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08 Associated changes in physiological parameters and spectral reflectance

indices in olive (Olea europaea L.) leaves in response to different levelsof water stress

P. SUN{, A. GRIGNETTI{, S. LIU{, R. CASACCHIA{, R. SALVATORI{,

F. PIETRINI§, F. LORETO§ and M. CENTRITTO*{{Institute of Forest Ecology, Environment and Protection, Chinese Academy of

Forestry, Beijing 100091, China

{CNR - Istituto sull’Inquinamento Atmosferico, via Salaria km 29.300, 00016

Monterotondo Scalo (RM), Italy

§CNR - Istituto di Biologia Agroambientale e Forestale, via Salaria km 29.300, 00016

Monterotondo Stazione (RM), Italy

(Received 1 December 2006; in final form 27 March 2007 )

This study aimed to determine whether modification of physiological parameters

could be detected remotely by monitoring the spectral reflectance of olive leaves

in response to different degrees of drought. Three different drought intensities

were simulated: (a) a mild drought by feeding abscisic acid to detached branches;

(b) a rapid and severe drought by detaching leaves and letting them dry over

several hours; (c) a relatively slow drought caused by withholding water to potted

olive plants. The three degrees of stress affected gas exchange and chlorophyll

fluorescence. When the inhibition of photosynthesis occurred within an hour it

was not accompanied by a parallel reduction in chlorophyll concentration in the

carotenoid to chlorophyll ratio. Consequently, changes in spectral reflectance in

the visible region, e.g. in PRI (photochemical reflectance index) and FRI

(fluorescence reflectance indices) were not significantly induced. In contrast,

when the inhibition of photosynthesis caused by slow developing drought was

prolonged (i.e. more than 24 hours) and led to a decrease in chlorophyll

concentration and to a simultaneous increase in carotenoid to chlorophyll ratio,

there were significant changes in the visible region of the leaf spectral reflectance

and, in turn, in PRI and FRI. We defined 16 new reference wavelengths, from

visible to SWIR regions, which are sensitive to both fast-developing and slow-

developing stresses. These reference wavelengths were used to develop an

algorithm, the Relative Reflectance Increment (RRI), that was linearly related to

changes in relative water content (RWC, r250.733). This algorithm showed that

the 1455 nm wavelength is highly affected by drought. This wavelength was

therefore used to elaborate the water content reflectance index that was inversely

related to RWC (r250.702).

1. Introduction

An important potential application of non-invasive remote-sensing techniques is to

monitor plant stress and photosynthetic status (Zarco-Tejada et al. 2003, Filella

et al. 2004, Dobrowski et al. 2005, Tian et al. 2005) and to provide information on

*Corresponding author. Email: mauro.centritto@ibaf.cnr.it

International Journal of Remote Sensing

Vol. 29, No. 6, 20 March 2008, 1725–1743

International Journal of Remote SensingISSN 0143-1161 print/ISSN 1366-5901 online # 2008 Taylor & Francis

http://www.tandf.co.uk/journalsDOI: 10.1080/01431160701373754

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the occurrence and intensity of water stress (Ustin et al. 1998, Li et al. 2001, Goward

et al. 2002). Remotely sensed vegetation indices, particularly those associated with

water and nutritional status and, consequently, with various physiological and

ecological phenomena, are increasingly required for the assessment of terrestrial

ecosystem momentum transfer (Sellers 1997, Szilagyi 2000) and carbon uptake

(Veroustraete et al. 2002).

A large number of spectral indices have been developed to estimate plant water

status and physiological conditions by using different approaches. This is because

drought is a complex syndrome affecting several leaf biophysical and biochemical

properties. Water is an important leaf component, accounting for 50–80% of the leaf

fresh weight. Water absorbs radiation from the near infrared region to the

shortwave infrared region, with centres at approximately 970, 1200, 1450, 1950 and

2250 nm (Carlson et al. 1971, Gausmann et al. 1971, Hunt et al. 1987, Rollin and

Milton 1998). Consequently, a change in leaf water content can result in fairly broad

spectral change in these regions (Hunt and Rock 1989, Yu et al. 2000, Ceccato et al.

2001, Imanishi et al. 2004).

The earlier literature describes many studies focused on the development of

spectral indices for the detection of water stress (Carlson et al. 1971, Gausman et al.

