Tapetal Cells Research Articles

Sporopollenin Biosynthetic Enzymes Interact and Constitute a Metabolon Localized to the Endoplasmic Reticulum of Tapetum Cells

The sporopollenin polymer is the major constituent of exine, the outer pollen wall. Recently fatty acid derivatives have been shown to be the precursors of sporopollenin building units. ACYL-COA SYNTHETASE, POLYKETIDE SYNTHASE A (PKSA) and PKSB, TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 have been demonstrated to be involved in sporopollenin biosynthesis in Arabidopsis (Arabidopsis thaliana). Here all these sporopollenin biosynthetic enzymes but TKPR2 have been immunolocalized to endoplasmic reticulum of anther tapetal cells. Pull-down experiments demonstrated that tagged recombinant proteins interacted to form complexes whose constituents were characterized by immunoblotting. In vivo protein interactions were evidenced by yeast (Saccharomyces cerevisiae) two-hybrid analysis and by fluorescence lifetime imaging microscopy/Förster resonance energy transfer studies in transgenic Nicotiana benthamiana, which were used to test the possibility that the enzymes interact to form a biosynthetic metabolon. Various pairs of proteins fused to two distinct fluorochromes were coexpressed in N. benthamiana leaf tissues and fluorescence lifetime imaging microscopy/Förster resonance energy transfer measurements demonstrated that proteins interacted pairwise in planta. Taken together, these results suggest the existence of a sporopollenin metabolon.

The Arabidopsis U–box/ARM repeat E3 ligase AtPUB4 influences growth and degeneration of tapetal cells, and its mutation leads to conditional male sterility

Pollen formation is a complex developmental process that has been extensively investigated to unravel underlying fundamental developmental mechanisms and for genetic manipulation of the male-sterility trait for hybrid crop production. Here we describe identification of AtPUB4, a U-box/ARM repeat-containing E3 ubiquitin ligase, as a novel player in male fertility in Arabidopsis. Loss of AtPUB4 function causes hypertrophic growth of the tapetum layer. The Atpub4 mutation also leads to incomplete degeneration of the tapetal cells and strikingly abnormal exine structures of pollen grains. As a result, although the Atpub4 mutant produces viable pollen, the pollen grains adhere to each other and to the remnants of incompletely degenerated tapetal cells, and do not properly disperse from dehisced anthers for successful pollination. We found that the male-sterility phenotype caused by the Atpub4 mutation is temperature-dependent: the mutant plants are sterile when grown at 22°C but are partially fertile at 16°C. Our study also indicates that the AtPUB4-mediated pathway acts in parallel with the brassinosteroid pathway in controlling developmental fates of the tapetal cells to ensure male fertility.

Microsporogenesis and Microgametogenesis of Male‐Sterile Mutant ms1 in Alfalfa

ABSTRACTThe use of male sterile lines is one of the ways to reduce the selfing rates and thus maximize heterosis in alfalfa (Medicago sativa L.). A spontaneous, stable male sterile mutant ms1 has been derived from M. sativa ‘Graze401’. In this paper, the microsporogenesis and microgametogenesis have been examined in ms1 and wild‐type plants under light and scanning electron microscopy. There were no significant differences of anther development between ms1 and the wild‐type plants until the end of the tetrad stage. The abortive development of anthers was initiated no later than the young microspore stage, as indicated by the appearance of large vacuoles in tapetal cells and the delayed tapetum degeneration leading to abortion of microsporogenesis. The shrunken shape and abnormal smooth surface of pollen walls in ms1 was observed, in which an incomplete exine formation on sterile microspores may indicate insufficient deposition of pollen kitt from the tapetal cells as well as microspores. Abnormalities of male meiosis in ms1 included lagged chromosomes, chromosome bridges, scattered chromosomes, micronuclei, and asynchronous chromosome segregations during meiosis II. We concluded that this new male sterile mutant is stable with normal stamen development but complete pollen abortion at a late stage of microsporogenesis.

