A double enhancement of greening in the cotyledons of cucumber seedlings by excision and red light

Cotyledons excised without the hypocotyl hook from 6-day-old etiolated cucumber ( Cucumis sativus L. var. Elem) seedlings accumulated a significantly higher amount of chlorophyll than cotyledons excised with hooks or intact cotyledons. It was found that maximum ehancement of greening was achieved after 2 h of dark incubation following excision. Pretreatments with red light effected an additive rise in chlorophyll level in subsequent white light after a dark incubation, suggesting that the effects of excision and phytochrome on greening act independently. Etiolated seedlings were variously dissected before greening and it was found that enhancement occurred only when cotyledons were excised at the level of the hypocotyl hook or above it. Similar results were obtained when the dissected plants were pre-treated with red light.     

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.     

Correlated Changes in the Activity,Amount of Protein,and Abundance of Transcript of NADPH:Protochlorophyllide Oxidoreductase and Chlorophyll Accumulation during Greening of Cucumber Cotyledons

Changes in the activity and abundance of NADPH:protochlorophyllide oxidoreductase (NPR) and the abundance of mRNA encoding it were examined during the greening of 5-d-old etiolated cucumber cotyledons under continuous illumination. To measure NPR activity in the extracts from fully greened tissues, we have developed an improved method of assay. Upon exposure of etiolated cotyledons to light, NPR activity decreased rapidly within the first 2 h of exposure. Thereafter, enzymatic activity increased transiently, reaching a submaximum level at 12 h, and decreased slowly. The level of immunodetectable NPR protein followed the same pattern of changes during 96 h of greening as observed for NPR activity. The NPR mRNA in etiolated cotyledons disappeared quickly in the 1st h of irradiation. However, the level of mRNA increased thereafter to reach 3-fold or more of the dark level at 12 h and then decreased. The changes in the activity, protein level, and mRNA level after the first rapid decreases corresponded chronologically and nearly paralleled the increase in the rate of chlorophyll accumulation. These findings suggest that the greening of cucumber cotyledons is regulated basically by the level of NPR protein without activation or repression of enzymatic activity and that NPR mRNA increased by light maintains the level of enzyme protein necessary for greening.     

Formation of Chlorophyll-Protein Complexes in Greening Cucumber Cotyledons in Light and then in Darkness

The changes in chlorophyll-protein complexes (CPs) in cucumbercotyledons during illumination and subsequent dark incubationwere studied by SDS-polyacrylamide gel electrophoresis. Whenetiolated cucumber seedlings were illuminated, chlorophyll wassynthesized and CPs were formed. In the early phase of greening(6 h of illumination), light-harvesting chlorophyll a/b-proteincomplex (LHCP) was the main GP. As the greening proceeded, P700chlorophyll a-protein complex (CP1) accumulated. When 6-h illuminatedseedlings were transferred to darkness, CP1 accumulated concomitantlywith a decrease in LHCP without new chlorophyll synthesis. Thechanges in the amounts of CPs in the dark became smaller withthe progress of greening and were not observed after 72 h ofillumination. These changes were confirmed by examining thechlorophyll/P700 ratio and the low temperature absorption spectrumof cotyledons. These results suggest that in the early phaseof greening, CPs were unstable and their chlorophyll moleculeseasily exchanged with those of other kinds of CPs. (Received October 14, 1982; Accepted December 1, 1982)     

Effect of the hypocotyl hook on greening in etiolated cucumber cotyledons

Four-day-old etiolated cucumber cotyledons (Cucumis sativus, L.) were excised and allowed to green in white fluorescent light at 28 C. Cotyledons excised with a full hypocotyl hook exhibited a lag phase of 1 hour before entering the rapid greening phase, whereas cotyledons excised without any hypocotyl hook exhibited a lag phase of 6 hours. Cotyledons excised with varying lengths of hypocotyl hook accumulated chlorophyll roughly in proportion to the hook length. When cotyledons were excised with a full hook and were partially or totally shielded from light with aluminum foil, the samples with the hook covered accumulated more chlorophyll than the wholly exposed samples. The samples with the cotyledons covered showed no net accumulation of chlorophyll irrespective of hook's exposure to light. These data suggest the contribution of some factor or factors by the hypocotyl hook which reduce the lag phase during greening.     

