Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses

ORIGINAL ARTICLE

Construction of cold induced subtracted cDNA libraryfrom Cicer microphyllum and transcript characterizationof identified novel wound induced gene

Rupesh Kumar Singh & Shweta Singh & Pankaj Pandey &

Sivalingam Anandhan & Danswrang Goyary &

Veena Pande & Zakwan Ahmed

Received: 23 February 2012 /Accepted: 18 June 2012# Springer-Verlag 2012

Abstract A forward cold induced subtracted cDNA librarywas constructed to identify the stress regulated genes in ahigh altitude cold desert-adapted species Cicer microphyl-lum, a wild relative of cultivated chickpea, distributed inwestern and trans-Himalayas. A total of 1,040 clones wereobtained from the subtracted cDNA library. These cloneswere screened for presence of insert with colony PCR. Atotal of 523 clones were picked by colony PCR amongwhich 300 clones were observed differentially expressedas per dot blot analysis. Differentially expressed clones weresequenced and assembled into clusters based on the pres-ence of overlapping, identical, or similar sequences. A totalof 283 ESTs were submitted in gene bank (accession numb-ers GO241043 to GO241326). BLAST analysis of these

ESTs revealed its similarity for regulatory proteins likekinases, metallothionin, and enzymes/proteins with un-known functions. A cDNA encoding wound induced likeprotein, identified from this cold induced subtraction cDNAlibrary, was full-length cloned using RACE and sequenced(accession number GQ914056). Southern blot of C. micro-phyllum indicated single copy of the gene in genome. Tran-script expression profiling of this gene by quantitative real-time PCR and northern blot confirmed its up-regulationduring low temperature stress. Further, in situ RNA hybrid-ization also revealed cold (4°C) induced expression of thegene.

Keywords Cicer microphyllum . Low temperature .

Suppression subtraction hybridization (SSH) .Woundinduced gene . qRT PCR . In situ hybridization

Introduction

Low temperature is one of the major environmental factorslimiting growth, productivity, and geographical distributionof crop plants. Being a polygenic trait, the complexity of theacclimation process is reflected by involvement of a largenumber of genes that are affected by low temperature, whichaccording to an estimate is up to 25% of the transcriptome inArabidopsis (Kreps et al. 2002). Molecular studies indicatethat cold acclimation involves major changes in expressionof cold-responsive COR genes. The alterations result invarious physiological and biochemical changes during theprocess of cold acclimation, and the combined effect of thegene products is manifested in the level of freezing toleranceobtained (Chinnusamy et al. 2003, 2007; Novillo et al.2004; Thomashow 2010). The activation of these CORgenes is controlled by a set of signaling pathways triggeredby exposure to the low temperature stimulus. Genes induced

Handling Editor: Bhumi Nath Tripathi

Electronic supplementary material The online version of this article(doi:10.1007/s00709-012-0429-z) contains supplementary material,which is available to authorized users.

R. K. Singh : S. Singh : P. Pandey : S. Anandhan :D. Goyary (*) : Z. AhmedDefence Institute of Bio-Energy Research, Ministry of Defence,Haldwani 263139 Uttarakhand, Indiae-mail: [email protected]

V. PandeDepartment of Biotechnology, Kumaon University,Nainital 263 001 Uttarakhand, India

Present Address:S. AnandhanDirectorate of Onion and Garlic Research Institute,Rajgurunagar,Pune 410 505 Maharashtra, India

Present Address:D. GoyaryDefence Research Laboratory, Ministry of Defence,Tezpur 784 001 Assam, India

ProtoplasmaDOI 10.1007/s00709-012-0429-z

during cold stress include those that encode enzymes requiredfor biosynthesis of osmoprotectants, lipid desaturases formaintaining membrane fluidity, protective proteins such asantifreeze proteins, dehydrins, chaperons, and proteins in-volved in signal transduction such as transcription factors,protein kinases, and phospholipase C (Fowler and Thoma-show 2002). Response to cold stress varies between speciesbelonging to tropical and temperate climate.

Cicer microphyllum is a high altitude legume plant wide-ly distributed in cold deserts of Leh and Ladakh in India(Chaurasia et al. 2007). Therefore, in order to study theresponses associated with cold adaptation, subtracted cDNAlibrary was constructed by using cold acclimated and con-trol poly(A)+ RNA of C. microphyllum. Herewith, we reportconstruction of cold induced subtracted cDNA library andidentification of differentially expressed clones in C. micro-phyllum to understand the molecular mechanisms in termsof transcript regulation underlying the cold acclimation.Further, a novel putative wound induced gene identified inthe subtracted library was full length cloned, copy numberstudied with southern hybridization, and the transcript ex-pression in C. microphyllum characterized. The purpose ofstudying this gene was to investigate whether wound in-duced gene have any role against the formation of icecrystals during cold stress which may also cause disruptionor wounds.

