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Measuring Arabidopsis, Tomato and Barley Leaf Relative Water Content (RWC)
拟南芥、番茄和大麦叶片中相对含水量(RWC)的测定   

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Abstract

Measuring leaf relative water content (RWC) is a reliable and simple way to assess the water status of a leaf without any need for special equipment. Similar to leaf water potential, leaf RWC gives a strong indication of the plant’s response to different environmental conditions; yet RWC has been shown to be a more stable parameter than leaf water potential (Sade et al., 2009; Sade et al., 2012). Although measuring RWC is destructive to the leaf, with proper planning, it need not affect the plant’s behavior. This note will focus on three different model plants which are representative of plants with various leaf shapes (e.g., Arabidopsis, tomato and barley). The technique for measuring RWC is the same for all three of these species (as well as for plants with many other types of leaves).

Keywords: Relative water content(相对含水量), Leaf water content(叶片水分含量), Drought stress(干旱胁迫), Salt stress(盐胁迫)

Materials and Reagents

  1. 4-week-old Arabidopsis plants grown under short-day conditions (8/16 L/D; 22/22 °C)
  2. 4-week-old tomato plants grown under moderate conditions (10/14 L/D; 25/20 °C)
  3. 4-week-old barley plants grown under short-day conditions (8/16 L/D; 16/10 °C)

Equipment

  1. Zipper-locked plastic bag (8 x 12 cm) for each sample
  2. Paper bag (8 x 12 cm) for each sample
  3. Sharp scissors
  4. Scalpel
  5. Paper towels
  6. Analytical balance with 0.1 mg readability
  7. 2-3 ml of 5 mM CaCl2 in DDW water for each sample
  8. Marker pen
  9. Plastic box that can be sealed tightly

Software

  1. Microsoft Excel

Procedure

  1. Grow the plants.
  2. Mark and weigh each plastic bag (BW) prior to the other measurements.
  3. Typically, leaves are measured before dawn and/or at midday (Matin et al., 1989). At the selected hour, select a fully expanded and mature leaf.
  4. For Arabidopsis and tomato, cut the leaf with a scalpel leaving a 1-cm-long petiole.
  5. For barley (or any other leaf without a petiole; e.g., rice, wheat), use scissors to cut a 6- to 10-cm-long piece of leaf blade. It is important to harvest leaf pieces of the same size and from the same area (typically 6-10 cm starting from the leaf tip). It is recommended to use at least 4-5 biological replications especially when measuring plants under different treatments (e.g. drought stress).
  6. Place the harvested leaf in a plastic bag immediately after cutting and close the bag (Figure 1).


    Figure 1. Representative leaves in the Zipper-locked plastic bag (8 x 12 cm). A. Arabidopsis mature rosette leaf; B. Tomato mature fully expanded leaflet; C. Barley 6- to 10-cm-long leaf blade end. Bottom and right part of the pic show a roller for estimation of plastic bag size.

  7. Make sure the bag is tightly sealed so that no vapor can escape. Put the bag in the closed (dark) box.
  8. Weigh each bag with the fresh leaf inside to determine the total fresh weight (TFW).
  9. Open the bag and gently make sure that the petiole is facing down. Insert 2-3 ml of 5 mM CaCl2 into the bag and make sure that only the leaf petiole is immersed in the solution (Figure 2). Close the bag and put the sample back in the box at room temperature.
  10. After 8 h, take the leaf out of the bag and put it between two paper towels to absorb excess water.
  11. Weigh the turgid leaf to determine the turgid weight (TW).
  12. Insert each sample into a paper bag and dry in a 60 °C dry oven for 3-4 days.
  13. Weigh the dried samples to determine the dry weight (DW).
  14. Calculate the relative water content of the leaf:

Notes

  1. It is very important to use high-quality zipper-locked bags, to prevent humidity and liquids from leaking out of the bag.
  2. Work quickly, but gently to maintain reliable results.
  3. Maintain the same order of sampling to ensure that all samples are measured using the same protocol.
  4. There are different zipper-locked bags that fit leaves of different sizes. Choose bags of a size appropriate for the species with which you are working.
  5. Make sure that only the petiole is immersed in the solution (or the cut part of the leaf in the case of grass species; Figure 2).


