Dissertations / Theses on the topic 'Xylogenesis'

Wood formation (xylogenesis) is a critical developmental process for all woody land plants. As an initial step to understand the molecular basis for temporal and spatial regulation of xylogenesis and the effect of the expression of individual genes on physical and chemical properties of wood, microarray and realtime RTPCR analyses were performed to monitor gene expression during xylogenesis under various developmental and environmental conditions. The specific objectives established for this study were: Objective 1. Microarray analysis of genes preferentially expressed in differentiating xylem compared to other tissues of loblolly pine (see Chapter II); Objective 2. Microarray analysis of seasonal variation in gene expression for loblolly pines (Pinus taeda L.) from different geographical sources (see Chapter III); Objective 3. Realtime RTPCR analysis of loblolly pine AGP and AGPlike genes (see Chapter IV). Based on the results from this study, candidate genes may be further studied for association with significant traits, used for genetic modification of wood properties, or included in future studies to further examine the molecular mechanisms of wood formation.

Pinus radiata is the dominant species of the plantations forests in New Zealand. The forest industry in New Zealand is heavily dependant on it. However, Pinus radiata can develop wood quality flaw called 'intra-ring checking'. The checks or splits appear in wood during kiln drying and usually affect the earlywood region of the wood. It lowers value of appearance grade timber leading to huge economic loses for the forest industry. This thesis presents a study that was undertaken as a part of ongoing collaborative work that is being carried out to understand wood quality issues in Pinus radiata, with a vision of improving its wood quality. This study was a part of that effort and was conducted with an aim to gain an insight into intra-ring checking, and the process of xylogenesis in Pinus radiata. The investigations for this study were carried out in two steps. The first step was to understand intra-ring checking. The location of intra-ring checking was determined by observing the checks using various microscopy techniques. Scanning electron microscopy confirmed that checking was as an intercell failure that usually occurs at the cm1/S1 boundary. A comparative study was also conducted to see if the checked wood had some inherent properties that made it more susceptible to checking. It was found that checking could be influenced by tracheid geometry and cell wall thickness. If the wood had large tracheids with thin walls, it was more likely to develop checks during drying. Lignin distribution in the cell wall layers was also seen to play an important role in checking. Lower lignin levels and disruption in the pattern of lignification of the cell wall layers increased the tendency of the wood to develop checks. Similarly, it the tracheids have larger pits then their tendency to check increases. Structural features that disrupt the uniformity of the interlocking pattern of the tracheid such as rays and resin canals could also play a role in checking. Checked wood tends to have more surface area occupied by ray tissue. However, resin canals do not seem to be directly involved in checking, though their arrangement could indicate disturbances during xylogenesis. The second step was to understand the process of xylogenesis in Pinus radiata especially with respect to the influence of auxin and boron on it. Nutrient and organ culture methods were manipulated and successfully used to study xylogenesis. An exhaustive comparative study was carried out to observe and measure selected wood properties. Microscopy and image analysis revealed that auxin and boron changes in the medium led to the alterations in the cell division, expansion and lignification. However, the analysis of the measurements and the observations displayed complex 'between-tree' and 'within-culture variations'. Clear trends did not emerge from the analysis hence, a confident conclusion on the association between auxin, boron and lignification could not be drawn from this organ culture study. The study has added to the knowledge about checking and wood properties associated with it. A new tool of organ culture had been established that can hlep future research on the process of xylogenesis in Pinus radiata.

Mestrado em Biologia Funcional - Instituto Superior de Agronomia
Portugal is one of the largest producers of pulp and paper derived from Eucalyptus globulus, which makes this a valuable species for the country. Wood is a complex and variable material, and its posttranscriptional regulation knowledge is only beginning. MicroRNAs (miRNA) are small size (21-24nt), endogenous non-coding RNAs, involved in post-transcriptional regulation. MiRBase v20 database encloses thousands of entries, however none from Eucalyptus. In this study we aim to validate E. globulus miRNAs candidates; to predict in silico and validate experimentally the miRNAs targets; and analyze the gene expression of validated targets. Four miRCa-02, miRCa-04, miRCa-08 and miRCa-09 candidates were validated by Northern blot and there in silico prediction revealed 42 target genes. Fourteen predicted target genes were tested through the RLM 5’-RACE methodology, but only three predicted targets were validated (Eucgr.E01509, Eucgr.C01382 and Eucgr.J02113 predicted target genes for miR171, miRCa-04 and miRCa-08, respectively). Expression of these three target genes analyzed by RT-qPCR suggests that the distinct expression levels found may be related with to wood formation in Eucalyptus globulus. For the first time, four Eucalytus miRNAs and their target genes were disclosed and validated by bioinformatic and molecular tools.

