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Observation of Chloroplast Movement in Vallisneria
苦草属中叶绿体运动的观察   

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Journal of Integrative Plant Biology
Jan 2015

Abstract

Chloroplasts accumulate to weak light and escape from strong light. These light-induced responses have been known from the 19th century (Böhm, 1856). Up to now, many scientists have developed different methods to investigate these dynamic phenomena in a variety of plant species including the model plant Arabidopsis thaliana, a terrestrial dicot (Wada, 2013). Especially, a serial recording to trace the position of individual chloroplast for the analysis of its mode of movement is critical to understand the underlying mechanism. An aquatic monocot Vallisneria (Alismatales: Hydrocharitaceae, Figure 1A) has contributed over a century to such investigation (Senn, 1908; Zurzycki, 1955; Seitz, 1967), because Vallisneria leaves have rectangular parallelepiped-shaped epidermal cells aligned orderly in a monolayer (Figure 1B), providing an excellent experimental system for microscopic studies. Here we describe a protocol for the up-to-date time-lapse imaging procedures to analyze Vallisneria chloroplast movement. Using this and prototype procedures, the relevant photoreceptor systems (Izutani et al., 1990; Dong et al., 1995; Sakai et al., 2015), association with actin cytoskeleton (Dong et al., 1996; Dong et al., 1998; Sakai and Takagi 2005; Sakurai et al., 2005), and regulatory roles of Ca2+ (Sakai et al., 2015) have been strenuously investigated.


Figure 1. Vallisneria plant. A. Whole plant body; B. A bright-field image of adaxial epidermal cells containing a large number of chloroplasts; C. Culture facilities.

Keywords: Live cell imaging (活细胞成像), Organelle movement (细胞器运动), Aquatic plants (水生植物), Light-induced responses (光诱导反应)

Materials and Reagents

  1. Petri dish
  2. Vallisneria plants
    Note: Young plants of Vallisneria of 20-30 cm long were purchased at a tropical-fish store and cultured in buckets (20 L) filled with tap water and with a layer of soil at the bottom (Figure 1C). The plants were grown under 12 h light/12 h dark regime at 20-24 °C.
  3. Vaseline (Wako Pure Chemical Industries, Siyaku, catalog number: 224-00165 )
  4. Potassium Chloride (KCl) (Wako Pure Chemical Industries, Siyaku, catalog number: 163-03545 )
  5. Sodium chloride (NaCl) (Nacalai tesque, catalog number: 31320-05 )
  6. Calcium Nitrate Tetrahydrate [Ca(NO3)2] (Wako Pure Chemical Industries, Siyaku, catalog number: 039-00735 )
  7. Magnesium Nitrate Hexahydrate [Mg(NO3)2] (Wako Pure Chemical Industries, Siyaku, catalog number: 134-00255 )
  8. PIPES (DOJINDO, catalog number: 345-02225 )
  9. Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, Siyaku, catalog number: 197-02125 )
  10. Artificial pond water (APW) (see Recipes)
  11. 100x APW mixture (see Recipes)
  12. 200 mM PIPES-NaOH (see Recipes)

Equipment

  1. Fluorescent lamps (National, model: FL20S-PG )
  2. Vacuum pump (ULVAC KIKO, model: DTU-20 )
  3. Microscope (OLYMPUS, model: BX50 )
  4. Neutral-density filters (Fujifilm Corporation)
  5. Interference filter (Toshiba, model: KL-55 )
  6. Cut-off filter (Toshiba, model: O-54 )
  7. Quantum sensor (LI-COR Biosciences, model: LI-190SA )
  8. Data logger (LI-COR Biosciences, model: LI-1400 )
  9. Charge-coupled device camera (QImaging, model: RETIGA 2000RV )

Procedure

  1. Plant material and pretreatment of specimens
    1. Young plants of Vallisneria of 20-30 cm long were purchased at a tropical-fish store and cultured in buckets (20 L) filled with tap water and with a layer of soil at the bottom (Figure 1C). The plants were grown under 12 h light/12 h dark regime at 20-24 °C. The light source was a bank of 20 W fluorescent lamps (50 μmol m-2 s-1, Figure 1C). The following steps A2-7 were carried out at 20-24 °C.
    2. At the end of the light period, healthy leaf segments of 10 cm long taken between 30 and 40 cm from the base of shoots, which had grown over 100 cm, were cut into smaller pieces about 4 mm in length and were put into artificial pond water (APW).
    3. The air trapped in the intercellular spaces was evacuated using a desiccator connected with a rotary vacuum pump for 20 min.
    4. Each 4 mm length leaf piece was further cut open and separated into adaxial half and abaxial half (Figure 2A; Sakurai et al., 2005).
    5. Each adaxial half was floated in a separate vessel with fresh APW and kept under another cycle of 12 h light/12 h dark regime.
    6. The leaf piece was mounted on a glass slide with a coverslip so that the epidermal cells could be seen from above, and secured with white Vaseline (Figure 2B).
    7. Each specimen was immersed in fresh APW in a Petri dish (Figure 2B) and kept under weak white light (2 μmol m-2 s-1) from the fluorescent lamps for 6 h. They were further kept in complete darkness for another 12-20 h.

