JXB Advance Access originally published online on April 10, 2007
Journal of Experimental Botany 2007 58(7):1771-1781; doi:10.1093/jxb/erm036
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Ultraviolet A-specific induction of anthocyanin biosynthesis in the swollen hypocotyls of turnip (Brassica rapa)
1College of Life Sciences, Northeast Forestry University, Harbin 150040, China
2Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo, 113-8657 Japan
* To whom correspondence should be addressed. E-mail: lyhshen{at}mail.hl.cn
Received 3 October 2006; Revised 5 February 2007 Accepted 6 February 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
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 [EC] ), chalcone synthase (CHS; EC 2.3.1.74 [EC] ), flavanone 3-hydroxylase (F3H; EC 1.14.11.9 [EC] ), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219 [EC] ), and anthocyanidin synthase (ANS; EC 1.14.11.19 [EC] ) 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 irradiation 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 |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
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 stimulates 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 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 anthocyanins 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 pigmentation 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 observed 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 penetrates 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 |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
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 supplemental 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 the swollen hypocotyls of the turnip ‘Tsuda’
To determine the effects of light quality on anthocyanin biosynthesis, 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 samples 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 irradiated 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 width=30 nm), red light at 13 W m–2 by using LEDs, SLA-580-JT (Rohm, 660 nm, half width=25 nm), far-red light at 14 W m–2 by using LEDs, L735AU (Epitex, Kanagawa, Japan, 735 nm, half width=304 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 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 cmx1 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, a section 1 cm or 2 cm long was excised from an upper or a bottom part of a hypocotyl, respectively. Two sections from independent seedlings were combined for each part and then anthocyanins were extracted similarly. The absorbance of the extracts at 530 nm was measured after they were diluted with 1% HCl methanol depending on the anthocyanin concentration. Anthocyanin content was expressed as optical density (OD530) per cm2 of peels of the swollen hypocotyls or OD530 per cm of the hypocotyls of young seedlings.
RNA extraction and northern blot analysis
Total RNA was extracted from the peeled surface tissue of 1–2 mm thickness by using Trizol or Concert (Invitrogen). The total RNA samples were used for northern hybridization or mRNA purification for microarray hybridization. The mRNA was purified by using Oligo(dT)-Cellulose Type 7 (GE Healthcare).
Northern hybridization was carried out for RNAs of genes encoding phenylalanine ammonia lyase (PAL; EC 4.3.1.5 [EC] ; DQ167187 [GenBank] ), CHS (EC 2.3.1.74 [EC] ; AY935256 [GenBank] ), flavanone 3-hydroxylase (F3H; EC 1.14.11.9 [EC] ; DQ167185 [GenBank] ), dihydroflavonol 4 reductase (DFR; EC 1.1.1.219 [EC] ; DQ167184 [GenBank] ), and anthocyanidin synthase (ANS; EC 1.14.11.19 [EC] ). A 10 µg aliquot of total RNA was separated on a 0.7% denatured agarose gel containing formaldehyde in 1x MOPS buffer. The RNA was transferred to a nylon membrane (MagnaGraph, Funakoshi) and subjected to northern blot analysis with probes labelled with digoxigenin (Roche) by PCR. Hybridizations and signal detections were performed according to the manufacturer's instructions (Roche).
Construction of subtractive cDNA libraries and cDNA microarrays
The mRNA extracted from hypocotyls of ‘Tsuda’ or ‘Yurugi Akamaru’, which were exposed to sunlight or shaded for approximately 2 months after the start of the swelling (3 months after sowing), were subjected to suppression subtractive hybridization (SSH) to obtain subtractive cDNAs by using a PCR-Select cDNA Subtraction Kit (Clontech). Whereas ‘Tsuda’ exhibits light-dependent anthocyanin biosynthesis, ‘Yurugi Akamaru’ exhibits anthocyanin biosynthesis even in the underground part of the hypocotyls, indicating that anthocyanin biosynthesis in this cultivar does not require light exposure. The subtraction was performed between shaded and unshaded ‘Tsuda’ and between ‘Tsuda’ and ‘Yurugi-Akamaru.’ The subtractive cDNAs were cloned into pBluescript II SK (+), resulting in subtractive cDNA libraries.
