Investigation on salt-response mechanisms in Arabidopsis thaliana from UniProt protein knowledgebase

ABSTRACT Salt stress negatively affects plant growth and crop productivity. As an ideal model pathway of salt tolerance in glycophyte. To better understand the molecular mechanisms of salt-response in glycophyte, 466 of 15,768 Arabidopsis thaliana proteins with the GO term of biological with known genetic background, Arabidopsis thaliana has been widely applied to disclose the process ‘response to salt stress’ were retrieved from UniPort and analyzed by bioinformatics tools of PANTHER, DAVID, KEGG, Cytoscape and STRING. Our results not only indicated the involvement of salt-responsive proteins in various pathways and interaction networks, but also demonstrated the more complicated cross-tolerances to both abiotic stresses (osmosis, water deprivation, abscisic acid, cold, heat, light and wounding) and biotic stresses (bacterium and fungus) and multiple subcellular locations of these salt-responsive proteins. Furthermore, protein activities of superoxide dismutase (SOD) and peroxidase (POD) in Arabidopsis thaliana were determined under salt, cold and osmotic stresses, which validated the hypothesis of cross-tolerance to multiple stresses. Our work will greatly improve the current knowledge of salt tolerance mechanism in glycophytes and provide potential salt-responsive candidates for promoting plant growth and increasing crop output.


Introduction
In the world, more than 20% irrigated lands are negatively affected by soil salinity, which seriously limits the plant growth and decreases the grain output (Zhao et al. 2013). Usually, osmosis tolerance, sodium ion exclusion and sodium ion accumulation tolerance are the main ways for plant to response to salt stress (Munns and Tester 2008). During this process, plant undergoes many changes including developmental, physiological, biochemical, and morphological (Abu et al. 2010).
Many plants have been used as research materials. Among them, arabidopsis is an ideal model plant, and great understandings of salt stress tolerance and adaptation have been obtained from arabidopsis (Ngara and Ndimba 2014). To discover novel phosphatidic acid-binding proteins associated with salt stress, an approach was launched in a previous research using lipid-affinity purification for the isolation of peripheral membrane proteins combined with mass spectrometry identification (McLoughlin et al. 2013). As a result, 42 phosphatidic acid-binding proteins had been identified including clathrin-assembly protein, glyceraldehyde 3-phosphate dehydrogenase and a phosphatidylinositol 4 kinase. Furthermore, a quantitative proteomic research using twodimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry had been performed to display the differentially expressed microsomal proteins under salt stress between Arabidopsis thaliana and Thellungiella salsuginea, and identified 36 significantly altered proteins. Gene ontology classification showed these proteins were involved in transport and carbohydrate metabolism (Vera-Estrella et al. 2014). In our previous works, 2D-DIGE has been carried out to investigate the differential proteins between wild-type Arabidopsis and its salt-tolerant mutant. A total of 19 altered proteins were identified by MALDI-TOF mass spectrometry, which is mainly associated with redox homeostasis, signal transduction, and carbohydrate metabolism (Guo et al. 2014b). Furthermore, phosphoproteome of Arabidopsis roots has been studied with Pro-Q Diamond staining, and the results showed nonsynchronous differences between total salt-responsive proteins and phosphorylated proteins. The differential proteins majored in signal transduction, and reactive oxygen species (ROS) scavenging (Guo et al. 2014a). Proteomic technology has greatly improved our knowledge of Arabidopsis salt-response mechanisms. But there are still limitations in our understanding of the salt tolerance process. Each research only provides limited information, and some proteomic data of studies are still not available. In order to better demonstrate the details of saltresponsive proteins in Arabidopsis, a more data-rich and accessible resource should be explored.
Therefore, in the present work, a more comprehensive dataset associated with salt tolerance was retrieved from the UniProt public database (release 2018_08, https://www. uniprot.org/). The UniProt knowledgebase provides the most abundant resources including more than 60 million protein sequences, of which over 500,000 sequences have been manually curated by scientists. Uniprot database is an ideal hub for the acquisition of protein information (Pundir et al. 2017;The UniProt Consortium 2017;Boutet et al. 2016;UniProt Consortium 2015). In 15,768 reviewed A. thaliana proteins, 466 salt-responsive proteins were selected, which significantly exceeds the number of salt-responsive proteins provided by any single previous research. Based on integrated bioinformatic analysis (PANTHER, DAVID, KEGG, Cytoscape, and STRING), the complicated crosstalk, interactions and pathways would extend our knowledge of salt response and adaption in Arabidopsis. Further experimental validation has been performed to confirm the prediction of bioinformatic analysis. Those salt-responsive proteins might be the potential targets for increasing crop output in the future research.

