weight basis were measured over seven stages of tomato fruit development and ripening. ... (1), pear (5, 6), and apple (7) during ripening and can be induced by ethylene to ..... account for the large change in respiration during the climacteric.
Plant Physiol. (1979) 63, 486-489 0032-0889/79/63/0486/04/$00.50/0
Changes in Activity of Ribulose-1,5-Bisphosphate Carboxylase/ Oxygenase and Three Peroxisomal Enzymes during Tomato Fruit Development and Ripening' Received for publication September 6, 1978 and in revised form November 13, 1978
BARRY A. MARTIN, JOHN A. GAUGER, AND N. EDWARD TOLBERT Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 ABSTRACT
that glycolate synthesis and oxidation may be a component of the respiratory climacteric. The activity of ribulose-P2 carboxylase/oxygenase and three enzymes associated with leaf peroxisomes, catalase, glycolate oxidase, and hydroxypyruvate reductase, were measured during tomato fruit development.
Ribulose-1,5-bisphosphate carboxylase/oxygenase, catalase, glycolate oxidase, and hydroxypyruvate reductase activities on a protein and fresh weight basis were measured over seven stages of tomato fruit development and ripening. Ribulose-1,5-bisphosphate carboxylase decreased steadily during fruit development from 23 ± 8 nmoles per minute per milligram MATERIALS AND METHODS protein at the mature green stage to 13.4 ± 2 at the table ripe stage. There was no change in partially purified preparations of the enzyme in the ratio Reagents. ['4C]NaHCO3 was from Amersham/Searle and riof carboxylase to oxygenase activity, which was about 10. Catalase activity bulose-P2 was synthesized enzymically (12). All other reagents reached a maximum during the climacteric,.simultaneously with increased were from Sigma. ethylene and CO2 formation. Glycolate oxidase activity decreased during Fruit. Four crops of tomato plants of varieties, Heinz 1350, rin early stages of development and was barely detectable at the climacteric. (from Dr. Herner, Department of Horticulture, Michigan State Hydroxypyruvate reductase, associated with serine formation by the glyc- University), and the breeding line (61-37, Fireball x Cornell 54erate pathway, increased in specific activity during early stages of tomato 149) from which rin originated, were grown over a 2-year period fruit ripening. In the fruit of the rin tomato mutant, which does not ripen in the greenhouse (20 C night, 25 C day). Only one flower (first or normally, none of these changes in enzyme activity occurred. second) per cluster was pollinated and tagged at anthesis, and fruit
During the climacteric period of tomato fruit ripening, CO2 evolution and 02 consumption increase 5- to 6-fold (3, 10, 18). Inasmuch as ripening can be delayed by decreasing 02 to 5% or increasing CO2 to 4% in the atmosphere (22), the activities of various oxidases have been investigated during fruit development. Lipoxygenase increases (19) and polyphenol oxidase decreases (14) in ripening tomatoes. Peroxidase increases in preclimacteric mangoes treated with ethylene (20). Microbodies or peroxisomes have been observed in tomato fruit (1), and peroxisomal catalase, which can act as a peroxidase (11), increases in activity in tomato (1), pear (5, 6), and apple (7) during ripening and can be induced by ethylene to increase in activity in preclimacteric mango (20). Glycolate metabolism, similar to that in leaf peroxisomes (24), has been proposed to be associated with the climacteric respiration in tomatoes (4) and pears (5). On a Chl basis, tomato fruit have substantial ribulose-P22 carboxylase/oxygenase in the outer wall of the pericarp (4, 15), and whole fruit flx CO2 by both this enzyme and P-enolpyruvate carboxylase (8). The oxygenase function of ribulose-P2 carboxylase/oxygenase in crude extracts from tomato fruit has been reported to increase relative to carboxylase activity during ripening (4), and glycolate has been observed to accumulate in tomatoes (4) and in strawberries and cherries (13) during ripening. These observations have led to the hypothesis
load was limited to 8 to 10 per plant. Data from fruit harvested during all seasons of the year have been averaged, although some differences over seasons were observed. Fruit was harvested at seven stages of development and ages: (I) cell division from 0 to 20 days; (II) cell expansion from 20 to 35 days; (III) mature green from 36 to 40 days; (IV) breaker from 40 to 45 days; (V) light pink from 43 to 48 days; (VI) dark pink from 45 to 50 days; and (VII) table ripe from 50 to 55 days. The fruit was quartered and cooled at 4 C for 1 h or placed whole into respirometers. Before homogenization, the seeds, placenta and locule walls were removed, and the fruit was rinsed in cold distilled H20, blotted dry, and cut into 1-cm3 pieces. Enzyme Assays. For extraction of peroxisomal enzymes 40 g of tissue was randomly chosen from each replicate