For constructing the microarray, the sequences of isolated clones from these libraries were clustered using the ‘blastclust’ program and approximately 2800 clusters were obtained. Basically, one clone from each cluster was chosen for spotting on microarrays as gene probes. The probes were amplified by direct PCR of the 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 microarray hybridization were performed by using an Expression Array Detection Kit for Microarray, Cy3/Cy5, 3DNA Array 900 (Genisphere), according to the manufacturer's instructions.
Hybridization design
The hybridization design is shown in Table 1. Direct comparisons were made between blue and dark, UV-A and dark, or UV-B and dark treatments. The dark control sample was labelled with Cy5 and the other samples were labelled with Cy3. In addition to these hybridizations, three concurrent split-control hybridizations of the dark control (Table 1, arrays 1–3) were made to correct gene-specific dye bias, according to Rosenzweig et al. (2004). Three biological replicates were made for each comparison (Table 1, arrays 4–12). For each biological replicate, RNA was extracted from more than five plants.
|
Scanning and image analysis
The microarray slides were scanned at 5 µm 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, span=0.5) and then to the absolute M values (scale smoothing, span=0.75) before excluding flagged data, according to Wit and McClure (2004). After print-tip loess normalization (span=0.75), 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 (approximately 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 |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
UV-A-specific induction of anthocyanin biosynthesis in the peel of swollen hypocotyls
To characterize the light-dependent regulation of anthocyanin biosynthesis in ‘Tsuda’, the effects of several wavelengths 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 conditions 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 significantly and had no effect on the anthocyanin accumulation (data not shown).
Irradiation of the swollen hypocotyls with visible light free of UV-A also failed to induce anthocyanin production, 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 UV-A exposure. In contrast, no induction in CHS expression 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 investigate the period of exposure required for anthocyanin 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 dependent 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 accumulated 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 biosynthesis, 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 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’ subtracted 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 hypocotyls of ‘Yurugi Akamaru’ (Library ST-Y); and (iv) sunlight-exposed or shaded swollen hypocotyls of ‘Yurugi Akamaru’ subtracted by shaded swollen hypocotyls of ‘Tsuda’ (Library Y-ST).
From these subtractive libraries, a total of 4200 clones were sequenced, and assembled by the CAP3 program (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 annotated. 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 remains the possibility that failure of anthocyanin production under blue or UV-B light could be due to insufficient light intensity, or contamination of other wavelengths of lights. Therefore, microarray analysis (GSE6876
[NCBI GEO]
) 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 biosynthesis, 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 endotransglycosylase/hydrolase, and aquaporin, and to glucosynolate metabolism, such as cytochrome P450 (CYP83A1), methylthioalkylmalate synthase, myrosinase-binding proteins, and myrosinase-associated proteins.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
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 expression of many genes that are involved in stress responses. In contrast, such responses were not observed for the 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.
Although UV-A at 7 W m–2 did not induce the same protective mechanisms as UV-B, this intensity was sufficient to induce anthocyanin production, which may contribute to the tolerance to UV irradiation. Anthocyanins in the outer layer of the tissue not only can absorb UV to protect the inner layer of the tissue, but also serve as a scavenger of reactive oxygen species (Gould et al., 2002). The production of anthocyanins by UV-A exposure may have contributed to the protection of the plant tissue from the potential damage by UV absorption, and this may be responsible for the observation that UV-A did not have a strong impact on gene expression profiles as observed for low fluence UV-B response.