Materials and methods

Plant material, treatment, and RNA isolation

Seeds of C. microphyllum were collected from DefenceInstitute of High Altitude Research, Leh, India and in vitrocultures were established from excised embryo (Singh et al.2007). The plants were transplanted in the pots filled withsoil mixture (vermiculite/peat moss/soil, 1:1:1) in cultureroom at 25°C with 16:8 h light and dark cycles. Plants wereexposed to cold stress at 4°C for 24 h. RNA was isolatedfrom 1 g each of control and stressed tissue by GTC method.The quality of RNAwas assessed by gel electrophoresis andquantitative estimation was done based on spectrophotomet-ric absorbance. Total RNA was treated with RNase-freeDNase (Fermentas Life Sciences) to remove genomicDNA contamination. Poly (A+) RNAwas enriched by usingPolyATtract® mRNA isolation system I (Promega, USA).

Suppression subtractive hybridization (SSH) and libraryconstruction

Subtractive hybridization was performed using the PCR-select cDNA subtraction kit (Clontech, USA) as per manu-facturer’s instructions. In brief, first and second strand

cDNA was synthesized from 2 μg of poly (A)+ RNA fromstressed (tester population) and control (driver population)tissue. The cDNA samples were digested with RsaI anddriver cDNA was ligated with adapters for further hybrid-ization reactions. In the first hybridization, an excess ofdriver cDNA was hybridized at 68°C for 8 h with testercDNA. In the second hybridization, both the reactions werehybridized together in the presence of fresh driver cDNA at68°C for overnight. After second hybridization, the sub-tracted product was amplified by PCR using oligonucleo-tides complementary to adapters. PCR was carried with aninitial denaturation at 75°C for 5 min followed by 27 cyclesof 94°C for 30 s, 66°C for 30 s, and 72°C for 1.5 min. Anested PCR reaction of 12 cycles at 94°C for 30 s, 68°C for30 s, and 72°C for 1.5 min was carried out. The final PCRproduct was electrophoresed on 2% agarose gel that showeda clear smear. The subtracted product was purified usingQIAquick PCR Purification kit (QIAGEN, Germany)digested with RsaI enzyme and ligated at SmaI digestedpBluescript KS (+) vector. Ligated products were trans-formed to DH5α cells and recombinants were selected onLuria Agar plate with ampicillin (100 μg/ml), IPTG(0.1 mM), and X-gal (40 μg/ml).

Screening of the subtracted library

The recombinant clones from the subtracted library werescreened for size of insert by colony PCR using M13 pri-mers. Amplification was performed with an initial denatur-ation at 95°C for 4 min, followed by 30 cycles of 95°C for30 s, 55°C for 1.0 min, and 72°C for 1.5 min, and a finalextension of 5 min at 72°C. PCR products were separated on1.0% agarose gel and recombinants were picked for differ-ential screening by dot blots.

Differential screening of the subtractive clones

Dot blots were prepared in duplicate in a lattice pattern byapplying a mixture of 5 μg PCR product of each positiveclone and 5 μl of freshly prepared 0.6 N NaOH to aHybond-N+ nylon membrane (Amersham Biosciences) andcross-linked using a UV cross-linker (Amersham Bioscien-ces) with a UV exposure (12,000 μJ/cm2). Formamide-based hybridization buffer (Sambrook et al. 1989) was usedfor hybridization at 42°C for 16 h. The blots were separatelyhybridized with labeled cDNA probes prepared frommRNA pool of the stressed and unstressed tissues. Afterhybridization, washing was performed in 2× SSC (salinesodium citrate), 0.1%w/v SDS (sodium dodecyl sulfate) for2×5 min, 0.5× SSC, 0.1%w/v SDS for 2×15 min followedby 0.1× SSC, 0.1% w/v SDS for 2×5 min at room temper-ature. Hybridized and washed blots were exposed to KodakX-ray film and stored at −80°C for autoradiography. The

R.K. Singh et al.

intensity of the dot blot was analyzed using AlphaEaseFC™software (Alpha Innotech Corporation).

Sequencing of cDNA clones and sequence analysis

Sequencing of differentially expressed clones was per-formed by outsourcing to Bangalore Genei, Bangalore, In-dia. Sequences were analyzed for homology with BLAST(Altschul et al. 1990) at NCBI.

