Planta (1991)185 :162-170

P l a n t a 9 Springer-Verlag1991

Photoresponses of transgenic tobacco plants expressing an oat phytochrome gene Alex C. McCormac, Joel R. Cherry*, Howard P. Hershey**, Richard D. Vierstra*, and Harry Smith Department of Botany, University of Leicester, Leicester LEI 7RH, UK Received 2 March; accepted 9 May 1991

Abstract. The physiological responses of transgenic tobacco ( N i c o t i a n a t a b a c u m L.) plants that express high levels of an introduced oat ( A r e n a s a t i v a L.) phytochrome ( p h y A ) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005--1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental F R light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both W T and transgenic seed, but addition of F R to the W L treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction of fluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form o f phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of darkgrown seedlings of both W T and transgenic lines was inhibited by continuous R or F R irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the W T seedlings had lost the FRmediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, F R - H I R response mediated by the overexpressed oat PhyA phytochrome. Light-grown W T seedlings exhibited typical shade-avoidance responses when treated with W L supplemented with high levels of FR radiation. * Present addresses: Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, and ** Agricultural Products Department, E.I. du Pont de Nemours & Co., P.O. Box 80402, Wilmington, DE 19880-0402, USA Abbreviations: FR = far-red light; PAR = photosynthetically active radiation; Pr, Pfr = red- and FR-absorbing forms of phytochrome; Ptot = total phytochrome; PhyA (PhyA)= gene (encoded protein) for phytochrome; R = red light; WL = white light; WT = wild type

Internode and petiole extension rates were markedly increased, and the chlorophyll a: b ratio decreased, in the low-R: F R treatment. The transgenics, however, showed no increases in extension growth under low-R: F R treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R : F R . Interpretation of these data is difficult. The depression of the chlorophyll a: b ratio by low R: F R was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat p h y A gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles. Key words: Gene (oat p h y A ) - Light (red: far red ratio) - N i c o t i a n a (transgenic, photoresponses) - Phytochrome - gene (in transgenic plant)

Introduction Phytochrome is a family of plant photoreceptors, encoded by at least three types of nuclear genes (Sharrock and Quail 1989). The members of the phytochrome family are responsible for acquiring from the natural light environment information that is crucially important for the normal development of the plant at all stages of its life-cycle (for a review, see Smith 1982). The realisation that phytochrome is encoded by a family of genes has led to the proposal that the members of the family may have differential roles in the regulation of development and metabolism (Sharrock and Quail 1989; Smith and Whitelam 1990; Smith et al. 1991). This concept offers the prospect o f resolving some o f the conflicts created by physiological investigations which have identified several different modes of phytochrome regulation of development, a multiplicity of control irreconcilable on the basis of a single photoreceptor. Direct evidence on whether or not different phytochromes have different roles is, how-

163

A.C. McCormac et al. : Photoresponses of transgenic tobacco ever, as yet unavailable. In this article, we address this question by the analysis o f the physiological responses of transgenic tobacco plants that constitutively express an introduced cereal p h y t o c h r o m e gene. We use here the terminology recently adopted regarding the naming of the different p h y t o c h r o m e genes and the gene products (see T h o m a s and Johnson 1991). Thus, phyA refers to the gene, and P h y A to the functional protein encoded by that gene. O f the three phy genes characterised by Sharrock and Quail (1989) in Arabidopsis, phyA was shown to be homologous with the genes previously characterised in oats (Hershey et al. 1985), rice ( K a y et al. 1989a) and peas (Sato 1988) and which are reasonably assumed to code for the species of phytochrome that accumulates in etiolated tissues, is downregulated by exposure to light, and is unstable in the far-red absorbing (Pfr) form. This form of p h y t o c h r o m e is variously known as Type I, or etiolated-tissue phytochrome, and can be distinguished from other forms of p h y t o c h r o m e on immunochemical grounds (Abe et al. 1985; Shimazaki and Pratt 1985; Tokuhisa and Quail 1985). It is not justified, however, to use the terms PhyA and Type I interchangeably, except where the identity of the gene product has been demonstrated; this restriction is adhered to here. Three reports have appeared of transgenic plants that express introduced cereal phyA genes. Keller et al. (1989) introduced an oat phyA gene into transgenic tobacco, Boylan and Quail (1989) introduced an oat phyA gene into transgenic tomato, and K a y et al. (1989b) introduced a rice phyA gene into transgenic tobacco. In all three cases, constitutive p r o m o t e r s were used, and high levels of expression of the introduced phyA genes were observed. In the oat gene transformations, m a j o r morphological phenotypic changes were observed, with transgenic plants exhibiting m a r k e d dwarfism and increased leaf and, for tomato, fruit pigmentation (Keller et al. 1989; Boylan and Quail 1989). Transgenic tobacco expressing the rice phyA gene did not exhibit a similar morphological phenotype, although alterations in photoregulated gene expression were observed (Kay et al. 1989b). The dwarfism caused by expression of an introduced phyA gene was interpreted by Keller et al. (1989) and Boylan and Quail (1989) as p r o b a b l y being a consequence of the abnormally high levels of functional Pfr established in the light-grown transgenic plants. This view is consistent with recent speculation (see, for example, Smith and Whitelam 1990; Smith et al. 1991) that Type I phytochrome, which accumulates in etiolated tissues to high levels, operates as an antenna, allowing rapid and sensitive detection of the a p p r o a c h of a seedling tip to the soil surface and the initiation of the developmental change towards the p h o t o a u t o t r o p h i c growth habit. I f this is so, then forms of p h y t o c h r o m e other than Type I m a y be responsible for the perception of the ratio o f red to far-red light (R: FR) in light-grown plants, and for the p h e n o m e n a of neighbour detection, proximity perception and shade avoidance that are of m a j o r importance in adaptation to the natural light environment (Holmes and Smith 1975; M o r g a n and Smith 1978; Smith 1982; Ballar6 et al. 1987, 1990; Casal and

