Isolation and Initial Characterization of Constitutive Nitrate Reductase-Deficient Mutants NR328 and NR345 of Soybean (Glycine max).

Abstract

Two nitrate reductase deficient mutants of soybean (Glycine max [L.] Merr. cv Bragg) were isolated from approximately 10,000 M(2) seedlings, using a direct enzymic assay in microtiter plates. Stable inheritance of NR345 and NR328 phenotypes has been demonstrated through to the M(5) generation. Both mutants were affected in constitutive nitrate reductase activity. Assayable activities of cNR in nitrate-free grown seedlings was about 3 to 4% of the control for NR345 and 14 to 16% of the control for NR328. Both mutants expressed inducible NR during early plant development and were sensitive to nitrate and urea inhibition of nodulation. These new mutants will allow an extension of the characterization of nitrate reductases and their function in soybean. Preliminary evidence indicates that NR345 is similar to the previously isolated mutant nr(1), while NR328 is different.

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PlantPhysiol. (1986) 81, 572-576
0032-0889/86/81/0572/05/$0 1.00/0
Isolation and Initial Characterization of Constitutive Nitrate
Reductase-Deficient Mutants NR328 and NR345 of Soybean
(Glycine max)1
Received for publication July 16, 1985 and in revised form February 28, 1986
BERNARD J. CARROLL* AND PETER M. GRESSHOFF
Botany Department, Australian National University, CanberraACT 2600, Australia
ABSTRACI
Two nitrate reductase deficient mutants of soybean (Glycine max IL.]
Merr. cv Bragg) were isolated from approximately 10,000 M2 seedlings,
using a direct enzymic assay in microtiter plates. Stable inheritance of
NR345 and NR328 phenotypes has been demonstrated through to the
M5 generation. Both mutants were affected in constitutive nitrate reduc-
tase activity. Assayable activities ofcNR in nitrate-free grown seedlings
was about 3 to 4% ofthe control for NR345 and 14 to 16% of the control
for NR328. Both mutants expressed inducible NR during early plant
development and were sensitive to nitrate and urea inhibition of nodula-
tion. These new mutants will allow an extension of the characterization
of nitrate reductases and their function in soybean. Preliminary evidence
indicates that NR345 is similar to the previously isolated mutantnri,
while NR328 is different.
cation of three biochemically distinct nitrate reductases in soy-
bean, and to the correlation of these activities with cNR and
iNR (27). Urea-grown wild-type plants contained two cNRs
designated cjNR and c2NR. the c,NR used both NADPH and
NADH, whereas c2NR used only NADH as an electron donor.
Only one iNR was identified in nitrate-grown nr1 plants (that
lacked cNR). Like c2NR, iNR used only NADH as an electron
donor (27).
Efficient procedures for assaying NR (16) and the availability
of chlorate as a positive selection agent (8, 13) have aided the
isolation of nitrate metabolism mutants in several higher plant
species (for review see Refs. 12 and 17). In legumes, NR-deficient
mutants have been isolated in pea (9, 16) as well as in soybean
(21). Such legume mutants are potentially useful for studying
nitrate inhibition of nodulation and nitrogen fixation (4-7). In
this paper, we describe the isolation and preliminary characteri-
zation oftwo independent NR-deficient mutants of soybean.
Generally, NR2 activity is only expressed when nitrate is
present in the growth medium (2), but soybean is an exception
to this generality and Harper (10) showed that leaf tissue from
inoculated plants grown without combined nitrogen expressed
NR activity. Similarly, it was demonstrated that leaf tissue from
urea-grown soybean plants had NR activity (18). Since this
activity was present in the absence of nitrate, it was termed
constitutive or noninducible NR (12). cNR activity is not ex-
pressed in soybean roots (15, 21) or soybean cell cultures (22),
and NR activity in these tissues is dependent on nitrate being
present. Thus, in addition to cNR, there is also iNR activity in
soybean. Multiple soybean NR activities have also been distin-
guished biochemically. Using column chromatography, Jolly et
al. (14) and Campbell (3) separated twoNR activities fromyoung
unifoliolate leaves of nitrate-grown soybean seedlings. The two
activities differed in affinities for nitrate and for the reduced
pyridine nucleotides NADH and NADPH. These NR activities
were also identified in cotyledons ofnitrate-grown seedlings (24).
