Estimating Chlorophyll Fluorescence Parameters Using the Joint Fraunhofer Line Depth and Laser-Induced Saturation Pulse ( FLD-LISP ) Method in Different Plant Species

A comprehensive evaluation of the recently developed Fraunhofer line depth (FLD) and laser-induced saturation pulse (FLD-LISP) method was conducted to measure chlorophyll fluorescence (ChlF) parameters of the quantum yield of photosystem II (ΦPSII), non-photochemical quenching (NPQ), and the photosystem II-based electron transport rate (ETR) in three plant species including paprika (C3 plant), maize (C4 plant), and pachira (C3 plant). First, the relationships between photosynthetic photon flux density (PPFD) and ChlF parameters retrieved using FLD-LISP and the pulse amplitude-modulated (PAM) methods were analyzed for all three species. Then the relationships between ChlF parameters measured using FLD-LISP and PAM were evaluated for the plants in different growth stages of leaves from mature to aging conditions. The relationships of ChlF parameters/PPFD were similar in both FLD-LISP and PAM methods in all plant species. ΦPSII showed a linear relationship with PPFD in all three species whereas NPQ was found to be linearly related to PPFD in paprika and maize, but not for pachira. The ETR/PPFD relationship was nonlinear with increasing values observed for PPFDs lower than about 800 μmol m−2 s−1 for paprika, lower than about 1200 μmol m−2 s−1 for maize, and lower than about 800 μmol m−2 s−1 for pachira. The ΦPSII, NPQ, and ETR of both the FLD-LISP and PAM methods were very well correlated (R2 = 0.89, RMSE = 0.05), (R2 = 0.86, RMSE = 0.44), and (R2 = 0.88, RMSE = 24.69), respectively, for all plants. Therefore, the FLD-LISP method can be recommended as a robust technique for the estimation of ChlF parameters.

