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.     

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.     

The persistence and lignin-like appearance of the primary cell wall of microsporocytes in male-sterile (CMS) sweet pepper,Capsicum annuum L

Using differential staining of cell walls the anatomy of microsporogenesis was investigated on cross-sections of anthers of fertile plants of the sweet pepperCapsicum annuum L. cv. Severka, as well as of its sterile analogues. The lignin-like staining as observed in the microsporocyte primary walls of fertile plants disappears with their getting independent in the course of meiosis. On the contrary, in sterile plants the stain increases in intensity, the thick-walled microsporocytes usually form a continuous block up to the period of tetradogenesis, and so far as microspores originate, they are not dissociated outwards. Moreover, in sterile anthers the external tapetum is usually not differentiated.     

水稻马协不育与可育花药ATPase定位（简报）

Pb3 (po4)2-precipitation was used to study the ATPase activities both in fertile and sterile anthers of rice (Oryza sativa L. cv. Marxie). At big-vacuole pollen stage, tapetum of the fertile anthers showed high ATPase activity in their nuclei. In fertile pollen, ATPase was localized on the outside surface of the exine and in the nucleus both at big-vacuole and at bi-nucleate pollen stage. At late bi-nucleate pollen stage, a large amount of Pb3 (PO4)2 precipitated in endintine of the fertile pollen. In sterile anthers, tapetum was fully degenerated at big-vacuole pollen stage. In sterile pollen, ATPase was localized both on the plasmamembrane and in intine. These phenomena lasted to the bi-nucleate pollen stage. In addition, most of the sterile pollen did not show well-developed endintine. Based on the above results, we suggested that abortive tapetum could not provide enough nutrition for pollen development, and the high ATPase activity both on plasma membrane and in intine would likely result in ATP shortage in sterile anthers.     

水稻马协不育与可育花药ATPase定位(简报)

马协不育系系朱英国等用农家品种“马尾粘”的不育株与“协青早选”测交和回交选育成功的。超微结构研究表明马协不育花药单核边位期绒毡层提前解体,液泡膜破裂;不育花粉外壁基粒棒少,且分布不均匀;线粒体有大的电子透明区,二核期不育花粉中央大液泡崩溃。不育花药绒毡层提前解体可能影响花粉外壁合成及花粉营养供应,大量研究表明绒毡层发育异常与花粉败育密切相关,而液泡膜的破坏会导致水解酶类的泄     

太谷核不育小麦可育花药内游离脯氨酸的来源、利用及与不育花药败育的关系

太谷核不育小麦营养体内的游离脯氨酸能迅速地运入并积累于花药中,花药有将谷氨酸转化成脯氨酸的能力。脯氨酸向花药内以主动运输为主,能被DNP所抑制,与旗叶的功能密切相关。在发育阶段Ⅰ(小孢子释放前后),脯氨酸已不能由营养体运入不育花药,运输的障碍在花丝或花药中,不育花药缺乏游离脯氨酸可能是主要原因。发育阶段Ⅰ的可育花药内,脯氨酸是合成蛋白质或其他大分子物质的原料。因而,脯氨酸的缺乏可能是加速不育花药小孢子败育和小孢子释放后急剧解体的重要原因。     

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.     

Hydroxycinnamic acid amides in fertile and cytoplasmic male sterile lines of maize

Hydroxycinnamic acid (HCA) amides in fertile and cytoplasmic male sterile lines of maize were determined in reproductive organs, developing grains and cobs. HCA amides occurred in large amounts in the anthers of fertile plants (line F7N) and were absent from the anthers of cytoplasmic male sterile lines (lines F7T and F7C). Restoration of fertility was associated with the production of these compounds (line FC31). Considerable variations were observed in the concentrations of HCA amides at different stages of growth and grain maturation. Changes of HCA amides in the grains which were to produce sterile plants followed a pattern similar to that obtained with the grains which were to produce fertile plants. Accumulation of HCA amides was substantially higher in fertile lines whatever their genotype (F7N, FC31 and F7T x FC31) than in sterile lines. Marked changes occurred in the HCA amide content of embryo and endosperm during grain development. Many changes in HCA amides were observed in cobs during development and maturation, but no substantial differences could be observed between fertile and sterile lines.     

