The expression of chloroplast genes during cotton embryogenesis

The expression of chloroplast genes during cotton embryogenesis was followed by measuring hybridization of cloned chloroplast genomic fragments to total RNA immobilzed on membrane filters. RNA was extracted from the cotyledons of cotton embryos at different stages of development ranging from very small embryos (5 mg) to the mature dry seed and from the cotyledons of seeds germinated for several days. The clones used in the hybridization reaction were those for 4 known chloroplast genes and one chloroplast sequence whose protein product is yet to be determined; all are from organisms other than cotton. The results of these dot blot hybridizations show that these genes are expressed as RNA constitutively during embryogenesis and that this expression increased between 5 and 20-fold (depending on the genomic sequence) during germination. In some cases it appears that these genes are expressed at a higher level in very young embryos than in more mature embryos. These results are surprising since cotton cotyledons are not exposed to light during embryogenesis and are not green at any embryonic stage.The extent of artifactual hybridization signals due to the presence of ctDNA in the RNA preparations was measured by treating the RNA on the filters with DNase I prior to hybridization with a labeled probe. To determine if the increased expression of these genes observed during germination is correlated with an increase in chloroplast genomes per cell, we followed the levels of ctDNA during embryogenesis and germination by hybridization of the clones to total DNA from different developmental stages. Although ctDNA per cell increases in the cotyledons during early germination in light or dark conditions, this increase is less than the increase observed in the mRNA levels, suggesting that the transcription rate increases or the mRNA degradation rate decreases for these genes during early germination.