The simultaneous measurement of leaf reflectance with chlorophyll fluorescence analysis using the already known mutant plant model facilitates the establishment of new reflectance parameters. The construction of new leaf reflectance parameters by our method allows real-time monitoring of changing plant behaviors under natural environmental conditions. Before beginning an experiment set up a measurement system as shown in this schematic.
With a sample stage, light source, PAM fluorometer, and spectral radiometer. Next attach a 10 cm squared steel plate with a one cm diameter hole to the custom made sample stage. And fit two thin fiber probes tightly together.
Wrap the probes with plastic tape. Then use a holder to clip the taped probes onto the sample stage. Position the probes vertical to the sample stage.
And attach a biforked light guide made of glass fibers to the halogen light source. Then eradiate the sample stage from both directions at approximately 45 degree angles. Adjusting the light source so that light uniformly illuminates the sample stage without casting shadows.
For simultaneous measurement of left reflectance and chlorophyll florescence analysis set up, in a dark room with green cellophane light place a test plant at the leaf holder of the sample stage and touch the leaf against the hole of steel plate on the stage. After turning off the weak green light turn on the PAM fluorometer and eradiate the leaf sample with a measuring light. Move the adjuster so that the florescence intensity measures approximately 100 and measure the distance between the probe and the leaf.
Fix the adjuster in place and record the value of the distance on the adjuster. Then turn off the measuring light and remove the test plant. For actinic light intensity measurement place a light quantimeter at the position where the sample leaf will be placed.
And irradiate light from the halogen light source to measure the light source intensity. Determine which positions of the light source dial generate intensity's of 30, 60, 120, 240, and 480 micromolar protons per meter squared per second. Place a white plate as a reflectant standard at the position of the leaf sample.
And turn on a spectral radiometer. Adjust the spectral reflectance between 350 and 850 nanometers. There are no spectral data at this time as there is no irradiating light.
Turn on the halogen lamp to irradiate with 480 micromolar photons per meter squared per second. The highest irradiance intensity in this test and adjust the detection strength of the radiometer to avoid saturation. Then record the spectral reflectance under illumination with 30, 60, 120, 240 and 480 micromolar photons per meter squared per second.
For simultaneous measurement of the leaf reflectance and chlorophyll florescence analysis transfer the plant from growth chamber to darkroom with the same temperature and humidity. After one hour place an Arabidopsis plant at the leaf sample position. And fix the sample leaf to the leaf so that the leaf surface is perpendicular to the detection probes.
Turn on the PAM fluorometer and initiate the curve recording. This value is called zero. Turn on the measuring light and wait approximately 30 seconds for the curve to respond.
This value was called F zero. Deliver a saturated pulse of 4, 000 micromolar photons per meter squared per second for 0.8 seconds from the PAM and obtain the highest value of the spike in the curve with increased fluorescence intensity. This value is called Fm.Then use the formula to calculate the maximum quantum yield of photo system two in the dark.
To measure photosynthetic behaviors in the steady state, after recording Fm irradiate the leaf sample with the 30 micromolar photons per meter squared per second. While turning on the spectral radiometer to monitor the leaf reflectance. Wait at least 20 minutes for the photosynthetic reaction to reach a steady state.
During this reaction period, the saturation pulse will be supplied at one minute intervals. The florescence intensity of the steady state is called Fs.The maximum florescence value achieved after 20 minutes of pulsed light is called Fm prime. Record the spectral reflectance at this point.
To calculate the photosynthesis activity at the steady state calculate the quantum yields of photo system two which can be estimated by irradiating with saturated pulses under actinic light. Estimate the linear electron flux from the photo system two reaction center. Then calculate the non photochemical quenching and calculate the photochemical reflectance index from 531 and 570 nanometers.
After acquiring Fs and the leaf reflectance turn off the actinic light and provide a saturating pulse at one minute intervals during the dark relaxation. The maximum florescence value induced by the saturation pulse under the dark is called Fm double prime. And save the Fm double prime data at two and 10 minutes after turning off the actinic light.
To calculate the parameters of the non photochemical quenching from the relaxation connectants, estimate the qE with the two minute dark adaptation Fm double prime data. Calculate the zanthoxylum dependent quenching, qZ, with the 10 minute dark adaptation Fm double prime data. Then calculate the photo inhibitory state.
As a next measurement turn on the actinic light at 60 micromolar photons per meter squared per second and repeat the same measurement. In this representative experiment, wild type and mutant Arabidopsis plants were compared. The change in photochemical reflectance index, PRI, calculated from the leaf reflectance, was plotted against the light dependent linear electron flow from photo system two as estimated by the PAM fluorometer.
In this experiment the change in the PRI was negatively correlated with the linear electron flow in wild type plants but not in mutant plants. qZ which represents the xanthophyll cycle was fractionated from the dark relaxation connectics of non photochemical quenching and was also plotted against the change in the PRI. In this analysis the qZ was also strongly correlated with the change in PRI for both plant strains, implying that the PRI reflects the xanthophyll cycle.
For simultaneous measurement of the same leaf area using the same light source or intensity use the same detection fiber in the fixing device to position the leaf vertically. Reflectance spectroscopy has a potential to be used in analysis of a variety of phenotypes, in wild type and mutant plants to elucidate plant molecular mechanisms.
