Cytoplasmic Male Sterile Plants Research Articles

Analysis of genetic diversity in cytoplasmic male sterility, and association of mitochondrial genes with petaloid-type cytoplasmic male sterility in tuber mustard (Brassica juncea var. tumida Tsen et Lee)

In our previous study, we bred a stable cytoplasmic male sterility (CMS) line of tuber mustard by using distant hybridization and subsequent backcrosses. In this CMS plants, all floral organs are normal except the anthers, which are transformed into petals or tubular structures. Recently, 2 mitochondrial genes-atpA and orf220-that are distinctively present in the CMS line of tuber mustard were cloned and partially characterized. In our study of genetic diversity analysis of CMS, 7 species of Brassica and Raphanus crops, which included 5 CMS lines and their respective maintainer lines, were used to compare the constitution of protein-coding genes in the mitochondrial genomes. In 4 of the 43 mitochondrial genes, namely, atpA, orf220, orf256, and orf305/orf324, polymorphisms were detected among the tuber mustard CMS line and its maintainer line. The results of a cluster analysis indicate that petaloid CMS phenotype of tuber mustard is a novel CMS type and is nearer to the nap CMS in Brassica napus at the phylogenetic level. The results of individual amplifications of these genes indicate the presence of 4 sequence-characterized amplified region (SCAR) markers, which enable rapid and reliable identification of this CMS. Expressions of the orf220 and orf256 genes were detected only in the CMS line, while expression of the orf305 gene was detected in the maintainer line. The different expression patterns of different mitochondrial-specific marker genes indicate that the quantity of mitochondrial proteins is differentially regulated during organ/tissue development in tuber mustard. The results of this study suggest that the above mentioned 4 mitochondrial genes are associated with the petaloid CMS phenotype in tuber mustard.

Genetic characterization of a new cytoplasmic male sterility system (hau) in Brassica juncea and its transfer to B. napus

A novel cytoplasmic male sterility (CMS) was identified in Brassica juncea, named as hau CMS (00-6-102A). Subsequently, the male sterility was transferred to B. napus by interspecific hybridization. The hau CMS has stable male sterility. Flowers on the A line are absolutely male sterile, and seeds harvested from the line following pollinations with the maintainer gave rise to 100% sterile progeny. The anthers in CMS plants are replaced by thickened petal-like structures and pollen grains were not detected. In contrast, in other CMS systems viz. pol, nap, tour, and ogu, anthers are formed but do not produce viable pollen. The sterility of hau CMS initiates at the stage of stamen primordium polarization, which is much earlier compared with the other four CMS systems. We have successfully transferred hau CMS from B. juncea to B. napus. Restorer lines for pol, ogu, nap, and tour CMS systems were found to be ineffective to restore fertility in hau CMS. Sixteen out of 40 combinations of mitochondrial probe/enzyme used for RFLP analysis distinguished the hau CMS system from the other four systems. Among these sixteen combinations, five ones alone could distinguish the five CMS systems from each other. The evidence from genetic, morphological, cytological and molecular studies confirmed that the hau CMS system is a novel CMS system.

An Alloplasmic Male-sterile Line of Brassica oleracea Harboring the Mitochondria from Diplotaxis muralis Expresses a Novel Chimeric Open Reading Frame, orf72

Nuclear so-called fertility-restorer genes reverse the pollen sterility of cytoplasmic male-sterile (CMS) plants caused by disturbed mitochondrial-nuclear interactions. We identified a CMS-associated chimeric mitochondrial gene in an alloplasmic CMS line of Brassica oleracea in the 'mur' system. This novel chimeric gene, orf72, was found in the mitochondrial genome of donor cytoplasm. It was located downstream of normal rps7 and contained part of atp9 (atp9-b). It was expressed specifically on the nuclear background of CMS B. oleracea, partially suppressed in the fertility-restored line and entirely suppressed in the cytoplasmic donor.

