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The ability to sense and respond to different environmental conditions allows living organisms to adapt quickly to their surroundings. In order to use light as a source of information, plants, fungi, and bacteria employ phytochromes. With their ability to detect far-red and red light, phytochromes constitute a major photoreceptor family. Bacterial phytochromes (BphPs) are composed of an apo-phytochrome and an open-chain tetrapyrrole, the chromophore biliverdin IXα, which mediates the photosensory properties. Depending on the photoexcitation and the quality of the incident light, phytochromes interconvert between two photoconvertible parental states: the red light-absorbing Pr-form and the far-red light-absorbing Pfr-form. In contrast to prototypical phytochromes, with a thermal stable Pr ground state, there is a group of bacterial phytochromes that exhibit dark reversion from the Pr- to the Pfr-form. These special proteins are classified as bathy phytochromes and range across different classes of bacteria. Moreover, the majority of BphPs act as sensor histidine kinases in two-component regulatory systems. The light-triggered conformational change results in the autophosphorylation of the histidine kinase domain and the transphosphorylation of an associated response regulator, inducing a cellular response. Spectroscopic analysis utilizing homologously produced protein identified PaBphP, the histidine kinase of the human opportunistic pathogen Pseudomonas aeruginosa, as a bathy phytochrome. Intensive research on PaBphP revealed evidence that the interconversion between its physiological active and inactive states is influenced by light and darkness rather than far-red and red light. In order to conduct a comprehensive systematic analysis, further bacterial phytochromes were investigated regarding their biochemical and spectroscopic behavior, as well as their autokinase activity. In addition to PaBphP, this work employs the bathy phytochromes AtBphP2, AvBphP2, XccBphP from the non-photosynthetic plant pathogens Agrobacterium tumefaciens, Allorhizobium vitis, Xanthomonas campestris, as well as RtBphP2 from the soil bacterium Ramlibacter tataouinensis. All investigated BphPs displayed a bathy-typical behavior by developing a distinct Pr-form under far-red light conditions and undergoing dark reversion to their Pfr-form. Different Pr/Pfr-fractions can be identified among the BphP populations in varying natural light conditions, including red or blue light. The Pr-form is considered as the active form due to autophosphorylation activity in the heterologously produced phytochromes when exposed to light. In the absence of light, associated with the development of the Pfr-form, the phytochromes exhibited disabled or strongly reduced autokinase activity. Additionally, light-triggered phosphorylation was observed for the response regulator PaAlgB, which is linked to the phytochrome of P. aeruginosa. This study presents the first comparative investigation of numerous bathy phytochromes under identical conditions. The work addressed a gap in the literature by providing quantitative correlation between kinase activity and calculated Pr/Pfr-fractions obtained from spectroscopic measurements. The biological role of PaBphP was partially elucidated through phenotypic characterization employing P. aeruginosa mutant and overexpression strains. The generation of a functional model was possible by considering the postulated functions of the other phytochromes found in the literature. In summary, bathy BphPs are hypothesized to modulate bacterial virulence according to the circadian day/night rhythm of their hosts. The pathogens are believed to reduce their virulence during daylight hours to evade immune and defense reactions, while increasing their virulence during the evening and night, enabling more effective infections.
Climate change will have severe consequences on Eastern Boundary Upwelling Systems (EBUS). They host the largest fisheries in the world supporting the life of millions of people due to their tremendous primary production. Therefore, it is of utmost importance to better understand predicted impacts like alternating upwelling intensities and light impediment on the structure and the trophic role of protistan plankton communities as they form the basis of the food web. Numerical models estimate the intensification of the frequency in eddy formation. These ocean features are of particular importance due to their influence on the distribution and diversity of plankton communities and the access to resources, which are still not well understood even to the present day. My PhD thesis entails two subjects conducted during large-scaled cooperation projects REEBUS (Role of Eddies in Eastern Boundary Upwelling Systems) and CUSCO (Coastal Upwelling System in a Changing Ocean).
