Roles Of AGPs During Embryo Development And Phenotype Analysis Of AtAGP11 Overexpression In Arabidopsis

Arabinogalactan proteins (AGPs) are one member of the proline/hydroxyproline-rich glycoprotein superfamily (HRGPs). Previous studies have shown that AGPs may play important roles in many aspects during plant growth and development. But, there were few studies on functions of AGPs in zygotic embryo development in Arabidopsis. In this studies, by using cytological and biophysics methods, included ovule isolation and in vitro ovule culture, transparentizing of cotyledons, immunofluorescent and immunoenzyme, fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) techniques and so on, AGPs functions in embryo germination, cotyledon formation, and cell wall deposition during embryo development of Arabidopsis. We also analyzed the phenotype of AtAGP11 overexpression in Arabidopsis. The main results are as follows:1. To investigate the roles of AGPs in embryo germination, we added 50μMβGlcY (a synthetic reagent that specifically binds to AGPs) was added to the medium, while media without and with 50μMβManY (an isomer of (3GlcY, can’t bind to AGPs) were used as two control tests. The results showed that the embryos germination frequency of the untreated ovules (control) was nearly two-fold higher than that of theβGlcY treated ovules after 10 days of culture. With culture time extended, such difference gradually reduced. And when cultured for 25 days, the difference of total embryo germination frequency between the control andβGlcY treated ovules was not obvious. Moreover, when PGlcY was replaced with PManY, the germination frequency of ovules was similar to that of the untreated control. These results indicate that a certain concentration of PGlcY can inhibit embryo germination in vitro. We suggest that AGPs may involve in the process of embryo germination.2. Addition of PGlcY reagent can also affect the cotyledon formation during embryo development in vitro. In the process of 3 days after pollination ovule culture without PGlcY reagent, there were some abnormal embryos with low frequency. But after treated with PGlcY, the percentage of germinated embryos with defects was much higher than that in untreated orβManY treated ovules. Those abnormal embryos were mainly behaving on the number, shape, arrangement and vasculature pattern of the cotyledons. The abnormal differentiated embryos were described as single cotyledon, asymmetry cotyledon, supernumerary cotyledon and unobservable cotyledon. We counted the numbers of different types of abnormal cotyledons and the results showed that the percentage of embryos with observable cotyledon (included single cotyledon, asymmetry cotyledon and supernumerary cotyledon) was 3.25 folds higher than the embryos with unobservable cotyledon when the ovules were cultured in medium with 50μMβGlcY. Furthermore, the results from transparentizing of cotyledons showed the big cotyledon maybe resulted from the combination of two normal cotyledons. These results indicated that the losing of AGPs functions may affect the cotyledon boundary establishment and the formation of cotyledon primordium.3. Moreover, the distribution of AGPs in globular-stage embryos and the changes of cell wall cellulose and pectin distribution in mature embryos were investigated. After 24h treated with PGlcY, the AGPs level reduced very much. By staining with Calcofluor White (CW), the cellulose fluorescent signals in the shoot apical meristem (SAM) region of an abnormal embryo were more intense than those in vivo and the cultured normal embryos, and immunoenzyme localization result showed that the quantity of pectin was evidently reduced in the whole body of abnormal embryos. This result was further proved by the FTIR data. It indicated that the losing functions of AGPs may disturb cell wall cellulose and pectin distribution.4. The results of semi-thin sections showed that the cells of abnormal embryo were relatively larger and the cell number was much less than that of normal embryo. The ultrathin sections displayed that the SAM cells in the abnormal embryos had weaker cytoplasm, more plastids and starch granules, and larger vacuoles. This implied that the blocking of AGPS functions byβGcY may inhibit cell division and led to cell differentiation in embryos.5. Addition, the phenotype of the AtAGP11 overexpression plants were detailed analyzed. In comparison with the wide type, the most significant phenotype was that the siliques were quite smaller and the ovules could not be fertilized in high levels of overexpression lines, but the development of pollen was normal. And in the medium expression levels of overexpression lines, some siliques showed the same phenotype as the high expression level lines, but some siliques could grow to produce seeds. Moreover, in high level lines, the stamen was lower than the pistil. The results of semi-thin section showed that the filaments cells arranged in confusion and cell length was shorter than in wide type plants. And, the study on crossing between the wide type and the overexpression plants showed that the pollens could germinate on the stigma, the ovules could fertilize and develop. We suggest that the AtAGP11 overexpression affects the filament cells growth and leads to the filament shorten, resulting in the sterile of the ovules.