ISSN 0026-8933, Molecular Biology, 2008, Vol. 42, No. 4, pp. 492–498. © Pleiades Publishing, Inc., 2008.

Original Russian Text © A. Maqbool, M. Zahur, M. Irfan, M. Younas, K. Barozai, B. Rashid, T. Husnain, S. Riazuddin, 2008, published in Molekulyarnaya Biologiya, 2008,
Vol. 42, No. 4, pp. 559–565.

GENOMICS. TRANSCRIPTOMICS. PROTEOMICS

UDC 577.21:579.23''315

Identification and Expression of Six Drought-Responsive

Transcripts through Differential Display
in Desi Cotton (Gossypium arboreum)1
A. Maqbool, M. Zahur, M. Irfan, M. Younas, K. Barozai, B. Rashid, T. Husnain, and S. Riazuddin
Center of Excellence in Molecular Biology, University of Punjab, 87-Canal Bank Road,
Thokar Niaz Baig Lahore (53700), Pakistan;
e-mail: tayyab@cemb.edu.pk
Received June 16, 2007; in final form July 09, 2007

Abstract—There is not enough information available on drought-modulated gene(s) in Gossypium arboreum,

which can be a valuable gene pool for improving modern cotton cultivars. In the present work, differential dis-
play reverse transcriptase PCR (DDRT) was used to compare overall differences in gene expression between
water-stressed and control plants. By screening 93 primer–pair combinations, the DDRT technique resulted in
upregulation of 30 cDNA transcripts. Through reamplification and quality control assay, ten cDNA transcripts
appeared false positive. The remaining 20-cDNA transcripts were extracted from the gel, reamplified, cloned
and sequenced. Homology search revealed that six transcripts showed significant homology with known genes.
Real-time RT-PCR showed that, among six transcripts, five showed significant overexpression in water-stressed
leaves as compared to control. This is an important finding since there are only few reports of universal stress
protein, and transposable elements are available in plants but none in cotton under drought condition.
DOI: 10.1134/S002689330804002X

Key words: differential display, drought, Gossypium, retrotransposon, transposon, universal stress protein

1 INTRODUCTION can cause genetic variation and alter gene expression,

Abiotic stresses, such as drought, salinity, extreme
temperatures, chemical toxicity and oxidative stress,
are serious threats to agriculture and result in the dete-
rioration of the environment. Abiotic stress is the pri-
mary cause of crop loss worldwide, reducing average
yields for most major crop plants by more than 50%
[1, 2]. To survive the environmental stresses, plants
respond and adapt through physiological, develop-
mental, and biochemical changes, including the
induction of gene expression and synthesis of a num-
ber of proteins [3]. As there is not a single gene
involved in a particular function of plant physiology,
studying differentially expressed genes may lead to
the identification of novel biomarkers [4].
Several stress proteins and soluble sugars have
been reported to act as protectants during cell dehy-
dration in many plants [5, 6]. The universal stress pro-
tein Usp is a small cytoplasmic protein whose expres-
sion is enhanced severalfold when cellular viability is
challenged with stress agents, suggesting that it
asserts a general “stress endurance” activity [7, 8].
Trehalose is one of the most effective osmoregulatory
sugars [9, 10] that can work as osmoprotectant during
desiccation stress [11]. Transposable elements, which
1 The text was submitted by the authors in English
are important in host adaptations to environmental
changes [12]. Some members of the water soluble
chlorophyll a/b binding proteins are induced after
drought and heat stress as well as leaf detachment in
plants [13]. Differential display of mRNA was
employed to identify such genes for comparing the
transcripts in several treatments simultaneously [14].
The objective of the present study was to identify
and isolate differentially expressed transcripts from
desi cotton under water stress. The identification of
uniquely expressed transcripts is an initial step for
identification of the novel gene and characterization
of their regulatory elements to design innovative strat-
egies for better understandings of pathways that plants
adapt under drought.
MATERIALS AND METHODS
Plant Material and Stress Treatment
Seeds of cotton cultivar (Gossypium arboreum
FDH–171, which is known for its drought tolerance)
was obtained from Cotton Research Substation Rai-
wind. Plants were grown in composite soil (peat, sand,
soil, 1 : 1 : 1) in CEMB greenhouse at temperature
25 ± 2°ë and relative humidity near 50%. Metal halide
illumination lamps (400 W) were used to supplement

