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Published online 7 November 2007
Published in Crop Sci 47:2437-2444 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Identification, Characterization and Expression of Drought Related alpha-Crystalline Heat Shock Protein Gene (GHSP26) from Desi Cotton

Asma Maqbool, Muzna Zahur, Muhammad Irfan, Uzma Qaiser, Bushra Rashid, Tayyab Husnain* and Shiekh Riazuddin.

Center of Excellence in Molecular Biology, University of the Punjab, 87-Canal Bank Road, Thokar Niaz Baig Lahore (53700) Pakistan

* Corresponding author (husnain_t{at}yahoo.com).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In response to water deficit stress, plants show quantitative and qualitative differences in gene expression. By using differential display and RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) techniques an alpha crystalline-type small heat shock protein gene (GHSP26) was isolated and characterized from Gossypium arboreum L. Alignments of 1108 bp genomic and 1026 bp cDNA sequences revealed that the GHSP26 gene comprises a single open reading frame of 230 amino acids and it contains a single intron. The gene product contains the highly conserved alpha crystalline region, spanning amino acid residues 133 to 217 and a Met-rich region from 94 to 117aa at the N terminus. Predicted amino acid sequence shares 65%, 63%, 59%, 58%, 56%, 55%, 53%, and 22% identities with Petunia hybrida, Nicotiana tabacum, Arabidopsis thaliana, Zea mays, Agrostis stolonifera, Triticum aestivum, Oryza sative, and Nitrosococcus oceani, respectively. Expression profile of the gene was studied from leaf, stem, and root tissues, through reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR analysis. The results indicated that the gene was expressed in all tissues tested in both fully hydrated and dehydrated plants. However, the gene was 100-fold more abundant in dehydrated leaves, while only two-fold abundant in stressed root and stem as compared to control tissues.

Abbreviations: CEMB, Center of Excellence in Molecular Biology • GH, gravimetric humidity • HSPs, heatshock proteins • LEA, late embryogenesis abundant • PCR, polymerase chain reaction • RACE, Rapid amplification of cDNA ends • RT-PCR, reverse transcriptase polymerase chain reaction • SHSP, small heat shock proteins • UTRs, untranslated regions

Identification, Characterization and Expression of Drought Related alpha-Crystalline Heat Shock Protein Gene (GHSP26) from Desi Cotton

Asma Maqbool, Muzna Zahur, Muhammad Irfan, Uzma Qaiser, Bushra Rashid, Tayyab Husnain* and Shiekh Riazuddin.

Center of Excellence in Molecular Biology, University of the Punjab, 87-Canal Bank Road, Thokar Niaz Baig Lahore (53700) Pakistan

* Corresponding author (husnain_t{at}yahoo.com).

In response to water deficit stress, plants show quantitative and qualitative differences in gene expression. By using differential display and RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) techniques an alpha crystalline-type small heat shock protein gene (GHSP26) was isolated and characterized from Gossypium arboreum L. Alignments of 1108 bp genomic and 1026 bp cDNA sequences revealed that the GHSP26 gene comprises a single open reading frame of 230 amino acids and it contains a single intron. The gene product contains the highly conserved alpha crystalline region, spanning amino acid residues 133 to 217 and a Met-rich region from 94 to 117aa at the N terminus. Predicted amino acid sequence shares 65%, 63%, 59%, 58%, 56%, 55%, 53%, and 22% identities with Petunia hybrida, Nicotiana tabacum, Arabidopsis thaliana, Zea mays, Agrostis stolonifera, Triticum aestivum, Oryza sative, and Nitrosococcus oceani, respectively. Expression profile of the gene was studied from leaf, stem, and root tissues, through reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR analysis. The results indicated that the gene was expressed in all tissues tested in both fully hydrated and dehydrated plants. However, the gene was 100-fold more abundant in dehydrated leaves, while only two-fold abundant in stressed root and stem as compared to control tissues.

