JIPB
Journal of Integrative Plant Biology
Genome‐wide identification, evolution, and expression analysis of RNA‐binding glycine‐rich protein family in maize Research Article
Jianhua Zhangy, Yanxin Zhaoy, Hailin Xiao, Yonglian Zheng and Bing Yue* National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. yThese authors contributed equally to this work. *Correspondence: [email protected]
Abstract The RNA‐binding glycine‐rich protein (RB‐GRP) family is characterized by the presence of a glycine‐rich domain arranged in (Gly)n‐X repeats and an RNA‐recognition motif (RRM). RB‐GRPs participate in varied physiological and biochemical processes especially in the stress response of plants. In this study, a total of 23 RB‐GRPs distributed on 10 chromosomes were identified in maize (Zea mays L.), and they were divided into four subgroups according to their conserved domain architecture. Five pairs of paralogs were identified, while none of them was located on the same chromosomal region, suggesting that segmental duplication is predominant in the duplication events of the RB‐GRPs in maize. Comparative analysis of RB‐GRPs in maize, Arabidopsis (Arabidopsis thaliana L.), rice (Oryza sativa L.), and wheat (Triticum aestivum) revealed that two exclusive subgroups were only identified in maize. Expression of eight ZmRB‐GRPs was significantly
INTRODUCTION Glycine‐rich proteins (GRPs) belong to a superfamily with a glycine‐rich domain arranged in (Gly)n‐X repeats. According to the presence of additional motifs and the arrangement of the glycine repeats, the GRP family was divided into four classes (Mangeon et al. 2010). Among them, members in Class IV are also known as RNA‐binding GRPs (RB‐GRPs). They include either an RNA‐recognition motif (RRM) or a cold‐ shock domain (CSD), in addition to the glycine‐rich domain, and CCHC zinc‐fingers may also appear in their structure (Mangeon et al. 2010). Moreover, there are four subgroups in the Class IV, IVa (in which one RRM motif is contained), IVb (one RRM and a CCHC zinc‐finger are involved), IVc (which contains a CSD and two or more zinc‐fingers), and IVd (which includes two RRMs) (Sachetto‐Martins et al. 2000; Fusaro et al. 2001; Bocca et al. 2005). The RRMs in the N termini of the four RB‐GRPs in Arabidopsis are high homologous, but the glycine‐rich domains in the C termini are different in length (Kim et al. 2010b). In the past 20 years, a number of RB‐GRPs were reported in different plants. For instance, eight and six RB‐GRPs were identified in Arabidopsis (Lorković and Barta 2002; Lorković 2009) and rice (Kim et al. 2010a), respectively. They would play important roles in plant growth, development, and stress resistance, because of the presence of RRM, CSD, or CCHC domains (Dreyfuss et al. 2002; Glisovic et al. 2008). More and October 2014 | Volume 56 | Issue 10 | 1020–1031
regulated by at least two kinds of stresses. In addition, cis‐ elements predicted in the promoter regions of the ZmRB‐GRPs also indicated that these ZmRB‐GRPs would be involved in stress response of maize. The preliminary genome‐wide analysis of the RB‐GRPs in maize would provide useful information for further study on the function of the ZmRB‐GRPs. Keywords: Gene expression; maize; motif prediction; phylogenetic analysis; RNA‐binding glycine‐rich proteins Citation: Zhang J, Zhao Y, Xiao H, Zheng Y, Yue B (2014) Genome‐wide identification, evolution, and expression analysis of RNA‐binding glycine‐rich protein family in maize. J Integr Plant Biol 56: 1020–1031. doi: 10.1111/jipb.12210 Edited by: Xiao‐Quan Wang, Institute of Botany, CAS, China Received Jan. 4, 2014; Accepted Apr. 25, 2014 Available online on Apr. 30, 2014 at www.wileyonlinelibrary.com/ journal/jipb © 2014 Institute of Botany, Chinese Academy of Sciences
more RB‐GRPs will be identified in plants with the rapid expansion of genome data in consideration of their critical role in plant development. In recent years, function of RB‐GRPs has been analyzed in various plants, and their expression was found to be regulated by different environmental stresses, such as low temperature, flooding, high temperature, high salt, UV radiation, heavy metal, and so on. For instance, in Arabidopsis, Sachetto‐Martins et al. (2000) found that the expression of RB‐GRPs was regulated by both biotic and abiotic factors; in the case of salt and dehydration, RB‐GRP2 promoted seed germination and growth of Arabidopsis (Park et al. 2009); AtGRP2, AtGRP7, AtRZ‐1a, and OsRZ2 could enhance cold and freezing tolerance in Arabidopsis and rice (reviewed by Jung et al. 2013). RB‐GRPs conferring stress tolerance might be through regulating the efficiency of mRNA translation as RNA chaperones in plants (reviewed by Kang et al. 2013). However, few RB‐GRPs were identified in maize to date, thus genome‐wide identification and characterization of RB‐GRPs in maize is very important. In this study, gene structure, phylogenetic relationships, motifs, and phosporylated residues of the 23 RB‐GRPs identified in maize were analyzed and predicted, and the expression patterns of eight ZmRB‐GRPs under different stress conditions were also surveyed. Results in this study would facilitate cloning and functional characterization of the RB‐GRPs in maize. www.jipb.net
RB‐GRP gene family in maize
