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- Materials and Methods
- Supporting Information
A vast amount (> 100 million tonnes) of nitrogen (N) fertilizer is applied to crops annually to maximize yield (FAO, 2006). However, in cereal production, only 40–50% of the applied N is actually taken up by the intended crop (Peoples et al., 1995; Sylvester-Bradley & Kindred, 2009). Given this low N uptake efficiency, we believe a better understanding of the N uptake process in cereals would help to identify the limiting factors contributing to poor N uptake efficiency and overall cereal nitrogen use efficiency (NUE). NUE, in this case, refers to grain yield per unit of available N in the soil (Moll et al., 1982; Dhugga & Waines, 1989; Good et al., 2004).
This study is focused on the uptake and use of nitrate (), as it is the predominant form of N in most high-input agricultural soils (Wolt, 1994; Miller et al., 2007). Plant uptake generally involves two types of transport system, one involving high-affinity (HATS) and the other low-affinity (LATS) transporters (Glass, 2003). In Arabidopsis, four transporters (NRTs) have been linked to uptake from the soil: NRT1.1 and NRT1.2 from the LATS class, and NRT2.1 and NRT2.2 from the HATS class (Tsay et al., 2007). NRT1.1 (Chl1) is unique among these in that it displays dual affinity towards depending on its phosphorylation status (Liu et al., 1999). Although we now have some fundamental knowledge about the functionality of these transporters, our understanding of their roles and of the regulation of uptake remains limited.
Certain aspects of the regulation of the Arabidopsis uptake system have been examined extensively. For example, the uptake capacity of HATS shows strong induction when plants are exposed to after a period of N starvation, and the uptake capacity is repressed following a period of sufficient (Minotti et al., 1969; Jackson et al., 1973; Goyal & Huffaker, 1986; Aslam et al., 1993; Henriksen & Spanswick, 1993; Zhuo et al., 1999). This strong induction and repression are reflected in the transcript levels of AtNRT2.1 and AtNRT2.2, which follow the induction and repression of the uptake capacity (Zhuo et al., 1999; Okamoto et al., 2003). Redinbaugh & Campbell (1993) referred to this pattern of induction and repression as the primary response. Whether this N response is relevant to longer time scales and to soil N characteristics of typical cropping soils has yet to be shown.
The relative roles of NRTs in the uptake of from the soil remain unclear, but circumstantial evidence has been used to postulate their activities. First, the concentration in agricultural soils is generally in the millimolar range (Wolt, 1994; Miller et al., 2007), well above the point at which the HATS system would be saturated (c. 250 μM) (Siddiqi et al., 1990; Kronzucker et al., 1995; Garnett et al., 2003). Second, the location of the transporters within a root suggests variable roles in uptake. AtNRT1.1 expression is localized in the tips of young roots (Huang et al., 1999; Guo et al., 2001), where roots first come into contact with the higher concentrations of unexplored soil, whereas AtNRT2.1 is localized in the cortex of older parts of the root, where external concentrations may be reduced following uptake at the root tip (Nazoa et al., 2003; Remans et al., 2006). Third, the pattern of NRT2 repression observed in roots exposed to sufficient N would seem to limit their relative importance to steady-state uptake in N-rich soils. Given this evidence, it has been proposed that the LATS system is most probably responsible for the majority of uptake from the soil (Glass, 2003).
Little is known about how uptake is actually managed over the lifecycle of the plant, with many studies on uptake focused on responses to perturbations, where external availability is varied in order to explore -dependent uptake responses. In one of the few published studies, Malagoli et al. (2004) measured the uptake capacity of the HATS and LATS in oilseed rape over time, and their response to various factors, and used this information, together with the modelling of field data, to suggest that HATS could supply most of the plants N requirements, even with high N availability. This work suggests that HATS are important in net uptake, necessitating a re-examination of the respective roles of these two transport systems. A detailed analysis of uptake capacity across the entire lifecycle is an important step towards the development of plants with enhanced N uptake capacity and efficiency, and may help to improve N fertilization practice where supply can be better matched to demand.
