THE EFFECT OF LIGHT INTENSITY AND SUCROSE FEEDING ON THE FINE STRUCTURE IN CHLOROPLASTS AND ON THE CHLOROPHYLL CONTENT OF ETIOLATED LEAVES

Changes in the fine structure of proplastids of etiolated leaves exposed to various conditions of light and darkness for 24 and 48 hours were investigated, and the chlorophyll content of the leaves so treated was determined in vivo. The light treatments were given while the leaves were floated on tap water or on a 0.2 M sucrose solution. Leaves floated on water under low light intensity (2 foot-candles) were low in chlorophyll and contained plastids with concentric rows of vesicles. Transferring the leaves back to darkness resulted in the disappearance of the concentric rigs and re-formation of vesicular centers together with straight rows of vesicles and tubules, evenly spaced throughout the stroma. Chloroplasts of leaves floated on a sucrose solution under low light showed large vesicular centers together with stacks of rows of elongated tubules. The same chloroplast structure was found in leaves floated on a sucrose solution in the dark, after having been exposed to weak light for 24 hours. Chlorophyll content in these leaves was the same as in leaves floated on water under high light intensity, where the chloroplasts had normal grana and lamellae. The effect of the investigated factors on plastid development is discussed.     

Transformation of plastids in the leaves of Acer negundo L. var. odessanum (H. Rothe).

The leaves of Acer negundo L. var. odessanum (H. Rothe), if permanently exposed to strong sunlight, do not green, but remain yellow and finally become bleached. In yellow leaves the plastids contain single thylakoids and no grana. In plastids of bleached leaves, however, only vesicles are present. The concentration of chlorophylls and photosynthetic activity are much lower in those leaves than in the green ones. If the illumination is reduced (e.g. by shading) both the yellow and the bleached leaves become greenish, and even fully green after a few days at a sufficiently low light intensity. The plastids of yellow-green leaves contain small grana. In dark green leaves the thylakoid system of the chloroplasts is normally developed forming true grana, regardless of whether the leaves were originally green, or became green by shading the yellow or bleached ones. Their pigment concentration and photosynthetic activity are also normal. If green leaves are exposed to sunlight they do not yellow or bleach. During a 3-week period the structure of the thylakoid system did not perceivably change, with the exception that large plastoglobules formed in the stroma.     

Air ion effects on EDTA-induced bleaching in green barley leaves

If green intact barley leaves are floated on the surface of a solution of EDTA (0.05 M, pH 7.0) and exposed to light, a severe chlorosis or bleaching is observed in the leaf tissues. This EDTA-bleaching effect can be quantitated in terms of the tissue content of chlorophylls a and b.The EDTA-bleaching effect is weakened by exposing to either negative or positive air ions. The addition of casein hydrolysate (2%) also protects intact green leaves against EDTA-bleaching.This protective effect of casein hydrolysate is significantly augmented by either negative or positive air ions. The leaf tissue content of protochlorophyll is not uniformly affected by EDTA or air ions.A hypothetical mechanism involved in the phenomena described has been proposed.

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Zusammenfassung Wenn heile grüne Gerstenblätter, die auf der Oberfläche einer Lösung von EDTA (0,05 M, pH 7,0) treiben, Licht ausgesetzt werden, tritt eine starke Chlorose oder Bleichung des Blattgewebes ein. Dieser EDTA-Bleichungseffekt kann quantitativ über den Gehalt an Chlorophyll a und b im Gewebe erfasst werden.Bei Einwirkung von positiven und negativen Luftionen wird der EDTA-Bleichungseffekt abgeschwächt. Die Zugabe von Caseinhydrolysat (2%) schützt heile grüne Blätter vor der Bleichung. Dieser Schutzeffekt des Caseinhydrolysats wird signifikant sowohl durch negative als auch durch positive Luftionen verstärkt.Der Anteil an Protochlorophyll im Blattgewebe wird durch EDTA und Luftionen nicht einheitlich beeinflusst.Eine Hypothese zur Erklärung dieser Phänomene wird vorgelegt.

