Ultrastructural expression of cytoplasmic male sterility in sugar beet (Beta vulgaris L.)

Summary The development of microspore mother cells (MMC) and tapetum in male-fertile and male-sterile anthers of Beta vulgaris L. was compared at the electron microscope level. These studies were complemented by morphometric analyses of mitochondria in both tissues through successive stages of microsporogenesis. The earliest irregularities in the ultrastructure of male-sterile anthers were noted within the tapetum at the tetrad stage. These disturbances were initially expressed by a slight reduction in mitochondrial size and the appearance of concentric configurations of endoplasmic reticulum. As development proceeded, a further decrease in mitochondrial size become more conspicuous and was accompanied by a reduction in ribosome population and a failure of the tapetum to produce Ubisch bodies. This failure to produce Ubisch bodies is reflected in the underdevelopment of sterile microspore exine.     

Histological aspects of microsporogenesis in fertile,cytoplasmic male sterile and restored fertilePetunia hybrida

Summary A comparative histological study is made of microsporogenesis in fertile, cytoplasmic male sterile and restored fertilePetunia. Microsporogenesis in sterile anthers proceeds normally until leptotene. The development of the restored fertile type at 25°C is normal until the tetrad stage. In both types sporogenesis arrests and the meiocytes, c.q. microspores ultimately degenerate. The first phenomena of deviation are found in the tapetum. The effects of degeneration on cellular structure, vacuolation and cytoplasmic organization of the tapetal and sporogenous cells are variable. The deposition of callose around the meiocytes appears independent of the process of degeneration. The absence of an increase in callase activity possibly explains the remnants of callose found at late stages of development. The failure of callose wall dissolution appears to be the result of metabolic abnormalities in the tapetum and is regarded as an indirect effect of sterility.     

Genetic analysis of instability inPetunia hybrida

Summary InPetunia hybrida frequent mutations of unstable alleles give rise to different types of periclinal chimeras. If genes expressed in the epidermis, such as the geneAn1 for flower colour, are concerned, mutations in the dermal layer of the shoot apex will result in changes in the phenotype but not in the offspring. Mutations in the subdermal layer will not lead to an altered phenotype, but to changes in the sporogenous tissues and, thus, to deviating segregations in progenies. Therefore, in crossing experiments with such an unstable mutant, it is always necessary to take the possibility into account that the plant may be a chimera, so as to prevent an incorrect interpretation of the recorded segregational ratios. Mutations of unstable alleles expressed in the mesophyll, such as geneYg3 for leaf colour, also give rise to chimeras. In such instances, however, a change in phenotype always involves a change in segregational ratios as well, since both the mesophyll and the sporogenous tissues are derived from the subdermal layer of the shoot apex.     

DNA synthesis and cytoplasmic differentiation in tapetal cells of normal and cytoplasmically male sterile lines of Petunia hybrida

Summary A new method is described by which tapetal cells may be isolated from anthers of cytoplasmic male sterile (CMS) and fertile lines of Petunia hybrida. Using a combination of stereometry and Feulgen densitometry it has been possible to chart DNA synthesis and nuclear division with great precision within these cells. Results are presented which show CMS lines both to synthesize far less DNA than the fertiles and also to undergo less cell division. These differences in DNA kinetics and cytokinesis are obvious long before any differences between sterile and fertile lines may be detected in the meiocyte mass. In addition to these difference in nuclear behaviour, the tapetal cells of CMS lines also synthesize far lower levels of rough endoplasmic reticulum than do their fertile counterparts. Again, this difference is evident at a very early stage of anther development when all other cell components, including mitochondria and plastids, appear normal. These data are discussed in terms of the very special differentiation undergone by tapetal cells in angiosperms, and the conclusions drawn are considered in the perspective of current hypotheses proposed to explain the operation of CMS.     

Pollen tube expression of pseudo-self-compatibility (PSC) inPetunia hybrida

Summary AnS _1.1 self-incompatible (SI) petunia plant which showed atypical seed set was found in an I₇ population. This plant showed a strong SI reaction when selfed but produced varying amounts of seed when used as the seed parent in crosses with unrelated individuals homozygous for the sameS allele. Reciprocal crosses yielded no seed indicating that the reaction was a stylar response. Self seed obtained by high temperature treatments produced 18 plants, all of which exhibited the parental characteristics, the ability to reject self pollen but accept, to varying degrees, pollen bearing the sameS allele from unrelated plants. Several petunias homozygous forS ₁, and exhibiting various levels of PSC as determined by self seed set, progeny tests and temperature treatments, were used as pollen parents. The mean seed set of these crosses produced a ranking of the pollen parents which reflected the PSC levels obtained by other methods. The behavior of the F₁ and F₂ populations suggests that the pollen discriminating ability may be a simply inherited, dominant character in these plants. The styles of these unusual petunias illustrate the participation of the pollen tube in determining PSC.Scientific Journal Series Paper Number 10.479 of the Minnesota Agricultural Experiment Station     

Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida

Summary Using an HPLC method it has proved possible to follow the levels of adenine and pyridine nucleotides in the anthers of normal and cytoplasmically male sterile (CMS) lines of Petunia hybrida. Well before the appearance of any structural differences, anthers of CMS plants begin to show lower ATP/ADP ratios. In anthers, as in other non-photosynthetic plant tissues, there is a strong correlation between the ATP/ADP ratio and levels of NADPH and, when NADPH was assayed in fertile and CMS anthers, very dramatic differences were discovered. In male fertile plants, the NADPH/NADP ratio differs strikingly between somatic and reproductive tissues and reaches a peak in anthers at the early prophase of meiosis. The ratio in male sterile anthers remains largely unchanged from that normally seen in somatic tissues over this period. A cytochemical localisation technique revealed that, at the stage of development in question, the major reserves of anther NADPH are held within the tapetal cells and that levels within CMS tapeta are strikingly lower than those of normal plants. These findings are discussed in the perspective of theories proposed to explain the operation of CMS, and also in terms of the now conclusive evidence that the genetical basis of CMS lies in changes in the organisation of the mitochondrial genome.     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

Cytoplasmic male sterility in Vicia faba L.

Summary In many higher plants, nucleo-cytoplasmic interactions lead to pollen abortion. In Vicia faba, cytoplasmic male sterility is unstable as the cytoplasm appears to shift from a sterile to a fertile state. In this report, five flower phenotypes are defined but the study is focussed on the progenies obtained from intermediate, semi-sterile plants with the same homozygous nuclear constitution during five successive generations. The results could be interpreted by quantitative modifications of at least four different kinds of cytoplasmic determinants.     

A new type of cytoplasmic male sterility in rye (Secale cereale L.): analysis of mitochondrial DNA

Summary Mitochondrial (mt) DNA of a new type of rye cytoplasm (Gülzow, G) that induces cytoplasmic male sterility (CMS) was analyzed and compared with rye mtDNAs of different origins MtDNA of the G type was easily distinguishable from mtDNA of another CMS source, Pampa (P) type, and from mtDNA of fertile lines with respect to restriction fragment patterns and hybridization with mitochondrial genes. The results of the molecular analyses indicate a close, but not identical relationship between the mtDNA of the G type cytoplasm and that of cv Pluto.     

Biochemical and functional characterization of ORF138, a mitochondrial protein responsible for Ogura cytoplasmic male sterility in Brassiceae

In cytoplasmic male sterility (CMS), original mitochondrial genes contribute to sex determinism by provoking pollen abortion. The function of the encoded proteins remains unclear. We studied the ORF138 protein, responsible for the 'Ogura' CMS, which is both used in hybrid seed production and present in natural populations. We analyzed the biochemical and structural properties of this protein in male-sterile plants and in E. coli. We showed that this protein spontaneously forms dimers in vitro. Truncated variants of the protein, containing either the hydrophobic or the hydrophilic moiety, also spontaneously dimerize. By fractionating mitochondria, we showed that ORF138 was strongly associated with the inner mitochondrial membrane of male-sterile plants. Our results also strongly suggest that ORF138 forms oligomers in male-sterile plant mitochondria. In E. coli, ORF138 was associated with the plasma membrane, as shown by membrane fractionation, and formed oligomers. The production of this protein strongly inhibited bacterial growth, but not by inhibiting respiration. The observed toxic effects required both the hydrophilic and hydrophobic moieties of the protein.     

A new source of cytoplasmic male sterility in maize induced by the nuclear gene,iojap

Summary Cytoplasmic male sterility (cms) was found in plants derived from the F₂ progeny of fertile, normal cytoplasm plants of the inbred R181 pollinated with a genetic stock carrying the recessive nuclear gene, iojap. The male sterile plants were maintained by back-crossing with the inbred W182BN which maintains all known sources of cytoplasmic male sterility. The new male sterile progeny were found to exhibit stable male sterility under field conditions in two environments. However, they were partially fertile in the hot, dry summer of 1983 at Aurora, NY. It was found that these lines were restored by lines that characteristically restore cms S group cytoplasms. Pollen phenotype studies indicated that the restoration was gametophytic in nature, also characteristic of the cms S group. Agarose gel electrophoresis of undigested mitochondrial DNA (mtDNA) from these steriles indicated that these lines have the S-1 and S-2 episomes characteristic of the cms S group. Restriction endonuclease digest patterns of mtDNA from these sterile lines digested with BamH I indicated that these steriles fit into the CA subgroup of the cms S group. The new source of cms has been designated cms Ij-1.     

