Tapetum determinant1 is required for cell specialization in the Arabidopsis anther

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. The microsporocytes generate microspores, whereas the tapetal cells support the development of microspores into mature pollen grains. Despite their importance to plant reproduction, little is known about the underlying genetic mechanisms that regulate the differentiation and interaction of these highly specialized cells in the anther. Here, we report the identification and characterization of a novel tapetum determinant1 (TPD1) gene that is required for the specialization of tapetal cells in the Arabidopsis anther. Analysis of the male-sterile mutant, tpd1, showed that functional interruption of TPD1 caused the precursors of tapetal cells to differentiate and develop into microsporocytes instead of tapetum. As a results, extra microsporocytes were formed and tapetum was absent in developing tpd1 anthers. Molecular cloning of TPD1 revealed that it encodes a small protein of 176 amino acids. In addition, tpd1 was phenotypically similar to excess microsporocytes1/extra sporogenous cells (ems1/exs) single and tpd1 ems1/exs double mutants. These data suggest that the TPD1 product plays an important role in the differentiation of tapetal cells, possibly in coordination with the EMS1/EXS gene product, a Leu-rich repeat receptor protein kinase.     

Overexpression of TAPETUM DETERMINANT1 alters the cell fates in the Arabidopsis carpel and tapetum via genetic interaction with excess microsporocytes1/extra sporogenous cells

Previously, we reported that the TAPETUM DETERMINANT1 (TPD1) gene is required for specialization of tapetal cells in the Arabidopsis (Arabidopsis thaliana) anther. The tpd1 mutant is phenotypically identical to the excess microsporocytes1 (ems1)/extra sporogenous cells (exs) mutant. The TPD1 and EMS1/EXS genes may function in the same developmental pathway in the Arabidopsis anther. Here, we further report that overexpression of TPD1 alters the cell fates in the Arabidopsis carpel and tapetum. When TPD1 was expressed ectopically in the wild-type Arabidopsis carpel, the number of cells in the carpel increased significantly, showing that the ectopic expression of TPD1 protein could activate the cell division in the carpel. Furthermore, the genetic analysis showed that the activation of cell division in the transgenic carpel by TPD1 was dependent on EMS1/EXS, as it did not happen in the ems1/exs mutant. This result further suggests that TPD1 regulates cell fates in coordination with EMS1/EXS. Moreover, overexpression of TPD1 in tapetal cells also delayed the degeneration of tapetum. The TPD1 may function not only in the specialization of tapetal cells but also in the maintenance of tapetal cell fate.     

Intra- and intertissular cytomictic interactions in the microsporogenesis of mono- and dicotyledonous plants

Comparative cytological analysis of intra- and intertissular cytomictic interactions in the microsporogenesis of mono- and dicotyledonous plants has been performed for two cellular systems: the microsporocytes and the tapetum. Cytomixis was shown to be more common for intratissular interactions, and cytomixis in the tapetum exhibited taxon-specific features, both structural and temporal. Nuclear migration in the microsporocytes mostly occurred during the zygotene–pachytene and exhibited certain synchrony with cytomixis in the tapetum. Intertissular cytomictic interactions (between the tapetum and the microsporocytes) were detected only in monocotyledonous plant anthers. Intertissular interactions may reflect more intense competition for space between the tapetum and the microsporocytes during the differentiation of anther tissues. The polyploid nuclei of the tapetum and the syncytia are powerful acceptors that can compete with the microsporocytes and attract the chromatin during translocation of the latter. The absence of intertissular interactions in dicotyledonous plants may be indicative of a better balance between the processes of differentiation of somatic and generative tissues of the microsporangium as compared to monocotyledonous plants.     

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.     

ABA and IAA control microsporogenesis in <Emphasis Type="Italic">Petunia hybrida</Emphasis> L.

The formation of fertile male gametophyte is known to require timely degeneration of polyfunctional tapetum tissue. The last process caused by the programmed cell death (PCD) is a part of the anther program maturation which leads to sequential anther tissue destruction coordinated with pollen differentiation. In the present work, distribution of abscisic acid (ABA) and indole-3-acetic acid (IAA) in developing anthers of male-fertile and male-sterile lines of petunia (Petunia hybrida L.) was analyzed by using the immunohistochemical method. It was established that the development of fertile male gametophyte was accompanied by monotonous elevation of ABA and IAA levels in reproductive cells and, in contrast, their monotonous lowering in tapetum cells and the middle layers. Abortion of microsporocytes in the meiosis prophase in the sterile line caused by premature tapetum degeneration along with complete maintenance of the middle layers was accompanied by dramatic, twofold elevation in the levels of both the phytohormones in reproductive cells. The data obtained allowed us to conclude that at the meiosis stage ABA and IAA are involved in the PCD of microsporocytes.     

Genetic control of male fertility in Arabidopsis thaliana: structural analysis of premeiotic developmental mutants

We have taken a mutational approach to identify genes important for male fertility in Arabidopsis thaliana and have isolated a number of nuclear male/ sterile mutants in which vegetative growth and female fertility are not altered. Here we describe detailed developmental analyses of four mutants, each of which defines a complementation group and has a distinct developmental end point. All four mutants represent premeiotic developmental lesions. In ms3, tapetum and middle layer hypertrophy result in the degeneration of microsporocytes. In ms4, microspore dyads persist for most of anther development as a result of impaired meiotic division. In ms5, degeneration occurs in all anther cells at an early stage of development. In ms15, both the tapetum and microsporocytes degenerate early in anther development. Each of these mutants had shorter filaments and a greater number of inflorescences than congenic male-fertile plants. The differences in the developmental phenotypes of these mutants, together with the non-allelic nature of the mutations indicate that four different genes important for pollen development, have been identified.     

