(Allocati et al., 2018). Clearly, this evolutionary divergence is well illustrated in our ML tree.
From the expression point of view, apart from the Ef1bγs andTQHODs , all other seven types (DHAR, Lambda, MGST, Phi, Tau, Theta, and Zeta ) of GST genes showed differential expression in response to UV-B stress (Table S3). Furthermore, even within the same type, the expression direction and levels of theseGST genes are largely different (Table S3). For instance, in theTau genes, we identified 35 up-regulated and six down-regulated genes, respectively (Table S3). This situation suggests a complex adaptation-specific expression of different GST genes under UV-B stress. In addition, the expression levels of the cotton GST genes under UV stress were differently altered, ranging from a 1,736-fold up-regulation (Gohir.A13G154000 ) to a 397-fold down-regulation (Gohir.D04G137100 ) (Table S3). As a ubiquitous gene family, the GST genes play a crucial role in ROS detoxification, and this differential expression pattern amongGST gene members under stress conditions is not uncommon in plants. In Medicago ruthenica , 66 GST genes were identified, of which 14 genes were remarkably affected under drought stress (Wang et al., 2021). In upland cotton, some authors (Li et al., 2019) found significant changes in the expression levels of 17GST genes between resistant and susceptible cultivars underVerticillium stress by more than 2.0-fold. Interestingly, in this study, six of the above 17 GST genes (Gohir.A02G027200 ,Gohir.A09G161200 , Gohir.A09G161400 ,Gohir.A09G161500 , Gohir.A11G225300 , andGohir.D13G156800 ) also showed significant changes in expression levels under UV-B stress. In particular, three genes (Gohir.A09G161200 , Gohir.A09G161400 andGohir.A09G161500 ) in a gene cluster on chromosome 09 of the upland cotton subgenome A, which have been shown to be involved inVerticillium resistance, were substantially up-regulated by 4–97 fold under UV-B stress (Table S3). These results indicate that someGST genes may have overlapping functions in response to different stresses. Similarly, the gene members in the other 11 structural enzyme families in the GSH metabolic pathway exhibited different expression levels and expression changes (up- or down-regulation) (Fig. 3), implying that there is a complex functional assignment and reprogramming among the gene members under UV-B stress.
Regulatory genes constitute a critical component of the signaling network by controlling the expression of structural genes involved in different defense pathways (Han et al., 2019). Several key regulatory genes such as WRKY , HSF , and ERF have been identified in the GSH metabolic pathway in A. thaliana andSolanum lycopersicum (Han et al., 2019; Jahan et al., 2019). Here, using the co-expression networks, we predicted that at least 98 regulatory genes might involve in the cotton GSH metabolic pathway, and 96 genes were differential expressed under UV-B stress, ranging from up-regulated 2,055-fold (Gohir.D07G155016 ,MYB14 ) to down-regulated 20-fold (Gohir.A09G003800 ,HSPC2 ) (Table S7). In addition, we found that among these regulatory genes, the WRKY gene family had the highest number of genes (24), followed by C2H2 (14), HSF (11), ERF (10), and nine other regulatory gene families (less than 10 genes) (Table S7). These regulatory genes are generally very active in regulating ROS homeostasis in response to other biotic and abiotic stresses (Akbar et al., 2020). For example, in sugarcane, the expression of several WRKY , NAC , and bHLH genes were significantly enhanced in resistance to mosaic virus (Akbar et al., 2020; Mijiti et al., 2022). In this study, we found a WRKY gene (Gohir.D10G039400 ) was up-regulated 159-fold under UV-B stress. In plants, WRKY is a major transcription factor (TF) family, and is involved in diverse biotic or abiotic stress responses as well as in developmental and physiological processes. In pepper, a WRKY gene (CaWRKY27b ) could not only promote the immune response to the infection of Ralstonia solanacearum , but also improve resistance to high-temperature and high-humidity stress (Yang et al., 2022). InA. thaliana , the homolog of the cotton WRKY gene, Gohir.D10G039400, displayed significant responses to multiple stresses such as osmotic and cold (Boro et al., 2022). Moreover, it has been found that the up-regulated expression of other WRKY genes in rice have enhanced resistance to UV-B stress (Wang et al., 2007). In this study, we also found a NAC gene (NAC081, Gohir.A05G382000 ) was up-regulated 1,187-fold under UV-B stress. NACis considered to be an important regulator of plant tolerance to abiotic stresses by enhancing the ability to scavenge ROS (Mijiti et al., 2022). Overexpressing a NAC4 gene of Tamarix hispida in Tamarix and Arabidopsis conferred salt and drought stress tolerance to these transgenic plants (Mijiti et al., 2022). These studies show that the regulatory network of the GSH pathway under UV-B stress may overlap with that under other stresses to some extent.
