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Plant Physiol. (1999) 119: 89-100
Age-Induced Protein Modifications and Increased Proteolysis in
Potato Seed-Tubers1
G.N. Mohan Kumar,
Robert L. Houtz, and
N. Richard Knowles*
Department of Agricultural, Food and Nutritional Science, 4-10
Agriculture/Forestry Center, University of Alberta, Edmonton, Alberta,
Canada T6G 2P5 (G.N.M.K., N.R.K.); and Department of Horticulture and
Landscape Architecture, University of Kentucky, Lexington, Kentucky
40546-0091 (R.L.H.)
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ABSTRACT |
Long-term aging of potato
(Solanum tuberosum) seed-tubers resulted in a loss of
patatin (40 kD) and a cysteine-proteinase inhibitor, potato
multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total
proteolytic activity in aged tubers was sensitive to
trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged
tubers were also inhibited by crude extracts or purified PMC from young
tubers, suggesting that the loss of PMC was responsible for the
age-induced increase in proteinase activity. Nonenzymatic oxidation,
glycation, and deamidation of proteins were enhanced by aging. Aged
tubers developed "daughter" tubers that contained 3-fold more
protein than "mother" tubers, with a polypeptide profile consistent
with that of young tubers. Although PMC and patatin were absent from
the older mother tubers, both proteins were expressed in the daughter
tubers, indicating that aging did not compromise the efficacy of genes
encoding PMC and patatin. Unlike the mother tubers, proteinase activity
in daughter tubers was undetectable. Our results indicate that tuber
aging nonenzymatically modifies proteins, which enhances their
susceptibility to breakdown; we also identify a role for PMC in
regulating protein turnover in potato tubers.
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INTRODUCTION |
Potato (Solanum tuberosum) seed-tubers are a model
system for studying the process of aging in plants. The tubers can be
stored (at 4°C and 95% RH) for to 3 years without a loss of
viability. However, storage (aging) beyond about 8 months effects a
progressive decline in apical dominance, rooting ability, and sprout
vigor (Kumar and Knowles, 1993a ). In addition to changes in growth
potential, aging is accompanied by increased respiration of tubers
(Kumar and Knowles, 1996a), oxidative stress (Kumar and Knowles,
1996b), lipid peroxidation (Kumar and Knowles, 1993b), and decreased
protein content (Kumar and Knowles, 1993c). Although protein loss is
partly due to reduced synthesis (Kumar and Knowles, 1993c), the
contribution of proteolysis and the mechanisms by which proteins become
damaged and subsequently targeted for degradation with advancing age
are unknown. Processes that may lead to protein degradation during aging include (a) increased accessibility of proteins to proteinases resulting from decompartmentation, (b) molecular modifications to
polypeptides that enhance proteolysis, and (c) increased activity of
proteinases (Dalling, 1987).
Oxidation, glycation, and isomerization/racemization of amino acid
residues of proteins have been identified as nonenzymatic mechanisms
that can adversely affect structure and function (Fig. 1), rendering proteins more susceptible
to proteolysis during aging (Dalling, 1987; Stadtman, 1992; Luthra and
Balasubramanian, 1993; Eckardt and Pell, 1995). Oxidative stress
contributes to the formation of carbonyl derivatives on amino acid
residues of proteins (Dalling, 1987; Oliver et al., 1987; Levine et
al., 1990). For example, carbonyl content and susceptibility of Rubisco
to proteolysis increased during oxidative stress (Ferriera and Shaw, 1989; Penarrubia and Moreno, 1990; Garcia-Ferris and Moreno, 1993; Eckardt and Pell, 1995). Similarly, oxidative stress caused by the
inhibition of catalase by aminotriazole in maize seedlings resulted
in a 2-fold increase in protein carbonyl content (Prasad, 1997). The
increased oxidative stress accompanying aging of potato tubers may
provide an ideal environment for oxidation of proteins.
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| Figure 1.
Schematic diagram showing several nonenzymatic
mechanisms that could affect protein structure and function in aging
potato tubers. Protein modifications that may accompany aging include
oxidation (increased carbonyl groups), glycation (reaction of amino
acids with reducing sugars leading to protein cross-linking), and
deamidation/isomerization/racemization of asparaginyl and aspartyl
residues. Although these molecular modifications can target proteins
for proteolysis, deamidation-mediated increases in isoaspartyl residues
creates substrates for PCMT-II, which can restore function of the
affected proteins. Effects of tuber age on such molecular modifications
are presented in Figures 9 and 10 and Table III.
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Amino groups of proteins can react with aldehyde or keto groups of
reducing sugars through a Schiff-base reaction, yielding brown
fluorescent pigments known as advanced glycation end products (Luthra and Balasubramaniyan, 1993). Proteins thus modified tend to
form cross-links (Fig. 1) that can destroy protein function (Wettlaufer
and Leopold, 1991). A number of age-related diseases in humans are
attributed to protein glycation. For example, in diabetics, elevated
blood Glc is associated with cataracts (Monnier et al., 1979),
accelerated aging, and vascular narrowing (Brownlee et al.,
1986; Cerami et al., 1987). In light of the substantial increase in
reducing sugar concentration of tubers during aging (Kumar and Knowles,
1993b), it was of interest to determine the extent of age-induced
protein glycation.
Proteins are also susceptible to nonenzymatic modification by
deamidation-mediated conversion of L-asparaginyl to
L-isoaspartyl residues (Fig. 1). Although proteins
containing isomerized residues can be targeted for degradation, they
are also substrates for PCMT (type II), which can restore protein
function. Repair to such damaged proteins involves methylation, using
AdoMet as a methyl donor. PCMT is a cytosolic "housekeeping" enzyme
with specificity for the recognition and repair of altered aspartyl
residues (Galletti et al., 1995), and has been detected in 45 plant
species belonging to 23 families (Mudgett et al., 1997). Changes in
PCMT activity with advancing tuber age were thus characterized as an
indicator of deamidation-mediated damage to proteins.
