https://orcid.org/0000-0001-7725-3899
Figures
Abstract
The NCED (9-cis-epoxy carotenoid dioxygenase) enzyme regulates the biosynthesis of abscisic acid (ABA), which is responsible for plant growth, development, and response to various environmental challenges. However, no genome-wide identification, characterization, functional regulatory element analysis, and expression profiles in response to different abiotic stresses of the NCED gene family have yet to be investigated in an economically important legume plant species, soybean (Glycine max L.). Through comprehensive analysis, 16 NCED genes (named GmNCED1 to GmNCED16) belonging to the RPE65 domain were identified in the soybean genome and found to be unequally distributed over 9 distinct chromosomes. The distinct intron-exon structures of GmNCED genes were categorized into six groups and shared a close relationship with the grapevine. Segmental gene duplication events and the purifying selection process were evident in GmNCED genes, according to evolutionary studies. Cis-acting regulatory element analysis revealed that GmNCED genes were largely associated with light response as well as stress response. ERF, MYB, bZIP, and LBD emerged as the major transcription factors in GmNCED genes. The protein-protein interactions demonstrated the close relationship between GmNCED and Arabidopsis thaliana proteins, while micro-RNA analysis revealed the involvement of GmNCED genes in plant growth and development as well as in the regulation of abiotic stress. The expression profiles of GmNCED2, GmNCED11, and GmNCED12 provided evidence of their engagement in dehydration and sodium salt stress, whereas GmNCED14 and GmNCED15 were up-regulated in drought stress. Moreover, the up-regulation of GmNCED13 and GmNCED14 genes in heat tolerant germinated seed stages at high temperature delta region. More specifically, GmNCED14 might be used as a novel candidate gene under drought stress, and influencing seed germination at high temperature. Overall, this study identified the crucial role of GmNCED in conferring resistance against abiotic stress such as dehydration, salt, and drought, and also uncovering the detailed regulatory mechanism of ABA biosynthesis during seed germination.
Citation: Islam MSU, Shing P, Ahmed M, Zohra FT, Rownaq A, Paul SK, et al. (2025) Genome-wide identification and characterization of NCED gene family in soybean (Glycine max L.) and their expression profiles in response to various abiotic stress treatments. PLoS ONE 20(3): e0319952. https://doi.org/10.1371/journal.pone.0319952
Editor: Niraj Agarwala, Gauhati University, INDIA
Received: March 28, 2024; Accepted: February 11, 2025; Published: March 25, 2025
Copyright: © 2025 Islam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Plants have developed a wide range of defense mechanisms and strategies to sustain, endure, and recover from environmental challenges such as water scarcity, saltiness, heat, cold stress, heavy metal toxicity, disease, and others. Among these strategies, abscisic acid (ABA) biosynthesis machinery is prerequisite for the plant growth and development including germination and seed dormancy, leaf senescence, the architecture of roots, regulation of stomata, and vegetable development and resistance to abiotic stress [1]. ABA can be synthesized in plants by direct or indirect pathways, with most higher plants using the indirect C40 pathway [2]. In the C40 pathway, zeaxanthin epoxidase (ZEP) enzyme converts zeaxanthin to violaxanthin and ultimately to neoxanthin. The conversion of violaxanthin and neoxanthin into xanthoxin is mediated by NCED enzyme [3]. Xanthoxin, the immediate precursor of ABA, is subsequently converted into abscisic aldehyde by the action of the short-chain alcohol dehydrogenase enzyme, ABA2. Ultimately, abscisic aldehyde is then oxidized by abscisic aldehyde oxidase to form abscisic acid [4,5].
NCED belongs to the CCD gene family, which typically contains the conserved domain REP65 [6,7]]. The maize V14 gene, expressed in embryos and roots, was the first NCED gene to be discovered and cloned [3,8]. Members of the NCED gene family have been studied in various plant species, including cotton (Gossypium hirsutum) [5], Arabidopsis (Arabidopsis thaliana) [9], grape (Vitis vinifera) [10], avocado (Persea americana) [11], and rice (Oryza sativa) [12]. In Arabidopsis, AtNCED6 is constitutively expressed in the endosperm and interacts with AtNCED9 to regulate seed dormancy and germination by regulating ABA levels [9,13–15]. When the NCED gene is expressed in seed, elevated levels of ABA is biosynthesized resulting in the seed retaining dormancy until favorable conditions for germination occur [16]. Conversely, AtNCED3 is primarily induced by water stress and regulates endogenous ABA levels in water-stressed environments [9,17,18]. In rice, OsNCED3 and OsNCED4 alter the plant and leaf morphology and enhance drought stress resistance [19–21]. Moreover, elevated levels of reactive oxygen species (ROS) signal drought stress [22]. Under this conditions, plants synthesize primary metabolites (PMs) and secondary metabolites (SMs) and exclusively store SMs products such as terpenes, phenolic compounds, glycine-betaine, and proline to counteract the drought [23]. Alternatively, the up-regulation of the NCED gene triggers the different signaling cascade pathways including mitogen-activated protein kinase (MAPK) to increase the production of ABA, thus boosting stomatal closure and minimizing water loss through transpiration [24–26]. This entire mechanism is regulated by various transcription factors (TFs) including MYB, NAC, ABF, DREB/CBF, MYC, ERF, and bZIP [27]. ABA regulation through NCED also influences root modulation and lateral root formation under such conditions [28].
Soybean (Glycine max L.), globally recognized oil crop species, belonging to the Fabaceae family, is important for both its economic and nutritional value. Soybean is an essential protein for both human and animal consumption, making its growth and productivity crucial for global food security [29]. However, abiotic factors such as drought, salt, and severe temperatures often severely impact on soybean cultivation [30]. In response to these challenges, plants activate complex stress-responsive ABA signaling-mediated pathways [31].
The objective of this study is to identify and annotate the GmNCED genes and unveil their involvement in ABA biosynthesis during the seed germination and in response to dehydration, sodium salt and drought stress. Comprehensive bioinformatics approaches were used to elucidate GmNCEDs physical and chemical properties, phylogenetic relationship, conserved domain, motifs, gene structural feature analysis, evolutionary relationships, chromosome mapping, gene duplication, subcellular localization, cis-acting regulatory elements, gene ontology, micro-RNA analysis along with performing RNA-seq analysis. Overall, this study will shed light on the mechanisms behind the functional diversity and the involvement of ABA biosynthesis during seed germination and stress responses aiding in the development of improved soybean cultivars in future breeding programs.
2. Methods and materials
2.1. Database searching and retrieval of GmNCED protein sequences
A. thaliana NCED DNA-binding domains were employed to extract NCED gene-encoding proteins in G. max from phytozome version 13 (https://phytozome-next.jgi.doe.gov/) using BLASTp (Protein-basic local alignment search tool) with an expected (E) threshold value of -1, comparison matrix (BLOSUM62), and other default parameters (S1 Data) [32]. SMART (Simple Modular Architecture Research Tool; http://smart.embl-heidelberg.de/) [33] and the NCBI CDD (Conserved Domain Database; https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) were used to analyze conserved domains with default parameters [34]. These proteins encoding typical RPE65 domains were renamed according to the order of their physical chromosomal positions.
