Mitochondrial Genome Organization and Cytoplasmic Male Sterility in Plants

J. Biosci., Vol. 18, Number 3, September 1993, pp 407–422. © Printed in India.

Mitochondrial genome organization and cytoplasmic male sterility in plants

C Κ Κ NAIR Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India

MS received 28 January 1993; revised 12 May 1993

Abstract. Plant mitochondrial genomes are much larger and more complex than those of other eukaryotic organisms. They contain a very active recombination system and have a multipartite genome organization with a master circle resolving into two or more subgenomic circles by recombination through repeated sequences. Their protein coding capacity is very low and is comparable to that of animal and fungal systems. Several subunits of mitochondrial functional complexes, a complete set of tRNAs and 26S, 18S and 5S rRNAs are coded by the plant mitochondrial genome. The protein coding genes contain group II introns. The organelle genome contains stretches of DNA sequences homologous to chloroplast DNA. It also contains actively transcribed DNA sequences having open reading frames. Plasmid like DNA molecules are found in mitochondria of some plants Cytoplasmic male sterility in plants, characterized by failure to produce functional pollen grains, is a maternally inherited trait. This phenomenon has been found in many species of plants and is conveniently used for hybrid plant production. The genetic determinants for cytoplasmic male sterility reside in the mitochondrial genome. Some species of plants exhibit more than one type of cytoplasmic male sterility. Several nuclear genes are known to control expression of cytoplasmic male sterility. Different cytoplasmic male sterility types are distinguished by their specific nuclear genes (rfs) which restore pollen fertility. Cytoplasmic male sterility types are also characterized by mitochondrial DNA restriction fragment length polymorphism patterns, variations in mitochondrial RNAs, differences in protein synthetic profiles, differences in sensitivity to fungal toxins and insecticides, presence of plasmid DNAs or RNAs and also presence of certain unique sequences in the genome. Recently nuclear male sterility systems based on (i) over expression of agrobacterial rol C gene and (ii) anther specific expression of an RNase gene have been developed in tobacco and Brassica by genetic engineering methods.

Keywords. Plant mitochondria; genome organization; cytoplasmic male sterility; mitochondrial DNA; mitochondrial genes; gene rearrangement.

1. Introduction

The subcellular organelle mitochondrion is the power house in the eukaryotic cell. It synthesizes ATP from stored energy in the form of fats, carbohydrates and proteins. Though the importance of mitochondrion in eukaryotic cell metabolism was recognized very early, it was only in 1960s the existence of a unique genetic system in this subcellular organelle was discovered. Since then, our understanding of the structure, information content and expression of mitochondrial genome has enormously expanded, especially in mammalian and amphibian systems where the entire mitochondrial genomes have been sequenced. Studies in plant mitochondrial genomes, though started late, have made rapid progress in recent years. The recent progress in plant mitochondrial genome analysis stems from the need to define and increase cytoplasmic genetic diversity in crops of commercial importance and to

407 408 C Κ Κ Nair understand the molecular basis of cytoplasmic mutations such as cytoplasmic male sterility, susceptibility to systemic insecticides and sensitivity to fungal toxins Lonsdale 1984, 1987 a; Leaver and Gray 1982; Laughman and Gabay-Laughman 1983; Douce 1985; Newton 1988; Levings and Brown 1989).

1.1 Size and organization

1.1a Size: Plant mitochondrial genomes are much larger and more complex than those of other aerobic organisms so far known. They vary in size from 200 kb in Brassica species to 2500 kb in musk melon (Ward et al 1981; Lonsdale 1984). The mitochondrial genomes of diptera, mammals, amphibian and fish range from 15 kb to 19 kb, while those of protists and fungi range from 15 kb to 108 kb (Bruijn 1983; Grivell 1983; Brown et al 1985; Braun et al 1992). Table 1 presents the sizes of some of the plant mitochondrial genomes.

Table 1. Sizes of plant mitochondrial genomes.

