Role of ethylene in cotyledon development of microspore-derived embryos of Brassica napus

Ethylene production during seed development in Brassica napus occurs first at 20 d after pollination (DAP), while a second greater peak occurs at 35 DAP. Because of the inaccessible location of the embryo within the maternal tissue, microspore-derived embryos (MDEs) of B. napus were used as a model for studying the role of ethylene during embryo development. The MDEs also produced a peak in ethylene evolution at 20 DAC (i.e. the early cotyledonary stage), dropping to minimal levels by 25-30 DAC. At 20 DAC the excised cotyledon evolved 85% of the ethylene found in the whole MDE. To determine the role of ethylene, MDEs were treated with aminoethoxyvinylglycine (AVG, an inhibitor of ethylene biosynthesis), CoCl(2) (an inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase), and silver thiosulphate (STS, an inhibitor of ethylene action). An inhibition in ethylene production or action at 20 DAC resulted in diminished lateral cotyledon expansion, due to a reduction in the lateral expansion of cells within the cotyledon. Recovery to 'control-type' levels of cotyledon cell expansion was achieved by application of ACC (the metabolic precursor of ethylene) to AVG-treated MDEs. Thus, ethylene production at 20 DAP likely controls cotyledon expansion during embryo development.     

Exogenous gibberellins inhibit coffee (Coffea arabica cv. Rubi) seed germination and cause cell death in the embryo

The mechanism of inhibition of coffee (Coffea arabica cv. Rubi) seed germination by exogenous gibberellins (GAs) and the requirement of germination for endogenous GA were studied. Exogenous GA(4+7) inhibited coffee seed germination. The response to GA(4+7) showed two sensitivity thresholds: a lower one between 0 and 1 microM and a higher one between 10 and 100 microM. However, radicle protrusion in coffee seed depended on the de novo synthesis of GAs. Endogenous GAs were required for embryo cell elongation and endosperm cap weakening. Incubation of coffee seed in exogenous GA(4+7) led to loss of embryo viability and dead cells were observed by low temperature scanning microscopy only when the endosperm was surrounding the embryo. The results described here indicate that the inhibition of germination by exogenous GAs is caused by factors that are released from the endosperm during or after its weakening, causing cell death in the embryo and leading to inhibition of radicle protrusion.     

Metabolic regulation in C4 photosynthesis: Phosphoenol pyruvate carboxylase and 3C intermediates of the photosynthetic carbon reduction cycle

Summary Gibberellins and auxins were extracted from embryos and suspensors of Phaseolus coccineus L. at two stages of development: A) heart-shaped embryo and B) cotyledonary embryo with suspensor in the initial stage of degeneration. The time interval between the two stages was 5–6 days.In both embryos and suspensors, gibberellin (GA)-like activity was found in three fractions: F-1 (ethyl acetate fraction at pH 8.0), F-2 (free GAs) and F-3 (bound GAs). At stage A, the total GA activity in the suspensor was about 30 times greater than in the embryo and the bound GAs contributed by about 90% to the total GA content. A dramatic decrease in level of bound GA-like substances was found in suspensors at stage B, when the level of total GAs in the embryo had increased to 10 times that at stage A. This might suggest a transport of GAs from the suspensor to the embryo. In both embryo and suspensor, qualitative changes in GAs with shift in activity of the fractions tested occurred at the two developmental stages.The methanolic extracts of stage A suspensors showed two inhibitors, one much more active than the other, and two large peaks of growth promoting activity at Rf 0.4–0.7; in stage A embryos, the general activity of the extracts was lower and the promoting effect was spread over Rf 0.3–0.9.The present results seem to support the view that the suspensor plays a role in embryogenesis by acting as a site of synthesis of growth regulators needed by the embryo.Abbreviations F-1 ethyl acetate fraction at pH 8.0 - F-2 free gibberellins - F-3 bound gibberellins - GA gibberellic acid - Stage A heart-shaped embryo - stage B cotyledonary embryo with suspensor in the initial stage of degeneration     

