/ . Biochem., 79, 277-282 (1976)
Wound-induced Phenylalanine Ammonia-Lyase in Potato Tuber Tissue Development of Enzyme Activity and Effects of Antibiotics Hiroshi HYODO Department of Horticulture, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka, Shizuoka 422 Received for publication, July 10, 1975
Phenylalanine ammonia-lyase [EC 4.3.1.5] activity increased rapidly after a 3-hr lag period in potato tuber {Solatium tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25°. The activity reached a maximum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.
Slicing a potato tuber causes marked synthesis of RNA and protein and a striking change in metabolic activity. A comprehensive review of the metabolic alterations and their regulation in potato tuber tissue during aging has been published by Kahl (1). Click and Hackett demonstrated that the rapid development of wound respiration in potato tuber slices was dependent on the synthesis of new RNA and protein (2). Changes in metabolism, with particular reference to protein metaboAbbreviation: PAL, phenylalanine ammonia-lyase [EC 4.3.1.5]. Vol. 79, No. 2, 1976
277
lism, in plant storage tissues after wounding and infection have been reviewed by Uritani (3). One of the marked changes in potato tuber after slicing is a production of phenylpropanoid compounds and the formation of associated enzymes. Zucker found that the activity of phenylalanine ammonia-lyase (PAL) [EC 4.3.1.5] greatly increased in wounded potato tuber tissue and the rise was further enhanced by exposing the tissue to light {4, 5). A rise in PAL activity upon mechanical injury or infection by pathogens occurs widely in plants. Induction of PAL and its regulation in plant tissues are dealt with in reviews by
278
H. HYODO
Amrhein and Zenk (6), Zucker (7), and Camm and Towers (8). This paper deals with the increasing activity of PAL after wounding and the effects of some antibiotics which inhibit nucleic acid and protein synthesis on PAL induction in potato tuber in response to wounding. A unique effect of chloramphenicol and mitomycin C on PAL induction is also discussed. MATERIALS AND METHODS Potato tubers (Solatium tuberosum L. cv. May Queen) dug in June from a field in the suburbs of Shizuoka and stored at 20° or 4° until needed were used as experimental materials. In some cases, cultivars, May Queen and Danshaku (Irish Cobbler), purchased from local markets were used. Tubers were surfacesterilized in ca. 0.1% sodium hypochlorite solution for 30 min and rinsed thoroughly with water. They were peeled and the inner pulp (without cortex) was bored out with a cork borer of 10-mm diameter. Tne cylinders were sliced into 2-mm thick disks. Twelve disks were placed on a filter paper pad in a 125-ml Erlenmyer flask. The pad had previously been wetted with 4 ml of 0.01 M phosphate buffer, pH 6.0, containing 1.6xl0" 4 M chloramphenicol to minimize the growth of bacteria. Occasionally the medium was supplemented with other compounds such as actinomycin D, cycloheximide, 6-methylpurine or mitomycin C. In most experiments, two polypropylene wells (Kontes Glass Co.) containing 0.25 M mercuric perchlorate solution and 20% KOH solution with filter paper wicks were included in the flask to absorb evolved ethylene and carbon dioxide, respectively. The amount of ethylene evolved from potato tuber disks was fairly low and the ethylene was almost completely trapped by this procedure (9). The flask was sealed with a rubber vaccine cap. Disks were incubated at 25° in the dark unless otherwise specified. Preparation of the disks and enzyme extraction were performed in normal room light. After incubation, the disks were taken out, blotted with filter paper and weighed. Ten disks (ca. 2 g) were homogenized in 15 ml of 0.025 M Tris-HCl buffer, pH 8.8, con-
