A model recently proposed by Siddiqi & Glass (Plant, Cell, and Environment 25, 1211–1217, 2002) attempts to reconcile discrepancies in the measurement of cytosolic nitrate concentrations ([NO3–]cyt) in plant root cells, specifically between low (∼ 4 mm) homeostatic values reported in studies using ion-specific microelectrodes on the one hand, and wide fluctuations in [NO3–]cyt reported in other studies, especially those using compartmental analysis by tracer efflux (CATE). Although Siddiqi & Glass concede that cytosolic NO3– homeostasis, as determined by microelectrodes, is at odds with certain experimental observations, they nevertheless promote a model that takes microelectrode readings at face value, and assert that the variations seen using CATE methodology are artefacts attributable to contributions from substantial, rapidly exchanging, and highly variable NO3– pools putatively residing in organelles such as plastids and the endoplasmic reticulum. We show here that such a model is not tenable, drawing upon experimental evidence from previous studies, and from a more comprehensive model that examines the characteristics and consequences of subcompartmented cytoplasmic exchange in root cells.
Unfortunately, although attractive, this hypothesis, and the classification and nomenclature associated with it, are unfounded. There is no factual basis, or apriori necessity, for the claim that techniques such as CATE fail to distinguish between cytosolic and organellar NO3– pools; nevertheless, Siddiqi & Glass take this claim as the basic premise of their arguments. The implications of such a statement are substantial, and must be challenged here. Some of the important issues at stake are: the thermodynamic gradients of NO3– across the plasma membrane as well as across internal membranes; substrate availability for nitrate reductase activity (often considered the limiting step in nitrate acquisition); the regulatory role of subcellular N pools in transport activity; the understanding of unique cellular properties of nitrate acquisition such as the induction and de-induction of transport and assimilation systems; and the more general understanding of cellular ionic interactions, given that nitrate can be one of the most abundant anions in the plant cell.
In this article, we will show that the classification of techniques into ‘group 1’ and ‘group 2’ methods requires reconsideration. More importantly, we will also show why the central claim of Siddiqi & Glass, about the errors associated with CATE studies and their results pertaining to variations in cytosolic NO3–, is not only not substantiated, but also fundamentally untenable.
The grouping of techniques into those that measure a purely cytosolic NO3– pool (group 1), and those that measure a combination of subcellular pools (group 2), is questionable. Several major flaws in this classification proposal are apparent, and will be discussed in turn here.
1Non-aqueous cell fractionation is considered by Siddiqi & Glass to be a group 2 technique, but, in fact, workers who use this technique go to substantial lengths to distinguish chemical pools in different subcellular organelles from one another, rather than presenting a combined reading. This is accomplished primarily by use of enzyme markers, following a well-established methodology (Martinoia, Heck & Wiemken 1981; Belton et al. 1985; Winter, Robinson & Heldt 1993, 1994). Surprisingly, in their only quantitative example, Siddiqi & Glass use ‘cytosolic’ values from one such study (in fact, these values are reported merely as ‘detection limits’ by personal communication in Winter et al. 1994), even after having stated that this technique, falling into group 2, does not distinguish ‘cytosolic’ from ‘cytoplasmic’ pools. It should be pointed out that for cellular compartments of small volume, such as the cytosol and the chloroplast stroma (as opposed to the vacuole) in many plant systems, even high nitrate concentrations would fall below the detection limits of this method (~30–46 mm in barley leaf cells), as would any variations in [NO3–]cyt occurring below this threshold. Detection limit values of this kind cannot be used in quantitative analyses such as presented by Siddiqi & Glass.
2A study based on 14N NMR methodology (Belton et al. 1985), on the other hand, was listed as using a group 1 technique. However, in this study, only a single NMR-spectrum peak for NO3– was observed in each plant system examined, and integration of this peak yielded values in excellent agreement with values for total tissue nitrate concentrations obtained from chemical analyses of plant tissue. Indeed, because of the large volume differences between cytosol and vacuole, cytosolic [NO3–] in mature maize and barley roots (plant systems comparable or identical to those used in studies with CATE and microelectrodes) could be as high as those in the vacuole (i.e. 15–90 mm!) and still remain undetectable by this method. Thus, as in the case of cell fractionation, listing this method as yielding ‘undetectable’ cytosolic NO3– levels is misleading, as it reflects more a limitation of sensitivity in technique than the lack of a substantial, and potentially variable, cytosolic NO3– pool.
