232

Prokaryotes

6 InteractionsBetweenPhotosyntheticNitrate

Assimilationand COrFixationin Cyanobacteria

T. CORONIL,
andM.G.GUERRERO'
C. LARA,J.M.ROMERO,

6.1 Introduction
As in other photosyntheticorganisms,carbon and nitrogen are two of the most abundant elementsin cyanobacteria(blue-greenalgae), accounting for about 5O% and
10%, respectively,of their dry weight. The major sourcesof carbonand nitrogenavailable to the generality of cyanobacteriaare the oxidized forms carbon dioxide and
nitrate, which are assimilatedphotosynthetically.CO2 fixation and nitrate assimilation actually representthe two major processes
in the photosyntheticmetabolismof
cyanobacteriawith CO2 fixation exceedingnitrate assimilationby just a factor of
two with regardto the consumptionof photosyntheticallygeneratedreducingpower
(Floreset al. 1983b:Guerreroand Lara 1987).
The assimilationof CO2 in cyanobacteriatakes place primarily through the well0:
known reductivepentosephosphatecycle.Photosyntheticnitrate assimilationincludes:
(1) entranceof nitrate into the cell, apparentlymediatedby an active transport system; (2) two-step reduction of nitrate to ammonium, calalyzedby ferredoxin-dependent nitrate reductaseand nitrite reductase;and (3) incorporation of ammonium to
carbon skeletonsvia the ATP-dependentglutamine synthetase(GS) and ferredofndependentglutamatesynthase(GOGAT) enzymesystem(Floreset al. 1983b;Guerrero
and l^ara1987).
The integration of photosynthetic carbon and nitrogen assimilationin greencells
standsas a challengingissuein photosynthetic metabolism.Although the relevance
of a balanced assimilationof these two bioelementsis self-evident,little is known
about the underlying control mechanisms.In this article we attempt to summarize
the presentstatusof knowledgeregardingthe interactionsbetweennitrate assimilation
and CO2 fixation in cyanobacteria,especiallywith respectto two different aspects:
(1) effects of CO2 fixation on nitrate assimilation;and (2) incidenceof nitrate and
ammoniumassimilationon CO, fixation..-+

'

Departamento de Bioquimica, Facultad d e Bio lo g ia, U ni versi dad de S evi l l a, A pdo. 1095,
4 1 0 8 0 S e v i l l a ,S p a in

InorganicNitrogenMetabolism
Editedby Ullrichet al.
@ Springer-Verlag
Berlin Heidelberg1987

46

C. Lara et al

Table 6.1. Effect of light intensity on the rate of COr, nitrate, and ammonium assimilation by
A. nidulons cells
Light intensity
(pE m-2 s-r )

34
57
92
140
260

Assimilationrate
(pmol mg-r chl h I )a
CO ,

NO;

NHo'

109
189
269
401
444

10
22
50

23
45
50
50

b+

)J

JZ

CO2 fixation was determined as raC-incorporation into acid-stable material at saturating IraC]NaHCO. (10 mM). Nitrate and ammonium uptake were determined as the disappearanceof the
ions from the medium in cell suspensionssupplied with 0.25 mM of either KNO, or NHoCI and
10 mM NaHCO3 (see Lara and Romero 1986, for details); chl = chlorophyll a.