1.1b Organization: Restriction endonuclease analysis of mitochondrial DNA from several plant species showed the presence of restriction fragments in submolar and supramolar amounts. This could be attributed to coincidental migration of restriction fragments and existence of an unusual genome organization. Electronmicroscopic studies on plant mitochondrial DNA isolates revealed occurrence of small linear and circular DNAs at high frequency and large circular molecules at a low frequency. These observations together suggested a multipartite genome organization in plant mitochondria with a master circle resolving into two or more subgenomic circles by recombination through directly repeated sequences (Leaver and Gray 1982; Laughman and Gabay-Laughman 1983; Lonsdale 1984 1987 a, b; Newton 1988; Levings and Brown 1989). Studies on mitochondrial DNA from Brassica campestris indicated that its genome is organized as a tripartite structure (Palmer and Sheilds 1984). A master circle of 218 kb recombines through a directly repeated 2 kb sequence forming two subgenomic circles of 135 kb and 83 kb each. In spinach mitochondria the repeat sequence is of 6 kb and the master circle is of 327 kb which gives rise to subgenomic circles of 93 kb and 134 kb (Stern and Palmer 1986). In maize and wheat mitochondria the genomes are more Mitochondrial genome organization and CMS in plants 409 complex. The wheat mitochondrial genome is 430 kb in size and contains atleast 10 repeat sequences (Lonsdale et al 1984). In maize the master circle is of 570 kb and contains 6 repeat sequences —five direct and one indirect —and gives rise to multiple subgenomic circles (Quetier et al 1985).

1.1c Genome rearrangements: Recombination involving direct repeats is also implicated in the rapid and extensive rearrangements that characterize the evolution of plant mitochondrial DNA (Small et al 1989; Fauron et al 1990). Though plant mitochondrial DNAs show exceptionally low rate of divergence at the primary sequence level. As a consequence of this evolutionary pattern, highly conserved coding sequences are often flanked by completely different sequences in mitochondrial DNA of different plant species (Palmer 1990). This results from rearrangements occurring close to the boundaries of coding regions. Mitochondrial genomes from closely related species often vary in linear gene order even though they have highly conserved primary sequences (Palmer 1990).

1.1d Plasmid like molecules: In addition to high molecular weight mitochondrial DNA, plasmid like molecules are present in mitochondria of plants. The presence of linear and circular DNA plasmids, and also double stranded RNAs have been reported in many higher plant mitochondria (Pring et al 1977; Palmer et al 1983; Schardl et al 1984; Sisco et al 1984; Douce 1985; Finnegan and Brown 1986; Newton 1988; Levings and Brown 1989; Canal et al 1991). Occurrence in higher frequency of certain plasmids have been reported in mitochondria of some cytoplasmic male sterile plants, although their relation to male sterile phenotype is not yet clear. Mitochondria of male sterile — cms S maize contain 6·4 kb and 5·4 kb plasmids while male sterile strain of Brassica species carries an 11·3 kb plasmid (Pring et al 1977; Palmer et al 1983). Though variations in mitochondrial plasmid profiles have been reported in normal and cytoplasmic male sterility (CMS) lines of sunflower, no correlation has been found between the presence of plasmids and CMS (Crouzillat et al 1987, 1989; Canal et al 1991).

1.1e Promiscuous DNA: Plant mitochondrial genome contains stretches of DNA sequences which are highly homologous (> 90%) to chloroplast DNA. These sequences include coding, noncoding, as well as non-transcribed regions of chloroplast genomes (Lonsdale 1985, 1987a,b; Newton 1988). It has been shown that the maize mitochondrial gene carries a 12 kb chloroplast DNA containing chloroplast 16S rRNA gene and two chloroplast tRNA genes (Stern and Lonsdale 1982; Stern and Palmer 1984, 1986; Wintz et al 1988). Also, a region homologuous to the large subunit gene of RUBISCO (rbcL) has been identified in mitochondrial DNA. Chloroplast related tRNAcys gene is transcribed in the mitochondria and the product has been identified (Wintz et al 1988). In mung bean, spinach, and pea also chloroplast DNA sequences are seen in mitochondrial DNA. The presence of these chloroplast DNA sequences in mitochondrial genome seems to be random. It is inferred that the transfer of these sequences from chloroplasts to mitochondria must have occurred via DNA transfer in recent past. In addition to the chloroplast specific sequences, DNA sequence homologies have also been detected between nuclear DNA and mitochondrial DNA (Kemble et al 1983 a, b: Timmis and Scott 1983: Scott and Timmis 1984). A few

