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Plant Mol Biol Rep (2010) 28:394–402 DOI 10.


A Putative Leucine-Rich Repeat Receptor-Like Kinase of Jute Involved in Stress Response
Md. Maksudul Alam & Sazia Sharmin & Zinnatun Nabi & Shakhinur Islam Mondal & Md. Shahidul Islam & Sarmah Bin Nayeem & Muhammad Shoyaib & Haseena Khan

Published online: 8 January 2010 # Springer-Verlag 2009

Abstract A putative leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene together with its 5′ and 3′ untranslated regions of jute (Corchorus olitorius L.) has been identified and sequenced. The gene is 3,371 bp long containing two exons and one intron. The coding sequence of the gene is 2,879 bp long encoding a peptide of 957 amino acids. The predicted protein contains several domains and motifs characteristic of a transmembrane protein kinase. It is complete with domains for an Nterminal leucine-rich repeat and a protein kinase core, an active site for serine/threonine protein kinase, an ATP

binding conserved site and a transmembrane region. Expression of the gene is induced by low temperature, high salt concentration, dehydration, abscisic acid treatment, and fungal infection, suggesting the involvement of the gene in multiple stress response pathways in jute (C. olitorius L.). A possible mechanism of the role of the gene in signal transduction and environmental stress response is discussed. To date, LRR-RLK is the only jute gene which has been completely sequenced and characterized. Keywords Jute . LRR-RLK . Gene prediction . Degenerate RT-PCR . RACE . Southern blot . Semiquantitative RT-PCR . RNA dot blot

Md. Maksudul Alam and Sazia Sharmin contributed equally to the study. M. M. Alam : S. Sharmin : H. Khan (*) Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka, Bangladesh e-mail: Z. Nabi Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada S. I. Mondal Department of Genetics, SUST, Sylhet, Bangladesh S. B. Nayeem Department of Applied Biotechnology, Uppsala University, Uppsala, Sweden M. Shoyaib Institute of Information Technology, University of Dhaka, Dhaka, Bangladesh M. S. Islam Bangladesh Jute Research Institute, Dhaka, Bangladesh

Introduction Jute is the principle coarse fiber crop grown for commercial purposes in many South Asian regions. United Nations has declared the year 2009 as the year of “natural fiber.” This crop has marketable significance for the generation of diversified value-added industrial products, in addition to its immense potential for industrial production of packaging material (Mondal 2000). Like other plants, jute has to cope with a variety of external and internal environmental changes compounded by the effects of climate change. Water, temperature, and light are some critical environmental factors for the survival of a plant. The ability of a plant to respond to these environmental factors is crucial for its proper development and survival. One of the major environmental stresses that any plant has to cope with is the change in osmotic potential. Knowledge of the molecular mechanism of signal transduction pathway leading to cellular response to osmotic stress is still minimal. However, increasing evidence suggests that plant receptor kinases (PRK) are involved in such

