The aerial plant body is derived from undifferentiated cells in the shoot apical meristem that in Arabidopsis thaliana is active throughout the plant life cycle. Upon transition to flowering the activity of the meristem is altered and the meristem starts to produce secondary inflorescences and floral meristems instead of leaves. Both the activity of the meristem and the decision of when to flower are processes strictly regulated by several mechanisms. In this thesis I describe the function of two genes that are active in the regulation of meristem function and in the regulation of when to shift to reproductive development. First, the Arabidopsis gene encoding TERMINAL FLOWER2 (TFL2), homologous to HETEROCHROMATIN PROTEIN1, was isolated and characterised. Mutations in TFL2 result in plants that are dwarfed, flowers early, have reduced sensitivity to day length and terminate the inflorescence in an apical flower. As homologues from other organisms TFL2 is active in gene regulation by gene repression. I show that the gene affect flowering time by the autonomous and the photoperiod pathways, two of four floral inductive pathways. TFL2 act to repress the activity of genes that are promoters of floral meristem identity and interacts genetically with factors known to alter the chromatin state. Further tfl2 is shown to have altered levels of and response to auxin. All together this shows that TFL2 is active as a regulator of several different processes during plant development. Second, I have characterised and studied the function of three genes encoding APETALA2 LIKE proteins in Norway spruce (Picea abies). In spruce these genes are expressed in meristems and reproductive tissues. When constitutively expressed in Arabidopsis two of the genes delays flowering time and alter the function of shoot apical and floral meristems. Together this suggests a function similar to the Arabidopsis homologues.

Photosynthesis is the most important process for nearly all life on earth. Photosynthetic organisms capture and transfer light energy from the sun into chemical energy which in turn provides a resource base for heterotrophic organisms. Natural light regimes are irregular and vary over magnitudes. At a certain light intensity, metabolic processes cannot keep up with the electron flow produced by the primary photoreactions, and thus reactive oxygen species (ROS) are produced. ROS are highly reactive and can damage the photosynthesis apparatus and hence plants have evolved several photoprotection mechanisms to avoid the formation of ROS. The aim of this thesis was to examine the ecological effects of variations in photoprotection in plants. In particular I wanted to study the effect on fitness and the interaction with herbivorous insects of plants with different ability in photoprotection. To study this I used wild-type and transgenic Arabidopsis thaliana plants and grew them under natural conditions in field experiments in our botanical garden in Umeå, northern Sweden. For the investigation of the plant-insect interaction, a specialist on Brassicaceae (Plutella xylostella – diamondback moth) and a generalist herbivore (Spodoptera littoralis - Egyptian cotton worm) were used. Plants that are genetically deficient in one of the photoprotection mechanisms showed reduced fitness under natural conditions. I could thus show that feedback de-excitation (FDE) is the most important photoprotection mechanism, because a lack of FDE showed the highest reduction in fitness. The comparison of field grown wild-type with FDE mutant plants, using molecular biology methods, revealed large changes in gene transcription and metabolic composition. In particular, the jasmonate pathway was upregulated in light stressed plants, especially in plants lacking FDE. Jasmonate in turn is known to be a chemical compound which induces herbivore resistance genes and other stress responses. Specialist and generalist insect herbivores responded differently in feeding (dual-choice and no-choice) and oviposition experiments with field grown plants that differed in FDE. Female diamondback moths were attracted by induced defense compounds whereas the larvae avoided these plants in feeding experiments. Generalist larvae preferred, and showed a higher survival rate, on less light-stressed plants compared to more light-stressed plants. Combining molecular biology with ecological experiments is a challenging task. To summarize my experiences, I have produced a guide for experiments on transgenic plants in common gardens. In future investigations it is important to examine natural variations in photoprotection to elucidate selection pressures on specific genes.

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.

