During the development and lifecycle of multicellular organisms, cells have to die, and this occurs by a process called programmed cell death or PCD, which can be separated from necrosis or accidental cell death (Pennell and Lamb, 1997). Senescence is the terminal phase in the development of an organism, organ, tissue or cell, where nutrients are remobilized from the senescing parts of the plant into other parts, and the cells of the senescing organ or tissue undergo PCD if the process is not reversed in time. Leaf senescence involves cessation of photosynthesis, loss of pigments and proteins, nutrient remobilization, and degradation of the plant cells (Smart, 1994). Initiation of leaf senescence is triggered by a wide range of endogenous and environmental factors, that through unknown pathways controls the process, and regulates the expression of senescence-associated genes (SAGs) (Buchanan-Wollaston, 1997). Autumn leaf senescence in deciduous trees is regulated by photoperiod and temperature, and is an attractive experimental system for studies on senescence in perennial plants. We have studied the process of autumn senescence in a free-growing aspen (Populus tremula) by following changes in pigment, metabolite and nutrient content, photosynthesis, and cell and organelle integrity. All data were combined in a cellular timetable of autumn senescence in aspen. The senescence process started on September 11 with degradation of pigments and other leaf constituents, and once initiated, progressed steadily without being affected by the environment. Chloroplasts were rapidly degraded, and mitochondria took over energy production after chlorophyll levels had dropped by 50%. At the end of remobilization, around 29th of September, some cells were still metabolically active and had chlorophyll-containing plastids. Over 80% of nitrogen and phosphorus was remobilized, and a sudden change in the 15N of the cellular content on September 29, indicated that volatile compounds may have been released. We have also studied gene expression in autumn leaves by analysing EST sequences from two different cDNA libraries, one from autumn leaves of a field-grown aspen and the other from young, but fully expanded leaves of a green-house grown aspen. In the autumn leaf library, ESTs encoding metallothioneins, proteases, stress-related proteins and proteins involved in respiration and breakdown of macromolecules were abundant, while genes coding for photosynthetic proteins were massively downregulated. We have also identified homologues to many known senescence-associated genes in annual plants. By using Populus cDNA microarrays, we could follow changes in gene expression during the autumn over four years in the same free-growing aspen tree. We also followed changes in chlorophyll content to monitor the progression of leaf senescence. We observed a major shift in gene expression, occuring at different times the four years, that reflected a metabolic shift from photosynthetic competence to energy generation by mitochondrial respiration. Even though autumn senescence was initiated almost at the same date each year, the transcriptional timetables were different from year to year, especially for 2004, which indicates that there is no strict correlation between the transcriptional and the cellular timetables of leaf senescence.

The physiological process of cold acclimation protects plants native to the temperate regions of the world from the deleterious effects of low and freezing temperatures. This is achieved by a series of transcriptional, regulatory, and metabolic changes that enable continued growth and survival. Within minutes of exposure to temperatures below ca. 10°C, a complex cascade of transcriptional events is initiated to accomplish these changes. The initial alarm phase favors the rapid induction of a library of stress proteins with protective functions (e.g. COR proteins). This is followed by a cold hardened phase, characterized by maximal freezing tolerance, which continues until either the stress is removed, or the plant's metabolic and/or developmental state can no longer support maximal resistance. We have studied some of the important transcription factors and transcriptional changes associated with the initial alarm and later hardened phases of cold acclimation in the herbaceous annual Arabidopsis thaliana and the woody perennial Populus spp. We confirmed the functionality of the CBF-mediated signaling cascade in Poplar overexpressing AtCBF1, but noted that regulon composition and