Microsatellite identification in Solanaceae crops associated with Nucleoside Diphosphate Kinase (NDK) specific to abiotic stress tolerance through in silico analysis Reena Rosy Thomas, M.K. Chandra Prakash, M. Krishna Reddy1, Sukhada Mohandas2 and Riaz Mahmood3 Section of Economics & Statistics Indian Institute of Horticultural Research, Hessaraghatta Bangalore -560089, India Email : reenart@hotmail.com ABSTRACT Abiotic stress often causes a series of morphological, physiological, biochemical and molecular changes that affect plant growth, development and productivity. To cope with abiotic stresses, it is necessary to understand plant responses to stresses that disturb homeostatic equilibrium at the cellular and molecular level. Genomic information on Capsicum annuum has been explored to identify microsatellite markers associated with abiotic stress tolerance and assign them to cognate functional groups related to specific stress responses. Several in silico methods have been used to identify simple sequence repeats associated with stress responsive gene candidates in Capsicum annuum. In this study, a microsatellite marker has been identified in Capsicum annuum associated with Nucleoside Diphosphate Kinase (NDK) having multiple environmental stress tolerance (oxidative, high temperature and salt stress) and which is also highly conserved in crops of Solanaceae. These are house-keeping enzymes that maintain intracellular levels of all nucleoside triphosphates (NTP) with the exception of adenosine triphosphate (ATP). These are also involved in phytochrome A response, UV-B signaling, auxin responses and oxidative stress signaling. Key words: Nucleoside Diphosphate Kinase (NDK), microsatellite, abiotic stress, Solanaceae J. Hortl. Sci. Vol. 8(2):195-198, 2013 1Division of Plant Pathology, 2Division of Biotechnology, Indian Institute of Horticultural Research, Bangalore - 560 089, India 3Dept. of Biotechnology, Kuvempu University, Shimoga - 577 451 INTRODUCTION Stress conditions such as drought, high salinity and extreme temperatures, are major factors affecting plant growth and crop productivity (Boyer, 1982) and are often inter-related. Exposure to adverse abiotic environmental conditions causes oxidative stress in plants by rapid and excessive accumulation of reactive oxygen species (ROS) in their cells (Foyer et al, 1994). Reactive oxygen species inactivate enzymes and damage important cellular components. ROS are responsible for protein, lipid and nucleic acid modification. As several stress responses are mediated through ROS, plants make use of common pathways that allow them to acclimatize to a range of different stresses and some changes in ROS metabolism cause enhanced tolerance and sensitivity. Therefore, to provide adequate protection against a hazardous environment, a common signaling system has evolved in plants and is known as cross-tolerance (Bowler and Fluhr, 2000). Plants respond to environmental stresses by activating related genes, to increase their tolerance to the latter. However, engineering of an individual stress-response gene has not been effective because many kinds of stress responses are necessary for plants to survive under various adverse conditions. An understanding of plant responses to abiotic stresses at the genomic level provides a foundation for identifying genes, microsatellite markers and associated elements. It has been reported in plants that nucleoside diphosphate kinases (NDKs) play a key role in signaling both stress and light. However, little is known about structural elements involved in their function. NDKs are ubiquitous enzymes that transfer phosphate groups from triphosphate nucleosides to nucleoside diphosphates (NDPs) (Parks and Agarwal, 1973). These enzymes play an important role in phytochromeA response, UV-B signaling, heat stress, and growth (Yano et al, 1995). Sequence of the NDK has been highly conserved throughout evolution. A single histidine residue is conserved in all known NDK isozymes and is involved in the catalytic mechanism. NDK2 plays a 196 regulatory role in H2O2-mediated mitogen-activated protein kinase (MAPK) signaling in plants, indicating that plant NDKs also have a diverse array of biological functions (Moon et al, 2003). Among various NDKs expressed in Arabidopsis thaliana, NDK2 is known to be involved in phytochrome-mediated signal transduction (Yang et al, 2003; Im et al, 2004). METERIAL AND METHODS With whole-genome sequencing initiatives, large