Isolation and characterisation of chlorate resistant mutants of barley

Abstract

The object of this study has been to characterise barley mutants which lack the functional nitrate reductase (NR) enzyme system. In the long term it is hoped that such studies will lead to improved nitrate utilisation and ultimately to improved quality barley protein. The progeny of nine chlorate resistant selections, in the barley cultivars Mavis Mink and Golden Promise, were studied. Four (R9201, R11301, R12202 and R12801) lacked NADH-NR and FMNH2-NR activities, the rest had the NR+ phenotype. None of the four were nitrate uptake mutants since they all possesed wild type or greater levels of nitrate. R9201, R11301 and the previously characterised R9401 (Bright et al, 1983) were not molybdenum uptake or Mo-accumulation mutants.

R9201, R11301 and R12202 lacked xanthine dehydrogenase (XDH) (an enzyme which contains the same molybdenum-containing cofactor, MoCo) activity suggesting that these lines have defective MoCo's, whilst R12801 possessed XDH activity indicating that it might have defective apoprotein subunits.

These four lines are similar to R9401 since they lack "NR activity and are unlike other previously selected ' barley NR mutants (Kleinhofs ' et al, 1980) which are leaky and possess up to 5% of the wild type (cv. Steptoe) in vitro NR activity.

R9201 and R11301, like R9401, were all caused by single recessive nuclear gene mutations.

The MoCo mutants, R9201, R9401, R11301 and R12202, could be divided into two groups on the basis of i) allelism, ii) presence or absence of wild type levels of dimeric NR and iii) the ability of their extracted MoCo's to reconstitute NADPH-NR in an extract of N. crassa nitl mutant (which supplies NR monomers) in the presence of excess molybdate.

R11301 is not allelic to R9201, whilst R9201 and R9401 are allelic. R9401 is also thought to be allelic to Az 34, a barley MoCo mutant isolated and characterised by Kleinhofs et al (1980). Az 34 has been designated nar2a and it is proposed that the allelic R9401 and R9201 should be classified as nar2b and nar2c respectively. It is possible that R11301 is either allelic with one of the other barley MoCo lines, nar3, nar4 or it is defective in a different MoCo gene. The same is also true for R12202.

R11301 was shown to possess inactive NR dimer at wild type concentrations, whilst R9201, R9401 and R12202 had little or no NR (inactive dimer) present under these conditions. R12801 possessed no dimer.

The MoCo extracted from R11301 was able to reconstitute the same level of NADPH-NR in the Hill extract as MoCo extracted from-the wild types. R9201, R9401 and R12202 lacked this ability.