taining 0.01 M 2-mercaptoethanol using a glass homogenizer with a variable speed motor. The homogenate was centrifuged at 6,000 X g for 10 min and the resulting supernatant was used for enzyme assay. The extraction and centrifugation were performed in the cold (0—4°) and the enzyme solution was stored in a refrigerator at 2° until use. PAL activity under these conditions decreased by approximately 20% in two days. PAL activity was determined spectrophotometrically by measuring the increase in absorbance at 290 nm of the reaction mixture using a Hitachi 139 spectrophotometer. Usually the assay mixture contained 200 //moles of Tris-buffer, pH 8.8, 60 //moles of L-phenylalanine and the enzyme preparation in a total volume of 6 ml. As a blank, a sample without phenylalanine was used. Incubation was carried out at 40°. Upon addition of the enzyme, the absorbance at 290 nm increased linearly for well over 3 hr, which was ascribed to linear formation of cinnamic acid. The activity was expressed as /imoles of cinnamic acid formed per hr per g fresh weight. The calculation was based on a molar extinction coefficient for cinnamate of 9,500. The UV spectrum of the product of the enzyme reaction was found to be identical with that of fr-aws-cinnamic acid, and with [uC]phenylalanine as a substrate the labeled product was isolated and identified as frans-cinnamic acid. For determination of the amount of actinomycin D which had penetrated into the disks, twelve disks were incubated on a filter paper pad wetted with 4 ml of buffer containing 800 tig of actinomycin D. After various times of incubation, the disks were taken out and blotted with filter paper. Ten disks were homogenized with 20 ml of methanol and the homogenate was filtered through a glass funnel. The residue was washed with 10 ml of methanol and the washing was combined with the filtrate. The combined solution was concentrated to dryness under a vacuum. The residue was dissolved in methanol and its absorbance at 442 nm was measured. The absorbance of the methanol extract from a zero-time sample was subtracted from those of incubated samples. The amounts of actinomycin D were calculated /. Biochtm.
WOUND-INDUCED PAL IN POTATO TUBER
279
on the basis of a molar extinction coefficient of 21,600 for actinomycin D. Actinomycin D was a generous gift of Merck Sharp & Dohme. All other chemicals were obtained commercially. RESULTS AND DISCUSSION PAL activity markedly increased during incubation of the disks with the medium in the dark at 25° (Fig. 1). The original activity in fresh potato tubers was exceedingly low and remained so during the period of storage. Upon slicing, the activity rose rapidly after a 3-hr lag period. It reached a maximum after incubation for about 40 hr. Subsequently during the course of incubation the activity declined gradually. The lag period and the maximum development seem to vary with the time and year of harvest, the maturity of tubers and the storage period after harvest. It is of interest that Rosenstock et al. reported that the ability of potato tubers to develop wound respiration gradually decreased during storage at 2°, 7, or 20° (10). As for the decline after reaching a maximum, the existence
20
30 40 Incubation lime (hr)
60
Fig. 1. The increase in PAL activity in potato tuber disks during incubation in the dark at 25°. Potato tuber disks (2x10 mm) were incubated on the medium for appropriate lengths of time and extracted for PAL assay. PAL activity was expressed as ^moles of cinnamic acid formed per hr per g fresh weight. Vol. 79, No. 2, 1976
of a PAL-inactivating system which may function in degrading PAL has been postulated (5). Light slightly enhanced the rise in PAL activity at 8 hr and 23 hr after slicing (Fig. 2). For these samples, disks were exposed continuously to white fluorescent light. The development was promoted by light (3,000 lux) to the extent of 46% at 8 hr and 22% at 23 hr of incubation. The conspicuous effect of light reported by Zucker (4) was not observed. It seems likely that, as reported by Sacher et al. (11), light is not essential for the synthesis of PAL in wounded tissue. As a matter of fact, when disks were prepared under dim green light and incubated in the dark afterwards, the PAL activity was similar to that of disks prepared under room light and incubated in the dark. The striking increase of PAL activity was completely prevented by the addition of antibiotics that inhibit RNA and protein synthesis, such as actinomycin D, cycloheximide, and 6methylpurine. These compounds were supplied to the medium at zero time of incuba-
8 hr 2 3 hr Incubation time
Fig. 2. Effect of light on the development of PAL activity in potato tuber disks during incubation. Incubation flasks were placed under white fluorescent lights (3,000 lux) at 29°. After incubation for 8hr and 23 hr, disks were taken out and extracted for PAL assay.
