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Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. This protocol, adapted from Zhang et al. (2011), describes a procedure for the isolation of rice protoplasts from green tissue and shows an efficient and rapid method for isolation of nuclei form these protoplasts which are commonly used in a variety of experimental procedures including the isolation of high-molecular-weight DNA (Watson and Thompson, 1986), in vitro DNA synthesis (Roman, 1980), isolation of labeled transcripts for differential screening of cDNA libraries (Somssich et al., 1989), preparation of nuclear extracts for in vitro transcription systems (Roberts and Okita, 1991), isolation of nuclear proteins (Harrison et al., 1992) and studies of protein targeting to the nucleus (Hicks and Raikhel, 1993).

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An Efficient Procedure for Protoplast Isolation from Mesophyll Cells and Nuclear Fractionation in Rice
水稻叶肉细胞原生质体分离和细胞核分级分离的有效方法

植物科学 > 植物细胞生物学 > 细胞分离
作者: Mehdi Jabnoune
Mehdi JabnouneAffiliation: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland
For correspondence: Mehdi.Jabnoune@unil.ch
Bio-protocol author page: a2028
David Secco
David SeccoAffiliation 1: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland
Affiliation 2: Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, Australia
Bio-protocol author page: a2029
Cécile Lecampion
Cécile LecampionAffiliation: Laboratory of Plant Genetics and Biophysics, CEA Institute of Environmental Biology and Biotechnology, Aix Marseille University, Faculty of Sciences, Marseille, France
Bio-protocol author page: a2030
Christophe Robaglia
Christophe RobagliaAffiliation: Laboratory of Plant Genetics and Biophysics, CEA Institute of Environmental Biology and Biotechnology, Aix Marseille University, Faculty of Sciences, Marseille, France
Bio-protocol author page: a2031
Qingyao Shu
Qingyao ShuAffiliation: Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou, China
Bio-protocol author page: a2032
 and Yves Poirier
Yves PoirierAffiliation: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland
Bio-protocol author page: a2033
Vol 5, Iss 5, 3/5/2015, 5464 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1412

[Abstract] Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. This protocol, adapted from Zhang et al. (2011), describes a procedure for the isolation of rice protoplasts from green tissue and shows an efficient and rapid method for isolation of nuclei form these protoplasts which are commonly used in a variety of experimental procedures including the isolation of high-molecular-weight DNA (Watson and Thompson, 1986), in vitro DNA synthesis (Roman, 1980), isolation of labeled transcripts for differential screening of cDNA libraries (Somssich et al., 1989), preparation of nuclear extracts for in vitro transcription systems (Roberts and Okita, 1991), isolation of nuclear proteins (Harrison et al., 1992) and studies of protein targeting to the nucleus (Hicks and Raikhel, 1993).

[Abstract]

Materials and Reagents

  1. 12-day-old plants with 1-2 cm leaves before flowering
  2. Cellulase RS (Yakult Pharmaceutical)
  3. Macerozyme R-10 (Yakult Pharmaceutical)
  4. D-Mannitol (Sigma-aldrich, catalog number: M4125 )
  5. KCl (Sigma-aldrich, catalog number: P9333 )
  6. MES hydrate (Sigma-aldrich, catalog number: M8250 )
  7. CaCl2 (Sigma-aldrich, catalog number: C1016 )
  8. BSA Bovine serum albumin (Sigma-aldrich, catalog number: A2153 )
  9. Spermidine (Sigma-aldrich, catalog number: S2626 )
  10. EDTA (Sigma-aldrich, catalog number: EDS )
  11. Sucrose (Sigma-aldrich, catalog number: 84097 )
  12. Triton X-100 (Sigma-aldrich, catalog number: X100 )
  13. DTT (Sigma-aldrich, catalog number: 43815 )
  14. Enzyme solution (ES) (see Recipes)
  15. Washing and incubation solution (WS1) (see Recipes)
  16. Washing solution (WS2) (see Recipes)
  17. MMG solution (see Recipes)
  18. Nuclei isolation solution (NIB) (see Recipes)

