Isolation and characterization of the cytoplasmic male sterility-associated orf456 gene of chili pepper (Capsicum annuum L.)
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Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T1 transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T1 plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper.
KeywordsArabidopsis thaliana Capsicum annuum L. Cytoplasmic male sterility (CMS) Mitochondrial mutant Mitochondrial targeting signal orf456
This research was supported by a grant from the Center for Plant Molecular Genetics and Breeding Research (CPMGBR) via the Korea Science and Engineering Foundation (KOSEF) and the Korea Ministry of Science and Technology (MOST). Dr. Yoo Jae Hyoung is acknowledged for the generous gift of plant material and discussions.
- Bowman JL (1993) Arabidopsis: an atlas of morphology and development. Springer, Berlin Heidelberg New York, pp 277–283Google Scholar
- Kim DH, Kang JG, Kim SJ, Kim BD (2001) Identification of cox2 and atp6 regions as associated to CMS in Capsicum annuum by using RFLP and long accurate PCR. J Kor Soc Hortic Sci 42:121–127Google Scholar
- Notsu Y, Masood S, Nishikawa T, Kubo N, Akiduki G, Nakazono M, Hirai A, Kadowaki K (2002) The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: frequent DNA sequence acquisition and loss during the evolution of flowering plants. Mol Genet Genomics 268:434–445Google Scholar
- Sane AP, Nath P, Sane PV (1994) Mitochondrial ATP synthase genes may be implicated in cytoplasmic male sterility in sorghum bicolor. J Biosci 19:43–55Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn, Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar