Promotion of flowering in the Pinaceae by gibberellins

Significant female flowering of 6- to 11-year-old seedlings and grafted ramets of sexually mature scions of lodgepole pine (Pinus contorta Dougl.) was promoted by both topical and spray applications of a gibberellin (GA) A_4/7 mixture (1.6 to c. 5 mg per plant in total) during that period (June to September) when sexual differentiation of lateral primordia would be expected to take place. Girdling was used in most experiments to enhance the GA_4/7 effect, as was the auxin, naphthaleneacetic acid (NAA). Average frequency of flowering branches on treated plants over all experiments ranged from 27 to 59% (control ranged from 0 to 36%) and average number of female strobili was increased from 2- to 6-fold by growth regulator treatment, relative to controls. Within an experiment, clonal or family frequency of flowering for treated plants ranged from 11 to 67% (controls were 0 to 28%), and number of female strobili was increased from 2- to 14-fold by growth regulator treatment, relative to controls. Movement of the flowering stimulus from the point of application was apparent in several experiments, the response in adjacent branches being correlated positively with increasing dosage of GA_4/7. Significant male flowering occurred only in one experiment, girdling and GA_4/7 treatment being promotive factors. The use of spray applications of GA_4/7+ NAA is warranted to induce early and enhanced flowering in lodgepole pine seedlings and vegetative propagules for genetic improvement programs.     

Indole-3-acetic acid in Douglas fir seedlings: A reappraisal

The use of ring-labelled, pentadeutero IAA as an internal standard in selected ion monitoring analysis of Douglas fir seedlings revealed an estimate of IAA which was nearly an order of magnitude smaller than that reported earlier.     

Effect of exogenous gibberellins on flowering in Pinus sylvestris grafts

Ethanolic sprays of gibberellins were applied to developing shoots of about 12-year-old Scots pine (Pinus sylvestris L.) grafts during the shoot elongation period in two consecutive years. Both male and female flowering was increased by these treatments in both years. The effect was particularly distinct in male flowering. However, different clones showed varying responses to the treatment. This variation was associated with both the genotype and the environmentally determined year-to-year fluctuation in flowering. Differences among clones were analysed further by introducing a model earlier developed for comparisons of the growth rhythm in various woody and herbaceous species.     

Flowering in tobacco needs gibberellins but is not promoted by the levels of active GA1 and GA4 in the apical shoot

Flowering of Nicotiana tabacum cv Xhanti depends on gibberellins because gibberellin-deficient plants, due to overexpression of a gibberellin 2-oxidase gene (35S:NoGA2ox3) or to treatment with the gibberellin biosynthesis inhibitor paclobutrazol, flowered later than wild type. These plants also showed inhibition of the expression of molecular markers related to floral transition (NtMADS-4 and NtMADS-11). To investigate further the role of gibberellin in flowering, we quantified its content in tobacco plants during development. We found a progressive reduction in the levels of GA1 and GA4 in the apical shoot during vegetative growth, reaching very low levels at floral transition and beyond. This excludes these two gibberellins as flowering-promoting factors in the apex. The evolution of active gibberellin content in apical shoots agrees with the expression patterns of gibberellin metabolism genes: two encoding gibberellin 20-oxidases (NtGA20ox1 = Ntc12, NtGA20ox2 = Ntc16), one encoding a gibberellin 3-oxidase (NtGA3ox1 = Nty) and one encoding a gibberellin 2-oxidase (NtGA2ox1), suggesting that active gibberellins are locally synthesized. In young apical leaves, GA1 and GA4 content and the expression of gibberellin metabolism genes were rather constant. Our results support that floral transition in tobacco, in contrast to that in Arabidopsis, is not regulated by the levels of GA1 and GA4 in apical shoots, although reaching a threshold in gibberellin levels may be necessary to allow meristem competence for flowering.     

Detection and identification of gibberellins in Douglas fir (Pseudotsuga menziesii) shoots

Extracts of Douglas fir ( Pseudotsuga menziesii [Mirb.] Franco) shoots were purified by reversed and normal phase HPLC; gibberellin (GA)-like compounds detected by radioimmunoassay with antibodies against GA₄ and the Tan-ginbozu dwarf rice micro-drop biossay were analyzed by GC-MS. Three major components were identified as GA₄, GA₇, and GA₉ while smaller amounts of GA¹, GA₃ and putative GA⁹-glucosyl ester were also present.     