Pinus radiata is by far the dominant species grown in New Zealand plantations as a renewable source of wood. Several wood quality issues have been identified in the material produced, including the high incidence of compression wood, which is undesirable for end users. At present our understanding of the complex array of developmental processes involved in wood formation (which has a direct bearing on wood quality) is limited. Hence, the forest industry is interested in attaining a better understanding of the processes involved. Towards this goal, and for reasons of biological curiosity, the experiments described in this thesis were carried out to investigate several aspects of xylem cell development. In an in arbor study, changes in the orientation of cortical microtubules and cellulose microfibrils were observed in developing tracheids. Results obtained provide evidence that cortical microtubules act to guide cellulose synthase complexes during secondary wall formation in tracheids. The mechanisms involved in controlling cell wall deposition in wood cells are poorly understood, and are difficult to study, especially in arbor. A major part of this thesis involved the development of an in vitro method for culturing radiata pine wood in which hormone levels, nutrients, sugars and other factors, could be controlled without confounding influences from other parts of the tree. The method developed was used in subsequent parts of this thesis to study compression wood development, and the influence of the hormone gibberellin on cellulose microfibril organisation in the cell wall. Results from the in vitro compression wood experiments suggested that: 1. when a tree is growing at a lean, the developing cell wall was able to perceive compressive forces generated by the weight of the rest of the tree, rather than perceive the lean per se. 2. ethylene, rather than auxin, was involved in the induction of compression wood. Culture of stem explants with gibberellin resulted in wider cells, with steeper cortical microtubules, and correspondingly steeper cellulose microfibrils in the S2 layer of developing wood cells. This observation provides further evidence that the orientation of microtubules guides the orientation of cellulose microfibrils. Overall, the work described in this thesis furthers our knowledge in the field of xylem cell development. The stem culture protocol developed will undoubtedly provide a valuable tool for future studies to be carried out.

Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen.

First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod.

A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient.

Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis.

Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our ownPopulusESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes.

Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified.

