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Article

Genome Wide Identification and Analysis of the R2R3-MYB Transcription Factor Gene Family in the Mangrove Avicennia marina

by
Seema Pradhan
,
P Sushree Shyamli
,
Sandhya Suranjika
and
Ajay Parida
*
Department of Plant and Microbial Biotechnology, Institute of Life Sciences, NALCO Square, Chandrasekharpur, Bhubaneswar-23, Odisha 751023, India
*
Author to whom correspondence should be addressed.
Agronomy 2021, 11(1), 123; https://doi.org/10.3390/agronomy11010123
Submission received: 25 November 2020 / Revised: 29 December 2020 / Accepted: 30 December 2020 / Published: 11 January 2021
(This article belongs to the Special Issue Addressing Abiotic Stress Responses in Plants: Emerging Biotechniques from Laboratory to the Field)
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Abstract

:
Drought and salinity stress have become the major factors for crop yield loss in recent years. Drastically changing climatic conditions will only add to the adverse effects of such abiotic stresses in the future. Hence, it is necessary to conduct extensive research to elucidate the molecular mechanisms that regulate plants’ response to abiotic stress. Halophytes are plants that can grow in conditions of high salinity and are naturally resistant to a number of abiotic stresses. Avicennia marina is one such halophyte, which grows in tropical regions of the world in areas of high salinity. In this study, we have analysed the role of R2R3-MYB transcription factor gene family in response abiotic stress, as a number of transcription factors have been reported to have a definite role in stress manifestation. We identified 185 R2R3 MYB genes at genome-wide level in A. marina and classified them based on the presence of conserved motifs in the protein sequences. Cis-regulatory elements (CREs) present in the promoter region of these genes were analysed to identify stress responsive elements. Comparative homology with genes from other plants provided an insight into the evolutionary changes in the A. marina R2R3 MYB genes. In silico expression analysis revealed 34 AmR2R3 MYB genes that were differentially regulated in the leaves and root tissue of A. marina subjected to drought and salinity stress. This study is the first report of the R2R3 MYB gene family in the A. marina genome and will help in selecting candidates for further functional characterisation.

1. Introduction

The phenomenon of global warming is responsible for drastic climate changes leading to an increase in the incidences of both biotic and abiotic stress conditions all over the world. In light of this worldwide problem, sustenance and enhancement of agricultural productivity in future is a global concern since such extremes in climatic conditions adversely affect the development, growth and productivity of major agricultural systems. Plants in general, evolve different mechanisms to cope with environmental stresses such as salinity, drought and low/high temperature. Among various strategies used for the development of abiotic stress tolerant plants, the identification of candidate genes and dissecting signaling mechanisms in abiotic stress responses have been widely attempted by researchers [1]. Recent studies have analysed the expression patterns of an array of genes regulating plant growth and development in order to counteract adverse environmental stress conditions [2,3]. Comprehension and analysis of the role of transcription factors is believed to be a more practical approach than focusing on individual gene components. Transcription factors (TFs) have been reported to play an important role in plant growth, development, and stress response through self-regulation [4,5,6,7]. They also regulate the expression of downstream target genes [5,6]. These abilities make them suitable candidates for genetic manipulation of complex stress tolerance traits. However, the difficulty often lies in the selection of a suitable gene for propagation. This problem is now alleviated by contemporary high throughput analyses which have now facilitated screening multiple gene families in silico to select the best candidate for further characterisation.
The MYB transcription factor family is one of the largest and functionally diverse TF families in plants and their role in plant development, abiotic stress response, secondary metabolism regulation, and hormone signal transduction have been well studied in Arabidopsis [4,8], Setaria italica [9], Vitis vinifera [10], Zea mays [11], Populus trichocarpa [12], Gossypium raimondii [13], and Oryza sativa [14]. Over 198 out of the 1600 transcription factors identified in Arabidopsis thaliana [15], belong to the MYB family [16]. The conserved MYB DNA-binding domain is essential for functioning of the MYB genes and is comprised of up to four imperfect repeats (R) of 50 to 53 amino acid sequences [17]. Depending on the number of repeats, MYB proteins have been grouped into four classes: 1R (R1/2, R3-MYB), 2R (R2R3-MYB), 3R (R1R2R3-MYB), and 4R (four R1/R2-like-MYB). In plants, R2R3-MYB proteins constitute the largest class of MYB transcription factors and have been further reassigned into 28 subgroups [18]. MYB gene in plants was first identified in Zea mays where a putative full set of R2R3-MYBs in the maize genome were identified comprising of a total of 157 typical R2R3-MYB encoding genes [11,19].
R2R3 MYB genes have been reported to act as regulators of abiotic stress response in economically relevant plants like cotton [20], wheat [4,21], apple [22], and rice [23], among many others. Moreover, studies have shown that ectopic expression of MYB genes resulted in enhanced tolerance to freezing, drought, and salt stress in non-halophytic plants [24,25]. In the model halophyte Thellungiella halophile, expressed sequence tags (ESTs) related to MYB proteins were reported [26] however, their roles in salt stress tolerance in halophytic plants have not been studied so far. The function of several other transcription regulators, such as the AP2 domain transcription factor and the homeobox-leucine rich protein has been extensively studied, with reference to stress tolerance in plants like Arabidopsis thaliana and Oryza sativa [27,28], whereas little is known about the function of MYB family transcription factors in the abiotic stress tolerance of halophytes [29].
Halophytes, owing to their capability to thrive under extremely saline conditions, are considered as one of the best source materials for identification of salinity tolerant genes. Avicennia marina, a salt-tolerant [30] mangrove species is one such halophyte and has been selected for the present study. Despite having information about a few candidate genes and their role in abiotic stress tolerance [31], information on molecular defense mechanism of halophytes against salt stress is scanty [32,33]. Therefore, the present study emphasizes the importance in assessing the diversity and potential role of MYB gene family in A. marina, a small evergreen tree that grows more than 10 m in height and is the most widely distributed mangrove species in the Indo-Western Pacific area belonging to Acanthaceae (Avicenniaceae) family. The plant is commonly known as the gray or white mangrove and is highly resistant to environmental stresses and can flourish under adverse environmental conditions of extreme tides, high salinity, high temperature, strong winds and anaerobic soil [34].
In this study, we have systematically investigated the R2R3 MYB gene family in Avicennia marina at the whole genome level, their classification into different groups, chromosomal distribution, presence of conserved motifs, phylogenetic relationship, and sequence homology with members of Arabidopsis thaliana and Oryza sativa. The study will provide us with an insight into the role of R2R3 MYB genes of A. marina in response to abiotic stresses like drought and salinity.

