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 .     

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.     

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.     

MICROSPOROGENESIS IN CITRUS LIMON (RUTACEAE)

Prior to meiosis tapetal cells become binucleate, and callose deposition separates spore mother cells from each other. No cytomictic channels are present during meiosis. Cytokinesis is simultaneous, by furrowing. The primexine and a rudimentary exine are laid down while the microspores are still in tetrads. After callose dissolution the released microspores gradually become vacuolate and the exine becomes more complex and massive. During the tetrad stage tapetal walls are gradually lost and orbicules are deposited outside the plasmalemma. This continues after microspore release. Later, at the vacuolate microspore stage, the tapetal cells become amoeboid and intrude among the microspores. Tapetal dissolution occurs just prior to the appearance of large amounts of starch and lipids in the microspores.     

Microspore separation in the quartet 3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation

Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development. Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging. QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica). The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity. In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings. Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage. Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

<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.     

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.     

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。     

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.     

铁皮石斛花粉块结构及发育过程的解剖学特征研究北大核心CSCD

兰科植物的有性生殖特殊,每朵花只有1个花药,且花粉有聚集成块发育的特征。为了揭示铁皮石斛花粉块的发育特征,该研究以野生铁皮石斛不同时期的花药为材料,采用半薄切片和植物组织化学方法对其发育过程进行解剖学观察分析,并对成熟花粉块进行离体培养,观察花粉管的萌发状况。结果表明:(1)铁皮石斛花药壁由1层表皮细胞,2层药室内壁细胞,1层中层细胞和1层绒毡层细胞组成。开花时,绒毡层细胞退化,中层细胞没有退化,药室内壁细胞则形成纤维状细胞壁;药室中的小孢子母细胞没有明显的胼胝质壁结构。(2)小孢子发生属同时型,减数分裂后四分体小孢子不分散,以四合花粉状态发育,并进一步连接形成花粉块。(3)在小孢子发育中,孢粉素覆盖在整个花粉块表面形成花粉外壁,但花粉块内部的花粉没有花粉外壁结构;在花粉块表面的花粉外壁上未见花粉萌发孔。(4)在花粉离体萌发实验中,具有花粉外壁的花粉块表面花粉未见萌发,仅由花粉块内部的花粉萌发出花粉管。     

Invasive tapetum and tricelled pollen inAmbrosia trifida (Asteraceae,tribeHeliantheae)

Staminate flowers of giant ragweed,Ambrosia trifida L. (Asteraceae, tribeHeliantheae, subtribeAmbrosiinae) were processed into resin and sectioned 1–2 µm thick. The invasive (amoeboid) anther tapetum remains parietal until microspores are released from tetrads, then it swells and invades the locule, merging gradually into a single protoplast that flows among the microspores. After the tapetal membrane ruptures at late microspore stage, tapetal debris fills the locule, then disappears as pollen matures. Pollen becomes tricelled before anthesis. The two sperm cell nuclei are slender and wormlike. The present report supports the two generalizations that invasive tapetum and tricelled pollen are attributes of theAsteraceae.     

Genetics and cytology of a genic male-sterile, female-sterile mutant from a transposon-containing soybean population

A male-sterile, female-sterile soybean mutant (w4-m sterile) was identified among progeny of germinal revertants of a gene-tagging study. Our objectives were to determine the genetics (inheritance, allelism, and linkage) and the cytology (microsporogenesis and microgametogenesis) of the w4-m sterile. The mutant was inherited as a single recessive nuclear gene and was nonallelic to known male-sterile, female-sterile mutants st2 st2, st3 st3, st4 st4, st5 st5, and st6 st6 st7 st7. No linkage was detected between the w4-m sterile and the w4w4, y10 y10, y11 y11, y20 y20, fr1 fr1, and fr2 fr2 mutants. Homologous chromosome pairing was complete in fertile plants. Chromosome pairing, as observed in squash preparation, was almost completely absent in sterile plants. Developmentally microsporogenesis proceeded normally in both the fertile and the w4-m sterile through the early microspore stage. Then the tapetal cells of the w4-m sterile surrounding the young microspores developed different-size vacuoles. These tapetal cells became smaller in size and separated from each other. Some of the microspores of the w4-m sterile also became more vacuolate prematurely and sometimes they collapsed, usually by the late microspore stage. In the w4-m sterile the microspore walls remained thinner and structurally different from the microspore walls of fertile plants. No pollen was formed in the mutant plants, even though some of the male cells reached the pollen stage, although without normal filling. The w4-m sterile was designated st8st8 and assigned Soybean Genetic Type Collection number T352.     

Functional analysis of a beta-1,3-glucanase gene (Tag1) with anther-specific RNA and protein accumulation using antisense RNA inhibition

A critical stage in pollen development is the dissolution of tetrads into free microspores. Tetrads are surrounded by a wall composed primarily of beta-1,3-glucan. At the completion of meiosis, tetrads are released into the anther locule after hydrolysis of the callose by a beta-1,3-glucanase complex. The cDNA corresponding to a beta-1,3-glucanase cloned from tobacco (Tag 1) represents a gene that is highly similar to other beta-1,3-glucanases and is expressed exclusively in anthers from the tetrad to free microspore stage of pollen development. Tag 1 protein was overexpressed in E. coli, accumulating in insoluble inclusion bodies. Polyclonal antibodies against Tag 1 recombinant protein identify a single 33 kD protein accumulating only in anthers at tetrad and free microspore stages where beta-1,3-glucanase activity is present. Transgenic plants expressing Tag 1 antisense RNA were produced. Although Tag 1 RNA and protein levels were greatly reduced, tetrad dissolution and pollen development were normal. These data indicate that under the conditions these tobacco plants were grown, wild type levels of Tag 1 protein are not necessary for male fertility.     

麻疯树花药发育过程中Ca2+的分布特征

采用超薄切片技术,在透射电镜下观察麻疯树(Jatropha curcasL.)花药发育过程中Ca2 的分布特征。在孢原细胞时期的花药中几乎看不到Ca2 沉淀,但花药维管束周围的细胞中有较多的Ca2 沉淀;到小孢子母细胞时期,细胞质中Ca2 沉淀依然较少,绒毡层壁上Ca2 沉淀明显增多;四分体形成时,小孢子细胞质和绒毡层细胞质中出现了较多的Ca2 沉淀;在小孢子发育早期,细胞质中Ca2 沉淀增加不明显,花粉壁部位累积有很多的Ca2 沉淀,绒毡层中Ca2 沉淀数量达到最多;到小孢子发育晚期,小孢子大液泡的液泡膜上有大量的Ca2 沉淀,绒毡层中Ca2 沉淀明显减少;随着二胞花粉中的大液泡消失,细胞质中积累淀粉粒以后,花粉中看到的Ca2 沉淀极少,同时,在花药维管束周围的薄壁细胞中,又出现了较多的Ca2 沉淀,表明花粉对Ca2 的需求可能降低。麻疯树花药发育过程中钙的动态分布特征暗示着钙参与了调控花粉发育过程,Ca2 的运输途径是由药隔薄壁组织运输到绒毡层,再进一步转移到小孢子表面和细胞质中,整个花药发育过程中,Ca2 沉淀表现为少—增加—减少的变化趋势。     

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。     

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。