INTRACELLULAR LOCALIZATION OF THE TASTE/ODOR METABOLITE 2-METHYLISOBORNEOL IN OSCILLATORIA LIMOSA (CYANOPHYTA)1

The monoterpene derivative, 2-methylisoborneol (MIB), is produced by many blue-green algae and often is responsible for the “musty” taste/odor in aquaculturally raised finfish and potable water supplies. Although previous researchers have suggested that taste/odor metabolites are partitioned among cell constituents and that coregulation with pigment biosynthesis occurs, no structural evidence for these hypotheses exists. MIB was localized in cells of Oscillatoria limosa (Roth) Agardh at the ultrastructural level using standard gold-labeled antibody techniques. There was no apparent relationship between age of the cells and MIB synthesis; cells that were and were not undergoing active division had similar amounts of MIB label. There was no consistent partitioning of MIB label within cells. However, occasionally, specific label was observed along photosynthetic lamellae, suggesting a potential linkage of MIB synthesis and/or binding to the pigment systems.     

Extent of polyteny in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster

In the polytene nuclei of germ-line cells (ovarian pseudonurse cells) of Drosophila melanogaster females mutant for otu ¹¹ (ovarian tumor), the pericentric heterochromatin is much more abundant than in somatic salivary gland cells. This is due to the degree of heterochromatin compaction (and consequently the level of underreplication) being lower in the nurse cells than in the salivary gland cells. The lower level of compaction probably results in a very low degree of position effect gene inactivation in the ovarian nurse cells.     

Differential molecular responses to abscisic acid and osmotic stress in viviparous maize embryos

Substantial quantities of mRNA encoding the abundant Em polypeptide accumulate, in planta, in developing embryos of maize (Zea mays L.). By contrast, accumulation of Em mRNA is only barely detectable in embryos with the vp-5/vp-5 genotype [an abscisic acid (ABA)-deficient viviparous phenotype]. Em mRNA is not detectable within viviparous embryos of the vp-1/vp-1 genotype that are non-responsive to ABA. Culture of immature wild-type and vp-5/vp-5 embryos in the presence of exogenous ABA or of an osmotically active agent prevents precocious germination and results in expression of the Em genes. When vp-1/vp-1 embryos are cultured under similar conditions, only the application of osmotic stress prevents precocious germination. However, Em mRNA does not accumulate either in ABA-treated or stressed, arrested embryos, indicating a requirement for ABA perception through a VP-1-mediated mechanism for Em gene expression. Nevertheless, vp-1/vp-1 embryos do show both ABA and stress responses at the molecular level. Treatment with ABA causes the accumulation of mRNA encoding a polypeptide of approx. 30 kDa, whilst osmotic stress induces the accumulation both of a 30-kDa polypeptide and a set of approx. 20-kDa polypeptides. This indicates the existence of discrete, parallel ABA and stress response pathways in developing maize embryos.Abbreviations ABA abscisic acid - cDNA copy-DNA - DAP days after pollination - kDa kilodaltons - MS Murashige and Skoog medium - LEA late embryogenesis abundant - NEpHGE non-equilibrium pH gradient gel electrophoresis - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinnia

In a culture system in which single cells isolated from the mesophyll of Zinnia elegans L. differentiate to tracheary elements (TEs), two inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5), L-α-aminooxy-β-phenylpropionic acid (AOPP) at 10 μM inhibited lignification without reducing the number of TEs formed. These inhibitors caused intracellular changes in peroxidase (EC 1.11.1.7) activities. The inhibitors increased the activity of peroxidases bound to the cell walls and especially the activity of peroxidase bound ionically to the cell walls. In contrast, the activity of extracellular peroxidase decreased. There were five isoenzymes, P1-P5, in the ionically bound peroxidase of cultured Zinnia cells. Among the isoenzymes, P4 and P5 appeared to be specific for TE differentation. Treatment with AOPP and AIP resulted in increases in the activities of P2, P4 and P5 isoenzymes, with the most prominent increase in P5 activity. The addition of lignin precursors, including coniferyl alcohol, to the AOPP-treated cells restored lignification, and suppressed the alteration of peroxidase isoenzyme patterns caused by AOPP. The relationship between the wall-bound peroxidases and lignification during TE differentiation is discussed in the light of these results.     

