Calcium changes during megasporogenesis and megaspore degeneration in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.)

Potassium pyroantimonate was used to localize loosely-bound calcium in young ovules of lettuce (Lactuca sativa L.) during megasporogenesis to investigate the relationship between ionically available calcium and megaspore degeneration. At the megasporocyte (megaspore mother cell) stage, few calcium precipitates were located in the ovule. Following meiosis in the megasporocyte, a linear tetrad of four megaspores is formed, with three of the four megaspores degenerating from the micropylar end inward. Only the chalazal-most megaspore continues to develop, becoming the functional megaspore. A decrease in amount of calcium precipitates in the megaspore, particularly in the nucleus, precedes the breakdown of the micropylar megaspores, which subsequently undergo structural disintegration and loss of recognizable cellular features. A partial recovery of calcium precipitates occurs during later degeneration. The functional megaspore retains a consistently higher concentration of calcium precipitates during development, which is retained in the developing embryo sac. This, to our knowledge, is the first report related to calcium dynamics during megaspore degeneration, and may facilitate future research aimed at elucidating the mechanisms of megasporogenesis.     

栽培甜菜大孢子发生的超微结构

栽培甜菜（Beta vulgaris）的大孢子发生为蓼型。减数分裂时,大孢子母细胞核中出现核液泡,形成联会复合体,细胞壁上有胼胝质加厚,并存在细胞质改组现象。大孢子母细胞减数第1次分裂形成二分体,2个细胞均被较厚的胼胝质壁包裹。合点端的二分体细胞中细胞器丰富,线粒体和质体的形态正常,表明完成了再分化。在大多数情况下,珠孔端的二分体细胞在减数第2次分裂前（或分裂的过程中）退化,合点端的细胞分裂产生大小不等的2个细胞,形成三分体。三分体合点端的大孢子体积较大,发育成单倍体的功能大孢子。     

栽培甜菜大孢子发生的超微结构

栽培甜菜(Beta vulgaris)的大孢子发生为蓼型。减数分裂时, 大孢子母细胞核中出现核液泡, 形成联会复合体, 细胞壁上有胼胝质加厚, 并存在细胞质改组现象。大孢子母细胞减数第1次分裂形成二分体, 2个细胞均被较厚的胼胝质壁包裹。合点端的二分体细胞中细胞器丰富, 线粒体和质体的形态正常, 表明完成了再分化。在大多数情况下, 珠孔端的二分体细胞在减数第2次分裂前(或分裂的过程中)退化, 合点端的细胞分裂产生大小不等的2个细胞, 形成三分体。三分体合点端的大孢子体积较大, 发育成单倍体的功能大孢子。     

Embryo sac development in Arabidopsis thaliana

Summary Aspects of megasporogenesis in Arabidopsis thaliana have been investigated using a variety of histochemical techniques to visualize general cell organization, DNA and callose in whole ovules and sections by bright field, fluorescence, differential interference contrast and scanning electron microscopy. The microtubular cytoskeleton has been studied using immunofluorescence localization of tubulin in sections and whole cells. The observations deviate from reports of preceding studies in that the megasporocyte was found to undergo both meiotic divisions followed by simultaneous cytokinesis (i.e. without an intermediate dyad stage) to give a multiplanar tetrad of megaspores. This represents a variation of monosporic development not previously described. Polarized distribution of organelles prior to meiosis ensures that the functional megaspore receives the largest share. Aberrant wall formation is common between degenerating megaspores. Localized callose deposition in the tetrad separates these cells from the active megaspore. Their pattern of degeneration and displacement is extremely flexible within the embryo sac space. The microtubular cytoskeleton is extensive and largely cytoplasmic, as distinct from cortical, throughout megasporogenesis. In the megasporocyte, megaspores and one-nucleate embryo sac, randomly oriented microtubules throughout the cells may serve to maintain cytoplasmic integrity and position organelles. Numerous microtubules (MTs) associate closely with the nucleus and some radiate from it, perhaps functioning in nuclear positioning. During meiosis MTs are restricted to the spindle configurations and later to the phragmoplasts which form between daughter nuclei. The lack of interphase cortical arrays suggests that the role of internal influences on cell shape is small.     