Subject I of my study was conducted within the multidisciplinary framework REEBUS to investigate the influence of eddies on the biological carbon pump in the Canary Current System (CanCS). More specifically, the aim was to find out how mesoscale cyclonic eddies affect the regional diversity, structure, and trophic role of protistan plankton communities in a subtropical oligotrophic oceanic offshore region.
Samples were taken during the M156 and M160 cruises in the Atlantic Ocean around Cape Verde during July and December 2019, respectively. Three eddies with varying ages of emergence and three water layers (deep chlorophyll maximum DCM, right beneath the DCM and oxygen minimum zone OMZ) were sampled. Additional stations without eddy perturbation were analyzed as references. The effect of oceanic mesoscale cyclonic eddies on protistan plankton communities was analyzed by implementing three approaches. (i) V9 18S rRNA gene amplicons were examined to analyze the diversity and structure of the plankton communities and to infer their role in the biological carbon pump. (ii) By assigning functional traits to taxonomically assigned eDNA sequences, functional richness and ecological strategies (ES) were determined. (iii) Grazing experiments were conducted to assess abundance and carbon transfer from prokaryotes to phagotrophic protists.
All three eddies examined in this study differed in their ASV abundance, diversity, and taxonomic composition with the most pronounced differences in the DCM. Dinoflagellates were the most abundant taxa in all three depth layers. Other dominating taxa were radiolarians, Discoba and haptophytes. The trait-approach could only assign ~15% of all ASVs and revealed in general a relatively high functional richness. But no unique ES was determined within a specific eddy. This indicates pronounced functional redundancy, which is recognized to be correlated with ecosystem resilience and robustness by providing a degree of buffering capacity in the face of biodiversity loss. Elevated microbial abundances as well as bacterivory were clearly associated to mesoscale eddy features, albeit with remarkable seasonal fluctuations. Since eddy activity is expected to increase on a global scale in future climate change scenarios, cyclonic eddies could counteract climate change by enhancing carbon sequestration to abyssal depths. The findings demonstrate that cyclonic eddies are unique, heterogeneous, and abundant ecosystems with trapped water masses in which characteristic protistan plankton develop as the eddies age and migrate westward into subtropical oligotrophic offshore waters. Therefore, eddies influence regional protistan plankton diversity qualitatively and quantitatively.
Subject II of my PhD project contributed to the CUSCO field campaign to identify the influence of varying upwelling intensities in combination with distinct light treatments on the whole food web structure and carbon pump in the Humboldt Current System (HCS) off Peru. To accomplish such a task, eight offshore-mesocosms were deployed and two light scenarios (low light, LL; high light, HL) were created by darkening half of the mesocosms. Upwelling was simulated by injecting distinct proportions (0%, 15%, 30% and 45%) of collected deep-water (DW) into each of the moored mesocosms. My aim was to examine the changes in diversity, structure, and trophic role of protistan plankton communities for the induced manipulations by analyzing the V9 18S rRNA gene amplicons and performing short-term grazing experiments.
The upwelling simulations induced a significant increase in alpha diversity under both light conditions. In austral summer, reflected by HL conditions, a generally higher alpha diversity was recorded compared to the austral winter simulation, instigated by LL treatment. Significant alterations of the protistan plankton community structure could likewise be observed. Diatoms were associated to increased levels of DW addition in the mimicked austral winter situation. Under nutrient depletion, chlorophytes exhibited high relative abundances in the simulated austral winter scenario. Dinoflagellates dominated the austral summer condition in all upwelling simulations. Tendencies of reduced unicellular eukaryotes and increased prokaryotic abundances were determined under light impediment. Protistan-mediated mortality of prokaryotes also decreased by ~30% in the mimicked austral winter scenario.
The findings indicate that the microbial loop is a more relevant factor in the structure of the food web in austral summer and is more focused on the utilization of diatoms in austral winter in the HCS off Peru. It was evident that distinct light intensities coupled with multiple upwelling scenarios could lead to alterations in biochemical cycles, trophic interactions, and ecosystem services. Considering the threat of climate change, the predicted relocation of EBUS could limit primary production and lengthen the food web structure with severe socio-economic consequences.
