Tapetal Cells Research Articles

Compositional Analysis of Grain and Forage from MON 87427, an Inducible Male Sterile and Tissue Selective Glyphosate-Tolerant Maize Product for Hybrid Seed Production

Conventional maize hybrid seed production has historically relied upon detasseling using either manual methods or semiautomated processes to ensure the purity of the hybrid cross. Monsanto Co. has developed biotechnology-derived MON 87427 maize with tissue-selective glyphosate tolerance to facilitate the production of hybrid maize seed. MON 87427 utilizes a specific promoter and intron combination to drive expression of CP4 EPSPS protein in vegetative and female reproductive tissues, conferring tolerance to glyphosate. This specific combination of regulatory elements also results in limited or no production of CP4 EPSPS protein in two key male reproductive tissues: pollen microspores, which develop into pollen grains, and tapetum cells that supply nutrients to the pollen. Thus, MON 87427 induces a male sterile phenotype after appropriately timed glyphosate applications. To confer additional benefits of herbicide tolerance and/or insect resistance, MON 87427 was combined with MON 89034 and NK603 by conventional breeding to develop MON 87427 × MON 89034 × NK603. The work described here is an assessment of the nutrient, antinutrient, and secondary metabolite levels in grain and forage tissues of MON 87427 and MON 87427 × MON 89034 × NK603. Results demonstrated that MON 87427 is compositionally equivalent to a near-isogenic conventional comparator. Results from this analysis established that the compositional equivalence observed for the single-event product MON 87427 is extendable to the combined-trait product, MON 87427 × MON 89034 × NK603. With increasing global demand for food production, the development of more efficient seed production strategies is important to sustainable agriculture. The study reported here demonstrated that biotechnology can be applied to simplify hybrid maize seed production without affecting crop composition.

Comparative Study on the Bisexual Flower and Unisexual Male Flower of Xanthoceras sorbifolia Bunge

Semi-thin sectioning and electron microscopy were used to observe and analyze the development processes in the bisexual flower and unisexual male flower of Xanthoceras sorbifolia Bunge. The results show that:(1) At the early microspore development stage,there is no obvious difference between these two different kinds of flowers. At the uninuclear phase of microspore development when the cell walls thicken,lip cells do not form in the bisexual flower,but develop in the unisexual male flower. At the mature microspore stage,the lip cells form completely and the anther forms a double heart-shape in the unisexual male flower. However,the anther has not broken completely and can not disperse pollen.(2) In the bisexual flower,the ovary has two rooms,the cells are closely packed,the stigmas are cylindrical with papillate cells,and there is a hollow canal inside the style. However,in the unisexual male flower,the development of stigmas cease without the formation of a hollow canal inside the style,the ovarian rooms shrink,and the embryo sac degenerates.(3) The tapetum cells of the unisexual male flower contain many protein bodies,amyloplasts,vacuoles,and Ubisch bodies,and the tapetum cells completely disappear at the later microspore development stage. However,these organelles become less abundant in tapetum cells of the bisexual flower,and the tapetum cells do not completely disappear.(4) The pollen cell wall thickens without germination apertures in the bisexual flower,whereas in the unisexual male flower there are three germination apertures on the pollen grains,and many vacuoles,protein bodies,and liposomes accumulate in the pollen grains.

Upregulation of the AT-hook DNA binding gene BoMF2 in OguCMS anthers of Brassica oleracea suggests that it encodes a transcriptional regulatory factor for anther development

Ogura cytoplasmic male sterility (OguCMS) is the most important CMS system used for F1 hybrid cabbage production worldwide. The anther abortion and defective pollen development exhibited in OguCMS are coordinately regulated by the mitochondrial male sterile gene orf138 and many nuclear transcriptional regulatory factors. AT-hook DNA binding proteins regulate cell-specific gene expression. In this study, we cloned the gene encoding the AT-hook DNA binding protein BoMF2 using the cDNA-AFLP TDF sequence, which was upregulated in OguCMS cabbage flower buds, as a querying probe. BoMF2 contains a 783-nt continuous complete open reading frame encoding a 260 amino-acid polypeptide. In vivo transient expression assays using GFP fusions showed that BoMF2 protein was located in the nucleus. BoMF2 was preferentially expressed in cabbage stamens, with a short expression window at anther development stage 7-8. However, in OguCMS flowers, BoMF2 expression continued into the mature pollen stage and was concomitant with the continued proliferation of tapetum cells exhibited in this mutant. Arabidopsis plants overexpressing BoMF2 showed significantly shorter siliques than the wild type, as well as decrease of pollen viability. These results suggest that BoMF2, a transcriptional regulatory factor, might regulate tapetum proliferation during anther development.

