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Article history:
Received 15 July 2015
Received in revised form 5 November 2015
Accepted 9 December 2015
Available online xxxx
Keywords:
Diffuse reectance
Diffuse transmittance
Absorptance
Leaf absorbance
Inltration
Chlorophyll uorescence
Collimated light
Diffuse light
Nonlinearity
a b s t r a c t
There are many studies showing the active optical reaction of a green leaf to the changing surroundings based on
chloroplast movement and their rearrangement in plant cells. These studies concentrated mostly on the effect of
one feature (leaf type, leaf side or light type) on the leaf optical spectra. We have measured the diffuse reectance
and transmittance spectra of tobacco green leaves in combination of 4 variants: in normal and water inltrated
leaves, in collimated or diffuse incident light, on both the adaxial and abaxial leaf sides, and for the face or side
chloroplast arrangement. A Simple Explicitly Non-Linear Empirical model for Leaf Optical Properties (SENLELOP
model) is used to theoretically describe, simulate and t the deviations from the LambertBeer's law causing
nonlinearity in the measured spectral changes. It is shown that the incident diffuse light is captured by the leaf
more effectively than the collimated light. The light incident from the adaxial leaf side is more effectively
absorbed than the same light incident from the abaxial leaf side. The air in intercellular spaces of natural leaf
increases about twice the beam path and strongly deepens the non-linearity of the absorption process when
compared with water inltrated leaf. The chloroplast arrangement in the palisade cells is reected in most of
the studied differences. The leaf absorbance changed in our case of tobacco leaves up to 30% when the
chloroplasts moved from the face to the side position. This change depends strongly on the wavelength and
quite slightly on the character of incident light. Further analysis predicts that in practice the effect of chloroplast
rearrangement on the reectance spectra is in dependence on the wavelength of the light about 25% in our case
of fully developed green leaves but can be higher in some cases. Thus it can affect values of some of the indices
used in the remote sensing.
2015 Elsevier Inc. All rights reserved.
1. Introduction
A green leaf of higher plants can, to some extent, react and adapt to
the changing light conditions. This reaction is aimed to optimize the absorption of light quanta both to maximize the energy utilization in photosynthesis and to avoid harmful over-excitation. The process of this
adaptation is rather complex occurring in different time-scales and
may cover a change in the leaf position, leaf structure (e.g., swelling),
chloroplast movement, pigment composition, the state transitions or
energy quenching. We concentrate in this paper on the effect of chloroplast arrangement on the optical properties of leaves.
The ability of leaves to adapt its inner chloroplast arrangement to the
light conditions also inuences the resulting optical properties of leaves,
i.e., the leaf spectra of reectance, transmittance, absorptance and uorescence. The leaf reectance is usually the basis of the image detection
of a canopy by the spectral or multispectral cameras, both in laboratory
and in remote sensing studies. This implies that the detected spectral
image can be also inuenced by the chloroplast arrangement in leaf
Corresponding author.
E-mail address: naus@prfnw.upol.cz (J. Nau).
http://dx.doi.org/10.1016/j.rse.2015.12.011
0034-4257/ 2015 Elsevier Inc. All rights reserved.
182
Fig. 1. Images of tobacco leaf parts from optical microscope using transmission (C) or uorescence (A, B, D) modes. A and B show side (A) and face (B) chloroplast positions as seen from
adaxial leaf side. C and D show leaf cross section with different architectures in the adaxial (top) and abaxial (bottom) leaf sides. The lighter dots in A, B, and D show uorescing
chloroplasts. The bar indicates 20 m.
that the palisade parenchyma cells enable deeper penetration of the collimated light (Vogelmann & Martin, 1993). On the other hand, the
spongy parenchyma may be more suited for absorption of the diffuse
light (Lee, Bone, Tarsis, & Storch, 1990). This leaf side is directed to the
soil or lower layers of the canopy and the incident light is mostly the diffuse one.
When evaluating the external reection Re, the Fresnel formulas
should be accordingly applied (see, e.g., Gerber et al., 2011; Gorton,
Craig, Brodersen, Williams, & Vogelmann, 2010; see the Supplementary
data). In this case, however, it should be mentioned, that the Fresnel formulas must be used in correct way to express the detected light intensity (see, e.g., Lucarini, Saarinen, Peiponen, & Vartiainen, 2005; Suila
& Nau, 2007).