Abbreviations: CEMB, Center of Excellence in Molecular Biology • GH, gravimetric humidity • HSPs, heatshock proteins • LEA, late embryogenesis abundant • PCR, polymerase chain reaction • RACE, Rapid amplification of cDNA ends • RT-PCR, reverse transcriptase polymerase chain reaction • SHSP, small heat shock proteins • UTRs, untranslated regions


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DIFFERENTIAL EXPRESSIONS of genes occur in response to abiotic stresses like drought, salt, or cold treatments. These genes include three major categories: (i) those that are involved in signaling cascades and in transcriptional control, such as MyC, MAP kinases and SOS kinase (Shinozaki and Yamaguchi-Shinozaki 1997; Munnik et al., 1999; Zhu 2001), phospholipases (Chapman 1998; Frank et al., 2000), and transcription factors such as HSF, and the CBF/DREB (Choi et al., 2000; Schöffl et al., 1998; Shinozaki and Yamaguchi-Shinozaki 2000; Stockinger et al., 1997); (ii) those that function directly in the protection of membranes and proteins, such as heatshock proteins (HSPs) and chaperones, late embryogenesis abundant (LEA) proteins (Bray et al., 2000; Ingram and Bartels 1996; Vierling 1991), osmoprotectants, and free-radical scavengers (Bohnert and Sheveleva 1998); (iii) those that are involved in water and ion uptake and transport such as aquaporins and ion transporters (Blumwald 2000).

Cells of most organisms induce a set of proteins, HSPs, when exposed not just to high temperature, but also to other stresses such as heavy metal contamination, water deficit, and presence of pathogens (Vierling 1991). The mechanism by which HSPs may affect such protection has not been determined in detail, but considerable recent data indicate that several HSPs function as ‘molecular chaperones’. Molecular chaperones are proteins that bind to partially folded or denatured substrate proteins and thereby prevent irreversible aggregation or promote correct folding of their substrates (Hartl et al., 1992; Hendrick and Hartl 1993; Landry and Gierasch 1994).

HSPs are generally designated by their approximate molecular weights in kDa as HSP110, HSP90, HSP70, HSP60, and Low Molecular Weight HSPs (15–30 kDa), the latter designated by small heat shock proteins (SHSP) (Sun et al., 2002; Waters et al., 1996; Vierling 1991). Although plants synthesize a similar set of high molecular weight HSPs, most of the translation capacity is devoted to the synthesis of the SHSPs (Mansfield and Key 1987). As far as it is known, all plant SHSPs are encoded by six nuclear gene families, each gene family corresponding to proteins found in distinct cellular compartments: cytosol (class I and class II), chloroplast, endoplasmic reticulum, mitochondria and membranes (Waters et al., 1996). Higher plants have at least 20 SHSPs and the same species may have up to 40 different SHSPs (Vierling 1991).

In an attempt to identify genes that are expressed during water deficiency, we have performed differential display and rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) analysis. The results of these approaches led to the novel observation that a gene encoding alpha-crystalline SHSP shows specific expression patterns during water stress.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material and Stress Treatment
Seeds of the cotton cultivar Gossypium arboreum FDH-171, which is known for its drought tolerance, were obtained from Cotton Research Sub station Raiwind. Plants were grown in composite soil (peat, sand, soil, 1:1:1) in the Center of Excellence in Molecular Biology (CEMB) greenhouse at the temperature 25 ± 2°C and relative humidity near 50%. Metal halide illumination lamps (400 W) were used to supplement natural radiation. Light radiation reached a maximum of 1500µmpl m–2s–1 at the top of canopy at midday. 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 molecular responses to water stress, 45-days-old seedlings were drought stressed for 10 d. Leaf samples were collected from stressed and irrigated plants in liquid N2 and stored at –70°C.