The red and near-infrared (NIR) emissions by Chl coupled with quenching mechanisms have been used to estimate photosynthetic levels for decades.The process of "quenching", occurs through photochemical and non-photochemical mechanisms.The active and passive remote sensing of ChlF was also frequently applied to detect plant stress on both leaf and canopy scales [16,[28][29][30][31][32][33][34][35].
By the development of active techniques based on the pulse amplitude-modulated (PAM) method, effective measurement of ChlF outside the laboratory became possible [12].The PAM technique with saturating light pulses allows the estimation of several ChlF parameters such as dark-adapted and light-adapted peak fluorescence (Fm and Fm', respectively) and steady-state ChlF (Fs) to estimate the quantum yield of photosystem II (Φ PSII ), the non-photochemical quenching (NPQ), and the photosystem II-based electron transport rate (ETR).Φ PSII , NPQ, and ETR are three widely used ChlF parameters, which are employed to measure photochemistry and the overall photosynthetic capacity of plants [9,24].
However, the PAM fluorometry method is limited to short-distance applications for individual leaves and small plants attributed to the need for an artificial saturation light pulse and accurate pulse-synchronized and modulated fluorimetric techniques.A method based on the laser induced Chl fluorescence transients (LIFT) was introduced as a tool to expand the application of active ChlF measurement methods to long-distance, canopy-scale studies [36,37].In this approach, low-intensity pulses, instead of a saturating pulse, are used to measure the fluorescence transient, which is interpolated to a maximum fluorescence level using a fluorescence model.Using LIFT, ChlF can be measured up to a distance of 50 m but there are still challenges for longer distance remote sensing applications using this method [36,38,39].
To address ChlF measurement shortcomings on larger scales, passive remote sensing of steady-state solar-induced ChlF (SIF) has recently attracted more attention [27].The advantage of the SIF method is that the technique does not require accurate pulse-synchronized and modulated fluorimetric techniques as needed in the PAM and LIFT methods.The Fraunhofer line depth (FLD) method [40] is the most widely used SIF technique to measure ChlF emission under solar light.Using the FLD method, absolute variations in steady-state ChlF can be estimated but ChlF parameters cannot be retrieved and consequently ETR cannot be estimated.At the ground level, two broad oxygen absorption bands of terrestrial atmosphere at 760 nm (O 2 A) and 686 nm (O 2 B) are usually used to estimate the steady-state SIF.These wavelengths can be used for SIF measurement on the ground and for low flying aircraft.For satellite observation, other wavelengths in the near-infrared region are recommended [41].SIF signals can be measured as a proxy of photosynthesis from air-borne devices for canopy and larger scale applications [27,34,[42][43][44][45][46][47][48][49][50][51].More recently, the development of space-borne SIF remote sensing capability of the GOSAT (Greenhouse gases Observing SATellite) has provided the opportunity for SIF measurement from space [26,52] and for global mapping of SIF in the far-red region from satellites [41, [53][54][55][56].The application of SIF to assess the global terrestrial carbon cycle through the gross primary production (GPP) estimation has increasingly drawn attention and several research efforts have been made over the recent years to study the mechanisms that link SIF to GPP [57][58][59][60] and photosynthesis [61][62][63].A new FLEX (Flourescence Explorer) carrying the FLORIS (FLuORescence Imaging Spectrometer) has already been planned to be launched in 2022 to provide high spectral (0.3 to 3 nm) and fine spatial resolution (300 m) SIF imagery [64].
Different plant species have different SIF magnitudes in both the red (O 2 B) and the far-red (O 2 A) regions.SIF signals in these regions depend on variations in leaf characteristics such as Chl content and leaf area index (LAI).Therefore, the effects of Chl content and LAI on SIF signals need to be identified and removed [48,49,58,65].Rascher et al. (2015) argued that SIF emission is related to the total absorbed radiation by Chl, the functional status of photosynthesis, and stress in the plants [48].Tubuxin et al. (2015) also confirmed that the SIF signal largely depends on apparent Chl content in both the O 2 A and O 2 B bands, especially the latter [65].
Mechanisms that control ChlF signals over the short-term are well known, however seasonal interactions of ChlF signals and photosynthesis are less understood.Still more research is required on both leaf and canopy scales to understand seasonal interplay of ChlF and photosynthesis using both active and passive methods especially when the aim is to relate the knowledge acquired using active techniques to passive techniques [6,66].
Both the PAM and FLD methods provide information on ChlF, however their measurement principles are different in wavelength and intensity.The ordinary FLD method is a method for steady-state ChlF measurement, and therefore it depends on wavelength and Chl content.In the FLD method, ChlF parameters cannot be measured and this makes the comparison of active and passive methods difficult [34,47].Seasonal steady-state ChlF measurement comparison of PAM and FLD methods using outdoor wheat plants in three different treatments showed a weak but statistically significant relationship between active and passive methods, but the FLD measurement presented higher seasonal variability compared to PAM [66].The authors suggest that these results indicate the complexity of measuring ChlF using passive techniques (FLD) as compared with active methods.
In our previous research [65], a new technique was introduced to estimate Φ PSII by joining the FLD method and the laser induced saturation pulse method in order to directly retrieve information on photosynthesis from SIF signals.We have named this method the Fraunhofer line depth and laser-induced saturation pulse (FLD-LISP).The advantages of the suggested method are: (i) ChlF parameters can be estimated using the SIF method under sunlight by the combined application of the FLD technique and the saturation pulse laser illumination; (ii) FLD-LISP signals and PAM retrieved ChlF signals can be better compared; and (iii) unlike PAM, the suggested method does not require synchronization with the measuring pulse because the measuring pulse for fluorescence measurement is not needed.Therefore, there is good potential for the application of this method for longer distance measurements in a new type of portable instrument.
In this paper, a more comprehensive study of the methodology suggested by Tubuxin et al. (2015) at the leaf level is presented.In the current study, we evaluated the performance of the FLD-LISP method to measure ChlF parameters of Φ PSII , NPQ, and ETR under different actinic photosynthetic photon flux densities (PPFD).These measurements were made in three plant species including paprika (C3 plant), maize (C4 plant), and pachira (C3 plant) at different growth stages of leaves using the O 2 A band (760 nm).The PAM method was used to verify and understand the FLD-LISP method.First the relationships between actinic PPFD and ChlF parameters retrieved from FLD-LISP and PAM methods were studied.Then the ChlF parameters measured using FLD-LISP were compared with those retrieved from the PAM method in the three plant species in different growth stages of leaves from mature to aging conditions.

