Endogenous gibberellins and kauranoids identified from developing and germinating barley grain

Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA₁ (trace), GA₄ (trace), GA₈ (trace), GA₁₂, GA₁₇, GA₂₀ (tentative) (trace), GA₂₅, GA₃₄, GA₄₈, 18-hydroxy-GA₄, 12-hydroxy-GA₉, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₁₇, GA₂₀, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA₁, GA₃ (possibly a contaminant), GA₁₇, GA₁₉, GA₂₀, GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1-Hydroxylated GAs previously found in wheat were not found in barley in this study.This work has been reported in a poster demonstration (Gaskin et al. 1982).     

Endogenous gibberellins and kauranoids identified from developing and germinating barley grain

Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA₁ (trace), GA₄ (trace), GA₈ (trace), GA₁₂, GA₁₇, GA₂₀ (tentative) (trace), GA₂₅, GA₃₄, GA₄₈, 18-hydroxy-GA₄, 12β-hydroxy-GA₉, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₁₇, GA₂₀, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA₁, GA₃ (possibly a contaminant), GA₁₇, GA₁₉, GA₂₀, GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1β-Hydroxylated GAs previously found in wheat were not found in barley in this study.     

Lysophosphatidylcholine biosynthesis in developing barley

Acetate-2-[¹⁴C] and choline-Me-[ ¹⁴C], absorbed through the stems of isolated barley heads, were used to label lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) of the endosperm tissue. Labelling of LPC occurred in barley heads at almost all stages of development but was at a maximum when the fr. wt of the seeds had attained ca 60–70% of their maximum wt. In time-course experiments labelling of PC from each substrate reached a maximum after 50 hr and then declined. Label in LPC, however, continued to accumulate throughout 72 hr. Stimulation of labelling of LPC from choline-Me-[¹⁴C] by sucrose was observed. A bound form of LPC (starch lipid) and a free form were distinguished by differential solvent extraction.     

Auxin is required for leaf vein pattern in Arabidopsis

To investigate possible roles of polar auxin transport in vein patterning, cotyledon and leaf vein patterns were compared for plants grown in medium containing polar auxin transport inhibitors (N-1-naphthylphthalamic acid, 9-hydroxyfluorene-9-carboxylic acid, and 2,3,5-triiodobenzoic acid) and in medium containing a less well-characterized inhibitor of auxin-mediated processes, 2-(p-chlorophynoxy)-2-methylpropionic acid. Cotyledon vein pattern was not affected by any inhibitor treatments, although vein morphology was altered. In contrast, leaf vein pattern was affected by inhibitor treatments. Growth in polar auxin transport inhibitors resulted in leaves that lacked vascular continuity through the petiole and had broad, loosely organized midveins, an increased number of secondary veins, and a dense band of misshapen tracheary elements adjacent to the leaf margin. Analysis of leaf vein pattern developmental time courses suggested that the primary vein did not develop in polar auxin transport inhibitor-grown plants, and that the broad midvein observed in these seedlings resulted from the coalescence of proximal regions of secondary veins. Possible models for leaf vein patterning that could account for these observations are discussed.     

Curvature in Arabidopsis inflorescence stems is limited to the region of amyloplast displacement

Gravitropic sensing in stems and stem-like organs is hypothesized to occur in the endodermis. However, since the endodermis runs the entire length of the stem, the precise site of gravisensing has been difficult to define. In this investigation of gravisensitivity in inflorescence stems of Arabidopsis, we positioned stems in a high gradient magnetic field (HGMF) on a rotating clinostat. Approximately 40% of the young, wild-type (WT) inflorescences, for all positions tested, curved toward the HGMF in the vicinity of the stem exposed to the field. In contrast, when the wedge was placed in the basal region of older inflorescence stems, no curvature was observed. As a control, the HGMF was applied to a starchless mutant, and 5% of the stems curved toward the field. Microscopy of the endodermis in the WT showed amyloplast displacement in the vicinity of the HGMF. Additional structural studies demonstrated that the basal region of WT stems experienced amyloplast displacement and, therefore, suggest this region is capable of gravity perception. However, increased lignification likely prevented curvature in the basal region. The lack of apical curvature after basal amyloplast displacement indicates that gravity perception in the base is not transmitted to the apex. Thus, these results provide evidence that the signal (and thus, response) resulting from perception in Arabidopsis inflorescence stems is spatially restricted.     