1971, Hunt et al. 1987, Pierce et al. 1990, Carter 1991, Cibula et al. 1992, Penuelas

et al. 1993, Carter 1993). Bowman (1989) showed that in cotton the reflectance in the

near-infrared region increased as relative water content decreased. Aoki et al. (1988)

reported a close relationship between the leaf water content (LWC) per unit leaf area

and the reflectance ratio R1650/R1430 for woody plants. Imanishi et al. (2004) used

the shift of red-edge position of vegetation reflectance spectra for detecting LWC in

Quercus glauca and Q. serrata. Yu et al. (2000), working on herbaceous and woody

plants (i.e. Glycine max, Zea mays, Liriodendron tulipifera, and Viburnum awabuki),

estimated the relationships between reflectance and many leaf water status

parameters during leaf dehydration experiments, i.e. relative water content

(RWC), LWC, specific leaf water content, leaf moisture percentage on fresh weight,

and relative leaf moisture percentage on fresh weight basis. Other studies developed

spectral indices for detecting water content as percentage of dry mass (Gao and

Goetz 1995, Ustin et al. 1998) or in terms of equivalent water thickness at leaf level

(Ceccato et al. 2001). However, although all these parameters describing plant water

status may be useful for many different purposes, only RWC has a real physiological

meaning, because it measures the amount of water available for transpiration in

leaves and, as such, it is universally used to assess plant water relations (Tyree and

Jarvis 1982, Kramer and Boyer 1995). Moreover, there have been surprisingly few

studies that have examined the relationships between these components of plant

water status and spectral reflectance indices in response to different intensities of

water stress.

By causing changes in leaf physiological parameters and pigments concentration,

water stress affects reflectance spectral properties related to these processes

(Penuelas and Inoue 1999, Sims and Gamon 2002, 2003, Dobrowski et al. 2005).

A number of sensitive spectral indices were found in the visible region to estimate

foliar pigment concentration (Yoder and Pettigrew-Crosby 1995, Penuelas and

Filella 1998, Blackburn 1999, Jago et al. 1999, Sims and Gamon 2003, Tian et al.

2005). The photochemical reflectance index (PRI, R5312R570/R531 + R570) is a useful

index for estimating xanthophyll cycle pigments (Gamon et al. 1992, Penuelas et al.

1994, Gamon et al. 1997) and carotenoid/chlorophyll ratios (Sims and Gamon 2002)

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and, thus, plant photosynthetic status, but its efficacy is still unclear for detecting

physiological changes in response to different intensities of water stress. Recently, an

increasing interest is devoted to steady-state chlorophyll fluorescence for real-time

monitoring of plant photosynthesis and water status (Zarco-Tejada et al. 2000a,

2000b, 2003, Dobrowski et al. 2005). Their findings opened up new possibilities in

monitoring vegetation physiological changes using passive remote sensing strategies.

However, because of the fact that the contribution of the fluorescence signal, namely

the steady-state fluorescence (Fs) to spectral reflectance is very low (Dobrowski

et al. 2005), the reliability of using these spectral indices to detect photosynthetic

status in response to different intensities of water stress is also unknown.

When using remote sensing, improved knowledge about the relationship between

leaf reflectance indices and different intensities of drought appears to be important.

In this paper we present and discuss the results obtained in experiments focused on

determining which particular wavelengths and spectral indices may be most effective

in detecting physiological changes induced by differences in drought occurrences. In

particular we address two main issues: (1) how spectral reflectance is modified when

plants suffer from different degrees of drought, and (2) whether it is possible to

remotely detect by sensitive spectral indices different physiological changes induced

by different stress. For this purpose, three kinds of water stress were imposed: (1) a

mild drought was simulated by feeding abscisic acid (ABA) to detached branches

(Davies and Zhang 1991); (2) the occurrence of rapid, severe drought was simulated

by detaching leaves and leaving them to dehydrate on the bench; (3) a relatively slow

drought occurrence was caused by withholding water to potted plants. We

hypothesized that: (1) ABA feeding would only affect CO2 diffusional limitations

to photosynthesis, without affecting leaf water status and concentrations of nitrogen

and pigments; (2) leaf fast dehydration would affect both diffusional limitations to

photosynthesis and leaf water status, without affecting concentrations of nitrogen

and pigments; and (3) the slow drying cycle would affect all these parameters.

2. Materials and methods

Two-year-old plants of olive (Olea europaea L.) were grown in the greenhouse of the

Institute of AgroEnvironmental and Forest Biology, National Research Council,

Monterotondo (RM), Italy. Plants were grown in 15 dm3 pots containing a sand–

perlite mixture (1:3). To avoid any water and nutrient limitation, the saplings were

watered every other day to pot water capacity and fertilised once a week, with

complete nutrient solution at low concentration not to restrict needs of the trees.

2.1 Experiments

Three experiments were conducted in summer to simulate different intensities of

water stress:

(1) ABA feeding — Mild water stress was simulated by feeding abscisic acid (ABA)

to branches detached from well-watered plants of olive. Branches were cut

under distilled water and cis–trans ABA (99% purity, Sigma) was then added to

the water supplied to the branches to bring its concentration to 1024 M. Control

branches were kept in distilled water throughout the experiment.

(2) Fast leaf dehydration — The occurrence of rapid, severe drought was simulated

on detached leaves from the top and bottom leaves of the plants. Fully

expanded leaves were detached from well-watered plants of olive and left on the

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lab bench for three hours at 25uC. The leaves were weighed every hour to

determine the rate of water loss.

