Lhl4 encodes a distant relative of light-harvesting Chl-a/b proteins in the green alga Chlamydomonas reinhardtii. Lhl4 mRNA markedly accumulated within 30 min after illumination and in proportion to the light intensity up to a fluence rate much higher than that required for photosynthesis. The high intensity light (HL)-induced accumulation of Lhl4 mRNA required continuous illumination, and the mRNA level rapidly decreased when the cells were placed in the dark. HL only slightly stabilized the mRNA, suggesting that the HL-induced expression of the Lhl4 gene is primarily regulated at the level of transcription. Blue light was more effective for inducing Lhl4 gene expression than green or red light, and far-red light had no effect. The action spectrum for Lhl4 gene expression was examined at wavelengths between 325 and 775 nm using the Okazaki Large Spectrograph. The obtained spectrum showed a distinct peak in the blue region (450 nm) and a shoulder in the UV-A region (375 nm). The curve in the spectrum rose steeply in the short wavelength UV region. In addition, we observed two minor peaks in the green (575 nm) and the red (675 nm) regions. The action spectrum suggests that a blue/UV-A light photoreceptor with a flavin-based chromophore participates in the HL response of Lhl4 gene expression. However, the hypersensitivity to near UV-B light suggests the involvement of an unidentified UV light perception system in the expression of the Lhl4 gene.
The light-harvesting antennae in higher plants and green algae are composed of several homologous light-harvesting Chl-a/b-binding (LHC) proteins. These LHC proteins all have three membrane-spanning helices containing Chl-binding sites. Additionally, various Lhc-like genes encoding distant relatives of LHC proteins have been identified (Jansson 1999). These LHC-like proteins possibly bind Chl due to the presence of the Chl-binding fold, although there is very limited biochemical evidence for Chl binding (Adamska et al. 1999). The best characterized LHC-like proteins are early light-inducible protein (ELIP), which has three membrane-spanning domains, and high light-inducible protein (HLIP), which has one membrane-spanning domain. The gene expression of these proteins is enhanced under high intensity light (HL) conditions, suggesting that they function to protect plants against light stress. It has been proposed that these proteins act by preventing the formation of reactive oxygen species by exciting free Chl as a transient pigment carrier or by dissipating excessive excitation energy in the photosystem (Adamska 1997, Montané and Kloppstech 2000, He et al. 2001, Havaux et al. 2003, Hutin et al. 2003). On the other hand, studies of deletion mutants have suggested that HLIPs are involved in the regulation of tetrapyrrole biosynthesis in response to pigment availability (Xu et al. 2002, Xu et al. 2004).
It has been reported that expression of ELIP is regulated by the redox state of the electron carrier in photosynthetic electron transport (Montané et al. 1998, Kimura et al. 2003) and by hydrogen peroxide (Kimura et al. 2001), which have been known to act as sensors in various kinds of light stress responses in plants. On the other hand, ELIP mRNA accumulates in response to blue or red light in etiolated pea seedlings (Adamska 1995). Enhancement of the expression of ELIP and HLIP genes by blue or UV-A light has been reported in mature green plants (Adamska et al. 1992a, Adamska et al. 1992b, Heddad and Adamska 2000) and in cyanobacteria (Dolganov et al. 1995, Salem and van Waasbergen 2004). Studies using light signaling mutants of Arabidopsis thaliana have indicated that phytochromes A and B participate in the induction of ELIP gene expression during photomorphogenesis in response to red/far-red light (Harari-Steinberg et al. 2001). However, no quantitative analysis of expression of these Lhc-like genes in response to the intensity and wavelength of light was carried out, and the photoreceptor mediating the blue/UV-A light responses has not been identified. A true action spectrum, a wavelength-dependent curve for a biological response based on the measurement of the light intensity–response curve at multiple wavelengths, can be correlated to the absorption spectrum of the photoreceptor mediating the response (Foster 2001). Therefore, determination of an action spectrum for expression of Lhc-like genes provides important information about the properties of photoreceptors involved in the light-dependent expression of these genes.