Chloroplast Biogenesis: XXII. Contribution of Short Wavelength and Long Wavelength Protochlorophyll Species to the Greening of Higher Plants

The contribution of short and long wavelength membrane-bound fluorescing protochlorophyll species to the over-all process of chlorophyll formation was assessed during photoperiodic growth. Protochlorophyll forms were monitored spectrofluorometrically at 77 K during the first six light and dark cycles in homogenates of cucumber (Cucumis sativus L.) cotyledons grown under a 14-hour light/10-hour dark photoperiodic regime, and in cotyledons developing in complete darkness. In the etiolated tissue, short wavelength protochlorophyll having a broad emission maximum between 630 and 640 nm appeared within 24 hours after sowing. Subsequently, the long wavelength species fluorescing at 657 nm appeared, and accumulated rapidly. This resulted in the preponderance of the long wavelength species which characterizes the protochlorophyll profile of etiolated tissues. The forms of protochlorophyll present in etiolated cucumber cotyledons resembled those in etiolated bean leaves in their absorption, fluorescence, and phototransformability. A different pattern of protochlorophyll accumulation was observed during the dark cycles of photoperiodic greening. The short wavelength species appeared within 24 hours after sowing. Subsequently, the long wavelength form accumulated and disappeared. The long wavelength to short wavelength protochlorophyll emission intensity ratio reached a maximum (~3:1) during the second dark cycle, then declined during subsequent dark cycles. Short wavelength species were continuously present in the light and dark. Primary corn and bean leaves exhibited a similar pattern of protochlorophyll accumulation. In cucumber cotyledons, both the short and long wavelengths species appeared to be directly phototransformable at all stages of photoperiodic development. It thus appears that whereas the long wavelength protochlorophyll species is the major chlorophyll precursor during primary photoconversion in older etiolated tissues, both long wavelength and short wavelength species seem to contribute to chlorophyll formation during greening under natural photoperiodic conditions.     

Stimulation by Ethylene of Chlorophyll Biosynthesis in Dark-grown Cucumber Cotyledons

Five-day-old etiolated cucumber (Cucumis sativus L. var. Alpha Green) cotyledons produced more chlorophyll over a 4-hour illumination period after a prolonged exposure (12 to 72 hours) in the dark to ethylene concentrations ranging from 0.1 to 10 μl/l. Intact seedlings and excised cotyledons responded in the same way to this treatment. This effect does not involve a shortening of the lag phase of chlorophyll accumulation. Exposure of cotyledons to ethylene during the illumination period did not produce the same stimulatory effect on chlorophyll synthesis and, under certain conditions, chlorophyll synthesis was slightly inhibited by exposure to ethylene in the light.     

Stimulation of cytokinins and chlorophyll synthesis in cucumber cotyledons by triadimefon

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanone] changes the morphology and partitioning of dry matter in cucumber ( Cucumis sativus L. cv. National Pickling) seedlings. The dry weights, potassium and cytokinin levels in the cotyledons and roots of the treated seedlings were higher, whereas the hypocotyl weights were lower than the controls. When etiolated intact seedlings or cotyledons excised from triadimefon-pretreated dark-grown seedlings were exposed to light, chlorophyll synthesis in the pretreated cotyledons was stimulated. Triadimefon does not have cytokinin-like activity in the cucumber cotyledon greening bioassay, but appears to induce the plants to produce more cytokinims, probably by stimulating root growth. Hence it is proposed that the stimulation of chlorophyll production by triadimefon in cucumber cotyledons is mediated by maintaining high levels of potassium and cytokinins in the cotyledons.     

A two-fold action of benzyladenine on chlorophyll formation in etiolated cucumber cotyledons

Treatment of 3-day-old excised etiolated cotyledons of cucumber ( Cucumis sativus L. cv. Aonagajibai) with benzyladenine (BA) in the dark stimulates chlorophyll (Chl) formation during the lag phase (designated as 'lag elimination') and accelerates the steady-state rate of Chl formation under subsequent continuous illumination with white light. The separation of this two-fold effect is possible using two different methods of BA treatment in darkness: a brief BA treatment followed by various periods of water treatment in darkness, or various periods of continuous dark BA treatment. In either treatment, BA rapidly eliminates the lag phase (the fast-appearing effect) and after a longer time period accelerates the steady-state rate (the late-appearing effect). With a brief BA treatment, both effects decay rapidly. In contrast, with continuous BA treatment, none of the effects decay after reaching their maxima, particularly in cotyledons excised 2 days after sowing and aged for a long period before the onset of BA treatment. These facts indicate that BA acts as a trigger in stimulating Chl formation. The relationship between the actions of BA and light is discussed.     

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.     

