Independent segregation of the plastid genome and cytoplasmic male sterility in Petunia somatic hybrids

Summary The chloroplast genomes of three sets of Petunia somatic hybrids were analyzed to examine the relationship between chloroplast DNA (cpDNA) composition and cytoplasmic male sterility (CMS). Chloroplast genomes of somatic hybrid plants were identified either by restriction and electrophoresis of purified cpDNAs or by hybridization of total DNA digests with cloned cpDNA probes that distinguish the parental genomes.The chloroplast genomes of a set of seven somatic hybrids derived from the fusion of Petunia CMS line 2423 and fertile line 3699 were analyzed. All seven plants were fertile, and all exhibited the cpDNA restriction pattern of the sterile cytoplasm. Similarly, four fertile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3677 were found to contain the CMS chloroplast genome. The cpDNA compositions of four fertile and two sterile somatic hybrids derived from the fusion of CMS line 3688 and fertile line 3704 were determined by restriction analysis of purified cpDNAs; all six plants exhibited the cpDNA restriction pattern of line 3704. Thus the CMS phenotype segregates independently of the chloroplast genome in Petunia somatic hybrids, indicating that CMS in Petunia is not specified by the chloroplast genome.     

Mechanism of male sterility in Petunia

Summary The free amino acid contents in the anthers of male fertile, cytoplasmic male sterile (cms) and genic male sterile (gms) petunia lines were compared at different developmental stages of the male gametophyte. Quantitative differences in the amounts of free amino acids were found between the fertile and male sterile lines and between the cms and gms lines. The differences between the sterile lines were correlated with the different developmental stages at which the breakdown in microsporogenesis occurred. In the Rosy Morn (RM) cms line, where breakdown of microsporogenesis occurred at the end of prophase 1, there was an associated increase in asparagine and decrease in the other amino acids. In the RM gms line, in which breakdown occurred at the tetrad stage, an accumulation of asparagine in the anthers corresponded with an accumulation of glutamine beginning at prophase 1. Compared with fertile anthers, the sterile anthers accumulated much proline at the early meiotic stages, but no -aminobutyric acid. Comparison of the free amino acids of the fertile and the male sterile lines indicates that certain biochemical events leading to breakdown of microsporogenesis precede the observed cytological breakdown. The results from adding asparagine and glutamine to extracts of anthers at different developmental stages suggest that the amino acid balance may contribute to the changes in pH in the fertile and male sterile anthers which we observed previously.Contribution from the Volcani Center, Agricultural Research Organization Bet Dagan, Israel. 1972 Series, No. 2083 E.     

Differential Mitochondrial Electron Transport through the Cyanide-Sensitive and Cyanide-Insensitive Pathways in Isonuclear Lines of Cytoplasmic Male Sterile, Male Fertile, and Restored Petunia

Three pairs of isonuclear lines of cytoplasmic male sterile (CMS) and fertile Petunia cells (Petunia hybrida [Hook] Vilm. and Petunia parodii L.S.M.) grown in suspension culture were examined for sensitivity to inhibitors of respiratory electron transport at time-points after transfer into fresh media. Cells from CMS lines differed from cells of fertile lines in their utilization of the cyanide-insensitive oxidase pathway. Under our culture regime, after approximately 3 days of culture cells from the CMS lines exhibited much lower cyanide-insensitive, salicylhydroxamic acid-sensitive respiration than cells from the fertile lines. This respiratory difference was shown to be specific to the mitochondrial alternative oxidase pathway by using other characteristic inhibitors of mitochondrial electron transport in experiments with isolated mitochondria. Immature anthers from CMS plants also showed lower alternative oxidase activity relative to anthers from male fertile plants, but no such difference was detected in leaf tissue, ovary or perianth tissue, or anthers collected just prior to anthesis. A cell line from a fertile plant carrying a nuclear fertility restorer gene and the CMS cytoplasm exhibited increased activity of the alternative pathway compared with the CMS lines.     

