Name: evaluation of photosynthetic behaviors by simultaneous measurements of leaf reflectance and chlorophyll fluorescence analyses
Uploaded: 2019-08-09T15:00:00
Description: Watch this Scientific Journal Video about Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses at JoVE.com

We are grateful to Dr. Kouki Hikosaka (Tohoku University) for stimulating discussions, assistance with a work space, and instruments for experiments. The work was supported in part by KAKENHI [grant numbers 18K05592, 18J40098] and Naito Foundation.

1. Cultivation of Arabidopsis plants
<ol>
	<li>Soak Arabidopsis thaliana seeds in sterilized deionized water in a microtube, and incubate for 2 days at 4 &#176;C in the dark.</li>
	<li>Place approximately four of the imbibed, cold-treated seeds onto the soil surface using a micropipette. Incubate the planted pots in a growth chamber with a 16 h light (120 &#956;mol photons m&#8211;2 s&#8211;1) and 8 h dark period at 22 &#176;C and 20 &#176;C, respectively.</li>
	<li>Grow one plant per pot...

<ol>
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R., Bj&#246;rkman, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mutants+define+a+central+role+for+the+xanthophyll+cycle+in+the+regulation+of+photosynthetic+energy+conversion.">Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion.</a> Plant Cell. 10 (7), 1121-1134 (1998).</li><li>Kohzuma, K., Hikosaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+validation+of+photochemical+reflectance+index+(PRI)+as+a+photosynthetic+parameter+using+Arabidopsis+thaliana+mutants.">Physiological validation of photochemical reflectance index (PRI) as a photosynthetic parameter using Arabidopsis thaliana mutants.</a> Biochemical and Biophysical Research Communications. 498, 52-57 (2018).</li><li>Hikosaka, K., Noda, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+leaf+CO2+assimilation+and+Photosystem+II+photochemistry+from+chlorophyll+fluorescence+and+the+photochemical+reflectance+index.">Modeling leaf CO2 assimilation and Photosystem II photochemistry from chlorophyll fluorescence and the photochemical reflectance index.</a> Plant, Cell and Environment. 42 (2), 730-739 (2019).</li><li>Brooks, M. D., Sylak-Glassman, E. J., Fleming, G. R., Niyogi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thioredoxin-like/&#946;-propeller+protein+maintains+the+efficiency+of+light+harvesting+in+Arabidopsis.">A thioredoxin-like/&#946;-propeller protein maintains the efficiency of light harvesting in Arabidopsis.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (29), E2733-E2740 (2013).</li><li>Nilkens, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+slowly+inducible+zeaxanthin-dependent+component+of+non-photochemical+quenching+of+chlorophyll+fluorescence+generated+under+steady-state+conditions+in+Arabidopsis.">Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1797 (4), 466-475 (2010).</li><li>Davis, G. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+photochemical+reflectance+index+provides+an+optical+indicator+of+spring+photosynthetic+activation+in+evergreen+conifers.">The photochemical reflectance index provides an optical indicator of spring photosynthetic activation in evergreen conifers.</a> New Phytologist. 206 (1), 196-208 (2015).</li><li>Miyake, C., Amako, K., Shiraishi, N., Sugimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acclimation+of+Tobacco+Leaves+to+High+Light+Intensity+Drives+the+Plastoquinone+Oxidation+System&#8212;Relationship+Among+the+Fraction+of+Open+PSII+Centers,+Non-Photochemical+Quenching+of+Chl+Fluorescence+and+the+Maximum+Quantum+Yield+of+PSII+in+the+Dark.">Acclimation of Tobacco Leaves to High Light Intensity Drives the Plastoquinone Oxidation System&#8212;Relationship Among the Fraction of Open PSII Centers, Non-Photochemical Quenching of Chl Fluorescence and the Maximum Quantum Yield of PSII in the Dark.</a> Plant and Cell Physiology. 50 (4), 730-743 (2009).</li><li>Munekage, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+electron+flow+around+photosystem+I+is+essential+for+photosynthesis.">Cyclic electron flow around photosystem I is essential for photosynthesis.</a> Nature. 429 (6991), 579-582 (2004).</li><li>Tubuxin, B., Rahimzadeh-Bajgiran, P., Ginnan, Y., Hosoi, F., Omasa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+chlorophyll+content+and+photochemical+yield+of+photosystem+II+(&#934;PSII)+using+solar-induced+chlorophyll+fluorescence+measurements+at+different+growing+stages+of+attached+leaves.">Estimating chlorophyll content and photochemical yield of photosystem II (&#934;PSII) using solar-induced chlorophyll fluorescence measurements at different growing stages of attached leaves.</a> Journal of Experimental Botany. 66 (18), 5595-5603 (2015).</li></ol>

In this study, we obtained additional evidence to show that PRI represents xanthophyll pigments by simultaneously measuring chlorophyll fluorescence and leaf reflectance.
A halogen light, which has wavelengths similar to sunlight, was adapted for use as an actinic light source to activate photosynthesis. We initially used a white LED light source to avoid thermal damage of the leaf surface, but this produced slow dark relaxation kinetics and exceptionally high qI (photoinhibitory quenching), p...

