A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Abstract

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.

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.…”

“…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. Research into this phenomenon has revealed much about the distribution and occurrence of CMS, the tissue-specificity of the phenotype (male gametophytic tissues only) (Jing et al, 2012;Hu et al, 2014), the modes of action behind CMS, and the cytonuclear cooperation that suppresses the phenotype (Carlsson et al, 2008). As more genomes (both nuclear and mitochondrial) and other "-omic" data become available (Du et al, 2016;Jacoby et al, 2016;Wang et al, 2016), the potential for increasing our understanding and manipulating this vitally important phenomenon will be enhanced.…”

“…Maladaptive retrograde signaling has also been implicated in mitochondrial interference with the rice nuclear gene COX11, resulting in the premature programmed cell death of tapetal cells and subsequent pollen abortion (Luo et al, 2013). Still, other species have the features and function of cytotoxic proteins (Dewey et al, 1987;Korth et al, 1991;Levings, 1993;Korth and Levings, 1993;Nakai et al, 1995;Duroc et al, 2005;Wang et al, 2006;Jing et al, 2012).…”

“…Most CMS-associated proteins possess transmembrane configurations capable of disrupting the mitochondrial membrane structure and/or altering the permeability and potential of mitochondrial membrane . These proteins can directly interfere with energy production (Jing et al, 2012;Wang et al, 2013c;Wu et al, 2013), induce the release of cytochrome C via accumulation of unusually large numbers of reactive oxygen species (ROS) (Yan et al, 2014), and stimulate premature programmed cell death in male reproductive tissues (Luo et al, 2013;Horn et al, 2014). Several CMS proteins have demonstrated toxicity, such as URF13 in CMS-T maize (Korth et al, 1991), ORFH79 in HL-CMS rice , Orf507 in CMS pepper , and ROS homeostasis associated protein in cotton .…”

Complete sequence of heterogenous-composition mitochondrial genome (Brassica napus) and its exogenous source

“…Generally, genes associated with CMS genes are located in the periphery of certain known mitochondrial genes and are cotranscribed with them [26]. T-urf13 ( orf115 ) was the first identified aberrant gene in the Texas (T)-cytoplasm of maize, which encodes a 13 kDa membrane-spanning polypeptide that depolarizes the mitochondria and leads to cell death [27-29].…”