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Plants have evolved many mechanisms to defend against herbivores and pathogens. In many cases, these mechanisms took other duties. One example of such a neofunction- alisation would be carnivory. Carnivory evolved from the defence against herbivores. Instead of repelling the predator with a bitter taste, the plant kills it and absorbs its nutrients. A second example can be found in the pollination process. Many of the genes involved here were originally part of defence mechanisms against pathogens. In this thesis, I study these two examples on a genomic and transcriptomic level. The first project, Genomics of carnivorous Droseraceae, aims at obtaining annotated genome sequences of three carnivorous plants. I assembled the genome of Aldrovanda vesiculosa, annotated those of A. vesiculosa, Drosera spatulata and Dionaea muscipula and com- pared their genomic contents. Because of the high repetitiveness of the D. muscipula genome, I also developed reper, an assembly free method for detection, classification and quantification of repeats. With that method, we were able to study the repeats without the need of incorporating them into a genome assembly. The second large project investigates the role of DEFL (defensin-like) genes in pollen tube guidance in tobacco flowers. We sequenced the transcriptome of the SR1 strain in different stages of the pollination process. I assembled and annotated the transcriptome and searched for differentially expressed genes. We also used a method based on Hidden- Markov-Models (HMM) to find DEFLs, which I then analysed regarding their expression during the different stages of fertilisation. In total, this thesis results in annotated genome assemblies of three carnivorous Droser- aceae, which are used as a foundation for various analyses investigating the roots of car- nivory, insights into the role of DEFLs on a transcriptomic level in tobacco pollination and a new method for repeat identification in complex genomes.
The Venus flytrap, \textit{Dionaea muscipula}, with its carnivorous life-style and its highly
specialized snap-traps has fascinated biologist since the days of Charles Darwin. The
goal of the \textit{D. muscipula} genome project is to gain comprehensive insights into the
genomic landscape of this remarkable plant.
The genome of the diploid Venus flytrap with an estimated size between 2.6 Gbp to
3.0 Gbp is comparatively large and comprises more than 70 % of repetitive regions.
Sequencing and assembly of genomes of this scale are even with state-of-the-art
technology and software challenging. Initial sequencing and assembly of the genome
was performed by the BGI (Beijing Genomics Institute) in 2011 resulting in a 3.7 Gbp
draft assembly. I started my work with thorough assessment of the delivered assembly
and data. My analysis showed that the BGI assembly is highly fragmented and
at the same time artificially inflated due to overassembly of repetitive sequences.
Furthermore, it only comprises about on third of the expected genes in full-length,
rendering it inadequate for downstream analysis.
In the following I sought to optimize the sequencing and assembly strategy to obtain
an assembly of higher completeness and contiguity by improving data quality and
assembly procedure and by developing tailored bioinformatics tools. Issues with
technical biases and high levels of heterogeneity in the original data set were solved
by sequencing additional short read libraries from high quality non-polymorphic DNA
samples. To address contiguity and heterozygosity I examined numerous alternative
assembly software packages and strategies and eventually identified ALLPATHS-LG
as the most suited program for assembling the data at hand. Moreover, by utilizing
digital normalization to reduce repetitive reads, I was able to substantially reduce
computational demands while at the same time significantly increasing contiguity of
the assembly.
To improve repeat resolution and scaffolding, I started to explore the novel PacBio
long read sequencing technology. Raw PacBio reads exhibit high error rates of 15 %
impeding their use for assembly. To overcome this issue, I developed the PacBio
hybrid correction pipeline proovread (Hackl et al., 2014). proovread uses high
coverage Illumina read data in an iterative mapping-based consensus procedure to
identify and remove errors present in raw PacBio reads. In terms of sensitivity and
accuracy, proovread outperforms existing software. In contrast to other correction
programs, which are incapable of handling data sets of the size of D. muscipula
project, proovread’s flexible design allows for the efficient distribution of work load on high-performance computing clusters, thus enabling the correction of the Venus
flytrap PacBio data set.
Next to the assembly process itself, also the assessment of the large de novo draft
assemblies, particularly with respect to coverage by available sequencing data, is
difficult. While typical evaluation procedures rely on computationally extensive
mapping approaches, I developed and implemented a set of tools that utilize k-mer
coverage and derived values to efficiently compute coverage landscapes of large-scale
assemblies and in addition allow for automated visualization of the of the obtained
information in comprehensive plots.
