Nitrate-induced changes in protein synthesis and translation of RNA in maize roots

Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [³⁵S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate-induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [³⁵S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vicinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots.     

Identification of a receptor protein in cotton fibers for the herbicide 2,6-dichlorobenzonitrile

The herbicide 2,6-dichlorobenzonitrile (DCB) is an effective and apparently specific inhibitor of cellulose synthesis in higher plants. We have synthesized a photoreactive analog of DCB (2,6-dichlorophenylazide [DCPA]) for use as an affinity-labeling probe to identify the DCB receptor in plants. This analog retains herbicide activity and inhibits cellulose synthesis in cotton fibers and tobacco cells in a manner similar to DCB. When cotton fiber extracts are incubated with [³H]DCPA and exposed to ultraviolet light, an 18 kilodalton polypeptide is specifically labeled. About 90% of this polypeptide is found in the 100,000g supernatant, the remainder being membrane-associated. Gel filtration and nondenaturing polyacrylamide gel electrophoresis of this polypeptide indicate that it is an acidic protein which has a similar size in its native or denatured state. The amount of 18 kilodalton polypeptide detectable by [³H]DCPA-labeling increases substantially at the onset of secondary wall cellulose synthesis in the fibers. A similar polypeptide, but of lower molecular weight (12,000), has been detected upon labeling of extracts from tomato or from the cellulosic alga Chara corallina. The specificity of labeling of the 18 kilodalton cotton fiber polypeptide, coupled with its pattern of developmental regulation, implicate a role for this protein in cellulose biosynthesis. Being, at most, only loosely associated with membranes, it is unlikely to be the catalytic polypeptide of the cellulose synthase, and we suggest instead that the DCB receptor may function as a regulatory protein for β-glucan synthesis in plants.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Identification of Proteins Correlated with Increased Freezing Tolerance in Bromegrass (Bromus inermis Leyss. cv Manchar) Cell Cultures

Cellular and extracellular protein profiles from Bromus inermis Leyss. cv Manchar cell suspension cultures cold hardened by low temperature and abscisic acid (ABA) treatment were analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cellular proteins (25, 165, 190, and 200 kilodaltons) increased by low temperature growth and cellular proteins (20, 25, 28, 30, 32, 37, 40, 45, 200 kilodaltons) increased by exogenous ABA treatment were identified. Low temperature treatment inhibited the synthesis of a 22 kilodalton protein and ABA treatment resulted in the synthesis of two extracellular proteins (17 and 21 kilodaltons). Low temperature and ABA-induced hardening conditions increased or induced a 25 and a 200 kilodalton protein. The 25 and a 30 kilodalton protein previously shown to be enriched by ABA-induced hardening conditions at both 3 and 23°C temperatures co-fractionated with the crude membrane fraction (30,000g sediment). The 200 kilodalton protein was detected in the 30,000g supernatant. Two-dimensional analysis of the crude membrane fraction resolved the 30 kilodalton protein band into a major polypeptide with an apparent isoelectric point of 6.85.     

Ethylene Biosynthesis-Inducing Xylanase : II. Purification and Physical Characterization of the Enzyme Produced by Trichoderma viride

The ethylene biosynthesis-inducing endoxylanase (EIX) from xylan-induced cultures of the fungus, Trichoderma viride, was purified to near homogeneity and compared with the EIX isolated from Cellulysin. Both enzymes migrate as 9.2 kilodalton proteins during gel filtration chromatography under nondenaturing conditions, but the mature polypeptide migrates as a 22 kilodalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the 22 kilodalton polypeptide is enriched by Gly, Ser, Thr, Trp, and Tyr, but depleted in Ala, Glx, Leu, and Lys. Both proteins lack sulfur-containing amino acids. The protein is glycosylated, and inhibition of EIX synthesis by tunicamycin suggests that at least some of the sugar moieties are linked to asparagine residues. EIX appears to be synthesized initially as a 25 kilodalton precursor protein that is processed to 22 kilodalton during secretion.     

