Chemical signals from host plant and sexual behavior in a moth.

In the phytophagous corn earworm, Helicoverpa (Heliothis) zea, females delay their reproductive behaviors until they find a suitable host on which to deposit their eggs. Perception of volatile chemical signals from corn silk triggers the production of sex pheromone followed by its release, which leads to mating. Several natural corn silk volatiles, including the plant hormone ethylene, induced pheromone production in H. zea females. Because H. zea larvae feed on the fruiting parts of a wide variety of hosts, ethylene, which is associated with fruit ripening, could act as a common cue.

Palmitic acid activation of peroxidase and its possible significance in mango ripening.

Palmitic acid stimulated the activity of mango peroxidase and reversed the inhibition due to the peroxidase inhibitor present in the preclimacteric fruit. The palmitic acid effect appeared to saturate in the range of 45 to 60 muM palmitic acid. Crude fatty acid extract of the mango exerted similar effect. The percentage stimulation was pH-dependent. Palmitic acid stimulated the enzyme by 18 percent at its optimum pH (5) but the stimulation was in excess of 63 percent at pH 2.5. At pH 2.5 the enzyme concentration versus velocity plot was non-linear and the activation by palmitic acid appeared to saturate between 32 and 48 muM concentration of the effector. The inhibition of the enzyme at and above 0.86 muM concentration of substrate (H202) was not found in the presence of palmitic acid. The effector also changed the heat inactivation kinetics of the enzyme and activated only two out of the four peroxidase isoenzymes present in the climacteric fruit extracts. The results presented indicate the regulatory nature of the enzyme and support its significance in fruit ripening.

8-azaguanine and flavinogenesis in Eremothecium ashbyii.

8-Azaguanine (10- minus 4 M) supplementation in synthetic medium inhibited flavinogenesis in Eremothecium ashbyii to far greater extent (68per cent) than the growth (25 per cent). That enzymes comprising the biosynthetic pathway of riboflavin are synthesized during early growth phase of the organism is supported by the data presented. 8-Azaguanine mediated inhibition in flavinogenesis was closely related with decreased levels of ribose-5'-phosphatase, ribose reductase and ribitol kinase, the enzymes involved in supplying ribitol for flavinogenesis. Addition of guanine and not ribitol during early growth phase to 8-azaguanine-added cultures released the inhibition of riboflavin synthesis and restored the enzyme levels in the presence of the antimetabolite.

Isolation and Identification of Ripening-Related Tomato Fruit Carboxypeptidase.

Tomato (Lycopersicon esculentum) fruit carboxypeptidase active on N-carbobenzoxy Z-L-phenylalanine-L-alanine was found to constitute a family of isoforms whose abundance changed differentially during ripening. A specific polyclonal antibody against the fruit carboxypeptidase was raised in rabbits and used to purify and identify the protein. The data from immunoaffinity chromatography, immunoinhibition studies, immunoprecipitation of the in vivo- and in vitro-labeled proteins, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of native isoforms strongly suggest that the fruit carboxypeptidases are monomers or oligomers of 68- and/or 43-kD subunits.

Tomato Fruit Carboxypeptidase (Properties, Induction upon Wounding, and Immunocytochemical Localization).

Carboxypeptidase activity was characterized during ripening and wounding of tomato (Lycopersicon esculentum) fruit. The fruit enzyme shares substrate specificity and susceptibility to the inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride with other plant carboxypeptidases. The abundance and stability of wound-induced carboxypeptidase were developmentally regulated. Oxidative stress caused by cupric ions impaired the membrane permeability in the slices from pink fruit, resulting in leakage of the carboxypeptidase into the medium and in its redistribution in the cell. The patterns of carboxypeptidase activity did not parallel the cupric ion effect on ethylene levels. Immunogold electron microscopy studies indicated that the fruit carboxypeptidase is associated with electron-dense inclusions in the vacuole.

Evidence for light-dependent and light-independent protein dephosphorylation in chloroplasts.

