Connecting grain yield and viability with photosynthetic electron transport in developing seeds
Lead Research Organisation:
Queen Mary University of London
Department Name: Sch of Biological & Behavioural Sciences
Abstract
It is critical for humanity that cereal crop yields increase, and this proposal addresses factors that contribute to grain yield and viability. Photosynthetic electron transport (PET) provides the energy to fix carbon in leaves, which is transported to support grain filling. Cereal floral organs are also green, and PET in developing seeds is particularly important for yield and viability, but poorly understood. We have developed techniques and genetic tools to help fill this knowledge gap.
Why barley seeds?: In the 1950s and 60s the "Green Revolution" saved millions from starvation by developing high yield cereal varieties, that were extremely efficient at transferring leaf photosynthate into developing seeds. Photosynthetic processes in cereal flower spikes are also important for high yield, and in particular the role of green tissues in developing cereal seeds remains poorly understood. Investigating this could inform breeding programs leading to further increases in grain yield. Studying barley is one of the fastest routes to improving cereal yield: In the UK and continental Europe, wheat is still the dominant cereal crop, but it is hexaploid (6 genome copies per cell) making it unwieldy as a genetic tool. Barley is the third most farmed cereal in Northern Europe and although closely related to wheat, is more genetically tractable as it is diploid (two genome copies per cell). Knowledge about barley can therefore also inform wheat breading programs, so work on barley is both rapid and high impact.
Why photosynthetic electron transport? Photosynthetic electron transport (PET) provides the energy for CO2 fixation, and is well understood in cereal leaves. By contrast, we know much less about PET in developing seeds, which is also important for viability and yield. Despite not efficiently exchanging O2 or CO2 with the atmosphere, the developing seed assembles and breaks down the apparatus for PET during its development. Two hypotheses have been proposed to explain this: 1) PET produces O2, preventing hypoxia in the seed and enabling respiration to support grain filling; 2) PET produces reactive oxygen species (ROS), which trigger hormone signaling pathways that control seed development and later seedling growth.
Is photosynthetic electron transport in seeds different from leaves? We previously found that TROL, a PET protein, is important for stress tolerance in Arabidopsis, a model plant. We investigated whether this finding could have agronomic importance by knocking out the 2 genes for this protein in barley. Surprisingly, loss of TROL did not affect PET in leaves, but did disrupt it in developing seeds. In comparison to wild type, the mutants also showed poor grain yield, and poor seed viability overall. In the work proposed here we will use these plants as a tool to understand how PET in the developing seed differs from PET in the leaf, and identify pathways and components that are uniquely important to seed PET.
Experiments proposed: Techniques to accurately measure PET require light transmittance through tissue, which is challenging in developing cereal seeds, as they are starchy, dense and scatter light. We have developed methods to accurately do this, and our preliminary results already indicate significant differences between leaf and seed PET. We will try to understand the basis of these differences by comparing the composition and structure of the PET apparatus in leaves and seeds. The TROL gene mutants already generated will be complimented with others to examine how different PET pathways contribute to seed yield and viability. Finally, we will determine whether seed yield and viability can be improved by stimulating TROL-dependent PET pathways at specific points in seed development. By understanding the triggers that regulate grain filling and viability, we hope to eventually identify ways in which cereal yields can be future-proofed against a changing environment.
Why barley seeds?: In the 1950s and 60s the "Green Revolution" saved millions from starvation by developing high yield cereal varieties, that were extremely efficient at transferring leaf photosynthate into developing seeds. Photosynthetic processes in cereal flower spikes are also important for high yield, and in particular the role of green tissues in developing cereal seeds remains poorly understood. Investigating this could inform breeding programs leading to further increases in grain yield. Studying barley is one of the fastest routes to improving cereal yield: In the UK and continental Europe, wheat is still the dominant cereal crop, but it is hexaploid (6 genome copies per cell) making it unwieldy as a genetic tool. Barley is the third most farmed cereal in Northern Europe and although closely related to wheat, is more genetically tractable as it is diploid (two genome copies per cell). Knowledge about barley can therefore also inform wheat breading programs, so work on barley is both rapid and high impact.