183
The light incident on the leaf surface may be of different spectral and
geometrical character and different polarization. The geometrical character of the incident light may change from a perpendicular collimated
light (a beam of parallel rays) to a completely diffuse light. The collimated light may make a different angle with the leaf normal, i.e., it may
come from different directions. Only several studies have concentrated
their attention on the comparison of leaf optical properties detected in
collimated and diffuse light (e.g. Brodersen & Vogelmann, 2010; Davis
et al., 2011; Gorton et al., 2010; Williams, Gorton, & Witiak, 2003).
Bird and Riordan (1986) found out that on a clear day light radiation
consists of 85% of direct (collimated) light and 15% of diffuse light. Diffuse light component increases with higher clouds presence and denser
vegetation dispersing the light. Experiments in low-intensity light
showed that in these conditions there is no type of radiation preferred.
On the other hand, photosynthesis was 15% higher in plants grown at
high-intensity direct light when compared to corresponding absorbed
amount of diffuse light (Brodersen & Vogelmann, 2010). These results
indicate that a leaf adapts both anatomically and biochemically to
light intensity during its development and thus it handles different absorbing properties to direct and diffuse light reaching it (Brodersen &
Vogelmann, 2010).
Every photosynthetic leaf cell contains usually dozens of chloroplasts situated between a central vacuole and a cell wall. Chloroplast arrangement is not stable, though, they respond to illumination reaching
the leaf surface. Chloroplast distribution thus depends on light conditions affecting the plant, i.e., on light intensity, direction, polarization
and spectral composition of incident illumination (Brugnoli &
Bjrkman, 1992; Davis et al., 2011; Kasahara et al., 2002; Nau et al.,
2010; Park, Chow, & Anderson, 1996; Vogelmann & Gorton, 2014;
Wada, 2013; Zurzycki, 1961).
Many papers have been devoted to description and signicance of
the chloroplast movement (e.g. Bana, Aggarwal, abuz, Sztatelman, &
Gabry, 2012; Brugnoli & Bjrkman, 1992; Davis et al., 2011; Kasahara
et al., 2002; Kniger, 2014; Kniger & Bolliger, 2012, Nau et al., 2010;
Park et al., 1996; Wada, 2013). There are two extreme types of the
movement. In case of high light intensity, the chloroplasts undergo the
so called avoidance movement placing the chloroplasts on the sides
(the side position) and let the light penetrate deeper into the leaf tissue,
see Fig. 1. On the other hand, in the case of low light intensity, the chloroplasts move to the periclinal cell walls (the face position) to enlarge
the light absorption (Bana et al., 2012; Gabry, 2004; Wada & Kong,
2011), see Fig. 1. This movement is in most cases in higher plants
evoked by the blue part of the incident light spectrum (Bana et al.,
2012; Gabry, 2004).
As a consequence of chloroplast movement, changes in optical leaf
properties occur. (Brugnoli & Bjrkman, 1992; Davis et al., 2011;
Kasahara et al., 2002; Nau et al., 2010; Park et al., 1996; Wada, 2013).
184
below). The disks were homogenized in 80% acetone with a low amount
of MgCO3 and then centrifuged (5 min at 3600 g). Absorption spectra of
the supernatant were measured with spectrophotometer Unicam
UV550 (Spectronic, Cambridge, UK). Concentrations of chlorophylls
(a + b) and of carotenoids were determined to be 25.9
3.42 g cm 2 and 6.5 0.70 g cm 2 (mean standard deviation,
n = 10), respectively. In such a way determined concentration of chlorophylls well agreed with measured SPAD-values (see below) as it was
previously calibrated for tobacco leaves by Nau et al. (2010).
2.3. Measurement of leaf optical properties
Perpendicular collimated and diffuse light were used to illuminate
the leaf. The portable spectroradiometer LI-1800 (LI-COR, Lincoln,
Nebraska, USA) with an integrating sphere LI-1800-12 were used for
measurements of diffuse reectance (RD) and transmittance (TD).
White light of illuminator was used for the measurement.
