Plant Physiol. (1988) 87, 395-4010032-0889/88/87/0395/07/$01 .00/0

RuBP Limitation of Photosynthetic Carbon Fixation duringNH3 Assimilation1INTERACTIONS BETWEEN PHOTOSYNTHESIS, RESPIRATION, AND AMMONIUM ASSIMILATIONIN N-LIMITED GREEN ALGAE

Received for publication December 16, 1987

IVOR R. ELRIFI, JODY J. HOLMES, HAROLD G. WEGER, WILLIAM P. MAYO, AND DAVID H. TURPIN*Queen's University, Kingston, Ontario, Canada, K7L 3N6

ABSTRACT

The effects ofammonium assimilation on photosynthetic carbon fixationand 02 exchange were examined in two species of N-limited green algae,Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturatingconditions, ammonium assimilation resulted in a suppression of photo-synthetic carbon fixation by S. minutum but not by C. pyrenoidosa. Thesedifferent responses are due to different relationships between cellularribulose bisphosphate (RuBP) concentration and the RuBP binding sitedensity of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In bothspecies, ammonium assimilation resulted in a decrease in RuBP concen-tration. In S. minutum the concentration fell below the RuBP binding sitedensity of Rubisco, indicating RuBP limitation of carboxylation. In con-trast, RuBP concentration remained above the binding site density in C.pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa withlow light resulted in an ammonium-induced decrease in RuBP concen-tration below the RuBP binding site density of Rubisco. This resulted ina decrease in photosynthetic carbon fixation. In both species, ammoniumassimilation resulted in a larger decrease in net 02 evolution than in carbonfixation. Mass spectrometric analysis shows this to be a result of an in-crease in the rate of mitochondrial respiration in the light.

The assimilation of ammonium by photosynthetic organismsresults in changes to both photosynthetic and respiratory carbonmetabolism. This is illustrated by the diversion of recent pho-tosynthate from the synthesis of starch to the production ofTCAcycle2 intermediates. The keto-acids thus produced are thenavailable for assimilation of ammonium into amino acids (1, 9,31). There is evidence that in natural environments algal growthmay be limited by inorganic nitrogen (7). A key adaptation underthese conditions is an increase in the saturated rate of nitrogenuptake and assimilation (29). It is therefore not surprising thatthe supply of nitrogen to N-limited algae affects photosyntheticand respiratory metabolism to a much greater extent than in N-sufficient algae (8, 16, 25).

In some species of N-limited microalgae, the rapid assimilationof inorganic nitrogen results in a decrease in the rate of photo-

' Supported by the Natural Sciences and Engineering Research Coun-cil of Canada.

2Abbreviations: TCA cycle, tricarboxylic acid cycle; a-KG, alpha-ketoglutarate; CABP, carboxyarabinitol bisphosphate; CPBP, carbox-ypentitol bisphosphate; DIC, dissolved inorganic carbon; RuBP, ribulosebisphosphate; Rubisco, ribulose bisphosphate carboxylase/oxygenase; PEP,phospho(enol)pyruvate.

synthetic carbon fixation (8, 10). Photosynthetic carbon fixationis controlled by many factors, including the quantity and acti-vation of Rubisco and the availability of its substrates, CO2, 02,and RuBP (5, 6, 17-20, 22, 26, 27). In a study with the N-limitedgreen alga Selenastrum minutum (8), it was shown that thesuppression of photosynthetic carbon fixation during the assim-ilation of inorganic nitrogen coincided with a decrease in RuBPconcentration. This implied that RuBP levels may be responsiblefor limiting carbon fixation. In Chlorella pyrenoidosa, however,nitrogen assimilation results in large decreases in RuBP with littleor no effect on photosynthetic carbon fixation (12). Given thisapparent discrepancy, the role of RuBP in determining the rateof photosynthetic carbon fixation during N-assimilation requiresfurther clarification.Net photosynthetic 02 evolution also exhibits a wide range of

