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remote sensing Article

Diurnal Cycle Relationships between Passive Fluorescence, PRI and NPQ of Vegetation in a Controlled Stress Experiment Luis Alonso *, Shari Van Wittenberghe, Julia Amorós-López, Joan Vila-Francés, Luis Gómez-Chova and Jose Moreno Image Processing Laboratory (IPL), University of Valencia, C/Catedrático José Beltrán, 2, Paterna, 46980 Valencia, Spain; [email protected] (S.V.W.); [email protected] (J.A.-L.); [email protected] (J.V.-F.); [email protected] (L.G.-C.); [email protected] (J.M.) * Correspondence: [email protected]; Tel.: +34-963-543-110 Received: 8 May 2017; Accepted: 21 July 2017; Published: 28 July 2017

Abstract: In order to estimate vegetation photosynthesis from remote sensing observations; some critical parameters need to be quantified. From all absorbed light; the plant needs to release any excess that is not used for photosynthesis; by non-photochemical quenching; by fluorescence emission and unregulated thermal dissipation. Non-photochemical quenching (NPQ) processes are controlled photoprotective mechanisms which; once activated; strongly control the dynamics of photochemical efficiency. With illumination conditions increasing and decreasing during a diurnal cycle; photoprotection mechanisms needs to change accordingly. The goal of this work is to quantify dynamic NPQ; measured from active fluorescence measurements; based on passive proximal sensing leaf measurements. During a 22-day controlled light and water stress experiment on a tobacco (Nicotiana tabacum L.) leaf we measured the diurnal dynamics of passive fluorescence (Chl F); the Photochemical Reflectance Index (PRI); the Absorbed Photosynthetically Active Radiation (APAR) and leaf temperature in combination with the actively retrieved non-photochemical quenching (NPQ) parameter. Based on a bi-linear combination of diurnal APAR and PRI (plane fit model) we succeeded to estimate NPQ with a RMSE of 0.08. The simple plane fit model estimation represents well the diurnal NPQ dynamics; except for the high light stress phase; when additional reversible photoinhibition processes took place. The present works presents a way of determining NPQ from passive remote sensing measurements; as a necessary step towards estimating photosynthetic rate. Keywords: drought; stress; non-photochemical energy dissipation; solar-induced fluorescence (SIF); photosynthesis; non-photochemical quenching (NPQ); Photochemical Reflectance Index (PRI); FLuorescence EXplorer; (FLEX)

1. Introduction Plants must deal with light in extreme ways. Both the quantity of solar radiation, varying over several orders of magnitude, as well as the temporal fluctuations, ranging from seconds to seasons, demand a high flexibility of plants to keep the light supply in balance with the demand for healthy metabolic growth. To do so, plants evolved with the remarkable capacity of dissipating absorbed solar energy in ways being profitable (harvesting light for growth) or protective (activating different mechanisms to avoid light damage). Absorbed photosynthetically active radiation (APAR) brings chlorophyll-a molecules to an excited and energetic singlet-state, ready to go back to its ground state through a combination of different mechanisms: photosynthesis (or Photochemical Quenching, PQ), non-photochemical quenching (NPQ), and fluorescence (F) emission. While fluorescence emission

Remote Sens. 2017, 9, 770; doi:10.3390/rs9080770

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Remote Sens. 2017, 9, 770

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is an unregulated process, non-photochemical fluorescence quenching (NPQ) involves controlled heat dissipation and becomes activated at a certain light excess [1,2]. During the early morning hours (or low illumination conditions in general) photoprotection is not activated, because the absorbed energy can still be efficiently trapped for photochemical use, driving the light and carbon reactions. Like this, absorbed solar energy can be harvested with an efficiency up to 90% [1]. Hence, typically during the first morning hours and towards sunset, the changes in the quantum yield of fluorescence are linearly controlled by PQ [3,4]. When light intensity further increases, the light and carbon reactions gradually become light saturated and the relative amount of energy emitted as F per energy unit absorbed energy, or F yield, increases. However, Chl F emission does not protect the plant from photodamage, so when incoming radiation increases even further, NPQ mechanisms are activated [1,2]. When the APAR becomes saturating for the safe and efficient use of the photosynthetic machinery, is hard to be defined on an absolute energy scale. This will depend on the physiological state of the plant, as well as on environmental parameters such as temperature [5]. Three major NPQ mechanisms are typically identified, i.e., the energy-dependent NPQ (qE), the photoinhibitory NPQ (qI), and the state-transitions NPQ (qT) [1]. The most important is the energy-dependent NPQ, which has as main function the protection of Photosystem II (PSII) from photoinhibition [6]. Once the NPQ protective mechanisms are activated, the changes in the quantum yield of photochemistry are controlled by the dynamics of NPQ [3]. The European Space Agency (ESA) selected FLuorescence EXplorer (FLEX) mission as 8th Earth Explorer, which is specifically designed to measure SIF, and will have an expected optimal overpass time during the phase in which photochemical and fluorescence yield are positively correlated, but dominated by NPQ [7]. Hence, knowledge on NPQ is necessary for establishing the unambiguous link between SIF and the quantum yield of photochemistry. From all three previously mentioned NPQ mechanisms, the energy-dependent NPQ or qE, regulated by lumen pH and the xanthophyll cycle pigments, represents the main component of NPQ [8], which is able to change within minutes after a light intensity change [1]. This reversible xanthophyll cycle can be detected by a small spectral change around 531 nm, translated to the widely used Photochemical Reflectance Index or PRI [9]. Furthermore, because xanthophyll cycle pigments adjust the energy distribution at the photosynthetic reaction centres, PRI has been used as a measure of photosynthetic light-use efficiency (LUE) and indicator of stress [10]. Moreover, PRI was found to track fluorometer-measured NPQ in several studies (for an overview, see [11]). In this meta-analysis [11], a non-significant PRI-NPQ relationship was reported at the short daily time scale for leaf level, while at the seasonal scale PRI could explain 77% of the variation in NPQ for herbaceous leaves. Several studies have reported good correlations between PRI and NPQ during different short-term light phases [12–14], however, also acknowledge some discrepancies due to other interacting NPQ mechanisms besides the xanthophyll cycle, such as photoinhibitory NPQ (qI), and the state-transitions NPQ (qT). Furthermore, these, and other relationships with PRI become weak over the whole growth cycle [13]. Upon stress events or seasonal development, when changes in the pigment pool (other than xanthophyll) and leaf structure occur, changing reflectance will affect the PRI. Under these circumstances, the relationship between PRI and the xanthophyll cycle gets modified. While PRI is an index related to the generally most important light-induced non-photochemical heat dissipation mechanism qE, Chl F is an actual energy emission that is physically linked to the photosynthesis process. Hence, both tell us something on the plants’ dissipation energy upon light absorption and can be passively measured by remote sensing. The FLEX mission will focus on the combined information of both parameters by carrying very high spectral resolution imaging sensors [7,15]. Therefore, understanding the dynamic diurnal relationships between Chl F, NPQ and PRI is a crucial point for establishing a better, solid knowledge on photosynthesis parameters as further mission products. In this study, we present a proximal sensing experiment at leaf level in which the diurnal behaviour of NPQ, APAR, PRI and F are investigated during long lasting physiological stress. With this, our


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Remote 2017,the 9, 770 3 of 16 aim is toSens. study optical diurnal dynamics and relationships between both actively and passively derived optical parameters, measurements which are currently lacking. Additionally, we want to passively derived optical parameters, measurements which are currently lacking. Additionally, we want explore the prediction of actively derived NPQ based solely on passively derived PRI and APAR. to explore the prediction of actively derived NPQ based solely on passively derived PRI and APAR.

