Mitochondrial ORF79 levels determine pollen abortion in cytoplasmic male sterile rice

Cytoplasmic male sterility (CMS) is an important agricultural trait characterized by lack of functional pollen, and caused by ectopic and defective mitochondrial gene expression. The pollen function in CMS plants is restored by the presence of nuclear‐encoded restorer of fertility (Rf) genes. Previously, we cloned Rf2, which restores the fertility of Lead Rice (LD)‐type CMS rice. However, neither the function of Rf2 nor the identity of the mitochondrial gene causing CMS has been determined in LD–CMS rice. Here, we show that the mitochondrial gene orf79 acts as a CMS‐associated gene in LD–CMS rice, similar to its role in BT–CMS rice originating from Chinsurah Boro II, and Rf2 weakly restores fertility in BT–CMS rice. We also show that RF2 promotes degradation of atp6–orf79 RNA in a different manner from that of RF1, which is the Rf gene product in BT–CMS rice. The amount of ORF79 protein in LD–CMS rice was one‐twentieth of the amount in BT–CMS rice. The difference in ORF79 protein levels probably accounts for the mild and severe pollen defects in LD–CMS and BT–CMS rice, respectively. In the presence of Rf2, accumulation of ORF79 was reduced to almost zero and 25% in LD–CMS and BT–CMS rice, respectively, which probably accounts for the complete and weak fertility restoration abilities of Rf2 in LD–CMS and BT–CMS rice, respectively. These observations indicate that the amount of ORF79 influences the pollen fertility in two strains of rice in which CMS is induced by orf79.     

Substoichiometric shifting in the fertility reversion of cytoplasmic male sterile pearl millet

Cytoplasmic male sterility (CMS) represents an important agricultural trait in pearl millet [Pennisetum glaucum (L.) R. Br.] with a value to the seed industry in facilitating economical hybrid seed production. Among the CMS systems available in millet, the A1 source is the most commonly used for hybrid production, but it can undergo low frequency reversion to fertility. Plant mitochondrial genomes are highly recombinogenic, becoming unstable and prone to ectopic recombination under conditions of tissue culture, somatic hybridization, or interspecific crossing. Similarly, CMS systems prone to spontaneous fertility reversion experience sporadic mitochondrial genome instability. We compared mitochondrial genome configurations between the male-sterile A1 line and fertile revertants of pearl millet to develop a model for millet mitochondrial genome reorganization upon reversion. Relative copy number of a subgenomic molecule containing the CoxI-1-2 junction region, a component of the recombination process for reversion, is amplified tenfold following reversion, relative to the CMS A1 line. We propose that increased copy number of this molecule in a small number of cells or at low frequency triggers a recombination cascade, likely during reproductive development. The proposed recombination process initiates with ectopic recombination through a 7-bp repeat to produce a novel CoxI-3-2 junction molecule and an unstable recombination intermediate. Subsequent intra-molecular recombination stabilizes the intermediate to form a new copy of CoxI accompanied by a deletion. This study furthers the argument that substoichiometric shifting within the plant mitochondrial genome plays an important role in the evolution of the mitochondrial genome and plant reproductive dynamics.     

High intraspecific diversity of Restorer‐of‐fertility‐like genes in barley

Nuclear restorer of fertility (Rf) genes suppress the effects of mitochondrial genes causing cytoplasmic male sterility (CMS), a condition in which plants fail to produce viable pollen. Rf genes, many of which encode RNA‐binding pentatricopeptide repeat (PPR) proteins, are applied in hybrid breeding to overcome CMS used to block self‐pollination of the seed parent. Here, we characterise the repertoire of restorer‐of‐fertility‐like (RFL) PPR genes in barley (Hordeum vulgare). We found 26 RFL genes in the reference genome (‘Morex’) and an additional 51 putative orthogroups (POGs) in a re‐sequencing data set from 262 barley genotypes and landraces. Whereas the sequences of some POGs are highly conserved across hundreds of barley accessions, the sequences of others are much more variable. High sequence variation strongly correlates with genomic location – the most variable genes are found in a cluster on chromosome 1H. A much higher likelihood of diversifying selection was found for genes within this cluster than for genes present as singlets. This work includes a comprehensive analysis of the patterns of intraspecific variation of RFL genes. The RFL sequences characterised in this study will be useful for the development of new markers for fertility restoration loci.     

