Planta (BEE.) 94, 282--290 (1970) 9 by Springer-Verlag 1970

The Photosynthetic Capacity of Pea Leaves with a Controlled Chlorophyll Formation* * * R. J. DOWDELL a n d A. D. D o D c z School of Biological Sciences, Bath University of Technology, Bath, England Received July 18, 1970

Summary. The relationship between chlorophyll content and photosynthesis as measured in whole leaves by COs uptake and by the component reactions of the electron transport chain of isolated chloroplasts, has been investigated. Leaves with a retarded chlorophyll formation, brought about by treatment with chloramphenicol, terramycin or by a low light intensity, were compared with control leaves (i) illuminated for a similar period of time, and (ii) with a similar chlorophyll content. There appeared to be no direct relationship between chlorophyll content and photosynthetic rate. I t is suggested that C02 uptake in low light treated leaves was limited by lack of enzymes, which are formed as a response to the supply of photosynthetic products. With terramycin and chloramphenicol the limiting factors may also be lowered enzyme levels, caused by specific protein synthesis inhibition. I t is suggested that a component of Light System I I required a high light intensity stimulation, and its formation was inhibited by ehloramphenicol. The synthesis of a substance linking Light Systems I and I I appears to be closely associated with chlorophyll formation, and could well be plastoquinone. Structural damage to the intermediate chain between Light Systems I and I I is also apparently induced by chloramphenieol. Introduction A d i r e c t r e l a t i o n s h i p b e t w e e n chlorophyll c o n t e n t a n d p h o t o s y n t h e t i c r a t e was e s t a b l i s h e d b y E m e r s o n (1929) a n d Fleischer (1935), in c o n t r a s t to W i l l s t ~ t t c r a n d Stoll (1918) a n d Gabrie]sen (1948). S u b s e q u e n t i n v e s t i g a t o r s h a v e s t u d i e d this r e l a t i o n s h i p b y following t h e d e v e l o p m e n t of p h o t o s y n t h e s i s in whole leaves (Rhodes a n d Y e m m , 1966) or i s o l a t e d chloroplasts d u r i n g t h e greening of e t i o l a t e d leaves (Dodge a n d W h i t t i n g h a m , 1966; G y l d e n h o l m a n d W h a t l e y , 1968). I n a n o t h e r p a p e r (Dowdell * This work was supported by a Science Research Council studentship granted to R. J. Dowdell and submitted for the degree of P h . D . of Bath University of Technology. ** The following abbreviations are used: ADP ~ adenosine diphosphate; ATP adenosine triphosphate; C M U = 3 (3-chlorophenyl)-l, 1-dimethylurea; D C I P = dichlorophenol indophenol; NADP = nicotinamide adenine dinucleotide phosphate; PMS=phenazine methosulphate; TRIS=2-amino-2-hydroxymethyl propane-I, 3-die1.