410 C Κ Κ Nair homologous sequences have been reported to be present in all the three genomes (Whisson and Scott 1985).

1.2 Genes and genetic capacity

The coding capacity of plant mitochondrial genomes appears to be exceptionally low in relation to their large size. Studies on in vitro protein synthesis using isolated mitochondria indicated that the number of polypeptides synthesized are comparable to that made by animal and fungal mitochondria (Hack and Leaver 1983). DNA cross hybridization studies demonstrated a core of conserved sequences which could represent the functional genes (Kemble et al 1983 a, b; Lonsdale 1987 a, b; Newton 1988).

1.2a Protein encoding genes: Using heterologous probes several genes have been identified in plant mitochondrial genome. Table 2 gives list of various mitochondrial genes identified.

Table 2. Mitochondrial genes.

1.2b Ribosomal RNA and tRNA genes: Apart from the protein coding genes the plant mitochondrial genome contains genes for 26S and 18S ribosomal RNAs and a complete set of tRNAs. Unlike mammalian and fungal mitochondrial genomes, plant mitochondrial genome contains a 5S rRNA gene which is always located 3' to the 18S rRNA gene (Bonen and Gray 1980; Stern et al 1982; Chao et al 1983; Boeshore et al 1985; Lonsdale 1987a, b; Newton 1988). The number of tRNA genes in plant mitochondria exceeds the 22 encoded by mammalian mitochondrial DNA. The tRNA genes are placed at several locations in the genome (Bonen and Gray 1980; Lonsdale 1987a,b). The initiator tRNA gene terminates one nucleotide 5' to the coding sequence of the 18S rRNA gene in wheat (Spencer et al 1984) while in

Mitochondrial genome organization and CMS in plants 411 maize the same genes are 70 nucleotides apart (Lonsdale 1987a,b). In soybean the initiator tRNA gene is located close to coxII gene (Grabau 1987).

1.2c Unidentified reading frames: Several actively transcribed DNA sequences having open reading frames have been identified, in plant mitochondria. The function of the products of these are not known. These genes are also found in the mitochondrial plasmids S1 and S2 occurring in cmsS cytoplasm of maize (Levings and Sederoff 1983; Paillard et al 1985).

1.2d Gene chimeras: These comprise stretches of DNA with open reading frames which consists of sequences derived from other genes (Dewey et al 1985; Rottmann et al 1987).

1.2e Nonfunctional genes: Plant mitochondrial genomes contain several nonfunctional genes which could have resulted from genomic rearrangements causing deletion of promoter region, loss of open reading frames and mutations leading to premature termination of translation (Schardl et al 1985; Houchins et al 1986).

1.2f Introns in protein coding genes: Introns having secondary structure typical of fungal group II type are found in many protein coding genes in plant mitochondria. These are summarized in table 3. None of the tRNA or rRNA genes in plant mitochondria contain introns.

Table 3. Protein genes carrying introns in plant mitochondria.