4.2 Homoeolog evolution in the GSH metabolic pathway ofG. hirsutum
Allopolyploidization is the integration of heterologous subgenomes into a common nucleus (Wendel et al., 2012). After polyploidization, subgenomic homoeologs usually do not evolve in parallel, and subgenome dominance or asymmetry is prevalent in allopolyploids (Chaudhary et al., 2009). Furthermore, from the point of view of the gene locus, unequal contribution by the At- and Dt-homoeologs to the cotton transcript pool of any single gene is common phenomena (Flagel & Wendel, 2010). In this study, we revealed that 64% (80 and 26.5% (26) of structural and regulatory gene loci (Table S3, S5) were duplicated by retaining both At- and Dt-homoeologs in the GSH metabolic pathway of G. hirsutum . However, the relative expression patterns of the doubled homoeologs at structural and regulatory gene loci are remarkably different.
In the case of structural genes of the GSH metabolic pathway, there is a general imbalance or biased evolution between At- and Dt-homoeologs at the expression level of each locus. A total of 72 structural homoeologous pairs were retrieved from cotton leaf transcriptomes under CK and UV-B stress (SRR22018192-SRR22018197) (Table S8). Of the 72 homoeologous pairs, only 12 structural pairs (Gohir.A09G192401 vs. Gohir.D09G186600, Gohir.A06G107600 vs. Gohir.D06G109900, Gohir.A06G069200 vs. Gohir.D06G067900, Gohir.A04G097000 vs. Gohir.D04G136800, Gohir.A04G097300 vs. Gohir.D04G137100, Gohir.A08G037800 vs. Gohir.D08G048100, Gohir.A08G181700 vs. Gohir.D08G200300, Gohir.A10G068200 vs. Gohir.D10G083000, Gohir.A11G157300 vs. Gohir.D11G164300, Gohir.A12G045000 vs. Gohir.D12G043800, Gohir.A13G152500 vs. Gohir.D13G156900, and Gohir.A07G027000 vs. Gohir.D07G031000 ) maintained the same relative expression ratios of At- to Dt homoeologs under UV-B stress (Table S8). This result suggests that in most cases, UV-B stimulation fail to have a concerted effect on At- and Dt- homoeologs at each structural locus. In other words, At- and Dt- homoeologs at most structural loci respond to UV-B stress independently of each other. Meanwhile, we also found that 10 homoeologous pairs from two structural gene families (sevenGSTs and three GPXs ) showed biased expression patterns (get At bias and get Dt bias) only under UV-B stress, suggesting an expression repartitioning between biparental homoeologs during UV-B defense.
