Wall development and its autofluorescence of sterile and fertileVicia faba L. Pollen

Summary The ultrastructural changes of the pollen wall of three types of fertile and one of sterileVicia pollen were related to the autofluorescence of the pollen wall, measured by a microspectroscopic method. Till the liberation of the microspores from the tetrad, the spectrum of the ectexine shows sometimes two maxima and has a very low intensity. After this period the endexine is formed and its spectrum has one maximum with a high intensity. The differences of the pollen wall between the sterile and fertile pollen exist of the presence of one spectral maximum during the tetrad stage, a thick endexine and the absence of the intine in the sterile pollen. The different types show much differences during the tetrad stage in the callose wall as well as the ectexine. The autofluorescence illustrates the complexity and specificity of the pollen wall development.     

Characterization of callase (β-1,3-d-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.     

Histological aspects of microsporogenesis in fertile,cytoplasmic male sterile and restored fertilePetunia hybrida

Summary A comparative histological study is made of microsporogenesis in fertile, cytoplasmic male sterile and restored fertilePetunia. Microsporogenesis in sterile anthers proceeds normally until leptotene. The development of the restored fertile type at 25°C is normal until the tetrad stage. In both types sporogenesis arrests and the meiocytes, c.q. microspores ultimately degenerate. The first phenomena of deviation are found in the tapetum. The effects of degeneration on cellular structure, vacuolation and cytoplasmic organization of the tapetal and sporogenous cells are variable. The deposition of callose around the meiocytes appears independent of the process of degeneration. The absence of an increase in callase activity possibly explains the remnants of callose found at late stages of development. The failure of callose wall dissolution appears to be the result of metabolic abnormalities in the tapetum and is regarded as an indirect effect of sterility.     

A histological comparison of the development of pollen and female gametophytes in fertile and sterile Cryptomeria japonica

To determine a possible mechanism causing male and female sterility in Cryptomeria japonica male and female cones were collected from a C. japonica, tree, ShinDai2, that lacks pollen release and fertile seeds and specimens were processed to examine the development of pollen and female gametophytes using light microscopy and field emission scanning electron microscopy. Pre-meiotic development proceeded normally, but the formation of aberrant meiotic products was observed in cones of both sexes. In sterile microsporangia, heterogeneous microspore populations ranging from monads to polyads gave rise to mature pollen grains of non-uniform size. These pollen grains were covered with an amorphous layer and adhered to each other. In addition, they remained in the microsporangia and were not released even after the onset of pollen dissemination from fertile trees. In the ovules of sterile female cones, megaspores with abnormal shapes, numbers, and sizes formed, and the development of female gametophytes was arrested at the free nuclear or archegonium formation stages. These gametophytes collapsed, and no fertile embryo was generated. Results indicate that meiotic defects are important in the sterility mechanism.     

丹参雄性不育系Sh-B的鉴定与花粉发育过程的解剖学研究

在显微水平上对新发现的丹参雄性不育系Sh-B花药发育过程进行了解剖学观察,并对其花粉活力和结实率进行了鉴定。结果显示:根据花器官及花药的形态、大小以及花丝的长度,可以将Sh-B不育株分为3个不育类型,即Sh-B1、Sh-B2和Sh-B3。这3种不育类型均属于雄性不育,其花丝不到正常可育株的1/2,花药干瘪而瘦小,内无花粉粒或花粉无活力;其根、茎、叶以及种子形态结构与正常可育植株基本相似。产生雄性不育的主要原因有:花粉囊药室内壁纤维层加厚,影响花药壁开裂;小孢子母细胞周围不产生胼胝质或产生的胼胝质很少;绒毡层细胞延迟解体;花粉粒畸形。在其花药发育的小孢子母细胞时期、四分体形成前期、单核期、双核期均可能产生雄性不育的小孢子或花粉粒。     

POLLEN AND TAPETUM DEVELOPMENT IN DESMODIUM GLUTINOSUM AND D. ILLINOENSE (PAPILIONOIDEAE; LEGUMINOSAE)

Each of the four microsporangia has three or four wall layers, a uninucleate tapetum of various cell shapes with nuclei that remain in prophase, and 12-24 pollen mother cells (PMCs). A sterile transverse septum sometimes bisects the microsporangium. PMCs secrete callose but not uniformly, and contact among them continues through meiosis. Simultaneous cytokinesis by furrowing isolates each microspore in callose, which later disperses. The separated microspores become vacuolate, undergo mitosis to become pollen, and later become filled with food reserves. Endothecial wall thickening and tapetal dissolution occur after pollen engorgement. Calcium oxalate crystals form in tapetal cells during the sporogenous stage, reach maximum size during early meiosis, and remain prominent until tapetal dissolution.     

