Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: molecular analysis of the cytoplasm of parental lines and corrected progeny

Summary Cytoplasmic differences between male-fertile and male-sterile Brassica napus as well as Raphanus sativus were investigated. Plastids of the male-fertile B. napus were found to differ from those of male-sterile B. napus and R. sativus with respect to DNA restriction enzyme patterns. Differences between male-fertile and male-sterile B. napus mitochondria were detected not only in the restriction fragment patterns of their DNA, but also at the level of expression by in organello translation of mitochondrial polypeptides.The chlorophyll deficiency obtained upon transferral of the male-sterility-conferring radish cytoplasm to a winter variety of B. napus had been corrected earlier through protoplast fusion. The cytoplasmic composition of the corrected lines was analysed using DNA restriction analysis and in organello translation. The stability of the recombined cytoplasm in the corrected lines was confirmed by analysis of the subsequent seed-derived generation.     

Influence of genotype and gelling agents on in vitro regeneration by organogenesis in sunflower

Fourteen recombinant inbred lines of sunflower (Helianthus annuus L.) and their parents (PAC-2 and RHA-266) were tested for their organogenesis ability. Seeds were surface sterilized and germinated on hormone free half strength MS basal medium containing 10 g l^-1 sucrose solidified with five different gelling agents: Phytagar (Gibco laboratoires) 3 g l^-1, Phytagel (Sigma) 3 g l^-1, Agarose (Sigma) 5 g l^-1, Arcagel (Sigma) 4 g l^-1 and Agar-Agar (Fisher France) 7 g l^-1. Cotyledons from 2-day-old seedlings were split in half and the four explants of each seed were cultived in 55 mm diameter petri dishes containing 10 ml of MS medium supplemented with 50 μM KNO₃, 1 μM myo-inositol, 5 μM casein hydrolysate, 4.4 μM of BA and 5.4 μM of NAA solidified with the same gelling agents. The experimental design was a randomized complete block with 3 replications. A replicate for each genotype consisted of ten petri dishes containing four explants. The statistical analysis showed significant differences among genotypes and gelling agents. Of the fourteen recombinant inbred lines tested `C93' presented the highest values for all regeneration traits in the five different media and it was better than the best parent. Agarose and Agar-Agar were more better than other gelling agents for shoot induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of an open reading frame related to the CMS-associated urf-s in fertile Petunia lines and species and in other fertile Solanaceae species

Summary In Petunia, a mitochondrial (mt) locus, S-Pcf, has been found to be strongly associated with cytoplasmic male sterility (CMS). The S-Pcf locus consists of three open reading frames (ORF) that are co-transcribed. The first ORF, termed Pcf, contains an unidentified reading frame urf-s that has been detected so far only in sterile Petunia lines and sterile somatic hybrids. In the study described here, a urf-s-related sequence was detected in seven different normal fertile Petunia lines and species as well as in additional members of the Solanaceae family by means of the polymerase chain reaction. The urf-s-related sequence identified in the fertile lines was termed orf152. In Petunia the nucleotide sequence of orf152 was found to be identical to the corresponding part of urf-s. However, the genome organization around orf152 was found to be different from that of urf-s. These results indicate that: (1) at least part of the urf-s sequence is present in fertile lines and species of Petunia and in other Solanaceae species; (2) the orf152 sequence of Petunia is not part of the Pcf ORF. The relevance of these findings to a better understanding of the evolution of the S-pcf locus in (S) cytoplasm in Petunia is discussed.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 3511-E, 1991 series     

The pollen-specific LIM protein PLIM-1 from sunflower binds nucleic acids in vitro

The protein PLIM-1 (formerly SF3) from sunflower is expressed exclusively in mature, free pollen. It contains two LIM domains associated with an acidic C-terminus comprising six copies of the pentapeptide motif (A,T,S) (E,D) TQN. We have expressed the pollen protein as well as some of its mutant forms inEscherichia coli and have used the bacterially produced proteins to study interactions with nucleic acids. Our studies show that the protein binds DNA and RNA in vitro to form large complexes, while mutant polypeptides containing either a single LIM domain or a destabilized first or second LIM domain do not. Although these data suggest that the biological function of PLIM-1 involves interactions with nucleic acids, its role in pollen development remains unclear.     

Effects of 1-aminocyclopropane-1-carboxylic acid and oxygen concentrations on in vivo and in vitro activity of ACC oxidase of sunflower hypocotyl segments

In vivo ethylene production by hypocotyl segments of sunflower seedlings and in vitro activity of 1-aminocyclopropane-1-carboxylic acid oxidase (formerly ethylene-forming enzyme) extacted from the same tissues increase with increasing concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and oxygen. ACC oxidase activity follows Michaelis-Menten kinetics. The apparent Km values of the enzyme towards ACC, estimated in vivo and in vitro, are respectively 219 M and 20.6 M. Both Km values towards O₂ are similar, ca 10.6–11.4%. A decrease in concentration in one of the substrates (ACC or O₂) results in an increase in in vivo apparent Km of ACC oxidase for the other substrate. On the contrary, Km values of the enzyme towards ACC or O₂ estimated in vitro are not dependent upon the concentration of the other substrate (ACC or O₂).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - MACC malonylate 1-aminocyclopropane-1-carboxylic acid - SD standard deviation     

Effect of denaturants on the oligomeric structure of the 11 S protein of sunflower (Helianthus annum)

The effect of sodium dodecyl sulphate, urea, guanidinium hydrochloride and heat on the oligomeric structure of the 11 S protein of sunflower has been determined. Sodium dodecyl sulphate directly dissociates the protein to 2 S subunits, whereas urea and guanidinium hydrochloride dissociate it through an intermediate 7 S protein. Heating the protein at 90‡C for 20 min caused dissociation of the 11 S protein, without any precipitation.     

