Plant Physiology and Biochemistry 77 (2014) 108e116

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Research article

Molecular cloning and expression analysis of mulberry MAPK gene family Congjin Wei a, b, Xueqin Liu a, b, Dingpei Long a, b, Qing Guo a, b, Yuan Fang a, b, Chenkai Bian a, b, Dayan Zhang a, b, Qiwei Zeng a, b, Zhonghuai Xiang a, b, Aichun Zhao a, b, * a b

State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 October 2013 Accepted 5 February 2014 Available online 14 February 2014

Mitogen-activated protein kinase (MAPK) cascades play an important role in regulating various biotic and abiotic stresses in plants. Although MAPKs have been identified and characterized in a few model plants, there is little information available for mulberry Morus sp. L., one of the most ecologically and economically important perennial trees. This study identified 47 mulberry Morus notabilis MAPK (MnMAPK) family genes: 32 MnMAPKKK, five MnMAPKK and ten MnMAPK genes, and cloned ten MnMAPK cDNA genes based on a genome-wide analysis of the morus genome database. Comparative analysis with MAPK gene families from other plants suggested that MnMAPKs could be divided into five subfamilies (groups A, B, C, D and E) and they could have similar functions in response to abiotic and biotic stresses. MnMAPK gene expression analysis of different stresses (high/low temperature, salt and drought) and signal molecules (ABA, SA, H2O2 and methyl jasmonate (MeJA)) revealed that all ten MnMAPK genes responded to high/low temperature, salt and drought stresses, and that nine of the ten MnMAPKs (MnMAPK7 excepted) could be induced by ABA, SA, H2O2 and MeJA, which suggested that MnMAPKs may play pivotal roles in signal transduction pathways. Our results indicated that almost all of the MnMAPKs may be involved in environmental stress and defense responses, which provides the basis for further characterization of the physiological functions of MnMAPKs. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Morus notabilis Mitogen-activated protein kinase (MAPK) Expression profile Abiotic stresses Biotic stresses

1. Introduction Plants often encounter a variety of adverse conditions, including: drought, high and low temperatures and high salt stress, during their growth and development (Meng and Zhang, 2013; Mizoguchi et al., 1997). Mitogen-activated protein kinase (MAPK) cascades play an important role in regulating various biotic and abiotic stresses during the plant life cycle. Those cascades are highly conserved signaling components in all eukaryotic cell signal transduction. They can be linked to different cell-membrane receptors and cell responses and play an important role in endogenous and exogenous signaling (Zhang et al., 2006). Plant MAPKs are similar to animal and yeast MAPKs (Nishihama et al., 1995). Their

* Corresponding author. State Key Laboratory of Silkworm Genome Biology, Southwest University, BeiBei, Chongqing 400716, PR China. Tel.: þ86 23 68251803; fax: þ86 23 68251128. E-mail addresses: [email protected], [email protected] (A. Zhao). http://dx.doi.org/10.1016/j.plaphy.2014.02.002 0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.

common features are that they have similar molecular weights (38e55 kD) and have 11 conserved subdomains (Hanks et al., 1988). MAPK cascades consist of three kinds of kinases: MAPKKK, MAPKK and MAPK, which are progressively phosphorylated by upstream stimulatory signals. TXY, an ATP phosphorylation site, is an essential factor present in the catalytic domain between the seventh and eighth sub-domains (Ichimura et al., 2002). Plant cells respond to environmental stresses (such as wounding, drought, temperature, etc.) and growth signals (such as auxin, ethylene, abscisic acid, etc.) via the receptor protein kinase (Jonak et al., 1999; Mishra et al., 2006). MAPKKK, MAPKK and MAPK are activated consecutively by the receptor protein kinase or by an upstream activator. MAPKKK is the most upstream in the cascade system and phosphorylates itself directly via stimulation of receptors, signal molecules or extracellular stimuli. The phosphorylated MAPKKK is able to activate the phosphorylation of its downstream factor, MAPKK. The MAPKK, which is phosphorylated at the Thr and Tyr residues of the activation loop for MAPKs, can activate MAPK phosphorylation (Kültz, 1998). When organisms are