492
 IDENTIFICATION AND EXPRESSION OF SIX DROUGHT-RESPONSIVE TRANSCRIPTS 493

Table 1. Primer sequences used for differential display PCR

5'Arbitrary 3'Anchored
Sequence Sequence
primers oligo-dT Primers

P1 5'-ATTAACCCTCACTAAATGCTGGGGA-3' T1 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTAA-3'
P2 5'-ATTAACCCTCACTAAATCGGTCATAG-3' T2 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTAC-3'
P3 5'-ATTAACCCTCACTAAATGCTGGTGG-3' T3 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTAG-3'
P4 5'-ATTAACCCTCACTAAATGCTGGTAG-3' T4 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTCA-3'
P5 5'-ATTAACCCTCACTAAAGATCTGACTG-3' T5 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTCC-3'
P6 5'-ATTAACCCTCACTAAATGCTGGGTG-3' T6 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTCG-3'
P7 5'-ATTAACCCTCACTAAATGCTGTATG-3' T7 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTGA-3'
P8 5'-ATTAACCCTCACTAAATGGAGCTGG-3' T8 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTGC-3'
P9 5'-ATTAACCCTCACTAAATGTGGCAGG-3' T9 5'-CATTATGCTGAGT-
GATATCTTTTTTTTTGG-3'
A1 5'-AAGCTTGATTGCC-3' B1 5'-AAGCTTTTTTTTTTTTTA-3'
A2 5'-AGCTTCAAGACC-3' B2 5'-AAGCTTTTTTTTTTTTTG-3'
A3 5'-AAGCTTTATTTAT-3'
A4 5'-AAGCTTCGACTGT-3'
A5 5'-AAGCTTGCCTTTA-3'
A6 5'-AAGCTTCTTTGGT-3'

natural radiation. Light radiation reached a maximum
of 1500 µmpl m–2s–1 at the top of the canopy at mid-
day. The volume of pure water added to the pots was
calculated periodically to maintain the pots of stressed
treatments at 5% gravimetric humidity (GH) and non-
stressed treatments at 15% GH. To monitor the molec-
ular responses to water stress, 45-day-old seedlings
were drought stressed for 10 days. Leaf samples were
collected from stressed and irrigated plants in liquid
N2 and stored at –70°ë.
Differential Display RT-PCR
Differential display of mRNA was performed
according to the method of [14]. Total RNA was
extracted from 1g leaf tissue by using the method of
[15]. DNAse-treated total RNA was reverse tran-
scribed with an anchored oligo-dT11 primer using a
RevertAidTM H minus first strand cDNA synthesis kit
(Fermentas) according to the manufacturer’s protocol.
Temperature changes were performed in a thermocy-
cler (MJ Research, Inc., model PTC–100TM).
A total of 11 anchored and 15 arbitrary primers
were used (Table 1). DDRT-PCR reaction was per-
formed in 20 µl volumes containing Taq Polymerase
1 unit, arbitrary primer 1 µM, anchored primer 1 µM,
dNTPs 0.05 mM, 1× PCR buffer, cDNA 3 µl, and
MgCl2 2.5 mM. Cycling conditions were the follow-
ing: initial denaturation at 94°ë for 4 min followed by
40 cycles of denaturation at 94°ë for 30 s, annealing
at 40°ë for 2 min, extension at 72°ë for 30 s, and final
elongation step at 72°ë for 5 min. The DDRT-PCR
mixture was denatured with an equal volume of gel
loading buffer [95% formamide, 0.1% xylene cyanole
FF, and 0.1% bromophenol blue] at 90°ë for 2 min.
The denatured products were separated by electro-
phoresis at 70 W constant powers on 6% denaturing
polyacrylamide gel. Silver staining of the polyacryla-

