IntroductionPotato (Solanum tuberosum L.), is the widely accepted and an important food crop ranked third after rice and wheat. It has potential in food security at global level due its high demand in human consumption as well as processing industry. The highly nutritious potato crop supplemented with proteins, carbohydrates, calcium and vitamins content is a good source of balanced diet. Potato is highly productive and adaptive crop as it can be grown in a short period of time and has the capability of adaptation in different intercropping systems as well as in diverse soil types (pH value of 5 to 7.5)1,2. In terms of production, total global output of 359 million tons was reported by more than 150 countries in 20203. Considering the productivity, China ranked the first followed by India owing to 48.2 million tons production annually in 20214. Keeping in view the rise in population growth, the potato demand is increasing at fast pace in the developing countries. Potato demand is accelerating at a great pace due to focus of general public demand towards nutritional shift to the processed and energy-rich food products. In this scenario, potato is considered to be staple food known to provide immense essential nutrients in terms of consumption1,5. To meet this demand, researchers are exploring the potato crop at the gene and genomics levels to tackle with the abiotic and biotic stresses.During the life cycle in potato, the plants are susceptible to multitude of abiotic and biotic stresses. Abiotic stresses such as salinity, cold, drought, excess water, and carbon dioxide stresses have a great effect on yield and growth as the prevailing climate crises are unpreventable6. On an average, 50% of the crop yield losses were estimated around the world primarily due to abiotic stresses7. World’s one third land area is affected by salt stress which leads to land degradation and lowers the soil quality causing a great decline in agricultural productivity or yield8,9,10. The total arable area under salt stress is expected to increase above 50% by 20508,9. To cope up with the abiotic stress, thorough knowledge of abiotic stress responsive genes is absolutely necessary to meet the competitive production of potato demand. The fluctuations in the environmental conditions and narrow genetic base among potato crop serve as a big challenge in the potato breeding research11. Earlier studies demonstrated the decline in biomass production, shoot length, and premature plant death due to salt stress12. In India, 6.74 million hectares of land is under the effect of salinity stress, of which, 0.15 million hectares in Punjab is covered by salt soil13. Salt stress in potato causes ion disequilibria and cellular functions alterations, which results in the increase of the mineral distribution swapping, respiration rate, cell membranes instability, integrity loss, and ultimately turgor pressure decline leading to yield loss8. Drought is a multipronged stress which has an adverse effect on almost all the plant processes and it causes reduction in total leaf area, stomatal closure and decrease in CO2 uptake which leads to inhibition of photosynthesis14,15. In India, 63 million hectares of land area is irrigated and 28% of cultivation land suffers water shortage and is considered drought-prone. Drought conditions trigger water potential loss in plant tissues due to moisture deficient soil is known as hydrological drought15. Drought and heat stresses are the main concerns in potato due to erratic rainfall, temperature rise, and dry spill period16. Low temperature is also one of the critical and major stresses in potato plants where the chilling effects occurs at temperature above 0° while frosting occurs at temperature below 0°. Cold stress cause significant loss in the plant productivity, water uptake and plant metabolic activities17. Cold stress causes increase in membrane viscosity, slows down cell metabolism, decreases energy dissipation and leads to free radical formation generating oxidative stress18. The plants perceive and respond to the low temperature stress that causes loss of water from the plant cells and to mitigate that stress enable the activation and suppression of stress-responsive genes to survive and thrive under unfavourable conditions19. All these abiotic stresses causes changes in gene expression resulting in sequential biochemical and physiological changes in the plant cells.Gene expression is a complex process which occurs at different