Final Report Summary - NITROSIGN (The molecular network linking nitrogen assimilation to growth)
The high yield of modern crop varieties is critically dependent on the supply of nitrogenous fertilizers, as nitrogen (N) is a key nutrient for plant growth and is normally limiting in most soils. Only 1/3 of applied N fertilizer is incorporated into grain, with the unused N representing a huge economic and environmental burden. Consequently research efforts are focused on understanding plant responses to N to enable the more efficient use of N fertilizers. The high yields of elite wheat varieties in response to nitrate fertilizers are due to higher N uptake efficiency (NupE) and N utilization efficiency (NUE). Breeding lines that maintain yields in lower N regimes and that have increased yields in response to N is a key objective in sustainable agriculture.
The Arabidopsis nitrate transporter encoding gene NRT2.1 encodes a high affinity nitrate transporter which is a major facilitator of high affinity nitrate uptake activity. Current research in this field shows that nitrate induces the expression of NRT2.1. Photosynthate sugars have a boost effect on inducing NRT2.1 expression regardless the presence of nitrate. It is thought that photosynthate sugars serve as fuel and signal for nitrate uptake and in turn nitrate assimilation to allow growth. The Bevan lab and other labs have shown independently that signals generated downstream of photosynthate sugar metabolism through the Oxidative Pentose Phosphate (OPP) pathway regulate NRT2.1 expression. However, the signals and genes that are responsible for this process are not known. This research project aims to identify key regulators that mediate sugar regulated NRT2.1 expression and further to unravel the detailed mechanisms that how sugar regulates this process. The knowledge obtained from this project will assist the improvement of crop nitrogen usage efficiency in the future.
The regulation of NRT2.1 expression is complex. Previous studies have reported various factors or genes affect NRT2.1 expression. During the first period of this project (year 1), I demonstrated that the key regulator Nin-Like Protein 7 (NLP7), previously shown to regulate NRT2.1 expression in response to nitrate, is also involved in sugar induced NRT2.1 expression. I then examined the role of NLP7 in this process by creating various deletion or mutation versions of NLP7 protein for functional analysis. In these analyses, bioimaging was used to assess protein abundance and subcellular location of NLP7. Luciferase reporter gene activity assays have been applied in root protoplasts to assess transcription activity of NLP7 and derivatives. Through these studies, we propose that sugar controls NLP7 protein abundance and most likely post-translational modifications are occurring on NLP7 in response to sugar signals.
During the second period of this project (year 2), I explored in detail the network controlling NRT2.1 expression in response to sugar signals. The questions I aimed to answer were: 1) what is or are the signals generated from OPP pathway controls NRT2.1 expression and which are the genes required for generating the signals? 2) What are the interacting protein partners of NLP7 that might regulate this activity? To address these key questions, I screened knockout lines of genes annotated as OPP pathway metabolic genes by testing the responsiveness of these lines to nitrate and sugars. I also screened candidate proteins by using split YFP vectors to identify potential interacting proteins of NLP7. With these investigations, I identified genes in the OPP pathway that affect NRT2.1 expression and one potential interacting partner of NLP7 regulating its activity. To follow up these discoveries, I wrote, with Prof. Bevan, a 3-year grant to the BBSRC, with myself as the Researcher Co-Investigator. This grant was funded (Ref. BB/M02184X/1), and in the future I will finalize the publications based on this project and employ proteomics methodology to identify interacting partners and study post-translational modifications of NLP7.
The Arabidopsis nitrate transporter encoding gene NRT2.1 encodes a high affinity nitrate transporter which is a major facilitator of high affinity nitrate uptake activity. Current research in this field shows that nitrate induces the expression of NRT2.1. Photosynthate sugars have a boost effect on inducing NRT2.1 expression regardless the presence of nitrate. It is thought that photosynthate sugars serve as fuel and signal for nitrate uptake and in turn nitrate assimilation to allow growth. The Bevan lab and other labs have shown independently that signals generated downstream of photosynthate sugar metabolism through the Oxidative Pentose Phosphate (OPP) pathway regulate NRT2.1 expression. However, the signals and genes that are responsible for this process are not known. This research project aims to identify key regulators that mediate sugar regulated NRT2.1 expression and further to unravel the detailed mechanisms that how sugar regulates this process. The knowledge obtained from this project will assist the improvement of crop nitrogen usage efficiency in the future.
The regulation of NRT2.1 expression is complex. Previous studies have reported various factors or genes affect NRT2.1 expression. During the first period of this project (year 1), I demonstrated that the key regulator Nin-Like Protein 7 (NLP7), previously shown to regulate NRT2.1 expression in response to nitrate, is also involved in sugar induced NRT2.1 expression. I then examined the role of NLP7 in this process by creating various deletion or mutation versions of NLP7 protein for functional analysis. In these analyses, bioimaging was used to assess protein abundance and subcellular location of NLP7. Luciferase reporter gene activity assays have been applied in root protoplasts to assess transcription activity of NLP7 and derivatives. Through these studies, we propose that sugar controls NLP7 protein abundance and most likely post-translational modifications are occurring on NLP7 in response to sugar signals.
During the second period of this project (year 2), I explored in detail the network controlling NRT2.1 expression in response to sugar signals. The questions I aimed to answer were: 1) what is or are the signals generated from OPP pathway controls NRT2.1 expression and which are the genes required for generating the signals? 2) What are the interacting protein partners of NLP7 that might regulate this activity? To address these key questions, I screened knockout lines of genes annotated as OPP pathway metabolic genes by testing the responsiveness of these lines to nitrate and sugars. I also screened candidate proteins by using split YFP vectors to identify potential interacting proteins of NLP7. With these investigations, I identified genes in the OPP pathway that affect NRT2.1 expression and one potential interacting partner of NLP7 regulating its activity. To follow up these discoveries, I wrote, with Prof. Bevan, a 3-year grant to the BBSRC, with myself as the Researcher Co-Investigator. This grant was funded (Ref. BB/M02184X/1), and in the future I will finalize the publications based on this project and employ proteomics methodology to identify interacting partners and study post-translational modifications of NLP7.