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     

Study of male sterility in Taiwania cryptomerioides Hayata (Taxodiaceae)

Summary. A study of male sterility over a period of three consecutive years on a conifer species endemic to Taiwan, Taiwania cryptomerioides Hayata (Taxodiaceae), was done for this article. With the aids of fluorescence and electron microscopic observations, the ontogenic processes in the fertile and sterile microsporangia are compared, using samples collected from Chitou Experimental Forest and Yeou-Shoei-Keng Clonal Orchard of the National Taiwan University, Nantou, Taiwan. The development of male strobili occurred from August to the end of March. Microsporogenesis starts with the formation of the archesporium and ends with the maturation of 2-celled pollen grains within the dehiscing microsporangium. Before meiosis, there was no significant difference in ultrastructure between the fertile and sterile microsporangia. Asynchronous pollen development with various tetrad forms may occur in the same microsporangium of either fertile or sterile strobili. However, a callose wall was observable in the fertile dyad and tetrad, but not in the sterile one. After dissolution of the callose wall, the fertile microspores were released into the locule, while some sterile microspores still retained as tetrads or dyads with intertwining of exine walls in the proximal faces. As a result, there was no well developed lamellated endexine and no granulate ectexine or intine in the sterile microspores. Eventually, the intracellular structures in sterile microspores were dramatically collapsed before anthesis. The present study shows that the abortion in pollen development is possibly attributed to the absence of the callose wall. The importance of this structure to the male sterility of T. cryptomerioides is discussed. Correspondence and reprints: Department of Life Science, National Taiwan University, 106 Taipei, Taiwan.     

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。     

GDSL esterase/lipases OsGELP34 and OsGELP110/OsGELP115 are essential for rice pollen development

Pollen exine contains complex biopolymers of aliphatic lipids and phenolics. Abnormal development of pollen exine often leads to plant sterility. Molecular mechanisms regulating exine formation have been studied extensively but remain ambiguous. Here we report the analyses of three GDSL esterase/lipase protein genes, OsGELP34, OsGELP110, and OsGELP115, for rice exine formation. OsGELP34 was identified by cloning of a male sterile mutant gene. OsGELP34 encodes an endoplasmic reticulum protein and was mainly expressed in anthers during pollen exine formation. osgelp34 mutant displayed abnormal exine and altered expression of a number of key genes required for pollen development. OsGELP110 was previously identified as a gene differentially expressed in meiotic anthers. OsGELP110 was most homologous to OsGELP115, and the two genes showed similar gene expression patterns. Both OsGELP110 and OsGELP115 proteins were localized in peroxisomes. Individual knockout of OsGELP110 and OsGELP115 did not affect the plant fertility, but double knockout of both genes altered the exine structure and rendered the plant male sterile. OsGELP34 is distant from OsGELP110 and OsGELP115 in sequence, and osgelp34 and osgelp110/osgelp115 mutants were different in anther morphology despite both were male sterile. These results suggested that OsGELP34 and OsGELP110/OsGELP115 catalyze different compounds for pollen exine development.     

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。     

Genetics and development of morphological and physiological characters of male sterility in Oenothera

Summary Male sterility in Oenothera is influenced by two nuclear genes,fr andster. Their function is independent of the plastomes. Development of anthers, fertile and sterile male, was studied by electron microscopy and histochemical methods. Both genes act on lipid metabolism but at different developmental stages. Infr/fr homozygotes the disturbance is expressed as a lack of sporopollenin in the exine, while amorphous lipid material is deposited in the loculus. Inster/ster homozygotes sporopollenin is formed normally in the endexine but the paracristalline structure of the ektexine is missing. In both mutants the disturbance leads to complete destruction of the pollen grain. The deviation from fertile pollen development is correlated with abnormalities of the tapetum and outer cell layers of the anther wall.     

