Physcomitrella patens has become a model system to study plant biology. 8 cellulose synthase (CesA) genes were identified by searching against Physcomitrella EST database. Two of these genes, PpCesA6 and PpCesA7 are the first full-length CesAs to be identified. These two genes are highly similar to each other, both on the cDNA and genomic DNA levels. They both have 13 introns and 12 exons. The first introns are more than 1kb. The proteins they encode both have 1096 amino acids. There are only three amino acid differences in the proteins they encode. PpCesA6 and PpCesA7 share 74% amino acid identity with Monterey pine (Pinus radiate) PrCesA10, 72% amino acid identity with quaking aspen (Populus tremuloide) PtrCesA6, 71% amino acid identity with maize (Zea mays) ZmCesA7 and three rice (Oryza sativa) CesAs, 65%-68% amino acid identity with Arabidopsis CesAs. The deduced proteins of PpCesA6 and PpCesA7 contain the D, D, D, QXXRW motif in the form of DDG, DCD, TED, QVLRW, which is the catalytic region of cellulose synthases. Two other pairs of CesA genes, PpCesA3 and PpCesA8, PpCesA4 and PpCesA10, also show high similarity. PpCesA2 and PpCesA9 are pseudogenes. By taking advantage of the high efficiency homologous recombination in Physcomitrella nuclear DNA, a C-terminus GFP fusion construct was produced for PpCesA6. Expression analysis showed that PpCesA6 is expressed in both protonemata and young gametophore. In protonemata, PpCesA6 is expressed in both chloronema and caulonema cells, but not in every cell. In young gametophore, PpCesA6 is expressed in axillary hairs and rhizoids. Confocal miscrocopy study shows that PpCesA protein is localized on the plasma membrane and it is randomly dispersed. The gene targeted knockout constructs of PpCesA6 and PpCesA7 were produced. The null mutants of PpCesA6 and PpCesA7 single knockout as well as double knockout were generated by the PEG (polyethylene glycol)-mediated protoplast transformation. Both single knockout mutants did not show obvious phenotypic differences from the wild type. The double knockout mutants had reduced stem length. The stem lengths of the wild type, PpCesA6 knockout mutant, PpCesA7 knockout mutant and double knockout mutant growing on BCD and BCDAT media were 3.93±0.45mm and 3.51±0.08mm, 3.82±0.46mm and 3.5±0.3mm, 3.65±0.68mm and 3.73±0.49mm, 2.75±0.22mm and 2.65±0.43mm, respectively. A cellulose synthase-like C gene (CslC4) was identified by searching against the Physcomitrella EST and genomic DNA databases. The protein it encodes is 694 amino acids. The D, D, D, QXXRW motif is in the form of DDS, DAD, VED, QQHRW. PpCslC4 genomic DNA has 4 small introns in the coding region. There is also one small intron at the 5'-UTR. The deduced PpCslC4 protein shows 72% similarity with PpCslC2 and PpCslC3, 65% similarity with PpCslC1. When compared with other organisms, PpCslC4 protein shows more than 60% similarity with Arabidopsis and Oryza sativa CslC proteins. A gene targeted knockout construct was produced for PpCslC4. The null mutants were generated by the PEG-mediated protoplast transformation. PpCslC4 mutant did not show any obvious phenotypic differences from the wild type.

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Plant Biology." Includes bibliographical references (p. 191-229).

No description available.

