Journal of
Experimental
Botany
Journal of Experimental Botany, Vol. 48, No. 309, pp. 943-949, April 1997
Priming and accelerated ageing affect L-isoaspartyl
methyltransferase activity in tomato (Lycopersicon
esculentum Mill.) seed
S.T. Kester, R.L. Geneve and R.L. Houtz1
Department of Horticulture and Landscape Architecture, University of Kentucky, Lexington,
KY 40546-0091, USA
Received 20 June 1996; Accepted 8 November 1996
Abstract
Damage and degradation of cellular proteins is
observed during age-induced seed deterioration, LIsoaspartyl protein methyltransferase (EC 2.1.1.77) is
an enzyme hypothesized to play a role in limiting and
repairing age-induced damage to proteins. Tomato
{Lycopersicon esculentum Mill. 'New Yorker') seeds
were assayed for changes in L-isoaspartyl methyltransferase activity during accelerated ageing and
after osmotic priming. Accelerated ageing of seeds for
1-4 d at 45 C and 100% relative humidity reduced
germination from 94% to 7 1 % , increased the mean
time of germination (MTC) from 2.4 to 5.8 d, and was
accompanied by a correlative decrease in L-isoaspartyl
methyltransferase activity (^ = 0.90). Aged and
untreated seeds were primed for 7 d at 20 °C in darkness using aerated solutions of 3% KNO3 or polyethylene glycol 8000 (PEG) with equivalent osmotic
potential ( —1.25 MPa). Priming with KNO3 decreased
the MTG, but did not improve germination percentage
for untreated seeds. Priming did not affect L-isoaspartyl methyltransferase activity in untreated seeds, but
restored activity in aged seeds primed in KNO3 to
levels near that of untreated seeds. Priming with PEG
did not effectively improve the MTG or increase
L-isoaspartyl methyltransferase activity. During germination, L-isoaspartyl methyltransferase activity
remained constant for 48 h post-imbibition and then
declined, suggesting that the enzyme was developmentally regulated and inactivated or degraded as
radicle emergence occurred.
Key words: L-lsoaspartyl methyltransferase, protein repair,
1
seed
priming,
esculentum.
ageing,
Lycopersicon
Introduction
Protein carboxyl methyltransferases are common enzymes
in prokaryotic and eukaryotic organisms. These enzymes
catalyse transfer of methyl groups from S-adenosylmethionine (AdoMet) to the carboxyl side chain of
amino acyl residues in a variety of target proteins. Protein
carboxyl methyltransferases have been shown to have
significance in modulation of bacterial chemotaxis and
neurosecretion in mammalian glands (Clarke, 1985). One
protein carboxyl methyltransferase that has been shown
to be important in the recognition and repair of proteins
damaged during cell ageing is L-isoaspartyl methyltransferase (EC 2.1.1.77). This protein methylates L-isoaspartyl
and, to a lesser extent, D-aspartyl residues in proteins
(Ota and Clarke, 1990). One aspect of the cellular ageing
process is the spontaneous deamidation, isomerization,
and racemization of L-asparaginyl and L-aspartyl residues
of proteins (Galletti et cil., 1995). These potentially detrimental residues accumulate during ageing and have been
shown to alter the structure and function of proteins
in vitro (Ota and Clarke, 1990). L-Isoaspartyl protein
methyltransferase recognizes L-isoaspartyl residues in a
wide variety of proteins with a high affinity (A^m = 20 ^M).
The resultant enzymatic reaction catalyses the transfer of
a methyl group from AdoMet. This is the first step in the
conversion of L-isoaspartyl to normal L-aspartyl residues
restoring enzymatic function to damaged proteins
(Johnson et ai, 1987). Protein carboxyl methyltransferase-mediated protein repair during cell ageing, has
been demonstrated in bacteria (Li and Clarke, 1992) and
To whom correspondence should be addressed. Fax: +1 606 257 2859. E-mail: rhoirtz.ca.uky.edu
© Oxford University Press 1997
accelerated
944
Kester et al.
human cells (Clarke, 1985; Lowenson and Clarke, 1992).
An E. coli mutant lacking protein carboxyl methyltransferase activity was not affected by the mutation during
the log phase of cell growth, however, cells in the stationary phase of growth had poor survival.