2.2 Determination of physio-chemical properties of GmNCED
The ProtParam online program (http://web.expasy.org/protparam/) was employed to determine the number of amino acid (a.a) residues, aliphatic index, molecular weight, instability index, isoelectric point (pI), and grand average of hydropathicity (GRAVY) of GmNCED proteins [35].
2.3. Phylogenetic relationship analysis between PpNCED, RcNCED, AtNCED, OsNCED, VvNCED, and GmNCED
NCED proteins derived from rose (Rosa chinensis) [36], grape vine (Vitis vinifera) [10], Arabidopsis (Arabidopsis thaliana) [9], peach (Prunus persica) [37], rice (Oryza sativa) [12], and soybean (Glycine max) were used to construct a phylogenetic tree (S2 Data). The MEGA11 software and ClustalW program were utilized to align the amino acid sequences and build the phylogenetic tree [38,39]. The NJ (neighbor-joining) method was used with 1000 bootstrap value and keeping other as default parameters. The constructed tree was uploaded to iTOL online tool version 6.7.4 (https://itol.embl.de/) for attractive visualization [40].
2.4. Gene structure analysis of GmNCED
CDS sequence (S3 Data) and genomic sequence (S4 Data) were uploaded to the Gene Structure Display Server database version 2.0 (GSDSv2.0; http://gsds.cbi.pku.edu.cn/) to analyze the GmNCED genes structure [41].
2.5. Conserved domain and motif analysis of GmNCED
InterPro database (http://www.ebi.ac.uk/interpro/) was employed to predict the conserved domains and visualized using TBtools software version 1.116 [42]. The structural motif of the GmNCED proteins was analyzed in Multiple EM for Motif Elicitation (MEME) tools of MEME-suite (https://meme-suite.org/meme/), selecting a maximum number of motifs 20 with other default parameters [43]. The motifs were visualized using the MEME online interface, employing the MEME and motif scanning approach.
2.6. Evolutionary divergence time and Ka/Ks ratio calculation in GmNCED
The GmNCED gene family Ka (non-synonymous) and Ks (synonymous) substitution ratio was computed using the Ka/Ks calculator (https://bio.tools/kaks_calculator) [44]. The duplication and time of divergence measured in million years ago (MYA) of GmNCED genes were determined using the formula T = Ks/2λ, where λ is equal to 6.5 × 10-9 [45]. The data was converted into log2 format in a heat map in TBtools to visualize the evolutionary relationship and divergence rate.
2.7. Collinearity and synteny analysis of GmNCED
Collinear and synteny analysis were performed based on the gene duplication events in GmNCED genes. Subsequently, the syntenic pairs with A. thaliana, O. sativa, and V. vinifera and collinear relationship within GmNCED genes were visualized in TBtools.
2.8. Chromosomal mapping and duplication analysis of GmNCED
The distribution of GmNCED genes across the chromosomes was mapped and visualized in the MapGene2Chrom online tool version 2.0 (MG2C; http://mg2c.iask.in/mg2c_v2.0/) [46]. The duplication of GmNCED genes was also illustrated.
2.9. Subcellular localization of GmNCED
The Wolf PSORT online tool (https://wolfpsort.hgc.jp/) was utilized to determine the subcellular localization of GmNCED genes [47]. The predicted protein signals were illustrated employing RStudio software version 2023.06.1 [48].
2.10. Cis-acting regulatory elements (CAREs) analysis of GmNCED
The 2000 bp from 5′ untranslated region (5′ UTR) was used to predict CAREs (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (S5 Data) [49]. The predicted CAREs were classified and visualized in heatmap using TBtools.
2.11. Gene ontology (GO) analysis of GmNCED
GO analysis was performed by extracting GO IDs from Plant Transcriptional Regulatory Map database (PlantRegMap; https://plantregmap.gao-lab.org/go.php) with p-value 0.01 and other default parameters [50]. Additionally, GO enrichment was visualized in chiplot online tool (https://www.chiplot.online/) [51].
2.12. Transcription factors (TFs) analysis of GmNCED
TFs binding site prediction in GmNCED genes was performed in the Plant Transcriptional Regulatory Map database (PlantRegMap; https://plantregmap.gao-lab.org/binding_site_prediction.php) using threshold p-value 1.0E-4 and other default parameter.
2.13. Regulatory network between TFs and GmNCEDs
Cytoscape software version 3.9.1 was utilized to construct and display the interaction network between TFs and predicted GmNCED genes [52].
2.14. Prediction of putative micro-RNAs (miRNAs) and network targeting GmNCED
The sequences of the putative miRNA of GmNCED were extracted from miRBase (https://mirbase.org/) [53]. The CDS sequences of GmNCED were uploaded to psRNATarget Server18 (https://www.zhaolab.org/psRNATarget/analysis?function=2) that target GmNCED genes keeping other parameters as default [54]. Furthermore, the interaction network of the predicted miRNAs and GmNCEDs was illustrated in cytoscape.
2.15. Protein-protein interaction (PPI) prediction of GmNCED
A. thaliana homologous proteins were used to predict the PPI network of GmNCED proteins in the string online tool version 12 (https://string-db.org/) [55]. The parameters of the string were kept as network type-full STRING network; the meaning of network edges-evidence; active interaction source-text mining, experiments, databases, co‑expression, neighborhood, gene fusion, co‑occurrence; minimum required interaction score-medium confidence parameter (0.4); maximum number of interactions display 1st shell-no more than 10, with 2nd shell was left blank; and enabling network display options as 3D bubble design.
2.16. Transcriptomic data analysis of GmNCED under salt, dehydration, and drought stresses
The RNA-seq data for salt and dehydration (GSE57252), drought stress (GSE69469) was extracted from the NCBI’s Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) [56]. A heat map was generated in TBtools to illustrate the expression pattern of GmNCED. The normalized FPKM (Fragments Per Kilobase per Million mapped fragments) value was log2 transformed to distinguish expression levels in the particular gene.
2.17. Transcriptomic data analysis of GmNCED during various seed developmental stages
The RNA-seq data included with two soybean lines; PI 587982A, a heat-tolerant landrace, and S99-11986, a conventional high-yielding adapted line) during three seed germination stages: (1) 6 h imbibed seed; (2) germinated seed; and (3) dry and mature seed, which were produced in two regions, considering the low-temperature south region, and heat stressed field located in the delta region [57]. The Sequence Read Archive (SRA) under bio-project PRJNA509794 was obtained from NCBI to perform the RNA-seq analysis. For trimming and quality control of the RNA-seq, trimmomatic package version 0.32 was used [58]. Subsequently, star package version 2.7.11b was utilized to align the RNA sequencing with the reference genome G. max [59]. Samtools package version 1.20 converted sequence alignment map (SAM) files to binary alignment map (BAM) files [60]. RSEM package version 1.1.17 was used to calculate the FPKM value [60,61]. Chiplot was used to visualize the expression pattern of GmNCED genes.
