Genome-wide analysis of the WRKY gene family in physic nut (Jatropha curcas L.)

Gene 524 (2013) 124–132

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Genome-wide analysis of the WRKY gene family in physic nut (Jatropha curcas L.) Wangdan Xiong a, b, Xueqin Xu a, b, Lin Zhang a, b, Pingzhi Wu a, Yaping Chen a, Meiru Li a, Huawu Jiang a, Guojiang Wu a,⁎ a b

Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China

a r t i c l e

i n f o

Article history: Accepted 15 April 2013 Available online 3 May 2013 Keywords: Abiotic stress Evolution Gene expression Physic nut WRKY transcription factor

a b s t r a c t The WRKY proteins, which contain highly conserved WRKYGQK amino acid sequences and zinc-finger-like motifs, constitute a large family of transcription factors in plants. They participate in diverse physiological and developmental processes. WRKY genes have been identified and characterized in a number of plant species. We identified a total of 58 WRKY genes (JcWRKY) in the genome of the physic nut (Jatropha curcas L.). On the basis of their conserved WRKY domain sequences, all of the JcWRKY proteins could be assigned to one of the previously defined groups, I–III. Phylogenetic analysis of JcWRKY genes with Arabidopsis and rice WRKY genes, and separately with castor bean WRKY genes, revealed no evidence of recent gene duplication in JcWRKY gene family. Analysis of transcript abundance of JcWRKY gene products were tested in different tissues under normal growth condition. In addition, 47 WRKY genes responded to at least one abiotic stress (drought, salinity, phosphate starvation and nitrogen starvation) in individual tissues (leaf, root and/or shoot cortex). Our study provides a useful reference data set as the basis for cloning and functional analysis of physic nut WRKY genes. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The WRKY transcription factors contain one or two WRKYGQK sequence domains followed by a zinc finger motif, which can bind to the W box of target genes, thus regulating their expression (Eulgem et al., 2000; Sun et al., 2003). The first WRKY gene to be identified, SPF1, which was found in sweet potato, was considered to play a potential negative role in the regulation of sucrose induced genes (Ishiguro and Nakamura, 1994). Since then, increasing numbers of WRKY genes have been recognized in plants. In the Arabidopsis genome, 72 WRKY genes have been predicted and they can be divided into three groups on the basis of the WRKY domain sequence (Eulgem et al., 2000). There are 109 WRKY genes in rice (Oryza sativa L.) (Ross et al., 2007), 57 in cucumber (Cucumis sativus var. sativus L.) (Ling et al., 2011), and 105 in poplar (Populus trichocarpa) (He et al., 2012). WRKYs play important roles in development and stress responses. Firstly, WRKYs have been proved to be involved in the processes of lateral root formation (Devaiah et al., 2007; Zhang et al., 2008), seed Abbreviations: ERK, extracellular signal-regulated protein; EST, expression sequence tag; ETI, effector-triggered immunity; LRR, leucine-rich repeat; MAP, mitogen-activated protein; NBS, nucleotide binding site; NJ, Neighbor-joining; PAMP, pathogen associated molecular pattern; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction; TPM, transcripts per million; UTR, untranslated region; WD, WRKY domain. ⁎ Corresponding author at: Xingke Road 723, Tianhe District, Guangzhou 510650, PR China. Tel.: +86 20 37252703. E-mail address: [email protected] (G. Wu). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.04.047

