Bark spectral signatures of one-year-old twigs of different shrubs mainly 
depend on their biochemical traits
Spektralni podpisi skorje enoletnih vejic različnih grmovnih vrst so večinoma 
odvisni od biokemijskih lastnosti skorje
Mateja Grašič*, Bojana Ropret, David Gradinjan, Alenka Gaberščik
Department of Biology, Biotechnical Faculty, University of Ljubljana,  
Večna pot 111, 1000 Ljubljana, Slovenia
*Correspondence: mateja.grasic@bf.uni-lj.si
Abstract: The interaction of bark with light depends on the optical properties of 
the bark, which are important for plant energy balance especially out of the vegetation 
season. Light reflected from bark also represents a kind of “bark spectral signature” 
that may be species-specific. This study examines some morphological, biochemical, 
and physiological traits that may affect the reflectance of the bark of 1-year-old twigs 
of different shrubs: Corylus avellana, Rosa canina, Ligustrum vulgare, Sambucus ni-
gra, Cornus sericea var. flaviramea, and Viburnum lantana. High variability was seen 
across these species for morphological, biochemical, and physiological traits, except 
for photochemical efficiency of photosystem II. The bark spectral signatures differed 
significantly across these species. The reflectance peak for C� sericea var. flaviramea 
was in red, for C� avellana in green, and the other species showed a wide peak from 
green to red light. Redundancy analysis revealed that UV-B–absorbing substances and 
anthocyanin content, along with outer and inner bark thickness, together explained 
61% of the reflectance spectra variability. Outer bark thickness was important for 
reflectance in UV, violet, and blue, while anthocyanins were important for reflectance 
in green and yellow. Chlorophyll b was negatively related to the reflectance of visible 
light. This study revealed great importance of biochemical traits for explaining bark 
reflectance. Differences in “bark spectral signatures” enable differentiation across 
species out of the vegetation season.
Keywords: bark, pigments, reflectance, UV-B–absorbing substances, woody plants
Izvleček: Interakcija skorje s svetlobo je odvisna od optičnih lastnosti skorje, ki so 
pomembne za energijsko ravnovesje rastlin zlasti izven vegetacijske sezone. Svetloba, 
ki se odbije od skorje, predstavlja tudi nekakšen “spektralni podpis skorje”, ki je lahko 
vrstno specifičen. V tej raziskavi smo proučili nekatere morfološke, biokemijske in 
fiziološke lastnosti, ki lahko vplivajo na odbojnost skorje enoletnih vejic različnih 
grmovnih vrst: Corylus avellana, Rosa canina, Ligustrum vulgare, Sambucus nigra, 
Cornus sericea var. flaviramea in Viburnum lantana. Opazili smo veliko variabilnost 
med proučevanimi vrstami glede njihovih morfoloških, biokemijskih in fizioloških 
lastnosti, razen fotokemične učinkovitosti fotosistema II. Med izbranimi vrstami so se 
 ACTA BIOLOGICA SLOVENICA 
 LJUBLJANA 2021               Vol. 64, Št. 1: 56–69
57Grašič et al.: Bark spectral signatures of shrubs
značilno razlikovali tudi spektralni podpisi skorje. Vrh odbojnosti pri vrsti C� sericea 
var. flaviramea je bil v rdečem, pri C� avellana v zelenem, druge vrste pa so pokazale 
širok vrh, ki se je raztezal od zelenega do rdečega spektralnega območja. Redundančna 
analiza je pokazala, da vsebnost UV-B–absorbirajočih snovi in antocianinov skupaj z 
debelino zunanje in notranje skorje skupaj pojasnjujejo 61 % variabilnosti odbojnih 
spektrov. Debelina zunanje skorje je bila pomembna za odbojnost v UV, vijoličnem 
in modrem spektralnem območju, vsebnost antocianinov pa za odbojnost v zelenem 
in rumenem spektralnem območju. Klorofil b je bil negativno povezan z odbojnostjo 
vidnega dela spektra. Ta raziskava je pokazala velik pomen biokemijskih lastnosti 
za razlago odbojnosti skorje. Razlike v “spektralnih podpisih skorje” omogočajo 
razlikovanje med vrstami izven vegetacijske sezone.
Ključne besede: lesnate rastline, odbojnost, pigmenti, skorja, UV-B–absorbira-
joče snovi
Introduction
Bark comprises tissues outside the vascular 
cambium, and consists of the inner bark, which 
contains secondary phloem, and the outer bark or 
periderm, produced by the cork cambium (Romero 
2014). Bark protects stems against physical dis-
turbances (e.g., fire) and environmental factors 
(e.g., extreme temperatures, drought), and it has 
high repair potential (Romero et al. 2009). In ad-
dition, the outer bark is an effective barrier against 
pollutants (Pfanz 1999), and enables transport 
of assimilates and water (Poorter et al. 2014). 
