Download Identification, characterization, and expression profiling of salt

Turkish Journal of Biology
Turk J Biol
(2016) 40: 889-898
© TÜBİTAK
doi:10.3906/biy-1504-30
http://journals.tubitak.gov.tr/biology/
Research Article
Identification, characterization, and expression profiling of salt-stress tolerant proton
gradient regulator 5 (PGR5) in Gossypium arboreum
1,
2
2
2
Muhammad Naveed SHAHID *, Adil JAMAL , Beenish AFTAB , Bahaeldeen Babiker MOHAMED ,
1
2
2
2
Javed Iqbal WATTOO , Muhammad Sarfraz KIANI , Bushra RASHID , Tayyab HUSNAIN
1
Institute of Molecular Biology and Biotechnology, University of Lahore, Lahore, Pakistan
2
Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
Received: 10.04.2015
Accepted/Published Online: 19.10.2015
Final Version: 21.06.2016
Abstract: Salinity is among the significant abiotic stresses adversely affecting plant development and yield for a wide range of crops.
For quantitative and qualitative expression studies of proton gradient regulator 5 (PGR5) under salt stress, differential display RT-PCR
and rapid amplification of cDNA ends (RACE) were performed on salt-stress responsive Gossypium arboreum. Alignment of genomic
and cDNA sequences revealed that the GPGR5 gene comprises a single open reading frame of 96 amino acids and contains no introns.
Alignment of cotton GPGR5 complete amino acid sequence with PGR5 from other plants revealed the following identities: Gossypium
raimondii (97%), Amaranthus hybridus (72%), Vitis vinifera (69%), Medicago truncatula (68%), Cucumis melo (62%), Arabidopsis
thaliana (62%), Portulaca oleracea (61%), Portulaca grandiflora (60%), and Zea mays (55%). The expression profile was studied in
different plant tissues (stem, leaf, and root) under the abiotic stresses salt, drought, and cold. The results showed a 7-fold increased
expression of GPGR5 in leaf tissue in salt stress and almost no induction of transcription in root and stem tissues in salt stress. This gene
has a good expression pattern under cold stress compared to salt and drought. In future studies, GPGR5 may be used as a prognostic
marker for the development of abiotic-stress–tolerant plants.
Key words: Gossypium arboreum, differential display PCR, RACE, proton gradient regulator 5, expression profiling
1. Introduction
Abiotic stresses like salinity, cold, drought, and high
temperature adversely affect the development and
productivity of crop plants. Salinity and drought are
becoming particularly widespread in many regions and
may cause serious salinization in more than 50% of all
arable lands by the year 2050 (Bray et al., 2000). Various
environmental stresses induce the expression of a variety of
genes with adverse effects in many plant species (Xiong et
al., 2002; Shinozaki et al., 2003; Bartels and Sunkar, 2005).
Studies have reported that the products of these genes
promote stress tolerance and regulate gene expression
through signal transduction pathways (Xiong et al., 2002;
Shinozaki et al., 2003).
Signal transduction networks for abiotic stress can be
divided into three major signaling types. The first involves
osmotic/oxidative stress signaling that uses MAPK
modules that are involved in the generation of reactive
oxygen species (ROS)-scavenging enzymes and antioxidant
compounds as well as osmolytes (Bartels and Sunkar,
2005). The second comprises Ca2+ dependent signaling
that leads to the activation of late embryogenesis abundant
*Correspondence: [email protected]
(LEA)-type genes that are involved in the production of
stress-responsive proteins of mostly undefined functions.
The third encompasses Ca2+ dependent salt overlay
sensitive (SOS) signaling, which is specific to ionic stress
and is involved in regulating homeostasis (Xiong et al.,
2002).
Proton gradient regulation 5 (PGR5) is a small nuclearencoded protein, essential for ferredoxin-plastoquinone
reductase (FQR)-dependent cyclic electron transport
(CET) in Arabidopsis thaliana and Synechocystis ps. PCC
6803 (Munekage et al., 2002, 2004; Yeremenko et al., 2005).
