Senescence of Oat Leaves - Plant Physiology

Plant Physiol. (1982) 70, 1592-1596 0032-0889/82/70/1592/05/$00.50/0

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 Received for publication April 5, 1982 and in revised form July 19, 1982

LIU-MEI SHIH,2 RAVINDAR KAUR-SAWHNEY, JURG FUHRER,3 SWAPNA SAMANTA, AND ARTHUR W. GALSTON

Department of Biology, Yale University, New Haven, Connecticut 06511 ABSTRACT 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 1,3diaminopropane (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. Chi 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 Chi in oat leaves. This resembles previously described effects of other polyamines. Addition of Mg{ 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 conta g Dap competitively reduces the effects of Dap. Thus, Dap, like other polyamins, seems to require an initial attachment to a membrane site shared with Ca2+ before exerting its andsenescence action.

Exogenous Dap, like Spd, inhibits senescence, possibly by preventing the increases in protease activity and ethylene production that precede Chl breakdown. Inhibition of senescence is possibly effected through an initial interaction between Dap and membranes.

MATERIALS AND METHODS Plant Material. The first leaf of 14-d-old seedlings ofoat (Avena sativa L., var. Victory) was used for most experiments. The seeds (Swedish Seed Company, Ltd., Svalov, Sweden) were grown in vermiculite in controlled growth rooms maintained at 24 ± 1 C and with a 16-h photoperiod of about 12,000 lux. Incubation of leaf segments. For measurements of protease activity, leaves were sterilized as described earlier (19). The lower epidermis was peeled off and 45-mm-long leaf segments were rinsed in distilled H20 and floated in Petri dishes, stripped side down, on 5 ml of 1 mm phosphate buffer (pH 5.7). All manipulations were performed aseptically in a laminar flow hood. For measurements of ethylene production, leaf segments were similarly handled and floated on solutions in bottles sealed with rubber serum caps. For estimating senescence by measurements of residual Chl content, the following materials were used: leaf segments from oats, barley (Hordeum vulgare cv Himalaya), corn (Zea mays cv Golden cross bantam), and wheat (Triticum aestivum cv YamDap,4 an oxidation product of the naturally occurring PAs, Spd, hill) and leaf discs (10 mm) from mature leaves of 1- to 2-monthand Spm, occurs in many cereal plants (28). The oxidation is old plants of bean (Phaseolus vulgaris cv Taylor's Horticultural), catalyzed by PA-oxidase, a cell wall-localized enzyme in oat leaves radish (Early Scarlet Globe), and broccoli (Waltham). Leaves of (17). Our recent observations (19) showed that endogenous levels these materials were floated on buffer in Petri dishes, with Dap, of Dap, like those of other PAs, decrease in attached oat leaves Spd (HCI salt, Sigma), and other effectors included as indicated with increasing age of seedlings and in excised leaves with increas- in the appropriate tables. Incubation was carried out at 25°C in ing time of dark incubation. This suggested that Dap, like other the dark or in the light in growth rooms as described above. PAs, may be involved in the control of senescence. Although other Determination of Protease Activity. Twelve leaf segments (45 PAs have been implicated in processes controlling cell growth (5, mm each) were homogenized in a prechilled mortar with 2 ml of 6, 8, 13) and senescence (2, 3, 9, 16, 18), virtually nothing has been cold 50 mm phosphate citrate buffer (pH 6.0). The homogenates reported about the biological activity of Dap in plant cells. were kept in the cold (4°C) for 0.5 h and then centrifuged at We now report that Dap accumulates following exogenous 12,000g for 15 min at 4°C. The clear supernatant fraction was application of Spd or Dap to excised dark-incubated oat leaves. assayed for protease activity using Azocoll (Calbiochem) as the substrate. The final l-ml reaction mixture contained 5 mg Azocoll, 'Supported by grant DAR 7813294 from the National Science Foun- 0.8 ml of 50 mm phosphate-citrate buffer (pH 4.2 for acid protease dation and 144-79 from the Binational Agricultural Research and Devel- and pH 6.6 for neutral protease), and 0.2 ml of the crude enzyme. For in vitro effects, the reaction mixture included 0.1 ml of various opment (BARD) agency to A. W. G. concentrations of Dap and Spd. The tubes (1.5-ml microcentri2 Present address: Calgene Inc., Davis, CA 95616. 3Present address: Institute of Plant Physiology, University of Bern, fuge) were stoppered, vortexed, and floated in a water bath equipped with a shaker and maintained at 43°C for 3 h. Controls Altenbergrain 21, CH-3013 Bern, Switzerland. 4Abbreviations: Dap, 1,3-diaminopropane; PA, polyamine; Spd, sper- were similarly prepared, with boiled enzyme or without enzyme. midine; Spm, spermine; ACC, I-aminocyclopropane-l -carboxylic acid; The reaction was terminated by immersing the tubes in an ice bath for I h, and the tubes were centrifuged to remove the Put, putrescine; AVG, aminoethoxyvinylglycine. 1592