development (Gonzalez et al., 2009; Luo et al., 2005) and leaf senescence (Besseau et al., 2012). Additionally, members of the WRKY protein are central to the innate immune systems of plants, such as the pathogen associated molecular pattern (PAMP) and effector-triggered immunity (ETI) based defense pathways and systemic resistance (Eulgem and Somssich, 2007). In Arabidopsis and rice, many WRKY genes have been shown to participate in responses to various abiotic stresses, such as threshold temperatures, cold, salinity, drought and low inorganic phosphate (Chen et al., 2009; Jiang and Deyholos, 2009; Li et al., 2011; Qiu and Yu, 2008; Wu et al., 2009). The physic nut (Jatropha curcas L.) is a small perennial tree or large shrub, which belongs to the Euphorbiaceae family. Physic nut is a drought-resistant, non-food oilseed plant that could meet many of the requirements for commercial biodiesel production. It is welladapted to semiarid and barren soil environments that are not suitable for cultivation of most crops (Makkar et al., 1997). Following the recent sequencing of the physic nut genome and the development of expressed sequence tag (EST) libraries by our group and others (Natarajan and Parani, 2011; Sato et al., 2011), it is now a useful model for studying the members of different families of transcription factor genes and their evolution. In this study, we searched the genome sequences in order to identify the WRKY genes in physic nut (JcWRKY). Subsequently, we characterized the motifs and exon–intron organization of these genes and subjected them to phylogenetic analysis. Finally, we analyzed the expression of the JcWRKY genes under normal growth conditions and under various abiotic stresses.

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Fig. 1. WRKY domains in proteins of the WRKY superfamily in physic nut. Conserved amino acid residues are shown in gray.

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Fig. 2. Gene structure of JcWRKY genes. The exons are shown as boxes (open reading frame in black, untranslated region (UTR) in gray), while the introns are represented by lines. *, intron in the 5′-UTR. **, intron in the 3′-UTR.

2. Materials and methods

2.2. Phylogenetic analysis

2.1. Sequence database searches

The conserved WRKY domains of WRKY genes were obtained using manual inspection of the WRKY family in the Pfam database, and multiple sequence alignment of WRKY domains was then carried out using CLUSTAL_X (Thompson et al., 1997). The phylogenetic tree was built using the neighbor-jointing method in MEGA 5.0 (Tamura et al., 2011). Bootstrap values were calculated with 1000 iterations.

Sequences of the Arabidopsis and rice WRKY domain proteins were downloaded from the Arabidopsis genome, TAIR 9.0 release (http://www.Arabidopsis.org/) and the rice genome annotation database (http://rice.plantbiology.msu.edu/, release 5.0), respectively. The castor bean WRKY protein sequences were downloaded from Phytozome (http://www.phytozome.net) (Chan et al., 2010). We searched for WRKY genes in the physic nut genome database of the Kazusa DNA Research Institute (http://www.kazusa.or.jp/ jatropha/) (Sato et al., 2011) and our own unpublished genome database. We used Arabidopsis WRKY proteins as query sequences to carry out Blastp and tBlastn searches against the physic nut genome sequences and predicted protein sequences. Sequences were selected for further analysis where the E value was less than − 10. Next, we corrected errors in annotation of WRKY coding domain sequences on the basis of the physic nut EST database available from GenBank (http://www.ncbi.nlm.nih.gov/) and our own physic nut and Jatropha integerrima L. EST datasets (unpublished data). The exon–intron structures of JcWRKY genes were determined by comparing the coding sequences and the corresponding genomic sequences using the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/).

2.3. Plant materials After disinfection with 1:5000 (v/v) KMnO4 solutions, seeds of the inbred physic nut cultivar GZQX0401 were planted in sand to germinate. When cotyledons were fully expanded, seedlings were transferred to trays containing a 3:1 mixture of sand and soil in a greenhouse illuminated with natural sunlight. After emergence of the first true leaf, the trays were irrigated with 1 L of Hoagland nutrient solution (pH 6.0) once every two days at dusk. Stress treatment was begun at the four-leaf stage (six weeks after germination). The plants assigned to the control and salinity treatments were then irrigated daily with complete Hoagland nutrient solutions, and the solutions plus 100 mM NaCl, respectively. For drought treatment, irrigation was withheld. Roots of both the treated and the control plants were sampled at 2 days after the onset of stress. Samples were frozen immediately in liquid nitrogen and stored at −80 °C prior to analysis.

