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.     

Accumulation and remobilization of amino acids during senescence of detached and attached leaves: in planta analysis of tryptophan levels by recombinant luminescent bacteria

The process of leaf senescence is biochemically characterized by the transition from nutrient assimilation to nutrient remobilization. The nutrient drain by developing vegetative and reproductive structures has been implicated in senescence induction. The steady-state levels of amino acids in senescing leaves are dependent on the rate of their release during protein degradation and on the rate of efflux into growing structures. To determine the possible regulatory role of amino acid content in leaf senescence, an in planta non-destructive, semi-quantitative method for the analysis of endogenous levels of free amino acids has been developed. The method is based on in vivo bioluminescence of amino acid-requiring strains of recombinant Escherichia coli carrying the lux gene. The luminescence signal was found to be proportional to the levels of added exogenous tryptophan and to the free amino acid levels in the plant tissues analysed. During the senescence of tobacco flowers and of detached leaves of oats and Arabidopsis, a progressive increase in the levels of free amino acids was monitored. By contrast to the detached leaves, the attached oat leaves displayed a decrease in the levels of free amino acids during senescence. In Arabidopsis, both the attached and detached leaves exhibited a similar pattern of gradual increase in amino acid content during senescence. The differences between the sink-source balance of the two species and the possible relationships between amino acid content and leaf senescence are discussed.     

Effects of Cycloheximide and Light on Leaf Senescence in Maize and Hydrangea

The senescence of maize and hydrangea leaves after detachmentand darkening was studied in terms of the loss of chlorophylland protein. Chlorophyll contents of the detached leaves decreasedin the dark in both plants. Cycloheximide at 0.1 mM effectivelyinhibited the loss of chlorophyll in maize, but did not do soin hydrangea. Continuous irradiation with white light of 4.6Wm^–2 prevented the loss of chlorophyll in hydrangea leaves,while it caused bleaching of maize leaves. Reducing agents suchas ascorbic acid and glutathione did not prevent the bleachingby light. In maize leaves, the amount of protein decreased inthe dark more slowly than that of chlorophyll, and cycloheximideslightly prevented the protein decrease. Continuous light irradiationof 4.6 Wm^–2 delayed the loss of protein more effectivelythan cycloheximide did. (Received January 31, 1981; Accepted May 21, 1981)     

Light-induced h secretion and the relation to senescence of oat leaves

When abraded oat (Avena sativa L. cv Victory) leaf segments are floated on KCl solution, white light causes acidification of the solution external to leaf tissue. The presence of mannitol amplifies the light-induced proton secretion. Mature leaves as well as young ones acidify the medium in light, while senescing leaves (after 3 to 4 days incubated in water in the dark) lose the ability to produce this response to light. The decrease in H⁺ secretion is already measureable after as little as 30 minutes in darkness, while the increase in proteolysis rate was detected only after 6 hours in dark. The decrease in capacity to secrete protons is one of the symptoms of leaf senescence. Moreover, fusicoccin mimics light in stimulating H⁺ pumping and delaying the senescence in the dark. On the other hand vanadate, an apparent inhibitor of plasma membrane H⁺ ATPase, blocks the acidification and promotes the chlorophyll and protein degradation in leaf segments during the 2-day period of incubation. These results, which show a parallel between cessation of H⁺ secretion and acceleration of senescence, may suggest a regulatory role for H⁺ secretion in leaf senescence.     

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 oat leaves: Changes in translatable mRNAs

Changes in translatable mRNA populations during the senescence of oat (Avena sativa L. cv. Victory) leaves were examined by analyzing the in vitro translation products of isolated RNA. Total RNA was isolated from oat leaves of 7-day-old seedlings, and also after these leaves were aged for different lengths of time under various conditions. Polypeptides from in vitro translations were separated by two-dimensional gel electrophoresis to estimate any changes in translatable mRNA populations associated with senescence. Corresponding leaf samples were monitored for loss of chlorophyll as a measure of the extent of senescence. The aging of excised leaves in the light for 4 days resulted in the disappearance or substantial quantitative decrease of a number of mRNA species, while only five new translatable mRNA species were produced. Three of these mRNAs were unique to aging of leaves under light. Two of these mRNA species were also produced during the early stages of senescence in attached leaves of seedlings grown under light. The translatable mRNA populations of leaves aged for 4 days either on intact seedlings or detached and kept in the light in the presence of kinetin were very similar. Aging of excised leaves in the dark on water for 24 h resulted in very extensive changes in translatable mRNA populations. Over thirty polypeptides disappeared or were substantially reduced in quantity, while about an equal number appeared de novo or were substantially increased in quantity. Aging of these leaves for an additional 24 or 48 h resulted in only a few additional changes in translatable mRNAs. The presence of kinetin during aging of excised leaves in the dark inhibited few of the numerous changes in mRNAs that occured during the first 24 h, but did inhibit most of the changes that occured after 48 or 72 h of aging in the dark. When leaves were first aged in the dark and then returned to light, most of the initial changes in translatable mRNAs expression were reversed. Such changes in mRNAs thus appear to be light-regulated and not necessarily associated with 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 ¹⁴C-sugars and ¹⁴CO₂. In the absence of CO₂, serine does cause chlorophyll loss in light and undergoes a decreased conversion to sugar.     

