Cell wall formation in wood development
Ewa Mellerowicz, Dept of Forest Genetics and Plant Physiology, SLU, tel .
E-mail:
and Björn Sundberg, Dept of Forest Genetics and Plant Physiology, SLU, tel .
E-mail:
The concept of the cell wall evolved from a passive structure that keeps cells from bursting to a living organelle that controls cellular processes. Cell wall directly affects plant growth and development through the regulation of cell size and shape (= cell growth). Cell growth is a function of water potential and the plasticity of cell wall. Various cell shapes are obtained when cell wall plasticity is locally modified. The wall plasticity depends on the abundance of cross - links of hemicelluloses, pectins and wall structural proteins that lock the cellulose microfibrils in place thus preventing further wall extension. These cross - links can be created and modified in the wall by the action of wall residing enzymes. Thus, these enzymes can directly affect cell growth.
Our group is studying involvement of wall residing enzymes, like XETs, expansins, cellulases and pectin methyl transferases, in wood formation processes in angiosperm trees. The importance of these proteins in modulation of cell wall structure is not at all known in the process of wood formation although they have been intensively studied in other plant growth processes. For example, we have recently discovered a novel role for XET in wood cells (Bourquin et al., Plant Cell, , vol.14: ). Also, the mechanism of the cell elongation during wood development is not well understood, yet the wood cell size and shape, and cell wall properties have a tremendous impact on the whole plant physiology by affecting water transport and, obviously, on the industrial fiber utilization.
At present, you can take part in one of the following projects listed below. Depending on the project, you will learn standard molecular biology techniques (northern, southern, western, PCR), methodologies related to using Arabidopsis mutants and transgenic plants to study gene function, and advanced microscopy techniques including confocal laser microscopy.
Project 1. Isolation and characterization of XET mutants in Arabidopsis.
Project 2. Role XET in wood formation determined by the analysis of transgenic plants.
Project 3. Studying mechanisms of cell elongation in primary and in secondary growth in Arabidopsis
Project 4. Effects of pectin methylesterification of cell wall exclusion limits and porosity of pit membranes - a confocal microscopy study.
Project 5. Cloning of "inducible vectors" to study effects of "lethal" cell wall related mutations
Project 6. Generation and characterization of PME overexpressing Arabidopsis.
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Hormonal control of wood development
Ewa Mellerowicz, Dept of Forest Genetics and Plant Physiology, SLU, tel .
E-mail:
and Björn Sundberg, Dept of Forest Genetics and Plant Physiology, SLU, tel .
E-mail:
Wood formation is a coordinated process whereby a cell grows and develops specialized cell wall depending on the long-range signals form the plant, short-range signals from the cell's neighbors and according to cell's competence to respond to these signals. Ethylene (ET), GA and IAA are most important signals in wood development. We are currently examining the role of ET in wood formation. You can select one of the following project:
Project 1: Biosynthesis of ethylene in wood
ET is one of the most important signaling molecules in the wood development but the source of ET in the wood is not known. ET is directly produced from ACC by the action of ACC oxidase, which in theory could be either a cell autonomous process occurring in each cell type where ET is needed or it can occur in some cell types only followed by diffusion of the ET to target cells. ACC precursor, in turn, can be either produced in the wood by ACC synthase residing there or it can be transported to the wood from roots or leaves. We have cloned ACC oxidase and ACC synthase cDNAs from the wood forming tissues of aspen. Your goal would be to analyze tissue- and cell type-specific expression of ACC oxidase and ACC synthase genes using in situ RT-PCR and to immunolocalize ACC oxidase in the wood forming tissues in order to obtain a more complete understanding of the source of ET in the wood. You will learn techniques such as western blotting, immunolocalization, in situ-RT-PCR TEM and confocal microscopy.
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Secondary metabolites as antifungal agents in boreal Ericaceous plants
Johanna Witzell, Dept of Forest Genetics and Plant Physiology, SLU, tel 786 93 92.
