Charles S. Levings

The Texas Cytoplasm of Maize: Cytoplasmic Male Sterility and Disease Susceptibility

The Texas cytoplasm of maize carries two cytoplasmically inherited traits, male sterility and disease susceptibility, which have been of great interest both for basic research and plant breeding. The two traits are inseparable and are associated with an unusual mitochondrial gene, T-urf13, which encodes a 13-kilodalton polypeptide (URF13). An interaction between fungal toxins and URF13, which results in permeabilization of the inner mitochondrial membrane, accounts for the specific susceptibility to the fungal pathogens.

Restriction endonuclease analysis of mitochondrial DNA from normal and Texas cytoplasmic male-sterile maize.

Mitochondrial DNA from normal and cytoplasmic male-sterile maize was digested with restriction endonucleases RI from Escherichia coli or dIII from Hemophilus influenzae. Electrophoresis of resulting fragments revealed distinctions between the two cytoplasmic types. These distinctions suggest that factors responsible or cytoplasmic male sterility are located in the mitochondrial DNA, and that the mitochondrial genome is not inherited paternally.

The tobacco mitochondrial ATPase subunit 9 gene is closely linked to an open reading frame for a ribosomal protein

SummaryA transcribed segment of mitochondrial DNA (mtDNA) from Nicotiana tabacum contains the F0-ATPase subunit 9 gene, an open reading frame with homology to the E. coli small subunit ribosomal protein S13 and an open reading frame with homology to a portion of the mammalian “URF 1” protein, recently shown to be a component of the NADH:ubiquinone reductase complex (NADH:Q 1). The transcriptional patterns of the tobacco ATPase 9 gene and S13-like open reading frame share eight RNA species indicating the two sequences are part of the same transcriptional unit. A maize mtDNA fragment contains the S13 homologous sequence and the NADH:Q 1 homologous sequence in an orientation similar to tobacco. The S13-like sequence is present as a single copy in maize and tobacco, as two copies in wheat, and is absent in pea and bean. We discuss the distribution and orientation of the S13-like and “URF 1”-like sequences and the possibility that they are active genes.

Cytoplasmic Reversion of cms-S in Maize: Association with a Transpositional Event.

Spontaneous reversion to fertility in S male-sterile cytoplasm of maize is correlated with the disappearance of the mitochondrial plasmid-like DNAs, S-1 and S-2, and changes in the mitochondrial chromosomal DNA. Hybridization data indicate that one of the plasmid-like DNAs, S-2, is prominently involved in the mitochondrial DNA rearrangements.

Nucleotide sequence of the S-1 mitochondrial DNA from the S cytoplasm of maize.

Mitochondria from the S male‐sterile cytoplasm of maize contain unique DNA‐protein complexes, designated S‐1 and S‐2. These complexes consist of double‐stranded linear DNAs with proteins covalently attached to the 5′ termini. To learn more about these unusual DNAs we have determined the complete nucleotide sequence of the S‐1 DNA molecule (6397 bp). The sequence of S‐2 has been previously determined. S‐1 and S‐2 are structurally similar and contain ˜1.7kb of sequence homology. S‐1 is terminated by exact 208‐bp inverted repeats that are identical with the terminal inverted repeats of S‐2. S‐1 and S‐2 also contain a 1462‐bp region of nearly perfect homology, which includes one of the terminal inverted repeats. The homology between the two molecules may be maintained, in part, by homologous recombination. S‐1 has three long unidentified open reading frames, URF2 (1017 bp), URF3 (2787 bp) and URF4 (768 bp). URF2 occurs in the 1462‐bp region of homology and is identical in length and location in both S‐1 and S‐2. Based on their structural organization and their viral‐like characteristics, we propose that S‐1 and S‐2 code for functions involved with their maintenance and replication.

