Nitrate reductase in cotyledons of cucumber seedlings as affected by nitrate, phytochrome and calcium

Regulation by the active form of phytochrome (P_FR) and the effect of Ca²⁺ was examined with nitrate reductase (NR) in etiolated cucumber ( Cucumis sativus cv. Beilpuig). Nitrate reductase activity (NRA) was studied in excised cotyledons of cucumber seedlings grown in distilled water and in darkness for seven days at 24 ± 0.5°C. All experiments were performed in the dark and a dim green safelight was used during analyses. In etiolated cucumber cotyledons NRA was induced by nitrate and a brief irradiation (15 min) with red light (R) resulted in 62% increase in NRA. This effect was nullified when R was followed immediately by a brief (5 min) far-red light (FR). NRA also showed a semidian (12 h) rhythmicity. Both P_FR, and nitrate effects were age dependent. Calcium seemed to be involved since the phytochrome effect was only observed when calcium was supplied in the external solution. The effect of R on NRA depended on the period of calcium nitrate incubation. An external supply of calcium ionophore mimicked the effect of R and, if supplied to R-irradiated cotyledons, produced a higher NR level than that caused by R alone. This suggested that intracellular free calcium was involved.     

Light inhibition of internode elongation in green plants

The clongation of the first internode of fully greenVigna sinensis L. is inhibited by white light (W). This inhibition is fluence-rate dependent between 0 and 70 Wm^–2. The kinetics of elongation rate in the light after darkness were investigated with linear displacement transducers. The internode elongation rate does not exhibit any endogenous rhythm. A rapid inhibition occurs during the first 2 or 3 h after the onset of light, and a second type of inhibition (slow reaction) increases from the beginning to the 8th hour of light. The rapid inhibition is not fluence-rate dependent between 20 and 70 Wm^–2, but the slow reaction is. There is no rapid inhibition in a low fluence rate white light to high fluence rate white light transition, only the slow reaction is observed. The responses to different wavebands, i.e., blue light (B), yellow and green light (YG), and red light (R), are the same for the two inhibition reactions. Each waveband used separately does not reproduce the full effect observed in W. Results show a stimulation with B, a greater inhibition activity with YG than with R, and a synergistic action of B and R which when given together lead to an inhibition similar to that obtained in W. Plants returned from the light to darkness progressively recover a high elongation rate without any latent period. The W light regulating internode elongation rate is mainly perceived by the growing internode itself.Abbreviations B blue light - D darkness - F far-red light - HW high fluence rate white light - LW low fluence rate white light - R red light - W white light - YG yellow and green light     

Internode length in Lathyrus odoratus. The involvement of gibberellins

Evidence was obtained by gas chromatography-mass spectrometry and gas chromatography-selected ion monitoring for the presence of gibberellin A₂₀), GA₁, GA₂₉, GA₈ and 2-epiGA₂₉ in vegetative shoots of tall sweet pea, Lathyrus odoratus L. Both tall (genotype L –) and dwarf (genotype II ) sweet peas elongated markedly in response to exogenous GA₁ attaining similar internode lengths at the highest dose levels. Likewise internode length in both genotypes was reduced by application of the GA biosynthesis inhibitor, PP333. The ratio of leaflet length to width was reduced by application of PP333 to tall plants and this effect was reversed by GA₁. When applied to plants previously treated with PP333, GA₂₀ promoted internode elongation of L – plants as effectively as GA₁, but GA₂₉ was not as effective as GA₁ when applied to II plants. In contrast, GA₂₀ and GA₁ were equally effective when applied to the semidwarf lb mutant but GA-treated lblb plants did not attain the same internode length as comparable GA-treated Lb – plants. The difference in stature between the tall and dwarf types persisted in dark-grown plants. It is concluded that GA₁ may be important for internode elongation and leaf growth in sweet pea. Mutant l may influence GA₁ synthesis by reducing 3β-hydroxylation of GA₂₀ whereas mutant lb appears to affect GA sensitivity.     

