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On the renewal of chlorophyll and protein in plants
Abstracts of Papers a 6t6 la meilleure. Les feuilles one eu un comportement & la pdndtration plus dlevd que les tiges, pour les dldments P, S e t Fe. L'gtge des feuilles ne semble pas avoir d'influense sur la pdndtration de Ca, S e t Fe. Pour tous les dldments, la face infdrieure des feuilles montre un meilleur pouvoir de pdndtration que la face supdrieure sauf dans le cas jeunes feuilles, pour l'dldment P. Contrairement £ ce qui se passe pour le Fe, la pdndtration de Ca, P e t S par les feuilles a 6td meilleure ~t la lumi6re. (2) Etude de la translocation et de la localisation des dldments apr+s absorption par racines, tiges, feuilles et p6tioles. Sauf pour la soufre, la plante nourrie par racines, accuse une concentration de l'61~ment beaucoup plus 61evde dans la racine que dans les autres organes. La partie mobilisde tend ~ se concentrer dans les feuilles. Pour les voles de pdndtration autres que les racines, on trouve systdmatiquement une plus grande concentration dans les feuilles. En gdndra], il semble que dans la plupart des cas, la varidtd sur M I X est plus favourable ~t une grande mobilitd des 61dments nutritifs que la vari6td sur M II. (3) Etude de la redistribution des dldments pendant 2 mois apr~s une absorption de 15 jours par pdtiole et feuilles. Le calcium ne se redistribue pas. 16°o du soufre semble migrer des feuilles vers les tiges (10.5°o) et les racines (5.5%). De m~me, 600o du fer semble migrer des feuilles vers les racines. (4) Etude de l'effet du lessivage des feuilles sur la perte en dldments. Suite ~t un lessivage d ' e a u courante (3 1./minute pendant 3 heures) les feuilles accusaient les pertes respectives: en pa2 (12.5%); Ca 45 (20%), S a5 (27.8%) et Fe s9 (0%).
181 M e a s u r e m e n t of I o n i c F l u x e s in R e d B e e t Tissues using Radioisotopes. G. E. BRIGGS, A. B. HOPE, a n d M . University of Cambridge, U.K.
G.
PITMAN,
The passage of solutes into the vacuoles of a piece of plant tissue from the surrounding solution can be analysed into at least two stages: diffusion through what has been called the Free Space and active transport from this into the vacuole. The movement of ions in the Free Space has been studied in the absence of accumulation by lowering the temperature, and using tissue that had previously accumulated salt to saturation. Diffusion constants relating to movement of ions into and through the disks have been
249
measured and are considered in relation to the properties of the Flee Space. Fluxes into and out of the vacoules were calculated from parallel measurements of the concentration and radioactivity in the solution external to the disks. The amount of cation in the Free Space was allowed for by making parallel experiments on tissue at a low temperature when the rate of accumulation was negligible. The fluxes have been measured for potassium and sodium in solutions containing one or both of these ions. 182
Investigations on Light-dependent Phosp h o r y l a t i o n in P l a n t s by M e a n s of P h o s p h o r u s - 3 2 . W . SI~IONIS, V e t e r i n a r y School, Hanover, Germany. To investigate the connection between photosynthesis and phosphate metabolism in plants, light-dependent phosphorylation and its relation to various external and internal factors was examined in unicellular algae (Ankistrodesmus)and higher plants (Elodea) by assimilating p32-marked orthophos -~ phoric acid in light and darkness. During these experiments it was found recently, that the assimilation of p32 into the phosphate fractions under investigation is varied by adding increasing amounts of low phosphate concentrations. Besides the phosphate contents of the algae themselves, this effect is of special importance when investigating more closely the light-dependent phosphorylation; i.e. when the CO2-relation of light-dependent phosphorylation is being studied into consideration the phosphate intake, marked variations of p32 assimilation were noted in light and darkness depending on the amount of phosphate added. The effects of inhibitors (iodoacetic acid and sodium fluoride) on light-dependent phosphorylation were studied next. By means of appropriate solvents radio paper-chromatographs were taken to obtain exact knowledge of the course of light-dependent phosphorylation. By these the dissimilation processes caused by the manner of killing the extracts were tested; these may overlap the actual process of assimilation to a marked degree. Only then were we able to establish and reproduce differences in pa2 assimilation in light and darkness. In these experiments special attention was paid to short-timed assimilation of pa2 into A T P and phosphoglyceric acid. 183
O n t h e R e n e w a l of C h l o r o p h y l l a n d P r o t e i n in P l a n t s . T. N. GODNEV, F. V. TtJRCHIN, a n d A. A. SHLYK, B y e l o r u s s i a n A c a d e m y o f Sciences, M i n s k , U . S . S . R . Although the idea of the renewal of chlorophyll belongs to the last century (X,VIESNER), it was proved
