Long- and short-term nitrate uptake regulation in maize

Abstract

Cereal crops supply a major proportion of the world’s food and their production capacity is tightly linked to nitrogen (N) fertiliser use. With on average less than half of the applied N being captured by crops, there is scope and need to improve N uptake in cereals. With nitrate (NO₃⁻) being the main form of N available to cereal crops there has been a significant global research effort to understand plant NO₃⁻ uptake. Despite this, our understanding of how the NO₃⁻ uptake system is regulated remains limited. To advance our understanding of the NO₃⁻ uptake system and its regulation, three knowledge gaps were identified and explored in this thesis. Firstly, there is an identified need to better understand the NO₃⁻ uptake system and the signalling molecules which modulate it. Secondly, with the literature containing alternative approaches to studying NO₃⁻ uptake, there is a need to appreciate how these studies relate to better leverage the existing literature. And finally, with strong transcriptional control governing the NO₃⁻ uptake system, new leads were sought for modulating transcription of NO₃⁻ transporter genes. To explore these knowledge gaps, dwarf maize (Zea mays L. var. Gaspe Flint) was grown hydroponically with either sufficient or limiting NO₃⁻ availability. During the vegetative growth period a subset of plants grown were moved from sufficient to limiting NO₃⁻ conditions and a range of physiological parameters were measured. The results showed: the high affinity NO₃⁻ uptake system (HATS) appears to contribute a major proportion of total NO₃⁻ uptake capacity and responds to N demand at external concentrations where it was previously thought to be saturated; NO₃⁻ itself appears to play a key role in modulating the NO₃⁻ uptake system, and; temporal variation of NRT transcripts are more variable than previously understood. The observed responses to reduction in NO₃⁻ revealed a series of responses leading to a new model for the control of the NO₃⁻ uptake system. Using the same growth system, plants were grown under steady state NO₃⁻ conditions and a starvation and re-supply (primary nitrate response – PNR) response was explored in parallel. The information generated provided data to relate the PNR literature to longer term steady state studies. The ZmNRT2.5 gene was highlighted as an interesting candidate for revealing cis-trans regulatory elements associated with low N responses. To explore this, a combined phylogenomics and co-expressed gene promoter analysis was undertaken. A number of evolutionarily and functionally conserved regions were identified in the ZmNRT2.5 promoter with six regions showing no resemblance to known transcription factor binding sites. These sequences provide a new resource for the discovery of cis-trans regulatory mechanisms associated with the low N expression of ZmNRT2.5. The findings in this thesis have identified key time points for future transcriptome analysis, and revealed putative cis-elements as new leads for discovering novel cis-trans regulatory elements associated with the regulation of NO₃⁻ uptake. Ultimately, further research may lead to the identification of key regulatory genes as candidates for the improvement of N uptake efficiency and overall N use efficiency in cereal crops.