Vol.2, No.7, 737-740 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.27092
Characterization of seed storage protein patterns of four
Iranian Pistachios using SDS-PAGE
Ali Akbar Ehsanpour*, Behrokh Shojaie, Fatemeh Rostami
Department of Biology, Faculty of ◌
ُScience, University of Isfahan, Isfahan, Iran; *Corresponding Author:
Received 6 March 2010; revised 10 April 2010; accepted 15 April 2010.
ABSTRACT
We used SDS-PAGE to evaluate and character-
ize the protein patterns of seed storage proteins
in four pistachios cultivars (Akbari, Ahmad
Aghaei, Fandoghi, and Kaleghouchi). Total pro-
tein content of pistachio seeds in all cultivars
did not show any significant difference. Results
of SDS PAGE pattern of a few protein bands
were up regulated whereas some other bands
showed down regulation. The identified protein
patterns may be used protein marker for pista-
chio cultivars.
Keywords: Pistachio; Protein Marker; SDS PAGE
1. INTRODUCTION
Pistacia vera L. (pistachio) (2n = 32) is belong to Ana-
cardiaceae family. Iran is one of the largest producer and
exporter of pistachio (300,000 tones per year) in the
world [1]. Pistachio seed is a very high source of protein,
lipid, and vitamins such as vitamin A, B1, B2C, and
Niacin. It has also a high percentage of potassium, cal-
cium and phosphorous. Iranian pistachios are different in
sizes and shape and they are divided into four major
groups including round (Fandoghi), jumbo (Kaleghouchi)
and long (Akbari, Ahmad Aghaei) (http://en.wikipedia.
org/wiki/Pistachio and http://www.sahravi.co m/pistachio
/iranian-pistachio.htm).
Plant storage proteins can be classified into two
classes; seed storage proteins (SSPs) and vegetative
storage proteins (VSPs). SSPs are a set of proteins that
accumulate at high levels in seeds during the late stages
of seed development, whereas VSPs are proteins that
accumulate in vegetative tissues such as leaves, stems
and tubers, depending on the plant species. SSP genes
were classic targets for work on plant molecular biology.
Their abundant expression in seeds allowed for easy
detection of the gene transcripts and cDNA cloning dur-
ing research on plant molecular biology in late 70’s to
early 80’s. Characterization of germplasm using bio-
chemical fingerprinting has got special attention due to
its increased used in crop improvement and the selection
of desirable genotypes for breeding crops. The use of
genetic markers and protein profiling has also been suc-
cessfully used to resolve the taxonomic and evolutionary
problems of several crop plants [2-6]. The seed storage
protein analyses helps in identification and characteriza-
tion of diversity in crop varieties, cultivars and their wild
varieties and also provides information on phylogenetic
relationship of the accessions. It is also known that
variation in protein bands provide information on the
relationship among the used seeds collected from vari-
ous geographical regions [6-8]. There are different
amounts of storage proteins in all plant seeds. They play
two main roles including nitrogen and energy source and
defense against insects and pathogens such as bacteria
and fungi.
Since, seed storage protein analysis can be a useful
tool for identification of species, varieties and cultivars,
in this study we investigate the protein pattern in four
Iranian pistachios seeds (Akbari, Ahmad Aghaei, Fan-
doghi, Kaleghouchi) in order to find protein bands as
markers for cultivar characterization.
2. MATERIALS AND METHODS
2.1. Plant Material
Fresh mature seeds of Pistachio cultivars including
Akbari, Ahmad Aghaei, Fandoghi, and Kaleghouchi
were harvested from pistachio garden in Ardestan,
Isfahan, Iran.
2.2. Extraction of Seed Proteins
Seed coats from fresh ten seeds from each cultivar were
removed and kernels were then grounded in liquid ni-
trogen with a mortar and pestle. The seed storage pro-
teins from each cultivar was extracted with cold acetone

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738
by stirring the mixture at 50 rpm at 4˚C for 48 h, and
subsequently the defatted powder of each cultivar of
pistachio was air dried at room temperature for 8 h. Next,
the dry powder was suspended in 1:20 (w/v) of 1 ml of
50 mM Tris-HCl buffer containing 1 mM DTT, 2 mM
EDTA, 2 mM 2-Mercaptoethanol, pH 7.5. The suspen-
sion was stirred at 50 rpm at 4˚C overnight and was cen-
trifuged for 25 min at 14000 rpm at 4˚C. The precipitate
was discarded and the supernatant was used for total
soluble protein (mg g1-dp or defatted powder) assay
according to modified Bradford method [9] described by
Olson and Markwell [10] using bovine serum albumin as
standard protein and SDS-PAGE analysis. SDS-PAGE
was performed using 12% separating and 5% stacking
gels [11]. After electrophoresis at 120 V, protein bands
were stained using silver nitrate and finally the relative
density of protein bands were analyzed by ImageJ (http:
//rsb.info.nih.gov/ij/). The intensity of protein bands in
different samples were analyzed by NTSYSPc2 program.
