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Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034, available FREE online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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3 Present address: Graduate School of Life Sciences, Tohoku University, Aoba, Sendai, 980-8578 Japan.
*Corresponding author: E-mail, itera@biol.s.u-tokyo.ac.jp ; Fax, +81-3-5841-4465 .
The literature and our present examinations indicate that
the intra-leaf light absorption profi le is in most cases
steeper than the photosynthetic capacity profi le. In strong
white light, therefore, the quantum yield of photosynthesis
would be lower in the upper chloroplasts, located near the
illuminated surface, than that in the lower chloroplasts.
Because green light can penetrate further into the leaf
than red or blue light, in strong white light, any additional
green light absorbed by the lower chloroplasts would
increase leaf photosynthesis to a greater extent than
would additional red or blue light. Based on the assessment
of effects of the additional monochromatic light on leaf
photosynthesis, we developed the differential quantum
yield method that quantifi es effi ciency of any
monochromatic light in white light. Application of this
method to sunfl ower leaves clearly showed that, in
moderate to strong white light, green light drove
photosynthesis more effectively than red light. The green
leaf should have a considerable volume of chloroplasts to
accommodate the ineffi cient carboxylation enzyme,
Rubisco, and deliver appropriate light to all the
chloroplasts. By using chlorophylls that absorb green light
weakly, modifying mesophyll structure and adjusting the
Rubisco/chlorophyll ratio, the leaf appears to satisfy two
somewhat confl icting requirements: to increase the
absorptance of photosynthetically active radiation, and to
drive photosynthesis effi ciently in all the chloroplasts. We
also discuss some serious problems that are caused by
neglecting these intra-leaf profi les when estimating whole
leaf electron transport rates and assessing photoinhibition
by fl uorescence techniques.
Keywords: Chlorophyll • Fluorescence • Palisade tissue •
Photoinhibition • Quantum yield • Spongy tissue.
Abbreviations: A , absorbance ; A n , net photosynthetic rate ;
E , excess energy ; F m ( F m
′ ) , maximum fl uorescence in the fully
relaxed state (in the light) ; F
s ′ , steady-state fl uorescence in
the light ; F v ( F v ′ ) , variable fl uorescence in the fully relaxed
state (in the light), F
v = F
m – F
0 ( F
v ′ = F
m
′ – F
0 ′ ) ; F 0 ( F
0 ′ ) , minimum
fl uorescence in the fully relaxed state (in the light) ; Φ , mean
quantum yield of monochromatic light in white light ; φ ,
differential quantum yield of monochromatic light in white
light ; PAM , pulse amplitude modulated ; PPFD , photo-
synthetically active photon fl ux density ; R , r e fl ectance ; RuBP ,
ribulose-1,5-bisphosphate ; T , transmittance.
Introduction
Absorbance spectra of chlorophylls or pigments extracted
from green leaves show that green light is absorbed only
weakly. Action spectra of photosynthesis for thin algal solu-
tions, transparent thalli of ordinary green algae, and leaves of
aquatic angiosperms also show that green light is less effec-
tive than red light. As has been pointed out by Nishio (2000) ,
these facts are often confused, and it is frequently argued
that green light is ineffi cient for photosynthesis in green
leaves. However, many spectra of absorptance (the absolute
value of light absorption) measured with integrating spheres
have shown clearly that ordinary, green leaves of land plants
absorb a substantial fraction of green light ( McCree 1972 ,
Inada 1976 , Gates 1980 ). It is also known that green light,
once absorbed by the leaves, drives photosynthesis with high
Green Light Drives Leaf Photosynthesis More Effi ciently than
Red Light in Strong White Light: Revisiting the Enigmatic
Question of Why Leaves are Green
Ichiro Terashima 1 , * , Takashi Fujita 1 , Takeshi Inoue 1 , Wah Soon Chow 2 and Riichi Oguchi 1 , 2 , 3
1 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033
Japan
2 Photobioenergetics Group, School of Biology, College of Medicine, Biology and Environment, The Australian National University,
Canberra, ACT 0200, Australia
Editor-in-Chief’s choice
684 Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
Special Issue – Mini Review
effi ciency ( Björkmann 1968 , Balegh and Biddulph 1970 ,
McCree 1972 , Inada 1976 ). On an absorbed quantum basis,
the effi ciency or photosynthetic quantum yield of green
light is comparable with that of red light, and greater than
that of blue light. The difference between the quantum
yields of green and blue light is particularly large in woody
plants grown outdoors in high light. The question of how
much green light is absorbed and used in photosynthesis by
the green leaves of land plants has therefore been solved. In
this mini-review, however, we aim at further clarifying
another important role of green light in photosynthesis,
by considering the intra-leaf profi les of light absorption
and photosynthetic capacity of chloroplasts. First, we
briefl y explain light absorption by the leaf. Secondly, we
examine the light environment within the leaf. Thirdly,
we compare the vertical, intra-leaf profi le of photosynthetic
capacity with that of light absorption. We also discuss
some serious problems with the use of pulse amplitude
modulated (PAM) fl uorometry in assessing leaf electron
transport rate and photoinhibition. Fourthly, we propose a
new method to measure the quantum yield of any mono-
chromatic light in white light, and demonstrate the effec-
tiveness of green light in strong white light. Based on these
arguments, we fi nally revisit the enigmatic question of why
leaves are green.
Absorption of light by the leaf
Lambert–Beer's law defi nes absorbance, A , as,
(1)
where I 0 is the intensity of the monochromatic light incident
on the surface of the optical cuvette, and I T is that of the
transmitted light. A is equal to ε cl , where ε is the absorption
coeffi cient of the pigment in the solution (m
2 mol –1 ), c is the
concentration of the pigment (mol m
–3 ) and l is length of
the light path (m). Transmittance, T , is defi ned as I T /I 0 . The
absorbance spectrum is the spectrum of A (or ε ) plotted
against the wavelength of monochromatic light. From Equa-
tion 1, it is obvious that the absorption spectrum does not
express absorptance straightforwardly. As we will see below,
it is dangerous to infer the absorptance of the leaf from the
apparent impression of the absorbance spectrum. Although
A is often called absorption, the common noun of the verb
‘absorb’, we use the term ‘absorbance’ in this article to avoid
confusion between absorption and absorptance.
As an optical system, the leaf differs from a pigment solu-
tion in two aspects: the concentration of pigments into
chloroplasts and the diffusive nature of plant tissues. The
fi rst factor decreases the opportunity for light to encounter
pigments and generally decreases light absorption, and has
been called the sieve or fl attening effect.
