Identification, phylogeny, and transcript profiling of ERF ... - Springer Link

Mol Genet Genomics (2010) 284:455–475 DOI 10.1007/s00438-010-0580-1

ORIGINAL PAPER

Identification, phylogeny, and transcript profiling of ERF family genes during development and abiotic stress treatments in tomato Manoj K. Sharma • Rahul Kumar • Amolkumar U. Solanke • Rita Sharma Akhilesh K. Tyagi • Arun K. Sharma

•

Received: 15 July 2010 / Accepted: 14 September 2010 / Published online: 5 October 2010 ! Springer-Verlag 2010

Abstract Ethylene responsive transcription factors have been shown to be intimately connected to plant development, defense responses and stress signaling pathways and in order to use them for plant improvement, we need to have better understanding of these proteins. In this study, 85 ERF genes have been identified from tomato using raw EST data in various public repositories. Phylogenetic analysis with tomato ERF domains revealed their distribution in all the groups, previously identified in model systems. MEME motif analysis resulted in identification of conserved domains, characteristic to member of each clade,

Communicated by S. Hohmann.

Electronic supplementary material The online version of this article (doi:10.1007/s00438-010-0580-1) contains supplementary material, which is available to authorized users. M. K. Sharma ! R. Kumar ! A. U. Solanke ! R. Sharma ! A. K. Tyagi ! A. K. Sharma (&) Department of Plant Molecular Biology, University of Delhi (South Campus), New Delhi 110021, India e-mail: [email protected]; [email protected] Present Address: M. K. Sharma ! R. Sharma Department of Plant Pathology, University of California, Davis, CA 95616, USA A. U. Solanke National Research Centre on Plant Biotechnology, IARI, New Delhi 110012, India A. K. Tyagi National Institute of Plant Genome Research, New Delhi 110067, India

in addition to ERF domain. Expression analysis during vegetative and reproductive stages of development using QPCR and tomato GeneChip" arrays, revealed their tissuespecific/preferential accumulation. In total, 57 genes were found to be differentially expressed during temporal stages of tomato fruit development. The expression analysis of 23 ERF family genes representing each clade in response to seven abiotic stress treatments revealed their differential expression in response to more than one abiotic stress treatments. Results suggest that ERF genes play diverse roles in plant’s life and comprehensive data generated will be helpful in conducting functional genomics studies to understand their precise role during plant development and stress response. Keywords Development ! Ethylene ! Ethylene response factor ! Expression ! Ripening ! Stress Abbreviations CAP3 Contig assembly program ERF Ethylene responsive factor EST Expressed sequence tag GEO Gene Expression Omnibus HMM Hidden Markov Model IMG Immature green MEME Multiple EM (Expectation Maximization) for Motif Elicitation NCBI National Center for Biotechnology Information NJ Neighbor-joining PTFD Plant Transcription Factor Database SGN SOL Genomics Network SMART Simple Modular Architecture Research Tool TFGD Tomato Functional Genomics Database TGI Tomato Gene Indices

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Introduction Being fixed in space, plants are subjected to various biological challenges from pathogens/pests and environmental extremes of temperature/water/nutrients with stupendous impact on plant’s survival, development, and productivity. In order to survive and flourish in multifarious environments, plants have evolved abstruse network of signaling at molecular, cellular, and system level. Gene regulation at the level of transcription is one of the major control points in biological processes and transcription factors (TFs) play a key role in this process. In Arabidopsis, more than 7% of the protein coding genes encode transcription factors (Riechmann et al. 2000; Guo et al. 2005; Iida et al. 2005) that reveal the complexity of the gene regulation at transcriptional level. Ethylene responsive transcription factors (ERF) have been shown to play a critical role during plant development and ability of plant to fight against ambiance (Brown et al. 2003; Chakravarthy et al. 2003; Agarwal et al. 2006; Chen et al. 2008a, b, c). Therefore, it is crucial to study the function of these genes to improve upon yield and making them better suited to diverse environmental conditions. ERFs are part of AP2 (APETALA2)/ERF super-family which also contains AP2 and RAV family genes and is characterized by the presence of the AP2/ERF DNA binding domain (Riechmann and Meyerowitz 1998; Sakuma et al. 2002). ERF family proteins contain only one AP2/ERF domain, whereas, AP2 family genes have two such domains. RAV family proteins contain an additional B3 DNA binding domain along with AP2/ERF domain. AP2/ERF domain consists of 60–70 amino acids and is involved in DNA binding. It was first identified in AP2 protein of Arabidopsis (Jofuku et al. 1994) and later in ethylene responsive element binding proteins (EREBP) from Nicotiana tabaccum (Ohme-Takagi and Shinshi 1995). Allen and coworkers (1998) described the threedimensional structure of AP2/ERF domain from AtERF1, using hetero-nuclear multidimensional Nuclear Magnetic Resonance. It consists of three anti-parallel b-sheets and an a-helix. In the DNA-TF complex, tryptophan and arginine residues present in the b-sheets have been found to make contact with the DNA in its major groove. Based upon the binding of ERF domain to DNA sequence element, ERF family has been further divided into two subfamilies, i.e., ERF and DREB. AP2/ERF domain of proteins belonging to ERF family binds to AGCCGCC box (Ohme-Takagi and Shinshi 1995; Hao et al. 1998), conversely, dehydration response element (DRE; TACCGACAT) containing Crepeat, is recognized by AP2/ERF domain of DREB family proteins (Jiang et al. 1996; Stockinger et al. 1997; Hao et al. 2002). ERF genes have been identified from several plant species (Nakano et al. 2006; Zhang et al. 2008a, b).

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Mol Genet Genomics (2010) 284:455–475

