Senescence and stomatal aperture as affected by antibiotics in darkness and light.

In leaves of barley (Hordeum vulgare), as previously found with oats (Avena sativa), a group of six antibiotics that interfere in different ways with the sequence DNA --> mRNA --> protein all delay senescence in the dark, acting to conserve chlorophyll (Chl) and protein and also to open the stomata. Among the active compounds is chloramphenicol, which had previously been reported to act only on procaryotes. It is now shown that all these compounds with senescence-delaying action in darkness have the opposite effect in light, accelerating Chl destruction and partially or completely closing the stomata. Leaves of the dicot Tropaeolum majus show most of the same responses, though the changes in protein and amino acids are more variable. The data as a whole support the previous conclusion that the synthesis of one or more proteins controls both the opening and the closing of the stomata. An additional compound, kanamycin, acts in the same way as the other six compounds on oats and barley, though its action on proteolysis is unclear. On Tropaeolum, however, it opens the stomata in both light and darkness. Anisomycin and ethidium bromide have comparably atypical effects. Thus, although changes in stomatal opening or closing in the majority of cases are closely linked to the breakdown or preservation of Chl, the occasional exception shows that the biochemical phenomena of senescence cannot be under the direct control of changes in stomatal aperture.

The dependence of stomatal closure on protein synthesis.

Seven different inhibitors of the synthesis of protein and RNA, all of which are found to delay the senescence of detached oat leaves in darkness, also cause the opening of the stomata in the dark. The concentration ranges for activity on the two processes agree closely. Four other compounds of similar effects on RNA and protein synthesis, but which are inactive on senescence, correspondingly fail to open the stomata. This not only strengthens the relationship between stomatal closure and senescence, but-more important-provides strong evidence that continued protein synthesis is necessary to keep foliar stomata closed.

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 alpha,alpha'-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.

Metabolism of Oat Leaves during Senescence : VIII. The Role of L-Serine in Modifying Senescence.

The mechanism whereby l-serine specifically promotes the dark senescence of detached oat (Avena) leaves has been examined. The fact that this promotion is strong in darkness but very weak in white light has been explained, at least in part, by the finding that added serine is partly converted to reducing sugars in light. Labeled serine gives rise to (14)C-sugars and (14)CO(2). In the absence of CO(2), serine does cause chlorophyll loss in light and undergoes a decreased conversion to sugar.As to the large promotion of protease activity which accompanies senescence in the dark, reported earlier, careful purification of the proteases shows that the l-[(14)C]serine is not incorporated into these enzymes, although it is incorporated into the total protein. Cycloheximide decreases the overall synthesis both of protease and of total protein, but again [(14)C]serine does not impart radioactivity to the purified acid proteases. Even when serine is simply added to the protease assay the proteolysis is significantly increased. It is concluded that serine promotes the protease activity by synergizing with the enzyme, or by activating an apoenzyme.

Interaction between Senescence and Wounding in Oat Leaves.

A study was made of the influence of wounding on the senescence of standard oat leaf segments in the dark. Wounding was by either subdividing the 3 centimeter long segments into 5 millimeter subsegments, gently scraping the adaxial surface of the segments with a sharp blade, making transverse linear cuts, or by making many small holes with a needle. Wounding considerably delayed the loss of both chlorophyll and protein in the dark and the amount of inhibition was roughly proportional to the intensity of wounding. With surface wounding, the inhibition of senescence was detectable from the first day of dark incubation; other methods caused moderate promotion of senescence for the first 2 days but decreased the loss of chlorophyll and protein thereafter. A number of senescence-modifying substances acted similarly on both unwounded and wounded segments, but the amount of chlorophyll and protein in the wounded segments was always more than in the respective controls. Cytokinins, however, provided an exception, since their effect was actually decreased by wounding. The proteases operating at pH 4.1 and 6.6 were both clearly less active in the wounded leaves than in controls. The possible mechanism of this inhibitory effect of wounding on senescence is discussed.

Lipid peroxidation forms ethylene from 1-aminocyclopropane-1-carboxylic acid and may operate in leaf senescence.

