Environmental and Experimental Botany 59 (2007) 293–298
Photosynthetic pigment contents in twigs of 24 woody species assessed
by in vivo reflectance spectroscopy indicate low chlorophyll levels
but high carotenoid/chlorophyll ratios
Efi Levizou a,b,∗ , Yiannis Manetas a
a
b
Laboratory of Plant Physiology, Department of Biology, University of Patras, Patras GR-265 00, Greece
Laboratory of Botany, Department of Biological Applications and Technology, University of Ioannina, Ioannina GR-451 10, Greece
Received 27 June 2005; received in revised form 25 November 2005; accepted 23 March 2006
Abstract
We have examined whether spectral reflectance indices used to non-destructively assess photosynthetic pigment levels and their ratios in leaves,
could also be used for the same purpose in peridermal twigs. Regression lines of selected indices versus actual pigment levels, obtained from
leaves and twigs of five species, suggested that semi-quantitative assessments are safe, provided that twig periderms could be easily removed.
Given that, we proceeded to our next objective of screening a large number of species (24), in order to characterize their photosynthetic pigment
profiles. Index comparisons between twigs and corresponding leaves indicated that twigs are characterized by lower levels of total chlorophyll and,
unexpectedly, higher carotenoid/chlorophyll ratios. Moreover, the exposed and shaded sides of twigs displayed similar values for both indices in
80% of the species, suggesting that shade may not be the only factor shaping pigment levels and ratios. We discuss our results arguing that the
distinct microenvironment within a twig may pose additional needs to the photosynthetic machinery, necessitating elevated carotenoid/chlorophyll
ratios.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Cortex; Periderm; Reflectance indices; Shade acclimation
1. Introduction
Although leaves are the main plant organs optimized for photosynthesis, active chloroplasts abound in many other organs
like petioles, twigs, stems and even flowers and roots, primarily designed for other functions (Aschan and Pfanz, 2003).
Thus, the so-called corticular photosynthesis occurs in the
chlorenchyma behind the periderm of stems and twigs. Since
periderms lack stomata and possess a high diffusive resistance
to gases, corticular photosynthesis practically re-fixes respiratory CO2 or that coming up with the transpiration stream
(Pfanz et al., 2002). The reported gross CO2 assimilation
rates in the cortex are low and seldom surpass those of CO2
production by respiration (Wittmann et al., 2001). Accordingly, corticular photosynthesis is considered as a mechanism
of CO2 recycling and O2 enrichment in the interior of the
stem, facilitating the avoidance of protoplasm acidification and
∗
Corresponding author. Tel.: +30 26510 97364; fax: +30 26510 97061.
E-mail address: [email protected] (E. Levizou).
0098-8472/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.envexpbot.2006.03.002
reducing the risk of anoxia or hypoxia (Pfanz et al., 2002).
Periderm displays considerable absorbance of visible radiation, transmitting only 10–50% of incident light, depending on
species and age of twigs (Kauppi, 1991; Aschan et al., 2001;
Manetas and Pfanz, 2005). Accordingly, corticular photosynthesis should display characteristics of the shade acclimation
syndrome. Indeed, light curves for both net CO2 assimilation
(Wittmann et al., 2001) and electron transport rates (Manetas,
2004a) attain saturation at low PAR and display low maximum
values.
Concerning photosynthetic pigments in a photon limited
environment, a general rule predicts that pigments engaged in
light capture are favored under shade, while those engaged in
protection against over-excitation may be down-regulated. Thus,
chlorophyll (chl) a/b ratios in shade leaves are low due to the
high ratios of light harvesting complexes per reaction centers
(Anderson, 1986). Moreover, the pool sizes of the potentially
photoprotective ␤-carotene and the components of the xanthophyll cycle tend to decrease in the shade (Demmig-Adams et
al., 1989; Thayer and Björkman, 1990; Rosevear et al., 2001).
Since the contents of photoselective xanthophylls (neoxanthin
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and lutein) are not appreciably affected by the light environment
(Demmig-Adams et al., 1989; Rosevear et al., 2001; Hansen et
al., 2002), the total carotenoid to chlorophyll ratios are reduced
in leaves under low light.