Using the developed tools to analyze preliminary assemblies and by combining my
findings regarding optimizations of the assembly process, I was ultimately able to
generate a high quality draft assembly for D. muscipula. I further refined the assembly
by removal of redundant contigs resulting from separate assembly of heterozygous
regions and additional scaffolding and gapclosing using corrected PacBio data. The
final draft assembly comprises 86 × 10 3 scaffolds and has a total size of 1.45 Gbp.
The difference to the estimated genomes size is well explained by collapsed repeats.
At the same time, the assembly exhibits high fractions full-length gene models,
corroborating the interpretation that the obtained draft assembly provides a complete
and comprehensive reference for further exploration of the fascinating biology of the
Venus flytrap.
Bacteria thrive and survive in many different environments, and as a result, they have developed robust mechanisms to adapt rapidly to alterations in their surroundings. The protection against osmotic forces is provided by mechanosensitive channels: their primary function is to maintain the integrity of the cell upon a hypoosmotic shock. The mechanosensitive channel of small conductance (MscS) is not only the smallest common structural unit of a diverse family that allows for a tailored response in osmoregulation; it is also the most intensively studied homologue. Mechanosensitive channels directly sense elevated membrane tension levels generated by increased pressure within the cell and open transiently. Escherichia coli has six paralogues that differ in their gating properties and the number of additional transmembrane (TM) helices. These TM helices, termed sensor paddles, are essential for sensing, as they directly contact the surrounding membrane; however, the role of the additional TM helices is still unclear. Furthermore, lipids occupy hydrophobic pockets far away from the membrane plane. A recent gating model for MscS states that increased membrane tension triggers the expulsion of lipids out of those pockets, modulating different conformational states of MscS. This model focuses on bound lipids, but it is still unclear to what extent the direct interaction with the membrane influences sensing and how relevant it is for the larger paralogues.
In the herein described work, structural studies on two larger paralogues, the medium-sized channel YnaI and the large channel YbiO were realised using electron cryomicroscopy (cryo-EM). Lipids were identified in YnaI in the pockets in a similar position and orientation as in MscS, suggesting a conserved sensing mechanism. Moreover, the copolymer diisobutylene/maleic acid (DIBMA) allowed the extraction of artificially activated YnaI from plasma membranes, leading to an open-like form of this channel. This novel conformation indicated that the pore helices bend at a GGxGG motif during gating, which is unique among the Escherichia coli paralogues, concomitant with a structural reorganisation of the sensor paddles. Thus, despite a high similarity of their closed states, the gating mechanisms of MscS and YnaI are surprisingly different. Furthermore, the comparison of MscS, YnaI, and YbiO accentuates variations and similarities between the differently sized family members, implying fine-tuning of channel properties in the pore regions and the cytosolic lateral entry sides into the channel. Structural analyses of MscS reconstituted into different systems showed the advantages and disadvantages of certain polymers and detergents. The novel DIBMA copolymer and the more conventional amphiphilic polymers, so-called Amphipols, perturb contacting transmembrane helices or lead to their denaturation. Due to this observation, the obtained structures of YnaI must also be cautiously considered. The structures obtained in detergents resulted in unaffected channels; however, the applicability of detergents for MscS-like channels is limited by the increased required sample concentration.
The role of lipids for gating MscS in the absence of a membrane was examined by deliberately removing coordinated lipid molecules from MscS using different amounts and kinds of detergent. The effects on the channel were inspected by cryo-EM. These experiments showed that closed MscS adopts the open conformation when it is enough delipidated by incubation with the detergent n-dodecyl-β-D-maltoside, and adding lipids to the open channel reverses this process. The results agree with the state-of-the-art model that the amount of lipid molecules in the pockets and grooves is responsible for the conformational state of MscS. Furthermore, incubation with the detergent lauryl maltose neopentyl glycol, which has stabilising and delipidating characteristics, resulted in a high-resolution structure of open MscS exhibiting an intricate network of ligands. Based on this structure, an updated gating model is proposed, which states that upon opening, lipids from the pockets migrate into the cytosolic membrane leaflet, while lipids from the periplasmic leaflet enter the grooves that arise between the sensor paddles.
In the framework of the presented doctoral thesis, the plant ubiquitous, non-selective vacuolar cation channel TPC1/SV was electrophysiologically studied in Arabidopsis thaliana mesophyll vacuoles to further enlighten its physiological role in plant stress responses. For this, the hyperactive channel version fou2 (D454N), gaining a non-functional vacuolar calcium sensor, strong retarded growth phenotype and upregulated JA signalling pathway, and eight fou2 reverting WT-like ouf mutants were used. Except of ouf4, all other seven ouf mutants carried a 2nd mutation in the TPC1 gene. Therefore, the TPC1 electrical features of all ouf mutants were electrophysiologically characterized with the patch clamp method and compared with fou2 and WT.
