DELLA-PIF Regulation of Nitrogen Assimilation: from Arabidopsis Model to Long-Term Translation to Crop Efficiency Gains

The famous cereal 'green revolution' of the 1960s/1970s increased crop yields, averted famine and fed a growing world population. Green Revolution Varieties (GRVs) of rice and wheat were the genetic foundation of the green revolution. GRVs carry mutant growth regulatory genes that confer dwarfism, and this dwarfism increases yield because it reduces loss due to 'lodging' (flattening of plants by wind and rain), hence causing the yield increases of the green revolution. However, the mutant growth regulatory genes also cause GRVs to be less efficient in assimilating the nitrogen (N) supplied to them in the form of fertilizer. As a result, N that is not assimilated by GRVs is dissipated into the wider environment, where it causes severe damage to terrestrial and aquatic ecosystems, together with atmospheric greenhouse-gas pollution that precipitates climate change. Because today's high-yielding crop varieties still depend upon the mutant dwarfing genes for their high yields, it is necessary to find ways of developing new crop varieties that retain the benefits of GRV dwarfism but that are more efficient in their use of N fertilizers (have improved N use efficiency, NUE).

Here we propose to exploit the rapid genetics and molecular biology of the genetic model Arabidopsis to make discoveries that will enable future enhancement of GRV NUE. The GRV dwarfing genes cause accumulation of a class of growth inhibitory proteins called DELLAs, and DELLAs also accumulate in the dwarf Arabidopsis GRV mutant model gai. Accumulated DELLAs inhibit the action of another class of regulatory proteins, the PIFs (or Phytochrome Interacting Factors). Our recent preliminary evidence from studies of Arabidopsis suggest that the inhibitory effect of DELLAs on PIFs may explain the reduced NUE of GRVs, and it is this novel and exciting finding that we exploit in this proposal.

We will therefore first further test our working hypothesis that interactions between DELLAs and PIFs affect the assimilation of N: that the DELLAs accumulated in GRVs and gai oppose PIF function, thus reducing N assimilation. If this hypothesis is correct, modulation of the DELLA-PIF relationship may provide a novel route towards improving GRV NUE. We have the following objectives:

A. Obtain an in-depth understanding of PIF-regulation of Arabidopsis and rice N assimilation - essentially performing genetic tests of the role of PIFs in regulation of N metabolism and assimilation in Arabidopsis and rice.
B. Determine how the DELLA-PIF interaction regulates the abundance of mRNA encoding nitrate reductase (NR), a key enzyme in N assimilation - this an exploration of how the DELLA-PIF interaction controls the expression of the gene encoding that enzyme.
C. Determine if the DELLA-PIF interaction also directly affects the abundance and/or specific enzymatic activity of the NR enzyme itself.
D. Determine if NUE can be increased despite retaining yield-enhancing dwarfism. This is important because it could lead to the development of crops which retain the high yields of current GRVs, but at reduced environmental cost. First, we will determine if increasing PIF activity might confer such benefits. However, because increasing the activity of PIFs themselves in GRVs might have additional unwanted consequences, we will additionally explore other routes (downstream of PIFs) to improving GRV NUE whilst retaining yield-enhancing dwarfism.

Inherent in our strategy is initial translation of findings from Arabidopsis model to crop (rice), exploiting our long-standing combined expertise in DELLA biology, model-crop translations, and whole genome sequence analysis. Our long-term aim (future proposals) is to use the fundamental understanding gained here in the development of rice and wheat GRVs having enhanced NUE, thus enhancing global food security and reducing agricultural environmental degradation.

We will test if DELLAs inhibit N assimilation by inhibiting PIF function, and if modulation of the DELLA-PIF relationship, or of other downstream factors, might enable increased GRV NUE. Fundamental studies in Arabidopsis will be combined with initial translational studies in rice, with the following objectives:

A. In-depth understanding of PIF-regulation of Arabidopsis and rice N assimilation. We will (A1) determine with an Arabidopsis PIF3-deficient mutant if PIF3 is an overall regulator of N metabolism, (A2) use CRISPR/cas9 mutagenesis to determine PIF function in regulating rice N assimilation.