Equipment

  1. Razor blades (No18 sterile stainless steel scalpel balde) (Swann Morton, catalog number: 0323 )
  2. Micro-dissecting forceps (Sigma-aldrich, catalog number: F3767 )
  3. Nunc petri dishes (diam 90 mm × H 15 mm, surface area size 58 cm2, vented) (Sigma-aldrich, catalog number: Z717223 )
  4. 50 ml conical tubes (Bioland scientific, catalog number: A02-04 )
  5. Orbitron Rotator I (115 V) (Boekel Scientific)
  6. Nylon mesh CX-60 (120 µm) and CX-400 (38 µm) (Carolina Biological Supplies, catalog number: 65-2222N )
  7. Glass funnel
  8. Hemocytometer [Mfr Part Number: 3100; Cell depth: 0.100mm +/- 2% (1/10mm); Volume: 0.1 Microliter] (PGC Scientific, catalog number: 1907353 ).
  9. Stereo Microscope (Olympus, catalog number: SZ51 )
  10. 25G5/8 gauge needle (Kendall, catalog number: KND8881511235 )

Procedure

  1. Prepare protoplast
    1. Weigh 1 g cut leaves, removing the petiole (see Figure 1A).
    2. On the flat glass, chop leaves into 0.5-1 mm strips with fresh razor blades or with flamed scissors without shredding.
      Note: Strips don’t need to be very small to avoid the accumulation of excessive cell debris on the filter. Leaves can be easily cut using a plate surface like a petri dish.
    3. Using a forceps, transfer immediately the plant material to a deep petri dish filled with washing solution WS1 (enough to cover the strips) and after gentle shaking (1-3 min by hand) remove all liquid by pipetting.
    4. Add freshly prepared enzyme solution (ES). 5 ml of ES is generally used per 1 g of cut leaf material. Incubate 4-5 h in the dark, at room temperature, with gentle shaking (60-80 rpm) in the rotator.

  2. Harvesting protoplasts
    1. After the enzymatic digestion, transfer the digestion mixture to a 50 ml Falcon tube, add an equal volume of WS2 solution and shake vigorously by hand for 10 sec.
    2. Release the protoplasts from undigested leaf tissue by filtering the mixture through 120 µm nylon mesh sieves on glass funnel into a round bottom tubes. Two sequential filtering steps, first using CX-60 (120 µm) and then CX-400 (38 µm), are sufficient for protoplasts being isolated from leaf tissue.
      Note: We usually go through a 3 step filtration procedure when working with suspension culture material since the sieves clog quickly. The sieves are used in the order CX-60, CX-150 (104 µm) and finally CX-400.
    3. Wash the protoplasts 3-5 times using 2 ml WS2 solution.
    4. Spin, after each washing, the tube for 10 min at 50 x g. Discard supernatant and wash protoplasts again with 2 ml WS2 solution.
    5. After washing, collect the pellets by centrifugation at 50 x g for 3 min with a swinging bucket rotor and re-suspend in MMG solution for counting or in NIB solution for the Isolation of nuclei.

  3. Evaluating the quality (viability) and counting the protoplasts

    Evaluating the quality
    The protoplast isolation procedure often damages or kills a percentage of cells. To determine the viability of the cells, we used fluorescein diacetate (FDA) staining to estimate the percentage of viable protoplast (green protoplast) in a preparation.
    1. Mix 0.25 ml of 0.05% fluoriscin diacetate (dissolved in acetone) with 20 ml of the appropriate isolation medium to make the ‘’stain’’ solution.
    2. Pipete 0.25 ml of the protoplast suspension into 1.0 ml of the stain solution.
    3. Swirl gently to mix and mount some of the protoplasts on a glass slide. First, look at the protoplasts using a 10 or 20 x objectives lens. Now view the protoplasts using the UV source (420- to 490 mn excitation). In the rice green tissue protoplasts, the chloroplasts could be easily identified by their typical chlorophyll autofluorescence under a confocal microscope, while they could not be clearly observed in etiolated protoplasts.
    4. Observe at least 50 protoplasts and calculate the percentage of living cells. Multiply the estimate of number of protoplasts by this percentage to obtain an estimate of the number of living protoplasts.
      Example: 5 x 105 protoplasts per milliliter multiplied by 0.95 viable protoplasts, equals 4.75 x 105 living protoplasts per milliliter. A good isolation will yield between 90 and 95 % viable protoplasts.

