Identification and characterization of RuvBL DNA helicase genes for tolerance against abiotic stresses in bread wheat (Triticum aestivum L.)

doi:10.21203/rs.3.rs-2229235/v1

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Identification and characterization of RuvBL DNA helicase genes for tolerance against abiotic stresses in bread wheat (Triticum aestivum L.)

https://doi.org/10.21203/rs.3.rs-2229235/v1

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published 27 Jul, 2023

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RuvBL helicase genes represent a conserved family of genes, which are known to be involved in providing tolerance against abiotic stresses like heat and drought in plants. We identified nine wheat RuvBL genes on nine different chromosomes, belonging to homoeologous groups 2, 3, and 4. Analysis of the structure and function of these genes revealed that the (i) length of genes ranged from 1647 to 2197 bp; (ii) genes exhibit synteny with corresponding genes in related species including Ae. tauschii, Z. mays, O. sativa, H. vulgare and B. distachyon; (iii) gene sequences were associated with cis-elements and transposable elements; (iv) the genes TaRuvBL1a-4A and TaRuvBL1a-4B also carried targets for a widely known miRNA, tae-miR164. Gene ontology revealed that these genes were closely associated with ATP-dependent formation of histone acetyltransferase complex. Analysis of the structure and function of RuvBL proteins revealed that (i) proteins were localized mainly in the cytoplasm; (ii) the protein encoded by the representative gene TaRuvBL1a-4A was shown to be involved in protein-protein interactions with ten other proteins; (iii) on the basis of phylogeny, RuvBL proteins were placed in two sub-divisions, namely RuvBL1 and RuvBL2, which were further classified into clusters and sub-clusters. In-silico expression analysis suggested that these genes were differentially expressed under heat/drought. The qRT-PCR analysis confirmed that expression of TaRuvBL genes differed among wheat cultivars with varying degrees of thermotolerance. This study advances our understanding of the biological role of wheat RuvBL genes and should help in planning future studies on RuvBL genes in wheat.

Wheat

Abiotic stress

RuvBL

Helicase

Common wheat (Triticum aestivum L.) is a major cereal and staple crop worldwide, providing food for humans and feed for animals (Gill et al. 2004; IWGSC 2018). However, wheat production is adversely affected by biotic stresses including diseases caused by insects, fungi, bacteria and viruses, and abiotic stresses including, heat, drought, cold, and salinity (Chen et al. 2015; Afzal et al. 2015; Kumar et al. 2020). Therefore, research on improvement of productivity, quality, and stress tolerance in wheat has been a priority area of research (Solangi et al. 2021). Among these stresses, heat and drought represent two major abiotic stresses, which limit plant growth and productivity (Nakashima et al. 2014). In the least developed and low/middle-income countries, drought alone causes 34% loss in crop/livestock production amounting to 37 billion US dollars (FAO 2021)¹. In many wheat-growing regions, heat waves and drought episodes occur concurrently and are the most devastating climatic stresses for wheat (Zampieri et al. 2017).

One of the major strategies used by plants to deal with abiotic stresses like heat and drought is the detection and repair of DNA damage, thus preserving genomic stability (Manova et al. 2015). The cellular mechanisms for detection and repair pathways involve a large number of genes including DNA helicases (Knoll et al. 2011; Wiedemann et al. 2018). These helicases are members of a protein family, which are known to provide tolerance against diverse abiotic stresses (heat, salinity, cold, and drought). The first DNA helicase was discovered in Escherichia coli in mid-1970s (Abdel-Monem et al. 1976). Following this, extensive research was undertaken on these proteins and their involvement in stress tolerance (Gorbalenya et al. 1989; Tuteja et al. 2009; Tuteja et al. 2015; Singh et al. 2020).

Genes encoding DNA helicases include a number of gene families including those encoding the following proteins: MCM6, XPB2, PDH45, p68, OsSUV3, OsBAT1, and RuvBL (Dang et al. 2011; Tuteja et al. 2014; Shivakumara et al. 2017; Passricha et al. 2019; Liu et al. 2020). Among these families of helicase genes, the genes belonging to RuvBL family encode proteins, which belong to AAA+ (ATPases associated with diverse cellular activities) superfamily of proteins. These AAA + proteins are multifunctional in nature and highly conserved, participating in a variety of functions including DNA repair, mitotic assembly, switching of histone variants, and assembly of telomerase core complex (Saifi et al. 2017).

The RuvBL helicases are characterized by the presence of a single domain TIP49/p-loop wih five conserved motifs: Walker A, Walker B, Sensor I, Sensor II, and Arginine finger (Tuteja et al. 1996). Among these, Walker A (GXXGXGKS/T) and Walker B (DEXX) are particularly conserved. Walker A motif contains the phosphate-binding loop (P-loop) of nucleosides, and Walker B motif is involved in metal (Mg²⁺) binding and nucleotide hydrolysis (Snider et al. 2008). The proteins encoded by genes of RuvBL family have been described under different names, such as RuvBL1 (TIP49a/b) in Arabidopsis (Holt et al. 2002), RuvB in bacteria (Dawid et al. 2004), pontin (TIP48)/reptin (TIP49) in humans (Choi et al. 2009) and mice (Boo et al. 2015), Rvb1 (TIP49a)/Rvb2 (TIP49b) in yeast, and RuvB1/RuvB2 in Plasmodium falciparum (Ahmad and Tuteja 2012). Monomers of RuvBL1 and RuvBL2 each form hexamers, which together generate a dodecamer, that is also described as a PAQosome (a multisubunit chaperone complex), which takes part in DNA repair (Fig. 1).

RuvBL1 and RuvBL2 have been discovered and characterized only in few plant systems like Arabidopsis with one RuvBL gene- RUVBL1 (TIP49a/b) (Fritsch et al. 2004; Zhang et al. 2017; Holt et al. 2002) and rice with four RuvBL genes- OsRuvBL1a, OsRuvBL1b, OsRuvBL2a, and OsRuvBL2b (Wang et al. 2011; Saifi et al. 2017; Saifi et al. 2018; Saifi et al. 2021). RuvBL genes have also been utilized for the production of transgenic pigeonpea, to demonstrate the function of these genes in providing tolerance against abiotic stresses (Singh et al. 2020). The characterization of the Arabidopsis RuvBL homologue AtTIP49 suggests that it is essential for growth, development of viability of both sporophyte and female gametophyte (Holt et al. 2002) and that RuvBL proteins interact with other proteins playing a major role in tolerance to abiotic stresses (Holt et al. 2002; Zhang et al. 2017; Saifi et al. 2017). However, these RuvBL genes remain to be explored in majority of plant systems. In wheat, no study of RuvBL genes has been undertaken so far.

In the present study, a comprehensive genome-wide analysis of wheat RuvBL genes and the corresponding proteins was carried out using recently released wheat whole-genome sequence data (IWGSC 2018). The study included the following aspects: (i) identification and analysis of structure of RuvBL1 and RuvBL2 genes and their mapping on individual chromosomes; (ii) identification of cis-regulatory elements (CREs) in promoter regions of RuvBL genes, tansposable elements (TEs) and microRNAs (miRNAs), if any associated with these genes; (iii) synteny analysis between relevant genomic regions in wheat and four other related species; (iv) gene duplications and annotations; (v) primary, secondary and three-dimensional (3D) structures of proteins including study of conserved domains and protein-protein interactions; (vi) sub-cellular localization of proteins; (vii) expression of genes in response to heat and drought treatments. Thus, the results of the present study improved our understanding of RuvBL family of genes in wheat, and provide a resource for planning future studies on this important family of genes with an ultimate aim of using these genes for developing high-yielding wheat cultivars with tolerance to abiotic stresses.

