Isolation and characterization of the cytoplasmic male sterility-associated orf456 gene of chili pepper (Capsicum annuum L.)

Abstract

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 coxll 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.

Mitochondrial-targeted Expression of orf456 Causes Male Sterility in Chinese Cabbage (Brassica rapa L.)

“…2c-4, c-5). These phenotypes of aberrant floral organs in transgenic B. rapa are similar with others studies (Yang et al 2010 andKim et al 2007). …”

“…In the previous study, a CMS-associated gene, orf456 was found at the 3' -end of the coxII gene, which was identified as a strong candidate for determining the malesterile phenotype in pepper (Kim et al 2007). When this gene was ectopically expressed in Arabidopsis, transgenic plants showed abnormal pollen development and sterility.…”

“…Then the second CMS-associated gene orf456 was identified through analysis DNA and RNA blot hybridization between male fertile and CMS lines of chili pepper (Kim et al 2007). The gene was located at the 3'-end of the coxII gene in CMS pepper.…”

“…The gene was located at the 3'-end of the coxII gene in CMS pepper. Later, it was shown that overexpression of orf456 severely induced male sterility in Arabidopsis (Kim et al 2007). To investigate the functional role of the ORF456 protein in B. rapa, we carried out transformation of B. rapa with orf456 fused to a mitochondrion-targeted coxIV gene.…”

Plant Mitochondrial Genome Evolution and Cytoplasmic Male Sterility

“…Novel and chimeric mitochondrial sequences are a frequent result of this recombination (Wise et al, 1987;Kennell and Pring, 1989;Wen and Chase, 1999;Gallagher et al, 2002;Okazaki et al, 2013;Yamagishi and Bhat, 2014;Tang et al, 2017), in which recombination sometimes leads to the creation of transcripts that interfere with normal male gametophyte development (Kitazaki and Kubo, 2010) via the generation of toxic and/or disruptive transmembrane proteins (Korth et al, 1991;Kim et al, 2007;Wan et al, 2007;Zhang et al, 2007;Yang et al, 2009;Gulyas et al, 2010;Jing et al, 2012;Flores-Renteria et al, 2013;Ji et al, 2013;Luo et al, 2013;Okazaki et al, 2013;Park et al, 2013;Hu et al, 2014). Surprisingly, such genes are not only abundant in many fertile plants, such as Arabidopsis thaliana Unseld et al, 1997), Beta vulgaris (Kubo et al, 2000), Oryza sativa (Notsu et al, 2002), Brassica napus (Handa, 2003), Zea mays (Clifton et al, 2004), Triticum aestivum (Ogihara et al, 2005), and Nicotiana tabacum (Sugiyama et al, 2005), but are also constitutively expressed.…”