Variation in the glucosinolate content of oilseed rape (Brassica napus L.) leaves

The glucosinolate content of oilseed rape {Brassica napus) leaves was monitored over the growth period 30–70 days after planting, and a comparison made between a single-low cultivar (low in erucic acid), Bienvenu, and a double-low cultivar (low in erucic acid and glucosinolate), Cobra. In older, fully-expanded leaves the glucosinolate concentration was very low (< 0.3 μmol/ml tissue water) and did not alter during the course of the experiment. In developing sixth leaves glucosinolate content increased rapidly and reached a maximum concentration (4–5 μmol/ml tissue water) 40 days after planting (6 days after leaf emergence). The concentration then declined, to about 1 μmol/ml after 60 days although the total glucosinolate content in leaves continued to increase until 50 days; much of the reduction in concentration was simply a result of leaf expansion. No major differences were seen between the two varieties in total glucosinolate content or in the individual compounds present. Cv. Cobra developed more quickly than cv. Bienvenu so direct comparison between leaves of the two cultivars was complex. When comparing the glucosinolate content of oilseed rape leaves, between cultivars or between treatments, it is vital to ensure that carefully matched leaves of comparable developmental age are selected.     

The effect of modifying the glucosinolate content of leaves of oilseed rape (Brassica napus ssp. oleifera) on its interaction with specialist and generalist pests

Twenty eight Brassica napus lines were developed which had contrasting leaf glucosinolate profiles to those found in commercial oilseed rape cultivars. The lines varied both in the total amount of aliphatic glucosinolates and in the ratio of different side chain structures. The lines were used in field experiments to assess the manner by which glucosinolates mediate the interactions between Brasssica and specialist pests (Psylliodes chrysocephala and Pieris rapae) and generalist pests (pigeons and slugs). Increases in the level of glucosinolates resulted in greater damage by adult flea beetles (P. chrysocephala) and a greater incidence of Pieris rapae larvae, but reduced the extent of grazing by pigeons and slugs. Decreasing the side chain length of aliphatic glucosinolates and reducing the extent of hydroxylation of butenyl glucosinolates increased the extent of adult flea beetle feeding. The implications of modifying the glucosinolate content of the leaves of oilseed rape and the role of these secondary metabolites in plant/herbivore interactions are discussed.     

Changes in glucosinolates during crop development in single- and double-low genotypes of winter oilseed rape (Brassica napus): II. Profiles and tissue-water concentrations in vegetative tissues and developing pods

The concentrations of glucosinolates in the tissue water of leaves, stems, floral buds and developing pods were measured at defined stages of development in four oilseed rape cultivars known to have different seed glucosinolate concentrations (Bienvenu, Ariana, Cobra and Capricorn). Five alkenyl, two aromatic and three indolyl compounds were identified in the vegetative and reproductive organs. Substantial differences developed in the profiles of compounds present during vegetative growth. The 2-hydroxy-3-butenyl glucosinolate was primarily associated with developing and mature seeds and germinated seedlings. The 4-pentyl glucosinolate occurred mainly during the later stages of vegetative growth in spring, when leaves contained higher concentrations than stems, and during the early stages of flowering. The 2-phenylethyl and 3-indolymethyl glucosinolates were present earlier in vegetative growth when similar concentrations were present in leaves and stems. Differences between cultivars in the tissue-water concentrations of individual glucosinolates were small during vegetative growth. The leaves of Cobra and Capricorn had smaller concentrations of the 3-pentyl glucosinolate than Bienvenu and Ariana especially early in spring, and the stems of Capricorn had smaller concentration of the 3-pentyl and 2-phenylethyl glucosinolates. Greater differences between cultivars developed during pod growth and involved large increases in the concentrations of the 2-hydroxy-3-butenyl and 3-butenyl glucosinolates, especially in Bienvenu and Ariana. The implications of these changes in the types and concentrations of glucosinolates in the different organs for the incidence of pests and diseases are discussed.     

Molecular markers for low-glucosinolate alleles in oilseed rape (Brassica napus L.)

Two recent studies have mapped QTLs associated with the level of seed glucosinolates in oilseed rape (Brassica napus L.). It was likely that the two most significant QTLs identified in each study were the same, as they were linked to RFLP alleles identified by common DNA probes. To investigate the utility of these probes in breeding programmes, they were used to study RFLPs in a range of low- and high-glucosinolate cultivars and breeding lines. It was shown that all low glucosinolate spring and winter cultivars possessed a specific RFLP fragment identified by probe wg3f7 which is linked to theGSL-1 QTL, and all high-glucosinolate cultivars possessed a specific RFLP fragment identified by probe wg7a8, which is linked to theGSL-2 QTL. Cultivar Ariana, which has intermediate levels of glucosinolates possessed the low-glucosinolate fragment atGSL-1 but the high-glucosinolate fragment atGSL-2. A similar result was found with the cvs. Martina and Bronowski which have intermediate and variable levels of glucosinolates. There were no other RFLP fragments identified by other DNA probes which were specific to either the low- or high-glucosinolate phenotypes. The use of probes wg3f7 and wg7a8 in selection of low-glucosinolate lines in breeding programmes is discussed.     

