According to the endosymbiosis theory, mitochondria originated from α-proteobacteria ancestors (Gray, 1999). During long-term evolution, most mitochondrial genes were transferred to the nucleus of the host cell (Timmis et al., 2004). Only a minor part of mitochondrial genes were retained in angiosperms and those genes are involved in the electron transport system or encode ribosomal proteins and tRNA (Kubo and Newton, 2008). After these mitochondrial genes are transcribed, their transcripts are processed mainly by nuclear-encoded proteins. The post-transcriptional processing generally includes intron splicing, RNA editing, cleavage, and maturation (Small et al., 2013; Hammani and Giege, 2014).
According to structural features and splicing mechanisms, mitochondrial introns in flowering plants were divided into group I and group II with the latter being more predominant (Bonen, 2008). Group II introns are involved in both cis-splicing and trans-splicing. Cis-splicing occurs within one pre-mRNA molecule, whereas the trans-splicing occurs between two pre-mRNA molecules (Sharp, 1987; Lasda and Blumenthal, 2011).
Pentatricopeptide repeat (PPR) proteins are the most studied RNA-binding proteins involved in splicing of numerous mitochondrial introns (Small and Peeters, 2000). PPRs can be divided into the major P and PLS subfamilies (Lurin et al., 2004). P-subfamily PPRs contain only canonical 35-amino-acid PPR (P) motifs whereas PLS-subfamily PPRs consist of ordered series of P, longer (L) and shorter (S) PPR motifs (Lurin et al., 2004; Shikanai and Fujii, 2013; Cheng et al., 2016). Many studies have shown that P-type PPRs are involved in splicing of mitochondrial introns. In Arabidopsis, ORGANELLE TRANSCRIPT PROCESSING43 (OTP43) (Falcon de Longevialle et al., 2007), OTP439, TANG2 (Colas des Francs-Small et al., 2014), ABA OVERLY SENSITIVE 5 (ABO5) (Liu et al., 2010), ABO8 (Yang et al., 2014), BSO-INSENSITIVE ROOTS6 (BIR6) (Koprivova et al., 2010), MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) (Haïli et al., 2016), SLOW GROWTH3 (SLO3) (Hsieh et al., 2015), Mitochondrial Intron Splicing Factor 26 (MISF26), MISF68 and MISF74 (Wang et al., 2018) were identified to be required for splicing of mitochondrial introns. Mutations of the above genes generally lead to delayed germination and retarded growth. Mutations of P-type PPRs in maize usually lead to lethal embryos within defective kernels or empty pericarps, in mutants such as defective kernel2 (dek2) (Qi et al., 2017), dek35 (Chen et al., 2017), dek37 (Dai et al, 2018), dek41/dek43 (Zhu et al., 2019; Ren et al., 2020), empty pericarp8 (emp8) (Sun et al., 2018), emp10 (Cai et al., 2017), emp11 (Ren et al., 2017), emp12 (Sun et al., 2019), emp16 (Xiu et al., 2016), emp602 (Ren et al., 2019) and ppr20 (Yang et al., 2020). In contrast to numerous P-subfamily PPR proteins reported in Arabidopsis and maize, few such proteins are reported in rice although its genome has 477 PPRs (O’Toole et al., 2008). FLOURY ENDOSPERM10 (FLO10), a mitochondrion-targeted P-type PPR protein characterized in rice, functions in regulating the trans-splicing of nad1 intron 1. Like mutants in Arabidopsis and maize, the flo10 mutant exhibited defective grain development evidenced by smaller opaque grains at maturity and obviously slower plant growth during the vegetative and reproductive stages (Wu et al., 2019).
Other proteins in addition to PPRs participate in splicing mitochondrial introns. ABO6 encodes a DEXH box containing RNA helicase that is involved in regulating the splicing of several genes of Complex I in mitochondria (He et al., 2012). Nuclear-encoded maturase nMAT1 (Nakagawa and Sakurai, 2006; Keren et al., 2012), nMAT2 (Keren et al., 2009), and nMAT4 (Cohen et al., 2014), DEAD-box protein PUTATIVE MITOCHONDRIAL RNA HELICASE2 (PMH2) (Köhler et al., 2010), RCC1 family protein RUG3 (Kühn et al., 2011), mitochondrial transcription termination factor mTERF15 (Hsu et al., 2014), and RAD52-like protein ODB1 (Gualberto et al., 2015) are also involved in splicing mitochondrial introns. Another protein family characterized by the plant organelle RNA recognition (PORR) domain also plays an important role in intron splicing in mitochondria and chloroplasts. The PORR domain was previously known as the “domain of unknown function 860” (DUF860) (http://pfam.xfam.org/family/PF11955) but was renamed as the PORR domain in 2009 (Kroeger et al., 2009). AtRPD1 (ROOT PRIMORDIUM DEFECTIVE 1), a member of PORR/DUF860 family, has a role in prearranging the maintenance of active cell proliferation during root primordial development. Disruption of the RPD1 gene caused embryogenesis arrest at the globular to transition stages. RPD1 is expressed in all organs of fourteen-day-old seedlings and the encoded protein is annotated to localize in mitochondria or plastids. In silico structural characterization of RPD1 and RPD1-like proteins suggested a possible involvement of RPD1 and RPD1-like proteins with winged helix proteins in various regulatory functions through DNA binding, RNA binding, and protein-protein interaction (Konishi and Sugiyama, 2006). Structural modeling suggests that PORR adopts a structure that has a surface reminiscent of helical repeat RNA-binding motifs such as the PPR motifs (Kroeger et al., 2009). At4g08940, encoding a PORR protein, responds to oxidative stress. The over-expressed transgenic plants of At4g08940 were more tolerant to paraquat and cold, and less tolerant to t-butyl hydroperoxide and salinity, but the underlying mechanism remains unknown (Luhua et al., 2008). ZmWTF1 (“what’s this factor?”), a chloroplast-targeted PORR protein, is required for the splicing of chloroplast-encoded introns. wtf1-1, a weaker mutant, showed a pale green phenotype whereas mutants wtf1-3 and wtf1-4 with null alleles were albinic (Kroeger et al., 2009). AtWTF9, a mitochondrially localized PORR protein, is required for rpl2 and ccmFC intron splicing. T-DNA insertion alleles wtf9-1 and wtf9-2 caused severely stunted shoots and roots; both homozygous mutants survived to flowering, but the flowers were small and produced only a few “milky” aborted seeds (Colas des Francs-Small et al., 2012). A recent study showed that mitochondrial heat shock protein 60 s (HSP60s) interact with WTF9 to regulate the intron splicing of ccmFC and rpl2. A retarded growth phenotype was observed in hsp60-3a-1hsp60-3b-1 which had small cotyledons, reduced root length and small stature (Hsu et al., 2019). ZmEMP6, a PORR protein located in mitochondria, is required in both endosperm and embryo development, but the intron(s) splicing was unclear (Chettoor et al., 2015). Up to now, no PORR protein has been identified in rice.
Here, we reported the isolation of FSE5 though map-based cloning in rice. The Fse5 allele encodes a mitochondria-localized PORR protein that is expressed constitutively in various tissues. Cis-splicing of nad4 intron 1 is abolished in the fse5 mutant and mitochondrial function and structure are disrupted consequently. Like other mutants causing defective splicing of mitochondrial intron(s) fse5 seed development and seedling growth were affected. Mature fse5 seeds showed a floury, shrunken phenotype, and either failed to germinate or produced weak seedlings that died within one month. These results indicated an essential role of FSE5 in seed development and subsequent seedling growth.