Plant Materials and Growth Conditions
In this experiment, paprika (Capsicum annuum cv., a C3 plant), maize (Zea mays L., a C4 plant), and pachira (Pachira aquatic Aubl, an indoor C3 plant) were used as plant materials.Paprika and maize were grown in an environmentally controlled growth chamber for 8-15 weeks.The plants were illuminated for 12 h each day with fluorescent and LED lights at a PPFD of 350 µmol m −2 s −1 .The air temperature of the growth chamber was 25.0 • C during the day and 20.0 • C at night with relative humidity set at ~70%.The seeds were planted in artificial soil (a mixture of vermiculite and perlite, 2:1, v/v) and were watered daily with a nutrient solution (1:1000 dilution of HYPONeX).Fully expanded mature and aging leaves at different growth stages were used in the experiments.Unlike the other two plants, the pachira plant was purchased and placed under indoor luminescent lamps at very low PPFD of ~50 µmol m −2 s −1 .

Measurement of ChlF Parameters Using the PAM Method
A JUNIOR PAM (Heinz Walz GmbH, Effeltrich, Germany) was used to measure the ChlF parameters of Φ PSII , NPQ, and ETR.Comparable vein-less sites of attached leaves were set vertically to incident light using a JUNIOR-B leaf clip.The glass fibre (fibre-PAM) was set at a 1 mm distance from the leaf and the blue LED (460 nm) saturation pulse PPFD was set at 10,000 µmol m −2 s −1 for 0.8 s.The PPFD measurement was performed at the 6th of the 12 steps (5 Hz).The leaf temperature and PPFD were recorded by sensors attached to a JUNIOR-B leaf clip and the corresponding data were transferred to a computer via a USB port using WinControl operating software.

Measurement of Spectral Radiant Intensity under Solar Light
An HR2000+ spectrometer (Ocean Optics, Dunedin, FL, USA) with spectral resolution of 0.065 nm at full-width at half-maximum (FWHM) was used for spectral radiant intensity measurement.The fibre optic (fibre-HR2000+) diffuser of the HR2000+ spectrometer was set at a 5 mm distance from the leaf surface, and it was held at 45 • to the fibre-PAM to reduce specular reflection from the leaf surface.The focal point of the fibre-HR2000+ was set at a distance of 2 mm from that of the fibre-PAM to avoid the influence of saturation pulse light and the shadow of the fibre-PAM.The integration time of the HR2000+ spectrometer was set at 0.2 s; more technical information is described in Tubuxin et al. (2015) [65].After the leaf spectral radiant measurement, a 90% White Card (Kodak, USA) set at the same angle and position as the leaf was used as the non-fluorescent reference standard.Both the spectra of the leaf and the non-fluorescent reference were recorded and transferred to a computer via a USB port using the OPwave+ operating software.A red laser (KaLaser, 660 nm, 200-250 mW) was used as the saturation pulse light, illuminating for 0.8 s at 60 • on the leaf from a distance of 40 cm and was set to have about a 3 cm diameter footprint.

Solar-Induced Chl Fluorescence Estimation Using FLD
Following Plascyk and Gabriel (1975) [40] and Moya et al. (2004) [46], the reflectance coefficient (R) and the solar-induced fluorescence intensity (F) were derived using the following equations: where M is the mean value of radiant intensity (digital number, DN) measured by the HR2000+ spectrometer, and the subscripts a and c mean the bands of the border (758.76 to 759.17 nm) and b and d mean the bands of the bottom (760.24 to 760.64 nm) of the O 2 A well (Fraunhofer line) from the non-fluorescent reference (a and b) and the leaf (c and d), respectively (see [65]).When the saturation laser pulse induced fluorescence intensity (Fm') under solar light is calculated by Equations ( 1) and ( 2 In this context, the radiant intensities Mc and Md apply to those from the leaf under both solar and laser lights.In the dark-adapted plants, the saturation laser pulse induced fluorescence intensity (Fm) was estimated from direct measurement at 760 nm using the spectrometer.