RESULTS RB‐GRPs in maize Consensus sequence of the RRM domain was used to query the maize genome annotation database using HMMER 3.0 package (Newberg 2009), a total of 336 non‐redundant RNA‐ binding protein (RBP)‐encoding genes were identified, and 169 maize RNA‐binding proteins (ZmRBPs) were annotated and classified based on sequence homology to the Arabidopsis RBPs (Table S1). Then, 23 RNA‐binding glycine‐rich proteins (ZmRB‐GRPs) were collected according to presence of (Gly)n‐X repeats in the 169 ZmRBPs. Because there was no annotation assigned to these newly identified genes, these ZmRB‐GRP genes were designated as ZmRB‐GRP1 to ZmRB‐GRP23 (Table 1). Subcellular location, isoelectric point (pI), and molecular weight (MW) of the ZmRB‐GRPs were shown in Table 1. Protein sequence analysis indicated that all of the 23 ZmRB‐ GRPs had a high conserved RRM domain, while their length varied from 155 to 700 amino acids (aa). Their MW varied from 15.4 to 74.5 kDa, and their pI ranged from 4.78 to 9.74. Subcellular localization predicted by WoLF PSORT showed that 15 of them were located in nucleus, three in cytoplasm, two in chloroplast, two in plastid, and one was targeted to mitochondria (ZmRB‐GRP7). Phylogenetic relationship and gene structure of the ZmRB‐GRPs The ZmRB‐GRPs were divided into four classes (a to d) based on their conserved motif composition as described previously
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(Mangeon et al. 2010) (Figure 1A). The classes a, b, and c contain six, six, and two ZmRB‐GRPs, respectively, and the remnant nine ZmRB‐GRPs were assigned to the Class d. In order to investigate the phylogenetic relationship among the ZmRB‐ GRPs, four neighbor‐joining (N‐J) phylogenetic trees for the four classes were built based on protein sequence alignment of the ZmRB‐GRPs (Figure 1B). To explore the evolutionary relationship, exon/intron structures of the ZmRB‐GRPs were analyzed by software GSDS (Figure 1C). The number of introns determined in the ZmRB‐GRPs ranges from 0 to 14. Most of the ZmRB‐GRPs have more than one intron; however, ZmRB‐GRP8, ‐13, ‐14, and ‐22 are intron less. Genes in the Class d have the largest number of introns, while those in the classes b and c contain less introns. Intragroup members share conserved gene structure; this indicates that they were derived from the same ancestor. Motif prediction of the ZmRB‐GRPs in maize To further understand the diversification of the ZmRB‐GRPs, databases Pfam (http://pfam.sanger.ac.uk/) and SMART (http://smart.embl‐heidelberg.de/) were used to predict putative motifs. Motifs in each ZmRB‐GRP are shown in Figure 1A. Proteins in the Class a contain one RRM, and those in the Class b include one RRM and a zinc finger. Class c consists of a CSD and more than one zinc finger. For the proteins in the Class d, two RRMs are included (Figure 1A). Motif prediction in these ZmRB‐GRPs also revealed that the intragroup ZmRB‐GRPs are conserved during evolution. Differences of motif composition
Table 1. Detailed information of the ZmRB‐GRPs Gene
Subcellular location
Size (aa)
pI
MW (kDa)
GRMZM2G150521 (ZmRB‐GRP1) GRMZM2G131167 (ZmRB‐GRP2) GRMZM2G042118 (ZmRB‐GRP3) GRMZM2G165901 (ZmRB‐GRP4) GRMZM2G080603 (ZmRB‐GRP5) GRMZM2G001850 (ZmRB‐GRP6) GRMZM5G874478 (ZmRB‐GRP7) GRMZM2G082931 (ZmRB‐GRP8) GRMZM2G083783 (ZmRB‐GRP9) GRMZM2G161242 (ZmRB‐GRP10) GRMZM2G053223 (ZmRB‐GRP11) GRMZM2G097775 (ZmRB‐GRP12) GRMZM2G389768 (ZmRB‐GRP13) GRMZM5G895313 (ZmRB‐GRP14) GRMZM2G139643 (ZmRB‐GRP15) GRMZM2G167356 (ZmRB‐GRP16) GRMZM2G064518 (ZmRB‐GRP17) GRMZM2G104481 (ZmRB‐GRP18) GRMZM2G152526 (ZmRB‐GRP19) AC198361.3_FGT004 (ZmRB‐GRP20) GRMZM2G167505 (ZmRB‐GRP21) GRMZM2G132465 (ZmRB‐GRP22) GRMZM2G131943 (ZmRB‐GRP23)
Cyto Chlo Chlo Nucl Nucl Cyto Mito Nucl Nucl Nucl Nucl Nucl Nucl Nucl Nucl Nucl Nucl Nucl Plas Nucl Nucl Plas Cyto
254 308 156 159 155 196 205 316 259 311 261 489 249 208 443 384 413 401 348 443 453 454 700
4.78 5.09 6.58 5.52 6.11 9.74 7.61 9.19 8.94 9.29 8.63 8.68 5.95 5.92 6.68 5.84 4.84 4.87 9.11 6.17 6.18 5.7 5.1
25.1 30.1 15.7 15.5 15.4 20.5 21.8 34.1 32.4 34.3 28.1 46.8 23.2 20.2 45.2 39.1 43.1 41.6 37.2 44.2 44.9 46.6 74.5
Information of the maize RB‐GRPs was from MaizeGDB (http://gbrowse.maizegdb.org) and their isoelectric points (pI) and molecular weights (MW) were computed by the online tool Expasy (http://expasy.org/tools). Subcellular location of the ZmRB‐ GRPs was predicted by WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html). Cyto, cytoplasm; nucl, nucleus; plas, plastid; mito, mitochondria; chlo, chloroplast. www.jipb.net
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Figure 1. Phylogenetic analysis, schematic diagrams of gene structure, and motif composition in the ZmRB‐GRPs (A) Distribution of conserved motifs in the ZmRB‐GRPs. The conserved motifs were identified by screening the Pfam database (http://pfam.janelia.org/) and displayed using DOG1.0 (http://dog.biocuckoo.org/). RRM, RNA‐recognition motif; CSD, cold‐shock domain; CCHC, zink knuckle or zink finger. (B) Phylogenetic trees of the ZmRB‐GRPs. The 23 ZmRB‐GRPs were divided into four classes based on their conserved motifs, and the trees were constructed with a complete alignment of the ZmRB‐GRPs in each class by the neighbor‐joining (N‐J) method with bootstrap analysis (1,000 replicates). Bootstrap values are indicated as percentages (when> 50%) along the branches. (C) Schematic diagram of gene structure was displayed by GSDS (http://gsds.cbi.pku.edu.cn/). October 2014 | Volume 56 | Issue 10 | 1020–1031
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Figure 1. Continued
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Figure 2. Structure of RNA‐recognition motifs (RRMs) in the ZmRB‐GRPs (A) Similarity of RRMs in the ZmRB‐GRPs. Multiple sequence alignment was conducted by T‐Coffee (http://www.tcoffee.org/). (B) The 3D structure of RRM domain in ZmRB‐GRP1. (C) The 3D structure of RRM domain in Rna15 (Pancevac et al. 2010). The two RRM domains (B, C) have high similarity (TM‐score ¼ 0.484, RMSD ¼ 3.790, IDEN ¼ 0.207, and Cov ¼ 0.642). The 3D structure of the ZmRB‐GRP1 RRM domain is developed using I‐TASSER (http://zhanglab.ccmb.med.umich.edu/I‐TASSER/) and exhibited by Chimera1.8.1 (http://www.cgl.ucsf.edu/chimera/). RNP1 and RNP2 are shown in purple and cyan, respectively.