2. Materials and Methods

2.1. Identification of R2R3-MYB Transcription Factors in A. marina Genome

The CDS and protein sequences for the annotated proteins of A. marina were downloaded from DRYAD [35]. The MYB consensus sequence [36] and the HMM profile for MYB DNA-binding domain from Pfam (https://pfam.xfam.org/family/PF00249) were used to identify putative MYB transcription factors in the peptide sequences of annotated CDS of A. marina. Thereafter, sequence identifiers provided by [14] were used to download the peptide sequences for R2R3-MYB transcription factors of rice and Arabidopsis from Rice Genome Annotation project (http://rice.plantbiology.msu.edu/) and TAIR (https://www.arabidopsis.org/) respectively. These sequences were aligned and the alignment file was used to build the HMM profile of R2R3-MYB domain using HMMER v3.3 (http://hmmer.org/) and this profile was used to identify the R2R3-MYB domain containing protein sequences amongst the MYB TFs in A. marina genome. Chemical properties of the protein sequences were evaluated with the ProtParam tool of ExPASy (https://web.expasy.org/protparam/; [37]) and subcellular localisation was predicted with BUSCA (http://busca.biocomp.unibo.it/; [38])

2.2. Chromosomal Location and Nomenclature

The standalone version of BLAST software (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/) was used to map the corresponding CDS sequences of the MYB TFs of A. marina onto the genome using Blastn. The output file was used to locate the positions of all predicted MYB genes on the various scaffolds of A. marina genome. The MYB coding genes were named sequentially based on their position on the chromosomes/scaffolds, starting from AmMYB1. Only the R2R3-MYB TFs were taken for further analysis and were localised onto the chromosomes using MapChart [39].

2.3. Phylogenetic Analysis

The protein sequences of the R2R3-MYB TFs were used to generate the phylogenetic tree on MEGA X software ([40]; https://www.megasoftware.net/). The sequences were aligned with ClustalW and the tree was constructed with the neighbour-joining method. Bootstrap value was set to 100 and branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The evolutionary distances were computed using the Jones-Taylor-Thornton (JTT) matrix-based method.

2.4. Exon-Intron Structure and Conserved Motif Analysis

The CDS sequences of R2R3 MYB TFs were mapped onto the genome of A. marina using Blastn and the start and end positions of the mapped gene positions were used to prepare the bed file for extracting the fasta sequences of the genomic regions. The fasta formatted CDS sequences and genomic sequences were uploaded to Gene Structure Display Server (http://gsds.gao-lab.org/) to generate exon-intron structure. Conserved motifs in the protein sequences of R2R3 MYB TFs were identified with MEME tool of MEME-suite (http://meme-suite.org/tools/meme) with the parameters set as Zero to one occurrences per sequence; motifs to be found = 15 and motif width = 6–200.

2.5. Duplication, dN/dS and Homology/Synteny Analysis

Protein sequences of R2R3 MYB TFs in A. marina were aligned with clustalW and duplicate genes were identified using MCScanX [41]. The ratios of non-synonymous (dN) and synonymous (dS) substitutions were calculated using PAL2NAL v14 (http://www.bork.embl.de/pal2nal/#Download) and CODEML program of PAML package (http://abacus.gene.ucl.ac.uk/software/paml.html). The R2R3 AmMYB genes were compared with the CDS sequences of R2R3 MYB genes of Arabidopsis thaliana and Oryza sativa and their genomic positions were recorded. The Circos tool (circos-0.69-9; http://circos.ca/) was used to generate the figure using the genomic positions and chromosome lengths.

