Funct Integr Genomics DOI 10.1007/s10142-014-0383-2
ORIGINAL PAPER
C2H2 type of zinc finger transcription factors in foxtail millet define response to abiotic stresses Mehanathan Muthamilarasan & Venkata Suresh Bonthala & Awdhesh Kumar Mishra & Rohit Khandelwal & Yusuf Khan & Riti Roy & Manoj Prasad
Received: 17 February 2014 / Revised: 27 May 2014 / Accepted: 1 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract C2H2 type of zinc finger transcription factors (TFs) play crucial roles in plant stress response and hormone signal transduction. Hence considering its importance, genome-wide investigation and characterization of C2H2 zinc finger proteins were performed in Arabidopsis, rice and poplar but no such study was conducted in foxtail millet which is a C4 Panicoid model crop well known for its abiotic stress tolerance. The present study identified 124 C2H2-type zinc finger TFs in foxtail millet (SiC2H2) and physically mapped them onto the genome. The gene duplication analysis revealed that SiC2H2s primarily expanded in the genome through tandem duplication. The phylogenetic tree classified these TFs into five groups (I–V). Further, miRNAs targeting SiC2H2 transcripts in foxtail millet were identified. Heat map demonstrated differential and tissue-specific expression patterns of these SiC2H2 genes. Comparative physical mapping between foxtail millet SiC2H2 genes and its orthologs of sorghum, maize and rice revealed the evolutionary relationships of C2H2 type of zinc finger TFs. The duplication and divergence data provided novel insight into the evolutionary aspects of these TFs in foxtail millet and related grass species. Expression profiling of candidate SiC2H2 genes in response to salinity, dehydration and cold stress showed differential expression pattern of these genes at different time points of stresses.
Mehanathan Muthamilarasan and Venkata Suresh Bonthala contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10142-014-0383-2) contains supplementary material, which is available to authorized users. M. Muthamilarasan : V. S. Bonthala : A. K. Mishra : R. Khandelwal : Y. Khan : R. Roy : M. Prasad (*) National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi 110 067, India e-mail: [email protected]
Keywords Foxtail millet (Setaria italica) . C2H2 type of zinc finger transcription factors . Genome-wide . Expression profiling . Phylogeny . Physical map . Comparative mapping
Introduction Foxtail millet [Setaria italica (L.) P. Beauv.] possesses several salient attributes such as small genome (~515 Mb; 2n=2×= 18) with a relatively lower repetitive DNA, an inbreeding nature and a short life cycle. Its genetic close relatedness to bioenergy grasses namely, switchgrass (Panicum virgatum), napier grass (Pennisetum purpureum) and pearl millet (Pennisetum glaucum) has made foxtail millet a tractable experimental model system to investigate architectural traits, evolutionary genomics and physiological aspects of bioenergy grasses and millets (Doust et al. 2009; Lata et al. 2013). In addition, its potential abiotic stress tolerance, particularly towards drought and salinity had drawn the interest of plant research community to examine the crop’s biology (Muthamilarasan et al. 2013). Considering the importance of foxtail millet, two independent groups namely the Beijing Genomics Institute (BGI) and China and US Department of Energy—Joint Genome Initiative (JGI) had sequenced its genome (Zhang et al. 2012; Bennetzen et al. 2012). The availability of genome sequence in public database has encouraged the millet research community to perform highthroughput investigations in both the aspects of structural and functional genomics (Pandey et al. 2013; Kumari et al. 2013; Jia et al. 2013; Suresh et al. 2013; Puranik et al. 2013; Muthamilarasan et al. 2014; Mishra et al. 2014; Gupta et al. 2014; Yadav et al. 2014; Yi et al. 2014; Khan et al. 2014). Transcription factors (TFs) and their corresponding cisregulatory sequences function as molecular controls for gene expression, regulating their temporal and spatial expression
Funct Integr Genomics
during various biological processes including stress responses (Puranik et al. 2012; Wang et al. 2013). Among the plant transcription factors, zinc finger proteins are the largest family (Takatsuji 1999). They constitute ~15 and ~13 % of transcription factors in Arabidopsis and rice, respectively (http:// planttfdb.cbi.pku.edu.cn/). The term “Zinc-finger” refers to a type of protein domain in which a zinc atom is surrounded by the cysteine residues and/or the histidine residues (Kiełbowicz-Matuk 2011). On the basis of number and location of characteristic residues, the zinc finger proteins are classified into C2H2, C2HC, C2HC5, C3HC4, CCCH, C4, C4HC3, C6 and C8 (Kiełbowicz-Matuk 2011). Of these, the C2H2 type of zinc finger proteins which are otherwise termed as TFIIIA-type zinc finger or the Krüppel-like zinc finger proteins are the most studied TFs. The C2H2-type TFs encompass the X2CX2–4CX12HX2–8H signature and constitute a redundant family of transcriptional regulators with varying numbers. Moreover, the C2H2-type zinc finger TFs vary extensively in structure and function, which ranges from DNA or RNA binding to protein-protein interactions (KiełbowiczMatuk 2011). Although, the C2H2-type zinc finger TFs were primarily identified to participate in plant growth and development (Xiao et al. 2009; Wang et al. 2009), a number of stress-responsive C2H2-type zinc finger TFs were also reported (Tian et al. 2010; Sun et al. 2010; Kodaira et al. 2011). Hence, considering the importance of C2H2-type zinc finger TFs in growth, development and stress-responsive pathways, a comprehensive investigation was conducted to (i) understand the gene structure, chromosomal location, genomic distribution and duplication of S. italica C2H2-type zinc finger TFs (SiC2H2s), (ii) identify its orthologous sequences in related grass species such as sorghum, maize and rice, (iii) derive the significance of evolutionary divergence between the paralogs and orthologs, (vi) in silico expression profiling in different tissues and (v) analyse the expression pattern of SiC2H2s at different time points during different abiotic stresses.
