The Growth Physiology of Coleoptiles in Continuous-Record Vessels: Anomalous Kinetics of Cycloheximide Action on Auxin-Controlled Growth

The interacting effects of auxin and cycloheximide upon the growth of Triticum coleoptiles depends upon the incubation conditions. In the growth vessel developed by Evans and Ray (1969) the action of cycloheximide is severe and the growth kinetics show a rapid nullification of auxin action by the inhibitor; hormone action is also totally prevented by a relatively short period of pre-treatment with cycloheximide. The kinetics of response to the two substances are quite different when coleoptiles are floating on the test solutions; here, cycloheximide is much less severe in its action. These differences are due to a heightened sensitivity to cycloheximide induced by exclusion of the air bubble from the coleoptile cylinder in the Evans and Ray chamber. The outer surface of the coleoptile wall is a less efficient respiratory surface than the inner one. When coleoptiles are filled with a stagnant solution insufficient oxygen can be taken up through the inner surface and the tissue therefore suffers oxygen deprivation. This exacerbates the action of cycloheximide as a potent respiratory inhibitor. The results raise questions concerning the use of conditions in which coleoptile sections are filled and submerged, which is a feature of recently introduced continuous-record growth vessels. In particular, interpretation of the interactions between cycloheximide and auxin under such conditions must be made cautiously.     

Expression of α-expansin genes in young seedlings of rice (Oryza sativa L.)

 Rice is the only cereal in which germination and coleoptile elongation occur in hypoxia or anoxia. Little is known of the molecular basis directly underlying coleoptile cell extension. In this paper, we describe the expression of α-expansin genes in embryos during seed development and young seedlings grown under various oxygen concentrations. The genes Os-EXP2 and Os-EXP1 were predominantly expressed in the developing seeds, mainly in newly developed leaves, coleoptiles, and seminal roots. These expansins expressed in the developing seeds may give cells the potential to expand after seed imbibition begins. In coleoptiles, Os-EXP4 and Os-EXP2 mRNAs were greatly induced by submergence, while they were weakly detected in aerobic or anoxic conditions. Under submerged soil conditions, the signals hybridized with probes Os-EXP4 and Os-EXP2 in coleoptiles were strongest when coleoptiles elongated in the water layer. These data show that expansin gene expression is highly correlated with coleoptile elongation in response to oxygen concentrations. The Os-EXP4 gene was also expressed in leaves, mesocotyls, and coleorhizas of young seedlings. The growth of these tissues was also correlated with the presence of expansins. Therefore, the evidence derived from this study clearly demonstrates that expansins are indispensable for the growing tissues of rice seedlings. Received: 23 December 1999 / Accepted: 24 February 2000     

Possible pathways of heat resistance induction in plant cells by exogenous nitrogen oxide

The mechanisms of exogenous nitrogen oxide (NO) influence on heat resistance of wheat coleoptiles have been studied. The treatment of plant cells with an NO donor (sodium nitroprusside) resulted in an increase in the generation of superoxide anion radicals (O ₂ ^·? ) after 10 min. The inhibitor of protein biosyn-thesis??cycloheximide??did not inhibit the O ₂ ^·? generation by coleoptiles induced by the NO donor, whereas the inhibitor of phosphatidic acid formation (butanol-1) inhibited it partially. Treatment of coleoptiles with calcium ionophore (A23187) or with an activator of the inositol cycle (inositol) compensated for the suppression effect of butanol-1 on NO-dependent O ₂ ^·? formation. Butanol-1 also leveled out the increase in the heat resistance of coleoptiles caused by the NO donor, whereas calcium ionophore and inositol almost completely removed the butanol-1 effect. The possible participation mechanisms of reactive oxygen species, phosphatidic acid, and calcium ions in the realization of NO physiological effects are discussed.     

