Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans

Summary A broad-spectrum mercury resistance locus (mer) from a spontaneous chloramphenicol-sensitive (Cm^s), arginine auxotrophic (Arg^–) mutant of Streptomyces lividan 1326 was isolated on a 6 kb DNA fragment by shotgun cloning into the mercury-sensitive derivative S. lividans TK64 using the vector pIJ702. The mer genes form part of a very large amplifiable DNA sequence present in S. lividans 1326. This element was amplified to about 20 copies per chromosome in the Cm^s Arg^– mutant and was missing from strains like S. lividans TK64, cured for the plasmid SLP3. DNA sequence analysis of a 5 kb region encompassing the whole region required for broad-spectrum mercury resistance revealed six open reading frames (ORFs) transcribed in opposite directions from a common intercistronic region. The protein sequences predicted from the two ORFs transcribed in one direction showed a high degree of similarity to mercuric reductase and organomercurial lyase from other gram-negative and gram-positive sources. Few, if any, similarities were found between the predicted polypeptide sequences of the other four ORFs and other known proteins.     

Cricket phonotaxis: localization depends on recognition of the calling song pattern

Summary Calling song with a carrier frequency of 5 kHz evokes positive phonotaxis in female crickets,Gryllus bimaculatus, when presented at an azimuth. In contrast, a continuous tone of 4.7 kHz in the same position when paired with calling song from above leads to negative phonotaxis. Under open-loop conditions, when a tethered animal runs on a paired tread wheel, characteristic curves are produced with the stable equilibrium point towards or away from the stimulus, respectively (Fig. 3).In order to understand this sign reversal at the neuronal level, directional characteristics of the ascending acoustic inter neurons AN1 and AN2 were measured using extracellular recordings from the cervical connectives.Taking the mean spike rate of the interneurons as a measure for their excitation, the function relating response magnitude to stimulus direction for calling song corresponds well to the behavioural characteristic curve (Fig. 5). The response function obtained using a continuous tone with simultaneous presentation of calling song from above is similar (Fig. 5) and hence does not correspond to the inverse behavioural characteristic curve.However, consideration of the extent to which the temporal parameters of the calling song (syllables and chirps) are reflected in the neuronal response (amplitudes of the Fourier components) leads to characteristic curves for AN1 and AN2 which are in good agreement with the behaviour for stimulation with calling song as well as for the continuous tone experiment (Fig. 8). In addition, the neural response curves correspond to the behaviour in showing smaller amplitudes when a continuous tone rather than the calling song is presented on the horizon (Fig. 8).From these data we conclude that the activity in interneurons AN1 and AN2 does not directly guide orientation in mating behaviour but first is filtered by a mechanism tuned to the frequency of syllables and/or chirps. According to this hypothesis recognition of conspecific song and localization proceed sequentially inGryllus.     

Physical and genetic map of the organomercury resistance (Omr) and inorganic mercury resistance (Hgr) loci of the IncM plasmid R831b

Tn7 insertion mutagenesis has been used to facilitate the generation of a physical (restriction endonuclease) and genetic map of the IncM plasmid, R831b. The only selectable phenotypes carried by this 90-kb conjugative plasmid are resistances to inorganic mercury [Hg(II)] and to organomercury compounds. Mutants in the Hgr locus of R831b complemented previously described mutants in the mer operon of the IncFII plasmid R100, indicating functional homology of the locus in each of these different plasmids. However, the R831b Hgr locus is not notably similar in restriction site pattern to either the mer operon of R100 or the mercury resistance transposon, Tn501. Although the enzymes they encode are co-ordinately regulated, the Omr locus of R831b maps approx. 13.5 kb away from the Hgr locus. Three insertions which affect neither phenotype lie between the Hgr and Omr loci; thus, the loci are separated both physically and genetically. One mutant was obtained which tentatively identifies the position of the Tra locus of R831b as adjacent to the Hgr locus.     

