Planta (BEE.) 75, 275--278 (1967)
Shor~ C o m m u n i c a t i o n
Glycollate Formation During the Photoassimilation of Acetate by Chlorella M. J. M n ~ T T and K. H. GOULDING School of Biological Sciences, University of Bradford, Great Britain Received April 8, 1967
Summary. When 3HJaC-acetate was supplied to Chlorella pyrenoidosa in the 3H light, glycollie acid became rapidly labelled with tritium and 14C. The ~ ratio of glyeollate was 10, whilst the ratio was 4 in the acetate added. Both 3H and 14C from acetate were present in glycollate before they were present in Calvin cycle intermediates, so that glycollate was not formed as a C2-fragment from the Calvin cycle. One of the first compounds to become labelled when Chlorella pyrenoidosa assimilates 14C-2-acetate in the light was glycol]ic acid (M]~ETT and GOULDINO, 1967). Kinetic analysis of 14C-2-acctate has shown that of the total 14C present in the soluble fraction of the cells, 60% was in succinic acid and 15% in glycollic acid after ten seconds (M]~RI~ETT and GOVLDI~G, 1967). The percentage of the total 1~C incorporated by the cells into succinic and glyco]lic acids decreased with time as 14C was passed to other compounds, because of this these acids were considered primary products of acetate assimilation. That glycollate was an early product of photosynthesis was established by ScHov, BENSO~, BASSHA• and CALWN (1950) but this is the first time that C. pyrenoidosa has been shown to form glycollate during the photoassimilation of acetate. Glycollate arising from the photoassimilation of acetate m a y be formed by the same biochemical pathway as glycollate derived from carbon dioxide in the light. Glycollate formation in photosynthesis results either from the action of transketolase on sugar phosphate intermediates of the Calvin cycle (WILSON and CALVin, 1955), or by the direct condensation of C1 units derived from carbon dioxide (Z]~LITC~, 1965). The fact that little laC was incorporated into phosphoglyceric acid while C. pyrenoidosa was metabolizing 14C-acetate in the light (GovLD~G and ME~RETT, 1966) made it unlikely that glycollate was formed via the Calvin cycle. Further experiments were carried out to see whether it was possible to demonstrate a pathway of glycol]ate formation during the photoassimilation of acetate that was not related to the Calvin cycle.
276
M.J. MERRETTand K. H. G o v ~ D ~ :
The possibility t h a t glycollic acid m i g h t be concerned with the t r a n s p o r t of h y d r o g e n d u r i n g t h e p h o t o a s s i m i l a t i o n of acetate was i n v e s t i g a t e d using t r i t i u m labelled acetate. Z~LITCH (1953) proposed t h a t glycollie acid migh~ be concerned i n the t r a n s p o r t of h y d r o g e n from photochemically reduced co-factors to i n t e r m e d i a t e s of photosynthesis. Chlorella pyrenoidosa (strain 211/8P/c.c.c.) was grown photoautotrophically, carbon starved and harvested exactly as described before (GouLDI:~Gand MEtCRETT, 1966; h [ E ~ T T and GOULDING, 1967). 20 ml of cell suspension (4 mg dry weight per ml) were transferred to the sampling apparatus (SYRETT, BOCKSand M~RETT, 1964) and allowed to equilibrate at a light intensity of 12,000 lux at 25~ and aerated at a rate of 7 ]itres per hour. 10 mins before zero time either 30 ~ moles sodium acetate pH 6.7 were added, followed at zero time by 10 ~ moles sodium acetate containing 50 t~e 3H-acetate and 50 ~c 14C-2-acetate or 30 ~ moles sodium acetate pH 6.7 plus 30 ~ moles sodium bicarbonate were added followed at zero time by 10 ~ moles sodium acetate containing 50 9c all-acetate and 50 tLe ~4C-2acetate plus 10 ~ moles sodium bicarbonate containing 50 ~c laC-bicarbonate. 2 ml of cell suspension were sampled at intervals into 8 ml of ethanol which was precooled to --40~C. The products of 3H-~4C-acetate assimilation were extracted, separated by chromatography, located by radioautography and identified as described previously (M~RRETTand GOULDING,1967). Radioactive areas from double-labelling experiments were cut from the chromatogram and divided into pieces no larger than 2.0 e m • 1.5 cm. These were transferred directly to glass counting vials containing 10 m] of scintillation solution made up of 0.5% 2,5-diphenyloxazole and 0.3% 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene in toluene. 