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confronted by stresses, such as: UV radiation, osmotic stress, heat stress, wounding and other hormones, MAPK is activated via a number of upstream signal molecules, which leads to the phosphorylation of downstream molecules by MAPK. MAPK cascades eventually transfer the external signal to the nucleus, which regulates the expression of the specific genes that cause physiological responses to occur (Pitzschke et al., 2009). To date, the responses of many MAPK genes to abiotic stress and molecular signaling pathways have been widely studied in several model plants (Jonak et al., 2002; Singh et al., 2012). Jammes et al. proved that two MAP kinases: MPK9 and MPK12, were preferentially expressed in guard cells and positively regulated ROS-mediated ABA signaling (Jammes et al., 2009). Arabidopsis MPK6 has been shown to be involved in a stress responses mechanism that modulated the biosynthesis of ethylene (Liu and Zhang, 2004). In rice, mitogen-activated protein kinase, OsMAPK5, was activated by ABA, various abiotic stresses and pathogen infection (Xiong and Yang, 2003). OsBIMK1 was rapidly activated after exposure to jasmonic acid and Pseudomonas syringae pv. syringae, and after wounding. These results suggest that OsBIMK1 plays an important role in rice disease resistance (Song and Goodman, 2002). More recently, transgenic Arabidopsis overexpressing GhMPK16 has been shown to have significant resistance to fungi, bacterial pathogens, drought and H2O2 (Shi et al., 2011) and activity at the transcript of CrMPK3 was induced by wounding, UV treatment and MeJA (Raina et al., 2012). To date, many studies have shown that the MAPK gene family is not only closely related to biotic and abiotic stresses, but is also involved in various important biological processes, such as growth and development. Genome-wide analysis of several plant genomes has identified MAPK families in a number of plant species. For example, Arabidopsis encodes 20 MAPKs compared with 26 MAPKs in apple and 21 MAPKs in poplar (Ichimura et al., 2002; Nicole et al., 2006; Zhang et al., 2013a). However, as far as can be ascertained, there have been no studies into the mulberry MAPK gene family at the whole genome level. Mulberry (Moraceae morus) is a perennial woody plant and is ecologically and economically important (Ramachandra Reddy et al., 2004). Mulberry leaves are the main source of food for the silkworm, Bombyx mori, and its fruit is very popular and nutritious (Singhal et al., 2010). In addition, mulberry can adapt to many different environments, including cold, waterlogged, drought and saline environments (Checker and Khurana, 2013), but there has been little research into its stress physiology, biochemistry and molecular biology. There is also very little mulberry MAPK information in the public databases (such as the NCBI, EMBL, etc.). After complete analysis of the Morus notabilis genome had been undertaken (He et al., 2013), it became possible to analyze the defense genes, such as MAPKs, involved in abiotic and biotic responses in mulberry at the genome wide level. This study provides a list of MAPK family members (32 MAPKKK, five MAPKK and ten MAPK genes), and cloned and sequenced all ten MAPK genes from M. notabilis. We classified these ten MAPKs into five groups according to their phylogenetic analysis. In addition, we investigated the conserved domains among the ten MAPKs by multi-alignment of protein sequences. Further expression profile analysis using the quantitative real timepolymerase chain reaction technique (qRT-PCR) showed that most of the MAPK genes from mulberry were induced by various stresses (high/low temperature, salt and drought) and signal molecules (ABA, SA, H2O2 and MeJA). Our data provide some insights into new potential functions of mulberry MAPKs and the research can be used as a basis for further characterization of the physiological functions of mulberry MAPKs.

