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.

Les triacylglycérol (TAG) est un métabolite hautement énergétique qui peut être facilement converti en biodiesel. Les TAG peuvent être produits à partir de plantes et de microalgues. L'étude des voies de signalisation de l'énergie peut offrir de nouvelles stratégies pour améliorer l'accumulation de biomasse et de TAG sans compromettre la croissance. Dans cette thèse, j'ai étudié le rôle de deux voies principales de signalisation énergétique: la voie du de guanosine ppGpp (guanosine penta(tétra) phosphate) dans le chloroplaste et la voie de TOR (Target of rapamycin) dans le cytosol. J'ai choisi de travailler sur la mousse Physcomitrella patens, un modèle d'eucaryote photosynthétique en raison de sa position évolutive entre les algues et les plantes vasculaires. Pour étudier le rôle du ppGpp, nous avons créé des lignées transgéniques exprimant de manière inductible une ppGpp synthase et sur-accumulant le ppGpp. J'ai trouvé que l'induction de SYN provoque une forte inhibition de la capacité photosynthétique en raison de l'inhibition de l'expression des protéines clés codées par les chloroplastes et aussi une réorganisation des membranes thylakoïdes. Pour l’étude de TOR nous avons traité la mousse avec des inhibiteurs de TOR et montré que cela provoque l’inhibition de la croissance de manière dose dépendante et l’accumulation de TAG. L’utilisation des marqueurs lipidiques a révélé la perte de petites vésicules associées à la croissance et l'accumulation de plus grandes structures de corps lipidiques. Des études supplémentaires permettront de développer des stratégies pour améliorer la production de biocarburants chez les organismes photosynthétiques. / Triacylglycerol (TAG) is a highly energetic metabolite that can be easily converted into biodiesel. TAG can be produced from both plants and microalgae. However, plants have low TAG yields in their dominant vegetative tissues. Microalgae can accumulate high amounts of TAG, but only under stress, leading to growth inhibition and limiting yield. The study and manipulation of stress and energy signaling pathways can offer new strategies to improve biomass and TAG accumulation without compromising growth. In this thesis, I studied the role of two major energy signaling pathways: the guanosine penta(tetra) phosphate (ppGpp) pathway in the chloroplast and the target of rapamycin (TOR) pathway in the cytosol. I chose to work on the moss Physcomitrella patens which is an interesting model of photosynthetic eukaryote because of its evolutionary position between algae and vascular plants. To study the role of ppGpp we created transgenic lines that inducibly express a ppGpp synthase and over-accumulate ppGpp. I found that ppGpp accumulation causes a strong inhibition of photosynthetic capacity due to the inhibition of the expression of key chloroplast encoded proteins, and also reorganization of the thylakoid membrane system into super grana. For the TOR pathway, we treated P. patens protonema with active site TOR inhibitors and showed that this cause growth inhibition in a dose dependent manner and is accompanied by TAG accumulation. The use of lipid dyes reveals a shift from small growth associated vesicles to a larger oil body structures after treatment. Further studies will allow the development of new strategies for improving biofuel production in photosynthetic organisms.

No description available.

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.

Zugl.: Karlsruhe, Univ., Diss., 2007 / Hergestellt on demand. - Auch im Internet unter der Adresse http://uvka.ubka.uni-karlsruhe.de/shop/isbn/978-3-86644-216-0 verfügbar