3. Results
3.1. Physicochemical characteristics of GmNCED
The physicochemical properties of GmNCED proteins were analyzed, revealing variation from 239 (GmNCED13) to 618 a.a (GmNCED12), with a mean length of 508.87 a.a (Table 1). The molecular weights of the encoded proteins estimated to range from 26559.57 (GmNCED13) to 69373.56 kDa (GmNCED12). The pI measurement indicated that 11 GmNCEDs had pI value less than 7.0, classifying them as acidic, while five GmNCEDs had pI value greater than 7.0, indicating an alkaline nature. According to the instability index analysis, there were five GmNCED proteins (GmNCED5, GmNCED8, GmNCED9, GmNCED11,GmNCED16) that had instability index value exceeding 40.0. Whereas, there were eleven GmNCED proteins (GmNCED1, GmNCED2, GmNCED3, GmNCED4, GmNCED6, GmNCED7, GmNCED10, GmNCED12, GmNCED13, GmNCED14,GmNCED15) that had instability index value lower than 40.0. The aliphatic index varied, with GmNCED7 contained the highest aliphatic of 84.70 and GmNCED10 and GmNCED11 possessing the lowest aliphatic index of 75.16. All the GmNCED proteins were found to be hydrophilic as indicated by their negative GRAVY score.
3.2. Phylogenetic relationship analysis between PpNCED, RcNCED, AtNCED, OsNCED, VvNCED, and GmNCED
A phylogenetic tree was constructed using the full-length a.a sequence between P. persica (12 PpNCEDs) R. chinensis (13 RcNCEDs), A. thaliana (5 AtNCEDs), O. sativa (5 OsNCEDs), V. vinifera (12 VvNCEDs), and G. max (16 GmNCEDs) (Fig 1). The phylogenetic tree categorized 63 NCED proteins into six distinct groups; A, B, C, D, E, and F. Group F consisted with the highest overall NCED protein count (22), while group A had the least number (5). Among GmNCED proteins distribution among groups, group C emerged with the highest number (4). Meanwhile, group D contained the least number (1) (S6 Data). But, no AtNCED or OsNCED proteins were found in group A, B, C, and E.
GmNCED was classified into 6 groups (A, B, C, D, E, and F), each marked by different colors and shapes. The red color star labeled the GmNCED. Whereas, RcNCED was labeled as triangular green, PpNCED was labeled as the light carmine pink triangle, VvNCED was labeled dark blue triangular, AtNCED was labeled as black triangular, and OsNCED was labeled as the magenta color triangle.
3.3. Gene structure analysis of GmNCED
The structural diversity of GmNCED genes were analyzed by comparing the distribution patterns of intron and exons. GmNCEDs genes exhibited a range of 0 to 13 introns and the highest intron count (43) observed in group C (Fig 2; S7 Data). GmNCED13, a member of group E, contained maximum number of introns (13). Additionally, no introns were found in group F. GmNCEDs genes exhibited a range of 1 to 14 exons, with group C consisting the largest number of exons (47). GmNCED13, from group E, contained the highest number of exons (14). The least number of exons were found in groups F, where GmNCED6, GmNCED10, GmNCED11, and GmNCED16 contained one exon each.
The grouping and colors of the GmNCED gene family members are based on the phylogenetic relationship. For the color bar, red represents exons, deep blue lines represent upstream/downstream and 0, 1, 2 represents intron phase.
3.4. Conserved domain and motif analysis of GmNCED
Conserved domain analysis revealed that all GmNCED proteins possessed an RPE65 domain (Fig 3). A total of 20 different conserved motifs were analyzed in the GmNCED proteins (Fig 4). All GmNCEDs, except for GmNCED13, possessed more than seven motifs. In contrast, GmNCED13 consisted of six motifs. Both GmNCED5 and GmNCED9 in group A shared the same motif patterns. Similarly, GmNCED1 and GmNCED12 in group B contained 12 motifs sharing similar motif structure. Further, motif 17 was only found in GmNCED1 and GmNCED12. The motifs varied among GmNCED14, GmNCED13, and GmNCED15 in group E and between GmNCED11 and GmNCED16 in group G. GmNCED10 contained the highest number of motifs (18), sharing all but motif 19 with GmNCED6. Motif 3 and Motif 9 were shared by all GmNCEDs except for GmNCED13. Additionally, all the motif logos varied in structure (S1 Fig).
The positions of the RPE65 conserved domain are demonstrated in sky blue color whereas the entire protein sequence of respective GmNCED is magenta colored.
Each motif is illustrated by a specific-colored box aligned on the right side of the figure. Different colors indicate individual motifs identified within each protein domain.
3.5. Evolutionary divergence time and Ka/Ks ratio calculation in GmNCED
The Ka value of GmNCED gene pairs varied between 0.01406467 and 0.31955, while the Ks value ranged from 0.05235362 to 0.671 (Fig 5). The ratio of Ka/Ks was within the range of 0.188673269 to 0.51972207. This indicated that they evolved primarily under purifying selection (S8 Data). The duplication time of the GmNCED gene pairs ranged from 3.990367378 to 51.14329268 MYA, with GmNCED11-GmNCED2 gene pairs showing evolutionary origin of 51.14329268 MYA.
The ratio of nonsynonymous (Ka) and synonymous (Ks) is represented by Ka/Ks. The time of divergence (measured in million years ago, MYA) is also represented. The different color bar represents the data range.
3.6. Collinear relationship analysis of GmNCED
The results indicated that single GmNCED gene was found on chromosome 5, 11, 13, and 15, while more than one GmNCED genes were found on chromosome 4 and 6 (Fig 6). In addition, GmNCED12 had a collinear gene pair with GmNCED1, GmNCED11 with GmNCED2, GmNCED3 with GmNCED7, GmNCED9 with GmNCED5, GmNCED6 with GmNCED10, and GmNCED15 with GmNCED13, resulting in a total of 6 pairs. Yet, GmNCED16, located on chromosome 15, did not have any collinear gene pairs.
Green color rectangles represent chromosomes of GmNCED. The purple red-colored lines linked between chromosomes represent collinear relations between the chromosomes.
3.7. Syntenic relationship analysis of GmNCED
The syntenic map was constructed between G. max (GmNCED) and of three other different plant species, two dicotyledonous notably V. vinifera (VvNCED) and A. thaliana (AtNCED), as well as the monocotyledonous O. sativa (OsNCED) (Fig 7). Unfortunately, no syntenic gene pairs were observed among GmNCED genes.
Red color rectangles represent the GmNCED chromosomes. Meanwhile, green rectangles represent AtNCED chromosomes. Furthermore, magenta color rectangles represent OsNCED chromosomes while blue color rectangles represent VvNCED chromosomes. The same color format is used to represent the syntenic relationship linkage between different species.
