The whole genome, gene, and protein sequences of the polyploid sugarcane cultivar R570 were downloaded from the Sugarcane Genome Hub. To identify PP2C genes in sugarcane, the signature PP2C domains from rice, barley, sorghum, and maize were obtained from the EKPD database (Wang et al., 2014). Additionally, the amino acid sequences of all reported PP2C genes from S. spontaneum were considered for identification (Huang et al., 2023). For the identification of candidate PP2C family protein sequences in sugarcane, multiple sequence alignment of all reported PP2C proteins from rice, maize, barley, S. spontaneum, and sorghum was performed using T-Coffee (Notredame et al., 2000). Subsequently, an HMM search was conducted on the whole-genome protein sequence of polyploid sugarcane cultivar R570 using the PP2C domain. Significant hits with an E-value <0.01 were further verified for the presence of the PP2C domain using Pfam and Conserved Domain Database (CDD) (Bateman et al., 2004; Marchler-Bauer et al., 2015). Finally, to maintain a uniform identification within the group, nomenclature was assigned to all identified PP2C genes in sugarcane hybrid cultivar R570 based on their chromosomal positions.

For evolutionary analysis, the protein sequences of identified PP2C genes in the sugarcane hybrid cultivar were aligned with PP2C protein sequences from maize, wheat, and S. spontaneum using T-Coffee with default parameters. A phylogenetic tree was constructed based on this alignment using the Maximum Likelihood (ML) method in MEGA11 with default parameters (Tamura et al., 2021). For comparison with a model organism, an additional phylogenetic analysis was performed using Arabidopsis PP2C genes in MEGA11 with 500 bootstrap replications. The generated tree was then annotated using iTOL (Letunic and Bork, 2024). To investigate the evolutionary relationship and genetic conservation of PP2C genes in sugarcane, a protein BLAST-based ortholog search was conducted using the PP2C protein sequences identified in the sugarcane cultivar R570. These sequences were mapped against its wild progenitors, S. officinarum and S. spontaneum.

The basic molecular features of PP2C proteins, including molecular weight, isoelectric point, and protein sequence length, were determined using the ProtParam tool (Gasteiger et al., 2003). Conserved motifs were identified through the MEME Suite (Bailey et al., 2009), with the number of motifs set to 10, while other parameters were kept at default. The exon-intron structure of the identified PP2C genes was analyzed using the Gene Structure Display Server (GSDS) (Guo et al., 2007). Subcellular localization predictions were performed using CELLO2GO (Yu et al., 2014). Conserved domains of PP2C proteins were predicted using the InterPro database (Hunter et al., 2009), and the results were visualized using TBtools (Chen et al., 2020) to provide a clear representation of domain structures.

The genomic features of all identified PP2C genes were extracted from the annotation file. Chromosomal mapping of these genes was performed using TBtools (Chen et al., 2020). Synteny analysis was conducted between the polyploid sugarcane cultivar and wild sugarcane (S. spontaneum AP85-441) using the Blastall program (Madden, 2013), followed by MCScanX (Wang et al., 2012). The synteny network was further visualized using Cytoscape (Kohl et al., 2011). A Circos plot was generated using TBtools (Chen et al., 2020) to illustrate tandem duplication among the identified PP2C genes in sugarcane. All collinear genes were analyzed for conserved domains, which were predicted using the InterPro database (Hunter et al., 2009) and visualized using TBtools (Chen et al., 2020). Evolutionary selection pressure among duplicated SoffiXPP2C gene pairs was assessed by calculating the non-synonymous (Ka) and synonymous (Ks) substitution rates using TBtools.

CLC Genomics Workbench was utilized to analyze in-house generated RNA-Seq data from the sugarcane hybrid cultivar Co 86032 to identify differentially expressed genes under control and oxidative stress conditions. A de novo transcriptome assembly approach was employed for the analysis. A total of 37,356 contigs were identified as differentially expressed. To investigate potential homology with PP2C genes, a pairwise alignment was performed between the FASTA sequences of these differentially expressed genes and 500 PP2C genes identified in sugarcane in the current study. Out of these, 34 SoffiXPP2C genes were found to align with five differentially expressed contigs. Further functional annotation of these five differentially expressed contigs was performed using UniProt. Gene Ontology (GO) analysis was carried out for 34 SoffiXPP2C genes. Pairwise sequence alignment using BLASTp was conducted between these 34 genes and the protein sequences from rice and sorghum. The GO terms (GO IDs) associated with the rice and sorghum homologs identified through BLAST were retrieved from Oryzabase and SorghumBase. These GO terms were then putatively assigned to the corresponding SoffiXPP2C genes based on sequence homology.

