Polar lobe specific regulation of translation in embryos of Ilyanassa obsoleta.

Comparisons of the patterns of synthesis of ¹⁴C-labeled proteins of normal embryos of Ilyanassa with the patterns of synthesis of ³H-labeled proteins of embryos from which the polar lobes had been removed at the trefoil stage were made by coelectrophoresis on polyacrylamide disc gels. Controls utilizing biochemical and morphological markers were performed to assure that normal and delobed embryos were developing at equivalent rates. The expression of significant differences in the patterns of protein synthesis were found between normal and delobed embryos, and these differences were not dependent upon concomitant RNA synthesis. These differences were observable as early as after only 24 hr of development, although organogenesis does not begin until much later in development. Therefore, the observed differences probably reflect determininative events. The results support the hypothesis that the developmental determinants of the polar lobe may include specific, preformed mRNA sequences, or specific regulators of translation.     

Post-transcriptional regulation of protein synthesis in Ilyanassa embryos and isolated polar lobes

The experimental removal of the polar lobe, an anucleate cytoplasmic protrusion formed in preparation for the first cleavage, from the egg of Ilyanassa obsoleta results in grossly abnormal embryonic development. In experiments reported here normal and delobed embryos, as well as isolated polar lobes, were incubated with [³⁵S]methionine for 4 hr beginning at the completion of the first cleavage or 21 hr later during epiboly. Proteins were extracted and examined by fluorography after resolution by two-dimensional polyacrylamide gel electrophoresis. In normal embryos the synthesis of several proteins begins or ends between the two stages investigated. In isolated polar lobes a subset of these developmental changes in protein synthesis occurs, indicating that the regulation of these events is independent of concomitant nuclear activity and probably involves selective regulation of the translation of mRNA stored in the eggs. The patterns of protein synthesis in normal embryos and delobed embryos are qualitatively extremely similar, though quantitative differences are also observed. No proteins can be detected which are synthesized exclusively in polar lobes.     

H1 histone subtypes and subtype synthesis switches of normal and delobed embryos of Ilyanassa obsoleta

Histone H1 subtype complexity and H1 histone subtype synthesis switches were characterized during the development of normal embryos of the mud snail Ilyanassa obsoleta. The effect of the removal of the third polar lobe on the normal H1 pattern of synthesis was then investigated in the delobed embryo to determine if classical polar lobe effects are accompanied by a perturbation of these patterns. SDS-gel electrophoresis and fluorography of radiolabeled 5% perchloric acid-soluble nuclear extracts resolved six H1 proteins designated bands 1-6. Bands 1-5 migrate as a cluster of individual bands with similar mobilities. Band 6 has a substantially slower mobility. The synthesis of band 6 is predominant during the first 6 hr post-trefoil. During cleavage and gastrulation bands 1 and 2 are predominant while band 3, 4, and 5 become predominant during organogenesis. In addition, it has been found that removal of the polar lobe delays the off-switch of the early bands 6, 1, and 2 and the on-switch of the late bands 3, 4, and 5. This must result in a different H1 composition in the chromatin of the two embryo types. Cell number data of normal and delobed embryos reveal that the delay in subtype synthesis switching is not caused by an overall delay of cell division in the delobed embryo. However, the data indicate that a subpopulation of cells may not divide, or may divide late, in the delayed embryo. The data also suggest that the D cell lineage may be involved in the control of histone synthesis switching in the A, B, and C cell lineages.     

Differences in Patterns of Newly Synthesized Proteins in Imbibing Embryos of After-ripened and Aged Seeds of Agrostemma githago and Comparison of Protein Synthesis In Vivo and In Vitro

Patterns of newly synthesized proteins in imbibing after-ripenedyoung and aged Agrostemma embryos show differences in the dynamicsof cotyledons and axes. In the course of imbibition the terminationof some syntheses in the embryonal parts of aged seeds is delayed.As was shown by in vitro translation, mRNAs coding for the synthesisof some proteins are still present in the embryos when proteintranslation has already finished. Key words: Protein pattern, in vivo protein synthesis, in vitro protein synthesis, after-ripened embryos, aged embryos, Agrostemma githago     

