Archesporial Cell Research Articles

Morphogenesis, megagametogenesis, and microgametogenesis in Actinidia arguta flower buds

In dioecious plants, understanding sexual differentiation is important for improving cultivation techniques. Flower bud morphogenesis and gametophyte formation underlie the mechanism of sexual differentiation. Actinidia arguta (hardy kiwi) is a unisexual plant, and there have been few studies on its flower bud differentiation or gametophyte formation. We studied flower bud morphogenesis and gametophyte formation in A. arguta using paraffin sections and found that the morphogenesis of male and female flower buds was divided into six stages: the undifferentiated stage, inflorescence initials differentiation stage, calyx primordium differentiation stage, petal primordium differentiation stage, stamen primordium differentiation stage, and pistil primordium differentiation stage. Morphogenesis in the male inflorescence can reach quaternary flower primordia, and morphogenesis in the female inflorescence can reach the tertiary flower primordia, however, no Stage 4 lateral flowers in the male inflorescence or Stage 3 lateral flowers in the female inflorescence were found in natural situation. The embryo sac in female plants was a monocellular Polygonum type, and the embryo sac development in male plants was incomplete; abortion occurred before the archesporial cells developed. The male gametophyte differentiation process in the male plant was normal, with pollen grains similar in shape to a rugby ball and arranged neatly in the anther. The pollen grain differentiation in female plants was abnormal; abortion occurred at the uninucleate microspore stage, and mature pollen grains were irregularly spherical.

Rank of the Subfamilies Loranthoideae and Viscoideae

The family Loranthaceae has been divided into the subfamilies Loranthoideae and Viscoideae (Engler, 1888-89 ; Danser, 1929, 1933a, b). These two subfamilies show great dissimilarity in their morphological as well as their embryological characters.In the Loranthoideae flowers may be unisexual or bisexual, but a calyculus is invariably present. The ovary may contain a lobed or unlobed mamelon, or the mamelon may be extremely reduced or absent. A collenchymatous pad or tube is invariably present in the ovary. The embryo sac is of the Polygonum type. Many embryo sacs may develop simultaneously in the same ovary and their tips grow up into the style and in some cases reach the stigma. The lower end of the sac sometimes produces a caecum. The endosperm is a composite structure formed by the fusion of all the endosperms developing in the same ovary. The first division of the zygote is always vertical and is followed by transverse divisions resulting in a long biseriate proembryo. The fruit shows 4 distinct layers and the viscid layer is situated outside the vascular bundles of the corolla.In the Viscoideae, flowers are unisexual and the calyculus is absent except in Viscum orientate and V. articulation. The central ovarian papilla has 2 or more archesporial cells, each of which divides to form two dyad cells. Of these the upper dyad cell is the larger and functions, while the lower soon degenerates. Thus the development conforms to Allium type. A peculiar feature is that after the 4-nucleate stage there is a slow but steady curvature of the embryo sac, which causes its lower end to bend out of the papilla and proceed upwards into the carpellary tissue. The egg apparatus differentiates from the quartet in the originally lower pole, which is later situated at a higher level than the upper. Generally only one embryo sac develops which is said to show inverted polarity. The endosperm is derived from the primary endosperm nucleus of a single embryo sac. The first division of the zygote is transverse except in Korthalsella. The embryo lacks a suspensor. The viscous part of the fruit is situated within the vascular bundles of the perigone.Thus, the Loranthoideae and the Viscoideae show important differences in floral structure, mode of development of the embryo sac, endosperm and embryo, and in the location of the viscid zone of the fruit. These differences adequately warrant the raising of the two subfamilies to the rank of fatnilies. As proposed by Miers (see Danser, 1929), the name Loranthaceae may be retained for the Loranthoideae, and the Viscoideae may be designated Viscaceae.

The adaptor protein ECAP, the corepressor LEUNIG, and the transcription factor BEH3 interact and regulate microsporocyte generation in Arabidopsis.

