Journal of Experimental Botany, Vol. 55, No. 396, pp. 517-523, February 1, 2004
© 2004 Oxford University Press
Plants and the Environment |
A transgene encoding a blue-light receptor, phot1, restores blue-light responses in the Arabidopsis phot1 phot2 double mutant
Received 22 July 2003; Accepted 15 October 2003
1 Research and Development Center for Higher Education, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan
2 Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan
* To whom correspondence should be addressed. Fax: +81 92 726 4758. E-mail: kenrcb{at}mbox.nc.kyushu-u.ac.jp
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Abstract |
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Phototropins (phot1 and phot2) are suggested to be multifunctional blue-light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses.
Key words: Arabidopsis thaliana, blue-light response, chloroplast movement, guard cells, phototropin, phototropism, stomatal movement, transformation.
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Introduction |
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Phototropins (phot1 and phot2) are autophosphorylating serine/threonine protein kinases that function as blue light (BL) receptors for phototropism, chloroplast movement, and stomatal opening in Arabidopsis (Briggs and Huala, 1999; Kagawa et al., 2001; Kinoshita et al., 2001). In addition to these BL responses, phototropins have also been suggested to play roles in leaf expansion (Sakai et al., 2001; Sakamoto and Briggs, 2002), rapid growth inhibition of hypocotyls (Folta and Spalding, 2001), and the destabilization of specific nuclear and chloroplast transcripts (Folta and Kaufman, 2003). Although phototropins mediate these BL responses over a wide range of BL fluence, it is not understood how the same photoreceptor mediates these diverse range of responses in plant cells. Furthermore, it has been suggested that BL-dependent stomatal opening is also regulated by a carotenoid zeaxanthin acting as a blue-light receptor (Zeiger and Zhu, 1998; Frechilla et al., 1999). The Arabidopsis mutant npq1, which is defective in zeaxanthin de-epoxidase, lacks the specific stomatal opening to BL in the presence of a red light (RL) background (Frechilla et al., 1999).
While a large body of biochemical and genetic studies using Arabidopsis mutants has suggested the involvement of phototropins in BL responses, it has not yet been determined whether a wild-type phototropin gene can complement the BL responses of a phototropin-deficient mutant, with the exception of phototropic responses. That is, transformation of an Arabidopsis phot1 mutant, that lacks phototropic activity, with the wild-type PHOT1 gene restored the phototropism (Huala et al., 1997; Christie et al., 2002; Sakamoto and Briggs, 2002), and it was further suggested that the phototropism required the autophosphorylating protein kinase activity of phot1, since transformation with a mutated phot1 devoid of light-inducible kinase activity failed to restore the phototropic curvature of seedlings (Christie et al., 2002). Based on these results, it was anticipated that phototropins also restore BL responses other than phototropism when the double mutant is transformed with the phototropin gene. Thus far, there has been no report of a successful transformation of the Arabidopsis phot1 phot2 double mutant with the phototropin gene which is really needed to confirm the role of phototropins in these BL responses in Arabidopsis.
In the present study, the phot1 phot2 double mutant was transformed with wild-type PHOT1 cDNA and the BL responses were analysed in transgenic plants. It was found that BL responses such as phototropism, chloroplast accumulation, leaf expansion, and stomatal opening were restored, although differentially, in transgenic plants, which differed in their expression of phot1. These results confirmed that these BL responses essentially require phot1 participation in Arabidopsis.
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Materials and methods |
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Plant materials and growth conditions
The homozygous recessive glabrous1 (gl1), phot2 single mutant (phot2-1) and phot1 phot2 double mutant (phot1-5 phot2-1) were used in this study (Kagawa et al., 2001). For investigation of stomatal opening and chloroplast movement, Arabidopsis plants were grown in soil for 4–5 weeks under white light (70 µmol m–2 s–1, 16/8 h light/dark cycles) at 24 °C in a growth room. For measurement of second positive curvature responses, seeds were planted in square Petri dishes in rows on half-strength Murashige and Skoog medium with 0.8% agar (w/v).
Transformation and detection of PHOT1 in transgenic plants
The cDNA encoding Arabidopsis full-length PHOT1 (41–3201 bp; GenBank accession no. AF030864
[GenBank]
) was cloned and fused to the 35S cauliflower mosaic virus (CMV) promoter, and inserted into the pBI121 vector. These plasmids were transformed into Agrobacterium tumefaciens strain C58. The floral dip method was used to transform the Arabidopsis phot1 phot2 double mutant (Clough and Bent, 1998). The transformants were screened on Murashige and Skoog plates supplemented with 75 mg l–1 kanamycin. PCR analysis was used to detect the presence of T-DNA in the kanamycin-resistant Arabidopsis. Genomic DNA was extracted from fresh leaves of 4–5-week-old plants by the method of Liu et al. (1995). PCR amplification of the PHOT1 gene was performed with the following specific primers: 5'-CCGGATCCC TCAAAAAACATTTGTTTGCAG-3' for the PHOT1 3', and 5'-GACGCACAATCCCACTATCCTTC-3' for the 35S promoter. Amplified DNA was separated by electrophoresis on 1% (w/v) agarose gel, stained with ethidium bromide, and detected by UV-light.