Expression levels of meristem identity and homeotic genes are modified by nuclear–mitochondrial interactions in alloplasmic male‐sterile lines of Brassica napus

Homeotic conversions of anthers were found in cytoplasmic male sterile (CMS) plants of Brassica napus derived from somatic hybrids of B. napus and Arabidopsis thaliana. CMS line flowers displayed petals reduced in size and width and stamens replaced by carpelloid structures. In order to investigate when these developmental aberrations appeared, flower development was analysed histologically, ultrastructurally and molecularly. Disorganized cell divisions were detected in the floral meristems of the CMS lines at stage 4. As CMS is associated with mitochondrial aberrations, ultrastructural analysis of the mitochondria in the floral meristems was performed. Two mitochondrial populations were found in the CMS lines. One type had disrupted cristae, while the other resembled mitochondria typical of B. napus. Furthermore, expression patterns of genes expressed in particular floral whorls were determined. In spite of the aberrant development of the third whorl organs, BnAP3 was expressed as in B. napus during the first six stages of development. However, the levels of BnPI were reduced. At later developmental stages, the expression of both BnAP3 and BnPI was strongly reduced. Interestingly the expression levels of genes responsible for AP3 and PI activation such as LFY, UFO and ASK1 were higher in the CMS lines, which indicates that activation of B-genes in the CMS lines does not occur as in B. napus. Disrupted and dysfunctional mitochondria seem to be one of the first aberrations manifested in CMS which result in a retrograde influence of the expression levels of genes responsible for the second and third whorl organ differentiation.

Presence of potential allergy‐related linear epitopes in novel proteins from conventional crops and the implication for the safety assessment of these crops with respect to the current testing of genetically modified crops

Mitochondria of cytoplasmic male sterile crop plants contain novel, chimeric open reading frames. In addition, a number of crops carry endogenous double-stranded ribonucleic acid (dsRNA). In this study, the novel proteins encoded by these genetic components were screened for the presence of potential binding sites (epitopes) of allergy-associated IgE antibodies, as was previously done with transgenic proteins from genetically modified crops. The procedure entails the identification of stretches of at least six contiguous amino acids that are shared by novel proteins and known allergenic proteins. These stretches are further checked for potential linear IgE-binding epitopes. Of the 16 novel protein sequences screened in this study, nine contained stretches of six or seven amino acids that were also present in allergenic proteins. Four cases of similarity are of special interest, given the predicted antigenicity of the identical stretch within the allergenic and novel protein, the IgE-binding by a peptide containing an identical stretch reported in literature, or the multiple incidence of identical stretches of the same allergen within a novel protein. These selected stretches are present in novel proteins derived from oilseed rape and radish (ORF138), rice (dsRNA), and fava bean (dsRNA), and warrant further clinical testing. The frequency of positive outcomes and the sizes of the identical stretches were comparable to those previously found for transgenic proteins in genetically modified crops. It is discussed whether novel proteins from conventional crops should be subject to an assessment of potential allergenicity, a procedure which is currently mandatory for transgenic proteins from genetically modified crops.

Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish.

Cytoplasmic male sterility (CMS) in plants is a maternally inherited inability to produce functional pollen, and is often associated with mitochondrial DNA abnormalities. Specific nuclear loci that suppress CMS, termed as restorers of fertility (Rf), have been identified. Previously, we identified an Rf for the CMS Kosena radish and used genetic analysis to identify the locus and create a contig covering the critical interval. To identify the Rf gene, we introduced each of the lambda and cosmid clones into the CMS Brassica napus and scored for fertility restoration. Fertility restoration was observed when one of the lambda clones was introduced into the CMS B. napus. Furthermore, introduction of a 4.7-kb BamHI/HpaI fragment of the lambda clone is enough to restore male fertility. A cDNA strand isolated from a positive fragment contained a predicted protein (ORF687) of 687 amino acids comprising 16 repeats of the 35-amino acid pentatricopeptide repeat (PPR) motif. Kosena CMS radish plants were found to express an allele of this gene possessing four substituted amino acids in the second and third repeats of the PPR suggesting that the domains formed by these repeats in ORF687 are essential for fertility restoration. Protein levels of the Kosena CMS-associated mitochondrial protein ORF125 were considerably reduced in plants in which fertility was restored, although mRNA expression was normal. Regarding the possible role for PPR-containing proteins in the regulation of the mitochondrial gene, we propose that ORF687 functions either directly or indirectly to lower the levels of ORF125, resulting in the restoration of fertility in CMS plants.

