Enhancing photosynthesis in crop plants: targets for improvement

Aerobic biological electron transfer systems avoid side-reactions with O₂ by redox tuning. Two physico-chemical numbers are key to these processes: i) -160mV, the redox midpoint potential of the O₂/O₂^-• couple, determining the favourability of superoxide formation; and ii) ~1eV, the amount of energy needed to convert ³O₂, molecular oxygen, to its highly reactive and damaging singlet form, ¹O₂. The occurrence of anomalously energy-inefficient reactions in aerobic bioenergetics can be understood in these terms. Protective and regulatory mechanisms can also be rationalised within this model (including an unexpected new sink-to-source regulatory mechanism in photosynthesis, which I will present). From this perspective Photosystem II (PSII) is “energy squeezed”, having insufficient energy in the 680nm photon to be able to achieve i) high quantum yield charge separation, ii) PSII chemistry (reduction of quinone and oxidation of water), and iii) the appropriate driving forces (over-potentials), while at the same time avoiding ¹O₂-mediated photodamage from back-reactions. PSII is particularly susceptible to photodamage since it must interface 1-photon-per-electron photochemistry with its multi-electron catalytic chemistry, inevitably generating intermediate states that back-react (i.e. S₂, S₃, Q_A- and Q_B-). This “energy squeeze” may set the “red-limit” for oxygenic photosynthesis. The existence of long-wavelength Chld-containing PSII in Acaryochloris appears to contradict this suggestion. We have suggested however that this improvement in efficiency is only possible due a decrease in the “energy headroom”, that part of the over-potential needed to deal with fluctuations in the environment (light intensity): i.e. efficiency gains are paid for by loss of resilience. If so, extending the spectrum of oxygenic photosynthesis to longer wavelengths will encounter problems. We are currently studying long-wavelength species to test these predictions.

Over the past two decades, together with several colleagues, I have used remotely sensed observations of oceanic chlorophyll and analogous observations of terrestrial plants to derive global production of the planet.  Simultaneously, we build and deployed dozens of high resolution fluorometers that measured the quantum efficiency of photosystem II across the world oceans from changes in the amplitude of variable fluorescence.  Over the past ca. 5 years we attempted to close the budget on the absorbed solar radiation absorbed by phytoplankton.  To this end, we built and deployed picosecond lifetime based instruments.  From tens of thousands of paired measurements of variable fluorescence amplitudes and lifetimes, we conclude that in the real world oceans only ~35% of absorbed solar photons are used for photochemistry, ~7% are dissipated as fluorescence, and the remainding ~58% are dissipated as heat.  The exceptionally high loss of absorbed solar radiation that is dissipated as heat appears to be a consequence of nutrient limitation of photosynthetic processes.  Two nutrients are especially identified: dissolved inorganic nitrogen and soluble iron.  The data suggest that these nutrients, which can be episodically supplied by turbulent mixing events (e.g., storms) allow for very rapid repair of the PSII reaction centers.  I will discuss how these processes in  phytoplankton potentially allow for pathways to enhance photosynthesis in crop plants.

Signalling between the chloroplasts and the nucleus is required to maintain photosynthesis and other functions. The regulated expression of nuclear genes by chloroplast signals is known as retrograde signalling. Redox signals arising in the chloroplast are considered to be an important in retrograde signalling but the mechanisms involved remain poorly defined.  Proteins such as WHIRLY1, which is a ssDNA-binding protein localised in the chloroplasts and nuclei, have the potential to fulfil redox signalling functions. WHIRLY1 is required for plastid genome stability and plastid gene transcription. We have characterised WHIRLY1 functions in barley using RNAi-knockdown lines (W1-1, W1-7 and W1-9) that have very low levels of HvWHIRLY1 transcripts. Leaves of the WHIRLY1-deficient plants establish photosynthesis more slowly than the wild type, but otherwise are similar to the wild type, Photosynthesis rates were similar in all lines but W1-1, W1-7 and W1-9 leaves had significantly more chlorophyll and less sucrose than the wild type. Transcripts encoding specific sub-sets of chloroplast-localised proteins such as ribosomal proteins, subunits of the RNA polymerase and the thylakoid NADH and cytochrome b6/f complexes were much more abundant in the W1-7 leaves than the wild type. The roles of WHIRLY1 in the acclimation of photosynthesis to stress will be discussed, together with possible retrograde signalling mechanisms.

Crop productivity needs to substantially increase to meet global food and feed demand of a rapidly growing world population. Agricultural technology developers are pursuing a variety of approaches based on both traditional technologies like genetic improvement, pest control and mechanization as well as new technologies like genomics, gene manipulation and environmental modeling to develop crops that are capable of meeting growing demand. Photosynthesis is a key biochemical process that many suggest is not yet optimized for industrial agriculture or the modern global environment. We are interested in identifying control points in maize photoassimilation that are amenable to gene manipulation to improve overall productivity. Our approach encompasses: developing and using novel gene discovery techniques, translating our discoveries into traits, and evaluating each trait in a stepwise manner that reflects a modern production environment. Our aim is to provide step change advancement in overall crop productivity and deliver this new technology into the hands of growers.