Smith 1989; Smith et al 1990). On this basis, the lightlability of Type I Pfr, and the down-regulation of Type I synthesis in the light, result in the rapid loss of Type I on de-etiolation to levels at which it presumably cannot interfere with R : F R perception by other p h y t o c h r o m e s normally present at very low concentrations. The heterologous P h y A in transgenic plants appears not to be subject to down-regulation of synthesis (Keller et al. 1989; Boylan and Quail 1989), presumably because o f the constitutive p r o m o t e r used, and therefore its level remains high even after de-etiolation. The fact that introduced PhyA is functional in an heterologous transgenic situation provides an opportunity to probe the physiological role o f this f o r m of phytochrome, and to begin to address the question o f where all phytochromes have interchangeable, or distinct, roles. In the experiments reported here, seeds, etiolated seedlings, and light-grown plants f r o m a transgenic tobacco line expressing high levels of oat p h y t o c h r o m e (Keller et al. 1989) have been subjected to physiological tests which reveal m a j o r perturbations o f the normal photobiological responses, some of which are difficult to interpret on the basis of current models. Materials and methods

Plant material. Seed used were of the wild-type (WT) Nicotiana tabacum L., cv. xanthi, and an homozygous isogenic line which had been transformed with the oat phyA gene fused to the constitutive cauliflower mosaic virus (CaMV-35S) promoter using standard Agrobacterium procedures (Keller et al. 1989), and referred to in the following text as the 9A4 line. Seed used in these experiments had been stored at room temperature for at least three months prior to sowing and were allowed to imbibe at 25~ C in white light (WL) on water-saturated filter paper. For use in experiments with lightgrown plants, germinated seed were transferred to potting compost and grown under continuous WL from fluorescent tubes (photosynthetically active radiation (PAR) = 130 ~mol photons, m -2 9s-1) at 25~ C until the third true leaf pair had expanded (approx. five weeks after sowing). At this stage plants were potted into 300-cm3 pots and transferred to the light treatments indicated.

Light sources. Red light (R) (total fluence rate 600- 700 nrn = 4.8 I~mol9m -2 9s- x) for inhibiton of seedling hypocotyl growth was provided by filtering the radiation from Thorn EMI (Birmingham, UK) Deluxe Natural 40-W fluorescent tubes through 1-cm-deep copper sulphate solution (1.5 % w/v) and one red (N o. 14) Cinemoid sheet (Rank Strand, Isleworth, Middlesex, UK). Far-red light (FR) (total fluence rate 700--800nm = l l g m o l - m - 2 . s - X ) was provided by water-cooled 100-W incandescent bulbs with a black acrylic filter (Plexiglas Type FRF 700; West Lake Plastics, Lem, Penn., USA). Two pairs of R: FR-treatment cabinets of similar design were used in this work, one used at low and one at high irradiances. In both pairs of cabinets, background WL was provided by banks of fluorescent tubes, and additional FR by filtering the radiation from interspersed banks of incandescent bulbs through combinations of red and green plastic filters, the radiant infra-red being removed by flowing "water windows". The design allows for uniform levels of PAR (400-700 nm) and wide ranges of R: FR. In the high-PAR cabinets, background WL was provided by 24 80-W Cool White fluorescent tubes, and FR was provided by filtering the radiation from 24 500-W Philips (Turnhout, Belgium) 7785R quartz-halogen lamps through 4 cm of cooled flowing water, one layer (3 ram) of red (Number 4400), and one layer of green (Number 6600) Perspe• (SBA, Leicester, UK). In the low-PAR cabinets, WL was from eight 40-W Cool White fluorescent tubes,

164

A.C. McCormac et al. : Photoresponses of transgenic tobacco ogy were observed in approx. 5% of the 9A4 population and these were excluded from calculations. R : FR 0.07 6.8 0.27 5,4

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(1)

Pfr/Ptot 0.31 0.71 0.55 0.71

Measurement of seedling hypocotyl growth. Dry seed were sown in a horizontal line on water-saturated 10" 12 cm 2 W h a t m a n (Maidstone, Kent., UK) 3MM paper. This was placed on a 3-ram-thick perspex plate and held vertically in a water-filled growth tray. The seed were allowed to imbibe for 24 h in the dark at 25 ~ C and then transferred to 4 ~ C for 10 d, still maintained in darkness. The seed were then either germinated at 25~ in WL ("light-grown seedlings") or maintained in darkness for a further 5 d ("dark-grown seedlings"). Subsequently, seedlings were transferred to continuous darkness, R or F R treatments for 48 h after which hypocotyl lengths were measured using a ruler to an accuracy of 0.5 mm. In the case of dark-grown seedlings, owing to non-uniformity of germination times, hypocotyl length was measured under a dim green light before and after the light treatments and growth expressed as the increase in length; only seedlings greater than 3 m m in length at the beginning of the light treatments were included. Light-grown seedlings were measured only after the R or F R period and growth was expressed as absolute hypocotyl length. Each replicate consisted of 20-25 seedlings and mean values were calculated from five such replicates.