Low chl a/b ratios have been consistently reported for twigs
of many plants (Pfanz et al., 2002). Yet, carotenoid to chl ratios,
estimated in crude extracts form three species, did not follow the predictions of a shade acclimation hypothesis. In one
case (Pilarski, 1999), the ratio was higher in twigs compared to
exposed leaves, while in two other cases the ratios were similar
(Wittmann et al., 2001). Moreover, a recent report on chromatographically analyzed photosynthetic pigments from twigs and
leaves of five species (Levizou et al., 2004b) indicated higher
carotenoid to chl ratios in twigs of four species and similar
ratios in the fifth species. These findings contradict expectations
and indicate that, in spite of the low light incident on corticular chlorenchyma, there may be other environmental conditions
within twigs that shape the unusual carotenoid to chl profiles.
One may argue that the sample size of the examined species is
low for generalization. However, screening many plant species
for photosynthetic pigment contents is impeded by the difficulties in extracting the hard twig material and the possibility
of co-extracting high amounts of phenolics, which may interfere with carotenoid estimation in the crude extracts at 470 nm
(Levizou et al., 2004a). On the other hand, chromatography can
bypass this problem, yet it is not recommended for large sample sizes in ecophysiological investigations as costly and time
consuming.
Pigments in an intact leaf can be non-destructively estimated
through optical methods. Thus, spectral reflectance is inversely
correlated to absorbance and, hence, to the chemical constituents
of a leaf and their concentrations. Accordingly, the intensity
of reflectance at specific spectral bands can give information
on leaf chemistry. Several reflectance indices have been developed for the estimation of total chls and the carotenoid/chl ratio
(Gitelson and Merzlyak, 1994; Peñuelas and Filella, 1998; Sims
and Gamon, 2002; Richardson et al., 2002). Reflectance spectroscopy has recently become popular in ecophysiological studies due to its simplicity, sensitivity, rapidity and non-destructive
nature (Fillela and Peñuelas, 1999; Carter and Knapp, 2001;
Richardson et al., 2001; Stylinski et al., 2002). Yet, it has not
been used with twigs up to now.
The present investigation had a dual scope. First, to examine
whether corticular pigment contents and their ratios could be
reliably assessed by in vivo reflectance measurements in twigs.
Provided that the method was indeed credible, we proceeded to
our second scope, i.e. screening a large number of plant species
in order to confirm (or reject) the unusual photosynthetic pigment profiles of twigs hitherto reported.
2. Materials and methods
The criteria used in preliminary trials to identify plant species
suitable for this study were a well-developed periderm that
could be removed, revealing intact green underlying tissues. In
addition, the green window after periderm removal should be
wide enough to accommodate the optical fibers used for spec-
tral reflectance measurements. Twenty-four species of woody
trees and shrubs were selected on the basis of the above prerequisites. The sampling site was within or in the vicinity of
the Patras University campus. On each sampling date (September/October 2004), 15–27-month-old leafy twigs were cut after
labeling the exposed (upper or south facing) side of the twig. At
this age periderm is developed as judged by its brownish color
and the visual presence of lenticels. Care was taken to sample
leafy twigs from the crown perimeter. The material was sealed
in plastic bags, transferred to the laboratory and kept in the dark
for at least 3 h before analysis. The following species were used:
Arbutus adrachne L., Arbutus unedo L., Citrus aurantium L.,
Cupressus sempervirens L., Elaeagnus angustifolius L., Ficus
carica L., Hedera helix L., Laurus nobilis L., Ligustrum japonicum Thunb., Melia azedarach L., Nerium oleander L., Phyllirea
latifolia L., Pinus halepensis Mill., Pinus nigra Arn., Pistacia lentiscus L., Populus deltoides Bartr., Prunus cerasus L.,
Punica granatum L., Pyrus piraster Burgsd., Quercus coccifera
L., Robinia pseudacacia L., Sorbus folgneri (C.K. Schneid.)