Histospecification and morphogenesis of anthers during development in Arabidopsis (Arabidopsis thaliana) are well understood. However, the regulatory mechanism of microsporocyte generation at the pre-meiotic stage remains unclear, especially how archesporial cells are specified and differentiate into 2 cell lineages with distinct developmental fates. SPOROCYTELESS (SPL) is a key reproductive gene that is activated during early anther development and remains active. In this study, we demonstrated that the EAR motif-containing adaptor protein (ECAP) interacts with the Gro/Tup1 family corepressor LEUNIG (LUG) and the BES1/BZR1 HOMOLOG3 (BEH3) transcription factor to form a transcription activator complex, epigenetically regulating SPL transcription. SPL participates in microsporocyte generation by modulating the specification of archesporial cells and the archesporial cell-derived differentiation of somatic and reproductive cell layers. This study illustrates the regulation of SPL expression by the ECAP-LUG-BEH3 complex, which is essential for the generation of microsporocytes. Moreover, our findings identified ECAP as a key transcription regulator that can combine with different partners to regulate gene expression in distinct ways, thereby facilitating diverse processes in various aspects of plant development.

Male gametogenesis in flowering plants

The life cycles of plants are characterized by significant alternations between the diploid sporophytic and the haploid gametophytic generations. Meiosis and fertilization are the prerequisites for achieving the alternation of generations. Diploid sporophytes undergo meiosis to produce haploid gametes, and male–female gametes fuse (double fertilization) to revert to the diploid sporophyte. The formation and development of male gametophytes are crucial for the alternation of generations in higher plants. During the long evolution of plants from aquatic to terrestrial, the way of sexual reproduction has undergone subversive innovations. From sperm swimming for fertilization to relying on the precise delivery of pollen tubes to female gametes for the fusion of the male–female gametes, higher requirements are placed on the male gametophytes’ development and fertilization process. The formation of male gametophytes has undergone significant changes to meet the needs for delivery and transportation of the male gametes. With the emergence of more and more evidence, the molecular mechanism underlying male gametophytes’ development, especially the initiation and specialization of germline cells has been better understood. Focusing on the latest studies, we reviewed and elucidated the critical proteins and factors involved in male gametophyte formation, highlighting the decisive role of auxin in archesporial cell specialization and the importance of microspore maturation in pre-mitosis, and analyzed the molecular mechanisms underlying male gametogenesis, with a view to providing insights for further exploration of male gametophytes formation in the future.

Development of anther and sectile pollinium in Herminium lanceum (Thunb. ex Sw.) Vuijk-a massulate orchid

Abstract The role of anther characteristics in orchid taxonomy, cladistic analysis, and understanding the development and evolution of various traits is significant. Therefore, Herminium lanceum (Thunb. ex Sw.) Vuijk was selected for the study due to the lack of information about these characteristics. In this taxon, anther primordium developed two thecae directed towards the lateral side, which produced wedge-shaped, rectangular or triangular massulae through repeated mitotic divisions of archesporial cells. The anther wall developed from the outer secondary parietal layer and inner secondary parietal layer, made up of epidermis, endothecium, a middle layer, and tapetum. Each theca had a central septum that divided it into two microsporangia. Tapetum of dual origin was observed. Cytokinesis produced isobilateral, tetrahedral, linear, T-shaped and decussate types of microspore tetrads. Each microspore had a smaller lenticular generative cell and a larger spherical vegetative cell, which were formed by the mitotic division of the nucleus. Additionally, the endothecium developed ring-shaped fibrous thickenings. Massulae of two locules were united to the reticulum and formed bipartite sectile pollinium.

3D cellular morphometrics of ovule primordium development in Zea mays reveal differential division and growth dynamics specifying megaspore mother cell singleness.