Genetic evidence for the presence of multiple NR activities in
soybean has been reported by Nelson et al. (21). They isolated
three allelic chlorate-resistant mutants (LNR-2, LNR-3, and
LNR-4) (26) that lacked cNR, but expressed iNR in root and
leaf tissue. Subsequent studies on the parent cultivar Williams
and the cNR mutant nr1 (formerly LNR-2) led to the identifi-
' Financial support was provided by the Australian Government for a
postgraduate research award to B. J. C. and by Agrigenetics Research
Associates.
2 Abbreviations: NR, nitrate reductase; cNR, constitutive nitrate re-
ductase; iNR, inducible nitrate reductase; EMS, ethylmethanesulfonate.
MATERIALS AND METHODS
Plant Material. M2 families of cultivar Bragg were screened
for cNR mutants. The families were derived from EMS muta-
genesis, which we described in previous publications (5, 7). For
comparison, Williams and nr, (formerly LNR-2) (21) were in-
cluded in some experiments. Seeds of nr1 were obtained from
Dr. S. Ryan (CSIRO, Canberra).
Plant Culture. All experiments were conducted in the glass-
house. Glasshouse temperatures were kept between 14 and 30°C
and incandescent bulbs extended the photoperiod to 16 h. M2
seeds were planted 1 cm below the surface in sand trays (63 cm
long, 23 cm wide, and 6.5 cm deep). The trays were inoculated
with Rhizobium japonicum strain CB1809 (= USDA 136), satu-
rated with N-free nutrient solution (6, 7) twice a week and
received tap water every other day. Ten to 16 d after planting,
unifoliolate leaves were screened for cNR activity (see below).
M2 variants were saved to produce M3 progeny which were
screened forcNR activity as described below. The stability ofthe
mutantcharacter was also verified in M4 and M5 seedlings, except
that these plants were cultured in 15 cm diameter pots of
vermiculite. M4 plants were also characterized for in vivo NR
activity after culture on nitrate, and for nodulation on various
nitrogen sources. In these experiments, plants were inoculated
with R.japonicum strainCB1809 and cultured in 15 cm diameter
pots ofriver sand (2 plants per pot). The pots were watered daily
with 600 ml of nutrient solution. KNO3 (2.5 mM) and urea (2.5
mm, i.e. 5 mM N) were added to the nutrient solution as required.
In Vivo cNR Screen. One unifoliolate leaf disc (4.5 mm
diameter) from each M2 seedling was screened for cNR activity
in a micro-well of a micro-test plate (96F with lid, Nunc (Inter
Med], Denmark). Each micro-test plate contained 96 wells. Prior
572

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CONSTITUTIVE NITRATE REDUCTASE MUTANTS OF SOYBEAN
to sampling the leafdiscs, 130glofNR assay solution (containing
50 mM KNO3) (23) was dispensed into each well using an eight-
tip micro-pipette (50-200 ul, serial no. 10379, Titertek). Leaf
discs were removed from seedlings and placed in the assay
solution and the plates were then incubated at 28°C for 3 to 4 h.
Subsequently, the activity was detected by adding 130,lofa 1:1
solution of 1% sulfanilamide in 3 N HCI and 0.02% N-naphthyl-
ethylene diamineHC 1 in distilled H20 to each well. This solution
turns pink in the presence of nitrite (28), and therefore decreased
pink coloration in the micro-well assay indicated decreased cNR
activity. The degree ofpink coloration is seen as shading around
the leaf discs in Figure 1. This screening method was very rapid
and inexpensive, and 600 seedlings could be easily screened by
one person in a day.
In vivo NR Assay. In vivo NR activity was determined by a
modification of the assay described by Nicholas et al. (23). Six
leaf discs (4.5 mm diameter) were sampled from unifoliolate
leaves and placed in chilled test tubes containing 5 ml of NR
assay solution. The assay solution was the same as that used by
Nicholas et al. (23) and contained either 0 or 50 mM KNO3. The
tubes were incubated in the dark in a 30°C water bath and
samples were removed over a 50 min incubation period for
nitrite determination (28). The data were expressed as nmol
NO2- produced leafdisc-' h-' or ,umol NO2-.g fresh weight
h-'. Each leafdisc weighed approximately 2.7 mg (fresh weight).