Overaccumulation of PGR5 in thylakoid membranes
enhances the activity of PSI CET and inhibits growth
under certain conditions due to decreased chloroplast
development (Okegawa et al., 2007). PGR5 does not have
any known metal-binding or transmembrane motifs and
is stable in mutant backgrounds lacking PSII, PSI, the
cytochrome b6/f complex, and ATPase, suggesting that
PGR5 is not a constituent of any of these major complexes
(Munekage and Shikanai, 2005). The exact localization of
PGR5 is in the thylakoid membrane (Okegawa et al., 2007).
Proton gradients are used by plants as the driving force of
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secondary transport systems that ultimately regulate ion
fluxes under stress conditions (Niu et al., 1995).
Plasma membranes and the vacuolar proton transport
system play a key role in salt-stress tolerance by sustaining
the transmembrane proton gradient that assures regulation
of ion fluxes and pH (Guern et al., 1989; Stevens and Forgac,
1997). Three protein/protein complexes exist for this
purpose: the plasma membrane (H+)-ATPase (P-ATPase)
and two vacuolar transport systems, an (H+)-ATPase
(V-ATPase), and a pyrophosphatase (PPiase). The plant
P-ATPase is represented by a gene family with more than
10 members, encoding proteins of approximately 100 kDa,
with homology to the yeast PMAs (Sussman, 1994; DeWitt
et al., 1996). This family takes part, as the major proton
pump, in the outer cell membrane, which is important
for several physiological functions (Michelet and Boutry,
1995). In addition, an increase in proton pump activity has
been observed under salt-stress conditions. Halophytic
plants have been shown to increase pump activity under
salt-stress conditions more than glycophytes (Weiss and
Pick, 1996), but little information is available about the
regulatory circuits that lead to either increased protein
amounts or activity during salt stress.
Salinity is one of the most significant abiotic stresses,
and it limits the productivity and geographical distribution
of plants. Approximately 20% of the world’s land mass and
nearly half of all irrigated lands are affected by salinity.
Salinity can cause ion imbalance, hyperosmotic stress,
and oxidative damage in plants. Cotton is one of the most
important fiber and oil crops, and in higher saline soil its
growth and yield are severely inhibited at the germination
and emergence stages (Ashraf, 2002).
The current study aimed to identify differentially
expressed genes under salt stress. DDRT and RACE
polymerase chain reaction (PCR) was performed for
transcript isolation and full-length gene amplification
studies. The outcomes of these studies showed that a gene
encoding a proton gradient regulation 5 (PGR5) protein
revealed specific changes in expression under salt stress.
2. Materials and methods
2.1. Cotton plants and salt-stress treatment
Seeds of a local variety of G. arboreum (FDH-171) that is
well known for abiotic stress resistance were taken from
the Cotton Research Substation, Raiwind. Seeds were
delinted and screened, as previous described (Shahid et al.,
2012). Seeds were germinated at 25 ± 2 °C in petri plates,
and seedlings were hydroponically grown with Hoagland’s
nutrient solution (Hoagland, 1950) in a greenhouse under
13 h light periods with 1500 µmol photon m–2 s–1 intensity
and at 50% humidity. At the 3–4 leaf stage, plants were
treated with 150 mM NaCl (Zhang et al., 2011) for 48 h,
and then leaves were harvested and stored at –80 °C until
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RNA extraction. Plants without NaCl treatment were
considered controls.