Gene sequence analysis

Identification of RuvBL genes in wheat and related species

Full-length cDNA sequences of rice RuvBL genes were retrieved (Saifi et al. 2017) and used as query for tBLASTx for the identification of RuvBL genes and their proteins in wheat and six related species (T. urartu, Ae. tauschii, T. turgidum, Z. mays, B. distachyon, and H. vulgare). The protein sequences identified thus were examined for the presence of complete TIP49/P-loop domain using conserved domain database (CDD) batch search tool available at NCBI (Marchler-bauer A et al. 2017)². RuvBL gene sequences were also used for comparative and evolutionary studies. All sequences (genomic, cDNA, coding DNA sequence (CDS), and protein) and important information of TaRuvBL genes including gene length, transcript length, CDS length, number of splice variants, and chromosomal positions were retrieved from EnsemblPlants database (Cunningham et al. 2022)³. A chromosome map depicting positions of TaRuvBL genes was prepared using chromosome map function of TBtools software (Chen et al. 2020)⁴. Wheat RuvBL genes were named following the nomenclature used for naming rice RuvBL genes (Saifi et al. 2017).

Structure of RuvBL genes

Gene structure analysis was conducted using the Gene Structure Display Server (GSDS) 2.0 (Ding et al. 2019)⁵. For this purpose, genomic and cDNA sequences of wheat RuvBL genes were used. Detailed information on number of exons and introns and their distribution in individual wheat RuvBL genes and intron phases was also collected. The three phases included the following: phase 0 (intron insertion between two codons), phase 1 (intron insertion after the first base of a codon), and phase 2 (intron insertion after the second base of a codon).

Analyses of synteny

Synteny was examined between wheat RuvBL genes and those from four related species (O. sativa, Z. mays, B. distachyon, and H. vulgare). For this purpose, the genomic files (in Fasta format) along with genome annotation files (in GFF3 format) for all the five species including wheat were downloaded from Phytozome database v13 (Goodstein et al. 2012)⁶. Synteny analysis was done using Multiple Collinear Scanning toolkit (MCScanX) available in TBtools software and syntenic plots were visualized using Dual Synteny Plot function of TBtools software (Chen et al. 2020).

Identification of TEs and miRNAs

Identification of TEs in TaRuvBL genes was undertaken using repeat masker (Chen et al. 2004)⁷ with default parameters. The putative miRNAs targeting TaRuvBL genes were searched using psRNATarget, a web-based server (Dai et al. 2018)⁸, with default e-value in the range of 0–3.

Promoter analysis- Cis regulatory element

For promoter analysis, 1.5 kb genomic sequence upstream of the start codon (ATG) of each TaRuvBL gene was extracted from the EnsemblPlants database (Cunningham et al. 2022)² and used as a promoter sequence for the identification of CREs. CREs in the promoter sequence of each TaRuvBL gene were identified using the PlantCARE database (Lescot et al. 2002)⁹. Only the response elements on the sense strand showing matrix values of ≥ 5 were accepted.

Protein sequence analysis

Physico-chemical properties, secondary structures, and conserved motifs

Major domains in TaRuvBL protein sequences were identified through the conserved domain (CD)-search program of conserved domain database (CDD) at NCBI (Marchler-BAUER A et al. 2017)¹⁰. The ProtParam tool (Garg et al. 2016)¹¹ of ExPASy was used to compute the following physico-chemical properties: (i) amino acid composition (%); (ii) molecular weight; (iii) isoelectric point (pI); (iv) number of positively/negatively charged residues; (v) instability index; (vi) aliphatic index; (vii) Grand Average of Hydropathy (GRAVY). Motifs in the sequences of proteins were identified using the MEME suite (Bailey et al. 2006)¹², using the following parameters: (i) maximum number of motifs = 10; (ii) optimum width of motif = 6–50; (iii) + nce/-nce of individual motif; (iv) number of sites in each motif = 2–600. All identified motifs were annotated using the InterProScan 82.0 database (Quevillon et al. 2005)¹³. Secondary structures were predicted using GOR4 (Garnier et al. 1996)¹⁴.

3D structure analysis, evaluation, and optimization

The 3D structures of putative proteins were predicted using homology modelling. PSI-BLAST was first carried out against protein data bank using 100 iterations and Swiss Model template library (Waterhouse et al. 2018)¹⁵. Suitable homologous templates (crystal and NMR 3D structures) were obtained on the basis of maximum identity, maximum score, and minimum e-value.

The best template for each protein from PSI-BLAST was selected in Swiss Model server to generate the 3D structures of the predicted proteins. The predicted 3D structures were verified by both geometric and energetic means using Structure Analysis and Verification Server (SAVES)¹⁶. The PROCHECK program was used to find out the relative proportion of the number of amino acids, which fall in favoured region, relative to other regions (Laskowski et al. 1993)¹⁷, VERIFY3D was used to determine the compatibility of an atomic model (3D) with its own amino acid sequence (Eisenberg et al. 1997)¹⁸. ERRAT was used to analyse the statistics of non-bonded interactions between different atom types (Colovos and Yeates 1993)¹⁹.

Structural simulation

Molecular dynamic simulation of 3D structure of RuvBL protein was performed utilizing GROMACS v2022.2²⁰ for studying the conformational changes and stability of the protein structure in biological environment. Energy minimization of RuvBL proteins was performed by embedding it into rhombic dodecahedron box with dimension of 1.0 nm × 1.0 nm × 1.0 nm using TIP3P water as solvent model. To ensure a complete coverage of solvent over RuvBL protein model, volume of box was further optimized accordingly. For the neuralization of this system, 96 Na⁺ ions were added to neutralize the negative charge (-96.000 e) on the system. To nullify steric clashes or inappropriate geometry, energy minimization of the system was performed before molecular dynamics by utilizing steepest descent minimization algorithm for 5000 iterations with a convergence threshold of 1000.0 kJ/mol/nm. In order to equilibrate the solvent and ions around RuvBL protein to start dynamics, NVT ensemble was first subjected to stabilize temperature at 300K utilizing velocity rescaling

thermostat for 100 ps; later, NPT ensemble was subjected to stabilize pressure at 1bar along with density of the system utilizing Parrinello-Rahman barostat for 100 ps. The well equilibrated system at desired temperature and pressure was subjected to production MD for 1000 ps. After MD production, trajectories of simulated RuvBL protein were corrected and then analysed for root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of Cα atoms and backbones of RuvBL protein structure in each trajectory with respect to corresponding time to analyse the stability of the simulated structure. The total energy and potential energy of the final simulated structure of RuvBL protein were also calculated.

Docking of proteins

Molecular docking of RuvBL1 and RuvBL2 proteins with DNA was performed using Hex (Ritchie and Kemp, 2000)²¹. DNA interation sites were also identified for hetero-12-mer RuBL protein using BindUP (Paz et al., 2016)²². Further, all interations were visualized using Molegro Molecular Viewer (Thomsen and Christensen, 2006)²³ visualization program.

Superimposition of 3D structures, sub-cellular localization and gene ontology

FATCAT server was used to check the accuracy and topology of simulated 3D structures of predicted proteins by aligning representative structures with the simulated 3D structures of proteins encoded by query genes belonging to rice (Saifi et al. 2017)²⁴. The similarity of the generated 3D structures was measured by comparing the RMSD value of the Cα atoms of the generated 3D structures to those of the corresponding 3D structures of the query genes. Sub-cellular localization and gene ontology (GO) analysis were conducted using Biomart tool available in EnsemblPlants (Cunningham et al. 2022)².

Interaction network analysis

STRING tool (Search Tool for the Retrieval of Interacting Genes) (Szklarczyk et al. 2019)²⁵ was employed to construct the protein association network between TaRuvBL and their interacting proteins. For this purpose, the confidence parameter was set to a threshold of 0.40. The number of interactors in the first shell was not > 10 with no interactor in the second shell. The amino acid sequences of proteins were used as query to analyse the network model of putative interacting partners.

Multiple sequence alignment and phylogenetic analysis

The conserved and coevolving amino acid residues among six species were identified through multiple sequence alignment (MSA) using Clustal-Omega program (Thompson et al. 2003)²⁶; the six species involved included the following: T. aestivum, O. sativa, Ae. tauschii, B. distachyon, Z. mays, and T. urartu. Coevolving residues were identified using mutual information (MI) between two amino acid positions in MSA using the MISTIC web server (Simonetti et al. 2013)²⁶. The MI between two positions (two columns in the MSA) reflects the extent to which knowledge about an amino acid at one position allows prediction of amino acid identity at another position. MI is thus a measure, which allows identification of sites of correlated and compensatory mutations in homologous proteins.

