Glutamine synthetase isoforms in Trientalis europaea: a biochemical and molecular approach

Abstract

Ion-exchange chromatography of extracts from Trientalis europaea L. leaf tissue have been shown to contain two distinct isoforms of glutamine synthetase (GS). However, analysis by Western blotting has shown that the first peak to elute contains a mixture of large and small GS subunits, whilst the second peak is comprised entirely of a smaller subunit. This is contrary to the widespread assumptions concerning plant GS biochemistry. Isolation of intact chloroplasts and subsequent extraction of GS, followed by ion-exchange chromatography, has shown that the first peak to elute contains a large subunit, and the second chloroplastic peak is composed entirely of the small subunit. This smaller subunit may be present due to it being encoded by a separate chloroplastic GS gene, or it may be present as a product of post-translational modification. DNA sequencing has been used to try and determine which of these may be occurring. The three partial DNA sequences (505 nucleotides) we have obtained from T. europaea have been compared with 64 other sequences available on the NCBI database, which have mainly been obtained from crop species. Neighbour joining and parsimony analysis (1000 bootstrap) has shown support (_∼30%) for the separation of plant GS from all other phyla. Within the plant phylum, there is total support for the separation of chloroplastic and cytosolic GS (100%), whilst the cytosolic sequences divide further into monocot and dicot species (77% support by NJ). Further subgroups of plants from the same families is also suggested. This is consistent with previous work containing fewer, but longer (_∼1000 nucleotides) GS sequences. The addition of GS sequences obtained from wild plant species, such as T. europaea, to the large amount of information already available on the database, will permit a better understanding of the evolution of this important enzyme.