Starch metabolism in developing embryos of oilseed rape

The aim of this work was to characterise the metabolism of starch in developing embryos of oilseed rape (Brassica napus L. cv. Topaz). The accumulation of starch in embryos in siliques which were darkened or had been exposed to the light was similar, suggesting that the starch is synthesised from imported sucrose rather than via photosynthesis in the embryo. Starch content and the activities of plastidial enzymes required for synthesis of starch from glucose 6-phosphate (Glc6P) both peaked during the early-mid stage of cotyledon development (i.e. during the early part of oil accumulation) and then declined. The mature embryo contained almost no starch. The starch-degrading enzymes α-(EC 3.2.1.1) and β-amylase (EC 3.2.1.2) and phosphorylase (EC 2.4.1.1) were present throughout development. Most of the activity of these three enzymes was extraplastidial and therefore unlikely to be involved in starch degradation, but there were distinct plastidial and extraplastidial isoforms of all three enzymes. Activity gels indicated that distinct plastidial isoforms increase during the change from net synthesis to net degradation of starch. Plastids isolated from embryos at stages both before and after the maximum starch content could convert Glc6P to starch although the rate was lower at the later stage. The results are consistent with the idea that starch synthesis and degradation occur simultaneously during embryo development. The possible roles of transient starch accumulation during embryo development are discussed. Received: 15 May 1997 / Accepted: 30 May 1997     

Carbon supply for storage-product synthesis in developing seeds of oilseed rape

The aim of this work was to find out how the sugars in the endosperm of oilseed rape contribute to the flux of oil synthesis. While the hexose content of the liquid endosperm decreased during development the sucrose content increased. It is important to understand the relative rates of use of the endosperm sugars for two reasons. Firstly we need to know which sugars are used, and at what stages in development, in order to understand the roles of enzymes involved in their metabolism. Secondly, changes in sugar concentration have been implicated in the regulation of expression of genes determining storage-product synthesis [see Weber, Borisjuk and Wobus (1997) Trends Plant Sci. 2, 169-174, for review]. The rate of consumption of sugar is one factor governing its concentration. We present data showing both the concentration-dependence of conversion of sugar to oil, and the in vivo concentrations of sugars; we relate these data sets to each other and discuss the effects of the intracellular pool of sucrose. Glucose, fructose and sucrose are all substrates for oil synthesis, but the rates of their use (particularly sucrose) are underestimated because of dilution by sucrose from the intracellular pool.     

Coordinate changes in carbon partitioning and plastidial metabolism during the development of oilseed rape embryos

Measurements of metabolic fluxes in whole embryos and isolated plastids have revealed major changes in the pathways of carbon utilization during cotyledon filling by oilseed rape (Brassica napus L.) embryos. In the early cotyledon stage (stage A), embryos used sucrose (Suc) predominantly for starch synthesis. Plastids isolated from these embryos imported glucose-6-phosphate (Glc-6-P) and partitioned it to starch and fatty acids synthesis and to the oxidative pentose phosphate pathway in the ratio of 2:1:1 on a hexose basis. Of the substrates tested, Glc-6-P gave the highest rates of fatty acid synthesis by the plastids and pyruvate was used weakly. By the mid- to late-cotyledon stage (stage C), oil accumulation by the embryos was rapid, as was their utilization of Suc for oil synthesis in vitro. Plastids from C-stage embryos differed markedly from those of stage-A embryos: (a) pyruvate uptake and utilization for fatty acid synthesis increased by respectively 18- and 25-fold; (b) Glc-6-P partitioning was predominantly to the oxidative pentose phosphate pathway (respective ratios of 1:1:3); and (c) the rate of plastidial fatty acid synthesis more than doubled. This increased rate of fatty synthesis was dependent upon the increase in pyruvate uptake and was mediated through the induction of a saturable transporter activity.     

