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Cellulose is the most abundant biopolymer on Earth and cell wall (CW) synthesis is one of the major carbon consumers in the plant cell. Structure and several interaction partners of plasma membrane (PM)-bound cellulose synthase (CESA) complexes, CSCs, have been studied extensively, but much less is understood about the signals that activate and translocate CESAs to the PM and how exactly cellulose synthesis is being regulated during the diel cycle. The literature describes CSC regulation possibilities through interactions with accessory proteins upon stress conditions (e.g. CC1), post-translational modifications that regulate CSC speed and their possible anchoring in the PM (e.g. with phosphorylation and S-acylation, respectively). In this thesis, 13CO2 labeling and imaging techniques were employed in the same Arabidopsis seedling growth system to elucidate how and when new carbon is incorporated into cell wall (CW) sugars and UDP-glucose, and to follow CSC behavior during the diel cycle. Additionally, an ubiquitination analysis was performed to investigate a possible mechanism to affect CSC trafficking to and/or from the PM. Carbon is being incorporated into CW glucose at a 3-fold higher rate during the light period in comparison to the night in wild-type seedlings. Furthermore, CSC density at the PM, as an indication of active cellulose synthesizing machinery, is increasing in the light and falling during the night, showing that CW biosynthesis is more active in the light. Therefore, CW synthesis might be regulated by the carbon status of the cell. This regulation is broken in the starchless pgm mutant where light and dark carbon incorporation rates into CW glucose are similar, possibly due to the high soluble sugar content in pgm during the first part of the night. Strikingly, pgm CSC abundance at the PM is constantly low during the whole diel cycle, indicating little or no cellulose synthesis, but can be restored with exogenous sucrose or a longer photoperiod. Ubiquitination was explored as a possible regulating mechanism for translocation of primary CW CSCs from the PM and several potential ubiquitination sites have been identified.. The approach in this thesis enabled to study cellulose/CW synthesis from different angles but in the same growth system, allowing direct comparison of those methodologies, which could help understand the relationship between the amount of available carbon in a plant cell and the cells capacity to synthesize cellulose/CW. Understanding which factors contribute to cellulose synthesis regulation and addressing those fundamental questions can provide essential knowledge to manage the need for increased crop production.
The energy required to drive photochemical reactions is derived from charge separation across the thylakoid membrane. As the consequence of difference in proton concentration between chloroplasts stroma and thylakoid lumen, a proton motive force (pmf) is generated. The pmf is composed out of the proton gradient (ΔpH) and membrane potential (ΔΨ), and together they drive the ATP synthesis. In nature, the amount of energy fueling photosynthesis varies due to frequent changes in the light intensity. Thylakoid ion transport can adapt the energy flow through a photosynthetic apparatus to the light availability by adjusting the pmf composition. Dissipation of ΔΨ reduces the charge recombination at the photosystem II, allowing for an increase in ΔpH component to trigger a feedback downregulation of photosynthesis. K+ Exchange Antiporter 3 (KEA3) driven K+/H+ antiport reduces the ΔpH fraction of pmf, thereby dampening a non-photochemical quenching (NPQ). As a result, it increases the photosynthesis efficiency during the transition to lower light intensity. This thesis aimed to find the answers for questions concerning KEA3 activity regulation and its role in plant development. Presented data shows that in plants lacking chloroplast ATP synthase assembly factor CGL160 with decreased ATP synthase activity, KEA3 has a pivotal role in photosynthesis regulation and plant growth during steady-state conditions. Lack of KEA3 in cgl160 mutant results in a strong growth impairment, as photosynthesis is limited due to increased pH-dependent NPQ and decreased electron flow through cytochrome b6f complex. Overexpression of KEA3 in cgl160 mutant increases charge recombination at photosystem II, promoting photosynthesis. Thus, during periods of low ATP synthase activity, plants benefit from KEA3 activity. The KEA3 undergoes dimerization via its regulatory C-terminus (RCT). The RCT responds to changes in light intensity as the plants expressing KEA3 without this domain show reduced photo-protective mechanism in light intensity transients. However, those plants fix more carbon during the photosynthesis induction phase as a trade-off for a long-term photoprotection, showing KEA3 regulatory role in plant development. The KEA3 RCT is facing thylakoid stroma, thus its regulation depends on light-induced changes in the stromal environment. KEA3 activity regulation overlaps with the stromal pH changes occurring during light fluctuations. The ATP and ADP has shown to have an affinity towards heterologously expressed KEA3 RCT. Such interaction causes conformational changes in RCT structure. The fold change of RCT-ligand interaction depends on the environmental pH value. With a combination of bioinformatics and in vitro approach, the ATP binding site at RCT was located. Introduction of binding site point mutation in planta KEA3 RCT resulted in antiporter activity deregulation during transition to low light. Together, the data presented in this thesis allowed us to assess more broadly a KEA3 role in photosynthesis adjustment and propose the models of KEA3 activity regulation throughout transition in light intensity.
