Journal of Experimental Botany, Vol. 58, No. 7, pp.

1771–1781, 2007
doi:10.1093/jxb/erm036 Advance Access publication 10 April, 2007

RESEARCH PAPER

Ultraviolet A-specific induction of anthocyanin

biosynthesis in the swollen hypocotyls of turnip
(Brassica rapa)

Bo Zhou1, Yuhua Li1,*,†, Zhiru Xu1, Haifang Yan1,2, Shinichiro Homma2 and Saneyuki Kawabata2,†
1
College of Life Sciences, Northeast Forestry University, Harbin 150040, China
2
Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan

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Received 3 October 2006; Revised 5 February 2007; Accepted 6 February 2007

Abstract
Ultraviolet A (UV-A)-mediated regulation of anthocyanin
biosynthesis was investigated in swollen hypocotyls of
the red turnip ‘Tsuda’. The shaded swollen hypocotyls
which contained negligible anthocyanin were exposed
to artificial light sources including low fluence UV-B,
UV-A, blue, red, far-red, red plus UV-A, far-red plus UV-A,
and blue plus red. Among these lights, only UV-A
induced anthocyanin biosynthesis and co-irradiation of
red or far-red with UV-A did not affect the extent of UV-A-
induced anthocyanin accumulation. The expression of
phenylalanine ammonia lyase (PAL; EC 4.3.1.5), chal-
cone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxy-
lase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase
(DFR; EC 1.1.1.219), and anthocyanidin synthase (ANS;
EC 1.14.11.19) genes was increased with time during
a 24 h exposure to UV-A. In contrast, irradiation with red,
blue, UV-B, and a combination of blue with red failed to
induce CHS expression. Microarray analysis showed
that only a few genes, including CHS and F3H, were
induced significantly by UV-A, while a separate set of
many genes was induced by low fluence UV-B. The UV-
A-specific induction of anthocyanin biosynthesis and
the unique gene expression profile upon UV-A irradia-
tion as compared with blue and UV-B demonstrated that
the observed induction of anthocyanin biosynthesis in
red turnips was mediated by a distinct UV-A-specific
photoreceptor, but not by phytochromes, UV-A/blue
photoreceptors, or UV-B photoreceptors.
Key words: Chalcone synthase, microarray, photoreceptor,
SSH, subtractive library.
Introduction
UV radiation from the sun induces various responses in
higher plants. While the greatest portion of UV-B (280–
320 nm) is absorbed by the ozone layer, UV-A (320–
400 nm) penetrates the atmosphere to reach the earth
surface. Typically, UV-A radiation in the temperate zone
is ;50 W m
2, while UV-B is 2 W m
2 during mid-
summer. High fluence UV-B causes serious damage to
DNA, membranes, and proteins. DNA is especially
sensitive to high fluence UV-B, resulting in the formation
of pyrimidine dimers (Taylor et al., 1997; Frohnmeyer
and Staiger, 2003). However, low fluence UV-B stim-
ulates distinct responses, such as the accumulation of
UV-absorbing pigments and expression of stress
response-related genes (Hahlbrock and Scheel, 1989; A-H
Mackerness et al., 2001; Brosché and Strid, 2003;
Frohnmeyer and Staiger, 2003). These responses are
considered to play a role as a protective mechanism
against potential damage by UV irradiation. Several
studies have suggested that these responses are mediated
by a UV-B receptor (Kim et al. 1998; Boccalandro et al.,
2001; Suesslin and Frohnmeyer, 2003).
Since most of the UV-A from the sun penetrates the
atmosphere, UV-A radiation at the earth’s surface is much
stronger than other wavelengths of UV rays. Thus, UV-A
may have significant influences on plants. Unlike the
responses to low fluence UV-B, however, such responses
have not been well studied. Since UV-A is in a range of
long-wave UV and thus has lower energy, it is expected
to cause mild or negligible damage to plants directly,
but may induce protective mechanisms against more

* To whom correspondence should be addressed. E-mail: lyhshen@mail.hl.cn

y
These authors contributed equally to this work.
Abbreviations: ANS, anthocyanidin synthase; CHS, chalcone synthase; CHI, chalcone isomerase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-
hydroxylase; PAL, phenylalanine ammonia lyase; SSH, suppression subtractive hybridization; UV-A, ultraviolet A, UV-B: ultraviolet B.

ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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1772 Zhou et al.
harmful short-wave UV as suggested for low fluence UV-
B. Low fluence UV-B and high fluence UV-A sometimes
caused similar responses, such as necrosis observed in
UV-sensitive Arabidopsis mutants defective in succinic-
semialdehyde dehydrogenase (Bouché et al., 2003). The
most common protective mechanism stimulated by low
fluence UV-B radiation is the accumulation of anthocya-
nins as observed in maize (Singh et al., 1999), rice
(Reddy et al., 1994), apple fruits (Arakawa et al., 1985,
1986; Arakawa, 1988; Dong et al., 1995), roses (Maekawa
et al., 1980; Nakamura et al., 1980; Mor and Zieslin,
1990), kangaroo paw (Ben-Tal and King, 1997), apple
flowers (Dong et al., 1998), and Arabidopsis (Kubasek
et al., 1992; Christie and Jenkins, 1996). Similarly, UV-A
induction of anthocyanin accumulation was also observed
in Arabidopsis (Christie and Jenkins, 1996; Fuglevand
et al., 1996), eggplant (Toguri et al., 1993), grape (Kataoka
et al., 2003), carrot cells (Takeda et al., 1994; Hirner and
Satz, 2000), and kalanchoë (Hoffmann, 1999). In addition,
it is known that many horticultural crops, including fruits
of eggplants (Matsumaru et al., 1971) and petals of
Primula malacoides (Kashiwagi et al., 1977), show poor pig-
mentation in a greenhouse covered with UV-A-absorbing
films, while the pigmentation was recovered when the
UV-A cut film was replaced with UV-B cut films, which
are commonly used for the production of these crops.
UV-A responses in Arabidopsis could be replaced by
blue light (Feinbaum et al., 1991; Kubasek et al., 1995;
Fuglevand et al., 1996), indicating the involvement of
UV-A/blue photoreceptors. However, the frequently ob-
served poor pigmentation in a greenhouse covered with
UV-A cut films is unlikely to be mediated by UV-A/blue
photoreceptors, because blue light of the sunlight pene-
trates the films. To determine whether UV-A induces
protective responses against the potential damage upon
UV irradiation as observed for low fluence UV-B in other
plants and to characterize the photoreceptor mediating
anthocyanin production, the responses of the swollen
hypocotyls of red turnips to different light conditions and
the subsequent changes in gene expression profiles by
using cDNA microarrays were investigated. Based on the
data, it is demonstrated that the swollen hypocotyls of
a red turnip cultivar ‘Tsuda’ accumulated anthocyanin in
response to UV-A irradiation, but failed to accumulate
under a UV-A-proof filter, indicating the involvement of
UV-A-specific photoreceptors in this response.
Materials and methods
Plant materials
Red turnips, Brassica rapa L. subsp. rapa ‘Tsuda’, were sown and
grown in 20 cm pots filled with soil in a greenhouse, in which the
temperature was maintained above 15
C at night and no supple-
mental lights were installed. The turnips exhibited swelling of
the tissue originating from the hypocotyl and the root. The upper
part of the swollen tissue that appears above the ground originates
from the hypocotyls, which can be distinguished from the root by
the absence of the lateral roots. Approximately 1 month after
sowing, the outer tissue of the hypocotyls split and the inner tissue
initiates swelling. The anthocyanins accumulate in the epidermis of
this swelling tissue. The anthocyanin accumulation occurs only in
the epidermis of the swollen tissue that appears above the ground in
the ‘Tsuda’ turnip.
When the hypocotyls started to swell, the upper part of
the swollen tissue appearing above the ground was covered
with aluminium foil and overlaid with soil to prevent exposure to
sunlight. Approximately 3 months after sowing, when the diameter
of the swollen tissue reached 2–3 cm, the plants were subjected to
light treatments as described below.
Effects of light quality on the anthocyanin biosynthesis in
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the swollen hypocotyls of the turnip ‘Tsuda’
To determine the effects of light quality on anthocyanin bio-
synthesis, three experiments were conducted. (i) The swollen
hypocotyls of the turnips were exposed to UV-B, UV-A, blue, red,
far-red, red plus UV-A, far-red plus UV-A, or blue plus red for 24 h
and then collected for anthocyanin measurements. Each treatment
consisted of eight replications. (ii) Shaded swollen hypocotyls
were exposed to UV-B, UV-A, blue, red, and blue plus red for
0, 6, 12, 18, 24, or 48 h, and total RNA was extracted from these sam-
ples for northern blot analysis. (iii) Shaded hypocotyls were exposed
to visible light, UV-A, or both, and then collected for anthocyanin
measurements. Each treatment consisted of 10 replications.
Effects of the intensity and the period of UV-A irradiation on
the induction of anthocyanin biosynthesis
(i) Shaded turnips were exposed to UV-A at 1.2, 2.4, or 7.2 W m
2
for 1, 3, or 6 h and then transferred to darkness. They were
collected at 24 h after the start of the UV-A treatment for the
measurement of anthocyanin content. (ii) Shaded turnips were
exposed to UV-A at 7.2 W m
2 for 3 h and transferred to darkness.
They were collected at the end of the UV-A pulse or 6, 12, 18, and
24 h after transfer to darkness. They were used for northern blot
analysis for the chalcone synthase gene (CHS).
Effects of light quality on anthocyanin contents in the
hypocotyls of seedlings
Two-day-old ‘Tsuda’ seedlings grown under darkness were irradi-
ated with UV-B, UV-A, blue, red, far-red light, or sunlight for 12 h
and kept in the dark for 24 h. The upper 1 cm or the bottom 2 cm
of the hypocotyls was collected and the anthocyanin content was
measured. Two seedlings were used for each replication. The
number of replications was eight.
Light treatments: The turnips were irradiated with UV-A by using
a fluorescent lamp, EFD15BLB or FL10BLB (Toshiba, wavelength
range of 310–410 nm with a peak at 352 nm). The intensity of
UV-A was set at 7 W m
2 (5 W m
2 at 320–380 nm) except for the
experiments where the intensity was varied. In some experiments,
the turnips were also irradiated with UV-B at 6 W m
2 (4 W m
2
at 280–330 nm) by using a handheld UV lamp, UVM16 (UVP,
wavelength range of 280–375 nm with a peak at 302 nm), blue light
at 12 W m
2 by using light-emitting diodes (LEDs), NSPB-510S
(Nichia, 465–475 nm, half width¼30 nm), red light at 13 W m
2
by using LEDs, SLA-580-JT (Rohm, 660 nm, half width¼25 nm),
far-red light at 14 W m
2 by using LEDs, L735AU (Epitex,
Kanagawa, Japan, 735 nm, half width¼304 nm), or visible light by
using metal halide lamps, Ecocera II (Type M400D, GS Yuasa,
Kyoto). The contaminant red light from the far-red LEDs was
 UV-A-specific induction of anthocyanins in Brassica rapa 1773
filtered by methacrylate plates (DELPET A72, Asahi Kasei). The
radiation from the Ecocera II light bulb covers a broad range of
wavelength, from UV-A to far-red, and has no particular peaks at
any wavelength. The light spectrum at 10 000 lux is [0.10 W nm
1/
400 nm, 0.10 W nm
1/450 nm, 0.12 W nm
1/500 nm, 0.10 W
nm
1/550 nm, 0.16 W nm
1/600 nm, 0.10 W nm
1/650 nm,
0.08 W nm
1/700 nm, and 0.04 W nm
1/750 nm]. The UV
radiation from the Ecocera II light bulb was filtered by an acrylic
plate (4 mm thickness, transmission threshold of 400 nm, Sumipex,
Sumitomo Chemical) to remove the UV-A rays.
Anthocyanin measurement
The surface tissue ;1–2 mm thick was peeled from the hypocotyl
part of the swollen tissue and trimmed to a 1 cm31 cm section.
These sections were soaked in 0.5–3 ml of methanol containing 1%
HCl for 12–24 h at 4
C. When young seedlings were used,
bacterial clones and purified by ethanol precipitation. The PCR
products were spotted on the microarrays in duplicate by Kaken
Genecs (Chiba, Japan).
The expression profile of genes in response to
blue, UV-A, or UV-B irradiation
Shaded hypocotyls of 3-month-old ‘Tsuda’ plants were exposed to
blue, UV-A, or low fluence UV-B, or kept in darkness each for
24 h. The microarray analysis was performed for the combinations
of blue versus dark, UV-A versus dark, and UV-B versus dark
treatments. Total RNA was extracted from the epidermis of the
swollen hypocotyls. The mRNA was purified by using Oligo(dT)-
Cellulose Type 7 (GE Healthcare). This purification step was not
repeated, so that the RNA sample contained a significant amount of
other RNAs. Approximately 100 ng of this crude mRNA was used
for each microarray hybridization. The cDNA synthesis and micro-