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- Materials and Methods
- Supporting Information
Across the lifecycle of Gaspe Flint, uptake capacity changed c. 10-fold irrespective of external N availability. This change was characterized by distinct peaks and troughs in uptake capacity, with a general trend towards decreased uptake capacity as plants grew to maturity, but correlation with plant N demand (Figs 2, 3). There was also clear evidence that uptake responded positively to reduced N supply, with increased uptake capacity in the lower N treatment (Fig. 2). The transcript profiles of the NRTs suggested that changes in uptake capacity, in response to supply and demand, were linked to changes in expression of the putative high-affinity NRTs ZmNRT2.1 and ZmNRT2.2. Their expression profiles, in response to N supply and time, provided strong correlative evidence of their in planta roles in uptake. When N supply was varied (N-inc or N-red), the commonality in change to ZmNRT2.1 and ZmNRT2.2 transcript levels and associated change in flux capacity further supported this role. We believe that the highly dynamic nature of N acquisition displayed here and the strong relationship to N provision provide new insights into the regulation of uptake which may lead to the manipulation of N uptake efficiency and, ultimately, NUE in plants.
uptake capacity responding to demand
The uptake capacity was extremely variable across the lifecycle. It has long been suggested that the growth rate determines the N uptake rate (Clement et al., 1978; Lemaire & Salette, 1984; Clarkson et al., 1986). The data presented here support this hypothesis, with the relative differences in growth rate between shoots and roots leading to variability in N demand and changes in uptake capacity (Figs 1, 3). In both treatments, we showed that uptake capacity increased with peaks in shoot growth and, consequently, N demand, but also decreased rapidly when shoot growth decreased, creating a characteristic trough in uptake capacity (Figs 1, 3). We propose that, during this period, the plants grown in 0.5 mM were responding to N limitation and it was plasticity in uptake capacity (HATS) that allowed sufficient N uptake to match the growth rate of the plants grown in 2.5 mM . This plasticity is highlighted by the rapid changes in uptake capacity observed in plants that were changed between treatments.
Transcript levels of ZmNRTs
The measurement of unidirectional influx at 50 and 250 μM was chosen to describe the uptake capacity of the HATS. Based on reliable estimates from the literature, the HATS for most plants are saturated at c. 250 μM (Siddiqi et al., 1990; Kronzucker et al., 1995; Garnett et al., 2003). Given the relatively high concentrations, at least in the 2.5-mM treatment, which were well above the point at which HATS would be saturated, it was anticipated that LATS would be responsible for much of the uptake. We also expected that there would be little variability in HATS activity based on the steady-state conditions in which we grew the plants, where constitutive HATS (cHATS) activity would be predicted to dominate, and induced HATS (iHATS) would be repressed after continued exposure to . However, this was not the case in either treatment, as evidenced by the influx analysis described above, and in the expression patterns of the NRT gene families, where the HATS responded intimately to supply and demand.
Previous evidence has suggested that HATS transcript levels are generally negatively regulated when N levels are high (e.g. 0.5–2.5 mM ) (Filleur et al., 2001; Okamoto et al., 2003, 2006; Santi et al., 2003; Liu et al., 2009). However, in this study, we found the opposite, where the baseline transcript levels of ZmNRT2.1 and ZmNRT2.2 were generally much higher than for any of the other transporters, regardless of the external N supply. Following the paradigm suggested by Glass (2003), the role of the HATS system is to acquire only when soil solution concentrations are low, well below the consistent levels of 0.5 or 2.5 mM used here. However, the high abundance of ZmNRT2.1 and ZmNRT2.2 transcripts, independent of the external N supply, suggests alternative roles for these gene products.
The high level of transcripts of the two putative HATS (ZmNRT2.1 and ZmNRT2.2) contrasts with the low transcript levels observed for the putative LATS, the ZmNRT1s, across the lifecycle. Despite differences in the abundance of LATS and HATS transcripts, there were some parallels in the expression patterns, particularly during the initial peak in uptake capacity (Figs 5, S5). These data support previous reports (Ho et al., 2009) of a possible link between NRT1 and NRT2 transport systems, although, in maize, the relationship may only extend to the early vegetative stage in which uptake capacity is at its maximum. Although the transcript levels of ZmNRT2.5 were very low, the observation that transcripts were only detected in the reduced treatment suggests that this putative transporter may play an important role in low N responses.
The delivery of into the xylem in Arabidopsis has been suggested to involve the NRT AtNRT1.5 (Lin et al. 2008). Unlike other ZmNRT1 genes, ZmNRT1.5A showed a similar transcript profile to ZmNRT2.1/2.2 and was responsive to the 0.5-mM treatment, this being consistent with a possible role in loading into the xylem in maize.
