Les modèles climatiques prédisent pour le futur une accentuation de la fréquence et de l'intensité des épisodes de sécheresse, ce qui affecterait sérieusement les écosystèmes forestiers. En conséquence, il y a une demande croissante pour du matériel végétal plus résistant à la sécheresse, et pour la compréhension des mécanismes génétiques et physiologiques de la tolérance des arbres à la sécheresse. Dans des conditions de sécheresse, la tension dans les vaisseaux du xylème augmente, et la cavitation peut se produire causant une embolie du vaisseau qui devient alors non fonctionnel. La vulnérabilité du xylème à la cavitation est corrélée à la tolérance à la sécheresse, indiquant l'importance de ce caractère pour la tolérance à la sécheresse. Cependant, peu était connu sur la variabilité de ce caractère au niveau intra-spécifique et ses bases génétiques étaient inconnues. Dans un premier temps, nous avons démontré que la vulnérabilité à la cavitation du xylème de peuplier (Populus tremula x Populus alba) s'acclimate à des conditions de sol plus sec et que ceci s'accompagne de changements dans la structure du xylème et d'expressions géniques. Ce processus d'acclimatation appuie l'hypothèse du rôle important joué par la vulnérabilité du xylème à la cavitation dans la tolérance à la sécheresse. Dans un second temps, nous avons étudié les bases structurales et génétiques de la vulnérabilité à la cavitation grâce à deux approches. La première a consisté à étudier les changements anatomiques et d'expressions géniques se produisant au cours de l'acclimatation de la vulnérabilité du xylème à la cavitation à des conditions plus sèches. Nous avons déterminé que l'augmentation de la vulnérabilité à la cavitation dans des conditions plus sèches est corrélée à une diminution du diamètre de la paroi de la ponctuation. Nous avons observé des changements d'expression géniques dans des conditions de sécheresse mais ceux ci n'ont pas pu être reliés à un changement de la vulnérabilité à la cavitation. Dans une seconde approche, nous avons utilisé dix lignées de peupliers dont l'expression de gènes impliqués dans le métabolisme de la lignine ont été modifiés et deux lignées surexprimant une pectine méthylestérase (PME) pour examiner le rôle de ces gènes dans la vulnérabilité à la cavitation. Chez les peupliers ayant un métabolisme des lignines modifié, nous avons également testé la relation entre les propriétés hydriques et mécaniques. Nous apportons des preuves que les lignines et les pectines (à travers les PME) sont impliquées dans la vulnérabilité à la cavitation et nos données sur les lignées transgéniques de peupliers ne soutiennent pas un lien exclusif entre les propriétés hydriques et mécaniques.