Effect of ultraviolet radiation on cell division and microtubule organization inPetunia hybrida protoplasts

Summary Mesophyll protoplasts isolated fromPetunia hybrida were subjected to UV radiation (280–360 nm) in an attempt to assess whether (a) UV radiation has an effect on cortical microtubule organization, (b) UV radiation affects the progression of protoplasts through the cell cycle, and (c) there is a connection between the effect of UV radiation on cell division and the polymerization state of the microtubules. The proto plasts were irradiated with the following UV doses: 4, 8, 12, and 24mmol photons/m², 30 min after isolation. Cell cycle analysis and immuno-localization of microtubules were carried out 0, 24, 48, and 72 h after irradiation. The length of cortical microtubules was determined after irradiation and in corresponding controls. We found that UV radiation induced breaks in cortical microtubules resulting in shorter fragments with increasing dose. Also, the protoplasts were delayed in their progression through the cell cycle, with G1 and G2 phases being affected as well as the S phase. The commencement of DNA synthesis in the irradiated protoplasts followed the re-establishment of a microtubule network. At 48 h after irradiation the protoplasts in all treatments, except for the 24 mmol/m², had cortical microtubules of similar length, and at 72 h after irradiation only the protoplasts that had received 24 mmol photons/m² had not started dividing.Abbreviations BSA bovine serum albumin - DMSO dimethyl sulfoxide - FDA fluorescein diacetate - MT microtubules - MTSB microtubule stabilizing buffer - PAR photosynthetically active radiation (400–700 nm) - PBS phosphate buffered saline - UV ultraviolet     

Mitochondrial DNA modifications associated with cytoplasmic male sterility in rice

Summary Mitochondrial DNA was isolated from fertile and cytoplasmic male sterile lines of rice. Restriction analysis showed specific modifications in the male sterile cytoplasm. In addition to the major mitochondrial DNA, three small plasmid-like DNA molecules were detected by agarose gel electrophoresis in both cytoplasms. An additional molecule was specifically found in the sterile cytoplasm. These mitochondrial DNA modifications support the hypothesis of the mitochondrial inheritance of the cytoplasmic male sterility in rice.     

Molecular analysis of cytoplasmic male sterility in chives (Allium schoenoprasum L.)

The mitochondria of chive plants with normal N or male-sterile S cytoplasms have been examined by restriction fragment analysis and Southern hybridizations of mitochondrial DNA (mtDNA) and in organello protein biosynthesis. Restriction fragment patterns of the mtDNA differed extensively between N-and S-cytoplasms. The percentage of fragments with different mobility varied between 44–48% depending on the restriction enzyme used. In contrast to mtDNA, the restriction fragment patterns of the chloropolast DNA from N- and S-cytoplasms were identical. The organization of the analyzed mitochondrial genes coxII, coxIII, nad1 and nad3 was different in N- and S-cytoplasms. Comparison of mitochondrial proteins analyzed by in organello translation revealed an 18-kDa protein present only in S-cytoplasm. The restorer gene X suppressed the synthesis of that protein in S-cytoplasm. Thus, the 18-kDa protein seems to be associated with the cytoplasmic male-sterile phenotype.     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

TheBrassica mitochondrial DNA plasmid and large RNAs are not exclusively associated with cytoplasmic male sterility

Summary More than 100 differentBrassica nucleo-cytoplasmic combinations were analysed for the presence or absence of the 11.3 kb mitochondrial plasmid. Contrary to some previous reports, no close association exists between the presence of the plasmid and cytoplasmic male sterility. Some novel abundant RNAs which copurified withBrassica mitochondria are described.     

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orf H522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two new types of cytoplasmic male sterility found in wild Beta beets

Summary Mitochondrial (mt) and chloroplast (ct) DNAs from sugar beet carrying normal fertile and different cytoplasmic male sterile (cms) cytoplasms were compared by restriction analysis and for the occurrence of minicircles. One of the cms materials had the Owen cms cytoplasm currently used for hybrid production in sugar beet; the other three cms materials were derived from wild Beta beets. The mtDNAs from two of the latter cms types (C 7051, C 8640) differed from both the Owen and the fertile cytoplasms in fragment patterns seen after restriction enzyme analysis and in minicircle composition. The third cms type (C 8684) differed from the Owen cytoplasm in mini-circle composition, but restriction enzyme analysis revealed no differences. The presence of the different minicircles was confirmed by Southern hybridization using minicircle-specific clones. All bands hybridized as predicted by gel electrophoresis except a band in the cms type C 8640, which migrated in a similar manner as the c.c.c. form of the a minicircle. This band hybridized only faintly to a minicircle a-specific probe and could be removed by treatment with nuclease S1. In contrast to the large mtDNA variation, restriction analysis of ctDNA detected little variation between cytoplasms. The molecular characterization of the new sources of cms supports the results of previous crossings. Two of the cytoplasms are not only of independent origin, but are also most likely functionally different and thus may be of value in future production of hybrid sugar beet varieties.