An enzyme system is described which oxidizes 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene under physiological conditions. It comprises linoleic acid, pyridoxal phosphate, manganese, and lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12). It requires oxygen and is specific for manganese; it can operate but only with greatly reduced yield in the absence of pyridoxal phosphate. An enzyme with the same properties was prepared from microsomal membranes of the seedling shoots of peas. Both have similar reactions to a variety of inhibitors and other reagents. The properties also resemble those of at least two of the in vivo systems recorded in the literature. Intact green oat leaves also contain a similar system. Because there is a growing body of evidence that ethylene formation is associated with cell membranes and because the yields of ethylene from the complete system are much higher than those recorded for other enzymes, it may be identical with the in vivo system acting in senescent leaves.

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.The respiration of freshly cut leaves is insensitive to KCN up to 8 millimolar, but sensitive to benzhydroxamate (BAM), 1 to 2 millimolar BAM causing 25% promotion and higher concentrations inhibiting. At the respiratory peak, however, part of the respiration becomes KCN-sensitive. Low concentrations of alcohols in darkness, or 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, in light, also render part of the respiration KCN-sensitive, but this sensitivity soon disappears again. Some 10 to 15% of the respiration is insensitive to both inhibitors. Thus, cyanide sensitivity comes and goes, while BAM sensitivity is always present. The current concept of the cyanide-resistant pathway as an overflow, therefore, does not fit well with behavior of these leaves. The respiratory rise in leaf senescence is similar to, but not identical with, the climacteric in ripening fruits and the aging phenomenon in tuber slices.

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 CO(2), 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 O(2), but it is greatly delayed in N(2); 100% N(2) 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 CO(2), 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 CO(2). Kinetin, which prevents chlorophyll loss in the dark, also prevents it in light minus CO(2). In general, therefore, the responses to light minus CO(2) are similar to the responses to darkness, and (with the exception of abscisic acid and kinetin) opposite to the response to light in air.

The role of ethylene in the senescence of oat leaves.

The evolution of ethylene, both from the endogenous source and from added 1-aminocyclopropane-1-carboxylic acid (ACC), has been followed in close relationship with the senescent loss of chlorophyll from seedling oat leaves. In white light, where chlorophyll loss is slow, the ethylene evolution increases slowly at first, but when the loss of chlorophyll becomes more rapid, ethylene evolution accelerates. CoCl(2) inhibits this increase and correspondingly maintains the chlorophyll content, with an optimum concentration of 10 micromolar. The rapid rate of chlorophyll loss in the dark is slightly decreased by 3-aminoethoxyvinyl glycine (AVG), by cobalt, and slightly stimulated by ACC. The slower chlorophyll loss in white light, however, is almost completely inhibited by silver ions, greatly decreased by cobalt and by AVG, and strongly increased by ACC. Since the chlorophyll loss is accompanied by proteolysis, it represents true senescence. Chlorophyll loss in light is also strongly antagonized by CO(2), 1% CO(2) giving almost 50% chlorophyll maintenance in controls, while in the presence of added ACC or ethylene gas, the chlorophyll loss is 50% reversed by about 3% CO(2). The ethylene system in leaves is thus more sensitive to CO(2) than that in fruits. Indoleacetic acid also clearly decreases the effect of ACC. It is shown that kinetin, CO(2), Ag(+), and indoleacetic acid, all of which oppose the effect of ethylene, nevertheless increase the evolution of ethylene by the leaves, and it is suggested that ethylene evolution may, in many instances, mean that its hormonal metabolism is being prevented.Abscisic acid somewhat increases ethylene evolution also, but its action in promoting senescence in light is antagonized only partially by Ag(+), Co(2+), or AVG. For this and a number of other reasons it is concluded that ethylene and abscisic acid both independently control leaf senescence in the light.

The influence of aliphatic alcohols on leaf senescence.

