Planta (1982)155:64 67
P l ~ n ~ J 9 Springer-Verlag 1982
Synthesis of polygalacturonase during tomato fruit ripening Gregory A. Tucker and Donald Grierson Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, U.K.
Abstract. The cell wall degrading enzyme polygalact-
uronase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in " N e v e r ripe" mutants and the lack of activity in "ripening inhibitor" mutants can be correlated to the levels of immunologically detectable polygalacturonase protein. Key words: Lycopersicon - Mutant (tomato) - Polygalacturonase - Ripening (fruit).
Introduction
Ripening results from a series of co-ordinated changes in the biochemistry of fruits which affect their colour, texture and flavour. These changes are associated with alterations in the activities of a large number of enzymes (Sacher 1973). Although no dramatic changes in the total protein content of fruits occur as they ripen there is much evidence that protein synthesis is required for ripening. For example the protein synthesis inhibitor cycloheximide retards ripening of pome (Frenkel et al. 1968) and banana fruits (Brady et al. 1970). Also there is enhanced incorporation of radioactive amino acids into protein from the onset of the climacteric until the respiratory peak in many fruit (Sacher 1966; Richmond and Biale 1966) including tomatoes (De Swardt et al. 1973). It is generally assumed that ripening requires the selective synthesis of a few critical enzymes (Hulme 1972) or specific isoenzymes. However, there is very little information Abbreviations: PG= polygalacturonase; N r tion; rin= ripening inhibitor mutation
0032-0935/82/0155/0064/$01.00
Never ripe muta-
about the way in which the various aspects of ripening are controlled and co-ordinated although ethylene is thought to play a role in the initiaion of these responses. During the ripening of tomato fruits several polysaccharide-degrading enzymes are known to increase in activity, including cellulase (Dickinson and McCollum 1964), pectinesterase (Hobson 1963) and polygalacturonase (PG) (Hobson 1964; Grierson etal. 1981). These last two enzymes are associated with cell walls and are believed to be involved in the substantial softening of the fruit which takes place during ripening. In mature green tomatoes there is no extractable PG activity. The enzyme appears at the commencement of colouration and then increases dramatically (Hobson 1963 ; Grierson et al. 1981). The activity arises from two isoenzymes (PG-1 and PG-2) which appear sequentially during ripening (Tucker et al. 1980). These isoenzymes contain similar polypeptides and it is probable that PG-1 is a dimer of PG-2 (Tucker et al. 1980). It is not known how the increase in enzyme activity is initiated or whether it is due to activation or synthesis of enzymes. The study of tomato ripening is aided by the availability of many mutants. Two such mutants are defective in PG activity. The " N e v e r ripe" (Nr) mutation results, in the homozygous condition, in a much lower PG activity than normal (Hobson 1967). This can be accounted for by the appearance of only one isoenzyme, equivalent to PG-1 from normal fruit (Tucker et al. 1980). The "ripening inhibitor" (rin) mutation results, in the homozygous condition, in the production of very little or no P G activity (Hobson 1980). In addition Nr fruit remain orange and soften slowly whilst rin fruit remain yellow and show very little softening (Tigchelaar et al. 1978). We present evidence here that in the case of PG, which is probably the major softening enzyme in tomatoes (Hobson 1965), the appearance of enzyme activity is due to de novo synthesis. Also the lack
G.A. Tucker and D. Grierson: Synthesis of tomato polygalacturonase
65
of P G activity in the m u t a n t s is correlated with a lack of i m m u n o l o g i c a l l y active protein.