responses to N assimilation. In some N-limited algae, N assim-ilation results in only a slight suppression of net 02 evolution(10), while in others it can result in net 02 consumption in thelight (8). Clearly, the effects on net 02 evolution are not re-stricted to photosynthetic 02 evolution, they also must involve02-consuming reactions.The purpose of this paper was to examine the effects of am-

monium assimilation on both photosynthetic carbon fixation andnet 02 evolution in two species of N-limited green algae. Underconditions of saturating light, one of these species (Selenastrumminutum) exhibited a decrease in photosynthetic carbon fixationduring ammonium assimilation while the other (Chlorella pyr-enoidosa) did not. Examination of RuBP concentration and theRuBP binding site density of Rubisco is consistent with the hy-pothesis that, under certain conditions, ammonium assimilationmay result in RuBP limitation of photosynthetic carbon fixation.Measurement of gross 02 exchange by mass spectrometry dem-onstrates that the effect of ammonium assimilation on net 02exchange is a result of changes in both the rate of gross 02evolution and mitochondrial respiration in the light.

MATERIALS AND METHODS

Chemostat Culture. Two species of green algae, Selenastrumminutum Naeg. Collins (UTEX 2459) and Chlorella pyrenoidosaChick (UTEX 343), were grown axenically in NO3- -limited che-mostat culture at a growth rate of 0.3 d- 1 (8).

Experimental Conditions. Cells were harvested from chemo-stats and transferred to a water-jacketed (20°C) experimentalcuvette (9). For most measurements, cells were used directlyfrom the chemostat (2.89 and 3.14 gg Chl-ml-' for C. pyren-oidosa and S. minutum, respectively). Cells were concentrated20-fold for measurement of Rubisco activity, RuBP concentra-tion, and RuBP binding site density. The rate of photosynthetic

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Plant Physiol. Vol. 87, 1988

carbon fixation was unaffected by cell concentration. Additionof ammonium was to a concentration of 50 pM unless otherwisespecified.Carbon Fixation. Culture samples were incubated in the pres-

ence of H14CO3- (Atomic Energy Commission of Canada, 50,uCi ml- 1) with or without NH4Cl enrichment. Aliquots werewithdrawn at discrete time intervals and the incorporation ofacid stable 14C was determined by liquid scintillation counting(8).Net 02 Evolution. Net 02 evolution was measured using a Clark

type electrode (Hansatech, King's Lynn, England) and methodspreviously described (9).PEP Carboxylase Assay. The PEP carboxylase activity in cell

extracts was determined as previously described (4). Photosyn-thesizing cells were withdrawn from the experimental cuvette,rapidly cooled to 4°C, and disrupted in a French Pressure Cell(18000 psi). Aliquots (100 ,ul) were placed into 380 ,ul of reactionbuffer (50 mm Hepes [pH 8.0], 10 mM MgCl2, 5 mM DTT, 1 mmNADH, 10 mm NaH14CO3 [1 ,uCi mmol- ], 5 mM PEP, and S

units of malate dehydrogenase, Sigma). Incubations were carriedout for 0.5, 1.0, and 5.0 min. Reactions were terminated byaddition of 500 ,ul of kill solution (80% aqueous ethanol, 5%HCOOH), and radiolabel was determined by liquid scintillationcounting. The accumulation of acid stable radiolabel was linearover this time period (data not shown).

Rubisco Assay. The activity of Rubisco in cell extracts was

determined using the method of Perchorowicz et al. (17). Cellextracts were obtained as outlined for the PEP carboxylase assay.Aliquots (100 ,ul) of pressate were preincubated for 10 min inassay buffer (50 mm Bicine [pH 8.0], 25 mM MgCl2, 1 mm DTr,0.25 mm EDTA, 10 mm NaH14CO3 [1 ,uCi ,umol1-]) to fullyactivate the enzyme. Reactions were initiated by addition of 0.8mM RuBP and were terminated by the addition of 500 p.l of killsolution. Radiolabel was determined by liquid scintillation count-ing.RuBP Assay. The concentration of RuBP in cell extracts of S.

