The elucidation of the binding of marijuana’s psychoactive compound, (-)-D9- tetrahydrocannabinol (THC), to specific membrane receptors, in the early 1990s, led to the identification of endogenous arachidonate-based lipids that activate cannabinoid receptors in mammals. While the metabolic and signaling pathway for these 20 carbon N-acylethanolamines (NAE) and their derivatives has been well characterized in mammals, thus far, only 12-18 carbon NAEs have been identified in plants and their metabolic pathway has been partly characterized. In plants, NAEs have been shown to modulate a number of physiological processes, including seed and seedling development and ability to respond to stress; however, the mechanisms by which they function remain to be elucidated. Our recent identification of a 20C NAE (arachidonylethanolamide) in moss provided us with an exciting possibility to identify receptor-mediated endocannabinoid signaling responses in plants that is akin to mammals. In this seminar, I will provide insights into the past, present and future aspects of plant endocannabinoid research.

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As one of the first land plant groups to diversify, mosses are central in understanding the origin, diversification, and early function of stomata. Unlike tracheophytes that have stomata on anatomically complex leaves and stems, mosses bear stomata exclusively on spore-bearing organs (capsules). However, stomata do not occur in all mosses and, indeed, are absence in the earliest-divergent mosses (Takakia, Andreaea, Andreaeobryum and Sphagnum), suggesting that stomata originated in mosses independently of other plants. The occurrence of structurally unique pseudostomata in Sphagnum further confounds the resolution of homology of moss stomata with those of other plants. The five studies included in this dissertation are aimed at clarifying the structure, development and evolution of moss stomata. The first study focuses on the sporophyte anatomy and stomatal ultrastructure in two structurally and phylogenetically divergent mosses, Oedipodium and Ephemerum. Oedipodium is the sister to peristomate mosses and the first extant moss with true stomata. This monospecific genus has an elaborated capsule with an extended apophysis bearing numerous long-pored stomata. In contrast, Ephemerum nests within the peristomate mosses and has a reduced capsule that lacks an apophysis and has a few round-pored stomata. Ultrastructure of stomata is similar in these two mosses and comparable to that of tracheophytes, except that the stomata of mosses are not as structurally distinct from epidermal cells as are tracheophyte stomata. Anatomical features such as the presence of a cuticle, water-conducting cells, and spongy tissues with large areas for gas exchange are more pronounced in Oedipodium sporophytes and support the role of stomata in gas exchange and water transport during development and maturation. The second study examines changes in pectin composition during development in the model moss Funaria. Stomatal movement in tracheophytes requires guard cell walls to be strong, yet flexible, because they have to undergo reversible deformation to open and close the pore. Pectins are necessary for wall flexibility and proper stomatal functioning in seed plants. In this study of Funaria, immunogold-labeling using five antibodies to pectin epitopes was conducted on guard cell walls during development to relate these features to the limited movement of stomata in moss. Movement of Funaria stomata coincides with capsule expansion when guard cell walls are thin and pectinaceous. Walls dramatically increase in thickness after pore formation and the pectin content significantly decreases in mature guard cell walls, suggesting that a decrease in flexibility is responsible for the inability to open a close previously reported in older moss guard cells. Because this was the first study to demonstrate changes in pectin composition during stomatal development in any plant, a similar study was done on Arabidopsis to identify the main types of pectins in guard cell walls. Localization of pectins in guard cell walls of Arabidopsis is similar to mosses in the stage they can move, with homogeneous walls rich in arabinan pectins that are required for wall flexibility. This study extends knowledge of pectin composition from stomata of the moss Funaria with limited stomatal movement to an angiosperm in which stomatal activity is crucial to the physiological health of the plant. The fourth study describes stomata development and internal changes in sporophyte anatomy that lead to formation of air spaces in the moss Funaria. Developing sporophytes at different stages were examined using light, fluorescence and electron microscopy; immunogold-labeling was used to investigate the presence of pectin in the newly formed cavities. Stomata in mosses do not develop from a self-generating meristemoid like in Arabidopsis, but instead they originate from a protodermal cell that differentiates directly into a guard mother cell. Epidermal cells develop from protodermal or other epidermal cells, i.e., there are no stomatal lineage ground cells. This developmental pattern is congruent with the presence of a gene ortholog of FAMA, but not SPCH and MUTE, in Physcomitrella. The final study in this dissertation focuses on the enigmatic Sphagnum. Although true stomata are absent in early-divergent mosses, Sphagnum has specialized epidermal cells, pseudostomata, that partially separate but do not open to the inside. To further understand the structure, function and evolution of pseudostomata, capsule anatomy and ultrastructure of pseudostomata were detailed. As in moss stomata, pseudostomata wall architecture and behavior facilitate capsule dehydration, shape change, and dehiscence, supporting this common function. Unlike other moss stomata, pseudostomata collapse along their ventral walls and they lack a substomatal cavity. Similarities to true stomata include two modified epidermal cells with specialized cell walls that separate by cuticle deposition and respond to drying. Pseudostomata may be interpreted as modified stomata that suppressed substomatal cavity formation, which in turn eliminated pore development. However, clarification of the homology of pseudostomata and moss stomata will require genomic studies integrated with physiological and structural data. The studies described in this dissertation significantly advance our understanding of moss stomatal development and structure, and provide a comparison point to better evaluate the evolution of stomata. Moss capsule anatomy coupled with the exclusive existence of stomata on capsules supports the concept that stomata in moss are involve in gas exchange but also facilitate drying and dispersal of spores. Changes in wall architecture coupled with a decrease in total pectin explain the inability of mature stomata to move. Development and distribution of stomata in Funaria provides evidence of a direct and less elaborated mechanism for stomatal development than described in Arabidopsis. Resolving relationships among early land plants, especially hornworts and mosses, the only bryophyte groups with stomata, is critical to understanding stomata evolution. Evaluated together, the results of this dissertation are consistent with a single origin of stomata in land plants.