In vitro propagation of Centaurea spachii from inflorescence stems

Procedures for micropropagation of Centaureaspachii (Compositae), an endangered rosulate plantendemic from the mediterranean Spain area, have beendeveloped using inflorescence nodal segments asexplants for in vitro establishment. Only 15%of explants remained contaminated using this materialto start the in vitro axenic cultures. Higherproliferation of shoots and multiplication coefficientwas obtained on Murashige and Skoog (MS) mineralmedium supplemented with 1.0 mg l^{minus 1}6-benzyladenine. However, shoot elongation decreasedwith the addition of this cytokinin.Rooting of shoots with only one auxin was very lowafter 6 weeks on the majority of rooting media tested.The best rooting result (60%) was obtained on MSmedium with a combination of 2 mg l^{minus 1}indole-3-acetic acid plus 2 mgl^{minus 1} indole-3-butyric acid. Moreover, in thisculture medium 50% of shoots rooted during the thirdweek of culture. High survival, over 80%, wasobtained when the plantlets were transferred togreenhouse conditions. The endangered Centaureaspachii can be successfully micropropagatedbeginning with a single inflorescence stem and withoutsignificant damage to the mother plant.     

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.     

The effect of zinc on the biosynthesis of tryptophan,andol auxins and gibberellins in barley

The action of zinc on the growth of barley and the biosynthesis of indol compounds and gibberellin-like substances was investigated in a number of concentrations of zinc from doses stimulating growth to toxic doses. The seeds were soaked before sowing in solutions of zinc sulphate (5.10^?5 to 5.10^?1% Zn), and the plants cultivated for 7 days in water. Lower concentrations of zinc increased both plant growth and the biosynthesis of tryptophan and auxins. At the optimum concentration of 5.10^?3% Zn this increase in tryptophan amounted to 241% of the variant without zinc; in substances with an R_F corresponding to indolyacetic acid, the increase determined by the biological test, was 207% as against the variant without zinc. Higher concentrations of zinc inhibited growth, the tryptophan content was decreased to below that of the control without zinc and the auxin content also fell to below the control values. Zinc also influenced the content of gibberellin-like substances in the plants. At a concentration of 5.10^?3% Zn the increase in the growth activity in the gibberellic acid area of the chromatogram was 294% of the variant without zinc. At toxic concentrations of zinc, the content of gibberellin-like substances fell to below that of the controls. The finding that zinc acts simultaneously on the biosynthesis of auxins and gibberellins is also evidence for the common action of growth substances of various chemical types on plant growth.     

molting defective is required for ecdysone biosynthesis

20-hydroxyecdysone was discovered as the major biologically active insect steroid hormone half a century ago, yet much remains to be learned about its biosynthesis and its activities. 20-hydroxyecdysone controls many biological processes, including progression between larval stages, entry to pupariation and metamorphosis. A number of genes required for 20-hydroxyecdysone production have been identified, including those encoding enzymes that mediate four of the late steps of biosynthesis. A second smaller group of low ecdysone mutants do not encode enzymes. Here, we report identification of one such gene, which we call molting defective, on the basis of its lethal phenotype. molting defective encodes a nuclear zinc finger protein required for ecdysone biosynthesis.     

UFO in the Arabidopsis inflorescence apex is required for floral-meristem identity and bract suppression

The UNUSUAL FLORAL ORGANS (UFO) gene of Arabidopsis encodes an F-box protein required for the determination of floral-organ and floral-meristem identity. Mutation of UFO leads to dramatic changes in floral-organ type which are well-characterized whereas inflorescence defects are more subtle and less understood. These defects include an increase in the number of secondary inflorescences, nodes that alternate between forming flowers and secondary inflorescences, and nodes in which a single flower is subtended by a bract. Here, we show how inflorescence defects correlate with the abnormal development of floral primordia and establish a temporal requirement for UFO in this process. At the inflorescence apex of ufo mutants, newly formed primordia are initially bract-like. Expression of the floral-meristem identity genes LFY and AP1 are confined to a relatively small adaxial region of these primordia with expression of the bract-identity marker FIL observed in cells that comprise the balance of the primordia. Proliferation of cells in the adaxial region of these early primordia is delayed by several nodes such that primordia appear “chimeric” at several nodes, having visible floral and bract components. However, by late stage 2 of floral development, growth of the bract generally ceases and is overtaken by development of the floral primordium. This abnormal pattern of floral meristem development is not rescued by expression of UFO from the AP1 promoter, indicating that UFO is required prior to AP1 activation for normal development of floral primordia. We propose that UFO and LFY are jointly required in the inflorescence meristem to both promote floral meristem development and inhibit, in a non-cell autonomous manner, growth of the bract.Shelley R. Hepworth and Jennifer E. Klenz contributed equally to this work.     

