Robert B. Goldberg

Arabidopsis LEAFY COTYLEDON1 Is Sufficient to Induce Embryo Development in Vegetative Cells

The Arabidopsis LEAFY COTYLEDON1 (LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box-binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.

Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development.

We studied the temporal and spatial regulation of three mRNA sequence sets that are present exclusively, or at elevated levels, in the tobacco anther. One mRNA set accumulates in the tapetum and decays as the tapetum degenerates later in anther development. The second mRNA set accumulates after the tapetal-specific mRNAs, is localized within the stomium and connective, and also decays as these cell types degenerate during anther maturation. The third mRNA sequence set persists throughout anther development and is localized within most anther tissues. A tapetal-specific gene, designated as TA29, was isolated from a tobacco genome library. Runoff transcription studies and experiments with chimeric [beta]-glucuronidase and diphtheria toxin A-chain genes showed that the TA29 gene is regulated primarily at the transcriptional level and that a 122-base pair 5[prime] region can program the tapetal-specific expression pattern. Destruction of the tapetum by the cytotoxic gene had no effect on the differentiation and/or function of surrounding sporophytic tissues but led to the production of male-sterile plants. Together, our studies show that several independent gene expression programs occur during anther development and that these programs correlate with the differentiated state of specific anther cell types.

Anther development: basic principles and practical applications.

Male reproductive processes in flowering plants take place in the stamen (Esau, 1977). This sporophytic organ system contains diploid cells that undergo meiosis and produce haploid male spores, or microspores. Microspores divide mitotically and differentiate into multicellular male gametophytes, or pollen grains, that contain the sperm cells. Figures 1 and 2 show that the stamen consists of two morphologically distinct partsthe anther and the filament. The filament is a tube of vascular tissue that anchors the stamen to the flower and serves as a conduit for water and nutrients. By contrast, the anther contains the reproductive and nonreproductive tissues that are responsible for producing and releasing pollen grains so that pollination and fertilization processes can occur within the flower. Figure 1 shows that anther development can be divided into two general phases. During phase 1, the morphology of the anther is established, cell and tissue differentiation occur, and microspore mother cells undergo meiosis. At the end of phase 1, the anther contains most of its specialized cells and tissues, and tetrads of microspores are present within the pollen sacs (Figure 1). During phase 2, pollen grains differentiate, the anther enlarges and is pushed upward in the flower by filament extension, and tissue degeneration, dehiscence, and pollen grain release occur (Figure 1). The cellular processes that regulate anther cell differentiation, establish anther tissue patterns, and cause the anther to switch from a histospecification program (phase l) to a cell degeneration and dehiscence program (phase 2) are not known. The developmental events leading to anther formation and pollen release are exquisitely timed and choreographed (Koltunow et al., 1990; Scott et al., 1991). Cell differentiation and dehiscence events occur in a precise chronological order that correlates with floral bud size (Koltunow et al., 1990; Scott et al., 1991). This permits the mechanisms responsible for cell-type differentiation, tissue degeneration, and cellspecific gene activation within the anther to be explored with relative Base. During the past few years, there has been an explosive burst of interest in anther biology, both as a system to dissect plant developmental processes at the molecular and genetic levels (Koltunow et al., 1990; Gasser, 1991; McCormick,

Anther developmental defects in Arabidopsis thaliana male-sterile mutants

Abstract We identified Arabidopsis thaliana sterility mutants by screening T-DNA and EMS-mutagenized lines and characterized several male-sterile mutants with defects specific for different anther processes. Approximately 44 and 855 sterile mutants were uncovered from the T-DNA and EMS screens, respectively. Several mutants were studied in detail with defects that included the establishment of anther morphology, microspore production, pollen differentiation, and anther dehiscence. Both non-dehiscencing and late-dehiscencing mutants were identified. In addition, pollenless mutants were observed with either apparent meiotic defects and/or abnormalities in cell layers surrounding the locules. Two mutant alleles were identified for the POLLENLESS3 locus which have defects in functional microspore production that lead to the degeneration of cells within the anther locules. pollenless3–1 contains a T-DNA insertion that co-segregates with the mutant phenotype and pollenless3–2 has a large deletion in the POLLENLESS3 gene. The POLLENLESS3 gene has no known counterparts in the GenBank, but encodes a protein containing putative nuclear localization and protein-protein interaction motifs. The POLLENLESS3 gene was shown recently to be the same as MS5, a previously described Arabidopsisthaliana male-sterility mutant. Three genes were identified in the POLLENLESS3 genomic region: GENEY, POLLENLESS3, and β9-TUBULIN. The segment of the Arabidopsisthaliana genome containing the POLLENLESS3 and β9-TUBULIN genes is duplicated and present on a different chromosome. Analysis of the POLLENLESS3 expression pattern determined that the 1.3-kb POLLENLESS3 mRNA is localized specifically within meiotic cells in the anther locules and that POLLENLESS3 mRNA is present only during late meiosis.

LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development.

The Arabidopsis LEAFY COTYLEDON2 (LEC2) gene is a central embryonic regulator that serves critical roles both early and late during embryo development. LEC2 is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination. We cloned the LEC2 gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors. We showed that LEC2 RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3. Ectopic, postembryonic expression of LEC2 in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings. Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development.

DEMETER, a DNA Glycosylase Domain Protein, Is Required for Endosperm Gene Imprinting and Seed Viability in Arabidopsis

We isolated mutations in Arabidopsis to understand how the female gametophyte controls embryo and endosperm development. For the DEMETER (DME) gene, seed viability depends only on the maternal allele. DME encodes a large protein with DNA glycosylase and nuclear localization domains. DME is expressed primarily in the central cell of the female gametophyte, the progenitor of the endosperm. DME is required for maternal allele expression of the imprinted MEDEA (MEA) Polycomb gene in the central cell and endosperm. Ectopic DME expression in endosperm activates expression of the normally silenced paternal MEA allele. In leaf, ectopic DME expression induces MEA and nicks the MEA promoter. Thus, a DNA glycosylase activates maternal expression of an imprinted gene in the central cell.

DEMETER DNA Glycosylase Establishes MEDEA Polycomb Gene Self-Imprinting by Allele-Specific Demethylation

MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.

Plant embryogenesis: Zygote to seed

Most differentiation events in higher plants occur continuously in the postembryonic adult phase of the life cycle. Embryogenesis in plants, therefore, is concerned primarily with establishing the basic shoot-root body pattern of the plant and accumulating food reserves that will be used by the germinating seedling after a period of embryonic dormancy within the seed. Recent genetics studies in Arabidopsis have identified genes that provide new insight into how embryos form during plant development. These studies, and others using molecular approaches, are beginning to reveal the underlying processes that control plant embryogenesis.

The Arabidopsis DELAYED DEHISCENCE1 Gene Encodes an Enzyme in the Jasmonic Acid Synthesis Pathway

delayed dehiscence1 is an Arabidopsis T-DNA mutant in which anthers release pollen grains too late for pollination to occur. The delayed dehiscence1 defect is caused by a delay in the stomium degeneration program. The gene disrupted in delayed dehiscence1 encodes 12-oxophytodienoate reductase, an enzyme in the jasmonic acid biosynthesis pathway. We rescued the mutant phenotype by exogenous application of jasmonic acid and obtained seed set from previously male-sterile plants. In situ hybridization studies showed that during the early stages of floral development, DELAYED DEHISCENCE1 mRNA accumulated within all floral organs. Later, DELAYED DEHISCENCE1 mRNA accumulated specifically within the pistil, petals, and stamen filaments. DELAYED DEHISCENCE1 mRNA was not detected in the stomium and septum cells of the anther that are involved in pollen release. The T-DNA insertion in delayed dehiscence1 eliminated both DELAYED DEHISCENCE1 mRNA accumulation and 12-oxophytodienoate reductase activity. These experiments suggest that jasmonic acid signaling plays a role in controlling the time of anther dehiscence within the flower.

Mutations in FIE, a WD Polycomb Group Gene, Allow Endosperm Development without Fertilization

A fundamental problem in biology is to understand how fertilization initiates reproductive development. Higher plant reproduction is unique because two fertilization events are required for sexual reproduction. First, a sperm must fuse with the egg to form an embryo. A second sperm must then fuse with the adjacent central cell nucleus that replicates to form an endosperm, which is the support tissue required for embryo and/or seedling development. Here, we report cloning of the Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) gene. The FIE protein is a homolog of the WD motif–containing Polycomb proteins from Drosophila and mammals. These proteins function as repressors of homeotic genes. A female gametophyte with a loss-of-function allele of fie undergoes replication of the central cell nucleus and initiates endosperm development without fertilization. These results suggest that the FIE Polycomb protein functions to suppress a critical aspect of early plant reproduction, namely, endosperm development, until fertilization occurs.