# Genome-Wide Analysis of Genes Encoding Methionine-Rich Proteins inArabidopsis and Soybean Suggesting Their Roles in the Adaptation of Plants to Abiotic Stress

Authors: Ha Duc Chu; Quynh Ngoc Le; Huy Quang Nguyen; Dung Tien Le
Journal: International Journal of Genomics
 (2016)

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## Abstract

Oxidation and reduction of methionine (Met) play important roles in scavenging reactive oxygen species (ROS) and signaling in living organisms. To understand the impacts of Met oxidation and reduction in plants during stress, we surveyed the genomes ofArabidopsis and soybean (Glycine max L.) for genes encoding Met-rich proteins (MRPs). We found 121 and 213 genes encoding MRPs inArabidopsis and soybean, respectively. Gene annotation indicated that those with known function are involved in vital cellular processes such as transcriptional control, calcium signaling, protein modification, and metal transport. Next, we analyzed the transcript levels of MRP-coding genes under normal and stress conditions. We found that 57 AtMRPs were responsive either to drought or to high salinity stress inArabidopsis; 35 GmMRPs were responsive to drought in the leaf of late vegetative or early reproductive stages of soybean. Among the MRP genes with a known function, the majority of the abiotic stress-responsive genes are involved in transcription control and calcium signaling. Finally,Arabidopsis plant which overexpressed an MRP-coding gene, whose transcripts were downregulated by abiotic stress, was more sensitive to paraquat than the control. Taken together, our report indicates that MRPs participate in various vital processes of plants under normal and stress conditions.


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## Body

## 1. Introduction

Under elevated ROS levels, free and protein-based Met are converted to methionine sulfoxide (MetO) which occurs in a diastereomeric mixture of methionine-S-sulfoxide (Met-S-O) and methionine-R-sulfoxide (Met-R-O) [1]. Oxidation of Met was reported to occur in various signaling proteins, thereby modulating their functions [1–9]. For example, calmodulin (CAM), a versatile protein involved in various signaling pathways, including ROS homeostasis inArabidopsis [10], is well known to have its methionine residue oxidation linked to protein dysfunction [11] and loss of protein stability [12]. Nevertheless, efforts to systematically identify all the proteins whose methionine residues are susceptible to oxidation yielded limited results due to the lack of viable tools, including an antibody specific to methionine sulfoxide [13–16]. Recently, Tarrago and colleagues proposed an affinity chromatography approach employing methionine sulfoxide reductase (MSR) to catch interacting partners. Using AtMSRB1 as bait, the authors isolated 24 interacting partners functioning in photosynthesis, translation, and protection against oxidative stress fromArabidopsis. The authors found a preference of proteins with higher Met content to bind to the bait, becoming isolated by this approach [17]. Quite recently, Jacques et al. used a newly developed technique called COFRADIC [18] for proteome-wide identification of Met oxidation sites inArabidopsis proteins. Their work revealed 500 sites of Met oxidation in 400 proteins of the plant [19].Organisms evolved two enzyme families to repair oxidized Met in proteins: methionine-S-sulfoxide reductase (MSRA) to reduce Met-S-O and methionine-R-sulfoxide reductase (MSRB) to reduce Met-R-O.In vivo modulation of MSR activities has been reported in yeast [20, 21], fruit flies [22], and mammals [23], which in turn affected their tolerance to oxidative stress and lifespan. In plants, Romero et al. demonstrated the role ofArabidopsis plastidial MSRAs in the defense against oxidative stress [24]. In another study, transgenic tomato overexpressing pepperMSR geneCaMSRB2 was found to protect against oxidative stress andPhytophthora pathogen infection [25]. Very recently, the role ofArabidopsis cytosolic AtMSRB7 and AtMSRB8 in conferring tolerance to oxidative stress was also demonstrated [26], whereas overexpression of AtMSRB1 and AtMSRB2 in plastids did not improve tolerance to high light stress [27].A number of studies have documented the role of enhancing expression of MSR-coding gene(s) in conferring stress tolerance to plants, but still little is known about the MSR targets which provide such tolerance. A notable study by Laugier et al. provided indirect evidence thatArabidopsis plastidial MSRBs confer tolerance to high light stress by acting on cpSRP43 and cpSRP54, thereby maintaining the integrity of the photosystem antenna under environmental constraints [28]. Another recent study by Lee and colleagues onArabidopsis cytosolic MSRB indicated that the enzyme conferred stress tolerance to the plants by acting on two glutathione transferases, GSTF2 and GSTF3 [29]. This study also suggested a list of potential substrates of AtMSRB7. Despite the fact that oxidation and reduction of Met residues in CAM and other calcium signaling proteins were experimentally verified to be involved in regulating the protein’s functions [2–4, 11, 12], they were not found among the potential candidates acquired by either the affinity chromatography approach or approaches that employed mass spectroscopy [17, 19, 29]. This line of evidence offers opportunity to argue that either the current approaches for proteome-wide identification of MSR targets pose technical limitations or the oxidation and reduction of Met in many proteins like CAM happen transiently, such that these techniques were unable to help in identifying them. To provide a complementary approach to identify possible targets of MSR in plants, in this work, we surveyed genomes of two dicots,Arabidopsis and soybean, to obtain polypeptides of more than 95 residues in length with more than 6% of Met in their sequences. We analyzed these genes in terms of functions, transcriptional responsiveness to stresses, and the conservation of Met residues in HMM profiles (a hidden Markov model-based profile of amino acid residues in a protein domain). A list of genes transcriptionally responsive to stresses with an HMM profile containing highly conserved Met is provided for experimental confirmation by the research community.


