Effects of light and spermine on senescence of hydrilla and spinach leaves

Light treatment markedly accelerated chlorophyll loss in Hydrilla (Hydrilla verticillata [L.f.] Royle) over dark treatment whereas such acceleration could not be observed in spinach (Spinacia oleracea L.) leaf segments. Spermine, a polyamine, retarded the loss of chlorophyll in the dark but markedly accelerated this loss in the light during senescence of Hydrilla leaves. However, such effect of spermine in the dark was not so pronounced in spinach. The loss of protein was slower in the light than in the dark in both the species. Spermine arrested the loss of protein (as in spinach) or even raised the protein level over initial (as in Hydrilla). Loss of both soluble and insoluble protein was slower in light than in darkness. Spermine treatment, either in light or darkness, markedly accelerated the loss of soluble protein but raised the level of insoluble protein over initial in both the species. The pattern of change in α-amino nitrogen in either species could be correlated well with that of protein level. In Hydrilla, light increased the soluble protein fraction over initial and this rise was prevented by cycloheximide and not by chloramphenicol. Also, spermine augmented the protease activity (both acid and neutral) while light retarded the rise in protease activity during senescence of either species. Although spermine treatment reduced the leaching of α-amino nitrogen and electrolytes in Hydrilla, it augmented the same in spinach.     

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.     

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.     

Effect of light and gibberellic acid on photosynthesis during leaf senescence of alstroemeria cut flowers.

The functioning of the photosynthetic apparatus during leaf senescence was investigated in alstroemeria cut flowers by a combination of gas-exchange measurements and analysis of in vivo chlorophyll fluorescence. Chlorophyll loss in leaves of alstroemeria cut flowers is delayed by light and by a treatment of the cut flowers with gibberellic acid (GA₃). The maximal photosynthesis of the leaves was approximately 6 μmol CO₂ m⁻² s⁻¹ at I 350 μmol m⁻² s⁻¹ (PAR) which is relatively low for intact C₃ leaves. Qualitatively the gas-exchange rates followed the decline in chlorophyll content for the various treatments, i.e. light and GA₃-treatment delayed the decline in photosynthetic rates. However, when chlorophyll loss could not yet be observed in the leaves, photosynthetic rates were already strongly decreased. In vivo fluorescence measurements revealed that the decrease in CO₂ uptake is (partly) due to a decreased electron flow through photosystem II. Furthermore, analysis of the fluorescence data showed a high nonphotochemical quenching under all experimental conditions, indicating that the consumption of reducing power in the Calvin cycle is very low. The chlorophyll, remaining after 9 days incubation of leaves with GA₃ in the dark should be considered as a 'cosmetic' pigment without any function in the supply of assimilates to the flowers.     

Modifications of chloroplasts of flax cotyledons treated with monuron: myelinoid figures formed under low light conditions

Abstract. Monuron treatment of flax cotyledons in the dark and at a low light intensity of 0.2 Wm⁻² caused a retardation of chlorophyll breakdown. With low light, carotenoid breakdown occurred more rapidly, suggesting a possible overloading of the carotenoid protective system, but in darkness chlorophyll loss was first. The ultrastructure of treated leaves was markedly altered. In the dark, monuron caused a swelling of the chloroplasts and a subsequent disruption of the thyl-akoids resulting in a concentric membrane formation in the stroma. Following envelope rupture the thylak-oid system was visible as a myelinoid figure in the cytoplasm.     

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.     

