BnC15 and BnATA20, the different putative components, control anther development in Brassica napus L

In Brassica napus, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes regulating anther cell differentiation and function. Here, we report two floral organ specific genes, BnC15 and BnATA20, derived from a B. napus two-line Rs1046A/B floral subtractive library. Although BnC15 and BnATA20 genes have a different expression pattern in anthers demonstrated by in situ hybridization and real-time PCR analysis, silencing of both genes in B. napus by antisense suppression resulted in pollen abortion after microspore release. Light and electron microscopy observation revealed the lack of plastoglobuli, lipid bodies and sporopollenin secreted from the tapetum leading to aberrations in exine sculpturing and the formation of a pollen coat. In addition, the microspores were squeezed to the irregular shape in the locule in the end. As shown by gene expression analysis in transgenic plants and the comparison of anther development between bnc15 or bnata20 mutants and Rs1046A, BnC15 and BnATA20 were positively regulated downstream of Rf gene controlling the fertility of Rs1046B in the same pathway. The results support the hypothesis that BnC15 and BnATA20 are crucial components of a genetic network that controls tapetum development and exine sculpturing.     

Zm401, a short-open reading-frame mRNA or noncoding RNA, is essential for tapetum and microspore development and can regulate the floret formation in maize

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. Previously, we cloned a pollen specific gene, zm401, from a cDNA library generated from the mature pollen of Zea mays. Expression of partial cDNA of zm401 in maize and ectopic expression of zm401 in tobacco suggested it may play a role in anther development. Here we present the expression and functional characterization of this pollen specific gene in maize. Zm401 is expressed primarily in the anthers (tapetal cells as well as microspores) in a developmentally regulated manner. That is, it is expressed from floret forming stage, increasing in concentration up to mature pollen. Knockdown of zm401 significantly affected the expression of ZmMADS2, MZm3-3, and ZmC5, critical genes for pollen development; led to aberrant development of the microspore and tapetum, and finally male-sterility. Zm401 possesses highly conserved sequences and evolutionary conserved stable RNA secondary structure in monocotyledon. These data show that zm401 could be one of the key growth regulators in anther development, and functions as a short-open reading-frame mRNA (sORF mRNA) and/or noncoding RNA (ncRNA).     

An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.     

拟南芥花药绒毡层细胞中具有基因簇特征的基因进化和功能分析

在植物基因组中, 除了同源基因成簇现象外, 近年来还发现一些具有共表达特性的异源基因也能够以基因簇形式存在, 但这些异源基因簇的进化和生物学功能尚不清楚。花药发育和花粉形成是植物进化出的特有的生殖生物学过程, 同时产生了一些在花药绒毡层中特异表达和特定功能的基因簇基因。该研究通过筛选和分析花药绒毡层中基因簇基因的分子特性、表达调控、基因年龄和基因重复进化等信息, 探讨花药基因簇基因与植物开花功能进化之间的关系。结果表明, 在拟南芥(Arabidopsis thaliana)中共筛选到84个(13个基因簇)花药绒毡层特异高表达的基因簇基因, 它们主要产生于串联重复事件, 76%的基因出现在开花植物分化后的阶段, 主要参与生殖发育、花粉鞘组成和脂代谢等生物学过程。研究初步解析了拟南芥花药绒毡层中基因簇基因的基本特征、生物学功能和基因进化机制, 为深入揭示植物基因簇基因的遗传学功能奠定了基础。     

ST273是拟南芥绒毡层和小孢子发育的必需基因

绒毡层在拟南芥花药花粉发育过程中具有重要作用,包括分泌降解胼胝质的胼胝质酶、为花粉壁的形成提供原料以及为小孢子发育提供营养物质.本文通过对拟南芥雄性不育突变体st273的分析,研究了ST273基因在花药花粉发育过程中的功能.st273是通过T-DNA插入诱变野生型拟南芥得到的一株突变体,遗传分析表明st273是单隐性核基因控制的.利用图位克隆的方法对不育基因ST273进行了定位,结果表明ST273基因与拟南芥第三条染色体上分子标记CIW11连锁.生物信息学分析发现该分子标记附近有一个调控花粉发育的基因TDF1.测序分析结果表明在st273突变体中,TDF1基因第三个外显子上459位的碱基发生了由G459变成了A459的单碱基变化,导致ST273基因该位点提前终止突变.等位分析结果表明st273与tdf1是等位突变体.st273突变体营养生长期发育正常,但生殖生长发育出现异常.亚历山大染色结果显示st273突变体花药中没有花粉.组织切片观察结果表明,突变体花药绒毡层异常肥大且空泡化,四分体不能正常释放小孢子,最终无法形成花粉.这些结果揭示了ST273蛋白质参与调控了绒毡层和小孢子发育过程.     

