Tapetal Cells Research Articles

The Post-meiotic Deficicent Anther1 (PDA1) gene is required for post-meiotic anther development in rice

To understand the molecular mechanism of male reproductive development in the model crop rice, we isolated a complete male sterile mutant post-meiotic deficient anther1 (pda1) from a gamma-ray-treated rice mutant library. Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene. The pda1 mutant anther seemed smaller with white appearance. Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis. However, the pda1 mutant displayed obvious defects in postmeiotic tapetal development, abnormal degeneration occurred in the tapetal cells at stage 9 of anther development. Also we observed abnormal lipidic Ubisch bodies from the tapetal layer of the pda1 mutant, causing no obvious pollen exine formation. RT-PCR analysis indicated that the expression of genes involved in anther development including GAMYB, OsC4 and Wax-deficient anther1 (WDA1) was greatly reduced in the pda1 mutant anther. Using map-based cloning approach, the PDA1 gene was finely mapped between two markers HLF610 and HLF627 on chromosome 6 using 3,883 individuals of F(2) population. The physical distance between HLF610 and HLF627 was about 194 kb. This work suggests that PDA1 is required for post-meiotic tapetal development and pollen/microspore formation in rice.

The Relationship between Tapetum Cells and Microspores Based on Protein Localization inFraxinus angustifolia(Oleaceae) Pollen Grains

This study contributes to the knowledge of the traffic of substances between tapetum cells and microspores through the localization of pollen proteins during microsporogenesis, by using immunocytochemical techniques with TEM. Because the Oleaceae genera, including Olea and Fraxinus, share a very similar protein profile, an Ole e 1–like protein (which cross‐reacts with the Olea europaea major allergen Ole e 1) was detected in Fraxinus angustifolia pollen grains. From the tetrad to mature–pollen grain stages, distinct labeling intensities were localized in the tapetal cells, anther locule, and orbicules as well as in the microspore and pollen cytoplasms and walls. The localization of this protein in the glycocalyx proves the special role of this layer in ectexine development. At exine deposition stages, the detection of immunogold particles in the anther locule and orbicules can be interpreted as showing an active transfer of these proteins from the tapetum cells to microspores. Moreover, at the bicellular stage, the diffusion of proteins from the pollen cytoplasm to the intine channels indicates their contribution to intine formation. These findings provide new evidence of the role of sporophytic and gametophytic tissues in pollen grain development. Moreover, the Ole e 1–like protein may have an important role in modifying pollen grain walls.

Down-regulation of the OsPDCD5 gene induced photoperiod-sensitive male sterility in rice

Programmed cell death (PCD) is a crucial process for plants during development and environmental stress. OsPDCD5, is an ortholog to mammalian programmed cell death 5 gene. Here we report that decreased expression of OsPDCD5 caused by antisense technology could induce pollen sterility in photoperiod-sensitive rice. The male sterility of transgenic plants is reversible, determined mainly by photoperiod. Transgenic plants are sterile under long-day photoperiod (≥13.5 h sunlight) with postponed heading time, while they can restore fertility under short-day photoperiod (below 13 h sunlight). Analysis of tissue sections of anthers displays the tapetum cells of the anther wall retard PCD process in transgenic plants under long-day photoperiod. The retarded PCD was also confirmed by DNA fragmentation. In F 1 hybrids made from transgenic plants with antisense- OsPDCD5 and japonica rice varieties, lower transcript inhibition could restore fertility, under certain photoperiod and temperatures. The surprising discovery of the photosensitive male fertility suggested that by using specific photoperiods, the male sterility (PGMS) could be used for commercial production of hybrid rice using a two-line breeding system. The transgenic hybrid strategy was easy to apply, reduced costs and shortened the breeding period. It provides great advantages for commercial applications in rice and other crops.

RA68 is required for postmeiotic pollen development in Oryza sativa

Postmeiotic development is a unique characteristic of flowering plants. During the development, microspores undergo two cycles of mitosis (PMI and PMII) and a subsequent maturation process to finally produce the mature pollen, but the mechanism underlying the development is still largely unknown. Here, we report on the roles of a novel gene, RA68, in postmeiotic pollen development in Oryza sativa. RA68 was expressed preferentially in shoots and flowers. In flowers, the transcript persisted from the floral organ differentiation to the mature pollen stages and showed preferential accumulation in male meiocytes, developing pollen and tapetal cells. RA68-deficient RNAi lines showed reduced seed setting and pollen viability but not an aberrant phenotype in vegetative organs. Knockdown of RA68 led to arrested PMI, smaller pollen grains with little or no starch, and aborted pollen but not severely distruped male meiosis. Additionally, no abnormality of anther wall development was observed in RA68-RNAi lines. RA68 may be required for postmeiotic pollen development by affecting PMI and starch accumulation.