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2. Materials and methods 2.1. Plant materials and stress treatments The M. notabilis leaves used for total RNA extraction were collected in Yingjing county of Ya’An city, Sichuan Province, China during May 2011. Morus multicaulis cv. Husang No. 32 mulberry seedlings were planted in a PQX type plant incubator with artificial intelligence capability (Ningbo Southeast Instrument Corporation, China) with a 22  C/26  C (night/day) and 8 h/16 h (night/day) temperature and light cycle. After about two months, mulberry seedlings that were about 25 cm tall were subjected to the abiotic stresses of: high temperature (40  C for 12 h), low temperature (4  C for 12 h), salt (200 mM NaCl for 2 d) and dehydration (drought for 10 d). There were also signal substance treatments, consisting of: abscisic acid (ABA) (400 mM ABA for 24 h), salicylic acid (SA) (2 mM SA for 24 h), hydrogen peroxide (H2O2) (10 mM H2O2 for 24 h) and methyl jasmonate (MeJA) (2 mM MeJA for 24 h), as described in previous papers (Caitriona Dowd et al., 2004; Zong et al., 2009). The leaves of the treated seedlings were preserved at 80  C for total RNA extraction. 2.2. Cloning and sequence identification of MAPKs from mulberry All of the logged MAPK amino acid sequences for several sequenced species were downloaded from the NCBI (http://www. ncbi.nlm.nih.gov/). The protein sequences were modeled in order to find mulberry MAPKs genes based on the M. notabilis database (http://morus.swu.edu.cn/morusdb/). Ten pairs of primers were designed by Primer Premier 5.0 (Premier Biosoft International, CA, USA) (Supplementary Table 1) to amplify the full-length coding sequence (CDS) of ten mulberry MAPK genes from the total cDNA in the M. notabilis leaves. The PCR conditions included an initial denaturation for 4 min at 94  C, followed by 30 cycles of: 94  C denaturation for 30 s, 45 s annealing at 60  C, 72  C elongation for 2 min and a final 72  C extension for 10 min. The PCR products were purified and cloned into the PMDÒ19-T Vector (TaKaRa, Dalian of China). The positive clones were sequenced by InvitrogenÔ from Life Technologies (Shanghai, China). The exon-intron structures of the mulberry MAPK genes were confirmed by aligning their coding sequences to their corresponding genomic sequences. The exonintron structure diagram was generated using the online Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn) and the exon position and gene length method. 2.3. Conserved domain and phylogenetic analysis We used the GeneDoc program, a full featured multiple sequence alignment editor, to investigate the amino acid sequences and kinase domains of MnMAPKs. The MAPK family protein sequence alignments and the phylogenetic tree were created using the MEGA5 program (Tamura et al., 2011). The phylogenetic trees for MAPKs from M. notabilis, Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Nicotiana tabacum and Fragaria vesca were constructed using the Neighbor-Joining (NJ) method and assessed by bootstrap analysis with 1000 resampling replicates. 2.4. Analysis of quantitative real-time PCR Total RNA was extracted from M. multicaulis cv. Husang No. 32 seedling leaves and preserved as outlined above. The first strand cDNA were synthesized using the Perfect Real Time version of the PrimerScriptÔ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Dalian, China). The primer pairs for the qRT-PCR analysis of ten mulberry MAPK genes were designed using Primer Premier

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Fig. 1. Phylogenetic relationships between MAPK families from M. notabilis. MnMAPK, MnMAPKK and MnMAPKKK are highlighted by green, red and black lines, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

5.0 (Supplementary Table 2). The amplification specificities of the designed qRT-PCR primer pairs were confirmed by semiquantitative PCR using the cDNA from M. multicaulis cv. Husang No. 32 as a template. The qRT-PCR reactions were performed using the Applied Biosystems StepOne Plus Real-Time PCR System (Applied Biosystems, USA). The reaction conditions were: 95  C initial denaturation for 30 s, 95  C denaturation for 5 s over 40 cycles and finally a 60  C extension for 30 s. The 20 ml qRT-PCR reaction solution contained 10 ml SYBR Green (Takara, Dalian, China), 0.4 ml of each primer, 0.4 ml Rox, 2 ml cDNA template and 6.8 ml ddH2O. The mulberry ribosomal protein L15 (RPL) gene, which was expressed at a constant level in all tissues, according to our obtained transcriptome data from different M. notabilis tissues, was used as a control for the qRT-PCR. Statistical analysis of the qRT-PCR data was performed using the software in Excel and SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA).