2.4. Collimated perpendicular light illumination
The collimated light was produced by the illuminator. The signal of
the sample was compared to the standard (LI-COR part # 991,804); a
small plate covered by puried barium sulfate which optimally reects
the light in a diffuse manner (diffuse reectance approaches 1) was
used as the standard. If the signal of the sample is Sv() and the standard
signal is Ss(), the nal signal RD() is:
RD
Sv
:
Ss
TD() is calculated using the following formula, where the standard signal is Ss() and the sample signal (the illuminator is behind
the sample) is STv():
T D
STv
Ss
SRS SRb
:
SRW SRb
inner reective surface of the sphere distributed the light rays randomly in the sphere. As a consequence, diffuse light was incident
on the sample.
The two spheres were attached together by the openings and the
sample was placed between them. When there was no sample, the detected signal STW() was proportional to the incident diffuse light. After
insertion of a leaf a spectrum STS() was measured. The diffuse transmittance under diffuse illumination, TDD(), was then calculated as:
T DD
STS
:
STW
185
where TD() and RD() are diffuse leaf transmittance and diffuse
leaf reectance, respectively. The word diffuse means that an integration sphere is used for the detection of reected or transmitted
light. Under such approach, all the light being reected to the hemisphere in front of leaf and transmitted to the hemisphere behind the
leaf is detected. The only error may come into consideration if some
light leaks through the leaf tissue out of the detection sphere.
186
I; x
10D :
I 0; x
10
Because the external reection of the cuvette Re() (in fact including
also the reection on the second cuvette wall) is mostly corrected by
using the reference cuvette with a solvent, the expression for the absorptance closer to reality should be:
A 1T Ri :
12
where DR() is a reference spectrum and D() the measured (calculated) spectrum after the change in light or leaf structural conditions. The
quantity S() means a change of the spectrum in relation to the original
(reference) one. In fact, there are two possibilities how to express the relation of two absorbances: i) One possibility is to use a known or supposed common reference spectrum obtained under nearly ideal
conditions, DR0(). The spectrum of diluted suspension of broken chloroplasts can serve for this purpose. This approach is suitable for the theoretical simulation of the studied effects. ii) The reference spectrum DR() is
that one of a pair of spectra being more similar to the ideal one. This sensitivity we will designate as the differential sensitivity Sd(). This Sd()
sensitivity is a more practical one, can be easily obtained, and was also
used by us (see Figs. 3-6). As the reference spectra we considered the
spectra obtained for inltrated leaf (Fig. 3), for collimated light (Fig. 4),
for abaxial leaf side (Fig. 5), and for face chloroplast position (Fig. 6).
3.4. Linear approximation
DDR
D
1;
DR
DR
11
Therefore, the Eq. (9) holds only for samples with negligible inner
reections. Generally, the analogy or differences between the method using integrating sphere and the spectrophotometric method
would desire a deeper analysis. However, this is not the goal of this
work.
Under the linear approximation we mean a validity of the linear expression for the absorbances in LambertBeer's law:
D C x;
13
DR R C R xR :
14
C x
1:
R C R xR
15
If we assume that there is no change in mean leaf pigment concentration (C = CR) and in the basic spectrum (() = R()), the differential sensitivity does not depend on the wavelength and it has a constant value:
Sd
x
1:
xR
16
Ideally, for the case when there is also no change between the length
path for the explored conditions and for the reference, the differential
sensitivity is zero upon the linear approximation.
3.5. Nonlinear approximation
However, the experimental data (Figs. 3-6) show that the differential sensitivity Sd() is not a constant, indicating that the linear approximation of the LambertBeer's law is not valid for leave tissues. The
deviation from the absorption linear law has been already studied and
considered (see, e.g., Lee et al., 1990; McClendon & Fukshansky,
1990a,b and references therein). We have revealed that the Sd() spectra bear inverse features of the reference spectra. This fact can be simulated by some kind of deviation from the linearity of the leaf absorbance.
The deviation can be expressed in the simplest way by introduction of a
weighted quadratic term in the expression of D():
D a C x bC x2 ;
17
for simplicity we assume that the reference absorbance has the linear
form as expressed by Eq. (14). It should be noted that any other more
complicated function describing well the experimental data could be
used instead of the quadratic function but we have chosen the quadratic
function as the easiest possibility. Moreover, the agreement between
theoretical model and experimental data is a support for our approach.