MOLECULAR BIOLOGY Vol. 42 No. 4 2008

494 MAQBOOL et al.
Table 2. Primer sequences used for Real Time PCR
Transcript Forward primer (5–3')
A6B2-2 CAGGGGACTCTTGTGGCTAA
P2T1-4 GGATTCCGGAAGCAACAGTA
P6T8-2 ATGGTGACCGAATTGACGTT
P5T4U-2 GAAGAGCTTCCCGGATTACC
P7T7-3 TCTGGGCTTGCCAAGTTATC
P2T9-3 CACCGTAGCCAACTCCAAGT
GAPDH TGGGGCTACTCTCAAAGGGTTG
mide gel was performed as described by the Bio-Rad
Silver Stain Handbook, with the modification that the
gel was fixed using 7.5% acetic acid (v/v) immedi-
ately after electrophoresis, and the developmental
reaction was stopped using 7.5% acetic acid (v/v) and
photographed using “GrabIT 2.5" software on a gel
documentation system.
Isolation, Reamplification, and Confirmation
of Differentially Expressed Transcripts
Bands that consistently appeared in treated sam-
ples were excised and extracted from gel by the crush
and soak method [16, 17]. The DNA was ethanol pre-
cipitated, redissolved in sterile distilled water and
reamplified in 25 µl PCR mixture using the same set
of primers that generated the differential display pro-
duct.
For each reamplified band, a quality control with-
out reverse transcriptase was added to monitor for
potential RNA contamination by residual genomic
DNA, which could be amplified during subsequent
PCR. Reamplified PCR products were separated on
agarose gel. Bands were cut and eluted from gel by
using DNA extraction kit (Fermentas) and cloned
employing the TA cloning kit (Invitrogen) according
to the manufacturer' instructions with minor modifica-
tions.
DNA Sequencing and Data Analysis
The sequencing reaction was performed with the
ABI prism Dye Terminator kit and ABI model 3100
automated DNA sequencer. Transcripts were
sequenced on both strands with M13 primers. The
nucleotide sequence or the deduced amino acid
sequence of each clone was compared with DNA,
EST, and protein sequences from various databases by
Reverse primer (5'–3') Amplicon length
ACCCTTTGAATGGTGCCAAG 125
GAACCAGCGGTGTAGCATTT 184
CACAATGGCCTGAATCCTTT 187
CCCAAGGAGACACACTAGGC 181
AGCCAACCTACCGTTCTTGA 182
GACTGAAGCCCCTGTCTTGA 184
TGAGAAATTGCTGAAGCCGAAA 162
means of the basic local alignment search tool
(BLAST) [18].
RT–PCR Analysis
RT-PCR reactions were performed using a pair of
specific primers for each transcript designed on the
basis of the G. arboreum cDNA sequence (Table 2).
The cotton Glyceraldehyde–3-phosphate dehydroge-
nase (GAPDH) was used as housekeeping control.
Thermal cycling conditions were 3 min of initial dena-
turation at 95°C followed by 40 cycles of denaturation
at 95°C for 30 s, annealing at 60°C for 30 s, extension
at 72°C for 45 s, and final elongation step at 72°ë for
10 min (semiquantitative RT-PCR).
Quantitative Real-Time RT–PCR
Real-time PCR reactions were carried out in an
iQ5 cycler (BIO-RAD) with a 96-well plate (Bio-Rad)
and using the IQTM SYBR Green Supermix (Bio-
Rad). Primers used were the same as for RT-PCR anal-
ysis. Different concentrations of the plasmid contain-
ing P2T1–4 were used as a standard to validate the
iQ5 Cycler reaction and to determine the quantifica-
tion range (standard curve). Fifty ng of cDNA was
used in each reaction. The reaction conditions were as
follows: initial denaturation at 95°ë for 3 min fol-
lowed by 40 cycles of denaturation at 94°ë for 30 s,
annealing at 60°ë for 30 s, extension at 72°ë for 45 s,
and final elongation step at 72°ë for 10 min. A melting
curve analysis was carried out by continuously moni-
toring fluorescence between 60 and 95°ë with 0.5°ë
increments every 30 s.
Statistical analysis of the real-time results was per-
formed using iQ5 software (Bio-Rad) version 1.0 on
the basis of CT values of the gene in different samples
converted to their linear form using the mathematical
MOLECULAR BIOLOGY Vol. 42 No. 4 2008
 IDENTIFICATION AND EXPRESSION OF SIX DROUGHT-RESPONSIVE TRANSCRIPTS 495

term 2CT [19], normalized with the GAPDH gene. 1 2 3 4 5 6 7 8 9 10 11 12

Analysis of variance (ANOVA) was performed to ana-
lyze significant difference in GHSP26 expression in
different tissues of control and treated plants.

