Antagonistic Regulation of Leaf Flattening by Phytochrome B and Phototropin in Arabidopsis thaliana

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1 Antagonistic Regulation of Leaf Flattening by Phytochrome B and Phototropin in Arabidopsis thaliana Toshiaki Kozuka 1, Noriyuki Suetsugu 2, Masamitsu Wada 2 and Akira Nagatani 1, * 1 Department of Botany, Graduate School of Science, Kyoto University, Kyoto, Japan 2 Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan *Corresponding author: , nagatani@physiol.bot.kyoto-u.ac.jp; Fax, (Received July 10, 2012; Accepted September 25, 2012) Light is one of the most important environmental factors regulating the growth and development of leaves. As the primary photosynthetic organs, leaves have a laminar structure in many dicotyledonous plants. The regulation of leaf flatness is a key mechanism for the efficient absorption of light under low light conditions. In the present study, we demonstrated that phytochrome B (phyb) promoted the development of curled leaves. Wild-type leaves gently curled downwards under white light, whereas the phybdeficient mutant (phyb) constitutively exhibited flatter leaves. In the wild type, leaf flattening was promoted by end-of-day far-red light (EODFR) treatment, which rapidly eliminates the active Pfr phytochrome. Interestingly, the curled-leaf phenotype in a phototropin-deficient mutant was almost completely suppressed by the phyb mutation as well as by EODFR. Thus, phototropin promotes leaf flattening by suppressing the leaf-curling activity of phyb. We examined the downstream components of phyb and phototropin to assess their antagonistic regulation of leaf flatness further. Consequently, we found that a phototropin signaling transducer, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3), was required to promote leaf flattening in phyb. The present study provides new insights into a mechanism in which leaf flatness is regulated in response to different light environmental cues. Keywords: Arabidospsis Auxin Leaf flatness NPH3 Phototropin Phytochrome. Abbreviations: EODFR, end-of-day far-red light; GFP, green fluorescent protein; NPH3, NON-PHOTOTROPIC HYPOCOTYL 3; PBG, GFP-tagged PHYB; phyb, phytochrome B; PIF, PHYTOCHROME INTERACTING FACTOR; RT PCR, reverse transcription PCR; SAS, shade-avoidance syndrome. Introduction Plants utilize multiple families of photoreceptors to recognize the surrounding light environment. These include phytochrome, phototropin, cryptochrome and ZEITLUPE (ZTL)/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1)/ LOV KELCH PROTEIN2 (LKP2) (Quail et al. 1995, Briggs and Christie 2002, Demarsy and Fankhauser 2009). Of these, phytochrome and phototropin are the best characterized. Plants sense the ratio of red to far-red light with phytochrome (Smith 1982) to regulate gene expression in the nucleus (Ni et al. 1998, Yamaguchi et al. 1999). In contrast, phototropin is a membrane-localized blue light photoreceptor that mediates responses such as phototropism, chloroplast movement, stomatal opening, leaf flattening and palisade development (Huala et al. 1997, Kagawa et al. 2001, Kinoshita et al. 2001, Sakai et al. 2001, Sakamoto and Briggs 2002, Kozuka et al. 2011). Light is an important environmental cue to which plants respond by regulating growth and development of leaves. The laminar structure of leaves in many dicotyledonous plants indicates that the regulation of leaf flatness is critical for effective light absorption. When Arabidopsis plants are grown under monochromatic red light, the leaves curl downwards; in contrast, flat leaves develop under monochromatic blue light (Inoue et al. 2008, Kozuka et al. 2011). Phototropin mediates this leaf-flattening response to blue light. Accordingly, a phototropin-deficient mutant exhibits a curled-leaf phenotype (Sakai et al. 2001, Sakamoto and Briggs 2002, de Carbonnel et al. 2010). Furthermore, leaf curling under red light can be explained by the loss of phototropin activation (Inoue et al. 2008, Kozuka et al. 2011). However, it remained possible that phytochrome is involved in this process. Arabidopsis contains two phototropins, phot1 and phot2 (Huala et al. 1997, Kagawa et al. 2001), both of which are widely expressed in nearly all leaf tissues (Sakamoto and Briggs 2002, Kong et al. 2006, Wan et al. 2008). It was recently reported that phot2 expression in the epidermis regulates leaf flattening (Kozuka et al. 2011). Thus, the epidermis is the site that regulates the degree of leaf flatness. In addition, a phototropin signaling transducer, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3), which physically interacts with phototropin (Motchoulski and Liscum 1999, Lariguet et al. 2006), is involved Special Focus Issue Regular Paper Plant Cell Physiol. 54(1): (2013) doi: /pcp/pcs134, available online at The Author Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please journals.permissions@oup.com 69