Differential Display Reverse Transcriptase Polymerase Chain Reaction
Differential display of mRNA was performed according to the method of Liang and Pardee (1992), with minor modifications. Total RNA was isolated from 1 g leaf tissue by using method of Jaakola et al. (2001). RNA quality and quantity was measured spectrophotometrically at 260nm and 280nm. The RNA was reverse transcribed with an anchored oligo-dT11 primer using RevertAid H minus first strand cDNA synthesis kit (Fermentas, Burlington, ON) according to the manufacturer's protocol. Temperature changes were performed in a thermo cycler (MJ Research, Inc., Waltham, MA, model PTC-100).

Differential Display Reverse Transcriptase Polymerase Chain Reaction (DDRT-PCR) was performed in 20ul volumes containing Taq Polymerase 1unit, arbitrary primer 1µM, anchored primer 1µM, dNTPs 0.05mM, and PCR buffer 1 X, cDNA 3µl, MgCl2 2.5mM in the thermocycler. A total of 15 arbitrary primers in combination with 11 anchoring primers were used. 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°C for 2 min. Denatured products (2ul) were separated by electrophoresis at 70W constant powers on 6% denaturing polyacrylamide gel and bands were detected by autoradiography. The experiment was repeated three times. Bands that consistently appeared in treated samples were excised and extracted from gel by crush and soak method (Maxam and Gilbert, 1977, 1980).

The DNA was ethanol precipitated, and reamplified in 25 µl PCR mixture using the same set of primers. Reamplified PCR product was analyzed on agarose gel. A band of expected size was cut and eluted from gel by using DNA extraction kit (Fermentas) according to manufacturer's protocol. Eluted DNA was cloned into PCR 2.1 vector (invitrogen). Plasmid DNA from at least four clones per transformation was isolated (Brinboim and Doly 1979). Insert of at least two (when both were of equal size), or all clones (when size of insert varied), were sequenced on both strands with M13 primers. The sequencing reaction was performed with the ABI prism Dye Terminator kit and ABI model 3100 automated DNA sequencer (Applied Biosystems, Foster City, CA). Database search for homology was performed using the BLAST tools provided by NCBI (http://0-www.ncbi.nlm.nih.gov.library.vu.edu.au/BLAST/ verified 18 Sept. 2007).

5'-Rapid Amplification of cDNA Ends (RACE)-PCR Analysis
RNA ligase-mediated 5' RACE was performed using the GeneRacer kit (Invitrogen Life Technologies, Carlsbad, CA). Three microgram of total RNA was used to generate RACE-ready first-strand cDNA. Gene-specific primers were designed and used for cDNA synthesis. For 5'-RACE PCR the forward GeneRacer 5' primer (5'-CGACTGGAGCACGAGGACACTGA-3') and reverse gene-specific primer HSP-1 (5'-CGAGTAGCCTCTGCCCTTACAACAGAC-3') were used. Touchdown PCR (Don et al., 1991) cycling parameters were used. Initial denaturation was conducted at 94°C for 2 min. Cycle 1 consisted of denaturation at 94°C for 2 min, annealing at 67°C for 30 s, and extension at 68°C for 1 min. Every five subsequent cycles, the annealing temperature was decreased by 1°C until 62°C was reached. The additional 30 cycles at an annealing temperature of 62°C were performed.

PCR products were resolved on 1% (w/v) agarose gel. A DNA fragment of the expected size was excised, purified, cloned, and sequenced.

Genomic DNA PCR Analysis
DNA was isolated from total leaf tissue by using the method of Saha et al. (1997).

Primers used for amplification reaction were:

GHSP-F
(5'- CTTCGACTGTATCTTGCTCATTTTC-3') and
GHSP-R
(5'-CCAAAGCTGGATTCCATATTAGAAG-3').

PCR reactions were performed in volumes of 25 µL and contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 U Taq DNA polymerase, 0.2 mM dNTPs, 10 ng of each primer, and 50 ng of DNA template. Initial denaturation was conducted at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min. A final extension at 72°C for 10 min was performed.

