An evolutionarily conserved mitochondrial orf108 is associated with cytoplasmic male sterility in different alloplasmic lines of Brassica juncea and induces male sterility in transgenic Arabidopsis thaliana

Abstract

Nuclear-mitochondrial gene interactions governing cytoplasmic male sterility (CMS) in angiosperms have been found to be unique to each system. Fertility restoration of three diverse alloplasmic CMS lines of Brassica juncea by a line carrying the fertility-restorer gene introgressed from Moricandia arvensis prompted this investigation to examine the molecular basis of CMS in these lines. Since previous studies had found altered atpA transcription associated with CMS in these lines, the atpA genes and transcripts of CMS, fertility-restored, and euplasmic lines were cloned and compared. atpA coding and downstream sequences were conserved among CMS and euplasmic lines but major differences were found in the 5' flanking sequences of atpA. A unique open reading frame (ORF), orf108, co-transcribed with atpA, was found in male sterile flowers of CMS lines carrying mitochondrial genomes of Diplotaxis berthautii, D. catholica, or D. erucoides. In presence of the restorer gene, the bicistronic orf108-atpA transcript was cleaved within orf108 to yield a monocistronic atpA transcript. Transgenic expression of orf108 with anther-specific Atprx18 promoter in Arabidopsis thaliana gave 50% pollen sterility, indicating that Orf108 is lethal at the gametophytic stage. Further, lack of transmission of orf108 to the progeny showed for the first time that mitochondrial ORFs could also cause female sterility. orf108 was found to be widely distributed among wild relatives of Brassica, indicating its ancient origin. This is the first report that shows that CMS lines of different origin and morphology could share common molecular basis. The gametic lethality of Orf108 offers a novel opportunity for transgene containment.

Cytoplasmic male sterility in Brassicaceae crops

“…The CMS B. juncea line derived from the somatic hybridization with M. arvensis carried the unaltered mitochondrial genome from M. arvensis in which orf108 is present (Prakash et al 1998). The finding is the first demonstration of a common molecular mechanism underlying CMS in lines of different origin (Ashutosh et al 2008, Kumar et al 2012). Apart from the above species, orf108 was also found in other wild species including B. oxyrrhina , Sinapis alba , En.…”

“…In B. juncea , the most important oil seed crop in Southern Asia, the cytoplasms of at least eight species induce CMS (Banga et al 2003, Kumar et al 2012, Landgren et al 1996, Malik et al 1999, Pathania et al 2003, Prakash and Chopra 1990, Prakash et al 2001, Rao et al 1994). In addition to D. muralis , four other cytoplasms induce CMS in B. rapa (Deol et al 2003, Matsuzawa et al 1999, Prakash and Chopra 1988, 1990, Tsutsui et al 2011).…”

“…Expression analyses suggested orf108 as the causal gene in these CMS sources (Kumar et al 2012). Orf108 was also found in male sterile B. juncea obtained by cell fusion between B. juncea and M. arvensis .…”

Mitochondrial Genetic Manipulation

“…Contrasting results were reported. Male sterile plants were obtained upon nuclear expression of the CMS-associated bean orf239 (He et al 1996), beet orf129 , chili pepper orf456 (Kim et al 2007), mustard orf220 , rice orf79 (Wang et al 2006) or wall-rocket orf108 (Kumar et al 2012). Conversely, similar attempts with the proteins associated with maize CMS-T, petunia CMS or radish and Brassica Ogura CMS failed to yield sterile plants (von Allmen et al 1991;Chaumont et al 1995;Wintz et al 1995;Duroc et al 2006).…”

“…Conversely, similar attempts with the proteins associated with maize CMS-T, petunia CMS or radish and Brassica Ogura CMS failed to yield sterile plants (von Allmen et al 1991;Chaumont et al 1995;Wintz et al 1995;Duroc et al 2006). Also puzzling, in some cases, expression of the CMS orf resulted in aberrant pollen function and a semi-sterile or male-sterile phenotype regardless of the presence or not of an MTS (He et al 1996;Kumar et al 2012). It seems, for instance, that the bean ORF239 protein can associate with the cell wall and therefore may cause pollen disruption even if it is not targeted to mitochondria.…”