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Fig. 3. Unrooted phylogenetic tree of the WRKY proteins of physic nut, Arabidopsis and rice. The amino acid sequences of the WRKY domains were aligned using CLUSTAL_X and the phylogenetic tree was constructed using the neighbor-joining method. The arrows show the locations of the subgroups IIf and IIg and the protein AtWRKY19 in the phylogenetic tree.

2.4. RNA isolation and RT-PCR

3. Results

Total RNA of roots was extracted using the CTAB method with some modifications (Chang et al., 1993). Briefly: the CTAB extraction buffer was spermidine-free and 1/4 volume 5 M KAc (pH 4.8) was added before the first centrifugation; 1/4 volume 8 M LiCl was used for precipitation; and the pellet was dissolved in guanidine thiocyanate solution [2.5 g guanidine thiocyanate in 6.6 mL CBS solution (42 mM sodium citrate and 8.38 g L − 1 N-lauroylsarcosine sodium)] instead of SSTE. The isolated RNA was subsequently treated with RNase-Free DNase I (Roche) before reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The first-strand cDNA was synthesized from 2 μg of total RNA, using M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. The specific primer sets are listed in Supplementary Table S1. A physic nut Actin gene was used as the internal control. The PCR reaction conditions were listed in Supplementary Table S1. A 10 μL sample of the PCR products was analyzed by electrophoresis on 1.5% agarose gel containing ethidium bromide.

3.1. Identification of WRKY family genes in physic nut To identify the WRKY members in the physic nut genome, the publicly available (Sato et al., 2011) and our own protein and genome sequences for this species were searched using the WRKY domains of Arabidopsis WRKY proteins as query sequences. As a result, a total of 58 WRKY genes were identified in the physic nut genome and the existence of the conserved WRKY motif was confirmed in each case using conserved domain analysis (http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi) (Marchler-Bauer et al., 2011). The obtained JcWRKY sequences were submitted to GenBank database, accession numbers were shown in Supplementary Table S2. The predicted JcWRKY proteins contain from 125 (JcWRKY58) to 797 (JcWRKY10) amino acid residues. The conserved WRKY domain (WD) is about 60 amino acid residues long; the 69 conserved WDs of JcWRKYs are presented in Fig. 1. In JcWRKY12, 13 and 14, the WRKYGQK motifs in the WDs are replaced by WRKYGKK, as is also the case in the rice proteins Os01g09100 (OsWRKY10) and Os01g51690

