Tree Physiology 27, 849–857
© 2007 Heron Publishing—Victoria, Canada
Leaf morphological and physiological adjustments to the spectrally
selective shade imposed by anthocyanins in Prunus cerasifera
A. KYPARISSIS,1 G. GRAMMATIKOPOULOS 2 and Y. MANETAS 2,3
1
Laboratory of Botany, Department of Biological Applications and Technology, University of Ioannina, 45110, Ioannina, Greece
2
Laboratory of Plant Physiology, Department of Biology, University of Patras, 26500 Patras, Greece
3
Corresponding author ([email protected])
Received May 16, 2006; accepted June 13, 2006; published online March 1, 2007
Summary Prunus domestica L. has green leaves, whereas
Prunus cerasifera Ehrh. var. atropurpurea has red leaves due
to the presence of mesophyll anthocyanins. We compared morphological and photosynthetic characteristics of leaves of these
species, which were sampled from shoots grafted in pairs on
P. domestica rootstocks, each pair comprising one shoot of
each species. Two hypotheses were tested: (1) anthocyanins
protect red leaves against photoinhibition; and (2) red leaves
display shade characteristics because of light attenuation by
anthocyanins. Parameters were measured seasonally, during a
period of increasing water stress, which caused a similar drop
in shoot water potential in each species. As judged by predawn
measurements of maximum PSII yield, chronic photoinhibition did not develop in either species and, despite the anthocyanic screen, the red leaves of P. cerasifera displayed lower
light-adapted PSII yields and higher non-photochemical
quenching than the green leaves of P. domestica. Thus, it appears that, in this system, anthocyanins afford little photoprotection.
As predicted by the shade acclimation hypothesis, red
leaves were thinner and had a lower stomatal frequency, areabased CO2 assimilation rate, apparent carboxylation efficiency
and chlorophyll a:b ratio than green leaves. However, red
leaves were similar to green leaves in conductivity to water vapor diffusion, dry-mass-based chlorophyll concentrations and
carotenoid:chlorophyll ratios. The data for red leaves indicate
adaptations to a green-depleted, red-enriched shade, rather
than a neutral or canopy-like shade. Thus, green light attenuation by anthocyanins may impose a limitation on leaf thickness. Moreover, the selective depletion of light at wavelengths
that are preferentially absorbed by PSII and chlorophyll b may
lead to adjustments in chlorophyll pigment ratios to compensate for the uneven spectral distribution of internal light. The
apparent photosynthetic cost associated with lost photons and
reduced leaf thickness, and the absence of a photoprotective
advantage, suggest that there are other, yet to be identified,
benefits for permanently anthocyanic leaves of P. cerasifera.
Keywords: chlorophyll fluorescence, CO2 assimilation, internal light environment, non-photochemical quenching, photoprotection, pigments, PSII yield, red leaves.
Introduction
Leaves in some plants appear red because of the accumulation
of anthocyanins that mask the green chlorophyll reflectance.
Although in some species the anthocyanic character is permanent, in most cases reddening is transient. Transient redness
may be developmentally or environmentally determined. In
the former case, redness appears in young or senescing leaves,
or both, of some species. Environmentally determined redness
occurs when anthocyanins accumulate in mature leaves as a
response to wounding (Stone et al. 2001), pathogen attack
(Hipskind et al. 1996), nutrient deficiencies (Hodges and
Nozzolillo 1996, Kumar and Sharma 1999), UV-B radiation
(Lindoo and Caldwell 1978, Mendez et al. 1999) or high light
in combination with low temperatures (Krol et al. 1995, Close
and Beadle 2003, Pietrini et al. 2002).
Because anthocyanins absorb light, they should restrict photosynthesis. Although this appears to be disadvantageous to a
healthy leaf under optimal conditions, it may be advantageous
under stressful conditions during specific phases of leaf development. Because the abiotic stresses that induce the synthesis
of anthocyanins predispose leaves to photoinhibition of photosynthesis (Long and Humphries 1994), it is reasonable to assume that anthocyanins have a photoprotective function, either
as passive light filters (Krol et al. 1995, Manetas et al. 2002,
Pietrini et al. 2002) or as antioxidants (Gould et al. 2002a,
Steyn et al. 2002). Additional hypotheses on the role of anthocyanins include an osmotic effect and a protective function
against UV-B radiation or herbivory (for recent reviews on
possible functions of leaf anthocyanins see Chalker-Scott
1999, Gould et al. 2002b, Steyn et al. 2002, Close and Beadle
2003 and Manetas 2006).
