Der radiale Wasserfluss durch die feinen Seitenwurzeln von Schilf- und Mais ist vom radialen Teilchenfluss entkoppelt. Der radiale Wasserfluss wird bereits im Kortex der Wurzel durch den Protoplasten kontrolliert, da die Strömung auf dem apoplastischen Zellwandweg um die Protoplasten herum gegenüber der Strömung durch die Protoplasten nicht signifikant ist. Der radiale Reflexionskoeffizient der Wurzeln wird durch den Reflexionskoeffizient der Plasmamembran bestimmt. Die Feinwurzeln von Schilf- und Mais besitzen einen Reflexionskoeffizienten für Salze, Zucker, Zuckeralkohole und Polymere der sich nicht signifikant von eins unterscheidet. An intakten Wurzeln wurde dies durch die Abwesenheit von solvent drag für NaCl und Mannitol bei der Steigerung des Wasserflusses und der gleich großen hydraulischen Wirkung von osmotischen und hydrostatischen Kräften auf die Exsudation nachgewiesen. Die radialen Wände der Endodermis von Schilf- und Maiswurzeln sind keine perfekte Diffusionsbarriere. Liegen die genannten Stoffe in einer signifikanten Konzentration in der Zellwand vor permeieren sie passiv unter Umgehung der Protoplasten durch die Endodermis in die Xylemgefäße. Auch die Epidermis/Hypodermis der untersuchten Wurzeln hat die Eigenschaft einer semipermeablen Membran in der osmotische Druckgradienten einen Volumenfluss erzeugen. Es wurden zwei Methoden etabliert, mit denen sich der osmotische Druck des Xylemsaftes in isolierten Feinwurzeln bestimmen lässt. Die Feinwurzeln unterschieden sich hinsichtlich des osmotischen Druckes ihres Xylemsaftes und ihrer radialen hydraulischen Leitfähigkeit stark. Die bekannte Fähigkeit der Schilfpflanzen Natriumionen an der Sprossbasis aus dem Xylem zu eliminieren muss um Chloridionen erweitert werden. Die hohe Permeabilität der Endodermis für NaCl verringert die osmotische Wirkung des Brackwassers auf die Wasseraufnahme. Die Entkopplung der Salzaufnahme vom Wasserfluss vermeidet eine exzessive Salzbelastung des Sprosses. / Radial Water fluxes are not coupled to the radial solute fluxes in fine lateral roots of mays and reed. The radial water flow is already controlled by the protoplast in the cortical parenchyma as the hydraulic conductivity of the cell wall path circumventing the protoplasts is negligible compared to hydraulic conductivity of the pathway through the protoplast. The radial reflection coefficient of the root is defined by the reflection coefficient of the plasma membrane. Therefore fine laterals of the common reed (Phragmites australis) and maize (Zea mays) therefore exhibit a reflection coefficient for salts, sugars, alditols and polymers that is not significantly different from unity. This conclusion was drawn from the absence of solvent drag for NaCl and mannitol with increasing water flux and by the observation of equality of the hydraulic effect of both osmotic and hydrostatic forces on the exudation flow in intact roots of both plants. The radial walls of the endodermis are no absolute barrier for diffusion of small osmolytes. In the presence of high cell wall concentrations, the abovementioned osmolytes passively permeated into the xylem vessels at high rates circumventing the protoplast. The epidermis/hypodermis exhibits a semipermeable barrier as well wherein osmotic forces can create a radial volume flux. Two methods were established that allow for the determination of the flow direction and the osmotic pressure of the xylem sap in isolated fine laterals. Laterals differed strongly regarding their hydraulic conductivity and the osmotic pressure of their xylem sap. The known ability of the reed plant to remove sodium ions from the ascending sap has to be expanded for chloride. The high permeability of the endodermis for NaCl reduces the osmotic force of the brackish medium on water uptake. Uncoupling of radial water from the solute fluxes avoids the excessive permeation of NaCl and its accumulation in the assimilating leaves at high rates of transpiration.