280
H. HYODO
tion. Cycloheximide was effective at a relatively low concentration, e.g., at a concentration of 1.8xlO-"M it inhibited the rise by 78% (Fig. 3). A higher concentration was required for the action of actinomycin D, i.e. 4.0xl0~ ! M actinomycin D was needed to prevent the increase completely (Fig. 3). A much higher concentration of 6-methylpurine was necessary for complete inhibition (Fig. 4). In any case, application of these inhibitors resulted in a
100-
I0"4 (M)
6 ' i
Fig. 3. Effects of various concentrations of actinomycin D ( x ) and cycloheximide (O) on the development of PAL activity. Disks were incubated in the dark at 25° for 21 hr (actinomycin D treatment) or for 18 hr (cycloheximide treatment). All additions were made at zero time. In each case, the PAL activity of the disks which received no inhibitor was taken as 100.
complete inhibition of the rise in PAL activity, suggesting that the rise was due to de novo synthesis of enzyme protein. This has been clearly shown by Sacher et at. (11) by DiO incorporation into PAL during aging. It was found by Kahl that slicing potato tubers resulted in marked formation of polyribosomes, which was completely blocked by administration of actinomycin D, and that protein synthesis was strongly dependent on the polyribosome formation (12, 13). The effects of delayed additions of actinomycin D and cycloheximide were investigated. The potato tubers used for these experiments exhibited a 3-hr lag period before the rapid rise in PAL activity. The incubation lasted seven and a half hour. Additions of cycloheximide at different times during the incubation period were very effective, although the effectiveness gradually declined, particularly in the later period (Fig. 5). However, the effect of actinomycin D was different, i.e. if additions were made during the lag phase it strongly inhibited the development of PAL, but if sup-
100Cydohexiinlde
IOO
o-20
7 (hr)
Fig. 5. Effect of delayed additions of actinomycin D and cycloheximide on the development of PAL activity. Potato tuber disks were incubated for seven and a half hr in the dark at 25°; at various times, disks were transferred to medium containing actinomycin D (4.0X10~°M) or cycloheximide (3.6 x 6-Metnytpurlne (M)
Fig. 4. Effect of 6-methylpurine on the development of PAL activity in potato tuber disks incubated in the dark at 25° for 19 hr. PAL activity of the disks which did not receive 6-methylpurine was taken as 100.
10"' M) and further incubated for the remaining time. When additions were made at zero time actinomycin D and cycloheximide suppressed the development of PAL activity almost completely. However, the extent of inhibition varied greatly between the two inhibitors in the case of delayed additions.
J. Biochem.
WOUND-INDUCED PAL IN POTATO TUBER
281
plied later it was ineffective, or slightly stimulative (Fig. 5). The requirement for DNAdirected RNA synthesis appeared to exist during the early hours of incubation. The possibility was examined that the ineffectiveness with delay in application of actinomycin D might be due to the time taken for penetration. The data indicated that penetration of actinomycin D into tuber disks almost reached a plateau within 1 hr, suggesting that penetration was not a limiting factor. Chloramphenicol included in the medium to prevent the proliferation of bacteria resulted in a marked stimulation of PAL formation. As seen in Fig. 6, at a concentration of 3x 10~5 M it nearly doubled the extent of the increase. Application of a wide range of concentrations up to as high as 1.6xlO"8M did not inhibit the development but in fact stimulated it. Chloramphenicol is known to be an inhibitor of protein synthesis in bacteria and plant organelles such as chloroplasts and mitochondria. In the potato tuber, PAL is found to be located in the soluble fraction but not in the organelles (14). As the increase of enzyme activity was prevented by cycloheximide but not by chloramphenicol, it can be supposed that PAL is synthesized by a cytoplasmic ribosomal system. However, it is not at all clear how chloramphenicol enhanced PAL