Phylogenetic analysis was conducted using protein sequences employing MEGA software v11.0 (Tamura et al. 2021). A phylogenetic tree was generated following neighbor joining method; a bootstrap involving 1000 iterations was used to construct an unrooted tree.

In-silico expression profiling of TaRuvBL genes

In silico expression analysis of TaRuvBL genes in different tissues and under heat and drought was carried out using the expVIP wheat expression database (Ma et al. 2017)²⁷. Tissue-specific expression included the following five tissues at three growth stages: (i) grain at 2, 14, and 30 days after anthesis (DAA); (ii) spike at two detectable nodes, namely flag leaf and anthesis; (iii) leaf at the following stages: seedling, tillering, and two DAA; (iv) stem at the following stages: two detectable nodes, 1 cm long spike, and at anthesis; (v) root at the following stages: seedling, three leaf, and flag leaf.

For the analysis of differential expression of TaRuvBL genes under heat and drought stress, transcriptome data (available on expVIP database) of wheat seedlings grown under control and heat/drought stress conditions (1 h and 6h) was used. Two fold change ( ≥ + 2 for upregulation and or ≤-2 for downregulation) was calculated by comparing the expression of a gene under control conditions and the expression of the same gene after heat/drought stress. Heat maps of the expression patterns were generated through the HeatmapIllustrator function of TB tools (Chen et al. 2020).

qRT-PCR analysis

Plant Growing Conditions

For qRT-PCR, following two wheat cultivars were used: HD2329 (heat sensitive) and Giza 168 (heat tolerant). Seeds were surface-sterilized in 70% alcohol for 5 min, treated with 0.1% mercuric chloride for 15 min, and washed three times (2 min each) with distilled water. After sterilization, seeds were germinated and grown in hydroponics = with half strength Hoagland’s solution (Hoagland and Arnon 1950). Seedlings were raised in a growth chamber at 22 ± 2°C, 16-h light, 8-h dark photoperiod, and 400-µmol m^− 2 s^− 1 light intensity. Heat stress (HS) was induced by exposing one-week-old seedling to a temperature of 42°C for six hours. The growing environment for control was set to 22°C. Each treatment (Control and HS treated) included three biological replicates. For RNA extraction, samples were promptly frozen using liquid nitrogen (–80°C).

RNA isolation and qRT-PCR

Total RNA was extracted from 100 mg tissue using TRizol reagent (Ambion). The cDNA was generated using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Quantitative expression was performed with a Quant Studio Real-Time PCR system (Applied Biosystems, U.S.A). Three technical replicates for each sample were used. The details of genome-specific primers are provided in Supplementary Table S1. The wheat GAPDH gene was used as an internal reference and the relative gene expression was estimated using 2^{− ΔΔCt} method (Livak and Schmittgen 2001).

Analysis of wheat RuvBL genes

Identification and structure

Following the whole-genome search, nine full-length wheat TaRuvBL genes were identified. The length of individual TaRuvBL genes ranged from 3301 bp (TaRuvBL2a-2B) to 7024 bp (TaRuvBL1b-3D). The length of corresponding cDNA sequences ranged from 1647 bp (TaRuvBL1b-3D) to 2197 bp (TaRuvBL1b-3A), while CDS lengths ranged from 1368 to 1491 nt. The information about these wheat RuvBL genes and rice orthologues is summarised in Table 1. The nine TaRuvBL genes were mapped one each on nine chromosomes of three homoeologous groups (group 2, group 3, and group 4). Eight genes were located on short arm (Five were on terminal regions while three genes were located on sub-terminal regions). Only one gene was located on the long arm (4A) (Fig. 2).

Table 1

Information about TaRuvBL genes, cDNA and CDS sequences relative to rice.
Gene (Length in bp)	cDNA /CDS (Length in bp)	Number of exon
Wheat genes
TaRuvBL1a-4A* (5148)	2122/1476	11
TaRuvBL1a-4B* (5322)	1962/1470	11
TaRuvBL1a-4D*(5389)	1850/1368	12
TaRuvBL1b-3A* (6334)	2197/1491	11
TaRuvBL1b-3B (6136)	1655/1368	12
TaRuvBL1b-3D (7024)	1647/1368	12
TaRuvBL2a-2A (3936)	1706/1425	2
TaRuvBL2a-2B (3301)	1720/1425	2
TaRuvBL2a-2D (3846)	1779/1425	2
Rice genes
OsRuvBL1a* (5353)	1723/1020	12
OsRuvBL1b (5036)	1826/1368	12
OsRuvBL2a (3518)	1774/1431	2
OsRuvBL2b ^# (519)	504/ 504	2
Note: CDS: coding DNA sequence; cDNA: complementary DNA; bp: base pair; *more than one splice variants ^# Incomplete domain.

Each TaRuvBL gene was a split gene, with number of introns ranging from 1 to 11, the number of exons being 2, 11 and 12 in three genes each. Only two of the three intron phases were present (six genes with phases 0 and 2, three genes with phase 2, and none with phase 1; Fig. 3).

Orthology of genes among eight species

In addition to wheat and rice, RuvBL genes were also identified in six other species, which included two diploid and one tetraploid wheat progenitors and three other species of the grass family (Table 2). Number of RuvBL genes were almost similar in all the eight species. Rice with four RuvBL genes was used as a reference species; one of these rice genes, namely OsRuvBL2b was an incomplete gene with its orthologue available only in maize and Brachypodium, the other species having orthologues of only three rice genes. Thus, rice and maize with four genes each and B. distachyon with only two RuvBL genes differ from the general pattern of three genes at the diploid level.

Table 2

Orthologs of RuvBL genes in eight grass species including hexaploid wheat, showing evolutionary relationship among the family (*Triticeae)*.
Species	Gene name
	RuvBL1a	RuvBL1b	RuvBL2a	RuvBL2b
O. sativa	OsRuvBL1a	OsRuvBL1b	OsRuvBL2a	OsRuvBL2b
T. aestivum	TaRuvBL1a-4A	TaRuvBL1b-3A	TaRuvBL2a-2A	-
	TaRuvBL1a-4B	TaRuvBL1b-3B	TaRuvBL2a-2B	-
	TaRuvBL1a-4D	TaRuvBL1b-3D	TaRuvBL2a-2D	-
Z. mays	ZmRuvBL1a	ZmRuvBL1b	ZmRuvBL2a	ZmRuvBL2b
H. vulgare	HvRuvBL1a	HvRuvBL1b	HvRuvBL2a	-
B. distachyon	-	-	BdRuvBL2a	BdRuvBL2b
T. uratu	TuRuvBL1a	TuRuvBL1b	TuRuvBL2a	-
Ae. tauschii	AetRuvBL1a	AetRuvBL1b	AetRuvBL2a	-
T. turgidum	TtRuvBL1a-4A	TtRuvBL1b-3A	TtRuvBL2a-2A	-
T. turgidum	TtRuvBL1a-4B	TtRuvBL1b-3B	TtRuvBL2a-2B	-

Synteny between different grass species

Four comparative syntenic maps (Fig. 4) were constructed to study the evolutionary relationships of TaRuvBL genes with those in H. vulgare, B. distachyon, O. sativa, and Z. mays (Fig. 4; Supplementary Table S2). Following syntenic relationships of wheat genes with genes of other species were observed: (i) five TaRuvBL genes syntenic with H. vulgare genes, (ii) three TaRuvBL genes syntenic with B. distachyon, (iii) three TaRuvBL genes syntenic with O. sativa, and (iv) three TaRuvBL genes syntenic with Z. mays.

Occurrence of transposable elements (TEs) within genes

In nine TaRuvBLgenes taken together, a total of 83 TEs belonging to Class I [retrotransposons (LTR and Non-LTR)] and Class II [DNA transposons (TIR and Non-TIR)] were identified (Fig. 5; Supplementary Table 3). The number of TEs per TaRuvBL gene ranged from four in TaRuvBL2a-2D to 14 in TaRuvBL1a-4D (Supplementary Table S3). TEs from the superfamily DNA/hAT (hobo/Ac/Tam3) were most frequent (17) followed by DNA/EnSpm/CACTA (14), LTR/Gypsy (14), DNA/MuDR (10), LTR/Copia (7), DNA/Mariner (6), NonLTR/L1 (5), DNA/Helitron (3), and NonLTR/Penelope/Athena (2). Some TEs (DNA/Ginger, DNA/harbinger, ERV/ERV3, NonLTR/Jockey, and Simple/Sat) were present only once.