6.2 Dependenceof InorganicNitrogenAssimilationon CO2 Fixation

@ tn ttte utilization of any form of inorganic nitrogen (either ammonium, nitrate, or
dinitrogen), an obligate requirementfor CO2 fixation products arisesat the level of
ammonium assimilation,as a consequence
of the substratedemandfor carbon skeletons, mostly as d-ketoacids,by the GS/GOGATcycle and relatedtransaminatingreactions. In this context are the observations
that sustainedammoniumuptakeinAnacystis
is dependentupon the availabilityof co2 (Boussibaet al. 1984)and that o,L-glyceraldehyde,a selectiveinhibitor of the reductivepentosephosphatecycle in Anacystis
(Romero et al. 1985), effectively inhibits ammonium uptake (Lara and Romero
1e86) .
A comparativestudy of the ratesof nitrate and ammonium utilization with those
of CO2 fixation in Anocystisnidulansasa function of light intensity hasrevealedthat
clear differences exist, however, between nitrate and ammonium utilization with
regard to their dependenceupon the provision of fixed carbon (Lara and Romero
1986). As shown in Table6.1, ammoniumutilization reachedits maximum rate at
low photon fluxes, at which CO2 fixation proceededat about one half of its maximal
velocity. This indicatesthat, on a quantitative basis,the dependenceof ammonium
assimilationupon CO2 fixation is only moderate,the former processreachingmaximal
rates at levels of CO2 fixation that, although far from maximal, ensurean adequate
provisionof a-ketoacids.In contrast,the ratesof nitrate uptake and CO2 fixation at
every light intensity correlatedvery closely(correlationcoefficient,0.996;0<0.001;
l-aru and,Romero 1986), both processesvirtually reachinglight saturationat about
250 pE m-2 s-l (Table 6.1). This positivecorrelationbetween the ratesof CO2
fixation and nitrate uptake is lost by treatment of the cells with the ammonium
assimilationinhibitor, L-methionineD,L-sulfoximine(MSX). In MSX-treated Anacystis
cells,nitrate entranceand reduction to ammonium reachessaturationat photon flux
valuesas low as 70 pE m-2 s-l , while the behaviorof CO2 fixation with regardto light
intensity remainsunchanged(l,ara and Romero 1986). It seemsthus clear that the

Interactions Between Photosynthetic Nitrate Assimilation and CO, Fixation in Cyanobacteria

47

tight coupling existing between the rates of CO2 fixation and nitrate utilization in
normal, untreated cells does not reflect the requirementfor nitrate assimilationof
either photosyntheticassimilatorypower or carbonskeletonsto incorporatethe resultof nitrate uptake
ing ammonium,but it rather representsanother type of dependence
upon CO2 fixation.
Some other observationsillustrate the strict, quantitative dependenceof nitrate
utilization on CO2 fixation. Nitrate uptake in A. nidulansreachesa half-maximumrate
Oz
at a CO2 concentration(3.7 pM) very closeto the K, for COz of CO2-dependent
inhibition of CO2 fixation
evolution(+ pM) (Floreset al. 1983c).Also, the progressive
by increasingconcentrationsof o,L-gryceraldehyderesults in a gradual decreasein
the rate of nitrateuptake(Romeroet al. 1985b).
As is the casefor eukaryoticmicroalgae(Syrett 1981), the CO2 requirementof
nitrate uptake in cyanobacteriacan be alleviatedor suppressedby altering the C/N
ratio of the cells. In this respect,N-starvedcyanobacterialcells exhibiting a high C/N
ratio are able to take up nitrate in the absenceof CO2 (Stevensand van Baalen1973;
Flores et al. 1983c), indicating that carbohydrateaccumulatedduring the N-starvation period can substitutefor fresh products of CO2 fixation in their positive effect
on nitrate uptake.
The strict carbon dependenceof nitrate utilization seemsto be relatedto the wellknown phenomenon of the ammonium-promotedinhibition of nitrate uptake (see
Guerreroet al. 1981;syrett l98l;I-Illrich 1983;Guerreroand [.ara1987;for reviews),
being regulatoryin nature. Studieswith inhibitors of ammonium assimilationsuchas
such as GOGAT,
MSX and azaserine,which inactivate GS and amide transferases
respectively,have shown that prevention of ammonium assimilationprotects nitrate
uptake from the negativeeffects of ammonium (Flores et al. 1980, 1983b),and
allowsthe processto proceedin the completeabsence
of CO2(Floreset al. 1983b,c).
Treatmentwith MSX or azaserinealso relievesthe inhibition of nitrate uptake caused
(Romero et al. 1985b)and, as disby the CO2-fixationinhibitor D,L-glyceraldehyde
cussedabove, releasesnitrate uptake from its close dependenceupon the CO2-fixation rate (Lara and Romero 1986). Under theseconditions,nitrate is taken up by the
cells in an uncontrolledmanner and reducedto ammonium which is excretedto the
medium. Thus, by preventing ammonium assimilation,nitrate uptake results freed
from both the inhibition by ammonium and the requirementfor CO2. Theseobservations suggesta common basisfor the mechanismunderlyingthe positiveeffect of CO2
and the negativeeffect of ammonium on nitrate uptake.This connectionis especially
evidentin HYl-treated cells. HYL (5-hydroxylysine)is a competitiveinhibitor of GS
which, in cellsof Anacystis,inducesa concentration-dependent
inhibition of ammonium
utilization. By treating,4. nidulanswith an appropriateconcentrationof HYL, it is possibleto obtain cellswith a moderateammoniumassimilationcapacity.Underconditions
of limited CO2 supply, addition of NaHCOgto HYl-treated cellsresultedin acceleration of CO2 fixation and in the simultaneousreleaseof nitrate uptake from the
ammonium inhibition, to reach rates similar to those of control cells not exposed
to ammonium (Romero et al. 1986). Theseresultsshow that CO2 actuallybehavesas
an antagonistof ammoniumin the modulation of nitrate uptake.
The data fit within the framework of a model for the regulationof nitrate uptake
involving products of both ammonium and CO2 assimilation(Flores et al. 1983c;