  2. Time-lapse light microscopy for observation of chloroplast movement
    1. The dark-adapted specimen was irradiated with blue light (488 nm, 80 μmol m-2 s-1) on the stage of a microscope from above through the objective lens, using the epi-illumination system with a dichroic mirror and a mercury lamp (Figure 2C). The fluence rate of blue light was adjusted manually with neutral-density filters.
    2. The specimen was observed with dim green light (550 nm, 10 μmol m-2 s-1) produced by combination of an interference filter (the maximal transmission is 35%) and a cut-off filter, according to Izutani et al. (1990), applied from below. Although many of the plant photoreceptors are known to absorb light in green region, we confirmed that light of 550 nm at this fluence rate barely affects chloroplast movement and cytoplasmic motility in Vallisneria (Izutani et al., 1990; Takagi et al., 2003).
      Note: The fluence rate of monochromatic light was measured with a quantum sensor and data logger.
    3. Before and during the actinic blue-light irradiation, optical images were captured digitally with a charge-coupled device camera at 5-sec intervals (Figure 2C).


      Figure 3. Observation of chloroplast movement. A. Scheme to prepare the adaxial leaf piece. Double-headed arrow; the longitudinal axis of leaf. Shadowed plane; cutting plane to make the adaxial and abaxial halves of leaf piece. B. Preparation of the specimen before the dark adaptation. APW; artificial pond water. C. Optical system. BL; blue light (488 nm, 80 μmol m-2 s-1), GL; green light (550 nm, 10 μmol m-2 s-1), ND; neutral density, CCD; charge-coupled device, PC; personal computer. D. Serial images of an epidermal cell arranged at 1-min intervals. At 0 min, the cell was exposed to blue light. Bar, 10 μm.

    4. The number and position of chloroplasts on each digital image were recorded for movement analyses. A series of digital images can also be assembled into a movie (Video 1).

      Video 1. Avoidance movement of chloroplasts in Vallisneria. A series of 5-sec interval images for 1 min before and 19 min during the blue-light irradiation were stacked into a movie (12 frame per sec). Bright flash in the movie means the start of the irradiation. Bar, 10 μm.

    5. When necessary, before the start of observation, specimens were perfused with APW containing various kinds of chemicals, for example, inhibitors of photosynthetic electron transport, cytoskeleton-disrupting reagents, and intracellular-calcium-transient inhibitors. All the procedures were carried out under dim green light (0.03 μmol m-2 s-1).

Notes

  1. Vallisneria leaf cells are extraordinarily sensitive to touch and chemicals. Such unintended stimuli should be excluded as much as possible.

Recipes

  1. Artificial pond water (APW)
    APW could be prepared in large volume and stored at room temperature.
    To prepare 10 L of APW, mix
    100 ml 100x APW mixture
    100 ml 200 mM PIPES-NaOH (pH 7.0)
    800 ml of deionized water
  2. 100x APW mixture
    100x APW could be stored at 4 °C.
    To prepare 1 L of 100x APW mixture, mix
    3.73 g KCl (final conc. 50mM)
    1.17 g NaCl (final conc. 20 mM)
    1.64 g Ca(NO3)2 (final conc. 10 mM)
    1.48 g Mg(NO3)2 (final conc. 10 mM)
  3. 200 mM PIPES-NaOH (pH 7.0)
    60.48 g PIPES solved in 1 L of deionized water, adjusted to pH 7.0 with NaOH
    It can be stored at 4 °C.

Acknowledgments

We thank Mr. Motoyuki Iida for taking pictures of culture facilities of Vallisneria plants (Figure 1C).