Physcomitrella patens, a haploid-dominant land plant, has increasingly become useful in molecular genetic studies and is a model for early land plant evolution. This thesis work explores the mucilage secretory hair ontology, development, and ultrastructure with microscopic methods. Axillary hair development parallels that of secretory tissues found in other mosses and ultrastructure shares important similarities with liverwort mucilage papillae. These mucilage secretory structures cover the developing apex and young leaves with mucilage for protection. Changes in the hair cell wall and mucilage secretion are mediated by pectin and wall modification. Using bioinformatic methods, this thesis also investigates protein-protein interactions in Physcomitrella to understand the molecular mechanisms governing pectin biosynthesis and modification.

Ocenění podniku působícího v oboru nákladní kamionové dopravy a komplexních logistických služeb prostřednictvím výnosové metody DCF ve variantě FCFF se zohledněním leasingového financování při výpočtu WACC.

Lipids are the main constituents of the cell membrane and maintain its fluidity. Plants undergo various changes in lipids under environmental stresses and alter the membrane fluidity and permeability. Membrane lipids mostly contain a polar or neutral head group and fatty acid tails that vary in length and degree of unsaturation. The composition of membrane affects its physicochemical properties and ability to tolerate stress. The moss Physcomitrium patens is an early land plant with unique ability to tolerate stressors like cold and dehydration. During its life cycle, for the most part, mosses remain as gametophytes, multiplying asexually. The period from germination of spores into filamentous protonema, which give rise to gametophyte is transient. They enter reproductive sporophytes stage only under cold temperatures. Because of the diverse roles of these developmental stages and the time span they are exposed to the environment, we hypothesized that these stages share distinct lipid content and composition. To this extent, using LC-MS/MS methods we carried out lipidomic analyses of five developmental stages of the moss. We identified and quantified the major and minor lipid classes (types) and their acyl composition of protonema, early, mid and late gametophyte and sporophyte tissues. Galactolipids, which typically occur in the plastid were predominant in green tissues and thus most abundant in the vegetative tissues but not in sporophytes. Throughout the life cycle, among the phospholipids, phosphatidylcholine was the abundant lipid, a feature that is typical of plant membranes. Sporophyte tissues, however, were distinct from gametophyte and protonema and also other vascular plants with high amounts of phosphatidic acid (PA). In plants, PA typically accumulates in response to stress; it is likely that the low temperature cue necessary for sporophyte formation is associated with spike in PA and needs further investigation. In comparing the acyl composition of the various lipid classes, we identified that in addition to 34C and 36C lipids, moss lipids also contain 38C and 40C, which are not represented in vascular plants. We predict that the occurrence of long-chain, highly unsaturated lipids might contribute to the dynamic nature of the membrane and stability under stress. This study serves as a primary resource to further investigate the role of specific lipids and acyl groups in maintaining membrane properties. Overall, it aids to our understanding of the evolution of stress tolerance in early land plants that coped through harsh environmental conditions during their transition from water to land.