Statistics. Data were analyzed by analysis of variance, using
the general statistical program Genstat (1).
'.
RESULTS
Selection and Stability of cNR Mutants NR328 and NR345.
Approximately 10,000 seedlings were screened for cNR activity
(Table I). These plants were derived from 1428 M2 families; 868
families came from EMS-population
families were from EMS-population 2. Ten seeds per M2 family
of population
planted for each M2 family in population 2, and approximately
7,550 and 2,450 M2 plants were screened in population
population 2, respectively. Thus, about three times as many M2
plants were screened in population I than in population 2 (Table
I).
Several M2 families apparently segregated for decreased cNR
activity, but only two M2 variants produced M3 progeny that had
little or no cNR activity. The two confirmed cNR-deficient
mutants were derived from M2 families 328 and 345, which
belonged to population 2 (Table I). In each ofthese families, one
out ofthe five plants screened was cNR-deficient. These variant
plants were designated NR328 and NR345. All three M3 plants
derived from NR328 had decreased cNR activity. Similarly,
NR345 was pure-breeding and all 12 M3 progeny had decreased
cNR activity (Fig. 1). Bragg had considerable cNR activity as
indicated by the deep shading in micro-wells containing a Bragg
leafdisc. In contrast, micro-wells with an nr1, NR328, or NR345
leafdisc had almost no color (Fig. 1), indicating that these lines
had decreased cNR activity. In vivo NR assays were also con-
1 and the remaining 560
1 were planted, whereas only five seeds were
1 and
ducted on unifoliolate leaftissue from these plantsand the results
are listed in Table II. NR328 had cNR activity significantly less
than Bragg, but significantly more than either NR345 or nr,.
Activity in NR328 was 16.2% that ofBragg, whereas NR345 and
nr, had negligible activity compared to Bragg.
In vivo cNR activity was also determined in unifoliolate leaves
ofM4 progeny, and the results obtained confirmed the M3 data
(data not shown). Mutants NR345 and nr, had very low activity
compared to the respective parent cultivars Bragg and Williams,
whereasNR328 had 14.1% ofthe wild-type activity. The stability
of the mutants was also verified in the M5 generation (data not
shown).
In Vivo Activity in Nitrate-Grown Plants. Bragg, NR328, and
NR345 plants were grown on nitrate and assayed forNR activity
21 d after planting. Both of the cNR mutants expressed iNR
activity (Table III). When NR activity was dependent on endog-
enous nitrate in the leaf tissue (minus NO3- assay), activity was
the same in Bragg, NR328, and NR345. However, with 50 mm
KNO3 added to the assay solution, Bragg NR activity was signif-
icantly higher than the activity in eitherNR328 orNR345 (Table
III).
Plant Fresh Weight Accumulation in N2-dependent and Ni-
trate-grown Plants. Table IV shows plant fresh weights ofBragg,
NR328, and NR345 plants after 7 weeks culture on N-free or
KNO3-supplemented nutrients. Two-way analysis of variance
showed that the effect ofnitrate was highly significant (calculated
F = 105.8 > F1,3o [0.05] = 4.17), but that the genotypes were not
significantly different and that they did not respond differently
to 2.5 mm nitrate being supplied.