Because of the effects of ethanol used as a solvent in other experiments, the action of aliphatic alcohols on leaf senescence in the dark has been studied systematically. These compounds both maintain chlorophyll and prevent proteolysis in the dark, much as do the cytokinins and other senescence-delaying substances. The activity of the straight-chain alcohols increases in a log-linear fashion with increasing chain length up to 1-octanol. Introduction of a branch in the chain or of a second OH group greatly decreases, or in some cases annuls, the antisenescence activity. In all cases, the action on senescence is closely (although not always exactly) paralleled by opening of the stomata. Abscisic acid and exposure to high concentrations of osmoticum, both of which close the stomata, antagonize the action of the alcohols. Some interactions with other agents are noted. The effects are compared with reported effects on seed germination, on hemolysis and animal membranes, and especially on permeability to K(+) ions, and a tentative basis for the mechanism of action is advanced.

Metabolism of Oat Leaves during Senescence: VI. CHANGES IN ATP LEVELS.

The ATP content of 7-day-old Avena sativa leaves during senescence in dark and in light, and after treatment with cytokinins and other reagents, has been determined by the luciferin-luciferase method. Special care was taken to avoid decomposition of the ATP, and a detailed procedure is presented for ATP analysis at the picomole level. Preliminary experiments with several inhibitors of photophosphorylation suggest, though not conclusively, that the delaying effect of light on senescence is mediated by photophosphorylation. The ATP values of the leaves senescing in darkness are found to increase in parallel with the large increase in respiratory rate, and kinetin prevents this increase just as completely as it prevents the respiratory rise. It is concluded that the respiratory increase in senescence cannot be simply due to uncoupling. In light the ATP level also rises, though more slowly, and again kinetin prevents this rise. l-Serine, which promotes dark senescence, does not significantly modify the dark ATP level, but both arginine and kinetin, which antagonize the action of serine on senescence, greatly lower the ATP level below that on serine alone. Cycloheximide has a similar effect, and the combination of cycloheximide and kinetin lowers the ATP level drastically. Fusicoccin, which opens stomata in the dark, correspondingly maintains the ATP at a low level. Thus, in general, a low level of ATP is associated with the prevention of dark senescence, i.e. probably with ATP utilization, and the ATP level at any time may thus be determined more by the rate of utilization than by the efficiency of respiratory coupling.

Changes in the abscisic acid content of oat leaves during senescence.

To investigate the possibility that experimental promotion of retardation of the senescence of oat leaves may be mediated by abscisic acid (AbA), determinations of AbA were made in leaves senescing under different conditions. The extracts were subjected to thin-layer chromatography, the spots were eluted and esterified, and the AbA was determined by gas chromatography (overall recovery, about 75%). In darkness, where the stomata are closed and senescence is rapid, the concentration of AbA increases to at least 5 times its initial value by the second day, the time when chlorophyll loss is most rapid. In light, where the stomata are open and senescence is very slow, no such increase occurs. But when, in light, the stomata are closed by floating the leaves on 1 M mannitol, the AbA level again increases to about 5 times the initial value; if the stoma response is prevented by kinetin, the increase in AbA is largely suppressed. Similarly, phenylmercuric nitrate, at a concentration that closes the stomata, causes a 4-fold increase in AbA. It is concluded that stomatal closure itself causes AbA accumulation and, thus, that AbA may indeed be the proximal cause of leaf senescence.

The effect of light on the production of ethylene from 1-aminocyclopropane-1-carboxylic acid by leaves.

White light inhibits the conversion of 1-amino-cyclopropane-1-carboxylic acid (ACC) in discs of green leaves of tobacco (Nicotiana tabacum L.) and segments of oat (Avena sativa L.) leaves by from 60 to 90%. Etiolated oat leaves do not show this effect. The general nature of the effect is shown by its presence in both a mono- and a dicotyledon. Since the leaves have been grown and pre-incubated in light, yet can produce from 2 to 9 times as much ethylene in the dark as in the light, it follows that the light inhibition is fully reversible. The inhibition by light is about equal to that exerted in the dark by CoCl2; it can be partly reversed by dithiothreitol and completely by mercaptoethanol. Thus the light is probably acting, via the photosynthetic system, on the SH group(s) of the enzyme system converting ACC to ethylene.

