Patterns of polypeptide synthesis in various maize organs under anaerobiosis

The pattern of protein synthesis was compared in several organs of maize (Zea mays L.) under aerobic and anaerobic conditions. Protein synthesis was measured by [³⁵S]methionine incorporation and analysis by two-dimensional native-SDS (sodium lauryl sulfate) polyacrylamide gel electrophoresis and fluorography. The aerobic protein-synthesis profiles were very different for root, endosperm, scutellum and anther wall. However, except for some characteristic qualitative and quantitative differences, the patterns of protein synthesis during anaerobiosis were remarkably similar for these diverse organs and also for mesocotyl and coleoptile. The proteins synthesized were the anaerobic polypeptides (ANPs) which have been previously described in anaerobic roots of seedlings. Leaves exhibited no detectable protein synthesis under anaerobic conditions, and died after a short anaerobic treatment. Evidence is presented that the ANPs are not a generalized response to stress. This indicates that the ANPs are synthesized as a specific response to anaerobic conditions such as flooding.Abbreviations ADH alcohol dehydrogenase - ANP anaerobic polypeptide - SDS sodium lauryl sulfate     

Synthesis of unique proteins at the onset of carbon starvation inEscherichia coli

Summary Escherichia coli bulk protein synthesis continued during the first 3–4 h of carbon starvation at 50–75% that of non-starved (growing) cells. Two-dimensional gel electrophoresis analysis of in vivo pulse-labelled proteins resolved at least 30 polypeptides with new or increased synthesis, relative to total protein synthesis, during this time. Among these polypeptides were several that were also synthesized by ethanol-treatedE. coli (heat-shock proteins). In addition, a number of unique polypeptides were synthesized by carbon-starved cells. These starvation proteins may be involved in survival of the starving bacteria.     

Variation in mitochondrial translation products in fertile and cytoplasmic male-sterile sugar beets

Summary Intact and functional mitochondria were isolated from sugar beet plants (Beta vulgaris L.) containing normal fertile (F) or cytoplasmic male-sterile (S₁–S₄) cytoplasms. Incorporation of ³⁵S-methionine by mitochondria isolated from both roots and leaves showed approximately 20 major and ten minor translation products. Comparison of the polypeptide synthesis patterns produced by leaf mitochondria from fertile plants of three different species within the genus Beta revealed several taxonomically related differences. Contrary to this, the patterns of polypeptides synthesized by mitochondria from roots and leaves of sugar beet plants containing the F and S₁–S₄ cytoplasms were very similar; in the S₁ and S₂ cytoplasms no qualitative, and only a few quantitative, differences from the F cytoplasm were observed. Thus, in these cases, cytoplasmic male sterility in sugar beet is not correlated with the constitutive expression of variant polypeptides. In the S₃ cytoplasm, however, an additional 6 kDa polypeptide was synthesized and in the S₄ cytoplasm an additional 10 kDa polypeptide was observed when compared with the F cytoplasm. The expression of cytoplasmic male sterility in sugar beet may be associated with these variant polypeptides. The mitochondrial polypeptides synthesized were identical in plants with different nuclear backgrounds but with identical S₁ cytoplasms. Mitochondria from plants with variants of the S₄ cytoplasm in the same nuclear genotype also showed identical patterns of polypeptide synthesis, including the synthesis of the 10 kDa S₄-specific polypeptide. Pulse-chase experiments did not affect the synthesis of this polypeptide.     

Protein synthesis in Petunia hybrida chloroplasts isolated from leaves and cell cultures

Isolation and incubation conditions were established for Petunia hybrida chloroplasts capable of performing in vitro protein and RNA synthesis. Under these conditions, chloroplasts from leaves as well as from the non-photoautotrophic mutant green cell culture AK-2401 are able to incorporate labeled amino acids into polypeptides. Intact chloroplasts can use light as an energy source; photosynthetically-inactive chloroplasts require the addition for ATP for this protein synthesis. Sodium dodecylsulphate polyacrylamide slab gel electrophoresis shows that in isolated leaf chloroplasts at least twenty-five radioactive polypeptide species are synthesized. The three major products synthesized have molecular weights of 52,000, 32,000 and 17,000. Coomassie brilliant-bluestained polypeptide patterns from plastids isolated from the mutant green cell culture AK-2401 differ considerably from those obtained from leaf chloroplasts. The pattern of radioactive polypeptides synthesized in these isolated cell culture plastids also shows differences. These results indicate that the difference in developmental stage observed between plastids from the cell culture AK-2401 and leaves is reflected in an altered expression of the chloroplast DNA.Abbreviations CAP D-threo-chloramphenicol - 2,4-D 2,4-dichlorophenoxyacetic acid - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecylsulphate     

