Studies on Greening of Etiolated Seedlings

Evidence is given that a selective light-pretreatment of the embryonic axis exerts a deep influence on the greening in primary leaves of 8-day-old etiolated bean seedlings (Phaseolus vulgaris cv. Limburg). After a subsequent dark incubation of sufficient length and a final exposure of the entire plants to continuous illumination the lag phase of chlorophyll synthesis is completely removed. In particular the highly meristematic hook tissue seems to be responsible for this light effect. Lengthening of the dark period following pre-irradiation increased the capability of chlorophyll production in the main white light period, reaching its maximum after about 12 hours of darkness. The period of dark incubation for elimination of the lag phase is considerably longer in plants with shielded leaves than the length of the lag phase in etiolated seedlings of the same age, exposed entirely to continuous light. This difference may be explained by the synergistic effect between leaves and embryonic axis. Evidence for this interorgan cooperation is given by experiments with a selective light-pretreatment of leaves and embryonic axis. After a 5 min pre-exposure to white light of whole plants the leaves of some of the plants were shielded and these plants received a further pre-illumination of 2 hours on their embryonic axis. In all the pre-irradiated, etiolated plants the lag phase of chlorophyll synthesis was eliminated during the main white light period, following a dark incubation of 2 hours. Additional and preferential light activation of the embryonic axis during the pretreatment had no significant effect on chlorophyll production during the white light illumination after a 2 hours dark incubation, but resulted in a lower yield of chlorophylls after 18 hours dark incubation compared to the white light controls, receiving no selective light-pretreatment on the embryonic axis. From our results we can decisively conclude that a simultaneous light-pretreatment of both, leaves and embryonic axis, is more effective and beneficial for building up a capacity of chlorophyll synthesis in the leaves than either a selective light-pretreatment of the embryonic axis alone or a simultaneous pre-illumination of leaves and embryonic axis, immediately followed by an additional preirradiation of the embryonic axis. Therefore, we think that several photoactive sites are involved in de-etiolation processes of intact, etiolated seedings. Light activation of the embryonic axis stimulates the development of this organ and contributes to the greening processes in the leaf. At the same time, by irradiating the leaf, light activates the photo-sensitive site in the leaf itself, which also develops a capacity for chlorophyll synthesis. Both photo-acts are cooperative, explaining the enhanced chlorophyll production. Additional pre-irradiation of the embryonic axis after a short illumination of whole plants favours its own development and reduces the synthetic capacity of the leaf. A prolonged far-red pretreatment induces qualitatively the same response as white light. We assume that these effects on lag phase removal and chlorophyll production, induced in etiolated, primary bean leaves by selective irradiation of the embryonic axis, is a phytochrome-mediated process. Our results indicate a transmission of light-induced stimuli from one organ to another.     

Mimicry by cytokinin of phytochrome-regulated inhibition of chloroplast development in etiolated cucumber cotyledons

A study of the kinetics of chlorophyll (Chl) synthesis in cotyledons of etiolated cucumber seedlings ( Cucumis sativus L . cv. Delilah) treated with 5×10^-5 M -ben-zyladenine (BA) showed that cytokinin, like a red light pulse, could inhibit as well as promote pigment accumulation depending on the length of the dark period following induction. Spraying intact, dark-grown seedlings with BA, 24 h prior to white light exposure, eliminated the lag phase in Chl synthesis, while treatment with hormone 72 h before greening not only delayed the onset of synthesis, but it also reduced the amount of Chl accumulated after 24 h continuous white light. Impairment of Chl formation was correlated with inhibited regeneration of protochlorophyll and delayed appearance of the light harvesting Chl alb polypeptide. Application of σ-aminolevulinic acid (15 m M ) 2 h before white light exposure shortened the lag phase in Chl synthesis in control as well as in inhibited cotyledons, but the adverse effect of the red light and BA treatments on long-term Chl accumulation (24 h) was not reversed. Application of glutamate did not stimulate Chl production. Simultaneous treatment with hormone and red light 72 h before greening enhanced their separate inhibitory effects on Chl synthesis, but when given together 24 h prior to white light, their promotive effects on pigment accumulation were not additive.     

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.     

The stimulating effect of a cold dark-pretreatment on the etioplast/chloroplast transformation of angiosperms. III. Involvement of the excision factor and of cold-retarded aging processes?