少花龙葵花药发育中多糖和脂滴的组织化学研究

对少花龙葵花药发育中淀粉粒和脂滴的分布和转化过程进行组织化学研究。结果显示：在造孢细胞时期幼小花药中没有营养物质积累；在小孢子母细胞时期，花药表皮和药室内壁细胞中出现淀粉粒，同时在绒毡层细胞中出现了较多的脂滴；在四分体时期，中层细胞中出现淀粉粒，而绒毡层细胞中的脂滴减少；到小孢子早期，花药表皮细胞中除淀粉粒外还出现了脂滴；但到小孢子晚期，药壁细胞中的淀粉和脂滴都明显减少；在二胞花粉早期，花粉开始积累淀粉粒，药壁细胞中的淀粉和脂滴几乎消失；在即将开花的成熟花粉中积累了大量淀粉粒和少量脂滴作为其存储物。 The distribution of polysaccharides and lipid drops in developing anther of Solanum photeinocarpum was studied. At the sporogenous cell stage, neither starches nor lipids were in anther wall cells. At the stage of microspore mother cells, some starches appeared in epidemic cells and endothecium cells and many lipids appeared in tapetal cells. At the tetrad stage, some polysaccharides appeared in middle layer cells. After microspores were released from tetrads, both starches and lipids appeared in epidemic cells, but both materials decreased at the late microspore stage. After the microspore division, early bicellular pollen began to accumulate polysaccharides, and the starches and lipids disappeared in anther wall cells. With the pollen development, many starches and some lipids were accumulated in the vegetative cell of the nearly mature pollen.

Screening of a New Chemical Hybridizing Agent in Wheat

To expand varieties of chemical hybridization agent and reduce their cost, We selected 15 varieties of chemical hybridization agent for treating wheat cultivar Xi'nong 1376 at three growth stages with four concentrations to survey the infertile effect, phytotoxicity and pollen abortion mechanism. The results showed that there were significant differences among the chemical hybridization agent treatments. At stages Feekes 7.5 and Feekes 9.5, abnormal vegetative growth or pollens abortion were not obvious. While at the stage of Feekes 8.5, the rate of male sterility of Xinong 1376 was 93.33% in 0.24 kg ha–1 T6 treatment, and the pistil and the plant showed normal development, we also achieved a high seed setting rate via crossing the male sterile plants with normal fertile lines by saturate pollination. Paraffin sections indicated that the degradable process of tapetum cells in the male sterile anther treated by T6-CHA was abnormal. The nucleus of tapetum cells degraded at uninucleate early stage, and then pollen nucleus degraded at uninucleate late stage. Tapetum cells, nucleus of pollen and nutrients were almost disappeared till trinucleate stage. Finally, a little residue of pollen wall left and pollen aborted. Therefore, we hypothesize that the male sterility induced by T6-CHA could be related to the degradation of tapetum cells in anther.

Some cytological and ultrastructural aspects of male sterility in Impatiens

Some cytological and ultrastructural aspects of tapetum and sporogenic cells, in both male-fertile and male-sterile <em>Impatiens</em>, were investigated and compared in order to find the cause of cytoplasmic male sterility.

Some cytological, ecological and evolutionary aspects of pollination

Different types of pollen dispersing units are described. The pollen of angiosperms may be dispersed in monads, tetrads, polyads, massulae or compact pollinia. The monads and tetrads may form larger clumps of pollen with the aid of the following substances: a) pollenkitt, a product of <em>in situ</em> degeneration of the tapetum; b) tryphine, a product of tapetal extra situm degeneration, found only in <em>Cruciferae</em>; c) elastoviscin, a highly viscous product of the degeneration of a limited number of tapetal cells, found only in certain <em>Orchidaceae</em>; d) viscin threads or sporopollenin filaments arising from the pollen exine. The number of pollen grains composing the clumps depends on pollen diameter and the cohesive forces holding the pollen grains together. The various types of pollen dispersing unit are discussed in relation to the type of pollination and number of ovules per ovary.

The metabolic incorporation of label from myoinositol-2-3H by the growing young anther of Lilium lorlgiflorum

Fine structural autoradiography involving the incorporation of myo-inositol-2-<sup>3</sup>H (MI-2-<sup>3</sup>H) unto lily anther during development has been used to explore the metabolic processes of microspore development and the interation between the devlaping microspore and degenerating tapetal cells.