Once light that is strongly absorbed by chlorophylls, such
as blue or red, encounters a chloroplast, most of the light
is absorbed. Let us make the drastic assumption that the
chloroplast is a sac containing a solution of chlorophylls at
a concentration of 100 mol m
–3
. This value is chosen because
(i) ordinary green leaves are a few hundred micrometers
thick; (ii) 50–80% of their volume comprises cells; and
(iii) chloroplasts occupy 5–10% of the cell volume. Given
that the values of ε for the mixture of chlorophylls at blue
and red wavelengths are > 1.0×10
4 m 2 mol –1 , and the chloro-
plast thickness is 2 µ m, then A of the chloroplast calculated
using Equation 1 is > 2. In other words, < 1% of the red or
blue light is transmitted through the chloroplast. On the
other hand, for wavelengths that are weakly absorbed, such
as green light, T is considerable. When ε for green light is
assumed to be 500 m
2 mol –1 , A and T would be 0.05 and
79.4%, respectively.
Using a simple model shown in
Fig. 1 , let us consider how
the sieve effect is infl uenced by wavelength. In the left-hand
cuvette, photosynthetic pigments are uniformly distributed,
whereas the right-hand model comprises one half-cuvette
with the pigments concentrated 2-fold and another half-
cuvette containing only the solvent. At wavelengths with
strong absorption, the loss of absorptance by the sieve effect
is large. On the other hand, at wavelengths of weak absorp-
tion such as green, the loss is marginal. The sieve effect,
therefore, strongly decreases absorptance at wavelengths of
strong absorption such as red and blue light. Because of this,
absorption spectra with strong sieve effects show fl attened
absorption peaks; hence the alternative term ‘fl attening
effect’.
Fig. 1 Model explaining the sieve effect on absorptance. Left: a cuvette
containing a pigment solution. Right: the pigment is concentrated in a
half-cuvette, while another half-cuvette contains only the solvent.
When the cuvette is uniformly irradiated with a strongly absorbed
monochromatic light, the decrease in absorptance by the sieve effect
was large (above), while in the case of weakly absorbed monochromatic
light the decrease in absorptance is small (below).
685
Why are leaves green?
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
The second point that distinguishes leaves from a simple
pigment solution is that leaf tissues are diffusive. This is due
to the fact that the leaf consists of cells and intercellular air
spaces. The refractive index, which depends on both the
material and wavelength of the light, of the bulk plant cells is
around 1.48, compared with 1.33 for water and 1.0 for air.
The diffusive nature of leaf tissues increases the light path
length (détour effect) and thereby the opportunity for light
to encounter chloroplasts, leading to the increase in absorp-
tance ( Vogelmann 1993 ). On the other hand, the diffusive
nature of the leaf tissues inevitably increases the refl ectance,
R , of the leaf to some extent. Leaves appear to minimize R of
the adaxial side by having a greater contact area between
the adaxial epidermis and palisade tissue cells per unit leaf
surface area than that between the abaxial epidermis and
spongy tissue cells. In some species, palisade tissue cells are
funnel-shaped, which further increases the contact area with
the epidermis ( Haberlandt 1914 ). By reducing the chances of
refraction at the interfaces between cells and air, R decreases
to a considerable extent (compare the differences in R
between the adaxial and abaxial sides).
The increase in absorptance due to light diffusion (détour
effect) is signifi cant in the spongy tissues in bifacial leaves
whose abaxial surfaces are paler than their adaxial surfaces
( Terashima and Saeki 1983 , Vogelmann 1993 ). In such leaves,
spongy tissues have cell surfaces facing various directions
and fewer chloroplasts (or chlorophyll) per unit mesophyll
volume. In leaves of Camellia japonica , a typical example,
lengthening of the optical path is more marked in the spongy
tissue than in the palisade tissue ( Terashima and Saeki 1983 ).
On the other hand, in spinach, where the difference in the
chlorophyll content per unit mesophyll volume between the
palisade and spongy tissues use is small, the optical path
length does not differ much between the tissues ( Vogelmann
and Evans 2002 ).
The consequence of lengthening the optical path can be
shown using the same model (
Fig. 2 ). In this model, the path
length increases by 3-fold (see Vogelmann 1993 ). At strongly
absorbed wavelengths, the increase in absorptance achieved
by lengthening the light path is 11% (while the increase in A
is, of course, 3-fold). In contrast, for weakly absorbed wave-
lengths such as green light, the increase in absorptance is
much greater.
In summary, for strongly absorbed light such as red or
blue, the sieve effect decreases absorptance considerably,
whereas the détour effect increases absorptance marginally.
On the other hand, for green light, loss in the effi ciency of
absorptance by the sieve effect is small, while gain in absorp-
tance by the détour effect is large. Consequently, green
leaves absorb much green light. Typical values of absorp-
tance at 550 nm range from 50% in Lactuca sativa (lettuce)
to 90% in evergreen broad-leaved trees ( Inada 1976 ). The
corresponding absorptance values for blue and red lights
range from 80 to 95%. Moreover, as already mentioned
above, it has been clearly shown that the quantum yield of
photosynthesis based on absorbed photosynthetically active
photon fl ux density (PPFD), measured at low PPFDs, was
comparable between green and red light. When measured in
leaves grown under natural conditions, particularly for those
of trees, the quantum yield of green light is considerably
greater than that of blue light ( Inada 1976 ), because some
fraction of blue light is absorbed by fl avonoids in vacuoles
and/or carotenoids in chloroplast envelopes. Moreover,
some carotenoids in thylakoid membranes do not transfer
energy to reaction centers, or transfer with an effi ciency sig-
nifi cantly less than 1.0 ( Akimoto and Mimuro 2005 ). For
example, one of the most abundant carotenoids in thyla-
koids, lutein, transfers its energy to chlorophyll with an effi -
ciency of 0.7 ( Akimoto et al. 2005 ). The effi ciency for
neoxanthin is even less, at most 0.09 ( Akimoto et al. 2005 ).
Accumulation of fl avonoids and carotenoids is well known
to increase in response to ultraviolet and/or strong light
( Lambers et al. 2008 ). This probably explains to a considerable
extent why the quantum yield of blue light is low.
Evans and Anderson (1987) reconstructed the absor-
bance spectrum of thylakoid membranes from those of the
chlorophyll–protein complexes and estimated the relative
excitation of PSII and PSI. Evans (1987) argued that
imbalance of PSII/PSI excitation would occur at wave-
lengths where light is absorbed by Chl b because energy is
preferentially transferred to PSII. This might also explain
why the quantum yield of blue light on an absorbed quan-
tum basis is low. If this effect is large, a decrease in the PSII
quantum yield (Genty's parameter, see below) might be
expected at wavelengths strongly absorbed by Chl b . In a
preliminary study with rice leaf discs illuminated with
monochoromatic lights at a low PPFD of 5–12 µ mol m –2 s –1 ,
Fig. 2 Model explaining the détour effect on absorptance. Left: no
détour effect. Right: the light path is lengthened 3-fold by the détour
effect. For strongly absorbed monochromatic light, the increase in
absorptance by the détour effect is small (above), while for weakly
absorbed light the increase is marked (below).
