Annals of Botany 101: 485–489, 2008
doi:10.1093/aob/mcm313, available online at www.aob.oxfordjournals.org
BOTANICAL BRIEFING
Cytosolic Nitrate Ion Homeostasis: Could it Have a Role in Sensing Nitrogen
Status?
A N T HO N Y J . M I L L E R* and S US A N J . S M I T H
Centre for Soils and Ecosystem Function, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
Received: 6 July 2007 Returned for revision: 14 August 2007 Accepted: 14 November 2007 Published electronically: 17 December 2007
† Background and Aims There is ongoing debate regarding homeostasis of cytosolic nutrient ion concentrations.
This deliberation centres on the question of whether homeostasis occurs for some nutrients and, if so, what are
the consequences for how plants sense their nutrient status. Particularly for nitrate, this controversy has focused
on the methods used and the cellular pools which they measure. Cytoplasm and cytosol have been distinguished
and it has been suggested that two ranges of nitrate values can be separated depending on whether the method separates the pools found in organelles.
† Scope The present study defines homeostasis of nutrient ions and discusses how whole organ averaging techniques
can hide important cellular differences that can help to explain some of the discrepancies between results reported by
various methods. These results are considered in relation to a possible role in signalling nutrient status, and have
relevance to other averaging techniques such as the use of ‘omics’ technologies.
Key words: Hordeum vulgare, homeostasis, cytosolic nitrate, compartmentation, ion-selective microelectrodes.
IN TROD UCT IO N
In biology, homeostasis is usually defined as an organism’s
ability to regulate its internal environment to maintain a
stable, constant condition. Within a cell, the cytoplasm performs many of the chemical reactions of life and maintaining the physico-chemical environment to optimize these
processes must be essential for continuing existence. For
this review, the cytoplasm is defined as the cytosol and
all the organelles within it, but excluding the vacuole.
Homeostasis of the cytosolic milieu might therefore seem
to be beyond doubt. However, the requirement for homeostasis is complicated by the fact that some key life processes
are located within membrane-bound organelles, such as respiration within mitochondria and transcription in the
nucleus. Compartmentation in organelles can act to
protect the rest of the cell from highly reactive oxygen
species that can be generated in the mitochondria and
chloroplasts. On the other hand, subcellular compartmentation may provide protection for these vital life processes
and thereby give an opportunity for some chemical variation in the cytosolic composition. Turgor, osmoregulation
and charge balance are the physiochemical requirements
within the cytosol, but these still allow some variation in
the types of cations and anions used to meet these
demands. Changes in the cytosolic concentrations of
specific ions could then directly reflect alterations in the
extracellular environment that can also provide a cellular
signal that perhaps influences transcription. The classic signalling ions are cytosolic Ca2þ and to a lesser extent Hþ.
These signalling ions are maintained with low internal concentrations and large gradients across the plasma membrane
between the cytosol and the apoplast. Sudden rapid transient changes in cytosolic Ca2þ can occur and these are
* For correspondence. E-mail [email protected]
generally accepted as having a signalling role in plant
cells, thereby providing a link between environmental
changes and cellular responses. More controversial are the
ideas that the cytosolic ionic environment is regulated for
homeostasis of other nutrient ions, such as nitrate or
potassium.
The controversy around the ideas that there is homeostasis of cytosolic nitrate comes from the fact that measurements of these parameters in planta seem to provide a
large range of values (Siddiqi and Glass, 2002; Britto and
Kronzucker, 2003). The methods for measuring these cytosolic ions seem to give measurements that depend, at least
in part, on whether single cells or whole tissues are
sampled. For nitrate two types of methods and results
have been defined: one group was considered to sample
the cytosol while the other was thought to measure the cytoplasm including the cytosol and the intracellular organelles
(Siddiqi and Glass, 2002). In a later paper this idea was disputed and it was argued that the compartmental tracer efflux
method showed a variable cytosolic nitrate pool with no
evidence for significant contributions to these kinetics
from organellar pools (Britto and Kronzucker, 2003). In
this method tissues are loaded with an isotopic tracer to
give a steady state (constant specific activity in all subcellular compartments). The tissues are then moved to an
unlabelled solution of the same composition as the
loading solution. This efflux solution is replaced periodically and the tracer content is measured. The kinetics of
the tracer loss from the tissue can be resolved into several
exponential components that each correspond to a different
tissue compartment: apoplast, cytoplasm and vacuole.
