The C-terminal kinase fragment of Arabidopsis phototropin 2 triggers constitutive phototropin responses

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1 The Plant Journal (2007) 51, doi: /j X x The C-terminal kinase fragment of Arabidopsis phototropin 2 triggers constitutive phototropin responses Sam-Geun Kong 1,, Toshinori Kinoshita 2, Ken-ichiro Shimazaki 2, Nobuyoshi Mochizuki 1, Tomomi Suzuki 1 and Akira Nagatani 1,* 1 Department of Botany, Graduate School of Science, Kyoto University, Kyoto , Japan, and 2 Department of Biology, Faculty of Science, Kyushu University, Ropponmatsu, Fukuoka , Japan Received 26 December 2006; revised 23 April 2007; accepted 3 May * For correspondence (fax ; nagatani@physiol.bot.kyoto-u.ac.jp). Present address: Division of Photobiology, National Institute for Basic Biology, Okazaki , Japan. Summary Phototropins mediate various blue-light responses such as phototropism, chloroplast relocation, stomatal opening and leaf flattening in plants. Phototropins are hydrophilic chromoproteins that are mainly bound to the plasma membrane. One of two phototropins in Arabidopsis thaliana, phot2, associates with the Golgi apparatus in a light-dependent manner. In this study, we analyzed the biological activities of the N-terminal photosensory and C-terminal kinase domains of phot2. For this purpose, these domains were fused to green fluorescent protein (GFP) and ectopically expressed in the wild-type and a phot1 phot2 double mutant of Arabidopsis. The kinase domain fused to GFP (P2CG) was localized to the plasma membrane and the Golgi apparatus, whereas the photosensory domain fused to GFP (P2NG) was uniformly localized in the cytosol. Hence, the kinase domain rather than the photosensory domain is responsible for the membrane association. Interestingly, the P2CG plants exhibited constitutive blue-light responses even in dark conditions, i.e. stomata were open and chloroplasts were in the avoidance position. By contrast, P2CG with a mutation that abolishes the kinase activity (P2C[D720/N]G) failed to exhibit these responses. phot2 kinase is therefore suggested to be correctly localized to functional sites in the cell and to trigger light signal transduction through its kinase activity. In contrast to P2CG, P2NG did not affect the phot2 responses, except for partial inhibition of the phototropic response caused by the endogenous phototropins. Keywords: phototropin, blue light, Golgi apparatus, GFP, Arabidopsis, kinase. Introduction As sessile organisms, plants have specific ways to respond to environmental stress and/or signals such as light, temperature, drought or high salt. To perceive light signals, plants have evolved several photosensory pigments, including red/far-red photo-reversible phytochromes, ultraviolet A- and blue light-absorbing phototropins, and cryptochromes (Chen et al., 2004). Phototropins have been identified in several plant species, from the green algae Chlamydomonas reinhardtii to higher plants (Briggs et al., 2001). In higher plants, phototropins mediate various physiological responses such as phototropism, chloroplast relocation, light-induced stomatal opening and leaf expansion (Briggs and Christie, 2002). There are two phototropins in Arabidopsis thaliana, phototropins 1 and 2 (phot1 and phot2). Although both phot1 and phot2 mediate phototropism, phot1 is far more sensitive to light than phot2 (Sakai et al., 2001). With regard to chloroplast relocation, phot1 and phot2 redundantly mediate the accumulation response to lower intensities of blue light (BL), whereas the avoidance response to higher intensities of BL is solely controlled by phot2 (Sakai et al., 2001). In the case of stomatal opening, the responses mediated by phot1 and phot2 are similar with respect to their relative contribution and light sensitivity (Kinoshita et al., 2001). Phototropins share highly conserved structural properties from unicellular green algae to higher plants. Phototropins consist of a photosensory N-terminal domain and a C-terminal Ser/Thr kinase domain (Briggs et al., 2001). The LOV1 and LOV2 domains, which are found within the N-terminal domain, are specialized PER/ANT/SIM (PAS) domains that bind the flavin mononucleotide (FMN) chromophore. Functional analyses of mutated phototropins have 862 Journal compilation ª 2007 Blackwell Publishing Ltd

2 phot2 kinase triggers physiological responses 863 demonstrated that both the light-induced photochemical reaction in LOV2 (Christie et al., 2002) and the C-terminal kinase activity (Kong et al., 2006) are necessary for a physiological response. The LOV domains play a major role in regulating the kinase activity of phototropin in response to light. The kinase fragment seperated from the N-terminal photosensory domain exhibits constitutive activity in vitro (Matsuoka and Tokutomi, 2005). The Ja helix, which is located between the LOV2 domain and the kinase domain, is involved in the lightdependent activation of the kinase (Harper et al., 2003, 2004). Furthermore, the isolated LOV2 domain interacts with the isolated kinase domain and regulates the kinase activity in vitro in a light-dependent manner (Matsuoka and Tokutomi, 2005). Biochemical analysis of phot1 has indicated that phototropin is a membrane-associated protein (Gallagher et al., 1988; Knieb et al., 2004). Green fluorescent protein (GFP) has been used successfully to demonstrate that both phot1 and phot2 are mainly localized in the plasma membrane region (Kong et al., 2006; Sakamoto and Briggs, 2002). As phototropins are not membrane-spanning proteins, the membrane association may be mediated by as yet unknown anchoring protein(s). Interestingly, a fraction of phot2 associates with the Golgi apparatus in response to BL (Kong et al., 2006). We have demonstrated that the kinase domain, but not the N-terminal photosensory domain, is responsible for the association with the plasma membrane and the Golgi apparatus in protoplasts (Kong et al., 2006). However, the mechanism by which light regulates the intracellular localization of phototropins is unknown. In the present study, we separately expressed the N-terminal photosensory and the C-terminal kinase domains of phot2 as fusion proteins with GFP in transgenic Arabidopsis. The results confirm that the kinase domain is responsible for the association with