Abolition of the tapetum suicide program ruins microsporogenesis

Microsporogenesis in angiosperms takes places within the anther. Microspores are surrounded by a layer of cells, the tapetum, which degenerates during the later stages of pollen development with cytological features characteristic of programmed cell death (PCD). We report herein that the expression of AtBI-1, which suppresses Bax-induced cell death, in the tapetum at the tetrad stage inhibits tapetum degeneration and subsequently results in pollen abortion, while activation of AtBI-1 at the later stage does not. Our results demonstrate that the PCD signal commences at the tetrad stage and that the proper timing of PCD in the tapetum is essential for normal microsporogenesis.     

The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development

In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.     

Male-sterility of thermosensitive genic male-sterile rice is associated with premature programmed cell death of the tapetum

The tapetum plays a crucial role in pollen development. This secretory tissue produces numerous nutritive proteins necessary for pollen maturation. The tapetum, whose cells undergo programmed cell death (PCD), is completely diminished by the time the pollen is fully mature. Our previous studies on a thermosensitive genic male-sterile (TGMS) rice (Oryza sativa L.) suggested that male-sterility was due to failure in pollen development. In this paper we describe how further analysis of the TGMS rice revealed that male-sterility is associated with premature PCD of the tapetum. Cytological observations of TGMS rice anthers at various developmental stages indicated that PCD initiates at an early stage of pollen development and continues until the tapetal cells are completely degraded, resulting in pollen collapse. Transmission electron microscopy showed the morphologically distinct hallmarks of apoptosis, including cytoplasmic shrinkage, membrane blebbing, and vacuolation. Identification of DNA fragmentation using the TUNEL assay supports the hypothesis that premature PCD is associated with male-sterility in the rice. The tissue-specific feature of the thermosensitive genic male-sterile phenotype is discussed with regard to PCD during anther development.     

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.     

Ultrastructural aspects and programmed cell death in the tapetal cells of Lathyrus undulatus Boiss

Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.     

不同倍性水稻亚种间杂种小孢子发生的细胞学观察

利用塑料半薄切片对水稻同源四倍体亚种间杂种F1及其对应的二倍体杂种F1小孢子母细胞减数分裂过程的细胞学变化进行观察研究.结果表明,同源四倍体水稻亚种间杂种造孢细胞期和小孢子母细胞期已经表现出较高频率的异常;减数分裂过程中,小孢子母细胞出现异常更加复杂,主要包括小孢子母细胞液泡化和退化两大类,这些异常是导致杂种花粉败育和花粉低育性的重要原因之一;此外,绒毡层异常也是导致杂种的花粉育性降低的因素.二倍体杂种小孢子母细胞在减数分裂过程的异常类型与水稻同源四倍体亚种间杂种基本相似,其绒毡层异常频率较低,对其花粉低育性影响不大.     

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.     

A comparative light and electron microscopic analysis of microspore and tapetum development in fertile and cytoplasmic male sterile radish

To gain further insight into the abortive stages and ultrastructural changes leading to pollen degeneration of a novel cytoplasmic male sterile radish 805A, we compared differences of cellular and subcellular structure of sterile anther with fertile anther by light and electron microscopy analysis. Two types of locule degeneration in sterile anther were detected, of which the time of degeneration occurred and completed was different. In type I, abnormality of pollen mother cells (PMCs) and tapetal cells, including condensation of cytoplasm and large vacuoles within tapetal cells, was shown at PMC stage. In type II, meiosis and early tetrad stage progressed normally except for large vacuoles that appeared in tapetal cells. Ultrastructural alterations of the cellular organization were observed in the type II locules, such as chromatin condensation at the periphery of the nucleus and degeneration of the karyotheca, compared with normal pollen development. The results suggested that the cytoplasmic male sterility anther degeneration was probably caused by dysfunctions of tapetum and vacuolation of tapetum, PMCs, and microspores. Thus, the identical factors, which induced CMS in the same cytoplasmic and nuclear genetic background, might affect development of tapetum and microspore at different stages during the cytoplasmic male sterile 805A anther development.     

大果白刺雄性不育系的细胞学研究

利用石蜡切片法对大果白刺花药进行细胞学研究,探讨雄性不育系发生败育的时期和方式以及雄性败育与药壁组织间的关系.结果表明:不育系小孢子母细胞形成前期,花药各部分结构发育正常.随着绒毡层的异常解体,多种异常现象相继出现,包括小孢子母细胞液泡化,中层、药室内壁、药隔细胞液泡化,细胞畸形,药壁细胞非正常解体等.退化后整个花药萎缩干瘪,不能开裂,无花粉.因此,大果白刺雄性不育系的绒毡层生理异常并提前退化是导致雄性不育的主要原因.     

Cytological mechanism of pollen abortion resulting from allelic interaction of F1 pollen sterility locus in rice (Oryza sativa L.)

Pollen abortion is one of the major reasons causing the inter-subspecific F₁ hybrid sterility in rice and is due to allelic interaction of F₁ pollen sterility genes. The microsporogenesis and microgametogenesis of Taichung 65 and its three F₁ hybrids were comparatively studied by using techniques of differential interference contrast microscopy, semi-thin section light microscopy, epifluorescence microscopy and TEM. The results showed that there were differences among the cytological mechanisms of pollen abortion due to allelic interaction at the three F₁ pollen sterility loci. The allelic interaction at S-a locus resulted in microspores unable to extend the protoplasm membrane with the enlargement of the microspore at the middle microspore stage and finally producing empty abortive pollen. The allelic interaction at S-b locus caused asynchronous development of microspores at the middle microspore stage producing stainable abortive pollen. The allelic interaction at S-c locus mainly led to the non-dissolution of the generative cell wall and finally caused the hybrid F₁ mainly producing stainable abortive pollen. Genotypic identification indicated that the abortive pollen were those with S ^j allele.     

