Enrichment of intrinsically disordered residues in ohnologs facilitates abiotic stress resilience in Brassica rapa

Arabidopsis thaliana and Brassica rapa are in the same evolutionary lineage, although the latter experienced an additional whole genome triplication event. Therefore, it would be intriguing to investigate the traits that gene duplication imposes to mediate plant stress tolerance. Here, we noticed that B. rapa abiotic stress resistance (ASR) genes which code at least one stress responsive domain have a significantly higher number of paralogs than A. thaliana. Analysing the disordered content of the ASR genes in both species, we found that intrinsically disordered residues (IDR) are specifically enriched in whole genome duplication (WGD) derived paralogs. Subsequently, domain similarity analysis between WGD pairs of both species has revealed that majority of WGD pairs in B. rapa did not share domains with each other. Furthermore, domain enrichment analysis has shown that B. rapa paralogs contain 36 distinct stress responsive enriched domains, significantly higher than A. thaliana paralogs. Next, we performed MSA to investigate the domain conservation between orthologs and ohnologs pairs, we found that 80.13% of B. rapa ohnologs acquire new domains, depicting the fact that ohnologs play a significant role in stress-related behaviours. The average IDR content of the ohnologs enriching new domains after gene duplication in B. rapa (0.19), is also significantly higher than A. thaliana (0.04). Interestingly, we also found that all of these attributes i.e., exhibiting higher number of WGD paralogs and enhancement of IDR in ASR genes of B. rapa compared to A. thaliana is exclusive for ASR genes only. No such significant differences were observed in randomly selected non-ASR genes between the two species. Together these results provide strong support for the hypothesis that augmentation of IDR content followed by a whole genome duplication event imposes the stress resistance potentiality in B. rapa. This research will shed light on the mechanism of how B. rapa is able to successfully adapt to stress over the evolutionary timescale.


Introduction
Brassica rapa L., a member of the family Cruciferae (Brassicaceae) is one of the most significant economic crops throughout the world since it is a major source of edible mustard oilseed. According to Al-Shehbaz (2012), the Brassicaceae family is one of the largest plant families, with 321 genera, 3,660 species, and 49 tribes. Each species has a variety of morphotypes that exhibit extreme features. Flowering plants frequently experience paleoploidization events, and the genes produced by these gene duplications have a profound evolutionary impact on plants. Gene duplication can occur through a variety of methods, including (a) Whole Genome Duplication (WGD), which causes an increase in ploidy level. (b) Tandem duplication, which happens when two similar alleles cross over unequally to duplicate a gene, (c) Transposed duplication: In this type of gene duplication, transposons are used to move the duplicated gene to a different chromosomal location. (d) Dispersed duplication (DSD), which results from arbitrary and questionable mechanisms that remains unclear and produces two gene copies that are neither next to one another nor located within the homologous chromosomal segment (Qiao et al. 2019).