686
I. Terashima et al.
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
we observed small reduction of the PSII reaction center
[decreased ( F m
′ – F s ′ )/ F m
′ mainly due to the decrease in photo-
chemical quenching] in two wavelength regions with peaks
at 470 and 650 nm, respectively, implying overexcitation of
PSII at these wavelengths. However, the decreases observed
were not enough to account for the large decrease in the
quantum yield of blue light.
Light environment within the leaf
Although there were some classical works, the light environ-
ment within the leaf was fi rst intensively studied in the early
1980s. The micro fi beroptic method is the most effi cient in
measuring the fl ux of light within a leaf ( Vogelmann et al.
1991 , Vogelmann 1993 ). Because the viewing angle of the
optical fi ber is narrow, the angular distribution of the light
fl ux, including backward scattering, is measured by inserting
the fi ber into the leaf from various directions. On the other
hand, it is not possible to measure the absorption profi le
using this method alone. Paradermal sectioning, i.e. section-
ing of the leaves parallel to the leaf epidermis, is suitable for
examining the optical properties of leaf tissues ( Terashima
and Saeki 1983 ). This sectioning method is also used to mea-
sure the profi les of photosynthetic properties within the leaf
( Terashima and Hikosaka 1995 ). Sectioning after exposure of
the leaf to
14 CO 2 has been used to reveal the photosynthetic
profi le in vivo across the leaf (for a review, see Nishio 2000 ).
Fukshansky and his colleagues applied the Kubelka–Munk
theory to predict the light environment within the leaf and
to characterize the optical properties of leaf tissues ( Richter
and Fukshansky 1996a , Richter and Fukshansky 1996b ).
Fluorescence techniques have also been used. Takahashi
et al. (1994) devised a method to illuminate a leaf segment
normal to its epidermis and measure fl uorescence from the
transversely cut surface of the segment in order to analyze
the light absorption gradient.
To analyze the light environment within the leaf in rela-
tion to photosynthesis, it is necessary to know the light
absorption profi le, not the light fl uxes per se. This is because
only those photons absorbed by pigments can work photo-
chemically (the law of photochemistry, see Clayton 1970 ).
There are no straightforward methods to measure the light
absorption profi le, but the method of Takahashi et al. (1994)
originally developed for rice leaves has been successfully
used for estimation of the light absorption profi les for leaves
of Rhizophora mucronata and C. japonica ( Koizumi et al.
1998 ), Spinacia oleracea ( Vogelmann and Evans 2002 ,
Vogelmann and Evans 2003) and Eucalyptus paucifl ora
( Evans and Vogelmann 2006 ).
Here, applying the Kubelka–Munk theory to the trans-
mittance and refl ectance data from paradermal leaf sections
( Terashima and Saeki 1983 ), we have reconstructed the light
environment and absorption profi le of a C. japonica L. leaf.
The method of Allen and Richardson (1968) was used to fi t
the data ( Gates 1980 ). For a leaf containing Chl a + b at
C mol m
–2 , the downward fl ux ( I ) and the upward fl ux ( J ) are
considered at the plane parallel to the irradiated leaf surface.
Let the cumulative Chl a + b from the surface to the plane be
c mol m
–2 . Then, introducing the absorption parameter,
k , and the scattering parameter, s , we obtain the following
set of differential equations:
(2)
Boundary conditions are
(3)
where R L is the refl ectance of the leaf, T L is the transmittance
of the leaf and R 0 is the refl ectance of the lower surface of the
leaf when light is applied from inside the leaf ( Gates 1980 ).
For the fi tting, we used data from paradermal sections of
C. japonica leaves with upper epidermes, and refl ectance
data from paradermal sections with lower epidermes
( Terashima and Saeki 1983 ). Through fi tting the data, k and
s for the palisade tissue and spongy tissues for 680 and
550 nm were obtained, respectively; 680 nm is the red
absorption peak of chlorophyll a in vivo, while 550 nm is
green light at which leaves show maximal T L and R L .
Fig. 3 shows fi tting of the Kubelka–Munk model to the
sections. The adopted k and s suffi ciently describe the opti-
cal properties of these leaf tissues.
Fig. 4 shows the down-
ward fl ux, I , the ratio of the upward fl ux to the downward
fl ux, J/I , the sum of both fl uxes, I + J , and absorption, k ( I + J ),
calculated for a model C. japonica leaf. The calculated values
showed abrupt changes at the interface between the pali-
sade and spongy tissues. This is because k and s for these
tissues differed. Both k and s for the spongy tissue were much
greater than those for the palisade tissue (see legend of
Fig. 3 ),
refl ecting the enhancement of absorption by the détour
effect and diffusive nature of the spongy tissue. If s were zero
or there were no scattering, the differential equations would
degenerate into the Lambert–Beer's law and k /2.3 would be
equal to ε (cf. log 10 e = 2.3). The value of k for the palisade
tissue at 680 nm, 10
4 , corresponds to ε of 4,300 m
2 mol –1 ,
which is about half the value for the red peak of chlorophyll
in the organic solvent. On the other hand, for 550 nm,
k /2.3 was 1,500, much greater than ε in the green region
(<500 m 2 mol –1 for the solution of chlorophylls) even for the
palisade tissue. These absolute values are somewhat greater
than those obtained for blue light (2,600–2,900 m
2 mol –1 )
and green light (1,000–1,300 m
2 mol –1 ) in spinach leaves
( Vogelmann and Evans 2002 ).
Our absorptance data agree well with a previous calculation
of the light absorption gradient, which was based purely on the
experimental data ( Terashima and Saeki 1985 ). When compared
687
Why are leaves green?
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
on a unit chlorophyll basis, the chloroplasts in the lower-
most part of the leaf absorb <10% of those in the uppermost
part, even at a wavelength of 550 nm at which the absorp-
tion gradient is most moderate. For spinach, various estima-
tions have been published. Using the method of Takahashi
et al. (1994) , Vogelmann and Evans (2002) and Evans and
Vogelmann (2003) indicated that, on a unit chlorophyll
basis, the chloroplasts in the lowermost part absorb about
10 and <20%, respectively, of the green light of those in the
uppermost part. For wavelengths with strong absorption,
such as red and blue, the fractions are much smaller. In
C. japonica , the absorption of 680 nm (red) light by the low-
ermost chloroplasts is <2% of the absorption by the upper-
most chloroplasts on a unit chlorophyll basis. For blue light
in spinach, the estimated absorption by the lowermost
chloroplasts was <5% of that of the uppermost ( Vogelmann
and Evans 2002 , Evans and Vogelmann 2003 ).
