NITRATE REDUCTASE-DEFICIENT MUTANTS IN PISUM SATIVUM

PNL Volume 13

1981

RESEARCH REPORTS

Warner, R. L., A. Kleinhofs, Washington State University, Pullman, WA USA

and F. J. Muehlbauer

Three nitrate reductase (NR)-deficient pea mutants were induced by sodium

azide in the cultivar 'Juneau' and were selected for a segregating M2 popula-

tion by a qualitative in vivo NR assay. The NR activities of mutants A300,

A317, and A334 are approximately 20, 1, and 5 percent, respectively, of the

wild-type. After several generations of seed increase in the greenhouse,

crosses were made among the mutants and Juneau; and the mutants were characterized

for NR-associated enzymatic activities.

NR analysis of the F1 and F2 seedlings indicated that the three mutants

are representative of two loci. Mutants A317 and A334 are allelic and have

provisionally been assigned the gene designation nar-1. Mutant A300 is repre-

sentative of a second locus and is designated nar-2. Segregation in the F2

from crosses of A300/A317 and A300/A334 indicated that nar-1 and nar-2

are not closely linked. Both nar-1 and nar-2 are codominant as indicated

by intermediate NR activity in the F1 hybrids of A300/Juneau, A317/Juneau,

and A334/Juneau.

Research with NR from fungi and higher plants has demonstrated that

NR-deficiency can be caused by mutations in the NR structural gene, mutations

in the NR regulatory gene, mutations in the gene(s) controlling synthesis

of molybdo-cofactor components of NR, and mutations in the gene(s) controlling

nitrate uptake. Enzymatic characterization of the NR partial activities

and of enzymes associated with NR can be used to predict the nature of nar-1

and nar-2 genes. Nitrate reductases from most organisms can use reduced

FMN, FAD, and viologens in addition to NAD(P)H to reduce nitrate in vitro.

Most NR-deficient mutants lack all these activities; however, some NR structural

gene mutations permit the synthesis of an enzyme which lacks NAD(P)H NR activity

but has FMHN₂ NR activity. However, the FMNH₂ NR activities of all three

mutants were lower than the respective NADH NR activities. Consequently the

FMNH₂ NR characteristic was not helpful in determining the nature of either

nar-1 or nar-2.

NR also has a diaphorase activity and can reduce other substrates such

as cytochrome c. Plants of course have cytochrome c reductases other than

NR; however, NR is the only enzyme with cytochrome c reductase activity that

is known to be induced by nitrate. All three mutants have at least some

nitrate inducible cytochrome c reductase activity which suggests that the

NR gene product is being synthesized in response to nitrate but lacks the

ability to reduce nitrate. This further suggests that neither nar-1 or

nar-2 are regulatory genes since NR regulatory gene mutations result in the

loss of the NR gene product which also eliminates the nitrate induced cyto-

chrome c reductase, component of the enzyme. Furthermore, all three mutants

have very high nitrate reductase activities (190-230% of the wild-type).

In fungi, NR and nitrite reductase are both controlled by the same regulatory

gene and loss of NR also results in the loss of nitrite reductase. However

NR regulatory gene mutants have not as yet been reported in higher plants

and the mechanism of control in higher plants may differ from fungi.

Another enzyme that can provide insight into the nature of the NR mutants

is xanthine dehydrogenase. Xanthine dehydrogenase is also a molybdo-enzyme

and appears to have the same molybdo-cofactor component as NR. The mutant

58 RESEARCH REPORTS

PNL Volume 13

1981

A300 does not have xanthine dehydrogenase activity which strongly suggests

that nar-2 is a gene involved with .the production of the molybdo-cofactor.

Although direct evidence is not yet available, nar-1 is most likely the

NR structural gene. The leaf nitrate concentrations in the mutants are 8

to 10 times greater than the wild-type indicating that nitrate uptake is

not a causal factor in the NR-deficiency. Since nar-1 does not have the

characteristics usually associated with NR regulatory gene mutants and nar-2

is a molybdo-cofactor gene, the only other known gene in which mutations

cause loss of NR activity is the NR structural gene.

Editor's Note: Happily, a number of workers have isolated mutants

that control nitrate reductase activity. Apparently, however, there

has not been any coordinated effort to name mutants or assign symbols

according to an agreed upon scheme. I strongly urge workers in this

area to communicate with each other and with Dr. Blixt who is overall

coordinator of gene symbols in Pisum. We want to avoid the duplication

of symbols or the use of different symbols for the same mutants. It

is much more convenient to avoid the problem in the first place than

to try to correct a situation that is entrenched in the literature.

Perhaps someone will volunteer to write an article summarizing the reports

on this subject to date?