Rice type I phytochrome regulates hypocotyl elongation in transgenic tobacco seedlings

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 88, pp , June 1991 Botany Rice type I phytochrome regulates hypocotyl elongation in transgenic tobacco seedlings (light regulation/transgenic plants/plant 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 ; and tthe Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY Communicated by Winslow R. Briggs, February 11, 1991 ABSTRACT We have examined the biological activity of rice type I phytochrome (PI) in transgenic tobacco seedlings. The progeny of four independent transformants that expressed the rice PI gene segregated 3:1 for shorter hypocotyl length under dim white light (0.04 W/m2). By contrast, this phenotype was not observed either in the dark or under white light at higher intensity (6.0 W/m2). This suggests that the phenotype is dependent not only on light but also on light intensity. The increased light sensitivity cosegregated with the kanamycin-resistance marker as well as with the rice PI polypeptides, indicating that this phenotype is directly related to the expression of the transgene. The transgenic plants showing short hypocotyls exhibited a reduced growth rate throughout the elongation period, and the resulting shorter hypocotyl length was attributable to shorter epidermal cell length but not to reduced cell number. Furthermore, successive pulse irradiations with red light elicited short hypocotyls similar to those obtained under dim white light, and the effect was reversed by immediate far-red light treatment, providing a direct indication that the phenotype is caused by biologically active rice PI. Therefore, the far-red-absorbing form of the introduced rice PI appears to regulate the hypocotyl length of the transgenic tobacco plants through endogenous signal-transduction pathways. This assay system will be a powerful tool for testing the biological activity of introduced phytochrome molecules. Phytochrome is a regulatory photoreceptor that plays a central role in linking external light signals to developmental responses in plants (1, 2). One of the underlying mechanisms of these developmental responses is the modulation of patterns of gene expression (3), and phytochrome has been shown to regulate the expression of many plant genes (4, 5). An understanding of the molecular mechanism of phytochrome action in vivo is therefore essential to elucidating the complex processes of plant growth and development. Phytochrome is a soluble chromoprotein consisting of an apoprotein (monomer, kda) covalently linked to a linear tetrapyrrole (6) and is located in the cytoplasm in vivo (7). Phytochrome is synthesized in the dark as the red light (Amax = 666 nm)-absorbing form (Pr), which is physiologically inactive. Absorption of red light by Pr converts the molecule to the far-red light (Amax = 730 nm)-absorbing form (Pfr), which is the biologically active conformation, and subsequent irradiation of Pfr with far-red light converts the molecule to the Pr form (8). Phytochrome responses are therefore elicited by red light and attenuated by far-red light. Immunological studies have indicated that more than one type of phytochrome exists in oat (9, 10) and in pea (11). More recently, this has been demonstrated at the molecular level by microsequencing of divergent pea phytochrome polypeptides (12) and by cloning of the divergent phytochrome The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. cdnas from Arabidopsis thaliana (13) and tobacco (S.A.K., M.D., and R. Kern, unpublished data). Thus, we have operationally designated a molecular form of phytochrome that is light-labile and most abundant in dark-grown tissue as type I phytochrome (PI) and one that is much less abundant and relatively stable irrespective of light conditions as type II phytochrome (12, 14, 15). It is possible to express a cloned phytochrome gene as well as its mutant forms in transgenic plants. As previously proposed (4), one can expect either to induce a dominant negative phenotype with a mutant molecule or to create an exaggerated phenotype by overexpression. This kind of experimental system can be readily used to probe functional domains of the introduced phytochrome. Such a system will also be useful in assigning distinct or overlapping roles for different types of the photoreceptor. Recently, we (16) and two other groups (17, 18) have overexpressed a monocot PI gene in a transgenic dicot background. The introduced PI showed normal Pr/Pfr photoconversion and was light-labile. Several resulting phenotypes were also observed; Keller et al. (17) and Boylan and Quail (18) noted semidwarfism and dark green leaves, whereas we observed altered patterns of endogenous Cab gene expression (16). However, the relationship between these phenotypes and the introduced phytochrome molecules has remained unclear. To investigate the utility of this approach further, we have generated several transgenic tobacco lines overexpressing rice PI. To assess the effects of the overexpression, we have designed an assay for hypocotyl length in transgenic tobacco seedlings. Using this assay, we have extended the previous studies by demonstrating that the short-hypocotyl phenotype is not only dependent upon light intensity but also regulated by the introduced rice PI. In addition, we have shown that rice PI