Development and disintegration of tapetum-specific lipid-accumulating organelles, elaioplasts and tapetosomes, in Arabidopsis thaliana and Brassica napus

The pollen coat covering the surface of pollen grains has many important roles for pollination. In Brassicaceae plants, the pollen coat components are synthesized and temporarily accumulated in two tapetum-specific organelles, the elaioplast and the tapetosome. Although many biochemical and electron microscopic analyses have been attempted, the structure and biogenesis of these organelles have not been fully elucidated. To resolve this problem, we performed live imaging of these organelles using two markers, FIB1a-GFP and GRP17-GFP. FIB1a is an Arabidopsis fibrillin, a structural protein of elaioplast plastoglobules. In transgenic Arabidopsis, fluorescence of FIB1a-GFP appeared in young elaioplasts, in which small plastoglobules were developing. However, the fluorescence disappeared in later stages, while enlargement of plastoglobules continued. GRP17 is an Arabidopsis oleopollenin, an oleosin-like protein in tapetosomes. Fluorescence microscopy of GRP17-GFP expressed in Arabidopsis and Brassica napus revealed that tapetosomes do not contain oleopollenin-coated vesicles but have an outer envelope, indicating that the tapetosome structure is distinct from seed oil bodies. Visualization of GRP17-GFP also demonstrated that the tapetal cells become protoplasts and migrate into locules before pollen coat formation, and provided live imaging of the foot formation between pollen grains and stigmatic papilla cells.

I. Primexine development in Passiflora racemosa Brot.: overlooked aspects of development

Developmental stages during the tetrad period were examined in detail by transmission electron microscopy with an emphasis on substructure. Our purpose was to find out whether the sequence of sporoderm developmental events provides additional evidence for our recent hypothesis on the underlying cause of exine ontogeny as a sequence of self-assembling micellar mesophases initiated by genomically given physicochemical parameters. Osmiophilic globules encrusting the surface of postmeiotic microspores and tapetal cells are temporary prepattern units which come first. The second prepattern structures are highly ordered bundles of microfilaments and microtubules which determine the position of microspore surface invaginations and clusters of the glycocalyx inside them. The first glycocalyx units are microgranules which during the middle tetrad stage rearrange into radially oriented rod-like units. The latter form lens-like clusters of the glycocalyx-1, located inside the invaginations. These clusters predestine the position of the future luminae in the exine reticulum. The second glycocalyx layer is laid down as a continuous layer over the whole microspore surface and has similar substructure, that is radial rods. Glycocalyx-2 is a framework for procolumellae which appear at the late tetrad stage. Therefore, the sequence of substructural units in the primexine is: globules, microgranules, rod-like units, and layers of radially oriented rods tightly packed in the periplasmic space. This sequence corresponds to the first three mesophases of self-assembling micelles: spherical micelles, cylindrical micelles, and layers of hexagonally packed cylindrical micelles (middle mesophase). We observed the same sequence in other species during primexine development, and the findings of this study provide new evidence for our hypothesis.

Distribution of insoluble polysaccharides, neutral lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. (Bignoniaceae)

In this study, distribution of polysaccharides, lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. was examined from sporogenous cell stage to mature pollen, using cytochemical methods. To detect the distribution and dynamic changes of insoluble polysaccharides, lipid bodies, and proteins in the anthers through progressive developmental stages, semi-thin sections of anthers at different developmental stages were stained with periodic-acid-Schiff (PAS) reagent, Sudan black B, and Coomassie brilliant blue, respectively, and examined under light microscope. Ultrastructural observations with TEM were also carried out to determine the storage form of starch in the connective tissue, and storage form of lipids in the tapetal cells. In sporogenous cell stage, anther wall contains numerous insoluble polysaccharides. However, from the sporogenous cell stage to the vacuolated microspore stage, the amount of insoluble polysaccharides in the anther wall decreases gradually. At bicellular pollen stage, tapetum degenerates completely and polysaccharides are not seen in the anther wall. Lipid bodies are observed in the cytoplasm of both middle layer and tapetal cells at tetrad stage, whereas they disappear in the vacuolated microspore stage. Compared with polysaccharides, proteins are limited in the anther wall at early stages of development. During pollen development, polysaccharides, proteins, and lipid bodies are scarce in the cytoplasm of sporogenous cells, but their amount increases at premeiotic stage. From tetrad stage to bicellular pollen stage, microspore cytoplasm contains variable amount of insoluble polysaccharide grains, lipid and protein bodies. At bicellular pollen stage, plentiful amount of starch granules are stored in the cytoplasm of the pollen grains. Proteins and lipid bodies are also present in the cytoplasm.