Geibberllin and temperature influence carbohydrate content and flowering in Phalaenopsis

When Phalaenopsis amabilis is grown under high temperature (30/25°C, day/night), flowering is blocked, and this can be reversed by gibberellin A₃ (GA₃) treatment. Associated with GA₃ treatment under high temperature are increases in sucrose, glucose and fructose as compared with warm-treated plants. Spraying with sucrose solution alone caused leaf epinasty in plants grown under high temperature. Epinasty was released by about 9 days of GA₃ treatment. In GA₃-treated plants under high temperatures, sucrose application to the source leaves led to an increase in sugar content in both leaves and inflorescence. In contrast, although in warm-treated plants sucrose application to the source leaves increased sugar content in the leaves, it did not increase sucrose content in the inflorescence. These results corroborate our hypothesis that in Phalaenopsis GA₃ stimulates sink activity in the apical meristem and promotes the translocation of sucrose from source leaves to the apex of the inflorescence, where it accumulates. GA₃ treatment led to an increase in sucrose synthase activity and had no effect on invertase activity.     

Karyotyping of three Pinaceae species via fluorescent in situ hybridization and computer-aided chromosome analysis

The positions of 18/25S rRNA genes, 5S RNA genes and of Arabidopsis-type telomeric repeats were localized by fluorescent in situ hybridization (FISH) on the chromosomes of three coniferous species; Picea abies, Larix decidua and Pinus sylvestris, each with 2n=24 chromosomes. Computer-aided chromosome analysis was performed on the basis of the chromosome length, the arm length ratio and the position of the hybridization signals. This enabled the chromosomes of the Norway spruce, 4 chromosomes of the European larch and 3 of the karyotype of the Scots pine to be individually distinguished. With respect to the chromosomal positions of rDNA and 5S rDNA loci, chromosome pair I of P. sylvestris is suggested to be homoeologous to pair II of P. abies, while another chromosome pair of P. sylvestris might be homoeologous to chromosome pair III of L. decidua.     

Promotion of Flowering in the Pinaceae by Gibberellins

Flowering was significantly promoted in 4-year-old grafts of mature coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) clones by exogenous gibberellins (GAs) A₄ and A₇ (as a mixture) applied alone and in combination with A₅ and A₉. Biweekly applications of 400 μg GA_4/7 per branch between late March and late June gave a 5-fold increase in ovulate and 3-fold increase in staminate strobilus production over untreated controls. ⁶N-benzyladenine and 2,3,5-triiodobenzoic acid applied in combination with GAs had no consistent effect on strobilus production. Non-destructive branch girdling, ineffective by itself as a cultural treatment, enhanced the GA benefit to flowering. Exogenous application of GA_4/7 is effective and appears to be a practical method for promotion of early and enhanced flowering in grafted Douglas-fir seed orchards.     

Promotion of Flowering in the Pinaceae by Gibberellins

Gibberellins (GAs) were effective in promoting flowering in sexually mature (45-year-old scions) grafts of loblolly pine (Pinus taeda L.). Seed-cone production was increased 12-fold in field-grown, grafted ramets by bi-weekly, May-September applications of 500 μg per branch of GA_4/7. Gibberellin A₃ was equally effective at 500 μg but not at 100 μg per branch, while GA₅ was ineffective at either concentration. A second study using potted, less-sexually mature (8-to 10-year-old scions) grafts gave a reduced level of seed cones in response to GAs. However, even on these younger grafts GA_4/7 was a significant promotive treatment, GA₃ being considerably less effective. Branch girdling, tested as an adjunct treatment, was ineffective. It is now apparent that exogenous applications of GA_4/7 are effective on a number of Pinaceae species, and their use to promote earlier and more abundant flowering in breeding orchards of grafted ramets for at least two species, loblolly pine and Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] is practical.     

Parasitism of seed of Douglas fir (<Emphasis Type=Italic>Pseudotsuga menziesii</Emphasis>) by the seed chalcid, <Emphasis Type=Italic>Megastigmus spermotrophus</Emphasis>, and its influence on seed hormone physiology

