ETR Measurement with FluorCam Introduction ETR, or electron transport rate, is a light-adapted parameter that is directly related to PSII operating efficiency (Y(II), Genty parametr, Fq´/Fv´, φPSII) by the equation, ETR = φPSII × PAR × aL × f PSII, where: φPSII= operating quantum efficiency of PSII PAR = photosynthetically active radiation (in µmol/m2/s) aL= leaf light absorption (between 0 and 1, usally about 0.8) f PSII= proportion of PSII over[PSII + PSI] (usually 0.5) Relative ETR measurement is achieved by multiplying Y(II) by the irradiation light level in the PAR range (400 nm to 700 nm) in µmol/m2/s, multiplied by the average ratio of light absorbed by the leaf 0.84, and multiplied by the average ratio of PSII reaction centers to PSI reaction centers, 0.50. Relative ETR values are used as valuable parametr for stress measurements when comparing one plant to another, as long as the plants to be compared have similar light absorption characteristics. ETR measurement in FluorCam (FC) In FluorCam ETR values are calculated based on the equation ETR = Y(II) × PAR × 0.8 × 0.5. To measure ETR parametr in FluorCam it is recommended to use Light Curve protocol (Fig. 1) that can be launched from the Protocol Menu. Light curve protocol for Actinic light 1 and Light Curve protocol for Actinic light 2 is available. FM Lss1 FM Lss2 FM Lss3 FM Lss4 FM Lss5 FM Lss6 FM Ft Lss1 Ft Lss2 Ft Lss3 Ft Lss4 Ft Lss5 Ft Lss6 F0 Saturating pulses Mf Actinic light of increasing intensities L1-L6 + Measuring flashes Fig.1 Schematics of Light curve protocol kinetics. Light Response Curves are a means of quantifying the rate of photosynthetic performance at different light irradiances. Light response curves (LRCs) of electron transport rate (ETR), effective quantum yield (Y(II)), and non-photochemical quenching (NPQ) against light intensity (PPFD) can be used as valuable tool to estimate the photosynthetic light-use efficiency in response to different stresses. Light Curve protocol in FC is designed to measure quenching analysis in light adapted state at different light irradiances (Fig. 2). As a result of the measurement, range of quenching analysis parametres are calculated for different light intensities such as Fv´/Fm´(Fv/Fm_Lss), QY_Lss (equal to Y(II), Fq´/Fm, φPSII, Genty parametr,...), NPQ_Lss, qP_Lss, qL_Lss, qN_Lss and ETR_Lss. In the Protocol Menu of FC Light Curve protocols are pre-defined with duration of actinic light exposure for 60 s at 6 irradiances that are gradually increasing (Fig. 2). The length of the irradiance period (actinic light exposure) and the number of irradiance steps can be optimised based on user´s needs. The user is adviced to contact PSI support firstname.lastname@example.org if modifications in the protocol are required such as duration of the actinic light exposure or number of irradiance steps. For example light curve protocols with 12 irradinace steps can be used for detail characterisation of photosynthetic performance during mutant screen in Arabidopsis thaliana (Ref. 1). L6 L5 L4 L3 L2 Increasing irradiance L1 Actinic light exposure Fig.2 Light curve protocol with schematics of actinic light exposure. Irradiance is gradually increasing from L1 to L6 actinic light exposure step. In the Light Curve protocol light irradiances are defined as percentage of the maximum light intesity (for Actinic light 1 or Actinic light 2), which is available for the given hardware of FluorCam configuration (Fig. 3). The user can use the pre-defined protocol settings for the light irradiance or modify the percentage numbers according to his/her specific requirements. Fig.3 Light curve protocol with highlighted commands for the light intensity settings- Light intesity is defined as percentage of the maximum light intensity (0-100%). For ETR calculation PAR light irradiance in µmol/m2/s is required. To convert the percentage of light intensity into light intensity expressed in µmol/m2/s the user will need to perform light calibration or use light calibration sheet provided by the manufacturer (Fig. 4). Light intesity for the given percentage of actinic light can be measured with Light Meter (e.g. LI-250A Light Meter from Li-Cor) or with spectroradiometer SpectraPen LM500. Please note, that the light calibration should be performed in defined distance from the light source. This distance should be always the distance, which is between the measured object (plant surface) and the light source. Actinic light 2 Intensity Act 2 [%] [µmol/m2/s] Actinic light 2 intensity 0 10 128 20 345 30 563 40 782 50 1001 60 1220 70 1436 80 1650 90 1863 100 2064 2500 Intensity [µmol/m2/s] 0 y = 212.35x - 269.4 R² = 0.999 2000 1500 1000 500 0 0 10 20 30 40 50 60 70 80 90 100 Act 2 [%] Fig.4 Example of light calibration for Actinic light 2. Quick Guide for ETR measurement with Light Curve Protocol 1. Select the Light Curve protocol you would like to use (with Act1 or Act2 light exposure) from Protocol Menu 2. To proceed with the measurement Light Calibration is required for conversion of light intensity expressed in the percentage into light intensity expressed in µmol/m2/s. 3. Perform the light calibration for the actinic light choosen for the Light Curve protocol, plot in the graph the light intesity in percentage and equivalent values in µmol/m2/s, add the linear trendline and calculate the equation (example is shown in fig. 4). 