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Chlorophyll Fluorescence Measurements in Arabidopsis Plants Using a Pulse-amplitude-modulated (PAM) Fluorometer
采用脉冲调幅(PAM)荧光计测定拟南芥植株的叶绿素荧光   

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Abstract

In this protocol, to analyze PSII activity in photosynthesis, we measure the Fv/Fm (Fv=Fm ± Fo) value (Fo and Fm are the minimum and maximum values of chlorophyll fluorescence of dark-adapted leaves, respectively). Fv/Fm is a reliable marker of photo- inhibition (Krause et al., 1988). Chlorophyll fluorescence in leaves was measured at room temperature using a photosynthesis yield analyzer (MINI- PAM, Walz, Effeltrich, Germany) and a pulse-amplitude-modulated (PAM) fluorometer (TEACHING-PAM, Walz, Effeltrich, Germany).

Keywords: PAM(帕姆), Chlorophyll(叶绿素), Fluorescence(荧光), Arabidopsis(拟南芥)

Materials and Reagents

  1. Arabidopsis plants
    Note: We plated Nossen ecotype seeds that had been surface-sterilized on germination medium (GM) agar plates (Motohashi et al., 2003) containing 1% sucrose, with the appropriate selection agent (antibiotic or herbicide) per specific genotype. Plants were kept at 4 °C for 3 days to improve germination rates and then grown in lighted growth chambers (CF-405, TOMY-Seiko, Tokyo, Japan) with approximately 75 μmol photon/m2/s at 22 °C under a 16 h-light /8-h dark cycle (long-day conditions) for 3 weeks.

Equipment

  1. Photosynthesis yield analyzer (Walz, MINI- PAM) (the equipment used in this protocol) (Figure 1)
    Compact design and easy operation are the most outstanding features of the MINI-PAM. This device is in particular well-suited for determination of quantum yield and photosynthetic electron transport rate (ETR). A flexible 5.5 mm glass fiberoptic was attached in the system and it can provide considerable high actinic intensities of white light. An optional 2 mm plastic fiberoptic (MINI-PAM/F1) is also used by excellent signal quality and can be attached to the cover of an optional gas-exchange system for measuring both CO2 and H2O exchange as well as fluorescence. For an exact measuring quantum flux density and temperature at precisely the fluorescence measuring spot, a useful leaf-clip holder is available as an accessory (Arabidopsis Leaf-Clip Holder, model: 2060-B). This leaf clip holder is especially developed for small leaves like an Arabidopsis leaf. With the help of the leaf clip holder, the photosynthetic active radiation (PAR) can be measured and an apparent electron transport rate (ETR) is calculated.
    A simple explanation of the equipment used can be found at the following URL: http://www.walz.com/downloads/manuals/mini-pam-II/MINI-PAM-II_Broschure.pdf. It should be noted that the current equipment being sold is the MINI-PAM II.


    Figure 1. Photosynthesis yield analyzer

  2. Pulse-amplitude-modulated (PAM) fluorometer (TEACHING-PAM) (Walz) (alternative equipment which can be used to measure chlorophyll fluorescence)
    Note: It is noted here that the MINI- PAM and TEACHING-PAM were developed for beginners; advanced researchers may utilize the larger PAM-2000 fluorometer (essentially the same instrument) to yield additional and more detailed results.

Procedure

Protocol for using the MINI-PAM as referenced from the official instruction manual (TEACHING-PAM has a similar protocol and as such is not included here). Basically the most relevant fluorescence parameters of MINI-PAM are automatically obtained by a single key operation within a second and up to 4,000 data sets are stored for future analysis:
(For reference, information regarding the MINI-PAM-II can be downloaded from the following URL: http://www.walz.com/downloads/manuals/mini-pam-II/MINI-PAM-II_Broschure.pdf.)

  1. Plants grown normally for 3 weeks are dark-adapted for 20 min before chlorophyll fluorescence measurements. *In our case, dark-adapted means the plants are kept either in a dark drawer (for plated plants) or covered with a large box (for potted plants), in both cases in rooms with dark curtains and no artificial light sources.
  2. Setup the MINI-PAM components. Additional peripheral components were connected to the four sockets at the side of the MINI-PAM Main Control Unit. PIN-assignments of “LEAF CLIP”, “RS 232”, “OUTPUT” and “CHARGE” indicate a Leaf Clip holder 2030-B, Computer control, Chart recorder and Battery Charger, respectively. The MINI-PAM was conceived as a typical stand-alone instrument for field experiments. Thus the actual measurement of the most relevant YIELD-parameter (quantum yield of photochemical energy conversion) just connected the fiberoprtics and leaf clip holder without conjunction with a PC and the WinControl software. So this protocol introduces the basic operation of the MINI-PAM without using computer control.
  3. Activate the MINI-PAM by pressing the “ON” button. Under standard conditions, the measuring light is on automatically.
  4. The AUTO-ZERO function (MODE-menu point 2) should be applied to determine the signal in absence of sample (background signal). To move to MODE-menu point 2, press “MODE” button (possible to omit) and “∧” button one times to select 2 of 51 points of the MODE-menu. Then push “SET” button to set the F value to zero (not stable, blinking) on measuring light (Figure 2).