## 2. Materials and Methods

### 2.1. Materials

Unless otherwise stated,Arabidopsis thaliana studied in this work is Columbia ecotype and soybean is ofWilliams 82 cultivar.Arabidopsis seed overexpressing MRP gene(s) was obtained fromArabidopsis FOX line library (RIKEN BioResource Center, Tsukuba, Japan). Briefly, full length cDNA ofArabidopsis were placed under the control of 35S promoter and then inserted into a hygromycin-resistant plasmid. The plasmids were then transformed intoArabidopsis plants using flower-dipping technique. The development of FOX line library and the line carrying At3g55240 were previously reported [30].


### 2.2. Growth ofArabidopsis and Stress Treatment

Arabidopsis seeds were germinated on 0.5x MS media with or without antibiotics. At 2 weeks old the plants were transferred to soil and allowed to grow at 24
±
2
°
C with 16-hour lighting. For paraquat leaf disc assay, 3-week-old rosette leaves were excised and placed on paraquat solutions of various concentrations; after keeping in the dark for 1 hour, the plates were kept at 24°C for 24 hours. The experiments were done in triplicate; each replicate consists of 3 plants.


### 2.3. Computational Analyses

Protein sequences were downloaded from the PHYTOZOME database (http://www.phytozome.net/) and searched for proteins of 95 residues or more whose sequences contain 6% or more Met by using a java script. The cutoff value for protein length was chosen after consulting reports on the distribution of protein sizes [31, 32]. Genes satisfying these conditions were named Met-rich proteins (MRP):AtMRPs for genes fromArabidopsis andGmMRPs for genes from soybean. Genes encoding MRPs were classified into functional categories using MAPMAN [33]. The PFAM database (http://pfam.xfam.org/) was used to search for HMM profiles as well as possible protein domains.To obtain transcription levels, microarray data from previous studies were mined, including datasets forArabidopsis under drought and salinity stress [34, 35] and soybean under experimental drought conditions [36]. In that study, for drought treatment ofArabidopsis, 2-week-old plants were transferred to soil and allowed to grow for one more week; the 3-week-old plants were then withheld from watering for 10 days. After the tenth day, rosette leaves were collected from both well-watered and drought-stressed plants, frozen in liquid nitrogen, and stored at −80°C until RNA extraction. For high salinity treatment, 10-day-old plants grown on GM media were transferred onto 0.5x MS plates without sucrose, containing either 0 mM (untreated) or 200 mM NaCl and maintained for a period of 24 h. Samples were collected in three biological replicates, frozen in liquid nitrogen, and stored at −80°C until used for RNA extraction. The drought treatment of soybean plants and data acquisition was described previously [36]. Data analyses were performed with functions integrated in MS EXCEL.


## 2.1. Materials

Unless otherwise stated,Arabidopsis thaliana studied in this work is Columbia ecotype and soybean is ofWilliams 82 cultivar.Arabidopsis seed overexpressing MRP gene(s) was obtained fromArabidopsis FOX line library (RIKEN BioResource Center, Tsukuba, Japan). Briefly, full length cDNA ofArabidopsis were placed under the control of 35S promoter and then inserted into a hygromycin-resistant plasmid. The plasmids were then transformed intoArabidopsis plants using flower-dipping technique. The development of FOX line library and the line carrying At3g55240 were previously reported [30].


## 2.2. Growth ofArabidopsis and Stress Treatment

Arabidopsis seeds were germinated on 0.5x MS media with or without antibiotics. At 2 weeks old the plants were transferred to soil and allowed to grow at 24
±
2
°
C with 16-hour lighting. For paraquat leaf disc assay, 3-week-old rosette leaves were excised and placed on paraquat solutions of various concentrations; after keeping in the dark for 1 hour, the plates were kept at 24°C for 24 hours. The experiments were done in triplicate; each replicate consists of 3 plants.


## 2.3. Computational Analyses

Protein sequences were downloaded from the PHYTOZOME database (http://www.phytozome.net/) and searched for proteins of 95 residues or more whose sequences contain 6% or more Met by using a java script. The cutoff value for protein length was chosen after consulting reports on the distribution of protein sizes [31, 32]. Genes satisfying these conditions were named Met-rich proteins (MRP):AtMRPs for genes fromArabidopsis andGmMRPs for genes from soybean. Genes encoding MRPs were classified into functional categories using MAPMAN [33]. The PFAM database (http://pfam.xfam.org/) was used to search for HMM profiles as well as possible protein domains.To obtain transcription levels, microarray data from previous studies were mined, including datasets forArabidopsis under drought and salinity stress [34, 35] and soybean under experimental drought conditions [36]. In that study, for drought treatment ofArabidopsis, 2-week-old plants were transferred to soil and allowed to grow for one more week; the 3-week-old plants were then withheld from watering for 10 days. After the tenth day, rosette leaves were collected from both well-watered and drought-stressed plants, frozen in liquid nitrogen, and stored at −80°C until RNA extraction. For high salinity treatment, 10-day-old plants grown on GM media were transferred onto 0.5x MS plates without sucrose, containing either 0 mM (untreated) or 200 mM NaCl and maintained for a period of 24 h. Samples were collected in three biological replicates, frozen in liquid nitrogen, and stored at −80°C until used for RNA extraction. The drought treatment of soybean plants and data acquisition was described previously [36]. Data analyses were performed with functions integrated in MS EXCEL.


## 3. Results and Discussions

### 3.1. Occurrence of Genes Encoding MRPs inArabidopsis and Soybean

An exhaustive search of genes encoding proteins longer than 95 residues and containing 6% Met or more resulted in 121 and 213 genes fromArabidopsis and soybean genomes, respectively. Functions of about 50% of those genes were not known. RNA transcription, protein modification, and calcium signaling were the three major functional categories of the MRPs analyzed (Figure 1), indicating the important roles of MRP-coding genes in overall cellular function. Smaller categories include RNA processing and metal transport. Specifically, there were 20 and 44 MRP-coding genes functioning in RNA transcription inArabidopsis and soybean, respectively, making it the most abundant category. The second most abundant category was protein modification, of which 12 and 15 genes were found inArabidopsis and soybean, respectively. There were 6 MRP-coding genes ofArabidopsis functioning in calcium signaling, whereas 31 MRP-coding genes found in soybean genome belonged to this category. Among MRP-coding genes identified, soybean has 10 genes distributed in 4 unique categories that were not presented inArabidopsis, namely, lipid metabolism (4 genes), amino acid metabolism (2 genes), hormones (2 genes), and redox regulation (2 genes).Figure 1
Distribution of genes encoding MRPs into various biological processes inArabidopsis (a) and soybean (b).
(a)
(b)


### 3.2. Stress-Responsive MRP-Coding Genes inArabidopsis

To obtain MRP-coding genes transcriptionally responsive to abiotic stresses, our published microarray data of drought and high salinity treatments of wild typeArabidopsis [34, 35] were analyzed and data mining was performed. Of the 121Arabidopsis genes, expression data of 108 genes were available (Table S
1 in the Supplementary Material available online at http://dx.doi.org/10.1155/2016/5427062). Further analyses indicated that 23 and 16 genes were induced and repressed more than 2-fold, respectively, by drought treatment. Under treatment by high salinity, 11 and 17 genes were induced and repressed more than 2-fold, respectively. Among drought- and salt-responsive MRP-coding genes, several genes were previously confirmed to be stress-inducible, of which the expressions of the 10 genes were responsive to both drought and high salinity (Table 1). Most of the genes were responsive to both stressors’ code for plant-specific proteins. AT4G33467, encoding an unknown protein, was the most induced gene. Its transcript level was upregulated more than 330-fold by drought. Among stress-repressed genes, AT3G55240 was the most downregulated. Its transcript level was repressed by 60- and 26-fold under drought and high salinity, respectively. The function of this gene is not yet known; however, it was reported that overexpression of this gene inArabidopsis led to the phenotype “pseudo-etiolation in light” [30].Table 1
Genes encoding AtMRPs whose expression levels were responsive to both drought and high salinity.
Number
Locus IDs
Met (%)
Length (a.a.)
Drought versus untreated1
Salinity versus untreated2
Gene descriptions
Fold change3
q-value
Fold change3
q-value
1
AT1G32560
6.02
134
135.33
0.002
3.31
0.005
LEA group 1 domain-containing protein
2
AT1G33860
8.55
153
2.37
0.092
2.16
0.003
Unknown protein
3
AT3G55240
6.12
95
−60.29
0.007
−26.88
0.001
Overexpression leads to pseudo-etiolation in light phenotype
4
AT3G59900
6.20
130
10.70
0.011
−2.57
0.015
(ARGOS); unknown protein [AT3G59900.1]
5
AT3G62090
6.38
346
64.56
0.020
2.28
0.002
PHYTOCHROME INTERACTING FACTOR 3-LIKE 2
6
AT4G12334
6.25
113
−9.79
0.003
−3.04
0.005
Pseudogene of cytochrome P450 family protein
7
AT4G33467
8.91
102
337.51
0.002
6.16
0.023
Unknown protein [AT4G33467.1]
8
AT4G34590
6.33
159
8.26
0.004
3.27
0.002
GBF6 (A. thaliana BASIC LEUCINE-ZIPPER 11)
9
AT5G42325
6.03
233
2.70
0.028
2.45
0.049
Transcription elongation factor-related
10
AT5G67390
7.43
176
−4.17
0.015
−4.15
0.001
Similar to unknown proteins (TAIR:AT1G69360.1)
1Two-week-old plants were transferred to soil and allowed to grow for an additional week; the plants were then withheld from watering for 10 days. After 10 d of water with holding, rosette leaves were collected from both well watered and drought-stressed plants in three biological replicates, frozen in liquid nitrogen, and stored at −80°C until used for RNA extraction [35]. 210-day-old plants grown on GM media were transferred onto 0.5 × MS plates without sucrose, containing either 0 mM (control) or 200 mM NaCl, and maintained for a period of 24 h. Three independent experiments were performed for each condition. The samples were collected as three biological replicates (10 plants/replicate), frozen in liquid nitrogen, and stored at −80°C until used for RNA extraction [34]. 3Stress responsive AtMRPs were defined as genes encoding MRP whose expression levels were induced or repressed 2-fold or more with an FDR corrected p value of less than 0.05.


### 3.3. Stress-Responsive MRP-Coding Genes in Soybean

To identify stress-responsive genes among MRP-coding genes, we performed data mining with the microarray experiments conducted earlier. In these experiments, drought treatments were carried out by withholding water. Leaves of V6 (vegetative) and R2 (reproductive) stages were collected and microarray analyses performed as reported [36]. Transcript levels of all 213 MRP-coding genes were obtained, of which 11 were repressed and 12 genes were induced under drought in V6trifolia. In reproductive leaves (R2trifolia), drought treatment induced 24 genes, whereas only 6 genes were repressed (Table S
2). A total of 13 MRP-coding genes were found to be transcriptionally responsive to drought in both vegetative and reproductive-stage leaves (Table 2). The gene Glyma04g37040, which encodes a calmodulin-binding protein CML38, was the most induced gene by drought and its transcript levels were induced 40- and 15-fold in R2 and V6trifolia, respectively. The most repressed gene by drought was Glyma02g10620, encoding a 98-residue protein of unknown function whose transcript levels were repressed 44- and 4-fold in V6 and R2trifolia, respectively.Table 2
Genes encoding GmMRPs whose expression is responsive to drought stress in V6 and R2 leaves.
Number
Glyma ID
Met
Length (a.a.)
V6trifolia
R2trifolia
Gene descriptions
Arabidopsis homologs
(%)
Fold change
q-value
Fold change
q-value
1
Glyma01g15910
8.08
100
3.63
0.045
4.96
0.042
No original description
2
Glyma01g15930
6.56
458
−20.34
0.007
−3.87
0.015
UNE10; transcription factor
AT4G00050
3
Glyma02g10620
7.22
98
−44.63
0.007
−4.04
0.053
Overexpression leads to pseudo-etiolation in light
AT3G55240
4
Glyma03g32740
6.04
481
−2.19
0.007
−2.02
0.030
PIF1, PIL5; transcription factor
AT2G20180
5
Glyma04g37040
7.91
140
15.03
0.012
40.08
0.005
Calmodulin-binding protein CML38
AT1G76650
6
Glyma06g39910
10.34
117
3.12
0.067
4.14
0.013
Calcium-binding EF hand family protein
AT4G27280
7
Glyma10g30380
7.43
149
7.53
0.013
5.27
0.026
calmodulin 5; calcium ion binding
AT2G27030
8
Glyma15g05510
7.37
96
2.93
0.025
2.41
0.023
No original description
9
Glyma16g02510
7.26
125
2.05
0.028
4.63
0.023
Calcium-binding protein, putative
AT2G46600
10
Glyma19g43580
6.7
210
−2.01
0.160
2.42
0.078
GIF, GIF1, AN3 (ANGUSITFOLIA3)
AT5G28640
11
Glyma20g00780
6.69
285
−3.03
0.046
−2.36
0.027
Contains homeodomain (InterPro:IPR009057)
AT1G10820
12
Glyma20g22280
6.59
426
2.25
0.046
2.99
0.056
PIF3, POC1, PAP3, transcription factor
AT1G09530
13
Glyma20g36730
7.89
153
3.06
0.042
2.29
0.129
calmodulin 5; calcium ion binding
AT2G27030


### 3.4. Common Stress-Responsive MRP-Coding Genes inArabidopsis and Soybean

InArabidopsis, 7 AtMRPs were upregulated and 3 other AtMRPs were downregulated under both drought and high salinity. In soybean drought, 8 and 5 GmMRPs were up- or downregulated, respectively, in both V6 and R2trifolia (Figure 2(a)). Among 6AtMRPs encoding calcium signaling proteins, three genes were transcriptionally responsive to either drought or salt (Table S
1). At the same time, 14GmMRPs encoding calmodulin-like proteins were transcriptionally responsive to drought in either V6 or R2trifolia or both. This data suggested that calcium signaling plays an important role in the plant’s signaling during abiotic stress exposure. In light of previous studies, it is very likely that Met oxidation and reduction of calmodulin may also contribute significantly to the plant’s signaling response to abiotic stresses. A number ofAtMRPs andGmMRPs encoding transcription factors were also responsive to abiotic stresses inArabidopsis and soybean (Tables 1 and 2), indicating the involvement of the MRPs in the important cellular activities.Figure 2
Venn diagram analyses of the expression of MRP-coding genes inArabidopsis and soybean under abiotic stresses (a), and HMM profile ofArabidopsis and soybean homologs share common responsiveness to drought (b) and their peptide sequence alignment (c).
(a)
(b)
(c)Further analysis of stress-responsive MRP-coding genes identified a gene coding for a plant-specific protein that has homologs in bothArabidopsis (AT3G55240) and soybean (Glyma02g10620). These genes encoded highly homologous proteins (>70% identity) of about 100 amino acid residues that share an HMM profile with several conserved Met residues in the N-terminal (Figures 2(b) and 2(c)). In a previous study, it was found that overexpression of this gene inArabidopsis caused a phenotype of pseudo-etiolation in light or leaf bleaching [30], although how such phenotype occurred was not explained. WhenArabidopsis was transformed with an RNAi construct to downregulate this gene, most of the transgenic plants died at a very early stage and the plants that survived did not show any reduction in the transcription levels, suggesting a vital function of this gene [30]. To determine if the gene At3g55240 is involved in redox stress responses, we acquired the overexpressor line from theArabidopsis FOX line library (RIKEN BioResource Center) and analyzed them. Growing of the plants on MS media and soil confirmed the pseudo-etiolation phenotype (Figure 3(a)). When treated with paraquat in a leaf disc assay, the overexpressor line exhibited higher sensitivity than the wild type control (Figure 3(b)), suggesting the gene may be involved in mediating redox stress responses. This gene and its soybean homolog were both repressed under abiotic stress; thus, increasing its expression may not provide benefit under stress.Figure 3
Phenotypes of anArabidopsis overexpressed a gene encoding a MRP under normal physiological condition (a) and under a paraquat leaf disc assay (b). WT, wild type control; RBC1,Arabidopsis plant overexpressed At3g55240 gene.
(a)
(b)


### 3.5. Stress-Responsivecis-Elements of the MRPs’ Promoter

To provide further evidence of the stress-responsiveness of MRP-coding genes, we searched for the presence of known stress-responsivecis-elements in the promoters of the genes inArabidopsis 1 kbs upstream of the transcriptional start sites. We found that promoters ofAtMRPs contain 23, 26, and 16cis-elements of ABRE, MYBR, and MYCR, respectively. On average there are 0.54cis-elements per AtMRP and 0.86cis-elements for each stress-responsiveAtMRP, indicating a slight enrichment of stress-responsivecis-elements among drought- and/or salt-responsiveAtMRPs.


### 3.6. Subcellular Localization of MRPs

Chloroplast and mitochondria are the two types of cellular organelles which generate high levels of ROS. Thus, identification of MRPs localizing to these organelles may shed light on their functions. Using amino acid sequences of AtMRPs and the prediction tools such as TargetP [37], pSORT [38], and CELLO [39], we identified all AtMRPs targeted to either chloroplast or mitochondria (Table S
1). Among 121 AtMRPs, 21 were predicted to target chloroplast by either ChloroP or pSORT or both; 9 were predicted to localize to mitochondria by either pSORT or CELLO or both.In conclusion, as a complementary approach to other studies on the identification of targets of Met oxidation and reduction in plants, here we found a large number of MRPs involved in important cellular processes such as RNA transcription control and calcium signaling. Several genes encoding these MRPs were transcriptionally responsive to abiotic stresses, such as drought and high salinity, suggesting their roles in the adaptation of plants to these stressors. The fact that promoters of the genes encoding stress-responsive MRPs are slightly enriched incis-element responsive to stresses and that product of these genes were predicted to localize in ROS-enriched organelles, chloroplast and mitochondria, further confirm their functions. Taken together, this work proposes unique evidence on methionine oxidation in proteins and its possible role in regulating the plant’s activities.


## 3.1. Occurrence of Genes Encoding MRPs inArabidopsis and Soybean

An exhaustive search of genes encoding proteins longer than 95 residues and containing 6% Met or more resulted in 121 and 213 genes fromArabidopsis and soybean genomes, respectively. Functions of about 50% of those genes were not known. RNA transcription, protein modification, and calcium signaling were the three major functional categories of the MRPs analyzed (Figure 1), indicating the important roles of MRP-coding genes in overall cellular function. Smaller categories include RNA processing and metal transport. Specifically, there were 20 and 44 MRP-coding genes functioning in RNA transcription inArabidopsis and soybean, respectively, making it the most abundant category. The second most abundant category was protein modification, of which 12 and 15 genes were found inArabidopsis and soybean, respectively. There were 6 MRP-coding genes ofArabidopsis functioning in calcium signaling, whereas 31 MRP-coding genes found in soybean genome belonged to this category. Among MRP-coding genes identified, soybean has 10 genes distributed in 4 unique categories that were not presented inArabidopsis, namely, lipid metabolism (4 genes), amino acid metabolism (2 genes), hormones (2 genes), and redox regulation (2 genes).Figure 1
Distribution of genes encoding MRPs into various biological processes inArabidopsis (a) and soybean (b).
(a)
(b)


## 3.2. Stress-Responsive MRP-Coding Genes inArabidopsis

To obtain MRP-coding genes transcriptionally responsive to abiotic stresses, our published microarray data of drought and high salinity treatments of wild typeArabidopsis [34, 35] were analyzed and data mining was performed. Of the 121Arabidopsis genes, expression data of 108 genes were available (Table S
1 in the Supplementary Material available online at http://dx.doi.org/10.1155/2016/5427062). Further analyses indicated that 23 and 16 genes were induced and repressed more than 2-fold, respectively, by drought treatment. Under treatment by high salinity, 11 and 17 genes were induced and repressed more than 2-fold, respectively. Among drought- and salt-responsive MRP-coding genes, several genes were previously confirmed to be stress-inducible, of which the expressions of the 10 genes were responsive to both drought and high salinity (Table 1). Most of the genes were responsive to both stressors’ code for plant-specific proteins. AT4G33467, encoding an unknown protein, was the most induced gene. Its transcript level was upregulated more than 330-fold by drought. Among stress-repressed genes, AT3G55240 was the most downregulated. Its transcript level was repressed by 60- and 26-fold under drought and high salinity, respectively. The function of this gene is not yet known; however, it was reported that overexpression of this gene inArabidopsis led to the phenotype “pseudo-etiolation in light” [30].Table 1
Genes encoding AtMRPs whose expression levels were responsive to both drought and high salinity.
Number
Locus IDs
Met (%)
Length (a.a.)
Drought versus untreated1
Salinity versus untreated2
Gene descriptions
Fold change3
q-value
Fold change3
q-value
1
AT1G32560
6.02
134
135.33
0.002
3.31
0.005
LEA group 1 domain-containing protein
2
AT1G33860
8.55
153
2.37
0.092
2.16
0.003
Unknown protein
3
AT3G55240
6.12
95
−60.29
0.007
−26.88
0.001
Overexpression leads to pseudo-etiolation in light phenotype
4
AT3G59900
6.20
130
10.70
0.011
−2.57
0.015
(ARGOS); unknown protein [AT3G59900.1]
5
AT3G62090
6.38
346
64.56
0.020
2.28
0.002
PHYTOCHROME INTERACTING FACTOR 3-LIKE 2
6
AT4G12334
6.25
113
−9.79
0.003
−3.04
0.005
Pseudogene of cytochrome P450 family protein
7
AT4G33467
8.91
102
337.51
0.002
6.16
0.023
Unknown protein [AT4G33467.1]
8
AT4G34590
6.33
159
8.26
0.004
3.27
0.002
GBF6 (A. thaliana BASIC LEUCINE-ZIPPER 11)
9
AT5G42325
6.03
233
2.70
0.028
2.45
0.049
Transcription elongation factor-related
10
AT5G67390
7.43
176
−4.17
0.015
−4.15
0.001
Similar to unknown proteins (TAIR:AT1G69360.1)
1Two-week-old plants were transferred to soil and allowed to grow for an additional week; the plants were then withheld from watering for 10 days. After 10 d of water with holding, rosette leaves were collected from both well watered and drought-stressed plants in three biological replicates, frozen in liquid nitrogen, and stored at −80°C until used for RNA extraction [35]. 210-day-old plants grown on GM media were transferred onto 0.5 × MS plates without sucrose, containing either 0 mM (control) or 200 mM NaCl, and maintained for a period of 24 h. Three independent experiments were performed for each condition. The samples were collected as three biological replicates (10 plants/replicate), frozen in liquid nitrogen, and stored at −80°C until used for RNA extraction [34]. 3Stress responsive AtMRPs were defined as genes encoding MRP whose expression levels were induced or repressed 2-fold or more with an FDR corrected p value of less than 0.05.


