Nitrate Reductase and Chlorate Toxicity in Chlorella vulgaris Beijerinck

Evidence is presented that chlorate is an extremely good analog for nitrate during nitrate uptake by intact barley (Hordeum vulgare cv. Fergus) roots. The depletion of C103-or NO3-from uptake media over 2 to 6 hours by seedlings was found to be dependent on combined N03-plus C103-concentrations, and total anion uptake was equivalent at different N03-/CI03-ratios. After loading barley seedlings with 3'CI03-for 6 hours, kinetic parameters were derived from the analysis of efflux of 13"CI chlorate into unlabeled solution. On the basis of this analysis, the half times for exchange for the cytoplasmic and vacuolar phases were 17 minutes and 20 hours, respectively.Data pooled from a number of different experiments were used to calculate kinetic constants (Km and V,,) for 36CI03-influx into barley roots at different external C103-/NO3-ratios, using short (10 minutes) influx times. There appeared to be no discrimination by the root cells between C103-and N03-. Lineweaver-Burk analysis of the interaction between nitrate and chlorate were characteristic of competitive inhibition at low nitrate concentrations (0-0.5 mM). At higher concentrations, in the range of >1 mm, similar interactions between these ions were evident.There is relatively little information available on the detailed kinetics of nitrate transport into barley plants. Even the more recent investigations (2, 7, 21) have described net nitrate flux into barley plants over periods of greater than 2 h duration. The use of the heavy isotope`5N03 as a tracer is not without difficulties, and again for whole plant studies uptake periods of 9 h or greater are no exception (1, 14). In part, this is due to the large internal pools of nitrogen (6). As far as the authors are aware N03 has so far only been used for the study of nitrate assimilation in tobacco culture cells (24) and soybean roots and nodules (23). Moreover, this isotope is not very convenient for routine experiments because of its extremely short half-life (10 min). Doddema and Telkamp (9) (Fig. 1, insert)

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 86%

Nitrate Uptake into Barley (Hordeum vulgare) Plants

Deane‐Drummond

Glass²

1982

“…Later work indicated that NR was specifically responsible for the reduction of C103 to C102- (17,25). Chlorate also competes with N03 for uptake in Hordeum vulgare and Arabidopsis thaliana roots (5,6).…”

mentioning

confidence: 99%

Soybean Mutants Lacking Constitutive Nitrate Reductase Activity

Nelson

Ryan²,

Harper³

1983

The objectives of this study were to select and initially characterize mutants of soybean (Gleine max L. Meff. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.In growth chamber studies, the fully expanded first trifoliolate leaf from N03-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity, the latter activity does not require the presence of NOs-in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (N03-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO3--grown wild-type plants. Root -(17, 25). Chlorate also competes with N03 for uptake in Hordeum vulgare and Arabidopsis thaliana roots (5, 6). Thus, C103-can act as an analog of NO3 during uptake, as well as during reduction by NR. The analog characteristic of C103 and the resultant toxicity symptoms associated with C103-reduction suggested that this ion could be used as a screening agent to identify organisms with decreased N03 uptake and metabolism. Although mechanisms of C103 toxicity other than that involving NR are possible (14,19,27), Cl03 resistant mutants which are NR deficient have been found in higher plants (7, 22,26) and plant cell lines (14,18,19). Soybeans (Glycine max L. Merr.) have been shown to display characteristic C103 toxicity symptoms (browning and stunting of leaves) (9, 28), and this was the basis for using a C103 screen to identify plants defective in N03 metabolism. This paper reports on the selection, from progeny of mutagenized seed, and initial characterization of three soybean lines which have decreased leaf NR activity compared with wild-type plants. MATERIALS AND METHODS

“…A possible explanation for the very low levels ofNR protein in chlorate-treated plants that have moderate levels of NR mRNA is that the normal metabolism of the plant cell is (32). Even if the NR gene was being induced by chlorate, the induction would be obscured by the inactivation of the enzyme by the reaction product chlorite.…”

Section: In Vivo Labelingmentioning

confidence: 99%

“…Although there are some exceptions (10,11), now most data strongly support Aberg's hypothesis and implicate NR2 as the enzyme that reduces chlorate to chlorite. Chlorate is a substrate of purified NR and a competitive inhibitor ofnitrate reduction (26,32). Conversely, nitrate acts as a competitive inhibitor of chlorate reduction, and cyanate, which inhibits NR in vitro, reduces chlorate toxicity in vivo (23).…”

mentioning

confidence: 99%

Effect of Chlorate Treatment on Nitrate Reductase and Nitrite Reductase Gene Expression in Arabidopsis thaliana

LaBrie¹,

Wilkinson²,

Crawford³

1991

The herbicide chlorate has been used extensively to isolate mutants that are defective in nitrate reduction. Chlorate is a substrate for the enzyme nitrate reductase (NR), which reduces chlorate to the toxic chlorite. Because NR is a substrate (NO3-)- (23). Finally, many chlorate resistant mutants have been isolated and characterized, and most of these mutants have been found to be impaired in nitrate reduction (38).The toxic effects of chlorate have been exploited not only as a means to eliminate plant life but also as a powerful tool to select for mutants with defects in nitrate reduction. Chlorate resistant mutants have been isolated from bacteria, fungi, and plants (21). These mutants are usually defective in NR due to lesions in either the NR structural gene or one of the six to seven genes that are required for the synthesis of a MoCo, an essential component of the NR holoenzyme (21). The first plant species that was used to isolate chlorate resistant mutants was Arabidopsis thaliana (4). Dozens of resistant mutants were isolated and found to comprise eight complementation groups (5). We have identified one of these loci, chl3, as the NR structural gene NIA2 (37). Other loci have been shown to be required for MoCo synthesis (e.g. B25) (7). Such mutants have been invaluable for studying the structure, function, and regulation of NR (8,12