Photosynthesis In Elodea canadensis Michx.

[Pages:3]Plant Physiol. (1977) 59, 1133-1135

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Photosynthesis In Elodea canadensis Michx.

FOUR-CARBON ACID SYNTHESIS1

Received for publication December 13, 1976 and in revised form February 18, 1977

DAVE DEGROOTE AND ROBERT A. KENNEDY Department of Botany, University of Iowa, Iowa City, Iowa 52242

ABSTRACT

Experiments to determine the early labeled photosynthetic products in Elodea canadensis show that after 2 seconds of exposure to NaH14CO3, 45% of the 14C incorporated is located in malate and aspar-

tate. Phosphoglyceric acid and sugars account for 27% of the label

during similar exposures. Equivalent amounts of organic acids and C3 cyde products are present after 8 seconds. Four-carbon acids remain relatively unchanged throughout the first 45 seconds of exposure, while sugars increase in a linear fashion. Enzyme asys indicate that ribulose diphosphate and phosphoenolpyruvate carboxylase enzymes are present in a ratio of approximately 2:1. It appears that E. canadensis is able to

synthesize significant amounts of four-carbon acids via (-carboxybtion

and this may play a role in maintaining a pH favorable for carboxylation in aquatic plants.

Recently, several studies have shown that aquatic plants can synthesize and metabolize four-carbon acids. Dohler (11) found that aspartate was the earliest labeled photosynthetic product in Anacystis nidulans after a few seconds of exposure to 14C, and three blue-green algal species, including Anacystis, were later observed to have up to five times more PEP carboxylase2 than RuDP carboxylase (9). Most recently, Benedict and Scott (5) reported that C4 acids were the earliest labeled photosynthetic products in Thallasia. In still other studies, Brown and associates (6) investigated primary photosynthetic products in two flowering plants, Egeria densa and Lagarosiphon major, which were growing in fresh water lakes in New Zealand. They noted that C4 acid production never exceeded 30% of the total label incorporated in either species, and that photorespiration rates were similar to those of C3 plants. In contrast, another aquatic angiosperm, Elodea, had a compensation point of zero under low 02 concentrations. Elodea also differed from the other two species with respect to light saturation point. It did not photosaturate up

to 3,000 ft-c while Egeria and Lagarosiphon saturated at ap-

proximately 1,000 ft-c. Along with the aquatic species just described, other plant

tissues or organs have been shown to utilize PEP carboxylase to synthesize C4 acids (13, 25). It is clear from these studies that certain aspects of the C4 pathway, notably the synthesis and metabolism of four-carbon acids, relatively high PEP carboxylase levels and the resultant low 13C/12C ratios, are present in

1 This study supported by National Science Foundation Grant BMS75-09931.

2 FDP: fructose 1,6-diphosphate; PEP carboxylase: phosphoenolpyruvate carboxylase; PEP: phosphoenolpyruvate; PGA: 3-phosphoglyceric acid; RuDP carboxylase: ribulose diphosphate carboxylase; RuDP: ribulose 1,5-diphosphate.

more than just C4 plants. The purpose of the present investigation was to examine the mechanism and pattern of CO2 assimilation in an aquatic plant species and to relate that information to the pathway of carbon assimilation in C4 plants.

MATERIALS AND METHODS

Elodea canadensis Michx. was grown in a greenhouse pond at 20 to 25 C with a pH of 8.5 to 9.2. Prior to use for incorporation studies, Elodea tissue was collected and placed in a two-liter flask containing 20 Mm NaH12CO3 (pH 6.5) and illuminated at 1,200 ,ueinsteins m-2 sec-' (2800 ft-c) at 20 C. This preincubation period was generally 30 to 45 min and experiments were initiated when a high rate of 02 evolution was observed from the cut end of the sprigs. Incorporations were carried out in a waterjacketed chamber containing 20 Mm NaH14CO3 (pH 6.5) with a light intensity of 1,200 Meinsteins m-2 sec-Q. Assimilation experiments were terminated by removing the leaves and immediately plunging them into liquid N2. For pulse-chase experiments, tissue was removed from the incorporation chamber after exposure to NaH14CO3 and placed in a flow through beaker. Unlabeled bicarbonate at the same concentration was then passed over the sprigs at a rate of 1 liter/min and the tissue was killed as described above. All subsequent extraction, separation, and identification procedures have been reported previously (17). Chlorophyll was determined according to Arnon (1) and total protein as given by Lowry et al. (19).