2. Materials and Methods 2. Materials and Methods A tobacco plant (Nicotiana tabacum L.) was grown in low light conditions (100 µmol m−2 s−1 ), A tobacco plant (Nicotiana tabacum L.) was grown in low light conditions (100 µmol m−2 s−1), while while being well-watered and supplied with fertilized soil. Our aim was to subject a leaf of the plant, being well-watered and supplied with fertilized soil. Our aim was to subject a leaf of the plant, mounted in a leaf clip holder, to two environmental stresses in a controlled way during a 22-day mounted in a leaf clip holder, to two environmental stresses in a controlled way during a 22-day experiment. First, by bringing the plant under high light intensity diurnal illumination, light stress was experiment. First, by bringing the plant under high light intensity diurnal illumination, light stress imposed. Additionally, we stopped watering the plant and the room temperature was kept constant was imposed. Additionally, we stopped watering the plant and the room temperature was kept at constant 25 ± 2 ◦ C, progressive drought stress. To stress. properly the leaf’sthe strain, at inducing 25 ± 2 °C,ainducing a progressive drought To evaluate properly evaluate leaf’sadditional strain, sensors were placed to control the air temperature, the relative humidity (Senserion’s SHT75), and the additional sensors were placed to control the air temperature, the relative humidity (Senserion’s soil moisture (Rika’s RK510). This set-up allowed us monitoring the increasing soil drought that was SHT75), and the soil moisture (Rika’s RK510). This set-up allowed us monitoring the increasing soil generated during the 22-day experiment (Figure 1). Due to unexpected interruption of the automatic drought that was generated during the 22-day experiment (Figure 1). Due to unexpected interruption measuring system,measuring three dayssystem, of the three experiment are missingdata (D8, D12 and D20). of the automatic days ofdata the experiment are missing (D8, D12 and D20).

Figure Soilmoisture moisture(%) (%) during during the the 22-day 22-day drought drought experiment. in in thethe graph areare thethe Figure 1. 1.Soil experiment.Marked Marked graph different phases identified and explained in the text below. different phases identified and explained in the text below.

This experiment was conducted in 2006 and partly presented in an earlier work that investigated This experimentofwas 2006 and presented in anformulated earlier work that investigated the comparability the conducted active and in passive Chl partly F measurements and the best satellite theoverpass comparability the activesensing and passive F measurements and formulated best satellite time forof the remote of Chl Chl F [16–18]. Here, we present new results the on the diurnal overpass time for the remote sensing of Chl F [16–18]. Here, we present new results on the diurnal interaction between the different energy dissipation mechanisms that can be measured through interaction different energy dissipation mechanisms that can be measured through active active andbetween passive the techniques. and passive techniques. Diurnal light cycles were generated by two high power white LED modules (Optospot Diurnal light cyclesS.p.A., wereMilano, generated two by high power white LEDLED modules OSP/LF6/L3, LC Relco Italy)by driven a computer controlled current(Optospot driver OSP/LF6/L3, LC Relco S.p.A.,Each Milano, Italy) driven by atocomputer controlled driver (Relco PTDCCD/15/350/S10). LED module, pointing the leaf surface, had aLED cyancurrent filter (K39912, Edmund Optics, York, UK)Each mounted front with a sharp cut-off 600 nm had to remove (Relco PTDCCD/15/350/S10). LED in module, pointing to the leafatsurface, a cyanthe filter incident light in the Chl F emission region. This illumination set-up, as illustrated in Figure 2, allowed (K39-912, Edmund Optics, York, UK) mounted in front with a sharp cut-off at 600 nm to remove a controlled predefined diurnal Photosynthetic Active set-up, Radiation (PAR) lightinpattern, thesimulating incident light in the Chl F emission region. This illumination as illustrated Figure 2, −2 −1 rangingsimulating from 0 to 1000 µmol m spredefined , updated every minute. This light pattern was controlled(PAR) through allowed a controlled diurnal Photosynthetic Active Radiation light − 2 − 1 the feedback of a micro-quantum sensor on the PAM-2000 leaf clip holder which at the same time pattern, ranging from 0 to 1000 µmol m s , updated every minute. This light pattern was controlled measured leaf temperature every 2 min withon a the contact thermocouple. One diurnal cycle of through the feedback of a micro-quantum sensor PAM-2000 leaf clip holder which at the same measurements lasted 11 h from which the first 80 min and last 40 min were dark. Hence, the time time measured leaf temperature every 2 min with a contact thermocouple. One diurnal cycle of from artificial sunrise to artificial sunset took 9 h. Net leaf temperature change (∆T) was calculated as measurements lasted 11 h from which the first 80 min and last 40 min were dark. Hence, the time the difference between the actual leaf temperature and the average leaf temperature during the dark from artificial sunrise to artificial sunset took 9 h. Net leaf temperature change (∆T) was calculated as period right before the start of the diurnal light cycle. the difference between the actual leaf temperature and the average leaf temperature during the dark The leaf’s diurnal fluorescence emission was simultaneously recorded by a pulse amplitude period right before the start of the diurnal light cycle. meter PAM-2000 (Waltz GmbH, Effeltrich, Germany) and a FieldSpec FR spectroradiometer (ASD Inc., Boulder, CO, USA). The fiber optic of the ASD spectroradiometer was aligned to point to the


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Remote Sens. 2017, 9, 770 of 16 same leaf spot where the PAM-2000’s measurements took place. Both fibers (with a similar FOV4 of

25°) were adjusted to the same relative inclination (30° from the leaf normal angle).

PAM fiber

ASD fiber LE D

D LE

cyan filter

cyan filter leaf

Figure 2. Picture (left) and diagram (right) of the experimental set-up. The symmetrical disposition Figure 2. Picture (left) and diagram (right) of the experimental set-up. The symmetrical disposition of of elements guarantee that both instruments measure the same spot under equal conditions (Adopted elements guarantee that instruments measure the same spot under equal conditions (Adopted from Amorós-López et both al. [16]). from Amorós-López et al. [16]).

Every 2 min the ASD FieldSpec FR spectroradiometer collected both the reflected radiance in the blue-green of thefluorescence visible (400–600 nm) and thesimultaneously full upward Chlrecorded F emission (650–850 The leaf’spart diurnal emission was byspectrum a pulse amplitude nm) with a spectral resolution of 3 nm and a sampling interval of 1 nm. Prior to the start of the meter PAM-2000 (Waltz GmbH, Effeltrich, Germany) and a FieldSpec FR spectroradiometer (ASD Inc., experiment, the irradiance at different intensities of the LED modules was toacquired a Boulder, CO, USA). The fiber optic of the ASD spectroradiometer was aligned point tousing the same Spectralon white reference to calculate the reflectance of the leaf in the blue-green part of the visible leaf spot where the PAM-2000’s measurements took place. Both fibers (with a similar FOV of 25◦ ) (400–600 nm). the diurnal reflectance datathe PRIleaf wasnormal calculated as: ◦ from were adjusted to From the same relativeleaf inclination (30(R) angle).