Key words:Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase

Apesar da importância econômica e ambiental que a madeira representa como fonte natural e renovável de energia e fibras, pouco é conhecido sobre os processos celulares, moleculares e bioquímicos envolvidos com a sua formação. Usando metodologias proteômicas como 2D-PAGE e espectrometria de massas foi iniciada a análise do proteoma do caule de Eucalyptus grandis em diferentes estádios de desenvolvimento (5 meses, 3 anos e 6 anos). O presente trabalho baseou-se especificamente na idade de cinco meses. As plantas tiveram suas folhas, raízes e cascas removidas e seus caules foram macerados em almofariz com nitrogênio líquido e as proteínas extraídas pelo método de extração fenólica. As proteínas foram separadas por eletroforese bidimensional em fitas IPG com gradiente de pH imobilizado linear de 4-7 na primeira dimensão e gel de poliacrilamida (12,5%) na segunda dimensão. A coloração dos géis foi realizada com coomassie G250. Foram detectados 438 spots e um total de 168 spots foram retirados do gel, digeridos com tripsina e submetidos ao sequenciamento por espectrometria de massas através do sistema LCMS/ MS. O sequenciamento por MS apresentou uma eficiência de 72,02% possibilitando a identificação de 121 spots, enquanto que 35 (20,83%) não apresentaram homologia com nenhuma base de dados. Entre as proteínas identificadas 22 foram representadas por mais de um spot, podendo indicar a ocorrência e eventos provenientes do splicing alternativo, modificações pós-traducional, variações alélicas de uma mesma proteína ou degradação da amostra. Entre os spots analisados, 22,02% estão relacionados com a produção de energia, (17,86%) metabolismo, (13,69%) processes celulares, (0,60%) transporte, (8,33%) componentes estruturais, (5,36%) metabolismo macromolecular, (4,17%) proteínas putativas, (20,83%) não apresentaram homologia com nenhuma base de dados e (7,14%) não demonstraram resultado. A comparação realizada entre o volume de 59 proteínas e os seus respectivos transcritos demonstrou que não existe correlação entre mRNA e as proteínas do caule. O método possibilitou uma rápida e precisa separação e identificação das proteínas do caule de Eucalyptus grandis que são diferencialmente expressas durante a fase de crescimento de cinco meses.
The process of wood formation is an important economical factor for the forestry industry and it is also of ecological importance, although little is known about the proteins involved in wood formation. The sequencing, identification and analysis of proteins provides such information of wood formation. Using proteomics techniques such as two-dimensional gel electrophoresis and mass spectrometry we have started a proteomic analysis of wood formation in Eucalyptus grandis at different stages of development (5 months, 3 and 6 years old). This work presents data related to the stage of 5 months. Using high resolution 2DE with linear pH gradient ranging from 4 to 7, a total of 438 spots were detected. However, only 168 spots were analyzed by LC ESIMS/ MS and 121 were identified (72.02%) while 35 (20.83%) presented no homology in the database used. Overall, 22 proteins appeared as multiple spots and accounted for most of the proteins found in the group. This observation may reflect post-translation modification, alternative splicing events, isozyme variation, allelic variation of the same protein, but also protein degradation. Over the 168 spots analysed, (22.02%) play a role in energy, (17.86%) metabolism, (13.69%) cellular processes, (0.60%) transport, (8.33%) structural components, (5.36%) macromolecular metabolism, (4.17%) putative protein, (20.83%) no homology and (7.14%) no result. For 59 proteins, the spot volume was compared with their respective transcript with mRNAs extracted from wood forming tissue. The method provided a faster and accurate tool for separation and identify of protein which are differentially expressed under different stages of development in Eucalyptus grandis.

La formation du bois (xylogénèse) produit une large partie de la biomasse de la planète et une ressource essentielle pour l'Homme. Les cellules du bois sont produites par division dans le cambium puis s'élargissent, forment une paroi épaisse lignifiée et meurent. Pendant l'année, ces processus sont définis par des dates, durées et vitesses qui caractérisent la dynamique intra-annuelle de la xylogénèse. Cette dynamique reste peu explorée alors que c'est un aspect clé, car c'est elle qui détermine la quantité et la qualité du bois produit et c'est sur elle que les facteurs de régulation agissent. Ce travail vise à améliorer nos connaissances sur la dynamique intra-annuelle de la xylogénèse. Pendant trois ans (2007-2009), la xylogénèse a été suivie pour 45 arbres de trois espèces de conifères (sapin pectiné, épicéa commun et pin sylvestre) dans les Vosges. Pour ça, des petits échantillons de bois ont été prélevés chaque semaine sur le tronc des arbres sélectionnés. Les échantillons ont été préparés au laboratoire, puis des sections anatomiques ont été réalisées pour observer la xylogénèse au microscope. Cette thèse a permis d'améliorer notre connaissance du fonctionnement de la xylogénèse, un système biologique d'une fascinante complexité. Nous avons caractérisé - grâce à l'innovation d'une méthode statistique performante - les aspects méconnus de la dynamique de différenciation des cellules du bois. Nous avons alors pu dévoiler les mécanismes par lesquels la dynamique de la xylogénèse donne forme à la structure du cerne, établir la dynamique intra-annuelle de l'accumulation du carbone dans le bois et évaluer les mécanismes de l'influence du climat sur la xylogénèse
Wood formation (xylogenesis) produces a large part of the biomass of this planet and provides a crucial resource to Mankind. Wood cells are produced by division in the cambium, after what they enlarge, build a lignified thick wall and die. During a year, these processes take place at certain dates, last for certain durations and go at certain rates. These dates, durations and rates characterize the intra-annual dynamics of xylogenesis. This dynamics remains poorly explored whereas it is a key aspect as it determines the quantity and quality of the produced wood and conveys the influence of intrinsic (gene, hormone) and extrinsic (environment) regulatory factors. This work aims to improve our knowledge on the intra-annual dynamics of xylogenesis. During three years (2007-2009), xylogenesis was monitored for 45 trees of three conifer species (silver fir, Norway spruce, and Scots pine) in northeast France. For that, small wood samples were collected weekly on tree stem. Samples were prepared at the laboratory, and anatomical sections were cut to observe xylogenesis under a light microscope. This thesis has improved our knowledge on the functioning of xylogenesis, a biological system of a fascinating complexity. We characterized - thanks to the development of an efficient statistical method - the little known aspects of wood cell differentiation dynamics. Based on this characterization, we eluded the mechanisms by which xylogenesis dynamics shapes tree ring structure, we established the intra-annual dynamics of carbon accumulation in wood and we evaluated the mechanisms of the climate influence on xylogenesis