Uptake of the fluorescent indicator atebrin into acidic vacuoles in the halotolerant alga Dunaliella satina

The fluorescent indicator atebrin (3-chloro-9-(4-diethylamino-1-methylbutyl)-7-methyoxy-acridine) is taken up by Dunaliella salina cells at alkaline external pH and accumulates in acidic vacuoles. The uptake is unaffected by light, by photosynthetic inhibitors, by protonophores or by ionophores; however, the dye can be released by amines, indicating that it is specifically accumulating in acidic vacuoles. Amines induce a biphasic enhancement of atebrin fluorescence — a fast phase, accompanied by redistribution within the cell, consistent with release of the dye from the vacuoles to the cytoplasm, and a slow phase, correlated with release of atebrin from the cells. These results are interpreted to indicate a slow equilibration of atebrin across the plasma membrane and a fast equilibration across the vacuolar membrane. Part of the dye cannot be released by the amines, and appears to be internally bound. Atebrin uptake is inhibited by cholesteryl hemisuccinate and is stimulated by lysophosphatidylcholine, indicating that modification of the lipid composition of the plasma membrane affects the permeability to atebrin. Analysis of the pH dependence of atebrin uptake indicates that the dye enters the cells by fluid-phase permeation. Different stresses enhance the rate of atebrin uptake and release, indicating that they modify plasma-membrane structure or composition. Atebrin may serve as a specific marker for acidic vacuoles, as an indicator for amine uptake, and as a probe for subtle changes in the permeability of the plasma membrane.Abbreviations Atebrin 3-chloro-9-(4-diethylamino-1-methylbutyl)-7-methoxy-acridine - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea - SF-6847 3,5-ditertbutyl-4-hydroxybenzylidenemalonitrile     

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca²⁺ uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 M CaCl₂ solution (pH 4.5) for 3 d. Subsequently, net Ca²⁺ fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca²⁺-selective microelectrode. The kinetics of Ca²⁺ uptake into cells of the root apex, for external Ca²⁺ concentrations from 20 to 300 M, were found to be quite similar for both cultivars in the absence of external Al; Ca²⁺ transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 M total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca²⁺ absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca²⁺ uptake over the entire Ca²⁺ concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca²⁺ uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 M) caused the K _m for Ca²⁺ uptake to increase with each rise in Al concentration, from approx. 100 M in the absence of Al to approx. 300 M in the presence of 10 M Al, while having no effect on the V _max. The same Al exposures had little effect on the kinetics of Ca²⁺ uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca²⁺ transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca²⁺-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.We would like to thank Dr. Lionel F. Jaffe, Director of the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA, for making his calcium-selective vibrating-mi-croelectrode system available for a portion of this work. The research presented here was supported in part by USDA/NRI Competitive Grant number 91-37100-6630 to Leon Kochian. Contribution from the USDA-ARS, U.S. Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, N.Y. This research was part of the program of the Center for Root-Soil Research, Cornell University, Ithaca, N.Y. Department of Soil, Crop and Atmosphere Science, paper No. 1741.     

Delineating species for conservation using mitochondrial sequence data: the taxonomic status of two problematic <Emphasis Type="Italic">Bombus</Emphasis> species (Hymenoptera: Apidae)

Across Western Europe and North America, many bumblebee species are currently undergoing drastic declines in their abundance and ranges, primarily as a result of habitat fragmentation. In contrast, a smaller number of species are seemingly unaffected by this and remain common. The UK Biodiversity Action Plan-designated Bombus ruderatus belongs to the former group while B. hortorum belongs to the latter. These two species are sympatric and remarkably similar in morphology. There are no diagnostic characters for workers and male genitalia are illustrated with the same diagram in standard keys. Isolated records of putative B. ruderatus occur amongst a mass of records for B. hortorum. This raises two important issues: first, are B. ruderatus and B. hortorum ‘good’ species? Second, if they are, can the uncertainty over their identification be resolved? We present COII and cytochrome b mtDNA sequence data from these and other Bombus species. Molecular data and coat colour characters are in concordance and confirm that B. ruderatus and B. hortorum should be regarded as separate species (although coat colour alone is an unreliable diagnostic character for many individuals). Confirmation of the specific status of B. ruderatus allows the work on the conservation of this species to continue.     

