Computational analysis of the methionine sulfoxide reductase gene family in soybean (glycine max) and their response in abiotic stresses

127 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2017-63 Chemical and Biological Science 2017, Vol. 62, Issue 10, pp. 127-133 This paper is available online at COMPUTATIONAL ANALYSIS OF THE METHIONINE SULFOXIDE REDUCTASE GENE FAMILY IN SOYBEAN (Glycine max) AND THEIR RESPONSE IN ABIOTIC STRESSES Chu Duc Ha 1 , Nguyen Thi Kim Lien 1 , Tran Thi Thanh Huyen 2 , Pham Thi Ly Thu 1 and Le Tien Dung 3 1 Agricultural Genetics Institute, Vietnam Academy of Agricultural Sciences 2 Faculty of Biology, Hanoi National University of Education 3 DEKALB Vietnam Company (MONSANTO) Abstract. In this study, we used various computational approaches to analyze the promoter regions of all members of the methionine sulfoxide reductase (MSR) family. The presences of many cis- regulatory elements related to the light responsiveness, tissue specific expression, hormonal and/or stress responsiveness indicated that GmMSR gene family may be involved in the various developmental processes, perhaps in the response to environmental conditions of soybean plants. Next, the available transcriptome databases were used to retrieve the expression profiles of GmMSR gene family in normal condition. All members of MSR gene family were highly expressed in various major tissues/organs. Interestingly, GmMSRB1/B3 and GmMSRA3 were exclusively expressed in the leaves and nodule, respectively. Finally, we analyzed the expressions of all MSR genes were changed under drought and salt stresses. GmMSRA4/B2/B5 genes were up-regulated in both drought and salt stresses. These genes can be used in genetic engineering of soybean plants against abiotic stresses. Keywords: Soybean, methionine sulfoxide reductase, bioinformatics, expression profile, abiotic stress. 1. Introduction Methionine (Met) is one of the most essential amino acids in plants. Met has been shown to be involved in antioxidant defense, catalysis, protein structure and regulation [1]. However, high accumulation of the reactive oxygen species (ROS) can modify protein via direct oxidation of Met residues, thus leading to the breakdown of the cell structure. The oxidation of Met can be reduced back to Met by the activities of enzyme methionine sulfoxide reductase (MSR) [2]. Received November 20, 2017. Revised December 9, 2017. Accepted December 15, 2017. Contact Chu Duc Ha, e-mail address: hachu_amser@yahoo.com Chu Duc Ha, Nguyen Thi Kim Lien, Tran Thi Thanh Huyen, Pham Thi Ly Thu and Le Tien Dung 128 MSR family can be classified into 2 isozymes, MSRA and MSRB, specific for the conversion of the -S- and -R- diasteromer forms of MetO, respectively [3]. Up to now, taking the advantages of omics era, MSR family has been identified and characterized in many plant species, such as Arabidopsis thaliana [2], rice (Oryza sativa) [4], tomato (Solanum lycopersicum) [5, 6] and maize (Zea mays) [7]. However, studies of MSR family in soybean, an economically important crop are still lacking. Recently, a group of Le reported the identification of 7 genes encoding MSRA and 5 genes encoding MSRB in the soybean genome [8]. But we do not know how GmMSR genes response to the adverse environmental conditions. In this study, the promoter regions of GmMSR genes were analyzed to find the cis- regulatory elements. The presence of stress-responsive elements might imply their roles in the regulation under stress conditions. The available transcriptome databases were then collected to search the expression profiles of GmMSR genes in various tissues/organs in normal conditions and under stress conditions. These computational analyses allow us to initially have a first glance into the expression of MSR gene family in soybean in response to stress conditions. 2. Content 2.1. Materials and methods * Materials Sequences of 7 GmMSRA and 5 GmMSRB genes were obtained from previous studies [8]. The soybean genome database of 'Williams 82' available in the Phytozome v12.0 [9] was also used to download interest sequences. * Methods - The prediction of cis- regulatory elements in the promoter regions of GmMSR genes: The promoter regions (1kb upstream from start codon site) of all GmMSR genes were obtained from the soybean genome database [10] available in the Phytozome v11.0 [9]. The presence of cis- regulatory elements (CREs) was determined through PlantCARE web server [11]. - The expression patterns of MSR gene family in various tissues in normal condition: Expression profiles of MSR genes were retrieved from the publicly available data [12]. Nine tissues were isolated from soybean cultivar "Williams 82" plants with three independent biological replicates for each tissue. Briefly, root hair cells and stripped roots were collected from 3-day-old seedlings, 84- and 120-HAS (hours after sowing) root hairs were harvested after being spraying with water. Root tips were dissected from 3-day-old seedlings, while other tissues, including 14-day-old shoot apical meristem (SAM, V2 stage), 18-day-old trifoliate leaves, stem and roots (V2 stage), flower (R2 stage), seeds and pods (R6 stage) were selected as previously described [12]. The normalized Solexa data of each GmMSR genes in different tissues were then obtained to draw a heat map. - The expression analysis of GmMSR genes under several abiotic stresses: Expression of MSR genes in drought, salinity were analyzed based on previous available microarray databases [13, 14]. All samples were soybean cultivar "Williams 82". Under drought treatment, the 4 th trifoliate leaves at V6 stage (28 days after sowing, containing 7 Computational analysis of the methionine sulfoxide reductase gene family in soybean (glycine max) 129 trifoliates) and the 3 rd trifoliate leaves at R2 reproductive stage samples were used to isolate the microarray analysis [14]. For salt stress, an RNA-Seq experiment was performed by using root samples at indicating times (0, 1, 6 and 10 hours) [13]. 