Involvement of UV-A-specific photoreceptor
At present, three types of photoreceptors have been characterized: (i) phytochromes which perceive red or far-red light (Quail, 1994); (ii) UV-A/blue photoreceptors including cryptochrome (Ahmad and Cashmore, 1993; Lin, 2000) and phototropin (Briggs and Christie, 2002); and (iii) the UV-B photoreceptor. Although the primary UV-A-responsive photoreceptor known is the UV-A/blue photoreceptor, all three types of these photoreceptors could potentially sense the UV-A signal. The measurement of absorption spectra of phytochrome suggested that it could also sense the blue/UV-A signal (Mancinelli, 1986). In fact, Pratt and Briggs (1966) reported in vivo conversion of phytochrome by blue light. Devlin and Kay (2000) proposed that phytochrome A acts as a photoreceptor in blue light input in circadian photoperception. For white cabbage seedlings, phytochrome alone appeared to be involved in anthocyanin production at a low fluence 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 production (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 biosynthesis 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 hypocotyls 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 phytochrome 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-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 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 |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
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.
|
Acknowledgements |
|---|
This study was funded by National Natural Science Foundation of China (30170785, 30040042).
|
Footnotes |
|---|
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.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
A-H Mackerness S, John CF, Jordan B, Thomas B. Early signaling components in ultraviolet-B responses: distinct roles for different reactive oxygen species and nitric oxide. FEBS Letters (2001) 489:237–242.[CrossRef][ISI][Medline]
Ahmad M, Cashmore AR. HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature (1993) 366:162–166.[CrossRef][Medline]
Arakawa O. Photoregulation of anthocyanin synthesis in apple fruit under UV-B and red light. Plant and Cell Physiology (1988) 29:1385–1390.
Arakawa O, Hori Y, Ogata R. Relative effectiveness and interaction of ultraviolet-B, red and blue light in anthocyanin synthesis of apple fruit. Physiologia Plantarum (1985) 64:323–327.[CrossRef]
Arakawa O, Hori Y, Ogata R. Characteristics of color development and relationship between anthocyanin synthesis and phenylalanine ammonia-lyase activity in ‘Starking Delicious’, ‘Fuji’ and ‘Mutsu’ apple fruits. Journal of the Japanese Society for Horticultural Science (1986) 54:424–430.[ISI]
Batschauer A, Rocholl M, Kaiser T, Nagatani A, Furuya M, Schäfer E. Blue and UV-A light-regulated CHS expression in Arabidopsis independent of phytochrome A and phytochrome B. The Plant Journal (1996) 9:63–69.[CrossRef][ISI]
Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society B (1995) 57:289–300.
Ben-Tal Y, King RW. Environmental factors involved in colouration of flowers of Kangaroo Paw. Scientia Horticulturae (1997) 72:35–48.[CrossRef]
Boccalandro HE, Mazza CA, Mazzella MA, Casal JJ, Ballaré CL. Ultraviolet B radiation enhances a phytochrome-B mediated photomorphogenic response in Arabidopsis. Plant Physiology (2001) 126:780–788.
Bouché N, Fait A, Bouchez D, Møller SG, Fromm H. Mitochondrial succinic-semialdehyde dehydrogenase of the 
-aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants. Proceedings of the National Academy of Sciences, USA (2003) 100:6843–6848.
Briggs WR, Christie JM. Phototropins 1 and 2: versatile plant blue-light receptors. Trends in Plant Science (2002) 7:204–210.[CrossRef][ISI][Medline]
Brosché M, Strid A. Molecular events following perception of ultraviolet-B radiation by plants. Physiologia Plantarum (2003) 117:1–10.[CrossRef]
Casati P, Walbot V. Gene expression profiling in response to ultraviolet radiation in maize genotypes with varying flavonoid content. Plant Physiology (2003) 132:1739–1754.
Casati P, Walbot V. Crosslinking of ribosomal proteins to RNA in maize ribosomes by UV-B and its effects on translation. Plant Physiology (2004) 136:3319–3332.
Christie JM, Jenkins GI. Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells. The Plant Cell (1996) 8:1555–1567.[Abstract]
Cuello J, Sanchez MD, Sabater S. Retardation of senescence by UV-A light in barley (Hordeum vulgare L.) leaf segments. Environmental and Experimental Botany (1994) 34:1–8.[CrossRef][ISI]
Devlin PF, Kay SA. Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity. The Plant Cell (2000) 12:2499–2509.
