MYB Transcription Factor Family in Pearl Millet: Genome-Wide Identification, Evolutionary Progression and Expression Analysis under Abiotic Stress and Phytohormone Treatments

Transcription factors (TFs) are the regulatory proteins that act as molecular switches in controlling stress-responsive gene expression. Among them, the MYB transcription factor family is one of the largest TF family in plants, playing a significant role in plant growth, development, phytohormone signaling and stress-responsive processes. Pearl millet (Pennisetum glaucum L.) is one of the most important C4 crop plants of the arid and semi-arid regions of Africa and Southeast Asia for sustaining food and fodder production. To explore the evolutionary mechanism and functional diversity of the MYB family in pearl millet, we conducted a comprehensive genome-wide survey and identified 279 MYB TFs (PgMYB) in pearl millet, distributed unevenly across seven chromosomes of pearl millet. A phylogenetic analysis of the identified PgMYBs classified them into 18 subgroups, and members of the same group showed a similar gene structure and conserved motif/s pattern. Further, duplication events were identified in pearl millet that indicated towards evolutionary progression and expansion of the MYB family. Transcriptome data and relative expression analysis by qRT-PCR identified differentially expressed candidate PgMYBs (PgMYB2, PgMYB9, PgMYB88 and PgMYB151) under dehydration, salinity, heat stress and phytohormone (ABA, SA and MeJA) treatment. Taken together, this study provides valuable information for a prospective functional characterization of the MYB family members of pearl millet and their application in the genetic improvement of crop plants.


Introduction
Environmental stresses and climate change pose a serious threat to global agricultural productivity. Plants face multiple stresses that challenge their growth and survival [1,2]. As a result, plants respond to these stress conditions, by activating various signaling pathways and by the synthesis of specialized metabolites. These responses are regulated by various transcription factor-encoding genes [3]. Transcription factors (TFs) bind to their cognate sites in the promoter region of the target gene and regulate the gene expression. Upon environmental stress on plants, TFs either induce or repress the expression of target genes [4,5]. Understanding the involvement of particular TF in various stress-signaling pathways will be helpful in developing stress-resistant and genetically modified (GM) crop plants with enhanced productivity. In recent times, several studies have identified and characterized various TF gene families, such as bZIP (basic leucine zipper), WRKY, MYB (v-myb avian myeloblastosis viral oncogene homolog), NAC, bHLH (basic helix-loop-helix), GRAS (Gibberellic-acid insensitive, repressor of GAI, and Scarecrow), THX (Trihelix) and ERF (Ethylene response factor), which are known to participate in multiple stresses and involved in phytohormonal signaling pathways [6,7].

Identification and Sequence Analysis of MYB TFs in Pearl Millet
Pearl millet protein and nucleotide sequences were downloaded from the genome database of pearl millet (http://cegsb.icrisat.org/ipmgsc/ (accessed on 23 May 2019)). In addition, publicly available transcriptome datasets on pearl millet were also used to identify the complete repertoire of MYB family members in pearl millet [28][29][30]. The MYB family protein sequences of rice were downloaded from Oryzabase (http://rice.plantbiology.msu. edu/ (accessed on 18 February 2020) [31] and GRASSIUS (Grass Regulatory Information Server) (accessed on 18 February 2020) [32]. Arabidopsis and foxtail millet MYB domain protein sequences were obtained from the plant genomics database, Phytozome [33]. The identification of MYB in pearl millet was initiated by constructing a hidden Markov model (HMM) profile of the reference sequences (rice, Arabidopsis and foxtail millet) through the hmmbuild program of the HMMER tool v3.2 [34], followed by a hmmsearch against pearl millet proteome. The initially identified sequences were then scanned against the HMM profiles obtained from the Pfam site [35]. All the non-redundant hits with an expected value cut-off of (0.01) were retained and the redundant sequences were removed. Further, the putative MYB genes were confirmed for the presence of MYB by using hmmscan (https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan (accessed on 4 March 2020), SMART (Simple Modular Architecture Research Tool) (http://smart.embl-heidelberg.de/ (accessed on 4 March 2020)) and CDD (Conserved Domains Database) (http://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 4 March 2020).
The prediction of the theoretical isoelectric point, protein molecular weight, instability index, GRAVY and aliphatic index for each protein was checked by using the Prot-Param tool (https://web.expasy.org/protparam/ (accessed on 2 April 2020). Further, the TMHMM-2.0 tool [36] was employed for the location of transmembrane helices in the PgMYB sequences. WoLF PSORT [37] was used for predicting subcellular localization sites in PgMYB sequences.

