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Composition of photosynthetic pigments and photosynthetic characteristics

in green and yellow sectors of the variegated Aucuba japonica ‘Variegata’
leaves

Article  in  Flora - Morphology Distribution Functional Ecology of Plants · December 2017

DOI: 10.1016/j.flora.2017.12.010

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 Flora 240 (2018) 25–33

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journal homepage: www.elsevier.com/locate/flora

Composition of photosynthetic pigments and photosynthetic characteristics T

in green and yellow sectors of the variegated Aucuba japonica ‘Variegata’
leaves
⁎
Qiang Zhanga, Min Zhangb, Yin Dingc, Peng Zhoub, Yanming Fanga,
a
Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, 159 Longpan Road, Nanjing,
210037, PR China
b
Jiangsu Academy of Forestry, Dong Shanqiao, Nanjing, 211153, PR China
c
State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, 22 Hankou Road, Nanjing, 210093, PR
China

A R T I C L E I N F O A B S T R A C T

Edited by Alessio Papini Aucuba japonica ‘Variegata’ is a widely used ornamental shrub with green-yellow variegated leaves. In this study,
Keywords: the formation of leaf variegation and photosynthetic characteristics in green and yellow sectors were in-
Aucuba japonica ‘Variegata’ vestigated. There were no marked anatomical differences in tissue organization between the green and yellow
Leaf variegation sectors. At the cellular level, it was observed that the chloroplasts in the yellow leaf tissue were vacuolated.
Photosynthetic pigments Besides, the pigment contents of the yellow leaf tissue were obviously lower than those in the green areas, and a
Chlorophyll fluorescence parameters very low intensity of chlorophyll auto-fluorescence was generated from the yellow areas. Furthermore, sig-
nificantly lower values of F0, Fm, Fv/Fm, ФPSII and non-photochemical quenching (NPQ) were noticed in yellow
sectors compared to the green ones, indicating that the yellow leaf tissue was less photoprotected than the green
area. In addition, the yellow sectors showed lower net photosynthesis and dark respiration rates compared to the
green leaf tissue. Immunofluorescence showed large amounts of ribulose-1, 5-bisphosphate carboxylase/oxy-
genase (Rubisco) in green leaf tissue, while only faint fluorescence was detected in the yellow sectors. As a
whole, the results of this study suggest that the leaf variegation of A. japonica ‘Variegata’ is “pigment type” and
that this pigment-related leaf variegation affects photosynthetic light use in the variegated leaves. These findings
also shed light on the coloration mechanism of ornamental plant foliage.

1. Introduction
Plant leaves are usually characterized by uniformly colored sur-
faces, but some plant species possess variegated leaves. The variegated
leaves display irregular spots or patches and regular patterns of color
distribution on the leaf surface (Sheue et al., 2012). A large number of
variegated plants are cultivated as garden ornamentals. They are also
relatively common in the forest understory, although few plant groups
have evolved variegation naturally (Esteban et al., 2008). In a study of
55 species belonging to 24 families, Hara (1957) recognized four types
of foliar variegation named “chlorophyll type,” “pigment type,” “air
space type,” and “epidermis type,” which fell into two groups: pigment-
related variegation and structural variegation.
The adaptive significance of leaf variegation is generally thought to
be defensive (Lev-Yadun et al., 2002). It was proposed that leaf var-
iegation may potentially provide defense from herbivory through
“dazzle effects” and “trickery coloration,” i.e., mottling on the leaves
disrupts the leaf outline creating a visual illusion and causing identifi-
cation problems for insects searching for specific leaf types (Campitelli
et al., 2008; Lev-Yadun, 2014). Aside from defending plants from her-
bivory, the “non-green” leaf sections may also be involved in increased
photoprotection. Generally, non-photochemical quenching (NPQ)
(Stern-Volmer non-photochemical quenching coefficient, Bilger and
Björkman 1991) is a method of photoprotection through the loss of
excess energy as heat. However, plants also have other mechanisms to
reduce excess energy from light capture. In variegated leaves, the
mottled sections reflect light more effectively than the green tissues
(Esteban et al., 2008).
Studies on the molecular mechanisms of leaf variegation have pro-
gressed considerably in recent years. A series of leaf-variegated mutants
of Arabidopsis thaliana have been investigated, which facilitated our
understanding of the molecular mechanisms involved in variegation.

