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Metabolism of sedoheptulose-C14 in plant leaves
Metabolism
of Sedoheptulose-04 in Plant Leaves’ N. E. Tolbert
From
the Biology
Division,
Oak
Ridge
Received
and L. P. ZiU National
October
Laboratory,
Oak
Ridge,
Tennessee
26, 1953
The importance of sedoheptulose in the path of carbon in photosynthesis was first shown by identifying its sugar phosphate as an early labeled product during light fixation of NaHCY40a by a varied group of photosynthetic organisms (1). During studies of the alternate or glucose shunt metabolism pathway in plants, sedoheptulose phosphate was identified as a product arising from pentose phosphates (2). Thus this 7-carbon sugar appears to function in one or more cyclic processes in the plant involving both photosynthesis and respiration. In animals and yeast, sedoheptulose 7-phosphate has been found to be an intermediate in the oxidation of hexose monophosphate by the alternate metabolism pathway (3-5). However, the further metabolism of sedoheptulose and the completion of the alternate glucose metabolism cycle has remained obscure. Recently, an enzyme from yeast, transaldolase, has been reported which is capable of breaking the ‘I-carbon chain of sedoheptulose 7-phosphate between the third and fourth carbons (6). An investigation is here reported of products formed from uniformly labeled sedoheptulose-Cl4 in plants during short-time experiments in vivo. The results indicate that sedoheptulose metabolism in plant depends greatly on the atmospheric conditions, and confirm the presence of a 4-carbon sugar phosphate as an intermediate product. PROCEDURE Approximately uniformly labeled sedoheptulose-Cl4 with a specific activity of 3.7 microcuries (Gc.)/mg. was prepared by biosynthesis (7). Leaves, removed from the greenhouse during the middle of the day, were set in a highly radioactive water solution containing l-2 mg./ml. of the sedoheptulose1 Work performed Energy Commission.
under
Contract
No. 392
W-7405-eng-26
for
the
U.
S. Atomic
SEDOHEPTULOSE-C’4
393
METABOLISM
Ci4so that only the bottom of the leaf was in the solution. The rate of movement of the sugar solution into the upper portions of the leaves was found to depend on the nature of the plant and the degree of wilting of the leaf. With a thin-window laboratory monitor, considerable Cl4 activity could usually be detected in the leaf tops within 2 min. after they had been placed in the solution. The leaves, dipping into the sedoheptulose solution, were kept in a glass chamber with a circulating atmosphere which could be varied, and the whole chamber was surrounded by a water cooling bath. Light intensities of 500 ft.-candles or higher were obtained from a Photoflood bulb. At the end of the experiment the leaves were ground in liquid nitrogen, and the powder was taken up in water and immeditely heated in a boiling water bath. This killing procedure is sufliciently rapid to prevent appreciable phosphatase action on phosphate esters. After centrifugation to remove the water-insoluble residue, the supernatant was reduced in vacua to a small volume. Products formed in the plant from the sedoheptulose were identified by two-dimensional paper chromatography (8). The solvents were phenol-water in the first direction and butanol-propionic acid-water in the second direction. Any products volatilized by this treatment would not be detected. Compounds were located on the chromatogram by No-Screen x-ray film, and the spots were counted with an end-window Geiger tube. Thus each spot was expressed as a percentage of the total activity on the chromatogram and each vertical line in Tables I, II, and III represents one chromatogram. The compounds were identified by specific color spray tests (9) and by elution and cochromatography with known compounds. Organic acids were rechromatographed in ether-acetic acid-water solvent (10). Phospoate esters of organic molecules were rechromatographed in phenol-formic acid-water (336 g. phenol, 10 g. formic acid, 172 g. water).2 The phosphate esters were also treated with phosphatase (Polidase), and the organic moiety was identified (1). The identification of products listed in the tables as areas was not carried further than a comparison of their Rf values with those given by Benson et al. (8). The labeled sucrose spot was eluted with water, and part of the eluate was rechromatographed with sucrose and part was hydrolyzed with Dowex 50 in the acid form for 30 min. at 100°C. followed by rechromatography with carrier glucose and fructose. The sugars were detected by aniline-trichloroacetic acid (TCA) spray test (II), and the radioactivity was found to coincide with the added carriers. Glucose and fructose were identified by their RI values from the extensive work on the use of chromatography in photosynthesis (8), and they were also eluted, rechromatographed with carrier, and detected by aniline-TCA spray. The same procedures were followed for identification of ribulosea and ribose. As in the case of glucose and fructose, the keto sugar, ribulose, had a sufficiently larger IZf value in both of the solvents being used to effect separation from the corresponding aldehyde sugar, ribose. Similar procedures were used to t,entatively identify the four carbon sugars. Erythrose4 and erythrulosea were detected on the chromatogram by aniline-TCA 2 Directions from R. H. Burris, University of Wisconsin. 3 Ribulose was kindly furnished by A. A. Benson. 4 Erythrose was kindly furnished by H. S. Isbell, and Chemical Company.
