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

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 [C. Lillo et al. (2003) Plant J 35:566-573]. When cut leaves or roots of this line (S-521) were placed in darkness in a buffer containing 50 mM KNO3, nitrite was excreted from the tissue at rates of 0.08-0.2 mumol (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 mumol (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.