Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: II. Oxygen and Temperature Dependency

Wound-induced ethylene synthesis by subapical stem sections of etiolated Pisum sativum L., cv. Alaska seedlings, as described by Saltveit and Dilley (Plant Physiol 1978 61: 447-450), was half-saturated at 3.6% (v/v) O₂ and saturated at about 10% O₂. Corresponding values for CO₂ production during the same period were 1.1% and 10% O₂, respectively. Anaerobiosis stopped all ethylene evolution and delayed the characteristic pattern of wound ethylene synthesis. Exposing tissue to 3.5% CO₂ in air in a flow-through system reduced wound ethylene synthesis by 30%. Enhancing gas diffusivity by reducing the total pressure to 130 mm Hg almost doubled the rate of wound ethylene synthesis and this effect was negated by exposure to 250 μl liter⁻¹ propylene. Applied ethylene or propylene stopped wound ethylene synthesis during the period of application, but unlike N₂, no lag period was observed upon flushing with air. It is concluded that the characteristic pattern of wound-induced ethylene synthesis resulted from negative feedback control by endogenous ethylene.

No wound ethylene was produced for 2 hours after excision at 10 or 38 C. Low temperatures prolonged the lag period, but did not prevent induction of the wound response, since tissue held for 2 hours at 10 C produced wound ethylene immediately when warmed to 30 C. In contrast, temperatures above 36 C prevented induction of wound ethylene synthesis, since tissue cooled to 30 C after 1 hour at 40 C required 2 hours before ethylene production returned to normal levels. The activation energy between 15 and 36 C was 12.1 mole kilocalories degree⁻¹.

     

Temperature Sensitivity of the Latent Phase in Ethylene-induced Elongation

The temperature sensitivity is reported for the latent period preceding ethylene-induced elongation in the adaxial half of the leaf petiole of Helianthus annuus. When intact plants were exposed to 10 μl of ethylene/l of air over the temperature range 18 to 35 C, the minimum latent time was 62 minutes at 28 C and the maximum was 132 minutes at 18 C. The temperature coefficient, Q₁₀, changed from 2.1 below 28 C, to 0.7 above. In 100 μl of ethylene/l of air, the latent time was reduced by 14% at 18 C, but was significantly increased at 28 and 38 C. These results show that the latent period in the elongation response of the petiole to ethylene cannot be reduced below about 60 minutes by raising either the leaf temperature or the atmospheric ethylene concentration.     

Ethylene production by callus and suspension cells from cortex tissue of postclimacteric apples

Cortex tissue from postclimacteric `Golden Delicious' apples (Malus domestica, Borkh.) stored at 0 C for 9 months after harvest were induced to form callus in vitro. Cell suspension cultures were subsequently formed from calli. Of five media tested, only the medium of Schenk and Hildebrandt (Can J Bot 1972, 50: 192) and that of Uchimiya and Murashige (Plant Physiol 1974, 54: 936) allowed callus formation. During growth both the callus and cell cultures produced ethylene in a pattern which showed a rapid rise and then a fall as the culture grew. ¹⁴C-Labeled methionine was converted to labeled ethylene by the cell suspension cultures, which also could be inhibited from producing ethylene by a rhizobitoxine analog or free radical scavengers. Ethylene production in these cultures, like that in intact fruit tissue slices, could be stimulated by IAA or suppressed by N⁶-(γ,γ-dimethylallyl) adenosine and GA₃.     

Effect of Ethylene on the Release of alpha-Amylase through Cell Walls of Barley Aleurone Layers

A large portion of the gibberellic acid (GA₃)-induced α-amylase in isolated aleurone layers is transported into the incubation medium. In the presence of GA₃ and ethylene, an even larger portion of the enzyme is found in the medium. Employing an acid washing technique developed by Varner and Mense (Plant Physiol 1972 49:187-189), it was observed that ethylene significantly reduces the amount of α-amylase trapped by the thick cell walls of aleurone layers. However, the amount of enzyme remaining in the cell (within the boundary of plasma membrane) is not affected by ethylene. Ethylene has no observable effect on membrane formation as measured by the incorporation of [³²P]orthophosphate into phospholipids. Because of these observations it is suggested that ethylene enhances the release of α-amylase, i.e. transport of α-amylase across cell walls, but not the secretion of α-amylase, i.e. transport of α-amylase past the barrier of plasma membrane. The possible mechanism of this ethylene effect is discussed.     