endogenous poplar CBF ortholog induction appeared to be tissue-specific. The lack of statistically significant DRE enrichment in the Poplar AtCBF1 regulons led us to investige cis-element abundance in the cold-associated transcription factor regulons of publicly available microarray data from Arabidopsis, leading to the development of a gene voting method of microarray analysis that we used to test for regulatory associations between transcription factors and their downstream cis-elements and gene targets. This analysis resulted in a new transcriptional model of the ICE1-mediated signaling cascade and implicated a role for phytochrome A. Application of this same method to microarray data from arabidopsis leaves developed at low temperature allowed us to identify a new cis-element, called DDT, which possessed enhancer-blocking function during the alarm stage of cold stress, but was enriched in the promoters of genes upregulated during the later cold hardened stages. As leaf growth and development at low temperature correlated with the enhancement freeze tolerance in Arabidopsis, we compared the transcriptomes of rapidly growing and fully grown poplar leaves at night (when both low temperatures and PhyA status might play important roles in nature), in the hopes of comparing this data with that of cold-treated leaves in the future. We identified the nocturnal mode of leaf growth in Populus deltoides as predominantly proliferative as opposed to expansive, and potentially linked to cellular carbohydrate status.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

The composition of lignin in the outer cell-wall layers of spruce and poplar has been studied and the data obtained have been compared with those of the mature reference wood in which the secondary cell wall predominates. Materials with exclusively or predominantly outer cell-wall layers were examined. Accurate data relating to the lignin monomer composition and the number of β-O-4´ bonds were obtained from pure middle lamella/primary cell wall lignin. Firstly, a 10 000 year old white spruce material, with most of the secondary cell wall missing, was studied. The aged lignin was composed of guaiacyl units only, and was slightly more condensed but otherwise similar to the reference lignin. Secondly, the developing xylem of a Norway spruce clone was analyzed during a growth season. In spring and early summer, growth is very rapid and the intention was to sample tissues in which the secondary cell-wall layers had not yet lignified, but where the outer layers at least had started to lignify. Microscopy, Klason lignin and carbohydrate analyses showed that the lignin in the developing xylem of samples from mid-June was located exclusively in the middle lamella. The lignin was more condensed, was composed of guaiacyl units only and contained more end-groups than the reference Norway spruce wood. Thirdly, the cambial tissues of a Balsam poplar clone were surveyed during a growth season. Both the phloem side and the xylem side of the cambial region were examined. The Klason lignin content and carbohydrate monomer distribution showed that in June and August the tissues on the phloem side contained material with mainly middle lamella/primary walls. In June, the xylem side in the cambial region contained mainly middle lamella/primary walls, and in August the secondary cell wall carbohydrates were being deposited. Both tissues contained lignin that was more condensed and had more end-groups than the reference lignin. In mid-June, the developing xylem had a ratio of syringyl to guaiacyl units of 0.6, whereas the ratio for the reference wood was 1.3. In the final study, lignin from the primary cell walls from a hybrid aspen cell suspension culture was investigated. The lignin contained only guaiacyl units which were more condensed than those observed in the reference poplar wood. / <p>QC 20100920</p>