Microsporogenesis in male sterile Rosmarinus officinalis L. (Lamiaceae), an ultrastructural study

Within an extensive reproductive biology program on Rosmarinus officinalis a study of microsporogenesis in male sterile plants has been carried out. Two events, related to the two types of male sterile flowers found in this species (MS; Male sterile and INT; Intermediate male sterile flowers), are described. The first event is characterized by the early appearance of necrotic areas in the anther tissues during the final differentiation phases of the MS flowers, before meiosis takes place. Initially, these necrotic areas are small, later on they enlarge affecting the sporogenous tissue and tapetum. All the anthers in which necrosis has begun finally become aborted and lack pollen grains. The second event is manifested in the anthers of the INT flowers. Previous to the release of the microspores from the callosic envelope, vacuolisation of the tapetal cells takes place. The tapetum does not properly carry out its secretory function. It accumulates some substances in vacuoles and starch granules in plastids. The microspores degenerate in the vacuolate stage. In the epidermal and endothecial cells modifications are observed which may be related to the carbohydrate metabolism. Indehiscent, whitish anthers containing inviable microspores result. We have not found significant differences between the mitochondria in the anthers of the fertile flowers and MS or INT flowers. But we suggest, for both events, a metabolic failure, possibly controlled by cytoplasmic genes as the origin of male sterility. Paracrystalline material has been detected in microspore nuclei of developing INT anthers, though it does not seem to be related to the expression of male sterility.     

Callose (β-1,3 glucan) is essential for <Emphasis Type="Italic">Arabidopsis</Emphasis>pollen wall patterning,but not tube growth

Background  

Callose (β-1,3 glucan) separates developing pollen grains, preventing their underlying walls (exine) from fusing. The pollen tubes that transport sperm to female gametes also contain callose, both in their walls as well as in the plugs that segment growing tubes. Mutations in CalS5, one of several Arabidopsis β-1,3 glucan synthases, were previously shown to disrupt callose formation around developing microspores, causing aberrations in exine patterning, degeneration of developing microspores, and pollen sterility.     

The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map‐based cloning and sequence analysis, we identified a 1,459‐bp deletion in an adenosine triphosphate (ATP)‐binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very‐long‐chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.     

云南紫稻细胞质雄性不育系花药发育过程中Ca2+的分布特点

运用焦锑酸钾沉淀法研究了云南紫稻细胞质雄性不育系和保持系花药在发育过程中Ca^2 的分布特点。结果表明,保持系的花粉母细胞和小孢子的胞质内部基本无Ca^2 的沉淀,后期花粉外壁出现Ca^2 的沉淀;保持系早期的绒毡层细胞形态正常,胞内有少量Ca^2 沉淀,后期绒毡层细胞开始凋亡,胞质凝集,胞内出现大量Ca^2 的颗粒。不育系花粉母细胞在减数分裂时期败育,胞质液泡化,内部出现大量Ca^2 的沉淀;不育系绒毡层细胞形态正常,胞内无Ca^2 的沉淀。绒毡层与花粉母细胞、小孢子之间出现大量Ca^2 颗粒。探讨了不育系花药花粉母细胞中以及与绒毡层细胞之间Ca^2 的异常积累与雄性不育的关系。     

白菜核雄性不育花药超微结构的研究

对白菜核雄性不育两用系的可育与不育花药进行了超微结构的比较观察。结果显示不育花药的造孢细胞核仁靠边分布:包裹小孢子母细胞的胼胝质厚薄不均匀,不完整等早期异常现象。减数分裂后,四分体细胞中常有多个细胞核。从四分体释放出的小孢子外壁的孢粉素物质不均匀沉积．呈不连续的单层异常结构。最后小孢子通过细胞质收缩方式败育。在可育花药中,绒毡层细胞在小孢子发育后期已显示出退化迹象,同时在细胞中开始积累脂类物质。但在同时期的不育花药中, 绒毡层细胞没有显示出退化的迹象,也不合成脂类物质。从时间上看,败育花药中小孢子母细胞及小孢子的异常在先,绒毡层细胞的异常在后。本研究揭示了白菜核雄性不育花药的超微结构特征, 对我们以前的光学显微镜观察结果予以补充和修正。     