Differentiation of spore mother cells marks the somatic-to-reproductive transition in higher plants. Spore mother cells are critical for fitness because they differentiate into gametes, leading to fertilization and seed formation. The female spore mother cell is called the megaspore mother cell (MMC) and is specified in the ovule primordium. The number of MMCs varies by species and genetic background, but in most cases, only a single mature MMC enters meiosis to form the embryo sac. Multiple candidate MMC precursor cells have been identified in both rice and Arabidopsis, so variability in MMC number is likely due to conserved early morphogenetic events. In Arabidopsis, the restriction of a single MMC per ovule, or MMC singleness, is determined by ovule geometry. To look for potential conservation of MMC ontogeny and specification mechanisms, we undertook a morphogenetic description of ovule primordium growth at cellular resolution in the model crop maize. We generated a collection of 48 three-dimensional (3D) ovule primordium images for five developmental stages, annotated for 11 cell types. Quantitative analysis of ovule and cell morphological descriptors allowed the reconstruction of a plausible developmental trajectory of the MMC and its neighbors. The MMC is specified within a niche of enlarged, homogenous L2 cells, forming a pool of candidate archesporial (MMC progenitor) cells. A prevalent periclinal division of the uppermost central archesporial cell formed the apical MMC and the underlying cell, a presumptive stack cell. The MMC stopped dividing and expanded, acquiring an anisotropic, trapezoidal shape. By contrast, periclinal divisions continued in L2 neighbor cells, resulting in a single central MMC. We propose a model where anisotropic ovule growth in maize drives L2 divisions and MMC elongation, coupling ovule geometry with MMC fate.

Anther development in Brachiaria brizantha (syn. Urochloa brizantha) and perspective for microspore in vitro culture.

Brachiaria, a genus from the Poaceae family, is largely cultivated as forage in Brazil. Among the most cultivated varieties of Brachiaria spp., B. brizantha cv. Marandu (syn. Urochloa brizantha) is of great agronomical importance due to the large areas cultivated with this species. This cultivar is apomictic and tetraploid. Sexual diploid genotype is available for this species. The difference in levels of ploidy among sexual and apomictic plants contributes to hindering Brachiaria breeding programs. The induction of haploids and double haploids is of great interest for the generation of new genotypes with potential use in intraspecific crosses. A key factor for the success of this technique is identifying adequate microspore developmental stages for efficient embryogenesis induction. Knowledge of the morphological changes during microsporogenesis and microgametogenesis and sporophytic tissues composing the anther is critical for identifying the stages in which microspores present a higher potential for embryogenic callus and somatic embryo through in vitro culture. In this work, morphological markers were associated with anther and pollen grain developmental stages, through histological analysis. Anther development was divided into 11 stages using morphological and cytological characteristics, from anther with archesporial cells to anther dehiscence. The morphological characteristics of each stage are presented. In addition, the response of stage 8 anthers to in vitro culture indicates microspores initiating somatic embryogenic pathway.

Ectopic expression of a male fertility gene, LOGL8, represses LOG and hinders panicle and ovule development

Grain number and seed-setting rate are components of crop yield. Cytokinin influences grain yield. However, emerging studies suggest that high cytokinin signals often lead to reduced branching or seed-setting rate, leading to reduced grain yield, although the mechanisms remain unclear. In this study, we identified and characterized the rice (Oryza sativa L.) gene LONELY GUY-LIKE 8 (LOGL8), based on analysis of the LOGL8-pm (promoter mutant of LOGL8) mutant, which harbors a T-DNA insertion in the promoter of this gene. The mutation in LOGL8-pm causes ectopic hyperexpression of LOGL8 in inflorescence organs, resulting in plants with smaller panicles and defective ovules lacking archesporial cells and integuments. Knockout of LOGL8 caused pollen abortion, leading to a reduced seed-setting rate. LOGL8 encodes a putative cytokinin-activating enzyme. Our results showed that LOGL8 directly catalyzes the biosynthesis of bioactive cytokinins. Therefore, we propose that the ectopic expression of LOGL8 disrupts cytokinin spatiotemporal distribution and causes inhibition of LONELY GUY (LOG), which affects panicle branching and female organ development. These findings reveal the important role of LOGL8 in male development, and highlight the delicate balance of local cytokinin levels during panicle branching and female organ development.

The OsIME4 gene identified as a key to meiosis initiation by RNA insitu hybridization.

