Research ArticleResearch Article

Maize NPF6 Proteins Are Homologs of Arabidopsis CHL1 That Are Selective for Both Nitrate and Chloride

Zhengyu Wen, Stephen D. Tyerman, Julie Dechorgnat, Evgenia Ovchinnikova, Kanwarpal S. Dhugga, Brent N. Kaiser

Published October 2017. DOI: https://doi.org/10.1105/tpc.16.00724

Abstract

Nitrate uptake by plant cells requires both high- and low-affinity transport activities. Arabidopsis thaliana nitrate transporter 1/peptide transporter family (NPF) 6.3 is a dual-affinity plasma membrane transport protein that has both high- and low-affinity functions. At-NPF6.3 imports and senses nitrate and is regulated by phosphorylation at Thr-101 (T101). A detailed functional analysis of two maize (Zea mays) homologs of At-NPF6.3 (Zm-NPF6.6 and Zm-NPF6.4) showed that Zm-NPF6.6 was a pH-dependent nonbiphasic high-affinity nitrate-specific transport protein. By contrast, maize NPF6.4 was a low-affinity nitrate transporter with efflux activity. When supplied chloride, NPF6.4 switched to a high-affinity chloride selective transporter, while NPF6.6 had only a low-affinity chloride transport activity. Structural predictions identified a nitrate binding His (H362) in NPF6.6 but not in NPF6.4. Mutation of NPF6.4 Tyr-370 to His (Y370H) resulted in saturable high-affinity nitrate transport activity and nitrate selectivity. Loss of H362 in NPF6.6 (H362Y) eliminated both nitrate and chloride transport. Furthermore, alterations to Thr-104, a conserved phosphorylation site in NPF6.6, resulted in a similar high-affinity nitrate transport activity with increased Km, whereas equivalent changes in NPF6.4 (T106) disrupted high-affinity chloride transport activity. NPF6 proteins exhibit different substrate specificity in plants and regulate nitrate transport affinity/selectivity using a conserved His residue.

INTRODUCTION

Nitrogen is required for plant growth and development. Nitrogen deficiencies in the soil can limit plant growth and harvestable yields and are often corrected through the application of nitrogen fertilizers, which total ∼119 million metric tons per year (FAO, 2014a). Maximizing the efficient use of nitrogen is a global pursuit because most agricultural crops display poor nitrogen use efficiencies, using only 25 to 50% of the applied nitrogen (Dhugga and Waines, 1989; Garnett et al., 2009; Moose and Below, 2009). Unused nitrogen fertilizers are mostly lost through leaching and runoff as well as escape into the atmosphere in the form of gases after denitrification by soil bacteria, and this leads to environmental pollution (Dhugga and Waines, 1989; Ramos, 1996; Stulen et al., 1998; Giles, 2005). To enhance the nitrogen use efficiency of crops, it is important to understand the mechanisms controlling nitrogen acquisition and utilization so that the overall genetic capacity for these traits can be maximized.

In most agricultural soils, microbial-mediated nitrate production by nitrification of reduced forms of nitrogen ensures that nitrate is the predominant ion available to plant roots (Dhugga et al., 1988; Garnett et al., 2009). Higher plants have multiple classes of nitrate transport protein families: nitrate transporter 1/peptide transporter family (NPF), nitrate transporter 2 family (NRT2), chloride channel family (CLC), and slow anion channel-associated homologs (Krapp et al., 2014). In general, nitrate uptake into plant roots involves two predominant physiological systems. At low external nitrate concentrations, a high-affinity transport system (HATS) is functional, which displays typical Michaelis-Menten saturation kinetics and operates at external concentrations of <0.25 mM. At higher concentrations, a low-affinity transport system (LATS) becomes active, where uptake increases linearly as external concentrations increase (Glass et al., 2002). In Arabidopsis thaliana and barley (Hordeum vulgare), HATS activity has been associated with the NRT2 class of nitrate transporters, where At-NRT2.1, 2.2, 2.4 and Hv-NRT2.1 were shown to be responsible for nitrate uptake at low concentrations (<0.25 mM) (Crawford and Glass, 1998; Tong et al., 2005; Li et al., 2007; Kiba et al., 2012). At higher concentrations, members of the NPF family together with NRT2 activities are responsible for net nitrate transport.

At-NPF6.3 has also been known as chlorate resistant1 (CHL1) or nitrate transporter1 (NRT1.1) and is the most studied member of the NPF family. It was first identified through a mutational genetic screen in Arabidopsis for resistance to chlorate, an analog of nitrate (Tsay et al., 1993). It has since been shown to be a dual-affinity transporter that transports nitrate in both the HATS and the LATS concentration ranges. This mechanism is believed to be regulated by the phosphorylation status of a conserved Thr residue (Thr-101) (Liu et al., 1999; Liu and Tsay, 2003). At-NPF6.3 is also considered to be a plasma membrane-localized nitrate transceptor, a transporter-substrate complex that transduces signals to the inside of a cell, where it participates in the primary nitrate signaling response in plant cells (Remans et al., 2006; Ho et al., 2009). In 2014, two structural models of At-NPF6.3 suggested contrasting roles of T101. Phosphorylation of Thr-101 either influenced its dimerization, a key process required for its affinity switch from high to low transport affinity, or altered its structural flexibility within the membrane, influencing nitrate transport activity (Parker and Newstead, 2014; Sun et al., 2014).

Maize (Zea mays) is a cereal crop predominantly cultivated in aerobic soils where nitrate is often the primary source of nitrogen available for growth. The annual global maize production is reported to be more than 109 million metric tons per year (FAO, 2014b). Although many physiological studies have investigated nitrate transport and assimilation in maize, little is known about the functional activity of individual maize nitrate transport proteins (Plett et al., 2010). In particular, it is not known whether some transporters possess multiple specificities for various anions.

Here, we report the characterization of two maize (Zm) homologs of Arabidopsis (At) NPF6.3: Zm-NPF6.4 and Zm-NPF6.6 (Plett et al., 2010). Both of these homologs are expressed in root and shoot tissues, with Zm-NPF6.6 responding to nitrogen provision. Zm-NPF6.4 and Zm-NPF6.6 transport nitrate and chloride at elevated concentrations. At low concentrations, Zm-NPF6.6 transports nitrate and Zm-NPF6.4 chloride. A predicted nitrate binding site (His-362), was found in Zm-NPF6.6 but not in Zm-NPF6.4 (Y370). Mutagenesis experiments revealed that His-362 is essential for nitrate transport. Conversion of Tyr-370 to a His residue in Zm-NPF6.4 (Y370H) created a high-affinity nitrate transport activity similar to that of At-NPF6.3, but eliminated chloride transport in a nitrate-dependent manner. The role of the conserved threonine residues, Thr-106 (Zm-NPF6.4) and Thr-104 (Zm-NPF6.6), as affinity switch mechanisms is less clear. Modification of Thr-104 in Zm-NPF6.6 to Ala or Asp resulted in a phosphorylation-independent but Thr-dependent control of nitrate affinity and transporter activity with no clear change between high- and low-affinity activity states. Alterations to Thr-106 of Zm-NPF6.4 to Ala or Asp disrupted high-affinity chloride transport in a phosphorylation independent manner. The phosphorylation-dependent affinity switch mechanism for nitrate transport, previously characterized for At-NPF6.3, is not evident in these maize homologs.