2 T. Kozuka et al. in the leaf-flattening response (Inoue et al. 2008, de Carbonnel et al. 2010). Phytochrome is reversibly converted between two spectral forms: the red-light-absorbing form Pr and the far-red-lightabsorbing form Pfr (Quail et al. 1995). When plants grow beneath neighboring plants, the ratio of red light to far-red light is substantially reduced (Kasperbauer 1971), and the equilibrium of phytochrome is consequently shifted toward the inactive Pr form. Additionally, shade-avoidance syndrome (SAS) responses are triggered, including the elongation of stems and petioles and accelerated flowering (Whitelam and Smith 1991, Devlin et al. 1999, Franklin et al. 2003). In Arabidopsis, the phytochrome family consists of five members, phytochrome A phytochrome E (Sharrock and Quail 1989, Clack et al. 1994). Among them, phyb primarily mediates the SAS response through transcriptional regulation (Whitelam and Smith 1991, Devlin et al. 2003). Although phyb Pr is evenly distributed in the cytosol, phyb Pfr accumulates and forms speckles in the nucleus (Yamaguchi et al. 1999). The PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors are destabilized by physical interactions with phyb Pfr (Ni et al. 1998, Al-Sady et al. 2008, Shen et al. 2008) probably at the sites of nuclear speckles (Chen et al. 2010). Under shade conditions, the absence of phyb Pfr allows for the accumulation of PIFs, which promote the expression of SAS-related genes (Hornitschek et al. 2012, Leivar et al. 2012, Li et al. 2012). End-of-day far-red light (EODFR) treatment exposes plants under day/night cycles to a pulse of far-red light at the end of the day and is a useful method for experimentally inducing the SAS (Smith 1982, Nagatani et al. 1991, Devlin et al. 1998). The active Pfr phytochrome immediately converts back to inactive Pr in response to EODFR, whereas no immediate conversion would occur in the absence of EODFR. Transcriptomic analyses revealed that the expression of many genes related to phytohormones, such as auxin, brassinosteroid, gibberellin, cytokinin, ABA and ethylene, is up-regulated in response to EODFR (Kozuka et al. 2010). Of these, auxin may have a role in leaf flattening. Indeed, the auxin-insensitive mutant massugu1 (msg1)/auxin response factor7 (arf7) exhibits a leaf-curling phenotype (Watahiki and Yamamoto 1997, Harper et al. 2000). Additionally, increased and decreased expression of IAMT1, which encodes an indole-3-acetic acid carboxyl methyltransferase, leads to upward and downward leaf curling, respectively (Qin et al. 2005). PhyB may therefore affect leaf flatness by regulating the auxin response in the leaf. In the present study, we investigated how phytochrome and phototropin coordinately regulate leaf flatness in response to light stimuli. Physiological and genetic approaches were successfully combined to find that phyb promotes leaf curling antagonistically to phototropins. We then investigated the molecular mechanism by which these photoreceptors interact with each other. Lastly, involvement of another signaling component, NPH3, in this response was examined. Results Promotion of curled leaf development by phyb Previous reports have shown that leaves curl downwards in wild-type Arabidopsis plants grown under monochromatic red light (Inoue et al. 2008, Kozuka et al. 2011). To assess the involvement of phytochrome in this process, we compared the extent of leaf flatness in the wild type and in phyb. Because the leaf blades of the phyb mutant fail to grow under monochromatic red light (Kozuka et al. 2005), wild-type and phyb plants were grown under continuous white light (70 mmol m 2 s 1 ). In the wild type, the fourth leaves were gently curled downwards, whereas flat leaves were observed in phyb (Fig. 1A). We determined the degree of leaf flatness, which can be expressed as the ratio of the straight-line distance between two leaf edges to the actual leaf width in the leaf transverse section (Kozuka et al. 2011). The values were approximately 0.67 and 0.94 in the wild type and phyb, respectively (Fig. 1B), confirming that the phyb leaves were flatter than those of the wild type. We also examined the development of curled leaves in transgenic plants overexpressing a green fluorescent protein (GFP)-tagged PHYB construct (PBG) (Yamaguchi et al. 1999) under the control of the Cauliflower mosaic virus (CaMV) 35S promoter in the phyb background (Pro35S:PBG/phyB lines). As expected, higher levels of PBG protein were observed in Pro35S:PBG/phyB leaves compared with