Protein carboxyl methyltransferase activity in plants
was initially reported in wheat germ extracts (Trivedi
et al., 1982; Johnson et al., 1991), and L-isoaspartyl
methyltransferase activity has recently been demonstrated
in a variety of plant parts and species (Mudgett and
Clarke, 1993). The highest level of activity was found in
seeds of the plant species evaluated. A wheat germ
carboxyl methyltransferase was purified that recognizes
L-isoaspartyl containing peptides but not D-aspartyl residues (Mudgett and Clarke, 1993). A full length cDNA
clone was obtained for wheat germ L-isoaspartyl methyltransferase that showed 31% and 50% homology with the
E. coli and mammalian protein carboxyl methyltransferase enzymes, respectively. The high activity of L-isoaspartyl methyltransferase in seeds led Mudgett and Clarke
(1993) to hypothesize that this enzyme may be important
for protein repair during seed storage.
Seed deterioration during storage is a complex physiological process leading to loss of viability and/or reduced
seed vigour. Environmental conditions and duration of
storage can reduce seed viability, but seed deterioration
can also be manifested by a loss in seed vigour in seed
lots with near 100% viability (Bewley and Black, 1994).
Seed deterioration has been shown to be a function of
changes in membrane integrity, chromosomal damage,
and loss of enzyme activity (Priestley, 1986).
Early metabolic events critical to germination include
ATP, RNA, and protein synthesis (Come and Corbineau,
1989). Not surprisingly, seeds with reduced viability are
impaired in these functions. Several mitochondrial
enzymes show reduced activity in deteriorated seeds
including cytochrome c oxidase, catalase, peroxidase and
several Krebs cycle enzymes (Priestley, 1986).
Deteriorated seeds also show a reduced capacity to synthesize new proteins after imbibition, demonstrated by
the inability of rye embryos to incorporate radiolabelled
precursors into protein (Sen and Osborne, 1977). Aged
seeds with reduced vigour but high viability had 80% less
protein synthesizing capacity than non-aged seeds. A
similar contrast was seen for mRNA content in wheat
embryos with different levels of vigour and viability
(Rushton and Bray, 1987). During imbibition, there is a
normal turnover of conserved mRNA stored in the dry
seed. Most mRNA stored in the seed are degraded rapidly
and new mRNA must be synthesized to complete germination (Cuming and Lane, 1979; Lalonde and Bewley,
1986). In deteriorated (aged) seeds, the ability to resynthesize mRNA is impaired. This loss in ability to synthesize new mRNA is thought to be in response to a general
failure in enzymes involved with DNA repair and damage
to DNA polymerase enzymes in stored seed (Osborne,
1980).
A procedure that can ameliorate the detrimental effects
of seed ageing is osmotic seed priming (Pandey, 1989).
Priming is accomplished by imbibing seeds in an osmotic
solution (—1.0 to 1.5 MPa) at cool temperatures (usually
at 10-20 °C). After priming seeds are permitted to enter
the lag stage of germination (stage with little or no fresh
weight increase prior to radicle emergence), but are then
desiccated back to approximately the original moisture
content before the radicle emerges (Bradford, 1986).
Upon subsequent rehydration, seeds show improved germination characteristics which include (1) reduced time
to radicle emergence, (2) synchronization of germination
within a seed lot, (3) greater percentage germination,
and (4) improved seed vigour in deteriorated seed lots
(Karssen et al., 1989).
The mechanisms for improved germination in primed
seeds are not completely understood (Karssen et al.,
1989). Seeds in the lag stage of germination are thought
to initiate metabolic processes required for germination
that are subsequently maintained in dried-primed seeds.
This extended time in the lag stage may also give seeds
additional time for cellular repair processes that ameliorate the detrimental effects of previous dry storage on
seeds. The objectives of this study were to examine the
relationships between L-isoaspartyl methyltransferase
activity and seed germination percentage and MTG, as
influenced by seed priming under normal and accelerated
ageing conditions.
Materials and methods
Chemicals
AdoMet was obtained from Sigma and was purified prior to
use as described by Chirpich (1968). [ 3 H-methyl]AdoMet was
from New England Nuclear (Boston, MA, 70-80 Ci m m o P ' ) .
Bio-Safe II scintillation fluid was from Research Products
International (RPI, Mount Prospect, IL). All other chemicals
were obtained from Sigma. All aqueous reagents and buffers
were prepared with Milli-Q deionized water (18 MQ cm" 1 ,
Millipore Corp., Bedford, MA).
Biological materials
A single seed lot of tomato seed (Ly copersicon esculentum Mill.
