Cytoplasmic-nuclear male sterility (CNMS), also called nuclear-cytoplasmic male sterility, is a form of plant male sterility caused by an interaction between cytoplasmic genetic factors and nuclear genes. It is usually associated with maternally inherited factors in the mitochondrial genome and with nuclear genes that either maintain sterility or restore male fertility. Plants with cytoplasmic-nuclear male sterility fail to produce functional pollen, anthers or male gametes, while female fertility is usually retained. In much of the English-language literature, cytoplasmic-nuclear male sterility is treated as a type or functional description of cytoplasmic male sterility (CMS). The term emphasizes that sterility is not determined by cytoplasm alone, but by disharmonious interaction between cytoplasmic sterility factors and the nuclear genome. Nuclear restorer-of-fertility genes, commonly abbreviated as Rf genes, can suppress or compensate for the effect of the sterility-associated mitochondrial factor and restore pollen fertility. Cytoplasmic-nuclear male sterility is important in plant breeding because male-sterile plants can be used as female parents without manual emasculation. It has been used in hybrid seed production in crops such as rice, maize, sorghum, pearl millet, rapeseed, sunflower, sugar beet, onion and pepper.
Concept and inheritance Cytoplasmic-nuclear male sterility differs from genic male sterility, which is controlled primarily by nuclear genes and generally follows Mendelian inheritance. In cytoplasmic-nuclear male sterility, the sterile phenotype results from the combination of a cytoplasmic sterility factor and a nuclear genetic background that fails to restore fertility. Most studied systems involve the mitochondrial genome rather than the chloroplast genome. In breeding literature, cytoplasm is often described as either normal cytoplasm (N) or sterile cytoplasm (S). A plant with S cytoplasm and no effective restorer gene is usually male sterile. A plant with S cytoplasm and an effective nuclear restorer gene may regain male fertility. A plant with N cytoplasm is normally male fertile in most nuclear backgrounds. Because mitochondria are usually transmitted through the egg cell in higher plants, the cytoplasmic component of sterility is maternally inherited, while nuclear restorer genes are inherited from both parents. Fertility restoration may be sporophytic or gametophytic. In sporophytic restoration, pollen fertility is determined mainly by the genotype of the plant producing the pollen. In gametophytic restoration, the genotype of each pollen grain influences its own development, and pollen grains lacking the restorer allele may abort. The number, dominance and effectiveness of restorer genes vary among crops and CMS systems.
Molecular basis The cytoplasmic factors in cytoplasmic-nuclear male sterility usually arise from the plant mitochondrial genome. Plant mitochondrial genomes are large, recombinogenic and structurally variable. Recombination and rearrangement can generate novel or chimeric open reading frames, many of which are located near, fused with or co-transcribed with genes involved in mitochondrial respiration or ATP synthesis. These abnormal mitochondrial sequences or proteins may interfere with anther development, pollen development, mitochondrial function or programmed cell death. Anther and pollen development are particularly sensitive to mitochondrial activity, energy supply and programmed cell death. The tapetum supplies nutrients and wall materials to developing microspores. Premature, delayed or abnormal tapetal degeneration can lead to pollen abortion. In sunflower, the PET1 CMS mitochondrial mutation is associated with premature programmed cell death and cytochrome c release. In wild-abortive rice CMS, the mitochondrial protein WA352 causes detrimental mitochondrial-nuclear interaction and abnormal cell death during anther development. Many cloned Rf genes encode mitochondrially targeted pentatricopeptide repeat (PPR) proteins. These proteins bind specific mitochondrial RNAs and influence RNA processing, cleavage, editing, stability or translation, thereby reducing the expression or accumulation of sterility-associated mitochondrial products. A PPR-containing Rf gene was identified in petunia, and PPR-type restorer genes have also been characterized in rice and the Ogura CMS system of Brassicaceae crops. Not all restorer genes encode PPR proteins. In T-cytoplasm maize, the nuclear restorer gene rf2 encodes a mitochondrial aldehyde dehydrogenase, indicating that fertility restoration can also involve mitochondrial metabolism and anther developmental pathways.
