Molecular analysis of the phytochrome deficiency in an aurea mutant of tomato

Summary The au ^w mutant allele of the aurea locus in tomato has previously been shown to cause deficiency for the phytochrome polypeptide (Parks et al. 1987). We have begun to characterize the molecular basis and consequences of this deficiency. Genomic Southern blot analysis indicates that there are at least two and probably more phytochrome polypeptide structural genes in tomato. RNA blot analysis shows that the au ^w mutant contains normal levels of phytochrome mRNA and in vitro translation of au ^w poly(A)⁺ RNA yields a phytochrome apoprotein that is quantitatively and qualitatively indistinguishable on SDS-polyacrylamide gels from that synthesized from wild-type RNA. These results indicate that the phytochrome deficiency in aurea is not the result of lack of expression of phytochrome genes but is more likely due to instability of the phytochrome polypeptide in planta. Possible reasons for such instability are discussed. Analysis of the molecular phenotype of aurea indicates that the phytochrome-mediated increase in the abundance of the mRNA encoding chlorophyll a/b binding protein (cab) is severely restricted in the mutant as compared with wild-type tomato. Thus, the au w strain exhibits defective photoregulation of gene expression consistent with its very reduced level of the phytochrome photoreceptor.     

Phytochrome modulation of ATPase activity in a membrane fraction from Phaseolus

Membrane-bound phytochrome and ATPase (ATP phosphohydrolase EC 3.6.1.3.) activity extracted from hypocotyl hooks of etiolated Phaseolus aureus Roxb. were both separated from solute proteins by gel filtration on Sepharose C1-2B. The amount of phytochrome detected in the membrane fraction was very small and was not significantly increased by red irradiation (in vivo or in vitro). Membrane-bound ATPase activity was modulated in vitro by the phytochrome in the membrane fraction, being lower after red light than after far-red light. This effect was potentiated by a preliminary light reaction which occurred only in vivo and, in continuous red light, required 60 to 90 s at 25°C. Thus a two minute, in vivo, red irradiation reduced membrane-bound ATPase activity to about half that of the etiolated state. Subsequently bound-ATPase activity was determined by the form of phytochrome (P_r or P_fr) irrespective of whether established in vivo or in vitro. These results indicate that binding or release (of enzyme, cofactors or inhibitors) is not involved in phytochrome modulation of enzyme activity in the membrane fraction.Abbreviations R red light - F far red light - P_r inactive form of phytochrome (_max=660 nm) - P_fr active form of phytochrome (_max=730 nm) - MOPS N-morpholino-3-propansulphonic acid     

Auxin-stimulated ATPase in membrane fractions from pumpkin hypocotyls (Cucurbita maxima L.)

Membrane fractions from Cucurbita maxima hypocotyls were isolated in a medium which inhibits the action of endogenous phospholipases. After removal of soluble phosphatases by Sepharose 2B-CL column chromatography, an auxin-stimulated ATPase activity was found in membrane fractions from linear sucrose gradients. In the presence of 10^-4 M phenylacetic acid (PAA), the stimulation by indol-3-acetic acid (IAA) exhibited a bimodal concentration dependence with maximal stimulation of about 50% at 10^-6 M IAA. Without PAA, only a high concentration of 10^-4 M IAA was stimulatory, whereas 10^-6 M IAA had no apparent effect and 10^-8 M IAA exhibited weak inhibition. PAA alone had only weak or no effects. The effects of IAA must be considered as hormone-specific. The ATPase activity in the presence of 10^-4 M PAA was activated only by 2,4-dichlorophenoxyacetic acid (2,4-D), an active auxin analogue, but not by the inactive stereoisomers, 2,3-D and 3,5-D. Comparison with marker enzyme profiles suggested that part of the auxin-stimulated ATPase was localized on plasma membranes as well as other compartments. Thus, the auxin-stimulated ATPase may become a useful tool in the investigation of the mechanism of action of auxin.Abbrevations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 3,5-D 3,5-dichlorophenoxyacetic acid - IAA indol-3-acetic acid - PAA phenylacetic acid - MES (2-(N-morpholino))-ethanesulfonic acid - EDTA ethylenediamine tetraacetic acid     

