Proteomic comparison of plastids from developing embryos and leaves of Brassica napus

Plastids are highly specialized organelles, responsible for photosynthesis and biosynthesis of various phytochemicals. To better understand plastid diversity and metabolism, a quantitative proteomic study of two plastid forms from Brassica napus (oilseed rape) was performed. Plastids were isolated from leaves (chloroplasts) of two-week-old plants and developing embryos (embryoplasts) three-weeks after flowering, using an approach avoiding protein storage vacuole contamination. Proteins from five different plastid preparations were prefractionated by SDS-PAGE and sectioned into multiple bands, and in-gel proteins were subjected to trypsin digestion. Tryptic peptides from each band were eluted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectra were searched against a comprehensive plant database. Proteins were quantified based on MS/MS spectral counting of unique, nonhomologous peptides. Functional classification and quantitative comparison of over 2000 redundant proteins (compiled to 675 nonredundant proteins) determined that light reaction proteins are more prominent in chloroplasts, while many Calvin cycle enzymes are more prominent in embryoplasts. Embryoplasts also contain a diversity of other metabolic enzymes undetected in chloroplasts. Many enzymes involved in de novo fatty acid and amino acid biosynthesis were detected in embryoplasts but not chloroplasts. Additionally, protein synthesis-related proteins were prominent in embryoplasts. Collectively, these results indicate that these two plastid types are distinct.     

Purification and proteomic characterization of plastids from Brassica napus developing embryos

Plastids are functionally and structurally diverse organelles responsible for numerous biosynthetic reactions within the plant cell. Plastids from embryos have a range of properties depending upon the plant source but compared to other plastid types are poorly understood and therefore, we term them embryoplasts. Isolating intact plastids from developing embryos is challenging due to large starch granules within the stroma and the prevalence of nonplastid, storage organelles (oil bodies and protein storage vacuoles) which compromise plastid integrity and purity, respectively. To characterize rapeseed embryoplasts it was necessary to develop an improved isolation procedure. A new method is presented for the isolation of intact plastids from developing embryos of Brassica napus seeds. Intactness and purity of embryoplast preparations was determined using phase-contrast and transmission electron microscopy, immunoblotting, and multidimensional protein identification technology (MudPIT) MS/MS. Eighty nonredundant proteins were identified by MudPIT analysis of embryoplast preparations. Approximately 53% of these proteins were components of photosystem, light harvesting, cytochrome b/f, and ATP synthase complexes, suggesting ATP and NADPH production are important functions for this plastid type.     

Export of acyl chains from plastids isolated from embryos of Brassica napus (L.)

We report the first measurements of the kinetics of transmembrane transport of acyl chains in plants. This was achieved by separating the period of in vitro synthesis of fatty acids from their export and by making use of acyl-CoA-binding protein (ACBP), which specifically binds long-chain acyl-CoAs. In the absence of added CoA but in the presence of ACBP, newly synthesised acyl chains accumulated as free fatty acids (FFAs) in plastids isolated from embryos of oilseed rape (Brassica napus L.). When CoA was added to plastids that had accumulated FFAs, the acyl chains were converted to acyl-CoAs that, in the presence of ACBP, were exported to the incubation medium. The rate of export was dependent on the CoA concentration and, at a saturating CoA concentration, was similar to the rate at which the fatty acids had been synthesised prior to CoA addition.     

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.     

Fatty acid breakdown in developing embryos of Brassica napus L

Developing Brassica napus embryos are primarily concerned with the accumulation of storage products, namely oil, starch and protein. The presence of fatty acid catabolic pathways in the background of this biosynthetic activity was investigated. Enzymes involved in the process of lipid mobilization, such as malate synthase and isocitrate lyase, are detectable towards the late stages of embryo development. [(14)C]Acetate feeding experiments also reveal that fatty acid catabolism becomes increasingly functional as the embryo matures.     

Photosynthesis by developing embryos of oilseed rape (Brassica napus L.)

