Xiuyan Yang

Nitraria sibirica Pall., a typical halophyte that can survive under extreme drought conditions and in saline-alkali environments, exhibits strong salt tolerance and environmental adaptability. Understanding the mechanism of molecular and physiological metabolic response to salt stress of plant will better promote the cultivation and use of halophytes. To explore the mechanism of molecular and physiological metabolic of N. sibirica response to salt stress, two-month-old seedlings were treated with 0, 100, and 400 mM NaCl. The results showed that the differentially expressed genes between 100 and 400 mmol L⁻¹ NaCl and unsalted treatment showed significant enrichment in GO terms such as binding, cell wall, extemal encapsulating structure, extracellular region and nucleotide binding. KEGG enrichment analysis found that NaCl treatment had a significant effect on the metabolic pathways in N. sibirica leaves, which mainly including plant-pathogen interaction, amino acid metabolism of the beta alanine, arginine, proline and glycine metabolism, carbon metabolism of glycolysis, gluconeogenesis, galactose, starch and sucrose metabolism, plant hormone signal transduction and spliceosome. Metabolomics analysis found that the differential metabolites between the unsalted treatment and the NaCl treatment are mainly amino acids (proline, aspartic acid, methionine, etc.), organic acids (oxaloacetic acid, fumaric acid, nicotinic acid, etc.) and polyhydric alcohols (inositol, ribitol, etc.), etc. KEGG annotation and enrichment analysis showed that 100 mmol L⁻¹ NaCl treatment had a greater effect on the sulfur metabolism, cysteine and methionine metabolism in N. sibirica leaves, while various amino acid metabolism, TCA cycle, photosynthetic carbon fixation and sulfur metabolism and other metabolic pathways have been significantly affected by 400 mmol L⁻¹ NaCl treatment. Correlation analysis of differential genes in transcriptome and differential metabolites in metabolome have found that the genes of AMY2, BAM1, GPAT3, ASP1, CML38 and RPL4 and the metabolites of L-cysteine, proline, 4-aminobutyric acid and oxaloacetate played an important role in N. sibirica salt tolerance control. This is a further improvement of the salt tolerance mechanism of N. sibirica, and it will provide a theoretical basis and technical support for treatment of saline-alkali soil and the cultivation of halophytes.

Plant salt tolerance relies on a coordinated functioning of different tissues and organs. Salinity tissue tolerance is one of the key traits that confers plant adaptation to saline environment. This trait implies maintenance low cytosolic Na⁺/K⁺ ratio in metabolically active cellular compartments. In this study, we used Nitraria sibirica Pall., a perennial woody halophytes species, to understand the mechanistic basis of its salinity tissue tolerance. The results showed that the growth of seedlings was stimulated by 100-200 mM NaCl treatment. The ions distribution analysis showed that the leaves acted as Na⁺ sink while plant root possess superior K⁺ retention. The excessive Na⁺ absorbed from soil was mainly transported to the shoot and eventually sequestrated into mesophyll vacuoles in the leaves. As a result, N. sibirica could keep optimal balance of K⁺/Na⁺ at a tissue- and cell-specific level under saline condition. To enable this, N. sibirica increased both vacuolar H⁺-ATPase and H⁺-PPase enzymes activities and up-regulated expressions of NsVHA, NsVP1 and NsNHX1 genes. Vacuolar Na⁺ sequestration in the leaf mesophyll mediated by NsVHA, NsVP1 and NsNHX1 reduced the Na⁺ concentration in cytosol and inhibited further K⁺ loss. Meanwhile, N.sibirica enhanced the TPK expression at the transcriptional level to promote K⁺ efflux from vacuole into cytoplasm, assisting in maintaining cytosolic K⁺ homeostasis. It is concluded that the tissue tolerance traits such as vacuolar Na⁺ sequestration and intracellular K⁺ homeostasis is critical to confer adaptation of N. sibirica to soil salinity.

MicroRNAs (miRNAs) are endogenous small RNAs that play a crucial role in plant growth, development, and environmental stress responses. Reaumuria soongorica is a typical halophyte that is widely distributed in saline–alkali desert regions. Under salt stress, R. soongorica can complete germination, a critical biological process in the life cycle of seed plants. To identify miRNAs and predict target mRNAs involved in seed germination during salt stress, nine small-RNA libraries were constructed and analyzed from R. soongorica seeds treated with various concentrations of NaCl. We also obtained transcriptome data under the same treatment conditions. Further analysis identified 88 conserved miRNAs representing 25 defined families and discovered 13 novel miRNAs from nine libraries. A co-expression analysis was performed on the same samples to identify putative miRNA–mRNA interactions that were responsive to salt stress. A comparative analysis of expression during germination under 273 (threshold) and 43 mM (optimal) NaCl treatments identified 13 differentially expressed miRNAs and 23 corresponding target mRNAs, while a comparison between 43 mM NaCl and non-salt-stress conditions uncovered one differentially expressed miRNA and one corresponding target mRNA. These results provide basic data for further study of molecular mechanisms involved in the germination of salt-stressed R. soongorica seeds, and also provide a reference for the improvement of salt tolerance during plant germination.