Mosses are bryophytes with a simple cellular organization and distinctive growth stages. With their unique lipid profile, most mosses are also tolerant to various stressors. A ubiquitous class of bioactive fatty acid ethanolamides in eukaryotes called N-acylethanolamines (NAEs) also occurs in the moss Physcomitrella patens. Unlike in higher plants, where saturated and unsaturated NAE types are limited to those with acyl chains 12C to 18C, P. patens also contains anandamide, NAE 20:4. In higher plants, NAEs are most abundant in desiccated seeds and mediate plant growth, development, cellular organization and response to stress, in an abscisic acid (ABA)-dependent or independent manner. In mammals, NAE 20:4 acts as an endocannabinoid ligand and mediates a multitude of physiological responses. This unique NAE type, NAE 20:4 is hypothesized to effect development, growth and cellular organization of P. patens. To determine the role of NAEs in moss development, NAE content and composition in protonema, early and late gametophyte stages, and sporophytes, will be quantified from their total lipid extracts, using selective lipidomics. The effects of anandamide on growth will be studied by culturing moss in the presence of exogenous NAE 20:4 in a dose-dependent manner. Temporal changes in growth patterns will be determined by the evaluation of digital images using Image tool. The effect of anandamide on cytoskeletal organization will be visualized by immunostaining the phyllodes exposed to NAE 20:4 and observing them under confocal microscope. Preliminary results indicate that the endogenous NAE content and composition is variable, depending on the developmental stage and that NAE 20:4 is a potent negative regulator of moss growth. More detailed studies are expected to provide novel insights into the role NAEs, specifically NAE 20:4 might play in mediating growth and development of seedless plants.

No description available.

Global demand for food, which is expected to double by 2050, presents plant biologists with a major challenge to generate higher yields on existing croplands. The long-term goal of the proposed research is to generate stress tolerant plants and improve crop productivity. Stress responses, in plants or animals, most often involve the activation stress-signaling pathways. One pathway is mediated by a group of bioactive lipid molecules, N-acylethanolamines (NAEs), and is highly conserved among eukaryotes. NAEs are composed mainly of amides of ethanolamine and fatty acids. In model Arabidopsis thaliana (At), NAEs inhibit germination and growth in seedlings via abscisic-dependent and independent mechanisms aimed at stress regulation. This work focuses on identifying the NAE pathway in the moss, Physcomitrella patens (P. patens), that shows higher resistant to stress than At and consequently the role played by NAE metabolites in the development of P. patens. Mosses are small seedless and fruitless early plants that have evolved successful mechanisms of surviving prolonged stresses during their transition from aquatic life to land. Previous studies have shown that P. patens is tolerant to high temperature, salinity and osmotic shock, to which most plants are fragile. I hypothesize that the NAE pathway is present in P. patens and that it plays a significant role in its development. NAE composition and metabolizing enzymes like FAAH and NAPE synthase will be measured to ascertain NAE pathway in P. patens. Further quantification of NAE content and composition during development will identify the key developmental time points that are associated with high metabolite levels. Total lipids will be extracted in triplicates from twelve tissue samples harvested at protonema stage; mature gametophyte stage which is subdivided into leafy tissue and rhizoids; antheridia and archegonia stage; zygotic stage and the mature and germinating developmental stages. The high lipid content of the mature spores was previously associated with extreme longevity in ephemeral habitats. It would be of interest to determine if such an association exists with NAE metabolite content. Key time points will then be used to determine the effect of exogenous NAE 12:0. Preliminary analysis shows that P. patens have higher levels of NAEs than other plants examined. Furthermore, fatty acid amide hydrolases (FAAH) that hydrolyses NAEs were identified in silico. Putative PpFAAH1 candidate showed 95 % homology with previously characterized AtFAAH in At. These results show the occurrence of NAEs in P. patens and a possible enzyme for its hydrolysis. Further confirmation of the NAE pathway in P. patens will be done by in silico identification of the precursor enzyme NAPE synthase. Understanding how P. patens tolerates stress using NAEs pathways by identifying key time points in its development will help other researchers know which specific NAEs affect the growth of P. patens and will facilitate the characterization of metabolic enzymes at specific times.

