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Developmental transitions are typically tightly controlled at the transcriptional level. Two of these transitions involve the induction of the embryo maturation program midway through seed development and its repression during the vegetative phase of plant growth. Very little is known about the factors responsible for this regulation during early embryogenesis, and only a couple of transcription factors have been characterized as repressors during the postgerminative phase. Arabidopsis 6b‐INTERACTING PROTEIN‐LIKE1 (ASIL1), a trihelix transcription factor, has been proposed to repress maturation both embryonically and postembryonically. Preliminary data also suggested that its closest paralog, ASIL2, might play a role as well. We used a transcriptomic approach, coupled with phenotypical observations, to test the hypothesis that ASIL1 and ASIL2 redundantly turn off maturation during both phases of growth. Our results indicate that, contrary to what was previously published, neither of the ASIL genes plays a role in the regulation of maturation, at any point during plant development. Analyses of gene ontology (GO)‐enriched terms and published transcriptomic datasets suggest that these genes might be involved in responses during the vegetative phase to certain biotic and abiotic stresses.

The LEAFY COTYLEDON1 (LEC1) transcription factor is a central regulator of seed development, because it controls diverse biological programs during seed development, such as embryo morphogenesis, photosynthesis, and seed maturation. To understand how LEC1 regulates different gene sets during development, we explored the possibility that LEC1 acts in combination with other transcription factors. We identified and compared genes that are directly transcriptionally regulated by ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3), BASIC LEUCINE ZIPPER67 (bZIP67), and ABA INSENSITIVE3 (ABI3) with those regulated by LEC1. We showed that LEC1 operates with specific sets of transcription factors to regulate different gene sets and, therefore, distinct developmental processes. Thus, LEC1 controls diverse processes through its combinatorial interactions with other transcription factors. DNA binding sites for the transcription factors are closely clustered in genomic regions upstream of target genes, defining cis -regulatory modules that are enriched for DNA sequence motifs that resemble sequences known to be bound by these transcription factors. Moreover, cis -regulatory modules for genes regulated by distinct transcription factor combinations are enriched for different sets of DNA motifs. Expression assays with embryo cells indicate that the enriched DNA motifs are functional cis elements that regulate transcription. Together, the results suggest that combinatorial interactions between LEC1 and other transcription factors are mediated by cis -regulatory modules containing clustered cis elements and by physical interactions that are documented to occur between the transcription factors.

Seed development is a complex period of the flowering plant life cycle. After fertilization, the three main regions of the seed, embryo, endosperm and seed coat, undergo a series of developmental processes that result in the production of a mature seed that is developmentally arrested, desiccated, and metabolically quiescent. These processes are highly coordinated, both temporally and spatially, to ensure the proper growth and development of the seed. The transcription factor, LEAFY COTYLEDON1 (LEC1), is a central regulator that controls several aspects of embryo and endosperm development, including embryo morphogenesis, photosynthesis, and storage reserve accumulation. Thus, LEC1 regulates distinct sets of genes at different stages of seed development. Despite its critical importance for seed development, an understanding of the mechanisms underlying LEC1's multifunctionality is only beginning to be obtained. Recent studies describe the roles of specific transcription factors and the hormones, gibberellic acid and abscisic acid, in controlling the activity and transcriptional specificity of LEC1 across seed development. Moreover, studies indicate that LEC1 acts as a pioneer transcription factor to promote epigenetic reprogramming during embryogenesis. In this review, we discuss the mechanisms that enable LEC1 to serve as a central regulator of seed development.

MicroRNAs (miRNAs) are important regulatory molecules in eukaryotic organisms. Existing methods for the identification of mature miRNA sequences in plants rely extensively on the search for stem-loop structures, leading to high false negative rates. Here, we describe a probabilistic method for ranking putative plant miRNAs using a naïve Bayes classifier and its publicly available implementation. We use a number of properties to construct the classifier, including sequence length, number of observations, existence of detectable predicted miRNA* sequences, the distribution of nearby reads and mapping multiplicity. We apply the method to small RNA sequence data from soybean, peach, Arabidopsis and rice and provide experimental validation of several predictions in soybean. The approach performs well overall and strongly enriches for known miRNAs over other types of sequences. By utilizing a Bayesian approach to rank putative miRNAs, our method is able to score miRNAs that would be eliminated by other methods, such as those that have low counts or lack detectable miRNA* sequences. As a result, we are able to detect several soybean miRNA candidates, including some that are 24 nucleotides long, a class that is almost universally eliminated by other methods.

