Mitochondrion role in molecular basis of cytoplasmic male sterility

Mitochondrion 19 (2014) 198–205

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Review

Mitochondrion role in molecular basis of cytoplasmic male sterility Renate Horn a,⁎, Kapuganti J. Gupta b, Noemi Colombo c a b c

Institute of Biological Sciences, Department of Plant Genetics, University of Rostock, Albert-Einstein-Str. 3, 18059 Rostock, Germany Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK Instituto de Genética, CICVYA, CNIA, INTA, CC 25 (1712), Castelar, Provincia de Buenos Aires, Argentina

a r t i c l e

i n f o

Available online 13 April 2014 Keywords: Cytoplasmic male sterility Restorer of fertility Mitochondria Chimeric open reading frame ATP level Reactive oxygen species

a b s t r a c t Cytoplasmic male sterility and its fertility restoration via nuclear genes offer the possibility to understand the role of mitochondria during microsporogenesis. In most cases rearrangements in the mitochondrial DNA involving known mitochondrial genes as well as unknown sequences result in the creation of new chimeric open reading frames, which encode proteins containing transmembrane domains. So far, most of the CMS systems have been characterized via restriction fragment polymorphisms followed by transcript analysis. However, whole mitochondrial genome sequence analyses comparing male sterile and fertile cytoplasm open options for deeper insights into mitochondrial genome rearrangements. We more and more start to unravel how mitochondria are involved in triggering death of the male reproductive organs. Reduced levels of ATP accompanied by increased concentrations of reactive oxygen species, which are produced more under conditions of mitochondrial dysfunction, seem to play a major role in the fate of pollen production. Nuclear genes, so called restorer-of-fertility are able to restore the male fertility. Fertility restoration can occur via pentatricopeptide repeat (PPR) proteins or via different mechanisms involving non-PPR proteins. © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial role in cytoplasmic male sterility . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mitochondrial DNA rearrangements and deletions leading to cytoplasmic male sterility . . . . . 2.1.1. Open reading frames derived from genes of complex I (NADH dehydrogenase) 2.1.2. Open reading frames derived from genes of complex IV (cytochrome oxidase) 2.1.3. CMS associated open reading frames derived from genes of complex V (F0F1-ATPase) . 2.1.4. Open reading frames involved in CMS derived from other sequences . . . . . . . . . 2.2. RNA editing and cytoplasmic male sterility . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Mitochondrial energy deficiency causes male sterility . . . . . . . . . . . . . . . . . . . . 2.4. Cytotoxic effects of CMS-specific proteins . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Role of reactive oxygen species in CMS . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Fertility restoration by nuclear genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Restorers-of fertility encoding PPR proteins . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Restorers-of-fertility encoding non-PPR proteins . . . . . . . . . . . . . . . . . . . . . . 3.3. Restorers of fertility encoding unknown proteins . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

⁎ Corresponding author. Tel.: +49 381 498 6170; fax: +49 381 498 6112. E-mail address: [email protected] (R. Horn).

http://dx.doi.org/10.1016/j.mito.2014.04.004 1567-7249/© 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

As the world population grows and we are facing a 70% increase of food demand over the next four decades (European Commission, 2011), the need to enhance plant performance and crop productivity while decreasing the ecological and environmental footprints of our agriculture is