RESULTS AND DISCUSSION

Gossypium genes, whose expression was regulated
by drought stress, were studied by differential mRNA
display. Differential display method reveals all
aspects of regulation (up and down), as well as the
absence/presence of bands, suggesting qualitative dif-
ferences, and signals with varying intensity, suggest-
ing quantitative difference [20].
With a total of 15 arbitrary primers in combination
with 11 anchored primers, 30 gene fragments were
found to be induced, whereas 65 were repressed in
response to water stress. Each experiment was Fig. 1. Differential display (DDRT-PCR) from leaves of
repeated by using total RNA from at least two inde- Gossypium arboreum. Odd numbered lanes represent con-
pendent preparations out of 30 apparently induced trol sample, while even numbered lanes represent water-
stressed samples. RT−PCR reactions were conducted using
fragments, ten were rejected as false positive [21] on anchored primers and arbitrary primers (Table 1). cDNA
reamplification and in quality control assay. The fragments, which appeared to be differentially expressed in
remaining were selected for further analysis due to treated samples, are indicated by arrows (lane 2: P5T4U-2;
ease of reamplification and cloning [22]. Search of lane 4: P2T1-4; lane 6: P6T8-2; lane 8: P7T7-3; lane 10:
A6B2-2; lane 12: P2T9-3).
GenBank databases revealed that six fragments,
whose size ranged between 300–600 bp (Fig. 1), gave
significant homologies with known genes (Fig. 2). in cotton varieties [5, 9]. This enzyme is involved in
The other 14 cDNA fragments showed very low or no the production of trehalose, a disaccharide known to
homology to known genes.
provide osmotic protection of cell membrane during
The expression levels of six transcripts were eval- dehydration. The transcript designated as P7T7-3 has
uated by real-time RT-PCR. The GAPDH gene was homology with light-harvesting chlorophyll a/b-bind-
used as the reference gene to normalize the expression
levels. The results showed that all transcripts showed
a different level of overexpression in water-stressed Table 3. Clone identification and homologies with known
leaves as compared to control. There are 98-fold more genes
expressions of P2T1-4 transcript, 240-fold of A6B2-2
transcript, 2.5-fold of P6T8-2 transcript, 680-fold of Clone
GenBank
Identifi- Size, bp Homology
P7T7-3 transcript, and 2.6-fold of P2T9-3 transcript Accession #
cation
as compared to the control one, while transcript
P5T4U-2 does not show significant difference in A6B2-2 581 EE298190 Universal stress protein
expression level of control and water-stressed leaves GenBankAccession
(Figs. 3 and 4). All of these transcripts were submitted # AB110169
to the NCBI GenBank EST database. Their accession P2T1-4 341 EE298191 Trehalose-6-phosphate
numbers are given in Table 3. synthase GenBankAc-
cession # AAX16015
A transcript designated as A6B2–2 has high
homology, with universal stress protein of Oryza P6T8-2 471 EE298192 Retrotransposon Gen-
sativa (Fig. 2d). All of these proteins belong to a uni- BankAccession
# ABD63156
versal stress protein family, which is known to be
induced in response to water stress and cell survival P5T4U-2 306 EE298193 Gag pol protein Gen-
under certain stress conditions [23–25]. It is a Ser and BankAccession
Thr phosphoprotein, which is phosphorylated by the # AAL59229
Tyr phosphoprotein TypA [26, 27]. The precise bio- P7T7-3 530 EE298194 Chlorophyll a/b binding
chemical function of UspA is unknown. The fragment proteins GenBankAc-
designated as P2T1-4 has homology with the Ginkgo cession # BAA32346
biloba trehalose–6-phosphate synthase enzyme P2T9-3 509 EE298195 Gag-polprotein Gen-
(Fig. 2b). The trehalose–6-phosphate synthase was BankAccession
already reported to be upregulated during water stress # AAL59229

