TÓM tắt TIÊNG ANH nghiên cứu sự biểu hiện của gen mã hóa methionine sulfoxide reductase trong điều kiện stress mặn, hạn ở cây arabidopsis và đậu tương

MINISTRY OF EDUCATION AND MINISTRY OF AGRICULTURE TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES CHU DUC HA EXPRESSION PROFILES OF THE GENES ENCODING ENZYME METHIONINE SULFOXIDE REDUCTASE IN ARABIDOPSIS AND SOYBEAN UNDER HIGH SALT AND DROUGHT STRESSES Specialization: Biotechnology Code: 9420201 SUMMARY OF THE PH.D THESIS HA NOI – 2018 The research work was conducted at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Supervisors: Dr Le Tien Dung Dr Pham Thi Ly Thu Critic 1: Critic 2: Critic 3: The thesis will be presented in the PhD dissertation committees of Vietnam Academy of Agricultural Sciences at: Vietnam Academy of Agricultural Sciences At in 2018 This thesis can be referred at: Vietnam National Library The Library of Vietnam Academy of Agricultural Sciences INTRODUCTION The rationale of the thesis Adverse environmental conditions, including biotic and abiotic stresses, can have negative effects on the growth and development of the plants Literally, the major mechanism of the attack(s) of stress(es) could be explained by the high accumulation of the reactive oxygen species (ROS) in the plant cells To respond to these stresses, the number of regulatory and functional proteins were determined to play important roles in the regulation of the growth and development of the plant Among them, enzyme methionine sulfoxide reductase (MSR), including methionine-S-sulfoxide Reductase (MSRA) and methionine-R-sulfoxide reductase (MSRB), is well-known to function in the reduction of the MetO into Met Previously, MSRs were determined to involve in the regulation of various biological processes, including the stress response, in the plant Therefore, the study on the MSR could get insight into the mechanism of the adaptation of plant to oxidative stress(es) and provide the candidate MSR-coding genes for the improvement of the stress tolerance in the plant by genetic engineering To address these questions, the model plant Arabidopsis thaliana and soybean (Glycine max) were used to carry out the experiments in the Ph.D thesis, namely "Expression analysis of the genes encoding methionine sulfoxide reductase in the salinity and drought stresses in Arabidopsis and soybean" The purpose of the thesis The aims of this Ph.D thesis are to analyze the roles of enzyme MSRs in the plant response to abiotic stress(es) and to figure out the potential proteins that need to repaired by MSR in plant cells To answer this question, these contents were described as below:  Identification and structural characterization of the Methionine-rich proteins in the model plant Arabidopsis thaliana  Identification and structural characterization of the Methionine-rich proteins in soybean  Identification and characterization of the genes encoding methionine sulfoxide reductase in soybean  Expression analysis of the genes encoding methionine sulfoxide reductase under the drought and salinity conditions in soybean plants Scientific and realistic meaning of the thesis Results obtained from the Ph.D thesis were an intensive understanding of the role(s) of the methionine-rich proteins in A thaliana, which get insight into the stress response in the plant via the methionine oxidation pathway Additionally, our identification and functional characterization of the genes encoding methionine sulfoxide reductase(s) in soybean could provide a basic understanding of the role(s) of these enzymes in the stress response, and thus, suggested the candidate genes for further genetic engineering for the improvement of the stress tolerance of chickpea plants The contributions of the thesis This Ph.D thesis was systematically analyzed the methionine-rich proteins in the model plant A thaliana and soybean, thus, here is the first study in Vietnam Furthermore, the stress-responsive methionine-rich protein encoded genes found in this thesis could provide reliable evidence of the protein susceptible to the methionine oxidation in the plant More significantly, the identification, annotation and characterization of the MSR gene family in soybean are the first reports in Vietnam Together with the previous study of GmMSRB, this thesis has successfully revealed the function of genes encoding MSR in soybean Interestingly, this thesis also provided several stress-responsive MSR gene for genetic engineering for the improvement of the stress tolerance in plants The structure of the thesis The Ph.D thesis was designed in 106 pages (excluding the Reference and Appendix) as following this order: Introduction (3 pages), Section I: Literature Review (34 pages), Section 2: Materials and Methods (13 pages), Section 3: Results and Discussion (54 pages), Conclusions and Recommendation (1 page) There were 186 references, including Vietnamese publications and 178 international publications were cited in the Ph.D thesis Additionally, there were 15 tables, 29 figures, appendix were presented in the Ph.D thesis More importantly, research articles were published based on the results of the Ph.D thesis SECTION LITERATURE REVIEW The Ph.D thesis were cited various references and summarized them in major contents as following: The impacts of the adverse environmental conditions on the