Gel filtration and absorption spectroscopic investigations on humic substances from organic fertilizers

Summary Three organic fertilizers: a farmyard manure (FYM), humified poplar barks (PBF) and spruce barks (SBF) were investigated by means of chemical analysis and by the study of humic substances extracted by 0.1M sodium pyrophosphate (pH=10.0). The organic carbon content and the C/N ratio were higher in SBF, probably as a consequence of the short fermentation period (5 months). Yields of organic substances removed by three consecutive extractions with Na₄P₂O₇ solution were in the order: PBF>FYM>SBF. Nominal molecular weight distribution of total extracts was studied using Sephadex G type gels; TRIS buffer (pH=9.0) was the eluant, since the use of water produced complex gel-solute interactions. It was shown that SBF had a higher content of small size particles than the other two products which exhibited quite similar elution curves. Slopes of log A(absorbance)vs log plots and ratio of absorbances at 465 and 665 nm (E₄/E₆) of fractions containing particle of lower size were the steepest and the highest, respectively.     

Overexpression of rice phytochrome A in tobacco seedlings modifies responsiveness but not competence to phytochrome

To analyse the control of rice phytochrome A (phyA) overexpression (wild type or variously mutated) on gene regulation, transgenic tobacco lines overexpressing various rice phyA constructs were crossed with transgenic tobacco lines containing mustard Lhcb1 or Chs1 promoters fused to the uidA reporter gene (-glucuronidase). It was demonstrated that the temporal pattern of competence to respond to phytochrome was not altered by rice phyA overexpression. Also, overexpression of rice phyA did not change the spatial pattern of gene expression. The responsiveness to red and far-red light, on the other hand, depended on the type of overexpressed rice phyA in a structure-function relation: the serine-to-alanine mutant mediated an enhanced response both under continuous red and far-red light, whereas the N-terminal deletion mutant showed a dominant negative effect under continuous far-red light and even after red light pulses. However, the effectiveness of rice phyA overexpression depended on the promoter construct and the developmental stage of the seedlings. The Lhcb1 promoter also conferred -glucuronidase activity in etiolated seedlings. This dark expression could be decreased by a long-wavelength farred light pulse given early in development (24 h after sowing), indicating that this phenomenon is under the control of stable types of phytochrome.Abbreviations Chs1 chalcone synthase - GUS -glucuronidase - Lhcb1 type 1 light-harvesting chlorophyll a/b-binding protein - NTD N-terminal deletion mutant of rice phyA - phyA phytochrome A - phyB phytochrome B - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - RW rice wild-type phyA - S/A serine-to-alanine mutant of rice phyA - XAN wild-type tobacco cv. Xanthi We thank N.-H. Chua (Rockefeller Univ., New York, USA) and J. Stockhaus (Heinrich-Heine-Universität, Düsseldorf, Germany) for providing seeds from tobacco lines overexpressing the diverse rice phyA proteins. The work was supported by a grant from the Human Frontier Science Program and a grant from Deutsche Forschungsgemeinschaft (SFB 388). K.E. is a recipient of a Landesgraduierten-förderung fellowship     

The filtration apparatus of Anostraca (Crustacea): species-specific setulation in the genus Streptocephalus

The structure of the setae, setules, spines and spinules on the limbs of males and females of Streptocephalus torvicornis, S. rubricaudatus and S. proboscideus are described, based on scanning electron micrographs. The data disclose significant variation among the three species in fine structure of the setules and inter-setular distance. The variation may be related to different trophic specialisations, facilitating the sympatric occurrence of congeneric species.     

Gel filtration of egg phosphatidylcholine vesicles

The abilities of Sepharose 2b (Pharmacia), Controlled Pore Glass (Electro-Nucleonics) and Bio-Gel A150m (Bio-Rad) to purify small unilamellar vesicles prepared by sonication and the ethanol-injection methods were compared. The Bio-Gel causes complete aggregation of the sonicated vesicles and partial aggregation of the ethanol-injection vesicles. Both Sepharose and Controlled Pore Glass are acceptable for purifying vesicles from multilamellar liposomes; however, neither will separate the vesicles from sonication by-products which might be formed.     