synthesis. In connection with this finding, it is of interest to note the reports by Shen (15) and Knypl (16) that chloramphenicol induced nitrate reductase activity in rice seedlings and in etiolated cucumber cotyledons, respectively. Application of mitomycin C, an inhibitor of DNA synthesis in bacteria (17), had an effect similar to that of chloramphenicol in that it strongly enhanced PAL formation (Fig. 7). In the presence of chloramphenicol, almost no effect of mitomycin C could be seen. The mode of action of mitomycin C remains to be clarified. Hadwiger and Schwochau studied the effects of a number of DNA intercalating compounds and antibiotics which acted on DNA during PAL induction in pea pods. They found that mitomycin C caused a marked stimulation, like actinomycin D (18). Asahi and Majima reported that the application of chloramphenicol (6 X10"8 M) to wounded tissue of sweet potato root strongly suppressed the increase in cytochrome oxidase [EC 1.9. 3.1] activity and the number of mitochondrial particles, whereas the rise in peroxidase [EC 1.11.1.7] activity was hardly affected (19). The effect of mitomycin C (3x 10~6 M) was similar, though smaller. It is possible that chloramphenicol and mitomycin C
I.O
s
—
£
1
3
itf
10°
Chloramphenicol (M) Fig. 6. Effect of chloramphenicol on the development of PAL activity. Tuber disks were incubated on media containing various concentrations of chloramphenicol for 24 hr in the dark at 25°. After incubation, disks were extracted for PAL assay. Vol. 79, No. 2, 1976
|+CP +MC
O
|-CP
1
|-CP
o
|+CP -MC
0J5-
_i
Fig. 7. Effects of mitomycin C and chloramphenicol on the development of PAL activity. Tuber disks were incubated on medium containing 1.6xlO~4M of chloramphenicol (+CP, -MC) or 3.0X10" S M of
mitomycin C (—CP, +MC), or both chloramphenicol and mitomycin C (+CP, +MC). Incubations were for 21 hr in the dark at 25°. Disks which received no chloramphenicol or mitomycin C served as controls ( - C P , -MC).
282
cause PAL synthesis in the wounded potato tuber tissue to rise by suppressing mitochondrial formation induced in the wounded tissue and thus providing the cytoplasmic ribosomal system with extra materials for enzyme synthesis. Slicing potato tubers causes an enhancement of DNA, RNA, and protein synthesis, followed by marked changes in metabolic activity (2, 5, 11-13, 20-25). Slicing (cutting) causes the formation of wounded tissue in that a surface layer is subjected to mechanical destruction and adjacent to this are inner cells to which a stimulus is transmitted from the injured cells. Wound reaction, typically the rise in metabolic activity, may be considered to be an active response of plants. It is of interest to determine what kind of substance can play a role as a trigger and be responsible for derepression of the gene activity in response to wounding. The author has examined the effects of plant hormones including ethylene, gibberellic acid, abscisic acid, auxin and kinetin, and growth retardants such as CCC and AMO-1618 on PAL induction in wounded potato tuber tissue. It was found that none of them was effective for the stimulation of PAL formation, but ethylene, IAA, and AMO1618 considerably suppressed its induction. The effects of polyamines such as putrescine, spermidine, and spermine were also investigated. It has been shown that the addition of putrescine resulted in a stimulation of PAL formation, whereas spermidine and spermine markedly reduced it. The mechanism and essential factor responsible for PAL induction in the wounded potato tuber tissue remain to be clarified. I thank MerCk Sharp & Dohrae Research Laboratories, Rahway, New Jersey for generous gifts of actinomycin D.
H. HYODO
REFERENCES 1. Kahl, G. (1973) Bot. Rev. 39, 274-299 2. Click, R.E. & Hackett, D.P. (1963) Proc. Natl. Acad. Set. 50, 243-250 3. Uritani, I. (1971) Ann. Rev. Phytopathol. 9, 211234 4. Zucker, M. (1965) Plant Physiol. 40, 779-784 5. Zucker, M. (1968) Plant Physiol. 43, 365-374 6. Amrhein, N. & Zenk, M.H. (1971) Z. Pflanzenphysiol. 64, 145-168 7. Zucker, M. (1972) Ann. Rev. Plant Physiol. 23, 133-156 8. Camm, E.L. & Towers, G.H.N. (1973) Phytochem. 12, 961-973 9. Hyodo, H. & Yang, S.F. (1974) Z. Pflanzenphysiol. 71, 76-79 10. Rosenstock, G., Kahl, G., & Lange, H. (1971) Z. Pflanzenphysiol. 64, 130-138 11. Sacher, J.A., Towers, G.H.N., & Davies, D.D. (1972) Phytochem. 11, 2383-2391 12. Kahl, G. (1971) Z. Naturforsch. 26b, 1058-1064 13. Kahl, G. (1971) Z. Naturforsch. 26b, 1064-1067 14. Camm, E.L. & Towers, G.H.N. (1973) Phytochem. 12, 1575-1580 15. Shen, T.C. (1972) Plant Physiol. 49, 546-549 16. Knypl, J.S. (1973) Planta 114, 311-321 17. Shiba, S., Terawaki, A., Taguchi, T., & Kawamata, J. (1959) Nature 183, 1056-1057 18. Hadwiger, L.A. & Schwochau, M.E. (1971) Plant Physiol. 47, 346-351 19. Asahi, T. & Majima, R. (1969) Plant & Cell Physiol. 10, 317-323 20. Brinkman, F.G., van der Plas, L.H.W., & Verleur, J.D. (1973) Z. Pflamsnphysiol. 68, 364372 21. Kahl, G., Lange, H., & Rosenstock, G. (1969) Z. Naturforsch. 24b, 911-918 22. Laties, G.G. (1965) Plant Physiol. 40, 1237-1241 23. Watanabe, A. & Imaseki, H. (1973) Plant. Physiol. 51, 772-776 24. Borchert, R. & McChesney, J.D. (1973) Develop. Biol. 35, 293-301 25. Willemot, C. & Stumpf, P.K. (1967) Plant Physiol. 42, 391-397