erythrulose
by
Sigma
394
N.
E.
TOLBERT
AND
L.
P.
ZILL
spray, since these 4-carbon sugars give a visible yellow color and a brilliant yellow fluorescence under short-wavelength ultraviolet radiation immediately after spraying and heating at 100°C. for 2 min. Under the chromatographic conditions, both tetroses had so nearly the same R/s that they were not well separated. These values in phenol-water solvent were about 0.77, and in butanol-propionic acidwater about 0.43. Thus all the sugars on the two-dimensional chromatograms were located on a narrow band out from the origin which passed first through the hexoses, then through ribose and ribulose, next through the tetroses, and lastly through dihydroxyacetone and glyceraldehyde. RESULTS
AND
DISCUSSION
The pattern for metabolism of sedoheptulose did not vary extensively among sugar beet, barley, or tobacco leaves, but did depend on the environmental conditions of the experiments. In Table I are tabulated the results from representative experiments with sugar beet leaves in the light, and in Table II are data with Wintex barley leaves. Somewhat similar results were obtained with tobacco leaves. Rates of conversion of sedoheptulose to other products can be estimated by noting the change in percentage of Cl4 in the compounds as the time of the experiments is increased. These rates are only indicative of the process involved, since they depend to a large extent on the rate at which the radioactive sedoheptulose solution moved into the leaf by translocation. Leaves of all three plants, when placed in the light and air, rapidly converted sedoheptulose-Cl4to sucrose labeled in both the glucose and fructose portions. This conversion was so rapid that intermediates did TABLE Percentage
of Total Metabolism
I
Cl4 in the Products from Sedoheptulose-04 bu Suaar Beet Leaves in the Liaht Experimental
compounds
Sedoheptulose Sucrose Raflinose area Upper phosphate Lower phosphate Alanine Glutamic Aspartic
Air
area area
conditions Nitrogen
N, and (21
31 min.
50 min.
55 mill.
120 min.
76.1 17.0 2.3 Trace Trace 4.6 0 0
31.9 59.0 0 0 0 0 2.1 3.2
93.7 0.4 Trace 1.7 0.4 3.8 0 0
47.0 41.8 0 Trace Trace 0 7.3 3.6
SEDOHEPTULOSE-C14
TABLE Percentage
of Total. Metabolism
395
METABOLISM
II
Cl4 in the Products by Barley Leaves”
from Sedoheptulose-Cl” in the Light
Experimental
I
conditions
Air 11 min.
Sedoheptulose Sucrose Ribose Upper phosphate area Lower phosphate area Alanine Glyceric acid Tetrose sugar area Glycolic acid Unknown (R, - 0.8) a Wintex
78 5 0 4 Trace 5 8 0 Trace 0
-
T 16 min. 48 16 0 10 Trace 5 9 0 Trace 8
-
82 min.
9 min.
35 23 0 7 Trace 8 2 5 Trace 17
75.0 0 0 4 2 10 7 0 Trace 0
-
--
-
Nitrogen
-
20min.
60 min.