stages of transcription, post-transcription, and translation. Translation initiation factors play an essential role in plant expression under different stress conditions20. Moreover, it is a high energy-demanding process and can be used as a major target for plant research in response to cellular stresses21. The abiotic stress conditions block the translation initiation to halt the production of functional protein. Therefore, the genes responsible for initiation of translation process can be targeted for enhancing sustainability and productivity of agricultural crops. The polysomic genetics complexity and heterozygosity in potato also hinders the phenotyping due to abiotic stresses11. So, the emphasis on the in-depth knowledge of regulatory pathway of eIF genes could be a contributing factor towards crop improvement. Several reports of expression and in-silico analysis for the identification of genes are available viz. heat shock transcription factor gene in rice22, phosphate transporter gene in sugarcane23, starch biosynthesis genes in potato24, flowering genes in citrus25, parthenocarpic fruit set‑related genes in cucumber26, and carotenoid cleavage oxygenase gene in citrus27. In eukaryotes, the occurrence of translation initiation factors has been reported to be responsible in the mitigation of abiotic stresses in various plant species such as Oryza sativa L.20 , Arabidopsis thaliana L.28 , and Mangifera indica L.29. The complex translational strategies serve as a challenging task to implement the genetic information using biotechnological interventions. The targeting of molecular genetic mechanism of translation initiation factor genes using in-silico analysis approach has the potential to refine gene expression and metabolic modulation. Initiation factors other than main eIF regulators are also crucial for the regulation of initiation process in translation under abiotic stresses30. The eukaryotic translation initiation factors are an essential machinery part of the translation process which are vital for plant developmental processes and permit stress endurance in plants in response to disease incidence31. The protein complexes which initiate the eukaryotic translation, function as input for regulation of post transcription processes and play a role in stabilization of ribosomal pre-initiation complexes32. The aim of the current investigation is to conduct the in-silico analysis of eIF genes in potato from ensemble database to delineate the regulatory network of abiotic stress tolerance. The expression of abiotic stress tolerance genes was studied through qRT-PCR studies. Additionally, the identified candidate genes could be exploited for mining the functional features beneath the gene and genomics level for crop improvement.Materials and methodsPlant materialThe potato tubers of two genotypes i.e. Lady Rosetta and Punjab Potato-101 were obtained from the Department of Vegetable Sciences, Punjab Agricultural University, Ludhiana. Punjab Potato-101, a short day and table purpose variety, has been developed by Punjab Agricultural University, Ludhiana, India in 2023. It is main season variety with early bulking potential and falls in 90 days maturity group. It is being well received by the growers and expected cover significant area under potato cultivation in the state. Lady Rosetta is a an early to medium maturity processing type variety widely popular among the growers and the processing industry. The nodal cuttings were used as explants and cultured on MS (Murashige and Skoog) media33 containing 2,4-D (2,4-Dichlorophenoxyacetic acid)34 for the callus formation. After 20 days, the grown callus was transferred to rooting media and further sub-cultured to grow seedlings. Overall, the potato tubers were grown into complete plantlets after 60 days. We also observed the morphological data of root length, fresh weight and dry weight per plant of both species (Table S1).Identification, sequence retrieval and chromosome localization of eIF genesThe literature was surveyed to identify the genes associated with abiotic stresses in potato. Total thirty-one genes from eIF family were found to be responsible for abiotic stress control in potato. Their coding, genomic, and amino acid sequences were retrieved using Ensemble plant database35. The chromosomal position of these 31 genes was arranged in an organized way on the chromosomes using Phenogram Plot (https://visualization.ritchielab.org/phenograms/plot) from short to long arm of