Materials and methods Plant material. The mutations Nr and rin have been bred into near-isogenic lines of tomatoes (Lycopersicon esculentum Mill var Ailsa Craig). Seeds of normal and mutant tomatoes homozygous for Nr and rin were obtained from Mr. L.A. Darby of the Glasshouse Crops Research Institute, Littlehampton, U.K. Plants were grown in straw bales in a glasshouse and fruit were collected just before being used for experiments. Enzyme extractions. Tomato pericarp was sliced into water (1 : 1 w/v) and briefly homogenised using a Polytron homogeniser (Kinematica GmbH, Luzern, Switzerland). The homogenate was centrifuged at 2,400 g for 10 rain and divided into pellet and supernatant fractions. Water soluble proteins were precipitated from the resultant supernatant by addition of 100% trichloroacetic acid to give a final concentration of 10%. The precipitate was washed twice with ethanol and finally dissolved in 10 mM Tris-HCi (tris(hydroxymethyl)aminomethane 2-amino-2-(hydroxymethyl)-1,3-propanediol HC1)) pH 8 containing 0.15 M NaCI for use on a 1.5% agar immunodiffusion gel. Control experiments with purified PG showed that precipitation with trichloroacetic acid has little effect on the subsequent recognition of the enzyme by antibody. Cell-wall-bound proteins were extracted from the initial pellet by washing in 1 M NaC1 pH 6 and precipitated with ammonium sulphate as described by Tucker et al. (1980). Washing the pellet extensively with water prior to the extraction into NaC1 did not alter the subsequent banding pattern of the cell-wall-bound protein extracts when analysed under denaturing conditions on polyacrylamide gels (unpublished results). The cell-wall-bound extracts were assayed for PG activity by the release of reducing groups (Tucker et al. 1980). The PG protein was measured by radioimmunoassay by competition between purified PG-2 labelled in vitro with lzsI, and tomato protein extracts, for antibody raised against PG-2 as described previously (Tucker et al. 1980). Lycopene measurements were made spectrophotometrically using acetone-hexane as described by Tomes (1963). Results Appearance o f wall-bound polygalacturonase during ripening. C e l l - w a l l - b o u n d p r o t e i n extracts were prepared f r o m m a t u r e green a n d red n o r m a l t o m a t o e s a n d f r o m o r a n g e Nr a n d yellow rin fruit. These extracts were analysed by p o l y a c r y l a m i d e gel electrophoresis u n d e r d e n a t u r i n g c o n d i t i o n s (Fig. 1). It has been s h o w n (Tucker et al. 1980) that u n d e r these conditions b o t h PG-1 a n d P G - 2 give a stained b a n d with a n a p p a r e n t m o l e c u l a r weight of 46,000. Purified P G 2 was r u n as a m a r k e r in Fig. 1. One of the m a i n differences between the extracts f r o m green a n d red n o r m a l fruit was the a p p e a r a n c e of a stained p r o t e i n b a n d in red fruit which m i g r a t e d to the same p o s i t i o n as purified P G . The e q u i v a l e n t b a n d was reduced in the extract f r o m Nr fruit a n d was a b s e n t f r o m the rin extract. This result correlates with the a p p e a r ance of P G activity d u r i n g n o r m a l ripening, with the reduced activity in Nr a n d the lack of activity in rin fruit.
Fig. 1. Polyacrylamide gel profiles of ceil-wall-bound proteins. Protein samples were suspended in a buffer (40 mM Tris-HC1pH 9.18 containing 50% (w/v) sucrose, i0% (w/v) sodium dodecylsulphate and 5% (v/v) mercaptoethanol) and boiled for 5 rain. Samples containing 50-100 ~tg were then fractionated in a 10-15% gradient polyacrylamide slab gel under denaturing conditions in 0.1% sodium dodecylsulphate. Track A contains purified PG-2 (PG). Tracks B-E contain total cell-wall-bound proteins from green normal, ripe normal, orange Nr and yellow rin fruit, respectively. Gels were stained in 0.1% Coomassie blue, 40% methanol, 7% acetic acid and destained in 30% methanol, 7% acetic acid
The above result could be explained if green, Nr a n d rin fruit c o n t a i n e d some P G p r o t e i n which was water soluble a n d n o t w a l l - b o u n d . However, water soluble extracts from m a t u r e green, orange N r or yellow rin fruit had n o detectable P G activity. Also, proteins recovered from the water soluble fraction by trichloroacetic acid precipitation showed no reaction with P G a n t i b o d y in i m m u n o d i f f u s i o n gels (unpublished results).
Changes in polygalaeturonase during ripening as measured by radioimmunoassay. A n a n t i b o d y has been raised, in rabbits, against P G - 2 a n d s h o w n to cross react with PG-1 by both r a d i o i m m u n o a s s a y a n d imm u n o d i f f u s i o n (Tucker et al. 1980). This was used to measure the synthesis of P G d u r i n g ripening. N o r real t o m a t o fruit were harvested at various stages of ripeness as j u d g e d by their colour d e v e l o p m e n t .
66
G.A. Tucker and D. Grierson: Synthesis of tomato polygalacturonase
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Fig. 2. Correlation between P G activity and P G protein in cell-wallbound extracts of normal fruit during ripening. Individual fruits harvested at various stages of ripeness were each assayed for lycopene content, P G enzyme activity ( o ) and P G protein measured by radioimmunoassay (e). Samples were ordered according to P G activity
Fig. 3. Radioimmunoassay of protein extracts from mutant tomato fruit. Total cell-wall-bound proteins were extracted from green (o) and red ( l ) normal fruit, and orange Nr (D) and yellow tin (e) fruit. These extracts were used to displace :25I-iabelled PG-2 in the radioimmunoassay. The P G specific activities of the extracts were 0, 0.001, 0.075 and 0.389 ~tmol galacturonic acid produced rain -1 m g -1 protein at 2 5 ~ for green, tin, Nr and red fruit, respectively
The pericarp of each individual fruit was weighed and homogenised in water. A sample, equivalent to 0.2 g pericarp, was taken and assayed for lycopene content. Cell-wall-bound proteins were than extracted from the remaining homogenate as described in Materials and methods. These extracts were then assayed for both P G activity and for PG protein. The results are presented in Fig. 2. It can be seen that PG activity, as detected by convential enzyme assays, increases dramatically as the fruit ripen. There is also a corresponding increase in the amount of P G protein, detectable by radioimmunoassay, as the fruit ripen. The results in Fig. 2 show that as fruits ripen, as indicated by their lycopene content, there is a large increase in both the P G activity and P G protein associated with the cell wall. This result suggests that the increased PG activity is caused by an increase in the number of P G enzyme molecules rather than an activation of any pre-existing enzyme.
