Formation of oleosomes (storage lipid bodies) during embryogenesis and their breakdown during seedling development in cotyledons of Sinapis alba L.

Electron microscopic and biochemical investigations of developing embryonic mustard cotyledons provided no evidence for the widely accepted hypothesis that oleosomes of fat-storing tissues originate from the endoplasmic reticulum and are surrounded by a unit- or half-unit membrane. In contrast, it was found that the first lipid droplets appear (about 12–14 d after pollination) in the ground cytoplasm near the surface of plastids. Subsequently these nascent lipid droplets, which lack any detectable boundary structure at this stage, become encircled by a cisterna of rough endoplasmic reticulum. At the same time an osmiophilic coat of about 3 nm thickness becomes detectable at the lipid/water interface. In the cotyledon cells of germinating seedlings a centrifugally moving front of fat degradation moves from the central vacuoles(s) towards the cell periphery, leaving behind collapsed coats of oleosomes which are depleted of their lipid contents (saccules). Although saccules appear tripartite in cross section, they are structurally different from endoplasmic reticulum membranes. The oleosome coats can be isolated from oleosome preparations by extracting lipids with organic solvents. The coat material is insoluble in detergents like Triton X-100 or deoxycholate and shows a tripartite, lamellar structure (similar to collapsed saccules) under the electron microscope. Upon dissolution with dodecylsulfate, polyacrylamide gel electrophoresis revealed a polypeptide composition (9 major bands) which is qualitatively different from that of the endoplasmic reticulum membrane. Also the buoyant densities of defatted oleosome coats and defatted endoplasmic reticulum membranes are very different. It is concluded that oleosome lipids accumulate in the ground cytoplasm and are bounded by a lamellar structure originating de novo from proteinaceous elements synthesized by specific regions of the endoplasmic reticulum.Abbreviation ER endoplasmic reticulum     

Carotenoid and chlorophyll biosynthesis in isolated plastids from mustard seedling cotyledons (Sinapis alba L.) during etioplast-chloroplast conversion

Etioplasts and etiochloroplasts, isolated from seedlings of white mustard (Sinapis alba L.) grown in continuous far-red light, and chloroplasts isolated from cotyledons and primary leaves of white-light-grown seedlings exhibit high prenyl-lipid-forming activities. Only the etioplasts and etiochloroplasts, and to a much lesser extent chloroplasts from cotyledons are capable of forming carotenes from isopentenyl diphosphate as substrate, whereas in chloroplasts from primary leaves no such activities could be detected. By subfractionation experiments, it could be demonstrated that the phytoene-synthase complex in etioplasts and etiochloroplasts is present in a soluble form in the stroma, whereas the subsequent enzymes, i.e. the dehydrogenase, cis-trans isomerase and cyclase are bound to both membrane fractions, the prolamellar bodies/prothylakoids and the envelopes. In good agreement with previous results using isolated chromoplasts and chloroplasts, it is concluded that the phytoene-synthase complex may change its topology from a peripheral membrane protein in non-green plastids to a tightly membrane-associated protein in chloroplasts. This change is apparently paralleled by altered functional properties which render the complex undetectable in isolated chloroplasts. Further experiments concerning the reduction of chlorophyll a containing a geranylgeranyl side chain to chlorophyll a indicate that the light-induced etioplast-chloroplast conversion is accompanied by a certain reorganization of the polyprenoid-forming enzymatic equipment.Abbreviations Chl a chlorophyll a - Chl_GG chlorophyll a containing a geranylgeranyl side chain - HPLC high-performance liquid chromatography - Tris 2-ammo-2-(hydroxymethyl)-1,3-propanediol     

Changes in adenine-nucleotide content in the apical bud of Sinapis alba L. during floral transition

