Introduction

As a gaseous phytohormone, ethylene is produced in most plant tissues. It is important in regulating plant growth and developmental processes, including seed germination, root initiation, root gravitropism, fruit ripening, flower and leaf senescence, abscission, and stress responses. In plant tissues, ethylene production is typically low but increases at developmental stages such as ripening and senescence and in response to mechanical and environmental stresses1. Nutrient stress is a complex phenomenon that leads to low or high availability of nutrients to the plant2. Different genes are also involved in plant responses due to nutrient stresses3. Hence, to understand the nutrient stress response mechanism of Musa acuminata in roots, it is important to identify and analyze the genes involved in ethylene biosynthesis. Furthermore, the ethylene biosynthesis (EB) genes in response to nitrogen (N) and potassium (K) stress have not yet been analyzed in Musa acuminata (banana) roots.

Ethylene also acts as an important modulator and inducer of plant aging4. Ethylene is biosynthesized from Methionine pathways in higher plants in which ACS and ACC oxidase (ACO) catalyze the reactions from S-adenosylmethionine (SAM) to 1-aminocyclopropane-1-carboxylate (ACC) and ACC to ethylene, respectively5,6. This ethylene biosynthesis pathway has been well explained and mapped in the Arabidopsis thaliana7. Using advanced molecular biology techniques, various plant species have been used to isolate cDNA and genomic clones for both enzymes, encoded by multigene families8,9,10,11,12,13. These cDNA clones have been used to characterize the expression of individual members in various tissues and in response to recognized ethylene biosynthesis-inducing stimuli7. In Arabidopsis, ACS2, ACS4 to ACS9, and ACS11 form functional homodimers, whereas unfunctional homodimer forms in ACS1. ACS3 is a pseudogene. Whereas ACS10 and ACS12 encode aminotransferase. All genes play a specific role in the plant during growth and development. It was studied that AtACS5 gene had the highest promoter activity in grown seedlings (2 weeks old). And this gene was localized at the reproductive stage from areoles where AtACS4 and AtACS7 were present in both veins and areoles14. Ethylene gas is a plant hormone and critical growth regulator. It synthesizes in response to biotic and abiotic stresses15,16,17,18,19. It also influences plant growth and developmental processes like germination, leaf and flower senescence and abscission, fruit ripening, and nodulation19,20. In most plant tissues, the level of ACS activity is almost directly proportional to the level of ethylene production21. The synthesis of ethylene involves simple yet highly regulated steps. ACS’s conversion of S-adenosyl methionine to ACC is a rate-limiting step in ethylene biosynthesis. ACC is converted to ethylene by ACC oxidase (ACO). 1-Aminocyclopropane-1-carboxylate oxidase (ACO), is an O2-activating ascorbate-dependent nonheme iron enzyme, which involves in the catalyses of ACC in the ethylene biosynthetic pathway and converts 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene22. An ethylene overproducer mutation, ETO1, negatively regulates ACS and ethylene production. ETO1 regulates the stability of 1-aminocyclopropane-1-carboxylate synthase (ACS) enzymes. It can also act as substrate-specific adapter connecting ACS enzymes like ACS5 to ubiquitin ligase complexes, leading to proteasomal degradation of ACS enzyme23,24. A constitutive triple-response phenotype in Arabidopsis was used to identify three mutants that were affected in the regulation of ethylene biosynthesis due to ethylene overproduction25,26. The post-transcriptional regulation of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) has been affected by the Arabidopsis ethylene-overproducing mutants ETO1, ETO2, and ETO3. ETO2 and ETO3 are dominant mutations, but ETO1 is inherited as a recessive mutation. The 12-amino-acid C-terminal region of ACS5 is predicted to be disrupted by the 1-bp insertion that causes the ETO2 mutation. Since the steady-state level of ACS5 mRNA is not increased by the ETO2 mutation, the mutation likely works post-transcriptionally27.

A comparative study of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), 1-aminocyclopropane-1-carboxylate oxidase (ACO), and Ethylene overproducer 1 (ETO1) genes from ethylene biosynthesis pathway in M. acuminata was performed with A. thaliana would provide a starting point for understanding how the M. acuminata gene family response under different stresses. Genome-wide analysis identified ACS, ACO, and ETO genes in M. acuminata. M. acuminata gene’s structures, functions, and conserved motifs were compared with A. thaliana’s ACS, ACO, and ETO gene to investigate their response under nitrogen (N) and potassium (k) stress. Furthermore, the expression of ACS, ACO, and ETO genes in M. acuminata in response to nitrogen (N) and potassium (k) stress was investigated. The present findings suggest the roles of ACS, ACO, and ETO genes in mediating abiotic stress and provide valuable information for further study on the function of ACS, ACO, and ETO genes in the growth, development, and stress responses of M. acuminata.

