澳作生态仪器有限公司 叶绿素荧光测量中的 PAR 测量 植物逆境生理生态研究方法专题系列 8 野外进行荧光测量时, 光照和温度条件变异性非常大 如果不进行光照和温度条件的控制或者精确测量, 那么荧光的测量结果将无法科学解释

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1 叶绿素荧光测量中的 PAR 测量 植物逆境生理生态研究方法专题系列 8 野外进行荧光测量时, 光照和温度条件变异性非常大 如果不进行光照和温度条件的控制或者精确测量, 那么荧光的测量结果将无法科学解释 Yield can vary significantly with light level and with temperature. Without controlling irradiation and temperature it is possible to misinterpret results. In fieldwork where both light and temperature can vary, a lower Yield measurement on one plant as compared to another could be misdiagnosed as stress when it may only be an increase in irradiation or a change in temperature on the leaf. When a PAR Clip is used to take Yield measurements, the combination can be formidable. PAR 叶夹可以在测量 Yield ETR 和 PAR 以及叶温等参数时将荧光仪探头固定在合适的距离和角度 A PAR Clip is a leaf cuvette that allows the holding of the sample leaf at a repeatable angle and distance from the measuring probe while measuring Yield, ETR, PAR, and leaf temperature. These values are usually contained in same data file with a time and date stamp. PAR (Photosynthetically Active Radiation) is irradiated light between the wavelengths of 400 nm and 700nm and it is measured very near the sample measuring area. When PAR is measured using a PAR Clip and the dimensions per square meter per second (μmol/m 2 /s) are used, the value becomes PPFD (Photosynthetic Photon Flux Density), (When using a PAR Clip the terms PAR and PPFD are interchangeable). 测量 Yield 和 ETR 时, 有些 PAR 叶夹可以利用荧光仪内置光源的数值, 所有样品都可以设置在同一光强条件 注意 : 使用内置光源时, 必须持续足够的时间以使植物达到光合作用稳态, 否则将会出错 研究显示,15-20 分钟的时 Some PAR Clips offer the option of using a predetermined light intensity level from an internal fluorometer light source to measure Yield and ETR. In this case, irradiation levels can be forced to be consistent for each sample. Note: When using internal illumination it is important to allow time for a plant to reach steady state photosynthesis before measurement or errors will result. Even small changes in irradiance level can change the measurement of both yield and ETR. Changes of as little as 100 umols can cause errors if one does not allow for photoprotective

2 间内, 植物会达到光合作用稳态 ( Maxwell and Johnson,2000) changes, and state transitions (Opti-Sciences unpublished). Maxwell and Johnson (2000) found that it takes between 15 minutes and 20 minutes for plants to reach steady state. OS5P 增强型便携多功能调制荧光仪配备的 PAR 叶夹, 可以使用内置光源, 并提供可信的 PAR 或 PPFD 值 这种情况下, PAR 传感器也必须在阳光下进行严格标定, 传感器位置和叶片的定位差异也需要严格校准 Some PAR Clips, such as the digital PAR clip for Opti-Sciences OS5p, allow PAR measurement while an internal light source is being used, thus providing a reliable PAR or PPFD values over time. Of course in this case the PAR sensor should be calibrated to the internal actinic illumination as well as to sun light. It should also be calibrated for the location difference between the PAR sensor and the leaf sample (Rascher 2000). Relative Electron Transport Rate or ETR. ETR 反应不同光照条件下 PSII 电子传递的速率 这是个相对值, 因为叶绿素荧光和气体交换的测量值不具有显著的相关性, 叶绿素荧光现象更多出现在植物上层叶片的光合作用测量过程中, 而底层的植物叶片光合作用也能够通过气体交换测量得到 (Schreiber 2004) ETR or Relative Electron Transport Rate is a parameter that is measured with a PAR Clip. ETR is a relative measurement that provides comparative electron transport rates for PSII at different light or radiation levels. It is considered relative because chlorophyll fluorescence does not correlate exactly with absolute gas exchange measurements. While most fluorescence occurs in the upper most layers of the leaf, gas exchange measures the effects of photosynthesis in deeper layers as well. (Schreiber 2004). ETR 和 C4 植物的碳同化具有线性相关关系 (Genty 1989), 与 C3 植物具有曲线相关关系 (Genty 1990) 每同化一个 CO2 分子或者还原一个 O2 分子, 需要传递 4 个电子 Even with this in mind, relative ETR can be extremely valuable. While exact correlation to gas exchange carbon assimilation is not possible, linear correlation