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Resume Si l'on place des feuilles d'orge saines et vertes à la surface d'EDTA (0,05 M, pH 7,0) et qu'on les expose à la lumière,on constate alors une forte chlorose ou décoloration du tissus foliaire. Cet effet décolorant du EDTA peut être déterminé quantitativement par la teneur en chlorophylle a et b du tissus.La présence d'ions positifs ou négatifs dans l'air diminue les effets décolorants d'EDTA. L'adjonction d'hydrolyse de caséïne (2%) protège les feuilles vertes et saines de la décoloration. Cet effet protecteur est renforcé de façon significative par des ions de l'air aussi bien positifs que négatifs.La teneur de protochlorophylle dans les tissus végétaux n'est pas uniformément influencée par l'EDTA ou l'ionisation de l'air. On présente enfin une hypothèse pour expliquer ces phénomènes.

     

Absence of nitrate reductase activity in San 9789 bleached leaves of barley seedlings (Hordeum vulgare cv. Midas)

Abstract San 9789 (norflurazone) blocks carotenoid synthesis which allows chlorophyll bleaching in the light, and has been used recently as a tool to study phytochrome responses without interference from photosynthetic pigments. By using this herbicide, we have found that nitrate reductase activity and light dependent nitrite reduction were lost simultaneously from achlorophyllous areas of barley leaves, with the green areas of the leaf tip still showing high activities. By contrast nitrate reductase is still present in the roots of herbicide treated plants. We suggest that intact chloroplasts are required for the presence of nitrate reductase in barley leaves.     

The effect of light on chlorophyll loss in senescing leaves of sycamore (Acer pseudoplatanus L.)

Breakdown of chlorophylls in attached senescing sycamore leaves held in darkness was significantly less over a 14-d period than that occurring in leaves exposed to natural light. Chlorophyll a declined more rapidly than chlorophyll b in both situations, the stability of the latter being particularly increased in darkness. The differences between dark-maintained leaves and those exposed to light with respect to soluble protein, cytoplasmic RNA, and free amino-nitrogen were much less marked. The data indicate that chlorophyll loss during senescence is, at least in part, the result of a direct photochemical degradation of the pigment.     

Air ion effects on RNAse activity in green barley leaves

Previously we reported that green barley leaves floated on the surface of a solution of EDTA and exposed to light undergo severe chlorosis. Such bleaching was substantially reduced by (+) or (–) small air ions. EDTA bleached leaves contained more RNA than did the controls. In the present work we found that (+) and (–) air ions markedly increased the RNAse activity of leaves floated on water in the presence or absence of light. EDTA in solution diminished RNAse activity and air ions do not affect this process. In the light there was a loss of RNAse protein; air ions do not influence the reaction. Even larger decreases in RNAse protein develop if EDTA is added to the water and here again air ions were without effect. RNA fractionation of leaves prior to exposure to water or light yielded a typical curve with four peaks representing: RNA, RNA-DNA, Ribosomal-DNA and Messenger-RNA. After 24 hours in water and light the peaks were much lower and were shifted to the right. Air ions did not effect the alternation in profiles. After 12 hours EDTA solution in the light produced 14 peaks instead of four. This profile disruption was eliminated by treatment with (+) or (–) air ions.

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Zusammenfassung Wir haben früher berichtet, dass grüne Gerstenblätter, die auf der Oberfläche einer EDTA-Lösung treiben, bei Belichtung eine schwere Chlorose aufweisen. Dieser Bleichungseffekt ist in Anwesenheit von (+) und (–) kleinen Luftionen erheblich vermindert. EDTA gebleichte Blätter enthalten mehr RNA als Kontrollen. Wir fanden in dieser Arbeit, dass (+) und (–) Luftionen die RNAse-Aktivität treibender Blätter auf Wasser mit und ohne Belichtung stark erhöhen. EDTA-Lösung verminderte die RNAse-Aktivität und darauf hatten Luftionen keinen Einfluss. Bei Lichtexponierung fiel der Anteil an RNAse-Protein; Luftionen beeinflussten diese Reaktion nicht. Der RNAse-Proteinanteil fiel weiter ab, wenn EDTA zugegeben wurde; auch darauf hatten Luftionen keinen Einfluss. Die RNA-Fraktionierung der Blätter vor dem Exponieren auf Wasser und im Licht ergab eine typische Kurve mit 4 Gipfeln (RNA, RNA — DNA, Ribosomen-DNA und Messenger-RNA). Nach 24 Stunden im Wasser und Licht waren die Gipfel flacher und rechtsverschoben, unabhängig von der Einwirkung von Luftionen. EDTA-Lösung und Licht bewirkten nach 12 Stunden 14 Gipfel anstatt 4. Diese Auflösung des Profils wurde durch Einwirkung von (+) und (–) Luftionen unterbunden.

