Integrated bioinformatics analysis reveals the response of Arabidopsis roots to salt stress through multiple gene expression omnibus datasets

ABSTRACT Plant growth and crop productivity has been adversely affected by salt stress around the world. Arabidopsis root is an important model to demonstrate how the glycophyte adapts to salt stress. To better understand the salt-responsive genes in Arabidopsis root, a rank aggregation method has been applied to disclose 836 dysregulated genes including 449 upregulated and 387 down-regulated genes out of more than 20,000 genes in NaCl-treated Arabidopsis root from the Gene Expression Omnibus (GEO) database. Then, the integrated approaches of PANTHER, DAVID, KEGG, Cytoscape and STRING have been used for bioinformatic analysis. Our results showed that these salt-responsive genes were involved in various gene ontology enrichments (transcription, oxidative stress, cell wall organization), differential pathways (MAPK signaling pathway, hormone signal transduction), and complicated interactions (cross-talking with wounding stress, abscisic acid stress, heat stress, water deprivation stress, cold stress). Furthermore, the activities of the oxidative stress related gene, peroxidase, were determined under salt stress, cold stress and water deprivation stress, which validated the bioinformatic outcome of cross-tolerance to different stresses. Our work will throw new lights on the response of Arabidopsis roots to salt stress, and provide potential salt-targets to improve plant growth and increase crop output.


Introduction
Soil salinity is a risk factor threatening plant growth and crop output (Guo et al. 2012a). Plants have developed many approaches including sodium exclusion and sodium ion accumulation tolerance to adapt to salt stress (Guo et al. 2012b). In order to demonstrate the mechanisms of saltresponse, many researches have been performed with the model plant Arabidopsis (Guo et al. 2019). Two-dimensional electrophoresis has been used to discover the differential Arabidopsis root proteins between wild type and salt-tolerance mutant, which identified 19 differential proteins related to signal transduction, redox homeostasis, and cell wall metabolism (Guo et al. 2014b). Furthermore, phosphorylation variations of proteins from Arabidopsis root under salt stress also have been determined by Pro-Q Diamond stain. Nonsynchronous differences between protein abundance and protein phosphorylation indicated the complicated mechanisms of root salt-responsive proteins. These altered proteins were associated with reactive oxygen species scavenging and metabolisms of the cell wall (Guo et al. 2014a). Compared with proteomic strategy, transcriptomic researches could provide complementary information in plant salt-response.
For the better understanding on salt-response in Arabidopsis root, differentially expressed genes (DEGs) in response to salt stress were retrieved from the gene expression omnibus database (GEO). Among more than 20,000 Arabidopsis genes, 836 dysregulated genes were screened based on a rank aggregation method, and further analyzed by the bioinformatic tools of PANTHER, DAVID, KEGG, Cytoscape, and STRING. The prediction of bioinformatic analysis was confirmed by experiment. The altered genes will facilitate the salt stress tolerance researches and provide new targets for the better explanation of the salt adaption in the plant root.

Pathway analysis
The pathways of salt-responsive Arabidopsis root genes were analyzed by the online program of KEGG (Kyoto Encyclopedia of Genes and Genomes, Release 94.0, 1 April 2020) (http://www.kegg.jp).

PPI network
The PPI interaction networks were constructed by the software of STRING (search tool for recurring instances of neighboring genes) database (Version 11.0, released 19 January 2019) (http://string-db.org/), and Cytoscape (an open source platform for complex network analysis and visualization, Version 3.7.2) (https://cytoscape.org/).

Growth conditions and harvest of Arabidopsis root
According to the previous publication (Guo et al. 2014b), the ecotype Col-0 Arabidopsis thaliana seeds were germinated on the normal medium plate with 22/20°C day/night, 8/16 h light/dark cycle, 60 μmol·m-2s-1 light intensity. After 7 days, the Arabidopsis seedlings were separately transferred into three stresses: cold-stress condition (8°C), water-deprivation stress condition (200 mmol·L-1 mannitol), and salt stress condition (150 mmol·L-1 NaCl) for another 3 days growth. Then the Arabidopsis roots were harvested and stored at -80°C.

