Glycolate Metabolism Low High CO2-Grown ... · GLYCOLATE METABOLISM IN CHLORELLA AND PAVLOVA linearincrease of10°C/minforboththeglycolate andamino acid fractions. TMS4-glycolate

$Page 1: Glycolate Metabolism Low High CO2-Grown ... · GLYCOLATE METABOLISM IN CHLORELLA AND PAVLOVA linearincrease of10°C/minforboththeglycolate andamino acid fractions. TMS4-glycolate$
Plant Physiol. (1989) 91, 1085-10930032-0889/89/91/1 085/09/$01 .00/0

Received for publication February 17, 1989and in revised form July 11, 1989

Glycolate Metabolism in Low and High CO2-GrownChlorella pyrenoidosa and Paviova lutheri as Determined by

180-Labeling1

Edward J. de Veau*2 and John E. Burris3Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

ABSTRACT

Photorespiration in Chlorella pyrenoidosa Chick. was assayedby measuring '10-labeled intermediates of the glycolate pathway.Glycolate, glycine, serine, and excreted glycolate were isolatedand analyzed on a gas chromatograph/mass spectrometer todetermine isotopic enrichment. Rates of glycolate synthesis weredetermined from 180-labeling kinetics of the intermediates, poolsizes, derived rate equations, and nonlinear regression tech-niques. Glycolate synthesis was higher in high C02-grown cellsthan in air-grown cells when both were assayed under the same02 and CO2 concentrations. Synthesis of glycolate, for both typesof cells, was sfimulated by high 02 levels and inhibited by highCO2 levels. Glycolate synthesis in 1.5% C02-grown Chlorella,when exposed to a 0.035% CO2 atmosphere, increased fromabout 41 to 86 nanomoles per milligram chlorophyll per minutewhen the 02 concentration was increased from 21% to 40%.Glycolate synthesis in air-grown cells increased from 2 to 6nanomoles per milligram chlorophyll per minute under the samegas levels. Synthesis was undetectable when either the 02 con-centration was lowered to 2% or the CO2 concentration was raisedto 1.5%. Glycolate excretion was also sensitive to 02 and CO2concentrations in 1.5% C02-grown cells and the glycolate thatwas excreted was '10-labeled. Air-grown cells did not excreteglycolate under any experimental condition. Indirect evidenceindicated that glycolate may be excreted as a lactone in Chlorella.Photorespiratory 18O-labeling kinetics were determined for Pav-lova Iutheri, which unlike Chlorella and higher plants did notdirectly synthesize glycine and serine from glycolate. This algadid excrete a significant proportion of newly synthesized glyco-late into the media.

The existence and the amount of photorespiration in fresh-water algae is uncertain (8). All the enzymes needed for thesynthesis and metabolism ofglycolate are present within mostspecies (29). Algae cultivated under high levels ofCO2 appear

'Supported in part by Grant No. 2466 from the U.S. Departmentof Agriculture Competitive Grants, The Pennsylvania State Univer-sity, and through the Ben and Helen D. Memorial Fund awarded toE. J. de V.

2 Present address: USDA, ARS, BARC-West, Plant PhotobiologyLaboratory, Bldg. 046A, Beltsville, Maryland 20705.

3 Present address: Commission on Life Sciences, National ResearchCouncil, Rm. 343, 2101 Constitution Avenue, NW, Washington DC20418.

to photorespire when transferred to low C02 levels and to doso in a manner similar to terrestrial C3 plants (33). They havehigh C02 compensation points (14), evolve large amounts ofC02 into C02-free air during illumination (14), and are pho-tosynthetically inhibited by 21% °02(18, 25). They also excretelarge amounts of glycolate (4, 7, 35). However, photorespira-

I tion appears to be suppressed and glycolate excretion is neg-ligible if the cells are grown under air-levels of 02 and C02(3, 4, 14, 25).

It is thought that algae grown under air-levels of C02actively pump in dissolved inorganic carbon, raising the in-tracellular level of inorganic carbon (2). This would presum-ably suppress the oxygenase function of ribulose bisphosphatecarboxylase/oxygenase, thus lowering the rate of glycolatesynthesis. However, it is not entirely clear if glycolate synthesisis completely suppressed. It has been reported that if cells aretreated with a glycolate metabolism inhibitor they excretealmost as much glycolate as if they had been grown underelevated C02 (31).The main objective of our research was to quantify and