Materials and methods

Database search and retrieval of sequence

377 Amino acid (AA) sequence of PLP_aminotran (CL0061)28 present in AtACS (Accession no. NP_191710.1), ACO (Accession no. NP_179549.1) and full-length AA sequence of ETO1 (Accession no. NP_190745.6) retrieved from the Arabidopsis thaliana proteome database at NCBI Gene bank and was used for the identification of ACS, ACO and ETO1 proteins-encoding genes in the banana proteome database at Phytozome https://phytozome.jgi.doe.gov/pz/portal.html using BLAST-P program29. The incorrect and redundant predicted sequences were manually removed, and then all putative MaACS, MaACO, and MaETO genes were further verified using Pfam database. The retrieved amino acid sequences were subjected to NCBI CDD (Conserved Domain Database) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)30 with the default parameters.

Determination of physical characteristics of banana proteins

Amino acid length, molecular weight, and theoretical pI of MaACS, MaACO, and MaETO proteins were predicted using ProtParam tool (http://web.expasy.org/protparam/)31. The information for gene IDs, chromosomal position, and gene and protein sequence was retrieved from Phytozome. All genes were renamed according to the order of their physical position. Subcellular localization of MaACS, MaACO, and MaETO was predicted using the online tool WoLF PSORT (https://wolfpsort.hgc.jp/)32.

Multiple sequence alignment and phylogenetic analysis

The amino acid sequences of ACS, proteins from banana, and arabidopsis were aligned using Clustal W version 2.133,34. The phylogenetic tree was constructed with MEGAx.0 using a neighbor-joining (NJ) method35. The bootstrap values were calculated for 1000 iterations. Similar protein alignment and phylogenetics analysis methods were used for ETO and ACO proteins. 14 MaACS genes and 12 Arabidopsis thaliana protein sequences were used for phylogenetic analysis. Similarly, for ACO, 10 MaACO genes and 5 Arabidopsis thaliana protein sequences, and for ETO, 3 MaETO genes and 5 Arabidopsis thaliana protein sequences were used.

Gene structure analysis and conserved motifs recognition

The genomic and coding sequences of identified genes were retrieved from the database to investigate the intron/exon arrangement of banana ACS, ACO, and ETO genes. These sequences were further used to draw the gene structure using Gene Structure Display Server (GSDS v2.036) (available at http://gsds.cbi.pku.edu.cn/). Multiple EM for Motif Elicitation (MEME) programs (http://meme.nbcr.net/meme/)37 was used to analyze the concluded protein sequences of the banana ACS, ACO, and ETO-1 with a maximum number of motif set as 20.

Gene duplication and calculation of Ka and Ks substitution rates

Putative Gene pair was generated using the knowledge obtained from phylogenetic, motif, and domain analysis of M. acuminata ACS, ACO, and ETO1 genes. These gene pairs were used to calculate the Ka and Ks values through tbtools38. The CDS, protein sequence, and gene pair of MaACS, MaACO, and MaETO were utilized. Using Ka/Ks ratio, molecular evolutionary rates of each gene pair were calculated. The Ka/Ks ratio less than 1 indicates the viability of purifying selection; however, Ka/Ks ratio greater than 1 indicates positive selection, and Ka/Ks = 1 indicates neutral selection39,40. Using the formula “t = Ks/2λ”, with λ (6.05 × 10−9), gene pair divergence was estimated to represent neutral substitution. The MaACS, MaACO, and MaETO like genes were mapped on scaffold using TBtool, and duplicated genes were connected on scaffold using red lines.

Promoter analysis

To analyze the organ-specific expression profile of MaACS, MaACO and MaETO at various development stages, the cis-regulatory element evaluation of recognized banana ACS, ACO, and ETO1 genes was performed by recovering 1000 base pairs sequence upstream from the starting site of banana genomic sequences (promoter region) using the Phytozome database. An ample number of cis-regulatory elements were analyzed in all the Musa acuminata 14 ACS, 10 ACO, and 3 ETO genes by employing the PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)41.