Metabolism of sugars in the endosperm of developing seeds of oilseed rape

The sugars in the endosperm of a developing seed have many potential roles, including the supply of carbon to the developing embryo and controlling gene expression in it. Our understanding of their metabolism is, however, fragmentary and is confined to a very few species (especially Vicia spp.). To develop a quantitative understanding of the regulation of sugars in seeds of oilseed rape (Brassica napus), we measured relevant enzyme activities, the sizes of the pools of sugars in the liquid endosperm, and the flux of sugars from the endosperm into the embryo. The concentrations of hexose sugars in the liquid endosperm decreased, and sucrose (Suc) increased through development. The overall osmotic potential also fell. The timing of the changes was not precise enough to determine whether they signaled the onset of rapid accumulation of storage products. Changes in endosperm invertase activity were complex and quantitatively do not explain the changes in sugars. The embryo can metabolize hexose sugars in addition to Suc, and possibly at higher rates. Therefore, in addition to invertase, the growing embryo itself has a potential to influence the balance of sugars in the endosperm. The activity of Suc synthase in the embryo was greater than that of invertase during development. This observation and a higher activity of fructokinase than glucokinase in the embryo are both consistent with the embryo using Suc as a carbon source.     

Lipid storage metabolism is limited by the prevailing low oxygen concentrations within developing seeds of oilseed rape

The aim of this study was to investigate whether endogenous restrictions in oxygen supply are limiting for storage metabolism in developing oilseed rape (Brassica napus) seeds. Siliques were studied 30 d after flowering, when rapid lipid accumulation is occurring in the seeds. (a). By using microsensors, oxygen concentrations were measured within seeds and in the silique space between seeds. At ambient external oxygen (21% [v/v]) in the light, oxygen fell to 17% (v/v) between and 0.8% (v/v) within seeds. A step-wise reduction of the external oxygen concentration led within 2 h to a further decrease of internal oxygen concentrations, and a step-wise increase of the external oxygen concentration up to 60% (v/v) resulted in an increase in internal oxygen that rose to 30% (v/v) between and 8% (v/v) within seeds. (b). The increase in oxygen levels in the seeds was accompanied by a progressive increase in the levels of ATP, UTP, and the ATP to ADP and UTP to UDP ratios over the entire range from 0% to 60% (v/v) external oxygen. (c). To investigate metabolic fluxes in planta, 14C-sucrose was injected into seeds, which remained otherwise intact within their siliques. The increase in oxygen in the seeds was accompanied by a progressive increase in the rate of lipid (including triacylglycerol), protein and cell wall synthesis, and an increase in glycolytic flux over a range from sub- to superambient oxygen concentrations. In contrast to lipid synthesis, starch synthesis was not significantly increased at superambient oxygen levels. The levels of fermentation products such as lactate and glycerol-3P increased only at very low (0%-4% [v/v]) external oxygen concentrations. (d). When 14C-acetate or 14C-acetyl-coenzyme A (CoA) was injected into seeds, label incorporation into triacylglycerol progressively increased over the whole range of external oxygen concentrations from 0% to 60% (v/v). (e). Stimulation of lipid synthesis was accompanied by an increase in sugar levels and a decrease in the levels of hexose-phosphates and acetyl-CoA, indicating sucrose unloading and the use of acetyl-CoA as possible regulatory sites. (f). Increased lipid synthesis was also accompanied by an increase in the maximal activities of invertase and diacylglycerol acyltransferase. (g). The developmental shift from starch to lipid storage between 15 and 45 d after flowering was accompanied by an increase in the seed energy state. (h). The results show that at ambient oxygen levels, the oxygen supply is strongly limiting for energy metabolism and biosynthetic fluxes in growing rape seeds, affecting lipid synthesis more strongly than starch synthesis. The underlying mechanisms and implications for strategies to increase yield and storage product composition in oilseed crops are discussed.     

Potential effects of global warming on oilseed rape pathogens in Northern Germany

This review analyses potential effects of global warming, i.e. higher mean temperatures, on the life cycle of five economically important oilseed rape pathogens in Northern Germany using a meta-analytical approach. First, the currently available but strongly fragmented knowledge about temperature influences on individual life cycle stages of the referred pathogens, such as survival, sporulation, infection and disease progress is summarised. These data are compared with current regional climate projections (REMO model) based on emission scenario A1B for the periods 2001–2030 and 2071–2100 in three different oilseed rape growing regions in Lower Saxony, Germany, using a baseline historical series of meteorological data collected from 1971 to 2000. Our analysis suggests that warming might lead to shifts in the future prevalence of these pathogens. Verticillium longisporum, Sclerotinia sclerotiorum and Alternaria brassicae could be particularly favoured, but beneficial effects may also occur for certain life cycle stages of Phoma lingam, such as infection and stem canker development. Pyrenopeziza brassicae might even lose importance under future warming. These changes may be particularly explicit in the long-term view (2071–2100). However, predictions on potential disease shifts are restricted by a lack of reliable, high-resolution future climate scenarios and complicated by the prospective adaptations of farming techniques and crop genotypes to climate change.     

