Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed.

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 data sets as an essential resource for the study of seed biology.

Identification and quantification of cyclolinopeptides in five flaxseed cultivars.

Cyclolinopeptides are a group of naturally occurring hydrophobic cyclic peptides found in flaxseed and flax oil that have immunosuppressive activity. This study describes the measurement of flaxseed cyclolinopeptide concentrations using an internal standard HPLC method. In addition, the concentration of cyclolinopeptides in the seed of Canadian flax cultivars grown at two locations over two years is reported. The data are consistent with the formation of flaxseed cyclolinopeptides from two ribosome-derived precursors. Each precursor protein includes the sequences corresponding to three cyclolinopeptides from which those cyclolinopeptides are presumably derived by precursor processing. The concentrations of cyclolinopeptides C and E, which are encoded by the same gene sequence, are highly correlated, and the concentrations of cyclolinopeptides D, F, and G, which are encoded by a second gene sequence, are also highly correlated. The strong correlation between the cyclolinopeptides arising from the same gene may prove to be important in understanding how peptide concentration is controlled. Additional research may lead to approaches to improve flax either as a platform for peptide production or as a source of oil with improved drying properties and flavor.

Gene expression analysis of flax seed development.

BACKGROUND: Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. RESULTS: We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. CONCLUSIONS: We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.

Topological orientation of acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) and identification of a putative active site histidine and the role of the n terminus in dimer/tetramer formation.

Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ∼50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation.

Arabidopsis LEAFY COTYLEDON2 induces maturation traits and auxin activity: Implications for somatic 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.

Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of embryo maturation and somatic embryogenesis.

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.