_
66.4 8.6 0 10.2 5.5 7.8 1.5 Trace Trace Trace
42.7 6.6 2.6 8.6 6.6 21.7 2.0 0 Trace 7.8 I
barley.
not accumulate significant amounts of radioactivity, and sucrose was the only major product. In sugar beet leaves the intermediary organophosphate compounds were hardly detectable on the chromatogram. Dependence of this conversion of sedoheptulose to sucrose on aerobic conditions is indicated by the appearance of only trace amounts of Cl4 in the sucrose in sugar beet leaves held in a nitrogen atmosphere and in the light. In a combined atmosphere of nitrogen and oxygen, however, the sedoheptulose was again rapidly converted in the light to sucrose in the sugar beet leaf. In experiments run in the light and with a nitrogen atmosphere, the conversion of sedoheptulose to sucrose was inhibited, so that the intermediate phosphate esters were formed in sufficient amounts to permit chromatographic identification. The leaves in the light and nitrogen atmosphere were probably not strictly anaerobic at the cellular level owing to some photosynthesis. Under these conditions the major products formed in cu. 1 hr. were alanine, glyceric acid, and compounds in the phosphate area. Chromatographic procedures used to separate all the plant constituents did not give sufficient separation of the phosphate esters, since they did not move far from the origin in either solvent. A generalization could be made that the upper phosphate area would contain the monophosphate esters of low-molecular-weight compounds
396
N.
E.
TOLBERT
AND
L.
I’.
ZILL
such as phosphoglyceric acid, and the lower phosphate area would contain higher molecular-weight phosphates such as sugar phosphates and the diphosphate esters. The phosphate areas were therefore eluted and treated with phosphatase, and the organic moieties were identified. In these short-time experiments a major product from such treatment of the upper phosphate area of all three plants cochromatographed with the tetrose sugars, erythrulose and erythrose. In short-time experiments in the light-andnitrogen atmosphere, other detectable sugars from the phosphate areas were glucose and fructose, but no ribose or ribulose. The alanine and glyceric acid, which were formed in large amounts in these short-time experiments, might have arisen in vivo from the 3-carbon product from the first cleavage of the original sedoheptulose. The results suggest that the initial breakdown products of sedoheptulose are a triose and a 4-carbon sugar phosphate. The triose was converted to alanine and glyceric acid, but the 4-carbon sugar phosphate accumulated as such, since the photosynthesis cycle could not function and aerobic respiration was blocked. Longer experiments with barley leaves were run in which the exposure time to sedoheptulose-Cl4 was 2 hr. in the light with a nitrogen atmosphere. In this series, the phosphate ester areas after phosphatase treatment gave large amounts of CY4-labeled compounds with R, values of ribulose, glucose, and moderate amounts of tetroses, ribose, fructose, sedoheptulose, and two unknown compounds which had chromatographic characteristics of sugar acids. The products are characteristic of the alternate glucose metabolism cycle and of the path of carbon in photosynthesis. These in vivo observations appear to be similar to the results of the investigation of an enzyme, transaldolase, which cleaves sedoheptulose 7-phosphate and was so named because it catalyzes a transfer of aldol linkages rather than hydrolytic cleavage (6). For that reaction glyceraldehyde 3-phosphate was used for combining with the dihydroxyacetone group of the first three carbons of sedoheptulose 7-phosphate, and preliminary evidence was presented for the existence of a tetrose from the lower carbons of sedoheptulose. In the plant the initial reaction also appears to be a similar break between carbon atoms 3 and 4. However, the in vivo data from these experiments may be interpreted in a manner analogous to the aldolase reaction with a t,etrose phosphate formation from the lower portion of the 7-carbon sugar. In the aldolase reaction with hexoses, the equilibrium lies toward the fructose 1 ,gdiphosphate. The reaction can be shifted in the other direction by removal of one
SEDOHEPTULOSE-C14
TABLE Percentage
III
of I’otal Cl4 in the Products from Sedoheptulose-(2’14 Metabolism in Leaves in the Dark Experimental
Compounds
Sugar Air
Sedoheptulose Sucrose Upper phosphate Lower phosphate Alanine Glutamic Aspartic &Alanine Asparagine Succinic acid Citric acid area Unknown (Rf a For
identification
397
METABOLISM
80 min.
58.5 1.3 3.9 0 Trace 14.3 10.4 3.9 0 5.2 Trace Trace
areaa areaa
0.8)
-i
beet
conditions Barley
Na 80 min.
Air 16 hr.