of three to eight fruits of the same age and homogenized for I min in 20 ml of 0.2 M phosphate buffer at pH 9.0 containing 2% (w/v) PVPP with a Waring Blendor at low speed. The homogenate was filtered through Miracloth, and the volume was recorded. Catalase, hydroxypyruvate reductase, and glycolate oxidase activities were measured spectrophotometrically (23). Catalase and hydroxypyruvate reductase activities were measured in the supernatant after centrifuging at 5,000g for 15 min. Due to extensive endogenous reduction of DCPIP in crude extracts of ripe fruit, it was necessary to run the glycolate oxidase assay on the protein precipitated from a 7-ml sample to which 5 ml of saturated (NH4)2SO4 at pH 7.1 had slowly been added with stirring. The precipitated protein was removed by centrifugation at 2,000g for 90 min, resuspended in 1 ml of 0.1 M K2HPO4 at pH 8.4, and stored frozen for assays the next day. Glycolate oxidase activity was also measured in some experiments as O2 uptake by the O2 electrode, and the results from both methods were similar. For extraction of ribulose-P2 carboxylase/oxygenase 70 g of the outer wall of the pericarp of tomato fruit was homogenized in 20 ml of a buffer at pH 10.5 containing 250 mm glycylglycine, 50 mM
1 This research has been supported by National Science Foundation Grant PCM 78 15891 and by the Union Carbide Corporation and published as Journal Article No. 8632 from the Michigan Agricultural Experiment Station. 2 Abbreviations: ribulose-P2: ribulose 1,5-bisphosphate; DCPIP: dichlorophenolindophenol; PVPP: polyvinyl polypyrrolidine. 486
Plant Physiol. Vol. 63, 1979
ENZYMIC CHANGES IN TOMATO FRUIT
487
MgCl2, and 2.5 mm Na2EDTA, to which 2 g PVPP and 0.40 g DTT were added before use. The tissues were homogenized for I cD min at low speed in a Waring Blendor in 4- to 15-s bursts. The 300 homogenate was filtered through Miracloth and eight layers of L cheesecloth and centrifuged for 15 min at 30,000g. The pH of the E supernatants, which ranged from 7.9 to 8.1, was raised to 8.4. 300 'E Aliquots were assayed for carboxylase activity and protein and .vC the remainder was used for partial purification of ribulose-P2 E° IVI carboxylase/oxygenase. Saturated (NH4)2SO4 at pH 7.1 was added F00' _E E310 Em to the extract until it was 37% saturated, and the precipitate was removed by centrifugation at 30,000g for 30 min and discarded. 2 o 2Za)v The ribulose-P2 carboxylase/oxygenase was collected between 37 _a and 50%o saturated (NH4)2SO4, and resuspended in 1 ml of 0.1 M 15 \ cs 15 nmt 25 1 1 at and nms EDTA. mm Bicine pH 8.4, MgCl2, DTT, 4 This preparation was desalted by passage through a Sephadex G9'I Glycolote Oxidase 25 column (1 x 50 cm) with a void volume of 15 ml and the first I~~~~ u U IV v m z 2 ml after the void volume was collected for assay. To avoid of Development Stage dilution and achieve higher activity some preparations were desalted by dialysis against four changes of the buffer. These ribuFIG. 1. Changes in specific activity of peroxisomal enzymes of Heinz lose-P2 carboxylase/oxygenase fractions were activated for 10 min 1350 tomato fruit during development. A description of the stages and at 30 C by incubation at a final concentration of 10 mm NaHCO3, ages of the fruit during development and ripening are cited under "Ma1.0 mm DTT, and 20 mM MgCl2. Then the carboxylase and terials and Methods." Catalase (-- ), hydroxypyruvate reductase E). oxygenase assays were run at the same time, but by different (A----A), and glycolate oxidase assays. The carboxylase assay, based on '4CO2 fixation, was performed as described previously (21). Ribulose-P2 oxygenase was based on ribulose-P2-dependent O2 uptake as measured in a Rank Brothers O2 electrode at 30 C in a final volume of 0.5 ml. The reaction mixture contained 0.1 M Bicine-KOH at pH 8.4, 20 mm MgCl2, 0.5 mm ribulose-P2, 0.2 mM EDTA, and I mm DTT. Reactions were initiated with 20 ,tl of the activated enzyme and S etalDse40 measured for 45 to 60 s. in were measured fruit T45Ethylene and CO2 evolution by placing sealed jars equipped with a septum, for I h and taking l-ml air El~~~~~~~~~~~ samples with a gas-tight syringe. The samples were injected in a Varian gas chromatograph equipped with a 90-cm alumina colE20300 umn, and the C2H4 was quantitated by peak height. CO2 evolution was measured by injecting samples into a Beckman CO2 analyzer Hi vate redutas (A---) an glcolae oida\Reductase-, which measured IR absorbance. Because substances are present in extracts of tomatoes which severely interfere with the standard Lowry procedure (17), protein ofdevelopment. Theactvityinyoungfuitfro earlyta002 concentration was determined by a modified Lowry method (2), which involved quantitative precipitation of the protein from the homogenates. a>
0
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-
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I
....