Chromosomal Localization, Gene Structure and Motif Analysis
The PgMYBs genes were mapped to the pearl millet chromosomes according to their physical positions (bp) with the help of MapInspect software v1.0 (http://mapinspect. software informer.com/ (accessed on 01st May 2020). The exon/intron structures of each MYB gene in the pearl millet were determined by the GSDS (Gene Structure Display Server) tool (accessed on 26 April 2020) [38]. In order to identify the conserved motifs in PgMYB sequences, the MEME suite (accessed on 18 April 2020) [39] was used with the following parameters: (20; maximum number of motifs, 6; minimum width and 50; maximum width). Further, the results obtained were visualized by TBtools [40].

Phylogenetic Analysis, Gene Duplication and Synteny Analysis
For the phylogenetic analysis, the MYB sequences of pearl millet, rice, Arabidopsis and foxtail millet were used. A multiple sequence alignment was done using MUSCLE with the default parameters [41]. The phylogenetic tree was constructed by the MEGA V7.0 (Molecular Evolutionary Genetics Analysis) program [42] using the neighbor-joining method (with 1000 bootstrap replicates), with the following parameters (Jones-Taylor-Thronton model; rates among sites: gamma distributed (G) and partial deletion of gaps). The MYB gene-duplication events were examined by Multiple Collinearity Scan toolkit (MCScanX) with the default parameters [43]. The synteny relationship between the PgMYB genes and the MYB genes from Oryza sativa, Arabidopsis thaliana and Setaria italica were visualized by AccuSyn software [44].

Cis-Acting Regulatory Elements Analysis (CREs)
The promoter sequences (2000 bp upstream region from translation start site) of all the identified PgMYB genes were extracted from the pearl millet genome database. The sequences were uploaded into the PlantCARE server (accessed on 23 April 2020) [45] Plants 2023, 12, 355 4 of 18 for identifying the cis-acting regulatory elements present in the promoter region of the identified PgMYBs.

In Silico Expression Analysis
For investigation of the expression profiles of the PgMYB genes, transcriptome datasets of pearl millet under drought and salinity stress (SRX3556461, SRX3556459, SRX6918725, SRX6918726) were downloaded from the SRA database of NCBI (https://www.ncbi.nlm. nih.gov/sra (23 February 2021)) [29,30]. First, the transcriptome datasets were aligned and mapped using Bowtie 2.0 tool, and the RSEM (RNA-Seq by Expectation-Maximization) software was used to quantify the RNA-seq reads [46][47][48]. Further, the differentially expressed genes were identified and MATRIX file was used to generate a heatmap using TBtools software.

