@phdthesis{Staiger2022,
  author    = {Staiger, Simona},
  title     = {Chemical and physical nature of the barrier against active ingredient penetration into leaves: effects of adjuvants on the cuticular diffusion barrier},
  doi       = {10.25972/OPUS-19937},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199375},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Agrochemicals like systemic active ingredients (AI) need to penetrate the outermost barrier of the plant, known as the plant cuticle, to reach its right target site. Therefore, adjuvants are added to provide precise and efficient biodelivery by i.a. modifying the cuticular barrier and increasing the AI diffusion. This modification process is depicted as plasticization of the cuticular wax which mainly consists of very long-chain aliphatic (VLCA) and cyclic compounds. Plasticization of cuticular waxes is pictured as an increase of amorphous domains and/or a decrease of crystalline fractions, but comprehensive, experimental proof is lacking to date. Hence, the objective of this thesis was to i) elucidate the permeation barrier of the plant cuticle to AIs in terms of the different wax fractions and ii) holistically investigate the modification of this barrier using selected oil and surface active adjuvants, an aliphatic leaf wax and an artificial model wax. Therefore, the oil adjuvant methyl oleate (MeO) and other oil derivatives like methyl linolenate (MeLin), methyl stearate (MeSt) and oleic acid (OA) were selected. Three monodisperse, non-ionic alcohol ethoxylates with increasing ethylene oxide monomer (EO) number (C10E2, C10E5, C10E8) were chosen as representatives of the group of surface active agents (surfactants). Both adjuvant classes are commonly used as formulation aids for agrochemicals which are known for its penetration enhancing effect. The aliphatic leaf wax of Schefflera elegantissima was selected, as well as a model wax comprising the four most abundant cuticular wax compounds of this species. Permeation, transpiration and penetration studies were conducted using enzymatically isolated cuticles of Prunus laurocerasus and Garcinia xanthochymus. Cuticular permeability to the three organic solutes theobromine, caffeine and azoxystrobin differing in lipophilicity was measured using a steady-state two-chamber system separated by the isolated leaf cuticles of the evergreen species P. laurocerasus and G. xanthochymus. Treating the isolated cuticles with methanol selectively removed the cyclic fraction, and membrane permeability to the organic compounds was not altered. In contrast, fully dewaxing the membranes using chloroform resulted in a statistically significant increase in permeance for all compounds and species, except caffeine with cuticles of G. xanthochymus due to a matrix-specific influence on the semi-hydrophilic compound. Crystalline regions may reduce the accessibility to the lipophilic pathway across the waxes and also block hydrophilic domains in the cuticle. Knowing that the aliphatic wax fraction builds the cuticular diffusion barrier, the influence of the adjuvants on the phase behaviour of an aliphatic cuticular wax as well as the influence on the cuticular penetration of AIs were investigated. Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) were selected to investigate the phase behaviour and thus possible plasticization of pure Schefflera elegantissima leaf wax, its artificial model wax comprising the four most abundant compounds (n-nonacosane, n-hentriacontane, 1-triacontanol and 1-dotriacontanol) and wax adjuvant mixtures. DSC thermograms showed a shift of the melting ranges to lower temperatures and decreased absolute values of the total enthalpy of transition (EOT) for all adjuvant leaf wax blends at 50 \% (w/w) adjuvant proportion. The highest decrease was found for C10E2 followed by MeO > OA and C10E8 > MeLin > MeSt. The aliphatic crystallinity determined by FTIR yielded declined values for the leaf and the artificial wax with 50 \% MeO. All other adjuvant leaf wax blends did not show a significant decrease of crystallinity. As it is assumed that the cuticular wax is formed by crystalline domains which consist of aliphatic hydrocarbon chains and an amorphous fraction comprising aliphatic chain ends and functional groups, the plasticizers are depicted as wax disruptors influencing amorphization and/or crystallization. The adjuvants can increase crystalline domains using the aliphatic tail whereas their more hydrophilic head is embedded in the amorphous wax fraction. DSC and FTIR showed similar trends using the leaf wax and the model wax in combination with the adjuvants. In general, cuticular transpiration increased after adding the pure adjuvants to the surface of isolated cuticles or leaf envelopes. As waxes build the cuticular permeation barrier not only to AIs but also to water, the adjuvant wax interaction might affect the cuticular barrier properties leading to increased transpiration. Direct evidence for increased AI penetration with the adjuvants was given using isolated cuticles of P. laurocerasus in combination with the non-steady-state setup simulation of foliar penetration (SOFP) and caffeine at relative humidity levels (RH) of 30, 50 and 80 \%. The increase in caffeine penetration was much more pronounced using C10E5 and C10E8 than MeO but always independent of RH. Only C10E2 exhibited an increased penetration