A number of photosystem II (PSII) associated proteins, including core proteins D1, D2 and CP43, and several proteins of the LHCII complex, are phosphorylated by a thylakoid-bound, redox-regulated kinase(s). We demonstrate here that the compound propyl gallate is an effective inhibitor of LHCII phosphorylation in vivo while having little effect on PSII core protein phosphorylation. Using this inhibitor, we demonstrate that LHCII dephosphorylation is insensitive to light in vivo. Taken together with our previous conclusion (Elich et al., EMBO J. 12 (1993) 4857-4862) that PSII core protein dephosphorylation is light-stimulated, our data suggest the presence of multiple phosphatases responsible for thylakoid protein dephosphorylation in vivo.

A functional tomato ACC synthase expressed in Escherichia coli demonstrates suicidal inactivation by its substrate S-adenosylmethionine.

1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme in the biosynthesis of the plant hormone, ethylene. We have isolated, sequenced and expressed a functional tomato (cv Pik-Red) ACC synthase gene in Escherichia coli. ACC synthase expressed in E. coli was inactivated by incubation with S-adenosylmethionine (SAM), the half-time of which was concentration dependent. Mixing the tomato fruit protein extract with the cell-free extract from transformed E. coli did not affect SAM-dependent inactivation of ACC synthase activity. Thus, single isoforms of the ACC synthase enzyme, which demonstrate the biochemical features expected of the tomato fruit enzyme, can be expressed in E. coli and their structure-function relationships investigated.

High level expression of "male specific" pheromone binding proteins (PBPs) in the antennae of female noctuiid moths.

Pheromone Binding Proteins (PBPs) are one branch of a multigene family of lepidopteran Odorant Binding Proteins (OBPs) that are known for their relatively high levels of expression in male antennae. However, PBP expression has been observed at low levels in female antennae of the Saturniidae, Bombycidae and Lymantriidae, and at relatively high levels in members of the Noctuiidae. The function of female PBP expression is unclear, as female lepidoptera are consistently noted for their failure to respond physiologically or behaviorally to sex-pheromone. In this study, the sexual dimorphism of PBP expression was examined in the noctuiid moths Helicoverpa zea, Heliothis virescens and Spodoptera frugiperda. A PBP cDNA clone was isolated from female H. zea, PBP-Hzea(f). Northern blot analysis indicated relatively high levels of PBP-Hzea(f) expression in both male and female antennae, though females consistently expressed about 50% that of males. Western blot analysis of male and female PBP expression supported these relative differences. Immunocytochemical analysis indicates discrete expression localized beneath olfactory sensilla of both male and female antennae. These results suggest female noctuiids possess the biochemistry to detect at least components of their sex-pheromone. Alternatively, these results may suggest that PBPs have a more general function in noctuiids, possibly reflecting behavioral and life history differences that distinguish this the Noctuiidae from other Lepidopteran families.

Ethylene binding during leaf development and senescence and its inhibition by silver nitrate.

Ethylene binding in situ was tested in leaves of tobacco (Nicotiana tabacum L. var. Xanthi) and of gynoecious and monoecious genotypes of cucumber (Cucumis sativus L.) during their development and senescence. Ethylene binding per gm fresh leaf remained constant during early stages of tobacco leaf development. It decreased in fully expanded leaves and during senescence of detached tobacco leaves. On a per leaf basis, ethylene binding increased as the leaf developed. The pattern for changes in ethylene binding by gynoecious and monoecious cucumber leaves were different from each other. Short-term treatment of detached tobacco and cucumber leaves with a solution of AgNO(3) (50 µM) markedly decreased their ability to bind ethylene concomitant to an inhibition in chlorophyll breakdown. The data support the hypothesis that ethylene-induced chlorophyll breakdown during leaf senescence may require the binding of ethylene to its receptor in vivo.

A wound-repressible glycine-rich protein transcript is enriched in vascular bundles of tomato fruit and stem.

The deduced protein sequence of a partial tomato cDNA clone is rich in the amino acid glycine and contains repeats of the amino acid sequence, (Gly)5Tyr(Gly)4-5Tyr(Gly)3ArgArgGlu. This protein sequence has significant similarity to a sorghum glycine-rich protein [S1, Cretin and Puigdomenech (1990) Plant Mol. Biol. 15: 783] and a maize embryo, abscisic acid-induced glycine-rich protein [Gomez et al. (1988) Nature 334: 262]. Tissue printing was used to localize the glycine-rich protein transcript in tomato fruit, stem and hypocotyl sections. The transcript is present throughout the tomato fruit pericarp but is enriched in the vascular bundles. In tomato hypocotyl tissue prints, the glycine-rich protein transcript as well as rRNA were localized within the vascular tissue. This shows that differential loading of RNA may occur when using the tissue printing technique on hypocotyl sections. Direct isolation and comparison of RNA from vascular-containing and non-vascular stem tissue confirmed, however, that the glycine-rich protein transcript is accumulated abundantly in the vascular tissue of tomato stem.