in different groups of the ZmRB‐GRPs suggest that their function was diversified over evolution. RRMs in the ZmRB‐GRPs were also analyzed by T‐Coffee (http://tcoffee.org/503/index.html). Most of the RRMs have high similarity and contain two consensus RNPs (Figure 2A). In addition, most of the RRMs are structured by two a‐helices and five b‐sheets (Figure 2B). The conserved b1‐a1‐b2‐b3‐a2‐ b4‐b5 topology is essential to RNA binding. To further understand the structure of the proteins, three‐dimensional (3D) structure of the RRM in ZmRB‐GRP1 was analyzed (Figure 2B); it has high similarity with Rna15 (TM score ¼ 0.484, RMSD ¼ 3.790, IDEN ¼ 0.207, and Cov ¼ 0.642) (Figure 2C). Chromosomal location and gene duplication of the ZmRB‐GRPs To explore genomic distribution of the ZmRB‐GRPs, cDNA sequence of each ZmRB‐GRP was used to search the MaizeGDB database (http://gbrowse.maizegdb.org) with BLASTn, and software MapInspect was used to display the location of each gene (Figure 3). The ZmRB‐GRPs distribute on all of the 10 chromosomes. Chromosomes 1 and 5 have a maximum of six and four ZmRB‐GRPs, respectively. Three ZmRB‐GRPs on chromosome 4, two each on chromosomes 3, 7, and 8 are found, respectively. One each distributes on the other four chromosomes (Figure 3). Three genes on chromosome 5, ZmRB‐GRP4, ‐10, and ‐21, are closely located to one another. Meanwhile, ZmRB‐GRP20 and ‐6 on chromosome 1 are also adjacent to each other. Distribution of paralogs could be used to analyze potential duplications and evolutionary patterns of the ZmRB‐GRPs. In this study, five pairs of paralogs were detected based on the bootstrap values in the phylogenetic analysis (Figure 1B). All of them randomly distribute on maize chromosomes, suggesting that segmental duplication is predominant in the expansion of the ZmRB‐GRPs in maize. October 2014 | Volume 56 | Issue 10 | 1020–1031
Evolutionary relationships of RB‐GRPs in maize, Arabidopsis, rice, and wheat Rapid development of plant genomics makes it possible to analyze the same gene family across different species. To investigate molecular evolution of RB‐GRPs in Arabidopsis, rice, wheat, and maize, protein sequences of 34 RB‐GRPs were phylogenetically analyzed. An N‐J distance phylogenetic tree was constructed with the alignment of all the known RB‐GRP amino acid sequences (Figure 4). The phylogenetic tree (Figure 4) shows that all the RB‐GRPs from the four higher plants are divided into four groups, A, B, C, and D. Among them, Group A forms the largest clade, containing 23 GR‐GRPs and accounting for 67.6% of the total RB‐GRPs, Group D constitutes the second largest clade, containing seven members. Groups B and C contain three and one member, only accounting for 8.8% and 2.9% of the total RB‐GRPs, respectively. Additionally, Groups B and C are exclusively derived from maize, implying that these RB‐GRPs turned up after maize appeared, or it may be due to the homologs of these genes not having been identified in other plants. On the other hand, Groups A and D are contributed by at least three species, indicating that these RB‐GRPs might have the same function in evolution. Prediction of phosphoraylation sites in the ZmRB‐GRPs Phosphorylation of the ZmRB‐GRPs was analyzed with P3DB (Figure 5). Most of the phosphorylated residues are located either ahead or behind the RRMs or CSDs, except for ZmRB‐ GRP13 and ‐16. No phosphorylated site was predicted in ZmRB‐ GRP3, ‐4, ‐5, ‐7, ‐12, and ‐14. All of the ZmRB‐GRPs in Class d were phosphorylated, while only half of the members in Class a were found to be phosphorylated. On average, 1.2, 3.5, 1.5, and 4.5 phosphorylated sites were found in each member of the four classes, respectively. Most of the phosphorylated residues were serine in the ZmRB‐GRPs of the Classes a and c, while phosphorylated threonine and tyrosine were www.jipb.net
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Figure 3. Physical locations of the ZmRB‐GRPs on maize chromosomes Chromosome number is indicated at the top of each chromosome. Chromosomal positions of the ZmRB‐GRPs are indicated by their names. The lines connect the corresponding five pairs of paralogous genes in duplicated blocks. All of them were clustered as segmental duplication.
detected in the members of the Classes b and d. In addition, in the ZmRB‐GRPs of the Classes a and b (except for ZmRB‐GRP10), most of the phosphorylated residues are located at the C termini, while in the members of the Classes c and d, phosphorylated residues are located either in the C termini or N termini. Expression profiles of the ZmRB‐GRPs in different tissues Two sets of data were used to analyze the expression patterns of the ZmRB‐GRPs in differentially developmental organs or tissues. The expressed‐sequence tag (EST) database of maize (http://blast.ncbi.nlm.nih.gov/dbEST/) was screened, which resulted in the identification of ESTs for most of the ZmRB‐ GRPs except for ZmRB‐GRP2, ‐3, and ‐5 (Table S4). ZmRB‐GRP1 was detected in all of the selected tissues. The others were found in more than one organ or tissue except mixed tissues. Meanwhile, the Nimblegen maize microarray data (ZM37) from PLEXdb (http://www.plexdb.org/) was also used to study the expression patterns of the ZmRB‐GRPs (Figure S1). Most of the ZmRB‐GRPs were highly expressed in all the tissues analyzed except for ZmRB‐GRP11, and ‐16, which illustrated that most of the ZmRB‐ GRPs play important roles in the whole lifetime of maize. Interestingly, of the ZmRB‐GRPs, ZmRB‐GRP5 had the highest expression level in all of the tissues. However, ZmRB‐GRP2 was expressed lower in the leaves compared to other tissues. Expression analysis of the ZmRB‐GRPs under different stress conditions In order to understand stress responsiveness of the ZmRB‐ GRPs, two genes from each class of the ZmRB‐GRPs were chosen for expression profile analysis of the ZmRB‐GRPs under different stress conditions, cold, salt, abscisic acid (ABA), and recovery‐of‐chilling, through real‐time quantitative reverse transcription polymerase chain reaction (qRT‐ www.jipb.net
PCR) (Figure 6). Under cold stress, ZmRB‐GRP13, ‐14, and ‐15 were downregulated, the other genes were upregulated at first then downregulated. When treated with salt, expression of ZmRB‐GRP13 and ‐21 did not change much, while that of the others was upregulated at first then downregulated. Expression of all eight genes was upregulated at first then downregulated under the stress of ABA, but that of ZmRB‐ GRP7 and ‐13 had the least changes. When the temperature increased from cold (10 °C) to normal (room temperature, 25 °C), ZmRB‐GRP4 and ‐7 were upregulated in general, expression of ZmRB‐GRP5, ‐8, and ‐13 was increased at first then decreased, but that of the others (ZmRB‐GRP14, ‐15, and ‐ 21) was not changed. In conclusion, these results implied that the ZmRB‐GRPs confer stress tolerance through regulating their expression. Cis‐elements in the promoter sequences of ZmRB‐GRPs Transcriptional factors (TFs) are essential for the regulation of gene expression, thus cis‐elements that interacted with specific TFs were predicted within the promoter sequences of the ZmRB‐GRPs by PlantPAN. A total of 20 cis‐elements were predicted to be associated with the ZmRB‐GRPs, and they were divided into three classes, namely ABA‐responsive elements (ABREs), drought‐responsive elements (DREs), and cold‐responsive elements (CREs) (Tables S2, S3). The average number of ABRE, CRE, and DRE in Class a is 2.0, 2.8, and 2.8, respectively, while that in Class b is 2.3, 3.2, and 3.8, respectively. In Class c, on average 1.5, 4.0, and 4.0 of ABREs, CREs, and DREs are contained, respectively, and in Class d, 2.0, 3.7, and 3.1 of ABREs, CREs, and DREs, respectively, are involved (Figure 7). Particularly, ZmRB‐GRP7 only possesses one CRE, and ZmRB‐GRP3, ‐12, and ‐19 only have one ABRE. October 2014 | Volume 56 | Issue 10 | 1020–1031
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Zhang et al. hupehensis Rehd (Wang et al. 2012), wheat (Nakaminami et al. 2005), tobacco (Brady et al. 1993), and petunia (Condit and Meagher 1987). Most of them were involved in plant growth, development, and various stress responses (reviewed by Jung et al. 2013; Kang et al. 2013). In maize, however, only a RB‐GRP, MA16, was characterized and found to be involved in a variety of stress responses (Ludevid et al. 1992). Thus genome‐ wide identification, evolution, and expression analysis of the RB‐GRP family in maize will facilitate understanding of the function of this gene family.
Figure 4. Phylogenetic relationships among the identified RB‐ GRPs in Arabidopsis, rice, wheat, and maize Phylogenetic tree of all the identified RB‐GRPs in the four species was constructed from a complete alignment of 34 GRP proteins by the neighbor‐joining method with bootstrap analysis (1,000 replicates). Arabidopsis, rice, wheat, and maize genes are indicated at the end of the branches, respectively. The scale bar corresponds to 0.2 estimated amino acid substitutions per site. Among the 34 RB‐GRPs, GRP162 was reported by Hu et al. (2012), and the other RB‐GRPs in wheat, rice, and Arabidopsis were collected from a report of Mangeon et al. (2010).