Abnormal Development of Tapetum and Microspores Induced by Chemical Hybridization Agent SQ-1 in Wheat

Chemical hybridization agent (CHA)-induced male sterility is an important tool in crop heterosis. To demonstrate that CHA-SQ-1-induced male sterility is associated with abnormal tapetal and microspore development, the cytology of CHA-SQ-1-treated plant anthers at various developmental stages was studied by light microscopy, scanning and transmission electron microscopy, in situ terminal deoxynucleotidyl transferasemediated dUTP nick end-labelling (TUNEL) assay and DAPI staining. The results indicated that the SQ-1-treated plants underwent premature tapetal programmed cell death (PCD), which was initiated at the early-uninucleate stage of microspore development and continued until the tapetal cells were completely degraded; the process of microspore development was then blocked. Microspores with low-viability (fluorescein diacetate staining) were aborted. The study suggests that premature tapetal PCD is the main cause of pollen abortion. Furthermore, it determines the starting period and a key factor in CHA-SQ-1-induced male sterility at the cell level, and provides cytological evidence to further study the mechanism between PCD and male sterility.     

New acritarch species from Holocene sediments in central West Greenland

Anther development, microsporogenesis, and microgametogenesis were studied using both light and TEM microscopy in the six accessible subdioecious/cryptically dioecious species of Consolea (Cactaceae). Anther wall development, microsporogenesis, and microgametogenesis are uniform in staminate flowers of all six species, and are typical for Cactaceae. Breakdown of microsporogenesis in male‐sterile anthers occurs early, at the onset of meiosis, and results in anthers bearing no pollen grains. The abortive process follows a common pattern in all investigated species. The tapetum is the first layer to deviate from normal male‐fertile anther development. Tapetal cells in male‐sterile anthers elongate at an early stage and have abundant rER with atypical configurations. Ultimately, the tapetum becomes hypertrophied and non‐functional. Male‐sterility in pistillate flowers appears to be directly related to these anomalies. In addition, other anther layers and tissues are affected, and normal patterns of programmed cell death (PCD) are disrupted. The relationship between these patterns and the pattern of PCD in normal male‐fertile anthers is discussed. We hypothesize a single origin for the cryptically dioecious/subdioecious breeding system of Consolea based on the uniformity of the anther's abortive processes in pistillate flowers.     

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。     

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.     

鹅掌楸花粉败育过程的超微结构观察

花粉败育是限制鹅掌楸〔Ｌｉｒｉｏｄｅｎｄｒｏｎｃｈｉｎｅｎｓｅ（Ｈｅｍｓｌ．）Ｓａｒｇ．〕生殖成功的重要因素之一。败育多数发生在四分体形成之前,少数发生在小孢子形成以后,是由于花粉发育过程中存在异常现象造成的。异常现象有７个方面：（１）造孢组织解体;（２）小孢子形成过程中胼胝质的积累与降解异常;（３）绒毡层发育异常;（４）小孢子母细胞胞质分裂异常;（５）小孢子解体;（６）生殖细胞败育;（７）药隔维管束韧皮部的伴胞解体。这些原因可引起花粉产量和质量降低,从而影响鹅掌楸生殖过程中的传粉受精及结籽的能力     

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     

ABA and IAA control microsporogenesis in <Emphasis Type="Italic">Petunia hybrida</Emphasis> L.

The formation of fertile male gametophyte is known to require timely degeneration of polyfunctional tapetum tissue. The last process caused by the programmed cell death (PCD) is a part of the anther program maturation which leads to sequential anther tissue destruction coordinated with pollen differentiation. In the present work, distribution of abscisic acid (ABA) and indole-3-acetic acid (IAA) in developing anthers of male-fertile and male-sterile lines of petunia (Petunia hybrida L.) was analyzed by using the immunohistochemical method. It was established that the development of fertile male gametophyte was accompanied by monotonous elevation of ABA and IAA levels in reproductive cells and, in contrast, their monotonous lowering in tapetum cells and the middle layers. Abortion of microsporocytes in the meiosis prophase in the sterile line caused by premature tapetum degeneration along with complete maintenance of the middle layers was accompanied by dramatic, twofold elevation in the levels of both the phytohormones in reproductive cells. The data obtained allowed us to conclude that at the meiosis stage ABA and IAA are involved in the PCD of microsporocytes.     

太空诱变玉米核不育材料花粉败育的细胞学观察(简报)

玉米是最早利用雄性不育系生产杂交种的作物之一。在玉米T型细胞质雄性不育杂交种遭受毁灭性病害侵袭之后,科学家认识到利用细胞质雄性不育制种存在潜在的遗传脆弱性,从此试图通过多种途径来创造新的雄性不育．并对雄性不育材料的遗传多样性进行研究。空间诱变育种是80年代于我国发展起来的新技术,在农作物品种改良和种质创新上已初见成效。[第一段]