Tissue-Specific Features of Pigment Biogenesis in Coleoptiles of Greening Etiolated Seedlings of Cereals

Biogenesis of the pigment apparatus was studied in coleoptiles of postetiolated barley seedlings (Hordeum vulgare L.) and triticale (Triticale), differing in chlorophyll content, during growing in a “ light-darkness” regime with a 16-h photoperiod. Photoactive protochlorophyllide with a fluorescence maximum at 655 nm (Pchlide655), which accumulates in coleoptiles of etiolated seedlings, was converted in the light into a chlorophyll pigment with a fluorescence maximum at 690 nm (excitation at 440 nm, temperature ?196°C). The spectral transition 690 nm → 675 nm forms was completed in darkness for 15 min illumination. There was almost no resynthesis of new portions of Pchlide655 in coleoptiles under darkness conditions, even after a 5–6-h darkness period after brief illumination of seedlings with flashes of white light. Chlorophyllide (Chlide) formed from Pchlide655 was not esterified and was destroyed both in the light (4 h, 1.0–1.5 klx) and darkness. In coleoptiles of greening etiolated seedlings, chlorophyll formation started only by 24 h of illumination. The instability of the chlorophyll pigment formed after etiolation indicates that plastids of coleoptiles do not contain the system of chlorophyll biosynthesis centers typical of leaves, which are bound to membranes and protect pigment from destruction.     

Effects of Light on the Enzymic Composition of Aegilops triaristata Seedlings

Electrophoretic patterns of enzymes, esterases, phosphatasesand peroxidases in hexaploid seedlings of Acgilops triaristatawere examined by horizontal starch gel electrophoresis. Electropherogramswere evaluated by gel densitometer traces. Comparisons weremade between organs of A. triaristata seedlings developed inlight and darkness. Each organ presented a characteristic enzymicpattern, while quantitative and qualitative differences wereobserved between organs developed in light and darkness. Thesame differences were also observed by an enzyme assay. Possiblecauses of these differences in enzyme activities, especiallyin coleoptiles, are discussed.     

Photogravitropic equilibrium in <Emphasis Type="Italic">Avena</Emphasis> coleoptiles: fluence rate–response relationships and dependence on dark adaptation and clinostating

Phototropism of Avena coleoptiles was measured in response to blue-light irradiation lasting between 2 and 24 h. During this time the coleoptiles established a bending angle of photogravitropic equilibrium that was dependent on the time of irradiation and also on the pretreatment in light or darkness prior to stimulation. The absolute threshold for the photogravitropic equilibrium in response to blue light was 10⁻⁸ μmol m⁻² s⁻¹. Photon fluence rate–response curves, which were generated after several hours of dark adaptation, had a characteristic shape with a prominent optimum in the middle of the dynamic range. Curves which were generated without prior dark adaptation displayed no such optimum. Clinostating dark-adapted coleoptiles caused an increase of sensitivity and responsiveness during a 2-h period of unilateral irradiation. The advantages and the drawbacks of long-term irradiation experiments for the investigation of phototropism and the generation of action spectra are discussed. Received: May 14, 2001 / Accepted: December 7, 2001     

On the developmental dependence between Cyanophora paradoxa and Cyanocyta korschikoffiana in symbiosis

The prokaryote Cyanocyta korschikoffiana was isolated from the eukaryote Cyanophora paradoxa. The synthesis of several thylakoid proteins in these cyanelles is influenced by light and darkness and is sensitive to cycloheximide, the inhibitor of the eukaryotic host's translation. The possibility of a direct coordination between the translations of the host and of the cyanelles is discussed.Abbreviations CHM treatment addition of cycloheximide - CPN chlorophylline - PBN phycobiline - SDS-PAGE sodium-dodecylsulphate-polyacrylamide gelelectrophoresis     

Effects of canavanine on IAA- and fusicoccin-stimulated cell enlargement, proton extrusion and potassium uptake in maize coleoptiles

In maize coleoptiles (Zea mays F₁ XL 640A, cv. Dekalb) canavanine and cycloheximide strongly and simultaneously inhibit cell elongation, H⁺ extrusion and K⁺ uptake, induced by IAA. They inhibit also, although to a much lesser degree, the same phenomena induced by fusicoccin. Cycloheximide severely depresses the incorporation of leucine into proteins, while canavanine leaves leucine incorporation almost unchanged. The data confirm that elongation, H⁺ extrusion and K⁺ uptake can be regarded as correlated processes; they also support the view that normal protein synthesis is essential for IAA-induced growth, while this requirement is only partial in growth stimulated by fusicoccin.     