Enzyme activity assay
According to the previous method (Guo et al. 2019), peroxidase (POD) activity of Arabidopsis root was assayed using the kit (Nanjing Jiancheng Bioengineering Institute). Briefly, the POD activity was determined based on the catalyzing H2O2 of absorbance change at 420 nm. The plots were drawn with the software of Graphpad Prism 8 for windows (Version 8.0).

Bioinformatics enrichment of salt-responsive DEGs in Arabidopsis root
Bioinformatics tools were used to analyze the identified 449 up-regulated and 387 down-regulated genes. Regarding biological processes, the significantly enriched processes of up-regulated DEGs were related to regulation of transcription, protein dephosphorylation, response to oxidative stress (Figure 4(A)), whereas, the significantly enriched processes of down-regulated DEGs were associated with cell wall organization, root hair elongation, sterol metabolic process (Figure 4 (B)). In terms of molecular function, the significantly enriched functions of up-regulated DEGs were related to transcription factor activity, glutathione transferase activity, kinase activity (Figure 4(C)), whereas, the significantly enriched functions of down-regulated DEGs were associated with phosphatase inhibitor activity, pectinesterase inhibitor activity, heme binding (Figure 4(D)). KEGG analysis indicated the up-regulated DEGs were involved in plant hormone signal transduction, metabolic pathways, MAPK signaling pathway, linoleic acid metabolism, glutathione metabolism, galactose metabolism, carotenoid biosynthesis, biosynthesis of secondary metabolites, amino sugar and nucleotide sugar metabolism, and the down-regulated DEGs mainly participated in pentose and glucoronate interconversions, and diterpenoid biosynthesis ( Figure 5).

PPI network and cross-talk of salt-responsive DEGs in Arabidopsis root
Totally, 449 up-regulated salt-responsive DEGs were connected with 879 edges (PPI enrichment p-value: < 1.0e-16) by STRING (Figure 6). In addition, the complicated crosstalks of up-regulated salt-responsive genes with wounding stress, cold stress, abscisic acid stress, heat stress, and water deprivation stress in Arabidopsis root were shown with Cytoscape (Figure 7).

POD activity determination in Arabidopsis root under salt, cold, and water deprivation stress
Our results showed POD was not only an up-regulated saltresponsive gene, but also was involved in other stresses. Therefore, POD activity of Arabidopsis root were determined under three stresses (salt, cold, and water deprivation stress) to confirm the confidence of bioinformatics prediction. The results indicated that compared with the stress-free control, POD activity was all increased at different time points (12, 24, 36, 48, 60, 72 h) under salt, cold, and water deprivation stresses (Figure 8).