In addition to reduced protein synthesis and enhanced susceptibility of
proteins to proteolysis, advancing tuber age may contribute to loss in
the ability to synthesize proteinase inhibitors and thus to protein
catabolism. Potato tubers contain a proteinase inhibitor, PMC (Rodis
and Hoff, 1984; Walsh and Strikland, 1993). With its multiple
inhibitory domains, PMC (85 kD) has the capacity for simultaneous
inhibition of several Cys-proteinase molecules (Walsh and Strickland,
1993). The effect of aging on PMC and proteinase levels is unknown.
Using potato as a model system, we examined potential mechanisms for
age-induced protein loss and the extent to which proteins become
nonenzymatically modified during aging.
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MATERIALS AND METHODS |
Tuber Aging
Certified potato (Solanum tuberosum L. cv Russet
Burbank) seed-tubers, obtained directly from a local grower at harvest,
were aged for 3 to 30 months in storage (at 4°C and 95% RH). These storage conditions were inhibitory to sprouting. Seed-tuber age was
calculated from harvest. For discussion, 3- to 6-month-old tubers are
considered to be physiologically "young" and tubers stored beyond
12 months are "old."
Protein Extraction and Analysis
Soluble proteins were extracted from the apical portion (periderm
included) of 6-, 18-, and 30-month-old tubers by homogenizing 20 g
of fresh tuber tissue with 20 mL of 50 mM potassium
phosphate buffer, pH 7.5, containing 2 µg mL 1
leupeptin, 2 µg mL1 antipain, 5 µg
mL1 aprotinin, 2 mM PMSF, 0.5%
(w/v) polyvinyl polypyrrolidone, and 2 mM
Na2S2O5.
The homogenate was filtered through Miracloth (Calbiochem) and
centrifuged at 30,000g for 30 min. All manipulations were at
4°C. Supernatants were stored at  80°C.
After determination of soluble protein (Bradford, 1976), proteins from
each of the three ages of tubers were mixed with equal volumes of
SDS-sample preparation buffer (62.5 mM Tris, pH 6.8, containing 2% [w/v] SDS, 5% [v/v]  -mercaptoethanol, 25%
[v/v] glycerol, and 0.01% [w/v] bromphenol blue) and were compared
after electrophoresis (Laemmli, 1970) on 10% polyacrylamide gels (27 µg of protein per lane).
Oxidized and Glycated Protein Determinations
Protein carbonyl content was determined as an index of protein
oxidation. Following derivitization of carbonyl groups with DNPH,
oxidized proteins in the 60% ammonium sulfate fraction were determined
spectrophotometrically. Carbonyl content was quantified using an
extinction coefficient of 22,000 M1
cm1 (Oliver et al., 1987; Levine et al., 1990).
Qualitative determination of oxidized proteins via western analysis
followed the methods of Levine et al. (1994). DNPH-treated protein
samples (60 µg of protein per lane) were resolved by SDS-PAGE (10%
acrylamide gels) and electroblotted to nitrocellulose membrane
(Laemmli, 1970; Kumar and Knowles, 1996a). Oxidized proteins were
probed with alkaline phosphatase-conjugated monoclonal anti-DNP
antibody (1:2,500, Sigma).
Glycated proteins (Amadori products) were quantified
spectrophotometrically (A435) using the
method of Wettlaufer and Leopold (1991). Quantification of glycated
protein was based on a standard curve using 0 to 15 µg of
5-hydroxymethyl-2-furaldehyde (Sigma).
Glycated proteins were also examined by boronate-affinity
chromatography. Tuber tissue (10 g fresh weight, as described above) from 3-, 15-, and 27-month-old tubers was homogenized using a mortar
and pestle with 10 mL of Tris buffer (100 mM, pH 7.4, containing 0.5 M NaCl, 1 mM EDTA, 0.1% [w/v]
NaN3, 1 mM DTT, 0.25 mM
PMSF, and 2 mM
Na2S2O5),
filtered through Miracloth, and centrifuged at 30,000g for
30 min. All manipulations were performed at 4°C. Glycated proteins in
the supernatant were isolated using a Glyco-Gel II column that
contained immobilized m-aminophenylboronic acid cross-linked
to 6% beaded agarose according to the manufacturer's instructions
(Pierce). The column (10-mL bed volume) was washed with 50 mL of 0.5%
(v/v) acetic acid and equilibrated with 50 mL of binding buffer (250 mM ammonium acetate, pH 8.05, containing 50 mM MgCl2, 0.02% NaN3, and 0.25 mM PMSF). The tuber extract (1000 µL) was loaded on the
column, and nonglycated proteins were removed by washing with 110 mL of
binding buffer. Glycated proteins were eluted with 50 mL of Tris buffer
(100 mM, pH 8.5) containing 200 mM sorbitol and
0.05% (w/v) NaN3. Protein in glycated and nonglycated
fractions was quantified by a modified Lowry method (Bensadoun and
Weinstein, 1976).