⁎
Corresponding author.
E-mail address: ymfangnfu@163.com (Y. Fang).

https://doi.org/10.1016/j.flora.2017.12.010
Received 30 September 2016; Received in revised form 20 December 2017; Accepted 21 December 2017
Available online 24 December 2017
0367-2530/ © 2017 Elsevier GmbH. All rights reserved.
Q. Zhang et al.
Among these mutants, the yellow variegated1 (var1) and var2 have been
extensively studied. The genes responsible, VAR1 and VAR2, encode the
FtsH metalloproteases FtsH5 and FtsH2, respectively (Chen et al., 2000;
Liu et al., 2010; Miura et al., 2007). FtsH is a thylakoid-localized pro-
tease that degrades several chloroplastic proteins. The results of Miura
et al. (2007) suggest that the balance between protein synthesis and
degradation in chloroplasts plays a crucial role in determining the
variegated phenotype in Arabidopsis leaves. In Nicotiana tabacum, it was
found that the suppression of FtsH gave rise to leaf variegation (Kato
et al., 2012). It was reported that mutation of Thylakoid Formation1
(THF1) could also lead to leaf variegation in Arabidopsis (Hu et al.,
2015; Wang et al., 2004). A more recent work showed that silencing the
DnaJ-like zinc finger domain protein PSA2 in A. thaliana resulted in
disturbance of chloroplast development and variegated leaves (Wang
et al., 2016a). Altogether, these studies suggest that the formation of
variegated leaves is associated with the mutation of genes involved in
the development of chloroplasts. However, one research group pro-
posed that leaf variegation of Clivia miniata var. variegata might be due
to differential DNA methylation in CCGG sites (Wang et al., 2016b).
Although leaf variegation could bring beneficial defensive effects to
the plants, it may also have adverse impacts. This disadvantage is re-
lated to the unavoidable decrease in light capture and the corre-
sponding decrease in photosynthetic rates (Konoplyova et al., 2008). In
addition, elevated photosynthetic rates were detected in green leaf
sectors of Arabidopsis variegation immutans (im), which was thought to
indicate a means of compensating for the lack of photosynthesis in the
white leaf sectors (Aluru et al., 2006). Recently, Borek et al. (2016)
examined the photosynthetic activity in variegated leaves of five cul-
tivars of Coleus × hybridus using chlorophyll fluorescence techniques.
They revealed heterogeneity in the capture, transfer, and dissipation of
excitation energy in differentially pigmented sectors of the leaves.
Chlorophyll a fluorescence has become a useful non-invasive tool
used to detect the response of plants to the ambient environment.
Among the fluorescence parameters, Fv/Fm (potential photochemical
efficiency of open PS II units determined in darkness) can be used as an
indicator of the photosynthetic activity of PS II. A persistent decline in
Fv/Fm values is a signal of photoinhibition of PS II and a decrease in
energy conversion efficiency (Krause and Jahns, 2004). Non-photo-
chemical quenching (NPQ) in PS II dissipates the excess energy as heat,
protecting leaves from light-induced damage (Johnson et al., 2011).
Thus, a high NPQ value signifies effective thermal energy dissipation.
To date, most of the information about leaf variegation was mainly
obtained from the study of herbaceous plants. By comparison, in-
formation obtained from the study of variegated woody plants is very
limited. To gain insights into the photosynthetic characteristics of the
variegated leaves of woody plants, Aucuba japonica ‘Variegata’ was used
as a model system in our study. A. japonica ‘Variegata’ is widely planted
in China as an ornamental garden plant. This species is best adapted to
shaded locations. Typically, the leaves of A. japonica ‘Variegata’ exhibit
an irregular pattern of yellow patches and spots. A previous study
showed that A. japonica ‘Variegata’ had a low light compensation point
and a relatively high light saturation point; furthermore, stomatal
conductance played a central role in the net photosynthetic rate in
growing seasons (Xu et al., 2009). The wild Aucuba japonica is an
evergreen dioecious shrub species located in the warm-temperate re-
gions of Japan (Abe, 2001). Anisophylly was found in this plant species
and was considered an effective means of minimizing self-shading (Ali
and Kikuzawa, 2005a). Moreover, plasticity in leaf-area density within
the crown was found in response to different light regimes (Ali and
Kikuzawa, 2005b). In our study, we explored the reason for the for-
mation of the variegated leaves of A. japonica ‘Variegata’. We also in-
vestigated the photosynthetic activities in differently colored parts
(green and yellow) of the variegated leaves by focusing our study on the
content and in situ localization of photosynthetic pigments in the green
and yellow sectors of A. japonica ‘Variegata’ leaves. The green and
yellowish areas of the leaves were investigated through chlorophyll
26
Flora 240 (2018) 25–33
fluorescence imaging techniques. Moreover, the expression and locali-
zation of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco)
in different sectors of the variegated leaves was also examined.
2. Materials and methods
2.1. Plant materials
The plant used in this study was Aucuba japonica ‘Variegata,’ which
has green leaves with irregular yellow patches and spots. A. japonica
‘Variegata’ was grown in a garden on the campus of Nanjing Forestry
University, China. The maximum photosynthetic photon flux density
was approximately 700 μmol/m2 s−1, and plants were watered every
2 days. Measurements were taken in June. Three plants were randomly
selected, and six leaves were sampled from each plant. Mature leaves
were collected in air-tight bags and immediately transferred to the la-
boratory for analysis.
2.2. Microscopic observation
The green and yellow parts of fully developed fresh leaves were
dissected and immediately embedded in OCT compound (Sakura
Finetek, CA, USA). Then, 20-μm cross-sections were cut using a Leica
CM1950 cryostat (Leica Biosystems Nussloch GmbH, Heidelberger,
Germany). Sections were mounted on poly-L-Lys-coated microscopic
slides, and images were captured using a Nikon Eclipse 50i light mi-
croscope equipped with a Nikon DS-Ri1 digital camera.
2.3. Histological analysis
For histological analysis, the green and yellow parts of fully devel-
oped leaves were dissected and processed according to the previously
described method (Voznesenskaya et al., 2004) with minor modifica-
tion. The dissected leaves were fixed in 4% (v/v) glutaraldehyde in
0.1 M phosphate buffer (pH 7.2) and postfixed in 4% (w/v) OsO4. After
a dehydration procedure with a graded ethanol series, leaf samples
were embedded in Epon 812 resin. Then, semithin sections were cut
and stained with 1% (w/v) Toluidine blue O in 1% (w/v) Na2B4O7 for
general histology or with periodic acid–Schiff reagent for the staining of
starch.
2.4. Transmission electron microscopy
The green and yellow parts of freshly harvested leaves were cut into
small pieces (2 mm × 3 mm) and fixed with 2.5% glutaraldehyde.
Samples were then postfixed with 1% OsO4, dehydrated in ethanol, and
embedded in Epon 812 resin. Ultrathin sections were stained with ur-
anyl acetate followed by lead citrate and examined with a JEM-1400
transmission electron microscope (JEOL, Japan) at 120 kV.
2.5. Pigment analysis
To determine the content of pigments in the leaves, the green and
yellow parts in the leaves were carefully dissected using microsurgical
scissors and forceps. The leaves for pigment analysis were sampled at
10 a.m. Subsequently, 0.2 g (fresh weight) of dissected green and 0.2 g
(fresh weight) of yellow leaves were each placed in 3 mL of 80%
acetone. The extraction was carried out in the dark for 24 h at 4 °C.
Chlorophyll concentrations were estimated according to Lichtenthaler
(1987). The following formula was used:
Chl a (mg/g) = 12.25A663.2−2.79A646.8
Chl b (mg/g) = 21.50A646.8−5.10A663.2
Total Chl (mg/g) = 7.15A663.2 + 18.71A646.8
Q. Zhang et al. Flora 240 (2018) 25–33