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Polyploidy or whole-genome duplication, among all these mechanisms, has greatly contributed to the morphological and physiological diversification over evolutionary history (Paterson et al. 2010;Soltis et al. 2009). While the vast majority of plant species underwent whole genome duplication, many species, including those in the Brassicaceae family, underwent triplication. The ancient whole genome duplications (WGDs) that occurred twice in the Brassicaceae genomes were followed by an additional genome triplication in the Brassica species between 14 and 24 million years ago (Koch et al. 2000;Lysak et al. 2005). Thus, compared to the model plant Arabidopsis thaliana, Brassica species had an additional whole genome triplication (WGT) event. Brassica species' whole genome sequencing revealed that WGT is crucial for the speciation and morphotype diversification of Brassica plants (Cheng et al. 2014). Prior research has shown that polyploidization increases gene expression and cell proliferation as well as a variety of cellular potentialities, including the development of cell signaling, homeostasis, and most importantly the capacity to withstand stress (Li et al. 2012;Yu et al. 2010). Numerous studies have shown that after polyploidization, several crop species become more tolerant to water shortage, temperature, and salt stress (Allario et al. 2013;Deng et al. 2012;Kasajima et al. 2010). Compared to their diploid relatives, polyploid plants like Brassica rapa and black locust (Robinia pseudoacacia) demonstrated better adaptation to salt stress (Allario et al. 2011;Meng et al. 2011;Saleh et al. 2008;Wang et al. 2013). Furthermore, tetraploid Dioscorea zingiberensis was more heat resistant than diploid varieties (Zhang et al. 2010a). One of the main topics of current research is to understand the underlying process that influences the ability to tolerate stress adaptation by multiplying genes. It is unavoidable that adapting to stressful situations calls for altered gene expression, which is gained through alterations in the genome's sequence. The duplicated gene undergo a variety of modifications or alterations, including (A) changes in the protein-coding region, such as alternative splicing (Zhang et al. 2010b), exon-intron gain or loss (Xu et al. 2012), and single nucleotide changes (Ali et al. 2012); and (B) changes in the regulatory regions, such as changes in cis elements (Arsovskiet al. 2015), small RNA binding sites , and methylation patterns . The other significant dynamic feature, which imposes a new dimension in the consequence of gene duplication pertaining to protein structure change, was described by Montanari et al. (2011). They have postulated that gain or losses of inherently disordered areas in the proteins of duplicated genes are responsible for functional divergence in duplicated genes (Montanari et al. 2011). Moreover, intrinsically disordered regions are extremely flexible due to their lack of a fixed 3D structure, which frequently allows for additional interactions with proteins and allows for the possibility of different biological activities (Brown et al. 2010;Nilsson et al. 2011). It has been observed that intrinsically disordered regions increased in paralogs after WGD or WGT events contribute to the stability of activities like the catalytic activity of proteins, metabolic and transport processes, and molecular binding in a variety of plant species, including Zea mays, Glycine max, and Populustrichocarpa (Yurela et al. 2018). We are therefore interested in investigating how WGD and intrinsically disordered residues (IDRs) interact to give B.rassica rapa better abiotic stress tolerance than A. thaliana. Here, we have observed that IDRs in B. rapa paralogs produced via WGD play crucial roles in directing stress tolerance traits in them.

Selection of abiotic stress responsive genes
The genes were chosen based on Pfam domains having stress response potentiality, the PfamA clan was downloaded from the database (https:// pfam. xfam. org), and the stress-specific Pfam IDs were manually curated based on prior research. A total of 229 Pfam domains (Table S1) were selected, with each having a stress function. We next mapped the A. thaliana genes that contained these 229 Pfam IDs from Ensembl Plants (https:// plants. ensem bl. org/ bioma rt/ martv iew, Release 52). A total collection of 2,047 abiotic stress responsive genes (ASR) of A. thaliana were retrieved after the elimination of redundant gene identifiers. We also collected certain random PFAM domains which do not perform any stress related functions to make a list of non-ASR genes. We obtained 1,469 A. thaliana genes after removing redundant genes.

Construction of Ortholog and paralog pairs
Phylogeny-based orthologs and paralogs predictions were made using MetaPhOrs (http:// ortho logy. phylo medb. org). MetaPhOrs uses the combined score (CS) for two sequences. CS is the proportion of trees that confirmed an orthologous link over all the trees that were used to infer a relationship between a particular protein pair: this value therefore spans from 0 (all trees anticipate a paralogy link between the sequences) to 1 (All trees predict an orthology relationship). The protein pair is mapped as an orthologous pair if CS is equal to or greater than a threshold (by default 0.5), if not they are viewed as paralogs (Pryszcz et al. 2011). Only 1,160 of the 2,047 A. thaliana genes could be mapped to the 1,798 B. rapa genes. A total of 1,160 genes in A. thaliana and 1,798 genes in B. rapa were considered as abiotic stress related genes for further downstream analysis (Table S2). From the MetaPhOrs data, the abiotic stress genes were divided into 1 3 singleton genes-those with no paralogs or just one and duplicated genes those with several paralogs. Finally, we received 1,093 duplicated genes in A. thaliana and 1,751 in B. rapa from the ASR genes, others are singleton genes (Table S2). For other non-ASR genes, we received a total of 1,036 A. thaliana duplicated genes, which were mapped with 1,582 B. rapa genes following similar protocol (Table S2). Ensembl plants (https:// plants. ensem bl. org/ index. html) was used to extract the coding and amino acid sequences of the stress genes and their paralogs.