Protein synthesis associated with quiescence and senescence in auxin-starved pear cells

Pear fruit cells (Pyrus communis L. cv Passe Crassane) stopped dividing when subcultured in a bioreactor under auxin starvation in the presence of 0.37 molar mannitol. The cessation of cell division was preceded by the accumulation of a specific basic polypeptide of 24 kilodalton. Readdition of 2.3 micromolar 2,4-dichlorophenoxyacetic acid (2,4-D) neither caused a resumption of cell division nor depressed the accumulation of this polypeptide. Under complete auxin starvation, cells began to die at day 18. In vivo radioactive labeling of proteins followed by two-dimensional electrophoresis showed that during auxin starvation the synthesis of some polypeptides including the 24 kilodalton one (referred to as homeostasis-related proteins, HRPs) was decreased while the synthesis of some others (referred as senescence-related proteins, SRPs) was increased. Readdition of 2.3 micromolar 2,4-D postponed the onset of cell death by 10 to 15 days while supplementation with 7.6 micromolar abscisic acid advanced cell death by 8 days. Two-dimensional analysis of protein synthesis indicated that both hormones interact on the synthesis of these two groups of polypeptides. The levels of most HRPs were maintained or increased in the presence of auxin, while the levels of the SRPs were decreased by auxin and increased by abscisic acid. Short and long-term effects of 2,4-D and abscisic acid on the synthesis of specific polypeptides were observed, allowing a discrimination between the direct and indirect effect of both hormones on the development of cell senescence.     

A New Chloroplast Protein Is Induced by Growth on Low CO(2) in Chlamydomonas reinhardtii

The biosynthesis of a 36 kilodalton polypeptide of Chlamydomonas reinhardtii was induced by photoautotrophic growth on low CO₂. Fractionation studies using the cell-wall-deficient strain of C. reinhardtii, CC-400, showed that this polypeptide was different from the low CO₂-induced periplasmic carbonic anhydrase. In addition, the 36 kilodalton polypeptide was found to be localized in intact chloroplasts isolated from low CO₂-adapting cultures. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO₂-grown C. reinhardtii cells.     

The Water Oxidation Complex of Chlamydomonas: Accumulation and Maturation of the Largest Subunit in Photosystem II Mutants

Photosystem II particles of Chlamydomonas reinhardtii contain three extrinsic polypeptides of 29, 20, and 16 kilodaltons, whose functions are incompletely defined. We prepared a monospecific polyclonal antibody against the 29 kilodalton protein and determined that it also specifically recognizes a protein of approximately 33 kilodaltons in thylakoid membrane fractions of several vascular plants, eukaryotic algae, and a cyanobacterium. The cross-reacting 33 kilodalton protein of pea was removed from inverted thylakoid vesicles by CaCl₂ washes demonstrating the structural relationship between the Chlamydomonas polypeptide and the largest subunit of the water oxidation complex of vascular plants. Functional identity of the Chlamydomonas polypeptide was confirmed by antibody inhibition of O₂ evolution in inverted pea vesicles. In contrast to wild-type cells, only low levels of the 29 kilodalton polypeptide are recovered with purified thylakoid membranes of the mutants examined. However, we show that the mature form of the 29 kilodalton polypeptide accumulates to wild-type levels in whole cell extracts of photosystem II deficient mutants and a water oxidation mutant of Chlamydomonas. Impaired membrane assembly has no effect on the maturation or stability of this component of the multi-subunit water oxidation complex.     

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures

Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of −20°C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [³⁵S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants.     

Biosynthesis of storage proteins in developing rice seeds

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10- to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76- and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.

The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with [¹⁴C]leucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kilodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.

In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.

     

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [γ-³²P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P₂, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [γ-³²P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [³²P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [γ-³²P]ATP or [³²P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.     