Zugl.: Karlsruhe, Univ., Diss., 2007

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The plant metabolite, strigolactone, has recently gained the status of phytohormone as the result of several studies that implicated its role in plant architecture. These studies would characteristically rely on the use of mutants, such as the rms lines that were generated in peas, that shared several characteristics. This method allowed for the identification of several genetic component of the shared pathway. It is now known that the biosynthesis of strigolactone is dependent on the sequential action of an isomerase (D27) and two carotenoid cleavage deoxygenases (CCD7 and CCD8). Furthermore, it is known that strigolactone perception is localised to the plant nucleus, where it interacts with an α/β-fold hydrolase (D14) which would concomitantly binds to target proteins. The F box protein (MAX2) is able to recognize this proteïen complex. Through a MAX2 dependent mechanism the target protein becomes tagged for proteolysis. However, this model, though intricate, has only really been shown in higher plants. The model bryophyte, Physcomitrella patens, serves as a useful tool in genetic studies due to its predisposition for homologous recombination. More recently it has also gained interest in studies pertaining to strigolactones, which has led to the generation of a Ppccd8Δ mutant. Compared to the wild type, the Ppccd8Δ line produces more protonemal tissue. Furthermore, exogenous strigolactones have also been shown to inhibit colony expansion. Here we shown that there is only a single candidate gene, PpMAX2, present in the P. patens genome that could serve as a homologue for the Arabidopsis thaliana MAX2. Furthermore, we show that a recombinant GFP:PpMAX2 localises to the nucleus of P. patens cells. A Ppmax2:: mutant was generated which, unexpectedly, did not show the phenotype of Ppccd8Δ. Ppmax2:: has an apparent inability to produce protonema and appears to rather dedicate its growth to the production of gametophores. A double mutant, Ppccd8Δ max2Δ was generated which also displayed the characteristic phenotype of Ppmax2::. It seems therefore that the activity of PpMAX2 is able to override that of PpCCD8. By employing a GUS reporter system, we showed that the promoter, PPpMAX2, is predominantly active within gametophore tissues. Taken together, these results suggest that the activity of PpMAX2 facilitates the transition of gametophore tissue to protonema tissue. Although exogenous strigolactones did not appear to affect the growth of the Ppmax2:: line as it did the PpWT or Ppccd8Δ lines, those responses that have been ascribed to strigolactones to date have mostly been observed in protonemal tissue. We therefore suspect that any strigolactone response that might have been elicited in Ppmax2:: would have been masked by its phenotype of predominantly protonemal tissue. We are therefore hesitant to make any sweeping statements in regards to the role PpMAX2 might have in strigolactone perception in P. patens. However, though we suspect that PpMAX2 might not be a true functional homologue for the characterised MAX2 homologues from higher plants, we suspect that it may well be the ancestral predecessor of MAX2. / AFRIKAANSE OPSOMMING: Strigolaktoon is ‘n metaboliet wat deur plante vervaardig word en is redelik onlangs as ’n fitohormoon geklassifiseer. Die klassifikasie as fitohormoon is die gevolg van verskeie studies wat strigolaktoon se rol in die plantstruktuur beklemtoon het. In hierdie studie is daar gebruik gemaak van mutante, soos onderandere die rms lyne, wat gegenereer is in ertjies, wat verskeie kenmerke deel. Sodoende is verskeie komponente van ’n gedeelde molekulêre padweg geïdentifiseer. Daar word tans verstaan dat die sintese van strigolaktoon afhanklik is van die stapsgewyse aksies van ’n isomerase (D27) en twee karotenoïedklewingsdeoksigenases (CCD7 en CCD8). Verder is dit bekend dat strigolaktoon waargeneem word in die plant nukleus deur te assosieer met ’n α/β-vou-hidrolase (D14) wat vervolgens met teikenproteïene bind. Die kompleks word deur ’n F-boks proteïen (MAX2) herken wat daartoe lei dat die teikenproteïen gemerk word vir proteolise; altans, dit is tans die model wat vir hoër plante aanvaar word. Die model briofiet, Physcomitrella patens, word dikwels aangewend in genetiese studies weens dit ’n hoër vatbaarheid vir homoloë rekombinasie het. Om P. patens te benut in navorsing wat die rol van strigolaktoon ondersoek is ook voordelig, aangesien daar reeds ’n Ppccd8Δ mutant beskikbaar is. In vergelyking met die wilde tipe, produseer Ppccd8Δ meer protonemale weefsel en blyk dit dat strigolaktoon die vermoë het om kolonie verspreiding te bekamp. Hier wys ons dat daar ’n enkele kandidaat geen, PpMAX2, in die genoom van die P. patens teenwoordig is wat as ’n homoloog vir die Arabidopsis thaliana MAX2 kan dien. Verder wys ons dat ’n rekombinante GFP:PpMAX2 proteïen wel na die selkern van P. patens selle lokaliseer. ’n Ppmax2:: mutant is gegenereer wat, onverwags, nie die fenotipe van Ppccd8Δ vertoon het nie. Ppmax2:: het ’n onvermoë om protonema te produseer en wy groei eerder aan die produksie van gametofiete. ’n Dubbele mutant, Ppccd8Δ max2Δ, is gegenereer wat ook die fenotipe van Ppmax2:: vertoon het; dus kom ons tot die gevolgtrekking dat die aktiwiteit van PpMAX2 dié van PpCCD8 oorheers. Deur gebruik te maak van ’n GUS verklikkersisteem kon ons aflei dat die aktiwiteit van die PPpMAX2 promotor hoofsaaklik tot die uitdrukking van PpMAX2 in gametofiet weefsel lei. Dit is moontlik dat die aktiwiteit PpMAX2 dus die oorgang van gametofoor weefsel na protonema weefsel te weg bring. Alhoewel strigolaktoon nie die groei van die Ppmax2:: lyn beïnvloed soos vir die PpWT of Ppccd8Δ lyne nie, vermoed ons dat die reaksie slegs in die protonemale weefsel waargeneem sal word. Daar kan tans nie met absolute sekerheid gesê word of PpMAX2 enigsins verbonde met strigolaktoon persepsie in mos is nie, tog vermoed ons dat PpMAX2 ’n primitiewe voorloper vir die gekarakteriseerde MAX2 homoloë van die hoër plante is.