in the radioimmunoassay (Fig. 3). The results show that rin extracts, like those from normal green fruit, displace very little PG-2. The displacement was so low that accurate determination of PG protein content could not be made. The Nr extracts contained less P G protein than extracts from normal fruit (Fig. 3), but the amount was measurable by radioimmunoassay. Measurements of PG activity and protein showed that Nr extracts have a specific activity for P G of 2-5 gmol galacturonic acid min- 1 mg- 1 protein. This was similar to the specific activities obtained in normal fruit extracts (Fig. 2). Thus in both Nr and rin mutants the reduced levels of PG activity can be correlated to a reduced amount of immunologically detectable PG protein.
Polygalacturonase in mutant fruit. Wall-bound extracts from Orange Nr and yellow rin fruit were tested for their ability to compete with radioiodinated PG-2
Discussion
The results in Fig. 1 show that a protein corresponding to P G appears in the cell wall fraction as fruit ripen. In ripe fruit the enzyme is a major protein of cell wall preparations. The results in Fig. 2 show
G.A. Tucker and D. Grierson: Synthesis of tomato polygalacturonase t h a t d u r i n g r i p e n i n g the a m o u n t o f P G e n z y m e activity a t t a c h e d to the cell walls is d i r e c t l y r e l a t e d to the a m o u n t o f p r o t e i n which reacts with a n t i b o d y raised a g a i n s t P G . This indicates t h a t there is no inactive f o r m o f the e n z y m e a t t a c h e d to the cell walls in u n r i p e fruit a n d t h a t the increase in e n z y m e activity is due to an increase in the n u m b e r o f enzyme molecules. This c o u l d be e x p l a i n e d either by the de n o v o synthesis o f e n z y m e d u r i n g r i p e n i n g o r b y the transfer o f s t o r e d e n z y m e f r o m the c y t o p l a s m to the cell walls. H o w e v e r , the i m m u n o d i f f u s i o n e x p e r i m e n t s with w a t e r soluble extracts f r o m green fruit gave no evidence for the presence o f P G a n d the latter p o s s i b i l i t y therefore a p p e a r s unlikely. One o t h e r possible explan a t i o n for these o b s e r v a t i o n s is t h a t the P G is p r e s e n t before r i p e n i n g b u t r e m a i n s tightly b o u n d to the cell walls o f green fruit a n d is released o n l y as the fruit ripens. H o w e v e r , a t t e m p t s to solubilise P G activity f r o m green cell walls b y a v a r i e t y o f m e t h o d s have failed ( u n p u b l i s h e d results). T h e results o f the e x p e r i m e n t s with m u t a n t fruit suggest t h a t the lack o f P G arises f r o m a p a r t i a l (Nr) o r c o m p l e t e (rin) failure in the synthesis o f P G . The tin m u t a t i o n m a y be in a s t r u c t u r a l gene for P G which results in an u n s t a b l e or altered P G p r o t e i n w i t h o u t e n z y m e activity a n d with no affinity for P G a n t i b o d y . H o w e v e r , since the rin m u t a t i o n has several different p h e n o t y p i c effects it seems m o r e likely t h a t the m u t a t i o n is in a r e g u l a t o r y gene which affects a n u m b e r o f r i p e n i n g - r e l a t e d changes, i n c l u d i n g P G synthesis. The r e d u c e d synthesis o f P G in N r m u t a n t s has p r e v i o u s l y been c o r r e l a t e d with the failure to p r o duce P G - 2 ( T u c k e r et al. 1980). H o w e v e r , i s o e n z y m e s 1 a n d 2 seem to be related, because they c o n t a i n very similar p o l y p e p t i d e s ( T u c k e r et al. 1980) a n d one i s o e n z y m e can be c o n v e r t e d to the o t h e r in vitro ( T u c k e r et al. 1981). The N r m u t a t i o n could, therefore, either be in a s t r u c t u r a l gene for P G - 2 , in a gene c o n t r o l l i n g rate o f e n z y m e a c c u m u l a t i o n o r in a gene g o v e r n i n g the i n t e r - c o n v e r s i o n o f i s o e n z y m e s 1 a n d 2. R i p e n i n g m a y require specific i s o e n z y m e forms. Thus the a b n o r m a l r i p e n i n g o f N r m a y be a t t r i b u t a b l e to the presence o f only one (PG1) i n s t e a d o f two, P G isoenzymes. The results in this p a p e r i n d i c a t e t h a t P G is one o f the critical enzymes t h a t are synthesised de n o v o d u r i n g ripening. T i g c h e l a a r et al. (1978) have suggested t h a t the a p p e a r a n c e o f P G activity m a y be the initial trigger o f fruit r i p e n i n g a n d t h a t ethylene synthesis a n d o t h e r events occur as a c o n s e q u e n c e o f P G activity. T h e lack o f b o t h P G synthesis a n d n o r m a l r i p e n i n g in the rin m u t a n t w o u l d s u p p o r t this theory. H o w e v e r , the N r m u t a n t also ripens a b n o r m a l l y b u t does s h o w