Rehder, Tamarix parviflora DC., Vitex agnus castus L.
2.1. Measuring spectral reflectance
All manipulations were performed under dim laboratory light
(PAR < 1 ␮mol m−2 s−1 , LI-185 Quantum sensor; Li-cor, Lincoln, NE, USA). In most cases, engraving the twig with a razor
blade facilitated periderm peeling off by hand. In more resistant species, the periderm was carefully scratched by the razor
blade until opening a window of suitable dimensions. Spectral
reflectance was measured with a portable diode-array spectrometer (Unispec; PP-Systems, Haverhill, MA, USA) equipped with
an internal halogen source and bifurcated fiber optic cables
directly attached to the sample (peeled twig or leaf). A spectralon standard (reflectance > 0.97 for the whole 400–1100 nm
range) was used as a reference and the obtained spectra
were dark-corrected for stray light after closing the instrument shutter. Measurements were performed on 24 species, 10
spots per twig (equally divided between exposed and shaded
side), 5 twigs/individual and 3 individuals/species. In leaves (5
spots/leaf, 5 leaves/individual, 3 individuals/species), only the
upper (exposed) surface was scanned.
The determined reflectance indices were the following, with
Rn denoting reflectance at λ = n:
750 −R705
1. Normalized difference index, NDI = R
R750 +R705 . This index
is positively correlated with chl concentrations (Gitelson and
Merzlyak, 1994).
531 −R570
2. Photochemical reflectance index, PRI = R
R531 +R570 . It is proposed as a measure of photosynthetic efficiency (Gamon et
al., 1992) and it is negatively correlated to the carotenoid/chl
ratio (Sims and Gamon, 2002).
2.2. Correlating reflectance indices with actual pigment
levels and ratios
A sub-sample consisting of five species (i.e. A. unedo, P.
lentiscus, P. deltoides, P. cerasus, Q. coccifera) was used to
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295
examine whether acceptable regression lines could be established between reflectance indices and actual pigment levels.
Reflectance indices were obtained from leaves and twigs and the
corresponding material was extracted in hot DMSO (dimethylsulfoxide) according to Wittmann et al. (2001) as modified
by Levizou, Petropoulou and Manetas (2004a). Whole twig
segments were used with no attempt to discriminate between
exposed and shaded twig sides. After centrifugation, chls a and
b in the crude extract were determined in the supernatant using
a Shimadzu UV-160A double beam spectrophotometer and
the equations of Wellburn (1994). Carotenoids were analyzed
with a Shimadzu LC-10 AD HPL chromatograph, equipped
with a non-endcapped Zorbax ODS (4.6 mm × 25.0 mm) column (Rockland Technologies, Chadds Ford, PA, USA), calibrated against purified ␤-carotene (Sigma Chemical, St. Louis,
MO, USA) and freshly prepared xanthophylls by TLC as
described by Kyparissis et al. (1995). Development was performed isocratically at 1 cm3 per min (20 min with acetonitrile:
methanol, 85:15 v/v, and 20 min with methanol:ethyl acetate,
68:32 v/v), according to Thayer and Björkman (1990). Pigments were detected by a Shimadzu SPD-M10AVP UV–vis
photodiode array detector and further analysed by a Shimadzu
Class-VP version 6.1 software package. Two to three separate
replications (i.e. extraction and chromatography) per species
and organ (i.e. a total of 23 chromatographic analyses) were
done.
2.3. Statistics
Since index differences between individuals were negligible, statistical analysis was based on single twigs (n = 15). In
addition, spectral reflectance of leaves, exposed twig side and
shaded twig side were obtained for the same twig. Accordingly, significance of differences in the indices between the
various organs and sides were assessed by paired t-test (SPSS
12.0). The same statistical package was used for computing
regression lines between indices and actual pigment levels and
ratios.
