Gibberellin Signal Transduction in Rice

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

Developing Inflorescences of Male and Female <Emphasis Type="Italic">Rumex acetosa</Emphasis> L. Show Differences in Gibberellin Content

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

Development of a one-step enzyme immunoassay for the quantitation of gibberellin A3 in barley seeds

The development of a sensitive and specific enzyme immunoassay for GA₃ is reported. This method was based on the use of peroxidase labelled GA₃ and immobilized antibodies. In order to obtain a rapid immunoassay, several steps of purification were analyzed to show their necessity. Barley seed extracts were assayed at different steps of purification to exhibit the effect of extract components on the assay. It was demonstrated that HPLC had to be performed when a selective quantitation of GA₃ was required. This assay allowed GA₃ to be measured with reproducibility as its unmethylated form and the quantitation of GA₃ in barley seeds with this enzyme immunoassay was correlated to a GC-MS method.Abbreviations GA₃ gibberellin A₃ - EIA enzyme immunoassay - DMF dimethylformamide - TEA tri(n)ethylamine - BSA bovine serum albumin - OVA ovalbumine - ECF ethylchloroformate - PB phosphate buffer     

Influence of electron transport proteins on the reactions catalyzed by Fusarium fujikuroi gibberellin monooxygenases

The multifunctional cytochrome P450 monooxygenases P450-1 and P450-2 from Fusarium fujikuroi catalyze the formation of GA14 and GA4, respectively, in the gibberellin (GA)-biosynthetic pathway. However, the activity of these enzymes is qualitatively and quantitatively different in mutants lacking the NADPH:cytochrome P450 oxidoreductase (CPR) compared to CPR-containing strains. 3beta-Hydroxylation, a major P450-1 activity in wild-type strains, was strongly decreased in the mutants relative to oxidation at C-6 and C-7, while synthesis of C19-GAs as a result of oxidative cleavage of C-20 by P450-2 was almost absent whereas the C-20 alcohol, aldehyde and carboxylic acid derivatives accumulated. Interaction of the monooxygenases with alternative electron transport proteins could account for these different product distributions. In the absence of CPR, P450-1 activities were NADH-dependent, and stimulated by cytochrome b5 or by added FAD. These properties as well as the decreased efficiency of P450-1 and P450-2 in the mutants are consistent with the participation of cytochrome b5:NADH cytochrome b5 reductase as redox partner of the gibberellin monooxygenases in the absence of CPR. We provide evidence, from either incubations of GA12 (C-20 methyl) with cultures of the mutant suspended in [18O]H2O or maintained under an atmosphere of [18O]O2:N2 (20:80), that GA15 (C-20 alcohol) and GA24 (C-20 aldehyde) are formed directly from dioxygen and not from hydrolysis of covalently enzyme-bound intermediates. Thus these partially oxidized GAs correspond to intermediates of the sequential oxidation of C-20 catalyzed by P450-2.     

Comparison of gas chromatography-mass spectrometry,radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.)

Quantitative estimates of gibberellin A₉ in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A₉, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW fresh weight - GA₉ gibberellin A₉ - GA₉–Me methylated GA₉ - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MID multiple-ion detection - RIA radioimmunoassay     

Convergent pathways of gibberellin A1 biosynthesis in Brassica

[²H, ³H]Gibberellin A₄ (GA₄) or [²H, ³H] GA₉ were applied to the shoot tips of seedlings of elongated internode (ein), a tall mutant of rapid cycling Brassica rapa. Following [²H]GA₉ application, [²H]GA₅₁, [²H]GA₂₀ and [²H]GA₄ were identified as products by GC-MS, while [²H]GA₃₄ and [²H]GA₁ were formed from [²H]GA₄. Other isotopically labelled products, including abundant putative conjugates, were also produced, but were not identified. Thus, in B. rapa, GA₁ biosynthesis involves the convergence of at least two metabolic pathways; it can be formed via GA₄ or GA₂₀, the latter of which can originate from GA₉ or from GA₁₉.     