2. Cytoplasmic male sterility

Male sterile plants fail to produce functional pollen grains. Male sterility can arise from alterations in nuclear or cytoplasmic genes. If the sterility trait is inherited in a non-Mendelian fashion it is called cytoplasmic male sterility (CMS). Cytoplasmic 412 C Κ Κ Nair genes are maternally transmitted. CMS is common in several plant species. It has been observed in about 140 species from 47 genera and 20 families (Hanson and Code 1985; Levings 1990; Braun et al 1992). CMS causes abortion of male gametophytes but does not affect female gametophytic development. CMS has been found to arise naturally as well as from intra- and interspecific crosses (Frank 1989; Levings 1990; Braun et al 1992). Extensive investigations on the molecular mechanisms of CMS in several plant species provided convincing evidence that the genetic determinants for this trait reside in the mitochondrial genome. However it is also certain that the nuclear genes control the expression of CMS phenotype (Newton 1988). CMS has been used conveniently in hybrid production since it eliminates the expense of hand emasculation procedures (Duvick 1965).

2.1 Origin of CMS

CMS results from either intraspecific crosses or from interspecific crosses (Hanson and Conde 1985). Back crossing the hybrid plant with the pollen parent and repeating this process for several generations, yields plants containing the cytoplasm of the original maternal parent and the nuclear genotype of the original male parent. In such plants difference in the interaction between nuclearly encoded and mitochondrially encoded gene products could lead to altered mitochondrial function. Nuclear-cytoplasmic incompatibilities could reflect nuclear gene effects on mitochondrial gene expression—transcription, processing of RNAs or translation. Alternately the enzymes of the inner mitochondrial membrane which contains subunits coded by both nuclear and mitochondrial genomes may be partially dysfunctional as a result of aberrant subunit interactions (Newton 1988).

2.2 Fertility restorer genes

A number of nuclear, restorer of fertility genes (Rf) which counteract or compensate for pollen sterility in CMS plants has been identified (Leaver and Gray 1982; Laughman and Gabay-Laughman 1983; Lonsdale 1984, 1987a, b; Newton 1988; Levings and Brown 1989; Braun et al 1992). Rf genes are useful when they are dominant since they make the Fl hybrid plant fertile. Multiple types of CMS caused by different defects in the mitochondrial genome are restored to fertility by different restorer genes. In some case a single CMS type is restored by a number of different restorer genes and it is not clear whether the various Rf genes are different alleles of the same gene (Braun et al 1992).

2.3 Variations in CMS types

Different CMS types are distinguished by the specific nuclear Rf genes which restore pollen fertility. Other characteristics which differentiates the CMS types include mitochondrial DNA restriction fragment length polymorphism, variations in mitochondrial RNAs as revealed in northern hybridization, differences in mitochondrial protein synthesis profile, differences in resistance to fungal infections, toxins and insecticides, histological variations, presence of certain unique sequences in mitochondrial DNA, and presence of DNA or RNA plasmids in mitochondria (Leaver and Gray 1982; Newton 1988; Johns et al 1992, Williams et al 1992). Mitochondrial genome organization and CMS in plants 413

2.3a Maize (Zea mays) CMS types: In maize the CMS types have been classified into three groups—cmsT, cmsC and cmsS (Laughman and Gabay-Laughman 1983; Newton 1988; Levings 1990; Braun et al 1992; Williams et al 1992).

(i) Maize cmsT: Maize plants carrying cmsT cytoplasm are susceptible to the fungal pathogen Bipolaris maydis race T. They are also sensitive to the carbamate insecticide methomyl. Fertility restoration in cmsT plants is brought about by two fertility restorer genes Rf1 and Rf2. The dominant alleles of both the genes must be provided by the nucleus for restoring fertility. The cmsT plants carry a mitochondrial gene designated as Turf13. This gene encodes a 13 kD polypeptide—Τ peptide (Forde et al 1986; Levings and Dewey 1988; Levings and Brown 1989; Levings 1990; Williams et al 1992). This polypeptide is located in the mitochondrial membrane and is shown to be responsible for conferring sensitivity to the fungal toxin and methomyl. The presence of the nuclear restorer genes Rf1 and Rf2 reduces the synthesis of the Τ peptide (Levings and Dewey 1988).