Let us suppose again that there is no change in mean leaf pigment
concentration (C = CR) and in the basic spectrum (() = R()) and
that the main cause of the spectrum change consists from a change of
the mean path length x. Then the differential sensitivity is:
Sd a
R C R x
R C R x2
b
1
R C R xR
R C R xR
18
and consequently
Sd a
x
R C R x2
b
1 a r b DR r2 1;
xR
xR
19
where
r
x
:
xR
20
We want to point out that the non-linearity of our model is not only
in x as might be concluded from Eq. (19), but explicitly also in () and C
(see Eq. (17)). We call the above mentioned Simple Explicitly NonLinear Empirical model for Leaf Optical Properties as SENLELOP model,
even if it is not a real structure-function-based model but a theoretical
description of experimental data. To our knowledge, the SENLELOP
model is the rst model which explicitly considers non-linearity, opposite to all published models so far (see Section 1.7). As our model well
describes experimental data (Figs. 3-6), the non-linearity should be
considered in more general models used in the remote sensing.
Although in a rather simplied way, we can now describe and discuss the differential sensitivity of selected pairs in terms of three parameters, basic weight of the linear term a, non-linear deviation b and the
relative change in the beam path r. However, we should rst relate
our r and b parameters to the known effect occurring in the leaves;
the sieve and detour effects (see Section 1.5). Even if we have to interpret our r and b parameters with some caution as their meaning is rather complex, in the rst approximation, we suggest to understand the r
parameter as reecting the detour effect (lengthening of the beam
path) and the b parameter as reecting the sieve effect. The suggested
meaning of the r and b parameters makes our non-linear approximation
more realistic.
3.6. Fitting the model parameters to experimental curves
Fitting of the parameters (a, r, b) of the model (Eq. (19); see below)
to experimental Sd() curves was performed using the solver function
of Microsoft Excel by estimating the parameters so that the criterion
function 2 is minimized. 2 is dened as a sum of squares of the difference between experimental Sd() and theoretical Sd() for given lambda. For a simplicity we assumed that a = 1 in all the cases and we tted
only values of b and r parameters.
3.7. Contribution of chlorophyll uorescence
187
188
Fig. 2. Typical spectra of diffuse reectance R (panel a) and diffuse transmittance T (panel b) measured with natural or inltrated Virgin tobacco leaf with its chloroplasts in the face position
upon illumination of the abaxial or adaxial leaf side by collimated light. The spectra then served for calculation of the typical spectra of diffuse absorptance A (panel c) and consequently of
the leaf absorbance D (panel d). The thin vertical line at 750 nm in panels a and c indicates a position of a hump in diffuse reectance R (panel a) and of a hollow in diffuse absorptance A
(panel c) caused by chlorophyll uorescence (see the text), which causes diffuse absorptance slightly below zero value as indicated by a thin horizontal line in panel c.
relative difference (sensitivity) of the absorbance of natural versus inltrated leaf shown in Fig. 2 was calculated using absorbances D[FDCN]
and D[FDCI] by the expression:
Sd FDCN=I FDCN=I DFDCNDFDCI=DFDCI:
In Table 1, for the sake of simplicity, only the symbols of the spectral
pairs are shown. To elucidate the way of interpretation we use, the effect of lengthening r and of non-linearity b on the Sd() spectrum should
be shortly explained. In a strongly simplied approach, an increase of
lengthening parameter r moves the sensitivity curve (understood as a
mean line) up to higher values whereas an increase in absolute values
of b enlarges the difference between the minima and maxima of the
Table 1
Results of the tting of the model parameters (b and r) to experimental Sd spectra. See the text for meaning of denotation of the variants.
