The Research On Physiological Pathways And Gene Regulation Network Associated With K~+ Absorption And Na~+ Expelling In Salt-tolerant Plant Thellungiella Salsuginea

Soil salinization or salt stress is one of the limiting factors to the reduction of crop production and quality. Soil salinization suppresses plant growth and seed germination, destroys ion balance (such as Na+/K+ balance), and then impairs cellular functions such as metabolism of sugar and amino acids, activities of antioxidant enzymes. Therefore salt stress becomes a limitation to the development of agricultural economy of China and the world. The unbalance of Na+/K+ is one of characteristics of soil salinization. Unbalanced Na+/K+ induced by soil salinization destroys the Na+/K+ harmony in plants and thus impairs their metabolism, which processes include ionic equilibrium and regulation of enzymatic activities.The Arabidopsis species of wide type Columbia (Col), high salicylic acid (SA) ecotype Cape Verde Islands (Cvi-0), NahG transgenic plants (with a lower SA content), and Thellungiella salsuginea (with much higher salt-resistance) were employed in this study. Col, Cvi-0, and NahG mutant seedlings were treated with 100 mM NaCl to analysis their resistance to unbalanced Na+/K+ conditions. Arabidopsis and Thellungiella were pretreated with 0.5 mM SA or PBZ (a SA inhibitor) before NaCl treatments to study their salt-resistances. Moreover, transcriptome sequencing methods and suppression subtractive hybridization (SSH) were employed to salt-stressed Arabidopsis and Thellungiella to explore the mechanism and the key genes related to the salt stress responses.(1) Lacking of SA contributes to salt tolerance, but not relates with the K+ absorption and Na+ expelling mechanism100 mM Na+ and 100 mM K+ treatments were administrated to Col, Cvi-0, and NahG seedlings, respectively. After being stressed 3 days, the leaf area (3rd true leaf, mm2), number of leaf, water content (percentage) and fresh weight (mg) of plant, Na+ and K+ contents (percentage) were detected. The results showed that the leaf area, number of leaf, and water content were suppressed by the salt stressed at different levels. After being treated with 100 mM Na+, Na+ content was significantly increased and K+ content was declined comparing with the control. However, when being treated with 100 mM K+, K+ content was significantly increased and Na+ content was declined comparing with the control. Additionally, both of 100 mM Na+ and 100 mM K+ administration showed the least impacts on the physiological indicators of NahG seedlings, compared with the others. This demonstrated that NahG plants showed better salt-resistance than the others. However, NahG plants have the normal levels of K+, similar to those of the wild-type plants. Thus its better salt-tolerance is not related with the K+ absorption and Na+ expelling mechanism.(2) Salicylic acid induces K+ absorption and Na+ expelling and therefore enhances plant's tolerance to salt stressArabidopsis and Thellungiella seedlings were pretreated with 0.5 mM SA and 0.5 mM PBZ before the 100 mM NaCl treatment. Physiological and biochemical indexes including water content, Na+ and K+ content, H2O2, soluble sugar concentration, malondialdehyde (MDA) content, and ion-leakage were detected. The results showed that SA pretreatment significantly reduced the changing levels of these indexes after the salt stress. For example, SA suppressed the salt-induced increasement of Na+ content, ion leakage (p<0.01) and MDA content (p<0.05); suppressed the salt-induced reduction of K+ content and water content (p<0.05). The indexes in Thellungiella were least changed by the salt-stress. Contrastingly, PBZ aggravated the damages of the salt-stress. SA pretreatment could facilitate the K+ uptake of (p<0.05) and Na+ expelling (p<0.05). Moreover, SA and PBZ generate the similar impacts on Thellungiella seedlings.(3) Transcriptome sequencing and suppression subtractive hybridization (SSH) analysis of salt-stressed ThellungiellaTranscriptome sequencing and suppression subtractive hybridization (SSH) were employed on Arabidopsis and Thellungiella before and after the stress. The bioinformatics analysis showed that Arabidopsis had more significantly changed genes than Thellungiella. The Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis showed that the differentially expressed genes in Arabidopsis and Thellungiella were related to hormone response, stress response and so on. The differentially expressed genes in Arabidopsis were enriched into the pathways such as P53 signaling pathway, autophagy, and FoxO signaling pathway. The differentially expressed genes in Thellungiella were enriched into the pathways including NF-kB signaling pathway, HIF-1 signaling pathway, MAPK signaling pathway and pentose phosphate pathway. Moreover, Arabidopsis and Thellungiella have some common differentially expressed genes related to the metabolisms of amino acid, sugar, plant hormone signaling pathways.We also found some specific genes in Thellungiella exponentially induced by the salt stress, such as C2H2 and WRKY transcription factors and some genes related to abscisic acid (ABA)-signaling. Additionally, the SA-signal marker genes NPR1 and PR1 were down-regulated by the salt-stress in Arabidopsis (Fold change, FC< 1), while up-regulated by the salt-stress in Thellungiella (FC> 3). The results imply that better salt-resistance of Thellungiella might be related to its higher expression levels of SA-related genes or higher-activated SA signals.