Genetic variation and structural diversity in major seed proteins among and within Camelina species.

MAIN CONCLUSION: Genetic variation in seed protein composition, seed protein gene expression and predictions of seed protein physiochemical properties were documented in C. sativa and other Camelina species. Seed protein diversity was examined in six Camelina species (C. hispida, C. laxa, C. microcarpa, C. neglecta, C. rumelica and C. sativa). Differences were observed in seed protein electrophoretic profiles, total seed protein content and amino acid composition between the species. Genes encoding major seed proteins (cruciferins, napins, oleosins and vicilins) were catalogued for C. sativa and RNA-Seq analysis established the expression patterns of these and other genes in developing seed from anthesis through to maturation. Examination of 187 C. sativa accessions revealed limited variation in seed protein electrophoretic profiles, though sufficient to group the majority into classes based on high MW protein profiles corresponding to the cruciferin region. C. sativa possessed four distinct types of cruciferins, named CsCRA, CsCRB, CsCRC and CsCRD, which corresponded to orthologues in Arabidopsis thaliana with members of each type encoded by homeologous genes on the three C. sativa sub-genomes. Total protein content and amino acid composition varied only slightly; however, RNA-Seq analysis revealed that CsCRA and CsCRB genes contributed > 95% of the cruciferin transcripts in most lines, whereas CsCRC genes were the most highly expressed cruciferin genes in others, including the type cultivar DH55. This was confirmed by proteomics analyses. Cruciferin is the most abundant seed protein and contributes the most to functionality. Modelling of the C. sativa cruciferins indicated that each type possesses different physiochemical attributes that were predicted to impart unique functional properties. As such, opportunities exist to create C. sativa cultivars with seed protein profiles tailored to specific technical applications.

SCARECROW-LIKE15 interacts with HISTONE DEACETYLASE19 and is essential for repressing the seed maturation programme.

Epigenetic regulation of gene expression is critical for controlling embryonic properties during the embryo-to-seedling phase transition. Here we report that a histone deacetylase19 (HDA19)-associated regulator, scarecrow-like15 (SCL15), is essential for repressing the seed maturation programme in vegetative tissues. SCL15 is expressed in and GFP-tagged SCL15 predominantly localizes to, the vascular bundles particularly in the phloem companion cells and neighbouring specialized cells. Mutation of SCL15 leads to a global shift in gene expression in seedlings to a profile resembling late embryogenesis in seeds. In scl15 seedlings, many genes involved in seed maturation are markedly derepressed with concomitant accumulation of seed 12S globulin; this is correlated with elevated levels of histone acetylation at a subset of seed-specific loci. SCL15 physically interacts with HDA19 and direct targets of HDA19-SCL15 association are identified. These studies reveal that SCL15 acts as an HDA19-associated regulator to repress embryonic traits in seedlings.

Analysis of expressed sequence tags in Brassica napus cotyledons damaged by crucifer flea beetle feeding.

The molecular basis of canola ( Brassica napus L.) susceptibility to the crucifer flea beetle (FB, Phyllotreta cruciferae Goeze) was investigated by comparing transcript representation in FB-damaged and undamaged cotyledons. The B. napus cotyledon transcriptome increased and diversified substantially after FB feeding damage. Twenty-two genes encoding proteins with unknown function, six encoding proteins involved in signaling, and a gene encoding a B-box zinc finger transcription factor were moderately or strongly changed in representation with FB feeding damage. Zinc finger and calcium-dependent genes formed the largest portion of transcription factors and signaling factors with changes in representation. Six genes with unknown function, one transcription factor, and one signaling gene specific to the FB-damaged library were co-represented in a FB-damaged leaf library. Out of 188 transcription factor and signaling gene families screened for "early" expression changes, 16 showed changes in expression within 8 h. Four of these early factors were zinc finger genes with representation only in the FB-damaged cotyledon. These genes are now available to test their potential at initiating or specifying cotyledon responses to crucifer FB feeding.

Repression of seed maturation genes by a trihelix transcriptional repressor in Arabidopsis seedlings.