Furthermore, if the compartmental volumes are known,
the specific activity within each compartment at the start
of the efflux period can be determined and used for calculation of the concentration. Britto and Kronzucker (2003)
# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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Miller and Smith — Homeostasis of Cytosolic Nitrate
went on to argue that only nitrate-selective microelectrode
measurements actually provide any evidence for cytosolic
nitrate homeostasis, and this technique may not be accurate.
M E T H O D S FO R M E A S U R I N G CE L L U L A R
CO M PA R T M E N TA L I O N CO N C E N T R AT I O N S ;
T I S S U E H E T E RO G E N E IT Y I S R E V E AL E D
BY SINGLE-CELL TECHNIQUES
The limitations of the compartmental tracer efflux method
(Miller and Smith, 1996) and microelectrodes (Britto and
Kronzucker, 2003) have been presented previously and so
will not be discussed in detail here. No method is ideal
and it is always better to compare the results obtained
using two different techniques for measuring the same cellular parameter. Very few papers have made this type of
comparison; some examples are single-cell sampling and
microelectrodes (Zhen et al., 1991) and 133Cs nuclear magnetic resonance (NMR) and microelectrodes (Radcliffe
et al., 2005). When these method comparisons have been
made there has been agreement between the results
obtained, arguing against the proposed inaccuracies associated with the nitrate-selective microelectrode technique
(Britto and Kronzucker, 2003). As discussed by Siddiqi
and Glass (2002), cellular nitrate reductase assays have
also provided measurements in a similar range to those
obtained using microelectrodes, but this technique has not
been used to demonstrate homeostasis.
When methods are used to focus on single cells they can
reveal heterogeneity between cellular nutrient pools that are
almost adjacent to one another. This is true for both leaves
and roots. Micropipette sampling of the vacuole of tissues
has revealed differences between epidermal and mesophyll
cells, and between abaxial and adaxial surfaces of leaves
(e.g. Fricke et al., 1994; Karley et al., 2000). In roots of
barley, Zhen et al. (1991) found that the cortical cells can
accumulate higher concentrations of nitrate than the epidermal cell layer. These two cell types are symplastically connected, but this might not result in a gradient of osmolarity
as concentrations of other ions such as organic anions could
provide a compensatory balance. For example, diurnal
changes in vacuolar nitrate involve compensatory changes
in organic anions such as malate (reviewed in Miller and
Cramer, 2004). Why epidermal and cortical cells accumulate differing vacuolar concentrations of nitrate remains
unknown; possibly this disparity may be explained by the
abundance or activity of vacuolar nitrate transporter(s)
between the two cell types. Furthermore, the time course
for remobilization of these two nitrate pools was different,
slower for the cortex than for the epidermis (van der Leij
et al., 1998). These double-barrelled microelectrode
measurements also showed that the cytosolic nitrate concentrations were very similar in the two tissues and that
they were maintained during the removal of the external
nitrate supply (van der Leij et al., 1998).
Nitrate-selective microelectrode measurements have been
used to measure the intracellular pools of nitrate in hydroponically grown barley roots growing in a range of external
nitrogen (N) supplies. These measurements showed that
cytosolic nitrate was maintained at a similar value that
was independent of 10 000-fold changes in the external
nitrate concentration (Fig. 1). In contrast, vacuolar nitrate
activity depended on the external supply and could be
almost two orders of magnitude different in individual
root cells growing under the same conditions. Ion-selective
microelectrodes report changes in the activity of an ion and
this is a more biologically useful measurement than concentration. Ion activity is equal to the chemical concentration
multiplied by the activity coefficient (for details, see
Walker et al., 1995). Some of these measurements required
the use of triple-barrelled microelectrodes incorporating
both nitrate- and pH-selective tips. The pH-selective
barrel was necessary to identify the compartmental location
of the electrode tip (Walker et al., 1995) and this was
important for conditions when the cytosol and vacuole contained very similar concentrations of nitrate (Fig. 1).
