Abstract
Asymmetric cell divisions occur widely during many developmental processes in plants. In most angiosperms, the first zygotic cell division is asymmetric resulting in two daughter cells of unequal size and with distinct fates. However, the critical molecular mechanisms regulating this division remain unknown. Previously we showed that treatment of tobacco zygotes with beta-glucosyl Yariv (βGlcY) could dramatically alter the first zygotic asymmetric division to produce symmetric two-celled proembryos. In the present study, we isolated zygotes and two-celled asymmetric proembryos in vivo by micromanipulation, and obtained symmetric, two-celled proembryos by in vitro cell cultures. Using suppression-subtractive hybridization (SSH) and macroarray analysis differential gene expression between the zygote and the asymmetric and symmetric two-celled proembryos was investigated. After sequencing of the differentially expressed clones, a total of 1610 EST clones representing 685 non-redundant transcripts were obtained. Gene ontology (GO) term analysis revealed that these transcripts include those involved in physiological processes such as response to stimulus, regulation of gene expression, and localization and formation of anatomical structures. A homology search against known genes from Arabidopsis indicated that some of the above transcripts are involved in asymmetric cell division and embryogenesis. Quantitative real-time PCR confirmed the up- or down-regulation of the selected candidate transcripts during zygotic division. A few of these transcripts were expressed exclusively in the zygote, or in either type of the two-celled proembryos. Expression analyses of select genes in different tissues and organs also revealed potential roles of these transcripts in fertilization, seed maturation and organ development. The putative roles of few of the identified transcripts in the regulation of zygotic division are discussed. Further functional work on these candidate transcripts will provide important information for understanding asymmetric zygotic division, generation of apical-basal polarity and cell fate decisions during early embryogenesis.
Highlights
Asymmetric cell division resulting in daughter cells differing in morphology, identify and function is a universal and fundamental mechanism for many developmental processes in angiosperms, and serves an essential role during embryonic and postembryonic development to generate cell diversity [1,2]
By using the well established procedures of micromanipulation combined with enzymatic maceration [37], we isolated the zygotes and two-celled proembryos from ovules at 84 h and 108 h after pollination, respectively (Figure 1A, B)
In order to reveal the molecular differences during the asymmetric and symmetric division of zygotes, the symmetric two-celled proembryos were collected from our in vitro culture system supplemented with 50 mM beta-glucosyl Yariv (bGlcY) reagent (Figure 1C), as described earlier [37]
Summary
Asymmetric cell division resulting in daughter cells differing in morphology, identify and function is a universal and fundamental mechanism for many developmental processes in angiosperms, and serves an essential role during embryonic and postembryonic development to generate cell diversity [1,2]. During development of stomata in leaves, asymmetric divisions of meristemoid mother cells generate smaller triangular meristemoid and larger sister cells, and the meristemoids generally undergo stem cell-like divisions, regenerating a larger sister cell with developmental plasticity and a smaller cell with meristemoid fate. The latter cell eventually differentiates into a guard mother cell to produce the two guard cells of the stomata [6]. After an ordered set of asymmetric divisions, the lateral root meristem initiates, subsequently grows out, and generates the lateral root [7]
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