Genome-wide analysis of bHLH and bZIP Transcription factors and their temporal expression under abiotic stress conditions in Groundnut (Arachis hypogaea L.) Текст научной статьи по специальности «Биологические науки»

Journal of Stress Physiology & Biochemistry, Vol. 18, No. 1, 2022, pp. 47-75 ISSN 1997-0838 Original Text Copyright © 2021 by Suchithra, Shafia Hoor and Nagesh Babu

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Genome-wide analysis of bHLH and bZIP Transcription factors and their temporal expression under abiotic stress conditions in Groundnut (Arachis hypogaea L.)

Suchithra B, Shafia Hoor F and Nagesh Babu R.*

1 School of Sciences, Department of Chemistry & Biochemistry, Maharani Cluster University, Bengaluru - 560001, Karnataka, India

*E-Mail: nageshbabur@gmail. com

Groundnut (Arachis hypogaea L.), is an important subsistence oil yielding crop of the semi-arid tropics and often exposed to several environmental cues (high temperature, drought & heavy metal). Transcription factors can control the expression of many target genes through specific binding to the cis-acting elements in the promoters of the target genes. The basic leucine zipper (bZIP) and basic helix-loop-helix (bHLH) represents one of the largest as well as most diverse transcription factor (TFs) families. They are known to play role in both stress as well as in various plant developmental processes. In this study, a comprehensive phylogeny, chromosomal location, conserved motif identification and expression profiles under high temperature and drought stress. of bZIP and bhLH TF gene family was carried in groundnut. A total of 151 bZIP and 39 bHLH transcription factors have been identified from groundnut. Expression analysis during high temperature and heavy metal stress conditions. Gene expression studies revealed differential expressions of bZIP and bhLH TFs suggesting the possible role in various stress mitigation and can serve as a candidate genes for improving abiotic stress tolerance and can be helpful in enhancing the crop productivity under stress conditions.

Key words: Groundnut, bZIP, bHLH, Abiotic stress

Received August 16, 2021

Plants are frequently being exposed to abiotic stresses such as drought, high salinity, high osmolarity, nutrient deficiency etc. These environmental factors negatively affect the plants leading to reduced growth and yield. Plants have evolved several defence mechanisms start from the alteration of gene expression and cellular metabolism to changes in plant growth, development, and crop yield (Akula Ramakrishna et al., 2011). Following exposure to abiotic stress specific ion channels and kinase cascades are activated, reactive oxygen species (ROS), phytohormones like abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) accumulate, and a reprogramming of the genetic machinery results in adequate defense reactions and an increase in plant tolerance in order to minimize the biological damage caused by the stress (Ines Ben Rejeb et al., 2014). Under stress conditions, plants synthesize ABA in various organs and initiate defense mechanisms, such as the regulation of stomatal aperture and expression of defense related genes conferring resistance to environmental stresses. Expression of functional proteins is largely controlled by specific transcription factors (TFs). Recent studies demonstrated that DREB1/ CBF, DREB2, AREB/ABF, and NAC have important roles in response to abiotic stresses in rice (Kazuo Nakashima et al., 2009). TFs like MYB, AP2/ERF, NAC, bZIP, bHLH and WRKY families act as the early responders to environmental signals and trigger the expression of stress-induced genes that are important for plants to be tolerant to abiotic stress.

Groundnut is one of the important legume crops of tropical and semiarid tropical countries (annual production of ~ 46 million tons) where it provides a major source of edible oil and protein. Groundnut kernels contain 47-53% oil and 25-36% protein. The genus Arachis belongs to family Fabaceae, sub family Papilionaceae, Tribe Aeschynomeneae, Subtribe Stylosanthinae. The genus Arachis has more than 70 wild species, of which only Arachis hypogaea L is domesticated and commonly cultivated. The Arachis genus is composed mostly of diploid species (2n = 2x = 20). A. hypogaea is an allotetraploid (AABB-type genome; 2n = 4x = 40), derived from a hybridization

event between two diploid species and polyploidization. Chromosomes are of mostly similar size and divided into A and B sub-genomes. Cytogenetic, phylogeographic and molecular evidence indicate A. duranensis and A. ipaensis as the donors of the A and B sub-genomes, respectively. In plant genomes approximately 7% of the coding sequences are assigned to transcription factors (TFs) (Soren Lindemose et al., 2013), and many of these are immediate-early abiotic stress-responsive genes (Kilian et al., 2012). A TF can control the expression of many target genes through specific binding to the cis-acting elements in the promoters of the target genes.

The basic leucine zipper (bZIP) transcription factor family is one of the largest and most conserved families, named according to the conserved bZIP domain that is composed of 60-80 amino acids and contains two functional regions: a basic region and a leucine zipper. The basic region is conserved and responsible for nuclear localization and DNA binding. The leucine zipper motif that consists of several repeats of leucine or other hydrophobic amino acids is involved in recognition and dimerization of bZIPs (Wei Hu et al., 2016). Recent studies show that bZIP TFs play crucial roles in various aspects of biological processes, including organ differentiation, embryogenesis, seed maturation, flower and vascular development. Increasing evidences have also indicated that bZIP TFs take part in the regulation of plants' response to biotic and abiotic stress. The basic helix-loop-helix (bHLH) proteins are a large superfamily of eukaryotic transcription factors, and play a central role in a wide range of metabolic, physiological, and developmental processes (Sonnenfeld et al., 2005). Their bHLH domain contains approximately 60 amino acids, including a basic region and a HLH region (Murre et al., 1989). The basic region, which consists of approximately 17 amino acids and is located at the N-terminus of the domain, is a DNA-binding region that allows HLH proteins to bind to a consensus hexanucleotide E-box (CANNTG) (Mark Eben Massari et al., 2000). The HLH region is composed of two amphipathic helices consisting of hydrophobic residues linked by a divergent (both in length and primary sequence) loop, and functions as a dimerization domain