2.6. Conserved Motif Identification in Promoter Region

A stretch of 2000 bp upstream of the transcription start site of A. marina R2R3 MYB TFs were extracted from the genome using BEDtools [42]. The motifs in promoter sequences were identified with PlantCARE [43]; http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

2.7. In Silico Expression Analysis

The short reads of Avicennia marina control leaf (Accession SRR2029733), control root (Accession SRR2029734), salt stressed leaf (Accession SRR2029735), salt stressed root (Accession SRR2029736), drought stressed leaf (Accession SRR2029738) and drought stressed root (Accession SRR2029739) were obtained from the SRA database of NCBI (https://www.ncbi.nlm.nih.gov/sra). These were aligned onto the CDS sequences of A. marina R2R3 MYB TFs and their normalised expression values were calculated using the tools provided in Trinity package v2.11.0 (https://github.com/trinityrnaseq/trinityrnaseq/releases; [44]). Detailed instructions for differential gene expression analysis can be found at: https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-Differential-Expression [45]. The TMM method was used for preparing expression matrix and the FPKM values were normalised by calculating the log2 values. Z score values were calculated for the log2 normalized FPKM according to the formulae described by [46] values to further remove bias andthese expression values were used to generate the heat map on MeV v4.8.1 (http://mev.tm4.org/; [47]).

3. Results

3.1. R2R3-MYB Transcription Factor Family in Avicennia marina

A total of 284 MYB TF proteins were identified in the genome of A. marina based on the presence of the MYB consensus sequence as well as the HMM profile of MYB-DNA binding domain. They were named AmMYB1-AmMYB284 based on their positions on A. marina chromosome/scaffold. Of these, 185 members were identified as R2R3 MYB based on their similarity to the R2R3 MYB proteins of rice and Arabidopsis (File S1). Analysis of the chemical properties of A. marina R2R3 MYB proteins revealed that their length ranged from 100 bp to 1748 bp, molecular weight ranged from 11.46 kDa to 191.24 kDa, and their isoelectric point ranged from 4.65 to 10.28 (Table 1). A majority of them were predicted to be localised to nucleus (89%) while a few were localised to chloroplast (7%). Four of the AmR2R3 MYB proteins (AmMYB41, AmMYB79, AmMYB130 and AmMYB133) contained a signal peptide implying that these are secretory proteins that localise to extracellular space while one member (AmMYB251) was localised to the mitochondrion (Table 1).

3.2. Phylogenetic Analysis and Classification of R2R3 MYB Genes

The phylogenetic tree was prepared based on the alignment of the R2R3 MYB proteins of A. marina amongst themselves. The tree was corelated with the gene structure (intron-exon arrangement) and the motif composition of these proteins. Based on the type of motifs present in the protein sequences, the R2R3 AmMYB were classified into 8 groups (I–VIII) and sub-groups (Figure 1a). Majority of the members belonged to group III and contained motifs 1, 2, 3, and 5 (motif information is provided in Figure S1) while some of the members also contained the motif 7. The similarities were also reflected in the exon-intron structure of the genes. For example, the members of group I were either intronless or consisted of only one intron. Similar results were found for most of the groups with the exception of few members which deviated from the group morphology. Interestingly, AmMYB63 and AmMYB137 did not contain motifs similar to the other AmR2R3 MYB but both had the R2R3 MYB domain and a number of distinct motifs (Figure S2).

3.3. Chromosomal Location and Gene Duplication

The recent A. marina genome assembly reports a chromosome level assembly with 32 super scaffolds representing the chromosomes [35]. The R2R3 MYB genes of A. marina were found to be located exclusively in these 32 chromosomes (Supplementary Table S1, Figure 2). Chromosomes 8 and 11 of A. marina contained the loci for highest number of R2R3 MYB genes (10 each) while chromosome 7 contained only one. Most of the chromosomes contained 3-7 R2R3 MYB genes implying that the distribution of AmR2R3 MYB genes is fairly uniform in all chromosomes. Analysis of duplication events revealed seven tandem duplication events and six collinear gene pairs that have resulted in the course of evolution of AmR2R3 MYB family (Figure 2). These gene pairs were analysed for the ratio of rates of non-synonymous (dN) to synonymous (dS) substitutions (Table 2). Generally, a value of <1 suggests purifying selection resulting in functional constraint, a value of >1 implies positive selection resulting in accelerated evolution and a value =1 implies neutral selection. It was observed that the value of dN/dS for all the gene pairs was <1 with the value for AmMYB225/AmMYB226 almost nearing 1 (Table 2).