A Siddiqi & Glass use detection-limit data reported in Winter et al. (1994; see above for a critique of this usage) to justify that the contribution of a plastidic nitrate pool could potentially double the estimates of what would be assumed to be a purely cytosolic pool obtained by CATE. However, although it is indeed well established that millimolar concentrations of NO3– can build up in chloroplasts (Schröppel-Meier & Kaiser 1988a, b), no such pools have been shown to exist in root proplastids (the pertinent compartment to be considering here), nor is there any reason to expect the existence of rapidly exchanging, high-capacity, nitrate storage functions in these organelles. Similarly, no such pools have been demonstrated to be associated with the endoplasmic reticulum, dictyosomes, nuclei, or any other major organelle in higher plant roots, other than the vacuole (Martinoia et al. 1981). Moreover, the requirement of the above proposal, that all variations in ‘cytoplasmic’ nitrate be due solely to variations in organellar pools, is unlikely to be true even in the case of chloroplasts; in fact, data from a significant number of studies estimating nitrate pools in the chloroplast, under various conditions, are remarkably consistent (typically 2–10 mm), and, importantly, these concentrations do not appear to vary as a function of external nitrate supply. In particular, Schröppel-Meier & Kaiser (1988a, b) have shown in spinach that, even when the external nitrate concentration is stepped up from 15 to 300 mm, the internal concentration in chloroplasts isolated from this material appears homeostatically held at ∼ 5 mm. This would render unlikely the possibility that plastids are responsible for the well-documented variability of ‘cytoplasmic’ nitrate pools, as determined by CATE and other methods. Interestingly, chloroplasts also act as a major storage pool for potassium, with stromal concentrations typically reaching double those found in the cytosol (Robinson & Downton 1984; Schröppel-Meier & Kaiser 1988a, b). Siddiqi & Glass use such considerations to explain why higher ‘cytoplasmic’[K+] values were found by Memon, Saccomani & Glass (1985) using CATE with 86Rb+ (a problematic tracer for K+– see Jeschke 1970; Jacoby, Abas & Steinitz 1973), than were found by Walker, Leigh & Miller (1996) using potassium-selective microelectrodes. However, our own extensive studies (unpublished), using CATE with 42K+ (an appropriate tracer, not afflicted with isotope discrimination), contradict this, having yielded cytosolic [K+] values closely in line with those determined for the same species (barley) in the study by Walker et al. (1996; see also Hajibagheri et al. 1988).
B A consequence of the proposal by Siddiqi & Glass, although not discussed explicitly by the authors, is that the presence of substantial, rapidly exchanging, extra-cytosolic nitrate pools renders the accumulation and release of tracer to and from the cytosol a non-exponential set of processes. Figure 1 shows how the patterns of labelling and release for the cytosol and a putative extra-cytosolic compartment are deflected from simple exponentiality, assuming that the nitrate contents in the cytosol and this compartment are equal, and that the fluxes across the plasma membrane and across the membrane separating the cytosol from the extra-cytosolic compartment are also equal. Not only do none of the four processes of labelling or elution obey first-order kinetics (see, for instance, Fig. 1, inset), but the apparent exchange ‘half-times’ falsely derived from efflux plots in such situations could be substantially longer than the apparent ‘half-times’ of the labelling process. Consequently, estimates of the specific activity of compartments within the cell that are releasing tracer to the external medium could be severely erroneous and, in turn, the widely used practice of tracer efflux analysis to determine proper loading and washing time courses for influx experiments would be invalidated. Although the present authors do not endorse this view, because such deviations from exponentiality have never been experimentally observed, we wish to point out that it is a necessary outcome of the proposal by Siddiqi & Glass. Only in the presence of very large fluxes between the cytosol and the extra-cytosolic compartment(s) would an approximation to exponentiality, and an equality between apparent labelling and elution half-times, take place, a condition that we will now address.