Previously we identified Lhl4, a novel Lhc-like gene in the green alga Chlamydomonas reinhardtii (Teramoto et al. 2004). We found that Lhl4 mRNA markedly accumulated after the transfer of the algal culture from low intensity light (LL) conditions to HL conditions. The Lhl4 protein was detected in the thylakoid fraction of HL-exposed algal cells, but not from LL-grown cells (unpublished data). These findings support the notion that the Lhl4 protein is involved in the protection of photosystems against light stress. The HL-induced accumulation of Lhl4 mRNA was most rapid and prominent among the members of the Lhc gene superfamily that we examined, including the ELIP and HLIP homologs in C. reinhardtii. The HL response was not inhibited by inhibitors of photosynthetic electron transfer, DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Furthermore, the induction of Lhl4 expression by the addition of hydrogen peroxide or methylviologen was very small. These results indicated that neither the redox state of the plastoquinone pool nor reactive oxygen species are main factors in the expression of Lhl4 in response to HL (Teramoto et al. 2004).
In the present study, we characterized the properties of the Lhl4 expression in response to light, including the effects of light intensity, light quality and illumination period. We also examined the stability of Lhl4 mRNA in the dark and under HL conditions. Furthermore, we measured the action spectrum for Lhl4 expression at wavelengths between 325 and 775 nm using the Okazaki Large Spectrograph at the National Institute for Basic Biology in Okazaki, Japan. The results show the wavelength-dependent expression of Lhl4, indicating the involvement of blue and/or UV light photoreceptor(s).
High intensity light-dependent expression of the Lhl4 gene
Fig. 1 shows the light intensity dependence of the expression of the Lhl4 gene in C. reinhardtii cells that had been dark adapted for 16 h and then exposed to different intensities of light (50–1,000 µmol m–2 s–1) for 30 min. Lhl4 mRNA accumulated in proportion to the intensity of light, and the induction started to be saturated at 1,000 µmol m–2 s–1. The light intensity required for saturation of Lhl4 expression was much higher than that for saturation of photosynthesis. These results are compatible with the view that Lhl4 gene expression is not directly related to the photosynthetic event.
As shown in Fig. 2, the level of Lhl4 mRNA markedly increased and reached a maximum after 30 min when the algal cells grown under LL at 10 µmol m–2 s–1 were exposed to HL at 1,000 µmol m–2 s–1. This finding agrees with the results of our previous study (Teramoto et al. 2004). We previously showed that Lhl4 mRNA then returned to a low level after 4–6 h even under HL conditions. Thus, it was possible that a short period of HL exposure is sufficient for the transient induction of Lhl4 expression. However, Lhl4 mRNA hardly accumulated when the cells were exposed to HL for 5 min followed by a 25 min incubation at LL (Fig. 2), indicating that continuous HL illumination is required for the accumulation of Lhl4 mRNA. Fig. 2 also shows that the level of mRNA following a 30 min exposure to HL rapidly decreased to near the initial very low level within 30 min of incubation in the dark. Therefore, HL could be required for stabilization of Lhl4 mRNA.
We therefore examined the effects of light on the stability of the Lhl4 mRNA (Fig. 3). Cells grown under LL were exposed to HL and then further incubated in the presence of actinomycin D, an inhibitor of transcription, in the dark or under HL conditions. The level of mRNA decreased with first-order kinetics in the dark and HL conditions. The degradation was faster in the dark (t1/2 = 8 min) than under HL conditions (t1/2 = 12 min), indicating that Lhl4 mRNA is somewhat more stable under the HL conditions. However, the stabilization by HL is too small to account for the marked accumulation of Lhl4 mRNA, indicating that the HL-induced expression of Lhl4 is primarily regulated at the transcriptional level.
Light quality-dependent expression of Lhl4 gene
We next examined the effects of light quality on Lhl4 expression. Fig. 4 shows the level of Lhl4 mRNA in algal cells exposed for 30 min to various monochromatic light-emitting diode (LED) lights (100 µmol m–2 s–1) or white light (1,000 µmol m–2 s–1) in the presence of DCMU and DBMIB at concentrations that completely inhibit the oxygen evolution activity of C. reinhardtii cells (Teramoto et al. 2004). We found that blue light is the most efficient for inducing Lhl4 expression. As shown in the inset in Fig. 4, the level of Lhl4 mRNA increased in proportion to the intensity of the blue light up to a high fluence rate (100 µmol m–2 s–1). Green light and, to a lesser extent, red light also promoted the accumulation of Lhl4 mRNA, but the effects were much weaker than those of blue light. In contrast, far-red light hardly induced the accumulation of Lhl4 mRNA.
