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Diffusional limitations to C4 photosynthesis

Periodic Reporting for period 2 - DILIPHO (Diffusional limitations to C4 photosynthesis)

Reporting period: 2019-09-01 to 2020-08-31

When Photosynthesis evolved in Bacteria some 3.5 billion years ago it was arguably the most important innovation in Earth's history. Photosynthetic organisms harvest light and use its energy for chemically reducing CO2 molecules to form the backbones of the plant body, reserves and reproductive organs. Through geological eras the atmosphere was progressively depleted of CO2 through burial of coal, oil and carbonate rock sediments. Around 30 million years ago, some plants evolved a biochemical pump, called C4 photosynthesis, that concentrates CO2 from the atmosphere in a partially sealed compartment inside the leaf. This Carbon Concentrating Mechanism (CCM) is an effective ‘turbocharger’ of the assimilatory machinery and confers higher productivity potential. C4 plants are of primary importance and leading grain (maize, sorghum), sugar (sugarcane), and biofuel (miscanthus) producers. Advanced breeding of C4 plants is currently impinged on negatively by lack of fundamental knowledge of C4 physiology. Importantly, it is unknown why C4 plants despite consuming less water and occupying drier habitats, are inherently more susceptible to water stress than C3 plants.
For C4 photosynthesis to operate, a substantial flow of metabolites is continuously exchanged between two partially isolated compartments in the leaf parenchyma arranged concentrically around veins. In the external layer, called mesophyll, CO2 is temporarily fixed into small organic acids composed of four atoms of carbon (hence the name C4). These C4 acids diffuse to the internal layer, called bundle sheath, through microscopic apertures called plasmodesmata, which connect adjacent cytoplasms in a water-based continuum. In the bundle sheath the C4 intermediates are decarboxylated, while the resulting C3 acids diffuse back to the mesophyll, where they are recycled. This gigantic flux of small metabolites needs to be continuously exchanged between the external and the internal layer through channels called plasmodesmata .
DILIPHO hypothesizes that hydraulic pressure within cells (called turgor) is required to keep the plasmodesmata section wide open. When water becomes less available (measured by water potential) turgor decreases and this would cause plasmodesmata to shrink. The consequent reduction in plasmodesmata cross section would reduce permeability to diffusion between mesophyll and bundle sheath, slowing down the exchange of metabolites, thus jamming the C4 machinery.
DILIPHO consists of three phases, two of which have now been completed. In the first conceptual phase (WP1) the fellow learned concepts of advanced Mathematics and Biophysics, prepared the following experiment and developed a mechanistic model to study metabolite transport at leaf level, DiliMOD. In the second phase (WP2) a dedicated and novel experiment was performed whereby a transient decrease in turgor was induced while photosynthesis and key physiological quantities were assessed in real time. In the third phase (WP3) the acquired data were analysed and interpreted using the novel model to test the hypothesis.
WP1 Development phase. This phase, was aimed at developing a prototype of DiliMOD and setting up the experiment. Firstly, the fellow gained new knowledge in the fields of mathematics through a dedicated course Mathematics and applications II (https://programsandcourses.anu.edu.au/2020/course/MATH1014). Secondly the fellow broadened his knowledge of system modelling by attending a specific course on System Modelling at the Deparment of Engineering at ANU (https://programsandcourses.anu.edu.au/2020/course/ENGN8120). Thirdly, the fellow developed the submodels that were assembled in a working prototype of DiliMOD. A novel model of electron transport chain of C4 photosynthesis was joined to the previously developed stoichiometric model of C4 photosynthesis. In this submodel diffusion responds to the geometry of plasmodesmata that is linked to the water status of the leaf (turgor). This work led to a functioning prototype of a dynamic biochemical model of C4 photosynthesis encompassing diffusional limitations in response to turgor (DiliMOD).
WP2 Experimental phase. This phase was carried out at the high quality lab at ANU using the state of the art measuring spectroscopes. The main hypothesis of DILIPHO was tested by measuring photosynthetic turgor response curves on major C3 and C4 crops. Gas exchange was measured concurrently to pulse modulated chlorophyll fluorescence and real time carbon and oxygen isotopic discrimination. A comprehensive dataset was acquired on two C4 species (maize and sorghum) and two C3 (wheat and sunflower). Alongside plant water relations were comprehensively characterised. The fellow developed a novel model of C4 photosynthesis to quantitatively interpret the results.
WP3 Implementation. This phase was based at UIB. In this phase the fellow used the newly developed model to acquire new knowledge on the physiology of C3 and C4 plants. Firstly, the acquired dataset was analysed using the new model to test the main hypothesis of DILIPHO. The hypothesis of DILIPHO was verified. A manuscript was prepared, is undergoing final checks and is expected to be submitted in October 2020. Further, six more manuscripts were prepared, four of which are currently being reviewed in top level journals.
The theoretical knowledge acquired during WP1 constitutes a solid foundation upon which the fellow will support future research and build a reputation as an independent group leader. The models developed by the fellow are highly innovative. On the one hand they will allow the fellow to tackle unanswered questions in plant biology, on the other, they will support the work of other researchers, either by collaborations, or by the release of advanced modelling tools. At EU level the skills acquired by the fellow are highly desirable to reinforce a scientific position which at the moment is lagging behind. In fact, EU level the only group operating in the field of theoretical physiology and photosynthesis modelling is that of X. Yin and co-workers in Wageningen and Leuven. Making available the newly acquired skills to the scientific community will on reinforce the reputation and the scientific maturity of the fellow, and will increase the potential of basic and applied European research, for instance in support of advanced crop breeding strategies.
The results are a novel understanding of C4 photosynthesis with novel modeling tools and a significant researchers network involved in advancing such understanding. The socio-economic and wider societal implications of the project would be revealed later on, after widespread testing of C4 physiology using the newly developed tools and ideas.