Secondary xylem, commonly known as wood, is essentially formed by highly lignified secondary cell walls of both fibres and vessels. The process of formation of secondary xylem is also termed xylogenesis. Secondary xylem derives from the vascular cambium, whose dividing cells undergo irreversible differentiation, under a strict temporal and spatial control.(...)

This diploma thesis is focused on microclimatic differences at treelines on north-facing and south-facing slopes and their effect on tree growth. Thesis contains an overview of factors affecting development and timing of xylogenesis. The effects of slope orientation on treeline in relation to exposure effect are mentioned as well. The aim of the practical part of diploma thesis was to determine development, timing and the impact of temperature characteristics on xylogenesis of Norway spurce (Picea abies) on two localities with opposite slope aspect. Locations were situated in the Důl Bílého Labe valley, Krkonoše Mountains. Xylogenesis was studied using sections of micro-cores. Microcores were sampled every 10 days during the growing season. They were subsequently analyzed in dendrochronological laboratory. My results show that influence of air temperature recorded at the start of cambial cell division and the development of enlarging cells in the first part of the growing season. Climatic characteristics of both sites was similar, larger differences in temperatures were recorded at the beginning and at the end of the reference period.

Dissertação de Mestrado em Ecologia apresentada à Faculdade de Ciências e Tecnologia
Climate change is widely recognized as a significant threat to biodiversity and ecosystem services which will have an impact on forests. Climate change forecasts a temperature increase, irregular precipitation and intense drought, conditioning tree growth. The Mediterranean region is no exception, with a prediction of more intense droughts and reduced precipitation. In the first stages of development, tree mortality is high and thus it is increasingly important to understand the impact of drought on saplings. Pinus pinaster is an important species in the Portuguese and Mediterranean forest. To understand the impact of water availability on growth parameters, xylogenesis and phloem development of P. pinaster saplings, a water manipulation experiment was performed using 2-year-old maritime pines. The saplings were submitted to a drought period but also to an extra irrigation period (after the drought) to verify if the saplings could recover from an intense drought period. Four treatments were established: CC (control + control); CI (control + extra irrigation); EC (water exclusion + control) and EI (water exclusion + extra irrigation).In general, the saplings that were submitted to a water exclusion period showed a lower diameter and height. Concerning the xylogenesis, no differences were observed among the treatments when comparing the number of cambial cells, cells in enlargement (E), in lignification (L) and mature (M). However, the treatments that had the extra irrigation after the drought period, showed a higher rate in the production of mature cells (M and ELM cells) and of phloem, with the EI treatment showing the fastest recovery. Furthermore, the extra irrigation treatments also produced more tracheids during that period.Overall, the fact that saplings did resist to a drought period is very important for restoration and reforestation programs under stressful and unfavorable conditions because the early life stages of trees are important for the initial establishment of a forest.
As alterações climáticas são reconhecidas como uma ameaça significativa à biodiversidade e aos ecossistemas. Prevê-se um aumento da temperatura, um regime de precipitação irregular e de seca intensa, condicionando o crescimento das árvores. A região do Mediterrâneo não é exceção, onde estas previsões se aplicam. A taxa de mortalidade nas árvores é elevada nas primeiras fases de desenvolvimento, por isso é crucial perceber como estas condições afetam árvores jovens e/ou plântulas. Para tal, foi desenhada uma experiência de manipulação de àgua. Utilizaram-se plântulas de pinheiro-bravo (Pinus pinaster), uma espécie importante na floresta portuguesa e mediterrânica, para entender o impacto da seca ao nível de crescimento, xilogénese e desenvolvimento do floema. Foi realizada uma experiência com plântulas de dois anos de idade sujeitas a diferentes regimes hídricos, um grupo foi submetido a uma seca mais intensa, seguida de um período de irrigação extra (após a seca) e o outro grupo serviu como controlo. A irrigação após a seca foi aplicada para perceber se as plântulas conseguem recuperar após um período de falta de água. Assim, foram estabelecidos quatro tratamentos: CC (controlo + controlo); CI (controlo + irrigação extra); EC (exclusão de água + controlo); EI (exclusão de água + irrigação extra). Em geral, as plântulas submetidas à exclusão de água apresentaram alturas e diâmetros reduzidos. Em relação à xilogénese, não houve diferenças significativas entre os tratamentos em relação ao número de células do câmbio, células em fase de expansão (E), lenhificação (L) e maduras (M). No entanto, os tratamentos com irrigação extra apresentaram uma elevada produção de células maduras (M) e de floema, com o tratamento EI a apresentar uma recuperação mais rápida. O facto de as plântulas terem resistido à seca é muito importante para a restauração e reflorestação de florestas em condições desfavoráveis e de stress, uma vez que as primeiras fases da vida das árvores são importantes para o estabelecimento inicial de uma floresta.

