The major antennal chemosensory protein of red imported fire ant workers.

Some chemosensory proteins (CSPs) are expressed in insect sensory appendages and are thought to be involved in chemical signalling by ants. We identified 14 unique CSP sequences in expressed sequence tag (EST) libraries of the red imported fire ant, Solenopsis invicta. One member of this group (Si-CSP1) is highly expressed in worker antennae, suggesting an olfactory function. A shotgun proteomic analysis of antennal proteins confirmed the high level of Si-CSP1 expression, and also showed expression of another CSP and two odorant-binding proteins (OBPs). We cloned and expressed the coding sequence for Si-CSP1. We used cyclodextrins as solubilizers to investigate ligand binding. Fire ant cuticular lipids strongly inhibited Si-CSP1 binding to the fluorescent dye N-phenyl-naphthylamine, suggesting cuticular substances are ligands for Si-CSP1. Analysis of the cuticular lipids showed that the endogenous ligands of Si-CSP1 are not cuticular hydrocarbons.

Characterization of new gibberellin-responsive semidwarf mutants of arabidopsis.

Chemical mutagenesis of Arabidopsis thaliana (L.) Heynh. yielded four semidwarf mutants, all of which appeared to be gibberellin (GA)-biosynthesis mutants. All four had atypical response profiles to C20-GAs, suggesting that each had impaired 20-oxidation. One mutant, 11.2, was shown to be allelic to ga5 and has been named ga5-2. It had altered metabolism of [14C]GA15 relative to that in wild-type plants and undetectable levels of C19-GAs in young stems, consistent with the known function of GA5 as a stem-expressed GA 20-oxidase. Two mutants (2.1 and 10.3), which had very short inflorescences and siliques, were allelic to each other but not to the known GA-responding mutants, ga1 to ga5. The locus defined by these two mutations is provisionally named GA6 and is purported to encode an inflorescence- and silique-expressed GA 20-oxidase. A double mutant, ga5-2 ga6-2, had an extreme dwarf phenotype with very short siliques. The fourth mutation, 1.1, gave a phenotype like ga5, but was not allelic to any of the known ga mutations. It has not yet been given a gene symbol pending further studies.

The Gibberellin Status of lip1, a Mutant of Pea That Exhibits Light-Independent Photomorphogenesis.

Dark-grown seedlings of the lip1 (light independent photomorphogenesis) mutant of Pisum sativum L. display many features of de-etiolated growth and are similar in many respects to wild-type (WT) seedlings grown in the light. The involvement of gibberellins (GAs) with the mutant phenotype was examined by applying GA1 and GA20 to the mutant and WT, and by quantifying endogenous GA1, GA8, GA19, GA20, and GA29 levels in the two genotypes. These experiments were conducted in both the light and the dark. In neither environment could GA application restore elongation in the mutant to that in GA-treated WT plants. Quantification of GAs provided further evidence that the mutant phenotype is not attributable to a deficiency in endogenous GA1. However, dark-grown lip1 seedlings contained lower levels of GA19 and higher levels of GA20 than dark-grown WT plants, whereas in the light, the effect of the mutation on the ratio of GA19 to GA20 was reversed. Thus, there appears to be a complex interaction between the lip1 mutation, the light regime, and the step GA19 to GA20.

Expression of the le Mutation in Young Ovaries of Pisum sativum and Its Effect on Fruit Development.

The effect of the le mutation on the growth and gibberellin (GA) content of developing fruits was investigated using the near-isogenic lines of Pisum sativum L. 205+ (LeLe) and 205- (lele). Although stem elongation is known to be reduced in 205- plants by approximately 65%, the growth of pods and seeds was unaffected by the le mutation. GA1, GA3, and GA20 stimulated parthenocarpic development of unpollinated ovaries on both 205+ and 205- plants. GA20 was less active on 205- ovaries than on 205+, whereas GA1 had similar, high activity in both lines. The activity of GA3 was even higher than that of GA1 in both lines. Decapitation of 205+ plants induced parthenocarpic development of unpollinated ovaries, but this treatment was much less effective on 205- plants. The contents of GA1 and GA8 in entire ovaries 6 d after anthesis, as well as in the pod and fertilized ovules, were substantially lower in 205- than in 205+ plants, whereas the reverse was true for the levels of GA20 and GA29. These results suggest that 3[beta]-hydroxylation of GA20 to GA1 is reduced in ovaries as well as in vegetative tissues. Thus, the le mutation appears to be expressed in young reproductive organs of the 205- line, even though it does not affect the fruit phenotype. Because the content of GA3 in the ovary was similar in the two lines, one explanation for the normal fruit size in the 205- line is that GA3 is the native regulator of pod growth. Alternatively, sufficient GA1 may still be produced in 205- fruits to maintain normal pod growth.