Patterns of protein synthesis during the germination of pea axes,and the effects of an interrupting desiccation period

As germination of axes of Pisum sativum L. seeds progressed, profound quantitative and qualitative changes occurred in the patterns of protein synthesis. This was shown by fluorography of gels following two-dimensional polyacrylamide gel electrophoresis separation of [³⁵S]methioninelabelled proteins. The effects of desiccation during germination on these in-vivo protein-synthesis patterns were followed. Desiccation differentially affected the synthesis of proteins. Usually, however, upon rehydration following desiccation the types of proteins being synthesized were recognizable as those synthesized earlier during imbibition of control, once-imbibed axes: seeds imbibed for 8 h, and then dried, did not recommence synthesis of proteins typical of 8-h-imbibed control seeds, but rather of 4-h-imbibed control seeds. Seeds imbibed for 12 h, and then dried and rehydrated, synthesized proteins typical of 4-h-and 8-h-control seeds. Thus drying of germinating pea axes caused the proteinsynthesizing mechanism to revert to producing proteins typical of earlier stages of imbibition. Drying during germination never caused the seed to revert to the metabolic status of the initial mature dry state, however.Abbreviation DR dried and rehydrated     

Detection of haemocyte proteins in the integument of the developing Mediterranean fruit flyCeratitis capitata

Summary Studies of the synthesis of integumental proteins during the feeding and non-feeding stages ofCeratitis capitata demonstrated stage specificity. The synthetic profile changed dramatically, showing a maximum of protein synthesis just before the larval wandering stage, followed by an abrupt decline. The comparison between synthetic and accumulation profiles indicated that some polypeptides must be internalized into the integument from the haemolymph. The major haemolymph proteins or arylphorins have already been documented to be incorporated into the integument. In the present work, we demonstrated the interalization of some haemocyte proteins into the integument. For that purpose, polyclonal antibodies were raised against total haemocyte proteins. Immunoblot analysis of haemocyte salt extractable proteins revealed that the protein bands at 36, 54, 58, 84, 110 and 130 kDa were immunoreactive with the total haemocyte antibodies. Cell-free protein synthesis, organ culture experiments and immunoblot analysis indicated that the 36-, 54- and 58-kDa polypeptides were synthesized only in the haemocytes and were probably internalized into the integument from the serum. The 36-kDa polypeptide was also demonstrated to be internalized into the fat body of white puparia. The immunofluorescence experiments suggested that the internalization of haemocyte proteins first occurs into the epidermal cells and then into the cuticle. The presence of haemocyte proteins in the integument was also demonstrated by immunofluorescence experiments in twoC. capitata mutants. These mutations affect the darkening and stiffening of the cuticle. The demonstration of 36-, 54- and 58-kDa haemocyte polypeptides in the integument reveals a hitherto unknown function of this cell type. Moreover, the demonstration of tyrosine binding to the 54- and 58-kDa polypeptides points to their potential involvement in the sclerotization process in the cuticle.     

Synthesis of ribulose 1,5-bisphosphate carboxylase by isolatedSorghum mesophyll chloroplasts

Chloroplasts isolated fromSorghum vulgare are active in light-dependent, organelle protein synthesis. Intact chloroplasts can use light as an energy source; photosynthetically inactive chloroplasts require the addition of ATP for this protein synthesis. Preincubation of chloroplasts in light at 25°C for 1 h depleted the endogenous templates completely; such preincubated chloroplasts translated exogenously added heterologous templates efficiently. When total cellular RNA fromChlorella protothecoides, a C₃ plant, was used as template for translation in a cell-free light-dependent system of isolated mesophyll chloroplasts fromSorghum vulgare, a C₄ type plant, polypeptides of 55 kDa (large subunit) and 15 kDa (small subunit) were detectable in the fluorographic profile of the newly synthesized proteins; these polypeptides were absent in the products obtained with endogenous RNA. Evidence for the fidelity of the system was obtained by immunological analysis of ribulose 1, 5-bisphosphate carboxylase obtained by the translation ofChlorella cellular RNAs.     

