Ying Bao*
School of Life Sciences, Qufu Normal University, Qufu Shandong 273165 China
* Correspondence: Ying Bao, baoying@qfnu.edu.cn
Abstract Increasing levels of UV-B radiation caused by the greenhouse effect has become an emerging threat to crop health and yield. The glutathione (GSH) metabolic pathway is generally involved in plant stress responses through scavenging accumulated reactive oxygen species, and is therefore believed to play an essential role in enhancing plant tolerance to UV-B stress. However, the complex evolutionary details of this pathway in polyploid plants, especially under UV-B stress, remain largely unknown. Here, using the important allotetraploid crop, Gossypium hirsutum, as an example, we comprehensively investigated the composition and phylogenetic relationships of genes encoding 12 key structural enzymes in this pathway, and compared the expression changes of all the relevant genes under UV-B stress (16 kJ m-2d-1) based on six leaf transcriptomes. Consequently, we identified 205 structural genes by genome-wide searching and predicted 98 potential regulatory genes under multiple stress conditions by co-expression network analysis. Furthermore, we revealed that 19 structural genes including five homoeologous pairs and 96 regulatory genes possessing 25 homoeologous pairs were reticulately correlated without homoeologous selection preference under UV-B stress. This result suggests a complex rewiring and reassignment between structural genes and their regulatory networks in the duplicated metabolic pathways of polyploid cotton. This study extends our understanding of the molecular dynamics of the GSH metabolic pathway in response to UV-B stress in G. hirsutum and, more broadly, in polyploid plants.
Key words: GSH, co-expression, regulatory network, UV-B.
Introduction
Ultraviolet-B (UV-B, 280–315 nm), a component of the solar electromagnetic spectrum that reaches the Earth’s surface, has received widespread attention in recent years because anthropogenic carbon dioxide emissions are destroying the ozone layer, resulting in increasing UV-B radiation (Liaqat et al., 2023). UV-B irradiation can cause significant alterations in plants morphology, physiological characteristics, and ultrastructure, leading to a reduction in plants biomass and yield (Kakani et al., 2003; Liaqat et al., 2023). At high flux rates, UV-B usually acts as a damaging agent causing harmfulness to plants biomolecules by generating excess reactive oxygen species (ROS). ROS comprising the singlet oxygen (1O2), superoxide (O2•-), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH) can trigger oxidation of lipid and protein, damage DNA and RNA, and reduce cell viability (Shi & Liu, 2021). In response to increased ROS, plants have developed various effective defense mechanisms by different antioxidants (Mahmood et al., 2019). Some antioxidants such as ascorbic acid, carotenoid, flavonoids, and phenolic have been proved to be effective to defense oxidative stress induced by UV-B radiation (Dias et al., 2020; Xie et al., 2022). These antioxidants are synthesized in several metabolic pathways. The gene composition, expression patterns and regulatory networks of the pathways under UV-B stress in plants are largely unknown.
Glutathione (γ-glutamyl-cysteinyl-glycine, GSH), a tripetide of cysteine, glutamic acid, and glycine, is an important antioxidant, radical scavenger, and antidote, which is essential for protecting cell, preserving enzymes activities and proteins functions, preventing cytoplasmic and outer membranes damage (Hasanuzzaman et al., 2019). GSH and glutathione S-transferase (GST) mediated antioxidant processes were enhanced after UV-B treatment (Yang et al., 2007). The GSH metabolic pathway is incorporated with multiple precursors and intermediates. GSH is synthesized from the amino acid substrates glutamate, cysteine (Cys) and glycine (Gly) by the consecutive reactions of glutamate cysteine ligase (GCL ) and glutathione synthetase (GS ) (Dorion et al., 2021). In addition, a γ-glutamyl transpeptidase (GGT ) can maintain GSH homeostasis by breaking down extracellular GSH and providing Cys, and promote intracellular de novo synthesis of GSH. The γ-glutamyl cyclotransferase (GGCT ) catalyzes the formation of 5-oxoproline from γ-glutamyl dipeptides, and with the help of 5-oxoprolinase (OXP ) to generate glutamate (Dorion et al., 2021).GGT can also initiate the metabolism of glutathione S-conjugates to mercapturic acids by transferring the γ-glutamyl moiety to an acceptor amino acid and releasing cysteinylglycine (Cys-Gly). Cyc-Gly is then broken down by leucine