cold and salt stress. and all plant LRR-RLKs analyzed to date possess serine/threonine (Ser/Thr) kinase activity (Anne and Steven 2004). In preparing the fungus for infection. and low temperature. Leucine-rich repeat (LRR)-containing PRKs represent the largest group of PRKs in the Arabidopsis thaliana genome with 216 members (Anne and Steven 2004). This library was enriched for simple sequence repeats. 150 μM ABA (15 ml per petri dish) was sprayed on 5-day-old seedlings.Plant Mol Biol Rep (2010) 28:394–402 395 signal transduction pathway (Stone and Walker 1995). we studied the expression of the gene in low temperature. 2003). and also upon fungal infection and abscisic acid treatment. such as stomatal cells (Robatzek et al. CTAB method (Doyle and Doyle 1987). which can be classified according to the organization of the LRRs in the extracellular domain (Shiu and Bleecker 2003). Degenerate RT-PCR and Degenerate PCR Methods and Materials All jute seeds were obtained from Bangladesh Jute Research Institute. D3R. thaliana induced by osmotic stress due to dehydration. a phytohormone that regulates adaptation of plants to various stresses. are important in the transduction of environmental and developmental signals (Tichtinsky et al. Primers All primers used in this study were custom synthesized from 1st BASE Singapore Ltd. 2003). For low-temperature treatment. a farmer popular variety. additional sequence of this gene was deduced using a combination of gene walking (with the help of degenerate primers) and both 3′ and 5′ rapid amplification of cDNA ends (RACE). 2006). One of the predicted gene sequences showed homology with an internal exon of LRR-RLK. One of the predicted exon (657 bp) showed homology with putative LRR-RLK of Arabidopsis and a number of other plants. To examine the role of LRR-RLK in jute. Expression of this gene is also induced by exogenous abscisic acid (ABA) (Hong et al. high salt. In order to study the effect of exogenous ABA on jute LRR-RLK expression. 1997). Hong et al. for jute (Corchorus olitorius L. until they were harvested on day 5. were then separated from the culture. including sclerotia. Sequences of these primers are listed in Table 1. Mycelia. 3-day-old seedlings were sprayed with 50 mM NaCl (10 ml solution per day per petri dish) for 2 days and then harvested. For inducing salt stress. Finkelstein et al. 2002. a number of exons have been identified. 50 mM manitol was sprayed on 3-day-old seedlings (10 ml solution per day per petri dish). such as ORF finder. high salt. dehydration. analyses of a jute genomic library containing 137 SSR enriched sequences. Using this sequence as a base. and a 1% suspension (in water) of this culture was sprayed on 3-day-old jute seedlings including the filter paper (on which the seedlings were grown) and kept at 30°C for 2 days (Bhowal et al. Seeds were grown on 3MM filter papers (Whatman) in a petri dish. Identification of Genes from SSR Genomic Library SSR sequences were analyzed for gene sequence using bioinformatics tools. 3-day-old seedlings were kept at 16°C for 2 days following 3 days of growth at room temperature. and GSP4F) were designed from the initially identified 656 bp internal exon. a central single pass transmembrane helix and an intracellular Ser/Thr kinase domain. GSP2R. Plant LRR-RLKs possess a functional cytoplasmic kinase domain. the conserved N-terminal domain of bacterial flagellin) has shown that the receptor is specifically localized to the cell periphery. (Singapore). LRR-RLKs containing an extracellular LRR and a Ser/Thr kinase domain are localized on the plasma membrane and in many plants. Four degenerate primers (D1F. Out of 137 SSR clones only 16 were identified to have gene sequences. Using gene find and homology search tools. which was then confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) followed by sequencing of the RT-PCR product. The PRKs can be classified on the basis of their extracellular domains (Shiu and Bleecker 2003). by regulating stomatal aperture and the expression of stressresponsive genes (Leung and Giraudat 1998. Construction of SSR Genomic Library A genomic library was prepared with the help of a commercial company. Himmelbach et al. GenScan. Total RNA was extracted from 5-day-old jute seedlings using TRIzol (Gibco BRL of Invitrogen. (1997) have identified a single LRR-RLK in A. Macrophomina phaseolina was grown for 7 days in a potato dextrose media (PDA). Recent study using GFP tagged FLS2 (an RLK with a leucine-rich repeat domain that is involved in the perception of flg22. 16 h prior to collection. particularly in the membranes of cells that could serve as a point of entry for pathogens. GSP3F. such as drought. Sequence determination revealed that the jute LRR-RLK contains an extracellular LRR domain (ligand binding). DNA was isolated from 5 day old seedlings by Four gene specific primers (GSP1R. To simulate the dehydration effect.) var O-4. D2F. Sequencing of the SSR positive clones was done using ABI PRISM 310 nucleotide sequencer. and BlastX. These represent 13 subfamilies (LRR I to XIII). and D4R) were also designed from the region of this gene . 2006). Life Technologies) following the instructions of the manufacturer. Vizon (Canada).

