2.1 Identification and retrieval of BBX genes

The family of BBX gene in A. thaliana was previously conveyed by Khanna et al., in 2009 in his paper “The Arabidopsis B-Box Zinc Finger Family”. To identified homology in sugarcane we extracted all the protein sequences of A. thaliana BBX gene family from the phytozome database (https://phytozome.jgi.doe.gov/) and picked AtBBX22 (AT1G78600) as a queries sequence for protein BLAST (BLASTP) search with an e-value threshold of < 1e-5 in the Sugarcane genome hub database (https://Sugarcane-genome.cirad.fr/). Following that, the identified hits were screened by using the different online available tools to ensure authenticity and the occurrence of the target domains: SMART (https://smart.embl-heidelberg.de/), Inter Pro Scan programme (https://www.ebi.ac.uk/interpro/), and Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/cdd/). The ExPASyProtParam tool (https://web.expasy.org/protparam/) was used to calculate the isoelectric point (PI), molecular weight (Da), and grand average of hydropathicity (GRAVY) of each Shbbx.

2.2 Phylogenetic analysis and sequence alignment

For Phylogenetic analysis between sugarcane and A. thaliana full length protein sequences of BBX genes was selected for better understanding between their evolutionary relationship. Protein sequences from both species were imported into MEGA X (Version 10.2.4) to achieve multiple sequence alignment with the default parameters in order to construct the phylogenetic tree. The obtained alignment data file was used as input for the construction of a phylogenetic tree in MEGA X (Version 10.2.4) using the neighbor-joining algorithm with a boot-strap value of 1000, but before that we compute pairwise distance of alignment file. The phylogenetic tree was then visualised with the ITOL (version 6.4.1) software. The MEME suite (https://meme.nbcr.net/meme/cgi-bin/meme.cgi) tool was used to create the sugarcane B-box domain motif logos.

2.3 Chromosomal location, gene structure, and duplication analysis

The sugarcane Genome hub (https://sugarcane-genome.cirad.fr/) platform was used to determine the chromosomal location of all the identified Shbbx genes in this study. Genes were mapped to individual chromosomes using map chart software (version 2.32) (Huang et al., 2012; Voorrips, 2002). Furthermore, the genomic sequences were aligned with their corresponding coding sequences in GSDS software (gene structure display server, https://gsds.cbi.pku.edu.cn) to determine the exon–intron organisation of Shbbx genes. Tandem duplications of adjacent genes were also identified if their similarities exceeded 70%. The ratio of Ka/Ks (evolutionary selection pressure) was used to express positive selection (with a ratio greater than 1), negative or stabilising selection (Ka/Ks less than 1), and neutral selection (Ka/Ks equal to 1), respectively, to interpret the evolutionary relationship of Shbbx genes. TBtools software (v1.09854) calculated the Ka (nonsynonymous substitutions) and Ks (synonymous substitutions) values.

2.4 Subcellular localization analysis

WoLF PSORT (https://www.genscript.com/psort/wolf_ psort.html) tool was used to identified the sugarcane BBX proteins location in different organelles. Furthermore, the results of the analysis were represented by a heatmap created with the TBtools software (v1.09854).

2.5 Codon analysis

The cDNA sequences of identified Shbbx genes were retrieved from the Sugarcane genome hub database (https://Sugarcane-genome.cirad.fr/) and stop codons were removed from each gene sequence. The multiple sequence alignment of the Shbbx genes was carried out by ClustalW in MEGA X (Version 10.2.4). After that the multiple sequence alignment result file served as input for the codon model selection analysis in classical datamonkey (https://classic.datamonkey.org/) online tool.

2.6 Gene ontology (GO) annotation

The Blast2GO server was used to perform GO annotation of Shbbx genes (Zou et al., 2018). The GO terms were divided into three categories: biological process, molecular function and cellular component. WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.pl) was used to plot GO classifications.

2.7 Protein structure analysis by homology modelling

The 3D structure of the Shbbx protein was predicted to probe the possible functions of the protein. 3D structures of Shbbx proteins were created by using homology modelling technique and the online available template-based program SWISS-MODEL (http://swissmodel.expasy.org) (R. Ma et al., 2021).

2.8 Structure validation

For the validation of protein structures, SWISS MODEL generated PDB file of the best models was used to create the Ramachandran plots using RAMPAGE server (https://zlab.umassmed.edu/bu/rama/) and assessing them under three factors as “favoured, allowed and outlier regions of amino acid residues”(das et al., 2019).

