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.laser-capture microdissection | mRNA localization | transcriptome

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.

The B3 domain protein LEAFY COTYLEDON2 (LEC2) is required for several aspects of embryogenesis, including the maturation phase, and is sufficient to induce somatic embryo development in vegetative cells. Here, we demonstrate that LEC2 directly controls a transcriptional program involved in the maturation phase of seed development. Induction of LEC2 activity in seedlings causes rapid accumulation of RNAs normally present primarily during the maturation phase. Several RNAs encode proteins with known roles in maturation processes, including seed-storage and lipid-body proteins. Clustering analyses identified other LEC2-induced RNAs not previously shown to be involved in the maturation phase. We show further that genes encoding these maturation RNAs all possess in their 5 flanking regions RY motifs, DNA elements bound by other closely related B3 domain transcription factors. Our finding that recombinant LEC2 specifically binds RY motifs from the 5 flanking regions of LEC2-induced genes provides strong evidence that these genes represent transcriptional targets of LEC2. Although these LEC2-induced RNAs accumulate primarily during the maturation phase, we show that a subset, including AGL15 and IAA30, accumulate in seeds containing zygotes. We discuss how identification of LEC2 target genes provides a potential link between the roles of LEC2 in the maturation phase and in the induction of somatic embryogenesis.Arabidopsis ͉ B3 domain E mbryogenesis in higher plants can be divided conceptually into two distinct phases. Early in embryogenesis, during the morphogenesis phase, the basic body plan of the plant is established with regional specification of apical-basal and radial domains from which morphological structures derive, fixation of polarity from specification of the shoot-root axis, and formation of embryonic tissue and organ systems (1-3). The morphogenesis phase is followed temporally by the maturation phase, although the two phases can overlap (4, 5). During the maturation phase, embryo cell-division rates decline markedly, embryo cells acquire the ability to withstand desiccation, and embryo cell growth occurs, with the accumulation of storage reserves that comprise lipids and proteins in Arabidopsis (6, 7). At the end of the maturation phase, the embryo becomes quiescent metabolically as the seed desiccates.The maturation phase can be viewed as an interruption of an ancestral life cycle, as occurs in lower plants, in which there are no periods of maturation or dormancy separating the end of embryogenesis and the beginning of postembryonic development (4). Evolution of this unique mode of embryogenesis has enabled higher plants to make seeds. The ability to make seeds has provided tremendous selective advantages that, in part, account for the success of the angiosperms (8, 9). Little is known at a mechanistic level about the processes by which the maturation phase has been integrated into the higher plant life cycle. LEAFY COTYLEDON2 (LEC2), along with ABA INSEN-SITIVE3 (ABI3), and FUSCA3 (FUS3), have been im...

LEAFY COTYLEDON1 (LEC1), an atypical subunit of the nuclear transcription factor Y (NF-Y) CCAAT-binding transcription factor, is a central regulator that controls many aspects of seed development including the maturation phase during which seeds accumulate storage macromolecules and embryos acquire the ability to withstand desiccation. To define the gene networks and developmental processes controlled by LEC1, genes regulated directly by and downstream of LEC1 were identified. We compared the mRNA profiles of wild-type and -null mutant seeds at several stages of development to define genes that are down-regulated or up-regulated by the mutation. We used ChIP and differential gene-expression analyses in seedlings overexpressing LEC1 and in developing and soybean seeds to identify globally the target genes that are transcriptionally regulated by LEC1 Collectively, our results show that LEC1 controls distinct gene sets at different developmental stages, including those that mediate the temporal transition between photosynthesis and chloroplast biogenesis early in seed development and seed maturation late in development. Analyses of enriched DNA sequence motifs that may act as-regulatory elements in the promoters of LEC1 target genes suggest that LEC1 may interact with other transcription factors to regulate distinct gene sets at different stages of seed development. Moreover, our results demonstrate strong conservation in the developmental processes and gene networks regulated by LEC1 in two dicotyledonous plants that diverged ∼92 Mya.

SignificanceWe 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.