The formation of asexual seeds in plants holds great promise as a breeding system for one-line hybrid rice. Entry into meiosis is a key developmental decision in gametogenesis, especially in formation of asexual seeds in plants. Apomeiosis in MeMCs can be achieved by identifying and manipulating meiosis-specific genes. Using methods based on insitu hybridization and expression analysis, we identified OsIME4 (inducerofmeiosis 4) sense and antisense transcripts involved in rice meiosis initiation, similar to initiation of meiosis in budding yeast. Our data suggest that the OsIME4 sense transcript, which encodes a putative mRNA N6-adenosine methyltransferase, keeps rice cells at mitosis stage through some form of epigenesis (DNA/RNA methylation), and the non-coding antisense transcript of OsIME4 converts the cell status from mitosis to meiosis by inhibiting expression (transcription and translation) of the sense transcript. We identified that the non-coding antisense transcript of OsIME4 converts archesporial cell status from mitosis to meiosis by inhibiting expression of the OsIME4 sense transcript in rice. Our results provide novel insights into meiosis initiation in rice and for engineering of apomixis in sexual crops by manipulating the OsIME4 sense and antisense transcripts, which has great promise for producing apomictic rice in the future.

The leucine-rich repeat receptor-like kinase OsERL plays a critical role in anther lobe formation in rice

In Arabidopsis, ERECTA (ER) subfamily of leucine-rich repeat (LRR) receptor kinases (LRR-RKs) play important roles in cell division and cell elongation. However, the functions of OsER genes in rice are still very much unknown. In this study, sixty-seven TILLING and four gene-edited mutants were identified for one of the three OsERs, OsERL, and used for functional analyses. Results showed that mutations in OsERL led to striking defects in anther development. Compete male sterility and reduced numbers of anther lobes, more severe than knockout mutants, were observed in mutants with amino acid substitutions in the kinase domain. Among alleles with amino acid changes in LRRs, only one mutation in the 16th LRR showed evident phenotype, suggesting a role of the LRR in ligand sensing. OsERL is expressed in shoot apcies, internodes and anthers, and within the anther OsERL is expressed in sporophytic and tapetal cells. Cell biological analyses revealed that mutations in OsERL led to defected periclinal division in archesporial cells in anthers, suggesting a critical role of OsERL in rice anther development.

Auxin guides germ-cell specification in Arabidopsis anthers

Germ cells (GCs) are the key carriers delivering genetic information from one generation to the next. In a majority of animals, GCs segregate from somatic cells during embryogenesis by forming germlines. In land plants, GCs segregate from somatic cells during postembryonic development. In a majority of angiosperms, male GCs (archesporial cells) initiate at the four corners of the anther primordia. Little is known about the mechanism underlying this initiation. Here, we discovered that the dynamic auxin distribution in developing anthers coincided with GC initiation. A centripetal auxin gradient gradually formed toward the four corners where GCs will initiate. Local auxin biosynthesis was necessary for this patterning and for GC specification. The GC determinant protein SPOROCYTELESS/NOZZLE (SPL/NZZ) mediated the effect of auxin on GC specification and modified auxin biosynthesis to maintain a centripetal auxin distribution. Our work reveals that auxin is a key factor guiding GC specification in Arabidopsis anthers. Moreover, we demonstrate that the GC segregation from somatic cells is not a simple switch on/off event but rather a complicated process that involves a dynamic feedback circuit among local auxin biosynthesis, transcription of SPL/NZZ, and a progressive GC specification. This finding sheds light on the mystery of how zygote-derived somatic cells diverge into GCs in plants.

Pollination related temporal sequences of reproductive development and tracing the path of pollen tubes in Cymbidium pendulum (Roxb.) Sw., an ornamental orchid

