Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.     

The induction of lincomycin resistance in Lycopersicon peruvianum and Lycopersicon esculentum

Lincomycin-resistant calli were induced from both Lycopersicon esculentum and Lycopersicon peruvianum using N-mitroso-N-methylurea (NMU) mutagenesis. From these calli lincomycin-resistant plants were regenerated. For L. peruvianum it was shown that the resistant plants could be divided in two classes with respect to their resistance to lincomycin and its derivative clindamycin. The first class comprised plants which were resistant to 500 mg/l lincomycin and showed no shoot or root formation in the presence of clindamycin; the second class consisted of plants resistant to 2000 mg/l lincomycin and these plants were able to form shoots and roots on clindamycin containing media. Lincomycin is an inhibitor of peptidyltransferase; chloroplast encoded parts of this enzymatic function are sensitive for this antibiotic. Reciprocal crosses between our lincomycin resistant and wild type L. peruvianum plants indicated a maternal inheritance of the mutation.     

Isolation and characterization of the membrane-bound cytochrome c-554 from the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus

The membrane-bound photooxidizable cytochrome c-554 from Chloroflexus aurantiacus has been purified. The purified protein runs as a single heme staining band on SDS-PAGE with an apparent molecular mass of 43 000 daltons. An extinction coefficient of 28 ± 1 mM^–1 cm^–1 per heme at 554 nm was found for the dithionite-reduced protein. The potentiometric titration of the hemes takes place over an extended range, showing clearly that the protein does not contain a single heme in a well-defined site. The titration can be fit to a Nernst curve with midpoint potentials at 0, +120, +220 and +300 mV vs the standard hydrogen electrode. Pyridine hemochrome analysis combined with a Lowry protein assay and the SDS-PAGE molecular weight indicates that there are a minimum of three, and probably four hemes per peptide. Amino acid analysis shows 5 histidine residues and 29% hydrophobic residues in the protein. This cytochrome appears to be functionally similar to the bound cytochrome from Rhodopseudomonas viridis. Both cytochrome c-554 from C. aurantiacus and the four-heme cytochrome c-558-553 from R. viridis appear to act as direct electron donors to the special bacteriochlorophyll pair of the photosynthetic reaction center. They have a similar content of hydrophobic amino acids, but differ in isoelectric point, thermodynamic characteristics, spectral properties, and in their ability to be photooxidized at low temperature.Abbreviations LDAO lauryl dimethyl amine-N-oxide - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - mV millivolt - E_m.8 midpoint potential at pH 8.0 - ODV optical density x volume in ml     

Fluorescence lifetimes of dimers and higher oligomers of bacteriochlorophyll c from Chlorobium limicola

Fluorescence lifetimes have been measured for bacteriochlorophyll (BChl) c isolated from Chlorobium limicola in different states of aggregation in non-polar solvents. Two different homologs of BChl c were used, one with an isobutyl group at the 4 position, the other with n-propyl. Species previously identified as dimers (Olson and Pedersen 1990, Photosynth Res, this issue) decayed with lifetimes of 0.64 ns for the isobutyl homolog, 0.71 ns for n-propyl. Decay-associated spectra indicate that the absorption spectrum of the isobutyl dimer is slightly red-shifted from that of the n-propyl dimer. Aggregates absorbing maximally at 710 nm fluoresced with a principal lifetime of 3.1 ns, independent of the homolog used. In CCl₄, only the isobutyl homolog forms a 747-nm absorbing oligomer spectrally similar to BChl c in vivo. This oligomer shows non-exponential fluorescence decay with lifetimes of 67 and 19 ps. Because the two components show different excitation spectra, the higher oligomer is probably a mixture of more than one species, both of which absorb at 747 nm.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - % MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGGipm0dc9vqaqpepu0xbbG8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeq4Xdm2aaW% baaSqabeaacaaIYaaaaaaa!3777!\[\chi ^2 \] chi-square - FWHM full-width at half-maximum     

Demography of agile gibbons (Hylobates agilis)

Demographic processes and the structure of a population of agile gibbons (Hylobates agilis) were investigated over 6 years in the Gunung Palung Reserve, Indonesia. Estimates of population size, density, and biomass revealed a population whose groups were stable in size and composition. Demographic processes place gibbons at risk, however, to short-term changes in their environment. Patterns of survival, fecundity, mortality, and dispersal combined to produce negative rates of growth. In addition, a top-heavy age-class distribution, with adults forming a large fraction of animals, makes it unlikely that this population could recover rapidly from a decline in numbers. Two behavioral factors, territoriality and monogamy, account for the size and stability of gibbon groups. Monogamy imposes limits on group size, while mating patterns and territoriality decrease the impact of sources of high mortality common in other primate species. These relationships underscore the fundamental importance of behavioral influence on demographic processes and social structure.     