INHIBITION OF LEAF SENESCENCE BY DAP

undigested Azocoll. The absorbance of the supernatant fractions was measured at 520 nm. Under these conditions, which were optimum for both acid and neutral proteases, the enzyme activity, expressed as units/mg protein.h, was linear with time and with concentration of crude extract. One unit of activity is the amount of enzyme which produces a AA of 0.1 in 1 h. Each assay was replicated three to four times. Determination of Ethylene Production. Air samples (1-5 ml) were withdrawn from the bottles with a syringe and injected into a gas chromatograph apparatus (Perkin Elmer, model F-l 1) equipped with an alumina column. Ethylene was identified and quantified by comparing the peaks with the retention time and peak heights of ethylene standards. Determination of PA Content. PAs were extracted from leaf segments (19) with 5% HC104, dansylated and separated on silica gel thin-layer plates, and quantified as described in earlier reports

(11, 19).

Determination of Chi Content. Chl extracted with hot 80%o ethanol was measured spectrophotometrically at 665 nm and expressed as percent of the initial value. Determination of Protein Content. The crude protease extract was used to measure soluble protein content according to the method of Lowry et al. (22), using BSA as a substrate. The data presented were from single experiments which are representative of 2 to 4 experiments. Each experiment was performed in duplicate.

RESULTS AND DISCUSSION Accumulation of Dap. When excised oat leaves were incubated on Spd-containing medium in the dark, there was a large accumulation of Dap, which increased with increasing concentration of Spd and time of treatment (Table I). Dap titer increased 44fold with 0.5 mm Spd and 66-fold with 1.0 mm Spd during 48-h treatments, while a 3-h exposure to 1.0 mm Spd increased the titer by only 9-fold. However, if the leaves were floated first on medium containing the PA oxidase inhibitor, hydroxyethyl hydrazine, and then on Spd, the accumulation of Dap in leaves was greatly reduced. Apparently, the accumulation of Dap occurs through a rapid uptake of Spd and its oxidation by PA oxidase, which is abundant in oat leaves (17, 28). Similar accumulation of Dap after exogenous Spd application was reported in the cyanobacterium Anacystis nidulans (27). The results in Table I further show that a high accumulation of Dap also occurs with exogenous application of Dap; this increases with increasing Dap concentration, being 35-fold in 0.5 mi Dap and as high as 120-fold in 1 mm treatments for 48 h. The accumulation must involve rapid and direct uptake of Dap since a 3-h treatment with 1 mm Dap caused a 26-fold Table I. Accumulation of Dap in Excised Oat Leaves Incubated in the Dark for 48 Hours on Medium Containing Spd or Dap PA Concn. Dap Content mM ,g/g fresh wt Control 3.9 175.7 Spd, 0.5 266.2 Spd, 1.0 Spd, 1.08 36.0 15.2 Sod + HEHb Dap, 0.5 137.9 482.7 Dap, 1.0 Dap, 1.08 110.5 Dap + HEHb 105.0 a Peeled leaves floated on Spd or Dap for 3 h, washed thoroughly, and floated on buffer for the remaining 45 h. h Peeled leaves floated on 2 mM HEH (hydroxyethyl hydrazine) for 2 h followed by HEH + I mm Spd or Dap for 3 h, washed thoroughly, and floated on buffer for the remaining 43 h.