'New Yorker') (Harris Seed Company, 60 Saginaw Drive,
Rochester, NY) was used for all experiments. All seeds were
surface disinfected with a 20% Clorox solution containing 0.1%
Tween 20 for 15 min followed by three rinses in distilled water.
Seeds were air-dried for 24 h prior to use and germinated by
placing 25 seeds on two pieces of germination paper (Anchor
Paper, Hudson, WI) in lOOx 15 mm Petri dishes. The paper
was wetted with 6 ml of sterile deionized water and sealed with
parafilm. Germination tests were performed at 30 °C in the
dark. There were 100 seeds per treatment and all experiments
were duplicated. Seeds with ^ 4 mm radicles were considered
germinated. The mean time to germination (MTG) was
Priming and accelerated ageing effect
calculated as days to 50% final germination (Alvarado et al.,
1987).
Extraction and assay of t-isoaspartyl methyltransferase
One gram of dry seed was homogenized with a polytron
(Brinkman Instruments, Westbury, NY) at a speed setting of
5, for 30 s in 5 ml of chilled extraction buffer (100 mM HEPESK + (pH 8.0), 10 mM 2-mecaptoethanol, 1 ^M leupeptin, 1 mM
phenylmethane sulphonylfluoride, 10 mM sodium hydrosulphite, and 10 mM sodium metabisulphite) containing 1.5 g of
buffer-hydrated polyvinyl-polypyrolidone (PVPP). The crude
homogenate was filtered through four layers of cheesecloth and
centrifuged at 2200 g for 20 min at 4 °C. The supernatant was
filtered through two layers of Miracloth (Calbiochem, La Jolla,
CA) and either assayed directly or stored at — 80 °C. LIsoaspartyl methyltransferase activity was determined by a
vapour-phase diffusion assay as described previously (Mudgett
and Clarke, 1993) using a synthetic peptide substrate VYP-{LisoAsp)-HA synthesized by the Macromolecular Structure
Analysis Facility at the University of Kentucky. The vapourphase diffusion assay measures methyl groups transferred to
the synthetic peptide from [ 3 H-methyl]AdoMet by releasing
them as [ 3 H]methanol after hydrolysis under alkaline conditions.
The reaction mixture contained 12^1 enzyme extract, 500/xM
synthetic peptide, 330 mM HEPES-K"1", (pH 8.0) and 10ftM
[ H-methyl] AdoMet (1.8 Ci mmol" 1 ) in a final reaction volume
of 40 fj.]. The reaction mixture was incubated at 30 °C for 1 h
followed by addition of 40 ^1 of 0.2 M NaOH containing 1%
sodium dodecyl sulphate (SDS). A 60 ^tl aliquot was placed on
pleated 3 mm filter paper (Whatman, Maldsone, Eng.) and
suspended over 5 ml of Bio-Safe II scintillation fluid in a 20 ml
scintillation vial. After 2 h, the filter paper was removed and
the [ 3 H]methanol captured in the scintillation solution from
the vapour-phase was determined by liquid scintillation
spectrometry. Endogenous L-isoaspartyl methyltransferase
activity in seed extracts was determined by omitting the
synthetic polypeptide substrate. Each assay was replicated and
experiments were repeated. Protein concentrations were determined by the Bradford dye-binding assay using BSA as a
standard (Bradford, 1976).
Protein electrophoresis, transfer, and imaging
SDS-polyacrylamide gel electrophoresis (PAGE) was performed
using a Bio-Rad Protean II unit as described by Laemmli
(1970) and Hames and Rickwood (1981). Proteins separated
by SDS-PAGE were transferred to polyvinylidene fluoride
(PVDF) membranes (Immobilon-P, Millipore Corp., Bedford,
MA) using a MiniBlot-SDE Transfer Apparatus (Millipore
Corp., Bedford, MA). Proteins were visualized by staining with
Coomassie brilliant blue R-250, and destaining with 40%
methanol:10% acetic acid. The incorporation of [3H-methyl]
groups from AdoMet into seed polypeptides was detected using
a Phospholmager (Model 425, Molecular Dynamics, Sunnyvale, CA). Tomato seed proteins were exposed to [3Hmethyl] AdoMet under the same conditions as for the
determination of L-isoaspartyl methyltransferase activity, except
that the concentration of AdoMet was reduced to 3.4 ^M and
the specific activity increased to 84.1 Ci mmol" 1 . After incubation at 30 °C for 60 min, 40 ^1 of SDS-PAGE sample preparation
buffer was added and the samples heated in boiling water for
3 min. Proteins in the samples were separated by SDS-PAGE
and electrophoretically transferred to PVDF membranes. After
transfer PVDF membranes were washed with methanol followed
by deionized water, and then dried at 50 °C. The dry PVDF
membranes were exposed for 7 d to a Phosphorlmager Tritium
945
screen prior to image acquisition. After imaging PVDF
membranes were soaked for 2 h in 300 mM TRIS-K + pH =
10.0 buffer, washed as before, and then reexposed to the Tritium
screen under the same conditions. A third image was also
acquired, but without prior treatment with the alkaline
TRIS buffer.