PCMT Activity
PCMT, a substrate-inducible enzyme (Mudgett and Clarke, 1994,
1996), was assayed in the absence of synthetic peptide substrate as an
index of endogenous proteins containing deamidated, isomerized, and/or
racemized asparaginyl and/or aspartyl residues in 6-, 18-, and
30-month-old tubers. PCMT activity was assessed in tubers stored at
4°C and in tubers acclimated to 23°C for 24 h. Ten grams (fresh weight) of tuber tissue from the apical end (see above) was
homogenized with 10 mL of 100 mM Hepes buffer, pH 7.5, containing 1 mM PMSF, 1 mM DTT, 5 g of
hydrated polyvinyl polypyrrolidone, 2 mM
Na2S2O5, and 10 mM
Na2S2O4. The homogenate was
filtered through Miracloth and centrifuged at 30,000g for 30 min. All
manipulations were performed at 4°C. The supernatant was frozen in
liquid nitrogen and stored at 80°C.
PCMT activity assays were according to the method of Mudgett and Clarke
(1993) with minor modifications. The reaction medium (40 µL),
consisting of 12 µL of enzyme extract, 10 µM
[3H-methyl]AdoMet (1.8 Ci mmol1, NEN;
70-80 Ci mmol1), and, where indicated, 500 µM synthetic peptide substrate
Val-Tyr-Pro-(L-isoAsp)-His-Ala (synthesized by the
Macromolecular Structure Analysis Facility, University of Kentucky,
Lexington) was incubated at 30°C for 60 min. The reaction was stopped
with 40 µL of 200 mM NaOH, containing 1% (w/v) SDS, and
60 µL of the reaction mixture was spotted onto 1.5- × 8-cm pleated
filter paper (3MM, Whatman) suspended in the neck of a 20-mL
scintillation vial containing 5 mL of Bio-Safe II scintillation
cocktail (Research Products International, Mount Prospect, IL).
After 2 h the paper was removed and the [3H]methanol
captured in the scintillation solution from the vapor phase was
determined (Kester et al., 1997).
Proteinase Extraction and Activity
Proteolytic enzymes were extracted from tuber tissue (a composite
of three tubers cut from the apical ends, periderm included) with 50 mM potassium citrate buffer, (pH 6.6, 1 g fresh weight mL1) containing 20 mM KCl, 2 mM
MgCl2, 5% (w/v) Suc, 1 mM DTT, 0.5% PVP, and
2 mM Na2S2O5 (Belles et
al., 1991) with a homogenizer. The homogenate was filtered through
Miracloth, centrifuged at 30,000g for 30 min, and the
supernatant was stored at 80°C for subsequent proteinase activity
assays. Insoluble protein was also quantified from these tuber
extracts. The pellet was suspended in 5 mL of buffer, as described
above, and starch was removed by centrifuging twice at 200g
for 5 min each time. The insoluble protein remaining in the supernatant
was pelleted at 30,000g for 30 min, and solubilized in 1 mL
of extraction buffer (described above), containing 0.2% (w/v) Triton
X-100. Protein was determined by the Bradford (1976) assay.
Proteolytic activity was assessed spectrofluorometrically with
FITC-casein (Sigma) as a substrate (Vera and Conejero, 1988; Belles et
al., 1991). The reaction mixture (2.6 mL), containing 150 mM potassium citrate buffer, pH 6.1, 1.5 mM
DTT, 513 µg of FITC-casein, and 800 µL of enzyme extract (2.1-2.9
mg of protein), was incubated at 37°C in the dark. At 0, 15, 30, 45, 60, and 90 min, 350 µL of the reaction medium was transferred to
Eppendorf tubes containing 100 µL of 40% TCA to stop the reaction.
The samples were held on ice for 30 min and centrifuged at
1640g for 20 min. The supernatant (300 µL) was mixed with
3 mL of 500 mM Tris-HCl buffer, pH 8.5, and emission at 520 nm (excitation at 500 nm) was measured on a spectrofluorometer (model
SF 330, Varian Instruments, Palo Alto, CA). The pH optimum for
FITC-casein hydrolysis by extracts from 30-month-old tubers was
determined by profiling activity from pH 4.0 to 9.0 in sodium
acetate/potassium citrate/Mes/Tricine (37.5 mM each)
buffer.
While FITC-casein-hydrolyzing activity was relatively low in extracts
from 6-month-old tubers, activity was high in extracts from 18- and
30-month-old tubers. The ability of extract from 6-month-old tubers to
inhibit the FITC-casein-hydrolyzing activity of extract from older
tubers was examined. Assays were as described above with the addition
of 6-month-old tuber extract as a source of proteinase inhibitor.
Extracts from 6-month-old tubers were included, such that the ratio of
protein in 18- and 30-month-old extracts to inhibitor protein
(6-month-old extract) remained constant at 2.7:1. In an additional
study, the heat lability (at 95°C for 10 min) of inhibitory factor(s)
in crude extracts from 6-month-old tubers was compared with that of PMC
in inhibiting FITC-casein hydrolysis by extracts from 30-month-old
tubers.
The relative contributions of different proteinases to proteolytic
activity was estimated using class-specific proteinase inhibitors. The
inhibition of FITC-casein hydrolysis by 1 mM PMSF, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 25 µM E-64, 180 µM leupeptin, 70 µM pepstatin, or 2 mM EDTA was assessed by
first incubating (at 4°C for 30 min) each inhibitor separately with extracts from 6-, 18-, and 30-month-old tubers. FITC-casein, DTT, and
potassium citrate buffer (see above) were then added to the samples and
the reactions allowed to continue at 37°C for 60 min. The proteinase
inhibitor concentrations indicated were concentrations before the
addition of the substrate and buffer. Reactions were stopped with 10%
(w/v) TCA, and FITC was quantified spectrofluorometrically as described
above. All inhibitors except PMSF and EDTA (Sigma) were prepared
according to the manufacturer's instructions (Calbiochem). PMSF and
EDTA were prepared in DMSO and distilled water, respectively.
