Expression of wild-type GBSS transgenes in the off-spring of partially and fully complementedamylose-free transformants of potato

Theamylose-free (amf) potato mutant can easily be complemented through introduction of the wild-type gene coding for granule-bound starch synthase (GBSS). After iodine staining the starch of theamf mutant is red whereas that of the wild type and the complementedamf mutant is blue. The level of complementation of selected transformants and their sexual off-spring after backcrossing withamf was investigated using sporophytic tuber cells and gametophytic microspore cells. Two diploid and two tetraploid transformants with full complementation demonstrated the expected segregation patterns of 1:1 (one active insert) or 3:1 (two independently segregating active inserts) in the microspores and in the F1 offspring based on staining of tubers. All expected genotypes in the F1 generation were found, based on microspore segregation patterns of the individual F1 plants. Two transformants with partial complementation (mixed phenotypes) were investigated. One of them, B1, was tetraploid and duplex for the GBSS insert, which had originated through mitotic doubling of the transformed diploid cells. In the F1 generation three phenotypic classes were found:amf, fully complemented and partially complemented. The latter two classes exist independently of a simplex or duplex gene status. The second transformant with partial complementation, B10, appeared to have a complex molecular composition. One cluster of five transgenes caused the partial complementation. Fully and partially complemented phenotypic classes were found after crossing B10 with theamf mutant. Indications were found that the ploidy level of the tissue in which the genes were introduced and expressed played an important role. Firstly, partial complementation was found after transformation of the diploid and not of the tetraploidamf genotypes. Secondly, the level of complementation was higher in tissue with lower ploidy levels, as illustrated by the colour of the starch inin vitro tubers (2x–4x cells) versus field-grown tubers (16x–64x).     

Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: Competition with H2PO 4 −

The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F₁ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H₂PO ₄ ^– is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15M). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theV _max, and an increase in theK _m for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H₂PO ₄ ^– is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H₂PO ₄ ^– interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F₁ remains at about 40% of the activity of the control in a concentration range of TFP of 40–100M, whereas in oxidative phosphorylation 14M TFP produces a 60% inhibition of phosphate uptake.     

Sucrose metabolism during endosperm development in wheat (Triticum aestivum)

This work reports changes in sucrose synthase and invertase activities throughout endosperm development in wheat, together with the associated substrates and metabolites, sucrose, UDP, glucose, fructose and UDP-glucose. Throughout endosperm development, sucrose synthase had consistently higher activity than invertase and indeed invertase activity did not change appreciably. The observed variation in pattern and amounts of glucose and fructose present during the mid- and late stages of endosperm development confirmed the suggestion that invertase was not the preferred pathway of sucrose catabolism. Kinetic parameters for sucrose synthase were determined in crude extracts. Estimates of UDP and sucrose concentrations suggest that sucrose synthase is unlikely to achieve its potential maximum velocity. This limitation may however be overcome in part by the apparent excess catalytic activity measured during endosperm development.     

Differential sensitivity of plant-associated bacteria to sulfonylurea and imidazolinone herbicides

The side effects of sulfonylurea and imidazolinone herbicides on plant-associated bacteria were investigated under pure culture conditions. Eighteen isolates, belonging to the genera Azotobacter, Azospirillum, Bacillus, Enterobacter Pseudomonas and Serratia, were exposed to four active compounds at concentration ranges similar to those in field soil. The sulfonylureas chlorsulfuron and rimsulfuron inhibited the growth of one of two Azospirillum and one of four Pseudomonas strains, while the imidazolinones imazapyr and imazethapyr were effective on two out of five Bacillus isolates. Surfactants in commercial formulation significantly enhanced rimsulfuron toxicity. With the exception of one Azospirillum strain, the differential tolerance of rhizobacteria to these herbicides was related to a differential sensitivity of their target, the activity of the first enzyme in branched-chain amino acid biosynthesis, acetohydroxyacid synthase (AHAS).Greenhouse pot studies were performed to assess the occurrence of inhibitory effects on bacterial growth in field conditions. Maize seedlings were bacterized with the two strains which had shown in vitro sensitivity to sulfonylureas. Following the application to the soil of a commercial formulation of rimsulfuron at rates of 0, 0.2 and 0.5 mol a.i. kg^–1, significative differences in the resulting degree of bacterial root colonization were found. Moreover, upon co-inoculation with two strains, one tolerant and one sensitive to the herbicide, the presence of rimsulfuron significantly enhanced root occupancy by resistant bacteria, suggesting that shifts in the microbial community structure of crop rhizosphere could indeed result as a consequence of weed control by AHAS inhibitors.Abbreviations AHAS acetohydroxyacid synthase - CETAB cetyltrimethylammonium bromide - ID₅₀ concentration causing 50% inhibition of enzyme activity - LD₅₀ concentration causing 50% decrease of growth constant value     

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163–168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.     