The discovery of a mammalian endocannabinoid, anandamide (N-arachidonylethanolamide; AEA or NAE 20:4) in Physcomitrella patens but not in higher plants prompted our interest in characterizing its metabolism and physiological role in the early land plant. Anandamide acts as an endocannabinoid ligand in the mammalian central and peripheral systems and mediates various physiological responses. Endocannabinoid signaling is terminated by a membrane-bound fatty acid amide hydrolase (FAAH). Based on sequence identity and in silico analyses, we identified nine orthologs of human and Arabidopsis FAAH in P. patens (PpFAAH1 to PpFAAH9). Predicted structural analysis revealed that all the nine PpFAAH contain characteristic amidase signature sequence with a highly conserved catalytic triad and share a number of key features of both plant and animal FAAH. These include a membrane binding cap, membrane access channel, substrate binding pocket and as well as potential for dimerization. Among the nine, gene expression levels for PpFAAH1 and PpFAAH9 were enhanced with exogenous anandamide treatment. Further cloning and heterologous expression, followed by radiolabeled in vitro enzyme assays revealed that PpFAAH1 activity was optimal at 37 °C and pH 8.0. Furthermore, PpFAAH1 showed higher specificity to NAE 20:4 than to other N-acylethanolamines such as NAE 16:0. Highest in planta amide hydrolase activity was noted in microsomes of gametophyte tissues, suggesting the possibility for membrane localization of active FAAH. Interestingly, when FAAH1 was overexpressed, the moss cultures not only showed reduced growth but their transition from protonemal stage to gametophyte was inhibited, which was rescued in part by exogenous AEA. Unlike overexpressors of AtFAAH1, which showed enhanced growth and hypersensitivity to abscisic acid, PpFAAH1 overexpressors showed tolerance to abscisic acid. Together, these data suggest that the occurrence of anandamide and distinct properties of PpFAAH1 in early land plants have physiological implications that are different from that of higher plants.

Fatty acid amide hydrolase (FAAH) belongs to a diverse class of enzymes in amidase signature family. In mammals, FAAH is targeted to affect neurological functions because it terminates endocannabinoid signaling by degrading anandamide, a 20C N-acylethanolamine (NAE 20:4). In higher plants, FAAH is known to modulate growth, development and stress tolerance by degrading 12-18C NAEs. Since anandamide was reported to exclusively occur in early land plants, we investigated its metabolic pathway in the moss Physcomitrella patens. Based on the highest identity with ratFAAH, we identified nine orthologs in moss, PpFAAH1 to PpFAAH9. Several bioinformatic tools were used to understand the structural basis of how catalytic residues fold for amidohydrolase activity. Based on these in silico analyses of PpFAAH homologs and their gene expression in response to saturated (NAE16:0) and unsaturated NAE (NAE 20:4) treatment, PpFAAH1 was selected for biochemical characterization. Heterologously expressed PpFAAH1 showed highest amidohydrolase activity at 37°C and pH 8.0. Both in vivo and in vitro studies showed that unsaturated NAE substrate is hydrolyzed faster than the saturated NAE (> 10-fold in vivo and 50-fold in vitro). Additionally, transgenic moss lines over expressing FAAH1 showed slower growth and disrupted gametophyte formation when compared to wild type. These data suggest that PpFAAH1-mediated NAE metabolism is likely involved in developmental transition in moss.

No description available.

Serine/Arginine-rich proteins (SR-proteins) have been well characterized in metazoans and in the flowering plant Arabidopsis thaliana. But so far no attempts on characterizing SR-proteins in the moss Physcomitrella patens have been done. SR-proteins are a conserved family of splicing regulators essential for constitutive- and alternative splicing. SR-proteins are mediators of alternative splicing (AS) and may be alternatively spliced themselves as a form of gene regulation. Three novel SR-proteins of the SR-subfamily were identified in P. patens. The three genes show conserved intron-exon structure and protein domain distribution, not surprising since the gene family has evidently evolved through gene duplications. The SR-proteins PpSR40 and PpSR36 show differential tissue-specific expression, whereas PpSR39 does not. Tissue-specific expression of SR-proteins has also been seen in A. thaliana. SR-proteins determine splice-site usage in a concentration dependent manner. SR-protein overexpression experiments in A. thaliana and Oryza sativa have shown alteration of splicing patterns of endogenous SR-proteins. Overexpression of PpSR40 did not alter the splicing patterns of PpSR40, PpSR36 and PpSR39. This suggests that they might not be a substrate for PpSR40. These first results of SR-protein characterization in P. patens may provide insights on the SR-protein regulation mechanisms of the common land plant ancestor.