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Investigation of water stress of cork oak leafs
and pine needles based on integral fluorescence
measurements and spectra characteristics
Andrei .B. Utkina*, Jorge Silva, Rui Vilarc, Alexander Lavrova, Nuno Santos a
a
INOV, Rua Alves Redol, 9, 1000-029, Lisbon, Portugal
b
University of Lisbon, Lisbon, Portugal
c
Instituto Superior Técnico, DepMat, Av. Rovisco Pais, 1049-001, Lisbon, Portugal
Abstract
Introduction
Idea to use laser induced fluorescence (LIF) from chlorophyll of plants for estimation of
its physiological status was firstly suggested and realized in [1] for lettuce. Later
investigations of physiological status for various species were conducted. For example
it is possible to mention work of Chappelle et al [2] who investigated corn and
soybeans, Edner et al [3], Saito et al [4] Astafurova et al [5], Richards et al [6], Fateeva
et al [7, 8], Zavorueva and Zavoruev [9], Grishin et al [10], Zuev, Zueva, and Grishaev
[11] and many others.
One of the specific problems that are investigated by LIF of plants is influence of water
deficiency on fluorescence spectra. This problem is examined for olive, rosemary and
*
Corresponding author. Tel.: +351-213100426; fax: +351-213100401. E-mail address:
andrei.utkin@inov.pt
1
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lavender by Nogues and Baker [13], for Phillyrea angustifolia L. (Oleaceae) by MunneBosch and Penuelas [14], for grass Setaria sphacelata by Marques da Silva e Arrabaca
[15] for maize by Xu et al [16] and for many other plants by various researches.
Estimation of physiological status of the plant is possible on the base of both the ratio
Fv/Fm (see later) and chlorophyll fluorescence spectra. There are two methods of
estimation of plant physiological status:
 Analysis of intensity of specific wavelength for control healthy plant and plant
with some physiological deficiency (water or nutrient stress, disease, etc.).
 Analysis of the ratio of signal intensities for two specific wavelengths for control
healthy plant and plant with some physiological deficiency.
In the present work second method (analysis of the ratio of signal intensities for two
specific wavelengths) is used for analysis of physiological status of leafs and needles in
the process of drying. In particular use of the ratio of fluorescence intensity at 685 nm to
740 nm that is suggested in several papers, see for example [3, 12], will be used in the
present work.
Instrumentation and material
Equipment for measurement of fluorescence spectra
The layout of the equipment for measurement of fluorescence spectra is shown in
Fig. 1. The source of the excitation of fluorescence is Nd:YAG laser that generates
20 mJ pulses at the second harmonic (532 nm) with repetition rate of 10 Hz and length
of 5 ns. The outgoing beam is directed to the vegetation. Fluorescence from vegetation
passes through longpass filter with cut-off wavelength of 550 nm, which prevents
detector from the damaging by the radiation at laser wavelength. Then radiation is
2
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collected by the collimator with optics diameter of 20 mm and transported by fiber optic
cable to Ocean Optics spectrometer. Collimator is designed for collecting without losses
fluorescence radiation from vegetation in the range of 650 to 800 nm. Fiber optic cable
transfers signal to Ocean Optics spectrometer. Spectrometer is controlled by PC
software. Synchronization pulse, that goes from laser to PC, fixes the time of begin and
end of spectrometer functioning to diminish the quantity of background radiation
entering in collimator. To diminish the signal noise 10 returns from 10 laser pulses are
averaged. Then they are stored in PC.
Fluorometry
Estimation of vegetation physiological status is based on Kautsky effect [17] that was
open up by Kautsky and Hirsh in 1931. In the modern treatment physiological status is
estimated in accordance with following procedure: Leaf (or needle) that is previously
kept in the darkness during approximately 10 minutes is lighted by bright red LED
(wavelength ~650 nm, intensity at leaf surface ~3000 to 6000 µmolm-2s-1) during ~60 s.
Fluorescence return from leaf, which is schematically shown in Fig. 2, increases from
zero to the ground value F0 very quickly (during ~10ns) and then during ~1 s
fluorescence increases to its maximum value Fm. Efficiency of photosystem is given by
Fv/Fm=(Fm-F0)/Fm. In the healthy leaves this value is approximately equals ~0.8
independently the species. Diminution of Fv/Fm testifies about existence some stress
condition of leaf.
Plant material
Ten leaves of mature cork tree and 40 needles of Mediterranean pine (four needles in
every packaging) were used in the process of 8 or 12 days of experiments. Every
3
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measurement included weighting of the pattern, measurement of Fv/Fm using PEA
device and obtaining of fluorescence spectrum. As the drying of cork tree leave is much
quicker than drying of needle measurements for the leaves were conducted 2 times per
day and for needle one time per day.
Experimental results
Experiments for cork oak were conducted in September 2009. Ten leaves were pulled
off from the tree branch. Then they were dried in the room with temperature about
240 C and relative humidity about 40%. Leaves weight, fluorescence spectra, and
parameter Fv / Fm were measured every day excepting Saturday and Sunday. Evolution
of fluorescence spectra of one of the leaves for 6 days (1st, 2nd, 3rd, 4th, 5th, and 8th) is
depicted in Fig. 3. Maximum of the spectrum in the first day is for far red
max  738 nm. As the leaf is drying maximum of the spectrum is changing a bit to the
short part of the range. In 8th day max is about 717 nm. It is seen that as the leaf is
drying the local maximum in the spectrum near wavelength of 685 nm is appearing.
Variation of Fv / Fm and I 685 / I 740 with time for cork oak leaves is presented in Fig. 4.
Fig. 3a.) vs time for cork oak. Averaging over 10 samples. The error bars denote the
standard deviation. The one of the purposes of the present investigation is to estimate
correlation between variation with time Fv / Fm and I 685 / I 740 . For calculation of
correlation coefficient formula from Korn and Korn [18] is used:
n
R