Bark physiological and biochemical traits vary 
significantly across species, and these specific 
bark traits define the bark interactions with the 
abiotic and biotic environments (Larcher 2003).
The surface structure of bark determines the 
composition and structure of epiphytic communi-
ties (Tewari et al. 2009). In various forest types, 
the suitability of bark for epiphytic lichen and 
bryophyte colonisation increases with increasing 
bark roughness (Black and Harper 1979). Ferren-
berg and Mitton (2014) demonstrated that smooth 
bark limits the attacks of insects specialised in 
attacking tree stems. Bark may also contain high 
concentrations of flavonoids, which are known 
to have multiple functions (Carrillo-Parra et al. 
2012). These substances protect plants from dif-
ferent biotic and abiotic stresses and act as filters 
for UV radiation, signal molecules, detoxifying 
agents, and antimicrobial compounds (Samanta 
et al. 2011).
The bark chlorenchyma is located under the 
rhytidomal or outer peridermal layers; it can harvest 
transmitted light and use the stem internal CO2 
for photosynthesis (Pfanz et al. 2002, Wittmann 
and Pfanz 2008, 2014). The transmission of light 
through the periderm is low and depends on the 
species and age of the branches or stems (Aschan 
et al. 2001). A study by Wittmann and Pfanz (2016) 
revealed that transmittance of photosynthetically 
active radiation (PAR) through the periderm of 
young stems varied between 8.5% and 42.0%, 
while PAR transmittance through the total bark 
ranged from 2.2% to 6.2%.
The fixation of CO2 via photosynthesis reduces 
internal carbon dioxide losses and thus contributes 
to a favourable carbon balance (Filippou et al. 2007, 
Eyles et al. 2009). Wittmann and Pfanz (2008) 
showed that photosynthesis of the bark of young 
stems can replace 60% to 90% of respiratory carbon 
losses. Assimilation of CO2 in stems has several 
advantages over photosynthesis in leaves, as stems 
lose less water and contain high concentrations of 
CO2, which reduce the level of photorespiration in 
stems (Damesin 2003). In young stems, respiration 
and the photosynthetic rate are generally higher 
than in older stems (Wittmann and Pfanz 2008). 
The amount of light that can reach different stem 
tissues affects plastid differentiation, and thus 
also their photosynthetic properties (Wittmann 
and Pfanz 2016). As branches grow older, the 
bark and the peridermal layer become thicker, 
and consequently the amount of chlorophyll and 
photosynthetic activity decrease (Pilarski 1999, 
Wittmann et al. 2001). Bark photosynthesis does 
not only have an important role in maintaining the 
carbon balance, but it also improves the oxygen 
content and enables undisturbed respiration of 
58Acta Biologica Slovenica, 2021, 64 (1), 56–69
metabolically active tissues (Wittmann and Pfanz 
2014). Green stems with poorly developed peri-
derm have functional stomata and can assimilate 
large amounts of carbon, especially during the 
period when the plants are leafless (Filippou et 
al. 2007). Also, in spite of the low light levels 
in winter, bark photosynthetic activity and res-
piration contribute to a more favourable carbon 
balance from late autumn to early spring, when 
photosynthetic activity in evergreen organs is 
reduced (Wittmann and Pfanz 2007) or is absent 
in deciduous species.
The light regime within the stem affects the 
differentiation of plastids and thus affects the 
photosynthetic properties of individual tissues 
in the stem (Wittmann and Pfanz 2016). Light 
penetration depends on the optical properties of 
the bark. Light reflected from bark represents a 
kind of “bark spectral signature” that is species-
specific and can reveal the physiological and 
biochemical properties of the bark (Tokarz and 
Pilarski 2005, Levizou and Manetas 2007). In 
deciduous species, the bark spectral signatures 
enable differentiation among woody species out 
of the vegetation season using remote sensing 
(Atkilt Girma et al. 2013), and host discrimination 
by bark and timber beetles (Campbell and Borden 
2005). Bark optical properties also contribute to 
the bark energy balance and prevent overheating 
(Henrion and Tributsch 2009).
In the present study, we examined different 
plant traits that affect bark reflectance of 1-year-old 
twigs of different woody species: Corylus avellana, 
Rosa canina, Ligustrum vulgare, Sambucus nigra, 
Cornus sericea var. flaviramea, and Viburnum 
lantana. We hypothesised that the bark biochemical 
traits and the bark thickness have important roles 
in shaping the bark reflectance. To show this, we 
analysed different pigments and UV-absorbing 
substances, and measured the inner and outer 
bark thicknesses, and the bark reflectance spectra.
Materials and methods
Plant species
The species studied were the woody plants 
Corylus avellana L. (Betulaceae), Rosa canina 
L. (Rosaceae), Ligustrum vulgare L. (Oleaceae), 
Sambucus nigra L. (Adoxaceae), Cornus sericea 
var. flaviramea L. (Cornaceae), and Viburnum 
lantana L. (Caprifoliaceae). All of these species 
are shrubs that are native to Europe, except for 
C� sericea, which is native to North America. 
