Physiological response and proteomics analysis of Reaumuria soongorica under salt stress

Effects of NaCl concentrations on growth indicators of R. soongorica seedlings

As shown in Table 1, when compared with control A (i.e., 0 mM NaCl), both the fresh weight and root/shoot ratio of R. soongorica in group B (i.e., 200 mM NaCl) were significantly higher. However, both fresh weight and root/shoot ratio gradually decreased in group C (i.e., 500 mM NaCl). When the NaCl concentration reached that of group C (i.e., 500 mM NaCl), the growth of R. soongorica was significantly inhibited. The fresh weight of above-ground and root tissues was respectively 43.82% and 50.99% that of the control, and these differences were significant (P < 0.05). Under the NaCl treatment with the B concentration, the water content of the above-ground and root tissues, as well as the total leaf area of leaves, exceeded that of the control. However, when the NaCl concentration was C, the water content of above-ground and root tissues were significantly lower than those of the control.

Effects of NaCl concentrations on relative conductivity and proline content of R. soongorica leaves

Under salt stress, Fig. 1-I shows the responsive changes in the relative conductivity and proline content of R. soongorica leaves. The relative conductivity decreased at first and then increased with an increasing NaCl stress concentration, and the differences were statistically significant. Meanwhile, leaf proline content also increased significantly (Fig. 1-II). These results indicated that R. soongorica could adapt to a salt stress environment by adjusting its leaf-level proline content. Under low salt stress, the cell membrane system of R. soongorica leaves was not damaged by stress, and its cell membrane had strong stability and could adequately adapt to a certain salt environment.

Number of differentially expressed proteins (DEPs)

Compared with group A, 47 DEPs were obtained from group B, of which 36 proteins were up-regulated and 11 proteins were down-regulated. Compared with group A, 177 DEPs were obtained from group C, with 126 of them up-regulated and the other 51 proteins down-regulated. Compared with group B, 136 DEPs were obtained from group C: 67 and 69 that were up- and down-regulated, respectively (Fig. 2-I). The identified proteins with significantly different expressions were statistically analyzed, and a certain number of proteins were found common among the three groups, as depicted in Fig. 2-II. Evidently, different proteins appeared in the three groups, and there were 12, 83, and 65 specific proteins in the A vs. B, A vs. C, and B vs. C comparison groups, respectively. In both A vs. B and A vs. C groups, 31 differential protein sites were found. Among these, 27 differential protein sites were up-regulated and 4 were down-regulated. In the A vs. B and B vs. C groups, there were 8 differential protein sites, 3 up-regulated and 1 down-regulated, while the other four DEPs showed opposite expression patterns in the two comparison groups. There were 67 differential protein sites in both A vs. C and B vs. C groups: 37 were up-regulated and 30 down-regulated. These results indicated significant differences in protein expression occurred between low salt (B) and high salt (C) conditions in R. soongorica seedlings. Notably, 83 proteins were only expressed in A vs. C under high salt stress, and this number significantly exceeded that of other comparison groups. This may point to the self-protection of plants under high salt stress by initiating greater levels of gene expression.

Hierarchical clustering analysis for DEPs under salt stress

As seen in Fig. 3, each column in the graphic represents a sample, and each row represents a protein; color represents the relative expression level of a given protein in the group of samples. On the left is the tree of protein clustering: the closer the branches of two proteins are, the closer their expression levels are, namely, the closer the trends in their variation. By analyzing the up-regulation and down-regulation of different proteins in different sample groups, we can tell that the similarity between the three repeated samples in each group is very high, which would support screening the DEPs accordingly.

Functional classification of DEPs according to the Clusters of Orthologous Groups (COGs) under salt stress

Figure 4 shows that under the salt stress response, DEPs are involved in different biological processes. These included RNA processing and modification, chromatin structure and dynamics, energy production and conversion, amino acid transport, nucleotide transport, and metabolism, among others. There were 29 differential proteins in the A vs. B comparison group that could be annotated and functionally classified by the COG database. These differential proteins were mainly involved in translation, ribosomal structure and biogenesis, function unknown, post-translational modification, protein turnover, and chaperones biological process, of which 21 were up-regulated and 8 were down-regulated. In the A vs. C comparison group, 112 differential proteins were annotated and functionally classified by COG database, these chiefly involved in translation, ribosomal structure and biogenesis, function unknown, general function prediction only, biological process of energy production and conversion, of which 78 and 34 respectively were up- and down-regulated. There were 86 differential proteins found in the B vs. C comparison group that could be annotated and functionally classified by the COG database. They were mostly involved in translation, ribosomal structure and biogenesis, general function prediction only, post-translational modification, protein turnover, and chaperones biological process, with 35 up-regulated and 51 down-regulated.

