1. Large experiment to be conducted at RERAF, Denmark
Introduction: The participants at the meeting in York all work on the effects of temperature on plant carbon metabolism. However, slight differences in growth facilities/plant material etc make it difficult in some cases for the knowledge in one system to be used to interpret the results of another system. It was agreed that the Research Network would conduct a large-scale collaborative project in Autumn to investigate the effects of exposure to low temperature on various aspects of plant carbon metabolism. The project will take advantage of the wide range of expertise offered by the meeting participants and the facilities offered by the Danish participants at the Ris° National Laboratory (Ris° Environmental Risk Assessment Facility; RERAF). The individual growth chambers at RERAF are large (24 m2) and have the capacity to provide both low and high temperatures as well as bright light. The project will enable the Network to obtain a more detailed understanding of how acclimation to low temperature affects several different processes that affect the carbon economy of plants.
Parameters to be measured: These include CO2 exchange (i.e. photosynthesis, photorespiration, leaf respiration in the light and dark, root respiration), O2 uptake (to investigate the regulation of respiration in leaves and roots), biomass allocation (e.g. ratio of leaf area to leaf mass, and root length to root mass in pre-existing and newly developed tissues), 13C-carbon transfer from the shoot to the root, mycorrhizal associations between fungi and plant roots, metabolite analysis (sugars, starch, amino acids and finally relative growth rate.
Plant Material: Three different groups of plants that are currently being used by different members of the research network will be used; transgenic Poplar (wild-type and SPS-over-expressers), Plantago lanceolata and wheat. The SPS transgenics are likely to differ in their ability for photosynthesis to acclimate to low temperatures. P. lanceolata was selected, as there is considerable background information on the effect of temperature on the carbon economy of P. lanceolata at York. Wheat was chosen as it is globally one of the most important crops and because it exhibits a contrasting degree of mycorrhizal infection than P. lanceolata.
2. Movement of Students and Post-doctoral Researchers in the Network
Characterisation of gas exchange parameters in transgenic Tobacco
Collaboration between Dr Beth Loveys and Dr Owen Atkin (University of York, United Kingdom) and Dr Kjell-Ove Holmstr÷m (University of Sk÷vde, Sweden).
Trehalose production increases freezing tolerance in plants. In this study, we compared the gas exchange properties of transgenic tobacco plants that differ in trehalose production. Trehalose expression was controlled by inducible promoters (were produced in Sk÷vde, Sweden) and sent to the York laboratory in September . Several lines expressing TPS1 and TPS2 under the control of Rab18 along with non-transformed controls and empty vector controls were included in the experiments. Exposure to drought was used as a screening treatment, as trehalose production increases drought tolerance as well as freezing tolerance.
Dr Kjell-Ove Holmstr÷m and Professor Palvaĺs group in Helsinki are also conducting additional collaborative work on trehalose biosynthesis in transgenic plants. Hopefully this may yield additional material of interest to members of the network.
An analysis of the underlying mechanisms that control the temperature sensitivity of respiration in potato leaf protoplasts
Student: Liz Covey-Crump, Dept of Biology, University of York
UK supervisor: Dr Owen Atkin
Swedish collaborator: Professor Per Gardestr÷m
This project will investigate the underlying mechanisms that control the temperature sensitivity of plant respiration in potato leaf protoplasts. The response of protoplast respiration to temperature will be characterised and the regulation of respiration at different temperatures will be investigated.
Work completed in
Ms Covey-Crump travelled to Umeň, Sweden in April for 2 weeks to test the feasibility of the planned experiments involving, measurement of respiratory metabolite levels over a range of temperatures and a top down metabolic study to determine control distribution at different temperatures in potato leaf protoplasts. This also enabled familiarisation with the techniques involved. Protoplast isolation and characterisation by measurement of chlorophyll content, respiration and photosynthesis was carried out. Rapid fractionation of protoplasts was performed at two temperatures. The fractions were tested for purity by measuring marker enzymes. The metabolites measured in each fraction were ATP/ADP, pyruvate and hexose-phosphates. The concentration of reduced and oxidised ubiquinone was determined using HPLC.
Research to be completed in March-May
The actual experiments will take place in Umeň, Sweden in March-May . This trip will be used to actually assess the effect of temperature on respiratory metabolite levels, using the above techniques. In addition, a study will be conducted using top down metabolic control analysis to determine quantitatively the distribution of control over respiratory flux and whether this varies with temperature. Collectively these studies will further elucidate the impact of temperature on plant respiration.