    Counting protoplasts
    1. Place a cover glass over the hemocytometer counting chambers.
    2.  Gently mix the protoplast sample thoroughly to insure a uniform distribution of the cells and withdraw a sample with a Pasteur pipet.
      Note: Dilute the sample if the cell count is greater than 107 cells/ml.
    3. Examine at the appropriate magnification (100 x should work for the protoplasts) and count the number of protoplasts in four, appropriately-sized grids in each chamber.
      Note: Include all cells touching the middle line along the left or top margin of a square, but not the right or bottom. At 100 x, a 1 mm3 square will fill the field.
    4. Count a total of 16 grids from 4 chambers
    5. Record the data in table 1 & 2 and calculate the number of cells per ml using the following equation (to be significant at the 5% level of confidence, the 8 counts cannot deviate more than 7% from the mean [Klein and Klein (1970)]): Cells/ml = Cells/square x 1 square/volume (mm3) x 1000 mm3/ ml x dilution factor

      Table 1: Hemocytometer data
      Grid size counted
      0.1 mm x _____ mm x _____mm
      volume of grid counted (mm3)

      Dilution (if any)


      Table 2: Protoplast density in a partially purified suspension of Red cabbage protoplasts measured with a hemocytometer
      Grid
      protoplasts grid-1
      protoplasts ml-1
      1


      2


      3


      4


      5


      6


      7


      8


      9


      10


      11


      12


      13


      14


      15


      16


      mean +/- std



  4. Isolation of nuclei
    1. Re-suspend pellet at 106/ml of cold NIB in 2 ml eppendorf tube or bigger tubes.
    2. Put on ice for 7 min.
    3. Fill a large syringes with protoplasts and pass it 4 times through a 25G5/8 gauge needle.
    4. Filter the lysate through 20 µm filter into a new tube. Centrifuge at 400 x g for 10 min.
    5. Discard supernatant
    6. Re-suspend in NIB + glycerol or SDS solution for SDS gel electrophoresis.

Representative data



Figure 1. Isolation of protoplasts from rice green tissue. A. A representative healthy 12-day-old rice seedling used for protoplast isolation. Black markers indicate the optimal sections of seedlings (stem and sheath) yielding protoplasts. B. Cut strips were digested by enzyme solution (ES). C. Protoplasts collected after the filtration step. D. Image of protoplasts obtained using a Leica digital camera under an Olympus microscope with a 40x objective.

Recipes

  1. Enzyme solution (ES)
    1.5% (w/v) cellulase R10
    0.4% (w/v) macerozyme R10
    0.4 M mannitol
    20 mM KCl
    20 mM MES (pH 5.5)
    Heat to 55 °C for 10 min, then cool to room temperature before adding 10 mM CaCl2
    0.1% BSA
    Note: For longer-term storage, the enzyme powder should be aliqouted and stored at -20 °C in tightly capped plastic tubes. For regular use, a frozen 5 g tube is "thawed" and stored at 4 °C.
  2. Washing and incubation solution (WS1)
    0.5 M mannitol
    4 mM MES-KOH (pH 5.5)
    20 mM KCl
  3. Washing solution (WS2)
    154 mM NaCl
    125 mM CaCl2
    5 mM KCl
    2 mM MES at pH 5.7
  4. MMG solution
    0.4 M mannitol
    15 mM MgCl2
    4 mM MES at pH 5.7
    Note: Solutions WS1, WS2 and MMG can be prepared before the experiment, autoclaved and stored at 4 °C. Just check the pH before using them.
  5. Nuclei isolation solution (NIB)
    0.1 mM spermidine
    10 mM MES-KOH (pH 5.5)
    2.5 mM EDTA
    10 mM NaCl
    10 mM KCl
    0.2 M sucrose
    0.15% Triton X-100
    2.5 mM DTT (add fresh just before using)

Acknowledgments

This work was funded by the Swiss National Foundation grant (31003A-12293 and 31003A-138339) and the Sino-Swiss Science and Technology Cooperation Program (IZLCZ3 123946 to YP and 2009DFA32040 to QS).

