凤仙花花药发育中多糖和脂滴组织化学研究

以不同发育时期的凤仙花花药为实验材料,采用组织化学方法,对花药发育中的结构变化及多糖和脂滴物质分布进行观察。结果表明:(1)凤仙花的花药壁由6层细胞组成,包括1层表皮细胞,2层药室内壁细胞,2层中层细胞和1层绒毡层细胞。其中绒毡层细胞的形态不明显,很难与造孢细胞区分,且在小孢子母细胞时期退化。(2)在小孢子母细胞中出现了一些淀粉粒,但减数分裂后,早期小孢子中的淀粉粒消失,又出现了一些小的脂滴;随着花粉的发育,小孢子形成大液泡,晚期小孢子中的脂滴也消失;小孢子分裂形成二胞花粉后,营养细胞中的大液泡降解、消失,二胞花粉中又开始积累淀粉;接近开花时,成熟花粉中充满细胞质,其中包含了较多的淀粉粒和脂滴。(3)在凤仙花的花药发育中,绒毡层细胞很早退化,为小孢子母细胞和四分体小孢子提供了营养物质;其后的中层细胞退化则为后期花粉发育提供了营养物质。     

MALE STERILITY IN SOYBEAN (GLYCINE MAX). I. PHENOTYPIC EXPRESSION OF THE ms2 MUTANT

The effects of a nuclear male-sterile mutant (ms2) of soybean, Glycine max (L.) Merr., on anther development were analyzed by means of light- and electron-microscopy. The structure of microspore mother cells (MMCs) in male-sterile plants was identical to that of male-fertile plants. Meiosis was completed, and tetrads of microspores formed. Microspores degenerated after the deposition of primexine and probacullae. The sheath of callose surrounding microspores did not dissolve. No structural abnormalities of the microspores were detected before the onset of degeneration. The tapetal and anther wall layers were characterized by aberrant development. Tapetal abnormalities included premature vacuolation, a persistent inner tangential cell wall, failure to differentiate normal concentrations of endoplasmic reticulum and dictyosomes, disruption of plastids, and premature degeneration. Malfunction of the tapetal layer preceded, and may have induced, microspore degeneration. Gross anther morphology was not influenced until advanced stages of development.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加。同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少。伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后。小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡。体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大液泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

Microsporogenesis and tapetal development in fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.)

Summary The development of sporogenous and tapetal cells in the anthers of male-fertile and cytoplasmic male-sterile sugar beet (Beta vulgaris L.) plants was studied using light and transmission electron microscopy. In general, male-sterile anthers showed a much greater variability in developmental pattern than male-fertile anthers. The earliest deviation from normal anther development was observed to occur in sterile anthers at meiotic early prophase: there was a degeneration or irregular proliferation of the tapetal cells. Other early aberrant events were the occurrence of numerous small vesicles in the microspore mother cells (MMC) and a disorganized chromatin condensation. Deviations that occurred in sterile anthers at later developmental stages included: (1) less distinct inner structures in the mitochondria of both MMC and tapetal cells from middle prophase onwards. (2) dilated ER and nuclear membranes at MMC prophase, in some cases associated with the formation of protein bodies. (3) breakdown of cell walls in MMCs and tapetal cells at late meiotic prophase. (4) no massive increase in tapetal ER at the tetrad stage. (5) a general dissolution of membranes, first in the MMC, then in the tapetum. (6) abortion of microspores and the occurrence of a plasmodial tapetum in anthers reaching the microspore stage. (7) no distinct degeneration of tapetal cells after microspore formation. Thus, it seems that the factors that lead to abortive microsporogenesis are structurally expressed at widely different times during anther development. Aberrant patterns are not restricted to the tetrad stage but occur at early prophase.     

莴苣花药发育过程中钙的分布特征

减数分裂前,莴苣花药中的钙颗粒很少。减数分裂后,花药绒毡层细胞中的钙颗粒明显增加, 同时在花药药室基质中也出现许多细小的钙颗粒。刚从四分体中释放出的小孢子内钙颗粒很少,伴随着花粉外壁物质在小孢子表面的沉积,钙颗粒开始积累在花粉壁部位。随后,小孢子中开始出现钙颗粒。当小孢子开始形成液泡后,钙颗粒向其中聚集,伴随着小液泡融合成大液泡,体积较大的钙颗粒主要集中在液泡中,而细胞质基质中的钙颗粒很少。随着二胞花粉中的大液泡消失,花粉细胞质中的钙颗粒变得很少。在以后的发育中,只有花粉壁中积累较多的钙颗粒。在莴苣花药发育过程中,钙与绒毡层细胞的退化和小孢子液泡形成以及二胞花粉中大波泡的消失有关。而花粉外壁表面积累丰富的钙与以后花粉的萌发有关。     

