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Isolation of Guard-cell Enriched Tissue for RNA Extraction
分离保卫细胞富集组织用于RNA提取   

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Abstract

This is a protocol for isolation of guard cell enriched samples from Arabidopsis thaliana plants for RNA extraction. Leaves are blended in ice-water and filtered through nylon mesh to obtain guard cell enriched fragments. With guard cell enriched samples, gene expression analysis can be done, e.g., comparing different gene expression levels in guard cells versus whole leaf to determine if a gene of interest is predominantly expressed in guard cells. It can also be used to study the effect of treatments or different genetic backgrounds in the regulation of the guard cell expressed genes.

Keywords: Arabidopsis thaliana(拟南芥), Guard cell isolation(保卫细胞分离), RNA(RNA)

Background

Isolation of guard cells for RNA extraction has traditionally relied on guard cell protoplast extraction (Leonhardt et al., 2004) or epidermal peels (Pandey et al., 2010). Manual dissection of guard cells from freeze-dried leaves has also been used (Bates et al., 2012). The protoplast procedure may introduce unwanted changes in gene expression from wounding effects and other methods are time consuming, require special equipment or expensive reagents (e.g., transcriptional inhibitors). Hence there is a need for a fast and simple protocol to isolate guard cells for gene expression analysis. Here we further describe a quick ice blender method for isolation of guard cell enriched tissue (Bauer et al., 2013), in the form of epidermal fragments without mesophyll and other vascular cells. This method uses a nylon mesh to collect epidermis from blended leaf tissue. The adequately large pore size of the nylon mesh allows mesophyll and other vascular cells to pass through while retaining the epidermal fragments. We propose that the critical factor in this protocol is the type of blender used to process the samples.

Materials and Reagents

  1. Razor blade
  2. 210 μm nylon mesh (A. Hartenstein, laborversand.de, catalog number: PAS1 )
  3. Plastic embroidery hoop from a handicraft store (Figure 1)
    Note: Nylon mesh is placed in the plastic embroidery hoop to provide a little tension to the mesh, to make it easier to pour the blended water and guard cell enriched fragments through the mesh.


    Figure 1. Nylon mesh with a plastic embroidery hoop

  4. Paper towel
  5. Small medical spatula or plastic spoon
  6. 1.5 ml tube or aluminum foil
  7. Arabidopsis plants
  8. Milli-Q or distilled water kept at 4 °C
  9. Crushed ice
  10. Liquid nitrogen

Equipment

  1. Blender (Braun JB 3060 or other 800 W blender) (Braun Household, model: JB 3060 )
    Note: A blender with lower effect has been tried, but failed to isolate proper fragments, probably due to insufficient power to fully crush the ice.
  2. Microscope (ZEISS, model: SteREO Discovery.v20 )

Procedure

Collect leaves from 4-7 week old Arabidopsis plants grown in soil at 12 h/12 h photoperiod, use 17-18 plants and 4-5 leaves per plant. If plants are smaller in size, use more leaves per plant.

  1. Remove the central vein (midrib) by razor blade. Collect about 1.0-1.5 g of leaves without midribs for the following procedures (Figure 2).


    Figure 2. Midrib is cut and removed with the razor blade

  2. Put the cut leaf blades in blender with 250 ml cold Milli-Q water and a handful of crushed ice.
    Blend 1 min (program 2 for the Braun blender), pour through mesh placed in a plastic embroidery hoop.
    Note: Pouring through mesh should be done slowly, so that the guard cell enriched fragments are collected in the middle of the mesh. This is especially important at step 7 after which the sample is collected.
  3. Wash the mesh with cold Milli-Q water above the blender cup so that the guard cell enriched fragments are transferred inside the blender cup. Add cold Milli-Q water up to 250 ml and handful of crushed ice.
  4. Blend 1 min (program 2), pour through mesh placed in a plastic embroidery hoop.
  5. Wash the mesh with cold Milli-Q water above the blender cup so that the guard cell enriched fragments are transferred inside the blender cup. Add cold Milli-Q water up to 250 ml and handful of crushed ice.
  6. Blend 1 min (program 2), pour through mesh placed in a plastic embroidery hoop.
  7. Remove the remaining dark green tissue fragments (small leaf pieces that have not been blended) from the light green epidermal fraction.
  8. Dry the underside of the mesh from excess water with paper towel about 10-15 sec and use a small spatula or plastic spoon to carefully scrape the sample into a 1.5 ml tube or aluminum foil (Figure 3).