RESULTS

Cloning and Sequence Analyses of Maize NPF6.4 and NPF6.6

Zm-NPF6.4 (GRMZM2G086496_T01) and Zm-NPF6.6 (GRMZM2G161459_T02) full-length cDNA clones were isolated using high-fidelity RT-PCR based on the sequence information at http://ensembl.gramene.org/Zea_mays/Info/Index. Both Zm-NPF6.4 and Zm-NPF6.6 are predicted to contain four exons and three introns, encoding proteins with 608 and 595 amino acids, respectively (Supplemental Figure 1A). Zm-NPF6.4 and Zm-NPF6.6 are members of the recently annotated NPF6 subfamily (Léran et al., 2014). Most of the NPF6 members are thought to be nitrate transporters (Chiu et al., 2004; Morère-Le Paven et al., 2011), including the first identified nitrate transceptor At-NPF6.3 (formerly At-NRT1.1 or CHL1) (Tsay et al., 1993; Liu et al., 1999; Liu and Tsay, 2003; Ho et al., 2009). The predicted protein sequences of Zm-NPF6.4 and Zm-NPF6.6 are 67% identical at the amino acid level and they both share 62% identity with At-NPF6.3. Several key residues are conserved among Zm-NPF6.4, Zm-NPF6.6, and At-NPF6.3 (Supplemental Figure 1B), including the affinity switch residue Thr-101, which in Zm-NPF6.4 is at position 106 and in Zm-NPF6.6 at position 104 (Liu and Tsay, 2003), and the proton binding motif EXXER and the salt bridge residues L164 and E476 (L172 and E493 in Zm-NPF6.4 and L169 and E479 in Zm-NPF6.6), which are important for the transport mechanism and the functional structure of At-NPF6.3 (Parker and Newstead, 2014; Sun et al., 2014). Like other characterized members of the NPF6 subfamily, Zm-NPF6.4 and Zm-NPF6.6 are predicted to contain 12 transmembrane domains and an ∼77-amino-acid-long cytoplasmic hydrophilic loop between transmembrane domains 6 and 7 (Supplemental Figure 1B).

NPF6.4 and NPF6.6 Are Nitrate Transporters

To characterize the nitrate transport properties of Zm-NPF6.4 and Zm-NPF6.6, purified cRNA of each gene was expressed in Xenopus laevis oocytes and analyzed using the two-electrode voltage clamp technique (Baumgartner et al., 1999). Zm-NPF6.4 and Zm-NPF6.6 cRNA-injected oocytes, but not water-injected controls, showed significant voltage-dependent inward currents in 10 mM external nitrate (pH 5.5) relative to a nitrate-free (but anion balanced) solution (Figures 1A and 1B). Inward nitrate elicited currents in both Zm-NPF6.4- and Zm-NPF6.6-injected oocytes were significantly reduced at alkaline pH (pH 7.5; Figures 1B and 1C). These pH-dependent currents are consistent with NPF proteins acting as proton-coupled nitrate transporters (Wang et al., 2012).

Figure 1.
Figure 1.

Nitrate Transport Activity of Maize NPF6.4 and NPF6.6.

(A) to (C) Nitrate-elicited current recordings in oocytes injected with water (A), NPF6.4 (B), or NPF6.6 (C). ΔI is the substrate-elicited current representing the current change following the switch from the basal solution to the nitrate solution. n = 6 oocytes.

(D) and (E) High-affinity (0.25 mM) and low-affinity (10 mM) nitrate transport activity of NPF6.4 and NPF6.6. n = 9 oocytes. For all the flux experiments, flux time is optimized to 1 h (Supplemental Figure 10).

(F) Nitrate efflux activity of NPF6.4 and NPF6.6. Efflux (retained 15N) was measured using 1 M 15NO3-injected oocytes (final internal oocyte concentration, 10 mM) placed in an efflux bath (ND96, pH 7.5) for 30 min. n = 8 oocytes.

(G) Kinetic analysis of NPF6.4 nitrate transport activity. Net nitrate uptake values of NPF6.4-injected oocytes from 25 μM to 30 mM (pH 5.5) were fitted by a linear regression (R2 = 0.94). Uptake values at low concentrations are shown in the figure insets. n = 9 to 10 oocytes.

(H) to (K) Kinetic analysis of nitrate transport activity of NPF6.6. Net nitrate uptake values were fitted by either a Michaelis-Menten plot (R2 = 0.95) (H) or a double reciprocal plot (I). As a comparison, net nitrate uptake values in the high-affinity range (from 25–300 μM) were fitted by a Michaelis-Menten curve in (J) and (K), and values in the low-affinity range (from 300 μM to 20 mM) were fitted either by a Michaelis-Menten curve (J) or by linear regression (R2 = 0.87) (K). n = 5 oocytes.

All values indicate means ± se. In (D) to (F), significance was determined using a one-way ANOVA (Supplemental File 1). In (G) to (K), data represent net nitrate uptake values with water-injected control values subtracted.

A unique characteristic of selected members of the NPF family is their ability to transport nitrate at both low and high concentrations. In plants, this trait has been observed in both Arabidopsis (At-NPF6.3) and Medicago truncatula (Mt-NPF6.8) (Liu et al., 1999; Morère-Le Paven et al., 2011). Similar to At-NPF6.3, Zm-NPF6.6-injected oocytes accumulated 15N-labeled nitrate at both low (0.25 mM) and high (10 mM) concentrations (Figures 1D and 1E). In contrast, Zm-NPF6.4 showed little high-affinity transport activity, whereas the rate of nitrate uptake in the low-affinity range (10 mM) was similar to that of At-NPF6.3 and half that of Zm-NPF6.6 (Figures 1D and 1E). The lack of high-affinity nitrate transport in Zm-NPF6.4 but not Zm-NPF6.6 was clearly evident across an extended range of nitrate concentrations, from 25 to 30 mM (Figures 1G to 1K). Nitrate uptake by Zm-NPF6.4 was found to be linear against increasing nitrate concentrations (0–30 mM, R2, 0.99). By contrast, Zm-NPF6.6 displayed clear saturable transport kinetics (Km, 210 µM, R2, 0.95) when using a Michaelis-Menten curve fit across the entire concentration range (Figure 1H).

We further tested this relationship using a double-reciprocal plot (Figure 1I) with a linear fit (R2, 0.99) and by separating the high (25–300 μM) and low-affinity (300 μM to 20 mM) ranges and plotting them with either a Michaelis-Menten curve fit across the high (Figures 1J and 1K; R2, 0.99) and low (Figure 1J; R2, 0.64) affinity ranges or with a linear regression across the low-affinity range (Figure 1K; R2, 0.87). The strength of the line fit in double reciprocal plot suggests that Zm-NPF6.6 operates in a monophasic manner across a wide range of concentrations with minimal shift between the high- and low-affinity activities, unlike the previously observation in At-NPF6.3 (Liu et al., 1999). Recent reports have indicated that At-NPF6.3 may also behave as a bidirectional transporter involved in nitrate efflux (Léran et al., 2013). We measured nitrate efflux from 15N-nitrate injected oocytes (initial internal nitrate concentration was 10 mM). After a 30-min efflux period into ND96 buffer (pH 7.5), Zm-NPF6.4-injected oocytes and the control, At-NPF6.3-injected oocytes displayed significant (P < 0.05) nitrate efflux compared with the water-injected control (Figure 1F), while Zm-NPF6.6 nitrate efflux was evident but not significant. However, Zm-NPF6.6 did display small outward currents (Figure 1C; pH 7.5), which could indicate a potential efflux activity.

NPF6.4 and NPF6.6 Also Transport Chloride

Electrophysiological studies in oocytes indicated that in solutions without nitrate, both Zm-NPF6.4 and Zm-NPF6.6 induced a shift in reversal potential relative to those injected with water (Supplemental Figure 2). We determined this shift was attributable to the presence of 1 mM chloride. We then tested whether chloride was also a substrate for both the Zm-NPFs. Using a 5 mM chloride-containing basal solution (pH 5.5), we observed chloride-elicited voltage-dependent inward currents in both Zm-NPF6.4- and Zm-NPF6.6-injected oocytes but not in the water-injected controls (Figure 2A). We then tested whether the electrical currents represented actual chloride flux into the oocytes. We found that 36Cl− uptake (1 mM, pH 5.5) occurred in both the Zm-NPF6.4- and Zm-NPF6.6-injected oocytes (Figure 2B). In the HATS activity range, Zm-NPF6.4-injected oocytes accumulated 36Cl− in a saturable manner (Km 390 µM; Figure 2C), while Zm-NPF6.6 uptake was predominantly linear (R2, 0.81; Figure 2D). The influx of 36Cl− was pH-dependent for both transporters, increasing at pH 5.5 and declining at the alkaline pH of 7.5 (Figure 2B), suggesting that chloride was transported along with protons.

Figure 2.
Figure 2.

Chloride Transport Activity of Maize NPF6.4 and NPF6.6.

(A) Chloride-elicited current recordings in oocytes injected with water, NPF6.4, or NPF6.6. ΔI is the substrate-elicited current representing the current change following the switch from the basal solution to the chloride solution. n = 5 oocytes.

