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J E Mullet

Publications and source records attributed to J E Mullet.

At least 91 records · Page 5Linked to original sources
PubMed1986

Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates.

Protein synthesis in isolated, intact pea chloroplasts was optimized and compared to translation within chloroplasts in vivo. Many polypeptides labeled with [35S]methionine in isolated intact chloroplasts did not comigrate with polypeptides which were labeled within chloroplasts in vivo. Antibodies to the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) immunoprecipitated [35S]-labeled large subunit plus several lower-molecular-mass translation products of isolated chloroplasts. The lower-molecular-mass soluble translation products synthesized in pulse-labeled chloroplasts were converted into full-length large-subunit polypeptides during a subsequent chase period. This result suggests that many of the polypeptides observed in pulse-labeled chloroplasts are incomplete translation products which are the result of ribosome pausing at discrete points along chloroplast mRNAs. The pulse-chase technique was used to follow synthesis of the 34.5-kDa precursor of the psb A gene product and its processing to the mature 32-kDa polypeptide in isolated chloroplasts. Chloroplast translation profiles obtained using the pulse-chase assay were very similar to translation profiles obtained in vivo thus extending the utility of protein synthesis in isolated chloroplasts.

Adenosine Triphosphate↗
PubMed1986

Increased Abscisic Acid Biosynthesis during Plant Dehydration Requires Transcription.

Excised pea plants were rapidly dehydrated to turgor pressures of 1.5 to 2.0 bars. After a 30 minute lag, abscisic acid (ABA) levels increased approximately 100-fold in the dehydrated plants. Pretreatment of plants with the transcription inhibitors actinomycin D or cordycepin or with an inhibitor of cytoplasmic protein synthesis prior to plant dehydration inhibited the synthesis of ABA. These results suggest that dehydration induced synthesis of ABA requires nuclear gene transcription.

Journal Article↗
PubMed1986

Pea chloroplast glutathione reductase: purification and characterization.

Glutathione reductase (EC 1.6.4.2) was purified from intact pea (Pisum sativum) chloroplasts by a method which includes affinity chromatography on ADP-agarose. Fractions from the affinity column which had glutathione reductase activity consisted of polypeptides of 60 and 32 kilodaltons. Separation of the proteins by electrophoresis on native gels showed that glutathione reductase activity was associated with 60 kilodalton polypeptides and not with the 32 kilodalton polypeptides. Antibodies to spinach whole leaf glutathione reductase (60 kilodaltons) cross-react with the chloroplast 60 kilodalton glutathione reductase but not the 32 kilodalton polypeptides. In the absence of dithiothreitol the 60 kilodalton polypeptides showed a shift in apparent molecular weight on sodium dodecyl sulfate gels to 72 kilodaltons. Dithiothreitol did not alter the activity of the chloroplast enzyme. Chloroplast glutathione reductase is relatively insensitive to NADPH.

Journal Article↗
PubMed1985

An in vitro system for accurate transcription initiation of chloroplast protein genes.

We have developed an homologous in vitro system from spinach chloroplasts that correctly initiates transcription of the plastid genes for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) and the beta subunit of the plastid ATPase (atpB). The transcriptionally active extracts from spinach chloroplasts require circular DNA templates for specific initiation. The RNA polymerase activity is insensitive to rifampicin. The extent of transcription in vitro is a function of the extract:template ratio. The efficiency of the rbcL transcription in vitro is more than one transcript per one hundred templates per hour.

Cell-Free System↗
PubMed1985

Accumulation of heat shock proteins in field-grown cotton.

Cotton (Gossypium hirsutum L.) plants grown under field water deficits exhibited an 80 to 85% reduction in leaf area index, plant height, and dry matter accumulation compared with irrigated controls. Midday photosynthetic rates of dryland plants decreased 2-fold, and canopy temperatures increased to 40 degrees C at 80 days after planting compared with canopy temperatures of 30 degrees C for irrigated plants. Leaves from dryland plants which had exhibited canopy temperatures of 40 degrees C for several weeks accumulated stainable levels of polypeptides with apparent molecular weights of 100, 94, 89, 75, 60, 58, 37, and 21 kilodaltons. These polypeptides did not accumulate in leaves from irrigated plants.Addition of [(35)S]methionine to leaves of growth chamber-grown cotton plants and subsequent incubation at 40 degrees C for 3 hours radiolabeled polypeptides with molecular weights similar to those that accumulate in dryland cotton leaves. These data suggest that the proteins which accumulate in water-stressed cotton leaves at elevated temperatures (40 degrees C) are heat shock proteins and that these proteins can accumulate to substantial levels in field-stressed plants.