Synthesis and secretion of salivary gland proteins in Drosophila gibberosa during larval and prepupal development

Summary The late larvae of Drosophila gibberosa Patterson and Mainland choose different pupariation sites than the larvae of Drosophila melanogaster Meigen. Since the larvae of D. gibberosa do not attach themselves to the substratum, the salivary glands contain only a small amount of the glue proteins before pupariation. Proteins comprising the salivary gland secretions of late larvae of these two species were compared and found to be qualitatively quite different. Only five polypeptides with the same molecular masses were identified in both species. The rate of protein synthesis in the salivary glands of D. gibberosa continued to increase through the late larval stage and pupariation. As a consequence, the total amount of protein contained in the salivary glands also continued to increase after pupariation. To demonstrate temporal changes in protein synthesis from 48 h before pupariation to 28 h after pupariation, newly synthesized polypeptides were pulse labeled by culturing salivary glands in vitro. The patterns of polypeptide synthesis fell into four major groups depending upon whether the synthesis of a protein stopped shortly after pupariation, stopped during late pupariation, increased at pupariation, or was initiated after pupariation. Changing patterns of protein synthesis are correlated with the known changes in gene puffing during this developmental period.     

Patterns of protein synthesis in the imaginal discs of Drosophila melanogaster: a comparison between different discs and stages

High-resolution two dimensional gel electrophoresis has been used to study the patterns of protein synthesis in imaginal discs of Drosophila melanogaster. In this paper we first compare the patterns of protein synthesis in wing, haltere, leg 1, leg 2, leg 3 and eye antenna imaginal discs of late third instar larvae. We have detected only quantitative changes: differences in 17 proteins among the different imaginal discs. In addition, we have analysed the variations in pattern of proteins in the wing disc of the last larval stage and early pupae as well as in wing discs cultured in vivo for 6 days. Variations in these patterns affect more than 20% of the proteins and involve both qualitative and quantitative changes. Some of the changes may correspond to protein phosphorylation. Correlations of these changes between discs and through development are also discussed. Correspondence to: F. Santaren     

Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum

Bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum are enclosed by a plant-derived peribacteriod membrane (PBM). The contents of the interstitial peribacteroid space (PBS) between bacteroid membrane and PBM were isolated by a controlled osmotic shock of PBM-enclosed bacteroids and analysed by two-dimensional gel electrophoresis. Silver staining revealed approximately 40 PBS polypeptides. Ex planta ³⁵S-methionine labeling of PBM-enclosed bacteroids revealed that about 90% of the PBS proteins are synthesized by the bacteroid. Approximately 30% of the PBS polypeptides are common between the PBS and the periplasmic space of free-living bacteria; one (38kDa) PBS protein is also excreted by free-living bacteria in the bacterial culture medium. At least four bacteroid-encoded PBS polypeptides were clearly identified as symbiosis-specific.     

Regulation of Protein Synthesis during Photomorphogenesis of Gametophytes of the Fern Onoclea sensibilis

Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with [³⁵S]methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation.     

Patterns of post-infectional protein synthesis in barley carrying different genes for resistance to the powdery mildew fungus

Summary Pairs of susceptible and resistant, near-isogenic cultivars ofHordeum vulgare which differ for the Mla, Mlk and Mlp genes for resistance toErysiphe graminis f. sp.hordei were inoculated with race 3 of this pathogen and patterns of protein synthesis associated with primary infection mapped using pulse-labelling with L-[³⁵S]methionine and 2-dimensional electrophoresis. Extraction of proteins with buffer containing detergent revealed the enhanced synthesis of 5 and 8 polypeptides at 25 and 30 h respectively after inoculation of barley carrying the Mla gene (cvMla). The enhanced synthesis of these same polypeptides together with 11 additional polypeptides was observed at 48 h and 72 h after inoculation of barley carrying either the Mlp (cvMlp) or Mlk (cvMlk) genes. The labelling of several major constitutive polypeptides was suppressed in cvMla at 24 h after inoculation; the labelling of six of these polypeptides was also suppressed in both cvMlp and cvMlk but not until 48 and 72 h after inoculation. These results indicate that changes occur in the synthesis of some common polypeptides following infection of cultivars carrying different resistance genes but the timing and extent of these changes varies with the resistance gene in the host.     