Comparison of the profi les of light absorption and
photosynthetic capacity
The profi les of photosynthetic capacity along the gradient of
light absorption have been reported for Spinacia oleracea
( Terashima and Hikosaka 1995 , Nishio 2000 , Evans and
Vogelmann 2003 ) and E. paucifl ora ( Evans and Vogelmann
2006 ). The differences in photosynthetic properties found
between the chloroplasts in the upper and lower parts of the
leaf are essentially identical to those found between sun and
shade leaves, or between sun and shade plants ( Terashima
and Hikosaka, 1995 ). Thus, the formation of an intra-leaf
profi le of photosynthetic capacity can be regarded as an
acclimation process ( Terashima et al. 2005 ). It is also worth
mentioning that we verifi ed acclimation of light sensitivity
of stomatal opening to the intra-leaf light environment with
Helianthus annuus leaves: stomata in the abaxial epidermis,
which are located in a light environment enriched in green
light, open in response to monochromatic green light,
whereas those in the adaxial epidermis do not ( Wang et al.
2008 ).
Based on observations of the differences in the shape of
light response curves depending on the direction of irradia-
tion, Oja and Laisk (1976) predicted the existence of an
intra-leaf gradient in photosynthetic capacity. The profi le in
photosynthetic capacity and the differentiation of optical
properties between palisade and spongy tissues are adaptive,
Fig. 3 Fitting of the Kubelka–Munk theory to the transmittance and refl ectance data of the paradermal sections of leaves of Camellia japonica .
Left: attenuance = –log
10 T of paradermal sections with adaxial epidermes. Although attenuance can be defi ned by the same mathematical
equation that defi nes absorbance ( A ), attenuance is used when the decrease in T due to R is substantial. The monochromatic light at 680 or
550 nm was applied to the adaxial epidermis. Right: refl ectance of the paradermal sections having the abaxial epidermes. Monochromatic light
was irradiated from the cut surface. Refl ectance was measured with an integrating sphere. Different symbols indicate different leaves. The
refl ectance of the abaxial epidermis was assumed to be 0.25, for both 680 and 550 nm. The boundary between the palisade and spongy tissues was
assumed to be at 0.37 mmol chlorophyll m
–2 (note the infl ections of the curves). k 680 and s 680 for the palisade tissue were 10,000 and 900, and k 680
and s 680 for the spongy tissue were 13,400 and 2,500, respectively. k 550 and s 550 for the palisade tissue were 3,400 and 1,100, and k 550 and s 550 for the
spongy tissue were 5,300 and 3,500, respectively. For the unit of these numbers, see the text. The data of transmittance and refl ectance were
adopted from Terashima and Saeki (1983) .
3
2
1
Attenuance (= –log T )
0
0 0.1 0.2 0.3
Cumulative chlorophyll a + b
(
mmol m–2 from the adaxial
)
0.4 0.5 0.6
30
20
10
Reflectance (%)
0
0 0.1 0.2 0.3
Cumulative chlorophyll a + b
(
mmol m–2 from the adaxial
)
0.4 0.5 0.
6
688
I. Terashima et al.
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
because these features improve the effi ciencies of both light
use and nitrogen use in photosynthesis ( Terashima and Saeki
1985 , Farquhar 1989 , Terashima and Hikosaka 1995 ). The
most effi cient situation is realized when the profi le of light
absorption and the profi le of photosynthetic capacity are
perfectly matched, and all the chloroplasts in the leaf behave
synchronously with respect to photosynthetic light satura-
tion ( Farquhar 1989 , Terashima and Hikosaka 1995 , Richter
and Fukshansky 1998 ).
Fig. 5 shows the gradients of Rubisco
content per Chl, and electron transport rate from water to
dichlorophenol indophenol per Chl. The gradients for elec-
tron transport rate were steeper than those for the Rubisco.
There appears to be a gradient in the balance between
ribulose-1,5-bisphosphate (RuBP) carboxylation and RuBP
regeneration capacities. Although the infl uence of light level
on this balance has not been studied intensively (see Evans
and Vogelmann 2003 ), it may be an important subject,
particularly with respect to photoinhibition (see below). The
gradients show that even chloroplasts in the lowermost part
of the leaf exhibit 20–40% of the maximal photosynthetic
capacity of the uppermost chloroplasts. Given that the pro-
fi le of photosynthetic capacity may be similar to that of light
absorption when bifacial spinach leaves are irradiated from
the adaxial side with green monochromatic light ( Evans and
Vogelmann 2003 ), and that T L values of ordinary green leaves
were at most 10–15% for PPFD ( McCree 1972 , Inada 1973),
the gradients of photosynthetic capacity are in most cases
more gradual than those of light absorption in situ.
Fig. 5 Intra-leaf profi les of photosynthetic capacities in leaves of
Spinacia oleracea . Green triangles, Rubisco content/Chl ( Terashima
and Inoue 1985 ); blue circles, Rubisco content/Chl ( Nishio et al. 1993 );
red squares, dichloroindophenol reduction rate/Chl ( Terashima and
Inoue 1985 ); and orange triangles, dichloroindophenol reduction rate/
Chl ( Terashima 1989 ). Quadratic equations were fi tted to the data.
Fig. 4 Light environment and light absorption profile within a
Camellia japonica leaf predicted by the Kubelka–Munk theory. The k
and s values fi tted to the data (Fig. 3) were used. I , downward fl ux;
J , upward fl ux; and k ·( I + J ), absorptance per unit chlorophyll (relative
value). The calculated results for 680 and 550 nm are shown together
with the mean values of 680 and 550 nm values (in gray). Unpublished
results of I. Terashima.
1
0.8
550 nm
550 nm
550 nm
680 nm
680 nm
680 nm
I
I+J
k(I+ J)
J/I
0.6
Downward flux (left axis)
Upward flux/ Downward flux (%, right axis)Downward flux + Upward flux, I+JLight absorption (= k(I+J), relative)
0.4
0.2
0
1.0
0.8
0.6
0.4
0.2
0
1.0
0.8
0.6
0.4
0.2
0
0 0.1 0.2 0.3
Cumulative chlorophyll a + b (mmol m–2)
0.4 0.5 0.6
20
10
0
689
Why are leaves green?
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
The underlying reason(s) for the more gradual gradient of
photosynthetic capacity have not been clarifi ed. It is likely,
however, that it is too costly for a given plant to be prepared
to acclimate to a very wide range of light environment. In
other words, in any given species, there is a limit to the
dynamic range of acclimational adjustment of chloroplast
properties to the light environment.