2.2. Differential display reverse transcriptase polymerase
chain reaction (DD-RT PCR)
Total RNA was extracted from 1 g of fresh leaf tissue with
little modification to the protocol described previously
(Jaakola et al., 2001). To remove DNA, RNA samples
were treated with DNase Free (Ambion). Then cDNA was
synthesized by using an oligo-dT (dT18) primer from a
cDNA synthesis kit (cat. no.: k 1631, Fermentas), and the
reaction was carried out according to the manufacturer’s
protocol. Differential display of mRNA was performed
using a previously described procedure (Liang and Pardee,
1992). Briefly, differential display reverse transcriptase
PCR was carried out in volume of 25 μL using 500 ng of
cDNA as a template. Each reaction mixture contained 1
unit of Taq DNA polymerase, 1 μM anchored primer, 1
μM arbitrary primers, 0.05 mM dNTPs, 1X PCR buffer,
and 2.5 mM MgCl2. The reaction was performed in
an ABI Gene Amp PCR system 9700 thermal cycler.
Seventeen arbitrary and 11 anchored oligo-dT primers
were employed randomly (Tawe et al., 1998; Maqbool
et al., 2008). DDRT-PCR products were denatured with
the same volume of gel loading buffer (95% formamide,
0.1% xylene cyanol FF, and 0.1% bromophenol blue) at 90
°C for 2 min. Denatured products (2 μL) were separated
by electrophoresis at 200 V constant electric power on
a 6% polyacrylamide/7 M urea DNA sequencing gel.
Polyacrylamide gels were silver-stained according to
the Bio-Rad silver-stain handbook and fixed with 8%
acetic acid (v/v) before being photographed with Grab
IT software v. 2.5 on a gel documentation system (UltraViolet Products). Each experimental reaction constituting
biological repeats was carried out, and those bands that
consistently appeared in stressed samples were excised and
extracted from the gel matrix using the crush-and-soak
method, as applied by Maxam and Gilbert (1977, 1980).
Extracted PCR product was then ethanol precipitated.
DNA was reamplified in 20 µL reactions using the same
combinations of primers. PCR products were resolved
on a 2% agarose gel, and bands of the required size were
cut and extracted with a GF-1 kit (cat no.: GF-GP-100)
following the manufacturer’s protocol. Isolated DNA was
cloned into a dual promoter pCR2.1 vector (Invitrogen),
and ligated plasmids were transformed in E. coli strain
Top-10 competent cells. Plasmid DNA was isolated from a
minimum of five clones for each transformation (Brinboim
and Doly, 1979). Inserts were confirmed by plasmid DNA
digestion, confirmatory PCR, and sequencing from both
strands with M13-primers. The sequencing reaction
was carried out using ABI Prism Dye Terminator kit.
The reaction was performed in ABI automated DNA
sequencer (model 3100) (Applied Biosystems, Foster City,
SHAHID et al. / Turk J Biol
CA, USA). Homology studies were carried out by BLAST
tool provided by the National Center for Biotechnology
Information (http://blast.ncbi.nlm.nih.gov/Blast).
2.3. 5ʹ-Rapid amplification of cDNA ends (RACE) PCR
analysis
RNA ligase-mediated 5′ RACE was carried out with Gene
Racer kit (Invitrogen). DNA-free total RNA (4 µg) was
applied to create first-strand cDNA with RACE PCR.
Primers for specific genes were designed to synthesize
cDNA. For 5′-RACE PCR, the forward Gene Racer 5′
primer
(5′-CGACTGGAGCACGAGGACACTGA-3′)
and
gene-specific
reverse
primer
PGR
(5′-TTGGCGAGTCACCACCACAACTGGA-3′)
were
used. The cycling parameters were as follows: initial
denaturation at 94 °C for 2 min, 5 cycles of denaturation
at 94 °C for 30 s, and annealing at 72 °C for 1 min; the
next 5 cycles were conducted with denaturation at 94 °C
for 30 s and annealing at 70 °C for 1 min. An additional 25
cycles were performed with denaturation at 94 °C for 30
s, annealing at 65 °C for 30 s, and extension at 68 °C for 1
min. Final extension was performed at 68 °C for 10 min.
PCR products were resolved on a 1% (w/v) agarose gel. A
DNA fragment of the expected size was excised, purified,
cloned, and sequenced.
