Isolation and characterization of the cytoplasmic male sterility-associated <Emphasis Type="Italic">orf456</Emphasis> gene of chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3′-end of the coxII gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45% of the T₁ transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T₁ plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene – from among the many CMS-associated mitochondrial genes – for determining the male-sterile phenotype of CMS in chili pepper. GenBank accession number DQ116040 (orf456 genomic sequence), DQ126683 (pepper coxII genomic sequence)     

Genetic characterization of a new cytoplasmic male sterility system (<Emphasis Type="Italic">hau</Emphasis>) in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">juncea</Emphasis> and its transfer to <Emphasis Type="Italic">B. napus</Emphasis>

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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Alloplasmic male-sterile <Emphasis Type="Italic">Brassica juncea</Emphasis> with <Emphasis Type="Italic">Enarthrocarpus lyratus</Emphasis> cytoplasm and the introgression of gene(s) for fertility restoration from cytoplasm donor species

A new cytoplasmic male sterility (CMS) source in Brassica juncea (2n = 36; AABB) was developed by substituting its nucleus into the cytoplasm of Enarthrocarpus lyratus (2n = 20; E(l)E(l)). Male sterility was complete, stable and manifested in either petaloid- or rudimentary-anthers which were devoid of fertile pollen grains. Male sterile plants resembled the euplasmic B. juncea except for slight leaf yellowing and delayed maturity. Leaf yellowing was due mainly to higher level of carotenoids rather than a reduction in chlorophyll pigments. Female fertility in male-sterile plants varied; it was normal in lines having rudimentary anthers but poor in those with petaloid anthers. Each of the 62 evaluated germplasm lines of B. juncea was a functional maintainer of male sterility. The gene(s) for male-fertility restoration ( Rf) were introgressed from the cytoplasm donor species through homoeologous pairing between A and E(l) chromosomes in monosomic addition plants (2n = 18II+1E(l)). The percent pollen fertility of restored F(1) ( lyr CMS x putative restorer) plants ranged from 60 to 80%. This, however, was sufficient to ensure complete seed set upon by bag selfing. The CMS ( lyr) B. juncea compared favourably with the existing CMS systems for various productivity related characteristics. However, the reduced transmission frequency of the Rf gene(s) through pollen grains, which was evident from the sporadic occurrence of male-sterile plants in restored F(1) hybrids, remains a limitation.     

Mitochondrially-targeted expression of a cytoplasmic male sterility-associated <Emphasis Type="Italic">orf220</Emphasis> gene causes male sterility in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Background  

The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility.     

Mitochondrial <Emphasis Type="Italic">atpA</Emphasis> gene is altered in a new <Emphasis Type="Italic">orf220</Emphasis>-type cytoplasmic male-sterile line of stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

The purpose of this research is to identify the probable mitochondrial factor associated with cytoplasmic male sterility (cms) by comparative analysis of cms and its isogenic maintainer lines in stem mustards. Dramatic variations in the morphology of floral organs were observed in cms stem mustard. Mitochondrial atpA gene was shown to be altered in cms compared with that in its maintainer line, of which mitochondrial atpA gene from its maintainer line was sequenced to encode 507 amino acids. It was indicative of high homology with mitochondrial atpA genes from other species, even as high as 94% in similarity with Oryza sativa in terms of amino acid constituents. However, only 429 amino acids were deduced in cms showing 83% similarity with atpA gene from its maintainer line. Two copies were observed in its maintainer line, but only one was found in cms. Such numerous differences of mitochondrial atpA gene between cms and its maintainer lines may not be the results of evolutionary divergence but the rearrangements of mitochondria. Expression of mitochondrial atpA gene was shown to be down-regulated in cms by using Northern blot. Consequently, mitochondrial ATP synthesis was severely decreased more than one fold in cms stem mustard indicating deficiency in mitochondrial ATP synthesis in this type of cms. Therefore, we deduced that mitochondrial atpA gene altered in cms could be associated with male-sterility in this type of cms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jing-Hua Yang and Yan Huai contributed equally to this work.     

Expression of a mitochondrial gene <Emphasis Type="Italic">orfH79</Emphasis> from the CMS-HongLian rice inhibits <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> growth and causes excessive ROS accumulation and decrease in ATP

Cytoplasmic male sterility (CMS) has often been associated with abnormal mitochondrial open reading frames (ORF), orfH79 is a mitochondria chimeric gene being responsible for the CMS trait in Honglian (HL) rice. Weakly expressed ORFH79 strongly inhibits the growth of yeast cells. In addition, the content of reactive oxygen species (ROS) in the transformants that expressed ORFH79 was increased by 31%, and ATP was decreased by 41% compared with the control. These results showed ORFH79 peptide is toxic to yeast cells.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.     