B. Determine how the DELLA-PIF interaction regulates NR mRNA abundance. We will (B1) use EMSA to determine if PIF3 binds candidate PIF-binding sites in the promoter of the NR-encoding AtNIA2 gene, (B2) use CHiP-PCR to determine if DELLAs inhibit binding of PIF3 to these sites, (B3) use CRISPR/cas9 to determine if these sites are necessary for AtNIA2 expression, and (B4) determine with transient assays if the DELLA-PIF interaction regulates AtNIA2 expression.

C. Determine protein-level DELLA-PIF interaction effects on NR/N assimilation. We will (C1) use commercially available antibodies and immuno-tagging techniques to determine the effects of the DELLA-PIF interaction on NR abundance, (C2) use electrophoretic, yeast 2-hybrid and proteomic techniques to determine if the DELLA-PIF interaction affects NR inactivation/activation by post-translational modification (and identify genes encoding NR-modifying functions in Arabidopsis and rice).

D. Determine if NUE can be increased despite retaining yield-enhancing dwarfism. We will (D1) determine if PIF overexpression enhances N assimilation in gai Arabidopsis and GRV rice, (D2) use an open-ended screen to identify (D3) new mutant genes conferring enhanced N assimilation on Arabidopsis gai (whilst retaining dwarfism).

The long term aim is to discover how to increase GRV NUE but retain yield-enhancing dwarfism.

Whilst the research in this project is largely fundamental in nature, and the immediate impacts of the project itself will largely be in the spheres of knowledge and people (see below), the longer-term aims are for the knowledge gained from the project to provide the foundation to major societal, economic and policy impacts centred around food security, sustainable agriculture and environmental protection.

Who might benefit from this research?

This fundamental research aims to increase the knowledge base underpinning long-term efforts to reduce the impacts of the interlinked problems of food security and environmental degradation that are world-wide in scope. Increased crop yields are needed to feed the growing human population. Yet increased crop yields have in the past been achieved by increasing fertiliser usage, and this increased usage increasingly damages the environment. We need to find ways of increasing crop yields without increasing fertiliser usage, and the research in this proposal aims to discover new pathways towards achieving this. With the long-term aim of improving the nutrient use efficiency of the world's major cereal crops, our research has the eventual potential to benefit the citizens of those many countries in the world where cereals (particularly rice or wheat) are major components of the diet. In addition, because the problems of food security and environmental degradation are global in nature, the potential beneficiaries from increased food production and availability, reduced social unrest consequent on food shortage, and a healthier environment with reduced threats to biodiversity are the entire peoples of the world. Before we can achieve these long-term aims we need to understand how plants coordinate growth with nutrient acquisition. Within the shorter-term scope of this project itself therefore, the major impact will be fundamental knowledge of how plants coordinate growth and nitrogen (N) metabolism and acquisition that will contribute to understanding of the research community as a whole (basic questions in bioscience/scientific advance), and to the wider body of scientific knowledge available to researchers, in particular to plant breeders. Some of the project involves initial steps of translation of that fundamental knowledge to rice breeders, with the eventual aim of developing Green Revolution Varieties (GRVs) with improved nitrogen use efficiency (NUE) (see also Pathways to Impact statement). The second major impact of this project is in terms of people: the research will contribute to long-term training of a postdoctoral scientist, thus enhancing the health of the science base for future scientific developments.


How might they benefit from this research?

The long-term aim of this research is to provide knowledge that will underpin the long-term development of cereal varieties that retain the yield benefits of current GRVs, but have increased NUE. Our new knowledge will in the longer term enable discovery of specific novel variant alleles that will be incorporated into rice and wheat breeding programmes. New commercial GRVs with increased NUE will be developed by breeders and released to market. Farmers will readily adopt these new varieties, as they will enable high yields at reduced input (fertiliser) cost. Reduced fertiliser use will reduce consequent environmental damage, thus benefitting the world as a whole. High yields will enhance global food supply, thus reducing the risk of food shortage, famine, and consequent global social unrest. Within the scope of this project itself, the major impacts will be that scientific understanding will be advanced, that the research community and plant breeders will benefit from access to our research, that the postdoc employed will be trained in cutting-edge scientific research methods. Our ways of maximising the realisation of these impacts is outlined in the Pathways to Impact statement.