telomere.Intron-exon organization of eIF gene family in potatoAll these collected sequence information was employed for the depiction of the intron-exon organization of the eIF gene family in the potato. Gene structure display server GSDS2.0 (https://gsds.gao-lab.org/) was used to depict the organization of gene structure into exon-intron regions36.Identification of conserved motifs and cis-response elementsThe promoter region of the eIF genes were surveyed for the identification of cis-response elements. NCBI database (https://www.ncbi.nlm.nih.gov/) was used for the retrieval of genomic sequence of each gene with promoter region of (> 300 bp) targeting upstream of the transcription start site. The complementary and non-complementary strands of the upstream region of the transcription start site was examined using New PLACE (https://www.dna.affrc.go.jp/PLACE/)37. The conserved motif locations were identified using the amino acid sequences of the eIF genes in the MEME suite (https://meme-suite.org/meme/tools/meme )38. The parameters include the discovered maximum numbers of motifs set at 10 with minimum and maximum width of 6 and 50, respectively, and site number range from 2 to 600 in each motif among the sequences.Phylogenetic analysisTo understand the evolutionary relationship, 19 eIF family genes from Arabidopsis thaliana L., 12 from S. lycopersicum L. and 31 from S. tuberosum L. were employed for construction of the phylogenetic tree. The multiple sequence alignment was done using amino acid sequences in the Clustal W (parameters include 10 gap opening and 0.1 gap extension penalty). The constructed tree depends upon the maximum likelihood method with 1000 bootstrapping replications and maximum thread set at 4 using MEGA11 software39. Further, iTOL Interactive Tree of Life (https://itol.embl.de/) was used for the visualization of the phylogenetic tree.Gene ontology (GO) and reactome database pathway analysisThe functional annotation of eIF genes in the BLAST2GO tool (https://www.blast2go/com/) was done through the use of retrieved amino acid sequences40. These annotated genes were categorized into biological processes, molecular function and cellular component. The annotated genes were used for pathway analysis for the prediction of enzymatic functions of genes41. The pathway analysis using reactome database was performed in the OmicsBox 3.4.5.Prediction of physico-chemical properties, three-dimensional structures and protein-protein interaction network of eIF proteinsThe physicochemical properties viz. amino acid length, the molecular weight, instability index, aliphatic index, isoelectric point (pI), and grand average hydropathy (GRAVY) were predicted using online Protparam expasy software (https://web.expasy.org/protparam/)42. The Pfam domain and the intercellular localization of the eIF proteins were identified using Pfam 37.0 (https://pfam.xfam.org/) and the ProtComp version 9.0 server (http://www.softberry.com/) tools, respectively43. The secondary structure of eIF genes analysed using Self-Optimized Prediction Method with Alignment tool (SOPMA, https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html )44. The information of the secondary structure of the eIF proteins including percentage of β-sheet, α-helix, random coils and turns were retrieved. The parameters with similarity threshold 8, output width of 70, and window width 17 were included to depict these four conformational states of the protein (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html). The three-dimensional structures of eIF proteins were built using homology modelling PHYRE 2 (http://www.sbg.bio.ic.ac.uk/~phyre2/)45,46. The amino acid sequences were uploaded under batch processing and modelling of the protein structures were done at > 90% confidence level in advanced model.Expression study of eIF gene family in potatoThe expression analysis of eIF genes linked with abiotic stress viz. salt, drought and cold stress treatments (SteIF3, SteIF4B-1, SteIF1a.1) was done in Punjab Potato-101 cultivar using qRT-PCR. The Lady Rosetta potato variety was used as a constitutive control. The housekeeping GAPDH gene was used to calculate the relative gene expression. The gene SteIF1a.1 belonging to Clade 1 and conserved pattern of motif locations permits interaction with the other initiation factors to streamline the initiation of translation was selected. We also found that the genes SteIF3 and SteIF4B-1 exhibit highest number of expressed genes in response