3.8. Chromosome mapping and duplications analysis of GmNCED
The analysis of chromosomal localization revealed that GmNCED genes were unequally distributed across the 9 different chromosomes (Fig 8). Chromosome 4 and 6 contained the highest number of GmNCED genes (3 each). Chromosome 4 (GmNCED4), chromosome 6 (GmNCED8), chromosome 12 (GmNCED14), and chromosome 15 (GmNCED16) were the independent chromosomes. Six segmental duplicated gene pairs; GmNCED11-GmNCED2, GmNCED10-GmNCED6, GmNCED15-GmNCED13, GmNCED12-GmNCED1, GmNCED9-GmNCED5, and GmNCED7-GmNCED3 were identified. Nevertheless, no evidence of tandem duplication was seen in the GmNCED gene family.
The number of distinct chromosomes is at the top of each chromosome bar. The chromosome scale is in millions of bases (Mb), indicating the length of each chromosome on the left. The chromosome is colored yellow, while sky blue lines indicate segmental duplications.
3.9. Subcellular localization analysis of GmNCED
The subcellular localization analysis revealed that the most of the GmNCEDs were located in the cytoplasm and chloroplast, with 13 GmNCED genes found in these organelles (Fig 9A). GmNCED4 was observed in highest number of organelles (6). Golgi apparatus and vacuole possessed one GmNCED each, which is GmNCED16, and GmNCED4 respectively. Additionally, fewer number of GmNCED genes were conserved in the nucleus, cytoskeletal, endoplasmic reticulum, and plasma membrane. Moreover, a significant percentage of GmNCED genes were also found in mitochondria, peroxisomal, and extracellular regions (Fig 9B). Moreover, the specific number of GmNCED genes that were found in a particular organelle was illustrated using a bubble plot (S2 Fig).
A. The heatmap represents the sub-cellular localization analysis of GmNCED genes. The names of each GmNCED gene are shown on the left side of the heatmap, while the names of the respective cellular organelles are shown at the bottom of the heatmap. The intensity of color on the right side of the heatmap indicates the presence of protein signals corresponding to the genes. B. The percentage distribution of GmNCED gene signal across various cellular organelles is represented by a bar diagram. The percentages of protein signals appearing in different cellular organelles are shown on the left side of the diagram.
3.10. Cis-acting regulatory elements (CAREs) analysis of GmNCED
46 CAREs were identified and classified into four distinct categories according to their functional regulation: light responsiveness, tissue-specific expression, phytohormone responsiveness, and stress responsiveness (Fig 10; S9 Data). Among these 46 CAREs, light responsiveness was the biggest group which included 20 elements such as GA-motif, Gap-box, GT1-motif, ACE, G-Box, G-box, AE-box, AT1-motif, ATCT-motif, Box 4, Box II, chs-CMA1a, GATA-motif, GTGGC-motif, I-box, LAMP-element, TCT-motif, MRE and TCCC-motif. Box 4 exhibited the highest number of cis elements in this category. The second-largest group was phytohormone responsiveness, which was composed of methyl jasmonate (MeJA) response elements (CGTCA-motif and TGACG-motif), salicylic acid (SA) response elements (TCA-element), gibberellin (GA) response elements (GARE-motif and TATC-box), abscisic acid (ABA) response elements (ABRE), anoxic specific inducibility (GC-motif), and auxin-responsive elements (TGA-element, AuxRR-core, and TGA-box). ABA response element (ABRE) was the largest group of phytohormone responsiveness-related cis-elements. The third largest group was tissue-specific expression which included A-box, ARE (anaerobic induction), AT-rich element (DNA binding protein), AT-rich sequence (maximal elicitor-mediated activation), CAT-box (meristem expression), CCAAT-box (MYBHv1 binding site), circadian (circadian control), GCN4_motif (endosperm expression), HD-Zip 1 (differentiation of the palisade mesophyll cells), MBSI (flavonoid biosynthetic gene regulation), MSA-like (cell cycle regulation), and O2-site (zein metabolism regulation). The most significant group was stress responsiveness, which included LTR (low-temperature responsive elements), TC-rich repeats (defense and stress-responsive elements), MYB (MYB binding sites involved in drought inducibility), and WUN-motif (wound-responsive elements).
The names of each GmNCED are shown on the left side of the heatmap. The number of putative cis-acting elements for each GmNCED gene is displayed on the right side of the heatmap and is represented by distinct colors. Functions associated with cis-acting elements of the corresponding genes, such as light responsiveness, tissue-specific expression, phytohormone responsiveness, and stress responsiveness, are shown at the bottom of the heatmap and labeled as red, yellow, green, and dark blue respectively.
3.11. Gene Ontology (GO) analysis of GmNCED
56 GO IDs were identified in GmNCED genes and classified into three categories according to their respective functions; biological process (BP), cellular component (CC), and molecular function (MF) (Fig 11; S10 Data). The biological process was found predominantly among the three categories, which comprised 41 GO IDs; GO:0055114 (p-value: 5.20E-14), GO:0016110 (p-value:1.10E-10), GO:0016118(p-value: 1.10E-10), GO:0016124 (p-value: 1.10E-10), GO:0016121(p-value: 4.40E-10), GO:0046247 (p-value: 4.40E-10), GO:0016119 (p-value: 1.30E-08), GO:0044710 (p-value: 1.70E-08), GO:0016122 (p-value: 3.10E-08), GO:0042214 (p-value: 9.00E-08), GO:0016106 (p-value: 1.10E-07), GO:0016115 (p-value: 1.10E-07), GO:0008300 (p-value: 1.30E-07), GO:0006714 (p-value: 3.60E-07), GO:0006721 (p-value: 6.10E-07), GO:0016108 (p-value: 7.80E-07), GO:0016116 (p-value: 7.80E-07), GO:0006720 (p-value: 2.30E-06), GO:1901334 (p-value: 5.20E-06), GO:1901336 (p-value: 5.20E-06), GO:1901600 (p-value: 5.20E-06), GO:1901601 (p-value: 5.20E-06), GO:0044242 (p-value: 1.10E-05) GO:0016114 (p-value: 1.50E-05), GO:0010223 (p-value: 3.80E-05) GO:0010346 (p-value: 3.80E-05), GO:0001763 (p-value: 4.20E-05), GO:0008299 (p-value: 5.30E-05), GO:0016042 (p-value: 7.90E-05), GO:0044699 (p-value: 0.00014), GO:0009926 (p-value: 0.00048), GO:0060918 (p-value: 0.00061), GO:0009914 (p-value: 0.00066), GO:0044255 (p-value: 0.00066), GO:0006629 (p-value: 0.00265), GO:0010016 (p-value: 0.00266), GO:0048646 (p-value: 0.00298), GO:0008610 (p-value: 0.00333), GO:0009414 (p-value: 0.00495), GO:0009415 (p-value: 0.00518), GO:0010817 (p-value: 0.00613). In addition, the cellular component included 8 GO functions; GO:0009570 (p-value: 0.00011), GO:0009532 (p-value: 0.00012), GO:0044434 (p-value: 0.00078), GO:0044435 (p-value: 0.00082), GO:0009507 (p-value: 0.00364), GO:0009536 (p-value: 0.00425), GO:0044446 (p-value: 0.00617), GO:0044422 (p-value: 0.00625) and the molecular component included 7 GO functions; GO:0016702 (p-value: 1.00E-30), GO:0016701 (p-value: 1.00E-30), GO:0051213 (p-value: 2.30E-29), GO:0016491 (p-value: 1.30E-15), GO:0010436 (p-value: 1.70E-07), GO:0045549 (p-value: 1.70E-07), GO:0003824 (p-value: 5.20E-05)
Classification of the GmNCED genes function are shown in circos plot. The number of genes involved under a certain GO ID, expected value, and rich factor are shown in a distinctive color. The scaling of the -log10 p-value is shown in three distinctive colors (red, yellow, and green).
