Possible mechanisms of light-induced chlorophyll degradation in senescing leaves of Hydrilla verticillata

Light treatment markedly accelerated the chlorophyll loss in senescing leaves of Hydrilla verticillata [(L.f.) Royle] as compared to dark treatment, whereas such acceleration could not be observed in senescing spinach (Spinacia oleracea L.) leaves. The light-induced cholorophyll loss in Hydrilla was retarded slightly by chloramphenicol and markedly by cycloheximide. Catalase (EC 1.11.1.6) activity did not change appreciably in Hydrilla leaves either in light or in darkness, while in spinach it declined markedly in the dark, and light retarded such decline. Peroxidase activity in Hydrilla showed faster increase in light than in darkness, while in spinach it increased only in light during senescence. The activity of phenol(pyrogallol)-specific peroxidase increased markedly in light, and that of ascorbate-specific peroxidase decreased slightly both in light and darkness during senescence of Hydrilla leaves. This rise in phenolspecific peroxidase activity was prevented by cycloheximide treatment. Pretreatment of Hydrilla leaves with monophenol (2,4-dichlorophenol) and o-diphenol (hydroquinone) accelerated and retarded, respectively, the light-induced cholorophyll loss. Pretreatment of Hydrilla leaves with H₂O₂ augmented the chlorophyll loss more markedly in light than in darkness. The endogenous level of H₂O₂ increased more in light than in dark during senescence of Hydrilla leaves. Treatment of Hydrilla leaves with 3-(3.4-dichlorophenyl)-l,l-dimethylurea. a photosystem II inhibitor, prevented both light-induced rise in H₂O: level and chlorophyll loss, but it was without effect in the dark. Retardation of light-induced chlorophyll loss occurred during senescence of Hydrilla leaves when light was given in different photoperiods in a 24-h daily cycle for 6 days instead of as continuous irradiance. There was a negative correlation between the length of the photoperiod and the extent of cholorophyll loss.     

Senescence of Hydrilla leaves in light and darkness

Senescence of isolated leaves of Hydrilla verticillata (L.f.) Royle was studied in both darkness and light (20 μmol m⁻² s⁻¹). Senescence in the dark followed a general pattern of deterioration, i.e., gradual loss of cellular macromolecules like chlorophyll, protein and RNA with a concomitant rise in α-amino nitrogen, protease activity and tissue permeability. In light, however, an accelerated loss of chlorophyll took place although protein loss and increase in protease activity were retarded. A higher level of α-amino nitrogen in leaves in the light than in darkness could be correlated with lower leaching of free amino acids in light. Light decreased tissue permeability, as evidenced by lower conductivity of the incubation medium. In the light, RNA increased over the initial level. Both soluble and insoluble carbohydrates declined in the dark. The decline of insoluble carbohydrate was retarded by light, whereas soluble carbohydrate showed an initial rise and then declined sharply in the light.     

Interaction of polyamines and light on biochemical processes involved in leaf senescence

Abstract The rapid senescence of detached oat leaves in darkness is first manifested by a sharp rise in RNase activity (about 50% within 1 h), then by a rise in protease activity (indicated by an increase in non-protein α-amino nitrogen within 6 h) and ultimately by chlorophyll degradation (beginning after 18 h). These degradative changes are delayed or prevented by low concentrations (1–10 mM) of the naturally-occurring polyamines cadaverine, putrescine, spermidine and spermine. The tetraamine spermine is generally more active than the triamine spermidine, which is in turn more active than the diamines putrescine and cadaverine. All the polyamines are more active than kinetin or cycloheximide. As little as 10 min of exposure to 1 mM spermine, especially at the beginning of the dark period, produces a marked retardation of chlorophyll degradation over a 48 h period, and 60 min of exposure saturates the effect. In the light, all polyamines promote, rather than retard, the disappearance of chlorophyll but, as in the dark, they inhibit the rise in RNase and non-protein α-amino nitrogen. The photobleaching of chlorophyll in the presence of polyamines is proportional to the duration of exposure to high irradiance (16.5 Wm^?2) fluorescent light. Such light is more effective toward the end of the 48 h post-excision test period than at the beginning. Calcium ion (1–10 mM) supplied together with the polyamines diminishes their action in dark and light, indicating probable involvement of an initial ionic attachment mechanism. The loss of chlorophyll from the leaves of four species of dicotyledonous plants (pea, bean, rape, tobacco) in the darkness is similarly retarded by 1–10 mM polyamines. In rape, the most rapidly senescing species, 1 mM spermine almost completely arrests chlorophyll degradation over a 96 h period. It is suggested that polyamine metabolism in plants may be related to normal physiological control mechanisms as in microorganisms and animals, and that polyamines could find use as anti-senescence agents for plants.     

