Heat-shock proteins during pollen development in maize

In contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat-shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat-shock responses. One-dimensional Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis technique (SDS-PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat-shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat-stress conditions; however, the consti-tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.     

Polypeptide induction by recombinant human IFN-gamma

Partially purified natural human IFN-gamma has been shown to induce the synthesis of a set of unique polypeptides that are not induced by comparable amounts of IFN-alpha and IFN-beta. In addition, a set of polypeptides is induced in common by all three IFNs. The present study utilized a pure recombinant IFN-gamma and a monoclonal antibody against IFN-gamma to investigate whether the unique polypeptide-inducing properties previously reported are due to IFN-gamma itself. With the possible exception of two polypeptides, the results demonstrate conclusively that IFN-gamma is the sole molecular species responsible for the induction of all polypeptides, including those whose induction pattern is unique to IFN-gamma. Very high doses of IFN-alpha and IFN-beta induce some of the latter set of polypeptides to a limited extent. Thus the differential effects of IFN-gamma compared to IFN-alpha and IFN-beta are, in part, quantitative rather than qualitative in nature.     

Membrane- and cell wall-associated heat shock proteins in two genotypes of barley seedlings

ABSTRACT

The heat-shock (HS) response of two genotypes (cv. Onice, winter type, and cv. Georgie, spring type) of barley (Hordeum vulgare L.) was compared. Protein synthesis was markedly reduced by HS in roots and coleoptiles of both genotypes. The reduction in cv. Onice was higher than in cv. Georgie. The pattern of cytosolic, membrane and cell wall HSPs was analysed by SDS-PAGE in coleoptiles and roots of the two genotypes. Differences and similarities in coleoptiles and roots of the same genotype and between the two genotypes were observed. In roots of the genotype Onice, LMW and HMW HSPs isolated from the cytosol, membranes and cell walls were resolved into a diverse array of polypeptides by two-dimensional gel electrophoresis. Present results confirm the de novo synthesis of cytosolic and membrane HSPs, and demonstrate, for the first time, their presence in cell walls.     

A dual role of 20-hydroxyecdysone in the control of protein synthesis related to DNA puff activity in the anterior region of Bradysia hygida (Diptera, Sciaridae) salivary gland

During the last 30 h of the larval stage, the salivary glands of Bradysia hygida show the amplification of some genes, resulting in the formation of two successive groups of DNA puffs, which direct the synthesis of two different sets of polypeptides. Incubation of anterior (S1) salivary gland regions, at age E7, beginning of first group of DNA puffs activity, in culture medium for 2 to 10 h results in a decrease in the synthesis of the polypeptides characteristic of this period. However, during subsequent incubation (from E7 to E7+12 h-24 h), when the second group of DNA puffs is active, S1 regions were able to synthesize some polypeptides characteristic of this period. The role of 20-OH ecdysone was studied, in vitro and in vivo, during these two periods of protein synthesis in S1 regions. The presence of the hormone was shown to be necessary to maintain, in vitro, the synthesis of the first set of polypeptides and was strongly inhibitory, in vitro and in vivo, to the synthesis of the second set of polypeptides. Thus, it is likely that the activity of the two distinct groups of DNA puffs is under opposite 20-OH-ecdysone control mechanisms.     

Proteomic Analysis of Allergen and Antinutritional Proteins in Wild and Cultivated Soybean Seeds

In this study, profiles of allergen and antinutritional proteins both in wild (Glycine soja) and cultivated (Glycine max) soybean seeds were compared. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the separation of proteins at two different pH ranges and applied a combined matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS) analysis for the identification of proteins. Although overall distribution patterns of the allergen (Gly m Bd 60K, Gly m Bd 30K, Gly m Bd 28K) and antinutritional proteins (trypsin inhibitors and lectin) appeared similar, there was remarkable variation in the number and intensity of the protein spots between wild and cultivated genotypes. The wild genotype showed fifteen polypeptides of Gly m Bd 60K and three polypeptides of trypsin inhibitors. The cultivated genotypes showed twelve polypeptides of Gly m Bd 60K and two polypeptides of trypsin inhibitors. In contrast, the cultivated genotype showed two polypeptides of Gly m Bd 30K and three polypeptides of lectin and the wild genotype showed two and one polypeptides of Gly m Bd 30K and lectin, respectively. Two polypeptides of Gly m Bd 28K were observed in both genotypes. This is the first study reporting the comparative analysis of allergen and antinutritional proteins in both wild and cultivated soybean genotypes using combined proteomic tools.     

Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli.

We investigated the regulation of the synthesis of the eight polypeptides of the Escherichia coli proton-translocating ATPase. A plasmid carrying the eight genes of the unc operon was used to direct in vivo and in vitro protein synthesis of the eight polypeptides. Analysis of these data indicates that the ATPase polypeptides are synthesized in unequal amounts both in vitro and in vivo. We identified several regions within the unc operon at which expression of a gene is either increased or decreased from that of the preceding gene. Since genetic information indicates a single polycistronic mRNA for all eight genes of this operon, the observed differential synthesis of the polypeptides is most likely the result of translational regulation. The effect of varying the temperature suggests that the secondary structure in the mRNA may affect the rate of translation initiation in the region between uncE and uncF.     

Regulation by ABA of osmotic-stress-induced changes in protein synthesis in tomato roots

Polypeptide synthesis and accumulation were examined in the roots of tomato seedlings exposed to a polyethylene glycol‐imposed water deficit stress. In these roots, the synthesis of a number of polypeptides was induced, while that of several others was enhanced or repressed. To examine the role played by abscisic acid (ABA) in co‐ordinating the accumulation of these proteins, water‐deficit‐stress‐responsive polypeptide synthesis was investigated in the roots of the ABA‐deficient mutant flacca. In the roots of this mutant, the ability to accumulate a complete set of water‐deficit‐stress‐responsive polypeptides was impaired, indicating that ABA is required for their synthesis. The role of ABA was further examined by exposing the roots of both genotypes to exogenous ABA, which, with one exception, elicited the accumulation of all water‐deficit‐stress‐responsive proteins. Polyethylene glycol‐induced polypeptide accumulation was accompanied by a 1·6‐fold increase in the level of endogenous ABA in the roots of wild‐type plants and a 5‐fold increase in the roots of flc. Thus, although the absolute level was lower than that of the wild‐type, flc has the capacity to accumulate ABA in its roots. When fluridone was used to prevent the biosynthesis of ABA, the accumulation of several water‐deficit‐stress‐responsive polypeptides was reduced further. The synthesis of polypeptides was also examined in the roots of salt‐treated seedlings. Salt altered the accumulation of several polypeptides, all of which were previously observed in water‐deficit‐stressed roots, indicating that their synthesis was the result of the osmotic component of the salt stress. However, the accumulation of these polypeptides was not impaired in flc roots, indicating that the role played by ABA in regulating their accumulation in salt‐and polyethylene glycol‐treated roots differs. As such, salt‐ and water‐deficit‐stress‐induced changes in gene expression may be effected by different mechanisms, at least at the level of polypeptide accumulation.     

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     

Differential translation in normal and adenovirus type 5-infected human cells and cell-free systems.

When uninfected or adenovirus 5-infected KB cells are exposed to hypertonic medium, the incorporation of radioactive amino acids into protein decreases in both, but more severely in the uninfected cells. Although the effect of hypertonic medium on the synthesis of specific polypeptides varies, the translation of viral polypeptides as a class is less inhibited. The same patterns of proteins are synthesized regardless of the solute used in the hypertonic medium. The mechanism by which hypertonic conditions exert their effect on whole cells was investigated in K cell-free systems. It was possible to simulate the differential patterns of protein synthesis obtained in whole cells in hypertonic medium by increasing ion concentrations in cell-free extracts which are capable of initiating polypeptide chains on exogenous templates. However, in cell lysates which only elongate proteins, the same patterns were not obtained. Certain host and viral polypeptides displayed striking responses to increased ionic conditions in whole cells and cell-free systems. The synthesis of a host 44K protein, actin, appeared to be most sensitive; lower-molecular-weight proteins were fairly resistant. Among the viral proteins, the synthesis of 100K was inhibited, but most notable was the marked resistance of the synthesis of polypeptide IX. Possible mechanisms for differential synthesis and their significance are considered.     

Protein synthesis in chloroplasts. VIII. Differential synthesis of chloroplast proteins during spinach leaf development

Excised primary leaves of spinach (Spinacia oleracea) incorporate [35S]-methionine into a number of chloroplast polypeptides. The ratio of incorporation of isotope into the large subunit of ribulose bisphosphate carboxylase relative to a thylakoid polypeptide (peak D) decreases during leaf development in whole leaves; this changing pattern of incorporation is also observed in isolated chloroplasts where these two polypeptides are the major products of protein synthesis. Chloroplast RNA prepared from developing leaves was translated in a reticulocyte lysate extract to yield full-length carboxylase large subunit and peak D polypeptides. The fidelity of translation of these two polypeptides was checked by partial protease digestion. Changes in the synthesis of the large subunit of the carboxylase and peak D in developing leaves are reflected in changes in the amount of translatable mRNA for these two polypeptides.     

