Copyright 0 1991 by the Genetics Society of America
Genetic and Biochemical Analysisof Intragenic Complementation
Events Among Nitrate Reductase Apoenzyme-Deficient
Mutants of Nicotiana plumbagingolia
F. Pelsy and M. Gonneau
Laboratoire de Biologie Cellulaire, INRA-Versailles, F-78026 Versailles Cedex, France
Manuscript received March 28, 1990
Accepted for publication October 1, 1990
ABSTRACT
Intragenic complementation has beenobserved between apoenzyme nitrate reductase-deficient
mutants (nia)of Nicotiana plumbuginqolia. I n vivo as in vitro,the NADH-nitrate reductase
(NR) activity
in plants heterozygous for two different nia alleles was lower than in the wildtype plant, but the
plants were able to grow on nitrate as a sole nitrogen source. NR activity, absent in extracts of
homozygous nia mutants was restored by mixing extracts from two complementing nia mutants.
These observations suggestthat N R intragenic complementationresults from either the formation of
heteromeric N R or from the interaction betweentwo modified enzymes. Complementationwas only
observed between mutants retaining different partial catalytic activities
of the enzyme.Results are in
agreement with molecular data suggestingthe presence of three catalytic domains in the subunit of
the enzyme.
I
NTRAGENIC complementation concerns the
interaction between two genomes which carry mutations located in the same functional unit. It is defined as the ability of the two mutated genomes to
function cooperatively in an heterozygous cell. T h e
mechanism of complementation involves interactions
between the products of the mutated genomes in the
absence of recombination of the genetic material.
A model has been proposed which postulates that
intragenic complementationmay occur when the gene
product is a multimeric enzyme composed of two or
more identical subunits. In such a case an heterozygous organism could produce a hybrid enzyme, containing polypeptide chains carrying different mutationalalterations, which mightresult in arestored
catalytic activity. Such an intragenic complementation
has been observed in a number of organisms: in vivo
as in vitro betweenalkalinephosphatase
structural
mutants of Escherichia coli (GARENand GAREN 1963;
SCHLESINCER
and LEVINTHAL 1963), betweenOAHOAS sulphydrylase mutants ofSaccharomyces cerevisiae
(D’ANDREA
et al. 1987) andin vitro complementation
of histidinol dehydrogenasemutanthavebeen
described in Salmonella typhimurium (LEE and GRUBMEYER 1987). Another so called “cross-feeding”mechanism of intragenic functional complementation could
also occur when the particular enzyme, such as the
HMG-CoA reductase (BASSONet al. 1987) orthe
tryptophan synthetase of
S. cerevisiae (DUNTZEand
MANNEY1968) catalyses a two step reaction with an
intermediary product which need not to be enzymebound for the second reaction to occur. In at least
one case, the yeast tryptophan synthetase encoded by
Genetics 127: 199-204 (January, 1991)
the TRPS gene, both mechanisms of intragenic complementation have been observed. Another well documented example of protein complementation is the
a-complementation of the E. coli /?-galactosidase (ZABIN 1982). In this complementation, the small amino
terminal a-fragment is able to restore high levels of
/?-galactosidase activity in astraindeletedforthe
corresponding region of the Lac Z gene.
NR is the first enzyme in nitrate assimilation pathway. All higher plant NRs characterized show homodimeric structure (for review, see CAMPBELL
1988).
Each monomer contains three prostheticgroups:
FAD, haem, and molybdenum cofactor which successively transfer two electrons from the initial donor
NAD(P)H to nitrate forits reduction to nitrite. These
three domains have been clearly evidenced in Arabidopsis by homology with proteinsequences in the
sequence data banks: the NAD(P)H/FAD binding domainhomologous tocytochrome b5 reductase,the
haem binding domain homologous tomammalian cytochrome b5 and the molybdenum cofactor domain
homologous to sulfite oxidase (CRAWFORDet al. 1988).
T h e prosthetic groups are housed in distinctfunctional domains covalently linked by protease sensitive
hinges (KUBO,OCURA
and NAKAGAWA
1988).
Nitrate reductase-deficient mutants affected in the
apoprotein structural NZA gene have been selected in
variousorganisms(for review, see WRAY1986). In
Nicotiana P l u ~ b u ~ n ~ Q al imodel
u , diploid plant species particularly well suited for genetical investigation
as only one gene encodes for the N R apoprotein, 65
mutants have beenclassified in the same NZA complementation group and the
possibility of intragenic com-
200
F. Pelsy and M. Gonneau
plementation were suggested by unexpected results
et al.
obtained during this genetic analysis (GABARD
1987). These mutantshave beenimmunologically and
biochemically characterized ( C H ~ R EetLal. 1990).