Glucosinolate biosynthesis in oilseed rape (Brassicanapus L.): studies with 35SO2-4 and glucosinolate precursors using oilseed rape pods and seeds

³⁵SO^2–₄ and glucosinolate precursors were administeredto investigate the capacity of oilseed rape intact whole pods,isolated pod walls and seeds to carry out reactions of the glucosinolatebiosynthetic pathway. Pod walls incorporated ³⁵S-label from³⁵SO^2–₄ into both the thio-glucose and sulphonate-sulphurof glucosinolates. Pulse-labelling experiments showed that podwalls were the predominant source of the glucosinolates accumulatedby the seeds. Isolated immatureseeds were capable of reductiveassimilation of ³⁵SO^2–₄ and incorporated ³⁵S into sulphur-containingamino acids and the sulphonate moiety of glucosinolates, butwere not able to incorporate ³⁵S into the thio-glucose of glucosinolates. Key words: Biosynthesis, glucosinolates, oilseed rape, pod walls, seeds     

Mapping of QTL for seed dormancy in a winter oilseed rape doubled haploid population

Following winter oilseed rape cultivation, considerable numbers of volunteer oilseed rape plants may occur in subsequent years in following crops. The appearance of volunteer oilseed rape plants is based on the capability of the seeds to become secondary dormant and to survive in this stage for many years in the soil. Genetic reduction of secondary seed dormancy in oilseed rape could provide a means to reduce the frequency of volunteer plants and especially the dispersal of transgenic oilseed rape. The objective of the present study was to analyse the inheritance of primary and secondary seed dormancy in a winter oilseed rape doubled haploid population derived from the cross Express 617 × R53 and to study correlations to other seed traits. Field experiments were performed in Germany for 2 years at two locations with two replicates. Seeds harvested from open pollinated plants were used for all analyses, including a laboratory test for seed dormancy. A previously developed molecular marker map of the doubled haploid population was used to map QTL of the relevant traits. For primary, secondary and total seed dormancy, the results showed significant effects of the genotypes and their interactions, with years and locations. Two, four and five QTL were detected for primary, secondary and total seed dormancy which explained 19, 35 and 42 % of the phenotypic variance, respectively. Results show that secondary seed dormancy is a heritable trait and that selection for low secondary seed dormancy is possible.     

Sources of uncertainty in the quantification of genetically modified oilseed rape contamination in seed lots

Testing of seed and grain lots is essential in the enforcement of GM labelling legislation and needs reliable procedures for which associated errors have been identified and minimised. In this paper we consider the testing of oilseed rape seed lots obtained from the harvest of a non-GM crop known to be contaminated by volunteer plants from a GM herbicide tolerant variety. The objective was to identify and quantify the error associated with the testing of these lots from the initial sampling to completion of the real-time PCR assay with which the level of GM contamination was quantified. The results showed that, under the controlled conditions of a single laboratory, the error associated with the real-time PCR assay to be negligible in comparison with sampling error, which was exacerbated by heterogeneity in the distribution of GM seeds, most notably at a small scale, i.e. 25 cm³. Sampling error was reduced by one to two thirds on the application of appropriate homogenisation procedures.     