Bisulfite interacts with binding sites of the auxin-transport inhibitor N-1-naphthylphthalamic acid

The affinity of the auxin-transport inhibitor N-1-naphthylphthalamic acid (NPA) for membrane particles as well as for solubilized binding sites from Cucurbita pepo L. hypocotyls was reduced by low concentrations of bisulfite (half-maximal inhibition at 2·10^-3–3·10^-3 M). Two membrane fractions obtained by sedimentation aided with polyethylene glycol showed differential sensitivity to bisulfite. Other oxidizing or reducing substances tested at 1 mM had no effect, except for N-ethylmaleimide (80% inhibition) and iodine (complete inhibition), both of which reduced the number of binding sites but not their affinity. Addition of bisulfite to either the isoalloxane ring of flavoproteins or to pyridoxal phosphate or quinones is proposed as a possible mechanism of action. Sulfur dioxide, at concentrations measured in polluted air, can lead to bisulfite concentrations in plant tissue sufficient to interfere with NPA-binding sites and hence with auxin transport.Abbreviations DTE dithioerythritol - DTT dithiothreitol - IC₅₀ concentration of half-maximal inhibition - NAA 1-naphthylacetic acid - NEM N-ethylmaleimide - NPA N-1-naphthylphthalamic acid - PEG polyethylene glycol, 6000 molecular weight     

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anion-exchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.     

Reduction of the filtration rate of Daphnia galeata by dissolved photosynthetic products of edible phytoplankton

The filtration rate of Daphnia galeata was determined in in situ experiments in Bautzen Reservoir and in laboratory experiments, where daphnids were exposed to filtrates that previously contained either natural phytoplankton or cultured eukaryotic algae (Scenedesmus obliquus or Asterionella formosa), respectively. Individual filtration rate (FR) was measured using fluorescent beads, taking into account ingested beads in the gut only. Compared to heated control treatments (100 °C), dissolved compounds released by the nutritious cultured algae during the preconditioning phase or by the natural phytoplankton assemblages from Bautzen Reservoir strongly reduced the filtration rate of D. galeata (down to 60%). Heating deactivated these dissolved compounds. A significant correlation was found between primary production measured in situ and the reduction of FR in the filtrate of reservoir water, indicating that extra-cellular products released during photosynthesis triggered the reduction of the filtration rate. The ratio of ingested to collected beads was used to quantify the proportion of food, which was not only collected but passed the mouth of D. galeata. The ratio of ingestion to collection was compared between filtered and unfiltered reservoir water both media identical with respect to the concentration of dissolved compounds, whereas other factors (e.g. food concentration, temperature, filtration rate) were different. The changes in this ratio between filtered and unfiltered reservoir water suggest that D. galeata is capable of a chemosensory control of the ingestion behaviour by detecting external metabolites.     

Gel filtration chromatography of triple-helical calf skin collagen

Gel filtration of type I collagen has been of limited use, because at low pH where the protein is not associated it binds to agarose gels, and at neutrality collagen has a tendency to form fibrils. The more porous polyacrylamide-based gels do not interact with collagen but cannot be used at very high flow rates because they are compressible. It was found that these difficulties are surmounted by use of Fractogel TSK HW-65F, a spherical gel made from a weakly hydrophilic vinyl polymer, and use of the buffer system 0.5 m urea, 0.117 m Tris-HCl, pH 7.3, which prevents fibril formation. The solvent has only a slight effect on the thermal stability of collagen, as determined by circular dichroism measurements. The recovery of native collagen, at 25°C, was at least 88% and that of partially unfolded collagen, at 35°C where it is about one-third unfolded, was 98%. The Fractogel TSK gels and the urea, Tris solvent system should be useful for both preparative work and for studies involving interaction of unaggregated type I collagen with smaller molecules at physiological pH.     