Significance We scanned soybean and Arabidopsis seed genomes for hypomethylated regions, or DNA methylation valleys (DMVs), present in mammalian cells. Seeds contain DMV regions that have, The precise mechanisms that control gene activity during seed development remain largely unknown. Previously, we showed that several genes essential for seed development, including those encoding storage proteins, fatty acid biosynthesis enzymes, and transcriptional regulators (e.g., ABI3, FUS3) are located within hypomethylated regions of the soybean genome. These hypomethylated regions are similar to the DNA methylation valleys (DMVs), or canyons, found in mammalian cells. Here, we address the question of the extent to which DMVs are present within seed genomes and what role they might play in seed development. We scanned soybean and Arabidopsis seed genomes from postfertilization through dormancy and germination for regions that contain

The funiculus anchors the structurally complex seed to the maternal plant, and is the only direct route of transport for nutrients and maternal signals to the seed. While our understanding of seed development is becoming clearer, current understanding of the genetics and cellular mechanisms that contribute to funiculus development is limited. Using laser microdissection combined with global RNA-profiling experiments we compared the genetic profiles of all maternal and zygotic regions and subregions during seed development. We found that the funiculus is a dynamic region of the seed that is enriched for mRNAs associated with hormone metabolism, molecular transport, and metabolic activities corresponding to biological processes that have yet to be described in this maternal seed structure. We complemented our genetic data with a complete histological analysis of the funiculus from the earliest stages of development through to seed maturation at the light and electron microscopy levels. The anatomy revealed signs of photosynthesis, the endomembrane system, cellular respiration, and transport within the funiculus, all of which supported data from the transcriptional analysis. Finally, we studied the transcriptional programming of the funiculus compared to other seed subregions throughout seed development. Using newly designed in silico algorithms, we identified a number of transcriptional networks hypothesized to be responsible for biological processes like auxin response and glucosinolate biosynthesis found specifically within the funiculus. Taken together, patterns of gene activity and histological observations reveal putative functions of the understudied funiculus region and identify predictive transcriptional circuits underlying these biological processes in space and time.

Significance We describe the spatial and temporal profiles of soybean and Arabidopsis seed methylomes during development. CHH methylation increases globally from fertilization through dormancy in all seed parts, decreases following germination, and targets primarily transposons. By contrast, CG- and CHG-context methylation remains constant throughout seed development. Mutant seeds lacking non-CG methylation develop normally, but have a set of up-regulated transposon RNAs suggesting that the CHH methylation increase may be a failsafe mechanism to reinforce transposon silencing. Major classes of seed genes have similar methylation profiles, whether they are active or not. Our results suggest that soybean and Arabidopsis seed methylomes are similar, and that DNA methylation does not play a significant role in regulating many genes important for seed development., We profiled soybean and Arabidopsis methylomes from the globular stage through dormancy and germination to understand the role of methylation in seed formation. CHH methylation increases significantly during development throughout the entire seed, targets primarily transposable elements (TEs), is maintained during endoreduplication, and drops precipitously within the germinating seedling. By contrast, no significant global changes in CG- and CHG-context methylation occur during the same developmental period. An Arabidopsis ddcc mutant lacking CHH and CHG methylation does not affect seed development, germination, or major patterns of gene expression, implying that CHH and CHG methylation does not play a significant role in seed development or in regulating seed gene activity. By contrast, over 100 TEs are transcriptionally de-repressed in ddcc seeds, suggesting that the increase in CHH-context methylation may be a failsafe mechanism to reinforce transposon silencing. Many genes encoding important classes of seed proteins, such as storage proteins, oil biosynthesis enzymes, and transcription factors, reside in genomic regions devoid of methylation at any stage of seed development. Many other genes in these classes have similar methylation patterns, whether the genes are active or repressed. Our results suggest that methylation does not play a significant role in regulating large numbers of genes important for programming seed development in both soybean and Arabidopsis. We conclude that understanding the mechanisms controlling seed development will require determining how cis-regulatory elements and their cognate transcription factors are organized in genetic regulatory networks.

We profiled soybean and Arabidopsis methylomes from the globular stage through dormancy and germination to understand the role of methylation in seed formation. CHH methylation increases significantly during development throughout the entire seed, targets primarily transposable elements (TEs), is maintained during endoreduplication, and drops precipitously within the germinating seedling. By contrast, no significant global changes in CG- and CHG-context methylation occur during the same developmental period. An Arabidopsis ddcc mutant lacking CHH and CHG methylation does not affect seed development, germination, or major patterns of gene expression, implying that CHH and CHG methylation does not play a significant role in seed development or in regulating seed gene activity. By contrast, over 100 TEs are transcriptionally de-repressed in ddcc seeds, suggesting that the increase in CHH-context methylation may be a failsafe mechanism to reinforce transposon silencing. Many genes encoding important classes of seed proteins, such as storage proteins, oil biosynthesis enzymes, and transcription factors, reside in genomic regions devoid of methylation at any stage of seed development. Many other genes in these classes have similar methylation patterns, whether the genes are active or repressed. Our results suggest that methylation does not play a significant role in regulating large numbers of genes important for programming seed development in both soybean and Arabidopsis. We conclude that understanding the mechanisms controlling seed development will require determining how cis-regulatory elements and their cognate transcription factors are organized in genetic regulatory networks.