R. Horn et al. / Mitochondrion 19 (2014) 198–205

one of the most important issues of our time. Hybrid technology is one of the tools to increase crop productivity (Kubo et al., 2011). The use of male sterility systems avoids the need for hand emasculation in hybrid production; thereby represents an important tool for development of hybrids. Male sterility can be caused either by interaction of mitochondrial genes with nuclear genes or nuclear genes alone. The former is known as cytoplasmic male sterility (CMS) and the latter as genic male sterility (GMS). Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants originating in the majority of cases from mitochondrial DNA rearrangements which results in plants unable to generate or shed functional pollen (Chase, 2007; Chen and Liu, 2014; Hanson and Bentolila, 2004; Horn, 2006; Schnable and Wise, 1998). The trait is widespread in flowering plants; more than 140 species are known to exhibit CMS (Laser and Lersten, 1972). CMS is exploited for hybrid production in a wide variety of crops such as sunflower, maize, onion, beet, sorghum, carrot, petunia, rapeseed, common beans, rice, cotton and wheat (Kubo et al., 2011; Schnable and Wise, 1998). CMS generated hybrids can show hybrid vigor, also termed heterosis, which describes the enhanced performance of hybrid progeny in comparison to the parental lines (Baranwal et al., 2012). For instance, in rice the yield has increased up to 20% in hybrids obtained by using CMS lines (Virmani, 2003). CMS mutants allow the genetic dissection of the mitochondrial role in the development of male reproductive organs. A better understanding of the molecular processes leading to pollen abortion will help us to use CMS more efficiently in hybrid breeding and to ensure reliable fertility restoration by restorer-of-fertility genes. However, it has to be kept in mind that mitochondrial genome organizations studied in cytoplasmic male sterility represent only a subset of possible mtDNA organizations, selected by breeders for male sterility (Horn, 2002). 2. Mitochondrial role in cytoplasmic male sterility 2.1. Mitochondrial DNA rearrangements and deletions leading to cytoplasmic male sterility Present mitochondrial genomes have evolved by rapid reshuffling of genomes during evolution from endosymbiotic organisms (Chang et al., 2011; Kubo and Newton, 2008). Today's plant mitochondrial proteins are encoded by the mitochondrial as well as the nuclear genome with an estimated 10% of nuclear genes targeted to mitochondria (Glaser and Soll, 2004). Mitochondria participate in retrograde signaling by sending signals to the nucleus to generate various proteins from the nuclear genome (Chase, 2007). Especially during mitochondrial biogenesis and substantial environmental changes a constant transcriptional synchronization between mitochondrial and cellular statuses is required (Roads, 2011; Schwarzländer and Finkemeier, 2013). CMS occurs due to incompatibility between nuclear and mitochondrial genomes. It is frequently observed as a result of interspecific crosses (Horn, 2002; Kofer et al., 1991), but can also occur spontaneously within a species. Hanson and Bentolila (2004) showed that interactions between mitochondrial and nuclear genes affect male gametophyte development. CMS is associated with rearrangements of mitochondrial genome (chimeric regions) via non-homologous recombination. On the other hand homologous recombinations can also occur between repeated sequences and lead to a multipartite organization and reorganization of mitochondrial genome (Sugiyama et al., 2005). Plant cells contain varying numbers of mitochondria. Sizes and shapes of mitochondria differ within a tissue or in the same cells. In some cases, mitochondrial population of a cell exists as a connected (i.e. fused) population, therefore mitochondrial genome rearrangements can spread to whole mitochondria population within a cell (Logan, 2006). The plant mitochondrial population might go through a ‘bottleneck’, meaning might be reduced before or during gametogenesis, as described in the female germ line in mammals (Taylor and Turnbull, 2005). Open reading frames associated with cytoplasmic male sterility often represent chimeric genes that seem to originate from multiple

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recombination events involving frequently known mitochondrial genes as well as their 5′- and 3′-flanking regions in addition to sequences of unknown origin (Chase, 2007; Chen and Liu, 2014; Hanson and Bentolila, 2004; Horn, 2006; Schnable and Wise, 1998). Most of these orfs have been identified based on restriction fragment length polymorphism by hybridization with known mitochondrial genes as probes followed by transcript analyses of the rearranged areas. However recently, whole mitochondrial genome sequence approaches have been applied (Bentolila and Stefanov, 2012; Park et al., 2013; Tanaka et al., 2012). In the following overview, CMS associated orfs will be sorted according to the mitochondrial genes involved. In the case of several genes participating in the creation of a new CMS orf, these orfs will be sorted according to the mitochondrial gene providing the promoter region that allows the transcription of the orfs. 2.1.1. Open reading frames derived from genes of complex I (NADH dehydrogenase) CMS phenotypes have been described involving nad7 and nad9. For the RT98A CMS line derived from Oryza rufipogon, orf113 shows completely identical sequences to nad9 in the region −151 to +11, whereas the rest consists of unknown sequences (Igarashi et al., 2013). In CMSI and CMSII in Nicotiana sylvestris no new open reading frames were created but a deletion of exons III and IV of nad7 in CMSI and the total elimination of nad7 in CMSII lead to cytoplasmic male sterility (Gutierres et al., 1997). 2.1.2. Open reading frames derived from genes of complex IV (cytochrome oxidase) A number of CMS associated open reading frames involve parts or modifications of genes of complex IV (cytochrome oxidase) or the promoter regions to express these orfs. In the 9E cytoplasm of Sorghum bicolor, cox1 is enlarged in the 5′- as well as in the 3′-region (Bailey-Serres et al., 1986). In Raphanus sativus DCGMS, orf463 consists also partially of cox1 sequences, but in addition contains 1261-bp unidentified sequences (Park et al., 2013). The new chimeric orfH79, identified for the Honglian (HL) cytoplasm in rice, shows homology to cox2 and orf107 (Yi et al., 2002). In I-12 CMS(3) derived from wild beet, the CMS-specific orf129 shows 37 bp similarity to the sequence of cox2 (Scox2-1) in Owen mitochondria (Senda et al., 1991); the remaining sequence is of unknown origin (Yamamoto et al., 2008). CMS-G in wild beets is characterized by a point mutation in the second exon of cox2, creating a premature STOP codon and thereby an eight amino acid shorter COX2 protein (Ducos et al., 2001). In the CMS-Peterson of chili pepper, orf507 is cotranscribed from the cox2 gene (Gulyas et al., 2010; Kim et al., 2007). However, the sequence of orf507 does not show any significant homology to the databases apart from a 51 bp of homology to the 5′-UTR of atp6. 2.1.3. CMS associated open reading frames derived from genes of complex V (F0F1-ATPase) The three mitochondrial genes, atp6, atp8, and atp9, which are involved in the majority of CMS so far investigated, represent subunits of the F0-sector of the F0F1-ATP synthase (Jia et al., 2007). These genes, especially atp8 and atp9, seem to represent hot spots of mitochondrial recombination. In the BT (Boro II)-type CMS cytoplasm of rice, orf79 is co-transcribed with a duplicated B-atp6 gene (Akagi et al., 2004; Wang et al., 2006). In the Owen CMS in sugar beet, preSatp6 representing the leader sequence of atp6 is fused in frame with the downstream atp6 (Yamamoto et al., 2005). In petunia, the pcf-gene consists of 5′-flanking and 5′-coding region of the atp9 gene, but also contains coding sequences of the cox2-gene as well as the sequences of an open reading frame urfS (Young and Hanson, 1987). In PEF1 cytoplasm of sunflower, a 0.5-kb insertion of unknown sequences in the 3′flanking region of the atp9 gene was identified as a cause for male sterility (de la Canal et al., 2001). In chive, the CMS1 configuration is derived in part from sequences of atp9 and atp6, with orf501 the most likely candidate of the three created orfs (Engelke and Tatlioglu, 2004; Potz and Tatlioglu, 1993). In maize T-cytoplasm, the adjacent atp6 gene allows

