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.     

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.     

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.     

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.     

Some new observations and interpretations on the vital staining of leaf epidermal tissue by neutral red

Summary The lower leaf epidermis from 5 plant species was stained with neutral red at 2 pH's (7.1 and 5.6) in the light and dark when the stomata were open or closed. At pH 5.6 no globule (= droplet) formation was observed in the guard cells whether the stomata were open or closed and cell walls possessed a high affinity for the stain. At pH 7.1 globules appeared in guard cells of open stomata, but not closed stomata, within 15 minutes. Anaerobic conditions prevented this globule formation. InZea mays, globules also appeared in subsidiary cells when the stomata were closed and in certain epidermal cells. Where globule formation did not occur increased diffuse staining of certain epidermal cells was considered to be the indication of cell integrity. In old leaf material very large numbers of dark blue globules appeared in epidermal cells ofCommelina diffusa, C. communis andSenecio odoris and this was associated with cell senescence.The staining characteristics were discussed in terms of cellular K⁺, Cl^–, tannin and flavonoid content and vacuolar pH.     

Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves : I. Inhibition of protease activity, ethylene production, and chlorophyll loss as related to polyamine content

Excision and dark incubation of oat (Avena sativa L., var. Victory) leaves cause a sharp increase in protease activity, which precedes Chl loss. Both these senescence processes are inhibited by exogenously applied 1,3-diaminopropane (Dap), which occurs naturally in leaf segments. The inhibition of protease activity is much greater in vivo than in vitro, suggesting inhibition of protease synthesis as well as protease action by Dap. Chl breakdown in leaves of radish and broccoli, which also senesce rapidly in the dark, is only slightly inhibited by DaP. These differences between cereal and dicotyledonous plants are correlated with the natural occurrence of Dap in cereals. In the light, Dap promotes, rather than retards, the loss of Chl in oat leaves. This resembles previously described effects of other polyamines. Addition of Mg²⁺ to the medium does not antagonize this effect. In the dark, the accumulated Dap also inhibits ethylene production and decreases titer of other polyamines. Addition of Ca²⁺ to the incubation medium containing Dap competitively reduces the effects of Dap. Thus, Dap, like other polyamines, seems to require an initial attachment to a membrane site shared with Ca²⁺ before exerting its antisenescence action.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

PHOTOCONTROL OF STOMATAL MOVEMENTS

1. Opening in light is a feature common to the majority of functional stomata, but the current argument is against the traditional view that light is the principal environmental promoter of opening, because stomata can open in the dark in response to CO₂ removal and/or temperature increase. In this review, evidence is provided that light is more efficient and effective than other physical factors in both producing and maintaining wide opening. However, light acts on stomata both directly and indirectly, in conjunction with changes in, for example, CO₂ balance, water regime and temperature of the leaf tissue. 2. Three general categories of light effects on stomata are recognized: (a) photosynthetic effects driven by metabolic processes, induced or enhanced by light, (b) hydrophotic effects mediating through light-induced changes in epidermal turgor, and (c) photothermal effects arising from light-dependent changes in leaf temperature. 3. Photosynthetic effects involve both CO₂ depletion, and starch mobilization, malate synthesis, H⁺ extrusion, and accumulation of K⁺ and C1^- in guard cells; these processes are triggered by light of different qualities: (a) Both blue and red light are involved in photosynthetic CO₂ fixation, utilizing energy from photosynthetic light reaction(s), which provides C precursors for synthesis of stornatal starch. (b) Blue light, but not red, enhances starch mobilization, PEP carboxylase activity and respiration. Accordingly, blue light is postulated to enhance hydrolysis of stornatal starch providing C₃ precursors for malate synthesis via PEP-fixation of endogenous CO₂; the active extrusion of H⁺, derived from malate, is coupled with K⁺ influx to guard cells. Malate and C1^- are competitive anions, for K⁺, and one begins to play a progressively more important role as the other becomes limiting; in intact leaves, however, malate plays a more decisive role. These processes are driven by the energy from blue-light-enhanced respiration. (c) Both photosynthetic fixation and PEP carboxylation act as CO₂ sensors, but the exact role of CO₂ in the stornatal mechanism has yet to be determined. 4. Hydrophotic and photothermal effects facilitate guard cell expansion by releasing epidermal pressure through enhanced evaporative water loss, and are, therefore, indirect effects of light; photothermal effects may also contribute to metabolic processes outlined in paragraph 3. 5. Stomatal closure in the dark accompanies starch synthesis, malate reduction, efflux of K⁺ and C1^- from guard cells, and accumulation of CO₂ in substomatal cavities. Malate may be converted to starch via C₂ compounds. Guard cells release K⁺ and C1- into apoplastic space, from which they are removed by neighbouring cells. The entry of K⁺ into neighbouring cells is supposed to be coupled with H⁺ extrusion. These processes are dependent on respiratory energy. 6. The differential abaxial and adaxial stomatal light responses are related to inherent metabolic differences between the two epidermes, but the biochemical basis is not known.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Chlorophyllase versus pheophytinase as candidates for chlorophyll dephytilation during senescence of broccoli