The seed consists of three distinct regions, the zygotic embryo and endosperm and the maternal seed coat. Development of the seed requires co-ordinated growth and differentiation within each of these regions. Although the major events that occur during seed development have been studied extensively at the anatomical, physiological and molecular level, a comprehensive understanding of gene activity in each seed region has been lacking. DNA microarrays and extensive sequencing of cDNA libraries permit genome-wide analyses of mRNA populations of biological samples. We review recent studies of genome-wide gene activity that illustrate approaches used to provide new insights into the processes that occur during seed development.

Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in Arabidopsis thaliana endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes as well as by Polycomb group proteins. Currently, only 11 imprinted A. thaliana genes are known. Here, we use extensive sequencing of cDNA libraries to identify 9 paternally expressed and 34 maternally expressed imprinted genes in A. thaliana endosperm that are regulated by the DNA-demethylating glycosylase DEMETER, the DNA methyltransferase MET1, and/or the core Polycomb group protein FIE. These genes encode transcription factors, proteins involved in hormone signaling, components of the ubiquitin protein degradation pathway, regulators of histone and DNA methylation, and small RNA pathway proteins. We also identify maternally expressed genes that may be regulated by unknown mechanisms or deposited from maternal tissues. We did not detect any imprinted genes in the embryo. Our results show that imprinted gene expression is an extensive mechanistically complex phenomenon that likely affects multiple aspects of seed development.

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

Two fundamental aspects of plant development are the maturation phase of embryo development in seed plants and totipotency via somatic embryogenesis (SE). The LEAFY COTYLEDON (LEC) transcription factors (TFs) establish environments that promote cellular processes characteristic of the maturation phase and the initiation of somatic embryo formation. Based on recent studies, we and others propose that specific target genes activated by the LEC TFs underlie, in part, their roles in the maturation phase and SE. We also propose that the effect of LEC TFs on the balance of abscisic acid to gibberellic acid might link their roles in totipotency and the maturation phase.

Seeds are essential for flowering plant reproduction because they protect, nourish, and contain the developing embryo that represents the next sporophytic generation. In addition, seeds contain energy resources that sustain the young sporophyte during germination before photosynthesis begins. In

Plants sense the foliar shade of competitors and alter their developmental programs through the shade-avoidance response. Internode and petiole elongation, and changes in overall leaf area and leaf mass per area, are the stereotypical architectural responses to foliar shade in the shoot. However, changes in leaf shape and complexity in response to shade remain incompletely, and qualitatively, described. Using a meta-analysis of more than 18,000 previously published leaflet outlines, we demonstrate that shade avoidance alters leaf shape in domesticated tomato (Solanum lycopersicum) and wild relatives. The effects of shade avoidance on leaf shape are subtle with respect to individual traits but are combinatorially strong. We then seek to describe the developmental origins of shade-induced changes in leaf shape by swapping plants between light treatments. Leaf size is light responsive late into development, but patterning events, such as stomatal index, are irrevocably specified earlier. Observing that shade induces increases in shoot apical meristem size, we then describe gene expression changes in early leaf primordia and the meristem using laser microdissection. We find that in leaf primordia, shade avoidance is not mediated through canonical pathways described in mature organs but rather through the expression of KNOTTED1-LIKE HOMEOBOX and other indeterminacy genes, altering known developmental pathways responsible for patterning leaf shape. We also demonstrate that shade-induced changes in leaf primordium gene expression largely do not overlap with those found in successively initiated leaf primordia, providing evidence against classic hypotheses that shaded leaf morphology results from the prolonged production of juvenile leaf types.

We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and INDUCER OF C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT-BINDING FACTOR EXPRESSION1/SCREAM2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development.

DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.

