Photoreactivation of nitrate reductase production in Nicotiana tabacum var. xanthi

338

Biochimica et Biophysica Acta, 407 (1975) 338--346 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98426

PHOTOREACTIVATION OF NITRATE REDUCTASE PRODUCTION IN NICOTIANA TABACUM VAR. X A N T H I

LAWRENCE A. WRIGHT, Jr and TERENCE M. MURPHY

Department of Botany, University of California, Davis, Calif. 95616 (U.S.A.) (Received May 15th, 1975)

Summary Ultraviolet (254 nm) irradiation of liquid-cultured tobacco cells inhibited the production of nitrate reductase; subsequent illumination with white light allowed a partial restoration of the synthesis of the enzyme (photoreactivation). Ultraviolet irradiation of these same cells also inhibited their ability to incorporate labeled uridine and labeled amino acids. Subsequent illumination with white light gave a partial restoration of the ability of the cells to incorporate uridine while a similar post-ultraviolet-irradiation treatment failed to restore the amino acid incorporation. The system in tobacco known to repair ultraviolet-damaged viral RNA thus does not seem to repair ultraviolet damage to the protein-synthesizing system of the cell. The photoreactivation of nitrate reductase production is best explained by the action of a DNA photorepairing system.

Introduction In higher plants there are two known photoreactivation systems that repair ultraviolet radiation-induced damage to nucleic acids. The first system works on DNA. The formation of cyclobutane-type pyrimidine dimers in DNA is responsible for a large part of the damage induced by ultraviolet in some organisms. Light-dependent monomerization of ultraviolet-induced dimers in DNA of Nicotiana tabacum and Ginkgo biloba has been demonstrated by Trosko and Mansour [1,2] and a photoreactivating enzyme that can monomerize dimers in vitro has been purified from pinto bean by Saito and Werbin [3,4] and from an orchid by Sutherland (personal communication). The second system works on RNA. Various plant RNA viruses are inactivated by irradiation with ultraviolet light, and most of these can be photoreactivated by their host plant [5,6]. Huang and Gordon [7] have suggested that the photoreactivation of RNA also involves the monomerization of pyrimidine

339 dimers. UltravioletAnactivated TMV RNA can be photoreactivate.d in vitro by an extract from N. tabacum var Xanthi nc illuminated with 365 nm light [8]. The relative physiological significance of these two repair systems is not well defined. Trosko and Mansour [1] correlated the induction and removal of DNA dimers in cultured tobacco cells with the inhibition and restoration of the growth rate of these cells. Murphy and Gordon [9] obtained indirect evidence that the system that photoreactivates ultraviolet-inactivated TMV RNA might also repair damage to the cell's own RNA. In theory, if ultraviolet-induced damage involved a block in the synthesis of proteins, either the DNA- or the RNA-specific system, or both, might be important in protecting plant cells from that damage. This present study investigates the inhibition of the production of an inducible enzyme, nitrate reductase, in cultured cells of Nicotiana tabacum var. X a n t h i nc by ultraviolet (254 nm) light. Subsequent illumination with white light showed a partial restoration of the ability of these cells to produce nitrate r e d u c t ~ e (photoreactivation). The inhibition of nitrate reductase production by ultraviolet radiation could be due to interference of either the transcriptional or translational process, or possibly to inhibition at both these levels of synthesis. Therefore, the photoreactivation of nitrate reductase production could be due to DNA photorepair, RNA photorepair or a combination of both repair mechanisms. In this paper we report the results of experiments designed to test the ability of tobacco cells to photorepair the transcriptional and translational processes. Materials and Methods Cell cultures

For incorporation experiments, suspended cell cultures of Nicotiana tabacum vat. Xa n th i nc were grown in either M-1D medium [10] or B5 medium [11] raider dim, indirect fluorescent lighting (less than 0.05 W/m 2 of warm white light) as was previously described [12]. For the nitrate reductase experiments, the cells were grown in the medium described by Linsmaler and Skoog [13] under direct fluorescent lighting (about 3 W/m 2 of warm white light). L e a f disks

Young leaves (lamina approximately 8 cm × 11 cm) were removed from N. tabacum var. X ant hi nc plants and allowed to wilt for 1--2 h. The abaxial

epidermis was then peeled off and disks, 5 mm in diameter, were punched from the peeled portions with a cork borer. The disks were floated, abaxial (peeled) surface down, on 20 ml of M-1D medium. Induction and determination o f nitrate reductase

