A proteomics approach to identify copper-regulated proteins - Mats Eriksson
Bidirectional hydrogenase - Anita Sellstedt
Biodiversity of Cyanobacteria and their adaptation to mosses in the Boreal forests - Anita Sellstedt
Changes in photosynthetic pigments during leaf senescence - Per Gardeström
Elucidating isoform specific functions of plasma membrane proton ATPase - Markus Müller
Genetic control of adventitious root formation in Arabidopsis thaliana - Catherine Bellini
Improved Plant Resistance - Gunnar Wingsle
Identification of ELD1 interacting proteins using two hybrid interaction studies - Rishi Bhalerao
Is high-PI Superoxide Dismutase a Glycosylated Monomer? - Gunnar Wingsle
Redox state of the mitochondrial ubiquinone-pool - Per Gardeström
Structure/function properties of UDP-glucose pyrophosphorylase - Leszek Kleczkowski
Uptake hydrogenase - Anita Sellstedt

Genetic control of adventitious root formation in Arabidopsis thaliana

Catherine Bellini, Dept. of Forest Genetics and Plant Physiology, SLUTel: 786 84 64

E-mail: 10 or 20 points project

Adventitious rooting is an essential step in forming root systems of lower vascular plants, monocots, and clonally propagated economically important horticultural and woody species. Adventitious roots may develop from different aerial organs (hypocotyl, stem, leaves) and from different tissues (pericycle, mesophyl, parenchyma, cambium, non-differentiated secondary phloem, protoxylem, epidermis. Adventitious root formation is a complex process, which is affected by multiple factors including phytohormones, phenolic compounds, nutritional status, associated stress responses such as wounding, and genetic characteristics. Adventitious rooting on vegetative explants allows clonal propagation and rapid fixation of superior genotypes prior to their introduction into production or breeding programs. This strategy is often used for long-lived woody species. Nevertheless, the inability to initiate adventitious roots remains an obstacle for elite genotypes of many crop and woody species. The mechanisms by which adventitious roots are formed are still not well understood.

As part of a strategy to identify genes involved in the regulation of adventitious root formation we decided to screen for suppressor mutations of the adventitious root phenotype of the superroot 2 mutant. sur2 mutants were isolated and characterized previously in our group (Delarue et al. Plant Journal 14, 603-611 ()). We have cloned the gene and showed that it encodes the cytochrome P450 CYP83B1, which catalysis the first step in the conversion of IAOx to indole glucosinolates (Barlier et al., Proc Natl Acad Sci U S A 97, 9-24. ()). This mutation leads to the accumulation of endogenous free IAA, which induces development of adventitious roots on the hypocotyl. sur2-1 homozygote seeds with EMS, and independent M2 lines produced. The screen already started and led to the identification of few potential repressors. We propose to the student to participate to the characterization of these newly identified mutants and to the screening for new suppressor mutations.

In parallel we have recently shown that genes from the auxin response factors family (ARFs) and auxin responsive GH3 genes may play a role in the regulation of either auxin metabolism or adventitious root formation. Mutants in these genes have been identified and need to be characterized in order to understand what is the contribution of these genes in the regulation of this aspect of plant development.

Supervisors: Catherine Bellini and John Bussel

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Biodiversity of Cyanobacteria and their adaptation to mosses in the Boreal forests

Anita Sellstedt, Dept of Plant Physiology, Umeå university, tel .
E-mail:

This title contains two projects1) investigation of the diversity of cyanobacteria in association with mosses in boreal ecosystems and 2) investigation of the adaptation of the cyanobacteria to the mosses in natural conditions.

Introduction

Recently we reported on a N-fixing association between a cyanobacteria (Nostoc spp.) and the ubiquitous boreal moss, Pleurozium schreberi Mitt. (DeLuca et al ), which largely explains the origin of nitrogen in the boreal forest. Our findings suggest that previous efforts have severely underestimated the potential N fixation rates in mosses in the boreal forest, as this newly found association may fix between 0.5 and 3.0 kg N2 ha-1 year-1 thereby leading to significant N accretion in this ecosystem (DeLuca et al ). This is a completely new finding, as this association has not been described as nitrogen fixing before.

The endobionts inhabiting P. schreberi are species of cyanobacteria, which are capable of forming associations with almost all groups of plants (Bergman et al., ) and they are also capable of fixing nitrogen (Sprent & Sprent, ). This is to advantage for many ecosystems since nitrogen is one of the most limiting essential elements in nature (Black, ). Looking into the associations, it has been shown that the cyanobacterial part of the association most often invades structures such as leaf cavities that are a normal part of the host plant (Solheim & Zielke, ). Mosses may also form casual associations with nitrogen-fixing cyanobacteria, which may be epiphytic. There are some reports on nitrogen fixation in Sphagnum (Granhall & Lindberg, ; Granhall & Selander, ), where the cyanobacteria are found in the hyaline cells (Meeks, ). And indeed, there is a report on nitrogen fixation in P. schreberi, but the attempt to find the cyanobacteria failed and the value was considered to be an anomaly (Alexander & Billington, )