油菜烯醇酶基因的克隆与分析

烯醇酶(enolase)是糖酵解途径中的一个重要酶类,它能够催化磷酸甘油酸酯(2-PGA)生成磷酸烯醇丙酮酸酯(PEP).我们通过RACE-PCR方法从油菜(Brassica napus L.)中克隆到了编码烯醇酶的全长基因.序列分析表明该基因全长cDNA为1 624bp,拥有一个由444个氨基酸组成的开放读码框,所编码的蛋白质分子量为47.38 kD,等电点为5.78.比较发现,油菜烯醇酶与已分离出的其他烯醇酶氨基酸序列有较高的同源性.Southern杂交结果显示烯醇酶以低拷贝形式在油菜基因组中存在.RT-PCR和Northern分析表明烯醇酶基因在100 mmol/L盐浓度胁迫条件下表达量上升,而在低温诱导时表达量下降.该研究表明所克隆基因是植物烯醇酶基因家族的新成员.     

油菜烯醇酶基因的克隆与分析(英文)

烯醇酶(enolase)是糖酵解途径中的一个重要酶类,它能够催化磷酸甘油酸酯(2-PGA)生成磷酸烯醇丙酮酸酯(PEP)。我们通过RACE-PCR方法从油菜(Brassica napus L. )中克隆到了编码烯醇酶的全长基因。序列分析表明该基因全长cDNA为1624bp,拥有一个由444个氨基酸组成的开放读码框,所编码的蛋白质分子量为47.38kD,等电点为5.78。比较发现,油菜烯醇酶与已分离出的其他烯醇酶氨基酸序列有较高的同源性。Southern杂交结果显示烯醇酶以低拷贝形式在油菜基因组中存在。RT-PCR和Northern分析表明烯醇酶基因在100mmol/L盐浓度胁迫条件下表达量上升,而在低温诱导时表达量下降。该研究表明所克隆基因是植物烯醇酶基因家族的新成员。     

Differential response of oilseed rape (Brassica napus L.) cultivars to low boron supply

Three experiments were carried out on an alluvial sandy loam (Udifluvent) at Tonglu, Zhejiang Province, P.R. China from 1992 to 1995, to determine the genotypic range in boron (B) efficiency of 16 oilseed rape (Brassica napus L.) cultivars, to identify the B-efficient cultivars and to identify specific responses which can be utilised for selection in a breeding program. The 16 cultivars which included high-quality and conventional types differed significantly in survival, plant height, leaf area, shoot dry weight and seed yield; however, the ranking of the cultivars for their seed yield or other plant traits differed with B treatment. With severe B deficiency (CaCl2 extractable B < 0.26 mg/kg) and no boron applied, none of the cultivars exhibited significant B efficiency, with seed yield <300 kg/ha. With moderate B deficiency (CaCl2 extractable B 0.34 mg/kg or 0.17 kg B/ha applied), seed yield varied significantly among the cultivars from 397 to 1889 kg/ha in year 1 and from 616 to 1260 kg/ha in year 3. Zhongyou 821 and 92-13 were the most B-efficient and Wanyou 324, Huashuang 2 and Su 2051 were the most B-inefficient cultivars under moderate B deficiency. Significant differences were found among the cultivars in leaf B concentration; however, there was no close relationship between leaf B concentration and seed yield responses to B of oilseed cultivars. Of all the growth parameters measured, leaf area was the early indicator best correlated with subsequent seed yield and may be useful for evaluating the response of cultivars to low B supply. Contrary to current opinion, it was also found that high-quality oilseed rape cultivars were not all sensitive to low B supply nor were all conventional cultivars B-efficient.     

Hormonal requirement and tissue competency for shoot organogenesis in two cultivars of Brassica napus

An investigation of the regeneration ability of explants taken from the floral stem of Brassica napus var. oleifera was performed in the winter cultivars Darmor and Bienvenu. Our purpose was to compare the regeneration ability of the two genotypes, to compare the competence of the different tissues of the stem and then to study histologically the regeneration of shoots. A strong genotypic effect was observed between the two cvs; Bienvenu had a poorer ability to produce shoots when cultured in the presence of benzyladenine: regeneration commenced later; the percentage of explants producing shoots and the number of shoots per regenerating explant were much lower. The comparison between the regeneration ability of different explants, i.e stem segments, internal stem segments, thin cell layer and peels, showed that the superficial tissues were able to regenerate roots but not shoots. Contrariwise, internal stem segments regenerated only shoots. The origin of shoots was investigated in stem segments of cv. Darmor. A kinetic histological analysis showed the basic role played by phloem and phloem-associated cells in shoot formation.     