Accelerated ageing
Seeds were aged according to Delouche and Baskin (1973).
One gram of surface disinfected seeds were suspended over
40 ml of deionized water on a wire mesh tray in a closed plastic
box (11x11 x 4 c m ) . Each box was placed in a water-jacketed
incubator maintained at 45 °C and near 100% humidity for up
to 4 d. Treated seeds were air-dried under a laminar flow hood
for 24 h (7% moisture) prior to germination or being assayed
for enzyme activity.
Osmotic seed priming
Aged and non-aged seeds were primed in aerated solutions of
K N 0 3 or polyethylene glycol 8000 (PEG) each at - 1 . 2 5 MPa
(Wartidiningsih et al., 1994). Seeds (0.5-1 g) were placed in
bags constructed of cheesecloth and suspended in 1 1 Erlenmeyer
flasks containing 500 ml of osmotic solution. Conditions for
priming were 20 °C in the dark for 7 d. Seeds were rinsed for
3 min under running distilled water and air-dried for 24 h prior
to germination or enzyme assay.
Results and discussion
The tomato seed lot used for these experiments exhibited
high viability (95% germination) and vigour (MTG =
2.8 d). During germination, L-isoaspartyl methyltransferase activity was initially high in dry and imbibed seeds,
but began to decline following radicle emergence
(Table 1). These data were in agreement with previous
observations by Mudgett and Clarke (1994) showing a
decline in enzyme activity as well as mRNA levels for
L-isoaspartyl methyltransferase during germination in
wheat seeds. Although the detection of enzyme activity
in extracts from dry seeds is not demonstrative evidence
for in vivo activity, there was a relatively high degree of
incorporation of 3H-methyl groups from AdoMet into
endogenous protein substrates (Table 1). This indicates
that there could be a number of damaged proteins in dry
Table 1. L-fsoaspartyl methyltransferase activity and germination
percentage in tomato seeds during germination at 30°C
Data presented for L-isoaspartyl methyltransferase activity are
means ± standard error.
Day after
imbibition
0
1
2
3
4
L-Isoaspartyl methyltransferase
activity
(pmoles min ~' mg" 1 protein)
Endogenous
Total
0.60±0.01
O.55±O.O1
0.50±0.01
0.39 ±0.01
0.37±0.01
5.00 ±0.03
5.13±0.09
4.53±0.14
4.15 ±0.02
3.16±0.44
Germination
(%)
0
0
64
88
90
946
Kester et al.
Table 2. L-Isoaspartvl methvltransferase activity, germination percentage and rate for aged and non-aged tomato seeds primed with
KNO3 or PEG
Seeds were pnmed in 3% KNO 3 or PEG-8000 of equivalent osmotic potential (-1.25 MPa) for 7 d. Seed aging was for 4 d at 45 °C and 100%
humidity. Data for L-isoaspartyl methyltransferase activity are means ±standard error.
Treatment
Non-aged
Primed
Primed
Aged
Primed
Primed
(KNO 3 )
(PEG)
(K.NO 3 )
(PEG)
L-Isoaspartyl methyltransferase activity
(pmoles min"1 mg"1 protein)
Germination
MTG
(d)
6.0±0.36
4.8±0.32
5.7±0 21
1.1 ±0.01
5 2±0.13
2 7±0.36
95
95
90
74
73
76
2.4
1.4
3.6
5.8
3.0
and imbibed seeds that are natural substrates for this
enzyme, and that L-isoaspartyl methyltransferase might
be a critical component of the germination process,
repairing proteins required for the initial stages of
germination.
Seeds subjected to accelerated ageing showed a 21%
reduction in germination percentage germination and an
increase in the MTG to 5.8 d (Table 2). This was accompanied by a substantial decrease in L-isoaspartyl methyltransferase activity (Table 2; Fig. 1). Aged seeds showed
a linear decrease in both viability and L-isoaspartyl
methyltransferase activity over 4 d of ageing (Fig. 1).
