Improving crop nitrogen (N) utilization efficiency (NUE) is of major importance in modern agriculture in order to reduce the amount of N fertilizer used for crop production. There is a high demand for development of crops which are able to produce high yields but with a concomitantly lower N fertilizer requirement. The
enzyme glutamine synthetase (GS) has been a major topic in plant nitrogen research for decades due to its central role in plant N metabolism. The cytosolic version of this enzyme (GS1) plays an important role in relation to primary N assimilation as well as in relation to N remobilisation from ageing plant parts. Thus, GS is highly involved in determining crop yield and NUE.
The major objective of this PhD project was to investigate the NUE properties of transgenic barley designed to constitutively overexpress a GS1 isogene (HvGS1.1). These transgenic lines exhibited an increased GS activity in root and stem during the vegetative growth stages and an increased GS activity in leaves during senescence compared to wildtype control. Furthermore, during the vegetative growth stages, there were distinct differences in N accumulation and biomass partitioning between transgenic lines and wildtype control. However, when grown to maturity the differences between transgenic lines and wildtype were highly dependent on the growth conditions applied. The transgenic lines had a higher N utilization efficiency
(NUtE) than wildtype control, but only when exposed to a mild N stress following the stem elongation stage, or when grown at a combination of high N supply and elevated atmospheric CO2. The transgenic plants may have been able to actively control the constitutively extra transcripts available, to generate active proteins
when necessary, but further studies are needed to establish the nature of this regulation.
A second part of the project was carried out at the Australian Centre for Plant Functional Genomic in Adelaide, Australia. Here, the N responsiveness and diurnal regulation of GS isogenes and putative nitrate transporters was investigated in wildtype barley. This study provided new insight into the adaptability of plants to fluctuations in N supply. There was a clear relationship between nitrate high affinity uptake and the transcript levels of high affinity transporter, HvNRT2s, suggesting that these may play a dynamic and important role in fine-tuning plant nitrate supply for N demand. Of the GS isogenes, only the transcript levels of root HvGS1.1 increased when plants were transferred from high to low N. This change coincided with an increase in total GS activity. Pronounced diurnal variation was observed for root nitrate transporter genes and GS isogenes in both root and shoot tissue. Overall, the variation in GS transcript levels did not directly translate into GS activity, highlighting the occurrence of post-transcriptional regulation.
Overall, the studies undertaken in this PhD project has provided new insight into the complex regulatory network that affects both N uptake and assimilation. Manipulating NUE by overexpressing GS1.1 is not straightforward, but will, due to potential post-transcriptional regulation, require a more targeted approach than the one utilized in the present study. Increasing our understanding of this regulation will be a prerequisite for future biotechnological use of GS1 genes to improve crop NUE.