Amplification of full-length cDNA of wound induced gene

Sequence of one of the differentially expressed cDNA clonesfrom the subtracted cDNA library revealed homology withputative wound induced gene whose full-length open readingframe (ORF) was amplified by rapid amplification of cDNAends (RACE) method. The sequence was complete from 5′end therefore remaining 3′ end sequence was investigated. Forthis, seedling of C. microphyllum was exposed to low temper-ature (4°C for 24 h) and RNAwas extracted from cold accli-mated leaf sample followed by mRNA purification. Reversetranscription and 3′ end amplification was carried out by usingGeneRacerTM RACE Ready cDNA Kit (Invitrogen). RACEwas performed by using 3′ adapter primer (5′-CGCTACG-TAACGGCATGACAGTG-3′) and 3′ gene-specific primer(5′-CCTCTGATTGCTTTGCATTC-3′).

Quantitative RT–PCR transcript expression analysis

C. microphyllum plants were subjected to low temperaturestress (4°C) for two different time durations (12 and 24 h)along with unstressed control. The leaf samples harvestedwere frozen with liquid nitrogen. Total RNA was isolatedusing RNeasy plant mini kit (Qiagen) and further treated withRNase free DNase. Total RNA was quantified (Qubit flour-ometer—Invitrogen) and equal quantity of RNAwas used forfirst strand cDNA synthesis. Two pairs of primers for detec-tion of putative wound induced gene (forward 5′-ATGAGTCCATCAAGCAGAGCAT-3 ′ and reverse primer 5 ′-TCATTTGCAGGTGCAAGGGTTG-3′) with an internalcontrol of 26S rDNA sequence (forward 5′-CACAATGATAGGAAGAGCCGAC-3′ and reverse primer 5′-CAAGGGAACGGGCTTGGCAGAAT-3′) were designed. Equalquantity of cDNA was used for quantitative PCR (Mx3005PStratagene/Agilent) with SYBR green master mix (Stratagene/Qiagen) and wound induced gene-specific primers in 25-μlreaction with PCR conditions of 95°C for 10 min followed by45 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s.The transcript expression was normalized with an internalcontrol (26S rDNA). The data obtained was presented as foldchange in transcript level with reference to control (calibrator).Similarly, the transcript abundance of wound induced gene ofC. microphyllum was also investigated after foliar spray of

salicylic acid (mock, 10 and 50 μM) and leaf samples collect-ed 24 h after spray. Control and treatment of experiments werereplicated three times. Data were analyzed using the statisticalprogram Cropstat (IRRI, Philippines) for the experiment laidin a completely randomized design.

Northern blot assay

C. microphyllum plants were subjected to low temperature(4°C) stress for 0 (control), 6, 12, and 24 h. Leaf sampleswere harvested and frozen with liquid nitrogen for totalRNA isolation by GTC method. The total RNA (10 μg)was quantified (Qubet flourometer—Invitrogen) and sepa-rated on 1% formamide denatured agarose gel. RNA wastransferred to Hybond-N+ membrane (Amersham Bioscien-ces) for 16 h (Sambrook et al. 1989) and cross-linked to themembrane using a UV cross-linker. The blot was hybridizedwith biotin labeled probe (Biotin Deca Lable DNA Label-ling Kit—Fermentas Life Sciences) and developed (BiotinChromogenic Detection Kit—Fermentas Life Sciences) asper manufacturer’s protocol. Northern blot experiment wasrepeated to confirm the results.

Whole plant in situ hybridization

Whole plant in situ hybridization was carried out for detec-tion of transcript distribution of wound induced gene in theplant. Whole plant was subjected to low temperature stress(4°C; 24 h), then shoots were taken out from stressed as wellas control plants. One positive and negative control was alsoconsidered for confirmation of the results. Plant tissues werethen fixed in ethanol and stored at −80°C for overnightfollowed by −20°C for 12 h and warmed up to room tem-perature for 12 h. The tissues were washed with RNAse-freewater and treated with bursting solution containing0.25 mM sodium azide, 0.1%w/v SDS, 5 mM EDTA, and1 mg/ml pronase. Tissues were then washed, baked, andhybridized with biotin labeled probe (Biotin Deca LableDNA Labelling Kit—Fermentas Life Sciences). Finally,the tissues were washed with 2× SSC solutions and devel-oped using Biotin Chromogenic Detection Kit (FermentasLife Sciences, USA) as per manufacturer’s instruction.