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the transcription of the chimeric T-urf13, which itself consists of coding and 3′-flanking region of the 26S rRNA gene as well as nine amino acids of unknown origin (Dewey et al., 1987). In sunflower PET1 cytoplasm, orfH522 consists of the first 57 bp of atp8 (formerly known as orfB) as well as unknown sequences. OrfH522 is co-transcribed with the atp1 on an additional larger transcript (Köhler et al., 1991). In maize CMSS, the co-transcribed open reading frames orf355 and orf77 are associated with cytoplasmic male sterility (Zabala et al., 1997). The orf77 contains three segments derived from atp9 (Zabala et al., 1997). Also in the A3 cytoplasm in S. bicolor, the CMS-specific orf107 shows a high homology to 5′-flanking and 5′-coding regions of the atp9 as well as to orf79 in the 3′-coding and -flanking regions (Tang et al., 1996). In the family of Brassicaceae, at least 11 CMS-type chimeric open reading frames associated with male sterility and co-transcribed with known mitochondrial genes have been characterized (Ashutosh et al., 2008; Jing et al., 2012). In most cases, this involves mitochondrial genes from complex V. In the Polima (Pol) cytoplasm orf224 is located upstream of and co-transcribed with the atp6 gene (Handa et al., 1995). This orf224 consists of the coding and 5′-flanking region of atp8, also known as orf158, a part of the exon 1 of the ribosomal protein S3 (rps3-gene) and unknown sequences (Handa et al., 1995). In the Shaan 2A cytoplasm orf224 only shows an additional single nucleotide polymorphism G-NA at position + 389 from Polima (Wang et al., 2002). In the nap CMS, orf222 is two codons shorter than orf224 due to a 6-bp deletion (L'Homme et al., 1997). The CMS-specific orf288 in the hau male sterility type in Brassica juncea is also co-transcribed with the atp6 gene (Jing et al., 2012). It represents a chimeric gene being identical to nad5 in the N-terminus and showing an additional homology to an uncharacterized ATP synthase C. For the TournefortiiStiewe CMS, the chimeric orf193, which exhibits partial sequence identity to atp6, is co-transcribed with atp9 and might also be translated uninterrupted into a chimeric 30.2 kDa protein (Dieterich et al., 2003). In the CMS-Peterson in chilli pepper, a second copy of the atp6 with a truncated 3′-region (ψatp6-2) might also be responsible for CMS (Ji et al., 2013). 2.1.4. Open reading frames involved in CMS derived from other sequences In the Ogura cytoplasm in rapeseed, the CMS-phenotype was correlated with orf138 (Bonhomme et al., 1992) and verified by a whole genome sequencing approach. The unique regions of the orf138 could have been created by the shuffling process of the mitochondrial genome of short homologous regions (Tanaka et al., 2012). The orf125 in the Kosena cytoplasm is very similar to the orf138 (Bellaoui et al., 1999; Krishnasamy and Makaroff, 1993). These two open reading frames only differ by two amino acid substitutions and a 39-bp deletion (Iwabuchi et al., 1999). Also in B. juncea carrying the Moricandia arvensis cytoplasm, CMS is associated with a novel orf108 that does not match any sequences in the databases (Ashutosh et al., 2008). The distronic transcript of orf108 and atp1 gene is processed in the presence of the fertility restorer, so that only the atp1 can be translated (Gaikwad et al., 2006). In Phaseolus vulgaris, the unique pvs-region of the CMS Sprite contains three open reading frames, orf98, orf97 and orf239 (Chase and Ortega, 1992; Johns et al., 1992). The pvs-region, which is flanked by the atpA gene and sequences of the cob gene, shows no homology to nuclear DNA, but to the intron of the plastidal tRNA alanine and a short part of the 5-kb repeat in maize. The development of the pvsregion is probably the result of several recombination events. The Wild Abortive (WA) cytoplasm in rice, WA352 consists of 512 bp identical to the 5′-region of rice orf284, whereas the 3′-region (583 bp) shows homology to rice orf288. So WA352 has no homology to genes involved in ATP production (Luo et al., 2013). 2.2. RNA editing and cytoplasmic male sterility RNA editing has been connected with cytoplasmic male sterility in a number of CMS types in various plant species (Castandet and Araya,