Cytoplasmic male sterility (CMS) in Lolium perenne L.: 1. Development of a diagnostic probe for the male-sterile cytoplasm

Analysis of reciprocal crosses between nonrestoring fertile genotypes and restored male-sterile genotypes of Lolium perenne confirmed the cytoplasmic nature of the sterility trait. This prompted a search for a molecular probe that could be used to distinguish between fertile and cytoplasmic male-sterile (CMS) cytoplasms. We describe the identification and cloning of a 4.5-kb BamHI-HindIII restriction fragment from the mtDNA of the CMS line. The cloned fragment (pCMS45) failed to hybridise to sequences in the mtDNA of fertile lines and was thus capable of unambiguously distinguishing between fertile and CMS cytoplasms. The use of pCMS45 as a diagnostic probe provided a simple test for positive identification of young non-flowering plants carrying the CMS cytoplasm and also permitted confirmation at the molecular level of the maternal transmission of the CMS trait suggested by the genetic data.     

A chimeric gene (orf256) is expressed as protein only in cytoplasmic male-sterile lines of wheat

Mitochondria derived from Triticum timopheevi have a chimeric gene, orf256, immediately upstream from coxI. Antibodies to a peptide corresponding to a part of the encoded amino acid sequence of orf256 detect a 7 kDa protein on western blots of mitochondrial proteins from cytoplasmic male-sterile (cms) wheat (T. aestivum nucleus, T. timopheevi mitochondria) but not in mitochondrial proteins from T. aestivum, T. timopheevi, or cms plants restored to fertility by introduction of nuclear genes for fertility restoration. The 7 kDa protein appears to serve as a marker for cms wheat. Its occurrence as an integral protein of the inner membrane may indicate a cms effect through an influence on mitochondrial membrane function.     

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.     

The control of flower initiation by gibberellin in Helianthus annuus L. (sunflower), a non-photoperiodic plant

The involvement of gibberellins in the control of flowering of sunflower was studied by direct application of GA₃ to the apex of the plants, analysis of the endogenous levels of gibberellin-like substances at different plant ages, and indirectly by the application of paclobutrazol, an inhibitor of gibberellin synthesis. GA₃ speeded-up flower initiation and floral apex development. The time of GA₃ application was more critical than the amount of GA₃ applied. The endogenous levels of gibberellin-like compounds increased significantly by day 15 after sowing. The application of paclobutrazol markedly delayed floral initiation and this effect was also depedent on plant age. Both GA₃ and paclobutrazol had their greatest effects between 10 and 20 days after sowing suggesting that an increase in gibberellins in that time period plays a role in floral initiation.     

Rapid hybridization-based assays for identification by DNA probes of male-sterile and male-fertile cytoplasms of the sugar beet Beta vulgaris L.

Summary Methods are described whereby hybridization of mitochondrial (mt) DNA with different DNA probes can definitely distinguish male-fertile and and male-sterile (cms) cytoplasms of sugar beet Beta vulgaris L. We have developed two types of miniassays. (1) Comparative methods requiring the isolation and restriction of total cellular DNA, hybridization with cloned mtDNA fragments from either fertile or male-sterile cytoplasms, and comparison of the hybridization patterns to the fertile-and sterile-specific patterns of mtDNA of sugar beet for the given mtDNA probe. For these analyses, we routinely used 1 g of plant material to determine the type of cytoplasm. (2) Noncomparative (plus-minus) methods requiring neither the isolation of pure DNA nor restriction, electrophoresis, or Southern blotting. Instead, alkaline-SDS plant extracts from as little as 50 mg of plant material were dot-blotted and hybridized with fertile-specific (mitochondrial minicircular DNA) and/or cms-specific probes (consisting of a 2.3-kb mtDNA sequence exclusively occurring in the cms cytoplasm). The assays are simple to perform, give definitive results, are nonde-structive to the plants, and may be used in mass screening of sugar beet populations for hybrid production or in in vitro culture processes.     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. I. The origin and distribution of Ogura male-sterile cytoplasm in Japanese wild radishes (Raphanus sativus L.) revealed by PCR-aided assay of their mitochondrial DNAs

Ogura male-sterile cytoplasm was surveyed in common Japanese radish cultivars and in wild radishes growing in various localities in Japan. Mitochondrial (mt) DNA rearrangement involving the atp6 gene was used as a molecular marker. To detect the mtDNA rearrangement, polymerase chain reactions (PCR) were designed to amplify the upstream region of the atp6 gene. The oligonucleotides homologous to the following three regions were synthesized: (1) trnfM, (2) ORF105 and (3) atp6. PCRs were conducted with a pair of the first and the third primers to detect normal mtDNA, and with the second and the third primers for Ogura-type mtDNA. All 15 Japanese cultivars yielded an amplification product which was the same as that of normal mtDNA, whereas some wild radishes gave the product specific to Ogura mtDNA. Twenty-four populations of wild radish were classified into three groups according to the frequency of Ogura-type mtDNA: (1) in ten populations, all four plants analyzed per population had normal type mtDNA, (2) in five populations, only plants with Ogura-type mtDNA were found, and (3) nine populations included both normal and Oguratype mtDNAs. There were no geographical restrictions and no cline in the distribution of the plants with Ogura-type mtDNA. These results suggested that the Ogura-type male-sterile cytoplasm originated in wild radishes.