Inhibition ofNodulation by Combined Nitrogen. A preliminary
experiment was conducted to ascertain the inhibitory effect of
2.5 mm nitrate or urea on nodulation ofthe parent cultivars and
the cNR mutants. As previously described for this system (6),
urea was more inhibitory than was nitrate. The absolute amount
ofnodulation per plant was not decreased by treatment with 2.5
mM nitrate (data not shown), but these plants were considerably
larger than N2-dependent plants (Table IV) and the nitrate
treatment did cause significant inhibition when nodulation was
expressed per g plant fresh weight (Table V). Two-way analysis
of variance indicated that the effect of combined nitrogen on
nodule number-g-' plant fresh weight was highly significant
(calculated F = 135.4 > F2,65 [0.05] = 3.15). Genotypic differ-
ences in this parameter were marginally significant (calculated F
= 2.87 > F4,65 [0.05] = 2.53). However, there was no significant
interaction between genotype and nitrogen source (calculated F
= 0.95 > F8,65 [0.05] = 2.10), indicating that nodule number in
the different genotypes was similarly affected by 2.5 mM nitrate
or urea. Essentially the same trend was observed for mg nodule
fresh weight-g-' plant fresh weight (Table V). The effect of
nitrogen source was highly significant (calculated F = 475.9 >
F2,67 [0.05] = 3.15). There was also a significant genotype effect
(calculated F = 10.99 > F4,67 [0.05] = 2.53) and nodule fresh
weight per plant biomass was consistently lower in NR328 than
in Bragg (Table V). However, the genotypes did not differ in
Table I. Frequencies ofChi-deficient Variants, nts, and cNR Mutants in Two Mutagenized Populations of
Soybean
Frequencyof
Frequencyof
No. ofM2 Families
Population
Screened for
Variants
Mutants in
in theM,?
EMS-1
0.9 x 10-2
3.6 x 10-4
EMS-2
aCited from Carroll et al. (5).
bBJ Carroll, DL McNeil, PM Gresshoff (unpublished data).
number of M2 families screened for cNR activity.
activity.
Approx. No. ofM2
Individuals
Screened forcNRd
No. ofcNR
Mutants
Recoverede
Chl-Deficient
nts
cNRc
the M2b
868
560
7,550
2,450
0
2
2.8 x 10-2
1.4 x 10'
CTotal
d Approximate number of M2 plants screened for cNR
' Number of mutants recovered from the cNR screen.
573

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CARROLL AND GRESSHOFF
.;*S. *
.*.0
0* 0
***
*e*
#0*
*
0**
FIG.
1. Rapid screening cNR assay on Bragg, nr1, and M3 progeny of
NR328 and NR345. For assay procedures see "Materials and Methods."
Each micro-well contained a unifoliolate leafdisc from a 14-d-old plant
that had been cultured on N-free nutrients. The degree ofshading around
the leaf disc reflects the amount of nitrite in each micro-well. Row 1,
Bragg; row 2, nr, (formerly LNR-2); row 3, NR328; row 4, NR345; and
rows 5 to 8, Bragg.
Table II. In vivo cNR Activity ofBragg, nr,, and M3 Progeny ofNR328
and NR345
Unifoliolate leaves were assayed (+ NO3 assay) 16 d after planting and
culture in sand trays. The trays were watered with N-free nutrient solution
as described in "Materials and Methods." Each entry in the table is the
mean ofthree or four assays on leaftissue from separate plants.
Genotype
NR Activity
nmol N02- * disc-' * h-'
Bragg
15.7
NR328
2.6
NR345
0.6
nr,
0.5
aRaw data required log, transformation to satisfy assumptions for
analysis of variance. The LSDo.05 (based on transformed data) was 0.48.
log (NR Activity)a
2.74
0.90
-0.88
-0.60
Table III. In vivo NR Activity ofNitrate-grown Bragg andM4 Progeny
ofNR328 and NR345
Unifoliolate leaves were assayed21 d afterplantingand culture inpots
ofriver sand. The pots were inoculated with R.japonicum strain CB1809
and watered daily with 2.5 mm KNO3-supplemented nutrient solution.
Leaf tissue was assayed with and without 50 mm KNO3 in the assay
solution. Each entry in the table is the mean of six to eight plants.