    Figure 2. F value blinks “0” after pressing the “SET” button

  5. Place a dark-adapted leaf sample on the measuring head of the Leaf Clip holder. The distance between sample and fiberoptics should be about 10-15 mm (Figure 3).
    We dark-adapt the plants by either putting them in drawers (for dished plants) or covering them with boxes (for potted plants) – in both cases dark curtains are used and all artificial lights are turned off. Temperature when measuring should be the same as the growth environment.


    Figure 3. A photo of the leaf clip holder and fiberoptics

  6. Just press the “START” button. Measuring the fluorescence parameters is proceeding automatically within seconds (see below).
    1. the minimum fluorescence in dark-adapted state (Fo) is sampled (displayed as ...F).
    2. a saturation pulse is applied.
    3. a saturation pulse induced maximum fluorescence in dark-adapted state (Fm) is sampled (displayed as …M).
    4. YIELD=(Fm-Fo)/Fm=Fv/Fm is calculated and shown on the display as …Y.
    5. When you use the Leaf Clip holder, the photosynthetically active radiation (PAR) and temperature at the same spot of a leaf where fluorescence is measured is also sampled (displayed as …L and …C, respectively).
    6. The apparent rate of electron transport (ETR) =YIELD x PAR x 0.5 x ETR-factor (0.84) is calculated (displayed as …E).
  7. The parameter indicated by the above is shown to a screen after measurement (Figure 4). The obtained data are stored in the MEMORY.
    Arabidopsis plants under normal growth condition shows an Fv/Fm value between 0.75 to 0.85. (If Fm/Fv is not between 0.75 and 0.85, it is highly likely that the sample Arabidopsis plants are in poor health or not properly grown.)


    Figure 4. A photo of the machine display after measurement

  8. If you want to know only the Fv/Fm value, following analysis is not needed.
    On the other hand, when leaf is illuminated, its fluorescence yield can change between Fo and Fm, which can be assessed after well dark-adaptation. Lower Fm value under light conditions may be caused either by photochemical quenching or by non-photochemical quenching (NPQ). The quenching coefficients are defined as follows:
    qP=(Fm’-F)/(Fm’-Fo)
    qN=(Fm-Fm’)/(Fm-Fo)
    NPQ=(Fm-Fm’)/Fm’
    A saturation pulse induced maximum fluorescence during light adaptation (Fm’) is sampled (displayed as …M).
  9. These quenching coefficients need to sample four values (Fo, Fm, F and Fm’). Others are calculated values by using these four parameters. The value of Fo and Fm were previously measured by using a dark-adapted leaf sample. Thus, these values need to store in the MINI-PAM system.
  10. The MODE-menu point 25 (Fo and Fm) should be applied to store the values of Fo and Fm (Figure 5). This function to sample Fo and Fm of a dark-adapted leaf by use of the SET-key. The stored Fo and Fm values are used for determination of qP, qN and NPQ.


    Figure 5. A photo of the display when setting Fo and Fm (“Mode” menu 25)

  11. Then light adapted leaf samples are prepared. A same as a dark-adapted leaf sample, press the “START” button on procedure 6. Measuring the fluorescence parameters under light condition is proceeding automatically within seconds and calculated qP, qN and NPQ as well as YIELD (Fv’/Fm’), ETR and PAR. The obtained data are stored in the MEMORY.
  12. Recall on display via MEM-key. Push “MEM” button and select measured sample by using “∧” and “∨” button.
  13. In the top line it can be seen the data set number and recording day time (Figure 6A). The bottom line shows YIELD (Y), ETR (E) and PAR (L).
  14. More information of data set can be displayed by pushing “SET” button. After the first SET, the top line shows the fluorescence yield measured briefly before the saturating light pulse (F), the maximum fluorescence (M) and temperature (C) (Figure 6B).
  15. After the second SET, the top line shows the quenching coefficients qP (P), qN (N) and NPQ (Q) (Figure 6C).


