Tree Physiology 27, 519–525
© 2007 Heron Publishing—Victoria, Canada
Alleviation of dormancy in walnut kernels by moist chilling is
independent from storage protein mobilization
ALI REZA EINALI1 and HAMID REZA SADEGHIPOUR1,2
1
Department of Biology, College of Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
2
Corresponding author (sadeghipour@gau.ac.ir)
Received February 20, 2006; accepted May 23, 2006; published online January 2, 2007
Summary We studied the effects of moist chilling and
warming on storage protein mobilization in walnut (Juglans
regia L.) kernels to assess the metabolic inhibition theory,
which states that dormant seeds are unable to utilize their own
food reserves and that cold conditions allow germination by ac-
tivating hydrolases involved in reserve mobilization. Stratify-
ing kernels at 5 °C for 40 days enhanced their germination.
During both cold stratification and warm incubation of kernels,
storage protein mobilization occurred in cotyledons rather than
axes. Kernel amino acid concentration increased during pro-
tein mobilization, with axes of warm-incubated kernels having
particularly high amino acid concentrations. Polyacrylamide
gel electrophoresis in the presence of sodium dodecyl sulfate
(SDS-PAGE) of the soluble protein fractions from both cold-
stratified and warm-incubated cotyledons revealed increased
band intensities of putative glutelins (19–22 and 32–35 kDa).
A very high molecular weight protease was detected by gelatin
SDS-PAGE that was most active at acid to neutral pHs in im-
bibed, cold stratified and germinated kernels suggesting the
protease(s) was synthesized earlier in the mature seeds. Thus,
in dormant walnut kernels there is no block to protein mobiliza-
tion, and imbibition alone is sufficient to initiate proteolysis.
Catalase activity was higher in warm-incubated kernels than in
cold-stratified kernels, suggesting that seed aging is hastened
under warm conditions and that cold stratification in walnut
kernels might involve activation of cellular repair mechanisms.
Keywords: amino acids, catalase, glutelin, Juglans regia, pro-
tease, stratification.
Introduction
Seed dormancy refers to a situation in which germination can-
not proceed despite favorable conditions. Breaking of dor-
mancy in some seeds is achieved by incubating them in moist
and cold (5 °C) conditions, a process termed cold stratifica-
tion. The nature of the cold-induced changes involved in the
alleviation of dormancy in stratified seeds is not well under-
stood.
Changes in the concentrations of phytohormones or changes
in embryo sensitivity to phytohormones, or both (Corbineau et
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al. 2002, Jacobsen et al. 2002, Schmitz et al. 2002), and
changes in enzymatic activities involved in seed reserve mobi-
lization and gluconeogenesis (Li and Ross 1990a, 1990b,
Ranjan and Lewak 1995, Bogatek et al. 2002) have been re-
ported. Seed storage proteins are also affected by cold stratifi-
cation. Mobilization of storage proteins is an important event
for seed germination because it provides amino acids for the
de novo synthesis of germination-specific proteins (Rajjou et
al. 2004). Ultrastructural studies have revealed the gradual
mobilization of protein bodies in the storage tissues of stratify-
ing seeds (Dawidowicz-Grzegorzewska 1989, Wang and
Berjak 2000). Increases in enzymatic activities related to pro-
teolysis along with the accumulation of free amino acids in
seeds during cold stratification have also been reported
(Zarska-Maciejewska and Lewak 1983, Ranjan and Lewak
1995, King and Gifford 1997, Stone and Gifford 1997,
Forward et al. 2001, Todd et al. 2001).
Based on the many studies described in the literature, the
metabolic inhibition theory was proposed (Ross 1984, Lewak
et al. 2000). This theory states that dormant seeds are metabol-
ically inhibited and are unable to utilize their own food re-
serves and that cold condition allows germination by remov-
ing this inhibition. The unequivocal acceptance of the meta-
bolic inhibition theory demands further studies on more
species.
Persian walnut (Juglans regia L.) is an important nut tree
species from temperate regions. Storage proteins constitute
17% of food reserves in the walnut kernel (Sze-Tao and Sathe
2000). For optimal germination, the nuts require stratification
for between 1 and 3 months. In the present study, walnut was
taken as a model system for examining the biochemical pro-
cesses involved during cold stratification in the context of the
metabolic inhibition theory.
