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GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression
GUS染色保卫细胞以鉴定保卫细胞中基因的表达   

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Abstract

Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both mesophyll and guard cells, it is hard to exhibit a clear expression of the gene in guard cells by a GUS staining image from leaf. To gain a clear guard cell GUS image of small G protein ROP7, a gene expressed in both mesophyll and guard cells, we peeled the epidermal strips from the leaf of 3-4 week-old plants. After removing the mesophyll cells, the epidermal strips were used for GUS staining. We compared the GUS staining images from epidermal strips or leaf of small G protein ROP7 and RopGEF4, a gene specifically expressed in guard cells, and found that GUS staining of epidermal strips provided a good method to show the guard cell expression of a gene expressed in both mesophyll and guard cells. This protocol is applicable for any genes that are expressed in guard cells of Arabidopsis, or other plants that epidermal strips can be easily peeled from the leaf.

Keywords: Guard cells(保卫细胞), Gene expression(基因表达), GUS staining(GUS染色), Epidermal strip(表皮条), Leaf(叶)

Background

Stomatal movements regulate the gas exchange between plants and environment, therefore, it is important to reveal the mechanism of the opening or closure of stomata. Determination of the guard cell expression of a gene is essential for studying its role in stomatal movements. There are several ways to identify the expression of a gene in guard cells. One way is to check the RNA expression of a gene in guard cells by RT-PCR (Jeon et al., 2008; Takimiya et al., 2013). To do so, the protoplasts of mesophyll and guard cells need to be separated. Another way is to check the GUS signal in guard cells of the transgenic plant expressing GUS driven by a gene’s native promoter. In some reports, the evidence of both RNA expression and GUS signal in guard cells were provided (Zheng et al., 2002; Jeon et al., 2008). As for the GUS signal in guard cells, if a gene is specifically expressed in guard cells, like OST1, MYB60, ROP11 and RopGEF4, a distinguished GUS signal in guard cells can be obtained from a GUS staining image with whole leaf (Mustilli et al., 2002; Li et al., 2012; Li and Liu, 2012; Rusconi et al., 2013). However, if a gene is expressed in both mesophyll and guard cells, like ROP10 and RopGEF2, the GUS signal in guard cells is hard to be distinguished from the mesophyll background (Zheng et al., 2002; Li and Liu, 2012). After GUS staining procedure, the leaf will become soft, and it is very difficult to peel the epidermal strips. Therefore, just after the leaf was excised from the plants, we peeled the epidermal strips from the leaf, and the strips were used for GUS staining after the mesophyll cells were removed. By this method, we obtained a clear guard cell GUS image of ROP7, a gene expressed in both mesophyll and guard cells.

Materials and Reagents

  1. Pipette tips
  2. 1.5 ml Eppendorf tubes
  3. Sterilized filter paper
  4. Plastic Petri dishes for plant culture
  5. Slide
  6. Cover glass
  7. 0.45 micron filter
  8. Aluminum foil
  9. Arabidopsis thaliana seeds of ROP7pro:GUS and RopGEF4pro:GUS lines
  10. 100%, 75%, 40%, 20%, 10%, 5% ethanol in water
  11. 50% glycerol (Sangon Biotech, catalog number: A100854 )
  12. 100% methanol
  13. 37% hydrochloric acid (12 N)
  14. Sodium hydroxide (AMRESCO, catalog number: 0583 )
  15. Ethylenediaminetetraacetate acid (EDTA) (AMRESCO, catalog number: 0322 )
  16. Triton X-100 (AMRESCO, catalog number: 0694 )
  17. Potassium ferricyanide (AMRESCO, catalog number: 0713 )
  18. Potassium ferrocyanide (Sigma-Aldrich, catalog number: P9387 )
  19. X-Gluc (Sigma-Aldrich, catalog number: B5285 )
  20. Dimethylformamide (AMRESCO, catalog number: 0464 )
  21. Sodium dihydrogen phosphate (NaH2PO4·H2O) (Sigma-Aldrich, catalog number: S9638 )
  22. Disodium hydrogen phosphate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: 431478 )
  23. 20% methanol in 0.24 N hydrochloric acid (see Recipes)
  24. 60% ethanol in 7% sodium hydroxide (see Recipes)
  25. GUS staining solution (see Recipes)