Tapetal dysplasia in a Swedish Vallhund dog

To describe the gross, histopathological, and ultrastructural findings in a dog with bilateral tapetal dysplasia. The globes of a 15-year-old neutered male Swedish Vallhund dog with a ventrally displaced tapetum in both eyes were fixed in 10% formalin and submitted to the Comparative Ocular Pathology Laboratory of Wisconsin for histological evaluation. Sections were stained with hematoxylin and eosin, Masson's trichrome, and Melan-A immunohistochemistry (IHC), and tissues were subsequently processed for transmission electron microscopy. Bilateral fundic and gross examination revealed a tapetal fundus inferior to the optic nerve head (ONH) and a nontapetal fundus with mild scattering of tapetal tissue superior to the ONH. Histologically, there was decreased pigmentation of the retinal pigment epithelium with only a few melanin granules in the peripheral retina. The affected tapetum was relatively acellular and fibrous with occasional tapetal cells scattered throughout the inner choroid or displaced into the vascular outer choroid. Special stains revealed that the tapetum was mostly composed of collagen (Masson's trichrome) and failed to express Melan-A (IHC) unlike a normal canine control tapetum. Ultrastructurally, the tapetum was markedly dysplastic both superior and inferior to the ONH with no uniformly arranged tapetal cells. The few cells identified within the tapetum contained irregularly arranged and disorganized electron-dense structures within their cytoplasm, which were interpreted as dysplastic tapetal rodlets. Based on microscopic and ultrastructural findings, this is the first report of tapetal dysplasia in a dog.

EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice

Programmed cell death is essential for the development of multicellular organisms, yet pathways of plant programmed cell death and its regulation remain elusive. Here we report that ETERNAL TAPETUM 1, a basic helix-loop-helix transcription factor conserved in land plants, positively regulates programmed cell death in tapetal cells in rice anthers. eat1 exhibits delayed tapetal cell death and aborted pollen formation. ETERNAL TAPETUM 1 directly regulates the expression of OsAP25 and OsAP37, which encode aspartic proteases that induce programmed cell death in both yeast and plants. Expression and genetic analyses revealed that ETERNAL TAPETUM 1 acts downstream of TAPETUM DEGENERATION RETARDATION, another positive regulator of tapetal programmed cell death, and that ETERNAL TAPETUM 1 can also interact with the TAPETUM DEGENERATION RETARDATION protein. This study demonstrates that ETERNAL TAPETUM 1 promotes aspartic proteases triggering plant programmed cell death, and reveals a dynamic regulatory cascade in male reproductive development in rice.

Post-meiotic deficient anther1 (PDA1) encodes an ABC transporter required for the development of anther cuticle and pollen exine in rice

The tapetum of the anther locule encloses the male reproductive cells and plays a supportive role for normal pollen development. However, the underlying mechanism remains less understood. Previously, we identified a complete recessive male sterile mutant, post-meiotic deficient anther1 (pda1), with abnormal postmeiotic tapetal development. In this study we comprehensively characterized pda1. Chemical analysis uncovered that pda1 anther had significant lower levels of cutin monomers and cuticular waxes. PDA1 gene encodes an ATP-binding cassette (ABC) half-transporter, namely OsABCG15, which is conserved from algae to higher plants. In situ RNA hybridization assay showed that PDA1 is strongly expressed in tapetal cells, and weakly in microspores during the anther development. Additionally, the expression of two pollen exine biosynthetic genes CYP704B2 and CYP703A3 was dramatically reduced in pda1 mutant anthers. Altogether, these observations suggest that the tapetum-expressed ABC transporter PDA1 plays a crucial role in secreting lipidic precursors from the tapetum to developing microspores and the anther epidermis.