We identified the Arabidopsis (Arabidopsis thaliana) tanmei/emb2757 (tan) mutation that causes defects in both embryo and seedling development. tan mutant embryos share many characteristics with the leafy cotyledon (lec) class of mutants in that they accumulate anthocyanin, are intolerant of desiccation, form trichomes on cotyledons, and have reduced accumulation of storage proteins and lipids. Thus, TAN functions both in the early and late phases of embryo development. Moreover, the TAN and LEC genes interact synergistically, suggesting that they do not act in series in the same genetic pathway but, rather, that they have overlapping roles during embryogenesis. tan mutants die as embryos, but immature mutant seeds can be germinated in culture. However, tan mutant seedlings are defective in shoot and root development, their hypocotyls fail to elongate in the dark, and they die as seedlings. We isolated the TAN gene and showed that the predicted polypeptide has seven WD repeat motifs, suggesting that TAN forms complexes with other proteins. Together, these results suggest that TAN interacts with other proteins to control many aspects of embryo development.

The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65-94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.

The MEA Polycomb gene is imprinted in the Arabidopsis endosperm. DME DNA glycosylase activates maternal MEA allele expression in the central cell of the female gametophyte, the progenitor of the endosperm. Maternal mutant dme or mea alleles result in seed abortion. We identified mutations that suppress dme seed abortion and found that they reside in the MET1 methyltransferase gene, which maintains cytosine methylation. Seeds with maternal dme and met1 alleles survive, indicating that suppression occurs in the female gametophyte. Suppression requires a maternal wild-type MEA allele, suggesting that MET1 functions upstream of, or at, MEA. DME activates whereas MET1 suppresses maternal MEA::GFP allele expression in the central cell. MET1 is required for DNA methylation of three regions in the MEA promoter in seeds. Our data suggest that imprinting is controlled in the female gametophyte by antagonism between the two DNA-modifying enzymes, MET1 methyltransferase and DME DNA glycosylase.

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.

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.

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 otherraspberry 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 RASPBERRY3protein 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.

Seeds play an integral role in the global food supply and account for more than 70% of the calories that we consume on a daily basis. To meet the demands of an increasing population, scientists are turning to seed genomics research to find new and innovative ways to increase food production. Seed genomics is evolving rapidly, and the information produced from seed genomics research has exploded over the past two decades. Advances in modern sequencing strategies that profile every molecule in every cell, tissue and organ of model and the emergence of new model systems has provided the tools necessary to understand all of the biological processes underlying seed development. Despite these advances, the analyses and mining of existing seed genomics data remain a monumental task for plant biologists. This review summarizes seed region and subregion genomic data that are currently available for existing and emerging oilseed models. We provide insight into the development of tools on how to analyze large-scale datasets.

Summary Embryogenesis is a critical period of the higher plant life cycle during which a single-celled zygote undergoes a series of differentiation events, leading to formation of a mature embryo that is generally desiccated and quiescent metabolically. Embryo development consists of two conceptually distinct phases: the early morphogenesis phase and the late maturation phase. Arabidopsis LEAFY COTYLEDON genes are central regulators of embryogenesis that play key roles in processes that occur during both the early and late phases. Evidence is discussed which suggests that the LEAFY COTYLEDON genes coordinate the morphogenesis and maturation phases and confer embryogenic competence to cells.

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.

Higher plant reproduction is unique because 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, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in Arabidopsis , f644 , that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant f644 egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the F644 gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that F644 is identical to MEDEA ( MEA ), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998) Science 280, 446–450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.

Embryogenesis is a critical stage of the sporophytic life cycle during which the basic body plan of the plant is established. Although positional information is implicated to play a major role in determining embryo cell fate, little is known about the nature of positional signals. Recent studies show that the monopterous and hobbit mutations reveal signaling during patterning of the embryonic axis. The LEAFY COTYLEDON1 and PICKLE genes have been implicated to play important roles in controlling embryo development.

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco ( Nicotiana tabacum ) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

A T-DNA-tagged, embryo-defective Arabidopsis thaliana mutant, fist, was identified and shown to exhibit defects in nuclear positioning and cell division orientation beginning at the four-cell stage of the embryo proper. Cell division orientation was randomised, with each embryo exhibiting a different pattern. Periclinal divisions did not occur after the eight-cell embryo proper stage and fist embryos lacked a histologically distinct protoderm layer. Terminal embryos resembled globular-stage embryos, but were a disorganised mass containing 30–100 cells. Some terminal embryos (5%) developed xylem-like elements in outer surface cells, indicating that the fist mutation affects radial pattern. A soybean β-conglycinin seed storage protein gene promoter, active in wild-type embryos from heart stage to maturity, was also active in terminal fist embryos despite their disorganised globular state. This indicated that some pathways of cellular differentiation in fist embryos proceed independently of both organised division plane orientation and normal morphogenesis. Endosperm morphogenesis in seeds containing terminal fist embryos was arrested at one of three distinct developmental stages and appeared unlinked to fist embryo morphogenesis. The β-conglycinin seed storage protein gene promoter, normally active in cellularised wild-type endosperm, was inactive in fist endosperm, indicating abnormal development of fist endosperm at the biochemical level. These data indicate that the fist mutation, either directly or indirectly, results in defects in cell division orientation during the early stages of Arabidopsis embryo development. Other aspects of the fist phenotype, such as defects in endosperm development and radial pattern formation, may be related to abnormal cell division orientation or may occur as pleiotropic effects of the fist mutation.