Journal Article↗
PubMed1983

The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts.

A positively charged amino acid sequence, located on the NH2 terminus of the polypeptides of the chlorophyll a/b light harvesting complex, stabilizes thylakoid membrane adhesion. Threonine residues in this segment are the site of light-induced, reversible phosphorylation; this covalent modification results in changes in excitation-energy distribution in chloroplast membranes. Removal of the positively charged peptide by treatment with trypsin or chemical modification of amino acids in the sequence disrupts thylakoid adhesion and inhibits regulation of excitation-energy distribution. Purified preparations of the chlorophyll a/b light harvesting complex consist of 2 major polypeptides of 27 and 26 kDa and 2 minor polypeptides of 29 and 25 kDa (based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Trypsin treatment of the isolated chlorophyll proteins decreases the apparent molecular mass of the 27- and 26-kDa polypeptides by 1-1.5 kDa and releases 3 peptides; [Lys, Arg], Ser-Ala-Thr-Thr-Lys-Lys, and Ser-Ala-Thr-Thr-Lys. These peptides probably form the overlap sequence, [Lys, Arg]-Ser-Ala-Thr-Thr-Lys-Lys. The polypeptides of the chlorophyll a/b light-harvesting complex were separated by isoelectric focusing into 5 chlorophyll protein fractions which had isoelectric points between 4.0 and 4.55. The 27-kDa polypeptides had an isoelectric point of 4.3, and bound 11 chlorophyll molecules/polypeptide.

Amino Acid Sequence↗
PubMed1982

Optimal conditions for post-translational uptake of proteins by isolated chloroplasts. In vitro synthesis and transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase.

Many polypeptides translated in the cytosol enter the chloroplast where they assemble into macromolecular complexes. The transport of these polypeptides into the plastid can be examined in vitro by mixing isolated chloroplasts with pea poly(A) RNA translation products. Following optimization of both translation in the wheat germ system and the conditions during in vitro uptake, we observe the post-translational transport of over 100 polypeptides; many remain in the soluble phase of the organelle while others integrate into the thylakoid membranes. Most products transported in vitro co-migrate with in vivo products on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, with the improved conditions, we demonstrate the transport of plastocyanin, ferredoxin-NADP+ oxidoreductase, and fructose-1,6-bisphosphatase into isolated plastids. While we have not been able to detect any cell-free translation product that is immunologically related to fructose-1,6-bisphosphatase, both plastocyanin and ferredoxin-NADP+ oxidoreductase are synthesized as precursors in vitro. These precursors are imported into the organelle where they are processed to the size of their mature counterparts. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the precursor to plastocyanin is 15,000 larger than the mature product and the precursor to ferredoxin-NADP+ oxidoreductase is 8,000 larger than the mature product.

Biological Transport↗
PubMed1981

A light-induced spin-polarized triplet detected by EPR in photosystem II reaction centers.

A light-induced spin-polarized triplet state has been detected in a purified Photosystem II preparation by electron paramagnetic resonance spectroscopy at liquid helium temperature. The electron spin polarization pattern is interpreted to indicate that the triplet originates from radical pair recombination between the oxidized primary donor chlorophyll, P-680+, and the reduced intermediate pheophytin, I-, as has been previously demonstrated in bacterial reaction centers. The dependence of the triplet signal on the redox state of I and the primary acceptor, Q, are consistent with the origin of the triplet signal from the triplet state of P-680. Redox-poising experiments indicate the presence of an endogenous donor (or donors) which operates at 3-5 K and 200 K. The zero field-splitting parameters of the triplet are very similar to those of monomeric chlorophyll a however, this alone does not allow a distinction to be made between monomeric and dimeric structures for P-680.

Chloroplasts↗
PubMed1981

Covalent modification of chloroplast photosystem II polypeptides by p-nitrothiophenol.

Illumination of the chlorophyll a/b light-harvesting complex in the presence of p-nitrothio[14C]phenol caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10-13 chlorophylls); 1/2 maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190-247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4-6 polypeptides plus 40-78 chlorophylls per complex. Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of p-nitrothio[14C]phenol caused inhibition of PS II activity and labeling of several polypeptides including those of 42-48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a 'reactive state' in which polypeptides of PS II are susceptible to p-nitrothiophenol modification.

Chlorophyll↗
PubMed1981

Reaction center triplet states in photosystem I and photosystem II.