2.4. Genomic DNA PCR analysis
Genomic DNA was isolated from cotton leaf tissue as
previously described (Saha et al., 1997). The following
primers were used for the amplification reaction: PGR-F
(5′-ACCCATCAAGCTTTACAACCAT-3′) and PGR-R
(5′-TGGTAGCAGAAGTACAGTGAAGG-3′).
The
reactions for PCR were carried out in a 25 µL reaction
volume using Taq DNA polymerase (Invitrogen),
according to the manufacturer’s protocol. Reactions were
prepared using 10X PCR buffer minus Mg (1X), 0.2 mM
dNTP mixture, 1.5 mM MgCl2, 0.5 µM of each primer,
and 1 unit Taq DNA polymerase. Initial denaturation was
conducted at 95 °C for 3 min, followed by 35 cycles of
denaturation at 95 °C for 45 s, annealing at 55 °C for 30 s,
and extension at 72 °C for 45 s with a final extension at 72
°C for 10 min.
2.5. Data analysis
The DNA sequence analysis was accomplished with the
BLAST search program (Altschul et al., 1990) and pairwise alignment algorithm (www.ebi.ac.uk/emboss/align).
To identify introns, exons, untranslated regions, and
poly-A tails the Softberry server was used (http://www.
softberry.com/berry.phtml). Nucleotide sequences were
translated with the open reading frame finder program
(http://www.ncbi.nlm.nih.gov/projects/gorf/orfig.cgi),
and molecular weight and subcellular localization were
determined using the Expasy server (http://expasy.
org/). Multiple sequence alignment was performed
using CLUSTALW (Thompson et al., 1994) with default
parameters through EMBnet (http://www.ch.embnet.org/
software/ClustalW.html). Black and gray shadings were
performed using BOXSHADE 3.21 to indicate conserved
amino acid residues (http://www.ch.embnet.org/software/
BOX_form.html).
A phylogenetic analysis was conducted with all publicly
available full-length PGR5 sequences for Gossypium
raimondii, Amaranthus hybridus, Vitis vinifera, Medicago
truncatula, Cucumis melo, A. thaliana, Portulaca oleracea,
Portulaca grandiflora, and Z. mays. A rooted neighborjoining phylogeny tree was created with MEGA software
package version 5.1 (http://www.megasoftware.net)
(Tamura et al., 2011) using the previously aligned amino
acid sequences with gaps treated as missing data. To root
the tree, the Nitosococcus oceani sequence was designated
as the outgroup. To determine the relative level of support
for the tree topology, bootstrap values were generated
from 1050 replicates.
2.6. Gene expression studies in Gossypium arboreum
under salt, drought, and cold stress
Seeds were germinated at 25 ± 2 °C, and seedlings were
hydroponically grown with Hoagland’s nutrient solution
(Hoagland, 1950) in a greenhouse under a 13 h photoperiod
(1500 µmol photons m–2 s–1) and 50% humidity. Some
seedlings were treated with 150 mM NaCl (Zhang et al.,
2011) for 48 h and some were drought stressed for 10
days (Maqbool et al., 2007) and cold-stressed <20 °C, 4
°C for 7 days (Chinnusamy et al., 2007). Following stress,
total RNA was isolated from leaf, stem, and root tissues
of both stressed and control plants. Extracted RNA (1
µg) was used to synthesize cDNA using oligo-dT primer
from RevertAid H Minus First Strand cDNA synthesis kit
(Fermentas), following the manufacturer’s protocol, after
DNase treatment. Both reverse RT-PCR and quantitative
real-time RT-PCR were carried out to study the expression
profile of the gene.
2.7. Reverse transcriptase PCR analysis
Reverse
transcriptase
PCR
was
performed
using
the
gene-specific
primers
Real-F
(5′-ACCCATCAAGCTTTACAACCAT-3′) and Real-R
(5′-TGGTAGCAGAAGTACAGTGAAGG-3′).