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a n d Dodge, 1970) we have a t t e m p t e d to find a correlation between chlorophyll c o n t e n t a n d the c o m m e n c e m e n t of photosynthesis i n leaves with a n artificially retarded rate of chlorophyll synthesis. I t is a p p a r e n t t h a t p r o p o r t i o n a l i t y between chlorophyll c o n t e n t a n d photosynthesis is only e v i d e n t when chlorophyll is the limiting factor. Thus a n y d e v i a t i o n during the greening of etiolated leaves m i g h t enable one to identify other c o n c u r r e n t l y developing factors. I n these experiments, the p h o t o s y n t h e t i c capacity of pea leaves h a v i n g a similar reduced chlorophyll c o n t e n t after a n identical period of i l l u m i n a t i o n , produced b y a v a r i e t y of t r e a t m e n t s was investigated. Two antibiotics, chloramphenicol (Margulies, 1962) a n d t e r r a m y c i n (Netien a n d Laeharme, 1955) a n d also light of low i n t e n s i t y (Huffaker et al., 1966) have been used to control chlorophyll formation. Photosynthetic a c t i v i t y as measured b y carbon dioxide u p t a k e of whole leaves, a n d of some c o m p o n e n t reactions of the electron t r a n s p o r t chain of isolated chloroplasts, are compared with results o b t a i n e d for two sets of control leaves. The u n t r e a t e d leaves were either i l l u m i n a t e d for a similar length of time b u t h a d a higher chlorophyll c o n t e n t or contained a similar chlorophyll c o n t e n t b u t were i l l u m i n a t e d for a shorter period of time. Materials and Methods Pea seeds (Pisum sativum, var. Meteor) were sown on damp expanded vermiculite and grown for 8 days at 20~ in continuous darkness. Antibiotic treatment of the etiolated plants was effeeted by supporting apical 3 em cuttings in an aqueous solution of the antibiotic in the medium of White (1943), which had been solidified with 1% agar and contained in erystallising dishes. The cuttings were then exposed to light of 3,500 lux intensity. In the experiments involving low light intensity the cuttings were supported in 1% agar containing White's medium, and the light intensity varied by covering the lids with layers of neutral density filter (Kodak, Ltd.). The total chlorophyll content of leaves and chloroplast suspensionswas estimated by the method of MaeKinney (1941). Chloroplasts were isolated as described by Hill and Walker (1959). Isolated chloroplasts containing approximately 15 tzg total chlorophyll were used in the following assays, the solutions for which were buffered with 90 [z moles TRIS-ehloride buffer, pH 7.7 and contained 10 ~ moles sodium chloride in a total of 3.0 ml distilled water. The reduction of 2 ~ moles potassium ferrieyanide was determined by the decrease in extinetion at 420 nm (Jagendorf and Margulies, i960). Metmyoglobin reduction in a reaction mixture containing 0.4 ~ moles metmyoglobin and 0.003 ~ moles PMS was estimated by an increase in extinction at 582 nm (Davenport, 1960). NADP and oxygen reduction with aseorbate-DCIP were carried out as described by Davenport and Dodge (1969). Cyclic photophosphorylation was determined in a reaction mixture containing 20 ~ moles magnesium chloride, 7.5 ~ moles disodium hydrogen phosphate, 0.075 ~ moles PMS, 7.5 ~x moles ADP, 30 ix moles glueose and 1,400 units hexokinase. After illumination for 5 mins at 10~ the reaction was stopped by the addition of 3.8 mg trichloroaeetie acid. The esterified phosphate was estimated by the method of Taussky and Short (1953).

284 R. J. Dowdell and A. D. Dodge: Photosynthetic Capacity of Pea Leaves An infra-red gas analyser (Grubb Parsons Ltd.) was used to estimate changes in the CO2 content of the air passing over leaf material contained within a 5 X 1.5 x 100 mm perspex chamber. The plant chamber was illuminated by a high intensity lamp (500 W), the evolved heat being absorbed by a screen of running water. Results

Chlorophyll Formation in Untreated Leaves. I t is well known that the rate of greening of etiolated leaves is to some extent controlled by the availability of nutrients (Kirk and Tilney-Bassett, 1967). After 50 hrs illumination, the chlorophyll content of the pea cuttings supplied with sucrose was 20% above the water controls, and those with White's medium 60% (Fig. 1). In all the subsequent experiments therefore, White's medium was included in the 1% agar. Chlorophyll Formation in Treated Leaves. a) Chloramphenicol. This antibiotic is reported to inhibit the synthesis of chlorophyll primarily by preventing the formation of d-aminolevulinic acid synthetase (Gassman and Bogorad, 1967a, 1967b) which is responsible for the production of an early precursor of the porphyrins. Fig. 2 demonstrated that in pea cuttings, increasing concentrations of chloramphenicol led to an increasingly intense inhibition of chlorophyll formation. b) Terramycin. Although the effect of terramycin on the metabolism of higher plants has received little attention, its mode of action in bacteria is better understood. I t is reported to have an affinity for magnesium and manganese ions (Gale and Fo]kes, 1953), in addition to inhibiting the formation of protein from m-RNA (Brody et al., 1954). Nctien and Laeharme (1955) demonstrated that radish seeds, when dusted with terramycin powder germinated into chlorophyll deficient seedlings. The results with pea cuttings (Fig. 2) confirmed that chlorophyll synthesis was inhibited by terramycin. c) Low light intensity. The synthesis of chlorophyll in etiolated leaves is limited by the intensity of the incident light. Although photoconversion of protochlorophyll was occurring at about 20 lux, an intensity of 300-400 lux appeared to be maximal for chlorophyll formation (Fig. 2). The increased synthesis of chlorophyll occurring above 1,000 lux was due to the provision of photosynthetic substrates. A Comparison o/ Photosynthetic Activity o/ Leaves with a Similar Chlorophyll Content. a) Photochemical activity of isolated chloroplasts. The photochemical activity of chloroplasts isolated from ehloramphenicol, terramycin or low light intensity treated leaves, all containing similar amounts of chlorophyll and illuminated for 48 hrs were compared with chloroplasts isolated from untreated plants which had been illuminated for 18 and 48 hrs. A chlorophyll content similar to 18 hr control plants,

