Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Dr. Yuhai Cui

2nd Supervisor

Dr. Susanne Kohalmi

Joint Supervisor

Abstract

Plants’ cell walls have unique chemical composition and features which enable them to play essential roles during plant development as shaping the cells and providing intercellular communication between adjacent cells. Polysaccharides, including callose, and glycoproteins are known as the main constituents of the cell wall. Callose, a linear β-1,3-glucan polymer, is accumulated at the cell plate during cytokinesis, in plasmodesmata, where it regulates cell-to-cell communication, in dormant phloem, where it seals sieve plates after mechanical injury and pathogen attack, and in male and female gametophytes. GLUCAN SYNTHASE-LIKE (GSL) genes in Arabidopsis comprise a family of 12 members. A new allele of GSL8, essp8, was identified as having seedling-lethal phenotype through a genetic screen for Arabidopsis mutants showing ectopic expression of seed storage proteins (essp). The gene responsible for the observed mutant phenotype was detected using a combination of bulked-segregant analysis, rough-mapping, and next-generation mapping. An EMS-induced point mutation was identified at an intron splice site of GSL8, predicted to introduce a premature STOP-codon. essp8 seedlings exhibit pleiotropic phenotypic defects, including disruption of root tissue patterning, dwarfism and seedling lethality. Histochemical detection of callose and cell-to-cell diffusion assays showed reduction of callose deposition at the cell plates and plasmodesmata, cytokinesis defects and significant increase in size exclusion limit of plasmodesmata in essp8 seedlings. Further investigation showed that the increase in size exclusion limit leads to an alteration in symplastic trafficking in primary roots of essp8 seedlings. Plasmodesmata defects in essp8 induce ectopic movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development. Attempts to identify the components of a hypothetical callose synthase complex revealed the interaction of GSL8 with two plasmodesmata-associated proteins, PLASMODESMATA-LOCALIZED PROTEIN 5 and β-1,3-GLUCANASE, as well as SUCROSE SYNTHASE 1, suggesting that they all might be parts of a single complex. The proposed putative complex might regulate callose deposition at the plasmodesmata and thereby determines the size exclusion limit. In summary, my findings suggest that GSL8 is required for cell wall integrity, maintaining the basic ploidy level and regulation of symplastic movement during early seedling development in Arabidopsis.

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