Earlier, AP2/ERF domain containing transcription factors were proposed to be plant-specific, but recently, their homologs have been identified from cyanobacterium Trichodesmium erythraeum, the ciliate Tetrahymena thermophila as well as viruses Enterobacteria phage Rb49 and Bacteriophage Felix 01 (Magnani et al. 2004). It has been suggested that AP2/ERF TFs may have originated by horizontal transfer of HNH-AP2 endonuclease from bacteria/viruses into plants via transpositions and homing processes. In addition to binding the promoters containing GCC box, Pti4 has also been shown to regulate the gene expression by directly interacting with non-GCC elements (Chakravarthy et al. 2003). ERF proteins have been shown to induce or repress gene expression in response to external or internal ethylene as well as trans-activation of other transcription factors (Fujimoto et al. 2000). ERF family genes have been shown to exhibit varied expression patterns during plant growth and development as well as in response to stress-related stimuli like ethylene, jasmonic acid, and salicylic acid (Fujimoto et al. 2000; Gu et al. 2000; Brown et al. 2003) or abiotic stress conditions (Chen et al. 2002; Tournier et al. 2003; Gutterson and Reuber 2004; Agarwal et al. 2006). When overexpressed, several ERF genes have been found to confer enhanced resistance to biotic (Onate-Sanchez and Singh 2002; Brown et al. 2003) or abiotic stresses (Sakuma et al. 2006; Chen et al. 2007; Chen et al. 2008a, b, c). Tomato is an important crop that belongs to Solanaceae, one of the largest and most important families of flowering plants. It includes fruit bearing vegetables (tomato and pepper), tuber-bearing potatoes, plants with edible leaves (Solanum aethiopicum, S. macrocarpon), medicinal (Datura), and ornamental plants (Petunia, Nicotiana) (Knapp 2002). In addition to its agricultural utility, tomato is an excellent model system to study hormonal and environmental signal transduction components active in fruit ripening, metabolic regulation, and plant–pathogen interactions (Alexander and Grierson 2002; Adams-Phillips et al. 2004; Arie et al. 2007). Till date, several ERF genes have been characterized in tomato that includes Pti4, 5, 6 (Zhou et al. 1997; Gu et al. 2002), Sl-ERF2 (Pirrello et al. 2006; Zhang et al. 2009), TERF1 (Huang et al. 2004), TSRF1 (Zhang et al. 2008b), JERF1 (Zhang et al. 2004a), JERF3 (Wang et al. 2004), and LeCBF1-3 (Zhang et al. 2004b). To explore further about the role of AP2/ERF genes in tomato, EST data available in public domain were analyzed resulting in the identification of 112 unigenes belonging to AP2/ERF superfamily of tomato. Out of these, 93 unigenes were found to contain single partial/complete AP2/ERF domain. In total, 85 unigenes containing single complete ERF domain were analyzed further. A comprehensive analysis of their structure and phylogeny was


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Materials and methods

neighbor-joining (NJ) (Saitou and Nei 1987) tree was generated in ClustalX with default parameters. Bootstrap analysis was performed using 5,000 replicates and tree thus obtained was viewed using Figtree (http://tree.bio.ed.ac.uk/ software/figtree/) or TREEVIEW software (Page 1996). Similarly, another tree was generated using ERF domains of tomato and characterized ERF proteins from other plant species. The conserved motifs, in addition to ERF domain, in tomato ERF protein sequences were identified using motif based sequence analysis tool, MEME Suite version 4.0.0 (Bailey and Elkan 1995) (http://meme.sdsc.edu/ meme4/cgi-bin/meme.cgi), with following parameters: optimum width 6–200 amino acid, any number of repetitions of a motif and maximum number of motifs set to 25. The resulting motifs were checked in NCBI and SMART databases for their significance.

Identification of ERF family genes in tomato genome

Collection of plant material

Tomato EST data were downloaded from various public repositories including SOL Genomics Network (SGN), Plant Transcription Factor Database (PTFD), National Center for Biotechnology Information (NCBI), and Plant Genome Database (PlantGDB). The data assembly was done using CAP3 (Huang and Madan 1999). Assembled sequences were translated with ESTSCAN (Iseli et al. 1999) using various matrices which included Arabidopsis, Oryza sativa, Zea mays, and Mus musculus. Hidden Markov Model (HMM) analysis was employed to identify ERF proteins from translated contigs (Altschul et al. 1997). To generate HMM profile, ERF protein sequences from various plant species, available in public databases, were downloaded and HMM profile was generated using either complete sequences or ERF domain sequences using HMMER 2.1.1 software package (Madera and Gough 2002). HMM search was performed on translated contigs to extract the ERF domain containing proteins. Name search using ERF tomato, ethylene responsive factor and EREBP as keywords in public databases helped in identification of more genes which could not be identified using HMM profile. Simple Modular Architecture Research Tool, SMART (Schultz et al. 1998; Letunic et al. 2009) was used to confirm the presence of AP2/ERF domain in resulting sequences. The ERF domain containing protein sequences from various sources were aligned using ClustalX 1.83 (Thompson et al. 1997) and redundant entries were removed.

Seeds of Solanum lycopersicum var. Pusa Ruby were grown in aseptic conditions as explained earlier (Sharma et al. 2009). Roots, cotyledons, and stem were harvested from 9-day-old seedlings. Flowers and fruit tissues of three different developmental stages [immature green (IMG), breaker (BK), and red ripe (RR)] were collected from mature tomato plants grown in culture room. After harvesting, tissue samples were frozen in liquid nitrogen and stored at -70#C.

Nine-day-old seedlings were subjected to various stress treatments. For cold or heat treatment, culture tubes containing tomato seedlings were transferred to a cold chamber maintained at 4 ± 1#C or an incubator at 42 ± 1#C, respectively. Submergence stress was given to seedlings by submerging the seedlings in sterile Milli-Q water. For desiccation stress, seedlings were removed from culture tube and kept on a 3-mm blotting paper. For mechanical wounding, each leaf/cotyledon of seedlings was punctured three times with needle and squeezed twice with forceps. For salt, ABA and oxidative stress, seedlings were removed from solid medium without disturbing roots and kept in liquid MS-B5 medium with 200 mM NaCl, 10 lM ABA, or 10 mM hydrogen peroxide, respectively. Tissue samples were harvested after 8 h of stress treatment and frozen in liquid nitrogen. Untreated seedlings were used as control.

Phylogenetic and MEME motif analysis

RNA extraction and microarray experiments

The ERF domains of all the Arabidopsis and tomato ERF proteins were extracted and aligned using ClustalX 1.83 program (Thompson et al. 1997). A combined un-rooted

Total RNA was extracted from three different stages of fruit development using hot phenol method (Reymond et al. 2000). Quality of RNA was checked on agarose

performed. The expression profiles of 44 ERF genes, represented on GeneChip" array, were analyzed in leaf, root tips and three temporal stages of fruit development. Furthermore, expression patterns of 41 genes, missing on the GeneChip", were analyzed by QPCR during eight stages of vegetative and reproductive development. Differential expression of representative 23 ERF genes from different phylogenetic clades were also analyzed in response to seven abiotic stress treatments. The data generated in this study will help in the selection of appropriate candidate genes for further functional characterization and understanding the precise regulatory checkpoints operating during development and stress response.