Immunohistochemistry of active gibberellins and gibberellin-inducible alpha-amylase in developing seeds of morning glory

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A(1)-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA(1) and/or GA(3) were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible alpha-amylase in this digestion. We isolated an alpha-amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA(3). RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA(1/3). An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA(1/3), was also localized around starch grains in the integument of developing young seeds. The localization of GA(1/3) in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds.     

Auxin signalling is involved in cadmium-induced glutathione-S-transferase activity in barley root

Transient exposure of barley roots to Cd, IAA or H₂O₂ for 30 min resulted in a significant root growth inhibition. Cd significantly increased the GST activity of roots 6 h after the end of short-term treatment. This increase was more relevant in root segment containing differentiation zone than in root segment just immediately behind the root apex. In contrast to Cd treatment, the short-term exposure of barley roots to IAA resulted in a significant increase of GST activity along the whole root tip and this increase was detectable already 3 h after the treatment with 10 μM IAA. Similarly to IAA, exogenously applied 10 mM H₂O₂ for 30 min caused significant increase of GST activity along the whole root tip 6 h after the treatment. This increase was already detectable 3 h after the exposure, but only in the differentiation zone of root tip. Auxin influx or signalling inhibitor considerable decreased the Cd- or IAA-induced GST activity in barley root tips. The strong activation of GST even after a brief exposure of barley roots to Cd support the crucial role of GST in the Cd-induced stress response in which presumably IAA and H₂O₂ play an important signalling role including the activation of GST.     

Circadian rhythm of circumnutation in inflorescence stems of Arabidopsis

We investigate the modulation of circumnutation in inflorescence stems of Arabidopsis to determine the circadian regulation of circumnutation. Under constant light conditions (LL), circumnutation speed in wild-type plants fluctuates, with the phase of the highest speed at subjective dawn; the period length is close to 24 h. toc1 appears to shorten the period and elf3 causes an arrhythmic phenotype in circumnutation speed in LL, suggesting that a common circadian clock may control both circumnutation speed and other circadian outputs. These results highlight for the first time a role for a circadian clock in the regulation of circumnutation based on genetic analysis of Arabidopsis.     

Gravitropic response of inflorescence stems in Arabidopsis thaliana.

We have characterized the gravitropic response of inflorescence stems in Arabidopsis thaliana. When the inflorescence stems were placed horizontally, they curved upward about 90 degrees within 90 min in darkness at 23 degrees C, exhibiting strong negative gravitropism. Decapitated stem segments (without all flowers, flower buds, and apical apices) also showed gravitropic responses when they included the elongation zone. This result indicates that the minimum elements needed for the gravitropic response exist in the decapitated inflorescence stem segments. At least the 3-min gravistimulation time was sufficient to induce the initial curvature at 23 degrees C after a lag time of about 30 min. In the gravitropic response of inflorescence stems, (a) the gravity perception site exists through the elongating zone, (b) auxin is involved in this response, (c) the gravitropic curvature was inhibited at 4 degrees C but at least the gravity perception step could occur, and (d) two curvatures could be induced in sequence at 23 degrees C by two opposite directional horizontal gravistimulations at 4 degrees C.     

Total protein release from barley aleurone layers as a bioassay for gibberellins

The effect of gibberellic acid on the secretion of proteins from barley (Hordeum vulgare L.) aleurone layers has been investigated for its suitability as a gibberellin bioassay. Concentrations from 10^–4 g/ml to 100 g/ml of GA₃ resulted in the release of proportionally increasing amounts of total protein. The release of proteins is not affected by indoleacetic acid and kinetin. This method has been applied and compared with the -amylase assay for the estimation of gibberellin in extracts of tomato fruits and maize seedlings.Abbreviations GA₃ gibberellic acid - IAA indoleactic acid - K kinetin     

TIGRINA d, required for regulating the biosynthesis of tetrapyrroles in barley, is an ortholog of the FLU gene of Arabidopsis thaliana

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control of steps prior to delta-aminolevulinic acid (ALA) formation. One of the first mutants with a defect in this control had been identified in barley. The tigrina (tig) d mutant accumulates 10-15-fold higher amounts of protochlorophyllide than wild type, when grown in the dark. The identity of the TIGRINA d protein and its mode of action are not known yet. Initially this protein had been proposed to act as a repressor of genes that encode enzymes involved in early steps of ALA formation, but subsequent attempts to confirm this experimentally failed. Here we demonstrate that the TIGRINA d gene of barley is an ortholog of the FLU gene of Arabidopsis thaliana. The FLU protein is a nuclear-encoded plastid protein that plays a key role in negative feedback control of chlorophyll biosynthesis in higher plants. Sequencing of the FLU gene of barley revealed a frame shift mutation in the FLU gene of the tig d mutant that results in the loss of two tetratricopeptide repeats that in the FLU protein of Arabidopsis are essential for its biological activity. This mutation cosegregates strictly with the tigrina phenotype within the F1 population of a heterozygous tig d mutant, thus providing additional support for the flu gene being responsible for the tigrina phenotype of barley.     

