Turkish Journal of Biology
Turk J Biol
(2013) 37: 1-10
© TÜBİTAK
doi:10.3906/biy-1111-23
http://journals.tubitak.gov.tr/biology/
Research Article
In vitro and in silico characterization of Solanum lycopersicum wound-inducible
proteinase inhibitor-II gene
1
2
3,
Faiza MUNIR , Syed Muhammad Saqlan NAQVI , Tariq MAHMOOD *
Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan
2
Department of Biochemistry, University of Arid Agriculture, Rawalpindi, Pakistan
3
Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan
1
Received: 15.11.2011
Accepted: 27.06.2012
Published Online: 10.01.2013
Printed: 01.02.2013
Abstract: Plant proteinase inhibitors (PIs) are antimetabolic defensive proteins conferring resistance in plants against a variety
of competing organisms such as bacteria, viruses, fungi, attacking nematodes, and insects. In the fields of plant biochemistry and
molecular genetics research, tremendous success has been achieved in generating transgenic crops that have defensive approaches
against biotic challenges. In this study, in vitro and in silico analysis was carried out for a wound-inducible PI-II gene isolated from 4
selected varieties (Roma, Nagina, Moneymaker, and Rio Grande) of Solanum lycopersicum L. Around 684 bp of PI-II gene was amplified,
sequenced, translated, modeled to protein structure, and phylogenetically analyzed. The sequence analysis by BLAST showed high
similarity scores (99%, 97%, 96%, and 94%) for Moneymaker, Roma, Rio Grande, and Nagina, respectively, with the original PI-II gene
sequence from Solanum lycopersicum var. cerasiforme (GenBank accession no. AY007240) selected for primer designing. Sequenced
data were translated to protein sequences, and translated sequences were modeled to 3-dimensional structures with iterative threading
assembly refinement (I-Tasser) software. Phylogenetic analysis was carried out using Molecular Evolutionary Genetic Analysis software.
Comparative phylogenetic analysis with 26 other complete coding sequences of PI from dicotyledonous plants was also done with in
vitro analyzed PI-II genes from selected tomato varieties. In silico insight into the phylogenetic evaluation revealed that 30 PIs from
different plants share a common root of evolutionary origin. Furthermore, 3-dimensional protein modeling by Ramachandran plot
analysis revealed that PI from S. lycopersicum ‘Roma’ has the best quality structure with 85% of residues in most allowed regions.
Key words: Solanum lycopersicum, proteinase inhibitor, wound-inducible, in vitro, phylogenetic tree
1. Introduction
In plants, antimicrobial defense systems are gifts of nature.
One of these systems comprises small antimicrobial
proteins known as proteinase inhibitors (PIs) (1) that
act as plant-defense–mediating proteins and contribute
an innate defense against attacking pathogens and
encroaching organisms such as infectious fungi, attacking
nematodes, and herbivores (2). Koiwa et al. (3) reported
4 major classes of PIs (serine, aspartic, cysteine and
metalloproteinase inhibitors) on the basis of target
reaction sites. Serine PIs are widespread in the plant
kingdom, and most of them have been characterized from
family Solanaceae members potato and tomato (4). The
second most studied class of PIs is cystatins, and among
these, heat-stable rice cystatins are the most important
(5). Margis-Pinheiro et al. (6) reported 9 cysteine PI genes
(PtCys1–PtCys9) from Populus trichocarpa. PIs play their
defensive role by obstructing metabolic proteins, which
results in poor digestion in plant pests (7,8).
*Correspondence: tmahmood@qau.edu.pk
Plants cope with attackers by generating countless
antimetabolic proteins that elicit noxious, revolting, and
antimetabolic effects on phytophagous competitors (9).
It has been observed that 1%–10% of the total protein
content of most storage organs, such as seeds and tubers,
are PIs that hinder the activity of different enzymes (10,11).
Nonstorage tissues such as leaves, flowers, and roots also
contain a large number of PIs (12–14). The tomato and
potato PI families have the best studied examples of genes
that are systemically expressed upon wounding. In potato,
proteinase inhibitor-II (PI-II) is a multigene family, and
its constitutive expression in tubers and floral buds and its
wound-inducible expression in leaves have been reported
(15,16). Plant PIs are developmentally regulated, and
distinct regulation patterns have been reported in response
to biotic and abiotic stresses. Studies have shown that
during seed germination and maturation, and also under
cold stress, expression of a wheat cystatin, TaMDC1, can be
observed (17). In another study, expression of strawberry
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MUNIR et al. / Turk J Biol
cystatin (Cyf1) was seen in vegetative organs such as leaves
and roots (18). Similarly, Lievens et al. (19) reported that
during nodulation of Sesbania rostrata, a PI belonging to
the Kunitz family (SrPI1) is expressed.
