Study On The Developmental Characters Of Chloroplasts In A Yellow Type Mutant Of Oilseed Rape Seedlings

Chloroplast biogenesis is a fascinating area of plant biology. The development of these photosynthetic apparatus is studied at the level of structure, biochemistry, genetic, and molecular biology. In this study, an oilseed rape mutant type Cr3529 was chosen to be the material to study the developmental characters of chloroplasts in etiolated mutant type.Firstly, the expression of the important transport protein Toc33/34 of chloroplast was analyzed. SDS-PAGE electrophoresis was introduced to separate different subunit proteins on envelopment membranes. Western blotting with Toc33 antibody were applied to determine Toc33/34 level in Cr3529. The results shew that only Toc33 can be detected by the Arabidopsis Toc33 antiserum both in the wild and in the mutant type. Abundance of Toc33 in both mutant and wild type is nearly the same but a little higher in the mutant type. Furthermore, Toc33 mRNA level is determined by RNA gel blot analysis. The results shew that Toc33 transcript level decreased in mutant cotyledon and different expression was found in hypocotyls and cotyledon. These results indicate that there is no change of Toc33, an important component of chloroplast outer envelope transport machinery, in the mutant type.Secondly, the content of total protein and chlorophyll were studied in different organs and different stages of the mutant oilseed rape. The results shew that (1) the quantity of total protein decreased in the mutant type, and so the LHCII accumulation, from the results of the SDS-PAGE and light absorption analysis. (2) The chla/chlb ratio of the mutant type increases nearly 30% in cotyledon and 20% in hypocotyls. (3) The accumulation of LHCII in hypocotyls in the mutant type increased when the equal total proteins were loaded. (4) Treated by Lincomycin, contrary to the wild type, the content of chlorophyll increased in the mutant type cotyledon. Chla and Chlb alsohave this change pattern. And the chla/chlb ratio increased in the mutant type, while it increased first and then decreased in the wild type. We speculate that, conversion efficiency of chla to chlb decrease in the mutant type, so the mutant type has much lower content of chlb, so the chlb-binding proteins are in a stage of quickly degredation and accumulates lower on the thylakoid membranes.Thirdly, the analysis of the thylakiod of the different organs and different stages of the oilseed rape indicate that (1) The similar light absorption pattern exhibited in both the cotyledon and the mature leaf, whether in the mutant or the wild type. And it is higher in the wild type and in the mature leaf. The absorptions at wavelength of 680~ 690nm and 440~450nm of Chla in the mutant type are weak, compared to those of in the wild type. In contrast to the wild type, the absorptions at wavelength of 680~ 660nm and 490~500nm of Chlb disappeared. (2) Room temperature fluorescence spectra of the wild type and the mutant type were recorded. Fluorescence emission bands observed with two excitation wavelengths of 436nm and 475nm belong to PSII activity. In mutant type, it decreased in the cotyledon; on the contrary, it increased in the mature leaf. (3) Chlorophyll-protein complexes from thylakoid membranes separated by mildy denaturing electrophoresis shows the accumulation of the LHCII and CP47 in the mutant decreased, but CP43 increased. The DCIP activity decreased too. (4) In the mutant type, the composition of LHCII has no difference. CD spectra at 685nm which belongs to the LHCII composition has Einstein shift too. These results indicate that, the composition of LHCII has no change, but the assembly of Chlorophyll-protein complexes changed.The polyribosome can show the level of translation, So the mRNA binding to the polyribosome was analyzed to study the translate ability. The results suggest that the efficiency of the translation of the mRNA decreased.