Regardless of function, anthocyanin accumulation may impose a double cost on the leaf: the cost of carbon skeletons diverted to pigment biosynthesis; and the photosynthetic cost resulting from competition between anthocyanins and chlorophylls for light capture. In addition, the shade imposed on
mesophyll chloroplasts by anthocyanins could alter their function and composition. For example, it has been reported that
red leaves have lower maximum CO2 assimilation rates
(Gould et al. 2002c), lower chl a:b ratios and a smaller pool of
850
KYPARISSIS, GRAMMATIKOPOULOS AND MANETAS
xanthophyll cycle components than green leaves (Manetas et
al. 2003). These characteristics form part of a large array of
traits that constitute the shade acclimation syndrome (Anderson 1986, Lambers et al. 1998). However, the magnitude of
imposed shade in anthocyanic leaves cannot be easily defined.
Based on an indirect method, Pietrini and Massacci (1998)
calculated that anthocyanins in red leaves may reduce the
photosynthetically active radiation reaching the chloroplasts
by as much as 40%. Gould et al. (2002c), who studied chlorophyll fluorescence profiles in red leaf sections, concluded that
it is mainly green light that is absorbed by anthocyanins, rendering it available for photosynthesis only in the uppermost
cell layers. Thus, there are considerable quantitative and qualitative differences between natural canopy shade and light attenuation by anthocyanic leaves. Understory leaves are subjected to photon fluence rates at least an order of magnitude
lower than those to which sun leaves are exposed, with only
brief exposure to direct sunlight due to sunflecks. Moreover,
natural shade is green and far-red enriched, whereas the mesophyll of a red leaf is exposed to light severely depleted in the
green portion of the visible spectrum (Karabourniotis et al.
1999, Gould et al. 2002c).
With this background, we undertook a comparative study of
Prunus domestica, possessing green leaves, and Prunus cerasifera, possessing red leaves. These species form compatible
grafts producing chimeric trees bearing green- and red-leaved
shoots on the same rootstock. We measured an array of morphological and photosynthetic parameters, during the Mediterranean summer, to investigate whether the presence of
anthocyanins: (1) reduces the photoinhibitory risk during the
seasonal increase in water stress; and (2) is related to leaf
structural and photosynthetic traits.
Materials and methods
Plant material and experimental design
Three specimens of Prunus cerasifera Ehrh. (var. atropurpurea) grafted on Prunus domestica L. rootstock (both Rosaceae) growing as ornamentals at the Patras University campus were used. Grafting was performed at the base of young
P. domestica rootstocks about 10 years before the present
study, producing 2-m-tall two-stemmed trees, one stem bearing green leaves (P. domestica), the other stem bearing red
(P. cerasifera) leaves. Both stems are winter deciduous. Developing leaves of the atropurpurea variety are bright red and mature leaves are dark red, with redness being apparent on both
sides of the leaf. All measurements were made on mature
leaves. Free-hand leaf cross sections viewed with the aid of a
microscope revealed that anthocyanins were located in the
mesophyll cells. Sampling was performed at 1-month intervals. During this period the plants received only natural precipitation. The canopies of both stems were relatively open
and not mutually shaded. All measurements were made on
clear days. Gas exchange and related parameters, as well as
chlorophyll fluorescence measurements in the light-adapted
state, were performed at various times during the day, on intact
leaves in the field, in natural light. Dark-adapted chlorophyll
fluorescence parameters were obtained on intact leaves at predawn. For destructive measurements (shoot water potential,
leaf thickness, leaf mass per area and leaf pigment analyses),
samples were collected at midday, placed in sealed plastic
bags and taken to the laboratory for immediate analysis.
Measurements
To determine shoot water potential (Ψ), shoots were recut under water with a razor blade and Ψ measured within 2–3 min
with a SKPM 1400 (Skye Instruments, Ltd., Llandrindod
Wells, U.K.) pressure chamber.
The thickness of veinless leaf areas was measured with a
friction-stop caliper (Mitutoyo, Japan). Leaf discs of known
areas were subsequently cut, dried in an oven at 80 °C and
weighed to determine dry mass. Leaf mass per area (LMA)
was then calculated.
Stomatal frequencies were determined by viewing freehand paradermal sections with the aid of an Axioplan microscope (Zeiss, Oberkochen, Germany).
Leaf pigments were extracted by grinding leaf tissue with a
mortar and pestle with pure methanol, a small amount of
washed sand and about 0.05 g of CaCO3. The extract was centrifuged and the chlorophylls and carotenoids in the supernatant determined spectrophotometrically based on the equations of Lichtenthaler and Wellburn (1983). Anthocyanins
were assessed by their peak absorbance (λ = 529 nm) after
acidification of the medium and correction for pheophytin
absorbance according to Lindoo and Caldwell (1978).