Peroxisome biogenesis requires that proteins be transported from their site of synthesis in the cytoplasm to their final location in the peroxisome matrix or membrane. Glyoxysomes are a class of peroxisomes found primarily in germinating seedlings and are involved in mobilizing fatty acids via the glyoxylate cycle and the β-oxidation pathway. We have used an in vitro assay to study the mechanism(s) of import of proteins into glyoxysomes. Results from this assay indicate that the transport process is time- and temperature- dependent and is specific for peroxisomal proteins. Isocitrate lyase, a glyoxysomal protein, and the leaf-type peroxisomal enzyme glycolate oxidase (GLO) were transported into pumpkin (Cucurbita pepo) glyoxysomes with no apparent differences in efficiency of import. Thus, this in vitro assay appears to be physiologically relevant and correlates well with expected in vivo conditions. Protein import was also energy-dependent and saturable. Nonradiolabeled GLO competed with radiolabeled, in vitro-synthesized GLO for components of the import machinery. Finally, pretreatment of the isolated glyoxysomes with protease virtually abolished subsequent import of GLO. Taken together, these results indicate that a proteinaceous receptor is involved in the import of peroxisomal proteins.

The overall goal of the project was to define the cellular processes that underlie embryo development in plants at a mechanistic level. Our studies focused on a critical transcriptional regulator, Arabidopsis LEAFY COTYLEDON (LEC1), that is necessary and sufficient to induce processes required for embryo development. Because LEC1 regulates lipid accumulation during the maturation phase of embryo development, information about LEC1 may be useful in designing approaches to enhance biofuel production in plants. During the tenure of this project, we determined the molecular mechanisms by which LEC1 acts as a transcription factor in embryos. We also identified genes directly regulated by LEC1 and showed that many of these genes are involved in maturation processes. This information has been useful in dissecting the gene regulatory networks controlling embryo development. Finally, LEC1 is a novel isoform of a transcription factor that is conserved among eukaryotes, and LEC1 is active primarily in seeds. Therefore, we determined that the LEC1-type transcription factors first appeared in lycophytes during land plant evolution. Together, this study provides basic information that has implications for biofuel production.

LEAFY COTYLEDON1 (LEC1) is a central regulator of seed development that plays a key role in controlling the maturation phase during which storage macromolecules accumulate and the embryo becomes tolerant of desiccation. We queried the genomes of seedless plants and identified a LEC1 homolog in the lycophyte, Selaginella moellendorffii , but not in the bryophyte, Physcomitrella patens . Genetic suppression experiments indicated that Selaginella LEC1 is the functional ortholog of Arabidopsis LEC1. Together, these results suggest that LEC1 originated at least 30 million years before the first seed plants appeared in the fossil record. The accumulation of Selaginella LEC1 RNA primarily in sexual and asexual reproductive structures suggests its involvement in cellular processes similar to those that occur during the maturation phase of seed development.

We analyzed DNA sequences that regulate the expression of an isocitrate lyase gene from Brassica napus L. during late embryogenesis and during postgerminative growth to determine whether glyoxysomal function is induced by a common mechanism at different developmental stages. beta-Glucuronidase constructs were used both in transient expression assays in B. napus and in transgenic Arabidopsis thaliana to identify the segments of the isocitrate lyase 5' flanking region that influence promoter activity. DNA sequences that play the principal role in activating the promoter during post-germinative growth are located more than 1,200 bp upstream of the gene. Distinct DNA sequences that were sufficient for high-level expression during late embryogenesis but only low-level expression during postgerminative growth were also identified. Other parts of the 5' flanking region increased promoter activity both in developing seed and in seedlings. We conclude that a combination of elements is involved in regulating the isocitrate lyase gene and that distinct DNA sequences play primary roles in activating the gene in embryos and in seedlings. These findings suggest that different signals contribute to the induction of glyoxysomal function during these two developmental stages. We also showed that some of the constructs were expressed differently in transient expression assays and in transgenic plants.