Detection of discrepancy of the profi les of light
absorption and photosynthetic capacity
When leaves are irradiated from the upper side, therefore,
there will be a situation in which the upper chloroplasts are
light saturated while the chloroplasts in the lower parts still
need additional light to reach saturation. In other words, the
quantum yield of photosynthesis differs within the leaf,
being less in the uppermost part. This discrepancy has been
detected by comparing the electron transport rates esti-
mated from the gas-exchange technique and from Genty's
parameter ( Tsuyama et al. 2003 ; for Genty's parameter, see
below). For upright or pendulous leaves, it has long been
known that the sharpest light response curves are obtained
when these leaves are irradiated equally from both sides
( Moss 1964 , Tanaka and Matsushima 1970 , Evans et al. 1993 ).
Thus, unilateral illumination should cause a considerable
discrepancy between the profi le of light absorption and the
profi le of photosynthetic capacity.
A more straightforward method to detect such a differ-
ence in light saturation would be to monitor fl uorescence
from both sides of the leaf, in order to assess the PSII quan-
tum yields or Genty's parameters for each side ( Genty et al.
1989 ).
After formulation by Genty et al. (1989) , the linear elec-
tron transport rate from water to NADP
+ for the whole leaf
has been frequently estimated as:
(4)
where α is the absorptance of the leaf, ϕ PSII is the fraction of
excitation energy allocated to PSII, F m
′ is the maximal fl uores-
cence in the light, and F s
′ is the fl uorescence level in the pres-
ence of actinic light. The last term, ( F m
′ – F s
′ )/ F m
′ , expresses the
quantum yield of PSII in actinic light and is often called
Genty's parameter.
Marked differences in Genty's parameter have already
been shown, for example for thick ( ∼ 300 µ m) horizontal
leaves of Eucalyptus maculata ( Evans et al. 1993 ). When the
adaxial surface was irradiated with strong white light,
( F m
′ – F s ′ )/ F m
′ estimated from fl uorescence signals from the
abaxial side was markedly greater than that estimated for the
adaxial side.
Fig. 6 shows an example for a relatively thin leaf
from a shade-grown Alocasia odora plant. At PPFDs
> 600 µ mol m –2 s –1 , ( F m
′ – F s
′ )/ F m
′ obtained from the upper side
was lower than that obtained from the lower side. These dif-
ferences are clear proofs of the discrepancies between the light
absorption profi le and that of the photosynthetic capacity.
Fig. 6 Light response curves of the rate of photosynthesis ( A n ) and quantum yield of PSII in the light in a leaf of a shade-grown Alocasia odora
plant. A n was measured with a portable gas-exchange system (LI-6400, Li-Cor, Lincoln, NE, USA) at a cuvette temperature of 25°C in air containing
380 µ l l –1 CO 2 . The leaf was sandwiched with two half-chambers that had transparent windows. White light from a metal-halide lamp was
provided via an optical fi ber with a rectangular light emitter. The fl uorescence signals were measured from the adaxial and abaxial sides with two
PAM fl uorometers (PAM 101/102/103, Walz, Effl tliche, Germany). The optical fi ber connected to one of the fl uorometers was placed in the
central space of the rectangular emitter for the actinic light. The other probe was placed below the lower half-chamber. Unpublished results of
T. Inoue.
4
3
2
Photosynthetic rate (mmol CO2 m–2 s–1)
1
0
0.8
0.6
(Fm
′–F
s
′) / Fm
′
0.4
0.2
0
0 200 400 600
abaxial side
adaxial side
PPFD (mmol m–2 s–1)
800 1000 1200
690
I. Terashima et al.
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Because ( F m
′ – F s
′ )/ F m
′ would not be uniform within the
leaf, indiscriminate use of the adaxial fl uorescence signals to
represent the whole leaf is very problematical.
Use of the adaxial fl uorescence signals in
photoinhibition studies
The fl uorescence method has been used in studies of photo-
inhibition for the past three decades. When PSII is damaged
by strong light, ( F m – F 0 )/ F m decreases ( Powles 1984 ). There
are two main hypotheses for the mechanism of photoinhibi-
tion: the excess energy hypothesis ( Weis and Lechtenberg
1989 , Osmond 1994 , Demmig-Adams et al. 1996 , Kato et al.
2003 ) and the two-step hypothesis ( Hakala et al. 2005 , Ohnishi
et al. 2005 , Nishiyama et al. 2006 ). The energy of photons
absorbed by PSII pigments is dissipated as either heat or
fl uorescence in PSII antennae, F 0
′ / F m
′ = 1 – F v
′ / F m
′ , drives pho-
tosynthesis, ( F m
′ – F s
′ )/ F m
′ (Genty's parameter), or migrates
to closed PSII reaction centers and is dissipated non-
photochemically, ( F s
′ – F 0 ′ )/ F m
′ (excess):
(5)
( Stefanov and Terashima 2008 ). Excess energy, E , can be
calculated as:
(6)
The excess hypothesis claims that the excess energy, the
energy migrated to closed PSII centers, is responsible for
photoinhibition. According to this hypothesis, photoinhibi-
tion would not occur when PSII reaction centers are open.
On the other hand, the two-step hypothesis claims that the
manganese cluster in the oxygen-evolving complexes is the
primary site of damage. The action spectrum for manganese
damage indicates that the effect is strongest in the UV region
and is progressively weaker in blue, green then red light
( Hakala et al. 2005 , Ohnishi et al. 2005 ). Once the oxygen-
evolving complex is damaged, damage to the PSII reaction
center occurs subsequently. These two hypotheses are
not necessarily mutually exclusive, and it is probable that
both mechanisms are important in nature (R. Oguchi and
W. S. Chow in preparation).
In the context of the topic of this review, it is important
to note that photoinhibitory damage does not occur uni-
formly throughout the leaf, irrespective of the mechanisms .
In the paradigm of the excess hypothesis, if ( F s ′ – F 0 ′ )/ F m
′ does
not differ much across the leaf, the extent of E should depend
on the depth within the leaf and the wavelength of the light,
as is apparent from Equation 6. For the two-step hypothesis,
if the fi rst step is irreversible, the damage to the manganese
cluster should depend on the absolute number of photons
(UV and blue) absorbed by chloroplasts. Therefore, the
damage to the manganese cluster should also differ depend-
ing both on the depth within the leaf and the wavelength.
In reality, ( F s ′ – F 0
′ )/ F m
′ would differ considerably across
the leaf [as has been argued above for ( F m
′ – F s
′ )/ F m
′ ; see also
Fig. 6 ], and the damage to the manganese cluster appears to
be reversible. These imply that photoinhibitory damage
should be greater in the chloroplasts near the irradiated
surfaces.
In the PAM system from Walz (Effeltrich, Germany), the
measuring beam is red light with an intensity peak around
650 nm. When the beam is irradiated from one side of the
leaf, the beam is absorbed mainly in the shallow part of the
mesophyll, and thereby fl uorescence emitted from chloro-
plasts near the irradiated surface is preferentially detected.