About 100 ml of suspended cells that had been growing in the medium described by Linsmaier and Skoog for about three weeks were transferred to 300 ml of fresh medium in order to induce the de novo synthesis of nitrate reductase [14,15]. The amount of nitrate reductase present in the cells at the various times after induction was determined by the following sampling procedure modified

340 from Filner [15] and Sanderson and Cocking [16]. 1.0 g (fresh-filtered weight) samples of cells were taken from each of the four flasks. The cells were suspended in 2.5 ml of 0.1 M Tris • HC1 buffer (pH 7.5) containing 0.001 M dithiothreitol and 0.001 M cysteine. HC1 and were then homogenized with 30 strokes of a m o t o r driven Thomas teflon-glass homogenizer. The homogenate was centrifuged for 20 min at 10 000 rev./min. The supernatant was purified by passing the 2.7 ml of enzyme extract through a column of 6.0 g of Sephadex G-25, coarse grade, and eluting with 0.1 M Tris. HC1 buffer (pH 7.5) containing 0.001 M cysteine • HC1. The first 7.5 ml of eluate were discarded and the next 5.4 ml were collected and used as the purified enzyme preparation. The extraction and treatments were carried out at 0 - 4 ° C . The nitrate reductase assay was that used by Filner [15]. One unit of nitrate reductase activity was defined as the a m o u n t which produced one pmole of nitrite per h per g of cells (fresh-filtered weight). The nitrite concentration was calculated from a standard absorbance curve made by assaying known amounts of NAN02.

Incorporation methoa For the uridine studies, 80--100 ml of cell suspension in M-1D medium were prelabeled with 0.1 #Ci of 14C.labele d amino acid mixture [12]. After a period of 1--2 h, 2.5 pCi of [3HI uridine were added to the prelabeled cells. Two to 3 h later, the cells were ready for ultraviolet irradiation and photoreactivation. After various periods of photoreactivation the cells were sampled [12] and their relative rates of trichloroacetic acid-insoluble [ 3H] uridine incorporation were calculated by dividing the 3H counts per min by the 14C prelabel counts per min. For the amino acid incorporation studies with cultured cells, 80--100 ml of cells in M-1D or B5 medium were prelabeled with 0.2--1.0 pCi of 3H-labeled amino acid mixture. After 1.5 to 2.5 h the cells were divided for ultraviolet irradiation. After irradiation, 0.2--1.0 pCi of 14C-labeled amino acid mixture diluted with 0.1 ml of 0.1% casein amino acids was added per 10 ml of cells. The cells were given photoreactivating light for 1.5 h; then t h e y were sampled for incorporation of labeled amino acids into trichloroacetic acid-insoluble compounds. The relative incorporation of amino acids was calculated by dividing the ~4 C counts by the 3H prelabel counts. For the amino acid incorporation studies with leaf disks, 80 leaf disks, floating on 20 ml of M-1D medium under 2 cool-white 20-watt fluorescent bulbs (5 W/m 2 incident irradiation), were prelabeled with 0.5--1.0 pCi of [14C]phenylalanine for 1 to 1.5 h. After ultraviolet irradiation, 1.0 pCi of 3 H-labeled amino acid mixture was added to each of the samples of leaf disks and the disks were then given photoreactivating illumination. After 1.5 h the leaf disks were placed into test tubes containing 1 ml of 10% trichloroacetic acid (2 disks per tube). The disks were broken apart by holding a spatula in the tube while placing the tube on a vortex mixer. Two ml of 5% trichloroacetic acid were added and the material was then sonicated and assayed for trichloroacetic acid-insoluble compounds. The relative incorporation of amino acids was calculated by dividing the 3H counts by the 14 C prelabel counts.

341 Irradiation The conditions for ultraviolet irradiation of the suspended cells have previously been described [12]. Leaf disks were placed in open dishes with their adaxial surfaces exposed to the ultraviolet light. The incident dose rate was 2 W/m 2. In all cases the irradiations were carried o u t in near darkness, the only light being that from a small photographic red safe-light ( D u p o n t S-55X safelight filter). Photoreactivation method The cells or leaf disks were divided into two equal groups. One group received ultraviolet irradiation, while the second group remained the un-irradiated control. Each group was then further subdivided into two groups. One control group was placed in a container that was covered with aluminum foil while the second control group was placed in a container covered with saran wrap. The ultraviolet-irradiated samples were divided in the same manner. The samples (control-light, control-dark, ultraviolet-light and ultraviolet-dark) were placed under warm-white fluorescent bulbs for photoreactivation illumination (3 W/m 2 for incorporation studies with cultured cells; 7 W/m 2 for incorporation studies with the leaf disks and for the nitrate reductase study). The samples were rotated at 80 cycles per min during the entire illumination period. Analysis o f the incorporation studies For each cultured cell experiment, 10 1-ml samples of cells were taken from each container and an average incorporation value was obtained for each treatment (control-light, control-dark, ultraviolet-light, ultraviolet~lark). The photoreactivated sector, fp, was calculated by comparing the fraction incorporated by illuminated cells (ultravioletAight/control-light) to the fraction incorporated b y the dark-treated cells (ultraviolet~dark/control-dark) according to the formula [17] :

fp=l--

log (ultravioletAight/control-light) log (ultraviolet-dark/control-dark)