This thesis deals with the influence of microclimatic conditions (air temperature near tree/shrub top, temperature of the stem and root zone) on the wood phenology of trees (Picea abies) and shrubs (Pinus mugo) growing on the same site in the alpine tree line (ATL) ecotone. Phenological and microclimatic research was carried out in the Giant Mountains at 1,370 m a. s. l. in the 2017 vegetation period, with the aim to compare the course of wood formation depending on the microclimate, to determine the temperature thresholds of wood growth and differences in the morphology of trees and shrubs. Based on the microclimate analysis of trees and shrubs, individual variables were determined (average, average minimum, average maximum, daily amplitude of air, stem and root zone temperatures) characterizing the growing season. The collection of micro-drills of trees and shrubs took place from April to October in an interval of 6 - 11 days, which were subsequently processed and examined in the laboratory. The following phenophases of wood development were distinguished: cambial, enlarging, wall-thickening, mature cells and cells of the previous ring. Subsequently, differences in the number of cells of individual phenophases were identified between trees and shrubs. Furthermore, the temperature thresholds for...

Mit molekularbiologischen und biophysikalischen Analysen sowie immunologischen Nachweisen wurden Grundlagen des kaliumabhängigen Holzwachstums erforscht. Aus Holz-Kambium-Bast-Gewebe wurden mit PTK2 (Populus tremula K+ channel 2), KPT1 (K+ channel Populus tremula 1) und PtKUP1 (Populus tremula K+ uptake transporter 1) drei Volllängen putativer Kaliumtransporter isoliert. PTK2 ließ sich anhand der abgeleiteten Aminosäuresequenz der AKT2/3-Unterfamilie und KPT1 dem KAT1-Subtyp der Shaker-Familie zuordnen, während sich PtKUP1 in die Familie der KT/KUP/HAK-Transporter einreihte. Ein direkter Zusammenhang zwischen Kaliumtransport und holzbildenden Zellen konnte durch Verringerung der Gefäßweiten und signifikante Schrumpfung der Streckungszone nach lokal begrenzter Applikation des Kaliumkanalblockers TEA+ sowie durch Kaliumlimitierende Bedingungen hergestellt werden. Die Transkripte von PTK2 und PTORK (Populus tremula outward rectifying K+ channel) wurden in den Leitgeweben des Stammes, dort besonders im Phloem und in Schließzellen lokalisiert. KPT1 wurde fast ausschließlich in den Schließzellen nachgewiesen. Die mRNA von PtKUP1 war ubiquitär in geringen Mengen vorhanden. Funktionell wurde PTK2 als schwach spannungsabhängiger, K+-selektiver, nicht-gleichrichtender Kaliumkanal charakterisiert. Wie AKT2/3-ähnliche Kaliumkanäle, wurde PTK2 durch Protonen und spannungsabhängig durch Calcium geblockt. KPT1 und PtKUP1 komplementierten einen Bakterienstamm in seiner Kalium-Aufnahmedefizienz und repräsentieren demnach Kalium-Aufnahmesysteme. In Protoplasten einer Pappel-Suspensionskultur konnte ein auswärtsgleichrichtender, kalium- und spannungsabhängiger, K+-Kanal nachgewiesen werden. Dieser Auswärtsgleichrichter zeigte langsame, sigmoidale Aktivierungskinetiken, ähnlich den Kaliumströmen PTORK-exprimierender Oocyten. Des Weiteren wurde in der Suspensionskultur ein einwärtsgleichrichtender, spannungsabhängiger K+-selektiver Kanal detektiert, der, wie PTK2 in Xenopus-Oocyten, spannungsabhängig durch Calcium geblockt wurde. Nach Zugabe extrazellulären Cäsiums kam der Einwärtsstrom vollständig zum Erliegen. Für alle drei Kaliumkanäle der Pappel sowie den Kalium-Carrier wurden die Promotorregionen isoliert. Sie enthielten