Carotenoids (mg/g) = (1000A470−1.82Chl a−85.02Chl b)/198

For the estimation of anthocyanin levels, the excised leaves were

extracted with 3 mL of 6 M HCl: H2O: MeOH (7: 23: 70) for 24 h at 4 °C.
Then, the methanol-HCl extracts were measured spectro-
photometrically as described by Hughes et al. (2007).

2.6. Laser confocal microscopy of pigments in leaf tissues

The in situ distribution of chlorophyll, carotenoids, and anthocya-

nins in the leaves was visualized with a Leica TCS SP5 confocal scan-
ning microscope (Leica Microsystems, Heidelberg GmbH, Mannheim,
Germany). The freshly excised leaf samples were embedded in OCT
compound (Sakura Finetek, CA, USA) and sectioned with a Leica CM
1950 cryostat. The cross sections (10 μm) were then mounted onto
poly-lysine-coated slides. Chlorophyll fluorescence was visualized with
a 633-nm helium-neon laser, and the emitted fluorescence was col-
lected between 650 and 700 nm (Egea et al., 2011). The auto-
fluorescence of carotenoid was excited using an argon laser (488 nm,
emission 500–600 nm). A 543-nm helium-neon laser was used to excite
anthocyanins (Gomez et al., 2011). The tissues were counterstained
with DAPI (4′, 6′-diamidino-2-phenylindole).