However, the cultivar C� sericea var. flaviramea 
was developed in Europe. The studied species are 
all deciduous, with the exception of L� vulgare, 
which is a semi-evergreen or deciduous shrub that 
is frequently grown as a hedge. C� avellana is an 
important component of hedgerows. R� canina is a 
climbing shrub that has stems, which are covered 
with small, sharp, hooked prickles. S� nigra is also 
very common in hedgerows and scrubland, and 
it is also widely grown as an ornamental shrub 
or small tree. C� sericea var. flaviramea is widely 
grown as an ornamental plant that grows best in 
moderate warmth in sunny places, although it can 
also tolerate shade. V� lantana is a tree-like shrub 
that is common along waysides and is also grown 
as an ornamental plant.
One-year-old and about 0.5 cm thick twigs 
from 10 plants of each species were sampled from 
open places in Ljubljana (46°3’N; 14°28’E) and 
Golnik (46°19’N; 14°19’E), and were processed 
on the day of sampling. The sampling and analysis 
took place before the development of leaves, from 
February to the end of March.
Morphological properties
Analysis of bark thickness was carried out 
on transverse sections of vital 1-year-old twigs. 
The measurements included the thickness of the 
inner and outer bark (periderm). The measure-
ments were performed using a light microscope 
(CX41; Olympus, Tokyo, Japan) equipped with a 
digital camera (XC30, Olympus) and the CellSens 
software (Olympus).
Biochemical properties
The chlorophyll a, chlorophyll b, and carot-
enoid contents were determined for bark extracts 
according to Lichtenthaler and Buschmann (2001a, 
b), with absorbance measured at 470 nm, 645 nm, 
and 662 nm using a UV/VIS spectrometer (Lambda 
25; Perkin-Elmer, Norwalk, CT, USA). Antho-
59Grašič et al.: Bark spectral signatures of shrubs
cyanin content was determined on bark extracts 
as described by Drumm and Mohr (1978), with 
absorbance measured at 530 nm. Total methanol-
soluble UV-B–absorbing and UV-A–absorbing 
substances (as a measure of total phenolics) were 
also extracted from fresh twigs, according to 
Caldwell (1968), with absorbance measured from 
280 nm to 319 nm, and from 320 nm to 400 nm, 
respectively. The extinction values were integrated 
for each UV region. The biochemical parameters 
are expressed per bark area.
Physiological properties
Chlorophyll fluorescence was measured using 
a portable chlorophyll fluorometer (PAM-2100; 
Heinz Walz GmbH, Effeltrich, Bavaria, Ger-
many). The potential and effective photochemical 
efficiency of photosystem (PS) II (Fv/Fm and 
Fq’/Fm’, respectively) were evaluated according 
to Schreiber et al. (1996), with the measurements 
performed on fresh twigs. Prior to the Fv/Fm 
measurements, twigs were kept in the dark for 
20 minutes. Fq’/Fm’ measurements were per-
formed at daylight at ˃1000 μmol m-2 s-1, with 
temperatures ranging from 10 to 15 °C. Fq’/Fm’ 
was calculated using equation 1,
  
(1)
where Fm’ is maximum fluorescence from dark- 
or light-adapted leaf, respectively (PS II centers 
closed), and F’ is fluorescence emission from 
dark- or light-adapted leaf, respectively (Brestic 
and Zivcak 2013).
Potential photochemical efficiency (Fv/Fm) 
was calculated using the equation 2,
 
  
(2)
where Fo is minimal fluorescence from dark-
adapted leaf (PS II centres open), and Fm is 
maximum fluorescence from dark- or light-adapted 
leaf, respectively (PS II centres closed) (Brestic 
and Zivcak 2013).
Optical properties
Bark reflectance was determined in the labora-
tory on the day of sampling. The bark was carefully 
removed from the twigs. The reflectance spectrum 
was measured from 290 nm to 800 nm, at a reso-
lution of ~1.3 nm, using a portable spectrometer 
(Jaz Modular Optical Sensing Suite; Ocean Optics, 
Inc., Dunedin, FL, USA; grating, #2; slit size, 
25 µm) with an optical fibre (QP600-1-SR-BX, 
Ocean Optics, Inc.) and an integrating sphere 
(ISP-30-6-R, Ocean Optics, Inc.). The reflectance 
spectrum was measured for the bark surface by 
illumination with a UV/VIS-near infrared (NIR) 
light source (DH-2000; Ocean Optics, Inc.). The 
spectrometer was calibrated to 100% reflectance 
using a white reference panel with >99% diffuse 
reflectance (Spectralon, Labsphere, North Sutton, 
NH, USA). More details on the procedure for 
measurement of optical properties can be found 
in Klančnik et al. (2015).