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increase and treatments with hydroxyethyl hydrazine did not alter the endogenous titer. Effect of Dap on Protease Activity. Excision and dark-incubation of cereal leaves cause a sharp rise in protease activity ( 18, 29). The activity (Table II) includes at least two proteases, showing pH optima at 4.2 and 6.6 with Azocoll as a substrate, supporting earlier reports (29). The exogenous application of Dap (in vivo) inhibited the rise in both acid and neutral proteases, the inhibition increasing with increasing Dap concentration. The increase in acid protease was higher than the neutral protease in control leaf segments during the 48-h senescence period, and the inhibition of acid protease by Dap (0.5 mM) was also more than the neutral protease (Table II). While these results support the contention that acid protease predominates during early stages of leaf senescence in cereal leaves, this is not the case in other plants such as tobacco and nasturtium (29). Similar inhibitory effects on in vivo protease activity were observed with Spd and Spm (20) and with cytokinins (29). The Dap-mediated inhibition of proteases is antagonized by Ca2"; for example, addition of 1 mm CaCl2 together with 1 mm Dap reduced the inhibition of acid protease by about half. These results suggest that protonated Dap (Dap2+) and Ca2+ may compete for the same electronegative sites on membranes. To determine whether Dap might retard senescence by directly inhibiting protease activity, 1 mm Dap was added in vitro to the enzyme extract during the assay of enzyme activity. Such in vitro addition of Dap was not as effective in decreasing protease activity as in vivo addition (data not shown). Rapid proteolysis is well documented to be one of the early events in cereal leaf senescence (25, 29). It is also known that cycloheximide at concentrations that inhibit protein synthesis prevents the rise of protease activity and the accompanying Chl loss. Consequently, the increase in protease activity has been suggested as due to its de novo synthesis (29). Thus Dap, which inhibits more than 90% of the rise in acid protease when applied exogenously, may retard senescence more by inhibiting the synthesis than the action of proteases. These results gain support from our work on inhibition of ACC synthase, the enzyme involved in ethylene biosynthesis (12) and from a recent report on the inhibition of protein synthesis by Dap in animal cells (30). It has also been suggested (7) that compartmentation of proteases may occur in lysosome-like organelles. Since PAs are known to stabilize membranes (5, 9, 24), Dap may act by limiting the release of proteases from these organelles. Effect of Dap on Chi Content. Excised oat leaf segments floated on medium containing Dap and incubated in the dark showed a striking decrease in Chl loss with increase in Dap concentrations (Table III). Even a short exposure to 1 mm Dap decreased Chl breakdown. In contrast, in the light, where Chl breakdown is generally lower than in the dark, Dap treatments promoted its breakdown (bleaching). These differential effects of Dap on Chl breakdown in the dark and in the light confirm our earlier results with other PAs (18). Further experiments are needed to determine the effects of Dap on protease activity and ethylene production in light which should help in understanding the differential effects of Dap on senescence in the dark and in the light. The inhibition of Chl breakdown in the dark by Dap was also examined in other cereals. Figure 1 shows that, as in oat leaves (Table III), increasing Dap concentrations cause progressive decreases in Chl loss in wheat, corn, and barley leaf segments. These findings, together with data showing decreases in Dap titer in senescing oat leaves (19), suggest that Dap may play a role in controlling senescence in cereal leaves. To examine whether Dap is similarly involved in the control of leaf senescence in dicotyledonous plants, we examined its effect on Chl breakdown in dark-incubated leaf discs from broccoli and radish, which also senesce rapidly. In these plants, Dap-treated leaf discs show a marked accumulation of Dap, but Dap was only

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Plant Physiol. Vol. 70,1982

SHIH ET AL.

Table II. Inhibition of Protease Activity by Exogenous Application of Dap to Excised Oat Leaves Peeled leaves floated on solutions containing Dap for 48 h in the dark. Initial values for acid and neutral proteases were 0.7 ± 0.07 and 1.3 ± 0.07 units/mg protein *h, respectively. Protease Activity Dap Concn. Acid Neutral mM units/mg protein- h % inhibition of increase units/mg protein h % inhibition of increase 0 (control) 3.5 ± 0.12a 0 3.1 ± 0.13 0 0.5 1.6 ± 0.03 68 2.3 ± 0.05 44 1.0 0.9±0.05 93 1.3±0.13 100 l.OmMDap+ 2.17 ±0.18 48 10 mM CaC12 a Mean ± SE.

Table III. Effect of Dap on Chl Content in Excised Oat Leaves Incubated in the Dark (2 Days) or Light (S Days) Initial value for Chl was 2.1 ± 0.06. Chl Content

Dap Concn.