Degradation of chlorophylls during senescence is a highly regulated process which requires the concerted action of several enzymes. Traditionally, it has been stated that the dismantling process of the chlorophyll molecule begins with a dephytilation step, followed by Mg²⁺ removal and other breakdown reactions. Recently, new evidence suggests the possibility of a rearrangement in the first two steps of this process, occurring Mg²⁺ removal prior to the loss of the phytol side chain. With the purpose of approximating to the real sequential order of these reactions and to assess if dephytilation occurs on intact (catalyzed by chlorophyllase) or Mg-free (catalyzed by pheophytinase) chlorophyll, expression of both genes was analyzed in broccoli tissue during senescence. Samples of broccoli florets treated with plant hormones, such as cytokinin and ethylene were utilized, as to assess the effect of such compounds on the expression of these genes. Results showed that chlorophyllase expression did not correlate to typical expression patterns for genes related to senescence, since a decrease in expression during senescence was found for one of the two chlorophyllase genes analyzed, and the hormonal-treatment effects on gene expression did not match those observed on chlorophyll content for both chlorophyllase genes. Pheophytinase expression patterns, on the other hand, displayed an increase in the first 3 days of induced senescence, followed by lower expression values towards the end of the experiment. Samples subjected to postharvest treatments mostly showed an inhibition of pheophytinase expression, especially in samples in which degradation of chlorophylls had been delayed. These results suggest that pheophytinase expression correlates to the visual manifestation of postharvest treatments, supporting the possibility that this enzyme is responsible for the dephytilation step in chlorophyll breakdown.     

Effects of growth regulators and light on chloroplasts NAD(P)H dehydrogenase activities of senescent barley leaves

The activities NADH and NADPH dehydrogenases were measured with ferricyanide as electron-acceptor (NADH-FeCN-ox and NADPH-FeCN-ox, respectively) in mitochondria-free chloroplasts of barley leaf segments after receiving various treatments affecting senescence. NADPH-FeCN-ox declined during senescence in the dark, in a way similar to chlorophyll and Hill reaction, and increased when leaf segments were incubated at light. These results suggest that NADPH-FeCN-ox is related to some photosynthetic electron transporter activity (probably ferredoxin-NADP⁺ oxidoreductase). In contrast, NADH-FeCN-ox is notably stable during senescence in the dark and at light. This activity increased during incubation with kinetin or methyl-jasmonate (Me-JA) but decreased when leaf segments were treated with abscisic acid (ABA). The effects of the inhibitors of protein synthesis cycloheximide and chloramphenicol suggest that the changes of NAD(P)H dehydrogenase activities may depend on protein synthesis in chloroplasts. In senescent leaf, chloroplast NADH dehydrogenase might be a way to dissipate NADH produced in the degradation of excess carbon which is released from the degradation of amino acids.Abbreviations ABA abscisic acid - DCPIP 2,6-dichlorophenol-indo-phenol - DOC deoxycholate - Me-JA methyl jasmonate - NADH-FeCN-ox NADH ferricyanide oxidoreductase - NADPH-FeCN-ox NADPH ferricyanide oxidoreductase     