influences hypocotyl length by altering cell length but not cell number. MATERIALS AND METHODS Plant Materials. Tobacco plants (Nicotiana tabacum cv. Xanthi) were transformed with the construct containing rice PI cdna fused to the cauliflower mosaic virus (CaMV) 35S promoter as described (16). The transformants, CR and CO, which showed the highest rice PI mrna accumulation among several independent transformants, were used in the present study. Preparation of the tobacco transgenic plants BN1 and BD1 (cv. SR1), which overexpress rice PI, have been described (16). Control transgenic tobacco plants p69 and 4C (cv. Xanthi) were transformed with constructs carrying the CaMV 35S promoter fused to the chloramphenicol acetyltransferase (CAT) gene or 83-glucuronidase (GUS) gene, respectively. Primary transgenic plants were desig- Abbreviations: CaMV, cauliflower mosaic virus; PI, type I phytochrome; Pfr, far-red-absorbing form of phytochrome; Pr, redabsorbing form of phytochrome. 5207

2 5208 Botany: Nagatani et al. nated the Ro generation. The R1 and R2 seeds were obtained by selfing the R0 and R1 plants, respectively. Samples of seeds from individual plants were first tested for kanamycin resistance by germinating the seeds on agar plates containing the antibiotic; seed populations that showed 3:1 segregation for kanamycin resistance were used. Growth Conditions. For hypocotyl length determination, the seeds were sown on 0.5% agar plates containing 0.1x Murashige and Skoog salt mixture (19). The plates were kept under continuous white light (6.0 W/m2) for 2 days to induce seed germination and then subjected to the light treatments for 5 days except where otherwise stated. To check the kanamycin resistance of the young seedlings after the light treatment, plants were transferred to 0.8% agar plates containing kanamycin (0.1 mg/ml) and Murashige's minimal organics medium (20) after the light treatment and grown for days at 250C under continuous white light. For immunoblot analysis, young plants were transferred to soil and grown for about 1 month under a 12 hr/12 hr light/dark cycle in a growth cabinet (Koitotron KG-206HL-D, Koito, Tokyo) at 25TC. The plants were then dark-adapted for 3 days before harvest to increase the amount of phytochrome. Light Sources. White light (6.0 W/m2) for inducing germination and growing plants on the agar plate was from white fluorescent tubes (FL20SS-W/18, Toshiba, Tokyo). Dim white light (0.04 W/m2) was obtained by attenuating the white light with three layers of filter paper (3 MM Chr, Whatman). Red light (0.4 W/m2) and far-red light (0.8 W/m2) were obtained from two fluorescent lamps (red, same as ones for white light; far-red, long-wavelength fluorescent lamp, FL20SFR74, Toshiba, Tokyo) filtered through one layer of acrylic (red, Shinkolite A102, Mitsubishi, Tokyo; far-red, Deraglass 102, Asahikasei, Tokyo). Dim green "safe" light was described elsewhere (21). Intensity of the light was determined with a radiometer (model 4090 radiant-power meter, Springfield Jarco, Yellow Springs, OH). Immunochemical Detection of Phytochrome. Crude extract was prepared from 1.5 g of young leaves of dark-adapted tobacco plants for immunochemical detection of phytochrome (21). The crude extract was concentrated to 0.2 ml Proc. Natl. Acad. Sci. USA 88 (1991) by (NH4)2SO4 precipitation (0.25 g/ml) and a 20-,u1 aliquot was then subjected to NaDodSO4/PAGE. The proteins in the gel were electrophoretically blotted onto nitrocellulose membrane and stained with the anti-rye phytochrome monoclonal antibody (mar14), which stained rice but not tobacco phytochrome (16), and the anti-pea PI monoclonal antibody (map5), which stained tobacco PI but not rice PI (16), as described (21). The extraction procedures were carried out under dim green safe light. RESULTS Segregation of the Short-Hypocotyl Phenotype Under Dim White Light. As described in our previous paper (16), we could not observe a clear morphological phenotype in mature plants of BN1 and several other transgenic tobaccos that accumulated high levels of rice PI. In these previous experiments the SR1 tobacco cultivar was used as the transgenic host. By contrast, the new transgenic lines, such as CR and CO, which were derived from the Xanthi cultivar, clearly showed shorter stem length and dark green leaves when grown under light/dark cycles (Fig. 1). However, these phenotypes were less clear under continuous white light (A.N., unpublished data), suggesting that these phenotypes are dependent on the ambient light intensity. To investigate this phenomenon we chose to examine young transgenic seedlings rather than mature plants, since the morphological variations observed among individual mature plants were usually much greater than those in younger plants. In addition, one can test larger numbers of individual plants in less time with young seedlings. When the R2 progeny of heterozygous R1 BN1 transgenic plants were grown under dim white light, the seedlings showed a 3:1 segregation of a short-hypocotyl phenotype from taller wild-type seedlings (Fig. 2B). Under dim white light, two populations of BN1 seedlings were evident, with mean peak heights of 3 mm and 8 mm. Under the same dim light conditions, control R1 transgenic (p69) and wild-type seedlings gave single populations with a mean peak height of 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.