E-mail: and
Anna Shevtsova, Dept of Forest Vegetation Ecology, SLU, tel 786 62 90, 070 - .
E-mail:
In several ecosystems, microorganisms have been shown to play a significant role in ecological changes. For instance, in boreal forests, parasitic fungi have been found to mediate changes in the species composition of herbaceous layer. The success of parasites on plants may be affected by the chemical quality of plants. Boreal Ericaceous plants contain phenolic secondary metabolites, which are potential antifungal agents. However, the exact effects of these chemicals on fungal parasites are still poorly known. The aim of this study is to test the direct (in vitro) effects of secondary metabolites of boreal Empetrum and Vaccinium plants on parasitic fungi in a series of biotests. Selected fungi will be cultivated on artificial medium, to which leaf extracts of Empetrum and Vaccinium and potentially antifungal plant secondary chemicals will be added in known concentrations, alone and in combinations. The growth characteristics (dry weight and colony diameter) of the fungi will be measured and related to secondary chemical concentrations and composition of the growth medium. The results are expected to elucidate whether or not the secondary chemicals present in Empetrum and Vaccinium leaves function as antifungal defensive chemicals in these plants.
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Proteomic approach to study proteins in the cyanobacterium Synechocystis with high homology to the Chlorophyll a/b binding proteins of plants.
Christiane Funk, Dept. of Biochemistry, Umeċ University, tel , fax .
E-mail:
Cyanobacteria are thought to be the evolutionary origin of the plant chloroplast. In the photosynthetic cyanobacterium Synechocystis sp. PCC five genes have been identified with homology to the chlorophyll a/b binding proteins (Cab proteins) of higher plants, although these cyanobacteria only bind chlorophyll a. The function of the small Cab-like proteins (SCPs) is not known. The SCP proteins were tagged with a His6 epitope in Synechocystis to faciliate the identification.
- What proteins build complexes with these SCPs ? The His-tagged SCPs will be purified using a Ni-column; co-purified proteins, that might build complexes with the SCPs can be identified using the proteomic approach (2D-gel electrophoresis and mass spectrometry).
- Do the SCPs bind pigments ? The SCPs will be overexpressed in E.coli. In vitro pigment reconstitution studies will show if these overexpressed proteins are able to bind chlorophyll or carotenoids and will fold in the presence of these pigments. The Biocore can be used for the same purpuse. Also mild isolation methods will be used to purify the SCPs in a pigment binding state.
Further information at: http://www.chem.umu.se/dep/biochem/forskning/cyano/
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Consequences of global warming on plant biochemistry in European shrublands
Annika Nordin, Dept of Forest Genetics and Plant Physiology,, SLU, tel , .
E-mail:
VULCAN - Vulnerability assessment of shrubland ecosystems in Europe under climatic changes - is a EU project investigating the effects of changes in the climate on the functioning of shrublands in order to support political decisions as well as management practices to sustain the quality of this habitat type in Europe. Project sites are located in Denmark, UK, Netherlands, Spain, Sardinia and Hungary. For more information see the VULCAN project homepage (http://www.vulcanproject.com/).
Within this project we now specifically want to investigate the effects of a warmer climate on the biochemistry of different shrubland plant species. Plant biochemistry plays a key role in ecosystem carbon and nitrogen cycling. Moreover, the biochemical quality of plant tissues determines plant susceptibility to attack from parasitic fungi as well as insect and mammalian herbivores. The results from the study will be evaluated in collaboration with other European researchers active in investigating other aspects of plant and soil ecology within the project.
The project will include a field trip during the vegetation period to visit the VULCAN project sites in Denmark, Netherlands and Wales to collect plant samples. At the laboratory in Umeċ the biochemical analysis of the plant material will take place. Specifically analysis of amino acids, proteins, soluble carbohydrates and phenolic compounds will be performed using HPLC (high performance liquid chromatography) and GC (gas chromatography) technique. The planned project is suitable for a student in biology, ecology, or biochemistry.