Proteinase activities were also compared on 10% polyacrylamide gels
(Laemmli, 1970) containing 0.1% (w/v) gelatin (Heussen and Dowdle,
1980; Michaud et al., 1993, 1994). Enzyme extracts were diluted 2-fold
in a sample preparation buffer (Michaud et al., 1994), incubated for 10 min at 37°C, and subjected to SDS-PAGE at 4°C (10% running gel)
for 60 or 120 min to resolve the low- and high-molecular-mass
proteinases, respectively (72 µg of protein per lane). The gels were
then washed in 2.5% (w/v) Triton X-100 for 30 min (23°C), and
incubated in 150 mM potassium citrate buffer, pH 6.1, containing 5 mM L-Cys and 0.1% (w/v) Triton
X-100 for 20 h (at 37°C). The gels were stained with Coomassie
blue, and gelatinolytic activity was assessed as achromatic zones on a
blue background following destaining.
Isolation of Patatin and PMC
Patatin was isolated from 6-month-old tubers to assess the effects
of tuber age on patatinolytic activity. Protein was precipitated from
tuber extract (see above) with 80% ammonium sulfate, solubilized in
SDS-PAGE sample preparation buffer (Laemmli, 1970), and 120 to 165 mg
was loaded onto a column (model 491, Prep Cell, Bio-Rad) containing
10% acrylamide. Electrophoresis was at a constant 200 V for 20 h.
Fractions were collected and analyzed for patatin by running 10-µL
aliquots on minigels. Fractions containing patatin were pooled and
concentrated by ultrafiltration. PMC was isolated from the peel of
young (5- to 7-month-old) tubers according to the method of Rodis and
Hoff (1984). Purity of the isolated PMC was verified via SDS-PAGE
(Laemmli, 1970) and western analysis (Walsh and Strickland, 1993).
PMC Inhibition of Age-Induced Patatinolytic Activity
We examined the ability of extracts from 26-month-old tubers to
degrade exogenous patatin in the presence and absence of PMC. The
reaction medium (250 µL) consisted of 100 µL of 150 mM
potassium citrate buffer, pH 6.1, 145 µg of patatin (see above), and
0 or 36 µg of PMC. The reaction was started by adding 50 µL (122 µg of protein) of the 26-month-old tuber extract, incubated at
37°C, and terminated at 0, 15, and 30 min with 10% (w/v) TCA (final concentration). The protein was then pelleted at 1640g for
20 min and separated by SDS-PAGE (Laemmli, 1970).
Tissue Prints of PMC
The tissue-printing technique of Varner (1992) was used to detect
PMC in 6-, 18-, and 30-month-old tubers. One-millimeter-thick longitudinal sections of tubers were cut and placed immediately on 0.25 µm nitrocellulose for 5 min. The blots were then blocked and probed
for PMC with anti-PMC antibody (Walsh and Strickland, 1993).
Mother/Daughter Tuber System
Tubers that had been stored for 29 months at 4°C (95% RH) were
placed in the dark at 23°C for approximately 2 months to develop daughter tubers. This system was then used to assess whether the older
mother tubers have the ability to produce daughter tubers with
polypeptide profiles, patatin content, proteinase activities, and PMC
levels comparable to those of younger mother tubers.
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RESULTS AND DISCUSSION |
Tuber Age Affects Total Protein, Patatin, and PMC Content
Soluble protein concentration (on a fresh-weight basis) decreased,
whereas insoluble protein increased as tubers aged from 6 to 30 months
(Table I). The loss of soluble proteins
was not simply a consequence of age-induced changes in tuber fresh
weight, as similar effects have been documented on a dry-weight basis (Kumar and Knowles, 1993c). The increase in insoluble protein suggests
that aging may induce molecular modifications to proteins that effect
reduced solubility (e.g. nonenzymatic glycation).
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Table I.
Changes in soluble and insoluble protein with
advancing tuber age
Soluble and insoluble protein were quantified in 30,000g
supernatants and destarched pellets, respectively. Insoluble protein
was solubilized with Triton X-100. Linear trends were significant
(P < 0.01) for soluble, insoluble, and total protein.
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Changes in soluble proteins over 20 d of sprouting were age
dependent. Tuber age influenced polypeptides with molecular masses of
110, 98, 85, 58, 40, 35, 25, and 20 kD and below (Fig.
2). The greatest effect of age was on
loss of patatin (40 kD, Fig. 2), the major storage glycoprotein that
makes up 20% to 40% of soluble protein in tubers (Galliard, 1971;
Racusen and Foote, 1980; Paiva et al., 1983; Bohac, 1991; Strickland et
al., 1995). Patatin is also a nonspecific lipid acyl hydrolase with
activity similar to that of the phospholipases A1,
A2, B, glycolipase, sulfolipase, monoacylglycerol lipase,
and esterase (Hirayama et al., 1975; Galliard, 1980; Strickland et al.,
1995).
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| Figure 2.
SDS-PAGE of soluble proteins (27 µg/lane) from
6-, 18-, and 30-month-old tubers during sprouting. Note the loss of the
85-kD PMC (black arrow) and 40-kD patatin (white arrow) with advancing
tuber age. PMC was present in 6-month-old tubers and nondetectable in
18- and 30-month-old tubers, as confirmed by western analysis with
anti-PMC (see Fig. 4B, inset). Aging also effected an increase in
glutathione reductase (58 kD, black arrowhead) as well as the loss of
several other unidentified proteins (e.g. 20 kD, white arrowhead).