Figure
3a
3a
3b
3b
3c
3c
3d
3d
Variant
SDC(N/I)
FDC(N/I)
SBC(N/I)
FBC(N/I)
SDU(N/I)
FDU(N/I)
SBU(N/I)
FBU(N/I)
b=
r=
0.242
1.724
0.199
1.961
0.234
1.400
0.231
1.498
0.191
1.556
0.081
1.552
0.250
1.083
0.147
1.209
Figure
4a
4a
4b
4b
4c
4c
4d
Variant
SD(U/C)N
FD(U/C)N
SB(U/C)N
FB(U/C)N
SD(U/C)I
FD(U/C)I
SB(U/C)I
FB(U/C)I
b=
r=
0.114
1.216
0.069
1.134
0.166
1.150
0.106
1.171
0.155
1.348
0.250
1.404
0.182
1.421
0.210
1.470
Figure
5a
5a
5b
5b
5c
5c
5d
5d
Variant
S(D/B)CN
F(D/B)CN
S(D/B)CI
F(D/B)CI
S(D/B)UN
F(D/B)UN
S(D/B)UI
F(D/B)UI
b=
r=
0.115
1.300
0.107
1.432
0.023
1.006
0.034
1.042
0.096
1.376
0.044
1.298
0.114
1.005
0.004
0.980
Figure
6a
6a
6b
6b
6c
6c
6d
6d
Variant
(S/F)BCN
(S/F)DCN
(S/F)BCI
(S/F)DCI
(S/F)BUN
(S/F)DUN
(S/F)BUI
(S/F)DUI
b=
r=
0.130
0.957
0.118
0.903
0.217
1.065
0.166
1.003
0.181
0.951
0.159
0.988
0.101
0.989
0.066
0.981
4d
189
Fig. 3. Experimental difference sensitivities Sd (symbols) showing the effect of leaf inltration where leaf absorbance of the inltrated leaf was considered as the reference. Panels a and b
show the Sd spectra for the collimated incident light whereas panels c and d show the Sd spectra for the diffuse incident light (see the left side captions). Panels a and c show the Sd spectra
for adaxial leaf side whereas panels b and d show the Sd spectra for the abaxial leaf sides (see the top captions). Red up- and blue down-triangles show the Sd spectra for face and side,
respectively, chloroplasts position whereas the black lines show the best t of the model (Eq. (19)) to the experimental spectra (see the legend inside the graph). Values of the model
parameters are summarized in Table 1. The range of Sd values is the same in all the panels. The error bars indicate standard deviations (SD) from 3 to 5 measurements and for the case
of clarity they are presented only as either +SD or SD.
Fig. 4. Experimental difference sensitivities Sd (symbols) showing the effect of geometrical character of the illuminating light where leaf absorbance upon collimated light was considered
as the reference. Meaning and description of the rest of the gure is in analogy with Fig. 3.
190
Fig. 5. Experimental difference sensitivities Sd (symbols) showing the effect of leaf side where leaf absorbance of the abaxial leaf side was considered as the reference. Meaning and
description of the rest of the gure is in analogy with Fig. 3.
sensitivity curve making the red and blue minima deeper (the curve is
less at). However, the two parameters partly correlate and so the effects of the curve shift and deeper prole are not fully independent.
Fig. 6. Experimental difference sensitivities Sd (symbols) showing the effect of chloroplast arrangement where leaf absorbance with the chloroplasts in their face positions was considered
as the reference. Meaning and description of the rest of the gure is in analogy with Fig. 3.
It might seem that our presentation (Figs. 3-6) repeats the results
and that the curves (spectra) intuitively thought to be mutually related.
Even if some spectra might look similar, no spectrum is presented twice.
As for mutual relationship of the spectra, we have analyzed this question carefully. The analysis leads to a complex mathematical expression
showing that there is a mutual relation between curves, however it is
very difcult to envisage without the mathematics. For instance a particular Sd() curve can be derived from three other curves in a nonlinear relation.
Because we have explored the effect of chloroplast arrangement for
the natural and inltrated leaf illuminated by the diffuse or collimated
light from adaxial or abaxial leaf sides and because of used denition
of differential sensitivity Sd(), it is easier and more illustrative to describe and discuss rst the effects of leaf inltration (natural versus inltrated leaf), geometrical character of the illuminating light (diffuse
versus collimated illumination), and leaf side (adaxial versus abaxial
leaf side) without discrimination between chloroplast arrangement.
The latter will be described and discussed at the end in a separate part.
4.1. Effect of leaf inltration (natural versus inltrated leaf), the role of
intercellular spaces
The absorbance of the inltrated leaf was taken as the reference
absorbance (see Fig. 3). As can be expected, the inltration of a leaf by
a liquid with the refraction index near to that of cytoplasm would
strongly simplify the optical situation and hence will cause a large difference between D() and DR() consequently leading to a big deviation
of Sd() from a constant value (Fig. 3), resulting in a high absolute values
of the non-linearity (b) and the path lengthening (r). Both the b and r
values (Table 1, row 1) were the largest when comparing the natural
with the inltrated state of the leaf than in other related pairs
(Table 1). The r value was approaching 2 in case of collimated light
and adaxial leaf side (Fig. 3a, Table 1). The non-linearity parameter b
reached also the highest absolute value of about 0.24. If we assume for
a while that the beam path is near to the leaf width of the inltrated
leaf then our results (r approaching 2) are in agreement with those of
McClendon and Fukshansky (1990b) who have found that the beam
path in leaves with a high chlorophyll content is about 2 to 3 times longer that the leaf width. Therefore, this pair may serve for a rough experimental estimation of the beam path length in a natural leaf. We recall
here the meaning of our beam path length; the concept concerns all
rays leaving the leaf (both transmitted and reected).