Every 2 min the ASD FieldSpec FR spectroradiometer collected both the reflected radiance in the R531 − R570 (1) PRI = blue-green part of the visible (400–600 nm) and the full+ upward R531 R570 Chl F emission spectrum (650–850 nm) with a spectral resolution of 3 nm and a sampling interval of 1 nm. Prior to the start of the experiment, One should note that this corresponds to the original formulation [9]. Nowadays it is becoming the irradiance at different intensities of the LED modules was acquired using a Spectralon white more common to find the PRI equation with the sign reversed so that an increase in photoprotective reference to calculate the as reflectance of of thethe leaf in the blue-green the visible (400–600 nm). mechanism is reflected an increase index; however, sincepart the of reverse formulation is still From the diurnal leaf reflectance (R) data PRI was calculated as: identified as PRI it might lead to confusion when compared to the original definition. Likewise, the PAM fluorometer measures,R531 every min, relative fluorescence values in the far− 2R570 PRI = red (Ft) by constant modulated light pulses. Since red light is used for the pulses and the detector is(1) R531 + R570 protected by a long-pass filter (λ > 700 nm), the PAM only provides fluorescence as a single broad band 710note to 850 the added to F signal excited by a modulated pulse. Because the F Onefrom should thatnm thisofcorresponds the original formulation [9]. Nowadays it is becoming measurement the relative to the given pulse light, the PAManFtincrease measurement is a relative more common toisfind the PRI increase equationdue with sign reversed so that in photoprotective value to some extent related Chl F yield. mechanism is reflected as an to increase of the index; however, since the reverse formulation is still Theascomparability between simultaneously measuredto active red-band (PAM) and passive identified PRI it might lead to confusion when compared the original definition. spectral (ASD) fluorescence was checked for all 2-min interval diurnal data of the Likewise, the PAM fluorometer measures, every 2 min, relative fluorescence22-day valuesexperiment. in the far-red The PAM F t measurements with arbitrary units were compared to both the simultaneous measured (Ft ) by constant modulated light pulses. Since red light is used for the pulses and the detector red fluorescence (RF, 650–705 nm)(λ yield andnm), far-red 705–790 nm) yield. As is protected by a long-pass filter > 700 thefluorescence PAM only(FRF, provides fluorescence as shown a single in previous research [19], the passively and actively retrieved FY was found comparable. The broad band from 710 to 850 nm of the added F signal excited by a modulated pulse. Because the F coefficient of determination between the RF Yield and Ft (R2 = 0.841) was expectedly lower compared measurement is the relative increase due to given pulse light, the PAM Ft measurement is a relative to one of the FRF Yield and Ft (R2 = 0.913) relationship. Spectral passive fluorescence yield (FY) was value to some extent related to Chl F yield. calculated as: The comparability between simultaneously measured active red-band (PAM) and passive spectral F diurnal data of the 22-day experiment. The PAM (ASD) fluorescence was checked for all 2-min interval (2) FY = APAR Ft measurements with arbitrary units were compared to both the simultaneous measured red fluorescence (RF, 650–705 nm) yield and far-red fluorescence (FRF, 705–790 nm) yield. As shown in previous research [19], the passively FY was APAR = and1actively − R2 −retrieved T · PAR · dλ,found comparable. The coefficient of determination between the RF Yield and Ft (R = 0.841) was expectedly lower compared to one(3) of the FRF Yield and Ft (R2 = 0.913) relationship. Spectral passive fluorescence yield (FY) was calculated as: with T R − min R

FY = APAR =

Z 650 400

F APAR

(1 − Rλ − Tλ )·PARλ ·dλ, with Tλ ≈ Rλ − min(R)

(2) (3)


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considering that the filter on the lamps completely blocks any light above 650 nm; and the transmittance has been approximated by the reflectance after subtracting the contribution of the light reflected by the epidermis before any absorption takes place (estimated as the minimum reflectance in the far blue wavelengths). At the beginning of every diurnal cycle, the dark-adapted leaf receives the first saturating pulse to determine the minimum and absolute maximum fluorescence (F0 and Fm respectively). Subsequently, the light source turns on and reproduces the diurnal cycle; meanwhile regular saturation pulses of 7000 µmol m−2 s−1 are given every 20 min, which induce maximum fluorescence (Fm 0 ), permitting the measurement of the non-photochemical fluorescence quenching (NPQ) as: NPQ =

Fm − Fm 0 Fm 0

(4)

with Fm 0 the maximum F at each saturation pulse [20]. By applying a saturation pulse only every 20 min during the 9 h diurnal cycle, the leaf spot has sufficient recovery time in between the flashes to relax rapid-responding NPQ mechanisms. Moreover, the 20 min separation between saturating pulses avoided the photoinhibitory quenching [21,22] that might appear with the use of flashes in short intervals. Hence, the photochemical quenching or the amount of open PSII centres at every consecutive pulse (34 pulses per diurnal cycle) remains only influenced by the adaptation to the available light intensity and environmental conditions. Besides, two additional saturating pulses were given in darkness, after completing the diurnal light cycle. This technique allows approximating the relaxation kinetics of the NPQ mechanisms, disentangling the energy-dependent quenching (qE), state-transition quenching (qT) and, photoinhibitory quenching (qI) based on their relaxation kinetics [6,23,24]. qE =

FE − Fm 0 Fm 0

qT =

FT − FE Fm 0

qI =

Fm − FT Fm 0

00

00

(5)

00

(6)

00

00

(7)

00

With FE and FT respectively the maximal F during the saturating pulses after 20 and 40 min in darkness at the end of the day. The Chl F acquisition by the spectrometer on the one hand and the fluorometer on the other hand were automatic and controlled from a desktop computer running a program in Matlab. The 2-min interval Chl F measurements of both instruments were synchronized with less than 5 s between passive and active measurements. The outcome of both passive and active measurements was evaluated for all 2- or 20-min intervals, and more specifically the morning measurements at 10:30, in order to interpret the corresponding physiology at the moment of planned FLEX satellite overpass over northern mid-latitudes. 3. Results In this section, we present the results from several aspects of the experiments. First, we address the diurnal evolution of NPQ and leaf temperature along the experiment through the different phases of stress and adaptation of the plant to the environmental conditions. Second, we describe the passively measured PRI and Chl F, and their response. Then, we show the different relationships between the passive parameters and NPQ, according to findings in the literature, found to be insufficient to represent and explain the totality of this experiment. Last, we propose a linear regression to a plane defined by experimental data of PRI, APAR and NPQ, that will allow estimating NPQ from passive measurements.


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3.1. 3.1.Diurnal DiurnalNon-Photochemical Non-PhotochemicalQuenching Quenching(NPQ) (NPQ) NPQ NPQmechanisms mechanismsare areactivated activatedwhen whenexcessive excessiveabsorbed absorbedlight lightneeds needstotobebedissipated, dissipated,showing showing high values at high illumination hours (Figure 3a). On the first day (D1), NPQ gets high values at high illumination hours (Figure 3a). On the first day (D1), NPQ getsquickly quicklyactivated activated rising risingtotohigh highvalues valuesdue duetotolight lightstress, stress,provided providedthat thatthe theplant plantwas wassubjected subjectedtotoaahigher higherillumination illumination than it was grown at. After a few days of light stress, NPQ slowly declines and remains stable than it was grown at. After a few days of light stress, NPQ slowly declines and remains stable until until D15, after which a slow steady increase is observed until the end of the experiment. Based D15, after which a slow steady increase is observed until the end of the experiment. Based on the on the NPQ relaxation partitioning technique [6,23], NPQ parameters (energy-dependent),qI NPQ relaxation partitioning technique [6,23], the the NPQ parameters qEqE (energy-dependent), qI(photoinhibitory (photoinhibitoryquenching) quenching)and andqT qT (state-transitions) (state-transitions) were were derived for the derived for the10:30 10:30measurement measurement (Figure 3b, Formulas (5)–(7)). We should stress out here that the behaviour of the different NPQ kinetics (Figure 3b, Formulas (5)–(7)). We should stress out here that the behaviour of the different NPQ iskinetics not stationary, but variable in the day. The energy-dependent NPQ, qE, has overall the largest share is not stationary, but variable in the day. The energy-dependent NPQ, qE, has overall the inlargest the NPQ dissipation the experiment. On D1,the however, the photoinhibitory quenching, share in thethroughout NPQ dissipation throughout experiment. On D1, however, the qI, is important asquenching, well, beforeqI, it is starts declining the following days. declining The energy-dependent NPQ qE photoinhibitory important as well, before it starts the following days. The declines as well after NPQ D2, but the as most important NPQ During relaxed phase energy-dependent qEremains declines well after D2, butmechanism. remains the mosttheimportant NPQ (D6–D11, Figure 3b), the 10:30 a.m. NPQ measurement reaches the lowest values in the experiment mechanism. During the relaxed phase (D6–D11, Figure 3b), the 10:30 a.m NPQ measurement reaches and becomes determinant for 84–90% of NPQ at this time of the NPQofstays lowof the qE lowest values in the experiment and qE becomes determinant forday. 84–90% NPQsimilarly at this time between D5 and D16. In the drought stress phase (D17–D22) total NPQ starts increasing again, due to the day. NPQ stays similarly low between D5 and D16. In the drought stress phase (D17–D22) total the increase of all three NPQ components. NPQ starts increasing again, due to the increase of all three NPQ components. 2.5 qE qI qT