then compare the photorespiratory rate ofair-grown and 1.5%C02-in-air-grown Chlorella pyrenoidosa as a function of gly-colate synthesis rates. Toward this goal, we developed a newassay utilizing 1802 to obtain '80-labeling kinetic curves ofthree photorespiratory intermediates, glycolate, glycine, andserine. This information, coupled with the pool sizes of theintermediates, was computer analyzed using nonlinear regres-sion to determine rates. The use of 180-labeling to determinephotorespiratory rates was first suggested and carried out byBerry et al. (5) for spinach. Later, Jolivet-Tournier and Gerster(17) used an '80-labeling method to determine photorespira-tion in maize. However, neither group of researchers ade-quately considered how the isotopic enrichment of a metab-olite pool changes with time. For example, Jolivet-Tournierand Gerster ( 17) derived their rate assuming that the isotopiccontent changes linearly with time, whereas in reality itchanges in exponential fashion (22, 28, 36). Previous studiesusing "1C to assay photorespiration in algae do not addressthe question of specific rates. Instead, they have investigatedprimarily the metabolic flow of carbon during glycolate me-tabolism (33). The '80-isotope has the added advantage inthat nearly 100% of the glycolate pool can becomes 180_labeled (5, 11, 12), whereas, in those studies using 14C only afraction ofthe glycolate pool becomes labeled. This is possiblydue to the participation of unlabeled storage carbohydrates inthe synthesis of ribulose bisphosphate (30). We have also

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Plant Physiol. Vol. 91, 1989

examined glycolate metabolism in Pavlova lutheri using the'8O-isotope.

MATERIALS AND METHODS

Both Chlorella pyrenoidosa Chick. and Pavlova lutheri(Droop.) Green were obtained from the University of Texasalgal culture condition, collection numbers UTEX1806 andLB 1293, respectively (27). The algae was grown separately inbatch culture under constant illumination (photon flux den-sity = 100 ,uE/m2/s) at 25°C. The medium used for Chlorellawas that of Sorokin and Krauss (26) while Pavlova wascultivated in IMR/2 (13) in artificial seawater (Food ChainResearch Group, Scripps Institution of Oceanography, CA).The pH ofthe cultivation media was 6.8 and 7.8 for Chlorellaand Pavlova, respectively. Pavlova was cultivated under airlevels of CO2.For the '80-labeling procedures of the algae, 3 mL of

appropriate cultivation medium was placed into either a 10mL (Chlorella) or a 5 mL (Pavlova) water-jacketed glasschamber maintained at 25°C. Figure 1 is a diagram of thealgal chamber. The chamber was sealed off with the siliconestopper and the magnetic stirrer was turned on. Both valveswere initially closed. The needle valve for the eighth-inch tubewas then opened. The air space was evacuated by the actionof the inlet vacuum pumps of the mass spectrometer. Thiscaused the gases dissolved in the medium to leave solution.Evacuation of the chamber was carried out for 1.5 min.

After evacuation, the needle value was closed and a mixtureof 02, C02, and argon was added via the injection port in thesilicone stopper. For the isotopic labeling of Chlorella, theconcentration of 02 added to the chamber was 2, 21, or 40%,enriched with 1802 (10-99.9% of total 02), while the CO2concentrations of the added gas was either 0.035 or 1.5%.Glycolate metabolism in Pavlova was assayed under an at-mosphere of 50% 02, enriched 70% with 1802, and 0.035%C02, with the balance argon.The gas mixture above the water level was allowed to

equilibrate with the gases dissolved in the aqueous mediumfor 15 min. Equilibrium was facilitated by the action of themagnetic stirrer. The bellows valve was then opened, thechamber was illustrated (photon flux density = 400 ,E/m2/s), and 0.25 mL of a concentrated cell suspension (total Chl= 1.5 ± 0.3 mg) was added to the chamber via the injectionport. The cells were in the logarithmic phase of growth andhad been concentrated by centrifugation. Photorespiratory'8O-labeling began with the addition of the cells. The com-position of the dissolved gas was periodically monitored dur-ing the labeling procedure by allowing dissolved gas to diffuseacross the teflon membrane on the one-quarter inch tube intothe mass spectrometer ( 16).

After the labeling period, the chamber was opened and thecell suspension was removed with a syringe. The cell suspen-sion was then filtered through Whatman No. 3 filter paperand frozen with an aerosol spray of dichlorodifluromethane.This operation took about 5 s to perform. The filter paperwas immersed in liquid N2 until the photorespiratory inter-mediates were extracted in 5 mm NaF. The filtrate was savedin a small vial and frozen until it could be analyzed forexcreted glycolate.The extraction, separation, and silylation of the photores-

A

Figure 1. Diagram of algal 180-labeling chamber. A, Air flow to massspectrometer; B, needle valve; C, one-eight inch stainless steeltubing; D, bellows valve; E, one-fourth inch stainless steel tubing; F,algal and gas injection port; G, silicone rubber stopper; H, water flowto circulating water bath; I, chamber air space; J, water-jacketedchamber; K, water layer; L, 'O' ring; M, water flow from circulatingwater bath; N, one-mil teflon membrane; 0, teflon-coated magneticstir-bar; P, magnetic stirrer motor.

piratory intermediates (glycolate, glycine, and serine) followeda modified procedure of that of Berry et al. (5) and has beenpreviously published (11, 12). The final yield of glycolate,glycine, and serine after silylation was 54 + 5%, 49 + 4%,and 67 ± 4%, respectively. Calculations ofthe pool sizes wereadjusted for the above losses. For the isolation of glycolateexcreted into the aqueous medium, the liquid was passedthrough a cation exchange column (Biorad AG 50W-X8,100-200 mesh, H+ form, 0.8 x 4.0 cm). The column waswashed with 10 mL of distilled water and the eluant wascollected and lyophilized. Checking this step with knownquantities of glycolate indicated that 70 ± 3% was recoveredafter lyophilization and silylation.