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Fig, 1. Spectral photon distributions of the light sources used for the mature-plant experiments. R : F R ratios of 6.8 (WL; dashed lines) and 0.07 (WL + F R ; solid lines) were delivered at P A R fluence rates (total integrated over the 400- to 700-nm wavelength interval) of 160, 75 and 55 pmol 9 m -2 9 s -1. Using separate light sources, R : F R ratios of 5.4 (WL; dotted lines) and 0.27 ( W L + F R ; dashed-dotted lines) were delivered at a P A R fluence rate of 20/tmol 9 m -2 9 s-1. R: FR was calculated as the ratio of photon fluence rates within the 654- to 664-nm and the 724- to 734-nm wavebands

and F R from eight quartz-halogen lamps using 4 cm of water and a single layer of red (No 14) and green (No 24A) Cinemoid as filters. Changes in fluence rate whilst maintaining a constant R : FR were achieved by altering the distance from the overhead light source (i.e. shelf height) within the growth cabinet.

Light measurements and Pfr/Ptot calculations. Fluence rates and spectral distributions of light sources were recorded by placing the cosine-corrected remote probe of a calibrated LI-1800 spectroradiometer (LI-COR, Lincoln, Neb. USA) horizontally at plant height. The P A R was measured as total photon fluence rate between 400 and 700 nm, and R: F R was calculated as the ratio of fluence rates over the 654- to 664-nm and 724- to 734-nm wavelength intervals. Phytochrome photoequilibrium (i.e., Pfr/Ptot calculations were performed by the method outlined by Hayward (1984), using a computer program which integrates the spectral photon-fluencerate data with the absorption coefficients and quantum efficiencies of photoconversion for the red-absorbing (Pr) and FR-absorbing/ Pfr forms of oat Type I phytochrome. The spectra, R: F R ratios and calculated phytochrome photoequilibria for the R:FR-ratio-treatment light cabinets are shown in Fig. 1. The corresponding integrated fluence rates for P A R are given in the text.

Measurements of plant morphology. All measurements were made two weeks after the beginning of the light treatments (when the seventh leaf pair was expanding) except for leaf angle (degrees from vertical) which was measured after 4 d. Plant height, internode length and petiole length were measured using a ruler divided to an accuracy of 1 mm. All leaf mesurements were recorded from the fifth pair; leaf area was measured using a LI-COR LI-3050A portable area meter. Total dry weight was measured after 48 h at 80 ~ C. Transgenic plants which appeared to have reverted to WT morphol-

Germination assay. Wild-type and transgenic seed were sown onto water-saturated W h a t m a n filter paper in 9-cm Petri dishes and immediately placed under the appropriate light conditions. The light treatments consisted of continuous WL illumination with and without supplementary FR, giving R : F R values of 0.07 and 6.8, respectively, and delivered at a PAR fluence rate of 160 or 55 ~tmol. m - 2 . s 1, respectively. Other seed were maintained in complete darkness apart from occasional observation under dim green light. Germination was assessed as emergence of the radicle or cotyledons after 10 d.

Measurement of chlorophyll levels. Chlorophyll was extracted from 0.16-cm 2 leaf discs by immersion in 1 cm 3 N,N-dimethylformamide for 48 h in the dark at 4 ~ C. Absorbance was read at 664 and 647 nm and chlorophyll a and b concentrations were calculated according to Moran (1982). hmmmochemical analysis of phytoehrome. Seedlings, grown on damp filter paper (Whatman 3MM) in the dark at 25 ~ C, were homogenised in buffer (1 cm 3 - gin- 1 fresh weight) of 50% (v/v) ethylene glycol in 100 m M (Tris[hydroxymethyl]amino-methane)HC1 (pH 7.8) containing 140 mM ammonium sulphate, 10 m M EDTA, 20 m M NazSO3, and 5 mM phenylmethylsulphonyl fluoride. After clarification by centrifugation, the supernatant was boiled for 2 rain with an equal volume of double-strength sodium dodecyl sulphate (SDS) sample buffer. Equal volumes were separated on an 8% SDS-acrylamide gel and electroblotted onto nitrocellulose (Hybond-C; Amersham International, Amersham, Bucks., UK). Phytochrome bands were detected by incubation with anti-phytochrome monoclonal antibodies which either specifically recognised monocotyledon phytochrome (antibody LAS41-mouse IgG1, raised against purified phytochrome from etiolated oats) or cross-reacted with both moncotyledon and dicotyledon phytochromes (antibody LAS32-mouse IgG1, raised against oat phytochrome) (Holdsworth 1987). Bands were visualised by secondary incubation with alkaline-phosphatase-conjugated goat anti-mouseimmunoglobulin antibodies and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) staining.

Results

Three principal comparisons of the photoresponses of w i l d - t y p e a n d t r a n s g e n i c t o b a c c o e x p r e s s i n g h i g h levels of oat phytochrome were performed. These comparisons

A.C. McCormac et al. : Photoresponses of transgenic tobacco

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Fig. 2. Effect of supplementary FR on WL-stimulated germination of WT and transgenic 9A4 tobacco seed. Percentage germination was assessed after dark-imbibed seed had been exposed to continuous illumination for 10d. R : F R ratios of 6.8 (WL) and (WL + FR) were delivered at PAR fluence rates of 160 (H) and 55 (L) gmol - m -2 9s- 1. Under W L + FR irradiation, FR fluence rates (integrated over the 700- to 800-nm wavelength interval) were 500 (H) and 172 (L)/tmol 9m -2 9s -1. Each value is the mean + S D of 3 sets of 50 seeds

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Fig. 4. Detection of oat phytochrome in transgenic tobacco by immunoblot analysis. Extractions of etiolated WT tobacco (WT) and the transformed line (9A4) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resulting electroblots were incubated with monoclonal antibodies either specific to oat phytochrome or capable of recognising both the introduced oat phytochrome and that of the host tobacco. Samples loaded in each lane contained equal amounts of total protein. Minor bands detected are considered to be degradation products of native phytochrome