甘蓝胞质雄性不育系及其保持系花药和叶片生化特性分析

对甘蓝胞质雄性不育系CMS158及其保持系花药激素、游离氨基酸和叶片叶绿素含量进行了比较研究。结果表明,不育系花药KT和IAA含量极显著低于保持系,GA含量不育系和保持系没有显著差异。游离氨基酸总量不育系花药极显著高于保持系,脯氨酸和胱氨酸含量不育系花药极显著低于保持系,赖氨酸含量两系无明显差异,苏氨酸在两系中均未检出,其余氨基酸含量均是不育系花药高于保持系;不育系花药中丝氨酸含量最高,保持系花药中脯氨酸含量最高,两系中天冬氨酸和脯氨酸含量的差异最大。叶片叶绿素含量不育系和保持系无显著差异。     

Proteomic approach for investigation of cytoplasmic male sterility (CMS) in Brassica

The alloplasmic ‘Tournefortii’ CMS system fromB. napus was chosen for a comprehensive investigation of the protein content of the mitochondrial compartment in male sterile and fertile near isogenic lines. As proteins involved in the male sterile phenotype may be expressed in a tissue-specific manner, mitochondrial preparations were made from different tissues and checked for the purity of the organelles with a new and highly sensitive method (BN-PAGE). InBrassica, only mitochondrial preparations from 4-5-day-old seedlings and from flower buds were found to be sufficiently pure. High resolution, two-dimensional gel electrophoresis of total protein from anthers and from mitochondrial protein of male sterile and fertile seedlings of near isogenic lines revealed distinct differences in the protein patterns. Several explanations for the occurrence of these differences, which may be the starting point for a detailed molecular analysis and a better understanding of the CMS syndrome, are discussed.     

Studies on the Free Amino Acid Content in Anthers of Malesterile and Fertile Plants of Taigu Wheat,with Special Reference to Free Proline Content

Biochemical analyses were made on anthers and pistils at various developmental stages of both male-sterne and fertile plants of Taigu wheat. Analyses ineluded total free amino aeids and free proline. The following results were obtained: 1. There was no significant difference between the content of free proline in anthers of male-sterile and fertile plants at reduction division of mierospore mother cells. 2. In anthers with early uninucleate miorospores, the content of free proline of fertile plants was remarkably higher than that of male-sterile plants. It is interesting to note that at this stage the content of free proline in fertile plants rose to 1.65% of the dry weight of the anther, constituting 50% of the total free amino acids, and amounted to 7-fold of that in male-sterile plants. This result is in line with the results obtained with most cytoplasmic malesterile plants reported by other workers, although malesterility in Taigu wheat is controlled by the nueleus. 3. In pistils, at the stages eorresponding to the early uninneleate and the binueleate stages of the pollen, the free proline content of fertile plants was twice as much as that of the male-sterile plants. This differenee disappeared gradually after fertilization. 4. Tile content of total free amino aoids did not fluetuate as much as the free proline content. There was no differenee in anthers of both types of plants during reduction division of mierospore mother cells. In anthers with early uninueleate pollen grains, total free amino acid content of fertile plants exceeded that of male-sterile plant, the difference levelled off at latter stages. In pistils, before fertilization, the content of total free amino acids of the fertile phmts was slightly higher than that of the male-sterile plants. After fertilization t}fis difference was no nmre noticeable.     

棉花洞A型核雄性不育系花药败育过程中的生化变化

研究了陆地棉洞A型核雄性不育系(抗A1,MA)不育和可育花药不同发育时期可溶性碳水化合物、游离氨基酸和IAA、GA3、ABA含量的动态变化。结果表明,不育花药中可溶性糖含量偏高,缺乏淀粉积累;有4种游离氨基酸含量在可育与不育花药问存在显著差异,其中不育花药天门冬氨酸含量偏高,这可能是其败育的原因之一,而脯氨酸、精氨酸、苯丙氨酸含量异常,则是不育花药败育的结果;在花药主要败育时期之前,不育花药中ABA含量极显著偏高,IAA、GA3含量极显著偏低。不育花药中IAA、GA3、ABA含量变化与洞A型核雄性不育系花药败育密切相关。     

太谷核不育小麦花药内游离脯氨酸和总氨基酸含量的变化及其与育性的关系

本文对显性单基因控制的太谷核不育小麦不同发育阶段的可育株和不育株的花药及雌蕊内游离肺氨酸和游离总氨基酸的含量进行了分析。结果表明:(1)在小孢子母细胞减数分裂期,不同育性花药之间游离脯氨酸的含量无明显差异,且含量较低。(2)在小孢子单核初期,可育花药内游离脯氨酸的含量显著高于不育花药,是不育花药的7倍,比减数分裂期增加20倍,高达其干重的1.65％,占其游离总氨基酸的50％。(3)在雌蕊中,游离脯氨酸的含量远远低于花药,不同育性植株之间差异不很明显。(4)关于游离总氨基酸的含量,在花药中减数分裂期,不同育性植株之间无明显差异;在小孢子单核初期,可育株高于不育株。在雌蕊中,相应于小孢子单核初期时,可育株稍高于不育株,受精后迅速趋于一致,但整个变化幅度不大。     

Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida

Summary Using an HPLC method it has proved possible to follow the levels of adenine and pyridine nucleotides in the anthers of normal and cytoplasmically male sterile (CMS) lines of Petunia hybrida. Well before the appearance of any structural differences, anthers of CMS plants begin to show lower ATP/ADP ratios. In anthers, as in other non-photosynthetic plant tissues, there is a strong correlation between the ATP/ADP ratio and levels of NADPH and, when NADPH was assayed in fertile and CMS anthers, very dramatic differences were discovered. In male fertile plants, the NADPH/NADP ratio differs strikingly between somatic and reproductive tissues and reaches a peak in anthers at the early prophase of meiosis. The ratio in male sterile anthers remains largely unchanged from that normally seen in somatic tissues over this period. A cytochemical localisation technique revealed that, at the stage of development in question, the major reserves of anther NADPH are held within the tapetal cells and that levels within CMS tapeta are strikingly lower than those of normal plants. These findings are discussed in the perspective of theories proposed to explain the operation of CMS, and also in terms of the now conclusive evidence that the genetical basis of CMS lies in changes in the organisation of the mitochondrial genome.     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

Amino acids in anthers of Milo and in cytoplasmic genetic male sterile sorghums (Sorghum bicolor L. Moench) of Indian origin

Summary Amino acid composition of proteins from anthers of milo and Indian origin male steriles were determined. Comparison of amino acid between A and B lines showed lower contents of histidine, threonine, glutamic acid, glycine, leucine and phenylalanine and higher contents of alanine, serine, proline and tyrosine in line A compared to line B. Alanine content in anthers of A lines was more than two fold higher than that in the anthers from B lines. Marked differences in amino acid composition of anthers of A and B lines are suggestive of their involvement in male sterility. Cytoplasmic male steriles of Indian origin M35-1A and M31-2A showed greater similarity but differed from milo, VZM2A and B.     

-Amino acid contents of mitochondria and some purple bacteria

The endosymbiont most likely to have given rise to mitochondria is an aerobic bacterium belonging to the α subdivision of the so-called purple bacteria such as Rickettsia, Bradythizobium and Agrobacterium [1 and 2]. Contents of the -enantiomers of serine, alanine, proline, glutamate and aspartate in rat liver whole mitochondria, mitochondrial outer membranes, inner membranes and matrix, soluble proteins and free amino acids were detected. These values for -amino acid content were compared with those in soluble proteins and free amino acids from the purple bacteria Paracoccus denitrificans, Pseudomonas aeruginosa and Escherichia coli, members, respectively of the α, β, and γ subdivisions, to find any similarity between mitochondria and these purple bacteria. A similarity was observed in protein -amino acid contents which were low (<1.5%, D-type/D-type+L-type) both in the membrane and soluble protein fractions from mitochondria and in soluble protein from bacteria. Oddly, substantial amounts of free -serine and free -aspartate (around 2%) were found for the first time in mitochondria. The contents of -serine and -aspartate were higher than those of -alanine, -proline and -glutamate. In purple bacteria, the concentration of -serine (<2%) was the lowest of the five amino acids examined, and those of -alanine (27–32%) and -glutamate (7–26%) were high. Therefore, no similarity was shown in the free -amino acid content between mitochondria and any of the three purple bacteria.     

Differences on the microsporogenesis and tapetal development of male fertile and cytoplasmic male sterile pepper (Capsicum annuum L.)