the levels of endogenous phyb (Fig. 1C). In these transgenic lines, the leaves were more severely curled than in the wild type (Fig. 1A, B). These observations indicate that in contrast to phototropins, phyb promotes leaf curling. Suppression of leaf curling by EODFR treatment EODFR treatment is a useful method for the elimination of the Pfr phytochrome form and the subsequent induction of the SAS (Smith 1982, Nagatani et al. 1991, Kozuka et al. 2010). We therefore examined the effect of EODFR on leaf flatness. Seedlings were grown under continuous white light for 7 d for complete de-etiolation. These seedlings were further grown under long-day conditions [14 h light (120 mmol m 2 s 1 )/ 10 h dark] for 23 d with or without EODFR treatment, and then the mature fourth leaves were used for the observation of leaf flatness. In the wild type, leaf flatness increased by 19% in response to EODFR. In contrast, phyb exhibited very flat leaves without EODFR and failed to respond to EODFR (Fig. 2). The curled-leaf phenotype in the Pro35S:PBG/phyB plants was substantially suppressed by EODFR (Fig. 2). Taken together, we concluded that the Pfr form of phyb promotes leaf curling. Relationship between phyb and phototropins in the regulation of leaf flatness Because phototropins (phot1 and phot2) promote leaf flattening (Sakamoto and Briggs 2002, Inoue et al. 2008, de Carbonnel et al. 2010, Kozuka et al. 2011), we assessed whether phyb promotes leaf curling through the reduction of phot1 and phot2 70

3 Promotion of leaf curling by phyb Fig. 1 Promotion of leaf curling by phyb. (A) Adaxial (top), abaxial (middle) and transverse (bottom) views of the fourth leaves of the wild type, phyb and Pro35S:PBG/phyB are shown. Each plant was grown under continuous white light for 30 d (see the Materials and Methods for details). Scale bars = 5 mm. (B) Leaf flatness was determined for the wild type, phyb and Pro35S:PBG/phyB, with growth conditions as described for Fig. 1A. The data indicate the means ± SD (n = 25). Asterisks indicate significant differences from the wild type (*P < 0.05, Student s t-test). (C) Immunoblotting detection of phyb was performed in the wild type, phyb and Pro35S:PBG/ phyb. Crude protein extracts were prepared from the third and fourth leaves of plants grown under continuous white light for 19 d. The blot was probed with an anti-phyb monoclonal antibody. Arrows indicate the positions of phyb and PBG. The molecular mass marker sizes in kilodaltons are shown on the right of the blot. expression. However, similar levels of phot1 and phot2 were observed when phyb and Pro35S:PBG/phyB were subjected to immunoblot analysis (Fig. 3). It is therefore unlikely that phyb promoted leaf curling by reducing phototropin expression. We then used genetic analysis to examine the relationship between phyb and phototropins in the regulation of leaf Fig. 2 The leaf-flattening response to EODFR. Leaf flatness was determined in the fourth leaves of the wild type, phyb and Pro35S:PBG/ phyb. Seven-day-old seedlings grown under continuous white light were further incubated under white light (14 h)/dark (10 h) cycles with (+) or without ( ) EODFR for 23 d. The data indicate the means ± SD (n = 23). Asterisks indicate significant differences from the EODFR control (*P < 0.05, Student s t-test). Fig. 3 Immunoblotting detection of phot1 and phot2. Crude protein extracts were prepared from the third and fourth leaves of wild-type, phyb and Pro35S:PBG/phyB plants, with growth conditions as described for Fig. 1C. The blot was probed with anti-phot1 and anti-phot2 polyclonal antibodies. Arrows indicate the positions of phot1 and phot2. Non-specific bands are marked with asterisks. The molecular mass marker sizes in kilodaltons are shown on the right of the blot. flatness with the aid of the phybphot1phot2 triple mutant. Surprisingly, the curled-leaf phenotype observed in phot1phot2 was almost completely suppressed by the phyb mutation (Fig. 4A, B). While phot1phot2 developed severely curled leaves under continuous white light as previously reported (Sakai et al. 2001, Sakamoto and Briggs 2002, de Carbonnel et al. 2010), phybphot1phot2 leaves were as flat as phyb leaves (Fig. 4B). Additionally, the curled-leaf phenotype was restored by introducing Pro35S:PBG into phybphot1phot2 (Fig. 4A, B). Thus, phyb was genetically epistatic to phot1phot2 with respect to the curled-leaf phenotype. We further examined how the elimination of Pfr by EODFR affected the curled-leaf phenotype in phot1phot2. As shown in 71