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。     

A cytological indicator allows rapid assessment of microspore maturity,leading to improved <Emphasis Type="Italic">in vitro</Emphasis> anther response in Indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The inadequate response of Indica rice (Oryza sativa L.) to anther culture hinders the utility of this technique for rice crop improvement. This study focused on intensifying the in vitro anther response of selected varieties from Sri Lankan germplasm by precisely targeting the most effective maturity stage of microspores for culture. Analysis of five varieties responsive to anther culture (“At 303”, “Bw 272-6B”, “Dahanala”, “Dikwee”, and “Herath Banda”), revealed that the microspore development stage at the time of culture was crucial for fine-tuning the callus induction response. The external guide used to determine microspore maturity which was the internode distance between the last two leaves of the panicle, could not be applied equally for all rice varieties. Anther response could be improved by, at least, four- to sixfold by precisely targeting anthers with late uninucleate microspores for culture. Close correlation was demonstrated between the stage of nuclear division in the microspores and the degree of starch accumulation. Therefore, the degree of starch deposition in the microspores can be used as an effective cytological marker, which can be applied more rapidly and reliably to assess microspore maturity, in place of the internode distance.     

山麦冬大小孢子发生及雌雄配子体发育研究

采用石蜡切片技术对百合科植物山麦冬大小孢子发生及雌雄配子体发育进行了观察研究。结果表明:(1)山麦冬花药具有4个花粉囊,花药壁的发育方式为基本型,花药壁完全分化时由表皮、药室内壁、中层及绒毡层组成。(2)绒毡层发育类型为分泌型,到四分体孢子彼此分离形成单细胞花粉阶段,绒毡层细胞开始解体退化,花粉成熟时绒毡层细胞完全消失;花粉母细胞减数分裂为连续型,四分体为左右对称形排列,成熟花粉为3-细胞花粉,单萌发沟。(3)子房3室,每室2枚胚珠,胚珠倒生型,双珠被,薄珠心,雌性孢原细胞不经过平周分裂而直接发育而成大孢子母细胞。(4)减数分裂后四分体大孢子呈线型或T型排列,合点端大孢子分化为功能大孢子,胚囊发育为蓼型;花粉母细胞减数分裂过程中,二分体、四分体细胞外方被胼胝质壁所包被,小孢子形成后胼胝质壁逐渐消失。该研究结果丰富了百合科植物生殖生物学研究的内容,也为探讨百合科植物的系统学研究提供了参考。     

Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features

Summary The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported. The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage. They start breaking down just before anther dehiscence. Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells. The wall-bearing tapetum phase ends at the tetrade stage. The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one. The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall. After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule. The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains. The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction.     

A contribution to the embryology of Desmodium motorium (Houtt.) Merr. (= D. gyrans (L.f.) DC.) (Fabaceae)

Abstract

The anthers are tetrasporangiate. The anther wall comprises epidermis, fibrous endothecium, middle layer and tapetal layer. The tapetum is of the Glandular type and its cells remain uninucleate. Meiosis in pollen mother cells is normal and simultaneous cytokinesis leads to the formation of tetrahedral and decussate microspore tetrads. The pollen grains are shed at 2-celled stage. The ovule is campylotropous, bitegmic and crassinucellate. Meiosis in megaspore mother cell results in the formation of linear or occasionally T-shaped megaspore tetrad. The chalazal megaspore develops into Monosporic Polygonum type of embryo sac. Endosperm development is of the Nuclear type.     

OsMAPK6, a mitogen‐activated protein kinase,influences rice grain size and biomass production