3. Results 3.1. Isolation and identification of mulberry MAPK family genes Based on an analysis of the morus genome database, 32 MnMAPKKK, five MnMAPKK and ten MnMAPK genes from M. notabilis were identified (Fig. 1). Ten MnMAPK genes were successfully cloned and sequenced. The gene data were deposited in GenBank, and their accession numbers are shown in Table 1. The information analysis of MnMAPK showed that the CDS sizes for MnMAPK genes ranged from 1107 bp to 1902 bp and they encoded proteins ranging from 368 to 633 amino acids (AA) in size. The deduced protein molecular weights were between 42.4 kD and 70.9 kD and the pIs ranged from 5.0 to 9.28 (Table 1). The exon-intron structure diagram for the MnMAPK genes is shown in Fig. 2.

Table 1 Descriptional information about the M. notabilis MAPK genes. Gene name

Genebank accession number

ORF length (bp)

Protein length (aa)

Molecular weight (kDa)

PI

MAPK group

T-loop

Gene IDa

MnMAPK1 MnMAPK2 MnMAPK3 MnMAPK4 MnMAPK5 MnMAPK6 MnMAPK7 MnMAPK8 MnMAPK9 MnMAPK10

KF683076 KF683077 KF683078 KF683079 KF683080 KF683081 KF683082 KF683083 KF683084 KF683085

1140 1182 1119 1134 1122 1119 1107 1755 1902 1665

379 393 372 377 373 372 368 584 633 554

43.5 44.9 42.9 43.2 42.9 42.5 42.4 66.2 70.9 62.9

5.59 5.57 6.09 6.08 5.00 6.31 7.63 9.28 6.47 8.56

A A B B B C C D E E

TEY TEY TEY TEY TEY TEY TEY TDY TDY TDY

Morus011199 Morus020377 Morus021480 Morus024227 Morus011188 Morus021919 Morus003624 Morus020749 Morus017390 Morus010052

a

Gene ID are from the morus database (http://morus.swu.edu.cn/morusdb/).

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Fig. 2. Exon-intron structures of mulberry M. notabilis MAPK genes. Exons and introns are indicated by green boxes and black horizontal lines, respectively. Numbers 0, 1 and 2 represent introns in phases 0, 1 and 2, respectively. The scale bar represents 1.0 kb. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.2. MnMAPK phylogenetic and domain analysis The MAPK family proteins in plants contain conserved domains (Mishra et al., 2006). The phylogenetic tree of three mulberry MAPKs families (32 MnMAPKKK, five MnMAPKK and ten MnMAPK genes) is shown in Fig. 1. Seven MnMAPK genes are clustered into one of the branches and the remaining three MnMAPK genes are clustered together with three MnMAPKKK genes. Some MnMAPKK genes are clustered individually and overall, the MnMAPKKKs are distributed across six branches. Ten MnMAPK genes underwent multiple sequence alignment (Fig. 3) by GeneDoc. The result showed that all the MnMAPK genes that shared the family joint structure contained 11 highly conserved protein kinase domains. Similarly, we found that a TXY motif phosphorylation site existed between the seventh and eighth sub-domains. A phylogenetic

analysis of the MAPK family proteins from A. thaliana, O. sativa, P. trichoderma, N. tabacum and F. vesca was constructed by multiple sequence alignment of their protein sequences using the NJ method. The MAPK families were divided into six groups (groups AeF) (Fig. 4). The MnMAPK genes were found in groups AeE, but there were no mulberry MAPKs in group F. The six groups were divided into two subtypes according to whether they had a TEY or TDY motif in their phosphorylation site (Ichimura et al., 2002; Pitzschke et al., 2009). The TEY type only existed in groups A, B, C and F, whereas groups D and E only contained the TDY motif (Fig. 3 and Table 1). The phylogenetic analysis of the six species indicated that genes that were clustered together might have similar functions. Interestingly, TEY type MAPKs from M. notabilis always clustered first with those of the strawberry, F. vesca, whereas TDY type MAPKs from M. notabilis always clustered first with those of

