Planta (Berl.) 124, 319--328 (1975) 9 by Springer-Verlag 1975
Red Light Induced Production of Gibberellin-like Substances in Homogenates of Etiolated Wheat Leaves and in Suspensions of Intact Etioplasts* 1%.J. Cooke**, P. F. Saunders, and R. E. Kendrick*** Department of Botany and Microbiology, University College of Wales, Aberystwyth, SY23 3DA, U.K. and ***Department of Plant Biology, The University, Newcastle upon Tyne, NE1 7RU, U.K. Received 19 April; accepted 29 April 1975 Summary. I-tomogenates of etiolated wheat leaves contain increased levels of acidic gibberellin (GA)-like substances following treatment with red light. Differential centrifugation of homogenates indicates that the response is confined to the 1000 g (or plastid) fraction. Irradiation of suspensions of intact etioplasts also increases the level of extractable GA-like activity. Phytochrome can be detected spectrophotometrically in preparations of etioplasts. The response in etioplasts can be inhibited by chloramphenicol, but not by cycloheximide, and partially by Amo 1618. The GA-like substances produced in etioplasts seem capable of passing into the surrounding medium within 20 rain.
Introduction W h e n segments of etiolated barley or wheat leaves are irradiated with red light, a rapid increase in the level of extractable gibberellin-like (GA-like) activity occurs (Reid et al., 1968; Beevers et al., 1970; Loveys and Wareing, 1971). This increase is t h o u g h t to be due to the stimulation of de novo GA synthesis and to the release of active GAs from a " b o u n d " form (Reid and Clements, 1968; Loveys and Wareing, 1971). Irradiation of homogenates of etiolated barley leaves has similarly been shown to increase the level of subsequently extractable GA-like activity (Reid et al., 1972). The production of GA-like substances in isolated wheat etioplasts is under p h y t o e h r o m e control (Cooke and Saunders, 1975). I n this paper we further characterise the GA production in homogenates of wheat leaves and demonstrate spectrophotometrically t h a t p h y t o c h r o m e is associated with isolated etioplasts. Materials and Methods Wheat fruits (Triticum aestivum, cv. Kolibri) were sown in moist vermiculite and grown in the dark for 7 days at 25 ~ C. Harvest and preparation of the tissue was performed under a dim green safelight. For the initial experiment, homogenates of 8 cm segments of leaf tissue were prepared in the dark using cold phosphate: citrate buffer, pH 6.0 (Reid et al., 1972). The technique of * Abbreviation: GA = GibbereIlin. ** Present address: Department of Plant Biology, The University, Newcastle upon Tyne,
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James and Das (1957) was adapted for differential centrifugation of homogenates, prepared in a medium as before but including 0.4 5{ sucrose and 0.01 5{ KC1. Centrifugation was carried out in the dark using an MSE 18 centrifuge, first at 1000 g for 10 rain and subsequently at 16 000 g for 15 rain. The pellets obtained in each case were washed in fresh extraction medium ~nd re-centrifuged at the appropriate speed. The temperature was maintained at 4~ throughout. The pellets were re-suspended before irradiation. Intact etioplasts were prepared from segments of leaves using a Sephadex G-50 column method (Wellburn and Wellburn, 1971). Fractions eluting from the column containing etioplasts were bulked, allowed to warm to 25~ and irradiated in glass dishes. The light sources have been described previously (Beevers et al., 1970; van Staden and Wareing, 1972). At the end of the light treatments, ice-cold absolute methanol was added to the extracts to give a final methanol concentration of 80%. Extracts were stirred for 30 min at 4 ~C, the methanol was removed in vacuo and the acidic and non-acidic ethyl acetate soluble fractions were obtained by methods commonly used in this laboratory (see Loveys and Wareing, 1971, for example). Ethyl acetate soluble fractions were then taken to dryness in vacuo and ehromatographed on 0.25 mm layers of silica gel GF 254 using the solvent system chloroform/ethyl acetate/acetic acid, 50:50: ]. After being thoroughly air-dried, chromatograms were divided into 10 equal Rf zones and bioassayed using the lettuce hypocotyl assay of Frankland and Wareing (1960). The lettuce variety "Artic King" was used. Phytochrome estimations were obtained at 0 ~ C using a modified Pcrkin-Elmer 156 dualwavelength spectrophotometer and calcium carbonate as a scattering agent (Kendrick and Smith, in press).
Results and Discussion E x p e r i m e n t s with Lea] Homogenates Fig. 1 illustrates the results of a n e x p e r i m e n t performed to e x a m i n e whether homogenates of etiolated wheat leaf tissue will respond to red light t r e a t m e n t i n a n analogous m a n n e r to t h a t reported b y R e i d et al., (1972) for barley. A h o m o g e n a t e of 30 g of leaf segments was prepared i n darkness, half oi which was t h e n i r r a d i a t e d with red light for 20 rain. I t is a p p a r e n t from Fig. 1 t h a t i I r a d i a t i o n increases the level of GA-like a c t i v i t y extractable from homogenates of wheat leaves. The GA-like substances present have similar Rf values to those reported i n barley leaf homogenates (Reid et al., 1972).