    Figure 6. A photo showing the display for “Mode” menu 25. A. The first display line shows data set number and recording day/time. The second line shows YIELD (Y), ETR (E) and PAR (L).  B. The first display line shows the fluorescence yield measured briefly before saturating light pulse (F), the maximum fluorescence (M) and temperature (C). C. The first display line shows the quenching coefficients qP (P), qN (N) and NPQ (Q).

  16. Repeat the same measurement at least four times and average results.

Notes

  1. In order to obtain reliably reproducible data, it is imperative that the plant growth environment be as uniform / consistent as possible. For example depending on light environment the value of a plant chlorophyll fluorescence will fluctuate. The amount of light a plant receives when next to the side light on the growth incubator is completely different from the light it receives when on the center of the shelf. For the reason it is important to shuffle the location of growth mediums, etc (Figure 7).


    Figure 7. A visual example of how growth mediums might be shuffled

  2. At least 5 replicates are measured, with final data being an average of these measurements. As measurement with the MINI-PAM is very easy and results are consistent over each measurement, measuring twice is enough to satisfy technical duplication requirements.

Acknowledgments

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (Japan) [Grants-in-Aid for Scientific Research (No.17681022 to R.M.)].
This protocol is modified and appended referencing the original, as featured in "Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins" (Satou et al., 2014).

References

  1. Krause, G. H., Grafflage, S., Rumich-Bayer, S. and Somersalo, S. (1988). Effects of freezing on plant mesophyll cells. Symp Soc Exp Biol 42: 311-327.
  2. Satou, M., Enoki, H., Oikawa, A., Ohta, D., Saito, K., Hachiya, T., Sakakibara, H., Kusano, M., Fukushima, A., Saito, K., Kobayashi, M., Nagata, N., Myouga, F., Shinozaki, K. and Motohashi, R. (2014). Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins. Plant Mol Biol 85(4-5): 411-428.
  3. Motohashi, R., Ito, T., Kobayashi, M., Taji, T., Nagata, N., Asami, T., Yoshida, S., Yamaguchi-Shinozaki, K. and Shinozaki, K. (2003). Functional analysis of the 37 kDa inner envelope membrane polypeptide in chloroplast biogenesis using a Ds-tagged Arabidopsis pale-green mutant. Plant J 34(5): 719-731.
  4. Valvekens, D., Montagu, M. V. and Van Lijsebettens, M. (1988). Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci U S A 85(15): 5536-5540.

简介

在这个协议中,为了分析光合作用中的PSII活性,我们测量Fv/Fm(Fv = Fm±Fo)值(Fo和Fm分别是暗适应叶的叶绿素荧光的最小值和最大值)。 Fv/Fm是光抑制的可靠标记(Krause等人,1988)。 在室温下使用光合作用产量分析仪(MINI-PAM,Walz,Effeltrich,Germany)和脉冲幅度调制(PAM)荧光计(TEACHING-PAM,Walz,Effeltrich,Germany)测量叶片中的叶绿素荧光。

关键字:帕姆, 叶绿素, 荧光, 拟南芥

材料和试剂

  1. 拟南芥植物
    注意:将已经在含有1%蔗糖的发芽培养基(GM)琼脂平板(Motohashi等人,2003)上表面灭菌的Nossen生态型种子以及每种特定基因型的合适的选择剂(抗生素或除草剂) 。将植物在4℃保持3天以提高发芽率,然后在发光生长室(CF-405,TOMY-Seiko,Tokyo,Japan)中生长,大约75μmol光子/m < > /s在22℃,16小时光照/8小时黑暗周期(长时间条件)下3周。

设备

  1. 光合作用产量分析仪(Walz,MINI-PAM)(本协议中使用的设备)(图1)
    紧凑的设计和易于操作是MINI-PAM最突出的特点。该装置特别适合于量子产率和光合电子传递速率(ETR)的测定。将柔性5.5mm玻璃光纤附接在系统中,并且其可以提供相当大的高光化强度的白光。一个可选的2 mm塑料光纤(MINI-PAM/F1)也使用优良的信号质量,可以连接到 用于测量CO 2和H 2 O交换以及荧光的任选的气体交换系统。对于在精确的荧光测量点处的精确的测量量子通量密度和温度,可使用有用的叶夹持器作为附件(拟南芥叶夹持器,型号:2060-B)。这种叶夹持器是专为小叶如拟南芥叶开发的。在叶夹夹持器的帮助下,可以测量光合有效辐射(PAR),并计算表观电子传输速率(ETR)。
    有关所使用设备的简单说明,请访问以下网址: http://www.walz.com/downloads/manuals/mini-pam-II/MINI-PAM-II_Broschure.pdf 。应该注意,目前出售的设备是MINI-PAM II