2012; Chase, 2007; Chase and Gabay-Laughnan, 2004). In S. bicolor A3Tx398 male sterile cytoplasm, RNA editing is significantly reduced for atp6 in anthers and again normal in fertility restored F1 and F2 plants (Howad and Kempken, 1997). Loss of RNA editing seems to be the cause for this type of cytoplasmic male sterility (Howad et al., 1999). In CMS-S in maize, RNA editing sites of atp9 present in orf77 are either not edited or inefficiently edited (Gallagher et al., 2002). In addition, an unexpected terminating editing results in a truncated orf77 encoding a 17 aa peptide being homologous to the ATP9 transmembrane domain. Also no RNA editing in the atp9 gene could be detected in the cytoplasmic male sterile soybean line NJCMS2A. This leads in this case to changes in the trans-membrane structure of the ATP9 protein (Jiang et al., 2011). Luo et al. (2013) has recently shown WA352 to be responsible for male sterility in the WA CMS system, although Das et al. (2010) had claimed an unedited orfB transcript as molecular cause for this CMS system in rice. In the Boro CMS, also in rice, RNA editing of the transcripts of both copies of atp6, N-atp6 and B-atp6, is reduced. The restorer gene RF1A increases atp6 mRNA editing levels of both N-atp6 and Batp6 RNA molecules. However, this does not seem to be essential for the fertility restoration, as RF1B does not show this effect (Wang et al., 2006). Investigating five isonuclear alloplasmic male sterile lines (IAMSLs) Hu et al. (2013) could show that nucleo-cytoplasmic interactions affect the RNA editing of cox2, atp6 and atp9. Genetically engineered male sterile tobacco plants could be produced by using the unedited atp9 from wheat (Hernould et al., 1993) and could be restored by inhibiting its expression with antisense RNA (Zabaleta et al., 1996). In rice, the male sterile ogri mutant, which is characterized by a mutated pentatricopeptide repeat-DYW protein, does not edit seven specific RNA editing sites in five mitochondrial transcripts nad2, nad4, cox2, cox3, and ccm (Kim et al., 2009). These examples show that RNA editing can be changed in cytoplasmic male sterile lines, but is not necessarily always the cause of CMS. However, these results all support the theory that nucleo-cytoplasmic conflicts may be the driving force for the emergence of RNA editing in plants (Castandet and Araya, 2012). Ultra-deep sequencing methods will help to further elucidate RNA editing events in mitochondrial genes (Suzuki et al., 2013). 2.3. Mitochondrial energy deficiency causes male sterility Mitochondria are essential organelles for cellular energy production because they harbor primary metabolic cycles such as TCA cycle, electron transport chain and ATP synthesis (Fernie et al., 2004). Considering that pollen development is dependent on a high energy supply, disturbances in mitochondrial functions could have dramatic effects on male fertility (Chen and Liu, 2014; Luo et al., 2013). In mitochondria energy is generated by the production of proton gradient via electron transport. The mitochondrial respiratory chain consists of four complexes: Complexes I (NADH dehydrogenase), II (succinate dehydrogenase), III (cytochrome c reductase), mobile electron protein cytochrome C and IV (cytochrome c oxidase). Complex V represents the F0F1–ATP synthase. Due to active electron transport via these complexes, a proton gradient is generated, which leads to ATP generation (Millar et al., 2011). Any disturbance of this electron transport chain impairs the energy production. In the Texas (T)-cytoplasm of maize, T-urf13 encodes a 13 kDa membrane-spanning polypeptide that depolarizes mitochondria, which leads to electron leakage and furthermore to cell death (Dewey et al., 1987; Rhoads et al., 1995; Wise et al., 1999a, 1999b). This contributes to a premature degradation of tapetum during the process of microsporogenesis, which further results in pollen abortion. Recently, Luo et al. (2013) reported that the WA352 gene product (Wild abortive) in wild rice interacts with the nuclear-encoded mitochondrial complex IV protein COX11 (protein subunit in cytochrome c oxidase). In CMSWA lines, WA352 accumulates in anther tapetum, thereby inhibiting COX11 which leads to an increased over-reduction of ubiquinone pool