Aliquots of the derivatized extracts were injected into anLKB 9000 combined gas chromatograph-mass spectrometer.The glass column used was 3% SP-2250 on 80/100 Supelco-port (Supelco, Bellefonte, PA). The flow rate of the heliumcarrier gas was 16 mL/min. The temperature program of thegas chromatograph oven was 100°C for 5 min followed by alinear increase of 10°C/min for both the glycolate and aminoacid fractions. TMS4-glycolate eluted at 145C, while TMS-glycine and TMS-serine eluted at 190 and 200°C, respectively.

DE VEAU AND BURRIS1 086

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GLYCOLATE METABOLISM IN CHLORELLA AND PAVLOVA

linear increase of 10°C/min for both the glycolate and aminoacid fractions. TMS4-glycolate eluted at 145°C, while TMS-glycine and TMS-serine eluted at 190 and 200°C, respectively.The portions of the mass spectra and the equations used to

determine molecular percent '8O-label were those of Berry etal. (5). Glycolate, glycine and serine standards were usedfollowing the method of Troxler et al. (34) to correct for thenatural abundance of C, N, 0, and Si isotopes. Since themolecular percent 1802/total 02 exposed to the algae varied,the enrichments of the intermediates were expressed as per-centages of the 1802 dissolved in the medium using the equa-tions of Jolivet-Tournier and Gerster (17). The equation is:

Relative enrichment = (molecular % '8O-labeledcompound/molecular % dissolved 802) x 100

The metabolic rates were determined by analyzing therelationship between '8O-enrichment and time for each pho-torespiratory metabolite pool and for excreted glycolate usingnonlinear regression. Pool sizes of the metabolites and theamount of excreted glycolate were determined by measuringthe area under the gas chromatograph tracing and comparingit to the area under the tracing of known amounts of therespective compounds. The rate equations for all the com-pounds were derived from the equation relating the instanta-neous change in concentration of a isotopically labeled me-tabolite over time as a function ofmetabolic rates offormationand degradation and the isotopic enrichment ofthe precursorand the production (22, 28, 36):

dB*/dt = riEa- r2Eb

where B* = concentration of isotopically labeled product; r,= rate of product synthesis; r2 = rate of product conversionto other metabolites; under steady state conditions r1 = r2; Ea= A*/A, the isotopic enrichment at time t, where A* is theconcentration of isotopically labeled precursor and A is thetotal precursor concentration; Eb = B*/B, the isotopic enrich-ment at time t, where B* is the concentration of isotopicallylabeled precursor and B is the total precursor concentration.The equation for '8O-glycolate formation is (12):

Ep = 1 - e(-.r/lp (1)

where Ep = P*/P, the isotopic enrichment ofglycolate at timet; P* is the concentration of 180-glycolate and P is the totalcellular pool of glycolate; r = rate of glycolate synthesis.Oxygen was assigned the role ofdirect precursor ofglycolate

instead of phosphoglycolate since the phosphoglycolate con-centration is small (29, 33) and therefore would not signifi-cantly influence the '8O-labeling ofglycolate (28). The isotopicenrichment and concentration of oxygen were assumed toremain constant. The glycolate pool was assumed to be insteady state, which means that the rate of glycolate synthesisequals the rate of its metabolism. Metabolism in this casemeans either glycolate excretion to the media and/or conver-sion to glycolate pathway intermediates.

The rate of photorespiratory glycine synthesis equals therate of glycolate synthesis in terrestrial plants (29). Photores-piratory serine synthesis rate equals one-half the glycolatesynthesis rate in these plants (29). However, algae may excretea portion of newly synthesized glycolate into the external

'Abbreviations: TMS, trimethylsilyl.

medium (7). Thus, any derived rate equations for the aboveamino acids must take glycolate excretion rates into effect.

If it is assumed that all of the above metabolites are insteady-state, the rates of glycolate synthesis and excretion areconstant, and the glycolate not excreted will be metabolizedentirely through a higher plant-like type photorespiratorymetabolism, then the rate equations for both glycine andserine are derived as follows.For glycine:

dG*/dt = (r - g)Ep - (r - g)Eg

where Eg = G*/G, the isotopic enrichment of glycine at timet; G* is the concentration of '8O-glycine and G is the totalcellular pool of glycine; g = rate of glycolate excretion.