Asymmetric cytokinesis guide the development of pseudomonads inRhynchospora pubera(Cyperaceae)

The late stages of microsporogenesis in the family Cyperaceae are marked by the formation of an asymmetrical tetrad, degeneration of three of the four nuclei resulting from meiosis and the formation of pseudomonads. In order to understand the cytological changes involved in the development of pseudomonads, a combination of 11 different techniques (conventional staining, cytochemistry procedures, immunofluorescence, FISH and transmission electron microscopy: TEM) were used to study the later stages of microsporogenesis in Rhynchospora pubera. The results demonstrated the occurrence of two cytoplasmic domains in the pseudomonads, one functional and the other degenerative, which are physically and asymmetrically separated by cell plate with an endomembrane system rich in polysaccharides. Other changes associated with endomembrane behaviour were observed, such as a large number of lipid droplets, vacuoles containing electron-dense material and concentric layers of endoplasmic reticulum. Concomitant with the isolation of degenerative nuclei, the tapetal cells also showed evidence of degeneration, indicating that both tissues under programmed cell death (PCD), as indicated by immunofluorescence and TEM procedures. The results are significant because they associate cellular polarisation and asymmetry with different cytoplasmic domains, and hence open new possibilities for studying cellular compartmentalisation and PCD.

Prediction of components of the sporopollenin synthesis pathway in peach by genomic and expression analyses

BackgroundThe outer cell wall of the pollen grain (exine) is an extremely resistant structure containing sporopollenin, a mixed polymer made up of fatty acids and phenolic compounds. The synthesis of sporopollenin in the tapetal cells and its proper deposition on the pollen surface are essential for the development of viable pollen. The beginning of microsporogenesis and pollen maturation in perennial plants from temperate climates, such as peach, is conditioned by the duration of flower bud dormancy. In order to identify putative genes involved in these processes, we analyzed the results of previous genomic experiments studying the dormancy-dependent gene expression in different peach cultivars.ResultsThe expression of 50 genes induced in flower buds after the endodormancy period (flower-bud late genes) was compared in ten cultivars of peach with different dormancy behaviour. We found two co-expression clusters enriched in putative orthologs of sporopollenin synthesis and deposition factors in Arabidopsis. Flower-bud late genes were transiently expressed in anthers coincidently with microsporogenesis and pollen maturation processes. We postulated the participation of some flower-bud late genes in the sporopollenin synthesis pathway and the transcriptional regulation of late anther development in peach.ConclusionsPeach and the model plant Arabidopsis thaliana show multiple elements in common within the essential sporopollenin synthesis pathway and gene expression regulatory mechanisms affecting anther development. The transcriptomic analysis of dormancy-released flower buds proved to be an efficient procedure for the identification of anther and pollen development genes in perennial plants showing seasonal dormancy.

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.

Anther development and cytochemistry in Asphodelus aestivus (Asphodelaceae)