The function of the tapetal tissue during microsporogenesis in Lilium

The main functional activity -of the tapetum in the <em>Lilium</em> anther is the synthesis of reserve lipids and carotenoid pigments. The fusion of these substances during tapetum desintegration results in the formation of pollenkitt Pollernkitt participates in the formation both of the exine and of sporopollenin-containing structures of the tapetal cell (orbicules, tapetal and peritapetal membranes) during the last steps of anther development.

Origin and structure of Ubisch bodies in Pinus sylvestris

In <em>Pinus sylvestris</em> Ubisch bodies are produced repeatedly, and each crop is formed at a distinct phase in the secretory cycles of tapetal cells. While each production has a Ubisch body wall similar to the then current state of the exine with regard to thickening and ornamentation, the survivers of previous productions do not change. Examples of all the structurally different Ubisch body wall forms can be seen when terminally, at the time of pollen shedding, the relict Ubisch bodies become spatially concentrated on the minimal surface area of the senescent cells of the tapetum. In angiosperms after one or a few periods of initiation Ubisch bodies may remain in association with the surface of tapetal cells where the Ubisch body wall undergoes changes like those of the maturing pollen exine. We conclude that as a consequence of Ubisch body detachment from the plasma membrane of tapetal cells there is in <em>Pinus sylvestris</em> no updated information for modification of the wall and the Ubisch body wall remains static.

A novel method to follow meiotic progression in Arabidopsis using confocal microscopy and 5-ethynyl-2'-deoxyuridine labeling.

BackgroundMeiosis progression in the more recent past has been investigated using 5-bromo-2′-deoxyuridine (BrdU) uptake by S-phase meiocytes undergoing DNA replication. BrdU uptake is detected by reaction with BrdU antibody followed by epifluorescent microscopy examination of chromosome spreads and/or squashes. We here report using confocal microscopic examination of intact meiocytes in conjunction with the new thymidine analog 5-ethynyl-2′-deoxyuridine (EdU). The simplicity of the EdU detection coupled with confocal examination of anthers provides a more exact temporal description of meiotic prophase I progression in Arabidopsis and opens up the possibility of examining the coordination of microsporocyte development with the other tissues of the anther.ResultsUsing our time course protocol, we have determined the duration of wild type Arabidopsis leptotene to be 5 h, zygotene -6 h, pachytene -10 h and a diplotene duration of approximately 1 h. We estimate G2 duration to be approximately 7 h based on the timing of the initial appearance of EdU signal in early leptotene meiocytes. In addition we have found that DNA replication in meiocytes is not done synchronously with the associated tapetal layer of cells. The EdU labeling suggests that S-phase replication of meiocyte DNA precedes the duplication of tapetal cell DNA.ConclusionsThe increased number of meiotic staging criteria that can be assessed in our confocal analysis, as compared to chromosome spreading or squashing, makes the identification of even the early and late portions of the prophase I substages attainable. This enhanced staging coupled with the ability to easily generate large data sets at hourly time points makes it possible to more exactly determine substage duration and to detect modest temporal abnormalities involving meiocyte entrance into and/or exit from leptotene, zygotene and pachytene. Confocal analysis also makes it possible to study the relationships between different cell types within the flower bud as meiosis proceeds.Electronic supplementary materialThe online version of this article (doi:10.1186/1746-4811-10-33) contains supplementary material, which is available to authorized users.

Cytological and Comparative Proteomic Analyses on Male Sterility in Brassica napus L. Induced by the Chemical Hybridization Agent Monosulphuron Ester Sodium

Male sterility induced by a chemical hybridization agent (CHA) is an important tool for utilizing crop heterosis. Monosulphuron ester sodium (MES), a new acetolactate synthase-inhibitor herbicide belonging to the sulphonylurea family, has been developed as an effective CHA to induce male sterility in rapeseed (Brassica napus L.). To understand MES-induced male sterility in rapeseed better, comparative cytological and proteomic analyses were conducted in this study. Cytological analysis indicated that defective tapetal cells and abnormal microspores were gradually generated in the developing anthers of MES-treated plants at various development stages, resulting in unviable microspores and male sterility. A total of 141 differentially expressed proteins between the MES-treated and control plants were revealed, and 131 of them were further identified by MALDI-TOF/TOF MS. Most of these proteins decreased in abundance in tissues of MES-treated rapeseed plants, and only a few increased. Notably, some proteins were absent or induced in developing anthers after MES treatment. These proteins were involved in several processes that may be crucial for tapetum and microspore development. Down-regulation of these proteins may disrupt the coordination of developmental and metabolic processes, resulting in defective tapetum and abnormal microspores that lead to male sterility in MES-treated plants. Accordingly, a simple model of CHA-MES-induced male sterility in rapeseed was established. This study is the first cytological and dynamic proteomic investigation on CHA-MES-induced male sterility in rapeseed, and the results provide new insights into the molecular events of male sterility.