Furthermore, the p-value in the BP, CC, and MF function categories were analyzed. Within the BP function category, GO:0010817 had the highest p-value of 0.00613, followed by GO:0009415 with a p-value of 0.00518, and then GO:0009414 with a p-value of 0.00495, and so on. Among the GO terms in the CC function category, GO:0044422 had the highest p-value of 0.00625, followed by GO:0044446 with a p-value of 0.00617, and subsequently GO:0044434 with a p-value of 0.00425. In the MF function category, GO:0003824 had the highest p-value of 0.000052, followed by GO:0010436 with a p-value of 0.00000017, and subsequently GO:0045549 with a p-value of 0.00000017. In BP, the single-organism process (GO:0044699, p-value: 0.00014), oxidation-reduction process (GO:0055114, p-value: 5.20E-14), and the single-organism metabolic process (GO:0044710, p-value: 1.70E-08) were predominantly seen. There were also a lot of different types of activities in the MF category. A few notable activities included oxidoreductase activity, acting on single donors and adding molecular oxygen (GO:0016702, p-value: 1.00E-30), oxidoreductase activity, which acts on single donors and adds molecular oxygen (GO:0016702, p-value: 1.00E-30), dioxygenase activity (GO:0051213, p-value: 2.30E-29), catalytic activity (GO:0003824, p-value: 5.20E-05) and oxidoreductase activity (GO:0016491, p-value: 1.30E-15).
3.12. Transcription factors (TFs) analysis of GmNCED
In this analysis, an overall 91 unique TFs were found which regulate the 16 GmNCED genes. The identified TFs were categorized into seven distinct families, including ERF, MYB, bZIP, LBD, C2H2, GATA, and TALE (Fig 12). Among them, four major families such as ERF, MYB, bZIP, and LBD included 39, 22, 11, and 8 TFs respectively accounting for 87.91% of overall 91 detected TFs.
The color intensity of the heatmap indicates the presence of TFs corresponding to the proteins. The TFs are distributed into 7 TF families recognized by distinctive color. The 7 TFs family are ERF, MYB, bZIP, LBD, C2H2, GATA, and TALE which are colored red, light green, light red, orange, yellow, lime, light green, and dark green respectively.
3.13. Regulatory relationship between TFs and GmNCED
The sub-network connection between TFs and GmNCED genes were predicted (Fig 13). The sub-network analysis findings indicated that ERF was associated with eleven GmNCED genes except GmNCED1, GmNCED3, GmNCED4, GmNCED7, and GmNCED14. In addition, MYB TF family was associated with GmNCED1, GmNCED2, GmNCED5, GmNCED6, GmNCED8, GmNCED9, GmNCED10, GmNCED11, and GmNCED12. Similarly, LBD TF family constructed the regulatory relationship with GmNCED2, GmNCED5, GmNCED6, GmNCED7, GmNCED9, GmNCED10 and GmNCED11. Furthermore, GATA and TALE TF family linked to GmNCED1, GmNCED2, GmNCED5, GmNCED6, GmNCED9, GmNCED10, GmNCED11, GmNCED12, GmNCED14, GmNCED15, GmNCED16 and GmNCED2, GmNCED5, GmNCED6, GmNCED9, GmNCED10, GmNCED11, GmNCED13 respectively. The C2H2 TF were associated with GmNCED2, GmNCED3, GmNCED4, GmNCED5, GmNCED6, GmNCED10, GmNCED11, GmNCED13, GmNCED14 and GmNCED15. However, there were only four GmNCED genes (GmNCED1, GmNCED6, GmNCED10, and GmNCED11) in the bZIP family.
The GmNCED is shown in round red. Whereas, the TFs are shown in green colors and different shapes. The 7 TF families ERF, MYB, C2H2, GATA, bZIP, LBD, and TALE have represented the shapes as diamond, parallelogram, rectangular, octagonal, triangle, hexagonal, and V respectively.
3.14. Prediction of putative micro-RNAs (miRNAs) and network targeting GmNCED
In this analysis, 126 mature miRNAs targeting all 16 genes of GmNCED were shown in the network illustration (Fig 14A; S11 Data) and the schematic diagrams indicate the GmNCED genes targeted by miRNAs (Fig 14B). The data analysis further showed that about 53 unique miRNA sequences were present. It was identified that 16 members of gma-miR166 targeted one gene in particular GmNCED9 (Table 2). Moreover, 9 members of gma-miR482 targeted GmNCED2, GmNCED6, and GmNCED10. Whereas, 6 family members of gma-miR159 targeted GmNCED3, GmNCED7 and GmNCED8 on the contrary 5 members of gma-miR9752 targeted 5 GmNCED genes (GmNCED1, GmNCED6, GmNCED10, GmNCED12 and GmNCED16). However, the majority of the miRNA targeted two or more genes for instance 3 members of gma-miR169 targeted GmNCED3 and GmNCED11. In the context of genes, GmNCED9 was targeted the most 19 times on the contrary, GmNCED8 was targeted 15 times. Whereas, GmNCED1, GmNCED3, and GmNCED6 were observed to be targeted up to 10 times.
A. Network illustration of predicted miRNA targets GmNCED genes. The red rectangle represents GmNCED genes while microRNA is labeled as sky blue ellipse. B. The schematic diagram indicates the GmNCED genes targeted by miRNAs. The green round rectangular is shown as exons of the respective gene, the blue round rectangular represents UTR, the straight black line represents intron and the red color small round rectangular represents miRNA.
