Changes in protein and amino acid content during anther development in fertile and cytoplasmic male sterile Petunia

Summary Development of anthers in cytoplasmic male sterile (CMS) Petunia diverges from the normal sequence of events early in meiosis. Quantitative and qualitative changes in morphology, proteins and free amino acid contents correlate with this divergence. In anthers of the fertile line (5719), total protein content increases, and SDS-PAGE protein patterns change as the anthers mature. Enhanced levels of three polypeptides with molecular weights of 64,000, 63,000 and 45,000 daltons characterize premeiosis in fertile anthers. Protein levels and patterns from anthers of the CMS line (5707) show little alteration during anther development. Protein synthesis seems to be at least partially blocked in the CMS microspore. The 63,000 and 45,000 dalton proteins are not present, and the absence of any unique protein(s) in the CMS line argues against a virus as the causal agent of CMS in Petunia. Analysis of free amino acids from anthers of the fertile line shows levels of proline and pipecolic acid 2–3 and 10–20 fold higher, respectively, than in the CMS line. The amino acids incorporated into proteins show no such differences; analysis of protein hydrolysates shows similar levels of each amino acid in both fertile and CMS lines at every developmental stage examined.     

Possible mitochondrial involvement in mechanism of cytoplasmic male sterility in maize (Zea mays L.)

The mechanism of cytoplasmic male sterility was investigated in maize by isolating mitochondria from seedlings and various anther stages and analyzing cytochrome oxidase and succinic dehydrogenase biochemically and electrophoretically. Sterile anthers exhibited a lack of biochemical activity and fewer isozymatic bands for cytochrome oxidase. No apparent differences were detected biochemically or electrophoretically between fertile and sterile anthers for succinate dehydrogenase.     

mRNA differential display between sterile and fertile anther of rice and analysis of cDNA differential fragments

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Differential proteomic studies of the genic male-sterile line and fertile line anthers of upland cotton (Gossypium hirsutum L.)

Pollen development is disturbed in the microspore development stage of the double-recessive nuclear male-sterile line ms5ms6 (Gossypium hirsutum L.). This study aimed to identify differentially expressed anther proteins and their potential roles in pollen development and male sterility. We compared the proteomes of sterile and fertile anthers of the double recessive nuclear male-sterile line ms5ms6. Approximately 1,390 protein spots were detected by two-dimensional differential gel electrophoresis. Proteins with altered accumulation levels in sterile anthers compared with fertile anthers were identified by mass spectrometry and the NCBInr and Viridiplantae EST databases. Down-regulated proteins in the sterile anthers included cytosolic ascorbate peroxidase 1 and glutaminyl-tRNA synthetase (glutamine-tRNA ligase). Several carbohydrate metabolism- and photosynthesis-related enzymes were also present at lower levels in the mutant anthers. By contrast, ATP-dependent RNA helicase eIF4A-13, NADH dehydrogenase subunit 1, enolase, gibberellin 20-oxidase, gibberellin 3-hydroxylase 1, alcohol dehydrogenase 2d, 3-ketoacyl-CoA synthase, and trehalose 6-phosphate synthase were expressed at higher levels in sterile anthers than in fertile anthers. The regulation of upland cotton pollen development involves a complex network of differentially expressed genes. This study provides the foundation for future investigations of gene function in upland cotton pollen development and male sterility.     

Mechanism of male sterility in Petunia: The relationship between pH,callase activity in the anthers,and the breakdown of the microsporogenesis

Summary In the locules of fertile Petunia hybrida anthers the in vivo pH during meiosis is 6.8–7.0 and no callase activity can be detected. Towards the end of the tetrad stage, the pH drops to 5.9–6.2 followed by a burst of callase activity. Subsequently, callose in the tetrad walls is digested and the quartets of microspores are released into the anther locules and develop into pollen grains. In the anther locules of one cytoplasmic male sterile (cms) Petunia type the pH drop and strong callase activity are already evident at early meiotic stages. Consequently, the callose already accumulated in the pollen mother cell (PMC) walls is digested and the PMC's cease to develop and are degraded. In another sterile genotype, the pH of the locule remains high (6.8–7.0), no callase activity is detected at the end of tetrad stage and the callose walls remain intact until a very late stage. It is suggested that the timing of callase activity is critical for the normal development of the male gametophyte and that faulty timing may result in male sterility. Measurements of pH in vivo and assays for callase activity in vitro indicate that the low pH is a precondition for the enzyme activity. Furthermore, it is suggested that the activation of callase in vivo is in some way connected with the changes in the pH of the locule.Contribution from The Volcani Institute of Agricultural Research, 1970 Series, No. 1709-E.Supported in part by grant No. FG-Is-171 from the United States Department of Agriculture, under P. L. 480.     