The Effects of Red, Far-red, and Blue Light on the Geotropic Response of Coleoptiles of Zea mays

The effects of red, far-red, and blue light on the geotropicresponse of excised coleoptiles of Zea mays have been investigated.Seedlings were grown in darkness for 5 or 6 days, exposed tovarious light treatments, and then returned to darkness fordetermination of the geotropic response. The rate of response of the coleoptiles is decreased after theyhave been exposed to red light (620–700 mµ, 560ergs cm^–2sec^–1 for the 24 hrs, but not for the 4hrs, preceding stimulation by gravity. Furthermore, their rateof response is greatly reduced if they are exposed to red lightfor 10 min and then returned to darkness for 20 hrs before geotropicstimulation. At 25° C an interval of 6 to 8 hrs elapses between a 10-minexposure to red light and the first detectable decrease in thegeotropic response of the coleoptile. This interval can be lengthenedby exposing the seedlings to low temperatures (0° to 2°C) after the light treatment but cannot be greatly shortenedby increasing the duration of exposure to red light. Using a standard procedure of exposing 5-day-old etiolated seedlingsto light for various times, replacing them in darkness for 20hrs and then determining the response of the coleoptiles to4 hrs geotropic stimulation, it has been found that: (a) Exposureto red light for 15 sec significantly decreases the geotropiccurvature of the coleoptiles and that further reduction occurson increasing the length of the light treatment to 2 and 5 min.(b) Far-red light has no effect on the geotropic response ofthe coleoptiles but it can completely reverse the effect ofred light. After repeated alternate exposure to red and far-redlight the geotropic response of the coleoptile is determinedby the nature of the last exposure, (c) Complete reversal ofthe effect of red light by far-red radiation only occurs whenexposure to far-red follows immediately after exposure to red.The reversing effect of far-red radiation is reduced if a periodof darkness intervenes between the red and far-red light treatments,and is lost after a dark interval of approximately 2 hrs. The effect of red light on the rate of geotropic response ofthe coleoptiles is independent of their age and length at thetime of excision. Blue light acts in a similar way to red light, but the seedlingsare less sensitive to blue than to red light. Coleoptiles grown throughout in a mixture of continuous, weak,red, and far-red light have a lower rate of geotropic responsethan etiolated coleoptiles.     

PHYTOCHROME CHANGES IN TISSUES OF DARK-GROWN SEEDLINGS REPRESENTING VARIOUS PHOTOPERIODIC CLASSES

Excised tissues of dark-grown seedlings representing long day, short day and daylength indifferent photoperiodic classes were assayed for nonphotochemical changes in phytochrome. In all tissues tested, these changes were qualitatively the same. A brief irradiation with red light was followed in darkness by a decrease in total phytochrome, the disappearance of P_FR, and an increase in detectable P_R. Within the limits of the tissues tested, the kinetics of phytochrome change can be assigned to three groups on the basis of rates. These groups are represented by coleoptiles, hypocotyls and epicotyls, and mesocotyls. The kinetics could not be distinguished on the basis of the photoperiodic class of the mature plant. The significance of these kinetics with respect to the photochemistry of phytochrome conversion is discussed.     

Stimulation of Avena coleoptile growth by reduction of oxygen supply

Summary The growth of Avena sativa L. coleoptiles was accelerated by reduction of the O₂ concentration in the surrounding atmosphere. Cell-wall extensibility was increased in close relation to the increase in elongation, 6–8% O₂ giving the optimal effect in either case. Growth promotion by reduced O₂ concentration and by auxin (indole-3-acetic acid; IAA) were additive, at least at lower auxin concentrations.This response to reduced O₂ concentration was also present in sections pretreated with cycloheximide for 2 h, although such sections showed no response to applied IAA. No significant change was observed in auxin transport under reduced O₂ supply. It is suggested that the acceleration of cell elongation and the loosening of cell wall by reduced O₂ supply are due to a mechanism which is not directly dependent on auxin-induced growth. We propose to designate this acceleration of growth under reduced O₂ concentrations as oxygen-sensitive growth.     

