Effects Of ZnO NPs On Distribution Of Ca²⁺ And Na⁺ In Protoplasts Of Aloe Cells After Freezing

Cold is the main factor that causes huge agricultural losses and plant deaths.Studies have shown that plants can sense frozen signals and transmit them to cells to activate responses,but the mechanism by which plants sense temperature is still unclear.There are mainly three methods for the study of cell freezing in the past:one is to measure the leakage of the electrolyte in the cell to assess the degree of cell membrane damage;the second is to compare the changes in the relative contents of DNA,ROS,MDA,etc.;and the third is to observe the ultrastructure of cell organelles.The change.This article mainly studies the distribution of ions in protoplasts under stress from a new perspective.Ion-selective microelectrode technology is mainly used to measure the ion flow of plant cells or tissues.This paper proposes a new method based on ion-selective microelectrode technology to detect ion concentration pulses during protoplast rupture to study the effect of freezing stress on ion in cells.The method can dynamically measure the ion pulse signals before and after the rupture near the protoplasts.The operation is convenient and the electrode detection range is large.For a single protoplast measurement,the anti-interference ability is strong and the experimental data obtained are accurate.We used Ca²⁺and Na⁺ion selective microelectrodes to study ion signal pulses of protoplasts from aloe protoplasts under freezing stress and found that there was a significant difference between frozen and untreated Ca²⁺signals.The Ca²⁺signaling image of the cryopreserved protoplast will have an apparent downward depression during the initial stage of the rupture,while Na⁺signal will not change.This phenomenon shows that the freezing stress will affect the distribution of Ca²⁺concentration in the cells.The concentration of Ca²⁺in the central part of the cells is high and the concentration of surrounding Ca²⁺is low.Ca²⁺distribution stratification began at a temperature of-7?,and this phenomenon has not disappeared after 5 hours of thawing.Using this method,we detected the concentrations signals of protoplast Ca²⁺and Na⁺in different concentrations of ZnO NPs?30,50,70,and 90 mg/L?.It was found that the Ca²⁺signal pulse increased dramatically in a short time after 18 h.It shows that under the stress of ZnO NPs,it will cause the concentration and distribution of Ca²⁺near the cell membrane,which will lead to a straight rise of the signal pulse.At the same time,the effects of ZnO NPs on the distribution of Ca²⁺and Na⁺in protoplasts before and after cryopreservation were studied.We first pretreated the protoplasts with ZnO NPs and then frozen them.When the protoplasts broke,the pulse curve of Ca²⁺concentration also appeared"depression"at the front edge.However,compared with the case without ZnO NPs pretreatment,the depth of the depression at the front edge of Ca²⁺concentration after ZnO NPs pretreatment was significantly reduced,indicating that Ca²⁺stratification has been alleviated.The shape of the Na⁺concentration pulse has not changed significantly,and its distribution in the protoplast has not been affected.We further examined the concentration of Ca²⁺and Na⁺signals of ZnO NPs on pre-freezing protoplasts.We found that not only did the depression of the Na⁺concentration pulse appear at the front edge,but also the“depression”did not appear at the front edge of the Ca²⁺concentration pulse,indicating that Ca²⁺in protoplasts.The stratification disappeared.It was shown that ZnO NPs have certain positive effects in preventing the decrease of Ca²⁺fluidity caused by freezing and maintaining the ability to regulate the distribution of Ca²⁺.This article provides a new idea for the comprehensive study of cell death mechanisms under freezing stress,and has important implications for understanding the physiological responses and cold-resistance of plants under freezing conditions.