A photosystem I (PS I) particle has been prepared by lithium dodecyl sulfate digestion which lacks the acceptor X, and iron-sulfur centers B and A. Illumination of these particles at liquid helium temperature results in the appearance of a light-induced spin-polarized triplet signal observed by EPR. This signal is attributed to the triplet state of P-700, the primary donor, formed by recombination of the light induced radical pair P-700+ A1- (where A1 is the intermediate acceptor). Formation of the triplet does not occur if P-700 is oxidized or if A1 is reduced, prior to the illumination. A comparison of the P-700 triplet with that of P-680, the primary donor of Photosystem II, shows several differences. (1) The P-680 triplet is 1.5 mT (15 G) wider than the P-700 triplet. This is reflected by the zero-field splitting parameters, which indicate that P-700 is a slightly larger species than P-680. The zero-field splitting parameters do not indicate that either P-700 or P-680 are dimeric. (2) The P-700 triplet is induced by red and far-red light, while the P-680 triplet is induced only by red light. (3) The temperature dependences of the P-700 triplet and the P-680 triplet are different.

Chloroplasts↗
PubMed1981

Identification of a 32-34-kilodalton polypeptide as a herbicide receptor protein in photosystem II.

Photosystem II particles which retained high rates of herbicide-sensitive activity were used to examine the site(s) of action of various herbicides. A polypeptide of 32-34 kdaltons was identified as the triazine-herbicide binding site based upon: (a) parallel loss of atrazine activity and the polypeptide during either trypsin treatment or selective detergent depletion of protein in the Photosystem II complex, and (b) covalent labeling of the polypeptide by a 14C-labeled photoaffinity triazine. In Photosystem II particles depleted of the 32-34-kdalton polypeptide, electron transport was still active and was slightly sensitive to DCMU and largely sensitive to dinoseb (urea and nitrophenol herbicides, respectively). On the basis of this result it is proposed that the general herbicide binding site common to atrazine, DCMU and dinoseb is formed by a minimum of two polypeptides which determine affinity and/or mediate herbicide-induced inhibition of electron transport on the acceptor side of Photosystem II.

Chloroplasts↗
PubMed1981

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid.

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 A subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 A CF1 are attached by stalks to an aggregate of CF0. The CF1-CF0 complex was incorporated into phospholipid vesicles; freeze-fracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; those averaged 96 A in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF0, was isolated from membranes by butanol extraction and was incorporated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 A in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF0 and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.

Adenosine Triphosphatases↗
PubMed1980

Simulation of grana stacking in a model membrane system. Mediation by a purified light-harvesting pigment-protein complex from chloroplasts.

An isolated light-harvesting pigment-protein complex contains polypeptides which bind chlorophyll a and b. The individual complexes can be purified from detergent-solubilized membranes. The isolated light-harvesting complex, when dialyzed to remove detergents, was examined by freeze-fracture electron microscopy. The material consisted of planar sheets of 80-Angstrom subunits which interacted via an edge-to-edge contact. Addition of cations caused the planar light-harvesting complex sheets to become tightly appressed in multilamellar stacks, with distinct subunits still visible within each lamellar sheet. A transition of particle organization from random to crystalline occurred in parallel with the cation-induced lamellar association. Treatment of the dialyzed light-harvesting complex subunits with low levels of the proteolytic enzyme trypsin removed a 2000 molecular weight segment of the major polypeptide of the light-harvesting complex and blocked all subsequent cation-induced changes in structural organization of the isolated light-harvesting complex lamellar sheets. To gain further evidence for mechanisms of cation effects upon the organization of the light-harvesting complex in native membranes, the light-harvesting complex was incorporated into uncharged (phosphatidylcholine) lipid vesicles. The protein complexes spanned the lipid bilayer and were arranged in either a random pattern or in hexagonal crystalline lattices. Addition of either monovalent or divalent cations to "low-salt" (20 mM monovalent cation) vesicles containing light-harvesting complex caused extensive regions of membrane appresion to appear. It is concluded that this cation-induced membrane appresion is mediated by surface-exposed segments of the light-harvesting complex since (a) phosphatidylcholine vesicles themselves did not undergo cation-induced aggregation, and (b) mild trypsin digestion of the surface-exposed regions of the light-harvesting complex blocked cation-induced lamellar appresion. The particles in the appressed vesicle membranes tended to form long, linear arrays of particles, with occasional mixed quasi-crystalline arrays with an angular displacement near 72 degrees. Surface-mediated interactions among light-harvesting complex subunits of different membranes are, therefore, related to changes in structural organization and interaction of the particles within the lipid phase of the membrane. Numerous previous studies have implicated the involvement of the light-harvesting complex in mediating grana stocking in intact chloro-last membranes. The data presented herein provide a simulation of the membrane appression phenomena using a single class of chloroplast-derived membrane subunits. The data demonstrate that specific surface-localized regions of the light-harvesting complex are involved in membrane-membrane interactions.

