PhD Research

My PhD research was conducted at Rothamsted Research (UK) with Dr Dimah Habash and Prof. Christine Foyer. It examined aspects of nitrogen use efficiency (NUE) in wheat (Triticum aestivum) particularly focusing on the remobilisation of nitrogen from senescing leaves to the grains and use of genetic modification to improve NUE. This project was part of a large EU funded project, which aimed at developing wheat with an enhanced NUE. Glutamine synthetase (GS: EC 6.3.1.2) is the major enzyme of ammonia assimilation in plants. I measured GS gene expression, polypeptide abundance and activity in a commercial wheat variety, Cadenza, and three Double Haploid (DH) lines derived from the cross Chinese Spring x SQ1, throughout development, as well as transformed plants from each of these lines expressing a cytosolic GS (Gln-α) gene from Phaseolus vulgaris. Real-time RT-PCR measurements revealed that wheat leaves, stems and roots expressed four main GS gene sub-families (GS2, GS1, GSr and GSe) at relatively high abundance. These genes showed differential patterns of expression with regard to tissue and development. GS2 was the main gene expressed in green wheat leaves while GSr was prevalent in roots. GS1 transcripts were induced in wheat flag leaves during senescence. This suggests a role for GS1 in N remobilisation for grain filling. To explore the regulation of GS and its role in NUE further the four wheat genotypes were transformed with a construct containing Gln-α under the  control of the promoter of the small subunit of ribulose-bisphosphate carboxylase/oxygenase from rice, to target the transgene specifically to photosynthetic tissues. Using particle bombardment nineteen independent transgenic lines were produced and characterised. The transgene was expressed mainly in leaves, at a level similar to that of the endogenous cytosolic GS genes. However, transgene expression had little effect on leaf GS polypeptide abundance or GS activity. These studies showed that the expression of Gln-α had little effect on the expression of native GS genes except for a marked decrease in GS2 and GS1 expression in at least two lines. This result might explain the absence of any marked increase in GS activity in the transgenic lines. This study confirmed the transition of ammonia assimilation from the chloroplast (GS2 expression) to the cytosol (GS1 expression) in wheat leaf senescence and demonstrated the presence of GSr transcripts in wheat leaves and roots throughout development. Moreover, these studies emphasise the complexity of GS regulation at multiple levels (from transcription to enzyme activity) throughout wheat development.

Stephanie Bernard
Postdoctoral Fellow

Ecology Department

Phone: 510-486-6125
Fax: 510-486-7152
Email: SMBernard@lbl.gov