ST273 Is Essential for Tapetum and Microspore Development in Arabidopsis thaliana*

In Arabidopsis, the tapetum plays important roles in anther and pollen development by providing enzymes for callose dissolution, materials for pollen wall formation, and nutrients for microspore development. This paper describes the functional analyses of the ST273 gene in anther and pollen development by using Arabidopsis male sterile mutant st273. Mutant st273 was identified from a T DNA insertion mutant population, and genetic analysis showed that st273 mutant was controlled by a single recessive nuclear gene. A map based cloning approach was used, and ST273 gene was mapped to be linked to a molecular marker CIW11 on chromosome 3. Bioinformatics analysis revealed that there is a TDF1 gene near the marker CIW11. Sequencing analysis indicated that st273 mutant had a G459 to A459 base pair change in the third exon of TDF1 gene, which resulted in premature termination mutation in this region. Allelism test indicated that ST273 and TDF1 belong to the same locus. The mutant plant grows normally during the vegetative growth stage, but show developmental defects at the reproductive growth stage. Alexander staining showed that there was no pollen in the mature anther locule. Cytology observation indicated that the mutant tapetum was enlarged and vacuolated, the tetrads could not release the microspores timely, and finally no pollen was formed in the anther. These results demonstrated that ST273 protein plays an important role in tapetum and microspore development.     

雄性不育与可育楸树花发育的细胞学比较研究

楸树（Catalpa bungei C.A.Meyer.）属紫葳科(Bignoniaceae)梓树属(Catalpa),落叶乔木,是我国特有的珍贵优质用材树种。本文用石蜡切片法对可育株和雄性不育株楸树的大、小孢子发生及雌、雄配子体发育过程进行了详细地比较观察。结果表明：可育株和不育株楸树雌蕊的发育基本相同,胚珠倒生,薄珠心,单珠被,胚囊发育为蓼型。可育株雄蕊花药四室,药隔薄壁组织发达;异型绒粘层,由药壁绒粘层和药隔绒粘层组成;花药壁表皮细胞在小孢子母细胞减数分裂前后开始径向伸长加厚,直到花药开裂并不降解,这可能与花药开裂有关;成熟花粉为四合花粉。雄性不育株花药的早期发育到次生造胞细胞时期与可育雄蕊的相同,小孢子母细胞减数分裂前绒毡层发育不充分;四分体时期,绒毡层细胞高度液泡化,细胞质稀薄,已提前降解,小孢子四分体因绒毡层结构和功能异常而不能正常发育,因此楸树雄性不育为结构型雄性不育。     

垂花悬铃花雌雄配子体发育研究

对垂花悬铃花雄配子体发育观察表明,其花药由表皮(1层)、药室内层(1层)、中层(2层)、绒毡层(1层)及造孢细胞组成,花药四室,药壁发育为双子叶型。雄配子体发育经由花粉母细胞减数分裂形成四分体,该四分体胞质分裂为同时型,四分体排列方式为四面体型,十字交叉型及左右对称型;小孢子再经有丝分裂形成营养核和生殖核,生殖核再经有丝分裂形成3-核花粉。花药壁层的变化,在单核小孢子期,表皮细胞解体,仅留下痕迹;中层在花粉母细胞期逐渐消失;药室内壁在单核小孢子期开始纤维化;绒毡层在单核小孢子期消失,属变形绒毡层。雌配子体发育观察表明,其子房上位,5室,每室1个胚珠,胚珠弯生,中轴胎座,大多数胚珠发育停留在珠心形成阶段,极少数珠心形成一群孢原细胞及单核、双核胚囊。     

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.     

