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Pal and ethylene content during maturation of red and golden delicious apples
~hytcxhem~stry,
Vol 27, No 4, pp 1001 1003, 1988
0
Prmted ,n Great Bntam
PAL AND ETHYLENE
CONTENT DURING GOLDEN DELICIOUS
SYLVIA M Department
of Horticultural
MATURATION APPLES
0031 9422/88 $300+000 Journals Ltd
1988 Pergamon
OF RED AND
and C. RICHARD UNRATH
BLANKENSHIP
Science, Box 7609, North
Carolina
State Umverslty,
Raleigh,
NC 27695, U S A
(Recewed 23 June 1987) Key Word Index--Ma/us
domestlca, Rosaceae,
anthocyamn;
rlpemng
Abstract-Phenylalamne ammonia-lyase actlvlty (PAL), a PAL inhlbltor (PAL-IS) and fruit internal ethylene levels were measured during apple maturation m Red and Golden Dehclous apples. In both cultlvars internal ethylene and PAL increased at approximately the same time Apple PAL-IS extract stimulated yeast PAL activity mihally, but increased yeast PAL mhlbitlon was observed as maturation progressed PAL mhlbltor levels were similar m both cuftlvars ‘The increase in yeast PAL mhlbltlon with maturation, increased red skm cofour development in Red Delicious and a comparable pattern of PAL actlvlty and mhlbltion in Red and Golden Delicious Indicates that PALIS was not closely involved with the regulation of anthocyamn synthesis m apple skm
INTRODUCTION
Phenylalamne ammonia lyase (E C 4 13.5) has been extensively reviewed [ 1,2] Ethylene enhances PAL activity m pea seedlings [3], carrots [4], and Swede and parsnip root tissue [S] PAL activity is stimulated by, but lags behind, ethylene production m irradiated grapefruit [6]. In cultured parsley cells, ethylene alone does not induce PAL [7] A protemaceous inhibitor of PAL activity is found m sunflower [S] and mhlbltors are present also m gherkin hypocotyls [9], sweet potato [lo] and apple skm [ll, 121. PAL activity 1s inhibited by the end product of the reaction, trans-cmnamlc acid [ 131 In apple skm, ethylene production is associated with anthocyamn accumulation and a concomitant increase m PAL activity m response to maturation, wounding and temperature [14-161. Low temperatature and light stimulate anthocyanm accumulation m apple skm, increase PAL activity, and lower PAL inhibitor activity [l 11. Faragher and Chalmers [ 171 concluded that while PAL influences anthocyanin accumulation m apples, there are other controlhng factors Tan [12] concluded that low temperature stimulates PAL actlvlty and decreases PAL mhlbltor, thus mcreasmg anthocyanin synthesis. The obJectIvea of this study were (i) to describe the sequence of changes of PAL, PAL-IS and ethylene levels in apple skin during fruit maturation on the tree, (u) to compare these changes in an anthocyamn producing Red Delicious apple with a non-anthocyamn producing Golden Delicious apple, and (m) determine if PAL mhibltlon is a major factor m the development of red skm colour in apples.
after which time It mcreasmgly inhibited the activity of the yeast PAL (Fig. 2). As internal ethylene and apple PAL actlvlty increased, mhlbitlon of the yeast PAL also increased. This Increasing mhlbltlon of yeast PAL actlvity with fruit maturation 1s m contrast to the conclusion of Tan [ 1 l] that PAL-IS 1s responsible for regulation of anthocyanin accumulation m apple. The use of yeast PAL m the assay 1s desirable due to Its high specific activity [8], however, yeast PAL may not clearly reflect the effect of the mhlbitor(s) on all PAL isozymes It has been reported that there may be more than one type of
1’0°
RESULTS AND DISCUSSION
PAL activity and internal ethylene level increased at approximately the same time in Red Dehclous (Fig. 1). Stimulation of PAL activity by ethylene in apples is m agreement with the findings of others [lS]. When apple PAL-IS extract was added to yeast PAL It did not greatly alter activity of the yeast PAL during the first sampling,
Days
after
full bloom
Fig 1 Changes m Internal ethylene concentration and PAL actwty of the skm of Red Delicious apples during maturatron
1001
S M BLANKENSHIP and C R UNKATH
1002
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-
IO
-
001
140$! y” -
IZO-
s c ::
100 -
;i’ a
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60 -
40 -
20 -
1 117
124
131
138
145
152