Orchids, known for their exquisite beauty, are highly priced as cut flowers and potted plants at national and International market. More than I lac manmade hybrids are produced every year at intra to pleurigeneric level. Therefore, it is of imperative interest to unravel the all aspects of orchids in details. In the present study, the flowers of Cymbidium pendulum, an ornamental important orchids, have been investigated. It is observed that the flowers of C. pendulum started wilting in 20 days without pollination and senesced within 8 days after the act of pollination, suggesting a rapid progression of senescence related events triggered by pollination. The placenta became meristematically active only after pollination. The ovule primordium consisted of an axial filament 5–7 cells high enclosed by epidermal cells. The uppermost cell of the nucellar filament differentiated into an archesporial cell at 20–26 days after pollination (DAP), which turned into megaspore mother cell at 26–30 DAP. The megaspore mother cell underwent meiosis resulting in the formation of a triad which converted in to an eight-nucleate polygonum type of embryo sac. Thus the total time taken to reach the organized embryo sac is between 55-60 days. The results were well confirmed by study with fluorescent microscope using fluorescent dye where the pollen tubes were observed to move through base of the column (6–10 DAP) and entered the ovarian locule (8–16 DAP) and grew on either side of the each placenta, stayed there till the embryo matures and then liberated male gametes for fertilization (54–58DAP). The fertilization occurred around 58–60 DAP. In the present studies, the detailed elucidation of post pollination reproductive development, along-with temporal sequences of path of pollen tubes, have generated valuable information, advantageously useful both for academicians and breeders.

The formation of integuments, megasporogenesis and megagametogenesis in Dendrobium catenatum, with special discussions on embryo sac types and section techniques

The formation of integuments, megasporogenesis and megagametogenesis in Dendrobium catenatum, an economically important orchid, are observed. After pollination, mitotic cell divisions of the placental epidermis result in the formation of a branching system of outgrowths. The tip of each branch consists of an archesporial cell derived from the differentiation of the terminal subepidermal nucellar cell. It differentiates directly into a megasporocyte. The first division of the meiosis of the megasporocyte produces a dyad approximately equal in size, in which the micropylar cell promptly degenerates. The second meiotic division of the remaining dyad cell results in the formation of two megaspores of unequal size. The larger chalazal cell becomes functional and eventually develops into a mature megagametophyte. The development of the megagametophyte conforms to the Monosporic Polygonum type. The final arrangement of the mature embryo sac conforms to a sevencelled/ eight-nucleate structure. The mature ovule is bitegmic, tenuinucellate and has an anatropous orientation. In the present study, we also discuss the differences between three main types of embryo sac development and the improvement of section techniques.

Why does only one embryo sac develop in the Paeonia ovule with multiple archesporium?

Structure of the multiple archesporium in an ovule, time and place of archesporial cell differentiation and their developmental potential have not been studied in detail. In Paeonia species supernumerary archesporial cells are formed and differentiate as multiple megasporocytes, but only one embryo sac usually develops into an ovule. The reasons leading to development of one gametophyte and the death of most megasporocytes are unknown. The morphological structure of the multiple archesporium in Paeonia veitchii and P.caucasica was studied using cytoembryological methods. We used staining with aniline blue and fluorescence microscopy for visualization of callose on the megasporocyte walls. All cells of the ovule in investigated Paeonia species are uniform and meristematic at the earliest development stage. The onset of archesporium differentiation correlates with inner integument initiation. The sporogenous complex includes ten to 25 cells which develop asynchronously. The cell located in the central part of the sporogenous complex is differentiated into a megasporocyte earlier than in neighbouring cells. Only this megasporocyte is enveloped in callose; it develops further through to meiosis and forms a female gametophyte. The other megasporocytes degenerate during ovule development. We consider that callose participates in the mechanism of 'lateral inhibition' during megasporocyte maturation. The cell located in the central part of the Paeonia ovule is the first to receive signals that stimulate the onset of megasporogenesis and formation of the callose wall. It is possible that callose participates in blocking of development signals to neighbouring megasporocytes, leading to the arrest of their development.

Characterization of floral morphoanatomy and identification of marker genes preferentially expressed during specific stages of cotton flower development.