Protein Synthesis and Differentiation During Pulmonate Development

An analysis of the changing patterns of protein synthesis duringearly development of Lymnaea palustris has been undertaken.An examination of the ingestion of capsule fluid protein suggeststhat after gastrulation 30 to 65% of the total embryo proteinis undigested food protein. Starch gel electrophoresis revealsa sudden increase in the number of hydrolases from four to twenty-siximmediately following the trochophore stage with the latterbeing present also in adult organs. Studies reported here andelsewhere demonstrate rhythmic changes in uridine incorporationduring early cleavage which peaks at the trochophore stage.Continuous treatment of embryos with 50 to 100µ.g of actinomycin-D(AMD) starting at the 2-cell stage slowed development throughthe trochophore stage but did not prevent normal larval organdevelopment. This AMD application reduced ³H uridine incorporationmore than 90% but did not appreciably alter the pattern of totalor ¹⁴C leucine pulse labeled peptides on sodium dodecyl sulfate(SDS) ultrathin slab electrophoresis gels. However, pronouncedand numerous changes in the patterns of both labeled and unlabeledpeptides were observed during development through 4 days withthe most notable alterations occurring at the 2.5 post-gastrulastage. This was true in normal and continuously treated AMDembryos. The morphological and biochemical data suggest Lymnaeaearly development is controlled by stable maternal messengerRNA.     

Mechanism of Action of Lycoricidinol, a Plant Growth Inhibitor Isolated from Lycoris radiata Herb.

We studied the action mechanism of lycoricidinol, a plant growthinhibitor isolated from Lycoris radiata Herb. Lycoricidinolinhibited protein synthesis in mung bean hypocotyls, but notRNA synthesis. Protein synthesis in Escherichia coli was notaffected by the inhibitor. Results of in vitro translation experimentswith the wheat germ system and the E. coli system indicatedthat lycoricidinol inhibited only eukaryotic but not prokaryotictranslation. Use of specific inhibitors of initiation and polypeptidechain elongation of polypeptide synthesis revealed that chainelongation was inhibited by lycoricidinol. ¹Permanent address: Department of Biology, Yonsei University,Seoul 120, Korea. (Received September 30, 1983; Accepted December 28, 1983)     

Natural Ageing: Poly(A) Polymerase in Germinating Embryos of Triticum durum Wheat

The viability of seeds is associated with ageing and storageconditions. A loss of viability is accompanied by slow germination,reduced growth, and a decline in protein and poly(A)⁺RNA synthesis.This paper reports on the activity of poly(A) polymerase indry and germinating embryos of Triticum durum Desf. cv. Cappellicaryopses of different ages and viability. The enzyme was presentas a single form during ageing and germination. The poly(A)polymerase was active at decreasing levels in all aged dry embryos,in parallel with loss of viability. Its activity strongly increasedduring the germination only in viable embryos. The observedincrease was due to de novo synthesis of the enzyme. Poly(A)polymerase synthesis was low during germination of less viableembryos and absent in older ones. Reduced poly(A) polymeraseactivity in dry or germinated wheat embryos may cause a shorteningof poly(A) chains in vitro and a decline in poly(A)⁺RNA synthesis.Copyright1995, 1999 Academic Press Triticum durum Desf. cv. Cappelli, wheat, embryo, natural ageing, poly(A) polymerase     

Protein synthesis in the embryos of Pinus thunbergii seed I. Influences of imbibition of seeds in the dark

The conditions and requirements of an in vitro protein-synthesizingsystem in the embryos of Pinus thunbergii seeds were studied.Even in the dry seed embryos, the ribosomes retained their syntheticcapacity. Even after imbibition in the dark, the ribosomes didnot show an increase in the activity of protein synthesis. Anincrease in the activity during dark imbibition was found inthe 100,000?g supernatant fraction. The activities of the cell-freesystems prepared from both embryos of dark-imbibed and dry seedswere dependent on the addition of poly U. This suggests thelack or inactivity of messenger RNA in these seed embryos. ¹ Present address: Faculty of Education, Utsunomiya University,Mine-machi, Utsunomiya 320, Japan. (Received July 19, 1976; )     

An Analysis of Seed Development in Pisum sativum: IV. COTYLEDON CELL POPULATIONS IN VIVO AND IN VITRO