 yi  y zi  z
i 1
n

 
2 n

 yi  y  zi  z
i 1

,
(1)
i
4
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where i is the number of the test, n maximum number of the tests (for our problem
n  6 ), yi  Fv / Fm i , zi  I 685 / I 740 i , and y and z are the sample means. For
experiments with cork oak we have R  0.99 . This result testifies that thee strong
unticorrelation exists between. So it is not necessary to measure both variables, we need
only to measure one of them. Previously it was confirmed that ratio Fv / Fm correlates
strongly with chlorophyll concentration. So, our results have showed that measurement
of I 685 / I 740 allows to estimate chlorophyll concentration.
Experiments for pine were conducted in December 2009. Forty needles (gathered in ten
bundles of needles, four needles in every bundle) were pulled off from the tree branch.
Then they were dried in the room with temperature about 200 C and relative humidity
about 60%. Needles weight, fluorescence spectra, and parameter Fv / Fm were measured
every day excepting Saturday and Sunday. As the drying of needles is slower for the
needle’s part near base and quicker near needle’s tip, measurements of spectra and
Fv / Fm were conducted three times for every needle bundle: near base, in the middle,
and neat tip. Evolution of fluorescence spectra of one of the needles for 8 days (1st, 3rd,
4th, 8th, 9th, 10th, 11th, and 12th) is depicted in Figs. 5 to 7. Maximum of
It is seen that, as the needles are drying the local maximum of fluorescence spectrum
near 685 nm is more pronounced.
Variation with time Fv / Fm and I 685 / I 740 .for three positions is presented in Figs. 8, and
9. Correlation coefficient between time dependence of Fv / Fm and I 685 / I 740 that was
calculated also using equation (1) equals, respectively, 0.91, 0.99, and 0.98 for positions
of observation: near needle base, in the middle, and neat tip. So, for needle we also may
conclude that ratio I 685 / I 740 may be used for estimation of chlorophyll concentration.
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In accordance with Barrs [19] relative water content in biology sample is calculated
using the equation:
 FW  DW
RWC  
 TW  DW