97.9 0 0 0 1.1 0 0 0 0 0 0 0
26.5 21.2 Trace Trace 6.8 7.6 3.8 1.5 6.1 0 2.3 19.7
see text.
of the cleavage products. In sedoheptuolse metabolism in the plant, the S-carbon compound appears to have been removed by conversion to alanine and to glyceric acid, whereas the 4-carbon sugar phosphate accumulated unchanged. The action of either one or both of these enzymes in vivo might account for the present results. In the dark under air, sedoheptulose was utilized in plant respiration, since the Cl4 activity appeared in products resulting from respiratory activity (Table III). However, sedoheptulose was not metabolized in the dark in a nitrogen atmosphere, which again indicates that the alternate sugar metabolism cycle is aerobic. The difference between the light and dark metabolism of sedoheptulose-04 in a nitrogen atmosphere might at first appear significant, since there was virtually no utilization of the sedoheptulose in the dark but a substantial utilization in the light. The results, however, may be explained on the basis that, in the light, cellular anaerobiosis was not realized in these short-time experiments owing to some photosynthesis while more anaerobic conditions were obtained in the dark. This explanation is supported by the fact that sedoheptulose was metabolized, however, in the dark in an oxygen-containing atmosphere of air or combined nitrogen and oxygen.
398
N. E. TOLBEHT
AND L. P. ZILL
Sedoheptulosan-Cl4 was also given to Wintex barley leaves in experiments identical to those with the free sugar. The sedoheptulosanCl4 was prepared from sedoheptulose-Cl4 by boiling with Dowex 50 in the acid form for 1 hr. (12), after which the two forms were separated by strip-paper chromatography using phenol-water solvent. The barley leaves were allowed to sit for 24 hr. in the sedoheptulosan-C*4.When the experiments were run in the dark, there was no utilization of the sedoheptulosan-Cl4 within the leaf, while a sedoheptulose-Cl4 control was almost completely utilized in respiration. In experiments run in the light, less than 2% of the sedoheptulosan-Cl4 was converted to four other spots on the chromatogram, and these compounds were all unknowns. SUMMARY
1. Sedoheptulose-Cl4 in leaves in the light and in air is rapidly converted to sucrose. In similar experiments in the dark the sedoheptulose is metabolized to respiratory products such as glutamic, aspartic, and succinic acids. 2. By short-time experiments in the light and in a nitrogen atmosphere, major early products formed from sedoheptulose-C?4are alanine, glyceric acid, and a 4-carbon sugar phosphate. The data suggest cleavage of sedoheptulose to a triose and a tetrose. 3. Sedoheptulosan-Cl4 is not metabolized in barley leaves. REFERENCES 1. BENSON, A. A., BASSHAM, J. A., CALVIN, M., HALL, A. G., HIRSCH, H. E., KAWAQUCHI, S., LYNCH, V., AND TOLBERT, N. E., J. Biol. Chem. 196, 703 (1952). 2. AXEL~OD, B., BANDURSKI, R. S., GREINER, C. M., AND JANG, R., J. Biol. Chem. 202, 619 (1953). 3. HORECKER, B. L., SMYRNIOTIS, P. Z., AND SEEGMILLER, J. E., J. Biol. Chem. 193, 383 (1951). 4. SEEGMILLER, J. E., AND HORECKER, B. L., J. Biol. Chem. 194, 261 (1952). 5. HORECKER, B. L., AND SMYRNIOTIS, P. Z., J. Am. Chem. Sot. 74,2123 (1952). 6. HORECKER, B. L., AND SMYRNIOTIS, P. Z., J. Am. Chem. Sot. 76,202l (1953). 7. TOLBERT, N. E., AND ZILL, L. P., Plant Physiol., in press. 8. BENSON, A. A., BASSHAM, J. A., CALVIN, M., GOODALE, T. C., HAAG, V. A., AND STEPKA, W., J. Am. Chem. Sot. 73, 1710 (1950). 9. BLOCK, R. J., LESTRANGE, R., AND ZWEIG, G., “Paper Chromatography.” Academic Press, Inc., New York, 1952. 10. DENISON, F. W., AND PHARES, E. F., Anal. Chem. 24, 1628 (1952). 11. HOUQH, L., JONES, J. K. N., AND WADMAN, W. H., J. Chem. Sot. 1960, 1702. 12. LA FOROE, F. B., AND HUDSON, C. S., J. Biol. Chem. 30.61 (1917).
