Transcriptomic Analysis of Early Flowering Signals in 'Royal' Flax.

Canada is one of the world's leading producers and exporters of flax seed, with most production occurring in the Prairie Provinces. However, reduced season length and risk of frost restricts production in the northern grain belt of the Canadian Prairies. To expand the growing region of flax and increase production in Canada, flax breeders need to develop earlier-flowering varieties capable of avoiding the risk of abiotic stress. A thorough understanding of flowering control of flax is essential for the efficient breeding of such lines. We identified 722 putative flax flowering genes that span all major flowering-time pathways. Frequently, we found multiple flax homologues for a single Arabidopsis flowering gene. We used RNA sequencing to quantify the expression of genes in the shoot apical meristem (SAM) at 10, 15, 19, and 29 days after planting (dap) using the 'Royal' cultivar. We observed the expression of 80% of putative flax flowering genes and the differential expression of only 30%; these included homologues of major flowering regulators, such as SOC1, FUL, and AP1. We also found enrichment of differentially expressed genes (DEGs) in transcription factor (TF) families involved in flowering. Finally, we identified the candidates' novel flowering genes amongst the uncharacterized flax genes. Our transcriptomic dataset provides a useful resource for investigating the regulatory control of the transition to flowering in flax and for the breeding of northern-adapted varieties.

Comparative analysis of cadmium uptake and distribution in contrasting canadian flax cultivars.

OBJECTIVE: Humans consume low quantities of cadmium (Cd), a non-nutritive and potentially toxic heavy metal, primarily via the dietary intake of grains. A trial experiment was conducted to investigate physiological and developmental differences in Cd content in four flax cultivars ('AC Emerson', 'Flanders', 'CDC Bethune', and 'AC McDuff') as part of a study to provide information that will assist in the breeding of low Cd-accumulating flax cultivars. Our objective was to identify varietal differences in the uptake and distribution of Cd in various tissues among flax cultivars grown in naturally Cd-containing soil in a controlled environment. RESULTS: Cadmium concentration was dependent on genotype, developmental stage, and tissue type, as well as their interaction. Cadmium concentration was higher in roots and leaves, relative to all other tissues, with a general trend of decreasing Cd content over time within leaves and stems. Notably, the concentration of Cd was higher in 'AC Emerson' relative to 'AC McDuff' across tissues and ages, including the seeds, while the concentration of 'Flanders' was higher than in 'AC McDuff' in seeds and other reproductive organs but similar in roots and leaves. The results suggest varietal differences in the mechanisms that determine Cd content in seeds.

Extractive silylation method for high throughput GC analysis of flaxseed cyanogenic glycosides.

The reported methods for the estimation of CG are indirect, long and tedious uses solvent extraction which results in lower recoveries due to several co-eluted components. The present study aims to develop and validate a high throughput method for the analysis of cyanogenic glycosides (CG) in flaxseed using extractive silylation. The experimental procedure comprised of preparation of trimethylsilyl (TMS) derivatives of CG, their quantitation through gas chromatography with flame ionization detector (GC-FID) and further characterization by LC-MS/MS. Different validation parameters determined in the experiment include the relative standard deviation both inter-day and intraday less than 5%, recovery in the range of 79.9-112.7%, limit of detection 4.72-6.43 µg/mL and limit of quantitation 14.31-19.50 µg/mL. Combinations of silylation reagent were screened in a central composite experimental design in order to examine their effect on the extraction recovery. Finally, the developed method was applied successfully to quantify CG in various flaxseed cultivars. Advantages of the extractive silylation are simple preparation, short reaction times and the combination of extraction and silylation in one step which indicate that the method has the potential to sensitively and accurately determine CG where large numbers of samples are being routinely analysed.

Photoperiod sensitivity of Canadian flax cultivars and 5-azacytidine treated early flowering derivative lines.