Thoughts on Cytoplasmic Male Sterility in cms-T Maize

Cytoplasmic male sterility (CMS) is a maternally inherited trait that suppresses the production of viable pollen grains. The Texas, or T, cytoplasm (cms-7) of maize, which was first described in the Golden June line in Texas (Rogers and Edwardson, 1952), carries the CMS trait. The Texas cytoplasm was an important discovery to geneticists and plant breeders because it eliminated the costly detasseling procedure used in maize hybrid seed production. Its commercial value has prompted many applied and basic studies of cms-T aimed at understanding CMS and its relationship with mitochondrial and nuclear genes. Although CMS has been observed in more than 150 plant species (Laser and Lersten, 1972), the mechanism by which it interrupts normal pollen development is not well understood. In this review, the basis of CMS in cms-T maize is considered. In cms-T; male sterility is characterized by the failure of anther exertion and pollen abortion. Female fertility is not affected by CMS, so male-sterile plants can set seed if viable pollen is provided. Comparisons of plants carrying the T and normal cytoplasms reveal very slight differences in severa1 other morphological characters (see below; Duvick, 1965). Initially, the three male-sterile cytoplasms of maize-cms-T, cms-C, and cms-S-were distinguished by specific nuclear genes, termed restorers of fertility (Rf), that suppress the male-sterile effect of the various cytoplasms and allow viable pollen production. For example, two genes, Rf7 and Rf2, acting jointly, restore pollen fertility to cms-7: Rf7 and Rf2do not restore pollen fertility to cms-C or cms-S; instead, different restorer genes are necessary to restore pollen fertility to these cytoplasms. Other characteristics also distinguish male-sterile and normal maize cytoplasms; they include mitochondrial DNA (mtDNA) restriction fragment length polymorphisms, variations in mitochondrial RNA (mtRNA), and differences in mitochondrial translational products (Pring and Levings, 1978; Leaver and Gray, 1982; Newton, 1988). Early on, these distinctions indicated that the various CMS types are based on different mechanisms and hereditary factors. The T cytoplasm is best known for the part it played in the U.S. epidemic of Southern corn leaf blight of 1969 and 1970 (Williams and Levings, 1992). In the two decades before the epidemic, cms-T had replaced detasseling as the chief method of pollen control in hybrid corn production (Wych, 1988), and by 1970, 85010 of the hybrid corn grown in the United States carried the T cytoplasm. After it was determined that cms-T was specifically susceptible to Bipolaris maydis race T, the organism responsible for the blight, its use by the hybrid seed corn industry was largely terminated (Ullstrup, 1972). Phyllosricra maydis, another fungal pathogen, is also specifically virulent on cms-7: Normally, B. maydis race T is a serious pathogen only on maize containing the T cytoplasm. Other male-sterile and normal maize cytoplasms support only limited colonization by the pathogen, and the lesions on the leaves remain small and isolated. By contrast, B. maydis lace T can quickly and completely colonize cms-Tmaize plants, causing extensive plant damage and sometimes death. Susceptibility of cms-Tto B. maydis race T is caused by mitochondrial sensitivity to a host-specific pathotoxin, designated BmT toxin, produced by the pathogen. P maydis also produces a pathotoxin that is structurally similar to the BmT toxin of B. maydis, to which cms-Tmitochondria are sensitive. Accordingly, susceptibility of cms-Tmaize to these fungal pathogens is due to mitochondrial sensitivity to the pathotoxins, whereas disease-resistant maize types have mitochondria that are insensitive to the pathotoxins.

Comparison of the mitochondrial genome of Nicotiana tabacum with its progenitor species.

SummaryMitochondrial DNAs from Nicotiana tabacum, an amphiploid, and its putative progenitor species, N. sylvestris and N. tomentosiformis were compared in structure and organization. By using DNA transfer techniques and cloned fragments of known genes from maize and N. sylvestris as labeled probes, the positions of homologous sequences in restriction digests of the Nicotiana species were analyzed. Results indicate that the mitochondrial DNA of N. tabacum was inherited from N. sylvestris. Conservation in organization and sequence homology between mtDNAs of N. tabacum and the maternal progenitor, N. sylvestris, provide evidence that the mitochondrial genome in these species is evolutionarily stable. Approximately one-third of the probed restriction fragments of N. tomentosiformis mtDNA showed conservation of position with the other two species. Pattern variations indicate that extensive rearrangement of mtDNA has occurred in the evolution of these Nicotiana species.