References

  1. Harrison, M. J., Choudhary, A. D., Lawton, M. A., Lamb, C. J. and Dixon, R. A. (1992). Analysis of defense gene transcriptional regulation. In: Bowles, D. J. (ed). Molecular Plant Pathology: A Practic Approach. Oxford University Press, pp 147-162.
  2. Hicks, G. R. and Raikhel, N. V. (1993). Specific binding of nuclear localization sequences to plant nuclei. Plant Cell 5(8): 983-994.
  3. Roberts, M. W. and Okita, T. W. (1991). Accurate in vitro transcription of plant promoters with nuclear extracts prepared from cultured plant cells. Plant Mol Biol 16(5): 771-786.
  4. Roman, R. (1980). Replication of DNA by nuclei isolated from soybean suspension cultures. Plant Physiol 66(4): 726-730.
  5. Somssich, I. E., Bollmann, J., Hahlbrock, K., Kombrink, E. and Schulz, W. (1989). Differential early activation of defense-related genes in elicitor-treated parsley cells. Plant Mol Biol 12(2): 227-234.
  6. Watson, J. C. and Thompson, W. F. (1986). Purification and restriction endonuclease analysis of plant nuclear DNA. Method Enzymol 118:57-79.
  7. Zhang, Y., Su, J., Duan, S., Ao, Y., Dai, J., Liu, J., Wang, P., Li, Y., Liu, B., Feng, D., Wang, J. and Wang, H. (2011). A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7(1): 30.

材料和试剂

  1. 开花前有1-2厘米叶的12日龄植株
  2. 纤维素酶RS(Yakult Pharmaceutical)
  3. Macerozyme R-10(Yakult Pharmaceutical)
  4. D-甘露醇(Sigma-aldrich,目录号:M4125)
  5. KCl(Sigma-aldrich,目录号:P9333)
  6. MES水合物(Sigma-aldrich,目录号:M8250)
  7. CaCl 2(Sigma-aldrich,目录号:C1016)
  8. BSA牛血清白蛋白(Sigma-aldrich,目录号:A2153)
  9. 亚精胺(Sigma-aldrich,目录号:S2626)
  10. EDTA(Sigma-aldrich,目录号:EDS)
  11. 蔗糖(Sigma-aldrich,目录号:84097)
  12. Triton X-100(Sigma-aldrich,目录号:X100)
  13. DTT(Sigma-aldrich,目录号:43815)
  14. 酶溶液(ES)(参见配方)
  15. 洗涤和孵育溶液(WS1)(参见配方)
  16. 洗涤液(WS2)(参见配方)
  17. MMG解决方案(参见配方)
  18. 核隔离溶液(NIB)(参见配方)

设备

  1. 剃刀刀片(No18无菌不锈钢手术刀)(Swann Morton,目录号:0323)
  2. 微解剖钳(Sigma-aldrich,目录号:F3767)
  3. Nunc培养皿(直径90mm×H 15mm,表面积大小58cm 2,通气)(Sigma-aldrich,目录号:Z717223)
  4. 50ml锥形管(Bioland scientific,目录号:A02-04)
  5. 轨道旋转器I(115V)(Boekel Scientific)
  6. 尼龙网CX-60(120μm)和CX-400(38μm)(Carolina Biological Supplies,目录号:65-2222N)
  7. 玻璃漏斗
  8. 血细胞计数[制造商零件编号:3100; 池深度:0.100mm +/- 2%(1/10mm); 体积:0.1微升](PGC Scientific,目录号:1907353)
  9. 立体显微镜(Olympus,目录号:SZ51)
  10. 25G5/8号针(Kendall,目录号:KND8881511235)