Stress treatments

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formaldehyde gel and quantified using nanodrop (ND-1000 spectrophotometer). Affymetrix GeneChip" Tomato arrays representing 10,024 probe sets were employed to study the transcriptome profiles during fruit ripening in tomato. cDNA preparation, labeling and hybridizations were carried out, as described earlier (Arora et al. 2007). GeneChip" Hybridization Oven 640 was used for hybridization for 16 h at 45#C and 60 rpm. Affymetrix fluidic station model 450 was used to wash and stain the arrays with streptavidin–phycoerythrin. Arrays were scanned using GeneChip" Scanner 3000. Microarray data analysis Raw microarray data for tomato leaf and root tips were downloaded from Gene Expression Omnibus (GEO) database of NCBI under the accession number GSE9683. The .CEL files representing three biological replicates of leaf, root tips and three stages of fruit development were imported in Array AssistTM data analysis software (Stratagene, Now Agilent Technologies Company). The data were normalized using Guanine Cytosine Robust MultiArray Analysis (GCRMA) algorithm, and log transformed. The expression values obtained in three biological replicates of each sample were averaged. The corresponding probe set IDs for ERF family genes were identified by BLAST analysis of the nucleotide sequences of ERF genes in probe set target sequence database (http://ted.bti.cornell. edu/cgi-bin/TFGD/array/blast.cgi). A cut off of C98% homology of target sequence to the query sequence was used to assign a probe set id to a particular gene. Cluster analysis on rows was performed using Euclidean distance metric and Ward’s linkage rule of hierarchical clustering. K-means clustering was performed using Array AssistTM to identify the patterns exhibited by ERF genes during temporal stages of fruit development. For the genes not represented on array, QPCR data was analyzed separately in Microsoft excel to identify patterns. Various patterns identified in both experiments were finally drawn in Adobe" illustrator. RNA isolation and quantitative real-time PCR analysis Total RNA was isolated using RNeasy" Mini Kit, from QIAGEN and DNAse treatment was given as per manufacturer’s instructions (Qiagen Inc., USA). Quantity and quality of RNA was checked by ND-1000 spectrophotometer (Nanodrop Technologies, USA) and agarose gel electrophoresis, respectively. Two micrograms RNA of each sample was reverse transcribed into cDNA using a high-capacity cDNA archive kit following instructions from manufacturer (Applied Biosystems). ABI Prism 7000 sequence detection system (Applied Biosystems) was used

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for QPCR (quantitative real-time PCR) amplification using default parameters. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as internal control (Iskandar et al. 2004; Jain et al. 2006; BarsalobresCavallari et al. 2009; Solanke et al. 2009). Primers for QPCR were designed in PRIMER EXPRESS version 2.0 software (PE Applied Biosystems, USA) using default parameters and checked for their uniqueness using BLAST tool against NCBI, SGN-unigene, and Tomato Gene Index databases. List of primers used for QPCR is given in Supplementary file S1. PCR reactions were carried out in 96-well optical reaction plates (Applied Biosystems, USA). Reaction was set up using 40 ng of cDNA sample as a template, 200 nM forward primers, 200 nM reverse primers, and SYBR green reaction mix (Applied Biosystems, USA). At least three biological and three technical replicates were performed for each reaction. The data was analyzed using DDCt method (Livak and Schmittgen 2001). For calculating relative expression values during developmental stages, the tissue exhibiting lowest expression level was used as control, whereas, for the stress conditions, unstressed sample was used as control. Data points in QPCR time course are plotted as means ± SE of three biological replicates.

Results Identification of AP2/ERF family genes in tomato EST data from various sources including SOL Genomic Network (2,21,718), Tomato Gene Indices (3,30,396), Plant-GDB (2,57,540), and NCBI (2,58,813) were assembled using CAP3 resulting in 38,272 unigenes including 31,970 contigs and 6,302 singlet sequences. Name search from public databases and HMM search of translated assembled EST data resulted in the identification of 112 AP2/ERF domain containing unique unigenes. Eleven unigenes contained two complete/partial AP2/ERF domains and hence were categorized in AP2 subfamily. Of the remaining 101 unigenes, containing single complete/partial ERF domain, five were showing higher homology with AP2 subfamily. Since it is possible that due to incomplete sequence of unigene, the second AP2 motif was not identified, hence they were also placed with AP2 subfamily genes. Although none of the unigenes had B3 DNA binding domain, characteristic of RAV family proteins, AP2/ERF domains of three unigenes were more similar to those found in RAV family proteins and therefore, were assigned to RAV family. Remaining 93 unigenes were assigned to ERF family. Out of 93 ERF proteins, 85 were found to contain complete AP2/ERF domain, whereas, remaining eight unigenes had an incomplete AP2/ERF domain and hence


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were not included in further analysis. The organization of AP2/ERF superfamily genes in tomato has been shown in Table 1 along with comparative distribution from soybean, Populus, Arabidopsis, and rice. The nucleotide and protein sequences of 85 tomato ERFs have been given in Supplementary file S2 and S3, respectively. Phylogenetic relationships between ERF family transcription factors in tomato and Arabidopsis To analyze the phylogenetic relationships between Arabidopsis and tomato ERF family proteins, a combined phylogenetic tree was generated using AP2/ERF domain sequences of all the ERF proteins. Multiple alignments indicated Gly-29 and Ala-37 are conserved in all the ERF family members of tomato and Arabidopsis (Supplementary file S4). Further, Gly-4, Arg-6, Arg-8, Gly-11, Glu-16, Ile-17, Arg-25, Try-27, Asp-42, and Asn-56 are also conserved in the ERF domains belonging to more than 95% ERF members of tomato and Arabidopsis. Immediate next position of Gly-29 and Ala-37 (completely conserved amino acids) is also occupied by neutral amino acids in ERF domains of Arabidopsis as well as tomato. As shown previously, ERF family genes were found to contain conserved alanine at position 14 and aspartic acid at position 19. Conversely, CBF/DREB family genes had valine at position 14 and glutamine acid at position 19. Though few genes lacked V14 or E19 but exhibited significant similarity with rest of the domain (Supplementary file S4). Eleven distinct groups could be identified in the phylogram and named as I–XI (Fig. 1). Group I to X corresponded to groups defined earlier in Arabidopsis and rice (Nakano et al. 2006). Earlier, few Arabidopsis proteins having low

homology to consensus sequence were categorized as VIlike (VI-L) and Xb-like based on the motif analysis (Nakano et al. 2006). We included these proteins in the phylogenetic reconstruction and they formed a separate group of ERF proteins. Twelve of tomato proteins also grouped with these proteins and the new group was named as group XI. Interestingly, all the groups had representative members from both the systems suggesting that major diversification of ERF clades predates the divergence of these species. Since, five ERF genes of tomato had been named previously from LeERF1 to LeERF5, to maintain uniformity, they were renamed as SlERF1 to SlERF5 and others were named from SlERF6 to SlERF85 in order of their placement in the phylogenetic tree. A separate tree was also generated using ERF domain sequences of tomato and characterized ERF proteins from various plant species reported in literature (Supplementary file S5). Though the major groups in both the phylogenetic trees were same, group five proteins were clubbed with DREB family members in the tree containing characterized proteins. However, inclusion of characterized genes helped in correlating the function with phylogenetic placement. Distribution of conserved motifs Complete ERF protein sequences of tomato were analyzed for the presence of conserved motifs using the Multiple EM (Expectation Maximization) for Motif Elicitation (MEME) analysis tool version 4.0.0. In total, 25 conserved motifs were identified and named 1–25. The distribution of these conserved motifs in proteins of respective clades in the phylogram has been presented in Fig. 2. The consensus