Material and methods
Plant material, stratification protocol and germination
studies
Freshly harvested nuts of Persian walnut (Juglans regia L.)
were procured from the Gorgan Office of Natural Resources
during October 2003 and 2004. After imbibing the nuts in tap
520 EINALI AND SADEGHIPOUR
water for 24 h, nuts were surface sterilized with 0.5% (w/v)
sodium hypochlorite solution for 15 min followed by four
washes with distilled water. To stratify the nuts, batches of
25 nuts (in triplicate) were wrapped in four layers of moist-
ened cheesecloth, placed in polyethylene bags and incubated
at 5 °C for periods up to 60 days. Every 10 days, triplicate
batches of stratified nuts were transferred to a well-irrigated
sand medium at 27 °C, and their germination recorded for a
period of 40 days. Kernels with a radicle length of about
10 mm were evident as bulges at the sand surface and were
considered germinated. Surface-sterilized, imbibed nuts kept
at 27 °C (non-stratified control) in a well-irrigated sand me-
dium served as non-stratified controls (hereafter called warm-
incubated nuts). Germination of the warm-incubated kernels
was recorded for a period of 40 days. Kernels were isolated
from both cold-stratified and warm-incubated nuts that
showed no signs of germination. The axis and cotyledons were
excised from each kernel with a razor blade and used for
subsequent biochemical analyses.
Total protein and amino acid determinations
For extraction of total protein, cotyledonary or axial tissue
(1 g) was defatted as described by Hara and Radin (1978). The
defatted powder (20 mg) was extracted in 0.5 ml of 60 mM
Tris-HCl buffer (pH 6.8) containing 10% (v/v) glycerol and 2
% (w/v) SDS (Stone and Gifford 1997) at 90 °C for 1 h. The
mixture was centrifuged at 10,000 g for 15 min and the clear
supernatant was used for total protein determination. Because
of interference by SDS in the Bradford (1976) protein assay,
the Markwell et al. (1981) method of protein determination
was used. Protein concentration was expressed per gram dry
mass of tissue.
For amino acid extraction, tissue pieces (1 g) from cotyle-
dons or embryo axes were ground with 5 ml of 80% (v/v) etha-
nol at 70 °C. After centrifugation at 3500 g for 10 min the
supernatant was saved and the pellet was re-extracted four
times with 80% ethanol. The extracts were pooled and reduced
to one fifth of their original volume by evaporation. The con-
centrated extract (5 ml) was then mixed with 1 ml of chloro-
form to partition pigments and lipids from the aqueous phase.
Total amino acid concentration in the aqueous phase was
quantified by the ninhydrin method of Yemm and Cocking
(1955) and expressed per gram dry mass of tissue.
Soluble protein extraction and analysis by SDS-PAGE
Soluble protein was extracted from kernels by grinding the tis-
sue (1 g) in a cold mortar with a pestle in 2 ml of grinding
buffer (100 mM Tris-HCl (pH 7.5), 0.4 M sucrose, 10 mM
KCl, 1 mM MgSO4, 1 mM EDTA, 1 mM PMSF, 0.6% (w/v)
PVPP, and 50 mM 2-mercaptoethanol). The homogenate was
filtered through four layers of cheese-cloth and the filtrate was
centrifuged at 10,000 g for 15 min. The supernatant, hereafter
called the soluble protein fraction, was assayed for protein by
the Bradford (1976) method. Aliquots of the supernatant were
separated by polyacrylamide gel electrophoresis in the pres-
ence of sodium dodecyl sulfate (SDS-PAGE) on a 15% resolv-
TREE PHYSIOLOGY VOLUME 27, 2007
ing gel as described by Fling and Gregerson (1986). After
electrophoresis, the gels were stained for proteins with Coo-
massie Brilliant Blue R-250.