minutum and C. pyrenoidosa was determined as previously de-scribed (8). Aliquots (750 ,ul) of photosynthesizing cells wereremoved from the experimental cuvette and placed directly inice-cold HC104 (10% final concentration). Illumination was pro-vided throughout the procedure. Samples were then neutralizedwith S M KOH and aliquots (100 ,ul) were assayed in reactionbuffer (50 mm Bicine [pH 8.0], 25 mM MgCl2, 1 mm DTT, 0.25mM EDTA, 10 mm NaH14CO3 [1 ,uCi ,gmol-1]) using activatedRubisco purified from commercially obtained spinach (8). Re-covery of an authentic internal standard of RuBP was alwaysgreater than 82%. Measured values were corrected accordingly.RuBP Binding Site Determination. The density of Rubisco

binding sites for RuBP was determined using CABP(5, 6, 20,26, 32). [14C]CPBP, an epimer mixture of [14C]CABP and [14C]-2-carboxyribitol bisphosphate, was prepared by a method sim-plified from Siegel and Lane (20). RuBP (86.25 ,umol) was re-

acted with a 10% excess of K14CN (95 ,umol, 8 ,uCi,u.mol- l, ICNRadiochemicals, Montreal, PQ) for 15 h at 30°C in 1.0 ml of 20mM HCO3 -/CO32- buffer. Formic acid (5.0 ml) was added, andthe mixture was evaporated under vacuum to dryness. The so-

lution was made to 0.1 M CPBP by adding 862.5 ,ul distilled H20,titrated to pH 10.0 with NaOH, and then adjusted to pH 7.5with HCl. CPBP was frozen in aliquots at - 20°C. Since the Kdof CABP is 105 times smaller than that of carboxyribitol bis-phosphate, it was assumed that CABP was the compound thatbound to Rubisco as an RuBP analog (32).

Cell extracts were prepared as previously described. Aliquots(100 p.l) were reacted with 100 to 200 pM [14C]CPBP in Rubiscoassay buffer. Antibodies to Rubisco obtained from rabbit serum

(11) were used to bind the Rubisco-[14C]CABP complex. Theantibody-Rubisco-CABP complex was precipitated using either

protein A-Sepharose (lot MH 02605, Pharmacia Chemicals, Swe-den) or Staphylococcus aureus (Staph A) cells (lot 745004, SigmaChemicals, St. Louis, MO). Both methods of immunoprecipi-tation resulted in removal of greater than 97% of Rubisco activityfrom the filtrate. The precipitate was recovered on Milliporefilters (0.4 ,um, EH), and washed twice with 0.85 M NaCl con-taining 20 mm NaHCO3 and 20 mM MgC12 (6). Radiolabel wasdetermined by liquid scintillation counting. Counts were cor-rected for CPBP adsorption to the filter. The accuracy of theCABP binding site determination was assessed by comparing theexperimentally obtained binding site density with that calculatedfor a known quantity of purified Rubisco. The quantification ofpurified Rubisco was undertaken using both the Bio-Rad proteinassay (Bio-Rad, Richmond, CA: bovine y-globulin standard) andthe absorption coefficient at 280 nm of 0.61 (mg ml- 1) -cm -

reported by Wishick et al. (30). The determination of the RuBPbinding site density with [14C]CABP gave results in excellentagreement with the other methods (Table I).Ammonium Uptake. Ammonium uptake was measured as dis-

appearance from the medium with an Orion 9512 NH3 electrode.Mass Spectrometry. 02 exchange was measured using a VG

Gas Analysis MM14-80SC mass spectrometer (Middlewich, Eng-land) as previously described (28). Mass/charge (m/z) ratios of32 (1602), 34 (160:180), 36 ('802), and 40 (40Ar) were measuredonce every 21 s in control and ammonium-enriched algal samples.Cell samples were illuminated at a saturating light intensity of165 ,E-m- 2.S- 1. The rates of gross 02 exchange were calculatedas previously described (28).Other Measurements. DIC was measured by IR gas analysis

(8). Chl was extracted in 100% methanol. To aid in Chl extractionfrom C. pyrenoidosa, 4% DMSO was added. Inhibitors wereused as previously described (8, 28).