Lignocellulosic plants are the most abundant source of terrestrial biomass and are one of the potential sources of renewable energy that can replace the use of fossil fuels. For a country such as Sweden, where the forest industry accounts for 10% of the total export, there would be large economical benefits associated with increased biomass yield. The availability of research on wood development conducted in conifer tree species, which represent the majority of the forestry in Sweden, is limited and the majority of research has been conducted in model angiosperm species such as Arabidopsis thaliana. However, the large evolutionary distance between angiosperms and gymnosperms limits the possibility to identify orthologous genes and regulatory pathways by comparing sequence similarity alone. At such large evolutionary distances, the identification of gene similarity is, in most cases, not sufficient and additional information is required for functional annotation. In this thesis, two high-spatial resolution datasets profiling wood development were processed; one from the angiosperm tree Populus tremula and the other from the conifer species Picea abies. These datasets were each published together with a web resource including tools for the exploration of gene expression, co-expression and functional enrichment of gene sets. One developed resource allows interactive, comparative co-expression analysis between species to identify conserved and diverged co-expression modules. These tools make it possible to identifying conserved regulatory modules that can focus downstream research and provide biologists with a resource to identify regulatory genes for targeted trait improvement. / Lignocellulosa är den vanligast förekommande källan till markburen biomassa och är en av de förnybara energikällor som potentiellt kan ersätta användningen av fossila bränslen. För ett land som Sverige, där skogsindustrin som står för 10 \% av den totala exporten, skulle därför en ökad produktion av biomassa kunna ge stora ekonomiska fördelar. Forskningen på barrträd, som utgör majoriteten av svensk skog är begränsad och den huvudsakliga forskningen som har bedrivits på växter, har skett i modell organismer tillhörande gruppen gömfröiga växter som till exempel i Arabidopsis thaliana. Det evolutionära avståndet mellan gömfröiga (blommor och träd) och nakenfröiga (gran och tall) begränsar dock möjligheten att identifiera regulatoriska system mellan dessa grupper. Vid sådana stora evolutionära avstånd krävs det mer än att bara identifiera en gen i en modellorganism utan ytterligare information krävs som till exempel genuttrycksdata. I denna avhandling har två högupplösta experiment som profilerar vedens utveckling undersökts; ett från gömfröiga träd Populus tremula och det andra från nakenföriga träd (barrträd) Picea abies. Datat som behandlats har publicerats tillsammans med webbsidor med flera olika verktyg för att bland annat visa genuttryck, se korrelationer av genuttryck och test för anrikning av funktionella gener i en grupp. En resurs som utvecklats tillåter interaktiva jämförelser av korrelationer mellan arter för att kunna identifiera moduler (grupper av gener) som bevaras eller skilts åt mellan arter över tid. Identifieringen av sådana bevarade moduler kan hjälpa att fokusera framtida forskning samt ge biologer en möjlighet att identifiera regulatoriska gener för en riktad förbättring av egenskaper hos träd.

PtaRHE1 is a poplar (Populus tremula x P. alba) gene encoding a REALLY INTERESTING NEW GENE (RING) domain-containing protein. RING proteins are largely represented in plants and play important roles in the regulation of many developmental processes as well as in plant-environment interactions. In this thesis, we present a functional characterization of PtaRHE1. To gain further insight into the role of this gene, molecular and genetic alteration approaches were used. The results of in vitro ubiquitination assays indicate that PtaRHE1 protein is a functional E3 ligase and this activity was shown to be specific with the human UbCH5a, among the tested ubiquitin-conjugating enzymes. Histochemical GUS stainings showed that the PtaRHE1 promoter is induced by plant pathogens and by elicitors such as salicylic acid and cellulase and is also developmentally regulated. In silico predictions and the transient expression of PtaRHE1-GFP fusion protein in N. tabacum epidermal cells revealed that PtaRHE1 is localized both in the plasma membrane and in the nucleus. The localization of expression of PtaRHE1 in poplar stem by in situ hybridization indicated that PtaRHE1 transcripts are localized within the cambial zone mainly in ray cells, suggesting a role of this gene in vascular tissue development and/or functioning. The overexpression of PtaRHE1 in tobacco resulted in a pleiotropic phenotype characterized by a curling of leaves, the formation of necrotic lesions on leaf blades, growth retardation as well as a delay in flower transition. Plant genes expression responses to PtaRHE1 overexpression provided evidence for the up-regulation of defence and/or programmed cell death (PCD) related genes. Moreover, genes coding for WRKY