C The reconciliation offered by Siddiqi & Glass between invariable ‘cytosolic’ readings by microelectrode methods, and variable ‘cytoplasmic’ readings by other methods, rests upon the assumption that any nitrate-pool variation observed occurs exclusively inside the extra-cytosolic compartment(s). Modelling the actual magnitude of variation seen experimentally (see Kronzucker, Glass & Siddiqi 1999 for a detailed data set on barley, used here for modelling purposes), this assumption requires that the apparent half-times of exchange made visible in tracer elution profiles must systematically change with external nitrate supply (and thus with the filling state of the extra-cytosolic compartment), unless the fluxes operating at the membranes bounding these extra-cytosolic compartments are of an unprecedentedly high magnitude (Fig. 2). Such changes in apparent ‘cytoplasmic’ NO3– half-times, however, have never been experimentally observed (see Britto & Kronzucker 2001; interestingly, the resilience of the half-time parameter across many conditions of NO3– provision is also acknowledged by Siddiqi & Glass). Comparing actual experimental values (from Kronzucker, Glass & Siddiqi 1999) for NO3– influx, half-time, and ‘cytoplasmic’ pool size in uninduced barley plants and plants cultured at a steady state of 10 mm NO3–, Fig. 2 shows that to account for the 38-fold increase in the ‘cytoplasmic’ pool size, fluxes of ∼ 800 µmol g−1(FW) h−1 would have to prevail across the extra-cytosolic compartment membranes to obtain a half-time commensurate with the one actually observed. Such large fluxes are without precedent in the plant ion transport literature, and, if they exist, should be the target of significant attention amongst transport physiologists in search of high-capacity NO3– membrane transporters capable of abstracting nitrate at enormously variable rates from a non-changing substrate pool (as implied by cytosolic NO3– homeostasis). However, until demonstrated, such flux events remain purely speculative.
4Although Glass & Siddiqi place CATE in ‘group 2’ without any evidence to support this categorization, they also place microelectrode methods in ‘group 1’, again without substantiation. If it is indeed true that substantial NO3– pools exist in plant cell compartments other than the cytosol or vacuole, it is plausible that microelectrodes, which at present are designed to only distinguish cytosol from vacuole, might furnish readings from such other compartments.
It emerges clearly that, both on experimental and theoretical grounds, the proposal by Siddiqi & Glass, which attempts to reconcile NO3– pool measurements obtained by microelectrodes and by CATE, is not tenable, and alternative explanations of the reported discrepancies must be sought. We here suggest that the lack of variability of NO3– pools in the cytosol as measured by the microelectrode method, in the face of substantial variations found by all other major methods, should give rise to doubt as to the veracity of the method in this particular context. Although it is true that microelectrodes afford some confidence because of the directness of the measurements involved, and indeed have provided subcellular pool size values for some ions (e.g. K+) that are in agreement with other methods, it should be kept in mind that electrical signals collected in such systems are not nearly as straightforward to interpret as is often suggested. Indeed, one leading expert in microelectrodes emphasized in a review on calcium electrodes, that work of this nature is ‘notoriously difficult, and prone to many pitfalls’ (Felle 1989). These problems include interference by ions other than the one of interest (Cuin et al. 1999; Carden, Diamond & Miller 2001), signal clarity and stability (Mertz & Higinbotham 1976; Beilby & Blatt 1986; see also Felle 1989), and calibration (Coombs, Miller & Sanders 1994; Miller & Smith 1996). Another problem, rarely considered, is the likelihood that the invasive nature of an electrode impalement will cause substantial ionic efflux from the cell compartment under observation (as is known for other kinds of physical perturbations; see Forde & Clarkson 1999), which could substantially influence the reading. The fact that NO3– readings obtained by microelectrodes appear to remain stable regardless of the level of NO3– supply (including the condition of prolonged NO3– withdrawal during which de-induction of nitrate transport and assimilation have been documented − see Siddiqi et al. 1989) adds additional uncertainty to nitrate-specific microelectrode methodology. This is particularly true given that the undiminished presence of NO3– as an induction signal, as implied by microelectrode results, challenges our understanding of the induction and de-induction of NO3– transport and assimilation (see, e.g. Forde & Clarkson 1999; Tischner 2000). CATE, on the other hand, has yielded eminently sensible results in this context (see, e.g. Kronzucker, Siddiqi & Glass 1997). It should also be noted that CATE has yielded compelling results regarding ecophysiological differences in nitrate acquisition among numerous plant species, especially trees (Kronzucker, Glass & Siddiqi 1995; Kronzucker, Siddiqi & Glass 1995, 1997; Min et al. 1999; Kronzucker et al. 2003), which reflect well-established differences in adaptations to ecological N niches. Until equally detailed studies are available from microelectrode work, dismissal of CATE appears premature, and is difficult to justify scientifically.
Received 11 September 2002;received inrevised form 11 October 2002;accepted for publication 14 October 2002