Southern blot analysis

Total genomic DNA was extracted and purified from 15-day-old seedlings of C. microphyllum using CTAB method(Doyle and Doyle 1987). Restriction digestion of purifiedgenomic DNA (10 μg) was performed with EcoRI andBamHI restriction enzymes. The digested product was sep-arated on 1% agarose gel, transferred to a Hybond-N+ mem-brane (Amersham Biosciences), and cross-linked using aUV cross-linker (Amersham Biosciences) with a UV

Construction of cold induced subtracted cDNA library from Cicer microphyllum

exposure (12,000 μJ/cm2). The blots were hybridized withprobe labeled using biotin labeling kit (Fermentas Life Sci-ences). The hybridization was performed at 42°C in aformamide-based hybridization buffer (Sambrook et al.1989). Blot was developed as per user manual of BiotinChromogenic detection kit (Fermentas Life Sciences).

Results and discussion

Construction, screening, and sequencing of subtractedcDNA library

A total of 1,040 clones were obtained from the subtractedcDNA library. These clones were screened for presence ofinsert with colony PCR (Electronic supplementary material1a). A total of 523 clones were picked by colony PCR amongwhich 300 clones were observed differentially expressed asper dot blot analysis (Electronic supplementary material 1b).The differentially expressed clones were sequenced and theobtained sequences were assembled into clusters based on thepresence of overlapping, identical, or similar sequences. Atotal of 283 ESTs were submitted in NCBI GenBank (acces-sion numbers GO241043 to GO241326). The remaining 17clones were not submitted to the gene bank due to their veryshort length and non-significant homology.

Similarity search

BLAST search of sequenced clones revealed similarity withknown genes. However, some of the ESTs revealed nosignificant similarity with that available in NCBI database.The ESTs showing significant similarity with previouslyreported genes in GenBank have been summarized in Ta-ble 1. The ESTs from forward subtracted library yielded 46single tons with an average length of 307 base pairs, whichcould be assigned putative functions on the basis of se-quence similarity to genes or proteins of known functionin the sequence data base. Detailed analysis revealed thatout of 283 transcripts up-regulated in cold stress, only 46transcripts (16.0%) are having a known function and theremaining 237 transcripts (84.0%) were of unknown func-tions. Most of the up-regulated transcripts were categorizedunder metabolism (4.0%), hypothetical protein (4.0%) fol-lowed by translation/post-translation (2.0%), housekeeping/cell development (2.0%), degradation (1.5%), transporter(1.5%), stress and signaling (0.7% each), and activator(0.4%). This result suggests the presence of putative novelgenes that may be specific to C. microphyllum and mightalso play important roles in cold stress adaptation (Fig. 1),corroborating with the previous report of 14% unknown/unclassified functions, induced upon dehydration stress infoxtail millet (Lata et al. 2010).

Many genes encoding cold stress responsive proteins wereobtained from forward subtracted library, such as cold accli-mation responsive protein, stress related protein of Zea mays;auxin induced protein and senescence related proteins havebeen identified as reported in early studies (Dhananjay et al.2007). These are proteins that have been shown previously tobe associated with cold stress responses on blueberry plants(Dhananjay et al. 2007) and in other plants (Hannah et al.2005). Many clones encoding putative transcription factorsand other proteins related to signal transduction were alsopresent such as zinc finger protein, putative myb-related pro-tein, and protein kinase family protein whose roles in coldacclimation responses are well known (Dhananjay et al. 2007;Mukhopadhyay and Tyagi 2004; Davletova and Mittler 2005;Urao et al. 1993). Identification of these genes in C. micro-phyllum may be validated functionally for application in en-gineering cold tolerance in crops. Besides, clones encodingspecific proteins like DNA binding proteins, calumenin ho-mologue, putative stress responsive protein, and serine-threonine kinase were also identified in cold subtracted li-brary. However, understanding their roles in cold tolerancewarrants further investigation.

Themostabundantclonefromthe forwardsubtracted librarywas found to be a gene encoding auxin induced protein (acces-sionnumberGO241313).Kyohei et al. (2009) has reported thatcold stress affects auxin transport rather than auxin signaling inArabidopsis thaliana. Furthermore, the inhibition of proteintrafficking by cold is independent of cellular actin organizationand membrane fluidity. These results suggest that the effect ofcold stress on auxin is linked to the inhibition of intracellulartrafficking of auxin efflux carriers. In the same direction, Lee etal. (2005) stated that a number of genes important for biosyn-thesis and signaling of plant hormones, such as abscisic acid,gibberellic acid, and auxin, are regulated bycold stresswhich ispotentially important in coordinating cold tolerance withgrowth and development. Bhoomica et al. (2007) constructedauxin induced cDNA library and concluded that a substantialnumber of genes are involved in hormone response, signalcascades, defense, anti-oxidation, programmed cell death/se-nescence, and cell division. In the present study, a senescenceassociated proteinwas observeddifferentially expressed in lowtemperature stress. Results demonstrated by Bhoomica et al.(2007) suggested that auxin regulated genes may also be in-volved in zygotic embryogenesis and developmental reprog-ramming processes, and may in fact be involved in multiplecellular pathways which are yet unknown and need furtherinvestigation.