Metabolism of Oat Leaves during Senescence : VII. The Interaction of Carbon Dioxide and Other Atmospheric Gases with Light in Controlling Chlorophyll Loss and Senescence

In air largely freed from CO₂, senescence of isolated oat (Avena sativa cv Victory) seedling leaves is no longer prevented by white light; instead, the leaves lose both chlorophyll and protein as rapidly as in the dark. Senescence in light is also accelerated in pure O₂, but it is greatly delayed in N₂; 100% N₂ preserves both protein and chlorophyll in light and in darkness. In light in air, most of the compounds tested that had previously been found to delay or inhibit senescence in darkness actually promote the loss of chlorophyll, but they do not promote proteolysis. Under these conditions, proteolysis can therefore be separated from chlorophyll loss. But in light minus CO₂, where chlorophyll loss is rapid in controls, two of these same reagents prevent the chlorophyll loss. Unlike the many reagents whose action in light is thus the opposite of that in darkness, abscisic acid, which promotes chlorophyll loss in the dark, also promotes it in light with or without CO₂. Kinetin, which prevents chlorophyll loss in the dark, also prevents it in light minus CO₂. In general, therefore, the responses to light minus CO₂ are similar to the responses to darkness, and (with the exception of abscisic acid and kinetin) opposite to the response to light in air.     

Effects of Polyamines on Chlorophyll and Protein Content, Photochemical Activity, and Chloroplast Ultrastructure of Barley Leaf Discs during Senescence

The polyamines putrescine, spermidine, and spermine prevent the loss of chlorophyll normally associated with senescence of excised leaf tissue maintained in darkness on water (control). Retention of chlorophyll in barley leaf discs was in the range of 90% 4 days after excision and placement on effective polyamine solutions. In contrast, the loss of soluble protein was hastened with 0.5 millimolar spermidine and spermine treatments but it was retarded by 0.5 millimolar putrescine.     

The senescence of detached leaves of tropaeolum

The senescence of detached Tropaeolum majus leaves was compared with that described earlier for Avena. Tropaeolum was chosen as being not only a dicot but also as having a nearly circular leaf, thus needing only the smallest minimum of wounding, since wounding delays the loss of chlorophyll and protein in darkness. Tropaeolum resembles Avena in that closing the stomata osmotically or with ABA causes rapid senescence in light. As in Avena also, n-hexanol and α,α′-dipyridyl delay senescence in darkness but cause `bleaching' of chlorophyll in light. Unlike Avena, however, kinetin and gibberellic acid, which delay senescence in the dark in both species, do so in Tropaeolum without causing any significant stomatal opening. The senescence of Tropaeolum leaves is actually promoted by fusicoccin, which powerfully delays senescence in Avena, although fusicoccin does cause stomatal opening in darkness in both species. Thus, many of the phenomena of senescence are alike in the monocot and dicot, but there are several significantly different responses to the senescence-modifying reagents. It is concluded that while stomatal closure accelerates senescence in both species, stomatal opening is not directly linked to the prevention of leaf senescence.     

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.     

Protein degradation in lemna with particular reference to ribulose bisphosphate carboxylase: I. The effect of light and dark