The relative significance for stem elongation and flowering in Lolium temulentum of 3β-hydroxylation of gibberellins

In previous experiments with many gibberellins (GAs) and GA derivatives applied to Lolium temulentum L., quite different structural requirements were evident for stem elongation on the one hand and for the promotion of flowering on the other. Whereas hydroxylation at carbons 12, 13 and 15 enhanced flowering relative to stem growth, the reverse was the case at carbon 3 (L.T. Evans et al. 1990, Planta 182, 97–106). The significance of hydroxylation at carbon 3 is examined in this paper. The application of inhibitors of 3β-hydroxylation, including C/D-ring-rearranged GAs, reduced stem growth but, in the case of the two acylcyclohexanediones, increased the flowering response when applied on the inductive long day. Later applications of the acylcyclohexanediones, made after floral initiation had occurred, were inhibitory to flowering, suggesting that subsequent inflorescence development requires 3β-hydroxylated GAs. Applications of the 3α-hydroxy epimers of GA₁, GA₃ and GA₄ gave slightly less promotion of flowering in comparison with the 3β-hydroxy GAs, but far less promotion of stem elongation, except in the case of 3-epi-GA₄, which was comparable to GA₄. The 3α-hydroxy epimer of 2,2-dimethyl GA₄ gave less promotion of flowering than its 3β-hydroxy epimer but almost no promotion of stem elongation. The 3α-hydroxy epimers of GA₃ and 2,2-dimethyl GA₄ did not act as competitive inhibitors of the stem elongation elicited by GA₃ and 2,2-dimethyl GA₄, respectively. These results extend the differences in GA structure which favour flowering as opposed to stem elongation, and indicate that 3-hydroxylation and its epimeric configuration are of much greater importance to stem elongation than to flower initiation in Lolium.     

Saponin-like substances inhibit α-galactosidase production in the endosperm of fenugreek seeds

When fenugreek (Trigonella foenum-graecum L.) endosperms plus testa (endosperms), which had been isolated from 5-h-imbibed seeds, were incubated for at least 2 h under germination conditions, they leaked substances which, like exogenous abscisic acid (ABA), inhibited the production of fenugreek endosperm -galactosidase. However, unlike ABA, 8 h treatment with these inhibitors had no effect on fenugreek endosperms which had been isolated from 15-h-imbibed seeds and leached for 2 h. This indicated that either their inhibitory action was on processes which were related to the production of -galactosidase and had been completed by this time, or that there might be factors present which inactivate these inhibitors. It was also concluded that the action of the endosperm leachate could not be attributed to the presence of ABA. The activity of the leachate decreased when it originated from endosperms imbibed for periods longer than 25 h and thin-layer chromatography (TLC) of extracts from these endosperms showed decreased contents of the leachable inhibitors as imbibition proceeded. From the seed leachate, which had a TLC pattern and inhibitory action similar to that of the endosperm, were isolated three substances which, when applied to endosperms, inhibited the production of -galactosidase activity. According to their chromatographic behaviour and their reaction with specific reagents, there are strong indications that these substances are saponins. These diffusible saponin-like substances were located in both endosperm and perisperm and their physiological role is discussed.Abbreviations ABA abscisic acid - PEG polyethylenglycol - TLC thin-layer chromatography We wish to thank the Alexander S. Onasis Public Benefit Foundation for a grant to K.Z. and Dr. J.S.G. Reid (University of Stirling, Scotland) for a kind gift of fenugreek seeds.     

Accumulation of the β-subunit of polygalacturonase 1 in normal and mutant tomato fruit

Polyclonal antiserum raised against the native PG1 isoform of tomato fruit (Lycopersicon esculentum Mill.) polygalacturonase [poly(1,4--d-galacturonide) glycanohydrolase, EC 3.2.1.15] bound to each of the subunits of the protein and also to a range of other fruit proteins. Affinity purification was used to remove antibody molecules that bound to the native form of the PG2 isoform. The resulting serum bound to native PG1, denatured PG2 and -subunits of PG1 but not to native PG2 or other fruit proteins. This anti-PG1 serum was used to monitor the occurrence of the PG1 -subunit and PG2 in detergent extracts of tomato tissues. The -subunit polypeptide was detected in pericarp but not locule tissue of fruit, including fruit of the rin and nor mutants. It increased in amount in the pericarp tissues from an early stage to the mature green stage, clearly prior to any appreciable accumulation of the PG2 subunit. The -subunit polypeptide was not detected in stem or leaf tissues. A PG2-specific antiserum was used to study the interaction of PG2 with the isolated -subunit. The PG2 isoform was bound to the -subunit over a wide range of salt concentrations and pH; the interaction was independent of the presence of reducing agents. It is concluded that strong non-covalent forces are involved in the interaction. The results are consistent with a model in which the -subunit is positioned in the cell wall structure and provides a specific binding site for the active PG2 subunit when this is synthesised during ripening.Abbreviations B breaker - MG mature green - M_r relative molecular mass - nor non-ripening mutant - PAGE polyacrylamide gel electrophoresis - PG polygalacturonase - rin ripening inhibitor mutant - SDS sodium dodecyl sulphate     

