Photosynthetic Carbon Metabolism of a Marine Grass1Received for publication January 20, 1976 and in revised form February 24, 1976
C. RoY BENEDICT AND JAMES R. ScorrDepartment of Plant Sciences Texas A & M University, College Station, Texas 77843
ABSTRACT
The 813C value of a tropical marine grass Thalassia testudinum is-9.04%.. This value is similar to the 813C value of terrestrial tropicalgrasses. The 813C values of the organic acid fraction, the amino acidfraction, the sugar fraction, malic acid, and glucose are: -11.2%.,-13.1%o, -10.1%o, -11.1%., and -11.5%., respectively. The 813C val-ues of malic acid and glucose of Thalassia are similar to the 813C valuesof these intermediates in sorghum leaves and attest to the presence ofthephotosynthetic C4-dicarboxylic acid pathway in this marine grass. Theinorganic HCO3- for the growth of the grass fluctuates between -6.7 to-2.7%o during the day. If CO2 fixation in Thalassia is catalyzed byphosphoenolpyruvate carboxylase (which would result in a -3%o frac-tionation between HCO3- and malic acid), the predicted 813C value forThalassia would be -9.7 to -5.7%o. This range is close to the observedrange of -12.6 to - 7.8%. for Thalassia and agree with the operation ofthe C4-dicarboxylic acid pathway in this plant. The early products of thefixation of HCO3- in the leaf sections are malic acid and aspartic acidwhich are similar to the early products of CO2 fiation in C4 terrestrialplants.
Electron microscopy of the leaves of Thalassia reveal thick waledepidermal cels exceedingly rich in mitochondrin and C3-type chloro-plasts. The mesophyil cells have many different shapes and surround airlacunae which contain 02 and CO2. The mesophyHl cells are highlyvacuolated and the parietal cytoplasm contains an occasional chloro-plast. This chloroplast contains grans but the lameUlar system is not asdeveloped as the system in epidermal chloroplasts. Extensive phloemtissue is present but the xylem elements are reduced in this aquatic grass.The vascular tissue is not surrounded by bundle sheath cels.
This work does not establish the exact relation between structure andfunction in Thalassia, but it does show the C4-type photosyntheticcarbon metabolism in this grss involves epidermal and mesophyfl cellsand internally produced 02 and CO2 in the air lacunae.
Tregunna et al. (20), Smith and Epstein (18), Bender (2), andTroughton (21) have shown a direct correlation between the813C values and the type of photosynthetic C metabolism. Plantswith C4 characteristics have 813C values of -9 to -19%o andplants with C3 characteristics have 813C values of -24 to -34%0.Whelan et al. (24) suggest fixation of CO2 by RuDP2 carboxylaseor PEP carboxylase may be responsible for the fractionation ofstable C isotopes by C3 and C4 plants. Bender et al. (3) andLerman et al. (11) indicate the wide range of 813C values ofCrassulacean acid metabolism plants is indicative of CO2 fixationthrough RuDP or PEP carboxylase. Wong et al. (25) have shown
I This work was supported by Grant A-482 from The Robert A.Welch Foundation.
2 Abbreviations: RuDP: ribulose-1 ,5-diphosphate; PEP: phospho-enolpyruvate; PGA: 3-phosphoglycerate; PDB: The 13C/12C ratio in thesample compared to a standard which is CO2 from the fossil carbonate ofBelemnitella americana.
the negative 813C values of the primitive photosynthetic bacte-rium Chromatium correlate with the operation of the Calvincycle in these microorganisms.
Craig (5), Parker (14), Parker and Calder (15), Smith andEpstein (18), Whelan (23), and Doohan and Newcomb (8) havereported the 813C values of a marine monocot Thalassia testu-dinum to be between -7.8 and -12.6%o. These values areindicative of a C4 metabolism. Another C4 characteristic of thisplant is its high productivity. Thayer et al. (19) showed theannual production of this grass ranges from 200 to 3000 g C/M2.The annual production of cultivated corn is 412 g C/M2. Jagels(10) and Doohan and Newcomb (8) have shown, however, theanatomy of the leaves of this tropical marine grass differs frommost terrestrial C4 grasses and lack bundle sheath cells. Thepurpose of this paper was to compare the 813C values of meta-bolic intermediates, CO2 fixation, and anatomy of this tropicalmarine grass to terrestrial C4 tropical grasses.
MATERIALS AND METHODS
Materials. Sodium 14C-bicarbonate was purchased from NewEngland Nuclear Corporation.