We present, here, theanalysis of intrageniccomplementation at the NZA locus. Plants resulting from the
sexual crosses between several homozygous nia mutants were able to grow on nitrate. T o test the hypothesis of protein-protein interactions between two
mutated inactive NR, protein extracts of both partners were mixed and a reconstituted NADH-NR activity were observed.
MATERIALS AND METHODS
Plant material and culture conditions: The nia mutants
were isolated from mutagenized haploid protoplasts of N .
plumbaginqolia cv Viviani as described by GRAFE,
MARIONPOLL andCABOCHE(1986) and by GABARD
et al. (1987).
Apoenzyme-deficient mutants were aseptically propagated
on solid BSR medium (GABARD
et al. 1987) containing 10
mM diammonium succinate as the nitrogen source and 10
mM KCI.
Genetic analysis of mutants: Since nia mutants plants do
not grow on their roots in the greenhouse they were routinely grafted onto N. tabacum cv Wisconsin-38 plants and
grown with standard nutrient solution containing nitrate
and ammonium(COICand LFSAINT1971). Flowering grafts
were used either for crosses between mutants or with the
NOT
wildtype.Collectedseedsweresterilized,sownon
medium and germinated under controlled conditions (GABARD et al. 1987). Heterozygous mutants carryingtwo distinct mutated nia alleles were transferedin the greenhouse
and their ability togrow on nitrate was tested.
Assay ofin vivo nitrate reductase activity:The measureet al.
mentswere done as previously described (GABARD
1987),but without propanol or Triton X-100 to minimize
the possible alteration of the complementing protein structure.
Assay of in vitro enzymatic NR activities: Soluble proteins were extracted from leaves and ammonium sulphate
precipitated. The overall or partial N R enzymatic activities
were measured according to a previously described proceet al. 1990).
dure (CH~REL
NADH-NR activity reconstitution procedure: Young
leaves were harvested after 8-hr illumination and stored at
-70°C beforeextraction, aspreviouslydescribed.
NO
NADH :nitrate reductase activity
was detected in individual
mutant extracts.Forreconstitutionexperiments,ammonium precipitated extracts of two different grafted homozygous mutant were mixedfor two hours at4" and NADHNR activity was measured by quantification of nitrite formation ( C H ~ R EetLal. 1990).
RESULTS
Genetic analysis of the NZA complementation
group: A total of 65 mutants have been confirmedto
belong to theNZA complementation group by genetic
analysis based on sexual crosses between nia E 2 3
mutants and homozygous or F1 plants (obtained after
cross of the homozygous mutants with the wild type
which results in a wild-type phenotype). T h e mutant
nia E 2 3 has been choosen as the reference nia mutant
as it is defective for production of the NR mRNA
TABLE 1
Example of genetic analysisfor the evidence of intragenic
complementation in the nia group
Group
Plants able to grow on
nitrate/total progeny
E23 Ro selfed
E77 Ro selfed
D5 1 Ro selfed
0/350 (0%)
0/248 (0%)
0/630 (0%)
E77 X E23 FI
D51 FI X E23 FI
E77 X D51
74/153 (48.4%)
156/203 (76.8%)
124/124 (100%)
[E77/D51] selfed
60/117 (51.2%)
Ro: grafted homozygous nia mutant. FI: heterozygous plants
obtained by crossing the homozygous nia mutant with the wild type.
Seeds were germinated on plates containing nitrate as sole nitrogen
source.
(POUTEAUet al. 1989)and does notrevert in the
progeny. All the nia mutants wereclassified according
to their biochemical properties (CHERELet al. 1990):
most of them (class 1) do not express immunodetectable NR protein with the monoclonal antibody ZM
96(9)25. Mutantswhich possess an immunodetectable
NR protein weresub-classified in two groups: mutants
which express partialnitrate reductaseactivities measured with reduced methyl viologen, FMNH2, reduced
bromphenol blue as artificial electron donors (class 2)
and mutantswhich only retain dehydrogenaseactivity
with artificial electron acceptor (cytochrome c) (class
3). Finally we also found mutants which have lost the
epitope recognized by the monoclonal antibody ZM
96(9)25 andso do notexhibit immunodetectable protein as tested in the sandwich ELISA measurement
but which still possess reduced bromphenol blue-nitrate reductase activity (class 4).