Involvement of polyamines in cytoplasmic male sterility of stem mustard (Brassica juncea var. tsatsai)

Polyamines (free and conjugates) and the activities of enzymes, arginine decarboxylase (EC 4.1.1.19) and ornithine decarboxylase (EC 4.1.1.17), which are involved in the polyamine biosynthesis, were examined at different stages of flower development in floral organs of CMS and normal stem mustard (Brassica juncea var. tsatsai) in order to establish the correlation of polyamines with cytoplasmic male sterility. A lower total fresh weight of all floral organs, especially stamens, in cytoplasmic male sterile plants was observed compared to normal plants. Putrescine was the predominant polyamine in various floral organs of cytoplasmic male sterile and normal plants. During the flowering stage, all free polyamines and conjugates examined were present at a lower level in floral organs of cytoplasmic male sterile plants compared to normal plants. The activities of arginine decarboxylase and ornithine decarboxylase were higher in normal plants than in cytoplasmic male sterile plants, increasing steadily in the gynoecium and sepals during flowering, but declining in the stamen and during petal flower opening. The possible correlation between these results and cytoplasmic male sterility in stem mustard are discussed.

Obtaining an ogura-type CMS line from asymmetrical protoplast fusion between cabbage (fertile) and radish (fertile)

Cytoplasmic male sterile (CMS) plants were obtained by asymmetrical protoplast fusion between red cabbage (fertile) and normal radish (fertile). The CMS plants showed restriction fragment length polymorphism (RFLP) patterns of mitochondrial (mt) DNA that were different from those of both parental lines. PCR analysis of mtDNA of the CMS plants and though Southern blot analysis showed that the mtDNA of the CMS line had the characteristics of the ogura CMS type. The results suggested that the orf138 gene and the ogura type atp6 gene are present in normal fertile cabbage and radish at a low copy level.

The nucleo-mitochondrial conflict in cytoplasmic male sterilities revisited.

Cytoplasmic male sterility (CMS) in plants is a classical example of genomic conflict, opposing maternally-inherited cytoplasmic genes (mitochondrial genes in most cases), which induce male sterility, and nuclear genes, which restore male fertility. In natural populations, this type of sex control leads to gynodioecy, that is, the co-occurrence of female and hermaphroditic individuals within a population. According to theoretical models, two conditions may maintain male sterility in a natural population: (1) female advantage (female plants are reproductively more successful than hermaphrodites on account of their global seed production); (2) the counter-selection of nuclear fertility restorers when the corresponding male-sterility-inducing cytoplasm is lacking. In this review, we re-examine the model of nuclear-mitochondrial conflict in the light of recent experimental results from naturally occurring CMS, alloplasmic CMS (appearing after interspecific crosses resulting from the association of nuclear and cytoplasmic genomes from different species), and CMS plants obtained in the laboratory and carrying mitochondrial mutations. We raise new hypotheses and discuss experimental models that would take physiological interactions between cytoplasmic and nuclear genomes into account.

Controlling the release of pollen from genetically modified maize and increasing its grain yield by growing mixtures of male-sterile and male-fertile plants

There is public concern about the consequences of pollen dispersal from genetically modified (GM) crops. There lease of viable pollen from GM maize can be controlled by growing mixtures of cytoplasmic male-sterile plants and male-fertile non-transformed pollinator plants. Our experiments indicate that such associations can bring about grain yields as high or even higher than those produced by pure male-fertile maize crops, especially when the male-sterile component is pollinated non-isogenically. The grain yield benefits from cytoplasmic malesterility and xenia as well as the fact that seed of male-sterile varieties can be produced cheaply and reliably in large quantities would facilitate the implementation of the proposed system in agricultural practice.