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).     

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。     

Distribution of cellulosic wall in the anthers of Arabidopsis during microsporogenesis

Key message

Cellulose-specific staining revealed that tapetal cells and microsporocytes lose cellulosic walls before the onset of meiosis. Cellulosic wall degradation in microsporocytes might be independent of tapetal cells (or TPD1).

Abstract

Some cell types in a variety of angiosperms have been reported to lack cell walls. Here, we report that the tapetal cells of the anther of Arabidopsis thaliana did not appear to have a cellulosic wall based on staining with Calcofluor and Renaissance 2200. During sporogenous cell formation, cellulosic wall was present in all anther tissues. However, before meiosis it was almost absent on the tapetal cells and on the microsporocytes. In a sporocyteless/nozzle (spl/nzz) mutant, which lacks several components (microsporocytes, tapetum, middle layer and endothecium), cellulosic wall was detected in all anther cells. In another mutant, tapetum determinant1 (tpd1), which lacks tapetum and has more microsporocytes, cellulosic wall was almost absent on the microsporocytes before meiosis, similar to the wild type. These results suggest that the tapetum cells and microsporocytes lose cellulosic walls during microsporocyte formation, and that cell wall degradation occurs downstream of SPL/NZZ and is independent of TPD1.     

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.     

A novel extinction screen in Arabidopsis thaliana identifies mutant plants defective in early microsporangial development

Few Arabidopsis mutants defective in early male or female germline development have been reported. A novel extinction screen has been devised which permits the identification of mutants deficient in the earliest stages of anther development. Using mutagenized plants carrying GUS reporter constructs driven by tapetal-specific promoters originally derived from Brassica genes, a wide spectrum of mutants have been identified in Arabidopsis, ranging from those defective in archesporial cell differentiation to others expressed later in development. Crosses between these lines and known anther development mutants have enabled the identification of lines carrying mutations in genes expressed during very early anther formation. Initial characterization reveals these early mutants fall into two classes, gne (GUS-negative) 1-like, and gne2-like. Members of the gne1 mutant class initiate all four layers of the anther wall and an appropriate number of sporogenous cells; however, as development proceeds the tapetal and middle-layer cells enlarge, eventually crushing the sporogenous cells. The gne2 class anthers are disrupted at an earlier stage, with the middle and tapetal layers failing to form, and an excess of sporogenous cells developing until the germline aborts late in meiosis II. Analysis of these mutants has already raised questions about the accuracy of current models of angiosperm anther development.     

Anther ontogeny in Campsis radicans (L.) Seem. (Bignoniaceae)

In this study anther ontogeny of Campsis radicans (L.) Seem. was investigated by transmission electron microscopy and light microscopy with special reference to the development of the anther wall. The anther wall formation follows the dicotyledonous type. The differentiation in anther starts with the appearance of archesporial cells which undergo periclinal divisions to give primary parietal layer to the epidermal site and the primary sporogenous cells to the inside. The primary parietal layer also divides to form two secondary parietal layers. Later, the outer secondary parietal layer (spl1) forms the endothecium and the middle layer by periclinal division whereas the inner one (spl2) directly develops into the outer tapetum forming the inner most layer of the anther wall. The sporogenous tissue is generally organized in two rows of cells with a horseshoe-shaped outline. The remainder of the tapetum lining the sporogenous mass is derived from the connective tissue. The tapetum thus has dual origin and dimorphic. Anthers are tetrasporangiate. The wall of the anther consists of an epidermis, endothecium, middle layer, and the secretory type tapetum. Tapetal cells are usually binucleated. Epidermis and Endothecium layers of anther wall remain intact until the end of anther and pollen development; however, middle layer and tapetum disappear during development.     

Microsporogenesis and microgametogenesis of Excentrodendron hsienmu (Malvaceae s.l.) and their systematic implications

Flowers, microsporogenesis and microgametogenesis of Excentrodendron hsienmu in opening-functional flowers and non-opening flowers were studied to investigate the evolutionary relationships of Excentrodendron . E. hsienmu is a dioecious species that blossoms every 3–4 years, although large numbers of flower buds develop every year. The anther is tetrasporangiate, the tapetum is of the secretory type, the microspore tetrads are mainly tetrahedral, and the pollen grains are two-celled when shed. Four to six microsporocytes are seen on the transverse section of the anthers, and cytokinesis is simultaneous. The development of the anther wall conforms to the basic type and the anther wall is five or six cells thick, with a fibrous endothecium. The difference between the opening-functional and the non-opening flowers is mainly in the thickness of the anther wall. Early megasporogenesis in staminate flowers up to megaspore mother cell or megaspore tetrads has been observed. Excentrodendron shares with Dombeyeae only plesiomorphic features, but differs in anther wall development type and thickness. Most features of Excentrodendron are shared with Pterospermum , including such synapomorphic features as basic type of anther wall development, five- to six-cell-thick anther wall, biseriate tapetum at some places, and degeneration of microsporocytes, suggesting placement near Pterospermum .   © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 447–457.