Leaf gas exchange was measured with a portable photosynthesis system (LCpro+, Analytical Development Company,
Hoddestdon, U.K.) functioning in open flow mode. Attached
leaves of various inclinations (i.e., different incident PPFs)
were measured in the field between 0900 and 1500 h, permitting the construction of net photosynthesis (An), stomatal conductance (gs) and internal CO2 concentration (Ci) versus incident PPF curves. Apparent carboxylation efficiency was calculated as An /Ci, based on measurements made at saturating
PPF (> 1400 µmol m – 2 s – 1).
Chlorophyll fluorescence in the field was measured in
leaves similar to those sampled for gas exchange with a portable pulse-amplitude-modulated fluorometer (MINI-PAM,
Walz, Effeltich, Germany) connected to a leaf clip (Leaf-Clip
Holder 2030-B). The PSII photochemical efficiency in the
light-adapted state was calculated according to Genty et al.
(1989) as:
∆F/Fm′ = (Fm′ – F)/Fm′
where F is the initial fluorescence yield of a light-adapted
sample before the application of a saturating light pulse, and
Fm′ is the maximum fluorescence yield of a light-adapted sample during the application of a saturating light pulse. Maximum (dark-adapted) PSII photochemical efficiency (Fv /Fm =
(Fm – Fo )/Fm )) was measured at predawn, where Fo is the initial fluorescence yield of a dark-adapted sample before the application of a saturating light pulse, and Fm is the maximum
fluorescence yield of a dark-adapted sample during the appli-
TREE PHYSIOLOGY VOLUME 27, 2007
LEAF ADJUSTMENTS TO A PERMANENT ANTHOCYANIC SHADE
cation of a saturating light pulse. Non-photochemical quenching (NPQ) was calculated from predawn Fm and light-adapted
Fm′ according to the formula (Maxwell and Johnson 2000):
NPQ = (Fm – Fm′)/Fm′
During the photosynthetic measurements (both gas analysis
and fluorometry), leaf temperatures were monitored by thermocouples.
Leaf optical properties were measured with an Optronic
(Orlando, FL) spectroradiometer equipped with a Taylor-type
integrating sphere and a stabilized quartz halogen source. For
reflectance (R), the leaves covered the exit port of the sphere.
For transmittance (T ), the leaves covered the entrance port.
Absorptance (A) was calculated as A = 1 – R – T. A Spectralon
standard (reflectance > 0.97) was used for calibration.
Statistics
Significance of differences in the measured parameters between green and red leaves was tested by a two-tailed MannWhitney test. For best fit lines, the PSII yield versus PPF and
the Ci versus PPF curves were treated as double exponential
decays, the An versus PPF and the gs versus PPF as hyperbolas
and the NPQ versus PPF as straight lines. An F-test assessing
the null hypothesis for one curve fitting both green and red
datasets was used. All statistical analyses were performed with
SPSS 9.0 statistical software (SPSS Inc., Chicago, IL).
Results
The green leaves of P. domestica and the red leaves of P. cerasifera had characteristic optical properties. The characteristic
green peak in reflectance and transmittance and the corresponding valley in absorptance of green leaves were absent in
red leaves. The spectral profiles in the red band were similar in
both leaf types (data not shown).
Red leaves were thinner and had lower LMA than green
leaves (Table 1). Stomata were observed only on lower leaf
surfaces, and their frequency was lower in red leaves than in
green leaves (Table 1). Compared with green leaves, red
leaves had reduced area-based chlorophyll and total carotenoid concentrations (Table 2). However, there were no differences between leaf types when pigment concentrations were
expressed on a dry mass basis (Table 2). The chlorophyll a:b
ratio was considerably lower in red leaves than in green leaves,
whereas the carotenoid:chlorophyll ratio was similar in red
and green leaves. As expected, anthocyanin concentrations
were much higher in red leaves than in green leaves (1.74 versus 0.06 absorbance units, normalized for 1 cm3 of extract
from 1 cm2 of leaf surface). Absorbance spectra of extracted
anthocyanins displayed a peak in the green band (529 nm) and
negligible absorbance in the red band of the spectrum (above
about 620 nm, results not shown).
Table 3 shows that, although shoot water potential declined
from early to late summer, the values for both red and green
stems were similar, except in early August, when red shoots
had a lower Ψ.
851
Table 1. Thickness, leaf mass per area (LMA) and stomatal frequency
of green and red leaves. The thickness, LMA and stomatal frequency
data are means ± SD of 60, 60 and 20 leaves, respectively, with 4–5
measurements per leaf. All differences were significant (P < 0.0001).
Because no differences between sampling dates were observed, results from all dates were pooled.
