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Real-time Analysis of Lateral Root Organogenesis in Arabidopsis
拟南芥侧根器官形成的实时分析   

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Abstract

Plants maintain capacity to form new organs such as leaves, flowers, lateral shoots and roots throughout their postembryonic lifetime. Lateral roots (LRs) originate from a few pericycle cells that acquire attributes of founder cells (FCs), undergo series of anticlinal divisions, and give rise to a few short initial cells. After initiation, coordinated cell division and differentiation occur, giving rise to lateral root primordia (LRP). Primordia continue to grow, emerge through the cortex and epidermal layers of the primary root, and finally a new apical meristem is established taking over the responsibility for growth of mature lateral roots [for detailed description of the individual stages of lateral root organogenesis see Malamy and Benfey (1997)]. To examine this highly dynamic developmental process and to investigate a role of various hormonal, genetic and environmental factors in the regulation of lateral root organogenesis, the real time imaging based analyses represent extremely powerful tools (Laskowski et al., 2008; De Smet et al., 2012; Marhavý et al., 2013; Marhavý et al., 2014). Herein, we describe a protocol for real time lateral root primordia (LRP) analysis, which enables the monitoring of an onset of the specific gene expression and subcellular protein localization during primordia organogenesis, as well as the evaluation of the impact of genetic and environmental perturbations on LRP organogenesis.

Materials and Reagents

  1. Arabidopsis seedlings (5-6 days old) expressing Green Fluorescent Protein (GFP) or analogous reporters (YFP, RFP, CFP, mCherry and others) (Chalfie et al., 1994; Shaner et al., 2007)
  2. MilliQ Water (H2O)
  3. Sucrose (VWR International, catalog number: 27483.294 )
  4. Murashige and Skoog basal salt mixture (MS salts) (Duchefa Biochemie, catalog number: M0221.0050 )
  5. 2- [N-morpholino] ethanesulfonic acid (MES) (Duchefa Biochemie, catalog number: M1503.0100 )
  6. Potassium hydroxide (KOH) (Merck KGaA, catalog number: 1.05021.1000 )
  7. Agar (LAB M, catalog number: MC029 )
  8. Ethanol (EtOH) (Sigma-Aldrich, catalog number: 32221 -2.5L)
  9. Seeds sterilization by ethanol (see Recipes)
  10. ½ MS media (see Recipes)
  11. Growth conditions (see Recipes)

Equipment

  1. Growth chamber to grow plant material
  2. Square plates 120 x 120 x 17 mm (Greiner Bio-One GmbH, catalog number: 688102 )
  3. Chambered cover glass (VWR, Kammerdeckgläser, Lab-TekTM, NuncTM - eine kammer, catalog number: 734-2056 )
  4. Inverted confocal microscope (Zeiss, model: LSM 700 )
    Note: Fully motorized X, Y, Z scanning stage is required to perform multi-position time-lapse experiment.
  5. Objectives: 20x [suitable to monitor early phases of the lateral root primordia (LRP) initiation, Figure 2], 40x or 60x (suitable to monitor LRP from the stage I onwards, Figure 3)
  6. Fluorescence signal detection system for GFP and other fluorescent reporters (Shaner et al., 2007)

Software

  1. Software operating the confocal microscope
  2. ImageJ (Abramoff et al., 2004)
  3. CellseT (Pound et al., 2012)
  4. Microsoft Excel

Procedure

  1. Sample preparation (Figure 1)
    1. Prepare chambered cover glass, wash with 70% ethanol and dry (Figure 1A).
    2. Pour 45 ml MS+ medium into the square plate and wait till it congeals (approx. 40 min at room temperature; solid medium should be 2-3 mm thin) (Figure 1B).
    3. Using the chambered cover glass cut out the block of solid MS media (Figure 1C-D).
    4. From the block of media cut off a ~3 mm wide strip (Figure 1C-D).
    5. Using the strip of media grease the chambered cover glass (Figure 1E).
    6. Transfer 10-15 seedlings inside the chamber, roots of individual seedlings must not overlap (Figure 1F).
    7. Cover seedlings with remaining block of media (Figure 1G).
    8. Close with the chambered cover glass lid (Figure 1H).