amounts of genomic sequence data are available in the public domain that serve as an attractive source of in silico mining of microsatellite sequences. However, finding potentially useful microsatellites that occupy specific genomic regions in plants, still remains a challenge. Availability of this information can facilitate discovery of microsatellites associated with abiotic stress tolerance, using in silico methods. Capsicum annuum EST database, consisting approximately 23000 sequences, has been explored for microsatellites having low-complexity repeats, for identification of markers associated with stress tolerance. Of the 23000 EST sequences, 2507 non-redundant EST sequences having repeats comprising of di, tri, tetra and penta SSRs were located using Repeat Finder programs. These 2507 EST sequences were converted to Textual Data in FASTA format. A computer program developed in Microsoft’s Visual studio 2010 in Windows platform has been coded specifically to read large-size of text files. Potential microsatellite markers comprising single, di and tri-nucleotide repeats were located in the text file, based on input data. These sequences were further subjected to in silico analysis for classifying the markers and to assign them to cognate functional groups related to specific stress responses. RESULTS AND DISCUSSION One of the 2507 non-redundant EST sequences with a length of 755bp from C. annuum, found to have a marker associated with Nucleoside Diphosphate Kinase (NDK), was subjected to BLAST analysis. The 755bp sequence of Capsicum annuum nucleoside diphosphate kinase mRNA sequence with a single nucleotide repeat sequence of 22bp of A repeats is given below: GGCACGAGATTTGCTAACTCATTCAGTAACATCAA AGAAGCAAGAAATGGAGCAAACTTTCATCATGATT AAGCCTGATGGTGTCCAACGTGGCCTCGTTGGTGA GATTATCGGCAGATTTGAAAAGAAAGGTTTCTCTT TGAAAGGTTTGAAGCTCATCACTGTGGATCGCGCT TTTGCTGAGAAGCATTATGCAGATTTGTCTGCTAAG CCTTTCTTTAATGGGCTTGTTGAGTACATTGTTTCT GGACCCGTTGTCTCTATGGTCTGGGAAGGTAAGGG TGTACTTACCACTGGCAGGAAGATCATTGGAGCAA CCAACCCCTTGGAATCTGCTCCTGGTACCATCCGTG GTGATTATGCTATTGACATTGGCAGGAATGTCATTC ATGGAAGTGATGCTGTTGAGAGTGCAAGGAAGGAGA TTGCTCTTTGGTTCCCCGAAGGAGTTGCAGAGTGG CAGAGCAGCCTTCACTGTTGGATCTACGAGTAGAAAA GTTCTATGAAAGATTTCATGGCCAGCCTCTTTGGTTG TAACTTATGAGTTTTGTTTGTCATTTAAGTCCAGAA GTAACTTAAGAGTTTTGTTCGTCATTTAAGTCCAG AAGTTAGATGTTTTTAAGATCTACTAGCGGTTCCCT ATTTGAAGAATATTTAAGTTGTGGTGTTTTATCTGTTG TGTTCCATGTGTTGCAATTTCTAGTAATTGAGCTTCCA CAATTTTTTAGCCGTCAAAAAAAAAAAAAAAAAAAAAA BLAST analysis shown in Fig. 1 revealed that the Nucleoside Diphosphate Kinase (NDK) sequence is evolutionarily conserved, conferring multiple environmental stress tolerance (oxidative, high temperature and salt stress). Further, the associated 22 base pair of single nucleotide ‘A’ repeat is also evolutionarily conserved in most of the Solanaceae crops (shown in green colour). This could be a potential microsatellite marker associated with NDK sequence. Data in Table 1 indicate that C. annuum sequence with a maximum identicality of >90% and E value of 0.0 Fig 1. BLAST analysis result shows that NDK sequence is highly conserved in crops of Solanaceae; Red color shows high sequence similarity and green colour shows associated marker of single nucleotide A repeats of 22 base pair length J. Hortl. Sci. Vol. 8(2):195-198, 2013 Reena Rosy Thomas et al 197 Table 1. Significant hit of nucleotide BLAST results against C. annuum 755bp NDK sequence Description Max Total Query E Max Accession score score coverage value identity number Capsicum annuum nucleoside 1395 1395 100% 0.0 100% AF108881.1 diphosphate kinase mRNA, complete cds Solanum chacoense cytosolic nucleoside 743 743 72% 0.0 91% DQ157699.1 diphosphate kinase mRNA, complete cds Nicotiana tabacum nucleoside diphosphate 719 719 72% 0.0 90% AY962601.1 kinase mRNA, complete cds Solanum lycopersicum cDNA, clone: 676 676 72% 0.0 89% AK320311.1 LEFL1007CH03, HTC in leaf Solanum lycopersicum cDNA, clone: 676 676 72% 0.0 89% AK246327.1 FC06DD10, HTC in fruit Lycopersicon esculentum clone 114282R, 676 676 72% 0.0 89% BT013034.1 mRNA sequence Solanum lycopersicum nucleoside diphosphate 636 636 68% 9e-179 89% NM_001247245. kinase (LOC544095), mRNA >emb|X75324.1| L. esculentum (Ailsa Craig) mRNA for nucleoside diphosphate kinase belongs to Solanaceae crops. It is also highly conserved in Solanum chacoense, Nicotiana tabacum, Solanum lycopersicum and Lycopersicon esculentum. Protein sequence similarity Protein sequence of NDK also blasted against