2.7. Chlorophyll fluorescence

Chlorophyll fluorescence imaging was performed using a CF Imager

(Technologica, Colchester, UK) following the manufacturer’s instruc-
tions. All measurements were performed on intact leaves. Leaves were
dark-adapted for 30 min before the measurements. CF images were
captured following the procedures shown in Table 1. The light resource
of CF Imager was provided by blue light. Fluorescence data were ana-
lyzed using FluorImager software. The formulas for the calculation of
each parameter are as follows (Borek et al., 2016):

Fv'/Fm' = (Fm'−Fo')/Fm'

ϕPSII = (Fm'−Ft)/Fm'

qP = (Fm'−Ft)/(Fm'-F0′).

NPQ = (Fm−Fm')/Fm'

2.8. Photosynthesis and respiration estimation

Fig. 1. Light micrographs showing transverse sections through green (B), yellow (C), and
transition regions (D) of the variegated A. japonica ‘Variegata’ leaves (A). Bars = 50 μm.
Photosynthetic oxygen evolution and respiratory O2 uptake of the (For interpretation of the references to colour in this figure legend, the reader is referred
green and yellow parts in A. japonica ‘Variegata’ leaves were measured to the web version of this article.)
with a Clark-type Liquid-Phase Oxygen Measurement system
(Chlorolab-2, Hansatech Instruments Ltd., King’s Lynn, UK) according
Cousins et al. (2003) with some modifications. Fresh leaves of green
to the manufacturer’s instructions. Leaf discs measuring 0.1 g were put
and yellow and the green-yellow transition regions were fixed in 4%
into 2 mL of bicarbonate buffer, and the dissolved O2 was measured.
paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.4. Samples
The analyses were carried out under 1000 μmol·m−2·s−1 illumination at
were then embedded in OCT compound (Sakura Finetek, CA, USA), and
a constant temperature of 25 °C. The net photosynthesis was obtained
10-μm cross-sections were cut using a Leica CM1950 cryostat. Subse-
by subtraction of dark respiration.
quently, the sections were adhered to poly-lysine-coated slides. Sections
were treated with 1% BSA in PBS for 15 min to block the non-specific
2.9. In situ immunolocalization
proteins. Then, the slides were incubated overnight at 4 °C with the
polyclonal primary rabbit anti-Rubisco antibody (Agrisera, Vännäs,
The immunolocalization of ribulose-1, 5-bisphosphate carboxylase/
Sweden). Sections were then washed with PBS and incubated with
oxygenase (Rubisco) in the leaves was performed in accordance with
Alexa Fluor™ 635 Goat anti-Rabbit secondary antibody (Invitrogen™,
Thermo Fisher Scientific Inc.) for 30 min at room temperature. Locali-
Table 1
Procedures for chlorophyll fluorescence imaging.
zation of Rubisco was visualized on a Leica TCS SP5 confocal micro-
scope.
Step Delay Action Cycles PPFD (μmol/m−2 s−1)

1 0s Change actinic 0 2.10. Statistical analysis

2 30 min Apply pulse 1 6164
3 1s Change actinic 500
4 30 min Apply pulse 6164 Statistical analysis was performed by one-way ANOVA using
SigmaStat V3.5 software. All data are presented as the mean ±