both the plasma membrane and the Golgi apparatus. Furthermore, the isolated phot2 kinase domain constitutively induced stomatal opening and the chloroplast avoidance response, even in darkness. By contrast, the same kinase domain, mutated to remove kinase activity, failed to induce these responses. Hence, the kinase activity appears to be essential for the response. In summary, the isolated phot2 kinase, which is known to be constitutively active as a kinase in vitro, localized to functional sites in the cell and was shown to trigger phototropin responses without light stimulus. Results Preparation of 35-P2NG, 35-P2CG and 35-P2C(D720/N)G transgenic plants We fused the N-terminal and C-terminal domains of phot2 with GFP (P2NG and P2CG, respectively) and expressed them under the control of the 35S promoter in Arabidopsis (Figure 1a). These transgenic lines are referred to as 35-P2NG and 35-P2CG, respectively. In addition, we expressed a kinase-negative form of P2CG, to which a missense mutation had been introduced (35-P2C(D720/N)G) (Kong et al., 2006). These lines were established in the wildtype background (designated 35-P2NG/WT etc.). We also expressed P2NG and P2CG in the phot1 phot2 double mutant background (designated 35-P2NG/p1p2 etc.). Both the 35-P2NG and 35-P2C(D720/N)G plants grew normally, except that seed setting was reduced to some extent (Figure 1b). By contrast, the 35-P2CG transgenic plants exhibited an adult phenotype. In these plants, several growth defects, such as reduction in rosette leaf size and shoot apical dominance, and male sterility, were observed (Figure 1b). These characteristics have been observed to some extent in transgenic Arabidopsis overexpressing Chlamydomonas phot (Onodera et al., 2005). Since few seeds were set by self-pollination of the homozygous 35- P2CG plants, 35-P2CG lines were maintained as heterozygous populations. Expression levels of the introduced proteins were determined by immunoblot analysis of protein extracts from rosette leaves using an anti-gfp antibody (Figure 1c,d). The levels of P2NG protein in 35-P2NG/WT and 35-P2NG/p1p2 plants were much higher than the level of the phot2 GFP fusion protein (P2G) in 35-P2G/p1p2 14-2, in which P2G overaccumulates by a factor of two compared with endogenous phot2 (Kong et al., 2006). In 35-P2CG/WT, 35-P2CG/ p1p2 and 35-P2C(D720/N)G/WT, the expression levels were relatively low in two independent lines for each construct. Based on serial dilution experiments, the levels were estimated to be about one quarter of that in 35-P2G/p1p (Figure 1d). Hence, the expression levels of P2CG and P2C(D720/N)G in these lines were approximately half of the level of endogenous phot2. Intracellular localization of the P2NG, P2CG and P2C(D720/N)G proteins We microscopically examined the intracellular localization patterns of the P2NG, P2CG and P2C(D720/N)G proteins. Cotyledon epidermal cells of 3-day-old dark-grown seedlings were observed with a confocal laser scanning microscope (Figure 2a). We also confirmed the localization patterns in the rosette leaf epidermis (data not shown). Consistent with the previous results for transient expression analysis in isolated protoplasts (Kong et al., 2006), P2NG was localized in the cytosol in 35-P2NG/WT seedlings (Figure 2a). By contrast to P2NG, P2CG fluorescence was observed mainly in the plasma membrane region in 35-P2CG/WT (Figure 2a). In addition, punctate structures were observed in some samples, even under dark conditions. However, the punctate pattern was less clear in

3 864 Sam-Geun Kong et al. (a) (c) (b) (d) Figure 1. Expression of phot2 derivatives in transgenic plants. (a) Schematic diagrams of the P2NG, P2CG and P2C(D720/N)G expression vectors. (b) Top views of 4-week-old 35-P2NG, 35-P2CG and 35-P2C(D720/N)G transgenic plants. (c) Immunoblot analysis of 35-P2NG/WT and 35-P2NG/p1p2 transgenic plants. Crude extracts (50 lg total protein) prepared from rosette leaves of 3-week-old plants were subjected to 7.5% SDS PAGE. The blot was probed with an anti-gfp monoclonal antibody. WT, wild-type. (d) Immunoblot analysis of 35-P2CG/WT, 35-P2CG/p1p2 and 35-P2C(D720/N)G/WT transgenic plants. The growth conditions and loading amounts are as for (c). For comparison, serial dilutions of extracts from 35-P2G/p1p plants were loaded. The blot was probed with an anti-gfp monoclonal antibody. WT, wild-type. 35-P2CG than in 35-P2G. P2C(D720/N)G was detected mainly in the plasma membrane region (Figure 2a), indicating that kinase activity is not required for plasma membrane localization. However, in most cases, the punctate pattern was not observed in 35-P2C(D720/N)G/WT. Hence, the kinase activity apparently enhances association with the Golgi apparatus. This result is consistent with a previous observation that P2G with the same mutation failed to exhibit the punctate staining pattern in transgenic Arabidopsis (Kong et al., 2006). We also examined the intracellular localization of P2NG, P2CG and P2C(D720/N) by protein fractionation. Protein extracts prepared from rosette leaves were fractionated into pellet and supernatant fractions and subjected to immunoblot analysis (Figures 2b,c). Consistent with the microscopic observations, both P2CG and P2C(D720/N)G were detected only in the pellet fraction. By contrast, P2NG was detected mainly in the supernatant fraction, with the remainder in the pellet fraction (Figure 2b,c). The latter signal is probably due to contamination by soluble proteins in the pellet fraction. We found that even GFP, a typical soluble protein, could be detected in this fraction (Figure S1). Endogenous phot2 bands were detectable on the blot probed with an antibody raised against the N-terminal domain of phot2 (Figure 2c). In all the lines, endogenous phot2 was detected mainly in the pellet fraction. Hence, expression of P2CG, P2C(D720/N)G or P2NG did not alter the intracellular distribution of the endogenous phot2. Stomatal opening in 35-P2CG plants Phototropin has light-dependent kinase activity (Christie et al., 1998; Sakai et al., 2001). Bacterially expressed phot2 kinase domains exhibit constitutive kinase activity in vitro (Matsuoka and Tokutomi, 2005). The intracellular localization pattern of P2CG resembled that of P2G under BL (see above). Hence, we examined the phototropin responses in the 35-P2CG lines.