Insect pollination enhances seed yield, quality, and market value in oilseed rape

The relationships between landscape intensification, the abundance and diversity of pollinating insects, and their contributions to crop yield, quality, and market value are poorly studied, despite observed declines in wild and domesticated pollinators. Abundance and species richness of pollinating insects were estimated in ten fields of spring oilseed rape, Brassica napus var. SW Stratos?, located along a gradient of landscape compositions ranging from simple landscapes dominated by arable land to heterogeneous landscapes with extensive cover of semi-natural habitats. In each field, we assessed the contribution of wind and insect pollination to seed yield, seed quality (individual seed weight and oil and chlorophyll contents), and market value in a block experiment with four replicates and two treatments: (1) all flowers were accessible to insects, self and wind pollination, and (2) flowers enclosed in tulle net bags (mesh: 1 × 1 mm) were accessible only to wind and self pollination. Complex landscapes enhanced the overall abundance of wild insects as well as the abundance and species richness of hoverflies. This did not translate to a higher yield, probably due to consistent pollination by honey bees across all fields. However, the pollination experiment showed that insects increased seed weight per plant by 18% and market value by 20%. Seed quality was enhanced by insect pollination, rendering heavier seeds as well as higher oil and lower chlorophyll contents, clearly showing that insect pollination is required to reach high seed yield and quality in oilseed rape. Our study demonstrates considerable and previously underestimated contributions from pollinating insects to both the yield and the market value of oilseed rape.     

Post-flowering nitrogen uptake leads to the genotypic variation in seed nitrogen accumulation of oilseed rape

Plant and Soil - The conventional management adopted in many Mediterranean olive orchards makes them more vulnerable to climate change and attacks by pathogens, due to the decreased chemical plant...     

Homoeologous loci control the accumulation of seed glucosinolates in oilseed rape (Brassica napus).

The genetic control of seed glucosinolate content in oilseed rape was investigated using two intervarietal backcross populations. Four QTLs segregating in the population derived from a Brassica napus L. 'Victor' x Brassica napus L. 'Tapidor' cross, together accounting for 76% of the phenotypic variation, were mapped. Three of these loci also appeared to control the accumulation of seed glucosinolates in a Brassica napus L. 'Bienvenu' x 'Tapidor' cross, and accounted for 86% of the phenotypic variation. The three QTLs common to both populations mapped to homoeologous regions of the B. napus genome, suggesting that seed glucosinolate accumulation is controlled by duplicate genes. It was possible to extend the comparative analysis of QTLs controlling seed glucosinolate accumulation by aligning the published genetic maps generated by several research groups. This comparative mapping demonstrated that high-glucosinolate varieties often carry low-glucosinolate alleles at one or more of the loci controlling seed glucosinolate accumulation.     

Large scale identification and quantitative profiling of phosphoproteins expressed during seed filling in oilseed rape

Seed filling is a dynamic, temporally regulated phase of seed development that determines the composition of storage reserves in mature seeds. Although the metabolic pathways responsible for storage reserve synthesis such as carbohydrates, oils, and proteins are known, little is known about their regulation. Protein phosphorylation is a ubiquitous form of regulation that influences many aspects of dynamic cellular behavior in plant biology. Here a systematic study has been conducted on five sequential stages (2, 3, 4, 5, and 6 weeks after flowering) of seed development in oilseed rape (Brassica napus L. Reston) to survey the presence and dynamics of phosphoproteins. High resolution two-dimensional gel electrophoresis in combination with a phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescence stain revealed approximately 300 phosphoprotein spots. Of these, quantitative expression profiles for 234 high quality spots were established, and hierarchical cluster analyses revealed the occurrence of six principal expression trends during seed filling. The identity of 103 spots was determined using LC-MS/MS. The identified spots represented 70 non-redundant phosphoproteins belonging to 10 major functional categories including energy, metabolism, protein destination, and signal transduction. Furthermore phosphorylation within 16 non-redundant phosphoproteins was verified by mapping the phosphorylation sites by LC-MS/MS. Although one of these sites was postulated previously, the remaining sites have not yet been reported in plants. Phosphoprotein data were assembled into a web database. Together this study provides evidence for the presence of a large number of functionally diverse phosphoproteins, including global regulatory factors like 14-3-3 proteins, within developing B. napus seed.     