Combined effects of temperature and CO2 on plant respiration
Student: Dan Bruhn Ris°
Danish supervisor: Dr. Teis N. Mikkelsen
UK collaborator: Dr Owen Atkin
The aim of this project is to investigate the mechanisms that control thermal acclimation of respiration in plants. This extends existing collaboration between Ris° and York and may also be extended to include research visits by Dan Bruhn to Estonia to conduct radiogasometric measurements to assess the changes that occur in the substrates used for respiration in the light and dark during the acclimation process in Plantago lanceolata. The collaboration with York will assess the effect of acclimation on the components of the mitochondria, in particular using the Q electrode to assess whether acclimation has changed the distribution of control over respiratory flux in the electron transport chain. A visit to Umeň may also occur in order for him to learn how to measure respiratory metabolites during the acclimation process. He is also likely to visit the lab of Tjeerd Bouma in The Netherlands to investigate whether acclimation of respiration involves changes in the costs associated with maintenance, growth etc.
Effect of temperature on leaf carbon metabolism in transgenic Arabidopsis
Student: Priscilla Gan, Dept of Biology, University of York
UK supervisor: Dr Owen Atkin
Swedish collaborator: Dr Vaughan Hurry
This project will determine underlying biochemical and physiological factors responsible for respiratory and photosynthetic acclimation to low temperature. The extent to which respiratory acclimation differs from photosynthetic acclimation (e.g. timing, requirement for the synthesis of new leaves or modification of existing leaves) will be assessed. Work will be conducted using transgenic Arabidopsis plants that differ in activity of key carbon metabolism enzymes involved in sucrose synthesis; sucrose phosphate synthase (SPS, anti-sense and overexpressed) and cytosolic fructose-1,6-bisphosphatase (F1,6BP, anti-sense) activity. The ability to acclimate to low temperatures is reduced in the antisense plants and enhanced in the over-expresser.
Research in in York, UK
The project will begin by assessing the impact of cold-acclimation on gas exchange properties of pre-existing, warm grown leaves and newly developed leaves formed in the cold. CO2 exchange measurements will be use to determine the effect of acclimation on key parameters of the Farquhar-type photosynthesis model, these being the maximum rate of carboxylation by the CO2 fixing enzyme, Rubisco (Vcmax) and the maximum rate of RuBP re-generation/photosynthetic electron transport (Jmax). The ratio of Jmax / Vcmax will be assessed ľ alterations in this parameter indicated changes in the relative allocation of nitrogen to carboxylation versus regeneration of RuBP. The effect of cold treatment on respiratory capacity will also be assessed using measurements of CO2 release (in darkness and in the light) and O2 uptake. Respiratory capacity will also be assessed in intact tissues via additions of uncoupling agents and exogenous substrates. Inhibitors of the cytochrome (COX) and alternative pathways (AOX) will be used to assess the effect of temperature on flux via these two pathways. Moreover, we will determine the effect of temperature of cytochrome c oxidase and fumerase activity in crude, whole leaf extracts. Finally, we will assess the effect of temperature on the amount of alternative and cytochrome oxidase protein using antibodies raised against AOX and COX. The impact of cold acclimation on biomass allocation, N-content/allocation and whole plant growth rates will be assessed. Importantly, the impact of low temperatures on the ratio of leaf area to leaf mass, nitrogen content and protein concentration of pre-existing and newly formed leaves will be determined.
Proposed research visit to Umeň, Sweden in
A 3 week trip to Umeň in is proposed for the student to learn how to do Western blots in order to establish that the plants being used are antisensed. The visit will also enable the student to be trained in how to assay the activity of key photosynthetic enzymes, such as Rubisco, stromal fructose-1,6-bisphosphatase, cytosolic (cytosolic fructose-1,6-bisphosphatase and sucrose-6-phosphate synthase. Subsequent visits will then enable the student to measure a range of photosynthetic enzymes and metabolites from plants grown under the different experimental conditions.
Analysis of Carbon Flux in Transgenic Arabidopsis at low temperature
Student: Peeter Tńlts, Estonia
Estonian supervisors: Dr Olav Keerberg & Dr. Tiit Pńrnik
Swedish collaborator: Professor Per Gardestr÷m
The Estonian members will expand their measurements of carbon flux through different control points in metabolism, utilising the SPS and cFBPase transgenic plants Arabidopsis available in Umeň. This work will mostly be conducted in Estonia. Plants with altered expression of key respiratory enzymes will be prepared both in Estonia and in Umeň. These plants will be analysed with respect to gas exchange in Estonia. Additional work, including studies of sub-cellular compartmentation of metabolites will be carried out in Umeň. This work may also be extended to incorporate the planned collaboration between Dr. Vaughan Hurry in Umeň and Dr. Tom Hamborg-Nielsonĺs group in Copenhagen, but this expansion will not be likely to occur until .