48

C. Lara et al.

NO ;

NH;

Glu t amin e

i n h i b i to ry C .N-m tabolites

co.
I

COr-fixation products

ot her C .N - c om pounds
l non- i nhi bi tor y l

Fig. 6.1. A working hypothesison the mechanismtbr the t'eedback

regulationof nitrate utilization
mediatedby organicN-compounds
and COr-fixationproducts(afterFloreset al. 1983c).Seetext
for details

Romeroet al. 1985b).Accordingto this scheme(Fig. 6.1),nitrateuptakeis underthe

negativefeedbackcontrol exertedby certainorganicnitrogenouscompoundsresulting
from nitrate assimilationvia ammonium. The intracellular concentration of these
inhibitory metaboliteswould be determinedboth by the supply of ammonium from
which they are generatedand by the provisionof certainCOz-fixation productswhich,
in the course of normal metabolism,would combine with the inhibitory ammonium
derivatives,removingthem while generatingother noninhibitory C,N-intermediates.
By
either lmpeding CO2 assimilationor limiting the rate of CO2 fixation through light or
CO2 availability, accumulationof the inhibitory metabolitesis favoredand total or
partial inhibition of nitrate uptake occurs.On the other hand,by inhibiting ammonium
assimilationand the generationof negativeeffectors,nitrate uptake is relievedfrom
the requirementfor CO2 fixation products,this responsebeingalsogradualdepending
on the extentof the inhibitionof ammoniumassimilation.
The operation of sucha regulatorysystemwould allow a fine modulation of nitrate
utilization exerted not only by accumulationof some of its own products,but also
through the availability of carbon metabolites,thus resulting in a tight control of
nitrate utilization by carbon assimilation.Some key issuesremain to be solved for
achievinga better understandingof this regulatorysystem,among them, the identity
of the ammonium derivative(s)actingasnegativeeffector(s),and the regulatedstep of
the process.
Glutamine appearsto be a good candidatefor the regulatorysignal,sinceits level
increases
in responseto ammoniumaddition,and treatmentof cellswith MSX, which
preventsinhibition of nitrate uptake, leadsto decreased
glutaminelevels(Floreset al.
1980, 1983b).We haverecentlyobservedthat in response
to ammoniumadditionto
Anacystiscellsactivelyutilizing nitrate the intracellularlevelof glutamineimmediately
increasedabout tenfold and, following ammonium depletion from the medium, fell
againto its originallevel(Coronil et al. 1986).A correlationis thus apparentin this
situation betweenthe inhibition of nitrate uptake and the high glutaminelevels.It is