Identification of duplication mode
Duplicated genes were categorised into distinct types of duplication pattern, such as WGD, Tandem, Transposed, Proximal, and Dispersed duplication, which have been detected using universal standards, using the Plant Duplicate Gene Database (PlantDGD; http:// pdgd. njau. edu. cn: 8080). The distribution of synonymous distances between paralog pairs, or synteny analyses, is the basis for this method of detecting genome duplications (Qiao et al. 2019). Based on the proportion of genes that duplicate in a specific way among all the genes that are duplicated, the percentage of genes with a particular mode of duplication has been computed. The list of duplicated genes with different modes is provided in Table S3.

Identification of intrinsically disordered regions
IUPred2 long version (IUPred2A.elte.hu) algorithm (Dosztanyi et al. 2005a) was used to predict structural disorder, which takes into account the pair-wise energy estimated from residue composition. We chose IUPred algorithm for disorder prediction as IUPred didn't train on any specific dataset and hence it offers an unbiased estimate of disorder score (Dosztanyi et al. 2005b). This predictor takes protein sequence as input and output a disorder probability in the 0.0-1.0 range for each residue. Using 0.5 as the threshold, a residue was classified as "D". The following parameters were extracted for each protein sequence using each of the above-mentioned meta predictors: (1) "Disorder" residue proportion in a protein, using 0.5 as a threshold; and (2) the number of disordered windows (regions with at least 30 consecutive disordered residues with the same threshold).

Sharing of domain architecture
For each pair of paralogs, a Domain sharing score (DSscore) was determined following the formula of Song et al. (2007).
where n1 denotes the proportion of domains in protein p1, n2 denotes the quantity of domains in protein p2, and a12 is the quantity of domains in proteins p1 and p2 that are domain from the same Pfam clan. Values ranged from 0 to 1, with 1representing pairs contains identical domain architecture while0 representing pairs that do not share the domain with each other.

Domain enrichment analysis
Using Fisher's exact test, the number of domains found in duplicated genes was compared to the number of domains found across the total genome to evaluate domain enrichment. The same gene's multiple instances of a certain domain were regarded as a single instance. In Fisher's exact, test adjusted p values of less than 0.05 were used to determine whether a domain was significantly enriched.

Statistical tests
Online Z-test was carried out for proportionality test between two groups. All other statistical analyses and Heatmap were performed using R.

Gene duplication profiles in abiotic stress responsive genes in A. thaliana and B. rapa
Gene duplication or Paleoploidization is the driving intensity that contributes to-the evolution of ultra-morphological and ecological diversity. It is a phenomenon which allows the genes to acquire novel features by neofunctionalization and sub functionalization (Paterson et al. 2010;Soltis et al. 2009). It has been reported earlier that the abiotic and biotic stress responsive genes were increased after whole genome duplication (WGD) (Rizzon et al. 2006). Now, a gene plays a specific function with the domains of its encoded protein as domains are the unique structural and/or functional units in a protein. Similar domains can be identified in proteins with a variety of functions, suggesting that domains can exist in a wide range of biological situations (Sharma and Pandey 2016). Also, the proteins in the gene family are playing various biological tasks by these additional domains. They are thought to mediate brief interactions between proteins and give them a critical function in numerous cellular processes that confer plants the ability to adapt and tolerate a variety of environmental situations (Mudgil et al. 2004;Prasad et al. 2010;Rivals et al. 2006;Sharma et al. 2014). Therefore, to further explore the role of gene duplication in stress responsive potentiality of B. rapa than A. thaliana, we assessed abiotic stress resistance (ASR) genes from A. thaliana using domain attributes of genes. We have considered only those genes which contain at least one stress tolerant domain enlisted in Table S1. Following these criteria, we have received (n = 2047) ASR genes in A. thaliana. Next, mapping the ASR orthologs in B. rapa, we have obtained (n = 1798) ASR genes. Successively, comparison on the number of paralogs of ASR genes in A. thaliana and their corresponding orthologs present in B. rapa has revealed that the proportion of ASR genes in B. rapa (n = 1,751 i.e., 97.38 %) undergo significantly (P = 0.00001, Z proportionality test) higher duplication events than the ASR genes in A. thaliana (n = 1,059 i.e., 94.2%). These observations also support the fact that Brassica species underwent five rounds of WGD whereas A. thaliana experienced two rounds of WGD (Lysak et al. 2005;Navabi et al. 2013). Thus, this result suggests that duplication may play possible roles in maintaining the stress resilience attribute in B. rapa. Formerly, it has been testified that mode of duplication among genes influence the functional role in a biased way (Qiao et al. 2018). Therefore, in our study, modes of duplication of the stress genes were derived and were classified into five different categories i.e., whole genome duplication (WGD), tandem duplication (TD), proximal duplication (PD), Transposed duplication (TRD) and Dispersed Duplication (DSD). The number of duplicate gene pairs for each category in each taxon was determined (Table S3). Figure 1, clearly shows depicts the percentages of ASR genes experiencing WGD is higher than other different modes of duplication in both species of ASR genes. It was also found that the percentage of ASR genes experiencing WGD is significantly (P = 0.00001, Z proportionality test) higher in B. rapa (n = 1,337 i.e., 76.35%) than that of A. thaliana (n = 421 i.e., 38.5%). To ascertain the role of WGD in abiotic stress resilience of B. rapa, we have checked WGD status in randomly chosen 1,036 and 1,584 non ASR genes in A. thaliana and B. rapa respectively. Interestingly, we observed that a similar proportion of genes had experienced WGD in A. thaliana (52.61%) and B. rapa (53.01%) [P = 0.42, Z proportionality test]. Since, WGD or polyploidization is the dominant feature of evolution in plants and it also creates an enormous number of duplicated genes by restoring, gaining or losing functions (Rizzon et al. 2006). Furthermore, previous assumptions suggest that WGD polyploidy allows the features like larger organs, stress tolerance, and changed flowering time to potentiate plant fitness or enable them to adapt to new ecological niches (MacKintosh and Ferrier 2017). Hence, we focus on ASR genes which underwent WGD events only and not the other modes like tandem duplication for our further analysis.