For each leaf disk experiment, 10 samples (2 disks per sample) were assayed for amino acid incorporation and an average value was obtained for each of the four treatments. The degree of photoreactivation (fo) was then determined by the same formula. The use of the formula is valid only if the following assumption is correct: the ultraviolet-inhibition of the process being studied follows exponential inhibition kinetics with increasing doses of ultraviolet radiation. This has been shown to be true for the incorporation of amino acids by the cultured tobacco cells [12]. Results Fig. 1 shows the induction kinetics for nitrate reductase production in our cultured tobacco cells. The cells were induced by transfer to fresh medium at day zero; the peak in enzyme activity occurred 3.5 days later. Irradiation with 1200 J / m 2 of ultraviolet light 9--10 h after the start of induction inhib-

342 3001500 Em = 200~100C

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Time (hours)

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Fig. 1. K i n e t i c s o f nitrate r e d u c t a s e p r o d u c t i o n in c u l t u r e d t o b a c c o cells. A t zero t i m e , a s t a t i o n a r y culture o f the cells w a s transferred to fresh m e d i u m . Fig. 2. P h o t o r e a c t i v a t i o n o f the u l t r a v i o l e t - i n h i b i t i o n o f nitrate r e d u e t a s e p r o d u c t i o n in c u l t u r e d t o b a c c o cells. Each p o i n t r e p r e s e n t s the average o f three e x p e r i m e n t s , T h e cells w e r e i n d u c e d at t i m e z e r o ; u l t r a v i o l e t irradiation w i t h 1 2 0 0 J / m 2 o c c u r r e d at 9 - - 1 0 h. T r e a t m e n t s : c o n t r o l - l i g h t ( o ) , c o n t r o l - d a r k ( e ) , u l t r a v i o l e t - l i g h t (~), u l t r a v i o l e t - d a r k (4).

ited the production of nitrate reductase; subsequent continuous illumination with white light led to a partial restoration of the ability of these cells to produce nitrate reductase (Fig. 2). The slopes suggest as much as 50% recovery. Cordycepin, a transcriptional inhibitor [ 1 8 ] , stopped the production of nitrate reductase; the greatest effect occurred when the inhibitor was added near the beginning of the induction period (Fig. 3). The kinetics of the inhibition of nitrate reductase production by addition of cordycepin at 24 h were quite similar to the inhibition observed when the cells were ultraviolet irradiated 9--10 h after induction (compare Fig. 3 with Fig. 2). This inhibitor study suggests that the site of the inhibition of nitrate reductase production by ultraviolet radiation is at the transcriptional level. The photorepair of nitrate

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343

reductase production could then be explained by the action of the DNA photorepairing enzyme. If the effects of ultraviolet and photoreactivating light on nitrate reductase production occur wholly or in part at the level of transcription, then it should be possible to demonstrate ultraviolet-inhibition and photoreactivation of RNA synthesis. The incorporation of [ 3 HI uridine by our cultured tobacco cells was approximately linear. Ultraviolet radiation with 1200 J/m 2 inhibited the incorporation of [ 3 H] uridine by about 60%. Subsequent illumination of ultravioletArradiated cells with continuous white light gave a partial restoration of the ability of these cells to incorporate uridine (Fig. 4). This restoration of uridine incorporation was used as a measure of the degree of DNA photo-