Table 1 Summary of AP2/ERF superfamily genes in tomato and their comparison with soybean, Arabidopsis, Populus, and rice Solanum lypersicon

Group AP2

Glycine maxa

Arabidopsis thalianab

Populus trichocarpac

Oryza sativad

16

26

18

26

nd

Double complete or incomplete AP2/ERF domains

11

8

14

26

nd

Single complete or incomplete AP2/ERF domains

5

18

4

–

nd

ERF

93

120

122

168

CBF/DREB

25

36

57

91

ERF

139

60

62

58

77

Others

–

–

7

–

–

Incomplete AP2/ERF domains

8

22

–

–

–

RAV Single complete or incomplete AP2/ERF domains Others Total

3

2

6

5

nd

–

–

1

1

–

112

148

147

200

nd

nd Not described a

Zhang et al. (2008b),

b,d

Nakano et al. (2006), cZhuang et al. (2008)

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Fig. 1 Phylogenetic analysis of tomato and Arabidopsis ERF proteins. The phylogenetic analysis of ERF domains of 85 unigenes of tomato and Arabidopsis ERF proteins (Nakano et al. 2006) has been presented in the form of a radial tree. The 11 clades have been marked. The dotted line marks distinction between ERF and DREB proteins. Scale bar represents 0.05 amino acid substitutions per site

sequences of these motifs are given in Supplementary file S6. Motif 1, 2, 3, 4, 5, 10, and 24 correspond to the AP2/ ERF domain region. Remaining 19 motifs were found to characterize the specific clades in the phylogentic tree (Fig. 2). Motif 1 is missing in group IX proteins; instead motif 10 representing last 11 amino acids of motif 1 is present along with motif 2 representing ERF domain in these proteins. Similarly, motif 2 is missing in group XI proteins, instead a smaller 21 amino acid partial region, named motif 4, is present in most of the proteins. Other

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members have even smaller 15 amino acid region, named motif 24 along with motif 1 representing ERF domain. There are few motifs which are specifically detected in members of a particular clade only. For example, motif 6 and 7 are present only in members of group III and group VII, respectively. Similarly motif 9, 10, 11, 12, 13, 18, 19, and 25 are characteristic to group VII, IX, III, IV, XI, VII, IV, and IX proteins, respectively. Few motifs are present in members of more than one clade, for example, motif 14, present in almost all the proteins of group VII was also


detected in one protein (SlERF85) of group X. Such examples may have resulted due to smaller rearrangements during recombination and evolution. The presence of characteristic motifs in members of a specific clade also strengthens the reliability of their placement in the phylogenetic tree. To look at taxonomic hierarchy of these conserved motifs, motif sequences were used as blast query in NCBI database and the summary of the organisms giving hit for the query is given in Supplementary file S7. Interestingly, motif 1, 6, 8–12, and 16 were specifically found in spermatophytes with motif 6, 8, 10–12, 15, and 16 only found in Magnoliophytes. Homologous sequences to motif 7, 17, 21, 22, and 24 were found in algae, bacteria, and animal kingdom also. Expression profiling of ERF genes during vegetative and reproductive development The expression profiles of 44 ERF family genes having corresponding probe sets on GeneChip" were analyzed during two stages of vegetative (leaf and root) and three stages of fruit development (IMG, BK, and RR). The hierarchical cluster display of their expression profiles have been shown in Fig. 3 and the average signal values of these genes from three biological replicates of each sample are shown in Supplementary file S8. The expression profiles of 12 genes exhibiting varied expression patterns have been validated using QPCR. As shown in Fig. 4, good correlation was found in the data obtained using both the techniques thereby strengthening the reliability of microarray data. The expression of these 12 genes was also checked in cotyledon, flower, and stem tissues. In addition, 41 genes for which corresponding probe set ID could not be identified on the chip, were analyzed for their expression profiles during eight stages of development including leaf, root, stem, cotyledon, flower and three stages of fruit development using QPCR (Fig. 5). In the QPCR analysis, genes exhibiting Ct values [36, were treated as nonexpressers. The expression of all the genes was normalized with reference to the expression of GAPDH gene. To check the relative expression levels in various developmental stages, the lowest expression level in any of the developmental stage or Ct value of 36 was considered as 1-fold. Interestingly, three genes (SlERF10, 41, and 54) were found to exhibit leaf-preferential accumulation, whereas, six genes (SlERF1, 26, 28, 60, 61, and 69) exhibited root tissue-specific/preferential expression. Transcripts of SlERF22 and 44 were mainly detected in leaf and root tissues. Similarly, SlERF4 and 33 expressed at high levels in cotyledons while SlERF67 exhibited stem-specific expression. Six genes including SlERF23, 24, 27, 38, 39, and 80 exhibited flower-specific/preferential accumulation.

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However, SlERF7, 13, 35, 36, 37, 40, and 63 were found to exhibit high-level expression in both vegetative and reproductive tissues. However, 10 genes (SlERF3, 15, 18, 19, 25, 53, 55, 59, 82, and 85), having corresponding probe sets IDs on GeneChip" were found to exhibit very low expression values suggesting them to exhibit very low level or no expression in the stages analyzed. It is possible that due to low-level expression of some genes, their transcripts could not be detected in the microarray data; however, QPCR being more sensitive technique could reveal expression data for such genes. For example, SlERF54 exhibiting very low-level expression in microarray data, showed significant accumulation in almost all the stages analyzed though, the expression was relatively higher in leaf tissue. SlERF34 and 46 did not show any expression in the selected stages. The NCBI ESTs corresponding to SlERF34 were mainly detected in libraries generated from callus, ovary, trichomes, shoot meristem and in response to Phytophthora infection. Although ESTs corresponding to SlERF34 are also available from fruit and leaf, we could not detect any expression of this gene in these stages. The ESTs corresponding to SlERF46 are from PAMP-elicited tomato leaf and callus. Based on the expression profiles of all the ERF genes during temporal stages of fruit development, 10 distinct patterns of their expression were recognized (Fig. 6). Eight genes (SlERF8, 17, 20, 31, 52, 58, 65, and 79), belonging to group I, exhibited high-level expression specifically in IMG stage of fruit development with no or negligible expression in BK and RR stage of fruit development. However, two genes (SlERF12 and 47) comprising group II accumulated specifically in BK stage of fruit development. Similarly, seven genes (SlERF4, 42, 49, 51, 61, 81, and 83) of group III were found to express only in RR stage of fruit development. Specific accumulation of their transcripts in different stages of fruit development indicates their involvement in stage-specific developmental activities. Group IV included five genes (SlERF5, 22, 36, 39, and 74) exhibiting uniform level expression in all three stages of fruit development. Eight genes (SlERF1, 7, 27, 28, 32, 60, 69, and 80) exhibiting high-level accumulation in IMG stage that gradually declines with development were placed in group V. Group VI genes (15 in number; SlERF9, 10, 11, 13, 16, 30, 38, 50, 57, 63, 70, 75, 76, 77, and 84) exhibited a very interesting expression profile with lowlevel expression in IMG stage that gradually increases in BK and RR stage in a temporal manner suggesting their involvement in fruit ripening. Interestingly, two genes, SlERF35 and 64 exhibited high expression in IMG stage that declined in BK stage but again accumulated in RR stage of fruit development. These have been placed in group VII. Conversely, group VIII genes (10 in number; SlERF2, 6, 14, 21, 29, 40, 43, 56, 62, and 68) exhibited

