Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.     

Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco

The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.     

The expression of alternative oxidase and alternative respiratory capacity in cytoplasmic male sterile common bean

Summary The alternative respiratory pathway is present in all plant species investigated to date. Yet, the role of the alternative pathway is not clear. Some evidence suggests an important role in pollen development. We undertook this study to investigate the expression of alternative oxidase, in comparison with expression of a component of cytochrome oxidase, during pollen formation in common bean (Phaseolus vulgaris L.). Expression was evaluated immunohistochemically. In addition, we compared both the alternative oxidase capacity in young seedling tissues and alternative oxidase expression in developing flower buds of isonuclear cytoplasmic male sterile and male fertile bean lines. We observed no evidence of an association between the abnormal pollen development of CMS bean and changes in alternative oxidase expression or capacity. We did observe a tissueand stage-specific pattern of expression of alternative oxidase, differing from the expression pattern of cytochrome oxidase subunit II, during anther development in normal bean lines. Although no association was evident between the cytoplasmic male sterility phenotype and differential expression of alternative oxidase, the regulated pattern of alternative oxidase expression in developing anthers does suggest that the alternative pathway may play a role in microgametogenesis and microsporogenesis.     

Ultrastructural aspects of cytoplasmic male sterility inPetunia hybrida

Summary Anther development of isogenic male fertile and cytoplasmic male sterile types ofPetunia hybrida cv. Blue Bedder is studied by electron microscopy. First deviation in sporogenesis of the sterile type, is observed during leptotene stage of the meiocytes. Initial aberration is represented by the presence of large vacuoles in the cytoplasm of the tapetal cells. These vacuoles reveal the first aspects of degeneration; no other ultrastructural differences are observed. Vacuolation is accompanied by the condensation of cytoplasmic organelles. The tapetal cells become distorted and ultrastructural aberrations in mitochondria do occur. The mitochondria elongate and contain several tubular cristae.Substantial evidence suggests, that cytoplasmic male sterility in petunia is encoded by the mitochondrial genome (Boeshore el al. 1983). However, before degeneration becomes manifest, no consistent ultrastructural differences in mitochondrial organization are observed.Abortion of the tapetum and the sporogenous tissue in cytoplasmic male sterile plants, generally follows a corresponding pattern. Ultimately, the cells are highly distorted, the nucleus is disrupted and the cytoplasm disorganized. Mitochondria and plastids degenerate and many lipid droplets are present.     

RNA sequence and the nature of the CuA-binding site in cytochrome c oxidase.

For cytochrome c oxidase subunit II (COXII), DNA and protein sequences suggest that Met-207 (bovine numbering) is conserved in all species except plants. Sequencing of plant mitochondrial COXII mRNAs now indicates that Met-207 is also conserved among plants as a result of a C-to-U type of RNA editing. Considering the strict evolutionary conservation of Met-207 and the homology of COXII to type I (blue) copper proteins and nitrous oxide reductase, we propose a model in which Met-207 is associated with the CuA-binding site (along with Cys-196, Cys-200 and His-204) and plays a role in determining its reduction potential and stability.     

Signal transduction pathways of plant mitochondria: Retrograde regulation

Characteristic features and functional role of mitochondrial retrograde regulation (MRR) are considered. It is emphasized that MRR is manifested at mitochondria dysfunctions induced by mutations, chemical agents, or stresses and poorly studied so far. The data concerning gene expression of alternative oxidase involved in the restoration of mitochondrial functions are presented. The phenomenon of cytoplasmic male sterility controlled by MRR and also MRR involvement in plant cell responses to biotic and abiotic stresses (pathogen attack; oxygen, heat, and oxidative stresses) are described. Identified and putative components of MRR signaling pathways are discussed. Unique properties of the plant mitochondrial genome are described.     

A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants

In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129 ) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST–ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.     

Cytoplasmic male sterility in plants: molecular evidence and the nucleocytoplasmic conflict

A much-debated issue in plant evolutionary biology concerns the maintenance of a high frequency of male sterility in natural populations. For the past decade, a theoretical framework has been provided by the concept of nucleocytoplasmic conflict. Recent molecular studies on cytoplasmic male sterility indicate that novel chimeric genes, resulting from duplications and rearrangements of mitochondrial DNA sequences, are involved In its control. Thus, male sterility, which is phenotypically the loss of the male function, is encoded by a new mitochondrial function at the molecular level. Molecular data are in agreement with theoretical models that consider cytoplasmic male sterility as a stage in the coevolution between nucleus and mitochondria, and not simply as a deleterious mitochondrial mutation.     

Programmed cell death and hybrid incompatibility

We propose a new theory to explain developmental aberrations in plant hybrids. In our theory, hybrid incompatibilities arise from imbalances in the mechanisms that cause male sterility in hermaphroditic plants. Mitochondria often cause male sterility by killing the tapetal tissue that nurtures pollen mother cells. Recent evidence suggests that mitochondria destroy the tapetum by triggering standard pathways of programmed cell death. Some nuclear genotypes repress mitochondrial male sterility and restore pollen fertility. Normal regulation of tapetal development therefore arises from a delicate balance between the disruptive effects of mitochondria and the defensive countermeasures of the nuclear genes. In hybrids, incompatibilities between male-sterile mitochondria and nuclear restorers may frequently upset the regulatory control of programmed cell death, causing tapetal abnormalities and male sterility. We propose that hybrid misregulation of programmed cell death may also spill over into other tissues, explaining various developmental aberrations observed in hybrids.     

