Analysis of phosphoglucomutase isoforms from physcomitrium patens

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Stellenbosch : Stellenbosch University
ENGLISH ABSTRACT: Starch is the main storage polymer found in most plants and plays a significant role in plant fitness. It is a polyglucan and is composed of two separate fractions named amylose and amylopectin. In photosynthetic tissues starch is synthesized in chloroplasts during the day and degraded at night to provide energy which supports metabolism. The pathways of starch synthesis and degradation have been extensively characterised in Angiosperms, however, in non-vascular plants these are less well understood. Physcomitrium patens is a Bryophyte that is an excellent non-vascular model plant due to its fully sequenced genome and the ease at which mutants can be created using homologous recombination. As Bryophytes have been classified as a transitionary species between water-based algae and land plants, research using them sheds light on how biochemical processes (such as starch metabolism) have changed during land colonisation. The first committed step of the starch biosynthetic pathway is the creation of ADP-glucose from glucose 1-phosphate (G1P). The formation of G1P is catalysed by plastidial isoforms of phosphoglucomutase (PGM) and these enzymes are encoded by a highly conserved family of genes. Previous studies in Angiosperms have demonstrated that mutating the plastidial isoform of PGM results in plants that accumulate almost no starch in all organs (Hanson & McHale, 1988; Harrison, et al., 2000; Vriet, et al., 2010). Cytosolic phosphoglucomutase isoforms are also present and the protein sequences of these are similar to those of plastidial isoforms. The first aspect of this project was to identify PGM genes in P. patens that demonstrate high similarity to PGM genes from Arabidopsis thaliana. Four were identified (named PpPGM1-4), and the amino acid sequences of the translated P. patens PGM polypeptides have high similarity to other phosphoglucomutases. The predicted intron-exon boundaries showed that PpPGM1 and PpPGM2 genes contain no introns whereas PpPGM3 and PpPGM4 contain seventeen introns each. Phylogenetic analysis of PGM sequences from red algae, Cyanobacteria and Viridiplantae demonstrated that sequences could be divided into three clades. One contained red algal and cyanobacterial sequences while the other two contained only Viridiplantae PGM’s. One of the Viridiplantae clades contained all isoforms that have been demonstrated experimentally to be plastidial (and PpPGM1 & 2), and the other all isoforms that have been demonstrated experimentally to be localised in the cytosol (and PpPGM3 & 4). The sub-cellular localization of the PGM protein was examined by transiently expressing PGM genes, which had been fused to a gene encoding the green fluorescent protein, in P. patens protoplasts. This demonstrated that PpPGM1 and PpPGM2 were imported into plastids and PpPGM3 and PpPGM4 were localised in the cytosol. All four genes were shown to encode active proteins and their expression restored the wildtype phenotype in an Escherichia coli pgm mutant. Furthermore, analysis of the PGM amino acid sequence showed that PpPGM1 and PpPGM2 both contain a SASHNP active site motif whereas in PpPGM3 and PpPGM4 this is TASHNP. Similarly, a metal binding motif differed between the four polypeptides, being DGDGD in PpPGM1 and PpPGM2 and DGDAD in PpPGM3 and PGM4. The known sugar binding sequence CGEESF was found in all four proteins. These versions of the active sites were conserved across the two Viridiplantae clades. The final aspect of this project was to create knockout mutants of the two plastidial PGM genes in P. patens to identify their effect on phenotype, this was attempted using PEG mediated homologous transformations, several plants survived selection but were demonstrated to be untransformed.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.
Thesis (MScAgric)--Stellenbosch University, 2023.