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

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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 [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S521) were placed in darkness in a buffer containing 50 mM KNO3, nitrite was excreted from the tissue at rates of 0.08–0.2 μmol (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 S521, although 20–40 μmol (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 S521 also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S521 was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.