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Considering its multiple roles as a storage protein and as a potential
antioxidant (Al-Saikhan et al., 1995) and its enzymatic involvement in
lipid metabolism, the loss of patatin may contribute to many of the
changes in tuber physiology that have been associated with advancing
age. These changes include increased peroxidation of membrane lipids
and reduced membrane integrity (Knowles and Knowles, 1989; Kumar and
Knowles, 1993b), increased oxidative stress (Kumar and Knowles, 1996b),
and loss of sprouting vigor (Kumar and Knowles, 1993a). Increases in
the relative levels of specific polypeptides with advancing tuber age
(e.g. 110, 58, and 35 kD; Fig. 2) could reflect a gel-loading bias
caused by the age-induced loss of patatin, because the gels were loaded on an equal-protein basis. However, increases in the concentrations of
these proteins over the sprouting interval were unique to the 30-month-old tubers. The 58-kD polypeptide has been previously identified as glutathione reductase (Kumar and Knowles, 1996b).
Another polypeptide lost during tuber aging was PMC (85 kD; Fig. 2), a
Cys-proteinase inhibitor (Rodis and Hoff, 1984) that can simultaneously
bind and inhibit the activities of as many as eight proteinase
molecules (Walsh and Strickland, 1993). The age-induced loss of PMC was
verified by tissue printing (Fig. 3) and
western analysis (see blot in Fig. 4B). Consistent with results from
SDS-PAGE (Fig. 2), PMC was present in the tissue print and western blot
from 6-month-old tubers, but was undetectable in those from 18- and
30-month-old tubers (Figs. 3 and 4).
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| Figure 3.
Tissue prints of PMC in longitudinal sections of
6-, 18-, and 30-month-old tubers. PMC is indicated by purple
coloration. Note the absence of PMC in the 18- and 30-month-old
tubers.
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| Figure 4.
A, FITC-casein hydrolyzing proteinase activity of
extracts from 6- ( ), 18- ( ), and 30-month-old ( ) tubers. B,
Inhibition of the proteinase activity in extracts from 18- () and
30-month-old () tubers with extracts from 6-month-old tubers. Data
are averages ± SE of three replicates (SE
values are eclipsed by the symbols in B). Inset shows immunoblots of
PMC (32 µg of protein per lane) in extracts from tubers, before and
after the addition of 6-month-old extract to extracts from 18- and
30-month-old tubers.
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Our previous studies have shown that tuber aging is accompanied by a
decrease in the capacity for protein synthesis and an increase in free
amino nitrogen (Kumar and Knowles, 1993c), suggesting that the
age-induced decline in protein content is mediated by increased
proteolysis. The loss of PMC could be a controlling factor in this
process. Therefore, we investigated the contribution of
proteolysis to the age-induced loss of proteins in general, and patatin in particular. We compared the proteolytic activity in
extracts from 6-, 18-, and 30-month-old tubers. Extracts from 6-month-old tubers hydrolyzed FITC-casein relatively slowly (Fig. 4A).
In contrast, FITC-casein-hydrolyzing activity in extracts from 18- and
30-month-old tubers was substantial and much greater than what could be
accounted for by age-induced declines in nonproteinase tuber proteins
(e.g. selective loss of patatin from older tubers). When compared on a
tuber fresh-weight basis, proteinase activity from 18- and 30-month-old
tubers was 1.6- and 10-fold greater, respectively, than those from
6-month-old tubers (data not shown).
Higher proteolytic activity in older tubers could have been the result
of increased synthesis and/or activation of proteinases, loss of
proteolytic inhibitors such as PMC, or a combination of these
during aging. Extracts from 6-month-old tubers effectively inhibited
the FITC-casein-hydrolyzing activities of extracts from 18- and
30-month-old tubers (Fig. 4B), reflecting the presence of a proteinase
inhibitor or inhibitors in younger tubers. Western analysis indicated
that PMC was present in 6-month-old tubers but was absent from 18- and
30-month-old tubers (Fig. 4B). To determine whether the inhibitory
effect of extracts from 6-month-old tubers was due to PMC, purified PMC
from 6-month-old tubers was assessed for its ability to inhibit
FITC-casein hydrolysis by extracts from 30-month-old tubers. PMC
was as effective as crude extracts from 6-month-old tubers in
inhibiting the proteinase activity in extracts from 30-month-old tubers
(Table II). Moreover, heat treatment of
crude extracts and PMC rendered both completely ineffective at
inhibiting the age-induced proteinase activity. The heat-labile nature
of PMC has been previously documented (Rodis and Hoff, 1984).
Collectively, these results indicate that the age-induced increase in
proteinase activity is a consequence of PMC loss during the aging
process.
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Table II.
Inhibition of proteinase activity in extracts from
30-month-old tubers by nonheated and heated crude extracts and PMC from
6-month-old tubers
FITC-casein hydrolysis was determined by incubating extracts from
30-month-old tubers at 37°C (60 min) with or without inhibitors.
Inhibitors included heat-treated (95°C, 10 min) or nonheated crude
extract (160 µg of protein equivalents) and PMC (65 µg of
protein equivalents) from 6-month-old tubers.
LSD0.05 = 15.8 units mg1
protein h1.
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Characterization of the proteinases in 6-, 18-, and 30-month-old tubers
provided further evidence that the age-induced increase in proteolytic
activity is primarily due to loss of PMC. Commercial Cys-type
proteinase inhibitors (E-64 and leupeptin) were most effective at
inhibiting FITC-casein-hydrolyzing activity of extracts from
30-month-old tubers, reducing activity by 90% to 93% (Fig. 5). Furthermore, the pH optimum for
FITC-casein hydrolysis by older tuber extracts was 6.1 (data not
shown), which is characteristic of potato Cys-type proteinases
(Kitamura and Maruyama, 1985). These results agree with those of Isola
and Franzoni (1993), who showed that the Cys-proteinases, which are
leupeptin-sensitive, were most active in protein degradation during
aging of potato slices. In contrast, Asp-type proteinases
(pepstatin-sensitive) dominated (74%) the total proteinase activity of
6-month-old tubers. Total proteinase activity in 18-month-old tubers
was equally sensitive to Asp- and Cys-type inhibitors. A shift
in the predominant proteinase from Asp- to Cys-type therefore
occurs during tuber aging, reflecting the age-induced loss of PMC.