Parasitism of Douglas fir (Pseudotsuga menziesii) megagametophytes by the seed chalcid, Megastigmus spermotrophus Wachtl, occurs naturally after pollination but before fertilization. In the absence of fertilization, the presence of insect larvae within the megagametophyte prevents abortion and the storage tissue continues to develop as if the seed had been fertilized. We investigated the effect of parasitism on the metabolism of abscisic acid (ABA), auxins, cytokinins, and gibberellins during early development of Douglas fir seeds. Hormones and hormone metabolites of infested and uninfested megagametophytes with or without pollination were analyzed by HPLC–ESI/MS/MS. At 1 week after Megastigmus introduction, the insect’s presence stimulated ABA accumulation in unpollinated megagametophytes compared to unpollinated, unparasitized megagametophytes. In pollinated material, parasitism did not stimulate ABA accumulation compared to levels present in unparasitized megagametophytes. In all four treatments, the metabolism of ABA occurred primarily through conjugation to the ABA glucose ester (ABAGE), while the 7′-, 8′- and, 9′-hydroxylation pathways were only minor. ABAGE levels declined with time in all treatments and this occurred to a greater extent in pollinated, parasitized megagametophytes, suggesting that the insect’s presence induced the dramatic decrease in ABAGE. Although there were temporal variations in the auxin, cytokinin, and gibberellin profiles of parasitized megagametophytes, the profiles were generally similar to those of unparasitized megagametophytes. Our results suggest that failure of parasitized megagametophytes to abort may be due to the insect inducing similar hormone profiles to those present during normal development of Douglas fir seed.     

Control of tuberisation in potato by gibberellins and phytochrome B

Phytochrome B-deficient plants exhibit increased gibberellin (GA) levels or responsiveness, which may contribute to their elongated growth and reduced chlorophyll levels. We have investigated the effects of applications of gibberellic acid and an inhibitor of gibberellin biosynthesis, ancymidol, on wild-type and phytochrome B-antisense potato (Solanum tuberosum ssp. andigena) plants. The results showed that some phenotypes of the phytochrome B-antisense plants, i.e. increased stem length and reduced chlorophyll, can be mimicked by treating wild-type plants with gibberellic acid. However, another phenotype, i.e. tuberisation response in long days, is mimicked by application of a GA biosynthesis inhibitor ancymidol, thus appearing to be the result of a reduction in the gibberellin levels. A simple increase in gibberellin levels or sensitivity is, therefore, not sufficient to explain the phenotype of the antisense plants.     

Changes in carbohydrate contents of <Emphasis Type="Italic">Zantedeschia</Emphasis> leaves under gibberellin-stimulated flowering

The effect of gibberellic acid (GA₃) on the carbohydrate accumulation in relation to vegetative growth of Zantedeschia ‘Black Magic’ plants undergoing transition to flowering was investigated. In response to GA treatment the carbohydrate level increased independently of earlier stimulation of shoot emergence. Under vegetative growth stage the content of reducing sugars of leaf blades was 2.5-fold higher than in control plants, and suggests the stimulation of photosynthetic activity. The changes observed during the flowering, in principle noted in petiole tissues, support the GA-effect on assimilate transport to the sink organs. Moreover, the high level of non-structural carbohydrates in petiole tissues, in particular reducing sugars, can be an effect of photosynthetic activity of these organs and/or essential for osmoregulation and high turgor pressure. The results indicate that apart from the influence on the shoot emergence, the GAs may stimulate the photosynthetic activity from the beginning of shoot growth and are thus responsible for the enhancement of callas flower yield.     

The differential effects of C-16,17-dihydro gibberellins and related compounds on stem elongation and flowering in Lolium temulentum

Plants of Lolium temulentum L. cv. Ceres grown under short days (SDs) can be induced to initiate inflorescences either by exposure to one long day (LD) or by single applications of some gibberellins (GAs), which also enhance the flowering response to one LD. Single doses of up to 25 μg per plant of C-16, 17-dihydro-GA₅ were about as effective as GA₅ for promoting flowering after one LD but inhibited stem elongation by up to 40% over three weeks. The promotion of flowering but not the inhibition of elongation by 16, 17-dihydro-GA₅ was reduced in SDs or in LDs low in far-red (FR) radiation. With shoot apices cultured in vitro, 16, 17-dihydro-GA₅ was more florigenic than GA₃ for apices excised after one LD of 14 h or more, but less florigenic for apices excised from plants in shorter days. 16, 17-Dihydro-GA₅ was ineffective compared with GA₁, GA₃ and GA₅ for α-amylase production by half-seeds of Lolium, a response concordant with its effect on stem elongation. As with GA₅, 16, 17-dihydro derivatives of GA₁, GA₃, GA₂₀ and several other GAs were more effective for flowering and less effective for stem elongation than the GAs from which they were derived. Hydroxylation at C-17 and/or C-16 generally reduced the effectiveness of 16, 17-dihydro-GA₅ for flowering. These results extend the known features of GA structure which favour flowering relative to stem elongation in L. temulentum. Moreover, C-16, 17-dihydro-GA₅ mimics, in its daylength- and wavelength-dependence and lack of stem elongation, characteristics of the LD stimulus in L. temulentum.     