Expression analysis

For expression profiling, Reads Per Kilo bases per Million mapped reads (RPKM) values from RNA-seq data were log2 transformed, and the p-value was calculated using t test. Expression patterns with hierarchical clustering are displayed in Heatmap Illustrator in TBtools38.

Putative microRNA target sites analysis

The micro-RNA (miRNA) datasets of Musa acuminata were retrieved from a knowledge-based database called Plant miRNA Encyclopedia (PmiREN, http://www.pmiren.com/)42. There, to find out the miRNAs that target the MaACS, MaACO, and MaETO genes, CDS sequences of all MaACS, MaACO, and MaETO genes were searched for the complementary sequences of miRNAs with the help of psRNA Target (https://plantgrn.noble.org/psRNATarget/analysis?function=3)43 with default parameters.

Ethical approval

It has been confirmed that the experimental samples of plants, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation with appropriate permissions from Authorities of Department of Horticulture, Faculty of Agricultural Sciences, University of the Punjab New campus, Lahore, Pakistan.

Results

Identification of the ACS, ACO and ETO1 genes in banana

ACS, ACO, and ETO genes, involved in the ethylene biosynthesis pathway, were detected in Banana Genome database. Accordingly, the initial analysis predicted 26, 18, and 8 protein sequences of ACS, ACO, and ETO, respectively. The proteins encoded by the same gene isoforms and proteins containing truncated ACS, ACO, and ETO DNA-binding domains were manually removed. Finally, 14 MaACS, 10 MaACO, and 3 MaETO non-redundant genes were identified and used for further analysis. The number of ACS genes was more than that of ACO and ETO.

The studied genes encoded proteins ranging from 443 to 2670 amino acids in length; MaACS3 was the smallest protein, whereas MaACO6 was the most significant protein. Molecular weight varied between 10.16 and 83.3. Isoelectric points ranged from 8.47 (MaACS6) to 4.79 (MaACO7) (Tables 1, 2, 3).

Table 1 Information about 14 MaACS genes discovered from the genome of M. acuminata, their orthologue in Arabidopsis and their functional annotation.
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Table 2 Information about 10 MaACO genes discovered from the genome of M. acuminata, their orthologue in Arabidopsis and their functional annotation.
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Table 3 Information about 10 MaACO genes discovered from the genome of M. acuminata.
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Comparative phylogenetic relatedness of banana ACS, ACO and ETO1 gene family with Arabidopsis

To investigate the evolutionary relationships between MaACS, MaACO and MaETO1 TFs and Arabidopsis thaliana, a neighbor-joining (NJ) phylogenetic tree was constructed by aligning their full-length protein sequences. The results showed that 14 MaACS proteins were distributed among 3 groups named I, II A, II B, and III (Table S1 and Fig. 1A).

Figure 1
figure 1

Phylogenetic analysis of Musa acuminata (MaACS) (A), MaETO (B) and MaACO (C) genes.

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Group I consisted of 3 Arabidopsis proteins while 3 belong to banana MaACS1, MaACS3. II-A group consisted of 5 Arabidopsis AtACS4, AtACS5, AtACS8, AtACS9, AtACS11 and 6 genes of MaACS4, MaACS5, MaACS6, MaACS7, MaACS8, MaACS9. II-B Group contained 2 Arabidopsis thaliana proteins AtACS12, AtACS10, while Musa acuminata consisted of MaACS12, MaACS13, MaACS14. Group III contained only 1 Arabidopsis protein AtACS7, whereas banana included MaACS10 and MaACS11.

The results for ETO1 depicted that 3 MaETO1 proteins were distributed among 3 groups named I, II, and III (Table S2 and Fig. 1B).

Group I consisted of 2 Arabidopsis thaliana AtETO1_951, AtETO1_959 proteins while 2 belong to banana MaETO1, MaETO2. II groups consist of only 1 Arabidopsis AtEOL2_925, and 0 banana genes. Group III contained only 1 Arabidopsis protein AtEOL1_888, whereas banana included MaETO3. According to ACO results, the division of MaACO into groups represents that group I contains 3 Arabidopsis thaliana proteins AtACO1, AtACO2, AtACO3, and 3 Musa acuminata genes, i.e. MaACO1, MaACO2, MaACO10. Group II consisted of 1 Arabidopsis thaliana protein AtACO5 and 2 Musa acuminata genes MaACO5 and MaACO6. Similarly, group III contain 1 Arabidopsis thaliana protein AtACO4 and 5 Musa acuminata genes MaACO3, MaACO4, MaACO7, MaACO8, MaACO9 (Table S3 and Fig. 1C).

