Plant Development: The SECrets of Arabidopsis embryogenesis

PLANT DEVELOPMENT

DETLEF WEIGEL

PLANT DEVELOPMENT

DETLEF WEIGEL

The SECrets of Arabidopsis embryogenesis The study of plant embryogenesis has come of age with the cloning of the first gene controlling pattern formation in the Arabidopsis embryo. Last year, I reviewed in these pages a string of recent papers providing the first in-depth analyses of several genes controlling patterning in the Arabidopsis embryo [1]. Just one year later, the first molecular analysis of one of these genes has been published, showing that plants are rapidly catching up with Drosophila and their ilk [2]. Those who thought that plant patterning would prove to be a simple 'me too' story, however, were wrong. Almost all of the known pattern-control genes of Drosophila encode either transcription factors or molecules involved in signal transduction cascades, but the EMB30/GNOM gene of Arabidopsis turns out to be related to a yeast gene involved in the transport of proteins through the Golgi apparatus. EMB30/GNOM first received considerable attention when Jiirgens and co-workers performed a saturation screen for mutations that disturb the body pattern of the, Arabidopsis seedling [3]. The premise underlying their approach was that the Arabidopsis seedling is developmentally equivalent to the first-instar Drosophilalarva, the cuticle pattern of which had been used some ten years earlier by Niisslein-Volhard, Wieschaus and Jurgens in their quest for the genes that are required for embryonic morphogenesis in the fly, but not for general cellular functions. The Arabidopsis mutagenesis was as successful as the Drosophila screen had been, although it soon became clear that Arabidopsis pattern genes do not fit simply into the Drosophila genes' straight-jacket of defining several independent morphogenetic activities. Gnom mutants, initially thought to be defective in a genetic activity specifically required for the development of terminal pattern elements of the seedling, such as cotyledons and roots, were found instead to be characterized by abnormal patterns of cell division [4]. Deviation of gnom mutants from the wild-type pattern of development becomes manifest as early as the first division. The first division in wild-type zygotes is asymmetric, resulting in a smaller apical cell and a larger basal cell, but the first division of gnom mutant zygotes generates two cells of nearly equal size. Subsequent divisions of gnom mutant embryos are randomly oriented, in contrast to the stereotypical pattern of pseudo-cleavage divisions that occur in the wild-type plant. The terminal-deletion phenotype of the fully-developed gnom mutant seedling, therefore, seems to be an indirect consequence of this abnormal sequence of early cell divisions. Mayer and co-workers [4] found that the wild-type GNOM gene is allelic to EMBRYONIC LETHAL 30 1040

(EMB30). Several years earlier, Meinke [5] had isolated the first emb30 allele, which in his interpretation failed to complete the last steps of embryonic development, including elaboration of the cotyledons and embryonic root. In addition, Meinke's group [6] had determined that emb30-1 embryos do not regenerate roots when placed on root-inducing media, in contrast to wild-type and other arrested mutants. These results implied that EMB30/GNOM is required at multiple stages during development, a suggestion that has now been endorsed by the results of Shevell and colleagues [2], who have found that the planes of cell division in mature emb30/gnom mutant seedlings are irregular, in addition to the irregularity that occurs in the early embryonic divisions. The result is that the tissue of the mutant seedling appears rather disorganized. However, not all stages of development are affected, as the gametes develop (probably) normally. In contrast to the situation in animals, the gametes of higher plants undergo several rounds of mitosis after the reduction division (meiosis), developing into haploid gametophytes. Although nobody has looked in detail at gametophyte development in emb30/gnom mutant plants, the normal meiotic segregation of wild-type and mutant gametes from heterozygotes indicates that the development of emb30/gnom mutant gametophytes proceeds normally. Shevell and colleagues [2] now report the initial molecular characterization of EMB30/GNOM. The first questions that they addressed were 'when and where is the gene expressed?' and 'what kind of protein does it encode?'. As the gene seems to be required at several stages of development, it was not entirely a surprise to discover that its RNA is detected throughout the plant's life cycle. But as the expression studies have been restricted so far to northern blot analyses, we do not yet know whether there are any spatial variations in the accumulation of EMB30/GNOM RNA or protein during development - for example, it is not known whether EMB30/GNOM is expressed more abundantly in dividing cells, or whether it is expressed at all in the gametophyte. More surprising than its ubiquitous expression was the finding [2] that the deduced amino-acid sequence of the EMB30/GNOM protein is extensively similar to that of the SEC7 protein of the budding yeast, Saccharomyces cerevisiae [7]. Both the EMB30/GNOM and SEC7 genes encode large proteins, of 1451 and 2009 amino acids,