Equipment

  1. Pipetman 100 μl (Gilson, catalog number: F123615 )
  2. Pipetman 1,000 μl (Gilson, catalog number: F123602 )
  3. Tweezers
  4. Brush pen
  5. Plant growth chamber (Percival Scientific, model: CU-36L5 ) and greenhouse
  6. Pots
  7. Incubator at 37 °C (SANFA, model: DNP-9052 )
  8. Microscope (ZEISS, model: Axio Imager A1 )
  9. Water purification system (deionized water) (EMD Millipore, model: Elix® Essential , 5 L)

Procedure

  1. Arabidopsis plants were grown according to Arabidopsis growing guide http://www.bio-protocol.org/e126.
  2. Harvest the fully expanded rosette leaves from 3-4 week-old plants, and peel the epidermal strips from the abaxial surface of the leaf (Figure 1A), remove the mesophyll tissue from the strips with a brush pen (Figure 1B). Epidermal strips or leaf are then immersed in 1.5 ml Eppendorf tubes with GUS staining solution (see Recipes), and keep the tubes in an incubator at 37 °C under darkness for 16-20 h.


    Figure 1. Images show the peeling of the epidermis from the abaxial surface of leaf (A) and removing the mesophyll tissue from epidermal strip by a brush pen (B)

  3. Replace the supernatant with 1 ml 100% ethanol and incubate for 30 min, repeat this step for 2 times.
  4. Replace the supernatant with 1 ml 75% ethanol, and incubate for 5 min.
  5. Replace the supernatant with 1 ml 20% methanol in 0.24 N hydrochloric acid, and incubate at 37 °C for 15 min.
  6. Replace the supernatant with 1 ml 60% ethanol in 7% sodium hydroxide, and incubate for 15 min.
  7. Replace the supernatant with 1 ml 40% ethanol, and incubate for 5 min; replace the supernatant with 20%, 10% and 5% ethanol gradually each for 5 min. After all these steps, the leaf tissue will turn to white, and keep the samples in 5% ethanol.
  8. Put one drop of 50% glycerol on the slide, and place the samples in the glycerol solution; put the cover glass on the samples.
  9. Observe the samples under a microscope and take pictures:
    1. Switch on the light source of the microscope, and rotate the nosepiece to the lowest-power objective.
    2. Place the slide on the stage of the microscope, and move the slide to center the specimen under the lens.
    3. Adjust the large coarse focus knob until the specimen is in focus.
    4. Scan the slide at low power objective (4x or 10x objective), center the part of the specimen that one wants to view, and then rotate the nosepiece to the 20x or 40x objective.
    5. Adjust the small fine focus knob until the cells are in focus, and adjust the diaphragm until the cells have clear and sharp contrast.
    6. Take a picture with the CCD.

Data analysis

As shown in Figure 2, the GUS signals in guard cells were very clear in the images of GUS staining from both the leaf (Figure 2C) and the epidermal strips (Figure 2D) of RopGEF4pro:GUS lines. The results were consistent with the specific expression of RopGEF4 in guard cells reported by Li and Liu (2012). However, the GUS signal was not clear when stained the whole leaf of ROP7pro:GUS line because of the strong background of mesophyll tissue (Figure 2A). When the epidermal strips were separated from mesophyll cells, we can see a clear guard cell GUS signal after GUS staining (Figure 2B). When this method is applied, it is better to use 3-4 week old plants of Arabidopsis, because the epidermal strips are easily peeled from the mesophyll tissue during this time. This method is applicable for any genes that are expressed in guard cells of Arabidopsis, or other plants that epidermal strips can be easily peeled from the leaf.