Another clone exhibited homology with cysteine proteaseand cathepsin-L like proteins (accession no. GO241197).Cysteine proteases of the papain super family have long beenrecognized for their role in intracellular and extracellularprotein degradation in a range of cellular processes (Bondand Butler 1987). Within the papain family, the cathepsins

R.K. Singh et al.

Table 1 List of subtracted cDNA clones showing similarity with NCBI database

Sl. no. Accession no. Size (bp) Homology E value

Activator

1 GO241313 487 Auxin-induced protein 6B 3e−31

Degradation

2 GO241318 146 Proteinase-like cysteine protease 3e−07

3 GO241197 340 cathepsin L-like (Salmo salar) 1e−10

4 GO241242 331 Ubiquitin family protein (Arabidopsis lyrata) 7e−07

5 GO241126 238 ATP-dependent Clp protease proteolytic subunit (ClpP4) (Arabidopsis thaliana) 8e−04

Metabolism

6 GO241127 232 Putative extracellular dioxygenase 2e−04

7 GO241128 226 Chloramphenicol acetyltransferase 2e−17

8 GO241324 402 Medicago truncatula Ser/Thr protein kinase (PRK), exostosin-like protein 7e−63

9 GO241303 257 Glutamate 2e−07

10 GO241289 482 Putative protocatechuate dioxygenase (Streptomyces avermitilis) 8e−14

11 GO241262 340 SAUR family protein (Populus trichocarpa) 6e−14

12 GO241215 156 GDP-L-galactose phosphorylase 2e−04

13 GO241187 180 Catalase (EC 1.11.1.6) CAT-2—maize (fragment) 1.4

14 GO241142 211 Class I chitinase 3e−06

15 GO241100 141 KAT2 (potassium channel in Arabidopsis thaliana 2) 0.001

16 GO241107 543 Chalcone reductase (Medicago sativa) 8e−78

Housekeeping/cell development

17 GO241299 716 Senescence-associated protein from Picea abies 1e−42

18 GO241191 442 Putative reverse transcriptase (Cicer arietinum) 8e−04

19 GO241185 329 Putative 60S ribosomal protein RPL10 (Novocrania anomala) 3e−12

20 GO241046 278 Ribosomal protein L19 (Triticum aestivum) 0.026

21 GO241129 90 SocE (Bacillus cereus) 0.81

Translation/post-translation

22 GO241074 144 Stress-responsive protein (Zea mays) 0.001

23 GO241147 198 MYB-like DNA-binding protein (Catharanthus roseus) 0.002

24 GO241161 217 Cold acclimation responsive protein BudCAR4 (M. sativa) 1e−05

25 GO241302 298 Zinc finger protein, putative from Ricinus communis 2e−22

26 GO241290 204 Signal peptidase I, putative (Ricinus communis) 8e−15

27 GO241293 215 Similar to digestive cysteine proteinase 2 (T. guttata) 2e−13

Transporter

28 GO241294 219 Nitrate transporter, putative (Ricinus communis) 6e−07

29 GO241227 123 Transporter, putative (Ricinus communis) 0.056

30 GO241123 157 Nitrate and chloride transporter (Glycine max) 8e−06

31 GO241051 219 Calumenin homologue (Ciona intestinalis) 0.005

Stress

32 GO241174 225 C. arietinum mRNA for class I type 2 metallothionein 1e−84

33 GO241284 526 Wound-responsive family protein (Arabidopsis thaliana) 5e−12

Signaling

34 GO241289 482 Putative membrane protein (Streptomyces scabiei) 2e−14

35 GO241095 109 Signal peptidase I, putative (Ricinus communis) 0.47

Hypothetical protein

36 GO241159 276 AT5G55660 (Arabidopsis thaliana) 4e−08

37 GO241087 136 Unnamed protein product (Ostreococcus tauri) 0.056

38 GO241143 159 Hypothetical protein (Arthrobacter sp. JEK-2009) 6e−19

39 GO241130 164 Unknown (Zea mays) .043

40 GO241167 708 Predicted protein (Populus trichocarpa) 0.002

Construction of cold induced subtracted cDNA library from Cicer microphyllum

can be subdivided into more than 10 subfamilies on the basisof their primary sequence and enzymatic activity (Santamariaet al. 1998). The family includes cathepsin B, C, L, and Z, allof which contain an essential cysteine residue in their activesite but differ in tissue distribution and in some enzymaticproperties, such as substrate specificity and pH stability (Tortet al. 1999). The role of these genes in low temperature stressis not clear and needs further investigation.

