Posttranslational Regulation of Nitrate Reductase in Higher Plants

Abstract

In many plants, nitrogen represents 2 to 6% of dry matter, most of it being present in the form of amino acids, proteins, or nucleic acids. In these organic compounds, nitrogen exists in its most reduced state (oxidation state -3). It is taken up from the soil primarily in the form of nitrate (oxidation state +5). Thus, nitrate has to be reduced by plants at the expense of reductants such as NADH or NADPH, requiring 8 mo1 of electrons [or 4 mo1 of NAD(P)H] per mo1 of nitrate. Reduction is a two-step mechanism. The first step, reduction of nitrate to nitrite (+3 oxidation state), is catalyzed by assimilatory NR, which is an NAD(P)H-dependent cytosolic enzyme. Nitrite is further reduced to ammonia (-3 oxidation state) in the plastids of leaves or roots by NiR.There are at least two important reasons why plants must exert control on the velocity of nitrate reduction. First, it is an energy-consuming process. As shown above, 8 electrons are required to reduce one nitrate to ammonium, but only 4 electrons are needed to reduce COz to the carbohydrate level. Accordingly, a C/N ratio of 10 in the plant biomass (a value found in many herbaceous plants) indicates that about 20% of the photosynthetically produced electrons are consumed for nitrate reduction. Plants have to avoid "luxury" consumption of nitrate and energy. Second, and perhaps more important, the primary product of nitrate reduction, nitrite (NO,-), is cytotoxic and regarded to be mutagenic as a result of the ability to diazotize amino groups. HNO, is also a weak acid that, in its undissociated form, can easily penetrate biomembranes, thereby leading to acidification of cells or subcellular compartments. Thus, it makes sense if nitrate reduction is controlled in such a way that it does not exceed nitrite and ammonium consumption. In fact, even under rapidly fluctuating environmental conditions, nitrite levels in plant tissues remain low (below 0.1 m). Apparently, the overall rates of nitrate and nitrite reduction always match the availability of energy and of carbohydrate, perhaps with the exception of some extreme conditions to be discussed below.Synchronization of nitrate reduction and carbon metabolism may occur at the level of transcription or translation of participating enzymes. The expression of NR genes at the transcription level is highly affected by nitrate, but also by light, plant hormones, and other factors (for review, see Solomonson and Barber, 1990;Lillo, 1994). The enzyme protein is rather short-lived, being degraded with a half-time of a few hours (Li and Oaks, 1993). This high tumover rate permits control of nitrate reduction, e.g. in response to nitrate availability (Li and Oaks, 1993). However, over the years, a number of situations have been described in which the level of extractable NRA did not match NR protein or the rate of nitrate reduction in vivo, indicating that yet other regulatory mechanisms might exist that modulate the catalytic activity of the protein (Lillo, 1994, and refs. cited therein). Such a newly discovered type of N...

Influence of malate and 2-oxoglutarate on the NIA transcript level and nitrate reductase activity in tobacco leaves

“…Third, malate led to a marked decrease of the NIA transcript level and NIA activity but did not markedly affect NIA activation (Figs 2,3 & 6). This contrasts with the response to stomatal closure or low CO 2 , which lead to post-translational inactivation of NIA (Kaiser & Förster 1989;Kaiser & Huber 1994). Fourth, a strikingly different response was found when 2-oxoglutarate was supplied (Figs 2, 4 & 6) even though both organic acids lead to a decrease in the transpiration rate.…”

“…1b, dotted line). Activation (the ratio of activity in the presence and absence of Mg 2+ ) increases after illumination and decreases after darkening as seen previously (Kaiser & Huber 1994;Scheible et al 1997b). Malate increased progressively throughout the light period and decreased at night (Fig.…”

“…Changes of NIA activity in this test are matched by changes in protein (Scheible et al 1997b). NIA activity was also assayed in the presence of 10 mm Mg 2+ as in Scheible et al (1997b) to allow inhibitory 14-3-3 proteins to bind to the phosphorylated form (Kaiser & Huber 1994;Moorhead et al 1996;Bachmann et al 1996).…”

“…Expression is totally inhibited when sugars fall below a critical threshold (Matt et al 1998). Reversible phosphorylation of ser 543 followed by Mg 2+ -dependent binding of inhibitory 14-3-3 proteins (Bachmann et al 1996;Moorhead et al 1996) facilitates rapid post-translational inactivation and degradation of NIA in the dark or when sugars are low (Kaiser & Huber 1994;Weiner & Kaiser 1999).…”

Nitrite–dependent nitric oxide production pathway: implications for involvement of active nitrogen species in photoinhibitionin vivo

“…also required to suppress nitrite accumulation in the tissues (Kaiser & Huber 1994), a potential cause of the oxidative damage mediated by active nitrogen species (Yamasaki & Sakihama 2000). Because the production of NO through the NRdependent nitrite pathway probably requires low or no activity of photosynthetic electron transport, oxidative damage induced by active nitrogen species could be not directly concerned with the high-light-induced photoinhibition.…”