DISCUSSION GRPs belong to a large GRP family, which exist ubiquitously in plant, bacteria, and humans, and are implicated in varied important biological processes. Up to now, the function of a number of GRPs has been studied in Arabidopsis (Yang and Karlson 2011), rice (Chaikam and Karlson 2008), Malus October 2014 | Volume 56 | Issue 10 | 1020–1031
RB‐GRPs in maize In this study, a total of 23 ZmRB‐GRPs were identified, which is much more than that identified in rapeseeds (Kim et al. 2012), Arabidopsis (Lorković and Barta 2002; Lorković 2009), and rice (Kim et al. 2010a). Conserved protein sequence analysis of the ZmRB‐GRPs indicated that there are four groups (Classes a, b, c, and d) in the maize genome. This was similar to that in other plant species (Mangeon et al. 2010). Although the ZmRB‐GRPs identified in this study contain glycine‐rich regions, no GRP domain was predicted in them by Pfam HMM file (http://pfam. janelia.org/). The HMM file in Pfam was built from the GRPs (especially from nodulin‐containing GRPs), which only represent a little part of the GRP superfamily rather than the RB‐GRP group. Thus no GRP domain in the ZmRB‐GRPs is possible. Intragroup ZmRB‐GRPs have conserved gene structure and motif composition, indicating that the ZmRB‐GRPs in the same group could have the same function and they would come from a common ancestor. For instance, ZmRB‐GRPs in the Class b include only one zinc finger, and that in the Class c consist of more than one zinc finger, while that in the Classes a and d contain no zinc finger. Besides, ZmRB‐GRPs in Class d contain two RRMs (Figure 1A, C), suggesting that they might change the conformation to bind RNA as U2AF65 doing in human (Daubner et al. 2013). However, ZmRB‐GRPs in the other classes only contain one RRM or CSD, suggesting that besides RNA recognition (Pancevac et al. 2010), they have the function of stress response. Comparative genomic analysis of RB‐GRPs was also carried out in maize, rice, wheat, and Arabidopsis, and generally four subgroups were divided (subgroups A, B, C, and D) (Figure 4). In the subgroups A and D, RB‐GRPs come from at least three plant species, which demonstrates these RB‐GRPs are derived from one ancestor and play the same role in different plants. Gene family expansion mainly caused by tandem duplication, segmental duplication and transposition events. In this study, five pairs of paralogous ZmRB‐GRPs are distributed on different chromosomes, suggesting that the ZmRB‐GRPs expand mainly through segmental duplications. In diploidized polyploid plants, segmental duplication is most frequently found (Cannon et al. 2004). RNA‐binding ability and compartmentalization of RBPs could be affected by phosphorylation and phosphorylation site. For instance, when serines in the RRM termini were phosphorylated, SRSF1 (aka ASF/SF2) appeared to induce a key molecular switch from intra‐ to inter‐molecular interactions during pre‐mRNA splicing (Cho et al. 2011). In addition, the phosphorylation of Nrd1 could enhance its localization to stress‐induced cytoplasmic granules in fission yeast (Satoh et al. 2012). In this study, most (18) of the 23 ZmRB‐GRPs were www.jipb.net
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Figure 5. Predicted phosphorylation sites in the ZmRB‐GRPs Phosphorylation analysis was carried out using P3DB (http://www.p3db.org/). The cutoff of specificity is 95% except for that of ZmRB‐GRP24 (99.9%) since there are too many phosphorylated sites in this gene. S, serine; T, threonine; Y, tyrosine.
predicted to contain 1 to 13 phosphorylation residues, and the residues and their locations were generally different in different classes (Figure 5), suggesting function of the genes in different classes are varied. As shown in Table 1, the ZmRB‐ GRPs targeted to different organelles, implying that the localization of them could be affected by the number or the variety of phosphorylated residues. Role of the ZmRB‐GRPs under stress tolerance Cis‐elements participate in controlling a lot of important cellular processes (Jiang et al. 2012), and they are essential for the regulation of gene expression. In this study, a total of 20 stress‐related cis‐elements localized in the promoters of the ZmRB‐GRPs were predicted (Tables S2, S3). Most of these cis‐ elements were found to be involved into stress tolerance in previous studies (Baker et al. 1994; Abe et al. 2003; Dubouzet et al. 2003). For example, ABI4, DPBFCOREDCDC3, ABREOSRAB21, ACGTABREMOTIFA2OSEM, and SBOXATRBCS were related to ABA response (Marcotte et al. 1989; Kim et al. 1997; Hattori et al. 2002; Niu et al. 2002; Acevedo‐Hernandez et al. 2005); DRECRTCOREAT, LTRECOREATCOR15, and MYCCONSENSUSAT were involved in cold, drought, light, and ABA response (Baker et al. 1994; Abe et al. 2003; Dubouzet et al. 2003); ABRELATERD1, ACGTATERD1, AtMYB2, AtMYC2, DRE2COREZMRAB17, MYB1AT, MYB2AT, MYB2CONSENSUSAT, MYBATRD22, MYBCORE, MYCATERD1, and MYCATRD22 were associated with drought‐dependent transcriptional control in gene expression (Abe et al. 1997; Busk 1997; Simpson et al. 2003). This implied that the ZmRB‐GRPs could respond to stress environments. Most of Arabidopsis and rice RB‐GRPs were involved in response to stresses (Lorković and Barta 2002; Lorković 2009; www.jipb.net
Kim et al. 2010a). In this study, expression of eight ZmRB‐GRPs selected from different subgroups was analyzed under four kinds of stress conditions. All of them significantly responded to two to four kinds of stresses, indicating that they would also be involved in the physiological processes of multiple stress responses of maize. It is interesting to notice that expression of ZmRB‐GRP7 with only one ABRE in its promoter was not affected by ABA treatment; expression of ZmRB‐ GRP13, ‐14, and ‐15 with the least number of CREs was downregulated by cold treatment; on the other hand, expression of ZmRB‐GRP4 and ‐5, which possess more ABREs and CREs in their promoter regions, was upregulated under the stresses of cold and ABA. Together, these results illustrate that the expression of most of the ZmRB‐GRPs could be regulated through cis‐elements under stress conditions, leading to tolerance to the stresses as reported previously (Nakaminami et al. 2006). However, their detailed roles in stress responses need to be further studied in future.