aminopeptidases (LAP ) to generate Cys or Gly, and Cys and Gly recycled to the process of GSH synthesis (Kumar et al., 2015). A study in Arabidopsis thaliana revealed that oxidative stress conditions caused by UV-B irradiation and disruption of the γ-glutamyl cycle resulted in a similar stress-induced response (Masi et al., 2016). GSH is then oxidized during peroxide disposal by glutathione peroxidases (GPX ) or dehydroascorbatereductase  (DHAR ) to GSSG (oxidized form of GSH) and is regenerated by glutathione reductase (GR ) from GSSG at the expense of NADPH (Dorion et al., 2021). Higher GSH content under stress is considered to maintain GSH/GSSG ratio. In this process, the orchestrated action of antioxidant enzymes, GPX and GR , are able to control the cellular concentration of O2•- and H2O2, thereby preventing the formation of reactive radicals (Dorion et al., 2021). Glucose-6-phosphate dehydrogenase (G6PDH ) and 6-phosphogluconate dehydrogenase (PGDC ) yield NADPH, a crucial cofactor of the enzyme GR converting GSSG into GSH (Cui et al., 2023). Some authors thought that elevated G6PDH activity played a key role in the control of GSH levels by utilizing NADPH under UV-B stress (Dorion et al., 2021; Cui et al., 2023). NADPH can be synthesized by isocitrate dehydrogenase (IDH ) (Moreno-Sanchez et al., 2018). GSH can also react with xenobiotics and endogenous compounds in reactions catalyzed byGST . Obviously, the GSH metabolic pathway including diverse structural enzymes is a very complicated process. Especially, several key structural enzymes in the pathway are encoded by multiple gene families, which undoubtedly adds complexity to the pathway. Furthermore, there is little information about the regulatory network for this pathway. Therefore, although the GSH pathway has been found to be associated with UV-B defense, the detailed characteristics of the structural and regulatory system of this pathway require further comprehensive study.
Gossypium L. is an economically important genus for providing natural fiber and seed nutrients that are widely used in textiles, industry, medicine, food and feed. A total of four species are domesticated in the genus, including two AA genome diploids (G. arboreum L. and G. herbaceum L.) and two AADD genome allotetraploids (G. barbadense L. and G. hirsutum L.). However, due to the high yield and wide adaptability, G. hirsutum , i.e. upland cotton, has been the major cotton species grown globally and accounts for more than 90% of the production share in the international cotton fiber market (Wendel et al., 2012). Therefore,G. hirsutum received more attention than the other three domesticated cottons in agriculture. In addition, this species, together with G. barbadense , can also provide a model system for studying the complex evolution of polyploid crops. G. hirsutum originated in approximately 1–2 million years ago by hybridization of two diploid species with AA and DD genomes and concomitant polyploidization (Wendel et al., 2012; Peng et al., 2022). As an allotetraploid species, upland cotton combines two sets of parental homoeologous genes (A- and D- subgenomic homoeologs, hereafter abbreviated as At-homoeolog and Dt-homoeolog, where t represents tetraploid) into a common nucleus, and thus possesses duplicated homoeologous pairs of both structural and regulatory genes in any metabolic pathway in most cases. This situation provides additional opportunities to rearrange or assign these duplicated homoeologous genes in the original parallel pathways, leading to neofunctionalization, subfunctionalization, non-functionalization, and complete loss of either or both of the diploid parental homoeologous genes (Chaudhary et al., 2009; Wendel et al., 2012). Obviously, clarifying the coexistence characteristics of these duplicated genes in the same pathway in G. hirsutum will provide more insights to further reveal the evolutionary patterns of other polyploid crops and enhance the effectiveness of molecular breeding in cotton.
With the changes in stratospheric ozone and climate over the past years (Liaqat et al., 2023), crops, including G. hirsutum, inevitably face an increasingly pronounced threat of UV-B radiation. Given that the consistent role of the GSH metabolism in antioxidants, in this study we sought to unravel the molecular basis of the pathway during UV-B stress resistance in upland cotton, and mainly attempt to explore four questions: (1) How many genes or gene members are in different structural enzyme families of the GSH pathway? (2) Which structural genes or members are differential expressed in the pathway during UV-B stress? (3)