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.