    Figure 3. Guard cell enriched sample that is collected into a 1.5 ml tube

  9. Freeze the sample in liquid nitrogen and keep at -80 °C. Alternatively, the sample can be immediately ground in mortar with liquid nitrogen and the powder used for RNA isolation.
    Note: If an estimation of guard cell enrichment is required, one easy control is to harvest a whole leaf sample from the same plants used for guard cell enrichment and freeze in liquid nitrogen and store at -80 °C. RNA can then be extracted in parallel with the guard cell samples. The level of guard cell enrichment can be estimated by real time quantitative PCR using guard cell preferentially expressed genes e.g., HT1 (AT1G62400), OST1 (AT4G33950) or GORK (AT5G37500).

Data analysis

Isolated guard cell enriched fragments can be visualized with a stereo microscope (Zeiss SteREO Discovery.v20), and no mesophyll fragments (green tissue) should be present (Figure 4A). While testing different blenders, successful isolation of guard cell enriched fragments was achieved with the Braun (800 W) blender but not with a blender with lower effect (Figure 4).


Figure 4. Isolated guard cell enriched fragments under a microscope. A. Sample isolated with the Braun blender; B. Sample isolated with a small 220 W blender.

To control the quality of guard cell enrichment, real-time quantitative PCR can be done with guard cell expressed genes like HT1 and GORK, by comparing their expression level in guard cell enriched sample versus a whole leaf sample (Figure 5; for details about the qPCR conditions see Jalakas et al., 2017).


Figure 5. Gene expression in Col-0 guard cell enriched sample compared to leaf sample. The mean of three biological replicates are shown; error bars depict ± SEM.

Acknowledgments

This protocol was adapted from the research article Bauer et al. (2013). This work was funded by the Estonian Ministry of Science and Education (IUT2-21 to H.K.), the European Regional Development Fund (Center of Excellence in Molecular Cell Engineering CEMCE to H.K.), the Academy of Finland (grant number #307335, Center of Excellence in Primary Producers 2014-2019 to M.B.).

References

  1. Bates, G. W., Rosenthal, D. M., Sun, J., Chattopadhyay, M., Peffer, E., Yang, J., Ort, D. R. and Jones, A. M. (2012). A comparative study of the Arabidopsis thaliana guard-cell transcriptome and its modulation by sucrose. PLoS One 7(11): e49641.
  2. Bauer, H., Ache, P., Lautner, S., Fromm, J., Hartung, W., Al-Rasheid, K. A., Sonnewald, S., Sonnewald, U., Kneitz, S., Lachmann, N., Mendel, R. R., Bittner, F., Hetherington, A. M. and Hedrich, R. (2013). The stomatal response to reduced relative humidity requires guard cell-autonomous ABA synthesis. Curr Biol 23(1): 53-57.
  3. Jalakas, P., Huang, Y.-C., Yeh, Y.-H., Zimmerli, L., Merilo, E., Kollist, H. and Brosché, M. (2017). The role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis stomatal responses is beyond ABA signaling. Plant Physiol 174: 665-671.
  4. Leonhardt, N., Kwak, J. M., Robert, N., Waner, D., Leonhardt, G. and Schroeder, J. I. (2004). Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant. Plant Cell 16(3): 596-615.
  5. Pandey, S., Wang, R. S., Wilson, L., Li, S., Zhao, Z., Gookin, T. E., Assmann, S. M. and Albert, R. (2010). Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action. Mol Syst Biol 6: 372.