enhancing effect positively related to RH. The role of the molecular structure of adjuvants in terms of humectant and plasticizer properties are discussed. Hence, the current work shows for the first time that the cuticular permeation barrier is associated with the VLCAs rather than the cyclic fraction and that adjuvants structurally influence this barrier resulting in penetration enhancing effects. Additionally, this work demonstrates that an artificial model wax is feasible to mimic the wax adjuvant interaction in conformity with a leaf wax, making it feasible for in-vitro experiments on a larger scale (e.g. screenings). This provides valuable knowledge about the cuticular barrier modification to enhance AI penetration which is a crucial factor concerning the optimization of AI formulations in agrochemistry.},
  subject      = {Adjuvans},
  language  = {en}
}
@phdthesis{BergmannBueno2021,
  author    = {Bergmann Bueno, Amauri},
  title     = {Ecophysiological adaptations of cuticular water permeability of plants to hot arid biomes},
  doi       = {10.25972/OPUS-16783},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167832},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Arid environments cover almost one-third of the land over the world. Plant life in hot arid regions is prone to the water shortage and associated high temperatures. Drought-stressed plants close the stomata to reduce water loss. Under such conditions, the remaining water loss exclusively happens across the plant cuticle. The cuticular water permeability equals the minimum and inevitable water loss from the epidermal cells to the atmosphere under maximally stomatal closure. Thus, low cuticular water permeability is primordial for plant survival and viability under limited water source. The assumption that non-succulent xerophytes retard water loss due to the secretion of a heavier cuticle is often found in the literature. Intuitively, this seems to be plausible, but few studies have been conducted to evaluate the cuticular permeability of xerophilous plants. In chapter one, we investigated whether the cuticular permeability of Quercus coccifera L. grown in the aridest Mediterranean-subtype climate is indeed lower than that of individuals grown under temperate climate conditions. Also, the cuticular wax chemical compositions of plants grown in both habitats were qualitatively and quantitatively analysed by gas-chromatography. In few words, our findings showed that although the cuticular wax deposition increased in plants under Mediterranean climate, the cuticular permeability remained unaltered, regardless of habitat. The associated high temperatures in arid regions can drastically increase the cuticular water permeability. Thereby, the thermal stability of the cuticular transpirational barrier is decisive for safeguarding non-succulent xerophytes against desiccation. The successful adaptation of plants to hot deserts might be based on finding different solutions to cope with water and heat stresses. Water-saver plants close the stomata before the leaf water potential drastically changes in order to prevent damage, whereas water-spender plants reduce the leaf water potential by opening the stomata, which allow them to extract water from the deep soil to compensate the high water loss by stomatal transpiration. In chapter two, we compare the thermal stability of the cuticular transpiration barrier of the desert water-saver Phoenix dactylifera L. and the water-spender Citrullus colocynthis (L.) Schrad. In short, the temperature-dependent increase of the cuticular permeability of P. dactylifera was linear over the whole temperature range (25-50°C), while that of C. colocynthis was biphasic with a steep increase at temperatures ≥ 40°C. This drastic increase of cuticular permeability indicates a thermally induced breakdown of the C. colocynthis cuticular transpiration barrier, which does not occur in P. dactylifera. We further discussed how the specific chemical composition of the cutin and cuticular waxes might contribute to the pronounced thermal resistance of the P. dactylifera cuticular transpiration barrier. A multitude of morpho and physiological modifications, including photosynthetic thermal tolerance and traits related to water balance, led to the successful plant colonisation of hot arid regions over the globe. High evaporative demand and elevated temperatures very often go along together, thereby constraining the plant life in arid environments. In chapter 3, we surveyed cuticular permeability, leaf thermal tolerance, and cuticular wax chemical composition of 14 non-succulent plant species native from some of the hottest and driest biomes in South-America, Europe, and Asia. Our findings showed that xerophilous flowering plants present high variability for cuticular permeability and leaf thermal tolerance, but both physiological features could not be associated with the species original habitat. We also provide substantial evidence that non-succulent xerophytes with more efficient cuticular transpirational barrier have higher leaf thermal tolerance, which might indicate a potential coevolution of these features in hot arid biomes. We further discussed the efficiency of the cuticular transpiration barrier in function to the cuticular wax chemical composition in the general discussion section.},
  subject      = {Plant cuticle},
  language  = {en}
}