Action spectrum for expression of Lhl4 gene
Fig. 5 shows the relative increase in the level of Lhl4 mRNA in dark-adapted algal cells that were exposed for 30 min to equal intensities (80 µmol m–2 s–1) of monochromatic light (375–775 nm) in the presence of DCMU and DBMIB under the Okazaki Large Spectrograph. The wavelength–response curve showed a peak in the blue light region (450 nm), and two minor peaks in the green and the red light regions (575 and 675 nm), of which the amplitude was about 25% of that of the blue peak. The presence of these peaks agrees with the observed accumulation of Lhl4 mRNA during illumination with LED light (Fig. 4). Notably, the response to 375 nm (UV-A) light is stronger than the response to blue light.
To evaluate the properties of the blue/UV-A light response of Lhl4 gene expression, we obtained a detailed and true action spectrum for the light-induced accumulation of Lhl4 mRNA at wavelengths between 325 and 510 nm. Dark-adapted algal cells were exposed to monochromatic light at various intensities for 30 min in the presence of DCMU and DBMIB using specially designed threshold boxes (Watanabe et al. 1982). Fig. 6 shows the light fluence–response curves for the accumulation of Lhl4 mRNA during exposure to the different monochromatic lights. The level of Lhl4 mRNA increased in proportion to the applied light intensity except at 325 nm, which tended to cause saturation of the mRNA level at much lower fluence rates than the other wavelengths.
Fig. 7 shows the action spectrum for Lhl4 expression, in which the relative quantum effectiveness for Lhl4 expression was plotted against the wavelength. The effectiveness was evaluated as the reciprocal of the fluence causing a fixed response (0.1 unit in Fig. 6) based on linear regression of the fluence–response curve. The obtained spectrum has a band in the blue light region that peaks at 450 nm and a shoulder in the UV-A light region at approximately 375 nm. The response curve measured at 80 µmol m–2 s–1 (Fig. 5) agrees well with the obtained action spectrum. Interestingly, the curve rose steeply in the UV light region (<350 nm); the amplitude of the spectrum at 325 nm rose to approximately 100-fold that for the blue peak (450 nm).
In the present study, we performed a detailed characterization of the properties of light-induced accumulation of Lhl4 mRNA in C. reinhardtii. The action spectrum showed that blue/UV-A light is responsible for the accumulation of Lhl4 mRNA, whereas wavelengths absorbed by photosynthetic pigments are much less effective for inducing the accumulation of this transcript. The results suggest that HL-dependent Lhl4 gene expression is predominantly mediated by a blue/UV-A light photoreceptor. As shown in Fig. 2, within 30 min of transferring the HL-exposed algal cells to darkness, the amount of Lhl4 mRNA decreased to near the original, very low level. This period of 30 min corresponds to the degradation rate of the mRNA in the presence of the transcriptional inhibitor, suggesting that the putative photoreceptor activated by illumination relaxes to the ground state within a few minutes after the transfer to darkness.
At present, it has been reported that C. reinhardtii has three types of photoreceptors that possibly respond to blue light. They include two types of flavin-containing photoreceptors, phototropin (Huang et al. 2002) and cryptochrome (Small et al. 1995), and retinal-containing rhodopsins (Sineshchekov et al. 2002). The action spectrum for Lhl4 expression showed a major peak in the blue region at 450 nm and a shoulder in the UV-A region at approximately 375 nm. The spectral features above 375 nm indicate that a photoreceptor with a flavin-based chromophore participates in Lhl4 gene expression. However, the response curve in the action spectrum continued to rise steeply below 350 nm, indicating that the expression is highly sensitive to short wavelength UV light. Flavin has a light absorption band in the UV region, but the intensity in the action spectrum at 325 nm was 100-fold higher than in the blue region. Apparently, any flavoproteins cannot account for such a difference in the absorption in the UV and blue regions. Therefore, it is likely that the short wavelength UV-induced expression of Lhl4 mRNA is mediated by a mechanism other than the blue light response. The expression of Lhc-like genes, including ELIP in mature green plants (Adamska et al. 1992a, Adamska et al. 1992b, Heddad and Adamska 2000) and HLIP in cyanobacteria (Dolganov et al. 1995, Salem and van Waasbergen 2004), was also reported to be enhanced by blue or UV-A light, although no quantitative analysis of the response to the light intensity and wavelength was carried out. The specific photoreceptor mediating the blue/UV-A light response has not been identified for the expression of ELIP and HLIP genes.