Plant Growth and Stress Treatments
Pearl millet seeds PRLT 2/89-33 were obtained from the International Crops Research Institute for Semi-Arid Tropics (ICRISAT) through a material transfer agreement (MTA). The seeds were sown in nutrient soil (1:1 mix of black and red soil) and allowed to grow in a greenhouse with a 16:8 h light:dark cycle at 28 • C (±2).
Four-week-old pearl millet seedlings were subjected to a drought condition by withholding water for 8 days, whereas the control plants were watered on alternate days. On the 9th day, both the control and treated plants were rewatered for their recovery. After treatment, well-expanded leaf samples of both the control and treated plants were collected on 0, 5, 7, 9 and 11 days, respectively. For salt stress, four-week-old seedlings were transferred to Hoagland solution containing 250 mM NaCl and, for the control, 1 2 strength Hoagland solution was used. Leaf samples were collected from both the control and treated plants at time points of 0 h, 3 h (early) and 24 h (late). For heat stress, plants were transferred to a 45 • C chamber for 12 h and leaf samples were collected at 0, 3, 12 and 24 h time points [28,49].
For the hormonal stress experiments, four-week-old seedlings were treated with 100 µM Abscisic acid (ABA), 100 µM Salicylic acid (SA) and 100 µM methyl jasmonate (MeJA) [50]. Leaf samples from both the control and treated plants were collected at different time points of 0 h, 3 h (early) and 24 h (late). The tissue-specific expression of the PgMYB genes was also studied by harvesting the leaf, stem and root from four-weekold plants under normal conditions. The harvested samples were snap-frozen in liquid nitrogen and stored at −80 • C until further analysis. All the samples were collected in biological triplicates.

Quantitative qRT-PCR Analysis
Total RNA was isolated from the samples by using a Spectrum Plant Total RNA Kit (Sigma-Aldrich, Lt. Louis, MO, USA) according to manufacturer's instructions. The RNA quality was checked on a 1.2% agarose gel with 18% formaldehyde. The purity and yield were estimated by a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA samples with a 260/280 nm ratio from 2.0 to 2.1 were used for further analysis. RNA purification was done by treating with DNAse I (Sigma-Aldrich, MO, USA) as per the manufacturer's protocol.
A cDNA synthesis was carried out by a First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). An expression analysis of selected MYB genes was performed using the QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All the primers used in this study were designed by the PrimerQuest tool of IDT. The qRT-PCR reaction mixture included a total volume of 20 µL containing 2 µL (20 ng) of cDNA, 10 µL of SYBR premix buffer (Mesa Green qPCR Master Mix (Eurogentec)), 1.0 µL each of forward and reverse primers (5 µM) and 6 µL of nuclease-free water. The qRT-PCR run profile was as follows: 95 • C for 10 min, followed by 40 cycles of 95 • C for 15 s, and 60 • C for 1 min. EF1α (elongation factor 1 alpha) and GAPDH (glyceraldehyde 3-phosphatedehydrogenase) [51] genes were taken for data normalization.

Identification and Physicochemical Characteristics of MYB TF Family in Pearl Millet
The HMM tool was used for the screening of MYB TF family members in the pearl millet. A total of 306 probable MYB members were identified using hmmsearch. All the redundant sequences were removed using the Expasy tool and the MYB DNA-binding domain (PF00249) was confirmed in the non-redundant putative MYB sequences using the hmmscan, SMART and CDD tools. Finally, a total of 279 MYB TFs were identified in the pearl millet and designated as PgMYB1 to PgMYB279, based on their chromosomal location and coordinates. Interestingly, the identified MYB members in pearl millet were higher in number compared to that of Arabidopsis (155), O. Sativa (197) S. bicolor (134), Soyabean (244), S. italica (209) and Z. maize (157) [52][53][54][55][56].
The physical and chemical properties, such as protein length, molecular weight (MWs), isoelectric point (PI), and subcellular localization of the identified PgMYBs, were analyzed (Additional Table S1). The amino acid length of the PgMYBs varies from 73 (PgMYB190) to 2299 (PgMYB250) amino acids and the relative molecular weight ranges from 7.46 kDa (PgMYB190) to 255.88 kDa (PgMYB250). Moreover, the theoretical isoelectric point (pI) of the identified PgMYB proteins ranges from 4.37 to 11.34, and the grand average of hydropathy (GRAVY) value of all the PgMYB proteins was found to be relatively low (<0), which suggests their hydrophilic nature. The prediction of their subcellular localization showed that most of the PgMYB proteins (234 PgMYBs) were located in the nucleus, which suggests their role as a TF. However, some PgMYB were localized in the chloroplast (23), cytoplasm (11), mitochondria (7), peroxisome (1) and plasma membrane (1). Further, PgMYB66 and PgMYB152 were predicted to contain transmembrane (TM) helices by the TMHMM server. (Additional Figure S1). Membrane-bound TFs (MTFs) are known to play a significant role in biotic and abiotic stress responses. The MTFs get activated by proteolytic cleavage during environmental stresses [57,58].

