Uptake and distribution of sinigrin in microspore derived embryos of Brassica napus L

In Brassica napus, glucosinolates are transported from all parts of the plant into the embryo during seed development. In this study we describe the uptake of the alkenyl glucosinolate sinigrin by microspore derived embryos from high and low glucosinolate genotypes. Microspore derived embryos develop completely isolated from maternal tissues unlike zygotic embryos, which contains glucosinolates transported into the embryo synthesised in the vegetative tissues. The sinigrin in the culture medium was almost completely absorbed by the embryos after three days of culture. The embryos of high and low glucosinolate genotypes were equally capable of absorbing sinigrin from the medium. A significant increase in different alkenyl glucosinolates following feeding of sinigrin suggests induction of biosynthetic enzymes in the embryos. Following excess feeding of sinigrin, we found a strong uptake against a concentration gradient and stable accumulation by the embryos. The glucosinolate was detected in single dissected cotyledons by a photometric test and by HPLC. This test could potentially be useful for screening mutants defective in glucosinolate uptake into the embryo.     

Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro

Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 g d^-1 during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10^-6 M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 g embryo^-1 d^-1) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.Abbreviations ABA abscisic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium lauryl sulfate     

Identification of differentially expressed genes in seeds of two near-isogenic Brassica napus lines with different oil content

The regulation of seed oil synthesis in rapeseed is largely unknown. In this study, we compared the gene expression during seed development between two lines of Brassica napus with a 10% difference in oil content. We isolated the immature seeds 15 and 25 days after flowering at periods preceding and including the major accumulation of storage oils and proteins. The differentially expressed gene clones between the two rape lines were isolated by subtractive suppression hybridization (SSH). All SSH clones were arrayed and screened by dot blot hybridization, followed by RT-PCR analysis for selected clones. A total of 217 cDNA clones corresponding to 30 genes were found to have a high expression in seeds with high oil content. Six genes were highly expressed in seeds with low oil content. Northern blot and enzyme activity analysis demonstrated a change in expression pattern of several genes. The results provide information on gene-encoding factors responsible for the regulation of oil synthesis. The possible role of these genes in seeds is discussed. The genes in this study may be suitable as novel targets for genetic improvement of seed oil content and may also provide molecular markers for studies of rape breeding.     

Oxidation of 8'-hydroxy abscisic acid in Black Mexican Sweet maize cell suspension cultures

In a biotransformation study to prepare deuterium labelled phaseic acid (PA) from deuterated abscisic acid (ABA), the product contained fewer deuterium atoms than expected. Thus, spectroscopic data of isolated deuterated PA prepared from biotransformation of (+)-5,8',8',8'-d4-ABA in maize (Zea mays L. cv. Black Mexican Sweet) cell suspension cultures showed 83% deuterium incorporation at the 8'-exo position. Also, metabolism studies of (+)-4,5-d2-ABA in maize resulted in the isolation of deuterium labelled ABA derivatives, namely PA, dihydrophaseic acid (DPA), 4'-O-beta-D-glucopyranosylDPA, 8'-hydroxyPA, 8'-hydroxyDPA and 8'-oxoDPA, as deduced from spectroscopic methods. These combined results suggested the presence of an aldehyde intermediate which is either: (a) reduced to 8'-hydroxyABA and cyclized to PA, or (b) is hydrated and cyclized to 8'-hydroxyPA or (c) is further oxidized to the acid and cyclized to 8'-oxoPA. The chemical synthesis of this intermediate, as well as its biotransformation in maize cell cultures is presented.     

Induction,purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus)

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.     

(n-7) and (n-9) cis-Monounsaturated fatty acid contents of 12 Brassica species

cis-Vaccenic acid or cis-11-octadecenoic acid, a C18:1 (n-7) isomer of oleic acid (C18:1 (n-9)) has been found in several oilseeds. It is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Delta9 desaturase and elongation by an elongase giving C18:1 (n-7). In this study, the fatty acid composition of 12 Brassica species was analyzed by GC-FID and confirmed by GC-MS. All species contained C18:1 (n-7), C20:1 (n-7) and C22:1 (n-7) fatty acid isomers, suggesting that C18:1 (n-7) was elongated. The levels of these fatty acids varied according to the species. C18:1(n-7)) represented from 0.4% to 3.3% of the total relative fatty acid contents of the seeds. The contents of C20:1(n-7) and C22:1(n-7) levels were lower than C18:1(n-7) contents; the relative fatty acid composition varied from 0.02% to 1.3% and from below the limit of detection to 1.3% for C20:1 (n-7) and C22:1 (n-7), respectively. The ratios of (n-7)/(n-9) ranged from 2.8% to 16.7%, 0.6% to 29.5% and 0% to 2.6% for C18:1, C20:1 and C22:2, respectively. Using statistical similarities or differences of the C18:1 (n-7)/(n-9) ratios for chemotaxonomy, the surveyed species could be arranged into three groups. The first group would include Brassica napus, B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The second group would include B. tournefortii, Raphanus sativus and Sinapis alba. The last group would include B. juncea, B. carinata and B. nigra with no similarity/relationship between them and between the other species. Results suggested that the level of C20:1 (n-7) influenced the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. On the other hand, palmitoleic acid (C16:1) levels, C16:1 being the parent of all (n-7) fatty acids, had no statistically significant correlation with the content of any of the fatty acids of the (n-7) or (n-9) family.     