Motive für licht- und temperaturabhängige Transkription, gewebespezifische Expression im Leitgewebe, in Schließzellen und in Wurzeln, sowie hormonabhängige Transkription. Die Genaktivitäten von PTORK und PTK2 wurden nach Transformation von A. thaliana mit geeigneten Promotor-GUS-Konstrukten im Phloem und Xylemparenchym von Blattstielen nachgewiesen. Erstmals für Pflanzen wurden mit PTORK und PTK2 Kaliumkanal-Proteine immunologisch durch Antikörper in Phloem- und Strahlzellen während des aktiven Holzwachstums lokalisiert. Während PTK2 gleichmäßig in den Strahlzellen verteilt war, wurde im gleichen Zelltyp für PTORK eine polare Anordnung zu den angrenzenden Gefäßen hin beobachtet. Um die verschiedenen Kaliumtransporter mit der kambialen Aktivität und dem Holzwachstum zu verknüpfen, wurden die Expressionsprofile mit jahreszeitlichen Änderungen der Kaliumgehalte im Stamm verglichen. Die Transkriptanalyse von PTORK, PTK2, KPT1 und PtKUP1 über den Zeitraum eines Jahres in Stamm- und Blattknospen zeigte eine transkriptionelle Korrelation von PTORK und PTK2 mit der saisonal begrenzten Holzbildung. Ihre hohen Transkriptmengen im Herbst lassen, zusammen mit ihrer Lokalisation im Leitgewebe und ihren funktionellen Eigenschaften, auf eine Beteiligung der beiden Kaliumkanäle an Speicherungsvorgängen in die lebenden Mark- und Baststrahlen im Herbst schließen. Im Frühjahr dagegen, wenn sich die Kaliumströme umkehren, um „sink“-Gewebe mit Kalium zu versorgen, wird das Kalium vermutlich hauptsächlich über PTK2, der dann maximal exprimiert wird, aus den Strahlen und Gefäßen zu den Meristemen in Stamm und Knospen transportiert. Die hauptsächlich in Schließzellen lokalisiert KPT1 wurde zur Knospenöffnung transient induziert. Damit könnte gesichert werden, dass ausreichend osmotisch aktives Kalium für Zellexpansion und Stomaöffnung in die Schließzellen gelangt. PtKUP1 war in allen Geweben während des gesamten Jahres niedrig exprimiert und sichert daher vermutlich eine Kaliumversorgung auch unter limitierenden Bedingungen. Zur Vermehrung und Schaffung neuen Pflanzenmaterials wurde eine sterile Agarkultur aus P. tremula x P. tremuloides sowie eine Pappel-Suspensionskultur aus oberirdischem, sich teilendem Sprossgewebe etabliert. Die Expressionsanalyse der Zellkultur deutete auf eine Ausstattung an Kaliumkanälen wie in Wurzelhaaren hin, mit hohen Transkriptzahlen für PTORK, geringer Expression von PTK2 und geringsten PtKUP1-Transkripten
The current work focussed on the elucidation of the molecular basis for K+-dependent wood formation. Using molecular techniques in combination with biophysical and immu-nological approaches the results can be summarised as follows: The cDNA’s of three putative potassium transporter, PTK2 (Populus tremula K+ channel 2), KPT1 (K+ channel Populus tremula 1) and PtKUP1 (Populus tremula K+ uptake trans-porter 1), were isolated from wood-cambium-phloem tissue. Based on the deduced amino acid sequences, PTK2 was assigned to the AKT2/3-subfamily and KPT1 to the KAT1-suptype of Shaker channels, while PtKUP1 was grouped into the family of the KT/KUP/HAK-transporters. The contribution of potassium channels for wood formation was shown by local application of the K+ channel blocker TEA+ as well as under limiting K+ conditions. Both treatments resulted in a decrease of vessel lumen area, a significant reduction of the zone of expanding xylem cells and a premature initiation of secondary cell wall synthesis. The transcripts of PTK2 and PTORK (Populus tremula outward rectifying K+ channel), were mainly