plants; Function(s) of the enzyme methionine sulfoxide reductase and methionine oxidation in plants; Methionine and the methioninerich proteins in plants; Potential application of the enzyme methionine sulfoxide reductase in the improvement of the stress tolerance in plants Briefly, the accumulation of the ROS in the organelles is well-established to cause the serious damages to the macromolecules in the plant cells [127] To adopt on the oxidative stress(es), many mechanisms were developed with the involvements of various distinct protein groups, such as enzymic and nonenzymic antioxidants [77] Among them, the repair of the protein oxidation was the important process that was attracted from the research community [47, 80, 83] For instance, approximately 68% macromolecules which easily oxidized by the ROS in the plant cell are reported as proteins [138] A hypothesis was thought that the oxidation of the methionine residues in the polypeptide sequences was a defense mechanism of the plant cells To address this question, three questions were raised that, how many proteins are susceptible with the methionine oxidation in the plant cells; what their general characteristics are; and how these proteins-encoded genes were responded under stress conditions On the other hand, the methionine oxidation could be repaired by the involvement of enzyme methionine sulfoxide reductases (MSRs), including two enzyme families, [145] Until now, the genes encoding MSR were studied in various plant species, including A thaliana [145] and several important crops, such as rice [65], tomato [41, 41], maize [186] Recently, an effort has been made to identify the MSR gene family in soja [159] Previously, Le et al also reported genes encoding GmMSRB in soybean [99] Unfortunately, no information of the GmMSRA gene family in soybean was reported Therefore, it is interesting to raise three questions that how many genes are encoding MSRA in soybean; what are their typical characteristics; how this gene family was expanded during the evolution and how these genes respond to stress conditions To sum up, all major contents above were presented in Section Based on the understanding provided in the literature review, it is strongly believed that the study on MSR and MRP in soybean are very important SECTION MATERIALS AND METHODS 2.1 Materials 2.1.1 Data for the computational approach The genome and proteome of A thaliana, ecotype Col-0 [91] The genome and proteome of soybean 'Williams 82' [149] 2.1.2 Materials for the experimental approach A thaliana ecotype Col-0 seeds were provided from RIKEN CSRS - Japan Arabidopsis seeds overexpressing At3G55240 were provided from RIKEN CSRS - Japan as previously reported [75] Soybean cultivar 'Williams 82' seeds were stored in Agricultural Genetics Institute - Vietnam 2.2 Duration and place of the study 2.2.1 Duration The Ph.D thesis was carried out from 2014 to 2017 2.2.2 Place International Laboratory for Cassava Molecular Breeding, National Key Laboratory for Plant Biotechnology, Agricultural Genetics Institute, Pham Van Dong Road, North Tu Liem District, Ha Noi, Viet Nam Department of Molecular Biology, Agricultural Genetics Institute, Pham Van Dong Road, North Tu Liem District, Ha Noi, Viet Nam Stress Adaptation Research Unit, RIKEN CSRS, Japan 2.3 Methods 2.3.1 Computational approach a Identification of the genes encoding the methionine-rich protein in Arabidopsis The current proteome database of A thaliana [91] was obtained to screen all proteins by a javascript with the criteria as follows: protein length > 95 amino acid residues and Met > % b Identification of the genes encoding the methionine-rich protein in soybean The current proteome database of soybean [145] was used to screen all proteins by a javascript with the criteria as follows: protein length > 95 amino acid residues and Met > % c Functional classification of the genes encoding MRP in Arabidopsis and soybean A list of identifiers of genes encoding MRP in Arabidopsis and soybean was obtained to analyze in the MAPMAN software [168] d Structural characterization of the MRP in Arabidopsis and soybean Amino acid compositions of MRPs were calculated by BioEDIT tool [68] The lengths of MRPs were identified in NCBI The subcellular localization of MRPs was predicted by ChloroP [49, 50], WoLF PSORT [73], CELLO [184] and Blast2GO Basic [35] e Prediction of the cis-regulatory elements in the promoter regions of the genes encoding MRPs in Arabidopsis An 1000-bp-upstream region in the promoter region of genes encoding MRPs in Arabidopsis was used to analyze by BioEDIT [68] to identify the presence(s) of the abscisic acid responsive element (ABRE) [84, 133], MYB recognition site (MYBR) and MYC recognition site (MYCR) [173] f Expression patterns of genes encoding MRP in Arabidopsis Expression patterns of genes encoding MRP in A thaliana were analyzed in the normal condition based on the microarray database namely GDS416 [26] and an RNA-seq database available in TraVA web-based tool [86], and under stress conditions based on the microarray databases [122, 123] g Expression patterns of genes encoding MRP in soybean Expression patterns of genes encoding MRP in soybean were analyzed in the normal condition based on the public information in Missouri University [104] and a microarray under drought stress [96] h Identification