Photoregulation of germination in seed of transgenic lines of tobacco and Arabidopsis which express an introduced cDNA encoding phytochrome A or phytochrome B

Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low redfar-red (RFR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-P_fr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.Abbreviations ABO seed transformed with Arabidopsis phyB - cDNA; CaMV cauliflower mosaic virus - FR far-red light - P_fr far-red-absorbing form of phytochrome - P_tot total phytochrome - P_fr/P_tot phytochrome photoequilibrium - R red light - RBO seed transformed with rice phyB cDNA - RFR quantum ratio of red and far-red light - WL white light - WL + FR whitelight supplemented with far-red light - WT wild type The authors wish to thank R.D. Vierstra (Department of Horticulture, University of Wisconsin-Madison, USA) for providing the transgenic tobacco line, and M.T. Boylan, D. Wagner and P.H. Quail (U.C. Berkeley/USDA Plant Gene Expression Center, Albany, Calif. USA) for providing the transgenic Arabidopsis lines. The work presented in this paper was funded by grants from the Agricultural and Food Research Council (H.S., A.C.M., G.C.W.).     

The fern as a model system to study photomorphogenesis

The fern gametophyte is a good model system for studying cell biological, physiological, and photobiological aspects of the fundamental processes of plant development and physiological phenomena, because of its autotrophic characteristics and its simple structure. The cells, moreover, are not surrounded by tissue, so observation and manipulation of the cells are very easy. Here I summarize a part of my knowledge of fern systems, which I have studied for nearly 40 years. Masamitsu Wada is the recipient of the BSJ Research Award for 2004.     

Immunoprecipitation of phytochrome from green Avena by rabbit antisera to phytochrome from etiolated Avena

Thirty-nine antiserum preparations from eight rabbits were screened for their ability to precipitate the immunochemically distinct phytochrome that is obtained from green oat (Avena sativa L.) shoots. The antisera were obtained from rabbits immunized with either proteolytically degraded, but still photoreversible, 60-kDa (kilodalton) phytochrome, or approx. 120-kDa phytochrome, both of which were purified from etiolated oat shoots. The ability of these antisera to precipitate phytochrome from green oats was independent of the size of phytochrome used for immunization. While crude antisera immunoprecipitated as much as 80% of the phytochrome isolated from green oat shoots, antibodies immunopurified from these sera with a column of highly purified, approx. 120-kDa phytochrome from etiolated oats precipitated no more than about 5–10%.Abbreviations kDa kilodalton - mU milliunit     

Effect of temperature on the filtration rate and percentage of assimilation ofSalpa fusiformis Cuvier (Tunicata: Thaliacea)

The effect of temperature on filtration and ingestion rates ofSalpa fusiformis Cuvier was determined over the temperature range 13–22°C at a constant algal concentration. Filtration and ingestion rates increased with temperature up to an optimum (19.7 to 20.2°C) beyond which they decreased. Food assimilation for this salp species was determined while fed onPhaeodactylum tricornutum Bohlin orHymenomonas elongata Stein at different algal concentrations. The percent assimilation varied between 28 and 39% (mean: 32%) with the diatomP. tricornutum as food, and between 39 to 81% (mean: 64%) with the flagellateH. elongata. Assimilation did not seem to depend on the algal concentration but it was greater with the flagellate than with the diatom, the latter having a higher ash content.     

Isolation of the factor VIII–von Willebrand factor complex directly from plasma by gel filtration

A high capacity gel filtration system was developed with the purpose of isolating factor VIII (FVIII) and von Willebrand factor (vWF) directly from plasma in significantly higher yields than obtained by cryoprecipitation, the technique most commonly used to recover FVIII–vWF from human plasma. After laboratory-scale gel filtration of plasma, a FVIII-containing fraction was collected containing about 90% of FVIII in the applied plasma and with almost tenfold higher purity than that obtained by cryoprecipitation. The gel filtration step has been scaled up for use as the initial step in the manufacturing process for a FVIII preparation (Nordiate).