The seed maturation program is repressed during germination and seedling development so that embryonic genes are not expressed in vegetative organs. Here, we describe a regulator that represses the expression of embryonic seed maturation genes in vegetative tissues. ASIL1 (for Arabidopsis 6b-interacting protein 1-like 1) was isolated by its interaction with the Arabidopsis thaliana 2S3 promoter. ASIL1 possesses domains conserved in the plant-specific trihelix family of DNA binding proteins and belongs to a subfamily of 6b-interacting protein 1-like factors. The seedlings of asil1 mutants exhibited a global shift in gene expression to a profile resembling late embryogenesis. LEAFY COTYLEDON1 and 2 were markedly derepressed during early germination, as was a large subset of seed maturation genes, such as those encoding seed storage proteins and oleosins, in seedlings of asil1 mutants. Consistent with this, asil1 seedlings accumulated 2S albumin and oil with a fatty acid composition similar to that of seed-derived lipid. Moreover, ASIL1 specifically recognized a GT element that overlaps the G-box and is in close proximity to the RY repeats of the 2S promoters. We suggest that ASIL1 targets GT-box-containing embryonic genes by competing with the binding of transcriptional activators to this promoter region.

Development of an Adonis aestivalis expressed sequence tag population as a resource for genes of the carotenoid pathway.

Expressed sequence tag (EST) libraries are a powerful tool for gene discovery in plants with genomes that are not fully sequenced. Adonis aestivalis (Ranunculaceae) is unusual among higher plants in that it accumulates large amounts of the valuable red ketocarotenoid astaxanthin in the flower petals, in addition to other carotenoids, and should therefore be a rich source of transcripts involved in carotenoid biosynthesis. To create a resource for gene discovery and increase our understanding of ketocarotenoid biosynthesis in plants, we sequenced 4189 cloned cDNAs from the floral tissues of A. aestivalis. The EST collection was annotated using sequence homology searches comparing the A. aestivalis ESTs with reference databases. ESTs involved in carotenoid biosynthesis were identified based on sequence homology to published sequences and by functional prediction using InterProScan. This study identified expressed transcripts for most of the known genes involved in the carotenoid pathway. Using an Escherichia coli functional expression system, two candidate transcripts from the A. aestivalis cDNA library were functionally characterized and identified as a novel beta-carotene 3-hydroxylase (CrtH2) and a beta-carotene monooxygenase. The expanded use of this EST collection combined with the E. coli expression system as presented here will greatly facilitate the cloning and characterization of candidate genes involved in carotenoid biosynthesis in plants.

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation.

Applications of buthionine sulfoximine (BSO), an inhibitor of GSH (reduced glutathione), which switches the cellular glutathione pool towards the oxidized form GSSG, positively influences embryo quality by improving the structure of the shoot apical meristem and promoting embryo maturation, both of which improve the post-embryonic performance of the embryos. To investigate the mechanisms underlying BSO-mediated improvement in embryo quality the transcript profiles of developing Brassica napus microspore-derived embryos cultured in the absence (control) or presence of BSO were analyzed using a 15,000-element B. napus oligo microarray. BSO applications induced major changes in transcript accumulation patterns, especially during the late phases of embryogenesis. BSO affected the transcription and activities of key enzymes involved in ascorbate metabolism, which resulted in major fluctuations in cellular ascorbate levels. These changes were related to morphological characteristics of the embryos and their post-embryonic performance. BSO applications also activated many genes controlling meristem formation and function, including ZWILLE, SHOOTMERISTEMLESS, and ARGONAUTE 1. Increased expression of these genes may contribute to the improved structural quality of the shoot poles observed in the presence of BSO. Compared to their control counterparts, middle- and late-stage BSO-treated embryos also showed increased accumulation of transcripts associated with the maturation phase of zygotic embryo development, including genes encoding ABA-responsive proteins and storage- and late-embryogenic abundant (LEA) proteins. Overall these transcriptional changes support the observation that the BSO-induced oxidized glutathione redox state allows cultured embryos to reach both morphological and physiological maturity, which in turn guarantees successful regeneration and enhanced post-embryonic growth.