In-depth Tissue-Level Dimerization Analysis of AtLEA proteins from Arabidopsis thaliana - Replicate 3

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: Exp3_PYF: Replicate 3 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).