Introduction
Photosynthesis involves a series of biophysical and biochemical processes in which light is used to provide the energy and reducing equivalents needed to convert CO2 into fixed carbon (carbohydrates).Both, the light-dependent and light-independent reactions required for photosynthesis involve proteins (enzymes, transporters and accessory proteins) all of which can, in principle, be targeted for improvement/optimisation. Indeed, many of these components have already been modified to boost the efficiency of photosynthesis with various degrees of success. Some of these approaches have achieved a ≥15% increase in photosynthetic efficiency in terms of biomass yields, but under controlled greenhouse conditions rather than the realistic environment proposed in PhotoBoost.
A key innovative concept of the PhotoBoost proposal is therefore to combine the ‘best of the best’ (i.e., the untapped potential offered by combining successful complementary individual strategies and novel strategies for the enhancement of photosynthesis), stacking all the improvements in a single plant line to exploit the additive effects and/or synergy achieved by maximising the efficiency of each photosynthetic component. The multiple components will also increase robustness because environmental stresses that inhibit one mechanism may not necessarily inhibit others, thus making the resulting new varieties not only more productive but also more suitable for a changing climate.
Strategies
Six individual strategies to boost the efficiency of photosynthesis and crop yields have been selected based on their impact on photosynthetic efficiency (strategies a–d, f) and water-use efficiency (strategy e). Photosynthesis is strongly related to water-use efficiency, defined as the amount of carbon assimilated as biomass or grain produced per unit of water used, so strategy e aims to improve water-use efficiency and increase drought tolerance by limiting stomatal conductance and the rate of transpiration.
- Optimisation of light reactions. This strategy targets the photoprotective mechanism that prevents the overexcitation of the photosynthetic antenna complexes. The mechanism has a slow adaptation to fluctuating light conditions, resulting in suboptimal photosynthetic yields at low light intensities due to transient shading caused by adjacent leaves or clouds. The photoprotective mechanism of non-photochemical quenching (NPQ) involves the conversion of exited electrons into heat. The process involves conformational changes in the light-harvesting proteins of photosystem II as well as epoxidases required for the regulation of this energy scavenging mechanism. Aiming for a faster transition between high NPQ activity under intense light and low activity under poor light, the overexpression of violaxanthin de-epoxidase (VDE), zeaxanthin epoxidase (ZEP) and light harvesting proteins increased CO2 uptake and dry biomass by 15% in tobacco under fluctuating light conditions
Kromdijk et al. (2016) Improving photosynthesis and crop productivity by accelerating recovery from photoprotection. Science 354, 857–861
- Integration of a CO2 concentration mechanism (CCM). This strategy will increase the CO2 levels near RuBisCO and thus mitigate the effect of RuBisCO oxygenase activity. The strategy is based on the targeting of algal carbonic anhydrases and bicarbonate transporters to different locations in the plastid. These algal components were chosen based on their ability to promote CO2 uptake at low and very low CO2 concentrations in the source organism. Expression of these enzymes individually increased photosynthetic efficiency and biomass accumulation by up to 48% in the greenhouse. The most suitable CCM components and combinations thereof will be identified by metabolic modelling and by using the photosynthetically active cell-free screening platform
Nölke et al. (2019) The integration of algal carbon concentration mechanism components into tobacco chloroplasts increases photosynthetic efficiency and biomass. Biotechnol J 1800170.
- Photorespiratory bypass. This strategy aims to reduce metabolic flux through the photorespiratory pathway by expressing a recombinant glycolate dehydrogenase polyprotein (DEFp) comprising all three subunits (D, E and F) of the first enzyme of the Escherichia coli glycolate oxidase pathway. In chloroplasts, the engineered enzyme converts glycolate to glyoxylate, which is oxidised further by endogenous pyruvate dehydrogenase, releasing CO2 in the vicinity of RuBisCO6. Alternatively, we will introduce the complete bacterial pathway by overexpressing two additional enzymes: glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). To minimise the glycolate flux of the native photorespiratory pathway, we will also knock out the expression of the plastid glycolate–glycerate transporter (PLGG1). Both strategies have been successful in Arabidopsis and tobacco. In potato, DEFp expression increased photosynthetic efficiency, leading to a 2.3-fold higher tuber yield under greenhouse conditions.
Nölke et al. (2014) The expression of a recombinant glycolate dehydrogenase polyprotein in potato (Solanum tuberosum) plastids strongly enhances photosynthesis and tuber yield. Plant Biotechnol J 12, 734–742.
- Optimisation of source–sink capacity. Crop yields are determined not only by photosynthetic carbon assimilation but also the translocation of assimilates (e.g., sucrose) and the sink strength. This strategy will simultaneously enhance the source and sink flux in potato under diverse environmental conditions by the mesophyll-specific overexpression of an enzyme to re-route photoassimilates to sink organs, and by the overexpression of two plastid metabolite translocators in tubers to increase the sink capacity. Using this approach, the consortium partners have doubled the tuber starch yield of potato plants. To enhance the drought resilience of potato, we will also use a CRISPR/Cas9 construct to knock out the phloem-mobile tuberisation signal SP6A, which is regulated by elevated temperatures.
Jonik et al. (2012) Simultaneous boosting of source and sink capacities doubles tuber starch yield of potato plants. Plant Biotechnol J 10, 1088–1098
- Water-use efficiency. This strategy will generate crops that require less water per unit mass of product, thus increasing the productivity of the crop under drought conditions. Sugars are known to promote stomatal closure, thereby coordinating photosynthesis with transpiration. To improve water-use efficiency, we will express a sugar-phosphorylating enzyme in the guard cells of potato and rice plants, which in previous studies reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency.
Lugassy et al. (2015) Expression of Arabidopsis hexokinase in citrus guard cells controls stomatal aperture and reduces transpiration. Front Plant Sci 16, 1114
- Oxygen scavenging. In this novel strategy, we will engineer a new oxygen scavenging pathway in potato and rice plastids to reduce the O2 concentration and boost the carboxylase activity of RuBisCO. The details are IP relevant and can´t be revealed here.
Modelling
Modelling photosynthetic metabolism. A major aim of the PhotoBoost project is the selection of the most suitable combinations of strategies a–f, including the most suitable enzymes, without causing unexpected pleiotropic effects such as growth inhibition. We will therefore use comprehensive, dynamic metabolic models of photosynthetic metabolism to connect photosynthetic mechanisms and crop yield predictions in a realistic environment. This will be achieved by taking the current complete model of C3 photosynthesis built by consortium partners and extending it to include stomatal dynamics and the limitations to CO2 diffusion through the stroma as well as environmental variations in light and temperature. The advanced models and simulations will allow us to predict the impact of strategies a–f and their combinations on photosynthesis, carbon partitioning and plant growth, and will also capture pleiotropic effects and identify potential bottlenecks in silico, to guide the rational selection of strategies for introduction into transgenic plants crop canopies, facilitating the identification of additional targets. And fine tuning the existing strategies.
Cell-Free
Generation of a cell free high-throughput enzyme screening and optimisation platform. We will fine-tune the selection of the most suitable components (enzymes and transporters) and combinations of strategies for the generation of transgenic plants by developing a high-throughput cell-free screening and optimisation platform based on photosynthetically active potato cells derived from green tissue. This will enable the rapid, multiplex analysis of many different combinations of proteins optimise stability, expression levels, and appropriate sub-cellular localisation.