Role of a GDSL lipase-like protein as sinapine esterase in Brassicaceae

The seeds of most members of the Brassicaceae accumulate high amounts of sinapine (sinapoylcholine) that is rapidly hydrolyzed during early stages of seed germination. One of three isoforms of sinapine esterase activity (BnSCE3) has been isolated from Brassica napus seedlings and subjected to trypsin digestion and spectrometric sequencing. The peptide sequences were used to isolate BnSCE3 cDNA, which was shown to contain an open reading frame of 1170 bp encoding a protein of 389 amino acids, including a leader peptide of 25 amino acids. Sequence comparison identified the protein as the recently cloned BnLIP2, i.e. a GDSL lipase-like protein, which displays high sequence identity to a large number of corresponding plant proteins, including four related Arabidopsis lipases. The enzymes belong to the SGNH protein family, which use a catalytic triad of Ser-Asp-His, with serine as the nucleophile of the GDSL motif. The corresponding B. napus and Arabidopsis genes were heterologously expressed in Nicotiana benthamiana leaves and proved to confer sinapine esterase activity. In addition to sinapine esterase activity, the native B. napus protein (BnSCE3/BnLIP2) showed broad substrate specificity towards various other choline esters, including phosphatidylcholine. This exceptionally broad substrate specificity, which is common to a large number of other GDSL lipases in plants, hampers their functional analysis. However, the data presented here indicate a role for the GDSL lipase-like BnSCE3/BnLIP2 as a sinapine esterase in members of the Brassicaceae, catalyzing hydrolysis of sinapine during seed germination, leading, via 1- O -sinapoyl-β-glucose, to sinapoyl- l -malate in the seedlings.     

Lipid biosynthesis in cultures of oilseed rape

Summary This review focuses on how microspore-derived (MD) embros and cell suspension cultures of oilseed rape have been used to advance our understanding of the biochemistry and molecular biology of lipid biosynthesis in plants. Both types of cultures are easily maintained and circumvent the difficulties associated with using developing seeds for investigations of lipid biosynthesis. Developing MD embryos exhibit a similar storage lipid accumulation profile and fatty acid composition to developing seed. The use of dihaploids derived from plantlets of MD embryos have accelerated breeding programs and have proven useful in the detection of recessive mutations. MD embryos and MD cell suspension cultures have been particularly useful in investigating the properties of key enzymes involved in triacylglycerol (TG) bioassembly. MD cell suspension cultures, however, offer the advantage of being able to study lipid metabolism in the absence of cellular differentiation. TG accumulation can be induced in MD cell suspension cultures by increasing the sucrose concentration of the growth medium thereby providing a useful system to investigate gene expression and the proteomics of lipid biosynthesis.     

Complex formation of myrosinase isoenzymes in oilseed rape seeds are dependent on the presence of myrosinase-binding proteins

The enzyme myrosinase (EC 3.2.3.1) degrades the secondary compounds glucosinolates upon wounding and serves as a defense to generalist pests in Capparales. Certain myrosinases are present in complexes together with other proteins such as myrosinase-binding proteins (MBP) in extracts of oilseed rape (Brassica napus) seeds. Immunhistochemical analysis of wild-type seeds showed that MBPs were present in most cells but not in the myrosin cells, indicating that the complex formation observed in extracts is initiated upon tissue disruption. To study the role of MBP in complex formation and defense, oilseed rape antisense plants lacking the seed MBPs were produced. Western blotting and immunohistochemical staining confirmed depletion of MBP in the transgenic seeds. The exclusive expression of myrosinase in idioblasts (myrosin cells) of the seed was not affected by the down-regulation of MBP. Using size-exclusion chromatography, we have shown that myrosinases with subunit molecular masses of 62 to 70 kD were present as free dimers from the antisense seed extract, whereas in the wild type, they formed complexes. In accordance with this, MBPs are necessary for myrosinase complex formation of the 62- to 70-kD myrosinases. The product formed from sinalbin hydrolysis by myrosinase was the same whether MBP was present or not. The performance of a common beetle generalist (Tenebrio molitor) fed with seeds, herbivory by flea beetles (Phyllotreta undulata) on cotyledons, or growth rate of the Brassica fungal pathogens Alternaria brassicae or Lepthosphaeria maculans in the presence of seed extracts were not affected by the down-regulation of MBP, leaving the physiological function of this protein family open.     