is possible with C4 plants (Genty 1989) and a curvilinear correlation is possible with C3 plants (Genty 1990). While four electrons must be transported for every CO2 molecule assimilated, or O2 molecule evolved, differences from gas exchange measurements can occur under conditions that promote of photorespiration, cyclic electron transport, and nitrate reduction (Schreiber 2004) (Baker, Oxborough 2004). For more detailed information concerning the relationship between fluorescence and gas exchange measurements again refer to another application note on Yield measurements. ETR 的计算公式在这个公式里, 假设叶片的平均光吸收率为 84%,PSII 可利用的光为光合有效辐射的 50% The equation for Relative ETR is ETR = (Yield or )F/Fm ) (0.84) (0.50) (PPFD or PAR) In this equation, Yield represents overall PSI and PSII yield. It assumes an average leaf light absorbance to be 84%, and the portion of light provided to PSII to be 50%. PPFD is PAR irradiation measured very near the leaf in micromoles or microeinsteins (equivalent units). The end

3 result is a close approximation of PSII ETR that can be used for relative evaluation of different samples. 研究发现, 陆生植物叶片的吸光率通常为 70% 和 90% 之间 研究表明,PSII 的吸光率为 40% 到 60% 之间 The absolute amounts for leaf and PSII light absorption can vary at steady state with species, stress and water content. Terrestrial leaf absorbance has been found to vary between 70% to 90% (Eichelman H., Oja V., Rasulov B., Padu E., Bichele I., Pettai H., Niinemets O., Laisk A. 2004), and the percentage of light absorbed by PSII has been found to range from 40% to 60% (Laisk A. and Loreto F. 1996), (Edwards GE and Baker NR 1993). Other areas of value: Besides the ability to control and relate yield measurements to PPFD, leaf temperature, and the ability to relate ETR to carbon assimilation, PAR Clips can provide additional specialized high value uses. PAR 叶夹除了能在测量 Yield 时能够控制和测量 PPFD 叶温 碳同化过程中的电子传递速率等参数外, 还可以提供很多更有效的信息 1 C4 植物的水分胁迫测量 2 光反应和暗反应之间的关系并非完全一致的 与气体交换测量结合起来,ETR 可用于反应反应光呼吸状况 C3 植物的水分胁迫和冻害胁迫以及轻微高温胁迫的研究 3 ETR 能够很好的反映重金属镍胁迫 4 ETR 与 PPFD 绘制光响应曲线, 可用于研究植物受限范围 确定植物逆境响应机理 对比阴影叶片和阳光下的叶片 确定植物突变异种以及研究逆境条件下植物光保护机制等等 1. C4 plant Water stress measurement The ratio of ETR to carbon assimilation, or ETR/A, is known to be consistent in C4 plants. This is not true in C3 plants. ETR requires a PAR Clip. (J Cavender-Bares & Fakhri A. Bazzaz 2004 ) (Cerovic 1996) For measurement of early water stress in C3 plants see (Burke 2007) or the Opti-Science App note on Water Stress. 2. The relationship between the light reaction and the dark reaction may not be straightforward under certain circumstances. In some cases light reaction electron transport from PSII is significantly higher than predicted by gas exchange measurements. When combined with gas exchange equipment, ETR can be used to improve the study of these effects in areas such as photorespiration (Roseqvist and van Kooten 2006), C3 plant water stress (Ohashi 2005), cold stress (Fryer M. J., Andrews J.R., Oxborough K., Blowers D. A., Baker N.E. 1998) and mild heat stress (Haldimann 2004). For more information on issues related to the relationship between fluorescence measurements and gas exchange, please review the OSI application note on Yield, value and limitations. 3. ETR is good indicator of Nickel stress and light reaction activity (Joshi & Mohanty2004), (Tripathy 1981). 4. Light response curves- These are charts that show the relationship of ETR and PPFD at different irradiation values at steady state photosynthesis. They can be used to study plant range limitations, identify stress resistant plants, study the response of shade leaves vs. sun leaves, identify mutants, study plant protective mechanisms under stress conditions, and other applications. (Rascher U.2000)

