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.     

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.     

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.     

Effects of Oxidative Stress Caused by Oxygen and Hydrogen Peroxide on Energy Metabolism and Senescence in Oat Leaves

In attached oat leaves the levels of adenine nucleotides decreasedduring leaf development and senescence. However, the energycharge (EC) only decreased from 0.90 in 4-cm leaves to 0.80in senescent leaves. In detached leaves the levels of adeninenucleotides increased for 48 h, in association with an increasein RNase activity and a decrease in levels of RNA. The EC remainedhigh until late senescence when levels of adenine nucleotidesfell to about 30% of initial values. A decrease in energy parametersinduced by transfer from light to darkness and from high (21%)to low (0.5% and anoxia) concentrations of oxygen resulted inan increase in membrane permeability. Oxidative stress (above 0.5% O₂ induced an increase in levelsof malondialdehyde (MDA) and then in permeability, associatedwith a decrease in levels of adenine nucleotides. Oxidativestress provoked by 0.05 and 0.10 M H₂O₂ caused a more rapiddecrease in energy parameters than O₂. Under oxidative stress(above 0.5% O₂) there is, first of all, an increase in membranepermeability and then a decrease in energy parameters, whichin turn are involved with senescence via increases in oxidationof membranes and degradation of energy-producing systems. (Received October 6, 1987; Accepted October 19, 1988)     

The Metabolism of Oat Leaves during Senescence: II. Senescence in Leaves Attached to the Plant

The course of senescence in the first leaves of light-grown Avena seedlings when attached to the plant has been compared with that previously studied in detached leaves and leaf segments. Proteolysis in the leaf, whether attached or detached, is accompanied by markedly polar basipetal transport of amino acids. This polar transport can be superimposed on the known transport of amino acids towards a locally applied cytokinin. In the intact plant, it results in a strong movement into the roots. The reducing sugars, which are set free in senescence, do not participate appreciably in this polar transport phenomenon.     

The Metabolism of Oat Leaves during Senescence: III. The Senescence of Isolated Chloroplasts

The changes in chlorophyll and protein in senescing chloroplasts isolated from the first leaves of 7-day-old oat (Avena sativa) seedlings have been investigated. In darkness the chlorophyll in these plastids is highly stable, losing only 5 to 10% of its content after 7 days at 26 C. This result contrasts with the behavior of chlorophyll in intact leaves, in which about 80% of the pigment would have disappeared in that time. The protein is less stable than the chlorophyll, though more stable than in the leaf; probably a small amount of protease is present in the plastids. Some protein is also being synthesized in the chloroplasts along with its breakdown; gains of up to 38% in protein and 13% in chlorophyll were observed under different conditions. l-Serine, which actively promotes senescence in the leaf, has only a very slight effect on the chloroplasts, and kinetin antagonizes it. Kinetin also has a small but significant effect in preserving the protein from breakdown. Acid pH somewhat promotes the breakdown, both of chlorophyll and protein. A loss of chlorophyll and protein comparable to that occurring in the senescence of the leaf could not be induced in the chloroplasts by suspending them in malate, in cytoplasmic extract, or in any of a number of enzymes tested alone. Incubation with a mixture of four enzymes was the only treatment which approximated the senescent process in the leaf, causing 34% loss of chlorophyll at pH 5 and 40% loss of protein at pH 7.4, both in 72 hours.In white light, the chlorophyll and the carotenoids, but not the protein, disappear rapidly. This disappearance was shown to be prevented in an atmosphere of nitrogen or in air by a number of reducing agents, of which ascorbic acid was the most effective. It is, therefore, ascribed to photooxidation rather than to normal senescence.     

燕麦叶片衰老与活性氧代谢的关系

燕麦连体叶片与高体叶片衰老中,过氧化氢酶和超氧物歧化酶(SOD)活性下降,脂类过氧化产物丙二醛(MDA)迅速积累,组织自动氧化速率显著加快。植物激素BA,GA_3,2,4—D及光、亚胺环己酮(CH),EDTA处理均不同程度地延缓离体叶片的衰老过程,同时抑制过氧化氢酶和SOD活性下降,阻止MDA的积累和组织自动氧化速率的提高.推测叶片衰老中活性氧起着重要的作用。     

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 ¹⁴C-sugars and ¹⁴CO₂. In the absence of CO₂, serine does cause chlorophyll loss in light and undergoes a decreased conversion to sugar.     