(3) Slow-developing water stress cycle — The olive saplings were kept at pot water

capacity until the beginning of June. Then half of the saplings (six plants) were

water-stressed by withholding water for a two-week drying cycle, simulating a

progressive decrease of water availability, while the other half of the saplings

were watered periodically to maintain the field capacity.

2.2 Gas exchange and fluorescence measurements

Leaf photosynthetic rates were measured by using a LI-6400-40 portable infrared

gas-analyser (Li-Cor, Lincoln, NE), allowing simultaneous measurement of gas

exchange and fluorescence. All gas exchange measurements were made between

10.00 and 15.00 h on the central section of a fully-developed leaf in ambient CO2

concentration. To enable measurements of photosynthetic rates at saturating

photosynthetically photon flux density (PPFD), leaves were illuminated using a red–

blue light source attached to the gas-exchange system and maintained at

1000 mmol m22s21. Leaf temperature was set at 25uC, and the relative humidity in

the leaf cuvette ranged between 47 and 50%. The fluorescence yield (i.e. the quantum

yield of PSII in the light, DF/F9m) was measured by using a saturating pulse

(10 000 mmol m22 s21) of white light (Genty et al. 1989).

2.3 Determination of relative water content, nitrogen and pigment concentrations

Leaf samples for relative water content (RWC), nitrogen and pigment concentra-

tions were taken immediately after the gas exchange measurements and spectral

measurements. Fully-developed leaves were recut under water and weighed to

determine leaf fresh mass (FM). After measuring leaf area, using a leaf area meter (LI

3100, LI-COR Inc., Lincoln, NE, USA), leaves were covered with a plastic bag and

allowed to rehydrate with the cut-end under water in a dark cold room at 5uC for

18 h. Immediately after rehydration, each leaf was weighed to determine the

saturated mass (SM), and then each leaf was oven-dried at 80uC for 48 hours to

determine dry mass (DM). Then specific leaf area (SLA) was calculated as dry mass

divided by the leaf area, whereas RWC was calculated as follows:

RWC~ FM{DMð Þ= SM{DMð Þ

The concentration of carotenoid and chlorophylls (Chl) a and b were measured in

intact leaf tissues by immersion in N,N-dimethylformamide (DMF). Leaf discs with

a total area of 1.5 cm22 were put in glass vials containing 2 cm3 DMF and

immediately placed in darkness at 4uC in an orbital shaker set to 100 rpm for 4 h

before absorbance (A) of the solution was read on a spectrophotometer (Perkin

Elmer, Norwalk, CT) at 663.8, 646.8, and 480 nm, using DMF as a blank. The

pigment concentrations were calculated according to the following equations

(Wellburn 1994):

Total chlorophyll azbð Þ~ 12|A663:8ð Þ{ 3:11|A646:8ð Þð Þ

z 20:78|A646:8ð Þ{ 4:88|A663:8ð Þð Þ

Carotenoid~ 1000|A480ð Þ{ 1:12|Chlað Þ{ 34:07|Chlbð Þð Þ=245

Nitrogen (N) concentration was measured following the wet digestion procedure for

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plant material (Allen 1989), on samples of freeze-dried, ground tissue, ranging in

mass between 95 and 105 mg. Nitrogen was measured colorimetrically with a flow-

injection analyser (FLOW 3000, Perstorp, Oregon, USA).

2.4 Spectral measurement and process

Spectral measurements were carried out in laboratory, by a portable spectroradiometer

(Field spec FR 350–2500 nm, ASD inc., USA), operating in the 350–2500 nm spectral

range. Fieldspec can acquire both radiance and reflectance data. The instrument

automatically calculates the reflectance value as the ratio between the incident

radiation, reflected from the surface target, and the incident radiation reflected by a

reference Spectralon panel. This material can be regarded as a Lambertian reflector.

Fieldspec may be operated with different lenses that control the field of view: in this

study an 8u lens was used to restrict the instrument field of view. Measurements were

taken in a dark room; the light source was an incandescent lamp. Reflectance spectra

were collected from a distance of 5 cm from the sample. Each spectral signature was

recorded as the average of 100 scans to reduce instrumental noise; a further check of

the stability of the reflected signal was performed by measuring the reflectance of the

reference panel at the beginning and end of each session. Reflectance spectra were pre-

processed by using the ViewSpecPro (ASD, Inc.) software.

Reference wavelengths were determined directly from the averaged reflectance

spectra of well-watered plants, while the inflection points were calculated by using

the first derivative reflectance spectra. Further spectral analysis and spectral indices

calculation of different drought treatments were performed on the basis of the

reference wavelengths. First derivative reflectance spectra were calculated as (Zarco-

Tejada et al. 2003):

LRi~ Riz1{Ri{1ð Þ= liz1{li{1ð Þ

where R is the reflectance and l is the wavelength.

The red edge (715 nm) was determined by identifying the peak wavelength in the

corresponding first derivative spectra between 680 nm and 750 nm.