Revealing a savior of manly vigor in plants

The superior performance of heterozygous hybrid progeny over its homozygous parents is widely exploited in crop production. Hybrid seed production is facilitated by the maternally inherited absence of viable pollen in cytoplasmic male-sterile plants, thereby eliminating the need of hand emasculation. Cytoplasmic male-sterility (CMS) occurs in many plant species, and is usually ascribed to mitochondrial defects. If seeds are the harvested products, the use of restorer genes suppressing the male-sterile phenotype in the hybrids is essential. These opposite players, called restorers of fertility (Rf), are able to avoid or overcome the mitochondrial defects and are encoded in the nuclear genome. However, the nature of these components and the mechanisms underlying the restoration of fertility through changing the expression of CMS-associated mitochondrial genes were unknown.

A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants.

Known in over 150 species, cytoplasmic male sterility is encoded by aberrant mitochondrial genes that prevent pollen development. The RNA- or protein-level expression of most of the mitochondrial genes encoding cytoplasmic male sterility is altered in the presence of one or more nuclear genes called restorers of fertility that suppress the male-sterile phenotype. Cytoplasmic male sterility/restorer systems have been proven to be an invaluable tool in the production of hybrid seeds. Despite their importance for both the production of major crops such as rice and sunflower and the study of organelle/nuclear interactions in plants, none of the nuclear fertility-restorer genes that reduce the expression of aberrant mitochondrial proteins have previously been cloned. Here we report the isolation of a gene directly involved in the control of the expression of a cytoplasmic male sterility-encoding gene. The Petunia restorer of fertility gene product is a mitochondrially targeted protein that is almost entirely composed of 14 repeats of the 35-aa pentatricopeptide repeat motif. In a nonrestoring genotype we identified a homologous gene that exhibits a deletion in the promoter region and is expressed in roots but not in floral buds.

A chimeric open reading frame associated with cytoplasmic male sterility in alloplasmic wheat with Triticum timopheevi mitochondria is present in several Triticum and Aegilops species, barley, and rye.

Mitochondrial DNA from Triticum timopheevi has a chimeric gene, orf256, upstream of coxI. This gene is cotranscribed with coxI in cytoplasmic male sterile plants and produces a 7-kDa protein which is not produced in fertile or fertility-restored plants. T. aestivum, the nuclear donor in sterile plants, does not have orf256. Analysis by polymerase chain reaction of DNA from barley, rye, Aegilops bicornis, Ae. searsii, Ae. sharonensis, Ae. speltoides, Ae. tauschii, T. monococcum, and T. turgidum was done with oligonucleotide primers designed to detect orf256 or coxI sequences. Except for T. turgidum, these plants have various elements of the orf256 sequence over a 1-kb length of DNA immediately upstream of coxI in exactly the same arrangement as is found in the coxI region of T. timopheevi. Only T. timopheevi and Ae. speltoides have orf256 transcripts, and only cytoplasmic male-sterile plants involving these two species as maternal donors produce a protein from orf256. Part of an orf256-like sequence is present in T. turgidum but is at least slightly different in arrangement relative to coxI, as compared with the sequence in T. timopheevi. Neither maize nor sorghum have the orf256 sequence.

Three QTL Regions for Restoration of Owen CMS in Sugar Beet

Three quantitative trati loci (QTL) regions for the restoration of Owen CMS (cytoplasmic male sterility) in sugar beet (Beta vulgaris L.ssp. vulgaris) were identified through analysis of three populations segregating for genes restoring the fertility of cytoplasmic male sterile plants. Two large populations, A and B, were studied by means of 51 and 42 polymorphic and evenly distributed restriction fragment length polymorphism (RFLP) markers, respectively. A smaller population, C, was analyzed by use of six RFLP markers. Composite interval mapping in Population A detected a strong signal on linkage group IV that explained 79% of the phenotypic variation. More detailed analysis revealed the presence of two linked QTL on this chromosome. In Population B, a QTL that explained 72% of the phenotypic variation was detected at one end of linkage group III. Single‐marker analysis of Population C resulted in significant signals for markers on linkage group IV, explaining 57% of the phenotypic variation. In each of the two large populations, what appeared to be minor alleles were also detected in the QTL regions.