Selection criteria of salt-responsive proteins from UniProt database
Based on the GO annotation in the UniProt database (release 2018_08), 466 proteins involved in the biological process term of 'response to salt stress' were recruited from 157,68 reviewed A. thaliana proteins. Further, to investigate the coss-talk of salt-responsive proteins with the proteins involved in other stresses, 142,283,242,323,340,137,162,451, and 129 reviewed proteins associated with the biological process term of 'response to osmotic stress,' 'response to cold,' 'response to water deprivation,' 'defense response to bacterium,' 'response to abscisic acid,' 'response to heat,' 'response to wounding,' 'defense response to fungus,' and 'response to light stimulus' were also selected respectively from the above A. thaliana UniProt knowledgebase (Supplementary Table 1).

Gene ontology classification
A web-accessible bioinformatic tool of DAVID (Database for annotation, visualization and integrated discovery, Version 6.8) (https://david.ncifcrf.gov/) and another online program PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system (Version 13.1 released 2018-02-03) (http://pantherdb.org/) were applied for gene ontology enrichment analysis of the recruited proteins. Each protein was classified into one category. The Venn diagram was prepared by an online tool of 'Calculate and draw custom Venn diagrams' (http://bioinformatics.psb.ugent.be/webtools/Venn/).

Pathway analysis
The pathways of salt-responsive A. thaliana proteins were assessed through the online program of KEGG (Kyoto Encyclopedia of Genes and Genomes, Version 88.0, released 1 October 2018) (http://www.kegg.jp).

Protein-protein interaction
The protein-protein interaction network of the recruited proteins was analyzed by the software of STRING (search tool for recurring instances of neighbouring genes) database (Version 10.5, released 14 May 2017) (http://string-db.org/), and an open-source software of Cytoscape (an open source platform for complex network analysis and visualization) (Version 3.6.1).

Arabidopsis thaliana material, growth conditions and harvest
According to the previous method (Guo et al. 2014a;Guo et al. 2014b), the seeds of A. thaliana (ecotype Col-0) were germinated in the normal MS medium-containing plate under the conditions of 8/16 h light/dark cycle, 22/20°C day/night, 60 μmol m −2 s −1 light intensity. After 8 days, the resultant A. thaliana seedlings were divided into four groups. The first group was transferred to the cold-stress condition (8°C, day and night), the second group was transferred into drought-stress condition (MS medium supplemented with 200 mmol L −1 mannitol), the third group was transferred into salt-stress condition (MS medium supplemented with 150 mmol L −1 NaCl), and the fourth group was transferred into the normal MS medium-containing plate under normal conditions. The A. thaliana seedlings of the above four groups continued to grow for 4 days. Then, A. thaliana roots and rosette leaves were harvested, and stored at -80°C for further analysis. Three replicates were performed for the above experimental design.

Enzyme activity assay
According to the determination protocol of superoxide dismutase (SOD) and peroxidase (POD) activities (Nanjing Jiancheng Bioengineering Institute) (Li et al. 2013), the above enzyme activities of A. thaliana roots and rosette leaves were assayed using the corresponding kit. The activity of SOD was defined as the inhibition of O 2 − production by the xanthine morpholine with xanthine oxidase. One unit of SOD activity was the SOD quantity used to inhibit 50% of nitrite reduction in 1 mL reaction solution, which was calculated by the change of absorbance at 550 nm. The POD activity was defined as the catalyzing H 2 O 2 change of absorbance at 420 nm. One unit was the amount of enzyme required to catalyze 1 µg substrate at 37°C. The graphs were drawn using the software of Graphpad Prism 6 for windows (Version 6.01).

