Differential Induction of Endoproteinases during Senescence of Attached and Detached Barley Leaves

Endoproteinase activities and species were compared during dark-induced senescence of attached and detached primary barley leaves by isoelectric focusing and polyacrylamide gel electrophoresis of cell-free extracts. Neither of the two major endoproteinases (EP1 and EP2) changed in amounts during senescence of attached leaves, nor did new endoproteinases appear. In contrast, during senescence of detached leaves, both EP1 and EP2 activities increased and four new species of endoproteinases appeared. Proteolytic activity was evenly distributed throughout attached leaves, but activity in the detached leaf increased sharply from the tip to the base with the four new higher molecular weight species of proteinases present only in the bottom half of the leaf nearest the cut end. Thus a wound response may be superimposed on the processes of senescence in detached leaves. Cycloheximide and kinetin both inhibited the increase of EP1, EP2, and the induction of the four new endoproteinases; chloramphenicol had no effect. Indications are that both the increases in activity and the induction of new species of proteinases were the result of activity of cytoplasmic ribosomes.

Hydrolysis of total protein and ribulose-1,5-bisphosphate carboxylase protein in vivo was somewhat faster in detached than attached leaves. The difference, however, was much less than would be expected from the great increase in proteolytic activity in detached leaves.

     

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.     

Hormonal Aspects of Senescence in Detached Tobacco Leaves

The objective of the present work was to describe the simultaneous changes in endogenous levels of cytokinins, abscisic acid, indoleacetic acid and ethylene in detached, senescing tobacco (Nicotiana rustica L.) leaves. These measurements were related to changes in chlorophyll contents, ¹⁴CO₂ fixation and proline contents — three parameters which have been considered to reflect senescence. Effects of exogenous hormonal treatments on these parameters, as well as on endogenous hormonal levels, provided further evidence for the interrelationships between hormones and for their roles in senescence. Starting with actively growing attached leaves and ending with well-advanced senescence in detached leaves, our data indicate a chronological sequence of three hormonal states: (a) cytokinins — high activity, abscisic acid, auxin and ethylene — low contents (actively growing, attached leaves); (b) cytokinins — low activity, abscisic acid — high, auxin and ethylene — low contents (apparent induction of senescence in detached leaves); and (c) cytokinins and abscisic acid — low, auxin and ethylene — high contents (senescence proper in detached leaves).     

Catalase, Peroxidase, and Polyphenoloxidase Activities during Rice Leaf Senescence

The activities of catalase, peroxidase, and polyphenoloxidase were studied in attached and detached rice (Oryza sativa L. cv. Ratna) leaves. Catalase activity decreased while peroxidase and polyphenoloxidase activities increased during senescence of both attached and detached rice leaves. Kinetic (5 mum) and benzimidazole (1 mm), which are known to delay the senescence of detached rice leaves, retarded the decrease of catalase activity during detached leaf senescence. On the other hand, these chemicals accelerated the increase of peroxidase and polyphenoloxidase activities over the water control. Total phenolics accumulated in detached and darkened rice leaves, but in attached leaf senescence in light no accumulation of phenolics was observed.     

The Control of Leaf Senescence in Kleinia articulata by Photoperiod

Experiments with attached and detached leaves of K. articulatahave shown that senescence rates are determined by the daylengthprevailing during early growth (leaf-expansion stage) and thatthis effect is lasting, long days leading to early leaf death.Daylength also affects longevity after full expansion has occurred,long days hastening senescence. Tests with numerous plant-growthregulators have revealed beneficial effects of gibberellic acid,while kinetin is detrimental to survival at 50 ppm. Entire detachedleaves and isolated petioles behave similarly to leaves on theplant, but leaf discs do not behave in the same way. Other parametersaffected by daylength include: leaf shape, the capacity of leavesto form roots, and enlargement of mesophyll cells normal tothe leaf surface.     

The Metabolism of Oat Leaves during Senescence: I. Respiration, Carbohydrate Metabolism, and the Action of Cytokinins