(Ferré D'Amaré et al., 1994). The HLH domain promotes protein-protein interactions and allows for the formation of homodimeric or heterodimeric complexes. Several previous studies showed that bHLH plays an important role in protecting plants from abiotic stresses. A novel bHLH transcription factor, PebHLH35, enhanced the drought tolerance of Populus euphratica (Dong et al., 2014). BrabHLH from Chinese cabbage participated in cold stress (Song et al., 2014), and the grapevine bHLH transcription factor confers tolerance to cold stress in Arabidopsis (Xu et al., 2014). Thus, bHLH TFs play an important role in various abiotic stresses. Agricultural production and quality are adversely affected by various abiotic stresses world-wide and this will be exacerbated by the deterioration of global climate. To feed a growing world population, it is very urgent to breed stresstolerant crops with higher yields and improved qualities against multiple environmental stresses. Our study provides detailed characterization of bZIP and bHLH TFs which can be used as candidate genes to develop stress tolerant varieties in groundnut.

MATERIALS AND METHODS

Plant materials and stress treatment

Seeds of groundnut (ICGV1119) were surface sterilized and grown under controlled conditions at 28 °C day/25 °C night with a 12-h light/12-h dark photo period. After 10 days of germination, heavy metal stress was imposed hydroponically for 3 days with 300|jM CdCl2 and for high temperature stress seedlings were exposed to high-temperature [42 °C for 2h (induction) followed by 48 °C for 6h]. After the stress treatment, control and stress exposed tissues were harvested immediately and stored at -80 °C for further analysis.

Identification, characterization and sub-cellular localization of bHLH and bZIP proteins

The bHLH and bZIP domain containing protein sequences of groundnut were retrieved from the Plant Transcription Factor Database ver. 2.0. and Arachis genome (Peanut Base) for the hidden Markov model (HMM) profile of the bHLH and bZIP domain downloaded from the Peanut database using HAMMER (ver. 3.0). All redundant sequences were removed and the collected data were further curated by examining the

presence of the conserved bHLH and bZIP domain with the help of Pfam (http://pfam.sanger.ac.uk/), SMART (http://smart.embl-heidelberg.de/) and InterProScan (http://www.ebi.ac.uk/Tools/InterProScan/) web server. The length, molecular weight and pI of each deduced polypeptide were calculated using ExpasyProtParam tool (http://web.expasy.org/protparam/). Further, WOLF PSORT (http://www.genscript.com/psort/wolf_psort.html) tool was used to predict the subcellular localizations.

Multiple Sequence Alignment and Phylogenetic Analysis

Amino acid sequences of bHLH and bZIP TFs belonging to groundnut were imported to BioEdit v7.2.5 (Hall 1999) and multiple sequence alignment was performed with bHLH and bZIP protein sequences using ClustalW with default parameters. The bHLH and bZIP sequences were imported into MEGA v6.06 (Tamura et al., 2013) to construct a phylogenetic tree.

Genome wide distribution, Gene structure and Conserved Motif analysis

The chromosomal location of bHLH and bZIP genes were obtained from Peanut base website (http://peanutbase.org/) and the map was generated using MapInspect

(http://mapinspect.software.informer.com/). Gene

Structure Display Server from Centre for Bioinformatics, Peking University, was used to display the intron exon junctions (http://gsds.cbi.pku.edu.cn/index.php). The genomic and mRNA sequences of bHLH and bZIP these were downloaded and used as query for generating its gene structure. A number of introns and exons were estimated based on this alignment and confirmed by the coordinates given in the sequences. The MEME Suite tool v4.9.1 (http://meme.nbcr.net/meme) was utilized for analysis of the conserved motifs.

Total RNA isolation and cDNA Synthesis and PCR amplification of bHLH and bZIP genes

Total RNA was isolated from control and stress treated shoot tissues using Trizol reagent and cDNA was synthesized by reverse transcription with 500ng of total RNA using PrimeScript RT Reagent Kit (Takara) according to the manufacturer's instructions. Gene specific primers for AdbHLH48, AibHLH22, AdbZIP12

and AibZIP15 are listed in Table 1. cDNA concentration was checked using Nanodrop 2000 (Thermo Scientific). PCR reactions were setup using Taq DNA Polymerase. Each PCR reaction included 2 Ml cDNA (1|jg), 1 unit Taq DNA Polymerase, 10mM dNTPs, 2.5 |l Taq Assay Buffer (10X), 0.5 |l gene specific forward primer (10 |mM), 0.5 |l reverse primer (10 |M), and made upto 25 |l with sterile water. The reactions conditions were 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 54 °C for 45s and 72 °C for 30s; 72 °C for 2 min.

