Coordinator’s
report: Biochemical mutants:
Barley
nitrate and nitrite reductase mutants
A.
Kleinhofs
Dept.
Crop and Soil Sciences and School of Molecular Biosciences
Washington
State University
Pullman,
WA 99164-6420
USA
Hard-copy edition pages 174 - 177.
Extensive genetic analysis of NR-deficient mutants
has been carried out in barley. The first barley NR mutants were reported in
1977 (Tokarev and Shumny, 1997; Warner et
al., 1997). A total of 23 NR-deficient mutants have been identified as
alleles of the nar1 locus coding for
the NADH-specific nitrate reductase EC1.6.6.1 (Kleinhofs et al., 1989). Greatly reduced, but not null NADH- and NAD(P)H-NR
activities were observed in young seedling shoots of all nar1 mutants. Both activities are due to the bispecific NAD(P)H-NR
and in some cases also due to a leaky mutation in the NADH NR gene. The
NAD(P)H-bispecific NR EC1.6.6.2 is found only in roots of wild-type barley
(Dailey et al., 1982). However, in the nar1
mutants it is present in both shoots and roots and accumulates to a high level
under field conditions. A single NAD(P)H NR-deficient mutant has been isolated
and designated nar7w (Warner et al., 1987). This mutant appears to be
similar to wild-type in phenotype but lacks the NAD(P)H-NR activity in roots.
Both nar1 and nar7 mutants are viable under Pullman, Washington field conditions
where nitrate is the principal nitrogen source. A constructed homozygous double
mutant is lethal under our field conditions, but does assimilate 15% of the
wild-type nitrate levels and can be grown to maturity in controlled conditions
with urea as a nitrogen source (Warner and Huffaker, 1989). Both NADH and
NAD(P)H NR structural genes have been cloned and characterized, genomic and
cDNA NADH NR accession numbers X57845 and X57844, respectively (Schnorr et al., 1991), genomic NAD(P)H NR
accession number X60173 (Miyazaki et al.,
1991).
Additional NR-deficient mutants are due to
mutations in the genes controlling the molybdenum cofactor essential for NR
activity (Bright et al., 1983;
Kleinhofs et al., 1989; Steven et al., 1989; Tokarev and Shumny, 1977).
These mutations are identified by the pleiotropic loss of NR and xanthine
dehydrogenase activities. Six different genes designated nar2, nar3, nar4, nar5, nar6, and nar8 have been identified in barley
(Kleinhofs et al., 1989 and
unpublished). Most of these mutants are conditional-lethal i.e. they are lethal
under field conditions, but most can be grown to maturity under controlled
conditions with a reduced nitrogen source. Most of the viable alleles are
probably due to a leaky mutation. The exception might be the three nar5 alleles, all of which grow rather
well under field conditions. Enzymatic activities characteristic of the
different alleles are described in Kleinhofs et al., 1989. Additional mutant descriptions are in Table 1.
A single nitrite reductase-deficient mutant nir1 (STS3999) has been described
(Duncanson
et al., 1993). The mutation is a
conditional-lethal and probably determines the formation of the nitrite
reductase apoenzyme. Three other alleles of nir1
(STA2760, STA1010 from cv. Klaxon and STA4169 from cv. Golden Promise) were
reported, but have not been described in detail (Wray 1993).
References:
1. Bright, S. W. J., P. B. Norbury, J. Franklin, D.
W. Kirk, and J. L. Wray. 1983. A conditional lethal cnx-type nitrate
reductase-deficient barley mutant. Mol.
Gen. Genet. 189:240-244.
2. Dailey, F. A., R. L. Waener, D. A. Somers, and
A. Kleinhofs. 1982. Characteristics of a nitrate reductase in a barley
mutant deficient in NADH nitrate reductase. Plant Physiology 69:1200-1204.3.
3. Duncanson, E., A. F. Gilkes, D. W. Kirk, A.
Sherman, and J. L. Wray. 1993. nir1, a
conditional-lethal mutation in barley causing a defect in nitrite
reduction. Mol. Gen. Genet.
236:275-282.
4. Kleinhofs, A., R. L. Warner, J. M. Lawrence, J.
M. Melzer, J. M. Jeter, and D. A. Kudrna. 1989. Molecular
genetics of nitrate reductase in barley. In:
Molecular and Genetic Aspects of Nitrate Assimilation. J. L. Wray and J. R.
Kinghorn (eds.) Oxford Univ. Press, New York. Chapter 13:197-211.
5.
Kleinhofs, A., R. L. Warner, and D. A. Kudrna. 2001.
(Unpublished).
6. Miyazaki, J., M. Juricek, K. Angelis, K. M.
Schnorr, A. Kleinhofs and R. L. Warner. 1991. Characterization and sequence of
a novel nitrate reductase from barley.
Mol. Gen. Genet. 228:329-334.
7. Schnorr, K. M., M. Juricek, C. Huang, D. Culley,
and A. Kleinhofs. 1991. Analysis of barley nitrate reductase cDNA and
genomic clones. Mol. Gen. Genet. 227:411-416.
8. Steven, B. J., D. W. Kirk, S. W. J. Bright, and
J. L. Wray. 1989. Biochemical genetics of further chlorate
resistant, molybdenum cofactor defective, conditional-lethal mutants of barley.
Mol. Gen. Genet. 219:421-428.