A method for quantifying changes in the cell population of Pisumsativum cotyledons during development is described. The methodis based on determining the frequency distribution for cellarea following the random sampling of a single-cell suspensionof cotyledon cells. The population profile of these cells changedprogressively and systematically from a single population, similarin size to meristematic cells, found in embryos less than 3.0mg in fresh weight, to a bimodal population in embryos greaterthan 100 mg fresh weight. This method was used to compare embryosof similar size from two genotypes near-isogenic except forgenes at the r locus. No significant differences were foundbetween the cell population profiles of embryos up to 30 mgfresh weight. However, a significant difference was found betweenembryos with fresh weights of 100 mg, the wrinkled (rr) linehaving a higher mean and maximum cell area (2 951 µm²and 9 240 µm² respectively) than the round (RR) line (2591µm²and 6470 µm²respectively). Comparisons were alsomade between cotyledon cell populations from round (RR) embryosgrown in vivo and in vitro. The most obvious differences werethe higher mean and maximum cell size of the large cell populationof in vitro grown embryos which were twice those found in vivo.Embryos grown to either 30 mg or 100 mg fresh weight in vitrohad a much greater proportion of large cells in the populationwith a corresponding reduction in total cotyledon cell number,compared with similar sized embryos grown in vivo. These data suggest that comparisons between different genotypes,or, between cultured and in vivoembryos, based on morphologicalsimilarities between embryos, may be invalid and subject tomisinterpretation. Key words: Pea, seed development, cell population     

Two-Dimensional Protein Patterns of Arabidopsis Wild-Type and Auxin Insensitive Mutants, axr1, axr2, Reveal Interactions between Drought and Hormonal Responses

In order to detect gene products involved in Arabidopsis droughtadaptive strategy, 2D-PAGE protein patterns of two auxin-insensitivemutants, axr1, axr2, differentially affected in specific droughtresponses, were compared to the wild-type Columbia ecotype,in well-watered and drought-stressed conditions. Coupled tocomputer analysis of polypeptide amounts, 2D-electrophoresisrevealed subtle changes in protein expression induced by progressivedrought stress and/or mutations affecting the auxin responsepathway. The differential protein patterns of axr1 and axr2 were consistentwith their contrasting drought responses. The specific leafand root protein patterns of axr1 showed that this mutationdisrupts drought responses related to auxin regulation. In particular,the near absence of drought rhizogenesis in axr1 was associatedwith a root protein pattern closer to the well-watered thanto the water-stressed axr2 and Columbia wild-type root proteinpatterns. Also, the largely different effects of axr1 and axr2mutations suggest that they affect different pathways in auxinresponse. Several sets of polypeptides, whose regulation wasaffected by drought and/or mutation, were thus detected. Thesepolypeptides could play a role both in the auxin and the droughtresponse pathways. Their identification, through microsequencing,should be most informative. ⁴Current address: Laboratoire de BioénergétiqueCellulaire, Département d'Ecophysiologie Végétaleet de Microbiologie, Centre d'Etudes de Cadarache, 13108 SaintPaul lez Durance, France     

The Effect of Salinity Stress upon Protein Synthesis of Germinating Wheat Embryos

Two differently salt-sensitive wheat genotypes were imbibedin 0·4 M NaCl for 72 h or, alternatively, for 48 h andthen transferred to water. Seed germination, fresh weight andprotein synthesis in embryos were determined. The followingdifferences were found in the synthesis of in vivo [³⁵S]methionine-labelledproteins during salt imbibition: (a) a general decrease or disappearanceof polypeptides specific to the radicle emergence phase in thesalt-sensitive genotype; (b) a new synthesis of polypeptideswhich are not found during water imbibition and are common toboth genotypes; (c) a differential synthesis of polypeptidesthat are unique to each cultivar. Upon return to water, salt-inducedproteins ceased to be synthesized while proteins associatedwith an advanced germination phase were actively produced. Theseresults suggest that the expression of 'salt stress' proteinsis related to the adaptation process of seeds to salinity aswell as to the genetic constitution of a selected salt-tolerantgenotype.Copyright 1993, 1999 Academic Press Triticum durum, wheat, embryo, salt stress, protein synthesis     

Response of Cultured Embryos of Phaseolus vulgaris and Hordeum vulgare to Farnesol

Excised embryos of Phaseolus vulgaris incubated in a mediumcontaining 10 mg dm^–3 farnesol showed enhanced root growthwhereas the leaves remained rudimentary At lower concentrationsof exogenous farnesol normal leaf development occurred and rootgrowth was comparable to untreated cultures. Enhanced root growthalso occurred when excised embryos of Hordeum vulgare were treatedwith farnesol but only at 10 mg dm^–3 and this treatmentdid not prevent leaf growth X-ray micro-probe analysis of leavesrevealed an increased phosphorus content in P vulgaris and adecreased sulphur content in H vulgare in comparison to untreatedplants. Hordeum vulgare L., barley, Phaseolus vulgaris, bean, embryo culture, farnesol, X-ray microprobe analysis, root growth     

Cell Morphogenesis and Macromolecule Synthesis during Phytochrome-controlled Germination of Spores of the Fern, Pteris vittata

