Phytochrome-controlled ethylene biosynthesis of intact etiolated bean seedlings

Intact etiolated bean (Phaseolus vulgaris L. cv. Limburgse vroege) seedlings were illuminated with red light (10.5 W·m^-2) for 10 min. After different time intervals ethylene production, and contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-1-carboxylic acid were measured. The red-light-induced decrease of ethylene production in 8-d-old intact etiolated bean seedlings was fast, strong and long-lasting ad was mediated through the phytochrome system. This effect appeared to be strictly age-dependent, as it could not be detected in plants younger than 6 d or older than 11 d.The capacity for the conversion of ACC to ethylene was not affected by red light. The inhibitory effect of the light treatment on ethylene production could be related to a reduced free-ACC content. This reduction was a consequence of a temporary non-reversible increase of ACC malonylation and a long-lasting, for a certain time reversible, inhibition of ACC synthesis. The effect of a brief irradiation with red light on the decrease of ethylene production and free-ACC content was completed after about 2 h. Reversibility by far-red, however, persisted for at least 3 h, and was lost between 3 and 6 h.Abbrevation ACC 1-aminocyclopropane-1-carboxylic acid - M-ACC 1-(malonylamino)cyclopropane-1-carboxylic acid     

Influence of ethylene produced by soil microorganisms on etiolated pea seedlings

There is indirect evidence that soil microorganisms producing ethylene (C(2)H(4)) can influence plant growth and development, but unequivocal proof is lacking in the literature. A laboratory study was conducted to demonstrate the validity of this speculation. Four experiments were carried out to observe the characteristic "triple" response of etiolated pea seedlings to C(2)H(4) microbially derived from l-methionine as a substrate in the presence or absence of Ag(I), a potent inhibitor of C(2)H(4) action. In two experiments, the combination of l-methionine and Acremonium falciforme (as an inoculum) was used, while in another study the indigenous soil microflora was responsible for C(2)H(4) production. A standardized experiment was conducted with C(2)H(4) gas to compare the contribution of the microflora to plant growth. In all cases, etiolated pea seedlings exhibited the classical triple response, which includes reduction in elongation, swelling of the hypocotyl, and a change in the direction of growth (horizontal). The presence of Ag(I) afforded protection to the pea seedlings against the microbially derived C(2)H(4). This study demonstrates that microbially produced C(2)H(4) in soil can influence plant growth.     

Dithiothreitol activation of some chloroplast enzymes in extracts of etiolated pea seedlings

Dithiothreitol treatment produces marked stimulation of the activity of the light-activated enzymes ribulose-5-P kinase, NADP-linked glyceraldehyde-3-P dehydrogenase, NAD-linked glyceraldehyde-3-P dehydrogenase, and NADP-linked malic dehydrogenase, and inactivation of the light-inactivated oxidative pentose phosphate pathway enzyme glucose-6-P dehydrogenase, in extracts of etiolated pea seedlings. It seems likely that light activation or inactivation of these enzymes can occur as soon as the photochemical apparatus becomes functional.     

Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a ²H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - FW fresh weight - GC/MS coupled gas chromatography/mass spectrometry - HPLC high-performance liquid chromatography     

The effects of inhibitors of protein synthesis on the differentiation of plastids in etiolated bean seedlings

Summary It has been shown that inhibitors of protein synthesis do not influence the breakdown of the crystal-lattice-like structure of the prolamellar bodies in the plastids when etiolated plants are exposed to light. The formation of grana and the greening of leaves are however considerably inhibited, depending on the concentration of the inhibitor used.     

Effects of red light and ethylene on growth of etiolated lettuce seedlings

Low concentrations of ethylene inhibit hypocotyl elongation of etiolated lettuce seedlings (Lactuca sativa cv. Grand Rapids), whereas red light does not inhibit it. The plumular hook tightens in response to either ethylene or red light. A combination of these two factors gives an additive response. Red light has no effect on ethylene production and red light will cause hook closure even under hypobaric pressure which removes endogenous ethylene. This suggests that ethylene and red light act independently in causing hook closure.     

Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings

The tonoplast ATPase from etiolated seedlings of Vigna radiata L. (mung bean) was isolated using a two-step detergent solubilization modified from Mandala and Taiz (S Mandala, L Taiz [1985] Plant Physiol 78: 327-333). After ultracentrifugation on 10 to 28% sucrose gradient, the ATPase showed a 31.6-fold purification over the initial specific activity of the starting tonoplast-enriched membranes. The purified ATPase used Mg²⁺-ATP as the preferred substrate. The tonoplast ATPase was isolated in a form with characteristics similar to that on its native membrane environment. Analysis by SDS-PAGE revealed two prominent bands with molecular weights of 78,000 (α subunit) and 64,000 (β subunit). The intensity of Coomassie blue staining showed a 1:1 stoichiometry for α and β subunits. The amino acid composition of α and β subunits also confirmed the suggested stoichiometry of the subunit composition of the tonoplast ATPase. Moreover, radiation inactivation analysis yielded a functional size of 414 ± 24 and 405 ± 25 kilodaltons for soluble and membrane bound tonoplast ATPases, respectively. It is possible that the functioning tonoplast ATPase may be in a form of αβ-heteromultimer.     

A brassinosteroid-cytokinin interaction on ethylene production by etiolated mung bean segments

Three cytokinins and their ribosides were tested in conjunction with brassinosteroid (BR) for their effects on ethylene production in etiolated mung bean ( Vigna radiata L. Rwilcz cv. Berken) hypocotyl segments. When varying concentrations of BR were tried in combination with a fixed amount of kinetin (10 μ M ), there was an increase in ethylene production with increasing concentrations of BR up to 3 μ M , and a decline beyond. A stimulation in ethylene production was observed with increasing concentrations of each of the cytokinins used. However, when applied in conjunction with 3 μ M BR, all cytokinin concentrations produced similar stimulatory patterns. When 3 μ M BR or 10 μ M 6-benzylaminopurine (BAP) was applied in conjunction with increasing concentrations of IAA, there was a stimulation in ethylene production with increasing concentrations of IAA. However, 3 μ M BR and 10 μ M BAP together with varying concentrations of IAA failed to alter the level of ethylene production.     

Multiple forms of hydroxycinnamate : CoA ligase in etiolated pea seedlings

A survey of a range of plant tissues showed that the hydroxycinnamate CoA ligase in crude extracts of pea shoots had a high relative activity towards sinapic and other methoxycinnamic acids, together with high activity with p-coumaric acid. The pea enzyme has been resolved by chromatography on DEAE-cellulose into two peaks which differ in their substrate specificity. The form which elutes at relatively low salt concentrations has a ratio activity towards p-coumaric and sinapic acids of about 1.8:1 while the form eluting at higher salt concentrations, although showing very high activity with p-coumaric acid, is inactive towards sinapic acid. The pattern of elution of these forms following gel filtration on Ultragel AcA 34 and Sephadex G100 suggests that these two isoenzymes which differ in ionic properties and substrate specificity can exist in two or three molecular weight forms and there is evidence that these forms are under certain circumstances interconvertible.     

Carbon isotope ratios of respired CO2 from castor bean, corn, peanut, pea, radish, squash, sunflower and wheat seedlings

During the first month after germination peanut and sunflowerseedlings exhibit a significant shift in the ¹³C/¹²C ratiosof respired CO₂ indicating thatcarbohydrate gives way to lipidas the respiratory substrate. Other species (castor bean, corn,pea,radish, squash and wheat) show no change in the ¹³C/¹²C ratio (Received March 9, 1971; )     

Control by phytochrome of C-sucrose incorporation into buds of etiolated pea seedlings

When etiolated pea epicotyls are excised immediately above the cotyledons and dipped basally into ¹⁴C-sucrose, their terminal buds respond to red light by increased growth (IG) and enhanced incorporation of sucrose (EIS). Both phenomena are phytochrome controlled, showing typical kinetics, reversal by far-red light, escape from photochemical control and limitation to leaf tissue. EIS is of greater magnitude, occurs more rapidly and is saturated by lower energies of red light than IG, suggesting its possible importance as a controlling reaction in phytochrome-mediated growth. Both IG and EIS are best shown in the presence of a long epicotyl derived from a 5 to 6-day-old seedling in the presence of about 0.1 m unlabeled sucrose in the medium.     