Deletion of the carboxyl-terminal region of 1-aminocyclopropane-1-carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme.

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is a key enzyme regulating biosynthesis of the plant hormone ethylene. The expression of an enzymatically active, wound-inducible tomato (Lycopersicon esculentum L. cv Pik-Red) ACC synthase (485 amino acids long) in a heterologous Escherichia coli system allowed us to study the importance of hypervariable COOH terminus in enzymatic activity and protein conformation. We constructed several deletion mutants of the gene, expressed these in E. coli, purified the protein products to apparent homogeneity, and analyzed both conformation and enzyme kinetic parameters of the wild-type and truncated ACC syntheses. Deletion of the COOH terminus through Arg429 results in complete inactivation of the enzyme. Deletion of 46-52 amino acids from the COOH terminus results in an enzyme that has nine times higher affinity for the substrate S-adenosylmethionine than the wild-type enzyme. The highly efficient, truncated ACC synthase was found to be a monomer of 52 +/- 1.8 kDa as determined by gel filtration, whereas the wild-type ACC synthase, analyzed under similar conditions, is a dimer. These results demonstrate that the non-conserved COOH terminus of ACC synthase affects its enzymatic function as well as dimerization.

Wound-regulated accumulation of specific transcripts in tomato fruit: interactions with fruit development, ethylene and light.

Regulation of three cDNA clones (pT52, pT53, and pT58) was analyzed in terms of wounding alone and wounding in conjunction with developmental and environmental cues (ripening, ethylene, and light) in tomato fruit tissue. The pT52-specific transcript level is induced by wounding in early-red and red stage fruit and by ethylene. The pT58-specific transcript level is also induced by wounding and ethylene in early-red stage fruit but is not induced by wounding in red fruit. The pT53-specific transcript level is repressed by wounding in early-red and red stage fruit. Like the pT52- and pT58-specific transcripts, the pT53-specific transcript is induced by ethylene. Furthermore, the level of the pT52-specific transcript is regulated by light. Analysis of unwounded tissue showed that the abundance of each cDNA-specific transcript changes during fruit ripening and that each of the transcripts is present in other plant organs as well. This analysis provides information about the interactions between developmental and environmental factors affecting these genes.

Intramembrane translocation and posttranslational palmitoylation of the chloroplast 32-kDa herbicide-binding protein.

The 32-kDa herbicide-binding protein, a component of photosystem II, is synthesized as a membrane-associated 33.5-kDa precursor within the chloroplast. We show that membrane attachment of the precursor and processing to the 32-kDa form occur in the unstacked stromal lamellae. Once processed, the 32-kDa protein translocates, within the thylakoids, to the topologically distinct stacked granal lamellae. Posttranslational palmitoylation of the processed 32-kDa protein is also shown to occur. This modification takes place in a membrane-protected domain and is mainly confined to the protein assembled in the granal lamellae, where functional photosystem II centers are concentrated.

Subcellular Distributions of Isoenzymes in Fruits of a Normal Cultivar of Tomato and of the rin Mutant at Two Stages of Development.

Fruits of tomato (Lycopersicon esculentum Mill.) cv. Rutgers and of a nearly isogenic stock containing the ripening inhibitor gene rin harvested at green (66% mature) and ripe (107% mature) stages were studied for the subcellular distribution of isoenzymes using isoelectric focusing. The enzymes studied were peroxidases, esterases, phosphatases, phosphorylase, malate dehydrogenases, and IAA oxidases. During ripening of normal fruit the activities in the supernatant fraction of all of these enzymes, except malate dehydrogenase, decreased. In the particulate fractions some enzymes decreased while others increased in activity. The rin gene inhibited only some of the changes which occurred during ripening of normal fruit. It is postulated that changes in the degree to which enzymes are bound to membranes comprise one of the mechanisms by which the activities of enzymes are controlled in tomato pericarp, and that these membranes remain intact during ripening.