Effects of the triazole plant growth retardant BAS 111¨W on gibberellin levels in oilseed rape, Brassica napus

Three-week-old shoots of the spring oilseed rape cv. Petranova ( Brassica napus L. ssp. napus ) were found by combined gas chromatography-mass spectrometry to contain GA₁, GA₈, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₂₉, 3-epi-GA₁ and a previously uncharacterised C₁₉ dicarboxylic acid that is probably structurally related to GA₂₄. Shoots of the winter cultivar Belinda, harvested at the early flowering stage, contained the same GAs with the exception of the C₁₉ dicarboxylic acid and, in addition, GA₃₄ and GA₅₁ were identified. All material contained higher levels of GA₂₀ than of GA₁; the ratio of GA₁ to GA₂₀ was highest in shoots containing the largest proportion of young immature tissues. Soil treatment of cv. Petranova seedlings with the growth retardant BAS 111¨W [1-phenoxy-5,5-dimethyl-3-(1,2,4-triazol-1-yl)-hexan-4-ol] caused 80% reduction in height 18 days after treatment and the levels of all GAs were 20% or less that of control plants. Foliar treatment at the same dosage reduced height by 50% and caused an 85% or greater reduction in the concentrations of the GA₁ precursors GA₂₀, GA₁₉ and GA₄₄. However, the levels of GA₁, GA₈ and GA₂₉ were affected to a much smaller extent. Foliar application of BAS 111¨W to cv. Belinda 1 month after sowing resulted in only a 20% height reduction at flowering, but no uniform decrease in the concentrations of endogenous GAs at this stage.     

Development and characteristics of myrosinase in Brassica napus during early seedling growth

Myrosinase (β-thioglucoside glucohydroase, E. C. 3.2.3.1) proteins with different physical, but similar kinetic characteristics exist in oilseed rape ( Brassica napus L. cv. Bienvenu) seedlings. Two protein fractions have been described which are immunologically, and therefore likely to be structurally, related. Myrosinase I, a dimeric 156 kDa glycosylated protein was purified to apparent homogeneity, and polyclonal antibodies were raised against this protein. Myrosinase II, in comparison, was significantly less glycosylated. The native protein had a molecular weight of approximately 188 kDa, with subunit M_r's of mainly 62 kDa and also 68 kDa. Total 'potential'enzyme activity (assayed in the presence of ascorbic acid activator) increased during early seedling growth. Immunoblot analysis of seedling proteins showed that this is directly related to an increase in the amount of myrosinase protein itself , predominantly myrosinase II proteins, which are not present in the dry seed. Myrosinase II protein is located exclusively in the cotyledons of 5-day-old seedlings, whilst myrosinase I is distributed throughout the seedling.     

Barriers to gene flow from oilseed rape (Brassica napus) into populations of Sinapis arvensis

One concern over growing herbicide-tolerant crops is that herbicide-tolerance genes may be transferred into the weeds they are designed to control. Brassica napus (oilseed rape) has a number of wild relatives that cause weed problems and the most widespread of these is Sinapis arvensis (charlock). Sinapis arvensis seed was collected from 102 populations across the UK, within and outside B. napus-growing areas. These populations were tested for sexual compatibility with B. napus and it was found that none of them hybridized readily in the glasshouse. In contrast to previous studies, we have found that hybrids can be formed naturally with S. arvensis as the maternal parent. Six diverse B. napus cultivars (Capricorn, Drakkar, Falcon, Galaxy, Hobson and Regent) were tested for their compatibility with S. arvensis but no cultivar hybridized readily in the glasshouse. We were unable to detect gene transfer from B. napus to S. arvensis in the field, confirming the extremely low probability of hybridization predicted from the glasshouse work.     

Dehiscence-related expression of an Arabidopsis thaliana gene encoding a polygalacturonase in transgenic plants of Brassica napus

Pod dehiscence in Arabidopsis thaliana is accompanied by an increase in the expression of a polygalacturonase (PG). The gene encoding this mRNA has been characterized and shown to have extensive homology to a similar PG gene from Brassica napus . The A. thaliana PG promoter was fused to β -glucuronidase (GUS) and the expression of this reporter gene analysed in transgenic B. napus plants. The GUS activity was detected throughout the dehiscence zone of pods from 35 d after anthesis and expression was restricted to those cells that undergo cell separation. Expression was also detectable at the junction between the seed and the funicular tissue and in mature anthers of flowers. Transgenic plants containing the PG promoter fused to barnase were sterile as a consequence of the anthers failing to undergo dehiscence. Fertilization of PG-barnase plants resulted in the development of pods that exhibited a reduced capacity to shatter. The role of PG during cell separation processes in plants is discussed.     