PCR amplifications were performed using the same gene specific primers on different templates: 1 PCR product generated from genomic DNA. For use as primers. Invitrogen Corporation. First round PCR of target cDNA was carried out by employing the primer GSP6R and AAP primer for 35 cycles (95°C for 1 min. 54°C for 50 s. and subsequent PCR amplification was done by employing primer pair GSP3F/D3R for 35 cycles (95°C for 40 s. Nested amplification of first round PCR product was carried out using primer pair GSP2R/D2F for 40 cycles (95°C for 40 s.ATG GGG AAG TCA CCT CT GAA G-3′ 5′-TAA GACAA GTC AAG CAC ACG TAA CG-3′ 5′-TAC CGA CCG AAA GTT GCA GA-3′ 5′-ACA GGA GGG AAA GAT TCT ACC AC-3′ 5′. Carlsbad. GSP6R. Amplification of genomic DNA isolated from jute seedlings was also carried out using two primer pairs GSP1R/D2F and GSP4F/D3R. The first strand cDNA was prepared from mRNA using the primer GSP5rt. first strand cDNA was synthesized from total RNA of jute seedlings by using primer D3R. The first strand cDNA was prepared from mRNA 2000 bp 1650 bp 1000 bp Fig. First round PCR of this cDNA was completed by primer pair GSP1R/D2F for 35 cycles (95°C for 40 s. and 72°C for 1 min). CA 92008. 1 Comparison of the cDNA and genomic region of jute LRRRLK gene. and M is 1 Kb plus DNA ladder . 55°C for 1 min. three gene specific primers. 55°C for 50 s. Nested PCR of the first round PCR product was done employing primer GSP7R and AUAP for 35 cycles (95°C for 1 min.396 Table 1 Primer sequences used in this study Primer GSP1R GSP2R GSP3F GSP4F GSP5rt GSP6R GSP7R GSP8F GSP9F GSP10R D1F D2F D3R D4R AP AAP AUAP Sequence Plant Mol Biol Rep (2010) 28:394–402 5′-CAG AAG AGA AAG TGA ACT GG-3′ 5′-ATT GCC CAT TTC TGA AGG AAT TGG-3′ 5′-TGG AAC ATG ACG AGA CCT TGT CT TC-3′ 5′-TGT CAA GAG CTT CCA CAG AAT ATG-3′ 5′-TAT CGA CAT GGG GAA GTC-3′ 5′. first strand cDNA was synthesized from total RNA using the primer GSP1R. and 72°C for 50 s). 2 PCR product generated from cDNA. and 72°C for 1 min). and 72°C for 1 min). and GSP7R. For deducing upstream coding sequences. 5′ RACE 5′ RACE approach was used to deduce 5′ end sequences. 58°C for 40 s. were custom synthesized. Purification of first strand cDNA and TdT tailing was carried out as recommended by the manufacturer (5′ RACE system. USA). 1600 Faraday Avenue. and 72°C for 2 min). 3′ RACE 3′ RACE was performed for finding 3′ end of the gene. Nested amplification of the PCR product was performed by employing primer pair GSP4F/D3R for 35 cycles (95°C for 40 s. Two gene specific primers GSP8F and GSP9F were custom synthesized.GAA CAA CTT CAT TCA TGG TTG GAC G-3′ 5′-CCM YSA TGG GGT TYG CTC TT-3′ 5′-TAY CYM AAR GGA WCT CTC CCT-3′ 5′-CCC GAA RCT GTA CAC GTC GCA-3′ 5′-TCS AYC GGY TTC TTC CCC GT-3′ 5′-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3′ 5′-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GG GII G-3′ 5′-GGC CAC GCG TCG ACT AGT AC-3′ conserved in other plant species. 59°C for 1 min. For determining downstream coding sequences. 58°C for 40 s. and 72°C for 2 min). GSP5rt.

c Hydrophobicity plot of the LRRRLK protein sequence .Plant Mol Biol Rep (2010) 28:394–402 397 Fig. a Amino acid sequence of LRR-RLK. various domains and motifs are indicated. 2 The protein sequence and the 3D structure of LRR-RLK.helixes are shown in orange and the β-sheets in green. Extracellular and intracellular regions are shown in black and red. respectively.0 server. The α. b 3D structure of the jute LRR-RLK as predicted by CPHmodels-3.