The transcript levels of ZmNRT3.1A were closest in terms of absolute levels to ZmNRT2.1/2.2. There is good evidence that AtNRT3.1 is essential to the function of the AtNRT2s (Okamoto et al., 2006; Orsel et al., 2006; Wirth et al., 2007). Based on transcript levels and the similarity in pattern across the lifecycle, this would also seem to be true for the maize homologues.
The regulation of uptake capacity
There is a correlation between the uptake capacity of HATS and the transcript levels of both ZmNRT2.1 and ZmNRT2.2. This has been found in plants other than maize, and has been proposed as evidence of the involvement of NRT2s in uptake (Forde & Clarkson, 1999; Lejay et al., 1999; Zhuo et al., 1999; Okamoto et al., 2003). Combined with the impairment of uptake associated with reduced transcript levels in Arabidopsis AtNRT2.1 and AtNRT2.2 knockout mutants (Filleur et al., 2001), this led to the proposal that uptake via AtNRT2.1 and AtNRT2.2 is regulated at the transcriptional level. However, transcript levels may not equate to levels of functional protein. Wirth et al. (2007) suggested that the NRT2s in Arabidopsis are long-lived proteins, and showed that the level of AtNRT2.1 protein was independent of transcript level or changes in uptake capacity, suggesting that there is considerable post-translational control of NRT2-mediated uptake.
The results presented here are compatible with a model that combines both transcriptional and post-translational control of uptake capacity (Fig. 9). In this model, the total concentration of ZmNRT2.1 and ZmNRT2.2 protein is predicted to be proportional to the sum of the ZmNRT2.1 and ZmNRT2.2 transcript levels at any given day plus, based on an estimated protein lifespan of NRT2 proteins of c. 5 d (Wirth et al., 2007), the sum of the transcript levels for the previous 4 d. This 5-d lifespan is based on Wirth et al. (2007), but estimates with a range of lifespans are shown in Fig. S7. This estimated protein concentration represents the maximal uptake capacity of NRT2.1 and 2.2 at a given day, the actual uptake capacity being dependent on the amount of post-translational inhibition, which could be through allosteric inhibition, phosphorylation or, given the results of Yong et al. (2010), perhaps a result of NRT2/NRT3(NAR2) complexes being removed from the plasma membrane.
As presented in Fig. 9, this model predicts that, up to day 15, the uptake capacity is equal to the potential uptake capacity, after which the actual uptake capacity measured is then reduced and becomes less than the potential uptake capacity. At day 22, the measured uptake capacity increases through the utilization of the potential uptake capacity without a transcriptional response. This changes at day 27 where, based on our model, the NRT2 protein levels are insufficient to provide the required uptake capacity, this leading to the transcriptional peak observed at day 29. In terms of the plants moved from 2.5 to 0.5 mM at day 15, the initial increase in uptake capacity seen at day 18 in Figs 4(b), 9(b) would be the result of a release of post-translational inhibition, and hence increased uptake capacity without a comparable increase in transcript levels (Fig. 6). The peak in NRT2.1 transcript levels at day 25 would be caused by the number of NRT2.1 proteins in these plants previously exposed to a much higher concentration not providing sufficient uptake capacity, even with no post-translational inhibition. This model predicts that transcription will provide the long-term regulation of uptake capacity, with short-term uptake capacity regulated via the post-translational regulation of the existing transport capacity, this short-term regulation being important for N homeostasis.
The current model of the regulation of uptake by the plant N status (tissue concentration of itself or a downstream assimilate, such as amino acids) has been described in numerous reviews (Cooper & Clarkson, 1989; Imsande & Touraine, 1994; Forde, 2002; Miller et al., 2008; Gojon et al., 2009). The two-component model of uptake capacity regulation described above requires two triggers in its regulation, one a transcriptional trigger and another that determines the extent of post-translational inhibition. Given the major drop in transcript levels beginning at day 18 until day 22, it may be that the trigger for the transcriptional response is the root amino acid/ level, which increases and reaches a peak at day 22 (Figs 7, 8). The decrease in uptake capacity beginning at day 15, which we propose is caused by an increase in post-translational inhibition, could be triggered by shoot amino acid/ levels which peak at this point.
NUE increases through increased uptake capacity with reduced N availability