Asphodelus aestivus Brot. (Asphodelaceae) anthers were analysed to provide a detailed understanding of the events that lead to pollen grain development, accompanied by cytochemical observations at different ontogenic stages. An anther locule of A. aestivus was bordered by 4 different layers: the tapetum, the middle layer, the endothecium, and the epidermis. At the tetrad stage, secretory tapetal cells enlarged maximally, while they showed degeneration at the young microspore stage. During the course of the degeneration, vacuolisation and reduction in the volume were conspicuous. Cytochemical analysis revealed that tapetal cells presented an intense reaction with regard to protein, insoluble polysaccharides, and lipids, although the rest of the wall cells reacted weakly throughout development. The entire wall layer cells were rich in starch grains as well. The ultrastructural outcomes confirmed that orbicules were embedded within the dissolving tapetal walls. Young pollen cytoplasm was rich in mitochondria and short ER cisterns, and encircled by the intine and exine. The exine of mature pollen was of the tectate-columellate type. The intine became thicker and consisted of 2 sublayers at the sulcus. The mature pollen had the following morphological characteristics: monosulcate, heteropolar, oblate-spheroidal, widely elliptical in polar view, rhombic, and large. The cytoplasm of a mature pollen grain was filled with starch grains, insoluble polysaccharides, and proteins.

Sugar Cane Genome Numbers Assumption by Ribosomal DNA FISH Techniques

Conventional cytological method is limited for polyploidy plant genome study, especially sugar cane chromosomes that show unstable numbers of each cultivar. Molecular cytogenetic as fluorescent in situ hybridization (FISH) techniques were used in this study. A basic chromosome number of sugar cane was estimated with three information; 1) number of 18S rDNA sites, 2) number of 5S rDNA sites and 3) total number of chromosomes. 18S and 5S rDNA were located by FISH techniques, the number of hom (e) ologous sites were illustrated in range of 7 to 9 and 13 to 15 sites. 110 chromosome numbers were shown in tapetal cells of flower buds of sugar cane. The implications of these results can predict about 14 basic chromosomes numbers but 5S rDNA seem reliable indicate for basic chromosome number and 18S rDNA were discussed about nucleolar dominance phenomenon of the sugar cane “KPS 00-25” cultivar

The promoter of an A9 homolog from the conifer Cryptomeria japonica imparts male strobilus-dominant expression in transgenic trees

KEY MESSAGE : GUS analysis in Cryptomeria japonica revealed that the CjMALE1 promoter is activated in the male strobilus of C. japonica. Toward the development of male sterile technology for Cryptomeria japonica, a male strobilus-dominant promoter of C. japonica was isolated. The CjMALE1 gene was isolated from a male strobilus-specific suppression subtractive hybridization (SSH) library, and the promoter was isolated by the TAIL-PCR method. To characterize the CjMALE1 promoter, β-glucuronidase (GUS)-fused genes were constructed and introduced into C. japonica using Agrobacterium tumefaciens. GUS expression from CjMALE1-2.5K (2,718bp fragment)::GUS C. japonica and CjMALE1-1K (1,029bp fragment)::GUS C. japonica was detected in the tapetum and microspore mother cells. These promoter fragments were comparably active in the pre-meiotic stage of the male strobilus of C. japonica. Our analysis showed that the 1,029bp promoter had all the cis-elements necessary for male strobilus-dominant expression of CjMALE1. When CjMALE1-1K::GUS was introduced into Arabidopsis, GUS expression was detected in the same spatiotemporal pattern as in C. japonica. These results suggest that the CjMALE1 promoter is subject to transcriptional regulatory systems consisting of cis- and trans-elements that have been highly conserved during evolution.

Ultrastructural study of pollen and anther development in Luehea divaricata (Malvaceae, Grewioideae) and its systematic implications: Role of tapetal transfer cells, orbicules and male germ unit

Developmental processes of microsporogenesis and microgametogenesis in Luehea divaricata (Malvaceae, Grewioideae) were analyzed with transmission electron microscopy. The species studied has perfect flowers. The young anthers are bithecal and tetrasporangiate; microspore mother cells undergo simultaneous meiosis, forming tetrads with a tetrahedral arrangement. The development of the anther wall conforms to the basic type and the tapetum is secretory. The results highlight the presence of multinucleate tapetal cells, which acquire ultrastructural features characteristic of transfer cells at the young pollen grain stage, and which are associated with the presence of orbicules. Tapetal transfer cells have not been previously investigated in detail for other species of angiosperms; their function during pollen development is discussed. The present work is the first contribution to the knowledge of ultrastructural pollen development in the genus Luehea, as well as in the subfamily Grewioideae.

