Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5207-5211, June 1991

Rice type I phytochrome regulates hypocotyl elongation in transgenic tobacco (light regulation/transgenic / development/growth regulation) AKIRA NAGATANI*, STEVE A. KAYt, MARIA DEAKt, NAM-HAI CHUAt, AND MASAKI FURUYA* *The Laboratory of Plant Biological Regulation, Frontier Research Program, RIKEN Institute, Hirosawa 2-1, Wako City, Saitama, Japan 351-01; and tThe Laboratory of Plant Molecular , The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399 Communicated by Winslow R. Briggs, February 11, 1991

ABSTRACT We have examined the biological activity of cDNAs from (13) and tobacco (S.A.K., rice type I phytochrome (PI) in transgenic tobacco seedlings. M.D., and R. Kern, unpublished data). Thus, we have The progeny offour independent transformants that expressed operationally designated a molecular form of phytochrome the rice PI segregated 3:1 for shorter hypocotyl length that is light-labile and most abundant in dark-grown tissue as under dim white light (0.04 W/m2). By contrast, this pheno- type I phytochrome (PI) and one that is much less abundant type was not observed either in the dark or under white light and relatively stable irrespective oflight conditions as type II at higher intensity (6.0 W/m2). This suggests that the pheno- phytochrome (12, 14, 15). type is dependent not only on light but also on light intensity. It is possible to express a cloned phytochrome gene as well The increased light sensitivity cosegregated with the kanamy- as its mutant forms in transgenic plants. As previously cin-resistance marker as well as with the rice PI polypeptides, proposed (4), one can expect either to induce a dominant indicating that this is directly related to the expres- negative phenotype with a mutant molecule or to create an sion of the transgene. The transgenic plants showing short exaggerated phenotype by overexpression. This kind of hypocotyls exhibited a reduced growth rate throughout the experimental system can be readily used to probe functional elongation period, and the resulting shorter hypocotyl length domains of the introduced phytochrome. Such a system will was attributable to shorter epidermal cell length but not to also be useful in assigning distinct or overlapping roles for reduced cell number. Furthermore, successive pulse irradia- different types of the photoreceptor. Recently, we (16) and tions with light elicited short hypocotyls similar to those two other groups (17, 18) have overexpressed a monocot PI obtained under dim white light, and the effect was reversed by gene in a transgenic dicot background. The introduced PI immediate far-red light treatment, providing a direct indica- showed normal Pr/Pfr photoconversion and was light-labile. tion that the phenotype is caused by biologically active rice PI. Several resulting were also observed; Keller et Therefore, the far-red-absorbing form ofthe introduced rice PI al. (17) and Boylan and Quail (18) noted semidwarfism and appears to regulate the hypocotyl length of the transgenic dark green , whereas we observed altered patterns of tobacco plants through endogenous signal-transduction path- endogenous Cab gene expression (16). However, the rela- ways. This assay system will be a powerful tool for testing the tionship between these phenotypes and the introduced phy- biological activity of introduced phytochrome molecules. tochrome molecules has remained unclear. To investigate the utility of this approach further, we have Phytochrome is a regulatory photoreceptor that plays a generated several transgenic tobacco lines overexpressing central role in linking external light signals to developmental rice PI. To assess the effects of the overexpression, we have responses in plants (1, 2). One of the underlying mechanisms designed an assay for hypocotyl length in transgenic tobacco of these developmental responses is the modulation of pat- seedlings. Using this assay, we