Heterogeneity in the distribution of genetically modified and conventional oilseed rape within fields and seed lots

The implementation of co-existence in the commercialisation of GM crops requires GM and non-GM products to be segregated in production and supply. However, maintaining segregation in oilseed rape will be made difficult by the highly persistent nature of this species. An understanding of its population dynamics is needed to predict persistence and develop potential strategies for control, while to ensure segregation is being achieved, the production of GM oilseed rape must be accompanied by the monitoring of GM levels in crop or seed populations. Heterogeneity in the spatial distribution of oilseed rape has the potential to affect both control and monitoring and, although a universal phenomenon in arable weeds and harvested seed lots, spatial heterogeneity in oilseed rape populations remains to be demonstrated and quantified. Here we investigate the distribution of crop and volunteer populations in a commercial field before and during the cultivation of the first conventional oilseed rape (winter) crop since the cultivation of a GM glufosinate-tolerant oilseed rape crop (spring) three years previously. GM presence was detected by ELISA for the PAT protein in each of three morphologically distinguishable phenotypes: autumn germinating crop-type plants (3% GM), autumn-germinating 'regrowths' (72% GM) and spring germinating 'small-type' plants (17% GM). Statistical models (Poisson log-normal and binomial logit-normal) were used to describe the spatial distribution of these populations at multiple spatial scales in the field and of GM presence in the harvested seed lot. Heterogeneity was a consistent feature in the distribution of GM and conventional oilseed rape. Large trends across the field (50 x 400 m) and seed lot (4 x 1.5 x 1.5 m) were observed in addition to small-scale heterogeneity, less than 20 m in the field and 20 cm in the seed lot. The heterogeneity was greater for the 'regrowth' and 'small' phenotypes, which were likely to be volunteers and included most of the GM plants detected, than for the largely non-GM 'crop' phenotype. The implications of the volunteer heterogeneity for field management and GM-sampling are discussed.     

Persistence of seeds from crops of conventional and herbicide tolerant oilseed rape (Brassica napus)

A series of rotation experiments at five sites over four years has explored the environmental and agronomic implications of growing herbicide tolerant oilseed rape and sugar beet. This paper reports on the population dynamics of volunteer rape (Brassica napus). The experiments compared four winter oilseed rape (WOSR) cultivars: a conventional cultivar (Apex) and three developmental cultivars either genetically modified (GM) to be tolerant to glyphosate or glufosinate, or conventionally bred to be tolerant to herbicides of the imidazolinone group. Seed losses at harvest averaged 3575 seeds m(-2) but ranged from less than 2000 up to more than 10000 seeds m(-2). There was a rapid decline in seed numbers during the first few months after harvest, resulting in a mean loss of seeds of 60%. In subsequent seasons, the seedbank declined much more slowly at four of the five sites (ca 20% per year) and the models predicted 95% seed loss after approximately 9 years. Seed decline was much faster at the fifth site. There were no clear differences between the four cultivars in either the numbers of seeds shed at harvest or in their subsequent persistence. The importance of the persistence of GM rape seeds, in the context of the coexistence of GM and non-GM crops and the role of good management practices that minimize seed persistence, are discussed.     

The transport of sugars to developing embryos is not via the bulk endosperm in oilseed rape seeds

The fate of sucrose (Suc) supplied via the phloem to developing oilseed rape (Brassica napus) seeds has been investigated by supplying [(14)C]Suc to pedicels of detached, developing siliques. The method gives high, sustained rates of lipid synthesis in developing embryos within the silique comparable with those on the intact plant. At very early developmental stages (3 d after anthesis), the liquid fraction that occupies most of the interior of the seed has a very high hexose-to-Suc ratio and [(14)C]Suc entering the seeds is rapidly converted to hexoses. Between 3 and 12 d after anthesis, the hexose-to-Suc ratio of the liquid fraction of the seed remains high, but the fraction of [(14)C]Suc converted to hexose falls dramatically. Instead, most of the [(14)C]Suc entering the seed is rapidly converted to products in the growing embryo. These data, together with light and nuclear magnetic resonance microscopy, reveal complex compartmentation of sugar metabolism and transport within the seed during development. The bulk of the sugar in the liquid fraction of the seed is probably contained within the central vacuole of the endosperm. This sugar is not in contact with the embryo and is not on the path taken by carbon from the phloem to the embryo. These findings have important implications for the sugar switch model of embryo development and for understanding the relationship between the embryo and the surrounding endosperm.