The effect of elevated levels of thyroxine on the aerobic capacity of locomotor muscles of the tufted duck,Aythya fuligula

Following extended periods of relative inactivity, or prior to migration, birds are able to increase the aerobic capacity of their locomotory muscles. Thyroid hormones may influence this process. A preliminary study was undertaken to assess the ability of elevated levels of thyroxine to increase the aerobic capacity of the locomotory and cardiac muscles of adult tufted ducks. Administration of thyroxine in the food for 8 weeks had little effect on body mass or on the masses of the pectoralis, semitendinosus and iliofibularis muscles, although there were increases in resting oxygen consumption and in the mass of the cardiac ventricles. The maximum activity of the aerobic enzyme, citrate synthase, was significantly greater in the left ventricle, liver, and iliofibularis muscles (P<0.005) of treated birds. However, while there was clearly no difference in activity in the semimembranosus leg muscle, that of the pectoralis was not quite significant (P=0.078). It is concluded that addition of supra-physiological levels of exogenous thyroxine may induce a differential increase in the maximum activity of citrate synthase in the locomotor muscles of the tufted duck, which is correlated with the fibre type composition of these muscles. These results are consistent with those found in studies on rats, with slow oxidative fibres being the most sensitive, and fast glycolytic fibres the least sensitive, to thyroxine treatment.Abbreviations BM body mass - CS citrate synthase - CYTOX cytochrome c oxidase - FG last glycolytic - FOG fast oxydative glycolytic - VO₂ oxygen consumption - SO slow oxidative - T₄ thyroxine - T₃ triiodothyronine     

Lack of coupling between secondary structure formation and collapse in a model polypeptide that mimics early folding intermediates, the F2 fragment of the Escherichia coli tryptophan-synthase beta chain.

The isolated, 101-residue long C-terminal (so called F2) fragment of the beta chain from Escherichia coli tryptophan synthase was shown previously to fold into an ensemble of conformations that are condensed, to contain large amounts of highly dynamic secondary structures, and to behave as a good model of structured intermediates that form at the very early stages of protein folding. Here, solvent perturbations were used to investigate the forces that are involved in stabilizing the secondary structure (monitored by far-UV CD) and the condensation of the polypeptide chain (monitored by dynamic light scattering) in isolated F2. It was observed that neither the ionic strength, nor the pH (between 7 and 10), nor salts of the Hofmeister series affected the global secondary structure contents of F2, whereas some of these salts affected the collapse slightly. Addition of trifluoroethanol resulted in a large increase in both the amount of secondary structure and the Stokes radius of F2. Conversely, F2 became more condensed upon raising the temperature from 4 to 60 degrees C, whereas in this temperature range, the secondary structure undergoes significant melting. These observations lead to the conclusion that, in isolated F2, there is no coupling between the hydrophobic collapse and the secondary structure. This finding will be discussed in terms of early events in protein folding.     

Neural and endothelial nitric oxide synthase activity in rat penile erectile tissue

NADPH-diaphorase (NADPH-D) activity and immunoreactivity for neural and endothelial nitric oxide synthase (nNOS and eNOS, respectively) were used to investigate nitric oxide (NO) regulation of penile vasculature. Both the histochemical and immunohistochemical techniques for NOS showed that all smooth muscles regions of the penis (dorsal penile artery and vein, deep penile vessels, and cavernosal muscles) were richly innervated. The endothelium of penile arteries, deep dorsal penile vein, and select veins in the crura and shaft were also stained for NADPH-D and eNOS. However, the endothelium of cavernous sinuses was unstained by both techniques. Fewer fibers were seen in the glans penis, those present being associated with small blood vessels and large nerve bundles near the trabecular walls. All penile neurons in the pelvic plexus, located by retrograde transport of a dye placed in the corpora cavernosa penis, were stained by the NADPH-D method. Essentially similar results were obtained with an antibody to nNOS. These data suggest that penile parasympathetic neurons comprise a uniform population, as all seem capable of forming nitric oxide. However, in contrast to the endothelium of penile vessels, the endothelium lining the cavernosal spaces may not be capable of nitric oxide synthesis.