Plant Physiol. (1 9 9 6 ) 11O : 883-892
Tomato Fruit Carboxypeptidase Properties, lnduction upon wounding, and lmmunocytochemical Localization Roshni A. Mehta, Robert D. Warmbardt', and Autar
K. Mattoo*
Plant Molecular Biology Laboratory, Beltsville Agricultural Research Center, United States Department of Agriculture/Agricultural Research Service, Beltsville, Maryland 20705 (R.A.M., A.K.M.); Department of Biological Sciences, University of Maryland, Catonsville, Maryland 21 228 (R.A.M.); and Plant Photobiology Laboratory, Beltsville Agricultural Research Center, United States Department of Agriculture/AgricuItural Research Service, Beltsville, Maryland 20705 (R.D.W.) In recent years, considerable attention has been given to regulatory mechanisms involved in the development and senescence of tomato fruit and to understanding how various stresses further affect these processes. Severa1 ripening-related genes and gene products have been identified and studied in tomato (Lycopersicon esculentum) fruit (Gray et al., 1992). Ripening and wounding of fruit tissues is associated with differential protein metabolism as well as differential gene expression (Mehta et al., 1991; Gray et al., 1992; Mehta, 1993). Therefore, protein turnover is highly regulated in fruits during ripening and in response to environmental stimuli. However, little is known about the mechanisms and type of proteases involved in protein metabolism of fruits. Tomato fruit contains a carboxypeptidase activity localized to the soluble fraction (Matoba and Doi, 1974); another report partially characterized a wound-induced carboxypeptidase from tomato leaf (Walker-Simmons and Ryan, 1980). We have identified this wound-inducible carboxypeptidase from tomato leaves to be a 69-kD protein and demonstrated that the leaf carboxypeptidase is specifically localized to the vacuoles of paraveinal mesophyll tissue (Mehta, 1993). The tomato fruit, on the other hand, contains severa1 immunologically related carboxypeptidases that are made up of 68- and 43-kD proteins (Mehta and Mattoo, 1996). We report here further characteristics of carboxypeptidases from tomato fruit and show that physical stress (wounding) causes induction of carboxypeptidase activity that is developmentally regulated, and a chemical stress inhibits its wound induction. We also demonstrate by immunogold EM that the fruit carboxypeptidase is localized within the vacuoles of the pericarp tissue.
Carboxypeptidase activity was characterized during ripening and wounding of tomato (Lycopersicon esculentum) fruit. l h e fruit enzyme shares substrate specificity and susceptibility to the inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride with other plant carboxypeptidases.l h e abundance and stability of wound-induced carboxypeptidase were developmentally regulated. Oxidative stress caused by cupric ions impaired the membrane permeability in the slices from pink fruit, resulting in leakage of the carboxypeptidase into the medium and in its redistribution in the cell. l h e patterns of carboxypeptidase activity did not parallel the cupric ion effect on ethylene levels. lmmunogold electron microscopy studies indicated that the fruit carboxypeptidase is associated with electron-dense inclusions in the vacuole.
Carboxypeptidases are proteases that sequentially hydrolyze amino acid residues from the C termini of proteins and have been implicated in the intracellular protein processing and turnover (Breddam, 1986; Preston and Kruger, 1986). In plants, carboxypeptidases were first detected and purified from the peel of citrus fruits (Zuber, 1964). Carboxypeptidases are ubiquitous and relatively abundant in most tissues of higher plants, including monocots, dicots, and gymnosperms (Mikola and Mikola, 1986). Their common properties include acidic p H optima, inactivation by DFP, insensitivity to chelating agents, and ability to liberate a11 kinds of C-terminal amino acid residues from Nsubstituted dipeptides, longer peptides, and proteins (Mikola and Mikola, 1986). Carboxypeptidases are usually glycoproteins that are sequestered in vacuoles in fungi (Valls et al., 1987) or in lysosomes of animals (Breddam, 1986). In plants, carboxypeptidases were shown to be localized in vacuoles on the basis of subcellular fractionation (Matoba and Doi, 1974; Breddam, 1986). The plant carboxypeptidases seem characteristically similar to the mammalian lysosomal carboxypeptidases A, B, and C (MacDonald and Schwabe, 1977), the acid carboxypeptidase of yeast, and carboxypeptidase Y (Hayashi, 1976).