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Nitrogen fixation and hydrogen metabolism in symbiotic and free-living bacteria
Supervisor: Anita Sellstedt, Dept of Plant Physiology, Umeċ university, tel .
E-mail:
Nitrogen is one of the most limiting essential elements for plants in nature. Of the diversity of procaryotic organisms, Rhizobium and Frankia are capable of biological nitrogen fixation, i.e. reduction of atmospheric nitrogen to ammonia assisted by the enzyme nitrogenase. These organisms also form symbioses with plants. The widespread soil filamentous bacterium Frankia infects a wide range of host families, all of them woody dicotyledonous plants. Although plants infected by the actinomycete Frankia (actinorhizal plants) rival legumes in the amount of nitrogen fixed, less research has been performed on them. One of the main reasons might be that it was not until that the first Frankia strain was available. Most Frankia differentiate into three different cell types, which can be observed in pure culture as well as in symbiosis, i.e. vesicles, hyphae and spores. However, in symbiosis the woody Casuarina do not develope vesicles, instead very specialized cell-walls containing lignin-like compounds has been observed. Different levels of effectiveness depending on both species combination and Frankia inocula has been shown. Studies of the regulation of the efficiency in nitrogen fixation is extremely important. By learning more about this regulation we can get a better knowledge of how to get efficient symbioses.
Student project 1: Uptake hydrogenase
Alnus - and Casuarina symbioses are shown to have excess of uptake hydrogenase, the hydrogen-oxidizing enzyme, in their nodules. Hydrogenase is believed to act as (i) protection of nitrogenase from oxygen and hydrogen and to (ii) reutilize some energy that is wasted as hydrogen is evolved from nitrogenase. Uptake hydrogenase activity was shown to be higher in the symbiosis compared to that in a free-living bacterium, and hydrogenase is a a very common characteristic in symbiotic Frankia. Only one symbiotic Frankia from Alnus has been shown to be Hup-, i.e. lacking uptake hydrogenase activity , with the larger hydrogenase subunit present but the small subunit missing or degraded. In free-living Frankia, the onset of nitrogen fixation was occurring before onset of uptake hydrogenase activity, indicating that hydrogen has to be evolved from nitrogenase before uptake hydrogenase starts to function. In N
2 fixing organisms, it has been suggested that occurrence of an uptake hydrogenase could be beneficial for the nitrogen fixation process (1) due to removal of hydrogen that is inhibiting the nitrogen fixing enzyme nitrogenase, (2) using H
2 as a respiratory substrate and thereby regaining some energy that is wasted via H
2 evolution and (3) using H
2 via the oxyhydrogen reaction to reduce oxygen levels and protect nitrogenase from oxygen inhibition (Dixon ). Support for these three suggestions has been obtained for Rhizobium. sp. Question: Is there any homology between uptake hydrogenase structural genes in Frankia and other organisms ?
Studies on homology between Frankia hup structural genes and other organisms will be made by use of a proteomics approach.
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Student project 2: Bidirectional hydrogenase
The metabolism of hydrogen by microorganisms has been known almost for a century. Biological production of hydrogen is thus one of the approaches used to obtain clean energy from biomass. The key enzymes involved in biological hydrogen metabolism are nitrogenases and hydrogenases. Nitrogenase is the enzyme catalyzing the reduction of atmospheric nitrogen to ammonium. Concomitantly with reduction of dinitrogen, there is also a reduction of protons to dihydrogen, resulting in evolution of hydrogen gas. Hydrogenases are only involved in biological hydrogen metabolism and can be divided in two functionally different groups; membrane associated uptake hydrogenase(s), which oxidizes hydrogen resulting in electrons and protons [H
2 ---> H e-], and reversible hydrogenase(s), mainly producing hydrogen gas [H e- ---> H
2]. In cyanobacteria both a membrane associated uptake hydrogenase and a soluble reversible hydrogenase have been described. Hydrogen is also evolved from nitrogenase. Unicellular cyanobacteria possessing nitrogenase have been shown to have a high hydrogen evolution . Method: How is the hydrogen evolution varying under different external conditions?
