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Cytoplasmic male sterility (CMS), a maternally inherited inability to produce functional pollen, has been observed in c. 200 species of higher plants (Hanson, 2004; Chase, 2007). Several CMS-associated genes have been found to be encoded by the mitochondrial genome. The CMS phenotype can be rescued by a class of nuclear-encoded genes termed ‘restorer of fertility’ (Rf) genes, highlighting the significance of the interactions of mitochondrial and nuclear gene products. Rf2 in T-CMS maize encodes an aldehyde dehydrogenase that accumulates in the mitochondria (Cui et al., 1996). Rf17, which has a partial acyl-carrier protein synthase (ACPS) domain, restores the fertility of CW-CMS rice via a retrograde regulation signal pathway (Fujii & Toriyama, 2009). The Rf2 gene in lead type-CMS (LD-CMS) in rice is a glycine-rich protein (GRP) (Itabashi et al., 2011). The remaining known Rf genes belong to the pentatricopeptide (PPR) family, a large family of genes that encode organelle-targeted proteins. These include PPR592 in petunia, Rfk1 in Kosena rapeseed, Rfo in radish, PPR13 in sorghum, Rf1a/Rf1b in Boro II type CMS (BT-CMS) rice and Rf5 in Honglian type CMS (HL-CMS) rice (Bentolila et al., 2002; Brown et al., 2003; Desloire et al., 2003; Kazama & Toriyama, 2003; Koizuka et al., 2003; Akagi et al., 2004; Komori et al., 2004; Klein et al., 2005; Wang et al., 2006; Hu et al., 2012).
The Rf genes act to reduce the accumulation of CMS-associated RNAs and/or proteins through different mechanisms, acting at the DNA, RNA, or protein level, or even by metabolic complementation. We consider three levels of regulation based on the changes in CMS genes in F1 hybrids, even though some Rf genes remain to be characterized. The first level is the DNA level. For example, Fr can restore the pollen fertility of a CMS common bean by decreasing the copy number of orf239 (Mackenzie & Chase, 1990; Arrieta-Montiel et al., 2001). The second level is the post-transcriptional level; most restoration processes can be categorized as this type. In T-CMS maize, for example, Rf1, Rf 8, and Rf * process the CMS RNA, T-urf13, to achieve restoration, while in S-CMS maize, Rf3 can alter the transcription pattern of orf355 and orf 77 (Wise et al., 1999). In rapeseed, the CMS RNAs orf224/atp6 in Polima CMS are processed in the presence of Rfp, and orf222 in napus CMS is processed in the presence of Rfn (Yuan et al., 2003; Geddy et al., 2005). In sunflower, PET1 might destabilize the CMS-associated transcript orf522 in a tissue-specific manner to restore male fertility (Moneger et al., 1994). The Petunia PPR592 not only reduces the transcript level of pcf but also affects its protein level (Hanson et al., 1999). The CMS RNA atp6-orf79 not only can be cleaved by PPR791 (RF1A) but also can be degraded by RF1B in Boro II CMS rice, while atp6-orfH79 in HL-CMS is cleaved by an RFC (Wang et al., 2006; Kazama et al., 2008; Hu et al., 2012). The destabilization and/or translational control of CMS proteins are additional potential mechanisms for restoration. The levels of the CMS proteins ORF125 and ORF138 are dramatically reduced in the presence of PPRB/ORF687 (Koizuka et al., 2003; Uyttewaal et al., 2008). In LD-CMS rice, RF2 might interact directly with the CMS-causing protein or form a multimolecular complex to achieve fertility restoration (Itabashi et al., 2011). The third level of regulation is metabolic complementation of the damage caused by CMS-associated proteins. For instance, URF13 expression in T-CMS maize is not reduced in the presence of Rf2, which encodes an ALDH protein that accumulates in the mitochondria (Chase, 2007). The pollen must eliminate the impairments caused by CMS genes by any means necessary in order to survive. Therefore, many types of Rfs have developed throughout evolution.
In a previous study, we showed that GRP162 is involved in the process of fertility restoration as a key component of the restoration fertility complex (RFC) in HL-CMS rice. Results established that GRP162 specifically bound to CMS RNAs directly. The expression of GRP162 was increased, and the localization of GRP162 was also markedly altered, in the presence of RF5 (Hu et al., 2012). Hence, we proposed that GRP162 could be rebuilt as a restorer of fertility gene with the addition of a mitochondrial transit peptide.
In this study, an artificial protein with CMS-associated transcript-binding activity was introduced into the mitochondria. The pollen was rescued in the presence of Mt-GRP162, as shown in a transgenic complementation assay. The results confirmed the mechanism of ORFH79 suppression by the artificial restorer fertility gene Mt-GRP162 and suggested a new pathway for fertility restoration. These findings also imply that GRP162 may not be the only essential factor for RNA cleavage in RFC.