Artificial light at night (ALAN), one form of human-induced rapid environmental change, is continuously spreading in space and time and increasing in intensity as part of the ongoing urbanization. A vast range of animals is known to be affected by ALAN as, among other things, it can mask natural light cues and change both the perceived as well as the actual predation risk. Since ALAN per se is restricted to the night, the majority of studies so far have focused on nocturnal species or behavioral changes during the night. How polyphasic species respond to ALAN has been largely overlooked, although they can possibly carry over effects of nighttime illumination into the day. Additionally, individuals within a species are known to consistently differ in their personality which includes risk-taking behavior. While this implies that ALAN can lead to varying anti-predatory responses in animals within a population, knowledge on this topic is still very limited. This thesis aims at investigating what initial behavioral reaction is caused by ALAN in polyphasic small mammals while also incorporating an animal’s personality. Nighttime and daytime activity, movement and foraging behavior of the bank vole (Myodes glareolus) were investigated in regards to effects of different light intensities and partial illumination in the laboratory. Additionally, changes in intra- and interspecific interactions of bank voles and striped field mice (Apodemus agrarius) subjected to ALAN were studied in experimental populations in semi-natural outdoor enclosures. Chapter I explores whether behavioral responses to ALAN of varying intensity are related to animal personality. Results showed that bank voles reduced movement and foraging already under dim light and that bold animals generally moved and foraged more than shy animals. Exclusively under bright illumination did bold animals exploit the food patches more than shy animals. The results demonstrate that bank voles are affected by light intensities prevalent in urban habitats. Additionally, certain light scenarios might lead to an advantage of and a shift towards certain personality types. Chapter II focusses on the effects of partial ALAN on foraging behavior of animals with varying animal personalities while extending the view towards possible carry-over effects of ALAN into the daytime. While bank voles reduced foraging behavior in illuminated areas at night, they increased foraging behavior in those areas at the subsequent day. Bold individuals generally had lower giving-up densities than shy individuals but this difference was especially pronounced during daytime at formerly illuminated food patches. Thus, ALAN can have carry-over effects into the daytime in polyphasic animals and thus has the potential to affect daytime intra- and interspecific interactions. Chapter III broadens the view from the individual to the population level. Experimental populations consisting of bank voles and striped field mice were established in large outdoor enclosures successively experienced natural and artificial light conditions at night. VHF telemetry data revealed that animals were predominantly active during the day under natural conditions. This difference between day and night vanished under ALAN. Additionally, conspecifics reduced home range overlap, proximity and activity synchrony while boldness was not associated with behavioral changed due to ALAN. The results suggest that ALAN has the potential to alter intraspecific interactions and thus can have fitness consequences on the population level. Overall, the present thesis shows that ALAN can affect nighttime and daytime behavior as well as intraspecific interactions of polyphasic small mammals. Differences in risk- taking behavior of individuals may vary in importance depending on other environmental variables. Thus, this thesis hopefully triggers broadening the view regarding the role of an animal’s personality in coping with ALAN and the effects on daytime behavior and diurnal species.