of the genes encoding MSRA in soybean At5G61640, AtMSRA1 in A thaliana [145], was used as a seed sequence to BlastP against the proteome database of soybean [149] in the Phytozome [60] The candidate proteins were then confirmed in the Pfam The gene, protein and locus identifiers, chromosomal distribution were identified in Phytozome [60] and NCBI [149] i Prediction of the gene duplication events The gene duplication events were predicted by the Plant Genome Duplication Database website [100] The identity of duplicated genes was calculated by the ClustalX tool [92] The number of nonsynonymous substitutions per nonsynonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks) were predicted by the DNAsp software [105] j Structural characterization of MSRA in soybean The lengths of MSRAs were identified in NCBI [149] The molecular weight and theoretical isoelectric point (pI) were calculated in Expasy [58] The subcellular localization of MSRAs was predicted by TargetP [49-51] The multiple alignments of the conserved domains were analyzed by ClustalX [169] Unrooted phylogenetic trees were constructed based on the Neighbor-Joining method in MEGA [89] k Prediction of the cis-regulatory elements in the promoter regions of the genes encoding MSR in soybean The presence(s) of the cis-regulatory elements were identified in the 1000bp-upstream promoter regions of the genes encoding MSR in soybean by using PlantCARE [101] l Expression patterns of the genes encoding MSRs in soybean Expression patterns of the MSR genes were analyzed in the normal condition and stress conditions based on the public transcriptome database [24, 96] m Design of primers for the qRT-PCR Primers for the quantitative realtime - PCR (qRT-PCR) were designed by using Primer3 [147] The specific primers were identified based on the multiple alignments of the genome of soybean [149] Fbox was used as a reference gene in this study [97] 2.3.2 Experimental approach a Evaluation of the morphology of the transgenic Arabidopsis line The transgenic Arabidopsis line and the control were sowed in the petri dishes containing ½ ( urashige-Skoog) agar with hygromycine [75], in the normal condition with the photoperiod 16 h light/8 h dark, temperature 24 ± oC [36] b Arabidopsis plant treatment and sample collection The transgenic Arabidopsis seeds were germinated on the petri dishes containing ½ with NaCl 175 mM Similarly, the 12-day-old plants were transferred to the petri dishes containing ½ with CdCl2 750 µ The paraquat treatment of the transgenic line was performed as previously described [182] c Soybean plant treatment and sample collection Soybean plants were treated with drought stress as following exactly previous study [98] For instance, the 12-day-old plants were kept on the bench at the indicating time, - - 10 h The controls were kept under the hydroponic condition at - 10 h For the salinity and ABA treatments, 12-day-old plants were treated in the solution containing NaCl 250 mM and ABA 100 μ , respecti ely The plants were then kept in the normal condition at the indicating time, - - 10 h The controls were kept under the hydroponic condition at - 10 h d Total RNA isolation and the cDNA synthesis Total RNA isolation was carried out based on the TRIZol-based approach (ThermoFisher Scientific, USA) For instance, total RNA was treated with the Ambion Turbo DNAse I kit (Ther isher cienti ic, a ) Treated RNA was then used to synthesize cDNA using ReverTra Ace qPCR RT kit (Toyobo, Japan) The procedures were followed the previous study [98] e The qRT-PCR and the data analysis The qRT-PCR steps were carried out by using the Stratagen MX3000P system (Agilent Technologies, USA) with the Thunderbird SYBR qPCR Mix kit (Toyobo, Japan) as previously described [98] The Δ-CT method was used to analyze the data as previously reported [98] SECTION RESULTS AND DISCUSSION 3.1 Identification and characterization of the methioinine-rich proteins in Arabidopsis 3.1.1 Identification, annotation of the genes encoding methioinine-rich proteins in Arabidopsis A total of 121 MRP was identified in this study All MRPs were noted to have the gene identifiers in the current genome assembly, no MRP gene was annotated in the unplaced scaffolds Particularly, the most of AtMRP genes were distributed on five chromosomes, while only two genes were noted to located on the mitochrondial To get insight into the AtMRPs, these genes were then used for functional classification based on the TAIR10 reference 3.1.2 Functional classification of the MRPs in Arabidopsis Our results showed that AtMRP genes may involve in various biological processes in Arabidopsis plants Particularly, 20 AtMRP genes were found to be associated with the transcription regulation Among them, several genes were noted to encode the transcription factors For instance, At4G34590 encodes leucine zipper 11, containing 6.33% Met, which was characterized to involve in the growth of the root system via the auxin signaling in A thaliana [180] At3G23050, encodes a member in the Indole-3-acetic acid family, was well-established to repress the expression of auxin-induced genes in plants Previously, At3G23050 was also known to involve in various biological processes related to the auxin signaling pathway, such as the light responsiveness [148], root development [137] and stress response