Plants. Plants of Thalassia testudinum were collected in Red-fish Bay, Port Aransas, Texas. The plant consists of an under-ground rhizome which produces shoots and roots. Most of theleaf samples were taken from the young tissue at the base of theleaf under or adjacent to the leaf sheath. Care was taken to washoff any epiphytic algae on these sections but compared to theolder leaf tissue the concentration of epiphytic algae is low ornegligible on young tissue under or adjacent to the leaf sheath.The leaf samples to be used for 813C analyses were dried orplaced directly in 95% ethyl alcohol. The leaf samples to be usedfor 14CO2 fixation and electron microscopy studies were broughtto the laboratory in chilled sea H20.CO2 Fixation. Leaf sections (1 x 1 cm) were cut from the base
of the leaf blades. The leaf sections were submerged in 4.9 ml ofsterile sea H2O or de-ionized H2O (pH 5.8) and equilibrated inthe light at an intensity of 1.15 x 104 ,ueinsteins/m2 - sec for 5 to10 min at 30 C. den Hartog (9) and Sculthorpe (16) have shownthe growth optimum of Thalassia is 30 C. Following the equili-bration period, 100 ,uCi of 14C-bicarbonate in 0.1 ml (specificradioactivity 1 mCi/0.142 mmol) was added to the incubationmedium. The leaf sections were allowed to fix the radioactivebicarbonate for various lengths of time, and the reactions wereterminated with boiling 80% ethyl alcohol.
Extraction of Radioactive Compounds. Following 14CO2 fixa-tion, the leaf sections were extracted 4 times with boiling 80%ethyl alcohol and 4 times with boiling 95% ethyl alcohol. Foreach extraction, the leaf sections were simmered on the hot platefor 10 to 15 min. The alcohol extracts were pooled, and formicacid was added to liberate the unreacted 14C-bicarbonate. Theextract was evaporated to dryness in vacuo. The residue waspartitioned between ethyl ether and H20. An aliquot of theether phase which contained pigments and lipids was added to ascintillation vial, evaporated to dryness with N2, dissolved inscintillation cocktail, and assayed for radioactivity. The H20
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phase was evaporated to dryness in vacuo. The residue wasdissolved in a known volume of H20, and an aliquot was assayedfor radioactivity. The H20-soluble extract was separated intobasic, acidic, and neutral fractions. The H20-soluble extract waspassed through a column (1 x 15 cm) of Dowex-50 (H+) resin.The basic fraction, consisting of '4C-amino acids, was elutedfrom the resin column with 50 ml of 1 N NH40H, and thesolution was evaporated to dryness. The effluent from theDowex-50 column was passed through a column (1 x 15 cm) ofDowex-1 (formate) resin. The acidic fraction, consisting of "4C-organic acids, was eluted from the resin column with 50 ml of 8 Nformic acid, and the solution was evaporated to dryness. Theeffluent from the Dowex-1 column, which consisted of 14C-sugars, was evaporated to dryness. The individual fractions weredissolved in H20, and an aliquot was assayed for radioactivity.
Chromatography. The amino acid, organic acid, and sugarfractions were resolved into individual components by two-di-mensional paper chromatography by the procedure of Benson etal. (4). The radioactive compounds were localized on the chro-matograms by radioautography on medical x-ray film. The "4C-compounds were cut from the chromatograms and assayed di-rectly for radioactivity by liquid scintillation counting of the filterpaper. For identification the radioactive compounds were elutedfrom the paper chromatograms and co-chromatographed withauthentic compounds followed by radioautography.813C Analysis. (500 g) Leaf sections were repeatedly extracted
with boiling 80% and 95% ethyl alcohol. The extracts werepooled and evaporated to dryness. The residue was partitionedbetween ethyl ether and H20. The H20-soluble phase was sepa-rated into amino acid, organic acid and sugar fractions by pas-sage through columns (2 x 25 cm) of Dowex resins by theprocedure described above. The individual fractions were evap-orated to dryness, dissolved in 5 to 10 ml H20 and 0.5 mlapplied stripwise to Whatman No. 3 filter papers. The extractswere separated by one-dimensional chromatography in the fol-lowing solvents: 1, equal volumes of 1-butanol-H20 (370:25 v/v) and propionic acid-H20 (180:220, v/v) for amino acids andorganic acids; and 2, ethyl acetate-acetic acid-H20 (150:50:100,v/v) for sugars. Malic acid, glucose, and fructose were analyti-cally separated by this chromatography. These compounds werelocated and eluted from the paper chromatograms, pooled andevaporated to dryness. The individual compounds were enriched1.1%o in 13C during the isolation procedure.Carbon isotope ratios were made on a 600 sector, Nier-type
mass spectrometer similar to the one described by McKinney etal. (12). All of the samples were converted to CO2 by combus-tion at 800 to 900 C over cupric oxide and in an excess 02atmosphere. The combustion products were circulated continu-ously by means of an electrically controlled Toepler pump. Afterremoval of H20 vapor and other condensable gases by passingthrough traps, the samples were cooled to dry ice temperaturesand the CO2 was distilled into a sample bulb at liquid N2 temper-atures.