白菜核雄性不育花药超微结构的研究

对白菜核雄性不育两用系的可育与不育花药进行了超微结构的比较观察。结果显示不育花药的造孢细胞核仁靠边分布;包裹小孢子母细胞的胼胝质厚薄不均匀,不完整等早期异常现象。减数分裂后．四分体细胞中常有多个细胞核。从四分体释放出的小孢子外壁的孢粉素物质不均匀沉积,呈不连续的单层异常结构。最后小孢子通过细胞质收缩方式败育。在可育花药中．绒毡层细胞在小孢子发育后期已显示出退化迹象,同时在细胞中开始积累脂类物质。但在同时期的不育花药中．绒毡层细胞没有显示出退化的迹象,也不合成脂类物质。从时间上看,败育花药中小孢子母细胞及小孢子的异常在先,绒毡层细胞的异常在后。本研究揭示了白菜核雄性不育花药的超微结构特征．对我们以前的光学显微镜观察结果予以补充和修正。     

The Arabidopsis U–box/ARM repeat E3 ligase AtPUB4 influences growth and degeneration of tapetal cells,and its mutation leads to conditional male sterility

Pollen formation is a complex developmental process that has been extensively investigated to unravel underlying fundamental developmental mechanisms and for genetic manipulation of the male‐sterility trait for hybrid crop production. Here we describe identification of AtPUB4, a U–box/ARM repeat‐containing E3 ubiquitin ligase, as a novel player in male fertility in Arabidopsis. Loss of AtPUB4 function causes hypertrophic growth of the tapetum layer. The Atpub4 mutation also leads to incomplete degeneration of the tapetal cells and strikingly abnormal exine structures of pollen grains. As a result, although the Atpub4 mutant produces viable pollen, the pollen grains adhere to each other and to the remnants of incompletely degenerated tapetal cells, and do not properly disperse from dehisced anthers for successful pollination. We found that the male‐sterility phenotype caused by the Atpub4 mutation is temperature‐dependent: the mutant plants are sterile when grown at 22°C but are partially fertile at 16°C. Our study also indicates that the AtPUB4‐mediated pathway acts in parallel with the brassinosteroid pathway in controlling developmental fates of the tapetal cells to ensure male fertility.     

Cytological investigation of male sterility in sorghum caused by a dominant mutation (<Emphasis Type="Italic">Ms</Emphasis><Subscript><Emphasis Type="Italic">tc</Emphasis></Subscript>) derived from tissue culture

Male sterility mutations are an important tool in the investigation of anther and pollen development and for obtaining hybrid seeds in plant breeding. Cytological analysis of microsporo- and microgameto-genesis in sorghum plants with dominant mutation of male sterility (Ms_tc) derived from tissue culture has been carried out. Using substitution backcrosses, this mutation was introduced first into the nuclear background of the fertile sorghum line SK-723 and from this line into Volzhskoe-4w (V-4w). The mechanism of Ms_tc action on anther and pollen development differed in different nuclear backgrounds. In SK-723, phenotypic expression of Ms_tc began before the beginning of meiosis, which resulted in degeneration of sporogenous tissue in some anthers and in significant disturbances of anther morphology. In microsporocytes that did not degenerate, the frequency of non-specific meiotic abnormalities characteristic of the fertile line SK-723 significantly increased. In addition, in the mutant plants, a number of specific meiotic abnormalities--almost complete desynapsis, and formation of syncytial structures--were observed, apparently the consequence of Ms_tc action. In mono- or bi-nucleate microspores, degenerative processes resulting in formation of empty or anomalously coloured pollen grains led to almost complete male sterility. In the V-4w nuclear background, changes in anther structure and meiotic disturbances were infrequent. The degenerative processes began at the uni- or binucleate microspore stage and resulted in formation of empty or abnormally coloured pollen grains, and in partial pollen sterility. Thus, the same nuclear male sterility-inducing mutation in different nuclear backgrounds affects different stages of pollen development.     

A COMPARATIVE LIGHT- AND ELECTRON-MICROSCOPIC STUDY OF MICROSPOROGENESIS IN MALE STERILE (MS,) AND MALE FERTILE SOYBEANS (GLYCINE MAX (L.) MERR.)