3.15. Protein-protein interaction (PPI) prediction of GmNCED
GmNCED was identified as string protein based on its higher homology with Arabidopsis. The result showed that 15 GmNCED proteins interacted with Arabidopsis proteins (S12 Data). Six GmNCED proteins (GmNCED3, GmNCED7, GmNCED8, GmNCED13, GmNCED14 and GmNCED15) were observed to homologous with AtCCD1 (Fig 15). Furthermore, AtCCD1 interacted with CCD7, CCD8, D27, LCY1, Z-ISO and BETA-OHASE2. On the contrary, GmNCED6, GmNCED10, GmNCED11, and GmNCED16 were observed to carry the same feature as AtNCED3. AtNCED3 linked with LCY1, CCD7, AAO3, Z-ISO and BETA-OHASE2. AtCCD7 and AtCCD8 were noticed to be homologous with two GmNCEDs GmNCED1, GmNCED12, and GmNCED5, GmNCED9 respectively. They both interacted with CCD1, CCD4, AA03, NCED3, LCY1, and Z-ISO. However, AtCCD4 was only homologous with GmNCED2.
The proteins were displayed at network nodes with the proteins in nodes, and the line colors indicate different data sources.
3.16. Transcriptomic analysis of GmNCED in dehydration, salt, and drought stress
The RNA-seq data showed the expression pattern of GmNCED genes under dehydration, salt, and drought stress. The expression analysis of the GmNCED genes under dehydration treatment (1hr, 3hr, and 6hr) showed that the most of the GmNCED genes were down-regulated compared to the control (Fig 16A; S13 Data). The expression of GmNCED15 was observed higher than any other gene though it had showed down-regulation. One gene in particular, GmNCED11, showed up-regulation of its expression in 1hr, 3hr, and 6hr water stress. Two genes (GmNCED6 and GmNCED10) showed little or no expression both in the control and treatment states. Under salt stress, the expression pattern in some instances showed similarity with water stress, however, the expression under salt stress showed some differences at 1hr, 3hr, and 6hr of sodium treatment. The expression of GmNCED2 at 6hr and 12hr sodium treatment showed the up-regulation of expression. GmNCED5 was observed to be up-regulated at 12hr though at 3hr and 6hr, the expression was down-regulated. GmNCED9 was observed to express higher than the control whereas GmNCED11 showed up-regulation of its expression in 1hr, 3hr, and 6hr sodium treatment revealing its ability to be expressed in salt conditions. Under drought stress, the expression of GmNCED genes in the drought treatment group divided into 4hr time intervals from lights came on (8:00 a.m. = Zeitgeber Time (ZT) 0), during a 24hr time course (ZT0, ZT4, ZT8, ZT12, ZT16, and ZT20) (Fig 16B; S14 Data). Under drought stress, the expression of most genes was observed insignificant as no real differences were monitored whether it was control or treatment group. However, some notable genes such as GmNCED2, GmNCED11, GmNCED14, and GmNCED15 showed up-regulation in the expression at different time intervals upon drought treatment.
A. The name of the respective GmNCED genes are shown on the left side of the heatmap. The bottom of the heatmap contains the control, dehydration, and sodium salt treatment at different hours. B. The name of the respective GmNCED genes are shown on the left side of the heatmap. The bottom of the heatmap contains the control and drought stress treatment at different hours. The FPKM value are transformed into the log2 format and are shown in the color gradient from low to high expression (green to red color) on the right side of both the heatmaps.
3.17. Transcriptomic analysis of GmNCED during seed developmental stages
The results from RNA-seq provided that in high temperature delta region, approximately 44% GmNCED genes expressed in high-yielding adapted 6hr imbibed seed where GmNCED2 and GmNCED15 expressed highly (Fig 17; S15). In contrast, 38% GmNCED genes expressed in heat-tolerant 6-hr imbibed Huang seed, with GmNCED2, GmNCED10 and GmNCED15 up-regulated in that region. When 6hr imbibed seed was produced in south region, 50% GmNCED genes expressed whereas heat-tolerant Huang showed expression of GmNCED genes approximately 38%. Meanwhile, GmNCED15 was the single up-regulated genes and rest of the GmNCED genes were down-regulated. When germinated seeds were cultivated in delta region, about 38% GmNCED genes expressed. However, the rate of expression was decreased to 31% when Huang was cultivated in same region with the up-regulation of GmNCED8, GmNCED13 and GmNCED14. In the southern region, expression rate of GmNCED gene in two types germinated seed were nearly equivalent. GmNCED14 and GmNCED15 exhibited up regulation in contrast GmNCED2, GmNCED6 GmNCED10, GmNCED13 and GmNCED16 exhibited down-regulation. The number of expressed GmNCED genes for mature seed of high-yielding and Huang cultivated in delta region were seven and five respectively. GmNCED13, GmNCED14 and GmNCED15 considered as up-regulated expressed genes. In the southern region, mature seed of high yielding and Huang expressed approximately 50% and 38% respectively where three up-regulated genes (GmNCED13, GmNCED14 and GmNCED15) and five down-regulated genes (GmNCED2, GmNCED6, GmNCED9, GmNCED10 and GmNCED16) were observed.
The name of the respective GmNCED genes are shown on the left side of the figure. The bottom of the heatmap contains different stages of high yielding and heat tolerant seed; 6hr imbibed seed, germinated seed, and mature seed produced in south and delta region. The color gradient (white to red color), on the right side of the heatmap, shows low to high expression.
4. Discussion
NCED genes are involved in the biosynthesis of ABA mediated stress response. Therefore, it is indispensable to be acquainted with the physiochemical properties of GmNCED proteins. The instability index indicated their overall stability, with values consistently below 40.0. Additionally, variations in the aliphatic index implied differences in their structural properties. In addition, all GmNCED proteins were hydrophilic. Rosaceae species revealed a relatively similar evolutionary relationship and demonstrated the expansion of the NCED gene family during the evolution of the Prunus genus [37]. An in-depth knowledge of a species evolutionary history is crucial step comprehending the evolutionary relationships of genes. In the phylogenetic tree, the proximity of genes within a cluster directly correlates with the similarity of their functions [62]. Most of the GmNCEDs were clustered with VvNCEDs, indicating GmNCEDs were closely related to the VvNCEDs. Additionally, the absence of AtNCED and OsNCED genes in multiple groups might indicate a divergence in the genetic composition of the species.
The seven groups varied in the intron-exon structure and number. The presence of many introns in GmNCED15 allows for the possibility of alternative splicing, indicating specific biological functions [63]. On the contrary, the absence of introns in group F indicated a simpler gene structure. It was found genes with longer introns have showed elevated expression [64]. GmNCED6, GmNCED10, GmNCED11, and GmNCED16 categorized as early response genes and activated more quickly because of fewer exons [65,66].
Moreover, all the GmNCED protein possessed RPE65 domain which is essential for the enzymatic oxidation of carotenoids [6]. Thus, GmNCED demonstrated a similar distribution pattern across different groups. GmNCED genes motif varied across different groups and were relatively similar in the same group. PbNCED genes motif arrangements were identical within the same subgroup but varied among different subgroups [67]. Moreover, the absence of motif 17 in GmNCED13 highlighted the distinct characteristics within group E. GmNCED5 and GmNCED9 in group A shared the same motif indicating that they might work together in related biological processes.