Diss. (sammanfattning). Umeå : Sveriges lantbruksuniv., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Boreal forests are often strongly nitrogen (N) limited. However, human activities are leading to increased N inputs into these ecosystems, through atmospheric N deposition and forest fertilization. N input into boreal forests can promote net primary productivity, increase herbivore and pathogen damage, and shift plant species composition and community structure. Genetic diversity has been suggested as a key mechanism to promote a plant species’ stability within communities in response to environmental change. Within any plant population, specific traits (e.g. growth and defense traits) can vary substantially among individuals, and a greater variation in traits may increase chances for the persistence of at least some individuals of a population, when environmental conditions change. One aspect of plant chemistry that can greatly vary among different genotypes (GTs) are condensed tannin (CTs). These secondary metabolites have been suggested to affect plant performance in many ways, e.g. through influencing plant growth, the interactions of plants with herbivores and pathogens, and through affecting litter decomposition, and hence the return of nutrients to plants. To investigate how genotypic variation in foliar CT production may mediate the effects that anthropogenic N enrichment can have on plant performance and litter decomposition, I performed a series of experiments. For these experiments, aspen (Populus tremula) GTs with contrasting abilities to produce foliar CTs (i.e. low- vs. high-tannin producers) were grown under 3 N conditions, representing ambient N (+0 kg ha-1), upper level atmospheric N deposition (+15 kg ha-1), and forest fertilization rates (+150 kg ha-1). This general experimental set-up was once established in a field-like environment, from which natural enemies were excluded, and once in a field, in which enemies were present. In my first two studies, I investigated tissue chemistry and plant performance in both environments. I observed that foliar CT levels decreased in response to N in the enemy‑free environment (study I), but increased with added N when enemies were present (study II). These opposing responses to N may be explained by differences in soil N availability in the two environments, or by induction of CTs after enemy attack. Enemy damage generally increased in response to N, and was higher in low-tannin than in high-tannin plants across all N levels. Plant growth of high‑tannin plants was restricted under ambient and low N conditions, probably due to a trade-off between growth and defense. This growth constraint for high‑tannin plants was weakened, when high amounts of N were added (study I and II), and when enemy levels were sufficiently high, so that benefits gained through defense could outweigh the costs of defense production (study II). Despite those general responses of low- and high‑tannin producers to added N, I also observed a number of individual responses of GTs to N addition, which in some case were not connected to the intrinsic ability of the GTs to produce foliar CTs. In study III, gene expression levels in young leaves and phenolic pools of the plants that were grown in the enemy‑free environment were studied. This study revealed that gene control over the regulation of the phenylpropanoid pathway (PPP) was distributed across the entire pathway. Moreover, PPP gene expression was higher in high-tannin GTs than in low‑tannin GTs, particularly under ambient N. At the low N level, gene expressions declined for both low- and high-tannin producers, whereas at the high N level expression at the beginning and the end of the PPP was upregulated and difference between tannin groups disappeared. Furthermore, this study showed that phenolic pools were frequently uncorrelated, and that phenolic pools were only to some extent related to tannin production and gene expression. In study IV, I investigated the decomposability of litter from the field plants. I found that N enrichment generally decreased mass loss, but there was substantial genetic variation in decomposition rates, and GTs were differentially responsive to added N. Study IV further showed that CTs only had a weak effect on decomposition, and other traits, such as specific leaf area and the lignin:N ratio, could better explain genotypic difference in mass loss. Furthermore, N addition caused a shift in which traits most strongly influenced decomposition rates. Collectively, the result of these studies highlight the importance of genetic diversity to promote the stability of species in environments that experience anthropogenic change. / Boreala skogar är ofta mycket kväve (N) begränsade. Men mänskliga aktiviteter leder till ökad N tillförsel i dessa ekosystem, både genom depostition av N från atmosfären och skogsgödsling. N-tillförsel i boreala skogar kan främja netto primärproduktionen men även leda till ökade skador från naturliga fiender (herbivorer och patogener) samt skiftningar i växtartsammansättning. Genetisk mångfald har föreslagits som en viktig mekanism för att främja en växtarts stabilitet inom samhällen som upplever miljöförändringar. Inom varje växtpopulation kan specifika egenskaper (t.ex. tillväxt och försvar) varierar kraftigt mellan individer och en större variation i egenskaper kan öka chanserna för att åtminstone några individer från en population överlever ifall miljöförhållandena förändras. En aspekt av växtkemi som i hög grad kan variera mellan olika genotyper (GT) är bladens kondenserade tanniner (KT). Dessa sekundära metaboliter har föreslagits påverka växtens prestationsförmåga på många sätt, t.ex. genom att påverka tillväxt, interaktioner mellan växter och herbivorer eller patogener och genom att påverka förna nedbrytning, och följaktligen återbördandet av näringsämnen till kretsloppet. För att undersöka hur genotypiska variation i KT produktion kan påverka de effekter som antopogent N kan ha på växtens prestationsförmåga och förna nedbrytning, utförde jag en serie experiment. Jag studerade olika asp (Populus tremula) GT med olika förmåga att producera KT (låg- och hög-tannin producenter). Växterna odlades i tre olika N förhållanden, som representerade ambient N nivå (+0 kg ha-1), atmosfärisk N deposition = låg nivå (+15 kg ha-1), och skogsgödsling = hög nivå (150 kg ha‑1). Dessa GT etablerades i en fält-liknande miljö där naturliga fiender uteslutits och i ett fält där naturliga fiender var närvarande. I mina första två studierna undersökte jag vävnadskemi och växternas prestationsförmåga i de båda miljöerna. Jag observerade att KT nivåerna sjönk till följd av N‑tillsats i den fiende-fria miljön (studie I), men ökade med N-tillsats ifall fiender var närvarande (studie II). Dessa motsatta reaktioner på N-tillsats kan förklaras av skillnader i N-tillgång mellan de två odlingsplatserna eller genom ökad KT produktion som respons på angrepp. Skador orsakade av herbivorer och patogener ökade generellt till följd av N‑tillsats och var högre i låg-tannin än i hög‑tannin producerande GT oavsett N‑förhållande. Tillväxten hos växter från hög‑tannin GT begränsades i ambient- och låg N-tillsats förhållanden, troligen på grund av att avvägning mellan tillväxt och försvar förskjutits emot försvar. Den begränsade tillväxten i hög-tannin växter minskade om stora mängder N tillsattes (studie I och II) och om antalet fiender var tillräckligt högt så att nyttan av försvaret kunde uppväga kostnaderna för försvarsproduktionen (studie II). Trots dessa generella respons hos låg- och hög-tannin GT till följd av N‑tillsats observerade jag även ett antal individuella respons hos GT som i vissa fall var orelaterade till växters förmåga att producera KT. I studie III undersöktes genuttrycksnivåer och fenolinnehåll i blad från växter som odladats i en miljö där naturliga fiender exkluderats. Denna studie visade att fenylpropanoidsyntesvägen (FPV) regleras genom kontroll av många av de undersökta FPV-generna. Dessutom var FPV genuttryck högre i hög-tannin GT än i låg-tannin GT, särskilt vid ambient N. Vid låg N-tillsats minskade genuttrycket av FPV-gener i både låg- och hög-tannin producenter, medan hög N-tillgång ledde till att gener i början och slutet av FPV uppreglerades och till att skillnaderna mellan tannin grupperna försvann. Dessutom visade studien att de separata fenol-poolerna ofta var okorrelerade med varandra och att fenol-poolerna bara till viss del var korrelerade med KT produktion och FPV-genutryck. I studie IV undersökte jag nedbrytningshastigheten för förnan från fältodlade aspar. Jag upptäckte att N-tillsats generellt minskade viktförlusten men att det fanns en betydande genetisk variation mellan GT och att dessa även var olika mottagliga för tillsatt N. Studie IV visade vidare att KT endast hade en svag effekt på nedbrytning och att andra egenskaper såsom specifik bladyta och lignin:N ratio kunde bättre förklara den genotypiska skillnaden i viktförlust. Dessutom orsakade N‑tillsats en förskjutning av vilka egenskaper som mest påverkade förnans nedbrytningshastighet. Sammanfattningsvis visar mina studier på vikten av genetisk mångfald för att främja växtartens stabilitet i miljöer som upplever antropogena förändringar.