Interestingly, the lengthening of beam path caused by the air spaces
and represented by the vertical shift of the Sd() curves, is lower when
the abaxial leaf side is illuminated as compared with the adaxial leaf
side both for collimated and diffuse light (compare Fig. 3 right and left
panels and Table 1, row 1). If we take into account the fact that the sensitivity means relative changes in absorbances, the above fact indicates
that some lengthening appears already in the inltrated state being
larger upon illumination of the abaxial leaf side then of the adaxial
leaf side. As it has been shown by Vogelmann and Martin (1993), the
palisade cells enable the beams to penetrate more easily into the leaf
structure. Therefore, it is tempting to expect a shortening of the beam
path when the leaves are illuminated from the adaxial as compared to
the abaxial leaf side. This may be true for the rays of the rst passage
(for explanation see Supplementary data), passing the leaf in transmittance mode and penetrating through the second boundary. However,
our absorbance comprises both rays transmitted by the leaf and those
returning back, which means also the rays of the second and following
passages. As it is shown in Fig. 2 in the near infrared region, the reectance at the adaxial leaf side is in a natural leaf larger than that of abaxial
leaf side; more rays return back at the adaxial leaf side. The fact that
there is a higher chlorophyll concentration at the adaxial side (e.g. Cui,
Vogelmann, & Smith, 1991; Lee et al., 1990) supports the results.
The above comparisons help us to understand the effect of air spaces
in the leaf structure. From the optical point of view, the intercellular air
191
spaces increase about twice the beam path and strongly deepen the
non-linearity of the absorption process.
192
It is of interest to mention that opposite results were found for sunower leaves by Gorton et al. (2010). The difference can be caused by
different architecture of sunower and tobacco leaves.
4.3. Effect of leaf side (adaxial versus abaxial leaf side), the optical polarity
of leaves
Next we have compared the optical properties of different sides of
the bifacial tobacco leaf. A different architecture of the two leaf sides is
shown in Fig. 1. The absorbance of the abaxial leaf side is taken as the
reference one (Fig. 5).
4.3.1. Inltrated leaf
The leaf inltration led to nearly negligible differences between optical parameters of adaxial and abaxial leaf sides (Fig. 5b, d) The b value
is quite near to zero (except the S(D/B)UI value its absolute b value is
0.114) and the r value is near to 1 in collimated light (Table 1, row 3).
This is to be expected as the water inltration and the surface lms of
water strongly enlarge the relative amount of beams transmitted
through the leaf and lowering the surface and internal reection (see
Fig. 2a, blue and red symbols). Then, in terms of the optical law of reversibility of the beam paths, the results obtained from the adaxial
and abaxial leaf side in case of completely homogeneous leaf interior
should not differ. As there remain some differences in the refraction indexes after inltration, a small effect of refraction and scattering may
cause the detected irregularities in the sensitivity curves and even an
asymmetrical passage of rays.
4.3.2. Natural leaf
On the other hand, the natural leaf revealed quite interesting behavior (Fig. 5a, c). It was found that the beam path in the leaf is larger both
for collimated and diffuse illumination (r about 1.2981.432; Table 1)
when the leaf is illuminated from the adaxial leaf side than from the abaxial side. In a possible explanation of this effect, we should take into account again the meaning of the beam path in our concept. As already
mentioned in Section 3.1, our calculation of leaf absorbance covers
both beams transmitted by the leaf and beams returning from the leaf
back to the space of illumination. Although the surface reection Re is
larger from the abaxial leaf side (Hlavinka et al., 2013; McClendon &
Fukshansky, 1990a), we recall here that the near infrared part (no pigment absorption) of the spectra of diffuse reectance and transmittance
show that more light is reected (sum of surface and inner reections)
from the adaxial leaf side than from the abaxial side (see Fig. 2a, green
and gray symbols). Most probably this reection is caused by the
inner reection originating from the interface between the palisade
and spongy parenchyma, the reection being mostly of total reection
character. This effect, together with different leaf structures on its two
sides (Fig. 1) cause that the light reected from the interface must
pass a larger path when reected from the adaxial than from the abaxial
leaf side. There is only a slight increase in the non-linearity when light
comes from the adaxial leaf side (Table 1, row 3).
