Molecular cloning of a cDNA fromBrassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis ofBrassica napus genomic DNA revealed the presence of 6 genes, 3 derived from theBrassica rapa parent and 3 fromBrassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.     

The microspore-derived embryo ofBrassica napus L. as a tool for studying embryo-specific lipid biogenesis and regulation of oil quality

Summary A time-course study of lipid accumulation in microspore-derived embryos and developing zygotic embryos of rapeseed (Brassica napus L. ssp.oleifera) is presented. Rapid storage fat (triacylglycerol) biosynthesis was induced in microspore-derived embryos of oilseed rape (cv Topas) when the embryos were transferred from standing cultures (10 ml) to fresh medium (75 ml) and shake cultured. Triacylglycerols accumulated, after a lag period of 7 days, at a linear rate of approximately twice that of the developing zygotic embryo. The fatty acid composition of triacylglycerols in microspore-derived embryos closely parallelled that of the developing zygotic embryos. In the microspore-derived embryos, the amount of phosphatidylcholine, the major substrate for the production of polyunsaturated fatty acids in oilseeds, remained constant during the linear phase of triacylglycerol production, whereas it increased steadily in the zygotic embryos. The fatty acid composition of individual cotyledons from microspore embryos shake cultured for 15 days was compared with that of individual mature seeds. Relative amounts of the major fatty acids, i.e. palmitic, oleic and linoleic acids, were essentially the same, whereas the microspore-derived embryos had about 35% less stearic acid and 35% more linolenic acid than the mature seeds. Variation in the amounts of oleic, linoleic and linolenic acids between seeds was similar to that found between cotyledons of microspore-derived embryos, whereas variation in palmitic and stearic acid levels was significantly lower between microsporederived cotyledons than between the seeds. The results indicate that microspore-derived embryos from shake cultures should be convenient for use in studying the regulation of oil biosynthesis and for rapidly screening for oil quality in genetically altered rapeseed.     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP-glucose-6-phosphate dehydrogenase, NADP-6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase, NADP-malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl-CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with ¹⁴C-labelled carbon precursors and the incorporation of ¹⁴C into starch and fatty acids was determined. ¹⁴C from glucose-6-phosphate (G-6-P), fructose, glucose, fructose-6-phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness- and ATP-dependent manner. The rate of starch synthesis was highest from G-6-P, although fructose gave rates which were 70% of those from G-6-P. Glucose-1-phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G-6-P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G-6-P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.     

黑胫病菌侵染过程中油菜响应基因的表达分析

油菜黑胫病是造成油菜产量损失的病害之一,致病菌为Leptosphaeria biglobosa.该研究采用形态学观察和转录组测序技术,分析油菜接种病原菌Leptosphaeria biglobosa 4、12、24、36、48和96 h后的表型及基因表达变化情况,以探讨响应死体营养型真菌L.biglobosa侵染时油菜...     

Transfer and segregation of triazine tolerant chloroplasts in Brassica napus L.

Summary Hypocotyl protoplasts of 45 different genotypes of German winter oilseed rape Brassica napus L. (double zero quality: high in yield, seeds low in erucic acid and glucosinolate content) were regenerated to plants. Triazine/triazinone (tri)-tolerant chloroplasts of the Canadian spring oilseed rape variety OAC Triton were introduced into some winter oilseed rapes by means of protoplast fusion. X-ray irradiation was used to limit the transfer of nuclear DNA of Triton protoplasts and to promote the selective transfer of tri-tolerant chloroplasts. Regenerated cybrid plants survived a treatment rate of 1000 g/ha metribuzin. The presence and segregation of the tri-tolerant chloroplasts in winter oilseed rape plants, regenerated from fusion products and their progeny, was investigated by restriction fragment length polymorphism (RFLP). Our results indicate that chloroplast segregation was not completed in plants regnerated from fusion products derived from X-irradiated OAC Triton mesophyll protoplasts and German winter oilseed rape hypocotyl protoplasts. In regenerants and their progeny both chloroplast types can still be present. Chloroplasts derived from wintertype protoplasts can outcompete tritolerant chloroplasts during plant development. In some instances, even progeny plants not kept under selective conditions (metribuzin) lost tri-tolerant chloroplasts. A homogenous population of tri-tolerant chloroplasts was necessary to obtain stable tri-tolerant winter oilseed rape plants.     