4 T. Kozuka et al. Fig. 4 Suppression of the phot1phot2 curled-leaf phenotype by phyb. (A) Adaxial (top), abaxial (middle) and transverse (bottom) views of the fourth leaves of phot1phot2, phybphot1phot2 and Pro35S:PBG/ phybphot1phot2 plants are shown. Each plant was grown as described for Fig. 1A. Scale bars = 5 mm. (B) Leaf flatness was determined for phot1phot2, phybphot1phot2 and Pro35S:PBG/phyBphot1phot2, with growth conditions as described for Fig. 1A. The data indicate the means ± SD (n = 25). Asterisks indicate significant differences from phot1phot2 (*P < 0.05, Student s t-test). (C) Immunoblotting detection of phyb was performed in the wild type, phot1phot2, phybphot1phot2 and Pro35S:PBG/phyBphot1phot2. Crude protein extracts were prepared from the third and fourth leaves of plants grown under continuous white light for 19 d. The blot was probed with an anti-phyb monoclonal antibody. Arrows indicate the positions of phyb and PBG. The molecular mass marker sizes in kilodaltons are shown on the right of the blot. Fig. 5, the phenotype was substantially suppressed by EODFR. The same effect was not observed in phybphot1phot2, and the introduction of Pro35S:PBG into the triple mutant (Pro35S: PBG/phyBphot1phot2 lines) restored the responsiveness to Fig. 5 Attenuation of the phot1phot2 curled-leaf phenotype by EODFR. Leaf flatness was determined for phot1phot2, phybphot1 phot2 and Pro35S:PBG/phyBphot1phot2, with growth conditions as described for Fig. 2. The data indicate the means ± SD (n = 23). Asterisks indicate significant differences from the EODFR control (*P < 0.05, Student s t-test). EODFR (Fig. 5). These findings indicate that the leaf-curling activity of phyb Pfr was exaggerated in the absence of phototropins. Taken together, we concluded that phototropins promote leaf flattening by suppressing the leaf-curling activity of phyb. We employed immunoblot analysis to determine whether phototropins reduced the expression level of phyb. However, phyb expression was not affected by the phot1phot2 mutations (Fig. 4C). Regulation of epidermal cell development by phyb and phototropins Because phot2 expression in the epidermis determines leaf flatness (Kozuka et al. 2011), leaf curling is most probably caused by an uneven expansion of leaf epidermal cells on the adaxial (upper) and abaxial (lower) sides. To test this hypothesis, we determined the epidermal cell area of 320 cells on the adaxial and abaxial sides of leaves from wild-type, phyb, phot1phot2 and phybphot1phot2 plants grown under continuous white light. In the adaxial epidermis, no differences were observed among these genotypes with respect to both the median cell area and the distribution of the number of cells within a given cell area (Fig. 6). In the abaxial epidermis, the median cell area was slightly reduced in phot1phot2. Notably, this reduction was not restored in phybphot1phot2 (Fig. 6), although the leaves were flat in this particular genotype (Fig. 4B). Thus, the median of cell area did not correlate with the curled-leaf phenotype. The cell area distribution was clearly reduced in the abaxial epidermis of phot1phot2 relative to the other genotypes (Fig. 6). For all genotypes, 320 cells were analyzed, and the numbers of abaxial epidermal cells >3,000 mm 2 were 94, 97 and 87 in the wild type, phyb and phybphot1phot2, respectively. 72

5 Promotion of leaf curling by phyb This number decreased to 50 in phot1phot2. Thus, curled leaf development may be associated with a decrease in the development of large cells in the abaxial epidermis, which may in turn be regulated by phyb and phototropins. Effect of phototropin on phyb speckle formation In the nucleus, phyb Pfr forms speckles (Yamaguchi et al. 1999), which are supposed to be the sites of light signaling (Chen et al. 2010). We therefore examined the speckling behavior of PBG in response to EODFR using the Pro35S:PBG/phyB line (Fig. 7A). In both the adaxial and abaxial epidermis, nuclear speckles were observed for at least 120 min after the plants were transferred Fig. 6 Size of adaxial and abaxial epidermal cells. The sizes of adaxial (left) and abaxial (right) epidermal cells were determined in the fourth leaves of the wild type, phyb, phot1phot2 and phybphot1phot2, with growth conditions as described for Fig. 1A. The box indicates the distribution of the number of epidermal cells. The horizontal line within the box represents the median value. The box delimits the 25th and 75th percentiles, and the bars indicate the 10th and 90th percentiles. Circles represent outlying values (n = 320 cells from 16 leaves). to darkness (Fig. 7A). In contrast, EODFR substantially reduced the number and size of speckles on both sides of the leaves (Fig. 7A). Although smaller speckles were observed in some cells 30 min after EODFR, no speckles were detected after 60 min. These observations indicate that speckle formation did not differ between the adaxial and