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and concomitant reactive oxygen species (ROS) production and triggers premature tapetal PCD and subsequent pollen abortion. Furthermore, the ORFH79 protein expressed in mitochondria in CMS-HL rice also leads to a reduced ATP/ADP ratio, decreased membrane potential, a lower respiration rate and an excessive accumulation of ROS, not only affecting the development of the male gametophyte, but also impairing root growth (Peng et al., 2010). It was demonstrated that ORFH79 interacts with the protein P61 (corresponding to QCR11 in yeast from complex III) impairing mitochondrial function (Wang et al., 2013a). In sunflower, ORFH522 shares an identical N-terminal amino acid sequence with ORFB. Therefore, ORFH522 competes with ORFB in binding to the ATP synthase (complex V) and reduces ATP synthesis mediated by the ATP–synthase complex (Sabar et al., 2003). Premature programmed cell death (PCD) of the tapetal cells occurs, which is trigged by a release of cytochrome c from the mitochondria (Balk and Leaver, 2001). However, the Ogura sterility-inducing protein forms a large complex without interfering with the oxidative phosphorylation components in rapeseed mitochondria (Duroc et al., 2009). Most CMS associated proteins – including URF13 in maize CMS-T, ORF138 in radish CMS-Ogura, ORF79 CMS-BT and ORFH79 in rice CMS-HL, and preSATP6 in sugar beet CMS-Owen – encode transmembrane proteins, which can be targeted to the inner mitochondrial membrane and thereby interfere with the proton gradient, subsequently affecting ATP synthesis. 2.4. Cytotoxic effects of CMS-specific proteins For a number of CMS-specific proteins overexpression in E. coli showed cytotoxic effects: orf79 of the BT (Boro II)-type CMS cytoplasm in rice (Akagi et al., 2004; Wang et al., 2006), orfH522 of the CMS PET1 in sunflower (Nizampatnam et al., 2009), and orf288 of the hau male sterility type in B. juncea (Jing et al., 2012). Transgenic expression of CMS associated open reading frames under the control of anther specific promoters induced male sterility in Arabidopsis in the case of orf108 (CMS B. juncea) and orf456 (CMS chili pepper) (Kim et al., 2007; Kumar et al., 2012). Transformation of tobacco with orfH522 of the CMS PET1 from sunflower also induced male sterility (Nizampatnam et al., 2009) as did transformation with orf239 of CMS Sprite from common bean. Also the transformation of rice with orf79 of the Boro II CMS caused gametophytic male sterility (Wang et al., 2006). In these cases, direct evidence was obtained that the CMS associated open reading frames are indeed causing male sterility. 2.5. Role of reactive oxygen species in CMS Reactive oxygen species (ROS) are byproducts during the operation of the electron transport chain in mitochondria. Major radicals are su• peroxide anion (O− 2 ), hydroperoxide radicals (OH ), and hydrogen peroxide (H2O2). At low concentrations ROS act as signals in plant stress and development, but if produced in high concentrations ROS induce programmed cell death (Lázaro et al., 2013). ROS triggers release of cytochrome c from mitochondria to cytosol, thus initiating programmed cell death. Degeneration of tapetum tissues in anthers by PCD at various developmental stage(s) is important for the occurrence of CMS (Hu et al., 2011). Mitochondrial protein dysfunction during CMS can lead to over-reduction of ubiquinone pool, which can enhance ROS production and subsequently interferes with pollen development as shown for CMS-WA and CMS-HL in rice (Luo et al., 2013; Peng et al., 2010). Also for pepper CMS-9704A, Deng et al. (2012) assume that chronic oxidative stress caused by abnormal increase in ROS and membrane lipid peroxidation leads to the abortion of microspores. In cotton, extraordinarily high amounts of ROS and lower activities of ROS scavenger enzymes were measured at the meiosis stage of the pollen mother cell (Jiang et al., 2007). Here the restorer gene plays an important role in keeping a dynamic balance between the production and elimination

