Embryogenesis is a fundamental developmental event in the life cycle of flowering plants. In higher plants, embryogenesis consists of two major phases: morphogenesis and maturation. Morphogenesis involves the establishment of the embryo’s body plan, while maturation includes cell expansion and accumulation of storage macromolecules prepared for embryo desiccation and germination as well as early seedling growth [1
]. Embryogenesis originates from the zygotic asymmetric division which results in the formation of a small cytoplasmically-dense apical cell and a larger vacuolated basal cell [4
]. These two distinct-sized daughter cells have different cell fates: the apical cell differentiates into an embryo proper that develops into most of the mature embryo, while the basal cell divides into the hypophysis and the suspensor [1
]. The hypophysis contributes to the formation of the root meristem within the embryo proper, while the suspensor is a highly specialized, terminally differentiated embryonic organ that plays structural and physiological roles in embryo development, and degenerates at the end of embryogenesis [6
]. The cause of the different developmental pathways of apical and basal cells remains to be researched.
The crucial concerns in plant embryogenesis research are unraveling the mechanisms that operate the processes of embryonic body plan establishment and different organ specification. The experimental manipulation for embryogenesis of angiosperms is difficult, particularly at the early stage when the embryo develops deeply inside maternal tissues [9
]. In recent years, the inaccessibility of some plant embryos has been overcome. Combined with the in vitro culture system, the isolated zygotes simulate normal developmental patterns and permit direct molecular analysis at any of the early embryonic stages [10
]. In the past few years, in our laboratory, the fertilized ovules [15
], zygotic embryos [17
] and even isolated zygotes [15
] were in vitro cultured and used to study developmental events of different staged embryos. Compared with Arabidopsis thaliana
, zygotes and proembryos in tobacco provide an ideal model system for investigating early embryo development, since they are relatively larger than those of Arabidopsis thaliana
and can be easily isolated [15
]. Recently, we extracted mRNAs from tobacco apical and basal cells to generate cDNA libraries and investigated the transcript profiles of the two daughter cells from zygotes by an expressed sequence tag analysis [20
]. The strategy of combining an in vitro culture system with genetic and molecular techniques should allow us to obtain new insight into early embryo development events and embryo cell fate decision in flowering plants.
Angiosperm embryo development follows a predictable pattern and numbers of cell divisions [3
]. In higher plants, cytokinesis is the process of separating cytoplasm through the formation of a new plasma membrane and cell wall between daughter cells [22
]. The major stages of plant cell division contain preprophase band (PPB) formation, phragmoplast (PHP) emergence, cell plate expansion and new cell wall location. Recent studies revealed that endocytic delivery of the cell surface material significantly contributes to cell plate formation during plant cell division [22
]. New cell wall components, such as pectin and wall-associated proteins (e.g. arabinogalactan proteins-AGPs), are transferred to the margin of the cell plate via endosomes [24
]. Eventually, the expanding cell plate fuses with the parental plasma membrane at the division site, and then separates the two daughter cells.
AGPs are a large family of highly glycosylated proteins thought to be involved in plant growth and development [27
]. In Arabidopsis
, the organ-, tissue- and cell-type specific expression patterns indicate that AGPs are markers of cell identity and fate decision [33
]. β-glucosyl Yariv (β-GlcY) reagent is used as an AGP inhibitor to determine AGP function by specific binding and blocking [36
]. Although the precise effect of the β-GlcY is unknown, it is possible that the formation of large complexes at the cell surface prevents the normal assembly of molecules into cell wall [38
]. In our laboratory, the previous studies have revealed that β-GlcY treatment can change the first zygotic division pattern from asymmetric to symmetric [15
]. The in vitro tobacco zygote culture system with β-GlcY treatment provided us with an ideal model which might shed light on our research in the early proembryo development of higher plants. In order to delve further into this important developmental event, the molecular and cellular studies were both carried out. Recently, our transcriptome study on β-GlcY treated zygotes and two-celled proembryos also revealed that β-GlcY affected the expression of some genes related to zygotic division and proembryonic development [39
As a continuous study of the previous work, in this paper, we used the tobacco in vitro zygote culture system and series of meticulous cell biology techniques to investigate the roles of AGPs in zygote and proembryo cell division. We adopted β-GlcY reagent to perturb AGPs function for in vitro cultured zygotes and proembryos of tobacco (Nicotiana tabacum L.). Both immunofluorescence detection with antibody JIM13 and staining with the β-GlcY reagent showed that AGPs distributed in the new cell plate during normal in vitro zygotic division. Cellular staining with Calcofluor white (CW) reagent (for observing cellulosic components), and fluorescent labeling with JIM5 and JIM7 monoclonal antibodies (for detecting low-esterified and high-esterified pectins, respectively) against both in vivo and in vitro proembryos demonstrated that β-GlcY treatment affected the location of cellulose deposits but not pectin polar distribution in the new cell wall. Furthermore, FM4-64 staining indicated that endocytic vesicles distributed in the cell plate when proembryo cells were dividing. The results imply that AGPs may be involved in the formation of new embryo cell plate. A possible model involving the relationship between AGPs and the other regulation factors was proposed and discussed.