Characterization and inheritance of a novel thermo-sensitive restoration of cytoplasmic male sterility in Capsicum annuum

Thermo-sensitive male sterility can be used for two-line hybrid production, if the fertility can be manipulated by temperature shifting. In this study, we investigated a novel thermo-sensitive cytoplasmic male-sterile (TCMS) line of pepper. The TCMS line was sterile at temperatures above 15°C but their fertility was restored when night temperatures were below 13°C. To elucidate the cellular mechanism acting in the TCMS line, histological analysis was performed using light microscopy and transmission electron microscopy (TEM). We demonstrated that stable male sterility in pepper might be induced due to the abnormal development of tapetal cells whereas defects of callose wall biosynthesis seemed to be the main reason for male sterility in TCMS plants. Meiotic cell division in sterile TCMS plants proceeded to the last stage, whereas normal sterile plants were arrested at the meiotic stage. Further analysis using TEM revealed that abnormal development and failure of timely programmed cell death of the tapetum led to secretory dysfunction of tapetal cells. Consequently, this secretory defect may have influenced callose wall deposition and pollen wall formation in sterile TCMS. To reveal genetic mechanisms influencing TCMS, inheritance and marker analysis was performed. Cytoplasmic male sterility (CMS)-associated marker tests showed that the TCMS line contains CMS cytoplasm. Inheritance patterns and Rf-linked marker analysis suggested that TCMS is controlled by an allele named RfTCMS located at the Rf locus. Restoring experiment at low temperature showed that the thermo-sensitive restorer, RfTCMS, may be another allele located on the Rf locus and is recessive to Rf and dominant to rf.

Classical macroautophagy in Lobivia rauschii (Cactaceae) and possible plastidial autophagy in Tillandsia albida (Bromeliaceae) tapetum cells

The tapetum in anthers is a tissue that undergoes programmed cell death (PCD) during the production of pollen. We observed two types of autophagy prior to cell death. In Lobivia rauschii (Cactaceae), tapetum cells showed plant-type autophagosomes-autolysosomes, which have been found previously exclusively in root meristem cells. The autophagic structures were formed by a network of tubules which apparently merged laterally, thereby sequestering a portion of the cytoplasm. The organelles observed in the sequestered material included multilamellar bodies, which have not been reported earlier in these organelles. By contrast, Tillandsia albida (Bromeliaceae) tapetum cells contained no such organelles but showed plastids that might possibly carry out autophagy, as they contained portions of the cytoplasm similar to the phenomenon reported earlier in Phaseolus and Dendrobium. However, the ultrastructure of the T. albida plastids was different from that in the previous reports. It is concluded that in L. rauschii classical plant macroautophagy was involved in degradation of the cytoplasm, while in T. albida such classical macroautophagy was not observed. Instead, the data in T. albida suggested the hypothesis that plastids are able to carry out degradation of the cytoplasm.

Epigenetic Changes Accompany Developmental Programmed Cell Death in Tapetum Cells

The tapetum, the nursing tissue inside anthers, undergoes cellular degradation by programmed cell death (PCD) during late stages of microspore-early pollen development. Despite the key function of tapetum, little is known about the molecular mechanisms regulating this cell death process in which profound nuclear and chromatin changes occur. Epigenetic features (DNA methylation and histone modifications) have been revealed as hallmarks that establish the functional status of chromatin domains, but no evidence on the epigenetic regulation of PCD has been reported. DNA methylation is accomplished by DNA methyltransferases, among which DNA methyl transferase 1 (MET1) constitutes one of the CG maintenance methyltransferase in plants, also showing de novo methyltransferase activity. In this work, the changes in epigenetic marks during the PCD of tapetal cells have been investigated by a multidisciplinary approach to reveal the dynamics of DNA methylation and the pattern of expression of MET1 in relation to the main cellular changes of this PCD process which have also been characterized in two species, Brassica napus and Nicotiana tabacum. The results showed that tapetum PCD progresses with the increase in global DNA methylation and MET1 expression, epigenetic changes that accompanied the reorganization of the nuclear architecture and a high chromatin condensation, activity of caspase 3-like proteases and Cyt c release. The reported data indicate a relationship between the PCD process and the DNA methylation dynamics and MET1 expression in tapetal cells, suggesting a possible new role for the epigenetic marks in the nuclear events occurring during this cell death process and providing new insights into the epigenetic control of plant PCD.

