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First published online January 22, 2004; 10.1104/pp.103.030650 Plant Physiology 134:790-800 (2004) © 2004 American Society of Plant Biologists High Pigment1 Mutation Negatively Regulates Phototropic Signal Transduction in Tomato Seedlings1School of Life Sciences, University of Hyderabad, Hyderabad-500046, India (A.S., R.K.B., R.S.); Division of Biological Regulation and Photobiology, National Institute for Basic Biology, Okazaki 444–8585, Japan (T.K., M.W., R.S.); and Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo 192–0397, Japan (M.W.)
Phototropins and phytochromes are the major photosensory receptors in plants and they regulate distinct photomorphogenic responses. The molecular mechanisms underlying functional interactions of phototropins and phytochromes remain largely unclear. We show that the tomato (Lycopersicon esculentum) phytochrome A deficient mutant fri lacks phototropic curvature to low fluence blue light, indicating requirement for phytochrome A for expression of phototropic response. The hp1 mutant that exhibits hypersensitive responses to blue light and red light reverses the impairment of second-positive phototropic response in tomato in phytochrome A-deficient background. Physiological analyses indicate that HP1 functions as a negative regulator of phototropic signal transduction pathway, which is removed via action of phytochrome A. The loss of HP1 gene product in frihp1 double mutant allows the unhindered operation of phototropic signal transduction chain, obviating the need for the phytochrome action. Our results also indicate that the role of phytochrome in regulating phototropism is restricted to low fluence blue light only, and at high fluence blue light, the phytochrome A-deficient fri mutant shows the normal phototropic response.
Plants use multiple photoreceptors to perceive changes in quality and quantity of light and to regulate growth and development. These photoreceptors sense UV-B, blue/UV-A, and red/far-red regions of the light spectrum. Blue light regulates a variety of physiological processes in plants and also in animals. Studies in higher plants have led to the identification of several types of blue light photoreceptors, all of which are flavin-containing proteins. Cryptochromes, which are related to the DNA repair enzyme DNA photolyase, function as the blue light photoreceptors for circadian rhythms and other light-regulated responses in plants, insects, and mammals (Lin and Shalitin, 2003  ). Plant phototropins, which are blue light-regulated protein kinases, control mechanical processes like phototropism and chloroplast movement in plants (Briggs and Christie, 2002). The red/far-red absorbing photoreceptors, phytochromes, which also have protein kinase activity, regulate a range of developmental processes, including seed germination, shade avoidance, and transition to flowering (Sharma, 2001; Quail, 2002).
The analysis of relationship between blue light intensity and phototropic curvature in several plant species has shown that phototropic responses can typically be classified as two types of positive phototropism based on their dependence on stimulus intensity, duration, and reciprocity; pulse-induced phototropism or "first-positive curvature," and time-dependent phototropism or "second-positive curvature" (Iino, 1990
The functional role of these two photoreceptors has been gradually understood by genetic and biochemical analysis of Arabidopsis mutants. The seedlings of phot1 mutant still show curvature in response to high-fluence unidirectional blue light; however, phototropic curvatures are significantly reduced in phot1phot2 double mutant seedlings (Sakai et al., 2001
The information on the downstream components involved in phototropin signaling is, at the moment, limited. Although several positive and negative regulatory components have been identified for phytochrome and cryptochrome, the numbers of loci identified regulating phototropic signal transduction are rather few. Only one mutant locus, nph3, has been identified that specifically disrupts phototropism under low fluence blue light (Liscum and Briggs, 1996
Although only UV-A/blue light is effective in inducing phototropism, in several species, a red light preirradiation-activating phytochrome significantly enhances the subsequent phototropic responsiveness (Janoudi and Poff, 1992
In species other than Arabidopsis, there is limited information available about the biochemical and genetic regulation of phototropism. Though tomato (Lycopersicon esculentum) has a range of phytochrome-deficient mutants that have been well characterized at physiological and genetic levels, their role in phototropism has not been examined. The most studied aurea (au) mutant is defective in phytochrome chromophore synthesis and is deficient in bulk pool of phyA (Sharma et al., 1993 The analysis of phototropism in tomato mutants can complement and extend the observations hitherto obtained only with Arabidopsis, particularly the role of different phytochrome species and interaction of blue light-mediated phototropism with an element located in signal transduction pathway of phytochrome. Here, we report experiments showing that the deficiency of phyA nearly abolishes phototropism under low fluence blue light. We also demonstrate a novel role for HP1, where it appears to act as a negative regulator of phot1-triggered signal transduction chain, revealing likely mode of participation of phytochrome in regulation of phototropism enhancement.