DATA Analysis
Sequences analysis was performed using the BLAST search program (Altschul et al., 1990) and Pair-wise alignment algorithm programs (www.ebi.ac.uk/emboss/align; verified 10 Sept. 2007). To find out the exon/intron boundaries, untranslated regions (UTRs), and poly-A tail, softberry server was used (http://www.softberry.com/berry.phtml). The conceptual translation of nucleotide sequence was made using the Open Reading Frame Finder program (ORF, www.ncbi.nlm.nih.gov/gorf/gorf.html; verified 10 Sept. 2007). Molecular weight and sub-cellular localization was determined by expasy server (http://expasy.org/ verified 18 Sept. 2008). Multiple sequence alignment was performed using the CLUSTALW (Thompson et al., 1994) with default parameters through EMBnet (http://www.ch.embnet.org/software/ClustalW.html; verified 10 Sept. 2007). Black and gray shadings were done with BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html; verified 10 Sept. 2007), indicating conserved amino acid residues.

A phylogenetic analysis was performed with all full length SHSP sequences publicly available for Arabidopsis thaliana, Oryza sative, Pisum sativum, Triticum aestivum, and nitrosococcus oceani. A rooted neighbor-joining phylogenetic tree was generated using the MEGA software package version 3.0 (http://www.megasoftware.net/ verified 18 Sept. 2007) (Kumar et al., 2004) from the previously aligned amino acid sequences. Gaps were treated as missing data. For rooting the tree, the Nitosococcus oceani sequence was designated as outgroup. To determine relative level of support for the tree topology bootstrap values were generated from 1050 replicates.

Expression Studies
Eight week-old cotton plants were subjected to water stress in the CEMB greenhouse under the same conditions as mentioned above. At the end of stress period, total RNA was extracted from leaf, shoot, and root samples of both control and water-stressed plants. Total RNA (1µg) was reverse transcribed with oligo-dT using RevertAid H minus first strand cDNA synthesis kit according to the manufacturer's protocol after DNase1 treatment. For expression studies both reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and quantitative real-time RT-PCR were performed.

RT–PCR Analysis
RT-PCR reactions were performed using a pair of gene specific primers:

real1F: 5'-CCTAAACGGTTGGCTATGGA-3'
real1R: 5'-CCTAAACGGTTGGCTATGGA-3'
designed on the basis of the G. arboreum cDNA sequence. The cotton Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as housekeeping control in RT-PCR experiment using the specific primers:
Gap-F: 5'-TGGGGCTACTCTCAAAGGGTTG-3'
Gap-R: 5'-TGAGAAATTGCTGAAGCCGAAA-3'

To quantify the transcripts of both genes, 35 cycles were performed using the following PCR conditions: 95°C for 5 min followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 45 s and final elongation step at 72°C for 10 min (semi-quantitative RT-PCR).

Quantitative Real-Time RT–PCR
For the real-time RT-PCRs, the Primer3 software (http://fokker.wi.mit.edu/primer3/input.htm; verified 18 Sept. 2007) (Rozen and Skaletsky 2000) was used to design several primer pairs for GHSP26 with a melting temperature of about 60oC, length 20 bp, and to generate amplicons between 110 and 140 bp. Specific PCR primers (Real-1F and Real-1R) were selected; the different melting curves were compared and the absence of non-specific bands was confirmed after electrophoresis in 1.8% w/v agarose gel. Efficiency of primer pairs was determined by running standard curves with five serial ten-fold dilutions of cDNA. Real-time PCRs were performed in an iQ5 cycler (BIO-RAD, Hercules, CA) with a 96-well plate (Bio-Rad) and using the IQTM SYBR_ Green Supermix (Bio-Rad). Different concentrations of the plasmid containing GHSP26 were used as a standard to validate the iQ5 Cycler reaction and to determine the quantification range (Standard curve). 50 ng of cDNA was used in each reaction. The reaction conditions were as follows: initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 45 s and final elongation step at 72°C for 10 min. A melting curve analysis was performed by continuously monitoring fluorescence between 60°C and 95°C with 0.5°C increments every 30s.