Mapping the genome of rapeseed (Brassica napus L.). II. Localization of genes controlling erucic acid synthesis and seed oil content

A F₁ microspore-derived DH population, previously used for the development of a rapeseed RFLP map, was analysed for the distribution of erucic acid and seed oil content. A clear three-class segregation for erucic acid content could be observed and the two erucic acid genes of rapeseed were mapped to two different linkage groups on the RFLP map. Although the parents of the segregating DH population showed no significant difference in seed oil content, in the DH population a transgressive segregation in oil content was observed. The segregation closely followed a normal distribution, characteristic of a quantitative trait. Using the program MAPMAKER/QTL, three QTLs for seed oil content could be mapped on three different linkage groups. The additive effects of these QTLs explain about 51% of the phenotypic variation observed for this trait in the DH population. Two of the QTLs for oil content showed a close association in location to the two erucic acid genes, indicating a direct effect of the erucic acid genes on oil content.     

Mapping loci controlling the concentrations of erucic and linolenic acids in seed oil of Brassica napus L.

The quality of plant oil is determined by its component fatty acids. Relatively high levels of linolenic acid reduce the oxidative stability of the oil, and high levels of erucic acid in the diet have been associated with health problems. Thus, oilseed Brassica napus cultivars with low linolenic and low erucic acid contents are highly desirable for edible oil production. In order to identify genes controlling the levels of erucic and linolenic acids, we analyzed the oil composition of 99 F₁-derived doubled haploid lines from a cross between cv Major (high levels of erucic and linolenic acids) and cv Stellar (low levels of both fatty acids). A molecular marker linkage map of 199 loci for this population was used to identify quantitative trait loci (QTL) controlling oil composition. We identified two regions that accounted for nearly all of the phenotypic variation in erucic acid concentration and one region that accounted for 47% of the variation in linolenic acid concentration. The QTL associated with linolenic acid concentration mapped near a RFLP locus detected by a cDNA clone encoding an omega-3 desaturase, suggesting that the low linolenic acid content of Stellar may be due to a mutation in this gene.     

Mapping of RFLP and qualitative trait loci in Brassica rapa and comparison to the linkage maps of B. napus,B. oleracea,and Arabidopsis thaliana

A linkage map of restriction fragment length polymorphisms (RFLPs) was constructed for oilseed, Brassica rapa, using anonymous genomic DNA and cDNA clones from Brassica and cloned genes from the crucifer Arabidopsis thaliana. We also mapped genes controlling the simply inherited traits, yellow seeds, low seed erucic acid, and pubescence. The map included 139 RFLP loci organized into ten linkage groups (LGs) and one small group covering 1785 cM. Each of the three traits mapped to a single locus on three different LGs. Many of the RFLP loci were detected with the same set of probes used to construct maps in the diploid B. oleracea and the amphidiploid B. napus. Comparisons of the linkage arrangements between the diploid species B. rapa and B. oleracea revealed six LGs with at least two loci in common. Nine of the B. rapa LGs had conserved linkage arrangements with B. napus LGs. The majority of loci in common were in the same order among the three species, although the distances between loci were largest on the B. rapa map. We also compared the genome organization between B. rapa and A. thaliana using RFLP loci detected with 12 cloned genes in the two species and found some evidence for a conservation of the linkage arrangements. This B. rapa map will be used to test for associations between segregation of RFLPs, detected by cloned genes of known function, and traits of interest.     

Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris

Capsella bursa-pastoris (L.) Medic (2n = 4x = 32) is a natural double-low (erucic acid < 1%, glucosinolates < 30 micromol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Hybridizations were carried out between two Brassica species viz. B. rapa (2n = 20) and B. napus (2n = 38) as female and C. bursa-pastoris as male parent to introduce these desirable traits into cultivated Brassica species. Majority of F(1) plants resembled female parents in morphology and only a few expressed some characters of male parent, including the white petals. Based on cytological observation of somatic cells, the F(1) plants were classified into five types: two types from the cross with B. rapa, type I had 2n = 27-29; type II had 2n = 20; three types from the crosses with B. napus, type III was haploids with 2n = 19; type IV had 2n = 29; type V had 2n = 38. One to two chromosomes of C. bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F1 plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in majority of F1 plants, even in Brassica-types and haploids, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum. The cytological and molecular mechanisms behind these results are discussed.     