NPQ (-)

2

1.5

1

0.5

(a)

D22

D21

D20

D19

D18

D17

D16

D15

D14

D13

D12

D11

D9

D10

D8

D7

D6

D5

D4

D3

D2

D1

0

(b)

Figure 3. Diurnal non-photochemical quenching (NPQ) of fluorescence during the 22-day experiment, Figure 3. Diurnal non-photochemical quenching (NPQ) of fluorescence during the 22-day experiment, the colour scheme indicating the days will be also used in further graphs (a), and NPQ partitioning the colour scheme indicating the days will be also used in further graphs (a), and NPQ partitioning at 10:30 a.m. according to the different quenching parameters: energy-dependent quenching qE, at 10:30 a.m. according to the different quenching parameters: energy-dependent quenching qE, photoinhibitory quenching qI and state-transition quenching qT (b). photoinhibitory quenching qI and state-transition quenching qT (b).

3.2. Leaf Temperature Change and Energy Dissipation 3.2. Leaf Temperature Change and Energy Dissipation Highest diurnal temperature changes (∆T) were found at the beginning of the experiment on Highest diurnal temperature changes (∆T) were found at the beginning of the experiment on (D1) (D1) and at the end of the experiment (D21–D22) (Figure 4a). During D21 and D22 a ∆T increase of and at the end of the experiment (D21–D22) (Figure 4a). During D21 and D22 a ∆T increase of 2.5 2.5 °C was recorded as the highest increase during the experiment. Leaf temperature is typically light ◦C was recorded as the highest increase during the experiment. Leaf temperature is typically light driven. However, on these high ∆T days, a steep increase in the morning is observed, which does not driven. However, on these high ∆T days, a steep increase in the morning is observed, which does not correspond to the diurnal illumination shape. Also, the afternoon leaf cooling effect on D21–D22 went correspond to the diurnal illumination shape. Also, the afternoon leaf cooling effect on D21–D22 went much slower, resulting in a higher leaf temperature at the end of the day. When normalizing ∆T by much slower, resulting in a higher leaf temperature at the end of the day. When normalizing ∆T by amount of PAR, these trends are better seen (Figure 4b). amount of PAR, these trends are better seen (Figure 4b).


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(a)

(b)

(a)

(b)

Figure 4. Diurnal leaf temperature change (∆T) with respect to before-dawn temperature during the

Figure 4. Diurnal leaf temperature change (∆T) with respect to before-dawn temperature during 22-day experiment (a) and temperature change normalized by PAR) (b); Diurnal PAR course is shown theFigure 22-day (a) and temperature change normalized by PAR) (b); Diurnal PAR course is 4. experiment Diurnal as a dashed line.leaf temperature change (∆T) with respect to before-dawn temperature during the shown as a dashed line. 22-day experiment (a) and temperature change normalized by PAR) (b); Diurnal PAR course is shown asDiurnal a dashedevolution line. of PRI, fluorescence at 740 nm (F740) and NPQ in relation to ∆T are shown in

Diurnal evolution fluorescence at 740 nm (F740) and NPQ in experiment, relation to ∆T are shown Figure 5a–c. PRI andof ∆TPRI, show a different relationship for each day of the in which the in Diurnal evolution of PRI, fluorescence at 740 nm (F740) and NPQ in relation to ∆T are shown 10:30 measurement does not follow any clear trend. F740for in function of of ∆Tthe at 10:30 shows a decreasing Figure 5a–c. PRI and ∆T show a different relationship each day experiment, in whichinthe Figure 5a–c. PRI and ∆T show a different relationship for each day of the experiment, in which the throughoutdoes the experiment, (whereas positive within each day), aftershows the light stress 10:30trend measurement not follow any clear trend. F740 in function of but ∆T only at 10:30 a decreasing 10:30 measurement not anyNPQ-∆T clear positive trend. F740 inrelationship function of ∆T atbe 10:30 shows anot decreasing has occurred (D9–D22). Forfollow the 10:30 a positive can seen, whenthe taking trend throughout thedoes experiment, (whereas within each day), but only after light stress trend throughout the experiment, (whereas positive within each day), but only after the light stress the light stress days into account (Figure 5c). Also, it can be observed here that the strength of the has occurred (D9–D22). For the 10:30 NPQ-∆T a positive relationship can be seen, when not taking has occurred (D9–D22). ForNPQ the 10:30 NPQ-∆T positive was relationship be seen, when notontaking coupling between ∆T and varies. A weakacoupling found oncan D1 and by extension D2– light stress days into account (Figure 5c). Also, it can be observed here that the strength of coupling D4, i.e., stress the light stress days with high NPQ. This steepit increase ∆T on D1 suggests a fast increaseof the light days into account (Figure 5c). Also, can be in observed here that the strength between ∆T and NPQ varies. A weak coupling was found on D1 and by extension on D2–D4, i.e., the in sensible heat ∆T dueand to NPQ an additional heat or and closure of the stomata, coupling between varies. A uncontrolled weak coupling wasdissipation found on D1 by extension on D2– light stress days with high NPQ. This steep increase in ∆T on D1 suggests a fast increase in sensible inhibiting the latent heat exchange through evapotranspiration. D4, i.e., the light stress days with high NPQ. This steep increase in ∆T on D1 suggests a fast increase heat due to an additional uncontrolled heat dissipation or closure of the stomata, inhibiting the latent in sensible heat due to an additional uncontrolled heat dissipation or closure of the stomata, heat exchange evapotranspiration. inhibiting thethrough latent heat exchange through evapotranspiration.

(a)

(a)

(b)

(c)

(b)

(c)

Figure 5. Relationship between diurnal leaf temperature change (∆T) and 2 min-interval PRI (a), F740 (b) and 20 min-interval NPQ (c); coloured bullets indicate the 10:30 measurement for each day.

Figure Relationship between diurnal leafStages temperature change PRI (a),(a), F740 Figure 5. 5.Relationship between diurnal leaf temperature change(∆T) (∆T)and and2 2min-interval min-interval PRI F740 3.3. Diurnal F740 and PRI at Different Stress (b) and 20 min-interval NPQ (c); coloured bullets indicate the 10:30 measurement for each day. (b) and 20 min-interval NPQ (c); coloured bullets indicate the 10:30 measurement for each day. To show the diurnal course of passive fluorescence, the far-red second peak fluorescence, located at Diurnal 740 nm F740 (F740), was used; however, it is Stages important to mention that we found a 1:1 correlation with 3.3. and PRI at Different Stress 3.3. Diurnal F740atand Stages 2 = 0.999),Stress fluorescence 760PRI nm at (RDifferent therefore all the following discussion and results are extendable To show the diurnal course of band. passive second peakevolutions fluorescence, located to usingcourse the O2-A Influorescence, general, dailythe Chlfar-red F and PRI typical are easy to Tomeasurements show the diurnal of passive fluorescence, the far-red second peak fluorescence, located at understand 740 nm (F740), was used; however, it is important to mention that we found a 1:1 correlation with and interpret. F740 reacts fast on the incoming light in the morning and rises to at 740 nm (F740), was used;2 however, it is important to mention that we found a 1:1 correlation awith fluorescence at 760 “solar nm (Rnoon” = 0.999), therefore all the following and results the are extendable maximum before (Figure 6). However, at a certaindiscussion point in the morning, in 2 = 0.999), fluorescence at 760 nm (Rthe therefore all thedaily following discussion and results areincrease extendable tolight measurements using O2-A band. In general, Chl F and PRI typical evolutions areenergyeasy to to becomes too excessive, activating other dissipation mechanisms. PRI, sensitive to the measurements using the O2-A band. In general, daily Chl F and PRI the typical evolutions are easy understand interpret. on the incoming light morning andstarting rises totoa to dependent and NPQ, decreasesF740 and reacts reachesfast a minimum commonly afterinsolar noon, before understand and interpret. F740 reacts fast on the incoming light in the morning and rises to a maximum maximum before “solar (Figure 6). However, at a are certain pointininscale the morning, theduring increase rise again. Yet, we cannoon” observe that these mechanisms variable and timing thein before “solar noon” 6). activating However, at a certain in the PRI morning, thesensitive increasefour in the light becomes light becomes too (Figure excessive, other dissipation mechanisms. PRI, to energystress experiment. According to the different trendspoint in diurnal we distinguished phases in toodependent excessive, activating other dissipation mechanisms. PRI, sensitive to the energy-dependent NPQ, NPQ, decreases and reaches a minimum commonly after solar noon, before starting to the experiment. decreases and reaches a minimum commonly after solar noon, before starting rise again. rise again. Yet, we can observe that these mechanisms are variable in scale andtotiming duringYet, thewe can observe that these mechanisms are variable in in scale and PRI timing during the stress stress experiment. According to the different trends diurnal we distinguished four experiment. phases in the experiment. According to the different trends in diurnal PRI we distinguished four phases in the experiment.


