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Cytoplasmic male sterility (CMS), a maternally inherited phenomenon existing widely in the plant kingdom (Lewis, 1941; Laser & Lersten, 1972; Janska & Mackenzie, 1993; Krishnasamy & Makaroff, 1994; Hanson & Bentolila, 2004), is an evolutionarily subtle means for plants to acquire foreign pollen to increase genetic diversity by preventing self-pollination (Dewey et al., 1986; Young & Hanson, 1987; Hanson, 1991; Smart et al., 1994). CMS has been a useful approach employed for heterosis in plant breeding programs.
Many studies suggest that the CMS-associated genes are always chimeric and cotranscribed with genes encoding the mitochondrial subunits of the electronic transport chain (ETC) or ATP synthase complex (Chase, 2007; Kubo & Newton, 2008; Arrieta-Montiel & Mackenzie, 2011) or truncated versions of the functional mitochondrial genes for ETC or ATP synthase complex. Based on these facts, two hypotheses have been proposed for the molecular mechanism of CMS (Budar et al., 2003; Chase, 2007).
The first hypothesis is known as the gain-of-function model. It emphasizes the potential involvement of the CMS gene in an unknown process that hurts the mitochondria. There are at least two examples that support this model. One is the Texas male sterile cytoplasm (T-cms) maize protein, T-URF13, which was thought to form a pore in the inner mitochondrial membrane to disrupt the membrane potential (Rhoads et al., 1995; Wise et al., 1998). Another example is the recently identified sterility-inducing protein, ORF138, in Ogura rapeseed. Similar to T-URF13 in maize, this protein is involved in an unknown complex mainly composed of itself that forms a pore in the inner mitochondrial membrane (Duroc et al., 2009).
The second model is termed loss-of-function, and is supported by more cases. Connett & Hanson (1990) first reported that different mitochondrial ETC complexes were observed between male sterile and fertile Petunia. A study of wild G CMS beets found instability of complex IV and a decrease of cytochrome c oxidase activity in mitochondria (Ducos et al., 2001). In sunflower, the CMS protein ORF522 has sequence identity with a subunit of ATP synthase. The authors speculated that ORF522 can compete with the normal subunit to impair the function of the ATP synthase complex (Sabar et al., 2003). All of these studies point to the association among the CMS protein and the ETC complexes of mitochondria. Thus, we speculate that the CMS proteins somehow bind to some mitochondrial proteins to achieve such an effect. However, to date, no candidate protein has been identified that directly interacts with CMS proteins.
Rice of Honglian (HL) CMS (HL-CMS), a gametophytic CMS derived from common wild rice (Oryza rufipogon), has been the main type of CMS used for hybrid rice production in China and southern Asia (Tan et al., 2012). The area cultivated with the HL-type hybrid has cumulatively reached almost 10 million hectares during the past several decades (Li et al., 2007). The pollen of HL-CMS halts development at the bicellular stage and exhibits spherical abortion (Supporting Information, Fig. S1). We have previously found that the chimeric gene, orfH79, is responsible for the male sterility in HL-CMS (Yi et al., 2002). A large accumulation of ORFH79 in mitochondria occurs during pollen development in HL-CMS lines (Hu et al., 2012), which consequently causes an increase in reactive oxygen species (ROS) and decrease in the ATP/ADP ratio in the anthers (Li et al., 2004; Wan et al., 2007; Peng et al., 2010). This suggests that severe energy deficiency in these mitochondria is possibly the biochemical cause of CMS, while the detailed mechanism of pollen disruption remains unclear. In this study, a subunit of mitochondria complex III, p61, was identified via bacterial two-hybrid library screening as a candidate protein that directly interacts with ORFH79. We further verified this interaction with other in vivo and in vitro assays. Our results shed much light on how the plant CMS genes function at the molecular level.
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The plant trait of CMS is usually determined by a mitochondrial gene and is recognized by the sterile pollen phenotype. Understanding the underlying mechanism of CMS is the basis for comprehending natural selection forces of CMS (Budar et al., 2003). When summarizing current studies on plant CMS, it is hard to unify our view on the mechanism of CMS. Both ‘gain-of-function’ and ‘loss-of-function’ models are supported by a lot of experimental evidence. Our study on Honglian CMS rice supports the loss-of-function model.
In our experiment, the macromolecular composition of these mitochodrial complexes resolved by BN-PAGE showed almost no difference between sterile lines and maintainer lines (Fig. 2b). Meanwhile, the ETC complex III activity of CMS lines was c. 45% lower than that of maintainer lines (Fig. 5a). These seemingly contradictory results in fact imply that ORFH79 binding to P61 reduced enzyme activity of ETC complex III, without affecting the overall composition of this complex. How this binding reduces enzyme activity is still unknown. One possibility is that the binding results in the malfunction of P61, which then leads to the decrease in enzyme activity. In yeast, the deletion of QCR10 (P61 homolog gene) significantly reduces the activity of ETC complex III; however, its deletion also significantly affects the composition of ETC complex III (Brandt et al., 1994). However, the HL-CMS lines and its maintainer basically showed no significant difference in vegetative growth. Thus, the deletion or RNAi of P61 in rice did not simulate the real situation of HL-CMS lines. We infer that ORFH79 somehow binds to P61 to affect the electron transfer in an unknown way, but does not affect the structure of the complex III dimer. Recent studies of plant mitochondrial supercomplexes indicate that the respiratory chain complexes (complexs I–IV) and the ATP synthase complex (complex V) are organized into different supercomplexes (Eubel et al., 2003; Dudkina et al., 2006; Rasmusson et al., 2008). ORFH79 may affect the formation of a supercomplex in which complex III participates.