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Resume On a rapporté dans un précédent mémoire que les feuilles vertes d'orge flottant à la surface d'une solution aqueuse de EDTA présentent des symptômes importants de chlorose dès qu'elles sont exposées à la lumière. Ces symptômes sont nettement moindres lorsqu'on introduit de petits ions (+) ou (–) dans l'air ambiant. Les feuilles chlorosées par EDTA contiennent plus de RNA que les feuilles de contrôle. Dans le présent travail, on démontre que les ions (+) et (–) de l'air augmentent de façon marquée la réduction du RNA dans les feuilles flottant sur l'eau et cela avec ou sans lumière. Une solution de EDTA diminue le phénomène et l'ionisation de l'air est sans effet. Par suite de l'exposition à la lumière, la proportion de la proteine issue de la dite réduction diminue et la présence d'ions dans l'air est sans effet. La proportion de la dite proteine diminue encore si l'on ajoute du EDTA et l'ionisation n'a également pas de répercussions sur ce phénomène. Lors de la distillation fractionnée du RNA tiré des feuilles avant leur traitement par l'eau et la lumière, on obtient une courbe typique avec 4 maximums correspondant à RNA, RNA-DNA, DNA ribosomal et RNA Messenger. Après 24 heures de traitement à l'eau et à la lumière, les pointes sont moins prononcées et déplacées vers la droite et cela quelle que soit l'ionisation de l'air. Un traitement de 12 heures à la solution de EDTA et à la lumière a eu pour effet de produire 14 maximums le long de la courbe au lieu de 4. Cette modification de la courbe a été supprimée par une ionisation positive ou négative de l'air ambiant.

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This work was supported by: (1) a grant (5 RO 1 AP00002-12) from the Air Pollution Control Office, Environmental Protection Agency, Department of Health, Education and Welfare and (2) the Bureau of Medicine and Surgery of the United States Navy.     

Control of senescence in marchantia by phytochrome

Mature green tissue of Marchantia polymorpha L. bleaches markedly when placed in continuous darkness for 4 days but remains green when given daily 1-hour photoperiods of white light. The tissue, however, is induced to bleach when each daily 1-hour photoperiod is terminated with a brief irradiation with far red light. The bleaching does not occur when each irradiation with far red light is followed by a brief irradiation with red light. The bleaching is taken as an index of senescence since the loss of chlorophyll in the bleached tissue is accompanied by a breakdown of cell organelles and cytoplasm. Phytochrome is clearly implicated in the control of senescence by light. It was also found that 5 minutes of red light given once a day was as effective as the 1-hour photoperiods with white light in preventing the bleaching and that bleaching was induced when each daily 5-minute irradiation with red light was followed by a 10-minute irradiation with far red light.     

Responses of stomata of barley and maize to phenylmercuric acetate

A reduction of stomatal aperture in light was found in leaves of maize after they had been treated with 10“3-5 m phenylmercuric acetate (PMA). Complete closure of the stomata in darkness was prevented, whilst there was total closure in the controls. Higher PMA concentrations had bigger effects. The relative water content (RWC) of barley tissues was slightly reduced 12 hours after treatment with PMA. The transpiration rate observed on PMA-treated barley plants was lower in light and higher in darkness than in untreated plants. Water saturation deficit (WSD) was higher by about 5%, and water holding capacity (WHC) lower (25%) than in untreated plants. The results suggest that the concentration of PMA normally applied as an antitranspirant is unfavourable for healthy growth of maize and barley.     