to salt, drought and cold stress in gene ontology studies. Therefore, we select these three candidate genes SteIF-1a.1, SteIF3 and SteIF4B-1 each from factor 1 A, 1B and 1 C that encodes translation initiation factor 1 A, Plant specific eukaryotic initiation factor 4B, and Eukaryotic translation initiation factor 3 subunit C respectively to perform the initiation of the translation process. Salt stress was given using 100 mM and 150 mM of sodium chloride (NaCl) dissolved in MS media were given to 15 days old in vitro cultured potato plants. Drought stress was applied through transferring of plants in MS media containing 2.5% and 5% (w/v) polyethylene glycol (PEG). The cold stress treatment was performed through the transfer of cultured plants to growth chambers with temperature of 4 °C and 8 °C under the light and photoperiodic conditions (12 h light and12 hr dark). In gene expression, the basis for setting different stress treatments (such as salt concentration, PEG concentration, temperature gradient) emanate from research findings in potato47,48,49. Three biological replicates from each genotype were used for each stress treatment. All the stress treatments were performed for 24 h (hr), 48 h and 72 h, respectively. The poly-house conditions with average temperature of 12–18 °C were maintained for the growth of potato cultivars at School of Agricultural Biotechnology, PAU, Ludhiana. After 60 days of germination, the collection of leaf samples was done in triplicates for RNA isolation using TriZol™ reagent procedure. cDNA synthesis kit (Thermoscientific First Strand) was used followed by quantification using gel electrophoresis and spectrophotometer (NanoDrop 2000D, Wilmington, USA). The primer designing for qRT-PCR was done using Primer3 tool (https://bioinfo.ut.ee/primer3/) (Table S2). Each reaction system constitutes 100ng of cDNA template concentration and the cycling parameters were pre-denaturation at 94 °C for 40 s, followed by 40 cycles of denaturation at 94 °C for 10 s, annealing at 57.2 °C for 30 s and final extension at 72 °C for 10 min50. Three biological and technical replicates for each sample were used. The change in fold change expression was calculated following Livak & Schmittgen51 approach. The post-hoc Tukey to compare the non-stressed vs. the stress plants (i.e. under salinity, cold and drought stress) were performed to determine the statistical significance analysis.Sequence alignment analysisAdditionally, a sequence alignment analysis of the promoter regions of selected candidate genes SteF1a.1, SteIF3 and SteIF 4B-1 was done through BLAST approach using potato genotypes i.e. Solanum tuberosum group Phureja, Solanum chacoense and Solanum candolleanum available in the SPUD database. We identified homologous genes and subsequently the promoter sequences of these homologous genes were retrieved. The key motifs and cis-response elements were identified using MEME tool and NEWPLACE tools respectively.ResultsIdentification and chromosomal locations of eIF genes in potatoA total of thirty-one (31) eIF genes associated with abiotic stress tolerance from the potato genome were identified from the survey literature (Table 1). These genes included Translation initiation factor 1 A (SteIF-1a, SteIF-1a.1, SteIF), Eukaryotic initiation factor 2B subunits alpha/beta/delta (SteIF-2balpha, SteIF-2balpha-2), Translation Initiation factor eIF-4e (SteIF(ISO)4E-1, SteIF(ISO)4E-2), Initiation factor 4G (St4F/eIF-4 F-1, St4F/eIF-4 F-2), Plant specific eukaryotic initiation factor 4B (SteIF-1), Translation initiation factor IF6 (SteIF-2, SteIF-4.1, StEMB1624), Translation elongation factor IF5A-like (SteIF5A-4, SteIF-5A5), Probable RNA-binding protein EIF1AD (SteIF-5), Eukaryotic translation initiation factor 3 subunit D (SteIF-3),, Initiation factor 2B-related (SteIF2b delta), Translation initiation factor IF2/IF5 (SteIF2beta, SteIF5, SteIF-1.1), Methylthioribose-1-phosphate isomerase (StCIG2), Translation initiation factor IF-1 (SteIF-chloroplastic), Plant specific eukaryotic initiation factor 4B (SteIF4B-1, SteIF4B-2), Translation elongation factor IF5A-like (SteIF5A3, SteIF5A-1/2), Eukaryotic translation initiation factor 3 subunit C (SteIF3), and Initiation factor 2B-related (SteIF2Bbeta).The Pfam domain of SteIF-2.1 and SteIF-3.1 were not predicted yet.The distribution of the all the eIF genes on the twelve chromosomes in potato were shown in Fig. 1a. The majority of the eIF genes (n = 7) were localized on chromosome 