Gibberellin biosynthesis in cell-free extracts from developing Cucurbita maxima embryos and the identification of new endogenous gibberellins

Gibberellin (GA) biosynthetic pathways from GA₁₂-aldehyde, GA₁₂ and GA₅₃ were investigated in cell-free systems from developing embryos of Cucurbita maxima L. Gibberellin A₁₂-aldehyde and GA₁₂ were converted to GA₂₅, putative 12α-hydroxyGA₂₅, GA₁₃ and GA₃₉ as main products. Minor products were GA₄, GA₃₄ and, when GA₁₂ was the substrate, putative 12α-hydroxyGA₁₂. The intermediates GA₁₅ and GA₂₄ accumulated at low protein concentrations. The influence of various factors on GA₁₂ metabolism was examined. At low 2-oxoglutarate and ascorbate concentrations, or at acid pH, 3β-hydroxylated products predominated, whereas with increasing 2-oxoglutarate and ascorbate concentrations, or at neutral pH, the yield of 12α-hydroxylated GAs increased. Gibberellin A₅₃ was metabolised mainly to the C₂₀-GAs GA₄₄, GA₁₉, GA₁₇, GA₂₃ and GA₂₈, with the C₁₉-GAs GA₂₀, GA₁ and GA₈ as minor products. Only C₁₉-GAs were 2β-hydroxylated, which is a main characteristic of the embryo systems. In addition to GA₁₃, GA₂₅, GA₃₉, GA₄₃, GA₄₉, GA₅₈, GA₇₄, 12α-hydroxyGA₂₅ and GA₃₉ 3-isovalerate, which were known previously from embryos of C. maxima, GA₁, GA₄, GA₁₇, GA₂₈, GA₃₇, GA₃₈, GA₄₈, GA₈₅, 12α-hydroxyGA₃₇ and putative 12α-hydroxyGA₄₃ were identified as endogenous components by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Evidence for putative 2β-hydroxyGA₂₈ and GA₂₃ was also obtained but it was less conclusive because of contamination.     

Effect of light and gibberellic acid on cell division in the first foliage leaf of durum wheat (Triticum durum Desf.)

The present paper is part of a research program which aims at a quantitative analysis of the effects of light and gibberellic acid (GA₃) on growth of the first foliage leaf in durum wheat (Triticum durum Desf.). Since leaf growth is the combined result of the increase in cell number (cell division) and cell enlargement, the influence of light and GA₃ treatment on cell division in the basal meristem of the first leaf in two cultivars, Cappelli and Creso, was investigated. Creso is a short-strawed cultivar carrying the Gai 1 gene which influences both plant height and insensitivity to applied GA₃. Cell division, as measured by mitotic index, was similar in darkness, continuous red light and dichromatic irradiation (far-red plus red), while lower mitotic rates were observed under continuous far-red light: this indicates that the response of cell division is modulated by a high-irradiance reaction of phytochrome in both cultivars. The two cultivars showed different responses to blue light. In Cappelli, blue light and dichromatic irradiation (blue plus red) gave lower mitotic indices than the dark control, indicating the action of a specific blue-light-absorbing photoreceptor, whereas in Creso the response kinetics to all light regimes which included blue light were more complex. On the basis also of the results obtained with GA₃ application in Cappelli, it appears that (i) the hormonal treatment is able to change the pattern of mitotic index only in the presence of the action of a blue-light receptor and (ii) the different responses of the two cultivars could be the result of different endogenous hormonal levels. The importance of the observations in relation to the data for first-leaf longitudinal growth reported in a previous paper (Baroncelli et al. 1984, Planta 160, 298–304) is discussed.Abbreviations BL blue light - D darkness - FR far-red light - GA gibberellin - GA₃ gibberellic acid - m.i. mitotic index - Norflurazon 4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-totyl-3(2H)) pyridazinone - R red light - WL white light - phytochrome photoequilibrium     

Biosynthesis of 12α-and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identification of new endogenous gibberellins