Days after full bloom
117
I24
131
I 138
Days after
I 145
I 152
I 159
fult blwm
Ftg 2 PAL actwlty of the yeast Rhodotorula y/utm~s as influenced by Red or Golden Dehclous PAL-IS extract and the per cent of the total Red Dehclous apple skm that exhIbIted red colour durmg maturdtlon
3 Changes m Internal ethylene concentration and PAL activity of the skm of Golden Dehcmus apples durmg maturation
mhrbttor m sunflower [S] and, perhaps, thts also occurs m apple skm. The hrgh PAL activity during the increase m internal ethylene had little effect on red colour development, smce the skm gradually developed red skm colour, even when PAL levels were low (Ftg 2) In Golden Dehctous PAL acttvtty and internal ethylene both Increased durmg maturatton (Ftg 3) Golden Dehctous often do not exhtbtt the consrstant increase m ethylene productton that occurs m Red Dehctous However, the onset ofethylene productton dtd occur at approxtmately the same time as an increase m PAL acttvtty Apple PAL acttvtty was comparable between Red and Golden Dehous The apple PAL-IS mmally stimulated the yeast PAL, but as maturation progressed, the yeast PAL acttvtty decreased (Fig 2) Overall PAL-IS levels were stmtlar in Red and Golden Dehctous apples during maturation (Ftg 2) The stmtlartty of PAL and PAL-IS acttvtty between Red and Golden Dehctous would amply that other steps m anthocyanm synthesis control red skm development The apple PAL-IS seems to be effecttve on the yeast PAL, but tts role m regulatton of rn IWO PAL acttvtty seems questtonable
ground m lOml50mM sodturn borate buffer. pH 8 8 with 5 mM 2-mercaptoethanol, 1 mM EDTA, and 0 25 gm PVPP The homogenate was centrifuged for 30mm at 20000 y and the supernatant collected The 30-50% (NH,),SO, fractton from the supernatant was obtained The resultmg pellet U’~Credissolved m the extraction buffer mmus PVPP, then dlalyscd against 50mM borate buffer, pH 8 8 The total assay \ol was 1ml and contamed 50~1 002 M phenylalanme In buffer, 300/d 50mM sodmm borate buffer with 5 mM 2-mercaptoethanol and 200~11 extract The blank contdmed all of the above except phenylalamne The assay was redd at 290nm at 25 over I hr PAL-IS was extracted and asaaycd by the methods of Tan [ll J against PAL enLyme from the yeast, Rhodotcrr& qlutrnls Internal C,H, was extracted under vacuum from 25 apples by the methods of Saltviet 1181 Duphcdte experiments were conducted with slmllar results and the d\erage of the 2 evperlments 1s presented
EXPERIMENTAL
Plant materuzl M&s domestlca c? Starkrlmson Dehclous or cv Golden Dehc~ous were harvested from commercial N C orchards Fruit was selected for umformlty of size, freedom from defects and slmllar posItIon on the trees Analyses were conducted wlthm 1 day of harvest The time period covered by this study represents the time period durmg which the apples were mltlally Immature, then attamed horticultural maturity, and commercial harvest was begun PAL assay 5g of skm from 3-5 apples was collected by cutting strips of skm at each 90’ from stem and calyx Skm was
REFERENCES
1 Jones, D H (1984) Phytochemlstry 23, 1349 2 Camm, E L and Towers, G H N (1977) Proqr Phytochern 4, 169 3 Hyodo, H dnd Yang, S F (1971) P/an! Ph~sml 47, 765 4 Sarkar, S K and Phan, C T (1974) PhyvoI Pkmt 32. 318 5 Rhodes, M J C and Wooltorton. I_ S C (1971) Phrrochrrnrslry 10, 1989 6 Rlov, J , Monsehse, S P and Kahan. R S (1970) Radmf Eozany 10, 281 I Chappell, J, Hahlbrock, K and Roller. T (1984) Pluma 161, 475. 8 Gupta, S. C and Creasy, L (I 984) PhwoI P/ant 62. 260 9 Billet, E E , Wallace, W , and Smith. H (19781 Btochlm Bqhys Acta 524, 219 10 Tanaka, Y., Matsushita, K dnd Urltam. I (1977) Plum Cell PhywI 18, 1209
PAL during apple maturation 11. Tan, S. C. (1979) J. Am. Sot. Ho&c. Sci. 104, 581. 12. Tan, S. C. (1980) AU. J. Plant Physiof. 7, 159. 13. BoIwell, G. P., Cramer, C. L., Lamb, C. J., Schuch. W. and Dixon, R. A. (1986) Pfunta 169, 97. 14. Chalmers, D. J. and Faragher, J. D. (1977) Aust. J. Plant Physiof. 4, 111.
1003
15. Faragher, J. D. and Brohier, R. L. (1984) Scientia Hart. 22, 89. 16. Faragher, J. D. (1983) J. Exp. Botany 34, 1291. 17. Faragher, J. D. and Chalmers, D. J. (1977) Aust. J. PIant Physiof. 4, 133. 18. Saitveit, M. E., Jr. (1982) Hort. Science 17, 878.
