Climate is changing and it is certain that this change is due to human activities. Atmospheric greenhouse gases have been rising in an unprecedented way during the last two centuries, although the land biosphere has dampened their increase by absorbing CO2 emitted by anthropogenic activities. However, it is unclear if this will continue in the future. This uncertainty makes it difficult to predict future climate changes and to determine how much greenhouse gas emissions must be reduced to protect climate. To understand the future role of plants in limiting the atmospheric CO2 level, the effect of increasing CO2 on plant photosynthesis and productivity has been studied. However, studies on trees showed contradictory results, which depended on the duration of the experiment. This revealed that an initial strong CO2 fertilization may be a transient response that disappears after a few years. Because climate changes over centuries, we must explore the response of vegetation to increasing CO2 on this time scale. Studying tree rings is a good alternative to impractical decade-long experiments, because trees have experienced the CO2 increase during the last 200 years and may already have responded to it. This thesis shows that the intramolecular distribution of the stable hydrogen isotope deuterium (deuterium isotopomer distribution, DID) of tree rings is a reliable tool to study long-term plant-climate adaptations. The premise for this is that the deuterium abundance in tree rings depends on environmental as well as physiological factors. Using newly developed methodology for DID measurements, the influences of both factors can be separated. Applied to tree rings, separating both factors opens a strategy for simultaneous reconstruction of climate and of physiological responses. The results presented show that DIDs are influenced by kinetic isotope effects of enzymes, allowing studies of metabolic regulation. We show that the abundances of specific D isotopomers in tree-ring cellulose indeed allow identifying environmental and physiological factors. For example, the D2 isotopomer is mostly influenced by environment, its abundance should allow better reconstruction of past temperature. On the other hand, the abundance ratio of two isotopomers (D6R and D6S) depends on atmospheric CO2, and might serve as a measure of the efficiency of photosynthesis (ratio of photorespiration to assimilation). The presence of this dependence in all species tested and in tree-ring cellulose allows studying adaptations of plants to increasing CO2 on long time scales, using tree-ring series or other remnant plant material. / Klimatet förändras och det är numera allmänt vedertaget att detta beror på människans aktiviteter. Halten av växthusgaser har stigit onormalt mycket under de senaste två århundradena och detta beror i största del på människans användning av fossila bränslen. Landbiosfären har hittills haft en buffrande effekt på klimatförändringen eftersom den tar upp och lagrar mycket av växthusgasen CO2. Det är dock osäkert om, och i så fall hur länge, denna effekt kvarstår. Detta gör det mycket svårt att förutsäga framtida klimatförändringar, och därmed hur mycket utsläppen av växthusgaser måste reduceras för att skydda klimatet. För att förstå växternas framtida förmåga att begränsa halten atmosfäriskt CO2 har man studerat effekten av förhöjda halter av CO2 på växters fotosyntes och produktivitet. Resultaten av dessa försök varierar i stor omfattning. Studier på träd odlade under höga halter CO2 indikerar att den initiala ökningen av en trädets produktivitet kan vara en temporär effekt som försvinner redan efter några år. Eftersom klimatförändringen sker under århundraden, måste även växternas anpassningar på förhöjd CO2 halt utforskas på denna tidsskala, men experiment som skulle ta tiotals år är opraktiska att utföra. Trädringar är ett bra sätt att studera sådana anpassningar, eftersom träd redan har upplevt de senaste två hundra årens ökning av koldioxid och dess trädringar därför kan innehålla information om en respons som redan skett. Denna avhandling visar att den intramolekylära fördelningen av den stabila väteisotopen deuterium i trädringar är en tillförlitlig metod för att studera växters anpassningsförmåga till långsiktiga klimatförändringar. Antagandet bakom denna strategi är att isotopfördelningen i trädringar beror på faktorer både från miljön och växtens fysiologi. Om båda faktorerna skulle kunna utvinnas från trädringar, skulle detta öppna en helt ny väg för parallell rekonstruktion av klimatet