3H and 1~C in these samples were counted using a Tricarb Liquid Scintillation counter with the following settings: - - red channel, window setting 50--220 with 23% gain, green channel 150--500 window setting and 4% gain. This gave a counting efficiency of 16.8% for aH in the red channel and 6.5% for 14C in the red channel. ~H was not counted in the green channel while laC was counted with 51.8% efficiency. Thus, laC was determined in the green channel, and aH in the red channel corrected for ~aC.~C and aH labelled compounds were obtained from the Radiochemical Centre, Amersham, Bucks. Results and Discussion W h e n a u t o t r o p h i c a l l y grown cells of C. pyrenoidosa were allowed to assilni]ate aH-14C-acetate i n t h e light r a p i d e q u i l i b r i u m of aH a n d 14C i n 3I-I glycollate was a t t a i n e d (Fig. ]) resulting i n a ~ ratio of 10 i n glyeollate 3H (Fig. 1) c o m p a r e d with a ~ ratio of 4 i n the acetate added. This shows t h a t the p a t h of h y d r o g e n d u r i n g the photoassimflation of acetate differs from the p a t h of h y d r o g e n derived from w a t e r i n p h o t o s y n t h e t i c carbon dioxide fixation, PL,~MONDO~ a n d BASSHAM (1966) h a v i n g f o u n d aH n o increase i n the ~a~- ratio of glycolate formed from aH20 a n d laCO2. W h e n HlaCO~ was a d d e d to the cells a t the same t i m e as aH-laC-aeetate, SH there followed a g r a d u a l decrease of the ~ ratio of glycollate (Fig. 1). Thus, t h e labelling of glyeollate with 14C from 14COz was a slower process
Glycollate Formation During the Photoassimilation of Acetate by Chlorella
277
than incorporation of ~4C from l~C-acetate into glycollate suggesting t h a t ~4C02 was not entering the same glycollate pool as t h a t incorporating SH and 14C from aH-14C-acetate. aH and 14C from the photoassimilation of aH-14C-acetate werepresent in glyeollate before intermediates of the Calvin cycle (Fig. 2). Equilibrium
10
~[~ 0
3H
~ig. i. ~
/0
60
30
Seo
300
ratio in g]ycollate formed during the photoassimilation of 3H-14C-aeetate
or from 3H-14C-acetate plus 14C-bicarbonate by C. 19yrenoidosa. o - - o Ratio in glyeollate from 3H-14C-2-~cetate. ~--o 1%atioin glycol]ate from ~H-14-C-2-acetate
plus 14C-bicarbonate
l
J
o
3
I 0
/0
30
6'0
/gO
o sec
200
~ i ~ I ~ I ~ h e 1~ ~or~ o r a~l o ~ o~ 3 ~ a n d 14~ ln~o ~ l y ~o] ]l ~ ~ eid ~ n d C a l v i n cy~]e l n ~ r l
mediates during the photoassimilation of 3H-14C-acet~te by Chlorella pyrenoidosa. o--o1~C in glycollate; .---. aH in glycoll~te; A--A 1~C in Calvin cycle intermediates; A--A 3H in Calivn cycle intermediates between 3H and 14C in glycollate was attained 30 seconds after the addition of acetate (Figs. 1 and 2) but incorporation of 14C and 3H into Calvin cycle intermediates was not detected until 60 seconds after addition of acetate (Fig. 2) when most of the label was still in phosphog]yceric acid. Thus, a mechanism operates in Chlorella for the formation
278
M.J. MERRETT and K. tt. GOULDING: Glyeollate Formation
of g l y c o l l a t e in t h e light which is n o t d e p e n d e n t on t h e Calvin cycle. 8H The difference in t h e ~ r a t i o of glycollate c o m p a r e d w i t h t h a t of t h e a c e t a t e rules o u t t h e f o r m a t i o n of glycollate b y t h e d i r e c t o x i d a t i o n of a c e t a t e v i a g l y e o a l d e h y d e . T h e p o s s i b i l i t y of a p h o t o c h e m i c a l l y p r o d u c e d r e d u c i n g c o m p o u n d f o r m e d b y t h e r e m o v a l of h y d r o g e n f r o m a c e t a t e r e a c t i n g w i t h c a r b o n d i o x i d e to p r o d u c e glycollic a c i d was n o t r u l e d o u t b y these e x p e r i m e n t s , a m e c h a n i s m suggested to a c c o u n t for t h e format i o n of glyeollate f r o m c a r b