Development of crop-specific transposable element (SINE) markers for studying gene flow from oilseed rape to wild radish

The screening of wild populations for evidence of gene flow from a crop to a wild related species requires the unambiguous detection of crop genes within the genome of the wild species, taking into account the intraspecific variability of each species. If the crop and wild relatives share a common ancestor, as is the case for the Brassica crops and their wild relatives (subtribe Brassiceae), the species-specific markers needed to make this unambiguous detection are difficult to identify. In the model oilseed rape (Brassica napus, AACC, 2n=38)-wild radish (Raphanus raphanistrum, RrRr, 2n=18) system, we utilized the presence or absence of a short-interspersed element (SINE) at a given locus to develop oilseed rape-specific markers, as SINE insertions are irreversible. By means of sequence-specific amplified polymorphism (SINE-SSAP) reactions, we identified and cloned 67 bands specific to the oilseed rape genome and absent from that of wild radish. Forty-seven PCR-specific markers were developed from three combinations of primers anchored either in (1) the 5- and 3-genomic sequences flanking the SINE, (2) the 5-flanking and SINE internal sequences or (3) the SINE internal and flanking 3-sequences. Seventeen markers were monomorphic whatever the oilseed rape varieties tested, whereas 30 revealed polymorphism and behaved either as dominant (17) or co-dominant (13) markers. Polymorphic markers were mapped on 19 genomic regions assigned to ten linkage groups. The markers developed will be efficient tools to trace the occurrence and frequency of introgressions of oilseed rape genomic region within wild radish populations.     

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5 sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of theRDPG1 5-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.     

Transformation of microspore-derived embryos of winter oilseed rape (Brassica napus L.) by usingAgrobacterium tumefaciens

Haploid microspore-derived embryos (MDEs) constitute a unique material for the introduction of new traits into winter oilseed rape (Brassica napus). MDEs have been transformed by usingAgrobacterium tumefaciens strains EHA105 and LBA4404, both carrying the binary vector pKGIB containing theuidA gene encoding β-glucuronidase (GUS) and thebar gene as a marker of resistance to phosphinotricin. Transformed embryos expressed GUS and regenerated plants that were resistant to herbicide Basta, as confirmed by a leaf-painting test. Progeny plants of the transformant T-39 were all transgenic, as they inherited T-DNA from their doubled haploid parental plant. Southern-blot analysis confirmed the integration and transmission of T-DNA into T₁ plants. Transformation of MDEs facilitates the obtaining of winter oilseed rape homozygous for the introduced genes.     

A major QTL on chromosome C05 significantly reduces acid detergent lignin (ADL) content and increases seed oil and protein content in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Key message

A reduction in acid detergent lignin content in oilseed rape resulted in an increase in seed oil and protein content.

Abstract

Worldwide increasing demand for vegetable oil and protein requires continuous breeding efforts to enhance the yield of oil and protein crop species. The oil-extracted meal of oilseed rape is currently mainly used for feeding livestock, but efforts are undertaken to use the oilseed rape protein in food production. One limiting factor is the high lignin content of black-seeded oilseed rape that negatively affects digestibility and sensory quality of food products compared to soybean. Breeding attempts to develop yellow seeded oilseed rape with reduced lignin content have not yet resulted in competitive cultivars. The objective of this work was to investigate the inheritance of seed quality in a DH population derived from the cross of the high oil lines SGDH14 and cv. Express. The DH population of 139 lines was tested in field experiments in 14 environments in north-west Europe. Seeds harvested from open pollinated plants were used for extensive seed quality analysis. A molecular marker map based on the Illumina Infinium 60 K Brassica SNP chip was used to map QTL. Amongst others, one major QTL for acid detergent lignin content, explaining 81% of the phenotypic variance, was identified on chromosome C05. Lines with reduced lignin content nevertheless did not show a yellowish appearance, but showed a reduced seed hull content. The position of the QTL co-located with QTL for oil and protein content of the defatted meal with opposite additive effects, suggesting that the reduction in lignin content resulted in an increase in oil and protein content.