Due to a missense mutation, ouf1 and ouf7 mutants harboured a truncated TPC1 channel protein, resulting in an impaired protein integrity and in turn loss of TPC1 channel activity. Accordingly, ouf1 and ouf7 mimicked the tpc1-2 null mutant with a WT- rather fou2-like phenotype. The ouf2 (G583D D454N) mutant exhibited inactive TPC1 channels, probably because the G583D mutation located in luminal part of the S11 helix caused (i) a shift of the activation threshold to much more positive voltages (i.e. to more than +110 mV) (ii) or channel blockage. As a result of the TPC1 channel inactivity, the ouf2 mutant also imitates the WT-like phenotype of the tpc1-2 null mutant. In the ouf6 mutant (A669V D454N) the 2nd reverting mutation selectively influenced fou2-like SV channel features. Both, the fast activation kinetics and reduced luminal calcium sensitivity were similar in ouf6 and fou2. However, deviations in both, the relative and absolute open channel probability, resulted in strongly reduced (80 %) current density at 0 mM and channel inactivity in the voltage range between -30 mV to +40 mV compared to fou2 and WT. Furthermore, the TPC1 channels in ouf6 exhibited a higher susceptibility to inhibitory luminal Ca2+ than fou2. As a result of these different effects, the TPC1 channel activity almost vanished at high luminal Ca2+ loads, what is very likely the reason that ouf6 lost the fou2-like phenotype. The ouf4 mutation did not change the fou2 TPC1-channel features like fast channel activation, single channel conductance and voltage-dependent gating behaviour. Nevertheless, the TPC1 current density was 80% less in ouf4 than in fou2. Since the TPC1 gene was not the target of the 2nd mutation, it can be assumed that it is modulated via external, yet unknown factor. In the ouf8 mutant the TPC1 channels additionally possess M629I mutation within the selectivity filter II resulting in a 50% decrease in the TPC1 unitary conductance. However, the slightly increased relative open channel probability of the TPC1 channels in ouf8 compared to fou2 appeared to be sufficient to compensate the reduced transport capacity of individual TPC1 channels. As a result, a similar macroscopic outward current density of ouf8 and fou2 was detected in the absence of vacuolar Ca2+. Furthermore, ouf8 mutation did not drastically change the typical fou2 TPC1 channel features such as fast activation, vacuolar calcium insensitivity and voltage dependency. However, a reversible block of the cytosol-directed potassium efflux at increased vacuolar calcium concentration in ouf8 mutant was found. Further inspection of transiently expressed TPC1 channel variants (M629I, M629T) on the single channel level suggest that Met629 of AtTPC1 in the channel pore region is crucial for the unitary channel conductance.
Taken together, current membrane recordings from ouf mutants revealed one common feature: All of them lacked or showed a strongly impaired ability for TPC1-mediated potassium release from the vacuole into the cytosol. Additionally, considering the detected dependence of the vacuolar membrane voltage on TPC1 activity, it thus seems that the TPC1-triggered vacuolar membrane depolarization caused by vacuolar K+ release plays a key role in generation of the fou2-like phenotype. Accordingly, one can conclude that TPC1-dependent vacuolar membrane depolarization and initiation of jasmonate production are likely linked. This statement is supported also by the complete restoration of WT-like plant phenotype and JA signalling in the ouf mutants. Finally, as a control element of the vacuolar membrane voltage TPC1 is probably upstream located in JA signalling pathway and therefore a perfect junction for linking multiple physiological stimuli and response to them.
Im Rahmen der vorgelegten Doktorarbeit wurde der in Pflanzen ubiquitär exprimierte, nicht-selektive vakuoläre Kationenkanal TPC1/SV elektrophysiologisch in Arabidopsis thaliana Mesophyllvakuolen untersucht, um seine physiologische Rolle in der pflanzlichen Stressantwort weiter aufzuklären. Hierfür wurde die hyperaktive Kanalvariante fou2 (D454N), die einen nicht-funktionalen vakuolären Calciumsensor, ein stark verzögertes Pflanzenwachstum und einen hochregulierten Jasmonsäure-Signalweg aufweist, sowie acht ouf Mutanten mit fou2-umkehrenden Phänotyp benutzt. Mit Ausnahme von ouf4 enthalten alle anderen ouf Mutanten eine weitere Mutation im TPC1-Gen. Daher wurden die elektrischen Eigenschaften von TPC1 in allen ouf Mutanten elektrophysiologisch mittels der Patch clamp Technik charakterisiert und mit fou2 und dem Wildtyp verglichen.