Ice structuring proteins (ISPs) naturally exist in many foods consumed as part of the human diet including plants or fish. ISPs from winter wheat grass have gained interest in the pharmaceutical and food industries as a non-toxic, natural and cost-effective product, which is easy to prepare as a crude extract. However, they have not been purified reproducibly and studied in detail to elucidate their structures, mechanism of actions and difference(s). ISPs from the apoplast region of cold acclimated winter wheat leaves were extracted through vacuum infiltration and purified using heat and ethanol precipitations, size exclusion and anionic exchange fast protein liquid chromatography techniques. The ISPs showed both significant inhibition of ice growth and thermal hysteresis activities. The non-acclimated apoplastic extracts from winter wheat leaves contained similar proteins without any abovementioned activities. The ISPs contained disulfide bridges, similar to thaumatin-like proteins (TLPs) and partially similar to ISPs from winter rye leaves and carrot. ISPs remained active after thermal treatment (i.e., pasteurization conditions) and over a wide range of pH (3-12). There are very few quantitative assays to measure the activity of antifreeze proteins (AFPs, or Ice Structuring Proteins, ISPs), which often suffer from various inaccuracies and inconsistencies. Some methods rely only on unassisted visual assessment. When microscopy is used to measure ice crystal size, it is critical that standardized procedures be adopted, especially when image analysis software is used to quantify sizes. Differential Scanning Calorimetry (DSC) has been used to measure the thermal hysteresis activity (TH) of AFPs. In this study, DSC was used isothermally to measure enthalpic changes associated with structural rearrangements as a function of time. Differences in slopes of thermograms between winter wheat ISP or AFP type I containing samples, and those without ISP or AFP type I were demonstrated. ISP or AFP type I containing samples had much higher slopes compared to those without ISP or AFP type I. Samples with higher concentration of ISP or AFP type I showed higher slope values. The proteinaceous activity of ISPs or AFP type I was confirmed by demonstrating changes in samples with and without proteases. A proposed mechanism of this method is discussed.

Plant oriented movements, or tropisms allow the plant to actively respond to environmental stimuli to get more light, better access to nutrients and to grow roots deeper into the soil. Gravitropism drives the growth of roots along the gravity vector. Perception of gravity is triggered by the sedimentation of statoliths in columella root cap, but the exact signalling pathway behind this process is not known. Perception of gravity results in an unequal redistribution of the phytohormone auxin in the outer cell layers which leads to different rate of growth on the root's upper and lower side and bending of the root. The changes in auxin redistribution are accompanied by changes in apoplastic pH. Knowing an exact pattern of these pH changes could shed light on the mechanisms laying behind the gravitropic response pathway. While microelectrodes can be used to measure pH precisely, they are not suitable for the long-term imaging of growing roots. In the past few years, several pH sensitive dyes and genetically encoded sensors emerged. These can be used for long-term live in vivo imaging of pH changes in growing roots. In this thesis, I analysed the performance of several published pH sensitive genetically encoded sensors and available dyes in the roots of Arabidopsis thaliana. I observed that dyes varied...

Almost all conifer ovules produce a liquid secretion as part of reproduction. This secretion, termed an ovular secretion, is produced during ovule receptivity and is involved in pollen capture and transport. Historically, examinations of the ovular secretion have focused on how they are part of pollination mechanisms. As a result, the chemical composition of the ovular secretion has not been examined systematically. Investigations into the constituents of the ovular secretion were limited to analyses for simple water soluble compounds such as sugars, minerals, amino acids and organic acids. More recently, the protein component of the secretion has been investigated using mass spectrometry-based proteomics. Proteins involved in processes such as carbohydrate modification, proteolysis, and defence have been identified in conifer ovular secretions. This biochemical complexity suggests a broader view of the function of the ovular secretion is warranted. However, protein identifications only provide putative information on function. Functional characterization of these proteins is needed in order to fully understand how they contribute to ovular secretion function. The research outlined in this dissertation describes the first functional characterizations of proteins found in conifer ovular secretions. Three proteins - invertase, chitinase, and thaumatin-like protein - were characterized in the ovular secretions of Douglas-fir (Pseudotsuga menziesii) and hybrid yew (Taxus × media). The Douglas-fir ovular secretion is capable of converting sucrose to glucose and fructose, confirming that invertases present in the secretion are functional. The invertase activity was maximal at pH 4.0. Activity was 77% of maximal at pH 4.5, the physiological pH. This indicates that post-secretory hydrolysis of sucrose occurs in situ in the Douglas-fir ovular secretion. Invertases in the ovular secretion are likely involved in controlling the movement of carbohydrates to developing pollen and could facilitate pollen selection. Chitinases present in the Douglas-fir ovular secretion are functional at physiological conditions. All three modes of chitinolytic activity, i.e. endochitinase, chitobiosidase and β-N-acetylglucosaminidase, were detected at physiological pH. β-N-acetylglucosaminidase activity was 80 % of maximal at physiological pH. Chitinases are pathogenesis-related proteins capable of hydrolysing chitin in fungal cell walls. These results suggest the ovular secretion is capable of defending the ovule against infection by phytopathogens. Thaumatin-like protein was immunolocalized to the cell wall and amyloplasts in Douglas-fir and yew nucellar tissue in a pattern consistent with a defensive role. It was also localized to the cell wall of fungal spores and germinating hyphae that were present in the micropyle of a yew ovule. These results provide additional evidence for an antifungal role for the ovular secretion. Functioning enzymes involved in pollen-ovule interactions and ovule defence are present in the conifer ovular secretion. The ovular secretion has functions beyond pollen capture. A revised functional model for the conifer ovular secretion is proposed. / Graduate / 2021-08-17

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