RESULTS

Light Saturated Photosynthesis and Rubisco Activity. Underlight saturating conditions (>100 ,E-m -2S -1) Selenastrum min-utum and Chlorella pyrenoidosa exhibited similar rates of carbonfixation (Table II). The addition of 50 p.M ammonium to S.

Table I. Evaluation of the Accuracy of the CABP Radioimmune Assayfor the Quantification of RuBP Binding Site Density of Purified

Spinach RubiscoThe conversion between protein concentration and binding site density

assumes 8 binding sites per holoenzyme.

Protein Binding SiteConcentration Density

ugllOO ,ul nmollOO ,ulAbs (280) 201.4 2.88aProtein assay 183.8 ± 8.9 2.63 ± 0.13aCABP assay 180.7 ± 4.la 2.58 ± 0.06

aThese numbers were calculated from the measured data.

Table II. Maximum Rates of Carbon Fixation, Rubisco Activity, andthe RuBP Binding Site Density in Cell Extracts of S. minutum and C.

pyrenoidosaS. minutum C. pyrenoidosa

Maximum rate of photosyntheticcarbon fixation(AmolCO2 * mg Chl *h -) 151 ± 6 169 ± 11

Rubisco activity(,umolCO2 * mg Chl' h ) 162 ± 15 210 ± 21

Binding site density(nmol * mg Chl) 59.8 ± 1.9 35.5 ± 1.0

396 ELRIFI ETAL.

PHOTOSYNTHESIS, RESPIRATION, AND NITROGEN ASSIMILATION

minutum resulted in nearly a 50% suppression of carbon fixationbut had no effect on C. pyrenoidosa (Fig. 1). Ammonium ad-dition had no effect on the extractable Rubisco activity in eitherspecies, the activity of which was slightly higher than the max-imum rate of photosynthetic carbon fixation (Table II).The control rates of photosynthetic 02 evolution were also

similar for both species (Fig. 1). The addition of 50 l.M am-monium to either species resulted in a decrease in net 02 evo-lution (Fig. 1). The magnitude of the decrease was greater in S.minutum than for C. pyrenoidosa.Ammonium Uptake. Under light saturating conditions, S. min-

utum and C. pyrenoidosa exhibited ammonium uptake rates of167 + 7 and 97 + 1 ,umol N mg-1 Chlh- , respectively (datanot shown).RuBP Concentration and RuBP Binding Site Density. In S.

minutum, the RuBP binding site density was 59.8 + 1.0 nmol-mg- I

Chl (Table II). The concentration of RuBP during steady statephotosynthesis was 109 + 1 nmol mg-I Chl. The addition ofammonium resulted in a decrease in the level of RuBP to 40 +1 nmol mg- I Chl, a value below the RuBP binding site density.This coincided with a decrease in photosynthetic carbon fixation(Fig. 2). In C. pyrenoidosa the RuBP binding site density was35.5 + 1.9 nmol mg-I Chl (Table II), a value significantly lessthan S. minutum. During steady state photosynthesis, the RuBPconcentration was 114 + 2 nmol mg-I Chl. This decreased to61 + 3 nmol mg-I Chl following ammonium addition, a con-centration greater than the density of RuBP binding sites.

Selenastrum

NH4 °o

1EA5

50-

00)E

0

E

40

30

20

10

0

Dark Carbon Fixation and PEP Carboxylase Activity. Additionof ammonium resulted in the stimulation of dark carbon fixationin both S. minutum and C. pyrenoidosa (Table III). The rate ofdark carbon fixation in the presence of ammonium was similarto the activity of PEP carboxylase measured in cell-free extractsof each species. However, ammonium addition had only a slightstimulatory effect on PEP carboxylase activity of cell extracts(Table III).02 Consumption in the Light and Dark. Addition of ammonium

in the dark resulted in a 153% and 129% stimulation of dark net02 consumption in C. pyrenoidosa and S. minutum, respectively(control rates were 34 + 1 and 91 + 4 ,umol 02 Chlh- , re-spectively).Mass spectrometry showed that 02 consumption in the light