Sequence‐level comparative analysis of the Brassica napus genome around two stearoyl‐ACP desaturase loci

We conducted a sequence‐level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl‐ACP desaturase‐encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (～56%) is lower than has been reported previously between A. thaliana and Brassica (～66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 ± 3.9% and 85.8 ± 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 ± 3.1% between the B. napus A genome and B. rapa, and 93.1 ± 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.     

The incidence of turnip yellows virus in oilseed rape crops (Brassica napus L.) in three different regions of England over three consecutive growing seasons and the relationship with the abundance of flying Myzus persicae

Turnip yellows virus (TuYV) is the most important virus infecting oilseed rape in the United Kingdom. The incidence and spatial distribution of TuYV in winter oilseed rape (WOSR) crops in three regions of England were determined over three growing seasons. Leaf samples were collected from three fields in each region, in autumn (November–December) and spring (April) of the three crop seasons and tested for virus presence by enzyme-linked immunosorbent assay. Infection was detected in all fields except one. Higher TuYV incidences were recorded in 2007–2008 (≤89%) and 2009–2010 (≤100%) crop seasons than in 2008–2009 (≤24%). Highest incidences were recorded in Lincolnshire (≤100%), followed by Warwickshire (≤88%), with lowest incidences in Yorkshire (1–74%). There was a significant increase in incidence detected between autumn and spring sampling in eight fields, a significant decrease in one field and no significant change in 18 fields. Rothamsted Insect Survey suction trap data for the aphid Myzus persicae in Lincolnshire, Warwickshire and Yorkshire revealed two peaks of flight activity in most years (2007–2009). The second peak (September–November) coincided with emergence of WOSR. The highest cumulative (August–November) trap catches in the three regions during the three crop seasons occurred in Lincolnshire and the lowest in Yorkshire; catches in autumn 2009 were highest and lowest in autumn 2008. Regression analysis revealed a highly significant association between the cumulative numbers of M. persicae caught in the suction traps closest to the crops between August and November each year and the incidence of TuYV detected in the WOSR crops in the autumn of each year. Results are discussed in the light of factors affecting the spread of TuYV and future possibilities for control.     

Expression of resistance to Leptosphaeria maculans in Brassica napus double haploid lines in France and Australia is influenced by location

Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus), worldwide, including Australia and France. The aims of these studies were first, to determine if higher levels of resistance to L. maculans could be generated in double haploid (DH) lines derived from spring‐type B. napus cv. Grouse, which has a good level of field resistance to blackleg; and second, to determine whether the resistance to blackleg disease of individual DH lines responds differentially to different L. maculans field populations within and between the two countries. DH lines were extracted from cv. Grouse and tested in field experiments carried out in both France and Australia against natural L. maculans populations. Extracting and screening DH lines were an effective means to select individual lines with greatly improved expression of resistance to blackleg crown canker disease in comparison with the original parental population. However, relative disease resistance rankings for DH lines were not always consistent between sites. The higher level of resistance in France was shown to be because of a high expression level of quantitative resistance in the French growing conditions. Big differences were observed for some DH lines between the 2004 and the 2005 field sites in Australia where the L. maculans populations differed by their virulence on single dominant gene‐based resistant lines derived from Brassica rapa ssp. sylvestris. This differential behaviour could not be clearly explained by the specific resistance genes until now identified in these DH lines. This investigation highlights the potential to derive DH lines with superior levels of resistance to L. maculans compared with parental populations. However, in locations with particularly high pathogen diversity, such as in southern Australia, multiyear and multisite evaluations should be performed to screen for the most efficient material in different situations.     

Changes in glucosinolates during crop development in single- and double-low genotypes of winter oilseed rape (Brassica napus): I. Production and distribution in vegetative tissues and developing pods during development and potential role in the recycling of sulphur within the crop

The distributions of glucosinolates and sulphur were measured in the vegetative and reproductive tissues in a series of single- and double-low cultivars of oilseed rape (Bienvenu, Ariana, Cobra and Capricorn) grown on a sulphur-sufficient soil at Rothamsted in 1987/88, and in crops of the cv. Libravo grown with none or 40 kg/ha of sulphur on a sulphur-deficient soil at Woburn in 1990/91. The glucosinolate measurements demonstrated large differences in the abilities of single- and double-low cultivars to synthesise glucosinolates, and showed that the biosynthetic differences were associated more with the developing pods than the vegetative tissues. It indicated that potential contribution of intact glucosinolates from vegetative tissues to the seed was likely to be small, but did not preclude the possibility that the vegetative tissues were a source of glucosinolate precursors. The sulphur measurements showed that the glucosinolates contained only a small proportion of the crop's total sulphur and that they were unlikely to be a major source of recyclable sulphur, even under conditions of severe sulphur deficiency.