The partial purification and characterisation of gibberellin 2β-hydroxylases from seeds of Pisum sativum

The gibberellin (GA) 2-hydroxylases in mature and immature seeds of Pisum sativum have been partially purified and characterised. The enzymes are unstable when stored below pH 7.0 or in the absence of a thiol reagent. The optimum assay pH is between 7.4 and 7.8 and activity is dependent upon the presence of -ketoglutarate, Fe²⁺ and ascorbate. The 2-hydroxylase activities for GA₁, GA₄, GA₉ and GA₂₀ are chromatographically inseparable and correspond to a protein of M_r 44000. The rate of GA 2-hydroxylation varies according to substrate and some evidence indicates that the 2-hydroxylase activities for GA₁ and GA₄ and for GA₉ and GA₂₀ may reside in different proteins. During pea seed maturation, the specific activity of the enzyme(s) increases dramatically and reaches a maximum at a time when endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ are also at their greatest concentration. This correlation is not the result of substrate induction of enzyme activity. Since the GA 2-hydroxylases operate at maximal rate at low substrate concentrations they are incapable of rapidly 2-hydroxylating excessive quantities of (exogenously applied) GA₁ or GA₂₀. On the basis of the kinetic parameters of the GA 2-hydroxylase activities, a generalised model is discussed for the control of the steady-state levels of bioactive hormone under normal physiological conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - HSS high-speed supernatant - LSS low-speed supernatant - PMSF phenylmethane sulphonyl fluoride     

Metabolism of gibberellins in a cell-free system from immature seeds of Phaseolus vulgaris L.

The biosynthetic steps from gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to C₁₉-GAs were studied by means of a cell-free system from the embryos of immature Phaseolus vulgaris seeds. Stable-isotope-labeled GAs were used as substrates and the products were identified by gas chromatography-mass spectrometry. Gibberellin A₁₂-aldehyde was converted to GA₄ via non-hydroxylated intermediates and to GA₁ via 13-hydroxylated intermediates. 13-Hydroxylation took place at the beginning of the pathway by the conversion of GA₁₂-aldehyde to GA₅₃-aldehyde. The conversion of GA₂₀ to GA₅ and GA₆ was also shown but no 2-hydroxylating activity was found. Endogenous GAs from embryos and testas of 17-dold seeds were re-examined by gas chromatography-selected ion monitoring using stable-isotopelabeled GAs as internal standards. Gibberellins A₉, A₁₂, A₁₅, A₁₉, A₂₃, A₂₄, and A₅₃ were identified for the first time in P. vulgaris, in addition to GA₁, GA₄, GA₅, GA₆, GA₈, GA₁₇, GA₂₀, GA₂₉, GA₃₇, GA₃₈ and GA₄₄, which were previously known to occur in this species. The levels of all GAs, except the 2-hydroxylated ones, were greater in the embryos than in the testas. Conversely, the contents of GA₈ and GA₂₉, both 2-hydroxylated, were much higher in the testas than in the embryos.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - GC-SIM gas chromatography-selected ion monitoring - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - m/z ion of mass     

The cytoskeleton of Cobaea seed hairs:

The cell wall of Cobaea scandens seed hairs developed in a characteristic sequence, with the deposition of a cellulose thread onto a pectic swelling layer was the final event. The cellulose thread was intracellularly accompanied by a band of 10–18 microtubules. During the formation of the swelling layer the microtubules were homogeneously distributed; they ran circumferentially normal to the cell axis. When cellulose-thread formation started, the microtubules became arranged in a helical band. The density of the microtubules varied during the different phases of development. The highest density was observed before cellulosethread formation and ranged from 6–15 m·m^-2. The length of the microtubules, 20–30 m, was determined by direct measurements, as well as estimated from the total microtubular length in a given area and the counted free ends. With the indirect immunofluorescence technique the microtubules of the band stained inhomogeneously. Those which were located at the edges of the band fluoresced more intensely than those of the central part. Attempts to visualize actin filaments in the hair cells with rhodaminyl-conjugated phalloidin resulted in a homogeneous staining of the area of the microtubular band, indicating that actin filaments may be present in this region. Though, in thin sections and dry-cleaved cells, filamentous structures were observed between the microtubules, caution is expressed that the observed fluorescence was, indeed, due to actin filaments. The role of the filamentous structures is discussed with respect to formation and maintenance of the microtubular band. Microtubules apparently did not cross coated pits which were visualized in the plasma membrane through the dry-cleaving technique.Abbreviations IFT indirect immunofluorescence technique - RP rhodaminyl-conjugated phalloidin - SEM scanning electron microscopy     