J. Biochem.
Wound-induced Phenylalanine Ammonia-Lyase in Potato Tuber Tissue Development of Enzyme Activity and Effects of Antibiotics Hiroshi HYODO Department of Horticulture, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka, Shizuoka 422 Received for publication, July 10, 1975
Phenylalanine ammonia-lyase [EC 4.3.1.5] activity increased rapidly after a 3-hr lag period in potato tuber {Solatium tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25°. The activity reached a maximum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.
Slicing a potato tuber causes marked synthesis of RNA and protein and a striking change in metabolic activity. A comprehensive review of the metabolic alterations and their regulation in potato tuber tissue during aging has been published by Kahl (1). Click and Hackett demonstrated that the rapid development of wound respiration in potato tuber slices was dependent on the synthesis of new RNA and protein (2). Changes in metabolism, with particular reference to protein metaboAbbreviation: PAL, phenylalanine ammonia-lyase [EC 4.3.1.5]. Vol. 79, No. 2, 1976
277
lism, in plant storage tissues after wounding and infection have been reviewed by Uritani (3). One of the marked changes in potato tuber after slicing is a production of phenylpropanoid compounds and the formation of associated enzymes. Zucker found that the activity of phenylalanine ammonia-lyase (PAL) [EC 4.3.1.5] greatly increased in wounded potato tuber tissue and the rise was further enhanced by exposing the tissue to light {4, 5). A rise in PAL activity upon mechanical injury or infection by pathogens occurs widely in plants. Induction of PAL and its regulation in plant tissues are dealt with in reviews by
278
H. HYODO
Amrhein and Zenk (6), Zucker (7), and Camm and Towers (8). This paper deals with the increasing activity of PAL after wounding and the effects of some antibiotics which inhibit nucleic acid and protein synthesis on PAL induction in potato tuber in response to wounding. A unique effect of chloramphenicol and mitomycin C on PAL induction is also discussed. MATERIALS AND METHODS Potato tubers (Solatium tuberosum L. cv. May Queen) dug in June from a field in the suburbs of Shizuoka and stored at 20° or 4° until needed were used as experimental materials. In some cases, cultivars, May Queen and Danshaku (Irish Cobbler), purchased from local markets were used. Tubers were surfacesterilized in ca. 0.1% sodium hypochlorite solution for 30 min and rinsed thoroughly with water. They were peeled and the inner pulp (without cortex) was bored out with a cork borer of 10-mm diameter. Tne cylinders were sliced into 2-mm thick disks. Twelve disks were placed on a filter paper pad in a 125-ml Erlenmyer flask. The pad had previously been wetted with 4 ml of 0.01 M phosphate buffer, pH 6.0, containing 1.6xl0" 4 M chloramphenicol to minimize the growth of bacteria. Occasionally the medium was supplemented with other compounds such as actinomycin D, cycloheximide, 6-methylpurine or mitomycin C. In most experiments, two polypropylene wells (Kontes Glass Co.) containing 0.25 M mercuric perchlorate solution and 20% KOH solution with filter paper wicks were included in the flask to absorb evolved ethylene and carbon dioxide, respectively. The amount of ethylene evolved from potato tuber disks was fairly low and the ethylene was almost completely trapped by this procedure (9). The flask was sealed with a rubber vaccine cap. Disks were incubated at 25° in the dark unless otherwise specified. Preparation of the disks and enzyme extraction were performed in normal room light. After incubation, the disks were taken out, blotted with filter paper and weighed. Ten disks (ca. 2 g) were homogenized in 15 ml of 0.025 M Tris-HCl buffer, pH 8.8, con-