Chromosomal Localization, Gene Structure and Motif Analysis of PgMYBs
The pearl millet genome comprises seven chromosomes with varying lengths: chromosome 3 being the longest (300.9 Mb), and chromosome 5 being the shortest (158.6 Mb). The identified 279 PgMYBs were mapped on all the chromosomes based on their positions and chromosomal coordinates in pearl millet genome using MapInspect software v1.0. The physical map positions demonstrated that the PgMYBs were unevenly distributed across all seven chromosomes ( Figure 1). The maximum number of PgMYB genes were located on chromosome 3 (49 PgMYBs), followed by chromosomes 1, 2 and 6 with 45 PgMYBs individually. Comparatively, fewer PgMYBs were located on chromosomes 5 (31) and 7 (21). The larger number of PgMYBs on chromosomes 3, 1, 2 and 6 indicate a possible hot spot region for MYB family member's distribution during the course of pearl millet evolution.
The diversification of gene structure is an important component of gene family evolution and also contributes to phylogenetic groupings. The exon/intron organization was analyzed to gain insights into the structural variation of PgMYB genes by the GSDS server. The number of introns in the PgMYB genes varied from 0 to 23; however, 21 PgMYBs did not contain an intron, while 56 PgMYBs had only one intron (Additional Figure S2). The maximum number of introns were observed in PgMYB264 (23), followed by PgMYB223 with 21 introns. The results indicate a high structural diversity of the PgMYB genes in pearl millet.
Motif 6, Motif 9, Motif 10, Motif 13 and Motif 15 were present in less than 10% of the PgMYB sequences. We also observed Motif 1, Motif 2, Motif 3, Motif 5 and Motif 7 were located towards the N-terminal, while Motif 4 and Motif 6 were found towards the Cterminal. Most of the PgMYB members from the same clade of the phylogenetic tree displayed similar motif compositions, which indicates their functional similarities within the same subgroup.  A total of 15 distinct conserved motifs were identified in the PgMYBs ( Figure 2). Motif 1, Motif 2, Motif 4 and Motif 5 were present in most of the PgMYBs, with conserved residues that are identical among all the R2R3-MYB domains, while Motif 3 was found to be a core conserved motif in the identified PgMYBs. Along with the typically conserved residue Trp (W), other residues such as Gly (G), Glu (E), Asp (D), Cys (C), Arg (R), Leu (L), Thr (T), Asn (N) and Lys (K) were also conserved. However, a few motifs such as Motif 6, Motif 9, Motif 10, Motif 13 and Motif 15 were present in less than 10% of the PgMYB sequences. We also observed Motif 1, Motif 2, Motif 3, Motif 5 and Motif 7 were located towards the N-terminal, while Motif 4 and Motif 6 were found towards the C-terminal. Most of the PgMYB members from the same clade of the phylogenetic tree displayed similar motif compositions, which indicates their functional similarities within the same subgroup.