Cotton
GAPDH (gyceraldehyde-3-phosphate dehydrogenase)
was used as an internal control in reverse transcriptase
PCR experiments by specific primers GAP-F
(5′-TGGGGCTACTCTCAAAGGGTTG-3′) and GAP-R
(5′-TGAGAAATTGCTGAAGCCGAAA-3′). Polymerase
chain reactions were performed in 25 µL volumes and
with initial denaturation at 95 °C for 3 min followed by
35 cycles of denaturation at 95 °C for 45 s, annealing at
55 °C for 30 s, and extension at 72 °C for 45 s with a final
extension at 72 °C for 10 min.
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2.8. Quantitative real-time RT PCR
For real-time RT PCR, several primer pairs were designed
for GPGR5 with a melting temperature of 59 °C and a
product size of 171 bp using Primer3 software (Primer3
Input v. 0.4.0; http://frodo.wi.mit.edu) (Rozen and
Skaletsky, 2000). The best primers were selected and named
Real-F and RealR. To check for nonspecificity of bands,
gradient PCR using different annealing temperatures was
performed, and PCR products were resolved on a 1% (w/v)
agarose gel. The efficiency of these primers was calculated
using a standard dilution curve with a large number of
technical replicates.
The Real Time ABI 7500 system (Applied Biosystems
Inc., USA) with Maxima SYBR Green/ROX qPCR Master
Mix (2X) (Fermentas) was used to perform real-time
PCR. A housekeeping gene, glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), was used to normalize realtime PCR data. RNA from different biological samples
was pooled, and then technical replicates were performed
in triplicate. Real-time PCR was performed with
denaturation at 95 °C for 3 min and at 95 °C for 30 s,
followed by annealing at 55 °C for 30 s, extension at 72 °C
for 45 s, and samples were cycled between the annealing
and extension conditions for an additional 40 cycles. A
final extension was performed at 72 °C for 10 min. Relative
gene expression analysis was carried out by SDS V3.1
software (Applied Biosystems Inc., USA).
3. Results and discussion
Cotton is a very important cash crop, and salinity severely
affects G. hirsutum; however, G. arboreum shows good
resistance against salt stress. The complete genome
sequence of G. arboreum has been predicted. This species
has 41,330 protein-coding genes (Li et al., 2014). To
identify salt-stress tolerance gene(s) from cotton plants, the
differential display technique was used; 25 cDNA bands that
consistently appeared were isolated, cloned, and sequenced.
Five bands showed good homology with known proteins,
while 20 bands were rejected due to no or low homology.
Four DNA fragments were shown in polyacrylamide gel
electrophoresis (PAGE) including P8B2-b (Figure 1)
which was previously reported at transcript level (Shahid
et al., 2012). Searching the GenBank database showed that
a 438 bp fragment corresponding to P8B2-b (Figure 2) has
high homology (5e–43, 127aa, 86%) with PGR5 of Cucumis
melo. The sequence of P8B2-b contains the 3ʹ UTR region
and poly-A tail but is truncated at the 5ʹ end due to the
differential display technique. There are many advantages
of DDRT-PCR and one advantage is that it requires a
small amount of total RNA. This method reveals aspects
of up- and downregulation and the absence or presence
of band fragments which indicates qualitative differences
and signals of different intensities that express quantitative
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C S
C S
C S
C S
DD-RT control and stressed
plants profile:
C = Control, S = Stressed
Indicate up -regulation of
transcripts under salt stress
Indicate down-regulation of
transcripts under salt stress
Indicate new (activated)
transcripts under salt stress
Figure 1. Differential display (DDRT-PCR) from leaves of
Gossypium arboreum. Odd numbered lanes represent control
sample, while even numbered lanes represent salt-stressed
samples. RT-PCR reactions were performed by using anchored
primers and arbitrary primers. cDNA fragments, which appeared
to be differentially expressed in treated samples, are indicated by
arrows.
differences. PCR reaction repetition for different primer
combinations allow us to access the different patterns of
band expressions and avoid genomic DNA contamination
in control and experimental reactions, which reduces the
chance of clone false positivity (Voelckel and Baldwin,
2003).