One of the major advances in agriculture has been the
introduction of genetically engineered insect-resistant
crops. The specificity of PIs in targeting definite groups
of insects can help in generating transgenic plants with
particular PIs that have inhibitory actions against specific
pests (20). For example, transformed white poplar (Populus
alba L.) plants developed using the Arabidopsis thaliana
cysteine PI gene were resistant against Chrysomela populi
beetle (21). Research data demonstrated that biotic stress
such as insect chewing results in the expression of plant
defensive proteins. For example, approximately 100 genes
in lima bean, Phaseolus lunatus L., can be expressed in
response to the chewing of the 2-spotted spider mite,
Tetranychus urticae (Koch) (22).
PI-I and PI-II type inhibitors are widespread in the
family Solanaceae, particularly in potato and tomato;
for this study, we selected the PI-II gene from Solanum
lycopersicum. We then focused on different tomato
varieties of commercial importance from Pakistan to
amplify and sequence this PI gene from different tomato
varieties. In order to characterize PI genes, the present
study was designed with the following main objectives:
amplification of a wound-inducible PI-II gene from
selected tomato varieties, analysis of the protein structures
encoded by these PI genes, sequencing of the amplified
genes, and in silico characterization of sequenced PI genes
from selected tomato varieties with 26 randomly selected
complete coding PI gene sequences from GenBank.
2. Materials and methods
2.1. Plant material
Seeds of 4 selected s. lycopersicum varieties (Roma, Nagina,
Moneymaker, and Rio Grande) were acquired from the
National Agriculture Research Center (NARC), Islamabad,
Pakistan. The seeds were germinated in small pots
containing manure soil in a growth room at 27 °C under
cool white fluorescent lights (2000 lux), 75% humidity, and a
photoperiod regime of 16 h of light and 8 h of dark.
2.2. Genomic DNA isolation and primer designing
DNA was extracted using the cetyltrimethylammonium
bromide (CTAB) method illustrated by Richards (23).
A pair of primers was designed using the bioinformatics
program Primer3 (http://frodo.wi.mit.edu/primer3/input.
htm). A sequence of the PI gene PI-II from the tomato s.
lycopersicum var. cerasiforme (accession no. AY007240)
was used for primer designing. The sequence of the
forward and reverse primers was as follows:
PI-II F:
PI-II R:
2
5’
5’
2.3. Amplification of PI-II gene from different tomato
varieties
To amplify a 684-bp gene, 25 µL of amplification reaction
containing 25 pmol of each primer, 2.5 µL of 10X PCR
buffer, 1.5 µL of 25 mM MgCl2, 1.5 µL of 2.0 mM dNTPs,
45 ng/µL of genomic DNA, and 1.5 U Taq polymerase
(Fermentas) was prepared. The amplification reaction
was conducted in a gradient MultiGene Thermal Cycler
(Labnet) programmed for 35 cycles of denaturation at 94
°C for 40 s, annealing at 55 °C for 40 s, and extension at 72
°C for 45 s, followed by a single-step final extension at 72
°C for 20 min. The amplification was confirmed on 1.5%
agarose gel prepared in 0.5X Tris acetate EDTA (TAE)
buffer.
2.4. DNA sequencing
The JETquick (Genomed) PCR Product Purification
Spin Kit was used to purify the PCR product. Purified
DNA product was used as a template for dye terminator
cycle sequencing reaction, and sequencing was done in a
Beckman and Coulter CEQ (8800) sequencer.
2.5. Sequence analysis
Different bioinformatics software, databases, and tools
were used for data analysis. Phylogenetic trees were
built by unweighted pair group method with arithmetic
mean (UPGMA) using Molecular Evolutionary Genetic
Analysis (MEGA) software version 4.0.02 (24). The
sequenced PI genes were translated to protein sequences
using the GENSCAN Web Server at MIT (http://www.
genes.mit.edu/GENSCAN.html). The protein sequences
were aligned using ClustalW (25). Iterative threading
assembly refinement (I-Tasser) software was used to
predict 3-dimensional protein structures from amino
acid sequences. In silico phylogenetic analysis was also
performed for a total of 30 PI gene sequences, including
the 4 sequenced PI genes from selected tomato varieties
used for the present research studies and 26 randomly
picked PI gene sequences (complete coding sequences)
selected from the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov). These 26
PI genes are complete protein coding sequences selected
from dicotyledonous plants.