Studies on the complex formed between glucagon and dicaprylphosphatidylcholine

The interaction between glucagon and dicaprylphosphatidylcholine (DCPC) was studied by fluorescence, circular dichroism and calorimetry, as well as by ¹H- and ³¹P-nuclear magnetic resonance. The water-soluble lipid-protein complex was also characterized by gel filtration and ultracentrifugation. The complex appeared to be monodisperse by sedimentation equilibrium measurements, with a molecular weight of (4.55 ± 0.57)·10⁴. This complex contained approximately 7 molecules of glucagon and 35 molecules of phospholipid. Proton-decoupled ³¹P-NMR spectra of the phospholipid in the lipid-protein complex display narrower resonances than those of sonicated vesicles of DCPC, and ¹H-³¹P coupling could be detected in proton coupled spectra. These NMR results, together with gel-filtration results, suggest that glucagon ‘solubilizes’ phospholipid aggregates, forming a lipid-protein complex which is smaller than sonicated preparations of DCPC. ¹H-NMR resonance of both the methionine methyl group (met-27) and the aromatic envelope of glucagon are broadened by the phospolipid, indicating that the C-terminal region and the aromatic residues are involved in the interaction with the phospholipid. Nuclear magnetic resonance titrations of the imidazole ring C(2) and C(4) protons of the histidine residue of glucagon show that DCPC lowers the pK of the imidazole. The alterations caused by the phospholipid in the far and near ultraviolet CD spectra of glucagon reflect, respectively, the increased helix content of the hormone and the fact that the aromatic residues are located in a more structured environment. The phospholipid also alters the fluorescence properties of glucagon, shifting the fluorescence emission maximum of the hormone to shorter wavelength, and enhancing its relative intensity. This suggests that the fluorophore is experiencing a more hydrophobic environment in the presence of the lipid. Binding of glucagon to the phospholipid was analysed by Scatchard plots of the enhancement of fluorescence caused by the phospholipid and showed that the equilibrium binding constants of glucagon to DCPC are (4.4 ± 0.5)·10⁴M^?1 and (7.5±0.5)·10⁴M^?1, at 15°C and 25°C, respectively. The average number of moles of phospholipid bound per mole of glucagon is 4.4±0.6. The isothermal enthalpy of reaction of glucagon with DCPC is ?20.5 kcal/mol of glucagon at 25°C and ?32.5 kcal/mol of glucagon at 15°C. The observed enthalpies can arise from glucagon-induced cyrstallization of the phospholipid, from the non-covalent interactions between the peptide and lipid as well as from the lipid-induced conformational change in the protein. These results demonstrate that, unlike the complexes formed between glucagon and phospholipids which form more stable bilayers, the complex formed between glucagon and DCPC is stable over a wide range of temperatures, including temperatures well above the phase transition.     

Genetic specificity of nitrogen nutrition in leguminous plants

Summary Mineral nitrogen did not increase grain yield and seed protein levels ofVicia faba L. andLupinus luteus L. in field trials and pot experiments. Fixed N₂ was substituted by mineral nitrogen in these cases because of inhibition of N₂ fixation by mineral nitrogen. Contrary to these results mineral nitrogen increased grain yields and seed protein amounts ofLupinus albus L.,Pisum sativum L., andGlycine max. (L.) Merr. The nitrogen effect was caused at an early stage by saving energy due to inhibition of N₂ fixation (measurement of gas exchange by means of IRGA). In case of the N application after flowering grain, yields and seed protein levels increased because the mineral N was an additional nitrogen source for plants. At this stage the plants had ceased fixing atmospheric nitrogen. The high sink activity of growing fruits induced a lack of assimilates in nodules (determined by means of¹⁴CO₂ application). The N effect was therefore the consequence of the lower assimilate pool for supplying root nodules in these plants in comparison withVicia faba L. andLupinus luteus L. Hence it follows that response to mineral nitrogen can be a criterion for discovering more effective Rhizobium-host combinations.     

Micelle formation of diacylglycerophosphocholines in organic solvents I. effects of the solvents on krafft points

Krafft points of diacylglycerophosphocholines (PC) were measured in alkanes-cyclohexane solutions by differential scanning calorimetry, and it was found that they were regularly increased following the increase in alkane content in the solutions and the chain length of the alkanes. From these results it was deduced that the mixing of PC with alkanes occurred in the gel state of the PC, but not in micelles at higher temperatures above the Krafft points. where micellar solutions are provided. The penetration of alkanes into gel state PC was found to be dominated by Langmuir type interaction, and the affinity of alkanes increases with increasing in chain lengths. Above the Krafft points, the micelle formation was confirmed by using the fluorescence probe technique.     