Our question is whether the stimulating effect of a cold dark-pretreatment on the process of de-etiolation in primary leaves of wheat seedlings under subsequent continuous white light is essentially mediated by the retarding effect of highly lowered temperatures on the following processes: aging and/or senescence, realization of the so-called excision factor in detached leaves, decrease of the cytokinin level in detached leaves. The strong stimulating effect of a cold dark-preatreatment remains inspite of progressive aging in parts of the leaves and a strong decrease of the capability of chlorophyll accumulation in detached in contrast to attached leaves. The strong stimulatory effect of a cold dark-pretreatment is not diminished by application of cytokinin or gibberellic acid. The stimulating effect of a cold dark-pretreatment is detectable over several days under continuous light, but it is lost during a warm dark-phase of a few hours duration between the cold dark-pretreatment and the white light phase.     

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.     

Effect of Age and Rehydration on Greening of Wheat Leaves

Photosynthetic pigment synthesis was enhanced upto 15 day withincreasing age of etiolated wheat (Triticum aestivum L. cv.Sonalika) seedlings. The adverse effect of water stress on chlorophyll(Chl) synthesis was observed even after complete rehydrationof the seedlings. Younger seedlings were more prone to stressthan the older ones. The older rehydrated seedlings showed stressrecovery ability in the post lag phase of greening. (Received August 15, 1985; Accepted October 21, 1986)     

Studies on Greening of Etiolated Seedlings II. Leaf Greening by Phytochrome Action in the Embryonic Axis

We could demonstrate that greening of primary bean leaves in etiolated seedlings of Phaseolus vulgaris cv. Limburg can be controlled by a selective light-pretreatment of the embryonic axis. This light-induced interorgan synergism proved to be a phytochrome-mediated process. The red/farred photoreversible effect on the embryonic axis seems to be primarily linked to changes in the energy metabolism of the primary leaves. Phototransformation of the protochlorophyll present and pigment synthesis are very dependent upon an adequate supply of biochemical energy. When the embryonic axis is selectively pre-exposed to red light for a short time, respiration is markedly enhanced in the leaves and photosynthesis starts immediately upon illumination of the etiolated leaves after an incubation period of optimal length in the dark. The stimulatory effect of the red pretreatment on leaf respiration and photosynthetic capacity could be abolished to the level of the dark controls by a subsequent far-red irradiation on the embryonic axis. It is therefore postulated that phytochrome plays a regulatory role in interorgan cooperation. The metabolic changes involved in photomorphogenesis of etiolated seedlings are closely related to changes in energy production. Our data indicate that the primary act of phytochrome becomes operative at the biochemical level by its directional influence on the energy balance of the cell and coordinates the use of metabolic energy within a tissue and between organs.     

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.     

The development of plastocyanin in greening bean leaves

The plastocyanin content of etiolated bean leaves (Phaseolus vulgaris L.) was measured, and the development of the protein in response to light was followed. Measurements were made by quantitative extraction of plastocyanin and a sensitive assay with an O₂ electrode. The electron-paramagnetic-resonance (e.p.r.) signal of oxidized plastocyanin was used as an independent check on the validity of the assay method, and on the thoroughness of extraction. After an initial lag period, the amount of plastocyanin in greening bean leaves increased to reach a maximum after 50h illumination. The chlorophyll/plastocyanin ratio reached a maximum value of 200 irrespective of the light intensity at which greening was carried out, suggesting that the synthesis of the two components is co-ordinated. Experiments involving treatment of etiolated seedlings with brief periods of light of different spectral composition indicated that phytochrome is involved in plastocyanin synthesis. The lack of inhibition of plastocyanin synthesis by specific inhibitors of chloroplast protein synthesis suggests that the protein is synthesized on cytoplasmic ribosomes. The data are discussed in relation to the development of ferredoxin in greening bean leaves.     

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.     

Protoplasts as a means of studying chloroplast development in vitro

Protoplasts obtained enzymically from etiolated primary leaves of oat were illuminated in vitro, and the process of etioplast chloroplast transformation followed. Chloroplast development proceeded up to 6 hours of incubation in the light (20 C). During this period, complete photosynthetic light and dark reactions were constituted, in addition to prolamellar body-degrading protease activity.     