2 (SteIF-2, SteIF-2.1, SteIF5, SteIF-3, SteIF-3.1, SteIF2b, d and StEMB1624); and on chromosome 12 with n = 7 (SteIF4.1, SteIF5A1/2, SteIF2b.a2, SteIF-1chl, St4FeIF1 and St4FeIF2). The genes were present over the entire regions of chromosomes in chromosome 2 and 3 while other genes were majorly located towards the end of the telomeric end of the chromosome 1, 4, 6, 7, 9 and 12. No genes were found on chromosome 11.Table 1 Detailed description of eIF genes of potato used in the present study with their gene identifier names.Full size tableThe exons and introns distribution of eIF genes using GSD2.0 software were revealed in Fig. 1b. The exon-intron organization of eIF genes in potato depicted that the shortest eIF gene was SteIF(ISO)4E-1 which was found to be less than 1 kb with only one exon and the longest StCIG2 comprises of more than 9 kb. The maximum number of exons (9) were included in St4F/eIF-4 F-1, StCIG2, SteIF2Bbeta and SteIF2beta genes and least number of exons (1) present in SteIF(ISO)4E-1, SteIF-1.1, SteIF-1-,chloroplastic, SteIF-3.1, SteIF5 and intronic sequence were not present in these genes. The longest intron was present in the StCIG2 gene. Two genes viz. SteIF-4.1 and SteIF-2b-delta have same similar number of exons (7) rather than their dissimilar length. These eIF genes represent maximum variation in the exon-intron organization. Similarly, the genes St4F/eIF-4 F-2, SteIF, SteIF1a.1, SteIF-1a, SteIF4B-2 with two exons, SteIF5, SteIF3, SteIF4B-1 with three exons, StEM1624 with four exons, and SteIF(ISO)4E-2, SteIF2b-alpha2, SteIF-5A5, SteIF5A-1/2, SteIF5A-4, SteIF5A-3 with five exons were represented.Fig. 1(a) Distribution of eIF genes on the 12 chromosomes of potato. Different genes are represented by different colours (b) Intron-exon organization of eIF genes in potato. Exons are shown by orange round corner rectangles and black curved lines represented by introns.Full size imageCis- response elements and conserved motifs identificationThe regulation of gene expression occurs by either repression or activation of the transcription. The cis- response elements occur in the promoter region of the genes for the regulation of the transcription through interaction with the transcription factors. About ten major cis response elements were identified using New PLACE (https://www.dna.affrc.go.jp/PLACE/). These cis response elements constitute ABRE box, TATA box, CRT box, GATA box, CAAT box, CBF box, G box, DRE box, MYB box and WRKY box were observed in the eIF genes majorly at the promoter region (Fig. 2a). The most abundant Abscisic acid Response Element (ABRE) box performs gene regulation linked with cold and osmotic stress response in plants52. Simultaneously, CBF box associated with the gene expression during cold induction in Arabidopsis thaliana L53. , DRE box (dehydration responsive elements) during up regulation in response to dehydration, low temperature and salinity stress54, MYB box found to be present on promoter regions of some abiotic stress controlling genes55. The majority of the eIF genes (36%) constituting CAAT box. Six genes i.e. SteIF(ISO)4E-2, SteIF5A-4, SteIF-3.2, SteIF-4.1, SteIF4B-1, SteIF4B-2 and StEMB1624 does not comprise any cis-response elements.Motifs refer to the short-conserved sequence pattern linked with a distinct structural site to perform distinct protein functions56. The motifs have the potential to predict the unidentified proteins57. The online MEME suite was used for the motif identification of all these eIF genes. Motifs discovered in the present study were aligned with the constructed phylogenetic tree (Fig. 2b). The motifs 1, 2, 3, 7 and 10 comprised largest motif whereas motif 9 exists with least frequency. Most of the genes i.e. SteIF-1a, SteIF-1a.1, SteIF, SteIF-5 A-4, SteIF-5A5, SteIF-5A3, SteIF-5 A-1/2 comprised of three motifs while SteIF(ISO)4E-1, SteIF(ISO)4E-2, St4F/eIF-4 F-1, St4F/eIF-4 F-2, SteIF5 constitute only one motif. The motifs locations for all the genes were observed on the positive strand.Fig. 2(a) The percent cis response elements in the eIF genes and functional annotation of cis-element. Different colours represent different cis response elements (b) Phylogenetic tree constructed using eIF genes from Arabidopsis thaliana L., S. lycopersicum L. and S. tuberosum L. and its alignment with the conserved motif identification of eIF genes in potato.Full size imageConstruction of phylogenetic treeThe eIF family genes retrieved from three plant species