3 A BN1 60 _ 40 n 40 11l9 2( 20 1 CA 0 g 80P-4 A 0-1 Z WT (SRI) O- 8- * 117 Il Hypocotyl length, mm Botany: Nagatani et al. B C FIG. 2. Hypocotyl elongation in the BN1 transgenic and control tobacco plants grown under different light conditions. Seeds of heterozygous R2 progeny of the BN1 plants were germinated under 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 length was determined 7 days after the sowing. As controls, the p69 plants and wild-type (WT) tobacco (cv. SR1) were tested. Numbers of plants tested are indicated in each panel. as no shorter population was observed when seedlings were grown in darkness (Fig. 2C). Similar segregation of the phenotype under dim white light was observed in several other transformants that overexpressed rice PI (Table 1). By contrast, the segregation was not clear under white light of higher intensity (Fig. 2A), suggesting that the phenotype is also intensity-dependent. To confirm that the phenotype was linked to the introduced gene, young seedlings exhibiting short hypocotyls and their taller siblings were transferred onto kanamycin plates to check the kanamycin resistance of the plants. Table 2 shows that the seedlings exhibited clear cosegregation of antibiotic resistance, which is a genetic marker for the introduced construct (16), with short hypocotyls. Furthermore, the short-hypocotyl phenotype also cosegregated with the accumulation of immunodetectable rice phytochrome polypep- Table 1. Segregation of short-hypocotyl phenotype and kanamycin (Kan) resistance in progenies of transgenic tobacco plants under dim white light Hypocotyl length* Kan resistancet x2 x2 Plants Short (3:1) R S (3:1) Transgenic Rice P1 BN1 83 (2.8) 33 (8.5) BD1 88 (5.3) 31 (13.5) CR 92 (6.0) 26 (12.5) CO 35 (4.4) 12 (9.8) Control p (11.1) > C (8.9) > Wild type SR (10.9) > >100 Xanthi (13.8) > >100 Germinated seedlings were grown under dim white light for 5 days to measure hypocotyl length. For kanamycin-resistance analysis, seeds were germinated and grown under white light on agar plates containing kanamycin. *No. of plants (average hypocotyl length, mm); x2 for 3:1 segregation is also given. tno. of resistant (R) or sensitive (S) plants. Proc. Natl. Acad. Sci. USA 88 (1991) 5209 Table 2. Kanamycin resistance of the progenies of transgenic tobacco plants Kanamycin Hypocotyl resistance* Plant (host) length R S Transgenic Rice PI BN1 (SR1) CR (Xanthi) Control p69 (Xanthi) Wild type SR1 Xanthi Short Short Plants were first tested for hypocotyl growth under dim white light on the agar plate without kanamycin and then transferred onto plates containing kanamycin. *No. of resistant (R) or sensitive (S) plants. tides, as detected by immunoblotting in the CR transgenic plants. As expected, all the CR progeny contained endogenous tobacco phytochrome polypeptides, as detected by immunoblotting with monoclonal antibody map5 (Fig. 3 Left). By contrast, mar14 detected rice phytochrome polypeptides only in the dwarfed progeny of CR and not in long CR or wild-type seedlings (Fig. 3 Right). The intensity of staining with mar14 indicated that the level of rice PI in the CR transgenic plants was comparable to that detected in the BN1 plant (16). Physiological Analysis of the Short-Hypocotyl Phenotype. A time course study of hypocotyl elongation in the CR transgenic plants under dim white light showed that the shorter hypocotyl length was attributable mainly to a slower growth rate throughout the elongation period; there was neither a delayed onset nor advanced cessation of hypocotyl elongation (Fig. 4). To further investigate the underlying cellular basis for the phenotype, we measured the epidermal cell length of the BN1 plants by light microscopy. Table 3 shows that in BN1 seedlings grown under dim light, the longhypocotyl population (10 mm) had a mean cell length of 479,um, whereas the short population (4 mm) had a mean epidermal cell length of only 222 Ztm. These data suggest that expression of the transgene resulted in a reduced hypocotyl length by changing the cell length under dim white light. Red/Far-Red Reversibility of the Response. The results described above strongly suggest that the short-hypocotyl Wild CR Wi] Id CR Type short long Tyype short long kda kda _~~~~q - Stained with map5 Staine -witm*a 4. Stained with mar14 FIG. 3. Detection of rice phytochrome polypeptide in the CR transgenic plants. Adult plants of the R1 progeny of CR plants grown from the seedlings that had been tested for hypocotyl elongation under dim white light were analyzed by immunoblotting with anti-pea phytochrome monoclonal antibody map5 (Left), which detects tobacco phytochrome, and mar14 (Right), which stains rice phytochrome (16). Arrow indicates the position of the 116-kDa marker (B8-galactosidase).