Aufgrund einer Missense-Mutation beinhalten die Mutanten ouf1 und ouf7 ein verkürztes TPC1 Protein, woraus eine gestörte Proteinintegrität resultiert und daraus wiederum ein Fehlen der TCP1-Kanalaktivität. Dementsprechend ähneln ouf1 und ouf7 der tpc1-2 Nullmutante mit einem WT- oder eher fou2-artigen Phänotyp. Wahrscheinlich weist die ouf2 (G583D D454N) Mutante einen inaktiven TPC1-Kanal auf, weil die G583D Mutation, die in einem luminalen Teil der S11 Helix sitzt, eine Verschiebung der Aktivierungsschwelle hin zu einer höheren Spannung (z. B. mehr als +110 mV) oder einen Kanalblock verursacht. Als Folge der TPC1 Kanal Inaktivität, ahmt die ouf2 Mutante auch den WT-ähnlichen Phänotyp der tpc1-2 Nullmutante nach. In der ouf6 Mutante (A669V D454N) beeinflusst die zweite Mutation selektiv die fou2-ähnlichen SV-Kanaleigenschaften. Sowohl die schnelle Aktivierungskinetik als auch die verringerte luminale Calciumsensitivität waren denen von ouf6 und fou2 ähnlich. Die Abweichungen in der relativen sowie der absoluten Offenwahrscheinlichkeit resultierten jedoch in einer stark reduzierten (80 %) Stromdichte bei 0 mM luminalem Calcium verglichen mit fou2 und dem WT, sowie einer Kanalinaktivität bei Spannungen zwischen -30 mV und +40 mV. Darüber hinaus zeigten die TPC1 Kanäle in ouf6 eine höhere Anfälligkeit für inhibitorisches, luminales Calcium als die in fou2. Das Ergebnis der beiden unterschiedlichen Effekte ist, dass die TPC1 Kanalaktivität bei einer hohen luminalen Calciumkonzentration fast verschwindet, woraus zu schließen ist, dass ouf6 den fou2-ähnlichen Phänotyp verlor. Die ouf4 Mutation veränderte nicht die fou2 TPC1 Kanaleigenschaften, wie die schnelle Kanalaktivierung, die Einzelkanalleitfähigkeit und das spannungsabhängige Verhalten. Nichtsdestotrotz war die TCP1 Stromdichte in ouf4 um 80 % geringer als in fou2. Da das TPC1 Gen nicht das Ziel der zweiten Mutation war, kann angenommen werden, dass es durch äußere, bisher noch unbekannte Faktoren, reguliert wird. In der ouf8 Mutante haben die TPC1 Kanäle zusätzlich eine M629I Mutation innerhalb des zweiten Selektivitätsfilters, welche in einem 50 % Rückgang der TCP1 Einzelkanalleitfähigkeit resultiert. Jedoch scheint die leicht erhöhte Offenwahrscheinlichkeit der TCP1 Kanäle in ouf8, verglichen mit fou2, ausreichend zu sein, um die reduzierte Transportkapazität der individuellen TPC1 Kanäle zu kompensieren. Schlussfolgernd wurde eine ähnliche makroskopische auswärts gerichtete Stromdichte des ouf8 und des fou2 in Abwesenheit vakuolären Calciums entdeckt. Des Weiteren änderte eine ouf8 Mutation die fou2 TPC1 Kanaleigenschaften wie eine schnelle Aktivierung, vakuoläre Calciuminsensitivität und die Spannungsabhängigkeit nicht drastisch. Jedoch wurde ein reversibler Block des Zytosol-gerichteten Kalium Ausstroms bei erhöhten vakuolären Calcium Konzentrationen in ouf8 gefunden. Eine weitere Betrachtung transient exprimierter TPC1 Kanalvarianten (M629I, M629T) auf Einzelkanalebene weist darauf hin, dass das Met629 des AtTPC1 in der Kanalporenregion entscheidend ist für die Einzelkanalleitfähigkeit.