Phylogenetic Relationship and Gene Duplication Events of PgMYBs
To explore the evolutionary relationships among the 279 PgMYBs, 126 OsMYBs and 141 AtMYBs, a phylogenetic tree was constructed by using MEGA7 software. We perceived from the phylogenetic tree ( Figure 3) that most of the PgMYBs were aligned in 18 (Group I-XVIII) subgroups with OsMYB and AtMYB. Among the 18 groups, Group XII found to be the largest, with 47 PgMYBs, followed by Group XIII (34 PgMYBs  Further, we performed a synteny analysis to identify the duplication events among the MYB family members of pearl millet, rice and foxtail millet using the MCScanX tool. We observed both orthologous and paralogous events across all seven chromosomes of the pearl millet. Moreover, the PgMYBs from chromosome 1, chromosome 3 and chromosome 6 of the pearl millet were predominantly involved in an orthologous relationship. A total of 198 paralogous pairs, including 22 tandem and 176 segmental events, were identified in the MYB family members of the pearl millet ( Figure 4). foxtail millet, which suggests their probable disappearance during the divergence of monocot and dicot. Similarly, nine collinear PgMYB pairs were found only with foxtail millet, but not with Arabidopsis and rice, which suggests these pairs may be formed during the millets' evolution; while six collinear PgMYB pairs were found with Arabidopsis and rice, but not with foxtail millet, which suggests their loss during the millets' evolution. The formation and loss of collinear pairs shows the evolutionary progression of the MYB family members in pearl millet [59,60].  A total of 204 PgMYBs were showing an orthologous relationship with the MYB family members of Arabidopsis, rice and foxtail millet. Foxtail millet showed the highest collinear relationship (70%) with 343 collinear genes, followed by Arabidopsis (48%) with 205 collinear genes and rice (28%) with 111 collinear genes (Additional Table S2). Interestingly, we found 9 collinear PgMYB pairs with rice and foxtail millet but not with Arabidopsis, indicating their probable formation after the monocot and dicot divergence, whereas, 14 collinear pairs of PgMYB occurred with Arabidopsis, but not with rice and foxtail millet, which suggests their probable disappearance during the divergence of monocot and dicot. Similarly, nine collinear PgMYB pairs were found only with foxtail millet, but not with Arabidopsis and rice, which suggests these pairs may be formed during the millets' evolution; while six collinear PgMYB pairs were found with Arabidopsis and rice, but not with foxtail millet, which suggests their loss during the millets' evolution. The formation and loss of collinear pairs shows the evolutionary progression of the MYB family members in pearl millet [59,60].

Cis-Acting Regulatory Elements Analysis in Promoter Regions of PgMYBs
The cis-acting regulatory elements' analysis of promoters helps in understanding gene regulation at the transcriptional level. Therefore, the putative cis-acting regulatory elements present in the 2000 bp upstream region of the identified PgMYBs were identified using the PlantCARE database. The results demonstrated the presence of versatile cisacting regulatory elements ( Figure 5

Cis-Acting Regulatory Elements Analysis in Promoter Regions of PgMYBs
The cis-acting regulatory elements' analysis of promoters helps in understanding gene regulation at the transcriptional level. Therefore, the putative cis-acting regulatory elements present in the 2000 bp upstream region of the identified PgMYBs were identified using the PlantCARE database. The results demonstrated the presence of versatile cis-acting regulatory elements ( Figure 5 sponses. The presence of versatile cis-acting regulatory elements in the presumptive promoter regions of the PgMYBs shows their functional diversity and their probable involvement in multiple biological plant processes [61,62].