In detail, we revealed that the expression change in the ratio of At- to Dt-homoeologs of each structural gene under UV-B stress was inconsistent compared with the CK condition, ranging from a 230-fold increase inGSTU8 (Gohir.A09G161200 vs. Gohir.D09G156900 ) to a 27-fold decrease in GGT1 (Gohir.A11G053200 vs. Gohir.D11G056700) (Table S8) . The GSTUs, belonging to a plant-specific subfamily of the GST gene family, are important structural genes in several metabolic pathways, and encode a common class of detoxifying and ROS-scavenging enzymes (Herrera-Vasquez et al., 2021). In A. thaliana , 17 GSTU genes were found to be up-regulated under UV-B stress, and nine of them were positively regulated by transcription factors TGA2/5/6 (Herrera-Vasquez et al., 2021). In this study, we identified 41 GSTU genes that were differentially expressed under UV-B stress (Table S3). These 41GSTUs included 10 homoeologous pairs (10 gene loci) (Table S3 ). Previous studies displayed that the GSTU genes were always up-regulated under stresses. Over-expression of a AtGSTU19 provided obvious tolerance to salt, drought and methyl viologen stresses in Arabidopsis (Xu et al., 2016). Here, under UV-B stress, we also found six down-regulated GSTUs (Table S3) . Notably, although both At- and Dt- homoeologs of the 10 cotton GSTU loci had consistent up- or down-regulated expression trends, the relative expression ratios of the two homoeologs were not consistent under UV-B stress, with a variety of relative expression patterns such as no bias, loss bias, At bias, and Get At bias (Table S8). Therefore, the At- or Dt- homoeologs of these GSTUs may play different roles to defense excess UV-B radiation independently. Further functional validation should be carried out in the future.
Previous studies have confirmed that in polyploid cottons, both homoeologs of some structural genes can also exhibit different expression in an environmental adaptation manner (Liu & Adams, 2007; Flagel & Wendel, 2010; Bao et al., 2019; Peng et al., 2022). For example, there is a cotton alcohol dehydrogenase gene, AdhA , whose At- and Dt-homoeolog show different expression levels under water-submersion, cold stress, and dark stress (Liu & Adams, 2007). Here, we also identified 19 loci where only a single At- or Dt- homoeolog was independently differentially expressed under UV-B stress (Table S8). Flagel et al. (2009) have suggested that expression divergence between homoeologs may be controlled by large-scale linkage in some genomic regions or small-scale fine regulation (Flagel et al., 2009). Sequence divergences in the promoters or other non-codon regions between two hetero-homoeologs of each structural gene locus in G. hirsutum provide more opportunities for regulatory changes (Chaudhary et al., 2009; Flagel et al., 2009; Bao et al., 2019). Thereafter, this inconsistent expression between duplicated homoeologs of certain structural loci may trigger further partitioning and redefinition of their functions, which in turn promotes the retention of those duplicated homoeologs during subsequent evolutionary processes (Liu & Adams, 2007).
In contrast, among the regulatory genes of the GSH metabolic pathway, the relative expression between At- and Dt-homoeologs at each locus tends to follow an unbiased pattern. Among 25 homoeologs pairs, 21 pairs (20 no bias, one A bias) maintained the same relative expression change of At- to Dt- homoeolog under both CK and UV-B stress conditions (Table S8). Given that both homoeologs of these regulatory genes are up-regulated in response to UV-B stress, this unbiased pattern of relative homoeolog expression suggests that UV-B stimulation acts equally on both homoeologs of each regulatory locus. Some authors used the gene balance hypothesis to explain that dose-sensitive genes (e.g., regulatory genes) are more likely to be retained as duplicates (Birchler & Veitia, 2007). Similar results have been found in polyploid wheat, where homoeologous copies of regulatory genes not only had highly similar co-expression patterns, but were also more likely to be retained than other genes (Evans et al., 2022).
In summary, we herein reveal the complex evolution of a duplicated GSH metabolic pathway in the allopolyploid G. hirsutum, which, rather than combining two diploid pathways in parallel in a common nucleus, influences the expression of structural homoeologs through a reticulated and cross-subgenomic regulatory system, and thereby increases the correlation and integrality of the duplicated pathway. Our present study not only explored the duplicated structural gene framework of the GSH metabolic pathway of G. hirsutum at whole genome-wide level, but also further predicted several potential reticulated regulatory systems in the pathway under UV-B stress. This study broadens our understanding of the molecular dynamics underlying the UV-B response inG. hirsutum and polyploid plants in general.