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Fig. 1A and B. The effect of red light irradiation on the level of GA-like activity extractable from homogenates of 15 g of etiolated wheat leaves. Lettuce hypocotyl bioassays of the acidic ethyl acetate soluble fractions following thin-layer chromatography. A Dark; B 20rain red light. Shaded portions of the histograms represent significant promotion at the 1% level of probability
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On the basis of this result, it was decided to fractionate the homogenate and examine each fraction for an effect of red light irradiation. 40 g of leaf tissue was homogenised and the 1000 g pellet, 16000 g pellet and supernatant obtained by eentrifugation in the dark. After re-suspending the pellets in fresh extraction medium, half of each fraction was given 20 rain of red light. The GA-like activity in each fraction was then determined and the results of the bioassays are presented in Fig. 2. I t is clear that although all three fractions of the homogenate appear to contain GA-]ike activity, the response to red light is confined almost entirely to the 1000 g fraction. This fraction will contain mainly plastids and thus it is reasonable to assume that the red light effect on GA levels in wheat leaves is largely mediated through the etioplasts. I t has previously been shown that suspensions of isolated intact etioplasts contain increased levels of GA-like substances following irradiation with red light and that this can be reversed by subsequent far-red light. This indicates phytoehrome control of GA production at the level of the etioplast in wheat leaves (Cooke and Saunders, 1975). Chromatographic evidence that the GA-like substances produced in suspensions of etioplasts are the same as those produced in leaf segments following red light was obtained in the following manner. A preparation of etioplasts was obtained from 80 g of wheat leaves. At the same time, 20 g of leaf segments were harvested. Half of each of these samples was irradiated, the etioplast suspension for 20 min with continuous red light and the leaf segments for 5 rain with red light, followed by 10 min in darkness (Loveys and Wareing, 1971). The acidic 22*
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Fig. 3A--H. A comparison of the GA-like substances present in l0 g of etiolated wheat leaves and in suspensions of etioplasts isolated from 40 g of wheat leaves. The acidic (A--D) and non-acidic (E--H) ethyl aceta.te soluble fractions were obtained from each extract, ehromatographed and bioassayed using the lettuce hypocotyl test. A, C, E and G leaf extracts; B, D, F and tI etioplast extracts. A, B, E and F Dark; C and G 5 rain red light, 10 min dark; D and H 20 rain red light. Shaded portions of the histograms represent significant promotion at the 1% level of probability
and non-acidic ethyl acetate soluble fractions were then partitioned from each extract. The techniques used for the extraction of the leaf segments have been described elsewhere (Loveys and Wareing, 1971). All of the fractions were ehromatographed and bioassayed. The chromatographic similarity of the acidic GA-like substances extractable from etioplasts and from leaf segments following red light treatment is apparent from Fig. 3. I n addition, at least part of the nonacidic GA-like activity present in dark-grown leaves would appear to be similar to t h a t occurring in intact etioplasts before irradiation. The polar non-acidic GAlike component present in dark-grown wheat leaves has been shown to be a " b o u n d " form of the hormone (Loveys and Wareing, 1971). I t could thus be suggested that the similar GA-like substance present in etioplasts prior to irradiation also represents a " b o u n d " form of GA. One effect of red light on suspensions of etioplasts is to cause the release of free, acidic GA-like substances from this "bound" form. Thus, the red light induced increase in GA levels in etiolated wheat leaves appears to be largely mediated through the etioplasts.
Experiments with Suspensions o/Etioplasts Fig. 4a is a representative electron micrograph of a portion of a typical etioplast suspension prepared by the technique of Wellburn and Wellbnrn (1971).
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Fig. 4. Electron micrographs of a portion of a typical etioplast preparation obtained using the technique of Wellburn and Wellbum (1971). a magnification • 10000, b magnification • 40000. Bar represents 1 tzm in each ease
The preparation can be seen to consist predominantly of etioplasts. Although not 100% intact, the majority of the etioplasts appear to possess a double outer membrane. There is very little contaminating material evident. The higher power micrograph (Fig. 4b) shows the internal fine structure of a typical etioplast. The stroma contains the prolamellar body in the paracrystalline state, in addition to densely staining "osmiophilie" granules and ribosomes. Similar preparations to this were used in the following experiments. I t has previously been reported that GA production in suspensions of isolated etioplasts is under phytoehrome control (Cooke and Saunders, 1975). Thus, it follows t h a t phytochrome must be associated with etioplasts. A suspension of etioplasts prepared in the normal manner was centrifuged for 10 min at 1000 g and the pellet obtained was resuspended in 2.0 ml of the extraction medium. This was then placed on a bed of calcium carbonate in a 1 em path length cuvette, mixed and the resultant suspension examined in a dual-wavelength spectrophotometer. Using measuring beams of 660 nm and 730 rim, it was possible to detect photo-reversible changes in the absorbanee of the sample following irradiation with actinic red and far-red light. A difference spectrum of the phytochrome associated with etioplasts is presented in Fig. 5. The peak position in the red region of the spectrum occurs at 660 nm and in the far-red region at 725 nm. The effect of pre-incubation with inhibitors of various kinds on the GA production induced by red light was investigated. Etioplasts were prepared as above and an equal volume of a 0.2 mg m1-1 solution of ehloramphenicol, eyeloheximide or Amo 1618 added to the suspension. Distilled water was added
324
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Fig. 5. Difference spectrum of the phytochrome present in a suspension of etioplasts prepared from 160 g of wheat leaves. The etioplasts were centrifuged down, re-suspended in 2.0 ml of extraction medium and the sample measured in a modified Perkin-Elmer 156 dualwavelength spectrophotometer. CaCOs was used as a scattering agent
