Comparison of IAA-Induced and Low Temperature-Induced GA(3) Responsiveness and alpha-Amylase Production by GA(3) Insensitive Dwarf Wheat Aleurone

Rht3-containing gibberellic acid (GA₃) insensitive deembryonated wheat (Triticum aestivum L. var Cappelle Desprez × Minister Dwarf) aleurone, that can be made responsive to GA₃ by low temperature, can also be rendered GA₃ sensitive by preincubation with indoleacetic acid (IAA). The IAA-induced response of the dwarf selection is concentration-dependent, relatively sensitive, and similar in magnitude to that induced by low temperature. Other auxins also induce GA₃ responsiveness to a greater or lesser degree. IAA has no apparent effect on the wild type (rht, tall) selection.     

Influence of Gibberellic Acid and the Rht3 Gene on Choline and Phospholipid Metabolism in Wheat Aleurone Tissue

The effect of gibberellic acid (GA3) on phospholipid metabolismand -amylase production was studied in aleurone tissue of twonear-isogenic lines of wheat (Triticum aesuvum L.). Incubationof embryoectomized seeds from a GA-responsive line (rht3, tall)with GA₃ caused the induction of -amylase activity after a lagphase of 30 h. In the case of embryoectomized seeds from a ‘GA-insensitive’line (Rh13, dwarf), however, the lag phase was extended up to50 h. During the first 14 h following imbibition, GA₃ inhibitedcholine uptake and its subsequent incorporation into phosphatidylcholine in the Rhr3 line but not in the rht3 line. GA₃ promotedphospholipid breakdown in both the lines during this period,however. GA₃ also terminated independent turnover of the cholineN-methyl groups in phosphatidyl choline and promoted turnoverof the whole choline headgroup. These results are discussedin relation to the possibility that phosphatidyl choline turnoveris an integral part of the GA₃ signal-transduction mechanismin aleurone tissue. Key words: GA3, Rht3 gene, choline, phospholipid     

Effect of Temperature on Gibberellin (GA) Responsiveness and on Endogenous GA(1) Content of Tall and Dwarf Wheat Genotypes

Near-isogenic wheat (Triticum aestivum L.) lines differing in height-reducing (Rht) alleles were used to investigate the effects of temperature on endogenous gibberellin (GA) levels and seedling growth response to applied GA₃. Sheath and lamina lengths of the first leaf were measured in GA treated and control seedlings, grown at 11, 18, and 25°C, of six Rht genotypes in each of two varietal backgrounds, cv Maris Huntsman and cv April Bearded. Endogenous GA₁ levels in the leaf extension zone of untreated seedlings were determined by gas chromatography-mass spectrometry with a deuterated internal standard in the six Maris Huntsman Rht lines grown at 10 and 25°C. Higher temperature increased leaf length considerably in the tall genotype, less so in the Rht1 and Rht2 genotypes, and had no consistent effect on the Rht1+2, Rht3 and Rht2+3 genotypes. In all genotypes, endogenous GA₁ was higher at 25°C than at 10°C. At 10°C the endogenous GA₁ was at a similar level in all the genotypes (except Rht2+3). At 25°C it increased 1.6-fold in the tall genotype, 3-fold in Rht1 and Rht2, 6-fold in Rht3, and 9-fold in Rht1+2. Likewise, the genotypic differences in leaf length were very conspicuous at 25°C, but were only slight and often unsignificant at 11°C. The response of leaf length to applied GA₃ in the Rht1, Rht2, and Rht1+2 genotypes increased significantly with lowering of temperature. These results suggest the possibility that the temperature effect on leaf elongation is mediated through its effect on the level of endogenous GA₁ and that leaf elongation response to endogenous or applied GAs is restricted by the upper limits set by the different Rht alleles.     