All other terms are as they were previously defined.A solution to the above equation is:

dG*/dt + (r - g)G*/G = (r - g)Ep

Multiply both sides by the integrating factor e(rg)t/G:[e(1g)'][dG*/dt + (r - g)G*/G] = [e(-g')/G](r - g)Ep

The substitution of Equation 1 for Ep, the integration ofboth sides with respect to t, and the simplification of termswill yield:

G *e(r-g)tIG = Ge(rg):/G 1 - [e(rIP)1

*P(r - g)/[P(r - g) - Gr]J + C

The constant C is found by setting t = 0 when G* = 0:

C = {P(r - g)/[P(r - g) - Gr] - Gj[e(g)11G][eg-r)t1G]Therefore by rearranging terms:

Eg = 1 + P(r - g)[egr)I/G -e('g)t/P]/[P(r - g) - Gr]

- e' r)t/G; when P(r - g) # Gr

Ifg = 0, then Equation 2 reduces to:

Eg = 1 + P(er/G -_ CrIP)/(P - G) -erIG

(2)

The rate equation for serine was derived assuming that itsrate of formation is one-half that of glycine:

dS*/dt = Eg(r - g)/2 - EA(r- g)/2

where Es = S*/S, the isotopic enrichment of serine at time t;S* is the concentration of '80-serine and S is the total cellularpool of serine.

All other terms are as they were previously defined.A solution to the above equation is:Rearrange terms and multiply both sides by the integrating

factor e(rg)t/2S:

e(g'2S[dS*Idt + S*(r - g)/2S] = [Eg(r -g)2]e'g)'12SThe substitution of Equation 2 for Eg, the integration of

both sides with respect to t, and the simplification and com-bining of terms yield:

S*e-g)tl2S = Se'(rg)t/2SI/ 1

+ [G2e('r)l/]/[P(r - g) - Gr](G - 2S)- eCrhP[P(r - g)]2[P(r - g) - Gr][P(r - g) - 2Sr]I + C