Fig. 1. Regression lines of reflectance indices vs. pigment levels and ratios. (A)
NDI vs. actual chl levels; (B) PRI vs. actual carotenoid/chl ratios. Values for
twigs are segregated in the left part of the curve (A) and the right part of the curve
(B). (t) and (l) denote twigs and leaves, respectively. When a linear regression
was attempted, corresponding statistical parameters for (A) were p < 0.001 and
r2 = 0.947 and for (B) p < 0.001 and r2 = 0.9.
3. Results
When reflectance indices were plotted against actual pigment concentrations and their ratios (obtained after extraction
and chromatography of leaves and twigs on five species) the
regressions deviated considerably from linearity (Fig. 1), being
represented by either a hyperbola (NDI) or an exponential decay
curve (PRI). It is clear, however, that the values for the leaves
and twigs segregated at the distant parts of the curves in all
cases, indicating that reflectance indices could be used for a
semi-quantitative empirical assessment of differences in pigment levels between leaves and twigs. Therefore, we proceeded
in screening a total of 24 species for these parameters, including also reflectance measurements from both the exposed and
shaded twig sides.
Fig. 2 shows that chl levels in leaves are always higher compared to twigs of the same species (p < 0.01). Yet, no significant
differences were found between the exposed and shaded twig
sides in 18 species. In five species, levels of chl were (E. angustifolius, P. cerasus, p < 0.01) or tended to be (A. adrachne, P.
halepensis, P. nigra, 0.05 < p < 0.1) higher in the shaded side of
twigs, while the opposite was observed in L. nobilis (p < 0.01).
As a whole, NDI values displayed a high interspecies variation.
For example, NDI for the two pine species leaves were lower
compared to twigs of several other species.
Overall, the ratio of carotenoids/chls was higher in twigs
(either exposed or shaded sides) compared to leaves of the same
species (Fig. 3). In 15 species the differences were statistically
significant (see figure legend for levels of significance). A further
group (A. unedo, A. adrachne and L. nobilis) displayed significant differences between leaves and the exposed twig side only.
In the remaining six species (F. carica, H. helix, M. azedarach, P.
cerasus, P. latifolia and T. parviflora), the trends were not statistically significant (Fig. 3). When the values for the exposed and
shaded twig sides were compared, no differences were found in
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Fig. 2. NDI (i.e. chl levels) in leaves (upper surface) and twigs (exposed and shaded side) of the indicated species. Values are means ± S.D. (n = 15). In all cases,
the difference between leaves and twigs were significant (p < 0.01). Differences between exposed and shaded twig sides were significant only in E. angustifolius, L.
nobilis and P. cerasus (p < 0.01), while in A. adrachne, P. halepensis, P. nigra marginal differences were obtained (0.05 < p < 0.1).
20 species, while in A. adrachne, E. angustifolius, L. nobilis and
S. folgneri the carotenoid/chl ratios were higher in the exposed
twig side.
4. Discussion
It is evident from Fig. 1 that high chl levels are associated with
high NDIs and high carotenoid/chl ratios are associated with low
PRIs. However, the considerable deviation from linearity when
both twig and leaf indices were included in the same graphs
does not allow a fully quantitative comparison. The reasons for
this divergence from linearity are not clear, yet the apparent
differences in the surface relief between an intact leaf and a
naked cortex are likely to affect reflectance indices (Peñuelas
and Filella, 1998). On the other hand, the clear segregation of
twigs and leaves indices towards the opposite edges along the
regression lines as well as the compatibility of the direction of
indices partition with actually measured pigment parameters,
definitely allow their semi-quantitative use.
The screening of a large number of species for chl levels
(index NDI, Fig. 2) simply confirmed earlier reports for lower
chl concentrations in twigs (Pfanz and Aschan, 2001). Curi-
ously though, the carotenoid/chl ratio (index PRI, Fig. 3) did not
conform to a shade acclimation hypothesis, being considerably
higher in the twigs of the majority of species examined (83%),
while the differences in the remaining species were not statistically significant. The result indicates that a similar recent report
based on extraction and chromatography of five species (Levizou
et al., 2004b) was not incidentally due to the small sample size.