Oxygen consumption and body temperature in yellow-bellied marmot populations from montane-mesic and lowland-xeric environments

Summary Yellow-bellied marmots characteristically live in montane-mesic environments, but in several areas in western North America, this species extended its range into lowland-xeric habitats. Body mass was significantly smaller in the lowland-xeric population from eastern Washington at 393 m than in the montane-mesic population from western Colorado at 2900 m. Oxygen consumption of marmots from montane-mesic and lowland-xeric environments was signiflcantly affected by ambient temperature (T_A) water regimen, population, and a population x water regimen x temperature interaction. Lowland-xeric animals had a higher metabolic rate at low T_As, but a lower metabolic rate at higher T_As than the montane-mesic aminals. Oxygen consumption was lower on a restricted-water regimen than on ad libitum water in both populations. Coefficients relating oxygen consumption to body mass were affected by T_A, water regimen, and population. These intraspecific coefficients are larger than the interspecific coefficients for all mammals. Body temperature (T_B) was affected significantly by T_A, water regimen, and population. T_A body mass, and a population x water regimen interaction significantly affected conductance. Conductance generally was higher in the lowland-xeric than in the montane-mesic marmots. Both populations increased conductance at high T_A, but the lowland-xeric population dissipated a much higher proportion of the heat by evaporative water loss (EWL) than did the montane-mesic population. Metabolic water production exceeded or equaled EWL at 5–20°C. Smaller body size, reduced metabolism at high T_A, and increased EWL at high T_A characterized the lowland-xeric population.Metabolic rates of yellow-bellied marmots were higher than predicted from body size during the reproductive season but decreased to 67% of that predicted from the Kleiber curve by late summer. Marmots minimize thermoregulatory costs by concentrating activity at times when the microclimate is favorable, by tolerating hyperthermia at high T_A in the field, and by having a conductance lower than that predicted from body size.Abbreviations DHC dry-heat conductance - EHL evaporative heat loss - EWL evaporative water loss - HP heat produced - T _A ambient temperature - T _n body temperature - M body mass     

Species diversity and seasonal dynamics of Acari on abandoned apple trees in southern Ontario,Canada

Foliage-inhabiting mites and associated insects were observed over a 3-year period on abandoned apple trees at two sites in southern Ontario. This study included species diversity and seasonal dynamics as well as the total habitat size and its seasonal fluctuations. Due to heavy feeding on the leaves in the early season by the fall cankerworm, at one observation site, the habitat available for foliage-inhabiting mites was regulated so that both the total number and the area of leaves on the tree did not increase significantly until early July. However, the influence of this on the population of the dominant mite species was minimal. The mite community was relatively stable over time. Four phytophagous species, representing a basic trophic level, were fed upon by a group of predacious mites. The phytophagous mites consisted of two rust-mite species (Diptacus gigantorhynchus sp. complex andEriophyes pyri sp. complex) and two tetranychid species (Bryobia rubrioculus andEotetranychus uncatus). On the basis of consistency in seasonal abundance,D. gigantorhynchus was the only prey species which could support the early-season population increase of the predacious mites. Ten species of predacious mites were recorded on the apple trees, nine phytoseiids and one stigmaeid; two separate species of these were common at each of two observation sites:Typhlodromus pomi and the stigmaeidZetzellia mali at one site; and two phytoseiid species,Phytoseius macropilis andAmblyseius finlandicus, at the other. There seemed to be a rather simple and stable prey/predator system at each site throughout the three seasons. Species dominance on these apple trees was investigated for the phytoseiids, especially from the perspective of the issue of one-species dominance.     

Assessment of methods to determine minimal cellulase concentrations for efficient hydrolysis of cellulose

Summary The enzyme loading needed to achieve substrate saturation appeared to be the most economical enzyme concentration to use for hydrolysis, based on percentage hydrolysis. Saturation was reached at 25 filter paper units per gram substrate on Solka Floc BW300, as determined by studying (a) initial adsorption of the cellulase preparation onto the substrate, (b) an actual hydrolysis or (c) a combined hydrolysis and fermentation (CHF) process. Initial adsorption of the cellulases onto the substrate can be used to determine the minimal cellulase requirements for efficient hydrolysis since enzymes initially adsorbed to the substrate have a strong role in governing the overall reaction. Trichoderma harzianum E58 produces high levels of -glucosidase and is able to cause high conversion of Solka Floc BW300 to glucose without the need for exogenous -glucosidase. End-product inhibition of the cellulase and -glucosidase can be more effectively reduced by employing a CHF process than by supplemental -glucosidase.Offprint requests to: C. M. Hogan