Vol 27, No 4, pp 1001 1003, 1988
0
Prmted ,n Great Bntam
PAL AND ETHYLENE
CONTENT DURING GOLDEN DELICIOUS
SYLVIA M Department
of Horticultural
MATURATION APPLES
0031 9422/88 $300+000 Journals Ltd
1988 Pergamon
OF RED AND
and C. RICHARD UNRATH
BLANKENSHIP
Science, Box 7609, North
Carolina
State Umverslty,
Raleigh,
NC 27695, U S A
(Recewed 23 June 1987) Key Word Index--Ma/us
domestlca, Rosaceae,
anthocyamn;
rlpemng
Abstract-Phenylalamne ammonia-lyase actlvlty (PAL), a PAL inhlbltor (PAL-IS) and fruit internal ethylene levels were measured during apple maturation m Red and Golden Dehclous apples. In both cultlvars internal ethylene and PAL increased at approximately the same time Apple PAL-IS extract stimulated yeast PAL activity mihally, but increased yeast PAL mhlbitlon was observed as maturation progressed PAL mhlbltor levels were similar m both cuftlvars ‘The increase in yeast PAL mhlbltlon with maturation, increased red skm cofour development in Red Delicious and a comparable pattern of PAL actlvlty and mhlbltion in Red and Golden Delicious Indicates that PALIS was not closely involved with the regulation of anthocyamn synthesis m apple skm
INTRODUCTION
Phenylalamne ammonia lyase (E C 4 13.5) has been extensively reviewed [ 1,2] Ethylene enhances PAL activity m pea seedlings [3], carrots [4], and Swede and parsnip root tissue [S] PAL activity is stimulated by, but lags behind, ethylene production m irradiated grapefruit [6]. In cultured parsley cells, ethylene alone does not induce PAL [7] A protemaceous inhibitor of PAL activity is found m sunflower [S] and mhlbltors are present also m gherkin hypocotyls [9], sweet potato [lo] and apple skm [ll, 121. PAL activity 1s inhibited by the end product of the reaction, trans-cmnamlc acid [ 131 In apple skm, ethylene production is associated with anthocyamn accumulation and a concomitant increase m PAL activity m response to maturation, wounding and temperature [14-161. Low temperatature and light stimulate anthocyanm accumulation m apple skm, increase PAL activity, and lower PAL inhibitor activity [l 11. Faragher and Chalmers [ 171 concluded that while PAL influences anthocyanin accumulation m apples, there are other controlhng factors Tan [12] concluded that low temperature stimulates PAL actlvlty and decreases PAL mhlbltor, thus mcreasmg anthocyanin synthesis. The obJectIvea of this study were (i) to describe the sequence of changes of PAL, PAL-IS and ethylene levels in apple skin during fruit maturation on the tree, (u) to compare these changes in an anthocyamn producing Red Delicious apple with a non-anthocyamn producing Golden Delicious apple, and (m) determine if PAL mhibltlon is a major factor m the development of red skm colour in apples.
after which time It mcreasmgly inhibited the activity of the yeast PAL (Fig. 2). As internal ethylene and apple PAL actlvlty increased, mhlbitlon of the yeast PAL also increased. This Increasing mhlbltlon of yeast PAL actlvity with fruit maturation 1s m contrast to the conclusion of Tan [ 1 l] that PAL-IS 1s responsible for regulation of anthocyanin accumulation m apple. The use of yeast PAL m the assay 1s desirable due to Its high specific activity [8], however, yeast PAL may not clearly reflect the effect of the mhlbitor(s) on all PAL isozymes It has been reported that there may be more than one type of
1’0°
RESULTS AND DISCUSSION
PAL activity and internal ethylene level increased at approximately the same time in Red Dehclous (Fig. 1). Stimulation of PAL activity by ethylene in apples is m agreement with the findings of others [lS]. When apple PAL-IS extract was added to yeast PAL It did not greatly alter activity of the yeast PAL during the first sampling,
Days
after
full bloom
Fig 1 Changes m Internal ethylene concentration and PAL actwty of the skm of Red Delicious apples during maturatron
1001
S M BLANKENSHIP and C R UNKATH
1002
I60-
-
IO
-
001
140$! y” -
IZO-
s c ::
100 -
;i’ a
eo-
60 -
40 -
20 -
1 117
124
131
138
145
152
Days after full bloom
117
I24
131
I 138
Days after
I 145
I 152
I 159
fult blwm
