Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress

Glycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of certain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild-type plants failed to germinate. At 100 mM NaCl, transformed plants grew well whereas wild-type plants did not do so. The transformed plants tolerated 200 mM NaCl, which was lethal to wild-type plants. After plants had been incubated with 400 mM NaCl for two days, the photosystem II activity of wild-type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild-type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.     

Active Site Mutations in Yeast Protein Disulfide Isomerase Cause Dithiothreitol Sensitivity and a Reduced Rate of Protein Folding in the Endoplasmic Reticulum

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a′, each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a′ domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.     

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 μM, 50% inhibition was observed at 4.8 μM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (K_j = 16 μM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate-fructose-1,6-bisphosphatase active site interaction.     

Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion

Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas.     

Genetic analysis of the yeastCandida maltosa by means of induced parasexual processes

The usefulness of hybridization by protoplast fusion and mitotic segregation for the genetic analysis of the imperfect fodder yeastCandida maltosa was tested. Mitotically stable fusion hybrids were obtained with frequencies between 10^–6 and 10^–7. Complementation tests were performed by protoplast fusion. Substances that are known to induce frequent mitotic segregation in other yeast species such as benomyl, p-fluorophenylalanine, and acriflavine were ineffective inC. maltosa. UV irradiation induced mitotic segregation in up to 10%. This agent induced mainly mitotic crossing over inC. maltosa. Our data enabled the construction of the linkage group I with the sequenceCEN-ade-26-pro-1.     

Corn leaf glutamate synthase: Purification and properties of the enzyme

An assay for ferredoxin-glutamate synthase is introduced thatuses an anion exchange resin to isolate the glutamate formedand subsequent determination with the ninhydrin procedure. Theenzyme was purified 200-fold from corn leaves by ammonium sulfatefractionation and chromatography on DEAE-cellulose, DEAE-Sephaceland ferredoxin- Sepharose. The purified enzyme had a specificactivity of 14 µmoles glutamate formed min^–1mg^–1protein. The enzyme has a molecular weight of 160,000. The pHoptimum for catalytic activity is 6.9. The isoelectric pointis at pH 4.2. The apparent Km values of the enzyme for L-glutamine,2-oxoglutarate and ferredoxin are 1,100, 240 and 1.7 µM.The enzyme has a high specificity toward these substrates witha stoichiometry between glutamate formation and glutamine consumption.Sulfhydryl reagents, bathophenanthroline, phthalein acids andazaserine produced strong inhibition of the enzyme activity. ¹Permanent address: Department of Agricultural Chemistry, KyotoUniversity, Kyoto 606, Japan. ²To whom inquiries should be addressed. (Received July 7, 1979; )     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of Chlamydia lipopolysaccharides.

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.     

The structure of the O-antigenic polysaccharide from lipopolysaccharide of Vibrio cholerae strain H11 (non-O1).

After acid degradation of the lipopolysaccharide (LPS) of Vibrio cholerae strain H11 (non-O1), a tetrasaccharide was obtained, the structure of which was determined by quantitative and methylation analyses, periodate oxidation, one- and two-dimensional NMR spectroscopy, and fast-atom-bombardment and four-sector tandem mass spectrometry as beta-D-GalANGro-(1-3)-beta-D-QuiNAc-(1-4)-alpha-D-GalANGr o-(1-4)-NeuAc, in which GalANGro is N-galacturonoyl-2-aminoglycerol and QuiN 2-amino-2,6-dideoxy-glucopyranose. In addition, the trisaccharide beta-D-GalANGro-(1-3)-beta-D-QuiNAc-(1-4)-D-altro-hept ulose and the disaccharide alpha-D-GalANGro-(1-4)-NeuAc were isolated from acid-degraded lipopolysaccharide; the occurrence of sedoheptulose in lipopolysaccharide has not been described before. Based on the result of methylation analysis showing that galacturonic acid was the terminal sugar of the polysaccharide chain, and on the assumption that the tri- and the disaccharide represented the reducing and the non-reducing ends of the polysaccharide, respectively, the chemical structure of the O-specific chain of V. cholerae H11 is proposed as alpha-D-GalANGro-(1-4)-alpha-NeuAc-(2-3)-beta-D-GalANGro-(1- 3)-beta-D-QuiNAc- (1-[4)-alpha-D-GalANGro-(1-4)-alpha-NeuAc-(2-3)-beta-D-GalANGro -(1-3)-beta-D- QuiNAc-(1-]n-(1-4)-D-altro-heptulose. However, other possible structures can not be ruled out since the tri- and the disaccharide could be localised at different positions.     

On coenzyme Q orientation in membranes: A linear dichroism study of ubiquinones in a model bilayer

Summary A general approach is developed to interpret linear dichroism (LD) spectra of ubiquinones (Q _n) in host bilayers. Information is reported in terms of guest-host mutual orientation and localization. The overall orientational anisotropy of guest ubiquinone molecules is described by a basic set of limiting orientation/localization modes. Assignments of the UV transitions of the ubiquinone chromophore were obtained by the liquid crystal-linear dichroism technique and molecular orbital (CNDO/S) calculations. The LD spectra of Q _n in the bilayers provided by the lyotropic nematic mesophase exhibited by water solutions of potassium laurate and decanol were interpreted on the basis of the above assignments. The resulting experimental evidence showed a multisite distribution in the host bilayer for the aromatic heads of all the investigated Q _n derivatives except Q₀. The orientational distribution suggested by the LD spectra fits the solubilization model recently proposed by G. Lenaz [J. Membrane Biol. (1988) 104:193–209] for ubiquinone in lipid membranes. Within this model Q _n molecules are located in the midplane and their headgroups oscillate transversally across the membrane. Q ₀ instead has a single site location, close to the polar bilayer interface. Experimental evidence that the headgroup carbonyls tend to grasp the polar interface of the host bilayer was also obtained. Orientation and location distributions of Q _n guest molecules are therefore likely to result from the tendency of their aromatic heads to grasp the polar heads of the host bilayer and from the concurrent tendency of their chains to settle into the hydrocarbon host interior.abbreviations AA average absorption - OD, OD optical densities for plane polarized radiations parallel () and perpendicular () to the sample optical axis - OD OD — OD - EPR electron paramagnetic resonance - LC-LD liquid crystal-linear dichroism - LD linear dichroism - LD _r reduced linear dichroism. - MO molecular orbital - N nematic - NMR nuclear magnetic resonance - S _jj order parameters of the directions j of the transition moments of the guest chromophore - S _ii order parameters of the orientational axes i of the guest molecule with respect to the magnetic field - S _ii order parameters of the axes i of the guest molecules with respect to the bilayer axis a - S _a order parameters of the host bilayer axis a with respect to the orienting magnetic field - _j,i deflection angles between the directions j and the axes i - O _i optical factors of the i axis see Eq. (A4)] - Q_n ubiquinone whose isoprenoid chain contains n isoprenoid units Dr. A. Rossi is gratefully acknowledged for the t.e.m. reduction of the spectra. Ubiquinone homologs were kind gifts from Eisai Co., Tokyo, Japan. This work was supported by M.U.R.S.T., and C.N.R. Target Project on Biotechnology and Bioinstrumentation, Rome, Italy.