abaxial sides under different light conditions. On the basis of the hypothesis that phototropins promote leaf flattening by suppressing the leaf-curling activity of phyb we examined whether phototropins affect the speckling behavior of PBG using Pro35S:PBG/phyBphot1phot2 (Fig. 7B). Speckles formed normally in Pro35S:PBG/phyBphot1phot2 under white light, and the speckles were as stable as observed in Pro35S:PBG/phyB (Fig. 7B). Furthermore, these speckles normally disappeared in response to EODFR (Fig. 7B). We therefore concluded that phototropins have no effect on phyb speckle formation. Involvement of NPH3 in the leaf-curling activity of phyb It has previously been reported that NPH3 functions in the leaf-flattening response downstream of phototorpins (Motchoulski and Liscum 1999, Inoue et al. 2008, de Carbonnel et al. 2010). To examine the relationship between phyb and NPH3 in the regulation of leaf flatness, we employed nph3 and the phybnph3 double mutant. Interestingly, the flat leaf phenotype of phyb was suppressed by the nph3 mutation, particularly in the peripheral regions (Fig. 8A, B). In nph3 and phybnph3, the peripheral region similarly curled downwards under continuous white light (Fig. 8A). No quantitative differences in leaf flatness were observed between nph3 and phybnph3 (P > 0.05, Student s t-test; Fig. 8B). These results suggested that nph3 was genetically epistatic to phyb with respect to the curled-leaf phenotype. Fig. 7 Time-course analysis of the PBG speckling behavior in response to EODFR. The PBG speckles were observed in adaxial and abaxial epidermal cells of Pro35S:PBG/phyB (A) and Pro35S:PBG/phyBphot1phot2 (B). The plants were grown under white light or darkness for the indicated number of minutes after the treatment with (+) or without ( ) EODFR. Scale bars = 10 mm. 73

6 T. Kozuka et al. Fig. 8 The curled-leaf phenotype in nph3. (A) Adaxial (top left), abaxial (top right) and transverse (bottom) views of the fourth leaves of nph3 and phybnph3 are shown. Each plant was grown as described for Fig. 1A. Scale bars = 5 mm. (B) Leaf flatness was determined for nph3 and phybnph3, with growth conditions as described for Fig. 1A. The data indicate the means ± SD (n = 25). (C) Leaf flatness was determined for nph3 and phybnph3, with growth conditions as described for Fig. 2. The data indicate the means ± SD (n = 23). Asterisks indicate significant differences from the EODFR controls (*P < 0.05, Student s t-test). Consistent with the above results, leaf flatness of nph3 was increased by only 10% in response to EODFR (Fig. 8C). In other words, the elimination of phytochrome Pfr by EODFR barely affected leaf flatness in nph3, compared with the wild type (Fig. 2). This is in marked contrast to the observation that leaf flatness was substantially increased in response to EODFR in phot1phot2. In summary, these findings suggest that phyb promotes leaf curling by suppressing the leafflattening activity of NPH3. We also observed that phybnph3 exhibited a weak response to EODFR, indicating that phytochromes other than phyb may also be involved in the regulation of leaf flatness. Analysis of gene expression in response to EODFR The expression levels of many genes are up-regulated in response to EODFR (Kozuka et al. 2010). We reasoned that some of these genes may be involved in the regulation of leaf flatness. Furthermore, phototropins may oppose the leafcurling activity of phyb by enhancing the expression of particular genes. To test this hypothesis, we determined the expression levels of EODFR-responsive genes in phot1phot2. We first examined expression of the marker gene for EODFR, ATHB2 (Carabelli et al. 1996). Its expression was slightly reduced in phot1phot2 regardless of EODFR (Fig. 9A). The difference observed was clearly too small to explain the curled-leaf phenotype observed in phot1phot2. Many auxin-related genes are up-regulated in response to EODFR (Kozuka et al. 2010). Interestingly, the auxin-insensitive mutant msg1/arf7 exhibits a curled-leaf phenotype (Watahiki and Yamamoto 1997, Harper et al. 2000). We therefore speculated that phyb Pfr may promote leaf curling by suppressing the expression of auxin-related genes. If this were the case, phototropins may conversely increase the expression of these genes. To test this hypothesis, we selected five EODFR-responsive and auxin-related genes, YUCCA9 (YUC9), PIN-FORMED3 (PIN3), IAA6, IAA19 and GH3.3, which are involved in auxin synthesis, polar transport, transcription and catabolism, respectively (Fig. 9). Although phot1phot2 exhibited a severe curled-leaf phenotype without EODFR (Fig. 5), the expression of these genes was only slightly reduced in phot1phot2 (Fig. 9A). Furthermore, all of these genes