Loss of Second-Positive Curvature in Phytochrome A-Deficient Seedlings Is Restored by Mutation at hp1 Locus To study how phytochromes are involved in regulation of phot1 function, we compared the phototropism in au mutant of tomato that is deficient in all phytochrome species with that of wild type (Fig. 1A). When exposed to continuous low fluence blue light, wild-type seedlings showed a lag phase of about 45 min and reached a maximum curvature by 2 h. Compared with wild type, the time-course profile of curvature was quite different in the au mutant. The au mutant exhibited delayed onset of phototropism with a prolonged lag phase of nearly 1.5 h and later sluggishly developed phototropic curvature. Even after 6 h of blue light exposure, the extent of curvature in au mutant was less than that observed for wild type at 2 h. Interestingly, the auhp1 double mutant showed phototropic curvature nearly comparable with the wild type, showing virtually total reversal of au effect.
Because the au mutant is likely deficient in all phytochrome species, the role of specific phytochromes in phototropism was examined by using the phyA-deficient fri mutant and the phyB1-defective tri mutant (Fig. 1B). In comparison with the au mutant, the fri mutant showed no phototropic curvature, even after a prolonged blue light exposure of 6 h. Interestingly, for this mutant as well, the frihp1 double mutant showed a time course of phototropic curvature nearly similar to wild type, showing that the hp1 mutation reversed the impairment of phototropism caused by fri mutation. Because the above results showed a role for phyA, we also examined a role for other phytochrome species by using tri mutant deficient in phyB1. Contrastingly, the tri seedlings showed phototropism comparable with wild type, whereas the fritri double mutant was nonphototropic, like the fri mutant (Fig. 1B, inset). The above results indicated that phyA-deficient seedlings are severely impaired in second-positive phototropism.
The second-positive curvature of plants has a distinctive feature in that it requires a continuous blue light irradiation for a minimum duration, which distinguishes it from first-positive curvature that can be induced by a short duration pulse of blue light. Because the hp1 mutation can overcome the phyA deficiency in au and fri seedlings, the influence of this mutation on the fluence response curve for phototropic curvature was determined. Dark-grown seedlings were exposed to a fixed fluence of 0.1 µmol m–2 s–1 for varying duration, and curvature was determined after 2 h. Figure 2A shows that the amplitude of first-positive curvature was much higher in the hp1 mutant compared with the wild type. Additionally, the fluence response profiles for hp1 mutant differed considerably from the wild type for the time threshold required to elicit second-positive curvature. Although a zone of indifferent phototropic responsivity between 100 and 1,000 s distinctly separated the first-positive curvature from second-positive curvature in the wild-type seedlings, the hp1 mutant seedlings initiated second-positive curvature without an intervening zone of indifferent phototropic responsivity. A comparison of phototropic curvature after a 10-min pulse of blue light clearly highlights this difference between the wild-type and hp1 mutants. On 10 min of blue light exposure, the hp1 mutant showed about 47° curvature, whereas the wild type showed only a 7° curvature (Fig. 2B). Examination of the time threshold in wild-type and hp1 seedlings for second-positive curvature showed that at least a 10-min continuous blue light exposure is needed to elicit the curvature in the wild type (Fig. 2B), whereas in the hp1 mutant, the time threshold is reduced to just 5 min (Fig. 2A). Preirradiation with a red, far-red, or red/far-red light 90 min before the onset of blue light exposure reduced the time threshold to 5 min in the wild-type seedlings. On the other hand, similar preirradiation of the hp1 seedlings with red, far-red, or red/far-red exposure had no effect on time threshold, and the values were nearly the same as the nonpreirradiated control (Fig. 2C).