Relation between senescence and stomatal opening: Senescence in darkness.

THE SENESCENCE (PROTEOLYSIS AND LOSS OF CHLOROPHYLL) OF ISOLATED LEAVES OF OAT SEEDLINGS IN THE DARK IS INHIBITED OR DELAYED BY COMPOUNDS OF SIX DIFFERENT TYPES: phenazine methosulfate, fusicoccin, alpha,alpha'-dipyridyl, cycloheximide, spermidine, and two cytokinins. In every case but the last, these compounds in optimum concentration caused the stomata to open and remain partly or completely open throughout the 72- or 96-hr experimental period. The cytokinins caused only a partial opening, which is ascribed to their exerting two different effects. Taken together with the previous report that five different treatments that accelerated or promoted senescence in the light caused stomatal closure or occlusion, these data establish a general parallel between stomatal aperture and senescence, with strong indication that the stomatal aperture is the causal factor. A possible explanation of the relationship is proposed.

Relation between leaf senescence and stomatal closure: Senescence in light.

Senescence of isolated oat seedling leaves, floating on water or solutions in white light, has been followed by the disappearance of chlorophyll and the liberation of free amino nitrogen. In parallel, measurements of stomatal aperture were made with a diffusion resistance porometer, and borne out also by changes in fresh weight. The stomata open as expected in the light but slowly begin to close after the first day; correspondingly, in the dark they close at once but gradually begin to open on successive days. Abscisic acid causes closure and this is accompanied by senescence. Phenylmercuric nitrate also causes closure and again the reaction is closely paralleled by senescence. Kinetin maintains stomatal opening even more than does light alone, and this is accompanied by complete prevention of chlorophyll loss for at least 5 days. Covering the leaf surface with a film of Vaseline, especially when detergents are added, accelerates senescence. Merely floating the leaf segments on hypertonic solutions of sucrose or mannitol suffices to bring the rate of senescence in light up to the rate in darkness. It is concluded that the effect of light in delaying senescence is primarily due to its effect on the stomatal aperture, and, more generally, that stomatal aperture is the principal controlling agent in leaf senescence.

Proteases of Senescing Oat Leaves: II. Reaction to Substrates and Inhibitors.

Two proteases isolated from senescent oat (Avena sativa) leaves have been subjected to further study. One of these, an acid protease active at pH 4.2, is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by iodoacetamide (IAc). The other, active at pH 6.6, is inhibited by both PMSF and IAc. These results, together with previously reported evidence that mercaptoethanol stimulates the activity of only the neutral protease, are taken to indicate that the acid protease is probably of the serine type, whereas the neutral enzyme is of the sulfhydryl type. Both enzymes are inhibited by irradiation in the presence of rose bengal, a selective histidine modification reagent. The acid protease was completely unaffected by chelators, but data on the neutral protease were equivocal.All protein substrates tested were attacked by both enzymes, though at strikingly different rates. Characterization of the digestion products, with denatured hemoglobin as substrate, indicated that the acidic enzyme is an endoprotease, while the neutral one is an exoprotease. Evidence is presented that these proteases undergo autolysis in vitro.

Proteases of senescing oat leaves: I. Purification and general properties.

Two proteases active in the senescing first leaves of oat seedlings (Avena sativa cv. Victory) have been purified approximately 500-fold by a combination of ammonium sulfate precipitation, affinity chromatography on hemoglobin-Sepharose, and ion exchange chromatography on DEAE-Sephadex. The enzymes show pH optima of 4.2 and 6.6 with denatured hemoglobin as substrate, and the molecular weights of both are about 76,000. Their optimum temperatures are close to 50 C. Small amounts of a third enzyme, active at pH 3.5, may also be present. The enzyme active at pH 6.6 shows evidence of a sulfhydryl residue in the active site.

Metabolism of Oat Leaves during Senescence: V. Senescence in Light.