Irradiance-dependent senescence of isolated leaves

Isolated leaves from pea ( Pisum sativum L. cv. Alaska or the genotype L-1107), oat ( Avena sativa L. cv. Victory), and fuchsia ( Fuchsia triphylla L. cv. Koralle) were retained at irradiances between 0 and 130 μmol m^-2 s^-1 PAR (photosynthetic active radiation). Irradiance-dependent CO₂ fixation was measured when the leaves were excised, and time-dependent changes in light compensation point were determined. If the irradiance was below the light compensation point for CO₂ fixation, the respiratory quotient was low, indicating that lipids were respired. The isolated leaves senesced at these low light levels. At higher light intensities the decrease in chlorophyll level was not accompainied by diminished protein level, and the respiratory quotient was close to unity. Only an irradiance equal to or slightly above the light compensation point maintained a stable chlorophyll level for a long time. This suggested that depletion of reserves in darkness or at low levels of irradiance is important for the initiation of the senescence syndrome. At high levels of irradiance, the decrement in chlorophyll level may be caused by photooxidation. Only in leaves placed under an irradiance close to the compensation point, was CO₂ fixation able to prevent aging of the leaves.     

Light-independent accumulation of chlorophyll a and b and protochlorophyllide in green barley (Hordeum vulgare)

Barley ( Hordeum vulgare L. cvs Clipper, Procter, Astrix) seedlings were transferred from daylight to darkness and changes in chlorophyll a , chlorophyll b , protochlorophyllide and chlorophyllide (μ leaf⁻¹) in either the first or second leaf determined spectrophotometrically after separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and light petroleum ether. Chlorophyll a and b as well as protochlorophyllide accumulated in the dark. The ratio of chlorophyll to protochlorophyllide formed in the absence of light was 18:1. 5-aminolevulinic acid (10 m M ) promoted the synthesis of chlorophyll a and b and protochlorophyllide. Pigment synthesis and response to 5-aminolevulinic acid addition was related to tissue age. Mature tissue in the apical third of the leaf accumulated most chlorophyll, but per μg chlorophyll present at the time of transfer to darkness, was less efficient than immature tissue towards the base of the leaf. Immature tissue was also most responsive to added 5-aminolevulinic acid. Chlorophyll synthesis in the dark was accompanied by chloroplast development. Chloroplasts in immature leaf tissue increased in size and extent of thylakoid development when transferred from daylight to darkness. The results indicate that chlorophyll synthesis and chloroplast membrane development in light-grown barley continue into the dark phase of the diurnal cycle. A light-independent protochlorophyllide reductase in light-grown barley seedlings is postulated.     

Changes in ribulose-1,5-bisphosphate carboxylase and its translatable small subunit mRNA levels during senescence of mustard (Sinapis alba) cotyledons

During the senescence of detached first leaves of oat ( Avena sativa L. cv. Victory) seedlings (grown in continuous light) the protein is hydrolyzed and the proteases increase, but the expected simple relation between these two factors is not always realized. The present experiments examine the timing, the influence of light and darkness and the action of the protein synthesis inhibitors cycloheximide (CHI) and cordycepin. Transfer from dark to light delays the breakdown of both chlorophyll (Chl) and protein, but some residual proteolysis is ascribed to the enzyme initially present. Transfer to CHI resembles transfer to light, while the action of cordyceptin is similar but much weaker. Repeated determinations of the acid protease, which is the most active one and the first to appear, show that this enzyme is formed in the light about as rapidly as in the dark, though with different kinetics. In spite of this there is little proteolysis in light in the first 5 days. One possible explanation of that could be that protein is rapidly resynthesized in light, but treatment with [¹⁴C]-leucine shows that such resynthesis is no faster in light than in darkness. It is therefore concluded that the protease initially does not have access to its substrates and, as a corollary, that the senescence process must be controlled by the gradual impairment of the vacuolar membrane, allowing protease to enter the cytosol and attack the proteins there and in the organelles. This concept is supported by many observations on the timing and on the known changes in membrane permeability during senescence.     