Expression analysis of bHLH and bZIP genes

All RNA samples were quantified by Nanodrop 2000 (Thermo Scientific). cDNA was synthesized by reverse transcription with 500ng of total RNA using PrimeScript RT Reagent Kit (Takara) according to the manufacturer's instructions. Gene specific primers for AdbHLH48, AibHLH22, AdbZIP12 and AibZIP15 were designed using Primer3 software (Table 1). qRT- PCR reactions were performed using SYBR Green PCR Master mix (Takara) on CFX96 Real Time PCR (Biorad). Each PCR reaction (10 |l) included 2 |l cDNA (100ng), 5|1 1x SYBR Green Master mix, 0.5 |l gene specific forward primer (10 |M), 0.5 |l reverse primer (10 |M), and 2 |l sterile water. The bHLH and bZIP expression was normalized against actin as reference gene. The reactions conditions were 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s, 54 °C for 45s and 72 °C for 30s. All reactions were run with three technical and the data was analyzed using 2-AACT method.

RESULTS AND DISCUSSION

Identification of bHLH, Protein features, multiple sequence alignment and Phylogenetic analysis

To identify all the bHLH transcription factors, we retrieved all the predicted bHLH genes from Plant TFDB and Peanut Base (http://peanutbase.org/). The keyword, HMM profile and BLAST search predicted that the groundnut genome encodes about 151 bHLH proteins. A total of 151 bHLH genes were identified from both A. duranensis and A.ipaensis. They were named as AdbHLHl to AdbHLH79, and AibHLHl to AibHLH72 respectively. Basic information like molecular weight and pI of AdbHLH are depicted in Table 2. The average polypeptide length was 351.21 residues with the length

ranging from 181aa (AdbHLH 77) to 665 aa (AdbHLH 67). The pI values range from 4.69 to 9.76. The sub-cellular localization results revealed that majority of the proteins were localized to nucleus and 2/76 were predicted to be localized in chloroplast and 1 in cytoplasm. Basic information like molecular weight and pI of AibHLH are depicted in Table 3. The average polypeptide length was 362.90 residues with the length ranging from 168aa (AibHLH 70) to 663 aa (AibHLH 18). The pI values range from 4.61 to 9.77. The sub- cellular localization results revealed that majority of the proteins were localized to nucleus and 1/76 were predicted to be localized in chloroplast. The multiple alignment of AdbHLH and AibHLH, proteins indicated that they share a highly conserved 7-9 domains consisting of N-terminal DNA binding domain and a variable C-terminal transcriptional regulation domain (Fig 1 and 2).

To examine the structure and phylogenetic relationships of groundnut bHLH TFs identified in our study, a combined phylogenetic tree was constructed with the aligned bHLH domains from groundnut. The relationship among the 79 AdbHLH and 72 AibHLH TFs was investigated through constructing phylogenetic trees using Neighbour Joining method and the tree topology revealed several pairs of bHLH proteins with a high degree of homology in the terminal nodes of each subfamily Fig 3 and 4. Examination of the phylogenetic tree emphasis that the groundnut AdbHLH TFs can be classified into seven major groups: Group 1 (17) Group 2 (15), Group 3 (11), Group 4 (7), Group 5 (11), Group 6 (13), Group7 (5). AibHLH can be classified into nine major groups: Group 1(17) , Group 2 (11), Group 3 (18), Group 4(4), Group 5(3),Group 6(7), Group7(3), Group8(1), Group 9(8).

Identification of bZIP, Protein features, Multiple sequence alignment and Phylogenetic analysis

To identify all the bZIP transcription factors, we retrieved all the predicted bZIP genes from Plant TFDB and Peanut Base (http://peanutbase.org/). The keyword, HMM profile and BLAST search predicted that the groundnut genome encodes about 39 bZIP proteins. A total of 39 bZIP genes were identified from both A. Duranensis and A.ipaensis. They were named as AdbZIP1 to AdbZIP18 and AibZIP1 to AibZIP21

respectively. Basic information like molecular weight and pI of AdbZIP are depicted in Table 4. The average polypeptide length was 293.4 residues with the length ranging from 146aa (AdbZIP 15) to 495 aa (AdbZIP 1). The pI values range from 5.03 to 9.9. The sub- cellular localization results revealed that majority of the proteins were localized to nucleus and 1/76 were predicted to be localized in endoplasmic reticulum. Basic information like molecular weight and pI of AibHLH are depicted in Table 5. The average polypeptide length was 296.4 residues with the length ranging from 145aa (AibZIP 18) to 800aa (AibZIP 8). The pI values range from 4.86 to 9.36. The sub- cellular localization results revealed that majority of the proteins were localized to nucleus and 2/76 were predicted to be localized in endoplasmic reticulum. The multiple alignment of AdbZIP and AibZIP indicated that they share 5 to 6 highly conserved domains consisting of N-terminal DNA binding domain and a variable C-terminal transcriptional regulation domain (Fig 5 and 6).

To examine the structure and phylogenetic relationships of groundnut bZIP TFs identified in our study, a combined phylogenetic tree was constructed with the aligned bZIP domains from groundnut. The relationship among the 18AdbZIP and 21AibZIP TFs was investigated through constructing phylogenetic trees using Neighbour Joining method and the tree topology revealed several pairs of bZIP proteins with a high degree of homology in the terminal nodes of each subfamily Fig 7 and 8. Examination of the phylogenetic tree emphasis that the groundnut AdbZIP is classified into 8 groups: Groupl (4), Group2 (1), Group3 (3), Group4 (2), Group5 (1), Group6 (1), Group7 (1), Group8 (5). AibZIP TFs are classified into 8 groups: Group1 (4), Group2 (2), Group3 (1), Group4 (4), Group5 (2), Group6 (1), Group7 (1), Group8 (6).

