Ethylene production by detached leaves infected with tobacco mosaic virus

VIROLOGY

40,

l-9

(1970)

Ethylene

Production

by

Detached

Tobacco Y. NAKAGAKI, Plant Pathology

Mosaic

T. HIRAI,

AND

Leaves

August

with

Virus’ MARK

Laboratory, Faculty of Agriculture, Nagoya University, of Biochemistry, University of Wisconsin, Madison, Accepted

Infected

A. STAHMANK Nagoya, Japan, Wisconsin

and Department

53706

4, 1969

Ethylene production by detached leaves infected with tobacco mosaic virus (TMV) with or without local lesions and the effect of exogenously introduced ethylene on TMV multiplication and lesion formation were investigated. Ethylene production was enhanced concurrently with local lesion development, but not with systemic infection. Increase in the rate of ethylene production was proportional to the number and size of lesions. This enhanced ethylene production reflects necrotization of the cells invaded by the virus; ethylene production was also enhanced by senescence and by chemical injury of leaves. Chemicals causing mild cell necrosis, such as Ni, Hg, and Cu salts, remarkably increased ethylene production with the development of toxic symptoms: Perchloric acid and trichloroacetic acid were less effective in increasing the rate of ethylene production. Ethylene production was inhibited by disodium ethylenediaminetetraacetate and by the protein synthesis inhibitors blasticidin S and puromycin. Exogenously introduced ethylene stimulated chlorophyll degradation and slightly inhibited peroxidase activity, but did not inhibit TMV multiplication or lesion formation.

dogenously produced ethylene and that abnormally enhanced ethylene occurred simultaneously with the appearance of necrotic local lesions, but, not with systemic infection. However, quantitative determination of ethylene production and its relation t.0 virus multiplication remain still unknown. This paper describes ethylene production by tobacco mosaic virus (TMV)-infected leaves, with or without necrosis, and the effect of ethylene on T&XIV multiplication and local lesion formation. The effect of various inhibitors on ethylene production is also included.

INTRODUCTION

Ethylene, a regulator for plant metabolism, is known as an endogenously produced substance which promotes fruit ripening and senescence of plant, tissues (Burg, 1962). Ethylene induced metabolic activation in sweet potato root tissues (Stahmann et al., 1966; Imaseki et al., 1968b) and is produced by injured or fungus-infected tissues (Williamson, 1950; Smith et al., 1964; Stahmann et al., 1966; Imaseki et al., 1968a-c). When exposed to ethylene, plants become epinastic and leaves eventually abscise, as had been observed in Physalis jloridana infected with potato virus Y (Ross, 1948). Other virus-induced epinastic symptoms were reported in rose wilt virus (Grieve, 1942, 1943). Ross and Williamson (1951) showed that epinastic symptoms were closely related with en1 Contribution No. 148, Plant Pathology oratory, Nagoya University.

MATERIALS

Plant materials and virus. Nicotiana glutinosa L., N. tabacum L., var. Samsun nc, N. tabacum L. var. Bright Yellow, and PhaseolusvuZgaris L. cv. Kairyo Otebo were

grown in a greenhouse. The ordinary strain of TMV was purified by the method of differential centrifugations. Tobacco half-

Lab1

Copyright

0

1970 by Academic

Press.

Inc.

AND METHODS

2

NAKAGAKI,

HIRAI,

leaves were dusted with Carborundum (500 mesh) and rubbed with 100 pg/ml of TMV suspended in M/l5 phosphate buffer (pH 7.0). Other half-leaves which were rubbed with the same buffer without virus served as controls, and untreated leaves as healthy controls. The leaves were rinsed with tap water immediately after inoculation. The upper surfaces of the primary leaves of 12-14-day-old bean seedlings were dusted with Carborundum and rubbed twice with a paint brush immersed in l-50 pg/ml of TMV. Heated virus solution was obtained by treating each concentration of TMV solution at 100” for 20 min. The amount of TMV produced in leaf disks was measured by the method of Taniguchi (1962); this consisted of the precipitation of viral nucleoprotein by ammonium sulfate and the measurement of the optical density at 260 mp. Incubation. An air-tight incubation chamber (inside volume 207 ml) was made by sealing the rims of two petri dish bottoms (9.7 cm in diameter) with a transparent vinyl tape (Imaseki et al., 1968c). The rim of one dish had a small notch so that an air sample could be withdrawn through the hole with a hypodermic needle. Half-leaves were placed in the chamber, which also contained filter paper and 5-10 ml of distilled water to maintain a high humidity. They were incubated in a growth chamber at 26” under continuous illumination (co. 5000 lux) from fluorescent lamps. Ethylene of 99.9 % purity in concentrations of 0.1-100 ppm was introduced into the sealed petri dishes through the hole by a hypodermic syringe. To treat leaves with injurious chemicals, small droplets (cu. 0.2-0.3 ~1) were placed by a glass capillary on the center of leaf disks in the chamber which contained filter paper and 5 ml of distilled water. These chambers were sealed and incubated at room temperature. Chemicals used were trichloroacetic acid (TCA), perchloric acid (PCA), CuC12, HgC12, and NiCL. Determination of ethylene. Ethylene was measured by a gas chromatography apparatus (Shimadzu GC-lC, Japan) equipped with a hydrogen flame ionization detector