MATERIALS AND METHODS Identification of ZmRB‐GRPs in maize Based on the characteristic of RB‐GRPs, a two‐step method was used to obtain RB‐GRPs in maize (Zea mays L.). First, all of the RRM or CSD domain‐containing proteins in maize genome were identified via screening the maize filtered‐gene set (ZmB73_5b_FGS_translations.fasta downloaded from www. maizesequence.org) with RRM, RRM‐related, and CSD hmm files (PF00076, PF04059, PF08777, PF10378, PF10598, PF13893, PF14259, and PF00313) from Pfam (http://pfam.sanger.ac.uk/) October 2014 | Volume 56 | Issue 10 | 1020–1031
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Figure 6. Expression profiles of the ZmRB‐GRPs under various stress conditions Expression analysis was carried out by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The y‐axis represents the relative expression levels of the ZmRB‐GRPs compared with that of actin1. The x‐axis represents different treat time in each group. Error bars represent standard deviations for three replicates. Real‐time PCR data were analyzed using the 2DDCt method as described previously (Livak and Schmittgen 2001). Expression of the eight ZmRB‐GRPs was investigated under four different stress conditions, cold, recovery‐of‐chilling, ABA, and NaCl. in the HMMER 3.0 package with the E‐value 200 bp, maximum identity>95% and E‐value
Journal of Integrative Plant Biology
Genome‐wide identification, evolution, and expression analysis of RNA‐binding glycine‐rich protein family in maize Research Article
Jianhua Zhangy, Yanxin Zhaoy, Hailin Xiao, Yonglian Zheng and Bing Yue* National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China. yThese authors contributed equally to this work. *Correspondence: [email protected]
Abstract The RNA‐binding glycine‐rich protein (RB‐GRP) family is characterized by the presence of a glycine‐rich domain arranged in (Gly)n‐X repeats and an RNA‐recognition motif (RRM). RB‐GRPs participate in varied physiological and biochemical processes especially in the stress response of plants. In this study, a total of 23 RB‐GRPs distributed on 10 chromosomes were identified in maize (Zea mays L.), and they were divided into four subgroups according to their conserved domain architecture. Five pairs of paralogs were identified, while none of them was located on the same chromosomal region, suggesting that segmental duplication is predominant in the duplication events of the RB‐GRPs in maize. Comparative analysis of RB‐GRPs in maize, Arabidopsis (Arabidopsis thaliana L.), rice (Oryza sativa L.), and wheat (Triticum aestivum) revealed that two exclusive subgroups were only identified in maize. Expression of eight ZmRB‐GRPs was significantly
INTRODUCTION Glycine‐rich proteins (GRPs) belong to a superfamily with a glycine‐rich domain arranged in (Gly)n‐X repeats. According to the presence of additional motifs and the arrangement of the glycine repeats, the GRP family was divided into four classes (Mangeon et al. 2010). Among them, members in Class IV are also known as RNA‐binding GRPs (RB‐GRPs). They include either an RNA‐recognition motif (RRM) or a cold‐ shock domain (CSD), in addition to the glycine‐rich domain, and CCHC zinc‐fingers may also appear in their structure (Mangeon et al. 2010). Moreover, there are four subgroups in the Class IV, IVa (in which one RRM motif is contained), IVb (one RRM and a CCHC zinc‐finger are involved), IVc (which contains a CSD and two or more zinc‐fingers), and IVd (which includes two RRMs) (Sachetto‐Martins et al. 2000; Fusaro et al. 2001; Bocca et al. 2005). The RRMs in the N termini of the four RB‐GRPs in Arabidopsis are high homologous, but the glycine‐rich domains in the C termini are different in length (Kim et al. 2010b). In the past 20 years, a number of RB‐GRPs were reported in different plants. For instance, eight and six RB‐GRPs were identified in Arabidopsis (Lorković and Barta 2002; Lorković 2009) and rice (Kim et al. 2010a), respectively. They would play important roles in plant growth, development, and stress resistance, because of the presence of RRM, CSD, or CCHC domains (Dreyfuss et al. 2002; Glisovic et al. 2008). More and October 2014 | Volume 56 | Issue 10 | 1020–1031
regulated by at least two kinds of stresses. In addition, cis‐ elements predicted in the promoter regions of the ZmRB‐GRPs also indicated that these ZmRB‐GRPs would be involved in stress response of maize. The preliminary genome‐wide analysis of the RB‐GRPs in maize would provide useful information for further study on the function of the ZmRB‐GRPs. Keywords: Gene expression; maize; motif prediction; phylogenetic analysis; RNA‐binding glycine‐rich proteins Citation: Zhang J, Zhao Y, Xiao H, Zheng Y, Yue B (2014) Genome‐wide identification, evolution, and expression analysis of RNA‐binding glycine‐rich protein family in maize. J Integr Plant Biol 56: 1020–1031. doi: 10.1111/jipb.12210 Edited by: Xiao‐Quan Wang, Institute of Botany, CAS, China Received Jan. 4, 2014; Accepted Apr. 25, 2014 Available online on Apr. 30, 2014 at www.wileyonlinelibrary.com/ journal/jipb © 2014 Institute of Botany, Chinese Academy of Sciences
more RB‐GRPs will be identified in plants with the rapid expansion of genome data in consideration of their critical role in plant development. In recent years, function of RB‐GRPs has been analyzed in various plants, and their expression was found to be regulated by different environmental stresses, such as low temperature, flooding, high temperature, high salt, UV radiation, heavy metal, and so on. For instance, in Arabidopsis, Sachetto‐Martins et al. (2000) found that the expression of RB‐GRPs was regulated by both biotic and abiotic factors; in the case of salt and dehydration, RB‐GRP2 promoted seed germination and growth of Arabidopsis (Park et al. 2009); AtGRP2, AtGRP7, AtRZ‐1a, and OsRZ2 could enhance cold and freezing tolerance in Arabidopsis and rice (reviewed by Jung et al. 2013). RB‐GRPs conferring stress tolerance might be through regulating the efficiency of mRNA translation as RNA chaperones in plants (reviewed by Kang et al. 2013). However, few RB‐GRPs were identified in maize to date, thus genome‐wide identification and characterization of RB‐GRPs in maize is very important. In this study, gene structure, phylogenetic relationships, motifs, and phosporylated residues of the 23 RB‐GRPs identified in maize were analyzed and predicted, and the expression patterns of eight ZmRB‐GRPs under different stress conditions were also surveyed. Results in this study would facilitate cloning and functional characterization of the RB‐GRPs in maize. www.jipb.net