Zymographic detection of kernel protease(s)
For zymographic detection of kernel protease(s), the soluble
protein fraction was prepared as described above except that
PMSF was excluded from the grinding buffer. Soluble protein
samples (100 µg) were separated by SDS-PAGE on a 12.5%
resolving gel containing 0.2% (w/v) gelatin as a protease sub-
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strate (Heussen and Dowdle 1980). Following electrophoresis
(70 V, 7 °C), the resolved protein bands were renatured by
transferring the gels to renaturing buffer (50 mM Tris (pH 7.5)
containing 2% (v/v) Triton X-100) for 30 min at 37 °C. Gels
were subsequently transferred to reaction buffer (50 mM Tris,
pH 7.5, or other buffers as indicated, containing 0.1% (v/v)
2-mercaptoethanol) for 3 h at 37 °C. Protease band(s) were vi-
sualized as transparent gelatin-free zones after staining with
0.1% (w/v) Amido Black in methanol:acetic acid:water
(3:1:6; v/v) followed by de-staining in methanol:acetic
acid:water (1.5:0.5:8; v/v).
Catalase extraction and assay
Catalase was extracted from cotyledonary and axial tissues as
described by Jordy et al. (2000) with minor modifications. Tis-
sue (1 g) was ground in a cold mortar with a pestle in 2 ml of
grinding buffer containing 0.1% (v/v) Triton X-100. The ho-
mogenate was filtered through four layers of cheese-cloth and
incubated at 4 °C for 1 h. The filtrate was then centrifuged at
10,000 g for 15 min and the resulting supernatant was assayed
for catalase activity according to the method of Luck (1962).
The reaction mixture in a final volume of 3 ml consisted of
50 mM phosphate buffer (pH 7), 12.5 mM H2O2 and enzyme
extract. The breakdown of H2O2 was recorded as a decrease in
absorbance at 240 nm, assuming an extinction coefficient (ε)
of 0.036 cm2 µmol – 1. Catalase activity was expressed as µmol
H2O2 consumed per minute per gram fresh mass of tissue.
Statistical analysis
Statistically significant differences at the 5% level were deter-
mined by the Duncan method.
Results
Effect of cold stratification on germination of walnut kernels
Non-stratified kernels that were imbibed for 40 days at 27 °C
showed germination percentages up to 20%. Cold stratifica-
tion increased both percentage and rate of walnut kernel ger-
mination (Figure 1). Germination percentage increased in ker-
nels cold-stratified for more than 20 days and the highest ger-
mination percentage (50%) and highest rate of germination
occurred in kernels that had been cold stratified for 40 days.
 PROTEIN MOBILIZATION IN STRATIFYING WALNUT KERNELS
Figure 1. Effect of cold stratification on germination percentage (䊏)
and germination rate (䉫) of walnut kernels. Every 10 days for a period
of 60 days, nuts were imbibed for 24 hours, surface sterilized, incu-
bated at 5 °C and then placed in a well-irrigated sand medium at
27 °C. Germination was recorded for a period of 40 days. Each value
is the mean ± SE of triplicate experiments each consisting of 25 nuts.
Changes in concentrations of total protein and amino acids
in stratified and warm-incubated walnut kernels
Total protein refers to proteins extracted in the presence of
SDS. Although SDS detergent cannot extract all proteins, the
yield of walnut proteins extracted in buffer containing SDS
was 10-fold higher than the amount of protein extracted in
buffer without SDS (data not shown). The total protein con-
centration of cotyledons decreased by about 40% during the
first 20 days of cold stratification, but the concentration re-
mained relatively constant during the remaining 40 days of
cold stratification (Figure 2A). The total amino acid concen-
tration of cotyledons increased slightly but not significantly
during cold stratification. Cold-stratification also affected pro-
tein and amino acid concentrations of embryonic axes (Fig-
ure 2B). Total protein concentration of the embryonic axes de-
creased during stratification and the greatest reduction oc-
curred in axes that had been stratified for 20 days. Axis amino
acid concentration increased from an initial concentration of
–1 –1
3.3 µmol g DW in imbibed axes to 8.4 µmol g DW in axes cold
stratified for 10 days, and thereafter it remained roughly un-
changed.