MicroRNA (miRNA) and their targets in genes

TaRuvBL genes were also examined for the presence of target sites of known miRNAs (Supplementary Table S4). Using E-value ≤ 3, two of the nine TaRuvBL genes (TaRuvBL1a-4A and TaRuvBL1a-4B) were found to have target sites for a solitary wheat miRNA (tae-miR164). Inhibition of expression of these two genes by wheat miRNA (tae-miR164) was found to be achieved through post-transcriptional cleavage.

Identification of cis-regulatory elements (CREs)

Based on the PlantCARE databases⁴, putative CREs involved in the regulation of gene expression were identified in the promoters of TaRuvBL genes. In brief, 37 CREs were identified, which included the following: (i) 11 elements for response to biotic and abiotic stresses; (ii) 19 elements for development and light responses, and (iii) seven elements for hormonal responses (Fig. 6; Supplementary Table S5). The number of CREs per TaRuvBL gene ranged from seven CREs in TaRuvBL1a-4B to 15 CREs in TaRuvBL1b-3A (Supplementary Table S5).

Analysis of RuvBL proteins

Characterization of proteins

The length of TaRuvBL proteins did not differ very much and ranged from 455 amino acids (aa) (TaRuvBL1b-3B and TaRuvBL1b-4D) to 496 aa (TaRuvBL1b-3A) (Table 3). The molecular weight of proteins ranged from 49.6 to 54.1kDa and isoelectric point (pI) ranged from of 5.36 to 5.81; proteins were within the acidic range, and aliphatic index (94.92-103.39) was stable over a wide range of temperatures. Grand average of hydropathy (GRAVY) ranged from − 0.045 to -193 (Table 3).

Table 3

A summary of the structures of wheat and rice RuvBL proteins, having unstable instability index aa = amino acid, AI = Aliphatic index, GRAVY = Grand average of hydropathy
Protein name (protein length as number of aa)	Mol wt (kDa)/pI	Domain type and location of aa	AI (GRAVY)
Wheat RuvBL proteins
TaRuvBL1a-4A (491)	53.8/5.81	TIP49/ P loop domain (14–401)	100.47 (-0.086)
TaRuvBL1a-4B (489)	53.6/5.73	TIP49/ P loop domain (14–399)	98.69 (-0.136)
TaRuvBL1a-4D (455)	49.6/5.36	TIP49/ P loop domain (14–365)	100.7 (-0.122)
TaRuvBL1b-3A (496)	54.1/5.72	TIP49/ P loop domain (14–406)	103.39 (-0.045)
TaRuvBL1b-3B (455)	49.7/5.63	TIP49/ P loop domain (14–365)	102.2 (-0.118)
TaRuvBL1b-3D (455)	49.7/5.66	TIP49/ P loop domain (14–365)	102.42 (-0.118)
TaRuvBL2a-2A (474)	51.7/5.5	TIP49/ P loop domain (18–370)	95.72 (-0.184)
TaRuvBL2a-2B (474)	51.6/5.46	TIP49/ P loop domain (18–371)	94.92 (-0.193)
TaRuvBL2a-2D (474)	51.7/5.46	TIP49/ P loop domain (1371)	95.32 (-0.189)
Rice RuvBLproteins
OsRuvBL1a (455)	49.6/5.45	TIP49/ P loop domain (14–365)	102.68 (-0.143)
OsRuvBL1b (455)	49.6/5.52	TIP49/ P loop domain (14–365)	102.46 (-0.141)
OsRuvBL2a (476)	31.8/5.7	TIP49/ P loop domain (1374)	96.34 (-0.15)

Functional domain and motifs

Each RuvBL protein including all TaRuvBL and OsRuvBL proteins contains a single TIP49/P-loop domain. The length of the domain did not differ much and ranged from 351 to 392 aa in wheat and 351 to 356 aa in rice (Table 3). A total of 20 distinct motifs (motif 1 to motif 20) were identified in each TaRuvBL protein (Fig. 7a). However, only 16 of the 20 motifs had each an E-value ≥ 10^− 3 (Supplementary Table S6), so that only these were examined further. Length of each of the 16 selected motifs ranged from 8 aa (motif 12, 13, and 14) to 50 aa (motif 1, 2, 4, 5, and 6) (Fig. 7b). Out of the 16 motifs also, 14 motifs were present in only 6 proteins (barring TaRuvBL2a-2A, TaRuvBL2a-2B, TaRuvBL2a-2D); motif 15 was present in only 3 proteins, (TaRuvBL2a-2D, TaRuvBL2a-2D, TaRuvBL2a-2D), and motif 16 was present in only 2 proteins (TaRuvBL1a-4A, TaRuvBL1a-4B). In three motifs (1, 2, and 3), a 4–6 aa long characteristic conserved sequence was observed; motif 1 had aa sequence “LATN/IATN” (Sensor1), motif 2 had “GPPATG” (Walker A), and motif 3 had “GEVH” (Walker B) (Fig. 7b). These sequences (Sensor 1, Walker A, and Walker B) were conserved across plant/animal proteins including humans. Out of 16 motifs, seven motifs (1, 2, 3, 4, 5, 6, and 8) were annotated as ATP binding, which is a characteristic feature of RuvBL proteins. Three motifs (2, 4, and 6) of the above seven motifs were also found to be involved in two NuA4 histone-acetyl transferase complexes, namely Ino80 complex, and R2TP complex. The remaining nine motifs (7, 9, 10, 11, 12, 13, 14, 15, and 16) were novel (Supplementary Table S6; Supplementary Figure S1)

Multiple sequence alignment (MSA) and coevolution

Similarity of amino acid sequences among wheat proteins ranged from 44.2–99.7% (Supplementary Table S7) and that between wheat proteins and rice proteins ranged from 33.3–94.07% (Supplementary Figure S2; Supplementary Table S7). MSA suggested the presence of all the five conserved motifs [Walker A, Walker B, Sensor I, Sensor II, and the Arginine finger (R) of AAA + family] (Supplementary Figure S2). Nine amino acids with highest MI were accepted to be highly conserved among wheat and rice (Supplementary Figure S3).

Secondary and tertiary (3D) structures

A comparison of the secondary structures of selected proteins suggested that α helix dominated in the secondary structure followed by random coil (Supplementary Table S8). Analysis of the Ramachandran plots indicated a high proportion of aa residues in the favoured region relative to those falling in the ‘allowed’ and ‘disallowed’ regions.

Similarity of 3D structures of TaRuvBL proteins with corresponding template proteins of rice ranged from 33.3–96.3%. All the RuvBL1 wheat proteins had homohexameric 3D structures whereas, RuvBL2 wheat proteins had monomeric 3D structure. Out of nine, six proteins had sequence similarity in the range of 71.52%-72.47%, while the other three proeins (TaRuvBL2a-2A, TaRuvBL2a-2B, TaRuvBL2a-2D) had sequence similarity in the range of 63.03%-63.25% (Supplementary Figure S4; Supplementary Table S9). Global model quality estimation (GMQE) of these 3D structures of TaRuvBLs ranged from 0.6 to 0.7, suggesting high-quality protein models. The QMEAN value ranged from − 0.65 to -0.85 (Supplementary Table S9). The quality factors (determined using ERRAT) ranged from 88.21–94.1% and 3D-1D score (determined using VERIFY3D) ranged from 76.04–81.7% (Supplementary Table S9) (Supplementary Figure S4).

