Browsing by Author "Tischner, R."

Aldehyde oxidase (AO; EC 1.2.3.1) isoforms in roots of barley plants grown on ammonium or nitrate as nitrogen sources were studied. Roots of ammonium-grown barley plants exhibited considerable levels of AO2, AO3, and AO4 activities after native PAGE. Significantly lower AO2 and AO3 activity bands were observed in roots of plants grown on nitrate. When abscisic aldehyde was used as a substrate a strong response of the AO2 band was observed as well as a faint reaction of the AO3 band, but no activity of AO4 was observed using this substrate. The 160 and 145 kDa polypeptides were detected in ammonium grown plants. Root extracts of nitrate-fed plants revealed only a minor 145 kDa protein band and none of the 160 kDa subunit was detected. The assembly of the AO3 heterodimer requires the simultaneous presence of 160 and 145 kDa subunits. Subunit analysis of AO2 and AO4 revealed homodimeric composition of 160 and 145 kDa, respectively. Western blot analysis revealed changing AO subunits levels during germination and plant development. Differential expression of AO subunits (160 and 145 kDa) and subsequent formation of isoforms, which differ in substrate specificity, distribution and fulfil different enzymatic reactions, may constitute an important regulatory mechanism in the plant.

The nitrate transporter from Chlorella sorokiniana (accession number AY026523) has been cloned by screening a cDNA library based on mRNA isolated after 30 min treatment of Chlorella with 5 mM nitrate and with a RT-PCR product (730 bp) as a probe. The Chlorella sequence has similarity to known nitrate transporters of the NRT2 family (high-affinity nitrate transporters). The cDNA clone was used for functional expression in Xenopus oocytes and a nitrate-dependent current was measured at pH 5.5 but not at pH 7.4. A second algal gene or a second gene product was not needed for functional expression in Xenopus. Inhibitor studies in Chlorella indicated that protein phosphorylation/dephosphorylation is involved in nitrate induction of ChNRT2.1. In addition to nitrate, ChNRT2.1 expression is induced by nitroprusside, a NO donor, and is affected by glucose.

Changes in the activity and subunit composition of cytosolic glutamine synthetase (GS 1; EC 6.3.1.2) and chloroplastic GS (GS 2) were studied in response to an internal (organ ontogeny) and external signal (N source: NO3- or NH4+). Maximum GS 1 activity of all organs examined was measured in the fibre roots, irrespective of the N source. The response of GS 1 to the N source was, however, organ specific. In the fibre roots, NH4+ nutrition resulted in a 2- to 7-fold (based on protein or freshweight, respectively) increase of CS 1 activity compared to NO3--grown plants. In contrast to the roots, GS I activity in the leaf blades was 2-fold lower with NH4+ nutrition, whereas only minor changes occurred in the petioles. CS 2 activity was highest in the mature and senescing leaf blade; activity was 2-fold higher with NH4+ than with NO3- nutrition, Not only activity, but also subunit composition of GS 1 changed during organ ontogeny as well as in response to the N source. In contrast to GS 1, only minor changes were evident in GS 2 subunit composition, despite significant changes in GS 2 activity. Up to 5 different GS 1 subunits of approximate to 41-43 kDa were separated; they were identical in all organs examined. GS 2 was composed of 4 different subunits of similar to 48 kDa.

A nitrate reductase (NR)-null mutant of Arabidopsis was constructed that had a deletion of the major NR gene NIA2 and an insertion in the NIA1 NR gene. This mutant had no detectable NR activity and could not use nitrate as the sole nitrogen source. Starch mobilization was not induced by nitrate in this mutant but was induced by ammonium, indicating that nitrate was not the signal for this process. Microarray analysis of gene expression revealed that 595 genes responded to nitrate (5 mm nitrate for 2 h) in both wild-type and mutant plants. This group of genes was overrepresented most significantly in the functional categories of energy, metabolism, and glycolysis and gluconeogenesis. Because the nitrate response of these genes was NR independent, nitrate and not a downstream metabolite served as the signal. The microarray analysis also revealed that shoots can be as responsive to nitrate as roots, yet there was substantial organ specificity to the nitrate response.

A method is presented to isolate mutants of Chlorella sorokiniana with defects in NO3- metabolism Three nitrite-reductase (NIR; E.C.1.7.7.1)-deficient mutants were obtained from 500 pinpoint-colony-forming clones. The final screening was performed using NO3-, NO2- or NH4+ as N-source. The mutants isolated absorb NO3- with rates close to those measured for the wild type and they excrete NO2- into the medium. The ratio between NO3- uptake and NO2- excretion was 1:1. The sensitivity of NO3- uptake to NH4+ was reduced in the mutant strains as it was in the N-starved wild type of Chlorella. Nitrate reductase (NR; EC 1.6.6.1) expression and NR activity were slightly reduced compared to the wild type due to feedback regulation in the mutant strains. No NIR protein was found in the three mutants. However, NIR activity was obtained (50% of the wildtype) for one mutant strain. The NIR-deficient mutants and the already available NR-deficient mutants will be promising tools for investigations of the nitrate assimilation pathway on the molecular level and for studies searching for signaling of C and N metabolism by inorganic N-compounds.

The effect of short-term low temperature treatment on nitrate reductase (NR, EC 1.6.6.1) activity, NR protein and NR transcript levels in excised leaves of winter wheat (Triticum aestivum L. cv. Sadovo-1) was investigated. NR activity, measured in the presence of Mg2+ (NRact), doubled within 2 h at 4degreesC, whereas NR activity, measured in the presence of EDTA (NRmax), did not respond to the cold treatment. Such an activation of NR occurred only if leaves were exposed to low temperature in the light but not in the dark. It was not affected by feeding cytoplasmic protein synthesis inhibitor, cycloheximide, or protein kinase inhibitor, staurosporin, but was completely prevented by okadaic acid, an inhibitor of protein phosphatases of the type 1 and 2 A. This inhibitory effect decreased gradually when okadaic acid-concentration in the nutrient solution was lowered below 1 muM and tended to disappear when leaves were fed with 10 nM okadaic acid. It was demonstrated that the cold-induced NR activation was dependent neither on cold-triggered calcium influx nor on high endogenous abscisic acid levels. The increased NRact in cold-exposed leaves was found to correlate with a higher level of NR transcript but not with an increased NR protein level. Feeding okadaic acid to these leaves prevented the cold-induced accumulation of NR mRNA. These data point to protein phosphatases of the type 2 A being involved in NR protein dephosphorylation and NR transcript accumulation as targets of activation by low temperature treatment.

The nitrogen compounds nitrate and ammonium are the minerals that plants need in large quantities and which limit their growth in temperate zones. The nitrate assimilation pathway starts with nitrate uptake followed by nitrate reduction resulting in ammonium which is fixed into the amino acids glutamine and glutamate in most plants. This review concentrates on nitrate uptake and nitrate reduction with respect to higher and lower plants. The physiology and the progress in molecular approaches of both processes are considered. For nitrate uptake the well-established uptake systems are discussed and special attention is drawn to nitrate sensing and the nitrate carrier. Knowledge, particularly on nitrate sensing is rare, but it seems to be the first step in a signal transduction chain triggered by nitrate. Therefore further work should consider this topic more frequently. For nitrate reductase the focus is on the posttranslational modification as a regulatory tool for nitrate assimilation, on the intersections of carbon and nitrogen metabolism and on the molecular approaches. A few remarks on how environmental conditions affect nitrate assimilation are also included. Further progress is needed to understand the transduction of positive and negative signals from the environment affecting the expression of genes coding for the nitrate assimilating pathway.