In Silico Expression Analysis of PgMYBs under Dehydration and Salinity Stress
The RNA-seq data was analyzed to assess the expression level of the identified 279 PgMYB genes under dehydration and salinity stress conditions in pearl millet. With respect to this, the publicly available transcriptome data and the Sequence Read Archive (SRA-NCBI) files were accessed and explored for the differential expression profiling of PgMYBs. As shown in Additional Figure S3a,b, most of the PgMYBs showed differential transcripts level under both dehydration and salinity stress. Specifically, upon dehydration stress, PgMYB2, PgMYB9, PgMYB61, PgMYB8, PgMYB106, PgMYB110, PgMYB114, PgMYB169, PgMYB198, PgMYB250, PgMYB269 and PgMYB275 showed a higher expression level, whereas the expression level of PgMYB138, PgMYB141, PgMYB228, PgMYB229, PgMYB245 and PgMYB246 was decreased. Similarly, under salinity stress, the expression level of PgMYB2, PgMYB9, PgMYB35, PgMYB49, PgMYB101, PgMYB102, PgMYB134, PgMYB146, PgMYB151, PgMYB241, PgMYB249, PgMYB267 and PgMYB271 was induced, while the transcripts accumulation for PgMYB126, PgMYB199, PgMYB201, PgMYB235 and PgMYB240 was reduced as compared to the control samples.
We tried to correlate the expression pattern of PgMYBs in dehydration and salinity stress. Here, we found similar upregulated expression profiles of PgMYB2 and PgMYB9 under both dehydration and salinity stresses, which suggests their probable involvement in abiotic stress responses. We also observed a few PgMYBs of the same phylogenetic clade or group showed a similar expression pattern under both dehydration and salinity stress, which suggests the similar functional profiling of subfamily members [15].

Relative Expression Analysis of PgMYBs
Previous studies have shown the role of MYB family members in regulating environmental stress responses [15]. We have selected 15 PgMYB genes (Additional Table S3)

In Silico Expression Analysis of PgMYBs under Dehydration and Salinity Stress
The RNA-seq data was analyzed to assess the expression level of the identified 279 PgMYB genes under dehydration and salinity stress conditions in pearl millet. With respect to this, the publicly available transcriptome data and the Sequence Read Archive (SRA-NCBI) files were accessed and explored for the differential expression profiling of PgMYBs. As shown in Additional Figure S3a,b, most of the PgMYBs showed differential transcripts level under both dehydration and salinity stress. Specifically, upon dehydration stress, PgMYB2, PgMYB9, PgMYB61, PgMYB8, PgMYB106, PgMYB110, PgMYB114, PgMYB169, PgMYB198, PgMYB250, PgMYB269 and PgMYB275 showed a higher expression level, whereas the expression level of PgMYB138, PgMYB141, PgMYB228, PgMYB229, PgMYB245 and PgMYB246 was decreased. Similarly, under salinity stress, the expression level of PgMYB2, PgMYB9, PgMYB35, PgMYB49, PgMYB101, PgMYB102, PgMYB134, PgMYB146, PgMYB151, PgMYB241, PgMYB249, PgMYB267 and PgMYB271 was induced, while the transcripts accumulation for PgMYB126, PgMYB199, PgMYB201, PgMYB235 and PgMYB240 was reduced as compared to the control samples.
We tried to correlate the expression pattern of PgMYBs in dehydration and salinity stress. Here, we found similar upregulated expression profiles of PgMYB2 and PgMYB9 under both dehydration and salinity stresses, which suggests their probable involvement in abiotic stress responses. We also observed a few PgMYBs of the same phylogenetic clade or group showed a similar expression pattern under both dehydration and salinity stress, which suggests the similar functional profiling of subfamily members [15].

Relative Expression Analysis of PgMYBs
Previous studies have shown the role of MYB family members in regulating environmental stress responses [15]. We have selected 15 PgMYB genes (Additional Table S3) based on in silico expression profiling, phylogenetic analysis, sequence homology and synteny analysis to explore their expression profiling in different tissues and under abiotic stress conditions.

Tissue-Specific Expression Profiling of PgMYBs
Tissue-specific expression analysis helps in understanding the role of a particular TF/gene in the growth and development of the plant. Therefore, we evaluated the spatial expression pattern of selected 15 PgMYBs in the leaf, stem and root tissues of pearl millet. Figure S4, the transcript level of PgMYB9, PgMYB44, PgMYB61, PgMYB88, PgMYB132, PgMYB151 and PgMYB198 was predominant in the leaf tissues, whereas the expression level of PgMYB49, PgMYB187 and PgMYB263 was significantly higher in the root tissues compared to the leaf and stem tissues. We also observed higher expression of four PgMYBs, namely PgMYB2, PgMYB134, PgMYB176 and PgMYB240, in both the leaf and root tissues. PgMYB229 was only expressed in stem tissues, predominantly. Taken together, the tissue-specific expression profiling of PgMYBs provides a basis for better understanding of pearl millet growth and development [63].

