| Pyropia haitanensis has important nutritional,economic and ecological values,and is a unique warm temperate macroalgae.P.haitanensis mainly lives in the intertidal zone and undergoes periodic low tide dehydration and high tide rehydration.P.haitanensis often suffers from hypersaline and hyposaline stress at low tides exposed to severe sunlight and rain.However,P.haitanensis can quickly return to normal growth after rehydration,which indicates that it has evolved a unique salt-tolerant mechanism in long-term natural selection.Many studies had shown that K~+/Na~+homeostasis plays an important role in salt-tolerant plants,seaweed and microalgae,but whether it participates and how to regulate the response of P.haitanensis to salt stress is not still clear.Therefore,the Z-61 strain of P.haitanensis was selected as the experimental material in this study.From the view of ion dynamic transport,the experiment combined with the pharmacological method of adding ion channel specific inhibitors to explore the mechanism of maintaining K~+/Na~+homeostasis and osmotic pressure regulation during the salt stress process.Moreover,this study analyzed the similarities and differences of hypersaline stress and hyposaline stress,and preliminarily estimated the salt tolerance mechanism of P.haitanensis,which provided a new theoretical basis for clarifying the stress resistance mechanism of P.haitanensis.The main experimental results are as follows:(1)Compared with the control group(30‰),100‰salinity stress had no significant effect on the maximum quantum yield(Fv/Fm)of P.haitanensis.Under 110‰salinity stress,the Fv/Fm of P.haitanensis gradually decreased to 0,but after returning to the control seawater,it can still return to the initial level.Thsi study refers to 110‰as the"sub-lethal"salt concentration.Under 120‰salinity stress,the Fv/Fm of P.haitanensis dropped sharply to 0,and it could not be recovered by transferring to the control seawater.Therefore,100‰and 110‰were selected as stress treatment conditions for subsequent ion homeostasis study.The results showed that the ionic toxicity of P.haitanensis under high salt stress was greater than that of osmotic stress,and osmotic stress had no significant effect on intracellular ion content.P.haitanensis can tolerate the environment of 100‰,mainly due to its strong ability to excrete Na~+and retain K~+,which can maintain intracellular K~+/Na~+stability.However,the K~+loss of P.haitanensis under 110‰was more serious than that of 100‰.K~+/Na~+ratio of P.haitanensis has been falling at 110‰,but it can still be maintained at around 1.28,which is to ensure that the intracellular K~+/Na~+homeostasis is not completely broken,and the algae can quickly recover after rehydration.Meanwhile,P.haitanensis reduces photosynthesis activity to prevent photodamage,ROS production and K~+/Na~+imbalance.Under 120‰salinity stress,intracellular Na~+of P.haitanensis content increased significantly in a short period of time.This leads to excessive Na~+of P.haitanensis causing damage to biological function,decreased Na~+efflux capacity,increased plasma membrane depolarization,and severe K~+leakage,eventually resulting in a K~+/Na~+ratio below 1.P.haitanensis is seriously damaged due to K~+/Na~+homeostasis,resulting in death.It can be seen that maintaining K~+/Na~+homeostasis is crucial for the response of hypersaline stress to P.haitanensis.Further analysis revealed that after amiloride and orthovanadate inhibited the activity of Na~+/H~+antiporter and plasma membrane proton pump,Na~+efflux system of P.haitanensis was damaged,resulting in the increase of intracellular Na~+,K~+efflux and K~+/Na~+ratio decrease significantly.However,tetraethylamine chloride(TEA)inhibited K~+leakage of P.haitanensis to some extent after inhibiting outward rectifying K~+channels(KORCs).This indicates that P.haitanensis can activate the plasma membrane H~+-ATPase to drive the Na~+/H~+antiporter under 100‰and 110‰stress,and discharge excess Na from the cytoplasm to the apoplast.This prevents Na~+from accumulating excessively in the cells,reduces plasma membrane depolarization caused by intracellular Na~+content increase,and decreases the loss of K~+from depolarization-activated KORCs,thereby maintaining K~+/Na~+homeostasis of P.haitanensis.In addition,under high osmotic conditions,P.haitanensis will rapidly synthesize osmotic pressure regulating substances such as proline,soluble sugar,betaine and trehalose to stabilize cell turgor and prevent excessive water loss.However,there was no significant difference in osmotic content between the 100‰and 110‰stress in P.haitanensis.Therefore,P.haitanensis can maintain a higher K~+/Na~+may be a crucial reason for the adaptation to 100‰salinity.(2)For hyposaline stress,P.haitanensis can tolerate 5‰low salt environment,and Fv/Fm has no obvious change.Under 0‰salinity stress,the K~+loss of P.haitanensis was serious,and it could not return to normal level after rehydration,which led to a significant decrease in Fv/Fm with stress time,indicating that the algae was irreversibly damaged.Meanwhile,Na~+content of P.haitanensis dropped sharply in a short time under 5‰and 0‰stress and rapidly regulated the osmotic pressure substances such as proline,betaine and trehalose to maintain cell swelling pressure,in order to cope with osmotic pressure stress in external water environment.In addition,ion channel inhibitors had no significant effect on the ion content and K~+/Na~+in P.haitanensis under hyposaline.The above results indicate that P.haitanensis preferentially efflux inorganic ions and reduce organic osmotic adjustment substances under hyposaline conditions to maintain the osmotic pressure balance between the intracellular and extracellular water environments.More importantly,maintaining a high K~+level may be one of the main reasons why P.haitanensis can adapt to the low salt environment.(3)Compared with the responses of P.haitanensis to hypersaline and hyposaline stress,it can be found that the damage of hypersaline stress to algae is mainly ion toxicity.The ability of P.haitanensis to maintain K~+/Na~+homeostasis plays an important role in its response to extreme hypersaline stress.P.haitanensis excretes intracellular Na~+and retains K~+mainly through plasma membrane H~+-ATPase,Na~+/H~+antiporter and ion channels.In contrast,the hyposaline stress in P.haitanensis is mainly caused by hypo-osmotic stress.The algae can respond to the extremely high osmotic potential difference between cells and the environment by rapidly regulating inorganic ions and organic osmotic regulators.In addition,the rapid loss of Na~+and K~+in P.haitanensis under low salt conditions is significantly different from that in hypersaline stress.This study preliminarily speculated that the two were mainly discharged through non-selective cation channels,and the specific mechanism needs further verification.In summary,P.haitanensis has a strong salt tolerance,and there is a clear difference between the mechanisms of hypersaline and hyposaline stress.This study provides a new perspective and theoretical basis for further clarifying the salt tolerance mechanism of P.haitanensis. |
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