Low Temperature-Induced GA(3) Sensitivity of Wheat : I. Characterization of the Low Temperature Effect on Isolated Aleurone OF KITE

Gibberellic acid (GA₃) sensitivity (measured as α-amylase production) of the isolated aleurone tissue/deembryonated seed of two wheat (Triticum aestivum L. var Kite and var Aroona) varieties each containing either one of the dwarfing genes, Rht1 and Rht2, was increased significantly as a result of low temperature treatment. The magnitude of the low temperature-induced increase occurred without any change in the lag time of α-amylase production. This low temperature induction of GA₃ sensitivity was found to be operative in aleurone tissue of only those varieties having at least one of the three Rht alleles. It is likely, therefore, that the low temperature treatment effect which `cures' or circumvents the genetic lesions manifest in the Rht1 and Rht2 genotypes is the same as that effective in the Rht3-containing genotype and probably involves an increase in hormone (GA₃) receptor sites. Furthermore, this increase appears to be a quantitative temporal one.     

Low Temperature-Induced GA(3) Sensitivity of Wheat : II. Changes in Lipids Associated with the Low Temperature-Induced GA(3) Sensisivity of Isolated Aleurone of Kite

Exposure of isolated aleurone tissue from the wheat (Triticum aestivum) variety Kite which contains the Rht2 allele, to low temperature (5°C) for 20 h prior to addition of exogenous GA₃, resulted in significant changes in the content of lipids, especially phospholipids. Significant low temperature-induced changes in both the head group and acyl contents of two phospholipids, phosphatidylcholine and phosphatidylethanolamine, were detected. More importantly, these changes displayed a very close temporal relationship with the low temperature-induced increase in GA₃ sensitivity. Further, this relationship was paralleled by a highly significant correlation between the changes in the phospholipids and the changes in α-amylase production. These results underline the possibility that the GA₃ receptor sites are membrane-based lipids.     

Salicylic Acid in Rice (Biosynthesis,Conjugation, and Possible Role)

Salicylic acid (SA) is a natural inducer of disease resistance in some dicotyledonous plants. Rice seedlings (Oryza sativa L.) had the highest levels of SA among all plants tested for SA content (between 0.01 and 37.19 [mu]g/g fresh weight). The second leaf of rice seedlings had slightly lower SA levels than any younger leaves. To investigate the role of SA in rice disease resistance, we examined the levels of SA in rice (cv M-201) after inoculation with bacterial and fungal pathogens. SA levels did not increase after inoculation with either the avirulent pathogen Pseudomonas syringae D20 or with the rice pathogens Magnaporthe grisea, the causal agent of rice blast, and Rhizoctonia solani, the causal agent of sheath blight. However, leaf SA levels in 28 rice varieties showed a correlation with generalized blast resistance, indicating that SA may play a role as a constitutive defense compound. Biosynthesis and metabolism of SA in rice was studied and compared to that of tobacco. Rice shoots converted [14C]cinnamic acid to SA and the lignin precursors p-coumaric and ferulic acids, whereas [14C]benzoic acid was readily converted to SA. The data suggest that in rice, as in tobacco, SA is synthesized from cinnamic acid via benzoic acid. In rice shoots, SA is largely present as a free acid; however, exogenously supplied SA was converted to [beta]-O-D-glucosylSA by an SA-inducible glucosyltransferase (SA-GTase). A 7-fold induction of SA-GTase activity was observed after 6 h of feeding 1 mM SA. Both rice roots and shoots showed similar patterns of SA-GTase induction by SA, with maximal induction after feeding with 1 mM SA.     

Genotypic variation in the responsiveness to GA3 within tall (rht1) and semi-dwarf (Rht1) spring wheat

The effect of GA₃ on coleoptile-and first leaf elongation of tall (rht1) and semi-dwarf (Rht1) nearly-isogenic genotypes, within each of 25 random F₉ wheat families, was determined on seedlings grown in a growth room at 18 °C. Conspicuous and very significant inter-family variation in the response of the first leaf to GA₃ application was found in both the rht1 and Rht1 genotypes. The magnitudes of the response of the different families within genotypes to GA₃ were not related to the leaf length of their untreated seedlings. It is suggested that, under given environmental conditions, background genotypic effects, inducing inter-family variation in responsiveness to GA₃, regulate the elongation growth up to the limits set by the Rht alleles.     