The interrelationship between the accumulation of lipids,protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Lipid accumulation during pollen and tapetal development was studied using cryostat sections of unfixed anthers from Brassica napus (rapeseed). Diamidino-2-henylindole (DAPI), a DNA fluorochrome, was used to stain the pollen nuclei in order to identify ten stages of pollen development in Brassica. Storage lipids (i.e. triacylglycerides) were stained using the fluorochrome Nile red. Pollen coat lipids are formed in tapetal plastids between the mid-vacuolate and early maturation pollen stages. The pollen coat components, including lipids and a proportion of the proteins, are derived from the remnants of the tapetum, after its rupture, during the second pollen mitosis. Quantitative microfluorometric analyses demonstrated four phases of lipid body accumulation or depletion in the developing pollen cytoplasm. The majority of storage lipids found in the cytoplasm of the mature pollen grain accumulated during the late vacuolate and early maturation stages when the pollen is bicellular. The level of acyl carrier protein, a protein integrally involved in lipid synthesis, was also found to be maximal in the developing pollen during the bicellular pollen stages of development. This coincided with the most active period of lipid accumulation. These data could indicate that the lipids of the pollen are synthesized in situ, by metabolic processes regulated by expression of genes in the haploid genome.To whom correspondence should be addressed     

Phylogenetic differences in cardiac activity, metal accumulation and mortality of limpets exposed to copper: A prosobranch-pulmonate comparison

Taxonomic relationships for pollutant tolerance in marine invertebrates are surprisingly poorly known, despite being potentially useful for pollution biomonitoring. A popular view is that cellular and molecular adaptations for natural stress may be important in tolerating pollution. We compared the physiological and mortality responses to copper (Cu²⁺) of limpets from two different lineages: the Prosobranchia (Patellogastropoda: Helcion concolor and Cellana capensis) and the Pulmonata (Siphonaria serrata and Siphonaria capensis). Copper tolerance was apparently more closely related to phylogenetically-based physiology, than to tolerance of desiccation and or heat. The Siphonaria limpets were nearly an order of magnitude more tolerant of copper than the patellogastropod limpets, even though S. serrata has the lowest intertidal distribution. Initial copper exposure (0.25 ppm Cu²⁺ for 2 h) induced heart rate depression in Siphonaria (to around 50% of the baseline rate), while their tissue copper concentrations remained at the relatively high control levels. Copper exposure (0.25 ppm Cu²⁺ for 2 h) had no effect on heart rate of the patellogastropod limpets, but led to a significant increase in tissue copper. These results suggest that enhanced copper tolerance by Siphonaria relates to cardiac depression and a concomitant metabolic depression. Such physiological attributes are implicated in prolonged behavioural isolation, involving pneumostome closure and shell clamping, which is likely to reduce the uptake of copper. Furthermore, better regulation of internal copper levels by Siphonaria, is suggested by their exclusive possession of blood haemocyanin. Dependence on relatively high aerobic metabolism by the patellogastropod limpets, would limit their capacity for isolation and pollutant avoidance.     

Characterization of natural variation for zinc, iron and manganese accumulation and zinc exposure response in Brassica rapa L.

Brassica rapa L. is an important vegetable crop in eastern Asia. The objective of this study was to investigate the genetic variation in leaf Zn, Fe and Mn accumulation, Zn toxicity tolerance and Zn efficiency in B. rapa. In total 188 accessions were screened for their Zn-related characteristics in hydroponic culture. In experiment 1, mineral assays on 111 accessions grown under sufficient Zn supply (2 μM ZnSO₄) revealed a variation range of 23.2–155.9 μg g⁻¹ dry weight (d. wt.) for Zn, 60.3–350.1 μg g⁻¹ d. wt. for Fe and 20.9–53.3 μg g⁻¹ d. wt. for the Mn concentration in shoot. The investigation of tolerance to excessive Zn (800 μM ZnSO₄) on 158 accessions, by using visual toxicity symptom parameters (TSPs), identified different levels of tolerance in B. rapa. In experiment 2, a selected sub-set of accessions from experiment 1 was characterized in more detail for their mineral accumulation and tolerance to excessive Zn supply (100 μM and 300 μM ZnSO₄). In this experiment Zn tolerance (ZT) determined by relative root or shoot dry biomass varied about 2-fold. The same six accessions were also examined for Zn efficiency, determined as relative growth under 0 μM ZnSO₄ compared to 2 μM ZnSO₄. Zn efficiency varied 1.8-fold based on shoot dry biomass and 2.6-fold variation based on root dry biomass. Zn accumulation was strongly correlated with Mn and Fe accumulation both under sufficient and deficient Zn supply. In conclusion, there is substantial variation for Zn accumulation, Zn toxicity tolerance and Zn efficiency in Brassica rapa L., which would allow selective breeding for these traits.     