Additionally, a number of MRP genes in Arabidopsis was also functionally categorized into some important biological processes, such as protein modification (12 AtMRPs - 11%), signaling (6 AtMRPs - 5%), metal transportation (6 AtMRPs - 5%), RNA processing (4 AtMRPs - 4%), cell cycle (3 AtMRPs - 3%), metal handling (2 AtMRPs - 2%), development and stress response shared AtMRP - 1% On the other hand, 56 AtMRPs have been not annotated in any specific functional categories Our results provided a list of potential proteins for further functionally characterizations 3.1.3 Structural characterization of MRP in Arabidopsis As the results, a total of 23, 26 and 16 ABREs, MYBRs and MYCRs has been identified in the promoter regions of MRP genes in A thaliana There were 65 CREs were found to locate on the promoter regions of 121 MRP genes (0.54 CRE per MRP gene) More importantly, the presence(s) of ABRE in promoter regions of MRP genes indicated that these genes might be involved in the ABA-dependent manner, while the occurrence(s) of MYBR and MYCR revealed that these MRP genes may respond to the abiotic stress [173] To sum up, our prediction strongly suggested the majority of MRP genes may involve in the stress response via the ABA-dependent and/or -independent pathways It is also very important to know the subcellular localizations of MRPs as suggesting their roles in the cells Our results suggested that 21 MRPs might be distributed in the chloroplast via the WolF PSORT and/or ChloroP tools, while MRPs could be found in the mitochondria through the WolF PSORT and/or CELLO software These predictions were also confirmed by the Blast2GO software 3.1.4 Expression patterns of the genes encoding MRPs in Arabidopsis Based on the gene identifier, expression patterns of 49 genes, covering 40.5% AtMRPs, have been found in three major organs, including leaves, shoots and flowers in Arabidopsis plants in the normal condition [26] Among them, six MRP genes were noted to strongly expressed in all organs For instance, 11 Next, the transgenic line was treated in the medium containing 175 mM NaCl As the results, both of the RBC1 and control plants were sensitive with the high salinity condition From the 6th to 8th day, the survival rate of the transgenic plants was significantly lowered than the controls (Figure 3.8) Day Day Day Day Day Day Day Day Figure 3.9 Evaluation of the physiological responses of transgenic Arabidopsis line and control plants to CdCl2 treatment Under the CdCl2 750 µ treatment, leaves of the transgenic plants were observed to transfer into the yellow After days of the treatment, the RBC1 line was obviously more sensitive than the control plants (figure 3.9) Thus, our results indicated that overexpression of At3G55240 showed the sensitivity to CdCl2 in A thaliana Figure 3.10 Evaluation of the physiological responses of transgenic Arabidopsis line and control plants to paraquat treatment 12 Under the paraquat treatment, overexpression of At3G55240 also exhibited the sensitivity in leaves as compared with controls (Figure 3.10) The transgenic leaves were found to lose the chlorophyll and be whitening faster than the control plants after 24 h of paraquat treatment These observations were also confirmed in all paraquat treatments (Figure 3.10) Taken together, overexpression of At3G55240 showed the sensitivity to the abiotic stresses, including salinity, CdCl2 and paraquat treatments In all experiments, the survival rates of the transgenic line were significantly lower than the control plants 3.2 Identification and characterization of the methioinine-rich proteins in soybean 3.2.1 Identification, annotation of the genes encoding methioinine-rich proteins in soybean According to the proteome of the soybean [149], a total of 213 MRPs has been identified All MRPs have the annotations, no gene encoding MRP was found in the unplaced scaffolds We also found that these MRP genes were located on the chromosomes with an uneven ratio, whereas no gene was found in the cytosol 3.2.2 Functional classification of the MRPs in soybean As the results, GmMRP genes were predicted to involve in various biological processes in the cells For instance, these processes could be metal handling, RNA processing, stress response, protein modification, transcription regulation, signaling, cell cycle, development, metal transportation and lipid processing Additionally, the approximately 43% MRP genes in soybean have been still the unknown function Thus, it would be very interesting to raise a question that these genes may involve in the stress response in soybean plants or not? Furthermore, the unknown-function MRP in soybean also could be used as the raw materials for further functional characterization 3.2.3 Expression patterns of the genes encoding MRPs in soybean Based on the transcriptome published by Libault et al (2010), 49 MRP genes were under the limitation of the detection in nine major organs in soybean plants in the normal condition [104] The remaining MRP genes tend to highly expressed in at least one major organ in the plant in the normal condition Among them, Glyma13g03910, encoding MRP related to the signaling pathway in the cell, was noted to specifically express in roots, shoot apical meristems (SAM), and green pods Two genes, Glyma10g29710 and 13 Glyma20g37600, encoding the proteins involved in the metal handling, also showed the high accumulation in the SAM Recently, one neighbor gene Atriplex canescens was well-established to involve in the metal resistance and abiotic stress(es) response in plant [158] Glyma18g44300, encoding lipid transporter, was specific in the root hairs and SAM, suggesting that lipid might be accumulated in the root hairs in the early germination stage Another example was Glyma15g05510, highly expressed in flowers, while its close-relationship gene, At1G25275 was known to involve in the light responsiveness in A thaliana [120] Our analysis suggested that Glyma15g05510 might involve in the flowering time in soybean via the light responsiveness Next, expression patterns of MRP genes were analyzed based on the public microarray in the drought condition [96] Our analysis showed that 11 and 12 genes were reduced and induced in leaves V6 in drought condition, respectively 24 and six genes were up- and down-regulated in leaves R2 in drought condition, respectively Among them, a total of 13 MRP genes were responsive in both leaves V6 and R2 under the drought condition (Table 3.7) Table 3.7 Expression patterns of MRP genes in leaves V6 and R2 in soybean under the drought condition # 10 11 12 13 Gene name Glyma01g15910 Glyma01g15930 Glyma02g10620 Glyma03g32740 Glyma04g37040 Glyma06g39910 Glyma10g30380 Glyma15g05510 Glyma16g02510 Glyma19g43580 Glyma20g00780 Glyma20g22280 Glyma20g36730 Met L Annotation 8,08 6,56 7,22 6,04 7,91 10,34 7,43 7,37 7,26 6,70 6,69 6,59 7,89 100 458 98 481 140 117 149 96 125 210 285 426 153 Unknown function UNE10 Unknown function PIF1 Calmodulin 38 Calmodulin Calmodulin Unknown function Calcium binding protein GIF, GIF1, AN3 Homeodomain PIF3 Calmodulin Drought condition LeavesV6 Leaves R2 3,63 4,96 -20,34 -3,87 -44,63 -4,04 -2,19 -2,02 15,03 40,08 3,12 4,14 7,50 5,27 2,93 2,41 2,05 4,63 -2,01 2,42 -3,03 -2,36 2,25 2,99 3,06 2,29 Met content (%),2Protein size (aa),3Public microarray database according to [96] The red and blue colors indicated the up- and down-regulated genes, respectively Among 13 genes described in Table 3.7, Glyma04g37040 encoding calmodulin-binding protein was highest expressed in leaves R2 and V6 by approximately 40-fold and 15-fold Glyma02g10620 encoding a 98-amino-cid- 14 residue protein was reduced in leaves V6 and R2 by approximately 44- and four-fold (Table 3.7) Additionally, Glyma19g43580 was reduced in leaves V6 but induced in leaves R2 under the drought condition At5G28640 in Arabidopsis, a neighbor gene with Glyma19g43580, was recently characterized to associate with the sugar homeostasis, and thus, involved in the cell differentiation in the leaves [114, 115] We hypothesized that the high accumulation of Glyma19g43580 in leaves under the normal condition might boost the sugar level in these tissues as a mechanism of the drought escape of the soybean plants It also very interestingly to raise a question of the functions and roles of the MRP genes in the stress response in soybean plants 3.3 In silico analysis of the genes encoding MSR in soybean 3.3.1 Genome-wide identification of the genes encoding MSR in soybean As the results, a total of genes encoding MSRA has been found in the soybean genome As compared with MSRAs in plant species, genes were found in A thaliana [145], while MSRA genes were identified in rice [65] Our analysis indicated that MSRA genes are a multiple gene family in plant species 3.3.2 Gene duplication in the MSRA gene family in soybean Based on the multiple alignments, three duplication events have been found in the MSRA gene family in soybean The identity of the duplicated genes varies from 73.76% (GmMSRA4/A7) to 96.00% (GmMSRA2/A5), while the pair of GmMSRA1/A6 shared the homology level of more than 90% Additionally, three duplicated pairs were distributed on the different chromosomes in the soybean genome For instance, GmMSRA1/A6 were located on the Chr and 16, GmMSRA2/A5 were mapped on the Chr2 and 14, and GmMSRA4/A7 were located on Chr and 18 Our results indicated that these genes were occurred from the segmental duplication events in the different chromosomes Previously, Le et al (2013) also reported two segmental duplication events in the MSRB gene family in soybean Particularly, GmMSRB2/B5 and GmMSRB3/B4 were located on the Chr 13/15, respectively [99] Taken together, our results revealed that the expansion of MSR gene family in soybean (including MSRA and MSRB) might be mostly explained by the segmental duplication events Furthermore, the Ka/Ks ratios of three duplicated pairs were less than 1, suggesting that the natural selection prevented the point mutations occurred in the MSRA gene family in soybean as previously described [103] 15 3.3.3 Structural characterization of the MSRA family in soybean The protein sizes of MSRAs were ranged from 194 (MSRA3) to 266 amino acid residues (MSRA2 and MSRA5) The molecular weights of MSRAs vary from 21.63 (MSRA3) to 29.87 kDa (MSRA2), while the pI was from 5.01 to 8.78, with the average pI was approximately 6.77 It should be remembered that the pI of a protein could suggest its subcellular localization [14] The pI values of 1, - , - , -A7 were acidic, suggesting that these proteins might be located on the cytosol Interestingly, the pI values of GmMSRA2 and GmMSRA5 were base, predicting that two proteins