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of ROS. ROS as a signal molecule is part of a complex network of signaling pathways within and across cells that needs to be further elucidated (Mittler et al., 2011). 3. Fertility restoration by nuclear genes The male sterile phenotype observed in CMS plants results from the interaction between the mitochondrial and the nuclear genomes. Deficiencies in pollen development caused by mitochondrial CMSassociated genes are suppressed by nuclear genes called restorerof-fertility genes (Rf genes) (Gabay-Laughnan and Laughnan, 1994). Restorer genes are specific for each type of male sterile cytoplasm and can be identified by crossing CMS plants with pollen donors of a different nuclear genotype. Rf genes are introgressed in breeding lines to ensure F1 hybrid seed production (Chase, 2007; Hanson and Bentolila, 2004). Most Rf genes are dominant; however, recessive, loss-of-function fertility restorer loci have been described in maize (Wen et al., 2003) and rice (Fujii and Toriyama, 2009) and a case of overdominance has been recently reported in radish (Wang et al., 2013c). Restoration systems are classified as being either sporophytic or gametophytic. Sporophytic restorers act prior to meiosis or in sporophytic tissues and gametophytic restorers act after meiosis in microspores or pollen grains. A diploid plant of genotype CMS Rf/rf will produce two classes of pollen grains: Rf and rf. In the case of a sporophytic restorer, both classes of gametes will be functional. By contrast, for a gametophytic restorer, only those gametes that carry the restorer will be functional (Schnable and Wise, 1998). Rf genes affect the expression of CMS associated genes at RNA and/or protein levels. Although several Rf genes have been cloned, the molecular mechanisms involved in restoration remain elusive. Most Rf genes cloned so far encode pentatricopeptide repeat (PPR) proteins, a highly expanded gene family in terrestrial plants (Barr and Fishman, 2010; Bentolila et al., 2002; Brown et al., 2003; Desloire et al., 2003; Hu et al., 2012; Jordan et al., 2010; Kazama and Toriyama, 2003; Klein et al., 2005; Koizuka et al., 2003; Komori et al., 2004; Wang et al., 2006; Wang et al., 2013b; Xu et al., 2009). PPR proteins, characterized by arrays of a degenerate 35 amino acid repeat – the PPR motif – repeated in tandem 2–26 times, are gene-specific RNA-binding proteins involved in all aspects of RNA processing that are found in plant organelles, such as RNA cleavage, splicing, stabilization, degradation, editing as well as in transcription and translation (Dahan and Mireau, 2013; Nakamura et al., 2012; Schmitz-Linneweber and Small, 2008). Restorer genes encoding PPR proteins might have evolved through the duplication of PPR-protein-encoding genes that are essential for normal mitochondrial gene expression, followed by functional divergence of the duplicate genes such that the product of one gene acts on transcripts of a CMS-determining locus (Chase, 2007). Rf-like PPR genes show a number of characteristic features compared with other PPR genes, including chromosomal clustering and unique patterns of evolution, notably high rates of nonsynonymous to synonymous substitutions, suggesting diversifying selection. Selection patterns on Rf-like genes reveal a molecular “arms-race” between the nuclear and mitochondrial genomes that has persisted throughout most of the evolutionary history of angiosperms (Fujii et al., 2011). However, Rf genes encoding non-PPR proteins have also been identified thus offering new insights in the mechanisms leading to fertility restoration (Cui et al., 1996; Fujii and Toriyama, 2009; Itabashi et al., 2011; Matsuhira et al., 2012). In this review restorer genes will be classified according to their products. 3.1. Restorers-of fertility encoding PPR proteins The first Rf gene encoding a PPR protein was cloned in petunia by positional cloning. The Rf locus is composed of duplicated genes containing a pentatricopeptide repeat (PPR) motif. One of the two PPR genes found in the Rf locus, denoted Rf-PPR592, encodes a 592-aa protein containing