温敏核不育小麦可育花药和败育花药发育观察

对温敏核不育小麦百农不育系（Bainong sterility,BNS）的可育和不育花药结构进行对比观察。在减数分裂期、小孢子早期和小孢子晚期,可育花药与不育花药的结构相同。小孢子分裂形成二胞花粉后,可育花粉中随着大液泡的分解,细胞质内含物增加,其中出现一些颗粒状物质。不育花药中,小孢子也可分裂形成二胞花粉,但营养细胞的大液泡不分解,细胞质也不增加,最终花粉中的细胞质消失,花粉败育。该种温敏核不育小麦的花粉败育时间发生在二胞花粉早期,可能和其大液泡没有适时分解有关。花粉败育时间的确定为进一步深入研究该种雄性不育小麦的败育机制打下了基础。     

Characterization of anther differentiation in cytoplasmic male sterile maize using a specific isozyme system (esterase)

Summary During anther development, characterized in maize plants with N cytoplasm, certain esterase isozymes in non-microspore cells decrease in amount with anther age and new isozymes appear in the developing microspores. In anthers from male sterile plants with cms T or cms C cytoplasm, neither of these changes in esterase patterns occurred. In anthers from plants with cms S cytoplasm, the decrease in the esterases of non-microsporogenous cells was observed but not the appearance of microspore esterases. In lines carrying cms S cytoplasm and nuclear restorer genes, esterase changes during anther development were as in normal fertile anthers. These results are discussed with respect to the phenomenon of cytoplasmic male sterility in the different maize genotypes.     

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.     

ORGANELLE SIZE AND NUMBER IN FERTILE AND T-CYTOPLASMIC MALE-STERILE CORN

Anthers of inbred F₄₄ fertile (N) and cytoplasmic male-sterile (T) corn plants (Zea mays L.) were compared cytologically. No differences between fertile and sterile anthers were observed in size and number of mitochondria or plastids until after the start of anther degeneration. A rapid division of mitochondria was observed, however, in the tapetum and sporogenous cells of both fertile and sterile anthers during early growth stages. This rapid increase in mitochondrial numbers per cell (some 20-to 40-fold) preceded tapetal breakdown in sterile anthers and did not occur in other anther cells or in plastids. Limited observations on the megagametophyte and nucellus revealed that mitochondria in ovules remain relatively constant in size and number during gametogenesis and do not undergo degeneration.     

小麦中雄性不育同源序列的分离、鉴定及表达分析

利用拟南芥中已克隆的雄性核不育基因MS2和水稻中假定雄性不育蛋白的保守区域,设计一对简并引物,并在太谷核不育小麦可育株及不育株花药中进行扩增,得到了一条134bp的片段。以该片段为基础,通过电子延伸得到一个长为1604bp的序列,该序列编码的氨基酸包含一段由200个氨基酸组成的雄性不育保守区。RT-PCR结果表明,该雄性不育同源序列只在小麦可育花药中表达,而在小麦败育花药、叶片和根中不表达,说明该雄性不育同源序列为花药发育特异基因。     

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     

Effects of Petunia cytoplasmic male sterile (CMS) cytoplasm on the development of sterile and fertility-restored P. parodii anthers

The developmental defects causing cytoplasmic male sterility in Petunia parodii are described in isonuclear fertile, sterile, and fertility-restored plants using both light- and scanning electron microscopy. The aberrant development of the sporogenous tissue and tapetal layer caused by the cytoplasmic male sterile cytoplasm in both Petunia hybrida and P. parodii nuclear backgrounds is similar in onset and progression. The degeneration of the sporogenous tissue and tapetal layer of sterile anthers is first apparent late in meiosis and results in highly abnormal sterile sporogenous tissue by tetrad stage of fertile anthers. The stomium and endothecium do not show major developmental differences between fertile and sterile anthers, but the inner connective tissue of sterile anthers contained calcium crystals not found at high abundance in fertile anthers. Ovoid bodies containing magnesium and phosphorus were seen only in the vascular bundles of fertile anthers. Material prepared for the scanning electron microscope by freeze drying showed better retention of fragile morphological features, while critical-point drying permitted examination of nonvolatile structures, such as cell walls.     

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.     