 100 ,

where DW is dry weight of the sample, TW is fully turgid fresh weight, and FW is
varying in the process of experiment weight of the sample. In the end of every series of
experiments biological sample is placed in water during two days (for leafs), or four
days (for needles), and after reweighting TW is obtained. DW is obtained after drying of
the samples at 800C during three days.
Statistical analysis
.
Discussion
.
Acknowledgement
The authors wish to thank Mr. Bruno Alves for his help in organizing the experiments.
This research was partially supported by the LIF-LIDAR project of the Portuguese
Ministry of Agriculture.
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9
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Leaf or
needle
Nd:YAG laser, 532 nm
Synch
Fluorescence from
vegetation
Longpass filter
Ocean Optics
Light gathering optics
spectrometer
Optical fiber
Control and data
acquisition
Fig. 1. Layout of the system for obtaining laser induced fluorescence spectra of vegetation
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Fluorescence signal, a.u.
Fm
5
4
3
2
1
0
1E-5
F0
1E-4
1E-3
0,01
0,1
1
Time, s
Fig. 2. A typical fluorescence
signal excited by red light from
dark adapted vegetation sample
11
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Normalized signal
1,00
8th day
5th day
0,75
4th day
3rd day
0,50
2nd day
1st day
0,25
0,00
650
700
750
800
Wavelength, nm
Fig. 3. Fluorescence spectra of the leaf of cork oak during 8 days.
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Fv/Fm
a
1.0
0.8
0.6
0.4
0.2
0.0
0
48
96
144
I685/I74
b
1.0
0
0.8
0.6
0.4
0.2
0.0
0
48
96
144
Time, hours
Fig. 4. Fv/Fm (a) and I685/I740 (b) vs time for cork oak. Averaging over
10 samples. The error bars denote the standard deviation
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Normalized signal
264 hours
240 hours
1.00
216 hours
0.75
192 hours
168 hours
0.50
72 hours
48 hours
0.25
0 hours
0.00
650
700
λ, nm
750
800
Fig. 5. Fluorescence spectra near needle’s base during 12 days (sample 06).
14
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Normalized signal
12th day
1.00
11th day
10th day
0.75
9th day
0.50
8th day
4th day
0.25
48 hours
1st day
0.00
650
700
750
800
λ, nm
Fig. 6. Fluorescence spectra in the middle of needle during 12 days (sample15).
15
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Normalized signal
1.00
264 hours
10th day
9th day
0.75
8th day
0.50
5th day
4th day
0.25
48 hours
1st day
0.00
650
700
750
800
λ, nm
Fig. 7. Fluorescence spectra near needle’s tip during 12 days (sample 24).
16
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Fv/Fm
a
0.8
0.6
0.4
0.2
0.0
0
48
96
144
192
240
0.8
b
0.6
0.4
0.2
0.0
0
48
96
144
192
240
0.8
c
0.6
0.4
0.2
0.0
0
48
96
144
Time, hours
192
240
Fig. 8. Fv/Fm vs time for pine needles. Averaging over 10 samples. The error bars denote the standard deviation
17
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I685/I740
a
1.6
1.2
0.8
0.4
0.0
0
48
96
144
192
240
192
240
1.6
b
1.2
0.8
0.4
0.0
1.6 0
48
96
144
c
1.2
0.8
0.4
0.0
0
48
96
144
192
240
Time, hours
Fig. 9. I685/F740 vs time for pine needles. Averaging over 10 samples. The error bars denote the standard deviation
18
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Fv/Fm
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
60
80
100
RWC, %
I685/I740
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
RWC, %
Fig. 10. Fv/Fm and I685/I740 vs RWC for cork oak. Results for ten samples. Every symbol co-ordinates
with specific sample.
19
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20
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21