Effects of Metal Chelators on Leaf Senescence in Maize and Hydrangea

The senescence of maize and hydrangea leaves after being detachedand kept in the dark was studied in terms of the loss of chlorophyll.Chlorophyll was more rapidly degraded in maize than hydrangeaduring the incubation period in the dark. The loss of chlorophyllin the dark was effectively inhibited in both plants by ,'-dipyridyland o-phenanthroline at concentrations between 0.01 and 0.1mM. Three other chelators of iron produced lesser inhibitionand only at higher concentrations. EDTA prevented the loss ofchlorophyll in maize leaves at concentrations above 10 mM, butdid not do so in hydrangea leaves. Detached leaves floated on EDTA, ,'-dipyridyl or o-phenanthrolinesolutions and exposed to light exhibited a marked bleaching.The bleaching was partially inhibited by applying ascorbic acid. (Received December 26, 1981; Accepted October 18, 1982)     

Effect of Kinetin on Ribosomes of Excised Barley Leaves

The changes in the amount and the composition of ribosomes in excised barley leaves floated on water or on 10 mg/l kinetin solution in the dark were examined. The rapid loss of polyribosomes and ribosomes in leaves floated on water was greatly retarded by kinetin. The ribosomes-polyribosomes which originally contained 49 per cent protein showed substantial decline in protein content in leaves floated on water but only slight decline in leaves floated on kinetin solution. It is suggested that kinetin by stimulating RNA synthesis and by suppressing the activities of rihonuclease and peplidase may preserve the ribosomes in excised leaves.     

Induction of Adventitious Buds on Undetached Leaves, Excised Leaves and Leaf Fragments of Heloniopsis orientalis

Adventitious buds were induced on intact, undetached leaves, isolated leaves, and both green and etiolated leaf fragments excised from young plants of Heloniopsis orientalis (Thunb.) C. Tanaka (Liliaceae) in darkness. Morphactin promoted bud initiation on undetached leaves. The regeneration loci on excised leaves were different in darkness and in light, and they were also modified by etiolation and by morphactin or benzyladenine. Experiments with pre-incubation in darkness, with successive treatments by sorbitol and sucrose, and with DCMU-treatment in light, led to the conclusion that bud formation on isolated leaves and leaf fragments is controlled by a photosynthetic system as well as the hormonal level.     

CYCLING OF STOMATAL APERTURE IN LEAVES OF PLANTS WITH CRASSULACEAN ACID METABOLISM UNDER CONSTANT CONDITIONS

When leaves of plants with C₃ metabolism are detached and held in darkness, they senesce and the stomata close. Because the relation of senescence and stomatal closure is very close, if not actually causal, the question arose as to whether in the leaves of plants with Crassulacean acid metabolism whose stomata open at night the relationship to senescence would be reversed. Detached leaves of four species of Hoya, floated on water in constant darkness or constant light, were found to show no large differences in stomatal aperture (measured as diffusion resistance) between those in the light or dark, but the aperture changed in a regular circadian rhythm. In some leaves the rhythm was simple, in others the peak showed small secondary peaks, but in all cases the values were nearly the same in the light as in the dark, throughout the cycle. Previous culture of the intact plants under normal day/night conditions gave results similar to those with plants that had had prolonged culture under constant light or darkness. In those cases when the stomata were more open in the dark, the chlorophyll content was greater than when the stomata were more open in the light; but when they were more open in the light, the chlorophyll content showed little difference between light and dark. When the leaves had only their petioles in water they showed greater senescence in the light than in the dark, and the stomata were more tightly closed in the light, especially at the apical ends. All four species of Hoya gave similar results. We deduce that senescence of these leaves is modified by stomatal aperture, and generally in the same direction as in C₃ leaves, but that in continuous light or darkness the primary control over the aperture is the endogenous cycle.     