Most non-stratified kernels incubated at 27 °C retained
white, sound-looking tissue for the first 16 to 20 days of warm
incubation, but thereafter many of the kernels turned rancid;
however, 20% of the warm-incubated kernels remained
healthy and germinated. Warm-incubated kernels that did not
show any morphological sign of germination were selected ev-
ery four days from the start of the warm-incubation period and
analyzed biochemically. Figure 3 shows the changes in total
protein and amino acid concentrations of cotyledons and axes
from warm-incubated kernels. Protein concentration of the
cotyledons decreased about 40% within the first 4 days of
warm incubation, but thereafter the cotyledonary protein con-
tent did not change significantly (Figure 3A). The concentra-
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Figure 2. Changes in total protein (䊏) and amino acid (䉫) concentra-
tions in cotyledons (A) and axes (B) during cold stratification of wal-
nut kernels. Each value is the mean ± SE of three separate extractions.
tion of amino acids in the cotyledons of warm-incubated ker-
nels was initially low but increased, reaching a maximum of
12.6 µmol g –1
DW after 12 days of warm incubation and thereafter
remained statistically unchanged. Despite the lack of signifi-
cant protein mobilization in axes from warm-incubated ker-
nels (Figure 3B), their amino acid concentration increased sig-
nificantly, peaking at 36 µmol g –1
DW at 16 days of warm incuba-
tion.
SDS-PAGE analyses of proteins from cold-stratified and
warm-incubated walnut kernels
The SDS-PAGE analyses of the total proteins extracted from
kernels with SDS-containing Tris buffer revealed no signifi-
cant differences in the protein profiles of the cold-stratified
and warm-incubated kernels (data not shown). In contrast, dif-
ferences in the soluble protein (i.e., proteins soluble in Tris
buffer without SDS) profiles of the cold-stratified and
warm-incubated kernels were detected by SDS-PAGE (Fig-
ures 4 and 5). Cold stratification and warm incubation of ker-
nels caused qualitative changes in the soluble protein profile of
the cotyledons but had little effect on the protein profile of the
axes (Figures 4 and 5). Polypeptides with molecular masses of
19–22, 32–35 and 42–49 kDa, which are representatives of
the major walnut kernel storage proteins, displayed increased
band intensities in cotyledons as incubation proceeded (Fig-
ures 4A and 5A). The band intensities of some cotyledonary
proteins, e.g., a 37 kDa polypeptide, declined during cold
stratification and it was undetectable after 4 days of warm in-
522 EINALI AND SADEGHIPOUR
Figure 3. Changes in total protein (䊏) and amino acid (䉫) concentra-
tions in cotyledons (A) and axes (B) of warm-incubated walnut ker-
nels. The bars show standard errors of the means of total protein and
amino acid measurements from three separate tissue samples.
cubation. In contrast, there were no apparent changes in the
band intensities of polypeptides with molecular masses of
19–22, 32–35 and 42–49 kDa in the axes during either cold
stratification or warm incubation (Figures 4B and 5B).
Zymographic detection of protease activity in walnut kernels
Use of a gelatin SDS-PAGE system to detect protease(s) likely
involved in protein mobilization in walnut kernels, led to the
TREE PHYSIOLOGY VOLUME 27, 2007
identification of a slowly migrating high molecular mass pro-
tease in imbibed, cold-stratified, as well as germinating, ker-
nels (Figure 6). The activity of this protease was detectable at
acid to neutral pHs with the greatest activity occurred at neu-
tral pHs. No activity was recorded at alkaline pHs (data not
shown). Proteases with molecular masses of 83 and 89 kDa
were detected in germinating kernels but not in imbibed or
cold-stratified kernels.
Catalase activity in cold-stratified and warm-incubated
walnut kernels
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Catalase activity started to increase in cotyledons following
exposure of kernels to cold conditions (Figure 7A) and a
nearly continuous increase in catalase activity occurred
throughout the stratification period. Catalase activity in the
axes, however, did not increase until the kernels had been cold
stratified for 40 days, but a significant increase in enzymatic
activity occurred thereafter. In warm-incubated kernels, cata-
lase activity developed rapidly within the first four days of in-
cubation in both cotyledons and axes (Figure 7B). Catalase ac-
tivity then remained nearly unchanged in both organs till
12 days of warm incubation when there was a significant in-
crease in enzymatic activity in the axes that peaked at 80 µmol
min – 1 g –FW
1
in 16–20-day-old axes.