have extended the previous terns of gene expression (3), and phytochrome has been studies by demonstrating that the short-hypocotyl phenotype shown to regulate the expression of many plant (4, 5). is not only dependent upon light intensity but also regulated An understanding of the molecular mechanism of phy- by the introduced rice PI. In addition, we have shown that tochrome action in vivo is therefore essential to elucidating rice PI influences hypocotyl length by altering cell length but the complex processes of plant growth and development. not cell number. Phytochrome is a soluble consisting of an apoprotein (monomer, 118-125 kDa) covalently linked to a linear (6) and is located in the cytoplasm in vivo MATERIALS AND METHODS (7). Phytochrome is synthesized in the dark as the red light Plant Materials. Tobacco plants (Nicotiana tabacum cv. (Amax = 666 nm)-absorbing form (Pr), which is physiologically Xanthi) were transformed with the construct containing rice inactive. Absorption of red light by Pr converts the molecule PI cDNA fused to the cauliflower mosaic virus (CaMV) 35S to the far-red light (Amax = 730 nm)-absorbing form (Pfr), promoter as described (16). The transformants, CR and CO, which is the biologically active conformation, and subse- which showed the highest rice PI mRNA accumulation quent irradiation of Pfr with far-red light converts the mol- among several independent transformants, were used in the ecule to the Pr form (8). Phytochrome responses are therefore present study. Preparation of the tobacco transgenic plants elicited by red light and attenuated by far-red light. BN1 and BD1 (cv. SR1), which overexpress rice PI, have Immunological studies have indicated that more than one been described (16). Control transgenic tobacco plants p69 type ofphytochrome exists in (9, 10) and in (11). More and 4C (cv. Xanthi) were transformed with constructs car- recently, this has been demonstrated at the molecular level rying the CaMV 35S promoter fused to the chloramphenicol by microsequencing of divergent pea phytochrome polypep- acetyltransferase (CAT) gene or 83-glucuronidase (GUS) tides (12) and by cloning of the divergent phytochrome gene, respectively. Primary transgenic plants were desig-

The publication costs of this article were defrayed in part by page charge Abbreviations: CaMV, cauliflower mosaic virus; PI, type I phy- payment. This article must therefore be hereby marked "advertisement" tochrome; Pfr, far-red-absorbing form of phytochrome; Pr, red- in accordance with 18 U.S.C. §1734 solely to indicate this fact. absorbing form of phytochrome. 5207 Downloaded by guest on September 24, 2021 5208 Botany: Nagatani et al. Proc. Natl. Acad. Sci. USA 88 (1991) nated the Ro generation. The R1 and R2 were obtained by (NH4)2SO4 precipitation (0.25 g/ml) and a 20-,u1 aliquot by selfing the R0 and R1 plants, respectively. Samples of was then subjected to NaDodSO4/PAGE. The in the seeds from individual plants were first tested for kanamycin gel were electrophoretically blotted onto nitrocellulose mem- resistance by germinating the seeds on agar plates containing brane and stained with the anti-rye phytochrome monoclonal the antibiotic; populations that showed 3:1 segregation antibody (mAR14), which stained rice but not tobacco phy- for kanamycin resistance were used. tochrome (16), and the anti-pea PI Growth Conditions. For hypocotyl length determination, (mAP5), which stained tobacco PI but not rice PI (16), as the seeds were sown on 0.5% agar plates containing 0.1x described (21). The extraction procedures were carried out Murashige and Skoog salt mixture (19). The plates were kept under dim green safe light. under continuous white light (6.0 W/m2) for 2 days to induce seed and then subjected to the light treatments RESULTS for 5 days except where otherwise stated. To check the kanamycin resistance of the young seedlings after the light Segregation of the Short-Hypocotyl Phenotype Under Dim