Project 1. Diversity of cyanobacteria

Knowledge of the diversity of cyanobacteria is extremely important since cyanobacteria inhabit many ecosystems and are early inhabitants of the earth and are therefore of evolutionary interest. Earlier classification systems for cyanobacteria relied primarily on morphological and anatomical characteristics of cells and colonies, and do not always give the proper identification. A splendid physiological classification, based pure cultures, was drawn up by Rippka et al. (), taking into account that anatomical characteristics varies greatly with growth conditions. However, it is well documented that genetic analysis has to be included for a proper identification of the cyanobacteria. For studies of biodiversity there are to date several genetic regions/ genes suitable and common used for identification and phylogenetic analysis, e.g. sequence analysis of genes encoding the small subunit ribosomal RNA (Wilmotte, ), analysis of the 16S-23SrRNA internal transcriber spacer (ITS) and analysis of functional genes such as the hetR and nifH gene(West & Adams, ; Rasmussen & Svenning, ). Characterization by DGGE has also successfully been used on cyanobacteria (Rasmussen & Svenning, ).

Question: What species of cyanobacteria are associated with the mosses in the boreal forest and surrounding ecosystems and are there free-living cyanobacteria as well?

Field samples collected at app. 20 sites from the boreal forest and surrounding ecosystems in Northern Sweden will be used in the studies of cyanobacterial biodiversity. The methods involved are PCR- amplification of the ITSregion and subsequent analysis. The biodiversity of cyanobacteria population will be studied in vivo using direct PCR-amplification on intact plant material (moss and cyanobacteria). Primers specific for cyanobacteria will be used for amplification of a partial region of the 16S rRNA gene followed by sequencing analysis. The identity of the sequences will be determined by BLAST search in the NCBI database.

Project 2. Adaptation of cyanobacteria to moss

Cyanobacteria are found world-wide in greatly diverse ecosystems from marine to terrestrial. The genus Nostoc dominates in terrestrial ecosystems and includes members which are able to form symbiosis with e.g. bryophytes, fungi, water ferns, gymnosperms and angiosperms (Bergman et al, ; Rai et al. ), Seeing that cyanobacteria are so widespread gives hope that they can be used to create artificial associations. So knowledge about the interaction and diversity is of great importance when looking into creation of artificial associations. In (Gentili et al, , manuscript) we successfully reconstituted a cyanobacterial species with Pleurozium schreberii, indicating that this is possible also in P. schreberii. Interestingly, artificial associations causing changes in cyanobacterial morphology was reported by Gusev et al (), and gigantic cells and decrease in the peptidoglycan layer were shown. Another interesting aspect of the association is the chemotactic attraction of the plant to the cyanobacteria. It has been shown that a 12 kDa compound released by the host termed HIF induced hormogonia formation in the cyanobacteria (e.g. Campbell & Meeks, ; Rasmussen et al. ; Watts, ).

Question: How do the cyanobacteria adapt to different temperatures?

2-D-gel electrophoresis analysis of cyanobacteria will be performed in order to identify proteins that are turned on during different temperature regimes. The peptides will be analysed by use of MALDI-Q-tof and the peptides identified will be run against NCBI protein database.

Physiological nitrogen fixation activity will be measured by use of gas-chromatography (Mattsson & Sellstedt, ).

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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 N₂ 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 H₂ as a respiratory substrate and thereby regaining some energy that is wasted via H₂ evolution and (3) using H₂ 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.

Supervisor: , Dept. of Plant Physiology, Umeå university

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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 [H₂ ---> H e-], and reversible hydrogenase(s), mainly producing hydrogen gas [H e- ---> H₂]. 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.

Supervisor: , Dept. of Plant Physiology, Umeå university

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Elucidating isoform specific functions of plasma membrane proton ATPase

Plasma membrane localized proton ATPases establish an electrochemical gradient across the plasma membrane, which is used for a broad spectrum of energy consuming transport processes. Especially nutrient uptake and transport from source to sink tissues are driven by this proton motive force. The protein is a single subunit membrane protein, that uses ATP as energy source to pump protons from the cytoplasm to the outside of the cell. A regulatory extension on the cytoplasmic side acts as autoinhibitory domain that de-represses the enzyme after a phosphorylation event at its penultimate threonin residue and subsequent binding of a 14-3-3 protein. In Arabidopsis thaliana, 11 genes of plasma-membrane proton ATPases (AHA) are known, their specific function in the large number of processes, however, remains unclear for most of these genes. This project shall give a deeper insight into isoform specific functions of plasma membrane proton ATPases in Arabidopsis.