Chlorophyll↗
PubMed1980

Chlorophyll proteins of photosystem I.

Data are presented which suggest the existence of a light-harvesting pigment-protein complex which is functionally and structurally associated with photosystem I (PSI) reaction centers. These observations are based on techniques which allow isolation of PSI using minimal concentrations of Triton X-100. Properties of density and self aggregation allowed purification of a "native" PSI complex.The isolated PSI particles appear as 106 A spherical subunits when viewed by freeze fracture microscopy. When incorporated into phosphatidyl choline vesicles, the particles lose self-aggregation properties and disperse uniformly within the lipid membrane.The isolated PSI preparation contains 100 +/- 10 chlorophylls/P(700) (Chl a/b ratio greater than 18); this represents a recovery of 27% of the original chloroplast membrane Chl. These particles were enriched in Chl a forms absorbing at 701 to 710 nm. Chl fluorescence at room temperature exhibited a maximum at 690 nm with a pronounced shoulder at 710 nm. At 77 K, peak fluorescence emission was at 736 nm; in the presence of dithionite an additional fluorescence maximum at 695 nm was obtained at 77 K. This dual fluorescence emission peak for the PSI particles is evidence for at least two Chl populations within the PSI membrane subunit. The fluorescence emission observed at 695 nm was identified as arising from the core of PSI which contains 40 Chl/P(700) (PSI-40). This core complex, derived from native PSI particles, was enriched in Chl a absorbing at 680 and 690 nm and fluorescing with maximal emission at 694 nm at 77 K. PSI particles consisting of the PSI core complex plus 20 to 25 Chl antennae (65 Chl/P(700)) could also be derived from native PSI complexes. These preparations were enriched in Chl a forms absorbing at 697 nm and exhibited a 77 K fluorescence emission maximum at 722 nm.A comparison of native PSI particles which contain 110 Chl/P(700) (PSI-110) and PSI particles containing 65 Chl/P(700) (PSI-65) provides evidence for the existence of a peripheral Chl-protein complex tightly associated in the native PSI complex. The native PSI subunits contain polypeptides of 22,500 to 24,500 daltons which are not found in the PSI-65 or PSI-40 subfractions. It is suggested that these polypeptides function to bind 40 to 45 Chl per structural complex, including the Chl which emits fluorescence at 736 nm.A model for the organization of Chl forms is presented in which the native PSI membrane subunit consists of a reaction center core complex plus two regions of associated light-harvesting antennae. The presence of energy "sinks" within the antennae is discussed.

Journal Article↗
PubMed1980

A developmental study of photosystem I peripheral chlorophyll proteins.

An isolated "native" photosystem I (PSI complex) contains three spectral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of these antennae ( approximately 45 Chl/P(700)) are associated with polypeptides of 21,500 to 24,500 daltons. The present study utilizes two developmental systems to verify this association.Chloroplasts were isolated from a Chl b-less barley mutant and from partially-developed cucumber cotyledons (greened under intermittent illumination [ImL] chloroplasts) and were compared to control chloroplasts isolated from wild-type barley and mature cucumber. Both the mutant and ImL chloroplasts exhibited a long wavelength fluorescence maximum at 724 nanometers at 77 K as compared to 735 to 738 nanometers emission maximum in the respective controls. Both the mutant and ImL chloroplasts were deficient in polypeptides of 21,500 to 24,500 daltons which were present in control membranes and in PSI fractions isolated from control membranes. In light-induced maturation of the ImL cucumbers, the synthesis of polypeptides in the 21,500 to 24,500 molecular weight range paralleled the appearance of PSI Chl species fluorescing at long wavelength ( approximately 735 nm).The PSI spectral properties of the control membranes were retained in isolated PSI particles containing 100 to 120 Chl/P(700) (PSI-110). Detergent extraction of PSI-110 removed polypeptides of 21,500 to 24,500 daltons plus approximately 45 Chl/P(700). The antennae-depleted PSI particle mimics PSI properties exhibited by incompletely differentiated mutant or ImL chloroplasts.

Journal Article↗