A comparison has been made of the progress of senescence in the first leaf of 7-day-old oat plants (Avena sativa cv. Victory) in darkness and in white light. Light delays the senescence, and intensities not over 100 to 200 ft-c (1000-2000 lux) suffice for the maximum effect. In such intensities, chlorophyll loss and amino acid liberation still go on in detached leaves at one-third to one-half the rate observed in darkness; however, when the leaves are attached to the plant, the loss of chlorophyll in 5 days is barely detectable. Transfer of the leaves from 1 or 2 days in the low intensity light to darkness, or vice versa, shows no carryover of the effects of the preceding exposure, so that such treatment affords no evidence for the photoproduction of a stable substance, such as cytokinin, inhibiting senescence. Light causes a large increase in invertaselabile sugar and a smaller increase in glucose, and application of 100 to 300 mm glucose or sucrose in the dark maintains the chlorophyll, at least partially. Correspondingly, short exposure to high light intensity, which increased the sugar content, had a moderate effect in maintaining the chlorophyll. However, 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) completely prevents the increases in sugars and yet does not prevent the effect of light on senescence, whether determined by chlorophyll loss or by protein hydrolysis. Light causes a 300% increase in the respiration of detached oat leaves, and kinetin lowers that only partly, but unlike the increased respiration associated with senescence in the dark, the increase in the light is fully sensitive to dinitrophenol, and therefore cannot be ascribed to respiratory uncoupling. The increased respiration in light is prevented by DCMU, parallel with the prevention of sugar formation. It is therefore ascribed to the accumulation of soluble sugars, acting as respirable substrate. Also, l-serine does not antagonize the light effect. For all of these reasons, it is concluded that the action of light is not mediated by photosynthetic sugar formation, nor by photoproduction of a cytokinin. Instead, we propose that light exerts its effect by photoproduction of ATP. The action of sugars is ascribed to the same mechanism but by way of respiratory ATP. This hypothesis unifies most of the observed phenomena of the senescence process in oat leaves, and helps to explain some of the divergent findings of earlier workers.

The retention of photosynthetic activity by senescing chloroplasts of oat leaves.

The retention of photosystems I and II and or RuDP carboxylase activity in chloroplasts isolated from the first leaves of Victory oat (Avena sativa L.) seedlings was followed as the chloroplasts senesced in darkness. Both photosystems (PS) I and II retained their full activity after 3 days at 1°C, while even after 7 days at 1°C around 80% of the activity was still present. After 3 days at 25°C, PS I lost only 20% and PS II 50% of the initial activity. Acid pH increased the rate of decay of both systems, PS II falling almost to zero after 3 days at pH 3.5 (at 25°C). The preparations were almost bacteria-free, and addition of antibiotics not only did not improve their stability, but accelerated the rates of loss of photosynthetic activity. This is held to indicate that the enzymes are undergoing some turnover even in isolated chloroplasts. If the leaves were allowed to senesce in the dark first and the chloroplasts then isolated, their photosynthetic activities had greatly decreased, showing that senescence is more rapid in situ than in isolation. Under these conditions PS I decayed more rapidly than PS II. Ribulosediphosphate carboxylase, as measured by CO2 fixation, declined more rapidly than the photosystems, though the addition of kinetin and indole-3-acetic acid somewhat decreased the rate of loss, at least for the first 24 h. When the intact (detached) leaves were held in the dark, the rate of oxygen evolution declined rapidly, but in monochromatic blue light (450 nm) at 25°C about 30% of the initial rate was retained after 72 h.

The Metabolism of Oat Leaves during Senescence: IV. The Effects of alphaalpha'-Dipyridyl and other Metal Chelators on Senescence.

The senescence of the first leaves of light-grown Avena seedlings when detached and placed in the dark is inhibited by alpha, alpha'-dipyridyl and alpha, alpha', alpha''-tripyridyl at concentrations between 10(-5) and 10(-4) M. Five other chelating agents exert similar inhibiting effects at concentrations 3 to 30 times higher. The senescence of etiolated leaves, as shown by loss of carotenoid and protein, is similarly inhibited. Ethylene-diaminetetraacetate has a similar effect in the dark, though only at 10 mM and above, but in the light it causes bleaching of chlorophyll. It is deduced that an iron-containing system plays an essential part in the initiation of the senescence process.