简介

这是用于从拟南芥植物中分离用于RNA提取的保护性细胞富集样品的方案。 将叶子混合在冰水中,并通过尼龙网过滤,获得保护细胞富集片段。 使用保卫性细胞富集的样品,可以进行基因表达分析,例如,比较保卫细胞中的不同基因表达水平与全叶,以确定感兴趣的基因是否主要在保卫细胞中表达。 它也可用于研究治疗或不同遗传背景对保卫细胞表达基因调控的影响。
【背景】RNA提取的保卫细胞的分离传统上依赖于保卫细胞原生质体提取(Leonhardt等人,2004)或表皮剥皮(Pandey等人,2010)。也已经使用了保护细胞从冷冻干燥的叶中的手工解剖(Bates等人,2012)。原生质体程序可能会从伤口效应引起基因表达的不必要的变化,而其他方法是耗时的,需要特殊的设备或昂贵的试剂(例如转录抑制剂)。因此,需要一种快速和简单的方案来分离保护细胞进行基因表达分析。在这里,我们进一步描述了以不含叶肉和其他血管细胞的表皮碎片的形式分离保卫性细胞富集组织(Bauer等人,2013)的快速冰混合器方法。该方法使用尼龙网从混合叶组织收集表皮。尼龙网足够大的孔径允许叶肉和其他血管细胞通过,同时保留表皮碎片。我们建议本协议中的关键因素是用于处理样品的混合器的类型。

关键字:拟南芥, 保卫细胞分离, RNA

材料和试剂

  1. 剃刀刀片
  2. 210μm尼龙网(A.Hartenstein,laborversand.de,目录号:PAS1)
  3. 来自工艺品店的塑料刺绣环(图1)
    注意:将尼龙网放置在塑料刺绣环中,为网状物提供一点张力,使其更容易倒入混合水中,并通过网格保护细胞富集的碎片。


    图1.带有塑料刺绣环的尼龙网

  4. 纸巾
  5. 小型医用刮刀或塑料勺子
  6. 1.5毫升管或铝箔
  7. 拟南芥植物
  8. Milli-Q或蒸馏水保持在4°C
  9. 碎冰
  10. 液氮

设备

  1. 搅拌机(Braun JB 3060或其他800 W搅拌机)(Braun家用,型号:JB 3060)
    注意:已尝试使用效果较差的搅拌机,但无法分离出适当的碎片,可能是由于没有足够的力量来完全粉碎冰块。
  2. 显微镜(ZEISS,型号:SteREO Discovery.v20)

程序

在12小时/ 12小时光周期内从4-7周龄的拟南芥植物中收集叶子,每株植物使用17-18株和4-5株。如果植物的尺寸较小,则每株植物需要更多的叶子。

  1. 用剃刀刀片去除中央静脉(中脉)。收集大约1.0-1.5 g的叶子,不需要上述步骤(图2)

    图2.使用剃刀刀片切割并删除中脉

  2. 将切割的叶片放入搅拌机中,加入250毫升冷的Milli-Q水和少量碎冰。
    混合1分钟(Braun搅拌机的程序2),通过网格倒入塑料刺绣箍中。
    注意:通过网格进行缓慢进行,使得保护细胞富集的碎片被收集在网格的中间。这在第7步特别重要,之后收集样品。
  3. 用搅拌杯上方的冷Milli-Q水清洗网孔,使保护细胞富集的碎片转移到搅拌杯内。将冷的Milli-Q水加至250毫升和少量碎冰。
  4. 混合1分钟(程序2),将网眼放在塑料刺绣箍中。
  5. 用搅拌杯上方的冷Milli-Q水清洗网孔,使保护细胞富集的碎片转移到搅拌杯内。将冷的Milli-Q水加至250毫升和少量碎冰。
  6. 混合1分钟(程序2),将网眼放在塑料刺绣箍中。
  7. 从浅绿色的表皮部分去除残留的深绿色组织碎片(尚未混合的小叶片)
  8. 用纸巾约10-15秒,用多余的水将网眼的下面干净,并用小铲或塑料勺子将样品小心地刮到1.5毫升管或铝箔上(图3)。

