During plant embryogenesis, a simple body plan is established that consists of shoot meristem, cotyledons, hypocotyl, root, and root meristem along the apical–basal axis and a concentric arrangement of epidermis, subepidermal ground tissue, and

Embryogenesis effects the transition from the fertilized egg to the new multicellular generation, the seedling, which displays the basic body plan and organization of the plant. An apical-basal pattern along the main body axis of the embryo

The regulation of maturation (MAf)- and late embryogenesis (MA)-specific gene expression in dicots involves factors related to AB13, a seed-specific component of the abscisic acid signal transduction pathways from Arabidopsis. In French bean (Phaseolus vulgaris), the ABI3-like factor, PvALF, activates transcription from MAT promoters of phytohemagglutinin (DLEC2) and P-phaseolin (PHSB) genes. We describe theIegulatorgf MAT2 (ROM2) as a basic leucine zipper (bZIP) DNA binding protein that recognizes motifs with symmetric (ACGT) and asymmetric (ACCT) core elements present in both MAT promoters. ROM2 antagonizes trans-activation of the DLEC2 promoter by PvALF in transient expression assays. Repression was abolished by mutations that prevented binding of ROM2 to the DLEC2 seed enhancer region. Momover, a hybrid protein composed of a PvALF activation domain and the DNA binding and dimerization domain of ROM2 activated gene expression, indicating that ROM2 recognizes the DLEC2 enhancer in vivo; consequently, ROM2 functions as a DNA binding site-dependent repressor. Supershift analysis of nuclear proteins, using a ROM2-specific antibody, revealed an increase in ROM2 DNA binding activity during seed desiccation. A corresponding increase in ROM2 mRNA coincided with the period when DLEC2 mRNA levels declined in embryos. These results demonstrate developmental regulation of the ROM2 repressor and point to a role for this factor in silencing DLEC2 transcription during late embryogenesis.

have been edited and the authors have corrected proofs, but before the final, complete issue is published online. Early posting of articles reduces normal time to publication by several weeks.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, which is a tissue that supports embryo development. MEDEA ( MEA ) encodes an Arabidopsis SET domain Polycomb protein. Inheritance of a maternal loss-of-function mea allele results in embryo abortion and prolonged endosperm production, irrespective of the genotype of the paternal allele. Thus, only the maternal wild-type MEA allele is required for proper embryo and endosperm development. To understand the molecular mechanism responsible for the parent-of-origin effects of mea mutations on seed development, we compared the expression of maternal and paternal MEA alleles in the progeny of crosses between two Arabidopsis ecotypes. Only the maternal MEA mRNA was detected in the endosperm from seeds at the torpedo stage and later. By contrast, expression of both maternal and paternal MEA alleles was observed in the embryo from seeds at the torpedo stage and later, in seedling, leaf, stem, and root. Thus, MEA is an imprinted gene that displays parent-of-origin-dependent monoallelic expression specifically in the endosperm. These results suggest that the embryo abortion observed in mutant mea seeds is due, at least in part, to a defect in endosperm function. Silencing of the paternal MEA allele in the endosperm and the phenotype of mutant mea seeds supports the parental conflict theory for the evolution of imprinting in plants and mammals. INTRODUCTION Flowering plant reproduction is characterized by the fertilization of two cells (reviewed in In Arabidopsis, the fertilized egg undergoes an asymmetric transverse cleavage to produce a small cytoplasmically dense apical cell and a large vacuolated basal cell. The apical cell divides many times and generates cells that comprise most of the embryonic structures found in the mature seed Embryo and endosperm are genetically identical except for their ratio of maternal-to-paternal genomes, which are 1:1 and 2:1, respectively. However, the pattern of endosperm development is quite distinct from that of the embryo. The fertilized central cell nucleus undergoes a series of mitotic divisions to produce a syncytium of nuclei that surround the embryo and fill the expanding central cell 1946 The Plant Cell begins, and endosperm cytoplasm and nuclei are sequestered into discrete cells The endosperm is thought to interact with the embryo during seed development. The chalazal-oriented portion of the endosperm is located next to a pad of maternal proliferative tissue that is adjacent to the vascular tissue. The relative position of these tissues has led to the suggestion that the chalazal-oriented endosperm may be involved in nutrient transfer into the developing seed The Arabidopsis MEDEA ( MEA ) gene encodes a SET domain Polycomb protein Genetic analysis has shown that only the maternal wildtype MEA allele, and not the paternal MEA allele, is required for proper embryo and endosperm development To understand the molecular basis for the parent-of-origin effects of mea mutations on reproductive development, we monitored expression of the maternal and paternal wild-type MEA alleles in the F 1 progeny of crosses between two Arabidopsis ecotypes. Here, we show that expression of the maternal MEA allele is predominant in the endosperm from seeds at the torpedo stage and later. By contrast, both maternal and paternal MEA mRNAs were detected in approximately equal concentrations in the embryo from seeds at the torpedo stage, and later, in seedling, leaf, stem, and root. Thus, the paternally derived MEA allele is silenced specifically within the endosperm. These results suggest that mea embryo abortion is due, at least in part, to a defect in endosperm function. Our results characterizing the phenotype of loss-of-function mea mutants RESULTS Strategy for Measuring Maternal-and Paternal-Specific MEA mRNA Levels To test the hypothesis that paternal gene silencing is responsible for parent-of-origin-specific effects of mea mutations on seed and plant development, we measured maternal and paternal MEA mRNA levels in reproductive and vegetative tissues. As shown in Pattern of Maternal and Paternal MEA Allele Expression during Plant Development Previously, it was shown that the MEA gene is expressed in siliques with developing seeds During double fertilization, the embryo inherits one paternal MEA allele and the endosperm inherits another paternal MEA allele. To determine whether one or both paternal MEA alleles are expressed during seed development, we harvested F 1 seeds from reciprocal crosses between L er and RLD plants at 6, 7, and 8 days after pollination. Seeds As shown in 1948 The Plant Cell dissected from seeds 7 days after pollination (data not shown). Taken together, these results show that expression of the paternal MEA allele is greatly reduced in the endosperm. To conclude that the paternal MEA allele is specifically silenced in the endosperm, we had to show that maternal MEA mRNA accumulates in the endosperm and not merely in the seed coat, which is maternal tissue. To address this issue, we crossed female RLD plants to male Ler plants and harvested F 1 seeds 7 days after pollination. Embryos were dissected from the F 1 seeds. Because the Arabidopsis seed coat has considerable tensile strength, it was possible to separate the seed coat from the endosperm. In this way, endosperm tissue that was visibly free from contaminating seed coat could be isolated ( Both maternal and paternal MEA alleles are expressed during embryogenesis (C) Accumulation of allele-specific MEA mRNA in dissected tissues. RLD females were crossed with Ler males. Seeds were harvested 7 days after pollination, corresponding to the walking stick embryo stage Paternal MEA Allele Silencing 1949 whether both maternal and paternal MEA alleles are expressed after germination in vegetative tissues, we performed reciprocal crosses between Ler and RLD plants, and RNA was isolated from F 1 seedling, rosette leaf, stem, and root tissue. As shown in DISCUSSION The Role of the Endosperm in Parent-of-Origin Effects on Seed Development Loss-of-function mutations in the MEA gene display parentof-origin effects on seed development Although our experiments suggest that paternal silencing in the endosperm is important, other mechanisms may also contribute to the parent-of-origin effects of mea mutations on seed development. First, it is possible that essential maternal MEA expression takes place before fertilization within the female gametophyte. Second, the paternal MEA allele may be silenced at an important period within the embryo before the torpedo stage, which is the earliest stage we were able to investigate

