Premature Tapetum Degeneration: a Major Cause of Abortive Pollen Development in Photoperiod Sensitive Genic Male Sterility in Rice

Photoperiod-sensitive genic male-sterile (PSGMS) rice ( Oryza sativa L.), a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell (PMC) stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL (TdT-mediated dUTP Nick End Labeling) assay, which detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the premature tapetum degeneration is in the process of PCD.     

Premature Tapetum Degeneration： a Major Cause of Abortive Pollen Development in Photoperiod Sensitive Genic Male Sterility in Rice

Photoperiod-sensitive genic male-sterile （PSGMS） rice （Oryza sativa L.）, a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell （PMC） stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL （TdT-mediated dUTP Nick End Labeling） assay, which detects DNA fragmentation resulting from programmed cell death （PCD）, indicating that the premature tapetum degeneration is in the process of PCD.     

荔枝雄花性别决定过程中细胞超微结构的变化

荔枝雄花雌蕊原基在大孢子母细胞减数分裂后开始衰退.内质网历经增生扩展,穿壁相连,同心缠绕,多条平行弯曲,不规则堆叠.内质网和高尔基体产生许多囊泡,囊泡在细胞内含物的降解和运输过程中起着重要的作用.线粒体在雌蕊原基细胞衰败的前、中期数量增加,后期分批降解.过氧化物酶体在雌蕊原基细胞衰败的中期紧挨核短暂出现.细胞核的染色质凝集断裂;核周腔扩大,形成胀泡;染色质趋边,外泄.细胞原生质表现出有序的、在膜包裹下的降解,首先是核糖体,而后依次是:过氧化物酶体、内质网、高尔基体、线粒体、核.雌蕊原基的衰败历程可能是一种程序性细胞死亡的过程.     

BCL-2 does not control programmed cell death in the IPLB-LdFB cell line from the insect Lymantria dispar

In the insect Lymantria dispar cell line IPLB-LdFB the presence of a Bcl-2-like molecule has been demonstrated. The Western blot analysis performed on the cells incubated with 2-deoxy-D-ribose (dRib), an apoptotic inducer, revealed that, in comparison with the control, the Bcl-2 expression was unaffected. Furthermore, incubation of the insect cells with an anti-Bcl-2 polyclonal antibody inhibited the apoptotic effect induced by dRib, and provoked mitochondrial membrane depolarization without any apoptotic phenomena. Similar behaviour was observed using the K+ ionophore valinomycin. From these findings, we hypothesize that the L. dispar Bcl-2-like protein is essential for maintenance of the mitochondrial membrane potential, but not, as usually thought, for the regulation of programmed cell death.     

植物细胞核雄性不育相关bHLH转录因子研究进展

雄性不育广泛存在于种子植物中。植物雄性不育不仅是植物生殖发育研究的重要内容,同时也可作为杂种优势利用的有效工具,因而具有重要的理论和应用价值。bHLH转录因子家族是植物中成员最多的转录因子家族,在植株的整个生长发育过程中起着重要的调控作用。本文介绍了拟南芥、水稻、玉米等几种重要模式植物bHLH转录因子调控雄蕊发育的作用机制,并重点阐述其功能异常引起细胞核雄性不育的分子机制,以期为作物育种与理论研究提供参考。     

小麦淀粉胚乳发育期间的程序性细胞死亡

小麦淀粉胚乳在发育过程中经历程序性细胞死亡(PCD).小麦淀粉胚乳的DNA在发育的特定阶段呈现梯状电泳条带,用乙烯处理使DNA片段化发生的时间提前,而且ABA处理虽然不能推迟DNA片段化的发生时间,但能减弱DNA片段化的程度.小麦淀粉胚乳细胞在PCD过程中出现某些动植物细胞凋亡的共同的结构变化特征,但也有一些独特的结构变化.如染色质凝聚后仅少数染色质块发生趋边化;细胞核在PCD过程中最先开始衰退,细胞核解体时胞质中有丰富的细胞器,细胞核解体后细胞并未死亡,在胞质中仍在合成和积累淀粉和储藏蛋白,直到细胞被淀粉充满,细胞才死亡;不形成凋亡小体,死亡的淀粉胚乳细胞成为营养物质的储藏库.因此小麦淀粉胚乳细胞的PCD是一种特殊形式的PCD.     