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Fig. 3. Relative effectiveness of R and FR irradiation for the inhibition of hypocotyl extension in etiolated (dark-grown) and de-etiolated (WL-grown) seedlings of WT (circles) and transgenic 9A4 (squares) tobacco. Values shown are the means :t: SE of 5 independent replicates of 20 seedlings

were selected to p r o b e the p o t e n t i a l p h y s i o l o g i c a l role o f the c o n s t i t u t i v e l y e x p r e s s e d P h y A in: (a), the r e g u l a t i o n o f seed g e r m i n a t i o n ; (b), the p e r s i s t e n c e o f a n F R m e d i a t e d h i g h - i r r a d i a n c e i n h i b i t i o n o f h y p o c o t y l extension in d e - e t i o l a t e d seedlings; a n d (c), the r e s p o n s e s o f l i g h t - g r o w n p l a n t s to the R : F R o f c o n t i n u o u s i r r a d i a tion.

(a) Light requirements for germination G e r m i n a t i o n o f b o t h W T a n d 9 A 4 seed was m a r k e d l y s t i m u l a t e d u n d e r W L c o m p a r e d to u n b r o k e n d a r k n e s s ;

over 80% o f all v i a b l e seed g e r m i n a t e d w i t h i n 5 d o f i m b i b i t i o n u n d e r W L c o m p a r e d to less t h a n 10% a f t e r two weeks w h e n m a i n t a i n e d in the d a r k . T h e r e was no significant effect o f v a r y i n g P A R fiuence r a t e b e t w e e n 55 a n d 160 g m o l ' m - 2 . s -1. G e r m i n a t i o n u n d e r c o n t i n u o u s F R - s u p p l e m e n t e d light ( R : F R = 0 . 0 7 ) was significantly d e p r e s s e d in b o t h W T a n d t r a n s g e n i c seed c o m p a r e d to n o n - s u p p l e m e n t e d w h i t e light ( R : F R = 6.8) at P A R = 160 ~tmol 9m z 9 s - t (Fig. 2). H o w e v e r , r e d u c tion & P A R to 55 g m o l 9m -2 9s -1, w i t h a c o n c o m i t a n t d e c r e a s e in F R - w a v e l e n g t h s t h e r e b y m a i n t a i n i n g R : F R at 0.07 ( i n t e g r a t e d fluence r a t e f o r 700-800 n m was red u c e d f r o m 500 to 172 g m o l 9m - z s - t ) , a c t e d to relieve this i n h i b i t i o n in the 9A4, b u t n o t W T seed (Fig. 2).

(b) Inhibition of hypocotyl 9rowth under continuous R and FR E x t e n s i o n - g r o w t h rates o f d a r k - g r o w n seedlings o f W T a n d 9 A 4 lines were s u b s t a n t i a l l y r e d u c e d w h e n i r r a d i a t e d w i t h c o n t i n u o u s R a n d F R w a v e l e n g t h s (Fig. 3). L i g h t - g r o w n seedlings o f the W T t o b a c c o d i s p l a y e d little o r no i n h i b i t i o n o f g r o w t h b y F R , b u t were inh i b i t e d b y R. T h e 9 A 4 seedlings, h o w e v e r , r e t a i n e d sensitivity to c o n t i n u o u s F R i r r a d i a t i o n f o l l o w i n g W L t r e a t m e n t (Fig. 3). T h e r e was n o m e a s u r a b l e difference between the a c t u a l rates o f h y p o c o t y l e x t e n s i o n in d a r k n e s s o f W T a n d t r a n s g e n i c e t i o l a t e d seedlings (2.9 4- 0.667 a n d 2.4 4- 0.567 m m 9 d - 1 respectively).

166 Table 1. Comparison of the morphology of the mature light-grown plants of WT N. tabacum var. xanthi and the oat-phyAexpressing 9A4 line. Light-grown plants were exposed for two weeks to continuous WL ( R : F R =6.8) at the PAR fluence rates shown. Measurements were taken when the plants were approx, seven weeks old, i.e. the seventh leaf pair was starting to expand. All leaf measurements were taken from the fifth leaf pair which was fully expanded at time of measurement. Values shown are the mean (+SE) of five replicates of five plants each

A.C. McCormac et al.: Photoresponses of transgenic tobacco PAR (lamol'm Z.s 1)

Character

160 75 55 160 Leaf area (cm 2) 75 55 160 Total leaf FW (mg) 75 55 160 FW density (mg-cm -2) 75 55 160 Total leaf DW (mg) 75 55 160 DW density (mg 9c m 2) 75 55 Total chlorophyll (lag cm -2) 160 75 55

Stem height (mm)

(c) Comparison of W T and transgenic green-plant morphology

Genotype WT

9A4

143.1 (9.82) 186.7 (10.33) 205.5 (6.20) 67.2 (4.54) 79.1 (5.32) 75.9 (4.97) 1977.8 (136.25) 2196.6 (92.57) 1979.8 (143.10) 29.2 (0.99) 27.1 (0.93) 26.0 (1.11) 300.1 (24.51) 204.1 (11.24) 139.7 (9.59) 4.5 (0.25) 2.6 (0.13) 1.9 (0.08) 51.6 (3.05) 72.0 (2.57) 61.1 (2.21)