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| Figure 5.
Effects of class-specific proteinase inhibitors on
the FITC-casein-hydrolyzing activities of extracts from 6-, 18-, and
30-month-old tubers. FITC-casein was added to tuber extracts containing
each inhibitor and hydrolysis was assessed after 60 min of incubation
at 37°C. Spectrofluorometric units are emission at 520 nm following
excitation at 500 nm. Proteinase inhibitors include PMSF and AEBSF
(Ser-type), E-64 and leupeptin (Cys-type), pepstatin (Asp-type),
and EDTA (metallo-type). Data are averages ± SE of
three replicates. Numbers in bars are percent inhibition relative to
control (no inhibitors present).
|
|
The age-induced loss of PMC and patatin, concomitant with increased
proteinase activity, suggests a role for PMC in the regulation of tuber
protein/patatin content. PMC and patatin were isolated from young
tubers (Fig. 6A). To determine whether
PMC can inhibit the patatinolytic activity of older tubers, extracts
from 26-month-old tubers were spiked with purified patatin with or
without PMC, and patatinolytic activity was assessed by SDS-PAGE.
Extracts from older tubers effectively degraded exogenous patatin
within 30 min of incubation (Fig. 6B). This proteolytic activity of the older tuber extracts was substantially inhibited when PMC was included
in the assay medium. Under similar conditions, extracts from younger
tubers did not catabolize patatin (data not shown), indicating a
potential role for PMC in regulating the degradation of patatin and
other tuber proteins.
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| Figure 6.
Ability of PMC to inhibit the patatinolytic
activity of extracts from 26-month-old tubers. A, SDS-PAGE of purified
PMC (black arrow) and patatin (white arrow) (3.5 and 7.6 µg per lane,
respectively) isolated from 6-month-old tubers. B, Patatin (white
arrow) was added to extracts from 26-month-old tubers, which were then
incubated for 0, 15 and 30 min (37°C) in the absence or presence of
PMC (black arrow). A total of 100 µg of protein containing 48 µg of
patatin and 12 µg of PMC (where indicated) at time zero was loaded in
each lane.
|
|
If PMC is involved in preventing patatinolytic activity in vivo, its
loss should precede that of patatin during the aging process. Evidence
for this is provided in Figures 2, 3, and 4B. As tuber age advanced
from 6 to 18 months, PMC decreased to nondetectable levels (Figs. 3 and
4B), while patatin, though significantly reduced in concentration, was
still present (Fig. 2). The mechanism by which PMC was lost during
tuber aging is unknown; however, while the proteinases in 30-month-old
tuber extracts did not inhibit the effects of exogenous PMC in
preventing FITC-casein-hydrolysis (Fig. 4B) and patatin degradation
(Fig. 6B) in short-term studies, extracts from 6- and 30-month-old
tubers were equally effective in degrading PMC into 65- and 20-kD
polypeptides over a 24-h incubation period (Fig.
7). Such partial proteolysis, however,
did not greatly alter the ability of PMC to inhibit patatin
degradation, presumably because PMC has multiple inhibitory domains and
its subunits are capable of retaining inhibitory activity even after
partial proteolysis (Walsh and Strickland, 1993). The age-induced loss
of PMC from tubers, which probably facilitates patatin breakdown, could
be due to a combination of reduced synthesis, complete proteolysis, and/or inactivation of PMC through a number of nonenzymatic mechanisms (e.g. glycation, oxidation, or isomerization/racemization).
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| Figure 7.
SDS-PAGE and immunoblots comparing the ability of
extracts from 6- and 30-month-old tubers to degrade PMC into 65- and
20-kD subunits. Extracts were spiked with 10 µg of PMC and incubated
for 0 and 24 h at 37°C prior to electrophoresis (64 µg/lane).
|
|
The nature of increased proteolytic activity associated with aging was
further examined by electrophoretic analysis of proteinases. Two
gelatinolytic proteinases (approximately 84 and 95 kD) were common
among the 6-, 18-, and 30-month-old tubers, increasing with advancing
age (Fig. 8). Tubers of all three ages
also had a 125-kD proteinase (Fig. 8B), but its activity in 6-month-old tubers was below photographic resolution. In 30-month-old tubers, additional proteinases with greatly enhanced activity were observed at
approximately 75, 90, and 100 kD. Similar increases in gelatinolytic activity were evident when gels were loaded on a tuber fresh-weight basis, eliminating gel-loading bias as an explanation for the increased
proteinase activity in older tubers. Age-induced proteinases could not
be detected in extracts from young tubers, even when protein loads were
3-fold greater than that of 30-month-old tuber extracts. All six
proteinases effectively degraded patatin on patatin-containing gels
(data not shown). Therefore, increased proteolytic activity in older
tubers was due to activation and/or synthesis of existing and novel
proteinases during aging, and protein loss with advancing tuber age was
correlated with the loss of PMC and increased activities of Cys-type
proteinases.
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| Figure 8.