Growth and anthocyanin synthesis in excised Sorghum internodes

Summary Ligh-induced anthocyanin synthesis in excised dark-grown internodes of Sorghum was depressed by the addition of auxin to the incubating medium at physiological concentrations. Both IAA and the synthetic auxin, 2,4-D, reduced anthocyanin yield. Similar results were obtained with isolated internode segments and in internodes incubated with coleoptiles (the major source of endogenous auxins) attached. Auxin increased the duration of the lag phase before anthocyanin synthesis began and reduced the rate during the subsequent linear phase. Elongation continued longer with IAA than without it and anthocyanin formation did not begin until extension growth had ceased or was slowing down in both cases; the rate of anthocyanin synthesis in the IAA solution remained depressed compared with that in buffer even after extension growth had ceased in both.At low concentrations IAA stimulated elongation growth without reducing anthocyanin yield and it is unlikely that the effect of IAA on anthocyanin synthesis results from the increased utilisation in growth of substrates needed for anthocyanin formation. The results of reciprocal transfer experiments from dark to light, and vice versa, showed that the action of IAA was associated with its presence in the incubating medium during the irradiation period. If present only in darkness, before or after transfer to light, IAA did not reduce anthocyanin formation; in the former case total yield was increased by IAA as a result of the stimulation of elongation growth, the concentration of anthocyanin remaining unchanged.GA₃ also decreased anthocyanin content; the effect was greater in sections incubated with coleoptiles attached and it is possible that GA₃ acts by increasing the concentration of endogenous auxins. However, CCC, which has been reported to decrease endogenous auxin levels, also reduced anthocyanin yield.The effect of IAA was not influenced by the presence of ascorbate in the incubating medium, nor did added ascorbate result in the formation of any acylated cyanidin derivative in internodes maintained in darkness.Possible relationships between light-induced anthocyanin formation and the photo-inhibition of elongation are discussed.     

Possible mechanisms of light-induced chlorophyll degradation in senescing leaves of Hydrilla verticillata

Light treatment markedly accelerated the chlorophyll loss in senescing leaves of Hydrilla verticillata [(L.f.) Royle] as compared to dark treatment, whereas such acceleration could not be observed in senescing spinach (Spinacia oleracea L.) leaves. The light-induced cholorophyll loss in Hydrilla was retarded slightly by chloramphenicol and markedly by cycloheximide. Catalase (EC 1.11.1.6) activity did not change appreciably in Hydrilla leaves either in light or in darkness, while in spinach it declined markedly in the dark, and light retarded such decline. Peroxidase activity in Hydrilla showed faster increase in light than in darkness, while in spinach it increased only in light during senescence. The activity of phenol(pyrogallol)-specific peroxidase increased markedly in light, and that of ascorbate-specific peroxidase decreased slightly both in light and darkness during senescence of Hydrilla leaves. This rise in phenolspecific peroxidase activity was prevented by cycloheximide treatment. Pretreatment of Hydrilla leaves with monophenol (2,4-dichlorophenol) and o-diphenol (hydroquinone) accelerated and retarded, respectively, the light-induced cholorophyll loss. Pretreatment of Hydrilla leaves with H₂O₂ augmented the chlorophyll loss more markedly in light than in darkness. The endogenous level of H₂O₂ increased more in light than in dark during senescence of Hydrilla leaves. Treatment of Hydrilla leaves with 3-(3.4-dichlorophenyl)-l,l-dimethylurea. a photosystem II inhibitor, prevented both light-induced rise in H₂O: level and chlorophyll loss, but it was without effect in the dark. Retardation of light-induced chlorophyll loss occurred during senescence of Hydrilla leaves when light was given in different photoperiods in a 24-h daily cycle for 6 days instead of as continuous irradiance. There was a negative correlation between the length of the photoperiod and the extent of cholorophyll loss.     