Relative Expression Analysis of PgMYBs under Abiotic Stress
To investigate the role of PgMYB genes under different abiotic stresses in pearl millet, the expression profile of 15 selected PgMYB genes was generated at different time points under dehydration, salinity and heat stress.
The PgMYBs showed a differential expression pattern under the dehydration stress condition ( Figure 6). Among the 15 selected PgMYBs, 13 PgMYB genes showed an upregulation pattern upon dehydration stress, whereas only 2 PgMYBs showed a downregulated expression pattern. Moreover, PgMYB2, PgMYB49 and PgMYB88 were significantly induced, while the expression level of PgMYB151 was prominently downregulated. Interestingly, we observed a recovery in the transcripts level of PgMYB44, PgMYB134, PgMYB151, PgMYB187 and PgMYB198 on 11th day (after rewatering). This suggests their possible involvement in the dehydration stress responses of pearl millet. We also noticed that qRT-PCR analysis data of most of the PgMYBs corroborated with the transcriptome profile under dehydration stress.
Plants 2022, 11, x FOR PEER REVIEW 12 of 19 Figure 6. Relative expression analysis of PgMYB genes under dehydration stress at 0th day, 5th day, 7th day, 9th day and 11th day time points. The X-axis represents different time points and the Yaxis indicates relative expression level. Significant difference in mean indicated by * p < 0.05 as obtained by Student's t-test. Figure 6. Relative expression analysis of PgMYB genes under dehydration stress at 0th day, 5th day, 7th day, 9th day and 11th day time points. The X-axis represents different time points and the Y-axis indicates relative expression level. Significant difference in mean indicated by * p < 0.05 as obtained by Student's t-test.
In the course of salinity stress, an upregulation of 7 PgMYBs and downregulation of 7 PgMYB was evinced at early or later time points (Figure 7). The PgMYBs showed a differential expression pattern at different time points, such as PgMYB2 and PgMYB151 showed changes in expression level at an early time point (3 h). Similarly, the expression level of PgMYB61, PgMYB132, PgMYB240, PgMYB263 and PgMYB198 was affected at later time points (24 h). Interestingly, we observed a significant downregulation of PgMYB9, PgMYB49, PgMYB187 and PgMYB229 at both the early (3 h) and late (24 h) time points. Most of the PgMYBs showed similar expression pattern in both the transcriptome data and relative expression profiling.

Relative Expression Analysis of PgMYBs upon Phytohormone Treatments
Phytohormones are well known for activating the specific signal cascades in response to various environmental stresses [21]. MYB TFs are reported to be involved in phytohormonal signaling pathways under various stress conditions [64]. Thus, in the present study, the expression pattern of PgMYB genes was evaluated in response to ABA, MeJA and SA treatment (Figure 9). Notably, PgMYB2, PgMYB88 and PgMYB263 were upregulated under dehydration, salinity and heat stress. Similarly, the transcript accumulation of PgMYB9 and PgMYB151 was reduced under the abiotic stress treatments. The differential expression profile under multiple stress conditions indicates their crucial role in abiotic stress responses in pearl millet. Several studies have demonstrated the role of MYB TFs in multiple abiotic stresses [19,52,53].