Differential gene expression in Arabidopsis wild-type and mutant anthers: insights into anther cell differentiation and regulatory networks

In flowering plants, the anther contains highly specialized reproductive and somatic cells that are required for male fertility. Genetic studies have uncovered several genes that are important for anther development. However, little information is available regarding most genes active during anther development, including possible relationships between these genes and genetically defined regulators. In Arabidopsis, two previously isolated male-sterile mutants display dramatically altered anther cell differentiation patterns. The sporocyteless (spl)/nozzle (nzz) mutant is defective in the differentiation of primary sporogenous cells into microsporocytes, and does not properly form the anther wall. The excess microsporocytes1 (ems1)/extrasporogenous cells (exs) mutants produce excess microsporocytes at the expense of the tapetum. To gain additional insights into microsporocyte and tapetum differentiation and to uncover potential genetic interactions, expression profiles were compared between wild-type anthers (stage 4-6) and those of the spl or ems1 mutants. A total of 1954 genes were found to be differentially expressed in the ems1 and/or spl anthers, and these were grouped into 14 co-expression clusters. The presence of genes with known and predicted functions in specific clusters suggests potential functions for other genes in the same cluster. To obtain clues about possible co-regulation within co-expression clusters, we searched for shared cis-regulatory motifs in putative promoter regions. Our analyses were combined with data from previous studies to develop a model of the anther gene regulatory network. This model includes hypotheses that can be tested experimentally to gain further understanding of the mechanisms controlling anther development.     

植物细胞核雄性不育基因研究进展

植物雄性不育既是研究植物生殖生物学重要的植物学性状也是研究作物杂种优势利用重要的农艺性状,在遗传和分子生物学中具有重要地位。以模式植物拟南芥和水稻为主,对植物雄性不育的控制基因和相关分子机理已有众多进展,按照花药发育时期和雄性败育的表现形式可以归纳为减数分裂异常、胼胝质代谢异常、绒毡层发育异常、花粉壁发育异常、花药开裂异常,以及其它类型的雄性不育。在不育相关基因中,导致胼胝质代谢异常、绒毡层发育异常和花粉壁发育异常的基因往往表现一因多效,一个相关基因的突变会产生复合表型。关于植物雄性不育相关基因的研究表明,雄性器官和小孢子形成过程中的任何相关基因的改变,均可导致雄性不育的产生。本文总结了植物核基因雄性不育的研究进展,以期促进不同物种间雄性不育基因的比较分析,使植物雄性不育研究更加深入。     

Pectin secretion and distribution in the anther during pollen development in Lilium

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium. Received: 3 July 2000 / Accepted: 19 October 2000     

西瓜小孢子囊发育及雄配子体发生的观察

西瓜(Citrullus lanatus)小孢子囊的孢原细胞出现在雄花原基出现后4—6天,孢原细胞数目推测只有一列;初生造孢细胞经过2—3次分裂,形成次生造孢细胞。开花前7—8天,小孢子囊发育健全,小孢子母细胞进入减数分裂期。同一花药不同花粉囊相同一药室,花粉母细胞减数分裂和小孢子的发育,并不是高度同步的。绒毡层为异型细胞,腺质绒毡层。雄配子体的发育开始于开花前6—7天,充分成熟的西瓜花粉已分裂为三细胞花粉。     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development

In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion.     

革苞菊胚胎学研究 Ⅰ.大、小孢子发生和雌、雄配子体发育

革苞菊为雌雄异株。在雄花中 ,花药 4室 ,药壁发育为双子叶型 ,由表皮、药室内壁 ,一层中层和绒毡层组成。绒毡层于小孢子四分体时期开始变形 ,其细胞原生质体向药室中移动 ,为变形绒毡层。小孢子孢原为多细胞 ,小孢子母细胞减数分裂产生四面体型的小孢子四分体。四分体胞质分裂为同时型。成熟花粉 3-细胞型。单核期的小孢子出现壁发育不良和巨大及空花粉现象。在雌花中 ,胚珠是倒生的 ,单珠被 ,薄珠心 ,珠被于孢原期已发育完整。大孢子孢原单细胞。由孢原细胞直接发育形成大孢子母细胞。 4个大孢子直线型 ,蓼型胚囊。于成熟胚囊期观察到发育异常的胚囊。通过对胚囊发育过程中营养物质消长规律的研究 ,讨论了环境与发育的相关性问题。