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19 Planta(Berl.),Bd.94

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C1 C2 CmTm LL C1 C2 CmTm LL Fig. 3. A comparison of the rate of isolated chloroplast electron flow as assayed by various electron aeceptors, and of whole leaf carbon dioxide uptake in treated and untreated pea leaves. The rates are expressed as ~moles electron acceptor reduced (A-D); t~moles phosphate esterified (E); Fmoles COn taken up (F) per g fresh wight per h; C1 untreated leaves, 48 hrs illumination; Ca untreated leaves, 18 hrs illumination; C M 1.5% chloramphenicol treated leaves, 48 hrs illumination; T M 0.7% terramycin treated leaves, 48 hrs illumination; E L low light intensity, 60 lax, 48 lu's illumination

was produced by 1.5% chloramphenieol, 0.7% terramycin and a light intensity of 60 lux. Ferrieyanide reduction (Fig. 3a) was inhibited by all treatments, when compared with both controls, the inhibition being less acute with terramycin treatment. Metmyoglobin reduction (Fig. 3 b) was markedly inhibited by chloramphenicol and to a lesser extent b y terramycin and low light. The 48 hr control metmyoglobin rate was significantly higher than the 18 hr control, in contrast to the two control ferrieyanide reduction rates, which were virtually identical. When the ascorbate-DCIP couple was used as electron donor, chloroplasts isolated from chloramphenicol treated leaves showed a marked

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increase in the rate of electron flow with either N A D P (Fig. 3e) or oxygen (Fig. 3d) as electron accepter. Low light treatment caused a greater inhibition of oxygen than N A D P reduction. I n comparison with the 18 hr control rates, cyclic photophosphorylation was enhanced b y ehloramphenicol and terramycin treatments (Fig. 3e). The results were however still lower t h a n the 48 hr controls, and as with the metmyoglobin results, this control was significantly higher than the 18 hr control. b) CO s uptake of whole leaves. A comparison was made of the photosynthetic rate of whole leaves treated in a similar manner to the previous section, as measured b y COs uptake. Fig. 3f demonstrates t h a t ehloramphenicol, terramycin and low intensity light caused a severe inhibition of photosynthesis. The rate of CO s uptake b y 18 hr control leaves was markedly less than those greened for 48 hrs. Discussion The CO s uptake rates of untreated leaves indicated t h a t after 18 hrs illumination chlorophyll was limiting photosynthesis. Furthermore leaves with a similar amount of chlorophyll, yet illuminated for 48 hrs under low intensity light showed an even more retarded CO2 uptake. There is good evidence t h a t the production of certain photosynthetic enzymes might be a response to the supply of photosynthetic products. Ziegler and Ziegler (1965) implicated N A D P H and A T P generation in the production of NADP-linked triosephosphate dehydrogenase while McMahon and Bogarad (1967) using CMU as an inhibitor of electron flow noted a retardation of ribose-5-phosphate isomerase production. I n contrast, Huffaker etal. (1966) found a good correlation between this enzyme and chlorophyll formation in barley leaves under a wide range of light intensities. I n the low intensity light treatments in these experiments it was evident t h a t the chloroplast was capable of electron transport, but a light intensity of 60 lux would almost certainly be insufficient for photosynthetic activity, and thus the lack of enzyme stimulation was seen as a low rate of CO s uptake. Bradbeer (1969) observed a lag of 15 hrs before the activity of NADP-linked triosephosphate dehydrogenase increased in Phaseolus leaves and has suggested the possibility of a similar form of enzyme stimulation. The rate of COs uptake in the terramycin treated leaves was significantly lower than the 18 hr control leaves which had a similar chlorophyll content. As electron flow rates in chloroplasts isolated from these treated leaves was hardly impaired, an interference of Calvin cycle enzymes is indicated. Although chloramphenicol treatment had some effect upon the electron transport a very poor rate of CO 2 uptake would again point to enzyme inhibition. Margulies (1964) has suggested 19"