The aim of this study was to assess the photosynthetic potentialof developing seeds of oilseed rape (Brassica napus L.) andto compare photosynthetic properties of embryo plastids withthose of leaf chloroplasts from the same species. Measurementsof CO₂-dependent O₂ evolution show that developing seeds ofB. napus are photosynthetically active in vitro. Essentially,all of the photosynthetic activity of the developing seed isaccounted for by the embryo. The rate of photosynthesis by developingembryos increased until the onset of desiccation, after whichit declined, so that by maturity embryos were no longer photosyntheticallyactive. Photosynthetic activity was positively correlated withchlorophyll content throughout development. Comparison of thephotosynthetic characteristics of leaf and embryo chloroplastsrevealed that rates of uncoupled electron transport were 2.5-foldgreater in those from the embryo. Light-saturated rates of CO₂-dependentO₂ evolution, per unit chlorophyll, and CO₂ saturation pointswere similar for chloroplasts from both tissues. However, light-saturationpoints and chlorophyll a/b ratios were lower for embryo thanfor leaf choroplasts. Embryos and embryo chloroplasts also containedconsiderably less ribulose 1,5-bisphosphate carboxylase/oxygenaseprotein per unit total protein, than leaves. Although excisedembryos were capable of high rates of CO₂-dependent O₂ evolution(90–100 mol mg^–1 chlorophyll h^–1) under asaturating photosynthetic photon flux density (PPFD), low transmittanceof light through the silique wall (30%), together with the highPPFD required to achieve light compensation points in developingseeds (500 mol m^–2 s^–1), suggests that photosynthesisin vivo is unlikely to make a net contribution to carbon economyunder normal environmental conditions. Key words: Embryo, development, photosynthesis, chloroplast, Brassica napus L.     

Mitochondrial metabolism in developing embryos of Brassica napus

The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-(13)C2]glucose + [U-(13)C6]glucose, [U-(13)C3]alanine, [U-(13)C5]glutamine, [(15)N]alanine, (amino)-[(15)N]glutamine, or (amide)-[(15)N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.     

Non-zygotic embryos of Brassica napus L. contain embryo-specific storage proteins

The storage-protein content of non-zygotic and zygotic embryos of B. napus was compared, using antibodies to guantitate 12S storage protein in extracts by rocket immunoelectrophoresis. Non-zygotic embryos were induced from microspores in anther culture and on the hypocotyls of zygotic embryos in culture. All embryo-like structures were found to contain 12S storage protein, whereas preculture anthers, anthers from which embryos had been removed, and regenerated shoots did not have detectable 12S storage protein. In zygotic embryos, 12S storage protein was first detected at the cotyledon stage, but microsporic embryos contained storage protein at the globular and heart stages. Storage protein levels in microsporic and hypocotyl embryos were low relative to those in zygotic embryos. The largest microsporic embryo had a storage protein concentration of 13 g mg^-1 fresh weight, almost 10 times lower than a mature zygotic embryo. Thus, although storage proteins are present in both zygotic and non-zygotic embryos, the timing and extent of accumulation differ.     

Metabolism of glucose 6-phosphate by plastids from developing pea embryos

The aim of this work was to investigate the metabolism of glucose 6-phosphate by plastids isolated from developing pea (Pisum sativum L.) embryos. Plastids metabolise exogenously supplied glucose 6-phosphate via the pathway of starch synthesis and the oxidative pentose-phosphate pathway. The flux through the latter pathway is greatly stimulated by the provision of glutamine and 2-oxoglutarate — the substrates of glutamate synthase — indicating that it is regulated by the demand for reductant within the plastid. Flux in the presence of glutamine and 2-oxoglutarate is about 20% of the maximum flux through the pathway of starch synthesis. There is no competition for glucose 6-phosphate between the two pathways at concentrations which are saturating for both. Isolated plastids do not convert glucose 6-phosphate to amino acids or fatty acids at significant rates under the conditins of our experiments.Abbreviations ADPG adenosine 5-diphosphoglucose We thank Mike Emes (Department of Cell and Structural Biology, University of Manchester, UK) for valuable advice during the course of this work, and for making unpublished information available to us. We also thank Mark Stitt (Botanisches Institut der Universität, Heidelberg, FRG) and our colleagues, particularly Kay Denyer and Lionel Hill, for their helpful and constructive criticism. This work was supported by funding from the European Community, under contract C11* 0417-UK (SMA).     

Fatty acid synthesis and the oxidative pentose phosphate pathway in developing embryos of oilseed rape (Brassica napus L.)