Proteins of common clade usually seem to show similarity in structure and functioning. So, all the ACS, ACO, and ETO1 like proteins of similar Clades may have similar structures and functions.

Gene structures and recognition of conserved motifs and domain

The organization of exon and intron helps verify the evolutionary relationship between genes or organisms48. Their distribution patterns and numbers are an evolutionary mark for a gene family. A comprehensive demonstration of Banana genes' exon–intron structures and phylogenetic revealed that the gene structure pattern was consistent with the phylogenetic analysis. The number of introns varied from 3 to 8 in MaACS gene, and no gene in MaACS is intron-less (Fig. 2A, Table S4).

Figure 2
figure 2

Phylogenetic relationship and gene structure of ACS, ETO and ACO genes from M. acuminata.

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In Group I, MaACS1 possesses 3 introns and 4 exons. Group IIA comprises of MaACS2 gene which has 3 introns and 4 exons. In group IIB, MaACS3 contain 5 introns and 6 exons, while MaACS4, MaACS5, MaACS6, MaACS7 genes contain 4 exons and 3 introns. In group III, MaACS11 gene contains 5 exons and 4 intron and MaACS13 gene has 9 exons and 8 introns, while, MaACS8, MaACS9, MaACS10, MaACS12 and MaACS14 consists of 4 exons and 3 introns.

According to Fig. 2B and Table S5, group I contain MaETO1 and MaETO2 genes consisting of 4 exons and 3 introns, while MaETO3 in group III has 4 exons and 3 introns. Similarly, MaACO1 and MaACO2 of group I contain 4 exons and 3 introns, while MaACO10 of group I have 2 exons and 1 intron. MaACO5 of group II comprises 2 introns and 3 exons, while MaACO6 contains 4 exons and 3 introns. MaACO3, MaACO8, and MaACO9 of group III contain 4 exons and 3 introns, while MaACO4 has 3 exons and 2 introns, and MaACO7 consists of 7 exons and 6 introns (Fig. 2C, Table S6).

The conserved motif analysis also verified the classification of MaACS genes. All MaACS protein sequences were loaded into the MEME analysis tool to identify the conserved motifs. As a result, twenty conserved motifs were observed, which were statistically significant with E-values less than 1 × 10−40 (Fig. 3). The motifs of MaACS proteins identified by MEME were between 15–50 amino acids in length. Motif 1, Motif 2, Motif 5, Motif 6, Motif 7 and Motif 9 are common in all groups. Group I and Group IIA had similar motif patterns. Motif 3 is also common in all groups except in AtACS-12_Type 2 gene of group IIB. All groups (I, IIA, IIB and III) contain M-4 and M-8 other than Group III (MaACS11) and G-I (MaACS3), respectively. Meanwhile, IIB group members have relatively complex motif patterns compared with Group IIA (Fig. 3A).

Figure 3
figure 3figure 3

The distribution of 10 motifs present in MaACS (A), MaETO (B) and MaACO (C) protein of banana.

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In MaETO proteins, all the motifs are common in all groups. Motif pattern is also the same in all groups. Figure 3B shows that MaETO gene structures are similar to the corresponding AtETO gene structures. TPR-1 domain is only present in AtETO genes. BTB_POZ domain is only present in MaETO3 genes, similar to atEOL-1 genes.

Motif analysis of MaACO genes shows that motif 3 is only present in MaACO7, MaACO8 MaACO9 of G-3. Motif 7 is only present in G-3 (MaACO3, MaACO4, MaACO7, MaACO8, MaACO9). MaACO10 contains only 5 motifs i-e. 1, 2, 3, 4 and 5. MaACO7 has repeats of motifs and domains. Both 2OG-Fell_Oxy and DIOX_N domains are present in all MaACO genes (Fig. 3C).