    Figure 2. Leaves in the Zipper-locked plastic bag after insertion of 2-3 ml of 5 mM CaCl2 into the bag. A. Arabidopsis mature rosette leaf; B. Tomato mature fully expanded leaflet; C. Barley 6- to 10-cm-long piece of leaf blade.
    Note the solution in the bottom part of the plastic bag.


  6. This protocol was used for measurements of 4 weeks old Tomato, Arabidopsis and Barley leaves under well irrigated conditions and short day photoperiod as stated in the Materials and Reagents section. However, it is suitable for different physiological age (as long as the leaves are fully expanded), different plants (as long as the morphological shape of the leaves is similar to those appear in this protocol), different photoperiod (both long and short day) and different irrigation regime (drought and salt).

Acknowledgments

This work was supported by the Israel Science Foundation (grant no. 1131/12) and the German-Israeli Project Cooperation (grant nos. FE 552/12–1 to A.R.F. and OR309/1-1.
This Protocol was adapted from Sade et al. (2014).

References

  1. Matin, M., Brown, J. H. and Ferguson, H. (1989). Leaf water potential, relative water content, and diffusive resistance as screening techniques for drought resistance in barley. Agron J 81(1): 100-105.
  2. Sade, N., Gebremedhin, A. and Moshelion, M. (2012). Risk-taking plants: anisohydric behavior as a stress-resistance trait. Plant Signal Behav 7(7): 767-770.
  3. Sade, D., Sade, N., Shriki, O., Lerner, S., Gebremedhin, A., Karavani, A., Brotman, Y., Osorio, S., Fernie, A. R., Willmitzer, L., Czosnek, H. and Moshelion, M. (2014). Water balance, hormone homeostasis, and sugar signaling are all involved in Tomato resistance to Tomato yellow leaf curl virus. Plant Physiol 165(4): 1684-1697.
  4. Sade, N., Vinocur, B. J., Diber, A., Shatil, A., Ronen, G., Nissan, H., Wallach, R., Karchi, H. and Moshelion, M. (2009). Improving plant stress tolerance and yield production: is the tonoplast aquaporin SlTIP2;2 a key to isohydric to anisohydric conversion? New Phytol 181(3): 651-661.

简介

测量叶片相对含水量(RWC)是一种可靠和简单的方法来评估叶片的水状况,而不需要特殊设备。 与叶水势相似,叶RWC给出植物对不同环境条件的响应的强烈指示; 然而RWC已被证明是比叶水势更稳定的参数(Sade等人,2009; Sade等人,2012)。 虽然测量RWC对叶片是破坏性的,但通过适当的规划,它不需要影响植物的行为。 本说明将集中于代表具有各种叶形状(例如拟南芥,番茄和大麦)的植物的三种不同的模型植物。 测量RWC的技术对于这三种物种(以及具有许多其它类型叶的植物)是相同的。

关键字:相对含水量, 叶片水分含量, 干旱胁迫, 盐胁迫

材料和试剂

  1. 在短日条件(8/16L/D; 22/22℃)下生长的4周龄拟南芥植物
  2. 在中等条件(10/14L/D; 25/20℃)下生长的4周龄番茄植物
  3. 在短日条件(8/16L/D; 16/10℃)下生长的4周龄大麦植物

设备

  1. 每个样品的拉链锁塑料袋(8 x 12厘米)
  2. 每个样品的纸袋(8 x 12厘米)
  3. 锋利的剪刀
  4. Scalpel
  5. 纸毛巾
  6. 具有0.1 mg可读性的分析天平
  7. 对于每个样品,在DDW水中2-3ml的5mM CaCl 2 2/
  8. 记号笔
  9. 可以牢固密封的塑料盒

软件

  1. Microsoft Excel

程序

  1. 种植植物。
  2. 在进行其他测量之前,请对每个塑料袋(BW)进行标记和称重
  3. 通常,在黎明和/或中午之前测量叶子(Matin等人,1989)。 在选定的小时,选择一个完全展开和成熟的叶子
  4. 对于拟南芥和番茄,用手术刀切割叶子,留下1厘米长的叶柄。
  5. 对于大麦(或任何其他没有叶柄的叶子;例如,大米,小麦),使用剪刀剪切6-10厘米长的叶片。 重要的是从相同面积(通常从叶尖开始6-10cm)收获相同大小的叶片。 建议使用至少4-5次生物复制,特别是当在不同处理(例如干旱胁迫)下测量植物时。
  6. 切割后立即将收获的叶子放在塑料袋中,然后关闭袋子(图1)。