Internode length in Pisum. Comparison of genotypes in the light and dark

There is a strong relationship across the full range of gibberellin deficient, internode length genotypes ( le, lh, is, na ) between internode length in the dark and in red or white light. Further, the new, more severe allele at the le locus. Ie ^d, is shown to influence growth in the dark as well as in the light. These results suggest that darkeness does not specifically overcome any of the steps blocked by the gibberellin (GA) synthesis genes contrasting with the conclusions drawn by other workers. Supporting this conclusion in relation to the Ie gene are results which show that, at least at certain dosage rates, dark-grown Ie na plants respond better to GA₁ than to GA₂₀ similar to the response previously reported in light grown plants.
The greater response by plants of the nana line NGB1766 ( na ) to GA₁ in the dark than in the light suggests that light may influence internode length by altering GA-sensitivity. These results are discussed in relation to previous views on the control of stem elongation by light.     

Temperature-dependent internode elongation in vegetative plants of Arabidopsis thaliana lacking phytochrome B and cryptochrome 1

 Vegetative plants of Arabidopsis thaliana (L.) Heynh. form a compact rosette of leaves in which internode growth is virtually arrested. Rapid extension of the internodes occurs after flower buds are present in the reproductive apex. Under natural radiation, continuous light from fluorescent lamps, or short photoperiods of light from fluorescent lamps, plants of the phyB cry1 double mutant (lacking both phytochrome B and cryptochrome 1) did not form normal rosettes because all the internodes showed some degree of elongation. Internode elongation was weak in the phyB single mutant and absent in the cry1 mutant, indicating redundancy between phytochrome B and cryptochrome 1. The absence of phytochrome A caused no effects. The failure to form normal rosettes was conditional because internode elongation was arrested at low temperatures in all the mutant combinations. In contrast, the temperature dependence of phytochrome B and cryptochrome 1 effects on hypocotyl growth was weak. The elongation of the internodes in phyB cry1 was not accompanied by early flowering as showed by the lack of effects on the final number of leaves. Apex dissection indicated that in phyB cry1 double mutants internode elongation anticipated the transition from the vegetative to the reproductive stage. Thus, stem growth in Arabidopsis thaliana is not fully dependent on the program of reproductive development. Received: 2 June 1999 / Accepted: 13 August 1999     

Internode length in Pisum. Action of gene lw

Jolly, C. J., Reid, J. B. and Ross, J. J. 1987. Internode length in Pisum. Action of gene lw.
Mutant K29 of Pisum sativum L. is shown to possess a recessive gene at a new locus, lw , which results in reduced internode length, delayed flowering and increased symptoms of water congestion compared with the parental cv. Torsdag. The interaction of gene lw with the internode length genes na, le, la and cry ⁵ is examined. Extracts from the shoots of Iw plants are shown to contain similar levels of gibberellin (GA)-like substances to comparable Lw plants, but Iw plants do not elongate to the same extent as Lw plants when treated with GA₁₉ GA₁₉, or GA₂₀. The effect of gene Iw is not graft-transmissible. Unlike essentially isogenic dwarf lines possessing the GA-synthesis genes le, Ih or Is, lw plants show a relative increase in elongation similar to Torsdag in response to photoperiod extensions from sources rich in far-red light. These results suggest that gene lw probably does not reduce elongation by influencing GA-synthesis and that the response to photoperiod extensions with far-red light may depend on the level of GA.     

An acylcyclohexadione retardant inhibits gibberellin A1 metabolism, thereby nullifying phytochrome-modulation of cowpea epicotyl explants

The regulation by phytochrome of stem elongation in light-grown plants depends on gibberellins (GAs). To investigate whether this is mediated by a change in GA metabolism, the effect of the GA biosynthesis inhibitor LAB 198 999 (an acylcyclohexadione derivative) on the end-of-day far-red (FR) response in cowpea ( Vigna sinensis L.) epicotyl explants has been investigated. Growth of epicotyl explants of light-grown seedlings was enhanced when treated with far-red light before incubation in the dark (end-of-day FR effect). Low doses of LAB 198 999 (0.05 and 0.5 μg explant⁻¹) reduced the effect of FR, whereas 5 to 50 μg explant⁻¹ stimulated elongation of both red light (R)- and FR-treated epicotyl explants while nullifying the differences between R and FR treatments. In paclobutrazol-treated epicotyl explants, FR enhanced the response to applied GA₁ and GA₂₀, whereas LAB 198 999 increased the activity of GA₁ and decreased that of GA₂₀, [³H]Gibberellin A₁, injected into the basal part of the epicotyl, was transported and metabolized mainly to [³H]GA₈ in the apical 20 mm of the epicotyl. The conversion of [³H]GA₁ to [³H]GA₈ was dramatically reduced by both end-of-day FR treatments and LAB 198 999 applications. In addition, both treatments enhanced epicotyl elongation. It is proposed that the regulation of cowpea epicotyl growth by phytocrome is mediated, at least partially, by modifying GA₁ degradation.     