Hormones in zygotic and microspore embryos of Brassica napus

A number of studies have used microspore-derived embryos (MDEs) as amodel for examining a range of processes, including hormonal regulation ofembryo development. We examined the hormonal physiology of MDEs with theprimaryobjective of testing the validity of using the MDE system as a model forhormonally-regulated development in zygotic embryos, through late stages. To dothis we identified and quantified endogenous levels of abscisic acid (ABA),indole-3-acetic acid (IAA) and a number of gibberellins (GAs), includingGA₁₉, GA₂₀, GA₁ and GA₈ in bothMDEsand zygotic embryos. The presence of GA₁₉, together with itsC₁₉ metabolites indicates that the early-13 hydroxylation pathway isoperative in both embryo systems. Gibberellins A₄ and GA₉were also identified, thereby confirming the presence of the earlynon-hydroxylation pathway in B. napus MDEs and zygoticembryos. In general, the pattern of change of hormone (ABA, IAA, GA₁and GA₂₀) content per embryo through embryogenesis was similar forMDEs and zygotic embryos. Indole-3-acetic acid and GA₁ increased toamaximum at day 30 after culture (DAC) before decreasing. Abscisicacid levels increased to a maximum at day 35, and declined in zygoticembryos but not in MDEs. GA₂₀ increased to the final harvest atmaturity, or day 40. The absolute content (g/embryo) of each hormone, howeverwas appreciably lower (5- to 15- fold) in the MDEs. This was not the result ofdilution into surrounding medium for ABA or IAA; GA₁, however, didaccumulate in the medium. Although there were absolute quantitative differencesin the levels of IAA and ABA found in the two embryo systems, the similaritiesin the pattern of hormone changes suggests that the MDE system can serve as auseful model for examining the physiological roles of hormones duringembryogenesis.     

Regulation of stem elongation in chinese cabbage by inflorescence removal and application of growth regulators

The effect of inflorescence removal on stem elongation in Chinese cabbage cv. Spring A was studied. Removal of the inflorescence before its visibility, or upon its appearance but before the beginning of bolting (stages 1–3), markedly reduced the stem length. Removal after the beginning of bolting (stage 5) had no effect on stem length. Application of GA₃ to the treated plants partially or fully restored the elongation of the flowering stem, whereas paclobutrazol inhibited the elongation of the treated, as well as the control stems. Indole-3-acetic acid (IAA) or kinetin was ineffective in restoring stem elongation of the plants from which the inflorescence had been removed. Inflorescences at stages 1–2 were found to secrete about 10 times more gibberellic acid (GA)-like activity compared with control apices or inflorescences at stage 5. It is suggested that the developing inflorescence is the major source of GAs which control stem elongation. However, shortly after the appearance of the inflorescence at the onset of bolting, stem elongation is no longer dependent on GAs derived from the apical inflorescence but require GAs from other sources.     

Regulation of stem elongation in chinese cabbage by inflorescence removal and application of growth regulators

The effect of inflorescence removal on stem elongation in Chinese cabbage cv. Spring A was studied. Removal of the inflorescence before its visibility, or upon its appearance but before the beginning of bolting (stages 1–3), markedly reduced the stem length. Removal after the beginning of bolting (stage 5) had no effect on stem length.Application of GA₃ to the treated plants partially or fully restored the elongation of the flowering stem, whereas paclobutrazol inhibited the elongation of the treated, as well as the control stems. Indole-3-acetic acid (IAA) or kinetin was ineffective in restoring stem elongation of the plants from which the inflorescence had been removed. Inflorescences at stages 1–2 were found to secrete about 10 times more gibberellic acid (GA)-like activity compared with control apices or inflorescences at stage 5.It is suggested that the developing inflorescence is the major source of GAs which control stem elongation. However, shortly after the appearance of the inflorescence at the onset of bolting, stem elongation is no longer dependent on GAs derived from the apical inflorescence but require GAs from other sources.Contribution from the Agricultural Research Organization, The Volcani Center Bet Dagan, Israel No. 2218-E, 1987 series.     

