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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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. 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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 ...

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