Interaction of gibberellins and phytochrome in the control of cowpea epicotyl elongation

The physiological response of cowpea ( Vigna sinensis L.) epicotyl explants to far‐red light (FR) and its interaction with gibberellins (GAs) have been investigated. The effect of FR and GA₁ varied with the age of the seedlings from which the explants were made: for FR, it decreased progressively with age (though the sensitivity of the epicotyls to FR did not change significantly until at least day 11), whereas it remained essentially constant for applied GA₁ between days 5 and 9 after sowing. This indicates that the loss of response to FR may be due to a decrease in endogenous GA levels in the epicotyl. For a range of GA₁ and GA₂₀ (0.01–1 µg explant⁻¹), both hormones were more active in FR than in R irradiated epicotyls, suggesting that phytochrome may affect GA sensitivity besides GA metabolism. The location of the epicotyl region most sensitive to FR (between 5 and 20 mm below the apex) was different from that to GAs (the upper 10 mm). Nevertheless, FR extended the region responsive to applied GAs, even in paclobutrazol‐treated epicotyls where elongation was due entirely to exogenous GAs. This means that modulation of epicotyl elongation by phytochrome, that occurs in a zone different from though overlapping with the GA‐sensitive subapical zone, is also mediated by GAs. Growth in the most FR‐sensitive region of the epicotyl stimulated by FR or GA₁ was due to cell elongation, and in the most GA‐sensitive region to both cell division and elongation. The effect of FR and GA₁ was negated by colchicine, indicating that microtubules may be involved in the response to both factors.     

The end-of-day far-red irradiation increases gibberellin A1 content in cowpea (Vigna sinensis) epicotyls by reducing its inactivation

It has been shown previously that gibberellins (GAs) mediate the phytochrome (Phy) control of cowpea ( Vigna sinensis L.) epicotyl elongation induced by end-of-day (EOD)-far-red light (FR). In the present work, the EOD-FR effect on GA metabolism and GA levels in cowpea has been investigated. GA₁, GA₈, GA₁₉ and GA₂₀ were identified in epicotyls, and GA₁, GA₁₉, GA₂₀ and GA₂₉-catabolite in leaves of 6-day-old cowpea seedlings. The content of GA₁ in the epicotyl paralleled the decrease of its growth rate, supporting the hypothesis that this is the GA bioactive in controlling cowpea epicotyl elongation. FR enhanced both the amount of [3H]GA₁ in the epicotyl produced from applied [3H]GA₂₀, and that of applied [3H]GA₁ that remained unmetabolized in epicotyl explants, suggesting that Phy may regulate the inactivation of GA₁. In agreement with this effect of light on GA₁ metabolism, the contents of GA₁ in the epicotyl remained higher in FR-treated than in R-treated explants. Moreover, in intact seedlings EOD-FR treatment increased both epicotyl elongation and GA₁ content in the responsive epicotyl, whereas it was not altered in the leaves. These results show, for the first time, that photostable Phys modulate the stem elongation in light-grown plants by locally controlling the GA₁ levels through regulation of its inactivation.     