Cold acclimation is a complex process where plants acquire increased freezing tolerance following exposure to low, non-freezing temperatures. Microtubules are dynamic components of the cytoskeleton that are essential for plant growth and development, and there are multiple lines of evidence indicating microtubules are involved in the acquisition of freezing tolerance. <p>The organization of microtubules (MTs) was tracked over the course of a cold acclimation period using GFP:TUB6 and fluorescent imaging tools. Experiments found that MTs undergo incomplete, transient disassembly following exposure to acclimating temperatures, which is accompanied by intranuclear tubulin accumulation and followed by MT reassembly. The importance of the observed changes to MT organization was examined with MT disrupting chemicals that caused reduced MT dynamics or induced transient MT disassembly similar to that of cold acclimation. Results of these experiments suggest that MT reorganization is important for cold acclimation, but the disassembly and reassembly do not directly control cold acclimation.<p>MT binding proteins are likely to play a key role in the low temperature response because they control MT activity and organization, participate in low temperature signal transduction pathways, and mediate interactions between various elements of this pathway. By employing a number of proteomics techniques we were able to identify 96 tubulin-binding proteins from untreated and short term cold acclimated Arabidopsis plants. Proteins both known to and predicted to bind to MTs and unexpected MT binding proteins were identified. The identified tubulin binding proteins have a range of cellular functions, including RNA transport and protein translation, stress responses, and functions related to various metabolic pathways, and cell growth and organization. <p>Exposure to low temperatures affected the binding of some of these proteins to MTs with the identified tubulin binding proteins potentially involved in the cold acclimation process and stress response through a number of possible pathways.<p>This study represents the first live cell imaging of MT reorganization in response to low temperatures and the first time microtubule binding proteins from whole plant protein extracts were identified using 1D gel LC-MS/MS analysis.

Adenosine (Ado) salvage is essential in plant development. The lack of Ado kinase activity (ADK) in Arabidopsis thaliana adk1 adk2 double mutants results in embryonic lethality; reduction of ADK expression causes a pleiotropic phenotype due to the accumulation of Ado inhibiting transmethylation activities. The phenotype of ADK mutants shows that this enzyme plays a critical role in Ado salvage but the functional significance of the other putative Ado recycling enzymes Ado deaminase (ADA) and Ado nucleosidase (ADN) in Arabidopsis thaliana have yet to be elucidated. ADA catalyzes the irreversible deamination of Ado to inosine. The locus At4g04880 (AtADA) of A. thaliana is annotated as encoding a putative ADA, based on its amino acid sequence similarity and the presence of important, conserved catalytic residues. However, indirect and direct spectrophotometric activity assays of the recombinant enzyme demonstrated that the gene product of this locus does not possess ADA activity; complementation experiments to test for the functionality of the AtADA product in A. thaliana and E. coli confirmed its lack of ADA activity. Instead, phylogenetic analysis revealed that AtADA belongs to the group of ADA-like (ADAL) proteins, a group closely related to ADAs that to date have not been shown to have ADA activity. AtADA is no exception as it also lacks ADA activity based on the in vivo and in vitro experiments outlined in this thesis. Thus, the locus At4g04880 should be re-annotated as ADAL. The question of the function of AtADAL cannot be answered as of yet; in general, the knockout of ADA gene product demonstrated that At4g04880 is not essential for Arabidopsis growth. Since no further ADA-related genes exist in the genome of Arabidopsis it is concluded that ADA activity is not present in this plant. ADN catalyzes the conversion of purine and pyrimidine ribosides to their corresponding bases; although it prefers Ado as a substrate it also acts on cytokinins. The activity of this enzyme has been described in several plant species but no corresponding genes have been identified to date. The genome of Arabidopsis was screened for ADN genes using an inosine-uridine nucleoside hydrolase sequence from the protozoa Crithidia fasciculata. Two genes, annotated as ADN1 and ADN2 were identified and their gene products were studied using a spectrophotometric assay. The substrate spectrum of ADN2 includes both purine and pyrimidine nucleosides but it prefers to utilize uridine. Thus, ADN2 is proposed to be involved in the purine and pyrimidine salvage in Arabidopsis but predominantly in uridine recycling. Recombinant ADN1 did not show activity on any of the tested substrates. Even though the in vivo role of both ADNs is still uncertain, due to their lack or low activity on Ado there may yet be the ADN gene in the Arabidopsis genome which likely acts on both adenosine and cytokinin ribosides.