Senescence-promoting effect of arabidopside A

Arabidopside A isolated from Arabidopsis thaliana is a rare oxylipin, containing 12-oxophytodienoic acid (OPDA) and dinor-oxophytodienoic acid (dn-OPDA) which are known as precursors of jasmonic acid (JA) and methyl jasmonate (MeJA). The senescence-promoting effect of arabidopside A was examined by an oat (Avena sativa) leaf assay under dark or continuous light condition. Arabidopside A promoted senescence of oat leaves, and the promoting activity was more effective than for JA and OPDA, and as strong as for MeJA, which was well known to be a senescence promoter. These results suggest that arabidopside A plays important roles in leaf senescence.     

The physiological significance of the leaf nodules of psychotria

Summary In 6-month growth experiments it was found that leaf-nodulatedPsychotria mucronata seedlings grown in N-poor soil showed a restricted growth and developed severe nitrogen-deficiency symptoms in the leaves. Plants in the same soil supplied with NO₃-N showed healthy growth and dark green leaves. Detached Psychotria leaves bearing leaf nodules exposed to an atmosphere containing N¹⁵-labelled nitrogen gas or acetylene gas gave no evidence of nitrogen fixation, either in the light or in the dark or in both in succession. Therefore nitrogen fixation is probably not associated with the leaf nodules. Chlorophyll retention was observed around the leaf nodules in senescent Psychotria leaves. Psychotria leaf-nodule discs placed on oat leaves cause chlorophyll retention in the oat leaves below the discs. As chlorophyll retention is a common bioassay for cytokinins, these results indicate that a cytokinin-like substance is involved. With the aid of autoradiography and C¹⁴-labelled α-amino-isobutyric acid it was shown that this amino acid accumulates in the leaf nodules. Such directed transport is also a property of cytokinin.     

Glyoxylate transamination in intact leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes were supplied with glycolate and one to three of the amino acids serine, glutamate, and alanine, and the amount of the respective α-keto acids formed in glyoxylate transamination was assayed. At 1 millimolar glycolate and 1 millimolar each of the three amino acids in combination, the transamination reaction reached saturation; reduction of either glycolate or amino acid concentration decreased the activity. The relative serine, glutamate, and alanine transamination at equal amino acid concentrations was roughly 40, 30, and 30%, respectively. The three amino acids exhibited mutual inhibition to one another in transamination due to the competition for the supply of glyoxylate. In addition to this competition for glyoxylate, competitive inhibition at the active site of enzymes occurred between glutamate and alanine, but not between serine and glutamate or alanine. Alteration of the relative concentrations of the three amino acids changed their relative transamination. Similar work was performed with intact oat (Avena sativa L.) leaf peroxisomes. At 1 millimolar of each of the three amino acids in combination, the relative serine, glutamate, and alanine transamination was roughly 60, 23, and 17%, respectively. Similarly, alteration of the relative concentration of the three amino acids changed their relative transamination. The contents of the three amino acids in leaf extracts were analyzed, and the relative contribution of the three amino acids in glycine production in photorespiration was assessed and discussed.     

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.     