Materials and methods Identification of C2H2-type zinc finger transcription factors in foxtail millet The whole proteome of S. italica comprising 40,599 predicted proteins was retrieved from Phytozome v9.1 (http:// phytozome.net/). The complete set of C2H2-type zinc finger protein sequences available in Plant Transcription Database (http://plntfdb.bio.uni-potsdam.de/v3.0; Pérez-Rodríguez et al. 2009) was downloaded, and HMM profile was prepared using HMMER suite (Eddy 1998). The HMM profile was then searched against the S. italica proteome data under default E value cut-off of 0.01. The raw alignments
were inspected manually to ensure reliability. The resultant protein sequences were analysed in PROSITE (Sigrist et al. 2013) and InterProScan (Zdobnov and Apweiler 2001) to confirm the presence of C2H2 zinc fingers. Analysis of gene structure, chromosomal location and duplication The amino acid sequences of S. italica C2H2-type zinc fingers (SiC2H2) were BLASTP searched against the foxtail millet genome in Phytozome to retrieve the respective genomic, transcript and CDS sequences. Further, chromosomal locations including the chromosome number, position of gene start, end and gene orientation were also identified. The SiC2H2 genes were then plotted onto the respective chromosomes of foxtail millet according to their ascending order of physical position (bp), from the short arm telomere to the long arm telomere. The alternate transcripts were removed and the resultant physical map was displayed using MapChart v2.2 (Voorrips 2002). Segmental duplication was analysed using the method of Plant Genome Duplication Database (Tang et al. 2008) by performing BLASTP search against the complete peptide sequences of S. italica and the first five matches with E value
ORIGINAL PAPER
C2H2 type of zinc finger transcription factors in foxtail millet define response to abiotic stresses Mehanathan Muthamilarasan & Venkata Suresh Bonthala & Awdhesh Kumar Mishra & Rohit Khandelwal & Yusuf Khan & Riti Roy & Manoj Prasad
Received: 17 February 2014 / Revised: 27 May 2014 / Accepted: 1 June 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract C2H2 type of zinc finger transcription factors (TFs) play crucial roles in plant stress response and hormone signal transduction. Hence considering its importance, genome-wide investigation and characterization of C2H2 zinc finger proteins were performed in Arabidopsis, rice and poplar but no such study was conducted in foxtail millet which is a C4 Panicoid model crop well known for its abiotic stress tolerance. The present study identified 124 C2H2-type zinc finger TFs in foxtail millet (SiC2H2) and physically mapped them onto the genome. The gene duplication analysis revealed that SiC2H2s primarily expanded in the genome through tandem duplication. The phylogenetic tree classified these TFs into five groups (I–V). Further, miRNAs targeting SiC2H2 transcripts in foxtail millet were identified. Heat map demonstrated differential and tissue-specific expression patterns of these SiC2H2 genes. Comparative physical mapping between foxtail millet SiC2H2 genes and its orthologs of sorghum, maize and rice revealed the evolutionary relationships of C2H2 type of zinc finger TFs. The duplication and divergence data provided novel insight into the evolutionary aspects of these TFs in foxtail millet and related grass species. Expression profiling of candidate SiC2H2 genes in response to salinity, dehydration and cold stress showed differential expression pattern of these genes at different time points of stresses.