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a section 1 cm or 2 cm long was excised from an upper or a bottom array hybridization were performed by using an Expression Array
part of a hypocotyl, respectively. Two sections from independent Detection Kit for Microarray, Cy3/Cy5, 3DNA Array 900
seedlings were combined for each part and then anthocyanins were (Genisphere), according to the manufacturer’s instructions.
extracted similarly. The absorbance of the extracts at 530 nm was
measured after they were diluted with 1% HCl methanol depending Hybridization design
on the anthocyanin concentration. Anthocyanin content was The hybridization design is shown in Table 1. Direct comparisons
expressed as optical density (OD530) per cm2 of peels of the swollen were made between blue and dark, UV-A and dark, or UV-B
hypocotyls or OD530 per cm of the hypocotyls of young seedlings. and dark treatments. The dark control sample was labelled with Cy5
and the other samples were labelled with Cy3. In addition to these
RNA extraction and northern blot analysis hybridizations, three concurrent split-control hybridizations of the
Total RNA was extracted from the peeled surface tissue of ;1–2 mm dark control (Table 1, arrays 1–3) were made to correct gene-specific
thickness by using Trizol or Concert (Invitrogen). The total RNA dye bias, according to Rosenzweig et al. (2004). Three biological
samples were used for northern hybridization or mRNA purification replicates were made for each comparison (Table 1, arrays 4–12).
for microarray hybridization. The mRNA was purified by using For each biological replicate, RNA was extracted from more than
Oligo(dT)-Cellulose Type 7 (GE Healthcare). five plants.
Northern hybridization was carried out for RNAs of genes
encoding phenylalanine ammonia lyase (PAL; EC 4.3.1.5;
DQ167187), CHS (EC 2.3.1.74; AY935256), flavanone 3-hydroxy- Table 1. The hybridization design of the microarray experiment
lase (F3H; EC 1.14.11.9; DQ167185), dihydroflavonol 4 reductase The epidermis of the swollen hypocotyls of 3-month-old ‘Tsuda’ turnip
(DFR; EC 1.1.1.219; DQ167184), and anthocyanidin synthase plants was exposed to blue light at 12 W m
2 (Blue), UV-A at 7 W
(ANS; EC 1.14.11.19). A 10 lg aliquot of total RNA was separated m
2 (UVA), or UV-B at 6 W m
2 (UVB), or kept in darkness (Dark).
on a 0.7% denatured agarose gel containing formaldehyde in 13 Direct comparisons were made between blue and dark (arrays 4–6),
MOPS buffer. The RNA was transferred to a nylon membrane UV-A and dark (arrays 7–9), and UV-B and dark (arrays 10–12). The
(MagnaGraph, Funakoshi) and subjected to northern blot analysis dark control samples were labelled with Cy5 and the other samples were
with probes labelled with digoxigenin (Roche) by PCR. Hybrid- labelled with Cy3. In addition to these hybridizations, three concurrent
izations and signal detections were performed according to the split-control hybridizations of dark control were made to correct gene-
specific dye bias (arrays 1–3). The numbers followed by the treatment
manufacturer’s instructions (Roche). names are the identifier of biological replicates for each treatment. The
column ‘Dye effect’ indicates the presence of gene-specific dye effects.
Construction of subtractive cDNA libraries and The last three columns indicate a design matrix representing the
cDNA microarrays contrasts between light treatments and dark control, by which statistical
analysis was performed using ‘limma’. 0, absent; 1, present (positive
The mRNA extracted from hypocotyls of ‘Tsuda’ or ‘Yurugi value for Cy3 versus Cy5).
Akamaru’, which were exposed to sunlight or shaded for approxi-
mately 2 months after the start of the swelling (3 months after Array mRNA sources Dye Design matrix
sowing), were subjected to suppression subtractive hybridization no. effect
(SSH) to obtain subtractive cDNAs by using a PCR-Select cDNA Cy3 Cy5 Blue– UVA– UVB–
Subtraction Kit (Clontech). Whereas ‘Tsuda’ exhibits light- Dark Dark Dark
dependent anthocyanin biosynthesis, ‘Yurugi Akamaru’ exhibits 1 Dark1 Dark1 1 – – –
anthocyanin biosynthesis even in the underground part of the 2 Dark2 Dark2 1 – – –
hypocotyls, indicating that anthocyanin biosynthesis in this cultivar 3 Dark3 Dark3 1 – – –
does not require light exposure. The subtraction was performed 4 Blue1 Dark1 1 1 0 0
between shaded and unshaded ‘Tsuda’ and between ‘Tsuda’ and 5 Blue2 Dark2 1 1 0 0
‘Yurugi-Akamaru.’ The subtractive cDNAs were cloned into 6 Blue3 Dark3 1 1 0 0
pBluescript II SK (+), resulting in subtractive cDNA libraries. 7 UVA1 Dark1 1 0 1 0
For constructing the microarray, the sequences of isolated clones 8 UVA2 Dark2 1 0 1 0
from these libraries were clustered using the ‘blastclust’ program 9 UVA3 Dark3 1 0 1 0
10 UVB1 Dark1 1 0 0 1
and approximately 2800 clusters were obtained. Basically, one 11 UVB2 Dark2 1 0 0 1
clone from each cluster was chosen for spotting on microarrays as 12 UVB3 Dark3 1 0 0 1
gene probes. The probes were amplified by direct PCR of the
1774 Zhou et al.
Scanning and image analysis
The microarray slides were scanned at 5 lm resolution, using
a ScanArray Express scanner (Perkin-Elmer). Scanner settings were
PMT (Photo Multiplier Tube) 65–87% for Cy3 and 63–82% for
Cy5, and laser power of 85–90%, depending on signal strength.
Spot images of probes were extracted and processed using
ScanArray Express software. The medians of the foreground
fluorescence intensities were calculated for each spot. Array quality
was examined initially by overall visual signal intensity, and then
by linear correlation assessment between the signal intensity of
duplicate spots within each slide. The correlation coefficient of log-
transformed signal intensity between duplicate spots was 0.72–0.94
for both dyes. In total, 12 successful microarrays were obtained.
Data processing
The output data of ScanArray Express software was processed
using Bioconductor packages in the R environment. Spots with
a foreground signal below three times the background signal in >10
arrays were flagged as low quality spots and excluded. Background
correction was not carried out. The M value was calculated as the
log2 ratio of the foreground signals. Spatial normalization was
applied to the M values (location smoothing, span¼0.5) and then to
the absolute M values (scale smoothing, span¼0.75) before
excluding flagged data, according to Wit and McClure (2004). After
print-tip loess normalization (span¼0.75), within-slide replicates
were collapsed by calculating the arithmetic average of replicate
spots for each probe. Finally, the gene-specific dye bias was
corrected by subtracting the average of the M value of the three
split dark controls for each probe.
The resultant nine processed data sets, three treatments and three
biological replicates, were subjected to a further statistical analysis
by using the ‘limma’ package (Smyth and Speed, 2003; Smyth,
2004; Smyth et al., 2005). A design matrix consisted of nine rows
representing arrays 4–12 (Table 1) and three columns representing
the dye-unbiased effect of the light treatment over the dark control
(Blue–Dark, UVA–Dark, or UVB–Dark in Table 1). Empirical
Bayesian methods were then used to calculate the moderated
F statistics. Benjamini and Hochberg’s method of controlling false
discovery rate (Benjamini and Hochberg, 1995) was used to adjust
P-values by using the ‘topTableF’ function of ‘limma’.
Bootstrap clustering