To study the specific function of certain members or groups of the proton ATPase family, we have choosen the RNAi approach. Small double stranded RNA fragments lead to destabilization of homologous RNA and as a result the expression of the targeted genes will be supressed. To create these RNA structures and to stably transform plants, numerous vector systems exist. We will use the Gateway^TM system to facilitate cloning. Since the RNAi approach is known to produce various degrees of silencing, we hope to be able to study effects of otherwise lethal mutations. In another approach, we want to study the tissue specific expression of AHA genes. Therefore we have generated AHA promoter::GFP-GUS fusions and transformed plants. From these plants we expect to get a comprehensive overview about the organ and cell-type specific expression of all AHA genes.

The candidate will participate in the microscopic analysis of promoter::GFP-GUS plants. This involves histological and microscopic methods, including confocal imaging. Further, the generation of RNAi plants will be continued, including molecular cloning, PCR, transformation of bacteria and plants. Depending on the progress of the project, analysis of RNAi plants, using real-time PCR and macroarray techniques will be performed. In the final step, the plants will be physiologically characterized.

Supervisor: , Dept. of Plant Physiology, Umeå University, phone: (090)-.

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Identification of ELD1 interacting proteins using two hybrid interaction studies

We have identified a potentially golgi localized Arabidopsis protein, ELD1 that appears to be important for cell elongation. However the exact function of this protein is unclear. In this project, yeast two hybrid system will be used to identify proteins that interact with ELD1. In a complementary proteomics approach Arabidopsis extracts will be used to immunoprecipitate ELD1 containing protein complexes followed by MALDI based identification of proteins interacting with ELD1. Furthermore, given the golgi localization of ELD1 and the phenotype of ELD1 mutant, the project will also involve analysis of cell walls from eld1 mutant using a panel of antibodies against specific cell wall components. In this project, you will get the opportunity to learn and use several techniques such as yeast two hybrid system and proteomics.

20 point or 10 point project

Supervisor: Dept. of Forest Genetics and Plant Physiology, SLU, Umeå

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Redox state of the mitochondrial ubiquinone-pool

We have adopted a method for determination of the redox state of mitochondrial ubiquinone (UQ) using extraction and HPLC separation (Wagner et al , Plant Physiol 108:277-283) to leaf protoplasts. The determination of the UQ redox level in chloroplast-containing cells depends on the successful removal of chloroplasts since the contamination of plastoquinone which interfere with the quantification of UQ. We have accomplished this by using the filtration step of “rapid fractionation” (Gardeström and Wigge , Plant Physiol 88:69-76). The first manuscript on UQ reduction level at different temperatures was recently submitted. The combined knowledge of redox status of NAD(P)(H) pools and the mitochondrial Q-pool will significantly extend our understanding of redox regulation in the plant cell.

The project will be to determine the UQ redox state in mitochondria of barley protoplasts incubated in photorespiratory and non-photorespiratory conditions. This would include a comparison of wt barley with a photorespiratory mutant deficient in glycine decarboxylation where results on subcellular NAD(P)(H) redox state is available (Igamberdiev and Gardeström , BBA :117-125)

Supervisor: , Dept. of Plant Physiology, Umeå university

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Changes in photosynthetic pigments during leaf senescence

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.

The project will be to study changes in photosynthetic pigments during the senescence process in Arabidopsis. Chlorophylls, carotenoids and xantofylls will be separated and quantified by HPLC. Two different ways to induce senescence will be compared 1) darkening of the entire plant and 2) darkening of individual leaves with the rest of the plant in normal light.

Supervisor: , Dept. of Plant Physiology, Umeå university

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Structure/function properties of UDP-glucose pyrophosphorylase

Leszek A. Kleczkowski, Dept of Plant Physiology, Umeå university, tel .
E-mail:

We have recently homology modeled a structure of UDP-glucose pyrophosphorylase (UGPase), a key protein of the production of glycogen, sucrose and cell wall polysaccharides (e.g. Kleczkowski et al. ). This is the first structural model of eukaryotic UGPase and it may be of key value in inferring structure/ function properties of other pyrophosphorylases. TheUGPase monomer is bowl-like, with an active site positioned at a central groove. The active site of UGPase contains several amino acid residues that were earlier shown to be important for substrate binding and catalysis of the enzyme. The protein has been proposed to be regulated by (de)oligomerization and, possibly, phosphorylation. The goal of this project is touse the derived model as a testable blueprint to verify details of the enzyme catalysis/ substrate binding and regulation. We can now rationally select amino acid residues, based on the model, modify them and test against the model with respect to enzyme activity and subunit integrity. An E. coli expression system will be used as in our previous study withbarley UGPase (Martz et al. ).

References

Kleczkowski LA, Geisler M, Ciereszko I, Johansson H () Plant Physiol 134: 912-918

Martz F, Wilczynska M, Kleczkowski LA () Biochem J 367: 295-300

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A proteomics approach to identify copper-regulated proteins.
Mats Eriksson, Dept of Plant Physiology, Umeå 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.

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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.

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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.

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