o n dioxide in p h o t o s y n t h e s i s b y WA~BUl~G a n d KarRPAHL (1960), TANNER, BROWN, EYSTEa a n d TlZEHARNE (1960), a n d ZELITCH (1965). This work was supported by a research grant from the Science Research Council. References GOVLDINo, K . H . , and M . J . MERRE~T: The photometabolism of acetate by Chlorella pyrenoidosa. J. exp. Bot. 17, 678--689 (1966). MERRETT, M. J., and K. H. GOULDING: Short-term products of 14C-acetate assimilation by Chlorella pyrenoidosa. J. exp. Bot. 18, ]28--140 (1967). PLA~O~DON, J. E., and J. A. BASSHAM: Glycolie acid labelling during photosynthesis with laCO2 and tritiated water. Plant Physiol. 41, 1272--1275 (1966). ScHou, L., A. A. BENSON, J. A. B A S S ~ , and M. CALVIN: The path of carbon in photosynthesis XI The role of glycolic acid. Physiol. Plantarum (Copenh.) 3, 487--495 (1950). SYRETT,P. J., S. M. BocI~s, and M. J. MERRETT: The assimilation of acetate by Chlorella vuIgaris. J. exp. Bot. 15, 3 5 4 7 (1964). TANNER, H. l~., T. E. BROWN, C. EYSTER, and R. W. TREIIARNE: A manganese dependent photosynthetic process. Biochem. hiophys. Res. Commun. 3, 205--210 (1960), WAEBUI~G,O., and G. KRIPPAttL: Glycolic acid synthesis in Chlorella. Z. Naturforsch. 15B, 197--199 (1960). W~so~, R. T., and M. CALVIN: The photosynthetic cycle: CO2 dependent transients. J. Amer. chem. Soc. 77, 5948--5977 (1955). ZELITCR, I. : Oxidation and reduction of glycolic and glyoxylic acids in plants I I Glyoxylic acid reductase. J. biol. Chem. ~01, 719--726 (1953). - - The relationship of glycolic acid synthesis to primary photosynthetic carboxylation reaction in leaves. J. biol. Chem. 240, 1869--1876 (1965). Dr. M. J. MERI~ETT University of Bradford School of Biological Sciences, Bradford 7, U.K.
Shor~ C o m m u n i c a t i o n
Glycollate Formation During the Photoassimilation of Acetate by Chlorella M. J. M n ~ T T and K. H. GOULDING School of Biological Sciences, University of Bradford, Great Britain Received April 8, 1967
Summary. When 3HJaC-acetate was supplied to Chlorella pyrenoidosa in the 3H light, glycollie acid became rapidly labelled with tritium and 14C. The ~ ratio of glyeollate was 10, whilst the ratio was 4 in the acetate added. Both 3H and 14C from acetate were present in glycollate before they were present in Calvin cycle intermediates, so that glycollate was not formed as a C2-fragment from the Calvin cycle. One of the first compounds to become labelled when Chlorella pyrenoidosa assimilates 14C-2-acetate in the light was glycol]ic acid (M]~ETT and GOULDINO, 1967). Kinetic analysis of 14C-2-acctate has shown that of the total 14C present in the soluble fraction of the cells, 60% was in succinic acid and 15% in glycollic acid after ten seconds (M]~RI~ETT and GOVLDI~G, 1967). The percentage of the total 1~C incorporated by the cells into succinic and glyco]lic acids decreased with time as 14C was passed to other compounds, because of this these acids were considered primary products of acetate assimilation. That glycollate was an early product of photosynthesis was established by ScHov, BENSO~, BASSHA• and CALWN (1950) but this is the first time that C. pyrenoidosa has been shown to form glycollate during the photoassimilation of acetate. Glycollate arising from the photoassimilation of acetate m a y be formed by the same biochemical pathway as glycollate derived from carbon dioxide in the light. Glycollate formation in photosynthesis results either from the action of transketolase on sugar phosphate intermediates of the Calvin cycle (WILSON and CALVin, 1955), or by the direct condensation of C1 units derived from carbon dioxide (Z]~LITC~, 1965). The fact that little laC was incorporated into phosphoglyceric acid while C. pyrenoidosa was metabolizing 14C-acetate in the light (GovLD~G and ME~RETT, 1966) made it unlikely that glycollate was formed via the Calvin cycle. Further experiments were carried out to see whether it was possible to demonstrate a pathway of glycol]ate formation during the photoassimilation of acetate that was not related to the Calvin cycle.