RNA interference studies have demonstrated that a phototropin is involved in regulation of the sexual life cycle in C. reinhardtii (Huang and Beck 2003, Ermilova et al. 2004). At present, there is little evidence for the involvement of phototropin in the expression of photosynthetic genes. In C. reinhardtii, blue light influences the expression of Lhc (Johanningmeier and Howell 1984, Kindle 1987) and gsa (Matters and Beale 1995), which encodes a chlorophyll biosynthetic enzyme. Experiments using a flavin antagonist have suggested the involvement of a flavoprotein in the expression of gsa (Herman et al. 1999), and a phototropin has been proposed to be involved in the blue light response of these genes in C. reinhardtii (unpublished observations commented on in Grossman et al. 2004).
In contrast to phototropin, the physiological function of cryptochrome in C. reinhardtii is largely unknown. The action spectrum for Lhl4 gene expression showed two small bands in the green (575 nm) and the red (675 nm) regions in addition to a major band in the blue region. Notably, the absorption spectra of cryptochromes of higher plants exhibit a small amount of absorption in the green/red region (500–700 nm) due to a neutral radical of flavin (Lin et al. 1995, Malhotra et al. 1995). Therefore, the two longer wavelength bands in the action spectrum may correspond to the absorption bands of cryptochrome, but we cannot exclude a contribution of some molecular species other than cryptochrome to these bands. The Lhl4 expression by blue light seemed to begin to be saturated at 100 µmol m–2 s–1 (Fig. 4), suggesting that the saturating expression level by blue light is lower than that by white light. If this is the case, the involvement of an additional photoreceptor would account for the response to white light. Further experiments using a much stronger monochromatic light source will be required to assess the level of saturation. Currently, a reliable in vivo action spectrum for cryptochrome is not available so that it is difficult to evaluate the involvement of this photoreceptor in Lhl4 gene expression based only on the action spectrum. In this context, it may be worth noting that the cryptochrome in C. reinhardtii (CPH1) was reported to accumulate in darkness and rapidly degrade under light (Reisdorph and Small 2004) like CRY2, one of the cryptochromes in A. thaliana (Lin et al. 1998). This light-induced degradation may be related to the transient induction of Lhl4 expression; an HL-induced increase in the level of Lhl4 mRNA was followed by a rapid decrease to a low level even under HL conditions (Teramoto et al. 2004).
UV light, especially UV-B light (280–320 nm), has various damaging effects on the growth and development of plants, and it also induces various defense systems (Jenkins 1997, Jansen et al. 1998, Brosché and Strid 2003). Some of these defense responses, such as biosynthesis of UV-screening flavonoids, are thought to be mediated by a UV-B photoreceptor, although a specific photoreceptor for UV-B light has not yet been identified. The high sensitivity of Lhl4 gene expression to near UV-B light may involve an unidentified UV-B photoreceptor. Because UV-B light is absorbed by various biomolecules, including DNA, proteins and lipids, and because it damages them, it is difficult to discriminate between the secondary effects of the UV-induced damage and the acclimation response through a specific photoreceptor. However, it is unlikely that the damaging effects are the main reason for expression of the Lhl4 gene because inhibition of protein synthesis suppresses the HL-induced accumulation of Lhl4 mRNA and because treatments with reactive oxygen species induce only a very low level of expression (Teramoto et al. 2004). A more detailed analysis of the response of Lhl4 gene expression to UV-B light will help to understand the properties of the UV light perception system. The photosynthetic machinery is one of the main targets for UV damage (Jansen et al. 1998). Thus, the hypersensitivity of Lhl4 expression to UV light could be related to participation of the Lhl4 protein in protecting the photosystem against damage under HL stress conditions.