     

The phloem mobility of glucosinolates

The transport properties of glucosinolates within Brassica napus are of interest as identification of the mechanism of transport could lead to lower levels being obtained in specific tissues such as the seeds. The phloem mobility of ³⁵S-gluconapin (but-3-enylglucosinolate) and ³⁵S-desulphogluconapin in oilseed rape plants has been inferred from tissue distribution patterns, as well as from observed coincident phloem mobility of ³H-gluconapin and ¹⁴C-sucrose. The measured relative phloem mobilities for sinigrin (2-propenylglucosinolate), ³H-gluconapin, ³⁵S-desulphogluconapin, ³⁵S-desulphosinigrin, ¹⁴C-tryptophan, ³H-AIB (-aminoisobutyric acid), and literature values for a reduced ³H-oligogalacutonide elicitor (degree of polymerization 6) and ¹⁴C-IAA (indolylacetic acid), have been compared with the predicted values obtained using the Kleier model for phloem mobility of xenobiotics. All the above compounds show phloem systemicity, demonstrated using the Ricinus assay, as predicted by the model. Log Kow (octanol-water partition coefficient) values for glucosinolates and desulphoglucosinolates measured at pH 4 and pH 7, and the effect of pH on uptake by oilseed rape embryos are provided as evidence against a weak acid trap mechanism acting in either the phloem mobility or the accumulation of glucosinolates in oilseed rape embryos. The phloem mobility of glucosinolates is explained by the intermediate permeability hypothesis. In conclusion, it would appear that glucosinolates like other groups of endogenous compounds have physicochemical properties allowing phloem mobility as predicted by the Kleier model.Keywords: Brassica napus, Ricinus communis, phloem mobility, glucosinolates, Kleier model.      

Comparison of the metabolic properties of plastids isolated from developing leaves or embryos of Brassica napus L

Plastids isolated from developing leaves and embryos of oilseed rape (Brassica napus L.) were incubated with substrates in the light or the dark, with or without exogenous ATP. Incorporation of HCO^-3, and carbon from a range of substrates into fatty acids and/or starch by leaf chloroplasts was absolutely light-dependent and was unaffected by provision of ATP. Incorporation of HCO^-3 into fatty acids and/or starch by embryo plastids was also light-dependent. However, the light-dependent rates attained, when expressed on a comparable basis, were less than 32% of those from Glc6P (plus ATP), which was the most effective substrate for starch and fatty acid synthesis. In the light alone the rates of carbon incorporation from Glc6P, pyruvate and acetate into fatty acids, and from Glc6P into starch by embryo plastids were less than 27% of the respective ATP-dependent (dark) rates. Light had no effect on these ATP-dependent rates of synthesis by embryo plastids. While transporter activities for both glucose and Glc6P were present in embryo plastids, leaf chloroplasts did not have the latter activity. It is concluded that light at in vivo levels can contribute energy to carbon metabolism in embryo plastids. However, this contribution is likely to be small and these plastids are therefore largely dependent upon interaction with the cytosol for the ATP, reducing power and carbon precursors that are required for maximal rates of starch and fatty acid synthesis.     

Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene

Summary Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter of cauliflower mosaic virus and an engineered napin (seed storage protein) gene with its own promoter (300 nucleotides 5 to the start of translation). Transformation of B. napus plants was confirmed by detection of NPT II enzyme activity, Southern blot analysis and inheritance of the kanamycin-resistance trait (NPT II gene) in the progeny. Expression of the engineered napin gene in embryos but not in leaves of transgenic plants was observed by Northern analysis. These data demonstrate that morphologically normal, fertile transgenic B. napus plants can be obtained using Agrobacterium as a gene vector and that developmentally regulated expression of reintroduced genes can be achieved.     

Mapping of QTL for the seed storage proteins cruciferin and napin in a winter oilseed rape doubled haploid population and their inheritance in relation to other seed traits

Key message

Cruciferin (cru) and napin (nap) were negatively correlated and the cru/nap ratio was closely negative correlated with glucosinolate content indicating a link between the two biosynthetic pathways.

