PNL Volume 13 1981
A CO-FACTOR DISTURBED NITRATE REDUCTASE DEFICIENT MUTANT
Feenstra, W. J. and E. Jacobsen University of Groningen, Haren, Netherlands
Nitrate reductase (NaR) of fungi consists of an apo-enzyme and a Mo-
bearing co-factor (see 3). Work on tobacco cell lines has shown that in a
higher plant the same structure can be present (4) . In order to obtain various
mutant forms of NaR we are currently selecting NaR deficient mutants of pea.
An NaR deficient pea mutant has already been isolated and partly described
(1,2). In that mutant, in vitro NaR activity is 5% of that of the parent
cv 'Rondo'. Sufficient plant growth and seed production is possible only on
an aerated liquid medium containing standard mineral solution (SMS, 1,2) with
NH4NO3 as N-source. Under these conditions NaR is sufficiently induced.
The apo-protein of NaR from Aspergillus nidulans shows cytochrome c
reductase (CR) activity sedimenting in sucrose gradient centrifugation with
a sedimentation value of 8 s whereas the co-factor is also used by the enzyme
xanthine dehydrogenase (XDH) (3). Therefore, the lesion in the enzyme NaR
can be determined by studying CR and XDH activities.
For the XDH activity determination a procedure adapted to pea has been
developed and will be published elsewhere. The XDH activity in mutant E1
was about 25% of the parent variety 'Rondo'. This result indicates that
mutant E1 is a co-factor mutant. For higher plants a fractionated CR activity
assay has been described (5) which appeared to be suitable for pea. In cv
Rondo grown on a medium with (NH4)2SO4 as N-source no NaR activity was found.
After fractionation a peak of CR activity with a sedimentation value of 4 s
was found. The same peak was found in mutant Ei grown under these conditions.
In an extract of cv Rondo grown on NH4NO3 as N-source NaR activity was found,
together with CR activity, the latter sedimenting with peaks at 4 s and 8 s,
the former with a peak of 8 s. Mutant E1 also showed two peaks of CR activity.
The 8 s CR activity was much higher than in cv Rondo. This shows that in
the mutant the apo-enzyme is still induced by nitrate. The increased activity
possibly is due to absence or delayed functioning of a feedback system.
These results corroborate the conclusion that mutant E1 is a co-factor
mutant. The designation cnx is based on the description of co-factor mutants
of A. nidulans (3).
The 8 s CR activity peak in the mutant was shifted to a slightly lower
s value. This lower value can directly be related to the co-factor deficiency
by the assumption that the NaR molecule is missing the co-factor. This would
mean that in mutant E1 the NaR molecule is defective in such a way that despite
absence of the co-factor the molecule is still able to reduce cytochrome c.
This assumption is in accordance with the scheme for the interactions of the
apo-enzyme and co-factor given by MacDonald et al. (3). The availability
of reliable tests both for CR activity and for XDH activity now enables us
to test NaR deficient mutants directly on apoprotein- and/or co-factor defi-
ciency. Using chlorate resistance (1) a number of NaR deficient pea mutants
have already been isolated. When sufficient seeds are available they will
1. Feenstra, W. J. and E. Jacobsen. 1980. Thero. Appl. Genet. 58:39-42.
2. Feenstra, W. J. and E. Jacobsen. 1980. PNL 12:11.
3. MacDonald, D. W. , et_ aj_. 1974. Molec. gen. Genet. 128:187-199.
4. Muller, R. R. and A. J. Muller. 1979. Molec. gen. Genet. 177:145-153.
5. Wallace, W. and C. B. Johnson. 1978. Plant Physiol. 61:748-752.