Statistical analysis of the real-time results was performed 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 term 2CT (Livak and Schmittgen 2001), normalized with GAPDH gene. Analysis of variance (ANOVA) was performed to analyze significant difference in GHSP26 expression in different tissues of control and treated plants.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To search for the genes involved in water deficit tolerance, differential display RT-PCR was used. The experiment was performed independently three times with fresh RNA extracts from leaf tissues. A total of 30 cDNA bands that consistently showed differential intensity in all experiments were isolated, cloned, and sequenced. Searches in the GenBank databases showed that one of these fragments (Fig. 1 ) A4B1 (858bp) has high homology (2e-66, 189aa, 78%), with an HSP of Petunia x hybrida. Due to the use of anchored oligo dT primers in differential display, the clone A4B1 contains the 3' UTR region and poly-A tail in the sequence but is truncated at its 5' end.


Figure 1
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  		Figure 1. Differential display profile of Gossypium arboreum with different set of primers. Result are distributed in three lines, each line representing different cDNA fragments amplified in water stressed and control plants. cDNA fragment A shows high homology to SHSP.

 
To obtain a full-length clone the 5' end of the gene was generated by RACE PCR from RNA extracts of stressed leaves. One gene specific primer (HSP-1) was designed using the cDNA sequence. The RACE product obtained was 325 bp long and contained an extra 168 bp at the 5' end, while 157 bp overlapped with the previously isolated 858 bp fragment (Fig. 2a ). A full-length cDNA clone was then obtained by overlapping the RACE product to the original cDNA isolated through differential display. This full-length cDNA sequence was deposited in GenBank with accession number DQ975269.


Figure 2
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  		Figure 2. (a) Schematic representation of three steps to obtain full length gene. Primer pairs used to obtain gene specific fragments are indicated. (b) Complete gene sequence. Solid black bars indicate exons; gray bar indicates intron while black lines indicate untranslated regions. The number of nucleotides in each intron and UTRs are indicated.

 
Sequence and Phylogenetic Analysis
To determine the intron/exon boundaries of the cotton GHSP26 specific primers were designed based on the cDNA sequence. A fragment of 1108 bp was obtained by genomic DNA PCR. This fragment was cloned, sequenced, and deposited in GenBank (accession number DQ975268). Identification of intron/exon boundaries was achieved by comparing the G. arboreum GHSP26 genomic sequence with the cDNA sequence using the NCBI BLAST pair-wise alignment algorithm programs (http://www.ncbi.nlm.nih.gov/BLAST/ verified 10 Sept. 2007). Accordingly, the GHSP26 was found to have two exons comprising the complete coding region (1026 bp) separated by one intron (82 bp). A fragment of 170 bp upstream to the initiation codon (ATG) corresponds to the 5' UTR, and a 162 bp fragment downstream to the termination codon (TAA) corresponds to 3' UTR. Poly-A tail spans a region of 16 bp ranging from 1092 to 1108 (Fig. 2b).

The polypeptide encoded by the GHSP26 is predicted to be 230 amino acids long. Molecular weight of the protein is found to be 26.1 Kda as predicted by SAP (http://www.isrec.isb-sib.ch/software/SAPS_form.html; verified 10 Sept. 2007) and ComputePl/Mw (http://expasy.org/tools/pi_tool.html; verified 10 Sept. 2007). The cellular localization is predicted to be chloroplast using WoLF PSORT Predict (http://wolfpsort.org/ verified 10 Sept. 2007) and ChloroP (http://www.cbs.dtu.dk/services/ChloroP/ verified 10 Sept. 2007) programs.