Chromosomal distribution and gene structure of bHLH and bZIP members

The genome of groundnut comprises of 20 chromosomes (10 from duranensis and 10 from ipaensis) varying in their length in which shortest being chromosome 8 and longest is the chromosome 3 in A. duranensis while in A.ipaensis, shortest being chromosome 4 and longest being chromosome 9. In

silico mapping of bHLH and bZIP indicated an uneven distribution of the genes on all the chromosomes. (Fig 9, 10, 11 and 12). The exact position (in bp) of each bHLH and bZIP genes on groundnut chromosomes is given in Table 2, 3 and 4. The gene structures were investigated through genomic annotation to determine the structural diversity. All bHLH and bZIP genes harbored at least two exons except few being the shortest not having intron. In addition, a separate phylogenetic tree was generated from the complete protein sequences of all the bHLH and bZIP genes (Fig 13, 14, 15 and 16).

Identification of conserved motifs

The MEME (Multiple Expectation Maximization for Motif Elicitation) server was used for exploring motif distribution in 79 AdbHLH, 72 AibHLH, 18 AdbZIP, and 21 AibZIP (Fig 17, 18 and 19). Five different conserved motifs were identified, of which most of them had at least three highly conserved motifs. The motif sequence logos are depicted in the Table 6, 7, 8 and 9. Some of these motifs have been characterized in animals regarding specificity in the DNA-binding sequence recognition and dimerization activities responsible for the activation or repression of target genes or for binding to small molecules. Multiple sequence alignment and identification of conserved motifs using MEME tool indicates that most of the bHLH and bZIP proteins possessed 5 to 6 sub-domains in the N termini that conferred the DNA-binding activities. The motif composition of these TF sequences may provide clues for further functional analysis of these TFs. However, the biological significance of most of the putative motifs remains to be elucidated.

PCR amplification of bHLH and bZIP genes

Total RNA was isolated from stress treated tissues (Fig 20 and 21). The PCR reaction mixtures were run on 1.5% Agarose gel prepared using 1X TAE buffer along with 100 bp ladder. A single band of AibZIP15 (704bp) and AdbZIP12 (709bp) was observed on gel. There was no PCR product for AdbHLH48 and AibHLH22 under high temperature stress (Fig 22b). Under heavy metal stress, AdbHLH48 (450bp) and AibZIP15 (709bp) were amplified and single band was observed on gel (Fig 22a). The band pattern is comparatively similar to the

results obtained by quantitative PCR analysis.

Expression profiles bHLH genes during high temperature and high metal stress

The plant specific bHLH TFs play important role in regulation of diverse biological processes, including development, growth, cell division and responses to environmental stimuli. To cope with these stresses, plants have evolved a range of physiological and biochemical responses and a complex of signalling transduction pathways (Rosa M. Pérez-Clemente et al., 2013). bHLH proteins are plant-specific TFs that have been shown to function in abiotic stress responses (Marie Pireyre et al., 2015). To investigate the responses of bHLH genes to high metal and high temperature stress, we analysed the expression profiles

Table 1. List of primers for RT-qPCR

of one bHLH gene from each genome and expressed the results as fold changes with respect to the control. During heavy metal stress, bHLH belonging to Group 5 and Group 1 such as AdbHLH48 and AibHLH22 genes were down regulated with folds 4.49 and 1.56 respectively (Fig 23). During high temperature, bHLH genes were up-regulated and AdbHLH48 and AibHLH22 were found to be induced by 16.9 and 0.93 folds respectively (Fig 24). During heavy metal stress, bZIP genes belonging to Group 4 and Group 1 such as AdbZIP12 and AibZIP15 showed up regulation by 9.25 and 10.01 folds respectively. Out of 2 bZIP genes AdbZIP12 was down regulated by 12.9 fold and AibZIP15 was up-regulated by 10.68 fold (Fig 24). AibZIP showed increased expression under both drought and high temperature stress.

Gene Name Fonvard Primer Reverse Primer Length (bp) Tm CC)