Other interesting clones from the subtracted library werecDNA encoding ion transporters and zinc finger familytranscription factors (accession no. GO241302). Results ofearlier microarray experiments in Arabidopsis also indicatedcold induced transcript expression of these two families(Hannah et al. 2005). In addition, Dhananjay et al. (2007)reported increased expression of zinc finger protein on ex-posure to cold stress in blueberry plants. XERICO is anArabidopsis zinc finger protein shown to confer droughttolerance through increased abscisic acid biosynthesis (Koet al. 2006). These results support the construction of theforward subtracted library enriched for genes whose expres-sion is up-regulated during cold acclimation.

One of the cDNA clones (accession no. GO241303)represented a glutamate-like gene which encoded the en-zyme glutamate synthetase. The enzyme plays a central rolein plant nitrogen metabolism and may be synthesized andmetabolized by a number of different pathways (Brian andLea 2007, 1998). Lam et al. (2006) stated that glutamatesignaling may be part of a much broader network of Nsignaling pathways that enable plant to monitor and adaptto changes in its N status and the N supply. H-proteinpromoter binding factor-1 was also identified in cold in-duced subtracted cDNA library. Joel et al. (2002) had dem-onstrated approximate 5-fold increase in transcript level ofthis gene after 3 h of cold stress (4°C) in roots and leavesseparately.

Another cDNA clone (accession no. GO241161) exhibitedhomology with cold acclimation responsive protein, whichbelongs to the dehydrin family. The expression of these genesis reported to increaseafter 2weeksof cold treatmentwithmoreexpression inradicle than incotyledon(Liuetal.2006).Further,function prediction suggested a role in protection ofmembranestructure and prevent macromolecular coagulation or

Table 1 (continued)

Sl. no. Accession no. Size (bp) Homology E value

41 GO241279 520 Unknown protein (Glycine max) 1e−06

42 GO241296 602 Hypothetical protein (Medicago truncatula) 5e−24

43 GO241322 499 Conserved hypothetical protein from Ricinus communis 2e−16

44 GO241326 542 Unknown protein from Arabidopsis thaliana 8e−04

45 GO241304 245 H-protein promoter binding factor−1 from A. thaliana 3e−07

46 GO241304 131 Hypothetical protein (Trifolium pretense) 0.004

Up-regulated transcripts in Cicer microphyllum in response to cold stress. The transcripts are listed according to their possible functions

Fig. 1 Pie chart depictingfunctional classification of 283differentially expressedtranscripts under cold stress inC. microphyllum identifiedfrom the libraries

R.K. Singh et al.

sequestrate calcium ions by association or disassociation withmembrane under low temperature conditions (Liu et al. 2006).

One of the cDNA clones (accession no. GO241187)exhibited homology with catalase enzyme encoding gene.There is increasing evidence that chilling elevates the levelsof active oxygen species (Wise and Naylor 1987), whichdamage cellular components (Elstner 1991; McKersie1991). Catalase activity is involved in active oxygen speciesdetoxification system in higher plants (Koh Iba 2002). Thehigher CAT activity in acclimated seedlings during chillingand the stress recovery suggested that CAT plays a majorantioxidant role, along with other antioxidant enzymes, inpre-emergent maize seedlings (Tottempudi 1997).

Other interesting clone (accession no. GO241142) was achitinase encoding gene. Chitinases are up-regulated by a vari-ety of stress conditions, both biotic and abiotic, and also regu-lated by phytohormones. Like other PR proteins, chitinasesplay a role in plant resistance against distinct pathogens andby reducing the defense reaction of the plant, chitinases allowsymbiotic interaction with nitrogen-fixing bacteria or mycor-rhizal fungi (Kasprzewska 2003). Recent studies (Maria et al.2006) showedenhanced resistance tobiotic andabiotic stress intransgenic tobacco plants overexpressing chitinases of fungalorigin.

cDNA clone (accession no. GO241126) encoding Clp pro-tease, one of the best-characterized energy-dependent pro-teases, was identified in subtracted library in this study. Clpproteasewas originally identified in vitro as anATP-dependentproteaseconsistingof twocomponents, theATPasesubunit andthe protease (ClpP) subunit (Hwanget al. 1988).Clpproteins indifferent bacteria are involved inmany stress responses such asheat, cold, high salt, low temperature, and UV light, and areoften strongly induced after brief exposure (Porankiewicz et al.1999; Skinner and Trempy 2001).Arabidopsis chloroplast Clpprotein expression and changes in Clp gene expression alongwith protein content have been earlier reported (Zheng et al.2002).