Seeds are complex structures that consist of the embryo, endosperm, and seed-coat regions that are of different ontogenetic origins, and each region can be further divided into morphologically distinct subregions. Despite the importance of seeds for food, fiber, and fuel globally, little is known of the cellular processes that characterize each subregion or how these processes are integrated to permit the coordinated development of the seed. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets offer the most comprehensive description of gene activity in seeds with high spatial and temporal resolution, providing unique insights into the function of understudied seed regions. Global comparisons of mRNA populations reveal unexpected overlaps in the functional identities of seed subregions. Analyses of coexpressed gene sets suggest that processes that regulate seed size and filling are coordinated across several subregions. Predictions of gene regulatory networks based on the association of transcription factors with enriched DNA sequence motifs upstream of coexpressed genes identify regulators of seed development. These studies emphasize the utility of these datasets as an essential resource for the study of seed biology.

The seed maturation programme occurs only during the late phase of embryo development, and repression of the maturation genes is pivotal for seedling development. However, mechanisms that repress the expression of this programme in vegetative tissues are not well understood. A genetic screen was performed for mutants that express maturation genes in leaves. Here, it is shown that mutations affecting SDG8 (SET DOMAIN GROUP 8), a putative histone methyltransferase, cause ectopic expression of a subset of maturation genes in leaves. Further, to investigate the relationship between SDG8 and the Polycomb Group (PcG) proteins, which are known to repress many developmentally important genes including seed maturation genes, double mutants were made and formation of somatic embryos was observed on mutant seedlings with mutations in both SDG8 and EMF2 (EMBRYONIC FLOWER 2). Analysis of histone methylation status at the chromatin sites of a number of maturation loci revealed a synergistic effect of emf2 and sdg8 on the deposition of the active histone mark which is the trimethylation of Lys4 on histone 3 (H3K4me3). This is consistent with high expression of these genes and formation of somatic embryos in the emf2 sdg8 double mutants. Interestingly, a double mutant of sdg8 and vrn2 (vernalization2), a paralogue of EMF2, grew and developed normally to maturity. These observations demonstrate a functional cooperative interplay between SDG8 and an EMF2-containing PcG complex in maintaining vegetative cell identity by repressing seed genes to promote seedling development. The work also indicates the functional specificities of PcG complexes in Arabidopsis.

To investigate if pollen possesses glyoxysomal function, we analyzed the activities of isocitrate lyase and malate synthase genes. Because the activities of these enzymes were exceedingly low in pollen extracts, we constructed fusion genes encoding [beta]-glucuronidase (GUS) that are regulated by isocitrate lyase or malate synthase promoters from Brassica napus L. to increase the sensitivity of our assays. Expression of the fusion genes in transgenic tobacco was qualitatively similar to that of the endogenous genes; GUS activity was low in dry seeds, maximal in seedlings, and very low or undetectable in leaves, indicating that the promoters are regulated correctly. We showed that isocitrate lyase and malate synthase genes are active at specific stages of pollen development and that their activities are not enhanced during pollen germination in transgenic tobacco. We also confirmed that the endogenous genes are active by showing that the corresponding mRNAs could be detected in pollen at specific stages of development. The activation of the isocitrate lyase and malate synthase genes suggests that glyoxysomal function is induced during pollen development.

We have analyzed the structure of genes encoding the glyoxylate cycle enzyme isocitrate lyase from Brassica napus L. and their expression during embryogeny and postgermination. Restriction mapping, nucleotide sequence, and DNA gel blot hybridization analyses of cDNA and genomic clones indicated that there are approximately six isocitrate lyase genes in the B. napus genome that can be divided into at least two subfamilies based upon their divergence in 5' and 3' untranslated regions. We showed previously that isocitrate lyase mRNA accumulates during late embryogeny and postgermination. Here, we present results which indicate that several isocitrate lyase genes are expressed at both stages of development. First, gene-specific probes were used to show that mRNAs encoded by representatives of both gene subfamilies accumulated in both late maturation stage embryos and in seedlings of B. napus. Second, a single B. napus isocitrate lyase gene, together with 3.5 kb and 1.4 kb of 5' and 3' flanking regions, respectively, was expressed in both embryos and seedlings of transgenic tobacco plants. The results indicated that accumulation of isocitrate lyase in late embryogeny and postgermination does not result from the alternate expression of distinct members of the gene family.