67 s o m e P G synthesis. Thus, unless P G - 2 is the key isoenzyme, results with the N r m u t a n t d o n o t fit this theory. To test T i g c h e l a a r ' s t h e o r y it w o u l d be useful to time a c c u r a t e l y the synthesis o f P G in n o r m a l fruit a n d relate this to o t h e r r i p e n i n g events n a m e l y ethylene p r o d u c t i o n a n d the c l i m a c t e r i c rise in r e s p i r a t i o n . E x p e r i m e n t s o f this type are being carried o u t in this laboratory. We would like to thank Mr. L.A. Darby of the Glasshouse Crops Research Institute for the kind gift of tomato seeds. Thanks also go to Dr. D.B. Crighton for the radioiodination of PG2. Finally we acknowledge the financial support of the Agricultural Research Council. References Brady, C.J., Palmer, J.K., O'Connell, P.B.H., Smillie, R.M. (1970) An increase in protein synthesis during ripening of the banana fruit. Phytochemistry 9, 1037 1047 De Swardt, G.H., Swanepoel, J.H., Dubenage, A.J. (1973) Relationships between changes in ribosomal RNA and total protein synthesis, and tire respiration climacteric in pericarp tissues of tomatoes. Z. Pflanzenphysiol, 70, 358-363 Dickinson, D.B., McCotlum, D.P. (1964) Cellulase in tomato fruits. Nature 203, 525-527 Frenkel, C., Klein, I., Dilley, D.R. (1968) Protein synthesis in relation to ripening of pome fruits. Plant Physiol. 43, 1146-1153 Grierson, D., Tucker, G.A., Robertson, N.G. (1981) The molecular biology of ripening. In: Biochemistry of fruit and vegetables, pp. 179 191, Friend, J., ed. Academic Press, London Hobson, G.E. (1963) Pectinesterase in normal and abnormal tomato fruit. Biochem. J. 86, 358-365 Hobson, G.E. (1964) Polygalacturonase in normal and abnormal tomato fruit. Biochem. J. 92, 324-332 Hobson, G.E. (1965) The firmness of tomato fruit in relation to polygalacturonase activity. J. Hortic. Sci. 40, 66 72 Hobson, G.E. (I967) The effect of alleles at the Nr locus on the ripening of tomato fruit. Phytochemistry 6, 1337 1341 Hobson, G.E. (1980) Effect of the introduction of non-ripening mutant genes on the composition and enzyme content of tomato fruit. J. Sci. Food Agric. 31, 578-584 Hulme, A.C. (1972) The proteins of fruits: their involvements as enzymes in ripening. A review. J. Food Technol. 7, 343 371 Richmond, A., Biale, J.B. (1966) Protein synthesis in avocado fruit tissue. Arch. Biochem. Biophys. 115, 211-214 Sacher, J.A. (1966) Permeability characteristics and amino acids incorporation during senescence (ripening) of banana tissue. Plant Physiol. 41, 701-708 Sacher, J.A. (1973) Senescence and postharvest physiology. Annu. Rev. Plant Physiol. 24, 197-224 Tigchelaar, E.C., McGlasson, W.B., Buescher, R.W. (1978) Genetic regulation of tomato fruit ripening. Hortscience 13, 508-513 Tomes, M.L. (1963) Temperature inhibition of carotene synthesis in tomato. Bot. Gaz. (Chicago) 124, 180-185 Tucker, G.A., Robertson, N.G., Grierson, D. (1980) Changes in polygalacturonase isoenzymes during the 'ripening' of normal and mutant tomato fruit. Eur. J. Biochem. 112, 119-124 Tucker, G.A., Robertson, N.G., Grierson, D. (I981) The conversion of tomato fruit polygalacturonase isoenzyme 2 into isoenzyme 1 in vitro. Eur. J. Biochem. 115, 87-90 Received 22 December 1981 ; accepted 16 February 1982
Note added in proof: Using gas chromatography and radioimmunoassay to measure ethylene and PG we have obtained evidence that during normal ripening PG synthesis begins 20-40 h after the start of ethylene evolution. These results, which will be published separately, indicate that the hypothesis of Tigchelaar et al. (1978) should be rejected.
P l ~ n ~ J 9 Springer-Verlag 1982
Synthesis of polygalacturonase during tomato fruit ripening Gregory A. Tucker and Donald Grierson Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough, Leicestershire LE12 5RD, U.K.