Ultrastructural studies on the sporogenous tissue and anther wall of Leucojum aestivum (amaryllidaceae) in different developmental stages

In this study, ultrastructures of anther wall and sporogenous tissue of Leucojum aestivum were investigated during different developmental stages. Cytomictic channels were seen between pollen mother cells during prophase I. Polar distribution was described in the organelle content of pollen mother cells and microspores in early phases of microsporogenesis and also in pollen mitosis. Active secretion was observed in tapetal cells. Previous reports about developmental stages of male gametophyte were compared with the results of this study.

The Arabidopsis LFR Gene Is Required for the Formation of Anther Cell Layers and Normal Expression of Key Regulatory Genes

The anther is the male reproductive organ in flowering plants. Although some genes were reported to be involved in anther development, the molecular mechanisms underlying the transcriptional regulation of these genes is unclear. lfr-2 (leaf and flower related-2), the null allele of Arabidopsis thaliana LFR (LEAF AND FLOWER RELATED), was male-sterile. The anthers of lfr-2 plants were defective in sporogenous cell formation, tapetum development, and pollen development. In agreement with these phenotypes, expression studies showed that LFR was expressed in all cell layers of the anther, and that expression was particularly strong in the tapetal cells and pollen grains. Quantitative RT-PCR analysis revealed that LFR is required for the normal transcription of some anther development-related genes, such as AMS, CALS5, and DYT1, MS1 and MS2, and ROXY2. Genetic analysis showed that SPL was epistatic to LFR while LFR was epistatic to DYT1. We propose that LFR may be a crucial component in the regulation of a genetic network that modulates anther development.

Some histochemical features of anther wall of Leucojum aestivum (Amaryllidaceae) during pollen development

In this study, polysaccharide and RNA contents of anthers were investigated on different phases of sporogenesis by using light microscopy techniques from histological and cytological point of view in Leucojum aestivum. Paraffin and semi-thin sections of anthers were stained with toluidine blue and PAS. Anthers were tetrasporangiate. The wall of the anther consists of an epidermis, endothecium, middle layer and glandular tapetum. During one nucleated microspore and mature pollen phase microspores and tapetum cells began to degenerate and they were become very rich of RNA in L. aestivum. And also RNA content was increased in endothecium and middle layer cells except the epidermis cells of anther wall. An increase in RNA content indicates cell activation. Polysaccharides were not seen in young anther wall but they were seen in older ones. They were generally condensed in the cell walls and especially in the cell walls of vascular bundles of connective tissue. This could be thought that insoluble polysaccharides were used in metabolic events in early developmental stages. Appearance of polysaccharides in late phases was indicated that polysaccharides were used in the formation of cuticule and differentiation of endothelium cell walls.

Pollen development of Cardiocrinum giganteum (Wall.) Makina in China

In this article, we studied the pollen morphology and wall development, microsporogenesis, male gametophyte development, and anther wall structure changes during pollen development of Cardiocrinum giganteum (Wall.) Makina from the genus Cardiocrinum (Endl.) Lindl. (Liliaceae) using paraffin sections, scanning and transmission electron microscopy, and fluorescence microscopy. The results showed that C. giganteum has oval-shaped pollen with a single sulcus and reticulate ornamentation. The exine is of the semi-tectum type and can be divided into the tectum layer, columellate layer and basal layer. Meiosis in the microsporocyte is accompanied by successive cytokinesis. The mature pollen is three-celled. The anther wall prior to maturity is built by one layer of epidermis, 1–2 layers of endothecium cells, 4–5 middle layers and 2 layers of tapetum, while upon maturity it is only built by one layer of epidermis, one layer of endothecium cells and one middle layer. The tapetal cells are secretory, with two or more nuclei. Ubisch bodies originate from rough endoplasmic reticulum except a few from mitochondria.