AND

STAHMANN

(Imaseki et al., 1967). Identification of ethylene was checked by comparing the retention time of authentic ethylene peaks with that of peaks of the sampled gas. The quantity of ethylene was determined from the area under the peak. Determination of chlorophyll content. Ten tobacco leaf disks (12 mm in diameter) were homogenized with 2 ml of distilled water and extracted with cold absolute acetone. The extract was centrifuged at 3000 rpm for 15 min, and the precipitate was again extracted with 80% acetone. The combined extracts were diluted, and the chlorophyll content was determined by the method of Arnon (1949). Assays of peroxidase activity. Ten leaf disks (12 mm in diameter) were chilled and ground with 4 ml of 0.1 M Tris-HCl buffer (pH 7.5). The homogenate was centrifuged at 15,000 g for 20 min, and the supernatant was used as a crude enzyme solution. Activity of peroxidase was assayed by the method of Shannon (1967), except that 0.2 ml of enzyme solutions, which were diluted to give a linear reaction rate for at least 2 min, were added at zero time and incubated for 20 sec. The increase in optical density at 460 rnp during 1 min was determined with a Gary model 14 spectrophotometer. Inhibitors and antibiotics used. The following inhibitors were dissolved in 0.01 M phosphate buffer (pH 5.5) : disodium ethylenediaminetetraacetate dihydrate (EDTA), sodium diethyldithiocarbamate (DIECA), and L-cysteine monohydrochloride (Cys HCl). Puromycin dihydrochloride (PM) was obtained from Nutritional Biochemical Corp., Cleveland, Ohio. Blasticidin S (BcS) was kindly supplied by Dr. Misato, Rikagaku Institute, Saitama, Japan. These antibiotics were dissolved in distilled water. RESULTS

Ethylene Production Local Lesions

by Detached Leaves with

Bean, N. glutinosa, and Samsun nc tobacco, all local lesions hosts, were inoculated with TMV and tested for ethylene production. Figure 1 presents the time course of ethylene production per leaf area during 72 hours after inoculation of bean

-----_-

-__-

--

BY DETACHED

^---I--^_-

3

LEAVES TABLE

1

EFFECT OF INOCULATION WITH DIFFERENT CONCENTRATIONS OF TMV AND HEAT-INACTIVATED TMV ON ETHYLENE PRODUCTION BY LEAVES OF Phaseolus vulgaris cv. KAIRYO OTEBO

TMV

TMV concentration Ethylene hdrnl) (m&‘cmz) 0 I 0

48 18 24 Hours after inoculation

72

I

FIG. 1. Time course changes of ethylene production by leaves of bean, Kairyo Otebo, following inoculation with TMV. 0, TMV 50 pg/ml; 0, TMV lOpg/ml; 0, rubbing with phosphate buffer; n , no rubbing. Average of 3 independent experiments, each of which consisted of 4 incubation chambers, 4 half-leaves each. Total accumulation of ethylene during the interval indicated.

