In-depth Tissue-Level Dimerization Analysis of AtLEA proteins from Arabidopsis thaliana - Replicate 1

Post 48-hour infiltration, leaves were examined utilizing a 3I Mariana Spinning Disc Confocal microscope equipped with a Zeiss Observer Z.1 Inverted base and a fluorescence spinning disk. In each experimental run, four plants were analyzed, each expressing one of three specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein: pYFN-4-/5pYFC-4-5 (representing the complete AtLEA4-5 protein), pYFN-4-51-77/pYFC-4-51-77 (encompassing the N-terminal region of AtLEA4-5), pYFN-4-578-158/pYFC-4-578-158 (comprising the C-terminal region of AtLEA4-5),pYFN-4-2/pYFC-4-2 (full length AtLEA4-2 protein), or pYFN-pYFC (serving as the control condition). Observations were conducted using a 20X—/0.8 NA air lens from Zeiss, under illumination provided by a pre-centered fiber illuminator from Intelligent Imaging Innovations, Inc. The detection of YFP fluorescence was facilitated by a 515 nm excitation laser and a 542/27 nm emission filter. Images were captured using an Andor Ixon 3 EMCCD camera (Model: DU-897E-CS0-#BV), adhering to protocols defined in SlideBook software version 6. Images were captured at specified intervals with exposure times ranging from 400 to 700 milliseconds. Each image consisted of a plane with a pixel size of 0.8 microns, spaced at 2 micrometer intervals. A total of 16 planes, correlating to an estimated depth of 32 micrometers per z-plane, were scrutinized. From each plant, up to three leaves were examined. The analysis included over 50 volumetric scenes per plant, with each scene encompassing a volume of 100 x 100 x 25 mm³. Image stacks were exported as *.tif Notation: Exp1_PYF: Replicate1 expressing one of four specific genetic constructs fused to complementary fragments of Yellow Fluorescent Protein. 45: Fused to the full length AtLEA4-5 protein (pYFN-4-/5pYFC-4-5). 4H: Fused to the N-terminal region of AtLEA4-5 (pYFN-4-51-77/pYFC-4-51-77). RC: Fused to the C-terminal region of AtLEA4-5 (pYFN-4-578-158/pYFC-4-578-158). control: The control condition (pYFN-pYFC). 42: Fused to the full length AtLEA4-2 protein (pYFN-4-2/pYFC-4-2).农杆菌介导渗透处理48小时后，采用搭载蔡司Observer Z.1倒置主机及荧光转盘模块的3I Mariana转盘式共聚焦显微镜（3I Mariana Spinning Disc Confocal Microscope）对叶片进行观测。 每次实验批次中，共分析4株植物，每株分别表达与黄色荧光蛋白（Yellow Fluorescent Protein, YFP）互补片段融合的特定遗传构建体之一，共计5组构建体：pYFN-4-/5pYFC-4-5（对应完整AtLEA4-5蛋白）、pYFN-4-51-77/pYFC-4-51-77（包含AtLEA4-5的N端区域）、pYFN-4-578-158/pYFC-4-578-158（包含AtLEA4-5的C端区域）、pYFN-4-2/pYFC-4-2（全长AtLEA4-2蛋白），以及pYFN-pYFC（作为对照处理）。 观测采用蔡司20×/0.8数值孔径（Numerical Aperture, NA）空气物镜，照明光源为Intelligent Imaging Innovations, Inc.生产的预对中光纤照明器。 YFP荧光的检测采用515 nm激发激光与542/27 nm发射滤光片完成。图像采集使用Andor Ixon 3型电子倍增电荷耦合器件（Electron Multiplying Charge Coupled Device, EMCCD）相机（型号：DU-897E-CS0-#BV），并遵循SlideBook软件v6.0定义的操作流程。 图像按指定间隔采集，曝光时长设置为400至700毫秒。每张图像对应一个成像平面，像素尺寸为0.8微米，平面间距为2微米。共分析16个成像平面，每个Z平面的估算深度为32微米。每株植物最多选取3片叶片进行观测。每株植物的分析包含超过50个体积成像场景，每个场景的体积为100×100×25立方毫米。图像栈以*.tif格式导出。 标注说明： Exp1_PYF：第1次生物学重复，表达与YFP互补片段融合的4种特定遗传构建体之一。 45：与完整AtLEA4-5蛋白融合（对应pYFN-4-/5pYFC-4-5）。 4H：与AtLEA4-5的N端区域融合（对应pYFN-4-51-77/pYFC-4-51-77）。 RC：与AtLEA4-5的C端区域融合（对应pYFN-4-578-158/pYFC-4-578-158）。 control：对照处理（对应pYFN-pYFC）。 42：与完整AtLEA4-2蛋白融合（对应pYFN-4-2/pYFC-4-2）。