Arabidopsis thaliana L., S. lycopersicum L. and S. tuberosum L. were used for constructing the phylogenetic tree (Fig. 3). The tree was categorized into three main clades- clade1 which includes 13 genes, clade 2 with 26 genes and clade 3 with 23 genes. Clade 1 constituting AteIF2, AteIF-2, SteIF-2-1, AteIF2alpha, AteIF-2.1, SteIF, SteIF1a.1, SteIF-1a, AteIF2/5, AteIF2/5 − 1, SteIF5, SteIF-1.1, SteIF-5 genes have interaction with the initiation factor IF1/IF2/IF5, and initiation factor 2B subunits alpha/beta/delta. Clade-2 consists of AteIF3C, AteIF-3C2, SleIF-3 C, SteIF3, AteIF5A-1, AteIf5A-1 2, AteIF5A-3, SleIF5A, SteIF5A-4, SleIF5A-3, SleIF5A-1, StEIF5A5, SleIF5A-4, SteIF5A-1/2, SleIF5A-2, SteIF5A-3, SteIF-2alpha, AteIF4B-1, AteIF-4B, SteIF4B-1, SteIF4B-2, SteIF-1, AteIF-2beta, SleIF2beta, SleIF2beta2, SteIF2beta genes mainly participates in the activation of translation elongation factors. Clade 3 constituting AteIF-2B, AteIF-2Balpha, SteIF-2balpha-2, StCIG2, SteIF2Bbeta, AteIF-4E, SteIF(ISO)4E-1, SteIF(ISO)4E-2, SteIF2b, delta, AteIF-3.7, SleIF3D, SteIF-3, SleIF-1,chloroplastic, SteIF-1, chloroplastic, AteIF-6, SleIF6, SteIF-4.1, SleIIF6.2, SteIF-2, StEMBL1624, St4F/eIF-4 F-1, St4F/eIF-4 F-2, SteIF-3.1 specifically involved in the translation initiation factor 4E for binding to the 5′ cap structure of messenger RNAs for the recruitment of ribosomes. The genes were grouped on the basis of their functions.Fig. 3Construction of phylogenetic tree among 62 eIF genes with bootstrap values retrieved from Arabidopsis thaliana L., S. lycopersicum L. and S. tuberosum L.Full size imageGO and reactome pathway analysisThe eIF genes functional annotation was performed using BLAST2GO platform categorized into three categories i.e. biological processes, molecular function and cellular components. The highest number of protein functions linked with molecular processes (18), biological processes (17) and cellular components (12) as shown in Fig. 4a. Among 31 eIF genes, the three genes SteIF-1, SteIF4B-1, SteIF4B-2 (GO:0003729) perform functioning in the eukaryotic translation initiation factor 4B were linked with the cellular components, and five genes SteIF(ISO)4E-1, SteIF(ISO)4E-2 (GO:0006417), SteIF5A-4, SteIF5A3, SteIF5A-1/2 (GO:0045905) known for the eukaryotic translation initiation factor activity during translation linked with the biological processes. All other genes SteIF-1a, SteIF-1a.1, SteIF (GO:0005737) that codes for eukaryotic translation initiation factor 1 A, SteIF-2balpha (GO:0005851) predicted: uncharacterized protein LOC107061760, SteIF-2balpha-2 (GO:0005829) translation initiation factor eIF-2B subunit alpha-like, SteIF(ISO)4 F-1, SteIF(ISO)4 F-2 (GO:0016281) eukaryotic translation initiation factor isoform 4G-1, SteIF-2 (GO:0005730) eukaryotic translation initiation factor 6 − 2, SteIF2b, delta (GO:0005829) translation initiation factor eIF-2B subunit delta-like isoform X1, SteIF-4.1, StEMB1624 (GO:0005730) eukaryotic translation initiation factor 6-2-like, SteIF2beta (GO:0005850) eukaryotic translation initiation factor 2 subunit beta-like, SteIF2beta (GO:0005634) methylthioribose-1-phosphate isomerase, SteIF-1,chloroplastic (GO:0005829) translation initiation factor IF-1, chloroplastic, SteIF3 (GO:0005852) eukaryotic translation initiation factor 3 subunit C-like, SteIF5 (GO:0005829) eukaryotic translation initiation factor 5-like, SteIF2Bbeta (GO:0005851) translation initiation factor eIF-2B subunit beta-like, SteIF-1.1 (GO:0005829) eukaryotic translation initiation factor 5-like belongs to the cellular component category.The highest gene numbers (n = 25) were reported in molecular function followed by biological process (n = 21) and cellular component (n = 8). Under molecular functions and biological processes, eIF genes viz. SteIF4B-1, SteIF3, SteIF-4.1, StEMB1624, SteIF-2balpha-2, SteIF4B-2, SteIF5, SteIF-1.1, St4F/eIF-4 F-2, St4F/eIF-4 F-1, SteIF2b, delta, SteIF-1, SteIF-2, SteIF2beta, SteIF5A3, SteIF2Bbeta, SteIF-1,chloroplastic, SteIF(ISO)4E-2, SteIF(ISO)4E-1, SteIF5A-1/2, SteIF, SteIF5A-4, SteIF-1a.1, SteIF-1a, SteIF-2balpha perform translation initiation factor activity. Under cellular component, the genes (n = 8) SteIF-4.1, StEMB1624, SteIF-2balpha-2, SteIF5, SteIF-1.1, SteIF-1-chloroplastic, SteIF2b, delta, SteIF-2 predominate in cytosol. Other eIF genes found to be localized in cytoplasm (n = 4), nucleolus (n = 3) and membrane (n = 1). Collectively, eIF genes mainly activate initiation factors of translation