Abstract

Canola-type oilseed rape (Brassica napus L.) is an economically important oilseed crop in temperate zones. Apart from the oil, the canola protein shows potential as a value-added food and nutraceutical ingredient. The two major storage protein groups occurring in oilseed rape are the 2 S napins and 12 S cruciferins. The aim of the present study was to analyse the genetic variation and the inheritance of napin and cruciferin content of the seed protein in the winter oilseed rape doubled haploid population Express 617 × R53 and to determine correlations to other seed traits. Seed samples were obtained from field experiments performed in 2 years at two locations with two replicates in Germany. A previously developed molecular marker map of the DH population was used to map quantitative trait loci (QTL) of the relevant traits. The results indicated highly significant effects of the year and the genotype on napin and cruciferin content as well as on the ratio of cruciferin to napin. Heritabilities were comparatively high with 0.79 for napin and 0.77 for cruciferin. Napin and cruciferin showed a significant negative correlation (?0.36**) and a close negative correlation of the cru/nap ratio to glucosinolate content was observed (?0.81**). Three QTL for napin and two QTL for cruciferin were detected, together explaining 47 and 35 % of the phenotypic variance. A major QTL for glucosinolate content was detected on linkage group N19 whose confidence interval overlapped with QTL for napin and cruciferin content. Results indicate a relationship between seed protein composition and glucosinolate content.     

Genetic diversity of oilseedBrassica napus germ plasm based on restriction fragment length polymorphisms

Oilseed rape (Brassica napus) is an important oilseed crop worldwide. Cultivars have been developed for many growing regions, however little is known about genetic diversity inB. napus germ plasm. The purpose of the research presented here was to study the genetic diversity and relationships ofB. napus accessions using restriction fragment length polymorphisms (RFLPs). Eighty threeB. napus accessions were screened using 43 genomic DNA clones which revealed 161 polymorphic fragments. Each accession was uniquely identified by the markers with the exception of the near-isogenic cvs Triton and Tower. The RFLP data were analyzed by cluster analysis of similarity coefficients and by principal component analysis. Overall, there were three major groups of cultivars. The first group included only spring accessions, the second mostly winter accessions and the third, rutabagas and oilseed rape accessions from China and Japan. These results indicate that withinB. napus, winter and spring cultivars represent genetically distinct groups. The grouping of accessions by cluster analysis was generally consistent with known pedigrees. This consistency included the grouping of lines derived both by backcrossing or self-pollination with their parents.     

Gene dispersal from transgenic crops

The risk of release of genetically modified oilseed rape (Brassica napus) was investigated in relation to interspecific gene flow with hoary mustard (Hirschfeldia incana). Microscopic studies showed polymorphism within the population of hoary mustard for pollen germination on oilseed rape flowers. The transgenic herbicide-resistant and a commercial cultivar of oilseed rape were not different for pollen behaviour and ovule fertilization. Pollen tube growth was slow and erratic in interspecific crosses. Fertilization efficiency of oilseed rape and hoary mustard pollen in interspecific crosses was 15% and 1.3%, respectively, of that in intraspecific crosses. This unequal efficiency in reciprocal crosses was confirmed by hybrid seed set in pods. There was no post-zygotic barrier to the development of hybrid embryos in hoary mustard pods. Up to 26 spontaneous hybrids per male sterile oilseed rape plant, and one per hoary mustard plant, were obtained in field experiments. Hybrids were identified by isozyme electrophoresis, morphology and cytology. All hybrids were triploid with 26 chromosomes, and had low fertility. They produced 0.5 seeds per plant after spontaneous backcrossing with hoary mustard. Some of these descendants were produced from unreduced gametes. Our results suggest that gene flow is likely to occur, but its actual frequency under crop growing conditions remains to be estimated.     

Crystal structure of the effector AvrLm4–7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins

The avirulence gene AvrLm4–7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4–7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4–7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well‐conserved among AvrLm4–7 homologs. Loss of recognition of AvrLm4–7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well‐conserved C–terminal motif or close to the glycine involved in Rlm4‐mediated recognition, resulting in the loss of Rlm7‐mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4–7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4–7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.     

Enzymatic fractionation of evening primrose oil by rape lipase: Enrichment of gamma-linolenic acid

Summary Rape lipase discriminates strongly against -linolenic acid (allcis-6, 9, 12–183; GLA). GLA in the fatty acids from evening primrose oil was concentrated from 9.5% to 62% with a 95% yield during esterification of these fatty acids with butanol catalyzed by rape lipase. Hydrolysis of evening primrose oil catalyzed by the rape lipase raised the GLA content in unhydrolyzed acylglycerols to 28%.