Zusammengefasst zeigt der über die Membran von ouf Mutanten gemessene Strom eine Gemeinsamkeit: Alle zeigten keinen oder einen stark beeinträchtigten TPC1-vermittelten Kaliumausstrom aus der Vakuole ins Zytosol. Unter Berücksichtigung der beobachteten Abhängigkeit der vakuolären Membranspannung von der TPC1 Aktivität, scheint es, als ob die durch TPC1 angeregte Depolarisation der Vakuolenmembran, welche durch die vakuoläre Kaliumfreisetzung bedingt wird, in der Ausbildung des fou2 Phänotyps eine Rolle spielt. Daraus lässt sich ableiten, dass die TPC1-abhängige Depolarisation der Vakuolenmembran und die Jasmonat Bildung vermutlich verbunden sind. Diese Behauptung wird auch gestützt durch die komplette Wiederherstellung des WT-ähnlichen Pflanzenphänotyps und des Jasmonsäure Signalwegs in den ouf Mutanten. Letztendlich ist TPC1 als kontrollierendes Element der vakuolären Membranspannung wahrscheinlich dem Jasmonsäure Signalweg vorgeschaltet und deswegen ein perfekter Knotenpunkt, der verschiedene physiologische Stimuli und ihre Antworten verbindet.
The phytohormone auxin performs important functions in the initiation of plant tissues and organs, as well as in the control of root growth in conjunction with external stimuli such as gravity, water and nutrient availability. These functions are based primarily on the auxin-dependent regulation of cell division and elongation. Important for the latter is the control of the cell turgor by the vacuole. As storage for nutrients, metabolites and toxins, vacuoles are of vital importance. Vacuolar stored metabolites and ions are exchanged across the vacuolar membrane with the cytoplasm via active transport processes as well as passively through ion channels. In their function as second messenger, calcium ions are important regulators but also subject to vacuolar transport processes. Changes in the cytosolic calcium concentration not only act locally, but are also associated with signal transduction over longer distances. In this work, electrophysiological methods were combined with imaging techniques to gain insights into the interaction between cytosolic calcium signals, vacuolar transport processes and auxin physiology in the intact plant organism.
Calcium signals are involved in the regulation of vacuolar ion channels and transporters. In order to investigate this in the intact organism, intracellular microelectrode measurements were performed in the model system of bulging Arabidopsis thaliana root hairs. By means of the two-electrode voltage-clamp technique, it could be confirmed that the vacuolar membrane is the limiting electrical resistance during intravacuolar measurements and thus measured ion currents actually represent only the currents across the vacuolar membrane. The already known time-dependent decrease of vacuolar conductivity during intravacuolar experiments could be further correlated with an impalement-related, transient increase of the cytosolic calcium concentration. Intravacuolar voltage-clamp experiments in root hair cells of calcium reporter plants confirmed this relationship between vacuolar conductivity and the cytosolic calcium concentration.
However, the vacuole is not just a recipient of cytosolic calcium signals. Since the vacuole represents the largest intracellular calcium reservoir, it has long been argued that it is also involved in the generation of such signals. This could be confirmed in intact root hair cells. Changes in the vacuolar membrane potential affected the cytosolic calcium concentration in these cells. While depolarizing potentials led to an increase of the cytosolic calcium concentration, hyperpolarization of the vacuolar membrane caused the opposite. Thermodynamic considerations of passive and active calcium transport across the vacuolar membrane suggested that the results described herein reflect the behaviour of vacuolar H+/Ca2+ exchangers whose activity is determined by the proton motive force.
In addition, cytosolic calcium has been shown to be a key regulator of a rapid auxin-induced signaling pathway that regulates polar transport of the hormone.
In the same model system of bulging root hairs it could be shown that the external application of auxin results in a very fast, auxin concentration- and pH-dependent depolarization of the plasma membrane potential. Synchronous with the depolarization of the plasma membrane potential, transient calcium signals were recorded in the cytosol. These were caused by an auxin-activated influx of calcium ions through the ion channel CNGC14. Experiments on loss-of-function mutants as well as pharmacological experiments showed that the auxin-induced activation of the calcium channel requires auxin-perception by the F-box proteins of the TIR1/AFB family.
Investigations of auxin-dependent depolarization as well as the auxin-induced influx of protons into epidermal root cells of loss-of-function mutants showed that the secondary active uptake of auxin by the high-affinity transport protein AUX1 is responsible for the rapid depolarization
Not only the cytosolic calcium signals correlated with CNGC14 function, but also the AUX1-mediated depolarization of root hairs. An unchanged expression of AUX1 in the cngc14 loss-of-function mutant suggested that the activity of AUX1 must be post-translationally regulated. This hypothesis was supported by experiments in which treatment with the calcium channel blocker lanthanum led to inactivation of AUX1 in the wild type.