Evidence that some of these nia mutants were able
to complement some but not all other mutants was
intriguing as they are shown to be true members of
the NZA complementation group. As far as possible
and accordingtotheirpoor
fertility and viability,
crosses between grafted nia homozygous (Ro) mutants
have been performed to obtain a homogeneous hetRo X F1 or F1 X F I
erozygousprogenyotherwise
crosses have been done. In these cases, the theoretical
proportions of the heterozygous [nialnia] seedling
represented respectively 50% and 25% of the total
progeny. When Ro X F1 or F1 X F, crosses were done,
the number of seedlings able to grow on nitrate was
considered only if the germinationlevels were respectively higher than 50% or 75%, to be sure that hetNR- phenotype
erozygotes [nia/nia] expressinga
were not hidden among the nongerminated seeds.
Whencomplementation was observed, the large
majority of heterozygous seedlings was able to grow
on nitrate and the growth of some of them did not
differfrom wild type. They were fertile and their
progenyafter self-pollination showed the expected
segregation of 50% NR- and 50% NR+ (Table 1). O n
20 1
Intragenic complementation at NZA
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F. Pelsy and M. Gonneau
nitrate medium, some complementing heterozygous
plants are not always as healthy as the wild type and
chlorotic phenotypes (+/-) intermediate between the
phenotype of the homozygous nia mutants (two white
cotyledons) and thatof the wild type (++) were sometimes observed (Table 2).
Complementationbetween two mutated NIA genomes was observed in crosses when the two parental
nia mutants were classified in different classes according to their retaining partial
activities. This ability can
be correlated to the functional domain conserved in
each mutant: when complementation occurred, it was
always observed in crosses between a mutant retaining
a NADH-dehydrogenaseactivity and a mutant retaining terminal nitrate reductase activities. Only the peculiar nia E56 is able to complement both kinds of
mutant.Moreover,thesecomplementationexperiments allow us to distinguish two subclasses among
the class 3 mutants: onone side nia E77, F19,I 9 which
complement class 2 mutants and on the other
side
mutants like nia E122, HZ2 or E87, biochemically
classified in the same class 3 which do not efficiently
complement any other class of mutants. These later
mutants may be defective not only in terminal reductase activity but also in other aspects of NR subunit
organization, such as their ability to assemble into
dimeric structures.The nia 0 8 0 and 12 mutants have
been classifiedinclass 2 regarding the genetic and
biochemical functionalcomplementations,although
neither immunodetectable NR protein nor partialcatalytic activities can be detected.
In class 4 mutants, the loss of the immunodetectable
properties is attributedtothe
modification of the
epitope ZM 96(9)25 located in the haem domain (M.
KAVANAGH, unpublished results). Forinstance, nia
E56 mutant represents a particularly interesting case:
this mutant does not express a protein immunodetectable with the monoclonal antibody ZM 96(9)25
but still possess reduced bromphenol blueNR activity
and a NADH-ferricyanide reductase activity (data not
shown). This mutant nia E56 is able to complement
mutants with either terminal nitrate reductase activities (class 2: nia D 5 1 , 0 5 7 , 0 6 4 . . .) or initial partial
catalytic (class 3: nia E77, F19, 19) activities. These
results show that the mutation on the nia E56 allele
of the NIA gene affects the haem domain.
Mutants nia A I , K 2 1 , E64 like nia E56 (class 4) do
not possess protein immunodetectable by ZM 96(9)25
antibody and express only a low bromphenol blue
nitratereductase activity. We do notobserve any
functional complementationbetweenthesemutants
and nia E56 as expected, nor with mutants with terminal catalytic activities. A weak complementation can
be observed with some of the class 3 nia mutants, for
instance the [ A I / E 7 7 ] heterozygote express a significant restauration (+/-, Table 2).
In vivo NR activity in heterozygous plants: All
TABLE 3
NADH-NR activity in heterozygous nia mutants and in the
corresponding mixed extracts
NADH-NR activity (% of wild type)
Reconstituted NR activity in
the corresponding mixture'
Heterozygote
In vivo"
In vitro'
[19/D51]
[ I9/D57 J
[I9/D64]
[E77/D51]
[E77/E56]
[E56/D64]
[E56/D51]
Wild type
13.7
2.1
5.08
3.4
0.9
0.7
5
100
1.46
0.12
0.19
6.4
1
1.8
8
0.19
3.8
18.9
100
1
2
9
37
100
In vivo nitrate reductase activity measured in young leaves of
heterozygous nia mutants cultivated on their roots in the greenhouse (wild type = 27.16 nmol NO;. min" .g" fresh weight).