responded quite normally to EODFR (Fig. 9A). Thus, phototropins do not appear to promote leaf flattening by up-regulating these auxin-related gene. Genes related to other hormones are also known to be up-regulated in response to EODFR (Kozuka et al. 2010). We examined the expression of 1-AMINO-CYCLOPROPANE-1- CARBOXYLATE SYNTHASE 8 (ACS8), ETHYLENE RESPONSE 2 (ETR2), CYTOKININ OXIDASE 6 (CKX6), PHYB ACTIVATION TAGGED SUPPRESSOR 1 (BAS1), GIBBERELLIN 20-OXIDASE 1 (GA20OX1) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), which are involved in ethylene synthesis, ethylene perception, cytokinin catabolism, brassinosteroid catabolism, gibberellin synthesis and ABA synthesis, respectively (Fig. 9B). As observed for the auxin-related genes (Fig. 9A), the expression of these genes was also unaffected in phot1phot2 (Fig. 9B). Altogether, these data indicate that phototropins do not promote leaf flattening by enhancing the expression of any of the major EODFR-responsive genes. Discussion The leaf-curling activity of phyb Pfr The present findings demonstrate the involvement of phyb in the regulation of leaf flatness. It is well established that phototropins promote leaf flattening in response to blue light (Sakamoto and Briggs 2002, Inoue et al. 2008, de Carbonnel et al. 2010, Kozuka et al. 2011). However, phototropins are not the sole photoreceptor for this response. The loss of phyb in the phyb mutant resulted in the development of completely flat leaves (Fig. 1A, B). Furthermore, elimination of phyb Pfr by EODFR promoted leaf flattening (Fig. 2). Altogether, these observations indicate that phyb Pfr exhibits substantial leaf-curling activity. 74

7 Promotion of leaf curling by phyb Fig. 9 Quantitative RT PCR analysis of shade-responsive genes. Total RNA was prepared from the third and fourth leaves of 19-day-old wild-type and phot1phot2 plants treated with (+) or without ( ) EODFR, as described for Fig. 2. (A) Data for the EODFR marker gene ATHB2, and the auxin-related genes YUC9, PIN3, IAA6, IAA19 and GH3.3. The data indicate the means ± SD (n = 6). (B) Data for the ethylene synthesis gene ACS8, ethylene receptor gene ETR2, cytokinin catabolic gene CKX6, brassinosteroid catabolic gene BAS1, gibberellin synthesis gene GA20OX1 and ABA synthesis gene NCED3. The data indicate the means ± SD (n = 6). When plants grow under neighboring plants, a decrease in the ratio of red to far-red light, primarily recognized by phyb, elicits the SAS responses, including stem elongation and accelerated flowering (Whitelam and Smith 1991, Devlin et al. 1999, Franklin et al. 2003). In leaves, the shade stimulus promotes petiole elongation and leaf-angle elevation (Vince-Prue et al. 1976, Nagatani et al. 1991, Vandenbussche et al. 2003, Kozuka et al. 2010). One critical consideration is that low light intensity in the shade most probably limits the photosynthesis (Smith 1982). It would therefore be reasonable for plants to promote leaf flattening under such conditions. Interaction between phyb and phototropins for the regulation of leaf flatness Our genetic analysis revealed that phyb acts downstream of phototropins in the regulation of leaf flatness, as the curled-leaf phenotype of phot1phot2 was almost completely suppressed by the phyb mutation (Fig. 4A, B). Accordingly, the elimination of phyb Pfr by EODFR greatly increased leaf flattening in the phot1phot2 background (Fig. 5). These findings demonstrate that phototropins promote leaf flattening by suppressing the leaf-curling activity of phyb (Fig. 10). Nevertheless, phototropins were not found to affect the expression level of phyb (Fig. 4C). Likewise, PBG speckle Fig. 10 Schematic model representing the signaling pathway for the regulation of leaf flatness by phyb, phototropin and NPH3. PhyB Pfr promotes leaf curling by inhibiting the leaf-flattening activity of NPH3, whereas phototropins oppose this negative effect of phyb on NPH3. formation in the nucleus was not affected in phot1phot2 regardless of EODFR (Fig. 7A, B). PhyB is believed to regulate gene expression in the nucleus directly (Ni et al. 1998, Yamaguchi et al. 1999), whereas phot1 and phot2 primarily 75

