The Molecular Mechanism Of Phsphatidic Acid-mediated Regulation Of Rice Potassium Channel OsAKT2

Potassium(K~+)is one of the essential elements for plant growth and development,and plays important physiological functions such as osmoregulation,maintenance of charge balance,regulation of metabolic enzyme activity and promotion of photosynthetic product transport.Plants rely on K~+transport proteins(potassium channels and potassium transporters)to mediate the uptake and long-distance transport of potassium ions in vivo.The study of K~+transport proteins’physiological functions and regulatory mechanisms has been a hot topic in plant physiology and molecular biology.Phosphatidic acid(PA)is not only an important component of cell membranes,but also acts as a second messenger to regulate plant growth and development,cytoskeleton dynamics,vesicle transport,and response to adversity stresses.In animal cells,PA has been reported to directly regulate a variety of potassium channel activities,but in plant cells,the mechanism of PA signaling-mediated potassium channel regulation is unclear.In this study,we revealed the molecular regulatory mechanism of phosphatidic acid on rice potassium channel OsAKT2 by electrophysiological assay,protein-lipid interaction analysis and phenotypic analysis,and the main findings are as follows:1.The using the Xenopus oocytes expression system and two-electrode voltage clamp experiments revealed that OsAKT2 is a weakly rectifying potassium channel and exogenous PA treatment significantly inhibited OsAKT2 currents;the inhibitory effect of PA on OsAKT2 was significantly time-and concentration-dependent;in addition,1-butanol(an inhibitor of PA synthesis)treatment significantly enhanced OsAKT2 currents;while co-expression of PLDs significantly inhibited OsAKT2 activity.2.The C-terminal structural domain of the intracellular region of OsAKT2 bound specifically to PA,but not to other phospholipid molecules(e.g.PC,PE,PI and DAG);PI and DAG had no significant effect on OsAKT2 activity.3.The possible amino acid sites of OsAKT2 binding to PA were verified using lipid-protein interaction analysis and electrophysiological experiments.Among them,mutations of 644R/645R and 755R/756R to neutral amino acids significantly reduced the binding ability of the mutant phenotype of OsAKT2 to PA and made it insensitive to PA treatment.In addition,190R/191K of the S4 transmembrane domain may also be involved in affecting PA-mediated regulation of OsAKT2.4.AtAKT2,the Arabidopsis homolog of OsAKT2,also has a strong PA-binding capacity and is significantly inhibited by PA.In this study,we found that endogenous PA levels were negatively correlated with the growth and development rate of Arabidopsis under short daylight,and given the positive regulation of AtAKT2 on Arabidopsis growth and development under energy-deprived conditions,it is hypothesized that PA signaling may affect Arabidopsis growth and development by regulating AtAKT2 activity.In summary,this study innovatively found that PA can directly bind and inhibit the channel activity of OsAKT2,with 644R/645R and 755R/756R in its intracellular region as PA binding sites,and that PA may affect plant growth and development under short daylight conditions by regulating AtAKT2 activity.This study reveals the direct interaction between phosphatidic acid and plant K~+channels,which not only expands the functional study of plant phospholipid signaling,but also has a pioneering significance for studying the mechanism of phospholipid signaling-mediated regulation of plant ion channels.