Proteolytic activity and nitrogen transfer in maize seeds during imbibition

Ethanol-soluble and insoluble nitrogen and protease activity in maize seeds during imbibition period of 6 to 60 h at 30 ± 2 °C were determined both in light and in the dark. In light, soluble and insoluble nitrogen in the embryo were similar to that in the dark. But the increase in soluble nitrogen in the endosperm up to 38 h was higher in light than in the dark. Decrease in insoluble nitrogen was correlated with increase in soluble nitrogen, the level always being higher in the dark. Light increased protease activity also in the endosperm. Among various light qualities, red light was most effective in inducing proteolysis, and loss of nitrogen from the endosperm. Further, the growth and organic nitrogen of primary leaves from seedlings raised from light pretreated seeds were better than those from dark pretreated ones.     

Interaction of cycloheximide and light on chlorophyll content and chloroplast ultrastructure inBrassica campestris

Cycloheximide retarded the loss of chlorophyll from detached komatsuna (Brassica campestris cv. Komatsuna) leaves during incubation in the dark but promoted its loss in light. Cycloheximide-induced chlorophyll bleaching in light was prevented by some active oxygen scavengers. Chloroplast envelopes of cycloheximide-treated leaves incubated in both the dark and light were destroyed within 48 h. The grana of cycloheximide-treated leaves incubated in light were dilated.     

Cysteine, γ-glutamyl-cysteine and glutathione contents of spinach leaves as affected by darkness and application of excess sulfur. II. Glutathione accumulation in detached leaves exposed to H2S in the absence of light is stimulated by the supply of glycine to the petiole

When illuminated leaf discs and detached leaves of spinach ( Spinacia oleracea L. cv. Estivato) were exposed to 0.4 and 0.25 μl 1^-1 H₂S, respectively, pool sizes of cysteine and glutathione increased. In the dark, apart from these compounds, the level of γ-glutamyl-cysteine also increased. Incubation of leaf discs with 1.0 m M buthionine sulfoximine (BSO) resulted in the accumulation of cysteine only, both in the light and in darkness. When glycine was supplied to the petioles of detached leaves exposed to H₂S in the dark, the accumulation of glutathione was stimulated, while γ-glutamyl-cysteine accumulation was prevented completely. Glycolate and glyoxylate, precursors of glycine in the glycolate pathway, had nearly the same effect as glycine. Although other amino acids were apparently taken up equally well as glycine when supplied to the petiole, they were much less effective, or not effective at all, in restoring glutathione synthesis in the dark. These results provide evidence, that H₂S-induced glutathione accumulation in spinach leaves in the dark is limited by the availability of glycine, giving rise to the accumulation of the metabolic precursor γ-glutamyl-cysteine.     

The influence of NaCI concentration on the amino acid and neutral soluble carbohydrate content of Dunaliella

Abstract. Two strains of Dunaliella , both grown at 25°C were analysed for their α-amino acid and neutral soluble carbohydrate content. Glycerol was found as a major cell constituent in both varieties; the amount was roughly proportional to the outside NaCI concentration between 0.5 and 3 kmol m⁻³, and varied from 3.4% to 45% of the total dry weight of packed cells. The sum of sugars and α-amino acids did not amount to more than 2% of the total dry weight at any NaCI concentration.     

Mobilization of sulphur in soybean cotyledons during germination

Soybean seeds ( Glycine max L. cv , Stephens) contain a large amount of sulphur (ca 40 μ mol seed⁻¹), mostly in the insoluble fraction in the cotyledons. During germination in nutrient solution lacking sulphur the amount of insoluble sulphur decreases to very low levels. This is accompanied by a transitory increase in the pool of soluble sulphur which then declines. All of the sulphur lost from the cotyledons is quantitatively recovered in the seedling. In the short term, the root and the stem are the most important sinks for sulphur from the cotyledons but as growth proceeds the shoot becomes the dominant sink for remobilized sulphur. Within the shoot most of the sulphur is recovered in leaves L1 and L2. The growth of L3 and, to a lesser extent, L2, was retarded due to sulphur insufficiency. The cotyledons of plants treated with 20 μ M sulphate also exhibited mobilization of sulphur from the insoluble fraction except that the maximum rate of loss of sulphur occurred somewhat later. Plants grown with sulphate exhibited a net gain of sulphur and did not exhibit sulphur insufficiency. In these plants, endogenous sulphur from the cotyledons was directed into L1–L3 and this sulphur remained within these leaves for the duration of the experiment. The delivery of exogenous sulphur (supplied as [³⁵S]sulphate via the roots) to the leaves increased with leaf number. In leaves L1–L3, the level of exogenous sulphur in any one leaf declined with time, indicating that this sulphur was remobilized and did not mix with the sulphur derived from the cotyledons. It was concluded that the cotyledons are an important source of sulphur to support early plant growth and development of soybean.     