of Sedoheptulose-04 in Plant Leaves’ N. E. Tolbert
From
the Biology
Division,
Oak
Ridge
Received
and L. P. ZiU National
October
Laboratory,
Oak
Ridge,
Tennessee
26, 1953
The importance of sedoheptulose in the path of carbon in photosynthesis was first shown by identifying its sugar phosphate as an early labeled product during light fixation of NaHCY40a by a varied group of photosynthetic organisms (1). During studies of the alternate or glucose shunt metabolism pathway in plants, sedoheptulose phosphate was identified as a product arising from pentose phosphates (2). Thus this 7-carbon sugar appears to function in one or more cyclic processes in the plant involving both photosynthesis and respiration. In animals and yeast, sedoheptulose 7-phosphate has been found to be an intermediate in the oxidation of hexose monophosphate by the alternate metabolism pathway (3-5). However, the further metabolism of sedoheptulose and the completion of the alternate glucose metabolism cycle has remained obscure. Recently, an enzyme from yeast, transaldolase, has been reported which is capable of breaking the ‘I-carbon chain of sedoheptulose 7-phosphate between the third and fourth carbons (6). An investigation is here reported of products formed from uniformly labeled sedoheptulose-Cl4 in plants during short-time experiments in vivo. The results indicate that sedoheptulose metabolism in plant depends greatly on the atmospheric conditions, and confirm the presence of a 4-carbon sugar phosphate as an intermediate product. PROCEDURE Approximately uniformly labeled sedoheptulose-Cl4 with a specific activity of 3.7 microcuries (Gc.)/mg. was prepared by biosynthesis (7). Leaves, removed from the greenhouse during the middle of the day, were set in a highly radioactive water solution containing l-2 mg./ml. of the sedoheptulose1 Work performed Energy Commission.
under
Contract
No. 392
W-7405-eng-26
for
the
U.
S. Atomic
SEDOHEPTULOSE-C’4
393
METABOLISM
Ci4so that only the bottom of the leaf was in the solution. The rate of movement of the sugar solution into the upper portions of the leaves was found to depend on the nature of the plant and the degree of wilting of the leaf. With a thin-window laboratory monitor, considerable Cl4 activity could usually be detected in the leaf tops within 2 min. after they had been placed in the solution. The leaves, dipping into the sedoheptulose solution, were kept in a glass chamber with a circulating atmosphere which could be varied, and the whole chamber was surrounded by a water cooling bath. Light intensities of 500 ft.-candles or higher were obtained from a Photoflood bulb. At the end of the experiment the leaves were ground in liquid nitrogen, and the powder was taken up in water and immeditely heated in a boiling water bath. This killing procedure is sufliciently rapid to prevent appreciable phosphatase action on phosphate esters. After centrifugation to remove the water-insoluble residue, the supernatant was reduced in vacua to a small volume. Products formed in the plant from the sedoheptulose were identified by two-dimensional paper chromatography (8). The solvents were phenol-water in the first direction and butanol-propionic acid-water in the second direction. Any products volatilized by this treatment would not be detected. Compounds were located on the chromatogram by No-Screen x-ray film, and the spots were counted with an end-window Geiger tube. Thus each spot was expressed as a percentage of the total activity on the chromatogram and each vertical line in Tables I, II, and III represents one chromatogram. The compounds were identified by specific color spray tests (9) and by elution and cochromatography with known compounds. Organic acids were rechromatographed in ether-acetic acid-water solvent (10). Phospoate esters of organic molecules were rechromatographed in phenol-formic acid-water (336 g. phenol, 10 g. formic acid, 172 g. water).2 The phosphate esters were also treated with phosphatase (Polidase), and the organic moiety was identified (1). The identification of products listed in the tables as areas was not carried further than a comparison of their Rf values with those given by Benson et al. (8). The labeled sucrose spot was eluted with water, and part of the eluate was rechromatographed with sucrose and part was hydrolyzed with Dowex 50 in the acid form for 30 min. at 100°C. followed by rechromatography with carrier glucose and fructose. The sugars were detected by aniline-trichloroacetic acid (TCA) spray test (II), and the radioactivity was found to coincide with the added carriers. Glucose and fructose were identified by their RI values from the extensive work on the use of chromatography in photosynthesis (8), and they were also eluted, rechromatographed with carrier, and detected by aniline-TCA spray. The same procedures were followed for identification of ribulosea and ribose. As in the case of glucose and fructose, the keto sugar, ribulose, had a sufficiently larger IZf value in both of the solvents being used to effect separation from the corresponding aldehyde sugar, ribose. Similar procedures were used to t,entatively identify the four carbon sugars. Erythrose4 and erythrulosea were detected on the chromatogram by aniline-TCA 2 Directions from R. H. Burris, University of Wisconsin. 3 Ribulose was kindly furnished by A. A. Benson. 4 Erythrose was kindly furnished by H. S. Isbell, and Chemical Company.