taining 0.01 M 2-mercaptoethanol using a glass homogenizer with a variable speed motor. The homogenate was centrifuged at 6,000 X g for 10 min and the resulting supernatant was used for enzyme assay. The extraction and centrifugation were performed in the cold (0—4°) and the enzyme solution was stored in a refrigerator at 2° until use. PAL activity under these conditions decreased by approximately 20% in two days. PAL activity was determined spectrophotometrically by measuring the increase in absorbance at 290 nm of the reaction mixture using a Hitachi 139 spectrophotometer. Usually the assay mixture contained 200 //moles of Tris-buffer, pH 8.8, 60 //moles of L-phenylalanine and the enzyme preparation in a total volume of 6 ml. As a blank, a sample without phenylalanine was used. Incubation was carried out at 40°. Upon addition of the enzyme, the absorbance at 290 nm increased linearly for well over 3 hr, which was ascribed to linear formation of cinnamic acid. The activity was expressed as /imoles of cinnamic acid formed per hr per g fresh weight. The calculation was based on a molar extinction coefficient for cinnamate of 9,500. The UV spectrum of the product of the enzyme reaction was found to be identical with that of fr-aws-cinnamic acid, and with [uC]phenylalanine as a substrate the labeled product was isolated and identified as frans-cinnamic acid. For determination of the amount of actinomycin D which had penetrated into the disks, twelve disks were incubated on a filter paper pad wetted with 4 ml of buffer containing 800 tig of actinomycin D. After various times of incubation, the disks were taken out and blotted with filter paper. Ten disks were homogenized with 20 ml of methanol and the homogenate was filtered through a glass funnel. The residue was washed with 10 ml of methanol and the washing was combined with the filtrate. The combined solution was concentrated to dryness under a vacuum. The residue was dissolved in methanol and its absorbance at 442 nm was measured. The absorbance of the methanol extract from a zero-time sample was subtracted from those of incubated samples. The amounts of actinomycin D were calculated /. Biochtm.
WOUND-INDUCED PAL IN POTATO TUBER
279
on the basis of a molar extinction coefficient of 21,600 for actinomycin D. Actinomycin D was a generous gift of Merck Sharp & Dohme. All other chemicals were obtained commercially. RESULTS AND DISCUSSION PAL activity markedly increased during incubation of the disks with the medium in the dark at 25° (Fig. 1). The original activity in fresh potato tubers was exceedingly low and remained so during the period of storage. Upon slicing, the activity rose rapidly after a 3-hr lag period. It reached a maximum after incubation for about 40 hr. Subsequently during the course of incubation the activity declined gradually. The lag period and the maximum development seem to vary with the time and year of harvest, the maturity of tubers and the storage period after harvest. It is of interest that Rosenstock et al. reported that the ability of potato tubers to develop wound respiration gradually decreased during storage at 2°, 7, or 20° (10). As for the decline after reaching a maximum, the existence
20
30 40 Incubation lime (hr)
60
Fig. 1. The increase in PAL activity in potato tuber disks during incubation in the dark at 25°. Potato tuber disks (2x10 mm) were incubated on the medium for appropriate lengths of time and extracted for PAL assay. PAL activity was expressed as ^moles of cinnamic acid formed per hr per g fresh weight. Vol. 79, No. 2, 1976
of a PAL-inactivating system which may function in degrading PAL has been postulated (5). Light slightly enhanced the rise in PAL activity at 8 hr and 23 hr after slicing (Fig. 2). For these samples, disks were exposed continuously to white fluorescent light. The development was promoted by light (3,000 lux) to the extent of 46% at 8 hr and 22% at 23 hr of incubation. The conspicuous effect of light reported by Zucker (4) was not observed. It seems likely that, as reported by Sacher et al. (11), light is not essential for the synthesis of PAL in wounded tissue. As a matter of fact, when disks were prepared under dim green light and incubated in the dark afterwards, the PAL activity was similar to that of disks prepared under room light and incubated in the dark. The striking increase of PAL activity was completely prevented by the addition of antibiotics that inhibit RNA and protein synthesis, such as actinomycin D, cycloheximide, and 6methylpurine. These compounds were supplied to the medium at zero time of incuba-
8 hr 2 3 hr Incubation time
Fig. 2. Effect of light on the development of PAL activity in potato tuber disks during incubation. Incubation flasks were placed under white fluorescent lights (3,000 lux) at 29°. After incubation for 8hr and 23 hr, disks were taken out and extracted for PAL assay.