1 087



~~~~~~~~~~~~~~~~~~PlantPhysiol. Vol. 91, 1989

The constant C was found by setting t = 0 when S* = 0 and

when it substituted into the above and terms are rearranged

then:

E= + G2r[e19-r)1/G - e(gr)1/2sII[P(r g) Gr]

(G 2S) + [P(r g)I2[e gr)1/2S e-rhl/p (3)

[P(r g) Gr][P(r g) 2Sr] _-(-rt2

when P(r g) Gr, P(r g) 2 Sr, and G 2S;

Ifg = 0 then Equation 3 reduces to:

E= + G2(ert/G_Crh/2S)/(P G)(G 2S) + p2(en/l2S-erlP)/(P G)(G 2S) Cr/l2S

The rate equation for the '80-labeling kinetics of excreted

glycolate is derived as follows:

dE*/dt = gEp MEe

where Ee = E*/E, the isotopic enrichment of excreted glyco-

late at time t, E* is the concentration of isotopically labeled

excreted glycolate and E is the total concentration of ex-

creted glycolate. E = gt; M = rate of metabolism of excreted

glycolate.

If it is assumed that once glycolate is excreted it is neither

taken back up by the alga nor metabolized by other microbes

within the medium during the labeling period, then M = 0

and:

dE"/dt = gEp

Equation is substituted for Ep and both sides are inte-

grated with respect to t:

L = gt + Pgcrt/P/r+c

To find C, t is set to 0 when E* = 0.

C = -Pg/r

Therefore:

E* = gt +Pg(ert/P Il)r

Since E = gt then:

Ee = + P(erh/P O)/r (4)

The photosynthetic rates for the algae were determined using

an oxygen electrode (Rank Bros., Cambridge, UK) following

the method of Delieu and Walker (10). The same temperature

and photon flux density were used as the '80-labeling proce-

dure. Prior to addition of the cells the medium was allowed

to equilibrate under laboratory concentrations Of02 and CO2.

Chl content in Chiorella was determined by filtering algal

suspensions onto Whatman No. 3 filter paper. The filter paper

was plunged into liquid N2 to facilitate cell destruction. The

filter paper with the algae was ground with a tissue homoge-

nizer in 10 mL of 80% v/v acetone in water under low light.

The acetone extract was transferred to a conical centrifuge

tube which was then covered with aluminum foil and placed

in a freezer to complete extraction. The following day the

centrifuge tube was spun to remove debris. The acetone

extract was transferred to a spectrophotometer cuvette and

the Chl content was determined using the wavelength and

equations of Arnon (1).

All statistical analyses were performed using SAS statistical

packages (24) on an IBM 3090-200 computer.

RESULTS

Algal cells undergoing photosynthesis and photorespiration

will simultaneously produce and consume 02. The 02 dis-

solved in the water is enriched with 1802 while the water

within and outside the cells is almost entirely H2160. Cells

will consume 1802 while they simultaneously produce 1602.

This would cause decreases in the percent relative isotopic

enrichment of 02. This is illustrated in Figure 2 for air-grown

C/hlorella. The decrease in percent 1802 was slight during the

labeling period because of the relatively large amount of 1802

present in the labeling chamber. The small change in isotopic

enrichment would have an insignificant effect on the labelingkinetics of the photorespiratory intermediates. The change in

isotopic composition during '80-labeling in high C02-grown

Chiorella and Paviova were similar to air-grown Chiorella and

are omitted here. The dissolved CO2 dropped less than 10%

in the first 2 min of the labeling period for all of the algae

while the dissolved 02 content remained constant (data not

shown).

Chiorellapyrenoidosa grown in air appeared to exhibit little

or no photorespiration.. Glycolate from this alga did not

become enriched with under an atmosphere of either 2%

02 and 0.035% CO2 or 21% 02 and 1.5% CO2 (Fig. 3).

Glycolate became enriched when the alga was exposed to

0.035% CO2 and either 21% or 40% 02, but maximum

100-

90-

z80-

90I80-

0(A1

"' 80-.--

z

0

100.

90.

80,

0---2 - --18 .TIME (MINUTES)

Figure 2. Time course of the change in percent relative 1802 enrich-

ment of dissolved 02 during 180-labeling of air-grown Chlorella. (A,

x, 5, K)) represent different trials. Initial 02 and CO2 concentrations

added to the air space above aqueous solution were, respectively:

A, 2% and 0.035%; B, 21% and 0.035%; 0, 40% and 0.035%; D,

21% and 1.5%.

Ao

......... . . . . .B

1088 DE VEAU AND BURRIS




100-

80

awA-j

Lwm

I-~z

wL

0

wL

0-

60'

40'~

20'

0 1 2 3 4 5 6 7 8 9 1'0

TIME (MINUTES)

Figure 3. Effects Of 02 and 002 concentration on 180-labeling of

glycolate in air-grown Chiorella. Y-coordinate is expressed as relative

molecular percent "10-enriched. The 02 and 002 concentrations

added to the air space were: 2% 02, 0.035%C002and 21%02 and

1.5%C002 (F-EJ), 21%02, 0.035% 002 (A----A), and 40% 02

and 0.035% 002 (X-.- -X).

isotopic enrichment was reached after 5 min. There was no

glycolate excretion or glycine or serine labeling under any of

the above atmospheres (data not shown). Glycolate did not

become isotopically labeled when the cells were exposed to

1802 in darkness (data not shown).

Chiorella grown in 1.5% enriched air did not incorporate

into any of the photorespiratory intermediates or excrete

glycolate when exposed to either 2% 02 and 0.035% CO2 or

21% 02 and 1.5% CO2 (Figs. 4-8). However, there was 180..

enrichment under the other two atmospheres. The amounts

of isotopic enrichment of the photorespiratory metabolites

and glycolate excretion were influenced by 02 concentration.

Glycolate did not become isotopically labeled when high GO2-

grown cells were exposed to 1802 in darkness (data not shown).

The pool sizes in Chiorella are shown in Table L. There