Uridine incorporation cultured cells (~ ~= 0.33 Sx= 0.09 "l

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Fig. 4. Photoreactivation of the ultraviolet-h~ibltion of uridine incorporation in liquid cultured tobacco calls. The cells were given 1200 J/m 2 of ultraviolet radiation; [ 3 H ] u r i d i n e was added at time zero, immediately following the irradiation period. Treatments: control-llsht (o), control-dark (e), ultravioletlight (4), ultravinlet-dazk (&). Fig. 5. A. P h o t o r e a c t i v a t i o n o f u r l d i n e i n c o r p o r a t i o n b y c u l t u r e d t o b a c c o cells. ( F o r t h i s s t u d y , 0 . 2 5 / ~ g o f chioramphenicol per 10 ml of medium was added after the irradiation to inhibit the growth of bacteria d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t ) . T h e cells w e r e given 9 0 0 J / m 2 o f u l t r a v i o l e t r a d i a t i o n . P h o t o r e a c t i rated sectors (fp) were calculated after 18 h of continuous photoreaetivating illumination. The positive m e a n f p v a l u e o f 0 . 3 3 i n d i c a t e s p h o t o r e p a l r o f t h e i n h i b i t i o n o f u r i d i n e i n c o r p o r a t i o n . B. P h o t o r e a c t i v a t l o n o f a m i n o a c i d i n c o z p o r a t i o n b y c u l t u r e d t o b a c c o cells. T h e cells w e r e given 6 0 0 J / m 2 o f u l t r a v i o l e t radlatio~n. P h o t o z e a e t i v a t e d s e c t o r s ( f p ) w e r e c a l c u l a t e d a f t e r 1 . 5 h o f c o n t i n u o u s p h o t o r e a e t i v a t i n g i l l u m i n a t i o n . T h e r a n d o m d i s t r i b u t i o n o f f p v a l u e s a r o u n d z e r o i n d i c a t e s t h e l a c k o f p h o t o r e a c t i v a t i o n . C. P h o t o r e a c t i v a t i o n o f a m i n o a c i d i n c o r p o r a t i o n b y t o b a c c o l e a f disks. T h e d i s k s w e r e g i v e n 1 2 0 0 J / m 2 o f ultraviolet radiation. Photoreactivated sectors (fp) were calculated after 1.5 h of continuous photor e a c t i v a t i n g i l l u m i n a t i o n . T h e r a n d o m d i s t r i b u t i o n o f fp v a l u e s a z o u n d z e r o i n d i c a t e s t h e l a c k o f P h o t o * reactivation.

344 reactivation. The photoreactivated sector (fp) for the experiment shown in Fig. 4 was 0.47 after 5 h of incorporation, suggesting a restoration of approximately one-half of the inhibition of uridine incorporation by ultraviolet radiation. In order to compare individual experiments, replicate samples were assayed after 18 h of continuous photoreactivating illumination and compared to dark-incubated controls. Photoreactivated sectors for the experiments were calculated as previously described and the data was graphed in a manner similar to that used by Hurter and co-workers [8]. Fig. 5A shows the results of 5 independent experiments with cultured cells. The mean fp was 0.33 with one standard error of +0.09. The photorepair of uridine incorporation is consistent with reports of a DNA photoreactivating system in these cultured tobacco cells. Even though the inhibitory effects of cordycepin and the uridine incorporation experiments show the possibility of transcriptional inhibition, it is still possible that a portion of the ultraviolet-inhibition and photoreactivation of nitrate reductase production is due to processes at the translational level. We have recently shown that ultraviolet radiation inhibits the incorporation of amino acids by cultured tobacco cells [12] and that as much as half of this inhibition could be due to inactivation of the polysomes by the ultraviolet radiation. If the damage to the polysomes were due to the formation of uracil dimers in m R N A or another R N A component, the TMV R N A photorepairing system, similar in action to the DNA photorepairing system, might monomerize these dimers with a concomitant repair of the ability of the RNA to function in the synthesis of proteins. Fig. 5B shows the results of 12 independent experiments which test for the ability of the cultured tobacco cells to photoreactivate the inhibition of amino acid incorporation by ultraviolet radiation. The cultured tobacco cells were first irradiated with ultraviolet light and then given photoreactivating illumination with white light for 1.5 h. Replicate samples were taken and average values were used to calculate the fp for the experiment. The mean fp was 0.15 with one standard error of -+0.15. Since the photoreactivation sectors were randomly distributed around a value within one standard error of zero, we conclude that there was no photorepair of the inhibition of amino acid incorporation by these cells. Photoreactivation of ultraviolet-damaged TMV RNA has been demonstrated in tobacco leaves [19], not tobacco callus; m a y b e the enzyme responsible for RNA photorepair is n o t present in cultured cells, but is present within the tobacco leaf cells. The incorporation of amino acids by tobacco leaf disks was also inhibited by ultraviolet radiation. Fig. 5C shows the results of 11 independent experiments testing for photoreactivation of amino acid incorporation by tobacco leaf disks. The mean fp was 0.10 with one standard error of + 0.07; there was no photorepair of the inhibition of amino acid incorporation by these tobacco leaf cells, either. From these results we make the following conclusions: (1) cultured cells of N i c o t i a n a t a b a c u m var. X a n t h i are able to photorepair the ultravioletinduced inhibition of amino acid incorporation into acid-precipitable material. Both cultured cells and leaf disks are unable to photorepair the ultravioletinduced inhibition of amino acid incorporation into acidprecipitable material. (3) The photorepair of nitrate reductase production by cultured tobacco cells is best explained by the action of the DNA photorepairing system.