Ribulose bisphosphate carboxylase from Lemna minor resembles the structure reported for the enzyme from other plants. When grown in the light, the enzyme appears to undergo little or no degradation, as measured by a double-isotope method. This situation is similar to that reported for wheat and barley, but is unlike that reported for maize, where the enzyme degrades at the same rate as total protein. Prolonged periods of darkness usually induce leaf senescence, characterized by the rapid degradation of chlorophyll and protein, with ribulose bisphosphate carboxylase undergoing preferential degradation. In L. minor there is selective protein degradation in the dark, but chlorophyll and ribulose bisphosphate carboxylase are stable when fronds are kept in the darkness for up to 8 days. It appears that Lemna is not programmed to senesce, or at least that darkness does not induce senescence in Lemna. Although there is no evidence for in vivo degradation or modification of ribulose bisphosphate carboxylase during prolonged periods of darkness, extracts from fronds which have been kept in the dark for periods in excess of 24 hours convert ribulose bisphosphate carboxylase to a more acidic form. The properties of the dark-induced system which acts on ribulose bisphosphate carboxylase, suggest that it may be a mixed function oxidase. The proposition that the selectivity of protein degradation is genetically determined, so that the rate at which a protein is degraded is determined by its charge or size, was tested for fronds grown in the light or maintained in the dark. There was no significant correlation between protein degradation and either charge or size, in light or dark.     

Biochemical Changes in Excised Leaves of Oryza sativa Subjected to Water Stress

Excised rice (Oryza sativa L. cv. Ratna) leaves were used to compare the changes in the levels of various biochemical intermediates and enzyme activities during senescence in turgid and water-stressed conditions. Chlorophyll, total protein and soluble protein content decreased but α-amino nitrogen content increased during the senescence of turgid leaves. In the leaves subjected to water stress, these changes were accelerated, the acceleration being greater with higher degree of water stress. Starch, soluble sugars, total carbohydrates and non-reducing sugar content decreased during senescence of turgid leaves. Water stress accelerated the changes in the levels of starch and non-reducing sugar, but the changes in the levels of soluble sugars and total carbohydrates were retarded. Reducing sugar content increased at first and then decreased in the turgid leaves, and water stress accelerated the change. The decline in the catalase activity and the increase in the peroxidase activity with time was faster in the water-stressed leaves than in the turgid leaves. Acid inorganic pyrophosphatase activity increased, but alkaline inorganic pyrophosphatase activity decreased during the senescence of turgid leaves, and such changes were accelerated by water stress. The results suggest that water stress does not accelerate all the processes connected with leaf senescence.     

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.     

Retarding effect of inhibitors of protein and RNA synthesis on chlorophyll and protein breakdown

Cycloheximide, ethionine,p-fluorophenylalanine, 6-azauracil, 5-diazouracil and vanillin, applied at relatively high concentrations, retarded the yellowing of kale (Brassica oleracea L. var.acephala) leaf discs in darkness, and stimulated it in light. All the compounds inhibited protein synthesis and retarded protein breakdown. Cycloheximide,p-fluorophenylalanine, diazouracil and vanillin also inhibited the incorporation of uracil-¹⁴C into RNA of senescing discs. Abscisic acid and 2-chloroethylphosphonic acid accelerated yellowing both in darkness and in light and stimulated the protein breakdown in senescing discs. Abscisic acid inhibited the chlorophyll, protein and RNA synthesis in detached, greening cucumber cotyledons. There was no direct correlation between the activity of a given compound as an inhibitor of yellowing in darkness, and the degree of inhibition of RNA synthesis. The arrest of yellowing in darkness is possibly a consequence of the retarded rate of protein breakdown. Yellowing in light, on the contrary, is dependent on the actual rate of protein synthesis.     

Relation between Respiration and Senescence in Oat Leaves

The respiration of excised oat (Avena sativa cv Victory) leaves and their sensitivity to inhibitors was followed during senescence under varied conditions. The respiration rate, which in controls reaches its peak on the third day in darkness, is lowered at the time of fastest loss of chlorophyll (as reported earlier) by seven unrelated reagents that all delay dark senescence. When senescence is delayed by white light or by cytokinins, the respiratory rise is correspondingly delayed. Kinetin and l-serine, which act as antagonists on senescence, also act as antagonists on the respiratory rate. However, an exception to this close correspondence between senescence and the respiratory rise is offered by the lower aliphatic alcohols, which delay dark senescence and yet accelerate the onset of the respiratory rise.     