Ftg 2 PAL actwlty of the yeast Rhodotorula y/utm~s as influenced by Red or Golden Dehclous PAL-IS extract and the per cent of the total Red Dehclous apple skm that exhIbIted red colour durmg maturdtlon
3 Changes m Internal ethylene concentration and PAL activity of the skm of Golden Dehcmus apples durmg maturation
mhrbttor m sunflower [S] and, perhaps, thts also occurs m apple skm. The hrgh PAL activity during the increase m internal ethylene had little effect on red colour development, smce the skm gradually developed red skm colour, even when PAL levels were low (Ftg 2) In Golden Dehctous PAL acttvtty and internal ethylene both Increased durmg maturatton (Ftg 3) Golden Dehctous often do not exhtbtt the consrstant increase m ethylene productton that occurs m Red Dehctous However, the onset ofethylene productton dtd occur at approxtmately the same time as an increase m PAL acttvtty Apple PAL acttvtty was comparable between Red and Golden Dehous The apple PAL-IS mmally stimulated the yeast PAL, but as maturation progressed, the yeast PAL acttvtty decreased (Fig 2) Overall PAL-IS levels were stmtlar in Red and Golden Dehctous apples during maturation (Ftg 2) The stmtlartty of PAL and PAL-IS acttvtty between Red and Golden Dehctous would amply that other steps m anthocyanm synthesis control red skm development The apple PAL-IS seems to be effecttve on the yeast PAL, but tts role m regulatton of rn IWO PAL acttvtty seems questtonable
ground m lOml50mM sodturn borate buffer. pH 8 8 with 5 mM 2-mercaptoethanol, 1 mM EDTA, and 0 25 gm PVPP The homogenate was centrifuged for 30mm at 20000 y and the supernatant collected The 30-50% (NH,),SO, fractton from the supernatant was obtained The resultmg pellet U’~Credissolved m the extraction buffer mmus PVPP, then dlalyscd against 50mM borate buffer, pH 8 8 The total assay \ol was 1ml and contamed 50~1 002 M phenylalanme In buffer, 300/d 50mM sodmm borate buffer with 5 mM 2-mercaptoethanol and 200~11 extract The blank contdmed all of the above except phenylalamne The assay was redd at 290nm at 25 over I hr PAL-IS was extracted and asaaycd by the methods of Tan [ll J against PAL enLyme from the yeast, Rhodotcrr& qlutrnls Internal C,H, was extracted under vacuum from 25 apples by the methods of Saltviet 1181 Duphcdte experiments were conducted with slmllar results and the d\erage of the 2 evperlments 1s presented
EXPERIMENTAL
Plant materuzl M&s domestlca c? Starkrlmson Dehclous or cv Golden Dehc~ous were harvested from commercial N C orchards Fruit was selected for umformlty of size, freedom from defects and slmllar posItIon on the trees Analyses were conducted wlthm 1 day of harvest The time period covered by this study represents the time period durmg which the apples were mltlally Immature, then attamed horticultural maturity, and commercial harvest was begun PAL assay 5g of skm from 3-5 apples was collected by cutting strips of skm at each 90’ from stem and calyx Skm was
REFERENCES
1 Jones, D H (1984) Phytochemlstry 23, 1349 2 Camm, E L and Towers, G H N (1977) Proqr Phytochern 4, 169 3 Hyodo, H dnd Yang, S F (1971) P/an! Ph~sml 47, 765 4 Sarkar, S K and Phan, C T (1974) PhyvoI Pkmt 32. 318 5 Rhodes, M J C and Wooltorton. I_ S C (1971) Phrrochrrnrslry 10, 1989 6 Rlov, J , Monsehse, S P and Kahan. R S (1970) Radmf Eozany 10, 281 I Chappell, J, Hahlbrock, K and Roller. T (1984) Pluma 161, 475. 8 Gupta, S. C and Creasy, L (I 984) PhwoI P/ant 62. 260 9 Billet, E E , Wallace, W , and Smith. H (19781 Btochlm Bqhys Acta 524, 219 10 Tanaka, Y., Matsushita, K dnd Urltam. I (1977) Plum Cell PhywI 18, 1209
PAL during apple maturation 11. Tan, S. C. (1979) J. Am. Sot. Ho&c. Sci. 104, 581. 12. Tan, S. C. (1980) AU. J. Plant Physiof. 7, 159. 13. BoIwell, G. P., Cramer, C. L., Lamb, C. J., Schuch. W. and Dixon, R. A. (1986) Pfunta 169, 97. 14. Chalmers, D. J. and Faragher, J. D. (1977) Aust. J. Plant Physiof. 4, 111.
1003
15. Faragher, J. D. and Brohier, R. L. (1984) Scientia Hart. 22, 89. 16. Faragher, J. D. (1983) J. Exp. Botany 34, 1291. 17. Faragher, J. D. and Chalmers, D. J. (1977) Aust. J. PIant Physiof. 4, 133. 18. Saitveit, M. E., Jr. (1982) Hort. Science 17, 878.