<Emphasis Type="Italic">Brassica napus</Emphasis> lines with rearranged <Emphasis Type="Italic">Arabidopsis</Emphasis> mitochondria display CMS and a range of developmental aberrations

Numerous Brassica napus (+) Arabidopsis thaliana somatic hybrids were screened for male sterility and aberrant flower phenotypes. Nine hybrids were selected and backcrossed recurrently to B. napus. The resulting lines displayed stable maternal inheritance of flower phenotypes. Nuclear and organellar genomes were characterized molecularly using RFLP analysis. No DNA from A. thaliana was found in the nuclear genome after six back-crosses, whilst the mitochondrial genomes contained rearranged DNA from both A. thaliana and B. napus. Each line tested had a unique RFLP pattern of the mitochondrial DNA (mtDNA) that remained unchanged between the BC(3) and BC(6) generation. The plastid genomes consisted of B. napus DNA. Five lines of the BC(5) generation were subjected to more comprehensive investigations of growth, morphology and fertility. On the basis of these investigations, the five CMS lines could be assigned to two groups, one represented by three lines displaying reduced vegetative development, complete male sterility, and homeotic conversions of stamens into feminized structures. The second group, represented by the other two lines, were not completely male-sterile but still displayed severely affected flower morphologies. These two lines did not display any reduction in vegetative development. For both groups only stamens and petals suffered from the morphological and functional aberrations, while the sepals and pistils displayed normal morphology. All plants were fully female-fertile. Different rearrangements of the mitochondrial genome disturbed nuclear-mitochondrial interactions and led to various types of aberrant growth and flower development. The existence of numerous CMS lines with different mitochondrial patterns involving a species with a sequenced genome offers new opportunities to investigate the genetic regulation of CMS and its associated developmental perturbations.     

Identification of a novel cytoplasmic male sterile line M2BS induced by partial-length <Emphasis Type="Italic">HcPDIL5-2a</Emphasis> transformation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A cytoplasmic male sterile line (designated as M2BS) was obtained from an indica rice maintainer M2B induced by partial-length HcPDIL5-2a (Hibiscus cannabinus protein disulfide isomerase-like) transformation. The anther of M2BS was short, slender, hygrophanous, and fissured. I2-KI staining method showed that there was typical and spherical abortion in pollen grains. M2BS was found abortive at middle and late stage of monocyte by the modified carbol fuchsin stained observation and paraffin section observation. The tapetum was observed pre-degenerated in M2BS. Hereditary analysis indicated that the male sterility of M2BS was a maternally inherited inability after six backcross generations with M2B and the combinations of M2BS hybridized with other two male fertile materials. The M2BS could be affirmed a cytoplasmic male sterile (CMS) type. Moreover, it was a transgenic plant confirmed by PCR, Southern blot and RT-PCR detection. M2BS could be distinguished from M2B and its CMS line M2A by RFLP analysis. The overall mitochondrial genome sequencing results showed, that in M2BS, the main differences of mitochondrial gene sequence were located in nad4, nad5, nad7, orf194 and intergenic region, relatively to those of M2A. The obtained results indicate that M2BS is a novel cytoplasmic male sterile line.     

Isolation and characterization of a male sterility gene homolog <Emphasis Type="Italic">BcMS2</Emphasis> from Chinese cabbagge-pak-choi that expressing in an anther-specific manner

A male sterility gene homolog, designated BcMS2, was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF093533. Comparison of BcMS2 gene with MS2 from Arabidopsis thaliana and MS2Bnap from Brassica napus revealed some differences in gene structure and evolution. The full genomic DNA sequence of BcMS2 was 2,576 bp in length containing 8 exons and 7 introns, more than those of MS2Bnap but less than MS2. RT-PCR showed that BcMS2 gene expressed only in stage III flower buds of male fertile Chinese cabbage-pak-choi 'ZUBajh97-01B' and there were no detection in all organs of Polima cytoplasmic male sterility (CMS) line 'Bpol97-05A' and Ogura CMS line 'Bogu97-06A'. Furthermore, RT-PCR revealed that BcMS2 expressed only in anthers of male fertile material and there were no expression in sepals, petals, filaments and pistils. These results suggested that BcMS2 was an anther-specific gene and might be essential for the fertility of Chinese cabbage-pak-choi.     

Cytological and ultra-structural study on microsporogenesis of cytoplasmic male sterility in <Emphasis Type="Italic">Raphanus sativus</Emphasis>

The cytological development of microspores and tapetum in cytoplasmic male sterile (CMS) line A₁₄ and its maintainer B₁₄ in radish were studied using light- and transmission electron microscopy (LM and TEM). The microspores of the CMS line began to abort soon after they were released from tetrads in pollen sacs with light microscopy investigation, while abnormal behavior of pollen mother cells (PMC) were observed during its meiotic stage in its ultra-structural study, including degeneration of organelles and irregularity of nuclear membrane. At the same time, development of tapetal cells was similar to that of the maintainer. With further development of the anther, the tapetal cells of CMS line showed an abnormal increase in size and other appearances, such as fewer organelles and indistinct cytoplasm. The microspores of the CMS line were always distinguishable from the maintainer line with irregular structure, more osphilic deposits and abnormal exine. It is inferred that abortion of microspores is attributed to mutation of genes controlling male sterility, which further leads to hypertrophy of tapetum and destruction of ultra-structure.     

Analysis of genetic diversity in cytoplasmic male sterility,and association of mitochondrial genes with petaloid-type cytoplasmic male sterility in tuber mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tumida</Emphasis> 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.     

Shared molecular characteristics of successfully transformed mitochondrial genomes in <Emphasis Type="Italic">Chlamydomonas</Emphasis><Emphasis Type="Italic">reinhardtii</Emphasis>

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.