Mehanathan Muthamilarasan and Venkata Suresh Bonthala contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10142-014-0383-2) contains supplementary material, which is available to authorized users. M. Muthamilarasan : V. S. Bonthala : A. K. Mishra : R. Khandelwal : Y. Khan : R. Roy : M. Prasad (*) National Institute of Plant Genome Research (NIPGR), Aruna Asaf Ali Marg, New Delhi 110 067, India e-mail: [email protected]
Keywords Foxtail millet (Setaria italica) . C2H2 type of zinc finger transcription factors . Genome-wide . Expression profiling . Phylogeny . Physical map . Comparative mapping
Introduction Foxtail millet [Setaria italica (L.) P. Beauv.] possesses several salient attributes such as small genome (~515 Mb; 2n=2×= 18) with a relatively lower repetitive DNA, an inbreeding nature and a short life cycle. Its genetic close relatedness to bioenergy grasses namely, switchgrass (Panicum virgatum), napier grass (Pennisetum purpureum) and pearl millet (Pennisetum glaucum) has made foxtail millet a tractable experimental model system to investigate architectural traits, evolutionary genomics and physiological aspects of bioenergy grasses and millets (Doust et al. 2009; Lata et al. 2013). In addition, its potential abiotic stress tolerance, particularly towards drought and salinity had drawn the interest of plant research community to examine the crop’s biology (Muthamilarasan et al. 2013). Considering the importance of foxtail millet, two independent groups namely the Beijing Genomics Institute (BGI) and China and US Department of Energy—Joint Genome Initiative (JGI) had sequenced its genome (Zhang et al. 2012; Bennetzen et al. 2012). The availability of genome sequence in public database has encouraged the millet research community to perform highthroughput investigations in both the aspects of structural and functional genomics (Pandey et al. 2013; Kumari et al. 2013; Jia et al. 2013; Suresh et al. 2013; Puranik et al. 2013; Muthamilarasan et al. 2014; Mishra et al. 2014; Gupta et al. 2014; Yadav et al. 2014; Yi et al. 2014; Khan et al. 2014). Transcription factors (TFs) and their corresponding cisregulatory sequences function as molecular controls for gene expression, regulating their temporal and spatial expression
Funct Integr Genomics
during various biological processes including stress responses (Puranik et al. 2012; Wang et al. 2013). Among the plant transcription factors, zinc finger proteins are the largest family (Takatsuji 1999). They constitute ~15 and ~13 % of transcription factors in Arabidopsis and rice, respectively (http:// planttfdb.cbi.pku.edu.cn/). The term “Zinc-finger” refers to a type of protein domain in which a zinc atom is surrounded by the cysteine residues and/or the histidine residues (Kiełbowicz-Matuk 2011). On the basis of number and location of characteristic residues, the zinc finger proteins are classified into C2H2, C2HC, C2HC5, C3HC4, CCCH, C4, C4HC3, C6 and C8 (Kiełbowicz-Matuk 2011). Of these, the C2H2 type of zinc finger proteins which are otherwise termed as TFIIIA-type zinc finger or the Krüppel-like zinc finger proteins are the most studied TFs. The C2H2-type TFs encompass the X2CX2–4CX12HX2–8H signature and constitute a redundant family of transcriptional regulators with varying numbers. Moreover, the C2H2-type zinc finger TFs vary extensively in structure and function, which ranges from DNA or RNA binding to protein-protein interactions (KiełbowiczMatuk 2011). Although, the C2H2-type zinc finger TFs were primarily identified to participate in plant growth and development (Xiao et al. 2009; Wang et al. 2009), a number of stress-responsive C2H2-type zinc finger TFs were also reported (Tian et al. 2010; Sun et al. 2010; Kodaira et al. 2011). Hence, considering the importance of C2H2-type zinc finger TFs in growth, development and stress-responsive pathways, a comprehensive investigation was conducted to (i) understand the gene structure, chromosomal location, genomic distribution and duplication of S. italica C2H2-type zinc finger TFs (SiC2H2s), (ii) identify its orthologous sequences in related grass species such as sorghum, maize and rice, (iii) derive the significance of evolutionary divergence between the paralogs and orthologs, (vi) in silico expression profiling in different tissues and (v) analyse the expression pattern of SiC2H2s at different time points during different abiotic stresses.
Materials and methods Identification of C2H2-type zinc finger transcription factors in foxtail millet The whole proteome of S. italica comprising 40,599 predicted proteins was retrieved from Phytozome v9.1 (http:// phytozome.net/). The complete set of C2H2-type zinc finger protein sequences available in Plant Transcription Database (http://plntfdb.bio.uni-potsdam.de/v3.0; Pérez-Rodríguez et al. 2009) was downloaded, and HMM profile was prepared using HMMER suite (Eddy 1998). The HMM profile was then searched against the S. italica proteome data under default E value cut-off of 0.01. The raw alignments
were inspected manually to ensure reliability. The resultant protein sequences were analysed in PROSITE (Sigrist et al. 2013) and InterProScan (Zdobnov and Apweiler 2001) to confirm the presence of C2H2 zinc fingers. Analysis of gene structure, chromosomal location and duplication The amino acid sequences of S. italica C2H2-type zinc fingers (SiC2H2) were BLASTP searched against the foxtail millet genome in Phytozome to retrieve the respective genomic, transcript and CDS sequences. Further, chromosomal locations including the chromosome number, position of gene start, end and gene orientation were also identified. The SiC2H2 genes were then plotted onto the respective chromosomes of foxtail millet according to their ascending order of physical position (bp), from the short arm telomere to the long arm telomere. The alternate transcripts were removed and the resultant physical map was displayed using MapChart v2.2 (Voorrips 2002). Segmental duplication was analysed using the method of Plant Genome Duplication Database (Tang et al. 2008) by performing BLASTP search against the complete peptide sequences of S. italica and the first five matches with E value