Interactions between Ethylene, CO(2), and ABA on GA(3)-Induced Amylase Synthesis in Barley Aleurone Tissue

Gibberellic acid-induced synthesis and release of α-amylase in barley aleurone tissue was inhibited by abscisic acid. This inhibition was relieved by simultaneous application of ethylene ranging in concentration from 0.1 to 100 microliters per liter. When CO₂ was applied, it eliminated the effect of 0.1 microliter per liter ethylene and reimposed the abscisic acid inhibition. All concentrations of CO₂ tested from 400 to 10⁵ microliters per liter counteracted the effect of 0.1 microliter per liter ethylene, but had no observable effect on any higher concentration of ethylene. The results indicate that some processes necessary for embryo growth may be subject to regulation by ethylene and carbon dioxide at naturally occurring concentrations of the gases.     

Modification of the Induction of Photosynthesis in Wheat by Glyphosate, an Inhibitor of Amino Acid Metabolism

Ireland, C. R., Pcrcival, M. P. and Baker, N. R. 1986. Modificationof the induction of photosynthesis in wheat by glyphosate, aninhibitor of amino acid metabolism.—J. exp. Bot. 37: 299–308 The effect of the herbicide glyphosate, an inhibitor of aminoacid metabolism, upon the kinetics of induction of photosynthesiswas investigated in wheat leaves. After treatment with a commercialpreparation of glyphosate the induction period for both photosyntheticcarbon assimilation and chlorophyll fluorescence emission wasincreased approximately by 50% after 2 d and by 100%) after5 d. The quenching of chlorophyll fluorescence became a slowerprocess and the S-M transient was diminished. In the steady-stateneither the rate of carbon fixation nor the level of fluorescenceemission were altered. The changes in induction kinetics precededthe occurrence of visible leaf damage. The fluorescence emissionwas analysed by a technique which estimates the redox stateof the PS2 primary electron acceptor, Q, and the photochemicaland non-photochemical components of fluorescence quenching.Glyphosate produced no fundamental change in the relationshipbetween the steady-state values of these parameters until thetime when extensive leaf damage occurred. Thus there was nodirect effect of glyphosate on the capacity for light captureor on the rate of photosynthetic electron transport. This wasconfirmed by the absence of any effect on the quantum efficiencyof oxygen evolution. The slower rate of fluorescence quenchingduring induction is attributed to a slower generation of thetrans-thylakoid pH. It is proposed that glyphosate affects photosyntheticinduction kinetics by an indirect modification of carbon metabolismwhich limits photosynthetic rate during this phase. The possibilityof screening for rapid effects of non-photosynthetic herbicides,such as glyphosate, by monitoring the kinetics of photosyntheticinduction, is raised. Key words: Photosynthesis, induction, glyphosate (N-phosphonomethyl-glycine), wheat (Triticum aestivum L.)     

Metabolism of Tritiated Gibberellin A(20) in Immature Seeds of Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A₂₀ ([³H]GA₂₀) was applied via the pedicel to immature pods and seeds of dwarf peas and three harvests were made at days 5, 10, and 23 (mature) after application. Of the five metabolites of [³H]GA₂₀, the three in highest yield were GA₂₉, an α,β-unsaturated ketone, and a compound (B), whose structure was only tentatively assigned. The metabolic sequence GA₂₀ → GA₂₉ → compound B → the ketone was indicated. The amount of [³H]GA₂₉ in both seeds and pods was highest at day 5 and declined to its lowest level at maturity. The amount of the [³H]ketone in the seed increased with time to its highest level at maturity. It is suggested that compound B and the ketone represent the major pathway of catabolism of GA₂₉, a 2β-hydroxylated GA of low biological activity, and that the ketone is not metabolized, or only slowly metabolized, during seed maturation.     