Interactions Between Photosynthetic Nitrate Assimilation and CO, Fixation in Cyanobacteria

49

interestingto note that no other amino acid,out of 20 more tested,accumulatedin

the cells shortly after ammoniumexposure(Coronil et al. 1986).It is tempting to propose glutamineas the negativeeffector of nitrate uptake, but steady-state
amino acid
analysisin normal untreated and HYl-treated cells exhibiting different capacitiesto
utilize nitrate in the presenceof ammoniumshow no correlationbetweenthe glutamine
level and the observedrates of nitrate uptake (unpublishedresults).The data speak
againstamino acids as negativeeffectorsof nitrate uptake, but do not excludethat a
transient increasein the glutamine pool might be the trigger promoting a metabolic
situation inhibitory for nitrate uptake, which could be maintainedeven after a substantialdecrease
in the pool ofglutamine had taken place.
As nltrate reductaseis not affectedby ammoniumin the short-term,nitrate entrance
to the cell has been suggestedto be the regulatedstepin nitrate utilization by cyanobacteria(Ohmori et aI. 1977;Floreset al. 1980, 1983b).This seemsto be the casefor the
diatomPhaeodactylurnin which nitrate accumulationis preventedby either ammonium
addition (R.C. Cresswelland Syrett 1979) or CO2 deprivation(P.J.Syrett, personal
communication). In vivo experimental separation of the entrance step from the
subsequentmetabolismof nitrate in cyanobacteriahas proven a difficult task. Very
recently. by performinghigh pressureliquid chromatographicanalysisof acid lysates
of A. nidulans cells after centrifugation through silicone oil, we have been able to
measureintracellularaccumulationof nitrate up to sixfold the externalconcentration
of the ion, indicativeof the existenceof an activetransportsystem.Preliminarydata
on the responsesof nitrate transport to ammonium and CO2 control indicate this to
be the target of the regulatorysystemof nitrate utilization in cyanobacteria(unpublishedresults).

6.3 Incidenceof Nitrogen Assimilationon COz Fixation

The assimilationof any form of inorganicnitrogen consumesassimilatorypower and
generatesorganic carbon-nitrogencompounds, utilizing CO2 fixation products as
amino acceptors.CO2 fixation might thus be affectedby nitrogenassimilationat two
different levels.A competition for reducingpower and/or ATP can resultin decreased
rates of CO2 fixation as a result of simultaneousnitrogen assimilation.On the other
hand, the utilization of e-ketoacidsin ammoniumassimilationcan affect qualitatively
the pattern of CO2 fixation products.Theseincidencesof inorganicnitrogen assimilation on carbon metabolismare still a matter of controversyin eukaryotic microalgae
(see Syrett 1981), but little has been done to investigatethe situationin cyanobacteria.
Recent resultsfrom our laboratory(Romero and Lara 1987)haveevidencedthat at
saturatinglight intensityandbicarbonateconcentration,the rateofoxygen evolutionby
intact A. nidulanscellsis increasedin responseto nitrate addition(about 2.4 mole extra
02 evolvedper mole nitrate taken up) without any depression
in the rate of roCO2
fixation, indicating that under theseconditions,the photosyntheticapparatusis able
to provideenoughassimilatorypower to maintainmaximal ratesof nitrate assimilation
and co2 fixation, without competition betweenthe two processes.
At limiting light
intensities,a moderate competition between nitrate utilization and CO2 fixation is