Green
Red
Leaf thickness
(mm)
LMA
(g dm – 2 )
Stomatal frequency
(no. mm – 2 )
0.245 ± 0.022
0.191 ± 0.027
0.921 ± 0.128
0.685 ± 0.064
141 ± 19
95 ± 14
Maximum (predawn) PSII photochemical efficiency was always within the range for healthy leaves (about 0.8), indicating that water stress did not cause chronic photoinhibition in
either red or green leaves. Compared with green leaves,
slightly, but significantly, lower values (2–3%) of Fv /Fm were
observed in red leaves towards the end of the dry summer (Table 4).
Measurements of the effective PSII yield made during the
day on various dates and plotted against incident PPF are
shown in Figures 1A–D. Datasets of both leaf types followed
the same curve in early June and early July. Later on, however,
the effective PSII yield of red leaves progressively declined.
We expected higher ∆F/Fm′ values in red leaves than in green
leaves because part of the incident light is lost to anthocyanin
absorption. Non-photochemical quenching was similar for
green and red leaves in early June (Figures 1E–H). With progressive water stress, NPQ increased gradually in both leaf
types. The increase, however, was more pronounced in red
leaves and the best fit lines for green and red leaves departed
considerably in late summer.
Net photosynthesis (An) was higher in green leaves than in
red leaves at all PPFs (Figures 2A–D). Yet, stomatal conductance was slightly higher in green leaves only in early August
(Figures 2E–H), probably because of the higher Ψ of greenleaved shoots observed on this date, although transpiration
rates were similar to those of red leaves (data not shown). Accordingly, the observed differences in An were not due to
stomatal limitations. As a result, internal CO2 concentrations
(Ci) were always higher in red leaves than in green leaves (Figures 2I–L). The combination of lower An and higher Ci in red
leaves resulted in a large reduction in apparent carboxylation
efficiency (Table 5). When An was expressed on a dry mass basis, the differences between green and red leaves were smaller
(Table 5). No temperature differences between the leaf types
were found during the experiment.
Discussion
Photoinhibitory risk and photoprotection
Both green and red shoots experienced progressive water
stress during the study, and the magnitude of stress, based on
midday shoot water potentials, was similar (Table 3). In addi-
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
852
KYPARISSIS, GRAMMATIKOPOULOS AND MANETAS
Table 2. Photosynthetic pigments expressed per leaf area or leaf dry mass and their ratios in green and red leaves. Data are means ± SD of eight independent measurements, with three leaves per measurement. Abbreviation: Chl = chlorophyll. Within a column, different letters indicate significant differences between green and red leaves (P < 0.05). Because no differences were observed between sampling dates, results from all dates
were pooled.
Green
Red
Chl a+b
(µg cm – 2 )
Chl a+b
(mg g –1 )
Total carotenoids
43.62 ± 4.23 a
31.58 ± 2.47 b
4.736 ± 0.440 a
4.610 ± 0.288 a
µg cm
–2
7.65 ± 0.63 a
5.83 ± 0.58 b
tion, leaf temperatures were similar in red and green leaves.
Hence, an interpretation of our results based on a differential
water or heat stress can be excluded.
Many native plants respond to the long Mediterranean summer by closing stomata, reducing photosynthetic and electron
transport rates and engaging photoprotective measures that,
nevertheless, may be insufficient to prevent chronic photoinhibition (Karavatas and Manetas 1999, Bombelli and Gratani
2003). Our test plants displayed progressive reductions in PSII
effective yield and gs, and increases in NPQ (Figures 1 and 2);
however, their photoprotective capacity was not exceeded, as
indicated by the absence of both a plateau in NPQ development (Figure 1) and photoinhibitory damage (Table 4), probably because the imposed stress was insufficient to drive the
photoprotective potential of our test plants to their limits.
Thus, our results fail to show whether anthocyanins can reduce
photoinhibition in P. cerasifera. Krol et al. (1995), Feild et al.
(2001), Manetas et al. (2002) and Pietrini et al. (2002) found a
positive correlation between anthocyanins and high light tolerance (but see Burger and Edwards (1996) for a different conclusion). However, no evidence that anthocyanins reduce the
risk of photoinhibition was obtained in field and greenhouse
experiments (Woodall and Stewart 1998, Dodd et al. 1998,
Lee et al. 2003, Karageorgou and Manetas 2006).
We predicted that, because part of the incident PPF is attenuated by anthocyanins and not used photosynthetically, a red
leaf will maintain a higher PSII yield and lower NPQ for a
given incident photon fluence rate of white light than a green
leaf. This was found in genotypes of corn responding to cold
stress by accumulating anthocyanin (Pietrini et al. 2002). In
our test system, however, PSII effective yield was lower and
NPQ was higher at the end of the summer in red leaves than in
Table 3. Midday water potentials of green- and red-leaved shoots on
the indicated dates. Data are means ± SD of 4–5 shoots. Within a row,
different letters indicate significant differences (P < 0.05).