Chalcone reductase was represented in cold subtracted li-brary (accession no. GO241107). The enzyme catalyzes a crit-ical biosynthetic branch point to impart additional ability inlegumes to synthesize a set of related deoxychalcone-derivedphytoalexins in response to herbivore or pathogen attack(Dixon and Paiva 1995). Earlier studies in Arabidopsis leaveshavedemonstratedthat levelsofflavanoids increase in responseto UVirradiation (Lois 1994). The importance of flavanoids inresponse to low temperature is not confirmed and still needscharacterization. Clone encoding putative myb-related proteinwasalsofound in forward librarywhich is reported toplaya role

Fig. 2 Nucleotide sequenceand its translated amino acidsequence of the putative woundinduced gene of Cicermicrophyllum

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Fig. 3 Quantitative real-time PCR analysis of fold change in transcriptabundance of putative wound induced gene during low temperaturestress in different time intervals (0, 6, 12, and 24 h). All values werenormalized with respect to level of housekeeping control 26rDNA

expression. Error bars indicates SE. Table indicates fold change intranscript abundance of putative wound induced gene during lowtemperature stress in different time intervals (0, 6, 12, and 24 h). Eachtreatment was replicated three times

Construction of cold induced subtracted cDNA library from Cicer microphyllum

in cold stress signaling in herbaceous plants (Urao et al. 1993).The transcription factor was also represented in cold inducedforward subtracted library in blueberry plant (Dhananjay et al.2007).Thus, the differential expressionof this geneduring coldstress indicates a role in low temperature stress.

Another cDNA clone (accession no. GO241324)showed homology with a plant receptor serine-threoninekinase which belongs to mitogen activated protein kinase(MAPKs) family. In Arabidopsis, at least three MAPKShave been found that are enzymatically activated by saltas well as by cold, wounding, and other environmentalsignals (Ichimura et al. 2000).

Wound induced gene, a possible part of complex woundsignaling network in plants, was represented in the sub-tracted library (accession no. GO241284). Wound signalmolecules are well known to promote rapid membraneassociated events such as depolarization of the membranewith a concomitant protein influx (Thain et al. 1995; Moyenand Johannes 1996) and elevation of intracellular levels ofcalcium. So, the wound signal transduction pathways musthave an association by mobilization of calcium from intra-cellular stores and by calmodulin-related activity (Leon et

al. 1998). In tomato, it has been reported that the expression ofa wound and systemin inducible calmodulin gene may beassociated with activation of wound responsive genes (Bergeyand Ryan 1999).

Full-length gene cloning of putative wound induced gene

Sequence analysis of the partial fragment (231 bp) of putativewound induced gene found during screening of cold inducedsubtracted cDNA library indicated the presence of initiationcodon. The lacking 3′ end sequence was amplified by rapidamplification of cDNA ends (RACE) method. The full-lengthORF of this gene was cloned and sequenced (accession num-ber GQ914056). The putative wound induced gene contains a279-bp-long open reading frame and encodes protein of 92amino acids (Fig. 2). Southern blot analysis revealed theoccurrence of a single copy of the isolated gene in the genome(Electronic supplementary material 2). Occurrence of woundinduced protein in the library for cold regulated genes wasconsidered unusual and further characterization was carriedout to confirm the regulation by low temperature stress.

Fig. 5 Whole plant in situhybridization of Cicermicrophyllum. a Negativecontrol. b Positive controlhybridized with biotin-labeled26rDNA sequence as probe. cUninduced plant hybridizedwith biotin-labeled wound in-duced cDNA as probe. dStressed plant (4°C/24 h) hy-bridized with biotin-labeledwound induced cDNA as probe

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Concentration of SA

Fig. 4 Quantitative real-time PCR analysis of fold change in transcriptabundance of putative wound induced gene in response to SA spray(mock, 10 and 50 μM). All values were normalized with respect tolevel of housekeeping control 26rDNA expression. Error bars

indicates SE. Table indicates fold change in transcript abundance ofputative wound induced gene in response to SA spray (mock, 10 and50 μM). Each treatment was replicated three times

R.K. Singh et al.