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14 PPRs. This protein is targeted to mitochondria and is able to restore fertility when transferred to rf rf CMS plants via the reduction of the CMS-associated protein PCF. Isolation of a recessive and nonfunctional homolog rf-PPR592 from a CMS plant indicates a deletion in the promoter area as the likely cause of its inability to restore fertility (Bentolila et al., 2002). The observed association of Rf-PPR592 with the pcf mRNA 5′-UTR supports a potential role in transcript 5′-end processing or in preventing proper translation of pcf mRNA (Gillman et al., 2007). The next Rf gene to be cloned was Rfo which restores fertility in Ogura cytoplasm of radish. The Rfo locus contains three genes organized in tandem, PPR-A, PPR-B, and PPR-C, out of which only PPR-B carries the restoration activity. Rfo was identical to Rfk1, which restores fertility of radish Kosena cytoplasm (Brown et al., 2003; Desloire et al., 2003; Koizuka et al., 2003). Using transgenic rapeseed plants expressing PPR-A and PPR-B, Uyttewaal et al. (2008) determined that the primary role of PPR-B is to inhibit ORF138 synthesis in the tapetum of young anthers. As it was demonstrated that PPR-B associated in vivo with orf138 mRNA but it had no effect on its accumulation, a role for PPR-B in the translational regulation of orf138 mRNA was postulated. Recently, RsRf3, a new restorer gene tightly linked to Rfo and encoding a PPR protein, has been reported in radish. In this case, the heterozygous RsRf3-1/ RsRf3-2 alleles are responsible for restoring fertility (Wang et al., 2013c). In BT-CMS rice it was shown that the classical Rf-1 locus consists of two closely linked Rf genes, Rf1a and Rf1b as members of a multigene cluster that encode PPR proteins. RF1A and RF1B block ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. Besides, independent of its cleavage of B-atp6/orf79, RF1A enhances atp6 mRNA editing levels of both N-atp6 and B-atp6 RNA molecules. This role appears to be redundant for the biological function of ATP6 and is not necessary for the fertility restoration (Akagi et al., 2004; Kazama and Toriyama, 2003; Komori et al., 2004; Wang et al., 2006). Fertility restoration in HL CMS rice involves an endonucleolytic cleavage upstream of orfH79. The restorer gene, Rf5, encodes a PPR protein identical to RF1A which was unable to bind to the CMS associated transcript atp6–orfH79 in vitro; however, a partner protein of RF5 (GRP162, a Gly-rich protein encoding 162 amino acids) was found to physically interact with RF5 and to bind to the CMS associated transcript via an RNA recognition motif. RF5 and GRP162 are both components of a restoration of fertility complex (RFC) that is 400 to 500 kD in size and can cleave CMS-associated transcripts in vitro. Results suggest that the critical function of RF5 is to assemble the RFC in mitochondria, wherein Rf5 and GRP coordinate the processing of transcripts. Direct interaction of a PPR protein with a Gly-rich protein to form a subunit of the RFC provides a new perspective on the molecular mechanisms underlying fertility restoration (Hu et al., 2012). In maize CMS-S cytoplasm, Rf3 restores fertility completely by processing all transcripts from orf355 and orf77 (Zabala et al., 1997). Xu et al. (2009) followed an in silico cloning approach and found that one of the three PPR genes (PPR-814a, PPR-814b, PPR-814c) could be considered as the candidate restorer gene for CMS-S. Sorghum cytoplasm A1 is restored by Rf1 and Rf2. Klein et al. (2005) by means of a positional cloning approach in conjunction with microcolinear analysis of sorghum and rice, postulated PPR13 as the potential candidate gene for Rf1. PPR13 was predicted to encode a mitochondrial-targeted protein containing a single exon with 14 PPR repeats. Jordan et al. (2010) used a fine-mapping population and postulated a PPR gene showing high homology with rice Rf1 as a candidate for Rf2 although additional fine mapping along with further experimental observations are necessary to confirm its identity. A new restorer gene, Rf5, which restores fertility in both A1 and A2 cytoplasms, has been mapped and a cluster of PPR proteins highly homolog to rice Rf1 and sorghum Rf2 was postulated as candidates for conditioning fertility restoration. The tight clustering of these genes makes conventional fine

mapping approaches inadequate to provide sufficient resolution. As a result, functional analyses will likely be required to determine the causal gene or genes (Jordan et al., 2011). Barr and Fishman (2010) fine mapped the nuclear restorer of cytoplasm-dependent anther sterility in Mimulus hybrids by identifying and targeting regions of the genome containing large numbers of candidate pentatricopeptide repeat genes (PPRs). Two tightly linked loci (Rf1 and Rf2) were identified, each of them spanning a physical region containing numerous PPRs with high homology to each other. Furthermore, these PPRs have higher homology to restorers in distantly related taxa than to PPRs elsewhere in the Mimulus genome, suggesting that the cytoplasmic male sterility (CMS)–PPR interaction is highly conserved across flowering plants (Barr and Fishman, 2010). 3.2. Restorers-of-fertility encoding non-PPR proteins The first cloned Rf gene was Rf2a, which restores fertility in maize CMS-T cytoplasm. Map-based cloning of Rf2a showed that it encodes an aldehyde dehydrogenase and acts by detoxifying toxic substances in the tapetum (Cui et al., 1996; Liu et al., 2001). This restoration mechanism does not affect the accumulation of toxic URF 13, but compensates for the metabolic dysfunctions caused by the CMS-associated gene product (Cui et al., 1996; Liu et al., 2001). The nuclear candidate gene for Rf17 in the CW-CMS/Rf17 rice system is RETROGRADE-REGULATED MALE STERILITY (RMS), which encodes a mitochondrial protein 178 aa in length of unknown function, with a segment partially similar to acyl-carrier protein synthase (ACPS). Gene silencing of RMS restored fertility to a CMS plant, whereas its overexpression in the fertility restorer line induced pollen abortion. The mRNA expression level of RMS in mature anthers depended on cytoplasmic genotype, suggesting that RMS is regulated via retrograde signaling. Unlike other PPR-encoding Rf genes which are involved in post-transcriptional RNA modification of the mitochondrial CMS-associated gene expression, loss-of-function of the RMS gene may provide a bypass to fertility restoration (Fujii and Toriyama, 2009). LD-CMS in rice is restored by Rf2, which encodes a protein consisting of 152 amino acids with a glycine-rich domain (GRP). Though typical GRPs frequently contain an RNA recognition motif (RRM), Rf2 does not possess such an RNA-binding motif or any other enzymatic domains. It has been postulated that the glycine-rich region of RF2 can directly interact with the CMS-causing protein and contribute to fertility restoration by itself. Alternatively, RF2 may interact with other proteins, possibly via the glycine-rich region, to form a multi-molecular complex, and then participate in fertility restoration (Itabashi et al., 2011). In sugar beet, the restorer gene Rf1 of Owen CMS has been recently cloned and its product resembled OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. This finding suggests that fertility restoration in sugar beet CMS involves a novel mechanism. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf1, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product (Matsuhira et al., 2012). 3.3. Restorers of fertility encoding unknown proteins In several CMS/Rf systems the effect of the restorer genes on the expression of the mitochondrial CMS-associated genes is known but the identity of the Rf genes has not been determined yet. Restorer genes affecting mitochondrial transcript processing have been described in maize (Dewey et al., 1987; Wise et al., 1999a, 1999b), sorghum (Tang et al., 1996), oil seed rape (Menassa et al., 1999), B. juncea (Ashutosh et al., 2008) and pepper (Kim and Kim, 2006). Instead, Rf1 restores fertility in PET1 sunflower by affecting transcript stability of PET1 chimeric transcripts post-transcriptionally via the regulation of their degradation