Fluorescence ratio indices (FRI) were calculated as FRIR690/R600 and FRIR740/R800

(Dobrowski et al. 2005), whereas the photochemical reflectance index (PRI) was

calculated as (R5312R570)/(R531 + R570) (Gamon et al. 1990, 1997). The structural

independent pigment index (SIPI, i.e. a measure of carotenoids to chlorophyll a

ratio) was calculated as (R8002R445)/(R8002R680), and the normalized phaeophy-

tinization index (NPQI, i.e. a measure of chlorophyll degradation) was calculated as

(R4152R435)/(R415 + R435) (Penuelas and Filella 1998). The water index (WI) was

calculated as R900/R970 (Penuelas and Filella 1998). Moreover, the relative depth

index (RDI) (Rollin and Milton 1998) was used to describe leaf water absorption at

about 1140 nm in relation to the maximum reference wavelength at 1098 nm (Rmax)

and minimum reference wavelength at 1202 nm (Rmin):

RDI~ Rmax{Rminð Þ=Rmaxð Þ|100

To quantify and compare the change of reflectance spectra at different reference

wavelengths in response to water stress, we calculated relative reflectance increment

(RRI):

RRIli~ Rli{Rlcð Þ=RlC

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where l is the reference wavelength, i is the number of hours after the stress onset,

and c is control. RRI was also used to identify the most sensitive wavelength over

the course of water stress. Water content reflectance index (WCRI) was developed

to describe leaf water absorption feature between NIR and SWIR regions.

WCRI~RlSWIR= RlNIR{RlSWIRð Þ

lSWIR is the reference wavelength where the reflectance minimum at 1455 nm, i.e. the

reflectance at which the maximum RRI was observed in SWIR region, and lNIR is

the wavelength at 1272 nm, i.e. the reference wavelength where a reflectance peak

occurs in the NIR region.

2.5 Statistics

Data were tested using a simple factorial ANOVA (two-way maximum interactions)

and Post-hoc Tukey’s test. The statistical analysis was performed by SigmaPlot

(Systat software, Inc., 2004).

3. Results

The reference wavelengths identified were: 524 nm, 555 nm, 572 nm, 673 nm in the

visible region; 715 nm, 919 nm, 958 nm and 1098 nm in the near infrared region

(NIR); 1140 nm, 1202 nm, 1225 nm, 1272 nm, 1391 nm, 1455 nm, 1502 nm, and

1656 nm in the shortwave infrared region (SWIR).

3.1 ABA feeding

ABA feeding did not induce any change in chlorophyll and N concentrations (data

not shown). RWC was not significantly increased (data not shown); however,

stomatal conductance and photosynthesis (figure 1a) and fluorescence yield (i.e. the

quantum yield of PSII in the light, DF/F9m) (figure 1b) decreased rapidly. A small

modification in the NIR of the spectral reflectance (figure 2a), which partly reflects

cellular scattering by leaf water content, was observed. This may reflect the slight

increase in RWC, whereas the changes in the photosynthetic properties were not

reflected by parallel modification in leaf spectral reflectance in the visible region

(figure 2a). The FRIR690/R600 and FRIR740/R800 and photochemical reflectance index

(PRI) showed no significant change during the ABA treatment (figure 3).

3.2 Fast leaf dehydration

In both top and bottom leaves, RWC decreased most rapidly during the first hour,

and then the water loss proceeded at slower rate (figure 4). Leaf dehydration

decreased leaf gas exchange properties significantly (data not shown), but did not

cause any changes in chlorophyll and N concentrations (data not shown). The

reduction of gas-exchange and fluorescence were not mirrored by changes in spectral

reflectance in the visible region (i.e. at wavelengths of 400–700 nm) (figure 2b).

Consequently, FRI and PRI were not affected by leaf dehydration (data not shown).

However, spectral changes in NIR and SWIR regions were consistent with changes

in RWC. RRI showed large changes especially at 715 and 1098 nm (NIR region) and

at 1391, 1455, 1502, and 1656 nm (SWIR region) (figure 5a). The changes of RRI in

the SWIR region, particularly at 1455 nm, were the most sensitive to leaf

dehydration (figure 5a). RRI increased markedly after the first hour of leaf

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dehydration (i.e. when water loss was particularly high, also causing a RWC

decrease of ,10%), reaching the value of ,0.3 at 1455 nm. Afterwards, RWC

decreased at a lower rate and this was mirrored by a lower rate of RRI increase. The

reflectance index reached a value of ,0.45 at 1455 nm after four hours of leaf

dehydration (figure 5a). RRI at 1455 nm was linearly correlated to RWC (r250.733)(figure 5b).