Interrelationships between trans-Plasma Membrane Electron/Proton Transfer Stoichiometry, Organic Acid Metabolism, and Nitrate Reduction in Dwarf Bean (Phaseolus vulgaris)

Iron deficiency in dwarf bean (Phaseolus vulgaris L.) induces an increased activity of a system in the rhizodermal cells, which reduces extracellular ferric salts, and an active proton efflux from the roots, which is coupled to accumulation of citrate and malate in the roots and subsequent export of these compounds in the xylem. During reduction of extracellular ferricyanide by Fe-deficient plants, the stoichiometry of electron transport to proton efflux is 2e⁻/1H⁺, and citrate and malate levels in the roots are strongly decreased. Reduction of ferricyanide by Fe-sufficient plants has no influence on root and shoot levels of citrate and malate, but in such plants the process is characterized by a e⁻/H⁺ efflux stoichiometry close to unity. Apparently, organic acid metabolism and transport are closely associated with the e⁻/H⁺ efflux ratio. To assess the significance of organic acid metabolism as one of the direct intracellular components of the induced unbalanced e⁻/H⁺ efflux by roots, we studied NO₃⁻ reduction in shoots and roots of Fe-deficient and Fe-sufficient plants. Nitrate reductase activity in the roots was positively correlated with the level of citrate and malate, whereas the enzyme activity in the leaves responded positively to the import of these organic acid anions.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Phosphatidate Kinase,A Novel Enzyme in Phospholipid Metabolism (Characterization of the Enzyme from Suspension-Cultured Catharanthus roseus Cells)

Phosphatidate kinase (adenosine 5[prime]-triphosphate:phosphatidic acid phosphotransferase), a novel enzyme of phospholipid metabolism, was detected recently in the plasma membranes of suspension-cultured Catharanthus roseus cells and purified (J.B. Wissing, H. Behrbohm [1993] Plant Physiol 102: 1243-1249). In the present work the properties of phosphatidate kinase are described. The enzyme showed a pH optimum of 6.1 and an isoelectric point of 4.8, and was rather stable in the presence of its substrates. Although the kinase accepted both ATP and GTP, with Km values of about 12 and 18 [mu]M, respectively, the only lipid substrate was phosphatidic acid; neither lysophosphatidic acid nor any other lipid tested was phosphorylated. With 32P- and 14C-labeled diacylglycerol pyrophosphate, the product of the enzyme, it was shown that the kinase catalyzes a reversible reaction. The activity of the extracted enzyme depended on the presence of surfactants such as Triton X-100 or [beta]-octylglucoside, whereas deoxycholate was strongly inhibitory. Kinetic analysis with Triton X-100/phosphatidate mixed micelles performed according to the "surface dilution" kinetic model showed saturation kinetics with respect to both bulk and surface concentration of phosphatidate. The interfacial Michaelis constant for phosphatidate was determined as 0.6 mol %.     

Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme)

The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle.     

Effect of High Temperature on the Cell Membrane,Membrane Lipid,and Ascorbic Acid Metabolism in Rauwolfia( Rauvolfia vericillata,Apocynaceae)

To explore the mechanism of growth retardation under high temperature in Rauwolfia ( Rauvolfia vericillata), the effects of high temperature on membrane integrity, lipid peroxidation , and antioxidant ascorbic acid were examined by measuring the electric conductivity, membrane lipid peroxide malondialdehyde (MDA), and changes of ascorbic acid in Rauwolfia leaves. The results showed that exposure to high temperature (44℃ and 50℃) resulted in increases in electric conductivity and MDA, and decrease in levels of abscorbic acid. Heat acclimation, pretreatment at 37℃, however, maintains ascorbic acid at higher levels than that without heat acclimation , which may be helpful for Rauwolfia to resist heat stress.     