were only slight changes in the size of the various poo1s

brought about by changes in gas composition. The concentra-

tion of the metabolites remained constant throughout the

labeling period (data not shown) indicating that the poois

were in steady state during this time.

The rate of glycolate excretion in high C02-grown cells was

determined by plotting the amount of glycolate detected in

the medium against time (Fig. 8) and then performing linear

regression on the time points from 0 to min. The excretion

rates were 27.9 ± 4.9 and 52.1 ± 8.0 nmol/mg Chl/min for

21% 02 and 40% 02, respectively. The corresponding corre-

lation coefficient were 0.59 and 0.65, respectively.

Glycolate synthesis rates for air-grown Chiorella under

atmospheres of 21 and 40% 02, CO2 concentration = 0.035%,

were 2.2 ± 0.3 (correlation coef'ficient = 0.74) and 5.7 ± 0.4

(correlation coefficient = 0.89) nmol/mg Chl/min, respec-

0

wL-J

1--z

LUJ

w

0 1 2 3 4 5 6 7 8 9 10

TIME (MINUTES)

Figure 4. Effects Of 02 and 002 concentration an 180-labeling of

glycolate in high C02-grown Chlorella. Y-coordinate is expressed as

relative molecular percent 180-enriched. The 02 and 002 concentra-

tions added to the air space were: 2% 02, 0.035% 002 and 21% 02

and 1.5% 002(f--l) 21% 02, 0.035% 002 (A- -A), and 40%

02 and 0.035% 002 (X- -X).

1

LUJ

LUJ

0-

TIME (MINUTES)

Figure 5. Effects Of 02 and 002 concentration on '80-Iabeling of

glycine in high C02-grown Chlorella. Y-coordinate is expressed as

relative molecular percent 180-enriched. The 02 and 002 concentra-

tions added to the air space were: 2% 02, 0.035% 002 and 21% 02

and 1.5% 002 (DI-EI), 21% 02, 0.035% 002 (A----A), and 40%

02 and 0.035% 002 (X- -X).

x

x

x

.0, a

. . . . . . . . . .

..I~~~~~~~~~~~~~~~0T~~~~~~~?-.7. I -.

1089



Plant Physiol. Vol. 91,1989

10 0 1 2 3 4 6 7 8 9 1b

TIME (MINUTES)

Figure 6. Effects of 02 and C02 concentration on 180-labeling ofserine in high C02-grown Chlorella. Y-coordinate is expressed asrelative molecular percent "8O-enriched. The 02 and C02 concentra-tions added to the air space were: 2% 02, 0.035% C02 and 21% 02and 1.5% C02 O), 21% 02, 0.035% C02 (A- --A), and 40%02 and 0.035% C02 (X-*-X).

-J

mSI--

zLJ

LUJ0.

0 1 2 3 4 5 6 7 8 9 10

TIME (MINUTES)Figure 7. Effects of 02 and C02 concentration on 180-labeling ofexcreted glycolate in high C02-grown Chlorella. Y-coordinate is ex-pressed as relative molecular percent 180-enriched. The 02 and C02concentrations added to the air space were: 21% 02 0.035% C02(A---A), and 40% 02 and 0.035% C02 (X- - -X).

TIME (MINUTES)

Figure 8. nMol glycolate excreted into aqueous medium versus timefor high C02-grown Chlorella dunng 180-labeling. The 02 and C02concentrations added to the air space were: 21% 02, 0.035% C02(A---A), and 40% 02 and 0.035% C02 (X---X).

Table I. Pool Sizes of the Photorespiratory Intermediates inChlorella (nmol/mg Chl)

Air-Growna High C02-Grown2% 02, 0.035% C02

Glycolate 34.6 ± 2.1 (7) 39.2 ± 3.2 (4)Glycine 80.4 ± 8.3 (7) 74.7 ± 6.2 (7)Serine 116.7 ± 11.2 (7) 111.3 ± 9.2 (7)

21% 02, 0.035% C02Glycolate 38.9 ± 3.5 (7) 44.2 ± 4.2 (5)Glycine 84.9 ± 7.4 (6) 86.0 ± 9.1 (5)Serine 125.9 ± 19.5 (6) 127.2 ± 12.7 (5)



aNumbers in parentheses = number of samples.

tively. The time points from 0 to 2 min were used in theregression equation because it was during this period that themost rapid changes in '80-enrichment occurred. The labelingkinetics were also assumed to have been least affected bychanges in CO2 concentration at these time points.The rates of glycolate synthesis in high C02-grown Chlo-

rella, as determined by the '80-labeling kinetics of glycolate,glycine, serine, and excreted glycolate, are listed in Table II.The time points 0 to 1 min were used for in the regressionequations for glycolate, glycine, and excreted glycolate. Thesepoints were selected because it was during this time that the

wLJwm

zL

0.

1 090 DE VEAU AND BURRIS




Table II. Rate of Glycolate Synthesis in High C02-Grown Chlorellaas Determined by 180-Labeling Kinetics of Glycolate and Calculatedfrom '80-Labeling Kinetics of Glycine, Serine, and ExcretedGlycolate (nmol/mg Chl/min)

Glycolate synthesis under 2% 02, 0.035% C02, and 21% 02,0.035% C02 atmospheres was not detected.

Rate Standard Error r2

21% 02, 0.035% C02 fromGlycolate 30.9 1.8 0.94Glycine 43.7 0.3 0.86Serine 38.8 0.2 0.94Excreted glycolate 18.2 0.5 0.98

40% 02, 0.035% C02 fromGlycolate 59.6 3.4 0.95Glycine 80.6 0.3 0.98Serine 92.0 1.1 0.88Excreted glycolate 34.6 0.76 0.97

most rapid change of "8O-enrichment occurred (Figs. 4, 5,and 7). Furthermore, the labeling kinetics were assumed tohave been least affected by changes in gas composition atthese time points. The glycolate excretion rates of high CO2grown cells also decrease after 1 min (Fig. 8). The time points0 to 5 min were used for serine. These points had to be usedbecause serine did not become labeled before 5 min.The rate of glycolate synthesis was affected by the gas

composition in high C02-grown Chlorella. It was highestunder a 40% 02, 0.035% CO2 atmosphere. It was also manytimes greater than the rate of glycolate synthesis in air-growncells exposed to similar 02 and CO2 levels. However, theextract rate of glycolate synthesis is uncertain. There was no

agreement between the rates determined from the amino acidsand the '8O-labeling data from intracellular and excretedglycolate, though the rates determined from glycine and serinedate were in agreement. The rate of glycolate synthesis as

