Confocal image_zeiss czi files

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>

为解析拟南芥花花序分生组织中均质化的分生细胞如何在分子层面开始获得独特身份并分化为花器官，我们在同步化的发育阶段中开展了时序单细胞核RNA测序（single-nucleus RNA sequencing, snRNA-seq）实验。该方法使我们能够刻画转录组动态变化，并探究在早期花发育过程中，细胞周期调控如何与发育程序相整合。我们的研究结果揭示了转录轨迹上的一个分叉点，该分叉将细胞周期进程与花器官分化分离开来。我们进一步鉴定出，<b>果荚成熟蛋白FRUITFULL（FRUITFULL, FUL）</b>对细胞周期抑制剂<b>KIP相关蛋白2（KIP-RELATED PROTEIN 2, KRP2）</b>的调控是该分叉点的关键节点，并在体内验证了其功能重要性。本数据集包含本项目中用于定量分析的所有共聚焦显微镜图像。<br><b><i>实验材料：</i></b><br>为阐明<b>FUL</b>对<b>KRP2</b>的直接调控作用，我们构建了两种不同的<i>KRP2–GFP</i>融合基因载体（分别包含或缺失FUL结合区域），并将其转化至哥伦比亚生态型（Col-0）和<i>ful-7</i>拟南芥背景中，由此获得四种基因型材料：<br>I. <i>pKRP2:KRP2–GFP:FUL_peak</i>（FGS1382）<br>II. <i>pKRP2:KRP2–GFP:FUL_peak ful-7</i>（FGS1383）<br>III. <i>pKRP2:KRP2–GFP:w/o_peak</i>（FGS1384）<br>IV. <i>pKRP2:KRP2–GFP:w/o_peak ful-7</i>（FGS1385）<br>为探究<b>FUL</b>在细胞周期中的作用，我们还将<b>植物细胞周期指示剂（Plant Cell Cycle Indicator, PlaCCI）</b>载体转化至Col-0和<i>ful-7</i>拟南芥中。该PlaCCI报告基因包含一个单一表达盒，由<i>pHTR13::HTR13–mCherry</i>、<i>pCDT1a::CDT1a–eCFP</i>和<i>pCYCB1;1::NCYCB1;1–YFP</i>组成，可共同用于区分不同的细胞周期时相。<br><b><i>成像与分析方法：</i></b><br>用于共聚焦显微镜成像的植株在生长室中培养至抽薹后两周内开放1~3朵花时，使用蔡司LSM 800共聚焦激光扫描显微镜以40倍物镜进行成像。成像前需小心去除老的花原基，保留最中心的组织；除PlaCCI植株外，其余样品用1 mg/mL碘化丙啶（propidium iodide, PI）染色5分钟。样品经清水短暂漂洗后即可用于成像。<br>1. 针对GFP阳性植株的扫描：使用单通道采集光路，以488 nm和561 nm激光分别激发PI和增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）的荧光；PI信号的发射光采集波段为595 nm~617 nm，eGFP信号的发射光采集波段为410 nm~532 nm。为在后续分析中获得更优质的细胞分割效果，层间扫描间隔设置为最优的0.66 μm。<br>成像完成后，将原始czi格式文件导入Fiji软件中，分别拆分PI和eGFP通道并保存为tif格式文件。后续的表达分析由MGX软件完成。<br>针对细胞分割，将PI通道的tif文件导入MGX并按以下步骤处理：<br>1) 堆栈/滤波/高斯模糊堆栈：0.3×0.3×0.3 μm³<br>2) 堆栈/ITK/分割/ITK自动种子分水岭算法：阈值Level=600<br>3) 选择“体积内标签删除”工具，点击生成的框体时按住Alt键。<br>4) 使用“体积内标签删除”或“体素编辑”工具，移除花序分生组织（inflorescence meristem, IM）以外的细胞。<br>5) 网格/创建/三维移动立方体算法：立方体尺寸=1.0 μm<br>6) 网格1/保存<br>随后，取消选择堆栈1中的“主图像”和“标签”通道，将eGFP通道的tif文件加载为主图像。接下来，为计算分割后细胞的eGFP信号强度，我们按以下步骤将eGFP信号投影至细胞网格上：<br>1) 网格/信号/信号投影：启用绝对值模式，最小距离Min Dist=0，最大距离Max Dist=3 μm<br>2) 网格/热图/测量/信号/总信号量<br>3) 网格/热图/热图保存<br>为获取每个细胞的信号值，我们根据此前研究中报道的分生组织细胞核尺寸（Laufs等，1998），将细胞机械表面3 μm半径范围内的信号进行投影。最终保存的热图文件包含了每个分割细胞的共聚焦成像eGFP信号绝对值。<br>2. 针对PlaCCI植株的检测：由于PI与单体红色荧光蛋白（mCherry）的波长存在重叠，因此省略PI染色步骤。使用三个独立的采集通道，分别获取该单一PlaCCI载体中三种荧光标记物的荧光信号。mCherry以561 nm激光激发，发射光采集波段为400 nm~630 nm。黄色荧光蛋白（yellow fluorescent protein, YFP）以488 nm激光激发，发射光采集波段为520 nm~580 nm。青色荧光蛋白（cyan fluorescent protein, CFP）以405 nm激光激发，发射光采集波段为410 nm~520 nm。根据此前研究中报道的分生组织细胞核尺寸（Laufs，1998），层间扫描间隔设置为3 μm以覆盖每个细胞核。<br>在Fiji软件中，裁剪出花序分生组织（IM）用于分析，并拆分各荧光通道。各通道的原始z层堆叠文件在MGX中按以下统一参数进行处理（改编自Vijayan等，2021）：<br>1) 堆栈/滤波/明暗调整：参数3<br>2) 堆栈/滤波/高斯模糊堆栈：0.2×0.2×0.2 μm³，重复执行两次<br>3) 堆栈/分割/局部最大值算法：xyz半径=1.8 μm；阈值=15000<br>4) 网格/创建/基于局部最大值的网格生成：半径=1.5 μm<br>5) 网格/热图/分析/三维细胞分析<br>6) 网格/属性/扩展CSV格式保存<br>通过上述处理，可获得Col-0与<i>ful-7</i>拟南芥中不同细胞周期时相的可比细胞核数量。根据PlaCCI标记物的细胞周期时相鉴定规则，pHTR13::HTR13-mCherry可标记S期+早G2期细胞，pCDT1a::CDT1a-eCFP可标记G1期细胞，pCYCB1;1::NCYCB1;1-YFP可标记晚G2期+M期细胞（Desvoyes等，2020）。因此，花序分生组织中不同细胞时相的比例可通过将各通道的总细胞核数除以对应单个通道的细胞核数计算得到。<br>综上，本研究证实FUL对<i>KRP2</i>的调控是协调早期花发育过程中细胞周期进程与发育命运决定的核心机制。