Efficient production of genetically engineered,male-sterile <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> using anther-specific promoters and genes derived from <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">B. rapa</Emphasis>

Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the β-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Secondary Metabolite Euplotin C Induces Apoptosis-Like Death in the Marine Ciliated Protist Euplotes vannus

ABSTRACT. The sesquiterpenoid euplotin C is a secondary metabolite produced by the ciliated protist Euplotes crassus and provides a mechanism for damping populations of potential competitors. Indeed, E. crassus is virtually resistant to its own product while different non-producer species representing an unbiased sample of the marine, interstitial, ciliate diversity are sensitive. For instance, euplotin C exerts a marked disruption of different homeostatic mechanisms in Euplotes vannus . We demonstrate by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay that euplotin C quickly decreases viability and mitochondrial function of E. vannus with a very high efficacy and at micromolar potency. In addition, euplotin C induces apoptosis in E. vannus as 4,6-diamino-2-phenylindole and terminal transferase dUTP nick end labeling staining show the rapid condensation and fragmentation of nuclear material in cells treated with euplotin C. These effects occur without detectable permeabilisation or rupture of cell membranes and with no major changes in the overall morphology, although some traits, such as vacuolisation and disorganized microtubules, can be observed by transmission electron microscopy. In particular, E. vannus show profound changes of the mitochondrial ultrastructure. Finally, we also show that caspase activity in E. vannus is increased by euplotin C. These data elucidate the pro-apoptotic role of euplotin C and suggest a mechanism for its impact on natural selection.     

镉诱导的茶树苗膜脂过氧化和细胞程序性死亡

在含镉的营养液中,茶树幼苗生长受到抑制。随培养时间延长,膜脂过氧化产物丙二醛含量持续升高;超氧物岐化酶（SOD）和过氧化氢酶（CAT）活性初期升高,而后分别在第4天和第2天开始下降。镉胁迫的第5-7天,一部分细胞陆续发生程序性死亡。其特征是：线粒体聚集于核周围,个数增加,嵴发达,而后衰亡。核仁消失,染色质凝结在核膜边缘,核萎缩,外层核膜局部扩张,形成胀泡。核以外溢、出芽和崩裂三种方式溃解。核是最后消亡的细胞器。程序性死亡的细胞局限于某些区域。镉胁迫下,幼苗膜脂过氧化可能是诱发PCD的主要原因。     

植物细胞程序性死亡──一个新兴的研究领域(英文)

近年来,越来越多的证据表明,植物细胞在生理、病理或逆境条件下可发生程序性死亡（ＰｒｏｇｒａｍｍｅｄＣｅｌｌＤｅａｔｈ,ＰＣＤ）。本文详细描述了植物ＰＣＤ的形态和生化特征、生理功能及其研究意义,并把这些方面与动物ＰＣＤ做了比较。另外,虽然植物ＰＣＤ的研究尚处于起步阶段,本文还是对其可能的信号传导机制、遗传调控以及ＰＣＤ的起源与进化作了探讨,并提出了植物ＰＣＤ的研究战略。具体说来有以下几个方面：１．形态和生化特征。目前,植物ＰＣＤ的研究主要还集中于形态和生化方面的描述。各种条件下的植物ＰＣＤ在形态和生化特征上都或多或少地与动物细胞凋亡存在差异,并不符合动物细胞凋亡定义的全部内容。并且不同植物ＰＣＤ类型相互之间也存在着较大的不同。尽管如此,动植物ＰＣＤ在形态和生化方面还是存在许多相似之处。无炎症反应、ＤＮＡ的特异片段化以及核酸内切酶和蛋白酶活性的升高在植物中也依然是区别ＰＣＤ与细胞坏死（ｎｅｃｒｏｓｉｓ）的形态和生化依据。２．分子水平上,植物ＰＣＤ也涉及到许多信号分子和特定基基参与调控的信号传导途径。但到目前为止,已分离的与植物ＰＣＤ直接相关的基因只有ＡＣＤ２、Ｄａｄ１等少数几个。尽管已证明一些信号分子如活性氧种     

Germ cell cluster formation and cell death are alternatives in caste-specific differentiation of the larval honey bee ovary

Summary

Caste-specific differentiation of the female honey bee gonad takes place in the fifth larval instar. In queen larvae most ovarioles exhibit almost simultaneous formation of numerous germ cell clusters within the first 20 h after the last larval molt. Ultrastructurally distinctive fusomal cytoplasm connects these cystocytes. Germ cell differentiation is accompanied by morphological changes in somatic components of the ovarioles, the follicle and the terminal filament cells. Subsequently, queen ovarioles elongate and differentiate basal stalks that coalesce in a basal calyx. A second round of mitotic activity was found to occur in the late prepupal and early pupal queen ovary. This round may elevate germ cell numbers composing each cluster to levels observed in follicles of adult honey bee queens. In contrast, germ cell cluster formation does not occur in most of the 120–160 ovarioles of the larval worker ovary, but instead many cells in such ovarioles show signs of impending degeneration, such as large autophagic bodies. DNA extracted from worker ovaries did not reveal nucleosomal laddering, and ultrastructurally, chromatin in germ cell nuclei appeared intact. In the 4–7 surviving ovarioles of the small worker ovary, germ cell clusters were found with ultrastructural characteristics identical to those in queen ovarioles. The temporal window during which divergence in developmental pathways of the larval ovaries initiates shortly after the last larval molt coincides with caste-specific differences in juvenile hormone titer which have long been considered critical to caste-specific morphogenesis.     