determined from the '8O-labeling kinetics of excreted glyco-late was too low to account for the rate of glycolate excretion.Possible explanations ofthis occurrence will be discussed later.The photosynthetic rate of air-grown cells was 1517 ± 165

nmol 02/mg Chl/min while the rate of photosynthesis of highC02-grown cells was 983 ± 90 nmol 02/mg Chl/min.

Glycolate extracted from Pavlova lutheri had appreciable18O-enrichment (Fig. 9). Since it took nearly 9 min for it toreach a maximum value, the rate of glycolate synthesis was

either slow or the pool size was large. Glycolate levels in thisalga were not determined so an estimation of glycolate syn-thesis cannot be determined. Pavlova did not, at least directly,synthesize glycine or serine from glycolate since neither 80.-

glycine nor 180-serine was isolated from this alga (data notshown). '8O-glycolate did appear in the medium (Fig. 9),indicating that this alga cannot fully metabolize glycolate.Glycolate did not become isotopically labeled when Pavlovawas exposed to 1802 in darkness (data not shown).

DISCUSSION

If Chlorella and Pavlova only synthesized glycolate throughribulose bisphosphate carboxylase/oxygenase then eventuallythe glycolate pool within the algae should reach 100% relative

w-m60.z0 40

ci.~~w~~~~~~20

20

0 3 6 9 12 15

TIME (MINUTES)Figure 9. 180-Labeling kinetics of intracellular and excreted glycolateof P. lutheri. Y-coordinate is expressed as relative molecular percent180-enriched. The concentration of 02 and C02 added to the chamberwere 50% and 0.035%, respectively. Intracellular glycolate (D-II)excreted glycolate (A- - -A).

"8O-enrichment. Alternate means of photorespiratory glyco-late synthesis that would not involve the incorporation of 180have not been described for any alga. Lorimer et al. (20)determined that glycolate synthesis in Chlorella fusca wasalmost exclusively through ribulose bisphosphate carboxyl-ase/oxygenase or at least through a pathway which involvedoxygen incorporation. However, the glycolate pool in noneof the algae reached full enrichment in our study (Figs. 3, 4,and 9). Lacking a known alternate means of glycolate synthe-sis, there can be at least two reasons for the failure of theglycolate pool to reach full "8O-enrichment. They are 1602dilution of 1802 during the labeling period or isotopic dilutionduring transfer of the algae from the labeling chamber to thefiltering and killing procedure.The first possible cause, isotopic dilution of 1802 by pho-

tosynthetically generated 1602, can be ruled out for this ex-periment. The algae were well mixed by the magnetic stir-barthroughout the labeling period. Thus, no localized depletionof percent 1802 should have occurred. The glycolate synthe-sized at any given time should reflect the molecular percentisotopic enrichment of02 at that time and the percent enrich-ment remained fairly constant throughout the labeling period(Fig. 2).The isotopic dilution of 1802 during transfer and filtering

of the algal cells seems to be the more likely reason for theapparent failure of the glycolate pool to achieve full 180-enrichment. An examination of the calculated rates of glyco-late synthesis in 1.5% C02-grown Chlorella support this con-tention. The rate of glycolate synthesis measured from the"8O-labeling kinetics of glycolate was 20 to 35% lower thanthat estimated from the "8O-labeling kinetics of glycine and

1091



DE VEAU AND BURRIS

serine (Table II). Glycolate would be more sensitive to the 5-s transfer and filtering time than the amino acids beingmetabolically upstream from glycine and serine and of a

smaller pool size (Table I).Further evidence for significant isotopic dilution during

transfer and filtering of the algal cells can be obtained byexamining the labeling kinetics ofthe two amino acids isolatedfrom high C02-grown Chlorella (Figs. 5 and 6). The twoamino acids show signs of increasing '8O-enrichment after a10-min labeling period, even though glycolate reached itsapparent maximum enrichment after about 2 min (Fig. 4).Since glycine and serine are less affected by the protractedtransfer and labeling time than glycolate, they will yield amore accurate estimation of the true glycolate synthesis rate.Further discussion ofthe glycolate synthesis rate in 1.5% CO2-grown Chlorella will omit the data obtained through thelabeling kinetics of cellular glycolate. A similar omissioncannot be done for air-grown cells since they did not excrete'8O-glycolate nor synthesize '8O-glycine and '8O-serine. Thus,for further discussion, glycolate rates of synthesis estimatedby '8O-labeling kinetics of glycolate need to be used for air-grown Chlorella.There is a third possible reason that glycolate from 1.5%

C02-grown Chlorella failed to reach full '80-enrichment aftera 10 min 1802 exposure. It is known that the adaptation ofhigh C02-grown algal cells to low CO2 begins immediatelyupon transfer (25), although complete adaptation is not fullyreached until several hours have past (32). This would lead toa lower rate of glycolate synthesis. A slowing of the rate ofglycolate synthesis can be seen in the 18O-labeling kinetics ofglycine (Fig. 5) and began to level off after 2 to 5 min of 1802exposure. The level of molecular percent '8O-enrichment ofthe glycine pool was significantly lower than the theoreticalmaximum of 100%. Glycolate excretion rates also began toslow down 1 to 2 min into the labeling period (Fig. 8). It isfor this reason that, unless otherwise indicated, the rates ofglycolate synthesis were determined using time points earlierthan 2 min.