leaves. It is evident that the increased rate of ethylene production was detectable concomitant with the appearance of visible lesions, while control and healthy control leaves showed no increase. Similar results were obtained with the other local-lesion hosts, N. glutinosa and Samsun nc tobacco. The rate of ethylene production was greater during the 1%24-hour period after inoculation; in this period local lesions appeared and enlarged. Ethylene production in bean leaves inoculated with different concentrations of TIVV was tested. Table 1 shows that ethylene production was proportional to the increase in the number of lesions that were caused by higher titers of TRW. Heatinactivated TMV, on the other hand, did not cause any increase in ethylene production nor did it cause lesion formation. Ethylene Production in Inoculated Leaves of Xystemic Infection Hosts Figure 2 presents the time course of ethylene production during 8 days after inoculation of detached leaves of tobacco, var. Bright Yellow, in which the virus multiplies systemically. Inoculated and control leaves showed no detectable increase in ethylene production during the first 4 days after inoculation. However, both leaves

1 5 10 20 50

Lesions (No./cmz)

0.55” 0.78 0.96 1.48 2.16 3.37

2.1 3.8 7.6 9.5 13.3

Ethy1cPe Ethylene pe$‘$on (ml/cm”) 0.37 0.25 0.19 0.22 0.25

0.60 0.59 0.59 0.61 0.62 0.63

0 Figures represent averages of 3 independent experiments, each of which consisted of 3-4 incubation chambers, 4 half-leaves each. Ethylene was measured 48 hours after inoculation.

1

1

2 345678 Days after Inoculation

FIG. 2. Time course changes of ethylene production by detached tobacco leaves, var. Bright Yellow, a systemic host, following inoculation with TMV. 0, TMV 100 pg/ml; 0, rubbing with phosphate buffer; n , no rubbing. Average of 3 independent experiments, each of which consisted of 4 incubation chambers, 2 half-leaves each.

became gradually yellowish with the progress of incubation time and the inoculated leaves produced higher amounts of ethylene in the later infection stagesthan the control leaves. Since yellowing of the leaves paralleled the production of ethylene, the increasedrate of ethylene production following senescence of detached leaves and virus infection probably reflects an induced

4

NAKAGAKI,

HIRAI,

AND

STAHMANN

premature senescencein the virus-infected leaves. This is supported by the data of Fig. 3, which show the ratio of ethylene production by leaf disks that were daily punched from healthy and inoculated leaves of intact plants. The ratio of ethylene production in infected to control leaf disks was not significantly altered during 7 days after inoculation, while the TMV titer increased.

incubated in the light. Noninoculated leaves incubated in the dark produced more ethylene than in the light, probably due to the senescenceof leaves, but much lessthan inoculated leaves.

E$ect of Darkness on Ethylene Production

FIG. 3. TMV multiplication and ethylene production by leaf disks of tobacco plants, var. Bright Yellow, which were punched from virus-infected or intact plants each day. 0, ratios of ethylene production of inoculated to noninoculated leaf disks after a 24-hour incubation period; 0, T&XV concentration. Average of 3 independent experiments, each of which consisted of 3 tobacco plants having 3 large leaves each. Ten leaf disks (12 mm in diameter) were punched from one leaf each, and 30 leaf disks were incubated in an incubation chamber.

Effect of Inhibitors on Ethylene Production

Table 3 shows the effect of inhibitors on ethylene production in Samsun nc and bean leaves inoculated with TlMV. Since peroxidase seems to play an important role in Ethylene Production in Leaves Treated with ethylene production, EDTA and DIECA, Chemicals inhibitors for peroxidase in vivo (Lieberman Figure 4 presents rates of ethylene pro- and Kunishi, 1967) and in vitro (Yang, duction by N. glutinosa leaves that were 1967), were tested for their effect on ethylene treated with chemicals as described in production, It was evident that EDTA at a Materials and Methods. Five percent TCA concentration of 10e3M was most effective and 5 % PCA, protein-precipitating agents, in inhibiting ethylene production, whereas formed a bright brown spot on leaf disks DIECA and CysHCl were almost ineffecwithin a few minutes after treatment, but tive. However, these chelators did not ethylene production was not enhanced inhibit lesion formation, except EDTA on because cells in the leaf tissues were killed bean, although lesions were less pigmented rapidly. When the chambers were opened than those on untreated leaves. and the excessive droplets on leaf disks were Puromycin (PM) and blasticidin S removed, ethylene production slightly in- (BcS), inhibitors of protein synthesis, creased; this might reflect slow progress of which caused no phytotoxicity on leaves at necrotic formation due to the chemicals low concentrations, inhibited lesion formainfiltrated into the tissues. Treatment with tion on Samsun nc and bean and ethylene 0.5 % CuCh and 0.5 % HgCL produced, 2-4 production, except PM on bean hours after treatment, dark green spots that changed to black granular spots and brown lesions. Ethylene production was enhanced during the first 24 hours and then decreased. One percent NiClz induced a mild cell necrosis and an increased ethylene production, which occurred during 2448 hours of incubation. Similar results were obtained with tobacco, Bright Yellow. It may be concluded that injury not resulting in quick killing of cells induces an enhancement of ethylene production. Days after Inoculation When inoculated leaves of N. glutinosa were placed in the dark, dark green spots appeared, while typical brown lesions developed in the light about 36-40 hours after inoculation. Ethylene production under both conditions was compared during 3 days after inoculation. The results are presented in Table 2. Virus-inoculated leaves produced much more ethylene when