Enrichment of intrinsically disordered regions in proteins encoded by duplicated pairs of ASR genes in A. thaliana and B. rapa
Ohnologs, the duplicates derived by whole genome duplication, were reported to encompass more intrinsically disordered residues than small scale duplicates (Banerjee et al. 2017). It was also reported in plant systems that the proteins of dehydrin family being almost entirely disordered could comprise in the response to drought and other environmental stresses (Mouillon et al. 2006). Thus, we have investigated intrinsically disordered regions in ASR gene coded proteins between the two species. We have considered the protein as intrinsically disordered if it contains at least one IDR comprising of 30 amino acid residues at a stretch (Uversky et al. 2019). When we have compared the presence of intrinsically disordered residues (IDRs) in ASR proteins undergo different modes of duplication pattern, we have noticed that IDRs are significantly more enriched in WGD derived duplicated proteins than tandem, dispersed, proximal and transposed duplicates in both species (Fig. 2). Thus, it could be established from the above facts that WGD is associated with the augmentation of IDRs in proteins. If so, it could also be assumed that ohnologs of B. rapa encode more intrinsically disordered proteins than A. thaliana since the former underwent three extra rounds of WGD. Consistent with our speculations, we too found that (n = 605 out of 1,337) 45.2 % of ohnolog pairs in B. rapa contain at least one intrinsically disordered region but in A. thaliana it is only 40.3 % pairs (n = 170 out of 421). The difference is significant at P < 0.0001 in Z-proportionality test. Moreover, we tested the IDR content in non-ASR sets. In this case, we found that (n = 161 out of 536) 30.03 % of ohnolog pairs in B. rapa contain at least one intrinsically disordered region but in A. thaliana it is only 27.7 % pairs (n =154 out of 554) but the difference is statistically non-significant [P = 0.20 in Z-proportionality test]. Thus, it could be suggested that IDR content in proteins could be an important attribute for enhanced stress tolerance in B. rapa. It is also noteworthy