treatment, plants were transferred to 0.8% agar plates con- White Light. As described in our previous paper (16), we taining kanamycin (0.1 mg/ml) and Murashige's minimal could not observe a clear morphological phenotype in mature organics medium (20) after the light treatment and grown for plants of BN1 and several other transgenic tobaccos that 10-14 days at 250C under continuous white light. For immu- accumulated high levels of rice PI. In these previous exper- noblot analysis, young plants were transferred to soil and iments the SR1 tobacco cultivar was used as the transgenic grown for about 1 month under a 12 hr/12 hr light/dark cycle host. By contrast, the new transgenic lines, such as CR and in a growth cabinet (Koitotron KG-206HL-D, Koito, Tokyo) CO, which were derived from the Xanthi cultivar, clearly at 25TC. The plants were then dark-adapted for 3 days before showed shorter stem length and dark green leaves when harvest to increase the amount of phytochrome. grown under light/dark cycles (Fig. 1). However, these Light Sources. White light (6.0 W/m2) for inducing germi- phenotypes were less clear under continuous white light nation and growing plants on the agar plate was from white (A.N., unpublished data), suggesting that these phenotypes fluorescent tubes (FL20SS-W/18, Toshiba, Tokyo). Dim are dependent on the ambient light intensity. To investigate white light (0.04 W/m2) was obtained by attenuating the white this phenomenon we chose to examine young transgenic light with three layers of filter paper (3 MM Chr, Whatman). seedlings rather than mature plants, since the morphological Red light (0.4 W/m2) and far-red light (0.8 W/m2) were variations observed among individual mature plants were obtained from two fluorescent lamps (red, same as ones for usually much greater than those in younger plants. In addi- white light; far-red, long-wavelength fluorescent lamp, tion, one can test larger numbers of individual plants in less FL20SFR74, Toshiba, Tokyo) filtered through one layer of time with young seedlings. acrylic (red, Shinkolite A102, Mitsubishi, Tokyo; far-red, When the R2 progeny of heterozygous R1 BN1 transgenic Deraglass 102, Asahikasei, Tokyo). Dim green "safe" light plants were grown under dim white light, the seedlings was described elsewhere (21). Intensity of the light was showed a 3:1 segregation of a short-hypocotyl phenotype determined with a radiometer (model 4090 radiant-power from taller wild-type seedlings (Fig. 2B). Under dim white meter, Springfield Jarco, Yellow Springs, OH). light, two populations of BN1 seedlings were evident, with Immunochemical Detection of Phytochrome. Crude extract mean peak heights of 3 mm and 8 mm. Under the same dim was prepared from 1.5 g of young leaves of dark-adapted light conditions, control R1 transgenic (p69) and wild-type tobacco plants for immunochemical detection of phy- seedlings gave single populations with a mean peak height of tochrome (21). The crude extract was concentrated to 0.2 ml 11 mm (Fig. 2B). The phenotype was clearly light-dependent,

FIG. 1. Adult transgenic tobacco plants expressing the rice PI gene. The R1 progeny of CR transgenic plants (three plants in the center) and the wild-type (cv. Xanthi) plants (left and right) were grown under light/dark cycles for several months in the growth cabinet. Downloaded by guest on September 24, 2021 Botany: Nagatani et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5209