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Fig. 2 Clade-wise distribution of conserved motifs in tomato ERF proteins. The motifs (1–25) identified using MEME search tool have been marked on protein sequences of respective clades. The length and order of the boxes corresponds to the actual length and position of

the conserved region in the protein sequence. Motifs 1, 2, 3, 4, 5, 10, and 24 correspond to AP2/ERF domain; motif 21 corresponds to EAR motif and others are unknown. For amino acid sequences of motifs, refer to Supplementary file S6

higher expression in BK stage as compared to IMG and RR stages. Five genes (SlERF37, 48, 71, 72, and 78) comprising group IX showed increase in expression from IMG to BK stage that persists till RR stage of fruit development.

Expression of tomato ERF genes in response to stress treatments

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Twenty-three genes (16 ERFs and seven DREBs) were selected representing all phylogenetic groups for their


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expression analysis during various abiotic stress conditions. Expression was analyzed in response to cold, heat, salt, dessication, mechanical, ABA, oxidative, and submergence stress. Figure 7 shows the expression patterns of selected genes during various stress conditions. All the analyzed genes exhibited differential accumulation in response to at least one abiotic stress treatments. Out of 16 ERF genes, 10 genes (SlERF1, 4, 5, 33, 50, 51, 61, 64, 73, and 77) were down-regulated by C3-fold in at least one of the stress conditions, whereas, only four genes (SlERF42, 52, 68, and 80) were up-regulated. Interestingly, SlERF68 was up-regulated C17-folds during salt and oxidation stress, however, SlERF80 was found to up-regulate C400folds during salt stress. Out of seven DREB genes, five genes (SlERF7, 9, 16, 26, and 30) were up-regulated significantly and two genes (SlERF14 and 21) were down-regulated in at least one of the stress conditions. The maximum differential expression in DREB genes was observed in response to salt, heat, and drought stress. Out of 10 genes up-regulated and three genes down-regulated in response to salt stress, five and two genes, respectively, belonged to DREB family. Similarly, three out of four genes induced in response to drought stress belonged to DREB family. Most of the ERF family genes (11 in number) were down-regulated in response to abiotic stress treatments, mainly heat stress. However, only two genes were up-regulated in response to heat stress which belonged to DREB family.

Discussion

Fig. 3 Hierarchical cluster display of expression profiles of 44 ERF genes in tomato. A gradient bar below represents the log2 normalized expression values. Darker shade indicates higher expression. The developmental stages analyzed have been marked on the top of heat map, leaf, root, IMG (immature green stage), BK (breaker stage), and RR (red ripe) stages of fruit development

Transcription factor proteins are foremost regulators of biological processes and have emerged as a powerful tool for the manipulation of complex metabolic pathways (Grotewolda 2008). In order to utilize these proteins for plant improvement, we need to have a better understanding about their role in gene regulatory networks. ERF family of transcription factors have a highly conserved signature element that includes an ERF domain responsible for DNA binding activity and are essential for normal plant development (Riechmann and Meyerowitz 1998; Ohta et al. 2001; Sakuma et al. 2002; Cao et al. 2006; Nakano et al. 2006). Systematic analysis of their phylogeny and expression will assist in mining candidate genes for detailed characterization. In this study, 93 ERF unigenes have been identified from EST data of tomato available in public domain, however, only 85 were found to contain complete AP2/ERF domain and so analyzed further.

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Fig. 4 Validation of microarray data using QPCR analysis. The expression data obtained using microarrays for selected 12 genes was validated using QPCR analysis. Three technical and three biological replicates were performed for each tissue. The expression was also analyzed in additional stages including leaf (L), root (R), flower (F), stem (S), cotyledon (C), immature green (IMG), breaker (BK), and

red ripe (RR). Y-axis represents the average normalized microarray expression values. The QPCR data values have been normalized to assist matching with the microarray profile. X-axis represents the developmental stages analyzed. Standard error bars have been shown for the data obtained using both the techniques

Organization of ERF family in tomato

of the amino acids at key positions in the ERF domain, i.e., 14 and 18 described earlier (Liu et al. 1998), genes containing single AP2/ERF domain are placed in ERF and DREB subfamilies. Recently, Goremykin and Moser (2009) used phylogenetic approach to classify ERF family genes in Arabidopsis. They also included group VI-like (VI-L) and Xb-like (described by Nakano et al. 2006) in their phylogenetic reconstruction which were placed in group XI and XII, respectively, in this study. Using similar approach, we could classify tomato ERF proteins into 11 groups. In total, 12 tomato ERFs were grouped with VIlike (VI-L) and Xb-like proteins of Arabidopsis that formed group XI. Since all the groups were represented in monocot

Earlier 122 and 120 genes were found to belong to ERF family in Arabidopsis and soybean, respectively (Nakano et al. 2006; Zhuang et al. 2008). Interestingly, though the genome size of tomato (950 Mb) is about seven times larger than that of Arabidopsis (145 Mb) (Mysore et al. 2001), the number of ERF family genes seems to be comparable in both the systems. Certainly, with the availability of whole genome sequence, the size of this family in tomato genome will be clearer, however, considering the large repertoire of EST data available, number of genes is not expected to change much. Based upon the conservation

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465

Fig. 5 Expression profiles of ERF genes analyzed using QPCR. The expression profiles of 39 genes analyzed using QPCR during seven stages of development have been presented. Y-axis represents relative expression values and X-axis represents stages of development as

follows: L leaf, R roots, C cotyledons, S stem, F flower, IMG immature green stages, BK breaker stage, and RR red ripe stage of fruit development. Error bars show the standard error between three replicates performed

as well as dicot systems (rice, Arabidopsis, and soybean), probably major diversification of this family predates monocot–dicot divergence. Structural analysis revealed

that most of the ERF proteins had conserved Ala-14 and Asp-19, whereas, DREB proteins had Val-14 and Glu/His19. A few members that did not show conservation at these