Sharing of domain architecture in WGD pairs of ASR genes in A. thaliana and B. rapa
Gene duplication plays an essential role in propelling evolutionary diversity by endowing functional and redundant DNA sequences as the raw material for evolution (Crow et al. 2005). Earlier, it was propounded that functional diversification of the persisting duplicated genes is one of the essential aspects that contribute to for the long-term evolution of polyploids (Blanc and Wolfe 2004). Since, domain is the crucial segment of a protein for contributing its function, we have measured domain sharing score (DS score) between WGD derived paralogous pairs of ASR genes by following the protocol of Song et al. (2007). The average domain similarity score is significantly higher (P = 0.003) in A. thaliana WGD pairs compared to B. rapa pairs (Fig. 3). We have found that in B. rapa, a major proportion of WGD pairs (n = 1,702; 82.54%) did not share a domain with each other whereas it is only (n = 331) 55.1% for A. thaliana. The difference in the proportion of genes in these two species is also significant at 0.99 confidence level. Next, we examined the enrichment of stress-specific domains obtained after WGD in both species by Fisher Exact T-test to get insight into the sorts of additional domains enriching in the paralogs after a duplication event. Figure 4 delineated the types of stress specific domains in gene pairs of two species. Interestingly,  we observed that a higher number of stress related domains are enriched in B. rapa (Fig. 4b) compared to A. thaliana (Fig. 4a) paralog pairs. Subsequently, we analysed the number of unique and common enriched domains those are gained in paralogs after duplication. The Venn-diagram (Fig. 5) showed the number of unique domains gained in paralogs is about eight times higher in B. rapa (n =36) compared to A. thaliana (n = 5). Thus, the prevalence of unique enriched domains in B. rapa paralogs makes it obvious that gene duplication plays a significant role in enhancing the stress-resistance potentiality of the B. rapa.

Conservation of stress related domains between A. thaliana and B. rapa
Protein domains are the part of a single amino acid chain and are present as small structural independent units. These small units often count on the coadjuvant effects to maintain their functions as a part in larger protein (Wang et al. 2021). They may be conserved within the species, as well as across different species. A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form functionally distinct molecules in a process known as domain shuffling (Aziz et al. 2004). Therefore, we looked at the enriched domain conservation using a MSA (Multiple sequence alignment) study in A. thaliana and B. rapa orthologs as well as ohnologs. The ortholog and ohnolog pairs which show exact sharing of domains are tagged as conserved pair. While comparing the pairs between both species, any domains if gained   Fig. 6a). Intriguingly, we noticed that the proportion of B. rapa orthologs which gained at least a new domain is significantly higher (n =593, 80.13%), than the proportion of A. thaliana (n = 381, 67.07%, Fig. 6b). The difference is significant with P < 0.0001 in Z-proportionality test. Moreover, it is also evident from Fig. 6b that the presence of conserved domains is much higher in ortholog pairs as opposed to ohnolog pairs. From this result, it could be concluded that the ohnologs in B. rapa predominately include many domains which are not present in A. thaliana. Moreover, B. rapa is anticipated to be more responsive to the gain and loss of stress-enriched domains due to its low conservation of ohnolog pairs. This indicates a selective pressure on orthologs than ohnologs to retain the domain architecture required for the proteins to perform a specific function. As a result, it could be affirmed that genome duplication plays a significant role in the acquisition of additional domains and subsequently increased stress functions.

Affirming the roles of IDRs in stress resistance potentiality of B. rapa
In the earlier section, we have evidenced that whole genome gene duplication causes a protein's inherently disordered residues to be enriched. After that, in order to confirm the potential contribution of IDRs to stress enrichment, we calculated the IDRs for all the ortholog and ohnolog gene pairs that had undergone domain gain, loss, and conservation. We observed that in contrast to A. thaliana ohnologs, B. rapa ohnologs experiencing domain gain have significantly higher average IDR (Fig. 7). Since, we noticed that a higher proportion of paralogs in B. rapa compared to A. thaliana experienced domain gain (Fig. 6), thus the enrichment of IDRs in B. rapa paralogs might signify the role of IDRs in attainment of a new domain. Notably, no IDRs are found in orthologous pairs which keep their domain conserved throughout evolution. From Fig. 7, it is also evident that compared to ortholog pairs, ohnolog pairs are enriched with more IDRs, thus we did a pair-wise comparison in terms of domain gain/ loss and IDR gain/loss in ohnologs pair which are diverged from A. thaliana to B. rapa. For this, we subtracted the total number of domain and IDR content from A. thaliana to B. rapa ohnologs to calculate gain and loss of domain and IDR in B. rapa. Quadrant plot (Fig. 8) clearly depicts that the population of genes in IDR and Domain gain quadrant is much higher than any of the other three quadrants. Thus, it could be anticipated that the IDRs provide the sites for enduring new domains for mediating functional divergence in the ohnologs and in turn help in the adaptation to stress activities. We strongly view this as evidence that IDR plays a major role to impart more stress resistance potentiality in B. rapa than A. thaliana to impose stress tolerance potentiality.