As expected from the argument above, large differences in
F v / F m between the leaf surfaces have been reported for leaves
of H. annuus that were unilaterally irradiated with strong
light ( Evans et al. 1993 ). When leaves of Capsicum annuum
were photoinhibited by irradiation to the upper side, the
fl uorescence signals from the upper side indicated consider-
able decreases in F v / F m , whereas F v / F m calculated from the
fl uorescence signals obtained from the abaxial side hardly
decreased (R. Oguchi and W. S. Chow in preparation).
To analyze photoinhibition within the leaf, the pulse-
modulated fi beroptic fl uorometer can be used. Schreiber
et al. (1996) devised this system (Microfi ber PAM) and dem-
onstrated several applications including the measurement
of the profi les of F v /F m within leaves of Syringa vulgaris . When
the leaf was irradiated from the adaxial side with strong
white light, the authors observed the lowest F v /F m in the
uppermost part of the mesophyll, and the value increased
with depth. Using C. annuum leaves, we also confi rmed the
same trend (
Fig. 7 , left). We further analyzed effects on the
photoinhibition profi le of treatments with broad-band red,
green and blue lights at the same PPFD (
Fig. 7 , right). Because
the manganese cluster would show strong absorption in UV
and blue regions, and the absorption by the manganese clus-
ter should decrease markedly with the increase in wave-
length, we might expect that the strongest photoinhibition
would occur with blue light, followed by green and then red
light. On the other hand, absorption of photons by the
chloroplasts near the irradiated surface should be greatest
when the leaf is irradiated by blue light, followed by red light,
and it should be lowest in green light. The results shown in
Fig. 7B may be explained by these two effects. The greatest
decrease in F v /F m in the uppermost part of the leaf was
observed with blue light, and F v /F m approached high levels at
depth. The second greatest damage to the surface chloro-
plasts was observed with red light, but the damage was con-
fi ned to the irradiated half of the leaf. On the other hand,
damage to the surface chloroplasts was least with green
light, but continued deep into the leaf, probably because
691
Why are leaves green?
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
suffi cient green light penetrated and was absorbed by the
chloroplasts in the abaxial side.
The Microfi ber PAM system uses photomultipliers to
detect fl uorescence. Although the system is operated in the
pulse amplitude modulation mode, strong background light
interferes with the measurements. It could be possible
to follow the recovery of fl uorescence yield after rapidly
turning off the actinic light by extrapolation . We would then
be able to estimate PSII quantum yield in the light and
thereby the profi le of photosynthetic capacity. However, we
are still awaiting suitable modifi cation of the system to allow
such an experiment.
As has been explained above, it is dangerous to assume
that the fl uorescence signals obtained from the irradiated
side of a leaf represent the quantum yield of the chloroplasts
within the whole leaf. In particular, when the chloroplasts
near the irradiated surface are photoinhibited, the mislead-
ing effect would be very large. It also causes some artifact in
estimating mesophyll conductance (or internal conduc-
tance), the conductance from the intercellular spaces to
chloroplast stroma for CO
2 diffusion, because the combined
gas-exchange and fl uorometry method for estimating this
conductance assumes that the quantum yield of PSII is uni-
form in the chloroplasts in the whole leaf. Because this prob-
lem is beyond the scope of this review, readers should refer
to appropriate papers (see Evans 2009 ).
In contrast to chlorophyll fl uorescence, radiation at
810 nm is hardly absorbed by chlorophyll, so measurements
based on an absorbance change at 810 nm associated with
the oxidation of P700 (the special chlorophyll pair in PSI)
monitor all P700 rather equally at all depths of a leaf. Using
the 810 nm signal to determine the relative content of PSII
after photoinhibition, Losciale et al. (2008) found a single
relationship that applied to leaves of diverse anatomy.
In situ quantum yield of monochromatic light
in white light
As Nishio (2000) clearly postulated, and as we have detailed
so far, red or blue light is preferentially absorbed by the chlo-
roplasts in the upper part of the leaf. Then, when PPFD is
high, the energy of these wavelengths tends to be dissipated
as heat by the upper chloroplasts, while green light drives
photosynthesis in the lower chloroplasts that are not light
saturated ( Sun et al. 1998 , Nishio 2000 ). However, there has
been no quantitative evaluation of this possibility. Here, we
propose a new method to quantify the quantum yield of
monochromatic light contained in white light.
Theory
We can measure the differential quantum yield of the mono-
chromatic light in any background white light at a PPFD of
I , φ λ
( I ), as in Fig. 8 . Initially, a leaf is illuminated with the
Fig. 7 Intra-leal profi les of F v / F m in leaves of Capsicum annuum . Leaf discs of C. annuum were treated with lincomycin, an inhibitor of protein
synthesis, and subsequently photoinhibited at 2,000 µ mol photon m
–2 s –1 at room temperature for 60 min by white light, broad-band blue (400–
500 nm), green (500–600 nm) or red (600–700 nm, right). A (left), results with white light; B (right), results with broad-band monochromatic
lights. A Microfi ber PAM (Walz, Effelt, Germany) having an optical microfi ber of 30 µ m diameter was used to measure intra-leaf profi les of F v / F m .
The micro optical fi ber was inserted into the leaf tissues with the aid of a three-dimensional water-pressure micromanipulator (WR-60, Narishige,
Tokyo, Japan). Unpublished data of R. Oguchi.
0.8 Control
White light
AB
0.6
0.4
PSII quantum yield (Fv/Fm)
0.2
00 100 200
Depth from the adaxial surface (mm)
300 0 100
Red
Green
Blue
200
Depth from the adaxial surface (mm)
300
692
I. Terashima et al.
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
white light at I . Then, weak monochromatic light at the
wavelength of λ (d I ) is added to the background white light.
The increment of the photosynthetic rate ( A n ), d A n , divided
by d I , d A n / d I , is defi ned as φ λ
( I ).
Let us then defi ne the mean quantum yield of monochro-
matic light at the wavelength of λ contained in the white
light at I as Φ λ
( I ). When the fraction of the PPFD of the
monochromatic light at λ to that of the whole white light is
r λ
, then r
λ · I · Φ λ
( I ) is the photosynthesis driven by the mono-
chromatic light at λ when the PPFD of the white light is I .
At a PPFD of I + ∆ I , the photosynthesis driven by the mono-
chromatic light at λ is r λ
· ( I + ∆ I ) · Φ λ
( I + ∆ I ), which can be
written as:
(7)
Rearrangement of this equation leads to:
(8)
Then, integrating φ λ
( I ) with respect to I , one obtains:
(9)
Thus, it is possible to estimate Φ λ
( I ), the mean quantum
yield of any monochromatic light in the white light.
