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

Summary

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.

Abstract

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.

Introduction

Leaf reflectance is used to remotely sense vegetation indices that reflect photosynthesis or traits in plants^1,2. 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 ....

Protocol

1. Cultivation of Arabidopsis plants

1. Soak Arabidopsis thaliana seeds in sterilized deionized water in a microtube, and incubate for 2 days at 4 °C in the dark.
2. 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 μmol photons m^–2 s^–1) and 8 h dark period at 22 °C and 20 °C, respectively.
3. Grow one plant per pot.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Halogen light source	OptoSigma	SHLA-150
Light quantum meter	LI-COR	LI-1000
PAM chlorophyll fluorometer	Walz	JUNIOR-PAM
PAM controliing software	Walz	WinControl-3.27
Reflectance standard	Labsphere, Inc.	SRT-99-050
Spectral radiometer	ADS Inc.	Field Spec3
Spectral radiometer controlling software	ADS Inc.	RS3