Measurement of stomatal movement
Stomatal movement in response to BL was measured by the gas exchange method. Gas exchange of Arabidopsis plants was carried out using LI-6400 open-flow systems (Li-Cor Inc., Lincoln, NE). Arabidopsis plants were removed from the soil, placed into a 14 ml Falcon tube with distilled water, and kept in darkness for 12 h before measurements. The leaf was clamped with a gas-tight Arabidopsis chamber (Li-Cor) designed for small leaves. The leaf temperature was maintained at 24 °C. Measurements were conducted under a constant CO2 concentration of 350 µl l–1 and a relative humidity of 55–60%. H2O and CO2 concentrations in the leaf chamber were measured using infrared gas analysers. Data were recorded at 10 s intervals and processed with a KaleidaGraphTM (Synerge Software, PA). The red light for background illumination was produced by a tungsten lamp (MHF-G150LR; Moritex, Tokyo, Japan) by passing the light through a red cut-off filter (Corning 2-61; Corning, MA). Blue light was provided by a metal halide lamp (LS-M250; Moritex) passed through a blue filter (Corning 5-60; Corning). Photon fluence rates were measured using a LI-250 light meter with an LI-190SA quantum sensor (Li-Cor).
Measurement of phototropic response
Measurement of phototropic curvature was performed as described previously (Lascève et al., 1999). To induce hypocotyls curvature, 3-d-old etiolated seedlings were irradiated for 16 h with unilateral blue light supplied by a 20 W blue fluorescent lamp (FL20SB-H; Matsushita, Tokyo, Japan) at a fluence rate of 1 µmol m–2 s–1. After illumination, the images of the seedlings were scanned using an image scanner (ES-2200; Epson, Tokyo, Japan). Curvatures were measured in degrees from the printout of the images as described previously (Sakai et al., 2001).
Measurement of chloroplast movement
For measurement of chloroplast movement, the plants were illuminated for 6 h with blue light at a fluence rate of 1 µmol m–2 s–1 supplied by blue light-emitting diode arrays with peak emissions of 470 nm (30 nm band width at half-peak height) (EYELA, Tokyo, Japan). After irradiation with BL, the micrographs were taken from the adaxial side of the leaves, which were pre-evacuated with a 3 ml syringe filled with distilled water.
Preparation of polyclonal antibodies against Arabidopsis PHOT1
The N-terminal region of PHOT1 was used to prepare polyclonal antibodies as antigens. The 543 bp DNA fragment encoding Met-1 to Ile-181 was amplified by PCR with the primers 5'-TTCCAT GGAACCAACAGAAAAACCATCGA-3' and 5'-TTCCATGGT TAAATCCCACTCCTCCCGCC-3'. The amplified DNA fragments were cloned into the pET30a(+) (Novagen, Madison, WI) and used to transform Escherichia coli BL21. These polypeptides were expressed as fusion proteins with a His tag and purified using His Bind Kits according to the manufacturer’s instructions (Novagen). The purified polypeptides were used to immunize rabbits.
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Results |
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Measurement of stomatal conductance in whole leaves
In this work, a gas exchange method was developed to measure stomatal opening in Arabidopsis whole plants as an alternative to determining stomatal aperture in epidermal peels. Despite the emergence of the gas exchange method as the preferred means of measuring stomatal movements, there have been relatively few studies applying this method to the model plant Arabidopsis (Lascève et al., 1999; Eckert and Kaldenhoff, 2000). The gas exchange method allows kinetic studies of stomatal movements in intact plants and therefore improves the reliance of measurements. In the present study, the dual beam protocol was applied to distinguish the stomatal responses specific to BL from RL (Ogawa et al., 1978). Figure 1 shows the light-induced changes in stomatal conductance, photosynthetic rate, and intercellular CO2 concentration in the Arabidopsis wild type. When the dark-adapted leaf was irradiated with a high fluence rate of red light (RL) at 600 µmol m–2 s–1, the photosynthetic CO2 uptake immediately increased and the intercellular CO2 concentration (Ci) immediately decreased, followed by an increase in stomatal conductance. After increasing for 40 min, stomatal conductance reached a steady-state level. Then a low fluence rate of BL (5 µmol m–2 s–1) was superimposed on the RL, and the stomatal conductance increased linearly with time for about 10 min. The average rate of increase in conductance by irradiation of BL was 0.115±0.024 mol H2O m–2 s–1 min–1, and this rate was about four times greater than that by irradiation with RL (Fig. 1). After turning off the BL, stomatal conductance decreased and eventually reached a steady-state level with a small dip. Although repeated application of BL under a background of RL caused almost the same changes in conductance, no change in conductance occurred when repeated applications of RL at the same fluence rate was used in place of BL (data not shown). Thus, the stomatal opening induced by weak BL irradiation over a background of strong RL irradiation was BL-specific and independent of photosynthesis, as previously described (Ogawa et al., 1978; Assmann, 1988).