Plants possess cell wall, a polysaccharide exoskeleton which encompasses all plant cells. Cell wall gives plant cells mechanical support, defines their shape, enables growth and water transport through a plant. It also has important role in communication with the external environment. Regulation of plant cell wall biosynthesis and cell and organ morphogenesis depends on cell’s ability to detect mechanical signals originating both from the external environment and from internal plant tissues. Thanks to the presence of the cell wall, all living plant cells develop constant internal pressure generated by the active water uptake, known as turgor pressure, which enables them to grow. Thus, actively growing cells in the tissue are exerting mechanical stress to each other. In order to properly coordinate cell growth, tissue morphogenesis and maintain cell-to-cell adhesion, plant cell have to detect these mechanical signals. That is performed by a group of still not well enough characterized plant mechanosensitive proteins. Mechanosensors are proteins capable of detecting changes in mechanical stress patterns and translating them into physiological and developmental outputs. One of plant mechanosensitive proteins, DEFECTIVE KERNEL1 (DEK1) has shown to be a very important in proper plant development. DEK1 bears similarity with animal cysteine proteases of Calpain superfamily. DEK1 is very important for plant development since all null alleles are embryo lethal. During the last 20 years of DEK1 studies, this protein has proven to be a very difficult for different molecular and biochemical manipulations. As a consequence, very little is known about its direct target proteins. Wang and co-workers (2003) and Johnson and co-workers (2008) have given a valuable contribution to biochemical understanding of DEK1 by determining that it functions as Cys-protease in similar way as animal calpains. However, a lot of indirect knowledge was gathered about the effects of disruption and modulation of DEK1 activity. DEK1 is important for proper organ development, epidermal specification, and maintenance. However, some studies have inferred that DEK1 affects expression of different cell wall related genes, and it regulates cell-to-cell adhesion in epidermal cells. This led to two extensive studies (Amanda et al., 2016, 2017) which demonstrated importance of DEK1 in regulation leaf epidermal cell walls in A. thaliana mature leaves and inflorescence stems. These studies demonstrated that DEK1 also influences cell wall thickness and cell-to-cell adhesion and that it could potentially regulate cell growth and expansion. Building up on this research, we decided to try to further characterize molecular and biomechanical aspects of DEK1 mediated cell wall regulation, with special emphasis on regulation of cellulose synthesis. We used two mutant lines, with modulated DEK1 activity, a constitutive overexpressor for DEK1 CALPAIN domain and a point mutant in CALPAIN domain, dek1-4. In Chapter 3 we demonstrated that DEK1 regulates dynamics of Cellulose Synthase Complexes (CSCs). Both lines showed decreased crystalline cellulose contents. This led us to investigate if velocity of CSCs in cotyledons, was affected, since it is known that changes in cellulose contents are often caused by defects in CSC. We found that bothDEK1 modulated lines we used have significantly decreased velocity of CSCs. We have also examined plasma membrane turnover rates of CSCs and found out that after photo-bleaching OE CALPAIN has much faster recovery rates compared to Col-0 wild type, while dek1-4 has lower exocytotic rates of CSCs, and much longer life-time of CSCs inserted into the plasma membrane. These results suggested that DEK1 regulates different aspects of CSC dynamics, possibly through interaction with different regulatory proteins. Decrease in cellulose contents we observed in DEK1 modulated lines, prompted us to investigate how this reflects biomechanics and structural properties of epidermal cotyledon cell walls of DEK1 modulated lines, which is described in Chapter 4. To achieve this, we developed a novel microdissection method for isolation and mechanical and structural characterization of native epidermal cell wall monolayers using atomic force microscopy (AFM). AFM force spectroscopy assays showed that both DEK1 modulated lines had stiffer cell walls compared to Col-0. This was awkward since we initially detected decrease in crystalline cellulose which implied decrease in cell wall stiffness. However, subsequent high-resolution AFM imaging has revealed that DEK1 modulate lines cells walls have their cellulose microfibrils organized in thicker bundles than Col-0. Also, polysaccharide composition analysis has revealed that DEK1 modulated lines have increased abundance of pectins, which could also be responsible for the observed increase in cell wall stiffness. Previous work has shown that different