Auxin-induced ethylene biosynthesis in subapical stem sections of etiolated seedlings of Pisum sativum L.

1-Aminocyclopropane-1-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections of etiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 mM ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-¹⁴C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections incubated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine - IAA indole-3-acetic acid - SAM S-adenosylmethionine - SMM S-methylmethionine     

Methionine metabolism and ethylene formation in etiolated pea stem sections

Stem sections of etiolated pea seedlings (Pisum sativum L. cv. Alaska) were incubated overnight on tracer amounts of l-[U-(14)C]methionine and, on the following morning, on 0.1 millimolar indoleacetic acid to induce ethylene formation. Following the overnight incubation, over 70% of the radioactivity in the soluble fraction was shown to be associated with S-methylmethionine (SMM). The specific radioactivity of the ethylene evolved closely paralleled that of carbon atoms 3 and 4 of methionine extracted from the tissue and was always higher than that determined for carbon atoms 3 and 4 of extracted SMM.Overnight incubation of pea stem sections on 1 millimolar methionine enhanced indoleacetic acid-induced ethylene formation by 5 to 10%. Under the same conditions, 1 millimolar homocysteine thiolactone increased ethylene synthesis by 20 to 25%, while SMM within a concentration range of 0.1 to 10 millimolar did not influence ethylene production. When unlabeled methionine or homocysteine thiolactone was applied to stem sections which had been incubated overnight in l-[U-(14)C]methionine, the specific radioactivity of the ethylene evolved was considerably lowered. Application of unlabeled SMM reduced the specific radioactivity of ethylene only slightly.     

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: III. Induction and Transmission of the Response

Increased ethylene synthesis was rapidly induced throughout the apical meristematic region of etiolated seedlings of Pisum sativum L., cv. Alaska by cuts made 1 centimeter from the apical hook. The wound signal was transmitted at about 2 millimeters per minute. Accumulation of substance(s) at the cut surfaces of excised sections, as the result of interrupted translocation, did not initiate or significantly contribute to wound-induced ethylene synthesis, nor was the cut surface the site of enhanced ethylene synthesis. Cutting subapical sections into shorter pieces showed that cells less than 2 millimeters from a cut surface produced about 30% less ethylene than cells greater than 2 millimeters from a cut surface.     

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.

     

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: I. Characterization of the Response

A rapidly induced, transitory increase in the rate of ethylene synthesis occurred in wounded tissue excised from actively growing regions of etiolated barley, cucumber, maize, oat, pea, tomato, and wheat seedlings. Cutting intact stems or excising 9-mm segments of tissue from near the apex of 7-day-old etiolated Pisum sativum L., cv. Alaska seedlings induced a remarkably consistent pattern of ethylene production. At 25 C, wound-induced ethylene production by segments excised 9 mm below the apical hook increased linearly after a lag of 26 minutes from 2.7 nanoliters per g per hour to the first maxium of 11.3 nanoliters per g per hour at 56 minutes. The rate of production then decreased to a minimum at 90 minutes, increased to a lower second maximum at 131 minutes, and subsequently declined over a period of about 100 minutes to about 4 nanoliters per g per hour. Removal of endogenous ethylene, before the wound response commenced, had no effect on the kinetics of ethylene production. Tissue containing large amounts of dissolved ethylene released it as an exponential decay with no lag period. Rapidly induced wound ethylene is synthesized by the tissue and is not merely the result of facilitated diffusion of ethylene already present in the tissue through the newly exposed cut surfaces. Previously wounded apical sections did not exhibit a second response when rewounded. No significant correlation was found between wound-induced ethylene synthesis and either CO₂ or ethane production.     

Enhancement of ethylene formation by selenoamino acids

Selenomethionine and selenoethionine enhanced ethylene production in senescing flower tissue of Ipomoea tricolor Cav. and in auxin-treated pea (Pisum sativum L.) stem sections. This enhancement was fully inhibited by the aminoethoxy analog of rhizobitoxine. Methionine did not have a comparable promotive effect, and ethionine partly inhibited ethylene production. When [¹⁴C]methionine was applied to flower or pea stem tissue followed by treatment with unlabeled selenomethionine or selenoethionine, the specific radioactivity of the ethylene evolved was considerably reduced. The dilution of the specific radioactivity of ethylene by selenomethionine, and in pea stem sections also by selenoethionine, was greater than the dilution by nonradioactive methionine at the same concentration. These results indicate that both selenoamino acids serve as precursors of ethylene and that they are converted to ethylene more efficiently than is methionine.     