Discussion
According to comparative genomic study, A. thaliana shared a similar evolutionary branch and experienced whole genome duplication about 20 million years ago (MYA), whereas the Brassica lineage underwent whole genome  (Lysak et al. 2005;Mun et al. 2009;Town et al. 2006;Yang et al. 2006;Zhao et al. 2013). The primary source of genetic innovation that emerged throughout genomic evolution is gene duplication. As a result of this genetic innovation, the DNA's structural makeup is altered, which might alter both the structure and function of the resulting protein. According to a recent study, the presence of this WGT leads to sequential genomic rearrangements, divergence, and speciation in Brassica species, making B. rapa more resistant to abiotic stress than A. thaliana (Kagale et al. 2014). It would thus be highly intriguing to explore the attributes imposed by gene duplication to mediate stress tolerance in plants. When we compared ASR genes from A. thaliana to their orthologs in B. rapa, we discovered that the majority of ASR genes in B. rapa (97.38 %) participate in gene duplication events. As a consequence, we noticed that A. thaliana has a much-reduced number of paralogs (average paralogs = 1.17) than B. rapa (average paralogs no = 2.63) which is consistent with the impacts of an additional wave of genome duplication in that species. This finding is supported by a recent work on A. thaliana, which proposed that many of the discovered duplicated gene pairs developing through the WGD process may be involved in growth, stress tolerance, and cell wall integrity pathways (De Smet et al. 2017). Whole-genome duplications, as opposed to small-scale gene duplications, duplicate whole pathways or networks and are crucial in the development of new features and enhanced biological complexity (Soltis and Soltis. 2016;Van de Peer et al. 2009;Vanneste et al. 2014). The process of gene duplication followed by gene loss or the evolution of new functionalities has received a great deal of attention. Gene duplication followed by structural changes has frequently eased their incorporation into a stress function (Lallemand et al. 2020). Genes may experience conformational changes upon duplication, either in the coding sections or regulatory areas like the promoter and small RNA binding sites. Large-scale structural changes that influence the organisation of domains or other similar sequence properties and have an impact on the final protein functionality can occur in coding areas Xu et al. 2012). Furthermore, proteins are commonly distinguished by the existence of several domains that interact with one another via intramolecular interactions. Additionally, proteins function in a dense intracellular space, transiently interacting with aplethora of other macromolecules . Previous evidence suggests that genomic recombination and chromosomal rearrangements during gene duplication might produce flexible or ductile areas in proteins that are referred to as intrinsically disordered proteins (Yruela et al. 2018;Yruela and Contreras-Moreira 2013). The discovery that up to 40% of eukaryotic proteins are intrinsically disordered, or have intrinsically disordered areas, and are highly dynamic entities without a well-defined three-dimensional structure has altered the structure-function paradigm and our knowledge of proteins (Malagrinò et al. 2022). There is profuse evidence that disordered residues confer flexibility to proteomes (Schad et al. 2011;Tompa 2002;Yruela and Contreras-Moreira 2012;Yruela et al. 2017), most importantly they contribute to organismic plasticity by facilitating complex gene regulatory network dynamics and protein multifunctionalities (Covarrubias et al. 2017;Wright 2002, 2005;Habchiet al. 2014;O'Shea et al. 2015;van der Lee et al. 2014;Wright and Dyson 2015;Xie et al. 2007;Yruela 2015). To further explore the role of WGD events in stress tolerance of plant we have analysed disordered content of ASR paralogs originated through different modes of duplication i.e. WGD, tandem, transposed, proximal and dispersed. We noticed that IDR content is explicitly enriched in WGD derived paralogs i.e., Ohnologs of A. thaliana and B. rapa than any other paralogs (Fig. 2). We demonstrated that WGT events in B. rapa showed enhanced IDRs in their proteomes, which may contribute to more successive adaptation to stress by comparing average IDRs in ASR genes of B. rapa and A. thaliana. Our findings back up previous research on a wellknown NAC family protein in A. thaliana, which found that differences in the IDR region contributed to functional divergences in NAC92 and NAC59, which are involved in senescence, salt stress responses, and lateral root development (Balazadeh et al. 2010;Yruela