Figure 2. GUS staining with the leaf (A) and epidermal strips (B) from ROP7pro:GUS line, and leaf (C) and epidermal strips (D) from RopGEF4pro:GUS line. Scale bar = 10 μm.

Notes

  1. Please be very careful to keep the epidermal strips in the tubes in each step, try to avoid losing the epidermal strips when removing the supernatant.
  2. The epidermal strips became soft and easily broken after GUS staining procedure, so be very careful to flatten the epidermal strip on the slide.

Recipes

  1. 20% methanol in 0.24 N hydrochloric acid
    Mix:
    20 ml of 100% methanol
    2 ml of 37% hydrochloric acid (12 N)
    Deionized water 78 ml
  2. 60% ethanol in 7% sodium hydroxide
    Dissolve 28 g sodium hydroxide in deionized water to 100 ml to make 28% sodium hydroxide
    Mix:
    60 ml of 100% ethanol
    25 ml of 28% sodium hydroxide
    15 ml of deionized water
  3. GUS staining solution
    100 mM sodium phosphate buffer (pH = 7.0)
    10 mM EDTA
    0.1% Triton X-100
    0.5 mM potassium ferricyanide
    0.5 mM potassium ferrocyanide
    1 mM X-Gluc
    1. Dissolve 0.8892 g X-Gluc in 100 ml dimethylformamide to make a 20 mM stock and keep in the dark at -20 °C
    2. Make 200 mM sodium phosphate buffer (pH 7.0): dissolve 1.08 g sodium dihydrogen phosphate (NaH2PO4·H2O) and 3.27 g disodium hydrogen phosphate (Na2HPO4·7H2O) in deionized water up to 100 ml
    3. Mix:
      50 ml of 200 mM sodium phosphate buffer (pH 7.0)
      2 ml of 500 mM EDTA solution
      0.5 ml of 20% (w/v) Triton X-100
      1 ml of 50 mM potassium ferricyanide
      1 ml of 50 mM potassium ferrocyanide
      5 ml of 20 mM X-Gluc
    4. Bring up to 100 ml with deionized water
    5. Filter-sterilize (0.45 micron filter) and keep at 4 °C wrapped with aluminum foil

Acknowledgments

This work was supported by the National Science Foundation of China (Grant No. 31571450 to Y.-L.C.). The protocol was modified according to Pecinka et al. (2009).

References

  1. Jeon, B. W., Hwang, J. U., Hwang, Y., Song, W. Y., Fu, Y., Gu, Y., Bao, F., Cho, D., Kwak, J. M., Yang, Z. and Lee, Y. (2008). The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening. Plant Cell 20(1): 75-87.
  2. Li, Z. and Liu, D. (2012). ROPGEF1 and ROPGEF4 are functional regulators of ROP11 GTPase in ABA-mediated stomatal closure in Arabidopsis. FEBS Lett 586(9): 1253-1258.
  3. Li, Z., Kang, J., Sui, N. and Liu, D. (2012). ROP11 GTPase is a negative regulator of multiple ABA responses in Arabidopsis. J Integr Plant Biol 54(3): 169-179.
  4. Mustilli, A. C., Merlot, S., Vavasseur, A., Fenzi, F. and Giraudat, J. (2002). Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14(12): 3089-3099.
  5. Pecinka, A., Rosa, M., Schikora, A., Berlinger, M., Hirt, H., Luschnig, C. and Mittelsten Scheid, O. (2009). Transgenerational stress memory is not a general response in Arabidopsis. PLoS One 4(4): e5202.
  6. Rusconi, F., Simeoni, F., Francia, P., Cominelli, E., Conti, L., Riboni, M., Simoni, L., Martin, C. R., Tonelli, C. and Galbiati, M. (2013). The Arabidopsis thaliana MYB60 promoter provides a tool for the spatio-temporal control of gene expression in stomatal guard cells. J Exp Bot 64(11): 3361-3371.
  7. Takemiya, A., Sugiyama, N., Fujimoto, H., Tsutsumi, T., Yamauchi, S., Hiyama, A., Tada, Y., Christie, J. M. and Shimazaki, K. (2013). Phosphorylation of BLUS1 kinase by phototropins is a primary step in stomatal opening. Nat Commun 4: 2094.
  8. Zheng, Z. L., Nafisi, M., Tam, A., Li, H., Crowell, D. N., Chary, S. N., Schroeder, J. I., Shen, J. and Yang, Z. (2002). Plasma membrane-associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis. Plant Cell 14(11): 2787-2797.