2.6. Protein structure modeling and analysis
Protein 3-dimensional models for the 4 translated PI
gene sequences were predicted using an online version
of I-Tasser (http://wwwzhanglab.ccmb.med.umich.edu/ITASSER). The protein structures were authenticated by
Ramachandran plot using the PROCHECK program
(http://www. ebi.ac.uk/thorton/software.html) (26).
TATCCATCATGGCTGTCCAC
AACACACAACTTGATCCCCACA
3’
3’
MUNIR et al. / Turk J Biol
3. Results and discussion
Isolated genomic DNA of 4 s. lycopersicum varieties
(Roma, Nagina, Moneymaker, and Rio Grande was
qualitatively analyzed by running it on 1% agarose gel.
The quantitative measurements were carried out by
spectrophotometry. The DNA concentration that resulted
in the best amplifications was 45 ng/µL. Amplification of
the PI-II gene resulted in a PCR product of approximately
684 bp on 1.5% agarose gel prepared in 0.5X TAE buffer.
The sequencing of PI-II genes from selected tomato
varieties indicated high quality results signified by sharp
peaks. The sequenced data from the tomato varieties
Moneymaker Roma, Rio Grande, and Nagina showed
99%, 97%, 96%, and 94% homology, respectively, with the
PI-II gene of S. lycopersicum var. cerasiforme (accession
no. AY007240), which was used for primer designing.
The sequences were submitted to GenBank and accession
numbers were acquired. The sequence similarity scores of
the in vitro analyzed PI-II gene from 4 tomato varieties
suggest that the nucleotide order for the sequenced PI
gene is common.
A phylogenetic tree was constructed for the 4
characterized PI gene sequences to establish the
evolutionary relationship with the original PI gene sequence
from which the primers were designed. The dendrogram
showed 2 major clusters, a and b (Figure 1). The PI genes
from S. lycopersicum varieties Roma (JN091682) and Rio
Grande (JN132113) in cluster a have close phylogenetic
affinity with the original PI sequence from S. lycopersicum
var. cerasiforme (AY007240). However, cluster b comprises
PI gene sequences from S. lycopersicum varieties Nagina
(JN132111) and Moneymaker (JN132112), showing a
direct evolutionary link with cluster a. The sequenced
nucleotides were translated into amino acid sequences by
the GENSCAN Web Server. Multiple sequence analysis
was performed for the 4 translated amino acid sequences
JN091682
AY007240
a
JN132113
JN132111
JN132112
0.08
0.06
0.04
0.02
b
0.00
Figure 1. Phylogenetic tree of 4 sequenced PI genes from S. lycopersicum ‘Roma’ (JN091682), S. lycopersicum ‘Nagina’ (JN132111),
S. lycopersicum ‘Moneymaker’ (JN132112), S. lycopersicum
‘Rio Grande’ (JN132113), and S. lycopersicum var. cerasiforme
(AY007240) using the UPGMA method of MEGA version 4.0.02.
with ClustalW (25). Conserved regions were highlighted
with different colors (Figure 2).
3.1. Protein structure analysis
Protein 3-dimensional structures (Figure 3) were
generated using I-Tasser version 1.1. The I-Tasser server
generates the 5 full length 3-dimensional models of each
query sequence along with the confidence score, evaluated
TM score (an algorithm that calculates the similarity of
topologies of 2 proteins or models), and root mean squared
deviation for the evaluations (27). From 5 predicted
structures for each PI protein, the best model was selected
after Ramachandran plot assessment. The finest model
was picked based on highest percentages of residues in
most allowed regions and lowest percentage scores in
disallowed regions. The confidence score values (C-scores)
predicted for the best selected PI protein structures by
I-Tasser were –1.320, –1.806, –0.928, and –1.262 for S.
lycopersicum varieties Roma, Nagina, Moneymaker, and
P1
P4
P3
P2
Roma
Rio Grande
Moneymaker
Nagina
MAVHKEVNFVAHLLIVLGMFLYVDAKACTRECGNLGFGICPRSEGSPLNPIFINCCSGYK
MPVHKEVNFVAYLLYVLGMFLYVDAKACTRECGNLGFGICPRSEGSPLNPIFINCCSGYK
MAVQQEVNFVAYLLIVHGMFLYVDAKACTRECGNLGFGICPRSEGSPLNPICINCCSGYK
MAVHKEVYFVAYRLIVLGMFLYVDAKACTRECGNLGFGICPRSEEKVPALIPHIAFNWLA
*.*::** ***: * * *************************** .