Statistical analysis
Statistical analysis was carried out in IBM 
SPSS statistics 22.0 using one-way multivariate 
analysis of variance followed by Duncan’s post-hoc 
multiple comparison tests. Prior to this analysis, 
normal distributions of the data were tested using 
Shapiro-Wilk tests in Past 3.14. We performed 
redundancy analysis (RDA) using CANOCO for 
Windows 4.5 programme package to determine 
whether variations in the reflectance spectra 
were related to morphological and biochemical 
parameters (ter Braak and Šmilauer 2002). The 
spectra were divided into spectral bands (i.e., 
UV-B, UV-A, violet, blue, green, yellow, red, and 
NIR) to obtain more detailed information about 
the correlations between explanatory and response 
variables. Forward selection of the explanatory 
variables was used to avoid co-linearity between 
the variables (Lepš and Šmilauer 2003). The level 
of significance was accepted at p ≤0.05. Non-
significant explanatory variables were excluded 
from further analysis. All of the variables in the 
RDA were standardised to eliminate the influence 
of magnitude differences between scales and units.
60Acta Biologica Slovenica, 2021, 64 (1), 56–69
Results
In the comparison of the biochemical traits, 
the greatest variability was seen for anthocyanin 
content (Tab. 1). The chlorophyll a to b ratio 
varied a lot across the species. The bark of C� 
sericea var. flaviramea differed the most from the 
other species, with the highest total chlorophylls, 
UV-absorbing substances, and anthocyanins, but 
not carotenoids. The peridermal layers had different 
thicknesses in the species studied. V� lantana had 
the thickest outer bark, while for S� nigra the inner 
bark was the thickest. R� canina and C� sericea 
var. flaviramea still had the epidermal layer and 
a very thin layer of periderm. Fv/Fm was high in 
the majority of species, however, Fq’/Fm’ was 
low and did not differ across the species.
The bark spectral signatures were very vari-
able. The bark reflectance differed the most in the 
visible part of the spectrum. C� sericea var. flavi-
ramea had high reflectance in the UV-B and NIR 
regions, and low reflectance in the visible region 
(Fig. 1). Its reflectance spectra had distinct peaks 
in the red region. However, the reflectance peak 
of other species was wide, and ranged from green 
to red, with the exception of C� avellana, which 
had a peak in the green region. The reflectance of 
V� lantana gradually increased along the spectra, 
with a slight depression in the red edge.
61Grašič et al.: Bark spectral signatures of shrubs
table 1:  Selected bark biochemical and physiological traits, along with bark reflectance and thicknesses for the 
  different woody species examined in this study.
tabela 1:  Izbrane biokemijske in fiziološke lastnosti ter odbojnost in debelina skorje različnih lesnatih vrst, 
 obravnavanih v tej raziskavi.
traits units Data measures according to species
Corylus
avellana
Rosa
canina
Ligustrum 
vulgare
Sambucus 
nigra
Cornus
sericea
Viburnum 
lantana
Biochemical
Chlorophyll a mg cm-2 0.012 ±0.001bc 0.022 ±0.002a 0.010 ±0.002b 0.016 ±0.002bc 0.019 ±0.012abc 0.014 ±0.009bc
Chlorophyll b mg cm-2 0.006 ±0.001a 0.013 ±0.001bc 0.007 ±0.003ab 0.011 ±0.003b 0.029 ±0.023c 0.017 ±0.014bc
Carotenoids mg cm-2 0.005 ±0.000ac 0.008 ±0.001b 0.003 ±0.001a 0.005 ±0.001c 0.006 ±0.003abc 0.006 ±0.001bc
Anthocyanins au cm-2 0.44 ±0.03a 0.84 ±0.17bc 0.23 ±0.07a 0.63 ±0.05b 1.48 ±0.53c 1.12 ±0.28c
UV-B–AS au cm-2 4.90 ±0.45a 9.64 ±0.93bc 7.69 ±1.11ab 10.64 ±1.43c 23.10 ±4.32d 12.27 ±1.42c
UV-A–AS au cm-2 4.09 ±0.37a 9.11 ±0.89bc 7.45 ±1.13ab 8.94 ±1.01bc 7.69 ±1.41b 10.89 ±1.76c
Bark thickness