      Figure 1. Sample preparation. A. Chambered cover glass. B. Solid MS+ medium 2 - 3 mm thin. C. Chambered cover glass used to cut the block of solid MS+ media. D. Small piece of MS+ media block is cut off. E. MS+ media block is used to grease the chambered cover glass. F. 10-15 Arabidopsis seedlings are transferred to the chamber. G. Arabidopsis seedlings are covered with remaining block of media. H. Closing with the chambered cover glass lid.

  2. Real-time confocal imaging
    1. Prepare inverted confocal microscope for use [set lasers (for GFP-488); objectives - 40x/1.20 W; image size - x: 114.09 µm, y: 114.09 µm; zoom - 1.4; scan mode - plane, time series; pixel dwell - 1.27 µs; master gain - 652; digital gain - 1.5; digital offset - 0.00; pinhole 70 µm; filters - SP 555; beam splitters - MBS: MBS 405/488/555/639 DBS1: 492 nm].
      Note: Parameters are to be adjusted according to specimen.
    2. Mount chamber with seedlings.
    3. Activate position list (list of marked positions of LRP).
    4. Find LRP at the stage of interest and focus at the middle plane of LRP (xylem pole strand adjacent to primordia must be in focus, Figures 2 and 3, time point 0). Mark position of the LRP. Move to next LRP and repeat the procedure. Optimal number of LRPs to be monitored is ~20 per experiment. To examine the process of founder cell (FC) specification and subsequent developmental phases we recommend to bend roots manually (Marhavy et al., 2013), to mark position of the root bent, focus on two xylem poles (Figure 2) and perform time-lapse imaging using objective 20x. To examine LRP development from stage I onwards we recommend performing time-lapse imaging using objective either 40x or 60x (dry, water or oil immersion).
    5. Activate time series.
    6. Set time intervals for scanning (typically 20 to 30 min). Keep in mind that with increasing number of LRPs over 30 you have to increase interval of scanning.
    7. Run time-lapse imaging. Typically, to follow LRP organogenesis from stage I till stage IV ~ 12 to 16 h observation time is needed. (In Arabidopsis thaliana LRP organogenesis involves eight developmental stages characterized by highly coordinated pattern of cell divisions and differentiation. Stage I: two pericycle founder cells divide asymmetrically to form primordia composed of up to ten short initial cells. Stage II: Initial cells divide periclinally forming an inner layer and an outer layer. Stages III and IV: The outer layer divides periclinally and the primordium consists of three layers (stage III) and later the inner layer undergoes a similar division, such that four cell layers are visible (stage IV). Stages V to VIII: Expansion and further division of the four layers eventually results in the emergence of the young lateral root from the parent tissue (the overlying tissue of the primary root) at stage eight. For details see Malamy and Benfey (1997).
    8. Process pictures for image analysis. Export confocal images in tif or jpg format; open images in ImageJ; proceed images to stack; and Save As an Avi format.

  3. Confocal imaging analysis
    1. To quantify the intensity of fluorescent reporter signal ImageJ might be used. Export and save the confocal pictures in TIF format to analyze data using ImageJ. Or, alternatively, import the confocal stacks into imageJ by the BioFormats plugin. Open image in ImageJ; using segmented line (width of the line adjusted accordingly); mark the area of interest and use function “Mean” to calculate average intensity in pixels. Copy the results “Mean” to Excel program for further processing.
    2. To determine polar localization of fluorescently labeled membrane proteins CellseT software might be used (Pound et al., 2012). The software is suitable to evaluate cell and tissue geometry as well as to quantify the intensity of fluorescent marker signal. Follow CellseT instructions for further details.
    3. To evaluate dynamics of LRP development time lapse series images might be processed into AVI file (Windows media player or comparable program).