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that chloramphenicol specifically b l o c k s ribulose-1, 5-diphosphate carboxylase production. In a consideration of some component parts of the photosynthetic electron transport pathway of chloroplasts isolated from these treated leaves, it is evident that the formation of a component of Light System 2 required a high intensity light stimulation, and was possibly inhibited b y ehloramphenicol. I t is suggested that the induction of this component could be linked to a functional photosystem, hence in illuminated leaves receiving light of 60 lux, Light System 2 was potentially operative but not functional. Metmyoglobin reduction, catalysed by PMS (Davenport, 1959) and involving both Light System 1 and 2 was markedly limited by chlorophyll content. However when the activity of Light System 2 (ferricyanide reduction) and Light System 1 (aseorbate oxidation) was estimated, the chlorophyll concentration was found to be in no way limiting. These results would suggest t h a t the limiting component was located on the electron transfer pathway between Light System 1 and 2 and that its formation was closely related to chlorophyll. I t was shown that PMS-mediated cyclic phosphorylation was retarded in a similar manner to metmyoglobin reduction, and this would implicate a site between the point of entry of cyclic electron flow, but before that of ascorbate-DCIP donation. Dodge and Whittingham (1966) using flax seedlings and Threlfall and Griffiths (1966) maize, observed a rapid formation of plastoquinone on the illumination of these etiolated seedlings and a correlation between this and chlorophyll production. These results suggest that the limiting factor implicated here could well be plastoquinone. I t appeared that terramycin treatment had little effect upon the electron transport pathways, in contrast to chloramphenicol which limited Light System 2 activity. The effect of chloramphenieol on this Light System is suggested to arise from the inhibition of the synthesis of a specific component. However the promoting effect of this treatment on the ascorbate-DCIP donation systems of NADP reduction and oxygen uptake might suggest the alternative possibility of a defective chloroplast structure. I t is of interest that when pea chloroplasts were submitted to disruptive treatments, such as ultrasonication or heat, Light System 2 activity was inhibited, but electron flow from ascorbate-DCIP enhanced (Davenport and Dodge, 1969). References Bradbeer, J. W. : The activities of the photosynthetic carbon cycle enzymes of greening bean leaves. New Phytol. 68, 233-245 (1969). Brody, T. ~., Hurwitz, R., Bain, J.A.: Magnesium and effect of tetracycline antibiotics on oxidative processes in mitoehonclria. Antibiot. and Chemother. 4, 864-870 (1954).