Cloning and functional annotation of rare mRNA species from drought-stressed hot pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

To better understand gene expression at very low levels, we have designed a method to eliminate cDNA clones representing abundant mRNAs. A cDNA library for drought-stressed hot pepper (Capsicum annuum) (Choi et al., 2002) underwent double-negative screening, once with probes made from a drought-stressed plant, the second time, with probes from a non-stressed plant. The cDNA clones that showed very weak or negative signals were isolated for further analysis, which resulted in 1399 cDNA clones from about 20,000 screened clones. When nucleotide sequences were determined, we obtained 1142 tentative unique genes, with a redundancy rate of 20.41%. An homology database search for the deduced amino acid sequences revealed that about 79% of the cDNA clones could not be matched for functioning with previously characterized sequences. However, when these uncategorized clones were subjected to classification based on functional domains, most could be cited. Notably, clones with possible functions in RNA transport, protein synthesis, and regulation of protein activity showed a dramatic increase in appearance while those coding for transposable elements, viral proteins, and plasmid proteins occupied a much smaller portion compared with those in theArabidopsis thaliana genome. In addition, those coding for proteins targeted to the endoplasmic reticulum were dramatically more abundant in our clones compared with theArabidopsis database.     

Protein polymorphism in sugarcane revealed by two-dimensional gel analysis

Summary In order to identify molecular markers for the analysis of the sugarcane genome, proteins extracted from apical segments of shoot tissues were resolved by a combination of equilibrium (IEF) and nonequilibrium (NEPHGE) two-dimensional polyacrylamide gel electrophoresis. A number of taxa of the Saccharum complex group (Saccharum species and the related genera of Andropogoneae) with presumed contributions to the sugarcane genome were surveyed. Protein profiles were compared to a reference map consisting of 1,482 protein spots from the noble cane,Saccharum officinarum L. Fifty-three polypeptides, representing about 3.6% of the total resolved spots, showed interspecific variation, whereas 78 polypeptides, about 5.3% of the total, showed intergeneric variation. Of the total polymorphic protein spots, qualitative (presence/absence) variation was more prevalent among the wild than among the cultivated species of the genusSaccharum, but the quantitative (spot intensity) variation was similar for both groups. The population of protein spots showing qualitative and quantitative variations was similar among the related genera of Andropogoneae. These polymorphic proteins can be used in genetic and evolutionary studies of the sugarcane genome.     

Localized synthesis of specific proteins during oogenesis and early embryogenesis inDrosophila melanogaster

Summary Protein synthesis in egg follicles and blastoderm embryos ofDrosophila melanogaster has been studied by means of two-dimensional gel electrophoresis. Up to 400 polypeptide spots have been resolved on autoradiographs. Stage 10 follicles (for stages see King, 1970) were labelled in vitro for 10 to 60 min with³⁵S-methionine and cut with tungsten needles into an anterior fragment containing the nurse cells and a posterior fragment containing the oocyte and follicle cells. The nurse cells were found to synthesize a complex pattern of proteins. At least two proteins were detected only in nurse cells but not in the oocyte even after a one hour labelling period. Nurse cells isolated from stages 9, 10 and 12 follicles were shown to synthesize stage specific patterns of proteins. Several proteins are synthesized in posterior fragments of stage 10 follicles but not in anterior fragments. These proteins are only found in follicle cells. No oocyte specific proteins have been detected. Striking differences between the protein patterns of anterior and posterior fragments persist until the nurse cells degenerate. In mature stage 14 follicles, labelled in vivo, no significant differences in the protein patterns of isolated anterior and posterior fragments could be detected; this may be due to technical limitations. At the blastoderm stage localized synthesis of specific proteins becomes detectable again. When blastoderm embryos, labelled in vivo, are cut with tungsten needles and the cells are isolated from anterior and posterior halves, differences become apparent. The pole cells located at the posterior pole are highly active in protein synthesis and contribute several specific proteins which are found exclusively in the posterior region of the embryo. In this study synthesis of specific proteins could only be demonstrated at those developmental stages which are characterized by the presence of different cell types within the egg chamber, while no differences were detected when stage 14 follicles were cut and anterior and posterior fragments analyzed separately. The differences in the pattern of protein synthesis by pole cells and blastoderm cells indicate that even the earliest stages of determination are reflected by marked changes at the biochemical level.     

