Submitted to: Annals Of Botany
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/6/2004
Publication Date: 12/15/2004
Citation: Karaca, M., Saha, S., Callahan, F.E., Jenkins, J.N., Read, J.J., Percy, R.G. 2004. Molecular and cytological characterization of a cytoplasmic-specific mutant in pima cotton (Gossypium barbadense L.). Euphytica. 139:187-197. Interpretive Summary: Chloroplast genomes are much simpler than the nuclear genome and provides a good model system for phylogenetic studies. Chloroplast genetic engineering (CpGE) is becoming the transformation method of choice for the next wave of transgenic products in crop plants. Manipulation of the chloroplast genome requires specific knowledge at the molecular level about genes controlling chloroplast development. Currently limited information is available on the genes associated with cotton chloroplast development. The molecular identification of chloroplast-associated genes in many crop species has been aided by the analysis of mutants. Recently, a maternally inherited morphological mutant (cyt-V) was reported in Pima (Gossypium barbadense L.) cotton, where the first to third leaves on the plant show a pale yellow to white color, followed by restoration of a normal green phenotype in the fifth to sixth leaves and in all subsequent leaves. Based on the results of electron microscope analyses we discovered that the abnormal morphological phenotype in the cyt-V mutant was associated with abnormal chloroplast development. Our results suggested that some chloroplast specific regulatory gene(s) might be involved in silencing the psbA gene in the yellow leaves of the cyt-V mutant. We identified the source and effect of the cyt-V mutant at the molecular level and may have discovered the regulatory mechanisms associated with the psbA gene chloroplasts of cotton.
Technical Abstract: The overall objective of this research was to identify the source and molecular effects of cyt-V, a cytoplasmic mutant of Gossypium barbadense. The maternally inherited cyt-V mutant was characterized at the morphological, cellular, genetic and molecular levels using reciprocal crosses and F2 generations. The yellow phenotype was specific only to leaves in the cyt-V mutant but the same phenotype was present in both leaves and cotyledons of v7v7, a nuclear virescent mutant suggesting that cyt-V and v7v7 had different organ-specific gene actions. Genetic analysis of F2 progenies further confirmed that cyt-V expression was absent in cotyledons but present in young true leaves. Chlorophyll and carotenoid levels of cyt-V and v7v7 mutant leaves were significantly lower than in normal leaves and this difference was increased by high light intensity. Ultrastructural analyses of chloroplast and mitochondria showed that chloroplasts of both cyt-V and v7v7 mutants lacked grana in the thylakoids, whereas no morphological differences were observed in the mitochondria of the mutant and normal plants. SDS-PAGE analysis of thylakoid preparations revealed decreases of several grana-localized PSII proteins. Application of the cDNA-AFLP method identified several differentially expressed transcripts in normal versus mutant plant leaves including one possessing a high sequence homology to a psbA gene (psbA encodes the PSII reaction center protein, D1). Western analysis further confirmed the absence of D1 protein in cyt-V yellow leaves. However, our results further showed the presence of D1 protein in the mutant mature leaves when they turned green. Results suggested that some chloroplast specific regulatory element(s) might be involved in a maturity specific silencing of the psbA gene in leaves of the cyt-V mutant. Preliminary observation also suggested that the mutant phenotype changed gradually from yellow to green based on light and temperature. Protein analysis further suggested that cyt-V mutant played an important role in both the regulation of plastid and nuclear gene expression. We elucidated for the first time the source of cyt-V was in chloroplast and discovered the regulatory mechanisms of transcription of cyt-V specific genes in cotton.