8 T. Kozuka et al. localize to the plasma membrane region (Sakamoto and Briggs 2002, Kong et al. 2006) and activate a kinase-based signal transduction pathway (Matsuoka and Tokutomi 2005, Kong et al. 2007). It is therefore unlikely that phototropins directly regulate the state or activity of phyb in the same cells. However, phytochrome has recently been shown to function outside of the nucleus for a certain response (Paik et al. 2012). In addition, phytochrome physically associates with phototropin in the plasma membrane region in Physcomitrella (Jaedickle et al. 2012). Hence, further investigation is necessary to determine if the interaction takes place in the same cell for the regulation of leaf flatness. In which tissue do phyb and phototropin regulate the leaf flatness? The histological analysis performed in the present study suggests that the curled-leaf phenotype of phot1phot2 may be due to a reduced number of large epidermal cells in the abaxial sides of leaves (Fig. 6). Furthermore, this defect in phot1phot2 was rescued by the phyb mutation (Fig. 6). It has been reported that expression of GFP-tagged phot2 in the epidermis, but not in the mesophyll, promotes leaf flattening (Kozuka et al. 2011). Thus, phototropins may cell-autonomously regulate epidermal cell development. In contrast, phyb in the mesophyll delays the flowering and inhibits hypocotyl elongation (Endo et al. 2005). Hence, phyb may suppress the development of large epidermal cells in the mesophyll cells. However, it remains possible that phyb directly interacts with phototropins and other factors such as NPH3 to regulate leaf flatness in the same cell (see above). Recently, tissue-specific promoters of the CHLOROPHYLL A/ B BINDING PROTEIN 3 (CAB3) and 3-KETOACYL-COA SYNTHASE 6 (CER6) genes have been used to express foreign proteins in the mesophyll and epidermis, respectively (Endo et al. 2007, Kozuka et al. 2011). In future studies, these promoters may be useful for experimentally determining the tissues in which phyb regulates leaf flatness. Involvement of NPH3 in the regulation of leaf flatness by phyb and phototropins NPH3, which physically associates with phot1 and phot2, functions as a signal transduction element for the leaf-flattening response (Motchoulski and Liscum 1999, Lariguet et al. 2006, Inoue et al. 2008, de Carbonnel et al. 2010). As previously reported, nph3 leaves curled downwards when grown under white light (Fig. 8A). Furthermore, the degree of leaf flatness in phybnph3 was comparable with that in nph3 (Fig. 8A, B). This strongly contrasts with the observation for phybphot1 phot2, which developed very flat leaves regardless of the loss of phototropins (Fig. 5). According to the present findings, phyb appears to promote leaf curling by inhibiting the leaf-flattening activity of NPH3 (Fig. 10). To our knowledge, this is the first report suggesting that NPH3 functions downstream of phyb, although it remains unclear how phyb inhibits the function of NPH3. The genetic analysis suggested that phototropins suppress the activity of phyb to promote leaf flattening. However, it remains unclear whether or not they interact with each other in the same cell (see discussion above). A model incorporating this hypothesis is as follows: phyb directly or indirectly inhibits NPH3 to promote leaf curling, whereas phototropins oppose this negative effect of phyb on NPH3 through physical interaction with NPH3 (Fig. 10). On the basis of this model, we investigated whether NPH3 protein levels were affected by phyb. However, we could not detect NPH3 in our conditions (data not shown). It was previously reported that the phosphorylation status of NPH3 is critical for its function (Pedmale and Liscum 2007). It would therefore be interesting to test whether phyb affects the phosphorylation of NPH3. Likewise, the effects of phyb on the intracellular localization of NPH3 should be examined in future studies. Possible involvement of auxin in the regulation of leaf flatness Although the expression of many hormone-responsive genes is regulated by phyb in Arabidopsis leaves (Kozuka et al. 2010), the present findings suggest that the suppression of the phyb leaf-curling activity by phototropins is not accomplished through direct regulation of the expression of these genes. This is consistent with a previous report which found that phototropins are not directly involved in transcriptional regulation (Ohgishi et al. 2004). Nevertheless, auxin and other phytohormones may play a role in the interaction between phyb and phototropins in the regulation of leaf flatness. Phototropins polarize PIN3 in endodermal cells toward the elongated lateral region to build an asymmetric auxin distribution that causes differential growth in the hypocotyl (Christie et al. 2011, Ding et al. 2011). In a similar manner, NPH3 may estabish an auxin gradient along the abaxial adaxial axis within leaves. In this model, altered expression of auxin-related genes may not be readily apparent in the whole leaf. It may neverthless have substantial morphological consequences. Clearly, phyb is one of the major factors determining the extent of basal auxin responses, and detailed gene expression analysis in different tissue components of the leaf represents one way of testing the above hypothesis. Materials and Methods Plant materials Arabidopsis thaliana wild type, phyb-9 (Chory et al. 1989), phot1-5 phot2-1 (Kinoshita et al. 2001) and nph3-6 (de Carbonnel et al. 2010) were in the Columbia background. The phyb-9 phot1-5 phot2-1 triple mutant was generated by crossing phyb-9 with phot1-5 phot2-1. phyb-9 and phyb-9 phot1-5 phot2-1 plants were transformed with the Pro35S:PHYB GFP (PBG) plasmid vector (Yamaguchi et al. 1999) to produce the Pro35S:PBG/phyB and Pro35S:PBG/phyBphot1phot2 lines, 76