© Current Biology 1994, Vol 4 No 11

DISPATCH respectively. The similarity between the two proteins is strongest in a 157-amino-acid region that is roughly in the middle of each protein (Fig. 1). The similarity extends throughout the proteins, but the degree of similarity is significantly lower outside the 157-aminoacid region. Several other proteins also have this region of similarity, for which Shevell and colleagues [2] propose the name 'Sec7 domain'. Among the genes encoding proteins with Sec7 domains is an anonymous Caenorhabditis elegans open reading frame, K06H7, and a human gene, B2-1, which is expressed in a natural killer T-cell line but not in a T-helper cell line [8]. Not all Sec7 domain proteins are as large as SEC7 and EMB30/GNOM B2-1 is only 398 amino-acids long. A recent search of the expressed sequence tags (EST) database produced several more Sec7 domain proteins, including at least four human genes and one from Caenorhabditis, indicating that there is likely to be a sizable gene family. As assessed by low-stringency hybridization, there is at least one more EMB30/GNOM homologue in the Arabidopsis genome [2]. That the Sec7 domain is a functionally important component of the EMB30/GNOM protein is confirmed by analysis of the emb30-1 mutation: an invariant glutamate that is conserved in all Sec7 domains is converted to a lysine in the mutant, and the function of the protein product is clearly altered in the mutant. Molecular analysis of additional emb30/gnom alleles should help to reveal other functional domains within the EMB30/GNOM protein. Mutant emb30/gnom alleles, of which over 30 have been isolated, fall into three classes, A, B and C. Trans-heterozygous combinations of alleles of the same class, as well as combinations of A and C or B and C alleles, exhibit the same phenotype as the parental alleles, whereas trans-heterozygotes of A and B alleles partially complement each other, suggesting that A and B mutations affect independently mutable functions within the protein [4]. (It has not been reported to which class the emb30-1 allele belongs, except that it is either A or C.) How does the function of SEC7 tie in with the effects of emb30/gnom mutations on the orientation of cell division? SEC7 is a cytosolic protein that is found associated with the Golgi complex [9]. Although deletion of the SEC7 gene is lethal [7], its inactivation in a temperaturesensitive mutant causes defects in protein secretion and glycosylation [10]. In addition, the number of cisternae within Golgi stacks is increased, compared to wild type. The underlying cause seems to be a defect in transport between different (but not all) compartments within the yeast Golgi apparatus. In emb30/gnom mutants, the Golgi apparatus is normal, as judged by transmission electron microscopy [2], but it is not known whether the mutants have defects in protein secretion, glycosylation or any other activity of the Golgi apparatus. Arabidopsis mutants deficient in N-linked glycosylation have been isolated -

Fig. 1. Sec7 and related proteins. (a) Schematic comparison of proteins with a Sec7 domain (blue). Note that the Sec7 domain of B2-1 is 156 amino acids long, whereas that of SEC7 and of EMB30/GNOM is 157 amino acids long. (b) Percentage of identical amino acids in four Sec7 domains. but the homozygous mutants are viable and do not show any gross developmental abnormalities [11], indicating that the emb30/gnom phenotype is not simply due to a non-specific glycosylation defect. A major function of the plant Golgi apparatus is to synthesize the polysaccharide components of the extracellular matrix that constitute the plant cell wall. Again, no obvious defects in the architecture of the cell wall have been reported in emb30/gnom mutants. And in contrast to the SEC7 gene, complete elimination of EMB30/GNOM is not lethal at the cellular level. The functional significance of the similarity between SEC7 and EMB30/GNOM remains, therefore, mysterious. As other Sec7 domain proteins are much smaller, it is possible that having the Sec7 domain alone does not mean that a protein functions within the Golgi apparatus. Apart from determining whether emb30/gnom mutants are defective in any of the known functions of the plant Golgi apparatus, such as glycosylation, secretion and polysaccharide synthesis, it will be interesting to learn whether EMB30/GNOM and SEC7 can substitute for one another, both in Arabidopsis and in yeast cells. In addition to testing the ability of the entire coding sequence of SEC7 to substitute for that of EMB30/GNOM (and vice versa), it might be worthwhile to exchange only the Sec7 domains between the two proteins and assess the functional consequences. As is so often the case with the cloning of genes, the initial molecular analysis of EMB30/GNOM has raised more new questions than it has answered old ones. Nevertheless, I look forward to the next chapter in what promises to become an exciting and unusual story.

