Characterisation of an Arabidopsis mutant with altered greening characteristics

Two plastid-to-nucleus signalling pathways had previously been identified by studies on genomes uncoupled (gun) mutants of Arabidopsis. A pathway responding to the state of plastid protein synthesis was disrupted in gunl mutants. Unlike the wild type, the mutants show expression of nuclear photosynthesis genes in the presence of norflurazon, which results in chloroplast photooxidation, or in the presence of lincomycin, an inhibitor of plastid translation. The aim of this study was to understand the role of plastid protein synthesis in plastid-to-nucleus signalling. Five putative gunl-like mutants from a new collection of Arabidopsis gun mutants with a green fluorescent protein (GFP) reporter gene under the control of a tobacco RbcS (encoding ribulose-1,5-bisphosphate carboxylase small subunit) promoter were examined further. One of the mutant lines, PR48.2N, showed two-fold higher transcript abundance of nuclear photosynthesis genes, RBCS and LHCBJ (encoding light-harvesting chlorophyll a/b-binding protein 1), compared to wild type with or without treatments of norflurazon or lincomycin. Pigment analysis of PR48.2N seedlings illuminated for 16 hours after being subjected to various lengths of dark treatment demonstrated that the mutant line accumulated less chlorophyll than wild type after short periods of darkness (2-4 days) but showed an enhanced ability to green after prolonged dark treatments (5-10 days). Consistent with the enhanced greening ability, transcript abundance of nuclear photosynthesis genes was higher and there was more thylakoid membrane in chloroplasts in greened PR48.2N seedlings after prolonged darkness compared to the wild type. Microarray analysis indicated that a group of transcripts encoding seed storage proteins, oleosins and late embryogenesis abundant proteins showed very low abundance in PR48.2N seedlings. The promoter regions of the genes shared some ciselements possibly involved in regulation by abscisic acid (ABA). However, the ABA content of PR48.2N seedlings was not significantly different to wild type, although the germination of mutant seeds was more sensitive to inhibition by ABA than the wild type. Expression of the GFP reporter gene in the presence of lincomycin and the enhanced greening ability of PR48.2N were shown to be inherited in a recessive manner by examining the segregation of these phenotypes in the F2 progeny of a PR48.2N (Ws) x wild type (Ler) cross. Genetic analysis using F2 individuals from agunl-1 x PR48.2N cross showed that the mutated genes were not allelic but might be interacting because putative double mutants with much higher GFP expression were observed. Preliminary data from PCR-based mapping methods using a small F2 mapping population suggest that the locus providing enhanced greening ability in PR48.2N may be in a region on chromosome V. PR48.2N phenotypes such as expression of the GFP reporter gene in the presence of norflurazon or lincomycin, enhanced greening ability, lower transcript levels of seed protein genes, and hypersensitivity to the ABA inhibitory effect on germination appeared to co-segregate in the mapping population. Although PR48.2N mutant was initially identified in a screen for gunl-like mutants, the evidence presented in this dissertation suggests that the product encoded by the gene mutated in PR48.2N may have a regulatory role in the transition from heterotrophic to photoautotrophic growth during early seedling development.