27
Q. Zhang et al.
standard error of mean (SEM) of three experiments. To determine
significant differences between green and yellow parts, the Duncan’s
multiple range test was used.
3. Results
3.1. Leaf anatomy and ultrastructure
Light-microscopic photographs showed that both the green and
yellow parts of the leaves were composed of two layers of palisade
parenchyma and approximately eight layers of spongy parenchyma
(Figs. 1 and 2). There were no obvious differences found in the ar-
rangement of cells between green and yellow parts; however, extremely
low chlorophyll content was already evident in the yellow parts (Fig. 1).
In addition, differences in the number and distribution of chloroplasts
were noticed. In the green leaf tissue, numerous chloroplasts were
present in the chlorenchyma, especially in the palisade cells
(Fig. 1Figs. 1B and Fig. 22A, B). The chloroplasts were found close to
28
Flora 240 (2018) 25–33
Fig. 2. General anatomy of green (A) and yellow (C)
sectors of the variegated A. japonica ‘Variegata’ leaf.
B and D are high-magnification images of the
squared regions (red boxes) in (A) and (C), respec-
tively. Sections were stained with Toluidine blue O,
and images were captured using a Nikon Eclipse 50i
light microscope. Bars = 100 μm. (For interpretation
of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
Fig. 3. PAS staining for starch localization in the
leaves. (A) green sector, (C) yellow sector. B and D
are high-magnification images of the squared regions
(red boxes) in (A) and (C), respectively. Red arrow
shows the starch grains accumulated in the chlor-
enchyma cells. Bars = 100 μm. (For interpretation of
the references to colour in this figure legend, the
reader is referred to the web version of this article.)
the cell walls. In the yellow leaf tissue, the chloroplasts were scarcely
observed in either the palisade cells or spongy cells (Fig. 1Figs. 1C and
Fig. 22C, D). Moreover, we noticed a clear border between the green
and yellow parts in the transition region of the leaves (Fig. 1D).
PAS staining showed a large number of starch grains accumulated in
the palisade and spongy cells in the green parts of the leaves (Fig. 3A,
B). By contrast, only a few starch grains were scattered in the chlor-
enchyma cells in the yellow parts (Fig. 3C, D).
The leaves were further investigated with TEM to compare differ-
ences in the cell structures of the green and yellow parts. As shown in
Fig. 4A, large numbers of grana stacks with normal appearance were
observed in the chloroplast of green tissue. By contrast, the yellow leaf
tissue had underdeveloped chloroplasts with dilated lamellae systems
(Fig. 4B).
3.2. Pigment contents
Chlorophylls, carotenoids, and anthocyanins were quantified in the
Q. Zhang et al. Flora 240 (2018) 25–33

red chlorophyll fluorescence. By contrast, only low intensity chlor-

ophyll fluorescence was observed in these areas in the yellow parts.
Interestingly, strong chlorophyll fluorescence was noticed in the site
corresponding to the stomata in the lower epidermis of the yellow parts.
Compared to chlorophyll fluorescence, only faint carotenoid fluores-
cence was seen in the chlorenchyma of the green parts. Strong fluor-
escence was found in both epidermises. The distribution of carotenoids
in the yellow parts showed a similar pattern to the distribution in the
green parts. Measuring fluorescence is a less direct method than pig-
ment extraction and is used as a gross comparison that highlights dif-
ferences in the locations of carotenoids between green and yellow spots.
Autofluorescence of anthocyanins was present in the leaf tissue con-
taining the palisade tissue and spongy parenchyma. There were no
differences in the distribution and intensity of anthocyanin fluorescence
between the green and yellow parts. Moreover, a sharp decrease in the
intensity of chlorophyll fluorescence was observed in the yellow part of
the transition region of the leaf (Suppl. Fig. 1).

3.4. Chlorophyll a fluorescence

Chlorophyll a fluorescence parameters in the green and yellow parts

Fig. 4. Electron microscopy of A. japonica ‘Variegata’ chlorenchyma cells in green (A) and
yellow (B) sectors. PG, plastoglobule; M, mitochondrion; GR, grana; TH, thylakoid. Scale
bar = 2 μm. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
were measured using a fluorescence imaging system (Suppl. Figs. 2–5).
The minimum level of fluorescence (Fo) and the maximum fluorescence
(Fm) were substantially higher in the green parts than in the yellow
parts (Table 3). Maximum photochemical efficiency of PSII (Fv/Fm) was
also significantly higher in the green parts, but there were no differ-
ences in Fvʹ/Fmʹ (maximum efficiency of PSII photochemistry in the
light) between the green and yellow parts. In contrast to these para-
meters, the operating efficiency of PSII (ϕPSII) and photochemical
quenching (qP) were significantly lower in the green parts than in the
yellow parts. Additionally, non-photochemical quenching (NPQ) in the
green parts was significantly high.
3.5. Photosynthesis and respiration in the leaves