To examine the differences in gene expression profiles between
the light treatments, probes which showed significant F statistics
at adjusted P <0.01 were subjected to multiscale bootstrapping
hierarchical clustering (Shimodaira, 2002, 2004) among arrays and
among probes. A package ‘pvclust’ (Suzuki and Shimodaira, 2006)
was used to perform hierarchical clustering and to calculate multiscale
BPs (bootstrap probability), and ‘scaleboot’ to calculate AU (approx-
imately unbiased P-value). One thousand bootstrap iterations and
multiscales of 0.55–9.00 for clustering of probes and 10 000
iterations and multiscales of 0.11–9.00 for clustering of arrays were
used to calculate significance scores for the clusters.
Results
UV-A-specific induction of anthocyanin biosynthesis
in the peel of swollen hypocotyls
To characterize the light-dependent regulation of anthocy-
anin biosynthesis in ‘Tsuda’, the effects of several wave-
lengths of light on anthocyanin biosynthesis in the
swollen hypocotyls of the ‘Tsuda’ turnip were compared,
by using artificial light sources of UV-B, UV-A, blue, red,
and far-red (Fig. 1). Of all light sources used, only UV-A
stimulated significant anthocyanin production, whereas the
amount of anthocyanin detected under other light con-
ditions was as low as that of hypocotyls kept under
darkness. In addition, no synergistic effect of red and
far-red was observed when they were co-irradiated with
UV-A. Co-irradiation with blue and red light also failed
to induce anthocyanin production.
The effect of UV-A was not attributable to contaminant
UV-B in the light source, because filtering the radiation
from the UV-A light source with the soda glass plate,
which absorbs UV-B, did not reduce the UV intensity
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significantly and had no effect on the anthocyanin accu-
mulation (data not shown).
Irradiation of the swollen hypocotyls with visible light
free of UV-A also failed to induce anthocyanin pro-
duction, and UV-A-dependent anthocyanin production
was not influenced by the co-irradiation with visible light
(Fig. 2). These results indicated that not only was
anthocyanin biosynthesis induced by UV-A, but UV-A
was also sufficient for full induction.
Concomitantly, expression of the structural genes in the
anthocyanin biosynthesis pathway was stimulated by UV-
A irradiation (Fig. 3). The transcript level of PAL and
CHS was detectable in darkness, but F3H, DFR, and ANS
were undetectable. The expression of PAL was stimulated
within 6 h of UV-A exposure, while the expression of
CHS, F3H, DFR, and ANS increased after 18–24 h of
0.5
Anthocyanin (OD• cm-2)
0.4
0.3
0.2
0.1
n.s. n.s. n.s. n.s. n.s.
n.s.
0
Dark FR Red Blue UV-A low high UV-A UV-A Blue
UV-B UV-B +FR +Red +Red
Fig. 1. Effect of irradiation with different wavelengths of lights on
anthocyanin accumulation in the epidermis of the swollen hypocotyls of
3-month-old ‘Tsuda’ turnip. The swollen hypocotyls were exposed to
UV-B at 6 W m
2, UV-A at 7 W m
2, blue light at 12 W m
2, red
light at 13 W m
2, far-red light at 14 W m
2, or a combination of red
or far-red (FR) with UV-A for 24 h, or collected without light exposure
(dark). Anthocyanin was extracted from the light-exposed part of the
epidermis of the swollen hypocotyls, and then the concentration was
determined. Vertical bars indicate 6SE (n¼8). *** and n.s. indicate that
the difference in means between a treatment and the dark control is
significant at P <0.001 and non-significant at P <0.05, respectively, by
Dunnet’s post hoc test using log-transformed values.
 UV-A-specific induction of anthocyanins in Brassica rapa 1775
1.2
1
Anthocyanin (OD• cm-2)
0.8
0.6
0.4
0.2
n.s.
0
UVA - - + +
VIS - + - +
Fig. 2. The interaction of irradiation with UV-A (UVA) and visible
light (VIS) in anthocyanin accumulation in the epidermis of the swollen
hypocotyls of 3-month-old ‘Tsuda’ turnip. The swollen hypocotyls were
exposed to combinations of continuous UVA at 0 (–) or 7 W m
2 (+)
and VIS at 0 (–) or 50 W m
2 (+) for 48 h. A part of the sample was
kept in darkness (–UVA, –VIS). Vertical bars indicate 6SE (n¼10).
*** and n.s. indicate that the difference in means between each
treatment and the dark control (–UVA, –VIS) is significant at P <0.001
and non-significant at P <0.05, respectively, by Dunnet’s post hoc test
using log-transformed values.
Fig. 3. Changes in transcript levels of genes involved in the antho-
cyanin biosynthesis pathway during continuous UV-A exposure in the
epidermis of the swollen hypocotyls of 3-month-old ‘Tsuda’ turnip. The
swollen hypocotyls were exposed to continuous UV-A at 7 W m
2 for
0–48 h. PAL, phenylalanine ammonia lyase; CHS, chalcone synthase;
F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol 4-reductase;
ANS, anthocyanidin synthase
UV-A exposure. In contrast, no induction in CHS expres-
sion was observed when the hypocotyls were irradiated
with red, blue, UV-B, or red plus blue (Fig. 4).
Short pulse UV-A can induce anthocyanin biosynthesis
in an irradiance-dependent manner
In low fluence UV-B responses, a very short period of
exposure could induce anthocyanin biosynthesis in rice
(Reddy et al., 1994) and maize (Singh et al., 1999). To
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Fig. 4. The effect of different light sources on the expression of CHS in
the epidermis of the swollen hypocotyls of 3-month-old ‘Tsuda’ turnip.
The swollen hypocotyls were exposed to UV-B at 6 W m
2 (UVB),
blue light at 12 W m
2 (Blue), red light at 13 W m
2 (Red),
or a combination of blue and red for 0–48 h, or exposed to UV-A at
7 W m
2 (UVA) for 24 h as the control.
investigate the period of exposure required for anthocya-
nin induction, we exposed turnip swollen hypocotyls to
UV-A of different light intensities for different periods of
time. As shown in Fig. 5, a 1 h pulse of UV-A at 7.2 W
m
2 could induce anthocyanin production. The extent of
the induction was dependent on UV-A intensity but not on
an exposure period of >1 h. UV-A irradiation at one-third
and one-sixth of the above intensity (1.2 W m
2 and
2.4 W m
2) failed to induce anthocyanin production, even
if the exposure was prolonged three and six times longer,
respectively. Therefore, a response to UV-A was de-
pendent on light intensity, but not on the total energy that
the hypocotyls perceived. Such a response is similar to the
high irradiance response (HIR) of phytochrome A.
As shown in Fig. 6, the transcripts for CHS were not
detectable at the end of a 3 h pulse of UV-A, but showed
a rise 6–12 h later, and then gradually decreased to an
undetectable level at 24 h after the end of the pulse.
Effect of light quality on anthocyanin biosynthesis in
young seedlings
It has been reported previously that turnip seedlings
produce anthocyanins in response to blue, red, and far-red
light (Siegelman and Hendricks, 1957). Therefore, the
effect of different light sources on anthocyanin production
in the ‘Tsuda’ seedlings was also investigated (Fig. 7).
The results showed that ‘Tsuda’ seedlings also accumu-
lated anthocyanins in response to UV-B and far-red light
as well as UV-A in the upper part of the hypocotyls
(Fig. 7A). Blue and red also induced anthocyanin bio-
synthesis, although to a lower extent. In contrast, the
bottom part, in which tissue swelling occurs as it grows,
did not show or showed only subtle anthocyanin
1776 Zhou et al.
0.4 0.5
0 W·m-2
0.35 1.2
A
Anthocyanin (OD• cm-2)