Discussion
The germination percentage of warm-incubated walnut ker-
nels was up to 20%, demonstrating that they are not deeply
dormant; however, cold stratification of the kernels enhanced
their germination to about 55%. Similar positive effects of
cold stratification on the germination percentage have been re-
ported for Corylus avellana L. (Andriotis et al. 2004) and
Pyrus serotina Rehd. seeds (Lin et al. 1994). Germination of
walnut kernels may not depend solely on the physiological
state of the embryo: the tissues surrounding the embryo may
Figure 4. Changes in the SDS-PAGE
pattern of soluble proteins extracted
from cotyledons (A) and axes (B) of
cold stratified walnut kernels. Soluble
proteins were extracted in Tris buffer
pH 7.5 without added SDS. Protein
samples (100 µg) were separated on a
15% resolving gel and subsequently
stained with Coomassie Brilliant Blue
R-250.
 PROTEIN MOBILIZATION IN STRATIFYING WALNUT KERNELS
also affect the germination process, as has been documented
for some other stratification-requiring seeds such as Chamae-
cyparis nootkatensis D. Don Spach (Ren and Kermode 1999).
Cold stratification of walnut kernels led to protein mobiliza-
tion and amino acid accumulation in both cotyledons and em-
bryonic axes (Figure 2). Protein mobilization in response to
cold stratification has been reported for several other species
(Ching 1968, Dawidowicz-Grezegorezewska 1989, King and
Gifford 1997). Because the mobilization of seed reserves and
Figure 6. Zymographic detection of protease activity in walnut ker-
nels by gelatin SDS-PAGE. Cotyledonary soluble proteins from im-
bibed (Lane 1), 20-day-stratified (Lane 2) and 10-day-germinated
(Lane 3) kernels were resolved on a 12.5% polyacrylamide gel con-
taining 0.2% gelatin. After renaturation of the resolved proteins at pH
7.5 in the presence of Triton X-100, the gels were incubated in reac-
tion buffers at pH 5.0 (A) and pH 7.5 (B) for 3 h at 37 °C. Transparent
gelatin-free zones representing protease activity were visualized by
staining gels with Amido Black.
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Figure 5. Changes in the SDS-PAGE
pattern of soluble proteins extracted
from cotyledons (A) and axes (B) of
warm-incubated walnut kernels. Solu-
ble proteins were extracted in Tris
buffer pH 7.5 without added SDS. Pro-
tein samples (100 µg) were separated
on a 15% resolving gel and subse-
quently stained with Coomassie Bril-
liant Blue R-250.
their subsequent metabolism in response to cold treatment oc-
curs primarily in embryonic axes rather than cotyledons, the
former organ is postulated to perceive the cold stimulus
(Dawidowicz-Grezegorzewska 1989, Bogatek et al. 1989, Li
and Ross 1990a, 1990b, Zarska-Maciejewska 1992, Andriotis
et al. 2004). However, the extent of protein mobilization was
similar in the embryonic axes and cotyledons of the stratifying
walnut kernels and so no cold-perceiving organ could be iden-
tified.
Most studies have shown a partial or complete absence of
reserve mobilization in stratification-requiring seeds main-
tained under warm conditions (Noland and Murphy 1984,
Dawidowicz-Grzegorzewska 1989, Li and Ross 1990a, An-
driotis et al. 2004); however, we found greater protein mobili-
Figure 7. Time course of changes in catalase activity in cotyledons
(䊏) and axes (䉫) from cold-stratified (A) and warm-incubated (B)
walnut kernels. Values are means ± SE obtained from three separate
extractions.
524 EINALI AND SADEGHIPOUR
zation in moistened warm-incubated walnut kernels than in
cold-stratified kernels (Figures 3A and 3B). Although the ex-
tent of protein mobilization was greater in cotyledons than in
axes under warm conditions, the axes accumulated high con-
centrations of free amino acids, up to 36 µmol g –1
DW . We suggest
that transport of amino acids from cotyledons to axes and the
failure of warm-incubated axes to metabolize amino acids
could account for this high concentration of amino acids. Evi-
dence in support of this suggestion comes from a study show-
ing that cold stratification increased the capacity of Acer
saccharum Marsh. embryo axes for protein synthesis, whereas
non-stratified warm-incubated axes had a low capacity for
protein synthesis (Hance and Bevington 1992).