The Effect of Benzimidazole and Red Light on the Senescence of Detached Leaves of Oryza sativa cv. Ratna

Excised rice leaves (Oryza sativa L. cv. Ratna) werefloated on a 10^–3M solution of benzirnidazole under dark or continuous red light. Compared to the water control a degradation of chlorophyll, protein, RNA, DNA and a decrease in the activity of alkaline inorganic pyrophosphatase was delayed at the same time as an increase of α-amino nitrogen and the activity of acid inorganic pyrophosphatase occurred, Benzimidazole was more effective under red light than in the dark in retarding senescence. The possible role of inorganic pyrophosphatases is discussed with respect to biosynthesis during leaf senescence.     

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.     

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.     

Inhibition of light-induced stomatal opening and of guard-cell-protoplast swelling in Vicia faba L. by tentoxin,an inhibitor of photophosphorylation

The fungal phytotoxin tentoxin and its natural derivative dihydrotentoxin impair light-induced stomatal opening in epidermal strips of broad bean (Vicia faba L.) incubated in a potassium-rich medium. Swelling of guard-cell protoplasts (GCPs) of the same species is inhibited in the presence of both substances. Swollen GCPs shrink after tentoxin or dihydrotentoxin treatment and these effects cannot be fully compensated by the phytoeffector fusicoccin. A comparison with the potassium carrier valinomycin shows that tentoxin acts in a different manner, because it is effective in the light only, whereas valinomycin causes shrinkage of GCPs also in the dark. Determination of adenine nucleotides in GCPs indicates a reduced ATP content and an enhanced ADP level after addition of tentoxin. At the same time, tentoxintreated GCPs contain more NADPH and less NAD⁺ than the control (NADP⁺ and NADH content does not differ). The results presented are consistent with the hypothesis that tentoxin closes stomata as a consequence of its inhibitory action on photophosphorylation.Abbreviations FC fusicoccin - GCP guard-cell protoplast - KIDA potassium iminodiacetate     

Role of Protein Synthesis in the Senescence of Leaves: II. The Influence of Amino Acids on Senescence

When the first leaf of the oat (Avena sativa) seedling is detached and placed in the dark, yellowing and proteolysis take place rapidly. The earlier finding that d-serine promotes this process has led to a further study of the controlling roles of several amino acids. Since the action of serine was found to be more powerful in presence of kinetin than alone, the effects of other amino acids have been restudied in presence of kinetin. Cysteine emerges as a moderately strong promotor of senescence, with glycine and alanine having definite but weaker effects. The serine effect is antagonized by arginine, especially in presence of kinetin, and so is the cysteine effect. This is considered to indicate that these two amino acids act in the same way. The antagonism exerted by arginine is in turn antagonized by canavanine. The protease activities at two pH regions which increase in the oat leaf during senescence react to both p-chlorimercuri-phenylsulfonate and to phenylmethyl-sulfonyl fluoride, and thus may contain both SH and OH groups. The amounts of both these enzyme activities formed in the leaf during 3 days in the dark are increased over 50% by pretreatment with serine, and this increase is very largely prevented by arginine. The amounts of soluble proteins left in the leaf vary as expected in the opposite sense. It is deduced that control of the new formation of proteases plays an important part in senescence. A suggestion is made as to the mechanism of control of senescence in leaves.     