Why photosynthetic electron transport? Photosynthetic electron transport (PET) provides the energy for CO2 fixation, and is well understood in cereal leaves. By contrast, we know much less about PET in developing seeds, which is also important for viability and yield. Despite not efficiently exchanging O2 or CO2 with the atmosphere, the developing seed assembles and breaks down the apparatus for PET during its development. Two hypotheses have been proposed to explain this: 1) PET produces O2, preventing hypoxia in the seed and enabling respiration to support grain filling; 2) PET produces reactive oxygen species (ROS), which trigger hormone signaling pathways that control seed development and later seedling growth.
Is photosynthetic electron transport in seeds different from leaves? We previously found that TROL, a PET protein, is important for stress tolerance in Arabidopsis, a model plant. We investigated whether this finding could have agronomic importance by knocking out the 2 genes for this protein in barley. Surprisingly, loss of TROL did not affect PET in leaves, but did disrupt it in developing seeds. In comparison to wild type, the mutants also showed poor grain yield, and poor seed viability overall. In the work proposed here we will use these plants as a tool to understand how PET in the developing seed differs from PET in the leaf, and identify pathways and components that are uniquely important to seed PET.
Experiments proposed: Techniques to accurately measure PET require light transmittance through tissue, which is challenging in developing cereal seeds, as they are starchy, dense and scatter light. We have developed methods to accurately do this, and our preliminary results already indicate significant differences between leaf and seed PET. We will try to understand the basis of these differences by comparing the composition and structure of the PET apparatus in leaves and seeds. The TROL gene mutants already generated will be complimented with others to examine how different PET pathways contribute to seed yield and viability. Finally, we will determine whether seed yield and viability can be improved by stimulating TROL-dependent PET pathways at specific points in seed development. By understanding the triggers that regulate grain filling and viability, we hope to eventually identify ways in which cereal yields can be future-proofed against a changing environment.
Technical Summary
Developing cereal seeds support starch synthesis through respiration, and current dogma states that a layer of photosynthetic tissue surrounding the endosperm prevents hypoxia by supplying O2. TROL is a tethering protein that anchors ferredoxin:NADPH oxidoreductase, the final enzyme in the photosynthetic electron transport chain (PET) to the thylakoid membrane. We have knocked out the two barley TROL genes using CRISPR-Cas9. Knock-out of TROL2 is lethal, but several independent trol1 lines are now available. Preliminary analysis of a representative line shows that, despite minimal impact on PET in leaves, there is a significant impact on PET in developing seeds and on seed viability. This implies a disproportionate role for TROL in developing cereal seeds. The work set out here aims to identify this role, by combining CRISPR-Cas9 genome editing of barley with cutting edge technology for measuring photosynthetic electron transport, and reactive oxygen species (ROS).
Because TROL is implicated in the cyclic electron transport (CET) pathway and an alternative CET pathway catalysed by the NDH complex is important for cereal grain yield in low light conditions, we will generate a mutant in the NDH-M gene. This will help us test whether the impact of TROL on seed viability is linked to CET in general, or is TROL specific. Seed development is regulated by the interplay between reactive oxygen species (ROS), and the auxin (ABA) and gibberellic acid (GA3) phytohormones. ROS are inevitably generated during PET, and we previously found that FNR:TROL interactions result in variable superoxide production in leaves. We will therefore investigate whether disrupted seed development in our mutants is related to hormone synthesis derived from altered ROS production. To do this we will detect ROS, ABA and GA3 in developing seeds using electron paramagnetic resonance spectroscopy, reporter dyes and reporter constructs combined with confocal microscopy.
Because TROL is implicated in the cyclic electron transport (CET) pathway and an alternative CET pathway catalysed by the NDH complex is important for cereal grain yield in low light conditions, we will generate a mutant in the NDH-M gene. This will help us test whether the impact of TROL on seed viability is linked to CET in general, or is TROL specific. Seed development is regulated by the interplay between reactive oxygen species (ROS), and the auxin (ABA) and gibberellic acid (GA3) phytohormones. ROS are inevitably generated during PET, and we previously found that FNR:TROL interactions result in variable superoxide production in leaves. We will therefore investigate whether disrupted seed development in our mutants is related to hormone synthesis derived from altered ROS production. To do this we will detect ROS, ABA and GA3 in developing seeds using electron paramagnetic resonance spectroscopy, reporter dyes and reporter constructs combined with confocal microscopy.
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| Guy Hanke (Principal Investigator) |