Electron Microscopy. The leaf sections were fixed in 1.5%glutaraldehyde in 0.1 M cacodylate buffer pH 7.2. The tissue wasthen exposed to a phosphate buffered osmium tetroxide fixation,dehydrated with ethanol and embedded in Epon. The sectionswere stained with uranyl acetate and lead citrate. The sectionswere photographed with a Hitachi HU-1lE electron micro-scope. The technique for light microscopy involved staining 2.5-,m sections from the Epon-embedded tissue with 1% methyleneblue buffered with 1% sodium borate at 80 C.
RESULTS AND DISCUSSION
The data in Table I show the 813C values of leaf blades andmetabolic intermediates of Thalassia. The 813C value of -9.04%0for the leaf blades compares favorably with the published valuesof -7.8 to -12.6%o for similar material (5, 8, 14, 15, 18, 23).
Table I. 8'3C Values of Metabolic Intermediates
Fraction sp 13C ValuesPDB
Whole Leaf - 9.04
Organic Acid Fraction -11.2
Amino Acid Fraction -13.1
Sugar Fraction -10.1
Malic Acid -11.1
Glucose -11.5
Fructose -11.1
These high 8'3C values are similar to the high 8'3C values ofterrestrial C4 plants (2, 18, 20, 21).The 6'3C values of the organic acid, amino acid, and sugar
fractions of the marine grass are -11.2%o, - 13.1%o, and-10.1%o, respectively. More specifically, the 813C values ofmalic acid and glucose are -11.1%o and -11.5%o. The 813Cvalues of the metabolic intermediates of Thalassia are similar tothe S'3C values of these intermediates in C4 plants. In sorghum(C4 plant) the 813C values of organic acid, amino acid, and sugarfractions are: -12.3%o, -12.7%o, and -9.9%. (24). In cotton(C3 plant) the S13C values of these fractions are: -24.8%.,-33.1%o, and -26.4%o (24). The 813C values of malic acid andglucose from leaves of sorghum and cotton are: -8.9%o, -9.9%o,-21%o, and -24.6%o, respectively.The pathway of synthesis of sugars from CO2 in terrestrial C4
plants is through malic acid by the Hatch and Slack pathway.The fact that the 813C values of malic acid and glucose insorghum are similar, reflects the synthesis of glucose from the 83-COOH group of malic acid by the C4-dicarboxylic acid pathway.If glucose was synthesized exclusively through the Calvin cycle insorghum leaves the 813C value of glucose would be about - 25%.,as found in C3 plants. The fact that the 813C values of glucose andmalic acid in Thalassia are similar to the S'3C values of thesecompounds in sorghum attests to the presence of the photosyn-thetic C4-dicarboxylic acid pathway in the marine grass.
Parker (14) has shown that the 8'3C value of the inorganic Cfrom Redfish Bay is -6.7%.. He further showed that the inor-ganic C of sea H20 in an experimental pond varied 4%. between12 noon and 6 PM due to a preferential use of '2C by photosyn-thesis in the marine plants and algae. Applying this 4%. variationto the 8'3C value of inorganic carbon in Redfish Bay, the inor-ganic C would vary between -6.7 to -2.7%o between 12 noonand 6 PM. In sea H20 at pH 8.1 to 8.6, 96% of the totalinorganic carbon is HC03- (14). The 8'3C value of dissolvedCO2 during this time would be -13.5 to -9.5%o (a correction of-6.8%. is applied to C isotope fractionation between gaseousCO2 and dissolved HC03- at 30 C, ref. 7).