Living systems incessantly engage in the regulation of their cellular processes to fulfill their biological functions. Beyond development-related adjustments or cell cycle oscillations, environmental fluctuations compel the system to reorganize metabolic pathways, structural components, or molecular repair and reconstitution mechanisms. These responses manifest across diverse temporal scales, necessitating an intricate regulatory orchestration. Time series experiments have become increasingly popular for charting the chronological order and elucidating the underlying mechanisms. In the era of high-throughput technologies, the majority of cellular molecules can be analyzed in one fell swoop, generating a comprehensive snapshot of the status quo of most present molecules. Methodological advancements also permit the monitoring not only of molecular abundances but also the functional status of transcripts and proteins. However, due to the still high efforts associated with such experiments, the number of measured time points and the replication of measurements remains limited. Resulting datasets contain signals from thousands of molecules, yet they are sparse in temporal resolution and are often imprecise due to biological variability and technical measurement inaccuracies.
This thesis explores the complexities arising from the examination of short time series data and introduces pioneering tools that offer fresh insights into the realm of biological time series analysis. The broad spectrum of analytic possibilities ranges from a molecule-centric investigation of individual time courses to a holistic aggregation of the system’s response to its main characteristics. By creating a modeling framework that applies domain-specific constraints, time-course signals can be transformed from a series of discrete data points into a continuous curve. These curves align with current biological conjectures about molecule kinetics being smooth and devoid of superfluous oscillations. Noise present at individual time points is judiciously accounted for during curve fitting, mitigating the impact of time points with high variance on the curve. Subsequent classification is based on the features of these curves (extreme points and inflection points) and ensures a reduction in data amount and complexity. Succinct labels assigned to each molecule's kinetics encapsulate the signal's most notable features. Besides this modeling approach, an innovative enrichment strategy is introduced, that is independent of prior data partitioning and capable of segregating the temporal response into its thermodynamically relevant components. This approach allows for a continuous assessment of each molecule's contribution to these components, obviating the need for exclusive allocation. The application of various analytical approaches to heat acclimation experiments in Chlamydomonas highlights the relevance and potential of time series experiments and specifically tailored analysis techniques. The integration of different system levels has led to the identification of regulatory peculiarities, such as an increased correlation between transcripts and corresponding proteins during acclimation responses. These and other insights may herald new avenues of research that could ultimately enhance plant robustness in the face of increasing environmental perturbations.
The growing popularity of time series experiments necessitates dedicated analytical approaches that empower researchers and analysts to decipher patterns, discern trends, and unravel the underlying structures within the data, facilitating predictions and the derivation of meaningful conclusions that could potentially build bridges between the interweaved systems levels.
Regulation of sucrose transport between source and sink tissues is critical for plant development and properties. In cells, the dynamic vacuolar sugar homeostasis is maintained by the controlled regulation of the activities of sugar importers and exporters residing in the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 protein, being the closest homolog to the proton/glucose symporter ERDL6, resides in the vacuolar membrane. We raised both, molecular expression and data deriving from non-aqueous fractionation studies indicating that ERDL4 was involved in glucose and fructose allocation across the tonoplast. Surprisingly, overexpression of ERDL4 increased total sugar levels in leaves, which is due to a concomitantly induced stimulation of TST2 expression, coding for the major vacuolar sugar loader. This conclusion is supported by the notion that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. That ERDL4 activity contributes to the coordination of cellular sugar homeostasis is further indicated by two observations. Firstly, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, secondly, the ERDL4 gene is markedly expressed during cold acclimation representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger size of rosettes and roots, a delayed flowering and increased total seed yield. In summary, we identified a novel factor influencing source to sink transfer of sucrose and by this governing plant organ development.
