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Chlorophyll Fluorescence Measurements in Arabidopsis Wild-type and Photosystem II Mutant Leaves
野生型拟南芥和光合系统II突变体的叶片中叶绿素荧光的测定   

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The Plant Journal
Apr 2014

Abstract

Chlorophyll fluorescence measurement is a widely used technique to determine photosynthetic performance. Light energy absorbed by a chlorophyll molecule can be dissipated by driving photochemical energy conversion, as heat in non-photochemical quenching processes, or it is re-emitted as fluorescence. The loss of light energy as chlorophyll fluorescence is primarily derived from photosystem II. Photosystem II is a thylakoid-embedded multiprotein complex which provides the high redox potential needed to oxidize water. Within photosystem II photons of light are captured and used to energize electrons. Energized electrons are fed into the linear electron transport chain and photosystem II replenishes lost electrons with electrons from splitting of water. Chlorophyll fluorescence yield can be quantified using a modulated fluorometer device. In such a device, a modulated measuring light beam (switched on and off at a high frequency) and the parallel detection of fluorescence exclusively excited by the measuring light allows chlorophyll fluorescence measurements in the presence of photosynthetic (actinic) light. In addition, high intensity, but short duration light flashes (saturating pulses) are used to determine maximum fluorescence yields in dark and light adapted leaves. In this protocol the procedure to receive a typical fluorescence graph of Arabidopsis wild-type leaves is given. Furthermore, this procedure can be used to identify Arabidopsis mutant plants affecting photosystem II, on the basis of the respective fluorescence graphs and values.

Keywords: Arabidopsis (拟南芥), Phtotosynthesis (phtotosynthesis), Photosystem II (光系统II), Chlorophyll fluorescence (叶绿素荧光), Pulse Amplitude Modulation PAM (脉冲振幅调制)

Materials and Reagents

  1. Arabidopsis wild-type plants (Ecotype: Col-0)
  2. Arabidopsis mutant plant of interest (Ecotype: Col-0)
  3. Stender Vermehrungssubstrat A210, 70 potting soil (Stender AG)
  4. Planting pots (7 x 6 x 6 cm)
  5. Plant tray and plastic wrap
  6. Tweezers (not sterile)

Equipment

  1. Cold room or refrigerator (4 °C)
  2. Growth chamber (12 h light/12 h dark with 21 °C/18 °C and a PFD of ~ 100 µmol/m2/s)
  3. Dual-PAM-100 for measuring chlorophyll fluorescence (Heinz Walz GmbH, model: Dual-PAM-100) connected to a PC and operated by the Dual-PAM software (see Note 1)
  4. Dark room

Software

  1. Dual-PAM software

Procedure

  1. Growing Arabidopsis plants
    1. Seeds of Arabidopsis wild-type and mutants lines are transferred to planting pots (approximately 20 seeds per pot) filled with completely soaked soil. Place a plastic wrap to cover the plants to keep necessary humidity.
    2. Put the pots in a tray in 4 °C for 3 days to synchronize germination.
    3. Transfer tray into a growth chamber with the plastic wrap still covering.
    4. Normally seeds germinate within 4-5 days, remove the plastic wrap 2 days afterwards.
    5. Transfer individual plants into fresh pots (one plant per pot) filled with fresh, soaked soil after 7 further days using slightly curved tweezers.
    6. Cultivate Arabidopsis plants in the same condition as before for another 1 or 2 weeks. See Note 2.

  2. Recording chlorophyll fluorescence
    1. Make sure that the Dual-PAM-100 device is properly connected to the PC and placed in a dark room.
    2. Place 3-4 week old Arabidopsis wild-type plants in a dark room for at least 30 min (this incubation is called dark adaptation). Perform subsequent steps in darkness. (Optional) Turn on green light for handling plants.


      Figure 1. Set up sample leaves between the measuring head of the Dual-Pam-100 device. For the protocol given here (e.g. duration time 6 min) a detached leaf is fixed between the measuring head (A). For modified protocols it might be useful to keep the plant intact by using an attached leaf between the measuring head (B).