RB‐GRP gene family in maize
RESULTS RB‐GRPs in maize Consensus sequence of the RRM domain was used to query the maize genome annotation database using HMMER 3.0 package (Newberg 2009), a total of 336 non‐redundant RNA‐ binding protein (RBP)‐encoding genes were identified, and 169 maize RNA‐binding proteins (ZmRBPs) were annotated and classified based on sequence homology to the Arabidopsis RBPs (Table S1). Then, 23 RNA‐binding glycine‐rich proteins (ZmRB‐GRPs) were collected according to presence of (Gly)n‐X repeats in the 169 ZmRBPs. Because there was no annotation assigned to these newly identified genes, these ZmRB‐GRP genes were designated as ZmRB‐GRP1 to ZmRB‐GRP23 (Table 1). Subcellular location, isoelectric point (pI), and molecular weight (MW) of the ZmRB‐GRPs were shown in Table 1. Protein sequence analysis indicated that all of the 23 ZmRB‐ GRPs had a high conserved RRM domain, while their length varied from 155 to 700 amino acids (aa). Their MW varied from 15.4 to 74.5 kDa, and their pI ranged from 4.78 to 9.74. Subcellular localization predicted by WoLF PSORT showed that 15 of them were located in nucleus, three in cytoplasm, two in chloroplast, two in plastid, and one was targeted to mitochondria (ZmRB‐GRP7). Phylogenetic relationship and gene structure of the ZmRB‐GRPs The ZmRB‐GRPs were divided into four classes (a to d) based on their conserved motif composition as described previously
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(Mangeon et al. 2010) (Figure 1A). The classes a, b, and c contain six, six, and two ZmRB‐GRPs, respectively, and the remnant nine ZmRB‐GRPs were assigned to the Class d. In order to investigate the phylogenetic relationship among the ZmRB‐ GRPs, four neighbor‐joining (N‐J) phylogenetic trees for the four classes were built based on protein sequence alignment of the ZmRB‐GRPs (Figure 1B). To explore the evolutionary relationship, exon/intron structures of the ZmRB‐GRPs were analyzed by software GSDS (Figure 1C). The number of introns determined in the ZmRB‐GRPs ranges from 0 to 14. Most of the ZmRB‐GRPs have more than one intron; however, ZmRB‐GRP8, ‐13, ‐14, and ‐22 are intron less. Genes in the Class d have the largest number of introns, while those in the classes b and c contain less introns. Intragroup members share conserved gene structure; this indicates that they were derived from the same ancestor. Motif prediction of the ZmRB‐GRPs in maize To further understand the diversification of the ZmRB‐GRPs, databases Pfam (http://pfam.sanger.ac.uk/) and SMART (http://smart.embl‐heidelberg.de/) were used to predict putative motifs. Motifs in each ZmRB‐GRP are shown in Figure 1A. Proteins in the Class a contain one RRM, and those in the Class b include one RRM and a zinc finger. Class c consists of a CSD and more than one zinc finger. For the proteins in the Class d, two RRMs are included (Figure 1A). Motif prediction in these ZmRB‐GRPs also revealed that the intragroup ZmRB‐GRPs are conserved during evolution. Differences of motif composition
Table 1. Detailed information of the ZmRB‐GRPs Gene
Subcellular location
Size (aa)
pI
MW (kDa)
GRMZM2G150521 (ZmRB‐GRP1) GRMZM2G131167 (ZmRB‐GRP2) GRMZM2G042118 (ZmRB‐GRP3) GRMZM2G165901 (ZmRB‐GRP4) GRMZM2G080603 (ZmRB‐GRP5) GRMZM2G001850 (ZmRB‐GRP6) GRMZM5G874478 (ZmRB‐GRP7) GRMZM2G082931 (ZmRB‐GRP8) GRMZM2G083783 (ZmRB‐GRP9) GRMZM2G161242 (ZmRB‐GRP10) GRMZM2G053223 (ZmRB‐GRP11) GRMZM2G097775 (ZmRB‐GRP12) GRMZM2G389768 (ZmRB‐GRP13) GRMZM5G895313 (ZmRB‐GRP14) GRMZM2G139643 (ZmRB‐GRP15) GRMZM2G167356 (ZmRB‐GRP16) GRMZM2G064518 (ZmRB‐GRP17) GRMZM2G104481 (ZmRB‐GRP18) GRMZM2G152526 (ZmRB‐GRP19) AC198361.3_FGT004 (ZmRB‐GRP20) GRMZM2G167505 (ZmRB‐GRP21) GRMZM2G132465 (ZmRB‐GRP22) GRMZM2G131943 (ZmRB‐GRP23)
Cyto Chlo Chlo Nucl Nucl Cyto Mito Nucl Nucl Nucl Nucl Nucl Nucl Nucl Nucl Nucl Nucl Nucl Plas Nucl Nucl Plas Cyto
254 308 156 159 155 196 205 316 259 311 261 489 249 208 443 384 413 401 348 443 453 454 700
4.78 5.09 6.58 5.52 6.11 9.74 7.61 9.19 8.94 9.29 8.63 8.68 5.95 5.92 6.68 5.84 4.84 4.87 9.11 6.17 6.18 5.7 5.1
25.1 30.1 15.7 15.5 15.4 20.5 21.8 34.1 32.4 34.3 28.1 46.8 23.2 20.2 45.2 39.1 43.1 41.6 37.2 44.2 44.9 46.6 74.5
Information of the maize RB‐GRPs was from MaizeGDB (http://gbrowse.maizegdb.org) and their isoelectric points (pI) and molecular weights (MW) were computed by the online tool Expasy (http://expasy.org/tools). Subcellular location of the ZmRB‐ GRPs was predicted by WoLF PSORT (http://www.genscript.com/psort/wolf_psort.html). Cyto, cytoplasm; nucl, nucleus; plas, plastid; mito, mitochondria; chlo, chloroplast. www.jipb.net
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Figure 1. Phylogenetic analysis, schematic diagrams of gene structure, and motif composition in the ZmRB‐GRPs (A) Distribution of conserved motifs in the ZmRB‐GRPs. The conserved motifs were identified by screening the Pfam database (http://pfam.janelia.org/) and displayed using DOG1.0 (http://dog.biocuckoo.org/). RRM, RNA‐recognition motif; CSD, cold‐shock domain; CCHC, zink knuckle or zink finger. (B) Phylogenetic trees of the ZmRB‐GRPs. The 23 ZmRB‐GRPs were divided into four classes based on their conserved motifs, and the trees were constructed with a complete alignment of the ZmRB‐GRPs in each class by the neighbor‐joining (N‐J) method with bootstrap analysis (1,000 replicates). Bootstrap values are indicated as percentages (when> 50%) along the branches. (C) Schematic diagram of gene structure was displayed by GSDS (http://gsds.cbi.pku.edu.cn/). October 2014 | Volume 56 | Issue 10 | 1020–1031
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Figure 1. Continued
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Figure 2. Structure of RNA‐recognition motifs (RRMs) in the ZmRB‐GRPs (A) Similarity of RRMs in the ZmRB‐GRPs. Multiple sequence alignment was conducted by T‐Coffee (http://www.tcoffee.org/). (B) The 3D structure of RRM domain in ZmRB‐GRP1. (C) The 3D structure of RRM domain in Rna15 (Pancevac et al. 2010). The two RRM domains (B, C) have high similarity (TM‐score ¼ 0.484, RMSD ¼ 3.790, IDEN ¼ 0.207, and Cov ¼ 0.642). The 3D structure of the ZmRB‐GRP1 RRM domain is developed using I‐TASSER (http://zhanglab.ccmb.med.umich.edu/I‐TASSER/) and exhibited by Chimera1.8.1 (http://www.cgl.ucsf.edu/chimera/). RNP1 and RNP2 are shown in purple and cyan, respectively.