The SDS-PAGE analyses revealed changes in the patterns of
cotyledonary soluble proteins during both cold stratification
and warm incubation (Figures 4A and 5A). In contrast, in
Malus domestica Borkh. seeds, major changes in the pattern of
soluble proteins were observed in axes rather than in cotyle-
dons and only during cold stratification (Eichholtz et al.
1983). Major polypeptides in the total soluble protein fraction
of walnut kernels had molecular masses of 19–22 and 32–
35 kDa (Figures 4 and 5). They represent putative walnut
glutelins (Sze-Tao and Sathe 2000), and those with molecular
masses of 42–49 kDa represent putative vicilins (Teuber et al.
1999). The increased band intensities of the putative glutelins
and vicilins in cotyledons might be attributed to the increased
solubility of these storage proteins as has been reported to oc-
cur during protein mobilization in germinating cereals (Ko-
brehel et al. 1992, Yano et al. 2001).
The gelatin SDS-PAGE technique revealed a high molecu-
lar mass protease most active at acidic to neutral pHs, along
with two proteases with molecular masses of 83 and 89 kDa
that were detected in germinating kernels but not in imbibed or
cold-stratified kernels (Figure 6). As both seed stored and ger-
mination-specific proteases have been reported previously
(Muntz et al. 2001), the high molecular mass protease and the
83 and 89 kDa proteases in walnut kernels might be seed
stored and germination-specific proteases, respectively, in-
volved in protein mobilization. High molecular mass proteases
most active at neutral to alkaline pHs have also been reported
in stratifying seeds of Pseudotsuga menziesii Franco (Forward
et al. 2001).
The current concept of tree seed dormancy removal by cold
stratification is based on the metabolic inhibition theory which
implies that reserve mobilization is inhibited in dormant seeds
and that cold treatment allows reserve mobilization to proceed
as a prerequisite for the germination process (Ross 1984,
Lewak et al. 2000). Evidently, in dormant walnut kernels there
is no block to storage protein mobilization because imbibition
alone is sufficient to initiate storage protein mobilization.
However, cold stratification may enhance the metabolism of
amino acids, e.g., arginine (King and Gifford 1997) by prefer-
entially increasing proteolysis and releasing amino acids to the
metabolic pathways needed for germination.
The higher catalase activity in both cotyledons and axes of
warm-incubated walnut kernels compared with stratified ker-
nels (Figures 7A and 7B) suggests that warm conditions en-
TREE PHYSIOLOGY VOLUME 27, 2007
hance seed aging. Increased catalase activity has also been re-
ported in warm-incubated Corylus avellana kernels and
attributed to the physiological process of seed aging (Li and
Ross 1990a). As suggested earlier for Picea mariana Mill.
seeds (Wang and Berjak 2000), cold stratification in walnut
kernels might also involve activation of repair mechanisms
that prevent seed aging.
Acknowledgments
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We thank the GUASNR Deputy of Research and Office of Higher Ed-
ucation for financial support to A.R.Einali in the form of grants for
M.Sc. research projects.
References
Andriotis, V.M.E., S.B. Smith and J.D. Ross. 2004. Phytic acid mobi-
lization is an early response to chilling of the embryonic axes from
dormant oilseed of hazel (Corylus avellana L.). J. Exp. Bot. 56:
537–545.
Bogatek, R., B. Zarska-Maciejewska, I. Sinska and S. Lewak. 1989.
The embryonic axis controls lipid catabolism in cotyledons of ap-
ple seeds during germination. Physiol Plant. 76:557–562.
Bogatek, R., D. Come, F. Corbineau, R. Ranjan and S. Lewak. 2002.
Jasmonic acid affects dormancy and sugar catabolism in germinat-
ing apple embryos. Plant Physiol. Biochem. 40:167–173.