The total adenylate pool of the apical buds of vegetative plants of Sinapis alba L. continuously grown in short days fluctuates over a 24-h cycle with the minimum occurring at the end of the dark period. In the buds of plants induced to flower by a single long-day treatment, total adenylate pool increases above the control level 16 h after the start of the long day, resulting mainly from a rise in ATP and ADP contents. This occurs 6 h after the increase in the soluble carbohydrate content previously shown to occur in the apical buds of plants induced to flower (Bodson 1977, Planta 135, 19–23). A transient rise of the energy charge occurs 22 h after the start of the inductive long day.Abbreviations LD long day - SD short day     

Regulation of the appearance of glutamine synthetase in mustard (Sinapis alba L.) cotyledons by light,nitrate and ammonium

During transformation of mustard seedlings cotyledons from storage organs to photosynthetically competent leaves, a process which occurs during the first 4 d after sowing, total glutamine-synthetase (GS, EC 6.3.1.2) activity increases from zero to the high level usually observed in green leaves. In the present study we have used ion-exchange chromatography to separate possible isoforms of GS during the development of the cotyledons. The approach failed since we could only detect a single form of GS, presumably plastidic GS, under all circumstances tested. The technique of selective photooxidative destruction of plastids in situ was applied to solve the problem of GS localization. It was inferred from the data that the GS as detected by ion-exchange chromatography is plastidic GS.The regulatory role, if any, of light, nitrate and ammonium in the process of the appearance of GS in the developing cotyledons was investigated. The results show that nitrate and ammonium play only minor roles. Light, operating via phytochrome, is the major regulatory factor.Abbreviations c continuous - D darkness - FPLC fast protein liquid chromatography - GS glutamine synthetase (L-glutamate:ammonia ligase, ADP forming, EC 6.3.1.2) - FR far-red light (3.5 W·m^-2) - NF Norflurazon - R red light (6.8 W·m^-2, _R=0.8)) - RG9-light long-wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Control by phytochrome of urate oxidase and allantoinase activities during peroxisome development in the cotyledons of mustard (Sinapis alba L.) seedlings

The peroxisomal enzyme, urate oxidase (EC 1.7.3.3), and the next enzyme of the urate pathway, allantoinase (EC 3.5.2.5), demonstrate a lightmediated rise of activity in the cotyledons of mustard (Sinapis alba L.). The capacity of the peroxisomes for urate breakdown, marked by the time course of urate oxidase, develops distinctly later than the two other peroxisome functions (fatty acid breakdown, glyoxysomal function; glycolate breakdown, leaf peroxisomal function). The light effect on urate oxidase and allantoinase is mediated through the phytochrome system in all three seedling organs (cotyledons, hypocotyl, radicle), as revealed by induction/reversion experiments with red/far-red light pulses and continuous irradiation with far-red light (high irradiance reaction of phytochrome). Both enzyme activities can be induced by phytochrome in the seedling cotyledons only during a sensitive period of about 48 h prior to the actual light-mediated rise of activity, making it necessary to assume the existence of a long-lived intermediate (transmitter) in the signal response chain connecting enzyme formation to the phytochrome system. Detailed kinetic investigation, designed to test whether urate oxidase and allantoinase are controlled by phytochrome via the same signal response chain (coordinate induction), revealed large differences between the two enzymes: (i) a different onset of the loss of reversibility of a red light induction by a far-red light pulse (=onset of transmitter formation=coupling point; 48 h/24 h after sowing for urate oxidase/allantoinase); (ii) a different onset of the response (=onset of competence for transmitter= starting point; 72 h/48 h); (iii) full loss of reversibility (=completion of transmitter formation) is reached at different times (independence point, 90 h/52 h). These differences show that phytochrome controls urate oxidase and allantoinase via separate signal response chains. While urate oxidase can be localized in the peroxisomal fraction isolated from crude organelle extracts of the cotyledons by density gradient centrifugation, most of the allantoinase activity found in the peroxisomal fraction did not appear to be an integral part of the peroxisome but originated presumably from adhering membrane fragments.Abbreviations AL allantoinase, EC 3.5.2.5 - CAT catalase, EC 1.11.1.6 - GO glycolate oxidase, EC 1.1.3.1 - ICL isocitrate lyase, EC 4.1.3.1 - UO urate oxidase, EC 1.7.3.3. P_r - P_fr red and far-red absorbing forms of phytochrome On the occasion of his 80th birthday we dedicate this paper to Prof. Dr. phil., Dr. mult. h.c. Kurt Mothes, pioneer in research on metabolism of urates     

