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     

Factors involved in the coordinate appearance of nitrite reductase and glutamine synthetase in the mustard (Sinapis alba L.) seedling

The extent to which the appearances of nitrite reductase (NIR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) are coordinated was studied in mustard (Sinapis alba L.) seedlings. It was established by immunotitration that the increased activities of NIR and GS in the presence of light and nitrate can be attributed to the de-novo synthesis of enzyme protein. The bulk of the NIR and GS was found in the developing cotyledons. In the absence of nitrate in the growth medium there was no coordinate appearance of NIR and GS. While light strongly stimulated the appearance of GS, the level of NIR was hardly affected and remained low. On the other hand, in the presence of nitrate in the medium the appearances of NIR and GS were strictly coordinated, the GS level being considerably above that of NIR. It is argued that phytochrome-controlled synthesis of GS in the absence of nitrate is part of the mechanism to reassimilate ammonium liberated during proteolysis of storage protein and metabolism of the resulting amino acids, whereas the strictly coordinated synthesis in the presence of light and nitrate indicates the dominance of nitrate assimilation under these circumstances. The fact that the level of GS was always considerably above that of NIR appears to be a safety measure to prevent ammonium accumulation.Abbreviations FR standardized far-red light (3.5 W·m^–2), to drive the high-irradiance reaction of phytochrome - GS glutamine synthetase, EC 6.3.1.2 - NIR nitrite reductase, EC 1.7.7.1 This work was supported by Heidelberger Akademie der Wissenschaften (Forschungsstelle Nitratassimilation).     

Appearance and disappearance of proteins in the shoot apical meristem of Sinapis alba in transition to flowering

Vegetative plants of Sinapis alba L. grown under short days were induced to flower by exposure to one long day or continuous long days. Irrespective of the number of long days, the first flower primordia were initiated by the shoot apical meristem 60 h after the start of the inductive treatment. An indirect histoimmunofluorescence technique was used to search in the apical meristem for three antigenic proteins which had been previously detected by immunodiffusion tests in the whole apical bud (Pierard et al. (1977) Physiol. Plant. 41, 254–258). One protein called protein A, present in the vegetative meristem, increased in concentration during the first 48 h following the start of the inductive treatment. It stayed constant up to 96 h and disappeared completely at a later time. Two other proteins called B and C, absent in the vegetative meristem, appeared in the meristem of induced plants between 30 and 36 h after the start of the inductive treatment and progressively accumulated at later times up to 240 h. These proteins appeared 8 h before the irreversible commitment of the meristem to produce flower primordia (point of no return) was reached and 24 h before start of flower production. These observations support an interpretation of floral evocation as consisting, at least partially, of an early and qualitative change in gene expression.Abbreviations AVB anti-vegetative-bud antiserum - ARB antireproductive-bud antiserum - IgG immunoglobulins G - TRITC tetramethylrhodamine isothiocyanate - GAR IgG goat antirabbit IgG - S₀ IgG non-immune rabbit IgG     

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     

Phytochrome action in light-grown plants: the influence of light quality and fluence rate on extension growth in Sinapis alba L.

Using light-grown plants of Sinapis alba an analysis has been made of the effect on extension growth of adding far red light to a background photosynthetic source. It has been possible to distinguish between the increase in fluence rate and the reduction of the amount of phytochrome present as Pfr, which are both consequences of the addition of supplementary far red light, and to determine that the response of increased extension growth is due only to the latter. It is shown that the degree of fluence rate dependency varies with photoequilibrium and the significance of this interaction is discussed in terms of the mode of action of phytochrome and of its role in the natural light environment.Abbreviations PAR photosynthetically active radiation - SAN 9789 4-chloro-5-(methylamino)-2-(,,-trifluoro-m-tolyl)-3(2H) pyridazinone - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - LER logarithmic extension rate     

Cyclic and noncyclic photophosphorylation during the ontogenesis of high-light and low-light leaves of Sinapis alba

Noncyclic electron transport to ferricyanide and photophosphorylation as well as the methylviologen mediated aerobic and anaerobic photophosphorylation with dichlorophenolindophenol-ascorbate as the electron donor of photosystem I were measured during the development of high-light and low-light adapted leaves of Sinapis alba. Anaerobic methylviologen-catalyzed phosphorylation is more than twice as high as aerobic phosphorylation. The difference between the rates of aerobic and anaerobic phosphorylation is sensitive to dibromothymoquinone. Thus, under anaerobic conditions, methylviologen mediates a cyclic phosphorylation including plastoquinone. All photochemical activities of high-light chloroplasts are about twice as high as that of low-light chloroplasts and show a permanent decline with increasing plant age. The lower activities of low-light chloroplasts correlate with a decrease of electron transport components, such as cytochrome f. This indicates that the number of electron transport chains is decreased under low-light conditions and more chlorophyll molecules interact with one electrontransport chain.Abbreviations Asc ascorbate - Chl chlorophyll a+b - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(dichlorophenyl)-1,1-dimethylurea - DCPIP dichlorophenolindophenol - HL high light - LL low light - MV methylviologen - PhAR photosynthetically active radiation - PS photosystem     