References

  1. Böhm, J. A. (1856). Beiträge zur näheren Kenntnis des Chlorophylls. S. B. Akad. Wiss. Wien, Math.-nat. Kl. 22: 479-498.
  2. Dong, X. J., Takagi, S. and Nagai, R. (1995). Regulation of the orientation movement of chloroplasts in epidermal cells of Vallisneria: cooperation of phytochrome with photosynthetic pigment under low-fluence-rate light. Planta 197: 257-263.
  3. Dong, X. J., Ryu, J. H., Takagi, S. and Nagai, R. (1996) Dynamic changes in the organization of microfilaments assosiated wite the photocontrolled motility of chloroplasts in epidermal cells of Vallisneria. Protoplasma 195: 18-24.
  4. Dong, X. J., Nagai, R. and Takagi, S. (1998) Microfilaments anchor chloroplasts along the outer periclinal wall in Vallisneria epidermal cells through cooperation of Pfr and Photosynthesis. Plant & Cell Physiology 39: 1299-1306.
  5. Izutani, Y., Takagi, S. and Nagai, R. (1990). Orientation movements of chloroplasts in Vallisneria epidermal cells: Different effects of light at low- and high-fluence rate. Photochem Photobiol 51: 105-111.
  6. Sakai, Y. and Takagi, S. (2005). Reorganized actin filaments anchor chloroplasts along the anticlinal walls of Vallisneria epidermal cells under high-intensity blue light. Planta 221(6): 823-830.
  7. Sakai, Y., Inoue, S., Harada, A., Shimazaki, K. and Takagi, S. (2015). Blue-light-induced rapid chloroplast de-anchoring in Vallisneria epidermal cells. J Integr Plant Biol 57(1): 93-105.
  8. Sakurai, N., Domoto, K. and Takagi, S. (2005). Blue-light-induced reorganization of the actin cytoskeleton and the avoidance response of chloroplasts in epidermal cells of Vallisneria gigantea. Planta 221(1): 66-74.
  9. Seitz, K. (1967). Wirkungsspektren für die Starklichtbewegung der Chloroplasten, die Photodinese und die lichtabhängige Viskositätsänderung bei Vallisneria spiralis. Z Pflanzenphysiol 56:246-261.
  10. Senn, G. (1908). Die Gestalts- und Lageveränderung der Pflanzen-Chromatophoren. Verlag von Wilhelm Engelmann, Leipzig (in Germany).
  11. Takagi, S., Kong, S. G., Mineyuki, Y. and Furuya, M. (2003). Regulation of actin-dependent cytoplasmic motility by type II phytochrome occurs within seconds in Vallisneria gigantea epidermal cells. Plant Cell 15(2): 331-345.
  12. Wada, M. (2013). Chloroplast movement. Plant Sci 210: 177-182.
  13. Zurzycki, J. (1955). Chloroplasts arrangement as a factor in photosynthesis. Acta Soc Bot Pol 24: 27-63.

简介

叶绿体累积到弱光并从强光中逃逸。这些光诱发的反应从19世纪就已知(Böhm,1856)。到目前为止,许多科学家已经开发了不同的方法来研究各种植物物种中的这些动态现象,包括模拟植物拟南芥(Arabidopsis thaliana),一种陆生双子叶植物(Wada,2013)。特别是,跟踪单个叶绿体的位置,用于分析其运动模式的连续记录对于理解底层机制至关重要。水生单子叶植物Vallisneria(Alismatales:Hydrocharitaceae,图1A)在一个世纪以来已经对这种研究作出贡献(Senn,1908; Zurzycki,1955; Seitz,1967),因为Vallisneria 叶具有在单层中有序排列的长方形表皮细胞(图1B),为显微镜研究提供了优良的实验系统。在这里我们描述了一个协议为最新的时间推移成像程序来分析Vallisneria 叶绿体运动。使用这个和原型程序,相关的光感受器系统(Izutani等人,1990; Dong等人,1995; Sakai等人, ,2015),与肌动蛋白细胞骨架的关联(Dong等人,1996; Dong等人,1998; Sakai和Takagi 2005; Sakurai等人,/sh>,2005),并且已经对Ca 2 + (Sakai等人,2015)的调节作用进行了深入研究。

< img src ="/attached/image/20151103/20151103015430_3278.jpg" alt ="" height ="269" width ="302"/>
图1. Vallisneria 。 A.整个植物体; B.含有大量叶绿体的近轴表皮细胞的明场图像; C.文化设施。