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.     

Rhythms in magnesium ion inhibition and hysteretic properties of nitrate reductase in the CAM plant Bryophyllum fedtschenkoi

Nitrate reductase (NR, EC 1.6.6.1) was tested in crude extracts of leaves from Bryophyllum fedtschenkoi plants growing under alternating light/darkness as well as in excised leaves kept in continuous light or darkness. In most extracts NR activity was inhibited 20–80% by 5 m M Mg²⁺ A light or darkness shift (30 min darkness) during the first part of the photoperiod gave an increase in the Mg²⁺ inhibition and a decrease in NR activity. Magnesium ion inhibition of NR also showed diurnal variations. Strongest inhibition was found in extracts made during the latter part of the photoperiod and start of the dark period. Pre-incubation of crude extracts with ATP increased Mg²⁺ inhibition, indicating that phosphorylation of NR is involved in regulation of NR in Crassulacean acid metabolism (CAM) plants. In continuous light an increase in Mg²⁺ inhibition occurred after 20 h and 40 h, indicating a rhythm in the phosphorylation of NR. A delay in the production of nitrite in the assay (hysteresis) was generally seen in extracts susceptible to Mg²⁺ inhibition. The rhythms related to NR activity showed the same period length (20 h) as the rhythm in CO₂ exchange. However, in contrast to the rhythm in CO₂ exchange, NR rhythms were strongly damped in continuous light. In constant darkness the rhythms were even more damped. The results show that post-translational modification of CAM NR is influenced by light/darkness and by an endogenous rhythm.     

Light effects on in vitro adventitious root formation in axillary shoots of mature Prunus serotina

Light effects on in vitro adventitious root formation in axillary shoots of a 95-year-old black cherry ( Prunus serotina Ehrh.) were examined using microcuttings derived from cultured vegetative buds. Three studies were performed: 1) complete darkness and 4 levels of continuous white light irradiance were tested at 70, 278, 555 and 833 μmol m⁻² s⁻¹; 2) white, red, yellow and blue light were tested to assess the importance of spectral quality; and 3) the effect of blue light at intensities of 7,15, 22 and 30 μmol m⁻² s⁻¹ was also studied, Measurements included rooting percentage, total number of roots per shoot, and shoot and root dry weight. There was a strong negative effect of white light intensity upon root formation. Blue light between 15 and 22 μmol m⁻²: s⁻¹ significantly retarded root formation and completely inhibited it at 36 μmol m⁻² s⁻¹. Shoots treated with yellow light exhibited the highest rooting percentage, mean number of roots per shoot, and root dry weight.     

Effect of benzimidazole and nickel ions in the control of Oryza sativa leaf senescence by red and far-red light

Changes in the levels of chlorophyll and protein were determined in detached rice leaves floated on water, benzimidazole (mM) and nickel chloride (mM) under continuous dark, red (R) and far-red (FR) illumination. Senescence was enhanced under FR and retarded under R illumination as compared to the dark treatment for leaves floated both on water and benzimidazole solution. Benzimidazole and nickel ions also delayed the senescence of leaves still further, although only to a limited extent under FR illumination. Protein changes showed similar trends to chlorophyll during senescence.