Establishment of microscale vegetation pattern in maritime chaparral after fire

We quantified microscale pattern in vegetation and seed assemblages along a 24 m transect before and for two years following a controlled burn in chamise (Adenostoma fasciculatum) chaparral in central coastal California. Our objective was to document scale-dependent correlation between pre-burn seed assemblages, pre-burn canopy cover, microtopography, soil temperatures during burning, post-burn seed assemblages and post-burn vegetation. Scale-dependent correlations among pre- and post-burn seed densities, maximum soil temperatures during burning, microtopography and post-burn vegetation were measured based on two-term local covariance analysis.Seed distribution varied among species prior to fire, with seeds of some annual species concentrated in gap areas. Maximum soil temperatures during burning ranged from less than 50 °C to 225 °C, and were generally lowest in gaps in the pre-burn canopy. These gaps were associated with local topographic depressions. After burning, readily germinable seeds were concentrated in or near gaps in the pre-burn canopy. Germination of different species was variously enhanced, diminished or unchanged by the passage of fire. Post-burn vegetation was very patchy, with some areas nearly devoid of seedlings and other areas, especially pre-burn canopy gaps, supporting numerous seedlings. Seedling recruitment patterns in the second year were generally highly correlated with patterns in pre-burn seed banks and first year vegetation. Although many species exhibited similar recruitment patterns, several different mechanisms may have been responsible for the origin of those patterns.     

胡萝卜人工种子在无菌条件下的贮藏及贮藏中种子活力变化

对用海藻酸钙包埋胡萝卜体细胞胚(0.6—2mm)制作的人工种子进行贮藏研究,发现低温及干燥均能在一定程度上抑制人工种子萌发。失水率达67%的种子,2℃贮藏60天后, 在发芽培养基上发芽率为100%,7天内的成苗率达80%。未经贮藏的对照种子发芽率为100%,成苗率为76%。无论在“种皮”还是在胚胎悬浮培养液中加入脱落酸或香豆素,都能抑制胚的生长,但也促进了胚的衰老。铝箔袋内密闭贮藏期间种子不萌发,但活力下降快。随着贮藏时间的延长,种子的活力指数、脱氢酶活性、氧吸收率都呈下降趋势。     

Contribution of buried seeds to revegetation after eruptions of the volcano Usu,Northern Japan

Soon after the 1977–78 eruptions of the volcano Usu, there were created many gullies in which former topsoil, i.e., well-developed soil accumulated before the eruptions, was eroded in the crater basin, whereas the outside of the gully was covered with thick volcanic deposits. The short-dispersal-seed plants were the most abundant in the inside of gully where the former topsoil was exposed, however, they have not immigrated from external environments. The germination tests of the seeds extracted from the former topsoil demonstrated that viable seeds were buried at 1683.3/m² for 9 years after the eruptions. At least, 12 herbaceous species, e.g.,Rumex obtusifolius, Geum macrophyllum var.sachalinense andPoa annua, were derived from the buried seeds in the former topsoil. Their contribution to revegetation was estimated to be 40.0% in the inside of gully where the former topsoil was exposed in 1983, suggesting that buried seeds are one of the most important seed source for revegetation even in the case of a volcano which produced pumice and ash by eruption.     

Labeling of Retina and Optic Tectum Phospholipids in Chickens Exposed to Light or Dark

The labeling of retina ganglion cell and optic tectum phospholipids was determined in chickens given an intraocular injection of 32P and then either exposed to light or maintained in the dark. Significantly higher labeling was found in the optic tectum phospholipids of light-exposed compared with dark-maintained animals after 3-24 h of labeling. In the ganglion cells, the labeling of phospholipids increased in dark with respect to light at 15 and 30 min of labeling; from 60 min to 24 h, the labeling of phospholipids was significantly higher in light with respect to dark, even if the precursor pool showed a higher labeling in dark at all times studied. When labeling was allowed to proceed in the dark for 30 min and then half of the animals were exposed to light for 15 min, the labeling of ganglion cell phospholipids of light-exposed animals was significantly higher than those of animals kept in the dark. No individual phospholipid accounted for the differences observed in the labeling of the total phospholipid pool. These results are interpreted as an increase in the biosynthesis of phospholipids in the ganglion cell somas in light with respect to dark.