erythrulose
by
Sigma
394
N.
E.
TOLBERT
AND
L.
P.
ZILL
spray, since these 4-carbon sugars give a visible yellow color and a brilliant yellow fluorescence under short-wavelength ultraviolet radiation immediately after spraying and heating at 100°C. for 2 min. Under the chromatographic conditions, both tetroses had so nearly the same R/s that they were not well separated. These values in phenol-water solvent were about 0.77, and in butanol-propionic acidwater about 0.43. Thus all the sugars on the two-dimensional chromatograms were located on a narrow band out from the origin which passed first through the hexoses, then through ribose and ribulose, next through the tetroses, and lastly through dihydroxyacetone and glyceraldehyde. RESULTS
AND
DISCUSSION
The pattern for metabolism of sedoheptulose did not vary extensively among sugar beet, barley, or tobacco leaves, but did depend on the environmental conditions of the experiments. In Table I are tabulated the results from representative experiments with sugar beet leaves in the light, and in Table II are data with Wintex barley leaves. Somewhat similar results were obtained with tobacco leaves. Rates of conversion of sedoheptulose to other products can be estimated by noting the change in percentage of Cl4 in the compounds as the time of the experiments is increased. These rates are only indicative of the process involved, since they depend to a large extent on the rate at which the radioactive sedoheptulose solution moved into the leaf by translocation. Leaves of all three plants, when placed in the light and air, rapidly converted sedoheptulose-Cl4to sucrose labeled in both the glucose and fructose portions. This conversion was so rapid that intermediates did TABLE Percentage
of Total Metabolism
I
Cl4 in the Products from Sedoheptulose-04 bu Suaar Beet Leaves in the Liaht Experimental
compounds
Sedoheptulose Sucrose Raflinose area Upper phosphate Lower phosphate Alanine Glutamic Aspartic
Air
area area
conditions Nitrogen
N, and (21
31 min.
50 min.
55 mill.
120 min.
76.1 17.0 2.3 Trace Trace 4.6 0 0
31.9 59.0 0 0 0 0 2.1 3.2
93.7 0.4 Trace 1.7 0.4 3.8 0 0
47.0 41.8 0 Trace Trace 0 7.3 3.6
SEDOHEPTULOSE-C14
TABLE Percentage
of Total. Metabolism
395
METABOLISM
II
Cl4 in the Products by Barley Leaves”
from Sedoheptulose-Cl” in the Light
Experimental
I
conditions
Air 11 min.
Sedoheptulose Sucrose Ribose Upper phosphate area Lower phosphate area Alanine Glyceric acid Tetrose sugar area Glycolic acid Unknown (R, - 0.8) a Wintex
78 5 0 4 Trace 5 8 0 Trace 0
-
T 16 min. 48 16 0 10 Trace 5 9 0 Trace 8
-
82 min.
9 min.
35 23 0 7 Trace 8 2 5 Trace 17
75.0 0 0 4 2 10 7 0 Trace 0
-
--
-
Nitrogen
-
20min.
60 min.