Field testing of transgenic rapeseed cv. Hero transformed with a yeast sn-2 acyltransferase results in increased oil content,erucic acid content and seed yield

A major goal of our research is to produce, by genetic manipulation, Brassica napus L. cultivars with higher levels of 22:1 in their seed oil than in present Canadian HEA cultivars developed through traditional breeding. Previously, we reported that transgenic expression of a mutated yeast sn-2 acyltransferase (SLC1-1) in industrial rapeseed cv. Hero resulted in increased seed oil content, increased proportions of erucic acid and increased average seed weight (Zou et al. 1997). Those results were reported only for plants grown in a controlled greenhouse setting. Here we report a summary of the results from two successive years of field trials with T₄ and T₅ generations of B. napus cv. Hero transformed with the SLC1-1 gene. These trials, conducted at Rosthern, Saskatchewan, in two very different growing seasons, show that the SLC1-1 transgenics clearly and consistently out-performed controls, with much increased oil and 22:1 contents, as well as yield, under varying field conditions.     

Formation of nodular structures on the non-legumes Brassica napus,B. campestris,B. juncea and Arabidopsis thaliana with Bradyrhizobium and Rhizobium isolated from Parasponia spp. or legumes grown in tropical soils

Strains of Bradyrhizobium formed nodule-like structures on Arabidopsis and species of Brassica in pots with sandvermiculite and in glass tubes on a nitrogen-free mineral salts agar. Broad-host-range Rhizobium strains NGR234 from Lablab purpureus and NGR76 from Phaseolus vulgaris formed similar nodule-like structures on Brassica spp. The size of these structures on plants in pots were large, often reaching 10 mm in diameter.The frequency of inoculated Brassica plants in pots with nodule-like structures was 25–50%, depending on the inoculum strain. The inheritable nature of factors involved in the formation of the nodule-like structures was demonstrated when the structures occurred on 100% of inoculated B. napus seedlings derived from plants with the nodule-like structures.Nodule-like structures occurred without, but not with, the application of a cellulase-pectolyase-PEG treatment to the roots. Attempts to isolate Bradyrhizobium or Rhizobium from the nodule-like structures failed. Internal infection of these structures could not be detected using either the light or electron microscope. The inoculum strains of root-nodule bacteria were detected in high numbers in the rhizosphere of plants 5 months after inoculation. On agar plates bacterial colonies could be seen, with undiminished growth, over the surface of the agar extending to the root surface. However, ground root tissue of Brassica was toxic to Bradyrhizobium strains. This suggested that Bradyrhizobium strains would not survive after infecting the roots of Brassica spp. Nitrogen fixation was associated with high rhizosphere populations of Azospirillum and not with Bradyrhizobium induced nodule structures of Brassica spp.     

Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.     

Effects of water deficit stress on growth,water relations and osmolyte accumulation in Medicago truncatula and M. laciniata populations

The effects of water stress were investigated in two Tunisian Medicago truncatula populations collected from arid (Mt-173) and sub-humid (Mt-664) climates and two Tunisian M. laciniata populations originating from arid (Ml-173) and semi-arid (Ml-345) regions. After a pre-treatment phase (24 days after sowing, DAS) of watering at 100% of field capacity (FC), the plants were either irrigated at 100% FC or at only 33% FC. After 12 days of treatment (36 DAS), one lot of dehydrated plants was rewatered at 100% FC. A final harvest was carried out after 24 days of treatment (48 DAS). Measured parameters were total dry weight (TDW), root shoot ratio (RSR), leaf relative water content (RWC), osmotic potential (OP), photosynthetic parameters (CO₂ net assimilation A, stomatal conductance g_s and transpiration E), malondialdehyde (MDA) concentration and leaf contents in inorganic (Na⁺ and K⁺) and organic solutes (proline and soluble sugars). Under water deficit conditions, compared to M. laciniata, M. truncatula populations showed a higher reduction in TDW, A, g_s and RWC associated with a higher increase in MDA concentration. Thus, the relative tolerance of M. laciniata populations to water shortage would be related to their lower intrinsic growth rate and stomatal control of gas exchange. TDW, A, g_s, E and RWC were more decreased by water deficit in Ml-345 than in Ml-173. Drought tolerance of Ml-173 was found to be associated with a more pronounced decrease of OP and a lower reduction in RWC due to the accumulation of solutes such as proline, soluble sugars and K⁺. In addition, Ml-173 showed the highest water use efficiency values (WUE) and the lowest MDA concentrations under water deficit stress.