2.9 Energy minimization of the predicted structures

Energy minimization was applied to predicted and evaluated structures in order to achieve the lowermost energy conformation by S P B D V (SWISS PDBVersion 4.1.0).

2.10 Molecular dynamics simulation

The model with the lowest energy was considered for molecular dynamics simulations. iMODS (https://imods.iqfr.csic.es/)was used to understand the best model stability in terms of the torsion angles between molecules.

3.1 Sugarcane BBX gene identification

BLAST searches were used to characterise the BBX gene family in sugarcane genome-wide analysis. The BBX genes discovered through similarity searches were confirmed the presence of number of B-box domain by the use of two different software’s are SMART (Simple Modular Architecture Research Tool) and Pfam databases. A local BLAST was performed in the sugarcane genome hub database to determine how many genes in the sugarcane genome encode BBX members with and without the CCT domain out of 21 Shbbxs genes. Nomenclature and consistency were maintained by naming these putative BBX genes as Shbbx1 to Shbbx21. Table 1 contains detailed information on 21 Shbbx genes, including “name of gene, length of protein, location of gene on chromosome, molecular weight, theoretical isoelectric point (pI) and GRAVY”. The 21 Shbbx proteins had varying molecular lengths and weights, with amino acid lengths ranging from 206 (Shbbx11) to 585 (Shbbx10). Shbbx had the smallest molecular weight (22.65 kDa), while Shbbx had the largest molecular weight (62.08 kDa). These Shbbx proteins' theoretical isoelectric points (pI) was ranged from 4.53 (Shbbx5) to 6.16. (Shbbx3). The hydrophilic nature of all Shbbx genes revealed that the gene's GRAVY value was less than zero (Table 1).WoLF PSORT predicted that the large number of Shbbx proteins would be present in the nucleus and extracellular space, but it also revealed that some Shbbx proteins show their presence in other subcellular compartments such as the chloroplast and cytoplasm (Table 1).

3.2 Protein sequence and phylogenetic analysis of the Shbbx gene family

Phylogenetic analysis was performed to understand the evolutionary relationship between the BBX genes of Sugarcane and A. thaliana. The phylogenetic analysis revealed 5 clades. The first, second, third, fourth, and fifth clade consists of 4, 2, 8, 9, and 23 genes respectively. Whereas 4 genes were not found in any clade. In the first clade, Shbbx20 showed association with 3 genes of A. thaliana. In contrast to the first clade, second clade genes belong only to Sugarcane. On the other hand, in the third clade, 4 genes of Sugarcane showed an evolutionary relationship with 4 genes of A. thaliana. Whereas, in clade 4, only 2 genes of Sugarcane i.e.., Shbbx6 and Shbbx4 were found. Maximum genes of Sugarcane i.e., 11genes were found in clade 5 (see Figs. 1,2).

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Fig. 1

Sugarcane and A. thaliana BBXs phylogenetic relationships. A phylogenetic tree was constructed using BBXs among sugarcane and A. thaliana. The brown colour signifies AtBBXs in A. thaliana and the colour blue Shbbxs in Sugarcane. The sequence alignments and phylogenetic tree were used to classify the 21 Shbbxs.

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Fig. 2

BBX proteins structure of sugarcane. Numbers In this figure show position of amino acid residues present in domains of sugarcane. The yellow rectangles signify B-BOX1domain and light green pentagons signify CCT domains in sugarcane. The bar scale represents 100 amino acids.

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3.3 Conserved domain analysis

Motifs serve an important function in transcriptional regulation. To understand the diversity and similarity of the identified Shbbx genes, the MEME suite tools were used. Motifs are generally important because of their relationship with protein structure and function, as specific motifs regulate protein networks. We discovered 15 conserved protein motifs in this study (supplement file 1). According to our findings, motifs 1 and 6 were found in all 21 genes of the BBX gene family. In contrast, motifs 3 and 4 were found in 11 and 10 genes, respectively. Similarly, motifs 2 and 9 were found in 9 different genes. As a result, motif 1 and 6 may be the primary domains representing the BBX gene family (Fig. 3).

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Fig. 3

MEME online tools were used to create a schematic representation of the amino acid motifs of BBX proteins. On the left, you'll find a list of BBX gene family. The length of the corresponding BBX protein is signified by the black solid lines. The various coloured boxes represent various motifs and their positions in the BBX protein sequences.