_
66.4 8.6 0 10.2 5.5 7.8 1.5 Trace Trace Trace
42.7 6.6 2.6 8.6 6.6 21.7 2.0 0 Trace 7.8 I
barley.
not accumulate significant amounts of radioactivity, and sucrose was the only major product. In sugar beet leaves the intermediary organophosphate compounds were hardly detectable on the chromatogram. Dependence of this conversion of sedoheptulose to sucrose on aerobic conditions is indicated by the appearance of only trace amounts of Cl4 in the sucrose in sugar beet leaves held in a nitrogen atmosphere and in the light. In a combined atmosphere of nitrogen and oxygen, however, the sedoheptulose was again rapidly converted in the light to sucrose in the sugar beet leaf. In experiments run in the light and with a nitrogen atmosphere, the conversion of sedoheptulose to sucrose was inhibited, so that the intermediate phosphate esters were formed in sufficient amounts to permit chromatographic identification. The leaves in the light and nitrogen atmosphere were probably not strictly anaerobic at the cellular level owing to some photosynthesis. Under these conditions the major products formed in cu. 1 hr. were alanine, glyceric acid, and compounds in the phosphate area. Chromatographic procedures used to separate all the plant constituents did not give sufficient separation of the phosphate esters, since they did not move far from the origin in either solvent. A generalization could be made that the upper phosphate area would contain the monophosphate esters of low-molecular-weight compounds
396
N.
E.
TOLBERT
AND
L.
I’.
ZILL
such as phosphoglyceric acid, and the lower phosphate area would contain higher molecular-weight phosphates such as sugar phosphates and the diphosphate esters. The phosphate areas were therefore eluted and treated with phosphatase, and the organic moieties were identified. In these short-time experiments a major product from such treatment of the upper phosphate area of all three plants cochromatographed with the tetrose sugars, erythrulose and erythrose. In short-time experiments in the light-andnitrogen atmosphere, other detectable sugars from the phosphate areas were glucose and fructose, but no ribose or ribulose. The alanine and glyceric acid, which were formed in large amounts in these short-time experiments, might have arisen in vivo from the 3-carbon product from the first cleavage of the original sedoheptulose. The results suggest that the initial breakdown products of sedoheptulose are a triose and a 4-carbon sugar phosphate. The triose was converted to alanine and glyceric acid, but the 4-carbon sugar phosphate accumulated as such, since the photosynthesis cycle could not function and aerobic respiration was blocked. Longer experiments with barley leaves were run in which the exposure time to sedoheptulose-Cl4 was 2 hr. in the light with a nitrogen atmosphere. In this series, the phosphate ester areas after phosphatase treatment gave large amounts of CY4-labeled compounds with R, values of ribulose, glucose, and moderate amounts of tetroses, ribose, fructose, sedoheptulose, and two unknown compounds which had chromatographic characteristics of sugar acids. The products are characteristic of the alternate glucose metabolism cycle and of the path of carbon in photosynthesis. These in vivo observations appear to be similar to the results of the investigation of an enzyme, transaldolase, which cleaves sedoheptulose 7-phosphate and was so named because it catalyzes a transfer of aldol linkages rather than hydrolytic cleavage (6). For that reaction glyceraldehyde 3-phosphate was used for combining with the dihydroxyacetone group of the first three carbons of sedoheptulose 7-phosphate, and preliminary evidence was presented for the existence of a tetrose from the lower carbons of sedoheptulose. In the plant the initial reaction also appears to be a similar break between carbon atoms 3 and 4. However, the in vivo data from these experiments may be interpreted in a manner analogous to the aldolase reaction with a t,etrose phosphate formation from the lower portion of the 7-carbon sugar. In the aldolase reaction with hexoses, the equilibrium lies toward the fructose 1 ,gdiphosphate. The reaction can be shifted in the other direction by removal of one
SEDOHEPTULOSE-C14
TABLE Percentage
III
of I’otal Cl4 in the Products from Sedoheptulose-(2’14 Metabolism in Leaves in the Dark Experimental
Compounds
Sugar Air
Sedoheptulose Sucrose Upper phosphate Lower phosphate Alanine Glutamic Aspartic &Alanine Asparagine Succinic acid Citric acid area Unknown (Rf a For
identification
397
METABOLISM
80 min.
58.5 1.3 3.9 0 Trace 14.3 10.4 3.9 0 5.2 Trace Trace
areaa areaa
0.8)
-i
beet
conditions Barley
Na 80 min.
Air 16 hr.