in different groups of the ZmRB‐GRPs suggest that their function was diversified over evolution. RRMs in the ZmRB‐GRPs were also analyzed by T‐Coffee (http://tcoffee.org/503/index.html). Most of the RRMs have high similarity and contain two consensus RNPs (Figure 2A). In addition, most of the RRMs are structured by two a‐helices and five b‐sheets (Figure 2B). The conserved b1‐a1‐b2‐b3‐a2‐ b4‐b5 topology is essential to RNA binding. To further understand the structure of the proteins, three‐dimensional (3D) structure of the RRM in ZmRB‐GRP1 was analyzed (Figure 2B); it has high similarity with Rna15 (TM score ¼ 0.484, RMSD ¼ 3.790, IDEN ¼ 0.207, and Cov ¼ 0.642) (Figure 2C). Chromosomal location and gene duplication of the ZmRB‐GRPs To explore genomic distribution of the ZmRB‐GRPs, cDNA sequence of each ZmRB‐GRP was used to search the MaizeGDB database (http://gbrowse.maizegdb.org) with BLASTn, and software MapInspect was used to display the location of each gene (Figure 3). The ZmRB‐GRPs distribute on all of the 10 chromosomes. Chromosomes 1 and 5 have a maximum of six and four ZmRB‐GRPs, respectively. Three ZmRB‐GRPs on chromosome 4, two each on chromosomes 3, 7, and 8 are found, respectively. One each distributes on the other four chromosomes (Figure 3). Three genes on chromosome 5, ZmRB‐GRP4, ‐10, and ‐21, are closely located to one another. Meanwhile, ZmRB‐GRP20 and ‐6 on chromosome 1 are also adjacent to each other. Distribution of paralogs could be used to analyze potential duplications and evolutionary patterns of the ZmRB‐GRPs. In this study, five pairs of paralogs were detected based on the bootstrap values in the phylogenetic analysis (Figure 1B). All of them randomly distribute on maize chromosomes, suggesting that segmental duplication is predominant in the expansion of the ZmRB‐GRPs in maize. October 2014 | Volume 56 | Issue 10 | 1020–1031
Evolutionary relationships of RB‐GRPs in maize, Arabidopsis, rice, and wheat Rapid development of plant genomics makes it possible to analyze the same gene family across different species. To investigate molecular evolution of RB‐GRPs in Arabidopsis, rice, wheat, and maize, protein sequences of 34 RB‐GRPs were phylogenetically analyzed. An N‐J distance phylogenetic tree was constructed with the alignment of all the known RB‐GRP amino acid sequences (Figure 4). The phylogenetic tree (Figure 4) shows that all the RB‐GRPs from the four higher plants are divided into four groups, A, B, C, and D. Among them, Group A forms the largest clade, containing 23 GR‐GRPs and accounting for 67.6% of the total RB‐GRPs, Group D constitutes the second largest clade, containing seven members. Groups B and C contain three and one member, only accounting for 8.8% and 2.9% of the total RB‐GRPs, respectively. Additionally, Groups B and C are exclusively derived from maize, implying that these RB‐GRPs turned up after maize appeared, or it may be due to the homologs of these genes not having been identified in other plants. On the other hand, Groups A and D are contributed by at least three species, indicating that these RB‐GRPs might have the same function in evolution. Prediction of phosphoraylation sites in the ZmRB‐GRPs Phosphorylation of the ZmRB‐GRPs was analyzed with P3DB (Figure 5). Most of the phosphorylated residues are located either ahead or behind the RRMs or CSDs, except for ZmRB‐ GRP13 and ‐16. No phosphorylated site was predicted in ZmRB‐ GRP3, ‐4, ‐5, ‐7, ‐12, and ‐14. All of the ZmRB‐GRPs in Class d were phosphorylated, while only half of the members in Class a were found to be phosphorylated. On average, 1.2, 3.5, 1.5, and 4.5 phosphorylated sites were found in each member of the four classes, respectively. Most of the phosphorylated residues were serine in the ZmRB‐GRPs of the Classes a and c, while phosphorylated threonine and tyrosine were www.jipb.net
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Figure 3. Physical locations of the ZmRB‐GRPs on maize chromosomes Chromosome number is indicated at the top of each chromosome. Chromosomal positions of the ZmRB‐GRPs are indicated by their names. The lines connect the corresponding five pairs of paralogous genes in duplicated blocks. All of them were clustered as segmental duplication.
detected in the members of the Classes b and d. In addition, in the ZmRB‐GRPs of the Classes a and b (except for ZmRB‐GRP10), most of the phosphorylated residues are located at the C termini, while in the members of the Classes c and d, phosphorylated residues are located either in the C termini or N termini. Expression profiles of the ZmRB‐GRPs in different tissues Two sets of data were used to analyze the expression patterns of the ZmRB‐GRPs in differentially developmental organs or tissues. The expressed‐sequence tag (EST) database of maize (http://blast.ncbi.nlm.nih.gov/dbEST/) was screened, which resulted in the identification of ESTs for most of the ZmRB‐ GRPs except for ZmRB‐GRP2, ‐3, and ‐5 (Table S4). ZmRB‐GRP1 was detected in all of the selected tissues. The others were found in more than one organ or tissue except mixed tissues. Meanwhile, the Nimblegen maize microarray data (ZM37) from PLEXdb (http://www.plexdb.org/) was also used to study the expression patterns of the ZmRB‐GRPs (Figure S1). Most of the ZmRB‐GRPs were highly expressed in all the tissues analyzed except for ZmRB‐GRP11, and ‐16, which illustrated that most of the ZmRB‐ GRPs play important roles in the whole lifetime of maize. Interestingly, of the ZmRB‐GRPs, ZmRB‐GRP5 had the highest expression level in all of the tissues. However, ZmRB‐GRP2 was expressed lower in the leaves compared to other tissues. Expression analysis of the ZmRB‐GRPs under different stress conditions In order to understand stress responsiveness of the ZmRB‐ GRPs, two genes from each class of the ZmRB‐GRPs were chosen for expression profile analysis of the ZmRB‐GRPs under different stress conditions, cold, salt, abscisic acid (ABA), and recovery‐of‐chilling, through real‐time quantitative reverse transcription polymerase chain reaction (qRT‐ www.jipb.net
PCR) (Figure 6). Under cold stress, ZmRB‐GRP13, ‐14, and ‐15 were downregulated, the other genes were upregulated at first then downregulated. When treated with salt, expression of ZmRB‐GRP13 and ‐21 did not change much, while that of the others was upregulated at first then downregulated. Expression of all eight genes was upregulated at first then downregulated under the stress of ABA, but that of ZmRB‐ GRP7 and ‐13 had the least changes. When the temperature increased from cold (10 °C) to normal (room temperature, 25 °C), ZmRB‐GRP4 and ‐7 were upregulated in general, expression of ZmRB‐GRP5, ‐8, and ‐13 was increased at first then decreased, but that of the others (ZmRB‐GRP14, ‐15, and ‐ 21) was not changed. In conclusion, these results implied that the ZmRB‐GRPs confer stress tolerance through regulating their expression. Cis‐elements in the promoter sequences of ZmRB‐GRPs Transcriptional factors (TFs) are essential for the regulation of gene expression, thus cis‐elements that interacted with specific TFs were predicted within the promoter sequences of the ZmRB‐GRPs by PlantPAN. A total of 20 cis‐elements were predicted to be associated with the ZmRB‐GRPs, and they were divided into three classes, namely ABA‐responsive elements (ABREs), drought‐responsive elements (DREs), and cold‐responsive elements (CREs) (Tables S2, S3). The average number of ABRE, CRE, and DRE in Class a is 2.0, 2.8, and 2.8, respectively, while that in Class b is 2.3, 3.2, and 3.8, respectively. In Class c, on average 1.5, 4.0, and 4.0 of ABREs, CREs, and DREs are contained, respectively, and in Class d, 2.0, 3.7, and 3.1 of ABREs, CREs, and DREs, respectively, are involved (Figure 7). Particularly, ZmRB‐GRP7 only possesses one CRE, and ZmRB‐GRP3, ‐12, and ‐19 only have one ABRE. October 2014 | Volume 56 | Issue 10 | 1020–1031
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Zhang et al. hupehensis Rehd (Wang et al. 2012), wheat (Nakaminami et al. 2005), tobacco (Brady et al. 1993), and petunia (Condit and Meagher 1987). Most of them were involved in plant growth, development, and various stress responses (reviewed by Jung et al. 2013; Kang et al. 2013). In maize, however, only a RB‐GRP, MA16, was characterized and found to be involved in a variety of stress responses (Ludevid et al. 1992). Thus genome‐ wide identification, evolution, and expression analysis of the RB‐GRP family in maize will facilitate understanding of the function of this gene family.
Figure 4. Phylogenetic relationships among the identified RB‐ GRPs in Arabidopsis, rice, wheat, and maize Phylogenetic tree of all the identified RB‐GRPs in the four species was constructed from a complete alignment of 34 GRP proteins by the neighbor‐joining method with bootstrap analysis (1,000 replicates). Arabidopsis, rice, wheat, and maize genes are indicated at the end of the branches, respectively. The scale bar corresponds to 0.2 estimated amino acid substitutions per site. Among the 34 RB‐GRPs, GRP162 was reported by Hu et al. (2012), and the other RB‐GRPs in wheat, rice, and Arabidopsis were collected from a report of Mangeon et al. (2010).