Abstract. The cell wall degrading enzyme polygalact-
uronase (E.C. 3.2.1.15) is not detectable in green tomatoes (Lycopersicon esculentum Mill). Activity appears at the onset of ripening and in ripe fruit it is one of the major cell-wall-bound proteins. Radioimmunoassay results, employing an antibody against purified polygalacturonase, suggest that during ripening the enzyme is synthesised de novo. Radioimmunoassay data also show that the low level of polygalacturonase in " N e v e r ripe" mutants and the lack of activity in "ripening inhibitor" mutants can be correlated to the levels of immunologically detectable polygalacturonase protein. Key words: Lycopersicon - Mutant (tomato) - Polygalacturonase - Ripening (fruit).
Introduction
Ripening results from a series of co-ordinated changes in the biochemistry of fruits which affect their colour, texture and flavour. These changes are associated with alterations in the activities of a large number of enzymes (Sacher 1973). Although no dramatic changes in the total protein content of fruits occur as they ripen there is much evidence that protein synthesis is required for ripening. For example the protein synthesis inhibitor cycloheximide retards ripening of pome (Frenkel et al. 1968) and banana fruits (Brady et al. 1970). Also there is enhanced incorporation of radioactive amino acids into protein from the onset of the climacteric until the respiratory peak in many fruit (Sacher 1966; Richmond and Biale 1966) including tomatoes (De Swardt et al. 1973). It is generally assumed that ripening requires the selective synthesis of a few critical enzymes (Hulme 1972) or specific isoenzymes. However, there is very little information Abbreviations: PG= polygalacturonase; N r tion; rin= ripening inhibitor mutation
0032-0935/82/0155/0064/$01.00
Never ripe muta-
about the way in which the various aspects of ripening are controlled and co-ordinated although ethylene is thought to play a role in the initiaion of these responses. During the ripening of tomato fruits several polysaccharide-degrading enzymes are known to increase in activity, including cellulase (Dickinson and McCollum 1964), pectinesterase (Hobson 1963) and polygalacturonase (PG) (Hobson 1964; Grierson etal. 1981). These last two enzymes are associated with cell walls and are believed to be involved in the substantial softening of the fruit which takes place during ripening. In mature green tomatoes there is no extractable PG activity. The enzyme appears at the commencement of colouration and then increases dramatically (Hobson 1963 ; Grierson et al. 1981). The activity arises from two isoenzymes (PG-1 and PG-2) which appear sequentially during ripening (Tucker et al. 1980). These isoenzymes contain similar polypeptides and it is probable that PG-1 is a dimer of PG-2 (Tucker et al. 1980). It is not known how the increase in enzyme activity is initiated or whether it is due to activation or synthesis of enzymes. The study of tomato ripening is aided by the availability of many mutants. Two such mutants are defective in PG activity. The " N e v e r ripe" (Nr) mutation results, in the homozygous condition, in a much lower PG activity than normal (Hobson 1967). This can be accounted for by the appearance of only one isoenzyme, equivalent to PG-1 from normal fruit (Tucker et al. 1980). The "ripening inhibitor" (rin) mutation results, in the homozygous condition, in the production of very little or no P G activity (Hobson 1980). In addition Nr fruit remain orange and soften slowly whilst rin fruit remain yellow and show very little softening (Tigchelaar et al. 1978). We present evidence here that in the case of PG, which is probably the major softening enzyme in tomatoes (Hobson 1965), the appearance of enzyme activity is due to de novo synthesis. Also the lack
G.A. Tucker and D. Grierson: Synthesis of tomato polygalacturonase
65
of P G activity in the m u t a n t s is correlated with a lack of i m m u n o l o g i c a l l y active protein.