Because au and fri seedlings showed impaired second-positive curvature, we examined whether phyA deficiency also similarly impaired first-positive curvature. The responsiveness of wild-type and mutant seedlings to the unidirectional blue light was examined by exposing seedling to short pulse(s) of blue light to induce first-positive curvature. A single pulse of blue light induced curvature of 17° in wild-type Ailsa Craig (AC) seedlings, whereas in the au mutant, the response was highly reduced with curvature of only 2.4° (Fig. 3A). The first-positive curvature was also extremely reduced in the fri mutant, and it showed only a 3° curvature toward the blue light (Fig. 3B). The responsivity of wild-type and mutant seedlings to multiple pulses of blue light that amplify first-positive curvature (Steintiz and Poff, 1986
These results highlight that fact that phyA deficiency impairs the phototropic responsivity of the seedlings to unidirectional blue light. In view of this, we compared the responsivity of tomato and its mutant seedlings with phytochrome-mediated enhancement of first-positive curvature. Figure 4A shows that a prior exposure of red or far-red light enhanced first-positive curvature in the au mutant, but compared with wild type, enhancement was drastically impaired. Even without a preirradiation, the hp1 mutant showed higher curvature than that of the wild type. However, the auhp1 mutant exhibited first-positive curvature similar to the au mutant. Figure 4B shows that the fri mutant displayed extremely reduced first-positive curvature. Interestingly, whereas the curvature of dark-grown seedlings of the auhp1 mutant was similar to the au mutant, the frihp1 seedlings showed higher curvature than the fri mutant. Moreover, a red preirradiation enhanced curvature in the fri mutant. Although the tri seedlings showed curvature similar to wild type, the fritri mutant seedlings exhibited curvatures similar to the fri mutant.
The above results indicate that phyA deficiency in the fri mutant impaired first- and second-positive responses initiated by blue light. It is now believed that the very high fluence blue light of prolonged duration can trigger phototropic responses via combined action of phot1 and phot2. The phototropic curvature was examined in the etiolated seedlings exposed to varying fluences of blue light for 2 h (Fig. 5A). The phototropic curvature of wild-type seedlings showed a dependence to light fluence, and the magnitude of curvature decreased with increasing fluence. A similar pattern was also observed for the hp1 and frihp1 double mutants. Contrastingly, au and fri seedlings did not show any phototropic curvature for the entire range of fluence used (0.1–40 µmol m–2 s–1). To examine whether the fri seedling respond to a higher fluence of light, wild-type and fri seedlings were exposed to 100 µmol m–2 s–1 blue light for 2 h. Interestingly, at this intensity, the curvature reappeared in wild-type seedlings, but the fri seedlings were still deficient in phototropism. The exposure of wild-type and fri seedlings to varying fluences of blue light for a prolonged duration of 12 h showed a contrastingly different pattern. Although the wild-type seedlings showed nearly the same curvature irrespective of fluence used, the magnitude of curvature in the fri seedlings increased with increase in fluence and was maximal at 100 µmol m–2 s–1 irradiation (Fig. 5B).
It is plausible that the loss of phototropic curvature in the seedlings deficient in phyA could have arisen due to reduction or loss of differential growth mediated by auxin. However, the examination of geotropic responsivity of these seedlings ruled out the above possibility. On horizontally orientating dark-grown seedlings, the kinetics of onset and increase of gravitropic curvature in wild-type seedlings were similar to that observed in au (Fig. 6A) and fri (Fig. 6A, inset) seedlings. The loss of phyA-mediated phototropic curvature appeared to be the feature of etiolated seedlings. The de-etiolation of seedlings under continuous white light for 24 h recovered phototropism in fri and au mutant seedlings similar to wild type, indicating that phyA deficiency does not block phototropic curvature after de-etiolation (Fig. 6B).
The possibility that the phyA deficiency in fri and au mutant seedlings might have affected the activity of phototropin molecule per se was examined by studying blue light-mediated phosphorylation of proteins in crude homogenates of tomato and its mutant seedlings (Short and Briggs, 1994
It is now known that the phot1 and phot2 control also control other mechanical responses such as movement of chloroplasts in plant cells in response to ambient light intensity. The possibility that the phytochrome deficiency impaired the process of chloroplast movement was examined by studying chloroplast accumulation and avoidance in tomato and its mutant seedlings. Figure 7B shows the accumulation as well as avoidance reaction of chloroplasts in the cotyledons of wild-type (AC) tomato seedlings under weak blue light and the strong blue light. Comparison of blue light-mediated accumulation of chloroplasts at low fluence light intensity and avoidance of chloroplast at high light intensity in au, fri, and frihp1 mutants with wild type revealed that chloroplast movement in above mutants was similar to that of wild type.