When the detached first leaves of green or etiolated oat (Avena sativa cv. Victory) seedlings senesce in the dark, their oxygen consumption shows a large increase, beginning after 24 hours and reaching a peak of up to 2.5 times the initial rate by the 3rd day. This effect takes place while the chlorophyll of green leaves, or the carotenoid of etiolated leaves, is steadily decreasing. Kinetin, at a concentration which inhibits the decrease in pigment, completely prevents the respiratory rise; instead, the oxygen consumption drifts downwards. Lower kinetin concentrations have a proportional effect, 50% reduction of respiration being given by about 0.1 mg/l. About one-fifth of the respiratory rise may be attributed to the free amino acids which are liberated during senescence; several amino acids are shown to cause increases of almost 50% in the oxygen consumption when supplied at the concentrations of total amino acid present during senescence. A smaller part of the rise may also be due to soluble sugars liberated during senescence, largely coming from the hydrolysis of a presumptive fructosan. The remainder, and the largest part, of the increase is ascribed to a natural uncoupling of respiration from phosphorylation. This is deduced from the fact that dinitrophenol causes a similar large rise in the oxygen consumption of the fresh leaves or of leaf segments kept green with kinetin, but causes only a very small rise when the oxygen consumption is near its peak in senescent controls. The respiration of these leaves is resistant to cyanide, and 10 mm KCN even increases it by some 30%; in contrast, etiolated leaves of the same age, which undergo a similar rise in oxygen consumption over the same time period, show normal sensitivity to cyanide. The respiratory quotient during senescence goes down as low as 0.7, both with and without kinetin, though it is somewhat increased by supplying sugars or amino acids; glucose or alanine at 0.3 m bring it up to 1.0 and 0.87, respectively.     

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.     

Stomatal Responses and the Senescence of Leaves

Guard cell responses were examined in green and senescing leavesof Victa faba using detached epidermal strips to eliminate influencesfrom the mesophyll. Stomatal opening was greater in epidermalstrips from mature leaves than from senescing leaves althoughthe latter still retained the ability to respond to CO₂ andto kinetin. It was concluded that the decline in stomatal activityduring senescence is an independent but parallel process tochanges occurring in the mesophyll. Vicia faba, leaf senescence, stomata, kinetin     

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)     

Effects of ABA on Senescence and Cellulase Activity of Detached Rice Leaves

The relationship of cellulase to detached leaf senescence of rice seedlings was investigated by examining the effect of ABA and 6-BA on changes in the level of cellulase of leaf segments during senescence. It was shown that the rise in cellulase activity increased with declining chlorophyll content, which was used as the senescence indicator during the senescence of detached rice leaves caused by ABA. The action of ABA took place only after a 48h lag period. ABA enhanced the cellulase secretion and increased the permeability of plasma membrane. A high level of cellulase activity in cell wall closely related to membrane permeability changes. The action of cellulase in the cell wall may cause depolymerization of β-1, 4-glucan in situ, thus speeding senescence. The 6-BA reverses completely or partly the increase in cellulase activity and tile permeability caused by ABA during the first two day, }) ut it antagonized hardly any of the ABA effect from the third day on, suggesting the onset of an irreversible stage in the senescence of detached rice leaves.     

Graft Transmission of Longday-induced Leaf Senescence in Kleinia articulata

Kulkarni, V. J. and Schwabe, W. W. 1985 Graft transmission oflongday-induced leaf senescence in Kleinia articulata.—J.exp. Bot. 36: 1620–1633. Senescence of attached and detached rooted leaves in Kleiniaarticulata can be regulated by daylength, accelerated by longphotoperiods LD (> 16 h) and retarded by short days SD (8h). Using detached, rooted leaves as stocks in leaf to leafgrafts, senescence was readily transmitted from a LD donor leafto a SD receptor leaf even if the receptor was retained in SD(D8).However, no transfer could be detected where it had to passthrough any stem tissue. Senescence was reversible up to a certain stage, beyond whichgrafts senesced, pointing to an accumulation of the graft transmissiblefactor to a threshold level that causes irreversible death. Key words: Kleinia, leaf senescence, senescence factor, daylength, graft transmission     

The Significance of Vascular Connection in Regulating Senescence of the Detached Flag Leaf of Wheat

The relationship between the accumulation of soluble substancesand the senescence of a detached flag leaf of wheat in the lightwas investigated. With respect to transport to and from theleaf, a detached flag leaf can be considered as either an ‘open’or a ‘closed’ system. A closed system was obtainedby cutting the flag leaf at the base of the leaf sheath, thussevering the vascular connection between the leaf and the stem.An open system was prepared by excising the wheat stem, belowthe flag leaf insertion node, thereby preserving vascular connectionsbetween the flag leaf, stem and ear. By varying the number ofnodes left on the stem, or having the ear either intact or detached,soluble carbohydrates and soluble nitrogenous compounds wereinduced to accumulate within the leaf blade at different rates.Treatments which restricted transport of carbohydrate and nitrogen,out of the detached leaf were found to promote senescence. Senescencewas most rapid when the leaf system was ‘closed’and was considerably delayed when the system was ‘open’and the ear intact. The onset of senescence was closely associatedwith the attainment of a threshold concentration of ethanol-solublecarbohydrate in the leaf, while the rate of senescence was modifiedby the number of attached nodes or by exogenous cytokinin treatments. Key words: Wheat, Flag leaf, Senescence     

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)     

Translocation and Metabolic Conversion of C-Labeled Assimilates in Detached and Attached Leaves of Phaseolus vulgaris L. in Different Phases of Leaf Expansion