Studies on the evolution of auxin carriers and phytotropin receptors: Transmembrane auxin transport in unicellular and multicellular Chlorophyta

The characteristics of transmembrane transport of ¹⁴C-labelled indol-3yl-acetic acid ([1-¹⁴C]IAA) were compared in Chlorella vulgaris Beij., a simple unicellular green alga, and in Chara vulgaris L., a branched, multicellular green alga exhibiting axial polarity and a high degree of cell and organ specialization. In Chara thallus cells, three distinguishable trans-plasmamembrane fluxes contributed to the net uptake of [1-¹⁴C]-IAA from an external solution, viz.: a non-mediated, pH-sensitive influx of undissociated IAA (IAAH); a saturable influx of IAA; and a saturable efflux of IAA. Both saturable fluxes were competitively inhibited by unlabelled IAA. Association of [³H]IAA with microsomal preparations from Chara thallus tissue was competitively inhibited by unlabelled IAA. Results indicated that up-take carriers occurred in the membranes at a much higher density than efflux carriers. The efflux component of IAA net uptake by Chara was not affected by several phytotropins (N-1-naphthylphthalmic acid, NPA; 2-(1-pyrenoyl)benzoic acid; and 5-(2-carboxyphenyl)-3-phenylpyrazole), which are potent non-competitive inhibitors of specific auxin-efflux carriers in more advanced plant groups, and no evidence was found for a specific association of [³H]NPA with Chara microsomal preparations. It was concluded that Chara lacked phytotropin receptors. Net uptake of [1-¹⁴C]IAA also was unaffected by 2,3,5-triiodobenzoic acid except at concentrations ( 10^–1 mol · m^–3) high enough to depress cytoplasmic pH (determined by uptake of 5,5-dimethyloxazolidine-2,4-dione). Chlorella cells accumulated [1-¹⁴C]IAA from an external solution by pH-sensitive diffusion of IAA across the plasma membrane and anion (IAA^–) trapping, but no evidence was found in Chlorella for the occurrence of IAA carriers. These results indicate that carrier systems capable of mediating the transmembrane transport of auxins appeared at a very early stage in the evolution of green plants, possibly in association with the origin of a differentiated, multicellular plant body. Phytotropin receptors evolved independently of the carriers.Abbreviations CPP 5-(2-carboxyphenyl)-3-phenylpyrazole - DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid We thank the Nuffield Foundation for the award of an Undergraduate Research Bursary to J.E.D.-F., Dr. G.F. Katekar, C.S.I.R.O., Canberra, Australia for generous gifts of phytotropins, and Mrs. R.P. Bell for technical support.     

Regulation of electron transport in maize mesophyll chloroplasts: The relationship between chlorophyll a fluorescence quenching and O2 evolution

The relationship between the redox state of the primary electron acceptor of photosystem II (Q_A) and the rate of O₂ evolution in isolated mesophyll chloroplasts from Zea mays L. is examined using pulse-modulated chlorophyll a fluorescence techniques. A linear relationship between photochemical quenching of chlorophyll fluorescence (q_Q) and the rate of O₂ evolution is evident under most conditions with either glycerate 3-phosphate or oxaloacetate as substrates. There appears to be no effect of the transthylakoid pH gradient on the rate of electron transfer from photosystem II into Q_A in these chloroplasts. However, the proportion of electron transport occurring through cyclic-pseudocyclic pathways relative to the non-cyclic pathway appears to be regulated by metabolic demand for ATP. The majority of non-photochemical quenching in these chloroplasts at moderate irradiances appeared to be energy-dependent quenching.Abbreviations and symbols PSII photosystem II - Fm maximum fluorescence obtained on application of a saturating light pulse - Fo basal fluorescence recorded in the absence of actinic light (i.e. all PSII traps are open) - Fv Fm-Fo - q_Q photochemical quenching - q_NP non-photochemical quenching - q_E energy-dependent quenching of chlorophyll fluorescence     

The effect of solubilisation on the character of an ethylene-binding site from Phaseolus vulgaris L. cotyledons