A B C Table 2. Kanamycin resistance of the progenies of transgenic tobacco plants BN1 60 _ Kanamycin n 4040|11l9 2( Hypocotyl resistance* 20 1 Plant (host) length R S CA 0 Transgenic g 80P-4 Rice PI A 0- 1 BN1 (SR1) Short 41 0 Long 0 37 CR (Xanthi) Short 36 0 Z WT 5 120 (SRI) Long 0 35 Control O8-- * 117Il2 p69 (Xanthi) Long 117 45 40- 1 Wild type 0 SR1 Long 0 36 Hypocotyl Xanthi Long 0 36 length, mm Hypocotyl length, mm Hypocotyl length, mm Plants were first tested for hypocotyl growth under dim white light FIG. 2. Hypocotyl elongation in the BN1 transgenic and control on the agar plate without kanamycin and then transferred onto plates tobacco plants grown under different light conditions. Seeds of containing kanamycin. heterozygous R2 progeny of the BN1 plants were germinated under *No. of resistant (R) or sensitive (S) plants. white light for 2 days and then grown under white light of 6.0 W/m2 (A), dim white light of 0.04 W/m2 (B), or in the dark (C). Hypocotyl tides, as detected by immunoblotting in the CR transgenic length was determined 7 days after the sowing. As controls, the p69 plants. As expected, all the CR progeny contained endoge- plants and wild-type (WT) tobacco (cv. SR1) were tested. Numbers nous tobacco phytochrome polypeptides, as detected by of plants tested are indicated in each panel. immunoblotting with monoclonal antibody mAP5 (Fig. 3 Left). By contrast, mAR14 detected rice phytochrome poly- as no shorter population was observed when seedlings were peptides only in the dwarfed progeny of CR and not in long grown in darkness (Fig. 2C). Similar segregation of the CR or wild-type seedlings (Fig. 3 Right). The intensity of phenotype under dim white light was observed in several staining with mAR14 indicated that the level of rice PI in the other transformants that overexpressed rice PI (Table 1). By CR transgenic plants was comparable to that detected in the contrast, the segregation was not clear under white light of BN1 plant (16). higher intensity (Fig. 2A), suggesting that the phenotype is Physiological Analysis of the Short-Hypocotyl Phenotype. A also intensity-dependent. time course study of hypocotyl elongation in the CR trans- To confirm that the phenotype was linked to the introduced genic plants under dim white light showed that the shorter gene, young seedlings exhibiting short hypocotyls and their hypocotyl length was attributable mainly to a slower growth taller siblings were transferred onto kanamycin plates to rate throughout the elongation period; there was neither a check the kanamycin resistance of the plants. Table 2 shows delayed onset nor advanced cessation of hypocotyl elonga- that the seedlings exhibited clear cosegregation of antibiotic tion (Fig. 4). To further investigate the underlying cellular resistance, which is a genetic marker for the introduced basis for the phenotype, we measured the epidermal cell construct (16), with short hypocotyls. Furthermore, the length of the BN1 plants by light microscopy. Table 3 shows short-hypocotyl phenotype also cosegregated with the accu- that in BN1 seedlings grown under dim light, the long- mulation of immunodetectable rice phytochrome polypep- hypocotyl population (10 mm) had a mean cell length of 479 ,um, whereas the short population (4 mm) had a mean Table 1. Segregation of short-hypocotyl phenotype and epidermal cell length of only 222 Ztm. These data suggest that kanamycin (Kan) resistance in progenies of transgenic expression of the transgene resulted in a reduced hypocotyl tobacco plants under dim white light length by changing the cell length under dim white light. Hypocotyl length* Kan resistancet Red/Far-Red Reversibility of the Response. The results described above strongly suggest that the short-hypocotyl x2 x2 Plants Short Long (3:1) R S (3:1) Wild CR Wi]Id CR Transgenic Type short long Tyype short long Rice P1 1 2 3 1 2 1 2 3 1 2 BN1 83 (2.8) 33 (8.5) 0.74 37 17 1.21 BD1 88 (5.3) 31 (13.5) 0.07 46 14 0.09 116 116 kDa kDa CR 92 (6.0) 26 (12.5) 0.55 42 14 0.00 _~~~~q - CO 35 (4.4) 12 (9.8) 0.01 63 17 0.60 Control Staine 4 p69 0 117 (11.1) >100 39 18 1.32 4C 0 101 (8.9) >100 46 13 0.28 -witm*A. Wild type SR1 0 118 (10.9) >100 0 55 >100 Stained with mAP5 Stained with mAR14 Xanthi 0 119 (13.8) >100 0 51 >100 FIG. 3. Detection of rice phytochrome polypeptide in the CR Germinated seedlings were grown under dim white light for 5 days transgenic plants. Adult plants of the R1 progeny of CR plants grown to measure hypocotyl length. For