Fig. 3. Protein sequence multi-alignment of the MAPKs from M. notabilis. Alignment was performed using the GeneDoc program. The subdomains are indicated with black Roman numerals (IeXI) on the top of each row. P-loop, C-loop and activation-loop motifs are indicated by boxes. The TXY phosphorylation sites are indicated by red asterisks. Conservative sequences are highlighted by black and gray shading. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Phylogenetic relationships between MAPK gene families from different plants. MAPK gene families are from M. notabilis, A. thaliana, O. sativa, P. trichocarpa, N. tabacum and F. vesca. Amino acid sequences were aligned using the Clustal-X computer software and subjected to phylogenetic analysis using the NJ method with 1000 resampling replicates. MnMAPKs are highlighted by the green line and triangles and the other MAPKs from A. thaliana, O. sativa, P. trichocarpa, N. tabacum and F. vesca are indicated by different colored lines and shapes as shown in the legend. The amino acid sequences of the MAPKs from A. thaliana, O. sativa, P. trichocarpa, N. tabacum and F. vesca were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Different groups are separated by black lines. Bootstrap values from 1000 replicates are indicated at each node. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the poplar, P. trichocarpa. In the MnMAPK gene family, the MnMAPKs consisted of six exons in groups A and B. Group C MnMAPKs consisted of two exons with strictly conserved sizes. Compared with the highly conserved group C, group D and E MnMAPKs possessed a complex distribution of exons and introns. MnMAPK8 and MnMAPK10 were both composed of nine exons, whereas MnMAPK9 possessed 11 exons. 3.3. Expression profiles of MnMAPK responses to various stresses and signals In order to explore the responses of mulberry MAPK genes to abiotic stresses, M. multicaulis cv. Husang No. 32 mulberry seedlings, after two months of growth, were subjected to high or low temperatures, drought and NaCl treatments, as described in the Materials and Methods. Then qRT-PCR was performed using the cDNAs obtained from the treated and untreated M. multicaulis cv. Husang No. 32 leaves as templates. The results of the qRT-PCR analysis for the different abiotic stresses are shown in Fig. 5. Compared with the controls, eight MnMAPK genes were significantly induced by 40  C high temperature stress. Four MnMAPKs (MnMAPK1, 5, 6 and 9) were up-regulated and the other four MnMAPKs (MnMAPK2, 3, 8 and 10) were down-regulated under 40  C high temperature stress. The expressions of MnMAPK1 and MnMAPK5 were significantly increased. The expressions of MnMAPK4 and MnMAPK7 changed less significantly (Fig. 5a). Under low temperature (4  C) stress treatment, the expression levels of two MnMAPKs (MnMAPK1 and MnMAPK5) were also up-regulated were significantly, which indicated that MnMAPK1 and MnMAPK5 might play important roles in temperature stress. Furthermore, four MnMAPKs (MnMAPK2, 3, 4