9 Promotion of leaf curling by phyb respectively, using the floral dip method (Clough and Bent 1998). Kanamycin-resistant (50 mg l 1 ) plants were selected on Murashige and Skoog agar, and T 3 and T 4 homozygous lines were used for all experiments. Growth and light conditions The seeds used in the experiment were sown on rockwool (Nitto Boseki) moistened with 0.5% (v/v) Hyponex solution (Hyponex) then incubated in darkness for 3 d at 4 C (Kozuka et al. 2011). The plants were grown under continuous white light (70 mmol m 2 s 1 ) for 7 d at 23 C followed by the experimental light treatment. Cool-white fluorescence tubes (FL20SSW/18; Toshiba) were used for the white light growth conditions. Far-red light was provided by light-emitting diodes with a maximum wavelength of 735 nm (IS-Series; CCS Inc.). EODFR treatment The EODFR treatment was performed as previously described (Kozuka et al. 2010). Seven-day-old seedlings grown under continuous white light conditions (70 mmol m 2 s 1 )at23 C were placed under a 14 h light (120 mmol m 2 s 1 )/10 h dark cycle with pulses of far-red light (50 mmol m 2 s 1 for 5 min) given at the end of the light period. Plants were treated with far-red light for 23 and 12 d for the anatomical and reverse transciption PCR (RT PCR) analyses, respectively. Anatomical analysis Leaves were numbered from the first rosette leaf that emerged after the cotyledons, and leaf flatness was determined in the fourth leaf. An approximately 3 mm thick transverse section was sliced at the central region of the leaf blade by handsectioning with a razor blade. Photographs of the sections were taken using a stereomicroscope (MZFL; Leica) and measured using ImageJ software ( NIH). The ratio of the straight-line distance between the two edges of the leaf blade to the actual width of the leaf blade along the curved surface was then determined, as previously described (Kozuka et al. 2011). To determine the epidermal cell area, the fourth leaves were fixed in formalin acetic acid solution (FAA) overnight at 4 C. These fixed leaves were cleared with chloral solution for 5 h at room temperature (Kozuka et al. 2005), and then stained with Safranin-O solution as previously described (Sugano et al. 2010) for observation under a microscope (Axioskop 2 plus system; Zeiss). The areas of the adaxial and abaxial epidermal cells were determined in each of 20 cells randomly selected from a small central region (0.125 mm 2 ) of either right or left halves of the leaf blade using ImageJ software ( For each condition, 16 leaves were observed. Immunoblot analysis Immunoblot analysis was performed as previously described (Kozuka et al. 2011). A 50mg aliquot of total crude proteins was prepared from the third and fourth leaves of plants grown under continuous white light (70 mmol m 2 s 1 ) for 21 d at 23 C then subjected to 7.5% SDS PAGE. The blots were probed with anti-phyb monoclonal (Nagatani et al. 1991), anti-phot1 polyclonal (Aihara et al. 2008) and anti-phot2 polyclonal (Kong et al., 2006) antibodies. Real-time RT PCR analysis Real-time RT PCR analysis was performed as previously described (Kozuka et al. 2010). Total RNA was prepared from the immature blades of third and fourth leaves treated or not with EODFR followed by 2 h incubation in darkness. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan [a Grant-in-Aid for Scientific Research on Priority Areas (No to A.N. and No to M.W.), a Grant-in-Aid for the Global COE Program Formation of a strategic base for biodiversity and evolutionary research: from genome to ecosystem (A06) to A.N., a Grant-in-Aid for Scientific Research on Innovative Areas (No to A.N. and No to M.W.)]; the Japan Society of Promotion of Science [a Grant-in-Aid for Scientific Research (B) (No to A.N.) and a Grant-in- Aid for Scientific Research (S) (No to M.W.)]. Acknowledgments We are grateful to Dr. K. Tamura and Dr. I. Hara-Nishimura (Kyoto University, Japan) for technical support, and to Dr. N Mochizuki and Dr. T. Suzuki (Kyoto University, Japan) for helpful discussions. References Aihara, Y., Tabata, R., Suzuki, T., Shimazaki, K. and Nagatani, A. (2008) Molecular basis of the functional specificities of phototropin 1 and 2. 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