MATERIALS A N D M E T H O D S Plant Material
Tomato (Lycopersicon esculentum cv Pik-Red) plants were grown either in a greenhouse under 16 h of daylight and 8 h of darkness at temperatures ranging from 25 to 3OoCor in the U.S. Department of Agriculture fields at Beltsville, MD.
Deceased. * Corresponding author; e-mail
[email protected]; fax
Abbreviations: DFP, diisopropyl fluorophosphate; 2-X-Y, Ncarbobenzoxy-2-X-Y, where X and Y are L amino acids.
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Unblemished, uniformly shaped fruit were surface sterilized with 70% ethanol and then thoroughly rinsed with sterile water. The fruits were sliced and the pericarp tissue was cut into smaller (1-5 cm) pieces. Pieces from at least six fruit were pooled for each experimental point to minimize any variation that may exist in the response of an individual fruit. A portion of the sliced tissue was frozen immediately in liquid nitrogen and used as an unwounded control. The wounding treatment consisted of incubating the sliced fruit pericarp in a sterile solution containing 0.4 M sorbitol and 0.01 M Mes-KOH, pH 6.0, under fluorescent white light (6 pmol mp2 spl) (Mehta et al., 1991). Samples were removed after various times of incubation and frozen in liquid nitrogen. A11 tissue samples were stored at -80°C until further use.
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Vienna, Austria) and collected on nickel grids for further processing. The following steps were done at i-oom temperature. Nickel grids with attached sections were first incubated in a blocking solution of PBS with 0.05% Tween 20 containing 1%BSA (Sigma) to prevent nonspecific binding of the antibodies. The sections were then drained and incubated for 1 h with rabbit anti-carboxypeptidase antibody (Mehta and Mattoo, 1996) diluted 1:500 in buffer. Control tissues were incubated with the nonimmlune rabbit IgG. Grids were then rinsed with PBS with Triton X-100 and incubated in 15 nm of gold-labeled, goat anti-rabbit IgG (Sigma) diluted 1:40 in buffer. The grids were washed with PBS followed by double-distilled water and counterstained with 2% (w/v) uranyl acetate in 50% ethanol for 15 min. Following immunolabeling, the thin sections were viewed and photographed with an HU-11E or H-500 transmission electron microscope (Hitachi, Tokyo, Japan).
Ethylene Measurement
Slices (0.5 cm in diameter) were cut from 1 g of fruit tissue and placed in 50-mL Erlenmeyer flasks containing 0.4 M sorbitol and 0.01 M Mes-KOH, pH 6.0, with or without 1 miv CuSO,. Incubation was carried out at 25°C. For determination of ethylene production, the flasks were stoppered between 0.5 and 1.5,2 and 3, 5 and 6, and 6 and 24 h. The flasks were flushed with air between the cumulative periods. The amount of ethylene that accumulated during each 1-h period was estimated by withdrawing a 3-mL gas sample from the head space of the flask with a hypodermic syringe and assaying with a gas chromatograph equipped with a flame ionization detector (model 3740; Varian Instrument Group, Harbor City, CA)2. Carboxypeptidase and Protein Assays
Carboxypeptidase activity and protein estimation were carried out as described by Mehta and Mattoo (1996).One unit of enzyme activity is defined as pmoles of Ala released per hour per milligram of protein at 30°C. The specific activity of carboxypeptidase was consistently found to be much higher in plants grown in the field compared to those grown in the greenhouse. However, highly similar patterns were obtained with fruit from plants grown either way. lmmunoelectron Microscopy
Small pieces (2 mm3) of tomato fruit tissue were fixed in a freshly prepared solution of 0.5% glutaraldehyde and 4% paraformaldehyde in 100 mM sodium phosphate buffer, pH 7.2, at 4°C for 18 h. The fixative was changed twice during this period. The tissue was then washed for 1 h in 100 mM phosphate buffer, dehydrated in a cold, graded ethanol series, and embedded and polymerized in L.R. White ”Hard” resin at 60°C for 48 h (Warmbardt, 1985). Ultrathin sections were cut with a diamond knife on an American optical ultracut ultramicrotome (Reichert-Jung, Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the products by the U.S. Department of Agriculture and does n o t imply its approval t o the exclusion of other products that m a y also be suitable.