Enzyme activity declined from 6.02 in non-aged seeds
to 0.99 pmol min" 1 mg" 1 protein in seeds aged for 4 d.
There was a strong positive correlation between enzyme
activity and germination percentage (7^ = 0.93), as well as
a strong negative correlation with MTG (r2 = 0.92). This
REGRESSION
•
y - 4 34(x) + 70 19 r2 -091
•
y = -066(x) + 597 Jr- 0 90
correlation between viability and vigour with L-isoaspartyl
methyltransferase activity suggests that L-isoaspartyl
methyltransferase activity is important in maintaining
seed quality. Although in this study naturally aged seeds
were not used, accelerated ageing has been used to make
seeds deteriorate quickly (Delouche and Baskin, 1973)
and accelerated ageing has been used previously to deteriorate tomato seeds for other physiological studies
(Argerich and Bradford, 1989).
The final germination percentage evaluated after 10 d
was not improved by any priming treatment (Table 2;
Fig. 2). However, priming with KN0 3 improved the
MTG in aged and non-aged seeds. PEG-8000 was not
effective in improving germination rate in tomato seeds,
similar to results from previous studies (Alvarado et al.,
1987; Argerich and Bradford, 1989). The improvement
in MTG but not germination percentage, has been
observed as the result of priming in many studies in
tomato as well as other species (Bradford, 1986). The
main benefit of priming in tomato seed is a reduction in
the time to germination (Karssen et al., 1989). This is
•
100
5.1
ion perceiitage
90
80
03
CO
T3
60
I
3
1
J
2 1
Davs of ageing
/ 0
L-isoaspartyl methyltransferase
pmol min "
mg protein
Fig. 1. Correlation between L-isoaspartyl methyltransferase activity,
germination percentage, and MTG for seeds deteriorated by accelerated
ageing 0 to 4 d at 45 C and 100% RH. Linear regression for
v = germination percentage ( • ) or >=mean days to germination ( • )
and x: = L-isoaspartyl methyltransferase activity
0
1
2
3
4
5
6
Days after imbibition
Fig. 2. The effect of seed priming with 3% K N 0 3 or PEG 8000 of
equivalent osmotic potential (-1.25 MPa) on percentage germination in
accelerated aged or non-aged tomato seed germinated at 30 °C over
10 d. Seeds were aged at 45 C and 100% RH for 4 d and then primed
for 7d at 20 CC. Data points are means ± standard error.
Priming and accelerated ageing effect
partly due to a reduction in the mechanical resistance
imposed on the radicle by endosperm tissue, and endo-j3mannanase activity was shown to be responsible for
alterations in cell wall properties associated with primingimproved germination (Haigh, 1988). Liptay and Zariffa
(1993) concluded that endosperm degradation and radicle
protrusion up to but not through the testa appeared to
be the result of priming in tomato. Primed tomato seeds
show increased synthesis of DNA (Bino et ai, 1992) and
ribosomal RNA (Coolbear and Grierson, 1979) prior to
germination. This indicates an increased capacity to synthesize new proteins during the early stages of germination
in primed seeds via accelerated turnover and replacement
of conserved messenger and ribosomal RNA.
Priming treatments did not affect L-isoaspartyl methyltransferase activity in non-aged seeds (Table 2). In aged
seeds L-isoaspartyl methyltransferase activity was reduced
from 6.0 to 1.1 pmol min" 1 mg - 1 protein and priming
947
with KNO3 restored enzyme activity to 5.2 pmol
m i n ^ m g " 1 protein, a level comparable to activity in
non-aged seeds. The MTG for germination for these seeds
was improved by 2.8 d over non-primed aged seeds. PEG
8000 priming was not as effective as priming with KN0 3
on either the rate of germination or increased L-isoaspartyl
methyltransferase activity (Table 2). It appears that an
increase in L-isoaspartyl methyltransferase activity cannot
be considered responsible for the priming effect seen in
non-aged seeds. However, this does not rule out a role
for this enzyme in protein repair during the extended lag
phase of priming. One of the proposed mechanisms for
priming is cellular repair during the lag period (Karssen
et al., 1989). In this study aged seed exhibited reduced Lisoaspartyl methyltransferase activity and priming either
provided a mechanism to recover enzyme activity or
initiated the synthesis of new enzyme.