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References 1. Weigel D: Patterning the Arabidopsis embryo. Curr Biol 1993, 3:443-445. 2. Shevell D, Leu W-M, Gilmour CS, Xia G, Feldmann KA, Chua N-H: EMB30 is essential for normal cell division, cell expansion, and cell adhesion in Arabidopsis and encodes a protein that has similarity to Sec7. Cell 1994, 77:1051-1062. 3. Mayer U, Torres Ruiz RA, Berleth T, Mis6ra S, Jrgens G: Mutations affecting body organization in the Arabidopsis embryo. Nature 1991, 353:402-407. 4. Mayer U, B0ttner G, Jorgens G: Apical-basal pattern formation in the Arabidopsis embryo: studies on the role of the gnom gene. Development 1993, 117:149-162. 5. Meinke DW: Embryo-lethal mutants of Arabidopsis thaliana: analysis of mutants with a wide range of lethal phases. Theor Appl Genet 1985, 69:543-552. 6. Baus AD, Franzmann L, Meinke DW: Growth in vitro of arrested embryos from lethal mutants of Arabidopsis thaliana. Theor Appl Genet 1986, 72:577-586. 7. Achstetter T, Franzusoff A, Field C, Schekman R: SEC7 encodes an unusual, high molecular weight protein required for membrane

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Liu L, Pohajdak B: Cloning and sequencing of a human cDNA from cytolytic NK/T cells with homology to yeast SEC7. Biochim Biophys Acta 1992, 1132:75-78. 9. Franzusoff A, Redding K, Crosby J, Fuller RS, Schekman R: Localization of components involved in protein transport and processing through the yeast Golgi apparatus. J Cell Biol 1991, 112:27-27. 10. Franzusoff A, Schekman R: Functional compartments of the yeast Golgi apparatus are defined by the sec7 mutation. EMBO J 1989, 8:2695-2702. 11. von Schaewen A, Sturm A, O'Neill J, Chrispeels MJ: Isolation of a mutant Arabidopsis plant that lacks N-acetylglucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans. Plant Phys 1993, 102:1109-1118. 8.

Detlef Weigel, Plant Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, LaJolla, California 92037, USA.

THE AUGUST 1994 ISSUE (VOL. 4, NO. 4) OF CURRENT OPINION IN GENETICS AND DEVELOPMENT included the following reviews, edited by Barbara J. Meyer and Janet Rossant, on Pattern Formation and Developmental Mechanisms: Pattern formation in plant development: four vignettes by Elliot M. Meyerowitz Dorsoventral patterning in Drosophila oogenesis by Trudi Schiipbach and Siegfried Roth Signal transduction and cell fate specification during Caenorhabditiselegans vulval development by David M. Eisenmann and Stuart K. Kim TGF-03 related genes in development by Nancy A. Wall and Brigid L.M. Hogan Wnt genes and vertebrate development by Brian A. Parr and Andrew P. McMahon The making of a maggot: patterning the Drosophila embryonic epidermis by Stephen DiNardo, Jill Heemskerk, Scott Dougan and Patrick H. O'Farrell Mechanisms of limb patterning by Randy L. Johnson, Robert D. Riddle and Clifford J. Tabin Neural induction in Xenopus by Richard M. Harland Retinoic acid and homeobox gene regulation by Alexander W. Langston and Lorraine J. Gudas Structure/function studies of lin-12/Notch proteins by Iva Greenwald Establishment of initial asymmetry in early Caenorhabditiselegans embryos by James R. Priess Genetic analyses of cell-matrix interactions in development by Richard O. Hynes Hams and Egls: genetic analysis of cell migration in Caenorhabditiselegans by Gian Garriga and Michael J. Stern Programmed cell death in Caenorhabditiselegans by Michael O. Hengartner and H. Robert Horvitz Axon guidance mechanisms in Caenorhabditiselegans by Joseph G. Culotti Axon guidance by diffusible repellants and attractants by Marc Tessier-Lavigne