Inner bark µm 84 ±7a 163 ±20c 173 ±18ac 210 ±17b 137 ±25c 169 ±40bc
Outer bark# µm 64 ±8a 33 ±1b 92 ±5a 165 ±10c 37 ±3b 284 ±32c
Physiological
Photochemical efficiency of PS II
Fq’/Fm’ au 0.12 ±0.05a 0.12 ±0.07a 0.14 ±0.11a 0.15 ±0.04a 0.11 ±0.03a 0.14 ±0.03a
Fv/Fm au 0.36 ±0.07ac 0.62 ±0.03b 0.65 ±0.06abd 0.71 ±0.03d 0.48 ±0.05c 0.69 ±0.05d
Reflectance %
UV-B 4.3 ±3.4abd 8.8 ±1.1b 4.9 ±2.2a 10.0 ±1.7bc 11.7 ±0.6c 0.3 ±1.8d
UV-A 6.6 ±2.2ab 9.3 ±1.0ab 7.7 ±1.2a 13.6 ±1.8c 9.2 ±0.5b 0.1 ±2.0c
Violet 14.1 ±8.7ab 12.6 ±7.3a 11.2 ±2.8a 13.8 ±2.3a 8.1 ±0.4b 3.6 ±2.0c
Blue 16.8 ±8.9ab 14.3 ±7.9a 14.2 ±3.0a 15.0 ±2.3a 7.6 ±0.4c 7.2 ±3.6bc
Green 23.5 ±7.4a 20.2 ±7.4a 23.5 ±3.6a 25.5 ±3.7a 7.3 ±0.5b 11.1 ±4.3b
Yellow 26.5 ±6.7ab 21.8 ±6.7ab 24.5 ±3.4a 23.3 ±3.2a 10.9 ±1.2c 15.5 ±4.9bc
Red 25.9 ±6.7a 20.3 ±6.6a 21.8 ±3.0a 19.2 ±2.8a 13.0 ±1.4b 19.1 ±5.4ab
NIR 49.0 ±7.5ab 56.4 ±4.9a 56.8 ±1.9a 55.7 ±6.2a 67.4 ±1.7c 43.8 ±7.5b
Data are means ±SD (n = 5-10 for each species); different superscript letters within each row indicate significant 
differences (p ≤0.05; Tukey tests); reflectance spectra represent means within 5-nm intervals (p ≤0.05, Tukey tests). 
Abbreviations: au, arbitrary unit; Fq’/Fm’, effective photochemical efficiency; Fv/Fm, potential photochemical efficiency; 
UV-B–AS, UV-B–absorbing substances; UV-A–AS, UV-A–absorbing substances; #epidermal layer with periderm.
62Acta Biologica Slovenica, 2021, 64 (1), 56–69
Figure 1:  Relative reflectance spectra of bark of the different shrub species.
Slika 1:   Relativni odbojni spektri skorje različnih grmovnih vrst.
Figure 2:  The strength of the associations between the bark properties, in terms of inner and outer bark thick-
nesses, anthocyanins, UV-B–absorbing substances (UV-B–AS), and different regions of the reflectance 
spectra. Grey diamonds, Corylus avellana; black squares, Rosa canina; black diamonds, Ligustrum 
vulgare; black triangles, Sambucus nigra; grey triangles, Cornus sericea var. flaviramea; grey squares, 
Viburnum lantana. The plot was generated using redundancy analysis.
Slika 2:   Moč povezav med lastnostmi skorje, in sicer med debelino notranje in zunanje skorje, antocianini, 
UV-B–absorbirajočimi snovmi (UV-B–AS) ter različnimi območji odbojnih spektrov. Sivi diamanti, 
Corylus avellana; črni kvadrati, Rosa canina; črni diamanti, Ligustrum vulgare; črni trikotniki, Sam-
bucus nigra; sivi trikotniki, Cornus sericea var. flaviramea; sivi kvadrati, Viburnum lantana. Graf je 
bil narejen v okviru redundančne analize.
63Grašič et al.: Bark spectral signatures of shrubs
RDA revealed negative relationships between 
the biochemistry and the reflectance in the visible 
region of the spectrum (Fig. 2). Here, UV-B–ab-
sorbing substances alone explained 36% of the 
variability of the bark reflectance spectra, antho-
cyanins alone explained 31%, while periderm and 
chlorophyll b alone explained 15% each. When 
considered together, UV-B–absorbing substances 
explained 38% of the variability (p = 0.001), and 
the outer bark thickness, inner bark thickness, and 
anthocyanin content explained an additional 12% 
(p = 0.001), 7% (p = 0.001), and 4% (p = 0.009) 
of the variability, respectively. With the measured 
parameters, as much as 61% of the reflectance 
spectra variability was explained.
In the explanation of the different parts of the 
reflectance spectra redundancy analyses, outer 
bark thickness was seen to have a crucial role in 
explaining the reflectance in the UV, violet, and 
blue regions. Furthermore, anthocyanins were 
important to explaining the reflectance in the green 
and yellow regions, while the UV-B–absorbing 
substances explained most of the reflectance in 
the red and NIR regions (Tab. 2).