The ability of contrasting species that differ in leaf structure and physiology to acclimate to changes in temperature
Post-doctoral researcher: unknown
UK supervisor: Dr Owen Atkin
Swedish collaborator: Dr Vaughan Hurry
Dr Atkin has written a grant application to the NERC in the UK in December to investigate the effects of temperature on photosynthesis and respiration in plants that differ in leaf life span. The response of plants to short-/long-term changes in temperature is critical in determining the distribution of plant species on earth. This project will determine whether trees, shrubs, forbs and grasses that exhibit contrasting leaf structure, chemical composition and physiology differ systematically in their degree of photosynthetic and respiratory acclimation to changes in temperature. The project will also investigate the extent to which the processes of photosynthetic and respiratory acclimation are co-ordinated. At the completion of this project we will have a more thorough understanding about the underlying processes involved in photosynthetic and respiratory acclimation. The project will also enable better predictions to be made of how current and future changes in temperature affect the exchange of CO2 between plants and the atmosphere. In the event that this grant is funded a post-doctoral research will be employed to work jointly between York and Umeň, although the individual will be based primarily in York.
Isolation of genes associated with cold acclimation in Birch
Post-doctoral researcher: Dr. Franciose Martz, Rovaniemi
Supervisor: Dr. Marja-Liisa Sutinen, Rovaniemi
Collaborator: Professor Tapio Palva, Helsinki
Dr. Martz visited Helsinki in March to isolate clones from a birch cDNA library made to cold-treated leaves. Four genes of interested have been isolated and their characterisation is now underway. A second stay in Helsinki is planned for January to complete the analysis of birch tissues exposed to low temperature. Depending on these results, additional visits may be planned later in the year.
Generation of transgenic Arabidopsis using specific low temperature promoters
Collaborator: Dr. Vaughan Hurry, Umeň
Collaborator: Dr Kjell-Ove Holmstr÷m, Sk÷vde
Results in Umeň have shown that in transgenic Arabidopsis, manipulating sucrose biosynthesis has significant impacts on speed and extent of cold acclimation, on the robustness of photosynthesis in response to fluctuating temperatures and on growth rates at low temperatures. We have therefore initiated a new project to extend this work to develop transformants over-expressing more than 1 of the strongly regulated enzymes involved in sucrose biosynthesis. We will also use both constitutive and cold regulated promoters to test whether putting the introduced transgenes under control of a low temperature promoter influences the positive acclimation effects we have observed. The genes of interest have been isolated in Umeň and transformation constructs will be made and tested in Sk÷vde. Plant transformation and subsequent analysis will be carried out in Umeň.
Whole plan freezing experiments with transgenic Arabidopsis
Collaborator: Dr. Vaughan Hurry, Umeň
Collaborator: Dr Einar Mńntylń, Reykjavik
To date all of our work assessing the effect of introduced transgenes on freezing tolerance has been assessed on the basis of freezing damage to individual leaves or leaf discs. While this is standard practice it is not ideal. The aim of this collaboration is to utilise the whole plant freezing facilities being established in Reykjavik to test the freezing tolerance of our transgenic lines. This wok is planned for the early autumn of .
Production of reactive oxygen species at low temperature
Collaborator: Professor I.M. M°ller, Ris°
Collaborator: Professor Per Gardestr÷m, Umeň
Collaborator: Dr. Owen Atkin, York
An increased production of reactive oxygen species (ROS) is a common feature in all types of plant stress. We are studying the production and detoxification of ROS by isolated plant mitochondria, one of the major sites of ROS production in the cell. The amount ROS will be measured in vitro as a function of assay temperature using a variety of mitochondrial substrates to locate the main sites of ROS production in the electron transport chain. We will also try to identify and quantify the mitochondrial ROS detoxification enzymes and a number of plant species will be tested, especially heat- or cold-stressed plants and cold-acclimated plants, to see if, and how, mitochondrial ROS production and detoxification is affected.
3. Exchange of Teachers
In this first year there has been no exchange of teachers to present lectures in undergraduate or graduate courses in network institutions. However, these activities are being planned for and will include (but are not limited too) teacher exchanges between York and Umeň to give lectures on Plant Physiology and Plant Ecophysiology. In addition, in Umeň we plan to invite lecturers to participate in our course in Plant Molecular Biology in the autumn of , with the aim of including a one-day mini-symposium on molecular approaches to modifying plant stress responses. The inclusion of this symposium was also delayed because of our institutional reorganization in Umeň