Molecular dynamic simulation and docking of RuvBL proteins

Assessment scores of structure model of RuvBL protein with 06 chains of TaRuvBL2a-2A and 06 chains of TaRuvBL1b-3D obtained from homology modelling using 4ww4.1 protein as template had Ramachandran score 94.56% by PROCHECK, quality factor 92.6665 by ERRAT, and 0.0% buried outlier protein atoms by PROVE. This model pass through MD-simulation to improve its structure and stability. Total energy and potential energies of the final simulated structure of RuvBL protein calculated were − 5.95113e + 06kJ/mol and − 7.30742e + 06kJ/mol respectively in comparison to initial model as -5.92421e + 06kJ/mol and − 7.27618e + 06kJ/mol respectively (Fig. 8a, b).

Docking was performed between DNA and TaRuvBL1b-3D (Fig. 9(a) with energy score of -4.020044e + 002, RMS of -1.00), and between DNA and TaRuvBL2a-2A (Fig. 9(b) with energy score of -2.824617e + 002, RMS of -1.00). Additionally, interaction of 12 DNA ligands with hetero-12-mer structure of RuvBL protein is shown in Fig. 9 (c, d), and its non-DNA iteraction sites were also extracted and shown in Fig. 9(e, f). Supplementary Table S10 is providing chain wise DNA interection sites and non-DNA interaction sites of hetero-12-mer structure of RuvBL protein.

Alignment and functional annotation of 3D structures

The 3D structures of wheat proteins were superimposed over the corresponding 3D structure of rice proteins (Supplementary Figure S5; Supplementary Table S11); 3D sructures with minimized energy were used for this purpose. The similarity of 3D structures of proteins with corresponding 3D structure of OsRuvBL2a protein ranged from 85.3%-95.02% with values for RMSD ranging from 0.45 Å to 3.05 Å. On the basis of similarity in 3D structure, wheat proteins were placed in the following categories: (i) those with significant similarity (85.3% to 90.44–85.3%) with OsRuvBL2a protein (RMSD, 3.05–2.83 Å); these proteins included following three wheat proteins: TaRuvBL1b-3A, TaRuvBL1b-3B, and TaRuvBL1b-3D; (ii) those with significant similarity (94.6-95.02%) with OsRuvBL1a protein (RMSD: 2.22–1.01) Å); these included following two proteins: TaRuvBL1a-4A, TaRuvBL1a-4B. Among these, proteins with maximum number of amino acids in favoured regions in the Ramachandran plot were used for superimposing on the Rice reference proteins (Supplementary Figure S5).

Sub-cellular localization and GO analysis

All TaRuvBL proteins were found to be located in the cytoplasm, although four proteins (TaRuvBL1b-3A, TaRuvBL2a-2A, TaRuvBL2a-2B, TaRuvBL2a-2D) were also found to be located in chloroplasts and mitochondria (Supplementary Table S12). Gene ontology (GO) of TaRuvBL genes suggested the following main biological functions; chromatin remodelling, box C/D snoRNP assembly, regulation of transcription by RNA polymerase II, histone acetylation, DNA duplex unwinding. Molecular functions of TaRuvBL genes included the following: DNA helicase, ATP binding, 5'-3' DNA helicase, and nucleotide binding (Supplementary Figure S6; Supplementary Table S13)

Protein–protein interactions

The interactions of TaRuvBL1a-4A with other genes were placed in the following three categories: (i) Known interactions that were experimentally determined and database annotated; (ii) Predicted interactions including those with neighbourhood on chromosome, gene-fusion and phylogenetic-co-occurrence; (iii) others with automated text-mining, homology, and co-expression (Fig. 10; Supplementary Table S14, S15). All the 10 interacting proteins belonged to categories (i) and (iii) (except homology); however only three proteins from category (ii) showed a close relationship (phylogenetic-co-occurrence) with the TaRuvBL1a-4A protein (Fig. 10; Supplementary Table S14, S15).

Phylogenetic analysis

On the basis of phylogenetic analysis, 34 RuvBL proteins belonging to eight species were placed in two subfamilies (i) RuvBL1 and (ii) RuvBL2. These subfamilies were further divided into clusters, RuvBL1 with five clusters (two big clusters, 7 + 8; and three relatively small clusters, 4 + 1 + 1) and RuvBL2 with two clusters (8 + 5). The details of the two subfamilies and the clusters in each subfamily are shown in Fig. 11.

In-silico expression analysis of genes

The expression patterns of TaRuvBL genes were examined in five different tissues (root, stem, leaf, spike, and grain) each sampled at three different developmental stages under normal conditions (Fig. 12a). Expression in seedlings was also examined under heat and drought stress. Following five genes showed high expression (TPM > 2) in grain/stem/leaf/root/spike: TaRuvBL2a-2A, TaRuvBL2a-2B, TaRuvBL2a-2D, TaRuvBL1b-3B, and TaRuvBL1b-3D, the very first of these genes (TaRuvBL2a-2A) showing the highest expression in all the tissues and at each developmental stage.

After 1h of heat stress, a solitary gene (TaRuvBL2a-2D) showed downregulation (-2.07) but, under the same duration of heat + drought stress, two genes (TaRuvBL2a-2D, TaRuvBL1b-3B) showed downregulation (-2 fold).

Expression patterns of genes under heat stress

On the basis of in silico expression analysis, three genes (TaRuvBL1a-4A, TaRuvBL1b-3B, and TaRuvBL2a-2D), one from each of the three homoeologous groups were selected for quantitative real-time PCR (qRT-PCR), to examine in planta changes of expression in response to heat stress. For this purpose, contrasting wheat genotypes (sensitive cv. HD2329 and tolerant cv. Giza) were used. Out of the three TaRuvBL genes, two (TaRuvBL1b-3B and TaRuvBL2a-2D) showed considerable differential expression (≥ 2 fold) under HS (Fig. 13). The expression levels for each of the three genes was relatively high in tolerant cv. Giza (Fig. 13). The above results suggest that at least some of the wheat RuvBL genes give higher expression under heat stress.

The RuvBL genes in eukarytoes resemble the RuvB helicase gene of E coli, and have therefore been described as RuvB like (RuvBL) genes. They encode proteins, each having both DNA-dependent ATPase and DNA helicase activities. These genes belong to the family of genes encoding AAA + family of ATPases associated with diverse cellular activities. The RuvBL protein associated with several multi-subunit transcriptional complexes, and also involves histone modification. In human beings, the RuvBLs have been shown to play important roles in essential signalling pathways such as the c-Myc and Wnt pathways, which are involved in chromatin remodelling, regulation of transcription and development, mitosis, DNA repair and apoptosis (Matias et al. 2006). RuvBL genes have also been shown to be involved in human cancers. In particular, proteins having RuvBL1/RuvBL2 ATPase activity, drive maturation of PAQosome (Particle for Arrangement of Quaternary Structure, a large multisubunit chaperone complex), DNA replication and radio-resistance in lung cancer (Yenerall et al. 2019). RuvBL1 is also an epigenetic factor promoting proliferation and inhibitory differentiation program in head and neck squamous cancers (Lin et al. 2020). RUVBL1/2-TTT complex is also involved in mTORC1-hyperactive cancer cells (Shin et al. 2020). Following are some other names for RuvBL1 proteins: RVB1, TIH1, ECP54, TIP49, ECP-54, INO80H, NMP238, PONTIN, TIP49A, NMP 238, and Pontin52.

Among higher plants, RuvBL genes have been studied in only two plant species, namely Arabidopsis (Holt et al. 2002; Schořová et al. 2019) and rice (Saifi et al. 2017). In pigeonpea, the RuBL genes have also been used for developing transgenics to examine the role of these genes in providing tolerance against salt stress (Singh et al. 2020). DNA helicases, like RuvBL, are crucial for nearly all DNA metabolic processes in plants, including pre-mRNA splicing. These act as molecular motor proteins that are crucial for numerous cellular functions (Tuteja and Tuteja 2004a). Mutations or complete absence of RuvBL genes or mutations in these genes have been shown to be lethal in yeast and Arabidopsis (Holt et al. 2002; Jonsson et al. 2001). The expression of these genes is necessary for the normal development of shoot meristem, as shown in Arabidopsis (Holt et al. 2002). Additionally, it is also known that NTP-dependent transcription activators are crucial for abiotic stress responses (Tuteja and Tuteja 2004b).