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In a first (D1–D5), the plant is still well-watered but under severe light stress (compared Remote Sens. phase 2017, 9, 770 8 of 16 to its growth under low light conditions around 100 µmol m−2 s−1 ). On D1 we see that, after 72 min In a first phase (D1–D5), the plant well-watered butm under light stressthe (compared −2 s−severe 1 (right of increasing light exposure, when PARisisstill around 400 µmol above illumination to its growth under low light conditions around 100 µmol m−2 s−1). On D1 we see that, after 72 min of level at which it was grown), PRI shows a steep decrease as a sudden response to the unexpected increasing light exposure, when PAR is around 400 µmol m−2 s−1 (right above the illumination level high light conditions (Figure 6a). Simultaneously, F740 emission reacts to this increase by showing at which it was grown), PRI shows a steep decrease as a sudden response to the unexpected high a sudden decrease in slope.6a). After a steep PRI decrease in thereacts morning hours, a further lesserasteep light conditions (Figure Simultaneously, F740 emission to this increase by showing decrease brings PRI to a minimum in the afternoon on D1. Hence, a rapid and subsequent sudden decrease in slope. After a steep PRI decrease in the morning hours, a further lesser steep slow decrease can brings be clearly distinguished onthe this day. Finally in theand later part of the day as decrease PRI to a minimum in afternoon on D1.recovering Hence, a rapid subsequent slow decrease can be clearly distinguished day. Finally thepursues later partthe of the daydecrease as incoming irradiation gets reduced. On on thethis following daysrecovering (D2–D4),inPRI steep irradiation gets period, reduced.reaching On the following (D2–D4), pursues the steep “solar decrease in theincoming morning for a longer the PRI days a much lowerPRI minimum around noon”, in the morning for a longer period, reaching the PRI a much lower minimum around “solar noon”, and resulting in a more symmetrical PRI diurnal progress with a deep PRI valley. Meanwhile, F740 and resulting in a in more symmetrical PRIway. diurnal progress with a deep valley. maximum decreases a corresponding With this response, PRIPRI and F740Meanwhile, seem to beF740 in phase, maximum decreases in a corresponding way. With this response, PRI and F740 seem to be in phase, i.e., both are simultaneously activated resulting in a synchronized F740 maximum and PRI minimum i.e., both are simultaneously activated resulting in a synchronized F740 maximum and PRI minimum around solar noon. On D5, after four days of high diurnal light exposure, a first step in the relaxation around solar noon. On D5, after four days of high diurnal light exposure, a first step in the relaxation of daily PRI PRI takes place. PRI does as much muchasasininprevious previous days, relaxation of daily takes place. PRI doesnot notdecrease decrease as days, andand this this relaxation effecteffect is also reflected in the riserise of of thethe maximum F740(Figure (Figure6a). 6a). is also reflected in the maximumdiurnal diurnal F740

(a)

(b)

(c)

(d)

Figure 6. Diurnal far-red fluorescence (F740) and the Photochemical Reflectance Index (PRI) during

Figure Diurnal fluorescence (F740) and the Photochemical Reflectance Index (PRI) during the6.light stress far-red phase (D1–D5) (a), the relaxed phase (D6–D11) (b), the mild drought stress (D13–D16) the light stress phase (D1–D5) (a), the relaxed phase (D6–D11) (b), the mild drought stress (D13–D16) (c) and the drought stress phase (D17–D22) (d); a vertical green dotted line marks the measurements (c) and the drought stress phase (D17–D22) (d); a vertical green dotted line marks the measurements at at 10:30 and a solid grey line indicates the course of PAR illumination as reference (units not given). 10:30 and a solid grey line indicates the course of PAR illumination as reference (units not given). During a second phase (D6–D11), identified as a relaxed state, PRI further relaxes (Figure 6b). The steep slope in the morning becomes attenuated, softening the PRI minimum. Due to this

During a second phase (D6–D11), identified as a relaxed state, PRI further relaxes (Figure 6b). The steep slope in the morning becomes attenuated, softening the PRI minimum. Due to this attenuated


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activation, a time shift takes place, postponing the PRI minimum to the afternoon. Meanwhile, Remote Sens. 2017,of 9, 770 9 of 16 the diurnal cycle F740 shows a moderate intensity decrease. During a third phase (D13–D16) drought stress is starting to increase and PRI is again decreasing attenuated activation, a time shift takes place, postponing the PRI minimum to the afternoon. faster in the morning (Figure 6c). However, due to a small shift in leaf position and lost measurements Meanwhile, the diurnal cycle of F740 shows a moderate intensity decrease. on D12, overall values higher drought on D13.stress Nonetheless, PRI values to decrease and on During aPRI third phaseare (D13–D16) is startingdaily to increase and PRIstart is again decreasing D16 faster a lowinPRI is steadily maintained during around noon. At and the lost same time, no change the morning (Figure 6c). However, duethe to ahours small shift in leaf position measurements in the cyclePRI of F740 observed during this mild drought stress phase. ondiurnal D12, overall valuesisare higher on D13. Nonetheless, daily PRI values start to decrease and on D16 a low steadily maintained duringprevailing the hours around noon. the same time,decreasing no change in From D16PRI on,isthe drought stress starts and the PRIAt starts rapidly day by the diurnal of F740 is observed during this mild stress phase. day early in thecycle morning (Figure 6d). The slopes heredrought are, however, less steep compared to the slopes From D16 on, the drought stress but starts prevailing and the PRI starts values rapidlyand decreasing bylower of the first light stress phase (D1–D4), PRI begins already at lower reachesday also day early in the morning (Figure 6d). The slopes here are, however, less steep compared to the slopes minima. Notable is the shift of the time at which the minimum PRI occurs each day. Minimal daily of the first light stress phase (D1–D4), but PRI begins already at lower values and reaches also lower PRI values of the entire experiment are reached on D22, with the minimum value one hour after solar minima. Notable is the shift of the time at which the minimum PRI occurs each day. Minimal daily noon. The afternoon recovery of PRI does not reach the early morning values, indicating that the PRI values of the entire experiment are reached on D22, with the minimum value one hour after solar protection mechanisms do not deactivate completely. Diurnal F740 shows a steady overall noon. The afternoon recovery of PRI does not reach the early morning values, indicating that decrease the that protection also reaches a minimum daily curve on the last experimental day. However, the strong time-shifts mechanisms do not deactivate completely. Diurnal F740 shows a steady overall decrease in the PRI minima D16 todaily D22 curve do not a counterpart F740, which the remains that also reachesfrom a minimum onfind the last experimentalinday. However, strongvery time-stable; in contrast the PRI minima fromphase D16 to(D1–D5) D22 do not findexperiment a counterpart in F740, which remains presented very this shifts falls in to the first of the when both parameters stable;shifts. this falls in contrast to the first phase (D1–D5) of the experiment when both parameters opposing presented opposing In general, we canshifts. observe that the diurnal PRI demonstrates a higher variability, both in relative In general, we can observe that the diurnal PRI demonstrates a higher variability, both in relative values as in diurnal shape, compared to the F740 diurnal variations. In the beginning of the experiment, values as in diurnal shape, compared to the F740 diurnal variations. In the beginning of the PRI showed a higher variation in-between days during the morning hours (on-set of PRI) compared to experiment, PRI showed a higher variation in-between days during the morning hours (on-set of PRI) the afternoon (recovery) hours. However, this was not the case during the last phase of the experiment. compared to the afternoon (recovery) hours. However, this was not the case during the last phase of This the indicates that This thereindicates is a marked differentiation in behaviour in regarding nature the of the stress. experiment. that there is a marked differentiation behaviourthe regarding nature of the stress.