(B) pH-dependent chloride transport activity of NPF6.4 and NPF6.6. n = 8 oocytes.

(C) and (D) Kinetic analysis of NPF6.4 and NPF6.6 chloride transport activity. n = 8 oocytes.

All values indicate means ± se. In (B), significance was determined using an unpaired t test. In (B) to (D), data represent net chloride uptake values with water-injected control values subtracted.

To examine the relationship between NO3− and Cl− transport in Zm-NPF6.4 and Zm-NPF6.6, we tested whether the two anions could compete with each other. For Cl− competition of 15NO3− uptake, we chose a nitrate concentration of 10 mM, where we previously showed both Zm-NPF6.4 and Zm-NPF6.6 proteins display nitrate transport activity (Figure 1D). When Cl− was added across a series of increasing Cl−/15NO3− (from 0% to 100%), Cl− significantly reduced the uptake of 15NO3− in Zm-NPF6.4-injected oocytes but had little impact on the Zm-NPF6.6-injected oocytes (Figure 3A). Conversely, NO3− was tested as a competitor to 36Cl− uptake. NO3− had little impact on 36Cl− uptake in Zm-NPF6.4 (Figure 3B), while NO3− had a significant impact on Zm-NPF6.6 (Figure 3B). The reduction of either nitrate or chloride uptake in Zm-NPF6.4- or Zm-NPF6.6-injected oocytes was not linked to the presence of sodium ions in the uptake buffers (Supplemental Figure 3). These results indicate that both transporters can transport either chloride or nitrate but Zm-NPF6.4 exhibits a chloride selectivity, while Zm-NPF6.6 is selective for nitrate.

Figure 3.
Figure 3.

Substrate Selectivity of Maize NPF6.4, NPF6.6, and NPF6.4:Y370H.

(A) Chloride selectivity in NPF6.4. Nitrate uptake values were fitted by linear regressions (y = −0.193x + 2.651 nmol h−1 oocyte−1 for Zm-NFP6.4, y = 0.006x + 4.088 nmol h−1 oocyte−1 for Zm-NFP6.6, and y = 0.003x + 2.537 nmol h−1 oocyte−1 for Zm-NFP6.4:Y370H). n = 8 oocytes.

(B) Nitrate selectivity in NPF6.6 and NPF6.4:Y370H. Chloride uptake values were fitted by linear regressions (y = 0.101x + 1.727 nmol h−1 oocyte−1 for Zm-NFP6.4, y = −1.213x + 1.712 nmol h−1 oocyte−1 for Zm-NFP6.6, and y = −1.478x + 2.776 nmol h−1 oocyte−1 for Zm-NFP6.4:Y370H). n = 7 oocytes.

Data represent net substrate uptake values with water-injected control values subtracted. Values indicate means ± se.

Maize NPF6s Transport Activity Is Not Regulated by Thr Phosphorylation

One of the interesting characteristics of the dual-affinity transceptor, At-NPF6.3, is the apparent regulation through its phosphorylation status. Following T101 phosphorylation, At-NPF6.3 becomes a high-affinity nitrate transporter, and when T101 is dephosphorylated, the protein has only low-affinity nitrate transport activity (Liu and Tsay, 2003). This response is also replicated through a signaling cascade regulating the expression of the HATS nitrate transporter At-NRT2.1 (Ho et al., 2009). In Zm-NPF6.6, we identified a Thr residue (T104) in transmembrane domain 3 corresponding to At-NPF6.3 (T101) (Supplemental Figure 1B). To test whether the T104 played a similar regulatory role in Zm-NPF6.6, two mutant Zm-NPF6.6 variants were constructed and were expressed individually in X. laevis oocytes, where the phosphorylation effect of Zm-NPF6.6 was altered using site-directed mutagenesis. Using a similar approach to Liu and Tsay (2003), Zm-NPF6.6 (T104A) was created to disrupt phosphorylation and a mimic for a phosphorylation event was created with Zm-NPF6.6 (T104D). Both mutants retained the high-affinity nitrate transport activity, but the Km for nitrate was shifted to a lower concentration relative to the unmodified control, from 260 to 160 µM and 150 µM, respectively (Figure 4A). The Thr residue is also conserved in the Zm-NPF6.4 (T106). We created similar mutants (Zm-NPF6.4:T106A and Zm-NPF6.6:T104D) to test whether the phosphorylation status of this Thr affected its chloride transport activity. Interestingly, both T106A and T104D eliminated the saturable curve observed in Cl− uptake with flux activity becoming linear as external chloride concentrations increased (Figure 4B).

Figure 4.
Figure 4.

Threonine Phosphorylation and Transporter Activity of Maize NPF6.4 and NPF6.6.

(A) Kinetic analysis of NPF6.6, NPF6.6:T104A, and NPF6.6:T104D nitrate transport activity. n = 8 oocytes. Net nitrate uptake values of NPF6.6, NPF6.6:T104A, and NPF6.6:T104D-injected oocytes from 25 μM to 20 mM (pH 5.5) were fitted by the Michaelis-Menten equation. Uptake values at low concentrations are shown in the figure inset.

(B) Kinetic analysis of NPF6.4, NPF6.4:T106A, and NPF6.4:T106D nitrate transport activity. n = 7 oocytes.

R2, Km, and 95% confidence level range of Km values are listed in tables below graphs. Data represents net chloride uptake values with water-injected control values subtracted. Values indicate means ± se.

In summary, these results suggest that alterations to either T104 in Zm-NPF6.6 or T106 in Zm-NPF6.4 influence transport activity in a phosphorylation-independent but threonine-dependent manner. The shift to a lower Km for nitrate in Zm-NPF6.6 may suggest T104 is important to the active site of the protein influencing nitrate binding and consequently transport rate. The alterations to T106 in Zm-NPF6.4 was more pronounced, possibly through a modified chloride binding, eliminating the saturable chloride transport pathway (Figure 2C) while enhancing the linear uptake pathway observed in Zm-NPF6.6 (Figure 2D).

The Y370H Mutation in NPF6.4 Resulted in High-Affinity Nitrate Activity and Selectivity

Two independent structural studies of At-NPF6.3 suggested that H356 was the potential substrate binding site and a regulator of the dual-affinity transport activity (Parker and Newstead, 2014; Sun et al., 2014). We observed that the His residue corresponding to His-356 in At-NPF6.3 was conserved in Zm-NPF6.6 (His-362) but not in Zm-NPF6.4, which contained a Tyr residue (Y370; Supplemental Figure 1B). Given the similar functional characteristics of At-NPF6.3 and Zm-NPF6.6 when expressed in X. laevis oocytes, it is possible that the substitution of the His residue by Tyr in Zm-NPF6.4 rendered the observed loss of the high-affinity nitrate transport activity and potentially its lack of selectivity for nitrate.

To test this hypothesis, we replaced Tyr-370 of Zm-NPF6.4 with His (Y370H). This resulted in saturable transport kinetics when exposed to a wide nitrate concentration range (0–20 mM, Km, 87 µM; Figure 5A). We also examined the influence of His introduction on the chloride transport affinity in Zm-NPF6.4. Interestingly, the saturable 36Cl− uptake of Zm-NPF6.4-injected oocytes was compromised by the Y370H mutation, resulting in a more linear chloride uptake (Figure 5B).

Figure 5.
Figure 5.

Nitrate and Chloride Transport Activity of Maize NPF6.4:Y370H.

(A) Kinetic analysis of NPF6.4:Y370H nitrate transport activity. Net nitrate uptake values of NPF6.4:Y370H-injected oocytes from 25 μM to 20 mM were fitted by the Michaelis-Menten equation. Uptake values at low concentrations are shown in the figure inset. n = 7 oocytes.

(B) Kinetic analysis of NPF6.4:Y370H chloride transport activity. Net chloride uptake values of NPF6.4:Y370H-injected oocytes from 50 μM to 1 mM were fitted by a linear regression analysis. n = 7 oocytes.

Data represent net substrate uptake values with water-injected control values subtracted. Values indicate means ± se.