Materials and methods Plant material. The mutations Nr and rin have been bred into near-isogenic lines of tomatoes (Lycopersicon esculentum Mill var Ailsa Craig). Seeds of normal and mutant tomatoes homozygous for Nr and rin were obtained from Mr. L.A. Darby of the Glasshouse Crops Research Institute, Littlehampton, U.K. Plants were grown in straw bales in a glasshouse and fruit were collected just before being used for experiments. Enzyme extractions. Tomato pericarp was sliced into water (1 : 1 w/v) and briefly homogenised using a Polytron homogeniser (Kinematica GmbH, Luzern, Switzerland). The homogenate was centrifuged at 2,400 g for 10 rain and divided into pellet and supernatant fractions. Water soluble proteins were precipitated from the resultant supernatant by addition of 100% trichloroacetic acid to give a final concentration of 10%. The precipitate was washed twice with ethanol and finally dissolved in 10 mM Tris-HCi (tris(hydroxymethyl)aminomethane 2-amino-2-(hydroxymethyl)-1,3-propanediol HC1)) pH 8 containing 0.15 M NaCI for use on a 1.5% agar immunodiffusion gel. Control experiments with purified PG showed that precipitation with trichloroacetic acid has little effect on the subsequent recognition of the enzyme by antibody. Cell-wall-bound proteins were extracted from the initial pellet by washing in 1 M NaC1 pH 6 and precipitated with ammonium sulphate as described by Tucker et al. (1980). Washing the pellet extensively with water prior to the extraction into NaC1 did not alter the subsequent banding pattern of the cell-wall-bound protein extracts when analysed under denaturing conditions on polyacrylamide gels (unpublished results). The cell-wall-bound extracts were assayed for PG activity by the release of reducing groups (Tucker et al. 1980). The PG protein was measured by radioimmunoassay by competition between purified PG-2 labelled in vitro with lzsI, and tomato protein extracts, for antibody raised against PG-2 as described previously (Tucker et al. 1980). Lycopene measurements were made spectrophotometrically using acetone-hexane as described by Tomes (1963). Results Appearance o f wall-bound polygalacturonase during ripening. C e l l - w a l l - b o u n d p r o t e i n extracts were prepared f r o m m a t u r e green a n d red n o r m a l t o m a t o e s a n d f r o m o r a n g e Nr a n d yellow rin fruit. These extracts were analysed by p o l y a c r y l a m i d e gel electrophoresis u n d e r d e n a t u r i n g c o n d i t i o n s (Fig. 1). It has been s h o w n (Tucker et al. 1980) that u n d e r these conditions b o t h PG-1 a n d P G - 2 give a stained b a n d with a n a p p a r e n t m o l e c u l a r weight of 46,000. Purified P G 2 was r u n as a m a r k e r in Fig. 1. One of the m a i n differences between the extracts f r o m green a n d red n o r m a l fruit was the a p p e a r a n c e of a stained p r o t e i n b a n d in red fruit which m i g r a t e d to the same p o s i t i o n as purified P G . The e q u i v a l e n t b a n d was reduced in the extract f r o m Nr fruit a n d was a b s e n t f r o m the rin extract. This result correlates with the a p p e a r ance of P G activity d u r i n g n o r m a l ripening, with the reduced activity in Nr a n d the lack of activity in rin fruit.
Fig. 1. Polyacrylamide gel profiles of ceil-wall-bound proteins. Protein samples were suspended in a buffer (40 mM Tris-HC1pH 9.18 containing 50% (w/v) sucrose, i0% (w/v) sodium dodecylsulphate and 5% (v/v) mercaptoethanol) and boiled for 5 rain. Samples containing 50-100 ~tg were then fractionated in a 10-15% gradient polyacrylamide slab gel under denaturing conditions in 0.1% sodium dodecylsulphate. Track A contains purified PG-2 (PG). Tracks B-E contain total cell-wall-bound proteins from green normal, ripe normal, orange Nr and yellow rin fruit, respectively. Gels were stained in 0.1% Coomassie blue, 40% methanol, 7% acetic acid and destained in 30% methanol, 7% acetic acid
The above result could be explained if green, Nr a n d rin fruit c o n t a i n e d some P G p r o t e i n which was water soluble a n d n o t w a l l - b o u n d . However, water soluble extracts from m a t u r e green, orange N r or yellow rin fruit had n o detectable P G activity. Also, proteins recovered from the water soluble fraction by trichloroacetic acid precipitation showed no reaction with P G a n t i b o d y in i m m u n o d i f f u s i o n gels (unpublished results).
Changes in polygalaeturonase during ripening as measured by radioimmunoassay. A n a n t i b o d y has been raised, in rabbits, against P G - 2 a n d s h o w n to cross react with PG-1 by both r a d i o i m m u n o a s s a y a n d imm u n o d i f f u s i o n (Tucker et al. 1980). This was used to measure the synthesis of P G d u r i n g ripening. N o r real t o m a t o fruit were harvested at various stages of ripeness as j u d g e d by their colour d e v e l o p m e n t .
66
G.A. Tucker and D. Grierson: Synthesis of tomato polygalacturonase
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Fig. 2. Correlation between P G activity and P G protein in cell-wallbound extracts of normal fruit during ripening. Individual fruits harvested at various stages of ripeness were each assayed for lycopene content, P G enzyme activity ( o ) and P G protein measured by radioimmunoassay (e). Samples were ordered according to P G activity
Fig. 3. Radioimmunoassay of protein extracts from mutant tomato fruit. Total cell-wall-bound proteins were extracted from green (o) and red ( l ) normal fruit, and orange Nr (D) and yellow tin (e) fruit. These extracts were used to displace :25I-iabelled PG-2 in the radioimmunoassay. The P G specific activities of the extracts were 0, 0.001, 0.075 and 0.389 ~tmol galacturonic acid produced rain -1 m g -1 protein at 2 5 ~ for green, tin, Nr and red fruit, respectively
The pericarp of each individual fruit was weighed and homogenised in water. A sample, equivalent to 0.2 g pericarp, was taken and assayed for lycopene content. Cell-wall-bound proteins were than extracted from the remaining homogenate as described in Materials and methods. These extracts were then assayed for both P G activity and for PG protein. The results are presented in Fig. 2. It can be seen that PG activity, as detected by convential enzyme assays, increases dramatically as the fruit ripen. There is also a corresponding increase in the amount of P G protein, detectable by radioimmunoassay, as the fruit ripen. The results in Fig. 2 show that as fruits ripen, as indicated by their lycopene content, there is a large increase in both the P G activity and P G protein associated with the cell wall. This result suggests that the increased PG activity is caused by an increase in the number of P G enzyme molecules rather than an activation of any pre-existing enzyme.