The ethylene-binding site (EBS) from Phaseolus vulgaris cv. Canadian Wonder cotyledons can be solubilised from 96,000 g pelleted material by Triton X-100 or sodium cholate. Extraction of 96,000 g pellets with acetone, butanol or butanol and ether results in a total loss of ethylene-binding activity. Like the membrane-bound form, the solubilised EBS has an apparent K_D(liquid) of 10^-10 M at a concentration of 32 pmol EBS per gram tissue fresh weight. Propylene and acetylene act as competitive inhibitors, carbon dioxide appears to promote ethylene binding and ethane has no significant effect. The solubilised EBS is completely denatured affect. The solubilised EBS is completely denatured after 10 min at 70°C, by 1 mM mercaptoethanol and 0.1 mM dithiothreitol, but not by trypsin or chymotrypsin. However, solubilisation decreases the rate constant of association from 10³ M^-1 s^-1 to 10¹–10² M^-1 s^-1 and hence does not permit experimental determination of the rate constant of dissociation. The pH optimum for ethylene binding is altered from the range pH 7–10 in the membrane-bound form to the pH range 4–7 in the solubilised form. The EBS appears to be a hydrophobic, intergral membrane protein, which requires a hydrophobic environment to retain its activity. Partitioning of the EBS into polymer phases is determined by the detergent used for solubilisation indicating that when solubilised, the EBS forms a complex with detergent molecules.Abbreviations EBS ethylene-binding site - PEG polyethylene glycol     

Autoradiographic studies on the role of plasmodesmata in the transport of gibberellin

Translocation of [¹⁴C]gibberellic acid into antheridial cells of Chara vulgaris L. was investigated in relation to the presence of symplasmic connections between the antheridium and the thallus. It was found that manubria, capitular cells, and antheridial filaments were about three-fold more strongly labelled in young antheridia connected to the thallus by plasmodesmata than in older antheridia in which spontaneous symplasmic isolation had occurred. Plasmolytically induced symplasmic isolation of young antheridia severely diminished the radioactivity of all the cells, down to the level characteristic for spontaneously isolated antheridia. It is concluded that plasmodesmata are the main channel of gibberellin transport into antheridia. The change in the character of symplasmic connections during the course of morphogenesis might, among other events, constitute a signal determining a shift of cell metabolism in a new direction, in response to a rapid change in gibberellin level.Abbreviations GA_(n) gibberellin (A_n) - GA₃ gibberellic acid - IAA indole-3-acetic acid This study was supported by the Polish Academy of Sciences research project CPBP 04.01.5.05.     

Effects of temperature on the regulation of photosynthetic carbon assimilation in leaves of maize and barley

The aim of this work was to examine the effect of temperature in the range 5 to 30 ° C upon the regulation of photosynthetic carbon assimilation in leaves of the C₄ plant maize (Zea mays L.) and the C₃ plant barley (Hordeum vulgare L.). Measurements of the CO₂-assimilation rate in relation to the temperature were made at high (735 bar) and low (143 bar) intercellular CO₂ pressure in barley and in air in maize. The results show that, as the temperature was decreased, (i) in barley, pools of phosphorylated metabolites, particularly hexose-phosphate, ribulose 1,5-bisphosphate and fructose 1,6-bisphosphate, increased in high and low CO₂; (ii) in maize, pools of glycerate 3-phosphate, triose-phosphate, pyruvate and phosphoenolpyruvate decreased, reflecting their role in, and dependence on, intercellular transport processes, while pools of hexose-phosphate, ribulose 1,5-bis phosphate and fructose 1,6-bisphosphate remained approximately constant; (iii) the redox state of the primary electron acceptor of photosystem II (Q_A) increased slightly in barley, but rose abruptly below 12° C in maize. Non-photochemical quenching of chlorophyll fluorescence increased slightly in barley and increased to high values below 20 ° C in maize. The data from barley are consistent with the development of a limitation by phosphate status at low temperatures in high CO₂, and indicate an increasing regulatory importance for regeneration of ribulose 1,5-bisphosphate within the Calvin cycle at low temperatures in low CO₂. The data from maize do not show that any steps of the C₄ cycle are particularly cold-sensitive, but do indicate that a restriction in electron transport occurs at low temperature. In both plants the data indicate that regulation of product synthesis results in the maintenance of pools of Calvin-cycle intermediates at low temperatures.Abbreviations Glc6P glucose-6-phosphate - Fru6P fructase-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - PGA glycerate-3-phosphate - p _i intercellular partial pressure of CO₂ - RuBP ribulose-1,5-bisphosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate We thank the Agricultural and Food Research Council, UK (Research grant PG50/67) and the Science and Engineering Research Council, UK for financial support. C.A.L. was supported by the British Council, by the Conselho Nacional de Desenvolvimento Cientiflco e Tecnologico (CNPq), Brazil and by an Overseas Research Student Award. We also thank Mark Stitt (Bayreuth, FRG) and Debbie Rees for helpful discussions.     