Callose in cell walls during megasporogenesis in angiosperms

Summary Callose was detected by fluorescence microscopy in megasporogenesis in all investigated species with mono- and bisporic embryo-sac development. Callose occurs first in the meiotic prophase in the chalazal part of the megasporocyte wall and by the first meiotic metaphase the whole cell is enveloped in a callose-containing wall. Later, there is a marked decrease of callose fluorescence, usually at the chalazal end of the megasporocyte. In Oenothera, where the micropylar megaspore is active, decrease of fluorescence takes place at the micropylar pole of the megasporocyte. Callose appears centrifugally in the cell plates forming eventually the walls dividing the megaspores. It disappears from the walls of the megaspores during degeneration and differentiation.     

Development and ultrastructure of the megagametophyte in Passiflora caerulea L. (Passifloraceae)

Megasporogenesis and megagametogenesis of Passiflora caerulea L. were studied using light and transmission electron microscopy. The archesporial tissue is generally formed by one cell. The megaspore mother cell gives rise to a linear tetrad of megaspores. The chalazal megaspore is the functional one, and originates a Polygonum -type female gametophyte. The antipodals are ephemeral. Abundant starch is found in the nucellar cells, specially the ones adjacent to the megagametophyte. The two synergids show ultrastructural differences, involving the filiform apparatus, the nucleolus and the endoplasmic reticulum; these differences suggest a functional differentiation, probably related to the reception of the pollen tube. This is the first report in angiosperms of substantial morphological differences between the two synergids.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 73–81.     

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.     

莴苣大孢子发生过程中钙的分布动态

莴苣胚囊发育为蓼型,减数分裂形成的4个大孢子中只有合点端的一个大孢子可继续发育,其余3个大孢子从珠孔端依次退化.大孢子母细胞中的钙沉淀颗粒很少,减数分裂后的四分体中的钙沉淀颗粒稍有增加.以后,4个大孢子中的钙沉淀颗粒在数量上有明显差异:即将退化的大孢子中钙明显减少,而未退化大孢子细胞质中则保持有较多的细小钙沉淀颗粒.大孢子的退化是一种细胞程序死亡现象,细胞中的钙浓度降低时可能启动了大孢子细胞的程序性死亡过程,而细胞中的钙浓度高时则保持大孢子细胞的继续发育.文章首次揭示了大孢子发生过程中钙的分布特征.     

珍稀濒危植物巴东木莲胚胎学研究

对巴东木莲(Manglietia patungensis Hu)的花发育以及胚胎发育过程进行了系统研究。巴东木莲花顶生,花器官头年年底开始分化到第二年3月分化出花被、雌雄蕊群直至6月发育成熟。雌蕊成熟时胚珠倒生,双珠被,厚珠心,大孢子四分体线形排列,合点端发育成功能大孢子,珠孔端的3个退化,大孢子为单孢子发生型,胚囊发育方式属蓼型;雄蕊花药外侧壁玫瑰红色,内侧有4个白色花粉囊,绒毡层有1层多核细胞,小孢子四分体排列方式多为左右对称形和交叉形,四面体形,偶为T字形和线形,成熟花粉粒为二细胞型。在巴东木莲花发育和大、小孢子发生以及雌、雄配子体形成过程中未见异常现象,因此笔者认为该物种的花器官发育以及雌、雄配子体发育并不构成导致该物种濒危的因素。     

银杏胚珠贮粉室的早期发育

对银杏 (GinkgobilobaL .)胚珠贮粉室的早期发育过程以及珠心细胞死亡的细胞学机制进行了研究。DNA电泳出现DNAladder和TUNEL标记说明参与形成贮粉室的珠心细胞死亡是程序性死亡 (PCD)过程 ,并且显示出在贮粉室形成中 ,PCD的发生有一定的空间分布式样。结合扫描电镜观察 ,贮粉室的早期发育可分为 4个阶段 :起始事件是位于珠孔端的 3至 4层珠心细胞纵向伸长 ;接着 ,位于珠心组织最上部 (珠孔端 )的珠心细胞启始死亡 ;然后 ,这些已经纵向伸长的珠心细胞向基地和侧向地逐渐死亡 ,形成一个空腔 ;最后 ,珠孔端珠心表皮细胞以开裂的方式与其余表皮细胞脱离而形成贮粉室的开口。大孢子母细胞时期 ,贮粉室尚未发生 ;四分体阶段 ,贮粉室已经开始形成 ;到雌配子体发育时期 ,贮粉室已经完全产生。反映大孢子发育和贮粉室发生的同步性。     