dek1 mutants and modulated lines have defects in cell-to-cell adhesion. This implied that DEK1 may be involved in sensing and/or maintaining cell wall integrity (CWI). We performed several growth assays to determine role of DEK1 in CWI, which is described in Chapter 5. We performed cellulose synthesis perturbation assays with cellulose synthesis inhibitor Isoxaben and obtained very interesting results. While OE CALPAIN plants were hypersensitive to Isoxaben, dek1-4 has shown complete insensitivity. Furthermore, a regular CWI maintenance response, reported in A. thaliana as result of compromised CWI, ectopic lignification in seedlings’ roots was absent in both DEK1 modulated lines we examined. We detected interesting growth response of DEK1 lines to NaCl and mannitol treatments as well. Although these findings are pointing out that DEK1 could be part of CWI signalling pathways, more experiments are necessary to fully elucidate possible role of DEK1 in CWI sensing and/or maintenance pathways, especially to check if DEK1 is interacting with Catharanthus roseus Receptor Like Kinase group of CWI sensors. Studies on 4-month old short day grown DEK1 modulated lines, have shown defects in branching, with development of fasciated stem branches in a DEK1 modulated line overexpressing CALPAIN domain (Amanda et al., 2017). This result pointed out to a possibility that DEK1 may regulate organ morphogenesis and patterning at the level of shoot apical meristem (SAM). Work towards elucidating role of DEK1 in SAM maintenance and organ patterning is detailed in Chapter 6. We determined that OE CALPAIN had significantly larger central zone of SAM as well as larger individual SAM cells in central zone, as well as higher distribution of cell sizes, implying possible cell expansion defects. dek1-4 did not exhibited changes in SAM central zone size or individual stem cell size, but it seemed that it had increased number of stem cells in SAM central zone. Both DEK1 lines had perturbation of phyllotaxis on SAM level, with disturbed divergence angles between floral primordia. Disturbed phyllotaxis was also observed between siliques, in mature plants. In addition to this, OE CALPAIN has exhibited occurrence of multiple (up to four) siliques growing from a single stem node. All this is pointing out that DEK1 might participate in hormone-signalling in the SAM.. DEK1 is a highly intriguing protein. However, since it is a unigene, and in addition to that, a regulatory protease, it probably participates in multiple signalling pathways, which makes understanding its function much more complicated.
Past and present biodiversity in northeastern Siberia inferred from sedimentary DNA metabarcoding
(2021)
The arctic-boreal treeline is a transition zone from taiga to tundra covering a vast area in Siberia. It often features large environmental gradients and reacts sensitively to changes in the environment. For example, the expansion of shrubs and a northward movement of the treeline are observable in Siberia as a response to the warming climate. The changes in vegetation across the treeline are known to influence the water chemistry in the lakes. This causes further alteration to the composition and diversity of sensitive aquatic organisms such as diatoms and macrophytes. Despite the rising awareness of the complex climate-feedback mechanisms of terrestrial plants, the understanding of their assembly rules and about responses of aquatic biomes in the surrounding treeline lakes is still limited. The goal of this thesis is to examine the previous and present biodiversity of terrestrial and freshwater biomes from the Siberian treeline ecotone, as well as their reactions to environmental changes. In particular, this thesis attempts to examine the performance of applying sedimentary DNA metabarcoding in terrestrial plants, aquatic macrophytes and diatoms, their spatial patterns along the environmental gradients and their temporal patterns throughout the climate transition from the late Pleistocene to Holocene. Sedimentary DNA metabarcoding combined with next-generation sequencing is applied as a primary tool to explore the composition and diversity of terrestrial plants, diatoms and aquatic macrophytes. The main study area is located in Chukotka of northeastern Siberia in the Arctic, a biodiversity hotspot due to its continental location and the diverse habitats of the glacial refugium. The modern diatom diversity was assessed with a specific diatom metabarcoding marker and morphological identification. Both approaches agree to a dominance of Fragilariaceae and Aulacoseiraceae, as well as on the environmental influential indicators of the diatom community. The high diversity of Fragilariaceae identified in the thermokarst lakes is found to follow the vegetation gradient along the treeline, suggesting that diatom metabarcoding can decipher relationships between diatom assemblage shifts and the