Through multiple sequence alignment of complete amino acids sequences the cotton GHSP26 shares identities ranging between 53% and 65% with the HSPs of other plants. Maximum homology is shown with heat shock protein of Petunia hybrida 65%, Nicotiana tabacum 63%, Arabidopsis thaliana 59%, Zea mays 58%, Agrostis stolonifera 56%, and Oryza sativa 53%. There is a consensus sequence of alpha-crystalline domain among all the amino acids sequences (Fig. 3 ), which is the heat shock domain. In the GHSP26 the alpha-crystalline domain is 74 amino acids long, ranging from 133 aa to 217 aa.


Figure 3
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  		Figure 3. Alignment of GHSP26 with related HSPs from other plant species. The deduced amino acid sequence of GHSP26 was aligned using clustalW with default parameters. Black and gray shadings indicate conserved amino acid residues.

 
From the phylogenetic tree (Fig. 4 ) GHSP26 is found to be closely related to small heat shock proteins of Petunia hybrida and Nicotiana tabaccum, and distantly related to Oryza sativa and Pisum sativum.


Figure 4
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  		Figure 4. A distance-based, neighbor-joining tree relating complete G. arboreum GHSP26 amino acid sequence to full length HSP amino acid sequences from plants. Sequences were aligned with the CLUSTALW. A neighbor-joining tree was constructed with the MEGA program. A bootstrap analysis (1050 replicates) was performed Nitrosococcus oceani (SHSP) was used as an outgroup.

 
Gene Expression
GHSP26 expression was determined in different plant tissues under different water treatments using semi quantitative RT-PCR and quantitative real-time RT-PCR. A 162 bp fragment of cotton Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as housekeeping control. A fragment of 118 bp amplified by RT-PCR shows that although GHSP26 is expressed in leaves, stems, and roots under well-watered and stressed conditions, a relatively higher expression was observed in water-stressed leaves, stems, and roots as compared to the control (Fig. 5 ).


Figure 5
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  		Figure 5. RT-PCR Expression analysis of cotton GHSP26 in stressed and control leaf, stem and roots. S: Stressed, C: Control; cotton GAP gene was used as housekeeping control; product for each sample was separated on a 1.8% (w/v) agarose gel.

 
There is 100 times greater expression of the gene in stressed leaves as compared to well-watered leaves, 2 times greater expression in both stressed root and stem as compared to their well-watered samples (Fig. 6 ). The expression profile of the gene depicts the most up-regulation in leaves as compared to stem and root. The estimation of the difference in expression concludes that GHSP26 in leaves is almost 40 times more abundant than in stem, while 30 times more abundant than in root (Fig. 6).


Figure 6
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  		Figure 6. Relative fold expression of GHSP26 in root, stem and leaves of well watered and stressed cotton plants through real-time PCR. Solid bars represent FAM (carboxyfluorescein) signals during the reaction.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
An important advantage of DDRT-PCR is that it requires small amounts of total RNA. The method reveals aspects of regulation (up and down) as well as the absence or presence of bands suggesting qualitative differences, and signals with varying intensities suggesting quantitative differences. Repetitive PCR reactions for each primer combination and treatment allow a more strict evaluation of the differential pattern and control for residual genomic DNA contamination which reduces the chance of cloning false positives (Voelckel and Baldwin 2003).

Through differential display of PCR several drought tolerant transcripts have been identified (Nepomuceno et al., 2002; Sharma and Kumar 2005). By means of this technique we have successfully identified A4B1, a novel Gossypium transcript of 855 bp that is induced by water deficiency. The fragment showed significant homology with SHSPs of plants.

Based on this 855 bp fragment a 1108 bp genomic segment, termed GHSP26, containing the complete coding region, intron and 5' and 3' UTRs, was obtained in three successive steps. The polypeptide encoded by the GHSP26 is predicted to be 230 amino acids long, comprising a single open-reading frame. Cellular localization programs predict that GHSP26 belongs to one of the chloroplast-localized SHSP families of plants (Vierling 1991, Helm et al., 1993, 1995).