Iron-rich particles in embryos of seeds from the family Pinaceae

Summary Iron-rich particles, previously reported in seeds of members of the genus Pinus, were found in radicle-hypocotyl tissues of dry embryos from eight other genera in the family Pinaceae. Thus, these Fe-rich particles are of common occurrence in seeds of this conifer family. These particles were most difficult to locate inPseudolarix amabilis, which has green embryos. Energy-dispersive X-ray analysis was used to determine the elements present in conifer Fe-rich particles and phytoferritin deposits in pea embryo axes. Ferich particles from all species studied contained mainly Fe and P but also contained considerable K and Mg. Abietoideae group I (genera Cedrus andAbies) had lower Fe P ratios compared to all the other combined subfamilies within the Pinaceae. Pea phytoferritin deposits contained markedly lower amounts of P relative to Fe based on peakto-background ratios and quantitative values calculated by using a ferric phosphate standard. We also found, for the first time, that pea phytoferritin contained considerable K. A strong similarity was found between the energy-dispersive X-ray analysis spectra from Ferich particles and portions of a laboratory-synthesized Fe, K, Mg phytate salt. Phytate is a common mineral-nutrient storage compound in seeds. The possibility of these Fe-rich particles being phytoferritin cannot be ruled out, but if they are phytoferritin, they have lower Fe P ratios than almost all other ferritins reported to date.Abbreviations EDX energy-dispersive X-ray     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Cytokinins and gibberellins in sap exudate of the oil palm

Exudates were collected from stumps of pre-anthesis inflorescences of oil palm and analysed for cytokinin and gibberellin content using combined HPLC-ELISA techniques. Three antisera, for zeatin-type, dihydrozeatin-type and isopentenyladenine-type cytokinins, were used in ELISAs to identify members of these three groups of cytokinins. Ribotides, 9-glucosides, free bases and ribosides were detected for each of the groups with zeatin riboside the most abundant cytokinin identified in the exudate. Isopentenyladenine-type and dihydrozeatin-type cytokinins were also identified but at lower levels. In addition, two monoclonal antibodies were used in the development of novel ELISAs for members of the 13-hydroxylated and non-13-hydroxylated families of gibberellins. The new ELISAs allow the determination of gibberellins in smaller amounts of tissue than are required for GC-MS. The most abundant gibberellins identified in exudates were GA19 and GA44, as well as other members of the early 13-hydroxylation pathway. Gibberellins were confirmed by GC-MS. The presence of these types of growth regulators in exudate supplying immature inflorescences suggest they have a role in growth and development of these structures.     

Stimulation of shoot elongation in Salix pentandra by gibberellin A9; activity appears to be dependent upon hydroxylation to GAl via GA20

Cessation of shoot elongation in seedlings of Salix pentandra L. is induced by short photoperiod. Gibbereliin A₉ (GA₉) applied either to the apical bud or injected into a mature leaf, induced shoot elongation under a short photoperiod of 12 h, and GA₉ could completely substitute for a transfer to a long photoperiod. When [³H]GA₉ or [²H₂]GA₉ was injected into a leaf, no [³H]GA₉ was detected in the elongating apex and only traces of [³H]GA₉ were found in the shoot above the treated leaf. By the use of gas chromatography-mass spectrometry (GC-MS), [²H₂]GA₂₀ was identified as the main metabolite of [²H₂]GA₉ in both the shoot and the treated leaf. In addition, [²H₂]GA₁ and [²H₂]GA₂₉ were also identified as metabolites of [²H₂]GA₉. These results are consistent with the hypothesis that exogenous GA, promotes shoot elongation in Salix through its metabolism to GA₂₀ and GA,.     

Isolation and characterization of polymorphic microsatellite markers in the endangered Nothotsuga longibracteata (Pinaceae)

Twelve polymorphic microsatellite loci were isolated and characterized from an AC‐enriched genomic library of Nothotsuga longibracteata. The average allele number of these microsatellites was 8.3 per locus, ranging from two to 13. The ranges of observed and expected heterozygosities were 0.03–0.97 and 0.09–0.88, respectively. These polymorphic markers provide useful tools for the study of evolutionary history and conservation genetics of N. longibracteata.     

Microbiological oxidation of sterols by Coriolus hirstus

Fermentation of cholesterol with a culture of Coriolus hirstus yielded a mixture containing 7-oxo-cholesterol and hydroxylated-cholesterol derivatives. Preparation of a possible precursor of antheridiol, 7-oxofucosterol, by this fungus was also examined.