ETHYLENE

PRODUCTION

Figure 5 presents time course changes in ethylene production in inoculated bean leaves treated with various concentrations of BcS. Lesions appeared about 18-20 hours after inoculation on both treated and un-

04

24 Incubation

48 Time

72

in Hours

FIG. 4. Effect of injurious chemicals on ethylene production by detached leaf disks of N. glutinosa. Twenty leaf disks (18 mm in diameter) were sealed in a 207-ml chamber and incubated for a given time. Droplets of chemicals (ca. 0.2-0.3 ~1) were placed on the center of disks with a glass capillary. 0, 5% TCA; W, 5% PCA; A, 0.5% HgC12; A, 0.5% CuCln; 0, 1% NiC12; 0, untreated. Arrows show the development of visible spots caused by the injurious chemicals. TABLE 2 EFFECTOFLI~HTANDDARKTREATMENTSONTHE INCREASEINETHYLENEPRODUCTIONBYLEAVES OF Nicotiana

glutinosa

Ethylene (mJ/cm2) Time after inoculation (hours)

Light

24 48 72

I N 1:N __-0.57a 0.60 0.95 3.75 0.61 6.15 8.38 0.63 13.31 -__~ 2.8

No. lesions per cm2

Dark I-

I -____ I 0.82 2.06 3.89 __-__

N

1:N I

0.85 0.96 1.20 1.71 1.81 2.15

a Figures represent average of 3 independent experiments, each of which consisted of 3 incubation chambers, 4 half-leaves each. Total accumulation of ethylene during the interval indicated. N: noninoculated, I: inoculated with TMV (10 rg/ml).

BY DETACHED

LEAVES

5

treated leaves. Ethylene production in the latter was enhanced after lesion appearance and increased linearly during 24-72 hours after inoculation, whereas 0.2 ppm BcStreated leaves produced relatively low levels of ethylene during the first 48 hours. The total number of lesions produced on 4 half leaves in an incubation chamber 3 days after inoculation was 200, 27, 0, and 472 by 0.01, 0.1, 0.2 ppm of BcS and untreated, respectively. Thus, 0.01 ppm of BcS caused no chemical injury and the decrease in ethylene production was almost proportional to the decreased number of lesions that were caused by the treatment. BcS (0.1-0.2 ppm) inhibited ethylene production during 24-48 hours and then caused a chemical injury that consisted of round transparent lesions on the blade of the half-leaves, leading to an enhancement of ethylene production 72 hours after inoculations. E$ect of Ethylene on TMV Multiplication, Lesion Formation, Chlorophyll Content, and Peroxidase Activity To test the possibility of ethylene playing a role in resistance against virus infection, the effect of ethylene, exogenously introduced to leaves, on TIMV multiplication and lesion formation was investigated. Inoculated and noninoculated tobacco leaf disks were incubated for 5 days in air containing various concentrations of ethylene. When incubated in high level of ethylene, tobacco leaves became yellowish 34 days or more after incubation and round or irregular brown lesionsdeveloped on yellowish leaves, usually on inoculated, and often on noninoculated, leaves. Table 4 indicates that high concentrations of exogenously introduced ethylene caused chlorophyll degradation, especially in inoculated leaf disks, whereas, in untreated controls, the rate of degradation was almost the same in inoculated and noninoculated leaves by 0.1-1.0 ppm ethylene treatments. Peroxidase activity per unit fresh weight of leaf disks was decreasedby lo-15 % by ethylene treatment. However, TMV multiplied to the same extent or a little more in treated leaf disks as compared with untreated. Table 5 in-

