质膜微区是细胞质膜上富含甾醇和鞘磷脂的微结构域, 参与信号转导、囊泡转运、胞吞和胞吐等众多生物学过程, 因此质膜微区动态过程是植物细胞生物学研究的重要领域之一。荧光探针结合荧光显微镜被广泛应用于检测植物活细胞状态。PA (push-pull pyrene)是一种基于芘的新型、高效且稳定的荧光探针, 但在植物活细胞成像研究中应用极少。该研究利用PA探针和激光共聚焦显微镜技术, 结合图像处理和极性归一化数值作图法对拟南芥(Arabidopsis thaliana)根中活细胞质膜的有序度进行了定量分析, 发现PA探针在拟南芥根细胞质膜中的液态有序相的发射光谱为500-550 nm, 液态无序相的发射光谱为580-700 nm。使用甾醇抽提剂MβCD处理野生型拟南芥可使质膜的有序度降低。缺乏甾醇合成关键的甲基转移酶双突变体smt2/smt3质膜的有序度与经甾醇抽提剂MβCD处理的野生型株系质膜一致。smt2/smt3突变体根毛细胞质膜的有序度低于野生型, 表明甾醇作为膜微区的关键组分在调节质膜的有序度上发挥重要作用。该研究为检测植物活细胞质膜动力学特征和质膜微区变化提供了一种直观且快速的检测手段。

Membrane microdomains, which are highly dynamic structures rich in sterols and sphingolipids on the plasma membrane, play crucial roles in various biological processes such as signal transduction, vesicle transport, endocytosis, and exocytosis. Consequently, the investigation of membrane microdomain dynamics is an important area of research in plant cell biology. Fluorescence probes combined with fluorescence microscopy are widely used to monitor the status of living plant cells. The PA probe (push-pull pyrene) is a novel, highly efficient and stable fluorescence probe based on pyrene: however, its application in imaging studies of living plant cells is limited. In this study, we used PA probes and laser scanning confocal microscopy, combined with image processing and the polar normalized value mapping method, to quantitatively analyze the order of the plasma membrane in Arabidopsis root cells. The results revealed that the emission spectrum of the liquid-ordered phase in the plasma membrane of Arabidopsis root cells labeled with the PA probe ranged from 500-550 nm, whereas the emission spectrum of the liquid-disordered phase ranged from 580-700 nm. Treatment of wild-type plants with the sterol extraction agent MβCD resulted in a decrease in plasma membrane order. In the smt2/smt3 double mutant lacking the key methyltransferase in sterol synthesis, the plasma membrane order was consistent with that of the wild-type plants after treatment with MβCD. In the smt2/smt3 mutant, the plasma membrane order of the root hair cells was lower than the plasma membrane order of the wild-type root hair cells, indicating that sterols, as key components of membrane microdomains, play an important role in regulating the order of the plasma membrane. This study provides a straightforward and rapid detection method for monitoring the dynamic characteristics of living plant cell membranes and changes in membrane microdomains.