3.3 Slow-developing water stress cycle

As no significant differences in the physiological parameters and the reflectanceindices measured on top and bottom leaves were found, the data were pooled. Water

stress induced large changes of photosynthesis, stomatal conductance and

fluorescence yield (table 1). These parameters were dramatically reduced after five

days of water stress (i.e. when the stress was moderate and RWC was slightly

affected), and further decreased at the end of the experiment, when RWC was

reduced by an average of 28% (table 1). The moderate water stress (after a five-

daytreatment) did not induce any change in total chlorophyll concentration,

whereas at the end of the experiment chlorophyll concentration was significantlyreduced by an average of 16% in top leaves and 23% in bottom leaves. Leaf

carotenoid to chlorophyll ratio increased significantly after 15 days. On the other

Figure 1. Time course of (a) photosynthesis (A) and stomatal conductance (gs) and (b)chlorophyll fluorescence (DF/F9m) in response to ABA feeding to branches detached fromwell-watered olive saplings. The measurement conducted immediately after the branches werecut and dipped into the pure water (time 0), and at one, two, three and four hours after ABAfeeding, respectively. Data are means of eight plants ¡1 SEM.

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hand, N concentration and SLA were not significantly affected by water stress cycle

in either leaf types (table 1).

The slow-developing water stress treatment dramatically affected the spectral

reflectance of olive leaves (figure 2c). As for the photosynthetic parameters, also PRI

(the photochemical reflectance index) and the R690/R600 ratio decreased gradually

from the onset of water stress to the end of the water stress cycle (table 1). However,

the R740/R800 ratio was not affected by water stress and, consequently, was not

correlated to changes of physiological properties. As water stress increased in

Figure 2. Modification of the reflectance spectra in (a) ABA treated leaves, (b) detachedleaves from well-watered olive saplings and (c) in leaves of olive saplings in well-wateredplants (control) and water-stressed plants after five and 15 days from the onset of water stress.In ABA treated and detached leaves, each reflectance spectra is a mean of 20 samples, whereasduring the water stress cycle each reflectance spectra is a mean of 48 samples (including bothtop and bottom leaves).

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Figure 3. Time course of (a) fluorescence reflectance indices and (b) photochemicalreflectance index (PRI) in response to ABA feeding to branches detached from well-wateredolive saplings. R690/R600, R740/R800 are reflectance indices calculated by using bands located atthe chlorophyll emission maxima normalized by bands not affected by chlorophyllfluorescence emission (600 nm and 800 nm) (Dobrowski 2005). Data are means of eightplants ¡1 SEM, three replicates of reflectance spectra were measured per leaf.

Figure 4. Time course of relative water content (RWC) during dehydration of leavesdetached from well-watered olive saplings. Data are means of 20 leaves ¡1 SEM.

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intensity, the red edge position was progressively shifted to shorter wavelengths and

NPQI was significantly decreased, whereas there was no effect on SIPI and WI

(table 1). The slow-developing water stress also significantly affected RRI,

particularly when it was measured at reference wavelengths in the visible region(figure 6). At the end of the drying cycle, RRI increased in all reference wavelengths,

showing, however, the strongest increase in both visible and SWIR regions. RRI

measured at 1455 nm was found again to be the most sensitive wavelength to water

stress. As seen in the leaf fast dehydration experiment (figure 5b), the increase of

RRI at 1455 nm might correlate with the strong decrease in RWC caused by severe

water stress (table 1). The reference wavelength at 1455 nm was also used to calculate

the water content reflectance index (WCRI). WCRI was inversely related to RWC

(r250.702) (figure 7a). However, the accuracy of this correlation was similar to thatfound in the linear relationship between the relative depth index (RDI) and RWC

(figure 7b).

Figure 5. Relative reflectance increment (RRI) changes at different reference wavelengthsover the course of leaf dehydration (a) and relationships between relative reflectanceincrement (RRI) at 1455 nm and relative water content (RWC) (b).

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4. Discussion

This study aimed to determine whether modification of physiological parameters

induced by water stress could be detected remotely by monitoring the electro-

magnetic energy (from visible to SWIR regions) reflected by olive leaves. There were

Table 1. Total chlorophyll (Chl, mg cm22) and nitrogen (N, g g21) concentration on leaf areabasis, leaf carotenoid to chlorophyll ratio (Carot/chl), relative water content (RWC), specificleaf area (SLA, cm2 g21), photosynthesis (A, mmol m22s21), stomatal conductance (gs,mol m22s21), fluorescence (DF/F9m), Water Index (WI), structural independent pigment index(SIPI), normalized phaeophytinization index (NPQI), relative depth index (RDI), red edgeposition, photochemical reflectance index (PRI), and fluorescence reflectance indices(FRIR690/R600 and FRIR740/R800) determined on leaves of olive saplings after five and 15days from the onset of water stress. Data, pooled from top and bottom leaves, are means of 48leaves (four per plant) ¡1 SEM. Letters (a, b, c) indicate significant differences at P,0.05 in

the same line.