276
M.J. MERRETTand K. H. G o v ~ D ~ :
The possibility t h a t glycollic acid m i g h t be concerned with the t r a n s p o r t of h y d r o g e n d u r i n g t h e p h o t o a s s i m i l a t i o n of acetate was i n v e s t i g a t e d using t r i t i u m labelled acetate. Z~LITCH (1953) proposed t h a t glycollie acid migh~ be concerned i n the t r a n s p o r t of h y d r o g e n from photochemically reduced co-factors to i n t e r m e d i a t e s of photosynthesis. Chlorella pyrenoidosa (strain 211/8P/c.c.c.) was grown photoautotrophically, carbon starved and harvested exactly as described before (GouLDI:~Gand MEtCRETT, 1966; h [ E ~ T T and GOULDING, 1967). 20 ml of cell suspension (4 mg dry weight per ml) were transferred to the sampling apparatus (SYRETT, BOCKSand M~RETT, 1964) and allowed to equilibrate at a light intensity of 12,000 lux at 25~ and aerated at a rate of 7 ]itres per hour. 10 mins before zero time either 30 ~ moles sodium acetate pH 6.7 were added, followed at zero time by 10 ~ moles sodium acetate containing 50 t~e 3H-acetate and 50 ~c 14C-2-acetate or 30 ~ moles sodium acetate pH 6.7 plus 30 ~ moles sodium bicarbonate were added followed at zero time by 10 ~ moles sodium acetate containing 50 9c all-acetate and 50 tLe ~4C-2acetate plus 10 ~ moles sodium bicarbonate containing 50 ~c laC-bicarbonate. 2 ml of cell suspension were sampled at intervals into 8 ml of ethanol which was precooled to --40~C. The products of 3H-~4C-acetate assimilation were extracted, separated by chromatography, located by radioautography and identified as described previously (M~RRETTand GOULDING,1967). Radioactive areas from double-labelling experiments were cut from the chromatogram and divided into pieces no larger than 2.0 e m • 1.5 cm. These were transferred directly to glass counting vials containing 10 m] of scintillation solution made up of 0.5% 2,5-diphenyloxazole and 0.3% 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene in toluene. 3H and 1~C in these samples were counted using a Tricarb Liquid Scintillation counter with the following settings: - - red channel, window setting 50--220 with 23% gain, green channel 150--500 window setting and 4% gain. This gave a counting efficiency of 16.8% for aH in the red channel and 6.5% for 14C in the red channel. ~H was not counted in the green channel while laC was counted with 51.8% efficiency. Thus, laC was determined in the green channel, and aH in the red channel corrected for ~aC.~C and aH labelled compounds were obtained from the Radiochemical Centre, Amersham, Bucks. Results and Discussion W h e n a u t o t r o p h i c a l l y grown cells of C. pyrenoidosa were allowed to assilni]ate aH-14C-acetate i n t h e light r a p i d e q u i l i b r i u m of aH a n d 14C i n 3I-I glycollate was a t t a i n e d (Fig. ]) resulting i n a ~ ratio of 10 i n glyeollate 3H (Fig. 1) c o m p a r e d with a ~ ratio of 4 i n the acetate added. This shows t h a t the p a t h of h y d r o g e n d u r i n g the photoassimflation of acetate differs from the p a t h of h y d r o g e n derived from w a t e r i n p h o t o s y n t h e t i c carbon dioxide fixation, PL,~MONDO~ a n d BASSHAM (1966) h a v i n g f o u n d aH n o increase i n the ~a~- ratio of glycolate formed from aH20 a n d laCO2. W h e n HlaCO~ was a d d e d to the cells a t the same t i m e as aH-laC-aeetate, SH there followed a g r a d u a l decrease of the ~ ratio of glycollate (Fig. 1). Thus, t h e labelling of glyeollate with 14C from 14COz was a slower process
Glycollate Formation During the Photoassimilation of Acetate by Chlorella
277