*
. .
60
60
60
60
P1
P4
P3
P2
Roma
Rio Grande
Moneymaker
Nagina
GCNYYNSFGKFICEGESDPKRPNACTFNCDPNIAYSRCPRSQGKSLIYPTGCTTCCTGYK
GCNYYNSFGKFICEGESDPKRPNACTFNCDPNIAYSRCPRSQGKSLIYPTGCTTCCTGYK
GCNYYNSFGKFICEGESDPKRPNACTFNCDPNIAYSRCPRSQGKSLIYPTGCTTCCTGYK
TREIYNSFGKFICEGESDPKRPNACTFNCDPNIAYSRCPRSQGKSLIYPTGCTTCCTGYK
: ********************************************************
120
120
120
120
P1
P4
P3
P2
Roma
Rio Grande
Moneymaker
Nagina
GCYYFGKDGKIVCEGESDEPQANMYPVM
GCYYFGKSGKIVCEGESDEPQANMYPVM
GCYYFGKDGKFVCEGESDEPKANMYPVM
GCYYFGKDGKFVCEGESDEPKANMYPVM
*******.**:*********:*******
148
148
148
148
Figure 2. Multiple alignment of 4 amino acid sequences by ClustalW. P1: S. lycopersicum ‘Roma’, P2:
S. lycopersicum ‘Nagina’, P3: S. lycopersicum ‘Moneymaker’, and P4: S. lycopersicum ‘Rio Grande’. Conserved regions are highlighted.
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MUNIR et al. / Turk J Biol
B
A
Beta sheet
Alpha helix
Coil
C
D
Figure 3. Predicted protein models of PIs from S. lycopersicum varieties. A: Roma
(JN091682), B: Nagina (JN132111), C: Moneymaker (JN132112), and D: Rio Grande
(JN132113). These 3-D structures were predicted using I-TASSER version 1.1.
Rio Grande, respectively. Protein 3-dimensional structures
are fundamental as the biological activity of a protein is
accomplished by its 3-dimensional structure (28). 3.2. Ramachandran plot analysis
The stereochemical quality and exactness of the predicted
PI proteins from the 4 tomato varieties (Roma, Nagina,
Moneymaker, and Rio Grande) under investigation were
analyzed through residue-by-residue geometry and overall
geometry of protein structures using the PROCHECK
program (http://www.ebi.ac.uk/thorton/software.html)
(26). Ramachandran plots were drawn for these protein
structures. In Ramachandran plots (Figure 4), the most
allowed regions are indicated by red patches, while
yellow areas show allowed regions. It was observed that
S. lycopersicum ‘Roma’ (JN091682) PI protein has 85.0%
fully allowed region, 11.7% additional allowed region,
2.5% generously allowed region, and 0.8% disallowed
region. In the case of S. lycopersicum ‘Nagina’ (JN132111),
plot analysis revealed 76.4% fully allowed region, 17.1%
additional allowed region, 4.9% generously allowed region,
and 1.6% disallowed region. However, in S. lycopersicum
‘Moneymaker’ (JN132112), the Ramachandran plot
showed 78.3% most favored region, 17.5% additional
allowed region, 3.3% generously allowed region, and
4
0.8% disallowed region. Finally, in S. lycopersicum ‘Rio
Grande’ (JN132113), there is 79.0% most favored region,
14.3% additional allowed region, 4.2% generously allowed
region, and 2.5% disallowed region. Assessment and
authentication results from Ramachandran plot analysis
showed that the PI protein structure of S. lycopersicum
‘Roma’ (JN091682) is a good quality structural model
with 85.0% of residues in the most favored region. Similar
structure modeling and Ramachandran plot analysis
was carried out to validate the structural and functional
analysis of cysteine protease and cystatin from tomato
(29). In another study, in silico studies were conducted
for structural modeling of antioxidant proteins of spinach
by Ramachandran plot analysis, and protein models were
validated by computational tools PROCHECK and WHAT
IF (30).