Gibberellin (GA) biosynthesis in cell-free systems from Cucurbita maxima L. endosperm was reinvestigated using incubation conditions different from those employed in previous work. The metabolism of GA₁₂ yielded GA₁₃, GA₄₃ and 12α-hydroxyGA₄₃ as major products, GA₄, GA₃₇, GA₃₉, GA₄₆ and four unidentified compounds as minor products. The intermediates GA₁₅, GA₂₄ and GA₂₅ accumulated at low protein concentrations. The structure of the previously uncharacterised 12α-hydroxyGA₄₃ was inferred from its mass spectrum and by its formation from both GA₃₉ and GA₄₃. Gibberellin A₃₉ and 12α-hydroxyGA₄₃ were formed by a soluble 12α-hydroxylase that had not been detected before. Gibberellin A₁₂-aldehyde was metabolised to essentially the same products as GA₁₂ but with less efficiency. A new 13-hydroxylation pathway was found. Gibberellin A₅₃, formed from GA₁₂ by a microsomal oxidase, was converted by soluble 2-oxoglutarate-dependent oxidases to GA₁ GA₂₃, GA₂₈, GA₄₄, and putative 2β-hydroxyGA₂₈. Minor products were GA₁₉, GA₂₀, GA₃₈ and three unidentified GAs. Microsomal 13-hydroxylation (the formation of GA₅₃) was suppressed by the cofactors for 2-oxoglutarate-dependent enzymes. Reinvestigation of the endogenous GAs confirmed the significance of the new metabolic products. In addition to the endogenous GAs reported by Blechschmidt et al. (1984, Phytochemistry 23, 553–558), GA₁, GA₈, GA₂₅, GA₂₈, GA₃₆, GA₄₈ and 12α-hydroxyGA₄₃ were identified by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Thus both the 12α-hydroxylation and the 13-hydroxylation pathways found in the cell-free system operate also in vivo, giving rise to 12α-hydroxyGA₄₃ and GA₁ (or GA₈), respectively, as their end products. Evidence for endogenous GA₂₀ and GA₂₄ was also obtained but it was less conclusive due to interference.     

Determination of femtomol quantities of gibberellic acid by radioimmunoassay

A highly sensitive and specific radioimmunoassay which allows the detection of as little as 5 fmol (2 pg) of gibberellic acid (GA₃) in crude plant extracts is described. Antisera of high affinity and titer were obtained by immunizing rabbits with a conjugate of carboxyl-coupled GA₃ and bovine serum albumin. [¹²⁵I]Gibberellic acid-[N-(p-hydroxybenzyl) putrescine]amide of high specific activity, used as the immunotracer, is readily displaced by gibberellic acid methyl ester but not by free gibberellic acid. Thus, methylation of extracts prior to analysis is required. The assay is very specific; besides GA₃, only the closely related GA₇ is highly immunoreactive. Various gibberellins, related compounds, as well as other classes of plant hormones do not interfere with the assay. Levels of immunoreactive gibberellins (GA₃, GA₇) in actively growing tissues, among them cell suspension cultures of 33 different species, were determined.Abbreviations ABA abscisic acid - GC-MS gas chromatography-mass spectroscopy - TLC thin-layer chromatography - GA gibberellin Part 17 in the Series: Use of Immunoassay in Plant Science     

Interactions between TGF-β1 and TGF-β3 and their role in medial edge epithelium cell death and palatal fusion in vitro