TaRuvBL genes in wheat and related species

The nine TaRuvBL genes identified in wheat during the present study differed among themselves (Fig. 2). Based on hexaploid nature of wheat genome and availability of four RuvBL genes in rice genome, normally, one should expect 12 genes in wheat, assuming that for each rice gene, there would be three genes in hexaploid wheat. However, in the present study only nine genes were available; this is not surprising, since one of the four genes in rice was incomplete (OsRuvBL2b), sugggesting that perhaps either this is under the process of degeneration/deletion or is the result of duplication (see later). Each of the seven plant species included in the present study had three RuvBL genes (T. aestivum and T. turgidum had three genes on each genome) except rice and maize, each with four genes (Table 2), suggesting deletion/neofunctionalization of RuvBL2b gene during evolution.

The nine TaRuvBL genes in wheat showed variation in the number of exons (Fig. 3). The number of exons in TaRuvBL genes ranged from 2 to 12. In the present study, we observed that homoeologues differed in the number of exons which is rather surprising. For example, TaRuvBL1a-4A and TaRuvBL1a-4B contain 11 exons each; while TaRuvBL1a-4D contains 12 exons. It seems that the first exon in the TaRuvBL genes present on A and B genomes split into two exons during evolution or vice versa. Variation of the first exon among homoeologues represents early stages of RuvBL gene sub-functionalization and neo-functionalization (Bartos et al. 2012). The presence of more than one splice variants in each of the following four genes suggest that alternate splicing of the transcripts of these genes perhaps resulted in multiple transcript variants (available at RefSeq, Jan 2016), TaRuvBL1a-4A, TaRuvBL1a-4A, TaRuvBL1a-4A, TaRuvBL1b-3A.

The avaibility of three RuvBL genes on each of the three wheat genomes (A, B, and D) are on almost same positions (terminal and subterminal regions of short arm) on three chromosomes of the same homoeologous group (Fig. 2) as also in each its progenitors. This suggests that no duplication/deletion events for RuvBL genes occurred during the evolution of hexaploid wheat (Table 2) and that TaRuvBL genes in wheat are largely conserved. The only exception was the gene TaRuvBL1a-4A, located on a long arm of chromosome 4A, which may be attributed to a pericentric inversion (4AS; 4AL) in 4A chromosome (Dvorak et al. 2018). This hypothesis receives support from the earlier observations, which suggested that chromosome 4A in hexaploid wheat was reversed following allopolyploidization (Devos et al. 1995; Dvorak et al. 2018; Zhou et al. 2020).

Orthologs of OsRuvBL2b were present only in maize and Brachypodium genome (Fig. 14) and absent in wheat genome as well as in genomes of its progenitors including T. urartu and Ae tauschii. It was also absent in the related diploid species H. vulgare. This suggests that the gene RuvBL2b must have been lost during evolution soon after divergence of rice and wheat from a common ancestor, but not before divergence of rice and maize from a common ancestor (Fig. 14).

In the present study, we also observed that no RuvbL gene was present on homoeologous chromosomes of rice and wheat, suggesting their diverse evolution. For example, TaRuvBL1a genes are present on each of the three chromosomes of homoeologous group 4 (4A, 4B, and 4D); these three genes are orthologues of the rice gene OsRuvBL1a located on rice chromosome 1, which is not homoeologous to chromosome of homoeologous group 4 of wheat (Ahn et al. 1993; Sorrells et al. 2003). Similarly, RuvBL1b genes are located on 3A, 3B, 3D chromosomes of wheat and an unrelated rice chromosome 7. TaRuvBL2a is located on wheat chromosomes 2A, 2B and 2D and the corresponding rice homoeologue, rice chromosome 6. No satisfactory explanation for this lack of conservation between crops is available, although structural variations (SVs) could be one possible reason (also see next section on syntney).

Evolution of RuvBL genes (synteny and phylogeny)

Synteny analysis gives information about the conservation of genes on the orthologous chromosomes of wheat and rice (Sorrells et al. 2003). In the present study, out of nine TaRuvBL genes, five genes exhibited syntenic relationship with genes in O. sativa, Z. mays, B. distachyon, and H. vulgare (Fig. 4; Supplementary Table S2). Three TaRuvBL genes present on homeologus group 2 (2A, 2B, and 2D) of wheat show synteny with all the four plant species (Z. mays, O. sativa, B. distachyon, H. vulgare). However, two different TaRuvBL genes present on chromosome 4B and 4D of wheat show synteny only with H. vulgare. Two genes of maize (ZmRuvBL2a and ZmRuvBL2b), each show synteny with only one homoeologous group (Group 2) of wheat genes (TaRuvBL2a-2A, TaRuvBL2a-2B, TaRuvBL2a-2D). This might be due to a duplication of RuvbL2a gene in maize which is also confirmed due to the high sequence similarity (96.9%) between ZmRuvBL2a and ZmRuvBL2b. In rice also duplication in OsRuvBL2a has occurred; however only truncated gene is present in rice. Therefore, it is possible that in rice and maize, gene duplication has taken place after their divergence from the common ancestor of wheat, rice, and maize. Thus, this homoeologuous group of wheat might have played a crucial role in the evolution of RuvBL genes. These results suggest that TaRuvBL genes in wheat might have originated from other related species instead of orthologous genes from rice and maize.

The above results also receive support from the results of phylogeny. In the phylogenetic tree, all the RuvBL proteins (ecoded by 34 RuvBL genes) belonging to eight plant species are distributed in two subfamilies (RuvBL1 and RuvBL2), which is in agreement with the earlier studies carried out in plants and animal systems (Wang et al. 2011; Saifi et al. 2017; Schorova et al. 2019). In the phylogenetic tree also, O. sativa and Z. maize proteins were clustered in separate groups as compared to proteins from T. aestivum, T. urartu, Ae. tauschi, and H. vulgare, showing their different ancestry. Therefore, the results from both, the synteny analysis and phylogenetic analysis are in agreement. These results suggest that the RuvBL genes in rice and maize and those of wheat and its progenitors followed different routes of evolution, after separating from common ancestry in the remote past.

Cis-regulatory elements (CREs) in promotor regions

Promoter analysis revealed the presence of a variety of CREs in nine TaRuvBL genes. These CREs included elements specific for response to development (tissue-specific), light (low temperature), and phytohormone, etc. (Supplementary Table S5). The light-responsive elements included Sp1, Box1, AE-box, I-box, and MRE. The phytohormone response elements included ABRE, ERE, as1, CGTCA, GARE, and TGA elements, where ABRE was present in maximum number. Stress-responsive elements included LTR, DRE, MYC, MYB, STRE, ARE, I-Box, MRE, WRE3, W-Box, MBS, and F- Box. Here, MYB and MYC occurred in highest frequency. However, development related CREs were relatively fewer in number. It appears that phytohormones control the expression of the majority of TaRuvBL genes; among these, auxin and strigolactone are known to form a dynamic feedback loop and modulate the distribution of each other (Hayward et al. 2009). It is also known that certain phytohormone responsive elements, such as ABRE, contribute to the majority in responses to heat and drought stress (Joo et al. 2013). Thus, ABRE elements along with other cis-acting elements that are available in TaRuvBL respond to phytohormones (e.g., MeJA, ABA, SA, GA, etc.), which may be regulating the expression of TaRuvBL genes under abiotic stress. Similarly, other response elements may have a role in the regulation of expression of TaRuvBL genes under the following conditions: (i) abiotic stress (low temperature), (ii) tissue-specific response, (iii) response to wound, and (iv) some physiological processes. Because of the lack of information on the regulation of expression of TaRuvBL genes in wheat, a more detailed discussion of our results on the occurrence of CREs in TaRuvBL promoters is not possible at this stage.

Transposons elements (TEs) within genes

In TaRuvBL genes, 83 TEs were identified, which included a maximum number of DNA/hAT followed by DNA/EnSpm/CACTA, LTR/gypsy, DNA/MuDR, LTR/copia, DNA/Mariner, NonLTR/L1, DNA/Helitron, and NonLTR/Penelope/Athena. In one of our earlier studies involving the identification and characterization of TaSDG genes, the TEs were found to differ in types and number (Batra et al. 2020). Therefore, it is apparent that even different related genes may harbour different types and numder of TEs (Lisch 2013). TEs can be activated upon different environmental cues in plants i.e., heat and drought stress (Cavrak et al. 2014; De Felice B et al. 2009).