    图1.拉链锁定塑料袋(8 x 12厘米)中的代表性叶子。 A.拟南芥成熟莲座叶; B.番茄成熟完全扩增 传单; C.大麦6-至10-cm长的叶片末端。底部和右边 pic的一部分显示用于估计塑料袋尺寸的滚筒
  7. 确保袋子密封,以免蒸气逸出。将袋子放在封闭(黑暗)的盒子里。
  8. 称重每个袋与新鲜叶里面,以确定总鲜重(TFW)
  9. 打开包,轻轻地确保叶柄朝下。将2-3ml的5mM CaCl 2插入袋中,并确保只有叶柄浸入溶液中(图2)。关闭袋子,将样品在室温下放回盒子中。
  10. 8小时后,将叶子从袋子里取出,放在两张纸巾之间吸收多余的水
  11. 称量膨胀叶以确定膨胀重量(TW)
  12. 将每个样品放入纸袋中,在60℃干燥炉中干燥3-4天
  13. 称重干燥的样品以确定干重(DW)
  14. 计算叶子的相对含水量:

笔记

  1. 使用高质量的拉链锁包,以防止湿气和液体从包装袋中泄漏是非常重要的。
  2. 快速工作,但轻轻地维持可靠的结果
  3. 保持相同的采样顺序,以确保所有样品都使用相同的协议进行测量
  4. 有不同的拉链锁袋适合不同大小的叶子。选择适合您工作物种大小的行李。
  5. 确保只有叶柄浸入溶液中(或在草种的情况下,切叶部分;图2)。


    图2.在将2-3ml的5mM CaCl 2溶液插入袋中后,在拉链锁定的塑料袋中的叶子。 A.拟南芥成熟莲座叶; B.番茄成熟完全扩增的传单; C.大麦6-至10厘米长的叶片。
    请注意塑料袋底部的解决方案。

  6. 该方案用于在良好灌溉条件和短日照光周期下测量4周龄番茄,拟南芥和大麦叶,如材料和试剂部分中所述。 然而,它适用于不同的生理年龄(只要叶充分膨胀),不同的植物(只要叶的形态形状类似于本协议中出现的那些),不同的光周期(长和短的天 )和不同的灌溉制度(干旱和盐)。

致谢

这项工作得到以色列科学基金会(授予号1131/12)和德以项目合作(授予FE 552/12-1给A.R.F.和OR309/1-1。)的支持。 本协议改编自Sade em et al。(2014)。

参考文献

  1. Matin,M.,Brown,J.H。和Ferguson,H。(1989)。 叶水势,相对含水量和扩散阻力作为耐旱性筛选技术 Agron J 81(1):100-105。
  2. Sade,N.,Gebremedhin,A.和Moshelion,M。(2012)。 冒险性植物:不对称行为是一种应激抵抗性状。 Plant Signal Behav 7(7):767-770。
  3. Sade,D.,Sade,N.,Shriki,O.,Lerner,S.,Gebremedhin,A.,Karavani,A.,Brotman,Y.,Osorio,S.,Fernie,AR,Willmitzer,L.,Czosnek ,H.和Moshelion,M.(2014)。 水平衡,激素内环境平衡和糖信号都涉及番茄抗性。番茄黄叶卷曲病毒。 植物生理 165(4):1684-1697。
  4. Sade,N.,Vinocur,B.J.,Diber,A.,Shatil,A.,Ronen,G.,Nissan,H.,Wallach,R.,Karchi,H.and Moshelion,M.(2009)。 提高植物胁迫耐受性和产量生产:是叶绿体水通道蛋白SlTIP2; 2是异羟基至异羟基的关键conversion? New Phytol 181(3):651-661。
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Copyright: © 2015 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. Sade, N., Galkin, E. and Moshelion, M. (2015). Measuring Arabidopsis, Tomato and Barley Leaf Relative Water Content (RWC). Bio-protocol 5(8): e1451. DOI: 10.21769/BioProtoc.1451.
  2. Sade, D., Sade, N., Shriki, O., Lerner, S., Gebremedhin, A., Karavani, A., Brotman, Y., Osorio, S., Fernie, A. R., Willmitzer, L., Czosnek, H. and Moshelion, M. (2014). Water balance, hormone homeostasis, and sugar signaling are all involved in Tomato resistance to Tomato yellow leaf curl virus. Plant Physiol 165(4): 1684-1697.
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