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(OsWRKY59) (Ross et al., 2007). All the JcWRKY genes have introns in the translated regions among them: 5 have one intron; 30 out of the 58 have two introns; 6 have three introns; 10 have four introns; and 7 have five introns. In addition, introns are found to be located in the 5′-untranslated region (UTR) of 5 genes and in the 3′-UTR of 3 genes. Alternative splicing of message RNAs are observed for 8 JcWRKY genes (Fig. 2). 3.2. Phylogenetic analysis of physic nut WRKY proteins To examine the phylogenetic relationships among the WRKY domain proteins of physic nut, Arabidopsis and rice, an unrooted tree was constructed from alignments of the conserved WRKY domain sequences (Fig. 3). The more conserved C-terminal WRKY domains of group I were used to construct the phylogenetic tree and several OsWRKYs containing incomplete WRKY domains were removed. The phylogenetic tree was constructed using MEGA 5.0 by employing the neighbor-joining (NJ) method. According to this tree, the WRKY proteins could all be assigned to one of the three previously defined groups, I–III (Eulgem et al., 2000). Eleven JcWRKY proteins were classed in the group I WRKY subfamily, members of which contain two WRKY domains, an N-terminal WD (NTWD) and a C-terminal WD (CTWD) (Eulgem et al., 2000). The AtWRKY19 (At4g12020) protein, which contains a plant MAP/ERK kinase–kinase domain, has no ortholog in either physic nut or rice (Fig. 3). Forty JcWRKY proteins were classed in the group II WRKY subfamily, which can be subdivided into additional seven subgroups (IIa–g). The group IIc proteins were close to group I in the phylogenetic tree, but they contain only one WRKY domain. The group IId proteins have an additional plant zinc cluster domain (pfam10533) upstream of the WRKY domain (Babu et al., 2006). Group IIg consisted of AtWRKY16 (At5g45050) and AtWRKY52 (At5g45260), which contain the NBS–LRR domain. Orthologs of these two proteins were not detected in physic nut or rice. Proteins in group IIf contain the fewest amino acid residues of all the 58 JcWRKYs. This WRKY subfamily is present in several other plant species (Supplementary Fig. S1), but no group IIf WRKY protein has been detected in Arabidopsis. The ortholog of JcWRKY58 in castor bean, Rc-scaffold28842, was identified by blasting its genome sequences, which was not predicted in its protein database (http://www.phytozome.net) (Supplementary Table S3). Seven JcWRKY proteins were classed into group III, in which the C2H2 zinc finger motif in other WRKY family proteins is replaced by a C2HC zinc finger motif (Fig. 1). The group III WRKY proteins could be subdivided into two subgroups (IIIa and IIIb). In comparison with physic nut, rice and Arabidopsis have more WRKY proteins in group IIIb. The number of WRKY proteins in the three species is compared in Table 1. The potential WRKY recent paralogs in physic nut were identified using the phylogenetic tree. They are JcWRKY25/26 in group IIc-1, 27/28 in group IIa, 34/36/37 in group IIb-1, and 51/52/53 in group IIIa. This result suggests that these genes have undergone duplication in the physic nut genome (Fig. 3). To test whether these duplicates arising from recent duplication events in the physic nut genome, we constructed another unrooted tree using JcWRKYs and WRKYs from the related species castor bean. This tree indicates that physic nut and castor bean have similar subfamilies of WRKY genes (Supplementary Fig. S2). The exceptions are that the JcWRKY55 ortholog in castor bean has undergone recent tandem duplication; and no castor bean ortholog of JcWRKY25 was detected, although other plants do possess such an ortholog (Supplementary Fig. S3). Thus, these potential WRKY recent paralogs in the physic nut genome probably arose from early gene duplication events. On the other hand, five scaffolds contained tandemly arrayed WRKY genes in the physic nut genome, members in four scaffolds falling into different subgroups of the WRKY family. Their orthologs also presented as tandem array in the cucumber and castor bean genome, except for the orthologs of JcWRKY12/15 in castor bean genome (Supplementary Table S4). In addition, JcWRKY20 and JcWRKY21 classed into subgroup IIc-3, while JcWRKY45 and JcWRKY47 into IIe-3. Clusters of JcWRKY20/45 and JcWRKY21/47 should arise from segmental duplication.

3.3. Expression analysis of JcWRKY genes We analyzed the expression of all JcWRKY genes under normal growth conditions in four different tissues: roots, stems (shoot cortex), leaves, and seeds (early development stage (S1), and filling and maturation stage (S2)) (Jiang et al., 2012). ESTs (expressed sequence tags) from three genes, JcWRKY13, 25 and 55, were not present in the physic nut EST database. However, their expression was detected at low levels in our transcriptomic expression database generated using next-generation sequencing-based digital gene expression tags (unpublished). These results suggest that all of the 58 JcWRKY genes are expressed in physic nut plants. Many JcWRKY genes were found to be expressed at differential abundance in different tissues according to the digital gene expression tags (Table 2; Supplementary Table S2). Eight genes (JcWRKY01, 04, 05, 06, 09, 10, 14 and 43) were highly expressed in all tissues tested. In developing seeds, three genes (JcWRKY14, 22 and 30) were expressed at relatively higher levels at the filling and maturation stage (S2) than at the early development stage (S1), while at least 8 WRKY genes were more highly expressed at S1 than at S2. Eight WRKY genes (JcWRKY07, 15, 17, 21, 23, 41, 45 and 46) were highly expressed only in roots, one (JcWRKY30) in leaves and one (JcWRKY54) in seeds of the tissues tested. Next, we examined the response of JcWRKY genes to four different abiotic stresses (drought, salinity, phosphate starvation and nitrogen starvation) using our next-generation sequencing-based digital gene expression tag database. Of the 58 JcWRKY genes, 47 showed differential expression in response to at least one stress in at least one tissue (Table 2, Supplementary Table S5). The number of genes showing at least a 2-fold increase or decrease in expression in response to drought, salinity, phosphate starvation and nitrogen starvation was 26, 30, 27 and 26, respectively. 17 WRKY genes responded to both drought and salinity stresses, and 16 for phosphate starvation and nitrogen starvation.