3.4. Diurnal NPQ in Relation to F690/F740, F740, FY740 and PRI 3.4. Diurnal in Relation to F690/F740, FY740 andused PRI red/far-red fluorescence peak ratio Based on NPQ spectral fluorescence theF740, commonly Based onAs spectral the commonly used red/far-red fluorescence ratio was was calculated. can befluorescence seen from Figure 7, the F690/F740 ratio shows somepeak significant diurnal calculated. As can be seen from Figure 7, the F690/F740 ratio shows some significant diurnal variation variation in function of NPQ, with an increasing ratio in the morning and a decreasing ratio in the in function of NPQ, increasing ratio in thethe morning and a decreasing ratiooverall in the afternoon. afternoon. During the with lightan stress days (D1–D4), F690/F740 ratio showed higher values During the light stress days (D1–D4), the F690/F740 ratio showed overall higher values compared to compared to the rest of the experiment. With increasing drought stress, the F690/F740 ratio measured the rest of the experiment. With increasing drought stress, the F690/F740 ratio measured at 10:30 at 10:30 decreases on D9–D11 (relaxation), and become even lower in the period D13–D22. However, decreases on D9–D11 (relaxation), and become even lower in the period D13–D22. However, there is there is few peak ratio variability between days both within the light stress phase and within the few peak ratio variability between days both within the light stress phase and within the drought drought showing poorindicator stress indicator for progressive stress. 7 we can stressstress phase,phase, showing a poor a stress for progressive stress. From FigureFrom 7 we Figure can further further observe that the peak ratio does not explain any variation in NPQ, but is able to separate observe that the peak ratio does not explain any variation in NPQ, but is able to separate light stress light stress from of states. from thethe restrest of states.

(a)

(b)

Figure 7. Diurnal relationship between F690 and F740 (a) and NPQ in relation to the F690/F740 peak

Figure 7. Diurnal relationship between F690 and F740 (a) and NPQ in relation to the F690/F740 peak ratio (b) during the 22-day experiment; colour bullets indicate the 10:30 measurement for each day. ratio (b) during the 22-day experiment; colour bullets indicate the 10:30 measurement for each day.

Remote Sens. Sens. 2017, 2017, 9, 9, 770 770 Remote

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During the 22-day stress experiment NPQ and PRI are strongly coupled, but not related through During the 22-day stress 8a), experiment NPQ regression and PRI areprovides stronglyan coupled, but (excluding not related the through a single relationship (Figure and a linear R2 of 0.69 light 2 of 0.69 (excluding the light astress singledays). relationship (Figure 8a), and a linear regression provides an R Even though there seems to be a non-linear correlation between both parameters for stress days). Even though there to be between both parameters for each individual day (except for seems day D1), thisa non-linear relationshipcorrelation shifts between days. Even more, when each individual day (except for day D1), this relationship shifts between days. Even more, when observing both parameters at a fixed instant in the diurnal cycle (10:30), it is clear that there is no observing parametersthe at astress fixedexperiment. instant in the diurnal some cycle observations (10:30), it is clear thatbethere is On no consistent both link throughout Although, can still made. consistent link throughout the stress Although, some observations be made. D1, when high light stress occurs, the experiment. coupling between NPQ and PRI is very low.can On still the following On D1, when high light stress occurs, the coupling between NPQ and PRI is very low. On the following days, a stronger daily coupling can be observed between both parameters, but the coupling slightly days, a stronger coupling can observed between both parameters, thestress coupling slightly weakens again daily toward the end ofbethe experiment (D19–D22). After thebut light days, NPQ weakens again towardconstant the end of the experiment (D19–D22). Afteraround the light stress days, NPQ decreases, decreases, remaining during D9–D16 with 10:30 values 0.8. During this mild drought remaining constant during D9–D16 values around 0.8. During mild however however PRI starts decreasing, and with upon10:30 an increased drought stress boththis NPQ anddrought PRI respectively PRI startsand decreasing, an increased drought stress both NPQ PRI respectively increase increase decrease.and Thisupon PRI trend can be observed by looking at theand marked 10:30 measurements, and decrease. This PRI trend can be observed by looking at the marked 10:30 measurements, which which show almost a day-by-day shift between D9 until D22. showWhen almostlooking a day-by-day shift between D9 until D22. at the relationship of NPQ with fluorescence yield at 740 nm (Figure 8c) it is found at (yielding the relationship NPQ with fluorescence yield days). at 740 nm (Figure 8c)atitlow is found to beWhen ratherlooking complex an R2 ofof0.59 excluding the light stress As FY is high light 2 of 0.59 excluding the light stress days). As FY is high at low light to be rather complex (yielding an R conditions, showing a first peak in the morning and a second but lower peak in the afternoon, diurnal conditions, showing a first peak in the morning andNPQ a second butalower peakdiurnal in the afternoon, FY shows an asymmetrical behaviour. In contrast, shows one-peak trend withdiurnal a peak FY shows an asymmetrical NPQ a one-peak diurnal trend with at a peak around noon or afternoon behaviour. depending In oncontrast, the timing of shows activation of the NPQ mechanisms high around noon or afternoon depending on the timing of activation of the NPQ mechanisms at high light light conditions. Hence, the NPQ-Yield F740 relationship is not straightforward. The observed conditions. the NPQ-Yield F740 is notexplain straightforward. The variation variation inHence, FY at 10:30 (0.27–0.37%) onrelationship D9–D22 cannot entirely the in observed between day NPQ in FY at 10:30 (0.27–0.37%) on D9–D22 cannot explain entirely the in between day NPQ variation. variation.

(a)

(b)

(c)

(d)

Figure 8. 8. Diurnal Diurnal relationships relationships during during the the 22-day 22-day experiment experiment of of NPQ NPQ and and PRI PRI (a), (a), PRI PRI and and F740 F740 (b), (b), Figure NPQ and Yield of F740 (c), and NPQ and F740 (d); coloured bullets indicate the 10:30 measurement NPQ and Yield of F740 (c), and NPQ and F740 (d); coloured bullets indicate the 10:30 measurement for for each the start of each is indicated by symbol the symbol at beginning the beginning of each curve. each day;day; the start of each day day is indicated by the at the of each curve.