濒危植物瑶山苣苔的大小孢子发生和雌雄配子体发育研究

利用石蜡切片技术对瑶山苣苔大小孢子发生、雌雄配子体发育及胚胎发育进行了细胞学观察,结果表明:瑶山苣苔胚珠倒生,单珠被,薄珠心,具珠被绒毡层。大孢子母细胞减数分裂形成的四分体呈直线排列,合点端的大孢子发育为功能大孢子,其余3个大孢子退化,胚囊为单孢子发生的蓼型胚囊发育方式。花药为四囊形,花药壁由外到内依次为表皮、药室内壁、中层和腺质绒毡层,小孢子形成时胞质分裂为修饰性同时型,小孢子四分体排列方式为四面体形,成熟花粉为2核细胞。胚乳发育为细胞型,在胚的发育过程中被吸收耗尽。瑶山苣苔大小孢子发生和雌雄配子体发育基本正常,不是导致其濒危的原因。但瑶山苣苔果实成熟时,胚仅发育至球形胚时期,需要经过一定时间休眠才能完成形态后熟,表明胚未发育完全可能是该物种天然更新困难的原因之一。     

彩色马蹄莲大小孢子发生和雌雄配子体发育的研究

选用石蜡切片法观察了彩色马蹄莲品种‘Majestic Red’的大小孢子发生及雌雄配子体发育的过程。研究结果表明：彩色马蹄莲的胚珠为倒生,具双珠被、厚珠心和珠被绒毡层。大孢子母细胞的减数分裂后形成的四分体为直线型或T型排列,合点端的大孢子发育成为功能大孢子,其余3个大孢子则退化,表明胚囊发育方式为单孢子发生的蓼型胚囊。观察到每个雄花花药多数,花粉囊呈蝶形,每侧有2个小孢子囊。花药壁由外到内分别为表皮、药室内壁、中层和绒毡层,其中绒毡层为变形绒毡层类型。在小孢子形成时,胞质分裂属于连续型,小孢子排列成十字形的四分体,成熟花粉则为二胞花粉粒。     

Cytokinin‐dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non‐overlapping T–DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM‐specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal‐localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.     

Localization of Callose during the Occurrence of Megasporocyte in Gastrodia elata Blume

The structure of ovule in Gastrodia elata Blume was very simple. Functional megaspore occurred at the chalazal end. Callose was absent at megasporocyte stage. It first appeared at the chalazal wall during the first meiotic prophase and exhibited continuous fluorescence. Soon later callose fluorescence disappeared in some part of the chalazal wall and many noncallosic dark areas took place, subsequently these nonfluorescence areas became larger and the callose fluorescence appeared discontinuous granulose distribution. This fluorescence maintained until the megaspore formed. The callose of micropylar wall appeared later and usually disappeared before megaspore formation. In the cross walls between the functional and the two degenarated megaspore callose fluorescence was very strong, continued and kept for a long time. But the side walls usually lacked callose. Accoding to the morphological character of simple ovule in G. eiata and the localization of acid phosphatase and polysaecharide grains, the transfer of vegetative materials from surrounding tissues into megasporocyte mainly passing through the chalazal end of megasporocyte. Thus a continuous callose wall deposited at the ehalazal end of megasporocyte, and it in reality caused the “isolation” of meiocyte. It was possible that a reduced form of callose disposition existed in parasetic orchids.     

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.     