The mean cytosolic nitrate activities for epidermal cells
of barley roots growing in 0.1 – 10 mM nitrate (supplied as
the calcium salt) are shown in Fig. 2. These measurements
suggest that there is homeostasis of cytosolic nitrate at
around 4 mM in cells 10– 20 mm from the root tip. When
roots were grown in 0.1 mM NH4NO3 the mean nitrate
F I G . 1. The relationship between external nitrate supply and nitrate
activity in the cytosol, epidermal vacuole or cortical vacuole of roots of
barley seedlings. Only epidermal cytosolic nitrate activities are shown,
but cortical cells were not significantly different (ANOVA and t-test,
data not shown). Barley seedlings (‘Klaxon’) were all grown in hydroponic
culture in modified Hoagland’s nutrient solution under the environmental
conditions described previously (van der Leij et al., 1998); nitrate was supplied at concentrations of 10, 1, 0.1 and 0.01 mM as the calcium salt. Root
cells were impaled 10–20 mm from the tip and mean values + s.d. are
shown with a sample size of between 15 and 65, with each value taken
from a similar number of roots. In all figures, error bars are unequal
because the concentrations are given from a log-linear conversion; see
Walker et al. (1995) for details.
Miller and Smith — Homeostasis of Cytosolic Nitrate
F I G . 2. Comparison of the mean cytosolic nitrate activity in barley root
epidermal cells with four different types of nitrogen supply. Barley seedlings (‘Klaxon’) were grown in hydroponic culture in modified
Hoagland’s nutrient solution under the environmental conditions described
previously (van der Leij et al., 1998). Nitrate was supplied at 10, 1 and
0.1 mM as Ca(NO3)2 or at 0.1 mM as NH4NO3 for 48 h before the microelectrode measurements. A two-way ANOVA and t-test were used to
.
compare data and only 10 mM NO2
3 and 0 1 mM NH4NO3 treatments
showed significant differences (P , 0.01). Root cells were impaled 10–
20 mm from the tip and mean values + s.d. are shown. For each N
supply, microelectrode measurements of between 11 and 34 cells were
obtained, with each value taken from a similar number of roots.
activity was lower than the values obtained for seedlings
growing in calcium nitrate, but this difference was only
statistically significant for seedlings supplied with 10 mM
nitrate (Fig. 2). As treatment with ammonium has been
reported both to decrease influx and to increase efflux of
nitrate (reviewed in Miller and Cramer, 2004) this topic
needs further investigation. As in experiments done by
feeding barley roots with glutamine (Fan et al., 2006), following the time course of ammonium treatments on single
cell recordings of cytosolic nitrate may help to explain the
interactions between these various forms of N supply.
Microelectrode measurements of the cytosolic nitrate
activity in cells 1 – 2 mm from the root tip showed that
these pools appeared to be more sensitive to changes in
the external supply (Fig. 3). This result also supports the
data obtained in maize roots using nitrate-selective microelectrodes and 133Cs NMR, which showed that root tips
(2 – 3 mm) behaved differently from the mature vacuolated
regions of the root (Radcliffe et al., 2005). The cytosol of
root tip cells seems to be more sensitive to changes in external supply of nitrate and this region of the root may be particularly important for sensing local nutrient availability.