NPQ and F740 measurements follow a daily loop with positive direction (Figure 8b). Similar as NPQ and F740 measurements follow a daily loop with positive direction (Figure 8b). Similar as in in the case with NPQ and PRI, these daily loops are not unique throughout the stress experiment. the case with NPQ and PRI, these daily loops are not unique throughout the stress experiment. The NPQ-F740 quenching slope is steeper during light stress, with the steepest slope on D1. The NPQ-F740 quenching slope is steeper during light stress, with the steepest slope on D1. Additionally, we have the same occurrence that one single F740 measurement does not match one single NPQ measurement over the entire experiment. As shown in Figure 6, F740 is high at the


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Remote Sens. 2017,we 9, 770 of 16 Additionally, have the same occurrence that one single F740 measurement does not match one11single

NPQ measurement over the entire experiment. As shown in Figure 6, F740 is high at the beginning of beginning of the experiment, lowers and increases slightly duringperiod the relaxed period afterafter lightwhich stress,a the experiment, lowers and increases slightly during the relaxed after light stress, after which a small further decrease continues until the end of the experiment. F740 and PRI show small further decrease continues until the end of the experiment. F740 and PRI show mutual diurnal mutual diurnal trends with a negative direction showing a low morning and afternoon coupling at trends with a negative direction showing a low morning and afternoon coupling at the beginning and the beginning and at the end of the8d). experiment (Figure retrieved 8d). Hence, retrieved PRIofor at the end of the experiment (Figure Hence, passively PRIpassively or fluorescence in any its fluorescence in any of its forms (F760, yield or peak ratio) are not capable to singlehandedly forms (F760, yield or peak ratio) are not capable to singlehandedly determine the dynamics of NPQ for determine dynamics of NPQ for the whole period. Therefore, it becomes necessary to find other the wholethe period. Therefore, it becomes necessary to find other relationships. relationships. 3.5. Estimation of Dynamic NPQ Based on Combined PRI and APAR 3.5. Estimation of Dynamic NPQ Based on Combined PRI and APAR The photoprotection mechanisms will react to the amount of light received by the plant, the health The photoprotection react to the amount of photoprotection light received bywill thebeplant, the status, and the availabilitymechanisms of resourceswill for photochemistry. Hence, activated health status, and theisavailability resources forcan photochemistry. photoprotection willgoes be when absorbed light in excess ofof what the plant deal with, and Hence, deactivated when the APAR activated absorbed light Provided is in excess of what the plant can deal with, and theit down to when manageable levels. that the photoprotective pigment pooldeactivated is reflectedwhen by PRI, APAR down to levels. Provided the aphotoprotective pigment seemsgoes reasonable to manageable try combining APAR and PRIthat to find relationship with NPQ. pool is reflected by PRI, seems9,reasonable try combining APARwith and PRI find relationship NPQ.how they InitFigure all data aretoplotted in a 3D graph, NPQtoon theaz-axis, clearlywith showing In Figure 9, all data are plotted in a 3D graph, with NPQ on the z-axis, clearly showing they fall across a tilted plane, except for the light-stress days D1–D4 (in dashed lines) that do not how follow the fall across a tilted plane, except for the light-stress days D1–D4 (in dashed lines) that do not follow trend of the rest of the data, as was also illustrated in Figure 8. In first approach, a basic plane was the trend of the rest the data, was also illustrated in Figure 8. In first approach, a basic plane was fitted excluding theoflight stressas days, fitted excluding the light stress days, NPQ = a + b·PRI + c·APAR, (8)

NPQ = + · PRI + · APAR, (8) 2 ·s/µmol m ·resulting s/μmol in the linear = 0.72906363; =− −16.781348; = 8.9183602 where a = 0.72906363; b = 16.781348; c = 8.9183602 ·10−4 ·m10

2 of 0.90. estimation with a RMSEof ofNPQ 0.08 and resulting in of theNPQ linear estimation withRa RMSE of 0.08 and R2 of 0.90.

(a)

(b)

Figure showing thethe relationship of NPQ (z-axis) withwith diurnal PRI PRI (x-axis) and and APAR (yFigure9.9.3D-plots 3D-plots showing relationship of NPQ (z-axis) diurnal (x-axis) APAR axis). The side view (a) shows how the data form all days fall approximately within a plane, except (y-axis). The side view (a) shows how the data form all days fall approximately within a plane, except for forthe thelight lightstress stressdays days(D1–D4) (D1–D4)represented representedinindashed dashedlines. lines.Also Alsoshown showniningrey greygrid gridisisthe theplane planefitfit for forthe theD5–D19 D5–D19data data(b), (b),for forclarity claritythe thelight lightstress stresslines linesare arenot notshown. shown.

From the graph in Figure 9a, we can see that morning and evening measurements (to the right From the graph in Figure 9a, we can see that morning and evening measurements (to the right hand of the plot) with low light conditions have a higher deviation from the plane fit surface, while hand of the plot) with low light conditions have a higher deviation from the plane fit surface, while the model predicts better for mid- and high-level light conditions. The fitting allows estimating NPQ the model predicts better for mid- and high-level light conditions. The fitting allows estimating NPQ for the whole development of the plant from its well adapted phase (from D5) to its severe drought (in D22) for the whole diurnal cycle as illustrated by Figure 10.


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for the whole development of the plant from its well adapted phase (from D5) to its severe drought (in D22) for the whole diurnal cycle as illustrated by Figure 10.


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(a)

(b)

Figure Figure 10. 10. Scatter Scatter plot plot of of PRI PRI and and APAR APAR with with isolines isolines from from estimated estimated NPQ NPQ (D5–D22), (D5–D22), excluding excluding the the light stress days (a). Daily average residuals of the NPQ estimation from the plane fit for light stress days (a). Daily average residuals of the NPQ estimation from the plane fit forD1–D22 D1–D22(b). (b). The The daily daily average average RMSE RMSE of of the the estimation estimation is is 0.1 0.1 or or below below for for the the whole whole experiment experiment except except for for the the first first four four days days during during the the light-stress light-stress phase. phase.

Note that the isolines in Figure 10a representing constant NPQ (in grey) run diagonally with Note that the isolines in Figure 10a representing constant NPQ (in grey) run diagonally with respect to the APAR and PRI axis, while the curves for each day cycle show a hysteresis evolution, respect to the APAR and PRI axis, while the curves for each day cycle show a hysteresis evolution, and and they shift from day to day covering the whole plot area. This implies that a single parameter they shift from day to day covering the whole plot area. This implies that a single parameter would not would not be sufficient to univocally determine the NPQ value on its own since there will be two be sufficient to univocally determine the NPQ value on its own since there will be two possible values possible values of NPQ for any single value of either parameter, APAR or PRI. Only when using the of NPQ for any single value of either parameter, APAR or PRI. Only when using the combination combination of both the duplicity is broken. Figure 10b shows the average RMSE of the NPQ of both the duplicity is broken. Figure 10b shows the average RMSE of the NPQ estimation using estimation using the plane fit for each individual day, with the error being close to or below 0.1, the plane fit for each individual day, with the error being close to or below 0.1, except for the light except for the light stress period D1–D4 that show much larger values, since they actually lie off the stress period D1–D4 that show much larger values, since they actually lie off the plane where the rest plane where the rest of the data are located (Figure 9a). It should be also noted that when evaluating of the data are located (Figure 9a). It should be also noted that when evaluating the NPQ prediction of the NPQ prediction of this model to the 10:30 measurements (the planed FLEX overpass), the result this model to the 10:30 measurements (the planed FLEX overpass), the result yields an R2 of 0.96 with 2 yields an R of 0.96 with a RMSE of 0.08. a RMSE of 0.08. 4. 4. Discussion Discussion Few Few studies studies have have tried tried to to obtain obtain knowledge knowledge on on the the dynamics dynamics of of photosynthetic photosynthetic behaviour behaviour and and photoprotection of plants based on spectral measurements. Atherton et al. [12] demonstrated photoprotection of plants based on spectral measurements. Atherton et al. [12] demonstrated the the possibility predict photochemical on PRI and fluorescence in possibility totopredict PSIIPSII photochemical yield yield based based on PRI and fluorescence dynamics indynamics combination combination with simple empirical models. They, however, also stressed out the sensitive behaviour with simple empirical models. They, however, also stressed out the sensitive behaviour of PRI, of PRI, wherefore linkNPQ withcan NPQ can change rapidly. During our 22-day experiment, diurnal PRIwherefore the linkthe with change rapidly. During our 22-day experiment, diurnal PRI-NPQ NPQ relationships indeed changed and moreover occurred. relationships indeed changed daily,daily, and moreover when when stress stress occurred. Based on the combination of empirical diurnal APAR, PRI, NPQ, F740 and ∆T, measured carefully Based on the combination of empirical diurnal APAR, PRI, NPQ, F740 and ∆T, carefully measured by active and passive techniques at the same leaf spot, we could follow physiological by active and passive techniques at the same leaf spot, we could follow physiological patterns in patterns the stress experiment. stress at the beginning of the experiment was by identified by the stressinexperiment. Light stressLight at the beginning of the experiment was identified high NPQ high NPQ values that showed a weak coupling with PRI, fluorescence or APAR, indicating the effect values that showed a weak coupling with PRI, fluorescence or APAR, indicating the effect of dynamic of dynamic photoinhibition. A similar observation of photoinhibition the between relationship photoinhibition. A similar observation of photoinhibition affecting the affecting relationship PRI between PRI and actual photochemical efficiency was mentioned by Ripullone et al. [25]. Hence, to and actual photochemical efficiency was mentioned by Ripullone et al. [25]. Hence, to understand understand and predict photosynthesis parameters based on passive data, extreme light stress as and predict photosynthesis parameters based on passive data, extreme light stress as created in this created in this experiment should avoided since vegetation is usually well adapted its natural experiment should be avoided sincebevegetation is usually well adapted to its natural lightto environment. light environment. Here, we nevertheless observed how photoprotection mechanisms (qE) quickly Here, we nevertheless observed how photoprotection mechanisms (qE) quickly take over from the take over from the photoinhibition (qI) (Figure 3b), bringing the plant in a relaxed and photoprotected state as is shown by an increase in qE and a decrease in PRI. Both F740 and NPQ are decreasing during the relaxed phase, suggesting an increase in photochemical efficiency, i.e., the leaf is starting to cope with the high illumination cycle thanks to earlier photoprotection by the repair cycle. Nonetheless, progressive drought is dominating the next