transcription factors as well as for MAPK, such as WIPK, were also found to be induced in the transgenic lines as compared to the wild type (WT). Taken together, our results suggest that the E3 ligase PtaRHE1 plays a role in the signal transduction pathways leading to defence responses against biotic and abiotic stresses. Identification of PtaRHE1 target(s) is required in order to fully assess the role of this E3 ligase in the ubiquitination-mediated regulation of defence response./<p>RÉSUMÉ<p><p><p>PtaRHE1 est un gène qui code pour une protéine possédant un domaine RING (REALLY INTERESTING NEW GENE) chez le peuplier (Populus tremula x P. alba). Les protéines de type RING sont très répandues chez les végétaux où elles jouent de rôles importants dans la régulation de plusieurs processus de développement et également dans les interactions plantes-environnement. Dans le cadre de ce travail, nous avons procédé à la caractérisation fonctionnelle du gène PtaRHE1. Dans le but de découvrir la fonction de ce gène, nous avons adopté une stratégie faisant usage d’approches moléculaires ainsi que de l’altération de l’expression génique. Les résultats obtenus montrent que la protéine PtaRHE1 est une E3 ligase et que cette activité enzymatique est spécifique à l’Ubiquitin-Conjugating enzym humaine UbCH5a. Les résultats du test histochimique GUS ont montré que le promoteur du gène PtaRHE1 est induit par des pathogènes et aussi par l’acide salicylique et la cellulase. Par ailleurs, ce promoteur est aussi régulé au cours du développement végétal. Les prédictions in silico et l’expression transitoire d’une fusion traductionnelle GFP-PtaRHE1, au niveau de l’épiderme des feuilles du tabac N. tabacum, ont révélé que la protéine PtaRHE1 se situe tant au niveau de la membrane cytoplasmique qu’au niveau du noyau. La localisation de l’expression du gène PtaRHE1, par les techniques d’hybridation in situ, montre que les transcrits de ce gène se retrouvent principalement au niveau des cellules de rayon, dans la zone cambiale, suggérant que ce gène pourrait jouer un rôle dans le développement ou la formation du tissu vasculaire. La surexpression du gène PtaRHE1 chez le tabac a conduit à l’obtention d’un phénotype pléiotropique caractérisé par un recroquevillement (incurvation) des feuilles, la formation des lésions nécrotiques sur le limbe, un retard de croissance ainsi qu’un retard dans la transition florale. L’analyse de la réponse de l’expression de différents gènes à la surexpression de PtaRHE1 a mis en évidence l’induction des gènes liés à la défense et ou à la mort cellulaire programmée. En outre, l’expression des gènes codant pour des facteurs de transcription WRKY et aussi des MAPKs, tel que WIPK, était aussi plus élevée chez les plantes transgéniques comparées au type sauvage. Les résultats de ce travail suggèrent que PtaRHE1, comme E3 ligase, pourrait jouer un rôle dans la transduction des signaux cellulaires conduisant aux réactions de défense contre les stress biotiques et abiotiques. L’identification de la (des) cible(s) de PtaRHE1 est indispensable pour la compréhension du rôle de cette protéine dans la régulation des réponses de défense par l’intermédiaire de l’ubiquitination.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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Primary cell walls are a composite of cellulose microfibrilsand hemicelluloses. Xyloglucan is the principal hemicelluloseof primary cell walls of dicotyledons. Xyloglucanendotransglycosylases (XETs) cleave and religate xyloglucanpolymers in plant cell walls. A XET (PttXET16A) from hybridaspen has been heterologously expressed and characterized inour lab. To study XETs enzymology on a molecular level a series ofnovel xyloglucan oligosaccharides (XGOs) have been synthesized.The chromogenic 2-nitrophenol XGO and fluorogenic XGOs havebeen used as kinetic probes for PttXET16A. The first 3-Dstructure of the XET and of the enzyme-substrate complexrevealed new insights into the requirements fortransglycosylation. Cellulose fibers are an important raw material for manyindustries. In a novel chemo-enzymatic approach, thetransglycosylating activity of XET was used for biomimeticfiber surface modification. The aminoalditol XGO derivate wasused as key intermediate to incorporate novel chemicalfunctionality into xyloglucan. TheXGO derivatives wereintegrated into xyloglucan with PttXET16A. The resultingmodified xyloglucan was used as a versatile tool fiber surfacemodification.

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