Measurements of hydrogenase activities will be recorded by use of a GC. Native (and SDS) -PAGE will be used in combination with Western immunoblots in order to verify the occurrence and to identify hydrogenases in organisms grown under different external conditions.
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Structure and function of the chlorophyll a/b-binding proteins
Stefan Jansson, Dept of Plant Physiology, Umeċ university, tel .
E-mail:
The light-harvesting chlorophyll a/b-binding proteins constitute a group of proteins that are the most abundant membrane proteins on earth. They co-ordinate the photosynthetic antenna pigments (chlorophylls and carotenoids) to ensure maximum efficiency in energy transfer, but they are also involved in both the long-term and short-term acclimation of the photosynthetic apparatus to different environmental condition. In the prime model plant,
Arabidopsis thaliana, 30 genes encodes proteins of this group and in our research group, we are systematically creating transgenic plants lacking the different gene products and analyse their phenotype. We also work with mutants affected in photosynthetic regulation. We analyse the plants using normal phyiological characterization (growth in different conditions, photosynthetic measurements) standard molecular techniques (nothern, Southern and immunoblotting), biochemistry (separation and analysis), fluorescence spectroscopy and ecological studies (fitness in different environments). You will in this project take part in this work, the precise tasks will depend both on the progress of the research and your personal interests.
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A novel protein uptake mechanism in Arabidopsis chloroplasts.
Jan Karlsson, Dept of Plant Physiology, Umeċ university, tel .
E-mail: and
Göran Samuelsson, Dept of Plant Physiology, Umeċ university, tel .
E-mail:
A new route for protein uptake into plastids was recently discovered by us. It involves uptake into the ER, glycosylation and further an unknown transport mechanism into the chloroplast. So far only one protein has been found to take this route. We propose a project to elucidate: what other proteins that takes this route, what is the molecular uptake mechanism for the uptake, and finally what information in these putative polypeptides guides them through the ER to the chloroplast. The project will involve protein separation on 2-D gels, mass-spectrometry, separation techniques, and bioinformatics.
Contribution of mitochondria and respiration to leaf senescence.
Per Gardeström, Dept of Plant Physiology, Umeċ university, tel .
E-mail:
Senescence is the final stage of plant development and can be induced by a number of both external and internal factors such as age, prolonged darkness, plant hormones, biotic or abiotic stress and seasonal responses. Regardless of how senescence is initiated it will eventually lead to cell death. In leaves an early event in leaf senescence is a decrease in photosynthesis which is accompanied by degradation of chlorophyll and the entire chloroplast. However, the cell degradation is initially only partial and compartmentation is maintained with intact mitochondria, peroxisomes and vacuoles. The aim of the project is to study leaf senescence using the model plant Arabidopsis thaliana. The focus will be on the involvement of mitochondria and respiration in the recovery of carbon and nutrients from senescing leaves. The hypothesis is that induction of senescence will lead to a change in mitochondrial function from mainly photorespiratory and redox regulation to ATP synthesis and production of carbon skeletons for retrieval of nitrogen. The project will combine a biochemical characterisation of mitochondria isolated from leaves under different stages of senescence with changes in mitochondrial protein content. Isolation of mitochondria from green and senescing leaves will be made using a new, very efficient grinding procedure. After purification using differential and Percoll gradient centrifugation respiratory properties will be determined using an oxygen electrode. From carefully purified mitochondria a proteomics approach will be taken to study changes in protein composition induced by senescence. Differences in the soluble protein composition between mitochondria from green and senescing leaves will be characterised by two-dimensional gel electrophoresis. Some proteins that increase or decrease will be identified using mass spectrometer techniques and available databases.