Therefore we have to admit that shade is not the only parameter
within twigs that shapes the unexpected carotenoid/chl ratios.
This is further strengthened by the absence of differences in PRI
between the exposed and shaded parts of the twigs.
Shade itself is qualitatively different in twigs compared
to leaves. Published transmittance spectra of isolated periderms indicate a preferential attenuation of blue radiation while
longer wavelengths are not appreciably impeded (Kauppi, 1991;
Solhaug et al., 1995; Manetas, 2004a; Manetas, 2004b; Manetas
and Pfanz, 2005). Accordingly, light in corticular chlorenchyma
is red-enriched. Judging from corresponding reports with leaves,
continuous irradiation with red compared to blue light results in
higher carotenoid/chl and lower chla/b ratios (Buschmann et al.,
1978; Lichtenthaler et al., 1980). The effect is possibly photomorphogenic and, concerning carotenoids, may improve the
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297
Fig. 3. PRI (i.e. carotenoid/chl ratio) in leaves (upper surface) and twigs (exposed and shaded side) of the indicated species. Values are means ± S.D. (n = 15).
Differences between leaves and twigs were significant (p < 0.05) in 15 species (C. aurantium, C. sempervirens, E. angustifolius, L. japonicum, N. oleander, P.
halepensis, P. nigra, P. lentiscus, P. deltoides, P. granatum, P. piraster, Q. coccifera, R. pseudacacia, S. folgneri, V. agnus castus) and between leaves and the exposed
twig side in further three species (p < 0.05 in A. adrachne, A. unedo, L. nobilis). The trends in the remaining species were not significant (see text for details).
Differences between exposed and shaded twig sides were significant only in A. adrachne, E. angustifolius, L. nobilis (p < 0.01) and S. folgneri (p < 0.05).
efficiency of energy capture in the blue region of the spectrum
where photons are less abundant.
Another peculiarity of stem internal microenvironment concerns partial pressures of CO2 . Since periderm is highly resistant
to gas diffusion, CO2 coming from respiration is trapped leading to extremely high concentrations, ranging from ca. 1–25%
(see Schaedle, 1975; Pfanz and Aschan, 2001 and the literature
there-in). Such high concentrations may surpass the buffering
capacity of protoplasm and chloroplast stroma leading to acidification (Yin et al., 1993; Bligny et al., 1997) and the inhibition
of sensitive enzymes of the reductive pentose phosphate cycle
(Pfanz and Heber, 1986). Therefore, the electron sink capacity of
the cycle may be reduced, raising the need for xanthophyll cyclemediated dissipation of extra excitation energy even at low light.
It has been reported in the case of leaves that reduction in Calvin
cycle activity by drought or cold elevates the pool sizes of the
xanthophyll cycle components (Kyparissis et al., 2000; MunnéBosch and Alegre, 2000). In twigs it has been shown that a high
CO2 stress suppresses linear electron transport rates along PSII
and enhances photoprotective non-photochemical quenching at
low irradiance (Manetas, 2004a).
If our interpretation is correct, one could expect analogous
photosynthetic pigment profiles in bulky tissues with high resistance to CO2 diffusion. Seeds and fruits, for example, contain
extremely high CO2 concentrations (Aschan and Pfanz, 2003;
Goffman et al., 2004) and high carotenoid/chl ratios have been
reported in one case (Roca and Minguez-Mosquera, 2001). The
relative importance of light quality, CO2 levels or another stem
internal factor (for example, low O2 partial pressures) in shaping
pigment profiles remain to be seen. We suggest that the neglected
area of corticular photosynthesis deserves more attention since
it occurs under conditions never encountered by a leaf.
5. Conclusions
The shade imposed to corticular chlorenchyma by the highly
light absorptive periderm may not be the main determinant of
twig photosynthetic pigment profiles. These are consistently and
unexpectedly characterized by high carotenoid to chlorophyll
ratios, possibly indicating the need for improved light harvesting
at specific spectral bands and/or increased photoprotection under
the extremely high CO2 levels encountered within twigs.
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