Molecular and cytological characterization of a cytoplasmic-specific mutant in pima cotton (Gossypium barbadense L.)

Abstract

A cytoplasmic mutant of Gossypium barbadense L., cyt-V was characterized at the morphological, cellular, genetic and molecular levels using comparison analysis with v 7 v 7, a nuclear virescent mutant to identify molecular effects of the cyt-V mutation. The yellow phenotype was specific only to leaves in the cyt-V mutant (CM-1-90) but the same phenotype was present in both leaves and cotyledons of v 7 v 7, a nuclear virescent mutant, suggesting that cyt-V and v 7 v 7, had different organ-specific gene actions. Chlorophyll and carotenoid levels of CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v 7 v 7 true leaves were significantly lower than in the true leaves of PS-7 × CM-1-90, v 7 v 7 × CM-1-90 and PS-7. Anatomical studies of chloroplast showed that CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v 7 v 7 lacked grana in the thylakoids of the mesophyll cells. This indicated that chlorophyll and carotenoid levels correlated with chloroplast structure. SDS-PAGE analysis of thylakoid preparations revealed decreases of several granalocalized PSII proteins in CM-1-90, CM-1-90 × PS-7 and CM-1-90 × v 7 v 7. cDNA-AFLP differential display studies identified several differentially expressed transcripts in the leaves of reciprocal crosses (PS-7 × CM-1-90, v 7 v 7 × CM-1-90 and CM-1-90 × PS-7 and CM-1-90 × v 7 v 7), including one possessing a high sequence homology to a psbA gene. Western blot analysis further confirmed the absence of D1 protein encoded by psbA in CM-1-90 × PS-7′ CM-1-90 × v 7 v 7 and CM-1-90 true leaves. Overall, we studied cyt-V and v 7 v 7 that both are developmental mutants, as all the virescents of cotton mutants, and as such it was difficult to separate cause and effect in the observation; however, we verified that the source of cyt-V mutation was in chloroplast and elucidated that its gene action was different from v 7 v 7. Results indicated that cyt-V is inherited as a single gene but it affects several chloroplast and nucleus-encoded genes. We identified several transcripts that associated with the cyt-V mutation. This study also suggested that chloroplast-encoded gene products might affect the expression of nuclear genes, possibly at the transcriptional stage.