We then performed substrate competition flux experiments between nitrate and chloride on Zm-NPF6.4Y370H-injected oocytes. The replacement of Tyr-370 with His abolished the chloride competition of 15N-nitrate uptake (10 mM; Figure 3A). The slope of the linear regression of Zm-NPF6.4 nitrate uptake against external Cl− concentration changed from −1.93 (Zm-NPF6.4) to 0.03 (Zm-NPF6.4:Y370H) nmol (NO3) oocyte−1 h−1 mM (Cl)−1, a value close to that of Zm-NPF6.6 (0.06; Figure 3A).

By contrast, the Y370H mutation introduced a strong nitrate competition against 36Cl− uptake (1 mM) in Zm-NPF6.4-injected oocytes (Figure 3B). His substitution in Zm-NPF6.4 reduced the uptake of Cl− by 1.48 nmol oocyte−1 h−1 per mmol of nitrate in the medium (Figure 3B), a trend similar to that displayed by Zm-NPF6.6 and At-NPF6.3 (Figure 3B; Supplemental Figure 4B).

In summary, Zm-NPF6.4:Y370H introduced high-affinity nitrate transport activity and changed the substrate selectivity of Zm-NPF6.4 to prefer nitrate over chloride. A reverse approach was tested with Zm-NPF6.6 in which H362 was changed to Tyr (H362Y). Unexpectedly, the mutation resulted in a loss of nitrate transport at 250 µM and 10 mM (Supplemental Figures 5A and 5B) and a loss of chloride uptake at 1 mM (Supplemental Figure 5C).

NPF6.4 and NPF6.6 Are Plasma Membrane Proteins

Both Zm-NPF6.4 and Zm-NPF6.6 have 12 predicted transmembrane domains, suggesting that both are membrane-associated proteins (Supplemental Figure 1B). We investigated the subcellular localization of Zm-NPF6.4 and Zm-NPF6.6 using both transient expression in onion (Allium cepa) epidermal cells (Figure 6A) and in maize mesophyll cells (Figure 6B) of yellow fluorescent protein (YFP) fused to the C terminus of Zm-NPF6.4 or Zm-NPF6.6 under the control of the cauliflower mosaic virus 35S RNA promoter. To help define localization, the enhanced cyan fluorescent protein plasma membrane marker (ECFP-Rop11) (Molendijk et al., 2008) was coexpressed with the Zm-NPF:YFP constructs. For both the proteins, YFP signal was localized to the cell periphery and, when tested in onion, overlapped with the ECFP signal (Figure 6A). In a high osmotic buffer, the YFP signal was primarily localized to the plasma membrane of onion epidermal cells with a slight presence in the Hechtian strands, which connect plasma membrane with the Hechtian reticulum of the cell wall (Figure 6A) (Lang-Pauluzzi and Gunning, 2000). In a corresponding study, YFP signal for both Zm-NPF6.4 and Zm-NPF6.6 were identified predominantly at the plasma membrane of transformed mesophyll cells (Figure 6B).

Figure 6.
Figure 6.

Subcellular Localization of Maize NPF6.4 and NPF6.6.

Maize NPF6.4 and NPF6.6 are localized on the plasma membrane of onion epidermal cells (A) and maize protoplasts (B). White or black arrows indicate YFP- or ECFP-stained Hechtian strands during plasmolysis of Zm-NPF6.6 and ECPF:Rop11. Bars = 100 μm in (A) and 10 μm in (B).

Nitrate Regulates NPF6.6 Expression in the Root

The expression patterns of Zm-NPF6.4 and Zm-NPF6.6 were examined in 11-d-old maize seedlings using quantitative RT-PCR. Plants were grown in 2.5 mM NH4NO3 for 7 d and then grown for 4 d either in a nitrogen-free nutrient solution or 2.5 mM NH4NO3 (Figure 7A). Zm-Actin, Zm-ElF1, and Zm-GaPDh were used as endogenous reference controls. In the presence or absence of nitrogen, Zm-NPF6.4 expression did not vary in either the root or the shoot tissues (Figure 7A; Supplemental Figure 6). In contrast, the expression of Zm-NPF6.6 was enhanced in roots with a nitrogen supply (Figure 7A), with no change in expression levels in the shoots. A subsequent experiment tested the induction of Zm-NPF6.6 by treating plant starved of nitrogen with either NO3−, NH4+, or Cl− (5 mM) for a 4-h period. Only NO3− induced Zm-NPF6.6 expression in roots (Figure 7B). The nitrate-induced upregulation of Zm-NPF6.6 was rapid, evident within 30 min before peaking and stabilizing after 120 min (Figure 7B, inset). In the shoot, Zm-NPF6.6 expression was very low relative to root tissues and did not respond to nitrogen treatment (Figure 7B).

Figure 7.
Figure 7.

Regulation of Maize NPF6.4 and NPF6.6 Expression.

(A) NPF6.6 was highly expressed in roots under high nitrogen conditions. Significance was determined using an unpaired t test. n = 4 plants.

(B) NPF6.6 expression responded to nitrate. Figure inset highlights the rapid induction of NPF6.6 by nitrate, starting at 30 min after resupply and peaking at 2 h. n = 4 plants.

Significance was determined using an unpaired t test (A) or a one-way ANOVA (B) (Supplemental File 1). Values represent means ± se.

Cellular localization of Zm-NPF6.4 and Zm-NPF6.6 was determined using in situ PCR with 7-d-old B73 maize tissues, including leaf blades and roots (Figure 8). Cross sections of leaves revealed Zm-NPF6.4 was expressed at low levels in stomata and bundle sheath cells. In contrast, Zm-NPF6.6 expression was more pronounced, with signal evident in multiple cell types of leaves. In root sections, both Zm-NPF6.4 and Zm-NPF6.6 showed staining across the section increasing in the stele relative to the cortex and epidermal cell layers (Figure 8).

Figure 8.
Figure 8.

Cellular Localization of Maize NPF6.4 and NPF6.6.

In situ PCR analysis of NPF6.4, NPF6.6, and 18S RNA gene expression in leaf and root tissues of 7-d-old B73 maize seedlings. Plant samples were stained for 3 h to develop sufficient signal intensity relative to the highly expressed 18S RNA control. ST, stomata; BS, bundle sheath.

DISCUSSION

Multiple NPF nitrate transporters have been isolated and characterized from model plant systems, including Arabidopsis, M. truncatula, and rice (Oryza sativa) (Tsay et al., 2007). The most well studied NPF is At-NPF6.3, which has been characterized as a bidirectional dual-affinity nitrate transceptor and as an auxin transporter. The protein has multiple activities, which include both high- and low-affinity nitrate transport, interactions with the regulatory control of high-affinity NRT2 expression, lateral root elongation, nascent organ development, and stomatal opening (Guo et al., 2001, 2003; Remans et al., 2006; Ho et al., 2009; Krouk et al., 2010; Léran et al., 2013). Our focus is to understand the mechanism of action of these types of transporters at biochemical and molecular levels in crop plants of economic and social impact. In this study, we identified and characterized in detail two maize NPF proteins that are homologs of At-NPF6.3. These homologs, Zm-NPF6.4 and Zm-NPF6.6, share 67% identity between themselves and ∼62% with At-NPF6.3.

Using a combination of approaches, including electrophysiological and chemical flux analysis in X. laevis oocytes, we demonstrated that, although Zm-NPF6.4 and Zm-NPF6.6 were both pH-dependent nitrate transporters (Figures 1B and 1C), they differed in their affinities for nitrate. Zm-NPF6.4 facilitated nitrate transport in a linear fashion reminiscent of a low-affinity transport pathway (Figure 1G). In contrast, Zm-NPF6.6 transported nitrate across a broad concentration range. Detailed analysis of both high- and low-affinity ranges (Figures 1H to 1K) would support the idea that transport follows typical Michaelis-Menten monophasic saturation kinetics without the distinct dual-affinity (high and low affinity) activity previously reported for At-NPF6.3 (Liu et al., 1999). We also tested for dual-affinity nitrate transport activity in At-NPF6.3, but failed to see a clear distinction between HATS and LATS activities (Supplemental Figure 7). Previous reports have also indicated At-NPF6.3 is a bidirectional transporter (Léran et al., 2013). When tested, we observed nitrate efflux activity with Zm-NPF6.4 and At-NPF6.3 but not Zm-NPF6.6. Interestingly, a pH-dependent chloride transport activity was also found in both Zm-NPFs (Figures 2A and 2B). Zm-NPF6.4 was able to transport chloride in a high-affinity saturable manner, whereas Zm-NPF6.6 displayed only linear low-affinity chloride transport activity (Figures 2C and 2D). This is direct evidence that NPF6.3 homologs are capable of chloride transport, a capacity first predicted by Schroeder (1994). Substrate competition assays indicated that Zm-NPF6.4 was more selective for chloride, whereas Zm-NPF6.6 showed a preference for nitrate (Figure 3). NPF proteins have been linked to plant hormone transport, including that of auxin in root tips by At-NPF6.3 (Krouk et al., 2010). Neither Zm-NPF6.4 nor Zm-NPF6.6 displayed significant uptake of auxin into oocytes under our experimental conditions (Supplemental Figure 11).