(ii) Maize cmsC: The cmsC type male sterile maize plants are restored to fertility by a single dominant nuclear gene Rf4 (Pour et al 1981). The cmsC mitochondria synthesize a unique 17·5 kD soluble peptide which appears to replace a 15·5 kD membrane bound polypeptide found in normal mitochondria (Newton 1988). Apart from this, the mitochondrial genome in cmsC has been shown to have mutations in the genes atp9, atp6 and coxII These mutations have resulted from gene rearrangements involving portions of mitochondrial genes and chloroplast DNA (Levings and Dewey 1988).

(iii) Maize cmsS: Fertility restoration in cmsS maize plants is bestowed by a dominant nuclear gene Rf3 (Laughman and Gabay-Laughman 1983; Braun et al 1992). In cmsS mitochondria synthesis of several novel but minor high molecular weight polypeptides are observed. Mitochondrial genomes in cmsS type plants are characterized by the presence of linear DNA plasmids S1 and S2 (Pring et al 1977). Plasmid S1 is 6397bp and S2 is 5453bp long (Levings and Sederoff 1983; Paillard et al 1985). The relationship between the presence of S1 and S2 plasmids and pollen sterility in cmsS is not understood. The mitochondria of plants restored to fertility by the restorer gene Rf3 retained the plasmids S1 and S2. Unlike cmsT and cmsC spontaneous reversion to fertility occurs in cmsS. This could be due to either nuclear mutations or gene rearrangements. In such revertants free S1 and S2 plasmids are not detected (Newton 1988; Braun et al 1992; Williams et al 1992). In one line of maize LBN, carrying the L subgroup of cmsS cytoplasm, two double stranded RNAs, LBN1 (2·9 kb) and LBN2 (0·84 kb) were detected in the mitochondria (Sisco et al 1984). Certain cmsS subgroups also contain single stranded RNAs in their mitochondria. The origin and possible function of the RNA plasmids are not known (Newton 1988;Braun et al 1992; Williams et al 1992).

2.3b CMS in French bean (Phaseolus vulgaris): CMS in P. vulgaris has been shown to be associated with the presence of a 3 kb unique mitochondrial sequence designated as pvs. This sequence encodes two open reading frames of 297 bp and 720 bp in length with portions derived from chloroplast genome. Fertility restoration is achieved by the nuclear restorer gene Fr (Johns et al 1992). 414 C Κ Κ Nair

2.3c CMS in Petunia: CMS in Petunia is restored by the dominant nuclear Rf gene. The mitochondrial genome of normal and CMS lines show differences in several restriction fragments (Newton 1988). One of the fragments unique for CMS has been shown to be generated through intergenic recombination at the atp9 locus (Rothenberg et al 1985). In CMS lines of Petunia a chimeric gene pcf having 354 codons formed by the fusion of 5'-noncoding region and amino-terminal region of atp9, a DNA segment within the coxII gene and an unidentified reading frame urfS has been identified (Young and Hanson 1987). The expression of pcf gene has been shown to be associated with CMS and is controlled by the restorer gene rf in Petunia (Nivison and Hanson 1989).

2.3d CMS in radish (Raphanus sativus): Ogura (1968) identified a CMS system –– ogu–in certain Japanese radish varieties. The ogu-CMS could be restored by other radish strains. Several DNA sequence rearrangements have been found in the mitochondrial genome in ogu-CMS radish (Makaroff and Palmer 1988; Makaroff et al 1991). Also differences in the transcript pattern for the mitochondrial genes atp6, atpA and coxII have been reported in case Qf ogu-CMS lines. An open reading frame containing 105 codons — orf105 — is found at the 5'-end of atp6 and is cotranscribed with atp6 (Makaroff and Palmer 1988; Makaroff et al 1989, 1990). A DNA sequence rearrangement of 120 bp at the 5'-end of the coding region of coxI gene resulting in differences in 5'-transcript terminus has been recently found in ogu- CMS line of radish (Makaroff et al 1991).