Formation of wheat protein bodies: Involvement of the Golgi apparatus in gliadin transport

Developing wheat (Triticum aestivum L.) endosperm was examined using ultrathin sections prepared from tissues harvested at 5, 9, 16 and 25 d after flowering. Protein bodies were evident by 9 d and displayed a variety of membranous structures and inclusions. The Golgi apparatus was a prominent organelle at all stages, and by 9 d was associated with small electron-dense inclusions. By immunocytochemical techniques, gliadin (wheat prolamine) was localized within these vesicles and in homogeneous regions of protein bodies, but not in the lumen of the rough endoplasmic reticulum. The protein bodies appear to enlarge by fusion of smaller protein bodies resulting in larger, irregular-shaped organelles. The affinity of the Golgi-derived vesicles for gliadin-specific probes during the period of maximal storage-protein synthesis and deposition indicates that this organelle includes the bulk, if not all, of the gliadin produced. The involvement of the Golgi apparatus in the packaging of gliadins into protein bodies indicates a pathway which differs from the mode of prolamine deposition in other cereals such as maize, rice and sorghum, and resembles the mechanism employed for the storage of rice glutelin and legume globulins.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - DAF days after flowering     

Biosynthesis of wax esters in tissues of Sinapis alba L. seeds

The biosynthesis of wax esters has been investigated in maturing seeds of Sinapis alba. Exogenous long-chain alcohols are incorporated exclusively into alkyl moieties of wax esters. Oxidation of the long-chain alcohols is not detected. Exogenous fatty acids are incorporated into acyl moieties of wax esters to a low extent. A reduction of fatty acids to alcohols is not observed. Synthesis of wax esters is localized exclusively in the testa; both outer and inner integument are equally active in wax ester biosynthesis. The biosynthesis of wax esters is specific with regard to both chain length and degree of unsaturation of long-chain alcohols. Exogenous and endogenous sterols are not esterified.     

Glycerolipid synthesis by homogenate and oil bodies from developing mustard (Sinapis alba L.) seed

[1-¹⁴C]Oleic acid and [14-¹⁴C]erucic acid were converted to their acyl-CoA derivatives and incorporated into acyl lipids by a homogenate from developing mustard (Sinapis alba L.) seed and oil bodies, as well as supernatant isolated by centrifugation at 20000 g. In both homogenate and oil bodies, the oleoyl moieties from exogenous [1-¹⁴C]oleoyl-CoA were most extensively incorporated into phosphatidic acids, but very little into phosphatidylcholines. The pattern of labelling of acyl lipids by oleoyl versus erucoyl moieties from either of the corresponding fatty acids, added individually or as a mixed substrate, indicates that oleoyl-CoA directly acylates sn-glycerol-3-phosphate to yield lysophosphatidic acids and phosphatidic acids that are subsequently converted to mono- and diacylglycerols. In contrast, erucoyl-CoA predominantly acylates preformed mono-and diacylglycerols containing oleoyl moieties to yield triacylglycerols containing erucoyl moieties.     

Amino-acid biosynthesis in the cotyledons of Sinapis alba L. in darkness and far-red light studied by deuterium labelling and mass spectrometry

The incorporation of deuterium from deuterium oxide into the free amino acids of the cotyledons of Sinapis alba L. was studied by gas chromatography-mass spectrometry and was similar, both qualitatively and quantitatively, after incubation of the seedlings in darkness or far-red light. The results support studies which show that phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is synthesised de novo, rather than activated, in response to far-red light.Abbreviations GC-MS Gas chromatography-mass spectrometry - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - HFB n-propyl heptafluorobutyryl n-propyl     

High irradiance response promotion of a subsequent light induction response in Sinapis alba L.