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466


Fig. 6 Expression patterns exhibited by ERF family genes during temporal stages of fruit development in tomato. In total, nine patterns observed have been shown. The numbers corresponds to tomato genes exhibiting a particular expression profile. The X-axis represents stages of fruit development,IMG immature green, B breaker, and RR red ripe

positions were categorized based on their placement in the phylogenetic tree. In the alignment of ERF domain from DREBs and ERFs, amino acid conservation patterns were also different to G29 and A37. Whereas in DREBs, G4 and A38 were conserved, R8, G11, E16, D18, and T30 were found to be conserved in AP2/ERF domain. Ala-37 in the tomato ERF domain is also conserved which has been suggested to play an important role in the stability of ERF domain or DNA binding with the DRE element or GCC box (Liu et al. 2006). Structural and functional divergence of tomato ERF genes Transcription factors generally contain functionally important conserved domains outside the DNA binding domain, which have been found to characterize the members of subgroups (De Bodt et al. 2003; Arora et al. 2007; Nijhawan et al. 2008). The distribution of the specific motifs in proteins belonging to specific clade in the phylogram was also observed in tomato ERF proteins which demonstrate the structural similarities among proteins within the same clade. These motifs may be playing an important role during gene regulation. For instance, Ohta and colleagues (2001) identified an ERF-associated amphiphilic repression (EAR) motif, which has been shown to function as repression domain. EAR motif sequence ‘‘L/FDLNL/FxP’’ was found in motif 21 listed in this study. EAR core motif has been identified in several repressor proteins including ZAT7, 10, 12, ERF3, AUX/ IAA, NIMIN1, HSI2, SUPERMAN (Arabidopsis), NRR (rice), ZFT1 (tobacco), and ZPT2-3 (Petunia) which play a variety of functions ranging from plant development to

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stress tolerance (Kazan 2006). An EAR motif containing C2H2 zinc finger protein (ZAT7), when over-expressed, enhanced tolerance toward salinity stress and plant growth was suppressed. Deletion of EAR motif from ZAT7 abolished salinity tolerance without affecting growth suppression, thereby indicating its role in providing salinity tolerance (Ciftci-Yilmaz et al. 2007). Recently, DEAR1, a DREB protein containing EAR motif, has been shown to mediate crosstalk between signaling pathways for biotic and abiotic stress responses (Tsutsui et al. 2009). In the phylogenetic tree of AP2/ERF domain of characterized ERF proteins from various plant species and tomato ERFs (Supplementary file S5), tomato proteins containing EAR motif were placed in the clade containing genes which have been shown to play important role during floral meristem development, organ identity and response to abiotic stress. Interestingly, SlERF38 (Fig. 5) and 39 (Fig. 4) were found to express at high levels in flower tissue and SlERF35–37 showed high expression levels during fruit development. Further, significant change in the transcript levels of SlERF39 was observed under abiotic stress conditions analyzed, thereby, suggesting that these genes (group VIII) could be actively involved in gene regulation during plant development and stress response. Motif 16 contain consensus sequence ‘‘PKK[PR]AGRKKF[RK]ETRHP.’’ The basic residue region ‘‘PKKP\RAGRKKFR’’ is located on the N-terminal region of the consensus sequence which might function as a nuclear localization signal (NLS) (El Kayal et al. 2006). The signature resembles nuclear transport signal described earlier (Stockinger et al. 1997) indicating that these ERFs may be involved in protein trafficking. Recently, this has been identified as essential signature for Arabidopsis CBF1 to bind DNA, thereby,


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Fig. 7 Expression analysis of 23 ERF family genes in response to abiotic stress treatments. Expression analysis of selected 23 tomato ERF family genes in response to seven abiotic stress treatments for 8 h was analyzed using QPCR. X-axis represents the stages analyzed

and Y-axis represents the fold changes. Standard error bars have been drawn among three biological replicates. CS cold stress, DS dehydration stress, SS salt stress, HS heat stress, MS mechanical stress, OxiS oxidative stress, and SubS submergence stress

indispensable for its transcriptional activity (Canella et al. 2009). A putative nuclear localization signal (KRKRK) has been identified in AP2/ERF proteins (van Raemdonck et al. 2005; El-Sharkawy et al. 2009). The KRKRK motif had been implicated in transcriptional activation by chicken thyroid hormone receptor a and influence DNA binding, dimerization, activation, and association of receptor with TFIIB (Hadzic et al. 1998). This signal has been identified in the members of group VII, as a part of conserved motif 18 in the N-terminal region, indicating their role in transcriptional activation. Recently, AP2 domain homologs have been identified in cyanobacterium (Trichodesmium erythraeum), ciliates (Tetrahymena thermophila), and viruses (Enterobacteria phage Rb49 and bacteriophage

Felix 01) (Magnani et al. 2004). Based upon NCBI search using conserved motif sequence as query, while many of them were found to be specific to spermatophytes, several motifs traced back to lower plants, bacteria, fungi, and animals. Presence of these conserved motifs in evolutionarily diverse organisms indicates that they play an important functional role, while spermatophyte-specific motifs may have been evolved later to cater specific functions. Various ERF encoding genes have been reported to play important roles during growth and development of the plant (Wilson et al. 1996; Chuck et al. 2002; Komatsu et al. 2003; Zhu et al. 2003; Marsch-Martinez et al. 2006) as well as in response to abiotic/biotic stress conditions (Zhou et al. 1997; Gilmour et al. 2000; Park et al. 2001; Gu et al.