och växters anpassning till det. Avhandlingen presenterar den första tekniken för att mäta isotopfördelningen av deuterium i växtglukos. Resultaten visar att deuteriumfördelningen hos växtglukos påverkas av enzymers isotopeffekter, vilket möjliggör att regleringen av växternas metabolism kan kartläggas. I avhandlingen bevisas att halten deuterium i skilda intramolekylära positioner (isotopomerer) av glukos från trädringcellulosa bestäms av miljöfaktorer respektive trädets fysiologi. T.ex. påverkas deuteriumhalten i position 2 (D2 isotopomer) av glukosmolekylen huvudsakligen av miljön, vilket kan användas för att förbättra temperaturrekonstruktioner från trädringar. Å andra sidan är kvoten deuterium mellan två andra positioner (D6R och D6S) relaterat till halten atmosfäriskt koldioxid, och kvoten skulle kunna användas som mått för fotosyntesens effektivitet, dvs. förhållandet mellan fotorespiration och fotosyntes. Närvaron av denna relation i trädringar och annat växtmaterial i alla de växter vi hittills studerat, öppnar en helt ny möjlighet att studera växters anpassning till den ökande mängden CO2 i atmosfären under århundraden.

Tropical seagrass ecosystems are highly productive and important for sustaining marine life and associated coastal societies. In this study, the diversity and role of nitrogen-fixing cyanobacteria associated with five common seagrass genera in coastal regions of the western Indian Ocean (WIO; Tanzania) were examined, as well as the impact of anthropogenic activities. Cyanobacteria were characterized morphologically and genetically (16S rRNA and nifH gene phylogeny), as were diel variations in nifH gene expression, NifH protein levels and nitrogenase activity. The results revealed that WIO seagrass beds supported rich cyanobacterial diversity and that these represented approx. 83% of total clones obtained (DNA and RNA nifH clone libraries). Non-heterocystous genera, such as Oscillatoria, Lyngbya, Leptolyngbya, Phormidium and Microcoleus dominated, while heterocystous morphotypes such as Calothrix were less frequent and unicellular morphotypes (e.g. Gloeocapsa, Chroococcus and Chroococcidiopsis) were few. Additionally, the phylogenetic analysis revealed several novel uncharacterized cyanobacterial clades. Cyanobacterial composition and nitrogenase activity varied over seasons and between the seagrass species. Day time nitrogenase activity originated primarily from heterocystous phylotypes, while non-heterocystous filamentous phylotypes fixed nitrogen at night. The highest activity in the diel cycle was 358 ± 232 nmol C2H4 g-1 h-1at 09.00 associated with epiphytes of the seagrass Cymodocea. Nitrogenase activity was consistently lower in anthropogenically disturbed (eutrophication) seagrass sites. Such data suggest that diazotrophic cyanobacteria may be a significant source of ‘new’ nitrogen in the often oligotrophic coastal regions of tropical oceans. It is also proposed that the rapid shifts in the cyanobacterial population and function found may also be used as early disturbance indicator in coastal management practices. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript. / SIDA SAREC Bilateral Marine Sciences Project / The Swedish Foundation for International Cooperation in Research and Higher Education

Since the first signs of life on planet earth, organisms have had to adapt to the daily changes between light and dark, and high and low temperatures. This has led to the evolution of an endogenous time keeper, known as the circadian clock. This biological timing system helps the organism to synchronize developmental and metabolic events to the most favorable time of the day. Such a mechanism is of considerable value to plants, since they in contrast to animals cannot change location when the environment becomes unfavorable. Thus is the ability to predict coming events of central importance in a plants life. This thesis is a study of the molecular machinery behind the clockwork in the small weed plant Arabidopsis thaliana as well as its close relative perennial; the woody species Populus. We have characterized a novel component of the circadian clock, EARLY BIRD (EBI). EBI is involved in transcriptional and translational regulation, via interaction with the known post-translational clock regulator ZEITLUPE (ZTL). In Populus, we describe the role of the circadian clock and its components with respect to entry and exit of dormancy and show that gene expression of the Populus LATE ELONATED HYPOCOTYL (LHY) genes are crucial importance for freezing tolerance