Date
June 5
July 1
August 1
September 2
Midday shoot Ψ (MPa)
mg g
0.831 ± 0.066 a
0.851 ± 0.073 a
Red leaves
–1.77 ± 0.15 a
–2.09 ± 0.14 a
–2.24 ± 0.11 a
–2.48 ± 0.08 a
–1.74 ± 0.07 a
–2.08 ± 0.25 a
–2.60 ± 0.05 b
–2.46 ± 0.06 a
Total carotenoids:Chla+b
3.18 ± 0.53 a
2.47 ± 0.21 b
0.18 ± 0.02 a
0.18 ± 0.01 a
green leaves, whereas similar values were obtained earlier in
the season for both leaf types (Figure 1). We conclude, therefore, that red leaves of P. cerasifera, despite their anthocyanic
screen, require a higher photoprotective potential than those of
the green-leaved P. domestica.
Properties of red leaves are partly compatible with the
classical shade acclimation syndrome
Some of our results indicated shade adaptation in red leaves
(see reviews by Anderson 1986, Lambers et al. 1998 and
Lichtenthaler et al. 2000). For example, red leaves of P. cerasifera were thinner and had a lower LMA, stomatal frequency,
chl a:b ratio and area-based net photosynthetic rate, and a considerably reduced apparent carboxylation efficiency compared
with green leaves (Tables 1, 2 and 5 and Figure 2). It is generally accepted that a thick leaf is less well adapted to shade because the attenuation of light as it passes through a thick leaf
may result in the irradiance impinging on some mesophyll
cells falling below the light compensation point. The need to
increase the probability of light capture in shade leaves results
in an increased ratio of light harvesting antennae per reaction
center, and, in turn, to lower chl a:b ratios. The redistribution
of tasks between light capture and electron flow leads to low
enzymatic activities in the reducing pentose phosphate cycle,
with the result that shade-adapted leaves display low areabased CO2 assimilation rates and low apparent carboxylation
efficiencies.
Although these components of the shade acclimation syndrome were found in red leaves, others were absent. For exam-
Table 4. Maximum dark-adapted PSII photochemical efficiency
(Fv /Fm) obtained from attached green and red leaves at predawn. Data
are means ± SD of 40–50 leaves for each date. Within a row, different
letters indicate significant differences between green and red leaves
(P < 0.05).
Date
Green leaves
Chl a:b
–1
June 5
July 1
August 1
September 2
TREE PHYSIOLOGY VOLUME 27, 2007
Predawn Fv /Fm
Green leaves
Red leaves
0.816 ± 0.007 a
0.834 ± 0.008 a
0.818 ± 0.016 a
0.819 ± 0.019 a
0.812 ± 0.007 a
0.831 ± 0.007 a
0.794 ± 0.032 b
0.803 ± 0.018 b
LEAF ADJUSTMENTS TO A PERMANENT ANTHOCYANIC SHADE
853
Figure 1. The PSII effective yield (∆F / Fm′; A–D) and non-photochemical quenching (NPQ; E–H) of green (䊊) and red (䊉) attached leaves plotted against incident photosynthetic photon flux (PPF). Measurements were performed between 0800 and 1200 h on leaves of various inclinations
and exposures.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
854
KYPARISSIS, GRAMMATIKOPOULOS AND MANETAS
Figure 2. Net photosynthetic rate (An; A–D), stomatal conductance (gs; E–H) and internal CO2 concentration (Ci; I–L) of green (䊊) and red (䊉)
attached leaves plotted against incident photosynthetic photon flux (PPF). Measurements were performed between 0900 and 1500 h on leaves of
various inclinations and exposures.
ple, shade leaves usually have similar area-based, but higher
dry-mass-based, total chlorophyll concentrations compared
with sun leaves, whereas the red leaves we studied had a total
chlorophyll concentration that was about 25% lower on a leaf
area basis, but similar on a dry mass basis, to that of green
leaves (Table 2). Because shade leaves require little photo-
TREE PHYSIOLOGY VOLUME 27, 2007
LEAF ADJUSTMENTS TO A PERMANENT ANTHOCYANIC SHADE
855
Table 5. Net photosynthetic rates expressed per unit leaf dry mass (An) and apparent carboxylation efficiency (A/Ci ) for measurements made at a
saturating photosynthetic photon flux (PPF > 1400 µmol m – 2 s – 1 ) in green and red leaves. Data are means ± SD of 10–20 leaves. For An, different
letters within a row indicate significant differences between green and red leaves (P < 0.05). For A/Ci, all differences are significant (P < 0.0013).
