Table_4_Characterization of plasma membrane proteins in stylosanthes leaves and roots using simplified enrichment method with a nonionic detergent.xlsx

Plant plasma membranes (PMs) play an important role in maintaining the stability of the intracellular environment and exchanging information with the external environment. Therefore, deciphering dynamics of PM proteome provides crucial information for elucidating cellular regulation in response to diverse stimuli. In the study, we developed a simplified method for enriching PM proteins in leaf and root tissues of a tropical forage Stylosanthes by combining differential centrifugation and Brij-58 treatment. Both immunoblot analysis and mass spectrometry demonstrated that the representation and abundance of PM proteins were increased in the enrichment fraction, and the contamination of other organellar proteins was decreased. A total of 426 and 388 proteins were predicted to be PM proteins in leaves and roots, respectively. Functional analysis classified these PM proteins into six major categories (transporter, enzyme, receptor, membrane structure protein, vesicular trafficking and chaperone), and orthologs of many PM proteins regulating the responses to abiotic and biotic stresses have been detected. In addition, the sequence analysis, subcellular localization and gene expression analyses of a newly identified receptor-like kinase, SgRKL1, has been performed. Together, these results show that the simplified PM enrichment method can be successfully applied to different plant tissue types and to study the dynamics of PM proteome of Stylosanthes in response to multiple stresses.

植物质膜（plasma membranes, PMs）在维持细胞内环境稳定以及与外界环境进行信息交换方面发挥着重要作用。因此，解析质膜蛋白质组的动态变化，可为阐明细胞应对各类刺激的调控机制提供关键信息。本研究通过差速离心结合Brij-58处理，建立了一种可富集热带牧草柱花草（Stylosanthes）叶片与根系组织中质膜蛋白的简化方法。免疫印迹分析与质谱检测均证实，富集组分中质膜蛋白的占比与丰度均有所提升，而其他细胞器蛋白的污染水平则有所降低。研究分别在叶片与根系中预测得到426种与388种质膜蛋白。功能分析将这些质膜蛋白划分为六大类别：转运蛋白、酶、受体、膜结构蛋白、囊泡转运蛋白与分子伴侣，且检测到诸多参与调控非生物与生物胁迫响应的质膜蛋白同源基因（orthologs）。此外，本研究还对新鉴定的类受体激酶SgRKL1开展了序列分析、亚细胞定位与基因表达分析。综上，上述结果表明，该简化质膜蛋白富集方法可成功应用于不同植物组织类型，且可用于研究柱花草应对多种胁迫时的质膜蛋白质组动态变化。