Effects of light and regulators on senescence-related changes in soluble proteins in detached oat (Avena sativa L.) leaves

The rate of senescence and the two-dimensional pattern of soluble proteins from detached oat leaves senescing in either darkness or light were analyzed, and compared to those of leaves in which senescence was delayed by application of the cytokinin benzyladenine or enhanced through the action of abscisic acid.Senescence of detached leaves in light did not differ significantly from senescence in attached leaves on intact plants. In darkness, protein was lost at a higher rate than in light, but several individual proteins showed relative increases. Notably, proteins previously characterized as high-molecular-weight proteins and senescence-associated proteins (Klerk et al., 1992) increased. Changes observed during incubation in light or darkness appeared to be related to this condition rather than the rate or progress of senescence. Cytokinins delayed and abscisic acid accelerated the changes in protein pattern compared to water. Beside changes previously identified in leaves senescing on the plant, detached leaves show alterations that reflect their condition of incubation rather than their developmental progress.Abbreviations 2D-PAG two-dimensional polyacrylamide gel electrophoresis - ABA abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - IEF isoelectric focusing - Rubisco ribulosebisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - Tris tris (hydroxymethyl) aminomethane     

The effect of light wavelength on the susceptibility of plants to virus infection

Plants exposed for 24–72 h to light of different wavelengths differed in their subsequent susceptibility to virus infection. French bean leaves were less susceptible to infection by tobacco necrosis virus and Nicotiana glutinosa leaves were less susceptible to infection by tobacco mosaic virus when previously exposed to blue or red light than when exposed to green light. These differences were most pronounced at low energy levels. Leaves exposed to each kind of light were less susceptible than those kept in darkness.     

Resilience and acclimation to bleaching stressors in the scleractinian coral Porites cylindrica

‘Resilience’, the capacity of the coral symbiosis with dinoflagellate algal symbionts (‘zooxanthellae’) to recover after bleaching, is a little-studied but crucial aspect of coral responses to bleaching stressors. This study investigated the response of the zooxanthella population in the coral Porites cylindrica after bleaching either naturally on a shallow subtidal reef or experimentally in response to elevated temperature and darkness. Coral resilience was influenced by the nature and duration of the stressor. Corals strongly bleached by natural stressors were less resilient than those that had been partially bleached; and a similar recovery profile was obtained for corals experimentally bleached by exposure to elevated temperature, in which recovery was slower for corals thermally-stressed 96 h than for 72 h. The opposite trend was evident for corals exposed to darkness, indicating that the bleaching trigger had a strong impact on coral resilience. When P. cylindrica recently recovered from bleaching was subjected to a repetition of bleaching stressors, it did not display acclimation, i.e. experience-mediated acquisition of resistance to bleaching stressors. The zooxanthella populations in all corals tested throughout the experiments were typed by PCR-RFLP as clade C, indicating that coral responses were not accompanied by any substantial change in zooxanthella composition at the cladal level.     

Loss of Ribulose 1,5-Diphosphate Carboxylase and Increase in Proteolytic Activity during Senescence of Detached Primary Barley Leaves

Symptoms typical of senescence occurred in green detached primary barley (Hordeum vulgare L.) leaves placed in darkness and in light. Chlorophyll, total soluble protein, ribulose 1,5-diphosphate carboxylase protein and activity each progressively decreased in darkness and to a lesser extent in light. In all treatments most of the total soluble protein lost was accounted for by a decrease in ribulose 1,5-diphosphate carboxylase protein, suggesting that the chloroplast was a major site of degradation early in senescence.     

Lipid transformations in greening and senescing leaf tissue

Analyses were made of chlorophyll a and b and fatty acids (18:3, 18:2, 18:1, 18:0, 16:2, 16:1, and 16:0) of greening and senescing leaf tissue. Those dark-grown tissues given a prior treatment of red, far red, or red followed by far red light showed similar increases in chlorophylls and linolenate (18:3) when exposed to continuous white light. In contrast, green barley (Hordeum vulgare L.) leaves placed in the dark lost chlorophylls and fatty acids, especially 18:3. Senescing cocklebur (Xanthium strumarium L.) leaf tissue showed a decline in chlorophyll and fatty acids, especially again 18:3. Abscisic acid, but not sucrose, accelerated these senescent changes. Radioactive acetate incorporation into the galacto-lipids and phospholipids of senescing cocklebur leaf tissue increased and then the radioactivity of the lipids decreased in senescent tissues.     

