The MYB gene family comprises one of the richest groups of transcription factors in plants. Plant MYB proteins are characterized by a highly conserved MYB DNA-binding domain. MYB proteins are classified into four major groups namely, 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB based on the number and position of MYB repeats. MYB transcription factors are involved in plant development, secondary metabolism, hormone signal transduction, disease resistance and abiotic stress tolerance. A comparative analysis of MYB family genes in rice and Arabidopsis will help reveal the evolution and function of MYB genes in plants.
Results
A genome-wide analysis identified at least 155 and 197 MYB genes in rice and Arabidopsis, respectively. Gene structure analysis revealed that MYB family genes possess relatively more number of introns in the middle as compared with C- and N-terminal regions of the predicted genes. Intronless MYB-genes are highly conserved both in rice and Arabidopsis. MYB genes encoding R2R3 repeat MYB proteins retained conserved gene structure with three exons and two introns, whereas genes encoding R1R2R3 repeat containing proteins consist of six exons and five introns. The splicing pattern is similar among R1R2R3 MYB genes in Arabidopsis. In contrast, variation in splicing pattern was observed among R1R2R3 MYB members of rice. Consensus motif analysis of 1kb upstream region (5′ to translation initiation codon) of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. Real-time quantitative RT-PCR analysis showed that several members of MYBs are up-regulated by various abiotic stresses both in rice and Arabidopsis.
Conclusion
A comprehensive genome-wide analysis of chromosomal distribution, tandem repeats and phylogenetic relationship of MYB family genes in rice and Arabidopsis suggested their evolution via duplication. Genome-wide comparative analysis of MYB genes and their expression analysis identified several MYBs with potential role in development and stress response of plants.
Background
Transcription factors are essential regulators of gene transcription and usually consist of at least two domains namely a DNA-binding and an activation/repression domain, that function together to regulate the target gene expression[1]. The MYB (my elob lastosis) transcription factor family is present in all eukaryotes. "Oncogene" vMYB was the first MYB gene identified in avian myeloblastosis virus[2]. Three vMYB-related genes namely c-MYB, A-MYB and B-MYB were subsequently identified in many vertebrates and implicated in the regulation of cell proliferation, differentiation, and apoptosis[3]. Homologous genes were also identified in insects, fungi and slime molds[4]. A homolog of mammalian c-MYB gene, Zea mays C1, involved in regulation of anthocyanin biosynthesis, was the first MYB gene to be characterized in plants[5]. Interestingly, plants encode large number of MYB genes as compared to fungi and animals[6–12]. MYB proteins contain a MYB DNA-binding domain, which is approximately 52 amino acid residues in length, and forms a helix-turn-helix fold with three regularly spaced tryptophan residues[13]. The three-dimensional structure of the MYB domain showed that the DNA recognition site α-helix interacts with the major groove of DNA[14]. However, amino acid sequences outside the MYB domain are highly divergent. Based on the number of adjacent MYB repeats, MYB transcription factors are classified into four major groups, namely 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB containing one, two, three and four MYB repeats, respectively. In animals, R1R2R3-type MYB domain proteins are predominant, while in plants, the R2R3-type MYB domain proteins are more prevalent[4, 7, 15]. The plant R2R3-MYB genes probably evolved from an R1R2R3-MYB gene progenitor through loss of R1 repeat or from an R1MYB gene through duplication of R1 repeat[16, 17].
In plants, MYB transcription factors play a key role in plant development, secondary metabolism, hormone signal transduction, disease resistance and abiotic stress tolerance[18, 19]. Several R2R3-MYB genes are involved in regulating responses to environmental stresses such as drought, salt, and cold[9, 20]. Transgenic rice over expressing OsMYB3R2 exhibited enhanced cold tolerance as well as increased cell mitotic index[21]. Enhanced freezing stress tolerance was observed in Arabidopsis over-expressing OsMYB4[10, 22]. Arabidopsis AtMYB96, an R2R3-type MYB transcription factor, regulates drought stress response by integrating ABA and auxin signals[23]. Transgenic Arabidopsis expressing AtMYB15 exhibited hypersensitivity to exogenous ABA and improved tolerance to drought[24], and cold stress[20]. The AtMYB15 negatively regulated the expression of CBF genes and conferred freezing tolerance in Arabidopsis[20]. Other functions of MYBs include control of cellular morphogenesis, regulation of secondary metabolism, meristem formation and the cell cycle regulation[15, 25–28]. Recent studies have shown that the MYB genes are post-transcriptionally regulated by microRNAs; for instance, AtMYB33, AtMYB35, AtMYB65 and AtMYB101 genes involved in anther or pollen development are targeted by miR159 family[29, 30].
MYB TF family genes have been identified in a number of monocot and dicot plants[9], and evolutionary relationship between rice and Arabidopsis MYB proteins has been reported[31]. We report here genome-wide classification of 155 and 197 MYB TF family genes in rice and Arabidopsis, respectively. We also analysed abiotic stress responsive and tissue specific expression pattern of the selected MYB genes. To map the evolutionary relationship among MYB family members, phylogenetic trees were constructed for both rice and Arabidopsis MYB proteins. Several over- represented cis-regulatory motifs in the promoter region of the MYB genes were also identified.
Results and discussion
Identification, classification and structural analysis of MYB family members
Genome-wide analysis led to the identification of 155 and 197 MYB genes in rice and Arabidopsis, respectively, with their mapping on different chromosomes (Additional file1: Table S1). We used previously assigned names to the MYB genes; for instance, AtMYB0 (GL1) name was accepted for the first identified R2R3 MYB gene; subsequently identified R2R3 MYB genes were named as AtMYB1, AtMYB2, etc. in Arabidopsis[31–34]. We classified MYB transcription factors in to four distinct groups namely “MYB-related genes”, “MYB-R2R3”, “MYB-R1R2R3”, and “Atypical MYB genes” based on the presence of one, two, three and four MYB repeats, respectively. Our analysis revealed that the MYB-R2R3 subfamily consisted of the highest number of MYB genes, with 56.77 and 70.05% of the total MYB genes in rice and Arabidopsis, respectively (Figure1a, b). In the R2R3-MYB proteins, N-terminal consists of MYB domains, while the regulatory C-terminal region is highly variable. Presence of a single MYB-like domain (e.g. hTRF1/hTRF2) in their C terminus is required for telomeric DNA binding in vitro[35]. Earlier study revealed that the R2R3-MYB related proteins arose after loss of the sequences encoding R1 in an ancestral 3R-MYB gene during plant evolution[36]. In contrast, only few MYB-R1R2R3 genes were identified in Arabidopsis and rice with 5 and 4 genes, respectively. The category “MYB-related genes” usually but not always contain a single MYB domain[17, 31, 36]. We found that “MYB-related genes” represented 40 and 26.39% of the total MYB genes in rice and Arabidopsis, respectively (Figure1a, b), and thus constituted the second largest group of MYB proteins in both rice and Arabidopsis. We also identified one MYB protein in rice and two MYB proteins in Arabidopsis that contained more than three MYB repeats and these belong to “Atypical MYB genes” group. The AT1G09770 in Arabidopsis and LOC_Os07g04700 in rice have five MYB domains and are called as CDC5-type protein, whereas AT3G18100 of Arabidopsis has four MYB domains and is named as 4R-type MYB (Table1; Additional file1: Table S1). The 4R-MYB proteins belong to the smallest class, which contains R1/R2-like repeats. MYB genes can also be classified into several subgroups based on gene function, such as Circadian Clock Associated1 (CCA1) and Late Elongated Hypocotyl (LHY), Triptychon (TRY) and Caprice (CPC)[15, 17, 37]. CPC and TRY belong to the R3-MYB group and are mainly involved in epidermal cell differentiation, together with ENHANCER OF TRY AND CPC1, 2 and 3 (ETC1, ETC2 and ETC3), and TRICHOMELESS1 and 2 (TCL1 and TCL2)[38–41]. Here, we observed that CCA1, CPC and LHY subgroups contain 23, 3 and 1 ‘MYB-related’ TF, respectively in Arabidopsis. To further understand the nature of MYB proteins, their physiochemical properties were also analyzed. The MYB proteins have similar grand average hydropathy (GRAVY) scores. Kyte and Doolittle[42] proposed that higher average hydropathy score of a protein indicates physiochemical property of an integral membrane protein, while a negative score indicates soluble nature of the protein. We observed that all MYB proteins in rice and Arabidopsis, except AT1G35516 had a negative GRAVY score, suggesting that MYBs are soluble proteins, a character that is necessary for transcription factors. Minimum and maximum score of GRAVY were recorded as −1.287 (LOC_Os02g47744) and −0.178 (LOC_Os08g37970) in rice, and −1.359 (AT5G41020) and 0.612 (AT1G35516) in Arabidopsis, respectively. We also calculated average isoelectric point (pI) value. The mean pI values for MYB-1R, R2R3 and R1R2R3 protein families were 7.55, 6.90 and 7.25 in rice, and 7.55, 6.89 and 6.80 in Arabidopsis, respectively. The average molecular weight of MYB-1R, R2R3 and R1R2R3 protein families were 31.128, 34.561 and 72.52 kDa in rice, and 34.186, 35.875 and 86.217 kDa in Arabidopsis, respectively (Additional file1: Table S1).
Figure 1
Chromosome-wise distribution ofMYBtranscription factor genes. a) rice, b) Arabidopsis. We classified MYB transcription factors in to four distinct groups namely “MYB-related genes”, “MYB-R2R3”, “MYB-R1R2R3”, and “Atypical MYB genes” based on the presence of one, two, three and four MYB repeats, respectively.
Table 1
MYB-domain based characterization and comparison ofMYBtranscription factor family genes in terms of GRAVY, molecular weight and cellular localization
RICE
MYB groups
No of genes
(%)
GRAVY
PI
Molecular weight
Localization
Min.
Max.
Avg.
Min.
Max.
Avg.
Min.
Max.
Avg.
MYB-related genes
62
40
−1.287
−0.201
−1.3875
3.99
12.26
8.125
7613.7
170921.8
89267.75
Nuclear
MYB-R2R3
88
56.77
−0.906
−0.178
−0.995
4.67
10.4
7.535
21605.3
75878.9
48742.1
Nuclear
MYB-R1R2R3
4
2.58
−0.691
−0.593
−0.9875
5.05
8.53
13.605
64100.1
109413.5
86756.8
Nuclear
Atypical MYB genes
1
0.64
−0.748
−0.748
−0.748
9.56
9.56
9.56
92424.6
92424.6
92424.6
Nuclear
ARABIDOPSIS
MYB groups
No of genes
(%)
GRAVY
PI
Molecular weight
Localization
Min.
Max.
Avg.
Min.
Max.
Avg.
Min.
Max.
Avg.
MYB-related genes
52
26.39
−1.359
0.612
−0.3735
4.75
6.62
2.375
7570.9
50112
3785.45
Nuclear
MYB-R2R3
138
70.05
−1.102
−0.471
−0.7865
4.16
10.24
7.2
27951.2
33239
13975.6
Nuclear
MYB-R1R2R3
5
2.54
−0.941
−0.774
−0.8575
5.43
9.22
7.325
50032.2
158268.4
79134.2
Nuclear
Atypical MYB genes
2
0.51
−0.941
−0.94
−0.9405
5.67
6.37
3.185
95766.5
96084.3
95925.4
Nuclear
Functional classification of MYB transcription factors
MYB proteins perform wide diversity of functions in plants. The R2R3-MYB proteins are involved in plant specific processes, such as control of secondary metabolism or cellular morphogenesis[43–49]. Gene ontology (GO) analysis suggested that R2R3-MYB genes, namely AtMYB16, AtMYB35, AtMYB5/AtMYB80, and AtMYB91 may regulate cell, anther, trichome and leaf morphogenesis, respectively. Likewise, R2R3-type genes, namely OsMYB16, OsMYB88, OsMYB117, LOC_Os01g50110 and LOC_Os03g38210 may regulate morphogenesis in rice. In addition to R2R3-type MYBs, two MYB-related genes, LOC_Os01g43180 and LOC_Os09g23200 may also regulate morphogenesis in rice. R2R3-type AtMYB10 and AT2G47210, MYB-related AT3G09600, and R1R2R3-type AtMYB3R4 genes were identified with GO function, such as N-terminal protein myristoylation, histone H3 acetylation, and regulation of DNA endoreduplication, respectively. Previous studies have shown that genes encoding 3R-MYB proteins have regulatory role in cell cycle control[28, 50]. We also found that AtMYB3R4 may be involved in cell cycle control (GO: 0007049). GO analysis of MYB proteins illustrated that 98.70% OsMYB and 98.47% AtMYB were fully involved in transcription activation, while rest of the MYB proteins were classified in to other GO functions, such as kinase activity, protein binding, transcription repressor activity, etc. GO analysis categorized rice LOC_Os01g62660 as signal transducer (GO: 0004871) and transcription activator. The R2R3-type AtMYB4 was classified into transcriptional repressor group. The AtMYB4 expression is down regulated by exposure to UV-B light, indicating that derepression of its target genes is an important mechanism for acclimation to UV-B in Arabidopsis[51, 52]. In our study, AtMYB34; a R2R3-type MYB protein, has been found with catalytic-kinase as well as transcription activator molecular functions as reported earlier[53, 54]. The AtMYB34 is also involved in defense response against insects[55]. In consistent with previous report[56], AtMYB23 was found to have protein binding (i.e. interaction with GL3) as well as DNA-binding functions.
The subcellular localization of MYB proteins was predicted using several localization predictor softwares. The predicted locations of the MYB proteins were also verified by gene ontology under keyword “GO cellular component” and species-specific localization prediction tools, e.g., AtSubP for Arabidopsis[57] to enhance the accuracy of prediction. Consensus outcome revealed that 98.71% OsMYB and all AtMYB proteins were found to be nuclear localized and confirmed by the presence of nuclear localization signal (NLS). The remaining two members of MYB proteins in rice were predicted to be localized in mitochondria and plasma membrane. A Complete list of functional assignment of MYB genes is given in Additional file2: Table S2.
Gene structure and intron distribution
To understand the structural components of MYB genes, their exon and intron organization was analyzed. We observed that 17 (10.96%) OsMYB and 9 (4.56%) AtMYB genes were intronless (Figure2), which is in conformity with the previous analysis[58]. To identify conserved intronless MYB genes, blastall (BLASTP) was performed between protein sequence of all the predicted intronless genes of rice and Arabidopsis, and vice versa. Expected cut-off value of 1e-6 or less was used to identify the conserved intronless genes. We found that 13 (76.47%) and 7 (77.77%) intronless OsMYB and AtMYB genes, respectively, were orthologs. Other intronless MYB genes that fulfilled the matching criteria, expected cut-off value of 1e-10 or less were referred to as paralogs. We observed that 4 (23.52%) and 2 (22.22%) intronless OsMYB and AtMYB genes, respectively, were paralogs (Additional file3: Table S3). This analysis showed that intronless genes of rice and Arabidopsis are highly conserved, and may be involved in similar regulatory functions in these plants[36, 58]. To explore the intron density in MYB genes with introns, we divided ORF into three zones, namely N-terminal, central and C-terminal zones. We observed that mid region had high density of introns, i.e., 43.99 and 50.63% in rice and Arabidopsis, respectively. The number of introns per ORF varied, with maximum of 12 and 15 introns in OsMYB4R1 and AT2G47210, respectively. Rice LOC_Os01g43180 and Arabidopsis AT3G10585 genes contain shortest introns with 37 and 43nt, respectively. Among all MYB genes, LOC_Os08g25799 of rice and AT1G35515 of Arabidopsis contained longest intron with an intron length of 5116 and 1621nt, respectively (Additional file4: Table S4). In order to gain insight into exon-intron architecture, the intron positions on MYB domains were investigated. In support with previous results[16, 59], we also noticed that a large number of rice (26.45%) and Arabidopsis (38.57%) R2R3-type domain containing proteins have a conserved splicing pattern with three exons and two introns. However, some R2R3-type MYB genes lack one intron either in R2 or R3 repeat in rice (23.22%) and Arabidopsis (25.88%) (Figure3). It has been proposed that the duplication of R2 in an early form of two repeat MYB proteins gave rise to the R1R2R3 MYB domains[17]. Hence, we also investigated the exon-intron structure of R1R2R3-type MYB proteins. We observed that 3R-MYB proteins contained conserved three exons-two introns pattern in R1 and R2 and one conserved intron in R3 repeat in Arabidopsis. Similarly, in rice, three out of five 3R-MYB genes have similar structure (Figure4; Additional file4: Table S4). These results indicate similar distribution of introns in MYB domain in both rice and Arabidopsis.
Figure 2
Chromosome-wise distribution of intronlessMYBgenes in rice and Arabidopsis.
Figure 3
Intron distribution within the MYB domains ofMYBgenes in rice and Arabidopsis. The graph shows dominantly two intron positions within the domain of MYB-related (a, c) and R2R3-MYB genes (b, d) in rice and Arabidopsis, respectively.
Figure 4
Conserved intron position within the MYB domain of R1R2R3-typeMYBgenes in rice and Arabidopsis. Vertical bar and arrow indicate conserved introns position. MSU Gene IDs in red letters represent genes with non-conserved intron position.
Chromosomal distribution, tandem repeats and duplication
The position of all 155 OsMYB and 197 AtMYB genes were mapped on chromosome pseudomolecules available at MSU (release 5) for rice and TAIR (release 8) for Arabidopsis (Figures5 and6). The distribution and density of the MYB genes on chromosomes were not uniform. Some chromosomes and chromosomal regions have high density of the MYB genes than other regions. Rice chromosome 1 and Arabidopsis chromosome 5 contained highest density of MYB genes, i.e. 21.93 and 28.93%, respectively. Conversely, chromosome 11 of rice and chromosome 2 of Arabidopsis contained lowest density of MYB genes, i.e. 2.58 and 12.69%, respectively. Distribution of MYB genes on chromosomes revealed that lower arm of chromosomes are rich in MYB genes, i.e. 65.16% in rice and 52.79% in Arabidopsis. Distribution pattern also revealed that chromosome 5 in rice, and chromosome 2 and 5 in Arabidopsis contained higher number of MYB genes with introns, i.e. 29.41 and 33.33%, respectively. Intronless MYB genes are absent in chromosome 4, 9, 10, 11 and 12 in rice, and chromosome 1 in Arabidopsis (Figure2). Distribution of MYB genes on chromosomal loci revealed that 11 (7.09%) in rice and 20 (10.15%) genes in Arabidopsis were found in tandem repeats suggesting local duplication (Table2). Chromosome 6 in rice and chromosome 1 in Arabidopsis contained higher number of tandem repeats, i.e. 7 genes and showed over-representation of MYB genes. Three direct tandem repeats were found on chromosome 6 (LOC_Os06g07640; LOC_Os06g07650; LOC_Os06g07660) in rice, and chromosome 1 (AT1G66370, AT1G66380; AT1G66390) as well as chromosome 5 (AT5G40330; AT5G40350; AT5G40360) in Arabidopsis. Four direct tandem repeats were also observed on chromosome 3 (AT3G10580, AT3G10585, AT3G10590 and AT3G10595) in Arabidopsis. Manual inspection unraveled 44 (28.38 %) and 69 (35.02%) homologous pairs of MYB genes in rice and Arabidopsis, respectively evolved due to segmental duplication. We also observed that two homologous pairs in Arabidopsis contained one MYB gene and other than that was not classified as MYB gene in TAIR (release 10) databases (Table3). About 44 (28.39%) OsMYB and 69 (35.02%) AtMYB genes showed homology with multiple genes including MYB genes from various locations on different chromosomes. It is widely accepted that redundant duplicated genes will be lost from the genome due to random mutation and loss of function, except when neo-or sub-functionalization occur[60, 61]. Rabinowicz et al. (1999) suggested that gene duplications in R2R3-type MYB family occurred during earlier period of evolution in land plants[62]. Recently, a range of duplicated pair of MYB genes in R2R3-type protein family has been identified in maize[63]. Among the tandem repeat pair (AT2G26950 and AT2G26960) in Arabidopsis, AtMYB104 (AT2G26950) is down-regulated by ABA, anoxia and cold stress, but up-regulated under drought, high temperature and salt, while AtMYB81 (AT2G26960) expression pattern was opposite to that of AtMYB104, i.e., AtMYB81 is up-regulated in response to ABA, anoxia and cold stress, but down regulated under drought, high temperature and salt stresses. Similar diversification was also observed in the duplicate pair (LOC_Os10g33810 and LOC_Os02g41510) in rice. OsMYB15 (LOC_Os10g33810) expressed in leaf, while LOC_Os02g41510 expressed in shoot and panicle tissue. These spatial and temporal differences among different MYB genes evolved by duplication indicate their functional diversification.
Figure 5
Distribution ofOsMYBgenes in rice genome. Arrow and star signs represent to tandem repeats and intronless genes, respectively.
Figure 6
Distribution ofAtMYBgenes in Arabidopsis genome. Arrow and star signs represent tandem repeats and intronless genes, respectively.
Table 2
Comparison of tandem repeatMYBgenes in rice and Arabidopsis based on cellular localization
Tandem repeat in rice
Blast 2 sequences alignment
TR_NO
TR_OsMYB_G1
TR_OsMYB_G2
OsMYB_G1
OsMYB_G2
Cellular localization G1
Cellular localization G2
Bit score
%identity
E-value
OsTR1
LOC_Os06g07640
LOC_Os06g07650
OsMYB
OsMYB
Nuclear
Nuclear
75.5
55%
2.00E-18
LOC_Os06g07650
LOC_Os06g07660
OsMYB
OsMYB
Nuclear
Nuclear
488
84%
2.00E-142
OsTR2
LOC_Os06g14700
LOC_Os06g14710
OsMYB
OsMYB
Nuclear
Nuclear
146
64%
2.00E-40
OsTR3
LOC_Os08g05510
LOC_Os08g05520
OsMYB
OsMYB103
Nuclear
Nuclear
19.2
25%
1.60E-01
OsTR4
LOC_Os09g12750
LOC_Os09g12770
OsMYB
OsMYB
Nuclear
Nuclear
55.8
40%
6.00E-13
OsTR5
LOC_Os12g07610
LOC_Os12g07640
OsMYB
OsMYB
Nuclear
Nuclear
105
45%
2.00E-27
Tandem repeat in Arabidopsis
Blast 2 sequences alignment
TR _NO
TR _AtMYB _G1
TR _AtMYB _G2
AtMYB _G1
AtMYB _G2
Cellular localization G 1
Cellular localization G2
Bit score
%identity
E-value
AtTR1
AT1G35515
AT1G35516
AtMYB8
AtMYB
Nuclear
Nuclear
No significant similarity found
AtTR2
AT1G66370
AT1G66380
AtMYB113
AtMYB114
Nuclear
Nuclear
212
80%
3.00E-60
AT1G66380
AT1G66390
AtMYB114
AtMYB90
Nuclear
Nuclear
220
87%
1.00E-62
AtTR3
AT1G69560
AT1G69580
AtMYB105
AtMYB
Nuclear
Nuclear
14.2
31%
5.3
AtTR4
AT2G26950
AT2G26960
AtMYB104
AtMYB81
Nuclear
Nuclear
358
50%
2.00E-103
AtTR5
AT3G10580
AT3G10585
AtMYB
AtMYB
Nuclear
Nuclear
172
64%
4.00E-48
AT3G10590
AT3G10595
AtMYB
AtMYB
Nuclear
Nuclear
56.6
27%
3.00E-13
AtTR6
AT3G12720
AT3G12730
AtMYB67
AtMYB
Nuclear
Nuclear
16.9
31%
4.40E-01
AtTR7
AT4G09450
AT4G09460
AtMYB
AtMYB6
Cytoplasmic
Nuclear
21.2
25%
1.40E-02
AtTR8
AT5G40330
AT5G40350
AtMYB23
AtMYB24
Nuclear
Nuclear
142
55%
5.00E-39
AT5G40350
AT5G40360
AtMYB24
AtMYB115
Nuclear
Nuclear
89.4
42%
8.00E-23
MYB coding sequence were aligned using BLAST 2 SEQUENCES to quantitate the sequence differences between the paired genes.
Table 3
Comparison of homologous pair ofMYBgenes of rice and Arabidopsis based on cellular localization
Duplications in rice
Blast 2 sequences alignment
HP_NO
OsMYB_HP_G1
OsMYB_HP_G2
OsMYB_G1
OsMYB_G2
Cellular localization G 1
Cellular localization G2
Bit score
%identity
E-value
OsHP1
LOC_Os01g06320
LOC_Os05g07010
OsMYB
OsMYB
Nuclear
Nuclear
160
81%
1.00E-38
OsHP2
LOC_Os01g18240
LOC_Os05g04820
OsMYB
OsMYB
Nuclear
Nuclear
1230
79%
0.00E+00
OsHP3
LOC_Os01g44370
LOC_Os05g50350
OsMYB
OsMYB
Nuclear
Nuclear
234
82%
8.00E-59
OsHP4
LOC_Os01g47370
LOC_Os05g49240
OsMYB
OsMYB
Nuclear
Nuclear
188
77%
3.00E-47
OsHP5
LOC_Os01g49160
LOC_Os05g48010
OsMYB
OsMYB
Nuclear
Nuclear
234
94%
2.00E-58
OsHP6
LOC_Os01g50720
LOC_Os05g46610
OsMYB
OsMYB
Nuclear
Nuclear
696
77%
0.00E+00
OsHP7
LOC_Os01g59660
LOC_Os05g41166
GAMYB
OsMYB
Nuclear
Nuclear
298
78%
1.00E-75
OsHP8
LOC_Os01g62410
LOC_Os05g38460
OsMYB3R-2
OsMYB
Nuclear
Nuclear
476
74%
8.00E-124
OsHP9
LOC_Os01g63460
LOC_Os05g37730
OsMYB
OsMYB
Nuclear
Nuclear
22
100%
6.80E-01
OsHP10
LOC_Os01g65370
LOC_Os05g35500
OsMYB3
OsMYB
Nuclear
Nuclear
636
88%
6.00E-168
OsHP11
LOC_Os02g09480
LOC_Os05g37730
OsMYB
OsMYB
Nuclear
Nuclear
32
87%
7.00E-04
OsHP12
LOC_Os02g14490
LOC_Os06g35140
OsMYB
OsMYB
Nuclear
Nuclear
548
73%
2.00E-143
OsHP13
LOC_Os02g40530
LOC_Os04g42950
OsMYB
OsMYB
Nuclear
Nuclear
284
94%
8.00E-72
OsHP14
LOC_Os02g41510
LOC_Os04g43680
OsMYB
OsMYB4
Nuclear
Nuclear
460
86%
3.00E-120
OsHP15
LOC_Os02g42870
LOC_Os04g45060
OsMYB
OsMYB
Nuclear
Nuclear
744
77%
0.00E+00
OsHP16
LOC_Os02g45080
LOC_Os04g47890
OsMYB
OsMYB
Nuclear
Nuclear
312
73%
6.00E-80
OsHP17
LOC_Os02g46780
LOC_Os04g50770
OsMYB
OsMYB
Nuclear
Nuclear
620
70%
2.00E-163
OsHP18
LOC_Os02g51799
LOC_Os06g11780
OsMYB
OsMYB93
Nuclear
Nuclear
442
80%
5.00E-115
OsHP19
LOC_Os02g54520
LOC_Os07g48870
OsMYB
OsMYB2
Nuclear
Nuclear
54
78%
1.00E-09
OsHP20
LOC_Os03g03760
LOC_Os10g39550
OsMYB
OsMYB
Nuclear
Nuclear
136
83%
3.00E-31
OsHP21
LOC_Os03g20090
LOC_Os07g48870
OsMYB112
OsMYB2
Nuclear
Nuclear
554
84%
2.00E-145
OsHP22
LOC_Os03g25550
LOC_Os07g44090
OsMYB
OsMYB
Nuclear
Nuclear
374
88%
1.00E-96
OsHP23
LOC_Os03g26130
LOC_Os07g43580
OsMYB
OsMYB30
Nuclear
Nuclear
384
82%
2.00E-99
OsHP24
LOC_Os05g04820
LOC_Os07g44090
OsMYB
OsMYB
Nuclear
Nuclear
422
83%
2.00E-109
OsHP25
LOC_Os05g10690
LOC_Os01g09640
OsMYB
OsMYB
Nuclear
Nuclear
232
83%
9.00E-58
OsHP26
LOC_Os05g49240
LOC_Os05g50340
OsMYB
OsMYB
Nuclear
Nuclear
104
72%
4.00E-24
OsHP27
LOC_Os06g43090
LOC_Os02g09480
OsMYB
OsMYB
Nuclear
Nuclear
616
71%
2.00E-162
OsHP28
LOC_Os06g45410
LOC_Os02g07770
OsMYB
OsMYB
Nuclear
Nuclear
180
90%
1.00E-43
OsHP29
LOC_Os06g45890
LOC_Os02g07170
OsMYB
OsMYB
Nuclear
Nuclear
98
81%
1.00E-21
OsHP30
LOC_Os07g02800
LOC_Os03g55590
OsMYB
OsMYB
Nuclear
Nuclear
162
91%
1.00E-38
OsHP31
LOC_Os08g25799
LOC_Os09g12750
OsMYB
OsMYB
Nuclear
Nuclear
682
80%
2.00E-180
OsHP32
LOC_Os08g25820
LOC_Os09g12770
OsMYB
OsMYB
Nuclear
Nuclear
616
73%
2.00E-162
OsHP33
LOC_Os08g33660
LOC_Os02g36890
OsMYB16
OsMYB
Nuclear
Nuclear
134
69%
4.00E-31
OsHP34
LOC_Os08g33660
LOC_Os04g38740
OsMYB16
OsMYB
Nuclear
Nuclear
136
80%
1.00E-31
OsHP35
LOC_Os08g33940
LOC_Os09g24800
OsMYB
OsMYB
Nuclear
Nuclear
838
76%
0.00E+00
OsHP36
LOC_Os08g43450
LOC_Os09g36250
OsMYB
OsMYB
Nuclear
Nuclear
76
71%
2.00E-15
OsHP37
LOC_Os08g43550
LOC_Os09g36730
OsMYB7
OsMYB
Nuclear
Nuclear
502
84%
1.00E-131
OsHP38
LOC_Os09g23200
LOC_Os08g33050
OsMYB
OsMYB
Nuclear
Nuclear
222
66%
2.00E-54
OsHP39
LOC_Os10g33810
LOC_Os02g41510
OsMYB15
OsMYB
Nuclear
Nuclear
374
81%
8.00E-97
OsHP40
LOC_Os10g33810
LOC_Os04g43680
OsMYB15
OsMYB4
Nuclear
Nuclear
384
82%
2.00E-99
OsHP41
LOC_Os10g39550
LOC_Os03g03760
OsMYB
OsMYB
Nuclear
Nuclear
384
81%
3.00E-99
OsHP42
LOC_Os11g03440
LOC_Os12g03150
OsMYB
OsMYB
Nuclear
Nuclear
1702
96%
0.00E+00
OsHP43
LOC_Os11g47460
LOC_Os12g37970
OsMYB
OsMYB
Nuclear
Nuclear
634
83%
2.00E-167
OsHP44
LOC_Os12g37690
LOC_Os11g45740
OsMYB78
OsMYB
Nuclear
Nuclear
226
88%
5.00E-56
Duplications inArabidopsis
Blast 2 sequences alignment
HP _NO
AtMYB _HP _G1
AtMYB _HP _G2
AtMYB _G1
ATMYB _G2
Cellular localization G 1
Cellular localization G2
Bit score
%identity
E-value
AtHP1
AT2G31180
AT1G06180
AtMYB14
AtMYB13
Nuclear
Nuclear
350
84%
2.00E-100
AtHP2
AT1G57560
AT1G09540
AtMYB50
AtMYB61
Nuclear
Nuclear
392
88%
7.00E-113
AtHP3
AT1G58220
AT1G09710
AtMYB1l
AtMYB
Nuclear
Nuclear
827
75%
0
AtHP4
AT1G26580
AT1G13880
AtMYB
No MYB
Nuclear
Nuclear
45.4
76%
4.00E-08
AtHP5
AT2G02820
AT1G14350
AtMYB88
AtMYB124
Nuclear
Nuclear
728
80%
0
AtHP6
AT3G12820
AT1G16490
AtMYB10
AtMYB58
Nuclear
Nuclear
293
79%
3.00E-83
AtHP7
AT1G17950
AT1G73410
AtMYB52
AtMYB54
Nuclear
Nuclear
381
88%
7.00E-110
AtHP8
AT1G79180
AT1G16490
AtMYB63
AtMYB58
Nuclear
Nuclear
346
84%
4.00E-99
AtHP9
AT5G61420
AT1G18570
AtMYB28
AtMYB51
Nuclear
Nuclear
99
86%
1.00E-24
AtHP10
AT1G74080
AT1G18570
AtMYB122
AtMYB51
Nuclear
Nuclear
305
81%
9.00E-87
AtHP11
AT5G07700
AT1G18570
AtMYB76
AtMYB51
Nuclear
Nuclear
185
71%
2.00E-50
AtHP12
AT5G60890
AT1G18570
AtMYB34
AtMYB51
Nuclear
Nuclear
206
77%
8.00E-57
AtHP13
AT1G74430
AT1G18710
AtMYB95
AtMYB47
Nuclear
Nuclear
351
82%
7.00E-101
AtHP14
AT1G74840
AT1G19000
AtMYB
AtMYB
Nuclear
Nuclear
233
85%
3.00E-65
AtHP15
AT1G35516
AT1G22640
AtMYB
AtMYB3
Nuclear
Nuclear
No significant similarity found
AtHP16
AT4G09460
AT1G22640
AtMYB6
AtMYB3
Nuclear
Nuclear
394
84%
1.00E-113
AtHP17
AT1G68320
AT1G25340
AtMYB62
AtMYB116
Nuclear
Nuclear
366
86%
3.00E-105
AtHP18
AT3G27810
AT1G25340
AtMYB21
AtMYB116
Nuclear
Nuclear
149
70%
7.00E-40
AtHP19
AT1G68670
AT1G25550
AtMYB
AtMYB
Nuclear
Nuclear
176
84%
8.00E-48
AtHP20
AT3G29020
AT1G26780
AtMYB110
AtMYB117
Nuclear
Nuclear
232
77%
8.00E-65
AtHP21
AT1G26780
AT1G69560
AtMYB117
AtMYB105
Nuclear
Nuclear
416
88%
3.00E-120
AtHP22
AT5G39700
AT1G69560
AtMYB89
AtMYB105
Nuclear
Nuclear
No significant similarity found
AtHP23
AT5G07690
AT1G74080
AtMYB29
AtMYB122
Nuclear
Nuclear
161
76%
2.00E-43
AtHP24
AT1G19510
AT1G75250
AtMYB
AtMYB
Nuclear
Nuclear
154
80%
4.00E-42
AtHP25
AT4G36570
AT1G75250
AtMYB
AtMYB
Nuclear
Nuclear
No significant similarity found
AtHP26
AT4G34990
AT2G16720
AtMYB32
AtMYB7
Nuclear
Nuclear
411
85%
1.00E-118
AtHP27
AT4G37260
AT2G23290
AtMYB73
AtMYB70
Nuclear
Nuclear
364
84%
1.00E-104
AtHP28
AT5G67300
AT2G23290
AtMYB44
AtMYB70
Nuclear
Nuclear
171
77%
3.00E-46
AtHP29
AT5G11050
AT2G25230
AtMYB64
AtMYB100
Nuclear
Nuclear
63.9
78%
1.00E-13
AtHP30
AT5G01200
AT2G38090
AtMYB
AtMYB
Nuclear
Nuclear
195
82%
1.00E-53
AtHP31
AT3G55730
AT2G39880
AtMYB109
AtMYB25
Nuclear
Nuclear
281
81%
2.00E-79
AtHP32
AT3G10760
AT2G40970
AtMYB
AtMYB
Nuclear
Nuclear
235
69%
8.00E-66
AtHP33
AT5G05090
AT2G40970
AtMYB
AtMYB
Nuclear
Nuclear
156
81%
5.00E-42
AtHP34
AT3G62610
AT2G47460
AtMYB11
AtMYB12
Nuclear
Nuclear
388
86%
9.00E-112
AtHP35
AT5G15310
AT3G01140
AtMYB16
AtMYB106
Nuclear
Nuclear
593
83%
2.00E-173
AtHP36
AT5G40350
AT3G01530
AtMYB24
AtMYB57
Nuclear
Nuclear
254
81%
1.00E-71
AtHP37
AT5G16600
AT3G02940
AtMYB43
AtMYB107
Nuclear
Nuclear
110
73%
7.00E-28
AtHP38
AT5G16770
AT3G02940
AtMYB9
AtMYB107
Nuclear
Nuclear
586
86%
3.00E-171
AtHP39
AT3G24120
AT3G04030
AtMYB3l
AtMYB
Nuclear
Nuclear
73%
86
1.00E-20
AtHP40
AT5G18240
AT3G04030
AtMYB
AtMYB
Nuclear
Nuclear
887
80%
0
AtHP41
AT5G49620
AT3G06490
AtMYB78
AtMYB108
Nuclear
Nuclear
396
83%
4.00E-114
AtHP42
AT5G02320
AT3G09370
AtMYB3R5
AtMYB3R3
Nuclear
Nuclear
610
85%
4.00E-178
AtHP43
AT5G04760
AT3G10580
AtMYB
AtMYB
Nuclear
Nuclear
105
71%
7.00E-27
AtHP44
AT5G05790
AT3G11280
AtMYB
AtMYB
Nuclear
Nuclear
455
80%
5.00E-132
AtHP45
AT5G06100
AT3G11440
AtMYB33
AtMYB65
Nuclear
Nuclear
710
78%
0
AtHP46
AT1G56160
AT3G12820
AtMYB72
AtMYB10
Nuclear
Nuclear
270
81%
2.00E-76
AtHP47
AT4G13480
AT3G24310
AtMYB79
AtMYB71
Nuclear
Nuclear
436
83%
2.00E-126
AtHP48
AT1G13300
AT3G25790
AtMYB
AtMYB
Nuclear
Nuclear
250
84%
4.00E-70
AtHP49
AT5G40360
AT3G27785
AtMYB115
AtMYB118
Nuclear
Nuclear
161
76%
3.00E-43
AtHP50
AT3G01530
At1g68320
AtMYB57
AtMYB62
Nuclear
Nuclear
239
81%
4.00E-67
AtHP51
AT5G14750
AT3G27920
AtMYB66
AtMYB0
Nuclear
Nuclear
320
80%
1.00E-91
AtHP52
AT5G40330
AT3G27920
AtMYB23
AtMYB0
Nuclear
Nuclear
379
85%
2.00E-109
AtHP53
AT5G59780
AT3G46130
AtMYB59
AtMYB48
Nuclear
Nuclear
237
86%
1.00E-66
AtHP54
AT5G59570
AT3G46640
AtMYB
AtMYB
Nuclear
Nuclear
313
85%
4.00E-89
AtHP55
AT5G62470
AT3G47600
AtMYB96
AtMYB94
Nuclear
Nuclear
527
88%
2.00E-153
AtHP56
AT5G65790
AT3G49690
AtMYB68
AtMYB84
Nuclear
Nuclear
494
87%
2.00E-143
AtHP57
AT4G37780
AT3G49690
AtMYB87
AtMYB84
Nuclear
Nuclear
246
79%
4.00E-69
AtHP58
AT4G22680
AT3G61250
AtMYB85
AtMYB17
Nuclear
Nuclear
147
70%
3.00E-39
AtHP59
AT1G01520
AT4G01280
AtMYB
AtMYB
Nuclear
Nuclear
272
83%
7.00E-77
AtHP60
AT4G21440
AT4G05100
AtMYB102
AtMYB74
Nuclear
Nuclear
385
89%
1.00E-110
AtHP61
AT5G52260
AT4G25560
AtMYB19
AtMYB18
Nuclear
Nuclear
407
79%
2.00E-117
AtHP62
AT5G55020
AT4G26930
AtMYB120
AtMYB97
Nuclear
Nuclear
283
82%
7.00E-80
AtHP63
AT2G20400
AT4G28610
AtMYB
No MYB
Nuclear
Nuclear
419
73%
7.00E-121
AtHP64
AT5G11510
AT4G32730
AtMYB3R4
AtMYB3R1
Nuclear
Nuclear
329
78%
3.00E-93
AtHP65
AT3G09600
AT5G02840
AtMYB
MYB (LCL1)
Nuclear
Nuclear
682
80%
0
AtHP66
AT3G10590
AT5G04760
AtMYB
AtMYB
Nuclear
Nuclear
51.8
76%
1.00E-10
AtHP67
AT5G23650
AT5G08520
AtMYB
AtMYB
Nuclear
Nuclear
139
72%
8.00E-37
AtHP68
AT5G65230
AT5G10280
AtMYB53
AtMYB92
Nuclear
Nuclear
534
84%
9.00E-156
AtHP69
AT3G50060
AT5G67300
AtMYB77
AtMYB44
Nuclear
Nuclear
265
82%
1.00E-74
The coding sequence were aligned using BLAST 2 SEQUENCES to quantitate the sequence differences between the gene pairs.
Cis-motifs in the MYB gene promoters
Discovery of regulatory cis-elements in the promoter regions is essential to understand the spatial and temporal expression pattern of MYB genes. Co-expressed genes may be regulated by a common set of transcription factors, and can be detected by the occurrence of specific cis-regulatory motifs in the promoter region. Hence, we analyzed the promoter regions of the drought up- and down-regulated MYB genes identified from our previous microarray data experiments[64]. Among the top five cis-motifs identified by this analysis, only CCA1 (TTWKTTWWTTTT) was the previously known cis-motif. Although, CCA1 cis-motif was reported as common feature of rice genome[65], we found CCA1 cis-motif only in genes that are down-regulated by drought stress (Figure7). The CCA1 motif was found in 94.74% of the drought down-regulated genes in rice. Furthermore, we investigated the group of R2R3-type MYB genes for the discovery of gene-specific new cis-regulatory element in both rice and Arabidopsis. Likewise, we discovered novel cis-motifs with no description in PLACE database, except for CCA1 motif in rice (Figure7). The CCA1 motif was found in 70.45% of the R2R3-type MYB genes in rice. The CCA1, a MYB-related TF, binds to CCA1 motif and regulate circadian clock controlled expression of genes in Arabidopsis[66]. To validate our prediction, we examined the diurnal or circadian clock controlled MYB expression using “Diurnal Version 2.0”[67]. About 47.74 and 90.86% MYB genes were found to be diurnal/circadian-regulated in rice and Arabidopsis, respectively (Additional file5: Table S5). Noticeably, we did not find any common motif between rice and Arabidopsis MYB promoter regions, indicating divergence in regulatory region of MYB genes between monocot and dicot species.
Figure 7
Conservedcis-motifs found in upstream promoter region ofMYBgenes in rice and Arabidopsis. a) Motifs from the promoter region of drought stress-regulated MYB genes in rice, b) Motifs from the group of R2R3-MYB genes in both rice and Arabidopsis.
Expression of MYB genes under abiotic stresses
To identify MYB genes with a potential role in abiotic stress response of plants, we analyzed the expression pattern of MYB genes in response to abiotic stresses. Expression of MYBs genes was examined from the availability of full-length cDNA (FL-cDNA) and Expressed Sequence Tag (EST) available at MSU and dbEST databases for rice and Arabidopsis, respectively[68]. It was found that 109 OsMYB genes in rice and 157 AtMYB genes in Arabidopsis had one or more representative ESTs. The LOC_Os10g41200 and AT5G47390 gene in rice and Arabidopsis had maximum number of ESTs, that is, 219 and 44, respectively. About 70% of rice MYB genes and 80% of Arabidopsis MYB genes appeared to be highly expressed as evident from the availability of ESTs for these genes (Additional file6: Table S6). Further, we assessed the expression levels of MYB genes under various abiotic stresses by PlantQTL-GE[69], GENEVESTIGATOR[70, 71] and our previous microarray data experiment (E-MEXP-2401) with rice cv. Nagina 22 and IR64 under normal and drought conditions (Additional file7: Table S7). In our previous microarray data experiments, we found that 142 (92.26%) MYB genes were expressed in seedlings of rice (Additional file8: Figure S1), of which 92 genes were differentially regulated under drought stress. In IR64, 30 genes were up-regulated (≥ 2.0 fold) and 30 genes were down-regulated (≤ 2.0 fold), while in Nagina 22, 22 genes were up-regulated (≥ 2.0 fold) and 19 genes were down-regulated (≤ 2.0 fold) under drought stress. The exploration of PlantQTL-GE for rice MYBs showed that 14 (9.03%) OsMYB genes were up-regulated under cold, drought and salt stress in rice, of which 10 are up-regulated under drought condition. These results suggest that large set of MYB genes may have a role in drought stress response in rice. Previous studies have shown that over-expression of MYB genes improved abiotic stress tolerance of rice and Arabidopsis[24, 72]. In addition to these, we have identified additional MYB genes that are regulated by drought and other stresses, and thus can be used as candidate genes for functional validation. The GENEVESTIGATOR analysis showed that 44.67, 41.12 and 56.85% AtMYB genes were down regulated and 47.21, 50.76 and 35.02% AtMYB genes were up regulated in cold, drought and salt stress, respectively (Additional file9: Figure S2a, b and c, Additional file10: Figure S3).
We analyzed expression patterns of 60 OsMYB and 21 AtMYB genes using QRT-PCR. These genes were selected based on phylogenetic analysis and one gene from each cluster was selected for expression analysis. Out of the 60 genes examined by QRT-PCR, 28 OsMYB genes were up-regulated (≥ 1.5 fold change) under drought stress in rice cv. Nagina 22 (Figure8). We also found that LOC_Os02g47744, LOC_Os12g41920 and LOC_Os06g19980 were highly up-regulated (≥ 4 fold change), indicating their potential role in drought stress. QRT-PCR analysis of 21 MYB genes in Arabidopsis revealed that 7 AtMYB genes were up-regulated (≥ 1.5 fold changes) and another 7 AtMYB genes were down-regulated (≤ 1.5 fold change) under drought stress (Figure8).
Figure 8
QRT-PCR expression analyses ofOsMYBandAtMYBgenes under drought stress in rice and Arabidopsis.
Tissue-specific expression
In rice, a tissue breakdown of EST evidence for MYB genes was analyzed using the Rice Gene Expression Anatomy Viewer, MSU database[73, 74]. In case of Arabidopsis, tissue-specific expressions of MYB genes were obtained from GENEVESTIGATOR tool[70, 71]. The expression patterns of MYB genes in different tissues are listed in Additional file11: Table S8. The results showed that large numbers of OsMYB genes (32.90%) were highly expressed in the panicle, leaf and shoots (Additional file12: Figure S4). EST frequency analysis suggested that OsMYB g enes, LOC_Os02g34630, LOC_Os08g05510, LOC_Os01g74590, LOC_Os02g09480, LOC_Os09g36730, OsMYB4, LOC_Os10g41200 and LOC_Os01g13740 are highly expressed in flower, anther, endosperm, pistil, shoot, panicle, immature seed and whole plant, respectively. In case of leaves, we observed that three MYB genes, i.e., OsMYB48, LOC_Os06g40710 and LOC_Os10g41200 showed highest levels of expression. In Arabidopsis, the following MYB genes expressed at a very high level: AtMYBCDC5 in callus and seed; AT1G19000 in seedling and stem; AT1G74840 in root and root tip; AT1G26580 in flower, AtMYB91 in shoot, and AtMYB44 in pedicel and leaves. In wheat, TaMYB1 showed high expression in root, sheath and leaf, while TaMYB2 expression was highest in root and leaf, but at low in sheath[75]. TaMYB1 and TaMYB2 showed a very high sequence similarity with AtMYB44 and OsMYB48, respectively. Our analysis also revealed that these two MYBs are highly expressed in leaf as in case of wheat. These analyses will be useful in selecting candidate genes for functional analysis of their role in a specific tissue.
Evolutionary relationship
To understand the evolutionary relationship among MYB family genes, phylogenetic trees were constructed using the multiple sequence alignment of MYB proteins[76]. The tree revealed that tandem repeat and homologous pairs were grouped together into single clade with very strong bootstrap support (Additional file13: Figure S5). These results further support gene duplication in rice and Arabidopsis during evolution which may allow functional diversification by adaptive protein structures[77]. It was also noticed that few “homologues pairs” (e.g. AT5G16600-AT3G02940 in Arabidopsis; LOC_Os12g07610- LOC_Os12g07640 in rice) and “tandem repeat pairs” (e.g. AT3G12720-AT3G12730 in Arabidopsis; LOC_Os06g14700-LOC_Os06g14710 in rice) were found in distinct clade, indicating that only few members had common ancestral origin that existed before the divergence of monocot and dicot. MYB proteins from rice and Arabidopsis with same number of MYB domains were grouped into a single clade. For instance, all the MYBs belonging to R1R2R3 family in both rice and Arabidopsis were clustered into single clade. Within the R2R3 clade, MYBs from rice and Arabidopsis were not found in distinct groups. These results suggest that significant expansion of R2R3-type MYB genes in plants occurred before the divergence of monocots and dicots, which in agreement with the previous studies[4, 62]. Finally, we observed that two CDC5-type and one 4-repeat MYB orthologs were clustered into single clade and might have been derived from an ancient paralog of widely distributed R2R3 MYB genes.
Conclusions
Our study provides genome-wide comparative analysis of MYB TF family gene organization, sequence diversity and expression pattern in rice and Arabidopsis. Structural analysis revealed that introns are highly conserved in the central region of the gene, and R2R3-type MYB proteins usually have two introns at conserved positions. Analysis of length and splicing of the intron/exon and their position in MYB domain suggested that introns were highly conserved within the same subfamily. Most of the MYB genes are present as duplicate genes in both rice and Arabidopsis. Phylogenetic analysis of rice and Arabidopsis MYB proteins showed that tandem repeat and homologous pair was grouped together into single clade. Consensus motif analysis of 1kb upstream region of MYB gene ORFs led to the identification of conserved and over-represented cis-motifs in both rice and Arabidopsis. The comparative analysis of MYB genes in rice and Arabidopsis elucidated chromosomal location, gene structure and phylogenetic relationships, and expression analysis led to the identification of abiotic stress responsive and tissue-specific expression pattern of the selected MYB genes, suggesting functional diversification. Our comprehensive analyses will help design experiments for functional validation of their precise role in plant development and stress responses.