Transient expression experiments in onion epidermal cells and maize mesophyll cells demonstrated that both Zm-NPF6.4 and Zm-NPF6.6 were plasma membrane proteins (Figure 6). Using in situ PCR, Zm-NPF6.4 and Zm-NPF6.6 were both shown to be expressed in root and shoot tissues (Figure 8). Zm-NPF6.4 had limited expression in bundle sheath cells in leaves, whereas Zm-NPF6.6 was much more highly expressed across most cell types of the leaf. In roots, both genes showed broad levels of expression across the root cellular profile, and both genes showed elevated levels of expression in the central stele. Quantitative measurements of mRNA abundance of Zm-NPF6.4 and Zm-NPF6.6 in root and shoot tissues revealed that Zm-NPF6.4 expression was relatively stable in root and shoot tissues and did not respond to either nitrogen starvation or nitrate and ammonium resupply (Figure 7; Supplemental Figure 6). In contrast, the expression of Zm-NPF6.6 was mainly limited to the roots and was responsive to nitrogen supply in that it was downregulated by nitrogen starvation but upregulated specifically by nitrate resupply (Figure 7B).

Thr Phosphorylation Disrupts Transport Activity in NPFs

It was shown that the phosphorylation status of Thr-101 in At-NPF6.3 regulates its dual-affinity transport activity (Liu and Tsay, 2003). Phosphorylation represses low-affinity activity, while dephosphorylation renders the high-affinity transport inactive (Liu and Tsay, 2003). Further evidence to support this hypothesis was observed in crystal structures of At-NPF6.3 (Parker and Newstead, 2014; Sun et al., 2014). Sun et al. (2014) reported that At-NPF6.3 was most likely a functional dimer in which phosphorylation of T101 acted as a dimerization switch favoring the monomeric form, possibly through the interactions between the N-terminal faces of the two monomers. Parker and Newstead (2014) suggested the phosphorylation of T101 localized in TM3 increased the structural flexibility of At-NPF6.3 by distorting the packaging of TM3 with TM1 and TM4 in the N-terminal bundle. Increased flexibility or a shift to the monomeric form was predicted to enhance the substrate transport rate of the protein and favor the activation of the high-affinity transport pathway. We introduced a similar phosphomimetic mutation (Thr to Asp) and the dephosphorylation mutation (Thr to Ala) into Zm-NPF6.6 and Zm-NPF6.4 (Figures 4A and 4B). In Zm-NPF6.6, both mutations retained similar saturable high-affinity activity, suggesting that T104 phosphorylation did not alter the transport-substrate binding directly. However, we observed a shift in the Km for nitrate for both mutants (from 260 µM to 160 and 150 µM) for T104D and T104A, respectively (Figure 4A). These changes in activity would appear to be independent of the phosphorylation status of T104. We then examined the chloride transport activity of Zm-NPF6.4 using similar modifications (T106A and T106D). With both mutations, the high-affinity saturable Cl− transport activity of Zm-NPF6.4 (Figure 2C) was replaced with a linear uptake pattern (Figure 4B), suggesting a direct impact on chloride binding.

These results contrast with the previous observation where a Thr-to-Ala mutation introduced in At-NPF6.3 abolished the high-affinity nitrate transport activity and a Thr-to-Asp mutation compromised the low-affinity activity (Liu and Tsay, 2003). The variation in response to phosphorylation status suggests further investigation is required to define whether other sites exist and how phosphorylation influences conformational change and its relationship between high- and low-affinity activities.

Key Residues Regulate the Transport Activity and Substrate Selectivity of NPFs

The ability of Zm-NPF6.4 and Zm-NPF6.6 to facilitate both nitrate and chloride transport highlighted the potential for structural conservation between the two proteins. In contrast, the differences in activity and substrate selectivity at lower concentrations indicated the proteins evolutionarily diverged at the substrate binding sites. Our findings reveal that Zm-NPF6.4 was similar to a high-affinity chloride transporter but also had the capacity to transport nitrate in a low-affinity manner, whereas Zm-NPF6.6 seemed to be a saturable high-affinity nitrate transporter with a low-affinity chloride activity. High-affinity activities dictated substrate selectivity in which Zm-NPF6.4 preferred chloride to nitrate while Zm-NPF6.6 was a nitrate-selective protein (Figure 3). We conclude that Zm-NPF6.4 is different from Zm-NPF6.6 and that Zm-NPF6.6 is most likely a distant but functional ortholog of At-NPF6.3.

In the crystal structure of At-NPF6.3, H356 was suggested to be a nitrate binding and/or recognition site, achieving the 2 H+/1 NO3− symport mechanism through proton binding at both the EXXER motif of TM1 and H356 directly (Parker and Newstead, 2014; Sun et al., 2014). Protonation of H356 facilitated nitrate binding in the pore region of the protein (Parker and Newstead, 2014; Sun et al., 2014). We found that the EXXER motif was conserved among At-NPF6.3, Zm-NPF6.4, and Zm-NPF6.6 (Supplemental Figure 1B), but an equivalent His (H362) was only identified in Zm-NPF6.6 (Supplemental Figure 1B). In contrast, Zm-NPF6.4 harbored a Tyr (Y370) in the same location (Supplemental Figure 1B). Structural threading of both Zm-NPF6.4 and Zm-NPF6.6 against the apoprotein model of At-NPF6.3 (Sun et al., 2014) revealed that both His-362 and Tyr-370 faced the proposed nitrate binding area of the protein (Supplemental Figures 8A and 8B). However, a substrate binding bond can only be predicted in Zm-NPF6.6 between the protonated H362 and nitrate (Supplemental Figure 8B), suggesting this His in Zm-NPF6.6 shares a similar nitrate binding role with that of H356 of At-NPF6.3. In contrast, the presence of Y370 in Zm-NPF6.4, might act to limit high-affinity nitrate transport because of restrictions in its ability to be protonated (Y370: pKa ∼10) or through limited electrostatic interactions with nitrate ions. Indeed, no Y370-nitrate interaction could be predicted in the Zm-NPF6.4 structure model (Supplemental Figure 8A). However, because Zm-NPF6.4 did transport chloride in a high-affinity manner, we suggest that Zm-NPF6.4 most likely retained a separate substrate binding and protonation site for chloride transport.

The transformation of a nitrate-specific transporter to a nonselective nitrate and chloride transporter was previously achieved by introduction of a single amino acid substitution in the substrate selective filter of the Arabidopsis vacuole nitrate accumulator At-CLCa (Bergsdorf et al., 2009; Wege et al., 2010). Following this lead, a His was introduced in Zm-NPF6.4 (Y370H). This mutation created a high-affinity nitrate transport activity (Km ∼87 µM) and changed the specificity of the transporter to prefer nitrate over chloride (Figures 3 and 5A), probably through a newly formed Y370H-nitrate interaction (Supplemental Figure 8C). These results further highlight the importance of the His residue in the binding pocket and for high-affinity nitrate transport. It is interesting to note that the majority of the characterized NPF nitrate transporters do not have a His (or other amino acids with a potential positive charge) at the equivalent site of At-NPF6.3:H356, as most identified residues are hydrophobic (Supplemental Figure 9). Further testing is required to confirm if saturable nitrate transport across NPF homologs is strictly dependent on the presence of a conserved His residue. This also raises the question on the necessity of a nitrate binding site for low-affinity anion transport. Our results with Zm-NPF6.4 would suggest a nitrate binding site might not be required for nitrate LATS activity. The ability to create high-affinity nitrate transport activity in Zm-NPF6.4 suggested that the nitrate binding His motif could be introduced into other NPF proteins, for example, the petiole-expressed At-NPF6.3 homolog, At-NPF6.2, which is currently characterized as a low-affinity transporter (Chiu et al., 2004). Structural predictions would suggest insertion of a His motif may function as observed with Zm-NPF6.4. (Supplemental Figures 8E and 8F).