3.3. Comparative phylogenetic analysis with other PI
gene sequences
Phylogenetic analysis was performed using MEGA version
4.0.02. The phylogram generated by cluster analysis of 30 PI
gene sequences (Table) (31–43) showed 2 major clusters, I
and II (Figure 5). The sequenced PI genes from the 4 tomato
varieties in our present research (accession nos. JN091682,
JN132111, JN132112, and JN132113) are present in cluster
MUNIR et al. / Turk J Biol
135
135
90
90
45
45
Psi (degrees)
180
Psi (degrees)
180
0
–45
0
–45
–90
–90
–135
–135
–180 –135 –90 –45 0
45 90
Phi (degrees)
A
135 180
135
135
90
90
45
45
Psi (degrees)
180
Psi (degrees)
180
0
0
–90
–90
–135
–135
–180 –135 –90 –45 0
45
Phi (degrees)
135 180
–45
–45
C
–180 –135 –90 –45 0
45 90
Phi (degrees)
B
90
135 180
D
–180 –135 –90 –45 0
45 90
Phi (degrees)
135 180
Figure 4. Ramachandran plot of predicted models of PIs (PI-II) from S. lycopersicum varieties. A: Roma
(JN091682), B: Nagina (JN132111), C: Moneymaker (JN132112), and D: Rio Grande (JN132113). Plots
were generated using PROCHECK program.
I. These PI genes are closely related to the PI gene sequence
from S. lycopersicum var. cerasiforme (accession no.
AY007240), from which the primers were designed. Cluster
I is divided in to 2 subclusters, A and B. Similarly, cluster
II has 2 subclusters, C and D. Overall, cluster I represents
the PI genes from 4 plant families, Solanaceae, Salicaceae,
Fabaceae, and Brassicaceae, and cluster II includes PI
genes from 3 plant families, Solanaceae, Fabaceae, and
Brassicaceae. In our study, cluster analysis showed that
5 PIs (accession nos. JN091682, JN132111, JN132112,
JN132113, and AY007240), all encoding PI-II protein in
cluster I, are 99% evolutionarily and genetically linked
with a genetic distance of 0.1% as indicated in the rooted
neighbor-joining tree (Figure 5). Furthermore, our results
revealed that 3 other PI genes (accession nos. AY204562,
AY204563, and AY059390) are 100% genetically allied,
and these 3 PIs encode trypsin inhibitor protein. Similarly,
2 PIs (accession nos. AY129402 and M15186) have 100%
genetic similarity.
The PI genes from the same plant may differ on the basis
of function and coding product. In our study there are 3 PI
genes (AM162666, AM162667, and AM162668) from the
family Brassicaceae; 2 of these (accession nos. AM162666
and AM162668) fall in cluster II, and 1 gene (accession no.
AM162667) falls in cluster I. The 2 PI genes in cluster II
encode the rapeseed trypsin inhibitor, while that in cluster
5
MUNIR et al. / Turk J Biol
Table. The GenBank accession numbers and sizes of 30 PI genes picked from different plants that were phylogenetically analyzed in the
current study.
Serial no.
Accession no.
