Tn10 mutagenesis in Azotobacter vinelandii

Summary The tetracycline-resistant transposon Tn10 and its high-hopper derivative Tn10HH104 were introduced into the Azotobacter vinelandii genome using suicide conjugative plasmids derived from pRK2013. Several types of mutants induced by either of these elements are described. Nif^- mutants (deficient in nitrogen fixation) were easily isolated, whereas the isolation of other mutant types (auxotrophs, sugar non-users) required special selection conditions. The characterization of the mutations as transposon insertions was often complicated and sometimes required a combination of genetic and physical tests. A common source of complication, the existence of double inserts, was found among the mutants induced by Tn10HH104 but not among those induced by Tn10. Both the high-hopper and the wild-type element proved to undergo secondary transpositions, albeit at different frequencies. Another type of complication, the existence of heterozygotes, occurred because of the high level of redundancy of the A. vinelandii genome.     

Further characterization of ribosome binding to thylakoid membranes

Previous work indicated more polysomes bound to pea (Pisum sativum cv Progress No. 9) thylakoids in light than in the dark, in vivo (LE Fish, AT Jagendorf 1982 Plant Physiol 69: 814-825). With isolated intact chloroplasts incubated in darkness, addition of MgATP had no effect but 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus, the major effect of light on ribosome-binding in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus, cycling of ribosomes is controlled by translation, initiation, and termination. Bound RNA accounted for 19 to 24% of the total chloroplast RNA and the incorporation of [³H]leucine into thylakoids was proportional to the amount of this bound RNA. These data support the concept that stroma ribosomes are recruited into thylakoid polysomes, which are active in synthesizing thylakoid proteins.     

Consequence of Absence of Nitrate Reductase Activity on Photosynthesis in Nicotiana plumbaginifolia Plants

Chlorate-resistant Nicotiana plumbaginifolia (cv Viviani) mutants were found to be deficient in the nitrate reductase apoprotein (NR⁻nia). Because they could not grow with nitrate as sole nitrogen source, they were cultivated as graftings on wild-type Nicotiana tabacum plants. The grafts of mutant plants were chlorotic compared to the grafts of wild type. Mutant leaves did not accumulate nitrogen and nitrate but contained less malate and more glutamine than wild leaves. They exhibited a slight increase of the proportion of the light-harvesting chlorophyll a/b protein complexes and a lowering of the efficiency of energy transfer between these complexes and the active centers. After a 3 second ¹⁴CO₂ pulse, the total ¹⁴C incorporation of the mutant leaves was approximately 20% of that of the control. The ¹⁴C was essentially recovered in ribulose bisphosphate in these plants. It was consistent with a decline of ribulose bisphosphate carboxylase activity observed in the mutant. After a 3 second ¹⁴CO₂ pulse followed by a 60 second chase with normal CO₂, ¹⁴C was mainly accumulated in starch which was labeled more in the mutant than in the wild type. These results confirm the observation that in the nitrate reductase deficient leaves, chloroplasts were loaded with large starch inclusions preceding disorganization of the photosynthetic apparatus.     

Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit

We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green `Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO₂ evolution by 400 micrograms per gram fresh weight galactose. 1-Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol.     

Biphasic fluence-response curves for phytochrome-mediated kalanchoë seed germination : sensitization by gibberellic Acid

The fluence-response curves for the effect of two red pulses separated by 24 hours on the germination of Kalanchoe blossfeldiana Poelln. cv Vesuv seeds, incubated on gibberellic acid (GA₃) are biphasic for suboptimal concentrations. The response in the low fluence range corresponds with a classical red/far-red reversible phytochrome mediated reaction. GA₃ induces an additional response in the very low fluence range, which is also phytochrome mediated. The sensitivity to phytochrome-far-red absorbing form (Pfr), however, is increased about 20,000-fold, so that even far-red fluences become saturating. Both in the very low and low fluence response range, the maximal responses induced by saturating fluences are modulated by the GA₃ concentration. GA₃ having no direct influence on the phytochrome phototransformations, alters the Pfr requirement and determines the responding seed population fraction in the very low and low fluence range. The effet of GA₃ appears to be on the transduction chain of the phytochrome signal.     

Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

Inhibition of pear fruit ripening by mannose

Softening of the flesh and the rise in ethylene evolution and respiration associated with ripening in pear (Pyrus communis L.) fruit was delayed when mannose was vacuum infiltrated into intact fruit. The extent of delay could be modified by altering the concentration or the volume of mannose applied to the fruit. Inhibition of ripening was associated with phosphorylation of mannose to mannose 6-phosphate (M6P), and accumulation of M6P was associated with lowered levels of inorganic phosphate (Pi), glucose 6-phosphate (G6P), and ATP in the fruit tissue. Subsequently, however, as the M6P was metabolized, the levels of Pi, G6P, and ATP increased and ripening processes were concomitantly released from inhibition. Hence, the degree of inhibition by mannose or the release from inhibition was related to the level of M6P in the fruit and its rate of metabolism. The data provide correlative evidence to support a view that one inhibitory effect of mannose is depletion of Pi in the cell as a result of phosphorylation of mannose to M6P. Inhibition of ripening by mannose was not alleviated by co-application of glucose as a competitive substrate for the hexokinase(s), or by Pi, presumably the depleted metabolite. Also, incubation of tissue disks with M6P resulted in inhibition of ethylene production and respiration. The structural analogs of mannose, glucosamine, and 2-deoxyglucose, which have been shown to mimic mannose action in several plant tissues, did not cause inhibition of ripening of pear fruit comparable with that associated with mannose. Both analogs stimulated respiration, and glucosamine caused only a small inhibition of softening and ethylene evolution. Another mannose analog, α-methylmannoside, did inhibit fruit ripening though to a lesser extent than mannose. Its influence was also associated with accumulation of M6P and a decrease of Pi levels. We conclude that the mannose effect may, in part, be due to M6P toxicity, as well as by depletion of Pi.     

Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX-2380)

We report the changes in the concentrations and ¹⁸O contents of extracellular CO₂ and HCO₃⁻ in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring ¹⁸O exchange between CO₂ and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO₂ hydration rate in different samples of cells that were grown on bubbled air (low-CO₂ conditions). This activity was only weakly inhibited by ethoxzolamide with an I₅₀ near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO₂-starved cells there is considerable generation of CO₂ from intracellular stores, a factor that can cause errors in measurement of net CO₂ uptake unless accounted for. It was demonstrated that use of ¹³C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO₂ readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO₃⁻ was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO₃⁻ from Synechococcus.     

Light and acetate regulate a mitochondrial malate dehydrogenase

A malate dehydrogenase was purified from the unicellular green alga Chlorogonium elongatum Dangeard. The enzyme was localized in the mitochondria by immunogold electron microscopy and was found to be present on the cristae. The concentration of the enzyme is regulated by acetate and light. In cells cultured heterotrophically with acetate as carbon source the activity and the concentration of the enzyme is 5- to 6-fold higher than in autotrophic cells. In mixotrophically cultured cells (light and acetate) the enzyme level attains only half of the value of that in heterotrophic cells. Acetate induces an increase of the enzyme concentration while light has an inhibitory effect on this process.     

A calcium-dependent but calmodulin-independent protein kinase from soybean

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≈2 micromolar). The protein kinase activity was stimulated 100-fold by ≥10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound ⁴⁵Ca²⁺ in the presence of KCl and MgCl₂, which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.