A low molecular weight proteome comparison of fertile and male sterile 8 anthers of Zea mays

During maize anther development, somatic locular cells differentiate to support meiosis in the pollen mother cells. Meiosis is an important event during anther growth and is essential for plant fertility as pollen contains the haploid sperm. A subset of maize male sterile mutants exhibit meiotic failure, including ms8 (male sterile 8) in which meiocytes arrest as dyads and the locular somatic cells exhibit multiple defects. Systematic proteomic profiles were analysed in biological triplicates plus technical triplicates comparing ms8 anthers with fertile sibling samples at both the premeiotic and meiotic stages; proteins from 3.5 to 20 kDa were fractionated by 1‐D PAGE, cleaved with Lys‐C and then sequenced using a LTQ Orbitrap Velos MS paradigm. Three hundred and 59proteins were identified with two or more assigned peptides in which each of those peptides were counted at least two or more times (0.4% peptide false discovery rate (FDR) and 0.2% protein FDR); 2761 proteins were identified with one or more assigned peptides (0.4% peptide FDR and 7.6% protein FDR). Stage‐specific protein expression provides candidate stage markers for early anther development, and proteins specifically expressed in fertile compared to sterile anthers provide important clues about the regulation of meiosis. 49% of the proteins detected by this study are new to an independent whole anther proteome, and many small proteins missed by automated maize genome annotation were validated; these outcomes indicate the value of focusing on low molecular weight proteins. The roles of distinctive expressed proteins and methods for mass spectrometry of low molecular weight proteins are discussed.     

水稻非花粉型细胞质雄性不育系及其保持系花药发育过程中Ca2+的分布变化

钙在高等植物中被称为第二信使,与植物的有性生殖有关。为了研究水稻（Oryza sativa L.）花药中钙的定位与花粉败育的关系,利用焦锑酸钾沉淀法研究了非花粉型细胞质雄性不育系G37A及其保持系G37B花药的发育过程及其细胞中Ca^2＋ 的分布变化。研究发现,在2个材料间花药中钙的分布存在大量差异。G37B的可育花药在花粉母细胞时期及二分体时期,很少看到有Ca^2＋的沉积;而在单核花粉时期,Ca^2＋沉积急速地增加,主要定位在绒毡层细胞、花粉外壁外层及乌氏体的表面;随后花药壁上沉积的Ca^2＋减少而花粉的外壁外层仍然有很多Ca^2＋沉积物。相反,G37A的不育花药在花粉母细胞时期和二分体时期有大量的Ca^2＋沉积在小孢子母细胞和花药壁,中间层和绒毡层特别多。在二分体时期之后,不育花药的Ca^2＋沉积减少,特别是绒毡层内切向质膜附近的Ca^2＋几乎消失。但是同时期的可育花药中,有大量的Ca^2＋沉积在绒毡层。不育花药的Ca^2＋沉积在开花几天后消失。根据研究结果推测在不育花药发育早期中更多的钙离子与花粉败育有一定的关系。     

光周期诱导光敏感核不育水稻花药蛋白变化的研究

采用双向电脉技术对不同光周期条件下,光敏感核不育水稻（农垦５８ｓ）的可育与不育花药蛋白的变化进行分析,发现花粉发育的不同阶段中,不育花药具有四个特异蛋白ｐＩ６．２／ｂＭＷ７０ＫＤ,ｐＩ６．２／ＭＷ６８ＫＤ,ｐＩ６．２／ＭＷ３８ＫＤ和ｐＩ７．４／ＭＷ３７ＫＤ．对游离组蛋白的分析表明．长日照诱导的不育花药中游离组蛋白的相对百分率均明显低于短日照下的可育花药．据此推测长日照诱导不百花药蛋白质组成和代谢变化．不育花药中游离组蛋白含量低,可能受ＤＮＡ合成数量少的影响．     

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.     

Ethylene is Involved in the Control of Male Gametophyte Development and Germination in Petunia

The time courses of 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production in developing anthers of petunia fertile and sterile lines and the effects of exogenously applied ethylene and an inhibitor of ethylene action, 2,5-norbornadiene (NBD), on male gametophyte development and germination were investigated. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during microspore development simultaneously with degeneration of both tapetal tissues and middle layers of the anther wall. The second peak coincided with maturation and dispersal of pollen grains. The mature pollen is characterized by a high ACC content (up to 300 nmol/g). Exogenously applied ethylene (1–100 ppm) induced degradation of gametophytic generation at the meiosis stage. NBD completely inhibited anther development at the early stages of its development and delayed anther dehiscence. In anther tissues of the petunia sterile line, tenfold higher ethylene production was observed at the meiosis stage compared to that in fertile male gametophytes and this correlated with degeneration of both microsporocytes and tapetal tissues. In vitro male gametophyte germination was accompanied by an increase of ethylene production, whereas NBD completely blocked male gametophyte germination. These results suggest that ethylene is an important factor in male gametophyte development and germination.     

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。