Protein-protein interaction network of A. thaliana salt-responsive proteins
A protein-protein interaction network was constructed by the online software of STRING. Totally, 416 of 466 saltresponsive proteins were connected with each other through 2,063 edges. PPI enrichment was statistically significant (p-value < 1.0e-16) (Figure 3).

Cross-tolerance visualization of A. thaliana salt-responsive proteins involved in the response to other stresses
Among 466 salt-responsive proteins, 260 proteins also responded to other stresses (eg. osmotic stress, cold, water deprivation, bacterium defense, abscisic acid, heat, wounding, fungus defense, and light stimulus). Figure 4 showed that 116  were involved in response to abscisic acid; 91 responded to water deprivation; 83 were also osmotic stress-related proteins; 82 were associated with response to cold; 36 saltresponsive proteins participated in defense response to bacterium; 29 were related to response to heat; 23 participated in response to wounding; 14 were related to defense response to fungus; and 14 were associated with response to light. The complicated cross-talk response of A. thaliana salt-responsive proteins with other stresses was presented in Figure 5.

Multiple subcellular localizations of A. thaliana saltresponsive proteins
Among the above 260 proteins involved in multiple stresses, 103 proteins had more than one subcellular localization ( Figure 6). For example, protein BTB/POZ and TAZ domain-containing protein 3 (BT3) could localize in the cytoplasm, nucleus, cell wall, endoplasmic reticulum, membrane, mitochondrion, plastid, symplast, thylakoid and vacuole; protein bifunctional enolase 2/transcriptional activator (ENO2) could be found in the cytoplasm, endoplasmic reticulum, membrane, mitochondrion, nucleus, plastid and symplast; and protein glyceraldehyde-3-phosphate dehydrogenase (GAPC1) could be mapped into the cytoplasm, endoplasmic reticulum, membrane, mitochondrion, nucleus, plastid, and vacuole (Figure 7). Those proteins were defined as double cross-talk proteins characterized by multi-location and multi-response to different stresses.

Antioxidative activity determination of A. thaliana under salt, cold, and osmotic stress
Under salt stress or other stresses, antioxidative activity is an important molecular function of these stress-responsive proteins. Our results showed POD and SOD were both saltresponsive proteins. Therefore, enzyme activities of POD and SOD in root and leaf were determined under three stresses (salt, cold, and osmotic stress). In comparison with the control, activities of POD and SOD were generally increased under salt, cold, and osmotic stresses at different check points Figure 3. Representative proteinprotein interaction networks of the salt-responsive proteins in A. thaliana. A total of 408 nodes were connected with 2063 edges (PPI enrichment p-value < 1.0e-16). The relationships were derived from the curated databases, experimentally determined, gene neighborhood, gene fusions, gene co-occurrence, textmining, co-expression, and protein homology. (12, 24, 36, 48, 60, 72, 84, 96 h), However, control treatments did not change the SOD and POD activities as shown in Figure 9.