Crude enzyme extracts were prepared by homogenizing Elodea tissue in a chilled mortar and pestle. The grinding buffer consisted of 195 mm K-phosphate (pH 7), 10 mM MgCl2, 20 mM 2-mercaptoethanol, 0.2 mm EDTA, and 16 g/1 PVP (3). The resulting homogenate was centrifuged at 10,OOOg at 12 C and the supematant was used for enzyme assays.

The ribulose diphosphate carboxylase (RuDP Case; EC 4.1.1.39) assay described by Chu and Bassham (2, 8) was used with some modifications. The reaction mixture contained 25 mM Tricine (pH 8), 10 mM MgCl2, 5 mm 2-mercaptoethanol, 5.5

mM RuDP, 0.05 mm NADPH, 1 mm FDP, 1.25 AM NaH"4C03

and 5 to 25 ,ul of enzyme extract. Final volume of the reaction mixture was 100 ,lI. A 5-min preincubation period was employed and reactions were initiated by the addition of RuDP. Assays were terminated after 1 or 2 min by the addition of 50 ,ul of glacial acetic acid. Aliquots of 50 ,l were taken, dried, and counted by liquid scintillation spectroscopy (15). Enzyme activities were expressed on the amount of Chl/g leaf tissue, rather than that contained in the enzyme extract.

Phosphoenolpyruvate carboxylase (PEP Case; EC 4.1.1.32) activity was assayed using a reaction mixture that contained 25 mM Tricine (pH 8.3), 10 mM MgC12, 5 mm 2-mercaptoethanol, 15 mM PEP, 15 mM sodium L-glutamate, 1.25 ,Mm NaH14CO3, and 5 to 25 ,l of tissue extract in a final volume of 100 ul. Assays were terminated as previously described.

1133

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DEGROOTE AND KENNEDY

RESULTS

Time course experiments of NaH14CO3 incorporation in Elodea indicate that after 2 sec the most heavily labeled product is malate (Fig. 1). Total C4 acids, malate plus aspartate, account for 45% of the label in this period, while PGA contains 15 % and sugars 12%. Malate alone increased to 40% of the total 14C fixed after 8 sec of exposure; PGA and sugars increase to 27 and 23%, respectively. After 45 sec, the malate pool remains essentially unchanged indicating that the turnover rate of the four-carbon acid pool may be slow.

PGA and sugars show curves which are typical of C3 pathway products in the first 45 sec of exposure to 14C. PGA drops sharply at first, while sugars continue to rise. There is a small increase in the PGA pool after 45 sec coinciding with a decrease in C4 acids. Sugars increase linearly in the period from 10 to 120

sec. Pulse-chase experiments again illustrate the slow turnover rate

of four-carbon acids (Fig. 2) (7, 16). The malate pool is almost completely retained after 60 sec in unlabeled bicarbonate. C3 products, on the other hand, show an expected increase in labeling during the chase phase.

Activities of PEP and RuDP carboxylase are shown in Table I. Spinach, a typical C3 species, has a RuDP to PEP ratio of 6; whereas corn, a C4 plant, has a ratio of 0.2. The RuDP to PEP carboxylase ratio in Elodea is intermediate between these two plants with a value of nearly 2.

