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)