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