The interaction of hormonal and environmental factors in leaf senescence

The work concerns the senescence of isolated young leaves of oats (Avena sativa) floated on water or solutions. Senescence is rapid in darkness but slow in white light; the effect of light is not due to photosynthesis, but is paralleled by stomatal opening. Closure of the stomata by osmotic or chemical means makes senescence in light proceed as fast as in darkness, while opening the stomata in darkness by cytokinins, fusicoccin,etc., delays senescence to rates typical of light. The osmotic closure in light is mediated by abscisic acid, and since this also accumulates in darkness it appears as a major factor controlling senescence. Efflux of ions into the solution; indicating increased permeability, occurs almost in parallel with senescence. Senescence in light is accelerated by 1-aminocyclopropane-l-carboxylic acid (ACC) and inhibited by cobalt, silver or aminoethoxyvinyl glycine (AVG) which interfere with ethylene production or action; however, ethylene’s role is unclear because some reagents, including kinetin, that delay senescence, actually increase ethylene production. At the endogenous level, therefore, ethylene may not be a limiting factor. Finally, a new ethylene-generating system is described in which the dehydrogenation of linoleic acid is coupled through manganese to the oxidation of ACC; it is probably activein vivo.     

Effects of Exogenous 1,3-Diaminopropane and Spermidine on Senescence of Oat Leaves : II. Inhibition of Ethylene Biosynthesis and Possible Mode of Action

The effects of the polyamines spermidine and 1,3-diaminopropane on ethylene biosynthesis and chlorophyll (Chl) loss were studied in peeled leaves of oat (Avena sativa L., var. Victory) incubated in the dark. Peeling off the epidermal cells induces an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity, resulting in an enhanced ACC and ethylene formation. Both polyamines inhibit ethylene biosynthesis from methionine by inhibiting ACC synthase activity and, more effectively, the conversion of ACC to ethylene. They also inhibit Chl loss occurring between 24 and 48 h of dark incubation; but, as shown by inhibitor experiments, inhibition of Chl loss does not result from inhibition of ethylene formation. Ethylene production and Chl loss, both associated with senescence, require membrane integrity; thus, treatments which promote deterioration of membranes inhibit both processes. Ca²⁺ in the incubation medium competitively reduces the polyamine-mediated inhibition of ACC conversion and Chl loss. The data suggest that polyamines initially attach to membranes, thereby inducing changes which, in turn, lead to inhibition of ethylene biosynthesis and retardation of senescence.     

Light promotes an increase of cytokinin oxidase/dehydrogenase activity during senescence of barley leaf segments

Following a study of the relationship between cytokinin oxidase/dehydrogenase (CKX) and senescence in darkened barley leaf segments, we have now investigated the influence of light on the in vitro activity of CKX. Seedlings of Hordeum vulgare L. were grown for 8 d under a light/dark regime of 18 h white light and 6 h darkness. Then apical parts of 7 cm length were cut from the first foliage leaves and their bases were placed in water. In segments kept in the dark, the CKX activity measured by cleavage of N₆-(Δ₂-isopentenyl)adenine rose from 0.1 pkat (g FW)⁻¹ to 0.8 pkat (g initial FW)⁻¹ within the first 4 d of incubation. In contrast, in segments kept under the light/dark regime it reached a value of 8.6 pkat (g initial FW)⁻¹ over the same time period. The chlorophyll a content declined slightly slower during light/dark cycling than in darkness. In contrast to segments and isolated laminae, corresponding attached laminae exhibited less CKX activity after 2 d under light/dark conditions than after 2 d in the dark. The activity in attached laminae of first foliage leaves of plants growing in light/dark cycling increased strongly only when the plants were older than 4 weeks. In line with this, the CKX activity in attached laminae of flag leaves of barley growing in fields increased in a late developmental state. The senescence of darkened isolated laminae of Zea mays L. and Phragmites australis (Cav.) Trin. ex Steudel was associated with an enhancement of CKX activity too. Because in most cases a positive correlation between CKX activity and senescence was found, it is likely that the enzyme promotes senescence by destroying cytokinins, which help to keep Poaceae leaves green. Light may promote not only cytokinin degradation but also the formation of bioactive cytokinins in leaf segments.