2.3e CMS in Brassica: In the genus Brassica, a number of CMS systems have been reported. Most of these originate by interspecific hybridization and result from nuclear cytoplasmic incompatibility (Braun et al 1992). The ogu cytoplasm of radish confers sterility in Brassica species. From studies on CMS revertants of Brassica napus/ogura cybrids Bonhomme et al (1991) observed that a 2·5 kb NcoI fragment of ogura-radish mitochondrial DNA is associated with ogu CMS in B. napus. However, this 2·5 kb NcoI fragment is not linked to apt6 or atpA genes (Williams and Levings 1992), Apart from ogu CMS a number of other CMS types have been characterized in Brassica. These include, mur CMS, nap CMS, pol CMS, nig CMS and ana CMS (Braun et al 1992). The mitochondrial genomic alterations associated with these CMS are not well understood. CMS induced by nap cytoplasm is observed only in a few B. napus nuclear genotypes and is found to be unstable under higher temperatures of growth (Fan and Stefansson 1986). Complete male sterility has been conferred in many Brassica species by the "Polima" or pol cytoplasm. Also, the pol cytoplasm induced male sterility is temperature stable. Mitochondrial DNA of pol CMS lines could be distinguished by restriction analysis (Erickson et al 1986; Witt et al 1991). In polCMS lines DNA rearrangements at upstream of atp6 generates a chimeric 224 codon open reading frame— orf224. Cotranscription of orf224 with atp6 gene giving a dicistronic transcript has been reported in pol CMS plants; nuclear restorer genes specifically alter the transcript pattern resulting predominantly in atp6 monocistronic transcript (Singh and Brown 1991). A number of restorer lines are available for pol CMS and pol cytoplasm is thought to be the most promising for hybrid rape seed production (Braun et al 1992). Mitochondrial genome organization and CMS in plants 415

2.3f CMS in rice (Oryza sativa): Male sterility in rice cmsBo has been shown to be associated with an additional chimeric gene urf-rmc which consists of 5'-flanking noncoding region of atp6 and an uncharacterized mitochondrial sequence (Kadowaki et al 1990). The transcript of this gene could code for 181 amino acids of atp 6 gene product and 9 amino acids of the uncharacterized region.

2.3g CMS in wheat (Triticum aestivum): In CMS wheat at the 5'-flanking region of coxI gene, presence of a chimeric open reading frame of 256 amino acids — orf256—has been reported (Rathburn and Hedgcoth 1991). The N-terminal amino acids coded by orf256 and coxI gene are similar. The sequence of the coxI coding region differs in normal and CMS wheat only with respect to the termination codon — TAA in normal and TAG in CMS line. A CMS unique 3'- flanking sequence is reported to begin at the G of the coxI termination codon. Rathburn and Hedgcoth (1992) studied restriction fragment length polymorphism in the mitochondrial genomes from fertile and CMS lines of wheat with probes made by labelling cloned sequences from maize episomes S 1 and S2. The restriction enzyme analysis did not show any detectable difference between fertile and CMS mitochondrial DNA around the S1-sequence. The S2-sequence has homology at two locations in the wheat mitochondrial genome and mitochondrial DNA fertile and CMS lines showed restriction fragment length polymorphism at one of the sites (Rathburn and Hedgcoth 1992).

2.3h CMS in sorghum (Sorghum bicolor): Alteration in the coxI gene has been reported in a CMS line of sorghum — CMS9E. The coding region of coxI gene in CMS9E is extended at the 3'-end by 303 nucleotides, resulting in an extension of 101 amino acids at the C-terminal end of the protein product (Bailey-Serres et al 1986a, b). Recently Chen et al (1993) have reported a novel chloroplast-DNA deletion in most of the CMS lines of sorghum. This deletion occurred in the middle of the gene rpoC2, coding for the ß"-subunit of RNA polymerase. How this deletion results in CMS is not understood.