than incorporation of ~4C from l~C-acetate into glycollate suggesting t h a t ~4C02 was not entering the same glycollate pool as t h a t incorporating SH and 14C from aH-14C-acetate. aH and 14C from the photoassimilation of aH-14C-acetate werepresent in glyeollate before intermediates of the Calvin cycle (Fig. 2). Equilibrium
10
~[~ 0
3H
~ig. i. ~
/0
60
30
Seo
300
ratio in g]ycollate formed during the photoassimilation of 3H-14C-aeetate
or from 3H-14C-acetate plus 14C-bicarbonate by C. 19yrenoidosa. o - - o Ratio in glyeollate from 3H-14C-2-~cetate. ~--o 1%atioin glycol]ate from ~H-14-C-2-acetate
plus 14C-bicarbonate
l
J
o
3
I 0
/0
30
6'0
/gO
o sec
200
~ i ~ I ~ I ~ h e 1~ ~or~ o r a~l o ~ o~ 3 ~ a n d 14~ ln~o ~ l y ~o] ]l ~ ~ eid ~ n d C a l v i n cy~]e l n ~ r l
mediates during the photoassimilation of 3H-14C-acet~te by Chlorella pyrenoidosa. o--o1~C in glycollate; .---. aH in glycoll~te; A--A 1~C in Calvin cycle intermediates; A--A 3H in Calivn cycle intermediates between 3H and 14C in glycollate was attained 30 seconds after the addition of acetate (Figs. 1 and 2) but incorporation of 14C and 3H into Calvin cycle intermediates was not detected until 60 seconds after addition of acetate (Fig. 2) when most of the label was still in phosphog]yceric acid. Thus, a mechanism operates in Chlorella for the formation
278
M.J. MERRETT and K. tt. GOULDING: Glyeollate Formation
of g l y c o l l a t e in t h e light which is n o t d e p e n d e n t on t h e Calvin cycle. 8H The difference in t h e ~ r a t i o of glycollate c o m p a r e d w i t h t h a t of t h e a c e t a t e rules o u t t h e f o r m a t i o n of glycollate b y t h e d i r e c t o x i d a t i o n of a c e t a t e v i a g l y e o a l d e h y d e . T h e p o s s i b i l i t y of a p h o t o c h e m i c a l l y p r o d u c e d r e d u c i n g c o m p o u n d f o r m e d b y t h e r e m o v a l of h y d r o g e n f r o m a c e t a t e r e a c t i n g w i t h c a r b o n d i o x i d e to p r o d u c e glycollic a c i d was n o t r u l e d o u t b y these e x p e r i m e n t s , a m e c h a n i s m suggested to a c c o u n t for t h e format i o n of glyeollate f r o m c a r b o n dioxide in p h o t o s y n t h e s i s b y WA~BUl~G a n d KarRPAHL (1960), TANNER, BROWN, EYSTEa a n d TlZEHARNE (1960), a n d ZELITCH (1965). This work was supported by a research grant from the Science Research Council. References GOVLDINo, K . H . , and M . J . MERRE~T: The photometabolism of acetate by Chlorella pyrenoidosa. J. exp. Bot. 17, 678--689 (1966). MERRETT, M. J., and K. H. GOULDING: Short-term products of 14C-acetate assimilation by Chlorella pyrenoidosa. J. exp. Bot. 18, ]28--140 (1967). PLA~O~DON, J. E., and J. A. BASSHAM: Glycolie acid labelling during photosynthesis with laCO2 and tritiated water. Plant Physiol. 41, 1272--1275 (1966). ScHou, L., A. A. BENSON, J. A. B A S S ~ , and M. CALVIN: The path of carbon in photosynthesis XI The role of glycolic acid. Physiol. Plantarum (Copenh.) 3, 487--495 (1950). SYRETT,P. J., S. M. BocI~s, and M. J. MERRETT: The assimilation of acetate by Chlorella vuIgaris. J. exp. Bot. 15, 3 5 4 7 (1964). TANNER, H. l~., T. E. BROWN, C. EYSTER, and R. W. TREIIARNE: A manganese dependent photosynthetic process. Biochem. hiophys. Res. Commun. 3, 205--210 (1960), WAEBUI~G,O., and G. KRIPPAttL: Glycolic acid synthesis in Chlorella. Z. Naturforsch. 15B, 197--199 (1960). W~so~, R. T., and M. CALVIN: The photosynthetic cycle: CO2 dependent transients. J. Amer. chem. Soc. 77, 5948--5977 (1955). ZELITCR, I. : Oxidation and reduction of glycolic and glyoxylic acids in plants I I Glyoxylic acid reductase. J. biol. Chem. ~01, 719--726 (1953). - - The relationship of glycolic acid synthesis to primary photosynthetic carboxylation reaction in leaves. J. biol. Chem. 240, 1869--1876 (1965). Dr. M. J. MERI~ETT University of Bradford School of Biological Sciences, Bradford 7, U.K.