4 phot2 kinase triggers physiological responses 865 Figure 2. Intracellular localization patterns of P2NG, P2CG and P2C(D720/N)G/WT proteins. (a) Confocal microscopic observation of P2G, P2NG, P2CG and P2C(D720/N)G fluorescence in the cotyledon epidermal cells of 3-day-old darkgrown transgenic seedlings. The lines shown are 35-P2G/p1p2 14-2, 35-P2NG/WT 14-4, 35-P2CG/ WT 2 and 35-P2C(D720/N)G/WT Samples were prepared in the dark, and the images in the top row are in the dark state. The samples were further scanned with laser BL five times at 1 min intervals and the images from the last scan were recorded (bottom row). Arrowheads indicate representative P2C(D720/N)G punctate stains. Bar = 10 lm. (b, c) Subcellular localization of P2NG, P2CG, P2C(D720/N)G and the endogenous phot2 protein in extracts from rosette leaves of 3-week-old plants. The total protein extracts (T) were fractionated into soluble (S) and pelleted (P) fractions by ultra-centrifugation ( g, 1 h at 4 C). Samples corresponding to 50 lg total protein in the T fractions were subjected to 7.5% SDS PAGE. The blots were probed with an anti-gfp monoclonal antibody (b) or anti-phot2 polyclonal antibody (c). WT, wild-type. (a) (b) (c) The stomatal apertures were measured in 35-P2CG/p1p2 under BL plus red light (RL) (Figure 3a). The stomatal aperture in the wild-type was wider under BL plus RL compared with RL alone (Figure 3a). By contrast, stomata were open regardless of the light conditions in 35-P2CG/ p1p2. A similar phenotype was observed in 35-P2CG/WT (Figure 3b). We confirmed that 35-P2G/p1p2 responded to BL normally. The results were not due to a permanent loss of the ability to close stomata in these plants because stomata closed normally in response to abscisic acid (ABA) (Figure 3c). Hence, it was concluded that P2CG could trigger a full stomatal response without BL stimulus. To examine whether or not kinase activity was required for stomatal opening by P2CG, the line expressing P2C(D720/N)G, a kinase-negative mutant of phot2 kinase, was examined. In contrast to P2CG, P2C(D720/N)G was not able to open stomata under RL (Figure 3b). Interestingly, it exhibited a dominant negative effect over the endogenous phototropins to reduce the stomatal opening in response to BL. Chloroplast relocation response in 35-P2CG plants We examined the chloroplast relocation in mesophyll cells in the 35-P2CG plants (Figure 4). For wild-type and 35-P2G/ p1p2 transgenic cells in the dark, a few chloroplasts were present on the upper cell surface of the mesophyll cells and most accumulated at the bottom, as previously reported (Suetsugu et al., 2005). As fewer chloroplasts were observed in the upper part of the wild-type cells in the dark, the edges of the cells were less clear under these conditions (Figure 4, top row). Under a low intensity of BL (0.5 lmol m )2 sec )1 ), chloroplasts accumulated on the upper cell surface due to the accumulation response in wild-type cells (Figure 4, middle row) as previously reported (Jarillo et al., 2001; Kagawa et al., 2001). Interestingly, the chloroplasts avoided the upper cell surface and accumulated on the side wall even under a low intensity of BL in 35-P2G/p1p2 transgenic cells. This is probably due to the overaccumulation P2G in this line. The avoidance response was observed under a high intensity of BL (50 lmol m )2 sec )1 ) in both lines (Figure 4, bottom row), as previously reported (Jarillo et al., 2001; Kagawa et al., 2001). In the phot1 phot2 double mutant, chloroplasts did not accumulate at the bottom and were positioned randomly under dark conditions (Sakai et al., 2001). Indeed, more chloroplasts were observed in the upper part of the cell in the phot1 phot2 double mutant than in the wild-type (Figure 4). The pattern did not change even when the leaves

5 866 Sam-Geun Kong et al. Figure 3. Stomatal apertures in 35-P2CG, 35-P2C(D720/N)G and 35-P2G/p1p2 plants. (a) Stomatal apertures in epidermal strips from wild-type (WT), phot1 phot2, 35-P2CG/p1p2 and 35-P2G/p1p2. The epidermal strips were irradiated with blue light (10 lmol m )2 sec )1 ) superimposed on red light (50 lmol m )2 sec )1 ) (gray bars) or with red light alone (black bars) for 3 h. (b) Stomatal apertures in epidermal strips from wild-type (WT), 35-P2CG/WT and 35-P2C(D720/N)G/WT. The epidermal strips were treated as for (a). (c) Abscisic acid (ABA)-induced stomatal closure. Plants were kept under light illumination for 3 h. Leaves were then blended, and epidermal strips were incubated for 3 h in the absence (gray bars) or presence of 20 lm ABA (black bars) under blue light (10 lmol m )2 sec )1 ) superimposed on red light (50 lmol m )2 sec )1 ). Data represent means and SD (n = 135) of three independent experiments. (a) were irradiated with BL. In 35-P2CG/WT, the distribution patterns were not affected by light (Figure 4). Under all conditions tested, the patterns resembled that under a high intensity of BL in the wild-type, namely the avoidance position. We confirmed that these patterns were also present in 35-P2CG/p1p2 (data not shown). By contrast to P2CG, P2C(D720/N)G did not affect chloroplast relocation in the wild-type background (Figure 4). Hence, kinase activity is required for P2CG to induce the chloroplast avoidance response, regardless of the light conditions. In summary, a constitutive chloroplast avoidance response was observed in P2CG plants. The avoidance distribution pattern was observed even in darkness. Kinase activity is required for this effect. In addition, no dominant negative effect of P2C(D720/N)G on the endogenous phototropins was observed in terms of chloroplast relocation. (b) (c) Elongation growth and