BACKGROUND: Early flowering and maturing flax (Linum usitatissimum L.) cultivars are better adapted than lines with a longer reproductive phase for the short growing season of the northern Canadian Prairies. We examined the role of long days (LD) and short days (SD) on the time taken to flower in five established flax cultivars and three mutant-derived F10 lines. The photoperiod sensitivity of these eight different genotypes was determined using a reciprocal transfer experiment involving weekly transfers between LD and SD environments. RESULTS: The genotypes tested had varying degrees of photoperiod sensitivity and demonstrated reduced time to flowering if exposed to LD environments prior to a critical time point. The duration of each of the three phases of vegetative growth differed among the genotypes studied. Transfers from SD to LD shortened the vegetative stage, reduced time to flowering, and extended the reproductive phase in the genotypes studied. Mutant-derived lines RE1/2/3 flowered significantly earlier compared to CDC Sorrel, CDC Bethune, Flanders, Prairie Thunder, and Royal. Modelling of the flowering times indicated that transferring the cultivars from SD to LD increased the photoperiod sensitive time; however, different reproductive phases for mutant lines were not defined as parsimonious models were not identified. Expression of the putative flax homologs for CONSTANS (CO), FLOWERING LOCUS T (FT), and GIGANTEA (GI) was examined in the leaves of Royal and RE1/2/3 plants at 10, 15, 19 and 29 days after planting. Expression of putative FT homologs was detected in all three early-flowering lines but expression was negligible, or not detected, in Royal. CONCLUSIONS: Models defining the three phases of reproductive development were established for the five cultivars studied; however, it was not possible to identify these phases for the three early flowering and photoperiod insensitive epimutant-derived lines. A putative flax homolog of FT, a key regulator of flowering time, is more highly expressed in RE plants, which may condition the day-length insensitivity in the early flowering 'epimutant' lines.

Proposed systematic nomenclature for orbitides.

Orbitides are short (5-11 amino acid residue), ribosomally synthesized homodetic plant cyclic peptides characterized by N-to-C amide bonds rather than disulfide bonds. Orbitides can be discovered using mass spectrometry of plant extracts or by identifying DNA sequences coding for the precursor protein. The number of orbitides that have been characterized to date, by a number of different research groups, is modest. The nomenclatural system currently used for the Type VI cyclic peptides has been developed in an ad hoc fashion and is somewhat arbitrary. We propose a systematic naming system specifically for the Type VI cyclic peptides that reflects the taxonomic name of the species producing the orbitides and a numbering system that enables systematic representation of amino acid residues and modifications. The proposed naming system emulates the IUPAC Nomenclature for Natural Products and UniProt, both of which use abbreviations of taxonomic names for the compounds in question. Nomenclature for post-translational modifications also follows the IUPAC precedent, as well as the cyclic peptide literature. Furthermore, the proposed system aims to maintain agreement with the precedents set by the pre-existing literature. An example of the proposed nomenclature is provided using the methionine-containing homodetic peptides of Linum usitatissimum (flaxseed).

Enhancing the carotenoid content of Brassica napus seeds by downregulating lycopene epsilon cyclase.

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.

Non-destructive diffraction enhanced imaging of seeds.

Techniques that make possible the non-destructive continuous observation of plant anatomy and developmental processes provide novel insights into these phenomena. Non-destructive imaging of seeds was demonstrated using the synchrotron-based X-ray imaging technique, diffraction enhanced imaging (DEI). The seed images obtained had good contrast and definition, allowing anatomical structures and physiological events to be observed. Structures such as hypocotyl-root axes, cotyledons, seed coats, air cavities, and embryo-less Brassica napus L. seeds were readily observed using DEI. Embryo axes, scutella, pericarp furrows, coleoptiles, and roots were observable over a time-course in individual germinating Triticum aestivum L. caryopses. Novel anatomical and physiological observations were also made that would have been difficult to make continuously using other techniques. The physical principles behind DEI make it a unique imaging technique. Contrast in DEI is the result of X-ray refraction at the density differences occurring at tissue boundaries, scatter caused by regions containing ordered molecules such as cellulose fibres, and attenuation. Sectioning of samples and the infusion of stains or other contrast agents are not necessary. Furthermore, as high-energy X-rays are used (30-40 keV), little X-ray absorption occurs, resulting in low levels of radiation damage. Consequently, studies of developmental processes may be performed on individuals. Individual germinating B. napus and T. aestivum seeds were imaged at several time points without incurring any apparent radiation damage. DEI offers a unique way of examining plant anatomy, development, and physiology, and provides images that are complementary to those obtained through other techniques.