Discussion
Salt stress is a nonnegligible issue that significantly limits plant growth on the earth. Many scientists have paid great attention to investigate the mechanisms of plant salt-tolerance and salt-adaption using genomic and proteomic   technologies (Shah et al. 2018;Wu et al. 2016). Arabidopsis thaliana is an ideal material to investigate the salt-responsive molecules (Guan et al. 2018;Zhang et al. 2018;Huang et al. 2018). But previous research only provided limited information. Therefore, more detailed data would extend our knowledge of plant salt-response pathways. Here, we screened 466 reviewed salt-responsive proteins from UniProt database, which contained the most comprehensive information of plant salt tolerance and adaption. Integrated bioinformatic analysis demonstrated the complicated pathways, protein interactions and cross-talks under different stresses in A. thaliana. Arginine and proline metabolism was a significantly enriched pathway. Among 466 salt-responsive proteins, 9 proteins, P-loop containing nucleoside triphosphate hydrolases superfamily protein (NOA1), aldehyde dehydrogenase 12A1 (ALDH12A1), aldehyde dehydrogenase 3H1 (ALDH3H1), aldehyde dehydrogenase 7B4 (ALDH7B4), arginine decarboxylase 1 (ADC1), arginine decarboxylase 2 (ADC2), delta1-pyrroline-5-carboxylate synthase 1 (P5CS1), ornithine-delta-aminotransferase (DELTA-OAT) and pyrroline-5-carboxylate (P5C) reductase (P5CR), were nodes of arginine and proline metabolism. The polyamines were catalyzed by ADC1 and ADC2, which were positively charged and involved in the binding with phospholipids, nucleic acids and membrane (Groppa and Benavides 2008). Previous studies have proved that polyamines are free radical scavenger and possess the molecular function of antioxidant activity. Therefore, proline metabolism might be an important way to protect A. thaliana from attaching free radicals under salt stress (Verbruggen and Hermans 2008).
Under salt stress, electron transport chain will be excessively attenuated, and the level of ROS dramatically increases,  such as the hydroxyl radicals, superoxide radicals, and hydrogen peroxide (Miller et al. 2010;Mittler et al. 2004). In order to maintain redox homeostasis in plant cell, ROS scavengingrelated enzymes are activated to decrease the oxidative perturbance, including superoxide dismutases (Sarker and Oba 2018), ascorbate peroxidase (Ribeiro et al. 2017), glutathione S-transferase (Chan and Lam 2014), and glutathione peroxidase (Islam et al. 2015). In our present work, many enzymes with ROS-scavenging activities (glutathione S-transferase family protein, ascorbate peroxidase 1, glutathione S-transferase 6, glutathione S-transferase 7, glutathione S-transferase PHI 2, glutathione S-transferase phi 8, and glutathione peroxidase 6) participated in the tolerance of salt stress in A. thaliana. To validate the outcome of bioinformatic analysis, the enzyme activities of POD and SOD were detected under salt stress, and the results indicated that these two proteins were indeed salt-responsive proteins, and might play an important role in the process of scavenging ROS.
Based on bioinformatic analysis of 466 salt-responsive A. thaliana proteins, some proteins were characterized by multiple subcellular locations. For example, BT3 and ENO2 each had 10 and 7 subcellular locations. The former might play roles in the process of gametophyte development (Robert et al. 2009), and the latter could influence metabolite synthesis under salt stress . The multiple subcellular localizations of these proteins might indicate their versatile functions under salt stress. Further studies are necessary to disclose the functions of these salt-responsive proteins under the stress.
Many genes and proteins have been found to participate in response to more than one stress, which is called cross-tolerance to multiple stresses. Cross-tolerance is a vital strategy for plants to survive under different stresses, and many proteins are involved in this process (Tuteja 2007). A previous study has demonstrated that approximately 75% of 195 salt-inducible genes are associated with drought or cold stresses (Seki et al. 2002). Our present work displayed that cross-tolerance is more universal and complicated. The salt-responsive proteins were not only involved in abiotic responses (osmosis, water deprivation, abscisic acid, cold, heat, light and wounding) but also associated with biotic responses (bacterium and fungus), which greatly extended the knowledge of salt tolerance mechanism in A. thaliana. The salt-responsive protein POD has been confirmed to be involved in the tolerance to the temperature stress (Neilson et al. 2010), and SOD was also related to other stresses (Zhao et al. 2013). In order to verify the above universality of cross-tolerance to multiple stresses, the enzyme activities of POD and SOD in root and leaf of A. thaliana were measured under salt, cold and osmotic stresses, and the results were in agreement with our hypothesis and validated the prediction of bioinformatics.

Conclusion
This work provided new understanding on the salt-response mechanisms of A. thaliana proteins and on the pathways associated with salt stress response. Those potential biomarkers identified were characterized by multiple subcellular locations and multiple stresses responses, which we named as the 'space-stress' double cross-tolerance effects. These results provided a clue that A. thaliana has developed an economical way to respond to various stresses. Further researches are therefore necessary to further elaborate the functions of these targets in the process of stress tolerance and adaption, which will be helpful to promote plant growth and to increase crop output.

Disclosure statement
No potential conflict of interest was reported by the authors.

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