Because the Chl a / b ratio and the carotenoids/chlorophyll
ratio are usually higher in sun-type chloroplasts, the quan-
tum yield of photosynthesis driven by blue light would differ
between sun and shade chloroplasts ( Lichtenthaler and Balari
2004 ). Thus, in this study, we compared the effects of green
light at 550 nm and red light at 668 nm. Judging from the
action spectra of green leaves ( McCree 1972 , Inada 1976 ), the
red light at 668 nm used in this study would not cause the
marked red-drop effect. Moreover, the measurements were
conducted in the presence of the background white light.
We obtained φ red and φ green in sunfl ower leaves irradi-ated
from the adaxial side and abaxial side, respectively. Typical
light response curves obtained by irradiating from the adax-
ial and abaxial sides of the same sunfl ower leaf are shown in
Fig. 9 . The different curves depending on the direction of
irradiation were reported for several species ( Moss 1964 , Oja
and Laisk 1976 , Terashima 1986 , Ögren and Evans 1993 ), and
the difference between the curves can be explained by the
profi le of photosynthetic capacity and the difference in opti-
cal properties between the palisade and spongy tissues.
When the leaf is irradiated from the lower side, light is pref-
erentially absorbed by the spongy tissue. Then, PPFDs have
to be increased to very high levels to deliver suffi cient light
energy to the upper chloroplasts for their light saturation
( Oja and Laisk 1976 , Terashima 1986 , Ögren and Evans 1993 ,
Terashima and Hikosaka 1995 , Sun and Nishio 2001 ).
When a sunfl ower leaf was irradiated from the adaxial
side, φ red was at fi rst greater than φ green ( Fig. 10 ). This can be
attributed to the difference in absorptance between these
two wavelengths. With the increase in PPFD of the white
light, both φ decreased but, as expected, the decrease
was more marked in φ red than in φ green . When the leaf was
irradiated from the abaxial side, φ red was greater only at the
lowest PPFD, and green light was more effective in higher
Fig. 8 The differential quantum yield measurement. In the presence
of white light at PPFD of I , weak monochromatic light (d I ) is given. The
ratio of increment of the photosynthetic rate (d A n ) to PPFD of the
monochromatic light (d I ), d A n /d I , gives the differential quantum yield
φ ( I ). When I = 0, the differential quantum yield is the same as the
ordinary quantum yield on an incident PPFD basis.
Fig. 9 Light response curves of the rate of net photosynthesis ( A n ) in a
leaf of Helianthus annuus obtained with irradiation to the adaxial or
abaxial side. The rate of photosynthesis was measured with a portable
gas-exchange system (LI-6400, Li-Cor), at a leaf temperature of 25°C in
air containing 390 µ l l –1 CO 2 . Vapor pressure defi cit was <0.7 kPa.
The light from a halogen lamp in a slide projector was delivered by a
tri-furcated optical fi ber. Helianthus annuus plants were grown at
350 µ mol m –2 s –1 at canopy height at 23°C with a photoperiod of 12 h.
Unpublished data of T. Fujita.
693
Why are leaves green?
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
PPFDs. We also noted that both φ red and φ green decreased
more abruptly than when irradiated from the adaxial side.
When the mean quantum yields, Φ , were compared in the
experiment in which the adaxial side was irradiated, Φ red was
greater at low PPFDs, but at PPFDs above approximately
450 µ mol m –2 s –1 , Φ green became greater than Φ red ( Fig. 11 ).
When irradiated from the abaxial side, Φ red was greater than
Φ green only at very low PPFDs. We initially expected that the
φ and Φ at very low PPFDs would be greater when the leaf
was irradiated from the adaxial side compared with the
abaxial side because, in ordinary bifacial leaves, R is smaller
and leaf absorptance is greater when irradiated from the
adaxial side. However, φ and Φ values at very low PPFD were
greater when the leaf was irradiated from the abaxial side.
In these measurements, we maintained the CO
2 concentra-
tion in the leaf cuvette at 390 µ mol mol –1 , and, as reported
by Wang et al. (2008) , the stomatal conductance at low
PPFDs was greater when the leaf was irradiated from the
abaxial side than when irradiated from the adaxial side (data
not shown). The difference in stomatal conductance caused
considerable differences in CO
2 concentration in the inter-
cellular spaces, which explains the present results. The data
shown in
Figs. 10 and 11 clearly demonstrate that green
light more effectively drove photosynthesis than red light in
the white light at high PPFDs.
If we analyze the changes in the PPFD and spectrum
of daylight with time of day in the natural environment,
it would be possible to compare the effi ciencies of green and
Fig. 10 Differential quantum yields for red and green monochromatic light in a leaf of Helianthus annuus . Left: data obtained by irradiation to
the adaxial side. Right: data obtained by irradiation to the abaxial side. The white light source was a halogen lamp delivered through a tri-furcated
optical fi ber. Red or green monochromatic light was obtained by passing the light from a halogen lamp through optical fi lters and delivered
through another tri-furcated optical fi ber. A hexagonal holder made of Plexiglas was used to hold six optical fi bers to secure uniform and constant
irradiation. The peak of the green monochromatic light was 550 nm with the half-band width of ±30 nm. The red light had a maximum at 668 nm.