also increased in response to ammonium addition. The controlrate of 02 consumption in the light by S. minutum was 78 + 19,umol mg-1 Chl-h-1. This increased to 202 + 21 ,umol mg-1Chlh-1 during NH3 assimilation. A previous study (28) has shownthat this increase in 02 consumption is due to an increase inmitochondrial electron transport. In C. pyrenoidosa, ammoniumaddition increased 02 consumption in the light from a controlrate of 40 ± 5 ,mol O2-mg ' to 155 + 3 ,umol O2-mg 1 Chl1h-1(Fig. 3). The addition of CN- resulted in a 81% inhibition of02 uptake (data not shown).

Interactions between Carbon Fixation and Ammonium Assim-ilation by C. pyrenoidosa at Different Light Intensities. Ammo-nium assimilation by C. pyrenoidosa at saturating light (>100

Time (min)FIG. 1. Short-term responses of light-saturated (160 ,E m-2.s- 1) net photosynthetic oxygen evolution and gross carbon fixation to NH4+ addition

in N-limited cultures of C. pyrenoidosa and S. minutum (,a = 0.3 d- 1). A, Effect of 50 ,iM NH4+ enrichment on oxygen evolution in S. minutum.The pre- and postenrichment rates were 138 + 5 and 7 + 1 ,umol 02 mg-' Chl h-1, respectively. B, Effect of 50 UM NH4+ enrichment on oxygenevolution in C. pyrenoidosa. The pre- and postenrichment rates of oxygen evolution were 150 ± 6 and 47 ± 3 ,umol 02 mg- ' Chl-h- l, respectively.C, Effect of 50 tsM NH4+ addition on carbon fixation in S. minutum. The pre- and postenrichment rates were 128 ± 3 and 63 ± 8 Amol CO2 mg- I

Chl-h-1, respectively. D, Effect of 50 IM NH4+ addition on carbon fixation in C. pyrenoidosa. The pre- and postenrichment rates were 122 ± 3and 120 ± 1 ,umol CO2 mg-l Chl-h-1, respectively.

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Plant Physiol. Vol. 87, 1988

I I~~~~~~~~~~~~~~~~~~~~~~*

Selenastrum

*o'o-mre-

A

NH4 A

v - w N

- E ; Em*----

le"" . "M-.

NHA C

OVOMPqvm0 afft00 a0 0toft0 ~ eom0m1.

NH4

NH

B

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Time (min)FIG. 2. Changes in photosynthetic carbon fixation and cellular RuBP concentration in response to NH4+ addition in N-limited cultures of C.

pyrenoidosa and S. minutum under light-saturating conditions (500 ,E.m2 S-1) A, The initial rate of photosynthesis in S. minutum was 110 +1,umol C02 mg-' Chl-h-1 and decreased to 61 ± 4 Amol C02 mg-1 Chl-h-1 following NH4+ addition. B, The initial rate of photosynthesis in C.pyrenaidosa was 117 ± 1 ,umol C02 mg-1 Chl-h-I and did not change following NH4+ addition. C, The average initial RuBP concentration in S.minutum was 109 ± 1 nmolmg-I Chl and decreased to 40 ± 1 nmol-mg-' Chl following NH4+ enrichment. The binding site density, indicatedby the dotted line, was 59.8 nmol-mg- Chl. The rate of NH4+ assimilation following NH4+ pulsing was 169 ,umol mg- I Chl-h- 1. D, The averageinitial RuBP concentration in C. pyrenoidosa was 114 ± 2 nmol mg - Chl and decreased to 61 + 3 nmol-mg Chl-' following NH4+ enrichment.The binding site density, indicated by the dotted line, was 35.5 nmol-mg- Chl. The rate of NH4+ assimilation following NH4+ pulsing was 92,umol-mg- Chl-h- .