Dark

mM 0 (control) 0.25 0.5 1.0 a Relative to initial value. b Mean ± SE. I

I

Light % lossa 77 73 41 13

A 665

0.5 ± 0.02b 0.6 ± 0.03 1.3 ± 0.06 1.9 ± 0.01

I

60

a 50 C

o 40 0

0

-J

30

0 20 0 0

WHEAT

U.-^ 10 I-

LEY

o I

I

0

0.25

0.50

1.2 ± 0.08 1.0 ± 0.04 0.4 ± 0.02 0.6 ± 0.02

exogenously to radish and broccoli probably indicate that it does not control senescence in dicots. Antagonism by Ca2" of Dap-Mediated Inhibition of Chi Loss. The data in Table IV show that the inhibition of Chl loss by Dap in leaves incubated in the dark and its increase in leaves exposed to light are greatly reduced by the simultaneous addition of CaCl2 to the incubation medium. These results support previous findings on the decrease by Ca2e of the Dap-mediated inhibition of protease activity (Table II). Likewise, in the light, where Dap promotes Chl loss, the presence of Ca2" partially inhibits the Dap effect. However, when the leaves were first floated on CaCl2 for 3 h and then on Dap for the remaining 45 h in darkness, no antagonism of Dap retardation of Chl loss was observed. Similar results were obtained when leaves were first floated on Dap and then on CaCl2. In both light and dark, Ca2" by itself had no effect on Chl loss when compared with controls. As in our earlier observations (18), these results indicate a competition by Ca2e and protonated PAs for attachment to membranes and entry into the cell. At physiological pH, the polyamines carry a net positive

I

I

70k w

% lossa 40 50 80 70

A6a

1.0

Table IV. Prevention by Calcium of Dap-Mediated Inhibition of Chi Loss Chl Loss Treatments Dark Light

(2 d)

(S d)

DAP Conc,mM FIG. 1. Effect of Dap on Chl content in excised cereal leaves incubated in the dark. Leaves were excised, peeled, and floated on I mm phosphate buffer or on buffer containing various concentrations of Dap and incubated for 48 h in the dark.

slightly effective in preventing Chl breakdown (data not shown). Furthermore, control leaf discs of broccoli and radish do not contain Dap, probably indicating the absence of PA oxidase. Thus, Dap which occurs naturally in cereal may control senescence there, but its absence and failure to act when applied

77 Control 40 73 CaCl2 (I mM) 44 13 76 Dap (I mM) 68 58 CaCl2 + Dap 5 CaCI2 (3 h)8 + Dap 27 Dap (3 h)b + CaCl2 a Peeled leaves floated on CaCI2 for 3 h, washed thoroughly, and floated on Dap for the remaining 45 h. b Peeled leaves floated on Dap for 3 h, washed thoroughly, and floated on CaCl2 for the remaining 45 h.

INHIBITION OF LEAF SENESCENCE BY DAP Table V. Effect of Dap on Ethylene Production and PA Titer in Excised Oat Leaves Incubatedfor 24 Hours in the Dark PA Titer Treatment Ethylene Production Put Spd Put nmol/gfresh wt. 24 h % inhibition ,ugigfresh wt % decrease Control 14.0 0 11.0 +1.0a 2.9 ± 0.87 0 Dap, 0.5 mM 2.2 84 5.2 ± 1.1 2.2 ± 0.35 53 Dap, 1.0 mM 1.0 93 3.6 ± 0.5 2.0 ± 0.1 67 a Mean ± SE.