Structural and functional similarities of delta-crystallin messenger ribonucleic acids from duck and chicken lenses

delta-Crystallin of the embryonic duck lens was compared with that of the embryonic chicken lens with respect to polypeptide composition, synthesis, and messenger ribonucleic acid (mRNA) sequences. Labeling experiments with [35S]methionine revealed that the duck delta-crystallin is composed of minor amounts of polypeptides with molecular weights near 50000 (50K) and 49000 (49K) and much greater amounts of polypeptides with molecular weights near 48000 (48K) and 47000 (47K), as judged by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. All four sizes of polypeptides were synthesized in similar relative proportions as found in vivo in a rabbit reticulocytes lysate supplemented with delta-crystallin mRNA isolated from the embryonic duck lens. Synthesis of the 48K and 47K delta-crystallin polypeptides was differentially reduced in duck lenses cultured in the presence of ouabain. This is similar to the differential reduction of synthesis of the lower molecular weight delta-crystallin peptides in embryonic chicken lenses demonstrated previously. R loops formed between duck or chicken delta-crystallin mRNA and a cloned chicken delta-crystallin cDNA and heteroduplexes formed between duck or chicken delta-crystallin mRNA and cloned chicken genomic DNAs containing delta-crystallin sequences showed that, except for the putative 5' leader sequence, the duck and chicken delta-crystallin mRNAs have extremely similar nucleotide sequences. These data indicate considerable conservation of delta-crystallin throughout the approximately 100 million years of divergence between ducks and chickens. The findings also suggest a possible relationship between the structure of delta-crystallin mRNA and the differential reduction in synthesis of the lower molecular weight delta-crystallin polypeptides in ouabain-treated lenses of ducks and chickens.     

Characterization of protein synthesis by isolated rice mitochondria

Summary Bacteria-free mitochondria were isolated from aseptically grown, etiolated and green seedlings of both cytoplasmic male-sterile (WA-type) and male-fertile rice (Oryza sativa L.). Protein synthesis in these isolated mitochondria was characterized by gel electrophoresis/fluorography and by the incorporation of [³⁵S]-methionine into protein. In the presence of cycloheximide, a set of some 25 discrete polypeptides and an electrophoretically unresolved population were synthesized. This pattern of protein synthesis in organello was essentially the same in mitochondria isolated from both male-fertile and malesterile cytoplasms. Our data does not preclude the possibility, however, that the WA-type CMS possesses a tissue-specific and/or a low abundance mitochondrial protein(s), whose synthesis eluded detection under our experimental conditions. The synthesis of the mitochondria-encoded polypeptides by isolated rice mitochondria was inhibited by chloramphenicol and incompletely inhibited by erythromycin. A minor chloramphenicol-insensitive, cycloheximide-sensitive translation activity was found consistently to copurify with the mitochondria. This activity generated a reproducible electrophoretic profile of a poorly resolved, weakly labelled population of polypeptides and of a few conspicuous polypeptides, including a 42 kDa species.     

Protein profiling in F<Subscript>1</Subscript> and F<Subscript>2</Subscript> generations of two tomato genotypes differing in ripening time

Pericarp polypeptide profiles were analyzed at three ripening stages in the F₁ hybrid and the F₂ population from the cross between the accessions: LA1385 (Lycopersicon esculentum var. cerasiforme) and 804627 (L. esculentum, a homozygous genotype for the nor mutant). Six polymorphic polypeptides were observed in LA1385, while no polymorphic polypeptides among ripening stages was observed in 804627. On the other hand, some polypeptides in the F₁ hybrid were not observed in the parents whereas others were present in both parental genotypes and were unnoticeable in the hybrid genotype. From a cluster analysis on the protein profiles of the F₂ population, the differential expression of proteins allowed to distinguish mature green (MG) stage from the others two stages, while for breaker stage (BR) and red ripe stage, the genetic background was more important in forming groups. The differential expression of proteins could be associated with fruit morphology traits such as a 72 kDa polypeptide present in MG stage with fruit diameter, height and mass and a 47 kDa polypeptide found in BR with fruit shelf life.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes.

We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.