Cytological Analyses on Development of Male and Female Gametophytes in an Interspecific Hybrid F_1 from Cucumis hystrix Chakr. × Cucumis sativus L.

An interspecific hybrid F1 of Cucumis hystrix Chakr. x Cucumis sativus L. （NC4406） was used to establish the developmental sequence and to characterize the male and female gametophytes at cytological level for further understanding of the phylogenic relationship and the mechanism of fertility or sterility in the interspecific hybrid F1 The development of male and female gametophytes was studied through meiotic analysis and paraffin section observation technique, respectively. Meanwhile, the fertility level was assessed through hybrid F1 backcrossing to cultivated cucumber 4406. Variable chromosome configurations were observed in the pollen mother cells （PMCs） of hybrid F1 at metaphase Ⅰ , e.g., univalents, bivalents, trivalents, quadravalents, etc. At anaphase Ⅰ and Ⅱ, chromosome lagging and bridges were frequently observed as well, which led to the formation of polyads and only a partial number of microspores could develop into fertile pollen grains （about 23.3%）. Observations of the paraffin sections showed numerous degenerated and abnormal embryo sacs during the development of female gametophytes, and only 40% of the female gametophytes could develop into normal eight-nuclear megaspore. On an average, 22.8 and 6.3 seeds per fruit could be obtained from the reciprocal backcross. The interspecific hybrid F1 of C. hystrix x NC4406 was partially fertile; however, the meiotic behaviors of hybrid F1 showed a high level of intergenomic recombination between C. hystrix and C. sativus chromosomes, which indicated that it plays an important role for introgression of useful traits from C. hystrix into C. sativus.

Pollen Mother Cell Miosis and Male Gametophyte Development of Pumpkin

[Objective] Pollen mother cell miosis and male gametophyte development of pumpkin were observed in this study, to provide some cytological basis for pumpkin anther or microspore culture. [Method] Ehrlich＇s hematoxylin staining-methyl salicylate clearing technique was used for observation and research of the variation of cell structure and chromosomal behavior during pollen mother cell miosis and male gametophyte development of ‘Tianhong＇ pumpkin. [Result] The meiosis in pollen moth- er cells of pumpkin was simultaneous cytokinesis. In the process of nuclear division, nuclear membrane and nucleolus of pumpkin pollen mother cells gradually disappeared in the metaphase I and reappeared in telophase I , phragmoplast formed between the two generated crescent-shaped nuclei without cell wall, the phragmoplast gradually disappeared in the metaphase II and reappeared in telophase II. Phragmoplast spread outward from the center of spindle during the second division was connected with that formed on the central interface of two nuclei during the first division, cell wall of microspores generated from periphery to center. Most of the tetrads contained four sub-cells while a few contained extra small cells. During the period of uniuclete microspore at periphery, the single nucleolus split into 2-3 or more small nucleoli, mature pollen grain was two-celled. Mononucleate pollen cells were mostly appeared in the flower buds with length of 1.0-2.0 cm, which could be used as an important indicator to collect materials for anther or microspore culture. [Conclusion] This study laid the foundation for research of the cytogenetics of pumpkin.

Development of a SCAR marker for male gametophyte of Gracilariopsis lemaneiformis based on AFLP technique

The red alga Gracilariopsis lemaneiformis(Bory) is an economically valuable macroalgae. As a means to identify the sex of immature Gracilariopsis lemaneiformis, the amplifi ed fragment length polymorphism(AFLP) technique was used to search for possible sex- or phase-related markers in male gametophytes, female gametophytes, and tetrasporophytes, respectively. Seven AFLP selective amplifi cation primers were used in this study. The primer combination E-TG/M-CCA detected a specifi c band linked to male gametophytes. The DNA fragment was recovered and a 402-bp fragment was sequenced. However, no DNA sequence match was found in public databases. Sequence characterized amplifi ed region(SCAR) primers were designed from the sequence to test the repeatability of the relationship to the sex, using 69 male gametophytes, 139 female gametophytes, and 47 tetrasporophytes. The test results demonstrate a good linkage and repeatability of the SCAR marker to sex. The SCAR primers developed in this study could reduce the time required for sex identifi cation of Gracilariopsis lemaneiformis by four to six months. This can reduce both the time investment and number of specimens required in breeding experiments.

Microsporogenesis and Male Gametophyte Development of Euphorbar pulcherrima

The microsporogenesis and male gametophyte development of Euphorbia pulcherrima were studied with paraffin section method. The results showed that the anther of Euphorbia pulcherrima had four chambers. The development of anther wall was dicotyledonous type; its wall consisted of epidermis, endothecium, middle layer and tapetum. The mature male gametophyte development experienced seven major stages as the followings： archesporial cells, aporgenous cells, microspore mother cells, tetrads of microspores （tetrahedral）, uninucleate microspores, 2-uncleate pollens and 2-celled pollen stage. The anther wall varied with the pollen development： the epidermis of anther ruptures at the anther mature stage; the middle layer disappears at tetrad stage; the endothelium became fibrous layer at the anther mature stage; the tapetum （glandular） was the most development at the microspore mother cells stage and disappeared at anther mature stage. The abnormal anther development was observed simultaneously.