      Figure 2. Real-time analysis of founder cell establishment and early phases of LRP initiation using DR5pro::N7:Venus auxin reporter (Heisler et al., 2005). Accumulation of the nuclear DR5pro::N7:Venus signal in two pericycle cells at 120 min. indicates establishment of FCs (yellow arrows). Anticlinal divisions occurring between 240-480 min give rise LRP at developmental stage A composed of 5 initial cells (green arrows). To observe FC establishment root were manually bent prior monitoring. Objective 20x used for observation. Time in minutes (upper right corner) is relative to root bending. Red asterisks indicate two xylem poles. Scale bar: 40 μm


      Figure 3. Real-time analysis of the LRP development using auxin efflux carrier PIN1::PIN1-GFP reporter (Benkova et al., 2003). PIN1::PIN1-GFP localizes to cell membranes (white arrows) and is expressed from LRP stage I onwards. In time interval 400-480 min LRP which composes of 5 initial cells (pink arrows) undergoes periclinal division (orange arrows) and transits to developmental stage II. Time in minutes (upper right corner). Red asterisks mark two xylem poles. Scale bar: 30 μm

Recipes

  1. Seeds sterilization by ethanol
    1. Transfer seeds in 2 ml Eppendorf tubes (maximum volume of seeds to be sterilized per tube should not exceed 3 mm from bottom).
    2. Add 1 ml 70% EtOH (technical grade is sufficient), shake for 5 sec and leave seeds to sediment for 10 min.
    3. Remove 70% EtOH.
    4. Wash seeds in 100% EtOH under the clean bench.
    5. Dry seeds under the clean bench.
  2. ½ MS media (1 L)
    1. Add 10 g sucrose
    2. Add 2.3 g MS Salts
    3. Add 0.5 g MES
    4. Adjust pH to 5.9 (KOH)
    5. Add 8 g agar (1,000 ml bottle)
    6. H2O
  3. Growth conditions
    1. Seeds of Arabidopsis were plated on square plates filled with MS+ medium (45.5 ml).
    2. Stratification for 2 days at 4 °C in dark.
    3. Seedlings were grown on vertically oriented plates in growth chambers under a 16-h-light/8-h-dark photoperiod at 18 or 21 °C.

Acknowledgments

We thank Matyas Fendrych for critical reading and comments. This work was supported by the European Research Council with a Starting Independent Research grant (ERC-2007-Stg-207362-HCPO) and the Czech Science Foundation (GA13-39982S) to Eva Benková. The protocol was developed based on previously published work of De Rybel et al. (2010) and Laskowski et al. (2008).