Omics-basierte Untersuchungen zum Wirkmechanismus des makrozyklischen Lactons Oxacyclododecindion
(2023)
Die Suche nach neuen therapeutischen Wirkstoffen ist, aufgrund der geringen Erfolgsquote vor allem
in späteren Phasen der Zulassung, nach wie vor eine große Herausforderung. Für einen möglichst
effizienten Entwicklung- und Zulassungsprozess zu erhöhen, ist eine möglichst genaue
Charakterisierung des Moleküls und der davon ausgehenden biologischen Aktivität sinnvoll. Eine
wertvolle Quelle an potenziellen Wirkstoffen stellen Sekundärmetaboliten aus Bakterien, Algen,
Pflanzen oder Pilzen dar. Im Rahmen dieser Arbeit wurden verschiedene, aus Pilzen isolierte Stoffe
näher untersucht, die durch eine phänotypische Wirkstoffsuche gefunden wurden. Im Gegensatz zu
einer Target-basierten Wirkstoffsuche, wird sich bei dieser Methode nicht auf ein bestimmtes
Zielprotein fokussiert, sondern in einem zellbasierten System nach Stoffen gesucht, die einen
phänotypischen Effekt hervorrufen. Das genaue Target bleibt daher zunächst unbekannt.
Hauptsächlich wurde das Makrolacton Oxacyclododecindion (Oxa) bzw. dessen Derivate untersucht.
In phänotypischen Analysen stellte sich Oxa bereits in der Vergangenheit als vielversprechender
Wirkstoff zur Behandlung inflammatorischer und fibrotischer Erkrankungen heraus. Verschiedene
Derivate des Naturstoffs wurden durch die Abteilung für Organische Chemie der Johannes
Gutenberg-Universität Mainz synthetisiert und im Rahmen dieser Arbeit auf ihre biologische Aktivität
hin untersucht. So konnte der Einfluss verschiedener Derivatisierungen auf die biologische Aktivität
analysiert und mit bereits publizierten Derivaten verglichen werden. So konnte die optimale Position
für weitere Funktionalisierungen des Moleküls gefunden werden.
Neben der Analyse globaler Effekte auf BEAS-2B und HepG2 stand vor allem die Identifikation
möglicher Targets im Vordergrund. Dazu wurden zunächst verschiedene Ansätze zur Identifikation
von Proteinen, die an das besonders aktive Derivat 14-Deoxy-14-methyloxacyclododecindion (DM-Oxa) binden, durchgeführt. So konnten bereits erste Kandidaten, insbesondere das
Hitzeschockprotein HSP90, als mögliche Interaktoren identifiziert werden. Dieses Protein hat einen
zentralen Stellenwert in zahlreichen zellulären Prozessen, wodurch es bereits als mögliches Target
für verschiedene Erkrankungen bekannt ist.
Um erstmals einen Einblick in die globalen Effekte von DM-Oxa auf die Genexpression und das
Proteom in verschiedenen Zelltypen zu erhalten, wurden sowohl mRNA-Sequenzierungs- als auch
MS-Proteomics-Ansätze durchgeführt. Somit konnten bereits bekannte Charakteristika verifiziert und
neue Einblicke in beteiligte Signalwege gewonnen werden. Im Rahmen dieser Analysen konnten
potenzielle Zielproteine von DM-Oxa identifiziert werden. Darunter beispielsweise die zentralen
Transkriptionsfaktoren FOXO3 und AP-1.
Auch die Rolle von HSP90 als möglicher Interaktionspartner von DM-Oxa wurde weiter beleuchtet.
Dazu wurde eine Co-Immunpräzipitation mit HSP90 Antikörpern durchgeführt, um Veränderungen in
der Abundanz von Proteinen, die an HSP90 binden, festzustellen. Solche Veränderungen könnten
auf die Blockade von Bindestellen durch DM-Oxa hinweisen. Tatsächlich wurde die Abundanz von
100 bekannten Interaktionspartnern durch DM-Oxa Behandlung signifikant beeinflusst, sodass die
Rolle von HSP90 als Oxa-Target weiter ins Zentrum rückt.
Neben der Analyse von Oxacyclododecindion und verschiedenen Derivaten wurden zwei weitere
bisher unveröffentlichte Naturstoffe aus einem Pilz der Gattung Alternaria näher untersucht. Diese
gehören zu Klasse der Perylenchinone und zeigten zum Teil toxische und oxidative Wirkung in
BEAS-2B-Zellen, was durch verschiedene Versuchsansätze nachgewiesen werden konnte.
