Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

Abstract

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme. When cut leaves or roots of this line (S(521)) were placed in darkness in a buffer containing 50 mM KNO(3), nitrite was excreted from the tissue at rates of 0.08-0.2 micromol (g FW)(-1) h(-1) for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1-3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S(521), although 20-40 micromol (g FW)(-1) nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S(521) also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S(521) was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.

Nitrite as the major source of nitric oxide production by Arabidopsis thaliana in response to Pseudomonas syringae

“…The conversion of NO À 2 to Å NO in plants has been attributed to NR [12,28]. However, in our experimental conditions, most of the NO À 2 -derived Å NO production cannot be attributed to NR because homogenates from plants lacking the two structural genes NIA1 and NIA2 also produced Å NO (Figs.…”

“…Other plant species also produce Å NO via NR when the tissue nitrite concentrations are high [12,33]. Likewise, transgenic plants expressing a permanently active NR enzyme have increased Å NO emissions [28]. The production of L L-citrulline from L L-arginine was increased in inoculated A. thaliana leaves from wild-type and nia1 nia2 double mutant plants, thus indicating the participation of an NOS-like enzyme in this response, as previously observed for other plant-pathogen interactions [4,5,8,11].…”

“…Since NR has been claimed to be an important source of Å NO in plants [12,28], we next examined the possibility that NO À 3 was the source of Å NO during the A. thaliana-P. syringae interaction. Homogenates from the leaf tissues of wild-type and NR-deficient mutant plants (controls or Psm-inoculated) were incubated with 100 mM phosphate buffer containing 1 mM NADH, an important cofactor for NR activity and the spin trap.…”

A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas

“…When the plant is photosynthetically active, nitrite produced by NR is immediately taken up by the chloroplast and reduced to ammonium (Rexach et al, 2000). However, nitrite accumulation can be expected when photosynthetic activity is low, so that NR-mediated conversion of nitrite to NO could take place in these conditions (Lillo et al, 2003;Lea et al, 2004) and NO could then repress NIA1 transcription, the whole process forming a feedback loop. This negative feedback loop could be part of a mechanism for protection against nitrite toxicity, so that further accumulation of nitrite would be blocked.…”

Nitrite Acts as a Transcriptome Signal at Micromolar Concentrations in Arabidopsis Roots

“…In contrast, NiR-deficient plants, which accumulate high nitrite levels, display chlorosis and dramatically reduced growth in tobacco (Nicotiana tabacum; Vaucheret et al, 1992) and rapid death in barley (Hordeum vulgare; Duncanson et al, 1993) if grown with nitrate. Such plants also produce high levels of nitric oxide (NO; Morot- Gaudry-Talarmain et al, 2002) because nitrite serves as a substrate for the formation of NO (Yamasaki et al, 1999;Rockel et al, 2002;Lea et al, 2004;Meyer et al, 2005;Planchet et al, 2005). It should be noted that nitrite can have a beneficial effect because it protects maize (Zea mays) roots from anoxia by reducing cytoplasmic acidosis (Libourel et al, 2006).…”