3.4. Promoter Motif Analysis

The promoter regions of genes contain conserved motifs which act as recognition and binding sites for various proteins. These interactions play an integral role in the regulation of gene expression and thereby affect the important biological processes in an organism. Therefore, conserved motifs in the promoter region of AmR2R3 MYB genes were analysed to evaluate their role in abiotic stress tolerance. The most frequently found motifs were core elements necessary for transcription such as CAAT-box (6126) and TATA-box (7599). A large number of cis-regulatory elements (CREs) related to stresss tolerance were identified (Figure 3, Table 3). Most abundant of these were the water and drought response elements MYB (872) and ABRE elements (668) followed by MYC (593), which is a drought response element, ARE (327) i.e., anaerobic-responsive elements and STRE (308) which is stress-responsive element. A number of other stress response CREs, such as ARE and ERE elements (oxidative stress responsive elements), W box (binding site for WRKY TFs), LTR (low temperature responsive elements), and DRE core (cold and dehydration responsive element), were also identified in the promoter regions of AmR2R3 MYB genes (Table 3).

3.5. Homology with R2R3 MYB Genes from Rice and Arabidopsis

Many transcription factor domains are known to be conserved across plant genera [48]. Probable reason for the high level of conservation could be the essential function of these genes in growth and development of plants. Thus, analysing the homology between different plants can provide an idea about the evolution of the gene family. Therefore, the AmR2R3 MYB proteins were compared with those from Arabidopsis and rice. It was interesting to note that none of the A. marina R2R3 MYB proteins grouped with the R2R3 MYB from rice and Arabidopsis (Figure 4a). The Am R2R3 MYB formed a distinctly separate clade while a number of members from Arabidopsis (with locus Ids starting with AT) and rice (with locus Ids starting with LOC_Os) showed similarity to each other. Thereafter, the AmR2R3 MYB genes (CDS sequences) were compared to the R2R3 MYB genes of rice (OsR2R3 MYB) and Arabidopsis (AtR2R3 MYB). We observed that 28 out of the 185 AmR2R3 MYB genes found homologous counterparts in AtR2R3 MYB genes and 27 found homologous counterparts in OsR2R3 MYB genes (Supplementary Table S2). Many of the AmR2R3 MYB genes had multiple homologs in Arabidopsis and rice (Figure 4b).

3.6. In Silico Gene Expression Analysis

In silico expression analysis was carried out using the RNA-Seq data for control and treated A. marina samples exposed to drought and salinity stress available at NCBI. The raw FPKM values were calculated using publicly available perl scripts and normalised by calculating their log2 values and z scores (Supplementary Table S3). The hierarchical clustering of values based on the pattern of expression clustered the AmR2R3 MYB genes into distinct blocks (Figure 5a). It was observed that drought stress had a more noticeable effect on the expression of AmR2R3 MYB genes as compared with salt stress. The differential expression was also more pronounced in the leaf tissue compared with root. AmMYB275, 61, 65, 237, 135, 190, 216, 1, 29, 209, 257, 112, 122, and 187 were upregulated in leaf and root tissues in response to drought stress and AmMYB267 was especially upregulated in root tissue (Figure 5b). On the other hand, AmMYB117, 158, 168, 183, 26, 80, and 140 were down regulated in leaf and root tissue in response to drought stress. AmMYB146, AmMYB164, and AmMYB51 were down regulated in leaf tissue while having no significant differential expression in root (Figure 5c). Salt stress had little effect on the expression of AmR2R3 MYB genes. Two of the members showed a noticeable change in expression in response to salt stress. AmMYB87 was upregulated in response to salt stress and downregulated in response to drought in both leaf and root tissue while the opposite was observed for AmMYB39 (Figure 5d).

4. Discussion

Plants response to environmental stress is one of the most extensively studied research area and assumes significance in developing strategies for addressing the growing concern of declining plant productivity in response to adverse climatic impacts. Abiotic stress conditions like drought and salinity are responsible for heavy yield losses in plants [49]. Therefore, understanding the molecular basis of abiotic stress response in plants has been a focal research area. Halophytes such as Avicennia marina typically grow in conditions of high salinity in various regions of the world [50]. Such plants can be a valuable resource for identifying genes which regulate their enhanced response to such stresses. The R2R3 MYB gene family is one of the most widespread family of transcription factors and transcription regulators with diverse functions, especially in abiotic stress response in plants [51]. In this study, we have undertaken the identification and analysis of the R2R3 MYB gene family in Avicennia marina which has led to a number of interesting observations.