Ka/Ks ratio calculation highlighted that the duplicated PbNCED gene pairs evolved by negative selection [67]. Ka/Ks analysis of the homologous GmNCED gene pairs revealed that all members of the GmNCED family engaged in purifying selection. This suggested that the GmNCED gene family was evolved to be highly conserved. Collinearity analysis revealed 6 collinear gene pairs in P. apricot, 6 in P. salicina, and 2 in A. thaliana [37]. Similarly, same numbers of GmNCED collinear gene pairs were identified, suggesting close genetic relationship among GmNCEDs. In contrast, GmNCED16 did not have any collinear gene pairs, suggesting that it might have a unique function or evolutionary history compared to the other GmNCED gene. But, no syntenic gene pairs were observed which implied a comparatively distant evolutionary relationship.
The uneven distribution of 16 GmNCED genes across 9 chromosomes implies potential functional diversification. Tandem, whole genome, and segmental duplications are the major driving mechanisms for the expansion of gene families in many plant species [68]. Eight gene pairs have undergone segmental duplication in M. albus SRS genes [69]. The prevalence of segmental duplications in this study among GmNCEDs implies the significance in contributing to the expansion of the gene family. Furthermore, the GmNCED gene family has been conserved over time, likely due to its important functional role. In addition, the absence of tandem duplication events supported the hypothesis that segmental duplications were important in the expansion of this gene family. Subcellular localization of certain proteins is crucial to many plant biological processes and activities [70,71]. Most of NCED proteins are found in chloroplast in rice and other species [72–74]. For instance, in Paeonia lactiflora PlNCED1 and PlNCED2, found in the nucleus and cytoplasm respectively, functions in regulating transcription factors serving as an active enzyme for ABA biosynthesis [75]. Similarly, in this study, most of the GmNCED members were located in the cytoplasm and chloroplast suggesting their potential role in regulating ABA biosynthesis.
Cis-elements are crucial in the regulation of gene expression, particularly in response to drought stress and hormone signal transduction [76]. The presence of a wide variety of CAREs in the 5′ UTR region of GmNCEDs implies their potential involvement in various stress responses and different plant hormone signaling pathways. The 5′ UTR region of GmNCED6 and GmNCED7 possessed CAREs including ARE, TCA-element, and ABRE, additionally, GmNCED7 also possess CGTCA-motif as well as TGACG-motif relevant to MeJA-response. In addition, MpNCED2 also included the CGTCA motif and the TGACG motif, which were important for the MeJA response [77]. Moreover, MBS CARE was found in GmNCED as drought inducibility response element [78]. The GmNCEDs were mostly involved in the oxidation-reduction process, oxidoreductase activities, single organism metabolic process, dioxygenase activities as well as various catalytic activities in GO. Furthermore, GmNCED proteins play essential role in various catabolic, metabolic, and biosynthetic processes. Moreover, oxidoreductase activity was identified in GmNCED, involving with key role in photosynthesis, respiration, and detoxification as well as in plant defense systems [79].
TFs perform an important role in controlling a broad range of functions, including biotic and abiotic stress responses, promoting development and growth, metabolic regulation, and defense against microbial infections. ERF, MYB, bZIP, LBD, C2H2, GATA, and TALE are some of the major TFs in GmNCED [80–82]. ERF (Ethylene Response Factor) TF is responsible for both ethylene signaling and the response pathway in plants. It is characterized by a single AP2 domain [83]. Moreover, certain AP2/ERF TF families exhibit the engagement abscisic acid (ABA) and ethylene (ET) synthesis, which stimulate the expression of stress-responsive (SR) genes that are either dependent or independent on ABA and ET [84]. The expression of an ethylene response factor (SlERF5/ERF5) has a role in enhancing the resistance of tomato plants to salt and drought [85]. MYB TFs accounted for approximately 9% of total TFs in Arabidopsis [86]. Thus, MYB family is associated with defense and stress responses, circadian rhythm, cell identity and fate, seed and floral development, and primary and secondary metabolic control [86,87]. The basic leucine zipper (bZIP) TF family is involved in a wide range of plant biological processes, including embryonic development, seed maturation, organ differentiation, floral formation, both abiotic and biotic stressors, and vascular development [88,89]. The LBD play a vital role in various aspects of plant development, such as the growth, initiation, metabolic regulation, and secondary growth of leaves, stems, roots, and corollas. Furthermore, nitrogen metabolism and anthocyanin metabolism are both influenced by LBD genes [90–93]. ERF and GATA showcased strongest association with the GmNCED genes. While bZIP TF had the weakest association. In addition, predicted GmNCED genes and their associated TFs exhibited a diverse spectrum of expression patterns.
The 20-22 base pair long noncoding RNA regarded as miRNA is responsible for the regulation of gene expression by binding with target mRNA. The binding initiates the translational inhibition or breakdown of the former [94,95]. The regulation of gene expression mostly affects the growth and development of plants as well as cell division and differentiation in abiotic stress. The miRNAs are also involved in the hormonal signaling pathway [96–98]. The miRNA166 is responsible for plant growth and development including cell division, differentiation, and various organ development as well as regulating biotic and abiotic stress [99,100]. The miRNA166 in soybean revealed that it might regulate the hormone gibberellic acid metabolism (anabolism and catabolism) to control plant height [101]. On the other hand, miR482 was associated with drought and salt stress [102]. However, miR482 in the soybean was reported to take part in nodulation by nitrogen fixation bacteria as well as fungi to resist disease against pathogens [103]. In soybean, miR9752 was perhaps hyper-methylated in nodule formation condition [104].