50

C. Lara et al.

apparent,nitrate inducing a depressionin the rate of laCOz fixation, in consistency

with the moderateextent of the nitrate-inducedstimulationof 02 evolution(lessthan
I mole extra 02 evolvedper mole nitrate taken up) observedunder theseconditions
(Romeroand Lara 1987).
In contrast, no competition is evident when ammonium is the nitrogen source
availableto the cells. In this case,even a consistentstimulation of CO2 fixation is
observedunder a wide range of light intensities,being particularly noticeableunder
light-saturatingconditions (Romero and Lara 1987). Simulation of CO2 fixation
by ammonium, both in the light and in the dark, has also been reported for the filamentous cyanobacteriumAnabaenavariabilis(Lawrie et al. 1976; Ohmori l98l) and
f or av ar iet y o f e u k a ry o ti c g re e n c e l l s (s e e Basshametal
l 93l .,andreferencestherei n).
In spinach cells, the stimulatory effect of ammonium is dependenton the CO2 concentration available,being maximal at saturatingCO2 (Woo and Canvin 1980). The
similarity of this behavior with that observedin Anacystis with regard to the light
intensity suggeststhat the ammonium-inducedstimulation of CO2 fixation may be
physiologically relevant under optimal conditions for light-dependentCO2 fixation. It would, therefore,appearthat when nitrate is the nitrogen sourceutilized by
the cells, the actual rate of COz fixation is, in fact, the balancedresultofthe negative
effect of nitrate reduction and the positive effect of the resulting ammonium, the
predominanceof one effect over the other being dependenton the light intensity.
While the basisof the positiveeffect of ammonium on CO2 fixation in cyanobacteria is not known, it doesnot seemto be causedby the cation itself, as it has been
proposedfor other green cells (Tillberget al.197l; Basshamet al. 1981), but to
require its assimilation,since it resultsabolishedwhen the cells are treated with the
ammonium assimilationinhibitor MSX (Ohmori 1981; Romero and Lara, 1987).
Actually, active ammonium assimilationinduced changesin the distribution of newly
fixed carbon into metabolite fractions, regardlessof the nitrogen sourceutilized by
the cells. Exposure of N-starvedAnabaena cylindrica to either N2 or ammonium
decreases
the incorporationof toC into sugarand sugarphosphateand increases
the
labeling of organic acids, especiallya-ketoacids(l^awrieet al. 1976). In N-sufficient
Anacystis niduluns cells,the situation is similar, but seemsto vary dependingon the
light intensity.Table 6.2 showsthe distributionof newly fixed carboninAnacystis
cells assimilatingcarbon and nitrogen at a light intensity of 57 1tEm-2 s-t , which
under our experimentalconditionsis limiting for both processes
(seeTable6.1).The
laC-labelingincorporated
into the acid-soluble
fraction increasedmarkedlyin cells
assimilatingeither nitrate or ammonium (Table 6.2). The magnitudeof the taC-shift
induced by the nitrogen source could be related to the rate of inorganic nitrogen
uptake, which, at this light intensity, was about twofold higher for ammonium than
for nitrate (Table6.1). Both nitrate and ammoniumeffectedan increasein the 1aClabelingof all the metabolitegroupsanalyzedin the acid-solublefraction, but the most
dramatic changeswere observedfor the case of the organic acid fraction, whose
labeling increasedup to fivefold in the presenceof nitrate and about 40-fold in the
presenceof ammonium(Table 6.2). Thesechangesin the distributionof carbonin
responseto inorganic nitrogen assimilationdid not take place in MSX-treatedcells
(datanot shown).

lnteractions Between Photosynthetic Nitrate Assimilation and CO, Fixation in Cyanobacteria

5l

Table 6.2. Effect of inorganic nitrogen assimilation on the distribution of newly fixed carbon
into metabolite fractions at limiting light intensity inA. nidulans cells
Fraction

Total
Ac id * o l u b l e
Sugar phosphate
Organic acids
Amino acids

In co r p o r a te d r a C (kdpm)a
Minus N

P/us NO.