Caspase activity is required for nephrogenesis in the developing mouse metanephros

Programmed cell death is a mechanism through which organisms get rid of unwanted cells and is thought to be an important process in organogenesis. Although large-scale cell death is observed in the developing kidney, the precise roles of cell death in kidney organogenesis remain to be elucidated. To address this question, we prevented cell death in metanephric explants by applying caspase inhibitors. Administration of caspase inhibitors (Z-D-CH2DCB and Ac-DEVD-CHO) effectively prevented the cell death that is normally observed in nondifferentiating mesenchymal cells. Both ureteric bud branching and nephrogenesis were prevented by caspase inhibition. Our results suggest that caspases are crucial in kidney organogenesis and cell death in the nondifferentiating mesenchyme.     

Ethylene and not embolism is required for wound-induced tylose development in stems of grapevines

The pruning of actively growing grapevines (Vitis vinifera) resulted in xylem vessel embolisms and a stimulation of tylose formation in the vessels below the pruning wound. Pruning was also followed by a 10-fold increase in the concentration of ethylene at the cut surface. When the pruning cut was made under water and maintained in water, embolisms were prevented, but there was no reduction in the formation of tyloses or the accumulation of ethylene. Treatment of the stems with inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine) and/or action (silver thiosulfate) delayed and greatly reduced the formation of tyloses in xylem tissue and the size and number of those that formed in individual vessels. Our data are consistent with the hypotheses that wound ethylene production is the cause of tylose formation and that embolisms in vessels are not directly required for wound-induced tylosis in pruned grapevines. The possible role of ethylene in the formation of tyloses in response to other stresses and during development, maturation, and senescence is discussed.     

Micropropagation of Limonium cavanillesii Erben, a threatened statice, from inflorescence stems

Micropropagation of Limonium cavanillesii Erben, a threatened and endemic statice species from Valencia Community (Eastern Spain), was successfully achieved using inflorescence stem pieces as initial explants. Segments 20 mm long from basal parts of immature inflorescences and with axillary buds were cut, sterilised and established in vitro. Shoots obtained from indifferentiated buds were sectioned and then transferred to Murashige and Skoog (MS) medium with 2 mg l^–1 kinetin to provide a plant stock.Shoot multiplication was achieved on MS medium with different cytokinins. The best results for shoot formation were obtained with 2–5 mg l^–1kinetin, 5 mg l^–1 6---dimethylallylaminopurine or 0.1 mg l^–1 6-benzylaminopurine, without significant differences between them. High shoot rooting (80–85%) was obtained within four weeks with indolebutyric acid or indoleacetic acid (0.1 or 0.5 mg l^–1), and also on medium without plant growth regulators. Plant survival to hardened greenhouse conditions was 90% four weeks after plantlet removal from in vitro conditions.This protocol for micropropagation of Limonium cavanillesii is very useful for conservation purposes of endangered statice species, because by using inflorescence stem as initial material it is easier to establish aseptic cultures while preserving the mother plant.     

Auxin is required for the assembly of A-type cyclin-dependent kinase complexes in tobacco cell suspension culture

Although activation of A-type cyclin-dependent kinase (CDKA) is required for plant cell division, little is known about how CDKA is activated before commitment to cell division. Here, we show that auxin is required for the formation of active CDKA-associated complexes, promoting assembly of the complex in tobacco suspension culture Bright Yellow-2 (BY-2) cells. Protein gel blot analysis revealed that CDKA levels increased greatly after stationary-phase BY-2 cells were subcultured into fresh medium to re-enter the cell cycle. However, these increasing levels subsided when cells were subcultured into auxin-deprived medium, and a subtle increase was observed after subculturing into sucrose-deprived medium. Additionally, p13(suc1)-associated kinase activity did not increase significantly after subculturing into either auxin- or sucrose-deprived medium, but increased strongly after subculturing into medium containing both auxin and sucrose. Using gel filtration, we found that p13(suc1)-associated kinase activity against tobacco retinoblastoma-related protein was maximal in fractions corresponding to the molecular mass of the cyclin/CDKA complex. Interestingly, this peak distribution of high molecular-mass fractions of CDKA disappeared after cells were subcultured into auxin-deprived medium. These findings suggest an important role for auxin in the assembly of active CDKA-associated complexes.