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences thst accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGLlL (for- AGAMOUS-like). AGL15 shows a nove1 pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL75 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGLl5 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGLlL is likely to be an important component of the regulatory circuitry directing seedspecific processes in the developing embryo.

OEP16, a channel protein of the outer membrane of chloroplasts, has been implicated in amino acid transport and in the substrate-dependent import of protochlorophyllide oxidoreductase A. Two major clades of OEP16related sequences were identified in land plants (OEP16-L and OEP16-S), which arose by a gene duplication event predating the divergence of seed plants and bryophytes. Remarkably, in angiosperms, OEP16-S genes evolved by gaining an additional exon that extends an interhelical loop domain in the pore-forming region of the protein. We analysed the sequence, structure and expression of the corresponding Arabidopsis genes (atOEP16-S and atOEP16-L) and demonstrated that following duplication, both genes diverged in terms of expression patterns and coding sequence. AtOEP16-S, which contains multiple G-box ABA-responsive elements (ABREs) in the promoter region, is regulated by ABI3 and ABI5 and is strongly expressed during the maturation phase in seeds and pollen grains, both desiccation-tolerant tissues. In contrast, atOEP-L, which lacks promoter ABREs, is expressed predominantly in leaves, is induced strongly by low-temperature stress and shows weak induction in response to osmotic stress, salicylic acid and exogenous ABA. Our results indicate that gene duplication, exon gain and regulatory sequence evolution each played a role in the divergence of OEP16 homologues in plants.

Seed storage proteins (SSPs) are important for seed germination and early seedling growth. We studied the accumulation and degradation profiles of four major Arabidopsis 12S SSPs using a 2-DE scheme combined with mass spectrometric methods. On the 2-DE map of 23 dpa (days post anthesis) siliques, 48 protein spots were identified as putative full-length or partial α, β subunits. Only 9 of them were found in 12 dpa siliques with none in younger than 8 dpa siliques, indicating that the accumulation of 12S SSPs started after the completion of cell elongation processes both in siliques and in developing seeds. The length and strength of transcription activity for each gene determined the final contents of respective SSP. At the beginning of imbibition, 68 SSP spots were identified while only 2 spots were found at the end of the 4 d germination period, with α subunits degraded more rapidly than the β subunits. The CRC α subunit was found to degrade from its C-terminus with conserved sequence motifs. Our data provide an important basis for understanding the nutritional value of developing plant seeds and may serve as a useful platform for other species.

have been edited and the authors have corrected proofs, but before the final, complete issue is published online. Early posting of articles reduces normal time to publication by several weeks.

Arabidopsis LEAFY COTYLEDON1 (LEC1) is a critical regulator required for normal development during the early and late phases of embryogenesis that is sufficient to induce embryonic development in vegetative cells. LEC1 encodes a HAP3 subunit of the CCAAT binding transcription factor. We show that the 10 Arabidopsis HAP3 (AHAP3) subunits can be divided into two classes based on sequence identity in their central, conserved B domain. LEC1 and its most closely related subunit, LEC1-LIKE (L1L), constitute LEC1-type AHAP3 subunits, whereas the remaining AHAP3 subunits are designated non-LEC1-type. Similar to LEC1, L1L is expressed primarily during seed development. However, suppression of L1L gene expression induced defects in embryo development that differed from those of lec1 mutants, suggesting that LEC1 and L1L play unique roles in embryogenesis. We show that L1L expressed under the control of DNA sequences flanking the LEC1 gene suppressed genetically the lec1 mutation, suggesting that the LEC1-type B domains of L1L and LEC1 are critical for their function in embryogenesis. Our results also suggest that LEC1-type HAP3 subunits arose from a common origin uniquely in plants. Thus, L1L, an essential regulator of embryo development, defines a unique class of plant HAP3 subunits.