Photosynthesis and stress in cyanobacteria
Dmitry Sveshnikov (Group of Prof. Gunnar Öquist), Dept of Plant Physiology, Umeċ university.
E-mail:
Photosynthetic machinery of higher plants is extremely flexible and adaptive to environmental changes such as nutrients deficiency, excessive light and temperature fluctuations. The issue is to understand how photosynthetic organisms avoid photooxidative destruction of photosystem II (PS II) and other cellular constituents under conditions when an ordered dissipation of excited chlorophyll through photosynthesis is impossible due to environmental constrains. Much of the research in the field is actually done on cyanobacteria, which contain almost the same photosynthetic apparatus as higher plants and allow more direct approach than plant chloroplasts. The project on iron deficiency in cyanobacteria is now in progress in the lab. While iron plays a key role in the photosynthetic apparatus, the lack of iron is a common encounter in the nature so living organisms have developed a number of strategies to deal with it. Structural and functional reorganisation under iron deficiency involves PS II core and cytochrome b6/f complexes. Among other effects, a component of PS II called CP43 is suppressed, and a homologous CP43’ is accumulated instead. A number of mutant strains with altered CP43’ expression activity is now available. The research goal is to reveal the localisation of the protein, its pigment content, and its exact function. Bench work includes culturing of Synechococcus strains under normal and iron-limited conditions, isolation of thylakoids (differential centrifugation) and photosystems (FPLC), biochemical characterisation of proteins (gel-electrophoresis, Western blotting) and pigments (HPLC), and biophysical measurements of photochemical activity of the photosystems (fluorescence techniques).
Pigment protein organization in winter stressed pines
Dmitry Sveshnikov (Group of Prof. Gunnar Öquist), Dept of Plant Physiology, Umeċ university.
E-mail:
You will further characterise the pigment organisation of winter stressed pine in relation to the heat dissipation ability. Our laboratory functions in tight collaboration with the University of Western Ontario, Canada, running a joint project on cold acclimation in pine needles. Winter reorganisation of the light-harvesting chlorophyll antennae upon degradation of photosystem II due to photoinhibition allows pine to maintain large reserves of chlorophyll in a quenched state thus avoiding severe photodestruction of pigments and thylakoids during the winter. This may be of great significance for the success of evergreens in cold climate and helps explain how conifers can compete with deciduous trees.
Carbohydrate Sensing and Signalling in Plants
Leszek A. Kleczkowski, Dept of Plant Physiology, Umeċ university, tel .
E-mail:
The studies focus on the role of sugars (e.g. sucrose, glucose) as transduction signals regulating activities of selected genes in plants, especially those encoding ADP-glucose pyrophosphorylase (AGPase), UDP-glucose pyrophosphorylase (UGPase) and sucrose synthase (SuSy), key enzymes of starch and sucrose synthesis. All these genes are potently and differentially regulated by sugars, possibly serving as important components of cell homeostatic response during stress conditions. The effects of sugars and the identities of factors involved in sugar transduction pathway(s) will be evaluated in Arabidopsis plants, both wild-type and mutants/ transgenics affected in sugar-signalling. Effects of stress conditions and the resulting changes in sugar levels will be evaluated with respect to regulation of AGPase/ UGPase/ SuSy genes and starch/sucrose metabolism.
I. Stress and sugar-signalling connection
In most stresses (e.g. cold exposure), sugar levels increase, providing possible signal(s) for gene responses during stress acclimation. Plants are able to sense changes in carbohydrate metabolism, and they relay this information, through distinct transduction pathway(s), to the nuclei where alterations in gene expression are brought about. This leads to the modulation of enzyme synthesis and metabolic activities of the plant. In the present study, stress responses of AGPase, UGPase, and SuSy genes will be compared with those induced by sugars in vitro.