Bradford, M. 1976. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of pro-
tein-dye binding. Anal. Biochem. 72:248–254.
Ching, T.M. 1968. Intracellular distribution of lipolytic activity in the
female gametophyte of germinating Douglas fir seeds. Lipids 3:
482–488.
Corbineau, F., J. Bianco, G. Garello and D. Come. 2002. Breakage of
Pseudotsuga menziesii seed dormancy by cold treatment as related
to changes in seed ABA sensitivity and ABA levels. Physiol. Plant.
114:313–319.
Dawidowicz-Grzegorzewska, A. 1989. Degradation of protein and
lipid bodies during dormancy removal in apple seeds. J. Plant
Physiol. 135:43–51.
Eichholtz, D.A., H.A. Robitaille and K.M. Herrmann. 1983. Protein
changes during the strafication of Malus domestica Borkh. seeds.
Plant Physiol. 72:750–753.
Fling, S.P. and D.S. Gregerson. 1986. Peptide and protein molecular
weight determination by electrophoresis using a high-molarity Tris
buffer system without urea. Anal. Biochem. 155:83–88.
Forward, B.S., T.J. Tranbarger and S. Misra. 2001. Characterization
of proteinase activity in stratified Douglas-fir seeds. Tree Physiol.
21:625–629.
Hance, B.A. and J.M. Bevington. 1992. Changes in protein synthesis
during stratification and dormancy release in embryos of sugar ma-
ple (Acer saccharum). Physiol. Plant. 86:365–371.
Hara, A. and N.S. Radin. 1978. Lipid extraction of tissues with a
low-toxicity solvent. Anal. Biochem. 90:420–426.
Heussen, C. and E.B. Dowdle. 1980. Electrophoretic analysis of
plasminogen activators in polyacrylamide gels containing sodium
dodecyl sulphate and copolymerized substrates. Anal. Biochem.
102:196–202.
Jacobsen, J.V., D.W. Pearce, A.T. Poole, R.P. Pharis and L.N. Man-
der. 2002. Abscisic acid, phaseic acid and gibberellin contents as-
sociated with dormancy and germination in barley. Physiol. Plant.
115:428–441.
 PROTEIN MOBILIZATION IN STRATIFYING WALNUT KERNELS
Jordy, M.N., S. Danti, J.M. Favre and M.L. Racchi. 2000. Histo-
logical and biochemical changes in Pinus spp. seeds during germi-
nation and post-germinative growth: triacylglycerol distribution
and catalase activity. Aust. J. Plant. Physiol. 27:1109–1117.
King, J.E. and D.J. Gifford. 1997. Amino acid utilization in seeds of
loblolly pine during germination and early seedling growth:
I. Argenin and arginase activity. Plant Physiol. 113:1125–1135.
Kobrehel, K., J.H. Wong, A. Balogh, F. Kiss, B.C. Yee and B.B. Bu-
chanan. 1992. Specific reduction of wheat storage proteins by
thioredoxin h. Plant Physiol. 99:919–924.
Lewak, S., R. Bogatek and B. Zarska-Maciejewska. 2000. Sugar me-
tabolism in apple embryos. In Dormancy in Plants. Eds. J.D. Vie-
mont and J. Crabbe. CAB International, Wallingord, U.K., pp
47–55.
Li, L. and J.D. Ross. 1990a. Lipid mobilization during dormancy
breakage in oilseed of Corylus avellana. Ann. Bot. 66:501–505.
Li, L. and J.D. Ross. 1990b. Starch synthesis during dormancy break-
age in oilseed of Corylus avellana. Ann. Bot. 66:507–512.
Lin, C-H., L-Y. Lee and M-J. Tseng. 1994. The effect of stratification
and thidiazuron treatment on germination and protein synthesis of
Pyrus serotina Rehd cv. Niauli. Ann. Bot. 73:515–523.
Luck, H. 1962. Methods of enzymatic analysis. 1st Edn. Ed.
H.U. Bergmeyer. Verlage Chemie, Weinheim, pp 885–894.
Markwell, M.A.K., S.M. Hass, N.E. Tolbert and L.L. Bieber. 1981.