kanamycin-resistance analysis, from the seedlings that had been tested for hypocotyl elongation seeds were germinated and grown under white light on agar plates under dim white light were analyzed by immunoblotting with anti-pea containing kanamycin. phytochrome monoclonal antibody mAP5 (Left), which detects *No. ofplants (average hypocotyl length, mm); x2 for 3:1 segregation tobacco phytochrome, and mAR14 (Right), which stains rice phy- is also given. tochrome (16). Arrow indicates the position of the 116-kDa marker tNo. of resistant (R) or sensitive (S) plants. (B8-galactosidase). Downloaded by guest on September 24, 2021 5210 Botany: Nagatani et al. Proc. Natl. Acad. Sci. USA 88 (1991)

1 20- t;15-

.,I -* 10- - -A U 0 Cl) >1 _q 0 5- U) 4- n0 V - _ 0) 2 4 6 8 10 12 zE Time after sowing, day z3 FIG. 4. Time course of hypocotyl elongation in the CR transgenic tobacco plants (0, *) and wild type (w, *). The seeds were kept under white light for 2 days to induce germination and then transferred to dim white light (a, i) or darkness (e, *). phenotype is dependent on the expression of the rice PI transgene. To confirm this, we examined the red/far-red reversibility of the response. First, we irradiated the seed- lings with successive pulses ofred light instead ofcontinuous a)M Ln a- - e In N- a, white light. Preliminary experiments showed that one or two Hypocotyl length, mm Hypocotyl length, mm red light pulses per day resulted in a single peak population with respect to hypocotyl length (A.N., unpublished data). FIG. 5. Hypocotyl length in the BN1 transgenic and wild-type More frequent irradiation, such as nine pulses per day, tobacco plants after treatment with various light pulses. The seeds of a but the seedlings BN1 plants (Left) and the wild type (WT; cv. SR1) (Right) were kept resulted in shorter hypocotyl length under white light for 3 days and then transferred into the dark. Three behaved as a single population. However, six red light pulses min of red light (R), 3 min of red light followed by 9 min of far-red per day gave two populations of seedlings with different light (R/F), 9 min of far-red light (F), or no light (D) was given to the hypocotyl lengths (Fig. 5). The shorter seedlings had a mean plants every 4 hr during the dark incubation. Hypocotyl length was hypocotyl length of 4.6 mm, while their taller siblings had a determined 8 days after the sowing. Numbers of plants tested are mean hypocotyl length of 10 mm. These values were com- indicated in each panel. parable to those found under dim white light (Table 1). The effects of the red light pulses could be reversed by (17, 18). Since we had used the SR1 cultivar oftobacco, while subsequent far-red illumination (Fig. 5). The hypocotyl Keller et al. (17) had used the Xanthi cultivar in their studies length in this case, which was 11.7 mm, was a little less than with oat phytochrome, the differences in our results could the hypocotyl length in the dark, which was 14.1 mm. In have been due to cultivar differences. When Xanthi was used addition, similar reversibility was also observed in the wild as a host for the same CaMV 35S promoter-rice phytochrome type, but to a lesser extent. The latter observation suggests construct, '30% ofthe resulting transgenic Ro population (55 that the shorter hypocotyl length observed in the wild-type plants total) exhibited dwarfism and dark green leaves in plants irradiated with pulses of red light was also phy- adult plants (S.A.K., unpublished data). We cannot yet tochrome-dependent. Thus, the phytochrome-dependent re- account for this difference between these two cultivars. sponse of the seedlings appeared to be exaggerated in those However, when we applied different illumination conditions that overexpressed the rice PI. These data directly demon- to growth, not only CR and CO (derived from cv. strate that the short hypocotyl length is caused by the Xanthi) but also BN1 and BD1 (derived from cv. SR1) clearly overexpression