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3.4 Chromosomal Localization, gene duplication and gene structure analysis of Shbbxs

All of the Shbbx genes were mapped onto different sugarcane chromosomes to determine their genomic distribution. Gene distribution was found to be uneven. Excluding the chromosomes 5 and 9, the 21 Shbbx genes were found on all sugarcane chromosomes. The maximum number of genes is found on chromosome number 10, where five genes are found, followed by chromosome number 4, where four genes are found. On Chromosomes 1 and 6 three genes have found while on chromosomes 7 and 8 two genes. The minimum number of genes were mapped on chromosomes 2 and 3, which each had one gene. Fig. 4 depicts the location of the Shbbx genes on the sugarcane chromosomes.

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Fig. 4

Sugarcane chromosomes contain BBX genes. The number of chromosomes (Chr 1-Chr 10) is designated at the top of each chromosome. The values following the BBX genes indicate their chromosomal location.

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Tandem and segmental duplications gone up significantly during the evolution and expansion of the gene family. Tandem duplication typically results in gene clusters, whereas segmental duplication may result in dispersed family members. Within the sugarcane genome, hardly two duplicated segments (Shbbx4/Shbbx6 and Shbbx9/Shbbx15) in the Shbbx gene family were found. Then, on chromosomes 4, 7, 10, 1, and 8, six pairs of tandem duplications (Shbbx7 / Shbbx13, Shbbx1 / Shbbx17, Shbbx2 / Shbbx8, Shbbx16 / Shbbx21, Shbbx3/ Shbbx5 and Shbbx18 / Shbbx19) in the Shbbx gene family were discovered (Shbbx (Supplementary file 2). The Ka/Ks ratios of the 21 Sugarcane BBX gene pairs were computed depending on the BBX family's comparative sequence homology, among which 10 Sugarcane homologous pairs gene were discovered, and the Ks values have been used to predict the time of Sugarcane BBX gene duplication activities. We accomplished a Ka/Ks (evolutionary selection pressure) analysis on Sugarcane Shbbx homologous gene pairs and observed that Shbbx17/Shbbx1 had the lowest Ka/Ks ratio (0.1798), along with Shbbx5/Shbbx3 (0.2786), and Shbbx21/Shbbx16 had the highest ratio (9567550). Sugarcane BBX homologues showed a Ka/Ks ratio of less than one, indicating strong purifying selection during the development of the sugarcane BBX gene family (Supplementary file 3). The ks values ​​derived from this analysis were used to calculate the time sugarcane took to mimic the developing BBX gene. According to the findings of this study (Maya), the replication time of the sugarcane B-box gene was mainly between 0 and 44 million years.

The intron/exon structure of the Shbbx gene were studied by matching cDNA sequences with corresponding genomic sequences for well understand their evolutionary relationships. As shown, the number of exons ranged from 1 to 6 (Fig. 5). Shbbx19 had the most exons (6), but Shbbx1, Shbbx17 and Shbbx18 only had one Shbbx exon. These data indicate that exon acquisition or loss occurred during the course of evolution.

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Fig. 5

GSDS-generated gene structure of the sugarcane BBX family. The red colour block represents the coding sequence (CDS), the blue colour block represents the genes' upstream or downstream, and the black line represents the intron. The length of the DNA sequences is indicated by the scale bar.

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3.5 Subcellular localization of sugarcane BBX protein

WoLF PSORT was used to predict sugarcane BBX protein subcellular localization (Table 1). The large number of Shbbx proteins were identified in the nucleus, with only a few found in another subcellular compartments like chloroplasts and cytoplasm. We used a heatmap to represent the subcellular localization of each gene, with the abundance of organelles in each gene represented by a graph scale ranging from 0 to 14 (Fig. 6). The colours red, green, and orange indicate high abundance, medium abundance, and low abundance, respectively. According to our findings, Shbbx4, Shbbx12, and Shbbx14 were abundant in the nucleus, whereas Shbbx2 was abundant in the chloroplast. Sugarcane has a medium to low abundance of the BBX gene in the remaining organelles.

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Fig. 6

The BBX genes' subcellular localization. The heatmap was created using WoLF PSORT data, and the colour scale depicts the abundance of organelles. Higher levels of expression were shown in blue, while lower levels were shown in dark red.