A Model for NPF Activity

To summarize our current findings, we propose a revised NPF activity model (Figure 9). High-affinity anion transport in NPF proteins requires both the proton-coupling mechanism and the presence of a substrate binding residue, such as H362 in Zm-NPF6.6 and H356 in At-NPF6.3 for nitrate in the pore region of the protein. For chloride transport, we suggest that Zm-NPF6.4 utilizes a different substrate binding residue or domain (described as X), which remains to be identified (Figures 9A and 9C). We predict that both residues are protonated and interact specifically with either nitrate or chloride, respectively, through a potential charge-charge interaction that stabilizes the anion and facilitates transport through the pore. The nitrate interactions of Zm-NPF6.6 and Zm-NPF6.4 (Y370H) may also be supported by the conserved substrate recognition residues E479 and E493, respectively (Supplemental Figures 8A and 8C), which are previously described in At-NPF6.3 (E476) as being localized on the C-terminal bundle of the protein (Parker and Newstead, 2014; Sun et al., 2014). We hypothesize that, when a nitrate selective His (Y370H) is introduced into Zm-NPF6.4 pore region, the chloride selectivity of residue X (Km ∼390 µM) is superseded by the high-affinity nitrate binding residue (Km ∼87 µM; Figures 5B and 9C ; Supplemental Figure 8C). A prerequisite for NPF activity is a proton-coupling (2H+ per NO3−) transport mechanism achieved through both the EXXER proton binding domain and the protonation of the substrate binding site. Together, these form the driving force for substrate transport in the direction of the favorable proton electrochemical gradient, which is less negative inside the cell (∼pH 7.1) than outside (∼pH 5.5). The conversion of H362Y in Zm-NPF6.6 disrupted both high- and low-affinity nitrate transport. We suggest the loss of the residue H362, which has an ionizable side chain with a pKa of ∼6.4, disrupted the proton coupling mechanism in Zm-NPF6.6 resulting in a total loss of function (Figure 9D; Supplemental Figure 8D).

Figure 9.
Figure 9.

NPF Substrate Selectivity Model.

The protonatable unknown residue X and H362 specifically bind chloride or nitrate in NPF6.4 (A) and NPF6.6 (B), respectively, creating an unique substrate selectivity for each of the two transporters. When a histidine is introduced into NPF6.4, the chloride selectivity is suppressed because H370 has a higher affinity for nitrate (∼87 μM) than that of residue X for chloride (∼390 μM) (C). In Zm-NPF6.6, in the absence of H362 and the alternative protonatable residue, the fundamental proton-coupling transport mechanism is undermined, resulting in loss of function (D).

Phosphorylation of T101 in At-NPF6.3 is thought to cause N-terminal bundle structural distortion in the intracellular side of the protein (Parker and Newstead, 2014). Such conformational change is not expected to alter the substrate binding properties of the protein (Parker and Newstead, 2014). Our results with Zm-NPF6.6:T104A/D show that the high-affinity activity facilitated by H362 nitrate binding remains active regardless of phosphorylation status albeit with a shift to a lower Km (higher affinity) for nitrate. In contrast, similar mutations of Zm-NPF6.4:T106A/D resulted in a loss of high-affinity chloride transport. Further investigation is required to both localize the chloride binding residue in Zm-NPF6.4 and understand how localized conformational change caused by T106 phosphorylation ultimately influences chloride binding.

Functional Roles of NPF6.4 and NPF6.6 in Maize Nutrient Transport

Based on the data from this study, we hypothesize that Zm-NPF6.4 is likely to be a component of the plant root chloride uptake system. Chloride is required by plants as an essential micronutrient for photosynthesis and as an osmoticum in turgor and osmoregulation (White and Broadley, 2001; Wege et al., 2017). In plant root cells, proton-coupled or pH-dependent chloride influx activities have been observed using electrophysiological studies (Felle, 1994; Tyerman and Skerrett, 1999). Several chloride permeable proteins have been cloned and characterized, including the recent NPF family members, At-NPF2.4 and At-NPF2.5, which are linked to root-to-shoot chloride translocation and chloride root exclusion, respectively (Li et al., 2016, 2017). However, the molecular identity of the root proton/chloride symport activity has remained elusive. Our results suggest that Zm-NPF6.4 is a plasma membrane-localized, high-affinity, proton-coupled, chloride-selective transporter in roots. It is likely to fulfill the role of plant root high-affinity chloride influx. The relative impact of Zm-NPF6.4 on net chloride uptake will be influenced partly by the role of Zm-NPF6.6, which can also transport chloride but which is mitigated by the antagonistic impact of nitrate. We found that chloride transport by Zm-NPF6.4 was not inhibited by increasing external nitrate concentrations up to 1 mM. This suggests that when the soil solution is dominated by nitrate, Zm-NPF6.4 can continue to accumulate chloride in the high-affinity range, whereas Zm-NPF6.6 continues to facilitate nitrate uptake.

A second function of Zm-NPF6.4 may be related to transport of chloride into guard cells. Stomatal opening is associated with proton-coupled chloride influx across the plasma membrane of guard cells (Cosgrove and Hedrich, 1991; Assmann and Wang, 2001; White and Broadley, 2001). However, the chloride transporter (a proton/chloride symporter) responsible for the chloride influx into guard cells has yet to be identified. Given the pH-dependent high-affinity chloride-selective transport activity of Zm-NPF6.4 together with its expression in maize leaves (Figure 8) (Sekhon et al., 2011), the data suggest Zm-NPF6.4 may be a candidate for chloride transport across the guard cell plasma membrane.

Zm-NPF6.6 is a high-affinity nitrate selective transporter predominantly expressed in the root and rapidly (within 30 min) responds to exogenous nitrate supply (Figures 1 and 7). This expression pattern is very similar to that of At-NPF6.3 (Tsay et al., 1993). In Arabidopsis, the dual-affinity At-NPF6.3 mainly contributes to the root LATS nitrate uptake and its rapid nitrate induction, as a part of the primary nitrate response. This response presumably allows the plant to absorb and utilize nitrate efficiently according to the localized nitrogen status (Huang et al., 1996; Ho et al., 2009; Glass and Kotur, 2013; Krapp et al., 2014). Although our data do not demonstrate a clear dual-affinity activity, we suggest that Zm-NPF6.6 plays a similar role in maize root nitrate uptake across both the HATS and LATS nitrate transport activity ranges.

At-NPF6.3 has also been characterized as a nitrate sensor that regulates gene expression linked to the primary nitrate response (Ho et al., 2009) and stimulates lateral root growth in response to nitrate through a nitrate-inhibited auxin transport mechanism (Remans et al., 2006; Krouk et al., 2010). Interestingly, a membrane targeting mutant of At-NPF6.3 (P492L) could still induce the expression of At-NRT2.1 during the primary nitrate response even without its membrane location or ability to transport nitrate or auxin (Bouguyon et al., 2015). This suggests that the role of At-NPF6.3 in modulating either lateral root elongation or gene regulation during the primary nitrate response involves two independent nitrate sensing/transduction mechanisms that coexist in At-NPF6.3 (Bouguyon et al., 2015). Zm-NPF6.6 did not transport auxin (Supplemental Figure 11), which may limit its role in lateral root development in response to nitrate supply. Both maize and Arabidopsis share a similar primary nitrate response where Zm-NRT2.1 expression in maize lateral roots is induced by nitrate (Liu et al., 2008). Whether Zm-NPF6.6 acts as a nitrate sensor through regulation of gene expression during the primary nitrate response is currently not known and requires further investigation.