RESULTS Carboxypeptidase Activity in Tomato Plant Tissues
Cell-free extracts were prepared from fruit (at different developmental stages), leaves, stems, and roots. Soluble proteins were precipitated by ammonium sulfate fractionation (40-80% saturation) and assayed after dialysis for carboxypeptidase activity using Z-Phe-Ala dipeptide as a substrate (Mehta and Mattoo, 1996). The carboxypeptidase activity was found to change with the stage of ripening of tomato, showing a gradual increase until the pink stage, after which it declined (Table I). The enzyme activity extracted from the stem and root was comparable to that in the pink fruit; however, that in the leaf had relatively the highest specific activity. Substrate Specificity
The specificities of the carboxypeptidases extracted from tomato fruit at different stages of ripening using various dipeptide substrates are compared in Table 11. A11 of the tissue extracts tested used Z-Phe-Ala as a substrate. Lower rates of hydrolysis were found for peptides having Gly and Pro at the C-terminal position. Fruit carboxypeptidase utiTable I. Specific activity o f carboxypeptidase in different tissues of tomato plant
Specific activity is defined as pmol of L-Ala released h-’ mg-’ protein at 30°C. Data are means ? SD of six independent preparations. Samples of fully developed leaf, stem, and root were excised from 2-month-old, mature, and fully developed tomato plants grown in the greenhouse Tissue
Specific Activitv
pmol h-
Fruit Green Breaker Pink Red Leaf Stem Root
2.91 3.14 3.81 2.25 5.33 3.67 3.75
mg?
protein
0.40
t 0.54 0.80 0.02 0.1 7 0.23 ? 0.75 ? -t ? -t
Tomato Fruit Carboxypeptidase
Table II. Relsive rates o f hydrolysis o f carbobenzoxy peptides by tomato fruit carboxypeptidases Substrates were dissolved in DMSO and made to a final concentration of 200 mM in 50 mM acetate buffer, p H 5.2, 1.5 mM EDTA. Mixtures of 0.1 mL of substrate solution and 0.1 mL of enzyme solution (5 pg of protein) were incubated at 30°C for 1 h. Relative activities are expressed as percentages of the activity with Z-Phe-Ala as the substrate. Actual rates for hydrolysis (pmol h-’ mg-’ protein) of Z-Phe-Ala were 0.81 (green), 4.1 3 (breaker), 5.09 (pink), and 0.89 (red), respectively. Relative Rates of Hydrolysis Substrate Creen
Breaker
Pink
Red
1 O0 102 98 78 4 O O O 4 4 4 O
1 O0 94 63 a9
1 O0 62 75 76
1O0
O O O O O O O
O
O 2 O O
Z-Phe-Ala Z-Phe-Leu Z-Pro-Leu Z-Pro-Phe 2-Cly-Tyr Z-Cly-Leu 2-Cly-Phe Z-Gly-Pro Z-Ala-Ala Z-Phe-G Iy Z-Ala-Gly Z-Glu-Phe
O
O O
O O O 30 O
40 72 29
41 12 4
16
lized the dipeptides in the order, Z-Phe-Ala > Z-Phe-Leu > Z-Pro-Leu > Z-Pro-Phe. We also found that, although tomato carboxypeptidase did not hydrolyze purified tomato proteinase. inhibitor, it readily hydrolyzed reduced, carboxymethylated, or denatured tomato or potato proteinase inhibitors (data not shown). Effect of lnhibitors
Z-Phe-Leu > Z-Pro-Phe > Z-Pro-Leu, indicates the importance of the penultimate amino acid at the C terminus for the enzyme specificity. Phe or Pro at the penultimate position favored hydrolysis. Similar results have been reported for Carboxypeptidase from watermelon (Matoba and Doi, 1975), tomato leaf (Walker-Simmons and Ryan, 1980), and tomato fruit (Matoba and Doi, 1974). These results corroborate the observation that plant carboxypeptidases exist in multiple molecular forms, which differ in their substrate specificity (Mikola and Mikola, 1986). Five carboxypeptidases from germinating wheat (Mikola and Mikola, 1984) and barley grains (Mikola, 1983) were purified on the basis of their selective substrate specificity. The apparent suppression of Carboxypeptidase activity, specifically in the pink fruit slices treated with cupric ions, was not corroborated by the immunogold EM. Thus, it would seem that the cupric ions may in fact induce carboxypeptidase activity and the protein, but the leakiness of pink fruit vacuolar membrane under these conditions complicates interpretation of data obtained because carboxypeptidase protein is released in the cytoplasm (immunogold EM data) or in the incubating medium (activity measurement data) with the result that the extractable
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Figure 6. (Legend appears on opposite page.)