II. Dissecting sugar signal transduction pathway(s) by using inhibitors
In plants, protein phosphorylation and/or dephosphorylation have been implicated in responses to many signals including light, pathogen invasion, and temperature stress. In many cases, sugars are believed to serve as triggers of the (de)phosphorylation events. In the present study, inhibitors of protein kinases (chelerythrine, staurosporine) and protein phosphatases (okadaic acid, calyculin, etc.) will be applied, together with sugars, to detached leaves, and the regulation of AGPase and SuSy gene expression will be compared to that in control plants. This is a very powerful method, which - if successful - allows for quick identification of a possible protein (de)phosphorylating event in any signal transduction pathway. What's in it for the student?: A student (10 or 20p project) can be involved in any of the aspects of the above-described projects. For instance, in project I, a feasible student assignment could involve an exposure of plants to low temperature conditions and studying expression of relevant genes and changes in starch/sucrose levels. In project II, a subject for student assignment could involve studying sugar regulation of AGPase/ UGPase/ SuSy genes in plants exposed to protein kinase/ phosphatase inhibitors. In all assignments, students will get a broad research background in plant biology, utilizing molecular biology, genetic, and biochemical approaches. All interested students are encouraged to contact Leszek Kleczkowski to discuss suitable projects.
A proteomics approach to identify copper-regulated proteins.
Mats Eriksson, Dept of Plant Physiology, Umeċ university, tel .
E-mail:
Copper is an essential constituent of a large number of enzymes, but at the same time, in high concentrations this metal is toxic to cells. Due to this, cells must have a precise system to tightly regulate its copper uptake and use and the expression of many proteins involved in this process. In the unicellular green alga Chlamydomonas reinhardtii we have identified the master regulator of copper-regulated gene-expression, Crr1, and we have a strain mutated in the gene coding for this protein. We know that Crr1 regulates at least seven genes and we have sequenced three of these. Our goal is to find, identify, and sequence all proteins regulated by Crr1 to learn more about the mechanisms plant cells are using to adapt to copper deficiency. In this project you will identify new proteins regulated by Crr1 through a proteomics approach. You will grow Crr1- and wild type Chlamydomonas cells in media with and without copper and compare their protein profiles by 2D electrophoresis. Differentially expressed proteins will be analysed by mass spectroscopy and identified by database searching. By doing this project you will get the possibility to learn all the techniques needed in proteomics, a field of science that will be one of the most important ones in the post-genomic era.
Is high-PI Superoxide Dismutase a Glycosylated Monomer?
Gunnar Wingsle, Dept of Forest Genetics and Plant Physiology, SLU, tel .
E-mail:
Superoxide dismutas (SOD) is an enzyme that’s converts superoxide to hydrogen peroxide. The enzyme has a central role in scavenging of reactive oxygen species as superoxide and the production of hydrogen peroxide. These components also have an impact in signalling and regulation of defence related genes. In plants several isoforms of SOD has been identified. We have indications that the usual dimeric form may consist of a monomer structure in a special high-PI SOD in Populus. In addition this type also shows an unresolved high molecular weight- a possible glycosylation ? This project will be focused on this special isoform where protein purification and analysis with mass spectrometer will be central tools to resolve these questions.
Improved Plant Resistance
Gunnar Wingsle, Dept of Forest Genetics and Plant Physiology, SLU, tel .
E-mail:
This project will be focused on improving the resistance in pine seedlings to abiotic and/or biotic stress. A big problem for plant nurseries is that plants being produced in greenhouses do not have the full potential to cope with the circumstances that the “real” environment produces outside. E.g high light, low temperatures and pathogens will cause big problems for the plants. In this project we will try to enhance the capacity of the plants to cope with these types of stresses. This will be performed by using signal components utilsed by the plants itself and also to use different stresses to prepare the plants for the subsequent stresses. Plant stress tools using e.g. fluorescence measurements will be important for this project.