charge and hence function as polyvalent cations. Cationic PAs bind to negative sites on membranes, ribosomes, and nucleic acids (1, 5, 8). Since Dap is a diamine and hence jositively charged, it may bind similarly. The antagonism with Ca + supports this view and indicates an initial ionic attachment of Dap to membranes. Further confirmation is obtained from our work on the antagonism by Ca2+ of the Dap- and Spd-mediated inhibition of ethylene production in oat leaf segments (12), aging apple tissue (4), and orange peels (10). The results suggest that the Dap-mediated retardation of senescence occurs by an initial binding of Dap to a membrane site which it shares with Ca2+, and that once both are inside the cell, Ca2e no longer interferes with Dap action. Dap-Mediated Inhibition of Chi Loss as Affected by Mg2e. The Dap-mediated increased Chl breakdown in leaf segments exposed to light may result from its action in displacing Mg2e from the pyrrole rings of the Chl molecule, similar to effects of EDTA and other chelators which remove Mg2+ (21). Addition of 1 or 10 mim MgCl2, however, did not decrease the Dap-mediated Chl breakdown in the light (data not shown). The bleaching effect caused by Dap and other PAs (18) may also involve a direct inhibition of a process responsible for Chl synthesis, since Dap is known to inhibit enzymes such as proteases (as demonstrated in the present study), ACC synthase (12), and ornithine decarboxylase (26). Effect of Dap on Ethylene Production and PA Titer. Since increased ethylene production is characteristic of senescing tissues and ethylene and PA biosynthesis have a common precursor in Sadenosylmethionine, we investigated the effect of Dap on their role in senescence. The results (Table V) show that exogenously applied Dap is a potent inhibitor of ethylene production. This marked decrease in ethylene production should result in an increase in PA titer through a diversion in S-adenosylmethionine metabolism. However, the results (Table V) show that exogenous Dap decreased Put and Spd levels, although it increased Dap titer (Table 1). Dap is known to inhibit ornithine decarboxylase activity in several animal systems (5, 26). This enzyme is rate limiting in some systems for the synthesis of Put, a precursor for Spd. Hence, the observed low titer of Put and Spd could result from the inhibition of ornithine decarboxylase (or possibly arginine decarboxylase) by the accumulated Dap in leaves floated on medium containing Dap. Furthermore, when leaf segments were floated on medium containing 0.2 mM AVG, a specific inhibitor of ethylene biosynthesis from methionine, ethylene production was almost completely inhibited, but there was only a slight effect on the endogenous levels of PAs, and no effect on prevention of Chl loss (data not shown). Thus, ethylene biosynthesis probably is not involved in Chl loss in darkness. These observations corroborate a recent report (14) and are further supported by data presented in the following paper (12). In contrast, PA synthesis has been shown to increase in senescing orange peels treated with AVG (10).

The data presented here thus demonstrate that Dap, like other naturally occurring PAs, is a potent inhibitor of dark-induced senescence. It inhibits the rise in protease activity, the loss in Chl, and ethylene production. Inhibition of the rise in protease activity seems to be, at least in part, due to a repression of the synthesis of the enzyme, and in part to direct inhibition of its action as reported

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for PA-mediated inhibition of protease (20) and RNase activities (15). Dap may bind to polyribosomes and affect protein synthesis which may account for the decrease in rise of protease activity. Antagonism by Ca2", however, suggests that the initial step in the retardation of senescence by Dap probably involves its attachment to membranes. Ethylene biosynthesis appears to be a valuable tool for studying the mode of action of Dap more specifically. The biosynthesis of ethylene from methionine includes two steps with probably regulated functions: (a) ACC synthesis from S-adenosylmethionine by de novo-synthesized ACC synthase (31); and (b) ACC oxidation, yielding ethylene by a constitutive membrane-associated system (23). If Dap inhibits protein synthesis and specific membrane functions, we would expect an inhibition of both steps. In the following paper, we have attempted to specify the nature of the inhibition and to examine whether the inhibition of ethylene production relates to the senescence-retarding effect of Dap. LITERATURE CITED 1. ABRAHAM AK, A PiHL 1981 Role of polyamines in macromolecular synthesis. Trends Biochem Sci 64: 106-107 2. ALTMAN A, U BACHRACH 1981 Involvement of polyamines in plant growth and senescence. In CM Caldarera, V Zappia, U Bachrach, eds, Advances in Polyamine Research, Vol. 3. Raven Press, New York, pp 365-375 3. ALTMAN A, R KAUR-SAWHNEY, AW GALSTON 1977 Stabilization of oat leaf protoplasts through polyamine-mediated inhibition of senescence. Plant Physiol 60: 570-574 4. APELBAUM A, AC BURGOON, JD ANDERSON, M LIEBERMAN, R BEN-ARIE, AK MArroo 1981 Polyamines inhibit biosynthesis of ethylene in higher plant tissue and fruit protoplasts. Plant Physiol 68: 453-456 5. BACHRACH U 1973 Function of Naturally Occurring Polyamines. Academic Press, New York 6. BAGNI N, D SERAFINI-FRAcAssINI, P TOMIGIANI 1981 Polyamines and growth in higher plants. In CM Caldarera, V Zappia, U Bachrach, eds, Advances in Polyamine Research, Vol. 3. Raven Press, New York, pp 377-388 7. BOLLER T, H KENDE 1979 Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol 63: 1123-1132 8. CoHEN SS 1971 Introduction to the Polyamines. Prentice-Hall, Inc., Englewood Cliffs, New Jersey 9. COHEN AS, RB Popovic, S ZALIK 1979 Effects of polyamines on chlorophyll and protein content, photochemical activity, and chloroplast ultrastructure of barley leaf discs during senescence. Plant Physiol 64: 717-720 10. EVEN-CHEN Z, AK MATToo, R GOREN 1981 Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4 - (14C) methionine into spermidine in aged orange peel discs. Plant Physiol 69: 385-388 11. FLORES HE, AW GALSTON 1982 Analysis of polyamines in higher plants by high performance liquid chromatography. Plant Physiol 69: 701-706 12. FUHRER J, R KAUR-SAWHNEY, L-M SHIH, AW GALSTON 1982, Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves. II. Inhibition of ethylene biosynthesis and possible mode of action. Plant Physiol 70: 1597-1600 13. GALSTON AW, R KAUR-SAWHNEY 1980 Polyamines in plant cells. What's New in Plant Physiol 11: 5-8 14. GEPSTEIN S, KV THIMANN 1981 The role of ethylene in the senescence of oat leaves. Plant Physiol 68: 349-354 15. KAUR-SAWHNEY R, A ALTMAN, AW GALSTON 1978 Dual mechanisms in polyamine-mediated control of ribonuclease activity in oat leaf protoplasts. Plant Physiol 62: 158-160 16. KAuR-SAWHNEY R, HE FLORES, AW GALSTON 1980 Polyamine-induced DNA synthesis and mitosis in oat leaf protoplasts. Plant Physiol 65: 368-371 17. KAUR-SAWHNEY R, HE FLORES, AW GALSTON 1981 Polyamine oxidase in oat leaves: a cell wall-localized enzyme. Plant Physiol 68: 494-498 18. KAUR-SAWHNEY R, AW GALSTON 1979 Interaction of polyamines and light on biochemical processes involved in leaf senescence. Plant Cell Environ 2: 189-196