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.     

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)     

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of ‘Lead Rice’ and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6–orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of ‘Chinsurah Boro II’. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6–orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.     

Reorganization of mitochondrial genomes of cytoplasmic revertants in cms-S inbred line WF9 in maize

Summary Cytoplasmic reversion to fertility in cms-S maize has been previously correlated with changes in mitochondrial genome organization, specifically with loss of the autonomously replicating linear plasmid-like DNAs, S1 and S2, and with accompanying alterations in the high molecular weight mtDNA (main genome) that specifically involved S1 and S2 sequences. These studies, however, dealt with cytoplasmic revertants occurring in the cms-VG M825 inbred line and in the cms-VG M825/Oh07 F₁ hybrid. This paper deals principally with patterns of mitochondrial DNA reorganization accompanying cytoplasmic reversion to fertility in the WF9 inbred line nuclear background. Here the free S1 and S2 plasmid-like DNAs are retained in the revertants. Mitochondrial DNA analysis by Southern hybridization using cloned fragments of S1 and S2 shows altered organization around S-homologous regions in the main mitochondrial genome of revertants as compared with that of the male-sterile parental controls, but the pattern of main genome changes involving these regions differs from that of the cytoplasmic revertants that occurred in M825 and M825/Oh07 backgrounds. Similar experiments using a clone of the cytochrome oxidase I (COX I) gene of maize as a probe indicate that reorganization in this region is also involved in the changes in mtDNA that accompany cytoplasmic reversion to male fertility in cms-S WF9. The heterogeneity in patterns of reorganization of the main mtDNA genome that accompany cytoplasmic reversion in the same and different nuclear backgrounds are discussed in relation to cytoplasmic male sterility (CMS).     

Convergence beyond flower morphology? Reproductive biology of hummingbird‐pollinated plants in the Brazilian Cerrado

Convergent reproductive traits in non‐related plants may be the result of similar environmental conditions and/or specialised interactions with pollinators. Here, we documented the pollination and reproductive biology of Bionia coriacea (Fabaceae), Esterhazya splendida (Orobanchaceae) and Ananas ananassoides (Bromeliaceae) as case studies in the context of hummingbird pollination in Cerrado, the Neotropical savanna of Central South America. We combined our results with a survey of hummingbird pollination studies in the region to investigate the recently suggested association of hummingbird pollination and self‐compatibility. Plant species studied here differed in their specialisation for ornithophily, from more generalist A. ananassoides to somewhat specialist B. coriacea and E. splendida. This continuum of specialisation in floral traits also translated into floral visitor composition. Amazilia fimbriata was the most frequent pollinator for all species, and the differences in floral display and nectar energy availability among plant species affect hummingbirds' behaviour. Most of the hummingbird‐pollinated Cerrado plants (60.0%, n = 20), including those studied here, were self‐incompatible, in contrast to other biomes in the Neotropics. Association to more generalist, often territorial, hummingbirds, and resulting reduced pollen flow in open savanna areas may explain predominance of self‐incompatibility. But it is possible that mating system is more associated with the predominance of woody hummingbird plants in the Cerrado plant assemblage than to the pollination system itself.     