Dong YH, Beuning L, Davies K, Mitra D, Morris B, Kootstra A. Expression of pigmentation genes and photo-regulation of anthocyanin biosynthesis in developing Royal Gala apple flowers. Australian Journal of Plant Physiology (1998) 25:245–252.[ISI]
Dong YH, Mitra D, Kootstra A, Lister C, Lancaster J. Postharvest stimulation of skin color in Royal Gala apple. Journal of the American Society for Horticultural Science (1995) 120:95–100.
Feinbaum Rl, Storz G, Ausubel FM. High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants. Molecular and General Genetics (1991) 226:449–456.
Frohnmeyer H, Staiger D. Ultraviolet-B radiation-mediated responses in plants. Balancing damage and protection. Plant Physiology (2003) 133:1420–1428.
Fuglevand GA, Jackson A, Jenkins GJ. UV-B, UV-A, and blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis. The Plant Cell (1996) 8:2347–2357.[Abstract]
Gould KS, McKelvie J, Markham KR. Do anthocyanins function as antioxidants in leaves? Imaging of H2O2 in red and green leaves after mechanical injury. Plant, Cell and Environment (2002) 25:1261–1269.[CrossRef]
Grill R. Photocontrol of anthocyanin formation in turnip seedlings I. Demonstration of phytochrome action. Planta (1965) 66:293–300.[CrossRef][ISI]
Hahlbrock K, Scheel D. Physiology and molecular biology of phenylpropanoid metabolism. Annual Review of Plant Physiology and Plant Molecular Biology (1989) 40:347–369.[CrossRef][ISI]
Hirner AA, Seitz HU. Isoforms of chalcone synthase in Daucus carota L. and their differential expression in organs from the European wild carrot and in ultraviolet-A-irradiated cell cultures. Planta (2000) 210:993–998.[CrossRef][ISI][Medline]
Hoffmann S. The effect of UV-radiation on colours of leaves and flowers of ornamental plants. Gartenbauwissenschaft (1999) 64:88–93.[ISI]
Holton TA, Cornish EC. Genetics and biochemistry of anthocyanin biosynthesis. The Plant Cell (1995) 7:1071–1083.[CrossRef][ISI][Medline]
Huang X, Madan A. CAP3: a DNA sequence assembly program. Genome Research (1999) 9:868–877.
Jenkins GI, Long JC, Wade HK, Shenton MR, Bibikova TN. UV and blue light signaling: pathways regulating chalcone synthase gene expression in Arabidopsis. New Phytologist (2001) 151:121–131.[CrossRef][ISI]
Kashiwagi I, Kobayashi Y, Matsukawa T. Effect of ultraviolet rays on the flower color of Primula malacoides I. Discoloration of flower color under a covering of plastic film absorbing ultraviolet rays. Journal of the Japanese Society for Horticultural Science (1977) 46:66–71.[ISI]
Kataoka I, Sugiyama A, Beppu K. Role of ultraviolet radiation in accumulation of anthocyanin in berries of ‘Gros Colman’ grapes (Vitis vinifera L.). Journal of the Japanese Society for Horticultural Science (2003) 72:1–6.[ISI]
Kim BC, Tenessen D, Last R. UV-B-induced photomorphogenesis in Arabidopsis thaliana. The Plant Journal (1998) 15:667–674.[CrossRef][ISI][Medline]
Koes R, Quattrocchio R, Mol J. The flavonoid biosynthetic pathway in plants: function and evolution. BioEssays (1994) 16:123–132.[CrossRef][ISI]
Kubasek WL, Shirley BW, McKillop A, Goodman HM, Briggs W, Ausubel FM. Regulation of flavonoid biosynthetic genes in germinating Arabidopsis seedlings. The Plant Cell (1992) 4:1229–1236.
Lercari B, Sodi F, Sbrana C. Comparison of photomorphogenic responses to UV light in red and white cabbage (Brassica oleracea L.). Plant Physiology (1989) 90:345–350.