'In vitro nitrate reductase activity measured in ammonium precipitated protein extracts of heterozygous nia mutants cultivated
o n their roots in the greenhouse (wild type = 32.35 nanomole
NO;. min-l. g" fresh weight).
Reconstituted NADH-NR activity measured after mixing in an
egal ratio ammonium sulfate precipitated protein extracts of homozygous grafted nia mutants (grafted wild type = 9.08 nanomole
NO;.min-I .g" fresh weight).
These results are themean of two distinct experiments.
seedlings of complementing heterozygous are able to
germinate on plates with nitrateas sole nitrogen
source.However, we can distinguish plants where
growth was stopped at the fourleaves stage and plants
able to develop normally in the greenhouse. The in
vivo and in vitro NADH-nitrate reductase activities of
these plantlets were examined (Table3).
T h e in vivo activities of leaves ofheterozygous complementing plants grown in greenhouse represent 115% of the wild type measured in the same conditions
and the in vitro activities determined after protein
extraction are in the range of 0.1-20% of the wild
type. The NR expressed in the heterozygous plants
could therefore bemore labile thanthe wild-type
enzyme. This wild-type NR activity is likely in excess
of that required for the normal growth since heterozygous plants in which the measured NR activity is
very low, grow as rapidly as the wild type (Table 3).
Reconstitution of the NR activity: T o test the
hypothesis of adirectinteraction
between inactive
subunits, functional NR activity reconstitution experiments were performedby mixing extracts of inactive
NR from leaves of several grafted homozygous nia
mutants. No NADH-NR activity was detected in the
extracts of homozygous mutants. NR activity was observed in the mixture of protein extracts of two complementing homozygous mutants. These results suggest that the complementation implies modified NR
protein interactions. When a protein extract from a
class 3 mutant such as nia E77, affected in the terminal
electron transfer steps, was mxed with a protein extract from a class 2 mutant affected for the
initial
steps like nia 0 5 1 , we reproducibly detected a significant NADH-NR activity. All the attempts performed
complementation
Intragenic
to detect NR activity in mixed extracts of nia mutants
have led to positive results only when the corresponding mutants were able to complement in vivo. When
mixing niaE56 with niaD51 proteinextractthe
reconstituted NADH-NR activity is much higher than
for any other reconstitutions. Such a result was expected according to the overexpression of the Nia
D5 1 NR protein and thenia E56 properties ( C H ~ R E L
et al. 1990).
DISCUSSION
In Nicotiana species only one NZA gene has so far
been found per haploid genome. T h e N . plumbaginifolia mutants of the NZA complementation group are
impaired in the NR apoenzyme in contrast to cnx
mutants affected in the molybdenum cofactor synthesis resulting in the lack of boththe NR andthe
xanthine dehydrogenase activities. T h e NIA complementation group is heterogenous in regards to the
presence of specific epitopes and to the partial NR
catalytic activities as expected for mutants affectedon
the NR structural gene.
Intragenic complementation among N . plumbaginifolia nia mutants can be observed when the parents
are affected on different functional domains of the
enzyme. Complementation is never observed between
mutants belonging to thesame biochemical class. The
systemic study of the NZA group showed that, when
complementation occurs, genetic results enable
to predict the biochemical classification and considering the
biochemical characteristics, the genetic complementation can be predicted even if it is sometimes very
poor. In Chlorella sorokiniana, the in vitro restoration
of a NR activity among nia mutants has been published recently (KNOBLOCHand TISCHNER
1989). In
vitro (FERNANDEZ and
CARDENAS
1981) and in vivo
(FERNANDEZ and
MATACNE1986) complementation
of assimilatory NAD(P)H-NR from mutantsof Chlamydomonas reinhardii has been reported. T h e authors
concluded thattheresultant
NR activity reflected
intergenic complementation due to the presence of
two genes coding fortwo types of subunit and forming
an heteromultimeric NR complex. Intheir model
eachsubunitcarries
separately the NAD(P)H-cytochrome c reductase activity and the reduced benzyl
viologen-nitrate reductase activities. Recently it has
been shown that in Chlamydomonas like other nitrate
reductase systems, theapoprotein is encoded by a
single gene (FERNANDEZ
et al. 1989). Therefore the
observedcomplementation has to be attributed to
intragenic rather than intergenic complementation as
reported here fora higher plant.