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.     

Membrane Permeability--Regulation by Exogenous Sugars during Senescence of Oat Leaf in Light and Darkness

The effect of sugars (sucrose, glucose and fructose) on normalphysiological changes during senescence of foliar segments ofAvena sativa cv. Suregrain was studied. In general applicationof sugars raised tissue permeability both in the light and indarkness. This change was associated with increases in endogenoussugars, hydroperoxide content and lipoxygenase activity. Inthe light it was also associated with low catalase activity.Sugars did not influence superoxide dismutase activity. In thelight, sugars accelerated senescence, measured as decreasesin chlorophyll and increases in soluble amino acids. In darknesssugars delayed senescence. The effect of sugars in the lightseemed to result from an increase in photo-oxidations associatedwith the increase in permeability. The delaying effect on senescence,found in darkness, seemed to result from an increase in respiratoryactivity plus the lack of (or combined with the lack of) photo-oxidations. (Received March 18, 1985; Accepted June 3, 1986)     

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 α, α′-dipyridyl and α, α′, α″-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.     

Regulation of Phospholipase D Activity by Light and Phytohormones in Oat Seedlings

The effect of synthetic analogs of phytohormones and red light absorbed by phytochrome on the phospholipase D activity (PLD) was studied in oat (Avena sativa L.) seedlings. ABA manifested a short-term stimulating effect on PLD activity in the green seedlings and inhibited phospholipase activity in the etiolated plants. Kinetin inhibited enzyme activity in the etiolated seedlings and did not affect its activity in light. GA did not markedly affect PLD activity in the etiolated plants and activated this enzyme in the green seedlings. Finally, IAA did not affect the enzyme activity. The relationship of the regulatory effects of phytohormones and light on PLD activity is discussed.     

Senescence in Detached Oat Leaves I. Changes in Free Amino Acid Levels

Changes in the levels of free amino acids have been measuredduring light and dark senescence of oat leaf segments. Leaveswere aged either on water, 5 ppm kinetin or 30 ppm abscisicacid. The patterns with which levels of individual amino acidschange differ a great deal in leaves senescing either in darkor light, signifying that different mechanisms may regulateoat leaf senescence in light and dark. Levels of serine andmost of the other amino acids that increase substantially duringdark senescence of oat leaves change parallel to mitochondrialrespiration. Kinetin depresses the rise in amino acids justas it does with respiration in the dark. The synthesis of serineproteases does not seem to be limited by the availability ofendogenous serine. The levels of glutamine increase dramaticallyin leaves kept in light (ca. 2,200% of initial value within7 days) but only a little in the dark, which may reflect a possiblerole of photorespiration during the senescence of oat leavesin the light. Abscisic acid enhances the release of amino acidsmore strongly in the light than dark. The senescence promotingeffect of abscisic acid in the light seems to bring about changesin amino acid levels similar to those that appear in leavessenescing on water in the dark. ¹ Present address: C.F. Kettering Research Laboratory, 150 EastSouth College Street, Yellow Springs, Ohio 45387, U.S.A. (Received June 24, 1981; Accepted October 30, 1981)     

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.     

Senescence in Detached Oat Leaves II. Identification and Changes in the Activity Levels of Aminopeptidases

Five aminopeptidases have been identified in the leaves of 7-dayold oat seedlings. Three aminopeptidases were able to hydrolyseL-Met ßNA, and two were active towards L-Arg ßNA.Only one of the enzymes (AP1) could cleave a variety of aminoacid residues. The enzymes active toward L-Met ßNA,L-Arg ßNA and L-Ala ßNA had pH optima 7.6,8.2 and 8.3 respectively. All the enzymes were fully activein the presence of various catalytic inhibitors tested, except1,10-phenanthroline which inhibited the activity of all theaminopeptidases, but only at a very high concentration (10mM).The activity levels of three types of aminopeptidases testedin the crude leaf extract decreased during the senescence ofoat leaves. ¹Present address: C. F. Kettering Research Laboratory, 150 EastSouth College Street, Yellow Springs, Ohio 45387, U.S.A. (Received November 4, 1982; Accepted June 1, 1983)     

Involvement of Oxidation, Proteolysis and Reductant Availability in the Regulation of in vivo Nitrate Reductase in Attached Oat Leaves during Growth and Senescence