Polarity of nuclear and organellar DNA during megasporogenesis and megagametogenesis in Plumbago zeylanica

Summary Megasporogenesis and megagametogenesis of Plumbago zeylanica were studied using isolated megasporocytes, megaspores, and embryo sacs labeled with Hoechst 33258 for nuclear and organellar (presumably plastid) DNA. Megasporogenesis conforms to the tetrasporic Plumbago type, producing a coenomegaspore with four megaspore nuclei. Organeller DNA is polarized in the micropylar end of the coenomegaspore and embryo sac, reflecting the site of egg cell formation. The three remaining nuclei are somewhat displaced to the chalazal pole, producing a variable number of accessory cells and a 4N secondary central cell nucleus. Ultimately, the mature embryo sac consists of two to five cells including an egg cell, a central cell, zero to two lateral cells, and zero to one antipodal cell depending on the degeneration of the lateral or chalazal nuclei during megagametogenesis.     

Origin of Megagametophytes with Supernumerary Egg Cells Occurring in Oryza sativa

Cytoembryological observations were attempted to reveal the cytological origin of megagametophyte with supernumerary egg cells. It was shown that all ovules underwent a normal megasporogenesis. The meiosis of megasporocyte consisted of two successive divisions, which gave rise to four haploid megaspores. It was the chalazal spore that developed to form the megagametophyte while the three micropylar megaspores degenerated quickly. After first mitosis in the functional megaspore the two nuclei were separated to the micropylar and chalazal poles by a large central vacuole, meanwhile a differential enlargement of the two-nucleate embryo sac was visualized. The micropylar side enlarged quickly and in contrast, the chalazal side remains almost unchanged. Immediately afterward, the second mitosis took place forming four-nucleate embryo sac. During the second mitosis, nucleus located in the narrow area of chalazal side divided transversely, with its upper sister nucleus migrating to the central or micropylar part of the embryo sac, while the nucleus in the micropylar side divided at an angle of about 45° against the micropylar-chalazal axis. Through the third mitosis, two patterns of nuclear arrangement deviating from polygonum were observed. (i) One nuclear distribution pattern was two, two, four respectively in chalazal, central and micropylar parts. And during maturation the four micropylar nuclei differentiated as egg apparatus consisting of two egg cells and two synergids. The two central nuclei, which presumably suppressed the movement of nucleus toward centre part from both micropylar and chalazal sides developed into central cell with two polar nuclei. And the two chalazal nuclei organized into antipodal cells. Rarely indeed, one nucleus of either chalazal or micropyle side did migrate to join the formation of central cell. (ii) The other nuclei arrangement pattern was two and six respectively positioned in chalazal and micropylar sides. During maturation, five micropylar nuclei differentiated into egg apparatus consisted of three egg cells and two synergids. The sixth one migrated to form the upper polar nucleus. The lower nucleus of the chalazal side developed into antipodal cell which divided quickly, and the upper nucleus became the lower polar nucleus.     

高等植物的PCD研究进展(一)

植物细胞程序性死亡(programmed cell death,PCD)已成为当前生物学的研究热点之一。植物PCD普遍存在于植物器官和个体生长发育过程及与环境相互作用过程中,具有重要的生物学意义。在高等植物生长发育过程中,根冠细胞、导管细胞、绒毡层细胞、胚乳细胞、胚柄细胞、糊粉细胞、大孢子细胞、助细胞和反足细胞等细胞在一定程度上均发生了PCD。另外,衰老也涉及PCD。本文综述了最近几年来与发育有关的PCD研究进展,主要包括高等植物细胞死亡的形式、起因及其PCD的形态、生化特征及高等植物营养器官(根、茎和叶)和生殖器官(花、果实和种子)在其生长发育过程中的PCD。文章最后还对植物PCD的进化和生物学意义作了进一步的讨论。 Abstract：Plant programmed cell death(PCD),the details of which are becoming a focus of intensive research in biology, is a ubiquitous phenomenon and plays an improtant biological role in the develpoment of organs and whole organisms and in interactions with the environment.During higher plant development,root cap cells,tracheary elements(TEs),tapetalcells,endosperm cells,suspensor cells,aleurone cells,megaspore cells,help cells and antipodal cells,etc.undergo PCD to some degree.In addition,senescence also involves PCD.This paper mainly reviewed PCD research progress in higher plant development in recent years,including forms and causes of cell death and PCD morphological and biochemical features in higher plants;PCD in development of nutritive organs(root ,stem and leaf) and reproductive organs(flower ,fruit and seed),evolution and biological rloes of plant PCD were further discussed in the paper.