protein database from NCBI, showed highest similarity with Arabidopsis thaliana (Fig. 2). Putative conserved domains are shown as small, red triangles against C. annuum sequences. It is seen that the sequence belongs to NDPk superfamily which has been highly conserved through evolution. Molecular graphic structure of NDK in Arabidopsis thaliana is shown in Fig. 3. NDK protein sequence of C. annuum, given below, has100% similarity to Arabidopsis thaliana. >gi|7643788|gb|AAF65509.1| nucleoside diphosphate kinase [Capsicum annuum] MEQTFIMIKPDGVQRGLVGEIIGRFEKKGFSLKGLKL ITVDRAFAEKHYADLSAKPFFNGLVEYIVSGPVVS MVWEGKGVLTTGRKIIGATNPLESAPGTIRGDYAID IGRNVIHGSDAVESARKEIALWFPEGVAEWQSSLHC WIYE Fig 2. Putative conserved domain and BLAST hits on protein sequence of C. annuum that shows high similarity with Arabidopsis thaliana Fig 3. molecular graphic structure of NDK in Arabidopsis thaliana J. Hortl. Sci. Vol. 8(2):195-198, 2013 Microsatellites in Solanaceae specific to abiotic stress tolerance 198 From Table 2, it is evident that C. annuum NDK protein sequence has >98% query coverage and is 80% identical to Arabidopsis thaliana sequences. Plant NDK plays a prominent role in plant defense mechanisms and involvement of NDK is associated with various stress mechanisms. It was proved that over expression of NDK resulted in tolerance against several environmental stresses such oxidative stress, high temperature and salt stress. C. annuum 755bp sequence having the maximum identicality with nucleotide and protein sequences in Solanaceae crops and Arabidopsis confirms similar structural and molecular functions. As the sequence of microsatellite marker is evolutionarily conserved across Solanaceae crops, this could be useful in selecting parental lines and developing abiotic-stress tolerant crops. REFERENCES Bowler, C. and Fluhr, R. 2000. The role of calcium and activated oxygen as signals for controlling cross- tolerance. Trends Pl. Sci., 5:241–246 Boyer, J.S. 1982. Plant productivity and environment. Science, 218:443–448 Foyer, C.H., Descourvieres, P. and Kunert, K.J. 1994. Protection against oxygen radicals: An important defense mechanism studied in transgenic plants. Plant Cell Environ., 17:507-523 Im, Y.J., Kim, J.I., Shen, Y., Na, Y., Han, Y.J., Kim, S.H., Song, P.S. and Eom, S.H. 2004. Structural analysis of Arabidopsis thaliana nucleoside diphosphate kinase- 2 for phytochrome-mediated light signaling. J. Mol. Biol., 343:659-70 Moon, H., Lee, B., Choi, G., Shin, D., Prasad, D.T., Lee, O., Kwak, S.S., Kim, D.H., Nam, J., Bahk, J., Hong, J.C., Lee, S.Y., Cho, M.J., Lim, C.O. and Yun, D.J. 2003. NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants. Proc. Nat’l. Acad. Sci., USA, 100:358–363 Parks, R.E.J. and Agarwal, R.P. 1973. Nucleoside diphosphokinases. In: The Enzymes, Boyer, P.D (Ed.). Academic, New York, USA, pp. 307–334 Yang, K.A., Moon, H.J., Kim, G.T., Lim, C.J., Hong, J.C., Lim, C.O. and Yun, D.J. 2003. NDP kinase 2 regulates expression of antioxidant genes in Arabidopsis. Proc. Jpn. Acad. Ser., B, 79:86–91 Yano, A., Umeda, M. and Uchimiya, H. 1995. Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ related alteration of NDK activities during rice seed germination (Oryza sativa L.). Pl. Mol. Biol., 27:1053–1058 Table 2. Protein BLAST result of C. annuum NDK protein sequence against Arabidopsis thaliana protein sequences Description Max Total Query E Max Accession score score cover value indent Nucleoside diphosphate kinase 1 255 255 100% 7e-87 80% NP_567346.1 [Arabidopsis thaliana] >gb|AEE82742.1| nucleoside diphosphate kinase 1 [Arabidopsis thaliana] RecName: Full=Nucleoside diphosphate kinase 1; 254 254 100% 9e-87 80% P39207.1 AltName: Full=Nucleoside diphosphate kinase I; Short=NDK I; Nucleoside diphosphate kinase [Arabidopsis thaliana] 250 250 98% 3e-85 80% CAA49170.1 Unknown protein [Arabidopsis thaliana] >gb 242 242 95% 7e-82 80% AAK48956.1 |AAL66933.1| unknown protein [Arabidopsis thaliana] Nucleoside diphosphate kinase [Arabidopsis thaliana] 223 223 100% 3e-74 74% CAA49173.1 Chain A, Crystal structure of Nucleoside Diphosphate 196 196 100% 1e-63 60% 1S57_A Kinase 2 from Arabidopsis> pdb|1S57|B Chain B, Crystal structure of Nucleoside Diphosphate Kinase 2 from Arabidopsis Nucleoside diphosphate kinase Ia [Arabidopsis thaliana] 196 196 100% 1e-63 60% AAC14280.1 (MS Received 07 February 2013, Revised 06 September 2013, Accepted 24 September 2013) J. Hortl. Sci. Vol. 8(2):195-198, 2013 Reena Rosy Thomas et al