Cytoplasmic male sterility plants are characterized by their inability to produce functional pollen. Interestingly, the CMS genes only disrupt pollen development, but vegetative development and female fertility are apparently unaffected. Previous work in our laboratory demonstrated that ORFH79 in HL-CMS could be detected in mitochondria from callus, root, leaf, and spikelet of CMS lines (Peng et al., 2010). The present results showed that P61 and OrfH79 had a coexpression pattern in seven different tissues of YA and enhanced expression at the post-meiosis stage of anther development (Fig. 6a). The analysis of microarray data indicates that the mitochondrial ETC genes accumulate during the mitosis stage of pollen (Fig. 6b). These results might explain why orfH79 only causes abnormal development of microspores. The leading hypothesis for CMS is that the developing microspores consume much more energy than any other tissues. The number of mitochondria increases significantly in the microspores and tapetum of the anther after the small gametophytic meiosis stage (Lee & Warmke, 1979). Some abnormal phenotypes caused by impairment of mitochondrial function also occur at this stage (Hanson & Bentolila, 2004). Two recent studies reporting that a mutation in flavoprotein subunit of complex II (Leon et al., 2007) and a mutation in a subunit of complex V (Li et al., 2010) both specifically cause abortive microspores support this hypothesis. Our findings here provide further supporting evidence. In fact, the microspores of HL-CMS rice could not enter into the second stage of mitosis, as they stopped at the bicellular stage (Fig. S1; Li et al., 2007). Although the microspores of HL-CMS could pass the meiosis and first mitosis stage, they showed abnormal mitosis during the highest energy-consuming stage (P2 and P3 in Fig. 6b) by the impairment of ORFH79.
Mitochondrial signals can influence the expression of nuclear genes, which is called mitochondrial retrograde regulation (Woodson & Chory, 2008). Although the energy limitation hypothesis more likely explains the tissue-specific injury caused by ORFH79 in HL-CMS, it is still not clear how the status of energy limitation leads to suspension of mitosis of pollen. In other words, the retrograde regulation of mitochondria in HL-CMS still requires resolution. ETC is considered an important source of oxygen radicals in mitochondria (Moller, 2001; Petrosillo et al., 2003; Brand, 2010). The impairment of complex III activity, as a result of ORFH79 binding, may increase the electron leakage from the electron transport chain to generate excessive ROS while reducing ATP production. Thus the excessive ROS and reduced ATP are likely the primary signals to be transferred to the nucleus, and ultimately lead to abnormal mitosis during the development of microspores (Moller & Sweetlove, 2010). Coincidently, a study in Drosophila melanogaster showed that shortage of ATP and overdose of ROS caused by disruption of the mitochondrial electron transport chain acted as mitochondrial retrograde signals to activate the G1-S checkpoint (Owusu-Ansah et al., 2008; Mandal et al., 2010). In the present paper, we investigated the expression of cell cycle-related genes in YA and YB. Their changes in YA imply that the pollen of HL-CMS may activate the G1-S checkpoint, as CDKA;1, the key regulator of the G1-S checkpoint of mitosis, exhibited a decrease in expression at RNA, protein, and protein modification (phosphorylation) levels (Fig. 7b).
Figure 7. Expression analysis of cell cycle-associated genes between the Honglian type cytoplasmic male sterility (CMS) line and the maintainer line of rice. (a) Real-time PCR quantification ( method) of rice homologs of FBL17, RHF, KRP6/7, and CDKA;1. The error bars represent the SDs of three replicates. (b) Western blot analysis with anti-KRP, anti-CDKA;1, and anti-phosphor-CDKA;1 antibodies. (c) The regulatory pathway of the G1-S checkpoint of the PM2 stage (the secondary mitosis of pollen development), which was modified from a previous version (Gusti et al., 2009). YB1, 2, 3, YB anthers at the unicellular, bicellular, and mature stages, respectively; YA1, 2, 3, YA anthers with the corresponding length to YB1, 2, 3.
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Based on these reports and our findings, we propose a hypothesis for the molecular mechanism of HL-CMS, as summarized in Fig. 8. CMS protein ORFH79 binds to ETC complex III and ultimately causes increased ROS and reduced ATP concentrations. This status of mitochondria is then sensed by the nucleus through a retrograde pathway. This information transferred from the organelle (mitochondrion) forces the nucleus to stop at the G1-S checkpoint of the cell cycle. As a result, the pollens of Honglian CMS rice exhibit an abortive phenotype.
Figure 8. Proposed model for Honglian cytoplasmic male sterile rice (HL-CMS). The CMS protein ORFH79 impairs the electron transport chain (ETC) by binding to P61 (complex III). This causes physiological changes that increase reactive oxygen species (ROS) and decrease ATP, resulting in retrograde signals from the mitochondrion to inhibit pollen development at the PM2 stage. GC, germ cell; VC, vegetative cell; PMI, pollen primary mitosis; PM II, pollen secondary mitosis. G1, S, and G2 indicate different stages of the cell cycle.
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Combined with the significantly different enzyme activity of ETC complex III between the CMS line and the maintainer line, we infer that ORFH79 binds to ETC complex III via P61, which subsequently suppresses its activity and ultimately causes the pollen abortion phenotype.