Anthocyanin (OD• cm-1)

0.3
2.4 0.4
7.2
0.25
0.3
0.2

0.15 0.2
0.1
0.1
0.05 n.s.
n.s. n.s. n.s. n.s. n.s.
0 0
Dark Sun- FR Red Blue UV-A UV-B
0L 1L+23D 3L+21D 6L+18D 24L light
Fig. 5. Effect of the period and intensity of UV-A exposure on antho-
0.5

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cyanin accumulation in the epidermis of swollen hypocotyls of 3-
month-old ‘Tsuda’ turnip. After the exposure to UV-A at 1.2 (light B
grey columns), 2.4 (dark grey columns), or 7.2 W m
2 (black columns)

Anthocyanin (OD• cm-1)

for 1, 3, or 6 h, the plants were placed in darkness for 23, 21, or 18 h
0.4
(1L+23D, 3L+21D, or 6L+18D, respectively). Another group of
samples was collected without light exposure (0L, white columns). 0.3
After these treatments, the anthocyanin content was determined immedi-
ately. Vertical bars indicate 6SE (n¼5–10). *** and n.s. indicate that the
difference in means between each treatment and the dark control (0L) is 0.2
significant at P <0.001 and non-significant at P <0.05, respectively, by
Dunnet’s post hoc test using log-transformed values.
0.1
n.s.
0
Dark Sun- FR Red Blue UV-A UV-B
light