Mesocotyl growth of etiolated seedlings ofAvena sativa andZea mays in relation to light and diffusible auxin

Diffusible auxin levels were measured in coleoptiles and mesocotyls of dark-grown seedlings ofavena sativa (cv. Spear) andZea mays (cv. Golden Cross Bantam) using theAvena curvature bioassay. The coleoptile tip was confirmed as the major auxin source in etiolated seedlings. Auxin levels were found to decrease basipetally in sequent sections of theAvena coleoptile but not to decrease in apical sections of increasing length. An inhibitor capable of inducing positive curvatures ofAvena test coleoptiles was discovered in diffusates from the mesocotyls of oat and corn seedlings. The amount of this inhibitor was correlated with the cessation of mesocotyl growth of oat seedlings grown in darkness, and with the inhibition of mesocotyl growth of corn seedlings exposed to red light.     

Metabolism of Indole-3-acetaldehyde

Infiltration of indolcacelaldehyde (IAAId) into living tissues of sonic lower and higher plants gives rise simultaneously to both indoleacetic acid (IAA) and Iryptophol (T-ol). But on a molar basis, there is no correlation between the products indicating a disimitation. Expressed juice of Avena coleoptiles by itself, exhibits only IAA forming activity. Approximately two moles of IAAld are consumed for each mole of IAA formed at pH 4.5, but only if necessary corrections are made for losses of substrate and products. Addition of reduced NAD or NADP readily induces tryptophol formation. But even at pH 4.5, adding reduced NADP causes greater tryptophol formation, leading to a marked divergence in the acid-alcohol ratio. Varying the pH in the presence of reduced coenzymes also alters the ratio, with alcohol formation predominating. NAD and NADP have no influence on the formation of IAA from IAAld by the whole cytoplasm of Avena coleoptiles. Whole cytoplasm of Asparagus shoots forms both IAA and tryptophol from IAAld, but in widely varying amounts, devoid of any suggestive stoichiometry between the products. With the acetone powder of Avena coleoptiles including the first leaf, data indicating an apparent disimitation of IAAld are obtainable only at pH 4.5. On altering the pH however, unequal amounts of the two products, namely IAA and tryptophol, are formed and hence a different ratio results. Acetone powders of wheat coleoptiles and Asparagus shoots do not yield data supporting disimitation either at pH 4.5 or 7.2. IAA formation in Avena is aerobic while tryptophol formation is seemingly independent of oxygen supply. The former activity is selectively abolished by 10^?3M dithionite while the latter activity suffers a similar suppression in the presence of 10^?3M manganese sulphate. Varying the IAAld concentration results in unequal amounts of the two products, revealing the dissimilar affinity of the two activities for the common substrate Saturation to a level of 30 percent with ammonium sulphate throws out most of the acid-forming activity whereas the alcohol-forming system appears mostly in the protein fraction precipitated between 30 and 40 percent saturation. The enzyme system of Avena coleoptiles oxidizing IAAld to IAA can also be easily separated by Sephadex gel filtration and its independent activity demonstrated in the total absence of tryptophol formation. Based on the heterogeneous properties of the two activities, metabolism of IAAld in Avena coleoptiles is believed to be mediated by two independent enzyme systems without the intervention of a mutase or a dismutation mechanism.     

Nitrate assimilation during the anaerobic germination of rice: expression of ferredoxin-dependent glutamate synthase

Ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) is the last enzyme involved in the pathway of nitrate assimilation in higher plants. This paper describes the synthesis and expression of the enzyme in anaerobic coleoptiles of rice (Oryza sativa L.) and its regulation by exogenous nitrate. The activity of Fd-GOGAT was strongly inhibited by cycloheximide between 4 and 9 d of anaerobic germination. The addition of nitrate slightly increased, in the first 5 h, the specific activity of Fd-GOGAT as well as the amount of a 160-kDa protein specifically immunoprecipitated with anti-Fd-GOGAT serum. Northern blot analysis, performed with a specific riboprobe, showed the presence of mRNA of the expected size and the inductive effect of nitrate. The role of Fd-GOGAT is discussed in relation to the anaerobic assimilation of nitrate by rice coleoptiles.Abbreviations CHX cycloheximide - Fd ferredoxin - GOGAT glutamate synthase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase The authors wish to thank Dr. J. Turner (Rothamsted Experimental Station, Harpenden, UK) for providing Fd-GOGAT antibody and Dr. H. Sakakibara (Nagoya University, Nagoya, Japan) for Fd-GOGAT clone. This research was supported by the National Research Council of Italy, special project RAISA, sub-projekt N. 2, paper N. 2174.     