Phytochrome action in light-grown mustard: kinetics,fluence-rate compensation and ecological significance

Internode extension in young, light-grown mustard plants was measured continuously to a high degree of resolution using linear voltage displacement transducers. Plants were grown in background white light (WL) and the first internode was irradiated with supplementary far-red (FR) from fibre-optic light guides, depressing the Pfr/P (ratio of FR-absorbing form of phytochrome to total spectrophotometrically assayable phytochrome) within the internode and causing an acceleration of extension rate. The internode was sensitive to periods of FR as brief as 1 min, with a sharp increase in extension rate occurring after the return to background WL only. The mean latent period of the response to FR was approx. 10 min. Periods of FR longer than approx. 35 min caused an apparently biphasic growth response, with an initial sharp acceleration in extension rate (Phase 1) being followed by a brief deceleration and a further acceleration to a more-or-less steady elevated rate, somewhat less than the first peak (Phase 2). With such longer-term FR, extension rate decelerated upon FR switch-off after a mean lag of approx. 6 min, achieving the prestimulation extension rate within 16 min. The magnitude of the FR-induced increase in extension rate, expressed as a percentage of the rate in WL alone, was an inverse, linear function of the phytochrome photoequilibrium (i.e. Pfr/P, measured in etiolated test material irradiated under the same geometry) over the range 0.17 to 0.63. This relationship was not significantly affected by variations in backround WL fluence rate over the range 50–150 mol·m^-2·s^-1 and was held both for Phase 1 and Phase 2 of the response. The data provide evidence for rapid coupling/uncoupling between phytochrome and its transduction chain in the light-grown plant and for fluence-rate compensation of the regulation of extension rate. The extensive linearity of the relationship between phytochrome photoequilibrium and proportional extension rate increment allows for fine tuning in shade avoidance. The results are discussed with respect to recent evidence on the nature of phytochrome in light-grown plants and in relation to the function of phytochrome in plants growing in the natural environment.Abbreviations FR far-red light - LVDT linear voltage displacement transducer - P total spectrophotometrically assayable phytochrome - PAR photosynthetically active radiation (400–700 nm) - Pfr FR-absorbing form of phytochrome - Pr R-absorbing form of phytochrome - R red light - WL white light     

Phytochrome in Norflurazon-treated seedlings of Pharbitis nil

The properties of phytochrome have been measured by dual-wavelength spectropho-tometry in the cotyledons of the short-day plant Pharbitis nil Choisy cv. Violet, where it is known to play a role in flower induction. In plants de-etiolated by a single white light period (4 h or longer), destruction of the far-red absorbing form of phytochrome (P_fr) was twice as rapid as after 10 min red light. A small fraction of P_fr was stable. After de-etiolation by a period of white light (6 h or longer) the rapid decrease of P_fr during the first 30 min was accompanied by a rapid increase of the red absorbing form of phytochrome (P_fr). This rapid increase of P_fr is probably due to dark reversion. Long term synthesis of phytochrome was inhibited by the presence of P_fr. Phytochrome synthesised in darkness showed the etiolated-plant type characteristics and underwent rapid destruction upon photoconversion to P_fr. The stable P_fr identified here is possibly that pool of phytochrome associated with the long term promotive process in flower induction, and the rapidly reverting P_fr is that pool associated with the night break inhibition of flowering.     

Photoperiodism and photocontrol of stem elongation in two photomorphogenic mutants of Pisum sativum L.

The photomorphogenic mutation lv in the garden pea (Pisum sativum L.), which appears to reduce the response to light-stable phytochrome, has been isolated on a tall, late photoperiodic genetic background and its effects further characterised. Plants possessing lv have a reduced flowering response to photoperiod relative to wild-type plants, indicating that light-stable phytochrome may have a flower-inhibitory role in the flowering response of long-day plants to photoperiod. In general, lv plants are longer and have reduced leaf development relative to Lv plants. These differences are maximised under continuous light from fluorescent lamps (containing negligible far-red (FR) light), and decrease with addition of FR to the incident light. Enrichment of white light from fluorescent lamps with FR promotes stem elongation in the wild type but causes a reduction in elongation in the lv mutant. This “negative” shade-avoidance response appears to be the consequence of a strong inhibitory effect of light rich in FR, revealed in lv plants in the absence of a normal response to red (R) light. These results indicate that the wild-type response to the R: FR ratio may be comprised of two distinct photoresponses, one in which FR supplementation promotes elongation by reducing the inhibitory effect of R, and the other in which light rich in FR actively inhibits elongation. This hypothesis is discussed in relation to functional differentiation of phytochrome types in the light-grown plant. Gene lw has been reported previously to reduce internode length and the response to gibberellin A₁, and to delay flowering. The present study shows that the lw mutation confers an increased response to photoperiod. In all these responses the lw phenotype is superficially “opposite” to the lv phenotype. The possibility that the mutation might primarily affect light perception was therefore considered. The degree of dwarfing of lw plants was found to depend upon light quality and quantity. Dwarfing is more extreme in plants grown under continuous R light than in those grown in continuous FR or blue light or in darkness. Studies of the fluence-rate response show that the lw mutation imparts a lower fluence requirement for inhibition of elongation by white light from fluorescent lamps. Dark-grown lw plants are more strongly inhibited by a R pulse than are wild-type plants but, as in the wild type, this inhibition remains reversible by FR. Light-grown lw plants show an exaggerated elongation response to end-of-day FR light. Taken together, these findings indicate that the lw mutant may be hypersensitive to phytochrome action.     

