Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^?1 h^?1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1μl L^?1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 μl L^?1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^–1 h^–1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1l L^–1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 l L^–1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

Inter-organ Control of Greening in Etiolated Cucumber Cotyledons

Inter-organ control of greening in etiolated cucumber (Cucumis sativus L. cv. Aonagajibae) cotyledons was investigated. Four- or six-day-old excised or intact etiolated cucumber cotyledons were illuminated under aerobic conditions. Excised cotyledons without hypocotyl hooks produced chlorophyll without a prolonged lag phase and the rate of chlorophyll formation was not depressed if they were illuminated immediately after excision. If the excised cotyledons were incubated in the dark before illumination, chlorophyll accumulation at the end of 6 h of continuous illumination was remarkably lowered as the dark period lengthened, especially in 6-day-old cotyledons. The rapid loss of chlorophyll-forming capacity of excised cotyledons during dark preincubation suggests a stimulatory effect of hypocotyls on the greening in cotyledons. The treatment of excised cotyledons with bleeding sap in the dark for 18 h resulted in the promotion of chlorophyll formation during subsequent continuous illumination. Partial fractionation of bleeding sap with organic solvents and paper chromatography indicates that the active substances showed the same behavior as cytokinins. These facts add weight to the hypothesis that cytokinins from roots flow into cotyledons and stimulate greening.     

Differential effects of benzyladenine and potassium on DNA, RNA, protein and chlorophyll contents and on expansion growth of detached cucumber cotyledons in the dark and light

Benzyladenine (BA) and KCl were applied to detached cucumber ( Cucumis sativus L. cv. Ohio) cotyledons in continuous light or in the dark with subsequent light. BA brought about an increase in fresh weight and in DNA, RNA and carotenoid contents in both treatments. KCl did not cause an increase in fresh weight and cellular constituents in the dark, but it did result in an increased fresh weight and DNA content after illumination or in continuous light. BA + KCl treatment resulted in increased carotenoid and DNA contents in the dark, and in increases in fresh weight and all cellular constituents upon subsequent exposure to light. The effects of BA and BA + KCl on growth and chlorophyll synthesis decreased with cotyledon age.
BA pretreatment in the dark eliminated the lag phase in chlorophyll synthesis and increased the rate of synthesis. Treatment in continuous light had little effect. KCl did not shorten the lag phase in chlorophyll synthesis, but it stimulated the rate of synthesis in the light. Dark pretreatment with BA + KCl markedly increased the effect of BA on chlorophyll synthesis. Chlorophyll content and fresh weight were higher in cotyledons treated with BA followed by KCl than in cotyledons treated in the reverse order. These results suggest that growth and greening in cucumber cotyledons are primarily controlled by BA and that KCl intensifies the BA effect after irradiation.     

Influence of chloroplast development on the activation of the diphenyl ether herbicide acifluorfen-methyl

The activity of acifluorfen-methyl (AFM); methyl 5-(2-chloro-4-[trifluoromethyl] phenoxy)-2-nitrobenzoate in excised cucumber cotyledons (Cucumis sativus L.) was examined. AFM induced membrane disruption, was significantly greater when etiolated cotyledons were illuminated 16 hours at 150 microeinsteins per square meter per second photosynthetically active radiation versus incubation under illumination of 4-fold greater intensity. These results were unexpected since the loss of membrane integrity is initiated by photodynamic reactions. Untreated, etiolated cotyledons were not able to accumulate chlorophyll under the higher light intensity while control and herbicide treated cotyledons greened significantly under the lower intensity illumination suggesting that some process associated with greening stimulated AFM activity. Inhibition of greening by cycloheximide also reduced AFM activity. Intermittent lighting induced greening in AFM treated cotyledons without causing any detectable loss of plasmalemma integrity. Utilization of this system for pretreatment of cotyledons prior to continuous illumination revealed that activity was greater when tissue was greened in the presence of AFM than when herbicide treatments were made after a greening period of the same duration. The results indicate that the pigments in situ in etiolated tissue are sufficient, without greening, to initiate membrane disruption by AFM. However, greening increases the herbicidal efficacy greatly. Furthermore, the stimulation appears to be due to specific interactions between AFM and the developing plastid and is not attributable solely to an increase in endogenous photosensitizers.     