The potential role of the plastidial oxidative pentose phosphate pathway (OPPP) in providing the NADPH for fatty acid synthesis in plastids from developing embryos of Brassica napus (L.) has been investigated. Measurements of distributions of enzyme activities in fractions obtained from homogenates of isolated embryos have revealed that the glucose 6-phosphate and 6-phosphogluconate dehydrogenases are present in both cytosol and plastid, as is ribose 5-phosphate isomerase. However, transketolase and transaldolase are most probably confined to the plastid, while ribulose 5-phosphate epimerase is essentially cytosolic, although a very small proportion of plastid-localized activity cannot be ruled out. The activity of the OPPP in intact plastids was measured by the release of (14)CO(2) from [1-(14)C]glucose 6-phosphate. Activity was detectable in the absence of electron sinks created by the addition of metabolites to the incubation media and was stimulated 1.3-, 3.2-, and 7.9-fold by the respective additions of glutamine plus 2-oxoglutarate, cofactors and substrates for fatty acid synthesis, or methyl viologen. An increase in OPPP activity in response to additions that are absolutely required for fatty acid synthesis in these isolated plastids provides direct evidence that these two processes are connected, most probably by NADP/NADPH metabolism. The OPPP activity with methyl viologen was more than twice that during fatty acid synthesis, suggesting that the latter is not limited by OPPP capacity. Light energy may also contribute to reductant provision and, consistent with the possibility of maintenance of a balance of NADPH from light and the OPPP, glucose 6-phosphate dehydrogenase activity in the isolated plastids was decreased by light or by DTT.     

A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos

Developing oilseeds synthesize large quantities of triacylglycerol from sucrose and hexose. To understand the fluxes involved in this conversion, a quantitative metabolic flux model was developed and tested for the reaction network of glycolysis and the oxidative pentose phosphate pathway (OPPP). Developing Brassica napus embryos were cultured with [U-13C6]glucose, [1-13C]glucose, [6-13C]glucose, [U-13C12]sucrose, and/or [1,2-13C2]glucose and the labeling patterns in amino acids, lipids, sucrose, and starch were measured by gas chromatography/mass spectrometry and NMR. Data were used to verify a reaction network of central carbon metabolism distributed between the cytosol and plastid. Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.     

Pyruvate-kinase isoenzymes from zygotic and microspore-derived embryos of Brassica napus

Polyclonal antibodies against castor-oil seed cytosolic and leucoplastic pyruvate kinases (PK_c and PK_p, respectively; EC 2.7.1.40) were utilized to examine the subunit compositions and developmental profiles of canola (Brassica napus L. cv. Topas) PK_c and PK_p over 6 d of seed germination and 35 d of culture of microspore-derived embryos. The PK_c from germinating seeds appears to be composed of a single type of 56-kDa subunit, whereas the enzyme from cultured embryos contains equal proportions of immunologically related 57- and 56-kDa subunits. The PK_p was immunologically undetectable in germinating seeds, while the enzyme from cultured embryos consisted of immunologically related 64- and 58-kDa subunits in a ratio of about 12, respectively. The large increase in PK activity that occurs between the second and fourth days of seed gemination is based upon de-novo synthesis of PK_c. Between 7 and 14 d of culture of microspore-derived embryos, the levels of PK_p and PK maximal activity increased approx. 3- and 2.5-fold, respectively. These increases were coincident with an approximately fourfold rise in the in-vivo pyruvate: phosphoenolpyruvate concentration ratio. Conversely, PK_c was not only far less abundant relative to PK_p, but its level remained constant over 35 d of microspore-embryo culture. Developing non-zygotic (microspore-derived) embryos strongly resembled ripening zygotic (seed) embryos in terms of PK specific activity as well as relative amounts and subunit compositions of PK_c and PK_p. The results indicate that the synthesis of PK isoenzymes in B. napus seeds is highly regulated and that this regulation follows a preset developmental program.Abbreviations IgG immunoglobulin G - IU international unit - PEP phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - PK(s) pyruvate kinase(s) - PK_c cytosolic pyruvate kinase - PK_p plastidic pyruvate kinase - PYR pyruvate Plant Research Centre contribution No. 1374We wish to thank Ms. Kathryn Hovey and Ms. Suzanne Belliveau (Agriculture Canada) for their expert assistance in the culturing and harvesting of microspore-derived embryos of canola. This work was supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada.     

Towards artificial seeds from microspore derived embryos of Brassica napus

Plant Cell, Tissue and Organ Culture (PCTOC) - Microspore derived embryos are haploid and their immediate diploidization generates doubled haploid homozygous plants, whereas a normal breeding...     