In recent decades, studies have shown that both TGF-β₁ and TGF-β₃ play an important role in the induction of medial edge epithelium (MEE) cell death and palatal fusion. Many of these experiments involved the addition or blockage of one of these growth factors in wild-type (WT) mouse palate cultures, where both TGF-β₁ and TGF-β₃ are present. Few studies have addressed the existence of interactions between TGF-β₁ and TGF-β₃, which could modify their individual roles in MEE cell death during palatal fusion. We carried out several experiments to test this possibility, and to investigate how this could influence TGF-β₁ and TGF-β₃ actions on MEE cell death and palatal shelf fusion. We double-immunolabelled developing mouse palates with anti-TGF-β₁ or anti-TGF-β₃ antibodies and TUNEL, added rhTGF-β₁ or rhTGF-β₃ or blocked the TGF-β₁ and TGF-β₃ action at different concentrations to WT or Tgf-β₃ null mutant palate cultures, performed in situ hybridizations with Tgf-β₁ or Tgf-β₃ riboprobes, and measured the presence of TUNEL-positive midline epithelial seam (MES) cells and MES disappearance (palatal shelf fusion) in the different in vitro conditions. By combining all these experiments, we demonstrate great interaction between TGF-β₁ and TGF-β₃ in the developing palate and confirm that TGF-β₃ has a more active role in MES cell death than TGF-β₁, although both are major inductors of MES disappearance. Finally, the co-localization of TGF-β₁, but not TGF-β₃, with TUNEL in the MES allows us to suggest a possible role for TGF-β₁ in MES apoptotic clearance.     

Biosynthesis of Biotin from Dethiobiotin by Intact Cells of Biotin-producing Bacterium

Intact cells of a biotin-producing bacterium, KY–21–1–25, were found to synthesize biotin from dethiobiotin. Optimal conditions for the biosynthesis of biotin from dethiobiotin by intact cells were investigated. Intact cells harvested from adenine-supplemented medium showed intensive biosynthesis. However, the biosynthesis of biotin by intact cells was strongly inhibited by the addition of adenine or adenosine. The inhibitory activity of adenine was about 10-fold greater than that of adenosine. Formation of several unidentified biotin-vitamers was observed in both reaction mixtures incubated with and/or without addition of adenine.     

Synthesis of gibberellin GA6 and its role in flowering of Lolium temulentum

The induction of flowering by one long day (LD) in the grass Lolium temulentum is most closely mimicked by application of the gibberellins (GAs) GA(5) or GA(6), both of which occur naturally. These gibberellins promote floral development but have little effect on stem elongation. Endogenous GA(5) and GA(6) contents in the shoot apex double on the day after the LD and, for GA(5) (and we presume for GA(6) as well) reach a concentration known to be inductive for the excised shoot apex in vitro. They are, therefore, strong candidates as LD floral stimuli in this grass. The synthesis of GA(6) and an examination of its florigenic properties in L. temulentum are described.     

The Interaction of Plant Growth Regulators and Vernalization on the Growth and Flowering of Cauliflower (Brassica oleracea var. botrytis)

The growth and flowering response of a cold-requiring cauliflower (Brassica oleracea var. botrytis cv. 60 day) to a range of temperatures under 10 h photoperiod and to growth regulator application were investigated. Endogenous gibberellin A₁(GA₁) concentrations were also assessed under these treatments. Flowering and growth of the inflorescence stalk were correlated with plant developmental stage at the time of a vernalizing cold treatment. Temperature and its duration also affected flowering and inflorescence development. The most effective temperature for inflorescence induction was 10 °C. Flowering did not occur in non-vernalized plants (25 °C) even though they had been treated with GA₃. Application of GA₃ promoted inflorescence stalk elongation greatly in vernalized plants (10 °C), but less so in partially vernalized plants (15 °C or 20 °C). Paclobutrazol (PP₃₃₃) sprayed at the 8–9 leaf stage significantly suppressed inflorescence stalk length and slightly delayed flower bud formation and anthesis. Vernalization at 10 °C increased endogenous GA₁ content in both leaves and the inflorescence stalk irrespective of GA₃ or PP₃₃₃ treatment. Application of GA₃ tended to increase GA₁ levels, while PP₃₃₃ significantly reduce GA₁, both irrespective of vernalization. Vernalization is an important factor for flowering, but not curd formation in this cauliflower cv. 60 day and GA₁ is likely a causal factor in inflorescence stalk elongation.     