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A typical response of photosynthesis and stomatal opening to light in the wild type and the phot1 phot2 double mutant is shown in Fig. 2. Both the wild-type and mutant plants had a positive conductance even after full adaptation to darkness, indicating that the stomata were at least partially opened. Furthermore, the level of conductance in dark-adapted leaves tended to be higher in the phot1 phot2 double mutant compared with the wild type. Irradiation of leaves with RL at a fluence rate of 600 µmol m–2 s–1 increased stomatal conductance both in wild-type and phot1 phot2 double mutant plants. Additional weak BL further increased stomatal conductance in the wild-type plant. By contrast, stomata in the double mutant did not respond to BL at all (Fig. 2). This defect in the BL-specific stomatal response was revealed for the first time by gas exchange analysis in a phot1 phot2 double mutant, and was in accord with those obtained previously in the epidermis and in whole plants (Kinoshita et al., 2001).
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Transformation of the Arabidopsis phot1 phot2 double mutant
The Arabidopsis phot1 phot2 double mutant was transformed using PHOT1 cDNA and the Agrobacterium-mediated floral dip method (Clough and Bent, 1998). Thirteen primary transformants were obtained with wild-type PHOT1. These plants were allowed to self independently, and four T2 lines (TF-A, TF-B, TF-C, and TF-D) that were homozygous for the PHOT1 transgene were selected and used throughout the study. The insertion of T-DNA in genomic DNA of these transgenic lines was confirmed by PCR (data not shown). Although the phot1 phot2 double mutant was also transformed with PHOT2 cDNA and 26 T-DNA inserted lines were obtained, it was not possible to find any lines that restored the targeted BL responses in those transgenic plants.
Expression of phot1 protein in transgenic plants
Expression of the Arabidopsis phot1 protein in transgenic plants was determined by western blot analysis using antibodies raised against the N-terminal region of Arabidopsis phot1. Determination of the phot1 protein was carried out in isolated microsomal membranes from dark-grown seedlings because of the relative abundance of phot1 protein in the membranes from etiolated seedlings (Sakamoto and Briggs, 2002). As shown in Fig. 3, the phot1 protein was detected in both the wild type and the transgenic line, TF-A. Phot1 was only detected in microsomal fractions, indicating that the phot1 expressed in the TF-A showed membrane-associated localization in etiolated seedlings of Arabidopsis, as also seen in the wild type (Christie et al., 2002; Sakamoto and Briggs, 2002). Although phot1 cDNA was expressed under the control of the CMV 35S promoter, the level of phot1 in the TF-A was unexpectedly lower and less than one-hundredth of that found in etiolated seedlings of the wild type. To date, the phot1 protein has been found to be absent in both the etiolated seedlings and mature leaves of three transgenic lines (TF-B, TF-C, and TF-D), even under enhanced conditions for detection using the chemiluminescence method (data not shown). However, because almost full and partial restoration of the phototropic response to BL were observed in TF-B and TF-C, respectively, as shown below, a certain quantity of phot1 protein should be present in these transformants. While the reason for the reduced level of phot1 in transgenic plants is not clear, low expression of phot1 has been reported when phot1 was expressed under the control of the 35S promoter (Christie et al., 2002).
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Restoration of stomatal opening in transgenic plants
It has been demonstrated that the stomata of transgenic plants opened in response to BL, and that this response was dependent on the fluence rate of BL (Fig. 4). Under the same conditions as described in Fig. 2, stomata in the TF-A clearly responded to weak BL by opening. By contrast, the TF-B responded to RL in the same manner as the wild type, but did not respond to BL. The stomatal response in the TF-A was dependent on the fluence rate of BL, and the sensitivity to BL was somewhat higher than those of either the wild type or phot2 single mutant. The fluence rates of BL required for a 50% increase in stomatal opening for the wild type and phot2 mutant were 0.78 and 0.54 µmol m–2 s–1, respectively, whereas less than 30% of these fluence rates (0.14 µmol m–2 s–1) was sufficient to cause the same response in the TF-A. While the reason for the hypersensitivity to BL in the TF-A is currently unknown, it should be noted that the stomatal responses to BL were not inferior to those of the wild type in spite of the considerably low expression of phot1 in the TF-A, as shown in Fig. 3. Such enhancement of BL sensitivity was obvious when a BL receptor cryptochrome was overexpressed under the control of the 35S promoter in Arabidopsis seedlings (Lin et al., 1998). However, despite the low concentration of phot1 in transgenic plants, the finding that transformation restored the responses indicates that phot1 plays an essential role in BL-specific stomatal opening. Furthermore, the full restoration of BL-dependent stomatal opening in the transgenic plants may exclude the participation of zeaxanthin as a BL-photoreceptor in Arabidopsis. The other transgenic lines (TF-C and TF-D like the transgenic line TF-B) showed a normal response to RL but were completely defective in BL-specific stomatal opening (data not shown).
