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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.

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.

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.

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.

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.

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.

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.

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.

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.

Chromosomes of Brassica oleracea (2n = 18) were dissected from the resynthesized amphidiploid B. napus Hakuran by repeated backcrosses to B. campestris (2n = 20), creating a series of monosomic alien chromosome addition line plants (2n = 21). Using morphological, isozyme and restriction fragment length polymorphism markers (RFLPs), 81 putative loci were identified. Of nine possible synteny groups, seven were represented in the 25 monosomic addition plants tested. Sequences homologous to 26% of the 61 DNA clones utilized (80% were cDNA clones) were found on more than one synteny group, indicating a high level of gene duplication. Anomalous synteny associations were detected in four 2n = 21 plants. One of these plants showed two markers from one B. oleracea chromosome associated with a second complete B. oleracea synteny group, suggesting translocation or recombination between non-homologous chromosomes in Hakuran or the backcross derivatives. The other three 2n = 21 plants each contained two or more B. oleracea synteny groups, suggesting chromosome substitution.

We investigated the control of genes expressed primarily during seed germination and postgerminative development in Brassica napus L. We identified cloned mRNA sequences which became prevalent within 1 day after the start of imbibition and were at low or undetectable levels in immature embryos, dry seeds, and leaves. Most postgermination-abundant mRNAs accumulated primarily, though not exclusively, in different parts of the seedling. Of the 14 cloned mRNAs, 8 were prevalent in cotyledons, 2 were abundant in seedling axes, and 4 were approximately equally distributed in both parts. We showed that although these mRNAs reached maximal levels in seedlings, the spatially regulated mRNAs were also detected at distinct embryonic stages; mRNAs prevalent in seedling axes accumulated primarily during early embryogenesis while cotyledon-abundant mRNA concentration increased during late embryogeny. We conclude that the temporal and spatial regulation of gene expression in seedlings reflects similarities and differences in the physiological functions of cotyledons and axes. Furthermore, the regulated expression of cotyledon-abundant genes during late embryogeny suggests that the mRNAs and possibly proteins may accumulate in preparation for subsequent seedling growth. Similarities in the accumulation of cotyledon-abundant mRNAs may indicate coordinate regulation of this gene set.

We have analyzed the nucleotide sequence and accumulation of an mRNA which is prevalent in seeds of Brassica napus L. During normal development, the mRNA begins to accumulate during late embryogeny, is stored in dry seeds, and becomes undetectable in seedlings within 24 hours after imbibition. Moreover, abscisic acid treatment of embryos precociously induces or enhances accumulation of the mRNA. Nucleotide sequencing studies show that the deduced 30 kDa polypeptide has an unusual primary structure; the polypeptide possesses direct amino acid sequence repeats and is virtually entirely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Based upon the expression pattern and predicted polypeptide sequence, we conclude that the mRNA is encoded by a late embryogenesis-abundant (Lea) gene in B. napus.

Arabidopsis APETALA2 (AP2) controls seed mass maternally, with ap2 mutants producing larger seeds than wild type. Here, we show that AP2 influences development of the three major seed compartments: embryo, endosperm, and seed coat. AP2 appears to have a significant effect on endosperm development. ap2 mutant seeds undergo an extended period of rapid endosperm growth early in development relative to wild type. This early expanded growth period in ap2 seeds is associated with delayed endosperm cellularization and overgrowth of the endosperm central vacuole. The subsequent period of moderate endosperm growth is also extended in ap2 seeds largely due to persistent cell divisions at the endosperm periphery. The effect of AP2 on endosperm development is mediated by different mechanisms than parent-of-origin effects on seed size observed in interploidy crosses. Seed coat development is affected; integument cells of ap2 mutants are more elongated than wild type. We conclude that endosperm overgrowth and/or integument cell elongation create a larger postfertilization embryo sac into which the ap2 embryo can grow. Morphological development of the embryo is initially delayed in ap2 compared with wild-type seeds, but ap2 embryos become larger than wild type after the bent-cotyledon stage of development. ap2 embryos are able to fill the enlarged postfertilization embryo sac, because they undergo extended periods of cell proliferation and seed filling. We discuss potential mechanisms by which maternally acting AP2 influences development of the zygotic embryo and endosperm to repress seed size.