Leaf reflectance is used to remotely sense vegetation indices that reflect photosynthesis or traits in plants1,2. The normalized difference vegetation index (NDVI), which is based on infrared reflection signals, is one of the most widely known vegetation indices for the detection of chlorophyll-related properties, and it is used in the ecology and agricultural sciences as an indicator of environmental responses in trees or crops3. In field studies, although many parameters (e.g., chlorophyll index (CI), water index (WI), etc.) have been developed and used, few detailed verifications of what these parameters directly (or indirectly) detect have been performed using mutants.
Pulse-amplitude modulation (PAM) analysis of chlorophyll fluorescence is an effective method to measure photosynthetic reactions and processes involved in photosystem II (PSII)4. Chlorophyll fluorescence can be detected with a camera and used for screening photosynthesis mutants5. However, camera detection of chlorophyll fluorescence requires complex protocols such as dark treatment or light saturation pulses, which are difficult to implement in field studies.
Leaf absorbed solar light energy is mainly consumed by photosynthetic reactions. By contrast, the absorption of excess light energy can generate reactive oxygen species, which causes damage to photosynthetic molecules. The excess light energy must be dissipated as heat through non-photochemical quenching (NPQ) mechanisms6. The photochemical reflectance index (PRI), which reflects light-dependent changes in leaf reflectance parameters, is derived from narrow-band reflectance at 531 and 570 nm (reference wavelength)7,8. It is reported to correlate with NPQ in chlorophyll fluorescence analysis9. However, since NPQ is a composite parameter that includes the xanthophyll cycle, state tradition, and photoinhibition, detailed validation is required to understand what the PRI parameter measures. We have focused on the xanthophyll cycle, a thermal dissipation system involving the de-epoxidation of xanthophyll pigments (violaxanthin to antheraxanthin and zeaxanthin) and a main component of NPQ because correlations between PRI and conversion of these pigments has been reported in previous studies8.
Many photosynthesis-related mutants have been isolated and identified in Arabidopsis. The npq1 mutant does not accumulate zeaxanthin because it carries a mutation in violaxanthin de-epoxidase (VDE), which catalyzes the conversion of violaxanthin to zeaxanthin10. To establish whether PRI only detects changes in xanthophyll pigments, we simultaneously measured PRI and chlorophyll fluorescence in the same leaf area in npq1 and the wild-type and then dissected NPQ at varying time scales of dark relaxation to extract the xanthophyll-related component11. These simultaneous measurements provide a valuable technique for the assignment of vegetation indices. Furthermore, since PRI correlates with gross primary productivity (GPP), the ability to assign PRI precisely to one component has important applications in ecology12.

<table>
	<tbody>
		<tr>
			<td>Halogen light source</td>
			<td>OptoSigma</td>
			<td>SHLA-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Light quantum meter</td>
			<td>LI-COR</td>
			<td>LI-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PAM chlorophyll fluorometer</td>
			<td>Walz</td>
			<td>JUNIOR-PAM</td>
			<td></td>
		</tr>
		<tr>
			<td>PAM controliing software</td>
			<td>Walz</td>
			<td>WinControl-3.27</td>
			<td></td>
		</tr>
		<tr>
			<td>Reflectance standard</td>
			<td>Labsphere, Inc.</td>
			<td>SRT-99-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectral radiometer</td>
			<td>ADS Inc.</td>
			<td>Field Spec3</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectral radiometer controlling software</td>
			<td>ADS Inc.</td>
			<td>RS3</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of photosynthetic behaviors by simultaneous measurements of leaf reflectance and chlorophyll fluorescence analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

Figure 1 presents a schematic diagram of the experimental set up for simultaneously measuring chlorophyll fluorescence and leaf reflectance. The fiber probes of the PAM and spectral radiometer were set perpendicularly to the leaf surface at the leaf holder on the custom-made sample stage, and a halogen lamp was used for actinic light irradiation from both left and right directions without casting any shadows. The PAM and leaf reflectance signals were detected...

Watch this Scientific Journal Video about Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses at JoVE.com

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

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.

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