## 3.3. Stress-Responsive MRP-Coding Genes in Soybean

To identify stress-responsive genes among MRP-coding genes, we performed data mining with the microarray experiments conducted earlier. In these experiments, drought treatments were carried out by withholding water. Leaves of V6 (vegetative) and R2 (reproductive) stages were collected and microarray analyses performed as reported [36]. Transcript levels of all 213 MRP-coding genes were obtained, of which 11 were repressed and 12 genes were induced under drought in V6trifolia. In reproductive leaves (R2trifolia), drought treatment induced 24 genes, whereas only 6 genes were repressed (Table S
2). A total of 13 MRP-coding genes were found to be transcriptionally responsive to drought in both vegetative and reproductive-stage leaves (Table 2). The gene Glyma04g37040, which encodes a calmodulin-binding protein CML38, was the most induced gene by drought and its transcript levels were induced 40- and 15-fold in R2 and V6trifolia, respectively. The most repressed gene by drought was Glyma02g10620, encoding a 98-residue protein of unknown function whose transcript levels were repressed 44- and 4-fold in V6 and R2trifolia, respectively.Table 2
Genes encoding GmMRPs whose expression is responsive to drought stress in V6 and R2 leaves.
Number
Glyma ID
Met
Length (a.a.)
V6trifolia
R2trifolia
Gene descriptions
Arabidopsis homologs
(%)
Fold change
q-value
Fold change
q-value
1
Glyma01g15910
8.08
100
3.63
0.045
4.96
0.042
No original description
2
Glyma01g15930
6.56
458
−20.34
0.007
−3.87
0.015
UNE10; transcription factor
AT4G00050
3
Glyma02g10620
7.22
98
−44.63
0.007
−4.04
0.053
Overexpression leads to pseudo-etiolation in light
AT3G55240
4
Glyma03g32740
6.04
481
−2.19
0.007
−2.02
0.030
PIF1, PIL5; transcription factor
AT2G20180
5
Glyma04g37040
7.91
140
15.03
0.012
40.08
0.005
Calmodulin-binding protein CML38
AT1G76650
6
Glyma06g39910
10.34
117
3.12
0.067
4.14
0.013
Calcium-binding EF hand family protein
AT4G27280
7
Glyma10g30380
7.43
149
7.53
0.013
5.27
0.026
calmodulin 5; calcium ion binding
AT2G27030
8
Glyma15g05510
7.37
96
2.93
0.025
2.41
0.023
No original description
9
Glyma16g02510
7.26
125
2.05
0.028
4.63
0.023
Calcium-binding protein, putative
AT2G46600
10
Glyma19g43580
6.7
210
−2.01
0.160
2.42
0.078
GIF, GIF1, AN3 (ANGUSITFOLIA3)
AT5G28640
11
Glyma20g00780
6.69
285
−3.03
0.046
−2.36
0.027
Contains homeodomain (InterPro:IPR009057)
AT1G10820
12
Glyma20g22280
6.59
426
2.25
0.046
2.99
0.056
PIF3, POC1, PAP3, transcription factor
AT1G09530
13
Glyma20g36730
7.89
153
3.06
0.042
2.29
0.129
calmodulin 5; calcium ion binding
AT2G27030


