NO emission from nitrate reductase

In leaves, NO emission from NR closely followed the NR activation state (Figure 1). NO emission was low in the dark (aerobic) and much higher in the light. Upon sudden light off, leaves often (but not always) produced a further transient increase in NO emission ("light-off peak", Figure 4). NO emission from leaves was at maximum under anoxia in the dark.

The factors that activated NR, like the 5'-AMP analogue AICAR, increased NO emission, whereas PP2A-inhibitors, which lead to a largely inactive NR prevented NO emission (Rockel et al. 2002). Thus, NO emission appeared to closely follow the NR activation state and the nitrite concentration in the tissue. As nitrite reduction was always blocked under anoxia, and NR was highly activated under these conditions, NO emission was always at maximum under anoxia because of the highly activated NR and the absence of nitrite reduction, which together led to massive nitrite accumulation in anoxic tissues (Rockel et al. 2002). Other reasons for the very high NO emission of plant tissues under anoxia might be the absence of reactive oxygen species (ROS), which can rapidly oxidise NO. Further evidence that NR (or nitrite) is a prerequisite for any NO formation in plants is based on the observation that plants grown on ammonium (which do not express NR and NiR) and which, accordingly, do not accumulate nitrite, would not emit NO even under anoxia (Figure 4). The same holds for NR-deficient mutants (not shown). On the other side, a tobacco transformant having normal NR activities but expressing NiR in antisense orientation so that less than 5% of the normal nitrite reduction capacity remained, always accumulated nitrite and produced very high NO emission (Morot-Gaudry-Talarmain et al. 2002).

Figure 4. Typical pattern of NO-emission from detached tobacco leaves. The upper curve shows data from nitrate-fed plants. NO emission is low in the dark (grey or black bars on top of the figure), up to 10-fold higher in the light and increases transiently after light off. This is due to a transient overshoot of nitrate reductase leading to some nitrite accumulation, which stops after NR has been down regulated in the dark, requiring 5 -15 min. In contrast to NR, nitrite reduction stops immediately after light off. NO emission in the dark is drastically stimulated under anoxia, (black bar) because NR is activated, NiR does not work, leading to a strong accumulation of nitrite. Probably, the absence of reactive oxygen species under anoxia also contributes to the high NO-emission under dark-anoxic conditions (compare to Figure 5). Also shown (lower curve with open symbols) is the complete absence of NO-emission from leaves of ammonium-grown hydroponic tobacco plants, which contain no measurable NR-activity (W.M. Kaiser, unpublished data).

Figure 4. Typical pattern of NO-emission from detached tobacco leaves. The upper curve shows data from nitrate-fed plants. NO emission is low in the dark (grey or black bars on top of the figure), up to 10-fold higher in the light and increases transiently after light off. This is due to a transient overshoot of nitrate reductase leading to some nitrite accumulation, which stops after NR has been down regulated in the dark, requiring 5 -15 min. In contrast to NR, nitrite reduction stops immediately after light off. NO emission in the dark is drastically stimulated under anoxia, (black bar) because NR is activated, NiR does not work, leading to a strong accumulation of nitrite. Probably, the absence of reactive oxygen species under anoxia also contributes to the high NO-emission under dark-anoxic conditions (compare to Figure 5). Also shown (lower curve with open symbols) is the complete absence of NO-emission from leaves of ammonium-grown hydroponic tobacco plants, which contain no measurable NR-activity (W.M. Kaiser, unpublished data).

Reduction of nitrite to NO (Figure 1) is not the only side reaction catalysed by NR. Indeed, it has been shown that NR also catalyses the reduction of molecular oxygen to superoxide (Ruoff and Lillo 1990, Barber and Kay 1996, Yamasaki et al. 1999, Yamasaki and Sakihama 2000), but the capacity of that reaction appears as low as that for NO formation (Figurel). Still, NO emission from an aerated solution of NR is decreased upon addition of H2O2 (Figure 5) and is increased upon addition of catalase and SOD or under anoxia (not shown).

This indicates that upon simultaneous production of reactive oxygen species and of NO, both products react with each other, probably by forming the highly toxic peroxynitrite. This leads to a partial "quenching" of apparent NO emission from the solution. The observation is also important because in cells, many other reactions can produce reactive oxygen species and can thus interfere with the measurement of apparent NO production.

Although we are just beginning to explore the secrets of NO formation in plants, it seems obvious that they possess multiple NO-producing systems (Neill et al. 2003). With the exception of NOS, all of them depend finally on nitrite as a substrate, and therefore on NR or on exogenously produced nitrite from soil-borne microorganisms (see above).

time (min)

Figure 5. A stirred solution of purified NR (maize) emits NO into the gas phase after addition of NADH and nitrite at time zero. The apparent NO production is inhibited by addition of H2O2 (100 pM). It should be noted, that normal NR activity is not inhibited by H2O2 (not shown). (W.M. Kaiser, unpublished data).

time (min)

Figure 5. A stirred solution of purified NR (maize) emits NO into the gas phase after addition of NADH and nitrite at time zero. The apparent NO production is inhibited by addition of H2O2 (100 pM). It should be noted, that normal NR activity is not inhibited by H2O2 (not shown). (W.M. Kaiser, unpublished data).

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