Development Of Embryo Sac In Eulaliopsis Binata And The Expression Of FIS-Class Genes

Eulaliopsis binata is a perennial grass belonging to the family Poaceae, and the proportion of apomixis embryo sac is more than 95%. Aposporous initiating cells (AI) via two mitotic divisions formed the Panicum type sac which contains 4-nuclei, embryo and endosperm autonomous development without requiring fertilization, seed formed via apomixis thus retain the genotype of the mother plant, it would provide a tremendous opportunity to maintain hybrid vigour over successive seed generations. Identification the sexual individual from facultative apomictic plants is the basic to study the apomixis genetic mechanism. Therefore, we use the tissue chemistry and embryology research, observed the development model of embryo sac in different populations'E. binata, moreover, the identified of sexual reproduction accession is a prerequisite for further genetic and molecular research of apomictic E. binate, and the separation of apomixis related gene.Some studies showed that the FIS-class mutations of Arabidopsis could lead to central cell autonomous division without fertilization, similar to fertilization-independent (autonomous) endosperm in apomixis plant. The study FIS-class gene function of autonomous endosperm E. binata, would easily understand the sexual reproduction and apomixis mechanism during early endosperm development. In this study, we use three FIS homologous in E. binata which were isolated in previous work, analysis of temporal and spatial expression of FIS-class genes during the process of growth and development of E. binata, provide the evidence of EbFIS function.The major findings are as follows:1 Callose deposition in E. binata embryo sac initiation cell during ovule early developmentCallose usually accumulates in the walls of cells undergoing megasporogenesis during sexual embryo sac initiation. We use the method of paraffin section combined with aniline blue staining, observed the beginning of sexual embryo sac in E. binata according to early development of the ovule binata whether callose fluorescence, as well as observed the apospory initial cell whether had callose. The results showed that abundant callose present in nucellar tissue during early development in the four populations E. binata (Xingzi 86.81%, Hengyang 95.05%, Xixia 96.77%, Xichuan 92.71%), indicated that the there were high proportion of initiation of sexual reproduction in E. binata. Callose was initially deposited in the walls of these sexual embryo sac cells, following the embryo sac mother cell via meiosis, callose also deposited in the megaspore tetrad walls. After meiosis, the distribution of callose at the micropylar end more than chalazal end until the functional megaspore selection callose wall began degradation, this consistent with the callose distribution patterns in the process of sexual reproduction in Polygonum-type embryo sac. Before and after meiosis of sexual embryo sac,1 or more aposporous initiating cells enlarged near the region of chalazal end nucellus, and these cells are no callose fluorescence, indicating that the wall of aposporous initiating without callose accumulation in E. binata.2 Sexual reproduction and apomictic embryo sac development patterns in E. binata2.1 The process of aposporous embryo sac development and the initial periodBy the observation of four E. binata population, the differentiation of apospory initial cells could derived from nucellar cells near the chalazal end adjacent to sexual cells at megaspore mother cell stage, or tetrad, and direct develop 1-nuclear embryo sac. Formation of apospory embryo sac with 4-nuclei after two mitotic, then the 4 nuclei further differentiation mature Panicum type sac with one egg cell, one synergid cell and two polar nuclei or one egg cell, two synergid cells.2.2 Process and the proportion of sexual embryo sacAccording to the cytological observation of Xingzi, Hengyang and Xixia populations during the embryo sac development, found that the formation of megaspore mother cell, the regular of meiosis, and linear megaspore tetrad, the functional megaspore via 3 mitosis, form typical Polygonum type embryo sac with 7 cells and 8-nuclei. The ratio of sexual reproduction mature embryo sac is 1% to 2%, and there were no different between four populations.During the embryo sac development, we did not found that one Polygonum type embryo sac and an aposporous Panicum type embryo sac in a same ovule, indicating sexual reproduction and apomixis had competition, and the aposporous reproductive development dominate, resulting the most sexual embryo sac abortion during the process of embryo sac development in apomixis E. binata.3 Spatial and temporal expression of FIS-class genesWe use the mRNA of EbFIE, EbEMFl, and EbEMF2 as probe; detect the temporal and spatial expression mode of three genes in root hair zone, leaves, tender and mature anthers, mature embryo sac, embryo and endosperm in different developmental stages and other organs. The results are as follows:3.1 FIE gene expression patterns FIE gene express in the maturation zone of root epidermal cells, cortical parenchyma cells and phloem vascular tissues; the expression in leaves was concentrated the phloem near lower epidermal. FIE gene expression in microspore mother cells, anther tapetum and vascular bundles were significantly higher. Following the development of male gametophyte, the expression level was gradually lower in the anther. In the mature embryo sac with little expression (compared with the control), and there was no significant difference between the sporophyte and gametophyte. FIE gene in different developmental stages of embryo and endosperm exist high expression after fertilization, and the hybridization signals were stronger in endosperm and embryo during late stage of embryonic development. EbFIE gene could expression in vegetative organs and reproductive organs, suggest that is constitutive expression, and it may be involved in multiple development processes of E. binata; express higher in embryo and endosperm show FIE gene indicated that it may had the function of influence the development of embryo and endosperm.3.2 EMF1 gene expression patternsEMF1 gene could express in all tissues of E. binata, and the higher hybridization signal were detected in the spherical and pear-shaped embryo during embryogenesis; expression pattern in male gametophyte was similar to EbFIE, mean that expressed strongly n in tapetum, pollen mother cell and anther during the stage of pollen mother cells, and lower in the early stage of pollen. Hybridization signal of EbEMFl in the vegetative organs was very strong, especially in roots; the main expression site is the root epidermal cells and phloem; while EbEMF1 expression in leaves was concentrated near the lower epidermis and phloem within the vascular bundle. The expression patterns of EMF1 gene showed that it may have some function in E. binata vegetative and reproductive growth.3.3 EMF2 gene expression patternsEMF2 gene has high expression in reproductive organs of E. binata, especially in the ovary after fertilization 5 days, the embryo and endosperm expression of the highest; The expression patterns of EMF2 gene in anthers was similar to EMF1; but the expression level in the vegetative organs of roots and leaves was lower than EMF1, only in root epidermal cells has lower expression with lighter color, indicating EMF2 gene may play a role of regulation embryo and endosperm development in E. binata.