In maize genome, four to nine TE families have been reported, including all major class I superfamilies (Gypsy, Copia, long interspersed nuclear elements (LINEs)), and DNA transposons. The promoters of many maize genes have been shown to be enriched (more than two-fold) with these TEs and these genes (with TEs) have been shown to be up-regulated in response to different abiotic stresses (Makarevitch et al. 2015).

An important class of TEs is also the hAT superfamily, identified in fungi, animals, and plants (Calvi et al. 1991; Rubin et al. 2001). In Arabidopsis a hAT-like transposon binds to the DNA repair gene Ku70, and it is essential for the development (Bundock and Hooykaas 2005). It is also highly conserved among monocots and eudicots, suggesting early specialization (Jesus et al. 2012). Previous studies have also reported that hAT-like elements are transcriptionally active in rice, grapevine, and sugarcane (Jiao and Deng 2007; Benjak et al. 2008; Jesus et al. 2012). CACTA is another type of TE which were identified during this study. In wheat, the percentage of CACTA TE is highest (15.5%) as compared to the other plant species (Appels et al. 2018). In rapeseed, it acts as an enhancer to increase silique length and seed weight (Shi et al. 2019). In Maize, insertion of CACTA-like transposon at the promoter of ZmCCT10 contributed to the flowering-time adaptation (Huang et al. 2018). In wheat, insertion of CACTAs in TaMFT and TaVrn1 genes into the third and first intron, respectively have been shown to increase seed dormancy (Wang et al. 2022).

miRNAs sequences in genes

The present study identified only one miRNA (tae-miR164) with its target sites in two TaRuvBL genes, namely TaRuvBL1a-4A and TaRuvBL1a-4B genes (Supplementary Table S4). It is well known that the miR164 family of miRNAs is highly conserved, and its members regulate conserved targets belonging to NAC transcription factor. There are at least two earlier studies, where targets of miR164 have been identified, and their interaction has been studied. (i) In Populaus euphratica, the peu-miR164a–e and its target gene POPTR 0007s08420 participate in abiotic stress response. Additionally, it is also reported that overexpressing PeNAC070 in Arabidopsis promoted the growth of lateral roots, delayed stem elongation, and increased sensitivity of transgenic plants to drought and salt stresses. This study helped in understanding the adaptability of P. euphratica to extreme drought and the salt environment by analysing tissue-specific expression patterns of miR164-regulated and specific promoter-regulated PeNAC genes. (ii) In an earlier study in wheat also, the target gene of tae-miR164 was found to be a gene encoding the transcription factor TaNAC21/22 of the NAM sub-family, involved in negative regulation of resistance against stripe rust, suggesting that the presence of miRNA provided resistance by controlling the expression of TaNAC21/22 (Feng et al. 2014); in this study in wheat, no effort was made to examine the role of tae-mi164 in tolerance to abiotic stresses like heat and drought. The present results, however, seem to have a parallel in Populus.

In the present study, only two of the nine genes, namely TaRuvBL1a-4A and TaRuvBL1a-4B were identified as the targets of a solitary miRNA. Obviously, tae-miR164 will reduce the expression of these two TaRuvBL genes, but how inhibition of the expression of these two target genes will influence tolerance to heat and drought is a subject of future study, particularly in view of the fact that in qRT-PCR analysis, the expression of three RuvBL genes, namely, TaRuvBL1b-3B, TaRuvBL1a-4A, TaRuvBL2a-2D, was higher in tolerant genotype (Giza) relative to that in the sensitive genotype (HD2329). However, since in Populus, peu-miR164a–e has been shown to have a role in tolerance against abiotic stresses, the role of tae-miR-164 in heat/drought response through inhibition of two TaRuvBL genes will be an interesting subject for future study.

Structure and function of proteins

TaRuvBL proteins (455 aa to 491 aa) have been shown to be long relative to OsRuvBL proteins (455 aa to 476 aa) which are more conserved. The variation in domain length also supports this observation, since domain length of TaRuvBL proteins varies from 351 to 387aa and in rice 351 to 356aa. This indicates evolutionary changes in wheat RuvBL proteins after speciation of wheat and rice from common ancestor. Grand Average of Hydropathy (-0.045 to -0.193) suggested hydrophilic nature of RuvBL proteins. These proteins function in cytoplasm, and most of the proteins functioning in cytoplasm are of hydrophilic in nature (Batra et al. 2017; Batra et al. 2019). This feature and higher aliphatic index of these genes (98.8 to 133.8) indicate their significance in high thermostability (Ikai 1980).

It may also be recalled that the presence of a solitary domain (TIP49/P-loop) in each of the TaRuvBL proteins, is a characteristic feature of all the RuvBL proteins of other species examined so far, including yeast, Arabidopsis, and rice (Fritsch et al. 2004; Holt et al. 2002; Gribun et al. 2008). This domain imparts nucleoside triphosphate hydrolase or P-loop NTPase activity and exhibits characteristic ATPase and DNA unwinding activities (Saifi et al. 2017). ATPase activity is involved in several cellular processes which initially protect cells from stress but eventually lead to cell death under continuous stress conditions (Saifi et al. 2019; Tuteja et al. 2015). Seven motifs (1, 2, 3, 4, 5, 6, and 8) were also annotated as ATP binding and ATP-dependent activity (GO: 0008094); Similar motifs were also reported in rice RuvBL proteins, which also exhibit the characteristic ATPase and DNA unwinding activities (Saifi et al. 2017). ATPase activity of RuvBL1 homologs from other systems was also reported in the presence of single-stranded DNA as well as double-stranded DNA (Gribun et al. 2008; Ahmad and Tuteja 2012).

Multiple sequence alignment suggested that all the nine TaRuvBL proteins carry some conserved amino acid sequences in their solitary characteristic domain (e.g., Walker A, Walker B, and Sensor1; Supplementary Fig. 1). Walker A contains the phosphate-binding loop (P-loop) of nucleosides, and Walker B is involved in metal (Mg2+) binding and nucleotide hydrolysis (Snider et al. 2008). Motifs 2, 4, and 6 are also involved in functions like NuA4 histone-acetyl-transferase complex (GO: 0035267), a complex having histone acetylase activity on chromatin, as well as ATPase, DNA helicase, and structural DNA binding activities. The complex is thought to be involved in transcription and DNA break repair (Utley et al. 2005).

The INO80 chromatin-remodelling complex, regulates the abundance and positioning of nucleosomes and DNA damage (Morrison et. al. 2017). R2TP complex (GO: 0097255) involved in RNA pol II assembly and apoptosis (Ahmad et al. 2013). The remaining motifs, namely 7, 9 and 10–16 are novel; their molecular characterization needs to be explored in future studies (Supplementary Figure S6, Supplementary Table S13).