relevant environmental changes. In particular, the metabarcoding approach detects diversification of fragilarioids in glacial lakes which is not visible using morphology. Sedimentary ancient DNA records indicate a vegetation mosaic of forb-dominated steppe-tundra during 28-19 ka, followed by a shift to dwarf-shrub tundra during 19-14 ka. During the most recent 14 thousand years, the vegetation consists of deciduous shrublands, then a change to boreal forest is observed. Investigations on the alpha diversity of the vegetation show that species richness is unexpectedly highest during pre-LGM, which is likely related to the extensive area that allows for more taxa. The optimum Holocene warming during 9-6 ka is not accompanied by a high richness as widely believed, but with an evenly distributed community by the fulfilment of erect shrubs. Furthermore, changes in taxonomic and phylogenetic diversity show complementary results in understanding community diversity. The composition and richness in the modern macrophytes community from Siberian Arctic and Chinese alpine are best co-influenced by July temperature and electrical conductivity.. Past macrophyte turnover during the late Pleistocene-Holocene is less noticeable in Siberia, whereas a pronounced community change from emergent to submerged plants is detected from Chinese alpine regions at about 14 ka due to increasing temperature and varying water conductivity. Finally, sedimentary DNA metabarcoding is a cost-effective and powerful proxy for ecological application, whereas completeness of the reference library, coverage and resolution of the metabarcoding marker are the major limitations of sedimentary DNA based diversity monitoring. The composition and richness in modern vegetation and macrophytes across broad spatial gradients is constrained by environmental variables, suggesting a potential usage for environmental monitoring. Diatom distributions are driven by different water variables along the treeline. Past records indicate that the shrub coverage has a noticeable influence on the assemblies of both terrestrial plants and aquatic macrophytes, though the shift in macrophyte community is relatively minor in the past 28 thousand years. In the long-term, the shrub expansion may eventually result in a genetically more diverse vegetation community but reduced species richness. When exceeding the optimal temperatures, further warming may lead to a decrease and putative loss of macrophytes and diatoms.
As the ongoing trend of developing smart materials that can reversibly switch geometry stimulated by environmental control addressed increasing attention in many research fields, especially for biomedical or soft robotic applications. Shape-memory polymers (SMPs), which can change shape, stiffness, size, and structure when exposed to an external stimulus, are intensively explored as encouraging material candidates for achieving multifunctionality, and for miniaturizing into micro-components to expand the applications. Besides, the geometrical design has gained growing attention for creating engineering applications, such as bi-stable mechanisms, and has the potential to be explored by implementing SMP for new functions. In this context, this thesis aimed to develop smart micro-/nano-objects based on SMP and explore new functions by geometrical design using SMP. Here, two types of stimuli-responsive objects capable of one-way temperature-memory effect (TME) or free-standing reversible actuation e.g., micro/nanofibers (i) and microcuboids (ii) at different aspects were explored. At first, it was hypothesized that the advanced atomic force microscopy (AFM) platform can be established to study individual polymeric micro-/nanofibers (i) in terms of incorporation and characterization of a reversible shape-memory actuation capability. Crystallizable material was chosen for preparing the fibers and the molecular alignment within the fibers among different diameters will influence the crystallization-induced elongation during cooling that determined the reversible effect. For the second type, microcuboids (ii), it was hypothesized that a programming and quantification approach can be developed to enable the realization and characterization of a one-way micro-TME and micro-shape-memory polymer actuation (SMPA) in microcuboids. The responsive temperature of one-way shape transformation can be tuned by programming temperature (Tp) and the separation temperature (Tsep) for post-programming can influence the actuation. Finally, a geometrical design with bi-stability was combined with SME to create new functions of shape actuation. It was hypothesized that the predicted bi-stable or mono-stable structures can be achieved with the aid of digital fabrication methods. Using shape-memory effect (SME), the alteration of bi-stable and mono-stable can initiate shape transformation with a larger magnitude and higher energy output.