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

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

Evidence that Ethylene is not Involved in Red-light-induced Stimulation of Chlorophyll Formation in Etiolated Cucumber Cotyledons

The involvement of ethylene in red-light-induced stimulationof chlorophyll (Chl) formation was studied because one of thered-light effects on Chl formation (the lateappearing effect)interacts with the ethylene effect in 3-day-old excised etiolatedcotyledons of cucumber (Cucumis sativus L. cv. Aonagajibai).Ethylene production by etiolated cotyledons of intact seedlingsin the dark is enhanced by a red-light pulse, but the effectdoes not occur in excised cotyledons. Application of ethylenein the dark to 3-day-old intact seedlings has little effecton Chl formation in the cotyledon during subsequent continuousillumination, although ethylene pretreatment of 5-day-old seedlingssignificantly stimulates Chl formation. Removal of endogenousethylene by mercuric perchlorate [Hg(ClO₄)₂] does not specificallysuppress the red-light action on Chl formation in both attachedand excised cotyledons. Inhibition of ethylene synthesis byaminoethoxyvinylglycine does not affect the red-light effecton Chl formation in excised cotyledons. These facts indicatethat ethylene does not operate as a mediator of red light instimulating Chi formation in either attached or excised cotyledons. (Received December 13, 1981; Accepted March 30, 1981)     

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.     

Hormonal Regulation, and Intracellular Localization of a 33-kD Cationic Peroxidase in Excised Cucumber Cotyledons

Ethylene enhanced chlorosis and levels of 33-kilodalton cationic peroxidase (33-CPO) in excised cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. Compared to other hormones, such as kinetin, indoleacetic acid, gibberellic acid, and abscisic acid, ethylene was the only effective promoter of 33-CPO synthesis. The hypothesis that peroxidase plays a role in chlorophyll degradation was tested by comparing levels of 33-CPO in cotyledons treated with compounds thought to either retard (kinetin, indoleacetic acid and gibberellic acid), or promote (abscisic acid and methyl jasmonate [MJ]) senescence. It was concluded that 33-CPO did not play a role in senescence since no direct correlation between chlorophyll content and 33-CPO was observed. MJ was as effective as ethylene in inducing senescence. However, ethylene did not appear to be involved in the action of MJ. Using immunocytochemistry, 33-CPO was found to be located primarily around starch grains and near the plasmalemma. High levels of 33-CPO were also found in cells destined to be vascular tissue.     

Stimulation of chlorophyll synthesis by benzyladenine and potassium in excised and intact cucumber cotyledons

Both benzyladenine (BA) and potassium (K) stimulated chlorophyll synthesis in cucumber ( Cucumus sativus L. cv. National Pickling) cotyledons. However, differences existed between the effects of BA and K. Stimulation of chlorophyll synthesis by BA (1 mg l⁻¹, 4.4 μ M ) was observed in excised cotyledons after 4 and 8 h of illumination but not after 24 h, whereas the stimulation caused by K (40 m M ) continued. In contrast to BA, K was unable to eliminate the lag phase of chlorophyll production, and it also required light for its stimulation of cotyledon expansion. Both BA and K were required to maximize cotyledon expansion and chlorophyll production. In intact plants, K was not limiting for chlorophyll synthesis since foliar or soil pretreatments with K did not markedly stimulate greening. Foliar pretreatment with BA stimulated chlorophyll levels in intact plants, whereas soil pretreatment with BA inhibited chlorophyll production, probably because BA was not readily transported from the roots to the shoot and created a "sink" effect. Inhibitor studies showed that stimulation by K of greening did not depend on RNA or chloroplastic protein synthesis to the extent that has been reported for BA. Thus it appears that BA and K stimulate chlorophyll synthesis via different mechanisms, although both cytokinins and K are essential for maximum rates of greening.     

Inhibition by calcium of senescence of detached cucumber cotyledons: effect on ethylene and hydroperoxide production

The effect of Ca on senescence was followed in detached cucumber (Cucumis sativus L.) cotyledons floating on various solutions in the dark. Compared with those in water, cotyledons in 10⁻⁴ molar CaCl₂ exhibited reduced chlorophyll loss and H₂O₂ production, reduced and delayed ethylene production, and did not undergo a burst in CO₂ production. In contrast, Mg had little effect on cotyledon senescence, whereas K stimulated chlorophyll loss but did not increase H₂O₂ accumulation of ethylene and CO₂ production. This reduction in the rate of senescence by Ca could also be achieved by increasing the endogenous levels of Ca in the cotyledons before excision, although the reduction was less than that with Ca in the external solution. The addition of H₂O₂ to the solutions on which cotyledons were floated stimulated chlorophyll breakdown, but effects on ethylene and CO₂ were not consistent.     