The 3D structures of nine TaRuvBL proteins showed high proportion of amino acid residues (> 90%), falling in the most favoured regions, suggesting that the predicted models are reliable (Batra et al. 2017; Kumar et al. 2018). We also found that the 3D structures of six TaRuvBL1 proteins (TaRuvBL1a-4A, TaRuvBL1a-4B, TaRuvBL1a-4D, TaRuvBL1b-3A, TaRuvBL1b3B, TaRuvBL1b-3D) had homo-hexameric structures whereas TaRuvBL2 proteins (TaRuvBL2a-2A, TaRuvBL2a-2A, TaRuvBL2a-2A) had monomeric structure (Supplementary Figure S4). In some earlier studies, it has also been emphasized that RuvBL1 is the eukaryotic homologue of the bacterial DNA-dependent ATPase and helicase RuvB (Putnam et al. 2001, Yamada et al. 2001), which assembles into functional homo-hexameric rings, and is the motor that drives branch migration of the Holliday junction in the presence of RuvA and RuvC during homologous recombination (Tsaneva et al. 1993). It was also proved experimentally (Matias et al. 2006) that crystal structure of the human AAA + Protein RuvBL1 had hexameric ring-shaped structure and is formed of ADP-bound RuvBL1 monomers (Matias et al. 2006). AAA + proteins generally form hexameric ring structures and contain conserved motifs for ATP binding and hydrolysis like the Walker A (P-loop) and Walker B box (Gorbalenya et al.1989; Schmid et al.1992), the Arg finger, and sensor residues. In the present study also, TaRuvBL proteins have been shown to consist of homohexameric rings, made up of six monomeric protein structure. In the present study, the 3D structure of OsRuvBL reference proteins was also examined (Supplementary Figure S4), where OsRuvBL1a and OsRuvBL1b were shown to exhibit homohexameric structure whereas OsRuvBL2a was shown to be monomeric structure. It confirms that protein structure of RuvBL family is conserved among species. Thus, the predicted 3D structure of these proteins provides an understanding to study the molecular functions of the RuvBL helicase protein family. Though monomeric subunits of RuvBL1b and RuvBL2a proteins are fully capable to perform ATPase and DNA helicase activities separately, the hetero polymorphic protein complex (heterododecamer) of these proteins may provide stability to RuvBL protein and may facilitate to provide DNA helicase activities at varying positions simultaneously. RuvBL1 and RuvBL2 interactions have also been reported earlier suggesting the formation of heterohexameric ring structure (Abrahao et al. 2021; Cheung et al. 2010).

Protein -protein interactions

Several studies have demonstrated that RuvBL proteins interact with other proteins to regulate diverse processes. For instance, A. thaliana mutants for these genes resulted in reduced telomerase activity and suggested the involvement of AtRuvBL proteins in plant telomerase biogenesis and show protein interaction with AtTERT, a high-molecular-weight protein (Schořová et al. 2019). Human RuvBL proteins have also been shown to interact with transcription factor MYC which is required for the expression of many genes involved in cell-cycle transition events and proliferation (Wood et al. 2000).

Protein interaction of represenatative protein TaRuvBL1a-4A is shown in (Fig. 10) (Supplementary table S14). The results of different protein interaction can provide valuable information in future for the prediction of working pathways and protein involved in it with TaRuvBL genes and for the further functional characterization of TaRuvBL genes.

In-silico expression and qRT-PCR

Five of the nine TaRuvBL genes exhibited high expression in root, spike, stem, leaf, and grain, at different developmental stages (Fig. 12a). Expression analysis of three representative genes was conducted using qRT-PCR. Two genes (TaRuvBL2a-2D, TaRuvBL1b-3B) were found to exhibit differential expression (upregulation) during qRT-PCR (Fig. 13). In another earlier study also, expression of OsRuvBL1a differed under abiotic stresses (cold, heat and salinity; Saifi et al. 2017), which was later confirmed using transgenic approach (Saifi et al. 2021). Similar results were also reported in Arabidopsis (Holt et al. 2002) and pigeon pea (Singh et al. 2020). TaRuvBL gene expression and upregulation of its transcript under abiotic stress conditions suggest its involvement in multiple cellular pathways. Further studies in wheat could be conducted to understand fully the role of TaRuvBL genes, and to utilize allelic variation in these genes for imparting heat stress to wheat cultivars

RuvBL (RuvB Like) genes derive their name from RuvB helicase gene of E coli and occur in the genomes of both plants and animals including humans. In humans, these genes are associated with several diseases including some cancers; in plants these genes provide tolerance against abiotic stresses like heat and drought. Keeping this in view, the RuvBL genes have been studied in Arabidopsis and rice and their utility demonstrated in pigeon pea through development of transgenics. In the present study, nine RuvBL genes were identified in wheat for the first time and named TaRuvBL genes that were found to be located on nine different chromosomes, one each assigned to chromosomes of three homoeologous grups 2, 3, and 4. The structure of TaRuvBL genes and that of the corresponding proteins is described in this article in some detail. The RuvBL proteins have ATPase activity; the monomer units associate into hexamers, followed by association of two hexamers into a dodecamer, also described as PAQosome (Particle for Arrangement of Quaternary Structure, a large multisubunit chaperone complex). A more detailed follow-up study of the structure of gene/protein may allow identification of causal polymophisms, which may then be used both for MAS and TALEN or CRISPR/Cas-mediated gene/base editing for developing thermotolerant and drought tolerant wheat cultivars.

Conflicts of interest

The authors declare no conflicts of interest.

Funding

No funding was received for conducting this study.

Author contributions

JC conceived and planned this study under mentorship of PKG. JC, TG, VG did the analysis. KS performed molecular dynamic simulation and docking and also wrote the related text in the manuscript. JC and SK prepared the first draft of the manuscript, which was improved by RB. PKG edited and finalised the manuscript.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Acknowledgements

Thanks, are due to Head, Department of Genetics and Plant Breeding, Ch Charan Singh University, Meerut, for providing the facilities for this work. Thanks, are also due to Dr. Sachin kumar, Assistant Professor, Department of Genetics and Plant Breeding, Ch Charan Singh University, Meerut, for providing seeds of a wheat genotype (Giza 168).

Ethics approval

The research was carried out in accordance with a named standard.

FAO: https://doi.org/10.4060/cb3673en
conserved domain database (CDD): https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml
Ensemble plant database: http://plants.ensembl.org/
TB tool: https://github.com/CJChen/TBtools/
GSDS: http://gsds.gao-lab.org/
Phytozome database v13: https://phytozome-next.jgi.doe.gov/
Repeat masker v4.0.5: www.repeatmasker.org/
psRNATarget: https://plantgrn.noble.org/psRNATarget/
PlantCARE database:http://bioinformatics.psb
ProtParam tool: htt psRNATarget psRNATarget ps://web.expasy.org/protparam
MEME suite: https://meme-suite.org/meme/tools/meme
InterProScan 82.0 database: https://www.ebi.ac.uk/interpro/search/sequence/
GOR4: https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html
Swiss Model: https://swissmodel.expasy.org/
SAVES: https://saves.mbi.ucla.edu/
PROCHECK: https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/
VERIFY3D: https://www.doe-mbi.ucla.edu/verify3d/
ERRAT: https://www.doe-mbi.ucla.edu/errat/
GROMACS: https://www.gromacs.org/
Hex: http://hex.loria.fr/
BindUP: https://bindup.technion.ac.il/
Molegro Molecular Viewer: http://molexus.io/molegro-molecular-viewer/
FATCAT: https://fatcat.godziklab.org/
STRING: https://string-db.org/
Clustal-Omega program: https://www.ebi.ac.uk/Tools/msa/clustalo/
MISTIC web server: http://mistic.leloir.org.ar/index.php
expVIP: http://www.wheat-expression.com/

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Identification and characterization of RuvBL DNA helicase genes for tolerance against abiotic stresses in bread wheat (Triticum aestivum L.)

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Material And Methods

Gene sequence analysis

Identification of RuvBL genes in wheat and related species

Structure of RuvBL genes

Analyses of synteny

Identification of TEs and miRNAs

Promoter analysis- Cis regulatory element

Protein sequence analysis

Physico-chemical properties, secondary structures, and conserved motifs

3D structure analysis, evaluation, and optimization

Structural simulation

Docking of proteins

Superimposition of 3D structures, sub-cellular localization and gene ontology

Interaction network analysis

Multiple sequence alignment and phylogenetic analysis

qRT-PCR analysis

Plant Growing Conditions

RNA isolation and qRT-PCR

Results

Analysis of wheat RuvBL genes

Identification and structure

Orthology of genes among eight species

Synteny between different grass species

MicroRNA (miRNA) and their targets in genes

Analysis of RuvBL proteins

Characterization of proteins

Functional domain and motifs

Alignment and functional annotation of 3D structures

Sub-cellular localization and GO analysis

Protein–protein interactions

Phylogenetic analysis

Expression patterns of genes under heat stress

Discussion

TaRuvBL genes in wheat and related species

Evolution of RuvBL genes (synteny and phylogeny)

Cis-regulatory elements (CREs) in promotor regions

Transposons elements (TEs) within genes

miRNAs sequences in genes

Structure and function of proteins

Protein -protein interactions

Summary And Conclusions

Declarations

Resources

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1