Simultaneous Measurement Using Both PAM and FLD-LISP Methods
Figure 1 shows a schematic diagram of the setup for the measurement of ChlF parameters using PAM and FLD-LISP.The experiments were carried out from 10 a.m. to 3 p.m. from August to October in an experimental room.Plants were moved from the growth chamber to the experimental room for adaptation at least one week before the experiments and were kept in the experimental room during the experiments.Air temperature in the experimental room was controlled but not recorded, however the leaf temperature was monitored by sensors attached to the JUNIOR-B leaf clip.The solar light entered through glass where its PPFD could have been variable by incident conditions via glass and cloudiness.In summer in Japan, the maximum solar light at a right-angled surface to the incident light can exceed 2000 µmol m −2 s −1 , so the incident light via glass reached about 2000 µmol m −2 s −1 in this experiment.The change from dark to light was carried out by rapid movement of a black box shown in Figure 1.The focal point (measurement area, white circle on the leaf) of fibre-HR2000+ was set at a distance of 2 mm from that of the fibre-PAM (light blue filled circle) to avoid the influence of saturation pulse light and shadow of the fibre-PAM.A time chart of the ChlF measurement using a combination of PAM and FLD-LISP methods is presented in Figure 2A.The set of sequential experiments included (i) dark adaptation of the clipped leaf area of the sample plant for 20 min; (ii) PAM measurement of maximum ChlF (Fm) during dark adaptation through switched on blue LED saturation pulse light (SL PAM ); (iii) after 50 s of the PAM measurement, FLD measurement of Fm using a HR2000+ spectrometer on red laser saturation pulse light (SL RL ); (iv) exposure to solar light for about 5 min; (v) PAM measurement of steady-state ChlF (Fs) and maximum ChlF (Fm') during the light adaptation; (vi) after 20 s of the PAM measurement, FLD measurement of Fs and Fm' using the HR2000+ spectrometer; and (vii) FLD measurement of the non-fluorescent reference (90% White Card) after about 20 s under solar light, immediately.The (v) and (vi) procedures were repeated 3 to 4 times at intervals of 40 s and it was found that the change in solar PPFD was negligible during the experiment.Several measures were taken throughout the FLD measurements to improve the Fm, Fm', and F S retrieval accuracies.First, optimal wavelength bands were selected for the mean values of radiant intensities (Ma, Mb, Mc, and Md) (See Section 2.4 and [65]).Second, in procedures (iii) and (vi) outlined above, Fm and Fm' were given by the mean value of three peak points during 0.6 s SL RL illumination and F S was calculated by averaging data during 10 s before the SL RL illumination using a median filter.It is known that the deviation in SIF retrieval using the FLD method is intrinsic to the method (not to the spectral resolution of the instrument, which is very high in this case) and linked to the reflectance spectral shape [67].However, a sequence of measurements within a day would be consistent (despite the offset) since the chlorophyll content does not substantially vary and the reflectance remains mostly stable.However, there will be deviations from one date to another where the chlorophyll content has changed, thus increasing the variance and inaccuracies.Therefore, the measurements in this paper can be considered appropriate.Nevertheless, more advanced methods [68] that cope with these effects will be necessary to increase the accuracy and reliability of the SIF measurements.After the measurement, the SPAD value (SPAD-502, Soil and Plant Analyzer Development, Konica Minolta, Tokyo, Japan) at the measured leaf area was estimated as an indicator of Chl content variations.These values were used to monitor leaf growth stages.For PPFD measurements during the experiments, an LI-250 light meter (LI-COR) and the PAM-attached sensor calibrated by LI-250 were used.

Methods for Calculating the ChlF Yield and Parameters
The ChlF yield (ΦF, the ratio of fluorescence photons to absorbed photons) was approximately calculated by ΦF = F/(PPFD × 0.84) where, F is the fluorescence intensity (rel.units), PPFD is the photosynthetic photon flux density (µmol m −2 s −1 ), and 0.84 is the leaf absorption coefficient.The ΦF can more accurately represent the relationship between the ChlF signal magnitude and PPFD than the ChlF intensity itself [2].Then Φ PSII and NPQ were calculated by the equations below [7,9,24]: where ΦFs is the steady-state ChlF yield from the light-adapted leaf (the mean value after median filtering of sequential data measured during 10 s just before the saturation laser pulse) and ΦFm' is the saturation pulse induced ChlF yield from the light-adapted leaf.
where ΦFm is the saturation pulse induced ChlF yield from the dark-adapted leaf.ETR was estimated using the equation given by Baker (2008) [1]:

Determination of the Saturation Light Pulse Intensity
To determine the saturation pulse intensity needed for this experiment, the ChlF yields (ΦF) in paprika leaves adapted under dark and solar lights at 700 and 1300 µmol m −2 s −1 PPFD were measured at different actinic light pulse intensities using the HR2000+ system.The leaf surface was illuminated by the blue LED of JUNIOR PAM.The fibre-HR2000+ was set at a 3 mm distance from the leaf surface, and it was held at 45 • to the fibre-PAM to fully detect the ChlF signal excited by the PAM actinic light.The light pulse intensities of PAM were successively raised from level 1 to 12 (25,45,66,90,125,190,285, 420, 625, 820, 1150, 1500 µmol m −2 s −1 , respectively) at a duration of about 2 s for each intensity and an interval of 30 s.The integration time of HR2000+ was set at 0.2 s and ΦF at 760.4 nm was obtained by averaging three peak points measured during 2 s.The number of measurements was three times in 20 min dark-adapted leaves and six times in 20 min light-adapted leaves, in which measurements were done four times at about 1300 µmol m −2 s −1 and two times at about 700 µmol m −2 s −1 .

Determination of the Saturation Light Pulse Intensity
We examined the relationships between the actinic PPFDs and ChlF yields in the dark and solar light adaptation periods for paprika leaves (Figure 3) as the saturation PPFD required to saturate the photosynthetic electron transport varies with the intensity of actinic light.Figure 3A,B show the results for a dark-adapted leaf (SPAD: 47.5, measured 3 times) and solar light-adapted leaves of 700 µmol m −2 s −1 (SPAD: 48.2, measured two times: ΦF1 and ΦF2) and 1300 µmol m −2 s −1 (SPAD: 42.6, measured four times: ΦF3, ΦF4, ΦF5, and ΦF6), respectively.
The ChlF yields of the dark-adapted leaf increased dramatically with a slight increase in PPFD and levelled off approximately at a PPFD of 800 µmol m −2 s −1 (dashed line in Figure 3A).The ChlF yields of the solar light-adapted leaf increased more gradually than those of the dark-adapted leaf, leveling off approximately at a PPFD of 1800 µmol m −2 s −1 (dashed line in Figure 3B).As a result, we confirmed the saturation of the photosynthetic electron transport at PPFD values over 1800 µmol m −2 s −1 at constant solar PPFD of 1300 µmol m −2 s −1 .Therefore, we decided to use the saturation pulse of the red laser that provides sufficiently strong PPFD of 6000 µmol m −2 s −1 for 0.8 s for paprika, maize, and pachira plants.This result can also be used in the PAM measurement.

Relationships between ChlF Parameters and Solar PPFDs
We examined the relationships between the PPFD and ChlF parameters of Φ PSII , NPQ, and ETR using PAM and FLD-LISP methods for paprika, maize, and pachira plants.The details of the measurement conditions, the number of samples, SPAD values, temperature of the abaxial side of the leaf, and solar PPFD are summarized in Table 1.The experiments were performed under steady weather conditions.Figures 4-6 present the results for the FLD-LISP measurements.The results of the PAM method exhibited very similar trends hence they were not presented in the plots.We also examined the relationships between SPAD values and ChlF parameters retrieved from FLD-LISP; no significant relationships were found confirming that the ChlF parameters are not affected by Chl content.
For the three plant species of paprika, maize, and pachira, the results of the relationships between Φ PSII , NPQ, and ETR and PPFDs using the FLD-LISP method are presented in Figures 4-6, respectively, while the R 2 values of the two methods are given in Table 1.As the solar PPFDs increased, Φ PSII decreased whereas NPQ increased exhibiting linear correlations for all three plant species, except that pachira's NPQ was over 1000 µmol m −2 s −1 (Figures 4A,B-6A,B, respectively).Compared to paprika, the Φ PSII and NPQ showed less variations for maize.The NPQ/PPFD relationship for pachira was non-linear, experiencing a sharp increase after the PPFD of about 1000 µmol m −2 s −1 .
For ETR, increasing trends were observed when the intensities of solar light were lower than about 800 µmol m −2 s −1 for paprika (Figure 4C), lower than about 1200 µmol m −2 s −1 for maize (Figure 5C), and lower than about 800 µmol m −2 s −1 for pachira (Figure 6C).In contrast, at solar light intensities greater than that stated above, ETR showed a lot of dispersions and the regression lines began to deteriorate.Table 1.Experimental conditions of the three plant species and the coefficients of determination (R 2 ) between ChlF parameters (Φ PSII , photochemical yield of photosystem II; NPQ, non-photochemical quenching; and ETR, photosystem II-based electron transport rate) and solar PPFD using the PAM and FLD-LISP methods.The results of the PAM method were very similar and the details are given in Table 1.(C) ETR of maize leaves estimated by the FLD-LISP method.The results of the PAM method were very similar and the details are given in Table 1.The results of the PAM method were very similar and the details are given in Table 1.