In conclusion, the light incident from the adaxial leaf side is more effectively absorbed than the same light incident from the abaxial leaf
side.
4.4. Effect of chloroplast arrangement (face versus side
chloroplasts positions)
Until now, we did dot discern between the sensitivities detected for
the face and side arrangements of chloroplasts in cells. These two positions give two curves (spectra) in Figs. 3-5. We will now discuss the differences between these two curves.
4.4.1. Effect of leaf inltration
The effect of chloroplast movement is demonstrated in the relation
of natural versus inltrated leaf (Fig. 3). The fact, that the chloroplast
avoidance movement was induced by blue light illumination of the adaxial leaf side before measurements of related spectra (see Sections 2.7
and 2.8) is reected in a rather different situation of the sensitivity
curves calculated for adaxial and abaxial sides in collimated light
(Fig. 3a, b). Whereas the adaxial leaf side revealed a distinct difference
in the sensitivity for the face and side chloroplast position (Fig. 3a),
the abaxial side showed nearly no difference (Fig. 3b). The side position
in the palisade cells allows the beams in natural leaves to shorten the
paths (Fig. 3a, Table 1) both in reected and transmitted beams when
illuminated from the adaxial leaf side.
Because the beams contributing to the calculated absorbance of the
abaxial leaf side contain a large part of the transmitted rays (see
Fig. 2b, infrared region, blue symbols), these rays change the direction
in the spongy parenchyma and enter the palisades in different directions. The side position of chloroplasts is now not so important and
the sensitivity curves do not differ signicantly.
4.4.2. Effect of geometrical character of the illuminating light
Changes in sensitivities caused by changes in chloroplast positions in
the cells are more pronounced when comparing the effect of the geometrical character of illuminating light in the case of the inltrated leaves
(Fig. 4c, d) than in the natural leaves (Fig. 4a, b) and in the case of adaxial
leaf side (Fig. 4a, c) than in abaxial leaf side (Fig. 4b, d). In the inltrated
leaf representing a more homogeneous space for light beam passage
than in the case of natural leaves, transition of chloroplasts from the
side positions into the face positions distort the ideal situation of the inltrated leaf more signicantly than in the case of the natural leaves.
4.4.3. Effect of leaf side
The effect of chloroplast movement may be demonstrated also when
comparing the sensitivity curves relating the adaxial to the abaxial leaf
side (Fig. 5). In case of the inltrated leaves (Fig. 5b, d), as already mentioned, the effect is very small and may be understood as insignicant
under our experimental conditions. However, in the natural leaves,
some distinct differences are visible. The movement of chloroplasts
from the face to side position lowers the difference in the mean beam
path between the abaxial and adaxial illumination (Fig. 5a). This can
be explained by the fact that the side position enables the perpendicular
collimated light penetrate the palisade cells more directly when illuminated from the adaxial side and shortens the overall light path.
The illumination by diffuse light (Fig. 5c, d) indicates no signicant
changes in the beam path judging from positions of the curves, however
the non-linearity is slightly higher in case of chloroplast side position;
the curves have deeper local minima. We suggest ascribing this nonlinearity increase to the chloroplast grouping effect in the side position.
It should be mentioned that this effect is visible not only for natural leaf
(Fig. 5c) but also in case of inltrated leaf (Fig 5d). This may be understood by a possibility that the diffuse light (Fig. 5d) meets more groups
of chloroplasts than the collimated one (Fig. 5b).
4.4.4. Change in leaf absorbance
For a moment, we can suggest to take the lengthening (parameter r)
as a measure of the increase in the absorption effectiveness. When the
sensitivity Sd() was calculated for comparison of side versus face position of natural leaves in collimated light, the face position being the reference one, the value of about 0.9 (0.903 for illumination from adaxial
leaf side) was obtained (Fig. 6; Table 1, row 4). This means that in this
special case the chloroplast rearrangement from the face to the side position lowers the absorption by about 10%. However, because the nonlinearity parameter b reached absolute values of about 0.12 in this
case, the side arrangement enlarges the non-linearity effect. Table 1,
row 4 shows an important fact that in all cases the side position revealed
a higher nonlinearity than the face position. This supports the notion
that the chloroplast grouping leads also to an increase of the nonlinearity in the light absorption. With some exception in the inltrated
leaves the side position reveals a tendency to shorten the light path in
the leaf (Table 1, row 4, the r parameter is slightly lower than 1).