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2002; Haake et al. 2002). ERF genes from various plant species whose biological functions have been reported are listed in Table 2. When the data available in literature and generated in this study was overlapped with the phylogenetic tree, certain interesting patterns emerged. Different clades of the family seem to have evolved to cater specific functions. Motif analysis also revealed certain motifs which were specific to each clade. These motifs may be important for a particular set of functions. For example, members of group VIII have been previously implicated in leaf petiole development and regulating meristem and floral organ identity (van der Graaff et al. 2000; Chuck et al. 2002; Kirch et al. 2003; Komatsu et al. 2003). In this study, two genes belonging to this clade, SlERF38 and 39 were found to exhibit flower-specific/preferential accumulation. Other genes belonging to this clade were also found to exhibit high-level transcript abundance in both vegetative and reproductive stages of development. Similarly, the genes belonging to group III have also been shown to play a role in GA biosynthesis and regulate plant growth and development (Wilson et al. 1996; Magome et al. 2004; Wang et al. 2008a, b). Most of the genes belonging to this clade were also found to express at high levels in reproductive tissues. Similarly, DREB family genes were mainly induced in response to heat, drought, and salt stress, whereas, most of the ERF family genes were down-regulated in response to abiotic stress treatments. Previous studies have shown that ERF family genes seem to have diverged as a result of duplication (Nakano et al. 2006; Zhuang et al. 2009). Interestingly, few genes lying in the same clade (SlERF9 and 11 of group IV; SlERF49–51 of group XI) were found to exhibit similar expression profiles. The conservation in their structure was observed in ERF domain as well as outside the domain suggesting that these could have probably resulted due to duplication. This information should be considered before validation of their functions as these may be redundant in their functions and hence, knocking out more than one gene may be required to decipher their functions. Role of ERF genes in fruit ripening Fruits being an important part of human diet have always been fascinating area to investigate. There have been attempts to manipulate the fruit ripening in tomato to increase its shelf life by manipulating the genes involved in cell wall biosynthesis and ethylene signaling. Since ethylene plays major role in ripening of fleshy fruits, understanding the key genes involved in ethylene biosynthesis and response is crucial to manipulate their expression which could in turn prevent losses due to over-ripening. ERFs regulate the final target gene expression of target genes in ethylene signal transduction pathway by binding

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to the GCC box in their promoter regions. These target genes in turn regulate the firmness, aroma, taste, color, and shelf life of the fruit (Nath et al. 2006). Overexpression of LeERF1 was found to show ethylene triple response on etiolated seedlings, however, antisense lines exhibited longer shelf life (Li et al. 2007). Similarly, overexpression of LeERF2 also stimulated premature seed germination by inducing mannanase 2 gene and enhanced formation of hook in etiolated seedlings (Pirrello et al. 2006). Recently, Zhang and coworkers (2009) have shown that LeERF2 is ethylene-inducible and acts as a positive regulator in the feedback loop of ethylene induction. Authors had shown LeERF2 regulates expression of ACS in tobacco and ACS as well as ACO3 in tomato suggesting novel function of ERFs in ethylene biosynthesis. These studies suggest that modulating the expression of ERF family genes may help in modulating the ethylene response to obtain desirable characteristics. Although Arabidopsis has been serving as a model system for most of the plant studies but for certain aspects, for example, fruit ripening in fleshy fruits, tomato has emerged as a model system to study climacteric fruit ripening. In fact, most of the ripening related studies have been carried out in tomato (Giovannoni 2004, 2007). Moreover, according to 2007 FAO statistics (www.faostat.fao.org), tomato comprises 28% (1.08 9 108 MT) of total world production (3.8 9 108 MT) of major fleshy fruits generating about 2.5 9 1010 international dollars (Bapat et al. 2010). In this study, 57 genes were found to exhibit differential expression in temporal stages of fruit development suggesting their possible involvement in fruit development and ripening. The genes belonging to group III, VI, and IX could be good candidates for further characterization and gain insight into mechanism of fruit ripening. However, their induction during ripening may be the repercussion of respiratory burst and other metabolic activities (Handa et al. 2007). Earlier, Tournier and coworkers (2003) analyzed the expression of LeERF1, 2, 3, and 4 (renamed SlERF1-4 in this study) in leaf, stem, buds, open flowers, and red fruits. Authors could find expression of SlERF1 in stem; however, with additional stages included, we could also detect its expression in root tips. Similarly, SlERF2, earlier shown to express in red fruits, was found to exhibit high accumulation in both BK and RR stages of fruit development along with significant expression in roots. SlERF3, previously detected in stem, leaf, and flowers was found to exhibit very low-level expression in the stages analyzed using microarrays. SlERF4 was earlier detected in leaves, however, we could find its higher accumulation in cotyledons with significant expression in leaf, flower, and stem suggesting that the data generated in this study not only conforms with the previous reports,


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Table 2 Summary of ERF transcription factor genes characterized in various plant species Gene name

Functions

Plant

References

Bolita

Cell expansion and proliferation

At

Marsch-Martinez et al. (2006)

OsDREB1A, B

High salinity and freezing

At

Ito et al. (2006)

TINY

Growth regulation

At

Wilson et al. (1996)

WIN1/SHNs

Wax accumulation

At

Aharoni et al. (2004), Broun et al. (2004)

WXP1, WXP2

Wax accumulation

At

Zhang et al. (2007a)

ABI4

Abscisic acid response, sugar signaling

At

Finkelstein et al. (1998), Arenas-Huertero et al. (2000), Huijser et al. (2000)

ABR1 AtERF14

Abscisic acid response Biotic stress

At At

Pandey et al. (2005) Onate-Sanchez et al. (2007)

AtERF38

Secondary wall metabolism

At

Lasserre et al. (2008)

AtERF4

Ethylene, jasmonic, and abscisic acid response

At

McGrath et al. (2005), Yang et al. (2005)

AtERF7

Abscisic acid response

At

Song et al. (2005)

CBF1-4/ DREB1A-D

Freezing, drought, salt tolerance

At

Liu et al. (1998), Gilmour et al. (2000), Haake et al. (2002)

DDF1

Salt tolerance, GA biosynthesis regulation

At

Magome et al. (2004)

DREB2A

Drought, freezing tolerance

At

Sakuma et al. (2006)

DREB2C

Thermotolerance

At

Lim et al. (2007)

ERF1

Disease resistance

At

Solano et al. (1998), Berrocal-Lobo et al. (2002)

ESR1/DRN

Organ identity

At

Banno et al. (2001), Kirch et al. (2003)

GmDREB2 GmSGR

High salt, drought Reduced ABA-sensitivity, enhanced salt sensitivity

At At

Chen et al. (2007) Wang et al. (2008a)

HvRAF

Salt tolerance and pathogen resistance

At

Banno et al. (2001), Kirch et al. (2003)

JcERF

Salt and freezing tolerance

At

Tang et al. (2007)

LEP

Leaf petiole development

At

van der Graaff et al. (2000)

LpCBF3

Freezing tolerance

At

Xiong and Fei (2006)

ORA59

Essential integrator of JA and ethylene signal transduction

At

Pre et al. (2008)

OsERF1

Growth and development

At

Hu et al. (2008)

OsDREB1F

Salt, drought, and low temperature tolerance

At, Os Wang et al. (2008b)

Ca-DREBLP1

Transcriptional activation in response to dehydration and Ca high salinity

CaERFLP1

Salt tolerance, disease resistance

Ca

Lee et al. (2004)

CaPF1

Freezing tolerance, disease resistance

Ca

Yi et al. (2004)

ORCA3

Indole alkaloid biosynthesis

Cr

van der Fits and Memelink (2000)

DvDREB2A

Heat, cold, drought, ABA, saline stress response

Dv

Liu et al. (2008)

