Characterization and Effect of Light on the Plasma Membrane H+-ATPase of Bean Leaves

Proton excretion from bean (Phaseolus vulgaris L.) leaf cells is increased by bright white light. To test whether this could be due, at least in part, to an increase in plasma membrane (PM) ATPase activity, PM vesicles were isolated from primary leaves by phase partitioning and used to characterize PM ATPase activity and changes in response to light. ATPase activity was characterized as magnesium ion dependent, vanadate sensitive, and slightly stimulated by potassium chloride. The pH optimum was 6.5, the K_m was approximately 0.30 millimolar ATP, and the activity was about 60% latent. PM vesicles were prepared from leaves of plants grown for 11 days in dim red light (growing slowly) or grown for 10 days in dim red light and then transferred to bright white-light for 1 day (growing rapidly). For both light treatments, ATPase specific activity was approximately 600 to 700 nanomoles per milligram protein per minute, and the latency, K_m, and sensitivity to potassium chloride were also similar. PM vesicles from plants grown in complete darkness, however, exhibited a twofold greater specific activity. We conclude that the promotion of leaf growth and proton excretion by bright white light is not due to an increase in ATPase specific activity. Light does influence ATPase activity, however; both dim red light and bright white light decreased the ATPase specific activity by nearly 50% as compared with dark-grown leaves.     

Ultrastructure of ascospore delimitation in freeze substituted samples ofAscodesmis nigricans (Pezizales)

Summary Freeze substitution proved to be a valuable technique for studying the early stages of ascosporogenesis inAscodesmis nigricans. Our observations indicate that the ascus vesicle originated from the ascus plasma membrane. Invaginations of the plasma membrane produced ascus vesicle initials consisting of two closely spaced unit membranes. The appearance of the outer leaflet of each of these membranes was identical to that of the inner leaflet of the ascus plasma membrane. Apparent points of continuity between ascus vesicle initials and the plasma membrane were observed. Ascus vesicle initials accumulated in the ascus cytoplasm near the plasma membrane and then coalesced to form the ascus vesicle, a peripheral, cylinder-like structure consisting of two closely spaced unit membranes that extended from the ascus apex to the ascus base. The ascus vesicle then became invaginated in a number of regions and subsequently gave rise to eight sheet-like segments, or ascosporedelimiting membranes, that encircled uninucleate segments of cytoplasm forming ascospore initials. Like the ascus vesicle, each ascospore-delimiting membrane consisted of two closely spaced unit membranes, the inner of which became the ascospore plasma membrane. The ascospore wall then developed between the spore plasma membrane and the outer membrane. Many details of ascospore maturation were clearly visible in freeze substituted samples.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss.

When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions. In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains of Pseudomonas putida serving as model GEMs. Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations. Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media. Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs. Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth). However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water. Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28- to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)     

T-DNA presence and opine production in tumors of Picea abies (L.) Karst induced by Agrobacterium tumefaciens A281

The hypervirulent Agrobacterium tumefaciens strain A281 formed frequent tumors (31%) on Picea abies (Norway spruce), an economically important tree species in Swedish forests. Three-month-old seedlings were inoculated and tumors were established that grew hormone-independently in culture. Tumors contained agropine and mannopine/mannopinic acid as determined by acid pH paper electrophoresis. In addition, DNA hybridization studies showed that the DNA from these tumor lines contained sequences homologous to Ti plasmid T-DNA, whereas wild-type spruce seedling DNA did not. These results suggest that Agrobacterium vectors can be used for gene transfer into this important forest species.     

Functional properties of the anaerobic responsive element of the maize Adh1 gene

The functional properties of the anaerobic responsive element (ARE) of the maize Adh1 gene have been analysed using a transient expression assay in electroporated maize protoplasts. The ARE functions in both orientations although inversion of the ARE sequence relative to the TATA box element produces slightly weaker promoter activity under anaerobic conditions and elevated expression under aerobic conditions. Promoter activity under anaerobic conditions is proportional to the number of complete ARE sequences in the Adh1 promotor. The ARE contains two sub-regions and dimers of sub-region II are as efficient as the wild-type sequence in activating gene expression under anaerobic conditions. However, sub-region I dimers do not appear capable of inducing gene expression in response to anaerobic stress. We conclude that sub-region II is essential for anaerobic induction of gene expression. Reporter gene expression remains constant when the spacing between sub-regions of the ARE is increased up to at least 64 bp, but increased spacing of 136 bp or greater abolishes expression in both aerobic and anaerobic conditions, indicating that a close association of the two sub-regions is required both for anaerobic responsiveness and for maximal levels of aerobic gene expression. When the ARE is placed upstream of position –90 of the CaMV 35S promoter, the ARE produces a high level of expression in both aerobic and anaerobic conditions. The general enhancement of gene expression driven by the hybrid ARE/35S promoter in aerobic conditions requires an intact sub-region II motif since mutation or deletion of sub-region II from the hybrid promoter reduces the level of expression to that observed for the truncated 35S promoter alone. In addition, mutation of the sub-region I sequences in the ARE/35S hybrid promoter does not significantly reduce expression in aerobic conditions, relative to pARE/35S(-90), suggesting that sub-region I does not contribute to this general enhancer function.     