References

  1. Abràmoff, M. D., Magalhães, P. J. and Ram, S. J. (2004). Image processing with ImageJ. Biophotonics Int 11(7): 36-43.
  2. Benková, E., Michniewicz, M., Sauer, M., Teichmann, T., Seifertová, D., Jürgens, G. and Friml, J. (2003). Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell 115(5): 591-602.
  3. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263(5148): 802-805.
  4. De Smet, I., White, P. J., Bengough, A. G., Dupuy, L., Parizot, B., Casimiro, I., Heidstra, R., Laskowski, M., Lepetit, M., Hochholdinger, F., Draye, X., Zhang, H., Broadley, M. R., Peret, B., Hammond, J. P., Fukaki, H., Mooney, S., Lynch, J. P., Nacry, P., Schurr, U., Laplaze, L., Benfey, P., Beeckman, T. and Bennett, M. (2012). Analyzing lateral root development: how to move forward. Plant Cell 24(1): 15-20. 
  5. De Rybel, B., Vassileva, V., Parizot, B., Demeulenaere, M., Grunewald, W., Audenaert, D., Van Campenhout, J., Overvoorde, P., Jansen, L., Vanneste, S., Moller, B., Wilson, M., Holman, T., Van Isterdael, G., Brunoud, G., Vuylsteke, M., Vernoux, T., De Veylder, L., Inze, D., Weijers, D., Bennett, M. J. and Beeckman, T. (2010). A novel aux/IAA28 signaling cascade activates GATA23-dependent specification of lateral root founder cell identity. Curr Biol 20(19): 1697-1706.   
  6. Dubrovsky, J. G. and Forde, B. G. (2012). Quantitative analysis of lateral root development: pitfalls and how to avoid them. Plant Cell 24(1): 4-14.
  7. Laskowski, M., Grieneisen, V. A., Hofhuis, H., Hove, C. A., Hogeweg, P., Maree, A. F. and Scheres, B. (2008). Root system architecture from coupling cell shape to auxin transport. PLoS Biol 6(12): e307.
  8. Malamy, J. E. and Benfey, P. N. (1997). Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124(1): 33-44.
  9. Marhavy, P., Bielach, A., Abas, L., Abuzeineh, A., Duclercq, J., Tanaka, H., Parezova, M., Petrasek, J., Friml, J., Kleine-Vehn, J. and Benkova, E. (2011). Cytokinin modulates endocytic trafficking of PIN1 auxin efflux carrier to control plant organogenesis. Dev Cell 21(4): 796-804.
  10. Marhavý, P., Vanstraelen, M., De Rybel, B., Zhaojun, D., Bennett, M. J., Beeckman, T. and Benková, E. (2013). Auxin reflux between the endodermis and pericycle promotes lateral root initiation. EMBO J 32(1): 149-158.
  11. Marhavý, P., Duclercq, J., Weller, B., Feraru, E., Bielach, A., Offringa, R., Friml, J., Schwechheimer, C., Murphy, A. and Benkova, E. (2014). Cytokinin controls polarity of PIN1-dependent auxin transport during lateral root organogenesis. Curr Biol 24(9): 1031-1037.
  12. Shaner, N. C., Patterson, G. H. and Davidson, M. W. (2007). Advances in fluorescent protein technology. J Cell Sci 120(Pt 24): 4247-4260.
  13. Pound, M. P., French, A. P., Wells, D. M., Bennett, M. J. and Pridmore, T. P. (2012). CellSeT: novel software to extract and analyze structured networks of plant cells from confocal images. Plant Cell 24(4): 1353-1361.

简介

植物在其胚后期内保持形成新的器官,例如叶,花,侧枝和根的能力。侧根(LR)源于几个获得创始细胞(FC)属性的周围细胞,经历一系列的背脊分裂,并产生几个短的初始细胞。起始后,发生协调的细胞分裂和分化,产生侧根原基(LRP)。原始状态继续生长,通过主根的皮层和表皮层出现,并且最终建立新的顶端分生组织,承担成熟侧根生长的责任[关于侧根根器官发生的各个阶段的详细描述,参见Malamy和Benfey(1997)]。为了检查这种高度动态的发育过程并且调查各种激素,遗传和环境因素在侧根器官发生的调节中的作用,基于实时成像的分析代表非常有力的工具(Laskowski等人 ,2008; De Smet等人,2012;Marhavý等人,2013;Marhavý等人,2014)。在这里,我们描述了用于实时侧根原基(LRP)分析的协议,其使得能够在原始器官发生期间监测特定基因表达和亚细胞蛋白定位的发生,以及评估遗传和环境扰动的影响对LRP器官发生的影响。

材料和试剂

  1. 表达绿色荧光蛋白(GFP)或类似报告物(YFP,RFP,CFP,mCherry等)的拟南芥幼苗(5-6天龄)(Chalfie等人, 1994; Shaner et al。,,2007)
  2. MilliQ水(H 2 O)
  3. 蔗糖(VWR International,目录号:27483.294)
  4. Murashige和Skoog基础盐混合物(MS盐)(Duchefa Biochemie,目录号:M0221.0050)
  5. 2- [N-吗啉代]乙磺酸(MES)(Duchefa Biochemie,目录号:M1503.0100)
  6. 氢氧化钾(KOH)(Merck KGaA,目录号:1.05021.1000)
  7. 琼脂(LAB M,目录号:MC029)
  8. 乙醇(EtOH)(Sigma-Aldrich,目录号:32221-2.5L)
  9. 种子用乙醇灭菌(见配方)
  10. ½MS介质(参见配方)
  11. 生长条件(参见配方)