Transcript expression analysis of putative wound inducedgenes in response to low temperature stress

The transcript quantitation of wound induced gene of C.microphyllum by real-time PCR confirmed an increase inaccumulation of transcripts after 12 h by 3-fold on exposureat low (4°C) temperature stress (Fig. 3). The increase wasconsiderable (11-fold) after exposure to 24 h of low temper-ature stress.

Whole plant in situ hybridization revealed that the tran-script was distributed in all tissues, i.e., roots, stem, andleaves. Further, increased transcript abundance was observedafter 24 h of low temperature stress at 4°C (Fig. 4). Northernblot experiment reconfirmed up-regulation of transcript ex-pression of putative wound induced gene during low temper-ature stress. Analysis of blot showed gradual increase in thetranscript level after 6, 12, and 24 h of low temperature stress(Electronic supplementary material 3).

All these experiments were repeated thrice to confirm theresults. Thus, the results conclusively prove the transcript up-regulation of putative wound induced gene by low tempera-ture stress in cold tolerant high altitude plantC. microphyllum.

The transcript regulation of wound induced gene of C.microphyllum was also investigated after foliar spray ofsalicylic acid (mock, 10 and 50 μM) which showed a2.75-fold increase in transcript abundance in comparisonto mock after spraying of 10 μM salicylic acid (Fig. 5).SA is a key endogenous signal involved in plant defenseresponses as well as flowering and thermogenesis (Dempseyet al. 1999; Raskin 1992). Yinong et al. (2004) suggestedthat salicylic acid plays an important role to modulate redoxbalance and protect rice plants from oxidative stress. Theseresults suggest that the putative wound induced gene isinvolved in salicylic acid mediated pathway and thus playa role in biotic and abiotic stress management.

Proteins encoded by these wound related genes may playsignificant functions in repairing damaged plant tissues, par-ticipating in the activation of wound defense signaling path-ways, and adjusting plant metabolism to the imposednutritional demands. Multiple signals and differential induc-tion of gene expression point to the existence of a complexwound signaling network in plants that, in addition, may havespecies-specific variations. Many structurally different mole-cules play regulatory roles in wound signaling, including theoligopeptide systemin (Pearce et al. 1991), oligosaccharidesreleased from the damaged cell wall (Bishop et al. 1981), andmolecules with hormonal activity such as jasmonates (Farmerand Ryan 1990), ethylene (O’Donnell et al. 1996), and absci-sic acid (Pena-Cortes et al. 1989). However, it has not beenpossible to identify and define unequivocally the nature of theprimary signals that trigger wound-activated defenseresponses. Frequently, the induction of wound responsesrequires the simultaneous action of different signals and

regulators and, quite often, the qualitative and quantitativeparticipation of any putative signal in the activation of woundresponses depends on the plant species as well.

The wound induced protein from C. microphyllum hasdisplayed cold responsive expression and salicyclic acidinduced expression. This indicated possible role of isolatedgenes in biotic and abiotic stress as well. This could bepossible that the protein might function in pathways whichare commonly required in both kinds of stress like antioxi-dant pathways. Otherwise, wound induction caused by lowtemperature stress could possibly signal the induction of thegene. Thus, the isolated gene may possibly be involved inmultiple stress responsive pathway like wound inductionand low temperature stress.

Conclusion

Low temperature induced genes were identified in a coldtolerant high altitude plant C. microphyllum using suppres-sion subtractive hybridization analysis. The data provided aclue on how C. microphyllum responds to and adjusts to lowtemperature stress by regulating the transcript expression ofthese stress responsive genes. Further studies on the novelcDNA clones with no significant similarity with databasemay promote the study to validate their roles in conferringtolerance during low temperature stress in C. microphyllum.One of the cDNA clones encoding putative wound inducedgene was full-length cloned and its transcript expression wascharacterized. Further, functional characterization of thisand other identified genes may be helpful in engineeringcold tolerance in sensitive crop plants. The outcome ofresearch on a high altitude cold tolerant plant C. micro-phyllum may be helpful in elucidating the physiological,biochemical, and molecular mechanisms involved in toler-ance of this plant in various environmental conditions.

Acknowledgment The authors thank the Director, DIHAR, Leh forproviding seeds of Cicer microphyllum. Dr. Patade Vikas Yadav isgratefully acknowledged for critical review during manuscript prepa-ration. Financial support to RKS, SS and PP in the form of ResearchFellowship by DRDO is gratefully acknowledged.

Certificate It is certified that the authors do not have any conflict ofinterest in publishing the manuscript.

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