Characterization and Complexity of Wheat Developing Endosperm mRNAs

Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA⁺ RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)⁺ RNAs, analyzed on isokinetic sucrose gradient with [³H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly ⅔ and ⅓ of the MB poly(A)⁺ RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins.     

Polymorphism in fowl serum albumin. VII. Distribution and activity of free and membrane-bound polysomes in developing fowl liver

The quantity and activities of membrane-bound and free polysomes in livers from chick embryos at successive stages of development were compared in cell-free protein-synthesizing systems. Membrane-bound polysomes increased 2-fold between 8 and 18 days of development, while total ribosome content remained constant. Free polysome activity also remained constant during this period, while that of membrane-bound (total--free) polysomes decreased, possibly because of an increase in ribonuclease activity in this fraction. Serum albumin biosynthesis occurred primarily on membrane-bound polysomes. With liver development, increased secretion of serum proteins may be correlated with synthesis of serum albumin on increasing numbers of membrane bound polyribosomes.     

The formation and translational activity of polysomes from developing lupin seeds

The formation and in vitro translational activity of total, free and membrane-bound polysomes from various stages of developing cotyledons of yellow lupin seeds (Lupinus luteus L. cv. Iryd) has been investigated. The early stages of seed formation were characterized by a low level of polysomes that progressively increased. The main features of the cotyledons at the middle phase of development were full expansion growth and the highest amount of polysomes observed in all three poly so me fractions. In The final stages of emhryogenesis. the seed dehydration was accompanied by-gradual loss of all types of polysomes, at which the membrane-attached formations were degraded earlier than the free ones. By means of a wheat germ-derived cell-free system for protein synthesis, a correlation was demonstrated between cotyledon growth, polysome formation and their capacity for protein synthesis in vitro. As compared to the free polysomes, both the total and membrane-bound formations were more active in protein synthesis in vitro. Analysis of the translational products by means of immunoprecipitation and gel electrophoresis followed by fluorography showed that only membrane-bound polysomes produced polypeptides of higher molecular weight, including subunits of a legumin-like protein.     

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.     

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate- cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S- translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.     

Storage Protein Synthesis in Maize: III. Developmental Changes in Membrane-bound Polyribosome Composition and in Vitro Protein Synthesis of Normal and Opaque-2 Maize

Zein synthesis accompanied an increase in large polyribosomes of maize (Zea mays) endosperm cells. The two classes of polyribosomes (free and membrane-bound) had dissimilar size class distributions. Membrane-bound polyribosomes were predominantly large size classes, which were not found in free polyribosomes. The ratio of large membrane-bound polysomes to total membrane-bound polysomes was highest when zein was being synthesized. Appearance of the large polysomes correlated with the onset of zein accumulation in vivo. These large size classes were nearly absent in the opaque-2 mutant at all stages of endosperm development. Similarly, rRNA content was reduced in the mutant from that in normal endosperm development. These differences were associated with reduced in vitro synthesis and in vivo accumulation of zein.     

The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons

Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.Polysomes were isolated from developing cotyledons 20–25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm³ (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.     

Polysome formation and stability in pea stem and root tissue

Polysome stability and the formation of various polysomal populations in pea stem and root tissue were examined. Both total ribosomal fraction and four polysome populations were isolated: FP (free polysomes), MBP (membrane-bound polysomes), CBP (cytoskeleton-bound polysomes) and CMBP (cytoskeleton-membrane-bound polysomes). The content of above mentioned populations decreased in roots and stems during germination. In both roots and stems a gradual decrease of FP participation in the total polysomal population was also observed during germination. On the other hand, an obvious increase in participation of CMBP population in the total polysomes pool was observed in later stages of germination. Increase of CMBP participation in pea root and stem tissues in later stages of germination is probably due to intensive enzymatic protein synthesis taking place in them. These proteins may participate in elongating growth of cells. The results of investigation on polysomes stability showed that total polysomes isolated from pea roots appeared to be more resistant to digestion by exogenous ribonuclease (EC 3.1.27.5) than polysomes isolated from stems. As protein-mRNA interactions are widely known and ribosomes are also very adhesive structures, numerous non-ribosomal proteins are present in the polysome preparations. We suppose that changes in proteins bound to polysomes indicated by us previously, significantly influence both the stability and also translatability of polysomes isolated from different plant organs.     