Characterization of the P5 subfamily of P-type transport ATPases in mice

In mammals, the most poorly understood P-type ATPases are those of the P(5) subfamily. To begin characterization of the mammalian P(5)-ATPases, BLAST searches of DNA sequence databases were performed. Five genes were identified in the mouse, human, dog, and rat genomes, and the coding sequences of the mouse genes, termed Atp13a1-Atp13a5, were determined. The intron/exon organization of Atp13a1 differs entirely from those of Atp13a2-5, which are closely related. Amino acid sequence comparisons between the five mouse and two yeast P(5)-ATPases suggest that Atp13a1 is orthologous to the yeast Cod1 gene and that Atp13a2-5 are orthologous to yeast Yor291w. Northern blot analysis showed that Atp13a1, Atp13a2, and Atp13a3 mRNAs were expressed in all mouse tissues, whereas Atp13a4 and Atp13a5 mRNAs were restricted to brain and stomach. While the substrate specificity of these transporters is unknown, their importance is underscored by the presence of homologs in fish, insects, worms, and other eukaryotes.     

基于比较代谢组学解析藤仓赤霉菌CGMCC 17793高产GA4机制

新型赤霉素(gibberellin) GA4因其独特的优势而具有广阔的应用前景。为探究GA₄生物合成调控机制,本研究以藤仓赤霉菌(Gibberella fujikuroi) GA₄生产菌株S (CGMCC 17793)和野生菌株Y (GenBank登录号：NRRL 13620)为研究对象,利用液相色谱-质谱联用(liquid chromatograph-mass spectrometer,LC-MS)代谢组学技术,结合主成分分析(principal component analysis,PCA)与偏最小二乘-显著性分析联合法(partial least squares-discrimination analysis,PLS-DA)等对两种菌株在同一发酵时间以及菌株S在不同发酵时间的差异代谢物进行筛选与鉴定,并利用KEGG数据库和MBROLE 2.0分析相关代谢通路。结果表明,与菌株Y相比,菌株S在发酵3、6、9 d时,显著上调和下调的代谢物分别为107种和66种、136种和47种、94种和65种。与生产菌株S发酵3 d时相比,6、9 d时显著上调和下调的代谢物分别为29种和40种、52种和67种。菌株S与菌株Y同一发酵时间的差异代谢物主要富集在氨基酸代谢、三羧酸循环(tricarboxylic acid cycle,TCA cycle)和萜类生物合成等通路。菌株S不同发酵时间的差异代谢物主要富集在氨基酸代谢与糖代谢等合成途径。通路注释与分析结果说明,菌株S通过促进氨基酸与糖代谢、TCA循环产生更多乙酰CoA,增强了甲羟戊酸途径,从而使萜类化合物合成的前体物质异戊烯焦磷酸(isopentenyl pyrophosphate,IPP)含量增加,最终提高了GA₄产量。本研究探究了藤仓赤霉菌GA₄代谢规律,为调控藤仓赤霉菌提高GA₄产量提供了理论基础。     

Thermodynamic and kinetic characterisation of individual haems in multicentre cytochromes c3

The characterisation of individual centres in multihaem proteins is difficult due to the similarities in the redox and spectroscopic properties of the centres. NMR has been used successfully to distinguish redox centres and allow the determination of the microscopic thermodynamic parameters in several multihaem cytochromes c₃ isolated from different sulphate-reducing bacteria. In this article we show that it is also possible to discriminate the kinetic properties of individual centres in multihaem proteins, if the complete microscopic thermodynamic characterisation is available and the system displays fast intramolecular equilibration in the time scale of the kinetic experiment. The deconvolution of the kinetic traces using a model of thermodynamic control provides a reference rate constant for each haem that does not depend on driving force and can be related to structural factors. The thermodynamic characterisation of three tetrahaem cytochromes and their kinetics of reduction by sodium dithionite are reported in this paper. Thermodynamic and kinetic data were fitted simultaneously to a model to obtain microscopic reduction potentials, haem-haem and haem-proton interacting potentials, and reference rate constants for the haems. The kinetic information obtained for these cytochromes and recently published data for other multihaem cytochromes is discussed with respect to the structural factors that determine the reference rates. The accessibility for the reducing agent seems to play an important role in controlling the kinetic rates, although is clearly not the only factor.