Biological Features of Flowering and Development of Male Gametophyte in Anthurium andreanum

The aim of this study is to follow each development stage of inflorescence in order to understand the biological feature of flowering and the development of male gametophyte in Anthurium andreanum “Arizona' and to try to find the optimum conditions for its pollination. The methods of dissection and paraffin section were adopted to examine the structural characteristics of anthurium’s tiny floret and the development of the microspore. All the florets of the anthurium arrange on the rhachis helically sub- tended by a colorful bract. Each tiny floret has one gynoecium, four tepals and four stamina. The bract and the florets show different colors during the whole blooming period. The ovary is bicarpellary and has two locules, each of which has one anatropous ovule. The placenta is of a central placentation type. The stylar canal cells not only can produce the secretory mucilage but also can release their own cytoplasm caused by their self-disintegration before the pistil reaches its maturity. The wall of the anther is composed of four layers: epidermis, endothecium, middle layer and tapetum. The tapetal cells and the middle layers’ cells degenerated completely dur- ing meiosis of microsporocytes. The pollen grains were 2-celled at the time of anther dehiscence. Early morning, when the inflores- cences stay at their fifth development stage, is the optimum opportunity for pistil to get pollen grains. The pollen-collection should be done at the end of the seventh stage.

Microsporogenesis and development of male gametophyte of B.rossica Fedtsch.et Flerov

There were four microsporanges in the mature anther of B. rossica Fedtsch. et Flerov. The development of the anther wall belonged to dicotyledonous type. The tapetum cell was dichotypic. Simultaneous cytocinesis in the microspore mother cells formed tetrad by reduction division and the array of microspore tetrads was different. There were tetrahedron type and bilateral symmetry type. The male gametophyte was 2-cell. The mature pollen was global and tricolporate. All those results were investigated under both light microscope （LM） and electron microscope （EM）

Epigenetic Repression of Male Gametophyte- Specific Genes in the Arabidopsis Sporophyte

Tissue formation, the identity of cells, and the functions they fulfill, are results of gene regulation. The male gametophyte of plants, pollen, is outstanding in this respect as several hundred genes expressed in pollen are not expressed in the sporophyte. How pollen-specific genes are down-regulated in the sporophyte has yet to be established. In this study, we have performed a bioinformatics analysis of publicly available genome-wide epigenetics data of sev- eral sporophytic tissues. By combining this analysis with DNase ! footprinting data, we assessed means by which the repression of pollen-specific genes in the Arabidopsis sporophyte is conferred. Our findings show that, in seedlings, the majority of pollen-specific genes are associated with histone-3 marked by mono- or trimethylation of Lys-27 （H3K27me1/ H3K27me3）, both of which are repressive markers for gene expression in the sporophyte. Analysis of DNase footprint profiles of pollen-specific genes in the sporophyte displayed closed chromatin proximal to the start codon. We describe a model of two-staged gene regulation in which a lack of nucleosome-free regions in promoters and histone modifications in open reading frames repress pollen-specific genes in the sporophyte.

Programmed cell death may act as a surveillance mechanism to safeguard male gametophyte development in Arabidopsis

Programmed cell death(PCD)plays an important role in plant growth and development as well as in stress responses.During male gametophyte development,it has been proposed that PCD may act as a cellular surveillance mechanism to ensure successful progression of male gametogenesis,and this suicide protective machinery is repressed under favorable growth conditions.However,the regulatory mechanism of male gametophyte-specific PCD remains unknown.Here,we report the use of a TdT-mediated dUTP nick-end labelingbased strategy for genetic screening of Arabidopsis mutants that present PCD phenotype during male gametophyte development.By using this approach,we identified 12 mutants,designated as pcd in male gametogenesis(pig).pig mutants are defective at various stages of male gametophyte development,among which nine pig mutants show a microspore-specific PCD phenotype occurring mainly around pollen mitosis I or the bicellular stage.The PIG1 gene was identified by map-based cloning,and was found to be identical to ATAXIA TELANGIECTASIA MUTATED(ATM),a highly conserved gene in eukaryotes and a key regulator of the DNA damage response.Our results suggest that PCD may act as a general mechanism to safeguard the entire process of male gametophyte development.

Roles of Pectin Methylesterases in Pollen-Tube Growth

Elongation of the pollen tube in pistil is essential for delivering sperms into the female gametophyte in sexual plant reproduction. Recently, a group of cell wall enzymes, pectin methylesterases （PMEs）, have been identified as playing an important role in this process. This article reviews the new understanding of the roles of PMEs in regulating pollen tube growth.

Ultrastructure analysis reveals sporopollenin deposition and nexine formation at early stage of pollen wall development in Arabidopsis

In angiosperm, pollen wall formation is a critical step for male gametophyte development. Pollen wall constitutes of the outer layer exine and the inner layer intine. Exine is further divided into sexine and nexine. In Arabidopsis, the general process of pollen wall formation has been reported. However, the nexine formation has not been revealed. Here, we observed the process of pollen wall formation in Arabidopsis thaliana using transmission electron microscope. After callose wall is formed, the primexine is present between plasma membrane and the callose layer in the tetrad. With plasma membrane undulation, sporopollenin precursors accumulated on the peak of undulated membrane which is further developed into probacula. The primexine determines plasma membrane undulation and sporopollenin accumulation based on previous analysis of an undulation-deficient mutant. Some materials obviously different from sporopollenin are filled between the primexine and plasma membrane. These materials cover all the surface of plasma membrane and gradually develop into nexine. After microspore is released from tetrad, the nexine layer is formed and the probacula is further developed into sexine with continued accumulation of sporopollenin. Based on these observations, we proposed a developmental model of early pollen wall formation.

AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis

In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood. in this study, we identified that the Arabidopsis Transmembrane Protein 18 （AtTMEM18） gene played important roles in pollen tube growth. The AtTMEM18 shares a high similarity with the Transmembrane 18 proteins （TMEM18s） that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtTMEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth. AtTMEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen-rescued assays showed that the mutation in AtTMEM18 also caused defects in roots, stems, leaves and transmitting tracts. AtTMEM18-GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtTMEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility.Furthermore, expression of the rice TM EM18-homologous protein （OsTMEM18） driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis.