Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis

  • Amit Katiyar1, 2,

    Affiliated with

    • Shuchi Smita1, 2,

      Affiliated with

      • Sangram Keshari Lenka1, 3,

        Affiliated with

        • Ravi Rajwanshi1, 4,

          Affiliated with

          • Viswanathan Chinnusamy5 and

            Affiliated with

            • Kailash Chander Bansal1, 2Email author

              Affiliated with

              BMC Genomics201213:544

              DOI: 10.1186/1471-2164-13-544

              Received: 14 February 2012

              Accepted: 1 October 2012

              Published: 10 October 2012

              Abstract

              Background

              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 (myeloblastosis) transcription factor family is present in all eukaryotes. "Oncogene" v MYB was the first MYB gene identified in avian myeloblastosis virus [2]. Three v MYB-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 [612]. 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 R1 MYB 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 OsMYB3R 2 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, 2528]. 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 file 1: 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 [3134]. 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 (Figure 1a, 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 (Figure 1a, 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 (Table 1; Additional file 1: 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) [3841]. 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 file 1: Table S1).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-544/MediaObjects/12864_2012_4562_Fig1_HTML.jpg
              Figure 1

              Chromosome-wise distribution of MYB transcription 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 of MYB transcription 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 [4349]. 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 file 2: 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 (Figure 2), 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 file 3: 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 file 4: 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%) (Figure 3). 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 (Figure 4; Additional file 4: Table S4). These results indicate similar distribution of introns in MYB domain in both rice and Arabidopsis.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-544/MediaObjects/12864_2012_4562_Fig2_HTML.jpg
              Figure 2

              Chromosome-wise distribution of intronless MYB genes in rice and Arabidopsis.

              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-544/MediaObjects/12864_2012_4562_Fig3_HTML.jpg
              Figure 3

              Intron distribution within the MYB domains of MYB genes 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.

              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-544/MediaObjects/12864_2012_4562_Fig4_HTML.jpg
              Figure 4

              Conserved intron position within the MYB domain of R1R2R3-type MYB genes 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 (Figures 5 and 6). 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 (Figure 2). 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 (Table 2). 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 (Table 3). 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.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-544/MediaObjects/12864_2012_4562_Fig5_HTML.jpg
              Figure 5

              Distribution of OsMYB genes in rice genome. Arrow and star signs represent to tandem repeats and intronless genes, respectively.

              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-544/MediaObjects/12864_2012_4562_Fig6_HTML.jpg
              Figure 6

              Distribution of AtMYB genes in Arabidopsis genome. Arrow and star signs represent tandem repeats and intronless genes, respectively.

              Table 2

              Comparison of tandem repeat MYB genes 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 of MYB genes 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 in Arabidopsis

                  

              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 (Figure 7). 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 (Figure 7). 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 file 5: 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.
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-544/MediaObjects/12864_2012_4562_Fig7_HTML.jpg
              Figure 7

              Conserved cis -motifs found in upstream promoter region of MYB genes 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 file 6: 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 file 7: Table S7). In our previous microarray data experiments, we found that 142 (92.26%) MYB genes were expressed in seedlings of rice (Additional file 8: 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 file 9: Figure S2a, b and c, Additional file 10: 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 (Figure 8). 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 (Figure 8).
              http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-13-544/MediaObjects/12864_2012_4562_Fig8_HTML.jpg
              Figure 8

              QRT-PCR expression analyses of OsMYB and AtMYB genes 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 file 11: Table S8. The results showed that large numbers of OsMYB genes (32.90%) were highly expressed in the panicle, leaf and shoots (Additional file 12: Figure S4). EST frequency analysis suggested that OsMYB genes, 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 file 13: 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.

              MYB annotation

              To identify number of domains present in MYB protein we executed domain search by Conserved Domains Database [78] ( http://​www.​ncbi.​nlm.​nih.​gov/​Structure/​cdd/​cdd.​shtml) and pfam database [79] ( http://​pfam.​sanger.​ac.​ukwith both local and global search strategy and expectation cut off (E value) 1.0 was set as the threshold for significance. Only significant domain found in rice and Arabidopsis MYB protein sequence were considered as a valid domain. To get more information about nature of the MYB protein, grand average of hydropathy (GRAVY), PI and the molecular weight were predicted by ProtParam tool available on Expert Protein Analysis System (ExPASy) proteomics server ( http://​www.​expasy.​ch/​tools/​protparam.​html). The subcellular localization of MYB proteins were predicted by Protein Localization Server (PLOC) ( http://​www.​genome.​jp/​SIT/​plocdir/​), Subcellular Localization Prediction of Eukaryotic Proteins (SubLoc V 1.0) ( http://​www.​bioinfo.​tsinghua.​edu.​cn/​SubLoc/​eu_​predict.​htm), SVM based server ESLpred ( http://​www.​imtech.​res.​in/​raghava/​eslpred/​submit.​html), and ProtComp 9.0 server ( http://​linux1.​softberry.​com/​berry.​phtml?​topic=​protcomppl&​group=​programs&​subgroup=​proloc). Further, species-specific localization prediction system was utilized for Arabidopsis (AtSubP, http://​bioinfo3.​noble.​org/​AtSubP/​) [57]. MYB protein function in term of their Gene Ontology (GO) was predicted by GO annotation search page available at MSU ( http://​rice.​plantbiology.​msu.​edu/​downloads_​gad.​shtml) and TAIR ( http://​www.​arabidopsis.​org/​tools/​bulk/​go/​index.​jsp) for rice and Arabidopsis, respectively. Localization consensus was predicted based on majority of result. The confidence level was acquired by assigning equal numeric value (e.g. one) to each general localization predictor and higher value to gene ontology (e.g. two) and species specific predictor (e.g. three).

              Identification of over-represented motifs

              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.

              Expression analysis

              Expression support for each gene model is explored through gene expression evidence search page ( http://​rice.​plantbiology.​msu.​edu/​locus_​expression_​evidence.​shtml) available at MSU for rice and GENEVESTIGATOR tool ( https://​www.​genevestigator.​com/​) for Arabidopsis. MYB genes for which no ESTs were found, blast (BLASTP and TBLASTN) ( http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi) search using NCBI databases was performed. Significant similarity of MYB genes with MYB genes of other plant species was searched. To measure the MYB expression level in abiotic stress plant QTLGE database was used ( http://​www.​scbit.​org/​qtl2gene/​new/​) for rice and GENEVESTIGATOR tool ( https://​www.​genevestigator.​com/​) for Arabidopsis. To identify tissue specific expression level of OsMYB genes in rice, highly expressed gene search ( http://​Rice.​plantbiology.​msu.​edu/​tissue.​expression.​shtml) available at MSU were used. For Arabidopsis, GENEVESTIGATOR tool ( https://​www.​genevestigator.​com/​gv/​user/​gvLogin.​jsp) was used.

              Plant materials and growth conditions

              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 file 14: 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

              EST: 

              Expressed Sequence Tag

              NCBI: 

              National Center for Biotechnology Information

              GEO: 

              Gene Expression Omnibus

              QRT-PCR: 

              Quantitative Reverse Transcription Polymerase Chain Reaction.

              Declarations

              Acknowledgements

              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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              This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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