Table 1 The number and the proportion of the main three groups and the number of their subgroups were marked in bold, as well as the total number of WRKY genes in physic nut, Arabidopsis and rice. Group

Subgroup

I II IIa IIb IIb-1 IIb-2 IIc IIc-1 IIc-2 IIc-3 IIc-4 IIc-5 IIc-6 IId IId-1 IId-2 IId-3 IIe IIe-1 IIe-2 IIe-3 IIf IIg III IIIa IIIb Total

JcWRKY

AtWRKY

OsWRKY

11 19% 40 69% 3 8 4 4 16 5 2 2 3 3 1 6 1 2 3 6 1 1 4 1 0 7 12% 5 2 58

14 19.4% 45 62.5% 3 8 4 4 18 7 2 3 2 3 1 7 3 2 2 7 2 2 3 0 2 13 18.1% 5 8 72

17 17.3% 47 48% 4 8 4 4 16 6 2 1 1 5 1 7 1 1 5 11 7 1 3 1 0 34 34.7% 8 26 98

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Table 2 Expression levels of JcWRKY genes in physic nut plants under normal condition (TPM) ** and changes under abiotic stress conditions. Greater than two-fold changes in expression level under drought (D), salinity (S), phosphate starvation (−P) and nitrogen starvation (−N) are indicated. Only genes with expression levels greater than 10 transcripts per million in the stress-treated plants are included. Gene