Gene duplication of banana ACS, ACO and ETO1 genes

The date of duplication of the gene was also estimated through MEGA-X using pairwise alignment that provided Ks and Ka values and then Ka/Ks was calculated manually (Fig. 4). Ks depicts the number of synonymous substitutions per synonymous site, whereas Ka shows the number of nonsynonymous substitutions per nonsynonymous site and the ratio of nonsynonymous (Ka) versus synonymous (Ks) mutation was represented by Ka/Ks. The speculative date for gene duplication of the paralogous group MaACS7_MaACS4 was calculated to be 99.11Mya, while for the other remaining 21 ACS paralogous pairs, the segmental duplication date of M. acuminata was estimated in the range from 46.08 to 88.78 Mya for paralogous pairs 15 and 11. All the paralogous groups in M. acuminata had a Ka/Ks ratio greater than 0.12, suggesting the possibility of considerable functional divergence after the duplication process (Fig. 4A, Table S7).

Figure 4
figure 4

Time of gene duplication estimated for different paralogous pairs of MaACS (A) and MaACO (B) genes based on Ks and Ka values.

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The date of gene duplication for ETO of M. acuminata was calculated 17.26 Mya for paralogous group MaETO2_MaETO1 (Table S8). Similarly, the gene duplication date for MaACO was calculated in 44 paralogous pairs. The segmental duplication date of banana was estimated from 0.45 to 24.13 Mya for paralogous pairs 38 and 14. In all paralogous groups of MaACO the Ka/Ks ratio is greater than 1.15 (Fig. 4B, Table S9).

Analysis of cis-regulatory elements

The spatial–temporal transcriptomic expression of genes is affected by the presence and organization of various cis-regulatory elements at the binding site of transcription factors on the promoter region. In-silico analysis of cis-regulatory elements can be employed to evaluate the putative functions of genes. Cis-regulatory elements related to vital physiological processes such as response to light, seed-specific, endosperm-specific, hormone-specific, meristem-specific, and stress were observed (Fig. 5). Mainly, 9 out of 14 MaACS, all 3 MaETO and 5 out of 10 MaACO genes possess element involved in light responsiveness, 6 MaACS genes possess a fragment of a conserved DNA module that takes part in light responsiveness, and 9 MaACS, 2 MaETO and All 10 MaACO elements involved in the abscisic acid response.1 MaACS and 1 MaETO gene possess elements involved in salicylic acid responsiveness, while 3 MaACS genes showed elements that show response in defense and stress, and 3 MaACS and 10 MaACO genes are related to meristem expression, 3 MaACS and 1 MaETO gene possess factors involved in low-temperature responsiveness, 4 MaACS, and 3 MaETO genes possess Auxin-responsive element, 2 MaACS, and 2 MaETO genes showed element specific to anoxic inducibility, 1 MaACS genes possess elements specific to seed regulation, 4 MaACS genes contain elements involved in endosperm expression, 6 MaACS, and 1 MaETO gene possess elements involved in element involved in gibberellin response, 6 MaACO genes essential for the anaerobic induction and all 10 MaACO genes involved in MeJA-responsiveness. The cis-regulatory elements identified among 14 ACS, 3 ETO, and 10 MaACO genes of banana and their functional annotation are shown in (Fig. 5, Tables S10, S11 and S12).

Figure 5
figure 5

Different cis-acting elements in putative MaACS (A), MaETO (B) and MaACO (C) promoters which are associated with abiotic stresses, hormone responses, growth and development.

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General miRNA expression dataset of Musa acuminata

Heat map for the expression profile of Musa acuminata genes in root under normal and low nitrogen and potassium shows that MaACS14 and MaACO6 expressed highly at a normal nitrogen supply level. Still, they do not respond significantly at low nitrogen levels. MaACO5, MaACO3, MaACO8 show very slight expression at normal nitrogen levels, while MaACS14 express slightly at low nitrogen levels. On the other hand, MaACS1 expresses well at low levels of potassium, whereas MaACO6 gives a significant response at normal levels of potassium. MaACS4, MaACO4 and MaACO5 show slight behavior in response to low potassium. (Fig. 6). The expression of each banana gene in roots is explained in Table 1.

Figure 6
figure 6

The heat map shows the expression profile of the M. acuminata ACS (B) and ACO (D) genes in root under the normal level of nitrogen (CR) and low level of nitrogen (NR) and ACS (A) and ACO (C) genes in roots under the normal level of potassium and low level of potassium in different organs. The expression levels of MaACS and MaACO genes are revealed by different colors, which increase from green to red.