Due to the advantages of the differential display
technique it was used to isolate and identify differentially
expressed genes against salt stress in barely (Lee et al.,
2009), wheat (Wang et al., 2005), sunflower (Saadia et al.,
2011), and Brassica (Qiu et al., 2009). Using the differential
display technique, a novel transcript, P8B2-b of 438 bp, was
isolated and identified from Gossypium arboreum under
salt stress. The transcript showed significant homology
with PGR5 genes of Theobroma cacao (90%), Camelina
sativa (90%), Arabidopsis thaliana (88%), Cicer arietinum
(86%), and Amaranthus hybridus (86%).
A
GPGR -F/GPGR-R (DNA) 669bp
PGR (cDNA RACE) 231bp
P8B2 (cDNA DDRT) 438bp
B
ATG
5 UTR (55bp )
TAA
coding reg ion (287bp)
AAAA
3 UTR (323bp)
Figure 2. a) The three steps involved to attain a full-sequence
gene. Primer pairs that were used to get gene-specific fragments
were shown. b) Representation of full-sequence gene. Solid black
bars showed exons, while black lines showed UTRs. The numbers
of nucleotides in UTRs were shown.
SHAHID et al. / Turk J Biol
To get a full-length gene from the cDNA clone, the
5ʹ end of the gene was produced using RACE-PCR of
the salt-stressed RNA sample. One primer for specific
gene PGR-P was designed using cDNA sequences.
The RACE-PCR product was 268 bp, and a full-length
gene was achieved by overlapping the RACE sequence
and the cDNA sequence isolated through DDRT-PCR.
Using the 438 bp transcript, a 669 bp genomic fragment,
GPGR5, with complete coding and 5ʹ and 3ʹ UTR region
was achieved through three successive steps (Maqbool
et al., 2007). This full-length sequence was submitted to
NCBI GenBank database (accession no.: JQ861978). A
complete open reading frame predicted a polypeptide of
96 amino acids encoding GPGR5. The GPGR5 gene of G.
arboreum was previously identified as transcript P8B2-b, a
homologue of C. melo PGR5, and was upregulated in saltstress conditions; however, its association with salt stress
was unknown until this study (Shahid et al., 2012). Using
WoLF PSORT Predict and ChloroP software, the cellular
localization was predicted to be the cytoplasmic. The exact
localization of PGR5 is in the thylakoid membrane as well
as the stroma lamellae (Yeremenko et al., 2005; Munekage
et al., 2010). The localization of PGR5 in stroma lamellae
indicates that PGR5-related cyclic electron transport takes
place in nonappressed thylakoid membranes (Munekage
et al., 2010).
3.1. Sequences and phylogenetic analysis
Specific primers were designed from the cDNA sequence
to find intron/exon boundaries in cotton GPGR5. A
fragment of 669 bp was obtained, cloned, and sequenced.
Confirmation of intron/exon boundaries was performed
by comparing the G. arboreum GPGR5 cDNA sequence
with genomic sequences using the NCBI BLAST pair-wise
alignment algorithm (http://embnet.vital-it.ch/software/
ClustalW.html), and no introns were found. A fragment of
55 bp upstream of the initiation codon (ATG) corresponded
to the 5ʹ UTR, and the 323 bp-fragment downstream of the
termination codon (TAA) corresponded to the 3ʹ UTR. A
poly-A tail of 14 bp corresponded to nucleotides 656–669
(Figure 2b). The polypeptide encoding from GPGR5
was predicted to be 96 amino acids long. The molecular
weight of GPGR5 was predicted to be 10.4 kDa by EnCor
Biotechnology Inc. (http://www.encorbio.com/protocols/
Prot-MW.htm) and Compute pl/Mw (http://expasy.org/
tools/pi_tool.html). The cellular localization was predicted
to be cytoplasmic using WoLF PSORT Predict (http://
wolfpsort.org/results/) and ChloroP (http://www.cbs.dtu.
