Land plants are characterized by the alternation of two generations: the haploid gametophyte and the diploid sporophyte. As land plants evolved from bryophytes to vascular plants, the sporophyte became the dominant generation in the life cycle. The sporophytes of bryophytes are developmentally simple structures characterized by determinate growth. In contrast, the sporophytes of vascular plants diverged to become highly variable and often complex structures with indeterminate growth. KNOTTED1-LIKE HOMEOBOX (KNOX) genes encode homeodomain containing transcription factors that are key regulators of sporophyte development. KNOX genes are divided into two subclasses, class 1 and class 2. The critical role of class 1 KNOX genes in the apical growth of all extant land plants studied to date, suggests that modifications to class 1 KNOX gene function may have played an important role in sporophyte evolution. However, the nature of any such modifications is largely unknown. In this study, a number of cross-species complementation experiments were carried out to determine the extent to which class 1 KNOX gene function is conserved between different land plant groups. The role of KNOX genes in hornworts was also investigated because hornworts are believed to be the sister group to vascular plants. The work presented in this thesis demonstrates that the function of class 1 KNOX genes is conserved between lycophytes and angiosperms. In contrast, the function of class 1 KNOX genes appears to have diversified between bryophytes and vascular plants. Collectively, these results indicate increased complexity of class 1 KNOX gene function during the evolution of land plants.

N-acylethanolamines (NAEs) are family of lipid-derived signaling molecules in many organisms, which include an endocannabinoid N-arachidonoylethanolamide (anandamide, AEA, NAE 20:4). NAEs, specifically AEA plays pivotal role in mammalian neurological and physiological functions; however their metabolism and functional implications in plants are yet to be fully discovered. Unlike seed plants, bryophytes possess unique fatty acid composition that includes 20:4 and 20:5, which prompted our search for endocannabinods in moss Physcomitrella patens. To this extent, we used targeted lipidomic analyses and discovered long-chain NAEs and their corresponding N-acyl-phosphatidylethalamine (NAPE) precursors in an early land plant species. In protonemal tissues N-arachidonyl-PE and N-20:5-PE contributed to about 49% and 30%, respectively. Matured gametophytes on the other hand showed a 12% increase in N-20:4-PE and 20% decline in N-20:5-PE, relative to NAPE content in protonema. In all haploid developmental stages analyzed NAE 20:4 levels contributed to ~ 23% of the total NAE while NAE 20:5 remained as a minor component (5%). Interestingly, in Selaginella moellendorffi, an early vascular plant N-18:2-PE species was most abundant; although minor amounts of N-20:3-PE, N-20:4-PE and N-20:5-PE were present, only a small quantity of NAE 20:4 was identified among the 20C NAEs. Both AEA and it corresponding fatty acid, arachidonic acid have growth inhibitory effects in a dose dependent manner. Biological implications of anandamide and its metabolic pathway in moss are under investigation. Our data reveals an evolutionarily conserved occurrence of NAE metabolites in early land plants, with an exclusive report of AEA presence in a bryophyte.

No description available.

Endocannabinoids are derivatives of arachidonate-based lipids that activatea network of signaling pathways in eukaryotes...

N-acylethanolamines (NAEs) are family of lipid-derived signaling molecules in many organisms, which include an endocannabinoid N-arachidonoylethanolamide (anandamide, AEA, NAE 20:4). NAEs, specifically AEA plays pivotal role in mammalian neurological and physiological functions; however their metabolism and functional implications in plants are yet to be fully discovered. Unlike seed plants, bryophytes possess unique fatty acid composition that includes 20:4 and 20:5, which prompted our search for endocannabinods in moss Physcomitrella patens. To this extent, we used targeted lipidomic analyses and discovered long-chain NAEs and their corresponding N-acyl-phosphatidylethalamine (NAPE) precursors in an early land plant species. In protonemal tissues N-arachidonyl-PE and N-20:5-PE contributed to about 49% and 30%, respectively. Matured gametophytes on the other hand showed a 12% increase in N-20:4-PE and 20% decline in N-20:5-PE, relative to NAPE content in protonema. In all haploid developmental stages analyzed NAE 20:4 levels contributed to ~ 23% of the total NAE while NAE 20:5 remained as a minor component (5%). Interestingly, in Selaginella moellendorffi, an early vascular plant N-18:2-PE species was most abundant; although minor amounts of N-20:3-PE, N-20:4-PE and N-20:5-PE were present, only a small quantity of NAE 20:4 was identified among the 20C NAEs. Both AEA and it corresponding fatty acid, arachidonic acid have growth inhibitory effects in a dose dependent manner. Biological implications of anandamide and its metabolic pathway in moss are under investigation. Our data reveals an evolutionarily conserved occurrence of NAE metabolites in early land plants, with an exclusive report of AEA presence in a bryophyte.

