Functional analysis of NLP7 for optimising nitrate responsive growth

Lead Research Organisation: John Innes Centre
Department Name: Cell and Develop Biology


Plants use nutrients from the soil, energy from sunlight, and air and water to produce all the metabolites they need for growth. In turn plants provide, directly and indirectly, essentially all of the nutrients that sustain humans. Nutrient acquisition from the soil requires special proteins called transporters that can take up trace levels of nutrients from the soil. Because soil composition varies, plants adapt their growth requirements to nutrient availability by altering the levels and activities of nutrient transporters so that plant growth is optimised even though nutrient levels change. Much has been learnt about the mechanisms involved in controlling nitrate uptake and utilisation for growth because it has a key bearing on the application of fertilisers. The high yields of crops are critically dependent on the application of high levels of fertilisers such as NPK to supply the main nutrients. About 1/3 of applied nitrogenous fertiliser is actually taken up and used by the plant, with the rest remaining bound in the soil or washed away into watercourses, where it is a major pollutant. Consequently the application of nitrate-containing fertilisers is limited by legislation, and may need to be reduced further. However, many modern crop varieties have been bred to produce high yields in response to high nitrate applications. Therefore we need to breed new crop varieties that can maintain high levels of productivity in relatively low nitrate levels. One way of approaching this challenge is to understand precisely how plants respond to nitrate levels in the soil and programme their metabolism and growth to take up and metabolise nitrate for growth. We have recently identified a genetic switch that coordinates this response. We have shown that this switch requires both nitrate and sugars to work, and that it may provide a way for plants to coordinate the supply of nitrate from the soil and energy and carbohydrates from photosynthesis to make nutrients for growth. In this research project we aim to understand this switch in a precise quantitative way, and then to subtly alter components to see if we can make a switch that has different quantitative responses to nitrate and sugar levels. We will then test if this alters plant growth responses to nitrate and illumination levels. In this way the proposed research will provide new knowledge and understanding that can be used to create new crop varieties that can grow and produce good yields from reduced inputs of fertilisers. This will help achieve more environmentally sustainable agricultural production systems for major crops.

Technical Summary

Recently Marchive et al described a very interesting genetic switch in Arabidopsis that responds to nitrate and integrates the expression of many nitrate uptake and assimilatory genes. They showed that nitrate rapidly stimulates the nuclear accumulation of the NIN- family transcription factor NLP7. It binds directly to the promoter of the nitrate transporter gene NRT2.1 and promotes high affinity nitrate uptake, and also activates the expression of many other genes that metabolise nitrate. We have shown that glucose has a key role in regulating NRT2.1 expression and in mediating NRT2.1 transporter activity post-transcriptionally (de Jong et al 2013). Recently we have also shown that glucose- mediated NRT2.1 expression is completely dependent on NLP7, and that glucose as well as nitrate is required for accumulation of NLP7 levels. We now want to understand more about the mechanisms involved in this interesting and important genetic switch, aiming to gain quantitative information about NLP7 levels and location, possible post-translational modifications and the role of other proteins required for NLP7 location, levels and activities. Using this functional information we will then assess mutant versions of NLP7 and interacting proteins for quantitative changes in nitrate- and glucose- responsive protein levels and localisation, and transcriptional activation of target genes in a high-throughput NRT2.1 expression system. Iterative trials aim to alter transcriptional responses of nitrate uptake and assimilation genes to nitrate and glucose. Finally mutant versions of NLP7 and interacting proteins will be assessed in plants for altered nitrate- and photosynthate- responsive growth. This will provide new understanding of a key mechanism that may be useful in optimising crop growth responses to nitrate.

Planned Impact

A. On the Researcher. This project provides outstanding opportunities for the researcher in terms of a very promising and productive project, training in biochemistry, proteomics and bioimaging, interactions with industry, transferable skills development and working with a large cohort of other early stage career scientists. The impacts include enhanced career opportunities, increasing the skills base of the UK, and preparation for possible career in industry.

B. On Industry. The genes and mechanisms we aim to discover have significant potential to help increase crop yield, through both GM and breeding routes. The industrial beneficiaries include Plant Biosciences Ltd who are JIC's partner in commercialisation and exploitation. The Impact Plan describes how we will communicate the outcomes of research with them, identify and protect innovative discoveries, and promote industrial engagement through patenting and licensing these discoveries.

C. On Producers and Consumers. Although the initial impacts of this proposed work will on the plant biotech sector remain uncertain and speculative until technologies based on our discoveries are made and assessed in crops, we strongly believe in the potential of the research outcomes to make important contributions to improving food security and the sustainability of agricultural production. This will ultimately benefit consumers by helping to develop new crops that are cheaper to produce, and will benefit the environment by reducing applications of nitrogenous fertilizers, leading to improved soil quality and water course pollution levels.

D. On BBSRC and policy makers. The project, through its impact plan, directly supports BBSRC and BIS strategic priorities in food security and sustainability by creating new knowledge to increase crop yields sustainably and to reduce the impacts of food production on the environment.


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Description we show that NPL7 interacts with alpha importin 1 in a nitrate responsive mechanism that then translocates NPL7 to the nucleus to activate nitrate assimilation. UPDATE: we have identified a CIP family protein kinase as an interactor with NLP7 in vivo. This phosphorylates a nuclear localisation motif and influences nitrate-mediate nuclear location and NRT2.1 expression. Currently this work is being organised into two publications.

This work came to an end without the possibility of publication due to the departure of the post-doc. She did not provide an outline manuscript or figures before leaving, therefore the project, which appeared to be very promising, is not able to be progressed towards publication as originally expected
Exploitation Route will need to be published and optimally the mechanism explored using synthetic biology approaches in wheat to optimise nitrate uptake and assimilation. UPDATE: the work in Arabidopsis will only be indirectly relevant to optimising N uptake in wheat. The mechanisms we have discovered will help understand how genetic variation in wheat influences N uptake efficiency
Sectors Agriculture, Food and Drink

Description NPL7 characterisation 
Organisation French National Institute of Agricultural Research
Department INRA Versailles
Country France 
Sector Academic/University 
PI Contribution In our BBSRC grant we are characterising the NIN-like transcription factor NLP7, which regulates NRT2.1, a high affinity nitrate uptake protein. We are identifying interacting proteins and protein modifications that are providing insights into how nitrate and sugars regulate nitrate uptake and assimilation
Collaborator Contribution Anne Krapp's group in Versailles has characterised the movement of NPL7 into the nucleus in response to nitrate. We are building on this knowledge to get a more detailed picture of NLP7 regulation
Impact In brief, we used a transgenic line expressing NLP7-GFP that complements the mutant to identify candidate interacting proteins and protein modifications. Importins and kinases were top candidates and their roles are being characterised genetically and biochemically, and by imaging
Start Year 2015