Discussion
In general, high variability was seen across 
the species for the morphological and biochemical 
traits, with less variability for the physiological 
traits. One of the important traits was the amount 
of photosynthetic pigments, which are responsible 
for bark photosynthesis (Pfanz 1999). The stems 
of woody plants usually contain much lower 
amounts of photosynthetic pigments than the 
leaves. However, in young stems with a thin layer 
of cork, the pigment content can be similar to that 
in leaves (Kocurek and Pilarski 2012). This was 
also the case in the present study, as we measured 
table 2:  Explained variance of the different reflectance spectra regions for the woody species according to the 
  morphological and biochemical parameters, as obtained by redundancy analysis.
tabela 2:  Pojasnjena varianca različnih spektralnih območij odbojnih spektrov za lesnate vrste glede na morfološke 
  in biokemijske parametre, ugotovljena s pomočjo redundančne analize.
Spectral region Bark trait Explained variance (%) p
UV Outer bark thickness 35 0.001
Inner bark 9 0.002
UV-A–AS 4 0.040
Anthocyanins 4 0.031
Violet, blue Outer bark thickness 39 0.001
UV-A–AS 6 0.030
Green, yellow Anthocyanins 48 0.001
Inner bark thickness 13 0.001
Carotenoids 8 0.002
Outer bark thickness 5 0.010
Chlorophyll b 4 0.032
Chlorophyll a 3 0.020
Red, NIR UV-B–AS 40 0.002
Outer bark thickness 7 0.010
Inner bark thickness 7 0.012
Abbreviations: p, p-value; UV-B–AS, UV-B–absorbing substances; UV-A–AS, UV-A–absorbing substances.
64Acta Biologica Slovenica, 2021, 64 (1), 56–69
comparable amounts of photosynthetic and other 
pigments per area, as seen for the leaves of dif-
ferent herbaceous species (Grašič et al. 2019a, 
b, c). The bark usually contains 90% of the total 
photosynthetic pigments, while the remaining 
10% is located in the deeper layers of the woody 
stem (Tokarz and Pilarski 2005). The main reason 
for this distribution is that the formation of bark 
reduces the intensity of radiation that reaches the 
chlorophyll layer and affects the content of photo-
synthetic pigments in the inner tissues (Pilarski et 
al. 2008). In the species studied here, there were 
differences across the levels of chlorophyll a, and 
even more pronounced differences for chlorophyll 
b, which resulted in variable chlorophyll a/b ratios 
that ranged from 2.00 in C� avellana to 0.65 in 
C� sericea var. flaviramea. According to these 
ratios, we cannot generally speak about a shady 
character of the bark inner environment. Shady 
character can only be confirmed for two species 
in this study, namely C� sericea var. flaviramea 
and V� lantana, with chlorophyll a/b ratios below 
1. Such ratios reveal higher contents of the acces-
sory pigment chlorophyll b (in comparison to the 
main pigment chlorophyll a), which is needed to 
increase light-use efficiency in shady environments 
(Larcher 2003).
The bark of C� sericea var. flaviramea was the 
thinnest, but it had the highest total chlorophylls, 
UV-absorbing substances, and anthocyanins, as 
was also evident from the noticeably red colour of 
the bark. In V� lantana, anthocyanin content was 
almost the same as in C� sericea var. flaviramea, 
even though its bark colour was completely differ-
ent, as also revealed from the reflectance spectra. 
The colour of anthocyanins varies according to the 
vacuolar pH, and therefore it cannot be implicitly 
assumed that non‐red tissues lack anthocyanins 
(Gould et al. 2000). Anthocyanins have multiple 
functions in plants. They can protect shade‐
adapted chloroplasts from exposure to high light 
intensity (Gould et al. 2000), and act as chemical 
repellents and visual signals. Therefore, they are 
also important in plant and animal interactions 
(Carrillo-Parra et al. 2012). Some anthocyanins 
also have significant antiviral, antibacterial, and 
fungicidal activities (Lev-Yadun and Gould 2008), 
which provides an advantage for plants with high 
anthocyanin levels. Their levels can also increase 
due to low temperatures, which was the case for a 
container-grown red maple in Georgia (Sibley et 
al. 1999). The anthocyanin production is related 
to the redox potential of the plastoquinone pool 
in the photosynthetic electron transport chain 
that regulates the anthocyanin biosynthesis genes 
(Kumar Das et al. 2011). In the study with green 
and red stems of C� stolonifera, Gould et al. (2010) 
showed high attenuation of PAR by anthocyanins, 
especially of green and yellow light, and pro-
nounced differences in photoprotective potential 
and anthocyanin concentrations in the red stems 
compared to the green stems. C� sericea var. 
flaviramea showed the highest reflectance in the 
UV and NIR regions, and the lowest reflectance 
in the visible regions. The reflectance peak of 
other species was wide, and ranged from green 
to red, except for C� avellana, which had a peak 
in the green region. The reflectance of V� lantana 
gradually increased along the spectra, with a slight 
depression in the red edge. These reflectances were 
related to bark morphology and biochemistry, as 
shown by RDA. Anthocyanins act as effective light 
screening substances (Kumar Das et al. 2011), as 
also seen in the present study. Negative relation-
ship was seen between the anthocyanins and the 
reflected light especially in the green, yellow, and 
blue regions. Their importance was also shown 
by RDA performed with specific regions of the 
spectrum, where anthocyanins explained 48% of 
the green and yellow reflectance spectra variability. 