Fig. 7. Effect of irradiation with different wavelengths of lights on

Fig. 6. Temporal changes in transcript levels of CHS in the epidermis
of the swollen hypocotyls of 3-month-old ‘Tsuda’ turnip after a pulse of
UV-A irradiation. The swollen hypocotyls were exposed to UV-A at
7 W m
2 for 3 h, and then placed in darkness for 0 (3L+0D), 6
(3L+6D), 12 (3L+12D), 18 (3L+18D), or 24 h (3L+24D).
accumulation in response to these lights, but exhibited
a UV-A-specific response as observed in the swollen
hypocotyls (Fig. 7B).
Construction of subtractive cDNA libraries
Subtractive cDNA libraries were constructed between
(i) sunlight-exposed swollen hypocotyls of ‘Tsuda’ sub-
tracted by shaded swollen hypocotyls of ‘Tsuda’ (Library
LT-ST); (ii) shaded swollen hypocotyls of ‘Tsuda’
subtracted by sunlight-exposed swollen hypocotyls of
‘Tsuda’ (Library ST-LT); (iii) shaded swollen hypocotyls
of ‘Tsuda’ subtracted by sunlight-exposed swollen hypo-
cotyls of ‘Yurugi Akamaru’ (Library ST-Y); and
(iv) sunlight-exposed or shaded swollen hypocotyls of
‘Yurugi Akamaru’ subtracted by shaded swollen hypo-
cotyls of ‘Tsuda’ (Library Y-ST).
anthocyanin accumulation in the upper 1 cm (A) or bottom 2 cm
(B) part of the hypocotyls of 3-d-old ‘Tsuda’ turnip seedlings. The
seedlings germinated under darkness were irradiated with UV-B at 6 W
m
2, UV-A at 7 W m
2, blue light at 12 W m
2, red light (Red) at
13 W m
2, far-red (FR) at 14 W m
2, or sunlight (Sunlight), or kept in
darkness (Dark) for 12 h. Vertical bars indicate 6SE (n¼8). ***, *, and
n.s. indicate that the difference in means between each treatment
and the dark control (Dark) is significant at P <0.001, at P <0.05, and
non-significant at P <0.05, respectively, by Dunnet’s post hoc test using
log-transformed values.
From these subtractive libraries, a total of 4200 clones
were sequenced, and assembled by the CAP3 pro-
gram (Huang and Madan, 1999), and 536 contigs and
1317 singletons were obtained. BLAST searches using the
‘RefSeq’ database at NCBI were conducted for these
assembled sequences, by which these clones were anno-
tated. Gene names were assigned using the E-value
threshold of E-15. Approximately 30% of total contigs
and singletons could not be annotated by this criterion,
and approximately 20% of the annotated sequences could
not be assigned to known functions. As for flavonoid
biosynthesis, PAL, CHS, F3H, DFR, and ANS were
isolated from both LT-ST and Y-ST libraries.
The expression profile of genes in response to
UV-A irradiation
Although the data presented above strongly suggested the
existence of a UV-A-specific photoreceptor, there still
 UV-A-specific induction of anthocyanins in Brassica rapa 1777
remains the possibility that failure of anthocyanin pro-
duction under blue or UV-B light could be due to
insufficient light intensity, or contamination of other
wavelengths of lights. Therefore, microarray analysis
(GSE6876) was conducted to see whether UV-A induces
the expression of a distinct gene set. A hierarchical
clustering of arrays using M values of the gene probes
which showed significant changes by F-statistics at an
adjusted P-value of 0.01 was conducted (Fig. 8). The
dendrogram showed that all the microarrays of UV-B
treatment were grouped into one cluster, while other
microarrays were grouped into a distinct cluster. This
indicated that UV-B induced a distinct set of genes which
were not induced by blue and UV-A treatments, and that
similar sets of genes were induced by blue and UV-A
treatments. A hierarchical clustering was also conducted
for microarray probes (Fig. 9). Several clusters were
identified by multiscale bootstrap clustering. Five groups
were selected by a criterion of AU >0.99, and the gene
expression profile for each group was shown in Fig. 9.
Many genes showed up-regulation (Group #5) or down-
regulation (Group #1) due to UV-B exposure. A smaller
set of genes showed up-regulation in response to UV-A
irradiation (Group #3). Genes for anthocyanin biosynthe-
sis, including CHS and F3H, were grouped into this
group (Table 2), while PAL was grouped into Group
#5 (see Supplementary Table S1 at JXB online). Other
genes of flavonoid biosynthesis spotted on the microarray
did not show significant changes, and were therefore
excluded.
The list of gene probes which showed significant
changes due to light exposure is shown in Supplementary
Table S1 at JXB online. Group #5 genes included genes
related to ethylene biosynthesis, reactive oxygen species
reactions such as glutathione S-transferase and superoxide
dismutase, and carbohydrate metabolism such as
chitinase and glyceraldehyde-3-phosphate dehydrogenase.
Down-regulated genes included genes related to tissue
or cell growth such as extensin, xyloglucan endotransgly-
cosylase/hydrolase, and aquaporin, and to glucosynolate
metabolism, such as cytochrome P450 (CYP83A1),
methylthioalkylmalate synthase, myrosinase-binding pro-
teins, and myrosinase-associated proteins.
Discussion
UV-A did not induce a broad range of stress
responses as observed for low fluence UV-B
Low fluence UV-B was found to stimulate the transcript
levels of a robust set of genes involved in stress responses
(Casati and Walbot, 2003, 2004; Ulm et al., 2004). In the
present experiment, UV-B irradiation altered the expres-
sion of many genes that are involved in stress responses.
In contrast, such responses were not observed for the
0.8
0.6
Height
0.4
1.00
0.90
0.97
0.2
0.96
Blue1
Blue3
Blue2
1.00
1.00 0.72
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UVA2
0.0
UVA1
UVA3
UVB2
UVB1
UVB3
Fig. 8. Dendrogram of bootstrapping hierarchical clustering of micro-
arrays with M value correlation distance between each pair
of microarrays by average linkage. The epidermis of the swollen
hypocotyls of 3-month-old ‘Tsuda’ turnip plants was exposed to blue
light at 12 W m
2 (Blue), UV-A at 7 W m
2 (UVA), or UV-B at 6 W
m
2 (UVB), or kept in darkness (Dark). The M value was calculated as
the log2 ratio between blue and dark (Blue1, Blue2, Blue3), UV-A and
dark (UVA1, UVA2, UVA3), and UV-B and dark (UVB1, UVB2,
UVB3). The numbers followed by the treatment names are the identifier
of biological replicates. The values at the nodes indicate the approx-
imately unbiased P-value (AU) for the clusters.
UV-A treatment, indicating that UV-A at 7 W m
2 did
not induce general protective mechanisms as observed for
UV-B in ‘Tsuda’ swollen hypocotyls. The damage due to
UV-A exposure is unlikely to be as severe as that caused
by low fluence UV-B, even if the intensity of UV-A was
equivalent to that of UV-B. The results did not support the
hypothesis that UV-A would induce similar protective
responses against potential damage upon UV exposure as
does the low fluence UV-B. UV-A intensity of sunlight
reached 50 W m
2 during mid-summer in the temperate
zone. Such a high intensity of UV-A may induce
protective mechanisms as observed for low fluence UV-B.
Instead of inducing expression changes in a robust set
of genes related to the stress response, UV-A induced
anthocyanin biosynthesis. The anthocyanin biosynthetic
pathway involves several enzymes including PAL, CHS,
CHI, F3H, DFR, ANS, and glycosyl- or acyl-transferases.
Genes encoding these enzymes have been isolated and
their developmental and tissue-specific expression patterns
have been investigated (Koes et al., 1994; Holton and
Cornish, 1995; Weisshaar and Jenkins, 1998). CHS plays
a key role in the regulation of anthocyanin biosynthesis in
many plant species (Jenkins et al., 2001; Wade et al.,
2001). The microarray analysis and subsequent northern
blot hybridization demonstrated the stimulation of PAL,
CHS, F3H, DFR, and ANS expression by UV-A.
1778 Zhou et al.
Group #1 Group #2 Group #3