Phytochrome A Is Required for Expression of First- and Second-Positive Curvature in Tomato Seedlings
Plant photoreceptors though independently affect many developmental responses; there is a growing body of evidence that the effect of individual photoreceptors can also be modulated by other photoreceptors (Mohr, 1994
In conformity with earlier observations, the results of this study demonstrate that in etiolated tomato seedlings, in addition to blue light photoreceptor, the activation of phytochrome is also necessary for the full expression of low fluence blue light-mediated phototropism. The marked reduction of first-positive and second-positive curvature in the au mutant of tomato, which by virtue of chromophore deficiency possess all phytochrome species at a reduced level, provides a good evidence for an active participation of at least one species of phytochrome in blue light-mediated phototropism in tomato. Among the phytochrome species present in tomato (Alba et al., 2000
Among first- and second-positive phototropic curvatures, the deficiency of phyA affects second-positive curvature most severely with near loss of phototropic curvature at low fluence blue light. Comparatively, fri and au mutant seedlings retain some remnants of first-positive curvature, as evident by presence of extremely weak curvature in response to single pulse of blue light. We reasoned that the above reduction in first-positive curvature results from a reduction in the active pool of phytochrome in mutants. In such a case exposure of seedlings to multiple blue light pulses interspersed with dark intervals, a treatment, which is known to significantly enhance first-positive curvature in plants (Steintiz and Poff, 1986
The phytochrome regulation of phototropism in tomato somewhat differs from Arabidopsis with respect to usage of different species of phytochromes. In tomato, only phyA seems to be used for the regulation of second-positive curvature. Even for the first-positive curvature, the severe impairment of response in the fri mutant indicates a major role of phyA. At the same time, additional phytochrome species may also participate in regulation of first-positive curvature as evident by slight stimulation of curvature by red light pretreatment of fri and fritri mutants. Contrastingly, in Arabidopsis, whereas only phyA is needed for amplification of the blue light-mediated first-positive curvature (Parks et al., 1996
The possibility that the loss of phytochrome in mutants may have in turn affected the level or function of blue light photoreceptors is least likely. The fritri double mutant of tomato, which is essentially blind in the red and far-red region of the spectrum, shows blue light-mediated anthocyanin induction, indicating a normal operation of blue photoreceptors even in absence of phyA and phyB (Kerckhoffs et al., 1997
Deficiency of phyA does not seem to affect other phototropin-mediated responses such as chloroplast movement (Wada et al., 2003
The precise role of phytochrome in enhancement of blue light-mediated phototropism is still not known and may be quite complex. There are number of photoresponses where coaction of blue light photoreceptors and phytochrome is needed to elicit the response (Mohr, 1994
The physiological studies on tomato have shown that mutation at the HP1 locus amplifies the phytochrome-mediated responses in the seedlings (Peters et al., 1989
The results of this study also provide a reasonable explanation for similar observation in Arabidopsis where it is observed that whereas red exposure to wild-type seedlings reduces the time threshold (Janoudi et al., 1992
Our studies complement and extend similar observations made in Arabidopsis where null alleles NPH4 are nonphototropic under low fluence blue light, but retain phototropic response to high fluence of blue light or to light conditions that simultaneously activate phytochrome and phot1 (Liscum, 2002
Plant Material The tomato (Lycopersicon esculentum) genotypes used were au, hp1, auhp1 and its isogenic wild type in the background AC, fri and its wild type in the background MM, and tri and its wild type in the breeding line GT; the frihp1 used was in the mixed background MM and AC, and the fritri used was in the mixed background MM and GT. Seed stock carrying the mutant lines were a generous gift from Prof. Maarten Koornneef and Prof. Richard E. Kendrick (Wageningen Universitat and Research Centrum, The Netherlands). The seeds used for experiments were of similar age and were a minimum of 2 years old. Seeds were surface sterilized by 1.5% (w/v) NaClO4 solution for 15 min, rinsed with distilled water, and were germinated on wet germination papers in the dark. The germinated seeds that showed emergences of radicle were individually transferred to screw caps (10-mm diameter x 5-mm height) filled with vermiculite/peat mixture. The seedlings were grown in darkness and used when hypocotyls were about 3 cm long, i.e. 4 d after germination. The seedlings for experiments on effect of varying fluence of blue lights were grown on 0.4% (w/v) agar in Eppendorf tubes. The plants for studies on chloroplast movements were grown in plastic cups filled with vermiculite and were irrigated with the tap water. The plants were then grown in a greenhouse and kept under weak white light conditions (5 µmol m–2) at least several hours before experiments. All of the experiments were carried out at 25°C ± 1°C and were repeated five to seven times. Unless otherwise indicated, all curvature data are mean values ± SE.