Changes in nitrate reductase (NR) activity during growth andsenescence of attached oat leaves were studied, in order tounderstand the influence of certain regulatory mechanisms onthe in vivo activity at each stage of foliar development. Theenzyme activity was greatest in 7-day-old leaves. It decreasedin young, still-growing 11-day-old leaves, was stable with the15th day, and decreased again at the onset of leaf senescence.The first decrease in NR activity coincided with the beginningof leaf unfolding and with the increase in endogenous oxidations,and also with the maximal activity of neutral proteases. Thisdecrease was prevented when plants had been exposed to low O₂pressure. High O₂ pressure impaired the NR activity increaseoccurred from 5 to 7 days in the control (21% O₂). Cysteineimproved NR activity at each stage of leaf development. Thesecond decrease in NR (during senescence) did not occur whenNADH was supplied to the assay medium (in vitro test). These results suggest that during growth, NR activity is regulatedby oxidation of SH groups and by the activity of neutral proteases.During senescence, reductant availability may also contributeto the regulation of NR. (Received February 24, 1987; Accepted August 10, 1987)     

Senescence of Rice Leaves XXIV. Involvement of Calcium and Calmodulin in the Regulation of Senescence

Effects of compounds that influenced calcium uptake and calmodulininhibitors on the senescence of detached rice leaves were examined.Chelators, ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraaceticacid (EGTA) and l,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraaceticacid (BAPTA), significantly promoted senescence of detachedrice leaves in the dark and light. The effect of EGTA can bereversed by treating detached rice leaves with calcium. Verapamil,a calcium channel blocker, and lanthanum chloride, a calciumantagonist, promoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. Calcium ionophore A23187 [GenBank] and ruthenium red, believed to raise cytosolic level of Ca²⁺,were quite effective in retarding dark-induced and ABA-promotedsenescence of detached rice leaves. Calmodulin inhibitors, W-7,compound 48/80, chlorpromazine and trifluoperazine, significantlypromoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. It is concluded that cytosoliclevel of Ca²⁺ may regulate senescence of detached rice leavesthrough a calmodulin-dependent mechanism. (Received June 13, 1990; Accepted August 3, 1990)     

Regulation of Nitrate Reductase Activity by Nitrate during Light-Dark Transition in Detached Oat Leaves

In oat (Avena sativa L. cv. Suregrain) leaf segments, light-darkmodulation of nitrate reductase (NR) activity could be observedonly when segments were kept in –NO₃ conditions. We presenthere evidence that nitrate would regulate NR activity by modulatingthe phosphorylation status of the enzyme. (Received June 19, 1995; Accepted August 14, 1995)     

4PU-30对水稻叶片衰老与内源激素的调控

4PU－30能显著地延缓水稻叶片衰老。根据叶片衰老过程中内源激素含量的变化,可明确减缓水稻叶片衰老期间内源ABA含量的增加和内源ZRs、GAs和IAA含量的减少,使叶片中保持有较低水平的ABA与较高水平的ZRs、GAs和IAA,是4PU－30延缓水稻叶片衰老的主要调控机理。     

烤烟叶片衰老期氨气挥发特征及其生理调控研究

以烤烟品种K326为试验材料,利用氨气收集装置测定烟叶的氨气挥发量,并利用谷氨酰胺合成酶(GS)抑制剂(Glufosinate)处理叶片和质外体提取等方法,研究了叶片氨气挥发及其与氮代谢相关生理指标的关系。结果表明:(1)随着叶片的衰老,氨气挥发量在叶龄70d时最大(10.96μg.m-2.h-1),与衰老初期(叶龄40d)相比增加了2.15倍;质外体NH4+浓度和pH、氨气补偿点逐渐上升,GS和硝酸还原酶(NR)活性下降,谷氨酸脱氢酶(GDH)活性升高,可溶性蛋白和总氮降解,叶片NH4+浓度升高。(2)GS抑制剂处理后,叶片组织NH4+浓度和氨气补偿点升高,氨气挥发量增大,与对照相比差异显著。(3)氨气挥发量与质外体NH4+浓度、质外体pH和氨气补偿点呈极显著或显著正相关,与GS活性呈显著负相关,与GDH活性呈显著正相关,与叶片组织NH4+浓度等其他指标相关性不显著。研究认为,烤烟叶片衰老期间氨挥发量大幅上升,挥发量的大小受气孔氨气补偿点、GS和GDH活性的直接调控,以及其他氮素代谢相关指标的间接调控,其中GS起主导作用。