Autonomous self‐pollination in the nectarless orchid Pogonia minor

The pollination biology of the nectarless orchid Pogonia minor was investigated in central Japan. The investigation revealed that the solitary flowers failed to attract pollinators, while high rates of fruit set were observed in the natural population. Comparable levels of fruit set were obtained in bagged, artificial self‐pollinated and artificial cross‐pollinated plants, indicating that the species is not pollinator‐limited for fruit set under natural conditions. Autonomous self‐pollination in P. minor resulted from a reduced rostellum, which allowed contact between the pollinia and the stigma. Self‐pollination is thought to be an adaptive response that provides reproductive assurance under conditions of pollinator limitation. Since pollen limitation is generally known to be frequent among deceptive orchids, strong pollen limitation is probably a driving force in the autonomous self‐pollination mechanism in the nectarless orchid P. minor.     

Cytoplasmic male-sterile synthetics: a new approach to the exploitation of heterosis in rape

In addition to their application in the production of F₁ hybrids in rape (Brassica napus L.), cytoplasmic male-sterility (CMS) systems may be used to produce synthetic varieties with much higher levels of heterozygosity than those expected in conventional rape synthetics. CMS synthetics are produced by compositing a CMS A-line with several male-fertile (MF) B-lines lacking nuclear alleles for fertility restoration, and increasing the resulting mixture by natural pollination. Over generations of increase, pollination of the CMS component by the MF component of the synthetic results in the progressive loss of A-line nuclear genes from the population. The initial proportions of CMS and MF plants are expected to be preserved over several generations of natural pollination if CMS and MF plants are equal in yield. Methods for estimating the heterozygosity level of CMS synthetics, taking into account the proportion of CMS plants, number of MF parents in Syn 0, and selfing rate (s) of MF plants, are presented. If completely inbred Syn 0 parents and s of 0 and 0.75 for CMS and MF plants respectively, are assumed, the heterozygosity level (1-F) of a synthetic derived from four inbred MF parents each comprising 6.25% of Syn 0 and one inbred CMS parent comprising 75% of Syn 0 is predicted to be 0.66 in Syn 5, compared to 0.30 in synthetics derived from four MF parents only. CMS synthetics offer a novel, low-cost approach to the exploitation of heterosis in rape and other species with mixed mating systems in which self-pollination predominates.     

The 5′-leader sequence of sugar beet mitochondrial <Emphasis Type="Italic">atp6</Emphasis> encodes a novel polypeptide that is characteristic of Owen cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is a mitochondrially encoded trait, which is characterized by a failure of plants to produce viable pollen. We have investigated the protein profile of mitochondria from sugar beet plants with normal (fertile) or CMS cytoplasm, and observed that a 35-kDa polypeptide is expressed in Owen CMS plants but not in normal plants. The variant 35-kDa polypeptide was found in CMS mitochondria placed in five different nuclear backgrounds. Interestingly, this polypeptide proved to be antigenically related to a 387-codon ORF (preSatp6) that is fused in-frame with the downstream atp6. The presequence extension of the atp6 ORF is commonly found in higher plants, but whether or not it is normally expressed has hitherto remained unclear. Our study is thus the first to demonstrate that the atp6 presequence is actually translated in mitochondria. We also observed that preSATP6 is a mitochondrial membrane protein that assembles into a homogeneous 200-kDa protein complex. In organello translation experiments in the presence of protease inhibitors showed a reduction in the abundance of mature preSATP6 with time, suggesting that the mature preSATP6 may be derived by proteolytic processing of a translation product of the preSatp6/Satp6 ORF.     

Genetic analysis of fertility restoration and accumulation of ORF125 mitochondrial protein in the kosena radish (Raphanus sativus cv. Kosena) and a Brassica napus restorer line

The genetics of fertility restoration (Rf) of kosena radish CMS has been characterized. The kosena CMS-Rf system is genetically the same as that of the ogura CMS-Rf system. Two dominant genes that act complementary to the restoration of fertility control fertility restoration in kosena CMS. One allele (Rf1) is associated with accumulation of the CMS-associated protein, ORF125. The interaction of Rf1 and another allele (Rf2) was essential for the restoration of fertility in radish, whereas Rf1 alone was sufficient for the complete restoration of fertility in the B. napus kosena CMS cybrid. Received: 13 August 1999 / Accepted: 16 September 1999     