NR Activity
Genotype
Minus NO3- assaya
Plus NO3 assayb
nmol NO2 disc'*h-'
Bragg
NR328
NR345
9.1
8.9
8.9
18.2
14.1
11.8
aNo significant difference between genotypes.
nificant and LSDo.05= 3.7.
sensitivity to combined nitrogen (calculated Finlem,ion= 1.74 <
F8,67 [0.05]=2.15). Urea-grown plants in these experiments had
considerable NR activity without nitratebeingadded to theassay
solution (data not shown). Evidently, in this sand culture, con-
siderable nitrification of urea had occurred. Nevertheless, it is
evident that cNR-deficiency did not confer nitrate-tolerant
nodulation in nr,, NR328, or NR345.
bF-statistic wassig-
Table IV. Effect ofNitrate on Plant Fresh weight Accumulation in
Bragg andM4 Progeny ofNR328 andNR345
Plants were inoculated with R. japonicum strain CB1809, cultured in
sand pots as described in "Materials and Methods" and harvested 7 weeks
after planting. Each entry in the table is the mean ofsix plants. Two-way
analysis of variance on the data indicated that the effect of nitrate
supplementation was significant, but that there was no difference between
the genotypes.
Plant Fresh Weight
Genotype
N-free
2.5 mM KNO3
g
Bragg
NR328
NR345
16.2
8.2
11.6
45.7
43.4
34.7
DISCUSSION
The results presented here show that it is possible to screen
successfully for soybean mutants in a specific enzyme activity.
Two cNR mutants, NR328 and NR345, were isolated using an
in vivoNR screen. An in vivo assay procedure hasbeen previously
used to isolate NR mutants in barley (29) and pea (16). In
contrast to the approach used to identify NR328 and NR345,
cNR soybean mutant nr, (formerly LNR-2) was isolated using
chlorate as a positive selection agent (21). The screening proce-
dure described here could also be useful in screening remutagen-
ized cNR-deficient lines for nitrate uptake and iNR mutants.
The direct assay forNR activity may have advantages overtesting
for chlorate resistance, in view of possible secondary effects of
chlorate (8).
NR328 and NR345 were recovered from separate M2 families.
Both these families segregated for the mutant character, indicat-
ing that the mutation arose from the mutagenesis treatment and
also that NR328 and NR345 were the result ofseparate mutation
events. The frequency ofnts (nitrate-tolerant symbiotic mutants),
cNR, and Chl-deficient mutants was consistently higher in pop-
ulation 2 than in population 1 (Table I). M2 selections NR328
and NR345 were pure breeding and homozygous for the respec-
tive mutations and the stability of the mutant characters have
been demonstrated through to the M5 generation. NR345 and
nr, had negligible cNR activity, whereas NR328 had approxi-
mately 15% ofthe wild-type activity (Table II). Constitutive NR-
deficient mutant nr, lacks both c,NR and c2NR activity that are
present in parent cultivar Williams (27). Studies on wild-type
Bragg grown in the absence of nitrate have shown that this
cultivar expresses cjNR and c2NR activity (19). It therefore
appears that NR345 described here also lacks c,NR and c2NR
activity. However, it is plausible that NR328 lacks only one of
the cNR activities or, alternatively, this mutant may be leaky
and have decreased amounts of both cNR activities. Allelism
tests are being conducted on nrr, NR328, and NR345. Crosses
have also been made between Bragg and the mutants to deter-
mine the nature of inheritance of the NR328 and NR345 char-
acters.
Both NR328 and NR345 expressed iNR activity (Table III).
When NR activity was dependent on endogenous nitrate (minus
NO3- assay), unifoliolate leaves of nitrate-grown Bragg, NR328,
and NR345 plants had the same NR activity. The addition of
nitrate to the assay medium resulted in Bragg having significantly
higherNR activity thanNR328 andNR345. It is likely, therefore,
that Bragg had more NR enzyme (presumably cNR) present in
situ, and that substrate supply limited activity in the minus N03
assay. The presence of iNR in NR328 and NR345 was also
reflected in the fact that nitrate stimulated plant fresh weight
accumulation in nitrate-grown NR328 and NR345 plants. Thus,
NR328 and NR345 are similar to nr,, in that iNR was expressed
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PlantPhysiol.Vol.81, 1986

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CONSTITUTIVE NITRATE REDUCTASE MUTANTS OF SOYBEAN
Table V. Effect ofKNO3 and Urea on Nodulation per Gram Plant Fresh weight in Bragg, Williams, nr,, and
M4 Progeny ofNR328 and NR345
Plants were harvested 7 weeks after planting and culture in pots of river sand. The pots were inoculated
with R. japonicum strain CB1809 and watered daily with 600 ml of nutrient solution. KNO3 (2.5 mM) and
urea (2.5 mM) were added to the nutrient solution as required. Each entry in the table is the mean ofthree to
six plants. Nitrogen source and genotype effects were significant but there was no significant interaction
between genotype and N source.