phototropism in 35-P2CG plants Phototropism, which is under the control of phot1 and phot2, is caused by asymmetric elongation growth between the side of a stem that is in the light and the shaded side (Iino, 2001). Phot1 also mediates very rapid inhibition of hypocotyl elongation, which is observed with 30 sec of illumination and disappears within min after the onset of light (Folta and Spalding, 2001). Hence, we first examined whether P2CG affected hypocotyl elongation in darkness (Figure 5a). It was found that the hypocotyls were shorter in 35-P2CG/WT than in the wild-type and 35-P2G/p1p2. As the phenotype was not observed in 35-P2C(D720/N)G/WT seedlings, the kinase activity of P2CG appears to be required for this phenotype. We then examined the phototropic response in 35-P2CG/ WT under different intensities of unilateral BL (Figure 5b). Interestingly, responses were reduced in 35-P2CG/WT for all the light intensities tested. As the response to lower intensities of BL is solely mediated by phot1 (Sakai et al., 2001), these data indicate that P2CG could interfere with a phot1-specific response. By contrast, P2C(D720/N)G/WT seedlings responded almost normally to the light, indicating that kinase activity is required for the inhibitory effect of P2CG on phototropism (Figure 5b). Auxin is involved in the phototropic response (Iino, 2001). Indeed, a gradient of auxin and auxin-dependent transcription precede this growth response (Esmon et al., 2006). Hence, we examined whether the auxin responses were altered in the 35-P2CG/WT seedlings. To monitor the response, we used the DR5::GUS line, in which expression of the reporter b-glucuronidase (GUS) is induced in

6 phot2 kinase triggers physiological responses 867 Figure 4. Chloroplast relocation in wild-type (WT), phot1 phot2, 35-P2CG/WT, 35-P2C(D720/ N)G/WT and 35-P2G/p1p2 transgenic plants. Leaves were detached from plants and darkadapted for h before BL treatment. The leaves were treated with 0.5 (LB) or 50 (HB) lmol m )2 sec )1 BL on agar plates, or not treated with BL (Dark) before observation. Lines shown are 35-P2CG/WT 2, 35-P2C(D720/N)G/WT 14-1 and 35-P2G/p1p Chlorophyll autofluorescence was detected with a confocal laser scanning microscope. Bar = 20 lm. response to auxin (Ulmasov et al., 1997). We crossed 35-P2CG/WT with DR5::GUS and examined the GUS expression pattern in seedlings treated with unilateral BL (Figure 5c). Surprisingly, GUS expression was much higher in 35-P2CG/WT than in wild-type. Although its physiological relevance remains unclear, the level and/or responsiveness to auxin were substantially disturbed in 35-P2CG/WT. Biological activities of P2NG We also examined whether the N-terminal domain had any physiological activity. The P2NG protein was effectively expressed in transgenic Arabidopsis (Figure 1). 35-P2NG/p1p2 seedlings were treated with unilateral BL (40 lmol m )2 sec )1 ) but no phototropic curvature was observed (Figure 6a), indicating that P2NG without the C-terminal kinase domain could not replace full-length phot2. We then examined whether P2NG interfered with the action of endogenous phototropin. Two independent 35-P2NG/WT lines were tested for the phototropic response to different fluence rates of BL. As shown in Figure 6(b), P2NG specifically reduced the phototropic response to lower fluence rates of BL (0.01 and 0.1 lmol m )2 sec )1 ), which is under the control of phot1 (Sakai et al., 2001). We also examined the chloroplast relocation response and the stomatal response in 35-P2NG/WT, but no effect on the endogenous phototropins was observed (data not shown). Discussion Plasma membrane association of phot2 The correct localization of a molecule in the cell is essential for its biological function. Both phot1 and phot2 are localized to the plasma membrane (Kong et al., 2006; Sakamoto and Briggs, 2002); in addition, phot2 is localized to punctate structures, presumed to be the Golgi apparatus, in a lightdependent manner (Kong et al., 2006). The present results demonstrated that P2CG was associated with the plasma membrane (Figure 2), while P2NG was distributed evenly in the cytoplasm. These observations are consistent with our previous results, obtained using a protoplast transient expression system (Kong et al., 2006). In addition, C-terminal kinase fragments of both Arabidopsis phot1 and the Chlamydomonas phototropin also localized to the plasma membrane in protoplasts (data not shown). Taken together, the results show that C-terminal kinase domain, but not the N-terminal photosensory domain, is responsible for the plasma membrane localization of phot2. As phot1 and phot2 are members of subfamily VIII of the AGC kinases (named after the camp-dependent protein kinase A, cgmp-dependent protein kinase G and phospholipiddependent protein kinase C) (Bögre et al., 2003), it would be interesting to determine whether other such kinases are also localized to the plasma membrane. Indeed, PINOID, a member of the AGC VIII kinase family, is localized to the cell periphery (Lee and Cho, 2006; Zegzouti et al., 2006). It is noteworthy here that the kinase activity was not required for the plasma membrane association of P2CG. The P2C(D720/N)G mutant, in which the kinase activity is abolished (Matsuoka and Tokutomi, 2005), still exhibited plasma membrane localization (Figure 2). This is consistent with the observation that phototropins are mainly localized to the plasma membrane even in the dark (Kong et al., 2006; Sakamoto and Briggs, 2002). In the dark, the kinase activity of phototropins is substantially reduced both in vitro (Christie et al., 1998; Sakai et al., 2001) and in vivo (Christie et al., 1998, 2002).