Table III. Effect ofAmmonium on Dark Carbon Fixation in WholeCells and the PEP Carboxylase Activity in Cell Extracts of S. minutum

and C. pyrenoidosa

S. minutum C. pyrenoi-dosa

Dark carbon fixationControl 1.1 ± 0.3 2.8 ± 0.2+ NH4+ 35.8 ± 1.9 35.5 ± 2.9

PEP carboxylase activityControl 38.1 ± 0.9 38.6 ± 1.9+NH4+ 43.6 ± 1.3 44.1 ± 4.2

,uEm -2-S 1) had no effect on photosynthetic carbon fixation.At light intensities in the light-limited region between 50 and 100,uEm -2-S 1, ammonium assimilation resulted in suppression ofphotosynthetic carbon fixation. Below this light intensity and inthe dark, the assimilation of ammonium stimulated carbon fix-ation (Fig. 4; Table III). The ammonium-enhanced rate of carbonfixation in the dark was similar to the maximum rate of PEPcarboxylase activity (Table III).

Effect of Ammonium Assimilation on RuBP Concentration ofC. pyrenoidosa at Low Light. At a light intensity of 80 ,uE-m-2 -1,the addition of ammonium resulted in a decrease in carbon fix-ation by C. pyrenoidosa (Fig. 4). This coincided with a drop in

n

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_= .c. _

._ CDc E

E 0cno0E& S

6

-50.

-100.

-150.

cwZLAJ . .

5 10 15 20

Time (min)

FIG. 3. Stimulation of mitochondrial oxygen consumption in the lightin response to NH4+ addition in C. pyrenoidosa. The initial rate of 02

consumption in the light was 40 + 5 ,umol 02-mg-' Chl h-' and in-creased to 155 + 3 ,tmol 0, mg- I Chl-h- following NH4+ enrichment.

150

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25 -

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Chlorella

NH+

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398 ELRIFI ETAL.

PHOTOSYNTHESIS, RESPIRATION, AND NITROGEN ASSIMILATION

200

Light Intensity400

(g E/m2 /s)

FIG. 4. A, Photosynthetic carbon fixation in C. pyrenoidosa as a func-tion of light intensity; B, the rate of photosynthetic carbon fixation inC. pyrenoidosa, following the addition of 50 ,UM NH4+, as a function oflight intensity; C, ammonium-induced suppression of photosynthetic car-

bon fixation as a function of light intensity in C. pyrenoidosa. A negativesuppression represents a stimulation of carbon fixation.

the concentration of RuBP below the RuBP binding site densityof Rubisco (Fig. 5).

DISCUSSION

Effects of N-Assimilation on Photosynthesis at Saturating Light.Carbon fixation. At saturating light (>100 ,uEm-2 -S 1), am-monium addition to N-limited Selenastrum minutum resulted ina major suppression in photosynthetic carbon fixation (Figs. 1and 4). This coincided with a decrease in RuBP below the RuBPbinding site density of Rubisco (Fig. 2). Such conditions implyRuBP was limiting photosynthetic carbon fixation (5, 6, 26).Previous work (8) has shown that both the decrease in RuBP

100

80 -

00 60-CD

o 40 -Ea

20 -

00 10 20

Time (min)

FIG. 5. Changes in and cellular RuBP concentration in response toammonium assimilation in N-limited C. pyrenoidosa under light-limitingconditions (80 uE m -2-S 1). The average initial RuBP concentration was89 + 1 nmol-mg -Chl and decreased to 29 ± 1 nmol-mg- I Chl followingammonium enrichment. The RuBP binding site density was indicated bythe dotted line and was 35.5 nmol mg - Chl.

and the resulting drop in photosynthetic carbon fixation are dueto ammonium assimilation and are not due to uncoupling ofphotosynthetic electron flow.