This study focused on the primary cell wall constituents and plasmodesmata (PD) density in three mosses and four liverwort apical cells (AC) and immediate derivatives. The three mosses have tetrahedral apical cells and the liverworts possess tetrahedral, hemidiscoid and lenticular AC geometries. The primary cell wall in the studied taxa is comprised of two layers. A fibrillar layer, which is the outer wall layer, contains compacted cellulosic fibrils, and represents the two adjacent primary walls and middle lamella, the latter of which is rarely discernible. An electron-lucent inner wall layer abuts the plasma membrane. This layer has faint fibrous materials that extend from the plasma membrane to the fibrillar layer. Generally, as the cell wall ages it thickens, the fibrillar layer increases in width while the electron-lucent wall stays more or less consistent in width. In the four liverworts, the most recent wall of the AC has the highest PD density in the apical region regardless of AC geometry. As the walls elongate, primary wall is laid down between PD, separating them and resulting in lower densities and wider PD diameters in older walls. The season of fixation and whether plants were studied from nature or culture have an influence on AC ultrastructure. A developmental study of Physcomitrella patents gametophores in four stages, bud, 2-leaved, 7-8-leaved and ~20-leaved, reveals that the primary cell wall constituents change slightly during development. Specifically, LM5 a RG-I pectin antibody against the galactan branch epitope is only localized in the fibrillar layer of young water-conducting cells in the 7-8-leaved and 20-leaved gametophores. LM20, an antibody against HG esterified pectins, does not localize in any of the cell walls during development. The distribution patterns for AGPs (JIM13 and LM2) are consistent during gametophore development and predominantly localize on the electron-lucent layer and wall/plasma membrane interface. However, LM2 is mainly localized on the fibrillar layer in 7-8-leaved cell walls. AGPs also localize on element of the cytoplasm. LM6, an antibody against an RG-I pectin with arabinan branch epitopes, also localizes AGPs and because it expressed similar distribution patterns as JIM13 and LM2 on the cell wall, it likely localizes AGP in Physcomitrella. In addition, LM6 localizes pectins on the fibrillar layer similar to LM5 and LM19 for HG unesterified pectins. Callose predominantly localizes at the PD neck region. This study provides the first documentation of changes in size and shape of AC with age in Physcomitrella patens gametophores. The PD densities of gametophytes examined in this study fall into the lineage-specific network of PD (LPD) group designated for sporophytes of monilophytes and Selaginella (heterosporous lycophyte) with single ACs. Takakia lepidozioides leafy shoot has a tetrahedral AC with a highly curved free surface. This peculiar moss has mucilage hair (MH) associated with axil of phyllids. Mucilage hair in both species are 3-celled with a forth epidermal cell as the base. However, occasional 2-celled MH is seen in T. ceratophylla. The ultrastructure of MH has similarities with other mosses and liverworts.