Multiscale structural basis of photoprotection in plant light-harvesting proteins

Plant productivity is defined by the ability to efficiently and safely harvest sunlight. Efficient light-harvesting is ensured by the 'antenna', a large assembly of chlorophyll-filled proteins that capture solar energy and deliver it to the few 'reaction centre' proteins that convert solar energy into chemical energy. However, light levels can to go from moderate to dangerously bright within minutes, leading to excess absorption of energy and damage to the delicate reaction centres. This is mitigated by Energy-dependent quenching (or 'qE'), a protective mechanism in which the antenna senses the high intensity of light and switches to a protective state. In this state excess energy is harmlessly dissipated (or 'quenched') before damage occurs.

Recently, it was shown that controlling qE at the molecular level is a very promising route to enhancing food production. This is somewhat hindered by the fact that, unlike other important biological mechanisms, the precise workings of qE are unclear. We do know that the major antenna protein, LHCII, plays a central role. High light intensity causes individual LHCII proteins to switch between energy-harvesting and energy-quenching states. They also collectively reorganize themselves to form large, clustered networks in the chloroplast membrane. qE is therefore a 'multi-scale' mechanism involving structural changes inside antenna proteins and in the whole antenna assembly. Unfortunately, we don't have a detailed picture of what these structural changes are which makes experimental data difficult to interpret and has led to many contradictory ideas of how qE works.

We will establish the mechanism of qE with greater accuracy than ever possible. We will use a 'bottom-up' approach, first studying how individual, isolated LHCII operate and using this to establish how they collectively operate as an entire 'antenna'. First we will predict the light-harvesting structure of LHCII. Since experimental techniques have failed to do this, we will instead use rigorous computational simulation. This allows us to mimic realistic conditions, in this case LHCII in a membrane under low light, and to see the movement and flexibility of the structure. Using the techniques of theoretical biophysics we will explain how this structure promotes efficient harvesting of energy rather than deliberate protective dissipation. We will then predict the potential protective, energy-dissipating structures of LHCII. The current 3D structure LHCII is often used as a prototype for the protective state but it is likely a poor approximation. We will use our bio-simulation approach to consider high light conditions, predict these structures, and fully characterize how they function protectively.

In the final part, we will consider the large networks of LHCII that occur in natural membranes by experimentation as well as theory. Using ultra-high resolution microscopy we will characterize the structures of these networks. At the same time, using high resolution fluorescence imaging, we will directly measure the rate at which energy is being quenched within them. When combined with our LHCII simulations we develop a complete picture of the qE mechanism: how it operates at the molecular level, how it is controlled by protein structure and external conditions, and how this effects the function of the plant antenna as a whole. This will finally answer a long-standing and controversial question in plant science. More importantly, it will provide a molecular foundation to our efforts to understand how plants capture, manage and transform the sun's energy.

The project objective is a structure-based molecular theory of the fast photoprotective mechanism (qE) in the PSII antenna of plants. Although now an immediate target for crop optimization efforts, qE is poorly structurally characterized and therefore highly controversial. We aim to resolve this with a multiscale approach, first establishing the structural changes which determine light-harvesting or quenching in individual LHCII sub-units and then combining these parameters with correlated AFM/FLIM to characterize large-scale photoprotective mechanisms within membranes.

The intrinsic switch in individual LHCII is too subtle to be resolved by structural experiment and so we will use a combination of Molecular Dynamics simulations and the PI's theory of carotenoid-mediated quenching to predict it. We will characterize how external conditions determine structure, how structure determines pigment interactions, energy transfer and quenching, and how these processes determine the functional and spectral properties of LHCII. By considering conditions and mutations that inhibit or promote quenched states we will reveal which interactions are responsible for energy quenching and the dynamics of their (de)activation.

Although quenching in isolated LHCII is a reductive model system, knowing its internal mechanics and kinetic parameters yields the basis for studying the basic functional units of in vivo qE: LHCII clusters that form in the thylakoid membrane. The Co-I's novel correlated AFM/FLIM approach allows us to directly visualize these and quantify their fluorescence kinetics. Combining this with our single molecule parameters we will construct the first structurally-informed theory of qE. We will determine which energy-quenching pathways are relevant in vivo, the concentration of these quenchers in the membrane and their relationship to the size and shape of the antenna superstructure. This will provide the core structural basis currently missing from this field.

This project will characterize the qE mechanism that allows plants to regulate their light-harvesting processes, switching between efficiency in low-light and protection in high light. Despite its importance to plant performance there is currently no definitive theory of qE due to a lack of structural information, both in terms of how individual LHCII light-harvesting proteins control their function by altering their shape and how the network of many of these proteins (the 'antenna') are rearranged and 'rewired' according to the light environment. Using a novel combination of bio-molecular simulation, theoretical bio-physics and super-resolution microscopy we will establish the light-harvesting and photoprotective structures of LHCII, the molecular mechanism at the centre of qE, and how these properties can be altered via structural modification. We have identified 4 impact objective.

1. Interaction with agritech: Working with A Ruban (QMUL) and S Santabarbara (CNR, Italy) the PI is developing tools for analysing the fluorescence measurements typically used in the field to quantify plant performance. A Ruban has links to the Waltz Heinz GmbH, developing systems for monitoring plant productivity, light-tolerance and photodamage through measurements of qE. Relating qE to macroscopic plant function is difficult given the lack of a coherent picture of what qE actually is. This project will address this and exploitation of the results will occur through the PI's current Royal Society International Exchange Grant which initiated this collaboration.

2. Future potential for understanding NPQ for crop productivity: Recently it was shown that qE is one of the key factors determining crop-plant productivity. Crude enhancement of qE in tobacco mutants resulted in a 15-20% yield increase in field conditions. However, without understanding the core qE mechanism, more refined approaches will be difficult. The project will produce a structure-based model of fundamental molecular processes of qE and its key structural features and experimental signatures. This involves characterizing altered structures of LHCII with enhanced qE properties. During the early stages of the project we will initiate communication with key research groups working in plant engineering to establish the data requirements of these efforts. At the culmination of the project we will initiate exchange with agritech companies such as Syngenta. The aim is to disseminate the key parameters of qE and our models of LHCII with enhanced NPQ features.

3. Developing the next generation of multi-disciplinary researchers: Bio-science is becoming a truly multi-disciplinary field. In particular, young researchers must be comfortable with both experiment and theory. The project is in collaboration with R Croce (Amsterdam) an expert in the spectroscopic techniques needed to support theoretical research in light-harvesting. Moreover, they will work very closely with the Co-I P Adams (Leeds) to carry out high level visualization measurements. By the end of the project the PDRA will be an expert bio-theoretician with the knowledge of the principals of spectroscopy and microscopy to allow them to design a comprehensive programme of research needed to support theoretical work.

4. Public communication and engagement: qE is a key bioenergetic mechanism but is generally unfamiliar to young science students. Theoretical approaches are also not seen as core to biology. We intend to take part in several out-reach activities, including the Leeds Festival of Science. These presentations have to be interactive and stimulating and we intend to achieve this with a simple animation and an interactive hydrological model of qE and light-harvesting that uses a network of water pipes and valves to illustrate how plants control the flow of solar energy into their biochemical processes.