Gibberellic Acid-enhanced Release of beta-1,3-Glucanase from Barley Aleurone Cells

A β-1, 3-glucanase of barley (Hordeum vulgare) aleurone cells accumulates when half-seeds are imbibed on water, and accumulation continues when the aleurone layers are incubated in buffer solution. The release of the enzyme is a gibberellic acid-dependent process, however. Although gibberellic acid stimulates glucanase release, it does not markedly affect the total amount of glucanase obtained from these cells when compared with water controls. β-1, 3-Glucanase release from aleurone cells is a function of gibberellic acid concentration and commences after a 4-hour lag period. Processes occurring during this lag period are also dependent upon gibberellic acid concentration. Removal of gibberellic acid from the incubation medium at the end of the lag period, however, does not affect subsequent release of glucanase. The release of glucanase from aleurone cells is an active process with a Q₁₀ greater than 3. Inhibitors of respiration and protein and RNA synthesis effectively inhibit the formation and release of glucanase. It is concluded that gibberellic acid functions primarily to enhance glucanase release rather than its formation.     

Mechanism of naphthaleneacetic Acid conjugation: no effect of ethylene

Formation of naphthaleneacetic acid-glucose (NAGLu) in detached leaves, floating on α-naphthaleneacetic acid-1-¹⁴C (NAA, 0.05 microcurie per milliliter, 3.1 μm)-buffer solution (phosphate-citrate, pH 4.2) began immediately while there was a 2- to 4-hour lag before NAA-asparatate (NAAsp) could be detected. Subsequent increase in the NAAsp conjugate reflected a decrease in free NAA to 1 to 2% of the total radioactivity taken up. Pretreatment with 31 μm¹²C-NAA for 18 hours doubled NAAsp formation after transfer for 4 hours to ¹⁴C-NAA. Pretreatment with ethylene, as ethephon (up to 400 milligrams per liter) or ethylene gas (10 microliters per liter), did not induce NAAsp formation. In the presence of NAA, ethylene had no effect on NAA conjugation. Similarly, CO₂ (5%) did not modify the formation of the conjugates. Rhizobitoxine (1.87 μm) inhibited NAA-induced ethylene production but did not prevent NAA-induced formation of NAAsp. We concluded that the conjugation of NAA with aspartic acid is not mediated by ethylene.     

Upsurge in respiration and peroxide formation in potato tubers as influenced by ethylene, propylene, and cyanide

A continuous application of ethylene (10 μl/l) and propylene (500 μl/l) to potato tubers (Solanum tuberosum L.) resulted in an upsurge of respiration and a concomitant rise in peroxides. When applied in 100% O₂, the effect of ethylene and propylene on respiration and peroxide formation was augmented. Hydrogen cyanide (500 μl/l) mimicked the action of ethylene and propylene inducing a respiratory rise and a corresponding increase in peroxides. As with ethylene, the effect of HCN was augmented in high O₂ tensions. The results support the suggestion that ethylene activates the cyanide-insensitive respiratory pathway.     

pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides

We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)₃, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)₃, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)₃, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)₃; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation.     

Effect of the Hypocotyl Hook on Chlorophyll Accumulation in Excised Cotyledons of Cucumis sativus L

Hypocotyl hooks have been shown to influence greening in excised cucumber (Cucumis sativus) cotyledons. The properties of the lag phase are greatly affected by the presence or absence of the hook tissue. A 45-second light pretreatment followed by 4 hours of darkness is sufficient to remove the lag phase from cotyledons with hooks, while hookless cotyledons require 2 hours of continuous illumination followed by 1 hour of dark incubation to break the lag phase. The effect of hooks on cotyledon greening is enhanced if the hooks are shielded from light. Cutting off the hooks after lag phase removal caused a marked decrease in chlorophyll accumulation in the cotyledons. These observations may indicate that the hypocotyl hooks produce a substance or substances needed in the greening process, which are translocated to the cotyledons. Indoleacetic acid, abscisic acid, gibberellin A₃, 6-benzylamino purine and δ-aminolevulinic acid do not show any activity; on the other hand, ethylene appears to replace partially the hypocotyl hooks.     

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells

The kinetics of accumulation (per milliliter of culture) of the α- and β- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The β-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the α-subunit holoenzyme(s). After 120 minutes, the α-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using ³⁵SO₄ and immunochemical procedures, the rate of synthesis of the α-subunit was shown to be greater than the β-subunit during the first 80 minutes of induction. The α- and β-subunits had different rates of degradation during the induction period (t_½ = 50 versus 150 minutes, respectively) and during the deinduction period (t_½ = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.     