280
H. HYODO
tion. Cycloheximide was effective at a relatively low concentration, e.g., at a concentration of 1.8xlO-"M it inhibited the rise by 78% (Fig. 3). A higher concentration was required for the action of actinomycin D, i.e. 4.0xl0~ ! M actinomycin D was needed to prevent the increase completely (Fig. 3). A much higher concentration of 6-methylpurine was necessary for complete inhibition (Fig. 4). In any case, application of these inhibitors resulted in a
100-
I0"4 (M)
6 ' i
Fig. 3. Effects of various concentrations of actinomycin D ( x ) and cycloheximide (O) on the development of PAL activity. Disks were incubated in the dark at 25° for 21 hr (actinomycin D treatment) or for 18 hr (cycloheximide treatment). All additions were made at zero time. In each case, the PAL activity of the disks which received no inhibitor was taken as 100.
complete inhibition of the rise in PAL activity, suggesting that the rise was due to de novo synthesis of enzyme protein. This has been clearly shown by Sacher et at. (11) by DiO incorporation into PAL during aging. It was found by Kahl that slicing potato tubers resulted in marked formation of polyribosomes, which was completely blocked by administration of actinomycin D, and that protein synthesis was strongly dependent on the polyribosome formation (12, 13). The effects of delayed additions of actinomycin D and cycloheximide were investigated. The potato tubers used for these experiments exhibited a 3-hr lag period before the rapid rise in PAL activity. The incubation lasted seven and a half hour. Additions of cycloheximide at different times during the incubation period were very effective, although the effectiveness gradually declined, particularly in the later period (Fig. 5). However, the effect of actinomycin D was different, i.e. if additions were made during the lag phase it strongly inhibited the development of PAL, but if sup-
100Cydohexiinlde
IOO
o-20
7 (hr)
Fig. 5. Effect of delayed additions of actinomycin D and cycloheximide on the development of PAL activity. Potato tuber disks were incubated for seven and a half hr in the dark at 25°; at various times, disks were transferred to medium containing actinomycin D (4.0X10~°M) or cycloheximide (3.6 x 6-Metnytpurlne (M)
Fig. 4. Effect of 6-methylpurine on the development of PAL activity in potato tuber disks incubated in the dark at 25° for 19 hr. PAL activity of the disks which did not receive 6-methylpurine was taken as 100.
10"' M) and further incubated for the remaining time. When additions were made at zero time actinomycin D and cycloheximide suppressed the development of PAL activity almost completely. However, the extent of inhibition varied greatly between the two inhibitors in the case of delayed additions.