P/as NHo*

t17
15
1. 5
0. 3
2. 6

99
29
3.7
1.6

t3l
59

l 1.l

4,tr

12.9
13.7

C"[ , * . r e s u b j e c t t o a p e r io d o f 1 8 m in o f CO, fixa tio n i n the presenceof 10 mM [t4C l N aH C O3

and, i n i t s c a s e ,0 . 2 5 mM KNO, o r 0 .2 5 m M NHo CI a t a light i ntensi ty of 57 pE m ' s ' . R eacti on
was stopped by HCI addition. The ta C-labeling incorporated into acid-stablematerial was measured
in the whole acid extract (total) and in the supernatant (acid-soluble fraction) of the centrifuged
sample (30 min, 12 000 g). The acid-soluble fraction was fractionated by a modification of the
procedure of Schiirmann ( 1969)

The resultsshowthat ammoniumassimilationresultsin a diversionof the carbonflow

(glycogen)to organicand aminoacidbiosynthesis.
from insolublecarbohydratereserves
A plausibleexplanationfor the positiveeffect of ammonium on COz fixation can be
raisedon thesegroundsas follows. Organicacids,especiallya-ketoacids,havea higher
oxidation level than carbohydrates.Thus, when ammoniumis beingassimilated.some
extra assimilatorypower would be releasedin the oxidativeconversionof sugarphosphate into the a-ketoacidsacting as amino acceptors.This assimilatorypower might
not only fulfill the requirementsof the GS/GOGAT cycle, avoidingcompetition with
the reductivepentosephosphatecycle, but also, dependingon which amino acidsare
This would explain
being synthesized,an excesscould be availablefor other processes.
why when ammonium assimilationis blocked by MSX, and therebythe utilization of
a-ketoacidsis hampered,the positive effect of ammonium on CO2 fixation and the
increasein t4C-labelingin solublemetabolitesare abolished(Romero and Lara 1987).
This hypothesis does not exclude other possibleammonium-promotedeffects that
could contribute to increasedCO2 fixation such as modulation of enzymesinvolved
in carbon metabolism by products of ammonium assimilation,a possibility that is
currentlybeinginvestigated
in our laboratory.

6.4 ConcludingRemarks
During the last 10 yearsa considerableadvancehas been made in the understanding
of the interactionsbetweencarbon and nitrogenassimilationin cyanobacteria.On the
one hand, the concertedaction of products of both nitrogen and carbonassimilation
in the control of nitrate utilization provides a clue on the ways by which the cell
balancesthe metabolismofcarbon and nitrogen,an outstandingaspectofcell physiology whose mechanismis still not understood.The emergingevidenceon the role of
nitrate transport as the controlled, rate-limiting step in nitrate utilization reinforces

52

C. Lara et al.: Interactions Between Photosynthetic Nitrate Assimilation and CO, Fixation

the importance of metabolic regulation at the level of substratesupply to the cell,

an idea that is gaining considerationamong biochemistsand physiologists(Kilberg
1986). Much researchis neededto elucidatethe nature of the regulatorysignalscontrolling nitrate uptake and the mechanismby which the activity of the nitrate translocator is modifred in responseto changesin the levelsof the regulatorymetabolites.
On the other hand, it seemsclear that not only COz fixation exertsa tight control of
inorganic nitrogen utilization, but also that nitrogen affectsthe rate of CO2 fixation
and the fate of newly fixed carbon.The prokaryotic cyanobacteriamay perhapsbe an
ideal system for the unravelingof the basic mechanismsof the ammonium-induced
stimulationof COz fixation and the partitioning of assimilatorypower betweenphotosynthetic CO2 fixation and nitrate assimilationunder light limiting conditions,which
hasresistedpreviousattemptswhen studiedin photosyntheticeukaryotes.
Acknowledgmer?/s. Research from the authors' laboratory has been supported by grants from
Comisi6n Asesora de Investigaci6n and Fundaci6n Ram6n Areces. We are grateful to M. Losada
for encouragement and help and to A. Friend and M.J. P6rez de Le6n for skillful secretarialassistance.

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