in the radioimmunoassay (Fig. 3). The results show that rin extracts, like those from normal green fruit, displace very little PG-2. The displacement was so low that accurate determination of PG protein content could not be made. The Nr extracts contained less P G protein than extracts from normal fruit (Fig. 3), but the amount was measurable by radioimmunoassay. Measurements of PG activity and protein showed that Nr extracts have a specific activity for P G of 2-5 gmol galacturonic acid min- 1 mg- 1 protein. This was similar to the specific activities obtained in normal fruit extracts (Fig. 2). Thus in both Nr and rin mutants the reduced levels of PG activity can be correlated to a reduced amount of immunologically detectable PG protein.
Polygalacturonase in mutant fruit. Wall-bound extracts from Orange Nr and yellow rin fruit were tested for their ability to compete with radioiodinated PG-2
Discussion
The results in Fig. 1 show that a protein corresponding to P G appears in the cell wall fraction as fruit ripen. In ripe fruit the enzyme is a major protein of cell wall preparations. The results in Fig. 2 show
G.A. Tucker and D. Grierson: Synthesis of tomato polygalacturonase t h a t d u r i n g r i p e n i n g the a m o u n t o f P G e n z y m e activity a t t a c h e d to the cell walls is d i r e c t l y r e l a t e d to the a m o u n t o f p r o t e i n which reacts with a n t i b o d y raised a g a i n s t P G . This indicates t h a t there is no inactive f o r m o f the e n z y m e a t t a c h e d to the cell walls in u n r i p e fruit a n d t h a t the increase in e n z y m e activity is due to an increase in the n u m b e r o f enzyme molecules. This c o u l d be e x p l a i n e d either by the de n o v o synthesis o f e n z y m e d u r i n g r i p e n i n g o r b y the transfer o f s t o r e d e n z y m e f r o m the c y t o p l a s m to the cell walls. H o w e v e r , the i m m u n o d i f f u s i o n e x p e r i m e n t s with w a t e r soluble extracts f r o m green fruit gave no evidence for the presence o f P G a n d the latter p o s s i b i l i t y therefore a p p e a r s unlikely. One o t h e r possible explan a t i o n for these o b s e r v a t i o n s is t h a t the P G is p r e s e n t before r i p e n i n g b u t r e m a i n s tightly b o u n d to the cell walls o f green fruit a n d is released o n l y as the fruit ripens. H o w e v e r , a t t e m p t s to solubilise P G activity f r o m green cell walls b y a v a r i e t y o f m e t h o d s have failed ( u n p u b l i s h e d results). T h e results o f the e x p e r i m e n t s with m u t a n t fruit suggest t h a t the lack o f P G arises f r o m a p a r t i a l (Nr) o r c o m p l e t e (rin) failure in the synthesis o f P G . The tin m u t a t i o n m a y be in a s t r u c t u r a l gene for P G which results in an u n s t a b l e or altered P G p r o t e i n w i t h o u t e n z y m e activity a n d with no affinity for P G a n t i b o d y . H o w e v e r , since the rin m u t a t i o n has several different p h e n o t y p i c effects it seems m o r e likely t h a t the m u t a t i o n is in a r e g u l a t o r y gene which affects a n u m b e r o f r i p e n i n g - r e l a t e d changes, i n c l u d i n g P G synthesis. The r e d u c e d synthesis o f P G in N r m u t a n t s has p r e v i o u s l y been c o r r e l a t e d with the failure to p r o duce P G - 2 ( T u c k e r et al. 1980). H o w e v e r , i s o e n z y m e s 1 a n d 2 seem to be related, because they c o n t a i n very similar p o l y p e p t i d e s ( T u c k e r et al. 1980) a n d one i s o e n z y m e can be c o n v e r t e d to the o t h e r in vitro ( T u c k e r et al. 1981). The N r m u t a t i o n could, therefore, either be in a s t r u c t u r a l gene for P G - 2 , in a gene c o n t r o l l i n g rate o f e n z y m e a c c u m u l a t i o n o r in a gene g o v e r n i n g the i n t e r - c o n v e r s i o n o f i s o e n z y m e s 1 a n d 2. R i p e n i n g m a y require specific i s o e n z y m e forms. Thus the a b n o r m a l r i p e n i n g o f N r m a y be a t t r i b u t a b l e to the presence o f only one (PG1) i n s t e a d o f two, P G isoenzymes. The results in this p a p e r i n d i c a t e t h a t P G is one o f the critical enzymes t h a t are synthesised de n o v o d u r i n g ripening. T i g c h e l a a r et al. (1978) have suggested t h a t the a p p e a r a n c e o f P G activity m a y be the initial trigger o f fruit r i p e n i n g a n d t h a t ethylene synthesis a n d o t h e r events occur as a c o n s e q u e n c e o f P G activity. T h e lack o f b o t h P G synthesis a n d n o r m a l r i p e n i n g in the rin m u t a n t w o u l d s u p p o r t this theory. H o w e v e r , the N r m u t a n t also ripens a b n o r m a l l y b u t does s h o w