Relative quantum responsivity curves for inhibition of hypocotyl elongation in Sinapis alba L. seedlings previously grown in white light confirm that a marked end of day inhibition response can be induced by a monochromatic light treatment (30 min) at the end of the light period. In dark grown seedlings, however, no growth inhibition can be induced by a 30 min monochromatic light treatment. A prerequisite for an induction response appears to be a pretreatment with continuous light. Far red light is most effective with blue and red light showing a lesser effectiveness. The light pretreatment also shows a marked fluence rate dependency with respect to its ability to allow an induction response to manifest itself. The pretreatment required shows all the characteristics of a classical HIR response. The appearance of the effect in plants treated with the herbicide SAN 9789 seems to exclude chlorophyll as being the photoreceptor.Abbreviations SAN 9789 4-chloro-5-(methylamino)-2-(, , -trifluoro-m-tolyl)-3(2H)-pyridazinone - RG9 light long wavelength far red light (Schott RG9 colour glass) - FR far red light - WL white light - BL blue light - RL red light - D darkness - P_tot total phytochrome - P_fr far red absorbing form of phytochrome - HSR high irradiance response     

Absence of fluence rate dependency of phytochrome modulation of stem extension in light-grown Sinapis alba L.

In background white light, supplementary far-red (_max 700 nm) is an order of magnitude less effective than supplementary far-red (_max 739 nm) in the stimulation of stem extension in Sinapis alba. The relationship between phytochrome photoequilibrium and extension rate increase for the two supplementary far-red treatments is, however, very similar. This evidence indicates that phytochrome cycling is not involved in the phytochrome control of stem extension in light-grown Sinapis alba and that the response to supplementary far-red light is not fluence rate (irradiance) dependent.Abbreviations Pfr far-red absorbing form of phytochrome - the phytochrome photoequilibrium (Pfr/Ptotal)     

Regulatory factors involved in gene expression (subunits of ribulose-1,5-bisphosphate carboxylase) in mustard (Sinapis alba L.) cotyledons

Phytochrome-controlled appearance of ribulose-1,5-bisphosphate carboxylase (RuBP-Case) and its subunits (large subunit LSU, small subunit SSU) was studied in the cotyledons of the mustard (Sinapis alba L.) seedling. The main results were as follows: (i) Control of RuBPCase appearance by phytochrome is a modulation of a process which is turned on by an endogenous factor between 30 and 33 h after sowing (25° C). Only 12 h later the process begins to respond to phytochrome. (ii) The rise in the level of RuBP-Case is the consequence of a strictly coordinated synthesis de novo of the subunits. (iii) While the levels of translatable mRNA for SSU are compatible with the rate of SSU synthesis the relatively high LSU mRNA levels are not reflected in the rates of in-vivo LSU or RuBPCase syntheses. (iv) Gene expression is also abolished in the case of nuclear-encoded SSU if intraplastidic translation and concomitant plastidogenesis is inhibited by chloramphenicol, pointing to a plastidic factor as an indispensable prerequisite for expression of the SSU gene(s). (v) Regarding the control mechanism for SSU gene expression, three factors seem to be involved: an endogenous factor which turns on gene expression, phytochrome which modulates gene expression, and the plastidic factor which is an indispensable prerequisite for the appearance of translatable SSU mRNA.Abbreviations CAP chloramphenicol - cFR continuous farred light - LSU large subunit of RuBPCase - NADP-GPD NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) - Pfr far-red-absorbing form of phytochrome - pSSU precursor of SSU - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - SSU small subunit of RuBPCase     

Identification of coiled bodies inBrassica napus nuclei during embryogenesis and early germination