K

14c 02

50 45-

40 - K yO

3530-

25-

20-

10 0

0 10-

a 5. 0

L) C

0

s 76

0

A_v- - - v II

8 10

30

0

0

Plant Physiol. Vol. 59, 1977

Ma]ate

Aspartate PEP

~~~~PEP

60

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DISCUSSION

In the present experiments, E. canadensis was found to have relatively higher levels of PEP carboxylase and more synthesis of

50-

45 -

0

1

40-

35Total C4 acids

30-

25 -

20-

Is -

0

0 0

I0-

5-

vv V 10 20 30 40 50

Mabw

Asportate

PEP v

100

120

0 -

1- 50-

0

45-

Sugars

40-

35-

30-

25-

d\ 20

15-

PGA

10- #

5-

\ #0

II

10 20 30 40 50

100 120

Time (Seconds)

FIG. 1. Time course of NaH14CO3 labeling of early photosynthetic products in E. canadensis expressed as a percentage of total 14C incorporated. The photosynthetic rate average 15 gmol/mg Chl * hr.

8 10

30

60

Time (Seconds)

FIG. 2. Pulse-chase experiment in which Elodea leaves were exposed

to NaH14CO3 for 8 sec followed by a NaH12CO3 chase for an additional

60 sec.

four-carbon acids in short time periods than would normally be

expected for a C3 plant. C4 acids were labeled twice as heavily as C3 products after 2 sec and there was 34% more PEP carboxylase relative to RuDP carboxylase in Elodea than in spinach. In

other studies, Elodea has been shown to have a low CO2 compensation point and high light saturation (6). Other characteristics of photosynthesis in this species illustrate distinct differences

between it and C4 plants. After the shortest exposure times to 14C02, labeled C4 acids contained less than 50% of the total radioactivity; whereas in C4 plants this percentage is generally

about 90% (12, 17). Also, from pulse-chase experiments, turnover of C4 acids did not occur as rapidly in this study as occurs in

C4 plants. The pattern of carbon metabolism in Elodea most closely

resembles that found in Anacystis by Dohler (11). In that study,

aspartate was the major labeled product of CO2 fixation experiments containing approximately 50 to 60% of the total "4C fixed in the first 1 to 2 min, while PGA had 15 to 25% of the label. Quite similar labeling patterns were also reported for Thallasia, a marine grass (5). What all three of these organisms have in common is an aqueous environment. The absolute amounts of carbon in an aqueous system are about 50 times those in air at 20 C but the form of carbon available, CO2 versus HCO3-, is very pH-dependent (6, 22, 24). Carbon availability is compounded by the fact that the diffusive pressure of CO2 in water is 100,000 times smaller than in air (0.16 cm2 sec-1 in air compared to 1.6 x

10-6 cm2 sec-1 in water) (22, 23). If RuDP carboxylase uses CO2

(10) and PEP carboxylase uses bicarbonate (20), then carboxylation of PEP and four-carbon acid formation would be favored

under conditions above a pH of 6.5. Since these pH levels are

frequent in ponds (6, 14), C4 acid synthesis would result in plants

which contained even moderate amounts of PEP carboxylase.

Plant Physiol. Vol. 59, 1977

FOUR-CARBON ACID SYNTHESIS IN ELODEA

Table I. Protein, chlorophyll, RuDP and PEP carboxylase enzyme activities for Elodea canadensis, Spinacia oleracea and Zea mays

Elodea Spinacia Zea

RuDP

PEP

RuDP/PEP

Carboxylase Carboxylase Ratio

lmol/mg Chl hr

27.9 + 2.2 14.21 + 2.5 1.96

Protein

mg/g fresh wt 38.1

66.6

11.1

6.00

69.3

31.5

147.9

0.21

130.5

Chlorophyll

0.72 2.10 2.31

2.5 2.9 3.44

1 135

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1Standard deviation. Standard error: 0.84 for RuDP Carboxylase and 0.72 PEP Carboxylase.

Two of the three plants discussed above, Elodea (Table I) and Anacystis (9), have been shown to contain relatively high levels of this enzyme.

The capability to synthesize and metabolize four-carbon acids (4), and the resultant low 13C/'2C ratios, does not mean that the three aquatic species discussed above possess other structural and functional features of C4 plants (18). From our observations (unpublished results), Elodea leaves are simply two cells thick and the midrib is the only vasculature present. Chloroplasts show extensive grana with no observable peripheral reticulum. In all respects they appear similar to C3 plant chloroplasts as earlier described by Muhlenthaler and Frey-Wyssling (21). Benedict and Scott (5) also found that C4 acid synthesis in Thallasia did not correlate with Kranz anatomy.

