Dynamic Characteristics Of Aquaporins In Guard Cells And Subsidiary Cells Of Plant Cells

Stomata as a channel of water transpiration and CO2 assimilation,play an important role in regulating plant growth development and in responding to environmental stresses.The stomatal movement of Arabidopsis thaliana is regulated by biological factors(such as pathogen invasion)and abiotic factors(such as light and drought).In pathogen-associated molecular pattern(PAMP),flg22 can induce the production of hydrogen peroxide,promote the transport of aquaporins to water,increase the protoplast water potential(pf)of guard cells and induce stomatal closure.Aquaporins play an important role in water transport between guard cells and accessory cells during stomatal closure.Up to now,the function of aquaporins has been studied extensively.However,the kinetics and regulation mechanism of aquaporins in transporting water to intracellular or extracellular membranes remained to be ftnther determined.This study used the model plant Arabidopsis thaliana(ecotype:Columbia-0)as material to investigate the role of AtPIP2;1 protein under different treatments.The stomatal opening measurements showed that after flg22 treatment,pip2;1 had a slower stomatal closure rate compared with WT and 3JS::GFP-PIP2;1.Laser scanning confocal microscopy(LSCM)observed that pip2;1 plasma membrane depolarization decreased.The expression of AtPIP2;1 mRNA and protein content did not change significantly after flg22 treatment by fluorescence quantitative PCR and Western blot detection.However,its subcellular transport changed significantly.It was further found that after flg22 treatment of Arabidopsis thaliana obvious internalizations occurred in guard cells.These results suggested that the early stage of flg22 infection in Arabidopsis thaliana can induce the transport of AtPIP2;1,thereby regulating stomatal dosure.Through the variable-angle total internal recflection fluorescence microscopy(VA-TIRFM),it was found that the fluorescent proteins of GFP-AtPIP2;1 showed a non-uniform dot-like distribution on the plasma membrane with a high degree of heterogeneity.Single particle tracking(SPT)analysis showed that the diffusion coefficient and diffusion rate of GFP-AtPIP2;1 increased significantly,and the residence time became longer in guard cells after flg22 treatment.While in the accessory cells the diffusion coefficient and the diffusion rate of GFP-AtPIP2;1 were lowered,and the residence time was shortened.When the cytoskeleton inhibitors were treated together with flg22,the lateral movement of GFP-AtPIP2;1 became faster than that of flg22 alone,and the change in guard cells was more significant than accessory cells.Cellulase in combination with flg22 also had effect on the movement of AtGFP-PIP2;1 protein in guard cells and accessory cells.In the process of stomatal closure,as compared with the effect of cell wall on the movement of GFP-AtPIP2;1 protein,disruption of microtubule skeleton can accelerate the movement of GFP-AtPIP2;1 protein.In conclusion,the current study has analyzed the dynamics of the AtPIP2;1 protein in Arabidopsis thaliana by cytological and biochemical methods.It was found that:(1)Aquaporin AtPIP2;1 was involved in regulating stomatal closure;(2)In the early stage of Arabidopsis thaliana stress,AtPIP2;1 altered the abundance of AtPIP2;1 through its internalization to regulate stomatal closure;(3)AtPIP2;1 protein lateral movement was regulated by both cytoskeleton and cell wall,and microtubule skeleton had a stronger effect on the movement of AtPIP2;1 protein in guard cellsthan that of accessory cells.These results revealed the dynamic characteristics of aquaporin PIP2;1 in different types of cells and the key factors regulating its dynamic characteristics,which provide theoretical reference for further elucidating the dynamic regulation mechanism of cell membrane proteins in different types of cells.