Use in three-line hybrid breeding The main agricultural use of cytoplasmic-nuclear male sterility is in three-line hybrid seed production. The system consists of a male-sterile line, a maintainer line and a restorer line. The male-sterile line, often called the A line, has sterile cytoplasm and a nuclear genotype that does not restore fertility. The maintainer line, or B line, has normal cytoplasm and a nuclear genotype similar to that of the A line, allowing it to pollinate and maintain the sterile line. The restorer line, or R line, carries one or more Rf genes and restores fertility in the hybrid progeny produced from the A line. The use of a CMS-based three-line system requires the identification of a stable CMS source, development of seed parents with complete sterility, maintenance of the A line, identification and improvement of restorer parents, and management of hybrid seed production. Isolation distance, removal of off-types, flowering synchrony between A and R lines, and the ratio of female to male rows are important components of hybrid seed production.
Yuan Longping and hybrid rice Yuan Longping played a major role in applying cytoplasmic-nuclear male sterility to hybrid rice breeding. In the 1960s, Yuan proposed that naturally occurring male-sterile rice plants could make large-scale hybrid rice production possible. In 1966, he published a report on male sterility in rice, linking the exploitation of heterosis in rice with the search for and use of male-sterile materials. In 1970, members of Yuan's research group found a male-sterile wild rice plant in Hainan. The material later became known as the wild-abortive, or WA, cytoplasm and provided an important cytoplasmic source for the development of three-line hybrid rice in China. By the early 1970s, male-sterile, maintainer and restorer lines had been assembled into a practical three-line breeding system. The successful use of WA-type CMS was a central step in the commercialization of hybrid rice in China. Yuan's contribution was not limited to the discovery and use of male-sterile materials. He and his collaborators helped develop an operational breeding and seed-production system that combined male sterility, maintainer relationships, fertility restoration and heterosis. Later hybrid rice research in China expanded from the three-line CMS system to two-line systems based on environment-sensitive genic male sterility, but the three-line system remained a major example of the practical use of cytoplasmic-nuclear male sterility.
Crop systems
Rice Rice has several well-studied CMS systems, including wild-abortive (WA), Honglian (HL) and Boro II (BT) types. In WA-type rice CMS, the mitochondrial gene WA352 encodes a protein that causes detrimental mitochondrial-nuclear interaction and abnormal cell death in anthers. The nuclear restorer gene Rf4 encodes a mitochondrially localized PPR protein that reduces WA352 transcript accumulation and restores fertility. In Honglian CMS, the rice PPR protein RF5 restores fertility through a complex with the glycine-rich protein GRP162 and affects sterility-associated mitochondrial transcripts. In Boro II CMS, sterility is caused by a cytotoxic mitochondrial peptide and is restored by related PPR motif genes through distinct modes of mRNA silencing.
Maize In maize, cytoplasmic-nuclear male sterility systems include T, C and S cytoplasms. T-cytoplasm maize was once widely used in hybrid seed production in the United States. However, T-cytoplasm plants were highly susceptible to the T race of the southern corn leaf blight pathogen. The 1970 southern corn leaf blight epidemic demonstrated the risk of relying heavily on a single cytoplasmic background and led to a major reduction in the commercial use of T cytoplasm.
Rapeseed, sunflower and other crops In rapeseed and other Brassicaceae crops, Ogura CMS is an important cytoplasmic source derived from radish cytoplasm. The restorer gene Rfo encodes a PPR protein that restores fertility in Ogura CMS. Other widely studied systems include PET1 CMS in sunflower, A1 CMS in sorghum, several CMS sources in pearl millet and S cytoplasm in onion. These systems differ in sterility source, restorer genetics, stability, ease of seed production and agronomic value.
Advantages and limitations The major advantage of cytoplasmic-nuclear male sterility is that it reduces the cost and labor of hybrid seed production. In self-pollinating or hermaphroditic crops, manual emasculation is inefficient and may lead to selfed seed contamination. A male-sterile female parent cannot produce viable pollen and therefore receives pollen from the selected male parent or restorer line. Several limitations affect its use. Sterility must remain stable across environments and genetic backgrounds. Restorer genes must be available and should not be closely linked with undesirable agronomic traits. Dependence on a single cytoplasmic source can narrow the genetic base of commercial cultivars and increase vulnerability to disease or environmental stress, as illustrated by the history of T-cytoplasm maize. Research on cytoplasmic-nuclear male sterility has also contributed to the study of organelle–nuclear coevolution, mitochondrial gene expression, RNA processing, anther development and plant reproductive evolution. Modern approaches include mitochondrial genome sequencing, molecular cloning of restorer genes, marker-assisted selection and genome editing.
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