An N-fix in time: circadian control of nodulation

Growth and development of plants, animals and microbes are under the control of a 24-hour biological clock known as the circadian clock, which enables them to coordinate their activities with diurnal changes in environmental conditions. The circadian clock sets in train how many important molecules are made, used and moved around cells and organisms at any time. When plants and microbes work together in symbiosis the timing of this movement of molecules between organisms is of key importance, because receiving them at the wrong time means that these important resources will not be utilised efficiently. As the world's population continues to grow we will need to produce 60% more food by 2050. Crop yields must increase without increasing expensive and environmentally costly fertiliser application to supply depleted soils with nutrients such as nitrogen and therefore research to understand how resource use can be maximized in plants is crucial.

One example is the symbiosis between legumes (peas, beans and lentils for example) and rhizobium, a nitrogen-fixing bacterium. Rhizobium fixes nitrogen from the atmosphere and passes it on to its plant host in the form of nitrate, thus reducing the need for nitrogen fertiliser. Since most soils are nitrogen-poor and the production of nitrogen fertiliser is incredibly energy- and environmentally-expensive, this symbiosis is crucial in agriculture. Legumes are also an important source of dietary protein for both humans and animals. Any way that we can enhance or improve the impact of nodulation would help to make legumes an even more useful crop to help us achieve sustainable ways of producing food.

We have discovered that part of the plant circadian clock called CCA1 affects the setting up of this symbiosis. In this project we will identify the specific stages of symbiosis that are affected by the clock. We will test whether the effects of CCA1 are linked to its function in the timing mechanism of the clock, and the role of molecules that might be affected by this. We will also test the role of CCA1 and of plant circadian rhythms in compatibility between the rhizobia symbiont and plant host. Together this research will enable us to design biotechnological treatments to improve nodulation in the future by working with the biological clock of the plant.

By determining how the plant circadian clock gene CCA1 affects nodulation in Medicago truncatula this work will identify novel avenues to improve nodulation and crop yield. Our data suggests the CCA1 effect could be mediated by regulation of a subset of the 700+ large family of Nodule-specific Cysteine Rich (NCR) small peptides. Aside from their known defensin-like role in controlling rhizobial population activity, our recent data shows that the expression of many NCRs are rhythmically expressed, regulated by differing levels of nitrogen, and affected by autoregulation of nodulation, consistent with additional functions in the regulation of development.

Our experiments to characterise the role of CCA1 in nodulation will use molecular biology, plant transcriptomics using RNAseq and proteomics to measure NCR peptide levels in nodules over timecourses in normal and non-24h light-dark cycles. Plant circadian rhythms in roots and nodules will be visualized using continual luciferase-imaging of a CCA1:luciferase line that we will construct. By studying the progression of nodulation in plants using microscopy alongside nodulation phenotypic and N-fixation activity assays we will be able to identify the stages of symbiosis establishment that are disrupted by cca1 mutation, and the associated mechanisms.

Our data will determine whether CCA1 is linked to its function in circadian timing and more specifically in the timing of NCR function. Modulating the timing of NCR expression or activation may provide avenues to control nodulation independently of other clock-promoted processes, to increase plant yield. It may also identify alternative targets for future manipulation, such as flavonoid signals that are involved in nodulation. This project will also test the role of CCA1 and of plant circadian rhythms in symbiont-host compatibility and will identify candidate mechanisms to inform the basis for rational selection of the most appropriate rhizobial inoculants in future.

As the world's population continues to grow we will need to produce 60% more food by 2050. Crop yields must increase without increasing expensive and environmentally costly fertiliser application to supply depleted soils with nutrients such as nitrogen. Moreover, CO2 emissions from the Haber-Bosch process that is required to produce nitrogen fertilisers are a major contributor to greenhouse gases. Legumes are an important source of dietary protein and are key crops for sustainable agriculture because they fix atmospheric nitrogen via symbiotic interactions with nitrogen-fixing rhizobia bacteria. Our recent work has uncovered a novel link between the plant circadian clock and nodulation. The research in this project aims to use characterization of this link in order to find novel ways to modulate the control of nodulation and increase nitrogen fixation and plant yield. We will carry out a range of activities to engage with a number of groups of interested parties, with details as set out in our Pathways to Impact:

The agricultural sector including seeds, chemical production and farmers will benefit directly from the longer-term impacts of this research since we will determine if modulating the function of circadian rhythms in plant hosts could enhance crop yield. As modulation of the underlying oscillator may have negative side-effects we will focus on understanding the role of rhythmically expressed signaling peptides that are nodule-specific and could act as growth-enhancers when applied to plants. Alternative, GM approaches would involve changing the timing of expression of these peptides to promote nodulation. Our work uses the model Medicago truncatula, but the information that we will gain will be broadly relevant since M. truncatula has a high degree of synteny with cultivated legume crops including the grassland crop Medicago sativa and soybean, a globally important crop. We aim to start developing these methods to enhance nodulation within 3-5 years of the project starting.

Maximising the potential of legume plants that do not require nitrogen fertiliser could have a large and long-term impact. Key end-users include farmers who want to reduce the application of fertilisers and irrigation to save money, organic farmers who want to understand how to increase crop yield without adding fertiliser, and those interested in producing novel crops such as Syngenta, an agri-tech company with strong ties to Warwick. These impacts are likely to be longer term (8+ years) but we will plan for these within the lifetime of the grant by talking to relevant industries and companies that Warwick already engages with.

The new types of scientific discoveries that we will make require research that brings together plant development, plant-microbe interactions and high-throughput molecular analyses. The PDRA and technician will benefit from training in these areas and their development and future career progression will be a key are of impact within the lifetime of the grant and beyond.

Members of the public who wish to continue to buy affordable food that is reliably available throughout the year will also benefit from this project. In order to sustainably increase food production we must grow crops that maintain high yields with fewer inputs. During this project we will generate fundamental knowledge that could help to improve the already substantial benefits that the growth of legume crops brings. This would also lead to a range of environmental and cost savings to farmers, the public and environmental policy makers. For example, there would be reduced river pollution due to lower nitrate fertiliser use and run-off, sustainable agricultural productivity, and also reduction in food miles to due higher efficiency farming methods. We expect to realise these benefits further ahead in time (10+ years) but will open a dialogue with the public and policy makers within the lifetime of the grant.