487
F I G . 3. Comparison of the mean cytosolic nitrate activity in barley root
tip cells supplied with four different types of nitrogen supply. Barley seedlings (‘Klaxon’) were grown in hydroponics and supplied with nitrate at
concentrations of 10, 1, 0.1 and 0.01 mM as Ca(NO3)2. The plants were
maintained in a modified Hoagland’s nutrient solution and grown under
the environmental conditions described previously (van der Leij et al.,
1998). A two-way ANOVA and t-test were used to compare data and the
10 and 1 mM treatments were significantly different from the 0.1 and
0.01 mM treatments (P , 0.01). Root cells were impaled 1– 2 mm from
the tip and mean values + s.d. are shown. For each nitrate supply microelectrode measurements of between nine and 22 cells were obtained,
with each value taken from a similar number of roots.
The cells 1 – 2 mm from the root tip do not have fully developed vacuoles and are likely to represent zones of both cell
division and elongation. This observation is consistent with
the idea that the vacuolar nitrate pool provides a reservoir of
stored nitrate that is used to maintain the cytosolic nitrate
concentration (van der Leij et al., 1998). However, more
detailed root tip measurements are needed to track down
the relationship between vacuole formation and the capacity
to regulate cytosolic nitrate. Taken together these data
provide strong evidence for regulation of the cytosolic
nitrate pool and therefore homeostasis in mature cells, but
this idea is complicated by the fact that there are important
differences between cell types. This tissue heterogeneity
may be hidden within whole root analysis methods such
as compartmental analysis, tracer efflux and ‘omics’ technologies. For sensing nutrient status, specific tissues such
as the root tip may be very important.
HO MEOSTA SI S O F CY TO SOL IC N ITR AT E –
CO U L D IT B E A SI GN AL ?
If the concept of homeostasis of cytosolic nitrate is
accepted, under what conditions might it change and can
it provide a cellular signal? To demonstrate a role in signalling, changes in cytosolic nitrate must be associated with
alterations in nutrient status. Each cell must optimize its
nutrient supply to achieve maximal growth and utilize all
available nitrate and this involves changes in gene
expression and the activation state of proteins. The trigger
for these intracellular responses could be changes in
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Miller and Smith — Homeostasis of Cytosolic Nitrate
cytosolic nitrate concentration reflecting the variations in
external concentration. Such changes have been measured
in Arabidopsis leaves in response to alterations in light
supply (Cookson et al., 2005) and in roots when the
amino acid glutamine was supplied exogenously (Fan
et al., 2006). In these two examples the cytosolic nitrate
pools were measured in vacuolated cells and the changes
were closely correlated with nitrate reductase activity. An
increase in nitrate reductase activity was correlated with a
decrease in cytosolic nitrate concentration and the reverse
was also true. Furthermore, in both cases the changes
occurred slowly when compared with calcium signalling
transients, but the slow response times of the nitrateselective microelectrodes (typically 30 –40 s half-times;
Cookson et al., 2005) may be limiting these measurements.
The results in Fig. 3 provide evidence that cells near the
root tip, in a region of tissue where the vacuole has not
fully developed, show that cytosolic nitrate activity is
more likely to change in response to changes in external
supply. Taken together these results argue for homeostasis
and alterations in the steady state that may be associated
with changes in plant cell N status.
One consequence of this homeostasis may be a requirement for cytosolic nitrate in all cells. For example, specialized cells and tissues such as pollen and endosperm may
contain nitrate when there is no obvious nutritional requirement for this form of N. Seed pools of nitrate have been
measured and shown to be important for germination (e.g.
Alboresi et al., 2005) and an Arabidopsis nitrate transporter
with strong expression in desiccating seeds has been identified (AtNRT2.7; Chopin et al., 2007). These facts argue for
an important role of seed nitrate and yet it represents a tiny
proportion (,1 in 1000) relative to the organic N in seed
(Chopin et al., 2007). The presence of some nitrate in the
environment may be essential for plant cell growth although
it is difficult to test this idea by supplying only organic
nitrogen and totally removing the ion from nutrient
supplies. As nitrate is present as a contaminant in other
salts and these are supplied in the millimolar range it is
therefore still available at micromolar concentrations via
the high-affinity uptake systems present in plant cell membranes. Testing the ability of other anions, such as chloride,
to replace nitrate in the cytosol may be one way of investigating the requirement for nitrate in the cytosol. The toxic
effects of treatments with NaCl on plants are chiefly considered as Naþ replacing Kþ but may also be due to chloride replacing nitrate in the cytosol and this is a topic worthy
of further investigation. Nitrification, the production of
nitrate from ammonium by bacteria, is well known; the
ability of plants to carry out this process is difficult to
measure, but it has been suggested and may be important
for the signalling role of nitrate.