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19. KAUR-SAWHNEY R, LM SHIH, HE FLORES, AW GALSTON 1982 Relation of polyamine synthesis and titer to aging and senescence in oat leaves. Plant Physiol 69: 405-410 20. KAUR-SAWHNEY R, LM SHIH, T CEGIELSKA, AW GALSTON 1982 InhibitiQn of protease activity by polyamines: relevance for control of leaf senescence. FEBS Lett, 145: 345-349 21. KOTAKA S, AP KRUEGER 1969 Some observations on the bleaching effect of ethylene-diaminetetraacetic acid on green barley leaves. Plant Physiol 44: 809-815 22. LowRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 23. MATToo AK, JE BARKER, E CHALUTZ, M LIEBERMAN 1977 Effect of temperature on the ethylene-synthesizing systems in apple, tomato, and Penicillium digitaturn Plant Cell Physiol 18: 715-719 24. NAIK BI, V SHARMA, SK SRIVASTAVA 1980 Interaction between growth regulator and polyamine effects on membrane permeability. Phytochemistry 19: 1321-1322

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25. PETERSON LW, RC HUFFAKER 1975 Loss ofnbulose 1,5-diphosphate carboxylase and increase in proteolytic activity during senescence of detached primary barley leaves. Plant Physiol 55: 1009-1015 26. Poso H, J JANNE 1976 Inhibition of polyamine accumulation and deoxyribonucleic acid synthesis in regenerating rat liver. Biochem J 158: 485-488 27. RAMAKISHNA S, L GUARINO, SS COHEN 1978 Polyamines of Anacystis nidulans and metabolism of exogenous spermidine and spermine. J Bacteriol 134: 744-750 28. SMITH TA 1980 Plant amines. In EA BelL BV Charlwood, eds, Secondary Metabolism (Encyclopedia of Plant Physiology, New Series, Vol 8). Springer-Verlag, Berlin, pp 433-460 29. THIMANN KV 1980 The senescence of leaves. In KV Thimann, ed, Senescence in Plants. CRC Press, Boca Raton, FL, pp 85-115 30. TuoMI K, A RAINA, R MANTYJARVI 1980 1,3-Diaminopropane rapidly inhibits protein synthesis and virus production in BKT-I cells. FEBS Lett 111: 329-332 31. Yu YB, DO ADAMS, SF YANG 1979 l-Aminocyclopropane- -carboxylate synthase, a key enzyme in ethylene biosynthesis. Arch Biochem Biophys 198: 280-286