and thereby survival at high latitudes. Furthermore, the input to the Populus clock is mediated via the phytochrome A (phyA) photoreceptor. / Liv på jorden har alltid behövt anpassa sig till de dagliga växlingarna mellan främst ljus och mörker. Detta har lett till evolutionen av en intern, biologisk klocka, känd som den circadianska klockan, efter latinets ”circa diem”, som betyder ”ungefär en dag”. Denna inre klocka hjälper organismer att styra biologiska processer till den tid på dygnet som är mest gynnsam för deras utveckling och överlevnad. Denna mekanism är av stort värde för växter, eftersom de inte kan söka skydd på mera lämpliga platser om de blir utsatta för olika former av stress. Det gör att förmågan att förutse kommande händelser är av yttersta vikt för växter. Denna avhandling är en studie av det molekylära nätverk som styr denna biologiska klocka i den lilla örtplantan Arabidopsis thaliana (backtrav), och den besläktade träd-arten Populus (hybrid-asp). Vi har karaktäriserat en ny komponent i den circadianska klockan i Arabidopsis, EARLY BIRD (EBI). EBI är involverad i transkriptionell och translationell reglering av klockan, via interaktion med den kända post-translationella klock-regulatorn ZEITLUPE (ZTL). I Populus har vi beskrivit den interna klockan och dess roll i processer som invintring, vinterdvala och återstart av tillväxt. LATE ELONATED HYPOCOTYL (LHY) generna i Populus är avgörande för förvärv av köld-tolerans och således överlevnad på högre latituder. Dessutom har vi visat att signaler till den circadianska klockan i Populus är medierade via fotoreceptorn phytochrome A (phyA).

During photosynthesis sunlight is absorbed by photosynthetic pigments and converted into organic compounds, such as carbohydrates. Photosynthesis needs to be highly regulated, since both too much and too little light are harmful to plant. If too little light is absorbed, a plant cannot store enough energy, which will have effects on growth and fitness of the plant. With too much light absorbed, a dangerous side reaction of photosynthesis, the production of reactive oxygen species can happen. These reactive oxygen species can damage the proteins in the chloroplast and the lipids of the chloroplast. To avoid the production of reactive oxygen species, plants have evolved many mechanisms, which act on different time-scales and different levels of organization. As a first measure, when the absorbed light is exceeding the capacity for its utilization, is to switch the light-harvesting antenna from efficient light harvesting to energy dissipation. This process is called feedback de-excitation (FDE). The protein PsbS is essential for this process as well as a functioning xanthophylls cycle with the enzyme violaxanthin de-epoxidase (VDE). I have investigated the effects of plants with changes in their ability to dissipate excess excitation energy in the model plants species Arabidopsis thaliana. Three genotypes with either increased or decreased capacity for FDE were used during my experiments. The first genotype over-expresses the PsbS gene, having approximately two-fold increased amounts of PsbS and FDE. The second is a PsbS deletion mutant with no PsbS protein and no FDE. The third genotype cannot perform the conversion of violaxanthin to zeaxanthin, because the enzyme VDE is missing. This mutant has some FDE left. Arabidopsis thaliana is an annual plant, which flowers only once in its lifetime. Therefore, when counting the seeds produced an estimation of fitness can be made from the amount of seeds produced. This was done during my experiments and shown that FDE is a trait and that plants with increased FDE have a higher fitness and vice versa. This was also the case for a collection of plants lacking a single protein from the light harvesting antenna. All of these genotypes had a fitness reduction, proving that their function is not redundant. In an attempt to explain why the fitness is reduced in plants with altered FDE, photosynthetic measurements, as well as a determination of the transcriptome and the metabolome was performed. Plants lacking FDE had higher levels of photoinhibition, leading both to lower rates of photosynthesis and to higher repair cost. This could in part explain the reduction in fitness. These plants also had major changes in their transcriptome and their metabolome. Primary metabolism was most effected, for example carbohydrate and amino acid metabolism. But there were also changes in secondary metabolism such as an up regulation of the biosynthesis of anthocyanins.