250
International Conference on Radioisotopes in Scient~e Research
to be correct only t h r o u g h the use of tracer technique. E. RtT a n d H. HussoN (1952) were the first to show the i n t r o d u c t i o n of C 14 into the composition of chlorophyll a n d carotinoids in the case of a short exposure of the p l a n t to C1~O2 a n d the absence of measureable changes in their quantities. I:. V. TURCHIN (1953), using N 15, o b t a i n e d for the first time d a t a on the rate of this process. I n t h a t same year, A. A. SHLYI< a n d T. N. GODNeV subjected chlorophylls a n d carotinoids labelled with C 14 to a n extremely careful double c h r o m a t o g r a p h y on saccharose a n d on p a p e r a n d proved definitely t h a t the presence of a n isotope in the composition of the pigments c a n n o t be explained by the occurrence of impurities. A calculation of the rate of the process by the exponential e q u a t i o n showed t h a t the period of semirenewal of chlorophyll a m o u n t s to a b o u t 13 days in the case of the m a t u r e leaves of tobacco. However, for certain other plants (for example, Ceratophyllum) a slightly different rate is observed. According to the data of F. V. T v a c m N , the period of semirenewal of the nitrogen of chlorophyll came to a b o u t 3 to 4 days for the young plants of oats, barley a n d rye. This difference is explained by the individual peculiarities of the metabolism of plants, their age a n d also ecological conditions. T h e peculiarities in the structure of p h o r b i n or phytol m a k e i m p r o b a b l e their renewal by individual atoms for groups of atoms. A c o m p a r a t i v e study of the distribution of radioactive c a r b o n of the chlorophylls a a n d b between p h o r b i n a n d phytol showed the impossibility of even a partial renewal of the pigments according to these big radicals. Apparently, the basic m e c h a n i s m of renewal consists in the decay of old molecules a n d the complete synthesis of new molecules of chlorophyll. This process is closely related to the renewal of protein. T h e rate of renewal of the nitrogen of protein proved to be of the same order as the renewal of chlorophyll a n d also a m o u n t s to a b o u t 3 days. I n the case of rye a n d barley in the dark, the renewal of chlorophyll does not take place, a l t h o u g h it continues to decay.
184 I n t e r r e l a t i o n of C h l o r o p h y l l C o m p o n e n t s a n d C a r o t i n o i d s in B i o s y n t h e s i s . A. A. SHLYK a n d T . N. GODNEV, I n s t i t u t e o f Biology, Academy of Sciences of the Byelorussian S.S.R., Minsk, U.S.S.R. T h e complex question of the interrelation of chlorophylls a a n d b a n d carotinoids in the process of biosynthesis can be solved by c o m p a r i n g their specific activities in the renewal dynamics. O n the examples of three plants it has been established t h a t the specific activites of c a r b o n in chlorophylls a a n d b differ considerably, a fact t h a t can be
a c c o u n t e d for by the u n e q u a l dilution of the newly formed molecules of the pigments present in the tissue. This difference was m a i n t a i n e d for a considerable time, thus proving t h a t in a p l a n t the two chlorophyll components are not rapidly transformed into each other. T h e splitting of the chlorophyll molecule into p h o r b i n a n d phytol a n d d e t e r m i n a t i o n of their radioactivity showed t h a t the c o m p o n e n t s a a n d b are only capable of slow exchange with phytol. Chlorophyll b, acquiring lower activity, c a n n o t be the predecessor in biosynthesis of chlorophyll a. Similarly, basically, distribution of the radioactivity of chlorophylls a a n d b a m o n g the individual parts of their molecules excludes the possibility of indep e n d e n t biosynthesis for b o t h pigments. This shows that they are formed from c o m m o n predecessors. Special experiments in which the renewal of chlomphyI1 was d e t e r m i n e d simuhaneously with its a c c u m u l a t i o n , have shown t h a t on the way from succinate to chlorophyll the c a r b o n becomes considerably diluted, in fact, m u c h more so than in the case of chlorophyll a. It can be supposed that such dilution is conditioned by the fact t h a t formation of chlorophyll b was not in parallel with that of chlorophyll a but occurred t h r o u g h it. T h e lower specific activity of the phytol c a r b o n as c o m p a r e d with t h a t of carotinoids carbon, observed in our experiments, makes it p r o b a b l e that in chloroplasts the chlorophyll phytol is not a predecessor of carotinoids. T h e appreciably higher specific activity of carotins in comparison with carotinols shows t h a t b o t h of these p i g m e n t groups are not subjected to such rapid reciprocal transformations which could be expected, assuming the participation of this system directly in photosynthesis.