In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos

Probes derived from cDNA clones of napin and cruciferin, the major storage proteins of Brassica napus, and in situ hybridization techniques were used to examine changes in the spatial and temporal distribution of storage protein messages during the course of embryogeny, with a special emphasis on the developing apical meristems. Napin mRNAs begin to accumulate in the cortex of the axis during late heart stage, in the outer faces of the cotyledons during torpedo stage and in the inner faces of the cotyledons during cotyledon stage. Cruciferin mRNAs accumulate in a similar pattern but approximately 5 days later. Cells in the apical regions where root and shoot meristems develop do not accumulate storage protein messages during early stages of embryogeny. In the upper axis, the boundary between these apical cells and immediately adjacent cells that accumulate napin and cruciferin mRNAs is particularly distinct. Our analysis indicates that this boundary is not related to differences in tissue or cell type, but appears instead to be coincident with the site of a particular set of early cell divisions. A major change in the mRNA accumulation patterns occurs halfway through embryogeny, as the embryos enter maturation stage and start drying down. Final maturation of the shoot apical meristem is associated with the development of leaf primordia and the accumulation of napin mRNAs in the meristem, associated leaf primordia and vascular tissue. Cruciferin mRNAs accumulate only in certain zones of the shoot apical meristem and on the flanks of leaf primordia. Neither type of mRNA accumulates in the root apical meristem at any stage.     

Compositional changes in developing rape seed (Brassica napus L.)

Summary The growth and composition of siliquas and seeds of oilseed rape was followed over 12 weeks from shortly after anthesis to maturity. Each plant produced 220 siliquas, this number being constant throughout development. Seed numbers per siliqua fell from 19 to 9 by week 5 and declined to 7 at maturity. Hull¹ and seed growth followed a sigmoid pattern, but were not in phase. Seed development could be divided into 3 phases: In Phase 1, seed weight was low and starch and ethanol soluble compounds accounted for 80% DM. Phase 2, seed growth increased and storage oil and proteins were deposited accounting for 40% and 20% DM respectively at the end of this stage. Starch, glucose and fructose were utilized in this process. Phase 3 was largely concerned with the deposition of oil and protein in fixed proportions. Seed weight more than doubled while DM composition remained constant. Sugars were transferred from the hull to the seed to support this growth.The proportion of hull lipids remained constant throughout development until shortly before maturity when MGDG and DGDG fell due to chloroplast breakdown as indicated by chlorophyll disappearance. The FA composition of the hull lipids resembled that of photosynthetic tissue. In the seeds, the neutral lipids increased from 20% of the total lipids in Phase 1 to 93% at maturity. The proportion of structural lipids declined as the storage lipids increased. In Phase 1 the FA composition of the lipid resembled that of photosynthetic tissue (high in C_16:0; C_18:2; C_18:3). In Phase 2, FA typical of storage triglycerides (C_20:1; C_22:1, appeared, C_18:1 transitorily increased, but C_18:2 and C_18:3 fell dramatically. In Phase 3, the content of C_22:1 continued to rise, but the proportions of the other FA remained constant.Abbreviations DM Dry matter - MGDG Monogalactosyldiglyceride - DGDG Digalactosyldiglyceride - NL Neutral lipid - PC Phosphatidyl choline - PE Phosphatidyl ethanolamine - C_16:0 Palmitic acid - C_18:1 Oleic acid - C_18:2 Linoleic acid - C_18:3 Linolenic acid - C_20:1 Eicosenoic acid - C_22:1 Erucic acid - FA Fatty acid     

Desiccation of microspore derived embryos of oilseed rape (Brassica napus L.)

Summary Microspore-derived embryos from Brassica napus L. were dried to less than 15% moisture and stored dry for a minimum of 7 days. Successful plant regeneration was observed when embryos at the cotyledonary stage of development were treated with 50 uM ABA for 7 days prior to desiccation. Solid agar or liquid medium gave similar results. The rate of drying of embryos after ABA pretreatment had only minor effects on embryo survival, but for untreated embryos, slow drying gave a small degree of survival. These results are very comparable to those with alfalfa somatic embryos, suggesting that the ABA treatment of cotyledonary stage embryos may be broadly used as a pretreatment for inducing the expression of desiccation tolerance in plant embryos.     

The role of plastidial transporters in developing embryos of oilseed rape (Brassica napus L.) for fatty acid synthesis

The phosphoenolpyruvate transporter (PPT) is one of several important transporters for channelling carbon intermediates utilized for fatty acid synthesis and other plastidial pathways from the cytosol into the plastid. In this paper we show results on how the activity of the PPT changes between two important, physiologically different developmental stages of oilseed rape embryos.     

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.     

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.