By introducing fluorescence protein sensors into live plants, a novel method that allows in planta measurement of NADPH level and NADH/NAD+ ratio in different cell types has been developed. These transgenic lines enable scientists to visualize the dynamic changes of these molecules in different subcellular compartments in real-time, to study photosynthesis and photorespiration.
Plants harvest energy from the sun and use this to fix CO2 from the atmosphere to produce complex organic molecules which are the basis for life on the earth. The process of photosynthesis takes place in leaves and other green parts of the plant where chloroplasts are main players of the process but the whole cell is involved. In plants, the shift between respiratory metabolism in the dark and photosynthetic metabolism in the light makes redox control of metabolism particularly complex. For an efficient process the redox states of all cellular compartments must be coordinated but is has been very difficult to obtain In planta data on this important aspect.
During C3 photosynthesis, for every 3 fixed CO2 molecules, about one O2 molecule is mistakenly fixed by Rubisco in chloroplasts. The recycling of the photorespiratory product involves reactions in both chloroplasts, peroxisomes and mitochondria. In connection to this it is commonly agreed that redox transfer between the compartments involved is important and that malate-OAA exchange contributes to this. However, the redox balance between the compartments is not well established and several suggestions can be found in the literature.
To study this question, an international team of researchers led by Dr. Boon Leong Lim of the School of Biological Sciences of the University of Hong Kong adopted fluorescent protein sensors to specifically monitor in planta dynamic changes in NADPH and NADH/NAD+ ratio in young leaves. The redox states of chloroplasts, cytosol and peroxisomes could be followed during transitions between dark and light with an emphasis on interplay between photosynthesis and photorespiration. Conventional detection methods require extraction and purification of these redox metabolites and subsequent determination by chemical methods. These methods have a few drawbacks as they are incapable of real-time, in planta measurements, nor measurement of these molecules in different cell types or different subcellular compartments. "Our novel technique can circumvent all of these problems. By employing these novel fluorescent protein sensors, we found that photorespiration supplies a large amount of NADH to mitochondria during photosynthesis, which exceeds the NADH-dissipating capacity of the mitochondrial respiratory chain. Consequently, the surplus NADH must be exported from the mitochondria to the cytosol through the mitochondrial malate-OAA shuttle. (Figure)", said Ms. Sheyli Lim, a PhD student and the first author of a manuscript published in Nature Communications. "Solving this question allows us to understand more about the energy flow between chloroplasts and mitochondria during photosynthesis, which could help us to booth the efficiency of photosynthesis in the future".
"The ability to get in vivo estimations on subcellular redox states gives important novel information on regulation of plant metabolism. The results highlight the close connection between the different subcellular compartments to achieve an efficient process. I have for a long time been studying the mitochondrial contribution to photosynthetic metabolism so for me this aspect has been most interesting" said co-author Prof. Per Gardeström of Umeå University.
Dr. Lim added: "We are the first group to introduce these three novel energy (ATP, NADPH, NADH/NAD+) sensors in plants. I wish they will have wide applications in researches regarding plant bioenergetics.".