dk/Services/ChloroP/). The nucleotide sequence of G.
arboreum was compared with G. raimondii. G. raimondii
species also has a total fragment of 669 bp, having coding
sequence of 369 bp from 115–483 bp. G. arboreum protein
has 96 amino acids, while G. raimondii has 122 amino
acids. Sequence alignment showed these sequences have
97% identities, while the first 89 amino acids are 100%
similar.
Multiple sequence alignment of complete amino acid
sequences revealed that the cotton GPGR5 has identities
with homologues in other plants ranging from 55% to
97%. Maximum homology was shown with PGR5 of other
plants such as Gossypium raimondii (97%), Amaranthus
hybridus (72%), Vitis vinifera (69%), Medicago truncatula
(68%), Cucumis melo (62%), Arabidopsis thaliana (62%),
Portulaca oleracea (61%), Portulaca grandiflora (60%),
and Zea mays (55%). The consensus sequence of proton
gradient regulator 5 homologues among all amino acid
sequences is shown in Figure 3. No conserved domains
have been identified in the subject sequence (http://www.
ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
Phylogenetically, GPGR5 of cotton is closely related to
the proton gradient regulator of Gossypium raimondii and
is distantly related to Pinus taeda and Zea mays (Figure
4). The deduced amino acid sequence of this gene has
high identity with Gossypium raimondii (XP012452997),
Amaranthus hybridus (BAE00072), Vitis vinifera
(XP002285437), and Medicago truncatula (XP003590158),
with greater conserved sequence at all 3ʹ ends compared
to 5ʹ ends. All of these plants have stress-related protein
PGR5 ranging from 13.4 to 13.7 kDa, while GPGR5 is
90.7 kDa, as determined by SAP (http://www.isrec.isbsib.
ch/software/SAPS_form.html). Studies of PGR5 revealed
that it is a small protein without any metal-binding
motifs. PGR5 is an important factor for the activity of the
ferredoxin-dependent cyclic electron transport around
PSI (Munekage et al., 2008). In recent studies, PGR5 has
been shown to interact functionally and physically with a
newly identified trans-membrane protein, PGRL1, which
is related to PSI in A. thaliana (Dalcorso et al., 2008). It
has been suggested that cyclic electron transport activity
is dependent upon formation of a PGR5-PGRL1 complex.
3.2. Gene expression in Gossypium arboreum under salt
stress
GPGR5 expression was examined under salt stress in
different plant tissues through semiquantitative RTPCR and quantitative real-time PCR. A 162 bp fragment
of the housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as an internal control.
Real-time primers producing a 171 bp product were used
to determine the relative expression of GPGR5 in control
and salt-stressed samples of leaf, stem, and root tissues.
Increment of gene expression in the salt-stressed leaf
sample was compared with the control leaf sample, and
there was no induction of expression in the stressed stem
and root samples compared with control (Figures 5A and
5B). The expression profile of GPGR5 gene increased in
leaves compared with stem and roots. Similarly, GPGR5
expression was higher in leaf samples compared with stem
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Figure 3. Gpgr5 alignment with others related PGR5s of different plant species. ClustalW software was used to align the deduced
amino acid sequence of GPGR5 with default parameters. Conserved amino acid residues were shown with black and gray shadings.
Cyanothece ssp
Pinus taeda
Zea mays
Cucumis melo
Vitis vinifera
Amaranthus hybridus
Arabidopsis thaliana
Medicago truncatula
Portulaca grandiflora
Portulaca oleracea
Gossypium arboreum
Gossypium raimondii
Figure 4. A distance-based, neighbor-joining tree relating the complete GPGR5 amino
acid sequence from G. arboreum to complete sequences of PGR5 from different plants.
