DISCUSSION
Nucleotide-blastn homologous sequence alignment revealed that the
sequences of OsGR3 , OsGPX1 , OsNR2 , OsGS1,and OsGOGAT1 of rice seedlings were markedly similar to those inArabidopsis thaliana , Zea mays , Hordeum vulgare ,
and Sorghum bicolor , with a consistency of more than 80%. Such
findings indicate that the sequences could be homologous sequences.
Based on chromosome mapping, the genes had a similar location
distribution on the chromosomes of rice, Arabidopsis thaliana ,Zea mays , Hordeum vulgare , and Sorghum bicolor ,
aligning with the results of other studies (Zhou et al., 2018). The
analysis of gene exon-intron structure showed that genes in rice,Arabidopsis thaliana , Zea mays , Hordeum vulgare ,
and Sorghum bicolor had similar gene structures, but different
fragment size, which may be caused by partial deletion or addition of
genes in the process of evolution (He et al., 2021; Li et al., 2021).
The proportions of intron phases 0, 1, and 2 to all introns were roughly
the same in rice, Arabidopsis thaliana , Zea mays ,Hordeum vulgare , and Sorghum bicolor , with a proportion of
56%-60% for intron phase 0. Therefore, the evolution of genes in rice,Arabidopsis thaliana , Zea mays , Hordeum vulgare ,
and Sorghum bicolor might be largely conserved and may carry out
similar functions. Other studies revealed a high proportion of intron
phase 0 and more conserved evolution of genes (Tyagi et al., 2017; Xie
et al., 2002). Overall, rice seedling genes (OsGR3 ,OsGPX1 , OsNR2 , OsGS1 , and OsGOGAT1 ) had
similar gene functions to Arabidopsis thaliana , Zea mays ,Hordeum vulgare , and Sorghum bicolor genes and may play
the same regulatory role in the synthesis of the AsA-GSH cycle and
nitrogen metabolism-related enzymes in various species.
In the AsA-GSH cycle, oxidative stress is alleviated by degrading
H2O2 and maintaining the cellular redox
balance through multi-step enzymatic reactions (Roychoudhury et al.,
2012). Nitrogen assimilation can directly affect crop biomass and grain
yield, and is closely related to crop growth and development. In this
study, NaCl stress significantly downregulated the expression ofOsGR3 , OsGPX1 , OsNR2 , OsGS1 , andOsGOGAT1 in seedlings, whereas AsA and GSH contents, enzyme
activities in the AsA-GSH cycle, and nitrogen metabolism were
significantly decreased. Such findings may be due to NaCl stress
downregulating the expression of enzyme genes in the AsA-GSH cycle and
nitrogen metabolism, resulting in changes in related enzyme activities,
which ultimately affect the ROS scavenging ability of the AsA-GSH cycle
and nitrogen metabolism efficiency. Similar results were obtained for
other crops under different stresses (Wang et al., 2012; Wang et al.,
2018; Zhang et al., 2020; Li et al., 2010). After treatment with
different concentrations of exogenous melatonin, the expression levels
of OsGR3 , OsGPX1 , OsNR2 , OsGS1 , andOsGOGAT1 in seedlings were upregulated, and the activities of
enzymes in the AsA-GSH cycle and nitrogen metabolism were significantly
enhanced, suggesting that exogenous melatonin treatment changed the
inhibitory effect of NaCl stress on seedlings to a certain extent. This
change significantly improved the ability of the AsA-GSH cycle to
scavenge ROS and the efficiency of nitrogen metabolism, which notably
improved the ROS scavenging ability of the AsA-GSH cycle and the
efficiency of nitrogen metabolism. In other studies of melatonin under
other stress conditions, melatonin resisted adverse stress by enhancing
the antioxidant capacity and nitrogen metabolism level of seedlings and
regulating the expression of antioxidant and nitrogen metabolism genes
(Xu et al., 2010; Zhang et al., 2017; Zhou et al., 2020). The results of
RT-qPCR and heat map analysis showed that exogenous melatonin regulated
the expression of OsGR3 , OsGPX1 , OsNR2 ,OsGS1 , and OsGOGAT1 in seedlings under NaCl stress,
affecting the activities of enzymes in the AsA-GSH cycle and nitrogen
metabolism, accelerating the AsA-GSH cycle and nitrogen generation
process, and reducing the damage caused by NaCl stress in seedlings.