of rice PI in transgenic tobacco. showed a short-hypocotyl phenotype (Table 1, Fig. 2). Thus, in assessing the biological activity of a particular phy- tochrome construct, the developmental stage of the plant as DISCUSSION well as the illumination conditions must be taken into con- In our initial experiments, we demonstrated an effect of sideration. overexpressed rice PI on circadian-regulated Cab gene Because the phenotype observed in CR and CO adult expression (16) but did not observe the morphological phe- plants is pleiotropic, we chose a single phenotype to assay for notypes in mature BN1 plants that had been noted by others rice PI function and found that the short hypocotyl length observed in the young seedlings of the transgenic plants was Table 3. Epidermal cell length of the BN1 transgenic light-intensity-dependent (Fig. 2). This intensity dependence tobacco plants of the phenotype reveals two important points. First, the short hypocotyl length must be directly related to the intro- Hypocotyl Cell length, duced phytochrome and is not due to a general metabolic Condition Plant length, mm Am disturbance caused by overexpression of the introduced Dim light BN1, short 4 222 ± 58 phytochrome. This is further supported by the red/far-red BN1, long 10 479 ± 81 reversibility ofthe phenotype (Fig. 5), which is the first direct Wild type 12 483 ± 106 indication that this phenotype is caused by the introduced Pfr Dark BN1 14 556 ± 87 molecules. Second, the increased amount of phytochrome in Wild type 16 636 ± 99 BN1, BD1, CR, and CO seedlings has sensitized the plants to Epidermal cell length of young seedlings grown under dim white respond to lower light intensities than the wild-type counter- light or in the dark for 5 days was determined by microscopic part. This finding supports the hypothesis that the accumu- observation (x 100). The epidermis was stripped for the observation. lation of high levels of phytochrome in etiolated seedlings is At least 30 cells were measured for each value. a mechanism for amplification of light signals at this crucial Downloaded by guest on September 24, 2021 Botany: Nagatani et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5211 stage of development. The increased sensitivity to light has grown under a range of light intensities. The activity of the physiological implications because it ensures that the plant introduced molecule can be quantitated by fluence-response can switch as quickly as possible from heterotrophic growth analysis. These data, coupled with exact measurements of during germination to photoautotrophic growth. the amount of expressed (spectrally and immunolog- The introduced rice PI appears to induce the short hypo- ically), will allow useful functional comparisons to be made cotyl length via normal physiological processes. This is between the wild-type and mutant phytochrome molecules. supported by two lines of evidence. The first comes from the Likewise, it will be ofparticular interest to study the range of developmental profile of the growing transgenic seedlings. phenotypes produced by overexpression ofdifferent forms of Wild-type seedlings grown in dim light commenced and the molecule, as this will be a powerful tool for their func- terminated elongation at the same stage as the plants in the tional dissection in vivo. dark. However, the elongation rate was reduced in dim light (Fig. 4, o and *). CR seedlings in dim light exhibited a more We thank Mss. Y. Kimura, K. Nakajima, K. Fujiwara, H. Oka- dramatic reduction of growth rate, suggesting that the phy- moto, E. Leheny, and I. Roberson for technical assistance. This tochrome effect was enhanced in CR plants (Fig. 4, 0 and *). work was supported in part by a grant to M.F. from Frontier Second, the epidermal cell length of the BN1 and the wild- Research Program; Grant-in-Aid 01621002 to A.N. from the Ministry type tobacco seedlings was in proportion to the measured of Education, Science, and Culture of Japan, and a grant from The hypocotyl length regardless of light conditions, indicating Rockefeller Foundation to N.