Table 2
The respiratory oxygen uptake and net photosynthetic oxygen
Pigment contents of green and yellow sectors.
evolution rates in the green and yellow parts of the leaves were mea-
Green Yellow sured (Table 4). These measurements showed that the rate of re-
spiratory O2 uptake in the green parts was significantly higher than in
−1 a
Chl a (mg g FW) 1.77 ± 0.144 0.03 ± 0.0003 b
the yellow parts. The gross photosynthesis rate in the green parts
Chl b (mg g−1 FW) 0.71 ± 0.061 a
0.02 ± 0.0002 b
Chl a + b (mg g−1 FW) 2.48 ± 0.203 a
0.05 ± 0.0017 b
tended to increase dramatically, which led to a 6.43-fold increase in
Chl a/b 2.51 ± 0.058 a
1.38 ± 0.150 b gross photosynthesis compared to the yellow parts. The net photo-
Car (mg g−1 FW) 0.40 ± 0.024 a
0.04 ± 0.002 b synthetic oxygen evolution was very high in the green parts, but in the
a
Chl a + b/Car 6.20 ± 0.269 1.25 ± 0. 070 b yellow parts, the value was negative. This suggests that the rate of re-
Ant (mg g−1 FW) 0.13 ± 0.004 a
0.14 ± 0.005 a
spiration was greater than the rate of photosynthesis in the yellow
Data were means ± SEM for 3 replicates of samples. Lowercase letters indicated sig-
parts.
nificant difference between green and yellow sectors of the same parameter.

green and yellow parts of the leaves (Table 2). The contents of chlor-
ophyll a, b and total chlorophyll in the green parts were almost two
orders of magnitude higher than those in yellow parts. The chlorophyll
a/b ratio was also significantly higher in the green parts. In addition,
the green parts contained many more carotenoids than the yellow parts.
A remarkable chlorophyll a + b/carotenoids ratio was noticed in the
green parts; however, there was no significant difference in antho-
cyanin content between the green and yellow parts.
3.3. Distribution of pigments in the leaves
The localizations of chlorophyll, carotenoids, and anthocyanins in
leaf tissues were examined using laser confocal microscopy. As shown
in Fig. 5, large numbers of chloroplasts accumulated in the palisade
tissue and spongy parenchyma in the green parts and exhibited intense
3.6. Expression and localization of rubisco in leaf tissues
In order to gain more insight into the photosynthetic characteristics
of different leaf sectors, the expression and localization of Rubisco in
the leaves were analyzed by immunofluorescence (Fig. 6). Rubisco was
detected in the chloroplasts of both the palisade cells and mesophyll
cells in the green parts. In the upper epidermis, Rubisco was almost
undetectable. Meanwhile, in the lower epidermis, it appeared to be
localized exclusively to the chloroplasts of the stomata cells. The in-
tensity of the fluorescence in the green parts was very strong. In con-
trast to the green parts, only dim fluorescence was detected in the
yellow parts of the leaves. Interestingly, an intense Rubisco fluores-
cence was observed in the chloroplasts of the stomata cells in the lower
epidermis of the yellow parts. In the transition region of the leaf, dis-
tinct differences in the expression and localization of Rubisco were
noticed between the green and yellow parts (Suppl. Fig. 6).

29
Q. Zhang et al. Flora 240 (2018) 25–33

Fig. 5. The in situ distribution of chlorophyll, car-

otenoids, and anthocyanins in the variegated A. ja-
ponica ‘Variegata’ leaves as visualized by confocal
laser-scanning microscopy. The autofluorescence of
chlorophyll, carotenoid, and anthocyanins was ex-
amined with a Leica TCS SP5 confocal scanning mi-
croscope. P, palisade tissue; S, spongy parenchyma;
DIC, differential interference contrast. Scale
bar = 100 μm.