Anandamide (N-arachidonoylethanolamide, AEA), a 20C polyunsaturated (PU) N-acylethanolamine (NAE) influences many neurological functions in mammals. Although 20C PU-NAEs are considered unique to animals, they were recently discovered in early land plants but their metabolism and functions remain unknown. Comprehensive lipidomic analyses of Physcomitrella patens revealed not only abundance of arachidonic acid (AA, 20:4) and eicosapentaenoic acid (EPA, 20:5) but also their corresponding ethanolamides (AEA and EPEA, respectively). While moss showed increasing AA with development, 14% and 24% in protonemata and gametophyte tissues, respectively, EPA decreased from 7% in protonemata to ~1.3 % in gametophytes. An increase in 20:4- and decrease in 20:5- ethanolamides and their corresponding membrane precursors, phosphatidylethanolamides, also was observed during gametophyte development. Pharmacological studies revealed that AEA specifically inhibits polarized tip growth, which justifies the low endogenous levels of AEA in protonemata. To further determine the physiological relevance of these 20C PU-NAEs, a fatty acid amide hydrolase that catabolizes NAEs has been heterologously characterized. Furthermore, generation of metabolite mutants with altered NAE levels is underway. Overall, we identified two novel NAEs, AEA and EPEA in Physcomitrella, which may play an important role in regulation of moss growth and development, although the underlying mechanism is still unclear.

The endogenous arachidonate-based lipids that activate cannabinoid receptors have been well characterized in mammals. In plants only 12-18 carbon fatty acid ethanolamides have been identifi ed so far and have been shown to modulate a number of physiological processes including seed and seedling development. However, since moss plants contain arachidonic acid, we hypothesized the occurrence of arachidonate-based metabolites in their tissues. Using selective lipidomics approach, we identifi ed the presence of anandamide or arachidonylethanolamide (a 20C polyunsaturated fatty acid ethanolamide) and its precursors, in Physcomitrella patens that were previously not reported in plants. Comprehensive lipid profi les for protonema and gametophyte tissues of moss also revealed the occurrence of other saturated and unsaturated fatty acid ethanolamides and a distinct phospholipid and galactolipid composition. Further studies showed that anandamide, like abscisic acid, inhibits the growth of gametophytes more severely than saturated fatty acid ethanolamides. Our current studies are focused on understanding the physiological and developmental role of polyunsaturated fatty acid ethanolamides in nonseed plants. In conclusion, discovery of anandamide in moss provided us with an exciting possibility to identify fatty acid ethanolamide metabolic pathway in early land plants and elucidate receptor-mediated endocannabinoid signaling responses in plants that is akin to mammals.

No description available.

The colonization of the arid continental surface by plants was one of the milestones in Earth's history. Morphological innovations, such as the origin of complex 3D tissues, allowed the successful colonization and radiation of plants on land. The epidermis is the outermost plant tissue that constitutes the interface between the plant and the environment. Thus, the evolution of epidermal cells was crucial for the adaptation of plants on the terrestrial arid environment. I undertook a combined approach that aims to understand the evolutionary trends that drove land plant colonization and the genetic mechanisms that underlie the development of the epidermis. This approach includes: 1) analyses of plant transcription factors (TFs) families distribution and diversification, with a particular focus on the basic Helix-Loop-Helix (bHLH) TF family, and 2) functional characterization of a putatively conserved bHLH TF subfamily involved in epidermal cell development in land plants. Here, I showed that there was a stepwise increase in the number of transcription factor (TF) families and bHLH subfamilies that predated the colonization of the terrestrial surface by plants. The subsequent increase in TF number on land was through duplication within pre-existing TF families and subfamilies. Moreover, a similar trend occurred in metazoan bHLH TF, suggesting that the majority of innovation in plant and metazoan TF families occurred in the Precambrian before the Phanerozoic radiation of land plants and metazoans. Furthermore, I demonstrated that the function of IIIf bHLH TFs in controlling the development of the epidermal cell layer is conserved between liverworts and angiosperms. This suggests that IIIf bHLH TFs are ancient and conserved regulators of epidermal cell development since the early colonization of the land by plants. Moreover, these bHLH TFs were recruited during the evolution of land plants to control the development of seemingly unrelated morphological characters in specific lineages of extant land plants. The recruitment of ancient developmental regulators to control distinct and unrelated developmental processes in land plants might underlie the huge morphological and taxonomic radiation of plants on land.

A cladistic analysis was carried out to resolve phylogenetic pattern among bryophytes and other land plants. The analysis used 22 taxa of land plants and 90 characters relating to male gametogenesis. Coleochaete or Chara/Nitella were the outgroups in various analyses using HENNIG86, PAUP, and MacClade, and the land plant phylogeny was unchanged regardless of outgroup utilized. The most parsimonious cladograms from HENNIG86 (7 trees) have treelengths of 243 (C.I. = 0.58, R.I. = 0.82). Bryophytes are monophyletic as are hornworts, liverworts, and mosses, with hornworts identified as the sister group of a liverwort/moss assemblage. In vascular plants, lycophytes are polyphyletic and Selaginella is close to the bryophytes. Lycopodium is the sister group of the remaining vascular plants (minus Selaginella). Longer treelengths (over 250) are required to produce tree topologies in which either lycophytes are monophyletic or to reconstruct the paraphyletic bryophyte phylogeny of recent authors. This analysis challenges existing concepts of bryophyte phylogeny based on more classical data and interpretations, and provides new insight into land plant evolution.