高温对萝芙木细胞膜、膜脂和抗坏血酸代谢造成的损伤

通过高温胁迫下萝芙木叶片电导率、膜脂过氧化物丙二醛(MDA)、自由基清除剂抗坏血酸的变化研究,探讨高温对萝芙木组织造成的损伤及自由基清除剂代谢的影响,从而揭示高温胁迫下萝芙木生长发育迟缓的原因.结果表明,热胁迫对萝芙木细胞膜造成损伤,使膜脂更多地被过氧化,抗坏血酸含量下降,致使萝芙木受到热损伤和氧化损伤,最终可能使其生长发育放缓,开花结果推迟.研究还表明,萝芙木经适当高温(37℃)热驯,能产生较多的抗坏血酸,以清除热胁迫诱导产生的氧自由基,降低氧化损伤程度.     

Phosphatidate Kinase,a Novel Enzyme in Phospholipid Metabolism (Purification,Subcellular Localization,and Occurrence in the Plant Kingdom)

Microsomal membranes from suspension-cultured Catharanthus roseus cells possess an enzymic activity that catalyzes the ATP-dependent phosphorylation of phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B. Wissing [1993] FEBS Lett 315: 95-99). This enzyme activity, PA kinase, was purified and characterized. Plasma membranes, obtained from C. roseus microsomes by aqueous two-phase partitioning, were extracted, and PA kinase was purified 3200-fold by applying different chromatographic steps that resulted in a specific activity of about 10 [mu]mol min-1 mg-1. Sodium dodecyl sulfate-gel electrophoresis of the fractions obtained from the final chromatographic step revealed a 39-kD protein that correlated with the enzyme activity; PA kinase activity could be eluted from this protein band. Subcellular localization, investigated with C. roseus cells, showed that the activity was confined to membrane fractions, and at least 80% was associated with plasma membranes. The data revealed the same distribution within the cellular membranes of PA kinase as reported for diacylglycerol kinase, which is a typical plasma membrane-located enzyme. Furthermore, PA kinase activity was detected in the calli of 16 different plant species and in the different organs of C. roseus plants and obviously occurs ubiquitously in the plant kingdom.     

Sulfuric Acid and Gibberellic Acid (GA3) Treatment Combined with Exposure to Cold Temperature Modulates Seed Proteins during Breaking of Dormancy to Germination in Tilia miqueliana

The Protein Journal - Tilia miqueliana produces woody seeds that exhibit deep dormancy. In this study, we used cell biology methods, including Paraffin section determination and Coomassie brilliant...     

Effects of Various Levels of CO(2) on the Induction of Crassulacean Acid Metabolism in Portulacaria afra (L.) Jacq

In response to water stress, Portulacaria afra (L.) Jacq. (Portulacaceae) shifts its photosynthetic carbon metabolism from the Calvin-Benson cycle for CO₂ fixation (C₃) photosynthesis or Crassulacean acid metabolism (CAM)-cycling, during which organic acids fluctuate with a C₃-type of gas exchange, to CAM. During the CAM induction, various attributes of CAM appear, such as stomatal closure during the day, increase in diurnal fluctuation of organic acids, and an increase in phosphoenolpyruvate carboxylase activity. It was hypothesized that stomatal closure due to water stress may induce changes in internal CO₂ concentration and that these changes in CO₂ could be a factor in CAM induction. Experiments were conducted to test this hypothesis. Well-watered plants and plants from which water was withheld starting at the beginning of the experiment were subjected to low (40 ppm), normal (ca. 330 ppm), and high (950 ppm) CO₂ during the day with normal concentrations of CO₂ during the night for 16 days. In water-stressed and in well-watered plants, CAM induction as ascertained by fluctuation of total titratable acidity, fluctuation of malic acid, stomatal conductance, CO₂ uptake, and phosphoenolpyruvate carboxylase activity, remained unaffected by low, normal, or high CO₂ treatments. In well-watered plants, however, both low and high ambient concentrations of CO₂ tended to reduce organic acid concentrations, low concentrations of CO₂ reducing the organic acids more than high CO₂. It was concluded that exposing the plants to the CO₂ concentrations mentioned had no effect on inducing or reducing the induction of CAM and that the effect of water stress on CAM induction is probably mediated by its effects on biochemical components of leaf metabolism.