7 868 Sam-Geun Kong et al. (a) (c) (a) (b) (b) Figure 6. Phototropic response in 35-P2NG lines. (a) Hypocotyl curvature for 35-P2NG/p1p2. Three-day-old dark-grown seedlings were treated with unilateral BL (40 lmol m )2 sec )1 ). Data represent means SD of at least 20 independent seedlings. WT, wild-type. (b) Hypocotyl curvature for 35-P2NG/WT. Three-day-old dark-grown seedlings were treated with various fluence rates of unilateral BL (0.02, 0.1, 2 or 40 lmol m )2 sec )1 ). Data represent means SD of at least 20 independent seedlings. WT, wild-type. Figure 5. Hypocotyl growth and phototropic responses in 35-P2CG/WT, 35- P2C(D720/N)G/WT and 35-P2G/p1p2 transgenic plants. (a) Hypocotyl lengths in etiolated seedlings of wild-type, the phot1 phot2 double mutant, 35-P2CG/WT, 35-P2C(D720/N)G/WT and 35-P2G/p1p2. Seedlings were grown on vertical agar plates for 3 days in darkness and the hypocotyl lengths were determined. Data represent means SD of at least 15 independent seedlings. WT, wild-type. (b) Hypocotyl curvature in 3-day-old dark-grown seedlings of 35-P2CG/WT and 35-P2C(D720/N)G/WT transgenic plants. The seedlings were treated with various fluence rates of unilateral BL (0.02, 0.1, 2 or 40 lmol m )2 sec )1 ) for 12 h. For 35-P2CG/WT, heterozygous populations were used. Each seedling was tested for P2CG expression after the measurement to exclude results on the segregating wild-type seedlings from the calculation. Data represent means SD of at least 20 independent seedlings. WT, wild-type. (c) Histochemical analysis of GUS expression in the DR5:GUS seedlings. Seedlings of 35-P2CG/DR5:GUS (left) and DR5:GUS (right) were treated with BL (40 lmol m )2 sec )1 ) as for (b) and then subjected to histochemical staining. Bar = 1 mm. It has not yet been elucidated how phototropins are recruited to the plasma membrane. Proteins need to interact with membrane lipid or membrane bound-macromolecules for plasma membrane association. Examination of the amino acid sequences of phototropins suggests that they do not have a membrane-spanning domain. Furthermore, biochemical analysis has indicated that phototropin is associated with the membrane by ionic interactions and/or hydrogen bonding (Knieb et al., 2004). NPH3, RPT2 and PKS1, the signaling components in the phototropic response, might anchor phototropins on the membrane. These proteins are associated with the plasma membrane (Inada et al., 2004; Lariguet et al., 2006; Motchoulski and Liscum, 1999). However, they physically interact with the N-terminal but not the C-terminal domain of phot1 according to analysis by the yeast two-hybrid system (Inada et al., 2004; Motchoulski and Liscum, 1999). Since phot2 appears to be recruited to the plasma membrane through its C-terminal domain (Kong et al., 2006; Figure 2), these proteins less likely act as the anchoring protein for phot2.