88.2 (2.34) 91.1 (8.28) 88.4 (9.11) 44.2 (2.51) 56.4 (2.39) 56.8 (3.43) 1465.0 (99.77) 1703.3 (157.25) 1651.0 (109.97) 32.3 (0.77) 30.2 (0.78) 29.6 (0.74) 226.8 (17.47) 1 3 6 . 7 (10.68) 1 0 0 . 4 (5.69) 5.0 (0.25) 2.4 (0.16) 1.7 (0.08) 71.5 (3.11) 83.0 (3.01) 70.0 (2.78)

80

General growth characteristics. T h e 9 A 4 t o b a c c o line was s h o w n b y K e l l e r et al. (1989) to h a v e e l e v a t e d levels o f s p e c t r o p h o t o m e t r i c a l l y active p h y t o c h r o m e c o m p a r e d to the W T in b o t h d a r k a n d l i g h t - g r o w n seedlings. I m m u n o b l o t a n a l y s i s has c o n f i r m e d t h a t this was also true for the t r a n s g e n i c p l a n t s used here, the p h y t o c h r o m e c o d e d for b y the i n t r o d u c e d o a t phyA gene being identified on i m m u n o b l o t s by a m o n o c l o n a l a n t i b o d y specific for o a t T y p e I p h y t o c h r o m e (Fig. 4). This effect on levels o f t o t a l p h y t o c h r o m e is a s s o c i a t e d with a distinctive m o r p h o l o g y o f the m a t u r e l i g h t - g r o w n p l a n t w h e n c o m p a r e d to t h a t o f the W T , m o s t n o t a b l e b e i n g the r e d u c e d stem height ( T a b l e 1 a n d C h e r r y et al. 1991). This is d u e to a reduction in i n t e r n o d e e x t e n s i o n (Fig. 5), r a t h e r t h a n i n t e r n o d e n u m b e r , as n o difference in the n u m b e r o f leaves was a p p a r e n t . T h e t r a n s g e n i c p l a n t s h a d s h o r t e r petioles (Fig. 6) a n d i n c r e a s e d c h l o r o p h y l l p e r unit l e a f a r e a ( T a b l e 1). T h e l e a f a r e a o f the 9 A 4 p l a n t s was also r e d u c e d w i t h a c o r r e s p o n d i n g decrease in t o t a l fresh weight, b u t a m o r e dense l a m i n a was m e a s u r e d as inc r e a s e d fresh w e i g h t p e r u n i t area. T h o s e traits unaffected b y the i n c r e a s e d p h y t o c h r o m e e x p r e s s i o n i n c l u d e d l e a f d r y - w e i g h t d e n s i t y ( T a b l e 1) a n d the c h l o r o p h y l l a : b r a t i o (Fig. 7).

Growth response to added FR. W i l d - t y p e p l a n t s d i s p l a y e d a large a n d r a p i d d e v e l o p m e n t a l r e s p o n s e to the a d d i t i o n o f F R w a v e l e n g t h s to b a c k g r o u n d W L ( R : F R = 0.07), the a n g l e o f l e a f o r i e n t a t i o n b e c o m i n g m o r e vertical by a p p r o x . 50 ~ w i t h i n the initial 24 h o f F R - i r r a d i a t i o n .

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Fig. 5. Internode extension of WT (circles) and transgenic 9A4 (squares) tobacco seedlings grown under continuous illumination with WL plus or minus additional FR. Two irradiation cabinets were used. One cabinet delivered fluences rates (400-700 nm) of 55, 75, or 160 # m o l - m - 2 . s 1 at an R : F R ratio of either 6.8 (open symbols) or 0.07 (closedsymbols). The other cabinet delivered a fluence rate (400-700 nm) of 20/~mol 9m-2 . S - 1 at an R : F R ratio of either 5.4 (open symbols) or 0.27 (closed symbols). General conditions of growth were not uniform between the two cabinets and hence direct comparison of mean values from each has no significance. The length of the internode above the ninth leaf was measured after two weeks growth in the respective treatments. Values shown are the means _+SE of five replicates of five plants each

A.C. McCormac et al.: Photoresponses of transgenic tobacco

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Fig. 6. Petiole extension of WT

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(squares) tobacco seedlings grown under continuous illumination with WL plus or minus additional FR. Treatments were as described in Fig. 5. Petiole length of the fifth leaf pair was measured after two weeks growth in the respective treatments. Values shown are the means + SE of five replicates of five plants each

Other developmental effects included an approximately threefold increase in internode length (Fig. 5) and petiole extension (Fig. 6), decreased chlorophyll content (51.6 gg 9cm -2 and 28.8 lag 9 cm -1 in W L and W L + FR, respectively) and a lowered chlorophyll a: b ratio (Fig. 7) relative to those plants maintained in W L ( R : F R = 6.8). There were no significant changes in leaf area or fresh weight (data not shown). These responses are typical of those reported during shade avoidance for a n u m b e r of other species. In contrast, plants of the 9A4 line, grown at the higher o f the fluence rates ( P A R = 160 lamol" m -2" s -1) showed little or no growth response to l o w - R : F R treatment (Figs. 5, 6). Internode growth under additional F R was not significantly different c o m p a r e d to that in WL, there was no change in leaf angle, and chlorophyll levels decreased only 20 % in contrast to 45% in the WT. Interaction between fluence rate and R: FR. Wild-type plants under W L showed a significant trend towards increased internode length and elongated petioles as fluence rate was decreased from 160 to 55 gmol" m -2 ' s- 1; those under FR-supplemented light showed no significant response to fluence rate (Figs. 5, 6). But consistently, within all three fluence-rate treatments, growth o f W T plants was stimulated by the addition of F R wavelengths. Plants of the 9A4 line displayed a similar trend under W L conditions (primarily in petiole length) but became increasingly dwarfed as the fluence rate of the F R supplemented light was reduced (Figs. 5, 6). The net result, therefore, was that at P A R = 55 g m o l . m -z -s -1 there was a highly significant ( P < 0 . 0 0 1 ) depression of