In the first part, the method to quantify the reversible SMPA of a single micro/nano crystallizable fiber with geometry change during the actuation was explored. Electrospinning was used to prepare poly (ε-caprolactone) (PCL) micro/nanofiber with different diameters, which were fixed by UV glue and crosslinked on the structured silicon wafer. Using AFM, the programming, as well as the observation of recovery and reversible displacement of the fiber, were performed by vertical three-point bending at the free suspended part. A plateau tip was chosen to achieve stable contact and longer working distance for performing larger deformation, enabling intensified reversible SMPA of single fibers. In this way, programming strains of 39 ± 1% or 46 ± 1% were realized for fiber with a diameter of 1 ± 0.2 µm and 300 ± 50 nm, which were bent at 80 °C and fixed at 10 °C. Values for the reversible elongation of εrev = 3.4 ± 0.1% and 10.5 ± 0.1% were obtained for a single micro and nanofiber respectively between 10 and 60 °C. The higher actuation effect observed for nanofiber demonstrated that the highly compact and oriented crystallites in nanofibers, which determined the pronounced εrev compared to the thick microfibers. Besides, a stable reversible actuation of a nanofiber can be tracked by AFM tip up to 10 cycles, indicating a sustainable application can be achieved on the fiber actuators. The findings obtained for cPCL micro-/nano-fibers will help design and evaluate the next generation polymeric microactuators or micromanipulators.
The second part of the thesis studies the shape-memory effect (SME) of a single individual SMP micro-object by controlling deformation temperatures during programming and actuation temperatures during reversible change. In this work, microcuboids of crosslinked poly[ethylene-co-(vinyl acetate)] (cPEVA) elastomers with 18 wt% vinyl acetate (VA) contents were successfully prepared by template-based replication from polydimethylsiloxane (PDMS) mold. The micro-TME and micro-SMPA were observed and studied based on micro-geometry change using optical microscopy (OM) and AFM. Different switching temperatures of shape recovery were achieved from 55 °C to 86 °C by tuning Tp from 55 °C to 100 °C, indicating a successful implementation of micro-TME on individual microcuboid. For micro-SMPA functionalization, microcuboids were deformed by compression at 100 °C and the change in single particle height was monitored during cyclic heating and cooling between various Tseps from 60 °C to 85 °C and 20 °C. The micro-SMPA on a single microcuboid was achieved with a reversible strain in the range of 2 to 7%, whereby higher compression ratio CR and Tsep induced prominent reversible strain. The results achieved in this work demonstrated the successful functionalization of microcuboids with different SMEs by controlling temperatures during programming and actuation processes. Based on these achievements, such micro-objects can be further designed as on demand switchable microactuators or release systems with adjustable working temperatures.
In the last part of the work, a new function of shape-memory polymeric bi-stable 3D structured film was designed and fabricated. The SME and geometrical design of compliant mechanics were merged to enable switching between bi-stable and mono-stable states, which generate snap movement that mimics the Venus flytrap. A truncated tetrahedron structure with a slope angle as a tunable parameter to alter the bi-stability was chosen for the study to combine with
SME. It was anticipated that the structured film designed with a slope angle of 30° exhibited mono-stable behavior, and such a structure with a slope angle of 45° exhibited bi-stable behavior. Then the structured SMP film of designed mono-stable shape was successfully fabricated using soft lithography based on 3D printed master molds supported from digital manufacturing. The structured mold was also used in programming the SMP film into the structure with a higher slope angle to attain bi-stability. Finally, the switching between bi-stable and mono-stable states was successfully realized using SME, which introduces snapping movement triggered by heat. The implementation of compliant mechanisms by the SME increased the magnitude of thermally induced reconfiguration without additional external force.
To sum up, the results of the thesis support the development of smart objects capable of one-way micro-TME, free-standing reversible actuation, or bi-stability mediated shape-memory reconfiguration. Electrospinning and template-based method were used for fabrication with good control of geometry and low size dispersity. Microscopy methods especially the AFM platform with decent sensitivity was developed for implementation as well as characterization of SME on individual micro-/nanoobjects. Implementation of bi-stability improves the shape transformation amplitude of thermally triggered SMP. These findings can give novel insights for designing polymer-based actuators or soft robotics.