Direct regeneration of shoots from immature inflorescence cultures of turmeric

Microspore-derived embryo (MDE) induction was evaluated following the culture of wheat (Triticum aestivum L.) anthers in a basal medium with a range of supplements. Several supplements were evaluated, including a commercial bovine haemoglobin solution (Erythrogen™), Ficoll and the co-polymer surfactant Pluronic F-68, but these did not enhance MDE induction. However, phenylacetic acid, replacing both 2,4-dichlorophenoxyacetic acid and kinetin, increased MDE induction by a factor of 1.6 (p<0.05), producing 257±29 (mean±s.e., n=3) MDEs per 100 anthers cultured. Twenty-four plants regenerated from MDEs induced via anther culture were used for ploidy determinations; 33% were sterile haploids, while the remainder were fertile. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

顺乌头酸酶基因在杀雄剂SQ-1诱导小麦雄性不育花药中的表达分析

采用RT-PCR技术,克隆了小麦胞质顺乌头酸酶基因(cACO)部分cDNA序列.该cDNA序列长1368bp,编码456个氨基酸,GenBank登录号为GU475062.半定量RT-PCR结果表明,在生理型不育和可育花药发育的单核早期至三核期,cACO基因的表达水平均表现为先升后降;在生理型不育花药发育的单核晚期cACO基因表达水平与同期可育花药相比显著升高,到二核期和三核期明显降低,ACO酶活性变化表现出相同趋势.这反映出在小麦生理型不育系中,cACO基因在花药败育关键期异常表达可能影响了花药发育过程中正常的能量供应和物质代谢,导致花粉发育能量不足和所需物质匮乏,从而导致了非遗传型花药败育现象.     

不同类型水稻不育系药隔发育过程中Ca2+的分布变化

采用焦锑酸钾沉淀法研究了不同类型水稻不育系与其相应保持系及其可育花药药隔发育过程中Ca^2＋的分布变化。结果显示在正常花药的发育过程中,药隔中的Ca^2＋呈以下变化规律：（1）花药发育早期药隔中的Ca^2＋沉淀很少;（2）随着花药的发育,药隔中的Ca^2＋沉淀增加,木质部细胞的次生加厚壁上有较多的Ca^2＋沉淀,连接组织中的Ca^2＋沉淀也大大增加;（3）到了花粉内容物充实后期及成熟花粉时期,药隔中的Ca^2＋又略有减少。而不育花药药隔中的Ca^2＋在花药发育的各时期均比可育花药要多。败育类型不同,药隔中出现大量Ca^2＋沉淀的时期也不同,无花粉型不育系在花粉母细胞时期药隔中就有大量Ca^2＋沉淀,而农垦58S从单核花粉时期约隔中才出现大量Ca^2＋沉淀。实验结果表明：Ca^2＋在不同类型的不育水稻中呈现出一定的规律性变化,药隔中Ca^2＋的异常分布可能与水稻花粉的败育有关。     

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     

Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice

Potassium antimonate was used to locate Ca²⁺ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion. Received: 30 May 1997 / Accepted: 5 July 1997     

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage. Supported by National Natural Science Foundation of CHINA (30170060)     

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.     

Proteolytic activity in anther extracts of fertile and cytoplasmic male sterile petunia

Proteolytic activity is compared in anther extracts from Petunia parodii fertile and cytoplasmic male sterile lines. It is characterized relative to developmental stage of the anthers, effect of variable incubation times, pH of isolation buffers, and degradation of marker proteins. In fertile anthers, proteolytic activity increases at the end of microsporogenesis and peaks early in microgametogenesis. Degradation is most severe in extracts of fertile anthers and in high molecular weight proteins and reaches its maximum within 20 minutes. Degradation of marker proteins is greatest at pH 5.6 to 8.0 in fertile anther extracts and is eliminated under strong acid conditions (pH 2.8 to 4.0) in both fertile and cytoplasmic male sterile anther extracts. Marker proteins degrade more severely in extracts of fertile anthers; however, the order of substrate sensitivity—myosin > phosphorylase b > bovine serum albumin and ovalbumin > β-galactosidase—is the same in extracts from fertile and cytoplasmic male sterile anthers.     

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。     

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.     

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orf H522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.