Seagrass meadows are highly productive, ecologically and economically valuable ecosystems. However, increased human activities along the coastal areas leading to processes such as eutrophication have resulted in the rapid loss and deterioration of seagrass ecosystems worldwide. This thesis focuses on the responses of seagrasses to increases in nutrients, subsequent increases in ephemeral algae, and changes in the physical-chemical properties of seawater induced by interaction with other marine biota. Both in situ and laboratory experiments conducted on the tropical seagrasses Cymodocea serrulata and Thalassia hemprichii revealed that increased concentrations of water column nutrients negatively affected seagrass photosynthesis by stimulating the growth of the epiphytic biomass on the seagrass leaves. Interaction between seagrasses and other marine organisms induced different responses in seagrass photosynthesis. Ulva intestinalis negatively affected the photosynthetic performance of the temperate seagrass Zostera marina both by reducing the light and by increasing the pH of the surrounding water. On the other hand, the coexistence of mussels Pinna muricata and seagrass Thalassia hemprichii enhanced the photosynthetic activity of the seagrass, but no effect on the mussels' calcification was recorded. This study demonstrates that seagrass productivity is affected by a multitude of indirect effects induced by nutrient over-enrichment, which act singly or in concert with each other. Understanding the responsive mechanisms involved is imperative to safeguard the ecosystem by providing knowledge and proposing measures to halt nutrient loading and to predict the future performance of seagrasses in response to increasing natural and human perturbations. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Papers 1, 3 and 4: Submitted. Paper 2: Manuscript. / Swedish Agency for Research Cooperation (Sida/SAREC) marine bilateral programme

När ett blad gulnar genomgår det både morfologiska och metaboliska förändringar. Denna process benämns senescence och en förbättrad förståelse av dess mekanismer är viktiga både ur ett grundvetenskapligt perspektiv och för potentiella bioteknologiska applikationer. Denna avhandling rapporterar om flera viktiga aspekter relaterade till de cellulära och metaboliska mekanismer som sker under senescencen med tonvikt på mitokondriernas bidrag till denna process. I ett första steg utvecklade vi metoder för att isolera antingen mycket funktionella mitokondrier eller mycket rena mitokondrier från blad av Arabidopsis thaliana. Dessa metoder användes sedan till för att studera mitokondriella bidrag till cellens redox balans och att uppskatta mitokondriernas kapacitet under senescence-processen. Framför allt jämfördes induktionen av senescencen berodende på olika mörkerbehandlingar av Arabidopsis. Jämförelse mellan individuellt mörklagda blad med hela mörklagda växter visade en betydande skillnad i metabolisk strategi mellan de två mörkerbehandlingarna. Genom att integrera data från mätningar av fotosyntes, respiration och konfokal mikroskopi med transcriptomics- och metabolomics-profiler föreslår vi att metabolismen hos blad från helt mörklagda växter antar ett ”stand-by läge” för att kunna bibehålla fotosynteskapaciteten så länge som möjligt. I kontrast till detta visar mitokondrier från individuellt mörklagda blad en hög aktivitet och kan därmed producera energi och kolskelett för degraderingen av cellkomponenter, vilket möjliggör återvinning av näringsämnen. Vi har även studerat dynamiken av det mikrotubulibaserade cytoskelettet under mörkerindicerad senescence. Mitokondriernas rörlighet påverkades av en tidig nedbrytning av mikrotubuli hos individuellt mörklagda blad men inte hos blad där hela växten mörkerbehandlats. Dessutom verkade ett flertal mikrotubuliassocierade proteiner (MAP’s) att vara involverade i buntningen av mikrotubuli runt kloroplasterna. Sammanfattningsvis belyser det arbete som presenteras i denna avhandling ett flertal viktiga steg med avseende på metabolisk anpassning och andra cellulär mekanismer i Arabidopsisblad som utsätts för långvarig mörkerbehendling. Specifikt föreslår vi att mitokondrierna bidrar med speciella och viktiga funktioner under bladens senescence eftersom