Date
June 5
July 1
August 1
September 2
An (µmol g –1 s –1 )
A/Ci (mol m – 2 s – 1 )
Green leaves
Red leaves
Green leaves
Red leaves
0.166 ± 0.039 a
0.113 ± 0.020 a
0.171 ± 0.060 a
0.095 ± 0.030 a
0.128 ± 0.033 b
0.087 ± 0.029 b
0.132 ± 0.062 a
0.093 ± 0.024 a
0.090 ± 0.035
0.045 ± 0.009
0.086 ± 0.044
0.048 ± 0.016
0.037 ± 0.012
0.023 ± 0.008
0.041 ± 0.021
0.030 ± 0.007
protection, their carotenoid:chla+b ratios are lower than those
of sun leaves, whereas the ratios were similar in the leaf types
we studied (Table 2). Furthermore, the lower area-based CO2
assimilation rates observed in shade leaves are fully compensated by the differences in LMA, so that sun- and shadeadapted leaves display similar An on a dry mass basis. In our
case, however, only part of the difference in An between leaf
types was explained by a difference in dry mass per unit leaf
area (Table 5). Finally, shade leaves display lower gs than sun
leaves, a difference we did not observe between red and green
leaves (Figure 2).
Effects of red-enriched, green-depleted shade
To explain the observed differences between the effects of
anthocyanin pigmentation and those attributed to the shade acclimation syndrome, we considered the kind of shade imposed
on the mesophyll of an anthocyanic leaf. Red anthocyanins absorb mainly in the green and less in the blue and yellow parts
of the spectrum, and they are transparent in the red and near infrared region; i.e., anthocyanins essentially eliminate the green
window of chlorophyll absorption. Anthocyanins thus appear
to provide only limited protection against photoinhibition,
whereas an effective sunscreen would be expected to match
the absorption spectrum of the target photodynamic chlorophyll molecules (Manetas 2006). Green light, with a lower
probability of absorption by chlorophyll, penetrates deeper
into the leaf and drives photosynthesis at a distance from the
exposed surface, whereas red light is absorbed in the more superficial layers, exhibiting a steep gradient across the leaf
(Gould et al. 2002c).
Nishio (2000), seeking an adaptive significance for the
green color of chlorophyll, argued that the green chlorophyll
window allows the leaf to acquire a mechanically acceptable
thickness, with successively deeper layers effectively using
different spectral bands for photosynthesis. We further argue
that green-light-absorbing anthocyanins impose a ceiling on
leaf thickness, making the development of additional layers
unproductive. This produces not a “shade leaf,” but a thinner
leaf, the missing layers being that part of a green leaf possessing more “shade” characteristics, i.e., its lower part. Gradients
in shade acclimation characteristics have been reported in successive paradermal leaf slices (Terashima and Inoue 1984).
This interpretation may explain the absence of differences be-
tween green and red leaves in dry-mass-based chla+b concentrations and carotenoid:chla+b ratios, and the lower
dry-mass-based CO2 assimilation rates. However, the presence
of green and yellow absorbing anthocyanins may disturb the
relative proportion of photons available for use by PSII and
PSI. Green and yellow light is better absorbed by PSII, whereas red and dark-red light is better absorbed by PSI (Melis et al.
1985). There are reports that growth of plants in red-enriched
and green- and yellow-depleted light results in a changed
photosystem stoichiometry by increasing the PSII:PSI ratio
(Chow et al. 1990). This adjustment is believed to be an acclimation to light quality to compensate for an uneven absorption
of light by PSI and PSII. As a result, the chl a:b ratio is decreased (Chow et al. 1990), as was the case in the anthocyanic
leaves we studied.
Our interpretation of a light-quality-dependent adjustment
of morphology and photosynthesis in red leaves is also corroborated by the work of Brown et al. (1995) and Yu and Ong
(2003), who found that acyanic (i.e., green-leaved) plants
grown in red light displayed lower CO2 assimilation rates,
electron transport rates and leaf mass per area than plants
grown in white light, whereas stomatal conductance and dry
mass-based chlorophyll concentrations were unaffected by the
light quality.
In conclusion, field evidence from predawn and daytime
chlorophyll fluorescence parameters did not allow confirmation of a photoprotective advantage of anthocyanic leaf pigmentation, even after prolonged water stress. Red leaves developed a higher intrinsic capacity for non-photochemical
quenching, despite their anthocyanic screen. However, we
cannot exclude a photoprotective function of anthocyanins in
other test systems or under different conditions. The assumed
shade acclimation of red leaves was supported only partly by
the comparative data collected from an array of characteristics
linked to the shade syndrome. The results were more compatible with mesophyll adjustments to an altered spectral distribution of internal light characterized by a relative lack of green
photons.