It has been suggested recently that shoot protein content
rather than NO2
3 acts as a signal to regulate dry matter partitioning between the shoot and root of higher plants
(Andrews et al., 2006). The shoot : root weight ratio is
used as an indicator of plant N status. When two cultivars
of rice with differing vegetative nitrogen use efficiencies
were compared, significant differences in steady-state leaf
cytosolic nitrate activity could be measured (Fan et al.,
2007). These results may indicate that it is this parameter,
rather than vacuolar nitrate (whole tissue nitrate), which
regulates shoot : root growth.
S E N S I N G AN D S O I L - AVA I L A B L E N
In temperate soils, N is mainly available to plants as nitrate
and its concentration can vary from a few micromols in
non-agricultural soils to several millimols after fertilizer
application (e.g. Miller et al., 2007). In laboratory experiments, one usually demonstrates the response of plants to
N by starving plants for several days and then resupplying
nitrate (e.g. Crawford, 1995). The treatment results in the
induction of genes and enzyme activities that are then
described as nitrate-induced (e.g. Wang et al., 2003).
However, in soil this situation is unlikely ever to occur
and plants must respond to changes in nitrate supply in
the environment to optimize nitrate uptake in order to
match supply with the demands of growth. Plants respond
to nitrate by altering gene expression for transporters and
assimilatory enzymes and by post-translational modifications to these same proteins (Miller and Cramer, 2004).
Plant root cells must therefore have some method not
only of sensing the presence of nitrate, but also of detecting
patches of relatively high concentration. The simplest
model to explain this would be for the cytosolic nitrate concentration of each cell to change in parallel with the external supply, and then changes in external nitrate could be
sensed in the cytosol or nucleus. Changes in cytosolic
nitrate concentration have been proposed as a possible feedback mechanism controlling uptake at the plasma membrane (King et al., 1993). This model may be refined as
the data shown in Fig. 3 suggest that the root tips, more generally perhaps the meristem, may be the particular sensing
region. As these changes in the external concentration
over the range 1 – 10 mM did not significantly change cytosolic nitrate activity (Fig. 3), there could still be a case for
an extracellular nitrate ‘sensor’ which responds in this range
(Redinbaugh and Campbell, 1991). Or perhaps roots are
insensitive to changes in this low-affinity range (see
Miller et al., 2007). During N deficiency the nitrate transporter proteins NRT1.1 and NRT2.1 seem to have a
sensing role and they are differentially expressed along
the root axis (Remans et al., 2006a, b) in a pattern that
may be important for maintaining cytosolic homeostasis.
CO N CLU DI NG R E MAR KS
These results support the idea that the cytosol is a carefully
regulated ionic environment with homeostasis not only of
pH, phosphate and calcium, but also of nitrate concentration. During alterations in environmental conditions,
cytosolic nitrate activity can change and these changes
might be a cellular signal. At low external concentrations
of nitrate below 1 mM in root tip cells where the vacuole
is not fully developed, cytosolic nitrate may be more sensitive to changes in supply. Finally, other single-cell methods
are needed to monitor cytosolic nitrate, and these may be
made available in the future through the development of
nitrate-sensitive dyes or fluorescence resonance energy
Miller and Smith — Homeostasis of Cytosolic Nitrate
transfer (FRET)-based systems for this ion (e.g. Looger
et al., 2005).
ACK NOW LED GE MENT
Rothamsted Research receives grant-aided support from the
Biotechnology and Biological Sciences Research Council
of the United Kingdom.
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