Generally, our results show (Table 1, row 4) that chloroplast
arrangement mainly effects the non-linearity (mean absolute value
of b parameter being 0.142) whereas the lengthening is almost unaffected (mean value of r parameter being 0.980). Considering our interpretation of the b an r parameters to reect the sieve and detour effects
(see Section 3.5), respectively, then our results agree with those of
Davis et al. (2011) who concluded that chloroplast movement manipulate the sieve but not the detour effect.
4.5. Quantication of the effect of chloroplast arrangement and predictions
for the reectance measurements
Fig. 6, presenting directly the effect of the chloroplast arrangement
on the spectra, shows that maximal absolute values of differential sensitivity, Sd(), are 0.20.3. This means that the leaf absorbance, D(), was
changed by chloroplast arrangement maximally by 2030%. However,
for practical reasons and application in remote sensing measurements,
it is more valuable to relate changes in D() to practically measured
quantity, leaf diffuse reectance RD(). This may also lead to specication of conditions under which the chloroplast rearrangement may be
most reected in the detected leaf diffuse reectance. In the Supplementary data we have estimated that in our case the chloroplast rearrangement can change the leaf reectance by about 2% in the blue and
red regions and by about 5% in the green region of light spectrum. All
the so far mentioned quantications are for our case of fully developed
green leaves.
As it is also shown in the Supplementary data, the highest changes in
the leaf reectance spectrum caused by chloroplast arrangement might
be found in spectral region between 500 and 650 nm and with leaves
having lower content of chlorophylls (young or senescent leaves, leaves
of lower nutrition supply, leaves of plants exposed to long lasting stress,
etc.) and/or leaves with high chloroplasts mobility. The decrease in
chlorophyll content causes a decrease in leaf absorbance, which, according to our derivation in the Supplementary data (equation S63), leads to
more pronounced changes in diffuse reectance. The decrease in chlorophyll content also causes more pronounced changes in leaf absorbance caused by chloroplast movement and thus also more
pronounced changes in diffuse reectance (Eq. S63). In connection to
that, Xiong et al. (2015) reported a more pronounced decrease in
SPAD-values, indicating a more pronounced changes in leaf absorbance,
caused by chloroplast movement for the leaves with a lower chlorophyll
content (caused by a low or no nitrogen supply) than for the leaves with
a higher chlorophyll content. Therefore, more signicant changes in the
leaf reectance caused by chloroplast arrangement than reported in our
case and expected in the other cases are justied. Taking all together, as
reectance signal is used for calculation of many indices in remote sensing, changes in chloroplast arrangement should thus be considered.
5. Conclusion
193
Rc
n12
n 12
A1
A2
A3
A4
Acknowledgments
This work was supported by the grant No. LO1204 (Sustainable Development of Research in the Centre of the Region Han) from the
2
p !2
1 4 cos n2 sin2
p
R F
2
cos n2 sin2
p !2 3
n2 cos n2 sin2 5
p
:
n2 cos n2 sin2
A5
194
The summated and weighted light path for rst passage is equal to:
Z2
sin cos
p1R F d
n2 sin2
X 2 t n
0
Z
2 t n
g 1 F d:
A6
X
xd
2 t n
X0
Z2
g 1 F d 1:098:
A14
We have designated:
sin cos
g 1F 1 F p 1R F
n2 sin2
xc 1Rc t:
Let us for a moment ignore the Fresnel reections, then the sum of
rst weighted light path is:
In summary, using the diffuse light instead of perpendicular collimated light should increase the geometrical beam path in the inltrated
leaf by a factor (assuming leaf n = 1.36).
Z2
X 1 2 t n
0
sin cos d
p :
n2 sin2
A7
A8
Z2
I w 2
sin cos d :
A9
p
X1
2n n n2 1 t:
X0
A10
A11
A12
R1 m1
1m R21
A13
and the beam path may reach higher values due to m being higher
than 1.
A15
xd
1:124:
xc
A16
195
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