Gelatinolytic proteinase activities of extracts
from 6-, 18-, and 30-month-old tubers (resolved by SDS-PAGE in gels
containing 0.1% gelatin). Proteins were electrophoresed for 60 min (A)
and 120 min (B) to resolve the total and high-molecular-mass
proteinases, respectively (72 µg/lane).
|
|
Age-Induced, Nonenzymatic Modifications of Tuber Proteins
Although proteolysis serves as a method for cellular housekeeping
by degrading defective proteins (Goldberg and Dice, 1974; Finley and
Chau, 1991; Hershko and Ciechanover, 1992), relatively few
modifications that target abnormal proteins for degradation have been
adequately documented. Defective proteins can arise through a number of
mechanisms, including biosynthetic errors (Jentsch, 1996), spontaneous
denaturation, and free-radical attack (Stadtman, 1992). Depending on
the type of damage, repair and restoration of protein conformation and
function are possible (Deshaies et al., 1988; Rothman, 1989); however,
degradation is the only fate for proteins synthesized from defective
mRNAs (Jentsch, 1996). Although defective proteins arise spontaneously
in every cell, the continued assault by free radicals on the
transcription/translation machinery (Desimone et al., 1996) of cells
under high oxidative stress produces unabated generation of abnormal
proteins. Failure to degrade such proteins results in their
accumulation to toxic levels (Vierstra, 1993) and limits the size of
the amino acid pool for protein turnover. An increase in free amino
acids with advancing tuber age, along with a reduced ability for
protein synthesis (Kumar and Knowles, 1993c) and increased proteolytic activity (Figs. 4, 6 and 8), indicates that tuber aging is associated predominantly with protein catabolism rather than with repair.
Nonenzymatic molecular modifications to proteins adversely affect their
structure and function, and have been associated with aging in
mammalian and other systems (Furth, 1988; Stadtman, 1988, 1992; Ota and
Clarke, 1990). For this reason, we examined the effects of tuber age on
protein oxidation, glycation, and susceptibility as substrates for PCMT
activity. Older tubers respire at a significantly higher rate than
younger tubers (Kumar and Knowles, 1996a) and are therefore subject to
increased oxidative stress (Kumar and Knowles, 1993b, 1996b) from the
accumulation of reactive oxygen species, emanating in part from
increased electron transport in mitochondria (Boveris et al., 1976;
Forman and Boveris, 1982). Reactive oxygen species effectively damage
various macromolecules, including proteins (Stadtman, 1992; Gebicki and
Gebicki, 1993; Sohal and Weindruch, 1996). Aging in animal systems is
accompanied by the accumulation of less active and in some cases
totally inactive enzymes, much of which has been attributed to
oxidative modifications (Oliver et al., 1987).
Protein oxidation is characterized by increased carbonyl content (Fig.
1) and, in general, such modifications target proteins for proteolysis
(Kyle et al., 1984; Dalling, 1987; Stadtman, 1992). For example,
increased carbonyl content resulting from oxidative stress effected
increased proteolysis of Rubisco (Ferriera and Shaw, 1989; Penarrubia
and Moreno, 1990; Garcia-Ferris and Moreno, 1993; Eckardt and Pell,
1995; Desimone et al., 1996). Carbonyl content of proteins from 18- and
30-month-old tubers was 17% and 61% higher, respectively, than that
of 6-month-old tubers (Fig. 9, compare
maxima at A380), reflecting increased
protein oxidation with advancing age. Western analysis showed
progressively greater proportions of oxidized proteins in the soluble
fraction as tubers aged from 6 to 30 months (Fig. 9). In fact, most of
the soluble polypeptides from 30-month-old tubers had increased
carbonyl content, reflecting the nonspecificity of protein oxidation.
Oxidation is therefore one mechanism by which proteins could be
targeted for degradation in aging tubers.
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| Figure 9.
Absorption spectra and immunoblots depicting the
effects of tuber age on protein oxidation. Carbonyl groups of oxidized
proteins were derivitized with DNPH and quantified at
A380. Oxidized proteins in the soluble-protein
fraction of 6-, 18-, and 30-month-old tubers over 20 d of
sprouting are shown in the western blot, where protein-carbonyl-DNP
derivatives were probed with monoclonal anti-DNP antibody.
|
|
Another nonenzymatic modification that effects decreased protein
function and increased catalysis in aging systems is glycation (Fig.
1), a consequence of free amino groups on proteins reacting with the
free carbonyl groups of reducing sugars. The resulting glycated
proteins often cross-link, leading to the accumulation of insoluble,
advanced-glycation end products (Furth, 1988). Tuber proteins may be
particularly susceptible to this type of damage, because reducing
sugars increase during aging, presenting the ideal environment for
protein glycation (Kumar and Knowles, 1993b). A linear increase in
glycated proteins (Amadori products) was evident as tubers aged from 6 to 30 months (Fig. 10). Increased glycation was not a
manifestation of the declining protein content associated with aging,
because isolation of the glycated protein by boronate affinity
chromatography showed that older tubers had 23% to 31% more glycated
protein than younger tubers, and the glycated fraction increased as a
percentage of total soluble protein on a tuber fresh-weight basis (Fig.
10, inset). According to Wettlaufer and Leopold (1991), the
nonenzymatic glycation of proteins during accelerated aging of soybean
seeds plays an important role in seed deterioration. These authors also
suggested that reducing sugars in seeds can reduce viability by
enhancing the glycation process. The negative correlation between
protein glycation and vigor of true seeds during aging is also apparent
for potato. The insoluble protein that accumulates in aging tubers may
consist of advanced glycation products, although this remains to be
established.
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| Figure 10.