AdbHLHJS ACGG AT CC TG ACC T GTTCC AACTGCTTG T ACTC G AGTCT ATG AG CTC CGGG ATGAG :s 63.0

AibHLH22 ACGG AT CC TCTCTTGC AG AGGGGAAAGA TACTCGAGCAAGTCTTGGGTTCACAGCA 2S 63.0

AdbZIPi: ACGG ATCC ACC AC C AGC AAATGTTC TCC TACTCGAGAGGCGCAAGAATTAGGAACA :s 63.0

AAZIP1Î ACGGATCCGGCGTC TTC AAGTGG AAC AT TACTCGAGAACTGGCTCC ATGAATG AC C :s 63.0

Protein Chromosome Chromosomal Location Deduced Polypeptide Subcellular

Number (bp) Localization

Start End Length pI MW

AdbHLH1 A01 11180803 11184851 450 5.9 56349.39 nucleus

AdbHLH2 A01 23818826 23821600 387 7.18 42810.87 Nucleus

AdbHLH3 A01 24107575 24110292 268 7.03 29696.69 Nucleus

AdbHLH4 A01 33747530 33755680 297 4.91 33725.91 Nucleus

AdbHLH5 A01 76355573 76358681 255 8.73 27535.72 Nucleus

AdbHLH6 A01 92963020 92966444 405 5.7 43265.18 Nucleus

AdbHLH7 A02 4990418 4992478 447 6.04 50189.76 Nucleus

AdbHLH8 A02 11490442 11491724 516 5.45 55840.51 Nucleus

AdbHLH9 A02 65874875 65881124 338 6.42 37356.38 Nucleus

AdbHLH10 A02 65875202 65880387 345 7.7 37894.06 Nucleus

AdbHLH11 A02 66678616 66681794 407 9.24 44298.24 Nucleus

AdbHLH12 A02 89000583 89007871 694 5.1 77631.15 Nucleus

AdbHLH13 A03 3170735 3173105 465 6.38 51017.44 Nucleus

AdbHLH14 A03 4396954 4399636 349 6.1 38798.89 Nucleus

AdbHLH15 A03 12136351 12138613 216 6.84 24041.54 Nucleus

AdbHLH16 A03 107170665 107172739 406 6.43 44670.22 Nucleus

AdbHLH17 A03 117229922 117230891 338 7.14 38190.66 Nucleus

AdbHLH18 A03 120673179 120676797 258 7 29055.83 Nucleus

AdbHLH19 A03 123213398 123215577 337 4.69 37951.67 Nucleus

AdbHLH20 A03 131184949 131187253 471 5.52 52619.98 Nucleus

AdbHLH21 A04 29987499 29997525 247 5.34 28285.34 Nucleus

AdbHLH22 A05 757006 758358 349 5.16 39414.5 Nucleus

AdbHLH23 A05 4471852 4474149 334 6.9 36880.17 Nucleus

AdbHLH24 A05 86030089 86031635 220 9.53 24900.43 Nucleus

AdbHLH25 A05 104078105 104082033 367 6.07 40422.45 Nucleus

Table 2. AdbHLH genes identified in Peanut, Chromosomal location, protein features and its localization prediction.