398 Plant Mol Biol Rep (2010) 28:394–402 .

Ricinus2-EEF52196. Primer3. These primers. Hybridization and detection was performed according to the guideline of the manufacturer under highly stringent conditions. CA.Pyrus3-AAT28309. corresponding to the predicted exon. when gel extracted and sequenced. They are ORF finder.1. were designed using Primer3. MRN The cDNA sequence of the following proteins were used in tree construction.1. MEGA4 and Mobyle portal. Gene specific primers.megasoftware. j-132-M13F-PUC (GI: 71082462) contains an exon of 656 bp. 0. For this analysis. Malus3-AAC36318.1. Pyrus1-AAT28307. Arabidop1-ACN59387.hmm.50 min).1. Ricinus3-EEF52194.1. Arabidop4-AAL12626. USA) using a cycle-sequencing protocol suggested by the manufacturer (ABI). Malus2-ABO61511.1.1. Semiquantitative RT-PCR Total RNA was isolated from jute seedlings and quantified using a nanodrop (ND-1000). PCR amplification of the cDNA was carried out for 25 cycle using primers GSP10R and GSP8F (95°C for 40 s. specific for the predicted exon.1. DNA Sequencing All DNA sequences were determined by an automated sequencer (model 381A. 11 175 033 910). RT-PCR amplification was performed in order to confirm this. Poly (A) RNA was isolated from total RNA using mRNA extraction kit (SIGMA. 55°C for 1 min. After a single band was isolated from agarose gel. Translate.The sequencing services were obtained from Macrogen online sequencing service of South Korea. was reverse transcribed.0. and 72°C for 1. b Phylogenetic analysis of jute LRRRLK performed on full-length cDNA sequences from various plants using the MEGA4. stringency washes with 0. Oryza4-BAB40094.hmm and GenScan confirmed that the 948 bp sequence of the SSR clone. 046k6879) and quantified. prepared from jute seedlings. and DNA was extracted using MinElute gel extraction kit following manufacturer’s (QIAGEN) instructions.1% SDS). Southern Blot The complexity of LRR-RLK gene in the genome of C. 11 175 033 910).1. and 72°C for 50 s). BlastX search of the exon sequence showed homology to a number of plants LRR-RLKs. and 72°C for 2 min).5% agarose gel for 40 min at 75 V. . Arabidop2-BAC42540. a Alignment of jute LRR-RLK with other homologous LRRRLKs from dicotyledenous plants. olitorius L. Correct sized bands were then cut from gel without exposing the same to UV light. Foster. CPHmodels-3. 1997) was performed by fixing 15 µg of total RNA in a positively charged nylon membrane (Amersham Hybond™-N). Oryza3-BAF30366. DNA Sequence Analysis The DNA sequences were analyzed with a number of freely available online software. Glycine1-AAF91322.3.0 program (http://www.html). Expression of β-actin was used as internal standard. Oryza2-ABA99940. Total RNA. GeneBank protein accession numbers: Corchorus— ACI42311. by employing the primer AP.1 of the manufacturer for highly stringent hybridization and detection conditions was followed (probe 10 ng/ml. The guideline Results Identification of an Internal Exon of Leucine-Rich Repeat Receptor-Like Protein Kinase Both GeneMark. This band. Arabidop3-ACN59227. Probe was synthesized from a RT-PCR product containing sequences from both exons using digoxigenin (DIG) nucleic acid labeling and detection kit (Roche. ClustalW. 3 Comparison of amino acid sequences of the jute LRR-RLK with other reported RLK proteins from higher plants available in the NCBI database.1. The housekeeping gene β-actin was used as the internal standard for reverse transcriptase polymerase chain reaction (RT-PCR).1. GenScan. Purification of PCR Products PCR product was run on 1. its concentration was checked by a nanodrop (ND-1000).1. First strand cDNA was synthesized from 50 ng of mRNA employing gene specific primer GSP10R. Malus4-ABO61514. Nested amplification of PCR product was carried out using primer pair GSP9F and AUAP for 40 cycles (95°C for 1 min. and Glycine2AAF91324. was examined by Southern blot analysis. gave exactly the same sequence as for the SSR clone (j-132-M13-PUC).1.1.5× SSC. 15 μg of EcoR1 and BamH1 digested genomic DNA was size-fractionated on 1% agarose gel and then transferred onto a positively charged nylon membrane (Amersham Hybond™-N). ABI Applied Biosystems. confirmed the presence of the exon by giving a band of the expected size. Ricinus1-EEF52195. RNA Dot Blot RNA dot blot (Hong et al.1. Pyrus2-AAT28308.1. Oryza1-BAF30367. 58°C for 1 min.Plant Mol Biol Rep (2010) 28:394–402 399 R Fig. InterProScan. First round PCR of the cDNA was performed by employing primer pair GSP8F and AUAP for 35 cycles (95°C for 1 min.1.. and the resulting single stranded cDNA was PCR amplified.1. Ricinus4-EEF40238. Malus1-ABO61511. Probe was synthesized using DIG nucleic acid detection and labeling kit (Roche. Arabidop5NP_172468. Inc. 58°C for 40 s.1. GeneMark. BLAST.