185 Radiochemical I d e n t i f i c a t i o n of D i g l y c e r o p h o s p h a t e and Its P r o b a b l e R o l e in Lipid S y n t h e s i s by P l a n t s . A. A. BENSON a n d B. MARt:O, D e p a r t m e n t of Agricultural and Biological Chemistry, Pennsylvania State University, University Park, Pennsylvania. T h e value of the radioactive tracer m e t h o d is d e m o n s t r a t e d by its application in the chemical identification a n d d e t e r m i n a t i o n of the metabolic significance of a new c o m p o u n d in green plants. R a d i o c h r o m a t o g r a p h i c analysis of extracts of the alga, Scenedesmus, labeled with pa2 revealed large a m o u n t s of a n u n k n o w n phosphorylated interm e d i a t e w h i c h decreased markedly u p o n illumination. D e t e r m i n a t i o n of its concentration a n d chemical d e g r a d a t i o n of tracer a m o u n t s of the u n k n o w n from p32_ a n d C14-1abeled algae resulted in its identification
181 M e a s u r e m e n t of I o n i c F l u x e s in R e d B e e t Tissues using Radioisotopes. G. E. BRIGGS, A. B. HOPE, a n d M . University of Cambridge, U.K.
G.
PITMAN,
The passage of solutes into the vacuoles of a piece of plant tissue from the surrounding solution can be analysed into at least two stages: diffusion through what has been called the Free Space and active transport from this into the vacuole. The movement of ions in the Free Space has been studied in the absence of accumulation by lowering the temperature, and using tissue that had previously accumulated salt to saturation. Diffusion constants relating to movement of ions into and through the disks have been
249
measured and are considered in relation to the properties of the Flee Space. Fluxes into and out of the vacoules were calculated from parallel measurements of the concentration and radioactivity in the solution external to the disks. The amount of cation in the Free Space was allowed for by making parallel experiments on tissue at a low temperature when the rate of accumulation was negligible. The fluxes have been measured for potassium and sodium in solutions containing one or both of these ions. 182
Investigations on Light-dependent Phosp h o r y l a t i o n in P l a n t s by M e a n s of P h o s p h o r u s - 3 2 . W . SI~IONIS, V e t e r i n a r y School, Hanover, Germany. To investigate the connection between photosynthesis and phosphate metabolism in plants, light-dependent phosphorylation and its relation to various external and internal factors was examined in unicellular algae (Ankistrodesmus)and higher plants (Elodea) by assimilating p32-marked orthophos -~ phoric acid in light and darkness. During these experiments it was found recently, that the assimilation of p32 into the phosphate fractions under investigation is varied by adding increasing amounts of low phosphate concentrations. Besides the phosphate contents of the algae themselves, this effect is of special importance when investigating more closely the light-dependent phosphorylation; i.e. when the CO2-relation of light-dependent phosphorylation is being studied into consideration the phosphate intake, marked variations of p32 assimilation were noted in light and darkness depending on the amount of phosphate added. The effects of inhibitors (iodoacetic acid and sodium fluoride) on light-dependent phosphorylation were studied next. By means of appropriate solvents radio paper-chromatographs were taken to obtain exact knowledge of the course of light-dependent phosphorylation. By these the dissimilation processes caused by the manner of killing the extracts were tested; these may overlap the actual process of assimilation to a marked degree. Only then were we able to establish and reproduce differences in pa2 assimilation in light and darkness. In these experiments special attention was paid to short-timed assimilation of pa2 into A T P and phosphoglyceric acid. 183
O n t h e R e n e w a l of C h l o r o p h y l l a n d P r o t e i n in P l a n t s . T. N. GODNEV, F. V. TtJRCHIN, a n d A. A. SHLYK, B y e l o r u s s i a n A c a d e m y o f Sciences, M i n s k , U . S . S . R . Although the idea of the renewal of chlorophyll belongs to the last century (X,VIESNER), it was proved
250
International Conference on Radioisotopes in Scient~e Research
to be correct only t h r o u g h the use of tracer technique. E. RtT a n d H. HussoN (1952) were the first to show the i n t r o d u c t i o n of C 14 into the composition of chlorophyll a n d carotinoids in the case of a short exposure of the p l a n t to C1~O2 a n d the absence of measureable changes in their quantities. I:. V. TURCHIN (1953), using N 15, o b t a i n e d for the first time d a t a on the rate of this process. I n t h a t same year, A. A. SHLYI< a n d T. N. GODNeV subjected chlorophylls a n d carotinoids labelled with C 14 to a n extremely careful double c h r o m a t o g r a p h y on saccharose a n d on p a p e r a n d proved definitely t h a t the presence of a n isotope in the composition of the pigments c a n n o t be explained by the occurrence of impurities. A calculation of the rate of the process by the exponential e q u a t i o n showed t h a t the period of semirenewal of chlorophyll a m o u n t s to a b o u t 13 days in the case of the m a t u r e leaves of tobacco. However, for certain other plants (for example, Ceratophyllum) a slightly different rate is observed. According to the data of F. V. T v a c m N , the period of semirenewal of the nitrogen of chlorophyll came to a b o u t 3 to 4 days for the young plants of oats, barley a n d rye. This difference is explained by the individual peculiarities of the metabolism of plants, their age a n d also ecological conditions. T h e peculiarities in the structure of p h o r b i n or phytol m a k e i m p r o b a b l e their renewal by individual atoms for groups of atoms. A c o m p a r a t i v e study of the distribution of radioactive c a r b o n of the chlorophylls a a n d b between p h o r b i n a n d phytol showed the impossibility of even a partial renewal of the pigments according to these big radicals. Apparently, the basic m e c h a n i s m of renewal consists in the decay of old molecules a n d the complete synthesis of new molecules of chlorophyll. This process is closely related to the renewal of protein. T h e rate of renewal of the nitrogen of protein proved to be of the same order as the renewal of chlorophyll a n d also a m o u n t s to a b o u t 3 days. I n the case of rye a n d barley in the dark, the renewal of chlorophyll does not take place, a l t h o u g h it continues to decay.