Chromosomal aberrations are manifold changes in the configuration of the DNA. Each cell in a tumor
may accumulate different karyotype changes, making it challenging to determine the causes and
consequences of this instability. Therefore, model systems have been developed in the past to
generate and study specific genome alterations. In this thesis, I present the results of my studies on
three types of chromosomal aberrations, all of which may contribute to tumor development or
progression.
Chromothripsis is a phenomenon that describes a one-off massive chromosomal disruption and
reassembly, perhaps arising via DNA damage micronuclei (MN). MN are small DNA-packed nuclear
envelopes. I tested potential causes of DNA damage in MN and found that the rupture of the MN
envelope and the entry of cytosolic fractions increase DNA damage in MN. Furthermore, I addressed
the question of what physiological consequences cell lines with an additional rearranged chromosome
have compared to those with an intact extra chromosome. Strikingly, the cells with more
rearrangements showed a functional advantage resulting in an improved fitness potential.
However, the engineering of polysomic cell lines with fully intact additional chromosomes increases
various cellular stress responses and reduces the proliferation capacity. To investigate how cancer cells
overcome the detrimental consequences of aneuploidy, I explored physiological adaptations of model
cells with a defined additional chromosome that underwent in vivo and in vitro evolution. Interestingly,
unfavorable phenotypes of aneuploid cells, such as the replication stress, were mitigated upon
evolution. Furthermore, I examined the replication on single molecule resolution, showing alteration
after evolution that might underlie the replication stress bypass or tolerance.
In contrast to these unbalanced forms of genomic aberrations, whole genome doubling (WGD) leads
to a full doubled chromosome set, which was shown to evolve into aneuploid karyotypes by
chromosomal instability (CIN), frequently by losing chromosomes. Cells that underwent WGD
accumulate DNA damage in the S phase. I performed a single molecule analysis on the DNA during the
first cell cycle after WGD to elucidate how the DNA damage arises and found that the number of active
origins is not sufficient to replicate the doubled amount of DNA in the first S phase after WGD faithfully.
This starts a genome-destabilizing cascade that eventually promotes tumorigenesis, metastasis, and
poor patient outcome.
Taken together, these studies provide insights into the causes and consequences of three types of
genomic aberrations: chromothripsis, polysomy, and WGD. However different these phenomena may
be, they share one common feature – they contribute to tumor development and progression.
Therefore, elucidating the aberrant cell functions caused by genomic aberrations contributes to a
better understanding of a cancer cell's nature and will perhaps help to find new cancer therapy targets.
Phycobilisomes (PBS) are the major light-harvesting complexes for the majority of cyanobacteria
and allow these organisms to absorb in the so-called green gap. They consist of smaller units called
phycobiliproteins (PBPs), which are composed of an α- and a β-subunit with covalently bound
linear tetrapyrroles (phycobilins). The latter are attached to the apo-PBPs by phycobiliprotein
lyases. Interestingly, cyanobacteria of the genus Prochlorococcus lack complete PBS and instead
use prochlorophyte chlorophyll-binding proteins (Pcbs), which effectively utilize the energy of the
blue light region. The low-light-adapted (LL) strain Prochlorococcus marinus SS120 has a single
PBP, phycoerythrin-III (PE-III). It has been postulated that PE-III is chromophorylated with the
phycobilins phycourobilin (PUB) and phycoerythrobilin (PEB) in a 3:1 ratio. Thereby, the function
of PE-III remains unclear so far, so that light-gathering function and also photoreceptor function
are discussed.