to the control suspensions and all of the suspensions were kept in the dark for one hour. At the end of this time, half of each preparation was irradiated with red light for 20 rain. The acidic ethyl acetate soluble fraction was obtained from each sample, ehromatographed and bioassayed. The results are given in Fig. 6, from which it is clear that whereas chloramphenicol completely inhibits red light stimulated production of GA-like substances, cycloheximide has little or no effect. Similar findings were reported by Reid et al., (1968), working with barley leaf segments. Amo 1618 appears to inhibit the production of the slower running peak of GA-like activity, whereas the other peak is unaffected. In the preceding experiments, no a t t e m p t was made to remove the inhibitor from the extracts prior to partitioning with ethyl acetate and thus any apparent effect on gibberellin levels could be due to the inhibitor running at the same Rf in the solvent system used and masking any GA-like activity. This applies particularly, perhaps, to the apparently differential effect of Amo 1618. To examine this possibility, the acidic ethyl acetate soluble fraction was prepared from 20 ml of a 0.2 mg m1-1 solution of Amo 1618, chromatogral0hed and bioassayed. One ml of a 1 ~zg m1-1 solution of GA 3 was added to each assay dish and it was found that the acidic ethyl acetate soluble fraction of Amo 1618 suppressed the growth of the lettuce hypocotyls induced by the added GA~ by less than 5 %. Moreover, the suppression was evident from Rf 0.1 to Rf 1.0 and thus it is unlikely that the effect of Amo 1618 in the present instance is due to the presence of the inhibitor at a particular Rf. The results of experiments with selective inhibitors of protein synthesis support the hypothesis that the plastid is the site of the red light effect. Chloramlohenicol, an inhibitor thought to act principally on plastid ribosomes, prevents the increase in gibberellin levels, whereas cycloheximide, which is believed to act mainly on cytoplasmic ribosomes, does not affect the red light
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response. Although doubts exist as to the specificity of these inhibitors (MacDonald et al., t966 ; MacDonald and Ellis, 1969), these results may indicate that protein synthesis is a pre-requisite of the response in etioplasts. The effect of Amo 1618 closely parallels that which we have found in wheat leaf tissue vacuum infiltrated with the inhibitor prior to irradiation and suggests t h a t de novo gibberellin synthesis m a y be at least partly involved in the red light effect. Suspensions of isolated chloroplasts and etioplasts have been shown to be capable of synthesising a wide range of compounds (Kindl, 1971 ; Wellburn et al., 1973; Buggy et al., 1974). Furthermore, several light-enhanced processes are known to occur in isolated etioplasts, including flavonoid synthesis (Weissenbock et al., 1972}, protein synthesis (Hearing, 1973) and synthesis of 6-methylsalicylic acid (Kannangara et al., 1971). There is substantial evidence for an association between gibberellins and chloroplasts in various species (Stoddart, 1968; Railton and Wareing, 1973; F r y d m a n and Wareing, 1973; gailton and Reid, 1974) and thus it m a y be that the plastids are the site of considerable gibberellinsynthesising capacity. An experiment was carried out to determine whether or not the gibberellins produced in etioplasts in response to red light remain within the etioplasts or pass into the surrounding medium. I n t a c t etioplasts were obtained from 120 g of leaf tissue and the suspension was divided into three samples. One of these served as a dark control and the other two were both given 5 rain of red light.
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One of the irradiated samples was left in darkness for 15 min whereas the other was left for 5 min in the dark and then centrifuged at 1000 g for 5 min, again with light excluded. The supernat&nt was decanted and following a further 5 rain in the dark, the pellet obtained from this centrifugation was resuspended in fresh extraction medium. The acidic GA-like substances were then partitioned from all of the samples. Fig. 7 shows that a large proportion of the GA-like activity produced by an etioplast suspension in response to red light is located in the supernatant following centrifugation, indicating that GA-like substances can pass fairly rapidly out of the etioplast following their production. The two peaks of acidic GA-like activity detectable on thin-layer chromatogr&ms of extracts seem to differ somewhat in their ability to pass h'om the etioplast. The faster running peak appears entirely in the supcrnatant following irradiation, whereas some of the activity of the slower running peak is retained within the etioplast pellet. Examination of a portion of the pellet obtained from the centrifugation at 1000 g of an irradiated etioplast suspension, using phase-contrast microscopy, indicated t h a t the majority of the etiopl&sts were still intact. Also, when a suspension of etioplasts, prepared in the dark, was given a 5 min centrifugation at 1000 g, no GA-like activity could be detected in either the pellet or the supernatant. Thus, it seems likely that the appearance of GA-like activity in the supernatant following irradiation of etioplast preparations is due to the red light treatment and not to rupture of the plastids during centrifugation. Since the plastid envelope is generally believed to be impermeable to terpenoids and related compounds, which would include gibberellins (Rogers et al., 1965), the results of this experiment suggest that red light irradiation alters the properties of the etioplast membrane, allowing GAs to be released. One current
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t h e o r y of t h e m e c h a n i s m of a c t i o n of p h y t o c h r o m e is t h a t a n e a r l y consequence of p h o t o t r a n s f o r m a t i o n involves a change in t h e functional p r o p e r t i e s of membranes of various kinds (Hendricks a n d B o r t h w i c k , 1967; Smith, 1970; Boisard et al., 1974). I t is t h u s t e m p t i n g to assume t h a t p a r t of t h e p h y t o e h r o m e associated with e t i o p l a s t s is l o c a t e d either in or on t h e e t i o p l a s t envelope, where it could induce changes in t h e p e r m e a b i l i t y of t h e envelope to GAs. The p h y t o chrome control of G A p r o d u c t i o n in etioplasts, however, is more c o m p l e x a n d involves control of t h e release of GAs from a " b o u n d " form as well as control of de novo G A biosynthesis. E x p e r i m e n t s are a t p r e s e n t being c o n d u c t e d to e l u c i d a t e these points of p h y t o e h r o m e control. R. J.C. acknowledges the award of a Science Research Council Stndentship and postdoctoral Fellowship. The assistance of Mr. R. Hewitt, University of Newcastle upon Tyne, in obtaining the electron micrographs is also gratefully acknowledged.