Stabilization of Thylakoid Membranes by Spermine during Stress-induced Senescence of Barley Leaf Discs

The effect of spermine on photochemical activity and polypeptide composition of chloroplasts from barley leaf discs during senescence in the dark was studied. Chloroplast membranes did not show photosystem II activity after spermine treatment when water was the electron donor, but in the presence of diphenylcarbazide, this activity was observed. The diphenylcarbazide-stimulated photoreduction of dichloroindophenol was 3-fold greater in leaf discs incubated for 72 hours in spermine than in water. Photosystem I activity was reduced by about 90% within the first 24 hours in the spermine-treated samples. This reduction, however, was not due to a decrease in the photosynthetic unit size. A preferential loss of polypeptides other than those associated with photosystem II was observed during senescence of the leaf discs in water, but this loss was reduced by spermine. Spermine treatment also prevented the appearance of several additional chlorophyll proteins found in the controls during senescence. The results have been interpreted on the basis of the interaction of spermine with thylakoid membranes resulting in stabilization of membrane function during senescence.     

Multiple actions of abscisic acid in senescence of oat leaves

The modifications induced by abscisic acid (ABA) on the senescence of oat leaves in darkness have been studied and are compared with its well-known effects in light. Contrary to the action in light, ABA preserves chlorophyll (Chl) in the dark almost as well as kinetin. Chlorophylla is decolorized more extensively thanb, and the content ofb is maintained by ABA almost at its initial level for 4 days. ABA also prevents proteolysis in darkness just as completely as chlorophyll loss, the relationship of both breakdown processes to ABA concentration being strictly log-linear over the range from 1 to 100 M. In line with this action, ABA inhibits formation of the neutral protease in the dark but not in the light. The data suggest that ABA and kinetin operate to preserve chlorophyll and protein by different mechanisms, since their actions are neither independent nor synergistic but actually interfere with one another. In this connection, protein values given by the Lowry and Bradford methods have been compared. In parallel with the effect on senescence, ABA slowly opens the stomata in the dark. This effect increases with time, and by day 3 the stomata in ABA are as open as in leaves on water in light. Thus all these effects of ABA in darkness are strikingly opposite to those commonly observed on leaves in natural lighting. In addition, ABA powerfully inhibits the formation of ethylene in the dark by the detached oat leaves, and this inhibition also tends to increase with time. Finally, a slight antagonism to ABA's action on senescence is exerted byp-coumaric acid in the light but not in the dark.     

Evidence for lack of turnover of ribulose 1,5-diphosphate carboxylase in barley leaves

Turnover of ribulose 1,5-diphosphate carboxylase in barley leaves (Hordeum vulgare L.) was followed over time in light and dark. The enzyme was degraded in prolonged darkness and was resynthesized after the plants were returned to light. Labeling with ¹⁴C showed that simultaneous synthesis and degradation (turnover) did not occur in light. In contrast, the remaining soluble protein was turned over rapidly in light. Although ribulose 1,5-diP carboxylase can be both degraded and synthesized, these processes seem not to occur simultaneously, but can be induced independently by changing environmental conditions.     

Effects of plant growth regulators on Hill activity of submerged aquatic plants during induced senescence

Effects of plant growth regulators on Hill activity during induced senescence of leaves of three submerged aquatic plants Vallisneria spiralis L., Hydrilla verticillata (L.f.) Royle and Potamogeton pectinatus L., and a terrestrial plant Spinacia oleracea L. were studied. Hill activity was reduced by 39.3, 42.7, 45.2 and 245.1 μmol DCIP (2,6-dichloroindophenol) (mg chl)^?1h^?1 in Vallisneria, Hydrilla, Potamogeton and Spinacia, respectively. During induced senescence of isolated mature leaves, Hill activity declined with increasing incubation time in all species. Kinetin (0.23 mM) treatment reduced the loss of Hill activity; while both 0.69 mM ehthrel and 0.075 mM ABA treatments decreased it in each species. The effect of kinetin was greatest in Spinacia, followed by Potamogeton, Hydrilla and Vallisneria, while the effect of either ethrel or ABA or both was greatest in Potamogeton, followed by Spinacia, Vallisneria and Hydrilla. Kinetin pre-treatment for an optimal period (12 h) followed by treatment with either ethrel or ABA partially removed the inhibitory effect of the latter on Hill activity. Pre-treatment of tissues with either ethrel or ABA solution, restricted to 12 h, followed by kinetin treatment markedly reduced the promotive effect of kinetin on the Hill activity of these species.     