and 8) were significantly down-regulated (Fig. 5b). We found that eight MnMAPKs were significantly induced by drought treatment (Fig. 5c). Among them, six MnMAPKs (MnMAPK3, 4, 6, 7, 8 and 9) showed positively regulated expressions, particularly MnMAPK7, which had very high expression level after 10 days of drought. Significant down-regulation occurred to two MnMAPKs (MnMAPK1 and MnMAPK2) and the remaining two MnMAPKs (MnMAPK5 and MnMAPK10) were slightly down-regulated. After salt stress treatment, three MnMAPKs (MnMAPK1, 9 and 10) were significantly upregulated. Four MnMAPKs (MnMAPK3, 4, 7 and 8) were significantly down-regulated (Fig. 5d). These results showed that MnMAPKs can be induced by a variety of abiotic stresses. Many studies have indicated that phytohormones and plant signaling molecules (ABA, SA, H2O2 and MeJA) are involved in plant environmental stresses and pathogen resistance signaling pathways (Pitzschke et al., 2009; Singh and Jwa, 2013). In order to explore the response of MnMAPK genes to these signaling molecules, we conducted a qRT-PCR analysis on treated and untreated mulberry material. The results of the qRT-PCR analysis are shown in Fig 6. Six MnMAPKs (MnMAPK1, 2, 4, 8, 9 and 10) were significantly up-regulated after ABA treatment (Fig. 6a). Only one mulberry MAPK gene, MnMAPK5, was significantly down-regulated. These results implied that MnMAPK1, 2, 4, 5, 8, 9 and 10 may be involved in ABA-dependent signaling pathways and MnMAPK3, 6 and 7 may be involved in ABA-independent signaling pathways. SA has been reported to improve plant tolerances to disease, salt and dehydration (Bartels et al., 2009). Our results showed that eight out of the ten mulberry genes were induced by SA (Fig. 6b). The expression level of MnMAPK1 was significantly up-regulated while that of another seven MnMAPKs (MnMAPK2, 3, 4, 5, 6, 9 and 10) were

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Fig. 5. Expression profiles of MnMAPKs in response to abiotic stress treatments. The qRT-PCR analyses were performed using total RNA extracted from mulberry leaves subjected to heat (40  C for 12 h) (a) and cold (4  C for 12 h) (b), drought (drought for 10 d) (c) and salt (200 mM NaCl for 2 d) (d) stresses. The error bars represent the standard error of means of three independent replicates. All data were normalized to the MnRPL expression level. The differences between the transcript levels of the treated samples and the control samples (0 h) were statistically significant (*P < 0.05, **P < 0.01).

down-regulated. Previous research has shown that H2O2 participated in the oxidative stress signal transduction pathway in plants (Zhang et al., 2006). All the MnMAPKs, except MnMAPK8, were induced by H2O2 treatment (Fig. 6c). The expression levels of MnMAPK1 and MnMAPK7 increased significantly, whereas the rest of the MnMAPKs (MnMAPK2, 3, 4, 5, 6, 9 and 10) declined significantly after H2O2 treatment. MeJA is an important endogenous signal molecule that is involved in plant stress tolerance. It plays an important role in disease defense and wound healing (Yue et al., 2012). The expression levels of MnMAPKs under MeJA treatment were analyzed in order to explore the MnMAPK responses to MeJA (Fig. 6d). The results showed that the expression levels of three MnMAPKs (MnMAPK1, 4 and 6) increased while five MnMAPKs (MnMAPK2, 3, 8, 9 and 10) decreased and two MnMAPKs (MnMAPK5 and 7) changed slightly. Table 2 shows that five MnMAPKs (MnMAPK1, 2, 3, 4 and 9) responded to more than seven kinds of stress. Four MnMAPKs (MnMAPK5, 6, 8 and 10) responded to five to six kinds of stress and one gene, MnMAPK7, responded to three kinds of stress. 4. Discussion Mitogen-activated protein kinase (MAPK) cascades play an important role in regulating various biotic and abiotic stresses in plants, including environmental stress responses, disease resistance and signal transduction pathways (Pitzschke et al., 2009). So far, a few model and crop plant MAPK families have been