8 phot2 kinase triggers physiological responses 869 Light-dependent Golgi association of phot2 A fraction of phot2 associates with the Golgi apparatus in response to BL (Kong et al., 2006). Intense punctate distribution is observed for P2CG expressed in protoplasts (Kong et al., 2006). In the present study, P2CG was detected in punctate structures in the cytoplasm even in the dark (Figure 2a). However, the structures were less prominent in 35-P2CG/WT than in 35-P2G/p1p2 plants. This could not be due to the presence of endogenous phot2, as a similar result was obtained for 35-P2CG/p1p2 (data not shown). More likely, the lower levels of P2CG expression than of P2G (Figure 1d) resulted in the weaker punctate staining. The punctate distribution was even less clear for P2C(D720/N)G (Figure 2), suggesting that kinase activity may enhance the formation of punctate structures. Consistently, full-length P2G lacking kinase activity (P2(D720/N)G) failed to associate with the Golgi apparatus in transgenic plants (Kong et al., 2006). However, this conclusion is weakened by the fact that the expression levels of P2CG and P2(D720/N)G were not identical. Phototropins are light-activated kinases (Christie et al., 1998; Sakai et al., 2001). Furthermore, the isolated kinase domain exhibits constitutive kinase activity in vitro (Matsuoka and Tokutomi, 2005). Hence, we can speculate that light activates the kinase activity of phot2, which in turn increases the affinity of phot2 for the Golgi apparatus (Kong et al., 2006). Alternatively, phot2 kinase may modify subcellular trafficking to alter its own subcellular localization. It is also possible that activation of the kinase may lead to a reduction in its affinity with the plasma membrane. In any case, auto-phosphorylation is probably not involved in such a process because the isolated kinase domain does not auto-phosphorylate (Matsuoka and Tokutomi, 2005). Identification of the substrate for the phototropin kinase is awaited. Stomatal opening Stomatal opening is controlled redundantly by phot1 and phot2 (Kinoshita et al., 2001). The present results indicated that stomata were open regardless of light conditions in P2CG (Figure 3). The kinase activity is required for this response because the stomata were closed under red light conditions in 35-P2C(D720/N)G plants. It should be noted here that the effects of P2CG and P2C(D720/N)G may be exaggerated by overaccumulation. The expression levels of P2CG and P2C(D720/N)G proteins were comparable to that of endogenous phot2 in rosette leaves as a whole (Figure 1). However, expression driven by the 35S promoter tends to be high in guard cells. Indeed, we observed relatively high GFP fluorescence in guard cells in these lines (data not shown). The simplest explanation for the above observation is that P2CG constitutively phosphorylates the target protein in guard cells to induce stomatal opening. Phototropins are involved in the phosphorylation and subsequent BL-dependent activation of guard cell H + -ATPases (Kinoshita and Shimazaki, 1999). However, it is unlikely that phototropin directly phosphorylates H + -ATPase (Kinoshita et al., 2001). Rather, as yet unknown plasma membrane factor(s) that regulate the phosphorylation of H + -ATPase might be phosphorylated by P2CG. P2C(D720/N)G not only failed to open stomata by itself but also inhibited opening by the endogenous phot2 (Figure 3). As phot1 and phot2 redundantly mediate stomatal opening, P2C(D720/N)G appears to inhibit functions of both phot1 and phot2. This dominant negative effect may be explained by competitive inhibition of the target phosphorylation. P2C(D720/N)G might inhibit auto-phosphorylation of the endogenous phototropins. Alternatively, P2C(D720/N)G may alter the localization of the endogenous phototropins. However, the latter is less likely because endogenous phot2 was found in the membrane fraction even in 35-P2C(D720/ N)G/WT (Figure 2c). P2CG was localized to the plasma membrane and punctate structures that were presumed to be the Golgi apparatus, whereas the punctate structures were less clear in P2C(D720/ N)G (Figure 2a). Nevertheless, P2C(D720/N)G exhibited a clear dominant negative effect over the endogenous phototropins in terms of stomatal opening (Figure 3). Hence, the plasma membrane may be more important than the Golgi apparatus as the functional site of phototropins for this response. This view is consistent with the fact that the phot1 GFP fusion does not exhibit punctate staining but mediates the stomatal opening (Sakamoto and Briggs, 2002). Chloroplast relocation response Chloroplasts exhibit photo-accumulation and avoidance responses depending on the light intensity (Jarillo et al., 2001; Kagawa et al., 2001). The former is mediated by both phot1 and phot2, whereas the latter is solely under the control of phot2 (Sakai et al., 2001). In 35-P2CG lines, chloroplasts were in the avoidance position even in the dark (Figure 4). Hence, P2CG constitutively triggered the avoidance response without the light stimulus. As is the case with the stomatal opening, kinase activity is required for this function as P2C(D720/N)G failed to induce the avoidance response (Figure 4). Although a dominant negative activity of P2C(D720/N)G was observed for stomatal opening (see above), P2C(D720/N)G did not affect the chloroplast relocation response by endogenous phototropins (Figure 4). This could be due to a relatively low level of P2C(D720/N)G expression in mesophyll cells compared with guard cells. Indeed, GFP fluorescence was barely detectable in mesophyll cells in 35-P2C(D720/N)G (data not shown). Alternatively, the primary actions of phototropins in chloroplast