Gene expression in the soybean seed axis during germination and early seedling growth

Copy-DNA clones have been obtained that distinguish eight messenger mRNAs, moderately abundant in the axes of the germinating soybean (Glycine max (L.) Merr.) seedling. These clones have been used to characterize the size of the mRNAs and to anlyze the accumulation of the mRNAs at different time points and in different parts of the axis during germination and early seedling growth. Three of the mRNAs accumulate to a substantial level by 9 h, a time point before either the beginning of growth or the accumulation of polyribosomes. Four other mRNAs reach a substantial level only at 24 h, a period when rapid seedling growth is occurring. Those mRNAs whose accumulation begins at 24 h were found only in the top (hypocotyl) half of the 24-h seedlings, while the remaining mRNAs were present also in the bottom half of the seedlings in different amounts. By 44 h, the bottom 0.5 cm of the seedlings, i.e., the region of meristematic growth, had little or none of the mRNAs, with the exception of one mRNA. These temporal and spatial observations indicate that many of the mRNAs are not involved simply in the general maintenance of ongoing cell proliferation, but that they may be related to differentiation during early seedling formation. Further, the early accumulating mRNAs may be functioning in regulating the onset of seedling growth.Abbreviations cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Formation of protein storage bodies during embryogenesis in cotyledons of Sinapis alba L.

An electron microscopic investigation of fine structural changes in post-meristematic cotyledon mesophyll cells during the period of storage protein accumulation (16–32 d after pollination) showed that the rough ER, the Golgi apparatus and the developing vacuome are intimately involved in the formation of storage protein bodies (aleurone bodies). At the onset of storage protein accumulation (16–18 d after pollination) storage protein-like material appears within Golgi vesicles and preformed vacuoles. At a later stage (24 d after pollination) similar material can also be detected within vesicles formed directly by the rough endoplasmic reticulum (ER). It is concluded that there are two routes for storage protein transport from its site of synthesis at the ER to its site of accumulation in the vacuome. The first route involves the participation of dictyosomes while the second route bypasses the Golgi apparatus. It appears that the normal pathways of membrane flow in the development of central vacuoles in post-meristematic cells are used to deposit the storage protein within the protein bodies. Thus, the protein body can be regarded as a transient stage in the process of vacuome development of these storage cells.Abbreviation ER endoplasmic reticulum     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric point To whom correspondence should be addressed.     

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     

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     

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     

Tissue-specific localization of heat-stress proteins during embryo development

We report on the stress-independent, tissue-specific expression of the heat-stress protein HSP17 in developing seeds of different plant species and on its intracellular localization. Though HSP17 expression during seed development seems to be a general phenomenon, the isoform patterns, the relative amounts in embryonic tissues and the intracellular localization show species-specific variations. In contrast to the results on the stressinduced protein forming large cytoplasmic aggregates (heat stress granules) the developmentally expressed HSP17 is mainly found in nuclei. But in addition, a considerable part is also detected in protein bodies of mature seeds of Lycopersicon esculentum and Vicia faba, but not of Zea mays. The mechanism of this transition into the vacuolar compartment remains to be investigated.Abbreviations 2D two-dimensional - HSE heat shock elements - HSP heat stress protein     

Expression of storage-protein genes during soybean seed development

Mature seeds of Glycine max (L.) Merr. contain two major storage proteins, a glycosylated 7S protein (conglycinin) and a non-glycosylated 11S protein (glycinin). Accumulation of these proteins and their mRNAs during seed development in cv. Provar was studied by SDS polyacrylamide gel electrophoresis and by Northern (DNA-RNA) hybridization. The 11S acidic and basic subunits and the 7S and subunits began to accumulate 18–20 d after pollination, shortly after the termination of cell division in developing cotyledons, whereas the 7S and 11S A-4 subunits were not detected until one to two weeks later, during the maturation phase of development. Messenger RNAs for 7S and 11S proteins were first detected 14–18 d after pollination, several days before the accumulation of storage proteins. Extracts from embryonic axes contained reduced levels of the 7S subunit, very little 11S protein, no detectable 7S or 11S A-4 subunits, and an additional 7S subunit not found in cotyledons. Soybean axes and cotyledons therefore differ in their synthesis of seed storage proteins.Abbreviations cDNA complimentary DNA - mRNA messenger RNA - SDS sodium dodecyl sulfate     

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