2.2. Results 2.2.1. Prediction of cis- regulatory elements in promoter regions of the GmMSR genes In previous studies, we revealed that there are 7 and 5 genes encoding MSRA and MSRB in the soybean genome, respectively [8]. To acquire the first look into the gene function and regulation, we firstly predicted the cis- regulatory elements in promoter regions of the GmMSR genes. The 1000-bp promoter regions of GmMSR genes upstream from start codon site were identified using the available genomic sequence information [10] and subjected to search against the PlantCARE database [11]. As shown in Figure 1, we found several core promoter elements, such as the TATA- box and CAAT-box [15]. The predicted cis- elements could be classified into several vital regulatory functions, including light responsiveness, tissue specific expression, hormonal and/or stress responsiveness. A majority of elements related to the light responsiveness we found in the promoter regions of all GmMSR genes were G-box, Box 4 and TCT- element [11], whereas the element regulated the circadian (LE-circadian) was located on only GmMSRB4 and GmMSRA1 [11]. In addition, we also identified several elements related to the tissue specific expression in the promoter regions of MSR gene family, including GCN4-motif, Skn-1 motif associated with endosperm expression [16], CAT- box and RY-element related to meristem and seed expression, respectively [17, 18]. Among them, CAT-box was predicted to be distributed only on the MSRB5's promoter, while the promoter regions of GmMSRA2/A5/A7 contained RY-element. We also found the presence of GCN4-motif and Skn-1 motif in the promoter regions of 7 genes, GmMSRA1/A3/A4/A6/A7 and GmMSRB1/B4. Our findings suggested that all members of MSR gene family may be responsive with light conditions and their expressions may be specific in several major organs/tissues. Figure 1. Prediction of cis- regulatory elements in the promoter regions of the GmMSR gene family in soybean Chu Duc Ha, Nguyen Thi Kim Lien, Tran Thi Thanh Huyen, Pham Thi Ly Thu and Le Tien Dung 130 Next, we scanned the presence of hormonal responsive elements in the promoter regions of GmMSR genes. As described in Figure 1, several regulatory elements associated with the gibberellins (GAs), salicylic acid (SA), auxin and methyl jasmonate acid (MeJA) responsiveness were found. The distributions of MeJA responsive elements in the promoter regions of GmMSRA1/A3/B5 genes revealed that these genes may be involved in the biotic stress responses in soybean plants [11]. Additionally, we also identified some GAs-, SA- and auxin- responsive elements in the promoter regions of GmMSR gene family. It means that MSR gene family might be associated with GAs-, SA- and auxin signaling networks in plants. To our interest, a number of well-known stress- responsive cis- elements were detected, including abscisic acid responsive element (ABRE), heat stress element (HSE), low temperature responsive element (LTRE), MYB binding site involved in drought responsiveness (MBS) and defense/stress-responsive element (TC-rich repeats) [11]. Most of MSR genes, except for GmMSRA2/A4/B2, contained at least 1 stress-responsive element (Figure 1), suggesting that MSR gene family may involved in the abiotic stress response in soybean plants. ABRE and MBS were found in the promoter regions of 4 genes, GmMSRA1/A3/A6/B1, indicating that these genes might be implicated in drought response via ABA-dependent pathway. In addition, LTRE was also found in the promoter regions of GmMSRB1 and GmMSRB5, suggesting that these genes might also be induced by cold stress. Taken together, our promoter analysis allowed us to get insight into the function and the regulation of MSR gene family in soybean. These data can be significant supports for us to explain their expression patterns in various tissues under various stress conditions. Furthermore, their promoter could be used in genetic engineering of soybean plants against abiotic stresses. 