The presence of both C3 and C4 cycle enzyme systems in some aquatic plants may mean that under various environmental conditions one, the other, or both enzymes may be actively fixing carbon. Functionally, this may be important in the maintenance of a cytoplasmic pH that is slightly acid. In the acidic condition, bicarbonate ions would be protonated to H2CO3 which in turn would increase the level of free CO2 that diffuses into the cell, thus creating a gradient of CO2 from the cytoplasm to the chloroplast that would facilitate Calvin cycle activity in that organelle. The slow turnover rate of four-carbon acids in the present experiments may also mean they represent a pool of CO2 that could be drawn upon by decarboxylation. Regardless of the functional significance it appears that several aquatic plants, as well as other assimilating plant tissues and organs (13, 25), are able to synthesize four-carbon acids. While these examples at first seem anomalous with respect to strict C3-C4 classification, they do not possess additional features of C4 plants and should not be confused with the latter.

Acknowledgment -We are grateful to R. M. Muir for the initial suggestion and his continuous support of this research.

LITERATURE CITED

1. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol 24: 1-15

2. BAHR JT, RG JENSEN 1974 Ribulose diphosphate carboxylase from freshly ruptured

spinach chloroplasts having an in vivo Km(CO2). Plant Physiol 53: 39-44 3. BALDRY CW, C BUCKE, J COOMBS 1971 Progressive release of carboxylating enzymes

during mechanical grinding of sugarcane leaves. Planta. 97: 310-319 4. BAssHA JA, AA BENSON, M CALVIN 1950 The path of carbon in photosynthesis. VIII.

The role of malic acid. J. Biol. Chem. 185: 781-787 5. BENEDICT CR, JR ScOTr 1976 Photosynthetic carbon metabolism of a marine grass. Plant

Physiol 57: 876-880 6. BROWN JMA, FI DRMoGooLE, MW TOWSEY, J BROWSE 1974 Photosynthesis and photores-

piration in aquatic macrophytes. In RL Bieliski, AR Ferguson, MM Cresswell, eds, Mechanisms of Regulation of Plant Growth. The Royal Society of New Zealand, Wellington pp 243-249 7. CHEN TM, RH BROWN, CC BLACK 1971 Photosynthetic '4CO2 fixation products and activities of enzymes related to photosynthesis in bermudagrass and other plants. Plant Physiol 47: 199-203 8. CHU DK, J BASSHAM 1975 Regulation of ribulose-1,5-diphosphate carboxylase by substrates and other metabolites. Plant Physiol. 55: 720-726 9. COLMAN B, KH CHENG, RK INGLE 1976 The relative activities of PEP carboxylase and RuDP carboxylase in blue-green algae. Plant Sci Lett 6: 123-127 10. COOPER TG, D FILMER, M WISHNICK, MD LANE 1969 The active species of "CO2" utilized by ribulose diphosphate carboxylase. J Biol Chem 244: 1081-1083 11. D6HLER G 1974 C4 Weg der Photosynthese in der Blaualge Anacystis nidulans. Planta 118: 259-269 12. HATCH MD, CR SLACK 1966 Photosynthesis by sugarcane leaves. A new carboxylation reaction and the pathway of sugar information. Biochem J 101: 103-111 13. HEDLEY CL, AO ROWLAND 1975 Changes in the activities of some respiration and photosynthetic enzymes during the early leaf development of Antirrhinum majus L. Plant Sci Lett 5: 119-126

14. HOUGH RA 1974 Photorespiration and productivity in submersed aquatic vascular plants. Limnol Oceanogr 19: 912-927

15. KENNEDY RA 1976 Relationship between leaf development, carboxylase enzyme activities and photorespiration in the C4 plant Portulaca oeracea L. Planta 128: 149-154

16. KENNEDY RA, WM LAETSCH 1974 Formation of '4C-labeled alanine from pyruvate during short term photosynthesis in a C4 plant. Plant Physiol 54: 608-611

17. KENNEDY RA, WM LAETSCH 1973 Relationship between leaf development and primary photosynthetic products in the C4 plant Portulaca okracea L. Planta 115: 113-124

18. LAETSCH WM 1974 The C4 syndrome: a structural analysis. Annu Rev Plant Physiol 25: 2752

19. LowRY OH, NJ ROSEBROUGH, AL FARR, Rl RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 262-275

20. MARUYAMA H, RL EASTERDAY, HC CHANG, MD LANE 1966 The enzymatic carboxylation

of phosphoenolpyruvate. J Biol Chem 241: 2405-2412 21. MUHLENTHALER VK, A FREY-WYSSLING 1959 Entwicklung und Strukur der Proplastiden.

Biophys Biochem Cytol 6: 507-512 22. NOBEL PS 1974 Biophysical Plant Physiology. WH Freeman & Co, San Francisco 23. RAVEN JA 1970 Exogenous inorganic carbon sources in plant photosynthesis. Biol Rev 45:

167-222 24. UMBREIr WW, RH BuRRs, JF STAUFFER 1959 Manometric Techniques. Burgess Publish-

ing Co, Minneapolis pp 18-27 25. WILLMER CM, WR JOHNSON 1976 Carbon dioxide assimilation in some aerial plant organs

and tissues. Planta 130: 33-37

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