Ethylene and the Responses to Light of Rice Seedlings

The growth of rice seedlings (Oryza satira L.) in the presence of ethylene caused a change in the response to light of coleoptile elongation. In plants grown in air without added ethylene coleoptile elongation was promoted by red, far-red and yellow-green light only in very young seedlings; in older plants irradiation inhibited the growth of the coleoptile. The effect of growing plants in the presence of ethylene was to prolong the period during which light promoted coleoptile growth. Elongation of the first internode was inhibited by light whether or not the seedlings were grown in the presence of ethylene. A correlation existed between the growth effect of an irradiation and the initial decay rate of phytochrome which was established by the treatment. Regardless of wave length, light sources whose intensities were adjusted to produce a decay rate of about 10% per hour or less induced a moderate rate of coleoptile elongation which persisted for a relatively long period. Irradiation with red or yellow-green light of higher intensity which produced a higher rate of phytochrome decay induced a higher rate of coleoptile elongation, but growth stopped after several hours. Other observations, however, showed that one cannot establish a general simple correlation between the rate of elongation of rice coleoptiles under light and the status of measurable phytochrome in the plant.     

Respiratory metabolism during cold storage of apple fruit. II. Alternative oxidase is induced at the climacteric

Rubbing applied to a young tomato internode inhibited the elongation of this internode and increased soluble peroxidase activity. These morphological and biochemical changes were observed both at the site of rubbing (local response) and in the neighbouring internode (systemic response). The cellular, biochemical, and molecular mechanisms leading to inhibition of internode elongation are not fully understood. It was proposed that mechanical stimuli increased the oxidation of IAA, via the induction of specific peroxidases and stimulated the lignification processes. In order to gain more information about the role of these enzymes, analysis of changes in peroxidase activities were performed. Qualitative analysis of isoperoxidases, by means of native cathodic PAGE, showed four induced isoforms termed C₁, C₂, C₃, and C₄. The major isoform (C₂) was purified to homogeneity and partially characterized. This isoform is probably unglycosylated, with a molecular mass of 36 kDa and a neutral pI of 7.1. The effects of pH and temperature on the activity were determined with guaiacol as electron donor. Optima were obtained at pH 5 and at a temperature of 55°C. The activity of the purified enzyme was not affected by Ca²⁺, Mg²⁺ and Mn²⁺ as was reported for some basic peroxidases. Analysis of substrate specificity revealed that this isoperoxidase acted on ABTS, o -dianisidine, pyrogallol, guaiacol, coniferyl alcohol (monolignol) and IAA but not on syringaldazine. Activitiy of C₂ isoperoxidase on coniferyl alcohol and IAA suggests a possible role of peroxidase C₂ in inhibition of internode elongation, observed in rubbed plants, probably via an increase in lignification processes and regulation of IAA levels in internode tissues.     