MOLECULAR BIOLOGY Vol. 42 No. 4 2008

496 MAQBOOL et al.

a: Amino acid sequence homology between clone P6T8-2 and retrotransposon (Retro);
Identities = 31/56 (55%), Positives = 40/56 (71%)
P6T8 421 S TMDGWKRYKDL L RRCPYHGL P LWVQVQT FHNGL NP L TRQM I NAAAGGT I NNKHL R 254
S D W+ RYKDL L R CP+ HGL W+ + T F+ NGL TR ++ AAAGG + NK +R
Retro 71 S L Y DAWERYKDL L RMCPHHGL E DWL I I HT FYNGL L Y NTRMT VDAAAGGA LMNKS VR 126
b: Amino acid sequence homology between clone P2T1-4 and trehalose-6-phosphate synthase (TPS);
Identities = 27/41 (65%), Positives = 34/41 (82%)
P2T1 7 SV I AE I F ACT VGQKPSMAKYYVDD I VEV I GL L RG I PEA T VQ 139
S I AE +F ACT VGQKPS AKYY+ DD VEV + + L + G + A+ Q
TPS 805 SP I AE VF ACT VGQKPSK AKYYL DD TVEV LR ML QG LAAA SDQ 845
c: Amino acid sequence homology between clone P2T9-3 and Gag-pol protein;
Identities = 70/112 (62%), Positives = 81/112 (72%)
P2T9 494 GFS L I VSPF TKMLRKGVP LNWTDAQQESFEK L KKV L TEAPV L I QL ESGKEF TVYSDASHV 315
GF S +V P T + L KG W +A Q F E+ L KK L T AP+ L + + K F VY DASH+
Gag-pol 787 GF S K LVKPL T SL L EKGKE FKWDEACQNCF EE L KKRL T T AP I LVMPD I HKGF DVYCDASHL 846
P2T9 314 GL GCV LMQEGKVVAYVSR * L K I HEANYPTHDL E L AT VVF A L K I WRT Y L YGEK 159
GL GCV LMQEGKV+ AY SR L + HE NYPTHDL E L A VV A L K I WR Y + G K
Gag-pol 847 GL GCV LMQEGKV I AYASRQL RKHEKNYPTHDL E L AAVVHA L K I WRHY M I GNK 898
d: Amino acid sequence homology between clone A6B2-2 and universal stress protein (USP);
Identities = 47/73 (64%), Positives = 66/73 (90%)
A6B2 1 DL VD I ASGQKQGT L VAK I YWGDARDK I CESVEDL K LDCL VMGSRGLGT I QRV L I GSVSNY 180
D++ D A+ Q + T +VAK +YWGDAR+ K +C+ + VE+ K +D L VMGSRGLG+ I QR+ L +GSV+ NY
USP 91 DMLD TAARQL E L T VVAK LYWGDAREK LCDAVEE QK I DT L VMGSRGLGS I QR I L LGSVTNY 150
A6B21 181 VMVNATCPVT I VK 219
V + NA+ CPVT + VK
USP 151 VL SNASCPVT VVK 163
e: Amino acid sequence homology between clone P7T7-3 and chlorophyll a/b binding proteins (chlo a/b);
Identities = 132/138 (95%), Positives = 136/138 (98%)
P7T7 21 AVWFKAGSQ I FSEGGL DY LGNPS L I HAQS I L A I WACQV I LMGAVEGYR I AGRP LGEVTDP 200
AVWFKAGSQ I FSEGGL DY LGNPS L +HAQS I L A I WACQV I LMGAVEGYR +AG P LGE+ TDP
chloa/b 129 AVWFKAGSQ I FSEGGL DY LGNPS L VHAQS I L A I WACQV I LMGAVEGYR VAGGP LGE I TDP 188
P7T7 201 L YPGGSFDP LGL ADDPET FAE L KVKE I KNGRL AMFSMFGF FVQA I VTGKGP L ENL ADHL A 380
L YPGGSFDP LGL ADDPE FAE L KVKE I KNGRL AMFSMFGF + VQA I VTGKGP L ENL ADHL A
chloa/b 189 L YPGGSFDP LGL ADDPEAFAE L KVKE I KNGRL AMFSMFGF YVQA I VTGKGP L ENL ADHL A 248

