Heterotrimeric G proteins in plant signal transduction : characterisation of tobacco and arabidopsis Gß subunits

Anderson, David John. (2002). Heterotrimeric G proteins in plant signal transduction : characterisation of tobacco and arabidopsis Gß subunits PhD Thesis, School of Biological Sciences, The University of Queensland.

       
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Author Anderson, David John.
Thesis Title Heterotrimeric G proteins in plant signal transduction : characterisation of tobacco and arabidopsis Gß subunits
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Associate Professor Jimmy Botella
Professor Robert Birch
Professor Christa Critchley
Total pages 180
Collection year 2002
Language eng
Subjects L
270202 Genome Structure
620199 Field crops not elsewhere classified
Formatted abstract

Heterotrimeric GTP-binding proteins or G proteins, consisting of Gα, Gβ, and Gγ subunits, are molecular switches involved in many eukaryotic signal transduction pathways. Current reports implicate G proteins in plant signalling associated with blue light, phytochromes, auxin, gibberellins, abscisic acid, and elicitor responses. We have sought to shed light on the role of G proteins in plants by investigating the tobacco and Arabidopsis Gβ subunits NtGβ1 and AtGβ1. Comparison of the four NtGβ1 cDNAs cloned to date showed that they almost certainly code for a single functionally distinct protein. Southern analyses of NtGβ1 indicated the presence of two highly similar genes in the allotetraploid genome of tobacco, one derived from the presumed diploid ancestor species Nicotiana sylvestris, and the other probably from another diploid Nicotiana species. Southern analysis of the AtGβl indicated that it is present in the Arabidopsis genome as a single copy gene with no close relatives, since confirmed by the genome sequencing project. These results suggest that NtGβ1 and AtGβ1 are the only prototypical Gβ subunits encoded within their genomes, in contrast to mammalian genomes, which contain five genes that result in six Gβ isoforms.

NtGβ1 expression was examined in seedlings as well as young and mature tobacco plants by northern and western analyses. Although NtGβ1 mRNA was expressed at varying levels in most tissues and stages tested, these did not correspond to protein levels, indicating that expression of NtGβ1 protein is tightly regulated, consistent with the known and anticipated signalling roles of plant G proteins. NtGβ1 protein was absent during germination and appeared in seedlings as the cotyledons emerged. NtGβ1 was expressed at differing levels in various young and mature plant tissues, being most abundant in regions of growth and development. Monitoring of young plants over a 24- hour period showed generally consistent protein levels that were not obviously light-regulated, despite an apparently photoperiod-dependent, rhythmic pattern of transcription. In contrast to a previous report (Kaydamov et al., 2000), no evidence was found for transcriptional induction or down-regulation of NtGβ1 expression in leaf disk assays with a variety of applied stresses and hormonal stimuli.

Northern analysis of AtGβ1 expression showed that transcripts are present in all tissues tested at varying levels. AtGβ1 expression was investigated in more detail using a promoter-intron-GUS fusion in transgenic Arabidopsis plants. Histochemical GUS detection revealed a complex expression pattern that covers all stages of development except for mature seeds. Expression occurred in vascular and immediately adjacent tissue, in much of the root system, and in the mesophyll tissue of first leaf-stage seedling cotyledons, suggesting a role in nutrient accumulation or transport, as already proposed for the Arabidopsis Gα subunit (Weiss et al., 1993). In accordance with a report that the Arabidopsis Gα subunit is involved in abscisic acid-mediated stomatal regulation (Wang et al., 2001), GUS was specifically expressed in guard cells. Highly localised expression was also found in the root cap and in trichomes, which may reflect roles in sensing external stimuli.

To determine the subcellular localisation of AtGβ1, a C-terminal GFP fusion was constructed and expressed in Arabidopsis. Examination of transgenic AtGβ1-GFP seedlings by confocal laser scanning microscopy showed that the fusion protein is present in the nucleus and also around the cell border. Comparison with non-targetted and endoplasmic reticulum-targetted GFP lines suggested that the cell border location is the plasma membrane. Although requiring confirmation by other methods, the results are consistent with expectations for Gβ subunits and agree with a report of Gβ localisation in tobacco (Peskan and Oelmüller, 2000).

Sense and antisense transgenic tobacco plants were generated in an attempt to alter NtGβ1 expression and induce a mutant phenotype. All transgenic plants appeared morphologically normal, and no significant changes in NtGβ1 protein levels were found among the large numbers of sense and antisense lines screened. However, northern analysis of representative lines demonstrated high levels of transgene transcription in most cases. Although no conclusions regarding function were possible, these results provide further evidence for the conclusion that NtGβ1 protein levels are tightly controlled. In summary, this thesis presents a detailed characterisation of the AtGβ1 and NtGβ1 genes, providing clues to the roles of G proteins in plants and a base of knowledge for new explorations.

Keyword G proteins
Transgenic plants
Plant genetic engineering

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
 
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Created: Fri, 24 Aug 2007, 17:50:26 EST