References
Anderson, J.M. 1986. Photoregulation of the composition, function,
and structure of thylakoid membranes. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 37:93–136.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
856
KYPARISSIS, GRAMMATIKOPOULOS AND MANETAS
Bombelli, A. and L. Gratani. 2003. Interspecific differences of leaf
gas exchange and water relations of three evergreen Mediterranean
shrub species. Photosynthetica 41:619–625.
Brown, C.S., A.C. Schuerger and J.C. Sager. 1995. Growth and
photomorphogenesis of pepper plants under red light-emitting-diodes with supplemental blue or far-red lighting. J. Am. Soc. Hortic.
Sci. 120:808–813.
Burger, J. and G.E. Edwards. 1996. Photosynthetic efficiency, and
photodamage by UV and visible radiation, in red versus green leaf
coleus varieties. Plant Cell Physiol. 37:395–399.
Chalker-Scott, L. 1999. Environmental significance of anthocyanins
in plant stress responses. Photochem. Photobiol. 70:1–9.
Chow, W.S., A. Melis and J.M. Anderson. 1990. Adjustments of
photosystem stoichiometry in chloroplasts improve the quantum
efficiency of photosynthesis. Proc. Natl. Acad. Sci. USA 87:
7502–7506.
Close, D.C. and C. McArthur. 2002. Rethinking the role of many
plant phenolics - protection from photodamage not herbivores?
Oikos 99:166–172.
Close, D.C. and C.L. Beadle. 2003. The ecophysiology of foliar
anthocyanins. Bot. Rev. 69:149–161.
Dodd, I.C., C. Critchley, G.S. Woodall and G.R. Stewart. 1998.
Photoinhibition in differently coloured juvenile leaves of Syzygium
species. J. Exp. Bot. 49:1437–1445.
Feild, T.S., D.W Lee and N.M. Holbrook. 2001. Why leaves turn red
in autumn. The role of anthocyanins in senescing leaves of redosier dogwood. Plant Physiol. 127:566–574.
Genty, B., J.M. Briantais and N.R. Baker. 1989. The relationship between the quantum yield of photosynthetic electron-transport and
quenching of chlorophyll fluorescence. Biochim. Biophys. Acta
990:87–92.
Gould, K.S., J. McKelvie and K.R. Markham. 2002a. Do anthocyanins function as antioxidants in leaves? Imaging of H2O2 in red
and green leaves after mechanical injury. Plant Cell Environ. 25:
1261–1269.
Gould, K.S., S. Neil and T.C. Vogelmann. 2002b. A unified explanation for anthocyanins in leaves? In Anthocyanins in leaves. Eds.
K.S. Gould and D.W. Lee. Advances in Botanical Research.
Vol. 37. Academic Press, London, pp 167–192.
Gould, K.S., T.C. Vogelmann, T. Han and M.J. Clearwater. 2002c.
Profiles of photosynthesis within red and green leaves of Quintinia
serrata A. Cunn. Physiol. Plant. 116:127–133.
Hipskind, J., K. Wood and R.L. Nicholson. 1996. Localized stimulation of anthocyanin accumulation and delineation of pathogen ingress in maize genetically resistant to Bipolaris maydis race O.
Physiol. Mol. Plant Pathol. 49:247–256.
Hodges, D.M. and C. Nozzolillo. 1996. Anthocyanin and anthocyanoplast content of cruciferous seedlings subjected to mineral
nutrient deficiencies. J. Plant Physiol. 147:749–754.
Karabourniotis, G., J.F. Bornman and V. Liakoura. 1999. Different
leaf surface characteristics of three grape cultivars affect leaf optical properties as measured with fibre optics: possible implication in
stress tolerance. Aust. J. Plant Physiol. 26:47–53.
Karageorgou, P. and Y. Manetas. 2006. The importance of being red
when young: anthocyanins and the protection of young leaves of
Quercus coccifera against insect herbivory and excess light. Tree
Physiol. 26:613–621.
Karavatas, S. and Y. Manetas. 1999. Seasonal patterns of photosystem
2 photochemical efficiency in evergreen sclerophylls and drought
semi-deciduous shrubs under Mediterranean field conditions. Photosynthetica 36:41–49.
Krol, M., G.R. Gray, V.M. Hurry, G. Öquist, L. Malek and N. Huner.
1995. Low-temperature stress and photoperiod affect an increased
tolerance to photoinhibition in Pinus banksiana seedlings. Can.