The observation of L-isoaspartyl methyltransferase
kDa
200 —
116 —
97
66 —
45 —
31 —
21.5 —
B
14 —
C
D
Fig. 3. SDS-PAGE of tomato seed polypeptides labelled with [3H-methyl]AdoMet. Tomato seed proteins were electrophoresed on 12% denaturing
acrylamide gels and stained with Coomassie blue R-250 (panel A), or transferred to a PVDF membrane and radioactivity visualized at three
successive weekly intervals by Phosphorlmaging (panels B, C and D). Lane 1, molecular mass standards, myosin (200 kDa), /5-galactosidase
(116 kDa), phosphorylase B (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor
(21.5 kDa), and lysozyme (14kDa); lanes 2-5, polypeptides from tomato seed subjected to accelerated ageing for4d (lane 2), accelerated ageing
for 4d and subsequently primed with 3% KNO3 (lane 3), imbibition for 24h (lane 4), and no treatment (lane 5). Arrows show position of
25.6 kDa and 17.1 kDa radiolabelled polypeptides.
948 Kester et al.
activity in the absence of the synthetic polypeptide
substrate VYP-(L-isoAsp)-HA suggests the presence
of endogenous protein substrates for L-isoaspartyl
methyltransferase as mentioned earlier (Table 1). The
endogenous tomato seed proteins susceptible to
methylation at L-isoaspartyl residues might be represented
by a general collection of many seed-localized proteins,
or alternatively by only a select few. This latter possibility
might be expected if L-isoaspartyl protein methyltransferase activity forms part of a critical protein repair
process, essential for restoration of specific enzymatic
activities associated with seed germination. An examination of tomato seed polypeptides radiolabelled by [3Hmethyl]AdoMet revealed that only two polypeptides were
appreciably labelled in the crude extracts from tomato
seed (Fig. 3 panel B). The two polypeptides with estimated molecular masses of 25.6 kDa and 17.1 kDa were
only visible in extracts from imbibed and dry seed (Fig. 3,
panel B, lanes 4 and 5, respectively). Extracts from seed
which had been aged, or aged and primed, did not reveal
any radiolabelled polypeptides (Fig. 3, lanes 2 and 3,
respectively). This could be a consequence of the loss of
endogenous protein substrates, or excessive loss of tritium
label during SDS-PAGE and electrophoretic transfer to
PVDF membranes under the alkaline conditions used.
That the incorporation of 3H from [3H-methyl]AdoMet
into tomato seed polypeptides was via base-labile carboxy-linkages is evident by the slow loss in radiolabel
signal during repeated imaging over several weeks,
especially after temporary incubation in an alkaline buffer
(Fig. 3, panels C and D). While the identity of the
radiolabeUed tomato seed polypeptides remains unknown,
automethylation of protein (D-aspartyl/L-isoaspartyl)
carboxy methyltransferase has been reported (Lindquist
and McFadden, 1994) and the molecular mass of wheat
L-isoaspartyl protein methyltransferase is 24.7 kDa
(Mudgett and Clarke, 1993), similar to the 25.6 kDa
radiolabelled tomato polypeptide. Additionally, the radiolabelled tomato polypeptide at a molecular mass of
17.1 kDa is similar in mass to calmodulin (17 kDa), a
known substrate for protein carboxyl methyltransferase
(Johnson et al., 1989). The base lability of the methyl
esters formed by carboxy-methylation of L-isoaspartyl
residues can be minimized by performing SDS-PAGE at
pH2.4 (Mudgett and Clarke, 1993; Fairbanks and
Avruch, 1972). However, fractionation of tomato seed
proteins using SDS-PAGE at pH 2.4 was not satisfactory
and resulted in poor resolution and separation of tomato
seed proteins (data not shown). Thus, the profile in Fig. 3
of [3H-methyl]AdoMet radiolabel incorporation into
tomato seed proteins is conservative, and other seedspecific proteins may have been labeled but are not
observable under the conditions described here.
This system affords an excellent model with which to
study the basic mechanisms underlying the loss of enzyme
activity during seed deterioration, and the recovery of
that activity during priming by an enzymatic protein
repair activity highly correlated with seed germination.
Acknowledgements
This research was supported
Department of Energy Grant
Hatch Project KY00590 to RL
lished as Kentucky Agricultural
No. 96-11-102.
in part by United States
DE-FGO5-92ER2OO75 and
Houtz. This article is pubExperiment Station Article
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