A separation defect of tapetum cells and microspore mother cells results in male sterility in Brassica napus: the role of abscisic acid in early anther development

Male sterility is an important contributor to heterosis in Brassica napus L. The B. napus line 7-7365ABC is a recessive epistatic genic male sterile (REGMS) three-line system. The 7-7365A line with the genotype Bnms3ms3ms4ms4RfRf is male-sterile, while the 7-7365B line with the genotype BnMs3ms3ms4ms4RfRf is male-fertile, and 7-7365C with homozygous recessive genotypes at the three loci shows male fertility because the loss function of Bnrf gene causes the inhibition of the genetic trait of the double mutant Bnms3 Bnms4. Histological studies addressing male sterility, transcriptional regulation pathways and the role of abscisic acid (ABA) in the anther development of REGMS plants are reported here. In the male-sterile line 7-7365A, tapetum cell and microspore mother cell separation were affected, and this led to failure of microspore release. The activity of polygalacturonase and the expression of the pectin methylesterase gene (AT3g06830) were significantly downregulated. Nine genes were downregulated in 7-7365A compared to 7-7365B and 7-7365C, including genes specifically expressed in tapetum (A3, A9, MS1) and the ABA-responsive gene KIN1. ABA concentration in 7-7365B was significantly higher than in 7-7365A and 7-7365C in young flower buds. Furthermore, temperature treatment made some sterile 7-7365A flowers become fertile. The stamens in these flowers produced viable pollen, and filament elongation was restored to its level in 7-7365C. We propose that ABA might control the expression of genes involved in cell separation during early anther development. The REGMS phenotype could be controlled by a primary pathway of male sterile metabolism positively regulated by the BnMs3 gene and a supplementary pathway negatively regulated by the BnRf gene.

Use of dihaploids in the breeding of Solanum tuberosum L. I. Cytological considerations

Chiasma frequency in 21 clones derived from crosses between Solanum tuberosum L. dihaploids or hybrid dihaploids and diploid species was estimated at diakinesis. The most common configurations at this stage were cross-, rod- and ring-shaped bivalents. The first two were considered to have one chiasma, the third one two chiasmata. Bivalents with three chiasmata were observed only at early diakinesis. The statistical analysis for chiasma frequency, carried out as a three level hierarchic analysis of variance with unequal sample sizes, showed that neither the variation among mothers nor that among fathers within mothers were statistically significant. The variation among clones within fathers was significant at the 0.001 level. Discussion on genomic relationships between S. tuberosum dihaploids and Solanum diploid and hexaploid species was based on the analysis of variance as well as on the occurrence of univalents, multivalents and laggards. Crossability and meiotic behaviour of the material studied may depend on genes controlling self- and cross-incompatibility, pairing and chiasma formation rather than on differences in the chromosomal structure of the genomes. The production of unreduced gametes was related to asynapsis and to nuclear restitution at AII. Endomitosis in tapetal cells was also recorded and mention was made of the similarity between chromosomes from asynaptic PMC's and those from endomitotic tapetum cells, especially when both have reached their maximal contraction. Chromosomes at endoprophase showed a quite sharp differential staining density pattern which may be useful for the identification of individual chromosomes. The origin of the common potato as well as the use of S. tuberosum dihaploids in the utilization of desirable genes present in some Solanum diploid and hexaploid species were discussed.

Reproductive biology and cytology of Hypericum brasiliense Choisy (Hypericaceae)

This is the first study of reproductive biology and cytology carried out with Hypericum brasiliense, a species with medicinal properties and potential agronomic interest. Three populations of H. brasiliense collected at Southeastern Brazil were studied. The results indicate that H. brasiliense is preferentially allogamous, self-compatible, facultative apomitic and anemophilous. Male sterility was observed in about 50% of individuals from the three populations. Anatomical studies evidenced structural abnormalities in anthers of male sterile flowers, showing enlarged tapetal cells and thick secretion deposits on the tapetal cell surfaces that may cause nutritional deficit for pollen mother cells. In cytogenetic studies several haploid chromosome numbers were observed like n = 4, 8, 9, 11, 16 and 17, including the presence of multivalents and micronuclei in tetrads, indicating the occurrence of abnormalities in the meiotic process of H. brasiliense. Despite these meiotic abnormalities the pollen viability and in vitro pollen germination rate observed in fertile flowers may be considered high. The diploid chromosome number 2n = 16 was observed, and the chromosomes in metaphase were small and similar. Fluorochrome staining techniques using DAPI and CMA3 were applied, with no positive bands observed.