In addition to anthocyanins, stems can contain high 
levels of other products of the phenylpropanoid 
pathway. The accumulation of UV-absorbing 
substances represents a primary mechanism by 
which plants can acclimatise to changing UV 
environments, including altered UV-B (Bornman 
et al. 2015). These phenolic substances strongly 
absorb in the UV region, while they reflect light 
of longer wavelengths. However, when a vari-
ety of vegetable crop plants were examined for 
tolerance to UV, it was shown that the levels of 
chlorophyll and UV-absorbing substances did not 
correlate directly with their sensitivity (Smith et 
al. 2000). The reflectance in the NIR region was 
explained by the UV-absorbing substances. The 
study of Kokaly and Skidmore (2015) showed 
that reflectance of different phenolic compounds 
in plants increases with increasing wavelength 
and reaches its peak at around 1600 nm. We ob-
tained positive relationship between the amount 
65Grašič et al.: Bark spectral signatures of shrubs
of UV-absorbing substances and the reflectance 
spectrum in the NIR region.
The presence of photosynthesis in green 
stems and in the bark of woody stems has been 
reported for different tree species (Pfanz 1999). 
Green stems without a well-developed periderm 
and with abundant, functional stomata can carry 
out photosynthesis (Nilsen 1995). The lack of 
stomata on woody stems results in the accumula-
tion of respiratory CO2, which supports CO2-rich 
and O2-poor environments (Mancuso and Marras 
2003). CO2 might also diffuse into the chloren-
chyma through the lenticels (Eyles et al. 2009). 
The highest photosynthetic activity takes place in 
the outer bark, which contains higher amounts of 
photosynthetic pigments compared to the inner 
bark, which has less chlorophyll and carotenoids, 
and mainly serves for storage of assimilates that 
are produced in the outer bark or in the leaves 
(Pilarski 1999). The photochemical efficiency of PS 
II in the bark has been reported to be much lower 
than in the leaves (Manetas and Yiotis 2009). The 
Fv/Fm measured in the present study ranged from 
relatively low in C� avellana (0.36) to relatively 
high in S� nigra (0.71). These efficiencies reveal 
the presence of strong and mild permanent stress, 
respectively. The mean for the photosynthetic 
apparatus for undisturbed functioning is close to 
0.83 (Björkman and Demmig 1987). Fq’/Fm’ was 
low and did not differ across these species (means 
from 0.11 to 0.15), which showed strong transient 
stress that was possibly the consequence of the 
low temperatures during the sampling period. 
Solhaug and Haugen (1998) reported that the low-
est photochemical efficiency of PS II in bark was 
detected during the winter period, due to the low 
temperatures. The chlorophyll fluorescence might 
also reflect the wood structural changes, such as 
changes in the bark chlorenchyma (Johnstone et 
al. 2014). High Fv/Fm along with efficient uptake 
of internal CO2 might support the maintenance of a 
favourable carbon balance in the plant, especially 
during winter (Ivanov et al. 2006).
conclusions
The parameters that affected bark reflectance of 
1-year-old twigs varied across the studied species. 
The present results revealed high importance of 
twig biochemistry for light reflectance. However, 
these relations may not be directly transferable 
to the plant level, since they depend on twig age 
and also on environmental conditions that may 
significantly alter plant traits. Therefore, additional 
research is needed to study this relation in twigs 
of different age, and in different times of the 
season, which is especially important in the case 
of deciduous species. Nevertheless, bark of the 
species examined here shows different spectral 
signatures, which enables differentiation across 
species out of the vegetation season.
Summary
Bark comprises tissues outside the vascular 
cambium, and consists of the inner bark, which 
contains secondary phloem, and the outer bark or 
periderm. Bark protects the stem from physical 
disturbances, environmental factors and pollut-
ants, and enables transport of assimilates and 
water. The physiological and biochemical traits 
of bark vary greatly between species. This study 
examined factors that affect bark reflectance of 
1-year-old twigs of different shrub species: Cory-
lus avellana, Rosa canina, Ligustrum vulgare, 
Sambucus nigra, Cornus sericea var. flaviramea, 
and Viburnum lantana. We hypothesised that bark 
biochemical properties and bark thickness play an 
important role in shaping bark reflectance and thus 
also the so-called “bark spectral signatures”. We 
analysed contents of various pigments and UV-
absorbing substances in the bark and measured 
bark thickness and bark reflectance spectra. Bark 
morphological and biochemical properties showed 
great variability between the species. The bark of 
C� sericea var. flaviramea differed the most from 
the other species, showing the highest amounts of 
total chlorophyll, UV-absorbing substances, and 
anthocyanins. The peridermal layer was developed 
differently in the studied species. V� lantana had 
the thickest outer bark, while the inner bark was 
thickest for S� nigra. R� canina and C� sericea 
66Acta Biologica Slovenica, 2021, 64 (1), 56–69
var. flaviramea had an epidermis and a very thin 
peridermal layer. Photosynthetic pigments in the 
bark allow the process of photosynthesis, which 
mostly takes place in the outer bark. Outer bark 
contains higher amounts of pigments compared 
to the inner bark, which serves primarily as an 
assimilate storage. Fv/Fm was relatively high in 
most of the studied species, while Fq’/Fm’ was 
relatively low and did not differ between the 
species. Redundancy analysis revealed associa-
tions between bark biochemical properties and 
reflectance in the visible part of the spectrum. 