Control 5-day stress 15-day stress

A 9.87¡0.98 a 1.21¡0.38 b 0.88¡0.29 cgs 0.240¡0.037 a 0.019¡0.003 b 0.015¡0.004 bDF/F9m 0.301¡0.023 a 0.176¡0.017 b 0.075¡0.016 cChl 40.9¡3.5 a 39.3¡4.9 a 34.2¡2.6 bN 1.20¡0.10 1.15¡0.20 1.15¡0.10Carot/Chl 0.215¡0.010 b 0.234¡0.015 b 0.292¡0.020 aRWC 0.93¡0.03 a 0.87¡0.02 b 0.68¡0.03 cSLA 39.6¡1.5 40.0¡1.6 38.9¡1.2WI 1.02¡0.01 1.01¡0.03 1.01¡0.02SIPI 0.969¡0.003 0.969¡0.002 0.972¡0.002NPQI 0.224¡0.017 a 0.155¡0.007 b 0.129¡0.010 cRDI 15.78¡0.42 a 16.06¡0.29 a 12.33¡0.59 bRed edge 716¡1 a 712¡1 b 707¡1 cPRI 26.9?1023¡0.3?1023 a 23.4?1022¡0.2?1022 b 27.7?1022¡0.4?1022 cFRIR690/R600 0.87¡0.03 a 0.80¡0.03 b 0.71¡0.03 cFRIR740/R800 0.88¡0.02 0.90¡0.04 0.89¡0.03

Figure 6. Relative reflectance increment (RRI) determined at different reference wave-lengths in water-stressed olive saplings after five and 15 days from the onset of water stress.Data are means of 48 leaves ¡1 SEM.

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no statistical differences in the physiological parameters and in the associated

reflectance spectrum in both top and bottom leaves, presumably because the plants

being young and with a limited canopy the bottom leaves had not yet developed as

shade leaves, and for this reason the data of the different leaf types were combined.

To establish reflectance indices and parameters that can be used for effectively

evaluating water stress, an algorithm (RRI) and 16 reference wavelengths were

developed (figures 5 and 6). Inhibition of gas exchange and fluorescence induced by

water stress which occurred over an hour timescale was not accompanied by parallel

reduction in chlorophyll concentration and an increase in the carotenoid to

chlorophyll ratio. Therefore, changes in spectral reflectance in the visible region

were not significantly induced (figures 2a and 2b), whereas simultaneous reductions

of the photosynthetic pigments and photosynthetic activity (table 1) induced by slow

water stress resulted in significant changes in the visible region of the leaf spectral

reflectance (figures 2c and 6). In an attempt to improve the remote detection of the

Figure 7. Relationships between relative water content (RWC) and (a) water contentreflectance index (WCRI) (r250.702) and (b) relative depth index (RDI) (r250.698) in well-watered plants (control) and water-stressed plants after five and 15 days from the onset ofwater stress.

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water status of the leaves a new water stress-related reflectance index (WCRI) was

developed (figure 7a).

A large number of spectral indices have been developed for estimating leaf

physiological status (e.g. Gamon et al. 1992, Gao and Goetz 1995, Ustin et al. 1998,

Yu et al. 2000, Zarco-Tejada et al. 2000a, Ceccato et al. 2001, Muttiah 2002,

Imanishi et al. 2004). RWC is universally used to detect the water status of plants

(Tyree and Jarvis 1982, Kramer and Boyer 1995), but until now correlations with

spectral indices have been made with other water status parameters (e.g. LWC per

unit leaf area, specific leaf water content, leaf moisture percentage on fresh weight,

relative leaf moisture percentage on fresh weight basis, etc.) which are not used in

studies on plant–water relations in the soil plant atmosphere continuum. Moreover,

because these indices have been tested only in a limited number of species, it is not

yet clear whether these indices are species-specific or whether their use can also be

extended to different plant functional types (Blackburn 1998, Sims and Gamon

2002, Bryant and Baird 2003). Furthermore, these indices may also show

intraspecific variations caused by different physiological conditions (Horler et al.

1983).

The term ‘reference wavelength’, as it has appeared in a number of previous

reports (Serrano et al. 2000, Sims and Gamon 2002, 2003), cannot be universally

applied for the effective evaluation of the water status of plants. Reference

wavelength was generally defined as a constant wavelength between 750 and 900 nm,

i.e. outside the chlorophyll absorption bands (Sims and Gamon 2002), and

consequently it has limited applications. For this reason, in this study, the

connotation of this term was extended by identifying a series of typical wavelengths

at the reflection peaks, valleys, and inflection points (maximum slope) of the control

leaf reflectance spectra from visible to SWIR regions. Thus, a new series of reference

wavelengths were defined and tested to see whether these wavelengths were suitable

indicators of different degrees of water stress. After identifying the reference

wavelengths, the corresponding reflectance values were determined in control

plants. These values were then used to compute the spectral reflectance changes in

water-stressed plants, allowing calculation of the Relative Reflectance Increment

(RRI) (figures 5 and 6). Differently from the spectral subtraction technique (Zarco-

Tejada et al. 2003), RRI shows the relative changes rather than absolute changes in

the spectral reflectance (Carter 1991). Consequently, even small changes in the

spectra can be effectively detected by RRI, for instance at low-reflective wavelengths

(i.e. visible region) where usually not large changes occur in response to water stress.