Sequence analysis of the full-length cDNA revealed an open reading frame of 2.or BamH1-digested genomic DNA (Fig. It is known that ABA is involved in a variety of stress responses Fig. and (e) a Ser/Thr kinase active site (Fig. which may have only a single copy in the jute genome as suggested by the Southern hybridization. The gene was then PCR amplified from both RNA and genomic DNA using three pairs of primers.879 bp encoding a putative protein of 957 amino acids with a predicted molecular weight of 106.dtu. (b) a phosphotransferase. After being exposed to low temperature. Significant increase was observed under low temperature. A nonrooted. we detected single bands in EcoR1. 3a). functions as a receptor protein kinase. 4). We have shown that the gene is induced by ABA. 6). 2008). and fungus for 2 days. was carried out on genomic DNA. (d) an intracellular kinase domain with ATP binding site. the transcript level increased under all stress conditions as well as in abscisic acid treatment (Fig. Integr8) against CATH structural domain database (Yeats et al. Thus. RefSeq. The remaining sequence at the two ends of the cDNA was deduced by 5′ and 3′ RACE. RNA Dot Blot RNA dot blot was performed to verify and strengthen the findings of semiquantitative RT-PCR.). The predicted protein also has 3D domain (Super family) structures similar to (a) a phosphorylase kinase.197 kDa. These PCR products were then sequenced. Protein sequence analysis using InterProScan revealed (a) a leucine-rich repeat N-terminal domain.400 Plant Mol Biol Rep (2010) 28:394–402 Deduction of Full-Length Coding Sequence of the Gene To deduce the full-length coding sequence of jute LRR-RLK. Hydrophobicity is needed for the localization of the protein on the plasma membrane and the signal peptide could possibly be removed after targeting. The predicted protein also contains a hydrophobic signal peptide which has a signal peptide cleavage site. this LRRRLK gene is most likely to be a single copy gene in the haploid genome of C. 4 Southern blot analysis of LRR-RLK: Both EcoR1 and BamH1 digested DNA gave a single band on Southern blot EcoR1 BamH1 5kb 4kb 3kb 2kb . DNA Gel-Blot Analysis The complexity of the LRR-RLK gene in the genome of C. The increase was several folds in case of abscisic acid treatment. (b) several leucine-rich repeats. 3D structure of jute LRR-RLK as predicted by CPHmodels) is shown in Fig 2b. 5). Discussion We report here the complete cDNA and genomic sequence of a putative LRR-RLK gene of jute (C. The tree was constructed using MEGA4 (Tamura et al. Gene3D assign sequence signatures to 3D domain structures by scanning protein sequences (UniProt. Comparison showed that the genomic region of LRR-RLK has only one intron of 108 bp. Phylogenetic clustering of jute LRR-RLK with two Arabidopsis proteins suggests that jute LRR-RLK is an ortholog of these two Arabidopsis paralogs. Only the primer pair flanking the intronic region gave differently sized bands (Fig. 2007). PCR. and fungal stress. an extracellular LRR domain. The sequence has been submitted to NCBI. high salt. and compared with the cDNA sequence. and (c) an LRR domain. Semiquantitative RT-PCR Semiquantitative RT-PCR analyses of the LRR-RLK transcripts showed that LRR-RLK is expressed at low levels in untreated jute seedlings. and a highly conserved kinase domain separated by a hydrophobic transmembrane helix suggests that the encoded protein. Another degenerate RT-PCR gave 745 bp sequences towards the 3′ end. Both the results were found to be similar (Fig. degenerate RT-PCR method was used in obtaining an 891 bp of additional upstream sequence of the predicted exon. bootstrap consensus plot generated using the maximum parsimony method with cutoff value for condensed tree set at 60 is shown in Fig. 2a). The deduced amino acid sequence of jute LRR-RLK was also aligned with other reported RLK protein sequences of dicotyledonous plants in the NCBI database (Fig. assembled. These 3D structures were predicted using Gene3D search of InterProScan. (c) a transmembrane helix. the accession number of which is GI: 209168628. olitorius (cultivar of jute) was examined by DNA gel-blot analysis. However. Presence of a signal peptide sequence. olitorius L. the increase was only slight under salt stress. using gene specific primers designed from sequence covering the complete cDNA. 3b.0 server (http://www. dehydration. 1). Using a cDNA probe that contained sequences from both exons. dehydration.