Induction of heat resistance of wheat coleoptiles by salicylic and succinic acids: Connection of the effect with the generation and neutralization of reactive oxygen species

The influence of salicylic (SaA) and succinic (SuA) acids on the generation of reactive oxygen species (ROS) and the heat resistance of wheat (Triticum aestivum L.) coleoptiles has been studied. The treatment of coleoptiles with 10 μM SaA or SuA results in the accumulation of hydrogen peroxide and enhanced formation of a superoxide anion radical. This effect was partially suppressed by both α-naphthol (the NADPH oxidase inhibitor) and salicylhydroxamic acid (peroxidase inhibitor). SaA and SuA cause an increase in the activity of antioxidant enzymes, such as superoxide dismutase, catalase, and soluble peroxidase, and improve the heat resistance of coleoptiles. Antioxidant ionol and inhibitors of the NADPH oxidase and peroxidase significantly reduce the positive influence of SaA and SuA on the heat resistance of wheat coleoptiles. ROS are considered to be intermediates for heat resistance induction in coleoptiles, treated with SaA and SuA; enhanced ROS generation can be caused by an increased activity of the NADPH oxidase and peroxidase.     

The isolation and physiological analysis of mutants of the moss,Physcomitrella patens,which over-produce gametophores

We have compared the effects of cycloheximide (CHI) and two other rapid and effective inhibitors of protein synthesis, pactamycin and 2-(4-methyl-2,6-dinitroanilino)-N-methyl proprionamide (MDMP), on protein synthesis, respiration, auxin-induced growth and H⁺-excreation of Avena sativa L. coleoptiles. All three compounds inhibit protein synthesis without affecting respiration. The effectiveness of the inhibitors against H⁺-excretion and growth correlates with their ability to inhibit protein synthesis. Both CHI and MDMP inhibit auxin-induced H⁺-excretion after a latent period of 5–8 min, and inhibit growth after a 8–10-min lag. These results support the idea that continued protein synthesis is required in the initial stages of the growth-promoting action of auxin.Abbreviations CHI cycloheximide - DMSO dimethyl sulfoxide - FC fusicoccin - IAA indole-3-acetic acid - MDMP 2-(4-methyl-2,6-dinitroanilino)-N-methyl proprionamide     

Fusicoccin-induced growth and hydrogen ion excretion of Avena coleoptiles: Relation to auxin responses

Summary The fungal toxin fusicoccin (FC) induces both rapid cell elongation and H⁺-excretion in Avena coleoptiles. The rates for both responses are greater with FC than with optimal auxin, and in both cases the lag after addition of the hormone is less with FC. This provides additional support for the acid-growth theory. The FC responses resemble the auxin responses in that they are inhibited by a range of metabolic inhibitors, but the responses differ in three ways. First auxin, but not FC, requires continual protein synthesis for its action. The auxin-induced H⁺-excretion is inhibited by water stress or by low external pH, while the FC-induced H⁺-excretion is much less sensitive to either. It is concluded that auxin-induced and FC-induced H⁺-excretion may occur via different mechanisms.Abbreviations FC fusicoccin - DNP dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - CHl cycloheximide - IAA indoleacetic acid     

Induced Protein Synthesis during the Adaptation to H2 Production in Chlamydomonas moewusii

Gas production by Chlamydomonas moewusii in the light has been followed by manometric techniques during the adaptation to anaerobiosis. The only detectable gases produced are CO₂ and H₂ CO₂ is produced at a rather constant rate whereas H₂ evolution increase with time. This increase of H₂ evolution during the adaptation period can be inhibited by cycloheximide and by chloral hydrate, two inhibitors of protein synthesis. If the inhibitors are added to already adapted cells there is no effect on H₂ evolution. Adapted cell suspensions are sensitive to oxygen. Incubation under O₂ for 10 min inhibits the H₂ evolution to 100%. After removal of oxygen the capability to evolve H₂ can be restored only by a new adaptation period. This second adaptation to H₂ evolution can also be inhibited by cycloheximide.