程序

  1. 准备原生质体
    1. 称取1g切叶,取出叶柄(见图1A)。
    2. 在平板玻璃上,用新鲜的剃刀刀片或用火焰剪刀将碎片切成0.5-1mm的条,而不撕碎。
      注意:条不需要非常小,以避免累积 过滤器上过多的细胞碎片。 叶子可以容易地使用a 板面如培养皿。
    3. 使用镊子,转移 立即将植物材料装入充满洗涤物的深层培养皿中 溶液WS1(足以覆盖条)和轻轻摇动后(1-3 min手动)通过移液除去所有液体。
    4. 添加新鲜 制备酶溶液(ES)。 通常每1g使用5ml ES 切叶材料。 在黑暗中,在室温下孵育4-5小时   在旋转器中轻轻摇动(60-80rpm)。

  2. 收获原生质体
    1. 酶消化后,转移消化混合物到50毫升   Falcon管,加入等体积的WS2溶液并剧烈摇动 用手10秒钟。
    2. 从未消化的叶中释放原生质体   组织通过120μm尼龙网筛过滤混合物 玻璃漏斗倒入圆底管中。 两个顺序过滤步骤, 首先使用CX-60(120μm),然后使用CX-400(38μm) 原生质体从叶组织分离。
      注意:我们通常去 当使用悬浮液时通过3步过滤程序 文化材料,因为筛网堵塞很快。 使用筛子 CX-60,CX-150(104μm)和最后的CX-400。
    3. 使用2ml WS2溶液洗涤原生质体3-5次。
    4. 在每次洗涤后,旋管在50×g下10分钟。 丢弃 上清液,再次用2ml WS2溶液洗涤原生质体。
    5. 洗涤后,通过在50×g离心3分钟收集沉淀   与摆动转子和重悬在MMG解决方案 计数或在NIB溶液中用于核的分离。

  3. 评价质量(生存力)和计数原生质体

    评估质量
    原生质体分离程序常常损伤或杀死一定百分比的细胞。 为了确定细胞的活力,我们使用荧光素二乙酸酯(FDA)染色来估计在制剂中的可行原生质体(绿色原生质体)的百分比。
    1. 将0.25ml 0.05%氟化二乙酸二乙酯(溶于丙酮中)与20   ml的合适的分离培养基以制备"染色"溶液。
    2. 将0.25ml原生质体悬浮液吸取到1.0ml染色溶液中。
    3. 轻轻旋转以混合和安装一些玻璃上的原生质体 滑动。首先,看看使用10或20 x物镜的原生质体 镜片。现在使用紫外光源观察原生质体(420至490 mn 励磁)。在稻绿色组织原生质体中,叶绿体 可以很容易地通过他们典型的叶绿素自体荧光鉴定  在共聚焦显微镜下,虽然他们不能清楚地观察 在原生质体中
    4. 观察至少50个原生质体和 计算活细胞的百分比。乘以估计 原生质体的数目按此百分比获得估计值 活原生质体数。
      例如:5 x 10 5 原生质体 毫升乘以0.95活原生质体,等于每毫升4.75×10 5个原生质体。良好的隔离将产生之间 90和95%活的原生质体

    计数原生质体
    1. 将盖玻片放在血细胞计数板上。
    2.  彻底混合原生质体样品,以确保制服 分布细胞并用巴斯德吸管取出样品。
      注意:如果细胞计数大于10 7 细胞/ml,则稀释样品。
    3. 检查在适当的放大倍数(100 x应该工作 原生质体),并计数四个原生质体的数量, 每个室中具有适当尺寸的网格。
      注意:包括所有单元格 触摸沿着正方形的左边或上边距的中间线,但是 不是右边或底部。 在100×,1mm×3的方形将填充场。
    4. 从4个室计数共有16个网格
    5. 将数据记录在表1& 2并计算单元格数   每毫升使用下列方程(在5%水平显着 的置信度,8个计数不能偏离平均值7%以上 [Klein和Klein(1970)]):细胞/ml =细胞/平方×1平方/体积 (mm <3/sup>)×1000mm 3 /ml×稀释因子