芝麻核雄性不育系ms86-1微粉发生的细胞学观察

芝麻(Sesamum indicum)核雄性不育系ms86-1姊妹交后代表现为可育、部分不育(即微粉)及完全不育(简称不育)3种类型。不同育性类型的花药及花粉粒形态差异明显。Alexander染色实验显示微粉植株花粉粒外壁为蓝绿色, 内部为不均一洋红色, 与可育株及不育株花粉粒的染色特征均不相同。为探明芝麻微粉发生机理, 在电子显微镜下比较观察了可育、微粉、不育类型的小孢子发育过程。结果表明, 可育株小孢子母细胞减数分裂时期代谢旺盛, 胞质中出现大量脂质小球; 四分体时期绒毡层细胞开始降解, 单核小孢子时期开始出现乌氏体, 成熟花粉时期花粉囊腔内及花粉粒周围分布着大量乌氏体, 花粉粒外壁有11–13个棱状凸起, 表面存在大量基粒棒, 形成紧密的覆盖层。不育株小孢子发育异常显现于减数分裂时期, 此时胞质中无脂质小球出现, 细胞壁开始积累胼胝质; 四分体时期绒毡层细胞未见降解; 单核小孢子时期无乌氏体出现; 成熟花粉时期花粉囊腔中未发现正常的乌氏体, 存在大量空瘪的败育小孢子, 外壁积累胼胝质, 缺乏基粒棒。微粉株小孢子在减数分裂时期可见胞质内有大量脂质小球, 四分体时期部分绒毡层发生变形, 单核小孢子时期有部分绒毡层开始降解; 绒毡层细胞降解滞后为少量发育进程迟缓的小孢子提供了营养物质, 部分小孢子发育为正常花粉粒; 这些花粉粒比较饱满, 表面有少量颗粒状突起, 但未能形成覆盖层, 花粉囊腔中及小孢子周围存在少量的乌氏体。小孢子形成的育性类型与绒毡层降解是否正常有关。     

Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall

The quartet (qrt) mutants of Arabidopsis thaliana produce tetrad pollen in which microspores fail to separate during pollen development. Because the amount of callose deposition between microspores is correlated with tetrad pollen formation in other species, and because pectin is implicated as playing a role in cell adhesion, these cell-wall components in wild-type and mutant anthers were visualized by immunofluorescence microscopy at different stages of microsporogenesis. In wild-type, callose was detected around the pollen mother cell at the onset of meiosis and around the microspores during the tetrad stage. Microspores were released into the anther locule at the stage where callose was no longer detected. Deposition and degradation of callose during tetrad pollen formation in qrt1 and qrt2 mutants were indistinguishable from those in wild-type. Enzymatic removal of callose from wild-type microspores at the tetrad stage did not release the microspores, suggesting that callose removal is not sufficient to disperse the microspores in wild-type. Pectic components were detected in the primary wall of the pollen mother cell. This wall surrounded the callosic wall around the pollen mother cell and the microspores during the tetrad stage. In wild-type, pectic components of this wall were no longer detectable at the time of microspore release. However, in qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation. This result suggests that failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, and indicates that QRT1 and QRT2 may be required for cell type-specific pectin degradation to separate microspores.     

Histology of sterile male and female cones in<Emphasis Type="Italic"> Pinus monticola</Emphasis> (western white pine)

Two years of histological samples were collected from a Pinus monticola Dougl. (western white pine) tree identified as not producing mature pollen or seed cones. Anatomical information was collected to the ultrastructural level, to assess possible mechanisms for pollen and cone abortion resulting in sterility. Development of male and female gametophytes in the sterile western white pine tree was arrested after meiosis and before further cell divisions could take place. Sterile male gametophytes (pollen grains) had poorly developed pollen walls and sacci, reduced and degenerative cytoplasm, and no evidence of stored starch grains. The pollen cone aborted prior to pollen dehiscence. Meiosis of the megaspore mother cell in the ovule produced four megaspores, but development was stopped at the functional megaspore stage. The seed cone aborted in the first year of growth before winter dormancy. Tapetal tissue in sterile microsporangia appeared similar to that of fertile microsporangia, until the vacuolate, uninucleate microspore stage. Tapetal cells and thecal fluid surrounding the sterile microspores persisted well past the time when microsporangia on fertile trees started the process of maturation and desiccation. At pollen dehiscence, sterile pollen cones did not release any pollen and the microsporangia were filled with a sticky fluid. The behaviour of the tapetum in P. monticola sterile cones is compared with reports of tapetal function and malfunction reported in studies of angiosperm and other gymnosperm species. The occurrence and timing of gametophyte abortion in both cone sexes suggests a genetic rather than environmental basis for the sterility mechanism.     