Storage Protein Synthesis in Maize: II. Reduced Synthesis of a Major Zein Component by the Opaque-2 Mutant of Maize

Two zein proteins (Z1 and Z2) represent the majority of the protein synthesized during maize endosperm development. Undegraded membrane-bound polysomes isolated from normal maize synthesized these proteins when incubated in a cell-free protein-synthesizing system from wheat germ. The proteins synthesized in vitro were similar to authentic zein in ethanol solubility and electrophoretic mobility. Zein synthesis was associated with large size classes of membrane bound polysomes in normal maize.Membrane-bound polysomes isolated from developing kernels of opaque-2 mutant synthesized less total zein in vitro, and dramatically reduced incorporation into the Z1 component. The reduction in total zein corresponded to a 50% reduction in the level of membrane-bound polysomes in opaque-2, and the near absence of the large polysome size classes, which synthesized zein in normal maize. We concluded that the opaque-2 mutation results in a decreased "availability" of the zein mRNAs, reflected in a reduced level of membrane-bound polysomes.     

Cell-free Synthesis of Globulin by Developing Oat (Avena sativa L.) Seeds

The primary storage protein synthesized during oat (Avena sativa L.) groat development is a globulin. Polysomes were isolated from oat groats 12 days after anthesis. These polysomes directed the incorporation of radioactive amino acids into protein in a cell-free protein synthesis system containing wheat germ supernatant. The Mg(2+) optimum was 4 mm, the pH optimum was 6-8, and the amount of amino acid incorporation depended on polysome concentration. Incorporation of amino acids was linear for about 10 min and approached a maximum after 20 min. Using the initiation inhibitor, T-2 toxin, it was determined that about 36% of the amino acid incorporation was due to the initiation of new polypeptide chains. The in vitro product co-electrophoresed with authentic oat groat globulin on polyacrylamide-sodium dodecyl sulfate (SDS) gels. The cyanogen bromide peptides of the in vitro product partially corresponded with those from authentic globulin when electrophoresed on polyacrylamide-SDS gels. These data suggest that the in vitro product is primarily oat globulin. The polysome population was separated into membrane-bound and free polysomes. Membrane-bound polysomes synthesized about twice the amount of protein as did free polysomes. Products synthesized in vitro on both types of polysomes were essentially the same.     

Both linked and unlinked mutations can alter the intracellular site of synthesis of exported proteins of Escherichia coli.

It previously has been demonstrated that synthesis of the periplasmic maltose-binding protein (MBP) and alkaline phosphatase (AP) of Eschericha coli predominantly occurs on membrane-bound polysomes. In this study, signal sequence alterations that adversely affect export of MBP and AP, resulting in their cytoplasmic accumulation as unprocessed precursors, were investigated to determine whether they have an effect on the intracellular site of synthesis of these proteins. Our findings indicate that export-defective MBP and AP are not synthesized or are synthesized in greatly reduced levels on membrane-bound polysomes. In some instances, a concomitant increase in the amount of these proteins synthesized on free polysomes was clearly discerned. We also determined the site of synthesis of MBP and AP in strains harboring mutations thought to alter the cellular secretion machinery. It was found that the presence of a prlA suppressor allele partially restored synthesis of export-defective MBP on membrane-bound polysomes. On the other hand, the absence of a functional SecA protein resulted in the synthesis of wild-type MBP and AP predominantly on free polysomes.     

Exogenous abscisic acid increases stability of polysomes in embryos of triticale caryopses during germination

Some posttranslational processes that occur in embryos of germinating triticale caryopses treated with different concentrations of abscisic acid (ABA) were examined. ABA increased the ratio of cytoskeleton-bound polysomes in the total population of polysomes and depressed the share of free and membrane-bound polysomes. Using exogenous RNase, stability of the total polysomal population as well as each polysomal fraction was investigated. The total extractable polysomes isolated from embryonic tissues of germinating triticale caryopses treated with ABA were more stable than the polysomes isolated from the control sample caryopses. The contribution of the polysomes that were not digested by RNase was increased by higher concentrations of ABA applied during germination. At high concentrations of ABA (50, 100 μM), the quantitative contribution of polysomes in the total ribosomal fraction was almost 100% of the amount of polysomes before digestion and the modifications observed consisted mainly of the shift of the so-called heavy polysomes towards light polysomes, containing a few ribosomes. Within each polysomal population, cytoskeleton-bound polysomes (CBP and CMBP) were the most stable, which may imply that the bonds between polysomes and these protein filaments, created in all eukaryotic cells increased their stability. It is assumed that mRNAs are stabilised or destabilised by interaction of proteins with their various sequences. A plant hormone may depress or elevate the quantities of these proteins, thus regulating the stability of different mRNAs. The results confirm the multi-faceted mechanism of ABA-induced response, where one of the constituents is the effect of ABA on the stability of mRNAs molecules. The co-ordinated regulation of mRNAs synthesis and their stability provide plants with improved adaptability.     