In order to augment accuracy, all experiments including
exaction and SDS-PAGE were repeated at least four
times. Data were subjected to ANOVA and the mean
differences were compared by Duncan test at P < 0.05.
3. RESULTS
To investigate variations among four Iranian pistachio
cultivars, seed storage proteins from cultivar Akbari (A),
Ahmad Aghaei (AA), Fandoghi (F) and Kaleghouchi (K)
were analyzed. As shown in Figure 1, no significant
difference in total protein content was observed in four
pistachio cultivars.
The SDS-PAGE protein patterns of four pistachio cul-
tivars showed changes in seven protein bands (Figure 2).
Subsequently, the relative levels of protein concentra-
0
50
100
150
200
250
300
350
400
450
AAA FK
Cultivar
Protein (mg / g dp)
a
a
a a
Figure 1. Total soluble protein in four pistachio cultivars Ak-
bari (A), Ahmad Aghaei (AA), Fandoghi (F) and Kaleghouchi
(K), defatted powder (dp). Values are the means ± SE. Similar
letters (a) indicate no significant difference (P < 0.05) based on
Duncan test in four pistachio cultivars.
tion of these seven protein bands were analyzed by Im-
age J program (Figure 3). Five protein bands (1, 2, 3, 4
and 5) with approximate MW 45, 33, 32, 27 and 20 kDa
respectively, showed maximum expression level in cul-
tivar AA. Protein bands of 6 and 7 with approximate
MW 16 and 15 kDa showed maximum expression level
in cultivar A, respectively. However, bands 3 and 4 were
not detectable in cultivars K and A, while they were de-
tected in the other cultivars. Protein bands 2, 5 and 6 had
lower expression level in cultivar F. The minimum level
of protein band 1 was observed in cultivar A and protein
band 7 in cultivar K.
Figure 2. SDS-PAGE pattern of four pistachio cultivars Akbari
(A), Ahmad Aghaei (AA), Fandoghi (F) and Kaleghouchi (K)
protein marker (M).
0
50
100
150
200
250
Band 1band 2band 3 band 4band 5band 6band 7
Band number
Relative levels of protein
expression
A
AA
F
K
Figure 3. Relative levels of protein expression of pistachio cul-
tivars Akbari (A), Ahmad Aghaei (AA), Fandoghi (F) and
Kaleghouchi (K). Values are the means ± SE of proteins bands
from three independent experiments.

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4. DISCUSSION
Electrophoresis of proteins is a powerful tool for identi-
fication of genetic diversity and the SDS-PAGE is par-
ticularly considered as a reliable technology because
seed storage proteins are highly independent of envi-
ronmental fluctuations [12,13]. Seed protein patterns can
also be used as a promising tool for distinguishing culti-
vars of particular crop species [14,15]. However, only a
few studies indicated that cultivar identification was not
possible with the SDS-PAGE method [16]. The SDS-
PAGE is considered to be a practical and reliable method
for species identification [17].
According to the results of the SDS-PAGE, the overall
pattern of seed storage-proteins showed the diversity of
pistachio cultivars. The diversity in seed storage proteins
has also been reported by Khan et al. for wheat varieties
[18]. Moreover, identification of three wheat genotypes
including ILC-195, CM-2000 and CM-98/99 has also
been reported by protein markers [19].
Since in mature seeds, type and amount of proteins are
more constant than other plant tissues [20] therefore, the
SDS-PAGE pattern of seed storage proteins of pistachio
showed polymorphism on the basis of difference in pro-
tein intensity among genotypes. The presence or absence
of protein bands has also been applied for detection of
polymorphism of Brassica cultivars [21].
The present investigation revealed variation in differ-
ent cultivars of pistachio seeds with regard to their total
seed protein profiles. Regarding interspecific variation
among cultivars this investigation revealed some varia-
tions. The genetic affinities within cultivars of the same
species generally corroborated the morphological analy-
sis. Similar to our finding the result of differentiation of
yellow sarson and brown seeded types of Brassica clear-
ly separated the yellow seeded and brown seeded varie-
ties by SDS PAGE [22]. However, we can conclude that,
SDS-PAGE can reveal the differences among seed stor-
age proteins of pistacia cultivars.
5. ACKNOWLEDGEMENTS
Authors wish to thank University of Isfahan for their support.
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