4.1. The Evolution of A. marina R2R3 MYB Gene Family

There are numerous parameters to assess the evolutionary history of gene families and phylogenetic relationships form the foundation of such analyses. The AmR2R3 MYB were grouped into eight groups and sub-groups based on the presence of similar motifs in their peptide sequences. A key factor that contributes to the evolution of a gene family is the duplication events. A number of tandem duplications as well as collinear AmR2R3 MYB gene pairs were identified and classified into negatively, positively and neutrally selected genes based on their dN/dS ratios. The AmR2R3 MYB genes were under negative or purifying selection. This type of expansion of a gene family ensures functional conservation of the genes in the course of evolution and is commonly observed in transcription factor families [52,53].
On one hand, the expansion of R2R3 MYB gene family in A. marina was found to be under negative selection leading to the conservation of gene function. However, contrasting results were obtained on analysing the phylogenetic association of the members between A. marina, Arabidopsis, and O. sativa. There was some intermixing observed for R2R3 MYB proteins from Arabidopsis and rice, both of which are glycophytes, while the R2R3 MYB proteins of the halophyte A. marina formed a clade that was distinctly separate from the counterparts in both Arabidopsis and rice. This outcome is fortified by the fact that Avicennia is a monotypic genus which implies that it would have a markedly different genetic composition to model plants like rice and Arabidopsis [50]. This supports the idea that the R2R3 MYB family in A. marina has evolved to help the plant to combat abiotic stress and is therefore crucial to its survival and growth in conditions of high salinity. However, a one on one BLASTn based comparison between the CDS sequences of R2R3 MYB from A. marina with those from Arabidopsis thaliana and Oryza sativa revealed 28 and 27 members of AmR2R3 MYB family to have similarities with AtR2R3 MYB and OsR2R3 MYB respectively. This suggests that although there may be similarities between the R2R3 MYB genes of A. marina with the other two plants at the nucleotide level, the results may not be reflected in phylogenetic tree generated using the corresponding proteins. This may be due to the fact that the algorithms for phylogenetic analysis consider only the best alignment scenarios when comparing gene sequences from multiple genera simultaneously. Therefore, AmR2R3 MYB formed a separate clade, as the protein sequences for R2R3 MYB from A. thaliana and O. sativa were more similar to each other than to A. marina.

4.2. AmR2R3 MYB in the Regulation of Gene Expression During Drought and Salinity Stress

The conserved DNA motifs in promoter regions of genes allow various regulators to bind and control gene expression [54]. Analysis of these motifs in AmR2R3 MYB gene promoter regions led to identification of a number of motifs involved in abiotic stress response. The “MYB” motif was found to be most abundant and has been reported to be involved in drought response by acting as a favourable binding site for bZIP TFs [55,56]. Similarly, “MYC” CRE is associated with response to drought stress and acts as a binding site for bHLH TFs [57,58]. “ABRE” and “DRE” motifs have been well characterised and known to regulate gene expression in response to drought stress in ABA-dependent and ABA-independent gene expression, respectively [59]. In addition to these, numerous other stress responsive elements, such as “STRE”, “LTR”, and “W box”, were also found which indicate that AmR2R3 MYB gene family plays a crucial role in abiotic stress tolerance in A. marina.
In silico gene expression analysis revealed a number of members of the AmR2R3 MYB family which are differentially expressed during drought and salinity stress. Interestingly, the expression of these genes was affected more by drought stress than salt and was more evident in leaf tissue rather than root. Roots are the first tissues to be affected by salinity [60] and the R2R3 MYB genes are known to be differentially expressed in plants like Arabidopsis and rice [3] under conditions of high salinity. However, the absence of such marked difference in expression of AmR2R3 MYB could be attributed to the natural adaptation of A. marina to regions of high salinity which allows it to tolerate higher levels of salinity. Drought stress, on the other hand, had a more noticeable effect on the AmR2R3 MYB, especially in the leaf tissue. Only two members, AmMYB87 and AmMYB39, showed considerable differential expression in response to salt stress and could be promising candidates for functional characterisation.

5. Conclusions

This study enumerates the unique characteristics of the R2R3 MYB gene family in the monotypic halophyte Avicennia marina. It sheds light on the evolution and functional diversification of the AmR2R3 MYB family based on comparisons with model plants such as Arabidopsis and rice. We have also identified important candidates that may be crucial to abiotic stress response in plants. This study can act as a foundation for selecting candidates for further functional characterisation.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4395/11/1/123/s1, Figure S1: Logos and sequences of the motifs identified in the AmR2R3 MYB peptide sequences, Figure S2: Motifs identified in the peptide sequences of AmMYB63 and AmMYB137, File S1: File containing the fasta formatted CDS and peptide sequences of AmR2R3 MYB genes predicted in this study, Table S1: Nomenclature of scaffold to designate chromosomes, Table S2: Chromosomal positions of homologous R2R3 MYB CDS sequences in A. marina–A. thalina and A. marina–O. sativa, Table S3: Z score values of Log2 normalised FPKM values from in silico expression analysis of AmR2R3 MYB genes.

Author Contributions

Conceptualization, A.P.; data curation, P.S.S. and S.S.; formal analysis, S.P., P.S.S., and S.S.; funding acquisition, A.P.; methodology, S.P.; supervision, A.P.; validation, S.P., P.S.S., and S.S.; writing—original draft, S.P.; writing—review and editing, P.S.S. and S.S. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the institutional grant from Department of Biotechnology, Government of India.

Data Availability Statement

Publicly available datasets were analyzed in this study. This data can be found here: Whole genome assembly and annotation files for Avicennia marina can be found at [35]. RNA-seq data used in gene expression analysis was downloaded from NCBI SRA database: Accession numbers SRR2029733, SRR2029734, SRR2029735, SRR2029736, SRR2029738, SRR2029739.

Acknowledgments

P.S.S. acknowledges the fellowship from DST-INSPIRE, Ministry of Science and Technology, Government of India. The authors acknowledge Department of Biotechnology, Government of India.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 11 00123 g001 550
Figure 1. Phylogenetic classification and motif analysis of the AmR2R3 MYB genes. (a) The phylogenetic tree was constructed using the MEGA X software with Neighbour-Joining algorithm with 100 bootstraps. The exon-intron structures of genes (b) and conserved motifs in protein sequences (c) are depicted alongside the relative phylogenetic positions of AmR2R3 MYB genes.
Figure 1. Phylogenetic classification and motif analysis of the AmR2R3 MYB genes. (a) The phylogenetic tree was constructed using the MEGA X software with Neighbour-Joining algorithm with 100 bootstraps. The exon-intron structures of genes (b) and conserved motifs in protein sequences (c) are depicted alongside the relative phylogenetic positions of AmR2R3 MYB genes.
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Figure 2. Chromosomal location and duplication events of AmR2R3 MYB. The AmR2R3 MYB genes were distributed on 22 pseudo-chromosomes of A. marina based on their physical positions. The tandemly duplicated genes are indicated with blue arrows. The red dashed lines between pseudo-chromosomes 18 and 30 indicate the collinear genes.
Figure 2. Chromosomal location and duplication events of AmR2R3 MYB. The AmR2R3 MYB genes were distributed on 22 pseudo-chromosomes of A. marina based on their physical positions. The tandemly duplicated genes are indicated with blue arrows. The red dashed lines between pseudo-chromosomes 18 and 30 indicate the collinear genes.
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Figure 3. Cis-regulatory elements in the promoter region of AmR2R3 MYB genes. The figure represents the number of each type of stress responsive motifs identified in the promoter sequence of AmR2R3 MYB.
Figure 3. Cis-regulatory elements in the promoter region of AmR2R3 MYB genes. The figure represents the number of each type of stress responsive motifs identified in the promoter sequence of AmR2R3 MYB.
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Figure 4. Phylogenetic relationship with R2R3 MYB from Arabidopsis and Oryza. (a) The AmR2R3 MYB proteins were compared to those from Arabidopsis (Ids starting with AT) and rice (Ids starting with Os_LOC) (b) Comparison of AmR2R3 MYB CDS sequences (Am1-32) homologous with the R2R3 MYB from Arabidopsis (At1-5) and rice (Os1-12) based on their chromosomal positions.
Figure 4. Phylogenetic relationship with R2R3 MYB from Arabidopsis and Oryza. (a) The AmR2R3 MYB proteins were compared to those from Arabidopsis (Ids starting with AT) and rice (Ids starting with Os_LOC) (b) Comparison of AmR2R3 MYB CDS sequences (Am1-32) homologous with the R2R3 MYB from Arabidopsis (At1-5) and rice (Os1-12) based on their chromosomal positions.
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Figure 5. In silico differential expression of AmR2R3 MYB genes in response to abiotic stress. (a) Hierarchical clustering of the AmR2R3 MYB genes based on their expression in leaf and root tissue under salinity and drought stress. (b) AmR2R3 MYB genes upregulated in A. marina tissues in response to drought stress (c) AmR2R3 MYB genes downregulated in A. marina tissues in response to drought stress and (d) AmR2R3 MYB genes differentially regulated in response to salt stress in A. marina.
Figure 5. In silico differential expression of AmR2R3 MYB genes in response to abiotic stress. (a) Hierarchical clustering of the AmR2R3 MYB genes based on their expression in leaf and root tissue under salinity and drought stress. (b) AmR2R3 MYB genes upregulated in A. marina tissues in response to drought stress (c) AmR2R3 MYB genes downregulated in A. marina tissues in response to drought stress and (d) AmR2R3 MYB genes differentially regulated in response to salt stress in A. marina.
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Table
Table 1. The length distribution and chemical properties of Am R2R3 MYB proteins.
Table 1. The length distribution and chemical properties of Am R2R3 MYB proteins.