Nodulation
Genotype
N-free
2.5 mm KNO3
2.5 mm urea
number.gplantfresh Wt-la
Bragg
6.5
2.9
0.9
NR328
5.1
2.5
0.7
NR345 10.1
2.7
0.7
Williams
2.2 0.9
nr,
5.3
1.7
0.9
a, bRaw data required log, and square root transformation, respectively, to satisfy assumptions for a two-
way analysis of variance.
Nodule Fresh Weight
N-free
2.5 mM KNO3
mg-g-' ofplantfresh Wtb
29.0
20.2
36.2
28.3
22.3
2.5 mm urea
69.4
53.8
87.2
53.1
66.4
6.7
0.9
4.5
4.5
5.4
5.1
and nitrate utilization was evident. However, as well as being
leaky, NR328 was also unique in that nitrate-grown plants
developed necrotic lesions on the distal margin of mature leaves
(D Whitmore Smith, BJ Caroll, unpublished data). This did not
occur in N2-dependent NR328 plants and thus the necrosis
observed in nitrate-grown plants was probably due to nitrate
accumulation. Necrosis of mature leaf margins was not evident
in nitrate-grown Bragg, NR345, or nrl plants and it is therefore
possible that iNR activity is impaired at some stages of devel-
opment in NR328. These hypotheses are currently being tested.
Mutant nr, has been extensively characterized. The mutant
character is inherited as a Mendelian monogenic recessive (26),
but it is not clear whether the mutation is located in a regulatory
gene or in a structural gene required for both c,NR and c2NR
activities (25). Mutant nr, also lacks in vivo NO(x) evolution
normally present in the wild-type (26). The NO(.) compound
evolved during an in vivoNR assay (11) has since been identified
as being predominantly acetaldehyde oxime (20). All F2 cNR-
deficient segregants derived from Williams x nr, crosses also
lacked acetaldehyde oxime evolution, and all cNR-positive
segregants (i.e. wild-types) evolved acetaldehyde oxime (26).
Furthermore, soybean root tissue (21) and cell cultures (22) lack
both cNR activity and in vivo acetaldehyde oxime evolution.
Although these genetic and physiological studies indicated that
in vivo acetaldehyde oxime evolution is associated with cNR
activity, the nature of this association has not been further
characterized. NR328 and NR345 may contribute to the knowl-
edge on acetaldehyde oxime evolution and its relationship to
cNR activity.
As reported by Ryan et al. (26), the nr, mutation did not affect
nitrate utilization or confer nitrate-tolerance to nitrogen fixation.
The results presented here also showed that cNR-deficiency in
NR328, NR345, and nr1 did not condition nitrate-tolerant
nodulation in these lines. Both nodule number and nodule
growth (Table V) in the mutants were as sensitive as the wild
type to nitrate and urea inhibition. Although nitrogen fixation
does not appear to be nitrate-tolerant in nr, (26), this area needs
to be investigated in NR328 and NR345. The cNR constitutes
approximately 12.5 to 20% oftotal NR activity in soybean (12),
but the physiological significance of this form of NR remains
obscure. The fact that nrl was selected on the basis of chlorate
resistance implies that cNR reduces nitrate in planta (assuming
nitrate and chlorate behave the same in this respect). However,
cNR activity does not appear to be regulated by nitrate (21) or
ammonia (19) and lack ofcNR activity does not impede nitrate
utilization (26). Further characterization ofNR328 and NR345
may lead to a better understanding ofthe role ofcNR in soybean.
Acknowledgments-Drs. D. McNeil, S. Ryan, D. Herridge, and J. Harper are
thanked for material and/or discussion. Mrs. A. Higgins, Mrs. K. Cowley, Mrs. J.
Bateman, Mrs. T. Raath, and Mr. K Mortimer are thanked for technical help.
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