DOI: 10.6084/m9.figshare.30073531
title: Confocal image_zeiss czi files

description: To understand how homogenized meristematic cells in Arabidopsis floral inflorescence meristems begin to acquire distinct identities and differentiate into floral organs at the molecular level, we performed time-series single-nucleus RNA sequencing (snRNA-seq) experiments across synchronized developmental stages. This approach allowed us to characterize transcriptome dynamics and to examine how cell-cycle regulation is integrated with developmental programs during early flower development. Our results revealed a bifurcation in transcriptional trajectories that separates cell-cycle progression from floral differentiation. We further identified regulation of the cell-cycle inhibitor <b>KIP-RELATED PROTEIN 2 (KRP2)</b> by <b>FRUITFULL (FUL)</b> as a key node at this bifurcation, and validated its functional importance in vivo.This dataset contains all confocal images generated for quantitative analyses in this project.<b><i>Materials:</i></b><br>To elucidate the direct regulation of <b>FUL</b> on <b>KRP2</b>, we generated two different <i>KRP2–GFP</i> constructs (with or without the FUL-binding region) and transformed them into both Col-0 and <i>ful-7</i> Arabidopsis backgrounds. This yielded four genotypes:I. <i>pKRP2:KRP2–GFP:FUL_peak</i> (FGS1382)II. <i>pKRP2:KRP2–GFP:FUL_peak ful-7</i> (FGS1383)III. <i>pKRP2:KRP2–GFP:w/o_peak</i> (FGS1384)IV. <i>pKRP2:KRP2–GFP:w/o_peak ful-7</i> (FGS1385)To examine the role of <b>FUL</b> in the cell cycle, we also introduced the <b>Plant Cell Cycle Indicator (PlaCCI)</b> construct into Col-0 and <i>ful-7</i>. The PlaCCI reporter contains a single cassette comprising <i>pHTR13::HTR13–mCherry</i>, <i>pCDT1a::CDT1a–eCFP</i>, and <i>pCYCB1;1::NCYCB1;1–YFP</i>, which together enable discrimination of distinct cell-cycle phases.<b><i>Imaging and analysis:</i></b>Plants chosen for confocal microscopy were grown in the growth chamber till 1~3 flowers opened (within two weeks after bolting) and imaged at 40x on a Zeiss LSM 800 confocal laser scanning microscope. Before imaging, the old floral buds were carefully removed, and the centermost part was kept and stained (not for PlaCCI plants) for 5 minutes by 1 mg/mL propidium iodide (PI). After being briefly rinsed by water, the sample tissue was used for imaging.1. For GFP-positive plants scanning, one track was used with 488 nm and 561 nm laser to excite the PI and eGFP fluorescences, and emission was collected from 595 nm to 617 nm for PI signal and from 410 nm to 532 nm for eGFP signal. To get better cell segmentation in further analysis, an optimal 0.66 μm interval was used between stacks.After imaging, the raw czi files were open in Fiji, and PI and eGFP channels were split and saved as tif files respectively. The following expression analysis was done by MGX. For cell segmentation, PI tif files were load in MGX and processed as follows:1) Stack/Filters/Gaussian Blur Stack: 0.3x0.3x0.3 μm32) Stack/ITK/Segmentation/ITK Watershed Auto Seeded: Level=6003) Select the “Delete label in volume” tool and press “Alt” when clicking the generated box.4) Select the “Delete label in volume” or “Voxel Edit” tool to remove the cells outside IM.5) Mesh/Creation/Marching Cubes 3D: Cube size=1.0 μm6) Mesh 1/SaveNext, <i>Main </i>and <i>Labels </i>in stack 1 were unselected, and the eGFP tif file was loaded as the Main figure. Then, to calculate the eGFP signal intensity for the segmented cells, I projected the eGFP signal onto the cell mesh by following steps:1) Mesh/Signal/Project Signal: Use absolute= Yes, Min Dist=0, Max Dist=3 μm2) Mesh/Heap Map/Measures/Signal/Signal Total3) Mesh/Heap Map/Heap Map SaveTo capture the signal for each cell, I projected the signals in the 3 μm radius range from the cell mech surface according to the general meristem nuclei size studied before (Laufs et al., 1998). Then, the saved heatmap file contains the absolute value of eGFP signal obtained by confocal microscopy for each segmented cell.<br>2. For PlaCCI checking, due to the overlap of wavelength between PI and mCherry, the PI staining step was skipped. Three individual tracks were used to collect the fluorescence signal for the three fluorescent indicators in the single PlaCCI construct. mCherry was excited with a 561 nm laser, and the emission was collected from 400 nm to 630 nm. YFP was excited with a 488 nm laser, and the emission was collected from 520 to 580 nm. CFP was excited with a 405 nm laser, and the emission was collected from 410 to 520 nm. To cover each nucleus, scanning intervals were 3 μm between stacks according to the meristem nuclei size studied before (Laufs, 1998).In Fiji, IMs were chopped out for analysis, and individual fluorescence channels were split. Raw z-stacks of each channel were processed in MGX with the same setting as follows (adapted from Vijayan et al., 2021):1) Stack/Filters/Bright Darken: 32) Stack/Filters/Gaussian Blur Stack: 0.2x0.2x0.2 μm3 (apply two times)3) Stack/Segmentation/Local Maxima: xyz radius=1.8 μm; threshold=150004) Mesh/Creation/Mesh From Local Maxima: radius=1.5 μm5) Mesh/Heat Map/Analysis/Cell Analysis 3D6) Mesh/Attributes/Save to CSV ExtendedThese processes generated comparable nuclei numbers of different cell cycle phases for Col-0 and <i>ful-7</i>. As identification of PlaCCI for dividing cells, pHTR13::HTR13-mCherry indicates S+early G2 cells, pCDT1a::CDT1a-eCFP indicates the G1 cells, and pCYCB1;1::NCYCB1;1-YFP represents to late G2+M cells (Desvoyes, 2020). Thus, the proportion of different cell phases in IM was calculated by dividing the summed nuclei number of all channels with the nuclei number from individual corresponding channels.<br>Together, these findings establish regulation of <i>KRP2</i> by FUL as a central mechanism that coordinates cell-cycle progression with developmental fate decisions during early flower development.<br>