    图3.将收集到1.5ml管中的保护细胞富集样品

  9. 将样品在液氮中冷冻并保持在-80°C。或者,样品可立即用液氮研磨,用于RNA分离的粉末 注意:如果需要对保卫细胞富集的估计,一个容易的控制是从用于保护细胞富集的同一植物中收获全叶样品并在液氮中冷冻并储存在-80℃。然后可以将RNA与保卫细胞样品并行提取。保护细胞富集水平可以通过使用保卫细胞优先表达基因例如HT1(AT1G62400),OST1(AT4G33950)或GORK(AT5G37500))的实时定量PCR来估计。

数据分析

可以用立体显微镜(Zeiss SteREO Discovery.v20)显现分离的保护细胞富集的片段,并且不应存在叶绿素片段(绿色组织)(图4A)。在测试不同的搅拌机时,用Braun(800W)搅拌机实现了成功分离富集保卫细胞的碎片,但是没有使用效果较差的搅拌机(图4)。


图4.在显微镜下分离的保护细胞富集碎片。 :一种。用Braun搅拌器分离样品; B.用小型220W搅拌机分离的样品。

为了控制保卫细胞富集的质量,可以通过比较其保守细胞富集的表达水平,通过保卫细胞表达的基因,如HT1和GORK 进行实时定量PCR样品与全叶样品(图5;有关qPCR条件的详细信息,参见Jalakas等人,2017)。


图5.与叶样品相比,Col-0保守细胞富集样品中的基因表达。显示三个生物重复的平均值;误差条描绘±SEM。

致谢

这个协议是从研究文章Bauer等人(2013)改编而成。这项工作由爱沙尼亚科学和教育部(IUT2-21 to HK),欧洲区域发展基金(CEMCE到香港分子细胞工程中心),芬兰科学院(拨款号码:307335,中心卓越初级生产者2014-2019至MB)。

参考

  1. Bates,GW,Rosenthal,DM,Sun,J.,Chattopadhyay,M.,Peffer,E.,Yang,J.,Ort,DR and Jones,AM(2012)。< a class =“ke-insertfile” href =“http://www.ncbi.nlm.nih.gov/pubmed/23185391”target =“_ blank”>拟南芥保守细胞转录组及其由蔗糖调节的比较研究 PLoS One 7(11):e49641。
  2. Bauer,H.,Ache,P.,Lautner,S.,Fromm,J.,Hartung,W.,Al-Rasheid,KA,Sonnewald,S.,Sonnewald,U.,Kneitz,S.,Lachmann, ,孟德尔,RR,Bittner,F.,Hetherington,AM和Hedrich,R。(2013)。  降低相对湿度的气孔反应需要保卫细胞自主的ABA合成。 23(1):53-57。
  3. Jalakas,P.,Huang,Y.-C.,Yeh,Y.-H.,Zimmerli,L.,Merilo,E.,Kollist,H.andBrosché,M.(2017)。< a class = “ke-insertfile”href =“https://www.ncbi.nlm.nih.gov/pubmed/28330935”target =“_ blank”>拟南芥中对ABA1(ERA1)的增强反应的作用气孔反应超出ABA信号。植物生理学174:665-671。
  4. Leonhardt,N.,Kwak,JM,Robert,N.,Waner,D.,Leonhardt,G.and Schroeder,JI(2004)。拟南芥保卫细胞的微阵列表达分析和隐性脱落酸过敏蛋白磷酸酶2C突变体的分离。 植物细胞 16(3):596-615。
  5. Pandey,S.,Wang,RS,Wilson,L.,Li,S.,Zhao,Z.,Gookin,TE,Assmann,SM and Albert,R。(2010)。  转录组数据的布尔建模揭示了异源三聚体G蛋白作用的新模式。 Mol Syst Biol 6:372.
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jalakas, P., Yarmolinsky, D., Kollist, H. and Brosche, M. (2017). Isolation of Guard-cell Enriched Tissue for RNA Extraction. Bio-protocol 7(15): e2447. DOI: 10.21769/BioProtoc.2447.
  2. Jalakas, P., Huang, Y.-C., Yeh, Y.-H., Zimmerli, L., Merilo, E., Kollist, H. and Brosché, M. (2017). The role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis stomatal responses is beyond ABA signaling. Plant Physiol 174: 665-671.
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