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(Gagliardi and Leaver, 1999; Monéger et al., 1994). Alternatively, Rf9 and Fr restore fertility in maize and bean respectively, by affecting mitochondrial DNA organization to reduce the amount of CMS-associated sequences (Gabay-Laughnan et al., 2009; Janska and Mackenzie, 1993; Janska et al., 1998). Present knowledge reveals a diversity of mechanisms involved in fertility restoration of cytoplasmic male sterility. Rf genes may affect the regulation of mitochondrial gene expression at DNA, RNA and protein levels or play a role in metabolic compensation. In this context it is conceivable that not a single but many gene families participate in the restoration process. PPR proteins have been claimed to be the main gene products related to fertility restoration, but as evidence accumulates their role needs to be reexamined as being a part of restoration complexes in which several partners act together. 4. Conclusions In most cases, multiple recombination events involving known mitochondrial genes as well as sequences of unknown origin create new open reading frame associated with cytoplasmic male sterility in higher plants (Horn, 2006). The deleterious effects of CMS proteins often causing ATP deficiencies might signal problems for the survival of the plants that might trigger a type of survival mode, in which the plant will primarily concentrate on the female part and the production of seeds. The hypothesis that disturbances of the respiratory chain can lead to male sterility was verified by Shaya et al. (2012) through transgenic expression of truncated mitochondrial genes (atp4, cox1 and rps3) within tapetum cells, which indeed induced male sterility. The CMS-specific proteins encoded by chimeric orfs often contain transmembrane domains and frequently include parts of mitochondrial genes that would allow interaction with the complexes of the respiratory chain or the F0F1–ATP synthase impairing the respiratory chain without leading to lethal effects in most parts of the plant. In addition, a large number of CMS associated orfs contain sequences of unknown origins whose role in creating the CMS phenotype has not been elucidated. In a few cases, the molecular mechanisms behind these interactions are getting unraveled at the moment. Wang et al. (2013a) could demonstrate for CMS-HL in rice that the interference of ORF79 with P61 leads to a decrease in enzyme activity of complex III, which further reduces the ATP production and results in an increase in ROS. There are more and more indications that mitochondria might be involved in triggering death of male reproductive organs (Chase, 2007), not only by the level of ATP (Li et al., 2013), but also via ROS production (Deng et al., 2012; Luo et al., 2013). ROS are more produced under conditions of mitochondrial dysfunction due to accumulation of electrons which further leads to overreduction of the ubiquinone pool (Chase, 2007). Future research has further to reveal the molecular mechanisms behind cytoplasmic male sterility and the interactions of proteins and processes leading to termination of the male gametogenesis, which are still far from being fully understood. References Akagi, H., Nakamura, A., Yokozeki-Misono, Y., Inagaki, A., Takahashi, H., Mori, K., Fujimura, T., 2004. Positional cloning of the rice Rf-1 gene, a restorer of BT-type cytoplasmic male sterility that encodes a mitochondria-targeting PPR protein. Theor. Appl. Genet. 108, 1449–1457. Ashutosh, Kumar, P., Dinesh Kumar, V., Sharma, P.C., Prakash, S., Bhat, S.R., 2008. A novel orf108 co-transcribed with the atpA gene is associated with cytoplasmic male sterility in Brassica juncea carrying Moricandia arvensis cytoplasm. Plant Cell Physiol. 49 (2), 284–289. Bailey-Serres, J., Hanson, D.K., Fox, T.D., Leaver, C.J., 1986. Mitochondrial genome rearrangement leads to extension and relocation of cytochrome c oxidase subunit I gene in Sorghum. Cell 47, 567–576. Balk, J., Leaver, C.J., 2001. The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release. Plant Cell 13, 1803–1818.

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