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photoinhibition (qI) (Figure 3b), bringing the plant in a relaxed and photoprotected state as is shown by an increase in qE and a decrease in PRI. Both F740 and NPQ are decreasing during the relaxed phase, suggesting an increase in photochemical efficiency, i.e., the leaf is starting to cope with the high illumination cycle thanks to earlier photoprotection by the repair cycle. Nonetheless, progressive drought is dominating the next two phases, reflected in a NPQ increase together with a PRI decrease. F740 also further decreases, although slightly. A lowered fluorescence intensity due to water-stressed trees has been observed by previous work of different authors [26,27], and has been explained by closure of the stomata and an increased photorespiration in C3 plants [28]. An increasing oxidative stress as consequence of the decreased CO2 availability induces NPQ mechanisms to rise again, quenching fluorescence. Since photosynthesis is inhibited by stomatal closure as suggested by the increased ∆T, the quenching relationship between NPQ and F740 results are slightly different on D21–D22, showing a steeper slope compared to the previous days (Figure 8b). In response to stomatal closure, sensible heat loss increases quickly through the increase of leaf temperature by preventing cooling by latent heat exchange. At canopy scale, canopy temperature is therefore generally found a better and quick drought stress indicator compared to fluorescence and PRI [27,29]. Hence, exploitation of remotely sensed canopy temperature allows detecting an impediment of photosynthesis due to stomatal closure. Fluorescence intensity, however, might not change significantly under these circumstances. Under severe drought stress and almost complete stomatal closure, photoinhibition will eventually occur [28], from which the beginning might be seen by a slow increase of the qI parameter towards the end of the experiment. Predicting NPQ based on PRI and APAR would only work when photoinhibition is not occurring, i.e., the photoprotective qE component is dominating in the NPQ and qI is negligible. Although the severe drought stress days (D21–D22) behave under inhibitory conditions for photosynthesis, the drought stress days were also well predicted by the plane fit model for NPQ (Figure 9). Hence, the model represents varying physiological conditions in which the dissipation mechanisms were altering under a progressive drought stress. The obtained relationship needs to be interpreted considering the conditions under which it holds: (1) the chlorophyll content did not present a strong change through the experiment since the plant was already mature when the experiment started and it was not yet decaying when it finished, therefore the changes to PRI can be mainly associated to changes in photoprotective pigments, (2) the sample was receiving direct light, with fixed illumination and sensing geometries, thus no structural effects were present, (3) a different ratio of carotenoids to chlorophylls might lead to different coefficients. If this relationship is to be extended to other phenological stages, species or to upscale to canopy level, it should be taken into consideration the sensitivity of PRI changes in chlorophyll content (regardless of having the same photoprotective state), the effect of the presence of shaded leaves (if the diffuse illumination is normalized by the global irradiance), or the change in the geometry (e.g., sun elevation through the year even at the same solar time). Some remote sensing studies have incorporated a correction for PRI, taking into account changing pigment pools. Rahimzadeh-Bajgiran et al. [13] corrected PRI with a spectral index for chlorophyll content, while Soudani et al. [30] corrected for the variation in the intercept of the PRI vs. APAR relationships to improve the multi-year PRI-LUE relationship. However, we would like to stress out that diurnal dynamics in PRI, NPQ, and photoprotection in general remains to be further understood. We remark that the proposed model shows an estimation for a limited range of NPQ under certain stable circumstances at leaf level. To understand and predict the dynamics of NPQ on a wider scale a further research is needed towards more species and environmental conditions. 5. Conclusions Non-photochemical dissipation mechanisms are the major energy dissipation mechanisms under sunny conditions, influencing the relationship between Chl F yield and photosynthetic yield. This 22-day experiment illustrated the high plasticity of pigments to adapt to photoprotection needs on


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a day-by-day basis. It was been seen in a change in PRI, starting at lower values each day and showing earlier or later decreases in the day depending on photoprotection needs and capacity. Results show it is feasible to estimate dynamic NPQ based on PRI and APAR, both retrieved from passive proximal sensing measurements. Diurnal NPQ-PRI relationships were not constant and can shift daily due to adaptation to changing stress conditions as shown in this light and drought stress experiment. Using APAR in combination with PRI data allowed the estimation of NPQ with good precision (RMSE = 0.08) through simple bi-linear regression. Under strong down-regulation of photosynthesis, e.g., photoinhibition due to unexpected high light conditions, the relationships between NPQ and PRI can however be strongly altered. It is important to remark that these findings are applicable to leaf level. To extend this approach to canopy level it should be necessary to carry out new proper experiments that also take into consideration other aspects that modify the response of PRI, such as structure, diffuse to direct illumination ratio, observational and illumination geometry. Nonetheless, strong NPQ levels or gradients, and thus effects on PRI, should be found at the higher canopy layers due to strong photoprotection needs of the top-of-canopy sunlit leaves. The presented dataset and results set up a better understanding of how passive measurements can lead to the estimation of NPQ, which is an essential energy dissipation mechanism, that together with fluorescence emission and leaf-air temperature gradient, will serve to close the link with photosynthesis. Acknowledgments: This study was funded by the project AVANFLEX (Advanced Products for the FLEX mission), ESP2016-79503-C2-1-P, National Program for the Promotion of Scientific and Technical Research of Excellence, Ministry of Economy and Competitiveness, Spain; S.V.W. is funded by an individual fellowship of the European Union’s H2020 Marie Skłodowska-Curie actions under the grant agreement FLUOPHOT nº [701815]. Author Contributions: L.A., J.A.-L., L.G.-C. and J.V.-F. conceived and designed the experiments; J.A.-L., L.G.-C. and J.V.-F. performed the experiments; L.A. and S.V.W. analysed the data; L.A. and S.V.W. wrote the paper; J.M. suggested and supervised the experiment, and provided insights on photosynthetic mechanisms. Conflicts of Interest: The authors declare no conflict of interest.

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