To confirm the presence of four selected PP2C genes SoffiXPP2C31, SoffiXPP2C75, SoffiXPP2C60, and SoffiXPP2C14 in sugarcane, PCR amplification was performed using genomic DNA as the template. Genomic DNA was extracted from leaf samples of five sugarcane cultivars (Co 86032, Co 11015, Co 91010, Co 0233, and Co 16001) using a standard DNA extraction method (CTAB). PCR amplification was performed in 20 μl reaction volumes containing PCR Master Mix, primers, and genomic DNA template. Amplified products were separated on 1% agarose gels stained with ethidium bromide and visualized under UV light.

Further to evaluate its expression in stress condition all this SoffiXPP2C genes (SoffiXPP2C31, 75, 60, and 14) were amplified in waterlogged stressed cDNA. The waterlogging condition in sugarcane genotypes was conducted in the green house at Sugarcane Research Station, Cuddalore. Three Clones/varieties of sugarcane like CoC 13339, C 16338 and C 2014-516 compared with tolerant (Co 62175) and susceptible (Co 86032). All plants were grown under well-watered but non-flooded conditions during the first one and a half months for proper establishment. Forty-five days old seedlings were subjected to waterlogging for twenty days continuously by maintaining the water level at 10cm above soil level. Simultaneously control pots were well watered (without waterlogging) and maintained with normal cultural practices.

The appropriate root and stem samples of Co 62175 (1), Co 86032 (4), CoC 13339 (5), C 16338 (8) and C 2014-516 (11) were collected from the field and stored in RNA later storage solution. The required materials for the RNA isolation were treated with 0.1% DEPC for overnight at 37°C, subsequently double autoclaved at 121°C for 20 min. The collected samples were ground to fine powder using liquid nitrogen. Total RNA isolation was carried out using RNeasy Plant mini kit. Integrity and quality of the RNA samples were checked by 1% (w/v). The quality and quantity of RNA were assessed using the Nanodrop spectrophotometer.

The PrimeScriptTM 1st strand cDNA synthesis kit (Takara Bio) was used for cDNA conversion. cDNA was quantified using nanodrop 2000 and diluted 100 ng/μL. The 25S rRNA gene was used as a reference for the stress responsive gene expression analysis. The normalization of cDNA samples performed using 25S rRNA primer. Quantitative PCR was performed for all the selected genes and also for the reference gene using TB GREEN^® premix Ex Taq™ II mix (TAKARA). Each reaction volume of 20 μL consists of 100 ng cDNA, TB GREEN master mix, 10 μM each of forward and reverse primers and nuclease-free water to make up the volume. The PCR cycle included a pre-incubation at 95°C for 1 minute followed by 40 cycles of amplification at 95°C for 5 seconds and required Tm for 30 seconds acquiring on cycle A Green channel and for Melt A Green ramp selected between 50°C -60°C. The Gain optimisation was selected at 1 to 3 range for Green channel.

The RT-qPCR experiments were performed using the Rotor-GeneQ Qiagen Real-Time PCR system (Qiagen, Hilden, Germany) and SYBR Green PCR Kit (TAKARA). The relative expression of gene in relation to stem, root & reference gene (25S RNA) are calculated on the basis of ‘delta delta Ct’ (ΔΔCt) values (Livak and Schmittgen, 2001). The most commonly used method for relative quantification is the 2(–ΔΔCt) method. Exported the raw Ct values (CT1 and CT2) from the real-time PCR analysis into Microsoft Excel sheet. Assigned sample number, gene name to each data set. Calculated the mean Ct value for the internal control gene and sample gene. The mean fold change (ΔΔCt), 2(–ΔΔCt) and standard deviation were calculated and interpreted in a graph with standard error values.