Thus, this new algorithm was used to identify the most sensitive wavelengths of the

spectral reflectance affected by water stress (figures 5a and 6) (Carter 1991).

Our study showed that the reflectance spectra in the visible region (figures 2a and

2b) and, consequently in PRI and FRI (figure 3), were not influenced when stomatal

closure and photosynthesis inhibition (figure 1) occurred within an hour and,

consequently, were not long enough to cause changes in the concentration of

photosynthetic pigments. This was the case of our experiments with ABA feeding

and fast leaf dehydration (data not shown). Similarly, Foley et al. (2006) also

showed that leaf reflectance in the visible region in five tropical trees was not

responsive to water content changes that occurred within an hour. This is often the

case in nature, when diurnal stresses develops in hot summer days, or in response to

vapour pressure deficit (Alvino et al. 1994, Centritto et al. 2000). It still remains

difficult to correctly assess these rapidly inducible and relieved physiological

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changes by remote indices. Recent studies suggested a strong relationship between

steady-state fluorescence (Fs) and photosynthesis under variable irradiance and

water stress conditions (Flexas et al. 2002, Dobrowski et al. 2005). These studies

may indicate the possibility to assess photosynthesis changes due to transient

stomatal closure by using FRI. However, both our ABA and fast leaf dehydration

experiments have demonstrated that FRI does not follow the decline of the

fluorescence parameter DF/F9m, which is also based on Fs measurements (Genty et

al. 1989). This is probably because ABA feeding (figure 3a) and fast leaf dehydration

(data not shown) did not induce significant changes in Fs, or small changes of Fs

signals have no effect on spectral reflectance index. The contribution of Fs to the

spectral reflectance is known to be very low (e.g. up to 2%, Zarco-Tejada et al.

2003), and this contribution may also be highly variable (Dobrowski et al. 2005).

Dobrowski et al. (2005) also attributed less than 1% of the canopy reflectance

change to Fs changes, in Vitis vinifera subjected to a diurnal heat and water stress.

Although Fs changes in response to transient water stress were detected in Acer

saccharum by using a time-decay experiment and induction with long-pass filters

(Zarco-Tejada et al. 2000a, 2000b), the estimation of photosynthetic status by FRI is

still a challenge.

On the other hand, when the rapid dehydration was associated with a decrease of

RWC, such as in the fast leaf dehydration experiment, this could be remotely

followed by measuring associated changes of the RRI in the NIR and SWIR areas

(figure 2b). Increased reflectance in reference wavelengths in both NIR and SWIR

regions of the leaf spectrum are known to indicate a decrease in leaf water content

(Carter 1991, Hunt and Rock 1989, Ceccato et al. 2001, Muttiah 2002). These

changes were indeed associated with the rapid dehydration of the leaf in a number of

different reference wavelengths (figure 5a). The most sensitive wavelengths to RWC

changes could be distinguished by RRI. The RRI measured at 1455 nm increased

faster than at other reference wavelengths, and was identified as the most sensitive

wavelength to RWC changes (figure 5b). This confirms earlier suggestions that the

wavelengths near 1450 nm are among the most sensitive bands to changes in water

content (Carter 1991, 1993). Reference wavelengths in the SWIR region (i.e.

1502 nm, 1391 nm and 1656 nm) were also found to be more sensitive than other

wavelengths in NIR region to RWC changes (figure 5a). This can be explained by

the strong water absorption capacity in the SWIR bands (Palmer and Williams

1974, Carter 1993).

The slow-developing water stress cycle experiment was perhaps the most

representative of natural conditions. This causes reductions of RWC associated

with changes in the structural components of the leaves, including pigments (table 1)

and other organic molecules (Bray 1997, Chaves and Oliveira 2004, Centritto 2005).

Whether a reflectance index was particularly suitable to monitor the occurrence of

physiological changes caused by this stress was investigated.

The photochemical reflectance index (PRI) was originally developed to estimate

rapid changes in the xanthophyll cycle that occur over a minute timescale and are

related to changes in photosynthetic light use efficiency (Gamon et al. 1992, 1997).

Differently from ABA feeding and rapid leaf dehydration, PRI showed a significant

decrease as water stress progressed (table 1). This may be related to the strong

inhibition in the photosynthetic activity due to prolonged drying over a day

timescale which led to a decrease in chlorophyll concentration and to a simultaneous

increase in the carotenoid to chlorophyll ratio (table 1 and Sims and Gamon 2002).