2.3i CMS in broad bean (Vicia faba): Unlike in other plants CMS in V. faba exhibits a unique correlation with the presence of spherical membrane bound particles of 70 nm diameter (Edwardson et al 1976). These particles are found to contain a 16·7 kb double stranded RNA along with a self coded replicase which is an RNA-dependent RNA polymerase (Lefebvre et al 1990). However, these particles are not located in the mitochondrion and their origin is not known. Fertility restoration in this CMS line is associated with loss of these cytoplasmic particles (Lefebvre et al 1990).

2.3j CMS in sugar beet (Beta vulgaris): In sugar beet CMS is associated with different forms of mitochondrial DNA and variation in the number and type of small supercoiled circular DNA plasmids. In addition to this two mitochondrially translated polypeptides of molecular weight 21 kDa and 32 kDa are absent in CMS lines of sugar beet (Mikami et al 1986). Restriction endonuclease digestion analysis of mitochondrial DNAs from CMS and normal fertile lines of sugar beet showed conspicuous differences in a number of restriction fragments, reflecting divergence 416 C Κ Κ Nair between the two genomes. Mitochondrial genes coxII and atpA from CMS and normal lines show distinct variations in their transcriptional patterns (Senda et al 1991).

2.3k CMS in sunflower (Helianthus annuus): A novel 16 kDa polypeptide has been identified in male sterile cytoplasm of sunflower (Horn et al 1991). Studies on mitochondrial genome analysis revealed that CMS lines contain a 5 kb fragment near the 5'-end of atpA locus and a 12 kb inversion adjacent to this (Sicullela and Palmer 1988). The transcript of the atpA gene was reported to be different in the CMS variety. The presence of an open reading frame orf-522 coding for a 19·5 kDa peptide has also been reported in this. The orf522 has been found to be cotranscribed with atpA and has partial sequence homology with the 5'-end of orfB of Oenothera (Kohler et al 1991).

2.4 Mechanism of CMS

Light and electron microscopic studies have revealed important differences between fertile and sterile anthers where the male reproductive process occurs. This organ is composed of several tissues and cell types. The innermost cell layer is a specialized tissue called the tapetum. The tapetum surrounds the pollen sac early in anther development, degenerates during later stages of development and is not present as an organized tissue in mature anther. The tapetal cells serve to nourish developing pollen by exporting nutrients and other molecules needed for pollen formation. Mitochondria in the tapetum and the adjacent cell layer of sterile anthers start to degenerate soon after meosis while in fertile anthers they remain intact. Mitochondrial degeneration in the tapetum of sterile anthers is the first sign of abnormality and it initiates events leading to pollen abortion. Pollen development requires higher levels of mitochondrial activity; even slight amount of mitochondrial dysfunction could result in pollen abortion. A 20 to 40- fold increase in mitochondrial number occurs during microsporogenesis in tapetal cell layer of the another. This indicates an increased requirement for mitochondria in pollen formation. Under these conditions a mitochondrial gene mutation or rearrangement may interrupt mitochondrial biogenesis in anther cells and account for CMS while in other less demanding developmental processes this mutation may not be limiting. Plant mitochondrial genome encodes polypeptides which are components of electron transport chain or F0-F1 ATPase. These are essential for respiration. Mutant forms of these peptides can cause respiration deficiencies. In humans it has been shown that mitochondrial gene mutations elicit harmful effects only in specific tissues (Wallace et al 1988). Similarly mitochondrial gene mutation or rearrangements in CMS varieties may express its adverse effects only in the tissue involved in pollen development where as other tissues remain unaffected. Mitochondrial activity could also be affected by the presence of an anther specific substance similar to the toxin of B. maydis race T. This anther specific substance could interact with polypeptides like turf13 to inhibit mitochondrial activity, similar to the toxin-turf13 interaction (Flavel 1974; Levings 1990). Mitochondrial genome organization and CMS in plants 417

