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Chlorophyll Content Assay to Quantify the Level of Necrosis Induced by Different R Gene/Elicitor Combinations after Transient Expression
通过测定叶绿素含量对瞬时表达后不同R基因/诱导子组合诱导产生的细胞坏死量进行定量测定   

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Abstract

This assay can be used to rapidly and accurately quantify levels of leaf necrosis induced after transient expression of R genes and elicitor combinations (Harris et al., 2013). It is based on the inverse correlation between level of necrosis and chlorophyll content in leaf tissue. It is adapted from the calculations described by (Strain et al., 1971).

Materials and Reagents

  1. 1.5 ml tube
  2. Leaf discs
    Note: Ensure that the leaf discs are fully submerged in the DMF solution.
  3. N, N-Dimethylformamide (DMF) (Sigma-Aldrich, catalog number: D4551-250 ml )

Equipment

  1. Spectrophotometer (Spectronic Biomate3) (Thermo Fisher Scientific, catalog number: 335904P )
  2. Glass spectrophotometer cuvettes (Sigma-Aldrich, catalog number: Z276898 )
    Note: Product Z276898 has been discontinued.

Procedure

  1. Place three leaf discs (e.g., 4 mm radius, from Eppendorf lid) into a 1.5 ml tube containing 1 ml of dimethylformamide (DMF). Use a cork borer to excise leaf disk. Include five replicates for each sample type. Ensure that the leaf discs are fully submerged in the DMF solution. Allow the chlorophyll to dissolve into the DMF solution by still incubation overnight at 4 °C.
  2. Mix 300 μl of sample solution with 600 μl of DMF in a fresh Eppendorf tube (2 volumes of DMF per volume of sample). Read the absorbance (A) in spectrophotometer at 647 nm and 664.5 nm wavelengths using a glass cuvette.
    Chlorophyll a content (µg/ml):
    = (12 x A664.5)-(2.79 x A647)
    Chlorophyll b content (µg/ml):
    = (20.78 x A647)-(4.88 x A664.5)
    Total chlorophyll content (µg/ml) = Chla + Chlb
    Sample area (for 3 leaf discs at 4 mm radius) (mm2) = 3 x πr2
    Total chlorophyll content (µg/mm2) = (Chla + Chlb)/Sample area

Notes

This protocol is most suitable for comparing treatments within an experiment, as the absolute chlorophyll content will vary between different sets of plants and over time. If measuring chlorophyll content after leaf infiltration assays, it is preferable to infiltrate all the treatment types to be compared on a single leaf. This can be repeated, in different configurations, on multiple leaves. To minimize the effects of inter-leaf variability, all the leaf discs from the same treatment type can then be pooled. A minimum of 15 leaf discs (3 per sample, five replicates) for each treatment type is required. While our experience with this protocol has been using leaves of Nicotiana benthamiana and N. tobacum, it has also been applied to (at least) Arabidopsis thaliana (Pruzinska et al., 2005) - as a proxy for leaf senescence - and could in principle be applied to many other plant species where chlorophyll content is of physiological relevance.

Acknowledgments

We would like to thank Sylvain Aubry for his inspiration in the establishing this protocol. Research in the Baulcombe laboratory is supported by the ERC Advanced Investigator grant ERC-2013-AdG 340642 TRIBE. C. J. H was supported by a BBSRC Ph.D. Studentship. D. C. B. is the Royal Society Edward Penley Abraham Research Professor.

References

  1. Harris, C. J., Slootweg, E. J., Goverse, A. and Baulcombe, D. C. (2013). Stepwise artificial evolution of a plant disease resistance gene. Proc Natl Acad Sci U S A 110(52): 21189-21194.
  2. Pruzinska, A., Tanner, G., Aubry, S., Anders, I., Moser, S., Muller, T., Ongania, K. H., Krautler, B., Youn, J. Y., Liljegren, S. J. and Hortensteiner, S. (2005). Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction. Plant Physiol 139(1): 52-63.
  3. Strain, H. H., Cope, B. T. and Svec, W. A. (1971). Analytical procedures for the isolation, identification, estimation and investigation of the chlorophylls. Methods Enzymol 23: 452-476.

