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.     

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.     

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     

Scanning electron microscopy of desiccation-tolerant and -sensitive microspore-derived embryos of Brassica napus L.

Desiccation tolerance can be induced in microspore-derived embryos of Brassica napus L. by application of abscisic acid (ABA). Scanning electron microscopy was employed to study and compare desiccation-tolerant and -sensitive microspore-derived embryos. The external surface of those desiccated embryos in which desiccation tolerance had been induced was uniformly shriveled due to severe dehydration, and their internal tissue system was well preserved. In contrast, in desiccation-sensitive embryos, dehydration caused tearing of the epidermis and collapse of the internal tissue system. After the desiccated embryos had been rehydrated, the size and external morphology of the desiccation-tolerant ones recovered to the pre-desiccation state within 1 day, whereas the sensitive ones did not recover or remained shriveled. The effect of ABA on the induction of desiccation tolerance is discussed. Received: 25 June 1998 / Revision received: 03 September 1998 / Accepted: 24 September 1998     

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.     

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.     

Effects of thyroid state on AMP-activated protein kinase and acetyl-CoA carboxylase expression in muscle.

AMP-activated protein kinase (AMPK) consists of three subunits: alpha, beta, and gamma. Two isoforms exist for the alpha-subunit (alpha(1) and alpha(2)), two for the beta-subunit (beta(1) and beta(2)), and three for the gamma-subunit (gamma(1), gamma(2), and gamma(3)). Although the specific roles of the beta- and gamma-subunits are not well understood, the alpha-subunit isoforms contain the catalytic site and also the phosphorylation/activation site for the upstream kinase. This study was designed to determine the role of thyroid hormones in controlling expression levels of these AMPK subunits and of one downstream target, acetyl-CoA carboxylase (ACC), in muscle. AMPK subunit and ACC levels were determined by Western blots in control rats, in rats given 0.01% propylthiouracil (PTU) in drinking water for 3 wk, and in rats given 3 mg of thyroxine and 1 mg of triiodothyronine per kilogram chow for 1 or 3 wk. In gastrocnemius muscle, all isoforms of AMPK subunits were significantly increased in rats given thyroid hormones for 3 wk vs. those treated with PTU. Similar patterns were seen in individual muscle types. Expression of muscle ACC was also significantly increased in response to 3 wk of treatment with excess thyroid hormones. Muscle content of malonyl-CoA was elevated in PTU-treated rats and depressed in thyroid hormone-treated rats. These data provide evidence that skeletal muscle AMPK subunit and ACC expression is partially under the control of thyroid hormones.     

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.     

甘蓝型油菜芥酸和二十碳烯酸含量的基因效应

以甘蓝型油菜的4种纯合芥酸基因型之间所有可能的6个杂交组合的P_1、P_2、F_1、P_2、B_1和B_2世代为材料,用生统遗传学方法研究了芥酸和二十碳烯酸的基因作用形式及效应。发现无论亲本是单基因差异还是二基因差异,F_1和F_2代的芥酸含量都接近中亲值,F_1略大于中亲值和F_(20)世代均值分析表明,芥酸含量的遗传符合加性显性模型,加性效应占绝对优势,显性效应不显著。用数量遗传学方法估计的芥酸基因数与已知的结果相近。     

Immunocytochemical localization of myrosinase in Brassica napus L.

The cytological and intracellular localization of myrosinase (EC 3.2.3.1) has been studied by immunochemical techniques using paraffin-embedded sections of radicles and cotyledons from seeds of Brassica napus L. cv. Niklas. For immunolabelling, sections were sequentially incubated with a monoclonal anti-myrosinase antibody and with peroxidase-and fluorescein-isothiocyanate-conjugated secondary antibodies. Enzyme and fluorescence label was present in typical myrosin cells both in radicles and in cotyledons. With higher magnification, fluorescence label revealed that the intracellular localization of myrosinase was associated with the tonoplast-like membrane surrounding the myrosin grains in the myrosin cells. The results also indicate that a large proportion of the positive myrosin cells are located in the second-outermost cell layer of the peripheral cortex region of the radicles.Abbreviations FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - PBS-T PBS with 0.5% (v/v) Tween-20 (polyoxyethylene sorbitane monolaurate) This work was supported by The Norwegian Research Council for Science and the Humanities. We wish to thank Professor Med. O.A. Haugen, Department of Pathology, University of Trondheim, Norway, for the skilful assistance provided regarding fixation and sectioning.     

Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as amodel for examining a range of processes, including hormonal regulation ofembryo development. We examined the hormonal physiology of MDEs with theprimaryobjective of testing the validity of using the MDE system as a model forhormonally-regulated development in zygotic embryos, through late stages. To dothis we identified and quantified endogenous levels of abscisic acid (ABA),indole-3-acetic acid (IAA) and a number of gibberellins (GAs), includingGA₁₉, GA₂₀, GA₁ and GA₈ in bothMDEsand zygotic embryos. The presence of GA₁₉, together with itsC₁₉ metabolites indicates that the early-13 hydroxylation pathway isoperative in both embryo systems. Gibberellins A₄ and GA₉were also identified, thereby confirming the presence of the earlynon-hydroxylation pathway in B. napus MDEs and zygoticembryos. In general, the pattern of change of hormone (ABA, IAA, GA₁and GA₂₀) content per embryo through embryogenesis was similar forMDEs and zygotic embryos. Indole-3-acetic acid and GA₁ increased toamaximum at day 30 after culture (DAC) before decreasing. Abscisicacid levels increased to a maximum at day 35, and declined in zygoticembryos but not in MDEs. GA₂₀ increased to the final harvest atmaturity, or day 40. The absolute content (g/embryo) of each hormone, howeverwas appreciably lower (5- to 15- fold) in the MDEs. This was not the result ofdilution into surrounding medium for ABA or IAA; GA₁, however, didaccumulate in the medium. Although there were absolute quantitative differencesin the levels of IAA and ABA found in the two embryo systems, the similaritiesin the pattern of hormone changes suggests that the MDE system can serve as auseful model for examining the physiological roles of hormones duringembryogenesis.     

Melliferous potential of Brassica napus L. subsp. napus (Cruciferae)

Morphophysiological characteristics of oilseed rape flowers, such as features of the nectaries, nectar production, and observations on honey bee visits and honey and seed yield were studied with the aim to evaluate the melliferous potential of this crop as well as its attractiveness to pollinators. Calculation of the theoretical maximal honey yield revealed that the actual amount of extracted honey was much lower than the potential yield, indicating that this bee pasture is underutilized. We found that honey bee pollination increased oilseed rape yield, i.e., seed production, by 12 % compared with the treatment in which pollinators were excluded.     

甘蓝型油菜花粉离体培养

通过对甘蓝型油菜花粉发育阶段和活力的检测确定花粉发育的时期,分离出单核晚期花粉进行离体培养.结果表明,(1)筛选出适合油菜小孢子花粉离体培养的液体培养基为T_1+怀特维生素(White's vitamins)+2%椰子汁+0.5 mol/L麦芽糖,在此培养基上花粉的成熟率可达25.1%,萌发率达6.3%.(2)筛选出适合成熟花粉离体萌发液体培养基为0.6 mol/L麦芽糖+1.6 mmol/L硼酸+2.9 mmol/L硝酸钙+29.6 μmol/L VB_1,在此培养基上,自然成熟花粉的萌发率可达75.2%.将离体培养成熟的花粉培养在萌发培养基,萌发的花粉占成熟花粉的66.3%.