Formation and Degradation of Protochlorophylls in Etiolated and Greening Cotyledons of Cucumber

The formation, degradation and phototransformation of protochlorophylls(Pchls) in the etiolated and greening cotyledons of cucumber(Cucumis sativus L.) werestudied using high-performance liquidchromatography. The pigment analysis of etiolated cotyledonsshowed the presence of four Pchls esterified with phytol, tetrahydrogeranylgeraniol(THGG), dihydrogeranylgeraniol (DHGG), and geranylgeraniol (GG).The content of Pchl THGG rapidly increased during dark developmentof seedlings and reached a maximal level at 4th day, then decreasedgradually. Unlike Pchl THGG, Pchl DHGG and Pchl GG showed asmall peak at 3rd day followed by a one-day lag, then accumulationbegan. The content of Pchl DHGG reached a maximal level at 12thday, then decreased rapidly, while Pchl GG continued to increaseand its maximal stage was not attained at 15th day. The contentof Pchl phytol remained very low during dark growth. These resultsmay indicate that with increasing age, the inactivation of hydrogenationof the alcohol moiety of Pchl proceeds stepwise at the sitesof Pchl THGG, Pchl DHGG and Pchl GG, in that order, withoutaffecting the esterification of Pchlide. The content of four Pchls remained unchanged before and after30-s illumination, indicating that none of the four Pchls istransformed to chlorophyll by light. Under continuous illumination,Pchls decreased exponentially or linearly at a rather slow rate.Thus, the four Pchls are not direct precursors for chlorophylland are metabolized slowly under greening. (Received December 6, 1982; Accepted April 13, 1983)     

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.     

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)     

Cytokinin-induced chlorophyll formation in cucumber cotyledons

Summary Cotyledons of cucumber plants grown in the dark for 7 days were treated with various concentrations of cytokinins for 14 h and then moved into light. After 3 h the treated cotyledons had up to 450% more chlorophyll than the water controls. This suggests that cytokinins have an important role in the formation of chlorophyll. The increase in chlorophyll level was proportional to cytokinin concentration and was apparent at concentrations as low as 0.001 mg/l. Sensitivity to cytokinins depended on the age of the cotyledons and the time of exposure to light. Gibberellic acid, indoleacetic acid, adenine and sucrose did not cause a similar increase in chlorophyll levels. This effect of cytokinins on chlorophyll formation is valuable as a simple, rapid bioassay for cytokinins.This study was supported by the National Research Council of Canada.     

Impaired chlorophyll formation in etiolated cucumber cotyledons following prolonged dark incubation after red light pretreatment

Red light (R) pretreatment of etiolated cucumber seedlings ( Cucumis sativus L. var. Elem) followed by prolonged dark incubation prior to white light (WL) exposure, had an adverse effect on the greening of the cotyledons. The effect was photoreversible by far-red (FR) light. Cotyledons which were dark incubated for 24 h following the R pulse greened more rapidly when exposed to WL than did the controls, while total chlorophyll (Chl) accumulation after 24 h in the light was about the same in both. However, after 48 h post-R dark incubation greening of the treated cotyledons was delayed, and their amount of Chl which accumulated after 24 h WL was about one half of that in non-treated seedlings. As the length of the post-R dark incubation period was extended Chl production became slower, so that after 96 h post-R dark incubation the Chl level in the treated cotyledons after 24 h WL was approximately 20% of the controls. No significant differences in amounts of protochlorophyll could be detected between seedlings preilluminated with R or R followed by FR. Seedlings 4-, 5- and 6-days-old at the time of R treatment showed similar degrees of impaired Chl synthesis following prolonged post-R dark incubation.     

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons were studied. Poly (A)⁺ RNA was prepared from etiolated cotyledons incubated with or without benzyladenine (BA) for various periods in the dark. Using nonequilibrium pH gradient electrophoresis-SDS polyacrylamide gel electrophoresis and isoelectric focusing-SDS polyacrylamide gel electrophoresis, both basic and neutral proteins translated in vitro were separated. About 240 spots were detected and 16 of them changed within 6 h after BA application. Some spots changed quickly (within 1–2 h). Among them, three were repressed markedly     

Comparative Effects of Abscisic Acid and Methyl Jasmonate in Greening Cucumber Cotyledons and Its Application to a Bioassay for Abscisic Acid

In the cucumber cotyledon greening system, abscisic acid (ABA)is more potent inhibitor of growth and chlorophyll productionand/or destruction than methyl jasmonate (MJ). The inhibitoryeffect of ABA is apparent within 5 h of exposure to light whereasMJ is ineffective at all concentrations tested (10^–6 to10^–3 M). With longer exposure of 24 h to light and inthe presence of 40 mM KC1, the inhibition of growth and chlorophyllproduction by ABA is more pronounced whereas MJ does not inhibitgrowth and inhibits chlorophyll levels only at the higher concentrations.Both benzyladenine and KC1 stimulate chlorophyll productionand increase the fresh weights of the cucumber cotyledons andeither one of these compounds reverse the inhibitory effectsof ABA. Inhibition of chlorophyll production by ABA is valuableas a simple and rapid bioassay for abscisic acid. Under similarconditions cytokinins increase chlorophyll production and hencethe cucumber cotyledon greening system is ideal for detectingboth ABA which inhibits and cytokinins which stimulate chlorophyllproduction. (Received December 6, 1982; Accepted June 9, 1983)     

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.