184 I n t e r r e l a t i o n of C h l o r o p h y l l C o m p o n e n t s a n d C a r o t i n o i d s in B i o s y n t h e s i s . A. A. SHLYK a n d T . N. GODNEV, I n s t i t u t e o f Biology, Academy of Sciences of the Byelorussian S.S.R., Minsk, U.S.S.R. T h e complex question of the interrelation of chlorophylls a a n d b a n d carotinoids in the process of biosynthesis can be solved by c o m p a r i n g their specific activities in the renewal dynamics. O n the examples of three plants it has been established t h a t the specific activites of c a r b o n in chlorophylls a a n d b differ considerably, a fact t h a t can be
a c c o u n t e d for by the u n e q u a l dilution of the newly formed molecules of the pigments present in the tissue. This difference was m a i n t a i n e d for a considerable time, thus proving t h a t in a p l a n t the two chlorophyll components are not rapidly transformed into each other. T h e splitting of the chlorophyll molecule into p h o r b i n a n d phytol a n d d e t e r m i n a t i o n of their radioactivity showed t h a t the c o m p o n e n t s a a n d b are only capable of slow exchange with phytol. Chlorophyll b, acquiring lower activity, c a n n o t be the predecessor in biosynthesis of chlorophyll a. Similarly, basically, distribution of the radioactivity of chlorophylls a a n d b a m o n g the individual parts of their molecules excludes the possibility of indep e n d e n t biosynthesis for b o t h pigments. This shows that they are formed from c o m m o n predecessors. Special experiments in which the renewal of chlomphyI1 was d e t e r m i n e d simuhaneously with its a c c u m u l a t i o n , have shown t h a t on the way from succinate to chlorophyll the c a r b o n becomes considerably diluted, in fact, m u c h more so than in the case of chlorophyll a. It can be supposed that such dilution is conditioned by the fact t h a t formation of chlorophyll b was not in parallel with that of chlorophyll a but occurred t h r o u g h it. T h e lower specific activity of the phytol c a r b o n as c o m p a r e d with t h a t of carotinoids carbon, observed in our experiments, makes it p r o b a b l e that in chloroplasts the chlorophyll phytol is not a predecessor of carotinoids. T h e appreciably higher specific activity of carotins in comparison with carotinols shows t h a t b o t h of these p i g m e n t groups are not subjected to such rapid reciprocal transformations which could be expected, assuming the participation of this system directly in photosynthesis.
185 Radiochemical I d e n t i f i c a t i o n of D i g l y c e r o p h o s p h a t e and Its P r o b a b l e R o l e in Lipid S y n t h e s i s by P l a n t s . A. A. BENSON a n d B. MARt:O, D e p a r t m e n t of Agricultural and Biological Chemistry, Pennsylvania State University, University Park, Pennsylvania. T h e value of the radioactive tracer m e t h o d is d e m o n s t r a t e d by its application in the chemical identification a n d d e t e r m i n a t i o n of the metabolic significance of a new c o m p o u n d in green plants. R a d i o c h r o m a t o g r a p h i c analysis of extracts of the alga, Scenedesmus, labeled with pa2 revealed large a m o u n t s of a n u n k n o w n phosphorylated interm e d i a t e w h i c h decreased markedly u p o n illumination. D e t e r m i n a t i o n of its concentration a n d chemical d e g r a d a t i o n of tracer a m o u n t s of the u n k n o w n from p32_ a n d C14-1abeled algae resulted in its identification