identified at the genome-wide level, including 20 MAPKs in Arabidopsis, 17 MAPKs in rice, 19 MAPKs in maize, 21 MAPKs in poplar, 16 MAPKs in tomato, 17 MAPKs in tobacco and 26 MAPKs in apple (Chen et al., 2012; Ichimura et al., 2002; Kong et al., 2012; Liu et al., 2013; Nicole et al., 2006; Zhang et al., 2013a,b). The number of MAPK family from mulberry is the smallest in several reported species. Interestingly, as a xylophyta, mulberry possesses only 10 MAPK genes, which is the same as humans (Krens et al., 2006b) and zebrafish (Krens et al., 2006a) (Table 3). The possible reason may be that the sequenced mulberry was a diploid mulberry (M. notabilis) from Sichuan Province, China, which has not undergone any genome-wide duplications (He et al., 2013). However, studies on MAPK functional characterization have mostly focused on model herbaceous plants (Hamel et al., 2012). Few studies have been reported for woody plants. As far as can be ascertained, only two poplar MAPKs and one MAPK in Poncirus trifoliata have been identified and they are involved in stressresponsive pathways (Hamel et al., 2005; Huang et al., 2011). Since herbaceous and woody plants are very different in many respects, such as their response to abiotic stresses and signal molecules, further functional research into MAPKs from woody plants is necessary. To date, information about mulberry MAPK genes has not yet been reported. In our study, we identified ten MnMAPKs based on the morus genome database. A comparison of the MnMAPKs sequences obtained from the sequenced clones with those of the morus genome database showed that the full-length cDNA of MnMAPK2 derived from morus genome database lacked

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Fig. 6. Expression profiles of MnMAPKs in response to abiotic stress treatments. The qRT-PCR analyses were performed using total RNA extracted from mulberry leaves treated with ABA (400 mM ABA for 24 h) (a), SA (2 mM SA for 24 h) (b), H2O2 (10 mM H2O2 for 24 h) (c) and MeJA (2 mM MeJA for 24 h) (d). The error bars represent the standard error of means of three independent replicates. All data were normalized to the MnRPL expression level. The differences between the transcript levels of the treated samples and the control samples (0 h) were statistically significant (*P < 0.05, **P < 0.01).

Table 2 MnMAPK gene expressions under different stress and signal molecule treatments.

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Table 3 Comparison of MAPKs from different plant and animal genomes.

MAPK Genome size (Mb) GWDa a b

M. notabilis

A. thaliana

O. sativa

P. trichocarpa

N. tabacum

F. vesca

M. domestica

Z. mays

Human

Zebrafish

10 330

20 115.4

17 430

21 378.5

17 450

10 240

26 603.9

19 730

10 750

10 313

NO

YES(2)

NO

YES(1)

NAb

NO

YES(2)

YES

NO

NO

GWD: Genome-Wide Duplications. NA: No Available.