Relationships between the ChlF Parameters of the Two Methods
For the three plant species, we also examined the relationships between the ChlF parameters of Φ PSII , NPQ, and ETR determined by PAM and FLD-LISP (Figure 7).The results of each plant showed very similar trends as seen in Figure 7 and the R 2 and root mean square error (RMSE) values are summarized in Table 2.The Φ PSII , NPQ, and ETR of both methods were very well correlated and the regression lines were y = 0.96x + 0.01 (R 2 = 0.89, RMSE = 0.05), y = 0.98x + 0.30 (R 2 = 0.86, RMSE = 0.44) and y = 0.88x + 10.06 (R 2 = 0.88, RMSE = 24.69),which were approximated by y = x, although the slope for ETR was 0.88, which was smaller than 0.96 for Φ PSII , and 0.98 for NPQ.Overall, the Φ PSII of the two methods showed very small deviations for the three plant species.However, the variations of NPQ increased at higher NPQ values.In addition, the FLD-LISP NPQ estimations were slightly higher than those made by PAM.  2.
Table 2. Coefficients of determination (R 2 ) and RMSE between the ChlF parameters (Φ PSII , NPQ, and ETR) measured by the PAM and FLD-LISP methods for the three plant species.This table is a summary of Figure 7.

Discussion
The FLD-LISP method was suggested as an effective method to measure ChlF parameters using the FLD approach.In this research, FLD-LISP was evaluated and the results were compared with ChlF parameters measured by the standard PAM method.First, the FLD-LISP ChlF yield was measured using the O 2 A band.To determine the saturation light pulse intensity to be used for FLD-LISP, the relationships between the actinic pulse PPFD and ChlF yields in dark-adapted and solar light-adapted paprika leaves were initially studied (Figure 3).The photosynthesis saturation PPFD of many leaves is in the range of 500-1000 µmol m −2 s −1 , which is considerably lower than the direct solar PPFD of 2000 µmol m −2 s −1 [69].Omasa et al. (2009) [18] presented that ChlF yields of the Boston fern leaf abaxial side, leaf mesophyll, and guard cell chloroplasts became constant at the saturation pulse PPFD of 800 µmol m −2 s −1 in dark-adapted conditions and 1300 µmol m −2 s −1 in light-adapted conditions.In this research a saturation pulse of >800 µmol m −2 s −1 in the dark and 1800 µmol m −2 s −1 under solar PPFD of 700 to 1300 µmol m −2 s −1 was sufficient to saturate the photosynthetic electron transport in paprika leaves (Figure 3).This is because adequately photoactivated PSII requires stronger light; the excess light energy is dissipated as heat [7,70].The relationship between PPFD and ChlF yield at 760.4 nm using FLD-LISP confirms the results of other studies.The saturation pulse of the red laser at the PPFD of 6000 µmol m −2 s −1 for 0.8 s was used for all three plant species in this research, which is considerably larger than the photosynthesis saturation PPFDs of many leaves [69].
The FLD-LISP method was applied to measure the ChlF parameters of Φ PSII , NPQ, and ETR in three plant species, paprika (C3 plant), maize (C4 plant), and pachira (indoor C3 plant), having different carbon fixation mechanisms and light tolerance.The relationships between PPFD and FLD-LISP derived ChlF parameters of Φ PSII , NPQ, and ETR for all three plant species were studied.
In general, under high PPFD, excess light energy is dissipated as heat through the NPQ process that is regulated by the acidification of the thylakoid lumen attributed to the accumulation of protons leading to a ∆pH [4], which results in an increase in NPQ.This is an indicator of the portion of the absorbed radiation not used for electron transport in photosynthesis [4,7,71].The regulatory protein PsbS and the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle are involved in this process [4].For both C3 and C4 plants, Φ PSII generally decreases whereas NPQ increases with increasing illumination.Also under the same level of illumination, leaves with a lower photosynthetic capacity exhibit lower Φ PSII and higher NPQ.These facts have been reported previously in various ChlF studies at leaf and cell levels [1,5,[72][73][74][75][76][77][78].The relationships between PPFD and the ChlF parameters of Φ PSII , NPQ, and ETR measured using FLD-LISP in all three plant species (Figures 4-6) were similar to the relationships obtained through the PAM measurement.This confirms that the FLD-LISP method is capable of measuring ChlF parameters accurately under different PPFDs.