-H.C. that the shorter hypocotyl length in dim light was attributable to shorter cell length in both plants (Table 3). However, the 1. Kendrick, R. E. & Kronenberg, G. H. M., eds. (1986) Photo- effect of light on the cell length was enhanced in BN1 plants. morphogenesis in Plants (Nijhoff, Dordrecht, The Nether- The CaMV 35S lands), p. 580. promoter, which was employed in the 2. Furuya, M. ed. (1987) Phytochrome and Photoregulation in present study, is known to potentiate transcription in many Plants (Academic, Tokyo), p. 354. plant cell types (22). Therefore, rice PI may have been 3. Mohr, H. (1966) Photochem. Photobiol. 5, 469-483. expressed in cells that do not normally express phytochrome. 4. Nagy, F., Kay, S. A. & Chua, N.-H. (1988) Trends Genet. 4, Even if this were true, the way in which the introduced 37-42. phytochrome altered the growth does not appear to be very 5. Gilmartin, P. M., Sarokin, L., Memelink, J. & Chua, N. H. different from the normal pathway. As discussed above, the (1990) Plant Cell 2, 369-378. transgenic plants responded to dim white light as the wild 6. Lagarias, J. C. & Rapoport, H. (1980) J. Am. Chem. Soc. 102, type might to light of higher intensity (Fig. 4, Table 3). In 4821-4828. the 7. Coleman, R. A. & Pratt, L. H. (1974) J. Histochem. Cytochem. addition, short-hypocotyl plants under dim white light 22, 1039-1047. were morphologically indistinguishable from the wild-type 8. Butler, W. L., Norris, K. H., Siegelman, H. W. & Hendricks, plants under light of higher intensity. This observation sug- S. B. (1959) Proc. NatI. Acad. Sci. USA 45, 1703-1708. gests that the overall pattern of growth is not greatly altered 9. Tokuhisa, J. G., Daniels, S. M. & Quail, P. H. (1985) Planta in the transgenic plants. 164, 321-332. It is also possible that the expression of the transgene 10. Shimazaki, Y. & Pratt, L. H. (1985) Planta 164, 333-344. encoding rice PI altered the stability of the endogenous 11. Abe, H., Yamamoto, K. T., Nagatani, A. & Furuya, M. (1985) tobacco phytochrome by retarding proteolysis ofthe latter or Plant Cell Physiol. 26, 1387-1399. reducing degradation of its mRNA. Increase in the tobacco 12. Abe, H., Takio, K., Titani, K. & Furuya, M. (1989) Plant Cell phytochrome level could result in an Physiol. 30, 1089-1097. increased sensitivity of 13. Sharrock, R. A. & Quail, P. H. (1989) Genes Dev. 3, 1745- the transgenic plants to light. However, this possibility is 1757. remote because the immunochemical experiments revealed 14. Furuya, M. (1989) Adv. Biophys. 25, 133-167. that in the transgenic plants the accumulation of the endog- 15. Tomizawa, K.-I., Nagatani, A. & Furuya, M. (1990) Photo- enous tobacco phytochrome is not significantly affected by chem. Photobiol. 52, 265-275. the presence of rice PI, as shown in Fig. 3 and also suggested 16. Kay, S. A., Nagatani, A., Keith, B., Deak, M., Furuya, M. & previously (16-18). Chua, N.-H. (1989) Plant Cell 1, 775-782. Taken together, these data suggest that Pfr molecules ofthe 17. Keller, J. M., Shanklin, J., Vierstra, R. D. & Hershey, H. P. introduced rice PI regulate the hypocotyl length of the host (1989) EMBO J. 8, 1005-1012. plants through endogenous 18. Boylan, M. T. & Quail, P. H. (1989) Plant Cell 1, 765-773. signal-transduction pathways. 19. Murashige, T. & Skoog, F. (1962) Physiol. Plant. 15, 473-497. We anticipate that the assay described here can be readily 20. Linsmaier, E. & Skoog, F. (1965) Physiol. Plant. 18, 100-127. used to probe functional domains of the introduced rice PI. 21. Nagatani, A., Kendrick, R. E., Koornneef, M. & Furuya, M. It is now possible to express mutant forms of the protein and (1989) Plant Cell Physiol. 30, 685-690. analyze their effects on the hypocotyl elongation of seedlings 22. Benfey, P. N. & Chua, N.-H. (1989) Science 244, 174-181. Downloaded by guest on September 24, 2021