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Fig. 7. Chlorophyll a: b ratio in leaves of WT (circles) and transgenic 9A4 (squares) tobacco seedlings grown under continuous illumination with WL plus or minus additional FR. Treatments were as described in Fig. 5. Total chlorophyll was extracted from the fifth

leaf pair after two weeks growth in the respective treatments, and the ratio of chlorophyll a:b calculated. Values shown are the means + SE of five replicates of five plants each the extension growth of the transgenic p h y t o c h r o m e expressers in response to lowered R : F R . This FR-mediated inhibition of extension growth was also observed in a separate experiment in which the R: F R quality of the light source was varied between 5.0 and 0.27, delivered at a P A R value of 20 lamol 9m - 2. s - 1 (Figs. 5, 6). Chlorophyll a." b ratio. Both W T and 9A4 lines exhibited identical decreases in chlorophyll a : b ratio under R : F R = 0 . 0 7 c o m p a r e d to R : F R = 6 . 8 (Fig. 7). This response was unaffected by fluence rate between 55 and 160 lamol, m - 2 - s -1 in both lines. An R : F R ratio of 0.27, delivered at a P A R value of 20 lamol 9 m - 2 . S - 1 however, proved ineffective in altering the chlorophyll a: b ratio relative to plants grown at an R: F R of 5.4, and this was seen in both W T and transgenic lines (Fig. 7).

Discussion The results of this series o f experiments demonstrate both expected and unexpected effects of the existence of supranormal levels o f a Type I p h y t o c h r o m e in transgenic plants. The fact that heterologous p h y t o c h r o m e m a y be functional in transgenic plants has already been demonstrated (Keller et al. 1989; Boylan and Quail 1989; K a y et al. 1989b), and has encouraged studies o f the detailed effects on the photophysiology of the transgenic seedlings (Cherry et al. 1991). Most of the observations reported here are interpretable on the basis of the persistence of the heterologous oat p h y t o c h r o m e after exposure of the transgenic tobacco seedlings to continuous WL. In the

168 WT plants, exposure to WL, and the consequent deetiolation, leads to a massive fall in the levels of Type I phytochrome, but in the transgenics, the use of a constitutive promoter presumably disrupts the normal down-regulation of phytochrome synthesis, allowing the persistence of high levels of oat PhyA in the light-grown plants (Cherry et al. 1991). The phenotypic effects of high levels of expression of an introduced phyA gene may be observed in three separate phenomena: changed photosensitivity of seed germination, the persistence of an FR-mediated response in de-etiolated seedlings resembling an etiolated-seedling high-irradiance response (HIR), and major perturbations of the responses of lightgrown plants to R : F R .

Photosensitivity of seed 9ermination. Both the WT and the transgenic 9A4 seed in this study germinated poorly in darkness, but were stimulated to maximum germination by exposure to continuous WL (Fig. 2); this is presumably a consequence of the establishment, in both lines of light-treated seed, of levels of Pfr that are above the saturation level for germination. Addition of FR to the continuous WL, providing an R: FR of 0.07, suppressed germination in both WT and 9A4 lines, a response that would be expected on the basis of the FR-mediated HIR inhibition of germination described by Bartley and Frankland (1982). In the WT, all FR fluence rates tested (172-500 ~tmol 9m - 2 . s-1) gave maximum inhibition of germination, whereas in the transgenics, reduction of FR fluence rate to 172 ~tmol 9m - 2 . S - 1 (at constant R: FR) partially relieved the inhibition of germination (Fig. 2). Bartley and Frankland (1982) proposed that the HIR inhibition of germination is dependent on two antagonistic components, one a function of Pfr concentration, and the other (known as H) a function of phytochrome cycling rate. Germination is promoted by high Pfr, and inhibited by high H. This model satisfactorily accounts for the behaviour of the transgenic 9A4 line if it is assumed that the heterologous oat phytochrome is capable of regulating germination. With much higher levels of total phytochrome, the concentration of Pfr in the transgenic seed would be much higher than in the WT at the same R : F R ; consequently, a higher cycling rate (H) would be required to achieve the same degree of inhibition of germination in the transgenic seed. These results indicate that transgenic phytochrome expressers will prove to be an excellent test for the Frankland model of the HIR regulation of germination. Persistence of FR-mediated 9rowth inhibition in de-etiolated seedlings. Perhaps the only photomorphogenic response that may be definitively attributed to the lightlabile Type I phytochrome is the FR-mediated HIR (Smith et al. 1991). The FR HIR can only be understood if it is mediated by a species of phytochrome that is unstable in the Pfr form, and this concept forms the basis of the principal hypothesis proposed to account for the phenomenon (Hartmann 1966). Thus, the F R - H I R is normally only observed in etiolated seedlings that contain high levels of light-labile Type I phytochrome (Mancinelli 1980). Light-grown plants commonly retain an