6

NAKAGAKI,

HIRAI,

AND STAHMANN

TABLE EFFECT

3

OF INHIBITORS

Inhibitor Noninoculated Inoculated Untreated DIECA 10-s iM CysHCl lo-3 M EDTA 10-a M Noninoculated Inoculated Untreated PM 25 wm BcS 0.05 ppm

ON ETHYLENE PRODUCTION BY LEAVES SAMSUN nc AND Phaseolus vulgaris cv. KAIRYO

-

13.63 9.84 12.10 8.10

P. vulgaris

Ethylene hWm3

(0) (28) (11) (40)

3.8 3.4 3.5 3.7

4.67 (0) 0.76 (83) 0.96 (79)

*so

Lesions (No./cm2) -

0.60 (0) (11) (8) (2)

2.05 1.89 1.93 1.35

-

0.61

tabacum v&r.

OTEBO

N. tabacum a,~ ethylene Lesions (no./cmz) bJ/cm2) 0.62

Nicotiana

OF

(0) (8) (6) (46)

8.3 8.5 8.4 5.3 -

0.60

1.3 (0) 0.5 (62) 0.6 (53)

(0) (-1) (0) (36)

4.8 (0) 1.6 (66) 2.2 (55)

1.19 (0) 1.46 (-22) 0.82 (31)

a Three days after inoculation with 10 pg/ml TMV. b Two days after inoculation with 50 rg/ml TMV. c Figures in parentheses show the percentage of inhibition. Figures represent average of 2 independent experiments, each of which consisted of 5 incubation chambers, 4 half-leaves each. TABLE EFFECT OF ETHYLENE ON CHLOROPHYLL CONTENT, TIVITY BY LEAF DISKS var. BRIGHT YELLOWS

4 TMV AND OF

Noninoculated

0

FIG. 5. Effect of blasticidin S (BcS) on ethylene production in leaves of bean, cv. Kairyo Otebo inoculated with TMV (10 rg/ml). Inoculated bean leaves were incubated in an incubation chamber containing filter paper and 10 ml of various concentrations of BcS. The amount of ethylene produced during l&hour incubation period after inoculation, before visible lesion appearance, is expressed as 100%. Average of 3 independent experiments, each of which consisted of 4 incubation chambers, 4 half-leaves each. Total accumulation of ethylene during the interval indicated.

dicates that ethylene, which was applied to tobacco leaf disks separately at different infection stages, did not inhibit TMV synthesis.

0.1 1.0 10 100

38 37 34 28 24

0.179 0.170 0.170 0.156 0.157

MULTIPLICATION, PEROXIDASE

Nicotiana

Actabacum

hocuhtedb

27 25 21 18 17

0.142 0.149 0.140 0.136 0.125

0.100 0.104 0.108 0.127 0.115

5 Inoculated and noninoculated tobacco leafdisks (30 X 12 mm in diameter) were incubated for 5 days in a chamber containing various concentrations of ethylene. Figures represent averages of 3 independent experiments. a Inoculation with 100 pg/ml TMV.

Bean half-leaves were preincubated with 0.1-100 ppm of ethylene for 6, 15, 24, 36, and 48 hours and then inoculated with 50 pg/ml of TMY. The leaves were transferred to ethylene-free petri dishes containing 10 ml of distilled water and incubated for 48 hours.

ETHYLENE

PRODUCTION

In another experiment, after inoculation with T&IV, bean leaves were incubated in various concentrations of ethylene for 2 days. Table 6 indicates that relative number of TABLE EFFECT

OF THE

MENT DISKS

ON OF

5

DURATION

TMV

OF ETHYLENE

TREAT-

MULTIPLICATION

BY

tabacum

Nicotiana

V&I-.

LEAF BRIGHT

BY

DETACHED

LEAVES

7

lesions (treated/untreated) produced on bean leaves increased a little by preincubation for 15-36 hours at lo-100 ppm of ethylene. However, as preincubation time was prolonged, the leaves became yellowish along the veins and eventually cells were killed. The number of lesions produced on inoculated leaves was not altered by the postinoculation treatment.

