Cytoplasmic male sterility in Petunia hybrida : a structural and histochemical analysis

This thesis presents an analysis of the structural and histochemical aspects of cytoplasmic male sterility (cms) in <u>Petunia</u><u>hybrida</u> . In petunia and in other crops, cms is the most commonly used tool for hybrid seed production. Application of the trait makes hybrid seed production possible without the need of emasculation of the maternal line. However, in spite of its economic importance, little is known on the primary causative factor and the initial step of pollen abortion in cms plants. Insights in the initial effects of cms may lead to a more comprehensive understanding of the regulation and expression of male sterility controlling genes, and, additionally, may possibly provide strategies for the introduction or induction of male sterility in crops in which cms systems are not available.In the first Chapter, some molecular aspects of cms are evaluated. Several lines of evidence indicate that the genetic determinants responsible for cms are carried by the mitochondrial genome. The mitochondrial involvement is found in a variety of plant species, including <u>Petunia</u><u>hybrida</u> . Most of the mitochondrial encoded polypeptides are components of complexes which are responsible for key steps in the process of oxidative phosphorylation and the generation of ATP. Correspondingly, mitochondria isolated from tissues of cms plants, may code for an aberrant polypeptide composition of components of one of these complexes. Nevertheless, more information on the expression of mitochondrial genes in different anther tissues at various stages of development is necessary before we can conclude whether or not the deviations in mitochondrial DNA are functionally associated with the non-formation of viable pollen.The initial abnormalities in anther development of cms plants are generally found in the tapetal tissue. Also in the cms form of <u>Petunia</u><u>hybrida</u> cv. Blue Bedder (BBS), the first symptoms of deviation are found in the tapetum (Chapter 2). Light microscopical analysis shows, that in BBS anthers, the tapetal breakdown begins at the prophase stage of the meiocytes. At the preceding stages of development, microsporogenesis in BBS anthers is normal and indistinguishable from the development in the male fertile counterpart (BBF). At the ultrastructural level, the initial aberration of BBS anthers is represented by the presence of large vacuoles in the cytoplasm of the tapetal cells (Chapter 3). At the leptotene stage of the meiocytes, these vacuoles are the first symptoms of degeneration. At later stages, the tapetal and sporogenous cells are highly distorted, the nucleus is disrupted and the cytoplasm disorganized. Mitochondria and plastids degenerate and many lipid droplets are present.Chapter 4 describes the way in which the biochemical and histochemical aspects of an enzyme system are influenced by the degeneration of the tapetal and sporogenous tissues. The Chapter gives information on the isoenzyme pattern, the activity, and the localization of esterases in anther tissues of cms and male fertile petunia cultivars. Esterases are rather unspecific, nuclear encoded enzymes occuring in all plant parts. The biochemical data show that, from the early meiosi S onward, esterase activity in cms-type anthers remains at a low level and hardly any new isoenzyme bands show up as compared to the situation in the male fertile counterpart. The histochemical determinations reveal, that in male fertile-type anthers, esterase activity is concentrated in the outer tapetal layer at late prophase and that it accumulates there till the early microspore stage. In anthers of cms plants, esterase accumulation in the tapetal cells ceases at the moment that tapetal breakdown becomes evident. These results suggest that the differences in total esterase activity and esterase isoenzyme patterns are an effect rather than a cause of the failing pollen formation.In cms forms of different species, aberrations in cytochrome c oxidase activity and other mitochondrial redox processes are associated with the cms plasmatype. A biochemical determination of the cytochrome c oxidase activity in anthers of <u>Petunia</u><u>hybrida</u> and <u>Zea</u><u>mays</u> is given in Chapter 5. The biochemical analysis is combined with a cytochemical localization of enzyme activity in mitochondria of sporogenous and tapetal tissues in both species. The data show that in anthers of different cms maize strains, the cytochrome c oxidase activity is reduced in comparison with the level found in male fertile-type anthers. Additionally, there are consistent cytochemical differences in the mitochondrial organization of cytochrome c oxidase activity between pollen of cms- S and male fertile maize plants. The aberrations in enzyme activity are observed at stages of development at which the structural aspects of degeneration are not yet evident. In fact, the deviation in cytochrome c oxidase may represent the initial symptom of male sterility in this maize type. Contrarily, in petunia, the first detectable differences in the mitochondrial enzyme activity occur only after the initial effects of tapetal degeneretion are apparent. Hence, in petunia, the decline in cytochrome c oxidase activity is the result rather than the cause of the proceeding process of degeneration.In Chapter 7 it is postulated that the cms specific deviations in the mitochondrial genome induce alterations in protein complexes which are essential for energy generating processes. Possibly, these aberrations adversely affect the energy status of cms cells. However, BBF and BBS plants possess similar growth characteristics, and, apparently, the viability of plants with cms plasmatype, is not diminished by the mitochondrial defects. In fact, abnormalities in the development of cms plants are only observed in particular anther tissues. These results may suggest that the aberrations in the mitochondrial genome are only expressed in the tapetal or sporogenous tissues at certain moments of development. However, this assumption is inconsistent with the fact that deviations in mitochondrial products are sometimes found in organelles isolated from vegetative parts of the plant. An alternative explanation for the tissue specific character of cms is, that the degeneration of the anther tissues is initiated by the specific metabolism of the cells. The adenylate energy charge ratios of petunia anther tissues is discussed in Chapter 6. As compared with petunia leaf tissue, the results give evidences for the particular metabolic state of the tapetal and sporogenous tissues. Examples of the metabolic activity in anthers of other plant species are evaluated in Chapter 7. Furthermore, the structural analyses as presented in the second and the third Chapter of this thesis, reveal that the cms petunia anther development is distorted at the moment at which there is a considerable rise in the metabolic activity of the tapetal cells of the male fertile counterpart. Possibly, during moments of energetic stress, the mitochondrial synthesis of energyrich products in tapetal cells of cms petunia is insufficient to meet the energetic demands for the normal functioning of the cells at that stage. Hence, as a result of defects in the mitochondrial genome, the tapetal, and consequently, the sporogenous tissues degenerate.