A simple and rapid procedure for high-yield isolation of essentially undegraded free and membrane-bound polysomes from rat liver

A procedure is described for the preparation of free and membrane-bound polysomes from rat liver. The procedure involves: differential centrifugation of liver homogenate to separate free and membrane-bound polysomes; treatment of the membrane-bound polysome fraction with a detergent to release bound polysomes from membranes; and magnesium precipitation of both classes of polysomes. Free and bound polysomes prepared in this manner were essentially undegraded and highly active in cell-free protein synthesis. The recovery of polysomes was nearly quantitative and the distribution between the free and membrane-bound state was 41 and 59%, respectively. Polypeptides synthesized in vitro by the free and membrane-bound polysomes were quite different. The majority (81-84%) of mRNA activities of two secretory proteins (albumin and transferrin) were recovered in the membrane-bound polysomes, whereas the majority (81-85%) of mRNA activities of two cytosolic [aldolase B, EC 4.1.2.13, and argininosuccinate synthetase, EC 6.3.4.5], one mitochondrial [ornithine carbamoyltransferase, EC 2.1.3.3] and one peroxisomal [catalase, EC 1.11.1.6] proteins were recovered in the free polysomes. A polysome class synthesizing ornithine carbamoyltransferase was purified 42-fold from the free polysomes by immunoprecipitation. The procedure is rapid (4-5 h) and reproducible, and provides a nearly quantitative means of separating the two classes of polysomes.     

Distribution of messenger ribonucleic acid in polysomes and nonpolysomal particles of sea urchin embryos: translational control of actin synthesis

We have used cell-free translation and two-dimensional gel electrophoresis to examine the complexities of the polysomal and cytoplasmic nonpolysomal [ribonucleo-protein (free RNP)] messenger ribonucleic acid (mRNA) populations of sea urchin eggs and embryos. We show that all species of mRNA detected by this method are represented in both the polysomes and free RNPs; essentially all messages present in polysomes are also in the free RNP fraction. However, the cytoplasmic distribution is clearly nonrandom since some templates are relatively concentrated in the free RNPs and others are predominantly in the polysomes. The polypeptides synthesized under the direction of unfertilized egg mRNA are qualitatively indistinguishable from those made by using embryonic mRNA, indicating that the complexity of the abundant class mRNA remains unchanged from egg through early development. However large changes in the abundancies of specific mRNAs occur, and changes are detected in the polysomal/free RNP distribution of some mRNAs through development. The differences in the realtive abundancies of specific mRNAs between polysomes and free RNPs and the developmental changes that take place indicate significant cytoplasmic selection of mRNA for translation. Three different forms of actin (termed alpha, beta, and gamma) were identified among the translation products. Messages for all three are present in the unfertilized egg and early cleavage embryo, yet the gamma form is preferentially located in the polysomes and the alpha and beta in the free RNPs. The relative concentrations of the three change greatly during development as do their relative distributions into polysomes and free RNPs. Examinations of in vivo labeled proteins largely support the in vitro findings. The results indicate that the synthesis of actin mRNAs increases greatly during development and that the expression of the actin mRNAs is partly controlled at the translation level during early development.     

Biosynthesis and secretion of collagen in normal and SV-40 virus-transformed human embryonal fibroblasts

The biosynthesis and secretion of collagen proteins was studied in cultures of normal human embryo fibroblasts at different passages and growth stages as well as in cultures of human embryo fibroblasts transformed by oncogenic virus SV-40. It was found that normal fibroblasts maintain at a constant level the collagen synthesis throughout 20 passages, which is typical of proliferating and resting cells. Virus-transformed cells produce 3-4 times less collagen proteins on a per cell count. Normal and transformed fibroblasts do not differ in terms of total protein synthesis. Secretion of collagen and non-collagen proteins in transformed cell cultures appeared to be much lower than in normal cell cultures. Study of synthesized proteins by polyacrylamide gel electrophoresis showed that both types of cells secrete collagen proteins predominantly as polymers containing interchain S-S bonds of 3-helix molecules. Study of the protein-synthesizing activity of two polysomal fractions, i.e. membrane bound and free polysomes, isolated from the cells of both types in a cell-free system showed that membrane-bound polysomes from transformed fibroblasts synthesize collagen much less actively in comparison with normal cells. However, in transformed cells free polysomes, in contrast with normal cells, are active participants of a cell-free collagen protein synthesis.     