A 36 Kilodalton Limiting-CO(2) Induced Polypeptide of Chlamydomonas Is Distinct from the 37 Kilodalton Periplasmic Carbonic Anhydrase

Chlamydomonas reinhardtii possesses a CO₂-concentrating mechanism, induced by limiting CO₂, which involves active transport and accumulation of inorganic carbon within the cell. Synthesis of several proteins is induced by limiting CO₂, but, of those, only periplasmic carbonic anhydrase has an identified function in the system. No proteins involved in active transport have yet been identified, but induced, membrane-associated polypeptides, such as the 36 kilodalton polypeptide focused on in this paper, would seem to be candidates for such involvement. The 36 kilodalton polypeptide was shown to be synthesized de novo upon transfer of cells to limiting CO₂. It was purified using SDS-PAGE and used to produce polyclonal antibodies. Antibodies were used to confirm the air-specific nature of the polypeptide, its strict association with membrane fractions, and the time course of its induction. Using the antibodies, a single, 36 kilodalton polypeptide was found to be specifically immunoprecipitated from in vitro translation products of poly(A⁺) RNA from cells only after exposure to limiting CO₂. The absence of translatable mRNA for this polypeptide in CO₂-enriched cells indicated that regulation occurs at the level of message abundance. The antibodies were also used to demonstrate the distinction between the limiting-CO₂ induced 36 kilodalton polypeptide and the similarly sized, limiting-CO₂ induced periplasmic carbonic anhydrase.     

Photosystem II in Guard Cells of Vicia faba: Immunological Detection

Antibodies were raised against individual polypeptides of the oxygen-evolving photosystem II (PSII) complex from mesophyll chloroplasts of Vicia faba (Long Pod). These antibodies were used to probe immunologically for the presence of the main structural components of the PSII complex in guard cell chloroplasts, using both immunofluorescence microscopy and Western blotting. Immunofluorescence of epidermal peels with antibodies raised against the extrinsic 33 kilodalton polypeptide, as well as the 47 and the 44 kilodalton subunits and the light-harvesting chlorophyll a/b protein, resulted in intense fluorescence indicating the presence of these polypeptide components in guard cell chloroplasts. Results obtained with Western blot analysis showed that the relative amounts of the 33 kilodalton and light-harvesting complex protein polypeptides are between 60 and 80% of that found in mesophyll cells (on chlorophyll basis). These results provide evidence for the existence of structural components associated with PSII activity in guard cell similar to those of mesophyll chloroplasts.     

Envelope synthesis during the cell cycle in Escherichia coli B/r

The rate of synthesis of envelope proteins and phospholipids during the cell cycle of Escherichia coli B/r has been studied using both synchronous cultures and random cultures, first labelled and then subsequently fractionated on an age basis by the membrane elution technique. The rate of total protein synthesis and of phospholipid synthesis, measured by incorporation of [2-³H]glycerol into whole cells, was found to increase exponentially throughout the cell cycle. Total envelope protein was also synthesized continuously throughout the cycle, but the rate of synthesis showed a stepwise pattern with a discrete doubling in rate in the first half of the cycle. Analysis of the pattern of synthesis of about 29 individual envelope polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography revealed that the great majority followed the pattern of the bulk measurements, with a discrete increase in rate of synthesis early in the cycle. One envelope polypeptide, molecular weight 76,000, was, however, only synthesized during a brief period, near the time of division of the bacteria. Pulse-chase studies of envelope polypeptide synthesis in synchronous cultures demonstrated that (1) synthesis and insertion of polypeptide into the envelope was always completed within the pulse period; (2) no post-synthetic modification of polypeptides was detected; (3) one group of polypeptides, including a major outer membrane protein, maintained a stable association with the envelope, whilst a second group displayed considerable “turnover”; (4) about 70% of newly synthesized 76,000 molecular weight protein was lost from the envelope during the succeeding generation.     