The equation as follows,

A total of 500 PP2C (Protein Phosphatase 2C) genes were identified in the sugarcane genome, with their respective gene IDs listed in Supplementary Table S1 . To identify these genes, multiple sequence alignment was performed with T-Coffee using the PP2C signature domain from S. spontaneum, rice, barley, sorghum, and maize, followed by an HMM search using the whole-genome protein sequence of the sugarcane under study. Sequences lacking complete conserved domains associated with the PP2C superfamily were excluded from further analysis. Ultimately, 500 genes of sugarcane hybrid cultivar under study containing the PP2C signature domain were selected for downstream analysis. In order to keep uniform identification, all these genes were renamed as SoffiXPP2C where SoffiX represents “Saccharum officinarum and Saccharum spontaneum hybrid” and PP2C represents “Protein Phosphatase 2C”, followed by a unique number assigned based on their order of position on the chromosome.

Phylogenetic analysis of the PP2C genes was performed using T-Coffee for multiple sequence alignment and MEGA 11 for tree construction, with PP2C genes from S. spontaneum, wheat, and maize as reference. Based on this analysis, all 500 genes were classified into 13 subfamilies (A–M) ( Supplementary Table S2 ). The distribution of the gene within its subfamily was uniform in the phylogenetic analysis involving sugarcane and wheat ( Supplementary Figure S3 ), as well as sugarcane and S. spontaneum ( Supplementary Figure S4 ). Gene subfamily assignments were made based on the combined results of these phylogenetic analyses.

Finally, a phylogenetic tree has been made considering only SoffiXPP2C genes ( Figure 1 ). The distribution of gene in phylogenetic tree has shown that all the genes were clustered together of assigned subfamily in previous analysis, except few. Among 13 subfamily largest subfamily was F with 74 genes, and smallest one was L with 18 genes. Subfamily A was second largest subfamily with 69 genes followed by E, D, G, K, C, I, B, J, H, and M which contains 57, 44, 39, 35, 27, 26, 25 and 21 genes respectively. Subfamily D and G contains equal number of genes i.e., 44 and subfamily H and M also contains equal number of genes i.e., 21. Additionally, phylogenetic analysis of 500 sugarcane PP2C genes was carried out using the reported PP2C genes of Arabidopsis thaliana ( Supplementary Figure S5 ). All sugarcane genes followed the previously established classification pattern of Arabidopsis and were clustered into 13 subfamilies, as reported by Xue et al. (2008). The distribution of genes among the subfamilies was largely consistent with prior classifications based on analysis with S. spontaneum PP2C genes. Only a few genes were found to be clustered differently in the analysis with Arabidopsis compared to that with S. spontaneum.

In the ortholog analysis, all PP2C genes identified in Sugarcane R570 showed significant identity to genes in both wild species, S. officinarum and S. spontaneum ( Supplementary Figure S6 ). Out of the 500 identified PP2C genes in R570, 215 genes (43%) showed 100% identity with S. officinarum, whereas only 14 genes showed 100% identity with S. spontaneum. Notably, 9 of these genes were common to both wild species. A total of 134 genes exhibited 99% identity with S. officinarum, while 110 genes showed 99% identity with S. spontaneum, with 43 genes being shared. Additionally, 75 genes displayed 98% identity with S. officinarum, while 114 genes showed the same identity level with S. spontaneum, and 16 of these were common to both. In the identity range of 50–90%, 20 genes matched with S. officinarum, whereas 112 genes matched with S. spontaneum, including 43 overlapping genes. No genes have detected identity less than 52% with S. officinarum, whereas up to 30% identity was observed with S. spontaneum.