    3. Take a middle aged (e.g. the seventh leaf from the apex by counting down) wild-type rosette leaf and fix it between Dual-PAM-100 measuring heads (Figure 1A). (Optional) Try to fix the leaf, still attached to the remaining plant, between the measuring heads (Figure 1B).
    4. Switch on the Dual-PAM software and define the settings. To measure chlorophyll fluorescence choose the <Fluo> and <SP-analysis> mode. A typical set up includes measuring light at 12 µmol/m2/s, the saturated pulse (SP) at 10,000 µmol/m2/s for 800 ms and the actinic light at 70 µmol/m2/s. Save user settings, thus allowing quick start.
    5. Open the <Slow Kinetics> window and apply measuring light for approximately 10 sec, fluorescence level is minimal (F0).
    6. Application of a saturated pulse will induce a maximal fluorescence level (Fm).
    7. After another minute switch on actinic light. Adjust the light intensity of the actinic light to growth light (see above). Perform actinic light treatment for 5 min, the level of fluorescence at that point is called Ft (= F0’ in <Report> window).
    8. A second saturated pulse is applied, which allows the measurement of the maximum fluorescence in the light (Fm’).
    9. Switch off actinic light, stop recording, export <Slow Kin. File> into a table calculation program (e.g. Excel) and recreate the graph (Figure 2A). Optional: Values can be averaged to recreate the graph. Repeat the measurement 3 times with different wild-type plants, but use leaves of corresponding age (e.g. the seventh leaf from the apex by counting down).
    10. Repeat the measurement with a leaf of the dark-adapted mutant plant (Figure 2B).

  3. Calculation of photochemical quenching parameters
    1. Open the <Report> window. Values for F0 and Fm are given. Calculate the maximum quantum yield according to the following formular (Maxwell and Johnson, 2000):    Fv/Fm = (Fm – F0)/Fm
    2. Values for Ft (= F0 in <Report> window) and Fm’ are given in <Report>. Calculate the effective quantum yield of PSII photochemistry (Maxwell and Johnson, 2000):
      ΦII = (Fm’ - Ft)/ Fm

Representative data


Figure 2. Fluorescence graphs of a wild-type (A) and a reference PSII mutant plant (B), see Note 3. Application of measuring light (ML) and saturated light pulses (SP) are indicated. Exposure to actinic light is shown and fluorescence parameters F0, Fm, Ft and Fm’ are denoted.

Notes

  1. For first time users it is recommended to start with the Junior PAM device, because of its easy set up and use (Heinz Walz GmbH, http://www.walz.com/products/chl_p700/junior-pam/introduction.html).
  2. It is important to grow healthy Arabidopsis plants, because any stress condition will eventually affect photosynthetic performance. Do not injure the root system upon transfer. Water plants approximately twice a week.
  3. A typical wild-type fluorescence graph is shown in Figure 2A. The fluorescence graph of a reference photosystem II mutant (psbo1/o2) is shown in Figure 2B. This mutant was generated by crossing psbo1-3 and psbo2-2, which carry a T-DNA insertion in exon 1 of PsbO1 and in the 5’ UTR of PsbO2, respectively (Figure 3A). Disruption of PsbO1 and PsbO2 entails a reduced PsbO protein level (Figure 3B). Particular mentionable is the drop of fluorescence below the F0 value after switching on actinic light. This phenomenon concomitant with a reduced Fv/Fm value is typically observed in PSII mutants (Peng et al., 2006; Armbruster et al., 2010; Schneider et al., 2014). Almost any PSII mutant with these characteristics is reduced in growth (Figure 3C).


    Figure 3. Generation of a reference PSII mutant plant (psbo1/o2) by crossing the homozygous T-DNA insertion line RATM12-1816-1 and homozygous T-DNA insertion line CSHL-ET9214 (A). The PsbO protein content is reduced in the psbo1/o2 mutant line as deduced from immunoblotting using a PsbO- and anLhcb2-specific antibody (B). Wild-type and psbo1/o2 mutant plants were grown for 4 weeks in a growth chamber (C).

Acknowledgments

This protocol was adapted or modified from previous work by Meurer et al. (1995). We thank Thilo Rühle for discussion and comments on the protocol. This work was carried out in the laboratory of Prof. Dario Leister (Biozentrum der LMU München, Department Biologie I, Munich, Germany) and supported by funds from the Deutsche Forschungsgemeinschaft (LE 1265/20-1) to D.L.

References

  1. Armbruster, U., Zuhlke, J., Rengstl, B., Kreller, R., Makarenko, E., Ruhle, T., Schunemann, D., Jahns, P., Weisshaar, B., Nickelsen, J. and Leister, D. (2010). The Arabidopsis thylakoid protein PAM68 is required for efficient D1 biogenesis and photosystem II assembly. Plant Cell 22(10): 3439-3460.
  2. Maxwell, K. and Johnson, G. N. (2000). Chlorophyll fluorescence--a practical guide. J Exp Bot 51(345): 659-668.
  3. Meurer, J., Meierhoff, K. and Westhoff, P. (1995). Isolation of high-chlorophyll-fluorescence mutants of Arabidopsis thaliana and their characterization by spectroscopy, immunoblotting and Northern hybridization. Planta 198, 385-396.
  4. Peng, L., Ma, J., Chi, W., Guo, J., Zhu, S., Lu, Q., Lu, C. and Zhang, L. (2006). LOW PSII ACCUMULATION1 is involved in efficient assembly of photosystem II in Arabidopsis thaliana. Plant Cell 18(4): 955-969.
  5. Schneider, A., Steinberger, I., Strissel, H., Kunz, H. H., Manavski, N., Meurer, J., Burkhard, G., Jarzombski, S., Schunemann, D., Geimer, S., Flugge, U. I. and Leister, D. (2014). The Arabidopsis Tellurite resistance C protein together with ALB3 is involved in photosystem II protein synthesis. Plant J 78(2): 344-356.