might be distributed on the membranes of the organelles (Table 3.10) Table 3.10 General characteristics of the MSRAs in soybean Gene name GmMSRA1 GmMSRA2 GmMSRA3 GmMSRA4 GmMSRA5 GmMSRA6 GmMSRA7 Gene identifier Glyma02g05550 Glyma02g46020 Glyma04g36480 Glyma08g42790 Glyma14g02705 Glyma16g24130 Glyma18g11110 Size 250 266 194 203 266 250 203 mW 26.17 29.87 21.63 22.78 29.69 28.11 22.76 pI 6.14 8.78 5.01 6.52 8.74 6.39 5.84 SL S* C* − − C* S* M Note: S: Secretory pathway; C: Chloroplast; M: Mitochondrion -: Unknown place; *: Reliability Prediction by TargetP revealed that GmMSRA1 and GmMSRA6 might be located on the secretory pathway, while GmMSRA2 and GmMSRA5 might be distributed on the chloroplast It should be noted that chloroplast is the organelle that ROS was highly accumulated under the abiotic stress(es) Therefore, GmMSRA2 and GmMSRA5 might involve in the reduction of the MetO, and GmMSRA1 and GmMSRA6 might be transported into many organelles to function on the repair of MetO in the cells Next, the phylogenetic tree was constructed based on the full-length protein sequences of MSRAs in soybean and A thaliana [145] As the results, the MSRA family in soybean could be classified into groups (Figure 3.13) Particularly, there are members in group 1, including MSRA2, -A3, -A4, -A5 and -A7, while group contains MSRA1 and MSRA6 Most of the members in the group should share similar structural characteristics The sizes of GmMSRA2 and GmMSRA5 were 266 amino acid residues with the mWs vary from 29.69 ~ 29.87 kDa, and the pI values are also similar (7.84 ~7.87) 16 Figure 3.13 A phylogenetic tree of the MSRA in soybean and A thaliana The conserved domains of MSRAs in soybean could be classified into groups as in the phylogenetic tree (Figure 3.13) All members in group shared the Cys residue with the catalytic function and two Cys residues with the resolving function The conserved domain was F[G/A]AGCW[G/S][V/A]E as previously confirmed in AtMSRA1, -A2, -A3 and -A4 in A thaliana [145] On the other hand, the conserved domain of group 2, including GmMSRA1, A6 and AtMSRA5 did not have the catalytic and resolving Cys residues 3.4 Structural characterization of the MSRA gene family in soybean 3.4.1 Prediction of the cis-regulatory elements in the promoter regions of MSRA gene family in soybean Previously, five genes encoding MSRB were reported in the soybean genome [99] Thus, MSR genes, including and genes encoding MSRA and MSRB have been genome-widely identified in soybean To get insight into the gene function and the regulation, the presence(s) of the CREs in the promoter regions of MSR genes were analyzed by using the PlantCARE web-based tool [101] Several typical core elements, such as TATA-box, CAAT-box were found in the promoter sequences of all MSR genes Generally, the CREs found in this study could be classified into three functional categories, including the CREs related to the light responsiveness, the CREs specific in the tissues and the CREs related to the stress and/or hormone responsiveness 3.4.2 Expression patterns of the MSR genes in soybean In the normal condition [104], expression profiles of the most of MSR genes were found, excluding GmMSRA5 had no information The majority of MSR genes were strongly and/or specifically expressed in at least one organ in 17 soybean plants Among them, MSRB1 and MSRB3 were noted to highly expressed in leaves, while MSRA3 were accumulated in the nodules The high accumulation of MSR in all major organs in soybean plants indicated that this enzyme family may function on the repair of the protein oxidation Furthermore, MSRA3, -B1 and -B3 were highly expressed in leaves and nodules which were predicted to accumulate the ROS during the adverse environmental conditions (Figure 3.15) Figure 3.15 Expression patterns of MSR genes during the development 84RH, 120RH: Root hairs havested after 84 and 120 hours of the germination.; SAM: Shoot apical meristerm; F: Flower; GP: Green pods; N: Nodules; L: Leaves; R: Roots; RT: Root tips; PKRM values were presented by the heatmap Under the drought condition [96], expression levels of all MSR genes were significantly changed For instance, five and one genes were induced and reduced in leaves V6 and/or R2 under the drought treatment, respectively In the leaves V6, MSRB3 and MSRA3 were up- and down-regulated under the drought condition, respectively On the other hand, GmMSRA4, -A7, -B2 and B5, and GmMSRA3 were induced and reduced in the leaves R2 under the drought condition, respectively (Figure 3.16A) Under the salinity treatment [24], the majority of MSR genes, excluding MSRA5, has the expression levels in the public RNA-seq data (Figure 3.16B) Several MSR genes were significantly altered in roots under the salinity 18 condition As compared with the controls, MSRA2 and MSRB1 were downregulated, whereas MSRA4, -B2 -B5 were noted to up-regulated in roots Figure 3.16 Expression patterns of the MSR genes under the (A) drought and (B) salinity conditions in soybean V6 and R2: Leave samples at V6 and R2 stages were treated in the drought condition; R-Na1hr, R-Na6hr and R-Na10hr: Root samples were havested at 1, and 10 hours after salinity treatment 3.4.3 Experimental validation of the expression levels of MSRB in soybean In the normal condition, MSRB1 was validated as the highest expressed gene by the