Table 3 variegated leaves, the green sectors have a wild-type genotype, while
Chlorophyll a fluorescence parameters of the green and yellow sectors in variegated the white sectors possess a mutant genotype (Chen et al., 2000). Leaf
Aucuba japonica ‘Variegata’ leaves. variegation of some species, including Begonia, Canna, and Polyanthes
Green Yellow
tuberose,is caused by somatic mutations, and these mutated plants are
maintained only through microtechniques used for plant regeneration
Fo 3734.33 ± 187.38a 653.53 ± 55.86b (Gottschalk and Wolff, 1983). Leaf variegation can also be induced by
Fm 19137.33 ± 646.72a 2803.67 ± 213.59b viral infection. Saitoh and Terauchi (2002) reported that virus-induced
Fv/Fm 0.81 ± 0.004a 0.77 ± 0.004b
silencing of the FtsH gene in Nicotiana benthamiana led to variegated
Fvʹ/Fmʹ 0.56 ± 0.009a 0.60 ± 0.023a
ΦPSII 0.46 ± 0.009b 0.56 ± 0.017a leaves similar to the var2 mutant of A. thaliana. Based on the work of
qP 0.82 ± 0.004b 0.94 ± 0.008a Hara (1957), foliar variegation is divided into two types: pigment-re-
NPQ 2.28 ± 0.086a 1.42 ± 0.173b lated and structural variegation. The above mentioned conclusions of
leaf variegation were mainly based on the study of herbaceous plants.
Different lowercase letters in the same line indicated significant difference between green
Here in our study, we investigated the mechanism of woody plant leaf
and yellow sectors (P < .05).
variegation. We first examined the anatomy of the variegated A. japo-
nica ‘Variegata’ leaves. The anatomies of the green and yellowish areas
4. Discussion
of the leaves did not show obvious differences (Figs. 1 and 2), whereas
the structure of the chloroplasts from yellow sectors appeared aberrant
Leaf variegations are characterized by the presence of white, yellow,
compared to those from the green sectors (Fig. 4). Thus, the mechanism
red, or purple sectors over the surfaces of normally green leaves (Aluru
causing variegation in the leaves of A. japonica ‘Variegata’ was different
et al., 2006; Esteban et al., 2008). Generally, leaf variegations are
from the structural variegation found in Begonia (Sheue et al., 2012)
considered to be a result of genotypic characteristics, somatic muta-
and Arum italicum (Rocca et al., 2011). The histological characteristics
tions, or viral infections. For example, in nuclear gene-induced

30
Q. Zhang et al. Flora 240 (2018) 25–33

Table 4 of the variegated leaves of A. japonica ‘Variegata’ were comparable to

Photosynthesis and respiration rate of variegated Aucuba japonica ‘Variegata’ leaves. those of the Chrysanthemum variety ‘NAU04-1-31-1′ (Chang et al.,
2013), the A. thaliana var1 (FtsH5) mutant (Sakamoto et al., 2002), and
Rate (μmol O2%h−1%g−1 FW)
the FtsH knock-down tobacco (Kato et al., 2012). In the yellow parts of
Dark respiration Gross photosynthesis Net photosynthesis the variegated ‘NAU04-1-31-1′ leaves, abnormal chloroplasts were
found. As with the variegated Chrysanthemum leaves, aberrant chlor-
Green −5.54 ± 0.324a 7.59 ± 0.224a 2.04 ± 0.127a
oplasts were present in the yellow areas of the variegated A. japonica
Yellow -2.29 ± 0.119b 1.18 ± 0.055b −1.10 ± 0.138b
‘Variegata’ leaves (Fig. 4), which may indicate that the variegation of A.
Data were means ± SEM (n = 3). Values with different lowercase letters in the same japonica ‘Variegata’ leaves is a result of disturbances in chloroplast
column were significantly different (Duncan’s multiple range, P < .05). development. Variegation mutants in A. thaliana are caused by defects
in chloroplast development through mutations in nuclear or organellar
genes (Aluru et al., 2006). The yellow variegated Arabidopsis mutant

Fig. 6. Immunolocalization of Rubisco in the var-

iegated A. japonica ‘Variegata’ leaves. (A-D) green
leaf sector, (F-I) yellow leaf sector. E and J are the
enlarged images of the squared regions (red boxes).
P, palisade tissue; S, spongy parenchyma.
Bars = 100 μm. (For interpretation of the references
to colour in this figure legend, the reader is referred
to the web version of this article.)