4. Go to Protocols window and insert the equation values from the graph into the protocol, where variable LightA and Light B is defined. See below and figure 5 for more information. Example of equation from Fig. 4: y = 212.35x - 269.4 R² = 0.999 Equation variable defined in the Light Curve protocol are: LightA=212.35 LightB=-269.4 Fig.5 Light curve prococol with highligted row where equation values from the light calibration curve should be inserted. 5. In the next step it is important to define the light intensity in percentage for the six irradiance steps the user wants to use for the light response curve measurement and determination of ETR. 6. Define the percentage of the light irradiance in the Light Curve protocol as shown in fig. 7. Fig.6 Light curve prococol with highligted row where light intensity is defined in percentage for the six irradinace steps. 7. The values in Light intensity row (as shown in fig. 6) must correspond to the values defined in the protocol body as shown in fig .3 and fig. 7. 8. Store the protocol for later use and measurements. 9. Go to Live window and optimise the camera settings as shutter and sensitivity to obtain suitable signal for minimal (F0) or instaneous (Ft) fluorescence of the object. Let measuring flashes switched on and adjust El. Shutter and Sensitivity in LIVE WINDOW. Suitable signal should be in the range of 200-500 digital units (dark blue or blue color of Extended spectrum 3_0_3, which is the most sensitive color scales for human eye). 10. Let measuring flashes switched on and adjust Actinic light intensity by trucking the bar. Use maximum light intensity of the desired actinic light used in the Light Curve protocol. Next switch ON Super in Light Sources panel in LIVE WINDOW and adjust it. Super light intensity should be saturating even at the highest actinic light intensities, therefore light intesity higher then highest actinic light intesity should be used. The saturating pulse is only switched for limited time (800ms), so it must be switched on each time the intensity is changed. 11. Finally, test image quality with all 3 lights (Flashes + Act + Super) switched on as described above. CCD camera is 12 bits, so fluorescence signal can be measured between 0 – 4096 digits. If the used settings are saturating for the tested object, the error message PIXELS OVERFLOW at the bottom of the LIVE WINDOW will warn you. In this case the dynamic range of the camera is exceeded and the signal above 4096 digits is not integrated. It is necessary to decrease Sensitivity or both Sensitivity + El. Shutter to reach ideally fluorescence signal of 2000-3000 digits when all light sources (Flashes + Act at maximum intensity used + Super) are switched ON. Fig.7 Protocol body with highlighted rows where percentage of light irradiance is defined and must be corresponding. 12. IMPORT the optimized camera and light settings to the protocol by clicking the button Use in the bottom of LIVE WINDOW. 13. To start the measurement click red flash icon, Start Experiment, in the top panel to run the measurement. 14. Calculated numeric ETR values for the individual light irrandiances can be after the analysis step exported, further processed and used to generate graph for the light response curves of electron transport rate (ETR) as shown in fig. 8. 40 35 30 ETR 25 20 15 10 5 0 0 500 1000 1500 2000 PAR (umol/m2/s) Fig.8 Light response curves of electron transport rate (ETR). References 1. Chen KM, Holmström M, Raksajit W, Suorsa M, Piippo M, Aro EM.(2010). Small chloroplast-targeted DnaJ proteins are involved in optimization of photosynthetic reactions in Arabidopsis thaliana. BMC Plant Biol. 2010 Mar 7;10:43. doi: 10.1186/1471-2229-10-43. 2. Zhang X, Wollenweber B, Jiang D, Liu F, Zhao J.(2008). Water deficits and heat shock effects on photosynthesis of a transgenic Arabidopsis thaliana constitutively expressing ABP9, a bZIP transcription factor. J Exp Bot. 2008;59(4):839-48. doi: 10.1093/jxb/erm364. Epub 2008 Feb 13. 05/2015 © PSI (Photon Systems Instruments), spol. s.r.o.