Seedlings OsGR3 , OsGPX1 , OsNR2 , OsGS1 , andOsGOGAT1 are essential for the life process of AsA-GSH cycle and
nitrogen metabolism under NaCl stress, which could affect the normal
growth and development of rice seedlings (Gu, 2019). Gene GO annotations
and KEGG metabolic pathways revealed that the genes were involved in
redox and other processes, including nitrogen metabolism, glutamate
metabolism, and glutathione metabolism, and regulated the synthesis of
key metabolic enzymes and combined with coenzymes. Such findings
indicate that these genes are crucial to the antioxidant system, ROS
scavenging, and metabolism. Based on the RT-qPCR and heat map results,
exogenous melatonin affected the process of the AsA-GSH cycle and
nitrogen metabolism by regulating the expression of OsGR3 ,OsGPX1 , OsNR2 , OsGS1 , and OsGOGAT1 in
seedlings under NaCl stress, reducing the content of ROS, improving the
utilization rate of nitrogen, and promoting the growth and development
of seedlings. Similar reports on beans and other crops have been
published (Wei et al., 2015; Erdal, 2019; ElSayed et al., 2021).
In this study, the plant height, root length, fresh weight, dry weight,
and chlorophyll content of seedlings were significantly decreased,
indicating that NaCl stress notably inhibited the growth of seedlings.
NaCl stress may inhibit the activities of enzymes in the AsA-GSH cycle
and nitrogen metabolism in seedlings, resulting in the accumulation of a
large amount of ROS in seedlings that could not be eliminated. The
content of MDA was significantly increased and the efficiency of
nitrogen assimilation slowed down, which markedly inhibited the growth
of seedlings. Related studies have reported that high salt stress is not
conducive to the growth of seedlings. In fact, high salt stress destroys
the structure of cells and significantly weakens their metabolic
activity (Zhan et al., 2019). On one hand, treatment with different
concentrations of exogenous melatonin improved the activity of enzymes
in the AsA-GSH cycle and nitrogen metabolism in seedlings, restored
normal ROS levels in seedlings under NaCl stress, decreased the content
of MDA in seedlings, and accelerated the efficiency of nitrogen
assimilation, thereby increasing the biomass of seedlings. On the other
hand, exogenous melatonin can reduce the degradation of chlorophyll in
seedlings under NaCl stress, improve the photosynthetic efficiency of
seedlings, and accelerate the generation rate of nutrients. Of note,
similar results were found in maize and cucumber seedlings. Overall,
exogenous melatonin promoted seedling growth by enhancing the activity
of enzymes in the AsA-GSH cycle, nitrogen metabolism, and photosynthesis
in seedlings (Li et al., 2021; Ahmad et al., 2021).
5 | CONCLUSIONS
In the present study, rice OsGR3 , OsGPX1 , OsNR2 ,OsGS1 , and OsGOGAT1 were found to have high homology with
similar genes in Arabidopsis thaliana , Zea mays ,Hordeum vulgare , and Sorghum bicolor based on sequence
comparison. The localization of genes on chromosomes and similar gene
structures were also revealed herein. Through GO annotation and KEGG
metabolic pathway analysis, the functions of genes and their
participation in metabolic processes were identified. Exogenous
melatonin regulates the expression of OsGR3 , OsGPX1 ,OsNR2 , OsGS1 , and OsGOGAT1 in seedlings under NaCl
stress to influence the efficiency of the AsA-GSH cycle and nitrogen
metabolism, reduce the ROS content of seedlings, improve nitrogen
utilization efficiency, enhance leaf photosynthetic rate, and promote
seedling growth.