References Beevers, L., Loveys, B., Pearson, J. A., Wareing, P. F. : Phytochrome and hormonal control of expansion and greening of etiolated wheat leaves. Planta (Berl.) 90, 286-297 (1970) Boisard, J., Marm~, D., Briggs, W . R . : I n vivo properties of membrane-bound phytochrome. Plant Physiol. 54, 272-276 (1974) Buggy, M. J., Britton, G., Goodwin, T. W. : Terpenoid biosynthesis by chloroplasts isolated in organic solvents. Phytochem. III, 125-129 (1974) Cooke, R . J . , Saunders, P. F. : Phyteehrome-mediated changes in extractable gibberellin activity in a cell-free system from etiolated wheat leaves. Planta (Berl.) 128, 299-302 (1975) Frankland, B., Wareing, P. F.: Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature (Lond.) 185, 255-256 (1960) Frydman, V.F., Wareing, P . F . : Phase change in Hedera helix L. L Gibberellin-like substances in the two growth phases. J. exp. Bot. 24, 1131-1138 (1973) Hearing, V. J. : Protein synthesis in isolated etioplasts after light stimulation. Phytochem. 12, 277-282 (1973) Hendricks, S. B., Borthwick, It. A. : The function of phytochrome in the regulation of plant growth. Proc. nat. Acad. Sci. (Wash.) 58, 2125-2130 (1967) James, W. 0., Das, V. S. R.: The organisation of respiration in chlorophyllous cells. New Phytol. 56, 325-345 (1957) Kannangara, C. G., Henningsen, K. W., Stumpf, P. K., vonWettstein, D. : 6-methylsalicylic acid synthesis by isolated barley chloroplasts. Europ. J. Biochem. 21, 344-348 (1971) Kendrick, R. E., Smith, It.: The assay and isolation of phytochrome. In: Chemistry and biochemistry of plant pigments (Goodwin, T. W., ed.), 2nd edit. London and New York: Academic Press (in press) Kindl, H. : Die Bildung eines Stilbens in isolierten Chloroplasten. Hoppe-Seylers Z. physiol. Chem. 852, 767-768 (1971) Loveys, B. R., Wareing, P. F. : The red light controlled production of gibberellin in etiolated wheat leaves. Planta (Berl.) 98, 109 116 (1971) MacDonald, I . R . , Bacon, J. S.D., Vaughan, D., Ellis, R . J . : The relation between ion absorption and protein synthesis in beet disks. J. exp. Bot. 17, 822-837 (1966) MacDonald, I. R., Ellis, R. J. : Does cycloheximide inhibit protein synthesis specifically in plant tissue ? Nature (Lond.) 222, 791-792 (t969) Railton, I. D., Reid, D. M.: Studies on gibberellins in shoots of light-grown peas. I. A reevaluation of the data. Plant Sci. Left. 2, 157-163 (1974) Railton, I. D., Wareing, P. F. : Effect of daylength on endogenous gibberellins in Solanum andigena. I I L Gibberellin production by the leaves. Physiol. Plant. (Copenh.) 29, 430-434 (1973)
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Reid, D. M., Clements, J. B. : RNA and protein synthesis: pre-requisites of red light induced gibberellin synthesis. Nature (Lond.) 219, 607-609 (1968) Reid, D. M., Clements, J. B., Carr, D. J. : Red light induction of gibberellin synthesis in leaves. :Nature (Lond.) 217, 580-582 (1968) Reid, D. M., Tuing, M. S., Durley, R. C., Railton, I. D.: Red-light-enhanced conversion of tritiated gibberellin A, into other gibberellin-like substances in homogenates of etiolated barley leaves. Planta (Berl.) 108, 67-75 (1972) Rogers, L. J., Shah, S. P. g., Goodwin, T. W. : Intracellular loealisation o~ mevalonic kinase in germinating seedlings: its importance in the regulation of terpenoid biosynthesis. Bioehem. J. 96, 7-8 (1965) Smith, H.: Phytochrome and photomorphogenesis in plants. Nature (Lond.) 227, 665-668 (1970) Staden, J. van, Wareing, P. F. : The effect of light on endogenous eytokinin levels in seeds of R u m e x obtusi/olius. Planta (Berl.) 104, 126-133 (1972) Stoddart, J. L. : The association of gibberellin-like activity with the chloroplast fraction o~ leaf homogenates. Planta (Berl.) 81, 106-112 (1968) Weissenbock, G., Fleing, I., Ruppel, H. G. : Untersuchungenzur Lokalisation yon Flavonoiden in Plas$iden. I. Flavonoide in Etioplasten yon Avena sativa L. Z. Naturforsch. 27, 1216-1224 (1972) Wellburn, A. R., Ashby, J. P., Wellburn, F. A. M. : Occurrence and biosynthesis of adenosine 3', 5' cyclic monophosphate in isolated Avena etioplasts. Biochim. biophys. Acts (Amst.) 320, 363-371 (1973) Wellburn, A. R., Wellburn, F. A.M.: A new method for the isolation of etioplasts with intact envelopes. J. exp. Bot. 23, 972-979 (1971)
Red Light Induced Production of Gibberellin-like Substances in Homogenates of Etiolated Wheat Leaves and in Suspensions of Intact Etioplasts* 1%.J. Cooke**, P. F. Saunders, and R. E. Kendrick*** Department of Botany and Microbiology, University College of Wales, Aberystwyth, SY23 3DA, U.K. and ***Department of Plant Biology, The University, Newcastle upon Tyne, NE1 7RU, U.K. Received 19 April; accepted 29 April 1975 Summary. I-tomogenates of etiolated wheat leaves contain increased levels of acidic gibberellin (GA)-like substances following treatment with red light. Differential centrifugation of homogenates indicates that the response is confined to the 1000 g (or plastid) fraction. Irradiation of suspensions of intact etioplasts also increases the level of extractable GA-like activity. Phytochrome can be detected spectrophotometrically in preparations of etioplasts. The response in etioplasts can be inhibited by chloramphenicol, but not by cycloheximide, and partially by Amo 1618. The GA-like substances produced in etioplasts seem capable of passing into the surrounding medium within 20 rain.