Response of net photosynthesis in bean (Phaseolus vulgaris) leaves to the elevation of the partial pressures of oxygen and carbon dioxide

Bean ( Phaseolus vulgaris L. cv. Golden Saxa) plants were grown under low artificial light or under natural daylight. The rate of net photosynthesis (P_N) was measured at: CO₂ partial pressure, p(CO₂), of 0.03, 0.09 or 0.15 kPa; O₂ partial pressure, p(O₂), of 2, 21 or 31 kPa and at light intensities of 350 or 1000 μmol m⁻² s⁻¹ (photosynthetically active radiation). In plants which had been grown under natural light, stimulation of P_N at 21 kPa p(O₂) was found only at elevated p(CO₂) and high light. It is proposed that this phenomenon is dependent on a high capacity of the photosynthetic apparatus to regenerate ribulose 1.5-bisphosphate.     

Effect of sink size on photosynthesis and carbohydrate content of leaves of three spring wheat varieties

Effects of CO₂ on stomatal movements of Commelina communis L. were studied with plants, epidermal strips and guard cell protoplasts. With plants, the stomatal response induced by a blue light pulse was studied for different ambient CO₂ concentration ranging from CO₂-deprived air to 100 Pa in darkness or under red light. It was observed that the blue light response could be obtained not only under a red light background but also in darkness and CO₂-free air, the two responses being quite similar.
With epidermal strips, the effect of CO₂ on ferricyanide reductase activity at the guard cell plasmalemma was studied by transmission electron microscopy. In the presence of ferric ions, reduced ferricyanide gives an electron dense precipitate of Prussian Blue. In darkness and air, no precipitate was observed. In darkness and CO₂-free air as well as under light and normal air, a precipitate was found along the plasmalemma of the guard cells, indicating a ferricyanide reductase activity. With guard cell protoplasts suspended in a medium either in equilibrium with air or in a CO₂-free medium the H⁺ extrusion induced by a blue light pulse added to a red light background was measured. A low CO₂ content was obtained by adding photosynthetic algae to the suspension of guard cell protoplasts. In a CO₂-free medium the rate of H⁺ extrusion was enhanced.
The results are discussed on the basis of a possible competition for reducing power between CO₂ fixation and a putative blue light dependent redox chain located on the plasma membrane.     

Involvement of ethylene in the abscission of flowers and petals of Leptospermum scoparium

The growth of cotyledons and primary leaves of I-day-old Sinapis alba L. plants were studied under various light conditions and action spectra produced. For both responses blue and red light are most effective and a strong fluence rate dependency can be observed. The red light effect appears to be mediated through phytochrome, that of blue light being due to a separate blue light receptor, although this receptor requires the presence of far-red absorbing phytochrome (P_fr) in order to be effective.     

Chromophore structure in phytochrome intermediates and bleached forms of phytochrome

Phytochrome (120 kdalton or 60 kdalton) was isolated from etiolated seedlings of Avena sativa L. cv. Pirol (Baywa München). Irradiation with red light of the P_r form at −23°C in aqueous medium or at −40°C in 66% glycerol leads to the intermediate meta-Rb. Acidification of the glycerol solution at −40°C leads to the absorption of the 15(E) phytochrome chromophore (= P_fr chromophore). Subsequent irradiation transforms this into the 15(Z) chromophore (= P_r chromophore). The presence of the 15(E) chromophore was demonstrated by the same methods also in phytochrome bleached either as P_fr in the dark by 4 M urea, methanol, acetone, ethylene glycol, 8-anilinonaphthalene-1-sulfonate, or as P_r by irradiation with red light in the presence of the same agents. Phytochrome bleached by sodium dodecylsulfate or by dehydration was also investigated. It was concluded that bleached phytochrome contains the P_fr chromophore without specific interaction with the protein.     

Internode length in Pisum. The lrs mutation reduces gibberellin response and levels

We describe a new mutation, lrs , which reduces internode length in Pisum sativum L. The mutation appears to act by reducing both GA synthesis and the response to GA₁. The levels of the 13‐hydroxylated GAs, GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, and GA₈ in the lrs mutant were greatly reduced compared with the wild‐type. The extent of the reduction in GA₁ content in the apical tissues would, at least in part, account for the dwarf phenotype of the mutant. The reduced GA responsiveness of the new mutant was indicated by the inability of applied GA₁ to remove the difference in elongation between lrs and LRS plants. The lrs mutant appears to be unique amongst internode length genotypes, possessing characteristics of both GA synthesis and GA response mutants.