UV-B–absorbing substances explained 38% of the 
variability of the reflectance spectra (p = 0.001), 
outer bark thickness explained 12% (p = 0.001), 
inner bark thickness 7% (p = 0.001), and anthocya-
nins explained an additional 4% (p = 0.009) of the 
variability. Therefore, these parameters together 
explained as much as 61% of the variability of 
the reflectance spectra. Bark of the studied spe-
cies showed different spectral signatures resulting 
from different bark physiological and biochemical 
properties. This allows us to differentiate between 
species out of the vegetation season.
Povzetek
Skorja zajema tkiva zunaj vaskularnega 
kambija in je sestavljena iz notranje skorje, ki 
vsebuje sekundarni floem, ter zunanje skorje 
ali periderma. Skorja ščiti steblo pred fizičnimi 
motnjami, okoljskimi dejavniki in onesnaževali ter 
omogoča transport asimilatov in vode. Fiziološke in 
biokemijske lastnosti skorje se med vrstami močno 
razlikujejo. V raziskavi smo proučili dejavnike, 
ki vplivajo na odbojnost skorje enoletnih vejic 
različnih grmovnih vrst: Corylus avellana, Rosa 
canina, Ligustrum vulgare, Sambucus nigra, Cor-
nus sericea var. flaviramea in Viburnum lantana. 
Predvidevali smo, da imajo biokemijske lastnosti in 
debelina skorje pomembno vlogo pri oblikovanju 
odbojnosti oziroma tako imenovanih “spektralnih 
podpisov” skorje. Analizirali smo vsebnost raz-
ličnih pigmentov in UV-absorbirajočih snovi v 
skorji ter izmerili debelino skorje in njene odbojne 
spektre. Morfološke in biokemijske lastnosti skorje 
so pokazale veliko variabilnost med vrstami. Naj-
bolj se je od ostalih vrst razlikovala skorja vrste 
C� sericea var. flaviramea, ki je vsebovala največ 
skupnih klorofilov, UV-absorbirajočih snovi in 
antocianinov. Proučevane vrste so imele različno 
razvito peridermalno plast. Najdebelejšo zunanjo 
skorjo je imela vrsta V� lantana, medtem ko je bila 
notranja skorja najdebelejša pri vrsti S� nigra. Vrsti 
R� canina in C� sericea var. flaviramea sta imeli 
prisotno povrhnjico in zelo tanko plast periderma. 
Fotosintezni pigmenti v skorji omogočajo proces 
fotosinteze, ki večinoma poteka v zunanji skorji. 
Ta vsebuje več pigmentov v primerjavi z notranjo 
skorjo, ki je namenjena predvsem shranjevanju 
asimilatov. Potencialna fotokemična učinkovitost 
FS II je bila pri večini proučevanih vrst razmeroma 
visoka, dejanska fotokemična učinkovitost FS II pa 
relativno nizka in se ni razlikovala med vrstami. 
Redundančna analiza je razkrila povezave med 
biokemijskimi lastnostmi in odbojnostjo skorje v 
vidnem delu spektra. UV-B–absorbirajoče snovi 
so razložile 38 % variabilnosti odbojnih spek-
trov (p = 0,001), debelina zunanje skorje 12 % 
(p = 0,001), debelina notranje skorje 7 % (p = 
0,001), vsebnost antocianinov pa še dodatne 4 
% (p = 0,009) variabilnosti. Omenjeni parametri 
so skupaj torej pojasnili kar 61 % variabilnosti 
odbojnih spektrov. Skorja proučevanih vrst je 
pokazala različne spektralne podpise, ki so 
posledica različnih fizioloških in biokemijskih 
lastnosti skorje. To nam omogoča razlikovanje 
med vrstami izven vegetacijske sezone.
Acknowledgements
The authors acknowledge financial support 
from the Slovenian Research Agency through 
core research funding for the programme Plant 
Biology (P1-0212) and the project Young Re-
searchers (39096). The authors would like to 
thank Dragan Abram for assistance with research 
work, and Christopher Berrie for revision of the 
English writing.
67Grašič et al.: Bark spectral signatures of shrubs
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