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The mature miRNAs sequences were retrieved from Plant MicroRNA Encyclopedia database. Later, those miRNAs that could potentially target MaACS genes were identified with the help of the psRNA Target online tool (https://plantgrn.noble.org/psRNATarget/analysis). Consequently, 49 miRNAs were found, targeting 8 out of 14 MaACS genes. The remaining 6 MaACS genes were not targeted by any of these miRNAs (Table 4). The number of miRNAs targeting these genes varies from 1 to 20 miRNAs per MaACS gene. MaACS 4, 9, 13, 14 are the genes targeted by only 1 miRNAs. On the other hand, MaACS 14 is targeted by 2 miRNAs. 3 miRNAs target MaACS 4. None of the gathered miRNAs targeted the remaining 6 MaACS genes. So, this indicates that MaACS 4 is the gene targeted by the maximum number of miRNAs. While discussing ba sed on groups, Group II A was targeted the most by these miRNAs, which was targeted by 3 miRNAs. On the other hand, Group A was targeted by only 3 miRNAs, which is the least among all (Table 4, Table S13).

Table 4 miRNA targets prediction of MaACS(A), MaACO(B) and MaETO(C).
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In MaACO, there are total 32 miRNAs that targeted 6 MaACO genes out of 10. The number of targeting miRNAs varies from 1 to 17. MaACO5 targeted 2 miRNAs, and the remaining 4 genes were targeted by 1 miRNA. So, this depicts that MaACO5 is the gene targeted by a maximum number of miRNAs (Table 4, Table S14). In ETO of banana, there are two 2 targeted miRNA for both MaETO1 and MaETO2. The total number of targets is 11, the maximum number of miRNA targets on the MaETO1 gene (Table 4, Table S15).

Discussion

The coordination of genes, hormones, and environmental factors made the ripening process successful57. Genes like ACS, ACO and ETO involved in ethylene biosynthesis pathway5,6 perform key role in ripening58. Banana genome database (https://phytozome-next.jgi.doe.gov/info/Macuminata_v1) implied to identify 14 ACS genes, 10 ACO genes and 3 ETO genes (Table 1) at the genome-wide level. The 14 banana ACS genes were classified into 4 subfamilies (Group I, IIA, IIB, III), whereas in ETO and ACO, there were divided into 3 subgroups (Group I, II, III) using the phylogenetic analysis (Fig. 1). The exon–intron structure and prediction can also be used as evidence for understanding the evolutionary relationships among genes or organisms48,59,60. The predicted exon–intron association revealed that all 14 genes have introns (Fig. 2A). The number of introns varied from 3 to 9 in MaACS gene (Fig. 2B), 3 to 4 in MaETO genes (Fig. 2B) and 1–6 in MaACO genes of banana (Fig. 2C). In general, it shows that banana MaACS genes in the same group share similar exon–intron structures (Fig. 2). Exon–intron having similar structures have also been noticed in Arabidopsis, rice and soybean61,62 which suggest that these structures are evolutionarily preserved.

Motif analysis shows that Motif 3 is common in all groups except in AtACS-12_Type 2 gene of group IIB. While, IIB group members have relatively complex motif patterns compared with Group IIA (Fig. 3A). Figure 3B shows that MaETO gene structures are similar to the corresponding AtETO gene structures. BTB_POZ domain is only present in MaETO gene that was vital for AtETO genes (Fig. 3B). Furthermore, MaACO7 has motifs and PLN02299 domain repeats, similar to CoACO1 and CoACO2 in Camellia oleifera63. PLN02403 domain is only present in MaACO5 and MaACO6 (Fig. 3C). The distribution of motifs among ACS proteins (Fig. 3) indicates evolutionary and structural relationships as deduced by the phylogenetic tree64,65,66. The motif data analysis by MEME (Fig. 3), and domain analysis using NCBI CDD distinct motifs were identified that were differentially distributed among MaACS (Fig. 3). Meanwhile, at least one or two conservative motif types and spatial distributions in MaACS were present in the same subfamily while some differences were present, implying certain functional similarities of banana ACS members within the same subfamily. In addition, MaACS genes showed structural conservation in subfamilies and were consistent with other plants such as Arabidopsis, rice, cotton and chickpea61,67,68,69,70. In addition, as predicted by in silico analyses, 3 deduced MaACS harbored NLSs to help localize them to the nucleus, but subcellular localization analysis using online tool WoLF PSORT (https://wolfpsort.hgc.jp/), supposed nucleolus localization in almost all MaACS protein except MaACS 12 and MaACS 14.