For the identified PP2C genes in sugarcane, key physical and biochemical properties were predicted, including gene and protein length (amino acids), molecular weight, isoelectric point (pI), and subcellular localization ( Supplementary Table S1 ). Gene length was determined from genome annotation files (GFF). The longest gene identified was SoffiXPP2C267 spanning 36,077 bp, while the shortest was SoffiXPP2C249, measuring 305 bp. The longest protein SoffiXPP2C436, consisting of 1,119 amino acids, whereas the shortest proteins, SoffiXPP2C242 and SoffiXPP2C249, each contained 101 amino acids. The highest molecular weight was observed in SoffiXPP2C436 at 127.29 kDa, while the lowest was in SoffiXPP2C249, measuring 10.96 kDa. The highest pI was recorded for SoffiXPP2C423 at 11.14, whereas the lowest was found in SoffiXPP2C213, at 0.13. The CDD algorithm confirmed the presence of the PP2C domain in all SoffiXPP2C members. Notably, the localization of this domain was highly conserved across various PP2C subfamilies.

The analysis of gene structure, in terms of exon numbers and exon-intron distribution, revealed a high degree of conservation within each SoffiXPP2C subfamily ( Supplementary Figure S1 ). The distribution of exons varied among the subfamilies, with subfamily A and C containing 1, 3, and 4 exons, while subfamily B had 1, 3, 4, and 5 exons. Similarly, subfamily D showed a distribution of 1, 2, 4, and 7 exons. Subfamily E contained 1 to 6 exons, and subfamily F exhibited a broader range, with 2 to 10 exons. The exon distribution in subfamily G included 3, 4, 10, and 12 exons, while subfamily H had 7 and 8 exons. Subfamily I displayed significant variation, with 10, 11, and 20 exons, whereas subfamily J had 3, 8, and 9 exons. In subfamily K, the exon count ranged from 1 to 12, and subfamily L had 3 and 14 exons. Subfamily M exhibited exon numbers ranging from 6 to 13. The highest exon count (20 exons) was observed in SoffiXPP2C436, SoffiXPP2C491, and SoffiXPP2C441, while the minimum exon count (1 exon) was found in 37 genes. A total of ten motifs were identified in the SoffiXPP2C genes using the MEME tool ( Supplementary Figure S2 ). Among them, Motifs 1, 3, 4, and 6 were present in all 500 SoffiXPP2C genes. Certain subfamilies exhibited identical motif compositions, with subfamilies A, E, F, G, I, and M sharing a completely similar motif pattern. Similarly, subfamilies H and J, as well as subfamilies C and L, displayed identical motif distributions. Notably, Motif 9 was exclusively present in subfamilies H and J, whereas Motif 7 was uniquely found in subfamilies B, H, and J.

The predicted subcellular localization of PP2C proteins indicated their predominant presence in the chloroplast (213 proteins), followed by the cytoplasm (156 proteins) and the nucleus (81 proteins). Analysis of conserved domains showed that sugarcane PP2C domain-containing proteins exhibit diverse structural features, including PP2C catalytic domains, PPM-type phosphatase domains, and PP2C signature motifs, which are present in almost all proteins. Additionally, several proteins also contain kinase domains, signal peptides, transmembrane regions, membrane-associated regions, extracellular domains, hydrophobic regions, disordered regions, and potential phosphorylation sites, highlighting their structural and functional complexity.

All 500 PP2C genes were identified and distributed across all 10 chromosomes and their sub genomes ( Figure 2 ; Supplementary Table S1 ). The highest number of genes (89) was found on chromosome 1 and its subgenome, followed by chromosome 3 and its subgenome (76 genes) and chromosome 2 and its subgenome (70 genes). Chromosome 5, 6, 7, 4, 8, 9 and 10 along with their subgenome contained 62, 38, 36, 34, 12, 11, 15 genes respectively. Additionally, genes were identified in various interspecific recombinant chromosome regions, including Chr5_9A (10 genes), Chr6_9A (9 genes), Chr7_10A (9 genes), Chr7os1 (4 genes), Chr8_10A (5 genes), and Chr8_5A (5 genes), while the lowest number of genes (1) was found on the chromosomes 10os1, 3os1, 3os2, and 9os1. In addition, 13 genes were identified in various scaffolds.