Photorespiratory glycolate synthesis in air-grown Chlorellais minimally affected by 02 concentration. If glycolate syn-thesis is expressed as a percentage of net photosynthesis thenglycolate synthesis is 0.2% versus 0.4% of net photosynthesisfor 21% and 40% 02 atmospheres, respectively (Co2 concen-tration = 0.035%). Since this alga does not excrete glycolate,all of the glycolate produced should be metabolized throughglycine and serine. The failure of these two amino acids tobecome isotopically labeled is most likely because of the lowrate of glycolate synthesis.

Glycine and serine '8O-labeling data from 1.5% C02-grownChlorella yield rates of glycolate synthesis that are in agree-ment with each other. However, the rate of glycolate produc-tion computed from the "8O-labeling of excreted glycolate isabout one-half lower than the rate estimated from the twoamino acids. It is too low to account for the glycolate excretionrate. One possible explanation for this is that glycolate isexcreted in the form of its lactone (7, 29). Since a lactone ofglycolate is unstable in an aqueous environment, it wouldrevert back to its charged form upon excretion. However, one

of its carboxyl oxygen would have been lost during lactone

formation. Thus, if '8O-glycolate is converted to a lactoneprior to excretion it has a 50% chance of losing the '8O-label.The calculated rate of glycolate synthesis for high C02-growncells under 21% 02 and 0.035% CO2 was 40.2 ± 1.3 nmol/mg Chl/min (correlation coefficient = 0.97) when we multi-plied the percent '8O-enrichment data by two prior to nonlin-ear regression. This rate was 83.4 ± 2.1 nmol/mg Chl/min(correlation coefficient = 0.98) for cells under 40% 02 and0.035% CO2. Both of these rates are in good agreement withthe rates estimated from the labeling kinetics of the aminoacids (Table II). This is the first piece of evidence, thoughindirect, the glycolate is excreted in the form of its lactone.

If the glycolate synthesis rates calculated from glycine andserine are averaged, then glycolate synthesis in high C02-grown Chlorella was about 41 nmol/mg Chl/min and 86nmol/mg Chl/min under 21% 02 and 40% 02, respectively.This is 15 to 20 times greater than the rates in air-grownChlorella under similar 02 concentrations. The glycolate syn-thesis rates of high C02-grown Chlorella were about 4 and9% of net photosynthesis under 21% 02 and 40% 02, respec-tively, values 20 times higher than comparable percentagesfrom air-grown cells.The lowered glycolate production in air-grown Chlorella

indicates that this alga has a lower photorespiratory rate than1.5% C02-grown cells. This is most likely the result of theactivity of the dissolved inorganic carbon pump suppressingthe oxygenase function of ribulose bisphosphate carboxylase/oxygenase (2).The lack of '8O-enrichment ofglycine and serine in Pavlova

suggests that this alga has a mechanism of glycolate metabo-lism different from Chlorella and terrestrial plants (19). Thismechanism could involve an 100% excretion of newly syn-thesized glycolate. Alternatively, a portion of newly synthe-sized glycolate could be diverted to a photorespiratory path-way which bypasses glycine and serine. Such a pathway hasalready been described for certain cyanobacteria (9), greenalgae (3), and diatoms (21). This alternate pathway involvesthe conversion of two glyoxylate molecules to tartronic acidsemialdehyde and CO2 via glyoxylate carboligase (3). Tar-tronic acid semialdehyde is then reduced by tartronic acidsemialdehyde-NADP-reductase to glycerate.The use of '8O-labeling of glycolate pathway intermediates

as a means to estimate photorespiration in algae has manyadvantages over other algal photorespiratory assays. The 180isotope is a specific tracer for the glycolate pathway (5). Theisotopic labeling kinetics curves of the intermediates can beused to determine rates of synthesis if they are coupled withthe appropriate rate equations and nonlinear regression tech-niques (22). The amount of isotopic recycling by photosyn-thetic tissue is negligible (23) and isotopic discrimination isinsignificant, at least for short-term studies (6, 15). Glycolatesynthesis can be measured under any concentration of02 andCO2. It is also one of the few assays which can detect andquantify photorespiration in algae suspended in an aqueousmedium. This is one of the first studies to demonstrate andquantify glycolate synthesis in algae under natural conditions.The principal disadvantage to this method is the requirementof a gas chromatograph/mass spectrometer, an expensiveinstrument which may not be available to every researcher.

1 092 Plant Physiol. Vol. 91, 1 989




The method is also not readily adaptable for field measure-

ments because of the required 180-labeling chamber, thesupporting apparatus for the storage and mixture of 1602,1802, CO2, and argon, and the mass spectrometer for themonitoring ofthe various gases. The 1802 gas is also expensive.Finally, the method is time-consuming since one must extract,isolate, and chemically prepare the photorespiratory metabo-lites prior to gas chromatograph/mass spectrometer analysis.Thus, the 180-labeling method may best be used in thoselaboratory studies which require detailed measurements ofglycolate synthesis.

ACKNOWLEDGMENTS

We would like to thank the following: the Food Science Depart-ment of The Pennsylvania State University and Dr. R. Gruver forthe use and maintenance of the gas chromatograph/mass spectrom-eter, Prof. D. Sibley for mathematical advice, Dr. K. Roeder forstatistical advice, Dr. B. Devlin for his assistance with the figures, andDr. J. M. Robinson for his assistance in the preparation of thismanuscript.

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