1
JN091682
Solanum lycopersicum
684
2
JN132111
Solanum lycopersicum
684
3
JN132112
Solanum lycopersicum
682
4
JN132113
Solanum lycopersicum
677
5
AB110700
Solanum lycopersicum
559
(31)
6
AY007240
Solanum lycopersicum
684
Xie and Wu (unpublished data)
7
L25128
Solanum lycopersicum
4272
(32)
8
AY129402
Solanum lycopersicum
1670
(33)
9
M15186
Solanum tuberosum
1241
(34)
10
U45450
Solanum tuberosum
2068
(35)
11
X04118
Solanum tuberosum
1914
(36)
12
X78275
Solanum tuberosum (Arran Banner) pin2-CM7 gene
584
(37)
13
Z12753
Solanum tuberosum
1695
(38)
14
Z13992
Solanum tuberosum
2330
Choi et al. (unpublished data)
15
AF330701
Ipomoea batatas
519
(39)
16
AF330702
Ipomoea batatas
524
(39)
17
AY059390
Phaseolus vulgaris
327
Yang et al. (unpublished data)
18
AY204563
Vigna unguiculata subsp. unguiculata
326
Yuan et al. (unpublished data)
19
AY204562
Vigna unguiculata subsp. unguiculata
326
Yuan et al. (unpublished data)
20
AF349441
Populus tremuloides
682
(40)
21
AY749108
Solanum americanum
1943
(41)
22
Z12824
Solanum tuberosum
1573
(38)
23
AM162668
Brassica napus
642
(42)
24
AM162667
Brassica napus
1023
(42)
25
AM162666
Brassica napus
1114
(42)
26
DQ412560
Vigna trilobata
678
Sinha et al. (unpublished data)
27
DQ417203
Vigna radiata
543
Sinha et al. (unpublished data)
28
DQ417204
Vigna unguiculata
663
Sinha et al. (unpublished data)
29
D17331
Solanum tuberosum
1103
(43)
30
D17332
Solanum tuberosum
1036
(43)
6
Source
Size (bp)
Reference
Present study
MUNIR et al. / Turk J Biol
JN132112 Solanum lycopersicum
AY007240 Solanum lycopersicum
JN091682 Solanum lycopersicum
JN132113 Solanum lycopersicum
JN132111 Solanum lycopersicum
Z13992 Solanum tuberosum
A
DQ417203 Solanum tuberosum
X04118 Solanum tuberosum
D17331 Solanum tuberosum
AF349441 Populus tremuloides
AF330701 Ipomoea batatas
I
AF330702 Ipomoea batatas
DQ412560 Vigna trilobata
DQ417204 Vigna unguiculata
D17332 Solanum tuberosum
Z12753 Solanum tuberosum
AM162667 Brassica napus
B
Z12824 Solanum tuberosum
U45450 Solanum tuberosum
AY749108 Solanum americanum
AB110700 Solanum lycopersicum
X78275 Solanum tuberosum
C
AY129402 Solanum lycopersicum
II
M15186 Solanum tuberosum
L25128 Solanum lycopersicum
AM162668 Brassica napus
AM162666 Brassica napus
D
AY204562 Vigna unguiculata
AY059390 Phaseolus vulgaris
AY204563 Vigna unguiculata
5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
Figure 5. A phylogenetic tree indicating relationship of 4 sequenced PI genes from
selected tomato varieties in current study with 26 randomly picked complete coding PI
gene sequences from different plants.
I encodes the rapeseed glutamyl endopeptidase inhibitor.
Although these PI genes are from the same plant (Brassica
napus), there is a difference in their function and coding
products. Genes closely related in terms of function are
evolutionarily linked.
Genes belonging to the same PI and plant family fall
into different clusters on the basis of size difference. From
the plant family Fabaceae, 2 PI genes (DQ412560 and
DQ417204) fall in cluster I, while 3 others (AY204562,
AY204563, and AY059390) are in cluster II. Although
these 5 PI genes are trypsin PIs from same plant family
(Fabaceae), they are in different clusters due to the
difference in their sizes (nucleotide length base pairs).
The 2 PI genes (DQ412560 and DQ417204) in cluster I
are closely related in terms of size at 678 and 663 bp in
length, respectively, while 3 other PIs that fall in cluster II
(AY204562, AY204563, and AY059390) are 326, 326, and
327 bp, respectively.
In silico phylogenetic evaluations can lead to
important insights in terms of evolutionary affinities
among investigated protein genes. Martinez et al. (44)
phylogenetically analyzed different plant cystatins
from rice, arabidopsis, and barley. In their study, 12
cysteine PI genes from rice, 7 from arabidopsis, and 7
from barley were analyzed in silico by constructing a
phylogenetic tree by neighbor-joining method using the
amino acid sequences of these 26 cystatin proteins. In
an earlier study, molecular and phylogenetic analysis of
the wound-inducible PI-I gene was carried out for the 7
direct ancestors of Lycopersicon esculentum: L. pennellii,
L. chilense, L. hirsutum, L. parviflorum, L. peruvianum
var. humifusum, L. cheesmanii, and L. peruvianum (45).