      表1:血细胞计数器
      网格大小计数
      0.1 mm x _____ mm x _____mm
      计数的网格的体积(mm <3)
      稀释(如果有)


      表2:用血球计测量的红球藻原生质体的部分纯化悬浮液中的原生质体密度
      网格
      原生质体网格 -1
      原生质体ml -1
      1


      2


      3


      4


      5


      6


      7


      8


      9


      10


      11


      12


      13


      14


      15


      16


      平均值+/- std



  4. 核的隔离
    1. 在2ml eppendorf管或更大的试管中,以10μL/ml的冷NIB重悬浮沉淀。
    2. 在冰上放置7分钟。
    3. 用原生质体填充大型注射器,并通过25G5/8号针穿过4次
    4. 通过20μm过滤器将裂解液过滤到新管中。 在400×g离心10分钟。
    5. 丢弃上清液
    6. 重悬在NIB +甘油或SDS溶液中进行SDS凝胶电泳。

代表数据



图1.从稻绿色组织中分离原生质体。A.代表性的健康的12日龄稻苗用于原生质体分离。 黑色标记指示产生原生质体的幼苗(茎和鞘)的最佳切片。 B.通过酶溶液(ES)消化切割的条带。 C.在过滤步骤后收集原生质体。 D.在具有40x物镜的Olympus显微镜下使用Leica数字照相机获得的原生质体的图像。

食谱

  1. 酶溶液(ES)
    1.5%(w/v)纤维素酶R10
    0.4%(w/v)macerozyme R10
    0.4M甘露醇 20 mM KCl
    20mM MES(pH 5.5) 加热至55℃10分钟,然后冷却至室温,然后加入10mM CaCl 2。 0.1%BSA
    注意:对于长期储存,应将酶粉末进行包装,并在密封的塑料管中-20°C储存。 对于常规使用,将冷冻的5g管"解冻"并在4℃下储存。
  2. 洗涤和孵育溶液(WS1)
    0.5 M甘露醇 4mM MES-KOH(pH 5.5)
    20 mM KCl
  3. 洗涤溶液(WS2)
    154 mM NaCl 125mM CaCl 2。 5 mM KCl
    2mM MES,pH5.5,
  4. MMG解决方案
    0.4M甘露醇 15mM MgCl 2·h/v 4mM MES,pH5.5
    注意:解决方案WS1,WS2和MMG可以在实验前制备,高压灭菌并在4℃下储存。 只需检查pH,然后使用它们。
  5. 核分离溶液(NIB)
    0.1mM亚精胺 10mM MES-KOH(pH5.5) 2.5mM EDTA
    10mM NaCl 10 mM KCl
    0.2 M蔗糖 0.15%Triton X-100 2.5mM DTT(在使用前加入新鲜)

致谢

这项工作由瑞士国家基金会拨款(31003A-12293和31003A-138339)和中瑞科学技术合作计划(IZLCZ3 123946到YP和2009DFA32040到QS)资助。

参考文献

  1. Harrison,M.J.,Choudhary,A.D.,Lawton,M.A.,Lamb,C.J.and Dixon,R.A。(1992)。 防御基因转录调控分析。 In:Bowles,D.J。(ed)。 Molecular Plant Pathology:A Practic Approach 。 牛津大学出版社,第147-162页。
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How to cite this protocol: Jabnoune, M., Secco, D., Lecampion, C., Robaglia, C., Shu, Q. and Poirier, Y. (2015). An Efficient Procedure for Protoplast Isolation from Mesophyll Cells and Nuclear Fractionation in Rice . Bio-protocol 5(5): e1412. DOI: 10.21769/BioProtoc.1412; Full Text



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