The interaction between nitrate and chloride uptake in plant roots has been well documented across a broad range of plant species (see reviews and references in Xu et al., 2000; White and Broadley, 2001; Wege et al., 2017). In most situations, nitrate is a strong antagonist of chloride uptake into plant roots (as shown in this study with Zm-NPF6.6 injected oocytes). For example, in barley, pre-exposure to nitrate (3 to 6 h) significantly reduces chloride uptake in the presence of external nitrate (Glass and Siddiqi, 1985). Interestingly, this inhibition is overcome when plants are not provided a nitrate pretreatment (Glass and Siddiqi, 1985). In another example, nitrate has been shown to be an effective competitor of chloride uptake in soybean (Glycine max), providing a possible mechanism that could be used to alleviate chloride toxicities (Guo et al., 2017). Dhugga et al. (1988) demonstrated that both nitrate and chloride uptake into excised maize roots can be inhibited by phenylglyoxal. Collectively, these physiological responses suggest that root influx of these two monovalent anions could be facilitated by the similar transport systems (Teakle and Tyerman, 2010).

In this study, we identified two maize NPF6 transporters, Zm-NPF6.4 and Zm-NPF6.6, which are permeable to both nitrate and chloride and have contrasting substrate selectivity. Possibly, the nitrate-induced and nitrate-selective Zm-NPF6.6 has an indirect responsibility for reducing chloride uptake in maize, particularly when roots are exposed to a high nitrate supply as has been shown indirectly in barley (Glass and Siddiqi, 1985). Furthermore, we were able to transform the chloride selective Zm-NPF6.4 into a nitrate-selective transporter by introducing a His-370 residue. This modification could be used to reduce chloride uptake if applied in planta. Despite maize being a generally chloride-tolerant species, a similar approach could be extended not only to other chloride susceptible crops, such as grapevine (Vitis vinifera), soybean, citrus (Citrus medica), and tomato (Solanum lycopersicum), but also to areas where chloride concentrations can become a problem to improve chloride tolerance (e.g., growth in arid areas subject to saline irrigation and/or rain-fed irrigation influenced by a nearby ocean).

In conclusion, we have determined that a His residue confers high-affinity nitrate transport activity in Zm-NPF6.6, and this residue can be introduced into a high-affinity chloride-specific transporter Zm-NPF6.4, changing its substrate selectivity from chloride to nitrate. This discovery has implications in altering chloride transport activity from the nitrate transporters possibly as a targeted trait to improve salt tolerance in plants. Furthermore, our results have advanced the understanding of the mechanism of nitrate and chloride transport in plant cells.

METHODS

Sequence Alignments

NPF protein amino acid sequences were obtained from The Arabidopsis Information Resource and maizesequence.org. NPF6 proteins were aligned using the Geneious R7 software alignment tool (Biomatters) with gap open penalty 12, gap extension penalty 3, and refinement iteration 2.

Seed Germination and Seedling Growth Conditions

Maize (Zea mays) B73 seeds were imbibed in aerated water for 4 h. Seeds were planted in wet diatomite rock and covered with aluminum foil and allowed to germinate (20°C for 4 d). Germinated seeds were transferred to the hydroponic system filled with growth basal solution (0.5 mM MgSO4, 0.5 mM KH2PO4, 25 µM H3BO3, 2 µM MnSO4, 2 µM ZnSO4, 0.5 µM CuSO4, 0.5 µM Na2MoO4, 1.05 mM KCl, 0.1 mM Fe-EDTA, 0.1 mM FeEDDHA, 1.25 mM K2SO4, 0.25 mM CaCl2, and 1.75 mM CaSO4, pH 5.9) supplemented with 2.5 mM NH4NO3 and grown for 7 d in a temperature-controlled growth chamber with a 16-h/8-h light/dark regime at 20°C. Individual plants were harvested, separated into root and shoot tissues, and rapidly frozen in liquid nitrogen and stored at −80°C.

RNA Extraction and Reverse Transcription

Frozen plant tissues were individually ground in a mortar and pestle cooled with liquid nitrogen. Total RNA was extracted from 100 mg of frozen ground tissue from individual plants using TRIzol reagent (Ambion) following the manufacturer’s instructions. RNAs were quantified and qualified using a ND-1000 spectrophotometer (Nanodrop). One microgram of total RNA from each sample was used for cDNA synthesis, using SuperScript III reverse transcriptase (Invitrogen) and the oligo(dT) primer, following the manufacturer’s instructions.

Molecular Cloning and Mutagenesis

Zm-NPF6.4 and Zm-NPF6.6 full-length coding sequences were amplified from maize B73 cDNA using Platinum Taq DNA polymerase (Invitrogen). cDNA was originally generated from purified total RNA using reverse transcriptase. Amplified PCR products were inserted into pCR8/GW/TOPO vector (Invitrogen) and verified sequences (B73 reference genome) subcloned into a Xenopus laevis oocyte expression vector, pGEMHE (Liman et al., 1992), using LR Clonase II (Invitrogen) following the manufacturer’s instructions. Sequences of the PCR primers used are listed in Supplemental Table 1. The pCR8-ZmNPF6.4:T106A, pCR8-ZmNPF6.4:T106D, pCR8-ZmNPF6.6:T104A, pCR8-ZmNPF6.6:T104D, pCR8-ZmNPF6.4:Y370H, pCR8-ZmNPF6.4:Y370A, pCR8-ZmNPF6.6:H362Y, and pCR8-ZmNPF6.6:H362A constructs were generated using Phusion polymerase (Thermo Scientific) alongside a point-directed mutagenesis PCR technique. Sequences of the primers used are listed in Supplemental Table 1.

Functional Analysis in X. laevis Oocytes

The pGEMHE-ZmNPF constructs were linearized using SphI or SbfI. cRNA was synthesized in vitro using the mMESSAGE mMACHINE T7 kit (Ambion). Oocytes were injected with 46 nL (23 ng) of cRNA or 46 nL water with a Nanoinject II microinjector (Drummond Scientific) and incubated for 2 d in a modified Ringers solution containing 96 mM NaCl, 2 mM KCl, 5 mM MgCl2, 5 mM HEPES, 0.6 mM CaCl2, 5% horse serum (Sigma-Aldrich), 1% penicillin-streptomycin (Sigma-Aldrich), and 50 mg/L tetracycline, pH 7.6. For all oocyte experiments, we routinely generated 10 to 20 individual cRNA injected oocytes per treatment to compensate for those that degrade after the injection procedure and postincubation period.

For electrophysiological studies, a method described by Huang et al. (1999) was modified and adopted. Briefly, oocytes were initially impaled by two microelectrodes to measure the membrane potential in the basal solution containing 0.15 mM Ca2+ and 3 mM MES, pH 7.5 adjusted with Bis-Tris propane, osmolality 230 mmol/kg adjusted with mannitol. Healthy oocytes with a membrane potential < −30 mV were then perfused in a similar basal solution with pH 5.5 for ∼5 to 10 min until the membrane potential became stable. Currents were recorded before and after changing the solution from basal solution to either nitrate or chloride solutions (basal solution supplemented with 10 mM HNO3 or 5 mM HCl, pH 5.5 or 7.5, osmolality 230 mmol/kg adjusted with mannitol). Oocytes were clamped with 600-ms pulses from −120 to +20 mV with 20-mV increments. Between each pulse, oocytes were clamped to −40 mV for 180 ms. Nitrate or chloride elicited currents were calculated by subtracting the current recorded in basal solution from the current recorded in nitrate or chloride solution, representing the actual current change caused by nitrate or chloride in the single oocyte. Currents were recorded just after first contact with nitrate or chloride solutions to minimize extended incubations which limits substrate elicited currents. Clamping and measurements were achieved using an OC-725 Oocyte Clamp (Warner Instrument) and Digidata 1440A digitizer (Axon) with the pCLAMP program, respectively.

For nitrate flux experiments, oocytes were washed three times with basal solution (pH 7.5) and transferred to the 15N-labeled (10%) nitrate solution (basal solution supplemented with 0.25 mM or 10 mM Na15NO3 [10%; Sigma-Aldrich], pH 5.5 or 7.5). After 1 h (nitrate uptake was in the linear phase; Supplemental Figure 10A), oocytes were washed three times with ice-cold basal solution (pH 7.5) and dried individually in tin capsules at 60°C for 3 d. The capsules were sealed and analyzed for the % nitrogen and the 14N/15N ratios using an isotope-ratio mass spectrometer (Sercon 20-20) coupled to a front-end elemental analyzer (Sercon). In the kinetic study, oocytes were incubated in nitrate solutions with 12 different concentrations (25 µM, 50 µM, 100 µM, 150 µM, 250 µM 400 µM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, and 30 mM, pH 5.5) for 1 h.