Adenosine (Ado) salvage is essential in plant development. The lack of Ado kinase activity (ADK) in Arabidopsis thaliana adk1 adk2 double mutants results in embryonic lethality; reduction of ADK expression causes a pleiotropic phenotype due to the accumulation of Ado inhibiting transmethylation activities. The phenotype of ADK mutants shows that this enzyme plays a critical role in Ado salvage but the functional significance of the other putative Ado recycling enzymes Ado deaminase (ADA) and Ado nucleosidase (ADN) in Arabidopsis thaliana have yet to be elucidated. ADA catalyzes the irreversible deamination of Ado to inosine. The locus At4g04880 (AtADA) of A. thaliana is annotated as encoding a putative ADA, based on its amino acid sequence similarity and the presence of important, conserved catalytic residues. However, indirect and direct spectrophotometric activity assays of the recombinant enzyme demonstrated that the gene product of this locus does not possess ADA activity; complementation experiments to test for the functionality of the AtADA product in A. thaliana and E. coli confirmed its lack of ADA activity. Instead, phylogenetic analysis revealed that AtADA belongs to the group of ADA-like (ADAL) proteins, a group closely related to ADAs that to date have not been shown to have ADA activity. AtADA is no exception as it also lacks ADA activity based on the in vivo and in vitro experiments outlined in this thesis. Thus, the locus At4g04880 should be re-annotated as ADAL. The question of the function of AtADAL cannot be answered as of yet; in general, the knockout of ADA gene product demonstrated that At4g04880 is not essential for Arabidopsis growth. Since no further ADA-related genes exist in the genome of Arabidopsis it is concluded that ADA activity is not present in this plant. ADN catalyzes the conversion of purine and pyrimidine ribosides to their corresponding bases; although it prefers Ado as a substrate it also acts on cytokinins. The activity of this enzyme has been described in several plant species but no corresponding genes have been identified to date. The genome of Arabidopsis was screened for ADN genes using an inosine-uridine nucleoside hydrolase sequence from the protozoa Crithidia fasciculata. Two genes, annotated as ADN1 and ADN2 were identified and their gene products were studied using a spectrophotometric assay. The substrate spectrum of ADN2 includes both purine and pyrimidine nucleosides but it prefers to utilize uridine. Thus, ADN2 is proposed to be involved in the purine and pyrimidine salvage in Arabidopsis but predominantly in uridine recycling. Recombinant ADN1 did not show activity on any of the tested substrates. Even though the in vivo role of both ADNs is still uncertain, due to their lack or low activity on Ado there may yet be the ADN gene in the Arabidopsis genome which likely acts on both adenosine and cytokinin ribosides.

Cold acclimation is a complex process where plants acquire increased freezing tolerance following exposure to low, non-freezing temperatures. Microtubules are dynamic components of the cytoskeleton that are essential for plant growth and development, and there are multiple lines of evidence indicating microtubules are involved in the acquisition of freezing tolerance. <p>The organization of microtubules (MTs) was tracked over the course of a cold acclimation period using GFP:TUB6 and fluorescent imaging tools. Experiments found that MTs undergo incomplete, transient disassembly following exposure to acclimating temperatures, which is accompanied by intranuclear tubulin accumulation and followed by MT reassembly. The importance of the observed changes to MT organization was examined with MT disrupting chemicals that caused reduced MT dynamics or induced transient MT disassembly similar to that of cold acclimation. Results of these experiments suggest that MT reorganization is important for cold acclimation, but the disassembly and reassembly do not directly control cold acclimation.<p>MT binding proteins are likely to play a key role in the low temperature response because they control MT activity and organization, participate in low temperature signal transduction pathways, and mediate interactions between various elements of this pathway. By employing a number of proteomics techniques we were able to identify 96 tubulin-binding proteins from untreated and short term cold acclimated Arabidopsis plants. Proteins both known to and predicted to bind to MTs and unexpected MT binding proteins were identified. The identified tubulin binding proteins have a range of cellular functions, including RNA transport and protein translation, stress responses, and functions related to various metabolic pathways, and cell growth and organization. <p>Exposure to low temperatures affected the binding of some of these proteins to MTs with the identified tubulin binding proteins potentially involved in the cold acclimation process and stress response through a number of possible pathways.<p>This study represents the first live cell imaging of MT reorganization in response to low temperatures and the first time microtubule binding proteins from whole plant protein extracts were identified using 1D gel LC-MS/MS analysis.