关键字:活细胞成像, 细胞器运动, 水生植物, 光诱导反应

材料和试剂

  1. 培养皿
  2. < em> Vallisneria植物
    注意:20-30厘米长的Vallisneria的幼苗在热带鱼店购买,并在装有自来水和底部一层土壤的桶(20L)中培养(图1C)。植物在12-24小时光照/12小时黑暗条件下在20-24℃生长。
  3. 凡士林(Wako Pure Chemical Industries,Siyaku,目录号:224-00165)
  4. 氯化钾(KCl)(Wako Pure Chemical Industries,Siyaku,目录号:163-03545)
  5. 氯化钠(NaCl)(Nacalai tesque,目录号:31320-05)
  6. 硝酸钙四水合物[Ca(NO 3)2](Wako Pure Chemical Industries,Siyaku,目录号:039-00735)
  7. 硝酸镁六水合物[Mg(NO 3)2](Wako Pure Chemical Industries,Siyaku,目录号:134-00255)
  8. PIPES(DOJINDO,目录号:345-02225)
  9. 氢氧化钠(NaOH)(Wako Pure Chemical Industries,Siyaku,目录号:197-02125)
  10. 人工池水(APW)(参见配方)
  11. 100x APW混合物(参见配方)
  12. 200 mM PIPES-NaOH(参见配方)

设备

  1. 荧光灯(国家,型号:FL20S-PG)
  2. 真空泵(ULVAC KIKO,型号:DTU-20)
  3. 显微镜(OLYMPUS,型号:BX50)
  4. 中性密度过滤器(Fujifilm Corporation)
  5. 干涉滤波器(东芝,型号:KL-55)
  6. 截止滤波器(东芝,型号:O-54)
  7. 量子传感器(LI-COR Biosciences,型号:LI-190SA)
  8. 数据记录器(LI-COR Biosciences,型号:LI-1400)
  9. 电荷耦合器件相机(QImaging,型号:RETIGA 2000RV)

程序

  1. 植物材料和标本的预处理
    1. 在a。处购买20-30cm长的
    2. 在......的最后 ?光照期,健康叶片段10厘米长采取30之间 并且距离生长超过100cm的枝条的根部40cm ?成为长度约4mm的小块并放入人工 池塘水(APW)。
    3. 被截留在细胞间隙中的空气 ?使用与旋转真空泵连接的干燥器抽空20小时 ?min。
    4. 每个4mm长的叶片进一步切开 分成近轴一半和下半轴(图2A; Sakurai等人, ?2005)。
    5. 将每个近轴半部浮在与之分开的容器中 新鲜APW并保持在12小时光照/12小时黑暗状态的另一个循环下。
    6. 将叶片用盖玻片安装在载玻片上 ?表皮细胞可以从上面看到,并用白色固定 凡士林(图2B)。
    7. 将每个样品浸入新鲜APW中 ?培养皿(图2B)并保持在来自荧光灯的弱白光(2μmolm -2 s -1 s -1)下6小时。他们进一步保持 完全黑暗另外12-20小时。

  2. 用于观察叶绿体运动的延时光学显微镜
    1. 用蓝光(488nm,80μm)照射暗适应样品 μmolm < - s s -1 )在显微镜的台上从上方通过 物镜,使用具有二向色镜的落射照明系统 ?和汞灯(图2C)。蓝光的流量率为 使用中性密度滤光片手动调整。
    2. 样品是 用浅绿色光(550nm,10μmolm -2 s -1 s -1 -1 )观察到,其通过 组合干扰滤波器( 最大透射率为35%)和根据Izutani等人(1990)从下面应用的截止滤光片。虽然 许多植物光感受器已知吸收绿色光 区域,我们证实在这个能量密度下550nm的光几乎没有 影响Vallisneria中的叶绿体运动和细胞质运动(Izutani等人,1990; Takagi等人,2003)。
      注意:流量率 的单色光用量子传感器和数据记录仪测量。
    3. 之前 ?并且在光化性蓝光照射期间,光学图像 使用电荷耦合器件相机以5秒数字捕获 间隔(图2C)。


      图3.叶绿体的观察 A.准备近轴叶片的方案。双头 箭头;叶的纵轴。阴影平面;切割平面 使叶片的近轴和下半部。 B.准备 ?样本在暗适应之前。 APW;人工池塘水。 C。 光学系统。 BL;蓝光(488nm,80μmolm -2 s -1 s -1),GL;绿色 光(550nm,10μmolm -2 s -1 s -1),ND;中性密度,CCD; 电荷耦合器件,PC;个人电脑。 D.序列图像的 表皮细胞以1分钟间隔排列。在0分钟时,细胞是 暴露于蓝光。柱:10μm