Summary Nucleolus-associated bodies characterize interphase nuclei of many plant species. The recent demonstration that such bodies contain small nuclear ribonucleoproteins as well as coilin clearly indicates that they belong to a larger family of nuclear structures, known as coiled bodies, that have been intensively studied in a variety of animal cell types. In a previous work, we have shown that coiled bodies were present in close association with the nucleolus inZea mays dry seeds as well as during subsequent stages of germination. This study reveals that similar nuclear structures were also present duringBrassica napus embryogenesis starting at the torpedo stage and that they were, likewise, generally located on the nucleolar surface. As in the case ofZ. mays, coiled bodies were observed in cells of dry seeds as well as in those of early germinating tissues. These bodies were labelled with monoclonal antibody K121, an antibody reacting with the unique 5-terminal cap structure containing 2,2,7-trimethylguanosine that characterizes small nuclear RNAs. Owing to their intimate association with the nucleolus in all stages studied, the possibility is considered that, in these plant cells, coiled bodies are assembled on an organizer element located within this organelle.Abbreviations BSA bovine serum albumin - IgM immunoglobulin M - NAB nucleolus-associated body - NAC nucleolus-associated chromatin - PBS phosphate-buffered saline - snRNA small nuclear ribonucleic acid - snRNP small nuclear ribonucleoprotein     

Phytochrome-induced flavonoid biosynthesis in mustard (Sinapis alba L.) cotyledons. Enzymic control and differential regulation of anthocyanin and quercetin formation

Phytochrome-induced increases in enzyme activities for phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone isomerase (EC 5.5.1.6), and in amounts of the related end products, anthocyanin and the flavonol, quercetin, were measured in cotyledons of mustard (Sinapis alba L.). There was no correlation between the activities of these enzymes and the rate of anthocyanin accumulation; however, some correlation was found with the quercetin accumulation rate. Since anthocyanin and flavonol accumulation is spatially separated in mustard (flavonols in the upper epidermis, anthocyanin in the lower epidermis), it was possible to measure anthocyanin-associated phenylalanine ammonia-lyase independently. This activity correlated well with the accumulation rate for anthocyanin during the first few hours after induction. The phytochrome effect on anthocyanin formation differed from that on quercetin formation: anthocyanin was strongly induced by continuous far-red light and by both continuous red light and red light pulses, whereas quercetin was only effectively induced by continuous far-red light.Abbreviations CHI chalcone isomerase - PAL phenylalanine ammonia-lyase     

Effect of light on the development of glyoxysomal functions in the cotyledons of mustard (Sinapis alba L.) seedlings

The degradation of storage fat in the cotyledons of mustard seedlings is unaffected by phytochrome and photosynthesis (irradiation with continuous red or far-red light from sowing of the seeds) although light imposes a strong constraint on the translocation of organic matter from the cotyledons into the seedling axis. Likewise, the development and disappearance of glyoxysomal enzyme activities (isocitrate lyase, malate synthase, citrate synthase) takes place independently of light. It is concluded that the mobilization of storage fat (fatcarbohydrate transformation) is independent of photomorphogenesis. The surplus of carbohydrate produced from fat in the light seems to be converted to starch grains in the plastids, which function as a secondary storage pool in the cotyledons.Abbreviations CS citrate synthase - ICL isocitrate lyase - MS malate synthase     

Sucrose increase during floral induction in the phloem sap collected at the apical part of the shoot of the long-day plant Sinapis alba L.

Sinapis alba L., a long-day plant, has been induced to flower either by a single 22-h-long photoperiod or by an 8-h short photoperiod displaced by 10 h in a 24 h cycle. The ehtylenediametetraacetate method previously used for leaf exudation was modified to collect phloem sap at the apical part of the shoot. Carbohydrates in the phloem sap have been analysed comparatively in vegetative and induced plants, using high-performance liquid chromatography and refractometry. Sucrose was the major sugar detected. A dramatic increase of its flux in the apical sap occurred early and transiently during the floral transition in plants induced by both long days and displaced short days. These results indicate a message-like role for sucrose since they fit nicely with previous observations indicating that an early event in the floral transition in S. alba is the accumulation of sucrose in the meristem.Abbreviations DSD displaced short day - LD long day - SD short day This work was supported by the Fonds pour la Recherche Fondamentale et Collective (FRFC) of Belgium (Grant n 2.9009.87) and the University of Liège (Action de Recherche Concertée 88/93-29). One of us (P.L.) is grateful to the I.R.S.I.A. for the award of a research fellowship.     