The action spectrum obtained in this study provides important information about the light perception system involved in the light-dependent expression of the Lhl4 gene. Our results indicate that Lhl4 is a potent probe for studying how the expression of Lhc-like genes responds to HL and UV light. Further analyses using C. reinhardtii RNA interference strains for phototropin and cryptochrome are needed to determine the involvement of these blue light photoreceptors in Lhl4 gene expression.
Materials and Methods
Cell culture and light treatments
The wild-type C. reinhardtii strain C-9 was obtained from the Institute of Applied Microbiology (IAM) culture collection at the University of Tokyo. C. reinhardtii cells were mixotrophically cultured in Tris-acetate-phosphate medium (Gorman and Levine 1965) under continuous LL (10 µmol m–2 s–1; white fluorescent bulbs) at 25°C with constant agitation. Aliquots of the cultures at the mid-logarithmic phase (1–3 × 106 cells ml–1) were subjected to various light treatments. Before light treatments, the cell culture was dark adapted for 16 h unless otherwise noted.
For the HL treatment, aliquots of the cultures were transferred to 100 ml glass test tubes (30 mm diameter), immersed in water at 25°C, and illuminated with a halogen lamp as previously described (Teramoto et al. 2004). For measurements of stability of the mRNA, the aliquots of the cell culture in the test tubes were exposed to HL for 20 min. Actinomycin D was added to the cell culture at 50 µg ml–1 and the culture was incubated further for 20 min. Finally, the algal cells were transferred to darkness or kept under HL and sampled every 6 min for a total of 30 min. We observed that 20 min incubation in the presence of actinomycin D under LL suppressed the accumulation of Lhl4 mRNA by the subsequent HL exposure, indicating that the inhibitor treatment really blocks RNA synthesis.
To examine the effects of light quality on Lhl4 gene expression, aliquots of the dark-adapted algal cell culture were transferred to glass Petri dishes (50 mm diameter) under dim red light, which had no observable effects on the level of Lhl4 mRNA, and then illuminated for 30 min from the bottom with a blue (λmax = 470 nm, Δλ1/2 = 33 nm), green (λmax = 520 nm, Δλ1/2 = 35 nm), red (λmax = 660 nm, Δλ1/2 = 27 nm) or far-red (λmax = 730 nm, Δλ1/2 = 43 nm) LED light (blue, red and far-red LED lights from Sanyo Electric Biomedical Co., Ltd, Tokyo, Japan; green LED from Hayashi Watch-Works Co., Ltd, Tokyo, Japan) in the presence of 10 µM DCMU and 0.4 µM DBMIB.
To determine the response and action spectra of Lhl4 gene expression, aliquots of the dark-adapted algal cell culture from a single batch were transferred to glass Petri dishes (25 mm diameter) without lids under dim red light, and then illuminated for 30 min simultaneously from the top with monochromatic light at various wavelengths between 325 and 775 nm using the Okazaki Large Spectrograph (Watanabe et al. 1982) at the National Institute for Basic Biology, Okazaki, Japan. Just before the illumination, DCMU and DBMIB were added at 10 and 0.4 µM, respectively. Cells were fixed by rapid addition of ethanol [final concentration of 50% (v/v)] at the designated illumination time, collected by centrifugation and stored at –80°C.
Stock solutions of DCMU (10 mM), DBMIB (0.4 mM) and actinomycin D (50 mg ml–1) were dissolved in dimethylsulfoxide. Equivalent amounts of dimethylsulfoxide alone had no observable effects on the level of Lhl4 mRNA.
Quantitative reverse transcription–PCR (RT–PCR)
Total RNA was prepared from the algal cells using an RNeasy mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s protocol. The amount of Lhl4 mRNA was examined by RT–PCR using a real-time PCR assay system as described previously (Teramoto et al. 2004).
We thank Professor M. Nishimura for use of the Okazaki Large Spectrograph. This research was supported by a Grant for the Frontier Research System at RIKEN and Grants-in-Aid for Young Scientists (15770037) from MEXT of Japan. Action spectroscopy in this study was carried out under the NIBB Cooperative Research Program for the Okazaki Large Spectrograph (5–502).
Present address: Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizu-cho Soraku-gun, Kyoto, 619-0292 Japan.