Effect of tuber age on protein glycation as
determined by boronate affinity chromatography (inset) and
thiobarbituric acid assay. The TBA data are averages ± SE of three replicates. Micrograms of hydroxymethyl
furaldehyde (HMF) equivalents are expressed per gram fresh weight ()
and per milligram protein ().
|
|
Molecular alterations affecting protein charge and/or structure also
yield dysfunctional proteins that may be targeted for degradation or
repair. One such nonenzymatic modification is the deamidation of
asparaginyl and the isomerization/racemization of aspartyl residues,
resulting in the formation of isomerized L-isoaspartyl or
racemized D-aspartyl residues (Fig. 1). Proteins with such
adducts accumulate in mammalian systems during aging (Galletti et al.,
1995). Deamidation affects Ca+2-binding domains
of calmodulin (Johnson et al., 1989) and results in functional
inactivation (Johnson et al., 1985, 1987). Functionality of deamidated
proteins, however, can be restored by methylation of isoaspartyl
residues by PCMT (Johnson et al., 1987; Li and Clarke, 1992), a
substrate-inducible enzyme (Mudgett and Clarke, 1994, 1996).
When PCMT activity was measured with a saturating level of
L-isoaspartyl-containing peptide substrate
(VYP-[L-isoAsp]-HA), activities from 6- and 30-month-old
tubers were comparable (30.5 pmol min1
mg1 protein on average). However, in the
absence of the synthetic peptide substrate, endogenous PCMT activity
increased linearly as tubers aged from 6 to 30 months (Table
III). Thus, the enhanced PCMT activity in
aged tubers was due to increased protein substrates containing altered
amino acid residues and not to changes in the level of PCMT, as
determined by total activity measurements with an artificial substrate.
This is in direct contrast to aging of barley seeds, in which PCMT
activity decreased with a concomitant increase in
L-isoaspartylated proteins over a 17-year period (Mudgett, et al., 1997). Accelerated aging of tomato seeds also lowered PCMT
activity, which was correlated with a loss of vigor (Kester et al.,
1997). To our knowledge, our study is the first report of an increase
in PCMT activity during aging of a vegetative plant tissue, a
consequence of the increasing level of damaged peptide substrate.
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|
Table III.
PCMT activity as affected by age and acclimation
temperature of potato tubers
PCMT was extracted from tubers directly from cold storage (4°C) and
after acclimation of tubers to 23°C for 24 h. Assays were
conducted at 30°C without exogenous polypeptide substrate. Main
effects of age (linear) and temperature were significant (P < 0.05). The age × temperature interaction was not significant.
|
|
Mother/Daughter Tuber System
Tubers stored for more than 20 months at 4°C developed daughter
tubers directly when placed in the dark at 23°C, providing an ideal
system for studying the effects of age on protein content and
metabolism (Fig. 11a). Daughter tubers
contained 3-fold more protein (on a fresh-weight basis) than their
29-month-old mother tubers, and the polypeptide profile of daughter
tubers (Fig. 11c) was comparable to that of 6-month-old tubers (Fig.
2). Tissue prints (Fig. 11b) and SDS-PAGE (Fig. 11c) showed that mother
tubers lacked PMC and patatin, whereas daughter tubers contained both proteins. Because daughter tubers are clones of mother-tuber tissue, aging did not compromise the fidelity of genes coding for PMC and
patatin. Our results suggest a new role for PMC in regulating the
levels of patatin and other tuber proteins through inhibiting proteolysis. Although gelatinolytic proteinase activity of extracts from 29-month-old mother tubers was consistent with that shown previously for 30-month-old tubers, proteolytic activity was absent from daughter tubers (compare Figs. 11d and 8A). The loss of PMC during
aging probably contributes to proteolysis of patatin and other tuber
proteins.
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| Figure 11.
a, Mother/daughter tuber system for studying the
effects of tuber age on protein metabolism. Mother tubers (M) were
stored at 4°C for 29 months, transferred to 23°C, and incubated in
the dark for approximately 2 months to induce daughter (D) tubers. b,
Tissue prints of PMC from longitudinal sections of 29-month-old mother
and daughter tubers. Purple color indicates the presence of PMC. c,
SDS-PAGE of proteins from mother and daughter tubers. Note the absence
of PMC (double-headed arrow) and patatin (arrow) from the 29-month-old
mother tuber, whereas daughter tubers contain high levels of both
proteins. d, Gelatinolytic proteinase activities in extracts from
mother tubers and their absence in extracts from daughter tubers.
|
|
In summary, our results demonstrate that: (a) the loss of patatin
contributes substantially to the age-induced decline in soluble protein
content of potato tubers; (b) aging results in an increase in
proteolytic activity and the appearance of several novel proteinases;
(c) PMC, a Cys-proteinase inhibitor effective against patatinolytic
proteinases in vitro, is lost with advancing seed-tuber age; (d)
age-enhanced proteolytic activity is mainly due to increases in
Cys-proteinases; (e) as tubers age there is a shift from Asp- to
Cys-type proteinases; and (f) the observed increases in protein
glycation, oxidation, and deamidation/isomerization/racemization with
advancing tuber age are potential targeting mechanisms for protein
degradation. To our knowledge, this is the first demonstration of the
plurality of nonenzymatic protein modifications that can be
definitively associated with aging in a nonsenescent plant system.
| |
FOOTNOTES |
1
This research was funded by the Natural Sciences
and Engineering Research Council of Canada (grant to N.R.K.) and by the
U.S. Department of Energy (grant no. DE-FG05-92ER26075 to R.L.H.).
*
Corresponding author; e-mail rknowles{at}afns.ualberta.ca; fax
1-403-492-4265.
Received May 28, 1998;
accepted September 15, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AdoMet, S-adenosyl-L-Met.
DNPH, 2,4-din-itrophenyl hydrazine.
E-64, trans-epoxysuccinyl-L-leucylamido (4-guanidino)
butane.
FITC, fluorescein isothiocyanate.
PCMT, protein carboxylmethyl
transferase.
PMC, potato multicystatin.
| |
ACKNOWLEDGMENT |
We thank T.A. Walsh (Biotechnology Laboratory, Dow-Elanco,
Indianapolis, IN) for providing PMC antisera.
| |
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