Leaf pretreatment with senescence retardants as a basis for oat protoplast improvement

Protoplasts obtained from oat leaves floated on buffer for 18hr show high nuclease activity, low rates of incorporation ofamino acids and nucleosides into macromolecules, and high ratesof spontaneous lysis. Addition to the leaf flotation mediumof the senescence retardants cycloheximide or kinetin, of thedibasic amino acids L-lysine or L-arginine, or of the diaminesputrescine or cadaverine reduces the rise in nuclease activityand spontaneous lysis of protoplasts, and increases the rateor extent of presumptive protein and nucleic acid synthesis.The diamines, which also retard chlorophyll degradation in theexcised leaves, appear to act both on the membrane and on systemscontrolling macromolecular synthesis and breakdown. By contrast,the senescence promoter L-serine hastens chlorophyll degradationfrom excised leaves and does not improve protoplasts derivedfrom those leaves. (Received July 4, 1977; )     

The exudation of solutes during senescence of oat leaves

Exudation of cations and of amino acids from detached oat seedling leaves ( Avena sativa L. cv. Victory) floated on water or solutions was measured under varied conditions. A small amount of exudation in the first 4 h, greater in the dark than in white light, is followed, one to 8 days later, by a relatively great increase which approximately accompanies senescence. This second increase is delayed by kinetin and is accelerated by abscisic acid or methyl jasmonate. both of which accelerate senescence. A nitrogen atmosphere (in darkness) causes immediate large exudation, mainly of potassium ions, and this is effectively delayed by light. There are two exceptions to the parallel between exudation and senescence; n-pentanol, which strongly delays senescence in darkness, nevertheless increases exudation both of potassium and of amino acids, probably due to a direct effect on the plasmalemma. Cycloheximide, which also delays senescence, increases the exudation somewhat. Kinetin prevents or delays exudation under all conditions. Thus, the permeability of the plasmalemma increases greatly along with other criteria of senescence, but this change is probably not the principal cause of the senescence syndrome. Some of the effects are considered to result from reduction in available energy and others from a direct influence on plasmalemma permeability.     

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

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

Amino Acid-Sugar Linkages in Rice Coleoptile Cell Walls

The nature of amino acid-sugar linkages in cell walls was investigatedin a monocotyledonous tissue, rice coleoptiles. The molar ratiosof aspartic acid, threonine, and serine in cell walls were decreasedby hydrazinolysis in coleoptiles grown both on and under water.The molar ratios of threonine and serine were decreased alsoby a NaOHNaBH₄ treatment, while the alanine content was increased,and -aminobutyric acid was not formed. The cell walls were treated with NaOH in the presence of NaB³H₄,hydrolyzed, then divided into amino acid and sugar fractions.Two distinct radioactive peaks were detected in the thin-layerchromatography of the amino acid fractions. One was identifiedas alanine derived from glycosylated serine; the other was confirmedto be an oxidation product of glucosaminitol. There was justone ³H-labeled product in the sugar fractions, galactitol. Theseresults suggest the presence of serine-O-galactose and asparagine-N-N-acetylglucosamine linkages in rice coleoptile cell walls. The existence of glucosamine linked to amino acids was furthersupported by the incorporation of ¹⁴C-glucosamine into cellwalls. These linkages were also detected in the cell walls ofa dicotyledonous tissue, Vicia epicotyls. (Received April 2, 1981; Accepted June 24, 1981)     

The Effects of Leaf Age and Senescence on the Distribution of Carbon in Lolium temulentum

Assimilate distribution in leaves of Lolium temulentum was establishedby root absorption of [¹⁴C]sucrose and after exposure to ¹⁴CO₂.Age determined the amount of carbon assimilated, with more labelbeing incorporated during expansion than at maturity. Duringsenescence ¹⁴C assimilation was much lower. Ethanol-solubleextracts from various tissues of root-labelled plants containedmost of the radioactivity chiefly in basic and acidic compounds.The neutral fraction was composed predominantly of sucrose. Sucrose was comparably labelled in leaves from plants fed equalamounts of either [¹⁴C]sucrose, glucose, or fructose and onlytraces of labelled monosaccharides appeared in extracts. Radioactive sucrose was translocated rapidly from mature leaveswhereas, in the expanding leaf, carbon incorporation was directedtowards growth and the greater proportion of label present atligule formation was in ethanol-insoluble material. Induced senescence, of a mature leaf fed during expansion, produceda rapid loss from the pool of insoluble ¹⁴C. This was accompaniedby a reduction in the contents of chlorophyll and soluble proteinand an accumulation of amino acids. The onset of senescencecaused changes in leaf sugar levels which were correlated withincreased rates of respiration.     