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Restoration of phototropism in transgenic plants
The phot1 phot2 double mutant shows neither phototropism nor chloroplast accumulation in response to BL at a low fluence rate, but displays a leaf curling not found in the wild type. PHOT1 cDNA under the control of the 35S promoter was used to determine whether these BL responses were restored in the transgenic plants used in this work.
As shown in Fig. 5A, both the transformant TF-A and the wild-type plants showed curvature of hypocotyls in response to BL at 1 µmol m–2 s–1. Almost full, and partial restoration of phototropic responses to BL were also observed in the TF-B and the TF-C, respectively, although phot1 was not detectable in these transgenic plants (Fig. 3). Among the four transgenic lines tested, only the TF-D plants showed no phototropic responses to BL.
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Restoration of chloroplast movement in transgenic plants
Next, chloroplast accumulation in transgenic plants was determined (Fig. 5B). When light-grown Arabidopsis plants were kept in darkness overnight, chloroplasts were present in the anticlinal walls of the mesophyll cells in both the wild-type and transgenic plants. In response to weak BL, chloroplasts moved, accumulated, and spread over the periclinal walls in the mesophyll cells in TF-A and TF-C. By contrast, TF-B and TF-D did not respond to BL as well as the phot1 phot2 double mutant.
Restoration of leaf expansion in transgenic plants
When transgenic plants were grown under a 16 h light cycle, the rosette leaves of TF-A were expanded and resembled those of the wild type, whereas those of the other transformants TF-B, TF-C, and TF-D showed strongly curled leaves resembling those of the phot1 phot2 double mutant (Fig. 5C). Thus, leaf expansion was controlled by BL through phot1 and restored by transformation of the double mutant with phot1 cDNA.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Despite the prevalence of genetic and biochemical studies using mutants, there has been no direct evidence that phototropins can complement BL responses in the phot1 phot2 double mutant. In this study it has been shown for the first time that a transgene encoding phot1 restored the BL-responses of phototropism, chloroplast movement, stomatal opening, and leaf expansion in the Arabidopsis phot1 phot2 double mutant. In addition to the defects of BL-responses in mutants (Huala et al., 1997; Kagawa et al., 2001; Kinoshita et al., 2001), these results strongly confirmed that phot1 participates in all BL responses as a BL-photoreceptor.
The BL responses of phototropic curvature and chloroplast accumulation were restored in three (TF-A, TF-B, and TF-C) and two (TF-A and TF-B) transgenic lines, respectively. By contrast, stomatal opening and leaf expansion were observed only in the TF-A line, which expressed the highest concentration of phot1 among transgenic plants. Thus, the restoration of individual responses was dependent on the transgenic lines, each of which showed a different level of expression of the phot1 protein. These results may reflect a divergent signalling pathway after BL reception by phot1 and differences in the autonomous functions of phot1 in the individual responses depend on the tissues and developmental stages in Arabidopsis. It is also indicated that the level of phot1 may determine whether or not each response, which has different threshold to BL fluence rate as described earlier (Sakai et al., 2001), could be restored in transgenic plants.
Unexpectedly, the level of phot1 expressed by the 35S promoter in transgenic plants was considerably lower than that of the wild-type plants. This low expression of phot1 may have been due to the control of phot1 expression by the 35S promoter, as suggested earlier (Christie et al., 2002). It should be noted, however, that a lesser amount of phot1, i.e. less than 1% of the wild-type value, was quantitatively sufficient to cause all the BL responses, as found in TF-A. Specifically, sensitivity to BL in the response of stomatal opening was higher in TF-A than in the wild-type plants. Such hypersensitivity to BL was reported in the inhibition of hypocotyl elongation when a BL receptor cryptochrome was overexpressed under the control of the 35S promoter in Arabidopsis seedlings (Lin et al., 1998). Therefore, it is possibly explained that the specific contents of phot1 in guard cells in TF-A are higher than those of the wild type, regardless of the low level in the whole plant, but it remains to be determined.
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Acknowledgements |
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We thank Professor Masamitsu Wada (Tokyo Metropolitan University) for kindly providing seeds of the Arabidopsis phot2 single mutant and phot1 phot2 double mutant.
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References |
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