4 5210 Botany: Nagatani et al. Proc. Natl. Acad. Sci. USA 88 (1991) t;15- -* 10- U 0 >1 5- _q.,i - -A Cl) 0 U) 4-0 0) n FIG. 4. V - _ Time after sowing, day 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 seedlings with successive pulses of red light instead of continuous white light. Preliminary experiments showed that one or two red light pulses per day resulted in a single peak population with respect to hypocotyl length (A.N., unpublished data). More frequent irradiation, such as nine pulses per day, resulted in a shorter hypocotyl length but the seedlings behaved as a single population. However, six red light pulses per day gave two populations of seedlings with different hypocotyl lengths (Fig. 5). The shorter seedlings had a mean hypocotyl length of 4.6 mm, while their taller siblings had a mean hypocotyl length of 10 mm. These values were comparable to those found under dim white light (Table 1). The effects of the red light pulses could be reversed by subsequent far-red illumination (Fig. 5). The hypocotyl length in this case, which was 11.7 mm, was a little less than the hypocotyl length in the dark, which was 14.1 mm. In addition, similar reversibility was also observed in the wild type, but to a lesser extent. The latter observation suggests that the shorter hypocotyl length observed in the wild-type plants irradiated with pulses of red light was also phytochrome-dependent. Thus, the phytochrome-dependent response of the seedlings appeared to be exaggerated in those that overexpressed the rice PI. These data directly demonstrate that the short hypocotyl length is caused by the overexpression of rice PI in transgenic tobacco. DISCUSSION In our initial experiments, we demonstrated an effect of overexpressed rice PI on circadian-regulated Cab gene expression (16) but did not observe the morphological phenotypes in mature BN1 plants that had been noted by others Table 3. Epidermal cell length of the BN1 transgenic tobacco plants Hypocotyl Cell length, Condition Plant length, mm Am Dim light BN1, short ± 58 BN1, long ± 81 Wild type ± 106 Dark BN ± 87 Wild type ± 99 Epidermal cell length of young seedlings grown under dim white light or in the dark for 5 days was determined by microscopic observation (x 100). The epidermis was stripped for the observation. At least 30 cells were measured for each value ze z3 a)m Ln a- - e In N- a, FIG. 5. Hypocotyl length in the BN1 transgenic and wild-type tobacco plants after treatment with various light pulses. The seeds of BN1 plants (Left) and the wild type (WT; cv. SR1) (Right) were kept under white light for 3 days and then transferred into the dark. Three min of red light (R), 3 min of red light followed by 9 min of far-red light (R/F), 9 min of far-red light (F), or no light (D) was given to the plants every 4 hr during the dark incubation. Hypocotyl length was determined 8 days after the sowing. Numbers of plants tested are indicated in each panel. (17, 18). Since we had used the SR1 cultivar of tobacco, while Keller et al. (17) had used the Xanthi cultivar in their studies with oat phytochrome, the differences in our results could have been due to cultivar differences. When Xanthi was used as a host for the same CaMV 35S promoter-rice phytochrome construct, '30% of the resulting transgenic Ro population (55 plants total) exhibited dwarfism and dark green leaves in adult plants (S.A.K., unpublished data). We cannot yet account for this difference between these two cultivars. However, when we applied different illumination conditions to seedling growth, not only CR and CO (derived from cv. Xanthi) but also BN1 and BD1 (derived from cv. SR1) clearly showed a short-hypocotyl phenotype (Table 1, Fig. 2). Thus, in assessing the biological activity of a particular phytochrome construct, the developmental stage of the plant as well as the illumination conditions must be taken into consideration. Because the phenotype observed in CR and CO adult plants is pleiotropic, we chose a single phenotype to assay for rice PI function and found that the short hypocotyl length observed in the young seedlings of the transgenic plants was light-intensity-dependent (Fig. 2). This intensity dependence of the phenotype reveals two important points. First, the short hypocotyl length must be directly related to the introduced phytochrome and is not due to a general metabolic disturbance caused by overexpression of the introduced phytochrome. This is further supported by the red/far-red reversibility of the phenotype (Fig. 5), which is the first direct indication that this phenotype is caused by the introduced Pfr molecules. Second, the increased amount of phytochrome in BN1, BD1, CR, and CO seedlings has sensitized the plants to respond to lower light intensities than the wild-type counterpart. This finding supports the hypothesis that the accumulation of high levels of phytochrome in etiolated seedlings is a mechanism for amplification of light signals at this crucial