Introduction W h e n segments of etiolated barley or wheat leaves are irradiated with red light, a rapid increase in the level of extractable gibberellin-like (GA-like) activity occurs (Reid et al., 1968; Beevers et al., 1970; Loveys and Wareing, 1971). This increase is t h o u g h t to be due to the stimulation of de novo GA synthesis and to the release of active GAs from a " b o u n d " form (Reid and Clements, 1968; Loveys and Wareing, 1971). Irradiation of homogenates of etiolated barley leaves has similarly been shown to increase the level of subsequently extractable GA-like activity (Reid et al., 1972). The production of GA-like substances in isolated wheat etioplasts is under p h y t o e h r o m e control (Cooke and Saunders, 1975). I n this paper we further characterise the GA production in homogenates of wheat leaves and demonstrate spectrophotometrically t h a t p h y t o c h r o m e is associated with isolated etioplasts. Materials and Methods Wheat fruits (Triticum aestivum, cv. Kolibri) were sown in moist vermiculite and grown in the dark for 7 days at 25 ~ C. Harvest and preparation of the tissue was performed under a dim green safelight. For the initial experiment, homogenates of 8 cm segments of leaf tissue were prepared in the dark using cold phosphate: citrate buffer, pH 6.0 (Reid et al., 1972). The technique of * Abbreviation: GA = GibbereIlin. ** Present address: Department of Plant Biology, The University, Newcastle upon Tyne,
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James and Das (1957) was adapted for differential centrifugation of homogenates, prepared in a medium as before but including 0.4 5{ sucrose and 0.01 5{ KC1. Centrifugation was carried out in the dark using an MSE 18 centrifuge, first at 1000 g for 10 rain and subsequently at 16 000 g for 15 rain. The pellets obtained in each case were washed in fresh extraction medium ~nd re-centrifuged at the appropriate speed. The temperature was maintained at 4~ throughout. The pellets were re-suspended before irradiation. Intact etioplasts were prepared from segments of leaves using a Sephadex G-50 column method (Wellburn and Wellburn, 1971). Fractions eluting from the column containing etioplasts were bulked, allowed to warm to 25~ and irradiated in glass dishes. The light sources have been described previously (Beevers et al., 1970; van Staden and Wareing, 1972). At the end of the light treatments, ice-cold absolute methanol was added to the extracts to give a final methanol concentration of 80%. Extracts were stirred for 30 min at 4 ~C, the methanol was removed in vacuo and the acidic and non-acidic ethyl acetate soluble fractions were obtained by methods commonly used in this laboratory (see Loveys and Wareing, 1971, for example). Ethyl acetate soluble fractions were then taken to dryness in vacuo and ehromatographed on 0.25 mm layers of silica gel GF 254 using the solvent system chloroform/ethyl acetate/acetic acid, 50:50: ]. After being thoroughly air-dried, chromatograms were divided into 10 equal Rf zones and bioassayed using the lettuce hypocotyl assay of Frankland and Wareing (1960). The lettuce variety "Artic King" was used. Phytochrome estimations were obtained at 0 ~ C using a modified Pcrkin-Elmer 156 dualwavelength spectrophotometer and calcium carbonate as a scattering agent (Kendrick and Smith, in press).
Results and Discussion E x p e r i m e n t s with Lea] Homogenates Fig. 1 illustrates the results of a n e x p e r i m e n t performed to e x a m i n e whether homogenates of etiolated wheat leaf tissue will respond to red light t r e a t m e n t i n a n analogous m a n n e r to t h a t reported b y R e i d et al., (1972) for barley. A h o m o g e n a t e of 30 g of leaf segments was prepared i n darkness, half oi which was t h e n i r r a d i a t e d with red light for 20 rain. I t is a p p a r e n t from Fig. 1 t h a t i I r a d i a t i o n increases the level of GA-like a c t i v i t y extractable from homogenates of wheat leaves. The GA-like substances present have similar Rf values to those reported i n barley leaf homogenates (Reid et al., 1972).