Nondwarf rice seedling bioassay for gibberellins

The use of nondwarf rice (Oryza sativa L.) cultivars treated with uniconazole as test plants for gibberellin (GA) bioassay instead of Tan-ginbozu dwarf rice variant was investigated. The sensitivity of six nondwarf rice cultivars to GAs was increased substantially by treatment of the seeds with uniconazole. The minimum detectable dose of a GA in the nondwarf cultivars treated with uniconazole was 1- to 1/10-fold of that in the nontreated Tanginbozu and 3- to 10-fold of that in uniconazole-treated Tanginbozu. The relative activity of several GAs on treated nondwarf rice cultivars was not largely different from that to Tan-ginbozu. Considering that seeds of nondwarf rice are available commercially, the nondwarf rice seedling assay would be useful as a simple assay for systematic analysis of GAs, and also as a routine teaching tool in high schools and universities.     

Uniconazole-induced tolerance of rape plants to heat stress in relation to changes in hormonal levels, enzyme activities and lipid peroxidation

Winter rape (Brassica napus L. cv. 601) seedlings were treated with 50 mg.l-1of foliar-applied uniconazole and then exposed to heat stress with a light/dark temperature regime of 43 °C/38 °C for 3 days at the stem elongation stage. Heat stressed plants contained lower endogenous GA3, IAA and zeatin contents than the controls, while ABA content and ethylene level were increased significantly. Uniconazole-treated plants had lower endogenous GA3 and IAA contents, and higher zeatin and ABA contents and ethylene levels. Leaf chlorophyll content and respiratory capacity of roots were reduced markedly after plants were subjected to heat stress, and foliar sprays of uniconazole retarded the degradation of chlorophyll and increased respiratory capacity of roots. Following exposure to heat, the activities of superoxide dismutase and peroxidase were significantly reduced. Uniconazole-induced heat tolerance was accompanied by increased activities of various antioxidant enzymes. Foliar applications of uniconazole reduced electrolyte leakage and malondialdehyde accumulation caused by heat stress, suggesting that uniconazole may have decreased heat-induced lipid peroxidation and membrane damage. Foliar sprays of uniconazole increased the tolerance of rape plants to heat stress.     

Gibberellin biosynthesis mutations and root development in pea

Dwarf mutants of pea (Pisum sativum), with impaired gibberellin (GA) biosynthesis in the shoot, were studied to determine whether the roots of these genotypes had altered elongation and GA levels. Mutations na, lh-2, and ls-1 reduced GA levels in root tips and taproot elongation, although in lh-2 and ls-1 roots the reduction in elongation was small (less than 15%). The na mutation reduced taproot length by about 50%. The roots of na plants elongated in response to applied GA(1) and recombining na with mutation sln (which blocks GA catabolism) increased GA(1) levels in root tips and completely restored normal root development. In shoots, Mendel's le-1 mutation impairs the 3beta-hydroxylation of GA(20) to the bioactive GA(1), resulting in dwarfism. However, GA(1) and GA(20) levels were normal in le-1 roots, as was root development. The null mutation le-2 also did not reduce root GA levels or elongation. The results support the theory that GAs are important for normal root elongation in pea, and indicate that a 3beta-hydroxylase gene other than LE operates in pea roots.     

NADP-Dependent Phenylacetaldehyde Dehydrogenase for Degradation of Phenylethylamine in Arthrobacter globiformis

The role of endogenous gibberellin (GA) in the flowering of the short-day plant, Pharbitis nil, was investigated by using uniconazole, which is a specific inhibitor of GA biosynthesis. Both the endogenous GA level and flowering response decreased with increasing concentration of uniconazole applied via the roots. The strongest inhibition of flowering was observed when uniconazole was applied one day before a 15-h dark treatment. The inhibition by uniconazole was overcome by an application of GAs to the plumules, the order of effectiveness of the endogenous GAs in P. nil being GA₁ ≧GA₂₀>GA₁₉≧GA₄₄>GA₅₃»GA_H. This is the first report of the correlation between the endogenous GA level and flowering response in P. nil. It was found that endogenous GAs were required for the flowering of P. nil during or just after the dark period.     