The main goal of this work was to characterize the assembly of PE-III and thus the function of the
six putative phycobiliprotein lyases of P. marinus SS120. Previous work found that the individual
lyases could not be produced in soluble form, so we switched to a dual pDuet™ plasmid system in
E. coli, which was successfully established. Investigation of the binding of PEB to Apo-PE
revealed that the CpeS lyase specifically chromophorylated Cys82 with 3Z-PEB. Unfortunately,
additional chromophorylation could not be observed using the pDuet system. Therefore, in a
second part of the work, the entire PE gene cluster from P. marinus SS120 was to be introduced
into E. coli and expressed. Although the gene cluster was successfully transcribed within E. coli,
no translation was observed, possibly due to incompatible translation initiation between
Prochlorococcus and E. coli. The introduction of a mini PE cluster (CpeAB) into the
cyanobacterium Synechococcus sp. PCC 7002 was also successfully performed, in which case
production of CpeB but not CpeA from Prochlorococcus was detected. Recombinant CpeB was
also detected together with intrinsic PBP in Synechococcussp. 7002, indicating structural similarity
and incorporation into PBS in Synechococcus sp. 7002. Overall, the obtained results suggest that a
cyanobacterial host is a good option for the studies on the assembly of PE-III from P. marinus and,
based on this, future work could aim at generating an artificial operon using synthetic biology to
achieve efficient translation of all genes.
Die steigende Verfügbarkeit von Smartphones und Tablet-PCs in der Schule bieten neue methodische und mediale Wege der Unterrichtsgestaltung, die zur Beurteilung der Lernwirksamkeit eine wissenschaftliche Betrachtung erfordern. Während zahlreiche Publikationen zu Augmented Reality (AR) im Bildungskontext existieren, fehlt es an einer differenzierten Betrachtung der Lernwirksamkeit AR-typischer Merkmale sowie breit angelegter Untersuchungsdesigns. Ziel dieser Arbeit ist es, die Lernwirksamkeit von AR in authentischen biologischen Unterrichtsszenarien multiperspektivisch zu betrachten. Zur Beurteilung der Lernwirksamkeit wurden Daten zu Lernzuwachs (LZ), Cognitive Load (CL), Nutzungserlebnis (UX), empfundener Lernunterstützung (ELU) sowie zur Immersion gemessen. Die Forschungsfragen beziehen sich auf den Einfluss der Art des Mediums, der Steuerung, des Triggers und der medialen Repräsentation auf die Lernwirksamkeit.
Die Untersuchungen wurden mithilfe eigens entwickelnder AR-Apps mit 769 Teilnehmenden aus rheinland-pfälzischen Gymnasien und zwei Universitäten durchgeführt. Neben dem Nachweis theoretischer Zusammenhänge der untersuchten Parameter mittels Strukturgleichungsmodellierung konnten mehrheitlich signifikante Unterschiede im LZ zugunsten von AR festgestellt werden. Darüber hinaus zeigten die Studien dieser Arbeit einen positiven Einfluss von AR auf den CL, sodass sich der Einsatz von AR nicht nachteilig auf die kognitive Belastung auswirkt. Neben des überdurchschnittlich bis exzellenten Abschneidens der AR-Apps im Benchmark-Vergleich (Vergleichsgruppe n = 20190 (Schrepp, 2019)), konnten positive Effekte der UX-Dimension Stimulation auf die Reduktion des lernhinderlichen Extraneous Cognitive Loads und die Steigerung des lernförderlichen Germane Cognitive Loads nachgewiesen werden. Hinsichtlich der ELU zeigten sich verschiedene mediale Präferenzen der Teilnehmenden, sodass durch die Verwendung von AR im Sinne eines wechselnden Medieneinsatzes die Bedürfnisse aller Lernenden abgedeckt werden kann. Weiterhin erreichten die Teilnehmenden die höchste der drei Ebenen der Immersion, sodass die Art des Triggers die 21 Immersionsfaktoren von Georgiou und Kyza (2017a) ergänzt.
Die Arbeit leistet auf Grundlage der Identifikation von AR-typischen Merkmalen einen Beitrag zum besseren Verständnis der lernwirksamen Potenziale von AR-basierten Lernumgebungen und zeigt darüber hinaus theoriebildende Implikationen zur Messung des CL auf. Ob sich mithilfe von AR der CL senken lässt, bedarf es Untersuchungen in Lernsettings, die einen hohen CL aufweisen. Weiterhin bietet der ARI-Fragebogen einen geeigneten Ausgangspunkt zur Erforschung AR-typischer Immersionsfaktoren. Trotzdem bedarf es weiterer Studien zur Validierung des ARI-Fragebogens und zur systematischen Untersuchung der Lernwirksamkeit von AR-typischen Merkmalen.