A high-throughput determination of metal concentrations in whole intact Arabidopsis thaliana seeds using synchrotron-based X-ray fluorescence spectroscopy.

The identification of genes involved in metal metabolism in plants requires the 'screening' of thousands of genetic variants. While inductively coupled plasma mass-spectroscopy has been used to identify variants with an altered total metal concentration, a more convenient high-throughput technique capable of examining individual seeds (or other tissues) would be useful. Here, the high brightness of synchrotron radiation has been utilised to examine relative metal concentrations in seeds of the genetically well characterised plant Arabidopsis thaliana. The relative concentrations of Mn, Fe, Ni, Cu and Zn in individual seeds were determined using a 500 microm x 500 microm beam. Metal concentrations were normally distributed, except where metal-containing dust contaminated the samples. Neither seed orientation nor genetic background (from three 'wild type' variants with different genetic lineages) had a significant affect on the Zn-normalised metal concentration. No advantages, such as the observation of tissue-specific metal accumulation, were obtained by using a 50 microm x 50 microm beam. A high-throughput proof-of-concept experiment was demonstrated that could be used to screen libraries of genetic variants for individuals with altered metal concentrations. Further work is required to standardise the technique before screening of libraries is possible.

A high- and low-temperature inducible Arabidopsis thaliana HSP101 promoter located in a nonautonomous mutator-like element.

Transcriptional activity of a 573-bp fragment of HSP101 (At1g74310) incorporated into a Mutator-like element (MULE) transposon was investigated in Arabidopsis thaliana Columbia. Sequence identity between the HSP101-MULE arrangement and a continuous segment of the original HSP101 promoter, 5' UTR exon, and open reading frame (ORF) was high (87%) but lower in the 5' UTR intron (69%). Collectively, the HSP101 ORF, the MULE 5' terminal inverted repeat (TIR), and the 1.3 kb immediately upstream of the TIR is located on chromosome IV, and we refer to it as HSP101B. Located within the HSP101B promoter, upstream of 2 heat shock elements (HSEs), are 4 COR15a-like low-temperature response elements (LTREs). The HSP101B ORF was transcribed in the leaves and influorescences of high-temperature stress (HTS) treated Arabidopsis thaliana but not in low-temperature stress (LTS) and control plants. Transiently transformed Arabidopsis seedlings, as well as stable transformed lines of Linum usitatissimum (flax) and Brassica napus (canola) containing a HSP101B promoter:GUS construct, showed either LTS-, or LTS- and HTS-, induced beta-glucuronidase expression. Results from PCR amplifications of HpaII- and MspI-digested Arabidopsis genomic DNA suggest that endogenous expression of HSP101B may be downregulated by partial methylation of the HSP101B sequence between the TIRs of the associated MULE.

High temperature stress of Brassica napus during flowering reduces micro- and megagametophyte fertility, induces fruit abortion, and disrupts seed production.

High temperature stress (HTS), during flowering, decreases seed production in many plants. To determine the effect of a moderate HTS on flowering, fruit and seed set in Brassica napus, plants were exposed to a HTS (8/16 h dark/light, 18 degrees C night, ramped at 2 degrees C h-1, over 6 h, to 35 degrees C for 4 h, ramped at 2 degrees C h-1 back to 23 degrees C for 6 h) for 1 or 2 weeks after the initiation of flowering. Although flowering on the HTS-treated plants, during both the 1 week and 2 week HTS treatments, was equal to that of control-grown plants, fruit and seed development, as well as seed weight, were significantly reduced. Under HTS, flowers either developed into seedless, parthenocarpic fruit or aborted on the stem. At the cessation of the HTS, plants compensated for the lack of fruit and seed production by increasing the number of lateral inflorescences produced. During the HTS, pollen viability and germinability were slightly reduced. In vitro pollen tube growth at 35 degrees C, from both control pollen and pollen developed under a HTS, appeared abnormal, however, in vivo tube growth to the micropyle appeared normal. Reciprocal pollination of HTS or control pistils with HTS or control pollen indicated that the combined effects of HTS on both micro- and megagametophytes was required to knock out fruit and seed development. Expression profiles for a subset of HEAT SHOCK PROTEINs (HSP101, HSP70, HSP17.6) showed that both micro- and megagametophytes were thermosensitive despite HTS-induced expression from these genes.