The effect of changing the composition of phosphatidylglycerol from thylakoid polar lipids of oleander and cucumber on the temperature of the transition related to chilling injury

The composition and phase behavior of some lipid classes and mixtures of thylakoid polar lipids were measured to investigate their role as determinants of the temperature of the transition associated with chilling injury. For Nerium oleander L., a plant which acclimates to growth temperature, a mixture of the phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG) showed transition temperatures of 22° and 10° C for plants grown at 45° and 20° C, respectively. This difference was similar to the 9 Celsius degrees differential in the transition of the polar lipids and indicated that the PG and-or the PG-SQDG mixture could be the major determinants of the transition temperature. Reconstitution of the PG-SQDG mixture from 20°-grown oleander with the galactolipids from 45°-grown plants, however, reduced the transition temperature by only 4 Celsius degrees. This indicates that some, low-melting-point lipids, which are structurally capable of forming a co-gel with the high-melting-point lipids, also play a role in determining the temperature of the transition and that the composition of these low-melting-point lipids also changes with growth temperature. More specific information on the role of PG was obtained using polar lipids from Cucumis sativus L., a chilling-sensitive plant. For this material the transition in the polar lipids was reduced from 9° to 5° and 4° C when the transition of the PG was reduced from 32° to 25° and 22° C. This was accomplished by reducing the proportion of disaturated molecular species in PG from 78 to 56 and 44 mol% by the addition of a fraction of the PG enriched in unsaturated molecular species. The data indicate that the transition temperature of the polar lipids of cucumber would be reduced to below 0° C, typical of a chillinginsensitive plant, when the transition temperature of PG was reduced to 15° C and this would occur at 21 mol% of disaturated molecular species. It is concluded that the transition in the thylakoid polar lipids, associated with chilling injury, involves both high- and low-meltingpoint lipids but can be reduced when the transition temperature of the high-melting-point component is reduced.Abbreviations DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PG phosphatidylglycerol - SQDG sulfoquinovosyldiacylglycerol     

Branchial and renal handling of urea in the gulf toadfish,Opsanus beta: the effect of exogenous urea loading

The objective of this study was to determine whether the pulsatile facilitated diffusion transport mechanism (tUT) found in the gills of the gulf toadfish (Opsanus beta) and the active secretion transporter thought to be present in its kidney could be saturated when faced with elevated plasma urea concentrations. Toadfish were infused with four consecutive exogenous urea loads at a rate of 0, 150, 300 and 600 micromol kg(-1) h(-1). Initial plasma and urine urea concentrations were 8.1+/-0.9 and 12.4+/-1.5 mmol l(-1), respectively, and steadily increased with increasing infused loads of urea to a maximum of 36.8+/-2.8 mmol l(-1) in the plasma and 39.8+/-6.5 mmol l(-1) in the urine. There was only a very weak relationship (r=0.17) between pulse size (measured as branchial excretion during pulsatile excretion of urea) and plasma urea concentration (slope=9.79 micromol-N kg(-1) per mmol-N l(-1); P<0.05) suggesting that the branchial excretion mechanism was already saturated at normal plasma urea concentrations. Urine flow rate (0.15+/-0.03 ml kg(-1) h(-1)) and glomerular filtration rate (0.025+/-0.004 ml kg(-1) h(-1)) remained constant throughout the experiment despite the increased volume load. Renal urea secretion rate maintained a strong linear relationship (r=0.84) to plasma urea levels (slope=0.391 micromol-N kg(-1) h(-1) per mmol-N l(-1); P<0.001) with no observable transport maximum, suggesting that the renal secretory transport mechanism was not saturated even at plasma urea levels well above normal, in contrast to the branchial excretion mechanism.     