9. Tokarev, B. I. and V. K. Shumny. 1977. Detection of
barley mutants with low level of nitrate reductase activity after the seed
treatment with ethylmethanesulphonate Genetika Moskva 13:2097-2103.
10. Warner R. L. and R. C. Huffaker. 1989. Nitrate
transport is independent of NADH and NAD(P)H nitrate reductases in barley
seedlings. Plant Physiology 91:947-953.
11. Warner, R. L., C. J. Lin, and A. Kleinhofs.
1977. Nitrate reductase-deficient mutants in barley. Nature 269:406-407.
12. Warner, R. L., K. R. Narayanan, and A.
Kleinhofs. 1987. Inheritance and expression of NAD(P)H nitrate
reductase in barley. Theor. Appl. Genet. 74:714-717.
13. Wray, J. L. 1993. Molecular
biology, genetics and regulation of nitrite reduction in higher plants.
Physiologia Plantarum 89:607-612.
Table 1. Barley nitrate and nitrite
reductase-deficient mutants.
|
Gene |
Sel. No. |
Location |
Viable? |
Parent cv. |
Mutagen |
References |
|
|
|
|
|
|
|
|
|
nar1a |
Az12 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4, 11 |
|
nar1b |
Az13 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4, 11 |
|
nar1c |
Az23 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1d |
Az28 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1e |
Az29 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1f |
Az30 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1g |
Az31 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1h |
Az32 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1i |
Az33 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1j |
Xno29 |
6(6H)-01 |
yes |
Winter |
EMS |
9 |
|
nar1k |
EMS29 |
6(6H)-01 |
yes |
Steptoe |
EMS |
4 |
|
nar1l |
EMS31 |
6(6H)-01 |
yes |
Steptoe |
EMS |
4 |
|
nar1m |
Az56 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1n |
Az57 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1p |
Az63 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1q |
Az64 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1r |
Az65 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1t |
Az67 |
6(6H)-01 |
yes |
Steptoe |
Azide |
5 |
|
nar1ab |
Az76 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1ac |
Az77 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1ai |
Az79 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1aj |
Az80 |
6(6H)-01 |
yes |
Steptoe |
Azide |
4 |
|
nar1ao |
BSMV1 |
6(6H)-01 |
yes |
Vantage |
BSMV |
5 |
|
|
|
|
|
|
|
|
|
nar2a |
AZ34 |
7(5H)-07 |
yes |
Steptoe |
Azide |
4 |
|
nar2ad |
R9401 |
7(5H)-07 |
no |
Maris Mink |
Azide |
1 |
|
nar2ag |
R9201 |
7(5H)-07 |
no |
Maris Mink |
Azide |
8 |
|
|
|
|
|
|
|
|
|
nar3a |
Xno18 |
1(7H)-05 |
no |
Winer |
EMS |
9 |
|
nar3b |
Xno19 |
1(7H)-05 |
no |
Winer |
EMS |
9 |
|
nar3x |
Az71 |
1(7H)-05 |
no |
Steptoe |
Azide |
4 |
|
|
|
|
|
|
|
|
|
nar4y |
Az72 |
2(2H-13 |
no |
Steptoe |
Azide |
4 |
|
|
|
|
|
|
|
|
Table
1. continuation.
|
Gene |
Sel. No. |
Location |
Viable? |
Parent cv. |
Mutagen |
References |
|
|
|
|
|
|
|
|
|
nar5o |
Az62 |
7(5H)-07 |
yes |
Steptoe |
Azide |
4 |
|
nar5s |
Az66 |
7(5H)-07 |
yes |
Steptoe |
Azide |
4 |
|
nar5u |
Az68 |
7(5H)-07 |
yes |
Steptoe |
Azide |
4 |
|
|
|
|
|
|
|
|
|
nar6v |
Az69 |
2(2H)-13 |
no |
Steptoe |
Azide |
4 |
|
|
|
|
|
|
|
|
|
nar7w |
Az70 |
6(6H)-09 |
yes |
Steptoe |
Azide |
4 |
|
|
|
|
|
|
|
|
|
nar8z |
Az73 |
6(6H)-01 |
? |
Steptoe |
Azide |
5 |
|
|
|
|
|
|
|
|
|
nar?ae |
R1103 |
2(2H)-13 |
no |
Maris Mink |
Azide |
8 |
|
nar?ak |
Az81 |
|
? |
Steptoe |
Azide |
5 |
|
nar?al |
Az82 |
|
? |
Steptoe |
Azide |
5 |
|
nar?am |
Az84 |
|
? |
Steptoe |
Azide |
5 |
|
nar?an |
Az85 |
|
? |
Steptoe |
Azide |
5 |
|
nar?ap |
Az86 |
|
? |
Steptoe |
Azide |
5 |
|
nar?aq |
Az87 |
|
? |
Steptoe |
Azide |
5 |
|
nar?ar |
Az88 |
|
? |
Steptoe |
Azide |
5 |
|
nar?as |
Az89 |
|
? |
Steptoe |
Azide |
5 |
|
nar?at |
Az94 |
2(2H)-07 |
? |
Steptoe |
Azide |
5 |
|
|
|
|
|
Steptoe |
|
|
|
nir1 |
STA3999 |
6(6H)-09 |
no |
Tweed |
Azide |
3 |