3. Male sterile plants by genetic engineering

Recently it has been demonstrated in tobacco and rape (B. napus) that male sterile plants can be made by introducing chimeric ribonuclease gene. In these transgenic plants the chimeric RNase gene is expressed within the anther. Selective destruction of the tapetal cell layer, preventing pollen formation resulted in the male sterility (Mariani et al 1990). Restoration of fertility in these genetically engineered rape plants was brought about by introducing a chimeric RNase inhibitor gene that expressed in the anther tapetal cell layer. This was achieved by crossing the male sterile plants with transgenic male fertile plants carrying chimeric tapetal cell specific RNase inhibitor gene. Fl progeny expressed genes, RNase and RNase inhibitor, and were found to be restored to pollen fertility by suppression of cytotoxic RNase activity in the anther by formation of cell specific RNase-RNase inhibitor complexes (Mariani et al 1992). Transgenic tobacco (Nicotiana tabaccum) plants, constructed by genetic engineering methods expressing the rolC gene of the T-DNA of Agrobacterium rhizogenes (plant pathogen causing hairy root disease) are shown to be male sterile (Schmulling et al 1988). Over expression of the rolC gene has also been shown to cause male sterility in other transgenic plants (Fladung 1990). The male sterility induced by rolC gene is of nuclear type and shows dominant mendelian inheritance. Recently restoration of fertility in rolC induced male sterility in transgenic tobacco plants by introduction of rolC-antisense genes has been demonstrated (Schmulling et al 1993).

4. Concluding remarks

The large size and complex organization confer a bewildering uniqueness to the mitochondrial genome of higher plants in the eukaryotic world. The protein coding capacity of this large organelle genome is comparatively low and the number of polypeptides coded are almost similar to that encoded by animal and fungal mitochondria of smaller size. It is not yet thoroughly established as to, (i) what function the extra load of DNA sequences in the mitochondrial genome render to the plant cell? and (ii) are these extra DNA sequences just remnants of genome evolution in the organelle or have arisen by sequence duplication and recombination? The plant mitochondrial genome offers an interesting system for basic studies in genetic recombination. The genome rearranges by recombination between repeated sequences but every repeated sequence does not participate in recombination. The mechanism of regulation of the recombinational process and the enzymes and protein factors participating in this process are not well understood. It is intriguing how the multipartite genome of the plant mitochondria is replicated and the functional genes equally partitioned in the daughter mitochondria. Clarification on the following aspects can throw more light towards the understanding of the phenomena: (i) Do all the subgenomic circles function as independent replicons or only the master genome replicates and gets distributed into the daughter mitochondria? (ii) Does the intra-genic recombination leading to multipartite genome organization occur after DNA replication and partitioning into the daughter mitochondria? 418 C Κ Κ Nair

An enormous fund of information exists to implicate mitochondrial gene mutations arising from recombinational events leading to CMS in higher plants. In almost all the plant species studied so far, except in some isolated instances such as V. faba, CMS has been considered to be the result of extensive intra- and inter- molecular recombination events involving repeated DNA elements in the mitochondrial genome. In different plant species the CMS phenotype manifests in a variety of ways due to alterations in the functioning of many different mitochondrial genes. The molecular events that restrict the phenotypic conse- quences of the defects in pollen development in CMS lines remain to be elucidated. Although several nuclear restorer genes are characterized and shown to have control of the expression of the CMS phenotype little information is available on the mechanism of restoration of fertility in CMS lines and control of nuclear- mitochondrial incompatibility by nuclear restorer genes and the regulation of mitochondrial biogenesis and function by nuclear genes at different developmental stages. Some of the questions that can be addressed could include: (i) Do the restorer genes play a role in transcription selectivity in the organelle gene regulation? (ii) Do they play any role either by themselves or in cooperation with mitochondrially coded maturases and other factors, in post-transcriptional selectivity such as RNA processing or RNA editing in the organelle? Future research in CMS and nuclear restorer genes would certainly expand our knowledge on the mechanisms underlying this agronomically important phenomenon as well as interorganellar communication.

Acknowledgements

The author thanks Dr Ν Κ Notani, former Director, Biomedical Group, for critical appraisal of the manuscript and valuable suggestions and Dr Β Β Singh, for his encouragement and support.

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