität
(2005)
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.
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.
Application of hybridisation capture to investigate complete mitogenomes from ancient samples
(2015)
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.
Analysis of structure and variability of major histocompatibility complex in different vertebrates
(2010)
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.
Rubisco catalyses the first step of CO2 assimilation into plant biomass. Despite its crucial role, it is notorious for its low catalytic rate and its tendency to fix O2 instead of CO2, giving rise to a toxic product that needs to be recycled in a process known as photorespiration. Since almost all our food supply relies on Rubisco, even small improvements in its specificity for CO2 could lead to an improvement of photosynthesis and ultimately, crop yield. In this work, we attempted to improve photosynthesis by decreasing photorespiration with an artificial CCM based on a fusion between Rubisco and a carbonic anhydrase (CA).
A preliminary set of plants contained fusions between one of two CAs, bCA1 and CAH3, and the N- or C-terminus of RbcL connected by a small flexible linker of 5 amino acids. Subsequently, further fusion proteins were created between RbcL C-terminus and bCA1/CAH3 with linkers of 14, 23, 32, and 41 amino acids. The transplastomic tobacco plants carrying fusions with bCA1 were able to grow autotrophically even with the shortest linkers, albeit at a low rate, and accumulated very low levels of the fusion protein. On the other hand, plants carrying fusions with CAH3 were autotrophic only with the longer linkers. The longest linker permitted nearly wild-type like growth of the plants carrying fusions with CAH3 and increased the levels of fusion protein, but also of smaller degradation products.
The fusion of catalytically inactive CAs to RbcL did not cause a different phenotype from the fusions with catalytically active CAs, suggesting that the selected CAs were not active in the fusion with RbcL or their activity did not have an effect on CO2 assimilation. However, fusions to RbcL did not abolish RbcL catalytic activity, as shown by the autotrophic growth, gas exchange and in vitro activity measurements. Furthermore, Rubisco carboxylation rate and specificity for CO2 was not altered in some of the fusion proteins, suggesting that despite the defect in RbcL folding or assembly caused by the fusions, the addition of 60-150 amino acids to RbcL does not affect its catalytic properties. On the contrary, most growth defects of the plants carrying RbcL-CA fusions are related to their reduced Rubisco content, likely caused by impaired RbcL folding or assembly. Finally, we found that fusions with RbcL C-terminus were better tolerated than with the N-terminus, and increasing the length of the linker relieved the growth impairment imposed by the fusion to RbcL. Together, the results of this work constitute considerable relevant findings for future Rubisco engineering.
Analysis of phosphorylation dynamics under nitrogen limitation and nitrate or ammonium resupply
(2009)
Analysis of organellar genome dynamics and development of methods for organelle transformation
(2010)
Analyse der Funktion der dualen Lokalisation der 3-Mercaptopyruvat Sulfurtransferase im Menschen
(2017)
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.
The importance of cryptic diversity in rotifers is well understood regarding its ecological consequences, but there remains an in depth comprehension of the underlying molecular mechanisms and forces driving speciation. Temperature has been found several times to affect species spatio-temporal distribution and organisms’ performance, but we lack information on the mechanisms that provide thermal tolerance to rotifers. High cryptic diversity was found recently in the freshwater rotifer “Brachionus calyciflorus”, showing that the complex comprises at least four species: B. calyciflorus sensu stricto (s.s.), B. fernandoi, B. dorcas, and B. elevatus. The temporal succession among species which have been observed in sympatry led to the idea that temperature might play a crucial role in species differentiation.
The central aim of this study was to unravel differences in thermal tolerance between species of the former B. calyciflorus species complex by comparing phenotypic and gene expression responses. More specifically, I used the critical maximum temperature as a proxy for inter-species differences in heat-tolerance; this was modeled as a bi-dimensional phenotypic trait taking into consideration the intention and the duration of heat stress. Significant differences on heat-tolerance between species were detected, with B. calyciflorus s.s. being able to tolerate higher temperatures than B. fernandoi.
Based on evidence of within species neutral genetic variation, I further examined adaptive genetic variability within two different mtDNA lineages of the heat tolerant B. calyciflorus s.s. to identify SNPs and genes under selection that might reflect their adaptive history. These analyses did not reveal adaptive genetic variation related to heat, however, they show putatively adaptive genetic variation which may reflect local adaptation. Functional enrichment of putatively positively selected genes revealed signals of adaptation in genes related to “lipid metabolism”, “xenobiotics biodegradation and metabolism” and “sensory system”, comprising candidate genes which can be utilized in studies on local adaptation. An absence of genetically-based differences in thermal adaptation between the two mtDNA lineages, together with our knowledge that B. calyciflorus s.s. can withstand a broad range of temperatures, led to the idea to further investigate shared transcriptomic responses to long-term exposure to high and low temperatures regimes. With this, I identified candidate genes that are involved in the response to temperature imposed stress. Lastly, I used comparative transcriptomics to examine responses to imposed heat-stress in heat-tolerant and heat-sensitive Brachionus species. I found considerably different patterns of gene expression in the two species. Most striking are patterns of expression regarding the heat shock proteins (hsps) between the two species. In the heat-tolerant, B. calyciflorus s.s., significant up-regulation of hsps at low temperatures was indicative of a stress response at the cooler end of the temperature regimes tested here. In contrast, in the heat-sensitive B. fernandoi, hsps generally exhibited up-regulation of these genes along with rising temperatures. Overall, identification of differences in expression of genes suggests suppression of protein biosynthesis to be a mechanism to increase thermal tolerance. Observed patterns in population growth are correlated with the hsp gene expression differences, indicating that this physiological stress response is indeed related to phenotypic life history performance.
Adaptation of nature conservation to global change: an ecosystem-based approach to priority-setting
(2013)
Leaves exhibit cells with varying degrees of shape complexity along the proximodistal axis. Heterogeneities in growth directions within individual cells bring about such complexity in cell shape. Highly complex and interconnected gene regulatory networks and signaling pathways have been identified to govern these processes. In addition, the organization of cytoskeletal networks and cell wall mechanical properties greatly influences the regulation of cell shape. Research has shown that microtubules are involved in regulating cellulose deposition and direc-tion of cell growth. However, comprehensive analysis of the regulation of the actin cytoskele-ton in cell shape regulation has not been well studied.
This thesis provides evidence that actin regulates aspects of cell growth, division, and direction-al expansion that impacts morphogenesis of developing leaves. The jigsaw puzzle piece mor-phology of epidermal pavement cells further serves as an ideal system to investigate the com-plex process of morphogenetic processes occurring at the cellular level. Here we have em-ployed live cell based imaging studies to track the development of pavement cells in actin com-promised conditions. Genetic perturbation of two predominantly expressed vegetative actin genes ACTIN2 and ACTIN7 results in delayed emergence of the cellular protrusions in pave-ment cells. Perturbation of actin also impacted the organization of microtubule in these cells that is known to promote emergence of cellular protrusions. Further, live-cell imaging of actin or-ganization revealed a correlation with cell shape, suggesting that actin plays a role in influencing pavement cell morphogenesis.
In addition, disruption of actin leads to an increase in cell size along the leaf midrib, with cells being highly anisotropic due to reduced cell division. The reduction of cell division further im-pacted the morphology of the entire leaf, with the mutant leaves being more curved. These re-sults suggests that actin plays a pivotal role in regulating morphogenesis at the cellular and tis-sue scales thereby providing valuable insights into the role of the actin cytoskeleton in plant morphogenesis.