Nitrite accumulation and nitric oxide emission in relation to cellular signaling in nitrite reductase antisense tobacco.

An antisense nitrite reductase (NiR, EC 1.7.7.1) tobacco ( Nicotiana tabacum L.) transformant (clone 271) was used to gain insight into a possible correlation between nitrate reductase (NR, EC 1.6.6.1)-dependent nitrite accumulation and nitric oxide (NO(.)) production, and to assess the regulation of signal transduction in response to stress conditions. Nitrite concentrations of clone 271 leaves were 10-fold, and NO(.) emission rates were 100-fold higher than in wild type leaves. Increased protein tyrosine nitration in clone 271 suggests that high NO(.) production resulted in increased peroxynitrite (ONOO(-)) formation. Tyrosine nitration was also observed in vitro by adding peroxynitrite to leaf extracts. As in mammalian cells, NO(.) and derivatives also increased synthesis of proteins like 14-3-3 and cyclophilins, which are both involved in regulation of activity and stability of enzymes.

Towards a better understanding of the genetic and physiological basis for nitrogen use efficiency in maize.

To enhance our understanding of the genetic basis of nitrogen use efficiency in maize (Zea mays), we have developed a quantitative genetic approach by associating metabolic functions and agronomic traits to DNA markers. In this study, leaves of vegetative recombinant inbred lines of maize, already assessed for their agronomic performance, were analyzed for physiological traits such as nitrate content, nitrate reductase (NR), and glutamine synthetase (GS) activities. A significant genotypic variation was found for these traits and a positive correlation was observed between nitrate content, GS activity and yield, and its components. NR activity, on the other hand, was negatively correlated. These results suggest that increased productivity in maize genotypes was due to their ability to accumulate nitrate in their leaves during vegetative growth and to efficiently remobilize this stored nitrogen during grain filling. Quantitative trait loci (QTL) for various agronomic and physiological traits were searched for and located on the genetic map of maize. Coincidences of QTL for yield and its components with genes encoding cytosolic GS and the corresponding enzyme activity were detected. In particular, it appears that the GS locus on chromosome 5 is a good candidate gene that can, at least partially, explain variations in yield or kernel weight. Because at this locus coincidences of QTLs for grain yield, GS, NR activity, and nitrate content were also observed, we hypothesize that leaf nitrate accumulation and the reactions catalyzed by NR and GS are coregulated and represent key elements controlling nitrogen use efficiency in maize.

A Transcriptionally Active State Is Required for Post-Transcriptional Silencing (Cosuppression) of Nitrate Reductase Host Genes and Transgenes.

Using tobacco nitrate reductase cosuppression as a model system of post-transcriptional gene silencing, we analyzed the influence of DNA and RNA dosages both together and independently. For this purpose, zero, one, two, or four active or transcriptionally silenced copies of a cauliflower mosaic virus 35S-Nia2 transgene were combined by transformation and subsequent crosses with zero, one, two, three, or four active, disrupted, or transcriptionally repressed copies of the wild-type host Nia genes. The analysis of the corresponding transgenic lines revealed that (1) the percentage of isogenic plants that are affected by cosuppression depends directly upon the relative dosage of both host gene and transgene; (2) transcriptional silencing of the 35S-Nia transgene impedes cosuppression; and (3) the absence of host gene transcription reduces the frequency of cosuppression or delays its triggering. Taken together, these results indicate that transgene DNA per se is not sufficient to trigger post-transcriptional cosuppression of nitrate reductase host genes and transgenes. The requirement for a transcriptionally active state is discussed with respect to both the RNA dosage and the DNA-DNA pairing hypotheses.

Abolition of Posttranscriptional Regulation of Nitrate Reductase Partially Prevents the Decrease in Leaf NO3- Reduction when Photosynthesis Is Inhibited by CO2 Deprivation, but Not in Darkness.

The activity of nitrate reductase (NR) in leaves is regulated by light and photosynthesis at transcriptional and posttranscriptional levels. To understand the physiological role of these controls, we have investigated the effects of light and CO2 on in vivo NO3- reduction in transgenic plants of Nicotiana plumbaginifolia lacking either transcriptional regulation alone or transcriptional and posttranscriptional regulation of NR. The abolition of both levels of NR regulation did not modify the light/dark changes in exogenous 15NO3- reduction in either intact plants or detached leaves. The same result was obtained for 15N incorporation into free amino acids in leaves after 15NO3- was supplied to the roots, and for reduction of endogenous NO3- after transfer of the plants to an N-deprived solution. In the light, however, deregulation of NR at the posttranscriptional level partially prevented the inhibition of leaf 15NO3- reduction resulting from the removal of CO2 from the atmosphere We concluded from these observations that in our conditions deregulation of NR in the transformants investigated had little impact on the adverse effect of darkness on leaf NO3- reduction, and that posttranscriptional regulation of NR is one of the mechanisms responsible for the short-term coupling between photosynthesis and leaf NO3- reduction in the light.