AccessionProtein Length (aa)Protein Molecular Weight (in kDa)Isoelectric Point (pI)Subcellular Localisation
AmMYB130734.686.20nucleus
AmMYB234938.135.21nucleus
AmMYB414616.729.68nucleus
AmMYB630434.178.90nucleus
AmMYB715617.6110.28chloroplast
AmMYB933537.075.93nucleus
AmMYB1030733.427.78nucleus
AmMYB1137342.586.08nucleus
AmMYB1228733.239.58nucleus
AmMYB1353359.687.63endomembrane system
AmMYB1410011.469.83nucleus
AmMYB1629531.798.65nucleus
AmMYB1744148.958.55nucleus
AmMYB1938641.646.17nucleus
AmMYB2021624.359.38chloroplast
AmMYB2127931.895.71nucleus
AmMYB2217320.095.85chloroplast
AmMYB2421423.679.54nucleus
AmMYB2547051.735.38nucleus
AmMYB2627531.237.69nucleus
AmMYB2937242.068.42chloroplast
AmMYB3027330.718.97nucleus
AmMYB3235338.867.55nucleus
AmMYB3332135.038.98nucleus
AmMYB3423525.839.30nucleus
AmMYB3627430.409.11nucleus
AmMYB3727531.305.64nucleus
AmMYB3827330.489.06nucleus
AmMYB3930133.268.78nucleus
AmMYB4029532.968.62nucleus
AmMYB4142046.476.13extracellular space
AmMYB4233037.257.88nucleus
AmMYB451159128.766.09nucleus
AmMYB4630734.109.86chloroplast
AmMYB4718921.439.52nucleus
AmMYB4933136.606.76nucleus
AmMYB5029131.098.55nucleus
AmMYB5137943.135.65nucleus
AmMYB5319522.899.31nucleus
AmMYB5425028.537.67nucleus
AmMYB5523627.156.19nucleus
AmMYB5731135.245.92nucleus
AmMYB5852959.047.61nucleus
AmMYB6029432.996.16nucleus
AmMYB6141145.866.19nucleus
AmMYB6227329.068.15nucleus
AmMYB6326128.935.22nucleus
AmMYB6535840.169.06nucleus
AmMYB6928030.619.05nucleus
AmMYB7132736.196.20nucleus
AmMYB7230432.816.45nucleus
AmMYB7328632.959.27nucleus
AmMYB7446551.356.38nucleus
AmMYB76959106.615.09nucleus
AmMYB7759763.176.22nucleus
AmMYB7928332.455.22extracellular space
AmMYB8020522.996.10nucleus
AmMYB8232035.696.27nucleus
AmMYB8331434.909.21nucleus
AmMYB8656861.704.88nucleus
AmMYB8718320.584.94nucleus
AmMYB8830834.328.86nucleus
AmMYB9118020.466.01nucleus
AmMYB9829533.907.06nucleus
AmMYB10029531.746.80nucleus
AmMYB10129032.416.72nucleus
AmMYB10233336.087.23chloroplast
AmMYB1051046115.145.10nucleus
AmMYB10657565.275.73nucleus
AmMYB10718120.946.36nucleus
AmMYB10835339.256.03nucleus
AmMYB11236741.385.93nucleus
AmMYB11627531.239.46nucleus
AmMYB11738543.539.31nucleus
AmMYB11832034.819.34nucleus
AmMYB11935339.438.04nucleus
AmMYB12020222.886.65nucleus
AmMYB12148252.817.55nucleus
AmMYB12233437.375.62nucleus
AmMYB12732836.676.70nucleus
AmMYB12843147.876.95nucleus
AmMYB12929932.849.75nucleus
AmMYB13012113.727.19extracellular space
AmMYB13125228.488.84nucleus
AmMYB13232936.005.09nucleus
AmMYB13332136.298.98extracellular space
AmMYB13438643.405.73nucleus
AmMYB13523225.946.24nucleus
AmMYB13725929.054.65chloroplast
AmMYB13855159.975.11nucleus
AmMYB13932136.136.21nucleus
AmMYB14027131.286.45nucleus
AmMYB14325128.416.13nucleus
AmMYB14622825.909.73nucleus
AmMYB14734338.227.92nucleus
AmMYB15130734.587.90nucleus
AmMYB15240343.715.25nucleus
AmMYB15334538.369.33chloroplast
AmMYB15537641.998.99nucleus
AmMYB15742447.746.14nucleus
AmMYB15826128.509.69nucleus
AmMYB15935239.466.45nucleus
AmMYB16125829.068.94nucleus
AmMYB16330233.139.38nucleus
AmMYB16442546.936.89nucleus
AmMYB16653958.424.87nucleus
AmMYB16733838.157.42nucleus
AmMYB16824228.699.55nucleus
AmMYB17224027.006.96nucleus
AmMYB17440545.338.93nucleus
AmMYB17535940.006.01nucleus
AmMYB17730834.258.47nucleus
AmMYB17833136.568.19nucleus
AmMYB18128230.999.48nucleus
AmMYB18253559.418.17nucleus
AmMYB18336141.329.15nucleus
AmMYB18430334.587.27nucleus
AmMYB18525328.509.15nucleus
AmMYB18741646.086.64nucleus
AmMYB18932335.538.94nucleus
AmMYB19032035.915.46nucleus
AmMYB19126630.009.12nucleus
AmMYB19246350.476.40nucleus
AmMYB194994110.875.20nucleus
AmMYB1961748191.246.20nucleus
AmMYB19833836.809.25nucleus
AmMYB19948254.225.75nucleus
AmMYB20139843.356.03nucleus
AmMYB20238642.747.23nucleus
AmMYB20431434.547.72nucleus
AmMYB20513515.6210.20nucleus
AmMYB20622825.459.08nucleus
AmMYB20831736.359.36nucleus
AmMYB20928831.918.84nucleus
AmMYB21134338.516.28nucleus
AmMYB21226930.885.79nucleus
AmMYB21328431.659.05nucleus
AmMYB21429431.428.96nucleus
AmMYB21628732.415.93nucleus
AmMYB21848354.266.23nucleus
AmMYB22034838.908.80endomembrane system
AmMYB22133637.148.12nucleus
AmMYB22231535.434.98nucleus