Discussion
Salt stress limits plant growth and decreases crop output on the earth. Many works have been done to improve the salttolerance of plants (Shah et al. 2018). Although several plants have been used as materials, Arabidopsis is the most ideal model to demonstrate the salt-response mechanisms . The root of Arabidopsis was an organ dealing with salt stress directly. Therefore, many researches about salt adaption in the Arabidopsis root have been performed. For example, phosphorylation dynamics in Arabidopsis roots proteins have been detected, and 77 novel phosphorylated sites were found, which might be involved in lipid signaling under abiotic stress responses (Vialaret et al. 2014). Another experiment about the Arabidopsis root response to various long-term salt stress indicated that significant changes in both physiological and proteome level have occurred (Carrera et al. 2018).
Although some work has explored the molecular mechanisms of plant salt-tolerance, more researches are still needed to provide further information. In the present work, we used an integrated approach to disclose how the plant deals with salt at the transcription level. In comparison with the proteomic data, we considered the transcription data could give the complementary information to facilitate our better understanding on the plant salt-response.
Here, to overcome and minimize the heterogeneity of results caused by different experimental conditions, 16 saltresponsive experiments in Arabidopsis root were retrieved from GEO database. To integrate the DEGs results more efficiently, the RRA method has been used to screen the DEGs list. As the result, 836 dysregulated genes including 449 up-regulated and 387 down-regulated genes were defined as the salt-responsive DEGs in the Arabidopsis root.
Based on the analysis of bioinformatics tools, both upregulated DEGs and down-regulated DEGs were involved in different biological process, molecular function and KEGG pathway. For example, in the up-regulated DEGs, response to oxidative stress was a significantly enriched biological process, and glutathione metabolism was a significantly enriched KEGG pathway. Usually, salt stress will attenuate the electron transport chain and produce excessive ROS, such as hydrogen peroxide, hydroxyl radicals, and superoxide radicals (Miller et al. 2010). ROS scavengingrelated enzymes including glutathione S-transferase family (Chan and Lam 2014) and POD (Islam et al. 2015) were activated to maintain the balance of redox homeostasis in plant cells. In the present work, POD, glutathione S-transferase tau 2 (GSTU2), glutathione S-transferase tau 3 (GSTU3), glutathione S-transferase tau 4 (GSTU4), glutathione S-transferase tau 5 (GSTU5), glutathione S-transferase tau 6 (GSTU6), glutathione S-transferase tau 9 (GSTU9), Glutathione Stransferase family protein (ERD9) were all up-regulated in response to the salt stress.
Based on the annotation of PPI network, some DEGs were involved in several stresses, which was named stress cross-talk (Tuteja 2007). A previous work has reported that some saltinducible genes were related to the cold stress (Seki et al. 2002). In the present work, the cross-talk of different stresses were more complicated than the previous finding. The saltresponsive DEGs were associated in wounding stress, cold stress, abscisic acid stress, heat stress, and water deprivation stress, which dramatically improved our understanding on salt-tolerance mechanisms in Arabidopsis root. To validate the cross-talk among different stresses, the enzyme activity of POD in root was assayed under salt stress, cold stress and water deprivation stress, individually. And the results agreed with the stress cross-talk prediction from the integrated bioinformatics analysis.

Conclusion
This work gave new light on the understanding of salt responses in Arabidopsis root. Those pathways, PPI networks, stress cross-talking would provide more important information to discover the potential salt-responsive biomarkers. In the future, more work is still needed to explore the functions of DEGs to better improve plant growth and crop output.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This project was supported by grants from Shandong Provincial Natural Science Foundation, China (ZR2010CQ024) and Shandong Provincial Universities Science and Technology Programme, China (J10LC72).

Notes on contributors
Meili Guo is an Associate Professor of Food and Biochemistry Engineering Department at Yantai Vocational College. Her research interest focuses on Arabidopsis growth and development, and the responses to abiotic stress responses.
Dr Xin Liu is an Associate Professor of Central Laboratory at the Affiliated Yantai Yuhuangding Hospital of Qingdao University. His research interest focuses on genomics, proteomics and bioinformatics. Lei Li is a lecturer of Medical Experiment and Test Center at Capital Medical University. Her research interest focuses on genomics, proteomics and bioinformatics.
Yusu Jiang is a lecturer of Food and Biochemistry Engineering Department at Yantai Vocational College. Her work focuses on investigations of plant growth and development, and stress physiology in plants.
Xuejuan Yu is an Associate Professor of Food and Biochemistry Engineering Department at Yantai Vocational College. Her research interest focuses on plant physiology.
Tao Meng is an assistant lecturer of Food and Biochemistry Engineering Department at Yantai Vocational College. His work focuses on plant physiology and plant abiotic stress responses.
Dr Chunxi Zhou is an Associate Professor of Beijing office at Bruker Daltonics Inc. His research interest focuses on genomics, proteomics and bioinformatics.