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Darbo objektas – Populus genties medžiai: Populus alba × Populus tremula (Nr. 20), Populus tremula x Populus alba ( Nr.21), P. tremuloides x P. tremula (Nr.8), P. tremuloides x P. tremula (Nr.3), P. tremuloides x P. tremula (Nr.9), Populus alba (Š16), P. berolinensis. Darbo tikslas – nustatyti tuopų genties medžių hibridų mikrodauginimo in vitro sąlygas, bei augalų- regenerantų išauginimą. Išskirti genominę DNR iš drebulių, naudojant genominės DNR išskyrimo rinkinį NucleoSpin Plant. Darbo metodai - Eksplantų paruošimas sterilinimui ir sterilinimas. Augalų regenerantų kultivavimo sąlygos. Maitinamosios terpės ruošimas, sterilinimas ir sudėtis. Mikroūglių perkėlimas į nesterilias sąlygas. DNR išskyrimas. Darbo rezultatai. Tuopų medžių eksplantų sterilinimui tikslinga naudoti aseptinių tirpalų kombinaciją be „ACE“, kad užtikrinti gyvybingų ir be užkrato eksplantų išlikimą. Grybine infekcija labiausiai užkrėsti berlyninės tuopos eksplantai: užkratas sudarė nuo 72 proc. iki 80 proc. priklausomai nuo eksplantų prigimties. Tuopų genties medžių genotipas turi įtakos morfogenezei audinių kultūroje. Geriausia maitinamoji terpė mikroūglų susidarymui yra Murashige Scoog (MS) su citokininu BA- 0,5 mg/l. Kaliaus susidarymas ir spalva priklauso nuo genotipo. Iš visų tuopų hibridų sparčiausiai augo hibridas Nr. 8 P.( tremuloides x P. tremula). Vidutinis jo augimo greitis- 8,3 mm per 10 dienų. Atlikti DNR tyrimai APPD metodu parodo, kad motinmedžiai ir išauginti augalai in vitro yra... [toliau žr. visą tekstą] / Aim of the work: Genus Populus trees: Populus alba × Populus tremula (Nr. 20), Populus tremula x Populus alba (Nr.21), P. tremuloides x P. tremula (Nr.8), P. tremuloides x P. tremula (Nr.3), P. tremuloides x P. tremula (Nr.9), Populus alba (Š16), P. berolinensis. Object of the work: Investigate poplar genus hybrids in vitro micro reproduction conditions and regenerate plants growth. Investigate poplar genomic DNA using NucleoSpin Plant Kit. Methods: Preparation of explants culture for sterile manner. Plants regenerate rear conditions. Growth media preparation and sterilization. Micro sprout input into unsterile conditions. DNA isolation. Results: For poplar trees explants sterilization is purposeful using aseptic solution combination without “ACE”, to vouch vital and half explants survival. Fungous infection found in berolinensis poplar explants: infections reach from 72 to 80 % depending from explants nature. Poplar genus genotype has impact on morphogenesis in tissue culture. The best growth media is Murashige Scoog (MS) with citotoksine BA- 0,5 mg/l. Callus formation and colaration depends on genotipe. Fastest growth had hybrid No. 8 ( tremuloides x P. tremula) from all poplar hybrids. His mean growth was 8.3 mm per 10 days. RAPD analysis shown that the parent trees and trees race in vitro are the similar (the same).

<p>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.</p><p>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.</p><p>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.</p>

<p>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.</p><p>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.</p><p>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.</p><p>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.</p>

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.