简介

保卫细胞中基因表达的测定对于研究气孔运动至关重要。 GUS染色是检测保卫细胞中基因表达定位的一种手段。如果在保卫细胞中特异性表达基因,则可以将整个子叶或玫瑰花叶用于GUS染色。然而,如果在叶肉和保卫细胞中表达基因,则难以通过来自叶的GUS染色图像在保护细胞中表达该基因的清楚表达。为了获得清晰的保护细胞GUS图像的小G蛋白ROP7 ,一种在叶肉和保卫细胞中表达的基因,我们从3-4周龄的植物的叶片剥离表皮条。去除叶肉细胞后,将表皮条用于GUS染色。我们比较了来自表皮条或小G蛋白ROP7和/或RopGEF4的叶片的GUS染色图像,其是在保卫细胞中特异性表达的基因,并且发现提供了表皮条带的GUS染色显示在叶肉和保卫细胞中表达的基因的保卫细胞表达的良好方法。该方案适用于在拟南芥保卫细胞中表达的任何基因,或表皮条可以容易地从叶上剥离的其它植物。
【背景】气孔运动调节植物和环境之间的气体交换,因此,重要的是揭示气孔的开启或关闭的机制。确定基因的保卫细胞表达对于研究其在气孔运动中的作用至关重要。鉴定基因在保卫细胞中的表达有几种方法。一种方法是通过RT-PCR(Jeon等人,2008; Takimiya等人,2013)检查保卫细胞中基因的RNA表达。为此,需要分离叶肉和保卫细胞的原生质体。另一种方法是检查表达由基因天然启动子驱动的GUS的转基因植物的保卫细胞中的GUS 信号。在一些报告中,提供了保卫细胞中RNA表达和GUS信号的证据(Zheng等人,2002; Jeon等人,2008)。对于保卫细胞中的GUS信号,如果基因在保卫细胞中特异性表达,例如,OST1 ,MYB60 ,ROP11和 RopGEF4 ,保卫细胞中的突出GUS信号可以从具有全叶(Mustilli等人,2002; Li等人)的GUS染色图像获得, ,2012; Li和Liu,2012; Rusconi等人,2013)。然而,如果在叶肉和保卫细胞中表达基因,如ROP10和RopGEF2,保卫细胞中的GUS信号难以与叶肉背景(Zheng 等人,2002; Li和Liu,2012)。 GUS染色程序后,叶片变软,剥离皮肤条很困难。因此,从植物切下叶子后,我们从叶子上剥下表皮条,去除叶肉细胞后,将条带用于GUS染色。通过这种方法,我们获得了在叶肉和保卫细胞中表达的基因的ROP7 的清晰的保护细胞GUS图像。

关键字:保卫细胞, 基因表达, GUS染色, 表皮条, 叶

材料和试剂

  1. 移液器提示
  2. 1.5 ml Eppendorf管
  3. 灭菌滤纸
  4. 用于植物培养的塑料培养皿
  5. 幻灯片
  6. 盖玻璃
  7. 0.45微米过滤器
  8. 铝箔
  9. ROP7pro的拟南芥种子:GUS和RopGEF4pro:GUS 线
  10. 100%,75%,40%,20%,10%,5%乙醇水溶液
  11. 50%甘油(Sangon Biotech,目录号:A100854)
  12. 100%甲醇
  13. 37%盐酸(12N)
  14. 氢氧化钠(AMRESCO,目录号:0583)
  15. 乙二胺四乙酸(EDTA)(AMRESCO,目录号:0322)
  16. Triton X-100(AMRESCO,目录号:0694)
  17. 铁氰化钾(AMRESCO,目录号:0713)
  18. 亚铁氰化钾(Sigma-Aldrich,目录号:P9387)
  19. X-Gluc(Sigma-Aldrich,目录号:B5285)
  20. 二甲基甲酰胺(AMRESCO,目录号:0464)
  21. 磷酸二氢钠(NaH 2 PO 4·H 2 O)(Sigma-Aldrich,目录号:S9638)
  22. 磷酸氢二钠(Na 2 HPO 4·7H 2 O)(Sigma-Aldrich,目录号:431478)
  23. 在0.24N盐酸中的20%甲醇(参见食谱)
  24. 7%氢氧化钠中的60%乙醇(见配方)
  25. GUS染色溶液(参见食谱)