Analysis of light-controlled accumulation of carotenoids in mustard (Sinapis alba L.) seedlings

Carotenoid accumulation in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by light. Besides the stimulatory function of phytochrome in carotenogenesis the experiments reveal the significance of chlorophyll accumulation for the accumulation of larger amounts of acrotenoids. A specific blue light effect was not found. The data suggest that light exerts its control over carotenoid biogenesis through two separate mechanisms: A phytochrome regulation of enzyme levels before a postulated pool of free carotenoids, and a regulation by chlorophyll draining the pool by complex-formation.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR high irradiance reaction (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - P_fof total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_fof], wavelength dependent photoequilibrium of the phytochrome system - red red light - fr far-red light     

The influence of light quality on the phytochrome content of light grown Sinapis alba L. and Phaseolus aureus Roxb.

The effect on the phytochrome system of light regimes establishing a range of photoequilibria was studied in two light grown dicotyledonous plants, both of which were treated with the herbicide SAN 9789 to prevent chlorophyll accumulation. In Sinapis alba L. cotyledons the results are comparable with phytochrome behaviour in etiolated mustard seedlings; the level of Pfr becomes independent of wave-length whereas the total phytochrome level is wave-length dependent. Contrasting properties are exhibited in Phaseolus aureus Roxb. leaves in which total phytochrome is unaffected by light quality; consequently the Pfr level is dependent on wavelength. Nevertheless, the amount of phytochrome in mung leaves increased after transfer to darkness suggesting that light still has a profound influence on the phytochrome system, even though light quality during the light period and prior to darkness does not.Abbreviations FR far-red light - WL white light - PAR photosynthetically active radiation - Pfr far-red light absorbing form of phytochrome - Pr red light absorbing form of phytochrome - Ptot total phytochrome level (=Pr+Pfr) - Pfr/Pfr+Pr - SAN 9789 4-chloro-5-(methylamino) 2(,, trifluoro-m tolyl)-3(2H)-pyridazinone     

Differential regulation by phytochrome of the appearance of plastidic and cytoplasmatic isoforms of glutathione reductase in mustard (Sinapis alba L.) cotyledons

An increase of glutathione reductase (GR; EC 1.6.4.2) activity during the transformation of mustard (Sinapis alba L.) cotyledons from storage organs to photosynthetically competent leaves was previously found to be controlled by light acting via phytochrome (Drumm, H., Mohr, H., Z. Naturforsch. 28c 559–563, 1973). Two isoforms of GR could be separated by disc electrophoresis. In the present study we have applied ionexchange chromatography to separate isoforms of GR during the development of the cotyledons. Furthermore, the technique of in situ photooxidation of plastids was used to distinguish between plastidic and cytoplasmatic isoforms. The isoform GR₂ is the plastidic enzyme, as shown by its sensitivity to photooxidative treatment, while GR₁ is a cytoplasmatic protein not affected by photooxidative treatment of plastids. Both isoforms are promoted by phytochrome but with different time courses. The appearance of GR₁ is independent of the integrity of the plastids, as one might expect. However, unexpectedly, the phytochrome-mediated re-appearance of GR₂ after a photooxidative treatment is much less affected by photooxidative destruction of the plastids, i.e. by the lack of a particular plastidic factor, than was to be expected from previous experience with typical plastidic proteins. An interpretation of this finding must await measurements at the level of GR₂ mRNA.Abbreviations c continuous - D darkness - FR far-red light (3.5 W·m^-2) - FPLC fast protein liquid chromatography - GR glutathione reductase (EC 1.6.4.2) - NF Norflurazon - R fed light (6.8 W·m^-2) - = Pfr/Ptot wavelength-dependent photoequilibrium of the phytochrome system