qPCR For instance, MSRB1 was strongly expressed in leaves in all stages in the normal condition (Figure 3.17) However, MSRB1 was not clearly expressed in the remaining organs Previously, MSRB1 was predicted to distribute on the chloroplast [99] Take together, our results revealed that MSRB1 was specific in leaves and may function on the oxidative stress response in the chroloplasts in leave tissues Figure 3.17 Validation of the expression patterns of MSRB genes in the normal condition Nine tissue samples, including leaves and roots of the seedlings, leaves V6 and R2, seeds R5 and R7, roots and root tips R2 were havested to examine the 19 expression profiles of MSR genes by using qRT-PCR Additionally, MSRB5 was lowest expressed in all tissue samples Our previous analysis also revealed that not many stress-responsive CREs were found in the promoter region of MSRB5 gene Expression patterns of MSRB5 were also not significantly altered in nine major organs in the normal condition [104] (Figure 3.15) Taken together, our data indicated that MSRB5 might play the less important role as compared with other member of MSRB gene family in soybean in the normal condition Figure 3.18 Validation of the expression patterns of MSRB genes in the drought condition (A): Validation of the expression profiles of MSRB genes in shoot samples under the drought treatment after - - 10 hours (B): Validation of the expression profiles of the MSRB genes in root samples under the drought treatment after - - 10 hours (C): Validation of the expression profiles of the MSRB genes in leaves V6 under the drought and well-watered (control) conditions (D): Validation of the expression profiles of the MSRB genes in leaves R2 under the drought and well-watered (control) conditions (E): Validation of the expression profiles of the 20 MSRB genes in root tips under the drought treatment Under the dehydration treatment, MSRB2 and MSRB5 were validated to up-regulate in leaves, whereas the remaining three MSR genes were not significantly altered (Figure 3.18A) For instance, no MSR genes were found to transcriptionally changed in roots under the dehydration treatment (Figure 3.18B) Interestingly, MSRB3 and two genes, MSRB2 and GmMSRB5 were up-regulated in leaves V6 and R2 under the dehydration treatment, respectively (Figure 3.18D), whereas no MSRB genes have been found to be responsive in roots (Figure 3.18E) As our previous discussion, expression profiles of the MSRB genes did not significantly change in roots in the normal condition (Figure 3.15) Taken together, our data indicated that MSRBs did not highly accumulate in root in the normal and dehydration conditions Figure 3.19 Validation of the expression patterns of MSRB genes in the salinity condition Expression profiles of MSRB genes in shoots (A) and roots (B) under the salinity condition after - - 10 hours Shoots have been havested at (S0s), (S2s) and 10 hourse (S10s) after the treatment Roots have been obtained at (R0s), (R2s) and 10 hours (R10s) after the treatment Our qRT-PCR confirmation showed that MSRB genes were up-regulated after h of the salinity treatment but seemed to reduced after 10 h of the treatment Among them, MSRB1 was noted to reduce in shoots after 10 h of the treatment (Figure 3.19A) MSRB5 was validated to up-regulate after h of the treatment but down-regulate after 10 h of the treatment Our data indicated that the majority of the MSRB genes, excluding MSRB2 seemed to up-regulated according to the duration of the treatment MSRB5 was significantly up-regulated in roots after 10 h of the salinity treatment (Figure 21 3.19B) Figure 3.20 Validation of the expression patterns of MSRB genes in the ABA condition (A): Expression profiles of MSRB genes in shoots under the ABA treatment (B): Expression profiles of MSRB genes in rootss under the ABA treatment To address the question of how MSRB genes respond to the adverse environmental conditions via the ABA-dependent and/or -independent pathways, the samples were havested to treat with ABA and used for the qPCR As provided in Figure 3.20A, all MSRB genes were tend to up-regulate in shoots, even no genes were responsive to the ABA This phenomenon was also recorded in the roots treated with ABA Interestingly, MSRB2 was found to highest induce in roots under the ABA treatment ( ình 20 ) Table 3.11 Summary of the expression levels of MSRB genes in soybean in the normal condition 22 Red color indicated the high-regulated genes Among them, MSRB1 was recently reported to locate on the chloroplast [99] Microarray analysis also revealed that MSRB1 was highly expressed in leaves and tend to induced in green pods [104] Our validation also confirmed the high expression levels of MSRB1 in leaves in the normal condition Additionally, MSRB3, predicted to locate on the chloroplast [99], was strongly expressed in leaves, roots and green pods in the normal condition Experimental validation confirmed that MSRB3 was up-regulated in leaves as our prediction This phenomenon was also recorded in MSRB4 as this gene was highly expressed in green pods and leaves in the normal condition (Table 3.11) Two remaining MSRB, including MSRB2 and MSRB5 were not specific in any major organs in soybean plants in normal condition Taken together, MSRB1, -B3 and -B4 were strongly expressed in leaves, suggesting these members might