A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco.

Abstract

In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239 gene constructs, with or without a mitochondrial targeting sequence, exhibited a semisterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the 0RF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that 0RF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.The mitochondrion, as a cellular center for energy metabolism, serves essential functions in the development of eukaryotic organisms. In plants, one of the developmental transitions that appears to be particularly influenced by mitochondrial function is male reproductive (pollen) development. Mutations in the mitochondrial genome most commonly result in the inability of the plant to shed viable pollen. This phenomenon, known as cytoplasmic male sterility (CMS) and observed in more than 150 plant species, has presented the opportunity to investigate the special functions provided by mitochondria at this crucial developmental stage.The association of CMS with abnormal mitochondrial gene expression has been established in many plant species including maize (1, 2), petunia (3, 4), sunflower (5-8), and common bean (9, 10). In all cases, pollen disruption in CMS lines is accompanied by the expression of novel mitochondrial DNA sequences that contain protein-coding open reading frames. To date, there are two important issues regarding CMS yet to be resolved. (i) Definitive evidence that these aberrant mitochondrial genes actually cause pollen disruption is not yet available in any plant system, and (ii) the molecular mechanisms effecting the disruption of pollen development are still not understood.In CMS common bean, male-sterile plants contain a novel mitochondrial DNA sequence (designated pvs) of unknown origin that is associated with the pollen sterility phenotype by various genetic criteria (10, 11). The 3.7-kb pvs sequence contains at least two open reading frames, ocf239 and orf98.

Interactions of Mitochondrial and Nuclear Genes That Affect Male Gametophyte Development

“…The protein product of orf239 was found to accumulate only in reproductive tissues of CMS plants (Abad et al, 1995;Sarria et al, 1998). ORF239 was further implicated as the cause of pollen disruption in common bean when tobacco plants expressing the gene from a nuclear transgene were found to be male sterile (He et al, 1996). Similar S158…”

“…The experiments with pvs may have succeeded in producing CMS plants because, remarkably, the gene product is found external to the mitochondria, associated with the cell walls of developing pollen (He et al, 1996).…”

Stoichiometric Shifts in the Common Bean Mitochondrial Genome Leading to Male Sterility and Spontaneous Reversion to Fertility

“…This conclusion is based on the fact that we were able, with a 90-min incubation of a defined population of mitochondria isolated from revertant seedling tissues, to observe a reversal of the reversion phenomenon in the form of amplification and expression of the pvs-orf239 sequence. Because we have, in previous studies, associated expression of the pvs-orf239 sequence with the sterility phenotype (Abad et al, 1995;He et al, 1996), we assume that the changes observed in vitro would have led to the reestablishment of the male-sterile phenotype in vivo. We are currently conducting experiments to test for the reversibility in vivo of the fertile revertant to the male-sterile phenotype.…”

“…We have detected translation of only pvs-orf239 , which produces a 27-kD polypeptide that accumulates in reproductive tissues (Abad et al, 1995) but is rapidly degraded in vegetative tissues (Sarria et al, 1998). Evidence suggests that the ORF239 product may induce the sterility phenotype (Abad et al, 1995;He et al, 1996). Mapping of the CMS common bean mitochondrial genome by overlapping cosmid clone analysis indicates that the genome is most likely organized as three interrecombining and highly redundant mitochondrial molecules of 394, 257, and 210 kb (Janska and Mackenzie, 1993).…”

Studies on the energy release of rice mitochondria under different conditions by means of microcalorimetry

“…Mutations in the mitochondrial genome most commonly result in the inability of the plant to shed viable pollen. This phenomenon is known as cytoplasmic male sterility (CMS) and is observed in more than 150 plant species [3]. The hybrid seeds can be produced with the help of male sterile lines, and the fine varieties of crops can be supplied by the hybrid seeds to raise the yield of crops.…”