Blue light for pulse treatments (0.1 µmol m–2 s–1) was obtained by passing light from a projector (150 W) through two CBS Blue filters (450 nm; Carolina Biological Supply, Burlington, NC). The continuous unidirectional blue light (0.1 µmol m–2 s–1) was provided by passing the output of a cool white fluorescent lamp (20 W) through a CBS blue filter (450 nm). The red light (25.0 µmol m–2 s–1) was obtained by passing light from a halogen lamp (150 W) through a CBS red filter (650 nm) and a 1-cm layer of water. Similarly, the far-red light (21 µmol m–2 s–1) was obtained by passing light from a halogen lamp (150 W) through a CBS far-red filter (750 nm) and a 1-cm layer of water. The photon fluence rate of light was measured by using a quantum photometer (SKY, Powys, UK).
The effect of different fluence of light was examined by exposing seedlings to varying fluences of light ranging from 0.1 to 40 µmol m–2 s–1 in a threshold box with seven chambers. The light source consisted of a 300-W Xenon lamp and the output of lamp was passed through a 10-cm layer of water and a broadband blue light filter. For 100 µmol m–2 s–1 light exposure, the light source consisted of a 1,000-W halogen lamp, and its light output was passed through a 10-cm layer of water and four layers of blue cellophane sheets (
Seedlings of right length and orientation were picked up, and adhering seed coats were removed under dim green safe light; thereafter, the seedlings were exposed to continuous or pulse(s) of unidirectional blue light. For continuous blue light treatment, the seedlings were exposed with cotyledons in front of the light source. For blue light pulse treatment, seedlings were exposed with hook in front of light source, because pulse treatment induced only small angles of curvature with cotyledons in front of the light source. At defined time points, seedlings were removed, placed on a transparency sheet, and photocopied on a photocopier (Modi-Xerox, Rampur, India). The angle of curvature was measured from photocopied images. For gravitropism experiments, seedlings were kept horizontally, and at different time intervals, the curvatures were measured similar to phototropism.
The in vitro phosphorylation was carried out essentially using the procedure outlined by Salomon et al. (1997
The chloroplast movement was examined in the cotyledons of 2-week-old tomato and its mutant seedlings. The cotyledons were evacuated with deionized water and the cells in the cotyledons were then observed under a microbeam irradiator (modified BX-50; Olympus, Tokyo) with a computer recording system. The monochromatic blue light was obtained through an interference filter (17-nm half-bandwidth centered at 449 nm). Other details were described in a previous paper (Kagawa and Wada, 2000
We thank Prof. M. Koornneef and Prof. R.E. Kendrick (Wageningen, The Netherlands) for providing the mutant seeds of tomato. Received July 22, 2003; returned for revision September 15, 2003; accepted November 16, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.030650.
1 This work was supported by University Grants Commission, New Delhi, India (research fellowship support to A.S.), by Council of Scientific and Industrial Research, New Delhi, India (to R.K.B.), by Department of Science and Technology, New Delhi, India (research grant to R.S.), and by a Japanese Society for the Promotion of Science, Tokyo, Japan Senior, Visiting Fellowship (to R.S.). * Corresponding author; e-mail rpssl{at}uohyd.ernet.in; fax 0091–40–23010120.
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ABSTRACT
RESULTS
max = 450 nm). 
-32P) ATP (specific activity of 4,000 Ci mol–1). One set of samples was irradiated with blue light at a fluence rate 2,000 µmol m–2 s–1 for 1 min and another set of samples was kept in the dark for control. Reactions were allowed to proceed at 25°C for 2 min and were then terminated by adding 10 µL of 4x SDS-PAGE sample buffer consisting of 200 mM Tris-HCl, pH 6.8, 8% (w/v) SDS, 40% (v/v) glycerol, 20% (v/v) 
-mercaptoethanol, and 0.4% (w/v) bromphenol blue followed by heating the sample for 4.5 min at 100°C. Aliquots from each sample containing equal amounts of protein were electrophoresed on 8% (w/v) SDS-PAGE followed by Coomassie staining and drying the gel. The radiolabeled protein bands were detected using a PhosphorImager (model no. STORM 840; Molecular Dynamics, Sunnyvale, CA). The molecular mass of the protein was determined by comparison with prestained molecular markers. 