| Popis: |
To identify novel and essential components of the plant membrane trafficking mechanisms, Arabidopsis membrane trafficking mutants from fluorescent protein-based forward genetic screens were characterized. First, four novel glutathione synthase (GSH2) mutant alleles featured swollen endoplasmic reticulum (ER)-derived bodies that accumulated a soluble secretory marker. Consistent with the role of GSH2 in glutathione biosynthesis, the loss-of-function mutant alleles exhibited gamma-glutamylcysteine (γ-EC) hyperaccumulation and glutathione deficiency. The aberrant ER morphology was ascribed to the γ-EC accumulation. Redox-sensitive fluorescent protein revealed that gsh2 seedlings maintained a reduced cytoplasm at steady state but were more sensitive to oxidative challenge. Second, Mut 21 was a conditional mutant that accumulated a secretory marker in the alkalized apoplast at restrictive temperature (31˚C). The mutant was identified as carrying a mutant allele of tuftelin-interacting protein 11 (TFIP11), which has been implicated in regulating redifferentiation and cell proliferation through a cytokinin signalling pathway. Hence, it was postulated that the changes in response to cytokinin affect auxin-mediated acidification of the apoplast. Third, Mut 43 was a conditional mutant that accumulated a soluble secretory marker in the ER and unidentified punctate structures at restrictive temperature, and exhibited perturbations in ER export of a soluble protein marker. Moreover, the mutant showed severe growth defects and abnormal radial root swelling in the apical elongation zone. A mutation identification method through deep-sequencing of the wild-type siblings in outcrossed heterozygous mutant families was developed and tested in Mut 43. At the time when this thesis was prepared, bioinformatic analysis has assigned Mut 43 to the bottom arm of chromosome two and predicted a 300kb mapping interval based on the observed bias in single nucleotide polymorphism ratios. This work demonstrates the feasibility of using forward genetics to study plant-specific aspects of membrane trafficking mechanisms and incorporates new technology to streamline the process of gene identification. |