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40
30
20
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Hydroxypyruvate
....
Glycolate Oxadose
......
RESULTS Peroxisomal Enzymes. Activities of the peroxisomal enzymes, catalase, glycolate oxidase, and hydroxypyruvate reductase, were measured at various stages of fruit development. The results are expressed on a protein basis (Fig. 1) and a fresh weight basis (Fig. 2). The data points show mean and standard deviation for the four stands of vines grown over the 2-year period. Standard deviation within one growth period was considerably less than the over-all standard deviation shown for the whole project. Catalase activity increased to a maximum at the light pink stage (V) after which it decreased during final maturation. This increase in catalase activity at stages IV and V occurred at the climacteric when ethylene production and CO2 evolution also increased, although protein in the ripening fruit had already reached minimal concentrations (Fig. 3). These results confirm those previously reported by Baker (1). The rin mutant tomato, which does not ripen normally or go through climacteric ethylene production (10), did not exhibit an increase in catalase activity at stages IV and V, although the parent variety did so (Fig. 4). Glycolate oxidase activity in the developing tomato fruit slowly decreased from 10 to 15 nmol min-' mg protein-' in the first stage to less than 1 nmol min-' mg protein-' in the ripening fruit. Leaves of most plants contain at least five times more glycolate
Stage FIG.
Heinz
of Development
Changes in peroxisomal enzyme activity per during development. Catalase ( o
2.
1350 fruit
vate reductase
(A-
oxidase activity
-A), and
glycolate oxidase
g fresh ),
weight
of
hydroxypyru-
(I-... E).
protein basis than tomato fruit in early stages activity in young fruit from early stages is measurable in crude extracts, but difficult to assay accurately from of development. Extracts of the ripening latter stages fruit after stage IV reduced DCPIP, the electron acceptor used in glycolate oxidase assay, at too high an endogenous rate to anow of
on a
development.
The
(IV-VII)
accurate
use
affect the
(NH4)2S04
of this assay for
glycolate-dependent oxidation.
In-
cyanide, metabisulfite, and Fe(N03)2 did not endogenous rate. If the enzyme was precipitated by
hibitors such
as
and redissolved in buffer,
as
described under "Mate-
rials and Methods," the
endogenous rates were eliminated, and satisfactory glycolate-dependent rates of DCPIP reduction were
obtained. By comparing the total glycolate oxidase activity in homogenates of young fruit, before and after the (NH4)2SO4 fractionation, at least 95% of the glycolate oxidase activity of the fruit was judged to have been recovered by the (NH4)2SO4 fractionation procedure.
488
MARTIN, GAUGER, AND TOLBERT /,
Ethylene
8
30
tisX
60
S!~ ~ ~vouto
CJ'
40
E
Q-~ ~~,\ _____ E ,0
I
m
m
II
T
V
Stage of Development
FIG. 3. Changes in respiration and protein of Heinz 1350 tomato fruit during development. CO2 evolution (A A) and ethylene evolution ([- -x) from intact fruit and water-soluble protein in extracts of the fruit (-*).
