Salt Stress Tolerance in Casuarina glauca: Insights from the Branchlets Transcriptome

Abstract

Climate change and the accelerated rate of population growth are imposing a progressive degradation of natural ecosystems worldwide. In this context, the use of pioneer trees represents a powerful approach to reverse the situation. Among others, N₂-fixing actinorhizal trees constitute important elements of plant communities and have been successfully used in land reclamation at a global scale. In this study, we have analyzed the transcriptome of the photosynthetic organs of Casuarina glauca (branchlets) to unravel the molecular mechanisms underlying salt stress tolerance. For that, C. glauca plants supplied either with chemical nitrogen (KNO₃⁺) or nodulated by Frankia (NOD⁺) were exposed to a gradient of salt concentrations (200, 400, and 600 mM NaCl) and RNA-Seq was performed. An average of ca. 25 million clean reads was obtained for each group of plants, corresponding to 86,202 unigenes. The patterns of differentially expressed genes (DEGs) clearly separate two groups: (i) control- and 200 mM NaCl-treated plants, and (ii) 400 and 600 mM NaCl-treated plants. Additionally, although the number of total transcripts was relatively high in both plant groups, the percentage of significant DEGs was very low, ranging from 6 (200 mM NaCl/NOD⁺) to 314 (600 mM NaCl/KNO₃⁺), mostly involving down-regulation. The vast majority of up-regulated genes was related to regulatory processes, reinforcing the hypothesis that some ecotypes of C. glauca have a strong stress-responsive system with an extensive set of constitutive defense mechanisms, complemented by a tight mechanism of transcriptional and post-transcriptional regulation. The results suggest that the robustness of the stress response system in C. glauca is regulated by a limited number of genes that tightly regulate detoxification and protein/enzyme stability, highlighting the complexity of the molecular interactions leading to salinity tolerance in this species.

Keywords: Casuarina glauca; Frankia; Illumina RNA-Seq; actinorhizal plants; salt-tolerance.

Figure 1

Differential gene expression in branchlets of Casuarina glauca plants nodulated by nitrogen-fixing Frankia casuarinae Thr (NOD⁺) or supplied with KNO₃ (KNO₃⁺). 200: 200 vs. 0 mM NaCl; 400: 400 vs. 0 mM NaCl; and 600: 600 vs. 0 mM NaCl, with a False Discovery Rate < 0.05. (A) Total number of DEGs. (B) Number of DEGs specific to each salinity condition relative to control.

Figure 2

Differential gene expression in Casuarina glauca plants supplied with KNO₃ (KNO₃⁺) or nodulated by nitrogen-fixing Frankia casuarinae Thr (NOD⁺), grown at 200 mM, 400 mM, and 600 mM NaCl. Total number of treatment-specific differentially expressed genes (DEGs) for each salt treatment relative to the control (0 mM NaCl). 200: 200 vs. 0 mM NaCl; 400: 400 vs. 0 mM NaCl; and 600: 600 vs. 0 mM NaCl. (A) KNO₃⁺ downregulated DEGs. (B) KNO₃⁺ upregulated DEGs. (C) NOD⁺ downregulated DEGs. (D) NOD⁺ upregulated DEGs.

Figure 3

Specific and overlapping genes between Casuarina glauca plants supplied with KNO₃ (KNO₃⁺) or nodulated by nitrogen-fixing Frankia casuarinae Thr (NOD⁺). Significant differentially expressed genes (DEGs), with a False Discovery Rate (FDR) < 0.05, were detected between the control (0 mM NaCl) and salinity-exposed plants: (A) 200 mM NaCl; (B) 400 mM NaCl; (C) 600 mM NaCl.

Figure 4

Pattern of expression of differentially expressed genes (DEGs) in non-nodulated Casuarina glauca plants supplied with KNO₃ (KNO₃⁺), grouped by predicted co-expressed gene clusters. Clustering analysis was performed by Clust tool with normalized reads from samples grown at 0, 200, 400, and 600 mM NaCl.

Figure 5

Pattern of expression of differentially expressed genes (DEGs) in Casuarina glauca nodulated by nitrogen-fixing Frankia casuarinae Thr (NOD⁺), grouped by predicted co-expressed gene clusters. Clustering analysis was performed by Clust tool, with normalized reads from samples grown at 0, 200, 400, and 600 mM NaCl.

Figure 6

COG distribution pattern in NOD⁺ plants (A) and non-nodulated KNO₃⁺ plants (B).