Cytochrome Oxidase Subunit II Gene from Carrot Contains an Intron

Introns in the cytochrome oxidase subunit II (COXII) gene of plant mitochondrial DNA (mtDNA) have been observed only in monocots. The COXII genes in dicots investigated to date do not contain introns. This is the first report of an intron in the COXII gene of a dicot. The presence of an intron in the carrot COXII intron was verified by restriction mapping and hybridization using specific maize and wheat COXII probes. Regions of the carrot COXII intron are homologous to the maize COXII intron and homologous to the wheat COXII intron-insert as demonstrated by hybridization. Homology of these regions was confirmed by sequencing portions of the gene. A comparison of the restriction map of the carrot COXII gene with the restriction maps of the COXII genes from pea, Oenothera, maize, wheat, and rice revealed that the carrot map coincides with the rice restriction map.     

Possible mitochondrial involvement in mechanism of cytoplasmic male sterility in maize (Zea mays L.)

The mechanism of cytoplasmic male sterility was investigated in maize by isolating mitochondria from seedlings and various anther stages and analyzing cytochrome oxidase and succinic dehydrogenase biochemically and electrophoretically. Sterile anthers exhibited a lack of biochemical activity and fewer isozymatic bands for cytochrome oxidase. No apparent differences were detected biochemically or electrophoretically between fertile and sterile anthers for succinate dehydrogenase.     

Plant Mitochondrial Genome Evolution and Cytoplasmic Male Sterility

Mitochondria are responsible for providing energy currency to life processes in the molecular form of ATP and are therefore typically referred to as the power factories of cells. Plant mitochondria are also relevant to the common phenomenon of cytoplasmic male sterility, which is agronomically important in various crop species. Cytoplasmic male sterility (CMS) is a complex trait that may be influenced by patterns of mitochondrial genome evolution, and by intergenomic gene transfer among the organellar and nuclear compartments of plant cells. Here, we review patterns and processes that shape plant mitochondrial genomes, some relevant interactions between organelles, and the general features shared by the majority of cytoplasmic male-sterile genes in plants to further the goal of understanding CMS.     

Plant mitochondria – past,present and future

The study of plant mitochondria started in earnest around 1950 with the first isolations of mitochondria from animal and plant tissues. The first 35 years were spent establishing the basic properties of plant mitochondria and plant respiration using biochemical and physiological approaches. A number of unique properties (compared to mammalian mitochondria) were observed: (i) the ability to oxidize malate, glycine and cytosolic NAD(P)H at high rates; (ii) the partial insensitivity to rotenone, which turned out to be due to the presence of a second NADH dehydrogenase on the inner surface of the inner mitochondrial membrane in addition to the classical Complex I NADH dehydrogenase; and (iii) the partial insensitivity to cyanide, which turned out to be due to an alternative oxidase, which is also located on the inner surface of the inner mitochondrial membrane, in addition to the classical Complex IV, cytochrome oxidase. With the appearance of molecular biology methods around 1985, followed by genomics, further unique properties were discovered: (iv) plant mitochondrial DNA (mtDNA) is 10–600 times larger than the mammalian mtDNA, yet it only contains approximately 50% more genes; (v) plant mtDNA has kept the standard genetic code, and it has a low divergence rate with respect to point mutations, but a high recombinatorial activity; (vi) mitochondrial mRNA maturation includes a uniquely complex set of activities for processing, splicing and editing (at hundreds of sites); (vii) recombination in mtDNA creates novel reading frames that can produce male sterility; and (viii) plant mitochondria have a large proteome with 2000–3000 different proteins containing many unique proteins such as 200–300 pentatricopeptide repeat proteins. We describe the present and fairly detailed picture of the structure and function of plant mitochondria and how the unique properties make their metabolism more flexible allowing them to be involved in many diverse processes in the plant cell, such as photosynthesis, photorespiration, CAM and C4 metabolism, heat production, temperature control, stress resistance mechanisms, programmed cell death and genomic evolution. However, it is still a challenge to understand how the regulation of metabolism and mtDNA expression works at the cellular level and how retrograde signaling from the mitochondria coordinates all those processes.     

Mitochondrial genome organization and cytoplasmic male sterility in plants

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 agrobacterialrol C gene and (ii) anther specific expression of an RNase gene have been developed in tobacco andBrassica by genetic engineering methods.     

The male sterile G cytoplasm of wild beet displays modified mitochondrial respiratory complexes

Cytoplasmic male sterility (CMS) in higher plants has been mainly studied in cultivated species. In most cases, pollen abortion is linked to the presence of an additional mitochondrial polypeptide leading to organelle dysfunction in reproductive tissues. In wild beet, both CMS and hermaphrodite plants coexist in natural populations. The G cytoplasm is widely distributed along the Western European coast, and previous genetic studies have demonstrated that this cytoplasm confers male sterility in beet. In the present study, we have identified two mutations of G mitochondrial genes, each of which results in the production of a respiratory chain complex subunit with an altered molecular weight; the NAD9 subunit has a C-terminal extension while the COX2 subunit has a truncated C-terminus. NADH dehydrogenase activity was unchanged in leaves, but cytochrome c oxidase activity was reduced by 50%. Moreover, Western blot analyses revealed that alternative oxidase was more abundant in male sterile G plants than in a fertile control (Nv), suggesting that this alternative pathway might compensate for the cytochrome c oxidase deficiency. Implications of respiratory chain changes and a putative link with CMS are discussed.