GhERF1

Salinity, cold and drought acclimation

Gh

Qiao et al. (2008)

GmEREBP1

Biotic stress

Gm

Mazarei et al. (2007)

GmRAV

Photosynthesis and senescence

Gm

Zhao et al. (2008)

Nud

Lipid biosynthetic pathway

Hv

Taketa et al. (2008)

MtSERF1

Somatic embryogenesis

Mt

Mantiri et al. (2008)

WXP1

Wax accumulation

Mt

Zhang et al. (2005)

NtERF5

Disease resistance

Nt

Fischer and Droge-Laser (2004)

OPBP1

Salt tolerance and disease resistance

Nt

Guo et al. (2004)

TaERF1

Multiple stress tolerance

Nt

Xu et al. (2007)

TaPIP1 Tsi1

Biotic stress Salt tolerance and disease resistance

Ta Nt

Dong et al. (2010) Park et al. (2001)

GbERF2

Biotic stress

Nt

Zuo et al. (2007)

JERF1

Salt and cold tolerance

Nt

Wu et al. (2007)

OsBIERF3

Salt and disease resistance

Nt

Cao et al. (2006)

Hong and Kim (2005)

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470


Table 2 continued Gene name

Functions

Plant

References

SodERF3

Salt and drought tolerance

Nt

Trujillo et al. (2008)

TiERF1

Biotic stress

Nt

Liang et al. (2008)

TSRF1

Absicic acid response

Nt

Zhang et al. (2008b)

FZP

Floral meristem identity

Os

Komatsu et al. (2003)

BFL1

Transition of spikelet to floret meristem

Os

Zhu et al. (2003)

Sub1

Submergence tolerance

Os

Xu et al. (2006)

ERF2

Seed germination

Sl

Pirrello et al. (2006)

JERF3

Salt tolerance

Sl

Wang et al. (2004)

LeERF3b

Drought, dessication and low temp response

Sl

Chen et al. (2008a, b, c)

Pti4

Disease resistance

Sl

Zhou et al. (1997), Gu et al. (2002)

Pti5 Pti6

Disease resistance Disease resistance

Sl Sl

He et al. (2001), Gu et al. (2002) Zhou et al. (1997), Gu et al. (2002)

TERF1

Salt tolerance

Sl

Huang et al. (2004)

CaPF1

Freezing, heat, heavy metal, and oxidative stress

St

Youm et al. (2008)

TaERF3

Defense

Ta

Zhang et al. (2007a, b)

Bd1

Floral meristem identity

Zm

Chuck et al. (2002)

At, Arabidopsis; Ca, Capsicum annum; Cr, Catharanthus roseus; Dv, Dendranthema vestitum; Gh, Gossypium hirsutum; Gm, Glycine max; Hv, Hordeum vulgare; Mt, Medicago truncatula; Nt, Nicotiana tabaccum; Os, Oryza sativa; Sl, Solanum lypersicon; St, Solanum tuberosum; Ta, Triticum aestivum; Zm, Zea mays

but also provide more comprehensive details of ERF family in tomato. The information generated in this study will help in the selection of candidate genes for further characterization. Since, high-level conservation exists in genic content as well as position in members of the Solanaceae, information generated in tomato can also be utilized in other Solanaceae members (Moore et al. 2005). Moreover, ERF genes have been shown to confer resistance in heterologous systems suggesting that the basic components of AP2 domain are conserved in different plant species (Jaglo et al. 2001; Gu et al. 2002; Wu et al. 2002). Further studies in these species will reveal the key players leading to phenotypic diversity and help understand the evolution of these agronomically important crops. Role of ERF family genes in abiotic stress response In order to adapt to a large number of biotic and abiotic stresses, plants respond at physiological as well as biochemical levels. Many transcription factor families have been shown to exhibit stress-responsive gene expression with significant overlap in response to various stress treatments, suggesting that signaling pathways involved in biotic and abiotic stress are interconnected (Kunkel and Brooks 2002; Singh et al. 2002; Fujita et al. 2006). We have also observed the induction of ERF family genes in response to more than one stress treatments suggesting a crosstalk between different stress signaling pathways.

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There is a complex regulatory network of signaling components that decide how a plant will respond to a stress treatment. Previous studies have shown that ERF domain binds to a cis-regulatory element, named GCC box in the promoter regions of the pathogenesis-related genes and regulates their expression in response to ethylene. Similarly, DREB domain binds to the DRE element and regulates expression in response to dehydration and lowtemperature. However, later Tsi1 (Tobacco stress-induced gene 1) was found to confer resistance to both pathogen as well as osmotic stress, when over-expressed. This finding suggests that an ERF domain can bind to both GCC box as well as DRE motif (Park et al. 2001). In the recent years, several other ERF proteins, capable of binding GCC box as well as DRE element, have been identified which include DREB2A (Sakuma et al. 2002), CBF1 (Hao et al. 2002), JERF1 (Zhang et al. 2004a), and BnDREBIII-1 (Liu et al. 2006). It suggests that DREB- and ERF-mediated signaling pathways are not exclusive and crosstalk exists between them. Overexpression of ERF family genes in Arabidopsis, tobacco, and tomato has been shown to confer increased resistance to biotic as well as abiotic stresses (Jaglo-Ottosen et al. 1998; Kasuga et al. 1999; He et al. 2001; Park et al. 2001; Berrocal-Lobo et al. 2002). In addition, the genes involved in jasmonate-responsive secondary metabolism have also been shown to be involved in AP2/ERF regulated defense response (van der Fits and Memelink 2000). Interestingly, members of both the groups were found to be differentially regulated in response to abiotic stress


treatments; however, DREB family genes exhibited maximum differential expression in response to dehydration, salt, and heat stress.

Conclusions ERF family genes play crucial role in developmental regulation as well as environmental stress response. It is essential to understand their transcriptional and translational regulation to utilize these genes for improving agronomic traits. The conservation in their structure in heterologous systems suggests them to play similar roles in diverse taxonomic groups. With the tomato genome being sequenced by an international consortium, this conservation can easily be utilized to gear up the pathway analysis in tomato. The expression data generated in this study reveals that several ERF family genes express in very narrow expression windows in developmental stagespecific manner. Further studies will need to be carried out to understand how the molecular functions of ERFs translate into diverse range of roles at the plant level. Data generated in this study after overlaying with the previous literature will add to our understanding of the complex regulatory networks operating during development and stress response and will open opportunities for applications in agriculture, environmental, and energy sectors. Successful field trial of submergence tolerant rice carrying Sub1 demonstrates one of the enormous potential implications of this family. Acknowledgments This work has been funded by Department of Biotechnology, Government of India. RK and AUS acknowledge Council for Scientific and Industrial Research (CSIR) for senior research fellowships.

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