Lin C. Plant blue-light receptors. Trends in Plant Science (2000) 5:337–342.[CrossRef][ISI][Medline]
Maekawa S, Terabun M, Nakamura N. Effects of ultraviolet and visible light on flower pigmentation of ‘Ehigasa’ roses. Journal of the Japanese Society for Horticultural Science (1980) 49:251–259.[ISI]
Mancinelli AL. Comparison of spectral properties of phytochromes from different preparations. Plant Physiology (1986) 82:956–961.
Matsumaru K, Kamihama T, Inada K. Covering materials with different transmission properties on anthocyanin content of eggplant pericarp. Environment Control in Biology (1971) 9:91–97.
Mor Y, Zieslin N. UV-induced blackening of rose petals. Environmental and Experimental Botany (1990) 30:455–462.[CrossRef][ISI]
Nakamura N, Nakamae H, Maekawa S. Effects of light and kinetin on anthocyanin accumulation in the petals of Rosa hybrida, Hort cv. Ehigasa. Zeitschrift für Pflanzenphysiologie (1980) 98:263–270.[ISI]
Pratt LH, Briggs WR. Photochemical and non-photochemical reactions of phytochrome in vivo. Plant Physiology (1966) 41:467–474.
Quail PH. Phytochrome genes and their expression. In: Photomorphogenesis in plants—Kendrick RE, Kronenberg GHM, eds. (1994) 2nd edn. London: Kluwer Academic Publishers. 71–104.
Reddy VS, Goud KV, Sharma R, Reddy AR. Ultraviolet-B-responsive anthocyanin production in a rice cultivar is associated with a specific phase of phenylalanine ammonia lyase biosynthesis. Plant Physiology (1994) 105:1059–1066.[Abstract]
Rosenzweig BA, Pine PS, Domon OE, Morris SM, Chen JJ, Sistare FD. Dye-bias correction in dual-labeled cDNA microarray gene expression measurements. Environmental Health Perspectives (2004) 112:480–487.[ISI][Medline]
Shimodaira H. An approximately unbiased test of phylogenetic tree selection. Systematic Biology (2002) 51:492–508.[CrossRef][ISI][Medline]
Shimodaira H. Approximately unbiased tests of regions using multistep–multiscale bootstrap resampling. Annals of Statistics (2004) 32:2616–2641.[CrossRef][ISI]
Siegelman HW, Hendricks SB. Photocontrol of anthocyanin formation in turnip and red cabbage seedlings. Plant Physiology (1957) 32:393–398.
Singh A, Selvi MT, Sharma R. Sunlight-induced anthocyanin pigmentation in maize vegetative tissues. Journal of Experimental Botany (1999) 50:1619–1625.
Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology (2004) 3. Article 3.
Smyth GK, Michaud J, Scott H. Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics (2005) 21:2067–2075.
Smyth GK, Speed TP. Normalization of cDNA microarray data. Methods (2003) 31:265–273.[CrossRef][ISI][Medline]
Suesslin C, Frohnmeyer H. An Arabidopsis mutant defective in UV-B light-mediated responses. The Plant Journal (2003) 33:591–601.[CrossRef][ISI][Medline]
Suzuki R, Shimodaira H. Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics (2006) 22:1540–1542.
Takeda J, Obi I, Yoshida K. Action spectra of phenylalanine ammonia-lyase and chalcone synthase expression in carrot cells in suspension. Physiologia Plantarum (1994) 91:517–521.[CrossRef]
Taylor RM, Tobin AK, Bray CM. DNA damage and repair in plants. In: Plants and UV-B: responses to environmental change—Lumsden PL, ed. (1997) Cambridge: Cambridge University Press. 53–75.
Tezuka T, Yamaguchi F, Ando Y. Physiological activation in radish plants by UV-A radiation. Journal of Photochemistry and Photobiology B: Biology (1994) 24:33–40.[CrossRef][ISI]
Toguri T, Umemoto N, Kobayashi O, Ohtani T. Activation of anthocyanin synthesis genes by white light in eggplant hypocotyl tissues, and identification of an inducible P-450 cDNA. Plant Molecular Biology (1993) 23:933–946.[CrossRef]