    图1。 光合作用产量分析器

  2. 脉冲幅度调制(PAM)荧光计(TEACHING-PAM)(Walz)(可用于测量叶绿素荧光的替代设备)
    注意:这里要注意的是,MINI-PAM和TEACHING-PAM是为初学者开发的;高级研究人员可以使用更大的PAM-2000荧光计(本质上是相同的仪器)产生更多更详细的结果。

程序

使用MINI-PAM的协议,从官方使用手册(TEACHING-PAM有一个类似的协议,因此不包括在这里)。基本上,MINI-PAM的最相关的荧光参数是通过一秒钟内的单次按键操作自动获得的,最多可存储4,000个数据集,以供将来分析:
(作为参考,有关MINI-PAM-II的信息可以从以下URL下载: http://www.walz.com/downloads/manuals/mini-pam-II/MINI-PAM-II_Broschure.pdf

  1. 在叶绿素荧光测量之前,正常生长3周的植物暗适应20分钟。 *在我们的情况下,暗适应意味着植物被保存在黑暗的抽屉(对于被镀的植物)或用一个大箱子(为盆的植物),在两个案件在有黑暗的窗帘,没有人为光源的房间。 br />
  2. 设置MINI-PAM组件。附加的外围组件连接到MINI-PAM主控制单元侧面的四个插座。 "LEAF CLIP","RS 232","OUTPUT"和"CHARGE"的PIN分配分别表示叶夹保持器2030-B,计算机控制,图表记录器和电池充电器。 MINI-PAM被认为是用于现场实验的典型的独立仪器。因此,最相关的YIELD参数(光化学能量转换的量子产率)的实际测量仅连接纤维和叶夹持器,而不与PC和WinControl软件结合。因此,该协议介绍了MINI-PAM的基本操作,而不使用计算机控制
  3. 通过按"ON"按钮激活MINI-PAM。在标准条件下,测量灯自动打开。
  4. 应该应用AUTO-ZERO功能(MODE-菜单点2)来确定没有样品(背景信号)的信号。要移动到MODE菜单点2,按"MODE"按钮(可以省略)和"∧"按钮一次,从MODE菜单的51个点中选择2个。然后按"SET"按钮将测量光的F值设置为零(不稳定,闪烁)(图2)。


    图2. F值在按下"SET"按钮后闪烁"0"

  5. 在叶夹支架的测量头上放置一个黑暗适应的叶样本。样品和光纤之间的距离应为约10-15 mm(图3) 我们通过将它们放入抽屉(用于凹陷的植物)或用盒子(用于盆栽植物)覆盖它们来黑暗适应植物 - 在这两种情况下,使用黑色窗帘并关闭所有人工灯。测量时的温度应与生长环境相同。


    图3。 叶夹持有者和光纤的照片

  6. 只需按下"开始"按钮。 测量荧光参数在几秒钟内自动进行(见下文)。
    1. 在暗适应状态(Fo)中的最小荧光被采样(显示为... F)
    2. 施加饱和脉冲。
    3. 在暗适应状态(Fm)中饱和脉冲诱导的最大荧光被采样(显示为... M)
    4. 计算YIELD =(Fm-Fo)/Fm = Fv/Fm,并在显示屏上显示为... Y。
    5. 当您使用叶夹夹,光合有效 辐射(PAR)和温度在叶片的同一点 荧光测量也被采样(显示为... L和... C, 分别)。
    6. 计算电子传输的表观速率(ETR)= YIELD×PAR×0.5×ETR-因子(0.84)(显示为... E)。
  7. 上述指示的参数显示在测量后的屏幕上(图4)。获得的数据存储在MEMORY中。
    在正常生长条件下的拟南芥植物表现出0.75至0.85之间的Fv/Fm值。 (如果Fm/Fv不在0.75和0.85之间,那么样品拟南芥植物很可能健康状况不佳或生长不良。)