Expression of ogu cytoplasmic male sterility in cybrids of Brassica napus

Summary A light and electron microscopic investigation revealed that ogu cytoplasmic male sterility (CMS) in cybrids of Brassica napus is primarily a deficiency of the tapetum and clearly time and site specific. Three patterns of ogu CMS were found, and specific conclusions drawn. First, the partially male fertile cybrid 23 was highly variable. It sometimes produced heterogeneous stamens with an endothecium formed exclusively around the fertile locules, thus delineating each microsporangium as a functional unit. The second type, including cybrids 27, 58 and 85, on the contrary, was stable and completely male sterile. In the four locules of normal length, microspores were observed to die at the vacuolate polarized stage while the tapetum disappeared prematurely through excessive vacuolization by the end of meiosis followed by a rapid autolysis during the tetrad or early free microspore stage. The subepidermal layer of the locule wall failed to form characteristic thickenings. The male-sterile stamens were completely indehiscent. At the time of anthesis they contained only collapsed empty exines adhering to each other. These cybrids, 27, 58 and 85, were closest to the ogu CMS trait of radish and seemed to be the best suited for further use in plant breeding. The third pattern was found in cybrids 77 and 118, which besides showing abortion of the microsporangia also showed a feminization of the stamens. We suggest that this feminization might be due to an alloplasmic situation associating Brassica napus nuclear genes with the mitochondrial DNA of radish.     

Effects of chilling on male gametophyte development in rice

Chilling during male gametophyte development in rice inhibits development of microspores, causing male sterility. Changes in cellular ultrastructure that have been exposed to mild chilling include microspores with poor pollen wall formation, abnormal vacuolation and hypertrophy of the tapetum and unusual starch accumulation in the plastids of the endothecium in post-meiotic anthers. Anthers observed during tetrad release also have callose (1,3-beta-glucan) wall abnormalities as shown by immunocytochemical labelling. Expression of rice anther specific monosaccharide transporter (OsMST8) is greatly affected by chilling treatment. Perturbed carbohydrate metabolism, which is particularly triggered by repressed genes OsINV4 and OsMST8 during chilling, causes unusual starch storage in the endothecium and this also contributes to other symptoms such as vacuolation and poor microspore wall formation. Premature callose breakdown apparently restricts the basic framework of the future pollen wall. Vacuolation and hypertrophy are also symptoms of osmotic imbalance triggered by the reabsorption of callose breakdown products due to absence of OsMST8 activity.     

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.     

华北落叶松花粉发育过程中的钙动态分布

运用焦锑酸钾沉淀法研究了华北落叶松(Larix principis-rupprechtii Mayr)小孢子发育过程中不同阶段Ca2 的分布情况.减数分裂时期,小孢子囊壁表皮和中层细胞的细胞壁及细胞间隙Ca2 分布较多,绒毡层只有外切向面的细胞膜有Ca2 分布,小孢子母细胞的各部位则很少有Ca2 ;四分体时期,包围四分小孢子的胼胝质壁上有大量的Ca2 分布,在四分孢子壁上也有较多沉淀;游离小孢子时期,钙离子在小孢子壁的分布较四分体时期有所减少,而到花粉成熟时又逐渐增多;从四分体到花粉成熟,乌氏体周围的Ca2 有增多的趋势.对四分体外壁Ca2 的大量分布与花粉壁的形成及信号物质在花粉表面贮存的关系,以及小孢子囊的外壁、绒毡层和乌氏体在Ca2 向花粉运输中所起的作用进行了讨论.     

Abnormal Vacuolization of the Tapetum During the Tetrad Stage is Associated with Male Sterility in the Recessive Genic Male Sterile <Emphasis Type="Italic">Brassica napus</Emphasis> L. Line 9012A

In the recessive genic male sterile line 9012A of Brassica napus, pollen development is affected during the tetrad stage. According to the light and electron microscopy analysis of tapetal cells and tetrads, the sterile tapetal cells swelled with expanded vacuoles at the early tetrad stage and finally filled the center of the locules where a majority of tetrads encased with the thick callose wall collapsed and degraded. We suggested that an absence of callase, which is a wall-degrading enzyme stored in the vacuoles of tapetal cells before secretion, resulted in the failure of tetrad separation. Moreover, transmission electron microscopy analysis showed that the secretory tapetal cells were not observed in sterile anthers, which indicated that the transition of the tapetum from the parietal type to the secretory type was probably aberrant. In plants, degeneration of the tapetum is thought to be the result of programmed cell death (PCD). PCD of tapetal cells was investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and signals indicative of deoxyribonucleic acid fragmentation were detected much earlier in sterile anther than in fertile anther. This suggests that tapetal breakdown does not occur by the normal procession of PCD and might be following an alternative mechanism of unscheduled apoptosis in line 9012A. This research supports the hypothesis that premature PCD is associated with male sterility in B. napus.     

Anatomical differences on development of fertile and sterile pollen grains of Prunus salicina Lindl.

