Resolving the key photoprotective switch in photosynthetic electron transport

We aim to understand the way in which plants are able to adapt to fluctuations in the environment by studying a specific example that has the potential to improve crop plant tolerance to stress. In the final step of photosynthetic electron transfer, the enzyme ferredoxin:NADP(H) oxidoreductase (FNR) uses photosynthetic electrons to reduce NADP+ to NADPH, which is then used in multiple reactions and is essential for C fixation. The amount of this enzyme has a strong effect (a high coefficient of control) on the entire pathway of photosynthesis (0.7 at low light and 0.94 at saturating light (1)). Interestingly, it has also been shown that the amount of FNR also strongly correlates with the ability to tolerate multiple environmental stresses in tobacco (2,3), although the reasons for this are not yet clear. One contributing factor could relate to the free radicals produced by photosynthetic electron transport (PET). We recently showed that variable FNR content and location results in disrupted free radical production, and that this could be responsible for "priming" the plant, and inducing defence mechanisms (4).

Although FNR has been well studied as an enzyme, its location within chloroplasts is highly dynamic, with many interaction partners. The reason for these multiple interactions, the activity of the enzyme at these different locations and the relationship of these complexes with the rest of the PET apparatus is not understood. There are three important recent developments that will enable us to answer these important questions. Firstly, we have produced transgenic Arabidopsis plants with FNR proteins localised to different complexes within the chloroplast (5). This means we can now compare the activity of the enzyme, and its associated metabolic pathways, when it is bound to different places. Introduction of cyanobacterial FNR to higher plants has been patented as a means of improving stress tolerance in crop plants, but the interactions of this prokaryotic enzyme in higher plant chloroplasts are unknown. Our novel plants will allow us to pinpoint the interactions responsible for stress tolerance. Secondly, new equipment has been developed that will allow us to monitor the activity of the enzyme inside a living leaf (6), which is much more accurate than working with semi-purified systems, where important components or regulatory events may be lost. Thirdly, we have promising preliminary results from a microscopy approach, that will help us image where in the chloroplast membranes these events occur. This is important, as many regulatory events in chloroplasts can only be understood in the context of spatial organisation between different parts of the organelle, or are too weak to detect with standard biochemical methods.

Using these tools we aim to discover how dynamic redistribution of FNR is able to regulate PET and promote stress tolerance. Plants have limited resources available to them, and must allocate these to ensure the greatest chance of survival and reproduction. Improving the efficiency of switching between protective states and assimilatory states will therefore improve the chances of the plant not only surviving stressful conditions, but conducting rapid photosynthesis afterward and achieving a high harvest index. Better understanding of this regulation may help to design or breed plants able to withstand specific stresses, or rapidly respond to the presence and absence of stresses in order to achieve survival but maintain high yields.

(1) Hajirezaei MR, et al. (2002) Plant J 29(3):281-93.
(2) Palatnik JF, et al. (2003) Plant J 35(3):332-41.
(3) Rodriguez RE, et al. (2007) Plant Physiol 143(2):639-49.
(4) Kozuleva M, et al. (2016) Plant Physiol 172: 1480-1493.
(5) Twachtmann M, et al. (2012) Plant Cell 24(7):2979-91.
(6) Klughammer C, et al. (2016) Photosynth Res 128(2):195-214.

We aim to better understand the regulatory mechanisms by which plants balance their response to environmental stress with investment in bioassimilation and crop yield. Work on photosynthetic electron transport and stress tolerance indicates that the redistribution of the enzyme ferredoxin:NADP(H) oxidoreductase (FNR) between different complexes on the chloroplast thylakoid membrane is an excellent candidate for a dynamic switch between assimilatory (linear electron flow) and protective (cyclic electron flow) states. We will use newly developed genetic resources: Arabidopsis thaliana plants with wild type levels of FNR, but localization of the enzyme at different membrane proteins, to determine the associations that promote involvement in either cyclic or linear electron flow and the interactions important for promoting stress tolerance. As well as methods we have developed to follow NADP(H) redox state in chloroplasts, and standard methods for measuring photosynthetic electron transport by chlorophyll fluorescence and the electrochromic band shift, we have been in consultation with Walz Heinz GmbH during the final stages of development of the Dual/KLAS NIR spectrophotometer capable of following ferredoxin redox state in leaves. This will give unique insight into FNR activity at different sub-chloroplast locations. Moreover, free radical evolution from membranes with differentially localised FNR will be compared by electron paramagnetic resonance spin trapping and responses to various stresses will be followed at the physiological and transcript level. Finally, we will use fluorescence immunolabelling to examine where different FNR interactions occur within the chloroplast, and develop this in fluorescence lifetime imaging microscopy (FLIM) and direct stochastic optional reconstruction microscopy (dSTORM) super-resolution microscopy approaches to examine the spatial aspect of FNR redistribution, and how this influences interaction with other membrane complexes.

It is essential to identify traits that enhance plant tolerance to abiotic stress. This project aims to understand the specific properties of a protein that has previously been shown to enhance stress tolerance in crop plants, and which is also essential for photosynthesis. This protein is encoded by several copies in crop plant genomes, and depending on the gene product can be localised to different places in the chloroplast. We aim to identify which locations are critical for photosynthesis, and which are important for stress tolerance.

The pressures of global warming, in combination with accelerating soil erosion and salinification mean that agricultural methods will come under extreme pressure to even maintain yields in the near to mid-future. Combined with a growing global population, this will inevitably lead to the large scale movement of people and global instability. It is therefore imperative to identify ways in which plants are able to respond to environmental stresses imposed upon them. Moreover, the capacity of a plant to balance the response to these stresses with investment in bioassimilation is essential in order to maintain high yield (harvest index). Understanding the control of these relative investments will enable the generation of improved crop plant varieties. For example, identification of which genes encode iso-proteins with photosynthesis associated localisation, and which genes encode iso-proteins with stress tolerance associated localisation could allow promoter modification to produce iso-genic crop plants (with no foreign genetic material) capable of rapid response to fluctuating environmental conditions. Alternatively, the advent of crop genome sequencing opens up the possibility of defining new quantitative trait loci. Knowledge of which genes encode isoforms associated with stress tolerance will provide the opportunity to selectively breed crops using traditional methods, combining loci that could only be predicted to act synergistically through biochemical, biophysical and cell biological studies such as that outlined in this proposal.

A PhD student currently working on this project is financially supported by Bayer Crop Science with an Otto Bayer Fellowship. There are no restrictions on intellectual property involved, but throughout the fellowship, and moving forward into the proposed granting period we will liaise with representatives at Bayer Crop Sciences regarding findings on the project, promoting our discoveries and exploring any potential agricultural applications.