mitokondriernas roll under långvarig mörkerbehandling av blad verkar bero på den totala statusen av metabolismen hos växten. / When switching from green to yellow, a leaf undergoes both morphological and metabolic changes. This process is known as senescence and improved understanding of its mechanisms is important both from a fundamental scientific perspective but also for biotechnological applications. The present thesis reports on several important aspects regarding the cellular and metabolic mechanisms occurring during leaf senescence with an emphasis on the mitochondrial contribution to this process. As a first step, we developed methods to isolate either highly functional crude mitochondria or highly purified mitochondria from leaves of Arabidopsis thaliana. These methods were further used to study mitochondrial contributions to cellular redox homeostasis and to estimate the mitochondrial capacities in leaves undergoing senescence. In particular, we compared the induction of senescence by different dark treatments in Arabidopsis. The comparison between individually darkened leaves and leaves from whole darkened plants revealed different metabolic strategies in response to darkness. Integrating data from measurements of photosynthesis, respiration and confocal laser microscopy with transcriptomics and metabolomics profiling, we suggested that metabolism in leaves of the whole darkened plants enter a “stand-by mode” with low mitochondrial activity in order to maintain the photosynthetic machinery for as long as possible. In contrast, mitochondria from individually darkened leaves are more active and may provide energy and carbon skeletons for the degradation of cell constituents, facilitating the retrieval of nutrients. We also investigated the dynamic of the microtubular cytoskeleton during dark-induced senescence. Mitochondrial mobility was affected by an early disruption of the microtubules in individually darkened leaves but not in whole darkened plants. In addition, several microtubules associated proteins (MAPs) seemed to be involved in the bundling of the microtubules around the chloroplasts. Altogether, the work presented in this thesis highlights several important steps regarding the metabolic adjustments and the cellular mechanisms in Arabidopsis leaves submitted to prolonged darkness. In particular, we suggest the mitochondria to fulfill specific and important functions during leaf senescence since the role of mitochondria in leaves experiencing prolonged darkness appears very dependant on the whole metabolic status of the plant.

Study of the Arabidopsis thaliana knockout mutant lacking Lhcb3 (koLhcb3) have revealed a close similarity to the wild type plants. Growth rate, NPQ, qP, Φ(PSII), circular dichroism spectra, pigment composition and content of LCHII trimers have been found to be unaffected by this mutation. The proteomic analysis shows only some minor increases in the amount of Lhcb1 and Lhcb2. PAM fluorometry revealed a significant increase in the rate of the state 1 to state 2 state transition in the koLhcb3. None the less, the extent of state transition is identical to wild type. Alterations in the PSII-LHCII supercomplex structure have been demonstrated as well. The M-trimer was found to be rotated ~21° CCW. This altered binding of the LHCII M-trimer is likely the cause of the altered affinity resulting in the increased rate of state transition. Proteomic analysis of the phosphorylation of LHCII revealed a significant increase in state 1 and 2 LHCII phosphorylation relative to wild type. Investigation whether phosphorylation or the altered LHCII binding is the cause of the accelerated rate of state transition have not been conclusive so far. A Lhcb6 depleted mutant (koLhcb6) showed a significant alteration of the PSII-LHCII supercomplex structure and photosynthetic acclimation processes. The LHCII M-trimer is depleted in the PSII-LHCII supercomplexes causing the state transition process to be “stuck” in state 2 and the mutants ability to preform NPQ is inhibited as well. The Lhcb6 protein was concluded to be essential for the binding of the LHCII M-trimer to the PSII core as well as energy transfer. The depletion of LHCII M-trimer was linked to the reduced ability to photoacclimate using NPQ as well.