Methods
Identification of MYB gene family in rice and Arabidopsis
To identify MYB transcription factor family genes, we searched and obtained genes annotated as MYB in MSU (release 5) for rice and TAIR (release 8) for Arabidopsis by using in-house PERL script along with careful manual inspection. The primary search disclosed 161 and 199 members annotated as “MYB” or “MYB-related genes” in MSU and TAIR database, respectively. We observed that some protein members lack MYB-DNA binding domain but still annotated as MYB protein family in MSU and TAIR database. We discarded these proteins based in the annotation in MSU (release 7) for rice and TAIR (release 10). Finally, we obtained 155 and 197 MYB genes in rice and Arabidopsis, respectively. The gene identifiers were assigned to each OsMYB and AtMYB genes to avoid confusion when multiple names are used for same gene. Uncharacterized MYB genes are denoted here by their locus id.
We discovered over represented cis-motif consensus pattern in 1 kb upstream sequence from translational initiation codon of MYB genes in both rice and Arabidopsis using the Multiple Expectation maximization for Motif Elicitation analysis tool[80] (MEME version 4.1.0,http://meme.sdsc.edu/meme/meme-intro.html). This program was used to search best 5 cis-motif consensus patterns of 8–12 bases width, with E-value < 0.01, only on the forward strand of the input sequences. Motifs graph were plotted according to their position within the region using WebLogo tool (http://weblogo.berkeley.edu/logo.cgi). Discovered motifs were analyzed using PLACE[81] (http://www.dna.affrc.go.jp/PLACE/). Diurnal and circadian controlled MYB expression was explored from “Diurnal Version 2.0” (Mockler lab;http://diurnal.mocklerlab.org/).
Phylogenetic analysis
To generate the phylogenetic trees of MYB transcription factor family genes, multiple sequence alignment of MYB protein sequence were performed using COBALT program[82] (http://www.ncbi.nlm.nih.gov/tools/cobalt/). COBALT program automatically utilize information about bona fide proteins (i.e. MYB domains in this case) to execute multiple sequence alignment and build phylogenetic tree. The dendrogram were constructed with the following parameters; method-fast minimum evolution, max sequence difference-0.85, distance- grishin (protein).
MYB localization, tandem repeat and duplication
To map the gene loci on rice and Arabidopsis chromosomes pseudomolecules were used in MapChart (version 2.2) program[83] for rice and chromosome map tool[84] for Arabidopsis available on The Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org/jsp/ChromosomeMap/tool.jsp). Tandem repeats were identified by manual visualization of rice and Arabidopsis physical map. Duplication or homologous pair genes were obtained by the segmental genome duplication segment (http://rice.plantbiology.msu.edu/segmental_dup/) and Arabidopsis Syntenic Pairs / Annotation Viewer (http://synteny.cnr.berkeley.edu/AtCNS/) in rice (distance = 500kb) and Arabidopsis, respectively. The tandem repeat and homologous pairs were aligned with the BLAST 2 SEQUENCE tool available on National Center on Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi/).
Gene structure analysis
To know more about intron / exon structure, MYB coding sequence (CDS) were aligned with their corresponding genomic sequences using spidey tool available on NCBI (http://www.ncbi.nlm.nih.gov/spidey/). To identify conserved intronless genes between rice and Arabidopsis, local protein blast (BLASTP) (http://www.molbiol.ox.ac.uk/analysis_tools/BLAST/BLAST_blastall.shtml) was performed for protein sequences of all predicted intronless genes in rice against all predicted intronless gene in Arabidopsis, and vice versa. Hits with 1e-6 or less were treated as conserved intronless genes and hits with 1e-10 or less were treated as paralogs. The cutoff of sequence identity was considered as ≥ 20% over the 70% average query coverage.
The plant materials used were drought tolerant rice (Oryza sativa L. subsp. Indica) cv. Nagina 22 and Arabidopsis thaliana ecotype Columbia. The seeds were surface sterilized. Rice seeds were placed on absorbent cotton, which was soaked overnight in water and kept in medium size plastic trays. Arabidopsis seeds were germinated on MS-agar medium containing 1% Sucrose and seven days old seedlings were transferred to soilrite for further growth. The rice and Arabidopsis seedlings were grown in a greenhouse under the photoperiod of 16/8 h light/dark cycle at 28°C ± 1 and 23°C ± 1, respectively.
Drought stress treatment
Drought was imposed to 3-weeks old rice seedlings[85] and 5-week-old Arabidopsis plants by withholding water till visible leaf rolling was observed. Control plants were irrigated with sufficient water. Plant water status was quantified by measuring relative water content of leaf. Control plants showed 96.89 and 97.49% RWC (relative water content), while stressed plants showed 64.86 and 65.2% RWC in rice and Arabidopsis, respectively.
Real-Time RT-PCR
Total RNA from rice and Arabidopsis were isolated by TRIzol Reagent (Ambion) and treated with DNase (QIAGEN, GmbH). The first strand cDNA of rice and Arabidopsis was synthesized using Superscript III Kit (Invitrogen) from 1 μg of total RNA according to manufacturer’s protocol. Reverse transcription reaction was carried out at 44°C for 60 min followed by 92°C for 10 min. Five ng of cDNA was used as template in a 20 μL RT reaction mixture. Sixty three pairs of rice and 51 pairs of Arabidopsis gene specific primers were used to study expression of MYB transcription factor. Gene specific primers were designed using IDT PrimerQuest (http://www.idtdna.com/scitools/applications/primerquest/default.aspx). Ubiquitin and actin primers were used as an internal control in rice and Arabidopsis, respectively. The primer combinations used here for real-time RT-PCR analysis specifically amplified only one desired band. The dissociation curve testing was carried out for each primer pair showing only one melting temperature. The RT-PCR reactions were carried out at 95°C for 5 min followed by 40 cycles of 95°C for 15s and 60°C for 30s each by the method described previously by Dai et al., 2007[24]. For qRT-PCR, QuantiFast SYBR Green PCR master mix (QIAGEN GmbH) was used according to manufacturer’s instruction. The threshold cycles (CT) of each test target were averaged for triplicate reactions, and the values were normalized according to the CT of the control products (Os-actin or Ubiquitin) in case of rice and Arabidopsis, respectively. MYB TFs expression data were normalized by subtracting the mean reference gene CT value from individual CT values of corresponding target genes (ΔCT). The fold change value was calculated using the expression, where ΔΔCT represents difference between the ΔCT condition of interest and ΔCT control. The primer sets used to study the MYB TFs expression profile are given in the Additional file14: Table S9.
Abbreviations
MSU:
Michigan State University
TAIR:
The Arabidopsis Information Resource
PERL:
Practical Extraction and Report Language
GO:
Gene Ontology
BLAST:
Basic Local Alignment Search Tool
MEME:
Multiple Expectation Maximization for Motif Elicitation
We thank Indian Council of Agricultural Research (ICAR) for supporting this work through the ICAR-sponsored Network Project on Transgenics in Crops (NPTC) and National Initiative on Climate Resilient Agriculture (NICRA). SKL gratefully acknowledge University Grants Commission (UGC) and Council of Scientific and Industrial Research (CSIR) for CSIR-UGC Junior and Senior Research Fellowship Grant. SS and RR acknowledge the senior research and research associate fellowship grant by Department of Biotechnology (DBT), Govt. of India, respectively. We thank Cathie Martin, John Innes Centre, Norwich Research Park, Colney, Norwich, UK, for her valuable suggestions on the data analysis and manuscript.
Authors’ Affiliations
(1)
National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute
(2)
National Bureau of Plant Genetic Resources, Indian Agricultural Research Institute Campus
(3)
Department of Biology, University of Massachusetts
(4)
Department of Biotechnology, Assam University
(5)
Division of Plant Physiology, Indian Agricultural Research Institute
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