인삼 융단조직의 프로그램 세포사에 관한 미세구조적 연구

Tapetum is the tissue in which nutrients are supplied to the developing microspore in angiosperm anther. At tetrad stage of microspore, the tapetal cells show maximum development, but they began to be degenerated by apoptotic programmed cell death (PCD) after sporopollenin accumulation in the pollen wall. The initial step of PCD was observed as vacuolar fusion. After that, cytoplasmic condensation and nuclear fragmentation followed. Lipid droplets are degenerated at a relatively late stage of PCD, and orbicular bodies are the last remains in tapetal cells. The cell wall was relatively resistant against vacuolar enzymes in tapetal cells; it was considered the last structure remaining during programmed cell death of tapetum in ginseng anther.

Cytological and ultra-structural study on microsporogenesis of cytoplasmic male sterility in Raphanus sativus

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.

Tissue-specific expression in transgenic rice and Arabidopsis thaliana plants of GUS gene driven by the 5′ regulatory sequences of an anther specific rice gene YY2

The identification and characterization of promoters, cis-elements and transcription factors are critical for studying gene expression during growth and development in any organism. Use of specific promoters is an absolute requirement for the expression of foreign genes in plants in a developmentally, spatially and/or temporally regulated manner. The YY2 cDNA has been shown to be expressed specifically in the tapetum cells of rice anther. In this study, a 968 bp upstream regulatory sequence (GenBank accession no. FJ957881) of the gene was isolated using genome walking method. The isolated fragment was shown to drive anther-specific expression of GUS reporter gene in rice as well as in the dicot plant Arabidopsis thaliana. Deletion analysis revealed that a ∼400 bp 5′ regulatory region is sufficient to confer anther-specific expression of GUS gene.

Analysis of gene expression profile in pollen development of recessive genic male sterile Brassica napus L. line S45A

Male sterility in a near-isogenic line S45AB after 25 generations of subcrossing is controlled by two pairs of duplicate genes. The genotype of S45A is Bnms1Bnms1Bnms2Bnms2, and that of S45B is BnMs1Bnms1Bnms2Bnms2, respectively. Histological observations revealed that abnormal anther development appeared in the tapetum and pollen exine during the tetrad stage. This male sterility was characterized by hypertrophy of the tapetal cells at the tetrad stage and a complete lack of microspore exine after the release of microspores from the tetrads. To elucidate the mechanism of this recessive genic male sterility, the flower bud expression profiles of the S45A and S45B lines were analyzed using an Arabidopsis thaliana ATH1 oligonucleotide array. When compared with the S45B line, 69 genes were significantly downregulated, and 46 genes were significantly upregulated in the S45A line. Real-time polymerase chain reaction (PCR) was then used to verify the results of the microarray analysis, and the majority of the downregulated genes in the S45A line were abundantly and specifically expressed in the anther. The results of the real-time PCR suggest that Bnms1 might be involved in the metabolism of lipid/fatty acids, and the homologous mutation of Bnms1 may either block the biosynthesis of sporopollenin or block sporopollenin from being deposited on the microspore surface, thus, preventing pollen exine formation. The role of Bnms1 in the regulatory network of exine formation is also discussed as well.

Disintegration of microsporocytes in a male sterile mutant of Brassica napus L. is possibly associated with endoplasmic reticulum-dependent autophagic programmed cell death

The mechanism of anther abortion in a male sterile (ms) line (Longyou 9S) of Brassica napus L. was evaluated by a combination of light and electron microscopies. Light microscopy showed that the tapetal cells of the ms line appeared smaller than those of its fertile line, sporogenous cells were rich in vacuoles, and pollen mother cells dismantled before the tetrad stage. Electron microscopy demonstrated that sporogenous cells were also rich in very long, plate-shaped endoplasmic reticulum cisternae that actively encircled portions of cytoplasm with organelles. In later stages, many large membrane-bound inclusions were observed in the sporogenous cells. These membrane-bound inclusions were lined by single or multiple layer(s) of the endoplasmic reticulum membranes and contained vacuoles, ribosome, plastid, mitochondria, small membrane-bound inclusion, and/or intact nuclei. In meiotic interphase and early meiosis prophase I, many vesicle aggregations and multivesicular bodies containing numerous vesicles appeared in pollen mother cells. More interestingly, some of the multivesicular bodies lay in deep cytoplasm or near cell wall, and some appeared fused with plasmalemma and released the inner vesicles out of plasmalemma. Such released vesicles gradually dispersed and later disappeared. The observations suggest that active endoplasmic reticulum-dependent autophagic programmed cell death and multivesicular body-dependent polar vesicle trafficking are probably present in the studied male sterile line.