The cytosolic loading of individual epidermal root cells with auxin resulted in the spread of lateral and acropetal calcium waves. These correlated with a shift of the auxin gradient at the root apex and thus supported a hypothetical calcium-dependent regulation of polar auxin transport. A model for a rapid, auxin-induced and calcium-dependent signaling pathway is presented and its importance for gravitropic root growth is discussed. Since AUX1-mediated depolarization varied with external phosphate concentration, the importance of this rapid signaling pathway is also discussed for the adaptation of root hair growth to an inadequate availability of phosphate.
The greatest problems faced during the 21st century is climate change which is a big threat to food security due to increasing number of people. The increase in extreme weather events, such as drought and heat, makes it difficult to cultivate conventional crops that are not stress tolerant. As a result, increasing irrigation of arable land leads to additional salinization of soils with plant-toxic sodium and chloride ions. Knowledge about the adaptation strategies of salt-tolerant plants to salt stress as well as detailed knowledge about the control of transpiration water loss of these plants are therefore important to guarantee productive agriculture in the future. In the present study, I have characterized salt sensitive and salt tolerant plant species at physiological, phenotypic and transcriptomic level under short (1x salt) and long-time (3x) saline growth conditions. Two approaches used for long-time saline growth conditions (i.e increasing saline conditions (3x salt) and constant high saline conditions (3x 200 mM salt) were successfully developed in the natural plant growth medium i.e soil. Salt sensitive plants, A. thaliana, were able to survive and successfully set seeds at the toxic concentrations on the increasing saline growth mediums, with minor changes in the phenotype. However, under constant high saline conditions they could not survive. This was due to keeping low potassium, and high salt ions (sodium and chloride) in the photosynthetic tissue i.e leaf. Similarly, high potassium and low salt ions in salt tolerant T. salsuginea on both saline environments were the key for survival of this plant species. Being salt tolerant, T. salsuginea always kept high potassium levels and low sodium (during 1x) and chloride levels (during both 1x and 3x) in the leaf tissue.
A strict control over transpirational water loss via stomata (formed by pair of guard cells) is important to maintain plant water balance. Aperture size of the stomata is regulated by the turgidity of the guard cells. More turgid the guard cells, bigger the apertures are and hence more transpiration. Under osmotic stress, the water loss is reduced which was evident in the salt sensitive A. thaliana plants under both short and long-time saline growth conditions. As the osmotic stress was only increased during long time saline growth conditions in T. salsuginea therefore, water loss was also decreased only under these saline conditions. Environmental CO2 assimilation also takes place via stomata in plants which then is used for photosynthesis. Stomatal apertures also influence CO2 assimilation. As the light absorbing photosynthetic pigments were more affected in A. thaliana, therefore photosynthetic activity of the whole plant was also reduced. Similarly, both short and long-time saline growth conditions also reduced the effective quantum yield of A. thaliana guard cells. Growth of the plant is dependent on energy which comes from photosynthesis. Reduced environmental CO2 assimilation would affect photosynthesis and hence growth, which was clearly observed in A. thaliana guard cells under long-time saline growth conditions.
Major differences in both guard cells types were observed in their chloride and potassium levels. Energy Dispersive X-Ray Analysis (EDXA) suggested strict control of chloride accumulation in T. salsuginea guard cells as the levels remain unchanged under all conditions. Similarly, use of sodium in place of potassium for osmotic adjustments seems to be dependent on Na+/K+ rations in both guard cell types. Increased salt ions and reduced potassium levels in A. thaliana guard cells posed negative effect on photochemistry which in turn increased ROS metabolism and reduced energy related pathways at transcriptomic level in this plant species. Moreover, photosynthesis was strongly affected in A. thaliana guard cells both at transcriptomic and physiological levels. Similarly, global phytohormones induced changes were more evident in A. thaliana guard cells especially on 3x salt medium. Among all phytohormones, genes under the control of auxin were more differentially expressed in A. thaliana guard cells which suggests wide changes in growth and development in this plant species under salinity.