J. Biochem.
WOUND-INDUCED PAL IN POTATO TUBER
281
plied later it was ineffective, or slightly stimulative (Fig. 5). The requirement for DNAdirected RNA synthesis appeared to exist during the early hours of incubation. The possibility was examined that the ineffectiveness with delay in application of actinomycin D might be due to the time taken for penetration. The data indicated that penetration of actinomycin D into tuber disks almost reached a plateau within 1 hr, suggesting that penetration was not a limiting factor. Chloramphenicol included in the medium to prevent the proliferation of bacteria resulted in a marked stimulation of PAL formation. As seen in Fig. 6, at a concentration of 3x 10~5 M it nearly doubled the extent of the increase. Application of a wide range of concentrations up to as high as 1.6xlO"8M did not inhibit the development but in fact stimulated it. Chloramphenicol is known to be an inhibitor of protein synthesis in bacteria and plant organelles such as chloroplasts and mitochondria. In the potato tuber, PAL is found to be located in the soluble fraction but not in the organelles (14). As the increase of enzyme activity was prevented by cycloheximide but not by chloramphenicol, it can be supposed that PAL is synthesized by a cytoplasmic ribosomal system. However, it is not at all clear how chloramphenicol enhanced PAL
synthesis. In connection with this finding, it is of interest to note the reports by Shen (15) and Knypl (16) that chloramphenicol induced nitrate reductase activity in rice seedlings and in etiolated cucumber cotyledons, respectively. Application of mitomycin C, an inhibitor of DNA synthesis in bacteria (17), had an effect similar to that of chloramphenicol in that it strongly enhanced PAL formation (Fig. 7). In the presence of chloramphenicol, almost no effect of mitomycin C could be seen. The mode of action of mitomycin C remains to be clarified. Hadwiger and Schwochau studied the effects of a number of DNA intercalating compounds and antibiotics which acted on DNA during PAL induction in pea pods. They found that mitomycin C caused a marked stimulation, like actinomycin D (18). Asahi and Majima reported that the application of chloramphenicol (6 X10"8 M) to wounded tissue of sweet potato root strongly suppressed the increase in cytochrome oxidase [EC 1.9. 3.1] activity and the number of mitochondrial particles, whereas the rise in peroxidase [EC 1.11.1.7] activity was hardly affected (19). The effect of mitomycin C (3x 10~6 M) was similar, though smaller. It is possible that chloramphenicol and mitomycin C
I.O
s
—
£
1
3
itf
10°
Chloramphenicol (M) Fig. 6. Effect of chloramphenicol on the development of PAL activity. Tuber disks were incubated on media containing various concentrations of chloramphenicol for 24 hr in the dark at 25°. After incubation, disks were extracted for PAL assay. Vol. 79, No. 2, 1976
|+CP +MC
O
|-CP
1
|-CP
o
|+CP -MC
0J5-
_i
Fig. 7. Effects of mitomycin C and chloramphenicol on the development of PAL activity. Tuber disks were incubated on medium containing 1.6xlO~4M of chloramphenicol (+CP, -MC) or 3.0X10" S M of
mitomycin C (—CP, +MC), or both chloramphenicol and mitomycin C (+CP, +MC). Incubations were for 21 hr in the dark at 25°. Disks which received no chloramphenicol or mitomycin C served as controls ( - C P , -MC).
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cause PAL synthesis in the wounded potato tuber tissue to rise by suppressing mitochondrial formation induced in the wounded tissue and thus providing the cytoplasmic ribosomal system with extra materials for enzyme synthesis. Slicing potato tubers causes an enhancement of DNA, RNA, and protein synthesis, followed by marked changes in metabolic activity (2, 5, 11-13, 20-25). Slicing (cutting) causes the formation of wounded tissue in that a surface layer is subjected to mechanical destruction and adjacent to this are inner cells to which a stimulus is transmitted from the injured cells. Wound reaction, typically the rise in metabolic activity, may be considered to be an active response of plants. It is of interest to determine what kind of substance can play a role as a trigger and be responsible for derepression of the gene activity in response to wounding. The author has examined the effects of plant hormones including ethylene, gibberellic acid, abscisic acid, auxin and kinetin, and growth retardants such as CCC and AMO-1618 on PAL induction in wounded potato tuber tissue. It was found that none of them was effective for the stimulation of PAL formation, but ethylene, IAA, and AMO1618 considerably suppressed its induction. The effects of polyamines such as putrescine, spermidine, and spermine were also investigated. It has been shown that the addition of putrescine resulted in a stimulation of PAL formation, whereas spermidine and spermine markedly reduced it. The mechanism and essential factor responsible for PAL induction in the wounded potato tuber tissue remain to be clarified. I thank MerCk Sharp & Dohrae Research Laboratories, Rahway, New Jersey for generous gifts of actinomycin D.
H. HYODO
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J. Biochem.