A comparative study of microsporogenesis in fertile and in male sterile (ms₁) soybean plants (Glycine max (L.) Merr.) was conducted by using various microscopic techniques. Once the developmental pattern for fertile microsporogenesis was established, it was compared with the developmental pattern in sterile plants to determine the time of microsporogenesis breakdown. Sterility of the ms₁ mutant is caused by failure of cytokinesis after telophase II. The four nuclei resulting from meiosis become enclosed in a single-celled structure, termed a coenocytic microspore. These microspores develop a pollen-like wall and become engorged with lipid and starch reserves. Coenocytic microspores usually degenerate after engorgement. This study of fertile and sterile (ms₁) microsporogenesis has shown that nuclear and cytoplasmic events must occur at precise times for the successful development of 1n pollen grains from 2n sporogenous cells. Any disruption during this process leads to 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.     

A case of early dissolution of the microsporocyte callose wall in male-sterile (CMS) sweet pepper (Capsicum annuum L.)

On squash preparations of anthers from pollen fertile and sterile plants of sweet pepper (Capsicum annuum L. cv. Severka) callose envelopes of microsporocytes, stained specifically with resorcin blue, were investigated microscopically. During normal course of microsporogenesis in fertile plants the envelopes remained intact up to the stage of microspore tetrads. Then callose begins to dissolve, and that from individual microspores towards the envelope periphery. In sterile analogues of the same cultivar the callose breakdown occurred precociously, usually in the course of the second, but sometimes as early as the first meiotic division of PMCs. Having completed meiosis sporadic microsporocytes formed microspore tetrads. Most PMCs contained an undivided four-nucleate protoplast rimmed with a narrow or wider unstained zone of dissolved callose. In certain cases more condensed callose septa pointing to the furrows on the surface of the PMC protoplast were well-observable in this lytic zone, as a residuum of normal mechanism of tetradogenesis.     

Temporal and spatial distribution of pectin epitopes in differentiating anthers and microspores of fertile and sterile sugar beet

We studied the possible involvement of several pectin epitopes in anther differentiation and microsporogenesis in fertile and cytoplasmically male sterile sugar beets. The spatial and temporal distribution of five structural motifs were traced with a panel of monoclonal antibodies in six stages: premeiosis, meiotic prophase, young and mature tetrads, young and expanding microspores. The composition of the walls of sporogenous cells and meiocytes differed than that in the tapetum, as evidenced by the presence of alpha-Fuc(1-->2)-beta-Gal and alpha-(1-->5)-L-Ara epitopes binding CCRC-M1 and LM6 antibodies. At meiotic prophase, the meiocyte walls were additionally marked by the appearance of poorly methyl-esterified domains of homogalacturonan and of (1-->4)-beta-Gal residues, detected by JIM5 and LM5. Some constituents of the meiocyte wall which reacted with JIM5 and JIM7 persisted on the surface of the special callose sheath during tetrad development. In newly formed primexine and exine layers of tetrads and microspores, epitopes that were bound by JIM5, JIM7 and LM5 were abundant. No differences in the deposition or relative abundance of pectins were found between fertile and sterile anthers until microspore release from the callose. Later, at the time of abortion, sterile microspores had much larger amounts of epitopes detected by JIM5 than their fertile counterparts.     

Cytological and ultra-structural study on microsporogenesis of cytoplasmic male sterility in <Emphasis Type="Italic">Raphanus sativus</Emphasis>

The cytological development of microspores and tapetum in cytoplasmic male sterile (CMS) line A₁₄ and its maintainer B₁₄ in radish were studied using light- and transmission electron microscopy (LM and TEM). The microspores of the CMS line began to abort soon after they were released from tetrads in pollen sacs with light microscopy investigation, while abnormal behavior of pollen mother cells (PMC) were observed during its meiotic stage in its ultra-structural study, including degeneration of organelles and irregularity of nuclear membrane. At the same time, development of tapetal cells was similar to that of the maintainer. With further development of the anther, the tapetal cells of CMS line showed an abnormal increase in size and other appearances, such as fewer organelles and indistinct cytoplasm. The microspores of the CMS line were always distinguishable from the maintainer line with irregular structure, more osphilic deposits and abnormal exine. It is inferred that abortion of microspores is attributed to mutation of genes controlling male sterility, which further leads to hypertrophy of tapetum and destruction of ultra-structure.