If all of the CO2 fixation by Thalassia is catalyzed by RuDPcarboxylase (which would result in a 18.3%o fractionation be-tween CO2 and PGA, 24) the 8'3C value of Thalassia would bebetween -31.8 to -27.8%.. On the other hand, if the CO2fixation of Thalassia is catalyzed by PEP carboxylase (whichwould result in a -3%o fractionation between HC03- and malicacid, ref. 24) the 813C value of Thalassia would be -9.7 to-5.7%O. This latter range is close to the observed 813C values ofThalassia (4, 14, 15, 18, 23) and is in agreement with theoperation of a C4-dicarboxylic acid pathway in this plant.The results in Figure 1 show the relative per cent of the total
14C-bicarbonate which is incorporated into amino acids, organicacids, and sugars. After 5 sec of incubation, 55% of the label isin the organic acids, 39% in amino acids, and 0% in the sugars.In 10 min there is a net synthesis of sugars. Chromatography andradioautography of the reaction products indicated that malicacid and aspartic acids were the earliest stable products of CO2fixation. After 5 sec of exposure of the leaf sections to H'4CO3-,
FIG. 1. Relative per cent of total radioactivity incorporated into leafsections. The rate of 14CO2 incorporation into the leaf sections was 1.008mg C/g dry wt- hr which compares with CO2 fixation in Zostera (13) of1.18 mg C/g dry wt * hr.
C)
0
0
0
-acx
a)-if
Time, minutes
FIG. 2. Relative per cent of total radioactivity incorporated intometabolic intermediates. Following the 5-min incubation period approx-imately 12% of the total radioactivity was recovered in unknown organicacids and 12% was recovered in unknown amino acids.
33% of the label is in malic acid, 30% in aspartic acid, and lessthan 2% in PGA. The results in Figure 2 show the relative percent of the total radioactivity which is incorporated into C4 acids(malic acid and aspartic acid), PGA, glutamic acid, citric acid,and hexose-phosphates. PGA is only slowly labeled in 1 min ofincubation and then shows a negative slope to 10 min of incuba-tion. The C4 acids show strikingly negative slopes of isotopeincorporation which indicates one site of CO2 incorporation. Thefirst labeled products of CO2 fixation are malic acid and asparticacid which is similar to the early products of CO2 fixation in C4plants.Jagels (10) has studied the anatomy of Thalassia in relation to
the mechanism of osmoregulation. The leaves have no cuticle orstomates. The mesophyll cells are undifferentiated and the re-duced vascular tissue is located between the fiber cells. There are
no bundle sheath cells around the vascular tissue. The epidermalcells are rich in chloroplasts and mitocfiondria, and containhighly invaginated plasmalemma which functions in salt secre-tion. No particular emphasis was placed on the type of chloro-plast in the epidermal cells and no chloroplasts were demon-strated in the mesophyll cells. We have studied the ultrastructureof Thalassia aiming toward a fuller description of the photosyn-thetic characteristics of the epidermal and mesophyll cells. Thisis important because (a) the 813C values and early products ofCO2 fixation indicate the presence of a C4-dicarboxylic acidpathway in this monocot, and (b) C4 metabolism in terrestrialgrasses is tightly associated with the mesophyll and bundlesheath cells of the leaves.A cross section of a leaf of Thalassia shows the epidermal cells
contain chloroplasts and enclose mesophyll cells and air lacunae.Figure 3 shows the cross sectional relation between the epider-mis, mesophyll, and air lacunae. The outer epidermal wall isthickened and the mesophyll cells have different morphologicalshapes. Different size air lacunae are present. The mesophyllcells surround the air lacunae. It has been shown (26) that the airlacunae contain 02 and CO2.The ultrastructure of an epidermal cell of Thalassia is shown in
Figure 4A. The cell is packed with chloroplasts. Higher magnifi-cations show many prominent mitochondria in the darker stain-ing layer of cytoplasm. The cell contains a prominent cell wallwith striations or layers. The outer epidermal wall is muchthicker than the walls between adjacent epidermal cells or be-tween epidermal and mesophyll cells. The cytoplasm and chloro-plasts contain abundant osmophillic granules and droplets. Theepidermal cells of Thalassia differ from the epidermal cells ofterrestrial C4 grasses which do not contain chloroplasts. Figure4B shows a higher magnification of an epidermal chloroplast.The chloroplast contains a well developed grana system andresembles the C3 type of chloroplast in the mesophyll cells of C4plants. There are osmophillic droplets in the chloroplasts but thechloroplasts do not contain starch granules. This electron pho-tomicrograph shows striations or layers in the cell wall. Alber-shiem (1) has concluded these repeating structures have theexpected dimensions of the primary wall in plants. Figure 4C isan electron photomicrograph of a mesophyll cell surrounding anair lacunae. The mesophyll cell is highly vacuolated and theparietal layer of cytoplasm contains an occasional chloroplast.Figure 4D shows a higher magnification of a mesophyll chloro-plast. The chloroplast does contain grana but the grana andstroma lamellae do not fill the chloroplast. The lamellar systemin these chloroplasts is not as well developed as the system inepidermal chloroplasts.