The object of this study was to characterize the pattern ofcell morphogenesis and synthesis of nucleic acids and proteinsduring phytochrome-controlled germination of spores of the fern,Pteris vittata. Phytochrome activation and germination wereinitiated in fully imbibed spores by exposure to a saturatingdose of red light. At timed intervals thereafter, spores werefixed in acrolein and embedded in glycol methacrylate for examinationin the light microscope. The first sign of germination, visiblein sections of the spore 12 h after irradiation, was the hydrolysisof storage protein granules. This was followed by a migrationof the nucleus from its central location to one side of thespore. Subsequently, the protoplast enlarged at the site ofthe nucleus and appeared outside the exine as a papillate structure.An asymmetrical division of the protoplast gave rise to a smallcolourless rhizoid cell and a large, chloroplast-containingprotonemal cell. During the early phase of germination, DNAwas synthesized both in the nucleus and cytoplasm as judgedby autoradiography of [³H]thymidine incorporation. [³H]Uridine,a precursor of RNA synthesis, was incorporated into the nucleolusand the rest of the nuclear material of germinating spores.Protein synthesis monitored by [³H]leucine incorporation occurredboth in the nucleus and cytoplasm during the early stage ofgermination, although a strictly cytoplasmic protein synthesiswas observed later. Addition of cycloheximide completely inhibitedgermination of photoinduced spores and incorporation of labelledprecursors of macromolecule synthesis into cellular components.Actinomycin D was much less effective as an inhibitor of germinationand, even in high concentrations of the drug which effectivelyinhibited DNA and RNA synthesis in spores, proteolysis and proteinsynthesis appeared normal. These findings are discussed withrespect to the regulation of nucleic acid and protein synthesisduring spore germination and the role of phytochrome in theprocess.     

Post-transcriptional regulation of protein synthesis during alfalfa embryogenesis: proteins associated with the cytoplasmic polysomal and non-polysomal mRNAs (messenger ribonucleoprotein complex)

Cytoplasmic polysomal and non-polysomal mRNA-associated proteincomplexes were isolated from, and characterized in, developingsomatic and zygotic embryos of alfalfa (Medicago sativa L.).³⁵S-methionine-labelled intact embryos were irradiated withultraviolet light (UV) in situ to cross-link mRNA and proteinsoccurring within one bond length, and the polysomal and non-polysomalfractions were extracted. Then the mRNA-protein complexes wereisolated from the fractions and separated using two cycles ofaffinity chromatography on an oligo(dT)-cellulose column. Followingdigestion with RNase A and T1 and micrococcal nuclease, mRNA-associatedproteins were separated by SDS-PAGE. Several polypeptides of 15–150 kDa were associated withthe polysomal and non-polysomal (ribonucleoprotein, mRNP) fractionsof alfalfa embryos after UV-cross-linking. Many of the polypeptidesassociated with the polysomal and non-polysomal mRNAs were qualitativelysimilar, although their concentration in the two fractions wasdifferent. However, some developmentally stage-specific polypeptideswere found to be associated with the non-polysomal mRNA fractionduring the early stages of embryogenesis (precotyledonary) ofsomatic embryos. Thus the presence of mRNPs during embryogenesishas been demonstrated, and proteins intimately associated withthe mRNAs identified. Key words: Embryogenesis, translational control, protein synthesis, messenger ribonucleoproteins, alfalfa (Medicago sativa L.)     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA     

An Analysis of Seed Development in Pisum sativum: VIII. DOES ABSCISIC ACID PREVENT PRECOCIOUS GERMINATION AND CONTROL STORAGE PROTEIN SYNTHESIS?

The influence of abscisic acid (ABA) on the precocious germinationand storage protein production of pea seeds has been examinedusing embryo and pod culture. The precocious germination ofembryos in culture could not be inhibited fully by ABA on apermissive medium (2% sucrose) even at 0.1 mol m^–3. However,increasing the sucrose concentration to 5% caused near completeinhibition when ABA was added to the medium. Embryos of differentweights cultured on a high osmoticum (mannitol-containing medium),equivalent to 10% sucrose, did not show any consistent differencein ABA content. When fluridone was added to a non-permissiveculture medium, no decrease in ABA content of the embryos couldbe observed and no precocious germination was induced. In contrast,fluridone was able to prevent the accumulation of ABA in seedspresent in pods cultured in its presence from an early stageof development. These seeds, however, grew normally and reachedmaturity, did not germinate precociously in vivo, were desiccationtolerant and still produced storage protein message whetheror not ABA was included in the culture medium. It does not appear,therefore, that ABA regulates normal development or storageprotein synthesis in pea embryos. Key words: Abscisic acid, peas, Pisum sativum, seed development