2.2.2. Expression of the GmMSR genes in different tissues during the development In order to understand the function of each GmMSR genes in soybean, we investigated their expression profiles using previous transcriptome data [12]. Nine major tissues/organs were isolated and analyzed, including root hair cells isolated 84 and 120 hours after sowing (84HAS RH, 120 HAS RH, respectively), root tip, root, mature nodules, leaves, shoot apical meristem (SAM), flower and green pods. As previously described, we followed the criteria of the classification of the tissue specificity of a gene [12]. A gene was defined to be preferentially expressed in one tissues (≥3- and <10-fold changes between the expression levels of the most highly expressed and second most highly expressed genes), specifically (≥10- and <100-fold change), very specifically (≥100- and <1000-fold change) and exclusively identified in one tissue (≥1000-fold change) [12]. In this study, expressions of most GmMSR genes can be found, except for GmMSRA5. A heat map was then constructed for the expression of 6 members of MSRAs and 5 members of MSRBs and shown in Figure 2. As a result, we found that all members of MSR gene family were highly expressed (fold change > 3) in various tissues. Interestingly, a majority of GmMSR genes were very specifically and/or exclusively expressed in at least one tissue. Among them, 2 genes - GmMSRB1 and GmMSRB3 were exclusively expressed in the leaves, while GmMSRA3 Computational analysis of the methionine sulfoxide reductase gene family in soybean (glycine max) 131 was the same in the nodule. In contrast, 3 genes - GmMSRA4/A6/A7 - were referred to be not very specific in any tissues as compared with others MSR genes. Overall, the role MSR gene family in the repair of oxidized proteins is well-established, and their strong expression in all tissues of soybean plants indicated that these enzymes are always synthesized in the cells to response to oxidative stresses. Furthermore, the very high accumulation of GmMSRA3/B1/B3 in leaves and nodules, where high level of ROS may be expected [13], suggesting that 3 members might play vital roles in repair oxidatively damaged proteins, perhaps also in response to stresses caused by adverse environmental conditions in soybean plants. Figure 2. Expression patterns of GmMSR gene family in various organs/tissues in soybean 2.2.3. Differential expression profile of MSR genes in soybean under abiotic stresses To investigate how MSR genes response to abiotic stresses in soybean, we analyzed the expression profiles of GmMSR genes under drought and salinity treatment based on the microarray data reported previously [14, 15]. The samples used in drought treatment were the 4 th trifoliate leaves at V6 stage and the 3 rd trifoliate leaves at R2 reproductive stage [15], whereas root samples at indicating times were harvested to carry out the RNA- Seq experiment for salt stress [14]. As a result, the expression patterns of GmMSR genes from the microarray data analyses are shown in heat maps (Figure 3). As shown in Figure 3A, expressions of all MSR genes were changed under drought treatment. Among them, 5 and 1 genes were induced (fold change > 1.5) and reduced (fold change < -1.5) in V6 and/or R2 trifolia, respectively. In V6 stage, GmMSRB3 was induced (fold change > 1.5), whereas GmMSRA3 was down-regulated under drought stress. In reproductive R2 stage, GmMSRA4/A7/B2/B5 genes were highly expressed, while GmMSRA3 was reduced. Next, we also found the significant changes of some GmMSR genes under salt stress (Figure 3B). As compared with non-stress condition, GmMSRA2/B1 genes were reduced in root, whereas 3 genes - GmMSRA4/B2/B5 - were up-regulated under salt stress. Chu Duc Ha, Nguyen Thi Kim Lien, Tran Thi Thanh Huyen, Pham Thi Ly Thu and Le Tien Dung 132 Figure 3. Expression profiles of MSR gene family under drought (A) and salinity stress (B) The reduction of GmMSRA3 in leave samples in drought stress, and the presence of ABRE in its promoter region, taken together, suggested that GmMSRA3 may response to drought stress through ABA-dependent pathway. GmMSRA4/B2/B5 were up-regulated in both drought and salt stresses, revealed that these genes would play a positive role in the repair of MetO in response to various abiotic stresses. As mentioned above, GmMSRB3 was also exclusively expressed in the leaves in normal condition and induced in leaves under drought stress. It might be noticeable if we discover the involvement of GmMSRB3 gene in the adaptation of soybean plants under drought stress. In further studies, the qRT- PCR experiments will be carried out to validate the expression profiles of GmMSR genes under different abiotic stresses. 3. Conclusion The promoter regions of all GmMSR gene family contained several core promoter elements. The presence of cis- regulatory elements related to the light responsiveness, tissue specific expression, hormonal and/or stress responsiveness indicated that GmMSR gene family may be involved in the various developmental processes, perhaps in the response to environmental conditions of soybean plants. Expression analysis of GmMSR gene family in normal condition revealed that all members of MSR gene family were highly expressed in various major tissues/organs. GmMSRB1/B3 and GmMSRA3 were exclusively expressed in the leaves and nodule, respectively. Expressions of all MSR genes were changed under drought and salt stresses. GmMSRA4/B2/B5 genes were up-regulated in both drought and salt stresses. These genes can be used in genetic engineering of soybean plants against abiotic stresses. REFERENCES [1] H. Weissbach, Etienne F., Hoshi T., Heinemann S.H., Lowther W.T., Matthews B., St John G., Nathan C., Brot N., 2002. 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