不同发育时期小麦种子活力的变化及其对环境温度的响应

以济麦22和山农23号为试验材料,利用标准发芽试验法对不同年份小麦种子发育过程中的种子活力变化进行研究,分析环境温度对不同发育时期小麦种子活力变化的影响,为早期小麦种子的利用及高活力种子的生产提供参考依据.结果表明: 伴随着小麦种子发育,鲜种子在花后26 d左右出现发芽能力,之后其发芽率整体呈上升趋势;干种子发芽势、发芽率和发芽指数在花后5～8 d迅速升高,之后保持相对稳定,活力指数主要受到幼苗单株干质量的影响而持续升高,一般在完熟前4～6 d达到最大值;不同发育时期小麦干种子的田间种植及其后代种子的活力测定表明,济麦22花后17 d以后的干种子田间出苗较好,并可成穗结实,其后代种子的发芽率和活力指数在不同样品间无显著差异.环境温度对不同发育时期小麦种子活力变化的影响显著,小麦花后日平均温度均值、日最高气温均值以及日最低气温均值均高,且花后日温差均值大的年份,种子发育时间短、百粒重及种子活力达到最大值的时间较早;反之,发育时间较长、百粒重及种子活力达到最大值的时间较晚,但完熟期积温高,种子活力较高.     

The influence of abscisic acid on different polysomal populations in embryonal tissue during pea seeds germination

The influence of abscisic acid (ABA) on the processes of formation of different polysomal populations, their structures and stability in embryonal tissue during pea seeds germination was studied. The contents of total ribosomal fraction increased in all samples up to 72 h of germination and then decreased. The contents of polysomal population (FP, MBP, CBP and CMBP) extracted from the embryonal tissue after 72 hrs of germination of pea seeds were then quantified. It turned out that in examined tissue of control sample, fraction of free polysomes (FP) was the most abounded. This population of polysomes in sprouts decreased after ABA treatment. FP content decreased even more when the higher ABA concentration was applied during germination. Similar changes were observed in the fraction of membrane-bound polysomes (MBP). Quite different tendencies were found, however, in forming population of the cytoskeleton-membrane-bound polysomes (CMBP). The CMBP population content in embryonal tissue increased in a dosage dependent manner with increasing concentration of ABA applied during seed germination. This indicates the important role of CMBP fraction in synthesis of specific proteins in embryos in the time when processes of seeds germination are retarded by ABA. In the final part we examined the stability of polysomes isolated from sprouts of germinating seeds in water and sprouts isolated from seeds treated with ABA (100 μM) during germination. Total polysomes isolated from embryonal tissue of germinating seeds treated with ABA showed much higher resistance to exogenous ribonuclease digestion than total polysomes of control sample. The obtained results suggest that ABA influence on different polysomal population formation also controls their stability.     

Translation and the cytoskeleton: a mechanism for targeted protein synthesis

This review describes the critical evidence that in eukaryotic cells polyribosomes, mRNAs and components of the protein synthetic machinery are associated with the cytoskeleton. The role of microtubules, intermediate filaments and microfilaments are discussed; at present most evidence suggests that polyribosomes interact with the actin filaments. The use of non-ionic detergent/deoxycholate treatment in the isolation of cytoskeletal-bound polysomes is described and the conclusion reached that at low salt concentrations this leads to mixed preparations of polysomes derived from both the cytoskeleton and the endoplasmic reticulum. At present the best approach for isolation of cytoskeletal-bound polysomes appears to involve extraction with salt concentrations greater than 130 mM after an initial non-ionic detergent treatment. Such polysomes appear to be enriched in certain mRNAs and thus it is suggested that they are involved in translation of a unique set of proteins. The evidence for mRNA localisation is presented and the role of the cytoskeleton in transport and localisation of RNA discussed. Recent data on the role of the 3 untranslated region in the targeting of mRNAs both to particular regions of the cell and for translation on cytoskeletal-bound polysomes is described. The hypothesis is developed that the association of polysomes with the cytoskeleton is the basis of a mechanism for the targeting of mRNAs and the compartmentalization of protein synthesis.Abbreviations CBP cytoskeletal-bound polysomes - FP free polysomes - MBP membrane-bound polysomes - ER endoplasmic reticulum