The characterization of the PP2C gene family in S. spontaneum identified PP2C genes exhibiting cold-stress-induced expression (Huang et al., 2023). Additionally, various studies have reported that wild sugarcane (S. spontaneum) is a drought-tolerant grass (Mirshad and Puthur, 2017). To understand the collinearity and gene conservation patterns of PP2C genes between the sugarcane hybrid cultivar genome under study and S. spontaneum, a synteny analysis was performed ( Figure 3 ; Supplementary Table S3 ). A total of 633 genes were found to be involved in synteny relationships, forming 1,988 pairs of associations and clustering into 62 synteny networks. Among these synteny pairs, 477 out of 500 PP2C genes were found to be associated with other genes from the sugarcane hybrid cultivar under study and S. spontaneum. A total of 156 genes from S. spontaneum were involved in synteny associations, of which 76 genes were identified as PP2C genes. All S. spontaneum genes involved in synteny relationships were located on five chromosomes and their subgenomes (1, 2, 3, 4, and 5). The gene IDs of PP2C genes in S. spontaneum, along with their locus IDs involved in synteny relationships, are listed in Supplementary Table S4 . Furthermore, in the sugarcane hybrid cultivar, all PP2C genes with chromosome annotations were included in the synteny analysis and found to be involved in synteny relationships.

Gene duplication events were analyzed to identify tandem duplication patterns in the PP2C gene family of sugarcane. Out of 500 genes, 107 were identified as duplicated, indicating the presence of paralogous genes ( Figure 4 ). A total of 105 duplicated genes were mapped across 45 chromosomes, including 8 chromosomes and their subgenomes, along with 5_9A, 8_5A, and 7os1. Additionally, two duplicated genes were found in scaffolds. Among the PP2C subfamilies, subfamily F exhibited the highest number of duplication events, with 23 genes involved, followed by subfamily E (18 genes), A (11 genes), D (11 genes), G (10 genes), H (8 genes), K (8 genes), I (5 genes), J (5 genes), M (5 genes), and L (3 genes).

Pairwise synonymous (Ks) and non-synonymous (Ka) substitution rate analysis was performed among duplicated SoffiXPP2C gene pairs, and the results indicated that most gene pairs showed Ka value 0, while Ks values were 0 or less than 1 ( Supplementary Table S8 ). Only two pairs had non-zero substitution ratios such as “SoffiXPP2C82 and SoffiXPP2C54” with a Ka/Ks value of 0.6759, and “SoffiXPP2C89 and SoffiXPP2C49” with a value of 0.12595. No gene pairs exhibited a Ka/Ks ratio.

A total of 34 out of 500 PP2C genes from the sugarcane hybrid cultivar were found to align with five differentially expressed contigs under oxidative stress conditions. Supplementary Table S5 presents these 34 SoffiXPP2C genes along with their alignment details with the five differentially expressed contigs. Supplementary Table S6 presents the differential expression data for five contigs that showed homology with 34 SoffiXPP2C genes in sugarcane hybrid cultivar. It details expression metrics such as Max Group Mean, Log₂ Fold Change, Fold Change, P-value, FDR P-value, and Bonferroni correction. Among these, Contigs joined_130 (SoffiXPP2C95, 105, 115, 126, 136, 142, 147, 156, and 351) exhibited the highest expression level (Max Group Mean = 17.11) with a Log₂ Fold Change of 0.6 and a statistically significant P-value of 0.00434. In contrast, Contigs joined_733 (SoffiXPP2C142) displayed a negative Log₂ Fold Change (-4.02) and a large negative Fold Change (-16.21), indicating strong downregulation. Contigs joined_346 (SoffiXPP2C10, 27, 42, 56, 71, 84, 278, 287, 296, 306, 317, 326, 448, 456 and 498) showed moderate upregulation with a Log₂ Fold Change of 0.71. On the other hand, Contigs joined_36 (SoffiXPP2C161, 187, 2023, 225) and Contigs joined_215 (SoffiXPP2C332, 339, 353, 357, 468, and 474) had a Max Group Mean of 0 and contained NaN values for differential expression metrics, indicating no detectable significant expression changes. The presence of NaN values suggests missing or unmeasurable expression levels, which may require further validation. Further annotation from UniProt revealed that four out of five differentially expressed contigs are associated with functions related to protein-serine/threonine phosphatases and the phosphatase 2C family. Additionally, they were identified as PPM-type phosphatase domain-containing proteins, RRM domain-containing proteins, splicing factor U2AF large subunit, and poly(A)-specific ribonuclease, while one contig was identified as glycerol kinase ( Supplementary Table S6 ).