Morphological and cytological study in a new type of cytoplasmic male-sterile line CMS-GIG2 in sunflower (Helianthus annuus)

With 3 figures and 1 table Cytoplasmic male sterility (CMS) is essential for sunflower hybrid production. CMS-GIG2, a new sunflower CMS type, was further confirmed by crossing with the maintainer and restorer lines for the CMS-PET1, both of which maintain the male sterility of CMS-GIG2. Meiotic division in CMS-GIG2 was observed with 4′6-diamidino-2-phenylindole dihydrochloride staining, indicating that microspore formation was disrupted before the meiotic cytokinesis was completed. Light microscopy observation showed that both middle layer and tapetal cells expand radially rather than degrade over time, followed by failure to form tetrads and normal microspores. This morphological defect leading to male sterility in CMS-GIG2 differs from that observed in the PET1 CMS type. CMS-GIG2 will certainly provide additional genetic diversity for sunflower hybrid breeding programmes.

Gibberellin Modulates Anther Development in Rice via the Transcriptional Regulation of GAMYB

Gibberellins (GAs) play important roles in regulating reproductive development, especially anther development. Our previous studies revealed that the MYB transcriptional factor GAMYB, an important component of GA signaling in cereal aleurone cells, is also important for anther development. Here, we examined the physiological functions of GA during anther development through phenotypic analyses of rice (Oryza sativa) GA-deficient, GA-insensitive, and gamyb mutants. The mutants exhibited common defects in programmed cell death (PCD) of tapetal cells and formation of exine and Ubisch bodies. Microarray analysis using anther RNAs of these mutants revealed that rice GAMYB is involved in almost all instances of GA-regulated gene expression in anthers. Among the GA-regulated genes, we focused on two lipid metabolic genes, a cytochrome P450 hydroxylase CYP703A3 and beta-ketoacyl reductase, both of which might be involved in providing a substrate for exine and Ubisch body. GAMYB specifically interacted with GAMYB binding motifs in the promoter regions in vitro, and mutation of these motifs in promoter-beta-glucuronidase (GUS) transformants caused reduced GUS expression in anthers. Furthermore, a knockout mutant for CYP703A3 showed gamyb-like defects in exine and Ubisch body formation. Together, these results suggest that GA regulates exine formation and the PCD of tapetal cells and that direct activation of CYP703A3 by GAMYB is key to exine formation.

Calcium Distribution in Developing Anthers of Lettuce (Lactuca sativa)

Potassium antimonite was used to locate calcium in the anthers of lettuce (Lactuca sativa). There are no calcium precipitates in young anthers. After meiosis of microspore mother cells, calcium precipitates first appear in the tapetal cells, from which some small secretive vesicles containing many calcium precipitates are secreted into the locule. At a late stage of the microspore, tapetal cells completely degenerate and their protoplasts move into the locule with many calcium precipitates. The calcium precipitates increase in the early microspores, and in the exine. When the microspores form some small vacuoles containing calcium precipitates, and those vacuoles then fuse to form a large one, the calcium precipitates evidently decrease. The large vacuole of bicellular pollen grain discomposes and calcium precipitates again appear in the cytoplasm and then decrease. When the pollen matures, most calcium precipitates are located in its exine with only a few in the cytoplasm.

