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Thermoluminescence (TL) Measurements in Tobacco Leaves
烟草叶片中的热致发光(TL)测定   

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Abstract

TL measurement is a useful tool for studying charge stabilization and subsequent recombination in photosystem II (PSII) in higher plants and cyanobacteria. Recombination of positive charges stored in the S2 and S3 oxidation states of the water oxidizing complex with electrons stabilized on the reduced QA and QB acceptors of PSII results in characteristic TL emissions. The TL intensity reflects the amount of recombining charges and the peak temperature is indicative of the energetic stabilization of the separated charge pair: the higher the peak temperature, the greater the stabilization. Illumination of single-turnover flash with the plant or cyanobacterial sample after a short dark adaptation induces a major TL band, called the B band which appears around at 30 °C and arises from S2/S3QB- recombination. If electron transfer between QA and QB is blocked by DCMU, the B band is replaced by the so-called Q-band arising from S2QA- recombination at around 10 °C. Illumination with a series of single-turnover flashes result in B bands oscillating with a period of 4, with a maximum at the second flash. Here we mainly described the measurements of TL B-band (charge reccombination of S2/S3QB-), Q-band (charge reccombination of S2QA-,) and period-four oscillation of the intensity of the B-band in tobacco leaves.

Materials and Reagents

  1. Tobacco leaves (Nicotiana tabaccum) (Wisconsin 38)
  2. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)
  3. MS medium
  4. 50 μM DCMU

Equipment

  1. Thermoluminescence extension of the Double-Modulated Fluorometer FL2000-S/F, consisting of Thermoregulator TR2000 (Photon Systems Instruments, Brno, Czech Republic)

Procedure

  1. The seeds of tobacco plants were allowed to germinate on MS medium, and then the plants were transferred to soil and grown for two weeks in a growth chamber at 25 ± 1 °C with PPFD of 100 μmol/m2/s, a relative humidity of 75–80%, and a photoperiod of 12/12 h light/dark. For TL Measurements, tobacco plants were adapted in dark for 30 min. The leaves (1.0 cm2) were detached and put on the sample pan, a drop of distilled water is needed between the metal disc surface and the sample to maintain good thermal connection, and then close the measuring chamber. If using larger leaves, make the appropriate cut with a punch-hole tool. In each measurement, equal areas of leaves are recommended. The temperature difference between the top and bottom areas of measured leaves should be as low as possible so as to minimize its heat capacity. The sample should be far away from the light.
  2. For measurement in charge recombination of S2/S3QB-, the samples were cooled to -5 °C and illuminated with one or multiple number of single-turnover flashes (single-turnover flash: Powerful and short enough, typically <5 μs, to induce one, and only one, charge separation in every PS II center). Then the samples were warmed up to 60 °C at a heating rate of 0.5-1 °C/s and the TL light emission was measured during the heating. A major TL band, called B-band, was observed (see Figure 1A). This TL B-band results largely from the recombination of the S2/3QB-charge pair.
  3. For measurement charge recombination of S2QA-, tobacco leaves were incubated in 50 μM DCMU in dark at room temperature for 30 min. Then the measurement was followed as described in step 2. A major TL band, called Q-band instead of B-band, was then observed (see Figure 1B). This TL Q-band is arising from S2QA- recombination.
  4. For the period-four oscillation of the intensity of B-band, the measurement was carried out as described in step 2 expect that leaves were illuminated with a series of single-turnover flashes. Sequences of 1, 2, 3 etc single turn-over flashes, followed by TL recording, result in B bands oscillating with a period of 4, with a maximum at the second flash (see Figure 1C).
  5. Decomposition analysis of the TL glow curves was performed by a non-linear, least squares algorithm that minimizes the χ2 function using a MicrocalTM OriginTM Version 6.0 software package (Microcal Software Inc., Northampton, MA).


    Figure 1. Thermoluminescence glow curves in tobacco leaves. A, B-band; B, Q-band; C, the period-four oscillation of the intensity of B-band

Recipes

  1. 50 μM DCMU (stock: 5 mM)

Acknowledgments

This protocol was adapted from Ding et al. (2012). This study was supported by the National Natural Science Foundation of China (30970218 , 30725024) to D.S. and L.C., the State Key Basic Research and Development Plan of China (2009CB118503) and Frontier Project of the Knowledge Innovation Engineering of Chinese Academy of Sciences (KSCX2-YW-N-042) to L.C., and Solar Energy Initiative of the Chinese Academy of Sciences to W.X.

References

  1. Ding, S. H., Lei, M., Lu, Q., Zhang, A., Yin, Y., Wen, X., Zhang, L., Lu, C.(2012). Enhanced sensitivity and characterization of photosystem II in transgenic tobacco plants with decreased chloroplast glutathione reductase under chilling stress. Biochim Biophys Acta 1817(11): 1979-1991.
  2. Ducruet, J. M., Vass, I. (2009). Thermoluminescence: experimental. Photosynth Res 101(2-3): 195-204.
  3. Rutherford, A. W., Crofts, A. R. and Inoue, Y. (1982). Thermoluminescence as a probe of photosystem II photochemistry: the origin of the flash-induced glow peaks. Biochim Biophys Acta 682(3): 457-465.