A.C. McCormacet al. : Photoresponses of transgenic tobacco R-mediated HIR, but lose the F R - H I R (Beggs et al. 1980; Holmes et al. 1982). In these experiments, continuous low-fluence-rate R and FR inhibited hypocotyl extension of etiolated seedlings of both WT and 9A4 lines (Fig. 3), typical of the HIR observed in many other species. Exposure of WT seedlings to 48 h of WL resulted in the loss of the FR-mediated inhibition of extension, although R continued to be inhibitory, as reported for mustard by Beggs et al. (1980). In contrast, de-etiolated transgenic seedlings continued to show inhibition of extension growth in response to prolonged FR irradiation (Fig. 3). These results are most simply interpreted on the basis of the retention in the transgenics of an F R - H I R mediated by the high levels of oat PhyA. This conclusion must be regarded as preliminary, however, since it is not yet known whether the heterologous oat PhyA is subject to first-order degradation of Pfr, a prerequisite of the Hartmann (1966) theory of the HIR. The observation of comparable rates of hypocotyl extension in etiolated seedlings grown in darkness, despite a 4.8-fold higher level of phytochrome in the etiolated transgenics compared to the WT (Cherry et al. 1991), provides support for the view that only Pfr is physiologically active in etiolated plants. As concluded from similar studies of phytochrome-deficient mutants (Adamse et al. 1988), it appears that the concentration of Pr does not directly influence extension rate in etiolated seedlings.

Response of light-grown plants to R: FR. The characteristic responses of light-grown plants to variations in R : F R have been amply described and shown to be mediated by phytochrome (Smith 1982; Casal and Smith 1989). In particular, shade-avoiding species exhibit marked acceleration of internode and petiole elongation when exposed to broad-band radiation rich in FR, as present within vegetation canopies (Morgan and Smith 1976, 1981). The WT tobacco in this study exhibited a strong shade-avoidance response, with up to a threefold increase in extension rate at R : F R = 0.07, compared to that at R: F R = 6.8 (Figs. 5, 6). The extension growth of the transgenic 9A4 line, however, was not stimulated by low R : F R at any PAR fluence rate used. This could be interpreted most simply in terms of the ninefold excess of phytochrome in the de-etiolated transgenics compared to the WT (Cherry et al. 1990). Moreover, at low fluence rates, the extension rate of the transgenic plants not only failed to be stimulated by lowered R: FR, but was significantly depressed (Figs. 5, 6). In no other case has a depression of extension rate by low R: FR been reported; even shade-tolerating species normally show weak extension-rate increases in response to low R: FR (Smith and Morgan 1983). These results are not easily interpreted on the existing data. One possibility that deserves further investigation is that the persistence of PhyA in lightgrown plants facilitates the continuation of photoresponses normally observed only in etiolated seedlings. One such response is the characteristic FR-mediated HIR, which might be expected to operate antagonistically to the normal stimulation of extension growth by

A.C. McCormac et al. : Photoresponses of transgenic tobacco supplementary F R , nullifying, or even reversing, shadeavoidance responses. O n this basis it m i g h t be speculated that, during n o r m a l development, removal o f P h y A during de-etiolation is i m p o r t a n t because it p r e s u m a b l y allows one (or more) other p h y t o c h r o m e species to operate as the R : F R sensor for shade avoidance. These results do n o t d e m o n s t r a t e definitely the persistence o f an F R - H I R in de-etiolated transgenic phyA expressers, but they are consistent with that idea. H o w e v e r , the direction o f the fluence-rate response o f 9A4 plants in F R supplemented light, i.e. increased dwarfism at decreased fluence rates, was the opposite o f that expected if a s t a n d a r d F R - H I R was operative, a result which as yet remains unexplained. One c o m p o n e n t o f the shade-avoidance s y n d r o m e that was unaffected by the presence o f the o a t phyA gene in the transgenic t o b a c c o was the chlorophyll a : b ratio (Fig. 7). This result is particularly i m p o r t a n t because it indicates that P h y A is n o t capable o f exercising c o n t r o l over all the p h e n o m e n a that are regulated by p h y t o c h r o m e in the light-grown plant. By extension, this implies that a n o t h e r f o r m o f p h y t o c h r o m e is responsible. Conclusions. A full p h o t o p h y s i o l o g i c a l analysis o f the transgenic p h y t o c h r o m e expressers will be a m a j o r task, and consequently these results should be regarded as preliminary indications. However, a l t h o u g h the results are n o t all amenable to simple interpretation, they are consistent with, but do n o t prove, a n u m b e r o f speculations that are currently in fashion. These include: (a) the F R - H I R is mediated by a P h y A ; (b) P h y A m u s t be r e m o v e d during de-etiolation in order that n e i g h b o u r detection a n d shade avoidance can operate effectively; (c) different species o f p h y t o c h r o m e m a y have different physiological roles. F u r t h e r investigation o f this last question will be assisted by the construction o f transgenic plants that overexpress, or underexpress, individual representatives o f the p h y t o c h r o m e gene family. This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.C.M. ; the production of the transgenic seed was funded by the U.S. Department of Energy (DE-F602-88ERI3968) to R.D.V., and by E.I. du Pont de Nemours; Dr. G.C. Whitelam is thanked for the provision of monoclonal antibodies for the immunoblot analyses.

References Abe, H., Yamamoto, K.T., Nagatani, A., Furuya, M. (1985) Characterization of green tissue-specific phytochrome isolated immunochemically from pea seedlings. Plant Cell Physiol. 26, 1387-1399 Adamse, P., Kendrick, R.E., Koorneef, M. (1988) Photomorphogenetic mutants of higher plants. Photochem. Photobiol. 48, 833-841 Ballar~, C.L., S~mchez, R.A., Scopel, A.L., Casal, J.J., Ghersa, C.M. (1987) Early detection of neighbour plants by phytochrome perception of spectral changes in reflected sunlight. Plant Cell Environ. 10, 551-557 Ballar6, C.L., Scopel, A.L., S~tnchez, R.A. (1990) Far red radiation reflected from adjacent leaves: an early signal of competition in plant canopies. Science 247, 329-332