Anatomical changes occurring during the microsporogenic development of P. salicina Lindl. were studied in male fertile and male sterile genotypes. Male fertile pollen grains showed three well determined pore regions, without ektexine. Intine was thick and surrounded the vegetative cell. Vegetative cells enclosed the generative cells; their cytoplasm was rich in plastids, abundant RER and active mitochondria. Development of sterile pollen was different from the meiosis step. Microspores did not show germination pores and ektexine was continuous around the whole grain. Pollen grains showed an atypical shape. The tapetum persisted after the tetrad stage and showed hypertrophy and vacuole development, resulting in abnormal microspore development. Only a few pollen grains and rudiments of collapsed microspores close to the anther wall were formed at anthesis.     

Tapetum-Specific Expression of the Gene for an Endo-{beta}-1,3-glucanase Causes Male Sterility in Transgenic Tobacco

A cDNA for a pathogenesis-related endo-ß-1,3-glucanaseisolated from soybean, was fused to an anther tapetum-specificpromoter (Osg6B promoter) isolated from rice and the resultingchimeric gene was introduced into tobacco. The Osg6B promoterbecame active in the anther tapetum during formation of tetradsand the tapetal glucanase activity in the transgenic plantscaused in a significant reduction in the number of fertile pollengrains. Most of the pollen grains were aberrant in shape, lackedgerminal apertures and aggregate of the pollen grains. Granulesof ß-1,3-glucan, which have not previously been reported,were often observed to adhere to the surface of the pollen grains.Further observations revealed that the callose wall was almostabsent in the pollen tetrads of transgenic plants. In wild-typeplants, by contrast, the tetrads were surrounded by callosethat was degraded soon after the tetrad stage to release freemicrospores. Thus, the introduced gene for endo-ß-1,3-endoglucanaseunder the control of the Osg6B promoter caused digestion ofthe callose wall at the beginning of the tetrad stage, a timethat was just a little earlier than the time at which endogenousglucanase activity normal appears. These results demonstratethat premature dissolution of the callose wall in pollen tetradscauses male sterility and suggest that the time at which tapetallyproduced glucanase is activate is critical for the normal developmentof microspores. (Received September 29, 1994; Accepted January 30, 1995)     

白菜核雄性不育两用系可育和不育花药ATPase的分布

对白菜核雄性不育两用系可育花药和不育花药的ATPase做了定位分析。可育花药的花粉母细胞核中积累了大量的ATPase反应颗粒,而细胞质中ATPase反应颗粒较少,但在线粒体中特异地聚集了一些大的ATPase反应颗粒。减数分裂后,小孢子细胞质中ATPase反应颗粒明显增加。随着小孢子发育,其细胞质中ATPase反应颗粒逐渐减少,但在线粒体中又特异性地聚集了较多的AT-Pase反应颗粒。当花药发育到二胞花粉时期,花粉和绒毡层细胞中的ATPase反应颗粒已很少了。不育花药的花粉母细胞中呈现较多的ATPase,然而在线粒体中很少。异常四分体小孢子细胞质中虽然有较多的ATPase反应颗粒,但还是通过细胞质收缩和质壁分离方式退化。对可育花药的花粉母细胞线粒体中特异出现的簇状ATPase分布现象进行了分析,讨论了不育花药中花粉母细胞线粒体ATPase的异常与花粉败育的可能关系。     

Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis

Male-sterile mutants are being studied to deepen our understanding of the complex processes of microsporogenesis and microgametogenesis. Due to difficulties associated with isolating the mutated gene, there is currently very little molecular information on the defects responsible for male sterility. As a first step in utilizing male-sterile mutants to better understand the bio-chemical and molecular processes that control pollen development, we have characterized a number of Arabidopsis thaliana lines that were generated by seed transformation and exhibit male sterility. We report here the identification and characterization of three male-sterile A. thaliana lines, all of which are tagged with T-DNA and show aberrant meiosis. A detailed cytochemical study was conducted on these lines to better understand the timing and nature of each mutation and to investigate how these mutations affect subsequent steps of pollen development. All three mutants undergo apparently normal morphogenesis until the onset of meiosis. In one line (6492) the mutation is most notable at the tetrad stage when up to eight microspores can be seen in each callose-encased tetrad. The resulting mutant microspores are of variable sizes and contain different amounts of DNA. Two other mutants (7219 and 7593) possess many common features, including variable developmental pathways, failure to produce callose, production of vacuolate, coenocytic (multi-nucleate) cells that are surrounded by persistent microsporocyte walls, and asynchronous patterns of development. Unlike the situation in wild-type plants, where developmental stages are correlated with bud length, such correlations are almost impossible with these two mutants. The sporogenous tissue within all three of these mutant lines collapses prior to anthesis.     

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