4 5 快速光曲线 --- 测量需要使用普通的暗适应叶夹 快速光曲线的典型应用是针对水生植物和冠层下植物等光照条件变异比较大的对象 6 白天无法通过气体交换测量的方法测量 CAM 植物, ETR 可用于研究其电子传递速率 5. Rapid light curves this is usually a partially dark adapted test that can take less than two minutes. Usually dark adaptation clips are used without a PAR Clip. Internal fluorometer look up tables usually supply PAR values. (With the OS5p and soon the OS1p, this test can be done using a PAR clip for actual measurement of PAR values.) Steady state photosynthesis is not reached, instead, measurements are made after 10 seconds, or 30 seconds or one or two minutes. Actinic light is stepped up or down. Data from several measurement curves at different times of day are required for reliable results as results change at different times of the day. This is a test that is typically used for aquatic plants and under canopy plants where light is constantly variable and other methods of testing prove difficult. Light fitting curves are typically used to determine cardinal point such as ETRmax (Rascher U. 2000), (Ralph P.J., Gademann R. 2005). It can be easy to misinterpret results (Rascher 2000). 6. CAM plant light reaction measurement. It is not possible to use gas exchange equipment during the daytime to measure photosynthesis in CAM plants. ETR may be used to study electron transport. (Edwards GE and Baker NR 1993), Rosenqvist E., van Kooten O., (2006), (Laisk A and Loreto F 1996). PAR and PPFD PAR 和 PPFD 是生物学领域常用的两个单位, 国际单位都是 umols.

5 Other important considerations when using a PAR Clip 使用 PAR 叶夹时需要考虑的其他事情 1 PAR 传感器余弦校正测量光合作用产量时, 需要在稳态下进行, 所以环境光或内置光源的 PAR 探头方向要保持不变, 以尽量减少误差 而采用了余弦校正的 PAR 光量子探头确保了叶片上方各个角度的入射光都能被测量 2 PAR 传感器定位误差自动校正 2000 年, 某科研工作者研究发现, 当 PAR 传感器和叶片的水平面之间不能完全吻合, 或者未能放置在测量光的中心点, 则 PAR 的测量结果将会产生 10% 的偏差 为了修正这个偏差,Optic 科学仪器公司用 Licor 190PAR 传感器进行了校准 将传感器与叶片置于同一平面, 并置于测量区域中心 测量太阳光时, 结果无显著偏差, 但当使用光纤发出的光束作为内置光化光光源时, 测量结果偏差最大可达 10% (Rascher,2000) 注 : 当使用内置光源测量光 1. Spatial error This is an error related to light angle incident on the sensor. It is made up of cosign error and angular error which are related to error caused by different radiation incident angles and the diffuse filter section used to correct for cosign error. The error is less at angles closer to normal or perpendicular. Opti-Sciences digital PAR Clips such as those used with the OS5p and OS1p chlorophyll fluorometers are Cosign corrected. At 60 degrees light irradiance is cut in half. 2. PAR sensor location error. While this is not significant when using a PAR Clip for ambient sunlight measurements, it can be significant when making measurements using a fluorometer with an internal light source. The height difference between the PAR sensor and the leaf can cause a significant error in the range of 10% (Rascher 2000). This can be corrected by calibrating the PAR Clip PAR sensor to a PAR sensor located at the leaf plane. The intensity relationship is linear (Rascher 2000). It can also be overcome by using internal fluorometer PPFD look-up tables for pre-measured values that correct for location error. However, if look up tables are used, the values can change over short periods of time due to heat, and over longer periods of time due to changes in lamps or LEDs. (The OS5p PAR Clip measures PPFD from internal actinic light sources. Internal look up tables are available and can be used when a PAR Clip is not being used. In the next software release for the OS5p and OS1p, separate calibrations will be made for sun light, the internal actinic LED, and the internal actinic halogen light sources at the leaf plane. There will also be a programmable factor that can be used for other laboratory light sources. 3. Spectral response error The type of illumination used can cause an error in PAR measurement. This is due to the different spectral radiation given off by the sun and sky, halogen lamps, arc lamps, or other lamps used in growth chambers and green houses. The most accurate way to measure PAR is with a spectroradiometer, an instrument that costs thousands of dollars.