The PPIs facilitate numerous biological mechanisms including cellular function coordination, signal transduction, and communication [105]. In this study, GmNCED protein interacted with CCD, D27, LCY1, Z-ISO, and BETA-OHASE2 protein family. CCD (Carotenoid Cleavage Dioxygenase), member of the CCO (carotenoid cleavage oxygenase) family, is closely related to NCED. They cleave carotenoids and form apocarotenoid molecules [106]. In response to stress signals, plants synthesize ABA, an apocarotenoid [107]. Iron-enriched D27 plant protein plays a crucial function in the synthesis of strigolactones hormone which is responsible for plant growth and development [108]. The carotenoid biosynthesis pathway relies on lycopene cyclases (LCY) enzymes, which are required to cyclize lycopene [109]. The RNA-seq has the central role in deciphering the complex analysis of gene expression in cells. It delivers an in-depth analysis of the genes that are actively being engaged and offers invaluable insight into the regulation and function of cells in different conditions such as various abiotic and biotic stress [110,111]. Most of the GmNCED showed down-regulation, however GmNCED11 in particular showed up-regulation indicating potential role in dehydration stress. Under sodium salt stress treatment, certain genes in different time conditions showed higher expression and down-regulation as well. For example, GmNCED2 showed up-regulation of expression in 6h and 12h whereas down-regulation at 3hr. NCED genes expression in cotton suggest the involvement of ABA biosynthesis [5]. Meanwhile, most of the GmNCED genes were down-regulated and few genes such as GmNCED2, GmNCED14, and GmNCED15 showed up-regulation in drought stress. Moreover, soybean genes might play significant role in regulating drought signals [112]. Global warming and climate change posse severe threat to the production of food [113]. Hence, heat tolerant crop species are turning out to be obligatory to maintain their production at large in the adverse condition [114,115]. Soybean, economical crop species, contributes as dietary component (complete protein source), animal feed and oil [116]. Although soybean is a heat tolerant vegetative propagated oil crop, it is sensitive to high temperature during reproductive process such as seed growth, development and maturation. Thus, the consequences are shrinkage in seed quality, ineffective germination, higher chances of pathogen infection and ultimately damage to the economic value [117,118]. The RNA-seq analysis demonstrated the expression of GmNCED genes in the heat tolerant and conventional high yielding during 6hr imbibed seed, germinated seed and mature seed. The overexpression of GmNCED15 during 6hr imbibed seed and mature seed showcased the heat stressed capability. Despite showing high expression in high yielding seed, GmNCED2 didn’t exhibit up-regulation when introduced into the Huang heat tolerant seed. However, overexpression of GmNCED13 and GmNCED14 during germinated seed stage provided the evidence that it might perform up regulation at heat stressed condition. ABA biosynthesis was involved in the seed development and germination stage during normal and heat stressed period [119]. The role of NCED gene in Arabidopsis revealed the seed specific expression in germination stage [13]. Hence, GmNCED14 revealed substantial expression in managing the drought stress and seed germination in high temperature [120]. Over all, this study indicated the involvement of GmNCED in the regulation of various abiotic stress such as dehydration, salt, and drought. Moreover, GmNCED genes expression in seed germination at heat stressed condition involved ABA biosynthesis.
5. Conclusion
This study characterized 16 GmNCED genes containing the RPE65 domain allocated throughout the 9 chromosomes. Most of the GmNCED genes were located in the chloroplast and cytoplasm. Besides, GmNCEDs were closely related to VvNCEDs according to the phylogenetic comparison. Diversification in gene structure and functional similarities within the GmNCED genes were discerned. GmNCED showcased segmental duplications and purifying selection process to establish their evolutionary significance. PPI exhibited the network within GmNCED. Most GmNCED genes were involved in biological functions such as metabolic, redox reaction, and biosynthetic in co-ordination with plant development and resistance to abiotic stress. CARE analysis revealed the involvement of GmNCED in drought stress. The miRNA regulated the expression of GmNCED against abiotic stress such as drought and slat as well as in seed germination stage. The RNA-seq data analysis confirmed the pivotal role of GmNCED2, GmNCED11, and GmNCED12 under dehydration and sodium salt stress. Furthermore, GmNCED14 and GmNCED15 up-regulated their expression more frequently than other GmNCED genes under drought stress. Moreover, the higher expression of GmNCED13 and GmNCED14 in heat stressed germinated seed at high temperature regions indicated the induction of the ABA biosynthesis pathway. Hence, GmNCED14 was up-regulated in both drought stress and seed germination stages elucidating the significance, it upholds in managing a challenged environment. Therefore, the findings from this study might provide a reliable and strong basis for functional characterization of GmNCED genes in wet lab conditions. Besides, this study contains useful information for the future breeding program aimed at improving the characteristics of this economically important soybean crop species.
Supporting information
S1 Data. Full-length protein sequences of GmNCED gene family.
https://doi.org/10.1371/journal.pone.0319952.s001
(TXT)
S2 Data. Full-length protein sequences of NCED gene families of G. max, R. chinensi, V. vinifer, A. thaliana, P. persica, and O. sativa plant species for constructing a phylogenetic tree.
https://doi.org/10.1371/journal.pone.0319952.s002
(TXT)
S3 Data. Full-length coding sequences of GmNCED gene families.
https://doi.org/10.1371/journal.pone.0319952.s003
(TXT)
S4 Data. Full-length genomic sequences of GmNCED gene families.
https://doi.org/10.1371/journal.pone.0319952.s004
(TXT)
S5 Data. The upstream promoter region (2.0 kb genomic sequences) of GmNCED gene families for the analysis of cis-acting regulatory elements.
https://doi.org/10.1371/journal.pone.0319952.s005
(TXT)
S6 Data. Distribution of GmNCED gene family members among groups based on phylogenetic analysis.
https://doi.org/10.1371/journal.pone.0319952.s006
(DOCX)
S7 Data. In silico predicted the number of introns and exons in GmNCED genes.
https://doi.org/10.1371/journal.pone.0319952.s007
(DOCX)
S8 Data. Time of gene duplication estimated for different pairs of GmNCED genes based on Ka and Ks values.
https://doi.org/10.1371/journal.pone.0319952.s008
(XLSX)
S9 Data. The predicted cis-acting regulatory elements of the upstream promoter region (2.0 kb genomic sequences) of the GmNCED gene.
https://doi.org/10.1371/journal.pone.0319952.s009
(XLSX)
S10 Data. The GO analysis of GmNCED gene families for the identification of gene functions.
https://doi.org/10.1371/journal.pone.0319952.s010
(XLSX)
S11 Data. The putative miRNA identification of GmNCED gene families.
https://doi.org/10.1371/journal.pone.0319952.s011
(XLSX)
S12 Data. Protein-protein interactions of GmNCED protein families.
https://doi.org/10.1371/journal.pone.0319952.s012
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S13 Data. Transcriptomic profiling of GmNCED genes in abiotic stresses, dehydration and salt.
https://doi.org/10.1371/journal.pone.0319952.s013
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S14 Data. The transcriptomic profiling of GmNCED genes in drought stress in FPKM values.
https://doi.org/10.1371/journal.pone.0319952.s014
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S15 Data. Transcriptomic profiling of GmNCED genes in seed developmental stage in heat stressed and control region.
https://doi.org/10.1371/journal.pone.0319952.s015
(XLSX)
S2 Fig. Bubble plot of subcellular localization of GmNCED genes.
https://doi.org/10.1371/journal.pone.0319952.s017
(TIF)
Acknowledgments
The authors are very grateful to the Laboratory of Functional Genomics and Proteomics, Department of Genetic Engineering and Biotechnology, Faculty of Biological Science and Technology, Jashore University of Science and Technology, Jashore 7408, Bangladesh for providing the opportunity to conduct this research. Special thanks to Mr. Tanzir Ahmed, Assistant Professor, Department of English, Faculty of Arts and Social Science, Jashore University of Science and Technology, Jashore 7408, Bangladesh for extensively editing the manuscript to avoid grammatical errors and reader clarity. The authors also acknowledge and appreciate the reviewers and the members of the editorial panel for their valuable comments and critical suggestions for improving the quality of this manuscript.
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