Changes in Nucleic Acid Metabolism of Excised Barley Leaves During Senescence and the Effect of Kinetin on these Changes

The changes in the amount, rale of synthesis and the nucleotide composition of different RNA fractions in excised barley leaves floated on water or kinetin (10 mg/l) in the dark were examined. In excised leaves floated on water all nucleic acid components declined and these declines were retarded by kinetin. Barley leaves floated on water showed a stimulation of ³²P incorporation into various RNA fractions within 48 hours followed by a decline after 96–144 hours. The leaves floated on kinetin, however, showed an even higher incorporation of ³²P into UNA by 48 hours which remained at a comparatively higher level throughout the experiment. In spite of the above changes in RNA synthesis significant differences in the ³²P sucrose gradient profiles or in the ³²P nucleotide composition of UNA from water and kinetin floated leaves were not noted. The results of this study show that important changes in nucleic acid metabolism occur during the early stages of leaf senescence and that alterations in nucleic acid metabolism during senescence and during kinetin treatment may involve quantitative and only subtle qualitative changes.     

Effect of vanadate on rhythmic leaflet movement in albizzia julibrissin

Vanadate (Na₃VO₄) selectively and reversibly affects the rhythmic movement of Albizzia julibrissin leaflets. Leaflets floated on 1 millimolar vanadate open at the same rate or more rapidly than controls, but closure is inhibited. After 6 to 24 hours incubation, the inhibition can be reversed by a 24-to 48-hour period on water or control buffer. Recovery is complete in light-dark cycles, and it is almost complete under free-running conditions (prolonged darkness). Leaflets floated on 10 millimolar vanadate do not open in darkness, but they open at a reduced rate in light. Concentrations of 100 micromolar or less are ineffective.     

Photosynthetic light-harvesting function of carotenoids in higher-plant leaves exposed to high light irradiances

Exposure of barley (Hordeum vulgare L.) leaves to strong white light (1500 μmol photons · m⁻² · s⁻¹) decreased the quantum yield of photosynthetic oxygen evolution in green light preferentially absorbed by carotenoids (Φ-510) but not in red light exclusively absorbed by chlorophylls (Φ-650). This phenomenon was observed to be (i) rapidly induced (within a few minutes), (ii) slowly reversible in darkness (within about 1 h), (iii) insensitive to dithiothreitol and (iv) maximally induced by photon flux densities higher than about 1000 μmol · m⁻² · s⁻¹. Determination of the carotenoid composition of the major light-harvesting complex of PSII (LHCII) and analysis of the thylakoid membrane lipid fluidity before and after strong illumination of barley leaves in the presence or the absence of dithiothreitol showed that the light-induced decrease in the Φ-510/Φ-650 ratio did not require the physical detachment of carotenoids from the pigment antennae. Compared to barley plants grown under moderate light and temperature conditions, plants grown in sustained high irradiance at elevated temperature exhibited (i) a lower Φ-510/Φ-650 ratio, (ii) a reduced size of the functional PSII pigment antenna in green light (but not in red light) and (iii) a marked increase in the amount of free carotenoids found in non-denaturing Deriphat-containing electrophoretic gels of thylakoid membranes. Similarly, the Φ-510/Φ-650 ratio of the LHCII-deficient chlorina-f2 barley mutant was very low compared to the wild type. Separation and quantification of the cis/trans carotenoid isomers of barley leaves revealed that strong illumination did not induce pronounced cis-trans isomerization of xanthophylls. Taken together, the data suggest that the efficiency of energy transfer from carotenoids to chlorophylls varies with the light environment both in the short term and in the long term, with excess light energy noticeably inhibiting the photosynthetic light-harvesting function of carotenoids. The photoprotective significance of this carotenoid decoupling from the chlorophyll antennae is discussed. Received: 28 July 1997 / Accepted: 25 October 1997