The Modulation Mechanism And Biological Effects Of He-Ne Laser On Salt Resistance Responses To Salt Stress In Tall Fescue Seedlings

Salt stress is one of the main environmental stressors that seriously inhibit the productivity, and the growth and development of crops and other plants throughout the world. Soil salinity usually causes soil degradation and decreases soil porosity that remarkably restricts water, nutrient substance, and oxygen gas availability which are responsible for plant growth and development, and fecundity. Furthermore, salt stress also results in the excessive accumulation of sodium（Na⁺） and chloride（Cl^-） ion in plants, which will generate cytotoxicity effects for living cells, finally reduce plant cell growth and survival.In this work, the modulation effects and molecular mechanism of He-Ne laser illumination on salt resistance response of salt stressed-tall fescue（Festuca arundinacea Schreb.） seedlings were studied and further analyzed. Tall fescue seedlings were illuminated with He-Ne laser(wavelength λ=632.8nm,beam diameter 100.0mm,irradiation dosage 5.0mW mmm^-2), or exogenous sodium nitroprusside（SNP, nitric oxide NO donor）, or white light(obtained by LED lamps, PAR=100μmol mm^-2 s-1) prior to salt stress（100.0mM NaCl supplemented in Hoagland’ nutrient solution）. Finally, the 28-d old of seedlings were harvested and stored in-80℃ for further determination and analysis. The protective effects of He-Ne laser illumination on nucleic genomic DNA structure and function damage of tall fescue seedlings was evaluated by agarose gel electrophoresis, nano-drop detector, and DNA apopoladder analysis technology. The changes in physicochemical properties and biological activities of cell wall polysaccharides from tall fescue seedlings was measured and analyzed by fourier transform-infrared spectroscopy（FTIR） system, UV-spectrum analysis, confocol laser scanning microscopy（CLSM） and high performance liquid chromatography（HPLC） technology in order to elucidate the molecular or physicochemical mechanisms of He-Ne laser modulation on adaptive responses to salt stress in tall fescue seedlings. The results were showed as followed.（1） Salt stress caused the significant inhibitory effects on tall fescue seedlings growth and development, and phenotypic morphogenesis, such as biomass decay and leaf and root development retardation. He-Ne laser irradiation can significantly alleviate these detrimental effects of salt stress on seedlings. Therefore, we supposed that suitable dosage and optimal treatment times of He-Ne laser irradiation would induce salt resistance responses to salinity conditions in plants.（2） We measured the changes of plant growth parameters, agronomic traits and phenotypic development, and compared the differences among seedlings from different treatment groups, including SNP treatment and common white light radiation group. Our data further established the favorable biological effects of He-Ne laser irradiation on salt stressed-seedlings.（3） He-Ne laser irradiation effectively protected tall fescue seedlings against oxidative damage and oxidative stress caused by salt stress. Endogenous nitric oxide（NO） signal molecule was involved in these physiological processes. He-Ne laser treatment significantly promoted the intracellular free Ca²⁺ levels elevating, that can bind calmodulin（CaM）, and activate NOS-like enzymatic activity, then cause endogenous NO release enhancement. Higher level of intracellular NO modulated plant antioxidant system, especially antioxidant enzyme activity, such as superoxide dismutase（SOD）, peroxidase（POD）, ascorbate peroxidase（APX）, further decreased reactive oxygen species（ROS） accumulation, finally alleviated oxidative stress for seedlings.（4） He-Ne laser irradiation resulted in gene encoding plant antioxidant enzyme expression un-regulation in salt stressed-tall fescue seedlings, which might also be beneficial to plant antioxidant system activation. In addition, we also examined that PhyB transcriptional level was activated by the laser, however, the interactive actions between PhyB expression up-regulation and plant salt resistance responses would be further investigated in the next study.（5） The another reason on the inhibitory effects of salt stress on plants is intracellular toxic ions（Na⁺, Cl^-） accumulation resulted in ionic toxicity effects. Our results showed that He-Ne laser irradiation detoxified the cytotoxicity effects of toxic ions on plant cells through improving the activities of plasma membrane（PM）-H⁺-ATPase and V-type-proton pump in tonoplasts.（6） Salt stress caused significant cell death phenomena both in leaf and root tissue, and cell apoptosis only in root tissue. The underlying mechanisms might be root directly meets nutrient solutions, resulting in toxic ions accumulation is more serious than in leaf tissue that caused cell viability in root was lower than in leaf. However, salt stress induced cell death phenomena both in leaf and root tissue, and cell apoptosis only in root tissue due to root has higher resistance than leaf. He-Ne laser irradiation can efficiently ameliorate these phenomena in leaf and root of tall fescue seedlings under salt stress conditions.（7） Salt stress caused nucleic genomic DNA damage, such as “DNA smear” and “DNA fragmentation” formation. He-Ne laser treatment reduced “DNA smear” and “DNA fragmentation” formation in seedlings, suggesting He-Ne laser irradiation protected nucleic DNA against salt stress, and maintained DNA structure and sequence integrity.（8） The results of cell wall polysaccharides physicochemical properties of tall fescue seedlings by FITR, UV-spectrometer and HPLC system showed that He-Ne laser irradiation also protected cell wall against physiological damage and physicochemical injury induced salt stress. So He-Ne laser irradiation maintained the higher physicochemical activities of cell wall due to helping cell wall reconstruction under unfavorable stressors in order to promote nucleic genomic DNA damage repair.In conclusion, He-Ne laser irradiation significantly activated plant antioxidant enzyme transcriptional levels and expression pattern, stimulated endogenous NO signal molecule release being independent in NOS activity, then improved plant antioxidant system in vivo, decreased the intracellular ROS accumulation and membrane injury, finally efficiently reduced oxidative damage and oxidative stress induced salt stress. At the same time, He-Ne laser also remarkably detoxified the ionic toxicity induced by salt stress via enhancement enzymatic activities of PM H⁺-ATPase and V-type proton pump in tonoplasts. Thus, He-Ne laser treatment significantly induced salt resistance responses of tall fescue seedlings to salt stress conditions.