Reproductive ecology of the carnivorous plant Pinguicula moranensis (Lentibulariaceae)

• The interaction of plants with pollinators can be a determinant of their reproductive fitness. However, information about the pollination biology of carnivorous plants is scarce. To increase knowledge of reproductive ecology of carnivorous plants we focused on Pinguicula moranensis. Specifically, based on the presence of large, zygomorphic and spurred flowers, we predicted higher reproductive fitness in cross‐pollinated than in self‐pollinated flowers.
• Within a plot of 51 m² we characterised the reproductive phenology, including flower lifespan and stigmatic receptivity. We identified pollinators and their movement patterns within the plot. Breeding system was experimentally evaluated using hand‐pollination (i.e. autonomous, self‐ and cross‐pollination).
• Flowers of P. moranensis were visited by long‐tongued pollinators, mainly members of the Lepidoptera. Hand‐pollination experiments confirmed our prediction and suggest that flower traits might favour cross‐pollination.
• We mainly discuss the implications of the patchy distribution of plants and behaviour of pollinators on gene movement in this plant species, as pollination between genetically related individuals could be occurring.

     

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.     

Fertile revertants from S-type male-sterile maize grown in vitro

Summary Plants were regenerated from callus cultures of maize inbred W182BN with the S(USDA) type of cytoplasmic male sterility (cms). Some regenerates from 16 of 18 separate cultures had fertile tassels. Many other regenerates, whose fertility could not be scored accurately because of abnormal plant morphology, produced fertile progeny after pollination with N cytoplasm W182BN. Revertant plants and/or progeny were obtained from all 18 cultures, which included the CA, D, LBN, and S sources of cmsS. More revertants were recovered from cultures maintained as callus for 12 months than from 3–4 month old cultures. Several types of evidence (absence of segregation for fertility after selfing or pollination of revertants with standard W182BN, pollen viability counts, failure of revertants to restore sterile cmsS lines to fertility, mitochondrial DNA analyses) indicated that the reversion to fertility involved cytoplasmic rather than nuclear alterations. All revertants examined lacked the S1 and S2 plasmid-like DNAs characteristic of the mitochondrial genome of sterile cmsS lines. Most callus cultures lost S1 and S2 after 13–20 months in vitro. No revertants were seen among thousands of W182BN cmsS plants grown from seed in the field or among plants from tissue cultures of W182BN with the C or T types of cms. The cytoplasmic revertants recovered from culture may be useful for the molecular analysis of cmsS.     

Spatiotemporal Variability in the Reproductive Success of the Continually Flowering Shrub Dillenia suffruticosa in Borneo

Continually flowering plants bloom continuously throughout the year, as often seen in plants distributed along the roadsides or in the understory layers in Southeast Asia's tropical rain forests. Dillenia suffruticosa (Griff. ex Hook. f. & Thomson) Martelli (Dilleniaceae) is one such continually flowering shrub that flowers during periods of community‐wide mass flowering, general flowering (GF), and non‐GF. During irregularly occurring GF periods, when species of all forest layers flower synchronously for several months, some pollinators migrate to the canopy layer, where GF promotes the pollination success of participating plants. Continually flowering plants share the available pollinator community with GF plants, and the reproductive success of continually flowering plants may be affected during the GF period. To assess the effects of GF on the reproductive success of a diverse range of continually flowering plants, we examined the differences in pollinator density and reproductive success between GF and non‐GF periods in D. suffruticosa at four different research sites. Although the seed set differed among the four research sites, pollinator density and fruit set did not differ between GF and non‐GF periods or research sites. Our results suggest that the reproductive success of D. suffruticosa was maintained at an approximately constant level, regardless of the flowering phenology of the canopy layer or other vegetation components.