169 Bartley, M.R., Frankland, B. (1982) Analysis of the dual role of phytochrome in the photoinhibition of seed germination. Nature 300, 750-752 Beggs, C.J., Holmes, M.G., Jabben, M., Sch/ifer, E. (1980) Action spectra for the inhibition of hypocotyl growth by continuous irradiation in light- and dark-grown Sinapis alba L. seedlings. Plant Physiol. 66, 615-618 Boylan, M.T., Quail, P.H. (1989) Oat phytochrome is biologically active in transgenic tomatoes. Plant Cell 1, 765 773 Casal, J.J., Smith, H. (1989) The function, action and adaptive significance of phytochrome in light-grown plants. Plant Cell Environ. 12, 855-862 Cherry, J.R., Hershey, H.P., Vierstra, R.D. (1991) Characterization of tobacco expressing functional oat phytochrome: Domains responsible for the rapid degradation of Pfr are conserved between monocots and dicots. Plant Physiol. (in press) Hartmann, K.M. (1966) A general hypothesis to interpret 'high energy phenomena' of photomorphogenesis on the basis of phytochrome. Photochem. Photobiol. g, 349-366 Hayward, P.M. (1984) Determination of phytochrome parameters from radiation measurements. In: Techniques in photomorphogenesis, pp. 159-173, Smith, H., Holmes, M.G., eds. Academic Press, London Hershey, H.P., Baker, R.F., Idler, K.B., Lissemore, J.L., Quail, P.H. (1985) Analysis of cloned cDNA and genomic sequences for phytochrome : Complete amino acid sequences for two gene products expressed in etiolated Arena. Nucleic Acids Res. 135, 8543-8559 Holdsworth, M.L. (1987). Characterisation of phytochrome using monoclonal antibodies. Ph.D. thesis, University of Leicester Holmes, M.G., Smith, H. (1975) The function of phytochrome in plants growing in the natural environment. Nature 254, 512514 Holmes, M.G., Beggs, C.J., Jabben, M., Sch/ifer, E. (1982) Hypocotyl growth in Sinapis alba L. : the roles of light quality and quantity. Plant Cell Environ. 5, 45-51 Kay, S.A., Keith, B., Shinozaki, K., Chye, M.-L., Chua, N.-H. (1989a) The rice phytochrome gene; structure, autoregulated expression, and binding of GT 1 to a conserved site in the 5' upstream region. Plant Cell l, 351-360 Kay, S.A., Nagatani, A., Keith, B., Deak, M., Furuya, M., Chua, N.-H. (1989b) Rice phytochrome is biologically active in transgenic tobacco. Plant Cell 1, 775-782 Keller, J., Shanklin, J., Vierstra, R.D., Hershey, H.P. (1989) Expression of a functional monocotyledonous phytochrome in transgenic tobacco. EMBO J. 8, 1005-1012 Mancinelli, A.L. (1980) The photoreceptors of the high irradiance responses of plant photomorphogenesis. Photochem. Photobiol. 32, 853-857 Moran, R. (1982) Formulae for determination of chlorophyllous pigments extracted with N,N-dimethylformamide. Plant Physiol. 69, 1376-1381 Morgan, D.C., Smith, H. (1976) Linear relationship between phytochrome photoequilibrium and growth in plants under simulated natural radiation. Nature 262, 210-212 Morgan, D.C., Smith, H. (1978) The relationship between phytochrome photoequilibrium and development in light grown Chenopodium album L. Planta 142, 187-193 Morgan, D.C., Smith, H. (1981) Non-photosynthetic responses to light quality. In: Encyclopedia of plant physiology, N.S., vol. 12A: Physiological plant ecology I, Lange, O.L., Nobel, P.S., Osmond, C.B., Ziegler, H., eds. Springer pp. 109 134, Berlin Heidelberg New York Sato, N. (1988) Nucleotide sequence and expression of the phytochrome gene in Pisum sativum: Differential regulation by light of multiple transcripts. Plant Mol. Biol. 11, 697-710 Sharrock, R.A., Quail, P.H. (1989) Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family, Genes Devel. 3, 1745-1757 Shimazaki, Y., Pratt, L.H. (1985) Immunochemical detection with

170 rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green A r e n a shoots. Planta 164, 333-344 Smith, H. Morgan, D.C. (1983) The function of phytochrome in Nature. In: Encyclopedia of plant physiology (N.S., vol. 16: Photomorphogenesis, pp. 491-517, Shropshire, W. Jr., Mohr, H . , eds. Springer, Berlin Heidelberg New York Smith, H., Whitelam, G.C. (1990) Phytochrome, a family of photoreceptors with multiple physiological roles. Plant Cell Environ. 13, 695-707 Smith, H., Casal, J.J., Jackson, G.M. (1990) reflection signals and the perception by phytochrome of the proximity of neighbouring vegetation. Plant Cell Environ. 13, 73-78

A.C. McCormac et al. : Photoresponses of transgenic tobacco Smith, H., Whitelam, G.C., McCormac, A.C. (1991) Do the members of the phytochrome family have different roles? Physiological evidence from wild-type, mutant and transgenic plants. In: Phytochrome properties and biological action, Thomas, B., Johnson, C.B., eds. Springer, Heidelberg (in press) Smith, H. (1982) Light quality, photoperception and plant strategy. Annu Rev Plant Physiol. 33, 481-518 Thomas, B., Johnson, C.B. (eds.) (1991) Phytochrome properties and biological action. Springer, Heidelberg (in press) Tokuhisa, J.G., Daniels, S.M., Quail, P.H. (1985) Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in lightgrown Arena sativa L. Planta 164, 321-332