The relationship between transpiration and chlorophyll synthesis in etiolated squash cotyledons

Etiolated cotyledons of squash (Cucurbita moschata Duch. var.melonaeformis Makino cv. Tokyo) treated with kinetin, KCl orfusicoccin in the dark increased both transpiration in the darkand chlorophyll synthesis after exposure to light. The minimumperiod of kinetin treatment to stimulate transpiration was similarto that to accelerate chlorophyll synthesis. On the other hand,treatment of cotyledons with vaseline or abscisic acid markedlyinhibited both transpiration and chlorophyll synthesis, suggestingthat cytokinin-induced stimulation of chlorophyll synthesiscould be mediated through the effect on the stomatal opening. (Received December 7, 1977; )     

Modulation of Chlorophyll, Carotene and Xanthophyll Formation by Penicillin, Benzyladenine and Embryonic Axis in Mung Bean (Phaseolus aureus L.) Cotyledons

Penicillin stimulated the synthesis of pigments in the cotyledonsof intact embryos and excised cotyledons of mung bean (Phaseolusaureus L.) and enhanced benzyladenine-induced accumulation ofchloroplast pigments. The presence of the embryonic axis duringlight exposure proved to be beneficial for chlorophyll synthesisby the cotyledons whereas its presence in dark germination producedan adverse effect. The possible involvement of nucleic acidand protein synthesis in light-regulated chlorophyll formationis suggested. The stimulating effect on pigment synthesis providedby penicillin in this system seems to involve a maintenanceof nucleic acid and protein synthesis. Phaseolus aureus L., mung bean, pigment synthesis, cotyledons     

Changes in Chlorophyll a and b Content in Dark-Incubated Cotyledons Excised from Illuminated Seedlings: THE EFFECT OF CALCIUM

Cucumber seedlings were illuminated for various time periods, cotyledons excised, placed in the dark, and changes in chlorophyll a and b content monitored. During the dark periods, chlorophyll b content decreased while chlorophyll a did not. When the illumination time was lengthened, the percentage of chlorophyll b decomposition from initial levels decreased. Ca²⁺ at 50 millimolar prevented the decrease in chlorophyll b and caused a decrease in chlorophyll a. The effect of Ca²⁺ decreased with increased illumination time. Cycloheximide and chloramphenicol inhibited chlorophyll b decrease, but did not induce chlorophyll a decrease.     

Evidence that Isolated Developing Chloroplasts Are Capable of Synthesizing Chlorophyll b from 5-Aminolevulinic Acid

Developing chloroplasts isolated from cucumber (Cucumis sativus L. var Beit Alpha) cotyledons are capable of incorporating [¹⁴C]5-aminolevulinic acid into chlorophyll (Chl) b and Chl a when incubated under photosynthetic illumination. Thin layer chromatography and high pressure liquid chromatography were employed to analyze the pigments. The specific radioactivity in Chl a was over three times higher than that found in Chl b. Both Chl a and b synthesizing activities in organello decayed rapidly at approximately the same rate. We conclude that concomitant synthesis of Chl a/b-binding apoprotein is not required for Chl b synthesis.     

Accumulation of photosynthetic pigments in Larix decidua Mill. and Picea abies (L.) Karst. cotyledons treated with 5-aminolevulinic acid under different irradiation

European larch (Larix decidua Mill.) and Norway spruce [Picea abies (L.) Karst.] synthesize chlorophyll (Chl) in darkness. This paper compares Chl accumulation in 14-d-old dark-grown seedlings of L. decidua and P. abies after shortterm (24 h) feeding with 5-aminolevulinic acid (ALA). We used two ALA concentrations (1 and 10 mM) fed to cotyledons of both species in darkness and in continuous light. The dark-grown seedlings of L. decidua accumulated Chl only in trace amounts and the seedlings remained etiolated. In contrast, P. abies seedlings grown in darkness were green and had significantly higher Chl content. After ALA feeding, higher protochlorophyllide (Pchlide) content was observed in L. decidua than in P. abies cotyledons incubated in darkness. Although short-term ALA feeding stimulated the synthesis of Pchlide, Chl content did not change significantly in cotyledons incubated in darkness. The Chl accumulation in cotyledons fed with ALA was similar to the rate of Chl accumulation in the controls. Higher Chl accumulation was reported in control samples after illumination: 86.9% in L. decidua cotyledons and 46.4% in P. abies cotyledons. The Chl content decreased and bleaching occurred in cotyledons incubated with ALA in light due to photooxidation. Analyses of Chlbinding proteins (D1 and LHCIIb) by Western blotting proved differences between Chl biosynthesis in L. decidua and P. abies seedlings in the dark and in the light. No remarkable increase was found in protein accumulation (D1 and LHCIIb) after ALA application. Our results showed interspecific difference in Chl synthesis between two gymnosperms. Shortterm ALA feeding did not stimulate Chl synthesis, thus ALA synthesis was not the rate-limiting step in Chl synthesis in the dark.     