Like all other chloroplast-localized SHSPs GHSP26 has a Met-rich region (amino acid 94–117) in the N-terminal domain. This region is conserved in the angiosperms' chloroplast SHSP and not found in any other plant or non-plant SHSP. It is predicted to form an amphiphatic alpha helix (Chen and Vierling 1991). BLAST analysis revealed the presence of a conserved alpha crystalline region, spanning residues 133 to 217 amino acid at C-terminal end. This domain is known as "heat shock domain" which is mainly involved in stress response (Caspers et al., 1995; Plesofsky-Vig et al., 1992). This domain contains five completely conserved (DMPGL) and 19 highly conserved amino acids. These findings correlate with the findings of Waters (1995) and Vierling (1991). The C-terminal domain is important in enabling SHSPs to form large oligomers and to interact with misfolded substrates (Ehrnsperger et al., 1998; Suzuki et al., 1998). Secondary structure predictions of the C-terminal domain from widely divergent SHSPs are very similar, even though the primary sequences are not highly conserved (Caspers et al., 1995).

The deduced amino acid sequence of the gene shares significantly high identity with Arabidopsis thaliana (CAA38036), Oryza sativa (BAA78385), and Nicotiana tabacum (BAA29064). All these proteins comprise alpha-crystalline-type HSPs, a family of small stress induced proteins ranging from 12 to 43 kDa, whose common feature is the alpha-crystalline domain. They are generally active as large oligomers consisting of multiple subunits. They are believed to be ATP-independent chaperones that prevent catastrophic aggregation of proteins and are important in refolding in combination with other HSPs (Jakob et al., 1993).

The phylogenetic tree resulting from the analysis of full length HSP amino acid sequences from Arabidopsis thaliana, Triticum aestivum, Oryza sativa and Pisum sativum indicates that G. arborium HSP26 is grouped with SHSPs from Nicotiana tabaccum and divergent from Triticum aestivum. The phylogenetic relationships of SHSPs revealed that gene duplication, sequence divergence, and gene conversion have all played a role in the evolution of the SHSPs which may have diversified in function as well as in sequence and cellular localization (Waters 1995).

The tissue specific study revealed distinct expression difference in leaf, root, and stem under water stress conditions. The expression was higher in leaf and very low elsewhere, suggesting a specific role of GHSP26 in chloroplast. Voloudakis et al. (2002) found that the HSPCB gene is mainly expressed in leaves of drought-tolerant cotton varieties under high water-stressed conditions (–0.3 MPa). Zheng et al. (2002) showed increasing amounts of all plastid ATP-dependant Clp proteins from stem to roots to leaves. Short-term moderate and severe stresses (desiccation, high salt, cold, heat, oxidation, wounding, and high light) all failed to elicit significant or rapid increases in any chloroplast Clp protein. However, increases in mRNA and protein content for chloroplastic molecular chaperones did occur during long-term high light and cold acclimation. They revealed the great complexity of Clp proteins within the stroma of plant chloroplasts. These proteins may also be involved in plant acclimation to different physiological conditions. Prändl et al. (1995) found the SHSP gene Athsp17.6 expressed in heat-shocked leaves but no expression in control leaves of Arabidopsis. In addition to thermal stress, studies in plants have indicated that HSPs are up-regulated in response to low water potential and to exogenously applied ABA (Coca et al., 1999; Pareek et al., 1995; Sun et al., 2001). The presence of GHSP26 transcripts in roots can be explained from the possible involvement of this gene with ABA, which is synthesized in all cells containing plastids (Bressan 1998). Our intention is to proceed with cellular localization, further elucidation of stress tolerance mechanisms at the transcriptional level, and transformation of GHSP26 in cotton.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge grants from Higher Education Commission and Ministry of Education, Government of Pakistan. The authors also acknowledge to Dr. Ahmad Ali Shahid, Dr. Zia-ur-Rehman, and Miss Saba Khaliq for their useful discussion and nice cooperation.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Received for publication March 1, 2007.


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