Characterization of anther and ovule developmental programs and expression analyses of stage-specific floral marker genes in Gossypium hirsutum allowed to build a comprehensive portrait of cotton flower development before fiber initiation. Gossypium hirsutum is the most important cotton species that is cultivated worldwide. Although cotton reproductive development is important for fiber production, since fiber is formed on the epidermis of mature ovules, cotton floral development remains poorly understood. Therefore, this work aims to characterize the cotton floral morphoanatomy by performing a detailed description of anther and ovule developmental programs and identifying stage-specific floral marker genes in G. hirsutum. Using light microscopy and scanning electron microscopy, we analyzed anther and ovule development during 11 stages of flower development. To better characterize the ovule development in cotton, we performed histochemical analyses to evaluate the accumulation of phenolic compounds, pectin, and sugar in ovule tissues. After identification of major hallmarks of floral development, three key stages were established in G. hirsutum floral development: in stage 1 (early-EF), sepal, petal, and stamen primordia were observed; in stage 2 (intermediate-IF), primordial ovules and anthers are present, and the differentiating archesporial cells were observed, marking the beginning of microsporogenesis; and in stage 6 (late-LF), flower buds presented initial anther tapetum degeneration and microspore were released from the tetrad, and nucellus and both inner and outer integuments are developing. We used transcriptome data of cotton EF, IF and LF stages to identify floral marker genes and evaluated their expression by real-time quantitative PCR (qPCR). Twelve marker genes were preferentially expressed in a stage-specific manner, including the putative homologs for AtLEAFY, AtAPETALA 3, AtAGAMOUS-LIKE 19 and AtMALE STERILITY 1, which are crucial for several aspects of reproductive development, such as flower organogenesis and anther and petal development. We also evaluated the expression profile of B-class MADS-box genes in G. hirsutum floral transcriptome (EF, IF, and LF). In addition, we performed a comparative analysis of developmental programs between Arabidopsis thaliana and G. hirsutum that considered major morphoanatomical and molecular processes of flower, anther, and ovule development. Our findings provide the first detailed analysis of cotton flower development.

Comparative Expression Profiling Reveals Genes Involved in Megasporogenesis.

Megasporogenesis is a key step during ovule development in angiosperms, but the small number and inaccessibility of these cells have hampered molecular and genome-wide studies. Thus, many questions remain regarding the molecular basis of cell specification, differentiation, and development in the female gametophyte. Here, taking advantage of the correlation between spikelet length and ovule development in rice (Oryza sativa), we studied the transcriptome dynamics of young ovules at three stages, the archesporial cell, the megaspore mother cell before meiosis, and the functional megaspore after meiosis, using expression profiling based on RNA sequencing. Our analysis showed that 5,274 genes were preferentially expressed in ovules during megasporogenesis as compared to ovules at the mature female gametophyte stage. Out of these, 958 (18.16%) genes were archesporial cell- and/or megaspore mother cell-preferential genes, and represent a significant enrichment of genes involved in hormone signal transduction and plant pathogen interaction pathways, as well as genes encoding transcription factors. The expression patterns of nine genes that were preferentially expressed in ovules of different developmental stages, including the OsERECTA2 (OsER2) receptor-like kinase gene, were confirmed by in situ hybridization. We further characterized the OsER2 loss-of-function mutant, which had an excessive number of female germline cells and an abnormal female gametophyte, suggesting that OsER2 regulates germline cell specification during megasporogenesis in rice. These results expand our understanding of the molecular control of megasporogenesis in rice and contribute to the functional studies of genes involved in megasporogenesis.

Anther and ovule development in Clematis terniflora var. mandshurica (Ranunculaceae)

Embryology can provide valuable information for determining taxonomic and phylogenetic relationships at the genus level. However, embryological information is fragmentary for the genus of Clematis. Herein, we studied the anther and ovule development of Clematis terniflora var. mandshurica using paraffin sections to examine microsporogenesis, microgametogenesis, megasporogenesis and macrogametogenesis in this plant. Tetrasporangiate anther, successive microsporocyte cytokinesis, and glandular tapetum, or occasionally amoeboid tapetum were observed in C. terniflora var. mandshurica. This plant’s microspore tetrads were mostly tetrahedral, and occasionally symmetrical, after meiosis. The anther wall was comprised of an epidermis and endothecium, both with fibrous thickenings in the mature anthers. Pollen grains were symmetrical, oblate, tricolpate, and two-celled. As for the ovary, it had one chamber with a few degraded ovules and a single normal one, which was anatropous, unitegmic, and tenuinucellate, and formed a Polygonum-type embryo-sac. Only one archesporial cell was present, and developed directly into a megaspore mother cell, which later formed a linear tetrad of megaspores. Larger dikaryocytic antipodal cells could also be observed in the mature embryo sac. This species might belong to a relatively primitive and transitional clade within the genus Clematis. To our knowledge, this study documented the embryological characters of C. terniflora var. mandshurica in detail for the first time and provided more information on the embryology of the genus Clematis.