67 s o m e P G synthesis. Thus, unless P G - 2 is the key isoenzyme, results with the N r m u t a n t d o n o t fit this theory. To test T i g c h e l a a r ' s t h e o r y it w o u l d be useful to time a c c u r a t e l y the synthesis o f P G in n o r m a l fruit a n d relate this to o t h e r r i p e n i n g events n a m e l y ethylene p r o d u c t i o n a n d the c l i m a c t e r i c rise in r e s p i r a t i o n . E x p e r i m e n t s o f this type are being carried o u t in this laboratory. We would like to thank Mr. L.A. Darby of the Glasshouse Crops Research Institute for the kind gift of tomato seeds. Thanks also go to Dr. D.B. Crighton for the radioiodination of PG2. Finally we acknowledge the financial support of the Agricultural Research Council. References Brady, C.J., Palmer, J.K., O'Connell, P.B.H., Smillie, R.M. (1970) An increase in protein synthesis during ripening of the banana fruit. Phytochemistry 9, 1037 1047 De Swardt, G.H., Swanepoel, J.H., Dubenage, A.J. (1973) Relationships between changes in ribosomal RNA and total protein synthesis, and tire respiration climacteric in pericarp tissues of tomatoes. Z. Pflanzenphysiol, 70, 358-363 Dickinson, D.B., McCotlum, D.P. (1964) Cellulase in tomato fruits. Nature 203, 525-527 Frenkel, C., Klein, I., Dilley, D.R. (1968) Protein synthesis in relation to ripening of pome fruits. Plant Physiol. 43, 1146-1153 Grierson, D., Tucker, G.A., Robertson, N.G. (1981) The molecular biology of ripening. In: Biochemistry of fruit and vegetables, pp. 179 191, Friend, J., ed. Academic Press, London Hobson, G.E. (1963) Pectinesterase in normal and abnormal tomato fruit. Biochem. J. 86, 358-365 Hobson, G.E. (1964) Polygalacturonase in normal and abnormal tomato fruit. Biochem. J. 92, 324-332 Hobson, G.E. (1965) The firmness of tomato fruit in relation to polygalacturonase activity. J. Hortic. Sci. 40, 66 72 Hobson, G.E. (I967) The effect of alleles at the Nr locus on the ripening of tomato fruit. Phytochemistry 6, 1337 1341 Hobson, G.E. (1980) Effect of the introduction of non-ripening mutant genes on the composition and enzyme content of tomato fruit. J. Sci. Food Agric. 31, 578-584 Hulme, A.C. (1972) The proteins of fruits: their involvements as enzymes in ripening. A review. J. Food Technol. 7, 343 371 Richmond, A., Biale, J.B. (1966) Protein synthesis in avocado fruit tissue. Arch. Biochem. Biophys. 115, 211-214 Sacher, J.A. (1966) Permeability characteristics and amino acids incorporation during senescence (ripening) of banana tissue. Plant Physiol. 41, 701-708 Sacher, J.A. (1973) Senescence and postharvest physiology. Annu. Rev. Plant Physiol. 24, 197-224 Tigchelaar, E.C., McGlasson, W.B., Buescher, R.W. (1978) Genetic regulation of tomato fruit ripening. Hortscience 13, 508-513 Tomes, M.L. (1963) Temperature inhibition of carotene synthesis in tomato. Bot. Gaz. (Chicago) 124, 180-185 Tucker, G.A., Robertson, N.G., Grierson, D. (1980) Changes in polygalacturonase isoenzymes during the 'ripening' of normal and mutant tomato fruit. Eur. J. Biochem. 112, 119-124 Tucker, G.A., Robertson, N.G., Grierson, D. (I981) The conversion of tomato fruit polygalacturonase isoenzyme 2 into isoenzyme 1 in vitro. Eur. J. Biochem. 115, 87-90 Received 22 December 1981 ; accepted 16 February 1982
Note added in proof: Using gas chromatography and radioimmunoassay to measure ethylene and PG we have obtained evidence that during normal ripening PG synthesis begins 20-40 h after the start of ethylene evolution. These results, which will be published separately, indicate that the hypothesis of Tigchelaar et al. (1978) should be rejected.