In photosynthesis absorbed sun light produces collective excitations (excitons) that form a coherent superposition of electronic and vibrational states of the individual pigments. Two-dimensional (2D) electronic spectroscopy allows a visualization of how these coherences are involved in the primary processes of energy and charge transfer. Based on quantitative modeling we identify the exciton-vibrational coherences observed in 2D photon echo of the photosystem II reaction center (PSII-RC). We find that the vibrations resonant with the exciton splittings can modify the delocalization of the exciton states and produce additional states, thus promoting directed energy transfer and allowing a switch between the two charge separation pathways. We conclude that the coincidence of the frequencies of the most intense vibrations with the splittings within the manifold of exciton and charge-transfer states in the PSII-RC is not occurring by chance, but reflects a fundamental principle of how energy conversion in photosynthesis was optimized.

Non-photochemical chlorophyll fluorescence quenching (NPQ) is broadly considered to reflect the major mechanism of rapid regulation of light harvesting which defends the photosystem II reaction center (RCII) against photodamage that leads to the photoinhibition of photosynthesis. Whilst there is a great deal of the knowledge concerning the elements that trigger, tune and actually cause the quenching, little is known about its protective efficiency, the critical light intensity that is ‘safe’ for the photosynthetic organism to live given that it has a capacity for a certain level of NPQ. A newly-developed methodology aimed at radically changing our understanding of the effectiveness of the NPQ process, by quantifying its photoprotective potential, will be presented. The technique is essential if we are to fully understand the trade-offs between the metabolic costs of photoinhibition and the reduction in quantum yield caused by engaging NPQ.  In this approach the value of photochemical quenching (qP) measured in the dark following illumination enables monitoring of the state of active PSII reaction centres in order to detect the early signs of photoinhibition. The method allows for the determination of the amplitude of photoprotective NPQ (pNPQ) and its potential to protect against photoinhibition. It also allows accurate quantification of the relationship between the protective component of pNPQ and actinic light intensity. This in turn makes possible the estimation of the maximum light intensity tolerated by the PSII reaction centers in a plant population, the photoptotective effectiveness of NPQ in plants with different levels of PsbS protein or zeaxanthin, and the fraction of captured energy that may be unnecessarily, or ‘wastefully’, dissipated.

Exposure of plants to excessive light intensities, particularly in combination with other abiotic stresses, such as elevated temperature and lack of water, causes a decline in the maximum rate of photosynthesis leading to a state of chronic photoinhibition. It is now well established that the oxygen-evolving photosystem II (PSII) complex of the thylakoid membrane is a primary target of light damage and that the D1 reaction centre polypeptide is most prone to irreversible damage. In vivo damaged PSII complexes can be efficiently repaired via the so-called PSII repair cycle which involves partial disassembly of the inactivated complex, proteolytic degradation of the damaged subunit and specific replacement by a newly synthesized copy followed by reassembly of the active complex. However, when the rate of PSII repair is unable to keep up with the rate of damage, there is a net loss of oxygen-evolving PSII complexes and decline of photosynthetic activity. Many of the proteases and accessory factors involved in the PSII repair cycle have now been identified and substantial progress has been made in understanding their function at the molecular level. In this talk, I will summarise the current view of PSII repair in cyanobacteria and chloroplasts and discuss the feasibility of improving the robustness of photosynthesis in crop plants at the level of the PSII repair cycle.

Protein levels of the plastid terminal oxidase PTOX increase upon abiotic stress. PTOX may protect the photosynthetic apparatus when electron transport is limited. However, overexpression of PTOX in Nicotiana tabacum, increased the production of reactive oxygen species and exacerbated photoinhibition.

The active site of PTOX comprises a non-heme diiron centre that catalyses the oxidation of plastoquinol and the reduction of O₂ to H₂O. We have performed for the first time a biochemical characterization of purified PTOX. The activity of PTOX was determined to be much higher than what had been previously estimated from chlorophyll fluorescence. The main reaction of PTOX is the reduction of O₂ to H₂O but PTOX generates O₂^•- in a side reaction in a substrate- and pH-dependent manner. PTOX activity in planta was investigated using Nicotiana tabacum expressing PTOX1 from Chlamydomonas reinhardtii. PTOX competed efficiently with photosynthetic electron flow. High CO₂ concentrations inactivated PTOX most likely because of an acidification of the stroma. Immunoblots showed that PTOX detached from the membrane in dark-adapted leaves or in the presence of uncouplers. A model is proposed in which the membrane association of PTOX is controlled by stromal pH. When the stromal pH is neutral, PTOX exists as a soluble form and is enzymatically inactive avoiding the interference of PTOX with linear electron flow. When the stromal pH is alkaline and the photosynthetic electron chain is highly reduced under stress conditions, PTOX binds to the membrane, has access to its substrate and can serve as safety valve.