Genetic manipulation of photoprotection and photooxidative stress tolerance in rice

All plants, including crop plants need to absorb light energy from the sun in order to grow, develop and eventually produce a harvestable product such as fruit or grain. Light is needed for plant development and it is also needed in photosynthesis where it is combined with carbon dioxide and water to synthesis sugars. The amount of light available cannot be controlled by the plants and depending on climatic factors, photosynthesis can be limited by light or it can absorb more than it needs. When too much light is absorbed, or 'harvested' there is a real danger that the energy will be passed to oxygen to form radicals which will damage plant tissues and even cause plant death. There are a number of mechanisms operating at the molecular level which sense the amount of surplus energy and 'dissipate' it harmlessly in a process called non photochemical quenching or NPQ. One mechanism involves the protein called PsbS which is present in all plants and acts as a 'switch' between light harvesting and energy dissipation. Another mechanism involves the synthesis of carotenoid molecules (specifically xanthophyll cycle XC carotenoids) which are colourful pigments (also present in all plants). They are also important antioxidants in the human diet. In plants they have a dual role: firstly they too regulate the process of NPQ, 'tuning' it to last a short or a long time. Secondly they are proven and powerful antioxidants in leaves, preventing damage to membranes. So far these molecules have only been investigated in the model plant Arabidopsis thaliana. There is a real need to investigate how these properties could be used in crop plants in order to improve growth and yield especially in stressful situations such as heat, drought or cold where, combined with high light, much damage from oxygen radicals can occur. This project uses a model crop, rice, in which the levels of PsbS and XC carotenoids have been manipulated by plant transformation procedures. Rice was chosen because it is easy to transform and has a sequenced genome. Plants with raised and lowered amounts of PsbS and raised and lowered amounts of XC carotenoids have been produced. The objectives of this proposal are to test the effects of these alterations on the efficiency with which light is absorbed and utilised by the plant. Are they at optimum levels or can we improve them? Secondly these plants, especially with raised levels of XC carotenoids should have an enhanced resistance to stress where membranes are the target, for example cold or heat and in the light. We will look for an enhanced tolerance to these stresses.. We will examine the biochemistry of plant membranes to find out how much more, or less, resistance exists. Lastly we will examine the growth rate and the potential for production of these plants in situations similar to growth in the field for grain production. We will find out whether the enhanced level of resistance to stress and the altered light use efficiency has a cost for the plant, or if it provides a real advantage. An important question to ask is whether the natural fluctuating levels of light we see outside in the field situations is efficiently converted by these processes or whether there is scope for improvement. There is good reason to believe that this project will show that we can make crop plants more resistant to environmental stress. Responses of plants to environmental stress should become more important as the impact of climate change is felt by agriculture. Additionally these processes should be of benefit to all crop plants including those which are used for energy crops or biofuels.

Rice plants (Oryza sativa) var. kaybonnet (japonica) were transformed. The genes used encoded for the thylakoid protein PsbS and the carotenoid biosynthesis enzyme beta-carotene hydroxylase (ChyB). Preliminary microarray studies in rice had shown that expression of these genes was consistent with the role in the optimisation of photoprotection. PsbS is a regulator of the photoprotective process non-photochemical quenching (NPQ). ChyB regulates the pool size of xanthophyll cycle (XC) carotenoids which have a role in NPQ regulation. However XC carotenoids are also antioxidants in leaves, reducing levels of lipid peroxidation under photooxidative stress conditions. Overexpression was achieved by using the genes in the sense direction under the control of the strong cestrum yellow leaf curling virus promoter. Reduced expression was achieved using an RNA interference approach with the same promoter. Transformation took place as part of a collaboration with Syngenta (Raleigh NC). Preliminary data showed that levels of NPQ, PsbS protein and XC carotenoid levels in leaves of rice transformants were equivalent to those achieved in published data for Arabidopsis thaliana. Evidence of altered tolerance to excess light stress was observed in whole rice plants. Techniques used will include gas exchange (leaf and whole plant), chlorophyll fluorescence, lipid peroxidation assays, pigment HPLC analysis, western blotting, PCR. Objectives: 1. Establish and quantify the kinetics of short-term responses of NPQ and leaf photosynthesis to alterations in light intensity for each transgenic type. 2. Define the temperature tolerance range under high light intensity for each transgenic type. Test the hypothesis that lipid peroxidation is a central mechanism of tolerance. 3. Quantify whole plant photosynthesis under high and fluctuating irradiance levels. 4. Quantify growth rate, biomass production capacity, leaf NPQ and radiation use efficiency under natural fluctuating light.