Generation of reactive oxygen species (ROS) is an important view point to evaluate heavy metal toxicity and resistance in plants. Arabidopsis thaliana is a fully sequenced model plant, and the characteristic between ecotypes due to adaptation towards varied environment can be used as a material for comparing physiological differences. In this experiments, two ecotypes of A. thaliana: Columbia (Col-0) and Landsberg erecta (Ler-0) is observed for the roots growth inhibition, plasma membrane integrity and lipid peroxidation after treated with different concentration of HgCl2 (0, 2, 4, 8 £gM), in attempt to compare the anti-oxidation defensive mechanism of two ecotypes and understand mercury-induced oxidative stress. ROS and Ca2+ generation is determined with CM-H2DCF-DA and Oregon Green 488 BAPTA-1 is under confocal microscopy. Some anti-oxidant enzymes such as superoxide dismutase (SOD EC 1.15.1.1), peroxidase (POD EC1.11.1.7) and ascorbate peroxidase (APX EC 1.11.1.11) are examined for the activity under protein gel electrophoresis. Experiment results showed that mercury-induced inhibition of root growth is more significant in Ler-0. ROS in roots of both ecotypes shows different trends under 8 £gM HgCl2 , however increment of ROS level below 4 £gM HgCl2 ; Ca2+ shows the similar result as ROS. Activity of SOD isoforms reached a peak at 2-4 £gM HgCl2. Expression of POD is correlated to the mercury concentration in both ecotypes. There are two types of APXs expression, one decreased as mercury concentration increased, another increased under 2 £gM HgCl2 and decreased as the concentration getting higher. According to the observation on expression of ROS generations and anti-oxidation system, we speculated that tolerance of Ler-0 towards mercury is weaker than Col-0. The results can be used as a basis for further discussion on influence of mercury towards different anti-oxidation enzymes and the signaling pathways.

Large, membrane-bound vacuoles of plant cells are suitable organelles for the compartmentation of ions. These vacuoles contain Na+/H+ antiporters for movement of Na+ within the organelle in exchange for H+. They provide an efficient mechanism to prevent the occurance of detrimental outcomes of Na+ accumulation in the cytosol. Identification of AtNHX1 gene that confers resistance to salinity by expressing a Na+/H+ antiport pump facilitates the understanding of the salt stress tolerance mechanisms of plants. The aim of the present study was to isolate and clone the Arabidopsis thaliana AtNHX1 coding sequence for transformation of Nicotiana tabacum plants via Agrobacterium tumefaciens mediated gene transfer. For this purpose, total RNA was isolated from Arabidopsis thaliana plants and cDNA synthesis was performed. AtNHX1 (1614bp) was amplified by using cDNA of Arabidopsis via specific primers. The amplified PCR product was verified by sequencing. AtNHX1 coding sequence was cloned into the plant transformation vector pCVB1 and 10 independent putative transgenic tobacco plants were obtained via Agrobacterium tumefaciens mediated gene transfer sysytem. Transfer of selected 8 putative transgenic plants to soil provided the regeneration of T1 seeds. Germination of the seeds under different salt treatments (0, 50, 100, 150, 200, 250 mM NaCl) was observed for evaluating the salt tolerance of transformed plants. The 82% and 60% of the transgenic T1 seeds were germinated on 150 mM NaCl and 200 mM NaCl containing media, respectively. In contrast the germination percentage of wild type tobacco seeds under 150 mM NaCl and 200 mM NaCl concentrations were 39% and 21%, respectively. The germination rate of the transgenic T1 seeds were significantly higher (p=0,001) when compared to the control seeds especially under high salt stress conditions (150 and 200 mM NaCl). Taken all together, our results demonstrated that the germination efficiencies and growth of the plants transformed with AtNHX1 were higher than the wild type tobacco plants under high salt concentrations.

In this thesis the effects of single photon and multi-photon excitation on protoplasts from Arabidopsis thaliana are compared. Time-lapsed micro-spectroscopy at high spatial resolution is employed to study the response of chloroplasts within the protoplasts from Arabidopsis thaliana. We have found that the fluorescence spectra of chloroplasts exhibits dramatic changes and the protoplasts are rapidly damaged under multi-photon excitation as a result of pulsed laser illumination. In contrast, single photon excitation of chloroplasts with cw laser is relatively inert to the vitality of the protoplasts. In addition to, we have built an ultrafast laser excited cryogenic micro-spectroscopy setup to study the photoluminescence of PPV thin film. We found that the spectrum of PPV¡¦s photoluminescence should shift toward longer wavelength and the non-radiative transition should be suppressed as a result of longer electron coherence length at low temperature.