The purpose of this thesis was to study the nucleic acids of Capsicum annuum plastids during fruit ripening. The ultrastructural characteristics of Capsicum annuum plastids were studied during the transition between chloroplast and chromoplast, revealing the complex changes in membrane structure. Ploidy changes in ripening plastids was followed using a new experimental system, the Leitz MPV3 microphotometer. The relative changes in fluorescence of DAPI-stained plastids was measured. Chromoplasts proved to contain a UV-absorbing substance which reduced the level of fluorescence. The amount of plastid DNA decreases from leaf chloroplasts to green pepper plastids, and possibly decreases further in the chromoplast. It was concluded that the system was unsuitable for use with plastids of changing pigment content. A ripeness index of pepper fruit was formulated using a general pigment extraction. Ripeness was assessed by the ratio of absorbance measured at 470 and 430nm of this extract. The use of an in vitro ripening system was investigated to counter the problems of availibility and uneven ripening of green-house ripened fruit. Similarity between the chloroplast and chromoplast genome was examined by comparison of Bam Hl digestion patterns. The resolved gel bands were identical. Clones were created containing Capsicum annuum chloroplast DNA using a modified lambda bacteriophage, lambdaL47 vector. The lambdacap clones were investigated using Bam Hl restriction endonuclease digests. The enzyme digests revealed fragments in the 4-8.5kb region, and l8kb upwards. It was concluded that the 4-8.5kb fragments comprised the foreign DNA insertion. Hybridization of chloroplast DNA with lambdacap clones gave low levels of hybridization, which was concluded to be due to impurities in the plastid DNA. Hybridization of lambdacap clones to each other revealed that 17 of the 22 clones are closely related. l6s rRNA extract hybridized with certain lambdacap clones, although clones containing known chloroplast genes for LSU, beta and alpha subunits of ATPase and cytochrome f did not. Collation of this evidence, together with the Bam Hl fragment patterns led to the conclusion that the lambda cap clones map in the small single copy region and part of the inverted repeat region. Study of gene expression during the chloroplast-chromoplast transition was studied by hybridization of RNA, isolated from pepper fruit ripened in vitro, with lambda cap clones. Evidence showed changes in expression accompanied fruit ripening.
|Date of Award||1984|