## 3.4. Common Stress-Responsive MRP-Coding Genes inArabidopsis and Soybean

InArabidopsis, 7 AtMRPs were upregulated and 3 other AtMRPs were downregulated under both drought and high salinity. In soybean drought, 8 and 5 GmMRPs were up- or downregulated, respectively, in both V6 and R2trifolia (Figure 2(a)). Among 6AtMRPs encoding calcium signaling proteins, three genes were transcriptionally responsive to either drought or salt (Table S
1). At the same time, 14GmMRPs encoding calmodulin-like proteins were transcriptionally responsive to drought in either V6 or R2trifolia or both. This data suggested that calcium signaling plays an important role in the plant’s signaling during abiotic stress exposure. In light of previous studies, it is very likely that Met oxidation and reduction of calmodulin may also contribute significantly to the plant’s signaling response to abiotic stresses. A number ofAtMRPs andGmMRPs encoding transcription factors were also responsive to abiotic stresses inArabidopsis and soybean (Tables 1 and 2), indicating the involvement of the MRPs in the important cellular activities.Figure 2
Venn diagram analyses of the expression of MRP-coding genes inArabidopsis and soybean under abiotic stresses (a), and HMM profile ofArabidopsis and soybean homologs share common responsiveness to drought (b) and their peptide sequence alignment (c).
(a)
(b)
(c)Further analysis of stress-responsive MRP-coding genes identified a gene coding for a plant-specific protein that has homologs in bothArabidopsis (AT3G55240) and soybean (Glyma02g10620). These genes encoded highly homologous proteins (>70% identity) of about 100 amino acid residues that share an HMM profile with several conserved Met residues in the N-terminal (Figures 2(b) and 2(c)). In a previous study, it was found that overexpression of this gene inArabidopsis caused a phenotype of pseudo-etiolation in light or leaf bleaching [30], although how such phenotype occurred was not explained. WhenArabidopsis was transformed with an RNAi construct to downregulate this gene, most of the transgenic plants died at a very early stage and the plants that survived did not show any reduction in the transcription levels, suggesting a vital function of this gene [30]. To determine if the gene At3g55240 is involved in redox stress responses, we acquired the overexpressor line from theArabidopsis FOX line library (RIKEN BioResource Center) and analyzed them. Growing of the plants on MS media and soil confirmed the pseudo-etiolation phenotype (Figure 3(a)). When treated with paraquat in a leaf disc assay, the overexpressor line exhibited higher sensitivity than the wild type control (Figure 3(b)), suggesting the gene may be involved in mediating redox stress responses. This gene and its soybean homolog were both repressed under abiotic stress; thus, increasing its expression may not provide benefit under stress.Figure 3
Phenotypes of anArabidopsis overexpressed a gene encoding a MRP under normal physiological condition (a) and under a paraquat leaf disc assay (b). WT, wild type control; RBC1,Arabidopsis plant overexpressed At3g55240 gene.
(a)
(b)


## 3.5. Stress-Responsivecis-Elements of the MRPs’ Promoter

To provide further evidence of the stress-responsiveness of MRP-coding genes, we searched for the presence of known stress-responsivecis-elements in the promoters of the genes inArabidopsis 1 kbs upstream of the transcriptional start sites. We found that promoters ofAtMRPs contain 23, 26, and 16cis-elements of ABRE, MYBR, and MYCR, respectively. On average there are 0.54cis-elements per AtMRP and 0.86cis-elements for each stress-responsiveAtMRP, indicating a slight enrichment of stress-responsivecis-elements among drought- and/or salt-responsiveAtMRPs.


## 3.6. Subcellular Localization of MRPs

Chloroplast and mitochondria are the two types of cellular organelles which generate high levels of ROS. Thus, identification of MRPs localizing to these organelles may shed light on their functions. Using amino acid sequences of AtMRPs and the prediction tools such as TargetP [37], pSORT [38], and CELLO [39], we identified all AtMRPs targeted to either chloroplast or mitochondria (Table S
1). Among 121 AtMRPs, 21 were predicted to target chloroplast by either ChloroP or pSORT or both; 9 were predicted to localize to mitochondria by either pSORT or CELLO or both.In conclusion, as a complementary approach to other studies on the identification of targets of Met oxidation and reduction in plants, here we found a large number of MRPs involved in important cellular processes such as RNA transcription control and calcium signaling. Several genes encoding these MRPs were transcriptionally responsive to abiotic stresses, such as drought and high salinity, suggesting their roles in the adaptation of plants to these stressors. The fact that promoters of the genes encoding stress-responsive MRPs are slightly enriched incis-element responsive to stresses and that product of these genes were predicted to localize in ROS-enriched organelles, chloroplast and mitochondria, further confirm their functions. Taken together, this work proposes unique evidence on methionine oxidation in proteins and its possible role in regulating the plant’s activities.



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*Source: 5427062-2016-08-18.xml*