Protein determination in membrane and lipoprotein samples: man-
ual and automated procedures. Meth. Enzymol. 72:296–303.
Muntz, K., M.A. Belozersky, Y.E. Dunaevsky, A. Schlereth and
J. Tiedemann. 2001. Stored proteinases and the initiation of storage
protein mobilization in seeds during germination and seedling
growth. J. Exp. Bot. 52:1741–1752.
Noland, T.L. and J.B. Murphy. 1984.Changes in isocitrate lyase activ-
ity and ATP content during stratification and germination of sugar
pine seeds. Seed Sci. Technol. 12:777–787.
Rajjou, L., K. Gallardo, I. Debeaujon, J. Vandekerckhove, C. Job and
D. Job. 2004. The effect of α-amanitin on the Arabidopsis seed
proteome highlights the distinct roles of stored and neosynthesized
mRNA during germination. Plant Physiol. 134:1598–1613.
Ranjan, R. and S. Lewak. 1995. Interaction of jasmonic and abscisic
acid in the control of lipase and proteases in germinating apple em-
bryos. Physiol. Plant. 93:421–426.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
525
Ren, C. and A.R. Kermode. 1999. Analyses to determine the role of
the megagametophyte and other seed tissues in dormancy mainte-
nance of yellow cedar (Chamaecyparis nootkatensis) seeds: mor-
phological, cellular and physiological changes following moist
chilling and during germination. J. Exp. Bot. 50:1403–1419.
Ross, J.D. 1984. Metabolic aspects of dormancy. In Seed Physiol-
ogy,Vol.2. Germination and Reserve Mobilization. Ed. D.R. Mur-
ray. Academic Press, New York, pp 45–75.
Schmitz, N., S.R. Abrams and A.R. Kermode. 2002. Changes in ABA
turnover and sensitivity that accompany dormancy termination of
yellow-cedar (Chamaecyparis nootkatensis) seeds. J. Exp. Bot.
53:89–101.
Downloaded from https://academic.oup.com/treephys/article-abstract/27/4/519/1666100 by guest on 09 May 2020
Stone, S.L. and D.J. Gifford. 1997. Structural and biochemical
changes in loblolly pine (Pinus taeda L.) seeds during germination
and early seedling growth: I.Storage protein reserves. Int. J. Plant
Sci. 158:727–737.
Sze-Tao, K.W.C. and S.K. Sathe. 2000. Walnuts (Juglans regia L.):
Proximate composition, protein solubility, protein amino acid com-
position and protein in vitro digestibility. J. Sci. Food Agric.
80:1393–1401.
Teuber, S.S., K.C. Jarvis, A.M. Dandekar, W.R. Peterson and
A.A. Ansari. 1999. Identification and cloning of a complementary
DNA encoding a vicilin-like proprotein, Jug r2, from English wal-
nut kernel (Juglans regia L.), a major food allergen. J. Allergy Clin.
Immunol. 104:1311–1320.
Todd, C.D., J.E.K. Cooke, R.T. Mullen and D.J. Gifford. 2001. Regu-
lation of loblolly pine (Pinus taeda L.) arginase in developing seed-
ling tissue during germination and post-germinative growth. Plant
Mol. Biol. 45:555–565.
Wang, B.S.P. and P. Berjak. 2000. Beneficial effects of moist chilling
on the seeds of black spruce (Picea mariana [Mill.] B.S.P.). Ann.
Bot. 86:29–36.
Yano, H., J.H. Wong, M-J. Cho and B.B. Buchanan. 2001. Redox
changes accompanying the degradation of seed storage proteins in
germinating rice. Plant Cell Physiol. 42:879–883.
Yemm, E.W. and E.C. Cocking. 1955. The determination of amino ac-
ids with Ninhydrin. Analyst 80:209–213.
Zarska-Maciejewska, B. and S. Lewak. 1983. The role of proteolytic
enzymes in the release from dormancy of apple seeds. Z. Pflan-
zenphysiol. 110:409–417.
Zarska-Maciejewska, B. 1992. Lipolytic activity during dormancy re-
moval in apple seeds. Plant Physiol. Biochem. 30:65–70.