    4. 数字和位置 ?记录每个数字图像上的叶绿体用于运动分析。 ?一系列数字图像也可以组合成电影(视频 1)。

      视频1.在Vallisneria中避免叶绿体运动。一系列5秒间隔的图像,1分钟前和19分钟 将蓝光照射堆叠成电影(每秒12帧)。 ?电影中的明亮闪光表示开始照射。酒吧; 10 μm
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      获取Adobe Flash Player

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    5. 必要时,在开始观察之前,标本 用含有各种化学品的APW灌注,例如, 光合电子传递的抑制剂,细胞骨架破坏 ?试剂和细胞内钙瞬变抑制剂。一切 程序在浅绿色光(0.03μmolm -2 -2 s -1 s -1)下进行。

笔记

食谱

  1. 人工池水(APW)
    APW可以大量制备并在室温下储存。
    要准备10升APW,混合
    100 ml 100x APW混合物
    100ml 200mM PIPES-NaOH(pH7.0) 800ml去离子水
  2. 100x APW混合物
    100x APW可以在4℃下储存。
    为了制备1L的100×APW混合物,混合
    3.73g KCl(最终浓度50mM) 1.17g NaCl(终浓度20mM) 1.64g Ca(NO 3)2(最终浓度10mM)
    1.48g Mg(NO 3)2(最终浓度为10mM)
  3. 200mM PIPES-NaOH(pH7.0) 60.48g溶于1L去离子水中的PIPES,用NaOH调节至pH7.0 它可以存储在4°C。

致谢

我们感谢Motoyuki Iida先生拍摄Vallisneria植物的培养设施的照片(图1C)。

参考文献

  1. B?hm,J.A。(1856)。 Beitr?gezurn?herenKenntnis des Chlorophylls。 S. B. Akad。 Wiss。 Wien,数学。 Kl。 22:479-498。
  2. Dong,X.J.,Takagi,S。和Nagai,R。(1995)。 对Vallisneria的表皮细胞中叶绿体的取向运动的调节:植物色素的合作与光合色素在低通量光下。 197:257-263。
  3. Dong,XJ,Ryu,JH,Takagi,S.and Nagai,R。(1996) 的表皮细胞中的叶绿体的光控制运动性。 195:18-24。
  4. Dong,XJ,Nagai,R。和Takagi,S。(1998)微丝锚定叶绿体沿着Vallisneria表皮细胞中的外周壁,通过Pfr和光合作用的合作。 Cell Physiology 39:1299-1306。
  5. Izutani,Y.,Takagi,S。和Nagai,R。(1990)。 叶绿体在 Vallisneria epidermal cells:Different effects of light at low and high-fluence rate。 Photochem Photobiol 51:105-111。
  6. Sakai,Y.and Takagi,S。(2005)。 重组的肌动蛋白丝锚定沿着Vallisneria的表皮细胞的背侧壁的叶绿体高强度蓝光。 Planta 221(6):823-830。
  7. Sakai,Y.,Inoue,S.,Harada,A.,Shimazaki,K.and Takagi,S。(2015)。 蓝光诱导的Vallisneria中的快速叶绿体解旋表皮细胞。 Integral Plant Biol 57(1):93-105。
  8. Sakurai,N.,Domoto,K.and Takagi,S。(2005)。 蓝光诱导的肌动蛋白细胞骨架的重组和叶绿体在表皮细胞中的回避反应em> Vallisneria gigantea 。 Planta 221(1):66-74。
  9. Seitz,K。(1967)。 Wirkungsspektrenfürdie Starklichtbewegung der Chloroplasten,die Photodinese und dielichtabh?ngigeViskosit?ts?nderungbei
  10. Senn,G.(1908)。 Die Gestalts- undLagever?nderungder Pflanzen-Chromatophoren。 (位于德国)。
  11. Takagi,S.,Kong,S.G.,Mineyuki,Y。和Furuya,M。(2003)。 通过II型植物色素调节肌动蛋白依赖性细胞质运动发生在几秒钟内Vallisneria gigantea 表皮细胞。 植物细胞 15(2):331-345。
  12. Wada,M。(2013)。 叶绿体运动 植物科学 210:177-182 。
  13. Zurzycki,J。(1955)。叶绿体排列作为光合作用的一个因素。 24:27-63。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sakai, Y. and Takagi, S. (2015). Observation of Chloroplast Movement in Vallisneria. Bio-protocol 5(21): e1646. DOI: 10.21769/BioProtoc.1646.
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