J. Bot. 73:1119–1127.
Kumar, V. and S.S. Sharma. 1999. Nutrient deficiency-dependent
anthocyanin development in Spirodela polyrhiza L.-Schleid. Biol.
Plant. 42:621–624.
Lambers, H., F.S. Chapin III and T.L. Pons. 1998. Plant physiological
ecology. Springer-Verlag, NewYork, pp 25-35.
Lee, D.W., J. O’Keefe, N.M. Holbrook and T.S. Feild. 2003. Pigment
dynamics and autumn leaf senescence in a New England deciduous
forest, eastern USA. Ecol. Res. 18:677–694.
Lichtenthaler, H.K. and A.R. Wellburn. 1983. Determinations of total
carotenoids and chlorophylls a and b of leaf extracts in different
solvents. Biochem. Soc. Trans. 11:591–592.
Lichtenthaler, H.K., F. Babani, G. Langsdorf and C. Buschmann.
2000. Measurement of differences in red chlorophyll fluorescence
and photosynthetic activity between sun and shade leaves by fluorescence imaging. Photosynthetica 38:521–529.
Lindoo, S.J. and M.M. Caldwell. 1978. Ultraviolet-B radiation-induced inhibition of leaf expansion and promotion of anthocyanin
production. Plant Physiol. 61:278–282.
Long, S.P. and S. Humphries. 1994. Photoinhibition of photosynthesis in nature. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45:
633–662.
Manetas, Y., A. Drinia and Y. Petropoulou. 2002. High contents of
anthocyanins in young leaves are correlated with low pools of
xanthophyll cycle components and low risk of photoinhibition.
Photosynthetica 40:349–354.
Manetas, Y., Y. Petropoulou, G.K. Psaras and A. Drinia. 2003. Exposed red (anthocyanic) leaves of Quercus coccifera display shade
characteristics. Funct. Plant Biol. 30:265–270.
Manetas, Y. 2006. Why some leaves are anthocyanic and why most
anthocyanic leaves are red. Flora 201:163–177.
Maxwell, K. and G.N. Johnson. 2000. Chlorophyll fluorescence—a
practical guide. J. Exp. Bot. 51:659–668.
Melis, A., A. Manodri, R.E. Glick, M.L. Ghirardi, S.W. McCauley
and P.J. Neale. 1985. The mechanism of photosynthetic membrane
adaptation to environmental-stress conditions—a hypothesis on the
role of electron-transport capacity and of ATP NADPH pool in the
regulation of thylakoid membrane organization and function.
Physiol. Veg. 23:757–765.
Mendez, M., D.G. Jones and Y. Manetas. 1999. Enhanced UV-B radiation under field conditions increases anthocyanin and reduces the
risk of photoinhibition but does not affect growth in the carnivorous
plant Pinguicula vulgaris. New Phytol. 144:275–282.
Nishio, J.N. 2000. Why are higher plants green? Evolution of the
higher plant photosynthetic pigment complement. Plant Cell Environ. 23:539–548.
Pietrini, F. and A. Massacci. 1998. Leaf anthocyanin content changes
in Zea mays L. grown at low temperature: significance for the relationship between the quantum yield of PSII and the apparent quantum yield of CO2 assimilation. Photosynth. Res. 58:213–219.
Pietrini, F., M.A. Iannelli and A. Massacci. 2002. Anthocyanin accumulation in the illuminated surface of maize leaves enhances protection from photo-inhibitory risks at low temperature, without
further limitation to photosynthesis. Plant Cell Environ. 25:
1251–1259.
Steyn, W.J., S.J.E. Wand, D.M. Holcroft and G. Jacobs. 2002. Anthocyanins in vegetative tissues: a proposed unified function in photoprotection. New Phytol. 155:349–361.
Stone, C., L. Chisholm and N. Coops. 2001. Spectral reflectance characteristics of eucalypt foliage damage by insects. Aust. J. Bot.
49:687–698.
Terashima, I. and Y. Inoue. 1984. Comparative photosynthetic properties of palisade tissue chloroplasts and spongy tissue chloroplasts
of Camellia japonica L.: functional adjustment of the photosynthetic apparatus to light environment within a leaf. Plant Cell
Physiol. 25:555–563.
TREE PHYSIOLOGY VOLUME 27, 2007
LEAF ADJUSTMENTS TO A PERMANENT ANTHOCYANIC SHADE
Woodall, G.S. and G.R. Stewart. 1998. Do anthocyanins play a role in
UV protection of red juvenile leaves of Syzygium? J. Exp. Bot.
49:1447–1450.
857
Yu, H. and B.L. Ong. 2003. Effect of radiation quality on growth and
photosynthesis of Acacia mangium seedlings. Photosynthetica 41:
349–355.
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com