Expression of male sterility in alloplasmic Brassica juncea with Erucastrum canariense cytoplasm and the development of a fertility restoration system

AbstractAn alloplasmic mustard, Brassica juncea, has been synthesized by placing its nucleus into the cytoplasm of the related wild species Erucastrum canariense to express cytoplasmic male sterility. To achieve this, the sexual hybrid E. canariense (2n=18, EcEc) ×Brassica campestris (2n= 20, AA) was repeatedly backcrossed to B. juncea (2n= 36, AABB). Cytoplasmic male‐sterile (CMS) plants were recovered in the BC4 generation. These plants are a normal green and the flowers have slender, non‐dehiscing anthers that contain sterile pollen. Nectaries are well developed and female fertility is > 90%. The fertility restoration gene was introgressed to CMS B. juncea from the cytoplasmic donor E. canariense through pairing between chromosomes belonging to B. juncea with those of the E. canariense genome. The restorer plants have normal flowers, with well‐developed anthers containing fertile pollen. Meiosis proceeds normally. Pollen and seed fertility averaged 90% and 82%, respectively. F1 hybrids between CMS and the restorer are fully pollen fertile and show normal seed set. Preliminary results indicate that restoration is achieved by a single dominant gene. The constitution of the organelle genomes of the CMS, restorer and fertility restored plants is identical, as revealed by Southern analysis using mitochondrial and chloroplast probes atp A and psb D, respectively.

A novel orfB-related gene of carrot mitochondrial genomes that is associated with homeotic cytoplasmic male sterility (CMS).

Three types of orfB-related genes, orfB-F1, orfB-F2 and orfB-CMS, were found in carrot mitochondrial genomes. OrfB-F1 has a structure similar to the reported orfB gene in sunflower, and orfB-F2 is a novel chimeric gene with about 200 bp of unknown sequence at the 3' end of the orfB-related sequence. All fertile plants examined contained either orfB-F1 or orfB-F2. OrfB-CMS is also a novel chimeric orfB-related gene with an additional 170 bp of unknown sequence at the 3' end. Fifteen carrot lines including petaloid CMS were examined by PCR analysis and all petaloid CMS plants were found to contain the orfB-CMS gene. The orfB-F2 and orfB-CMS genes coexist in three lines, and these lines exhibit a CMS phenotype, suggesting that the CMS phenotype is associated with orfB-CMS and is independent of the presence of orfB-F2. Interestingly, differences in predicted amino acid sequence between orfB-CMS and orfB-F2 were very limited and restricted to the carboxy-terminal region of the protein. The orfB-related genes were transcribed as expected from their DNA structures, but orfB-CMS protein accumulated only in floral organs of the CMS lines. Four RNA editing sites were common to orfB-CMS and orfB-F2, and C-to-U editing occurred in both floral and leaf organs for orfB-CMS. These results strongly suggest that the orfB-CMS gene is closely associated with the petaloid phenotype and its expression is not regulated by RNA editing, but by post-transcriptional events.