For nitrate efflux experiments, oocytes were injected with 46 nL 15N-labeled (10%) nitrate (1 M) to achieve a final intracellular nitrate concentration of 10 mM. Oocytes were incubated in ND96 (pH 7.5) solution for a 30-min efflux period and then collected from the bath solution. Retained 15N nitrate in each oocyte was analyzed as described above.

For chloride flux experiments, similar protocols were used as described in the nitrate flux experiments. Before each experiment, radioactive Na36Cl (Amersham Biosciences) was added into the uptake solution to a final concentration of 0.1% (v/v). After 1 h incubation (incubation time was optimized; Supplemental Figures 10B and 10C), each oocyte was washed three times with ice-cold basal solution (pH 7.5) and dissolved in 100 μL of 10% SDS in a 5 mL scintillation vial. Incorporated radioactivity of each oocyte was quantified using a liquid scintillation analyzer (TRI-CARB 2100TR). In the kinetic study, oocytes were incubated in chloride solutions with eight different concentrations (50 µM, 150 µM, 300 µM, 500 µM, 700 µM, 1 mM, and 2 mM, pH 5.5) for 1 h.

For 15NO3−/Cl− competition flux experiments, oocytes were incubated in nitrate solution (10 mM) with Cl− in a series of increasing Cl−/15NO3− ratios (from 0% to 100%) for 1 h. For 36Cl−/NO3− competition flux experiment, instead of using 10 mM, 1 mM NaCl was added into the uptake solution to minimize the impact of endogenous X. laevis oocyte Cl− channels on the measured Zm-NPF chloride uptake (Machaca and Hartzell, 1998; Weber, 1999). The rest of the method is similar for both nitrate or chloride flux experiments.

To measure auxin influx, the method from Yang et al. (2006) was modified and adopted. Oocytes were incubated in modified Ringers solution (pH 7.5) supplemented with 1 µM 3H-IAA (10%; Perkin-Elmer) for 1 h. Incorporated radioactivity of each oocyte was quantified using a liquid scintillation analyzer (TRI-CARB 2100TR).

Subcellular Localization

Biolistic transformation of onion epidermal cells was performed as described (Campos-Soriano et al., 2011; Chiasson et al., 2014). Briefly, Zm-NPF6.4 or Zm-NPF6.6 was subcloned into the pBS-35S-attR-YFP vector and coated onto 0.6-µm gold microcarriers along with a plasma membrane marker, ECFP:Rop11 (Subramanian et al., 2006; Molendijk et al., 2008). Then, the DNA-coated gold microcarriers were bombarded into onion epidermal cells using a biolistic PSD-1000/He particle delivery system (Bio-Rad). After 12 to 24 h, onion peels were visualized using by a LSM 5 PASCAL laser scanning microscope (Zeiss) through two band-pass filters, 470 to 500 nm for ECFP and 570 to 590 nm for YFP. For further confirmation of the plasma membrane localization, plasmolysis was induced by treating the onion epidermal cell with 0.75 M mannitol for 15 min.

Transient expression of Zm-NPF6.4 and Zm-NPF6.6 in maize mesophyll protoplasts was performed according to Yoo et al. (2007). Briefly, mesophyll protoplasts were isolated from pooled etiolated leaves of 12-d-old maize plants and diluted to 6 × 106 protoplast/mL. Protoplasts were then transformed with pBS-35S-YFP-ZmNPF6.4 or pBS-35S-YFP-ZmNPF6.6 constructs using electroporation (2 × 4 ms at 400 V) and resuspended to 2 × 105 protoplast/mL. After 24 h dark incubation, protoplasts were visualized using a Leica SP5 II confocal microscope with an emission filter setting of 470 to 500 nm.

Quantitative RT-PCR

B73 seedlings were grown as described above. Seven-day-old seedlings were removed from the NH4NO3 solution and rinsed with reverse osmosis water before transferring to the basal solution for a 4-d nitrogen starvation treatment. The seedlings were then transferred to resupply solutions (basal solution supplemented with either 5 mM NO3−, NH4+, or Cl−) for 4 h before harvest. Maize root and shoot tissues from individual plants were separated at the coleoptile/mesocotyl interface, ground in liquid nitrogen, and stored at −80°C. RNA extraction and reverse transcription were conducted as described above. One microliter of synthesized cDNA and 4 µM forward and reverse primers was added in each reaction with SYBR Green real-time PCR master mixes (Life Technology) according to the manufacturer’s instructions. Reactions were performed in a QuantStudio 12K flex real-time PCR system (Life Technology) with an initial denaturation of 95°C for 20 s, followed by 40 cycles of (9°C for 1 s and 55°C for 20 s). One additional melting curve step was added at the end of qPCR reaction to ensure the quality of reaction. qPCR primers of Zm-NPF6.4 and Zm-NPF6.6 and three endogenous reference genes, Zm-Actin, Zm-ElF1, and Zm-GaPDh, were designed using Primer 3 software and primer efficiency were tested with threshold of 90%. Relative expression level of each gene was calculated using equation by 2−ΔCt, where ΔCt is the sample Ct subtracted by the geometric mean of the Ct of three reference genes. Sequences of the primers used are listed in Supplemental Table 1.

In Situ PCR

Zm-NPF6.4 and Zm-NPF6.6 in situ mRNA transcripts were amplified using the method with modifications of Athman et al. (2014). Maize leaf and root samples from 7-d-old seedlings were immersed in fixative containing 63% (v/v) ethanol, 5% (v/v) acetic acid, and 2% (v/v) formaldehyde for 3 and 4 h, respectively. Then samples were embedded into 5% (w/v) agarose and sectioned to 50 µm. PCR was performed with in situ PCR primers listed in Supplemental Table 1. The PCR involved a cycling parameter of 30 s initial denaturation at 98°C, 30 cycles of 98°C for 10 s, 58°C for 25 s, 72°C for 5 s, and a final extension at 72°C for 5 min. The samples were stained using BM purple AP substrate (Roche) for 3 h to develop sufficient stain (a 1.5-h staining period was found insufficient for both Zm-NPF6.4 and Zm-NPF6.6 but not the 18S control). After staining, the sections were washed and then mounted in 40% (v/v) glycerol and observed on a Leica DM2500M microscope.

Accession Numbers

Sequence data for the genes described in this article can be found in the Arabidopsis TAIR database (https://www.arabidopsis.org) or Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html) under the following accession numbers: At-NPF6.3 (AT1G12110.1), Zm-NPF6.4 (GRMZM2G086496_P01), and Zm-NPF6.6 (GRMZM2G161459_P02).

Supplemental Data

Acknowledgments

This work was supported by an Australian Research Council Linkage Grant (LP110200878) awarded to B.N.K., S.D.T., and K.S.D.

AUTHOR CONTRIBUTIONS

Z.W., B.N.K., S.D.T., and K.S.D. designed the research. Z.W., E.O., and J.D. performed the research. Z.W., B.N.K., S.D.T., and K.S.D. contributed resources and analyzed the data. Z.W. and B.N.K. wrote the article.

Footnotes

  • www.plantcell.org/cgi/doi/10.1105/tpc.16.00724

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Brent N. Kaiser (brent.kaiser{at}sydney.edu.au).

  • ↵1 Current address: International Maize and Wheat Improvement Center (CIMMYT), El Batan, C.P. 56237, Mexico.

  • Received September 20, 2016.
  • Revised July 21, 2017.
  • Accepted September 5, 2017.
  • Published September 8, 2017.

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Maize NPF6 Proteins Are Homologs of Arabidopsis CHL1 That Are Selective for Both Nitrate and Chloride
Zhengyu Wen, Stephen D. Tyerman, Julie Dechorgnat, Evgenia Ovchinnikova, Kanwarpal S. Dhugga, Brent N. Kaiser
The Plant Cell Oct 2017, 29 (10) 2581-2596; DOI: 10.1105/tpc.16.00724
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