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line pepper were studied using transmission electron microscopy. The results showed that CMS line anthers appeared to have much greater variability in developmental pattern than male fertile maintainer line ones. The earliest deviation from normal anther development occurred in CMS line anthers at prophase I was cytomixis in some microspore mother cells (MMCs), and vacuolisation in tapetal cells. Then, MMCs in CMS line anthers developed asynchronously and a small part of ones at the different stage degenerated in advance appearing to have typical morphological features of programmed cell death (PCD). Most MMCs could complete the meiosis, but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei, and some degenerated ahead of time as well. Tapetal cells in CMS line anther degenerated during meiosis, and were crushed at the tetrad stage, which paralleled the collapse of pollens. Pollen abortion in CMS line anthers happened by PCD themselves, and the premature PCD of tapetal cells were closely associated with male sterility.     

Intraspecific larval competition in two solitary parasitoids, Apoanagyrus (Epidinocarsis) lopezi and Leptomastix dactylopii

Analysis of total aromatic amino acid (free and bound) in some cucumber accessions selected previously for resistance to western flower thrips, Frankliniella occidentalis (Pergande) [Thysanoptera: Thripidae], indicated that low concentrations of these essential nutrients, relative to total leaf protein, were correlated with a reduction in damage by the insect. Further analysis of samples of four important horticultural crops (lettuce, tomato, pepper and cucumber) with unknown levels of resistance to thrips showed a significant genotypic variation in the concentrations of total aromatic amino acids relative to the total leaf protein. Accessions from each crop with low or high concentrations of aromatic amino acids in proteins were exposed to thrips larvae. Regression analysis showed a highly significant positive correlation between aromatic amino acid concentration in leaf protein and thrips damage, regardless of crop species. It is concluded that higher concentrations of aromatic amino acids in plant proteins are important for successful thrips development. These results provide plant breeders with a promising tool for indirect selection without using undesirable insect bioassays.     

Characterization of embryo globulins encoded by the maizeGlb genes

Two of the most abundant proteins in maize embryos are saline-soluble, water-insoluble globulins. One is aM _r 63,000 protein encoded by theGlbl gene and the other is aM _r 45,000 component encoded by theGlb2 gene. Both proteins accumulate to high levels during embryo development and are rapidly degraded during the early stages of seed germination. Amino acid composition analysis indicates that these proteins may serve as storage reserves to provide sources of nitrogen and carbon to the germinating embryo. Amino-terminal sequence analysis of the finalGlb1 gene product, GLB1, and its immediate precursor, GLB1′, indicates that the latter is proteolytically cleaved near the amino terminus to form GLB1. In addition to these biochemical studies, we describe the identification of a novel maize variant which lacks the protein product of theGlb2 gene. This contribution from the University of Illinois Agricultural Experiment Station was supported by grants from The Standard Oil Corporation, a wholly owned subsidiary of BP America, Inc., and the U.S. Department of Agriculture (No. 88-37262-3427).     

A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca2+-binding proteins

Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was specifically allergic to Brassica pollen. These clones were divided into two groups, I and II, based on the sequence similarity. All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity. Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca²⁺-binding sites of Ca²⁺-binding proteins such as calmodulin. However flanking sequences were distinct from that of calmodulin or other Ca²⁺-binding proteins. RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen. The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the IgE of a non-allergic patient. The cDNA clones reported here, therefore, represent pollen allergens of Brassica species.     

Biochemical analysis of four species ofCassia L. pollen

The present paper describes the comparative biochemical studies in terms of quantitative analyses of carbohydrates, lipids, proteins, free amino acids, nucleic acids, minerals, ash and moisture as well as the identification of free amino acids of pollen of four species ofCassia L. (C. alata L,C. fistula L,C. occidentalis L andC. siamea Lam.). A significant variation in the chemical constituents was observed among the four species.C. occidentalis showed the highest levels of carbohydrate (15.15%) and protein (22.45%), andC. siamea had the lowest levels of carbohydrate (7.15%), lipid (6.2%) and protein (13.85%).C. alata andC. fistula showed intermediate results. However,C. alata showed the highest amount of free amino acids (3.8%) and the least of 1.42% was found inC. fistula. Thin layer chromatography (TLC) of free amino acids of the four species showed some homology in their amino acid content, of which proline, glutamic acid, methionine and phenyl-alanine were the most dominant. The level of nucleic acids and minerals was found to be comparatively low.C. siamea andC. alata showed an exceptionally high level of ash content (8.6 and 8.8%, respectively) while moisture content varied from 8 to 11%.     

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny

Summary A circular supercoiled mitochondrial DNA plasmid P₁ (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P₂) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P₁ was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P₁ and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P₁ and P₂. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.