9 870 Sam-Geun Kong et al. relocation and stomatal opening may be quite different. It should be noted here that we might have overlooked the effects of P2C(D7120/N)G on chloroplast relocation because the method employed was not very quantitative. Hence, it remains possible that P2C(D720/N)G affects the chloroplast relocation to some extent. Hypocotyl growth P2CG inhibited hypocotyl elongation (Figure 5a). A preliminary analysis suggested that cell elongation was affected in the 35-P2CG seedlings, although the difference between 35-P2CG and wild-type was relatively small (data not shown). Furthermore, P2CG caused aberrant expression of the DR5::GUS reporter gene (Figure 5c), indicating that the levels of auxin and/or the responsiveness to auxin were disturbed in the 35-P2CG seedlings. Consistent with this view, adult 35-P2CG plants exhibited a pleiotropic phenotype including small leaves, reduced apical dominance, reduced internode elongation and reduced fertility. These deficiencies are reminiscent of those observed in the transgenic plants ectopically expressing the protein kinase PINOID (Benjamins et al., 2001; Christensen et al., 2000). Considering the structural similarity between P2CG and PINOID, they may disturb the auxin functions through similar mechanisms. Interpretation of the phototropic response in 35-P2CG/ WT seedlings is difficult because of the above phenotype. Asymmetrical cell elongation plays a critical role in this response (Iino, 2001). A gradient of auxin and auxindependent transcription precede this growth response (Esmon et al., 2006). Hence, apparent deterioration in the phototropic response in 35-P2CG/WT seedlings could be due to the general reduction in the elongation growth. Nevertheless, the result is compatible with the interpretation that overexpression of P2CG triggers the response elicited by light in the lit side of the wild-type hypocotyl. If such responses are triggered in both sides of the hypocotyl, the phototropic response is severely disturbed. Factors such as NPH3, RPT2 and PKS1, which act downstream of phototropins to induce the phototropic response (Lariguet et al., 2006; Motchoulski and Liscum, 1999; Sakai et al., 2000), might be involved in the process by which P2CG inhibits the phototropic response. As these proteins are associated with the plasma membrane (Inada et al., 2004; Lariguet et al., 2006; Motchoulski and Liscum, 1999), they could be either a direct target for phosphorylation by phototropins or tightly connected to such reactions. Although these factors preferentially bind to the N-terminal rather than the C-terminal domain of phot1 (Inada et al., 2004; Motchoulski and Liscum, 1999), it remains possible that P2CG transiently interacts with these factors on the plasma membrane and phosphorylates them. Dominant negative activity of P2NG Despite higher accumulation levels of P2NG in 35-P2NG lines (Figures 1 and 2), we could not observe an effect on the phototropin responses except that P2NG weakly inhibited the response towards weak BL (Figure 6). Hence, the physiological activity of P2NG was quite limited compared with that of P2CG. This is not very surprising for various reasons, such as the intracellular distribution pattern of P2NG being very different from that of full-length phot2 (Figure 2). The inhibitory effect of P2NG on phototropism may be explained by a possible interaction with downstream factors such as NPH3, RPT2 and PKS1 (Lariguet et al., 2006; Motchoulski and Liscum, 1999; Sakai et al., 2000). These factors have been shown to interact with the N-terminal domain of phot1 using the yeast two-hybrid system and in vitro (Inada et al., 2004; Motchoulski and Liscum, 1999). As the phot2 N-terminal domain exhibits high homology with that of phot1 (Briggs et al., 2001; Kasahara et al., 2002), P2NG might interact with NPH3, RPT2 and/or PKS1 to interfere with phototropin signal transduction. Alternatively, P2NG might affect the phototropic response through interaction with the kinase domains of the endogenous phototropins (Matsuoka and Tokutomi, 2005). It is also possible that P2NG formed a heterodimer with the endogenous phototropins (Salomon et al., 2004), reducing their activity. Concluding remarks Phototropins behave as light-activated kinases both in vivo and in vitro (Christie et al., 2002). Mutated phototropins, in which the kinase activity is abolished, fail to mediate the light responses (Christie et al., 2002; Kong et al., 2006). The present results have further advanced this view. The isolated kinase domain triggered phototropin responses without the light stimulus. Hence, the photosensory domain appears to act negatively to suppress the signaling under dark conditions. This view is consistent with the observation that the LOV2 domain fragment inhibits the activity of the phot2 kinase fragment in vitro (Matsuoka and Tokutomi, 2005). In addition, the present results demonstrate that the kinase domain is responsible for the correct intracellular localization of phot2. Thus, 35-P2CG and 35-P2C(D720/N)G plants may be powerful tools to elucidate the signal transduction mechanism of phototropins in future studies. Experimental procedures Plant materials and growth conditions The wild-type (gl-1, ecotype Columbia) and phot-deficient mutants, phot1-5, phot2-1, phot1-5 phot2-1, phot1-5 phot2-2, have been described previously (Kong et al., 2006). The transgenic plants,

10 phot2 kinase triggers physiological responses P2G/p1p2 1-4 and 14-2, and 35-GFP/WT (Kong et al., 2006), were used as controls. For the expression of P2NG, P2CG and P2C(D720/ N)G, wild-type plants and phot1 phot2 double mutants were transformed with the vectors described below. The 35-P2CG/WT 15 line was crossed with the DR5:GUS reporter line (Ulmasov et al., 1997) to examine auxin-induced gene expression in 35-P2CG/WT. As few (if any) seeds set by self-pollination in the homozygous P2CG plants, they were maintained as heterozygous populations. The plants were grown on 0.6% agar plates containing halfstrength Murashige and Skoog (MS) medium with 2% w/v sucrose, or in pots of soil, under 16 h day/8 h night cycles, essentially as described previously (Kong et al., 2006), except for the stomatal opening experiments. The average light intensity at the top of the pots was 80 lmol m )2 sec )1. For stomata aperture measurements, plants were grown on half-strength MS medium supplemented with 1% w/v sucrose at ph 5.7 for 10 days (at 24 C with constant light at 50 lmol m )2 sec )1 ). The plants were then transferred to soil irrigated with mineral nutrients under white fluorescent