设备

  1. Pipetman 100μl(Gilson,目录号:F123615)
  2. Pipetman 1000μl(Gilson,目录号:F123602)
  3. 镊子
  4. 刷笔
  5. 植物生长室(Percival Scientific,型号:CU-36L5)和温室

  6. 37℃的培养箱(SANFA,型号:DNP-9052)
  7. 显微镜(ZEISS,型号:Axio Imager A1)
  8. 水净化系统(去离子水)(EMD Millipore,型号:Elix Essential,5 L)

程序

  1. 根据拟南芥生长指南 http://www.bio-protocol.org/e126
  2. 从3-4周的植物中收获完全膨大的玫瑰花叶,并从叶片的背面剥离表皮条(图1A),用刷笔从条上除去叶肉组织(图1B)。然后将表皮条或叶浸入具有GUS染色溶液的1.5ml Eppendorf管中(参见食谱),并将管保持在37℃,黑暗下16-20小时的培养箱中。


    图1.图像显示表皮从叶(A)的背面剥离,并通过刷笔从表皮条上去除叶肉组织(B) />
  3. 用1 ml 100%乙醇取代上清液,孵育30 min,重复此步骤2次
  4. 用1 ml 75%乙醇取代上清液,孵育5 min
  5. 用0.24N盐酸的1ml 20%甲醇替换上清液,37℃孵育15分钟
  6. 用1%60%乙醇的7%氢氧化钠代替上清液,孵育15分钟
  7. 用1 ml 40%乙醇取代上清液,孵育5 min;用20%,10%和5%乙醇将上清液逐渐取代5分钟。在所有这些步骤之后,叶组织将变成白色,并将样品保持在5%乙醇中
  8. 将一滴50%甘油置于载玻片上,并将样品置于甘油溶液中;把盖玻片放在样品上。
  9. 在显微镜下观察样品并拍照:
    1. 打开显微镜的光源,并将鼻梁旋转到最低功率的物镜上。
    2. 将幻灯片放置在显微镜的舞台上,并移动幻灯片将样本放置在镜片下方。
    3. 调整粗调焦点旋钮,直至样本对焦为止。
    4. 以低功率物镜(4x或10x物镜)扫描幻灯片,将要查看的样品的一部分居中,然后将鼻梁旋转至20x或40x物镜。
    5. 调整小调焦旋钮,直到细胞处于对焦状态,并调节光阑,直到细胞具有清晰锐利的对比度
    6. 用CCD拍照。

数据分析

如图2所示,保护细胞中的GUS信号在来自叶(图2C)和RopGEF4pro:GUS 线的表皮条(图2D)的GUS染色的图像中非常清楚。结果与Li和Liu(2012)报道的保卫细胞中RopGEF4的特异性表达一致。然而,由于叶肉组织的强烈背景,GAS信号在染色整个ROP7pro:GUS 线条时不清楚(图2A)。当表皮条与叶肉细胞分离时,GUS染色后可以看到明显的保护细胞GUS信号(图2B)。当应用这种方法时,最好使用拟南芥3-4周龄的植物,因为在此期间表皮条很容易从叶肉组织中剥离。该方法适用于在拟南芥保卫细胞中表达的任何基因,或其他植物,表皮条可以容易地从叶上剥离。