In contrast to these results, ammonium assimilation by C.pyrenoidosa did not affect carbon fixation at saturating light (Fig.1B). Although the assimilation of ammonium resulted in a de-crease in the cellular RuBP concentration, it remained above thebinding site density of Rubisco (Fig. 2). Therefore, photosyn-thetic carbon fixation remained RuBP saturated.The mechanism accounting for the decrease in RuBP during

ammonium assimilation is still unknown. One hypothesis is thatduring ammonium assimilation Calvin cycle intermediates areexported from the chloroplast to provide carbon skeletons forN-assimilation. If the removal of triose phosphates from theCalvin cycle exceeded one-third the rate of carbon fixation, RuBPregeneration and RuBP concentrations would decrease. Previouswork with S. minutum has shown that the rate of carbon flowinto amino acids during ammonium assimilation is greater thanthat which could be supported by steady state photosynthesisalone (9) and that the additional carbon requirements are metby starch degradation in the light (our unpublished data; 14).Although this is a necessary condition of this hypothesis it doesnot constitute proof. It is possible that other factors regulateCalvin cycle activity during ammonium assimilation and hencecontribute to the decrease in RuBP.The underlying cause of the different responses exhibited by

these two organisms may in part be explained by both the lowerrate of ammonium assimilation by C. pyrenoidosa and its lowerRuBP binding site density. The lower rate of ammonium assim-ilation would result in lower rates of carbon flow into aminoacids and possibly lower demands on Calvin cycle intermediates.Although the drop in RuBP is extremely rapid, the imbalancebetween RuBP regeneration and consumption is short lived. Withinminutes of ammonium addition, a new steady state RuBP con-centration is achieved, and RuBP consumption and RuBP re-generation must again be in equilibrium. Although we are unableto provide experimental support for a detailed mechanism of theeffects of ammonium assimilation, it is apparent that changes inthe RuBP pool relative to the RuBP binding site density providean explanation for the species-specific differences in the effectsof ammonium assimilation on photosynthetic carbon fixation.Net 02 Evolution. The effects of ammonium assimilation on

net 02 evolution are markedly different from those on carbonfixation. In S. minutum the 50% inhibition of carbon fixationwas accompanied by a 95% inhibition in net 02 evolution (Fig.

Chlorella

-

a t)o0x E

_

2o

ca

C) -"00

E

r-

,o t)

.x E

c cm0 0

E=L

.- # . w Chlorella

. ., .

NH4I~ I

100

a0

0.

C')an

Q1IQ0

0

399

Plant Physiol. Vol. 87, 1988

1). Net 02 evolution, however, is the sum of gross 02 evolutionfrom water photolysis and gross 02 consumption. Previous workby Weger et al. (28) has shown that mitochondrial electron trans-port chain activity increased during ammonium assimilation. Thisserved to oxidize the NADH produced by the TCA cycle duringammonium assimilation. The result was an increase in respiratory02 consumption in the light. This accounted for the larger de-crease in net 02 evolution than in carbon fixation in S. minutum(28).

In C. pyrenoidosa, ammonium addition did not affect pho-tosynthetic carbon fixation but resulted in a 69% decrease in net02 evolution (Fig. 1). An increase in mitochondrial respirationduring ammonium assimilation, similar to that observed in S.minutum, may explain the ammonium-induced decrease in net02 evolution in the absence of any effect on carbon fixation. Ifthis were the case, respiratory 02 consumption would be sup-ported by increased NADH production by the TCA cycle. Infact, early work on this organism by Kanazawa et al. (12) showedthat the assimilation of nitrogen resulted in an increase in therate of TCA cycle activity in the light. Figure 3 shows that 02consumption in the light by C. pyrenoidosa increases from ap-proximately 40 ,umol O2 mg-1Chl-h-1 to 155 gmol mg-1 Chl-h-during the assimilation of ammonium. This 02 uptake was cy-anide sensitive (81%), implying that it was at least partially dueto the activity of cytochrome oxidase.The possibility that reactions other than mitochondrial respi-