Endopeptidase activity and nitrogen mobilization in senescing leaves of Nicotiana rustica in light and dark

The time course of endopeptidase activity (digestion of azocasein at pH 4.6) in leaves of intact plants of Nicotiana rustica L. was studied and related to changes in the contents of chlorophyll, total nitrogen and soluble and insoluble protein nitrogen. Endopeptidase activity increased several fold during senescence. However, the course of protein degradation did not reflect the steep slope of azocaseolytic activity. When single mature leaves were darkened, senescence proceeded faster than in illuminated leaves but the amount of nitrogen mobilized and translocated did not differ greatly between darkened and illuminated leaves. However, in contrast to leaves in light, azocaseolytic activity did not increase.
Gelatin zymograms obtained using isoelectric focusing of extracts of mature leaves showed several bands in the pH 4.0 to 6.5 region of the gels. During senescence in both light and dark the position and number of bands remained largely unchanged. In leaves in light, the activity of endopeptidases focusing in the range pH 4.1 to 5.0 increased greatly. In leaves in dark, however, no major changes in activity could be detected. The results suggest that in tobacco leaves endopeptidase activity normally increases considerably during senescence but this increase is not a prerequisite for an effective protein degradation.
Separation and analysis of free amino acids showed that during senescence in light the levels of all amino acids decreased considerably. In leaves senescing in the dark there were large increases in the levels of glutamine and asparagine, concomitant decreases in glutamate and aspartate, and considerable increases in all other amino acids.     

Nucleic Acid Metabolism in the Developing Primary Leaf and Senescing Coleoptile of Germinating Oats

Nucleic acid metabolism in the coleoptile and primary leaf tissues of the germinating oat (Avena sativa L.) seedling was studied. The concentrations of the different species of nucleic acid present at various stages of development were determined and the amounts of each compared. All species of nucleic acid in the coleaptile increased as the tissue elongated; but, with the onset of senescence all species decreased, especially rRNA. Exposing dark grown coleoptiles to light did not modify their capacity to synthesize nucleic acids. In the rapidly developing leaf, all species of nucleic. acid increased throughout early germination. A general enhancement in the synthesis of all species of nucleic acids resulted when dark-grown-leaves were exposed to light. Furthermore, the tRNA/DNA ratio remained constant in both tissues during development, whereas the rRNA/DNA ratio changed.     

Changes in metabolism of cell wall polysaccharides in oat leaves during senescence: relevance to the senescence-promoting effect of methyl jasmonate

Changes in cell wall polysaccharides in oat (Avena sativa L.) leaf segments during senescence promoted by methyl jasmonate (JA-Me) were studied. During the incubation with water at 25 °C in the dark, the loss of chlorophyll of the segments excised from the primary leaves of 8-day-old green seedlings was found dramatically just after leaf excision, and leaf color completely turned to yellow after the 3- to 4-day incubation in the dark. Application of 10 µM JA-Me substantially promoted the loss of chlorophyll corresponding with the chloroplast degradation. Cell wall polysaccharides in oat leaf segments mainly consisted of hemicellulosic and cellulosic ones. During the process of leaf senescence, the amount of hemicellulosic I and II, and cellulosic polysaccharides decreased, but little in pectic polysaccharides. JA-Me significantly enhanced the decrease in cellulosic polysaccharides, but little in hemicellulosic ones. Arabinose, xylose and glucose were identified as main constituents of neutral sugars of hemicellulosic polysaccharides. The neutral sugar compositions of hemicellulosic polysaccharides changed little during leaf senescence both in the presence or absence of JA-Me. These facts suggest that JA-Me affects sugar metabolism relating to cellulosic polysaccharides during leaf senescence.     

EFFECTS OF CHLORAMPHENICOL AND KINETIN ON UPTAKE AND INCORPORATION OF AMINO ACIDS BY TOBACCO LEAF DISKS

The effects of chloramphenicol and kinetin on uptake and incorporationof ³⁵S-methionine and some ₁₄C-amino acids have been investigatedin leaf-disks of Nicotiana rustica in light and dark. Chloramphenicolin a concentration of 1 mg per ml inhibits the uptake of aminoacids from 30 to 60 per cent compared with the water control.The incorporation of amino acids into bulk protein is stronglyinhibited in light (40 to 70 per cent), but only to a smalldegree in dark (10 to 20 per cent), as revealed also by ¹⁴CO₂-photosynthesisof the disks and following treatment with chloramphenicol indark. The stimulating effect of kinetin on uptake and incorporationof amino acids is dependent upon its concentration (10^–5to 10^–6 M ; but 10^–4 M solution inhibits stronglyboth uptake and incorporation). The stimulation seems to influencemore incorporation than uptake processes. Possible interactionsof chloramphenicol and kinetin in the protein metabolism oftobacco leaves have been discussed. (Received April 27, 1964; )