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Davenport, H. E.: Relationship between photosynthesis and the Hill reaction. Nature (Lond.) 184, 524-526 (1959). A protein from leaves catalysing the reduction of metmyoglobin and TPN by illuminated chloroplasts. Biochem. J. 77, 471-477 (1960). - - Dodge, A. D. : The effect of ultrasonic and heat treatment on some chloroplast reactions. Phytochem. 8, 1849-1857 (1969). Dodge, A. D., Whittingham, C. P.: Photochemical activity of chloroplasts isolated from etiolated plants. Ann. Bot. 80, 711-719 (1966). Dowdell, R. J., Dodge, A. D. : In press (1970). Emerson, R. : Relation between maximum rate of photosynthesis and chlorophyll concentration. J. gen. Physiol. 12, 669-622 (1929). Fleischer, W. E.: The relation between chlorophyll content and rate of photosynthesis. J. gem Physiol. 18, 573-597 (1935). Gabrielsen, E. K. : Effects of different chlorophyll concentrations on photosynthesis in foliage leaves. Physiol. Plant. (Cph.) 1, 5-37 (1948). Gale, E. F., Folkes, J. F." Assimilation of amino acids by bacteria. Biochem. J. 58, 493-498 (1953). Gassman, M., Bogorad, L. : Control of chlorophyll production in rapidly greening bean leaves. Plant Physiol. 42, 774-780 (19679). - - - - Studies on the regeneration of protoctdorophyllide after brief illumination of etiolated bean leaves. Plant Physiol. 42, 781-784 (1967b). Gyldenholm, A. O., Whatley, F. R. : The onset of photophosphorylation inchloroplasts isolated from developing bean leaves. New Phytol. 67, 461-468 (1968). Hill, R., Walker, D. A.: Pyoeyanine and phosphorylation in chloroplasts. Plant. Physiol. 84, 240-245 (1959). Huffaker, R. C., Oberdorf, R. L., Keller, C. J., Kleinkopf, G. E.: Effects of light intensity on photosynthetic carboxylative phase enzymes and chlorophyll synthesis in greening leaves of Hordeum vulgare. Plant. Physiol. 41, 913-918 (1966). Jagendorf, A.T., Margulies, M.M.: Inhibition of spinach chloroplast photochemical reactions by p-chlorophenyl-1, 1-dimethylurea. Arch. Biochem. 90, 184-195 (1960). Kirk, J. T. O., Tilney-Bassett, R. A. E. : The plastids, 455-457. London and San Francisco: Freeman and Co. 1967. MacKinney, G. : The absorption of light by chlorophyll solutions. J. biol. Chem. 140, 315-322 (1941). McMahon, D., Bogorad, L.: Inhibition of photosynthetic enzyme induction by inhibitors of photosynthesis. Fed. Proc. 26, 807 (1967). Margulies, M. M.: Effect of chloramphenicol on light dependent development of seedlings of Phaseolus vulgari8 var. Black Valentine, with particular reference to the development of photosynthetic activity. Plant. Physiol. 87, 473-480 (1962). - - Effect of chloramphenicol on light dependent synthesis of proteins and enzymes of leaves and chloroplasts of Phaseotus vulgaris. Plant. Physiol. 89, 579~585 (1964). Netien, G., Laeharme, J. : Recherche sur raction de la tetramycine clans la formation des pigments de la plantule de radis. Bull. Soe. Chim. biol. (Paris) 87, 643-653 (1955). Rhodes, M. J.C., u E . W . : The development of chloroplasts and photosynthetic activities in young barley leaves. New Phytol. 6~, 331-342 (1966). Taussky, H.H., Shorr, E.: Semimicro quantitative determinations of inorganic phosphorus. J. biol. Chem. 2{}2, 675-685 (1953). -

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290 R. J. Dowdell and A. D. Dodge: Photosynthetic Capacity of Pea Leaves Threlfall, D.R., Griffiths, W . T . : Biosynthesis of terpenoid quinones. In: Biochemistry of chloroplasts, vol. I I (T. W. Goodwin, ed.), p. 255-271. London and New York: Academic Press 1967. White, P. R.: Nutrient deficiency studies and an improved inorganic nutrient for the cultivation of excised tomato roots. Growth 7, 53-65 (1943). Willst~tter, R., Stoll, A. : Untersuchungen fiber die Assimilation der Kohlens~ure. Berlin: Julius Springer 1918. Ziegler, H., Ziegler, I.: Der Einflul3 der Belichtung auf die NADP-abh~ngige Glycerinaldehyde-3-Phosphat-Dehydrogenase.Planta (Berl.) 65, 369-380 (1965). A. D. Dodge School of Biological Sciences Bath University of Technology Bath, England

R. J. Dowdell's present address Agricultural Research Council Letcombe Laboratory Wantage, Berkshire, England