Grain size is an important agronomic trait in determining grain yield. However, the molecular mechanisms that determine the final grain size are not well understood. Here, we report the functional analysis of a rice (Oryza sativa L.) mutant, dwarf and small grain1 (dsg1), which displays pleiotropic phenotypes, including small grains, dwarfism and erect leaves. Cytological observations revealed that the small grain and dwarfism of dsg1 were mainly caused by the inhibition of cell proliferation. Map‐based cloning revealed that DSG1 encoded a mitogen‐activated protein kinase (MAPK), OsMAPK6. OsMAPK6 was mainly located in the nucleus and cytoplasm, and was ubiquitously distributed in various organs, predominately in spikelets and spikelet hulls, consistent with its role in grain size and biomass production. As a functional kinase, OsMAPK6 interacts strongly with OsMKK4, indicating that OsMKK4 is likely to be the upstream MAPK kinase of OsMAPK6 in rice. In addition, hormone sensitivity tests indicated that the dsg1 mutant was less sensitive to brassinosteroids (BRs). The endogenous BR levels were reduced in dsg1, and the expression of several BR signaling pathway genes and feedback‐inhibited genes was altered in the dsg1 mutant, with or without exogenous BRs, indicating that OsMAPK6 may contribute to influence BR homeostasis and signaling. Thus, OsMAPK6, a MAPK, plays a pivotal role in grain size in rice, via cell proliferation, and BR signaling and homeostasis.     

PERSISTENT TAPETAL CELL1 encodes a PHD-finger protein that is required for tapetal cell death and pollen development in rice

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.     

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 Study of Anther and Pollen Development in Male Fertile and Male. Sterile Green Onion (Allium fistulosum L.)

Anther and pollen development in male-fertile and male-sterile green onions was studied. In the male-fertile line, both meiotic microspore mother ceils and tetrads have a callose wall. Mature pollen grains are 2-celled. The elongated generative cell with two bended ends displays a PAS positive cell wall. The tapetum has the character of both secretory and invasive types. From microspore stage onwards, many oil bodies or masses accumulate in the cytoplasm of the tapetal cells. The tapetum degenerates at middle 2-celled pollen stage. In male-sterile line, meiosis in microspore mother cells proceeds normally to form the tetrads. Pollen abortion occurs at microspore with vacuole stage. Two types of pollen abortion were observed. In type I, the protoplasts of the microspores contract and gradually disintegrate. At the same time the cytoplasm of microspores accumulates oil bodies which remain in the empty pollen. The tapetal cells behave normally up to the microspore stage and early stage of microspore abortion, but contain fewer oil bodies or masses than those in the male-fertilt line. At late stage of microspore abortion, three forms of the tapetal ceils can be observed: (1) the tapetal cells with degenerating protoplasts become flattened, (2) the tapetal cells enlarge but protoplasts retractor, (3) the cells break down and tile middle layer enlarges. In type Ⅱ, the cytoplasm degenerates earlier than the nucleus of the microspores and no protoplast is found in the anther locule. There are fibrous thickenings iii the endothecium of both types. It is difficult to verify whether the tapetum behavior and pollen abortion is the cause or the effect.     

CYCLIC INVASION OF TAPETAL CELLS INTO LOCULI DURING MICROSPORE DEVELOPMENT IN NYMPHAEA COLORATA (NYMPHACEAE)

In work with Nymphaea colorata Peter three distinct intervals were recorded during which tapetal cells (protoplasts) protruded into anther locules either as bridges and partitions or as invasive cells between or around tetrads of microspores. Before these intervals and between and after them, tapetal cells, while variable in shape, were noninvasive. Observations were based on sections of over 60 fixed and epoxy-embedded anthers covering the relatively brief interval from the end of meiosis through the vacuolate microspore stage. The progression of development, from early microspore stages through the microspore vacuolate period, is illustrated by transmission electron micrographs showing change in proexine and exine size and complexity. Our results indicate cycles of tapetal cell differentiation and dedifferentiation in this species.     

巴戟天花药发育过程中多糖和脂滴分布特征

巴戟天花药发育中多糖和脂滴类物质的分布呈现一定的规律：减数分裂之前,花药壁的绒毡层细胞中有少量脂滴,其他细胞中脂滴和淀粉粒都很少。四分体时期,四分体小孢子中开始出现脂滴,绒毡层细胞中的脂滴较以前增加,其他细胞中的脂滴和淀粉粒仍然很少。小孢子早期,游离小孢子在其表面形成了花粉外壁,靠外壁下方有一层周缘分布的多糖物质。绒毡层细胞中的脂滴明显减少。发育晚期的小孢子中形成一个大液泡,细胞质中出现淀粉粒;同时在药壁和药隔组织中也出现了淀粉粒。此时绒毡层退化。在二胞花粉早期,花粉中积累了大量淀粉粒和一些脂滴。但在成熟的花粉中（二胞花粉晚期）,淀粉粒消失,只有一定数量的脂滴保留。巴戟天成熟花粉中积累的营养物质主要为脂滴。     

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.