through RNA binding in the cytosol conferring towards abiotic stress tolerance in plants.Reactome database pathway analysis showed the proteins linked with the initiation step of the translation process in the cytosol (Fig. 4b). This pathway demonstrated that the translation initiation complex formation resulting in the generation of ceruloplasmic mRNA associated with phosphor-L-13a to process the ribosomal scanning. This is a complex process that involved the series of enzymatic activities. Mainly eIF3 and eIF1A genes bind to the 40 S ribosomal subunit for the 43 S pre-initiation complex formation. Lastly, met-tRNAi binds where for the formation of ternary complex that determines the efficacy in the initiation step of translation. The pathway mainly showed the proteins linked with the regulation of expression of translation initiation factors.Fig. 4(a) Gene ontology analysis annotated eIF genes into biological process, molecular function and cellular component (b) Reactome database pathway showing the proteins involved in initiation step of the translation initiation factor.Full size imagePhysico-chemical properties and homology modelling of eIF genes related proteinsThe physico-chemical properties of eIF genes were depicted using ProtParamExPasy software (Table S3). SteIF3 protein had maximum length of 914 amino acids whereas SteIF-3.1 has minimum length of 50 amino acids. pI of all the proteins falls in the range of 4.67–10.1. The molecular weight of eIF proteins observed to be higher than 5 kDa. The proteins viz. SteIF-1a, SteIF-1a.1, SteIF-2balpha, SteIF, SteIF(ISO)4E-1, SteIF(ISO)4E-2, St4F/eIF-4 F-1, St4F/eIF-4 F-2, SteIF-1, SteIF2.1, SteIF-3, SteIF2b delta, SteIF2beta, SteIF2Bbeta, SteIF-chloroplastic, SteIF4B-1, SteIF4B-2, StEMB1624, SteIF3 had more than 40 instability index and hence considered as unstable. The proteins viz. SteIF-2b alpha-2, SteIF-2, SteIF5A-4, SteIF-5, SteIF3.1, SteIF4.1, StCIG2, SteIF-5A5, SteIF5A-1/2, SteIF5, SteIF-1.1 had less than 40 instability index and hence, reported as stable proteins. GRAVY (Grand Average of Hydropathy) index of all proteins fall in the range of -0.957 to 0.102 while aliphatic index of the proteins fall in the range of 42.44–105.39.The homology modelling was done using Phyre software for the 3D structures prediction of the eIF proteins (Fig. S1). The details of PDB header, template proteins, Gene identifier names and confidence (%) was shown in Table S4. Majority of eIF proteins have 100% confidence except SteIF-3.1, SteIF-1, SteIF4B-2, SteIF-2balpha, SteIF(IS)4E-1, SteIF-2.1 and SteIF4B-1. On the basis of PDB header, the template proteins of the models were reported as translation and RNA binding proteins. Besides translation, eIF proteins perform multitude of functions performing role in apoptosis, immune system, ribosome, isomerase, cell invasion, viral protein, translation regulator, membrane protein, structural protein and viral protein activities. The secondary structure analysis was done through protein homology. The average number of random coils (53.82%) followed by α-helix (30.69%) and β turns (15.31%) among all the eIF proteins are described in Fig. S2 (a). Only SteIF-2b alpha exhibited conformational state turns of 5.67%. The different conformational stages in the secondary structure of all proteins are shown in Fig. S2 (b).Expression analysis of eIF genesThree eIF genes associated with abiotic stresses (SteIF1a.1, SteIF3, SteIF4B-1,) were analysed under salt, drought and cold stress conditions through qRT-PCR experiment in the present study (Fig. 5). The housekeeping GAPDH gene was used for normalization and as constitutive gene internal control in this analysis. During qRT-PCR analysis, GAPDH is the most frequently used endogenous control due to its consistent expression at different time points and varying manipulations to ensure the reproducibility of the experiment58. It was observed that SteIF1a.1 showed highest 25.45 fold change expression at 24 h under salt stress (100mM NaCl) conditions. SteIF3 showed highest 8.1 fold change expression at 24 h under drought stress (2.5% PEG) conditions The SteIF4B-1 gene showed the high fold change expression under cold stress treatment (4 °C) at 72 h. The results showed that SteIF1a.1, SteIF3 and SteIF4B-1 genes are the prominent genes showing high change in fold expression under salt, drought and cold stress conditions, respectively, combating towards abiotic stress in potato. We also observed significant difference at P