1994). Expression levels of β-actin served as an internal control . 2008). (2003) have shown that ABA signaling occur at the plasma membrane in extracellular ABA perception. this could possibly be due to the fact that the gene is induced more by water deficit than Normal LRR-RLK Low T osmotic stress or the salt concentration (50 mM) used in this study was not high enough to elicit sufficient osmotic stress. Moreover. PKABA1 (Holappa and Walker-Simmons 1995).Plant Mol Biol Rep (2010) 28:394–402 Normal LRR-RLK β-Actin Low T Dehydration High Salt Fungus ABA 401 Fig. Jute seedlings were exposed to various stresses (as indicated in the figure) for a specified period (see “Methods and Material” section). The LRR sequence is known to be involved in strong protein–protein interactions (Kobe and Deisenhofer 1995). 2008). as has been suggested for a wheat protein kinase. It is possible that the LRR-RLK interacts with ABA through ABA-binding protein in the extracellular region. The increased expression of the gene in fungal stress may be due to the accumulation of ABA at the infection site (Schimdt et al. 1997). it is possible that induction of the LRR-RLK gene is due to a common osmotic stress and that the gene is involved in an osmotic rather than in a general stress response. Alternatively. 4 and 5). Thus. The presence of a transphosphorylase domain in LRR-RLK suggests that this protein could be activated by interreceptor transphosphorylation followed by oligomerization as in the case of brassinosteroid receptor in plants (Xiaofeng et al. 1997). Zeevaart and Creelman 1988. 1997). Trewavas and Jones 1991. Some polypeptides induced by ABA and osmotic stress. such as maize RAB-17 (Plana et al. as in the case of some animal receptor kinases (Hong et al. 1997). Although the LRR-RLK gene appears to be involved in a general stress response. Dehydration causes osmotic stress by a direct loss of water (Hong et al. this observation suggests that LRR-RLK may function in the transmission of various ABA-mediated environmental stress and defense response signals. 6 RNA dot blot showing similar expression pattern of jute LRR-RLK as observed by semiquantitative RT-PCR. Yamazaki et al. although the adaptive. regulation of the gene by a variety of stresses and ABA may indicate that the gene is involved in a general stress response. one possible common physiological and physical environment that plant cells experience as a result of dehydration. 1991) and tomato dehydrin (Godoy et al. 1997). 5 RT-PCR analyses of the jute LRR-RLK transcripts induced by low temperature dehydration. high salt and low temperature is water deficit and/or osmotic stress. Skriver and Mundy 1990. Expression levels of β-actin served as an internal control (Osakabe et al. ABA has also been shown to be involved in an osmotic stress response that is caused by several environmental stressors (Hong et al. and abscisic acid. an ABA-specific binding protein from broad bean leaves was purified and shown to be functionally an ABA receptor in guard cell protoplasts (Zhang et al. This study also shows that the gene is induced by several abiotic (dehydration. High salt causes osmotic stress by reducing water potential (Hong et al. A number of evidences suggest that protein phosphorylation may be involved in the pathway (Hong et al. Specific protein kinases that can phosphorylate these proteins in vitro have Fungus ABA Dehydration High Salt β-Actin Fig. fungus. 2005). high salt concentration. Davies et al. high salt. and molecular responses to osmotic stress have been studied intensively in plants (Mansfield 1988. 1994). are accumulated in a phosphorylated form. The expression of LRR-RLK is significantly low in salt stress compared to other stresses (Figs. a protein–protein interaction involving homoor hetero-oligomerization of LRR-RLK on the outside of the plasma membrane may occur. Cold stress is also known to cause osmotic stress by reducing the water supply from the roots to green tissues (Yamaguchi-Shinozaki and Shinozaki 1993). Bray 1994. physiological. and low temperature) and biotic (fungal) stresses. Considering LRR-RLK to be a receptor protein kinase. 2002). Knowledge of the molecular mechanisms that operate in the signal transduction pathway of cellular response to osmotic stress is still minimal. In addition.

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