lamps (14 h day/10 h night cycles, 50 lmol m )2 sec )1 ). The temperature was maintained at 24 C and the relative humidity at 50%. Plasmid constructions and production of transgenic plants The DNA fragments P2N:GFP, P2C:GFP and P2C(D720/N):GFP (Kong et al., 2006) were cloned into the P 35S -nost/ppzp211 transformation vector to give the transformation vectors, 35-P2NG, 35-P2CG and 35-P2(D720/N)G, respectively (Figure 1a). The vectors were used for Arabidopsis transformation by the Agrobacterium-mediated floral dip method (Clough and Bent, 1998). Transformed plants were selected on agar medium containing 25 mg l )1 kanamycin. Independent transformants were maintained up to the T 3 generation. Immunoblot analysis Immunoblot analysis was performed essentially as described previously (Kong et al., 2006). Protein extracts were prepared from the rosette leaves of 3-week-old transgenic plants grown on agar plates. Protein fractionation was carried out essentially as described previously (Kong et al., 2006). Equal volumes corresponding to 50 lg total protein were taken from the soluble and pelleted fractions and subjected to 7.5% SDS PAGE. Immunoblot analysis of GFP fusion proteins was performed using an anti-gfp monoclonal antibody (Nacalai, or an anti-phot2 polyclonal antibody (Kong et al., 2006). Confocal laser scanning microscopy and image analysis Confocal laser scanning microscopy and image analysis were carried out essentially as described previously (Kong et al., 2006). The observation filter sets were nm for GFP and >610 nm for chlorophyll (Olympus; The confocal images were processed using Adobe Photoshop 7.0 (Adobe Systems, Stomatal opening Measurements of stomatal aperture were performed according to a previously described method, with modifications (Kinoshita et al., 2001). As a homozygous population of 35-P2CG was not available, we chose plants that had small rosette leaves (Figure 1b) from heterozygous populations. For other lines, homozygous seed populations were used. Leaves from 4- to 5-week-old plants were blended in distilled water in a Waring Commercial blender (Waring Commercial, Waring Products Inc., waringproducts.com) for 15 sec. The blended material was filtered through a 58 lm nylon mesh. The epidermal strips were suspended in 3 ml of basal reaction mixture (5 mm MES-BTP (MES, 2-[N-morpholino]ethanesulphonic acid; BTP 1,3-bis[tris(hydroxymethyl)- methylamino]-propane), ph 6.5, 50 mm KCl, 0.1 mm CaCl 2 ) in Petri dishes (20 mm diameter). Strips were then illuminated with BL (10 lmol m )2 sec )1 ) superimposed on background red light (50 lmol m )2 sec )1 ) for 3 h in the absence or presence of 20 lm ABA. After treatment, the strips were collected on a 58 lm nylon mesh, and placed onto a microscope slide with a cover glass. Stomatal apertures in the abaxial epidermis were measured at 600 magnification. Data represent means SD (n = 135) for three independent experiments. All measurements were taken between 08:00 and 14:00 h, at 24 C. Chloroplast relocation response Four-week-old plants were dark-adapted for h at 23 C before observation. As a homozygous population of 35-P2CG was not available, we chose plants that had small rosette leaves (Figure 1b) and exhibited high GFP fluorescence from heterozygous populations. For other lines, homozygous seed populations were used. The rosette leaves were inspected for chloroplast position in the mesophyll cells on the adaxial side of the leaves. The chloroplast positions were determined essentially as described previously (Kong et al., 2006), except that chloroplasts were observed by confocal laser scanning microscopy instead of conventional microscopy. A few optical sections (at 4 lm intervals) corresponding to the upper part of the mesophyll cells were electronically overlaid. Phototropism Hypocotyl curvature was assayed essentially as described previously (Kong et al., 2006). As a homozygous population of 35-P2CG was not available, heterozygous populations were used. After measurement of the curvature, 35-P2CG seedlings were transplanted and grown further to examine whether or not each individual expressed P2CG. For 35-P2NG and 35-P2C(D720/N)G, homozygous seed populations were used. Histochemical GUS assay Histochemical GUS enzyme activity detection (Gallagher, 1992; Jefferson, 1987) was performed as follows. The GUS substrate, 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-Gluc; Rose Scientific, was dissolved in dimethylformamide at 50 mg ml )1. The solution was added to GUS histochemical staining buffer (50 mm Na 2 HPO 4, ph 7.0, 10 mm Na 2 EDTA, 0.5% Triton X-100) to give a final concentration of X-Gluc of 0.5 mg ml )1. Seedlings were soaked in the buffer and evacuated twice to allow full permeation of the GUS substrate. After incubating overnight at 37 C in an air incubator, the buffer was replaced with fixative solution (ethanol:water:acetic acid, 8:1:1 by volume) (Tanaka et al., 2002). The seedlings were then observed under a stereoscopic microscope. Acknowledgements We thank Professor Masamitsu Wada for allowing S.-G.K. to complete this work in his laboratory, and BioMed Proofreading for

11 872 Sam-Geun Kong et al. English proofreading. This work was partially supported by a Grantin-Aid for Scientific Research (B) (to A.N.), a Grant-in-Aid for Scientific Research on Priority Areas (to A.N.), and a Grant-in-Aid for 21st Century Centers of Excellence Research, Kyoto University (A14) (to A.N.). S.-G.K. was supported in part by the research fellowship from the Japan Society for the Promotion of Science for Foreign Researchers (P06181). Supplementary material The following supplementary material is available for this article online: Figure S1. Immunoblot detection of P2NG and GFP in pelleted and soluble fractions. Protein extracts were fractionated by high-speed ultracentrifugation and subjected to the immunoblot analysis. This material is available as part of the online article from References Benjamins, R., Quint, A., Weijers, D., Hooykaas, P. and Offringa, R. (2001) The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. 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