rations were involved in the ammonium-induced 02 uptake wasevaluated. A role for photorespiratory 02 consumption was elim-inated by carrying out these experiments at high levels of CO2(2, 3). Other workers have suggested that the Mehler reactionmay play an important role in 02 consumption in the light bygreen algae (23, 24). This suggestion was evaluated by inhibitinglinear photosynthetic electron flow, and hence the Mehler re-action, with DCMU. The addition of ammonium resulted in asimilar increase in 02 consumption indicating that, under ourconditions, the Mehler reaction does not contribute significantlyto ammonium-induced 02 consumption (28). We therefore con-clude that mitochondrial 02 consumption in the light increasesdramatically during ammonium assimilation by C. pyrenoidosa.The increase in mitochondrial 02 consumption during am-

monium assimilation by C. pyrenoidosa (Fig. 3, 110 ,umol mg-1Chl h- 1) accounts almost entirely for the 103 umol mg- 1 Chl-h- I

decrease in net 02 evolution during ammonium assimilation (Fig.1). Our conclusion is that the suppression of net 'photosynthetic'02 evolution during ammonium assimilation by C. pyrenoidosais not caused by a decrease in the rate of water splitting but byan increase in mitochondrial respiration. This interpretation isconsistent with the lack of an ammonium effect on light saturatedcarbon fixation in this species.

Induction of Photosynthetic Suppression in C. pyrenoidosa.Photosynthetic carbon fixation remains RuBP-saturated duringammonium assimilation by N-limited C. pyrenoidosa at saturat-ing light. This implies that the rate of RuBP regeneration remainshigh enough to ensure a saturating pool of RuBP during am-monium assimilation. If the rate of RuBP regeneration could belowered, it may be possible to produce cells of C. pyrenoidosawhich would exhibit a decrease in photosynthetic carbon fixationduring the assimilation of ammonium. The simplest way to dothis is by lowering the light intensity (26). The relationship be-tween photosynthetic carbon fixation and light intensity for C.pyrenoidosa is given in Figure 4. Carbon fixation by C. pyren-oidosa was unaffected by ammonium assimilation at light in-tensities above 100 ,gE-ms-2S-. At light intensities below 50,uEm -2-S 1, and in the dark, ammonium increased the rate ofcarbon fixation (Fig. 4; Table 3). This is presumably due to theincrease in PEP carboxylase activity required during ammoniumassimilation (8, 9, 12) and accounts for the substantial rate of

dark carbon fixation in ammonium-treated cells (Table 3). Be-tween 50 and 100 AEm -2.S- 1, the addition of ammonium re-sulted in a large suppression in photosynthetic carbon fixation.The suppression in carbon fixation at this light intensity wasaccompanied by a decrease in the RuBP concentration belowthe RuBP binding site density of Rubisco (Fig. 5). We concludethat under light-limiting conditions the assimilation of ammo-nium by C. pyrenoidosa may result in the limitation of photo-synthetic carbon fixation by RuBP.

CONCLUSION

The results presented in this paper suggest that ammoniumassimilation by N-limited green algae may affect the observedrates of photosynthesis in at least two ways. If the rate of RuBPregeneration is compromised and the RuBP pool decreases belowthe RuBP binding site density of Rubisco, photosynthetic carbonfixation will decrease. All things remaining equal, there wouldbe a corresponding decrease in gross 02 evolution. Measuredrates of net 02 evolution may also be affected independently ofchanges in photosynthetic carbon fixation. The increase in TCAcycle activity required to provide keto-acids for amino acid syn-thesis coincides with an increase in NADH oxidation via themitochondrial electron transport chain. This increases the rateof gross 02 consumption in the light and results in additionaldecreases in the measured rate of net 02 evolution.

Acknowledgments-We especially wish to thank Dr. S. Hemmingsen for pro-viding us with antibodies to Rubisco. We thank Drs. J. A. Berry and W. P. Plaxtonfor their advice on CPBP synthesis and Rubisco immunoprecipitation, respectively.D. G Birch is gratefully acknowledged for his assistance with mass spectrometryand figure preparation.

LITERATURE CITED

1. BASSHAM JA, PO LARSEN, AL LAWYER, KL CORNWELL 1981 Relationshipsbetween nitrogen metabolism and photosynthesis. In JD Bewley, ed, Nitro-gen and Carbon Metabolism. Junk, London, pp 135-163

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