Influence of ethylene on nucleic acid synthesis in etiolated Pisum sativum

Ethylene applied to intact etiolated seedlings of Pisum sativumcv. Alaska inhibits incorporation of ³H-thymidine into DNA insubsequently excised plumular and subapical tissue segmentsbut has no influence on incorporation of ³H-uridine into RNA.The effect on DNA synthesis begins about 2 hr after ethyleneis applied, and intensifies progressively. A similar inhibitionof DNA synthesis occurs when ethylene is applied directly toplumular sections cut from control plants, but not with subapicalsegments under these conditions. Inhibition of DNA synthesisby ethylene is reversed by benzyl adenine in plumular sections.Brief exposure of dark grown seedlings to red light causes asubsequent increase in DNA synthesis in plumular tissue. Thechanges in DNA synthesis in tissues exposed to ethylene, benzyladenine and red light are correlated with the effects of thesetreatments on the mitotic index. (Received March 12, 1973; )     

Effects of Protein Synthesis Inhibitors on ent-Kaurene Biosynthesis during Photomorphogenesis of Etiolated Pea Seedlings

Excised shoot tips from 10-day-old etiolated pea (Pisum sativum L. cv. Alaska) seedlings were incubated in solutions of chloramphenicol, cycloheximide, and lincomycin at different concentrations during periods of 0, 4, 8, and 12 hours of irradiation with high intensity white light. Enzyme extracts were prepared from the whole shoot tips and compared with extracts from nontreated shoot tips for their capacity to synthesize ent-kaurene from mevalonate. In control samples, kaurene synthesis increased during the first 8 hours of irradiation and decreased after 12 hours. Chlorophyll content increased steadily up to 12 hours of irradiation. Chloramphenicol and cycloheximide reduced both kaurene synthesis and chlorophyll formation to a similar extent during all periods of irradiation, the reduction being greatest after 8 hours of irradiation. Lincomycin, a specific inhibitor of the formation of chloroplast ribosomes in detached pea shoot tips, did not significantly affect kaurene synthesis activity but strongly inhibited chlorophyll formation. It is tentatively concluded that the increase in kaurene synthesis activity during normal photomorphogenesis in pea seedlings is due to photoinduction of de novo synthesis of one or more proteins involved in the biosynthetic pathway from mevalonate to kaurene.     

Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth

Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect—90% inhibition—after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the K_m^app of arginine decarboxylase for its substrate and reduced the V_max^app by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed.     

Involvement of Ethylene in Phytochrome-mediated Carotenoid Synthesis

Accumulation of carotenoid pigments in the shoot apex of etiolated pea (Pisum sativum cv. Alaska) seedlings is completely prevented by ethylene. Under certain conditions carotenoid synthesis is normally controlled by endogenously produced ethylene. The gas completely inhibits carotenoid synthesis induced either by continuous white light or brief illumination with red light, but only partially inhibits light-induced chlorophyll formation. Far red illumination followed by red illumination reverses the action of red light on carotenoid synthesis. Red light-induced carotenogenesis is partly or wholly caused by phytochrome-mediated inhibition of ethylene biosynthesis.     

Effects of ethylene and gibberellic Acid on cellular growth and development in apical and subapical regions of etiolated pea seedling

Subhook swelling of 4-day-old etiolated pea seedlings (var. Alaska), caused by 0.5 microliter per liter ethylene, was prevented by preincubation and continued growth in 0.1 mm gibberellic acid (GA). The subhook region exhibited normal elongation and cell size and volume. However, inhibition of elongation and cessation of cell division caused by 0.5 microliter per liter ethylene in the apical hook region of the etiolated pea stem were not overcome by GA. Most of the arrested cells were in G(2). These data suggest a possible interaction of GA and ethylene in cell enlargement in the subhook region of the etiolated pea seedlings. They also suggest a different mode of action by ethylene in the apical hook region where the ethylene effect was not counteracted by GA.     

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     

Effects of aqueous extract of rice plants (Oryza sativa L.) on seed germination and radicle elongation of Monochoria vaginalis var. plantaginea

Laboratory experiments were conducted to examine the allelopathic potentials of rice plants (Oryza sativa L.) on seed germination and radicle elongation of Monochoria vaginalis (Burm. f.) Presl var. plantaginea (Roxb.) Solms-Laubat. To compare the relative allelopathic potentials of rice plants at different growth stages, aqueous extracts of dried tissues were obtained from the seeds, husks, and seedlings harvested 10, 20, 40 and 120 days after seeding. When M. vaginalis seeds were incubated in solutions containing aqueous extracts, significant increases in the germination rate were observed for all the aqueous extracts except for the extract of 10 d-old shoots at higher dosages. When partitioned with ethyl acetate, the germination-promoter(s) in the aqueous extracts remained in the aqueous phase while some inhibitors were extracted with ethyl acetate. In contrast, aqueous shoot extracts added to the incubation media at the same dosage for promotion of seed germination inhibited radicle elongation of M. vaginalis, indicating that the sensitivity of the weed varies between these two growth stages.     