f: Amino acid sequence homology between clone P5T4U-2 and Gag-pol protein;
Identities = 57/78 (73%), Positives = 68/78 (87)
P5T4U 55 EFRDVFPEE L PGL PPNREVEFG I DL L PGTAPVS I APYRMAPKEXVE L KAQ I QE L L DRGF I 234
E+ DVFPEE L PG+ PP+ R+ + EF I + L L PGTAP+ S PYRM K+ VE L K Q I + E L L + + GF I
Gag-pol 511 EYPDVFPEE L PGMPPDRD I EFS I E L L PGTAP I S KRPYRMDVKDL VE L KKQ I E E L L EKGF I 570
P5T4U 235 RPSVSPWGAPV L FV K KK D 288
RPS SPWGAPV L FV KK D
Gag-pol 571 RPSSSPWGAPV L FV N KK D 588

Fig. 2. Amino acid sequence homology of six clones with known genes. The deduced amino acid sequences were aligned using
NCBI BLAST Pairwise Alignment algorithm programs (http://www.ncbi.nlm.nih.gov/BLAST/).

ing proteins of photosystems II of Cryptomeria japon- are induced after drought and heat stress as well as
ica (Fig. 2e). It was reported that some members of the after leaf detachment [13].
chlorophyll a/b-binding protein belonging to the The deduced amino acid sequences of three tran-
WSCPs (water soluble chlorophyll proteins) family scripts designated as P5T4U-2 have homology with

MOLECULAR BIOLOGY Vol. 42 No. 4 2008

 IDENTIFICATION AND EXPRESSION OF SIX DROUGHT-RESPONSIVE TRANSCRIPTS 497

P2T1-4 A6B2-2 P7T7-3 P2T9-3 P5T4U-2 P6T8-2

1.2

1.0

0.8

0.6

0.4

0.2

0
Stressed Control Stressed Control Stressed Control Stressed Control Stressed Control Stressed Control

Fig. 3. Relative expression of transcripts under water-stressed and control conditions. GAPDH gene was used as internal control
and gene expression is indicated as a fold-increase relative to the same transcript under control conditions.

Gag-pol of Zea mays (Fig. 2f), P2T9-3 has homology
with Gag-pol protein of Zea mays (Fig. 2c), and
P6T8-2 has homology with retrotransposon gag pro-
tein of Asparagus officinalis (Fig. 2a). Transposable
elements, which can cause genetic variation and alter
gene expression, are important in host adaptations to
environmental changes [12].
Expression of plant retrotransposons has been
reported to be activated at the transcriptional level in
response to different biotic and abiotic stresses [28].
For example, expression of the Tnt1 and Tto1 ret-
rotransposons are induced by wounding, methyl jas-
monate, CuCl2, and salicylic acid [29].
Two partial cDNA clones, similar to two different
types of transposable elements, the activator-like
transposable element, and a polyprotein (reverse tran-
scriptase) that represent genes differentially expressed
in response to drought and/or salinity stress were
reported in sunflower [30]. Further characterization of
these genes will provide information on the role of
1 2 3 4 5 6 7 8 9 10 11 12 13 14
P2T1-4 A6B2-2 P7T7-3 P2T9-3 P5T4U-2 P6T8-2
Fig. 4. RT-PCR Expression analysis of cotton transcripts in
water-stressed and control leaves. Odd numbers indicate
water-stressed samples and even numbers to control sam-
ples. Cotton GAPDH gene was used as housekeeping con-
trol; product for each sample was separated on a 1.8% (w/v)
agarose gel.
transposable elements in the host plant’s adaptation or
response to environmental stress.
Identification of these stress-regulated transcripts
is an initial step towards cloning and characterization
of full-length cDNAs and promoter regions. Such
studies should identify common and/or unique regula-
tory elements and, thus, provide insight into the mech-
anism of a gene’s individual expression, as well as its
potential role in stress response. This information will
in turn help us to understand better signaling and
interactions between the major drought stress-
response pathways.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the grants
from the Higher Education Commission and Ministry
of Education, Government of Pakistan.
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