345 Discussion The ability of cultured tobacco cells to photoreactivate production of a specific protein emphasizes the importance of the photoreactivation process in higher plants. Trosko and Mansour [1] were able to show that DNA photoreactivation could play a significant role in maintaining the growth potential of a cultured cell population. The photorepair of nitrate reductase production demonstrates that the photorepair process may be important for the maintenance of cellular activity even in non-growing cells (for instance, in mature leaves). The existence of a DNA photorepairing system in higher plants that can photoreactivate the inhibition of uridine incorporation by ultraviolet is in agreement with other work. Trosko and Mansour [1,2] reported that cultured cells of N. tabacum var. X a n t h i and G. biloba are able to remove ultravioletinduced DNA pyrimidine dimers from the DNA during treatment with light, and Saito and Werbin [3] were able to isolate a DNA photoreactivating enzyme from the young leaves of Phaseolus vulgaris var. Pinto. The presence of DNA photoreactivation in our cultured tobacco cells could account for the observed photoreactivation of nitrate reductase production if the transcription of messenger RNA is the limiting factor. Previous workers have shown that the induction of nitrate reductase in higher plants involves de novo synthesis [14,15]. Our experiments with cordycepin (Fig. 3) confirm that the induction depends on synthesis of RNA. The inhibition of nitrate reductase production by ultraviolet irradiation closely resembled the type of inhibition obtained by the addition of cordycepin, which indicates that the inhibition of nitrate reductase production by ultraviolet radiation is at the transcriptional level. The possibility existed, however, that some photoreactivation of nitrate reductase production is due to a photorepairing system that functions not at the transcriptional level but at the translational level. This possibility was suggested b y photoreactivation experiments with viral RNAs on higher plants [5,6,19] and by the competition experiments [9] in which ultraviolet-irradiation of tobacco leaves inhibited their ability to photoreactivate ultravioletinactivated TMV RNA, presumably by forming a competitor for RNA-photoreactivating enzyme. However, we were unable to demonstrate the existence of a repair system in tobacco that can photoreactivate ultraviolet-damage to the translation process. There are several possible explanations for the lack of detectable photorepair of the ultraviolet-induced inhibition of amino acid incorporation in cultured tobacco cells and leaf disks. (a) There may be no RNA photoreactivating system present in either the cultured cells or the leaves of X a n t h i tobacco that is able to photorepair ultraviolet-induced lesions in the plant's own RNA. This could imply that the system that photoreactivates TMV RNA is virus-specific. If this is true, its function is still a mystery. (b} The type of damage that was sustained by the plant protein-synthetic apparatus under our conditions of irradiation may not have been photorepairable. A significant portion of the damage may be due to components that do not depend on RNA structure [12]. The damage to RNA may have involved pyrimidine hydrates instead of dimers, or it may have involved an interaction with proteins in the cell that in some

346

way inhibited the photorepair process, just as TMV coat protein can inhibit the photorepair of ultraviolet-irradiated TMV RNA [20] or PVX RNA [21]. Damage induced by different conditions of irradiation (higher wavelengths or lower dose rates) might yet be photoreactivable. We note, however, that Murphy and Gordon [9] used irradiation conditions similar to ours, and their "competitive" effect was itself [Alotoreactivable. (c) The ultraviolet light used in our experimen*~, may have ciamaged ~he RNA-photoreactivating enzyme itself, thereby renderin~ it inactive. Tyrrell et al. [22] have suggested that DNA-photoreactivating enzyme is damaged by exposure to high doses of near-ultraviolet (365 nm) light. The ultraviolet-~-hibition of the RNA-photoreactivating enzyme would explain the ultraviolet-inhibition of TMV RNA photorepair observed by Murphy and Gordon [9], though not the subsequent light-dependent repair of this inhibition. (d) The photorepair that occurred in our amino acid incorporation experiments may have been too low to detect. We must conclude, in view of this and other work [23], thaL ~ T ~ nhotorepair in higher plants has little, if any, effect in protecting plan~ cells from 254 nm-ultraviolet radiation. Acknowledgements This study was supported by NSF Grant No. GB-30317. The authors thank Prof. B. Bonnet and Prof. T. Rost for valuable suggestions and Prof. M.P. Gordon for criticizing the manuscript. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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