Studies on peroxidase isolated from etiolated Alaska pea seedlings I. General properties

Two kinds of peroxidase were isolated chromatographically frometiolated Alaska pea seedlings. One had absorption maxima at421 and 530 mµ, and the other, at 405, 500 and 640 mµ.From their affinities for cyanide and migrating patterns onelectrophoresis, the latter was concluded to be a modified formof the former. They were named peroxidase b and b', respectively.Both oxidized IAA, the optimal pH value for the reaction being3.0. (Received April 20, 1970; )     

The effect of brassinosteroid on auxin-induced ethylene production by etiolated mung bean segments

Brassinosteroid, an analogue of brassinolide, (BR) (2α, 3α, 22β, 23β-tetrahydroxy-24β-methyl-B-homo-7-oxa-5α-cholestan-6-one), was tested in conjunction with indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), indole-3-propionic acid (IPA), indole-3-pyruvic acid (IPyA), indole-3-aldehyde (IAld), indole-3-carbinol (ICB) or tryptophan (TRP) for its effects on ethylene production by etiolated mung bean (Vigna radiata (L.) Rwilcz cv. Berken) hypocotyl segements. The enhancement of ethylene production due to BR was greatest in conjunction with 1 μM IBA, 2,4-D, IAA, or NAA (these increases were 2580, 2070, 890, and 300%, respectively). When increasing concentrations of IBA, 2,4-D, IAA, or NAA were used, there was a decrease in the percentage stimulation by BR. Both IPyA and IPA had different optimal concentrations than the other auxins tested. Their BR-enhanced maximum percentage stimulations (1430 and 1580%) were greatest with 5 μM IPya and 10 μM IPA, respectively. There was a marked reduction in the percentage stimulation by BR with either 100 μM IPyA or IPA. The inactive indoles (IAld, ICB, or TRP) did not synergize with BR at any of the concentrations tested. Four hours following treatment those segments in contact with 1 μM BR with or without the addition of 10 μM IAA began to show a stimulation in ethylene production above the control and this stimulation became greater over the following 20 h. It was necessary for BR to be in continual contact with the tissue to have a stimulatory effect on auxin-induced ethylene production. When segments excised from greater distances below the hypocotyl hook, were treated with either IAA alone or in combination with BR, there was a decrease in ethylene production with increasing distance. There was no effect of hypocotyl length on BR stimulation of auxin-induced ethylene production; however, there was a definite decrease in ethylene production when IAA was applied alone.     

Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction     

On ethylene and stem elongation in green pea seedlings

Maximum elongation of excised internodal stem sections of light-grown pea (Pisum sativum L.) seedlings occurred at 10⁻⁵ molar indoleacetic acid (IAA), with submaximal responses occurring at 10⁻⁴ and 10⁻³ molar. Accompanying elongation at concentrations of IAA of 10⁻⁶ to 10⁻³ molar was production of ethylene, with the amount increasing up to 10⁻⁴ molar IAA and then becoming nearly constant. Elongation of light-grown sections was not inhibited by exogenous ethylene up to 10,000 ppm in the presence of 10⁻⁵ molar IAA. Marked (up to 50%) inhibition of elongation of internodal segments in situ was observed after treating whole light-grown seedlings with exogenous ethylene for 20 hours. It is concluded that ethylene is not responsible for the submaximal elongation responses of green pea stem sections at high auxin concentrations, but that IAA per se is accountable.     

PIP1 aquaporins,sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm³) and “heavy” (≥1.146 g/cm³) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.     

Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings.

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.     

Regulation of ethylene biosynthesis after short-term heat treatment in etiolated pea stems

Incubation of plant tissues at a constant elevated temperature greatly inhibits both basal and wound ethylene production. However, recovery from heat treatment is relatively rapid and is followed by stimulated ethylene production. The present investigation examines the kinetics of ethylene production after short-term heal treatment and the regulation of heat-altered ethylene production. Subapical stem segments of 7-day-old etiolated pea L. cv. Alaska) seedlings were analyzed for ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) oxidation, and ACC and l-(malonylamino)cyclopropane-l-carboxylic acid (MACC) content after a 2-min 40°C heat pulse. The short-term heat pulse transiently inhibited ethylene production and ACC oxidation accompanied by a slight ACC accumulation within a 30-min time period. Conjugation to MACC did not appear to play an integral role in heat-regulated ethylene production. It was concluded that the major factor affecting ethylene production after heat treatment is the temporary inactivation of ACC oxidation. The possible roles of ACC synthase, ACC oxidase and lipoxygenase in regulating ethylene production after heat treatment are discussed.