Both Φ PSII /PPFD and NPQ/PPFD relationships were found to be linear for paprika and maize under ordinary sunlight intensity (Figures 4A,B and 5A,B).For maize, the Φ PSII values were higher and the NPQ values were lower especially in high PPFDs in comparison with paprika.These results are acceptable as paprika is a C3 plant whereas maize is a C4 plant [79,80].For pachira, more variations were observed for NPQ when the PPFD was higher than 1000 µmol m −2 s −1 .Especially, when Φ PSII decreased to smaller values, the NPQ consequently increased to a large extent (Figure 6A,B).These results are expected because pachira is an indoor C3 plant adapted to low light.The ETR/PPFD relationship was nonlinear in all plants.The maximum ETR for maize was about twice as that for paprika and pachira.Increasing trends were observed for PPFD values lower than about 800 µmol m −2 s −1 for paprika and pachira (Figures 4C and 6C) and lower than about 1200 µmol m −2 s −1 for maize (Figure 5C), but the ETR decreased at high PPFD.This phenomenon may be caused by photoinhibition under high solar light because the plant materials were cultured at low PPFD under fluorescent and LED lights.Some of the variations seen in Figures 4-6 might have also been related to the effects of temperature and other environmental conditions.
Finally, ChlF parameters measured in different growth stages and light conditions using FLD-LISP and PAM were compared.The Φ PSII , NPQ, and ETR of both methods were very well correlated confirming good accuracy of FLD-LISP to estimate ChlF parameters regardless of differences in plant growth stages (variable Chl content) and in the response of the species to light.The Φ PSII of the two methods were better correlated compared to NPQ (Figure 7A,B).NPQ values derived from FLD-LISP were slightly higher than those obtained from PAM.This can be related to the difference in the method used for ΦFm and ΦFm' measurements (see Materials and Methods section).The variations of ETR increased at higher ETR values and the slope of regression equation was 0.88 for ETR (Figure 7C).This may have been caused by differences in light calibration between FLD-LISP and PAM.
The results presented in this research confirm the capability of FLD-LISP to measure ChlF parameters and the photosynthesis capacity of plants.The potential sources of error using this method as described by Tubuxin et al. (2015) [65] can be (i) noise in the spectrometer; (ii) solar light fluctuations including incident light variations through the glass during the measurement; (iii) differences in the angle of the uneven leaf surface and non-fluorescent reference; and (iv) the inaccuracy of the PPFD measurement on the leaf.In addition, variations in temperature and environmental conditions may affect the accuracy of the measurements.These sources of error need to be considered for the future applications of the suggested methodology.Several measures were taken in this research to improve the FLD method and to increase the ChlF retrieval accuracy.The results indicated that the applied measures were effective as good relationships were observed between the ChlF parameters measured using FLD-LISP and those retrieved from PAM, provided that they were measured simultaneously within a short time frame.At the same time, the presence of samples with a range of chlorophyll content might have resulted in an increased variance in the results due to the limitations of the standard FLD method.However, techniques such as improved FLD (iFLD) or spectral fitting [67,68,81] can be other improved alternatives for the FLD retrieval and are recommended to be evaluated and used in future applications of the developed FLD-LISP method.

Conclusions
To address the shortcomings of the SIF method, including the inability to measure the ChlF parameters and being affected by the plant's Chl content, we have introduced a new technique using a joint Fraunhofer line depth and laser induced saturation pulse method (FLD-LISP) to measure photosynthesis signals directly [65].In this paper a more comprehensive study of FLD-LISP was presented to better evaluate the performance of the suggested methodology for the measurement of ChlF parameters.We evaluated the performance of FLD-LISP to measure Φ PSII , NPQ, and ETR under different actinic PPFDs in three C3 and C4 plant species having different responses to light.The PAM method was used to verify and understand the FLD-LISP.The effect of PPFD on Φ PSII , NPQ, and ETR exhibited the same trend regardless of the method used to estimate ChlF parameters in all three plant species.The Φ PSII , NPQ, and ETR values derived using FLD-LISP and PAM were very well correlated, confirming the good accuracy of FLD-LISP to estimate ChlF parameters regardless of growth stages (variable Chl content) and differences in species confirming the usefulness of FLD-LISP to measure ChlF parameters and plant photosynthesis activity.
Finally, the FLD-LISP method does not need accurate pulse-synchronized and modulated fluorimetric techniques as the PAM and LIFT methods do.For long-distance (canopy scale) measurement of ChlF parameters using FLD-LISP, the combination of FLD-LISP and LIFT's lighting technique, which is interpolated to a maximum fluorescence level using a series of low-intensity laser pulses and a fluorescence model, would be recommended.
), the radiant intensities Ma and Mb from the non-fluorescent reference under only solar light are used because the radiant intensities in the a and b bands of the O 2 A are not affected by the red laser pulse.