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3.6 Codon analysis of sugarcane BBX protein

To observe whether the Shbbx genes have been subjected to adaptive evolution, an investigation of variation in selective pressure was carried out. A codon-based MSA was evaluated for multiple models by using Datamonkey web server (https://classic.datamonkey.org/). In total, codon model selection procedure had examined 15052 models. The best model (log(L) = -19293.9, mBIC = 39638.65), has 4 rates (Supplementary file 4). This model provides an improvement of 377.44 log(L) and 697.55 mBIC points over a single rate model, where all non-synonymous substitutions occur at the same rate. According to the table (2) dN/dS ratio of 0.67 has highest rates in class (32), Stanfel class changes (15), polarity changes (14) and charge changes (11) (Supplementary file 5). show correlation between inferred rates and biochemical properties Fig. 7.

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Fig. 7

codon analysis of BBX gene family members in Sugarcane.

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3.7 GO annotation analysis

To understand the function of BBX gene found in sugarcane, GO annotation was used. Based on the GO footnote analysis, 21 Shbbx gene family were distributed into 13 functional categories from three major ontologies are: cellular components, molecular function, and biological processes (Supplementary file 6). Cell parts and organelles were the most common in these categories. Examination of molecular function annotations revealed that the majority of Shbbx is involved in binding of DNA, along with chromatin binding. Examination of the cellular component annotations show that BBX proteins were found primarily in the nucleus.

3.8 Protein structure analysis by homology modelling

The tertiary structure of the protein was predicted using the Swiss model chosen to analyze the function of the protein. We selected Shbbx20 for subclass I, Shbbx17 for subclass II, tertiary structural homology using pair and multiple alignments. The 3D structures of the five subclasses showed different tertiary structure (Fig. 8). The Shbbx20 sequence comprises of 3 helices, 6 sheets and 6 amino acid residues particularly bind to zinc ions (C.65, C.62, C.85, C.82, C.34, C.74). The Shbbx17 sequence contained two helices and triple sheets and five amino acid residues that particularly bound to Zn ions (C.39, C.20, C.36, C.17, C.28). The Shbbx5 sequence comprised of two coils, two folded shells, and five amino acid residues particularly bound to Zn ions (C.23, C.40, C.43, C.20, C.32). The Shbbx4 sequence contained only four coils, six folded strands, and five zinc-binding amino acid residues (C.17, C.51, C.48, C.68, C.71). The Shbbx14 sequence comprised mainly of four helices, five folded sheets, and five amino acid residues linked by Zn ions (C.81, C.58, C.78, C.70, C.17).

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Fig. 8

Five sugarcane BBX protein sequences in three dimensions. SWISS-MODEL software was used to create the models. The red areas represent -folds, while the bright blue areas represent -helices. Cysteines bound to a zinc ion are indicated by small yellow spikes surrounding a blue ball.

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Ramachandran plot structure validation: To analyse the anticipated models, the PDB files for the Shbbx genes were obtained from the SWISS MODEL and put forward to the online database SAVES 6.0, which created the Ramachandran plot (Fig. 9). In this graph, the residues of amino acids were dispersed in 3 zones. Favored, allowed regions and generously allowed regions were the three distinct regions in (supplementary file 7).

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Fig. 9

Ramachandran plots for different Shbbx genes.

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Energy minimization: The energy minimizations of the predicted models were accomplished using the SPBDV9 software. The energy minimization values were compared to find the best and foremost predicted models. Shbbx 17 (E = -809.338KJ/mol) was the least energetic model shown in (supplementary file 8).

MD simulation: After comparing the energy minimization outcomes, we can see that the best predicted protein model (Swiss-Model) has the lowest value for minimization. As a result, the SWISS-MODEL was further interpreted for molecular dynamics simulation in order to examine the stability and physical activities utilizing the iMODS webserver's normal mode analysis (NMA). Even with significantly large macromolecules, iMODS enabled the exploration of such modes and produced plausible transition paths between two homologous structures. The unique internal coordinate formulation increases NMA's efficiency and broadens its applicability while maintaining stereochemistry implicitly.

Each normal mode's variance is inversely related to its eigenvalue. Individual (red) and cumulative (green) variances are represented by coloured bars (supplementary file 9 A). The elastic network model specifies which atom pairs are linked by springs. Each dot in the graph represents a spring between the corresponding atom pair. The stiffness of the dots is indicated by their colour; darker greys indicate stiffer springs and vice versa (supplementary file 9B). All above mentioned points denote the higher stability of the complex.