A Moricandia arvensis– based cytoplasmic male sterility and fertility restoration system in Brassica juncea

A cytoplasmic male-sterility system has been developed in mustard (Brassica juncea) following repeated backcrossings of the somatic hybrid Moricandia arvensis (2n=28, MM)+B. juncea (2n=36, AABB), carrying mitochondria and chloroplasts from M. arvensis, to Brassica juncea. Cytoplasmic male-sterile (CMS) plants are similar to normal B. juncea; however, the leaves exhibit severe chlorosis resulting in delayed flowering. Flowers are normal with slender, non-dehiscent anthers and excellent nectaries. CMS plants show regular meiosis with pollen degeneration occurring during microsporogenesis. Female fertility was normal. Genetic information for fertility restoration was introgressed following the development of a M. arvensis monosomic addition line on CMS B. juncea. The additional chromosome paired allosyndetically with one of the B. juncea bivalents and allowed introgression. The putative restorer plant also exhibited severe chlorosis similar to CMS plants but possessed 89% and 73% pollen and seed fertility, respectively, which subsequently increased to 96% and 87% in the selfed progeny. The progeny of the cross of CMS line with the restorer line MJR-15, segregated into 1 fertile : 1 sterile. The CMS (Moricandia) B. juncea, the restorer (MJR-15), and fertility restored F1 plants possess similar cytoplasmic organellar genomes as revealed by ‘Southern’ analysis.

CMS due to tapetal failure in cybrids between Nicotiana tabacum and Petunia hybrida

Cytoplasmic hybrids (cybrids) between the two sexually incompatible species Nicotiana tabacum and Petunia hybrida were constructed. Three green plants were obtained after fusion of leaf protoplasts from a cytoplasmic chlorophyll deficient mutant of tobacco, with iodoacetamide inactivated protoplasts of P. hybrida. All regenerated plants were phenotypically similar to tobacco, but male and female sterile. Chromosome and isoenzyme analyses of the nuclear genome, and restriction and blot hybridization analyses of the organelle composition revealed that the regenerated cybrids possessed nuclear genome of N. tabacum, chloroplasts from P. hybrida and recombinant chondriomes. In vitro culture of ovules from one cybrid plant pollinated by N. tabacum resulted in the regeneration of cytoplasmic male sterile progeny plants. Cross-section of anthers from these CMS plants showed that male sterility was due to a failure of tapetum development.

Cell type-specific loss of atp6 RNA editing in cytoplasmic male sterile Sorghum bicolor.

RNA editing and cytoplasmic male sterility are two important phenomena in higher plant mitochondria. To determine whether correlations might exist between the two, RNA editing in different tissues of Sorghum bicolor was compared employing reverse transcription-PCR and subsequent sequence analysis. In etiolated shoots, RNA editing of transcripts of plant mitochondrial atp6, atp9, nad3, nad4, and rps12 genes was identical among fertile or cytoplasmic male sterile plants. We then established a protocol for mitochondrial RNA isolation from plant anthers and pollen to include in these studies. Whereas RNA editing of atp9, nad3, nad4, and rps12 transcripts in anthers was similar to etiolated shoots, mitochondrial atp6 RNA editing was strongly reduced in anthers of the A3Tx398 male sterile line of S. bicolor. atp6 transcripts of wheat and selected plastid transcripts in S. bicolor showed normal RNA editing, indicating that loss of atp6 RNA editing is specific for cytoplasmic male sterility S. bicolor mitochondria. Restoration of fertility in F1 and F2 lines correlated with an increase in RNA editing of atp6 transcripts. Our data suggest that loss of atp6 RNA editing contributes to or causes cytoplasmic male sterility in S. bicolor. Further analysis of the mechanism of cell type-specific loss of atp6 RNA editing activity may advance our understanding of the mechanism of RNA editing.

Nuclear fertility restorer genes map to the same linkage group in cytoplasmic male-sterile bean

Fertility restoration in cytoplasmic malesterile plants (CMS) by nuclear restorer genes is one of the few useful systems for studying nuclear-mitochondrial interactions in higher eukaryotes. In CMS bean there exist multiple independently identified nuclear genes that restore fertility. Two restorer genes (Fr and Fr 2) have been characterized previously. We have genetically characterized two additional restorer genes; both restorers are single genes that behave similarly to Fr 2. We compared the linkage relationship of all four independently identified restorer genes to understand the relationship among these loci further. All four genes map to the same linkage group. It was not possible to distinguish between the two newly identified restorer genes and Fr 2. We suggest that they may be allelic.