Regulation of cell divisions and differentiation by MALE STERILITY32 is required for anther development in maize

Male fertility in flowering plants relies on proper division and differentiation of cells in the anther, a process that gives rise to four somatic layers surrounding central germinal cells. The maize gene male sterility32 (ms32) encodes a basic helix-loop-helix (bHLH) transcription factor, which functions as an important regulator of both division and differentiation during anther development. After the four somatic cell layers are generated properly through successive periclinal divisions, in the ms32 mutant, tapetal precursor cells fail to differentiate, and, instead, undergo additional periclinal divisions to form extra layers of cells. These cells become vacuolated and expand, and lead to failure in pollen mother cell development. ms32 expression is specific to the pre-meiotic anthers and is distributed initially broadly in the four lobes, but as the anther develops, its expression becomes restricted to the innermost somatic layer, the tapetum. The ms32-ref mac1-1 double mutant is unable to form tapetal precursors and also exhibits excessive somatic proliferation leading to numerous, disorganized cell layers, suggesting a synergistic interaction between ms32 and mac1. Altogether, our results show that MS32 is a major regulator in maize anther development that promotes tapetum differentiation and inhibits periclinal division once a tapetal cell is specified.

Tapetal and parenchymatic anther tissues participate in polyad adhesive production in Calliandra brevipes (Leguminosae)

Calliandra brevipes Benth. is an American shrubby species largely used for ornamental purposes. Like all other Calliandra species it has heteromorphic pollen grains shed in ellipse-shaped, calymmate polyads and a unique mode of pollen presentation by producing a sticky substance called “pollen adhesive”. The present study aimed to investigate in detail the origin of polyad adhesive in C. brevipes. Serial microtome sections of anthers in various developmental stages were used and histochemical tests applied to detect the adhesive production sites and investigate the subcellular characteristics of the anther cells under transmission electron microscopy. The pollen adhesive in C. brevipes is not only produced by parenchymatic cells of the anther transversal septum, as it is described for Calliandra angustifolia, but parenchymatic cells and tapetal cells next to the polyad apical pollen grain also participate in the pollen adhesive production. The cytoplasm of the degenerating tapetum cells contains oleoplasts and fibrogranular material inside the vacuoles which mixes with the adhesive produced by adjacent parenchymatic cells and which therefore contributes to its composition. Vacuoles containing fibrogranular material are very similar to those found in tryphine producing tapetal cells, and the subcellular structures of parenchymatic and tapetal cells are similar to each other. The fact that the pollen adhesive becomes solid in contact with the environment is attributed to dehydration and the presence of the protein fibrogranular material and lipid substances since resin could not be found in its composition. It seems that the sites of pollen adhesive production in Calliandra may vary among its members. Studies of polyad adhesive production in the genus should be standardized in order to verify the information already available in the literature.

Distribution of cellulosic wall in the anthers of Arabidopsis during microsporogenesis

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). 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.

Maize Male sterile 8 (Ms8), a putative β-1,3-galactosyltransferase, modulates cell division, expansion, and differentiation during early maize anther development

Precise somatic and reproductive cell proliferation and differentiation in anthers are crucial for male fertility. Loss of function of the Male sterile 8 (Ms8) gene causes male sterility with multiple phenotypic defects first visible in the epidermal and tapetal cells. Here, we document the cloning of Ms8, which is a putative β-1,3-galactosyltransferase. Ms8 transcript is abundant in immature anthers with a peak at the meiotic stage; RNA expression is highly correlated with protein accumulation. Co-immunoprecipitation coupled with mass spectrometry sequencing identified several MS8-associated proteins, including arabinogalactan proteins, prohibitins, and porin. We discuss the hypotheses that arabinogalactan protein might be an MS8 substrate and that MS8 might be involved in maintenance of mitochondrial integrity.

The ATP‐binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map-based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development.