The vast majority of all mitochondrial proteins are synthesized in the cytosol. These proteins carry characteristic targeting motifs within their sequence, which allows for the binding of chaperones, that in turn usher precursors to the mitochondrial surface for import and assembly. Though, our understanding of these early reactions is still lacking, recent efforts have shown that the ER surface can facilitate the import of mitochondrial proteins (ER-SURF) with the help of the J-protein Djp1. Close cooperation of organelles in form of membrane contact sites is crucial for cellular function. The aim of my work was to investigate whether ER-mitochondria contact sites are critical for the transfer of proteins from the ER to mitochondria.
Several contact sites have been characterized between ER and mitochondria in S. cerevisiae. One contact site is called the ER mitochondria encounter structure (ERMES) and another is partly formed by Tom70. Owing to the high propensity of suppressor mutations in ERMES, I employed a knockdown approach to deplete this contact site. Using an inducible CRISPR interference (CRISPRi) system, I could rapidly and efficiently deplete Mdm34, which is a part of ERMES. I could show that depletion of Mdm34 had a synthetic negative effect in combination with a deletion of TOM70. Loss of both contact sites led to a strong decrease of many mitochondrial proteins in the whole cell proteome. Using affinity purification of ER and mitochondria in conjunction with mass spectrometry I could demonstrate that a specific set of mitochondrial proteins are enriched on the ER upon loss of Mdm34 and Tom70, which mainly were proteins of the inner membrane e.g., Oxa1 and Cox5A. Moreover, I was able to validate that the import of these proteins was hampered upon loss of both contact sites. Also, in vivo the biogenesis of Oxa1 was impeded upon single loss of Mdm34 or Tom70 and strongly impaired if both were lost. Analysis of the maximum hydrophobicity of inner membrane proteins in the ER-SURF set revealed on average a significantly higher peak compared to other inner membrane proteins. I could show that deleting or swapping the transmembrane domain of Cox5A would make it contact site independent or reliant on contact sites respectively, as revealed by an in vitro import assay.
In this study I was able to demonstrate the involvement of membrane contact sites in ER-SURF and identify a list of putative clients. Furthermore, I could show that hydrophobicity of the transmembrane segment of inner membrane proteins is one determinant for ER-SURF dependence.
Drought is a significant environmental factor that can impair plant growth and development, leading to reduced crop productivity or even plant death. Maintaining sugar distribution from source to sink is crucial for increasing crop production under water limitation conditions. Numerous studies have suggested that nutrition fertilization, especially potassium (K), can enhance plant growth and yield production. To investigate the mechanism of K in sugar long-distance transportation under drought stress, we established a soil-grow system and a hydroponic-grow system with varying amounts of potassium supplementation and analyzed the biochemical and molecular responses in Arabidopsis and potato plants under drought stress conditions. Our findings showed that excess potassium fertilization limited sucrose metabolism, leading to lower drought tolerance in Arabidopsis in both grow systems. However, higher potassium supplementation altered sugar relocation and potassium movement, resulting in an increase in starch yield production in both potato plants with different sink strength capacities. We also proposed that a low amount of sodium increases Arabidopsis drought tolerance under low potassium conditions since a low amount of sodium can improve the control of osmotic potential, leading to more water being retained in plant cells.
Silicon (Si) has received considerable attention recently for its potential in mitigating drought stress, although the effects vary among different plant species. To investigate the mechanism of Si in drought stress tolerance, we applied monosilicic acid in hydroponic media and then applied PEG8000 to simulate drought stress. Our findings revealed that Si-dependent drought mitigation occurred more in the shoot than in the root of Arabidopsis, and we observed silicon accumulation in the shoot of Arabidopsis. In Si-treated plants, more glucose was accumulated in the vacuole, leading to better osmotic potential control under drought stress. RNA sequencing analysis showed that Si altered the activity of sugar transporters and the sugar metabolism process, and increased photosynthesis. However, Si-dependent regulation in sugar transporter showed different responses in potato. Understanding the mechanism of Si in potato requires further studies. Overall, our dissertation provides important information for clarifying the mechanism of Si in drought stress, which forms the basis for further investigation.