Endogenous Gibberellin-Like Substances in Somatic Embryos of Grape (Vitis vinifera x Vitis rupestris) in Relation to Embryogenesis and the Chilling Requirement for Subsequent Development of Mature Embryos

Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera × Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4°C, and subsequently incubated at 26°C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H₂O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO₂ partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H₂O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4°C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H₂O-soluble fraction declined throughout chilling. Activity in the GA-precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26°C after chilling enhanced activity in the free GA and H₂O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26°C with (±) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H₂O-soluble fractions.

Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA₄ and GA₉ in ABA-treated, chilled embryos was also confirmed by GLC-MS.

     

Gibberellins and seed development in maize. II. Gibberellin synthesis inhibition enhances abscisic acid signaling in cultured embryos

Abscisic acid (ABA) is required for seed maturation in maize (Zea mays L.) and other plants. Gibberellins (GAs) are also present in developing maize embryos, and mutual antagonism of GAs and ABA appears to govern the choice between precocious germination or quiescence and maturation. Exogenous ABA can also induce quiescence and maturation in immature maize embryos in culture. To examine the role of GAs versus ABA in regulating maize embryo maturation, the effects of modulating GA levels were compared with those of ABA in embryos cultured at successive stages of development. The effects of GA synthesis inhibition or exogenous GA application differed markedly in embryos at different stages of development, indicating changes in both endogenous GA levels and in the capacity for GA synthesis as embryogenesis and maturation progress. In immature embryos, the inhibition of GA synthesis mimicked the effects of exogenous ABA, as shown by the suppression of germination, the acquisition of anthocyanin pigments, and the accumulation of a variety of maturation-phase mRNAs. We suggest that GA antagonizes ABA signaling in developing maize embryos, and that the changing hormone balance provides temporal control over the maturation phase.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.     

The interaction of light quality and irradiance with gibberellins, cytokinins and auxin in regulating growth of Helianthus annuus hypocotyls

A reduced red to far-red (R/FR) light ratio and low photosynthetically active radiation (PAR) irradiance are both strong signals for inducing etiolation growth of plant stems. Under natural field conditions, plants can be exposed to either a reduced R/FR ratio or lower PAR, or to a combination of both. We used Helianthus annuus L., the sunflower, to study the effect of reduced R/FR ratio, low PAR or their combination on hypocotyl elongation. To accomplish this, we attempted to uncouple light quality from light irradiance as factors controlling hypocotyl elongation. We measured alterations in the levels of endogenous gibberellins (GAs), cytokinins (CKs) and the auxin indole-3-acetic acid (IAA), and the effect of exogenous hormones on hypocotyl growth. As expected, both reduced R/FR ratio and lower PAR can significantly promote sunflower hypocotyl elongation when given separately. However, providing the reduced R/FR ratio at a low PAR resulted in the greatest hypocotyl growth, and this was accompanied by significantly higher levels of endogenous IAA, GA1, GA8, GA20 and of a wide range of CKs. Providing a reduced R/FR ratio under normal PAR also significantly increased growth and again gave significantly higher levels of endogenous IAA, GAs and CKs. However, only under the de-etiolating influence of a normal R/FR ratio did lowering PAR significantly increase levels of GA1, GA8 and GA20. We thus conclude that light quality (e.g. the R/FR ratio) is the most important component of shade for controlling hypocotyl growth and elevated growth hormone content.     

水稻茎伸长生长与植物激素

赤霉素（GA）,生长素（IAA）,脱落酸（ABA）和乙烯影响水稻茎（或节间）的伸长,其中赤霉素与水稻茎伸长生长的关系最密切。GA1是植物体内刺激茎伸长的至关重要的赤霉素,GA3已作为最常用的外源激素诱导水稻的节间伸长。水稻茎秆的伸受激素浓度和敏感性的双重控制,激素浓度或敏感性任一方的改变都有可能导致株高的变异。赤霉素如此显著地促进茎的伸长可能与增加细胞分裂和促使细胞壁松弛有关。而生长素主要促进细胞伸长。植物激素促进水稻茎长的分子机理的研究已有较大的进展,预期这方面的研究和应用在未来几年内将有新的突破。