AdbHLH26 A05 105232729 105237613 539 5.16 58266.45 Nucleus

AdbHLH27 A06 11815778 11820126 357 5.69 39118.08 Nucleus

AdbHLH28 A06 109557765 109560470 419 5.65 45509.36 Nucleus

AdbHLH29 A06 110137735 110139757 277 5.85 31321.61 Nucleus

AdbHLH30 A07 9293133 9294428 256 8.76 28456.33 Nucleus

AdbHLH31 A07 55234108 55235523 417 6.44 52504.98 Nucleus

AdbHLH32 A07 57713413 57715466 397 7.08 43397.54 Nucleus

AdbHLH33 A07 68460163 68461453 321 4.83 36494.42 Nucleus

AdbHLH34 A07 70853348 70871615 350 4.88 39768.86 Nucleus

AdbHLH35 A07 75244942 75246494 313 9.18 34370.98 Nucleus

AdbHLH36 A08 15304566 15307528 343 4.86 39261.21 Nucleus

AdbHLH37 A08 17382662 17385316 393 5.85 42849.76 Nucleus

AdbHLH38 A08 23771934 23774264 328 5.57 37212.97 Nucleus

AdbHLH39 A08 24276717 24279598 401 5.68 44863.38 Nucleus

AdbHLH40 A08 29959576 29960976 236 6.01 26574.31 Nucleus

AdbHLH41 A08 43112521 43115242 400 8.88 44306.71 Nucleus

AdbHLH42 A09 1141726 1145405 357 6.77 40041.13 Nucleus

AdbHLH43 A09 4405214 4406928 366 5.31 41366.62 Nucleus

AdbHLH44 A09 9616546 9619558 580 6.45 63460.81 Nucleus

AdbHLH45 A09 116722167 116723907 291 7.76 31338.87 Chloroplast

AdbHLH46 A10 4762764 4765178 368 7.66 41676.67 Nucleus

AdbHLH47 A10 22120099 22122002 236 9.28 26260.28 Nucleus

AdbHLH48 A10 104379283 104380857 238 7.86 26566 Nucleus

AdbHLH49 A01 90749358 90752534 256 4.87 29223.97 Nucleus

AdbHLH50 A03 19592294 19594435 221 6.67 25350.15 Nucleus

AdbHLH51 A03 38984268 38987093 528 9.04 59651.37 Nucleus

AdbHLH52 A03 121854601 121856521 327 6.41 36595.78 Nucleus

AdbHLH53 A04 20876962 20880191 487 5.13 54204.89 Nucleus

AdbHLH54 A05 1099767 1101296 336 6.28 37941.85 Nucleus

AdbHLH55 A05 5650995 5652478 335 4.96 37474.88 Nucleus

AdbHLH56 A05 5676022 5677733 322 6.57 36193.44 Nucleus

AdbHLH57 A06 1704105 1705517 307 5.86 35063.41 Nucleus

AdbHLH58 A06 4585188 4586562 278 5.36 31533.59 Nucleus

AdbHLH59 A07 63884568 63887247 332 6.06 36849.39 Nucleus

AdbHLH60 A07 70852467 70853604 193 9.76 21638.45 Nucleus

AdbHLH61 A08 13571762 13573816 325 7.22 35758.02 Nucleus

AdbHLH62 A08 31926538 31927699 228 7.07 26186.85 Nucleus

AdbHLH63 A09 36737760 36739645 303 9.3 34193.65 Nucleus

AdbHLH64 A10 57409973 57414243 311 6.38 35382.97 Nucleus

AdbHLH65 A02 4340844 4342187 473 5.51 53353.28 Nucleus

AdbHLH66 A03 6936674 6945870 279 7.7 31084.6 Nucleus

AdbHLH67 A06 2305528 2307525 665 6.22 72541.1 Nucleus

AdbHLH68 A07 66084895 66087281 262 8.84 28851.52 Nucleus

AdbHLH69 A08 32827123 32829163 272 8.61 30093.18 Nucleus

AdbHLH70 A09 96870471 96872697 223 6.33 25042.36 Nucleus

AdbHLH71 A09 112385877 112387278 311 5.3 34671.55 Nucleus

AdbHLH72 A09 112389260 112390463 302 5.2 33731.5 Nucleus

AdbHLH73 A09 120376856 120378791 361 6.31 40610.33 Nucleus

AdbHLH74 A03 19352315 19353746 208 5.69 23649.96 Cytoplasm

AdbHLH75 A05 105520640 105522451 262 6.01 29705.07 Nucleus

AdbHLH76 A09 110546064 110547184 275 6.99 31371.18 Nucleus

AdbHLH77 A10 2767037 2768803 181 9.26 20830.97 Nucleus

AdbHLH78 A04 62752020 62756877 662 5.54 74386.17 chloroplast

AdbHLH79 A06 14352907 14359399 572 8.25 64663.36 Nucleus

Table 3: AibHLH genes identified in Peanut, Chromosomal location, protein features and its localization prediction.