Leaf excision greatly affected the actual levels of ¹⁴C-assimilates in laminas and petioles of primary bean leaves (Phaseolus vulgaris L.) following a transport period. However, it did not affect the percentage of starch in the insoluble residue; starch decreased from 20% of the insoluble residue after exposure to ¹⁴CO₂ to 3% after 5 hr in both attached and detached leaves. The transition from import to export of attached and detached leaves was at the same stage, i.e., when the cotyledons were 63 to 85% depleted. The composition of the ¹⁴C-assimilates in importing leaves was different from that in exporting leaves. In the former, only 5% of the soluble label was free sugar, while 74% was free sugar in the latter. The failure of importing leaves to export was not due to the labeled substances being nontransferable. Extracts from importing leaves applied to exporting leaves were exported; these extracts were high in amino acids and organic acids but low in free sugar. However, exporting leaves exposed to ¹⁴CO₂ appeared to export sugars more readily than amino acids. Cotyledon excision did not delay transition of leaves from import to export. Actually, excision seemed to enhance slightly the transition of the primary leaves from import to export.     

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。     

A Role for Glutamine Synthetase in the Remobilization of Leaf Nitrogen during Natural Senescence in Rice Leaves

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of corresponding mRNAs were determined in leaves of hydroponically grown rice (Oryza sativa) plants during natural senescence. The plants were grown in the greenhouse for 105 days at which time the thirteenth leaf was fully expanded. This was counted as zero time for senescence of the twelfth leaf. The twelfth leaf blade on the main stem was analyzed over a time period of −7 days (98 days after germination) to +42 days (147 days after germination). Total GS activity declined to less than a quarter of its initial level during the senescence for 35 days and this decline was mainly caused by a decrease in the amount of GS2 polypeptide. Immunoblotting analyses showed that contents of other chloroplastic enzymes, such as ribulose-1,5-bisphosphate carboxylase/oxygenase and Fd-glutamate synthase, declined in parallel with GS2. In contrast, the GS1 polypeptide remained constant throughout the senescence period. Translatable mRNA for GS1 increased about fourfold during the senescence for 35 days. During senescence, there was a marked decrease in content of glutamate (to about one-sixth of the zero time value); glutamate is the major form of free amino acid in rice leaves. Glutamine, the major transported amino acid, increased about threefold compared to the early phase of the harvest in the senescing rice leaf blades. These observations suggest that GS1 in senescing leaf blades is responsible for the synthesis of glutamine, which is then transferred to the growing tissues in rice plants.     

Effect of Oxygen Concentration on C-Photoassimilate Transport from Leaves of Salvia splendens L

Partitioning and transport of recently fixed photosynthate was examined following ¹⁴CO₂ pulse-labeling of intact, attached leaves of Salvia splendens L. maintained in an atmosphere of 300 microliters per liter CO₂ and 20, 210, or 500 milliliters per liter O₂. Under conditions of increasing O₂ (210, 500 milliliters per liter), a smaller percentage of the recently fixed ¹⁴C in the leaf was allocated to starch, whereas a greater percentage of the fixed ¹⁴C appeared in amino acids, particularly serine. The increase in ¹⁴C in amino acids was reflected in material exported from source leaves. A higher percentage of ¹⁴C in serine, glycine, and glutamate was recovered in petiole extracts when source leaves were maintained under elevated O₂ levels. Although pool sizes of these amino acids were increased in both the leaves and petioles with increasing photorespiratory activity, no significant changes in either ¹⁴C distribution or concentration of transport sugars (i.e. stachyose, sucrose, verbascose) were observed. The data indicate that, in addition to being recycled intracellularly into Calvin cycle intermediates, amino acids produced during photorespiration may also serve as transport metabolites, allowing the mobilization of both carbon and nitrogen from the leaf under conditions of limited photosynthesis.     

The Importance of Roots in Regulating the Senescence of Soybean Primary Leaves

The role of roots in regulating primary leaf senescence of 14-day-old soybean seedlings was investigated. Compared with intact seedlings, the senescence of primary leaves is accelerated by removal of the root system but delayed if apical bud and the first trifoliate leaf are removed. No difference in senescence was found between intact seedlings and seedlings without roots, apical bud, and first trifoliate leaf. Lateral roots seem to play a predominant role in regulating primary leaf senescence. However, neither root nodules nor primary root play any function in senescence. Results indicate that benzyladenine (BA) at optimal concentration (2 mg/1) completely replaces the roots to prevent the senescence of primary leaves, whereas gibberellic acid (GA) and abscisic acid (ABA) accelerate. The effect of indole-3-acetic acid (IAA) to replace roots in preventing senescence depends on the season the young seedlings are grown. Additional, though indirect, information of acropetal transport of ABA is provided. In conclusion, it seems that cytokinins in lateral roots play a predominant role in leaf senescence and the normal supply of root cytokinins is important in leaf metabolism.