In N . plumbagin$olia heterozygous NZA plants, the
NADH-NR activity either tested in vivoor in vitro was
much lower than that of the wild type. We assume
that the amount of functional hybrid enzyme in heterozygous plants is low or that theenzyme is particu-
at NZA
203
larly sensitive to degradation and difficult to extract
in its active form. It should be noted however that
this low expression of NR activity is nevertheless high
enough to support growth of specific heterozygotes
on nitrate as sole nitrogen source.
In the amphidiploid species N . tabacum, a partial
complementation in hybrids between different n i a l ,
nia2 double mutants has been shown (A. J. MULLER
and R. R. MENDEL,personal communication). Genetic
analysis indicated that this complementation may result from interaction betweenthe productof the NZAl
allele and that of the NZA2 allele. In these heterozygous plants invivo and invitro NR activities are
respectively around 50% and 3% that of the wild type.
As in N . plumbagin$olia, these activity levels are lower
thanthat of the wild type mainlyin the invitro
experiments.
Even though if the hypothesis of a “cross-feeding’’
mechanism or of exchange of domains between complementaryaffectedsubunitscannotbe
completely
excluded, aggregation of mutated subunits likely allows the restoration of the electron flow from NADH
to nitrate. The reconstitution experiments are also in
favor with this hypothesis especially since the restoration of NADH-NR activity is very rapid aftermixing
of the extract. However, we do not know precisely
the size of the functionalhybridprotein
andthe
stability of the complex. T h e mechanism of restoration of NR activity may be different in vivo in the
leaves of complementing plants and in vitro during
the reconstitution experiments. In vivo activity could
result from interaction betweentwo de novo translated
polypeptidic chains althoughthe reconstituted NR
activity could be attributed to intermolecular interactions between inactive NR enzymes. Moreover, as
it is possible to reconstitute in vitro the NADH-NR
activity, it can be ruled out that oneof the NZA allele
in the heterozygote could be reactivated at the transcriptional or translational level by the product of an
other gene acting in trans.
It was shown that the native NR in Chlorella is an
isologous tetramer which dissociates to anactive dimer
at low proteinconcentrations(HOWARD and SOLOMONSON 1982). On an electrophoresis gel in native
conditions a general two band pattern has been observed with squash purified NADH:NR whichmay
reflect a tendency of purified NR to polymerize (REDINBAUGH and CAMPBELL 1983, 1985). A similar observation has beenmadefor
maize NR (CH~REL,
GROSCLAUDE
and ROUZE1985). Evidence for a very
stable interchaindisulfide bond in higher plant nitrate
reductases have been shown recently (HYDEet al.
1989). This interchain disulfide bond seems to be
located in the molybdopterinbindingdomain
and
probably plays a role in nitrate reductase stability but
is not essential forthe enzymatic activity. As the
terminal activities are dependentof the presence of a
204
F. Pelsy and M. Gonneau
functional MoCo which could be involved in the dimerization of the enzyme (MENDEL and MULLER
1979), the mutants with a terminal nitrate reductase
activity are possibly dimeric. On the other hand, the
molecular organization of mutant protein with diaphorase activity is still uncertain. In a native NR, each
complete subunit appears able to catalyze the nitrate
reduction independently as shown by SOLOMONSON
and MCCREERY(1986). Our results of NR activity
reconstitutionprovide evidence that functionaldomains may interact through intermolecular contacts
inwhich theelectronscan
flow fromonesubunit
damaged in one domain to an other onein which the
corresponding domain is functional. The property of
nitrate reductase to receive electrons from artificial
donors or to transfer them to artificial acceptors assumes a relatively external localization of the prosthetic groups and indicates that the electrons can still
flow in or out of the subunits.
We wish to thank MICHEL
CABOCHEfor useful advice and critical
readingofthemanuscript.
T h e technicalassistanceofALINE
DOUARD and
JACQUES GOUJAUD
is gratefully acknowledged. This
work was partially funded by a grant from theMRES (1279A).
LITERATURE CITED
BASSON,M. E., R. L. MOORE,J. O’REARandJ.RINE,1987
Identifying mutations in duplicated functions in Saccharomyces
cerevisiae: recessive mutations in HMG-CoA reductase genes.
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