Control of development of amylolytic and proteolytic activities in cotyledons of germinating black gram seeds

The effect of polyamines and related metabolites on several parameters of leaf senescence was followed in detached radish ( Raphanus sativus L. var. radicular cv. "Giant Butter") leaves floated on test solutions in darkness. Leaf senescence was accompanied by a marked loss of chlorophyll, which started at 24–48 h of incubation. The polyamines, spermine and spermidine, and the diamines, putrescine and cadaverine, were highly effective in arresting chlorophyll loss over a period of at least 96 h. l -arginine, and especially l -ornithine, were less active. Polyaminens prevented the marked chlorophyll loss in dark-incubated leaves, but did not compensate for the moderate chlorophyll loss when the leaves were aged in light. Polyamines were also highly effective in retarding earlier events of leaf senescence, prior to chlorophyll loss: both protein degradation and ribonuclease activity were inhibited by spermidine. Chlorophyll and protein loss in dark-or light-incubated suspensions of either "intact" or disrupted chloroplasts was not affected by polyamines. – It is concluded that polyamines are highly effective in preventing chlorophyll loss from detached leaves, possibly by controlling early senescence-linked events which occur in darkness rather than by direct inhibition of chlorophyll degradation.     

Characterization of Senescence in Lemna gibba G3: A Determinate Growth System

Frond senescence in Lemna gibba G3 was characterized, and itscontrol by light, ABA and kinetin investigated. The plant exhibitsa determinate growth pattern with a frond producing a set numberof daughter fronds before undergoing senescence and death regardlessof whether or not it flowers. When a frond was cut in half,the distal half (half frond) which lacks any meristem underwentrapid senescence as compared with intact fronds. In both intactand half fronds, the onset of senescence was accelerated byABA and retarded by kinetin. Continuous white light acceleratedsenescence in both intact and half fronds over the dark controls.Under different photoperiodic light regime, the pace of daughterfrond production is accelerated in proportion to the lengthof light period. In half fronds, however, very short photoperiodiclight treatments (e.g. 1L: 23D or 3L: 21D) rather delayed senescenceover the dark controls. Two separate light control systems operatingin opposite directions in Lemana senescence appear to exist. ¹Present address: Department of Biology, Yonsei University,Seoul 120-749, Korea ²Present address: U.S. Department of Agriculture, Aero SpaceBuilding, Rm. 323, 901 D Street, S.W., Washington, D.C. 20251-2200, U.S.A. (Received July 13, 1989; Accepted May 8, 1990)     

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     

Thigmomorphogenesis: XXIII. Promotion of foliar senescence by mechanical perturbation of Avena sativa and four other species

Mechanical perturbation (MP, gentle tubbing) promoted the senescence of detached oat ( Avena sativa L. cv. Victory) leaf segments in the dark. The promotion of senescence increased with increase in the number of rubbings and could be seen after 24 h of dark incubation; the maximum effect was reached on day 3. The effect (% of control) of MP on the loss of protein was greater than the effect on chlorophyll (Chl) loss on day 1. However, on day 3 the effect of MP on the loss of Chl became greater than the effect on the loss of protein. Ethephon and 1-aminocyclopropane-1-carboxylic acid (ACC) marginally promoted the loss of Chl by both control and rubbed oat leaf segments, and the effect was additive with MP. Chloramphenicol (CAP), spermine, aminoethoxyvinylglycine (AVG) and Ca²⁺ marginally delayed the loss of Chl and protein in both control and rubbed segments. Kinetin greatly retarded the senescence of all segments. Even in the presence of these substances, the amounts of Chl and protein in the rubbed segments were always less than in their respective controls, thus retaining the effect of the MP. However, abscisic acid (ABA) and cycloheximide (CHI) caused the rubbed oat leaf segments to retain more Chl and protein than their respective control segments. The effect of CHI was actually enhanced by MP. Rubbing promoted the senescence of attached leaves of oats ( Avena sativa L. cv. Victory), maize ( Zea mays L. cv. Early Belle) and pumpkin ( Cucurbita pepo L. cv. Jack-o-lantern) cotyledons in the dark. Rubbing promoted the senescence of oat leaf segments even in light, although to a lesser extent compared to the effect in the dark. The senescence of leaves of pumpkin and cocklebur ( Xanthium strumarium Wallr. var. Pennsylvanicum ) in situ was also enhanced by MP.