DISCUSSION GRPs belong to a large GRP family, which exist ubiquitously in plant, bacteria, and humans, and are implicated in varied important biological processes. Up to now, the function of a number of GRPs has been studied in Arabidopsis (Yang and Karlson 2011), rice (Chaikam and Karlson 2008), Malus October 2014 | Volume 56 | Issue 10 | 1020–1031
RB‐GRPs in maize In this study, a total of 23 ZmRB‐GRPs were identified, which is much more than that identified in rapeseeds (Kim et al. 2012), Arabidopsis (Lorković and Barta 2002; Lorković 2009), and rice (Kim et al. 2010a). Conserved protein sequence analysis of the ZmRB‐GRPs indicated that there are four groups (Classes a, b, c, and d) in the maize genome. This was similar to that in other plant species (Mangeon et al. 2010). Although the ZmRB‐GRPs identified in this study contain glycine‐rich regions, no GRP domain was predicted in them by Pfam HMM file (http://pfam. janelia.org/). The HMM file in Pfam was built from the GRPs (especially from nodulin‐containing GRPs), which only represent a little part of the GRP superfamily rather than the RB‐GRP group. Thus no GRP domain in the ZmRB‐GRPs is possible. Intragroup ZmRB‐GRPs have conserved gene structure and motif composition, indicating that the ZmRB‐GRPs in the same group could have the same function and they would come from a common ancestor. For instance, ZmRB‐GRPs in the Class b include only one zinc finger, and that in the Class c consist of more than one zinc finger, while that in the Classes a and d contain no zinc finger. Besides, ZmRB‐GRPs in Class d contain two RRMs (Figure 1A, C), suggesting that they might change the conformation to bind RNA as U2AF65 doing in human (Daubner et al. 2013). However, ZmRB‐GRPs in the other classes only contain one RRM or CSD, suggesting that besides RNA recognition (Pancevac et al. 2010), they have the function of stress response. Comparative genomic analysis of RB‐GRPs was also carried out in maize, rice, wheat, and Arabidopsis, and generally four subgroups were divided (subgroups A, B, C, and D) (Figure 4). In the subgroups A and D, RB‐GRPs come from at least three plant species, which demonstrates these RB‐GRPs are derived from one ancestor and play the same role in different plants. Gene family expansion mainly caused by tandem duplication, segmental duplication and transposition events. In this study, five pairs of paralogous ZmRB‐GRPs are distributed on different chromosomes, suggesting that the ZmRB‐GRPs expand mainly through segmental duplications. In diploidized polyploid plants, segmental duplication is most frequently found (Cannon et al. 2004). RNA‐binding ability and compartmentalization of RBPs could be affected by phosphorylation and phosphorylation site. For instance, when serines in the RRM termini were phosphorylated, SRSF1 (aka ASF/SF2) appeared to induce a key molecular switch from intra‐ to inter‐molecular interactions during pre‐mRNA splicing (Cho et al. 2011). In addition, the phosphorylation of Nrd1 could enhance its localization to stress‐induced cytoplasmic granules in fission yeast (Satoh et al. 2012). In this study, most (18) of the 23 ZmRB‐GRPs were www.jipb.net
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Figure 5. Predicted phosphorylation sites in the ZmRB‐GRPs Phosphorylation analysis was carried out using P3DB (http://www.p3db.org/). The cutoff of specificity is 95% except for that of ZmRB‐GRP24 (99.9%) since there are too many phosphorylated sites in this gene. S, serine; T, threonine; Y, tyrosine.
predicted to contain 1 to 13 phosphorylation residues, and the residues and their locations were generally different in different classes (Figure 5), suggesting function of the genes in different classes are varied. As shown in Table 1, the ZmRB‐ GRPs targeted to different organelles, implying that the localization of them could be affected by the number or the variety of phosphorylated residues. Role of the ZmRB‐GRPs under stress tolerance Cis‐elements participate in controlling a lot of important cellular processes (Jiang et al. 2012), and they are essential for the regulation of gene expression. In this study, a total of 20 stress‐related cis‐elements localized in the promoters of the ZmRB‐GRPs were predicted (Tables S2, S3). Most of these cis‐ elements were found to be involved into stress tolerance in previous studies (Baker et al. 1994; Abe et al. 2003; Dubouzet et al. 2003). For example, ABI4, DPBFCOREDCDC3, ABREOSRAB21, ACGTABREMOTIFA2OSEM, and SBOXATRBCS were related to ABA response (Marcotte et al. 1989; Kim et al. 1997; Hattori et al. 2002; Niu et al. 2002; Acevedo‐Hernandez et al. 2005); DRECRTCOREAT, LTRECOREATCOR15, and MYCCONSENSUSAT were involved in cold, drought, light, and ABA response (Baker et al. 1994; Abe et al. 2003; Dubouzet et al. 2003); ABRELATERD1, ACGTATERD1, AtMYB2, AtMYC2, DRE2COREZMRAB17, MYB1AT, MYB2AT, MYB2CONSENSUSAT, MYBATRD22, MYBCORE, MYCATERD1, and MYCATRD22 were associated with drought‐dependent transcriptional control in gene expression (Abe et al. 1997; Busk 1997; Simpson et al. 2003). This implied that the ZmRB‐GRPs could respond to stress environments. Most of Arabidopsis and rice RB‐GRPs were involved in response to stresses (Lorković and Barta 2002; Lorković 2009; www.jipb.net
Kim et al. 2010a). In this study, expression of eight ZmRB‐GRPs selected from different subgroups was analyzed under four kinds of stress conditions. All of them significantly responded to two to four kinds of stresses, indicating that they would also be involved in the physiological processes of multiple stress responses of maize. It is interesting to notice that expression of ZmRB‐GRP7 with only one ABRE in its promoter was not affected by ABA treatment; expression of ZmRB‐ GRP13, ‐14, and ‐15 with the least number of CREs was downregulated by cold treatment; on the other hand, expression of ZmRB‐GRP4 and ‐5, which possess more ABREs and CREs in their promoter regions, was upregulated under the stresses of cold and ABA. Together, these results illustrate that the expression of most of the ZmRB‐GRPs could be regulated through cis‐elements under stress conditions, leading to tolerance to the stresses as reported previously (Nakaminami et al. 2006). However, their detailed roles in stress responses need to be further studied in future.
MATERIALS AND METHODS Identification of ZmRB‐GRPs in maize Based on the characteristic of RB‐GRPs, a two‐step method was used to obtain RB‐GRPs in maize (Zea mays L.). First, all of the RRM or CSD domain‐containing proteins in maize genome were identified via screening the maize filtered‐gene set (ZmB73_5b_FGS_translations.fasta downloaded from www. maizesequence.org) with RRM, RRM‐related, and CSD hmm files (PF00076, PF04059, PF08777, PF10378, PF10598, PF13893, PF14259, and PF00313) from Pfam (http://pfam.sanger.ac.uk/) October 2014 | Volume 56 | Issue 10 | 1020–1031
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Figure 6. Expression profiles of the ZmRB‐GRPs under various stress conditions Expression analysis was carried out by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The y‐axis represents the relative expression levels of the ZmRB‐GRPs compared with that of actin1. The x‐axis represents different treat time in each group. Error bars represent standard deviations for three replicates. Real‐time PCR data were analyzed using the 2DDCt method as described previously (Livak and Schmittgen 2001). Expression of the eight ZmRB‐GRPs was investigated under four different stress conditions, cold, recovery‐of‐chilling, ABA, and NaCl. in the HMMER 3.0 package with the E‐value 200 bp, maximum identity>95% and E‐value