Cross-Correlation Functions for a Neuronal Model

Cross-correlation functions, R_XY(t,τ), are obtained for a neuron model which is characterized by constant threshold θ, by resetting to resting level after an output, and by membrane potential U(t) which results from linear summation of excitatory postsynaptic potentials h(t). The results show that: (1) Near time lag τ = 0, R_XY(t,τ) = f_U [θ-h(τ), t + τ] {h′(τ) + E_U [u′(t + τ)]} for positive values of this quantity, where f_U(u,t) is the probability density function of U(t) and E_U [u′(t + τ)] is the mean value function of U′(t + τ). (2) Minima may appear in R_XY(t,τ) for a neuron subjected only to excitation. (3) For large τ, R_XY(t,τ) is given approximately by the convolution of the input autocorrelation function with the functional of point (1). (4) R_XY(t,τ) is a biased estimator of the shape of h(t), generally over-estimating both its time to peak and its rise time.     

Abscission: role of cellulase

Cellulase (β-1,4-glucan-glucanohydrolase EC 3.2.1.4) activity increased during abscission and was localized in the cell separation layer of Phaseolus vulgaris L. cv. Red Kidney (bean), Gossypium hirsutum L. cv. Acala 4-42 (Cotton) and Coleus blumei Benth. Princeton strain (Coleus) abscission zone explants. Cellulase activity was optimum at pH 7, was reduced by one-half after heating to 55° for 10 min, and was associated with the soluble components of the cell. Explants treated with aging retardants (indoleacetic acid, ⁶N-benzyladenine, and coumarin), CO₂, actinomycin D or cycloheximide had less cellulase activity than untreated controls. Ethylene increased cellulase activity of aged explants after a 3-hr lag period but had no effect on cellulase activity of freshly excised explants. It was concluded that 1 of the roles of ethylene in abscission is to regulate the production of cellulase which in turn is required for cell separation.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase

F_OF₁ ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of F_OF₁. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-β, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. b_I and b_II). The results show the b dimer to be located at a non-catalytic α/β cleft, with b_I close to subunit α, whereas b_II is proximal to subunit β. Furthermore, b_I can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-b_I-α, b_II-β, α-b_I-b_II-β, and a-b_I-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that b_I is functionally related to the single b subunit present in mitochondrial ATP synthase.     

Mitochondrially Mediated Integrin αIIbβ3 Protein Inactivation Limits Thrombus Growth

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin α_IIbβ₃ or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin α_IIbβ₃ and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin α_IIbβ₃ epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin α_IIbβ₃ function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β₃ cytoplasmic domain, coincided closely with integrin α_IIbβ₃ inactivation. Calpain inhibition blocked integrin β₃ cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β₃ and associated regulatory proteins, resulting in integrin α_IIbβ₃ inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.     

Endogenous Gibberellins from Sporophytes of Two Tree Ferns, Cibotium glaucum and Dicksonia antarctica

Gibberellin A₁ (GA₁), 3-epi-GA₁, GA₄, GA₉, 11α-hydroxyGA₁₂, 12α-hydroxyGA₁₂, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₅, GA₃₇, GA₄₀, GA₅₈, GA₆₉, GA₇₀, and GA₇₁ have been identified from Kovats retention indices and full scan mass spectra by capillary GC-MS analyses of purified extracts from sporophytes of the tree fern, Cibotium glaucum. Abscisic acid, dihydrophaseic acid, an epimer of 4′-dihydrophaseic acid, and the epimeric ent-6α, 7α, 16α, 17-(OH)₄ and ent-6α, 7α, 16β, 17-(OH)₄ derivatives of ent16, 17-dihydrokaurenoic acid, in addition to the epimeric 16α, 17- and 16β, 17-dihydroxy-16, 17-dihydro derivatives of GA₁₂, were also identified in extracts of C. glaucum. An oxodihydrophaseic acid and a hydroxydihydrophaseic acid were also detected. In extracts of sporophytes of Dicksonia antarctica, GA₄, GA₉, 12α- and 12β-hydroxyGA₁₂, GA₁₅, GA₂₅, and GA₃₇ were identified by the same criteria, as well as abscisic acid, phaseic acid, 8′-hydroxymethylabscisic acid and dihydrophaseic acid. This is the first time that GA₄₀ has been identified in a higher plant; it is also the first report of the natural occurrence of the two gibberellins, 11α- and 12β-hydroxyGA₁₂. The total gibberellin (GA) content in C. glaucum (tall) was at least one order of magnitude greater than that of D. antarctica (dwarf) based on total ion current response in GC-MS and bioassay data. Abscisic acid was a major component of D. antarctica and the oxodihydrophaseic acid was a major component of C. glaucum.     

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

The DnaX complex (DnaX₃δδ′χψ) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β₂, onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β₂ binding (determined functionally) is diminished 12–30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β₂. DNA synthesis activity can be restored by increased concentrations of β₂. In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β₂ loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.