简介

TL测量是研究在高等植物和蓝细菌中的光系统II(PSII)中的电荷稳定化和随后的重组的有用工具。存储在水氧化络合物的S 2和S 3氧化态中的正电荷与稳定在还原的Q A和Q上的电子的重组PSII的受体导致特征TL发射。 TL强度反映重组电荷的量,并且峰值温度指示分离的电荷对的能量稳定化:峰值温度越高,稳定性越大。在短暗适应之后用植物或蓝藻样品的单周转闪光的照射诱导主要的TL带,称为B带,其出现在30℃左右,并由S sub2/S 3 - 重组。如果Q A和Q B之间的电子转移被DCMU阻断,则B带被从S 2和S 4产生的所谓Q带代替,在大约10℃下的Q sub-A sub-Sub复合。具有一系列单次翻转闪光的照明导致B波段以4的周期振荡,在第二次闪光时最大。在这里,我们主要描述了TL B带的测量(S sub 2/S sub 3 Q sub Sub Sub B Subband)的电荷重新组合)的Q波段(S <2> Q - 的电荷重组)和B波段的强度的周期四振荡,带在烟草叶。

材料和试剂

  1. 烟草叶(Nicotiana tabaccum)(Wisconsin 38)
  2. 3-(3,4-二氯苯基)-1,1-二甲基脲(DCMU)
  3. MS培养基
  4. 50μMDCMU

设备

  1. 由Thermoregulator TR2000(Photon Systems Instruments,Brno,Czech Republic)组成的双调制荧光计FL2000-S/F的热释光延伸

程序

  1. 使烟草植物的种子在MS培养基上发芽,然后将植物转移到土壤中,并在25±1℃下在生长室中生长两周,PPFD为100μmol/m 2相对湿度为75-80%,光周期为12/12h光/暗。对于TL测量,将烟草植物在暗处适应30分钟。将叶(1.0cm 2)分离并放在样品盘上,在金属盘表面和样品之间需要一滴蒸馏水以保持良好的热连接,然后关闭测量室。如果使用较大的叶片,请使用穿孔工具进行适当的切割。在每次测量中,推荐使用相等面积的叶片。被测叶片的顶部和底部区域之间的温度差应该尽可能低,以使其热容量最小化。样品应远离光源。
  2. 为了测量S 2/S 3/QB - 的电荷复合,将样品冷却至-5℃,并用一个或多个单次翻转闪光次数(单次翻转闪光:强大且足够短,通常<5μs,以在每个PS II中心感应一次且仅一次电荷分离)。然后将样品以0.5-1℃/s的加热速率升温至60℃,并测量TL光发射 。观察到主要的TL带,称为B带(参见图1A)。该TL B带主要来自S sub 2/Sub 3+ QB - 电荷对的重组。
  3. 为了测量S Sub2 Sub Q Sub的电荷重组,将烟草叶在50μMDCMU中在黑暗中在室温下孵育30分钟。然后如步骤2中所述进行测量。然后观察到称为Q带而不是B带的主TL带(参见图1B)。该TL Q带由S Q A - 重组产生。
  4. 对于B波段强度的周期四振荡,如步骤2所述进行测量,期望叶子用一系列单周期闪光照射。 1,2,3等序列的单次翻转闪光,接着TL记录,导致B波段以4的周期振荡,在第二次闪光时最大值(见图1C)。
  5. 通过非线性最小二乘法算法对TL发光曲线进行分解分析,该算法使用Microcal TM sup/TM原始 TM最小化χ 2 > 6.0版软件包(Microcal Software Inc.,Northampton,MA)。


    图1.烟草叶中的热致发光曲线。 A,B带; B,Q带; C,周期四阶振荡的B带强度

食谱

  1. 50μMDCMU(原液:5mM)

致谢

该方案改编自Ding等人(2012)。 本研究得到中国国家自然科学基金(30970218,30725024)DS,LC,中国国家重点基础研究与发展规划(2009CB118503)和中国科学院知识创新工程前沿项目(KSCX2 -YW-N-042)到LC,以及中国科学院太阳能倡议到WX

参考文献

  1. Ding,S. H.,Lei,M.,Lu,Q.,Zhang,A.,Yin,Y.,Wen,X.,Zhang,L.,Lu,C。 在冷冻胁迫下叶绿体谷胱甘肽还原酶减少的转基因烟草植物中光系统II的增强的灵敏度和表征< a>。 1817(11):1979-1991。
  2. Ducruet,J.M.,Vass,I。(2009)。 Thermoluminescence:experimental 。 Photosynth Res 101(2- 3):195-204。
  3. Rutherford,A.W.,Crofts,A.R.and Inoue,Y。(1982)。 热释光作为光系统II光化学的探针:闪光诱导辉光峰的起源 。 Biochim Biophys Acta 682(3):457-465。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ding, S., Wen, X. and Lu, C. (2013). Thermoluminescence (TL) Measurements in Tobacco Leaves. Bio-protocol 3(5): e408. DOI: 10.21769/BioProtoc.408.
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