Phytohormone, ABA is vital for closing the stomata under abiotic stress conditions. Increased levels of ABA during saline conditions led to efflux of potassium and counter anions (chloride, malate, nitrate) from the guard cells which caused the outward flow of water and hence reduction in turgor pressure. Reduced turgor pressure led to reduced water loss and CO2 assimilation especially in A. thaliana. Guard cells of both plant species synthesized ABA during saline conditions which was reflected from transcriptomic data and ABA quantification in the guard cells. ABA induced signaling in both plant species varied at the ABA receptor (PYL/PYR) levels where totally contrasting responses were observed. PYL2, PYL8 and PYL9 were specific to A. thaliana, furthermore, PYL2 was found to be differentially expressed only under 3x salt growth conditions thus suggesting its role during long term salt stress in this plant species. Protein phosphatases, which negatively regulate ABA signaling on one hand and act as ABA sensor on the other hand were found to be more differentially expressed in A. thaliana than T. salsuginea guard cells, which suggests their diverse role in both plant species under saline conditions. Differential expression of more ABA signaling players in long time saline conditions was prominent which could be because of darkness, as it is well known that rapid closure of stomata under dark conditions require ABA signaling. Moreover, representation of these components in dark also suggests that plants become more sensitive to dark under saline conditions which is also evident from the transpiration rates.
Altogether, increased salt ions in A. thaliana guard cells and leaves led to pigment degradation and ABA induced reduction in transpiration which in turn influenced its growth. In contrast, T. salsuginea is the salt excluder and therefore keeps low levels of salt ions especially the chloride both in leaves and guard cells which mildly affects its growth. Guard cells of A. thaliana encounter severe energy problems at physiological and transcriptomic level. Main differences in the ABA signalling between both plant species were observed at the ABA receptor level.
A part of the plant kingdom consists of a variety of carnivorous plants. Some trap their prey
using sticky leaves, others have pitfall traps where prey cannot escape once it has fallen inside.
A rare trap type is the snap-trap: it appears only twice in the plant kingdom, in the genera
Aldrovanda and Dionaea. Even Charles Darwin himself described Dionaea muscipula, the
Venus flytrap, with the following words “This plant, commonly called Venus' fly-trap, from the
rapidity and force of its movements, is one of the most wonderful in the world”. For a long
time now, the mechanisms of Dionaea’s prey recognition, capture and utilization are of
interest for scientists and have been studied intensively.
Dionaea presents itself with traps wide-open, ready to catch insects upon contact. For this,
the insect has to touch the trigger hairs of the opened trap twice within about 20-30 seconds.
Once the prey is trapped, the trap lobes close tight, forming a hermetically sealed “green
stomach”.
Until lately, there was only limited knowledge about the molecular and hormonal mechanisms
which lead to prey capture and excretion of digestive fluids. It is known that the digestion
process is very water-consuming; therefore, the interplay of digestion-inducing and digestion inhibiting
substances was to be analyzed in this work, to elucidate the fine-tuning of the
digestive pathway. Special attention was given to the impact of phytohormones on mRNA
transcript levels of digestion-related proteins after various stimuli as well as their effect on
Dionaea’s physiological responses.
Jasmonic acid (JA) and its isoleucine-conjugated form, JA-Ile, are an important signal in the
jasmonate pathway. In the majority of non-carnivorous plants, jasmonates are critical for the
defense against herbivory and pathogens. In Dionaea, this defense mechanism has been
restructured towards offensive prey catching. One question in this work was how the
frequency of trigger hair bendings is related to the formation of jasmonates and the induction
of the digestion process. Upon contact of a prey with the trigger hairs in the inside of the trap,
the trap closes and jasmonates are produced biosynthetically. JA-Ile interacts with the COI1-
receptor, thereby activating the digestion pathway which leads to the secretion of digestive
fluid and production of transporters needed to take up prey-derived nutrients. In this work it
could be shown that the number of trigger hair bendings is positively correlated with the level
and duration of transcriptional induction of several digestive enzymes/hydrolases.
Abscisic acid (ABA) acts, along with many other functions, as the plant “drought stress
hormone”. It is synthesized either by roots as the primary sensor for water shortage or by
guard cells in the leaves. ABA affects a network of several thousand genes whose regulation
prepares the plant for drought and initiates protective measurements. It was known from
previous work that the application of ABA for 48 hours increased the required amount of
trigger hair bendings to achieve trap closure. As the digestion process is very water-intensive,
the question arose how exactly the interplay between the jasmonate- and the ABA-pathway
is organized, and if ABA could stop the running digestion process once it had been activated.