97.9 0 0 0 1.1 0 0 0 0 0 0 0
26.5 21.2 Trace Trace 6.8 7.6 3.8 1.5 6.1 0 2.3 19.7
see text.
of the cleavage products. In sedoheptuolse metabolism in the plant, the S-carbon compound appears to have been removed by conversion to alanine and to glyceric acid, whereas the 4-carbon sugar phosphate accumulated unchanged. The action of either one or both of these enzymes in vivo might account for the present results. In the dark under air, sedoheptulose was utilized in plant respiration, since the Cl4 activity appeared in products resulting from respiratory activity (Table III). However, sedoheptulose was not metabolized in the dark in a nitrogen atmosphere, which again indicates that the alternate sugar metabolism cycle is aerobic. The difference between the light and dark metabolism of sedoheptulose-04 in a nitrogen atmosphere might at first appear significant, since there was virtually no utilization of the sedoheptulose in the dark but a substantial utilization in the light. The results, however, may be explained on the basis that, in the light, cellular anaerobiosis was not realized in these short-time experiments owing to some photosynthesis while more anaerobic conditions were obtained in the dark. This explanation is supported by the fact that sedoheptulose was metabolized, however, in the dark in an oxygen-containing atmosphere of air or combined nitrogen and oxygen.
398
N. E. TOLBEHT
AND L. P. ZILL
Sedoheptulosan-Cl4 was also given to Wintex barley leaves in experiments identical to those with the free sugar. The sedoheptulosanCl4 was prepared from sedoheptulose-Cl4 by boiling with Dowex 50 in the acid form for 1 hr. (12), after which the two forms were separated by strip-paper chromatography using phenol-water solvent. The barley leaves were allowed to sit for 24 hr. in the sedoheptulosan-C*4.When the experiments were run in the dark, there was no utilization of the sedoheptulosan-Cl4 within the leaf, while a sedoheptulose-Cl4 control was almost completely utilized in respiration. In experiments run in the light, less than 2% of the sedoheptulosan-Cl4 was converted to four other spots on the chromatogram, and these compounds were all unknowns. SUMMARY
1. Sedoheptulose-Cl4 in leaves in the light and in air is rapidly converted to sucrose. In similar experiments in the dark the sedoheptulose is metabolized to respiratory products such as glutamic, aspartic, and succinic acids. 2. By short-time experiments in the light and in a nitrogen atmosphere, major early products formed from sedoheptulose-C?4are alanine, glyceric acid, and a 4-carbon sugar phosphate. The data suggest cleavage of sedoheptulose to a triose and a tetrose. 3. Sedoheptulosan-Cl4 is not metabolized in barley leaves. REFERENCES 1. BENSON, A. A., BASSHAM, J. A., CALVIN, M., HALL, A. G., HIRSCH, H. E., KAWAQUCHI, S., LYNCH, V., AND TOLBERT, N. E., J. Biol. Chem. 196, 703 (1952). 2. AXEL~OD, B., BANDURSKI, R. S., GREINER, C. M., AND JANG, R., J. Biol. Chem. 202, 619 (1953). 3. HORECKER, B. L., SMYRNIOTIS, P. Z., AND SEEGMILLER, J. E., J. Biol. Chem. 193, 383 (1951). 4. SEEGMILLER, J. E., AND HORECKER, B. L., J. Biol. Chem. 194, 261 (1952). 5. HORECKER, B. L., AND SMYRNIOTIS, P. Z., J. Am. Chem. Sot. 74,2123 (1952). 6. HORECKER, B. L., AND SMYRNIOTIS, P. Z., J. Am. Chem. Sot. 76,202l (1953). 7. TOLBERT, N. E., AND ZILL, L. P., Plant Physiol., in press. 8. BENSON, A. A., BASSHAM, J. A., CALVIN, M., GOODALE, T. C., HAAG, V. A., AND STEPKA, W., J. Am. Chem. Sot. 73, 1710 (1950). 9. BLOCK, R. J., LESTRANGE, R., AND ZWEIG, G., “Paper Chromatography.” Academic Press, Inc., New York, 1952. 10. DENISON, F. W., AND PHARES, E. F., Anal. Chem. 24, 1628 (1952). 11. HOUQH, L., JONES, J. K. N., AND WADMAN, W. H., J. Chem. Sot. 1960, 1702. 12. LA FOROE, F. B., AND HUDSON, C. S., J. Biol. Chem. 30.61 (1917).