75-
tslase
7soO
c
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(0
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50
Fruit Age (Days from Anthesis) FIG. 4. Changes in enzyme activity per g fresh weight of tomato fruit * ) and the normal line 16-37 (O A O). Catalase of the rin variety (@A A, - -A), and (0-0, 0-- 0), hydroxypyruvate reductase ( A-
glycolate oxidase
(EO-[0,
-
-U).
The activity of hydroxypyruvate reductase on a protein basis reached a maximum at stages III and IV for mature green fruit entering the breaker stage (Fig. 1). This increase in hydroxypyruvate reductase activity was much less, or not evident, on a weight basis (Figs. 1 and 2) due to the rapid decrease in protein content during this period (Fig. 3). Changes in Ribulose-P2 Carboxylase/Oxygenase. Sufficient carboxylase activity was present in crude extracts of green tomato fruit grown in the summer for reliable assays. The specific activity of ribulose-P2 carboxylase in the mature green fruit stage (III) was 23 + 8 nmol min-m mg protein-'. Activity declined to 16 ± 4 at the breaker stage (IV) and to 13 ± 2 at the table ripe stage (VII). The values were 20- to 50-fold less than ribulose-P2 carboxylase activities of approximately 600 nmol min-' mg protein-' found in crude extracts of the leaves. Purified preparations of the enzyme from spinach leaves have specific activities ranging from 1,500 to
Plant Physiol. Vol. 63, 1979
2,000 nmol min-' mg protein'-. In fruit grown in the greenhouse in the winter, lower specific activities were observed than for fruit from the summer period. In crude extracts of tomato fruit, assays for ribulose-P2 oxygenase activity were unreliable for two reasons. There was insufficient activity, and the endogenous rates were so high that ribulose-P2dependent oxidation was too small relative to the endogenous rate to be measured accurately. It was necessary to make an ammonium sulfate fractionation of the extracts from tomato fruit in order to eliminate the endogenous rate and to concentrate the protein for accurate measurements of the oxygenase activity. The ratio of ribulose-P2 carboxylase to ribulose-P2 oxygenase activity in the (NH4)2SO2 preparation from tomato fruit was approximately 10 and did not significantly change at different stages of fruit development. The ratio was the same as in pure enzyme preparations from tobacco and spinach leaves that were used as standards for comparison.
DISCUSSION Ribulose-P2 carboxylase/oxygenase was present in extracts from the outer wall of the pericarp of tomato fruit as observed by other investigators (4, 15). The specific activity of ribulose-P2 carboxylase/oxygenase in green tomato fruit was low and declined further with fruit ripening. It is unlikely that this low activity could account for the large change in respiration during the climacteric. No difference in the ratio of the carboxylase to oxygenase activity was detected at different stages of development by our preparation and assay conditions, and the ratio of 10 was the same as for the purified enzyme from tobacco and spinach leaves. While tomato fruit contain catalase and glycolate oxidase, indicative of the glycolate pathway of photorespiration, the low and declining activity of ribulose-P2 carboxylase/oxygenase during fruit maturation and ripening suggests that this pathway could have little effect on the climacteric respiration. In fact, actual ribulose-P2 oxygenase activity in vivo might even be less at the climacteric than in our assay, because of high internal CO2 which would inhibit the oxygenase activity. Glycolate oxidase activity at the climacteric had reached its lowest level of activity. Catalase activity in the fruit did not decrease and in fact increased in specific activity during the climacteric. However, nothing in our data indicates that this increase in catalase could be associated with increased H202 production from glycolate oxidation. Contrary to the decrease in glycolate oxidase during fruit development, hydroxypyruvate reductase, associated with the glycerate pathway in the peroxisomes for serine formation and metabolism, increased during the mature green stage and remained high in the breaker stage. An increase in hydroxypyruvate reductase was not observed in the nonripening rin mutant (Fig. 4). Hydroxypyruvate reductase is found in leaf peroxisomes (23) and is presumed to be located in the peroxisomes of the tomato fruit. Some increase in specific activity of hydroxypyruvate reductase before the climacteric might maintain serine and glycine production by the glycerate pathway during ripening (24) at a time when glycolate oxidase activity was decreasing. This increase in hydroxypyruvate reductase might account for the large pool of serine present in the breaker stage fruit (9, 25). Serine, aspartate, and glutamate are the only amino acids to increase during this period, and serine added to apple discs at the climacteric hbits ethylene production (16). There is no suggestion in the literature that serine in leaf peroxisomes could account for an increase in synthesis catalase, which might have been expected to decrease, since the glycerate pathway is not known to involve H202 production. Acknowledgments-We thank Dr. Dilley, Department of Horticulture, for the use of his gas and CO2 analyzer and Dr. R. Herner, Department of Horiculture, for seeds and fruit of rin and the parent tomato. In our laboratory, N. Selph, J. Uhlig, N. P. Hall S. McCurry, and C. Paech helped with enzyme assays and control preparations of ribulose-P2 carboxylase/
chromatography oxygenase
and ribulose-P2.
