Mitochondrial genome organization and expression associated with cytoplasmic male sterility in sunflower (Helianthus annuus).

A comparative investigation of the organization and expression of the mitochondrial genome in fertile and cytoplasmic male sterile (CMS) sunflower (Helianthus annuus) has been undertaken. A region of mitochondrial genome variation between the two phenotypes has been located in the 3' flanking region of the gene encoding the alpha subunit of the F1 ATPase (atpA). Physical mapping and sequence analysis have been used to show that a rearrangement involving an inversion and an insertion has occurred immediately downstream of the atpA coding region in the mitochondrial DNA from sterile sunflower. This rearrangement has resulted in the creation of a new open reading frame (ORFc) which is co-transcribed with atpA in sterile sunflower. In organello labelling of mitochondrial translation products from the two types of sunflower shows that a 15 kDa protein is synthesized by the mitochondria from sterile sunflower but not by those from fertile plants. The ORFc sequence could encode this 15 kDa protein which may be causally related to the CMS phenotype.     

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orf H522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.