Use of an Acylcyclohexanedione Growth Retardant, LAB 198 999, to Determine Whether Gibberellin A(20) Has Biological Activity per se in Dark-Grown Dwarf (le) Seedlings of Pisum sativum.

Dwarf (le(5839)) seedlings of Pisum sativum respond to gibberellin A(20) (GA(20)) in the dark, although the same dosage of GA(20) applied to light-grown le(5839) seedlings elicits no growth response. The acylcyclohexanedione growth retardant, LAB 198 999, which is known to inhibit gibberellin oxidation and in particular 3beta-hydroxylation such as the conversion of GA(20) to GA(1), also inhibits the growth response of dark-grown dwarf (le(5839)) seedlings to GA(20). Thus, the biological activity of GA(20) in the dark appears to be a consequence of its conversion to GA(1), even though it is known from studies with light-grown seedlings that the le mutation reduces the conversion of GA(20) to GA(1).

Immunochromatographic purification of gibberellins from vegetative tissues of Cucumis sativus L: Separation and identification of 13-hydroxy and 13-deoxy gibberellins.

Immunological methods are described for the separation and purification of 13-hydroxy and 13-deoxy-gibberellins of Cucumis sativus. Qualitative and quantitative data show that 13-deoxygibberellins predominate over 13-hydroxygibberellins in stems and leaves of this species.

Gibberellins in developing fruits of Pisum sativum cv. Alaska: Studies on their role in pod growth and seed development.

Gibberellins A1, A8, A20 and A29 were identified by capillary gas chromatography-mass spectrometry in the pods and seeds from 5-d-old pollinated ovaries of pea (Pisum sativum cv. Alaska). These gibberellins were also identified in 4-d-old non-developing, parthenocarpic and pollinated ovaries. The level of gibberellin A1 within these ovary types was correlated with pod size. Gibberellin A1, applied to emasculated ovaries cultured in vitro, was three to five times more active than gibberellin A20. Using pollinated ovary explants cultured in vitro, the effects of inhibitors of gibberellin biosynthesis on pod growth and seed development were examined. The inhibitors retarded pod growth during the first 7 d after anthesis, and this inhibition was reversed by simultaneous application of gibberellin A3. In contrast, the inhibitors, when supplied to 4-d-old pollinated ovaries for 16 d, had little effect on seed fresh weight although they reduced the levels of endogenous gibberellins A20 and A29 in the enlarging seeds to almost zero. Paclobutrazol, which was one of the inhibitors used, is xylem-mobile and it efficiently reduced the level of seed gibberellins without being taken up into the seed. In intact fruits the pod may therefore be a source of precursors for gibberellin biosynthesis in the seed. Overall, the results indicate that gibberellin A1, present in parthenocarpic and pollinated fruits early in development, regulates pod growth. In contrast the high levels of gibberellins A20 and A29, which accumulate during seed enlargement, appear to be unnecessary for normal seed development or for subsequent germination.

Gibberellins in dark- and red-light-grown shoots of dwarf and tall cultivars of Pisum sativum: The quantification, metabolism and biological activity of gibberellins in Progress no. 9 and Alaska.

The stem growth in darkness or in continuous red light of two pea cultivars, Alaska (Le Le, tall) and Progress No. 9 (le le, dwarf), was measured for 13 d. The lengths of the first three internodes in dark-grown seedlings of the two cultivars were similar, substantiating previous literature reports that Progress No. 9 has a tall phenotype in the dark. The biological activity of gibberellin A20 (GA20), which is normally inactive in le le geno-types, was compared in darkness and in red light. Alaska seedlings, regardless of growing conditions, responded to GA20. Dark-grown seedlings of Progress No. 9 also responded to GA20, although red-light-grown seedlings did not. Gibberellin A1 was active in both cultivars, in both darkness and red light. The metabolism of [(13)C(3)H]GA20 has also been studied. In dark-grown shoots of Alaska and Progress No. 9 [(13)C(3)H]GA20 is converted to [(13)C(3)H]GA1, [(13)C(3)H]GA8, [(13)C]GA29, its 2α-epimer, and [(13)C(3)H]GA29-catabolite. [(13)C(3)H] Gibberellin A1 was a minor product which appeared to be rapidly turned over, so that in some feeds only its metabolite, [(13)C(3)H]GA8, was detected. However results do indicate that the tall growth habit of Progress No. 9 in the dark, and its ability to respond to GA20 in the dark may be related to its capacity to 3ß-hydroxylate GA20 to give GA1. In red light the overall metabolism of [(13)C(3)H]GA20 was reduced in both cultivars. There is some evidence that 3ß-hydroxylation of [(13)C(3)H]GA20 can occur in red light-grown Alaska seedlings, but no 3ß-hydroxylated metabolites of [(13)C(3)H]GA20 were observed in red light-grown Progress. Thus the dwarf habit of Progress No. 9 in red light and its inability to respond to GA20 may be related, as in other dwarf genotypes, to its inability to 3ß-hydroxylate GA20 to GA1. However identification and quantification of native GAs in both cultivars showed that red-light-grown Progress does contain native GA1. Thus the inability of red light-grown Progress No. 9 seedlings to respond to, and to 3ß-hydroxylate, applied GA20 may be due to an effect of red light on uptake and compartmentation of GAs.