<p>Concentrations of various metals are elevated above background levels in several intertidal areas along the Tanzanian coasts. However, there is little available information concerning the toxicity of these metals and how the uptake of these metals by bioindicators are influenced by external factors, such as heavy rains and increased coastal eutrophication, which tend to fluctuate.</p><p>The present study focused on the uptake and toxicity of Cu and Zn in two common macroalgal species,<i> Padina gymnospora</i> (Phaeophyta) and<i> Ulva</i> <i>reticulata</i> (Chlorophyta). Laboratory studies were performed where metal content, growth (DGR), maximal quantum yields (Fv/Fm) and protein expression patterns (in <i>Ulva</i>) were measured as a response to exposure to Cu and Zn. The levels of metals accumulated in algal tissues correlated well to exposure concentrations and the longer the exposure time, the greater the uptake. However, an increased nutrient load (tested on <i>Padina</i>) or dilution of the seawater (tested on<i> Ulva</i>) affected both uptake of metals and their toxic effects. Here, DGR was more affected than Fv/Fm, suggesting DGR to be the more sensitive indicator of Cu and Zn toxicity. As shown by 2-D gel electrophoresis, more than ten proteins were up-regulated in <i>U. reticulata</i> after being exposed to Cu (1μg/L), while at higher concentrations (10 and 100 μg/L) of Cu numerous proteins were down-regulated.</p><p><i>P. gymnospora </i>was also used as a bioindicator to monitor long-term (1994–2005) and seasonal in-year variations in heavy metal concentrations in the Zanzibar Channel. No clear overall trends were revealed, but analysis of the combined dataset clearly pinpointed the most contaminated sites. It was concluded that seasonal and long-term variations, as well as environmental conditions need to be taken into consideration when using macroalgae as bioindicators.</p>

<p>The aquatic plant <i>Ipomoea aquatica</i> is a popular vegetable in Southeast Asia, often cultivated in nutrient rich and polluted waters. The overall aim of this thesis was to estimate potential risks for human health and reduced plant growth due to accumulation and toxicity of total-Hg, methyl-Hg, Cd and Pb.</p><p>In plants from cultivations in Thailand, the concentrations of Cd and Pb in the shoots were well beneath recommended maximum values for human consumption, but at some sites the Hg concentrations were high. It was demonstrated that <i>I. aquatica</i> has the capacity to accumulate much higher Cd and Pb concentrations in the shoots than found in field-cultivations, before exhibiting toxic symptoms. The Hg concentrations, however, occasionally reached levels that are toxic for the plant. Up to11% of total-Hg was methyl-Hg, the most toxic Hg species, though at one site it was 50-100%. To study if methyl-Hg is formed in <i>I.</i> <i>aquatica</i>, plants were exposed to inorganic Hg through the roots. Of the Hg that reached the young, metabolically active parts of the shoots, a part was transformed to methyl-Hg. A major proportion of absorbed metals was retained in the roots, which had a high tolerance for high internal metal concentrations. </p><p>The nutrient level did influence accumulation and effects of Hg, Cd and Pb in<i> I. aquatica</i>. Low external nutrient levels resulted in increased metal accumulation in the shoots and in metal-induced toxic effects in the plant at low external metal levels. A generous supply of sulphur or nitrogen induced formation of thiol-rich peptides in <i>I. aquatica</i>, compounds that have a metal detoxifying effect in plants. </p><p>To conclude, the levels of Cd and Pb in field cultivated <i>I. aquatica</i> do not pose any apparent threat to human health or risk for reduced plant growth. The levels of Hg however, were high at some sites and could be a health threat, for children and foetuses in particular, and especially considering the presence of methyl-Hg. The use of fertilizers is favourable as it reduces the risk for increased metal concentrations in <i>I.</i> <i>aquatica</i> and for reduced crop yields. </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>