Characterisation of sucrose phosphate synthase isoenzymes from Arabidopsis thaliana
The dicot model plant Arabidopsis thaliana (A. thaliana) contains four copies of the sucrose phosphate synthase (SPS) gene. In this project, the quantitative real-time PCR (qPCR) technique was validated to investigate the differential expression patterns of the four SPS transcripts in A. thaliana. Based on this validation, a set of experimental conditions were selected to study the differential expression of the four SPS transcripts. The level of the AtSPS1 and AtSPS5a transcripts changed in response to the hormone abscicic acid (ABA). In the absence of sucrose, AtSPS5a expression increased 2-fold, and when seedlings were heat treated under light without sucrose the transcript was elevated up to ~14-fold after 3 h. Cold-acclimated plant leaves had elevated levels of all four isoenzymes after 40 days, with AtSPS5b showing the highest expression. The AtSPS5b transcript responded to heat stress under light but not in the dark. Heat stress, under dark conditions, in the absence of sucrose, repressed the levels of all transcripts. Tissue-specific expression of the four isozymes was also investigated. The AtSPS4 and AtSPS5a transcripts showed highest expression in leaf tissue. Publicly available microarray data were collated and compared with the gene expression patterns seen above in different developmental stages and selected hormone and environmental treatments. Meta-analysis of microarray data showed that AtSPS4 was down regulated in the majority of the datasets. The AtSPS5b transcript was elevated in response to light, nutrient and cold, suggesting its involvement in sucrose sensing and signalling. The AtSPS1 isoenzyme is expressed the least in almost all tissues examined, especially in seedlings. In both qPCR and DNA-microarray analysis, the AtSPS5a transcript showed the highest expression in all tissues, which is in line with the trend seen in literature. The SPS promoters were also investigated for unique elements to enhance the understanding of the expression patterns seen in qPCR analysis and DNA microarrays. The AtSPS1 and AtSPS4 promoters were the smallest and longest defined promoters, respectively, without predicted ORFs or micro RNAs. Analysis of the elements suggested that AtSPS5a promoter may be involved in circadian control and AtSPS5b in low-temperature response. Protein phylogeny was investigated using the current list of full length, partial length as well as microbial SPS protein sequences from the publicly available databases to construct a revised SPS phylogenetic tree. SPS phylogenetic subfamilies are still subdividing within each of the four families, and this may become more prominent as more sequences of monocots become available. Microbial phylogeny was not well-defined, with each clade having representatives from different microbial families, suggesting a more complex phylogenetic arrangement implicating horizontal gene transfer.
Advisor: Eaton-Rye, Julian; McRae, Elspeth
Degree Name: Doctor of Philosophy
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: qPCR; sucrose phosphate synthase; gene expression; carbohydrate metabolism; multigene family; phylogeny
Research Type: Thesis