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Fig. 1A and B. The effect of red light irradiation on the level of GA-like activity extractable from homogenates of 15 g of etiolated wheat leaves. Lettuce hypocotyl bioassays of the acidic ethyl acetate soluble fractions following thin-layer chromatography. A Dark; B 20rain red light. Shaded portions of the histograms represent significant promotion at the 1% level of probability
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On the basis of this result, it was decided to fractionate the homogenate and examine each fraction for an effect of red light irradiation. 40 g of leaf tissue was homogenised and the 1000 g pellet, 16000 g pellet and supernatant obtained by eentrifugation in the dark. After re-suspending the pellets in fresh extraction medium, half of each fraction was given 20 rain of red light. The GA-like activity in each fraction was then determined and the results of the bioassays are presented in Fig. 2. I t is clear that although all three fractions of the homogenate appear to contain GA-]ike activity, the response to red light is confined almost entirely to the 1000 g fraction. This fraction will contain mainly plastids and thus it is reasonable to assume that the red light effect on GA levels in wheat leaves is largely mediated through the etioplasts. I t has previously been shown that suspensions of isolated intact etioplasts contain increased levels of GA-like substances following irradiation with red light and that this can be reversed by subsequent far-red light. This indicates phytoehrome control of GA production at the level of the etioplast in wheat leaves (Cooke and Saunders, 1975). Chromatographic evidence that the GA-like substances produced in suspensions of etioplasts are the same as those produced in leaf segments following red light was obtained in the following manner. A preparation of etioplasts was obtained from 80 g of wheat leaves. At the same time, 20 g of leaf segments were harvested. Half of each of these samples was irradiated, the etioplast suspension for 20 min with continuous red light and the leaf segments for 5 rain with red light, followed by 10 min in darkness (Loveys and Wareing, 1971). The acidic 22*
R.J. Cooke et al.
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Fig. 3A--H. A comparison of the GA-like substances present in l0 g of etiolated wheat leaves and in suspensions of etioplasts isolated from 40 g of wheat leaves. The acidic (A--D) and non-acidic (E--H) ethyl aceta.te soluble fractions were obtained from each extract, ehromatographed and bioassayed using the lettuce hypocotyl test. A, C, E and G leaf extracts; B, D, F and tI etioplast extracts. A, B, E and F Dark; C and G 5 rain red light, 10 min dark; D and H 20 rain red light. Shaded portions of the histograms represent significant promotion at the 1% level of probability
and non-acidic ethyl acetate soluble fractions were then partitioned from each extract. The techniques used for the extraction of the leaf segments have been described elsewhere (Loveys and Wareing, 1971). All of the fractions were ehromatographed and bioassayed. The chromatographic similarity of the acidic GA-like substances extractable from etioplasts and from leaf segments following red light treatment is apparent from Fig. 3. I n addition, at least part of the nonacidic GA-like activity present in dark-grown leaves would appear to be similar to t h a t occurring in intact etioplasts before irradiation. The polar non-acidic GAlike component present in dark-grown wheat leaves has been shown to be a " b o u n d " form of the hormone (Loveys and Wareing, 1971). I t could thus be suggested that the similar GA-like substance present in etioplasts prior to irradiation also represents a " b o u n d " form of GA. One effect of red light on suspensions of etioplasts is to cause the release of free, acidic GA-like substances from this "bound" form. Thus, the red light induced increase in GA levels in etiolated wheat leaves appears to be largely mediated through the etioplasts.
Experiments with Suspensions o/Etioplasts Fig. 4a is a representative electron micrograph of a portion of a typical etioplast suspension prepared by the technique of Wellburn and Wellbnrn (1971).
Production of GA-like Substances
323
Fig. 4. Electron micrographs of a portion of a typical etioplast preparation obtained using the technique of Wellburn and Wellbum (1971). a magnification • 10000, b magnification • 40000. Bar represents 1 tzm in each ease
The preparation can be seen to consist predominantly of etioplasts. Although not 100% intact, the majority of the etioplasts appear to possess a double outer membrane. There is very little contaminating material evident. The higher power micrograph (Fig. 4b) shows the internal fine structure of a typical etioplast. The stroma contains the prolamellar body in the paracrystalline state, in addition to densely staining "osmiophilie" granules and ribosomes. Similar preparations to this were used in the following experiments. I t has previously been reported that GA production in suspensions of isolated etioplasts is under phytoehrome control (Cooke and Saunders, 1975). Thus, it follows t h a t phytochrome must be associated with etioplasts. A suspension of etioplasts prepared in the normal manner was centrifuged for 10 min at 1000 g and the pellet obtained was resuspended in 2.0 ml of the extraction medium. This was then placed on a bed of calcium carbonate in a 1 em path length cuvette, mixed and the resultant suspension examined in a dual-wavelength spectrophotometer. Using measuring beams of 660 nm and 730 rim, it was possible to detect photo-reversible changes in the absorbanee of the sample following irradiation with actinic red and far-red light. A difference spectrum of the phytochrome associated with etioplasts is presented in Fig. 5. The peak position in the red region of the spectrum occurs at 660 nm and in the far-red region at 725 nm. The effect of pre-incubation with inhibitors of various kinds on the GA production induced by red light was investigated. Etioplasts were prepared as above and an equal volume of a 0.2 mg m1-1 solution of ehloramphenicol, eyeloheximide or Amo 1618 added to the suspension. Distilled water was added
324
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Fig. 5. Difference spectrum of the phytochrome present in a suspension of etioplasts prepared from 160 g of wheat leaves. The etioplasts were centrifuged down, re-suspended in 2.0 ml of extraction medium and the sample measured in a modified Perkin-Elmer 156 dualwavelength spectrophotometer. CaCOs was used as a scattering agent