<i>OsMAPK6</i> Affects Male Fertility by Reducing Microspore Number and Delaying Tapetum Degradation in <i>Oryza sativa</i> L.

The mitogen-activated protein kinase (MAPK) cascade is important in stress signal transduction and plant development. In the present study, we identified a rice (Oryza sativa L.) mutant with reduced fertility, Oryza sativa mitogen-activated protein kinase 6 (osmapk6), which harbored a mutated MAPK gene. Scanning and transmission electron microscopy, quantitative RT-PCR analysis, TUNEL assays, RNA in situ hybridization, longitudinal and transverse histological sectioning, and map-based cloning were performed to characterize the osmapk6 mutant. The gene OsMAPK6 was expressed throughout the plant but predominantly in the microspore mother cells, tapetal cells, and microspores in the anther sac. Compared with the wild type, the total number of microspores was reduced in the osmapk6 mutant. The formation of microspore mother cells was reduced in the osmapk6 anther sac at an early stage of anther development, which was the primary reason for the decrease in the total number of microspores. Programmed cell death of some tapetal cells was delayed in osmapk6 anthers and affected exine formation in neighboring microspores. These results suggest that OsMAPK6 plays pivotal roles in microspore mother cell formation and tapetal cell degradation.     

Cell death upon H(2)O(2) induction in the unicellular green alga Micrasterias

In the present study, we investigated whether the unicellular green alga Micrasterias denticulata is capable of executing programmed cell death (PCD) upon experimental induction, and which morphological, molecular and physiological hallmarks characterise this. This is particularly interesting as unicellular freshwater green algae growing in shallow bog ponds are exposed to extreme environmental conditions, and the capacity to perform PCD may be an important strategy to guarantee survival of the population. The theoretically 'immortal' alga Micrasterias is an ideal object for such investigations as it has served as a cell biological model system for many years and details on its growth properties, physiology and ultrastructure throughout the cell cycle are well known. Treatments with low concentrations of H(2)O(2) are known to induce PCD in other organisms, resulting in severe ultrastructural changes to organelles, as observed in TEM. These include deformation and part disintegration of mitochondria, abnormal dilatation of cisternal rims of dictyosomes, occurrence of multivesicular bodies, an increase in the number of ER compartments, and slight condensation of chromatin. Additionally, a statistically significant increase in caspase-3-like activity was detected, which was abrogated by a caspase-3 inhibitor. Photosynthetic activity measured by fast chlorophyll fluorescence decreased as a consequence of H(2)O(2) exposure, whereas pigment composition, except for a reduction in carotenoids, was the same as in untreated controls. TUNEL positive staining and ladder-like degradation of DNA, both frequently regarded as a hallmark of PCD in higher plants, could only be detected in dead Micrasterias cells.     

Programmed cell death promotes male sterility in the functional dioecious Opuntia stenopetala (Cactaceae)

Background and Aims

The sexual separation in dioecious species has interested biologists for decades; however, the cellular mechanism leading to unisexuality has been poorly understood. In this study, the cellular changes that lead to male sterility in the functionally dioecious cactus, Opuntia stenopetala, are described.

Methods

The spatial and temporal patterns of programmed cell death (PCD) were determined in the anthers of male and female flowers using scanning electron microscopy analysis and histological observations, focusing attention on the transition from bisexual to unisexual development. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assays were used as an indicator of DNA fragmentation to corroborate PCD.

Key results

PCD was detected in anthers of both female and male flowers, but their patterns differed in time and space. Functionally male individuals developed viable pollen, and normal development involved PCD on each layer of the anther wall, which occurred progressively from the inner (tapetum) to the outer layer (epidermis). Conversely, functional female individuals aborted anthers by premature and displaced PCD. In anthers of female flowers, the first signs of PCD, such as a nucleus with irregular shape, fragmented and condensed chromatin, high vacuolization and condensed cytoplasm, occurred at the microspore mother cell stage. Later these features were observed simultaneously in all anther wall layers, connective tissue and filament. Neither pollen formation nor anther dehiscence was detected in female flowers of O. stenopetala due to total anther disruption.

Conclusions

Temporal and spatial changes in the patterns of PCD are responsible for male sterility of female flowers in O. stenopetala. Male fertility requires the co-ordination of different events, which, when altered, can lead to male sterility and to functionally unisexual individuals. PCD could be a widespread mechanism in the determination of functionally dioecious species.     