Using Non-invasive Micro-test Technology (NMT), the Na⁺, K⁺ and H⁺ flux profiles in the root meristem regions were investigated in Nitraria sibirica Pall. seedlings under different NaCl concentrations. NaCl stress increased the K⁺ and Na⁺ contents in the roots of N. sibirica seedlings. NaCl stress significantly increased the steady Na⁺ efflux from the N. sibirica seedling roots. Steady K⁺ effluxes were measured in the control roots (without NaCl) and in the roots treated with 200 mM NaCl, and no significant differences were observed between the two treatments. The steady K⁺ efflux from roots treated with 400 mM NaCl decreased gradually. NaCl treatment significantly increased the H⁺ influx. Pharmacological experiments showed that amiloride and sodium vanadate significantly inhibited the Na⁺ efflux and H⁺ influx, suggesting that the Na⁺ efflux was mediated by a Na⁺/H⁺ antiporter using energy provided by plasma membrane H⁺-ATPase. The NaCl-induced root K⁺ efflux was inhibited by the K⁺ channel inhibitor tetraethylammonium chloride (TEA), and was significantly increased by the H⁺-ATPase inhibitor sodium vanadate. The NaCl-induced K⁺ efflux was mediated by depolarization-activated outward-rectifying K⁺ channels and nonselective cation channels (NSCCs). Under salt stress, N. sibirica seedlings showed increased Na⁺ efflux due to increased plasma membrane H⁺-ATPase and Na⁺/H⁺ antiporter activity. High H⁺ pump activity not only restricts the Na⁺ influx through NSCCs, but also limits K⁺ leakage through outward-rectifying K⁺ channels and NSCCs, leading to maintenance of the K⁺/Na⁺ balance and higher salt tolerance.

Nitraria sibirica Pall., a typical halophyte of great ecological value, is widely distributed in desert, saline, and coastal saline-alkali environments. Consequently, researching the salt tolerance mechanism of N. sibirica Pall. has great significance to the cultivation and utilization of salt-tolerant plants. In this research, RNA-seq, digital gene expression (DGE), and high flux element analysis technologies were used to investigate the molecular and physiological mechanisms related to salt tolerance of N. sibirica Pall. Integrative analysis and de novo transcriptome assembly generated 137,421 unigenes. In total, 58,340 and 34,033 unigenes were annotated with gene ontology (GO) terms and mapped in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. Three differentially expressed genes (DEGs) libraries were subsequently constructed from the leaves of N. sibirica Pall. seedlings under different treatments: control (CK), light short-term salt stress (CL2), and heavy long-term salt stress (CL6). Eight hundred and twenty-six, and 224 differentially expressed genes were identified in CL2 and CL6 compared to CK, respectively. Finally, ionomic analysis of N. sibirica Pall. seedlings treated with 0, 100, 200 or 300 mM concentrations of NaCl for one day showed that the uptake and distribution of Ca, Cu, Fe, Mg and K in different organs of N. sibirica Pall. were significantly affected by salt stress. Our findings have identified potential genes involved in salt tolerance and in the reference transcriptome and have revealed the salt tolerance mechanism in N. sibirica Pall. These findings will provide further insight into the molecular and physiological mechanisms related to salt stress in N. sibirica Pall. and in other halophytes.

MicroRNAs (miRNAs) are a class of endogenous small RNAs with important roles in plant growth, development, and environmental stress responses. Ulmus pumila L., a deciduous broadleaved tree species of northern temperate regions, is widely distributed in central and northern Asia and has important economic and ecological value. With the spread and aggravation of soil salinization, salt stress has become a major abiotic stress affecting the normal growth and development of U. pumila. However, the influence of salt stress on U. pumila miRNA expression has not been investigated. To identify miRNAs and predict their target mRNA genes under salt stress, three small RNA libraries were generated and sequenced from roots of U. pumila seedlings treated with various concentrations of NaCl corresponding to no salt stress, light short-term salt stress, and medium-heavy long-term salt stress. Integrative analysis identified 254 conserved miRNAs representing 29 families and 49 novel miRNAs; 232 potential targets of the miRNAs were also predicted. Expression profiling of miRNAs between libraries was performed, and the expression of six miRNAs was validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Our findings provide an overview of potential miRNAs and corresponding targets involved in regulating U. pumila salt defense responses. These results lay the foundation for further research into molecular mechanisms involved in salt stress resistance in U. pumila and other Ulmaceae species.