Zygotic embryogenesis in higher plants describes the developmental period in which the zygote undergoes a series of differentiation events, leading to the formation of a mature embryo. Establishment of the major embryonic organs and shoot and root apical meristems occur though partitioning events along the apical–basal axis, and many of these events are guided by the hormone auxin. Formation of the three embryonic tissue systems occurs along a radial axis perpendicular to the apical–basal axis. The mature zygotic embryo is generally developmentally arrested, metabolically quiescent and enclosed within maternal tissues of the seed. Somatic cells can be induced to divert from their normal fate and develop into embryos in a process termed somatic embryogenesis. Auxin and other plant hormones appear to play critical roles in inducing embryogenic competence. Zygotic and somatic embryogenesis represent parallel developmental programs in which cells acquire embryogenic cell fate and develop into mature embryos. Key Concepts: Embryo development can be divided into two phases: the morphogenesis phase in which the basic body plan of the embryo is established, and the maturation phase in which the embryo becomes tolerant of desiccation and accumulates storage macromolecules such as lipids, proteins and starch. During its development, the embryo is divided into distinct domains along its apical–basal and radial axes. Plant embryo formation represents a series of partitioning events in which organs and tissues are formed from larger domains. Cell fate within the embryo is largely dependent on the position of the cell within the embryo body. Plant cells are totipotent and have the ability to regenerate a fully differentiated organism. Somatic embryos follow similar developmental patterns to their zygotic counterparts, and they occur naturally or are induced in culture. Establishment of embryogenic competence occurs before the formation of the somatic embryo. Keywords: apical–basal axis; auxin; meristems; patterning; radial axis; totipotency; zygote

The plastid is an organelle vital to all photosynthetic and some non-photosynthetic eukaryotes. In the model plant Arabidopsis thaliana, a number of nuclear genes encoding plastid proteins have been found to be necessary for embryo development. However, the exact roles of plastids in this process remain largely unknown. Here we use publicly available datasets to obtain insights into the relevance of plastid activities to A. thaliana embryogenesis. By searching the SeedGenes database (http://www.seedgenes.org) and recent literature, we found that, of the 339 non-redundant genes required for proper embryo formation, 108 genes likely encode plastid-targeted proteins. Nineteen of these genes are necessary for development of preglobular embryos and/or their conversion to globular embryos, of which 13 genes encode proteins involved in non-photosynthetic metabolism. By contrast, among 38 genes which are dispensable for globular embryo formation but necessary for further development, only one codes for a protein involved in metabolism. Products of 21 of the 38 genes play roles in plastid gene expression and maintenance. Examination of RNA profiles of embryos at distinct growth stages obtained in laser-capture microdissection coupled with DNA microarray experiments revealed that most of the identified genes are expressed throughout embryo morphogenesis and maturation. These findings suggest that metabolic activities are required at preglobular and throughout all stages of embryo development, whereas plastid gene expression becomes necessary during and/or after the globular stage to sustain various activities of the organelle including photosynthetic electron transport.

Most of the transcription factors (TFs) responsible for controlling seed development are not yet known. To identify TF genes expressed at specific stages of seed development, including those unique to seeds, we used Affymetrix GeneChips to profile Arabidopsis genes active in seeds from fertilization through maturation and at other times of the plant life cycle. Seed gene sets were compared with those expressed in prefertilization ovules, germinating seedlings, and leaves, roots, stems, and floral buds of the mature plant. Most genes active in seeds are shared by all stages of seed development, although significant quantitative changes in gene activity occur. Each stage of seed development has a small gene set that is either specific at the level of the GeneChip or up-regulated with respect to genes active at other stages, including those that encode TFs. We identified 289 seed-specific genes, including 48 that encode TFs. Seven of the seed-specific TF genes are known regulators of seed development and include the LEAFY COTYLEDON ( LEC ) genes LEC1, LEC1-LIKE, LEC2 , and FUS3 . The rest represent different classes of TFs with unknown roles in seed development. Promoter-β -glucuronidase ( GUS ) fusion experiments and seed mRNA localization GeneChip datasets showed that the seed-specific TF genes are active in different compartments and tissues of the seed at unique times of development. Collectively, these seed-specific TF genes should facilitate the identification of regulatory networks that are important for programming seed development.

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.

F2 segregation analyses of DNA restriction fragment length polymorphisms (RFLPs) detected between two cultivars of canola ('Westar' × Topas') was used to construct a detailed genetic map of Brassica napus. RFLP markers were from a seedling-specific cDNA library. They were either randomly selected or previously characterized as seedling-specific cDNA clones. The 120 loci so far mapped in B. napus covered 1413 recombination units. They are assembled into 19 linkage groups. Seventeen loci were found unlinked to any other markers. Few polymorphisms were detected with the seedling-specific cDNAs and only seven loci could be mapped in this cross. Duplication of RFLP loci was extensive and reflects the amphidiploid nature of this species. However, several rearrangements of the linear order of duplicated loci could be seen. This RFLP study in B. napus provides important information on genome organization of functional DNA sequences and complements our current RFLP mapping effort in Brassica oleracea. The genetic markers of this map are currently being used in several breeding applications, such as tagging important agronomic traits and fingerprinting breeding lines and cultivars of canola, a major oilseed crop.Key words: genetic mapping, Brassica napus, breeding, restriction fragment length polymorphisms.