简介

叶绿素荧光测量是一种广泛使用的技术来确定光合性能。由叶绿素分子吸收的光能可以通过驱动光化学能量转化,作为非光化学淬灭过程中的热量而消散,或者其作为荧光再发射。作为叶绿素荧光的光能的损失主要来自光系统II。光系统II是类囊体嵌入的多蛋白复合物,其提供氧化水所需的高氧化还原电位。在光系统II中,光子被捕获并用于激发电子。激发的电子被馈送到线性电子传输链,并且光系统II用来自水分裂的电子补充丢失的电子。可以使用调制的荧光计设备定量叶绿素荧光产量。在这种装置中,调制的测量光束(以高频率打开和关闭)和仅由测量光激发的荧光的平行检测允许在光合(光化)光的存在下进行叶绿素荧光测量。此外,高强度但短持续时间的光闪烁(饱和脉冲)用于确定在黑暗和光适应叶中的最大荧光产量。在该方案中,给出接受拟南芥野生型叶的典型荧光图的程序。此外,该程序可用于基于各自的荧光图和数值来鉴定影响光系统II的拟南芥突变植物。

关键字:拟南芥, phtotosynthesis, 光系统II, 叶绿素荧光, 脉冲振幅调制

材料和试剂

  1. 拟南芥野生型植物(生态型:Col-0)
  2. 拟南芥突变体植物(生态型:Col-0)
  3. Stender Vermehrungssubstrat A210,70盆栽土(Stender AG)
  4. 种植盆(7 x 6 x 6厘米)
  5. 植物托盘和塑料包装
  6. 镊子(非无菌)

设备

  1. 冷室或冰箱(4℃)
  2. 生长室(12小时光/12小时黑暗,21℃/18℃,PFD为约100μmol/m 2/s)
  3. 用于测量叶绿素荧光的双-PAM-100(Heinz Walz GmbH,型号:Dual-PAM-100),连接到PC,并通过Dual-PAM软件(见注1)操作。
  4. 暗室

软件

  1. 双PAM软件

程序

  1. 种植拟南芥植物
    1. 转移拟南芥属的种子野生型和突变体系 以种植完全填充的盆(每盆约20粒种子)   浸泡土壤。 放置塑料包装覆盖植物以保持必要   湿度
    2. 将盆放在4℃的托盘中3天,以同步发芽
    3. 将托盘转移到生长室中,塑料包装仍然覆盖
    4. 通常种子在4-5天内发芽,2天后取出塑料包装。
    5. 转移单个植物到新鲜的盆(每盆一个植物) 用新鲜的,浸泡的土壤7天后使用轻微弯曲 镊子
    6. 在与之前相同的条件下培养拟南芥植物另外1或2周。 见注2。

  2. 记录叶绿素荧光
    1. 确保Dual-PAM-100设备已正确连接到PC,并置于黑暗的房间中。
    2. 将3-4周龄的拟南芥野生型植物置于暗室中 至少30分钟(这种孵育称为暗适应)。 执行 后续步骤在黑暗中。 (可选)打开绿色指示灯 处理植物

      图1.在Dual-Pam-100设备的测量头之间设置样品叶。对于这里给出的方案(如持续时间6分钟) 固定在测量之间 头(A)。 对于修改的协议,保持植物可能是有用的 通过使用测量头(B)之间的附着叶完好
    3. 中间年龄(例如第七片叶子   顶点通过倒计数)野生型玫瑰花叶和修复它之间 双PAM-100测量头(图1A)。 (可选)尝试修复 叶,仍附着于剩余植物,在测量头之间   (图1B)。
    4. 打开Dual-PAM软件并定义 设置。 为了测量叶绿素荧光,选择< Fluo> < SP分析> 模式。 典型的设置包括测量光 在12μmol/m 2/s/s下,10000μmol/m 2/s/s的饱和脉冲(SP)持续800ms   和70μmol/m 2/s/s的光化性光。 保存用户设置,因此 允许快速启动。
    5. 打开<慢动力学> 窗口 并应用测量光约10秒,荧光水平 是最小的(F <0> )。
    6. 饱和脉冲的应用将诱导最大荧光水平(F
    7. 又一分钟后开启光化灯。 调整灯光 光化光对生长光的强度(见上文)。 执行 光化光处理5分钟,荧光水平 点在< Report>窗口中称为F (= F 0
    8. 施加第二饱和脉冲,其允许测量光中的最大荧光(F m)。
    9. 关闭光化灯,停止录制,导出<慢Kin。 文件> 插入表计算程序(例如 Excel),然后重新创建 图(图2A)。 可选:可以对值进行平均以重新创建 图形。 用不同的野生型植物重复测量3次, 但使用相应年龄的叶(例如来自顶点的第七叶)   通过倒计时)。
    10. 用暗适应突变植物的叶重复测量(图2B)。