et al. 2017). The structurefunction continuum notion, which says that distinct biological functions of proteins require distinct 3D-structures, has captivated scientists' attention for more than a century. Thus, it is noteworthy to mention that protein-protein interactions are always mediated by a protein domain which dictate how proteins carry out their intended tasks. To support this idea, we examined the domain sharing status of the two species. We found that a significant portion of WGD pairs in B. rapa did not share a domain between them, potentially leading to an increase in functional divergence (Fig. 3). Similar predictions were also made in Chinese White Pear (Pyrus bretschneideri) as the MYB gene family functional divergence was caused by a lack of sharing of domains in the MYB binding  . Moreover, domain enrichment analysis has portrayed that paralogs in B. rapa contain 36 unique enriched domains which is much greater than the paralogs in A. thaliana (Fig. 5).For instance, the domain-PF00249 which functions in Myb-like DNA-binding domain (Feller et al. 2011), and PF03106 WRKY domain functions as heat shock protein (Kohan-Baghkheirati and Geisler-Lee 2015). PF01061-Drought induced 19 protein (Di19) is a zinc-binding domain which functions in enduring drought stress (Wu et al. 2022) and PF00313a 'Cold-shock' DNA-binding domain functions with adapting to cold stress (Zhang et al. 2020), The presence of such Pfam domains in paralogs aids in the conferring of stress traits in B. rapa as opposed to A. thaliana. To explore the event of gene gain, loss and conservation between orthologs and ohnologs pairs, we performed MSA. Our analysis (Fig. 6) evidenced that 80.13 % of B. rapa ohnologs acquire new domains which lends support to our hypothesis that ohnologs play an important role in stressrelated behaviours. The enduring view, however, has been challenged by the expanding awareness of the important functional role of IDRs, demonstrating unequivocally that structure-free IDPs/IDPRs are functional and capable of engaging in biological activities and pulling off unimaginable feats that are incredibly implausible for organised proteins (Deiana et al. 2019). It was previously revealed that IDRs can be found in the binding domain of single-domain interface hub proteins to pursue a variety of functionalities (Kim et al. 2008;Podder and Ghosh 2010;Singh et al. 2007). As a result, we additionally investigated the IDR content of the genes encoding the enriched domain. We found that the average IDR for B. rapa ohnolog pairs is substantiallyhigher (0.19) than for A. thaliana ohnolog pairs (0.04, Fig. 7) which have undergone domain gain after gene duplication. Interestingly, no IDR enrichment was found in the orthologous pairs containing the conserved domain. Moreover, we also noticed that most of the ohnologs in B. rapa which attain extra domain from A. thaliana ohnologs after duplication are also augmented with more IDRs (Fig. 8). This indicates that genes with disordered residues are more likely to develop a certain function. It is also noteworthy to mention that no such augmentation of WGD derived paralogs and IDR content were observed in the case of randomly selected non-ASR genes. Thus, these findings offer compelling evidence for the evolution of IDRs with WGD events and its consequences for stress tolerance. This investigation led us to the conclusion that IDRs in proteins may independently regulate the capacity for stress tolerance in B. rapa ohnologs. Numerous studies have established the importance of intrinsically disordered residues in driving a number of biological activities. However, the critical role that these residues play at the genome level in plants' ability to adapt to stress has not yet been investigated. In order to better understand the molecular mechanisms imposing stress tolerance potentiality in plants, our discoveries will open new avenues in the discipline of plant science.

Conclusions
After the phenomenon of whole genome triplication (WGT) Brassica species experienced an additional round of whole genome duplication compared with the model plant A. thaliana., the occurrence of this WGT confer B. rapa more abiotic stress resistant than A. thaliana by successive genomic rearrangements, divergence and speciation. Based on our study, it is reasonable to confirm that accompanying with WGD events, IDRs mediate stress adaption potentiality in B. rapa. We have illustrated the fact that the structural flexibility of IDRs expedites the stress tolerant attributes in ohnologs by accommodating stress specific domains in B. rapa. This study will open new avenues in understanding over evolutionary time, how duplication confers stress adaptation potentiality in B. rapa.