简介

该测定可用于快速和准确地定量R基因和激发剂组合的瞬时表达后诱导的叶坏死的水平(Harris等人,2013)。 它是基于坏死水平与叶组织中叶绿素含量之间的逆相关性。 它根据(Strain等人,1971)描述的计算改编。

材料和试剂

  1. 1.5 ml管
  2. 叶片
    注意:确保叶片完全浸没在DMF溶液中。
  3. N,N-二甲基甲酰胺(DMF)(Sigma-Aldrich,目录号:D4551-250ml)

设备

  1. 分光光度计(Spectronic Biomate3)(Thermo Fisher Scientific,目录号:335904P)
  2. 玻璃分光光度计比色杯(Sigma-Aldrich,目录号:Z276898)
    注意:产品Z276898已停产。

程序

  1. 将三片叶片(例如,4mm半径,来自Eppendorf盖)放入含有1ml二甲基甲酰胺(DMF)的1.5ml管中。使用软木钻孔器切除叶盘。每个样品类型包括五个重复。确保叶圆片完全浸没在DMF溶液中。在4℃下孵育过夜,使叶绿素溶解在DMF溶液中
  2. 将300μl样品溶液与600μlDMF在新鲜的Eppendorf管中混合(每体积样品2倍体积的DMF)。使用玻璃比色皿读取分光光度计在647 nm和664.5 nm波长的吸光度(A)。
    叶绿素 a 内容(μg/ml):
    =(12×A <664.5) - (2.79×A 647
    叶绿素 b 内容(μg/ml):
    =(20.78×A 647) - (4.88×A 664.5
    总叶绿素含量(μg/ml)= Chl a + Chl b 样品面积(对于4mm半径的3片叶片)(mm 2)= 3×πr2
    总叶绿素含量(μg/mm 2 )=(Chl a + Chl b )/示例区域

笔记

该方案最适合于比较实验中的处理,因为绝对叶绿素含量将在不同组的植物之间和随时间变化。如果在叶浸润测定后测量叶绿素含量,优选在单叶上渗透所有待比较的处理类型。这可以在不同的配置中在多个叶子上重复。为了使叶间变异性的影响最小化,然后可以汇集来自相同处理类型的所有叶盘。每种治疗类型需要至少15个叶盘(每个样品3个,重复5次)。虽然我们对这个协议的经验已经使用烟草的叶片 和 N。 tobacum ,它也已被应用于(至少)拟南芥(Pruzinska等人,2005) - 作为叶衰老的代表,并且可以主要应用于许多其他叶绿素含量具有生理相关性的植物物种。

致谢

我们要感谢Sylvain Aubry在建立这个协议的灵感。 Baulcombe实验室的研究由ERC高级研究者奖励ERC-2013-AdG 340642 TRIBE支持。 C. J. H由BBSRC博士支持。学生。 D. C. B.是皇家学会爱德华·彭利亚伯拉罕研究教授。

参考文献

  1. Harris,C.J.,Slootweg,E.J.,Goverse,A.and Baulcombe,D.C。(2013)。 植物病害抗性基因的逐步人工进化。 Proc Natl Acad Sci USA 110(52):21189-21194。
  2. Pruzinska,A.,Tanner,G.,Aubry,S.,Anders,I.,Moser,S.,Muller,T.,Ongania,KH,Krautler,B.,Youn,JY,Liljegren,SJand Hortensteiner,S 。(2005)。 拟南芥叶片衰老中的叶绿素分解。参与去细胞反应的叶绿素分解代谢物和叶绿素分解代谢酶的表征。植物生理学139(1):52-63。
  3. Strain,H.H.,Cope,B.T.and Svec,W.A。(1971)。 叶绿素的分离,鉴定,估计和研究的分析程序。 Methods Enzymol 23:452-476
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引用:Harris, C. J. and Baulcombe, D. C. (2015). Chlorophyll Content Assay to Quantify the Level of Necrosis Induced by Different R Gene/Elicitor Combinations after Transient Expression. Bio-protocol 5(23): e1670. DOI: 10.21769/BioProtoc.1670.
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