Effect of chlorsulfuron on ethylene evolution from sunflower seedlings

The effect of the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy - 6 - methyl -1, 3,5 - triazin - 2 - yl)aminocarbonyl]benzenesulfonamide) on ethylene production in light-grown sunflower (Helianthus annuus L.) seedlings was examined. Application of chlorsulfuron to the apex stimulated ethylene production in all tissues examined: cotyledons, hypocotyls, and roots. The greatest stimulation occurred in the upper portion of the hypocotyl adjacent to, and including, the cotyledonary node. Ethylene evolution from hypocotyls excised from treated seedlings was stimulated over control levels 1 day after herbicide application and reached a maximum (approx. 75 x control or 17 nl/g f wt/h) 2 to 3 days after treatment. Labeling and inhibitor studies indicated that the ethylene produced was derived primarily from methionine. Chlorsulfuron treatment stimulated the rate of accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as well as the ability of the tissue to convert exogenous ACC to ethylene. Chlorsulfuron had little effect on ethylene production when administered to the hypocotylsin vitro. Removal of the cotyledons from treated seedlings reduced the rate of ethylene evolution from the hypocotyls. These results suggest that stimulation of ethylene production in sunflower hypocotyls by chlorsulfuron is not a wound response but rather is dependent on factors derived from the cotyledons.     

Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

Synthesis and characterization of water-soluble two-photon excited blue fluorescent chromophores for bioimaging.

We report here the synthesis and characterization of a new type of non-ionic blue fluorescent water-soluble chromophores specifically designed for two-photon absorption microscopy. The water solubility is induced by introduction of short oligo(ethylene glycol) monomethyl ether moieties. This work has led to low molecular weight dyes with efficient two-photon absorption cross sections and high fluorescence quantum yield in organic solvents as well as in aqueous solutions.     

磷石膏浸提液对豌豆种子生理及幼苗生长的影响

利用不同浓度的磷石膏浸提液处理豌豆种子,测定豌豆种子淀粉酶活性、可溶性糖、种子生命力、吲哚乙酸含量、吲哚乙酸氧化酶活性和发芽率、苗高、植株鲜重。结果表明:磷石膏浸提液处理后,豌豆种子可溶性糖含量和吲哚乙酸含量分别比对照增加6.7%～43.3%和9.4%～40.8%。豌豆幼芽中α-淀粉酶活性和吲哚乙酸氧化酶活性分别比对照高出8%～64%和15.2%～30.9%;发芽率、苗高和植株鲜重分别比对照提高10%以上。表明磷石膏能促进豌豆萌发和生长。     

Effects of ethylene, kinetin, and calcium on growth and wall composition of pea epicotyls

Ethylene supplied with indoleacetic acid at 0.1 and 1 mum inhibited elongation and enhanced swelling in epicotyls of decapitated and derooted pea seedlings (Pisum sativum L., var. Alaska). These growth responses were correlated with the development of cell walls rich in weak acid-extractable materials and pectic uronic acids. Ethylene had no effect on the formation of hemicellulose, or hemicellulosic uronic acid. Ethylene stimulated the formation of residual materials at 0.1 mum indoleacetic acid but had little effect at 1 mum. With indoleacetic acid at 10 mum, ethylene modified neither the growth or wall composition appreciably. Growth and wall composition in intact seedlings were modified in similar fashion by ethylene. In intact seedlings ethylene promoted the development of walls high in weak acid-extractable materials and pectic uronic acid. These effects were less impressive in the first 24 hours than in the second 24 hours when the control plants suffered a net loss of these constituents. Ethylene considerably inhibited the formation of hemicellulose and residual wall materials in the apical sections but promoted it in the basal sections of the intact seedlings.Measurements of ethylene production by decapitated and derooted pea seedlings suggest that Ca(2+) and kinetin do not promote swelling through an effect on the formation of ethylene.We propose that cells of ethylene-treated pea epicotyls lack polarity because their walls are abnormally rich in pectic substances.     

Oxygen-induced changes in pulmonary phospholipids in the rat.

The composition and synthesis of alveolar and lung tissue phospholipids were investigated in normal and oxygen-poisoned rat lungs. Sixty-hour exposure to oxygen increased the total amount of phospholipids in the endobronchial extracts and lung tissue. Phosphatidyl glycerol was identified in both endobronchial extracts and lung tissue. The amount of unsaturated fatty acids in surfactant lecithin and phosphatidyl glycerol was slightly increased in oxygen-poisoned lungs whereas the composition of phospholipids in the endobronchial extracts was not affected by oxygen. After intraperitoneal administration of [32P]phosphate the specific activities of surfactant lecithin and phosphatidyl glycerol were clearly lower in oxygen-treated animals whereas the specific activities of lung tissue lecithin and phosphatidyl glycerol remained unaffected. The synthesis of lecithin from [14C]methionine through N-methyltransferase pathway was markedly depressed in lung slices but increased in liver tissue taken from oxygen-poisoned rats and incubated under oxygen indicating a difference between lung and liver methyltransferase enzymes. In conclusion, the present work suggests impaired synthesis and removal of alveolar phospholipids in oxygen-poisoned rats.     

Mechanisms of hormone action: use of deuterated ethylene to measure isotopic exchange with plant material and the biological effects of deuterated ethylene

We observed no exchange between deuterated ethylene (C₂D₄) and the hydrogen of pea seedlings (Pisum sativum L. cv. Alaska). This suggests that bonding forces in which exchange could readily occur are not important in the physiological action of ethylene. Deuterated ethylene was just as effective as normal ethylene in inhibiting the growth of pea root sections. These results indicate that splitting carbon to hydrogen bonds did not occur during ethylene action.