to the control suspensions and all of the suspensions were kept in the dark for one hour. At the end of this time, half of each preparation was irradiated with red light for 20 rain. The acidic ethyl acetate soluble fraction was obtained from each sample, ehromatographed and bioassayed. The results are given in Fig. 6, from which it is clear that whereas chloramphenicol completely inhibits red light stimulated production of GA-like substances, cycloheximide has little or no effect. Similar findings were reported by Reid et al., (1968), working with barley leaf segments. Amo 1618 appears to inhibit the production of the slower running peak of GA-like activity, whereas the other peak is unaffected. In the preceding experiments, no a t t e m p t was made to remove the inhibitor from the extracts prior to partitioning with ethyl acetate and thus any apparent effect on gibberellin levels could be due to the inhibitor running at the same Rf in the solvent system used and masking any GA-like activity. This applies particularly, perhaps, to the apparently differential effect of Amo 1618. To examine this possibility, the acidic ethyl acetate soluble fraction was prepared from 20 ml of a 0.2 mg m1-1 solution of Amo 1618, chromatogral0hed and bioassayed. One ml of a 1 ~zg m1-1 solution of GA 3 was added to each assay dish and it was found that the acidic ethyl acetate soluble fraction of Amo 1618 suppressed the growth of the lettuce hypocotyls induced by the added GA~ by less than 5 %. Moreover, the suppression was evident from Rf 0.1 to Rf 1.0 and thus it is unlikely that the effect of Amo 1618 in the present instance is due to the presence of the inhibitor at a particular Rf. The results of experiments with selective inhibitors of protein synthesis support the hypothesis that the plastid is the site of the red light effect. Chloramlohenicol, an inhibitor thought to act principally on plastid ribosomes, prevents the increase in gibberellin levels, whereas cycloheximide, which is believed to act mainly on cytoplasmic ribosomes, does not affect the red light
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response. Although doubts exist as to the specificity of these inhibitors (MacDonald et al., t966 ; MacDonald and Ellis, 1969), these results may indicate that protein synthesis is a pre-requisite of the response in etioplasts. The effect of Amo 1618 closely parallels that which we have found in wheat leaf tissue vacuum infiltrated with the inhibitor prior to irradiation and suggests t h a t de novo gibberellin synthesis m a y be at least partly involved in the red light effect. Suspensions of isolated chloroplasts and etioplasts have been shown to be capable of synthesising a wide range of compounds (Kindl, 1971 ; Wellburn et al., 1973; Buggy et al., 1974). Furthermore, several light-enhanced processes are known to occur in isolated etioplasts, including flavonoid synthesis (Weissenbock et al., 1972}, protein synthesis (Hearing, 1973) and synthesis of 6-methylsalicylic acid (Kannangara et al., 1971). There is substantial evidence for an association between gibberellins and chloroplasts in various species (Stoddart, 1968; Railton and Wareing, 1973; F r y d m a n and Wareing, 1973; gailton and Reid, 1974) and thus it m a y be that the plastids are the site of considerable gibberellinsynthesising capacity. An experiment was carried out to determine whether or not the gibberellins produced in etioplasts in response to red light remain within the etioplasts or pass into the surrounding medium. I n t a c t etioplasts were obtained from 120 g of leaf tissue and the suspension was divided into three samples. One of these served as a dark control and the other two were both given 5 rain of red light.
326
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One of the irradiated samples was left in darkness for 15 min whereas the other was left for 5 min in the dark and then centrifuged at 1000 g for 5 min, again with light excluded. The supernat&nt was decanted and following a further 5 rain in the dark, the pellet obtained from this centrifugation was resuspended in fresh extraction medium. The acidic GA-like substances were then partitioned from all of the samples. Fig. 7 shows that a large proportion of the GA-like activity produced by an etioplast suspension in response to red light is located in the supernatant following centrifugation, indicating that GA-like substances can pass fairly rapidly out of the etioplast following their production. The two peaks of acidic GA-like activity detectable on thin-layer chromatogr&ms of extracts seem to differ somewhat in their ability to pass h'om the etioplast. The faster running peak appears entirely in the supcrnatant following irradiation, whereas some of the activity of the slower running peak is retained within the etioplast pellet. Examination of a portion of the pellet obtained from the centrifugation at 1000 g of an irradiated etioplast suspension, using phase-contrast microscopy, indicated t h a t the majority of the etiopl&sts were still intact. Also, when a suspension of etioplasts, prepared in the dark, was given a 5 min centrifugation at 1000 g, no GA-like activity could be detected in either the pellet or the supernatant. Thus, it seems likely that the appearance of GA-like activity in the supernatant following irradiation of etioplast preparations is due to the red light treatment and not to rupture of the plastids during centrifugation. Since the plastid envelope is generally believed to be impermeable to terpenoids and related compounds, which would include gibberellins (Rogers et al., 1965), the results of this experiment suggest that red light irradiation alters the properties of the etioplast membrane, allowing GAs to be released. One current
Production of GA-like Substances
327
t h e o r y of t h e m e c h a n i s m of a c t i o n of p h y t o c h r o m e is t h a t a n e a r l y consequence of p h o t o t r a n s f o r m a t i o n involves a change in t h e functional p r o p e r t i e s of membranes of various kinds (Hendricks a n d B o r t h w i c k , 1967; Smith, 1970; Boisard et al., 1974). I t is t h u s t e m p t i n g to assume t h a t p a r t of t h e p h y t o e h r o m e associated with e t i o p l a s t s is l o c a t e d either in or on t h e e t i o p l a s t envelope, where it could induce changes in t h e p e r m e a b i l i t y of t h e envelope to GAs. The p h y t o chrome control of G A p r o d u c t i o n in etioplasts, however, is more c o m p l e x a n d involves control of t h e release of GAs from a " b o u n d " form as well as control of de novo G A biosynthesis. E x p e r i m e n t s are a t p r e s e n t being c o n d u c t e d to e l u c i d a t e these points of p h y t o e h r o m e control. R. J.C. acknowledges the award of a Science Research Council Stndentship and postdoctoral Fellowship. The assistance of Mr. R. Hewitt, University of Newcastle upon Tyne, in obtaining the electron micrographs is also gratefully acknowledged.