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The decrease in chlorophyll concentration was mirrored by the parallel decrease in

NPQI, which is according to Penuelas and Filella (1998) a measure of chlorophyll

degradation, whereas there was no correlation between the biochemical measure-

ments of the carotenoid to chlorophyll ratio and the indirect measurements of the

carotenoid to chlorophyll (i.e. SIPI). However, a decrease of photosynthetic light

use efficiency was also observed in response to the slowly developing water stress, as

inferred from the photosynthesis reduction at a PPFD of 1000 mmol m22s21 and by

the reduction of the fluorescence index DF/F9m at the same light intensity in turn

probably linked to an increased dissipation by xanthophyll deep-oxidation

(Demmig-Adams and Adams 2000, Demmig-Adams 2003). Thus, the influence of

these two components of PRI could not be separated.

The FRIR690/R600 also showed reductions as the water stress progressed and may

reflect a corresponding decrease in DF/F9m (table 1). This, in turn, may indicate a

significant decrease of Fs as found by Dobrowski et al. (2005). In contrast, the

FRIR740/R800 did not show any change throughout the course of water stress cycle

(table 1). Thus wavelengths in the NIR region may not only refer to water stress but

may also indicate structural changes over the 15-day water stress cycle, such as

ontogenetic variations of SLA (table 1) or dry mass (Muttiah 2002).

As also indicated by the fast-developing stress experiment, the 1455 nm

wavelength is highly sensitive to water stress (figures 5a and 2b). However, because

of the limited time series of data in the slow-developing water stress experiment, the

relationship between RRI (at 1455 nm) and RWC, as shown in figure 5b for the fast-

developing stress, could not be estimated. Therefore an index based on the 1455 nm

response, the WCRI, was developed for detecting RWC and, consequently, the

intensity of water stress. Simultaneously, the RDI index was applied to the

estimation of RWC. RDI was measured in the NIR region and was found to be

positively correlated in field conditions to the percentage of canopy water content in

a grassland (RDI centred upon 1150 nm and normalized with the 1116 nm band)

(Rollin and Milton 1998), and to the percentage of leaf water contents in wheat

subjected to different irrigation treatments (RDI centred upon 965–1085 nm spectral

regions and normalized with the 1192–1282 nm spectral regions) (Zhao et al. 2004).

A similar relationship with RWC was found when using both WCRI (figure 7a) and

RDI (figure 7b). However, this relationship was clear at low RWC values (after a 15-

day stress), while it was weak during the first phase of the stress (i.e. high RWC

values). Thus, both indices still need to be refined in detecting plant water status by

remote sensing. However, we speculate that because WCRI is centred on a band (i.e.

1455 nm wavelength) which is very sensitive to water stress, it could result in a more

useful index to estimate leaf water status in olive and similar sclerophyllous plants

by proximal sensing. However, because of the scattering due to atmospheric

pollution (Cartalis and Varotsos 1994, Kondratyev and Varotos 2001), also

absorption by the atmospheric water vapour is very strong at 1455 nm.

Consequently, WCRI may prove to be inadequate in assessing water stress from

airborne and satellite platforms.

It should be noted that the physiological significance of the reflectance indices is

dependent not only on physiological activities but also on the density of vegetation.

There are suggestions from previous studies that NIR wavelengths, e.g. water

indices namely R900/R970 (Penuelas et al. 1993) and the ratio between 1150 and

1260 nm (Sims and Gamon 2003), penetrate further into canopies and thus might

allow better estimation of water content in thick canopies. However, the surface

Effects of drought on leaf spectral reflectance in olive 1739

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layer of the canopy is also the photosynthetically active layer, and thus RWC (and

associated reflectance indices such as the WCRI) of surface canopies may still be an

important indicator of physiological drought in thick canopies.

To summarize, the following questions have been investigated: (1) how spectral

reflectance is modified when plants are subject to different degrees of drought, and (2)

whether it is possible to remotely detect by sensitive spectral indices physiological

changes induced by different degrees of stress. In this study, 16 new reference

wavelengths, a new water stress-related reflectance index (WCRI) and a new algorithm

(RRI) were developed to improve detection of water stress by spectral reflectance

changes. When the inhibition of gas exchange and chlorophyll fluorescence induced by

water stress was not accompanied by parallel reduction in pigment concentrations,

changes in spectral reflectance in the visible region were not significantly induced.

However, changes of leaf water status induced by both fast-developing and slow-

developing stress were detected, and reduction of both photosynthetic pigments and

photosynthetic activity induced by slow-developing water stress were also detected by

monitoring changes of the leaf spectral reflectance and this may have interesting

applications for both proximal and remote sensing of water stress.

Acknowledgements

This research was funded by the national Natural Science Foundation of China

(NSFC) (No. 30471383), national Key Fundamental Research Plan of China

(No. 2002CB111504), key joint research project of NSFC (No. 30590383), key

Laboratory of Forest Ecology and Environment of State Forestry Administration,

and by the Agreement of Scientific Cooperation between the Consiglio Nazionale

delle Ricerche of Italy and the Chinese Academy of Forestry of P.R. China. We

would like to thank Dr. Maria Soprano for the nitrogen analysis, Dr. Ulo Niinemets

and Prof. Josep Penuelas for helpful comments. We thank two anonymous reviewers

for their constructive and helpful comments on the manuscript.

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