Gibberellins in immature seeds and dark-grown shoots of Pisum sativum : Gibberellins identified in the tall cultivar Alaska in comparison with those in the dwarf Progress No. 9.

Gibberellins (GAs) A17, A19, A20, A29, A44, 2ßOH-GA44 (tentative) and GA29-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA19 which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA1, GA8, GA20, GA29, GA8-catabolite and GA29-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA8, GA20, GA29 and GA29-catabolite, and the presence of GA1 was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA1 in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA20, GA29 and GA29-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 10(2)- to 10(3)-fold less than the levels in developing seeds. The 2-epimer of GA29 is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.

Identification and localization of gibberellins in maturing seeds of the cucurbit Sechium edule, and a comparison between this cucurbit and the legume Phaseolus coccineus.

Twenty known gibberellins (GAs) have been identified by combined capillary gas chromatography-mass spectrometry in extracts from less than 10 g fresh weight of maturing seeds of the cucurbit Sechium edule Sw. The GAs are predominantly 3- and-or 13-hydroxylated. This is the first reported identification of non-conjugated 13-hydroxylated GAs in a cucurbit. Gibberellin A8 and gibberellin A8-catabolite are the major GAs in terms of quantity and are largely accumulated in the testa. The catabolites of 2ß-hydroxylated GAs are α,ß-unsaturated ketones which no longer possess of a γ-lactone. They were hitherto known only in legumes. The presence of GA8-catabolite as a major component of Sechium seeds indicates that the distribution of these GA-catabolites may be more widespread than previously envisaged. The localization of known GAs in maturing seeds of the legume Phaseolus coccineus L. was found to resemble closely that in Sechium. Gibberellin A8, a putative conjugate of GA8 and GA8-catabolite are accumulated in the testa. The localization in the testa of end-products of the GA-biosynthetic pathway, which was first observed in maturing seeds of Pisum sativum, and is now described in Phaseolus and Sechium, may be a general feature of seed development.

Metabolism of gibberellins by immature barley grain.

Gibberellins A1, A4, A9, A12-aldehyde, A20 and A51, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented.

The localization, metabolism and biological activity of gibberellins in maturing and germinating seeds of Pisum sativum cv. Progress No. 9.

Gibberellin A20 (GA20), GA29 and GA29-catabolite were quantified in cotyledons, embryonic axes, and testas of Pisum sativum cv. Progress No. 9 throughout the final stages of seed maturation and during germination. Stable isotope-labelled GAs were used as internal standards in conjunction with combined gas chromatography-mass spectrometry. Gibberellin A20 and GA29 were mainly located in the cotyledons of maturing seeds, and GA29-catabolite was predominantly located in the testa. Stable isotope- and radio-labelled GA20 and GA29 were fed to both intact seeds developing in vivo, and to isolated seed parts cultured in vitro. The combined results of in-vivo and in-vitro feeds indicated that GA20 is metabolised to GA29 in the cotyledons, that GA29 is transported from the cotyledons to the testa, and that GA29 is metabolised to GA29-catabolite in the testa. Although the metabolism of GA20 in the cotyledons and of GA29 in the testa has been shown definitively, the mobility of GA29 has not yet been demonstrated directly. During seed desiccation and germination GA29-catabolite and products arising from it are transferred from the testa into the embryo. There is no evidence of a physiological function for GA29-catabolite in germination or early seedling growth. Use of a growth retardant indicates that seedling growth, but not germination, is dependent on de-novo GA biosynthesis.

Metabolism of [(13)C 1]gibberellin A 29 to [ (13)C 1]gibberellin-catabolite in maturing seeds of Pisum sativum cv. Progress No. 9.