5 Botany: Nagatani et al. stage of development. The increased sensitivity to light has physiological implications because it ensures that the plant can switch as quickly as possible from heterotrophic growth during germination to photoautotrophic growth. The introduced rice PI appears to induce the short hypocotyl length via normal physiological processes. This is supported by two lines of evidence. The first comes from the developmental profile of the growing transgenic seedlings. Wild-type seedlings grown in dim light commenced and terminated elongation at the same stage as the plants in the dark. However, the elongation rate was reduced in dim light (Fig. 4, o and *). CR seedlings in dim light exhibited a more dramatic reduction of growth rate, suggesting that the phytochrome effect was enhanced in CR plants (Fig. 4, 0 and *). Second, the epidermal cell length of the BN1 and the wildtype tobacco seedlings was in proportion to the measured hypocotyl length regardless of light conditions, indicating that the shorter hypocotyl length in dim light was attributable to shorter cell length in both plants (Table 3). However, the effect of light on the cell length was enhanced in BN1 plants. The CaMV 35S promoter, which was employed in the present study, is known to potentiate transcription in many plant cell types (22). Therefore, rice PI may have been expressed in cells that do not normally express phytochrome. Even if this were true, the way in which the introduced phytochrome altered the growth does not appear to be very different from the normal pathway. As discussed above, the transgenic plants responded to dim white light as the wild type might to light of higher intensity (Fig. 4, Table 3). In addition, the short-hypocotyl plants under dim white light were morphologically indistinguishable from the wild-type plants under light of higher intensity. This observation suggests that the overall pattern of growth is not greatly altered in the transgenic plants. It is also possible that the expression of the transgene encoding rice PI altered the stability of the endogenous tobacco phytochrome by retarding proteolysis of the latter or reducing degradation of its mrna. Increase in the tobacco phytochrome level could result in an increased sensitivity of the transgenic plants to light. However, this possibility is remote because the immunochemical experiments revealed that in the transgenic plants the accumulation of the endogenous tobacco phytochrome is not significantly affected by the presence of rice PI, as shown in Fig. 3 and also suggested previously (16-18). Taken together, these data suggest that Pfr molecules of the introduced rice PI regulate the hypocotyl length of the host plants through endogenous signal-transduction pathways. We anticipate that the assay described here can be readily used to probe functional domains of the introduced rice PI. It is now possible to express mutant forms of the protein and analyze their effects on the hypocotyl elongation of seedlings Proc. Natl. Acad. Sci. USA 88 (1991) 5211 grown under a range of light intensities. The activity of the introduced molecule can be quantitated by fluence-response analysis. These data, coupled with exact measurements of the amount of expressed protein (spectrally and immunologically), will allow useful functional comparisons to be made between the wild-type and mutant phytochrome molecules. Likewise, it will be of particular interest to study the range of phenotypes produced by overexpression of different forms of the molecule, as this will be a powerful tool for their functional dissection in vivo. We thank Mss. Y. Kimura, K. Nakajima, K. Fujiwara, H. Okamoto, E. Leheny, and I. Roberson for technical assistance. This work was supported in part by a grant to M.F. from Frontier Research Program; Grant-in-Aid to A.N. from the Ministry of Education, Science, and Culture of Japan, and a grant from The Rockefeller Foundation to N.-H.C. 1. Kendrick, R. E. & Kronenberg, G. H. M., eds. (1986) Photomorphogenesis in Plants (Nijhoff, Dordrecht, The Netherlands), p Furuya, M. ed. (1987) Phytochrome and Photoregulation in Plants (Academic, Tokyo), p Mohr, H. (1966) Photochem. Photobiol. 5, Nagy, F., Kay, S. A. & Chua, N.-H. (1988) Trends Genet. 4, Gilmartin, P. M., Sarokin, L., Memelink, J. & Chua, N. H. (1990) Plant Cell 2, Lagarias, J. C. & Rapoport, H. (1980) J. Am. Chem. 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