In the present work it could be shown that the application of ABA on intact traps prior to
mechanically stimulating the trigger hairs (mechanostimulation) already significantly reduced
the transcription of digestive enzymes for an incubation time as short as 4 h, showing that
already short-term exposure to ABA counteracts the effects of jasmonates when it comes to
initiating the digestion process, but does not inhibit trap closure. Incubation for 24 and 48
hours with 100 μM active ABA had no effect on trap reopening, only very high levels of 200
μM of active ABA inhibited trap reopening but also led to tissue necrosis. As the application
of ABA could reduce the transcription of digestive hydrolases, it is likely that Dionaea can stop
the digestion process, if corresponding external stimuli are received.
Another factor, which only emerged later, was the effect of the wounding-induced systemic
jasmonate burst. As efficient as ABA was in inhibiting marker hydrolase expression after
mechanostimulation in intact plants, the application of ABA on truncated traps was not able
to inhibit mechanostimulation-induced marker hydrolase expression. One reason might be
that the ABA-signal is perceived in the roots, and therefore truncated traps were not able to
react to it. Another reason might be that the wounding desensitized the tissue for the ABAsignal.
Further research is required at this point.
Inhibitors of the jasmonate pathway were also used to assess their effect on the regulation of
Dionaea´s hunting cycle. Coronatine-O-methyloxime proved to be a potent inhibitor of
mechanostimulation-induced expression of digestive enzymes, thus confirming the key
regulatory role of jasmonates for Dionaea´s prey consumption mechanism.
In a parallel project, the generation of in vitro cultures from sterilized seeds and single plant
parts proved successful, which may be important for stock-keeping of future transgenic lines.
Protoplasts were generated from leaf blade tissue and transiently transformed, expressing the
reporter protein YFP after 24 h of incubation. In the future this might be the starting point for
the generation of transgenic lines or the functional testing of DNA constructs.
In contrast to the well described molecular basis for S-type anion currents, the genes underlying R-type anion currents were unknown until 2010. Meyer S. and colleagues (2010) showed that, localized in the guard cell plasma membrane, AtALMT12 is an R-type anion channel involved in stomatal closure. However, knocking out AtALMT12 did not fully shut down R-type currents; the almt12 loss-of-function mutant has residual R-type-like currents indicating that ALMT12 is not the only gene encoding Arabidopsis thaliana R-type channels (Meyer S. et al., 2010). This PhD thesis is focussed on understanding the properties, regulation and molecular nature of the R-type channels in Arabidopsis thaliana plants. To fulfil these aims, the patch clamp technique was used to characterize electrical features of R-type currents in various conditions such as the presence/absence of ATP, variation in cytosolic calcium concentration or the presence of cytosolic chloride. Electrophysiological study revealed many similarities between the features of Arabidopsis thaliana R-type currents (Col0) and residual R-type currents (the almt12 loss-of-function mutant). Strong voltage dependency, channel activity in the same voltage range, position of maximal recorded current and blockage by cytosolic ATP all pointed to a shared phylogenetic origin of the channels underlying these R-type currents. Expression patterns of the ALMT family members for Col0 and the almt12 mutant revealed ALMT13 and AMT14 as potential candidates of the R-type channels. Electrical characterization of Col0, almt12 and the two double loss-of-function mutants (almt12/almt13 and almt12/almt14) strongly suggest that ALMT13 mediates the calcium-dependent R-type current component that is directly regulated by cytosolic calcium. Additionally, similarly to ALMT12, ALMT14 could participate as a calcium-independent R-type anion channel. Differences in response to the cytosolic calcium concentration between ALMT12, ALMT13 and ALMT14 suggest their possible involvement in different signalling pathways leading to stomatal closure. Moreover, a study performed for the two Arabidopsis thaliana ecotypes Col0 and WS showed drastically increased ALMT13 expression for WS, which is related to R-type current properties. The WS ecotype has calcium-dependent R-type current behaviour, while it is calcium-independent in Col0. Furthermore, this plant line showed lower peak current densities compared to Col0 and almt mutants. These facts strongly suggest interaction between ALMT12 and ALMT13, with ALMT13 as a repressor of the ALMT12. Acquired patch clamp data revealed sulphate-dependent increases in ALMT13 current. This could be caused by changes in absolute open probability and/or permeability for sulphate and possibly chloride and links ALMT13 with sulphate-mediated stomatal closure under drought stress. It was then confirmed that ATP affects R-type currents. In contrast to Vicia faba, ATP was identified as a negative regulator of the Arabidopsis thaliana R-type anion channels. The effect of ATP is ambiguous but there is a high probability that it is a result of direct block and phosphorylation. However, the phosphorylation site and place of ATP binding needs further investigation.
The story of the ALMT family, as examined in this thesis, sheds light on the complexity of the stomatal closure process.