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 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.
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.
1. Cultivation of Arabidopsis plants
2. Setting up the sample stage, photosynthetic instruments, and light source
NOTE: For this protocol, a custom-built sample stage was used for fixing leaves and detection probes (Figure 1).
3. Setting up simultaneous measurements of leaf reflectance and chlorophyll fluorescence
NOTE: All steps are performed in the dark room to avoid the detection of light other than actinic light. A weak-green light (e.g., green-cellophaned light) should be turned off before the actual measurements.
4. Simultaneous measurements of leaf reflectance and chlorophyll a fluorescence, and calculation of photosynthetic parameters
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 using the software of the separate systems. This experimental system was used to compare Arabidopsis wild-type (Col) and npq1 mutant (lack zeaxanthin) plants (Figure 2). The ΔPRI calculated from the leaf reflectance was plotted against light-dependent linear electron flow from PSII estimated by the PAM (Figure 2A). PRI is reported to be affected not only by xanthophyll but also by carotenoids16. The PRI was corrected by being PRI at each light intensity minus PRI at the lowest light intensity (ΔPRI) to observe only light-dependent PRI changes11. The results showed that ΔPRI was negatively correlated with LEF in wild-type plants, but not in npq1. We also dissected qZ, which represents the xanthophyll cycle, from the dark relaxation kinetics of NPQ and plotted it as ΔPRI in Figure 2B. The results show that qZ is strongly correlated with ΔPRI (r2 = -0.87, p-value < 0.001), implying that PRI reflects the xanthophyll cycle.
Figure 1: Schematic diagram of the experimental system for simultaneous measurement of chlorophyll a fluorescence and leaf reflectance. Details are described in the Protocol section. A plant pot was positioned by a lab jack (solid double-headed arrow). A halogen lamp was used to irradiate various light intensities to activate photosynthesis (thin solid arrow). Chlorophyll a fluorescence signals were detected using a system of pulse amplitude modulation (PAM); the red line indicates the fiber probe from the PAM chlorophyll fluorometer. The leaf reflectance was detected by a spectral radiometer under the light illumination; the blue line indicates the fiber probe from the spectral radiometer. The measuring light (dotted arrow) and the short-saturated light (thick solid arrow) also were provided by the PAM chlorophyll fluorometer. The saturated lights were pulsed with 1 min intervals during light adaptation for 20 min and dark relaxation for 10 min. Please click here to view a larger version of this figure.
Figure 2: Changes in photosynthetic parameters in wild-type Columbia (black squares), and npq1 mutant (red squares) Arabidopsis plants. ΔPRI (PRI at each irradiance intensity minus PRI at the lowest intensity of 30 µmol photons m-2 s-1) was plotted against (A) the rate of linear electron flux (LEF), and (B) qZ after dark relaxation for 10 min. The irradiance intensity of the actinic light was 30, 60, 120, 240, and 480 µmol photons m-2 s-1. Data points and error bars represent means ± SD for n=3. The line in B is a regression curve that applies for all data points. Please click here to view a larger version of this figure.
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), possibly by photodamaging PSII. We therefore adapted the halogen lamp with a built-in cold filter to reduce heat production. This light source did not cause any abnormalities in dark recovery or qI.
The most important variable in our method is the positional relationship between the leaf, the light source, and the detection probes. We have tested measuring the chlorophyll fluorescence and leaf reflectance from various diagonal angles with light irradiating from directly above to the leaf. However, the intensity of the detection signals differed depending on the angle. To avoid this variability, the probes were fixed vertically above the leaf sample (Figure 1). The light source was delivered using bifurcated fibers that irradiated the leaf surface from both the left and right sides to generate a uniform irradiating light (Figure 1).
Studies of leaf reflectance have been primarily used in ecology to determine various plant vegetation indices in field settings, such as differences between plant species, nutritional conditions, or seasonal changes. However, few studies have tested and verified these vegetation indices in model plants such as Arabidopsis and tobacco, whose mutants could possess a wealth of genetic information and omics analyses data. Verifying and developing vegetation indices for these plants could identify novel photosynthetic parameters represented in innovative vegetation indices, which would contribute to the discipline of ecology.
This study focused on the dark relaxation kinetics of NPQ to verify the xanthophyll cycle behavior. New photosynthesis-related parameters are currently under development for chlorophyll fluorescence analysis (e.g., estimations of the redox state of the plastoquinone pool (qL) or the activity of cyclic electron flow around PSI17,18). The simultaneous measurement of chlorophyll fluorescence and leaf reflectance in related Arabidopsis mutants will advance research into the molecular mechanisms of photosynthesis and help to utilize this knowledge in field studies. A recent study reported that chlorophyll fluorescence in plants can be remotely sensed from leaf spectral reflectance. The parameter calls solar-induced chlorophyll fluorescence (SIF) is measured utilizing a Fraunhofer line, dark lines absorbed by Oxygen, under solar light 12,19. If the currently developed vegetation indices were reassigned using these techniques, it would be possible to remotely assess photosynthetic responses in plants without using special treatments such as saturated pulses or dark adaptation.
The authors have nothing to disclose.
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.
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 |