localised in the phloem of the vascular tissue and in guard cells while KPT1 was restricted to guard cells. In contrast, the mRNA of PtKUP1 was ubiqui-tously present at low levels. PTK2 was functionally characterised as a weakly voltage-dependent, K+-selective channel mediating potassium currents into and out of the cell. Reminiscent to AKT2/3-like channels, PTK2 was sensitive to extra cellular protons and blocked by calcium in a voltage-dependent manner. KPT1 and PtKUP1 were able to com-plement a potassium uptake deficient strain of E. coli. Therefore KPT1 und PtKUP1 repre-sent potassium uptake transport proteins. Protoplasts from poplar suspension cultures dis-played an outward rectifying, potassium- and voltage-dependent, K+-selective channel. This outward rectifier exhibiting slow sigmoidal activation kinetics, reminiscent of PTORK when heterologously expressed in Xenopus oocytes. In addition, an inward rectify-ing, voltage-dependent, K+-selective channel was detected in suspension cultured cells, showing a voltage-dependent calcium block like PTK2 expressed in Xenopus oocytes. The currents carried by this channel were completely abolished after application of extra cellu-lar caesium. The promotor regions of all three poplar potassium channels and the potas-sium transporter were isolated. Sequence analysis revealed signal motifs for temperature- and light-dependent regulation as well as tissue-specific expression in vascular tissue, guard cells and roots and motifs for hormonal dependent transcription. Transformation of A. thaliana with promotor-GUS-constructs showed that PTORK and PTK2 gene activities were predominantly observed in the phloem and xylem parenchyma of petioles. For the first time in plants the K+ channel proteins PTORK and PTK2 were immunologically local-ised with antibodies in phloem and rays during the active wood forming period. In contrast to PTK2 which was equally distributed within ray cells PTORK was polar arranged to neighbouring vessels. In order to link the different potassium transporters to cambial activ-ity and wood formation expression profiles were compared to seasonal changes of potas-sium content of the stems. The annual expression analysis of PTORK, PTK2, KPT1 und PtKUP1 in stems and buds revealed a correlation of PTORK und PTK2 with seasonally limited wood formation. Their induction in autumn, as well as their localisation in vascular tissues and their functional properties, indicated an involvement of both potassium chan-nels in loading the still living pit and phloem rays in autumn. In contrast in spring, when K+ fluxes reverse from the rays and vessels to the meristematic tissues to ensure cambial activ-ity, this ion is probably transported via PTK2, which is maximal expressed at this time. KPT1, which is mainly localised in guard cells, was induced transiently when buds open and therefore probably ensures that sufficient, osmotic active potassium comes into guard cells for expansion and stomatal opening. Finally, PtKUP1 may represent a gene that is important for potassium nutrition under limiting conditions since transcript levels were low in all tissues throughout the year. A sterile agar culture of P. tremula x P. tremuloides was generated for propagation and a sterile poplar suspension culture was established from over ground dividing stem tissue. The K+-channel expression profile of the cell culture was similar to that described for root hairs showing high transcript levels of PTORK, little ex-pression of PTK2 and minimal amounts of PtKUP1 transcripts