AmMYB22511312.9810.13nucleus
AmMYB22610812.3110.05nucleus
AmMYB22721124.327.01nucleus
AmMYB22833437.685.34nucleus
AmMYB22938541.706.28nucleus
AmMYB23026529.929.43chloroplast
AmMYB23125128.285.48nucleus
AmMYB23232535.587.76nucleus
AmMYB23335139.476.55nucleus
AmMYB23426931.229.30chloroplast
AmMYB23514816.5410.15nucleus
AmMYB23647852.718.68nucleus
AmMYB23730234.656.17nucleus
AmMYB24024427.545.62nucleus
AmMYB24125829.426.08nucleus
AmMYB24235440.167.17nucleus
AmMYB24430332.149.21nucleus
AmMYB24561267.247.47nucleus
AmMYB24732135.669.07nucleus
AmMYB24834238.215.63nucleus
AmMYB25041746.736.56nucleus
AmMYB25131334.578.99mitochondrion
AmMYB25325228.079.09nucleus
AmMYB25530834.728.42nucleus
AmMYB25639943.215.59nucleus
AmMYB25731234.837.53chloroplast
AmMYB25941546.148.70nucleus
AmMYB26021723.939.15nucleus
AmMYB26233437.348.15nucleus
AmMYB26338342.638.94nucleus
AmMYB26535139.185.54nucleus
AmMYB26633238.238.40nucleus
AmMYB26725128.397.31nucleus
AmMYB26851956.127.25chloroplast
AmMYB27137742.755.76nucleus
AmMYB27433236.776.87nucleus
AmMYB27533037.516.73chloroplast
AmMYB27737040.707.61nucleus
AmMYB27947252.657.11nucleus
AmMYB28032535.509.14nucleus
AmMYB28321924.827.87nucleus
AmMYB2841743191.105.83nucleus
Table
Table 2. The ratio of non-synonymous to synonymous substitution rates (dN/dS) in the AmR2R3 MYB genes.
Table 2. The ratio of non-synonymous to synonymous substitution rates (dN/dS) in the AmR2R3 MYB genes.
Tandem Duplication Events
Gene PairdNdSdN/dSType of selection
AmMYB222/AmMYB2250.34812.7080.1285Purifying
AmMYB225/AmMYB2260.11590.11960.9684Purifying/Neutral
AmMYB240/AmMYB2410.34530.91790.3761Purifying
AmMYB266/AmMYB2670.56211.91590.2934Purifying
AmMYB33/AmMYB340.45987.2480.0634Purifying
AmMYB53/AmMYB540.28370.66260.4281Purifying
AmMYB80/AmMYB820.619723.71590.0261Purifying
Collinear duplications
AmMYB151/AmMYB2550.10090.60720.1662Purifying
AmMYB152/AmMYB2560.18610.4410.4221Purifying
AmMYB155/AmMYB2570.29130.70770.4116Purifying
AmMYB157/AmMYB2590.1620.56260.2879Purifying
AmMYB158/AmMYB2600.27320.82140.3326Purifying
AmMYB159/AmMYB2620.15360.52620.292Purifying
Table
Table 3. Cis-regulatory motifs in promoter regions of AmR2R3 MYB genes.
Table 3. Cis-regulatory motifs in promoter regions of AmR2R3 MYB genes.
Name of the MotifMotif SequenceFunction
ABRETACGTG; ACGTG; CACGTA; CACGTG; CGCACGTGTC; CGTACGTGCA; AACCCGGcis-acting element involved in the abscisic acid responsiveness
AREAAACCAcis-acting regulatory element essential for the anaerobic induction
CGTCA-motifCGTCAcis-acting regulatory element involved in the MeJA-responsiveness
DRE coreGCCGACcis-acting regulatory element involved in cold and dehydration response
EREATTTTAAAcis-acting regulatory element involved in oxidative stress response
LTRCCGAAAcis-acting element involved in low-temperature responsiveness
MBSCAACTGMYB binding site involved in drought-inducibility
MYBTAACCA; CAACCA; CAACAG; CAACTG; TAACTG; TAACCAcis-acting element involved in drought responsiveness
MYCCATGTG; CATTTG; TCTCTTA; CAATTGcis-acting element involved in drought responsiveness
STREAGGGGcis-regulatory element able to mediate transcriptional induction by different forms of stress
W boxTTGACCcis-regulatory element that acts as a binding site for WRKY TFs
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Pradhan, S.; Shyamli, P.S.; Suranjika, S.; Parida, A. Genome Wide Identification and Analysis of the R2R3-MYB Transcription Factor Gene Family in the Mangrove Avicennia marina. Agronomy 2021, 11, 123. https://doi.org/10.3390/agronomy11010123

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Pradhan S, Shyamli PS, Suranjika S, Parida A. Genome Wide Identification and Analysis of the R2R3-MYB Transcription Factor Gene Family in the Mangrove Avicennia marina. Agronomy. 2021; 11(1):123. https://doi.org/10.3390/agronomy11010123

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Pradhan, Seema, P Sushree Shyamli, Sandhya Suranjika, and Ajay Parida. 2021. "Genome Wide Identification and Analysis of the R2R3-MYB Transcription Factor Gene Family in the Mangrove Avicennia marina" Agronomy 11, no. 1: 123. https://doi.org/10.3390/agronomy11010123

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