4
2

2
0

0
-2

-2

-2
-4

-4

-4
Log2(Cy3/Cy5)
Blue UVA UVB Blue UVA UVB Blue UVA UVB

4 Group #4 Group #5

4
2

2
0

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-2

-2
-4

-4
Blue UVA UVB Blue UVA UVB

Fig. 9. M values of probes in each of the significant clusters at P >0.99 based on bootstrapping hierarchical clustering with M value correlation
distance between each pair of probes by average linkage. The epidermis of the swollen hypocotyls of 3-month-old ‘Tsuda’ turnip plants was exposed
to blue light at 12 W m
2 (Blue), UV-A at 7 W m
2, or UV-B at 6 W m
2, or kept in darkness for 24 h. Microarray hybridization was performed
between blue/dark (Blue), UV-A/dark (UVA), or UV-B/dark (UVB). The y-axis indicates M values calculated as the log2 ratio of light treatment
signals to the dark control signals. Values are means of three microarray replicates. Bootstrap clustering with 1000 iterations and AU (approximately
unbiased P-value) threshold of 0.99 identified five major clusters (Group #1–5), and several subclusters (not shown).

Table 2. The list of probes in Group #3 in Fig. 9 Although UV-A at 7 W m
2 did not induce the same
The epidermis of the swollen hypocotyls of 3-month-old ‘Tsuda’ turnip protective mechanisms as UV-B, this intensity was
plants was exposed to blue light at 12 W m
2 (Blue), UV-A at 7 W sufficient to induce anthocyanin production, which may
m
2, or UV-B at 6 W m
2, or kept in darkness for 24 h. Microarray
hybridization was performed between blue/dark (Blue), UV-A/dark contribute to the tolerance to UV irradiation. Anthocya-
(UVA), or UV-B/dark (UVB). M values are the log2 ratio of light nins in the outer layer of the tissue not only can absorb
treatment signals to the dark control signals. Means of three microarray UV to protect the inner layer of the tissue, but also serve
replicates are indicated. The top hit results of the blast search using the
‘RefSeq’ database with an E-value threshold of E-15 are indicated as a scavenger of reactive oxygen species (Gould et al.,
(Annotation/top blast hit). 2002). The production of anthocyanins by UV-A exposure
may have contributed to the protection of the plant tissue
Probe M value Annotation/top
ID blast hit from the potential damage by UV absorption, and this
Blue UVA UVB may be responsible for the observation that UV-A did not
have a strong impact on gene expression profiles as
BR3105D05 0.317 2.042 0.946 Myrosinase-associated
protein, putative observed for low fluence UV-B response.
(AT4G01130)
BR3105D04 0.759 3.100 1.545 CHS (chalcone
synthase) Involvement of UV-A-specific photoreceptor
BR3105H01 0.459 2.072 1.292 CHS (chalcone At present, three types of photoreceptors have been
synthase)
BR8871 1.647 3.857 2.069 CHS (chalcone characterized: (i) phytochromes which perceive red or far-
synthase) red light (Quail, 1994); (ii) UV-A/blue photoreceptors
BR8691 0.886 2.016 0.634 CHS (chalcone including cryptochrome (Ahmad and Cashmore, 1993;
synthase)
BR9398 0.391 2.719 1.156 CHS (chalcone Lin, 2000) and phototropin (Briggs and Christie, 2002);
synthase) and (iii) the UV-B photoreceptor. Although the primary
BR10900 1.103 3.190 1.899 Invertase/pectin UV-A-responsive photoreceptor known is the UV-A/blue
methylesterase inhibitor
family protein photoreceptor, all three types of these photoreceptors
(AT5G62350) could potentially sense the UV-A signal. The measure-
BR8668 0.245 1.791 1.299 F3H (naringenin ment of absorption spectra of phytochrome suggested that
3-dioxygenase/flavanone
3-hydroxylase) it could also sense the blue/UV-A signal (Mancinelli,
BR3101E06
0.107 1.008 0.765 RNA-binding/nucleic 1986). In fact, Pratt and Briggs (1966) reported in vivo
acid-binding conversion of phytochrome by blue light. Devlin and Kay
(AT3G10845)
BR8879 0.849 1.692 1.496 Unknown protein (2000) proposed that phytochrome A acts as a photorecep-
(AT3G24100) tor in blue light input in circadian photoperception. For
BR10780 0.314 1.341 0.345 Unknown protein white cabbage seedlings, phytochrome alone appeared to
(AT3G54260)
be involved in anthocyanin production at a low fluence
 UV-A-specific induction of anthocyanins in Brassica rapa 1779
rate of UV (Lercari et al., 1989). In addition, as the UV-B
photoreceptor molecule has not been isolated, it has not
been characterized yet.
The studies using artificial light sources demonstrated
the absence of red, far-red, blue, and UV-B induction of
CHS and anthocyanin production. It was also indicated
that anthocyanin production induced by the UV-A lamp
was not induced by possible contaminant UV-B. Whereas
one-third the intensity of UV-A light failed to induce
anthocyanin production, UV-A filtered with a glass plate,
which completely absorbs UV-B (<320 nm), could induce
anthocyanin production. An LED light of 360 nm UV-A,
which did not contain UV-B, induced anthocyanin pro-
duction (data not shown). The effect of longer wavelength
UV-A at 395 nm (data not shown) and visible light free of
UV (Fig. 2) was also examined, and it was found that
neither of them could induce anthocyanin biosynthesis.
All of these results indicate that the UV-A-dependent
anthocyanin biosynthesis in swollen hypocotyls of turnip
is mediated by a UV-A-specific photoreceptor, but not by
blue or UV-B photoreceptors. Such responses were not
observed in UV-A-dependent anthocyanin biosynthesis in
