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Title: Regulation of expression of genes encoding the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase in Phaseolus vulgaris L.
Author: Sawbridge, Timothy Ivor
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1993
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The expression of the three known rbcS genes of Phaseolus vulgaris L. was examined under different growth conditions in the primary leaves using gene-specific oligonucleotide probes. These experiments demonstrated that the three rbcS genes had different quantitative levels of expression but very similar patterns of expression under the majority of conditions examined. The one exception to this was when plants that had been light-adapted were returned to darkness. Under these conditions, mRNA levels of one of the genes (rbcS2 ) increased after prolonged (4 days) dark adaptation. Whether this observation represents a real difference in the pattern of expression of the rbcS2 gene relative to the other two genes is discussed. The expression of the gene family was also examined using non-discriminating oligonucleotide and cDNA probes. These studies showed that there is an underlying ontogenetic control of rbcS expression in the primary leaves of dark-grown Phaseolus vulgaris plants. This pattern of expression is modified by the presence of light, and the extent of the 'modification' is dependent on the fluence rate of light with which the plant is illuminated. The control of rbcS mRNA levels in the light-grown plant in response to an increased fluence rate of light was also examined. It was found that exposure of 16 day old plants grown under a low (15 ?mol/m2/s) fluence rate of white light to an increased fluence rate (150 mumol/m2/s) of white light for 2 days resulted in a substantial increase in the rbcS mRNA level. Similar experiments using increased fluence rates of different light qualities (blue-enriched and red) indicated that a blue light photoreceptor was involved in mediating the increase in rbcS mRNA levels. Measurements of oxygen evolution and stomatal resistance s howed that the different qualities of light did not result in differential rates of photos ynthesis and stomatal opening under the different light qualities, supporting the idea, that it was a blue light photoreceptor that was mediating the increase in rbcS expression. An increase in rbcS expression in response to an increased fluence rate of blue-enriched light, which was not apparent with an identical fluence rate of red light, was found to occur in primary leaves of different ages, showing that the response was not limited to one developmental stage of the primary leaf. The photoregulation of transcription of the rbcS genes was examined. Plants were grown in a similar manner to those used for experiments to measure the steady state mRNA level, and moved to an increased fluence rate of blue-enriched light. Leaves were harvested over a timecourse and nuclei extracted from them for use in run- on transcription assays. These experiments showed that the blue light treatment resulted in an increase in the transcription rate of the rbcS genes that peaked at around 12 hours and fell by 24 hours. The effect of other light qualities on the transcription of rbcS genes was also examined after a 12 hour treatment. These experiments indicated that, when equal fluence rates of light were used, an increased fluence rate of blue-enriched light was the most effective in increasing the transcription of rbcS genes. These results indicated that the increase in steady-state rbcS mRNA seen after treatment with an increased fluence rate of blue-enriched light occurred as a result of a blue photoreceptor mediating an increase in rbcS transcription. As expected from the steady-state mRNA data, an equal fluence rate of white light gave a smaller increase in rbcS transcription than the blue-enriched light, but, some what unexpectedly, an increased fluence rate of red light also gave a small increase in transcription of the rbcS genes, a response that was not evident when steady-state rbcS mRNA levels were studied. Possible reasons for this are discussed. A method to determine the relative levels of the mRNA of the three rbcS genes in a single reaction was developed. This was done because such information could not be obtained from SI-nuclease analysis with a single probe due to the similarity between the sequences of the three genes. The method involved using the polymerase chain reaction with three gene-specific 3' primers, and a single non-discriminating 5' primer, following a reverse transcriptase reaction carried out with the three gene-specific 3' primers and total RNA. These primers should theoretically have allowed the amplification of three PCR products of different sizes. This was demonstrated to be the case, and the method was shown to produce similar measurements of rbcS mRNA levels to hybridisation of probes to a northern blot. The results demonstrated that the method could prove useful with some further refinement which, unfortunately, time did not allow. One problem with the method is that the estimates of the three genes' relative mRNA levels differ from those found both through the use of northern analysis with gene-specific oligonucleotide probes and examination of the frequency of the corresponding cDNAs in a cDNA library. Possible reasons for this discrepancy are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available