The ratio of Ka/Ks provides an understanding of the selection pressure on substituting amino acids. Less than one ratio of Ka/Ks (Ka/Ks < 1) suggests the possibility of purifying selection, whereas more than one ratio of Ka/Ks (Ka/Ks > 1) suggests the likelihood of positive selection39,71,72. Generally, evaluation of selective pressure provides a particular lead for amino acid sequence altered in a protein and is also necessary for interpreting functional residues and protein shifts73. Ka/Ks ratios of the sequences from the different banana MaACS groups vary remarkably, while in ETO, only one gene showed Ka/Ks ratio. Despite the differences, all the estimated values of Ka/Ks were less than 1, suggesting that all the ACS sequences in each group undergo strong purifying selection pressure and positive selection might have acted on only a few sites during the process of evolution. In MaACO, all the estimated values of Ka/Ks which were more than one (> 1) shows the possibility of existence of significant positive selection after duplication (Fig. 4).

Heat map for the expression profile of Musa acuminata genes in root under normal and low nitrogen and potassium shows that MaACS14 and MaACO6 expressed highly at normal nitrogen supply. Whereas, MaACS1 represents well at low potassium levels, whereas MaACO6 gives a significant response at normal levels of potassium (Fig. 5). In contrast, ACS genes exhibited low expression under potassium and cadmium stress74. Similarly, ACS1, ACS4 and ACS7 expression were enhanced in response to UV treatment in tomatoes, while ACS3, ACS5 and ACS6 showed no variation75.

MicroRNAs are very important regulators of plants that regulate almost every biological process, ranging from growth and development to combating pathogens and maintaining proper internal conditions, as miRNA affects many genes of specified functions43,76,77,78,79. miRNAs are highly conserved among different species, as each microRNA performs a specific function, regardless of the type of species in which they were observed. In Musa acuminata, MaACS4 is targeted by 3 miRNA i.e. Mac-miR159, Mac-miR319, and Mac-miRN2010, which are induced in cold stress and repressed in heat stress49, dehydration, salinity, or ABA52 and involved in root formation and cell elongation51. Mac-miR396 is an important miRNA that targets MaACS6, MaACS8 and MaACS14 that activate in cold stress and repress in heat stress49. It also regulates the transition of Arabidopsis root stem cells by transit-amplifying cells to form a regulatory circuit by repressing GRF50. MaACS14 is targeted by 2 miRNAs, Mac-miRN2002 and Mac-miR396, which are important for root stem cells in Arabidopsis50. MaACO5 is targeted by 2 miRNA Mac-miR391 and Mac-miR172, which are involved in the ripening process of banana53. Mac-miR172 is a known MiRNA of banana53, has maximum targets on MaACO5 and does not target any other protein in MaACO. In MaETO, both MaETO1 and MaETO2 are targeted by 2 miRNA. Mac-miR171 and Mac-miR428 both target on MaETO1, in which Mac-miR171 inhibits translation in plants54, and Mac-miR428 is expressed in neural and ectoderm tissue but was not expressed in blastula55. While Mac-miR N2009 and Mac-miR528 target MaETO2. Mac-miR528 targets many genes encoding copper‐containing proteins and polyphenol oxidase (PPO), and is downregulated in cold stress56. It is reported that three specialized miRNAs (miR173, miR390 and miR828) have been identified and well characterized in Arabidopsis80,81,82. Only miR390 is present in MaACO7 and MaACO9. This might suggest that Mac-miR396 in MaACS genes are important for root cell elongation and maintaining the regulatory mechanism between root and stem.

Conclusion

A comprehensive analysis of genes (ACS, ACO, and ETO1) involved in ethylene biosynthesis in the Musa acuminata (Banana) genome was discussed in this study. The 15 genes of ACS, 10 genes of ACO and 3 genes of ETO1 were categorised into subgroups. The structural and functional properties of each MaACS, MaACO and MaETO member were characterized under Nitrogen (N) and Potassium (K) stress in plant roots, where, MaACO6 expressed highly at both normal and low level of N and P, and MaACS14 expressed well at low nitrogen level, while, MaACS4, MaACO4 and MaACO5 responded to low potassium. Most genes were involved in root cell formation and maintaining the regulation mechanism between roots and stem, suggesting their role in plant root growth and development. The detailed computational inspection of Banana ACS, ACO and ETO proteins revealed in the current study might be selected for cloning purposes at the molecular level, portraying gene expression and studying their interaction with different transcription factors.