References Beevers, L., Loveys, B., Pearson, J. A., Wareing, P. F. : Phytochrome and hormonal control of expansion and greening of etiolated wheat leaves. Planta (Berl.) 90, 286-297 (1970) Boisard, J., Marm~, D., Briggs, W . R . : I n vivo properties of membrane-bound phytochrome. Plant Physiol. 54, 272-276 (1974) Buggy, M. J., Britton, G., Goodwin, T. W. : Terpenoid biosynthesis by chloroplasts isolated in organic solvents. Phytochem. III, 125-129 (1974) Cooke, R . J . , Saunders, P. F. : Phyteehrome-mediated changes in extractable gibberellin activity in a cell-free system from etiolated wheat leaves. Planta (Berl.) 128, 299-302 (1975) Frankland, B., Wareing, P. F.: Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature (Lond.) 185, 255-256 (1960) Frydman, V.F., Wareing, P . F . : Phase change in Hedera helix L. L Gibberellin-like substances in the two growth phases. J. exp. Bot. 24, 1131-1138 (1973) Hearing, V. J. : Protein synthesis in isolated etioplasts after light stimulation. Phytochem. 12, 277-282 (1973) Hendricks, S. B., Borthwick, It. A. : The function of phytochrome in the regulation of plant growth. Proc. nat. Acad. Sci. (Wash.) 58, 2125-2130 (1967) James, W. 0., Das, V. S. R.: The organisation of respiration in chlorophyllous cells. New Phytol. 56, 325-345 (1957) Kannangara, C. G., Henningsen, K. W., Stumpf, P. K., vonWettstein, D. : 6-methylsalicylic acid synthesis by isolated barley chloroplasts. Europ. J. Biochem. 21, 344-348 (1971) Kendrick, R. E., Smith, It.: The assay and isolation of phytochrome. In: Chemistry and biochemistry of plant pigments (Goodwin, T. W., ed.), 2nd edit. London and New York: Academic Press (in press) Kindl, H. : Die Bildung eines Stilbens in isolierten Chloroplasten. Hoppe-Seylers Z. physiol. Chem. 852, 767-768 (1971) Loveys, B. R., Wareing, P. F. : The red light controlled production of gibberellin in etiolated wheat leaves. Planta (Berl.) 98, 109 116 (1971) MacDonald, I . R . , Bacon, J. S.D., Vaughan, D., Ellis, R . J . : The relation between ion absorption and protein synthesis in beet disks. J. exp. Bot. 17, 822-837 (1966) MacDonald, I. R., Ellis, R. J. : Does cycloheximide inhibit protein synthesis specifically in plant tissue ? Nature (Lond.) 222, 791-792 (t969) Railton, I. D., Reid, D. M.: Studies on gibberellins in shoots of light-grown peas. I. A reevaluation of the data. Plant Sci. Left. 2, 157-163 (1974) Railton, I. D., Wareing, P. F. : Effect of daylength on endogenous gibberellins in Solanum andigena. I I L Gibberellin production by the leaves. Physiol. Plant. (Copenh.) 29, 430-434 (1973)
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Reid, D. M., Clements, J. B. : RNA and protein synthesis: pre-requisites of red light induced gibberellin synthesis. Nature (Lond.) 219, 607-609 (1968) Reid, D. M., Clements, J. B., Carr, D. J. : Red light induction of gibberellin synthesis in leaves. :Nature (Lond.) 217, 580-582 (1968) Reid, D. M., Tuing, M. S., Durley, R. C., Railton, I. D.: Red-light-enhanced conversion of tritiated gibberellin A, into other gibberellin-like substances in homogenates of etiolated barley leaves. Planta (Berl.) 108, 67-75 (1972) Rogers, L. J., Shah, S. P. g., Goodwin, T. W. : Intracellular loealisation o~ mevalonic kinase in germinating seedlings: its importance in the regulation of terpenoid biosynthesis. Bioehem. J. 96, 7-8 (1965) Smith, H.: Phytochrome and photomorphogenesis in plants. Nature (Lond.) 227, 665-668 (1970) Staden, J. van, Wareing, P. F. : The effect of light on endogenous eytokinin levels in seeds of R u m e x obtusi/olius. Planta (Berl.) 104, 126-133 (1972) Stoddart, J. L. : The association of gibberellin-like activity with the chloroplast fraction o~ leaf homogenates. Planta (Berl.) 81, 106-112 (1968) Weissenbock, G., Fleing, I., Ruppel, H. G. : Untersuchungenzur Lokalisation yon Flavonoiden in Plas$iden. I. Flavonoide in Etioplasten yon Avena sativa L. Z. Naturforsch. 27, 1216-1224 (1972) Wellburn, A. R., Ashby, J. P., Wellburn, F. A. M. : Occurrence and biosynthesis of adenosine 3', 5' cyclic monophosphate in isolated Avena etioplasts. Biochim. biophys. Acts (Amst.) 320, 363-371 (1973) Wellburn, A. R., Wellburn, F. A.M.: A new method for the isolation of etioplasts with intact envelopes. J. exp. Bot. 23, 972-979 (1971)