设备

  1. 生长室生长植物材料
  2. 方形板120×120×17mm(Greiner Bio-One GmbH,目录号:688102)
  3. 玻璃盖玻璃(VWR,Kammerdeckgläser,Lab-Tek TM ,Nunc TM -eine kammer,目录号:734-2056)
  4. 反相共聚焦显微镜(Zeiss,型号:LSM 700)
    注意:需要完全电动的X,Y,Z扫描级来执行多位置时间推移实验。
  5. 目标:20x [适合于监测侧根原基(LRP)起始的早期阶段,图2],40x或60x(适于从阶段I向前监测LRP,图3)
  6. 用于GFP和其他荧光报道分子的荧光信号检测系统(Shaner等人,2007)

软件

  1. 操作共焦显微镜的软件
  2. ImageJ(Abramoff等人,2004)
  3. CellseT(Pound et al。,2012)
  4. Microsoft Excel

程序

  1. 样品制备(图1)
    1. 准备带盖的玻璃盖,用70%乙醇洗涤并干燥(图1A)。
    2. 将45ml MS + 培养基倒入正方形板中,并等待直到它 凝固(在室温下约40分钟;固体培养基应为2-3   mm薄)(图1B)。
    3. 使用带腔盖玻片切出固体MS介质块(图1C-D)。
    4. 从介质块切下〜3mm宽的条(图1C-D)。
    5. 使用带状介质润滑室内盖玻璃(图1E)。
    6. 在室内转移10-15棵幼苗,单个幼苗的根不能重叠(图1F)
    7. 用剩余的培养基覆盖幼苗(图1G)
    8. 用带盖的玻璃盖关闭(图1H)。


      图1.样品制备。 A.盖好的盖玻璃。 B.固体MS + 培养基2-3mm薄。 C.用于切割块的盖玻片 固体MS + 培养基。 D.小块MS + 媒体块被切断。 E. MS + 介质块用于润滑带腔盖玻片。 F. 10-15个拟南芥幼苗转移到室中。 拟南芥幼苗用剩余的培养基覆盖。 H.闭上   有盖的玻璃盖。

  2. 实时共焦成像
    1. 准备反向共聚焦显微镜使用[设置激光(GFP-488); 目标 - 40x/1.20 W; 图像尺寸-x:114.09μm,y:114.09μm; zoom - 1.4; 扫描模式 - 平面,时间序列; 像素驻留 - 1.27μs; 主增益 - 652; 数字增益 - 1.5; 数字偏移 - 0.00; 针孔70   μm; 过滤器 - SP 555; 光束分离器 - MBS:MBS 405/488/555/639 DBS1: 492nm]。
      注意:参数将根据样本进行调整。
    2. 安装室与幼苗。
    3. 激活位置列表(LRP的标记位置列表)。
    4. 在感兴趣的阶段找到LRP,并在中间平面处聚焦 LRP(邻近原基的木质部杆状链必须在焦点上,图2 和3,时间点0)。 标记LRP的位置。 移动到下一个LRP和 重复该过程。 要监测的LRP的最佳数量为〜20   实验。 检查创始人细胞(FC)规范的过程 和随后的发展阶段,我们建议手动弯曲根 (Marhavy等人,2013),以标记根弯的位置,重点在两个 木质部杆(图2),并使用物镜进行延时成像 20x。 为了检查从I期起的LRP发展,我们建议 使用物镜执行延时成像40x或60x(干, 水或油浸)。
    5. 激活时间系列。
    6. 设置时间 扫描间隔(通常为20至30分钟)。 记住与 增加LRP的数量超过30你必须增加间隔 扫描。
    7. 运行延时成像。 通常,遵循LRP 器官发生从I期到IV期〜12〜16 h观察时间 是必需的。 (In拟南芥)LRP器官发生涉及八个 发育阶段的特征在于高度协调的细胞模式   分裂和分化。 阶段I:两个周长创始细胞 不对称地分裂形成由多达十个短线组成的原基 初始细胞。 阶段II:初始细胞分裂形成 内层和外层。 阶段III和IV:外层 并且原始由三层组成(阶段 III)并且稍后内层经历类似的分割,使得 四个细胞层是可见的(阶段IV)。 阶段V至VIII:扩展和   进一步划分四层最终导致出现   的年轻侧根从母体组织(上覆组织 的主根)。 详情见Malamy和Benfey (1997)。
    8. 处理图片以进行图像分析。 出口共焦 图像以tif或jpg格式; 在ImageJ中打开图像; 继续图像 堆栈 和另存为Avi格式。