31
Q. Zhang et al.
Var2, for example, is caused by a nuclear recessive gene mutation. The
plastids in the white sectors of this variegation mutant appeared va-
cuolated and abnormal (Chen et al., 1999).
The difference in the colors in variegated leaves depends on the
presence or absence of chlorophyll, carotenoids, and anthocyanins
(Esteban et al., 2008). Lower contents of chlorophyll a and b, car-
otenoids, and anthocyanins were detected in the light-green leaf sec-
tions compared with the dark-green leaf sections of Cyclamen purpur-
ascens (Klančnik et al., 2016). In Pulmonaria officinalis L. and Arum
italicum, the amounts of chlorophylls and carotenoids in the variegated
pale-green sectors were appreciably lower than in full-green sectors
(Esteban et al., 2008; Rocca et al., 2011). Total chlorophyll in the
yellow sectors of the variegated A. japonica ‘Variegata’ leaves was ob-
viously lower than in the green areas (Table 2). This phenomenon was
also observed in the chlorophyll-less gold-colored leaves of Ligustrum
vicaryi (Yuan et al., 2010) and the leaves of chimera Hosta “Gold
Standard” (Yu et al., 2016). The lower content of chlorophyll in the
non-green patches could decrease photosynthetic rates (Esteban et al.,
2008). In the present study, lower photosynthesis and respiration rates
and lower starch accumulation were found in the yellow sectors of the
variegated A. japonica ‘Variegata’ leaves. We also investigated the in situ
localization of the pigments in the variegated leaves, which showed
that only a very low intensity of chlorophyll auto-fluorescence was
generated from the yellow leaf tissues (Fig. 5). A similar result was
observed in the yellow sectors of variegated leaves of the Chry-
santhemum variety ‘NAU04-1-31-1′ (Chang et al., 2013).
It was assumed that the photosynthetic performance of the var-
iegated leaves decreased due to the reduction of light capture in the
chlorophyll-less leaf areas (Konoplyova et al., 2008). In recent years,
the chlorophyll a fluorescence techniques have been used widely in
monitoring the photosynthetic performance of plants in response to
various biotic and abiotic stresses (Baker, 2008; Borek et al., 2016). In
this study, we utilized a noninvasive method to examine the actual state
of the photosynthetic apparatus in the variegated leaves. It revealed
that the values of F0, Fm, Fv/Fm, and NPQ in the yellow sectors were
very low when compared to the green areas. Decline in these chlor-
ophyll fluorescence (CF) parameters, as described here, has also been
observed in the variegated leaves of Coleus × hybridus hort. cultivars
(Borek et al., 2016). Another report showed that the Fv/Fm and Y(II)
(effective PS-II quantum yield) values were lower in the variegated
leaves of Hedera helix, Ardisia pusilla, and Scindapsus aureus compared to
their green counterparts (Khalekuzzaman et al., 2015). A dramatic
decline in Fv/Fm was also observed in the light areas of leaves of Ficus
pumila ‘Sonny’ (Sheue et al., 2012). The lowered Fv/Fm in the yellow
sectors indicated loss of PSII efficiency, which may be a result of a
decline in the PSII centers capable of photochemistry (Baker and
Oxborough, 2004). In contrast, a conflicting result of qP was noticed in
the yellow sectors of A. japonica ‘Variegata’ leaves, which could be due
to differences between the plant species. The high ΦPSII together with
low NPQ in these non-green areas indicated that the yellow sectors
were less photoprotected than the green leaf tissues.
The photosynthetic activity in the variegated A. japonica ‘Variegata’
leaves was further examined by measuring O2 evolution. As expected,
the net photosynthesis rate in the green tissues was much higher than
that in the yellow sectors. Contrary to our results, no significant dif-
ferences in photosynthesis between the pale-green and dark-green areas
of the variegated Cyclamen hederifolium leaves were found (Konoplyova
et al., 2008). This may be due to the different types of foliar variega-
tion. Another interesting finding in our study was the lower dark re-
spiration rates found in the yellow sectors. This phenomenon was also
observed in the white sectors of the variegated leaves of 12 plant spe-
cies including dicots and monocots (Toshoji et al., 2012). It was sug-
gested that the lower dark respiration rates in the white sectors were
probably due to a lesser demand for respiratory energy to maintain cell
function in these areas (Toshoji et al., 2012).
In the yellow spots of the variegated A. japonica ‘Variegata’ leaves,
32
Flora 240 (2018) 25–33
obviously lower metabolic activity was noticed, which was character-
ized by lower photosynthesis and respiration rates, starch accumula-
tion, and chloroplast organization. The low photosynthesis rate was due
to low contents of photosynthetic pigments in yellow spots. In addition,
the abnormal chloroplasts in yellow parts inevitably affected starch
accumulation. It was supposed that the low energy demand and limited
carbohydrate supply from dysfunctional chloroplasts might account for
the lower dark respiration rates in white sectors of variegated leaves
(Toshoji et al., 2012).
In summary, the results of this study show that the leaf variegation
of the woody plant A. japonica ‘Variegata’ was “pigment type.” Defects
in chloroplast development play a pivotal role in the formation of leaf
variegation in this plant species. This pigment-related leaf variegation
brought a photosynthetic cost to the variegated A. japonica ‘Variegata’
leaves.
Acknowledgments
This study was funded by the National Natural Science Foundation
in China (31300510 and 31300515), the Collaborative Innovation Plan
of Jiangsu Higher Education, and the Priority Academic Program
Development of Jiangsu Higher Education Institutions(PAPD).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.flora.2017.12.010.
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