Involvement of polyamines in the inhibiting effect of injury caused by cutting on K+ uptake through the plasma membrane

The inhibition of K⁺ uptake through the plasma membrane resulting from injury caused by cutting, or from application of polyamines (PAs), has been investigated in root segments of maize (Zea mays L.) and pea (Pisum sativum L.). It was found, for both treatments, that K⁺ uptake recovered if the segments were washed for 2 h. The K⁺ uptake inhibited by cutting and that inhibited by spermidine treatment were stimulated to the same extent by fusicoccin. In addition, there was a correlation between the extent of the recovery of K⁺ uptake caused by washing and the distribution, along the root axis, of both PAs and the activities of enzymes responsible for PA degradation. In apical segments of maize, where the PA content and the activity of the degradative enzyme polyamine oxidase (EC 1.5.3.3) were higher than in the more distal segments, the recovery of K⁺ uptake caused by washing was also higher. On the other hand, the opposite trend was observed in root segments of pea, where the PA content and the activity of the degradative enzyme diamine oxidase (EC 1.4.3.6) were higher in distal segments in which K⁺ uptake was greatly stimulated by washing. The effect of the amine-oxidase inhibitor, aminoguanidine, indicates that the degradation products of PAs are involved in the mechanism of inhibition of K⁺ uptake by PAs. The data also seem to indicate that PAs and their degradation products are responsible for the inhibition of K⁺ uptake occurring as a result of injury sustained by cutting roots into segments.Abbreviations DAO diamine oxidase - FC fusicoccin - PA polyamine - PAO polyamine oxidase - PUT putrescine - SPD spermidine     

Diverse effects of formate on the dissimilatory metabolism of nitrate in Pseudomonas denitrificans ATCC 13867: Growth,nitrite accumulation in culture,cellular activities of nitrate and nitrite reductases

The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate.     

Effects of temperature on the activity of phosphoenolpyruvate carboxylase and on the control of CO2 fixation in Bryophyllum fedtschenkoi

The phosphorylation state and the malate sensitivity of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in Bryophyllum fedtschenkoi Hamet et Perrier are altered by changes in the ambient temperature. These effects, in turn alter the in-vivo activity of the enzyme. Low temperature (3 °C or less), stabilizes the phosphorylated form of the enzyme, while high temperature (30 °C) promotes its dephosphorylation. The catalytic activity of the phosphorylated and dephosphorylated forms of PEPCase increases with temperature, but the apparent K_i values for malate of both forms of the enzyme decrease. Results of experiments with detached leaves maintained in darkness in normal air indicate that the changes in malate sensitivity and phosphorylation state of PEPCase with temperature are of physiological significance. When the phosphorylated form of PEPCase is stabilized by reducing the temperature of leaves 9 h after transfer to constant darkness at 15 °C, a prolonged period of CO₂ fixation follows. When leaves are maintained in constant darkness at 15 °C until CO₂ output reaches a low steady-state level and the PEPCase is dephosphorylated, reducing the temperature to 3 °C results in a further period of CO₂ fixation even though the phosphorylation state of PEPCase does not change.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylaseWe thank the Agricultural and Food Research Council for financial support for this work.     

Receptor-mediated endocytosis of storage proteins by the fat body of Helicoverpa zea

The selective uptake of storage proteins by the fat body of the corn earworm Helicoverpa zea is mediated by a membrane-bound receptor protein. In this study, the major storage proteins of this insect species, arylphorin and very high density lipoprotein, were directly labeled with colloidal gold-particles of different size. After the fat body had been incubated with the labeled storage proteins, the distribution of these proteins was examined by electron microscopy. Both storage proteins were found at the extracellular side of coated pits and within coated vesicles. Moreover, fusion products of several coated vesicles such as endosomes and multivesicular bodies contained both proteins in their lumen. Ultimately, the proteins accumulated in electron-dense storage granules. Equal numbers of either storage protein were present in each organelle, supporting the notion that a single receptor mediates the uptake of both proteins. In contrast, only small numbers of gold-labeled immunoglobulin G molecules were found in the organelles, indicating that the protein uptake is specific for storage proteins. The results show that storage protein uptake in this lepidoteran species occurs in a process of receptor-mediated endocytosis that is similar to the well-established uptake of specific proteins into mammalian tissues.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.Abbreviations kDa kilodalton - LHCP-2 light-harvesting chlorophyll a/b-binding protein - M_r relative molecular mass - PSII photosystem II - SDS sodium dodecyl sulphate