Protein Chromosome Chromosomal Location Deduced Polypeptide Subcellular

Number Start End Length pI MW Localization

AibHLHl B01 636283 640342 520 5.9 56387.44 nucleus

AibHLH2 B01 30217938 30223799 354 4.75 39553.40 nucleus

AibHLH3 B01 107608827 107611906 255 8.73 27506.73 nucleus

AibHLH4 B01 136640565 136641988 255 4.87 29061.79 nucleus

AibHLH5 B02 77755308 77758153 413 9.27 44795.72 nucleus

AibHLH6 B02 94625362 94627028 189 6.5 21513.15 nucleus

AibHLH7 B03 108120840 108123374 271 8.77 29901.1 nucleus

AibHLH8 B03 121291052 121294003 258 7.04 28944.73 nucleus

AibHLH9 B03 123867236 123869445 347 4.7 39115.85 nucleus

AibHLH10 B03 132161677 132163387 508 5.45 56536.24 nucleus

AibHLH11 B04 28150372 28160597 219 6.86 25266.9 nucleus

AibHLH12 B05 746211 747857 350 5.06 39482.53 nucleus

AibHLH13 B05 4501469 4503100 304 7.2 33763.84 nucleus

AibHLH14 B05 98520553 98525231 536 5.15 57911.12 nucleus

AibHLH15 B05 109099855 109104474 367 6.07 40381.4 nucleus

AibHLH16 B06 3996822 4000058 209 9.69 23867 nucleus

AibHLH17 B06 4110261 4113865 362 5.85 39529.32 nucleus

AibHLH18 B06 18357835 18358780 663 6.22 72369.88 nucleus

AibHLH19 B06 134206069 134209265 420 5.65 45537.41 nucleus

AibHLH20 B07 9285262 9287119 256 8.6 28370.24 nucleus

AibHLH21 B07 33652692 33654351 357 4.71 40780.16 nucleus

AibHLH22 B07 62509004 62511083 395 7.07 43147.23 nucleus

AibHLH23 B07 123477966 123480961 311 4.88 35544.09 nucleus

AibHLH24 B07 125315732 125318601 380 5.74 41449.09 nucleus

AibHLH25 B08 990433 991857 405 6.35 52694.21 nucleus

AibHLH26 B08 1799227 1801832 330 5.44 37429.29 nucleus

AibHLH27 B08 2084005 2086453 402 5.68 44927.41 nucleus

AibHLH28 B08 7512500 7513857 180 8.8 20429.6 nucleus

AibHLH29 B08 89807832 89809286 329 4.91 37237.77 nucleus

AibHLH30 B08 128695301 128698133 369 8.19 41646.29 nucleus

AibHLH31 B09 276697 278046 344 4.61 39789.54 nucleus

AibHLH32 B09 1342273 1346258 242 9.2 27208.04 nucleus

AibHLH33 B09 139937689 139941958 327 8.82 34926.71 nucleus

AibHLH34 B09 6839044 6841764 354 6.97 39996.66 nucleus

AibHLH35 B10 131077378 131078628 238 8.46 26579.05 nucleus

AibHLH36 B01 636822 639231 520 5.9 56387.44 nucleus

AibHLH37 B01 773401 775837 451 5.37 51213.51 nucleus

AibHLH38 B01 29853579 29855816 365 7.18 40438.28 nucleus

AibHLH39 B01 134857606 134860895 407 5.7 43503.43 nucleus

AibHLH40 B01 137028861 137032298 416 9.75 46883.57 chloroplast

AibHLH41 B02 6253314 6255779 446 6.13 50118.64 nucleus

AibHLH42 B02 102553396 102557165 661 4.89 73703.12 nucleus

AibHLH43 B02 105759050 105761496 338 6.81 38166.63 nucleus

AibHLH44 B03 5875560 5878134 463 6.38 50834.15 nucleus

AibHLH45 B03 10090832 10092450 279 7.7 30985.46 nucleus

AibHLH46 B03 14817318 14819570 217 5.99 24186.71 nucleus

AibHLH47 B03 41339015 41343427 539 8.88 60474.21 nucleus

AibHLH48 B03 122440911 122442823 327 6.03 36538.68 nucleus

AibHLH49 B04 20530498 20533734 487 5.13 54204.89 nucleus

AibHLH50 B05 1081535 1082506 342 6.24 38412.28 nucleus

AibHLH51 B05 5834411 5835652 335 5 37457.8 nucleus

AibHLH52 B06 13767672 13769322 289 5.2 32933.15 nucleus

AibHLH53 B06 20280531 20282138 530 6.11 58644.05 nucleus

AibHLH54 B06 134873226 134875351 362 4.9 41028.16 nucleus

AibHLH55 B07 38152793 38153907 266 8.24 29235.88 nucleus

AibHLH56 B07 42703139 42704374 332 6.06 36819.32 nucleus

AibHLH57 B07 121797374 121799212 310 8.77 34239.27 nucleus

AibHLH58 B07 125783661 125785901 272 4.79 30831.18 nucleus

AibHLH59 B09 269215 270201 192 9.77 21471.28 nucleus

AibHLH60 B09 44277247 44279136 303 9.15 34152.55 nucleus

AibHLH61 B09 131488064 131489865 369 6.16 41501.24 nucleus

AibHLH62 B09 146220285 146221793 272 6.53 31041.7 nucleus

AibHLH63 B10 72699993 72703002 305 6.56 34721.31 nucleus

AibHLH64 B02 4649893 4651764 656 6.38 72947.22 nucleus

AibHLH65 B02 14875849 14877865 516 5.45 55794.42 nucleus

AibHLH66 B04 76925964 76930950 662 5.59 74255 nucleus

AibHLH67 B06 2329181 2336236 569 7.11 64242.7 nucleus

AibHLH68 B08 7512297 7514586 180 8.8 20429.6 nucleus

AibHLH69 B09 118317111 118319428 220 6.4 24837.11 nucleus

AibHLH70 B03 21876207 21877320 168 8.31 19027.77 nucleus

AibHLH71 B05 145697971 145699460 264 5.75 29877.09 nucleus

AibHLH72 B03 125089589 125093842 480 5.93 54008.05 nucleus

Figure 1. Multiple alignment of 79 AdbHLH TFs of groundnut

Figure 1. Continued

Figure 1. Continued

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Group 4 Group 3

Figure 3. Phylogenetic relationship and gene structure of the bHLH genes. Phylogenetic tree was constructed with MEGA 6.0 on a multiple alignment of 79 amino acid sequences of bHLH genes from Arachis duranensis Exon/ intron structure of bHLH genes are represented by boxes and black lines, respectively.

Group 3

Figure 4. Phylogenetic relationship and gene structure of the bHLH genes. Phylogenetic tree was constructed with MEGA6.0 on a multiple alignment of 72 amino acid sequences of bHLH genes from Arachis ipaensis.

Table 4. AdbZIP and AibZIP proteins identified in Peanut, Chromosomal location, protein features and its localization prediction.

Protein Chromosome Number Chromosom (b ial Location P) Deduced Polypeptide Subcellular Localization Nucleus