Gene ontology (GO) enrichment analysis for DEPs under salt stress

After their GO annotation, differential proteins were classified according to the functional categories of molecular function (MF), cell component (CC), and biological process (BP). Major biological functions performed by the DEPS could be determined by a GO significance analysis. In the A vs. B control group analysis, 276 GO items were obtained (P < 0.05), consisting of 194 BP items, 31 CC items, and 51 MF items, with 14 differential proteins annotated by GO. These DEPs were mainly enriched in translation, response to external stimulus, intracellular structure, ribosome and structural constituent of ribosome, and metabolic process, etc. In the A vs. C control group analysis, 495 GO items were obtained (P < 0.05), namely 274 BP items, 88 CC items, and 133 MF items, with 82 differential proteins annotated by GO. These DEPs were mainly enriched in translation, biosynthetic process, ribosome, membrane, structural constituent of ribosome, oxidoreductase activity, and in other ways. In the B vs. C control group analysis, 390 GO items were obtained (P < 0.05), comprising 213 BP items, 78 CC items, and 99 MF items, with 50 differential proteins annotated by GO. These DEPs were mainly enriched in translation, photosynthesis, cytoplasm, small ribosomal subunit, structural constituent of ribosome, and RNA binding, etc. (Fig. 5).

KEGG pathway enrichment analysis of DEPs

In the face of salt stress, protein functioning depends on the synergistic action of multiple proteins, resulting in significant changes in terms of their abundance. Pathway analysis can provide a more comprehensive and systematic understanding of the biological process each protein is relevant to, and thus point to and reveal the metabolic network of salt stress. In order to further understand the biological functions of the uncovered DEPs, their KEGG enrichment analysis was performed. These results showed that (Fig. 6) in the A vs. B comparison group, differential proteins were significantly enriched (P < 0.05) to six metabolic pathways (sesquiterpenoid and triterpenoid biosynthesis, glucosinolate biosynthesis, plant–pathogen interaction, ribosome, etc.). The differential proteins of A vs. C comparison group were significantly enriched to 16 metabolic pathways (linoleic acid metabolism, C5-dibasic acid metabolism, porphyrin and chlorophyll metabolism, etc.). Finally, the differential proteins in the B vs. C comparison group were significantly enriched to 13 metabolic pathways (glucosinolate biosynthesis, nitrogen metabolism, SNARE interactions in vesicular transport, etc.).

Identification of protein–protein interaction (PPI) networks among DEPs

To investigate the biological function and regulation of DEPs in R. soongorica leaves under salt stress, and to uncover those key proteins related to salt tolerance. For this, a composite score of PPIs (protein interactions) greater than 0.4 was used to determine the interaction network. As Fig. 7 shows, five histone interactions were identified in the A vs. B comparison group, and a total of 17 DEPs were involved in the protein interaction network of seedlings under salt stress. The RPS23B (40S ribosomal protein S23-2) and RACK1C (receptor for activated C kinase 1C) had 7 and 6 node connections, respectively. The node connections of AT5G18380 (40S ribosomal protein S16-3), RPL2-A (50S ribosomal protein L2), and EMB3010 (40S ribosomal protein S6-2) numbered 5 in each case. For P5CR (pyrroline-5-carboxylate reductase), NUDT3 (nudix hydrolase 3), and RPL12-A (60S ribosomal protein L12), each had 3 node connections. In the A vs. C comparison group, 87 DEPs were involved in the protein interaction network under salt stress, and 18 of them had more than 10 node connections, with the most node lines obtained for RPS13 (30S ribosomal protein S13, 21), RPS10 (30S ribosomal protein S10, 21), RPL27 (50S ribosomal protein L27, 17), PPOX2 (protoporphyrinogen oxidase 2, 17), RPL29 (50S ribosomal protein L29, 14), and RPL2-A (50S ribosomal protein L2, 14), etc. In the B vs. C comparison group, 61 DEPs were involved in the protein interaction network under salt stress; 14 of them had more than 10 node connections, with the most node lines found for RPS13 (30S ribosomal protein S13, 20), CFBP1 (fructose-1,6-bisphosphatase 1, 18), RPS10 (30S ribosomal protein S10, 17), RPL27 (50S ribosomal protein L27, 15), RPS11C (40S ribosomal protein S11-3, 14), and RPL5 (50S ribosomal protein L5, 13), etc.

Screening of key DEPs in leaves of R. soongorica seedlings under salt stress

Based on the COG database, 29, 112, and 86 DEPs in A vs. B, A vs. C, and B vs. C comparison groups could be annotated and functionally classified. Combined with the differential protein interaction regulatory network, protein points with a node connection number > 1 were selected and repeated proteins in each comparison group were integrated to further screen out the 72 DEPs. The abundance of 34 DEPs increased and 38 DEPs decreased under the salt stress treatments. The most varied proteins were involved in translation, ribosomal structure and biogenesis, amounting to 20 of them, of which 17 belonged to the ribosomal protein family (RP). Six ribosomal proteins (RPL2-A, RPL12A, RPS23B, RPS6B, RPL30A, and RPS16C) were up-regulated, while the expression levels of another 11 (RPL5, RPS13, RPS10, RPS2D, RPS11C, RPS20B, RPL21A, RPL27, RPS9, RPL29, and RPL7AA) were down-regulated under the NaCl stress. These results showed that R. soongorica seedlings could tolerate stress by synthesizing and degrading proteins in response to salt stress conditions. Furthermore, some proteins (GUN4, MRL1) with pronounced expression differences but not any reported functions were also found. These will be investigated in planned future work. The score of each protein was calculated by Mascot search software²². If the score was more than 60, the protein was considered reliable. Finally, four categories of concern were determined and their related DEPs artificially grouped: those proteins related to plant energy and metabolism, those proteins associated with photosynthesis, those proteins related to plant defense and stress resistance, and those participating in protein synthesis, processing, and degradation (Table 2).

Source: Ecology - nature.com