Kainate activated single channel cnrrents as revealed by domoic acid

We have studied the properties of kainic acid receptor-activated channels using domoic acid as an agonist. Similarities of the electrophysiological, pharmacological and noise properties of domoic acid and kainic acid-evoked currents confirm that domoate is a potent and specific agonist of the kainate receptor. Single-channel properties of domoic acid-evoked currents were directly determined from outside-out membrane patches for the first time, and results were compared with those obtained by fluctuation analysis of macroscopic currents. Small conductance cationic-selective channels of 4 pS and a mean open time of 2 to 3 ms were detected using both methods. Offprint requests to: O. Moran     

Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiotrichum curvatum

Summary From six unsaturated fatty acid auxotrophs (Ufa mutants) of the oleaginous yeast Apiotrichum curvatum blocked in the conversion of stearic to oleic acid, were isolated revertants able to grow in the absence of unsaturated fatty acids, in a search for strains that can produce cocoa butter equivalents. A broad range in the percentage of saturated fatty acids (%SFA) was observed in the lipids of individual revertants (varying from 27%–86% SFA), compared with the wild-type (44% SFA). Further analysis of fatty acid composition indicated that: (i) not all six Ufa mutants had the same genetic background and (ii) one specific Ufa mutation could be reverted in more than one way. Revertants that produced lipids with a %SFA>56%, were examined further. These strains were cultivated for 50 generations and half of them produced lipids with high %SFA after that time and were defined as stable. The viability of revertant strains with extremely high %SFA (>80%) may be explained by our finding that polar lipids, which are part of yeast membranes, contained much more polyunsaturated fatty acids and a significantly lower %SFA than neutral (storage) lipids. One revertant (R25.75) was selected that was able to produce lipids in whey permeate at a rate comparable with wild-type A. curvatum and with a fatty acid composition and congelation curve comparable with cocoa butter. Offprint requests to: A. Ykema     

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996     

DEVELOPMENTAL STABILITY IN LEAVES OF CLARKIA TEMBLORIENSIS (ONAGRACEAE) AS RELATED TO POPULATION OUTCROSSING RATES AND HETEROZYGOSITY

Four natural populations of Clarkia tembloriensis, whose levels of heterozygosity and rates of outcrossing were previously found to be correlated, are examined for developmental instability in their leaves. From the northern end of the species range, we compare a predominantly selfing population (t? = 0.26) with a more outcrossed population (t? = 0.84), which is genetically similar. From the southern end of the range, we compare a highly selfing population (t? = 0.03) with a more outcrossed population (t? = 0.58). We measured developmental stability in the populations using two measures of within-plant variation in leaf length as well as calculations of fluctuating asymmetry (FA) for several leaf traits. Growth-chamber experiments show that selfing populations are significantly more variable in leaf length than more outcrossed populations. Developmental instability can contribute to this difference in population-level variance. Plants from more homozygous populations tend to have greater within-plant variance over developmentally comparable nodes than plants from more heterozygous populations, but the difference is not significant. At the upper nodes of the plant, mature leaf length declines steadily with plant age, allowing for a regression of leaf length on node. On average, the plants from more homozygous populations showed higher variance about the regression (MSE) and lower R² values, suggesting that the decline in leaf length with plant age is less stable in plants from selfing populations than in plants from outcrossing populations. Fluctuating asymmetry (FA) was calculated for four traits within single leaves at up to five nodes per plant. At the early nodes of the plant where leaf arrangement is opposite, FA was also calculated for the same traits between opposite leaves at a node. Fluctuating asymmetry is significantly greater in the southern selfing population than in the neighboring outcrossed population. Northern populations do not differ in FA. Fluctuating asymmetry can vary significantly between nodes. The FA values of different leaf traits were not correlated. We show that developmental stability can be measured in plants using FA and within-plant variance. Our data suggest that large differences in breeding system are associated with differences in stability, with more inbred populations being the least stable.