YELLOW

DISCUSSION

Treated

Ethylene

period

bpm)

(days)

Untreated period (days)

TMV concentration ODdlO o/0 disks

0 1 10 100

5 4 4 4

0.594 0.605 0.684 0.545

100 101.7 115.1 91.7

0 1 10 100

5 2 2 2

0.731 0.709 0.685 0.633

100 97.0 93.7 87.0

0 1 10 100

5 0 0 0

1.003 1.082 1.271 1.153

100 107.9 126.7 115.0

a Figures represent averages of 3 independent experiments with 30 leaf disks each. Thirty leaf disks (12 mm in diameter) were incubated for 5 days in an incubation chamber containing various concentrations of ethylene or ethylene-free air, and 10 leaf disks each were used for determination of virus content. TABLE

6

EFFECT OF EXOGENOUSLY INTRODUCED ON LOCAL LESION FORMATION ON

dgaris Etbylene C!Jpm)

1 10 100

cv.

KAIRYO

Number

of local

Preincubation

(hr)

ETHYLENE

Phaseolus

OTEBO lesionsa Postincubation (hr)

6

15

24

36

48

48

103 114 106

125 117 138

124 137 152

122 120 119

90 75 60

88 98 92

Q Per cent of untreated. Preincubation: incubation before inoculation. Postincubation: incubation after inoculation. Average of 3 independent experiments, each of which consisted of 4 incubation chambers, 4 half-leaves each.

Ross and Williamson (1951) demonstrated that abnormally high levels of ethylene were produced with necrotic local lesions, but not with systemic infection. In the present experiments, ethylene production was enhanced only after the development of lesions, but not before lesion appearance nor in the systemic hosts showing no senescence.It seems,therefore, that the increase in ethylene production is correlated with the necrotization of cells invaded by viruses. The rate of ethylene production increased with increasing number of lesions and then decreased at the later infection stages. Therefore, increased ethylene production does not reflect rapid virus synthesis, but reflects the development of necrotic local lesions. Induction of cell necrosis by chemicals revealed that ethylene production was enhanced in leaves mildly injured by chemicals such as NiC&, HgC12, and CuC12, but not as much in leaves drastically damaged by TCA and PCA. Thus, the presence of injured but not necrotic cells may be necessary for ethylene production. These results are consistent with those in injured and black rot-infected sweet potatoes (Imaseki et al., 1968a, c). Much evidence has accumulated on the close relation between virus-induced lesion development and activation of oxidative enzymes (Farkas et al., 1960; Kikuchi and Yamaguchi, 1960; Farkas and Stahmann, 1966; Farkas and Solymosy, 1965; John and Weintraub, 1967; Solymosy and Farkas, 1963). Enhanced ethylene production around local lesions, in senescent tissues, or in tissues injured by poisons may be correlated with increased oxidative activities (Farkas and Stahmann, 1966; Novacky and Hampton, 1968; Solymosy et al., 1967). In recent

8

NAKAGAKI,

HIRAI,

AND STAHMANN

years, enzymatic formation of ethylene from GRIEVE, B. J. (1942). Further observations on rose wilt virus. Proc. Roy. Sot. Victoria 54,229-238. methional by extracts of cauliflower (Mapson and Wardale, 1967) or by extracts of GRIEVE, B. J. (1943). Mechanism of abnormal and pathological growth. A review. Proc. Roy. Sot. pea seedlings (Ku et al., 1967) has been Victoria 55, 109-132. described and horseradish peroxidase was IMASEKI, H., ASAHI, T., and URITANI, I. (1967). shown to participate in this ethylene bioInvestigations on the possible inducers of metasynthesis. Among various inhibitors tested, bolic changes in injured plant tissues. In “BioEDTA was most effective in inhibiting chemical Regulation in Diseased Plants or Inethylene production. PM and BcS also jury” (T. Hirai, J. Hidaka, and I. Uritani, eds.), inhibited ethylene production as well as pp. 189-201. Phytopathology Society of Japan, Tokyo. lesion formation. Therefore, ethylene proH., TERANISHI, T., and URITANI, I. duction seems to be mediated through de IMASEKI, (1968a). Production of ethylene by sweet potato novo synthesis of oxidative enzymes, probaroots infected by black rot fungus. Plant Cell bly peroxidase. Physiol. 9, 769-781. Stahmann et al. (1966) suggested that IMASEKI, H., UCHIYAMA, M., and URITANI, I. exogenously introduced ethylene can induce (1968b). Effect of ethylene on the inductive inan increase in peroxidase and polyphenol crease in metabolic activities in sliced sweet oxidase activities in sweet potato tissues, potato roots. Agr. Biol. Chem. 32,387-389. leading to a blocking of further penetration IMASEKI, H., URITANI, I., and STAHMANN, M. A. (1968c). Production of ethylene by injured of the tissues by pathogens. In tobacco sweet potato root tissue. Plant Cell Physiol. 9, leaves, exogenously applied ethylene slightly 757-768. inhibited peroxidase activity, but did not JOHN, V. T., and WEINTRAUB, M. (1967). Phenoaffect the rate of TMV multiplication. lase activity in Nicotiana glut&rosa infected Preincubation or postincubation in ethylene with tobacco mosaic virus. Phytopathology 57, of infected leaves with local lesions did not 154-158. decrease the number of lesions caused by KIKUCHI, M., and YAMAGUCHI, A. (1960). PolyTMV. Thus, in virus infection, ethylene phenol oxidase activity of Nicotiana glutinosa seems not to exert an inhibitory action on leaves infected with tobacco mosaic virus. TMV synthesis, but is correlated with the Nature 187, 1048-1049. necrotization of tissues caused by virus Ku, H. S., YANG, S. F., and PRATT, H. K. (1967). Enzymic evolution of ethylene from methional infection. ACKNOWLEDGMENTS The authors are indebted to Professor Dr. I. Uritani and Dr. H. Imaseki, Laboratory of Biochemistry, Nagoya University, for helpful discussions and for supplying ethylene gas. REFERENCES D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidases in Beta vul-

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garis. BURQ,

Plant

Physiol.

24,1-15.

S. P. (1962). The physiology of ethylene formation. Ann. Rev. Plant Physiol. 13,%X-302. FARKAS, G. L., and SOLYMOSY, F. (1965). Host metabolism and symptom production in virusinfected plants. Phytopathol. 2. 53,85-93. FARKAS, G. L., and STAHMANN, M. A. (1966). On the nature of changes in peroxidase isozymes in bean leaves infected by southern bean mosaic virus. Phytopathology 56,669-677. FARKAS, G. L., KIR~LY, Z., and SOLYMOSY, F. (1960). Role of oxidative metabolism in the localization of plant viruses. Virology 12, 40% 421.

by a pea seedling extract. Arch. Biochem. Biophys. 118, 756-758. LIEBERMAN, M., and KUNISHI, A. T. (1967). Originsof ethyleneinplants.l;n “Biochemical Regulation in Diseased Plants or Injury” (T. Hirai, J. Hidaka, and I. Uritani, eds.), pp. 165-179. Phytopathology Society of Japan, Tokyo. MAPSON, L. W., and WARDALE, D. A. (1967). Biosynthesis of ethylene. Formation of ethylene from methional by a cellfree enzyme system from cauliflower florets. Biochem. J. 102, 574585. NOVACKY, A., and HAMPTON, R. E. (1968). Peroxidase isozymes in virus-infected plants. Phytopathology

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38,930-932.

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ETHYLENE

PRODUCTION

W. H., MEIGH, D. F., and PARKER, J. C. (1964). Effect of damage and fungal infection on the production of ethylene by carnation. Nature 204,92-93. SOLYMOSY, F., and FARKAS, G. L. (1963). Metabolic characteristics at the enzymatic level of tobacco tissues exhibiting localized acquired resistance toviralinfection. ViroZogy21,210-221. SOLYMOSY, F., SZIRMAI, J., BECZNER, L., and FARKAS, G. L. (1967). Changes in peroxidaseisozyme patterns induced by virus infection. Virology 32, 117-121. STAHMANN, M. A., CLARE, B. G., and WOODBURY, W. (1966). Increased disease resistance and enSMITH,

BY DETACHED

LEAVES

9

zyme activity induced by ethylene and ethylene production by black rot infected sweet potato tissue. Plant Physiol. 41,1505-1512. TANICUCHI, T. (1962). A rapid method for microanalytical determination of the amount of tobacco mosaic virus in plant tissues. Nature 194, 708. WILLIAMSON, C. E. (1950). Ethylene, a metabolic product of diseased or injured plants. Phytopathology 40, 205-208. YANG, S. F. (1967). Biosynthesis of ethylene. Ethylene formation from methional by horseradish peroxidase. Arch. Biochem. Biophys. 122, 481-487.