Effect of phytochrome activation followed by prolonged dark incubation on chloroplast development in etiolated cucumber cotyledons

The inhibitory effect of a short red light pulse followed by prolonged dark incubation on chlorophyll accumulation in etiolated cucumber cotyledons ( Cucumis sativus L. cv. Elem) was reflected in the development of the internal membrane system of the mesophyll plastids. Dark incubation for 24 h after phytochrome activation produced the characteristic accelerating effect OB chlorophyll synthesis and chloroplast development. However, longer intervening dark periods (48, 72 and 96 h) before white light exposure resulted not only in a diminished capacity to concentrate chlorophyll, but also in an impaired ability to form grana. The absence of stacking was consistent with a high chlorophyll a to chlorophyll b ratio.     

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.     

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.     

Regulation of 5-aminolevulinic Acid synthesis in developing chloroplasts : I. Effect of light/dark treatments in vivo and in organello

Intact chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons regenerated protochlorophyllide (Pchlide) in the dark with added cofactors from either exogenous glutamate or endogenous substrates. No other intermediates of the chlorophyll biosynthetic pathway accumulated. When inhibitors of 5-aminolevulinic acid (ALA) dehydratase were added, the Pchlide that failed to form was replaced by an excessive amount of ALA. When greening seedlings were returned to the dark, ALA-synthesizing activity in the isolated chloroplasts decreased dramatically and recovered if the dark-treated seedlings were again exposed to continuous white light prior to chloroplast isolation. Both the decline and the recovery of ALA-synthesizing activity were complete in approximately 50 minutes. Changes in chloroplast structure during in vivo light to dark and dark to light transitions (as evidenced by electron microscopy) were much slower. Exposing isolated chloroplasts from dark-treated seedlings to short white flashes before incubation transformed nearly all the endogenous Pchlide, but hardly stimulated ALA synthesis, suggesting that Pchlide does not act as a feed-back inhibitor on ALA synthesis. Chloroplasts isolated from dark-treated tissue did not form Pchlide from glutamate when incubated in the dark with added cofactors; moreover, the endogenous Pchlide did not turn over in organello. However, these chloroplasts did synthesize Pchlide from added ALA at the normal rate and synthesized ALA from glutamate at a reduced, but still significant, rate. Mg chelation was not affected by in vivo dark treatment.     

The effect of simulated microgravity on formation of the pigment apparatus in etiolated barley seedlings

The effect of horizontal clinorotation on the dynamics of the accumulation of the main photosynthetic pigments in the greening of 6-day-old etiolated barley seedlings has been studied. The content of protochlorophillide, the direct precursor of chlorophyll a, in clinorotated seedlings in the dark was 9–20% lower than in the control group. After exposure of barley seedlings to light for 12 h under clinorotation, chlorophyll accumulation lagged behind the control by 45% and reached the control value after 48–72 h. The total content of carotenoids increased many fold during greening; at the first stage the carotenoid level in clinorotated seedlings was less than in the control. The synthesis rates of δ-aminolevulinic acid and δ-aminolevulinate dehydratase activity in clinorotated seedlings were slower than in the control after 24 h of greening and after 72 h of greening reaching the control values. The activity of Mg-protoporphyrin IX chelatase catalyzing the incorporation of Mg ions in the structure of chlorophyll a, did not change when exposed to clinorotation. The results we obtained show inhibition of the initial stages of chlorophyll biosynthesis in the conditions of simulated microgravity. The light, to a certain extent, decreases the negative effect of microgravity on the formation of the photosynthetic apparatus in plants.     

A single red-light pulse leads to a block of greening in the high-pigment-1 mutant of tomato

A single pulse of red light (R) given to 4-d-old etiolated high-pigment-1 (hp-1) mutant tomato (Solanum lycopersicum L.) seedlings followed by a 3-d dark period is demonstrated to result in a block of greening in subsequent white light. Wild-type seedlings green normally under this regime. The block of greening in the hp-1 mutant depends on the length of the dark period before and after the R pulse and operates via the low-fluence-response mode of phytochrome action. This block of greening takes place in hp-1 double mutants lacking either phytochrome A or phytochrome B1, but is absent in the hp-1 triple mutant lacking both phytochromes A and B1. These observations enable a screen to be devised for new phytochrome B1 mutants either within the photoreceptor or mutants defective in phytochrome B1-signalling steps which result in loss of capacity to green, by mutagenising the phytochrome A-deficient hp-1, fri double mutant. Received: 20 February 1998 / Accepted: 18 June 1998