Relative Expression Analysis of PgMYBs upon Phytohormone Treatments
Phytohormones are well known for activating the specific signal cascades in response to various environmental stresses [21]. MYB TFs are reported to be involved in phytohormonal signaling pathways under various stress conditions [64]. Thus, in the present study, the expression pattern of PgMYB genes was evaluated in response to ABA, MeJA and SA treatment (Figure 9). The ABA treatment led to the upregulation of PgMYB2 and PgMYB134 and do regulation of PgMYB9, PgMYB61 and PgMYB240 at 3 h and 24 h time points. We have observed the upregulated expression of eight PgMYBs at the early time point (3 h), w PgMYB151 showed downregulation at the early time point (3 h) upon ABA treatm Furthermore, it was shown that the MYB family members are involved in the ABA pendent signal pathway and activate antioxidant enzymes to improve plant stress t ance [16]. Similarly, under dehydration stress and ABA treatment, PgMYB2, PgMY PgMYB49, PgMYB88, PgMYB134, PgMYB187, PgMYB198 and PgMYB229 were indu meanwhile, PgMYB9 and PgMYB151 were downregulated. Therefore, these PgM might play an important role in providing drought tolerance through the ABA sign pathway in pearl millet, though further validation is necessary to confirm these find The significant role of jasmonates and salicylic acid in stress responses has been well documented [65]. Upon treatment with MeJA, the transcript accumulation of mo the PgMYBs was reduced; however, the expression level of PgMYB134, PgMYB The ABA treatment led to the upregulation of PgMYB2 and PgMYB134 and downregulation of PgMYB9, PgMYB61 and PgMYB240 at 3 h and 24 h time points. We have also observed the upregulated expression of eight PgMYBs at the early time point (3 h), while PgMYB151 showed downregulation at the early time point (3 h) upon ABA treatment. Furthermore, it was shown that the MYB family members are involved in the ABA-dependent signal pathway and activate antioxidant enzymes to improve plant stress tolerance [16]. Similarly, under dehydration stress and ABA treatment, PgMYB2, PgMYB44, PgMYB49, PgMYB88, PgMYB134, PgMYB187, PgMYB198 and PgMYB229 were induced; meanwhile, PgMYB9 and PgMYB151 were downregulated. Therefore, these PgMYBs might play an important role in providing drought tolerance through the ABA signaling pathway in pearl millet, though further validation is necessary to confirm these findings.
The significant role of jasmonates and salicylic acid in stress responses has been very well documented [65]. Upon treatment with MeJA, the transcript accumulation of most of the PgMYBs was reduced; however, the expression level of PgMYB134, PgMYB198, PgMYB229 and PgMYB240 was increased. Meanwhile, the SA treatment caused down-regulation of the majority of the PgMYB genes in the pearl millet. The transcript level of PgMYB229 was upregulated at both the 3 h and 24 h time points. The early upregulated response (at 3 h) of nine PgMYBs was also observed in response to an exogenous SA application. However, at the 24 h time point, the expression level of these nine PgMYBs was reduced. The differential expression profile of PgMYBs upon exogenous phytohormone treatments indicates a possible involvement of the PgMYBs in phytohormone stress signaling for implying the plant's tolerance [66].
Taken together, the differential expression analysis of PgMYB genes under abiotic stress and phytohormone treatment indicates their probable involvement in different signaling pathways linked to stress responses in pearl millet.

Conclusions
In the present study, a total number of 279 putative PgMYBs were identified and distributed unevenly across seven chromosomes of the pearl millet genome. The phylogenetic analysis, motif conservation and gene structure analysis provided insights into the structural diversity of PgMYBs. The tandem and segmental duplication events suggest the expansion of the MYB family in pearl millet. In addition, the transcriptome data and relative expression profiling of selected PgMYBs in different tissues and upon various stress treatments enabled us to understand their role in pearl millet development and stress responses. The majority of PgMYB genes also showed a differential expression profile under abiotic stress, as well as upon phytohormone treatments, which suggest their probable involvement in various phytohormone-signaling pathways for mediating the stress responses in pearl millet. Therefore, our work provides a comprehensive understanding of the MYB family members and their functional role in pearl millet. The information obtained will be useful for understanding the detailed evolutionary progression and functional characterization of candidate PgMYB TFs in plants. Further, the identified candidate PgMYBs could contribute significantly towards the development of engineered, multiple stress-tolerant crop plants to ensure better crop productivity.