  3. 光化学淬灭参数的计算
    1. 打开< Report> 窗口。 给出了F <0>和 m 的值。 根据以下公式计算最大量子产额 (Maxwell和Johnson,2000):   < f>< f>/f>/f</sub> =(F sub)/sub>
    2. 值  在< Report>中给出了在< Report>窗口中的 t (在窗口中的F <  计算PSII光化学的有效量子产率(Maxwell 和Johnson,2000):

代表数据


图2.野生型(A)和参考PSII突变植物(B)的荧光图,参见注释3. 指示测量光(ML)和饱和光脉冲(SP)的应用。显示了暴露于光化性光,并且荧光参数F0,Fm,F↑和F m分别为表示。

笔记

  1. 对于第一次用户,建议从Junior PAM设备开始,因为它易于安装和使用(Heinz Walz GmbH, http://www.walz.com/products/chl_p700/junior-pam/introduction.html )。
  2. 重要的是生长健康的拟南芥植物,因为任何胁迫条件将最终影响光合作用的性能。转移时不要损伤根系。水厂每周大约两次。
  3. 典型的野生型荧光图示于图2A中。参考光系统II突变体(psbo1/o2)的荧光图示于图2B中。通过将 psbo1 和 psbo2-2 杂交产生该突变体,其携带在PsbO1的外显子1中的T-DNA插入 (图3A)的5'非翻译区(5'UTR)。中断Psb 和 PsbO2 会降低PsbO蛋白水平(图3B)。特别值得注意的是在接通光化性光之后荧光下降到低于F 0值。在PSII突变体中通常观察到伴随降低的F vv/F m值的这种现象(Peng等人,2006; Armbruster >等人,2010; Schneider等人,2014)。几乎任何具有这些特征的PSII突变体生长减少(图3C)

    图3.通过使纯合的T-DNA插入系RATM12-1816-1和纯合的T-DNA插入系CSHL-ET9214杂交而产生参考PSII突变体植物( psbo1/o2 。使用PsbO-和anLhcb2特异性抗体(B),从免疫印迹中推导出的psbo1/o2 突变体系中Psb0蛋白含量降低。野生型和psbo1/o2 突变体植物在生长室(C)中生长4周。

致谢

该协议根据Meurer等人(1995)的以前的工作进行了修改或修改。我们感谢ThiloRühle对协议的讨论和评论。这项工作在Dario Leister教授(Biozentrum der LMUMünchen,Department Biologie I,Munich,Germany)的实验室中进行,并由Deutsche Forschungsgemeinschaft(LE 1265/20-1)至D.L.的资金支持。

参考文献

  1. Armbruster,U.,Zuhlke,J.,Rengstl,B.,Kreller,R.,Makarenko,E.,Ruhle,T.,Schunemann,D.,Jahns,P.,Weisshaar,B.,Nickelsen, Leister,D。(2010)。 拟南芥类囊体蛋白PAM68是有效的D1生物发生和光系统II所必需的 植物细胞 22(10):3439-3460。
  2. Maxwell,K。和Johnson,G.N。(2000)。 叶绿素荧光 - 实用指南 J Exp Bot 51(345):659-668。
  3. Meurer,J.,Meierhoff,K.and Westhoff,P。(1995)。 拟南芥的高叶绿素荧光突变体的分离及其通过光谱的表征,免疫印迹和Northern杂交。 Planta 198,385-396。
  4. Peng,L.,Ma,J.,Chi,W.,Guo,J.,Zhu,S.,Lu,Q.,Lu,C.and Zhang,L。(2006)。 低PSII ACCUMULATION1参与拟南芥中光系统II的有效装配。植物细胞18(4):955-969。
  5. Schneider,A.,Steinberger,I.,Strissel,H.,Kunz,HH,Manavski,N.,Meurer,J.,Burkhard,G.,Jarzombski,S.,Schunemann,D.,Geimer, ,UI和Leister,D。(2014)。 拟南芥 Tellurite抗性C蛋白与ALB3一起参与光系统II蛋白质合成。植物杂志78(2):344-356。

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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Steinberger, I., Egidi, F. and Schneider, A. (2015). Chlorophyll Fluorescence Measurements in Arabidopsis Wild-type and Photosystem II Mutant Leaves. Bio-protocol 5(14): e1532. DOI: 10.21769/BioProtoc.1532.
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