Gene Ontology (GO) analysis was conducted for 34 SoffiXPP2C genes based on their sequence homology with rice and sorghum ( Supplementary Table S7 ). Six homologous genes from each of rice and sorghum were identified corresponding to the SoffiXPP2C gene set. The GO terms associated with these homologs were retrieved and used to infer the putative functions of the SoffiXPP2C genes. GO term assignments for mapping of functional annotations to the GO terms were performed using the Go.db package in R ( Figure 5 ). A large number of Gene Ontology (GO) terms fall under the biological Process (BP) category, indicating functional roles in response to stress, biotic and abiotic stimuli, metabolic processes (including carbohydrate metabolism, glycerol-3-phosphate metabolism, and biosynthetic pathways), as well as catabolic and general cellular processes. In the Molecular Function (MF) category, the GO terms suggest involvement in phosphatase activity (protein serine/threonine phosphatase, hydrolase, and kinase activities) along with catalytic and transferase functions. For the Cellular Component (CC) category, the associated genes are primarily localized to the nucleus, cytosol, and plasma membrane.

PCR amplification of four sugarcane PP2C genes SoffiXPP2C31, SoffiXPP2C75, SoffiXPP2C60, and SoffiXPP2C14 was performed to confirm the presence of the target genes in genomic DNA and to assess their expression under waterlogging stress conditions using cDNA synthesized from treated samples ( Figure 6 ). For genomic DNA amplification, leaf samples from five sugarcane cultivars Co 86032, Co 11015, Co 91010, Co 0233, and Co 16001 were used. All four genes showed successful amplification across all cultivars producing bands of different sizes: SoffiXPP2C31 (594 bp), SoffiXPP2C75 (595 bp), SoffiXPP2C14 (503 bp), and SoffiXPP2C60 (599 bp), confirming their presence in the genome. For cDNA amplification, the stem and root samples from five sugarcane cultivars Co 62175, Coc 1339, Coc 1339, C 2014 516, and C 16338 were analysed. SoffiXPP2C31 showed amplification in both stem and root samples of all cultivars producing bands of 594 bp. SoffiXPP2C75 was amplified in all samples except the stem cDNA of cultivar C 2014 516 producing bands of 595 bp. SoffiXPP2C14 showed amplification in all samples except the root cDNA of cultivar C 2014 516 producing bands of 503 bp. SoffiXPP2C60 amplification was observed only in the root cDNA of cultivar C 2014 516 producing bands of 599 bp ( Figure 6 ).

Gene expression profiling of roots and stem samples of sugarcane was conducted through qRT-PCR. The samples were taken from the waterlogging stress experiments. The relative expression levels of SoffiXPP2C75, SoffiXPP2C60, SoffiXPP2C31 and SoffiXPP2C14 genes with the endogenous control 25s were examined using relative quantification or comparative CT method. Fold expression [2^ (-ΔΔC T)] of the samples related to respective genes are interpreted ( Table 1 ).

From the fold expression values of each sample both up regulated (greater than 1) and down (lesser than 1) regulated performance of gene has been observed.

The root sample of Co 62175 (1R) showing up regulation for all the genes except SoffiXPP2C14. The stem sample of Co 62175 (1S) showing up regulation for all the genes except SoffiXPP2C60. The root and stem sample of this genotype showing relatively higher expression in SoffiXPP2C60 and SoffiXPP2C14 respectively. The root and stem sample of Co 86032 (4R & 4S) showing down regulation for all the four genes. The root sample of CoC 13339 (5R) up regulated in all genes except SoffiXPP2C14. The root sample of C 16338 showing up regulation in SoffiXPP2C14 and all other genes are down regulated. The stem sample of C 2014-516 (11s) showing up regulation for all the four genes with higher expression in SoffiXPP2C75 gene. The gene expression indicates that the SoffiXPP2C genes differentially expressed in different cultivars in root and stem samples ( Figures 7 , 8 ).