Plant Physiol. Vol. 63, 1979
ENZYMIC CHANGES IN TOMATO FRUIT
LITERATURE CIMD 1. BAKck JE 1973 Microbodies and catalase of tomato fruits. Plant Physiol 51: S-18 2. BENSADOUN A, D WEINSTEIN 1976 Assay of proteins in the presence of interfering materials. Anal Biochem 700: 241-250 3. BIALE JB 1960 Respiration of Fruits. Encycl Plant Physiol 12: 536-592 4. BRAVDo B, A PALGI, S LuRs, C FRENxn 1977 Changng ribulose diphosphate carboxylase/ oxygenase activity in ripening tomato fruit. Plant Physiol 60: 309-312 5. BRENNAN T, C. FRENxEL 1977 Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol 59: 411-416 6. EzELL BD, F GEsHARDT 1938 Oxidase and catalase activity of Bartlett pears in relation to maturity and storage. J Agric Res 56: 337-386 7. EzsiI BD, F GERHARDT 1942 Respiration and oxidase and catalase activity of appes in relation to maturity and storage. J Agric Res 65: 453-470 8. FAINEAu J, D LARAL-MARTIN 1977 Light versus dark carbon metabolism in cherry tomato fruits. II. Plant Physiol 60: 877-880 9. FREEMAN JA, CG WOODBRIDGE 1961 Effects of maturation ripening and truss position on the free amino acid content in tomato fruits. Am Soc Hort Sci 76: 515-524 10. HERNER RC, KC Ssmx 1973 Ethylene production and respiratory behavior of the rin tomato mutant. Plant Physiol 52: 38-42 11. HoLMEs R, C MAsTrES 1973 Peroxisomes: new aspects of cell physiology and biochemistry. Physiol Rev 57: 816-883 12. HoREcICER BL, J HuRwTz, A WEISSBACH 1958 Ribulose diphosphate. Biochem Prep 6: 83-90
489
13. HULME AC, LSC WOOLTORTON 1958 The acid content of cherries and strawberries. Chem Ind 22: 659 14. KIDSON EB 1958 "Cloud" or vascular browning in tomatoes. IV. NZ J Agric Res 1: 896-902 15. LARAL-MARTIN D, J FARinEAu, J DLAmoND 1977 Light versus dark carbon metabolism in cherry tomato fruits. I. Plant Physiol 60: 872-876 16. LIsEBtEN M, A Kumxssn, LW MAPSON, DA WARDALs 1966 Stimulation of ethylene production in apple tissue slices by methionine. Plant Physiol 41: 376-382 17. LowRY OH, NJ ROSEBROUGH, AL F,Rx, Rl RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275 18. LYONS, JM, HK PRATT 1964 Effect of stage of maturity and ethylene treatment on respiration and ripening of tomato fruits. J Am Soc Hon Sci 84: 491-500 19. MAPSON LW, DA WaxAnDs 1971 Enzymes involved in the synthesis of ethylene from methionine, or its derivatives, in tomatoes. Phytochemistry 18: 29-39 20. MATTOo AK, W MODI 1969 Ethylene and the ripening of mangoes. Plant Physiol 44: 308-310 21. McCuRRY SD, NE TOLBERT 1977 Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by xylukose-1,5-bisphosphate. J Biol Chem 252: 8344-8346 22. MIzUNO S 1971 The effect of carbon dioxide and oxygen on the ripening of tomatoes at 200C. J Jap Soc Hort Sci 40: 292-299 23. TOLBERT NE 1971 Isolation of leaf peroxisomes. Methods Enzymol 23: 665-682 24. TOLBERT NE 1971 Microbodies-peroxisomes and glyoxysomes. Annu Rev Plant Physiol 22:
45-74 25. Yu MH, LE OLSON, DK SALuNKHE 1967 Precursors of volatile components in tomato fruit. I. Phytochemistry 6: 1457-1465