图2.来自 ROP7pro:GUS 线和叶(A)的叶(A)和表皮条(B)的GUS染色, C)和表皮条(D)来自 RopGEF4pro:GUS 线。比例尺= 10μm。

笔记

  1. 请仔细保持每个步骤中的表皮条在管中,尽量避免在去除上清液时丢失表皮条。
  2. GUS染色手术后,表皮条变软,容易断裂,所以要非常小心地将载玻片上的表皮条铺平。

食谱

  1. 在0.24N盐酸中的20%甲醇
    混合:
    20毫升100%甲醇
    2ml 37%盐酸(12N) 去离子水78 ml
  2. 60%乙醇在7%氢氧化钠中 将28 g氢氧化钠溶于去离子水中至100ml,制成28%氢氧化钠 混合:
    60毫升100%乙醇
    25毫升28%氢氧化钠
    15毫升去离子水
  3. GUS染色溶液
    100mM磷酸钠缓冲液(pH = 7.0)
    10 mM EDTA
    0.1%Triton X-100
    0.5 mM铁氰化钾
    0.5mM亚铁氰化钾
    1mM X-Gluc
    1. 将0.8892g X-Gluc溶于100ml二甲基甲酰胺中以制备20mM储备液,并保持在-20℃的黑暗中
    2. 制备200mM磷酸钠缓冲液(pH7.0):溶解1.08g磷酸二氢钠(NaH 2 PO 4·H 2 O)和3.27 g磷酸氢二钠(Na 2 HPO 4·7H 2 O)在去离子水中最多可加入100ml
    3. 混合:
      50ml 200mM磷酸钠缓冲液(pH7.0)
      2 ml 500 mM EDTA溶液
      0.5ml 20%(w / v)Triton X-100
      1毫升50毫摩尔铁氰化钾 1毫升50毫摩尔亚铁氰化钾 5 ml 20 mM X-Gluc
    4. 用去离子水加至100毫升
    5. 过滤灭菌(0.45微米过滤器)并保持在4℃用铝箔包裹

致谢

这项工作得到了中国国家科学基金(授予号为31571450)的支持。该方案根据Pecinka等人(2009)进行了修改。

参考

  1. Jeon,BW,Hwang,JU,Hwang,Y.,Song,WY,Fu,Y.,Gu,Y.,Bao,F.,Cho,D.,Kwak,JM,Yang,Z.and Lee, (2008)。拟南芥小G蛋白ROP2被保护细胞中的光激活,并抑制光诱导的气孔开放。植物细胞<20>(1):75-87。
  2. Li,Z.和Liu,D。(2012)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/22500990”target =“_ blank” > ROPGEF1和ROPGEF4是拟南芥ABA介导的气孔闭合中ROP11GTPase的功能调节剂。 FEBS Let t 586(9):1253-1258。 br />
  3. Li,Z.,Kang,J.,Sui,N。和Liu,D。(2012)。 ROP11 GTPase是拟南芥中多个ABA反应的阴性调节因子。 J Integr Plant Biol 54(3) :169-179。
  4. Mustilli,AC,Merlot,S.,Vavasseur,A.,Fenzi,F.and Giraudat,J。(2002)。 拟南芥 OST1蛋白激酶介导了脱落酸对气孔孔径​​的调节,并在活性氧生产的上游起作用。 >植物细胞 14(12):3089-3099。
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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. Liu, Z., Wang, W., Zhang, C., Zhao, J. and Chen, Y. (2017). GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression. Bio-protocol 7(14): e2446. DOI: 10.21769/BioProtoc.2446.
  2. Wang, W., Liu, Z., Bao, L. J., Zhang, S. S., Zhang, C. G., Li, X., Li, H. X., Zhang, X. L., Bones, A. M., Yang, Z. B. and Chen, Y. L. (2017). The RopGEF2-ROP7/ROP2 Pathway Activated by phyB Suppresses Red Light-Induced Stomatal Opening. Plant Physiol 174(2): 717-731.
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