CLUSTALW software was used to align the sequences. MEGA program 5.1.0 was
used to construct a neighbor-joining tree. A bootstrap analysis (1050 replicates) was
conducted. Cyanothece sp. (PGR5) was used as an outgroup.
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Relative Expression [fold]
Figure 5. a) Reverse transcriptase PCR expression studies of cotton GPGR5 in salt-stressed and control leaf, stem, and root.
S: stressed, C: control. Cotton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as internal control.
Samples were separated on 1.5% (w/v) agarose gel. b) Relative fold expression of GPGR5 in leaf, stem, and root of control
and salt-stressed G. arboreum plants with real-time PCR. Solid bars represent FAM signals during the reaction, while error
bars depicted standard error of replicates mean values (±SE, n = 3).
Figure 6. a) RT-PCR expression analysis of Gossypium arboreum gene GPGR5 in control, salt, drought, and cold-stress leaf
samples. Cotton glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as housekeeping control. Samples
were run on 1.5% agarose gel. b) Relative fold expression of GPGR5 in control, salt-, drought-, and cold-stressed leaves of
cotton plants with q-PCR. Solid bars represent FAM signals during the reaction, while error bars depicted standard error of
replicates mean values (±SE, n = 3).
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and roots (Yao et al., 2011). The expression of GPGR5 was
studied under conditions of different abiotic stresses such as
salt, drought, and cold. The expression in salt, drought, and
cold-stressed samples was 6.8-, 3.8-, and 9.4-fold, respectively
(Figures 6A and 6B). Estimation of gene expression
concluded that it is 68%, 38%, and 94% in salt, drought, and
cold-stressed leaf samples, respectively. Differential gene
expression was also studied against cold, salt, and drought
stress in rice (Ramamoorthy et al., 2008). Drought, salinity,
and low temperature are often interconnected and may
induce similar cellular damage. Drought and/or salinization
are manifested primarily as osmotic stress, resulting in the
disruption of homeostasis and ion distribution in the cell
(Serrano et al., 1999; Zhu, 2001b). As a result, these diverse
environmental stresses often activate similar cell-signaling
pathways (Shinozaki and Yamaguchi-Shinozaki, 2000; Knight
and Knight, 2001; Zhu, 2001c, 2002) and cellular responses,
such as the production of stress proteins, upregulation of
antioxidants, and accumulation of compatible solutes.
The expression analysis of GPGR5 revealed that
transcription of this gene was upregulated in salt-stressed
plant leaf tissues and showed no significant alteration in
control leaf tissues. Plant tolerance to salt is governed by
multiple processes including detoxification, osmotic and
ionic homeostasis, growth regulation, signal perception,
transduction, and altered gene expression (Grover et al.,
2001; Zhu, 2001a). The Arabidopsis mutants (psad1 and
psae1) also show greater levels of ferredoxin and of the
PPP7/PGR5 complex, supporting the role of PPP7 and
PGR5 in the switch from linear to cyclic electron flow
depending on the redox state of the chloroplast. Tissuespecific expression analysis revealed variation in GPGR5
expression among stem, leaf, and root. Strong expression
for GPGR5 was observed in leaf tissues, and very low
expression was observed in stems and root tissues. Abiotic
stresses (salt, cold, and drought) were applied to analyze
the expression of this gene in leaf tissue. Expression
signals were strongly induced under conditions of cold
stress, while they were repressed after salt stress. Results
suggest that GPGR5 gene triggers well under salt stress
and under cold environmental conditions. This gene may
be transformed in plants to help them cope with abiotic
stresses and to increase their tolerance.
Acknowledgment
The authors gratefully acknowledge the Higher Education
Commission (HEC) of Pakistan for the partial funding to
support and carry out this research project.
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