6 响应曲线 快速光响应曲线 非序列光响应曲线 荧光淬灭以及光合作用产量时, 都需要修正传感器定位误差 Optic 公司的仪器可以进行自动校准 3 光谱误差自动校正不同的光源产生不同的光谱 Optic 公司利用 Licor 190 系列 PAR 传感器对自己生产的传感器在太阳光 内置卤素灯 内置 LED 等几种不同光源下进行了校准, 以减小光谱误差 同时校准系数也考虑了在实验室内使用外置光源时 注 : 选择了光源后, 偏差校准即自动进行 8 稳定的光化光光源为了获得可靠的荧光淬灭 光响应曲线 快速光响应和非序列光响应曲线, 可靠稳定的光化光光源是一个必要条件 不稳定的光强将会导致显著的测量偏差 大多数内置光化光源会因为长时间使用的发热效应而强度逐渐降低 OS5P 使用了稳定的卤素灯作为内置光化光源, 在 30 分钟的连续使用后, 光强只降低不到 2%! 此外, 在进行荧光淬灭和各种类型光响应曲线的测量时, Optic 公司的 PAR 叶夹可以实时测量并记录内置光源 To provide a field portable, reliable compromise, various photodiodes are used to measure PPFD with fluorometers and PAR Clips. The Opti-Sciences sensor underestimates PPFD in the blue range and overestimates PPFD in the red range as compared to an ideal PAR sensor. Because the Sun and built in halogen light sources used to drive photosynthesis offer a wide spectrum of light over the PAR range, these spectral errors offset each other, minimizing overall spectral error. 4. Absolute error - this is caused by error in the special halogen light source used by National Institute of Standards and Technology. 5. Stability error Solid state sensors do not change significantly over time, it is recommended that a PAR Clip Calibration should occur every two years. 6. Temperature error in a range of 15 degrees centigrade to 35 degrees centigrade the error is very small. 7. Readout error caused by the digital readout is very small. 8. Internal actinic lamp temperature stability error. This is most important when using internal look up tables for irradiation intensity at the leaf. Par Clips that actually measure PPFD from an internal source during measurement will detect variation in light level due to increased instrument heat levels. The PAR output of most internal fluorometer illuminators will change over time due to increased heat caused by the lamp. This can also be of concern when making light curves and even rapid light curves. Lamp output falls off most dramatically during the first few minutes after turning on the internal actinic illuminator. If lookup tables are used for PAR or PPFD values, as when dark adaptation clips are used for light curves or rapid light curves, the values may not be accurate if the light source intensity changes. When using the OS5p it is recommended that the Halogen actinic illuminator be used for longer light curves, rapid light curves, dark light kinetic tests, and quenching tests. The Halogen light was designed to be very stable over time. Tests have shown that there is less than a 2% variation of intensity over time. The LED actinic light source can be used for shorter yield measurements.