function on the repair of MetO in leave tissues Two genes, MSRB2 and MSRB5 might play less important role as compared with other members of MSRB genes Table 3.12 Summary of the expression profiles of MSRB genes in soybean under the drought condition Red and blue colors indicated the up- and down-regulated gene ABRE: Abscisic acid responsive element [TC] n: TC-rich repeats 23 Of our interest, our analyses also provided some reliable indication based on the in silico and experimental analyses The summary of the expression profiles of MSRB genes in soybean under the drought condition was represented in Table 3.12 For instance, MSRB3 was predicted and confirmed to be responsive in leaves V6 under the drought condition (Figure 3.18C) but not altered by ABA (Figure 3.20) Table 3.13 Summary of the expression profiles of MSRB genes in soybean under the salinity condition Red and blue colors indicated the up- and down-regulated gene ABRE: Abscisic acid responsive element [TC] n: TC-rich repeats Our results indicated that expression level of MSRB4 did not significantly change in any conditions, even this gene seemed to highly express in green pods and leaves in the normal condition Thus, MSRB4 was not responsive in the drought and salinity treatments Our microarray analyses and qRT-PCR validations strongly confirmed that MSRB genes did not significantly change in roots under the adverse envinromental conditions MSRB5 gene was not noticeably expressed in the normal condition but was considered as the most responsive gene in the stress condition(s) MSRB1 was the highest expressed gene in the normal condition, but was reduced in the salinity treatment In this study, we provided a list of candidate MSR genes for further studies for the improvement of the stress tolerance in soybean plants 24 CONCLUSIONS AND RECOMMENDATIONS Conclusions A total of 121 MRPs was identified in A thaliana MRP genes were involved in various important biological processes in the cells In silico analysis indicated that At3G55240 was the most reduced MRP genes in drought and salinity conditions by approximately -60 and -26-fold, respectively Overexpression of At3G55240 caused the sensitivity of the transgenic Arabidopsis plants to abiotic stress(es) In this study, 213 MRPs were identified in soybean The genes encoding MRPs were found to mostly involve in the RNA processing and signaling In silico analysis indicated that the genes encoding MRP were highly expressed in at least one major organ in the normal condition We also found that 13 MRP genes were responsive in leaves R2 and V6 under the drought treatment A total of genes encoding MSRA has been identified and characterized in soybean Among them, MSRA1 and MSRA6 were predicted to located on the secretory pathway, while MSRA2 and MSRA5 were found in the chloroplast The coverage of the cis- regulatory elements in the promoter regions indicated that MSRA and MSRB genes might be light responsiveness, hormone and stress responsiveness Our microarray analysis showed that genes encoding MSRAs and MSRBs were significantly altered in various organs in the normal and stress conditions The validation also confirmed that MSRB genes were not responsive in roots under the stress treatments Most importantly, MSRB5 was considered as the most responsive gene in the adverse envinronmental conditions found in this study Recommendations It would be very interesting to functionally characterize some unknown MRP genes in A thaliana and soybean and the promoter regions containing high accumulation of the stress-responsive CREs It is also significant to analyze the expression level of MSRA genes in soybean under abiotic stress condition(s) LIST OF PUBLISHED WORKS RELATED TO THE DISSERTATION Chu Duc Ha, Le Thi Ngoc Quynh, Pham Thi Ly Thu, Nguyen Quang Huy, Le Tien Dung, (2015), “On the roles of genes encoding methionine rich proteins in Arabidopsis thaliana in response to abiotic stresses”, Vietnam J Biol, 37(4): tr 487-495 Chu Duc Ha, Nguyen Thi Kim Lien, Pham Thi Ly Thu, Le Tien Dung, (2016), “Identification of the members of the methionine-S-sulfoxide reductase family in soybean (Glycine max)”, J Vietnam Agri Sci Tech, 9(70): tr 27-31 Ha Duc Chu, Quynh Ngoc Le, Huy Quang Nguyen, Dung Tien Le, (2016), “Genome-wide analysis of genes encoding methionine-rich proteins in Arabidopsis and soybean suggesting their roles in the adaptation of plants to abiotic stress”, International J Genom, 2016: pp 1-8 Ha Duc Chu, Kim-Lien Nguyen, Yasuko Watanabe, Dung Tien Le, LamSon Phan Tran, (2016), “Expression analyses of soybean genes encoding methionine sulfoxide reductase under various condition suggest a possible role in the adaptation to stress”, Appl Biol Chem, 59(5): pp 681-687 ... development of the plant Among them, enzyme methionine sulfoxide reductase (MSR), including methionine- S -sulfoxide Reductase (MSRA) and methionine- R -sulfoxide reductase (MSRB), is well-known to function... enzyme methionine sulfoxide reductase and methionine oxidation in plants; Methionine and the methioninerich proteins in plants; Potential application of the enzyme methionine sulfoxide reductase. .. Identification and characterization of the genes encoding methionine sulfoxide reductase in soybean  Expression analysis of the genes encoding methionine sulfoxide reductase under the drought and salinity