Arabidopsis or grape berry, where anthocyanin biosynthe-
sis was induced by blue or white light as well as by
UV-A.
This conclusion was verified by microarray analysis. In
microarray analysis, gene expression profiles were clearly
different between UV-A and UV-B as indicated by
bootstrapping hierarchical clustering (Fig. 8). Because the
UV-B radiation altered the gene expression profiles as
shown for other plants exposed to UV-B (Casati and
Walbot, 2003; Ulm et al., 2004), the fluence rate of the
radiation in this study was within an appropriate level to
induce general UV-B responses. The increased genes
upon UV-B exposure included those related to stress
responses as observed for maize (Casati and Walbot,
2003) and Arabidopsis (Ulm et al., 2004). Ulm et al.
(2004) found the presence of at least two separate UV-B
perception responses: one for short-wave UV-B and the
other for long-wave UV-B in studies using high density
microarray analysis in Arabidopsis seedlings. While
a large set of genes were up- or down-regulated by UV-B
of shorter wavelength, only a few genes were up- or
down-regulated by UV-B of longer wavelength (Ulm
et al., 2004). These observations supported the separation
of responses by UV-B and those by longer wavelength
UV.
The gene expression profiles were the closest between
blue and UV-A treatments. The similar expression
profiles, as shown in Fig. 8, suggest that the UV-A/blue
photoreceptor was involved in the regulation of these
genes and that blue light intensity was sufficient to induce
such responses. However, the expression of anthocyanin
biosynthesis genes, i.e. CHS and F3H, was up-regulated
only by UV-A exposure (Fig. 9; Table 2). The different
profiles of gene expression under irradiation by UV-A,
low fluence UV-B, and blue light indicated the existence
of separate regulatory mechanisms stimulated by these
light sources.
Additional experiments using seedlings provided further
evidence for a UV-A-specific photoreceptor. The light
responses of turnip seedlings have been extensively
studied before. Anthocyanin biosynthesis in the hypo-
cotyls of turnip seedlings was shown to be induced by
blue, red, and far-red (Siegelman and Hendricks, 1957), and
controlled by the red/far-red reversible reaction of phyto-
chrome with co-action of blue light (Grill, 1965). The
present experiment using turnip seedlings showed that the
upper part of the seedling hypocotyls displayed far-red-
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dependent anthocyanin biosynthesis, while the bottom
part where swelling occurs did not respond to these light
wavelengths, but did respond to UV-A. Because both the
upper and bottom part of the hypocotyls were exposed to
the same intensity of light, subtle anthocyanin induction in
the bottom part by blue, far-red, and UV-B cannot be
attributable to insufficient light intensities. The results
provide evidence of discriminating UV-A responses from
other light responses. The different responses between
previous reports (Siegelman and Hendricks, 1957; Grill,
1965) and the present experiment would be because of the
difference in the part of the hypocotyls used.
The separation of UV-A-dependent activity from blue
light-dependent activity has been shown in several studies.
Fuglevand et al. (1996) observed that pretreatment with
blue light stimulated UV-B induction of CHS expression
in Arabidopsis, while UV-A did not. Takeda et al. (1994)
reported that UV-A ranging from 330 nm to 360 nm
induced CHS expression, while UV-A with a wavelength
longer than 400 nm did not. These findings imply the
existence of distinct UV-A-specific photoreceptors
(Young et al., 1992; Cuello et al., 1994; Tezuka et al.,
1994; Batschauer et al., 1996). However, evidence for
their existence has not been provided, because their results
can be attributable to inappropriate light intensities or
contaminant irradiation of other light wavelengths. The
present study demonstrated UV-A-specific induction of
anthocyanin biosynthesis and provided the evidence for
the presence of a UV-A-specific photoreceptor.
Whereas several horticultural crops appear to have
UV-A-specific regulatory mechanisms of anthocyanin
biosynthesis, the use of red turnip for the study of this
UV-A-specific response has several advantages. The
induction of anthocyanin biosynthesis in turnip was clear,
and no induction by other photosensing systems seems to
be involved. Such a response was also observed in the
bottom part of the hypocotyls of seedlings, which are
more easily prepared for experiments. In addition, turnip
can be propagated within approximately a 3 month life
cycle under growth condition of controlled temperature
and photoperiod. Since turnips are taxonomically close to
1780 Zhou et al.
Arabidopsis, a genomic database obtained in Arabidopsis
provides useful information about this plant. More
importantly, an international genome project is underway
for Chinese cabbage, a subspecies of Brassica rapa to
which the turnip belongs.
Supplementary data
The Supplementary data mentioned herein can be found at
JXB online.
Table S1. The list of probes which showed significant
changes in M values in response to light exposure as
compared with dark control.
Acknowledgement
This study was funded by National Natural Science Foundation of
China (30170785, 30040042).
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