Embryogenesis in higher plants consists of two major phases, morphogenesis and maturation. Morphogenesis involves the establishment of the embryo's body plan, whereas maturation involves cell expansion and accumulation of storage macromolecules to prepare for desiccation, germination and early seedling growth. Arabidopsis mutants showing defects in embryogenesis have provided information for understanding the events that govern embryo formation through molecular, genetic and biochemical analyses. Thus, many of the processes that underlie embryogenesis are beginning to be understood. In this chapter, we focus on genes that play key roles in the morphogenesis phase of Arabidopsis embryogenesis.

LEAFY COTYLEDON2 (LEC2) is a central regulator of embryogenesis sufficient to induce somatic cells to form embryos when expressed ectopically. Here, we analyze the cellular processes induced by LEC2, a B3 domain transcription factor, that may underlie its ability to promote somatic embryogenesis. We show auxin-responsive genes are induced after LEC2 activation in seedlings. Genes encoding enzymes involved in auxin biosynthesis, YUC2 and YUC4 , are activated within 1 h after induction of LEC2 activity, and YUC4 appears to be a direct transcriptional target of LEC2. We also show ectopic LEC2 expression induces accumulation of seed storage protein and oil bodies in vegetative and reproductive organs, events that normally occur during the maturation phase of embryogenesis. Furthermore, LEC2 activates seed protein genes before an increase in RNAs encoding LEC1 or FUS3 is observed. Thus, LEC2 causes rapid changes in auxin responses and induces cellular differentiation characteristic of the maturation phase. The relevance of these changes to the ability of LEC2 to promote somatic embryogenesis is discussed.

We have analyzed the accumulation of the glyoxylate cycle enzymes isocitrate lyase and malate synthase in embryos and seedlings of Brassica napus L. The two enzyme activities and proteins begin to accumulate during late embryogeny, reach maximal levels in seedlings, and are not detected in young leaves of mature plants. We showed previously that mRNAs encoding the two enzymes exhibit similar qualitative patterns of accumulation during development and that the two mRNAs accumulate to different levels in both embryos and seedlings (L. Comai et al. , 1989, Plant Cell 1, 293–300). In this report, we show that the relative accumulation of the proteins and activities do not correspond to these mRNA levels. In embryos and seedlings, the specific activities of isocitrate lyase and malate synthase are approximately constant. By contrast, the ratio of malate synthase protein to mRNA is 14-fold higher than that of isocitrate lyase. Differences in the translational efficiencies of the two mRNAs in vitro do not appear to account for the discrepancy between mRNA and protein levels. Our results suggest that translational and/ or post-translational processes affect differentially the accumulation of the proteins.

Previous studies in our laboratory have shown that the Dolichos biflorus plant contains two similar lectins, a seed lectin and a stem and leaf lectin called DB58, that are present at different stages in the plant's life cycle. We have now established that each of these lectins is encoded by a separate gene by isolating these lectin genes from a library of D. biflorus nuclear DNA. Restriction mapping and nucleotide sequencing analyses show that the seed lectin and DB58 genes are located in the same transcriptional orientation within 3-kilobase pairs of one another. The lectin genes contain no introns and show greater than 90% nucleotide sequence identity in their protein coding and untranslated regions. This sequence similarity extends to both the 5' and 3' flanking regions of the genes; the major exception is that a 116-base pair segment located at position -215 to -100 from the transcription start site of the seed lectin gene is missing in the 5' flanking region of the DB58 gene. The possible significance of this segment with respect to the differential expression of these genes is discussed.

Plant somatic cells have the remarkable ability to regenerate an entire organism. Many species in the genus Kalanchoë , known as “mother of thousands,” develop plantlets on the leaf margins. Using key regulators of organogenesis ( STM ) and embryogenesis ( LEC1 and FUS3 ) processes, we analyzed asexual reproduction in Kalanchoë leaves. Suppression of STM abolished the ability to make plantlets. Here, we report that constitutive plantlet-forming species, like Kalanchoë daigremontiana , form plantlets by coopting both organogenesis and embryogenesis programs into leaves. These species have a defective LEC1 gene and produce nonviable seed, whereas species that produce plantlets only upon stress induction have an intact LEC1 gene and produce viable seed. The latter species are basal in the genus, suggesting that induced-plantlet formation and seed viability are ancestral traits. We provide evidence that asexual reproduction likely initiated as a process of organogenesis and then recruited an embryogenesis program into the leaves in response to loss of sexual reproduction within this genus.