  3. 共焦成像分析
    1. 为了量化荧光报告信号ImageJ的强度可能 用过的。 导出并保存在TIF格式的共聚焦图片进行分析 数据使用ImageJ。 或者,或者,导入共焦堆栈 imageJ由BioFormats插件。 在ImageJ中打开图像; 使用分段 线(线的宽度相应调整); 标记感兴趣的区域   并使用函数"Mean"计算像素中的平均强度。 复制 结果"平均值"到Excel程序进行进一步处理。
    2. 至 确定荧光标记的膜蛋白的极性定位 可以使用CellseT软件(Pound等人,2012)。 软件是 适合评估细胞和组织几何以及量化   荧光标记信号的强度。 按照CellseT instructions   进一步详情。
    3. 评价LRP开发时间的动态 连续系列图像可能会被处理为AVI文件(Windows媒体 播放器或类似程序)。


      图2.实时分析 使用DR5pro :: N7:Venus生长素报道子(Heisler等人,2005)建立起始细胞建立和LRP起始的早期阶段。 累积 在120分钟时在两个周围细胞中的核DR5pro :: N7:Venus信号。 表示FC的建立(黄色箭头)。斜角分裂 发生在240-480分钟之间,在发育阶段A产生LRP 由5个初始细胞(绿色箭头)组成。观察FC建立 根在手动弯曲之前监测。目标20x用于 观察。以分钟为单位的时间(右上角)是相对于根 弯曲。红色星号表示两个木质部极。比例尺:40μm


      图3.使用生长素流出的LRP开发的实时分析 (PIN1 :: PIN1-GFP )的PIN1 :: PIN1-GFP 细胞膜(白色箭头),并从LRP表达 第一阶段。在时间间隔400-480分钟内,LRP组成为5 初始细胞(粉红色箭头)经历周壁分裂(橙色 箭头),并转入发育阶段II。时间(分钟) 右边角)。红色星号标记两个木质部极。比例尺:30μm

食谱

  1. 种子用乙醇灭菌
    1. 在2ml Eppendorf管中转移种子(每根管灭菌的种子的最大体积不应超过离底部3mm)。
    2. 加入1ml 70%EtOH(工业级是足够的),摇动5秒,并留下种子沉淀10分钟
    3. 除去70%EtOH。
    4. 在干净的工作台下在100%EtOH中洗涤种子
    5. 干种子在干净的长凳下。
  2. ½MS介质(1 L)
    1. 加入10克蔗糖
    2. 加入2.3克MS盐
    3. 添加0.5 g MES
    4. 将pH调节至5.9(KOH)
    5. 加入8克琼脂(1000毫升瓶)
    6. H 2 O
  3. 生长条件
    1. 将拟南芥种子铺在填充有MS +培养基(45.5ml)的方板上。
    2. 在4℃黑暗中分层2天
    3. 幼苗在生长中在垂直取向的板上生长 室在18或21℃的16-h光/8-h-暗光周期下。

致谢

我们感谢Matyas Fendrych的批评性阅读和评论。 这项工作由欧洲研究理事会支持,起始独立研究资助(ERC-2007-Stg-207362-HCPO)和捷克科学基金会(GA13-39982S)给EvaBenková。 该方案是基于先前发表的De Rybel等人(2010)和Laskowski等人(2008)的工作开发的。

参考文献

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引用:Marhavý, P. and Benková, E. (2015). Real-time Analysis of Lateral Root Organogenesis in Arabidopsis. Bio-protocol 5(8): e1446. DOI: 10.21769/BioProtoc.1446.
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