FIG. 3. Cross sectional view of epidermis, mesophyll, and air lacunaein a leaf of Thalassia. E: epidermal cell; M: mesophyll cell; AL: airlacunae. x 160.
Plant Physiol. Vol. 57, 1976 PHOTOSYNTHETIC CARBON METABOLISM.N .,.r,rE-J@-! a* k.-s , t- _ -f-,,, _, .e , s X . _._S o* . ... *. .L . . os .* w ............................. ;e i.S .-. ,. .^ t^!_- .......................... _ ... s r- ^U . s1s K w . ° . _ K __ _v r _ { | :-_ , _ffi _> a :S | c;_ F _ _ = _ . ._:25ZE11 S iX- ! ,,sp>;|;, . : ^ w___S 31N ] _ 1|1 h1 E r ;_ _11S! _ \ w
x ir_s- }!sb, s. ,. .Pts. _L _ '
S-,<t a B~~~~
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FIG. 4. Fine structure of Thalassia. A: An electron photomicrograph of an epidermal cell. OW: outer cell wall; E: epidermal cell; M: mesophyllcell. x 6,020. B: An electron photomicrograph of an epidermal chloroplast. WS: wall striations; G: grana; C: cytoplasm. x 23,800. C: An electronphotomicrograph of a mesophyll cell. M: mesophyll cell; C: parietal cytoplasm; MC: mesophyll chloroplast; E: epidermal cell; AL: air lacunae.x 2,800. D: An electron photomicrograph of a mesophyll chloroplast within the parietal cytoplasm. C: parietal cytoplasm; G: grana; SG: starchgrain; S: stroma x 25,160.
Figure 5 is an electron photomicrograph of the vascular tissuebetween the fiber cells and epidermal cells of Thalassia. Thevascular tissue is not surrounded by bundle sheath cells. Thephloem tissue is extensive but like many submerged aquaticplants the xylem tissue is greatly reduced (16). Highly vacuo-lated mesophyll cells are present between phloem cells and fibercells. There does not seem to be any anatomical arrangement ofvascular bundles and bundle sheath cells of the Kranz anatomyof terrestrial C4 plants.
In conclusion, the 813C analysis of the metabolic intermediatesand CO2 fixation studies show that the photosynthetic carbon
metabolism of Thalassia is similar to C4-type metabolism. Yetthe electron microscopy studies show the anatomy of the tropicalmarine grass is decidely different than tropical C4 grasses. It isimportant to note that Shomer-Ilan et al. (17) have shown thatSuaeda monoica lacks bundle sheath cells but is a C4 plant. TheC4 metabolism in these succulent leaves probably involves twotypes of chlorenchymatic cells. This work does not establish therelationship between structure and function in Thalassia butmany features are noteworthy. The epidermal cells appear tohave active photosynthetic and mitochondrial metabolism asso-ciated with osmoregulation (10). These cells may be the primary
FIG. 5. Electron photomicrograph of a cross section of Thalassiathrough the vascular tissue. F: fiber cells; X: xylem element; P: phloemtissue; E: epidermal cell; M: mesophyll cell. x 2,800.
Plant Physiol. Vol. 57, 1976
CO2 fixation site and CO2 uptake may be associated with osmo-
regulation. The CO2 fixation products in the epidermal cells maybe transported to the mesophyll cells but Jagels (10) was unableto detect plasmodesmata between these cells. The mesophyllcells contain a small population of chloroplasts and probablycould directly fix CO2. The leaves contain a well developedsystem of air lacunae surrounded by mesophyll cells. The inter-nal gas (CO2 and 02) production and storage in the air lacunaeswells the leaf blades 200 to 250% during the day (26). Themesophyll cells could probably refix any respired CO2 in theselacunae. Thalassia lacks bundle sheath cells but photosyntheticcarbon metabolism in the epidermal and mesophyll cells to-gether with the recycling of CO2 in the air lacunae may be a
system to account for the high productivity of this tropicalmarine grass.
Acknowledgments -The authors gratefully acknowledge W. W. Wong and W. M. Sackett.Department of Oceanography, Texas A & M University, College Station, Texas 77843 for the
813C analyses.
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