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J. Exp. Bot.-1996-Miller-843-54
J. Exp. Bot.-1996-Miller-843-54
Journal of Experimental Botany, Vol. 47, No. 300, pp. 843-854, July 1996 Experimental
Botany
REVIEW ARTICLE
Anaerobic NR assay
Introduction
The anaerobic NR method has been used to estimate the
The measurement of cytosolic and vacuolar nitrate con- 'metabolic' nitrate pool and is based on measurement of
centration is essential for determining the mechanism of the nitrite formation in the absence of external nitrate,
nitrate transport at both the plasma membrane and under conditions (anoxia, darkness) intended to inhibit
tonoplast because the electrochemical gradient of this ion nitrite reduction (Ferrari et cil., 1973). This technique
across each of these membranes determines whether the depends on the cytosolic location of NR, and the resulting
uptake of nitrate into the cell and into the vacuole occurs limited access of the enzyme to its substrate, nitrate. The
by passive or active mechanisms. Clarkson (1986) con- cessation of nitrite formation is regarded as the indication
cluded that measurements of cytosolic nitrate are a pre- of exhaustion of the 'metabolic' nitrate pool which is
1
To whom correspondence should be addressed. Fax: +44 1582 760981. E-mail tony.miller©bbsrc.ac.uk
Abbreviations: JG'/F, free-energy change for H+/NOj~-symport; F, Faraday constant; pHc, cytoplasmic pH; pHo, external pH; pINOJ,,, -log10 cytosolic
NO^" concentration; pmf, proton motive force; pINOJo, -tog10 externaJ NO^~ concentration; NR, nitrate reductase; NMR, nuclear magnetic resonance.
Solution Cytosol
assumed to be synonomous with cytosolic nitrate. of ions (MacRobbie, 1971). For this method, tissues are
Cytosolic nitrate values obtained using this type of loaded to a steady-state (constant specific activity in all
f
i •
influx of nitrate, a single open anion channel with an energy required to maintain a cytosolic nitrate concentra-
efflux rate of 107 ions s~' will deplete the cytosolic nitrate tion of 4 mol m~3 can thus be calculated for different
concentration from 4 mol m~3 to mmol m~3 levels (pass- values of n. By calculating the free energy at different
ive nitrate distribution) in 50-200 s. external nitrate and pH values, the ability of different
symport mechanisms to maintain cytosolic nitrate can be
Active nitrate transport assessed. The results of such calculations are shown
Active transport is required at the plasma membrane and plotted in Fig. 2. For the calculations it was assumed that
the tonoplast of epidermal cells in both maize and barley the cytosolic nitrate concentration remained at 4 mol m" 3
roots to maintain the measured intracellular concentra- at all external nitrate concentrations (see above), that the
tions of nitrate. The proton electrochemical gradient cytosolic pH was 7.2 (Miller and Smith, 1992) and was
across both the tonoplast and the plasma membrane can insensitive to external changes pH (see Kurkdjian and
provide the energy for the transport of nitrate. Active Guern, 1989, and references therein), and that the mem-
nitrate transport at the plasma membrane is thought to brane potential was -70 mV (Zhen et al., 1991). Although
occur by symport with protons. Measurements in maize it remains to be established that all parameters would
and barley of the nitrate-elicited changes in electrical remain constant under the combination of conditions
potential difference across the plasma membrane support assumed, the calculations give an estimate of the ability
a proton symport model (McClure et al., 1990; Glass of the different mechanisms to account for the observed
et al., 1992). These measurements suggest that the sym- cytosolic nitrate concentration.
and this may be the reason for controlling cytosolic determining the levels of cytosolic nitrate even though in
concentration. At high concentrations the ion has non- many species NR activity increases with nitrate supply
specific chaotropic effects (Griffith et al., 1986; Weiser (Aslam et al., 1993; Fedorova et al., 1994). However,
and Bentrup, 1994), while even at concentrations below under anaerobic conditions NR activity increases and
10 mol m~\ nitrate specifically inhibits proton pumping may then influence cytosolic nitrate levels, and this is the
by the vacuolar ATPase (Wang and Sze, 1985). basis for one of the methods for measuring cytosolic
nitrate and under these conditions nitrate supply deter-
Mechanisms for regulating cytosolic nitrate mines NR activity (King et al., 1992). Measurements of
cytosolic nitrate in these NR-deficient mutants will estab-
Cytosolic nitrate concentration in a root cell must be lish if assimilation has any role in regulating cytosolic
determined by several processes, including transport at nitrate activity.
both the plasma membrane and tonoplast, assimilation Xylem loading may be involved in the regulation of
and symplastic transport to the xylem parenchyma for cytosolic nitrate concentration because excised roots show
transport to the shoot. a decrease in cytosolic nitrate (Zhen et al., 1992), but
Cytosolic nitrate concentration must be maintained by excised roots can continue to produce xylem exudate
the steady-state between processes at both the plasma which contains nitrate (Behl etal., 1988). It seems unlikely
membrane and the tonoplast. At the plasma membrane, that the only mechanism maintaining cytosolic nitrate
the steady-state between influx and efflux will not only concentration should be dependent on transpiration