EST

Root

Stem

Leaf

S1

S2

Root

Stem

Leaf

JcWRKY01

+

D

S

JcWRKY02

+

−P

D

JcWRKY03

+

S

JcWRKY04

+

−P

JcWRKY05

+

JcWRKY06

+

JcWRKY07

+

D, S, −N

D

JcWRKY08

+

S, −P

S, −N, −P

JcWRKY09

+

S, −N

JcWRKY10

+

JcWRKY11

+

D, −N, −P D, −N

D, S, −N, −P

JcWRKY12

+

S,-P

-P

JcWRKY13

−

JcWRKY14

+

JcWRKY15

+

JcWRKY16

+

JcWRKY17

+

JcWRKY18

+

JcWRKY19

+

S

JcWRKY20

+

−N

JcWRKY21

+

S

−N

JcWRKY22

+

−P

S, −N

JcWRKY23

+

−P

S

JcWRKY24

+

JcWRKY25

−

JcWRKY26

+

−P

JcWRKY27

+

S, −N, −P −N

D, S, −P

JcWRKY28

+

S, −P

D, S, −P

JcWRKY29

+

JcWRKY30

+

D, −N, −P

S, −N, −P

JcWRKY31

+

S, −N

JcWRKY32

+

JcWRKY33

+

S

JcWRKY34

+

S

JcWRKY35

+

S

15

D, S D, −N S S, −N

D

−N

S

S, −N

D

D, −P

D, S, −N

D

S, −N

D

S, −N S, −N

not available 20

D

5

0.1

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Table 2 (continued) Gene

EST

Root

Stem

Leaf

S1

S2

Root

Stem

Leaf

JcWRKY36

+

−P

JcWRKY37

+

−P

JcWRKY38

+

JcWRKY39

+

JcWRKY40

+

S

JcWRKY41

+

−N

JcWRKY42

+

JcWRKY43

+

JcWRKY44

+

−P

JcWRKY45

+

D, S

JcWRKY46

+

D, −N, −P

JcWRKY47

+

D, −N

JcWRKY48

+

S, −N, −P

JcWRKY49

+

JcWRKY50

+

−N, −P

JcWRKY51

+

−P

JcWRKY52

+

JcWRKY53

+

JcWRKY54

+

JcWRKY55

−

JcWRKY56

+

S, −N, −P

D, S, −N, −P

JcWRKY57

+

D, S, −N, −P −N

D, −N, −P

JcWRKY58

+

−N, −P

−P

−N

−N

D

D D, S

D, S, −N, −P

D D, −N

D, −P

D

−P

D

S D, −P

−N

−P

−P not available

20

15

5

0.1

**TPM (number of transcripts per million tags).

To assess whether the digital expression data could be confirmed by an alternate method, the expression levels of group IIe genes in roots under drought and salinity stresses at the 2 d point were tested by semi-quantitative RT-PCR, because these genes were all expressed in roots and their expression levels were regulated under drought and/or salinity stress (Table 2). We performed semi-quantitative RT-PCR on these 6 genes and the results were shown in Fig. 4. The RT-PCR results of these 6 genes showed general agreement with their transcript abundance changes determined by the digital gene expression tag profiling experiments, suggesting the reliability of the digital expression data. However, moderate discrepancies between the expression levels and TPM (number of transcripts per million tags) values were observed in JcWRKY49, which was shown to be significantly up-regulated under drought and salinity stresses by RT-PCR (Fig. 4), but not by TPM values (Supplementary Table S5). This may be due to its low expression level in the tested tissue. Thus, the JcWRKY genes shown differential expression levels in response to the tested stresses were listed only when their TPM values were larger than 5 (Table 2).

4. Discussion A total of 58 WRKY genes were identified in physic nut, and all of these genes were expressed in one or more tissues according to our data. The JcWRKY proteins could all be assigned to one of the three previously described three groups (Eulgem et al., 2000), and each group could be subdivided into a variable number of subgroups. Of all the JcWRKY proteins, 19% fell into group I and 69% into group II. These ratios were similar to those found in the AtWRKY family (Table 1). In subgroup IIc5 (JcWRKY12, 13 and 14), the WRKYGQK motifs in the WDs are replaced by WRKYGKK, which was the same case in Arabidopsis and rice (Ross et al., 2007). Former studies showed that WRKYGKK type failed to bind to W box (van Verk et al., 2008; Zhou et al., 2008). The subgroup IIc5 experienced a variation in sequence in evolution, which would be diversified in function. Physic nut and rice both have the WRKY group IIf, which was not detected in Arabidopsis (Fig. 3). It also exists in many other plant species (Supplementary Table S3; Supplementary Fig. S1). This result

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Fig. 4. Expression analysis of group IIe WRKY genes in roots under drought and salinity stress. Roots were sampled from 2 days after the onset of stress. Control plants (CK); drought (D); salinity (S). A, semi-quantitative RT-PCR analysis. Lower panel: samples normalized with respect to expression of the physic nut Actin gene. B, expression changes obtained using our digital gene expression tag profiling database. Scale = log2 (stress/CK).