Half-lives of oat mRNAs in vivo and in a polysome-based in-vitro system

We have used a cell-free polysome-based in-vitro mRNA-degradation system to investigate the halflives of plant cell mRNAs. In order to establish the fidelity of the in-vitro system, we used cordycepin to determine the in-vivo half-lives of -tubulin and actin mRNAs in the primary leaves of 4-d-old etiolated oat (Avena sativa L.) seedlings. The in-vitro rank order of half-lives for phytochrome A (45 min), -tubulin (105 min), and actin (220 min) mRNAs mimicked the in-vivo rank order. A pulse of red light given to excised etiolated primary leaves caused an in-vivo reduction in the half-life of -tubulin mRNA. The selectivity of the polysome-based system was further demonstrated by the decrease in the half-life of -tubulin mRNA (from 105 min to 60 min) induced by a pulse of red light given to the etiolated oat seedlings prior to isolation of polysomes. Red light did not affect the apparent half-lives of phytochrome A or actin mRNAs.Abbreviations cab gene for chlorophyll-a/b-binding protein - kb(p) kilobase (pair) - phyA gene for type-I phytochrome protein - rbcS gene for ribulose-1,5-bisphosphate-carboxylase small-subunit We thank Dr. Richard B. Meagher for the pSAc3 actin clone. We thank Dr. Cecil Stewart for the use of his density-gradient fractionator, and Dr. Virginia Crane for instruction in using the fractionator. We also appreciate the helpful comments provided by the other members of the laboratory during the course of this research: Dr. Isaac John, Dr. Iffat Rahim, Linda Barnes, Bruce Held, David Higgs, and Theresa Tirimanne. This work was supported by USDA grants CRGO 88-37261-4196 and 91-37304-6397, and the Iowa State University Biotechnology Program.     

Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit

The present study describes the biochemical characteristics of an acid -fructosidase (EC 3.2.1.26) purified from the fruit of sweet pepper (Capsicum annuum L.). The soluble form, which constitutes more than 95% of the total activity at pH 4.5, hydrolyzes sucrose, raffinose, and stachyose. Its pH and temperature optima are 4.5 and 55 °C, respectively. Metal cations such as Ag⁺ and Hg²⁺ strongly inhibit its activity, suggesting the presence of at least one sulfhydryl group at the catalytic site. After purification of the enzyme by means of ammonium sulfate fractionation, gel chromatography (diethyl-aminoethyl-Sephacel, hydroxylapatite, concanavalin A-Sepharose), and preparative gel electrophoresis, the purified enzyme was shown to be a 42 kDa glycoprotein interacting specifically with concanavalin A. After complete chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight of the constitutive polypeptide was estimated to be 39 kDa. The enzyme glycans were characterized using both affino- and immunodetection. The enzyme has at least two N-linked oligosaccharide sidechains, one of the high-mannose type, and the other of the complex type. The high-mannose glycan has a low molecular weight (1 kDa), and is responsible for the interaction between the enzyme and concanavalin A. The complex-type glycan has an estimated molecular weight of 2 kDa. It contains one 1 2-linked xylose residue, probably one fucose residue 1 3-linked to the chitobiose unit, and no terminal galactose residue. The two glycans, associated to the 39 kDa polypeptide, constitute the acid -fructosidase of the sweet-pepper fruit.Abbreviations F -fructosidase - ConA concanavalin A - DEAE diethylaminoethyl - DTNB dithionitrobenzoic acid - endo F endo--N-acetylglucosamidase F - endo H endo--N-acetylglucosamidase H - NEM N-ethylmaleimide - PCMB parachloromercurobenzoate - PNGase glycopeptide-N-glycosidase - TFMS trifluoromethane sulfonic acid This work was partly supported by a grant from the Commission Permanente de Coopération Franco-Québécoise to L. Faye, and S. Yelle. D. Michaud was a recipient of a graduate scholarship from the Natural Science and Engineering Research Council of Canada.