A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines

BAHD acyltransferases catalyze the acylation of many plant secondary metabolites. We characterized the function of At2g19070, a member of the BAHD gene family of Arabidopsis thaliana. The acyltransferase gene was shown to be specifically expressed in anther tapetum cells in the early stages of flower development. The impact of gene repression was studied in RNAi plants and in a knockout (KO) mutant line. Immunoblotting with a specific antiserum raised against the recombinant protein was used to evaluate the accumulation of At2g19070 gene product in flowers of various Arabidopsis genotypes including the KO and RNAi lines, the male sterile mutant ms1 and transformants overexpressing the acyltransferase gene. Metabolic profiling of flower bud tissues from these genetic backgrounds demonstrated a positive correlation between the accumulation of acyltransferase protein and the quantities of metabolites that were putatively identified by tandem mass spectrometry as N(1),N(5),N(10)-trihydroxyferuloyl spermidine and N(1),N(5)-dihydroxyferuloyl-N(10)-sinapoyl spermidine. These products, deposited in pollen coat, can be readily extracted by pollen wash and were shown to be responsible for pollen autofluorescence. The activity of the recombinant enzyme produced in bacteria was assayed with various hydroxycinnamoyl-CoA esters and polyamines as donor and acceptor substrates, respectively. Feruloyl-CoA and spermidine proved the best substrates, and the enzyme has therefore been named spermidine hydroxycinnamoyl transferase (SHT). A methyltransferase gene (At1g67990) which co-regulated with SHT during flower development, was shown to be involved in the O-methylation of spermidine conjugates by analyzing the consequences of its repression in RNAi plants and by characterizing the methylation activity of the recombinant enzyme.

A Novel Fatty Acyl-CoA Synthetase Is Required for Pollen Development and Sporopollenin Biosynthesis inArabidopsis

Acyl-CoA Synthetase (ACOS) genes are related to 4-coumarate:CoA ligase (4CL) but have distinct functions. The Arabidopsis thaliana ACOS5 protein is in clade A of Arabidopsis ACOS proteins, the clade most closely related to 4CL proteins. This clade contains putative nonperoxisomal ACOS enzymes conserved in several angiosperm lineages and in the moss Physcomitrella patens. Although its function is unknown, ACOS5 is preferentially expressed in the flowers of all angiosperms examined. Here, we show that an acos5 mutant produced no pollen in mature anthers and no seeds by self-fertilization and was severely compromised in pollen wall formation apparently lacking sporopollenin or exine. The phenotype was first evident at stage 8 of anther development and correlated with maximum ACOS5 mRNA accumulation in tapetal cells at stages 7 to 8. Green fluorescent protein-ACOS5 fusions showed that ACOS5 is located in the cytoplasm. Recombinant ACOS5 enzyme was active against oleic acid, allowing kinetic constants for ACOS5 substrates to be established. Substrate competition assays indicated broad in vitro preference of the enzyme for medium-chain fatty acids. We propose that ACOS5 encodes an enzyme that participates in a conserved and ancient biochemical pathway required for sporopollenin monomer biosynthesis that may also include the Arabidopsis CYP703A2 and MS2 enzymes.

Scanning electron microscopy of nuclear division stages in Vicia faba and Haemanthus cinnabarinus

Fixation of developing anthers of Vicia faba and Haemanthus cinnabarinus in acetic-ethanol 1:1 and subsequent isolation of pollen mother cells and tapetal cells made it possible to obtain nuclear division stages free from cytoplasm by heating in 45% acetic acid. After transfer through equal parts of ethanol and glacial acetic acid and further to pure ethanol the specimens were dried from liquid CO2, by the critical point method, They were then coated with gold or gold-palladium in a vacuum evaporator and examined in a scanning electron microscope. The preparations permit detailed studies of the threedimensional arrangements and shapes of chromosomes during nuclear divisions.

The GhACS1 gene encodes an acyl‐CoA synthetase which is essential for normal microsporogenesis in early anther development of cotton

Microsporogenesis, associated with the functional expression of many genes, is a highly programmed and regulated process in flowering plants. To elucidate the roles of genes during anther development, two anther-specific cDNAs (designated GhACS1 and GhACS2) encoding acyl-CoA synthetases (ACSs) were isolated from a cotton (Gossypium hirsutum) flower cDNA library. Subsequently, the corresponding GhACS1 gene was isolated from a cotton genomic DNA library. Real-time quantitative RT-PCR and northern blot analyses revealed that GhACS1 transcripts were predominantly accumulated in the developing anthers of cotton. The specificity of GhACS1 expression in primary sporogenous cells (PSCs), pollen mother cells (PMCs), microspores, and tapetal cells was demonstrated by in situ hybridization as well as histochemical assay of GUS expression controlled under the GhACS1 promoter. High levels of GhACS1 activity are crucial for fatty acid metabolism in PSCs, PMCs, microspores and particularly tapetal cells. Reduction of ACS enzymatic activity by suppressing GhACS1 expression severely affected the tapetal cells and consequently blocked normal microsporogenesis in early anther development. Aberrant and defective microspores were generated in the transgenic anthers. As a result, the transgenic plants failed to produce functional pollen grains and were male-sterile, suggesting that the GhACS1 gene is required for normal microsporogenesis in early anther development of cotton.

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility.