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Inhibition of endogenous ethylene biosynthesis by L-canaline in leaves of Poa pratensis infected by Bipolaris sorokiniana
Physiological
and Molecular
Inhibition L-canaline Bipolaris
Plant Palhology
( 1994)
301
44, 30 I-309
of endogenous ethylene biosynthesis by in leaves of Poa pratensis infected by sorokiniana
C. F. HODGES and D. A. Departmenl
of Hor!icultrre,
(Acceptedfor
publication
CAMPBELL
Iowa Stale Unioersi&v, Ames, Iowa 50011, U.S.A. March
1994)
L-canaline was evaluated for its elfcct on hyphal growth of B$olaris sorokiniana and on ethylene biosynthesis in leaf blades of Poa pratensir infected by B. sorokiniana. L-canaline was slightly inhibitory of hyphal growth at 10e4 M; concentrations of IO-s, IO-s, 10-r’ and 10-r’ M had no effect. Application of L-canaline to leaf blades of P. prafensis did not inhibit infection or lesion development by B. sorokiniana. The ability of L-canaline to decrease ethylene biosynthesis in inoculated leaf blades varied with concentration, time, and progressive development of the disease. L-canaline at IO-“ and 10-s M decreased ethylene biosynthesis in inoculated leaf blades at each 24-h sampling period for 96 h. L-canaline at 10-s M decreased ethylene biosynthesis through 72 h after inoculation, and at 10-t’ M it decreased ethylene through 48 h after inoculation. As lesion development progressed from 48 to 96 h, the more dilute concentrations of L-canaline failed to decrease ethylene biosynthesis; 10-s M failed at 96 h and 10-r” M failed at 72 and 96 h. Lcanaline at IO-” M did not decrease ethylene at any time. Chlorophyll retention at 96 h after inoculation by leaf blades treated with L-canaline at IO-” and 10-s M was 80 and 81 o/0 of the control, respectively. More dilute concentrations did not increase the retention of chlorophyll by inoculated leaves during pathogenesis.
INTRODUCTION
L-a-amino-y-[aminooxyl-n-butyric acid (L-canaline) is hydrolytically cleaved by arginase from the non-protein amino acid 2-amino-4-(guanidinooxy)-butyric acid (Lcanavanine) [21]. Both L-canaline and L-canavanine are toxic to someinsects [21-231 and L-canaline inhibits the action of pyridoxal phosphate-dependent enzymes [3,18, 201. Inhibition of pyridoxal phosphate-dependent enzymes prevents ethylene biosynthesis in higher plants [2, 12, 13,291. The biosynthesis of ethylene in higher plants progressesfrom methionine (Met) + S-adenosyl-L-methionine (AdoMet) + l-aminocyclopropane- 1-carboxylic acid (ACC) + C,H, [29] and the conversion of AdoMet to ACC is mediated by the pyridoxal phosphate-dependent enzyme ACC synthase [I]. Of the pyridoxal enzyme inhibitors, L-canaline has had relatively little study compared to aminooxyacetic acid (AOA) and aminoethoxyvinylglycin (AVG) [2, 12,291. Abbreviations used in text: ACC, I-aminocyclopropane-I-carboxylic methionine; AOA, aminooxyacetic acid; AVG, aminoethosyvinylglycin; [aminooxyl-n-butyric acid; L-canavanine, 2-amino-4-(guanidinooxy)-butyric methionine; MSD, minimum significant difference; NBD, 2,5-norbornadiene. 0885-5765/94/0400301+09 20
$08.00/O
acid;
AdoMet, S-adenosyl-LL-canaline, L-a-amino-yacid; Chl, chlorophyll; Met,
0
1994 Academic
Press Limited MPP44
C. F. Hodges and D. A. Campbell 302 Plants infected by fungal pathogens often produce endogenousethylene in excessof that in healthy plants [I&17]. Endogenous ethylene of Poa pratensis L. leaf blades infected by Bipolaris sorokiniana (Sacc.) Subram. and Jain increases and peaks at 48-72 h after inoculation and the increase is correlated with a lossof chlorophyll (Chl) during pathogenesis [8,9]. Exposure of inoculated leaf blades to hypobaric pressure [9,14] or to 2,5-norbornadiene (NBD) [8] decreasesethylene-induced Chl lossduring pathogenesis.Hypobaric pressurereduces ethylene retention by inoculated leaf blades and thereby prevents its mode of action [5, 71. NBD does not prevent the biosynthesis of ethylene, but it too effectively prevents the mode of action of ethylene [26,27]. Neither hypobaric evacuation of ethylene from infected tissue, nor the use of NBD provide a practical meansof decreasing ethylene biosynthesisand subsequent Chl loss from infected leaf blades during pathogenesis; i.e., the former method is restricted by physical requirements and, the latter method, by the volatile nature of NBD. Preliminary evaluations of pyridoxal enzyme inhibitors applied to the leaves of intact P. pratensis showed AVG, and to lesserextent AOA, to induce visible chlorosis of older leavesof the shoots; this chlorosis was not observed on leaves treated with L-canaline. This characteristic of L-canaline suggestedthat it might control ethylene biosynthesis during pathogenesisin this host-pathogen interaction. Such a responsecould minimize chlorosis as a symptom of infected leaves and possibly provide a new approach to diseasesymptom management. This study was initiated to determine the direct effect of L-canaline on the growth of B. sorokiniana, to determine the effect of foliar application of L-canaline on ethylene biosynthesis in leaf blades infected by B. sorokiniana, and to determine the effect of subsequent Chl lossfrom leaf blades during pathogenesis.
MATERIALS
AND METHODS
Plant materials P. pratensis L. cv. Newport was vegetatively propagated in a steamed loam-peat soil
substrate (2 : 1, v/v) in 7.6-cm square plastic pots. All plants were grown for a minimum of 60 days in the greenhouseunder natural light at a temperature range of 22-32 “C. Plants were fertilized weekly (N-P-K, 15-15-15) and maintained with a single tiller with four leaves. Only plants with the youngest visible leaf at least 10 cm long were used. B. sorokiniana (Sacc.) Shoem. was grown for 20 days on 20 ml of 3 o/oBacto agar in 15 x 100 mm sterile plastic Petri dishes before use. Treatments and inoculations
L-canaline (dipicrate salt) (Sigma Chemical Co., St Louis, Missouri 63178) was used at concentrations of 10e4, 10e6, 10-s, 10-l’ and lo-” M for all treatments and was evaluated for its effect on hyphal growth of B. sorokiniana, inhibition of ethylene biosynthesis in leaf blades of P. pratensis inoculated with B. sorokiniana, and for Chl retention in treated and inoculated leaf blades. Hyphal growth of B. sorokiniana on L-canaline was determined by preparing 3.0 o/o Bacto agar containing the various concentrations of L-canaline. The agar was autoclaved (20 min at 1.4 kg cm-’ at 121 “C) and placed on a magnetic stirrer for lo-15 min to cool, and then L-canaline was added to make each dilution. Twenty
Inhibition
of ethylene
biosynthesis
by L-canaline
303
millilitres of each dilution were poured into 15 x 100 mm plastic Petri dishes and solidified. Hyphae of B. sorokiniunawere introduced to the L-canaline agar plates by cutting 5-mm cores from 12-day-old cultures of the pathogen on 3 o/oBacto agar and placing the cores in the centre of the L-canaline agar plates. Hyphal growth was measured at 9 days by averaging three random diameter measurementsof the hyphal colonies and expressing their area as cm‘. The hyphal plates with each L-canaline treatment were replicated 10 times in a randomized complete block. An ANOVA and Waller-Duncan K-ratio I-test (P = O-05) were performed to determine minimum significant differences (MSD). The effect of L-canaline on endogenous ethylene biosynthesis in P. pratemis leaf blades inoculated with B. sorokiniana was determined by inserting four leaf blades of a single shoot into a previously described inoculation apparatus [9, 191, with their upper epidermis exposed for treatment and inoculation. A lo-cm section of each leaf blade was atomized with 2.4 ml of the appropriate dilution of L-canaline (an estimated 0.4 ml covered the surface of the four leaves). Two applications of L-canaline were made; the first 72 h before inoculation, and the second at the time of inoculation. Treated leaf blades were inoculated by placing three equally spaced 5-mm cylinders of B. sorokiniana on 3% Bacto agar (cut with a cork borer) onto their surface. Each cylinder was placed with the hyphae against the leaf blade surface and O-1 ml of sterile distilled water was applied to provide a water continuum between the hyphae and leaf blade surface. Inoculated plants and controls were placed in incubators at 22 “C with a 9-h photoperiod (40 pmol m-* s-l) and the leaf blades of each plant were analysed for ethylene at 24, 48, 72 and 96 h after inoculation, All treatments were replicated three times, and an ANOVA and Waller-Duncan K-ratio i-test (P = O-05) were performed to determine MSD within each sampling period (24, 48, 72 and 96 h) for ethylene content. Ethldene anal_),sis
The four leaf blades of each plant were removed from the inoculation apparatus, combined, and the endogenous ethylene extracted. Ethylene was determined by a modification of the vacuum extraction method for leaves developed by Beyer & Morgan [4], The modification consisted of the use of several culture tubes (25 x 200 mm) for collecting ethylene from multiple setsof leaf blades simultaneously [9]. A 100 pl sample of the gasesreleasedfrom leaves was removed from the collection tubes with a gas-tight syringe and injected into a Varian 3700 GC with a 2-m activated alumina column (60/80 mesh) and flame ionization detector (FID). Injector and detector temperatures were both 250 “C, oven temperature was 110 “C, and carrier gas (He) flow rate was 30 cm3 min-‘. The signal of the FID was connected to a Cary model 401 electrometer coupled to a Spectra-Physics 4100 computing integrator. After calibration, sample data were plotted and quantified by the computing integrator and expressedas ~1 1-l ethylene. Chlorophyll
analysis
Chlorophyll content of the four 10 cm leaf blade sectionsof each plant subjected to the various treatments was determined by a modification of the method of Knudson et al. [IO]. Only the leaf blades from the 96 h treatments were analysed for Chl content. 20-Z
C. F. Hodges
304
and D. A. Campbell
Leaves were cut into small pieces after ethylene extraction, washed three times in distilled water to remove (NH,),SO, from the ethylene extraction procedure, and freeze-dried (48 h, -48 “C, 50 pl Hg). After dry weight determination, the leaf blade tissuewas placed in 10 ml of 99 o/oethanol and the Chl extracted in the dark for 24 h. The 24-h ethanol extractions were repeated three times and combined (30 ml total). Chlorophyll absorbency was measuredspectrophotometrically at 665 and 649 nm, and calculations were made to determine total Chl [IO]. Chlorophyll concentrations were determined as pg mg-’ leaf d. wt, and the Chl toncentration of inoculated and/or treated leaf blades was expressed as a percentage of the Chl in non-treated, noninoculated leaf blades. An ANOVA and Waller-Duncan K-ratio l-test (P = O-05) were performed to determine MSD between treatments for Chl content at 96 h after inoculation. RESULTS Hyphal growth
The mean area of hyphal growth of g-day-old control cultures of B. sorokiniana on 3 y. Bacto agar was 39.9 cm’ (Fig. 1). L-canaline at 10m4M induced a slight, but significant inhibition of hyphal growth (36.6 cm’). Hyphal growth on L-canaline at 10e6, lo-‘, lo-lo and 10-l” M ranged from 39.4 to 40.8 cm’ and did not differ from the control.
L
MSD
= 2.69
35 2 5
30
’
25
5l
E
2o 15
FIG. 1. Effect of various dilutions of L-canaline (CAN) on the hyphal growth of Bipolaris mrokiniaaa in t&o on 3 o/0 Bacto agar. An ANOVA and Wailer-Duncan K-ratio f-test (P = @OS) were performed to determine minimum significant differences (MSD).
inhibition
of ethylene
biosynthesis
305
by L-canaline
Ethylene production
Endogenous ethylene levels in non-treated, non-inoculated control leaf blades remained relatively constant from one 24-h sampling period to another; mean levels of ethylene at 24, 48, 72 and 96 h ranged from 0.29 to 0.31 @I-’ [Fig. 2(a-d)]. Endogenous MSD
= 0.64
(b)
0.8 0.6 0.4 0.2 0.0 z
2 ii
1.6 1.4 1.2
MSD = 0.46
MSD = 0.52
Cd)
1.0 i 0.8 0.6 0.4 0.2 0.0
Treatment FIG. 2. Endogcnous ethylene of the internal atmosphere of Pea ,!wafensis leaf blades infected by Bipoloris soroil-~~krt~o (B.S.). Leaf blades were treated with IO-“, IO-s, 10e8, IO-r0 or IO-‘? M Lcanalinc (CAN), and ethylene content was determined at (a) 24, (b) 48, (c) 72 and (d) 96 II aftct inoculation as pathogcnesis progressed. An ANOVA and Wailer-Duncan K-ratio f-test (P = 0.05) were performed to dctcrminc minimum significant differences (MSD) at each 24-h sampling period.
ethylene in non-inoculated leaf blades remained unchanged or decreasedin responseto the various concentrations of L-canaline. Ethylene content of non-inoculawd leaf blades was decreased by IO-“ M L-canaline at 24, 48, 72 and 96 h, and by IO-” M at 24, 48 and 72 h. L-canaline at 10-s M decreased the ethylene content of non-inoculated leaf blades only at 72 h and L-canaline at lo-” and 10-l’ M had no effect on the ethylene content. Endogenous ethylene in non-treated, inoculated leaf blades increased progressively from 24 to 48 h, peaked at 72 h (1.66 ~1 l-l), and declined after 96 h [Fig. 2(a-d)]. Ethylene content of inoculated leaf blades treated with lo-” and 10d6M L-canaline did not differ from that of the non-treated, non-inoculated leaf blades at 24 h [Fig. 2(a)]. Inoculated leaf blades treated with more dilute concentrations of L-canaline ( IOVH,
C. F. Hodges 120
MSD
and D. A. Campbell
= i.64
Treatment Fro 3. Chlorophyll content of Pan prokwi~ leaf blades treated with IO-“, IO-s, IO-*, 10-t’ or 10-r’ M L-canaline (CAN) and inoculated with Bipoloris sorokiniana (B.S.). Leaf blades of each shoot were pooled, and chlorophyll (Chl) content was determined at 96 h after inoculation. All treatment values were calculated against the Chl content of non-inoculated, non-treated control leaves (8.6 pg mg-’ leaf d. wt = 100°/a). An ANOVA and Wailer-Duncan K-ratio f-test (P = 0.05) were performed to determine minimum significant differences (MSD).
10-l’ and lo-‘* M) had ethylene levels greater than that of non-treated, non-inoculated leaf blades at all 24-h sampling periods [Fig. 2(a-d)]. Inoculated leaf blades treated with L-canaline at lo-’ and 10m6M had ethylene levels below that of non-treated, inoculated leaf blades at each 24-h sampling period [Fig. 2(a-d)]. Inoculated leaf blades treated with L-canaline at lo-* M also decreased ethylene relative to the non-treated, inoculated leaf bladesat all 24-h sampling periods, except at 96 h [Fig. 2(d)]. I noculated leaf blades treated with L-canaline at 10-l’ M decreasedethylene relative to the non-treated, inoculated leaf blades at 24 and 48 h [Fig. 2(a and b)]; at 72 h, the ethylene was greater than that in the non-treated, inoculated leaf blades, and at 96 h, it did not differ from the non-treated, inoculated leaf blades [Fig. 2(c and d)]. The ethylene responsein inoculated leaf blades treated with L-canaline at lo-‘* M did not differ from, or was greater than, that of the non-treated, inoculated leaf blades [Fig. 2(a-d)].
Inhibition
of ethylene
Leaf ctdo~oplyll
biosynthesis
by L-canaline
307
conlenl
Non-treated, non-inoculated leaf blades had a mean Chl content of 8.6 l.tg mg-’ leaf d. wt (= 1009,) at 96 h. The Chl content of non-inoculated leaf blades treated with the various concentrations of L-canaline did not differ from that of the non-treated, non-inoculated leaf blades (Fig. 3). Chlorophyll content of non-treated leaf blades inoculated with B. sorokiniana was 63% of that in non-treated, non-inoculated leaf blades 96 h after inoculation (Fig. 3). Retention of Chl by inoculated leaf blades treated with 1OeJ and 10-OM L-canaline was 89 and 81 %, respectively, and significantly greater than that in the non-treated, inoculated leaf blades. Chlorophyll retention by inoculated leaf blades treated with lOmEM L-canaline increased slightly but not significantly so relative to the non-treated, inoculated leaf blades, and Chl retention in inoculated leaf blades treated with L-canaline at 10-i’ and 10-l’ M did not differ from the non-treated, inoculated leaf blades (Fig. 3). DISCUSSION
L-canaline decreasesbiosynthesisof endogenousethylene and subsequent lossof Chl by P. ,ha/ensis leaf blades infected by B. sorokiniana without preventing typical infection and lesion development by the pathogen. The toxic nature of L-canaline [Z&23] did not affect the growth of P. pralensis. After removal of treated and infected leaf blades for ethylene and Chl analyses, the plants were returned to the greenhouse where their typical growth pattern was unaffected over a 3-6-month period. Hyphal growth of B. sorokiniana is slightly inhibited by L-canaline at lo-’ M, but the inhibition did not affect the infection process.More dilute concentrations of L-canaline had no effect on hyphal growth. L-canavanine affects the development of some fungi by inhibiting hyphal growth and sporulation, altering morphology, and inducing mutations [6, II, 24,2.5, 281, but there are no reported observations on the effects of L-canavanine on B. sorokiniana, and L-canaline has not been evaluated for its effects on the growth and development of fungal organisms. The ability of L-canaline to decreaseethylene biosynthesis in P. pralensis leaf blades in response to infection by B. sorokiniana is a function of concentration, time and the progression of the pathogen in the infected tissue. Only concentrations of L-canaline at lo-’ and low6M were effective at decreasing biosynthesis of ethylene in infected leaf blades over the entire 96 h of the study. The more dilute concentrations of L-canaline neither decreasedethylene production nor the development of the pathogen in the leaf blades. The ethylene levels found in this study in the infected leaf blades were derived from the combined internal atmospheresof the four inoculated leaf blades of each plant. In this host-pathogen interaction, ethylene produced in leaf blades-in responseto infection varies with leaf age [8, 1.51.Typically, ethylene biosynthesis is greatest in the youngest inoculated leaf blade and decreasesin each older inoculated leaf blade. The oldest leaf blades typically respond to infection with the lowest ethylene surge, although they showed the greatest sensitivity to ethylene and respond with the greatest loss of Chl [8,15]. The inability of older leaves to produce high levels of ethylene in responseto infection combined with the greater lossof Chl suggeststhat their level of metabolic activity is much below that of younger leaves. These leaf-age related responsessuggest
308
C. F. Hodges
and D. A. Campbell
that the ability of L-canaline (and perhaps other pyridoxal enzyme inhibitors) to control ethylene biosynthesisand Chl lossin infected leaf blades could differ in leaves of different ages. The observations made in the present study suggest, however, that symptom expression in the form of leaf chlorosis might be substantially reduced by physiologically blocking ethylene biosynthesisduring pathogenesis.Continued work in this area might provide methods specifically designedfor the management of symptom expressionby diseasedplants. Such a management system could be especially useful for foli’ar diseaseslike B. sorokinian~leaf spot which a’re difficult to control but where the aesthetic value of the grassplant is of major importance. This is paper No. J-15615 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011 (Project 3193). Supported, in part, by a grant from the United States Golf Association, Green Section. REFERENCES 1. Adams DO, Yang SF. 1979. Ethylene biosynthesis: identification of I-aminocyclopropane-lcarboxylic acid as an intermediate in the conversion of methionine to ethylene. Proceedings of fhe N&ml Academy ojSciences of the U.S.A. 76: 170-l 74. 2. Amrhein N, Wenker D. 1979. Novel inhibitors of ethylene production in higher plants. Plant and Cell Physiolou 20 : 1635-l 642. 3. Be&r T, Churchich JE. 1976. Reactivity of phosphopyridoxal groups of cystathionase. journal of Biological Chemislry 251: 5267-5271. 4. Beyer EM Jr, Morgan PW. 1970. A method for determining the concentration of ethylene in the gas phase of vegetative plant tissue. Plan/ Physiology 46: 352-354. 5. Burg SP, Burg EA. 1965. Gas exchange in fruits. Plan1 Physiology 18: 870-874. 6. Cbilds EA, Ayres JC, Koehler PE. 1971. Differentiation in AspergillusJlavus as influenced by Lcanavanine. Mycologia 63: 181-184. 7. Davis JE, McKetta JJ. 1960. Solubility or ethylene in water. Journal of Chemical Engineering Data 5: 374-375. 8. Hodges CF. 1990. Endogenous ethylene response and chlorophyll loss in sequentially older leaves oTPoa profensis infected by Bipolaris sorokiniana. 30um41 of Plan! Physiology 136: 670-674. 9. Hodges CF, Coleman LW. 1984. Ethylene-induced chlorosis in the pathogenesis ofBipolaris sorokiniana leaf spot of Pea prulensis. Plua~ Physiology 75 : 462-465. IO. Knudson LL, Tibbitts TW, Edwards GE. 1977. Measurement of ozone injury by determination of chlorophyll concentration. Plant P/~siolo~ 60: 606-608. Il. Lewis CM, Tarrant GM. 1971. Induction of mutation by 5-Ruorouracil and amino acid analogs in Uslilago maydis. Adulation Research 12: 349-356. 12. Lieberman M. 1979. Biosynthesis and action ofethylene. Annual Review of Plan1 Plysiolo~ 30: 5X3-591. 13. Murr DP, Yang SF. 1975. Inhibition of in uiuo conversion of methionine to ethylene by L-canaline and 2,4-dinitrophenol. Plan1 Physiology 55: 79-82. 14. N&en RN, Hodges CF. 1983. Hypobaric control of ethylene-induced leaf senescence in intact plants of Phaseolus vulgaris L. Planl Physiolou 71: 96- 10 I. 15.Nilsen KN, HodgesCF, Madsen JP. 1979. Pathogenesis of Drechslera sorokiniana leaf soot on progressively older leaves of Pea pensis as influenced by photoperiod and light quality. Phys;ologicol Plant Pathology 15: 171-176. 16. Pegg GF. 197%. The involvement of ethylene in pathogenesis. In: Heitefus R, Williams PH, eds. Physiological Plan1 Patholoar, Encyclopedia of Plant Plysiology. New Series, Vol. 4. New York: Springer Verlag, 582-59 1. 17. Pegg GF. 1981. The involvement of growth regulators in the diseased plant. In: Ayres PG, ed. Effecb of Disease on the Physiology of the Growing Plonf. Societyfor Experimental Biology, Seminar Series II. New York: Cambridge Universitv Press. 149-177. 18. Rahiala E-i, Kekomi M, J&me J, Riina A, Riiihii NCR. 197 I. Inhibition of pyridoxal enzymes by L-canaline. Biochimica et Biop&ca Acla 227: 337-343. 19. Robinson PW, Hodges CF. 1976. An inoculation apparatus for evaluation ofBipolurir sorokiniana lesion development on progressively older leaves of Pea pralensis. Phylopafholo~ 66: 360-362.
lnhibition
of ethylene
biosynthesis
by L-canaline
309
20. Rosenthal GA. 1981. A mechanism ofl-canaline toxicity. European Journal oJEiochemi.rfry 114: 301-304. 21. Rosenthal GA. 1990. Metabolism of L-canavanine and L-canaline in leguminous plants. Pfarf Pl~siology 94 : l-3. 22. Rosenthal GA. 1991. The biochemical basis for the deleterious effects of L-canavanine. PhyW~~isfry 30: 1055-1058. 23. Rosenthal GA, Betge MA, Bleiler JA. 1989. A novel detoxification of L-canaline. Biochemical System&s and Ecology 17: 203-206. 24. Samborski DJ, Forsyth FR. 1960. Inhibition of rust development on detached wheat leaves by metabolites, antimetabolites, and enzyme poisons. Conadion Journal of Botany 38: 467-476. 25. Shepherd CJ, Mandryk M. 1964. Effects of metabolities and antimetabolites on the sporulation of Peronospora labacina Adam. on tobacco leaf disks. Arrslralian journal of Biological Sciences 17: 878-891. 26. Sisler EC, Pain A. 1973. Effect of ethylene and cyclic olefins on tobacco leaves. Tobacco Science 17: 68-72. 27. Sisler EC, Yang SF. 1984. Anti-ethylene effects of cis-2-butene and cyclic olefins. Phylochemisfry 23: 2765-2768. 28. Toshikazu T, Irikura H, Naito N. 1971. Inhibition of uredosorus formation of Puccinia coronak~ by plant growth regulators and antimetabolites. Technical Bullelin of I;hcul(y of Agriculfure, Kagawa Universi~ 23: 42-50. 29. Yang SF, Hoffman NE. 1984. Ethylene biosynthesis and its regulation in higher plants. Annual Reoiew of Plant Plpiology 35 : l55- 189.
and Molecular
Inhibition L-canaline Bipolaris
Plant Palhology
( 1994)
301
44, 30 I-309
of endogenous ethylene biosynthesis by in leaves of Poa pratensis infected by sorokiniana
C. F. HODGES and D. A. Departmenl
of Hor!icultrre,
(Acceptedfor
publication
CAMPBELL
Iowa Stale Unioersi&v, Ames, Iowa 50011, U.S.A. March
1994)
L-canaline was evaluated for its elfcct on hyphal growth of B$olaris sorokiniana and on ethylene biosynthesis in leaf blades of Poa pratensir infected by B. sorokiniana. L-canaline was slightly inhibitory of hyphal growth at 10e4 M; concentrations of IO-s, IO-s, 10-r’ and 10-r’ M had no effect. Application of L-canaline to leaf blades of P. prafensis did not inhibit infection or lesion development by B. sorokiniana. The ability of L-canaline to decrease ethylene biosynthesis in inoculated leaf blades varied with concentration, time, and progressive development of the disease. L-canaline at IO-“ and 10-s M decreased ethylene biosynthesis in inoculated leaf blades at each 24-h sampling period for 96 h. L-canaline at 10-s M decreased ethylene biosynthesis through 72 h after inoculation, and at 10-t’ M it decreased ethylene through 48 h after inoculation. As lesion development progressed from 48 to 96 h, the more dilute concentrations of L-canaline failed to decrease ethylene biosynthesis; 10-s M failed at 96 h and 10-r” M failed at 72 and 96 h. Lcanaline at IO-” M did not decrease ethylene at any time. Chlorophyll retention at 96 h after inoculation by leaf blades treated with L-canaline at IO-” and 10-s M was 80 and 81 o/0 of the control, respectively. More dilute concentrations did not increase the retention of chlorophyll by inoculated leaves during pathogenesis.
INTRODUCTION
L-a-amino-y-[aminooxyl-n-butyric acid (L-canaline) is hydrolytically cleaved by arginase from the non-protein amino acid 2-amino-4-(guanidinooxy)-butyric acid (Lcanavanine) [21]. Both L-canaline and L-canavanine are toxic to someinsects [21-231 and L-canaline inhibits the action of pyridoxal phosphate-dependent enzymes [3,18, 201. Inhibition of pyridoxal phosphate-dependent enzymes prevents ethylene biosynthesis in higher plants [2, 12, 13,291. The biosynthesis of ethylene in higher plants progressesfrom methionine (Met) + S-adenosyl-L-methionine (AdoMet) + l-aminocyclopropane- 1-carboxylic acid (ACC) + C,H, [29] and the conversion of AdoMet to ACC is mediated by the pyridoxal phosphate-dependent enzyme ACC synthase [I]. Of the pyridoxal enzyme inhibitors, L-canaline has had relatively little study compared to aminooxyacetic acid (AOA) and aminoethoxyvinylglycin (AVG) [2, 12,291. Abbreviations used in text: ACC, I-aminocyclopropane-I-carboxylic methionine; AOA, aminooxyacetic acid; AVG, aminoethosyvinylglycin; [aminooxyl-n-butyric acid; L-canavanine, 2-amino-4-(guanidinooxy)-butyric methionine; MSD, minimum significant difference; NBD, 2,5-norbornadiene. 0885-5765/94/0400301+09 20
$08.00/O
acid;
AdoMet, S-adenosyl-LL-canaline, L-a-amino-yacid; Chl, chlorophyll; Met,
0
1994 Academic
Press Limited MPP44
C. F. Hodges and D. A. Campbell 302 Plants infected by fungal pathogens often produce endogenousethylene in excessof that in healthy plants [I&17]. Endogenous ethylene of Poa pratensis L. leaf blades infected by Bipolaris sorokiniana (Sacc.) Subram. and Jain increases and peaks at 48-72 h after inoculation and the increase is correlated with a lossof chlorophyll (Chl) during pathogenesis [8,9]. Exposure of inoculated leaf blades to hypobaric pressure [9,14] or to 2,5-norbornadiene (NBD) [8] decreasesethylene-induced Chl lossduring pathogenesis.Hypobaric pressurereduces ethylene retention by inoculated leaf blades and thereby prevents its mode of action [5, 71. NBD does not prevent the biosynthesis of ethylene, but it too effectively prevents the mode of action of ethylene [26,27]. Neither hypobaric evacuation of ethylene from infected tissue, nor the use of NBD provide a practical meansof decreasing ethylene biosynthesisand subsequent Chl loss from infected leaf blades during pathogenesis; i.e., the former method is restricted by physical requirements and, the latter method, by the volatile nature of NBD. Preliminary evaluations of pyridoxal enzyme inhibitors applied to the leaves of intact P. pratensis showed AVG, and to lesserextent AOA, to induce visible chlorosis of older leavesof the shoots; this chlorosis was not observed on leaves treated with L-canaline. This characteristic of L-canaline suggestedthat it might control ethylene biosynthesis during pathogenesisin this host-pathogen interaction. Such a responsecould minimize chlorosis as a symptom of infected leaves and possibly provide a new approach to diseasesymptom management. This study was initiated to determine the direct effect of L-canaline on the growth of B. sorokiniana, to determine the effect of foliar application of L-canaline on ethylene biosynthesis in leaf blades infected by B. sorokiniana, and to determine the effect of subsequent Chl lossfrom leaf blades during pathogenesis.
MATERIALS
AND METHODS
Plant materials P. pratensis L. cv. Newport was vegetatively propagated in a steamed loam-peat soil
substrate (2 : 1, v/v) in 7.6-cm square plastic pots. All plants were grown for a minimum of 60 days in the greenhouseunder natural light at a temperature range of 22-32 “C. Plants were fertilized weekly (N-P-K, 15-15-15) and maintained with a single tiller with four leaves. Only plants with the youngest visible leaf at least 10 cm long were used. B. sorokiniana (Sacc.) Shoem. was grown for 20 days on 20 ml of 3 o/oBacto agar in 15 x 100 mm sterile plastic Petri dishes before use. Treatments and inoculations
L-canaline (dipicrate salt) (Sigma Chemical Co., St Louis, Missouri 63178) was used at concentrations of 10e4, 10e6, 10-s, 10-l’ and lo-” M for all treatments and was evaluated for its effect on hyphal growth of B. sorokiniana, inhibition of ethylene biosynthesis in leaf blades of P. pratensis inoculated with B. sorokiniana, and for Chl retention in treated and inoculated leaf blades. Hyphal growth of B. sorokiniana on L-canaline was determined by preparing 3.0 o/o Bacto agar containing the various concentrations of L-canaline. The agar was autoclaved (20 min at 1.4 kg cm-’ at 121 “C) and placed on a magnetic stirrer for lo-15 min to cool, and then L-canaline was added to make each dilution. Twenty
Inhibition
of ethylene
biosynthesis
by L-canaline
303
millilitres of each dilution were poured into 15 x 100 mm plastic Petri dishes and solidified. Hyphae of B. sorokiniunawere introduced to the L-canaline agar plates by cutting 5-mm cores from 12-day-old cultures of the pathogen on 3 o/oBacto agar and placing the cores in the centre of the L-canaline agar plates. Hyphal growth was measured at 9 days by averaging three random diameter measurementsof the hyphal colonies and expressing their area as cm‘. The hyphal plates with each L-canaline treatment were replicated 10 times in a randomized complete block. An ANOVA and Waller-Duncan K-ratio I-test (P = O-05) were performed to determine minimum significant differences (MSD). The effect of L-canaline on endogenous ethylene biosynthesis in P. pratemis leaf blades inoculated with B. sorokiniana was determined by inserting four leaf blades of a single shoot into a previously described inoculation apparatus [9, 191, with their upper epidermis exposed for treatment and inoculation. A lo-cm section of each leaf blade was atomized with 2.4 ml of the appropriate dilution of L-canaline (an estimated 0.4 ml covered the surface of the four leaves). Two applications of L-canaline were made; the first 72 h before inoculation, and the second at the time of inoculation. Treated leaf blades were inoculated by placing three equally spaced 5-mm cylinders of B. sorokiniana on 3% Bacto agar (cut with a cork borer) onto their surface. Each cylinder was placed with the hyphae against the leaf blade surface and O-1 ml of sterile distilled water was applied to provide a water continuum between the hyphae and leaf blade surface. Inoculated plants and controls were placed in incubators at 22 “C with a 9-h photoperiod (40 pmol m-* s-l) and the leaf blades of each plant were analysed for ethylene at 24, 48, 72 and 96 h after inoculation, All treatments were replicated three times, and an ANOVA and Waller-Duncan K-ratio i-test (P = O-05) were performed to determine MSD within each sampling period (24, 48, 72 and 96 h) for ethylene content. Ethldene anal_),sis
The four leaf blades of each plant were removed from the inoculation apparatus, combined, and the endogenous ethylene extracted. Ethylene was determined by a modification of the vacuum extraction method for leaves developed by Beyer & Morgan [4], The modification consisted of the use of several culture tubes (25 x 200 mm) for collecting ethylene from multiple setsof leaf blades simultaneously [9]. A 100 pl sample of the gasesreleasedfrom leaves was removed from the collection tubes with a gas-tight syringe and injected into a Varian 3700 GC with a 2-m activated alumina column (60/80 mesh) and flame ionization detector (FID). Injector and detector temperatures were both 250 “C, oven temperature was 110 “C, and carrier gas (He) flow rate was 30 cm3 min-‘. The signal of the FID was connected to a Cary model 401 electrometer coupled to a Spectra-Physics 4100 computing integrator. After calibration, sample data were plotted and quantified by the computing integrator and expressedas ~1 1-l ethylene. Chlorophyll
analysis
Chlorophyll content of the four 10 cm leaf blade sectionsof each plant subjected to the various treatments was determined by a modification of the method of Knudson et al. [IO]. Only the leaf blades from the 96 h treatments were analysed for Chl content. 20-Z
C. F. Hodges
304
and D. A. Campbell
Leaves were cut into small pieces after ethylene extraction, washed three times in distilled water to remove (NH,),SO, from the ethylene extraction procedure, and freeze-dried (48 h, -48 “C, 50 pl Hg). After dry weight determination, the leaf blade tissuewas placed in 10 ml of 99 o/oethanol and the Chl extracted in the dark for 24 h. The 24-h ethanol extractions were repeated three times and combined (30 ml total). Chlorophyll absorbency was measuredspectrophotometrically at 665 and 649 nm, and calculations were made to determine total Chl [IO]. Chlorophyll concentrations were determined as pg mg-’ leaf d. wt, and the Chl toncentration of inoculated and/or treated leaf blades was expressed as a percentage of the Chl in non-treated, noninoculated leaf blades. An ANOVA and Waller-Duncan K-ratio l-test (P = O-05) were performed to determine MSD between treatments for Chl content at 96 h after inoculation. RESULTS Hyphal growth
The mean area of hyphal growth of g-day-old control cultures of B. sorokiniana on 3 y. Bacto agar was 39.9 cm’ (Fig. 1). L-canaline at 10m4M induced a slight, but significant inhibition of hyphal growth (36.6 cm’). Hyphal growth on L-canaline at 10e6, lo-‘, lo-lo and 10-l” M ranged from 39.4 to 40.8 cm’ and did not differ from the control.
L
MSD
= 2.69
35 2 5
30
’
25
5l
E
2o 15
FIG. 1. Effect of various dilutions of L-canaline (CAN) on the hyphal growth of Bipolaris mrokiniaaa in t&o on 3 o/0 Bacto agar. An ANOVA and Wailer-Duncan K-ratio f-test (P = @OS) were performed to determine minimum significant differences (MSD).
inhibition
of ethylene
biosynthesis
305
by L-canaline
Ethylene production
Endogenous ethylene levels in non-treated, non-inoculated control leaf blades remained relatively constant from one 24-h sampling period to another; mean levels of ethylene at 24, 48, 72 and 96 h ranged from 0.29 to 0.31 @I-’ [Fig. 2(a-d)]. Endogenous MSD
= 0.64
(b)
0.8 0.6 0.4 0.2 0.0 z
2 ii
1.6 1.4 1.2
MSD = 0.46
MSD = 0.52
Cd)
1.0 i 0.8 0.6 0.4 0.2 0.0
Treatment FIG. 2. Endogcnous ethylene of the internal atmosphere of Pea ,!wafensis leaf blades infected by Bipoloris soroil-~~krt~o (B.S.). Leaf blades were treated with IO-“, IO-s, 10e8, IO-r0 or IO-‘? M Lcanalinc (CAN), and ethylene content was determined at (a) 24, (b) 48, (c) 72 and (d) 96 II aftct inoculation as pathogcnesis progressed. An ANOVA and Wailer-Duncan K-ratio f-test (P = 0.05) were performed to dctcrminc minimum significant differences (MSD) at each 24-h sampling period.
ethylene in non-inoculated leaf blades remained unchanged or decreasedin responseto the various concentrations of L-canaline. Ethylene content of non-inoculawd leaf blades was decreased by IO-“ M L-canaline at 24, 48, 72 and 96 h, and by IO-” M at 24, 48 and 72 h. L-canaline at 10-s M decreased the ethylene content of non-inoculated leaf blades only at 72 h and L-canaline at lo-” and 10-l’ M had no effect on the ethylene content. Endogenous ethylene in non-treated, inoculated leaf blades increased progressively from 24 to 48 h, peaked at 72 h (1.66 ~1 l-l), and declined after 96 h [Fig. 2(a-d)]. Ethylene content of inoculated leaf blades treated with lo-” and 10d6M L-canaline did not differ from that of the non-treated, non-inoculated leaf blades at 24 h [Fig. 2(a)]. Inoculated leaf blades treated with more dilute concentrations of L-canaline ( IOVH,
C. F. Hodges 120
MSD
and D. A. Campbell
= i.64
Treatment Fro 3. Chlorophyll content of Pan prokwi~ leaf blades treated with IO-“, IO-s, IO-*, 10-t’ or 10-r’ M L-canaline (CAN) and inoculated with Bipoloris sorokiniana (B.S.). Leaf blades of each shoot were pooled, and chlorophyll (Chl) content was determined at 96 h after inoculation. All treatment values were calculated against the Chl content of non-inoculated, non-treated control leaves (8.6 pg mg-’ leaf d. wt = 100°/a). An ANOVA and Wailer-Duncan K-ratio f-test (P = 0.05) were performed to determine minimum significant differences (MSD).
10-l’ and lo-‘* M) had ethylene levels greater than that of non-treated, non-inoculated leaf blades at all 24-h sampling periods [Fig. 2(a-d)]. Inoculated leaf blades treated with L-canaline at lo-’ and 10m6M had ethylene levels below that of non-treated, inoculated leaf blades at each 24-h sampling period [Fig. 2(a-d)]. Inoculated leaf blades treated with L-canaline at lo-* M also decreased ethylene relative to the non-treated, inoculated leaf bladesat all 24-h sampling periods, except at 96 h [Fig. 2(d)]. I noculated leaf blades treated with L-canaline at 10-l’ M decreasedethylene relative to the non-treated, inoculated leaf blades at 24 and 48 h [Fig. 2(a and b)]; at 72 h, the ethylene was greater than that in the non-treated, inoculated leaf blades, and at 96 h, it did not differ from the non-treated, inoculated leaf blades [Fig. 2(c and d)]. The ethylene responsein inoculated leaf blades treated with L-canaline at lo-‘* M did not differ from, or was greater than, that of the non-treated, inoculated leaf blades [Fig. 2(a-d)].
Inhibition
of ethylene
Leaf ctdo~oplyll
biosynthesis
by L-canaline
307
conlenl
Non-treated, non-inoculated leaf blades had a mean Chl content of 8.6 l.tg mg-’ leaf d. wt (= 1009,) at 96 h. The Chl content of non-inoculated leaf blades treated with the various concentrations of L-canaline did not differ from that of the non-treated, non-inoculated leaf blades (Fig. 3). Chlorophyll content of non-treated leaf blades inoculated with B. sorokiniana was 63% of that in non-treated, non-inoculated leaf blades 96 h after inoculation (Fig. 3). Retention of Chl by inoculated leaf blades treated with 1OeJ and 10-OM L-canaline was 89 and 81 %, respectively, and significantly greater than that in the non-treated, inoculated leaf blades. Chlorophyll retention by inoculated leaf blades treated with lOmEM L-canaline increased slightly but not significantly so relative to the non-treated, inoculated leaf blades, and Chl retention in inoculated leaf blades treated with L-canaline at 10-i’ and 10-l’ M did not differ from the non-treated, inoculated leaf blades (Fig. 3). DISCUSSION
L-canaline decreasesbiosynthesisof endogenousethylene and subsequent lossof Chl by P. ,ha/ensis leaf blades infected by B. sorokiniana without preventing typical infection and lesion development by the pathogen. The toxic nature of L-canaline [Z&23] did not affect the growth of P. pralensis. After removal of treated and infected leaf blades for ethylene and Chl analyses, the plants were returned to the greenhouse where their typical growth pattern was unaffected over a 3-6-month period. Hyphal growth of B. sorokiniana is slightly inhibited by L-canaline at lo-’ M, but the inhibition did not affect the infection process.More dilute concentrations of L-canaline had no effect on hyphal growth. L-canavanine affects the development of some fungi by inhibiting hyphal growth and sporulation, altering morphology, and inducing mutations [6, II, 24,2.5, 281, but there are no reported observations on the effects of L-canavanine on B. sorokiniana, and L-canaline has not been evaluated for its effects on the growth and development of fungal organisms. The ability of L-canaline to decreaseethylene biosynthesis in P. pralensis leaf blades in response to infection by B. sorokiniana is a function of concentration, time and the progression of the pathogen in the infected tissue. Only concentrations of L-canaline at lo-’ and low6M were effective at decreasing biosynthesis of ethylene in infected leaf blades over the entire 96 h of the study. The more dilute concentrations of L-canaline neither decreasedethylene production nor the development of the pathogen in the leaf blades. The ethylene levels found in this study in the infected leaf blades were derived from the combined internal atmospheresof the four inoculated leaf blades of each plant. In this host-pathogen interaction, ethylene produced in leaf blades-in responseto infection varies with leaf age [8, 1.51.Typically, ethylene biosynthesis is greatest in the youngest inoculated leaf blade and decreasesin each older inoculated leaf blade. The oldest leaf blades typically respond to infection with the lowest ethylene surge, although they showed the greatest sensitivity to ethylene and respond with the greatest loss of Chl [8,15]. The inability of older leaves to produce high levels of ethylene in responseto infection combined with the greater lossof Chl suggeststhat their level of metabolic activity is much below that of younger leaves. These leaf-age related responsessuggest
308
C. F. Hodges
and D. A. Campbell
that the ability of L-canaline (and perhaps other pyridoxal enzyme inhibitors) to control ethylene biosynthesisand Chl lossin infected leaf blades could differ in leaves of different ages. The observations made in the present study suggest, however, that symptom expression in the form of leaf chlorosis might be substantially reduced by physiologically blocking ethylene biosynthesisduring pathogenesis.Continued work in this area might provide methods specifically designedfor the management of symptom expressionby diseasedplants. Such a management system could be especially useful for foli’ar diseaseslike B. sorokinian~leaf spot which a’re difficult to control but where the aesthetic value of the grassplant is of major importance. This is paper No. J-15615 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011 (Project 3193). Supported, in part, by a grant from the United States Golf Association, Green Section. REFERENCES 1. Adams DO, Yang SF. 1979. Ethylene biosynthesis: identification of I-aminocyclopropane-lcarboxylic acid as an intermediate in the conversion of methionine to ethylene. Proceedings of fhe N&ml Academy ojSciences of the U.S.A. 76: 170-l 74. 2. Amrhein N, Wenker D. 1979. Novel inhibitors of ethylene production in higher plants. Plant and Cell Physiolou 20 : 1635-l 642. 3. Be&r T, Churchich JE. 1976. Reactivity of phosphopyridoxal groups of cystathionase. journal of Biological Chemislry 251: 5267-5271. 4. Beyer EM Jr, Morgan PW. 1970. A method for determining the concentration of ethylene in the gas phase of vegetative plant tissue. Plan/ Physiology 46: 352-354. 5. Burg SP, Burg EA. 1965. Gas exchange in fruits. Plan1 Physiology 18: 870-874. 6. Cbilds EA, Ayres JC, Koehler PE. 1971. Differentiation in AspergillusJlavus as influenced by Lcanavanine. Mycologia 63: 181-184. 7. Davis JE, McKetta JJ. 1960. Solubility or ethylene in water. Journal of Chemical Engineering Data 5: 374-375. 8. Hodges CF. 1990. Endogenous ethylene response and chlorophyll loss in sequentially older leaves oTPoa profensis infected by Bipolaris sorokiniana. 30um41 of Plan! Physiology 136: 670-674. 9. Hodges CF, Coleman LW. 1984. Ethylene-induced chlorosis in the pathogenesis ofBipolaris sorokiniana leaf spot of Pea prulensis. Plua~ Physiology 75 : 462-465. IO. Knudson LL, Tibbitts TW, Edwards GE. 1977. Measurement of ozone injury by determination of chlorophyll concentration. Plant P/~siolo~ 60: 606-608. Il. Lewis CM, Tarrant GM. 1971. Induction of mutation by 5-Ruorouracil and amino acid analogs in Uslilago maydis. Adulation Research 12: 349-356. 12. Lieberman M. 1979. Biosynthesis and action ofethylene. Annual Review of Plan1 Plysiolo~ 30: 5X3-591. 13. Murr DP, Yang SF. 1975. Inhibition of in uiuo conversion of methionine to ethylene by L-canaline and 2,4-dinitrophenol. Plan1 Physiology 55: 79-82. 14. N&en RN, Hodges CF. 1983. Hypobaric control of ethylene-induced leaf senescence in intact plants of Phaseolus vulgaris L. Planl Physiolou 71: 96- 10 I. 15.Nilsen KN, HodgesCF, Madsen JP. 1979. Pathogenesis of Drechslera sorokiniana leaf soot on progressively older leaves of Pea pensis as influenced by photoperiod and light quality. Phys;ologicol Plant Pathology 15: 171-176. 16. Pegg GF. 197%. The involvement of ethylene in pathogenesis. In: Heitefus R, Williams PH, eds. Physiological Plan1 Patholoar, Encyclopedia of Plant Plysiology. New Series, Vol. 4. New York: Springer Verlag, 582-59 1. 17. Pegg GF. 1981. The involvement of growth regulators in the diseased plant. In: Ayres PG, ed. Effecb of Disease on the Physiology of the Growing Plonf. Societyfor Experimental Biology, Seminar Series II. New York: Cambridge Universitv Press. 149-177. 18. Rahiala E-i, Kekomi M, J&me J, Riina A, Riiihii NCR. 197 I. Inhibition of pyridoxal enzymes by L-canaline. Biochimica et Biop&ca Acla 227: 337-343. 19. Robinson PW, Hodges CF. 1976. An inoculation apparatus for evaluation ofBipolurir sorokiniana lesion development on progressively older leaves of Pea pralensis. Phylopafholo~ 66: 360-362.
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of ethylene
biosynthesis
by L-canaline
309
20. Rosenthal GA. 1981. A mechanism ofl-canaline toxicity. European Journal oJEiochemi.rfry 114: 301-304. 21. Rosenthal GA. 1990. Metabolism of L-canavanine and L-canaline in leguminous plants. Pfarf Pl~siology 94 : l-3. 22. Rosenthal GA. 1991. The biochemical basis for the deleterious effects of L-canavanine. PhyW~~isfry 30: 1055-1058. 23. Rosenthal GA, Betge MA, Bleiler JA. 1989. A novel detoxification of L-canaline. Biochemical System&s and Ecology 17: 203-206. 24. Samborski DJ, Forsyth FR. 1960. Inhibition of rust development on detached wheat leaves by metabolites, antimetabolites, and enzyme poisons. Conadion Journal of Botany 38: 467-476. 25. Shepherd CJ, Mandryk M. 1964. Effects of metabolities and antimetabolites on the sporulation of Peronospora labacina Adam. on tobacco leaf disks. Arrslralian journal of Biological Sciences 17: 878-891. 26. Sisler EC, Pain A. 1973. Effect of ethylene and cyclic olefins on tobacco leaves. Tobacco Science 17: 68-72. 27. Sisler EC, Yang SF. 1984. Anti-ethylene effects of cis-2-butene and cyclic olefins. Phylochemisfry 23: 2765-2768. 28. Toshikazu T, Irikura H, Naito N. 1971. Inhibition of uredosorus formation of Puccinia coronak~ by plant growth regulators and antimetabolites. Technical Bullelin of I;hcul(y of Agriculfure, Kagawa Universi~ 23: 42-50. 29. Yang SF, Hoffman NE. 1984. Ethylene biosynthesis and its regulation in higher plants. Annual Reoiew of Plant Plpiology 35 : l55- 189.