Half the maximum transmittance occurred at 641 and 690 nm. The PPFDs of the white light were 0, 40, 100, 150, 200, 450, 700, 950 and 1,200 µ mol
photons m
–2 s –1 . The light response curves obtained by irradiation with the white light are shown in Fig. 9. The PPFD of the monochromatic light
was either 50 µ mol m –2 s –1 (for the white light at 0, 40, 100 and 150 µ mol m –2 s –1 ) or 150 µ mol m –2 s –1 (for the white light at 200, 450, 700, 950 and
1,200 µ mol m –2 s –1 ). The differential quantum yield is plotted against the PPFD (PPFD of the white light + half the PPFD of the monochromatic
light). The data were obtained with the same leaf of H. annuus that was used for Fig. 9. Once A n attained a stable value in the white light at the
ambient CO
2 concentration ( C a ) at 390 nm, then monochromatic light was added, keeping the intercellular CO
2 concentration ( C i ) constant by
manipulating C a . Therefore, C i was constant for three measurements, in the white light, the white light + red monochromatic light and the white
light + green monochromatic light. Quadratic or quadruple equations were fitted to the data. φ green,adax ( I ≤ 180 µ mol
m
–2 s –1 ) = –1.0938·10 –7 I 2 + 1.7422·10 –5 I + 3.5598·10 –2 ( r 2 = 0.803); φ green,adax ( I ≥ 180 µ mol m –2 s –1 ) = 1.1978·10 –8 I 2 – 4.5847·10
–5 I + 4.3115·10 –2 ( r 2 = 0.999);
φ red,adax ( I ≤ 180 µ mol m –2 s –1 ) = –3.8099·10
–8 I 2 – 6.3947·10
–6 I + 4.0115·10 –2 ( r 2 = 0.980); φ red,adax ( I ≥ 180 µ mol m –2 s –1 ) = 4.1802·10
–14 I 4 – 1.4411·10
–
10 I 3 + 1.9467·10
–7 I 2 + 1.264·10
–10 I + 5.7508·10
–2 ( r 2 = 0.999); φ green,abax ( I ≤ 280 µ mol m –2 s –1 ) = –1.0914·10
–8 I 2 – 6.1311·10
–5 I + 3.9941·10
–2 ( r 2 = 0.968);
φ green,abax ( I ≥ 280 µ mol m –2 s –1 ) = 1.8263·10
–8 I 2 – 4.4363·10
–5 I + 3.2675·10
–2 ( r 2 = 0.990); φ red,abax ( I ≤ 280 µ mol m –2 s –1 ) = 2.0084·10
–7 I 2 – 1.556·10
–
4 I + 4.4579·10 –2 ( r 2 = 0.985), and φ red,abax ( I ≥ 280 µ mol m –2 s –1 ) = 2.0539·10 –8 I 2 – 4.6917·10
–5 I + 2.8126·10 –2 ( r 2 = 0.988) Unpublished data of T. Fujita
and I. Terashima.
0.05
irradiation from
the adaxial side
irradiation from
the abaxial side
Differential quantum yield
(mol CO2 mol–1 photon)
0.04
0.03
0.02
0.01
0
fred
fred
fgreen fgreen
PPFD (mmol m–2 S–1)
0 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 1400
694
I. Terashima et al.
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other monochromatic lights in an ecological context. The
method enables us to measure in situ quantum yield and
opens the way to obtaining ecologically meaningful action
spectra. Further studies are, of course, awaited.
Although the light absorption profi les calculated by
Nishio (2000) are spurious ( Vogelmann and Evans 2002 ), his
argument has nevertheless been proven experimentally to
be correct using our differential quantum yield method.
Namely, red light is more effective than green light in white
light at low PPFDs, but as PPFD increases, light energy
absorbed by the uppermost chloroplasts tends to be dissi-
pated as heat, while penetrating green light increases photo-
synthesis by exciting chloroplasts located deep in the
mesophyll. Thus, for leaves, it could be adaptive to use chlo-
rophylls as photosynthetic pigments, because, by having
chlorophyll with a ‘green window’ the leaves are able to
maintain high quantum yields for the whole leaf in both
weak and strong light conditions.
Some green algae such as Codium fragile and Ulva pertusa ,
inhabiting the deepest part of the green algae zonation,
appear very black, because they contain a keto-carotenoid,
siphonaxanthin, which absorbs green light with a peak at
535 nm and transfers energy to chlorophylls with an effi -
ciency of 1.0 ( Kageyama et al. 1977 , Akimoto et al. 2004 ,
Akimoto et al. 2007 ). Because the peak of available PPFD
shifts toward blue wavelengths with depth of sea-water, it
has been argued that siphonaxanthin is a useful carotenoid to
absorb green light. If leaves of land plants had black chloro-
plasts with siphonaxanthin, the leaves could close the
so-called ‘green window’ and increase their absorptance.
If the carboxylation enzyme, Rubisco, were very effi cient,
land plants would indeed be able to have thin black leaves.
However, having the ineffi cient Rubisco as their primary car-
boxylation enzyme, leaves receiving high light need consid-
erable chloroplast volumes to contain it ( Terashima et al.
2005 , Terashima et al. 2006 ). Moreover, to supply CO
2 effi -
ciently to the chloroplasts, the leaf also needs a large cumu-
lative cell surface area per leaf area, so the chloroplasts must
be distributed throughout the leaf ( Terashima et al. 2001 ,
Terashima et al. 2005 , Terashima et al. 2006 ). Given these
constraints, it would be ideal to have chlorophyll that enables
considerable light absorptance, due to the high absorptivity
of blue and red light, but also penetration of green light to
the lower chloroplasts. As Nishio (2000) argued, this may
explain why land plants adopted Chl a and b from green
algae but did not develop other pigment systems.
If a gradient in the ratio of Rubisco to photosynthetic pig-
ments freely changes in response to PPFD, leaves could exist
with black chloroplasts containing both chlorophylls and
siphonaxanthin. When light absorption is plotted against
the cumulative black pigment content for such leaves, the
gradient would be very steep, because absorption coeffi -
cients would now be high for green as well as blue and red
light. In the upper chloroplasts, the ratio of Rubisco to black
pigments would then need to be very large but to decrease
drastically with depth. Noting that the dynamic range of accli-
mational modifi cation of chloroplast properties is limited
within a given species, it would be impossible to counterbal-
ance the profi le of light absorption by drastically changing the
Rubisco/black pigment ratio. It is, therefore, worth mention-
ing again that, by having chlorophylls with a ‘green window’
to the most abundant photosynthetically active wavelengths
of solar radiation, green leaves have succeeded in moderating
the intra-leaf light gradient to a considerable extent.
Funding
The Japan Society for Promotion of Science (JSPS, grant
No. 16207002); University of Tokyo; JSPS research fellowship
for young scientists (to R.O.).
Acknowledgements
We thank the Namoto Trading Co. for the loan of a Microfi -
ber PAM system, Professor Y. Oka and Dr. H. Abe of our
Department, Graduate School of Science, The Univeristy of
Tokyo, for the loan of a micromanipulator and kindly adjust-
ing it for the Microfi ber PAM system. We are also grateful to
Mr. S. Otsuka and Mr. Y. Nanjo of the Workshop, Graduate
School of Science, The University of Tokyo, for skillfully man-
ufacturing various parts for the present optical system. We
also thank Professor Mamoru Mimuro, Hall of Environmental
Research, Kyoto University, for his recent articles on energy
transfer from carotenoids to chlorophyll and his kind advice,
and Dr. J. R. Evans for his constructive and provocative
Fig. 11 Calculated mean quantum yield of monochromatic light in
white light, Φ ( I ). The values were obtained by integration of each of
the curves shown in Fig. 10 with respect to I from 0 to I . Unpublished
results of T. Fujita and I. Terashima.
695
Why are leaves green?
Plant Cell Physiol. 50(4): 684–697 (2009) doi:10.1093/pcp/pcp034 © The Author 2009.
comments and editing of the text. We are grateful to Profes-
sor Amane Makino, the editor, and another anonymous
reviewer for encouraging comments.
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(Received January 4, 2009; Accepted February 24, 2009)
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