Start End Length (aa) PI MW

AdbZIPl A06 67093845 67094858 495 7.07 54713.19

AdbZIP2 A07 72015371 72018639 322 5.94 35914.02 Nucleus

AdbZIP3 A10 95293270 95294273 316 5.03 33962.22 Nucleus

AdbZIP4 A10 104633025 104633360 212 5.7 24810.41 Nucleus

AdbZIP5 A01 104038428 104038856 217 8.48 24519.65 Nucleus

AdbZIP6 A02 45495034 45497618 443 6.06 48807.58 Nucleus

AdbZIP7 A10 104137408 104138555 800 5.84 86930.94 E.R

AdbZIP8 A03 21151650 21152954 304 5.65 33778.3551 Nucleus

AdbZIP9 A08 1090955 1091461 168 7.1 19345.84 Nucleus

AdbZIP10 A08 32533691 32536893 332 5.3 37390.56 Nucleus

AdbZIP11 A05 4161574 4162581 224 9.9 24845.75 Nucleus

AdbZIP12 A06 104086507 104089154 373 5.18 40440.98 Nucleus

AdbZIP13 A07 23354880 23357810 225 9.13 25954.56 Nucleus

AdbZIP14 A03 4,793,469 4,793,906 163 6.12 18187.23 Nucleus

AdbZIP15 A06 7,343,727 7,344,167 146 5.78 16384.81 Nucleus

AdbZIP16 A03 134,132,232 134,132,612 160 6.14 17947.95 Nucleus

AdbZIP17 A05 4,161,574 .4,162,581 224 9.9 24845.75 Nucleus

AdbZIP18 A06 16,421,562 16,422,038 158 8.91 18493.87 Nucleus

AibZIPl B03 135171129 135171510 155 6.22 17528.52 Nucleus

AibZIP2 B04 130023980 130024291 224 4.98 26262.69 Nucleus

AibZIP3 B05 101761857 101762086 234 4.86 26920.53 Nucleus

AibZIP4 B05 1567450 1572508 388 5.72 41074.03 Nucleus

AibZIP5 B07 106034150 106034580 164 7.1 18889.34 Nucleus

AibZIP6 B08 25788300 25790824 307 7.21 34005.71 Nucleus

AibZIP7 B10 119022617 119023360 327 5.11 35139.44 Nucleus

AibZIP8 B10 130798698 130800592 800 5.89 86979.07 E .R

AibZIP9 B10 131256375 131256710 216 6.04 25239.89 Nucleus

AibZIP10 B03 8405762 8407205 271 6.21 29420.98 Nucleus

AibZIP11 B10 130798698 130799838 800 5.89 86979.07 E .R

AibZIP12 B01 704446 705030 194 5.85 22774.32 Nucleus

AibZIP13 B01 26679598 26679918 183 9.36 21497.39 Nucleus

AibZIP14 B02 54142154 54144616 296 5.72 33288.03 Nucleus

AibZIP15 B03 7502032 7502526 164 6.12 18274.30 Nucleus

AibZIP16 B03 23484533 23487428 344 5.66 39240.76 Nucleus

AibZIP17 B08 11006331 11009532 331 5.38 37397.60 Nucleus

AibZIP18 B09 21764782 21765285 145 5.61 16555.98 Nucleus

AibZIP19 B06 128409215 128411175 372 5.18 40414.98 Nucleus

AibZIP20 B06 :9,088,845 9,089,285 146 5.78 16384.81 Nucleus

AibZIP21 B07 :106,033,813 106,034,307 164 7.10 18889.34 Nucleus

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Figure 8. Phylogenetic relationship and gene structure of the bZIP genes. Phylogenetic tree was constructed with MEGA6.0 on a multiple alignment of 21 amino acid sequences of bZIP genes from Arachis ipaensis

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Figure 14. Phylogenetic relationship and gene structure of the bHLH genes. Phylogenetic tree was constructed with MEGA6.0 on a multiple alignment of 72 amino acid sequences of bHLH genes from Arachis ipaensis Exon/intron structure of bHLH genes are represented by boxes and black lines, respectively

ce:

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Figure 16. Phylogenetic relationship and gene structure of the bZIP genes. Phylogenetic tree was constructed with MEGA6.0 on a multiple alignment of 21 amino acid sequences of bZIP genes from Arachis ipaensis Exon/intron structure of bZIP genes are represented by boxes and black lines, respectively

Figure 17. Schematic representation of conserved motifs in the AdbHLH proteins predicted by MEME. Each motif is represented by a number in the colored box. The black lines represent non-conserved sequences.

Name

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AibHLH3

AibHLH4

AibHLH5

AibHLH6

AibHLH7

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AibHLH9

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AibHLH42

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AibHLH44

AibHLH45

AibHLH46

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AibHLH50

Motif Location

AibHLH51 AibHLH52 AibHLH53 Ai ЬНLH54 Ai ЬНLH55 Ai ЬНLH56 Ai ЬНLH57 AibHLH58 AibHLH59 Ai bHLH60 AibHLH61 AibHLH62 Ai bH LH 63 Ai bHLH64 Ai bH LH 65 AibHLH66 Ai bHLH67 Ai bHLH68 Ai bHLH69 AibHLH70 AibHLH71 AibHLH72

Figure 18. Schematic representation of conserved motifs AibHLH proteins predicted by MEME. Each motif is represented by a number in the colored box. The black lines represent non-conserved sequences.

Figure 19. Schematic representation of conserved motifs in the AdbZIP and AibZIP proteins predicted by MEME. Each motif is represented by a number in the colored box. The black lines represent non-conserved sequences.

Table 6: Conserved motif logos identified in AdbHLH proteins using MEME tool

Table 7: Conserved motif logos identified in AibHLH proteins using MEME tool

Table 8: Conserved motif logos identified in AdbZIP proteins using MEME tool

Name

Motif Logo

QQOKLCEAAVLN QQKKKSAL RTFTA F

Table 9: Conserved motif logos identified in AibZIP proteins using MEME tool

Name

Motif Logo

5

3

4

5

Figure 20: Control and heavy metal treated seedlings

Figure 21: Control and high temperature stress treated seedlings

A B

Figure 22: A) Agarose gel electrophoresis of PCR amplified products under high temperature stress. B) Agarose gel electrophoresis of PCR amplified products under heavy metal stress

Cadmium Stress

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CONCLUSION

Plants growing in their natural habitats are often challenged simultaneously by multiple stress factors, both abiotic and biotic. Several families of plant TFs play significant roles in translating abiotic stress signals into changes in gene expression. So far, research into TFs that regulate abiotic stress responses has mainly focused on single TFs and their isolated function. The present study comprising genome- wide analysis of bHLH and bZIP TFs, detailed protein features, motif composition, multiple sequence alignment, phylogenetic analysis, gene structure, chromosomal location and expression analysis under high temperature and heavy metal stress provides valuable data for further functional analysis to develop multi stress tolerant varieties in groundnut.

CONFLICTS OF INTEREST

The authors declare that they have no potential conflicts of interest.

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Figure 24: Expression profile of AdbZIP and AibZIP genes obtained by RT-qPCR of high temperature treated shoot samples.

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