15 nucleotide bases and MnMAPK8 existed in two splicing forms (Supplementary Fig. S1). All the other sequenced MnMAPKs corresponded accurately to those predicted from the morus genome database. When comparing the structure of MnMAPKs genes with other plants, all the group A MAPKs consisted of six exons in mulberry, Arabidopsis, apple and poplar. Group B genes contained six exons in mulberry, tomato and poplar, while AtMPK5, AtMPK11 and AtMPK13 consisted of four exons in Arabidopsis. Group C MnMAPKs consisted of only two exons with highly conserved sizes, which is similar to the other plants, including Arabidopsis, poplar and apple. Groups D, E and F MnMAPKs were complex in mulberry, Arabidopsis, poplar, tobacco and apple, despite having the same number of exons. A phylogenetic tree of the MAPK family proteins from M. notabilis, A. thaliana, O. sativa, P. trichocarpa, N. tabacum and F. vesca was constructed using multiple sequence alignment. The phylogenetic tree divided the MAPK proteins into six subgroups named A to F (Fig. 4) on the basis of previous reports (Zhang et al., 2013b). At present, most molecular biology research and reports focus on groups A and B and the genes in these two groups have been widely studied (Beckers et al., 2009). MAPK cascades, in particular those dependent on group A MPK3/MPK6, have been shown to be important regulators in response to pathogen infections and abiotic stress in plants (Beckers et al., 2009). MnMAPK1 and MnMAPK2, belonging to group A, were not only induced by ABA, SA, high/low temperature and drought, but also responded to MeJA and H2O2, which indicated that MnMAPK1 and MnMAPK2 might also have important functions in wound-induced and oxidative stress situations. Interestingly, MnMAPK1 responded to various stresses and phytohormone stimuli and was significantly up-regulated. However, under drought stress its expression level declined. The expression of MnMAPK2 was almost always downregulated after a variety of treatments. This showed that two genes in group A may have opposite expression patterns in response to stress. Group B genes include three mulberry genes: MnMAPK3, MnMAPK4 and MnMAPK5. Group B has not been as well studied as group A, but previous research has shown that group B AtMPK4 participated in environmental stress responses, cell division and disease resistance. It also plays an important role in abiotic stress responses (Kosetsu et al., 2010). MAPK9 and MAPK12 from Arabidopsis have also been shown to participate in biotic stress responses and stomata closure (Jammes et al., 2009). Similarly, MnMAPK3 and MnMAPK5 from group B were also induced by stress and phytohormones, which suggested that they may be involved in abiotic stress responses. To date, the research into MAPK genes in group C has been much less than for groups A and B. In recent years, a limited number of studies have found that the MAPKs in group C also participated in environmental stress responses and in the transmission of signaling molecules (ABA, H2O2, etc.) (Yue et al., 2012). MnMAPKs in group C include: MnMAPK6 and MnMAPK7. They were also induced by H2O2, high temperature, drought and salt. In group D and E MAPK genes, the activation site for encoding the amino acid sequence was the TDY motif and the functions of the two subfamilies have been mentioned in only a few studies

(Shi et al., 2011). Recently, MPK9, a group D MAPK in Arabidopsis that is highly homologous with MnMAPK9, was found to be preferentially expressed in guard cells and was up-regulated downstream of the reactive oxygen species (ROS) in guard cell abscisic acid (ABA) signaling (Jammes et al., 2009). MnMAPK8, MnMAPK9 and MnMAPK10 in groups D and E have similar expression patterns under various stress treatments. All were highly induced by ABA, SA, H2O2 and MeJA. Our results showed that groups D and E mulberry MAPK genes may be associated with signal transduction and in responses to wounding. There have been very few reports about the function of group F MAPKs and no MnMAPKs have been found in this group. In summary, our results indicated that MnMAPKs may play vital roles in biotic and abiotic stress responses by mulberry trees. Further studies into MnMAPK functions and signal transduction mechanisms will need to be conducted. Transgenic approach is a good method for studying the functions of mulberry MAPK genes and we will continue to use these approaches in the future. 5. Conclusion In conclusion, we identified 47 mulberry Morus notabilis MAPK (MnMAPK) family genes: 32 MnMAPKKK, five MnMAPKK and ten MnMAPK genes, and cloned ten MnMAPK cDNA genes. MnMAPKs could be divided into five subfamilies (groups A, B, C D and E). Further expression profile analysis using qRT-PCR showed that most of the MAPK genes from mulberry were induced by various stresses (high/low temperature, salt and drought) and signal molecules (ABA, SA, H2O2 and MeJA). This new information provides some insights into new potential functions of mulberry MAPKs, which provides the basis for further characterization of the physiological functions of MnMAPKs. Acknowledgments This work was supported by grants from the National Hi-Tech Research and Development Program of China (No. 2013AA100605-3), the Chongqing Science & Technology Commission (No. cstc2012jjA80039 and cstc2012jjys80001) and the China Agriculture Research System (No. CARS-22-ZJ0102). Contributions Zhao A. and Xiang Z. supervised the project; Zhao A. conceived and designed the study; Wei C. performed mainly experiments; Liu X., Long D., Guo Q., Fang Y., Bian C., Zhang D. and Zeng Q. provided to assist in the experiment. Wei C. and Zhao A. wrote the paper. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.02.002.

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