Transceptor-mediated nitrogen sensing in legumes

Legume plants have the special ability to interact with soil bacteria called rhizobia. Rhizobia colonise plant roots and are taken up inside the cells of special outgrowths called nodules. Inside the nodule, the rhizobia use sugars provided by the plant as energy to convert nitrogen gas from the air into ammonia which is a form of nitrogen the plants can use as fertilizer (fixed nitrogen). This process of bacterial colonization, nodule formation and nitrogen fixation is called nodulation. Since plants cannot fix nitrogen themselves, and the availability of fixed nitrogen is a major limiting factor for plant growth, nodulation provides a big advantage for legumes in nitrogen poor soils. In agricultural settings this means that legumes like pea, soybean or lucerne require less artificial fertilizer. Another advantage is that legumes leave more nitrogen in the soil than other crops, a quality which has led to legumes having a special role in agriculture as they can be grown in rotation with non-legume plants like wheat or vegetable crops in order to provide these crops with extra nitrogen. In fact, legumes sometimes are grown solely for the purpose of increasing the levels of fixed nitrogen in the soil for the next crop as a type of 'green manure'. Aside from the benefits to soil fertility, legume seed crops such as soybean, field bean, and navy bean are valuable as a protein source and are grown throughout the world. Either as a rotation crop or as a green manure legumes require less industrial fertilizer inputs which are expensive and harmful to the environment, which makes legumes a key component of sustainable agriculture.

Nitrogen fixation of legumes in agricultural systems is sometimes very low. The main reason for this is that high levels of soil nitrogen inhibits nodulation. Legumes have evolved to utilize fixed nitrogen available from the soil (such as ammonia or nitrate) rather than fix nitrogen by nodulation that would require using its own sugars. In order for the legume plant to choose between using fixed nitrogen from the soil or to fix nitrogen by nodulating it must be able to sense soil nitrogen. The aim of this study is to identify the legume genes involved in the sensing of external soil nitrogen. We hypothesize that if we create legume plants with a reduced ability to sense fixed nitrogen they will continue to nodulate and fix nitrogen even when soil nitrogen is abundant. A gene called NRT1.1 was identified as being important for nitrogen in the non-legume plant Arabidopsis. Interestingly, legumes appear to have multiple copies of NRT1.1. It is possible that since legumes nodulate and produce their own fixed nitrogen they may have developed a more sophisticated nitrogen sensing system. To investigate this possibility, we will test each of the legume NRT1.1 genes to see if they are important for sensing fixed nitrogen. To do this we will identify and characterize legume mutants that have non-functional NRT1.1 genes. In particular, we will test these mutants to see if they are still able to nodulate and fix nitrogen even when there are high levels of fixed nitrogen available. In addition, we will biochemically characterize these mutants and the NRT1.1 proteins. Finally, we will use a similar strategy in an important UK crop, peas. Peas that can nodulate and fix nitrogen in nitrogen rich soils could be grown in rotation with crops such as wheat to lessen the need for industrial nitrogen inputs thereby lowering costs and decreasing environmental damage.

Legumes engage in a root symbiosis called nodulation with rhizobial bacteria. In this interaction rhizobia are taken up into root nodule cells where they fix N. Nodulation and N-fixation are suppressed by high levels of soil N. In Arabidopsis, recent work has shown that soil nitrate availability is sensed by the nitrate transporter NRT1.1. This plasma membrane protein can transport nitrate in both high (micromolar) and low (mM) affinity ranges. In Arabidopsis NRT1.1 is phosphorylated at Thr101 in response to changes in external nitrate supply, switching between high and low concentration ranges. NRT1.1 was recently shown to transport auxin and nrt1.1 mutants showed altered root architectural response to nitrate availability. Medicago truncatula has three NRT1.1 homologues, two of them are expressed in roots and have a conserved Thr101, and one of these is induced in N treated nodules. Non-legumes and the primitive legume Chamaecrista fasciculata have only one copy of NRT1.1. Our goals include the electrophysiological characterization of the M. truncatula, and C. fasciculata NRT1.1 homologues. We will test their ability to transport nitrate (high and low affinity), auxin and other substrates and determine their phosphorylation status under different N regimes. Medicago NRT1.1 mutants will be obtained to test their role in N suppression of nodulation. The ultimate aims of the study are to 1) identify and characterize components of the legume N-sensing apparatus 2) understand the links between N perception and nodulation 3) gain insights into the evolution of N-sensing in legumes and 3) to develop a pea that can fix N at high soil nitrate levels. The last aim has important applications in sustainable agriculture, for instance as green manures in crop rotation to lower N fertilizer inputs for wheat but also for legume crops like pea and soybean where early N applications that are used to establish the crop can also inhibit nodulation.

The beneficiaries of the proposed research include the private sector, farmers, and the public.

The Private Sector/Plant Breeding

This line of research has long term commercial potential in the seed industry. Knowledge obtained from this study has the potential to lead to the development of improved green manure cultivars for use in wheat rotations. Such a cultivar would continue to fix N even in high N soils resulting in increased N availability to the subsequent crop. If mutation of one or more NRT1.1s is sufficent to achieve this aim, nitrogen tolerant legumes could be created using a non-genetically modified approaches such as TILLING or the TAL-effector nuclease (TALEN) gene-knockout technology which has recently been developed for plants. Alternatively, it may be found to be more expedient to introduce NRT1.1 variants using transgene technology into the mutants to produce the desired level of nitrogen insensitivity without directly altering nitrate transport functions. While we expect that achieving our research aims is practical, it can be expected that any application of this research will require considerable efforts by pea breeders and agronomists to refine the technology in order to produce a useful cultivar, so these benefits, and the related benefits to farmers and the public sector (below) will take 10-20 years to be realized.

Farmers

Nitrogen fixing legumes like pea and field bean add nitrogen to agricultural soils which results in less N fertilizer required for subsequent crops grown in the same field. By increasing nodulation and nitrogen fixation in legumes in fertilized soils more effective green manures could be developed that leave more residual nitrogen in the soil. Therefore this research could lead to decreased use of industrial fertilizers which require consumption of fossil fuels. Farmers will benefit through reduced costs directly, through buying less fertilizer, and indirectly, by reducing their energy bills by having to use fertilizer application equipment less.

The Public sector

As a consequence of the growing world population it has been predicted food costs will steadily increase. This initiative has the potential to reduce fertlizer inputs for wheat which is the most important crop in the UK and the throughout the world. Such an advance in sustainability would contribute to food security and benefit society in general. Lowered fertilizer requirements will result in a reduction in environmental impact, and allow policy setters to achieve environmental targets quicker (as set out in documents such as the BBSRC policy papers), reduce cost for water companies to remove polluting chemicals (ie. nitrate) from water sources, reduce eutrophication of water sources etc; the general public will have cheaper, safer food. The proposed research thereby contributes to the BBSRC's strategic priorities on 'Crop Science (food security)'.

The public will benefit directly from the engagement of the research participants in activities designed to inform and educate school children and adults on how agricultural research is carried out and its importance. This will happen throughout during the four year funding period. In addition the PDTF will develop highly transferable complementary skills including project management, public speaking and written communication skills, and learn to work as part of a group.