Commitment to vegetalized development in sea urchin embryos

Summary Strongylocentrotus purpuratus embryos were reared in 0.025 M LiCl, which causes commitment to vegetalized development within 5 h after treatment begun at fertilization. Treated and control embryos were labelled with³⁵S-methionine for 3 h intervals from 2–14 h, solubilized, and subjected to 2-dimensional polyacrylamide gel electrophoresis. Comparison of autoradiographs of the gels, in which over 400 proteins can be detected, indicate that while LiCl treatment causes a short delay in the initiation or cessation of synthesis of a few proteins, no qualitative or major quantitative differences can be detected between control and treated embryos. Normal gastrulae and vegetalized exogastrulae labelled 38 h after fertilization have several differences in patterns of protein synthesis. We conclude that the early determinative events involved in vegetalization are not reflected in detectable differences in the pattern of protein synthesis.     

Protein analysis during the ontogeny of normal and male sterile stamenless-2 mutant stamens of tomato (Lycopersicon esculentum Mill.)

The levels and synthesis of proteins during the ontogeny of normal and male sterile stamenless-2 (sl-2/sl-2) mutant stamens of tomato (Lycopersicon esculentum) were examined. The mutant stamens contained low levels of soluble protein which were related to reduction in protein synthesis. The mutant stamens, however, possessed many polypeptides similar to the normal and synthesized a 53-kd polypeptide at stages when there are abnormalities in tapetum development. The mutant stamens also possessed a 23-kd and some low molecular weight polypeptides that were considered as degradative proteins. Normal stamens exhibited the synthesis of many polypeptides not found in the mutant, from microspore mother cell to the preanthesis stages. In addition, at the time of pollen maturation there was a greater synthesis of several polypeptides, particularly those of 42 and 37 kd. Although the causative mechanisms of male sterility in the sl-2/sl-2 mutant are not known, the synthesis, and the lack, of specific polypeptides reported here appears to be associated with pollen degeneration.This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to V.K.S.     

Changes in protein and amino acid content during anther development in fertile and cytoplasmic male sterile Petunia

Summary Development of anthers in cytoplasmic male sterile (CMS) Petunia diverges from the normal sequence of events early in meiosis. Quantitative and qualitative changes in morphology, proteins and free amino acid contents correlate with this divergence. In anthers of the fertile line (5719), total protein content increases, and SDS-PAGE protein patterns change as the anthers mature. Enhanced levels of three polypeptides with molecular weights of 64,000, 63,000 and 45,000 daltons characterize premeiosis in fertile anthers. Protein levels and patterns from anthers of the CMS line (5707) show little alteration during anther development. Protein synthesis seems to be at least partially blocked in the CMS microspore. The 63,000 and 45,000 dalton proteins are not present, and the absence of any unique protein(s) in the CMS line argues against a virus as the causal agent of CMS in Petunia. Analysis of free amino acids from anthers of the fertile line shows levels of proline and pipecolic acid 2–3 and 10–20 fold higher, respectively, than in the CMS line. The amino acids incorporated into proteins show no such differences; analysis of protein hydrolysates shows similar levels of each amino acid in both fertile and CMS lines at every developmental stage examined.     

Effects of 2,4-D removal on the synthesis of specific proteins by Catharanthus roseus cell cultures

Total and neosynthesized proteins of periwinkle cell suspensions (Catharanthus roseus) were first investigated in cells grown in a 2,4-D-containing medium. Analysis of total (silver-stained) proteins by two-dimensional gel electrophoresis revealed that the levels of seventeen polypeptides were altered during the growth cycle of the cells. Analysis of in vivo [³⁵S]-methionine labeled polypeptides revealed differences in the synthesis of at least 35 polypeptides. Three polypeptides with molecular masses of 30, 35 and 39 kDa appeared to be specific markers of the early stationary phase. In a second sequence of experiments, cells were grown in a 2,4-D-free medium. Alterations in protein synthesis were observed: several polypeptides were expressed earlier in the 2,4-D-starved cells than in control cells; the synthesis of at least two specific polypeptides was increased in cells grown in 2,4-D-free medium, whereas the synthesis of three other polypeptides (molecular masses 33, 34 and 52.5 kDa) was switched on in these cells. As previous studies showed that 2,4-D depletion increased the alkaloid production in C. roseus cells, the present results may suggest that these polypeptides are implicated in the regulation of the alkaloid pathway.