In another report, it was observed that HvCPI-4 from
Hordeum vulgare and OC-XII protein from Oryza sativa
are closely allied with the highest similarity scores, while
the Arabidopsis cystatins were found scattered in the
resulting tree and appeared to be functionally distant from
rice and barley proteins. In yet another study, Martinez et
al. (46) conducted the phylogenetic analysis of 17 cysteine
PI proteins from different plants, and it was found that
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MUNIR et al. / Turk J Biol
cysteine PI from Malus domestica had a close phylogenetic
affinity with the functionally analyzed strawberry FaCPI-1
in terms of antifungal properties. Phytocystatins have
not been reported from any other cultivated fruit crop
except apple (Malus domestica). Among plants, proteinase
inhibitors PI-I and PI-II belonging to the serine PI family
have been widely investigated. Recently, 9 cysteine PI genes,
designated as PtCys1–PtCys9, have been documented
from Populus trichocarpa. The location of these genes in the
genome is as follows: chromosome 1 (PtCys1 and PtCys2),
chromosome 2 (PtCys3), chromosome 3 (PtCys4),
chromosome 6 (PtCys5 and PtCys6), chromosome 9
(PtCys7), chromosome 14 (PtCys8), and chromosome 16
(PtCys9) (6). Similar phylogenetic analysis was conducted
for 15 PIs from the mustard inhibitor family using the
neighbor-joining cluster analysis method (42). In that
study, 3 PI genes out of 15 mustard PIs from Brassica napus
were characterized in vitro, and phylogenetic analysis was
conducted with 12 other coding PI genes from the mustard
inhibitor family selected from GenBank. Baloğlu et al.
(47) constructed a neighbor-joining phylogenetic tree to
investigate evolutionary association among 14 different
Ran binding proteins from different plant species.
In our study, tomato varieties were selected from
different regions of Pakistan. Rio Grande, Roma, and
Nagina belong to Punjab Province while Moneymaker
originates in the northern areas of Pakistan. In Pakistan,
tomato varieties are generally categorized into 2
distinct types: determinate tomatoes and indeterminate
tomatoes. Determinate varieties flower and set fruit all
at once, followed by dropping. Determinate varieties are
compact plants, and they flower at the ends of shoots,
which determines their length. Rio Grande and Roma
are good examples of determinate varieties in Pakistan.
Indeterminate varieties continue to grow throughout
the season. Their flowers grow along vines that do not
determine their length; indeterminate varieties require
support and pruning. The best example of an indeterminate
tomato variety in Pakistan is Moneymaker. Indeterminate
varieties have a higher yield potential than determinate
varieties. Postharvest research work conducted on tomato
cultivars at different research institutes in Pakistan such as
the NARC in Islamabad, the University of Agriculture in
Faisalabad, the Sindh Horticulture Agriculture Research
Institute in Mirpurkhas, and the Ayub Agriculture
Research Institute in Faisalabad reported that the 2 tomato
varieties Rio Grande and Roma were high-yielding with
a longer postharvest life; hence, these are commercially
valuable tomato cultivars in Pakistan.
Specific environmental conditions in a particular
region can explain some genetic variations among the 4
genetically analyzed tomato varieties. In another study it
was reported that changes in gene structure and function
may occur due to transposable elements through insertion,
excision, and transposition (48). Various important
molecular phenomena reported in the potato type II (pot
II) PI family (tandem duplication, domain swapping, and
fold circular permutation) can be used for evolutionary
studies in the gene family (49,50). According to Mello
et al. (51), gene mutations, as internal gene duplications,
may be the reason behind the evolution of the family of
Bowman–Birk inhibitors (BBIs), revealing great variability
in BBIs from monocotyledonous plants. The 2 legume
species Glycine soja and Glycine max were placed under
phylogenetic study utilizing the gene sequences from a
multigene PI family that illustrated evolutional propinquity
between these 2 legume strains (52).
In the current study, the PI-II gene from tomato varieties
was analyzed through extensively studied members of
the family Solanaceae. From our phylogenetic results, we
conclude that PIs from the same plant family may separate
into different clades on the basis of differences in coding
products. Genes closely related in terms of translated
products/functioning are evolutionarily associated.
Ramachandran plot analysis of predicted proteins depicted
good quality structures. The finest PI protein structural
model was from S. lycopersicum ‘Roma’ (JN091682) with
the highest percentage (85.0%) of residues in the most
allowed region. Our future aim is to use the PI-II gene
from S. lycopersicum characterized in our present study to
generate valuable transformed crop plants with improved
defensive chemistry. We are working to express this PIII gene under the control of a powerful tissue-specific
promoter to generate transgenic potato plants.
Acknowledgments
The authors are deeply thankful to the Pakistan Science
Foundation, Islamabad, and the Higher Education
Commission, Islamabad, Pakisan, for financial assistance.
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