Figure 1 .
Figure 1.Schematic diagram for the measurement of ChlF (Chlorophyll Fluorescence) parameters using PAM (Pulse Amplitude-Modulated) and FLD-LISP (Fraunhofer Line Depth and Laser-Induced Saturation Pulse) methods.The focal point (measurement area, white circle on leaf) of fibre-HR2000+ was set at a distance of 2 mm from that of the fibre-PAM (light blue filled circle) to avoid the influence of saturation pulse light and shadow of the fibre-PAM.Red filled area on the leaf is the footprint of the red laser beam.A black box was used to switch between dark condition and solar light.The timing and duty ratio of the red laser radiation were maintained by a laser pulse controller.

Figure 2 .
Figure2.Typical measurement diagrams of ChlF parameters using PAM and FLD-LISP.(A) a time sequence of lighting for leaf radiance spectral measurements using a HR2000+ spectrometer and PAM measurement during the dark and solar light adaptation periods.SL RL is the saturation light pulse (PPFD, Photosynthetic Photon Flux Density): 6000 µmol m −2 s −1 ) using a red laser (660 nm) for measurement using HR2000+ and SL PAM is the saturation light pulse (blue LED, PPFD about 10,000 µmol m −2 s −1 ) for measurement using JUNIOR PAM (Heinz Walz GmbH, Effeltrich.Germany); (B) temporal changes in ChlF yield (ΦF: near 760.4 nm) estimated by FLD-LISP, where ΦFm and ΦFm' are the laser saturation pulse-induced ChlF yields in dark-adapted and light-adapted leaves, respectively.The thick line is the mean value after median filtering during 10 s before the SL RL illumination for estimating the steady-state ChlF yield (ΦFs).

Figure 3 .
Figure 3. Relationships between light pulse PPFD and ChlF yield (ΦF: near 760.4 nm) of paprika leaves.(A) results in the dark-adapted leaf (SPAD value of 47.5, measured by SPAD-502, Soil and Plant Analyzer Development, Konica Minolta, Tokyo, Japan) measured three times (ΦF1 to ΦF3); (B) results in the light-adapted leaf (SPAD value of 42.6) measured twice (ΦF1 and ΦF2) at solar PPFD of 700 µmol m −2 s −1 and four times (ΦF3 to ΦF6) at 1300 µmol m −2 s −1 .The JUNIOR PAM actinic light system was used as the light pulse source.Dashed lines represent the minimal PPFD for the saturation light pulse.

Figure 4 .
Figure 4. Relationships between solar PPFD and ChlF parameters (A) Φ PSII ; (B) NPQ; and (C) ETR of paprika leaves estimated by the FLD-LISP method.The results of the PAM method were very similar and the details are given in Table1.

Figure 5 .
Figure 5. Relationships between the solar PPFD and the ChlF parameters (A) Φ PSII ; (B) NPQ; and(C) ETR of maize leaves estimated by the FLD-LISP method.The results of the PAM method were very similar and the details are given in Table1.

Figure 6 .
Figure 6.Relationships between the solar PPFD and ChlF parameters (A) Φ PSII ; (B) NPQ; and (C) ETR of pachira leaves estimated by the FLD-LISP method.The results of the PAM method were very similar and the details are given in Table1.

Figure 7 .
Figure 7. Relationships between the ChlF parameters estimated by the two methods (PAM and FLD-LISP) (A) Φ PSII ; (B) NPQ; and (C) ETR.The diamonds, circles, and triangles represent the results of paprika, maize, and pachira, respectively.R 2 and RMSE of all samples are shown in the figures and the results are summarized in Table2.