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Tomato Fruit Carboxypeptidase
carboxypeptidase activity is only 50% of that i n t h e untreated, control tissue. These results demonstrate differentia1 effects of physical versus chemical stress on t h e distrib u t i o n of carboxypeptidase in. t h e cell. Thus, whereas physical w o u n d i n g does n o t impair t h e vacuolar membrane, chemical stress does, suggesting further that w o u n d i n g is a less intense stress on t h e fruit pericarp t h a n t h e cupric i o n treatment (oxidative conditions). The increase seen i n carboxypeptidase activity during ripening of tomato fruit suggests that the enzyme m a y act t o d e g r a d e the intracellular proteins and generate free a m i n o acids, a s h a s been proposed for the e n z y m e d u r i n g seed germination (Mikola a n d Mikola, 1986). A t present, little is known a b o u t t h e expression of fruit carboxypeptid a s e genes a n d their role in development a n d responses t o stress. Wound-induced increase i n a plant carboxypeptid a s e h a s been reported for tomato leaf carboxypeptidase (Walker-Simmons a n d Ryan, 1980). The induction appears t o be related to t h e large accumulation of t h e proteinase inhibitors induced by w o u n d i n g (Walker-Simmons a n d Ryan, 1980). It is possible that a n increase i n t h e carboxypeptidase activity d u r i n g w o u n d i n g is reflective of localized cell d e a t h responses i n d a m a g e d tissue a n d the events that occur d u r i n g senescence. Thus, t h e enzyme m a y be involved i n salvaging proteins from d a m a g e d cells a n d senescing tissue, constituting a n important aspect of plant life cycle. It is also possible that t h e carboxypeptidase activity is p a r t of a regulated increase i n intracellular protein turnover designed t o s u p p l y free a m i n o acids for synthesis of proteinase inhibitors. This role for t h e enzyme d u r i n g w o u n d i n g is indirectly substantiated by o u r finding that the e n z y m e is exclusively localized i n t h e vacuoles of m e s o p h y l l cells. T h e p r o t e a s e i n h i b i t o r s i n d u c e d b y w o u n d i n g also accumulate i n t h e vacuoles; they do not inhibit plant proteases b u t block animal a n d funga1 digestive proteases (Walker-Simmons and Ryan, 1980). Thus, it may be worthwhile t o consider the possibility that t h e fruit carboxypeptidase m a y h a v e a n additional function a s a defense against pathogen attack.
ACKNOWLEDCMENTS
We thank Dr. Arkesh Mehta for valuable discussions, Drs. M. Tucker and E. Herman for constructive comments on the original manuscript, members of the Mattoo group for a critica1 reading of the manuscript, and Michael Reinsel and Malendia Maccree for assistance with photography.
Figure 6. (Figure appears on opposite page.) Redistribution of carboxypeptidase in oxidatively stressed fruit tissue. Tomato pericarp tissue sections from green (A-D) and pink (E-H) fruit were treated with antibodies to carboxypeptidase followed by goat anti-rabbit IgG conjugated to 15 n m of colloidal.gold. Portions of unwounded (A and E), wounded (6 and F), wounded plus copper-treated for 3 h (C and G), and wounded and treated with copper for 1.5 h and then incubated without copper for 1.5 h (D and H) are shown. A, X8,640; 6, X10,640; C, X10,640; D, X10,260; E, X8,360; F, X8,360; C, X8,360; H, X7,980. Marker bars = 1 .O mm. ER, Endoplasmic reticulum; M, mitochondrion; N, nucleus; P, plastid; V, vacuole; W, cell wall.
Received May 26, 1995; accepted November 18, 1995. Copyright Clearance Center: 0032-0889/96/ll0/0883/10.
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