Drosophila Bcl-2 proteins participate in stress-induced apoptosis, but are not required for normal development

Many developing tissues require programmed cell death (PCD) for proper formation. In mice and C. elegans, developmental PCD is regulated by the Bcl-2 family of proteins. Two bcl-2 genes are encoded in the Drosophila genome (debcl/dBorg1/Drob-1/dBok and buffy/dBorg2) and previous RNAi-based studies suggested a requirement for these in embryonic development. However, we report here that, despite the fact that many tissues in fruit flies are shaped by PCD, deletion of the bcl-2 genes does not perturb normal development. We investigated whether the fly bcl-2 genes regulate non-apoptotic processes that require caspases, but found these to be bcl-2 gene-independent. However, irradiation of the mutants demonstrates that DNA damage-induced apoptosis, mediated by Reaper, is blocked by buffy and that debcl is required to inhibit buffy. Our results demonstrate that developmental PCD regulation in the fly does not rely upon the Bcl-2 proteins, but that they provide an added layer of protection in the apoptotic response to stress.     

Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots

Establishing the mechanisms regulating the autolysis of xylem tracheary elements (TEs) is important for understanding this programmed cell death process. These data demonstrate that two paralogous Arabidopsis thaliana proteases, XYLEM CYSTEINE PROTEASE1 (XCP1) and XCP2, participated in micro-autolysis within the intact central vacuole before mega-autolysis was initiated by tonoplast implosion. The data acquisition was aided by the predictable pattern of seedling root xylogenesis, the availability of single and double total knock-out T-DNA lines, anti-sera that recognized XCP1 and XCP2, and the microwave-assisted processing of whole seedlings prior to immunolabeling and observation in the transmission electron microscope. During secondary wall thickening, XCP1 and XCP2 (in wild type), XCP1 (in xcp2 seedlings) or XCP2 (in xcp1 seedlings) were imported into the TE central vacuole. Both XCP1 and XCP2 heavily labeled dense aggregates of material within the vacuole. However, because of XCP1 deficiency in xcp1 and xcp1 xcp2 TEs, non-degraded cellular remnants first accumulated in the vacuole and then persisted in the TE lumen (longer than in the wild type) after the final mega-autolysis was otherwise complete. This delayed TE clearing phenotype in xcp1 was rescued by complementation with wild-type XCP1. Although TEs in the xcp2 single knock-out cleared comparably with wild type, the non-degraded remnants in xcp1 xcp2 TEs were more densely packed than in xcp1 TEs. Therefore, XCP2 has a minor but distinct role in micro-autolysis. After tonoplast implosion, XCP1 and XCP2 remained associated with disintegrating cellular material as mega-autolysis, aided by additional lytic enzymes, destroyed the bulk of the cellular contents.     

Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.     

Reactive oxygen species contribute to the release of volatile organic compounds from Chlamydomonas reinhardtii during programmed cell death

Acetic acid at pH 5.0 can induce programmed cell death (PCD) in Chlamydomonas reinhardtii cells, and abundant volatile organic compounds (VOCs) were released during the process. In this study, the caspase‐3‐like activity was determined during the PCD, and it was increased significantly after 1 h. During the PCD, the dynamic release of VOCs from the cells was analyzed, and the emissions of total VOCs were raised markedly and reached the highest level at 2 h. Among the seven types of VOCs, such as alkanes, alkenes, terpenoids, alcohols, aldehydes, ketones and esters, three oxygenated compounds (aldehydes, ketones and esters) showed the most significant increase. O₂^‐· and H₂O₂ were rapidly accumulated to high levels in the cells at the beginning of the PCD, but their content was reduced during the process. The activities of antioxidant enzymes were reduced gradually and even disappeared completely, demonstrating that the reduction of reactive oxygen species (ROS) may not be scavenged by the antioxidant enzyme system. ROS have an intense oxidation and scavenging ability to volatile compounds, and the oxidation results in the production of oxygenated compounds. Therefore, the abundant production of oxygenated compounds indicated that ROS may play an important role in the dynamic release of VOCs from C. reinhardtii cells during PCD.     

Cell renewal and apoptosis in macrostomum sp. [Lignano

In platyhelminths, all cell renewal is accomplished by totipotent stem cells (neoblasts). Tissue maintenance is achieved in a balance between cell proliferation and apoptosis. It is known that in Macrostomum sp. the epidermis undergoes extensive cell renewal. Here we show that parenchymal cells also exhibit a high rate of cell turnover. We demonstrate cell renewal using continuous 5'bromo-2-deoxyuridine (BrdU) exposure. About one-third of all cells are replaced after 14 days. The high level of replacement requires an equivalent removal of cells by apoptosis. Cell death is characterized using a combination of three methods: (1). terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), (2). specific binding of phosphatidyl-serine to fluorescent-labelled annexin V and (3). identification of apoptotic stages by ultrastructure. The number of cells observed in apoptosis is insufficient to explain the homeostasis of tissues in Macrostomum. Apoptosis-independent mechanisms may play an additional role in tissue dynamics.