Protein phosphorylation: Localization in regenerating optic axons

A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration (Larrivee and Grafstein, 1989). (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of [³H]proline and³²P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the [³H]proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the³²P label, suggesting that phosphorylation was largely independent of synthesis. (2) To deterine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced [³H]proline labeling of total protein by 88% and³²P labeling by 49%. Among the individual proteins [³H]proline labeling was reduced by 90% or more in 18 cases but³²P labeling was reduced only by 50% or less. (3) When³²P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.Abbreviations SDS sodium lauryl sulfate - GAP growth associated protein - TCA trichloracetic acid - kD kilodalton     

Novel light-regulated chloroplast thylakoid membrane protein

A 64 kilodalton chloroplast membrane polypeptide was dependent on growth irradiance with 10-fold greater quantities of the protein present in barley (Hordeum vulgare) grown under 500 micromoles of photons per square meter per second compared with growth at 50 micromoles per square meter per second. The concentration of the protein was sensitive to changes in irradiance, with a slow time course for the response (days) similar to other reported light acclimation processes. The polypeptide also was observed in maize (Zea mays), oats (Avena sativa), and wheat (Triticum aestivum), but not in soybean (Glycine max Merr). The 64 kilodalton polypeptide did not correspond to any thylakoid membrane protein with an assigned function, so its structural or regulatory role is not known.     

Mode of action of abscisic Acid in barley aleurone layers : induction of new proteins by abscisic Acid

As part of a continuing effort to elucidate the mode of action of abscisic acid (ABA) in barley (Hordeum vulgare L. cv Himalaya) aleurone layers, we have investigated the induction of several polypeptides by ABA in this tissue. There were nine ABA-induced polypeptides as observed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and considerably more (at least 16 spots) on a two-dimensional gel. These proteins started to show enhanced synthesis 2 to 4 hours after ABA treatment, and their synthesis continued for at least 48 hours. In vitro translation using total RNA isolated from ABA-treated aleurone layers indicated that translatable mRNA levels of these proteins essentially paralleled the levels of in vivo synthesized proteins. The most abundant of the ABA-induced proteins was a 29 kilodalton polypeptide which was also synthesized in tissue incubated without ABA. In vivo synthesis of this protein declined as ABA concentration was decreased, with 1 nanomolar ABA approaching control level. Cell fractionation experiments located the 29 kilodalton major ABA-induced protein in 1,000g and 13,000g pellets; most other induced proteins were in the 80,000g supernatant. The 29 kilodalton protein appeared to be sensitive to degradation by sulfhydryl type proteases. As expected, the induction of these proteins by ABA was suppressed by gibberellic acid. Phaseic acid, the first stable metabolite of ABA, suppressed the gibberellic acid-enhanced α-amylase synthesis but was unable to induce the ABA-induced proteins. None of the ABA-induced proteins were secreted into the incubation medium. A 36 kilodalton ABA-induced protein showed cross-reactivity with antibody against a barley lectin specific for glucosamine, galactosamine, and mannosamine.     

Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development

Oat (Avena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [³⁵S]sulfate into protein. Incorporation of [³⁵S]sulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the α and β globulin subunits were present 2 to 4 DPA, but the full complement of globulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis.     

Purification of an h-translocating inorganic pyrophosphatase from vacuole membranes of red beet

An H⁺-translocating inorganic pyrophosphatase (PPase) was isolated and purified from red beet (Beta vulgaris L.) tonoplast. One major polypeptide of molecular weight 67 kilodalton copurified with fluoride-inhibitable PPase activity when subjected to one-dimensional polyacrylamide gel electrophoresis. Overall, a 150-fold purification of the PPase was obtained, from the tonoplast fraction, through anion exchange chromatography of the detergent-solubilized membranes followed by ammonium sulfate precipitation and gel filtration chromatography. The purified polypeptide showed no cross-reactivity with antibodies raised against the 67 kilodalton subunit of the tonoplast ATPase.