Abscission: the role of ethylene modification of auxin transport

The role of ethylene-mediated reduction of auxin transport in natural and ethylene-induced leaf abscission was studied in the cotton (Gossypium hirsutum L., cv. Stoneville 213) cotyledonary leaf system. The threshold level of ethylene required to cause abscission of intact leaves was between 0.08 and 1 μl/l with abscission generally occurring 12 to 24 hours following ethylene fumigation. The threshold level of ethylene required to reduce the auxin transport capacity in the cotyle-donary petiole paralleled that required for stimulation of abscission. In plants where cotyledons are allowed to senesce naturally there is a decline in auxin transport capacity of petioles and increase in ethylene synthesis of cotyledons. The visible senescence process which precedes abscission requires up to 11 days, and increases in ethylene production rates and internal levels were detected well before abscission. Ethylene production rates for entire cotyledons rose to 2.5 mμ1 g⁻¹ hr⁻¹ and internal levels of 0.7 μl/l were observed. These levels appear to be high enough to cause the observed decline in auxin transport capacity. These findings, along with those of others, indicate that ethylene has several roles in abscission control (e.g., transport modification, enzyme induction, enzyme secretion). The data indicate that ethylene modification of auxin transport participates in both natural abscission and abscission hastened by exogenous ethylene.     

Induction of 33-kD and 60-kD Peroxidases during Ethylene-Induced Senescence of Cucumber Cotyledons

Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25, 23, 20, 16, 12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pI = 8.9) and 60 kilodaltons (anionic, pI = 4.0). The observation that [³⁵S]Na₂SO₄ was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues.     

Effect of 4-Thiouridine on Photosystems of a Higher Plant

Effect of 4-thiouridine, which was proved to inhibit selectively and “light-reversibly” the synthesis of chloroplast ribosomal RNAs in radish cotyledons, on the photo-induced development of photosystem I, II and a complete electron transport chain was investigated with plastids obtained from 4-thiouridine treated dark-grown radish cotyledons after various times of development in the light. It was demonstrated that the 4-thioridine treated chloroplasts showed a higher activity of photoreduction than the control untreated chloroplasts in every system on a chlorophyll basis during the development after 24 hr illumination. This specific activity decreased in both chloroplasts, as the chloroplasts matured with the time of illumination. The activity per g of fresh cotyledons treated with 4-thiouridine, especially in the early stage of development, was lower than that of ones untreated with the drug because total chlorophyll content was poor, but the activity of the former was enhanced with the increase of total chlorophyll content upon illumination while the activity of the latter decreased on 24 hr illumination. Moreover, Hill reaction measurements showed that 4-thiouridine treated chloroplasts were saturated at lower light intensity than untreated ones inspite of the same content of chlorophyll in both the chloroplasts: photoreduction of NADP⁺ was saturated at 3000 lux for the former and at 5000 lux for the latter. Based upon these results, specific development of the chloroplast is discussed.     

Involvement of ethylene in chlorophyll degradation in peel of citrus fruits

The effect of ethylene on chlorophyll degradation in the peel of Robinson tangerine (X Citrus reticulata Blanco) and calamondin (X Citrofortunellamitis [Blanco] Ingram and Moore) fruits was studied. The chlorophyll degrading system in the peel of these two citrus species was not self-sustaining but required ethylene to function. Chlorophyll degradation ceased immediately when fruit were removed from ethylene and held in ethylene-free air at 0.2 atmospheric pressure. However, at atmospheric pressure, chlorophyll degradation continued for 24 hours in the absence of exogenous ethylene. Although chlorophyllase levels were negatively correlated with chlorophyll content in the peel (r = −0.981; P < 0.01), the level of chlorophyllase activity did not change when fruit were removed from ethylene, even though chlorophyll degradation had stopped. From these observations, it was concluded that ethylene is necessary for chlorophyll degradation in the two species of citrus studied, but its primary role is not solely for the induction of chlorophyllase activity.