Overexpression of Populus euphratica PePKS5 enhancing salt tolerance of Arabidopsis thaliana

Objective Salt stress is an adverse environmental factor affecting plant growth and development. As an important component of plant salt overly sensitive signal transduction pathway, protein kinase PKS5 plays an important regulatory role in plant response to salt stress. The aim of this study was to investigate the regulation mechanism of PePKS5 gene in plant tolerance to salt stress at physiological and molecular levels.

Method The PePKS5 gene from Populus euphratica was cloned and overexpressed in Arabidopsis thaliana to obtain a T₃ generation transgenic A. thaliana homozygous. We observed the salt tolerance phenotype of transgenic A. thaliana lines under salt stress, measured their peroxidase (POD), catalase (CAT), and superoxide dismutase activities, as well as the expression levels of salt stress responsive genes. Non-invasive micro measurement technology was used to measure the dynamic ion flow of Na⁺ and K⁺ in the root tips of transgenic A. thaliana lines, and the Na⁺ and H₂O₂ contents in the root tips of transgenic A. thaliana lines were observed using laser confocal microscopy. We measured the physiological indicators such as chlorophyll fluorescence parameters and photosynthetic parameters of transgenic A. thaliana seedlings cultured in soil after salt treatment, and revealed the physiological regulatory effect of PePKS5 gene on A. thaliana under salt stress. A subcellular localization vector was constructed and the subcellular localization of PePKS5 protein was observed by transient transformation of tobacco.

Result (1) The CDS sequence of PePKS5 gene encoded 417 amino acids, and the amino acid sequence of PePKS5 was the most similar to PtPKS5 of Populus tomentosa. (2) PePKS5 was localized in cytoplasm and nucleus. (3) The expression of PePKS5 gene in leaves of P. euphratica was up-regulated after 6 h of NaCl treatment, and gradually recovered to normal level after 72 h. (4) Under salt stress, the survival rate and root length of PePKS5 gene overexpressing A. thaliana lines (PePKS5-OE10 and PePKS5-OE15) were significantly higher than those of wild-type (WT) and vector control (VC) lines. (5) Compared with WT and VC lines, the activity of POD and CAT and the expression of APX1 and CAT2 in PePKS5-OE10 and PePKS5-OE15 lines were increased. (6) Under salt stress, the Na⁺ efflux and K⁺ influx of PePKS5-OE10 and PePKS5-OE15 lines were significantly higher than those of WT and VC lines, and the accumulated Na⁺ and H₂O₂ concentration in roots tips were significantly lower than those of WT and VC lines. (7) Under salt stress, the relative chlorophyll content, maximum photoquantum efficiency of PSⅡ, actual photosynthetic quantum yield and relative electron transport rate of PePKS5-OE10 and PePKS5-OE15 lines were higher than those of WT and VC lines. (8) Under salt stress, the net photosynthetic of PePKS5-OE10 and PePKS5-OE15 lines was higher than that of WT and VC lines, while the intercellular CO₂ concentration, stomatal conductance and transpiration rate were lower than those of WT and VC lines.

Conclusion Overexpression of PePKS5 gene in P. euphratica can enhance the Na⁺ efflux ability of A. thaliana, maintain the balance of reactive oxygen species and relatively stable photosynthetic capacity, thus improving the salt stress tolerance in A. thaliana.