The metabolism of GA29 during seed maturation in Pisum sativum cv. Progress No. 9 was further investigated. [17-(13)C1]GA29 was metabolised to a GA-catabolite (structure 3), with incorporation of the [(13)C] label from the GA29 substrate into the GA-catabolite being demonstrated by GC-MS. Quantitation of the GA-catabolite using GC-MS was achieved by adding GA-catabolite, labelled with [(18)O], to seed extracts as an internal standard. At least 50% conversion of [(13)C1]GA29 to [(13)C1]GA-catabolite was demonstrated with the build up of exogenous [(13)C1]GA-catabolite strictly paralleling the accumulation of native GA-catabolite. These results strongly suggest that conversion of GA29 to the GA-catabolite is a natural metabolic step occurring during the final stages of seed maturation. 25 µg per seed of native GA-catabolite was recorded in 37 day old seeds. Some problems encountered in the analysis of extracts containing the GA-catabolite are discussed briefly.

The identification of gibberellins in immature seeds of Vicia faba, and some chemotaxonomic considerations.

GA17, GA19, GA20, GA29, GA44 and 13-hydroxy-GA12, now named GA53, were identified by GC-MS in immature seeds of Vicia faba (broad bean). Also identified were a GA catabolite, two polyhydroxykauranoic acids, and abscisic, phaseic and dihydrophaseic acids. The GAs of Vicia are hydroxylated at C-13, in common with those of other legumes. However the GAs of Vicia are not hydroxylated at C-3, nor do they appear to be readily conjugated. In these respects Vicia resembles Pisum, another member of the tribe Viciae. Vicia differs from Phaseolus and Vigna, of the tribe Phaseoleae, in both these respects.

Metabolism of gibberellin A29 in seeds of Pisum sativum cv. Progress No. 9; Use of [(2)H] and [ (3)H]GAs, and the identification of a new GA catabolite.

The metabolism of GA29 in maturing seeds of Pisum sativum cv. Progress No. 9 was further investigated, and the utility of (2)H-labelled GAs in conjuction with GC-MS is illustrated. Using [2α-(2)H1]GA29 as an internal standard, endogenous GA29 was shown to reach a maximal level (ca. 10 µg/seed) 27 days from anthesis, and to decline to ca. 1.6 µg/seed in mature seeds. In a time-course feed the metabolism of [2α-(2)H1] [2α-(3)H1]GA29 applied to 27 day old seeds, and of endogenous GA29, was compared from the (1)H:(2)H ratios in the recovered GA29. Although both [2α-(2)H1] [2α-(3)H1]GA29 and endogenous GA29 were metabolised to the same limited extent to a putative conjugate, in the main metabolic process endogenous GA29 was preferentially converted to an untraceable (i.e. unlabelled) metabolite. In contrast, endogenous GA29 and [1ß,3α-(2)H2] [1ß,3α-(3)H2]GA29, derived from [1ß,3α-(2)H2] [1ß,3α-(3)H2]GA20 in a time-course feed, were metabolised in an identical manner. In the latter case isotope loss precluded identification of the metabolite. The structure (8) has been assigned to a GA catabolite present in maturing seeds and seedlings of pea. The isotope data are consistent with this compound being the hitherto untraced metabolite of GA29 in pea.

Further studies on the metabolism of gibberellins (GAs) A9, A 20 and A 29 in immature seeds of Pisum sativum cv. progress No. 9.

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under "summer" and "winter" conditions the metabolism of [(3)H]GA9 in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [(3)H]GA9→[(3)H]GA20 is restricted to early stages of seed development. 2ß-Hydroxylation of [(3)H]GA9→2ß-OH-[(3)H]GA9 has only been observed at a stage of development after endogenous GA9 has accumulated. 2ß-OH-GA9 has been shown to be endogenous to pea and is named GA51. H2-GA31 and its conjugate have not been shown to be present in pea and may be induced metabolites of [(3)H]GA9. The metabolism of GA20→GA29 is used to illustrate a technique of feeding [(2)H][(3)H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [(3)H]GA20→[(3)H]GA29, and the virtual non-metabolism of [(3)H]GA29 have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.

The biological activities of some new gibberellins (GAs) in six plant bioassays.

The biological activities of GA40, GA43, GA46, GA47, GA51 and GA4 20,4-lactone were tested over a wide range of concentrations in six plant bioassays. GA4 20,4-lactone showed the highest activity. Of the two 2α-hydroxylated compounds GA47 showed moderately high activity, and GA40 was slightly active. The 2ß-hydroxylated compunds GA43, GA46 and GA51 were virtually inactive.