    图4。 测量后机器显示的照片

  8. 如果只想知道Fv/Fm值,则不需要进行以下分析。
    另一方面,当叶被照亮时,其荧光产量可以在Fo和Fm之间变化,这可以在完全黑暗适应之后评估。在光条件下较低的Fm值可以由光化学淬灭或通过非光化学淬灭(NPQ)引起。淬火系数定义如下:
    qP =(Fm'-F)/(Fm'-Fo)
    qN =(Fm-Fm')/(Fm-Fo)
    NPQ =(Fm-Fm')/Fm'
    在光适应(Fm')期间饱和脉冲引起的最大荧光被采样(显示为... M)
  9. 这些淬火系数需要采样四个值(Fo,Fm,F和Fm')。其他是通过使用这四个参数的计算值。 Fo和Fm的值先前通过使用暗适应叶样品测量。因此,这些值需要存储在MINI-PAM系统中。
  10. 应该应用MODE-菜单点25(Fo和Fm)来存储Fo和Fm的值(图5)。此函数通过使用SET键来采样暗适应叶的Fo和Fm。存储的Fo和Fm值用于确定qP,qN和NPQ

    图5。 设置Fo和Fm("模式"菜单25)时显示的照片

  11. 然后制备适应光的叶样品。与暗适应叶样品相同,按程序6上的"开始"按钮。在光条件下测量荧光参数是自动进行的 秒和计算的qP,qN和NPQ以及YIELD(Fv'/Fm'),ETR和PAR。获得的数据存储在MEMORY中。
  12. 通过MEM键显示。按"MEM"按钮,使用"∧"和"∨"按钮选择测量样品
  13. 在顶行中,可以看到数据集编号和记录日时间(图6A)。底线显示YIELD(Y),ETR(E)和PAR(L)
  14. 通过按"SET"按钮可以显示数据集的更多信息。在第一次SET之后,顶行显示在饱和光脉冲(F),最大荧光(M)和温度(℃)(图6B)之前短暂测量的荧光产量。
  15. 在第二次SET之后,顶行显示淬灭系数qP(P),qN(N)和NPQ(Q)(图6C)。


    图6。 显示"模式"菜单25的显示照片 A.第一个显示行显示数据集编号和录制日期/时间。第二行显示YIELD(Y),ETR(E)和PAR(L)。 B.第一显示线显示在饱和光脉冲(F),最大荧光(M)和温度(C)之前短暂测量的荧光产量。第一显示行显示淬灭系数qP(P),qN(N)和NPQ(Q)。

  16. 重复相同的测量至少四次和平均结果。

笔记

  1. 为了获得可靠的可再现数据,必须使植物生长环境尽可能均匀/一致。 例如,根据光环境,植物叶绿素荧光的价值将波动。 植物在靠近生长培养箱上的侧灯时接收的光的量与在搁架的中心上接收的光完全不同。 因为重要的是洗涤生长介质的位置,等(图7)。


    图7。 生长介质如何洗牌的视觉示例

  2. 测量至少5个重复,最终数据是这些测量的平均值。由于使用MINI-PAM的测量非常容易,每次测量的结果都是一致的,测量两次就足以满足技术重复要求。

致谢

这项工作得到教育,文化,体育,科学和技术部(日本)[科学研究助理(No.17681022至R.M.)]的支持。
该协议被修改并附加参考原始的,如"Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins"(Satou等人,2014)。

参考文献

  1. Krause,G.H.,Grafflage,S.,Rumich-Bayer,S。和Somersalo,S。(1988)。 冷冻对植物叶肉细胞的影响 Symp Soc Exp Biol < em> 42:311-327。
  2. Satou,M.,Enoki,H.,Oikawa,A.,Ohta,D.,Saito,K.,Hachiya,T.,Sakakibara,H.,Kusano,M.,Fukushima,A.,Saito, Kobayashi,M.,Nagata,N.,Myouga,F.,Shinozaki,K.and Motohashi,R。(2014)。 拟南芥白色或转绿色突变体的转录组和代谢组的综合分析被破坏的核编码叶绿体蛋白。 Plant Mol Biol 85(4-5):411-428。
  3. Motohashi,R.,Ito,T.,Kobayashi,M.,Taji,T.,Nagata,N.,Asami,T.,Yoshida,S.,Yamaguchi-Shinozaki,K.and Shinozaki,K。 37 kDa内包膜多肽在叶绿体生物发生中的功能分析,使用Ds-标记的拟南芥淡绿色突变体。植物J 34(5):719-731。
  4. Valvekens,D.,Montagu,M.V.and Van Lijsebettens,M。(1988)。 根瘤土壤杆菌 - 介导的拟南芥的转化 >通过使用卡那霉素选择来检测根外植体。 Proc Natl Acad Sci USA 85(15):5536-5540。
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引用:Motohashi, R. and Myouga, F. (2015). Chlorophyll Fluorescence Measurements in Arabidopsis Plants Using a Pulse-amplitude-modulated (PAM) Fluorometer. Bio-protocol 5(9): e1464. DOI: 10.21769/BioProtoc.1464.
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