7 9 操作误差尽量避免在多云或者有较多光斑的地方进行叶绿素荧光测量 ; 测量时候不能改变叶片的朝向以及光照的区域, 对叶片的外界刺激以及呼吸影响都应避免 9 User error can be unlimited. Trying to measure samples that have momentary flecks of sunlight and short periods of shade caused by wind or cloud cover can cause momentary PAR measurement to be almost meaningless. For best results it is important not to change the orientation of a leaf and to avoid shading the sample measuring area with the PAR clip or by other means. Extraneous reflections and breathing on the sample should also be avoided (Rosenqvist and van Kooten 2006). Failing to clean the sensor can also cause errors. 10 当使用一个小的光量子传感器测量光照时候, 尤其是当处于其他植物的冠层下方时, 测量值可能会因为阴影 光斑等各种原因而造成误差 为了解决这个问题, 研发者想了很多方法, 譬如延长传感器, 增加传感器的测量点, 然后计算多点的平均值 11 众多用户反馈, 很多荧光仪的 PAR 叶夹由于设计上的问题常常在不恰当的时候被打开, 10 Under Canopy Errors occur when using a single small sensor to measure radiation within a crop canopy, in or under other plant canopies, or within a growth chamber. The values measured on a given plane below canopy can vary considerably due to shadows, reflections, and sunflecks. Neglecting this factor in measurements can introduce errors up to 1000%. A Study comparing instrumentation and methods for under canopy irradiation measurements claim that accuracy is within 20% except when gaps in the canopy are non-random (Welles and Cohen 1996). Many schemes and methods have been used to solve the issues related to under canopy light irradiation PAR measurement. Some involve meter long sensors and others use point sensors. In these cases averaging the PAR value over time and in some cases averaging yield over time is a method that has worked. 11 Mechanical issues Feedback from customers of various brands of instruments has also added concern about PAR clips opening at unwanted times, such as when used for high leaf measurement while

8 影响了正常的测量 Optic 公司专门针对这个问题设计了在底部打开的 PAR 叶夹, 不仅不容易打开, 而且方便针对较高处的叶片测量 适用于 OS5P 和 OS1P reaching over ones head,andwhen used with a tripod for quenching studies. The new Standard PAR clip has been designed to open from the bottom and not the top as other PAR clips have done. This prevents the opening of the clip with high leaf measurements caused by tension of the fiber optic sensor on the clip, and it also prevents the opening of the PAR clip on tripods due to fiber optic weight. 参考文献 References: Allen J. F., Mullineaux C.W., (2004) Probing the mechanism of State Transitions in Oxygenic Photosynthesis by Chlorophyll Fluorescence Spectroscopy, Kinetics and Imaging. From Chapter 17, Chlorophyll a Fluorescence a Signature of Photosynthesis,edited by George Papaqeorgiou and Govindjee, published by Springer 2004, PO Box 17, 3300 AA Dordrecht, The Netherlands, pages Baker N. R., Oxborough K., (2004) Chlorophyll fluorescence as a probe of photosynthetic productivity. From Chapter 3, Chlorophyll a Fluorescence a Signature of Photosynthesis, edited by George Papaqeorgiou and Govindjee, published by Springer 2004, PO Box17, 3300 AA Dordrecht, The Netherlands, pages Burke J. (2007) Evaluation of Source Leaf Responses to Water-Deficit Stresses in Cotton Using a Novel Stress Bioassay, Plant Physiology, Jan. 2007, Vol 143, pp Cavender-Bares J. & Fakhri A. Bazzaz 2004 From Leaves to Ecosystem: Using Chlorophyll Fluorescence to Assess Photosynthesis and Plant Function in Ecological Studies. Jeannine Cavender Bares, Fakhri A. Bazzaz, From Chapter 29, Chlorophyll a Fluorescence a Signature of Photosynthesis, edited by George Papaqeorgiou and Govindjee, published by Springer 2004, PO Box 17, 3300 AA Dordrecht, The Netherlands, page Cerovic Z, Goulas Y. Gorbunov M, Britantais J-M, Camenen L, & Moya I., (1996) Fluoresensing of water stress in plants. Diurinal changes of mean lifetime and yield of chlorophyll fluorescence, measured simultaiously at distance with a Lidar and modified PAM-fluorometer, in maize, sugar beet and Kalanchoe. Remote Sens Environment 58: Cheeseman J.M., Herendeen L.B,. Cheeseman A.T., Clough B.F., (1997) Photosynthesis and photoprotection in mangroves under field conditions. Plant Cell Envir 20:

9 Earl H., Said Ennahli S., (2004) Estimating photosynthetic electron transport via chlorophyll fluorometry without Photosystem II light saturation. Photosynthesis Research 82: , Edwards GE and Baker NR (1993) Can Co2 assimilation in maize leaves be predicted accurately from chlorophyll fluorescence analysis? Photosynthesis Res 37: Fryer M. J., Andrews J.R., Oxborough K., Blowers D. A., Baker N.E. (1998) Relationship between CO2 assimilation, photosynthetic electron transport and active 02 metabolism in leaves of maize in the field during periods during periods of low temperature. Genty B., Briantais J. M. & Baker N. R. (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence, Biochimica et Biophysica Acta 990, Genty B, Harbinson J., Baker N.R. (1990) Relative quantum efficiencies of the two photosystems of leaves in photo respiratory and non-photo respiratory conditions. Plant Physiol Biochem 28: 1-10 Haldimann P, & Feller U. (2004) Inhibition of photosynthesis by high temperature in oak (Quercus pubescens L.) leaves grown under natural conditions closely correlates with a reversible heat dependent reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase. Plant, Cell and Environment (2004) 27, Maxwell K., Johnson G. N, (2000) Chlorophyll fluorescence a practical guide. Journal of Experimental Botany Vol. 51, No. 345, pp April 2000 Joshi M.K., Mohanty P. (2004): Chlorophyll a fluorescence as a probe of heavy metal ion toxicity in plants. In: Papageorgiou G.C. (eds.): Chlorophyll Fluorescence: A Signature of Photosynthesis Advances in Photosynthesis and Respiration. Springer, Dordrecht: Laisk A and Loreto F (1996) Determining photosynthetic parameters from leaf CO2 exchange and chlorophyll fluorescence. Ribulose-1,5-bisphosphate carboxylase / oxygenase specificity factor, dark respiration in the light, excitation distribution between photosystems, alternative electron transport rate, and mesophyll diffusion resistance. Plant Physiol 110: Maxwell K., Johnson G. N, (2000) Chlorophyll fluorescence a practical guide. Journal of Experimental Botany Vol.51, No. 345, pp April 2000 OHASHI Y., NAKAYAMA N., SANEOKA H., FUJITA K., (2006) Effects of drought stress on photosynthetic gas exchange, chlorophyll fluorescence and stem diameter of soybean plants. BIOLOGIA PLANTARUM 50 (1): , 2006 Rascher U (2000). Evaluation of instant light-response curves of chlorophyll fluorescence parameters obtained with a portable chlorophyll fluorometer on site in the field U. RASCHER, M. LIEBIG & U. LÜTTGE Plant, Cell and Environment (2000) 23, Ralph P.J., Gademann R. (2005) Rapid light curves: A powerful tool to assess photosynthetic activity. Aquatic

10 Botany 82 (2005) Rascher U (2000). Evaluation of instant light-response curves of chlorophyll fluorescence parameters obtained with aportable chlorophyll fluorometer on site in the field U. RASCHER, M. LIEBIG & U. LÜTTGE Plant, Cell and Environment (2000) 23, Rosenqvist E., van Kooten O., (2006) Chlorophyll Fluorescence: A General Description and Nomenclature. From Chapter 2 Practical Applications of Chlorophyll Fluorescence in Plant Biology. by Jennifer R. DeEll (Editor), Peter M.A. Toivonen (Editor) Kluwer Academic Publishers group, P.O Box 322, 3300 A.H. Dordrecht, the Netherlands, pages Schreiber U, Pulse-Amplitude-Modulation (PAM) Fluorometry and Saturation Pulse Method: An Overview From Chapter 11, Chlorophyll a Fluorescence a Signature of Photosynthesis, edited by George Papaqeorgiou and Govindjee, published by Springer 2004, PO Box 17, 3300 AA Dordrecht, The Netherlands, page Tripathy BC, Bhatia B., Mohanty P., (1981) Inactivation of chloroplast photosynthetic electron transport activity by Ni ++. Biochim Biophys Acta 638: Wells M., Cohen S. (1996)Canopy structure measurement by gap fraction analysis using commercial instrumentation Jon Journal of Experimental Botany, Vol. 47, No. 302, pp , September 1996