We identified two Arabidopsis embryo mutants, designated as raspberry1 and raspberry2, by screening T-DNA-mutagenized Arabidopsis lines. Embryogenesis in these mutants is indistinguishable from that of wild-type plants until the late-globular stage, after which raspberry1 and raspberry2 embryos fail to undergo the transition to heart stage, remain globular shaped, and proliferate an enlarged suspensor region. raspberry1 and raspberry2 embryo-proper regions enlarge during embryogenesis, become highly vacuolate, and display prominent convex, or "raspberry-like" protuberances on their outer cell layers. In situ hybridization studies with several embryo cell-specific mRNA probes indicated that the raspberry1 and raspberry2 embryo-proper regions differentiate tissue layers in their correct spatial contexts and that the regulation of cell-specific genes within these layers is normal. Surprisingly, a similar spatial and temporal pattern of mRNA accumulation occurs within the enlarged suspensor region of raspberry1 and raspberry2 embryos, suggesting that a defect in embryo-proper morphogenesis can cause the suspensor to take on an embryo-proper-like state and differentiate a radial tissue-type axis. We conclude that cell differentiation can occur in the absence of both organ formation and morphogenesis during plant embryogenesis and that interactions occur between the embryo-proper and suspensor regions.

We identified a T-DNA-generated mutation in the chaperonin-60␣ gene of Arabidopsis that produces a defect in embryo development. The mutation, termed schlepperless (slp), causes retardation of embryo development before the heart stage, even though embryo morphology remains normal. Beyond the heart stage, the slp mutation results in defective embryos with highly reduced cotyledons. slp embryos exhibit a normal apical-basal pattern and radial tissue organization, but they are morphologically retarded. Even though slp embryos are competent to transcribe two late-maturation gene markers, this competence is acquired more slowly as compared with wild-type embryos. slp embryos also exhibit a defect in plastid development-they remain white during maturation in planta and in culture. Hence, the overall developmental phenotype of the slp mutant reflects a lesion in the chloroplast that affects embryo development. The slp phenotype highlights the importance of the chaperonin-60␣ protein for chloroplast development and subsequently for the proper development of the plant embryo and seedling.

We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/beta-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote.

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We identified a new gene that is interrupted by T-DNA in an Arabidopsis embryo mutant called raspberry3. raspberry3 has "raspberry-like" cellular protuberances with an enlarged suspensor characteristic of other raspberry embryo mutants, and is arrested morphologically at the globular stage of embryo development. The predicted RASPBERRY3 protein has domains found in proteins present in prokaryotes and algae chloroplasts. Computer prediction analysis suggests that the RASPBERRY3 protein may be localized in the chloroplast. Complementation analysis supports the possibility that the RASPBERRY3 protein may be involved in chloroplast development. Our experiments demonstrate the important role of the chloroplast, directly or indirectly, in embryo morphogenesis and development.The molecular and cellular mechanisms that program the series of events leading to the development of a plant embryo are not well understood. Embryogenesis is a complex process that requires regulation of cell-specific and housekeeping genes within the embryo proper and neighboring seed tissues (e.g. endosperm) surrounding the embryo (Goldberg et al., 1994). In Arabidopsis, it has been estimated from genetic studies that there are about 4,000 essential genes and about 40 embryonic patterning genes that are required for normal plant embryogenesis (Jü rgens et al., 1991). The regulation of these genes must be tightly coordinated and controlled in a spatially and timely manner starting from the zygote to the mature embryo in dormant seeds (Goldberg et al., 1989; West and Harada, 1993;Jü rgens, 1995;Jü rgens et al., 1995). How the plant embryo achieves this coordination is not yet known and is a major question of plant developmental biology.A genetic approach is one of the strategies adopted to begin to understand the process of plant embryogenesis. There are more than 2,000 Arabidopsis embryo mutants that have been isolated by several laboratories over the last 10 years using ethyl methanesulfonate mutagenesis, T-DNA insertional inactivation, and transposon tagging (Errampalli et al., 1991;Mayer et al., 1991;Meinke et al., 1994;Schwartz et al., 1994;Yadegari et al., 1994; Altmann et al., 1995; Devic et al., 1996;McElver et al., 2001). The largest class of mutants within these collections are those that remain morphologically globular in shape (Errampalli et al., 1991;Mayer et al., 1991;Meinke et al., 1994;Schwartz et al., 1994;Yadegari et al., 1994). The raspberry (rsy) mutants, including rsy1 and rsy2, belong to this class (Yadegari et al., 1994).Several embryo mutants have been characterized, leading to the identification of embryo genes and what their functions are during embryo development. Some genes encode proteins that are involved in transcription or that are associated with transcription factors (Aida et al., 1997;Hardtke and Berleth, 1998;Li and Thomas, 1998;Lotan et al., 1998;Stone et al., 2001). Others encode proteins that are important for cell division, cell polarity, differentiation (Berleth and Jü rgens, 1993;Shevell et al., 1994;...