Molecular basis of disease susceptibility in the Texas cytoplasm of maize.

The Texas, or T, cytoplasm (cms-T) of maize (Zea mays L.) carries a cytoplasmic ally inherited male sterility. This cytoplasmic male sterility (CMS) suppresses the production of viable pollen grains and is inherited in a non-Mendelian manner. cms-T was first described in the line Golden June in Texas [32, 69]. The discovery of the Texas cytoplasm was important to maize geneticists and breeders because they wished to use CMS in maize hybrid production. Hybrid maize, first double-cross hybrids and later single-cross, was used to exploit hybrid vigor to achieve higher yields and greater uniformity. Many applied and basic studies have been devoted to cms-T to understand CMS and its relationship with mitochondrial and nuclear genes.

The plant mitochondrial genome and its mutants

Organization and Structure Mitochondrial genomes of higher plants are substantially larger and more variable than those of other organisms. Among the score of plant species investigated, mitochondrial genomes range from 250 to 2500 kb. Within a single family, the cucurbits, 7 to 8 fold size diversity was demonstrated (muskmelon 1600 mdal, cucumber 1000 mdal, zucchini squash 560 mdal and watermelon 220 mdal; Ward et al., Cell 25, 793-803, 1981). These large genomes are composed chiefly of unique sequences (>90%). This remarkable variation is in marked contrast with other mitochondrial genomes (e.g., animals 15-l 8 kb, fungi 18-78 kb, protists 15-47 kb) and the chloroplast genomes of higher plants (120-l 80 kb). Why plant genomes are so large and variable is not understood. It is generally agreed that they may code for a few additional genes not found in other organisms but certainly nowhere near the number needed to account for the extra mitochondrial DNA (mtDNA). “Selfish DNA” would not easily account for the large amounts of nonrepeated, high-complexity DNA encountered in plant mitochondria. It has also been suggested that the extra DNA could serve a “filler” function; however, as yet no mitochondrial functions have been associated with “filler” DNA (Ward et al., op. cit.). The organization of the mitochondrial genome of higher plants is puzzling. Electron microscopy studies characteristically reveal a majority of large linear molecules and lesser amounts of open circular and covalently closed circular molecules (Dale, In Mitochondrial Genes, Slonimski et al., eds., Cold Spring Harbor Laboratory, pp. 471-476, 1982). Generally, the frequency of the circular molecules is low, ranging from about 5% in maize and soybeans to upwards of 40% in tobacco cell cultures. Surprisingly, the circular molecules are most often heterogeneous in both size and relative abundance. For example, in maize at least seven discrete molecular size classes were observed that range from 1.8 kb to as much as 68 kb, and in abundance from 48% to less than 1% of the circles. Molecular heterogeneity appears widespread among plants, since equivalent variation is reported in potatoes, oenothera, Virginia creeper, tobacco, flax and sugar beets. Minicircles and/or minilinears (less than 2.5 kb) are prevalent and are commonly visualized by gel electrophoresis in many species, e.g., maize, tobacco, beans, teosinte. Multimeric series of plant mitochondrial DNAs are reported in several plant species, especially among the smaller size classes. That Minireviews

Fungal toxins bind to the URF13 protein in maize mitochondria and Escherichia coli.

Expression of the maize mitochondrial T-urf13 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide. Similar effects of pathotoxins and methomyl are observed when T-urf13 is cloned and expressed in Escherichia coli. An interaction between these compounds and the membrane-bound URF13 protein permeabilizes the inner mitochondrial and bacterial plasma membranes. To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein. Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E. coli expressing URF13. Binding analysis in E. coli reveals cooperative toxin binding. A low level of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria. Competition and displacement studies in E. coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites. Two toxin-insensitive URF13 mutants display a diminished capability to bind toxin in E. coli, which identifies residues of URF13 important in toxin binding. A third toxin-insensitive URF13 mutant shows considerable toxin binding in E. coli, demonstrating that toxin binding can occur without causing membrane permeabilization. Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URF13.