suggests that the IIf subfamily was lost in Arabidopsis. In contrast, AtWRKY19 in group I and AtWRKY16 and AtWRKY52 in group IIg appear to be unique to Arabidopsis. These proteins contain an additional MAP/ERK kinase kinase domain or NBS–LRR domain. Their orthologs were not detected in physic nut, rice or the gene sequence database available for other plant species. These results imply that they are novel genes which have arisen in Arabidopsis and related species, or (less likely) have been lost from other plants during their evolution. The physic nut has fewer group III genes than Arabidopsis and rice (Table 1; Fig. 3). This is probably because group III genes underwent ancient and recent gene duplication in Arabidopsis and rice and were retained in their genomes during their evolution (Zhang and Wang, 2005). As in the cases of the C. sativus and P. trichocarpa genomes, several ancient duplicates of genes in this group were retained in the physic nut genome (He et al., 2012; Ling et al., 2011). Thus, fewer group III genes exist in the physic nut (7), C. sativus (7) and P. trichocarpa (10) genomes than in those of Arabidopsis (13) and rice (34). Although physic nut has four putative recent paralogs based on the phylogenetic tree, constructed using the WRKY genes from physic nut, Arabidopsis and rice (Fig. 3), physic nut and castor bean have the similar members in each subfamily, with the exception of gene JcWRKY25/26 (Supplementary Fig. S2). The JcWRKY25 and JcWRKY26 orthologs are present in poplar, grape and soybean genomes (Supplementary Fig. S3). This result indicates that JcWRKY25/26 does not represent a recent duplication. In contrast, they arose from an early duplication event before the separation of dicotyledons (Supplementary Fig. S3). Besides the above mentioned four gene pairs, several tandem duplicates of WRKY genes in the physic nut genome are also present in the castor bean and cucumber genomes. These results indicate that the expansion of WRKY genes within dicots includes tandem duplication and others, such as segmental duplication. Many ancient WRKY duplicates are retained in the physic nut genome. In contrast, no recent gene duplication of WRKY genes is apparent in the physic nut genome. The WRKY family genes play important roles in plant development and biotic and abiotic stress responses, including lateral root formation, seed coat and trichome development, leaf senescence, pathogeninduced defense programs, and responses to drought, cold and nutrient deficiency (Agarwal et al., 2011; Chen et al., 2012). Differential expression of WRKY genes between different tissues was detected in physic nut plants (Table 2). Genes highly expressed in specific tissues may be important for their development and metabolism. In Arabidopsis, AtWRKY44 (TTG2) and AtWRKY10 (MINI3) regulate seed coat development and seed size (Johnson et al., 2002; Luo et al., 2005). JcWRKY04, the ortholog of AtWRKY44 and AtWRKY10, was highly expressed early in early seed development (Table 2) (Jiang et al., 2012). AtWRKY23

mediates auxin distribution during the process of root development, and its ortholog JcWRKY22 is highly expressed in root, implying that it may have a similar function (Grunewald et al., 2012). AtWRKY70 and AtWRKY54, co-operate as negative regulators of leaf senescence (Besseau et al., 2012). The ortholog gene JcWRKY56 was expressed highly in leaf but to a lower level in root and stem in physic nut (Table 2), suggesting that it may play a role in leaf development, possibly including senescence. Overexpression of OsWRKY45 (Os03g21710) enhanced drought tolerance in Arabidopsis (Qiu and Yu, 2008). Its ortholog, JcWRKY56, was upregulated under drought and salinity stress in physic nut. AtWRKY75 is a modulator of Pi-starvation responses as well as root development (Devaiah et al., 2007). JcWRKY17, the ortholog of AtWRKY75 in physic nut (Fig. 3), is highly expressed only in roots, suggesting that it plays a role in root development in physic nut. Moreover, expression of the JcWRKY17 gene was up-regulated under phosphate starvation stress. These results suggest that the functions of at least some WRKY orthologs are conserved across different plant species. To date, the responses and functions of most WRKY genes in abiotic stresses are not well understood. Many JcWRKY genes showed distinct patterns of expression under different abiotic stresses, suggesting that they have different functions during stress in the physic nut (Table 2; Fig. 4). Thus, the expression patterns of the JcWRKY gene family reported here may facilitate a more comprehensive understanding of the specific functions of WRKY family genes. 5. Conclusions In conclusion, a total of 58 JcWRKY genes were identified in the physic nut genome. There was no recent gene duplication among the JcWRKY genes. Analysis of transcript abundance of JcWRKY gene products were tested in different tissues under normal growth conditions. Forty-seven JcWRKY genes responded under the tested abiotic stresses. Our study provides a useful reference data set as the basis for cloning and functional analysis of physic nut WRKY genes. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2013.04.047. Conflict of interest The authors declare that they have no conflict of interests. Acknowledgments The work was supported by grants from the National Basic Research Program of China (973 Program) (2010CB126603), the

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Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-J-28), and the CAS/SAFEA International Partnership Program for Creative Research Teams.

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