SRNA-FISH: versatile fluorescent insitu detection of small RNAs in plants.

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent insitu hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent insitu detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA-FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co-localization of miR2275 and a 24-nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric insitu hybridization, sRNA-FISH signals can be imaged using super-resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level.

Sporogenesis and gametophytes development in Datura stramonium L. (Solanaceae)

Datura stramonium L., belonging to Solanaceae, is a medicinal plant with bioremediation potency that has variable characters and problematic taxonomy. The present study provides additional information on embryological characters in the D. stramonium that can provide useful information about the taxonomic of this species. The information is important for understanding the sexual reproduction process of this medicinal plant and also its phylogenetic position. The embryological properties, including sporogenesis and gametophyte development, of D. stramonium were examined by light, stereo, fluorescence and scanning electron microscopes. Results showed that each flower contained 5–6 tetrasporangiated stamens. The microsporogenesis and male gametogenesis characters of D. stramonium included dicotyledonous-type of anther wall formation, bi-nucleated and secretory tapetal cells, simultaneous cytokinesis in pollen mother cells, tetrahedral and tetragonal microspore tetrads and two-celled mature pollen grains. Ovary is formed by two carpels and contained numerous ovules with axile placentation. The anatropous, tenuinucellate and unitegmic ovules contained three archesporial cells. Only a single chalazal megaspore of the linear tetrad is functional. Polygonum-type embryo sac development, the presence of an endothelium, fusion of three antipodal cells and then formation of embryo without fertilization and cellular endosperm formation are the most important features in D. stramonium. The present findings reveal valuable developmental features can be used to discriminate this medicinal and environmentally important species and its phylogenetic relationships within the family Solanaceae.

Cytological and iTRAQ-based quantitative proteomic analyses of hau CMS in Brassica napus L

Hau cytoplasmic male sterility (CMS) is a new type of CMS that was originally identified in Brassica juncea and subsequently transferred to B. napus and B. rapa. To further elucidate the molecular mechanism underlying hau CMS in B. napus, semithin section analysis and iTRAQ-based differential proteomic analysis were performed to compare the hau CMS and its maintainer line. Cytological analysis revealed that abnormal anther development in the hau CMS line was arrested during the differentiation of stamen archesporial cells. qRT-PCR analysis showed that the sterility gene orf288 was expressed at substantially higher levels in CMS anthers than in anthers with restored fertility. In comparison with the maintainer line, a total of 186 differentially abundant proteins were identified in the CMS line, 58 of which exhibited increased accumulation and 128 exhibited decreased accumulation. Bioinformatics analysis showed that proteins involved in carbohydrate and energy metabolism, such as those involved in oxidative phosphorylation, glycolysis/gluconeogenesis and pyruvate metabolism, exhibited decreased accumulation in the hau CMS line, whereas those involved in oxidative stress, antagonism of cell death and protein processing exhibited increased accumulation in the hau CMS line, indicating the potential roles of carbohydrate metabolism and energy supply in the regulation of hau anther abortion. Biological significanceCytoplasmic male sterility (CMS) is one of the most efficient ways to produce hybrid seeds in crops. CMS is mainly caused by mitochondrial mutation and has been an important model for investigation of cytoplasmic and nuclear interactions in various plant species. Hau is a new type of CMS line in Brassica with completely abortive anthers. Although studies have been conducted to identify the key genes associated with CMS, the molecular mechanisms underlying hau CMS remain unclear. In this study, cytological, molecular, and proteomic approaches were used to reveal the mechanism underlying hau CMS in B. napus. Based on a comparison of the protein expression profiles of the hau CMS line and its maintainer line to elucidate the mechanisms underlying hau CMS, a potential protein regulatory network is proposed herein. These results may provide new insights into the molecular basis of hau CMS in B. napus.