Respiration in Cells and Mitochondria of Male-Fertile and Male-Sterile Nicotiana spp.'

Three cytoplasmic male-sterile Nicotiana cultivars together with corresponding male-fertile progenitors and restored lines were investigated in order to find possible correlations between respiratory characteristics and male sterility. Oxygen consumption measurements were performed on cells from suspension cultures as well as on mitochondria isolated from green leaves. Inhibitors, which have been reported to specifically block either the cytochrome (KCN) or the alternative (propyl gallate and salicylhydroxamic acid [SHAM] respiratory pathways, were used in order to measure the capacity and activity of the two pathways. One of the inhibitors, SHAM, was found unsuitable to measure the activity of the altemative pathway due to the lack of specificity of SHAM for this pathway. A great difference in the capacity of the alternative pathway was detected between the two types of cell materials tested. Mitochondria isolated from green leaves showed a capacity of the alternative pathway of 5 to 20% of total mitochondrial repiration, while the capacity of cells from suspension cultures generally ranged from 50 to 80%. In addition to this, in organello synthesis of mitochondrial proteins revealed differences between mitochondria isolated from green leaves and from cell suspensions. No correlation, however, could be found between respiratory characteristics and male sterility. showed a capacity of this pathway of about 20% of the total respiration. The existence of an alternative respiratory pathway in higher plants is well documented (21, 23, 31). It can be detected through its resistance to cyanide and sensitivity to inhibitors such as SHAM, propyl gallate and disulfiram. Lambers (20) suggested that the alternative pathway functions as an overflow mechanism when the cytochrome pathway is saturated. An additional terminal oxidase responsible for the activity of this alternative respiratory pathway has been identified (6, 11, 12). This study was performed to investigate whether a difference in the capacity of the alternative respiratory pathway could be detected between male-fertile and alloplasmic malesterile Nicotiana materials. The genetic background of CMS is well defined in the chosen materials making comparative measurements between male-sterile cultivars and male-fertile progenitor lines possible. Besides this, cultivars restored to fertility were included in the study. The Nicotiana materials used have been analyzed earlier for variations in mitochondrial DNA and proteins (15). MATERIALS AND METHODS Plant Material CMS,2 a widespread phenomenon among higher plants, is utilized by plant breeders in the production of hybrid seeds. Mitochondria are known to contain the cytoplasmic factor involved in CMS, and specific alterations in both DNA and proteins have been detected in male-sterile lines (16, 18). To detect the basic causes of the aberrant reproductive development, which are still not known, CMS has been studied both structurally and biochemically. For example, tapetal cell degeneration has been noted by Bino (4, 5) in Petunia and by Warmke and Lee (35) in Zea mays. Biochemical changes in male-sterile plants have been reviewed by Kaul (18). Whether such alterations are the cause or the result of abnormal development in sterile plants is difficult to establish. Musgrave et al. (26) reported on a major difference in respiratory behavior between male-fertile and male-sterile plants from several species. All sterile plants investigated were found to lack the alternative respiratory pathway, while fertile lines ' Supported by the Swedish Natural Science Research Council and the U.S. Department of Agriculture. 2 Abbreviations: CMS, cytoplasmic male sterility; SHAM, salicylhydroxamic acid. 367 Three sets of materials were used. Each set consisted of four lines: a male-fertile Nicotiana tabacum cultivar, a male-fertile cytoplasmic donor species (N. repanda, N. suaveolens, or N. debneyi), the male-sterile cultivar that resulted from an introgression of the nucleus of the male-fertile N. tabacum cultivar into the cytoplasm of the respective donor species, and a corresponding cultivar with restored fertility. In each of the three sets, the nuclear background of the male-sterile, male-fertile, and restored cultivars of N. tabacum was identical, except for the presence, in restored lines, of genetic material originating from the respective donor species. These Nicotiana materials and their suitability for studies of CMS were described in more detail by Hakansson et al. (15). Isolation of Mitochondria Isolation of washed mitochondria for respiratory measurements was carried out according to Schwitzguebel and Siegenthaler (29) with the following modifications. Young leaves were harvested from 10 to 14 week old plants grown in the greenhouse. The leaves with a total fresh weight of 20 to 25 g were ground in several batches with a mortar and pestle www.plantphysiol. org on July 22, 2018 Published by Downloaded from Copyright © 1990 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 93,1990 together with extraction medium. Mitochondria were suspended in a small volume of0.3 M mannitol buffer, containing 0.1 mm EDTA, 20 mm Hepes (pH 7.2) and 0.1% BSA (w/v). Protein and Chi Estimation Protein and Chl content were measured in the final mitochondrial suspension, as well as in the chloroplast fraction. Proteins were measured using the Bio-Rad protein assay based on the dye-binding method of Bradford (7) and Chl was estimated by the method of Arnon (1).